©[2020] Anika Haq ALL RIGHTS RESERVED

©[2020]

Anika Haq

ALL RIGHTS RESERVED

APPLICATION OF SOLUBILITY-PHYSICOCHEMICAL-THERMODYNAMIC (SPT)

THEORY FOR DESIGNING A TOPICALLY APPLIED THYMOQUINONE POLYMER

FILM TO TREAT INFECTED WOUNDS

By

ANIKA HAQ

A dissertation submitted to the

School of Graduate Studies

Rutgers, The State University of New Jersey

In partial fulfillment of the requirements

For the degree of

Doctor of Philosophy

Graduate Program in Pharmaceutical Sciences

Written under the direction of

Bozena Michniak-Kohn

And approved by

New Brunswick, New Jersey

May, 2020

ii

ABSTRACT OF THE DISSERTATION

APPLICATION OF SOLUBILITY-PHYSICOCHEMICAL-THERMODYNAMIC

(SPT) THEORY FOR DESIGNING A TOPICALLY APPLIED

THYMOQUINONE POLYMER FILM TO TREAT INFECTED WOUNDS

By ANIKA HAQ

Dissertation Director:

Professor Bozena Michniak-Kohn

Skin has significant barrier properties that inhibit the passive transport of many

active molecules. Different strategies are developed to overcome this skin barrier such as,

chemical enhancement techniques using penetration enhancers and targeted drug delivery

using topical and/or transdermal formulations. Usually these approaches are tested using

human or animal skin. Human skin is not easily accessible and animal skin has significant

biological and barrier differences when compared with human skin. Due to these issues the

possibility of having a synthetic skin membrane is an attractive option. In this thesis, firstly,

we investigated different formulations containing various enhancers from the aspect of

their ability to enhance or reduce the delivery of nicotine through human cadaver skin and

correlated that to Strat-M® synthetic membrane to examine the usefulness of this

membrane as a convenient screening tool to investigate topically applied formulations and

TDDS (Transdermal Delivery System). Formulations containing nicotine and a chemical

penetration enhancer (CPE) were used for evaluating drug penetration to understand how

iii

each enhancer impacts the permeability of nicotine as a model compound. The permeability

measurements of human cadaver skin and Strat-M® membrane were performed with Franz

diffusion cell methods accompanied by HPLC analysis. The study demonstrated the good

correlation (R2=0.99) of the permeability data obtained through human cadaver skin and

Strat-M® membrane. Our data suggests that although Strat-M® lacks the highly organized

stratum corneum (SC) intercellular structure and provided higher nicotine flux compared

to human cadaver skin where the highly structured SC significantly reduced nicotine

permeability, both membranes still provided similar enhancement factors for a given

enhancer. These studies suggest that the Strat-M® synthetic membrane lipid composition

probably closely mimics that of human cadaver skin based on the data obtained. The time

point correlation between Strat-M® and human cadaver skin were in the range 0.90-0.99.

This work suggests that some of the main transport mechanisms for drug diffusion and

permeation of Strat-M® membrane could be similar to an ex vivo human skin model.

Secondly, we report on that the overall mechanism of action of skin penetration

enhancers is best explained by the Solubility-Physicochemical-Thermodynamic (SPT)

theory. The SPT theory puts forward the concept that the mode of action of enhancers is

related to solubility parameters, physicochemical interactions and thermodynamic activity.

We have discussed these concepts by using experimentally derived permeation data,

various physicochemical and solubility parameters (ingredient active gap (IAG), ingredient

skin gap (ISG), solubility of active in the formulation (SolV) and the formulation solubility

in the skin (SolS)) generated by using FFE (Formulating for EfficacyTM - ACT Solutions

Corp) software. Our data suggests that there is an inverse relationship between measured

flux and IAG values given that there is an optimum ingredient skin gap, SolV and SolS

iv

ratio. The study demonstrated that the flux is actually proportional to a gradient of

thermodynamic activity rather than the concentration and maximum skin penetration and

deposition can be achieved when the drug is at its highest thermodynamic activity. This

work will connect the solubility and physicochemical properties of the active and

enhancers/ingredients with the thermodynamic activity of the model drug used in order to

explain the mode of action of enhancers in a given formulation with that specific drug.

Thirdly, we studied the effect of an ethanol and propylene glycol donor solvent

system along with various compositions of receptor solvents to investigate the feasibility

of transdermal delivery of thymoquinone (TQ). The effects of penetration enhancers on the

in vitro skin permeation and TQ skin absorption were studied using human cadaver skin in

Franz diffusion cells. The permeation of saturated solutions of TQ was investigated with

5% v/v of each of the following enhancers: Azone (laurocapram), Transcutol® P (Tc), oleic

acid, ethanol, Polysorbate 80 (Tween 80), and N-methyl-pyrrolidone (NMP). Our data

suggests that Azone, oleic Acid and Tc were able to provide adequate TQ flux and may be

the agents of choice for use in a novel transdermal formulation of TQ. These penetration

enhancers were also able to generate TQ reservoirs in the skin that may be useful to provide

sustained release of TQ from the stratum corneum over longer periods of time. The study

also demonstrated pull or drag effect of permeation enhancers and vehicle on TQ skin

deposition. These studies suggest that ethanol was able to pull more drug into the skin and

all the enhancers used in this study showed low “pulling” effect. Rather these enhancers

(Azone, oleic acid and Tc) showed enhanced permeation as the enhancers has permeation

enhancing effect. Finally, we synthesized and characterized a biocompatible novel topical

polymeric film system that has the potential to deliver antibacterial/anti-inflammatory

v

agent thymoquinone (TQ) directly to the skin target site and that may be useful for the

treatment and management of wound infections. The polyvinyl pyrrolidone (PVP) matrix-

type films containing TQ were prepared by the solvent casting method using dibutyl

phthalate as a plasticizer and Azone (laurocapram) as a penetration enhancer. The

developed films were evaluated for thickness, drug content uniformity, weight variation,

flatness, folding endurance, percentage of moisture content and uptake which were found

to 1.17 ± 0.04 mm, 100 ± 6.4 %, 82.04 ± 1.9 mg, 100%, 68 ± 2.38, 14.12 ± 0.42 %, and

2.26 ± 0.47 % respectively. FESEM photograph of the film showed polymer networks

inside the film and a homogeneous dispersion of drug inside the polymer networks. In vitro

skin permeation studies on human cadaver skin produced a mean flux of 2.3 µg/cm2/h. In

vitro scratch assay results revealed that 100 ng of TQ had significant wound closure activity

in human dermal fibroblast cells compared to both control (p = 0.0014) and positive control

(p = 0.0004). Using human keratinocyte cell line, 100 ng TQ group showed 85% wound

closure activity at day six which was significantly higher (p = 0.0001) than the control

group. In a zone-of-inhibition (ZOI) assay, the presence of TQ-containing films completely

wiped out Staphylococcus aureus in a 10 cm in diameter TSA (Tryptone soya agar) plates

while 500 ug/mL gentamicin containing filters gave 10 mm of ZOI. In an ex vivo model,

the presence of TQ-film eradicated the bacterial colonization on human cadaver skin.

Furthermore, in the BALB/c mice wound model, TQ-films showed significant activity in

controlling Staphylococcus aureus infection and promoting wound closure compared to

control film. These results indicate, TQ/PVP films developed in this study have potential

for the treatment and management of wound infection.

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Acknowledgments

I am grateful to all of those with whom I have had the pleasure to work during my Ph.D.

dissertation studies and other related projects.

I would like to express my sincere gratitude to my advisor Dr. Bozena Michniak-Kohn,

Professor of Pharmaceutics, Ernest Mario School of Pharmacy for the continuous support

and motivation. Thank you for teaching me how to be an independent researcher and for

providing the scientific platform that helped me to build my knowledge on skin research

through attending numerous seminars, building networks and collaborations that further

developed into research projects. Thank you for trusting me and for encouraging me to

pursue my passion in science.

I am also very grateful to Dr. Suneel Kumar at Department of Biomedical Engineering,

Rutgers University for assisting me with in vitro and in vivo wound healing study and for

teaching me all the required techniques for the animal study. A sincere and heartfelt

gratitude to Dr Yong Mao, Research Associate Professor, New Jersey Center for

Biomaterials for sharing your valuable input in the bacterial study design. Both of your

invaluable knowledge, experience and expertise in your respective fields taught me various

necessary research skills. I am truly inspired by both of your pleasant personality and

scientific mind.

I would like to thank my thesis committee members, Dr. Tamara Minko, Distinguished

Professor and Chair, Ernest Mario School of Pharmacy and Dr. Leonid Kagan, Associate

Professor, Ernest Mario School of Pharmacy for their insightful comments,

vii

encouragement, valuable time and guidance throughout my thesis defense process. A

special thanks to Dr. Francois Berthiaume, Professor, Department of Biomedical

Engineering to allow me to collaborate with his research team in the wound infection

project and for providing me with all the necessary tools to conduct the animal study.

I would especially like to thank Mark Chandler (President, ACT Solutions Corp.) my

outside thesis committee member. As my mentor, he has shown me, by his example, what

a good scientist and person should be. Thank you for inspiring me with your positive

attitude and energy.

I would like to express my gratefulness to Dr. Tony Kong, Director, Graduate Program in

Pharmaceutical Science and Ms. Hui Pung, Senior Program Coordinator of Pharmaceutical

Sciences Graduate Program for their help and support throughout my graduate studies.

My sincere thank also goes to Dr. Firouz Asgarzadeh, Dr. Simone Carvalho, Mitul Patel,

Kenneth Banks and Joe Abrantes, who provided me an opportunity to join their team as

intern at Evonik Corporation, and who gave access to the laboratory and research facilities.

Without their precious support and knowledge sharing it would not be possible to conduct

my internship project.

I want to take this opportunity to thank the past and current members of Center for Dermal

Research (CDR): Ben Goodyear, Dina Ameen, Jemima Shultz, Anna Froelich, Rose

Soskind, Sonia Trehan, Parinbhai Shah, Vinam Puri and Julia Zhang; whom I have had the

joy of working with. Thank you all for being the awesome lab mates. You all made my

Ph.D. journey memorable and fun.

viii

I would also like to thank the Center for Dermal Research (CDR) for providing funding for

this research and the Berthiaume laboratory for funds for the animal studies. In addition, I

acknowledge the Division of Life Sciences, School of Arts and Sciences and Ernest Mario

School of Pharmacy at Rutgers University for providing me with a Teaching Assistantship

from 2016 to 2020.

Finally, I would like to express my love and respect for my parents for raising me as a

strong woman and as an empathetic human being. Thank you for believing in me and for

guiding me through my life. You both are my role models. You both encourage me to be

the best version of myself and to live my life up to my full potential while caring and

serving for others. You taught me to believe in the power of being happy as a whole with

everyone in my life. Most importantly, I wish to thank my loving and supportive husband,

Atik, my two beautiful sisters, Lima and Tanny, and my two wonderful nephews, Tahsin

and Tahan. You all are the blessings of my life and a great source of comfort. You all

provide unending inspiration.

ix

Table of Contents

Abstract of the Dissertation…………………………………………………………… ii

Acknowledgments………………………………………………………………………. vi

List of Tables …………………………………………………………………………. xvii

List of Figures ………………………………………………………………………… xix

Chapter 1: Background and Specific aims …………………………………………… 1

1.1. Skin physiology …………………………………………………………………….. 1

1.2. Artificial membrane ………………………………………………………………… 8

1.3. Skin penetration enhancement …………………………………………………….. 11

1.4. Topical and transdermal drug delivery ……………………………………………. 18

1.5. Thymoquinone …………………………………………………………………….. 21

1.6. Specific aims ……………………………………………………………………… 22

Chapter 2: Evaluating Strat-M® Synthetic Membrane as a Screening Tool for

Topical/Transdermal Formulation ………………………………………………….. 27

2.1. Introduction ………………………………………………………………………... 27

2.2. Materials and Methods ……………………………………………………………. 30

2.2.1. Materials ………………………………………………………………………. 30

2.2.2. Preparation of formulations …………………………………………………… 31

2.2.3. In vitro skin permeation test (IVPT) studies ………………………………….. 31

x

2.2.4. High performance liquid chromatography (HPLC) …………………………… 32

2.2.5. Data analysis ………………………………………………………………….. 33

2.2.6. Statistical analysis …………………………………………………………….. 33

2.3. Results and discussion ……………………………………………………………. 33

2.3.1. Effect of Azone ……………………………………………………………….. 34

2.3.2. Effect of Propylene glycol ……………………………………………………. 36

2.3.3. Effect of Eucalyptol and Tween 80 …………………………………………… 37

2.3.4. Effect of N-methyl pyrrolidone ………………………………………………. 38

2.3.5. Enhancement factor ………………………………………………………….. 38

2.3.6. Correlation of Strat-M membrane to human cadaver skin ……………………. 39

2.4. Conclusions ………………………………………………………………………. 40

Chapter 3: Solubility-Physicochemical-Thermodynamic Theory of Penetration

Enhancers Mechanism of Action …………………………………………………… 44

3.1. Introduction ………………………………………………………………………. 44

3.1.1. Theoretical background ………………………………………………………. 47


3.2.1. Materials ………………………………………………………………………. 51

3.2.2. Preparation of formulation and solubility determination ……………………… 52

3.2.3. Permeation procedure for enhancer studies …………………………………… 52

xi

3.2.4. Skin deposition study ………………………………………………………… 53

3.2.5. High performance liquid chromatography (HPLC) ………………………….. 53

3.2.5.1. Nicotine ………………………………………………………………….. 54

3.2.5.2. Thymoquinone …………………………………………………………… 54

3.2.6. Calculated solubility and physicochemical parameters and permeation data … 54

3.2.7. Data and statistical analysis …………………………………………………… 55

3.3. Results and discussion …………………………………………………………….. 55

3.3.1. Nicotine ………………………………………………………………………. 55

3.3.2. Thymoquinone ………………………………………………………………... 65

3.3.3. Concentration dependency of Oleic acid ……………………………………... 74

3.4. Conclusions ……………………………………………………………………….. 77

Chapter 4: Effects of Solvents and Penetration Enhancers on Transdermal Delivery

of Thymoquinone: Permeability and Skin Deposition Study ……………………… 78

4.1. Introduction ……………………………………………………………………….. 78


4.2.1. Materials ……………………………………………………………………… 80

4.2.2. Solubility determination ……………………………………………………… 81

4.2.3. In vitro skin permeation test (IVPT) studies ………………………………….. 81

4.2.4. High-performance liquid chromatography (HPLC) method development and

validation for TQ ……………………………………………………………… 82

4.2.4.1. Method characteristics ……………………………………………………. 82

4.2.4.2. Standard solutions and calibration curve …………………………………. 82

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4.2.4.3. Method validation ……………………………………………………… 83

4.2.5. Determination of TQ concentration in the skin ……………………………… 83

4.2.6. Data and statistical analysis …………………………………………………… 84

4.3. Results and discussion ……………………………………………………………. 85

4.3.1. HPLC method validation ……………………………………………………... 85

4.3.2. Thymoquinone solubility study ……………………………………………… 88

4.3.3. Effect of propylene glycol and ethanol donor solvent ……………………….. 89

4.3.4. Effect of receiver solvent composition ………………………………………. 94

4.3.5. Effect (pull or drag) of permeation enhancers and vehicle on TQ skin deposition

……………………………………………………………………………….. 96

4.4. Conclusions ………………………………………………………………………. 99

Chapter 5: Thymoquinone Loaded Polymeric Films and Hydrogels for the

Treatment of Wound Healing and Bacterial Skin Infections…………………….. 100

5.1. Introduction ……………………………………………………………………… 100

5.2. Materials and Methods …………………………………………………………… 104

5.2.1. Materials …………………………………………………………………….. 104

5.2.2. Fourier Transform Infrared (FTIR) analysis ……………………………....... 105

5.2.3. Fabrication of films …………………………………………………………. 105

5.2.4. Preparation of TQ hydrogel formulations ………………………………........ 106

5.2.5. Field Emission Scanning Electron Microscopic (FESEM) studies …………. 106

5.2.6. Physicochemical characterization of films ………………………………..... 107

5.2.6.1. Film thickness …………………………………………………………… 107

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5.2.6.2. Drug content uniformity ……………………………………………… 107

5.2.6.3. Weight variation ……………………………………………………… 108

5.2.6.4. Flatness ……………………………………………………………….. 108

5.2.6.5. Folding endurance ……………………………………………………. 108

5.2.6.6. Percentage of moisture content ………………………………………. 108

5.2.6.7. Percentage of moisture uptake ……………………………………….. 109

5.2.7. Physicochemical characterization of the prepared hydrogels ……………... 109

5.2.7.1. Visual inspection ……………………………………………………… 109

5.2.7.2. pH determination ……………………………………………………... 109

5.2.7.3. Spreadability test …………………………………………………….. 109

5.2.7.4. Drug content uniformity ……………………………………………… 110

5.2.8. Rheological characterization of hydrogel formulation …………………… 110

5.2.8.1. Oscillation stress sweep ……………………………………………... 110

5.2.8.2. Frequency sweep …………………………………………………….. 110

5.2.9. In Vitro skin permeation studies …………………………………………. 111

5.2.10. High-performance liquid chromatography (HPLC) …………………….. 111

5.2.11. Skin deposition study …………………………………………………… 112

5.2.12. Stability study …………………………………………………………... 112

5.2.13. In vitro antibacterial activity of TQ films and hydrogels ………………. 113

5.2.14. Ex vivo antibacterial activity of TQ films and hydrogels using human

cadaver skin explants ………………………………………………….. 113

5.2.15. Cyto-compatibility study ……………………………………………… 114

5.2.16. Scratch assay for wound closure activity ……………………………… 114

5.2.17. In vivo bacterial skin infection study …………………………………. 115

xiv

5.2.18. Behavioral response of mice ……………………………………………… 116

5.2.19. Histopathological examination …………………………………………… 117

5.2.20. Data and statistical analysis ……………………………………………… 117

5.3.Results and discussion …………………………………………………………… 118

5.3.1. Fourier Transform Infrared (FTIR) Spectroscopic studies ……………....... 118

5.3.2. Physicochemical characterization of films ………………………………… 118

5.3.3. Characterization of the TQ hydrogels ……………………………………... 121

5.3.4. In Vitro skin permeation and deposition studies …………………………... 125

5.3.5. Stability study ……………………………………………………………… 125

5.3.6. Cyto-compatibility study …………………………………………………... 127

5.3.7. In vitro and Ex vivo bacterial inhibition study ……………………………. 128

5.3.8. Scratch assay for wound closure activity ……………………………......... 130

5.3.9. Wound healing and anti-bacterial activity of TQ film in vivo ……………. 132

5.3.10. Histological examination ………………………………………………… 136

5.4. Conclusions ……………………………………………………………………... 137

Appendix A: Development of Lidocaine Loaded EUDRAGIT® RLPO Transdermal

Patch Application …………………………………………………………………... 139

A.1. Introduction ……………………………………………………………………. 139

A.2. Materials and Methods ………………………………………………………… 141

A.2.1. Materials …………………………………………………………………….. 141

A.2.2. Preparation of lidocaine loaded transdermal patches ……………………… 142

A.2.3. Patch characterization ……………………………………………………….. 144

xv

A.2.3.1. Thickness …………………………………………………………… 144

A.2.3.2. Weight variation ……………………………………………………. 144

A.2.3.3. Content uniformity …………………………………………………. 144

A.2.4. High performance liquid chromatography (HPLC) ………………........... 144

A.2.5. Mechanical properties …………………………………………………… 145

A.2.6. Loop tack test or adhesive strength study ……………………………….. 145

A.2.7. Rheology ………………………………………………………………… 146

A.2.7.1. Oscillation stress sweep …………………………………………….. 146

A.2.7.2. Frequency sweep ……………………………………………………. 146

A.2.8. In vitro release study …………………………………………………….. 147

A.2.9. Water vapor transmission of transdermal patch system ………………… 147

A.2.10. Shower resistance study ……………………………………………….. 148

A.2.11. SEM-EDS (Scanning Electron Microscopy Energy

Dispersive Spectroscopy) ……………………………………………… 148

A.2.12. Differential scanning calorimetry studies ……………………………… 149

A.2.13. Data and statistical analysis ……………………………………………. 149

A.3. Results and discussion …………………………………………………………. 149

A.3.1. Appearance and patch thickness ………………………………………… 150

A.3.2. Content uniformity ………………………………………………………. 150

A.3.3. The effect of Eudragit® RLPO in adhesive and cohesive strength ……… 151

A.3.4. The effect of drug loading and Eudragit® RLPO on the

mechanical properties of transdermal patches …………………………. 153

A.3.5. The effect of drug loading on rheological behavior

xvi

of the formulations ……………………………………………………. 155

A.3.6. In vitro release study …………………………………………………….. 157

A.3.7. Scanning electron microscopy …………………………………………... 159

A.3.8. Differential scanning calorimetry ……………………………………….. 161

A.3.9. WVP evaluation …………………………………………………………. 164

A.3.10. Evaluation of shower effect on the patches …………………………… 165

A.4. Conclusions …………………………………………………………………. 166

References ………………………………………………………………………… 168

Thesis summary and future perspectives ………………………………………. 177

xvii

List of Tables

Table 1.1. Regional variations in drug permeability of stratum corneum. 4

Table 1.2. A list of various skin models and membranes used for skin research. 8

Table 1.3. Examples of chemical penetration enhancers on the bases of their 13

structure.

Table 2.1. Penetration parameters of nicotine through Strat-M® membrane after 35

8 hours.

Table 2.2 Penetration parameters of nicotine through human cadaver skin after 35

8 hours.

Table 3.1. Hansen solubility parameters and molar volume for nicotine and different 57

solvents/enhancers.

Table 3.2. Physicochemical parameters of nicotine and different 58

enhancers.

Table 3.3. Penetration parameters of nicotine through human cadaver skin after 65

8 hours.

Table 3.4. Hansen solubility parameters and molar volume of thymoquinone and 67

different solvents/enhancers.

Table 3.5. Physicochemical parameters of thymoquinone and different enhancers. 67

Table 3.6. Penetration parameters of thymoquinone through human cadaver skin 70

(N=5) after 24 hours.

Table 3.7. Summary of the solubility study results showing the effect of 5% 71

penetration enhancers on the solubility of TQ using propylene glycol. The values

represent the concentration of TQ ± SD (N=3) in mg/mL at 48 hours.

Table 4.1. Intra-day variability of TQ standard solutions of three separate runs 87

in one day.

Table 4.2. Inter-day variability of TQ standard solutions of two separate runs 87

in two days.

Table 4.3. Summary of the Solubility Study Results. The values represent the 88

concentration of TQ ± SD (N=3) in mg/mL at 48 hours.

xviii


(N=5) after 24 hours using propylene glycol vehicle.


(N=5) after 24 hours using ethanol vehicle.


(N=5) after 24 hours using ethanol vehicle and ethanol : PBS pH 7.4 (60:40)

receptor solvents.

Table 5.1. Composition of TQ topical hydrogels (% w/w). 107

Table 5.2. Physicochemical properties of TQ films (data shows 121

mean of five determinations with ± standard deviation).

Table 5.3. Physicochemical properties of TQ topical hydrogel formulations 122

(F1- F10).

Table 5.4. Penetration parameters of thymoquinone through human cadaver 126

skin (N=5) after 8 hours.

Table A.1. Lidocaine-loaded patch composition % (w/w) at different drug 143

loading ranging from 4% to 20% (Formulation A-D).

Table A.2. Physical and mechanical properties of transdermal 151

patches containing lidocaine. Data represents N=3, mean ± SD.

xix

List of Figures

Figure 1.1. Structure of the skin. 2

Figure 1.2. Anatomy and physiology of the skin. 3

Figure 1.3. Schematic representation of penetration pathways. 5

Figure 1.4. Scanning electron microscopic image of a cross-section of Strat-M™. 9

The first (i), second (ii), and third layer (iii) of Strat-M™.

Figure 1.5. Transmission scanning electron microscopic observation of a 10

cross-section of Strat-M™. The first (i), second (ii), and third layer (iii) of

Strat-M™. Lipids in the layer were stained with a black color. Arrows show the

lipid region.

Figure 1.6. Lipophilic and hydrophilic pathways of drug penetration and mode of 12

action of penetration enhancers.

Figure 1.7. Franz diffusion cell. 14

Figure 1.8. A typical plot of permeation study. 17

Figure 1.9. Schematic representation of the process involved in drug transport from 19

topical or transdermal formulation.

Figure 1.10. Nigella sativa, black cumin seed and chemical structure of 21

thymoquinone.

Figure 2.1. Multilayered structure of Strat-M® membrane. 29

Figure 2.2. Cumulative amounts of nicotine per cm2 of membrane/skin permeated 36

after 8 hours through Strat-M® membrane (n=6) and human cadaver skin (n=6)

samples.

Figure 2.3. Schematic representation of Azone disrupting the stratum corneum 37

intercellular lipids.

Figure 2.4. Human cadaver skin and Strat-M® membrane enhancement factors for 39

different formulation enhancers.

Figure 2.5. Nicotine penetration profiles of transdermal formulations (A) human 41

cadaver skin, (B) Strat-M® membrane, and (C) time point correlations between the

amounts of drug penetrated through human cadaver skin and Strat-M® membrane.

Means plus minus S.D. and correlation coefficients.

xx

Figure 2.6. Correlation of flux between Strat-M® synthetic membrane and human 42

cadaver skin.

Figure 3.1. Nicotine permeation profiles of transdermal formulations (A) with the 59

Franz diffusion cell method using human cadaver skin, (B) the correlations between

the calculated and measured permeation of nicotine.

Figure 3.2. Position of the active nicotine and penetration enhancers/ingredients in 60

3D Hansen Space.

Figure 3.3. The influence of physicochemical interactions (IAG, SolV) 64

between penetration enhancer and active (Nicotine) on the driving force for diffusion

and, the influence of various physicochemical and solubility parameters (ISG, SolS)

of the formulation on the skin affinity of the penetrant is illustrated. (A) The

possible mechanism of action of skin penetration enhancers; (B) a representation

of the active-enhancer and stratum corneum interactions promoting partitioning

into the stratum corneum.

Figure 3.4. Thymoquinone permeation profiles of transdermal formulations 68

(A) with the Franz diffusion cell method using human cadaver skin, (B) the

correlations between the calculated and measured permeation of Thymoquinone.

Figure 3.5. Position of the active Thymoquinone and penetration enhancers/ 69

ingredients in 3D Hansen Space.

Figure 3.6. The influence of physicochemical interactions (IAG, SolV) 73

between penetration enhancer and active (Thymoquinone) on the driving

force for diffusion and, the influence of various physicochemical and solubility

parameters (ISG, SolS) of the formulation on the skin affinity of the penetrant

is illustrated. (A) The possible mechanism of action of skin penetration enhancers;

(B) a representation of the active-enhancer and stratum corneum interactions

promoting partitioning into the stratum corneum.

Figure 3.7. Amount of Thymoquinone detected at 24 hours in human cadaver skin. 75

Figure 3.8. Thymoquinone permeation profile in propylene glycol vehicle containing 76

different concentration of Oleic Acid. Time points were measured at 3, 4, 6, 8, 10, 12

and 24 hours. Each point represents the mean ± S.D. of five experiments. ***p<0.02.

Figure 4.1. Thymoquinone chromatogram peak at retention time of 4.2 min. 85

Figure 4.2. Thymoquinone standard curve for HPLC assay. 86

Figure 4.3. Thymoquinone permeation profile in propylene glycol vehicle. 90

Time points were measured at 3,4,6,8,10,12 and 24 hours. Each point represents the

xxi

mean ± S.D. of five experiments.

Figure 4.4. Thymoquinone permeation profile in ethanol vehicle. Time points were 91

measured at 3,4,6,8,10,12 and 24 hours. Each point represents the mean ± S.D. of five

experiments.

Figure 4.5. Thymoquinone permeation profile in ethanol vehicle and ethanol:PBS 93

(pH 7.4) receptor. Time points were measured at 3,4,6,8,10,12 and 24 hours. Each

point represents the mean ± S.D. of five experiments.

Figure 4.6. Amount of TQ (PG vehicle) detected after 24 hours in human cadaver 96

skin (N=5, mean ± SD).

Figure 4.7. Amount of TQ (ethanol vehicle) detected after 24 hours in human 98

cadaver skin (N=5, mean ± SD).

Figure 4.8. Amount of TQ (ethanol : PBS receptor) detected after 24 hours 99

in human cadaver skin (N=5, mean ± SD).

Figure 5.1. Physicochemical characterization of TQ films. (A) FTIR spectrum of 119

TQ pure drug, PVP, physical mixture of drug and polymer, freshly prepared

films containing drug and polymer, stored films containing drug and polymer; (B)

control films (i); Field emission scanning electron microscopic (FESEM) images

showing surface morphology of control film (ii-iii) at different magnifications; and

(C) TQ films (i); FESEM images showing surface morphology of TQ films (ii-iii)

at different magnifications.

Figure 5.2. Rheological characterization of TQ hydrogel formulations (F1-F10). 124

(A) Oscillation frequency sweep data. The elastic modulus (i); The viscous modulus

(ii) were plotted against angular frequency. TQ permeation and skin deposition from

film and gel formulations (B). TQ permeation profile for different hydrogel formulations

(i). Time points were measured at 1, 2, 3, 4, 5, 6 and 8 hours. Each point represents the

mean ± S.D. of five experiments; TQ permeation from film formulation across human

cadaver skin (mean ± S.D., n=5) (ii); Amount of TQ detected after 8 hours in human

cadaver skin (N=5, mean ± SD) using different TQ hydrogel formulations (iii).

Figure 5.3. Cytocompatibility study of TQ film. Cell viability of TQ film with HDF 128

and HaCat cells using alamarBlue® assay.

Figure 5.4. Bacterial inhibition study. (A) Inhibition of bacterial growth on agar plate 129

by Control negative (i); Gentamicin positive control 50 µg/mL (ii) right upper and

500 µg/mL (ii) right lower; Control film (iii); TQ hydrogel (iv) and TQ film (v)

against Staphylococcus aureus; (B) Ex vivo antibacterial activity by Control (i);

Control film (ii); Gentamicin sulfate USP, 0.1% marketed cream (iii); TQ hydrogel

(iv); TQ film (v) and Log of bacterial reduction with different treatment groups (vi).

Data represent mean ± SD of four replicates. ***p = < 0.001 and ^^^p = < 0.05.

xxii

Figure 5.5. Effect of different treatment groups on the wound healing of 131

keratinocytes and fibroblasts. (A) Representative micrographs from control, 1 ng/mL

and 100 ng/mL of TQ, showing the original wound and the wound after 6 days;

(B) Quantitative analysis of wound closure as a function of time. The wound area

was determined as the wound area at a given time relative to the original wound area.

Data are presented as the means ± SD (n=5-6). ***p<0.001 (control vs 100 ng/mL)

and ^p<0.05 (Control vs 1 ng/mL). (C) Representative micrographs from control, 1

ng/mL and 100 ng/mL of TQ, showing the original wound and the wound after 24 hour;

(D) Quantitative analysis of wound closure as a function of time. The wound area

was determined as the wound area at a given time relative to the original wound area.

Data are presented as the means ± SD (n=6). **p<0.01 and ***p<0.001 (control vs

100 ng) and ^p<0.05 (Control vs 1 ng); (E) Quantitative measurement of cells

number migrating in the corresponding scratched wound areas at different

treatment groups. The values plotted were means of 6 determinations (𝑛 = 6).

***p<0.001 (100 ng/mL vs control/1 ng/mL/10 ng/ml).

Figure 5.6. Macroscopic observations, wound closure and bacterial reduction. 134

(A) Photographs of wounds in BALB/c mice in which the wounds received TQ

loaded film and Gentamicin. The animal with bacterial wounds and wound with no

bacterial served as control group and animal with control film served as a vehicle

control. Representative photographs of the wounds were taken at 0, 3, 7, 10, 14,

and 21 days post-wounding; (B) Log of bacterial reduction at each time point (Day 1,

2, 3 and 7) using different experimental groups. Data are presented as the means ± SD

(n=2-4). ***p<0.001 (Bacterial wound vs TQ Film) and ^^^p<0.001 (Bacterial wound

vs Gentamicin); (C) Percentage of wound closure in all experimental groups at 0, 3, 7,

10, and 14 days post-wounding.

Figure 5.7. Masson’s trichrome staining of the different samples at day 21 post- 137

wounding (control wound (i); Gentamicin sulfate USP, 0.1% marketed cream (ii);

bacterial wound (iii); control film (iv); TQ film (v); TQ film + dose (vi)) indicates

epidermis and indicates dermis.

Figure A.1. Schematic representation of solvent evaporation method. 143

Figure A.2. Peak adhesive force of different formulations containing Eudragit® 152

RLPO and different concentration of Lidocaine. Data represents mean ± SD (n=3).

Figure A.3. Cohesive properties (A) formulation with EUDRAGIT® RLPO, 153

(B) formulation without EUDRAGIT® RLPO.

Figure A.4. The effect of different formulations on tensile stress. Data represents 154

mean ± SD (n=3), ***p < 0.005.

Figure A.5. The effect of different formulations on % Elongation. Data represents 154

mean ± SD (n=3), ***p < 0.005.

xxiii

Figure A.6. Rheological behavior of different drug loaded patch formulations in 156

terms of oscillation frequency sweep data (A) the elastic or storage modulus and

(B) the viscous or loss modulus were plotted against angular frequency.

Figure A.7. In vitro release profile of lidocaine from experimental and marketed 157

patch formulations in phosphate buffer at pH 7.4 (N=3).

Figure A.8. Higuchi release kinetics, in phosphate buffer at pH 7.4 after 25 h 159

(N=3).

Figure A.9. SEM-EDS photographs of (A) lidocaine 4% transdermal patch (B) 160

lidocaine 10% transdermal patch after 30 days and (C) marketed 5% lidocaine patch.

Figure A.10. Differential scanning calorimetry profiles of different components 162

in transdermal patches: (A) Pure lidocaine; (B) 1:1:1 physical mixture of

EUDRAGIT® RLPO, HPMC and chitosan; (C) 1:1:1:1:1 physical mixture

of EUDRAGIT® RLPO, HPMC, chitosan, TEC and lidocaine; (D) Ex lidocaine

patch 10% and [E] Ex lidocaine patch 20%.

Figure A.11. Effect of shower or 40 psi water pressure on marketed (A-C) 166

and experimental patches (D-F).

1

Chapter 1. Background and Specific aims

1.1.Skin physiology

The skin is a complex arrangement of structures and has a multifunctional role-

provides a physical barrier to the environment by acting as a protective barrier against the

ingress of foreign material, maintains homeostasis and thermoregulation by limiting the

loss of water, electrolytes, and heat and prevents microbial colonization [1]. It is the largest

organ in the body and occupies about 16% of the total body weight of an adult and has a

surface area of about 2 m2 [2]. It weighs approximately 3-5 kg, twice as much as the brain.

Even though it is structurally continuous throughout the body, skin varies in thickness

according to the function, age of the individual and area of the body (on the eyelids, the

skin is only 0.5 mm thick, whereas on the soles of the feet it can has the thickness of 3-4

mm). In general, skin is 1-2 mm thick. In about one square centimeter of skin there are 10

hair follicles, 100 sweat glands, 15 sebaceous glands, 12 nerves, 360 cm of nerves and 3

blood vessels [3]. Hair, nails, sweat glands and sebaceous glands are considered to be skin

appendages or derivatives. The skin is a multilayered organ (Figure 1.1 and 1.2) and can

be considered to have four distinct mutually dependent tissue layers [4]:

1. Non-viable epidermis (stratum corneum)

2. Viable epidermis

3. Viable dermis

4. Subcutaneous connective tissue (hypodermis)

Its multilayered structure reflects the barrier properties of the skin (Figure 1.2) and each

layer is known to represent different levels of cellular or epidermal differentiation.

2

Figure 1.1. Structure of the skin [5].

Non-viable epidermis (stratum corneum)

The stratum corneum (SC) is the uppermost layer and consists of 10-15 layers

of corneocytes and varies in thickness from approximately 10-15 µm in the dry state to

40 µm when hydrated. These corneocytes are denucleated, nonliving, flattened cells-

34-44 µm long, 25-36 µm wide, 0.5- 0.20 µm thick with a surface area of 750 to 1200

µm2.

3

Figure 1.2. Anatomy and physiology of the skin [6].

So, the SC is comprised of multi-layered “brick and mortar” like structure of keratin-

rich corneocytes (bricks) embedded in a complex matrix of organized lipid bilayers

(mortar) composed primarily of free fatty acids (10-15%), long chain ceramides (40-

50%), cholesterol (25%) and 5% of other lipids (triglycerides, cholesterol sulfate and

sterol/wax esters) [7-11]. The barrier property of the SC is governed by the presence of

79-90% of protein and 5-15% of lipids and was first established in the 1940s [12]. The

protein part of the SC primarily contains approximately 70% of α-keratin, 10% of β-

keratin and 5% of the cell envelope. The SC is lipophilic and contains 13% of water

and the skin’s hydrophilic properties increase from the surface as its depth increases.

4

The SC is crucially important in controlling the percutaneous absorption of most drugs

and other chemicals. Table 1.1 represents the regional variations in drug permeability

through SC.

Table 1.1. Regional variations in drug permeability of stratum corneum [13].

It is generally thought that drug can be penetrated by three pathways in the SC (Figure 1.3)-

1. Transcellular Route: most direct route and require transport through densely packed

keratin-filled corneocytes followed by multiple transfers between the corneocytes

and the lipid filled intercellular areas.

2. Paracellular Route: the most common penetration pathway of drug molecules. In

this pathway, drug remains in the lipid moiety and stay around keratin and follows

a tortuous diffusion pathway.

3. Transappendgeal Route: the route via skin appendages (hair follicles, sweat glands)

which form shunt pathways through the intact epidermis.

5

Figure 1.3. Schematic representation of penetration pathways [6].

Viable epidermis

Below the SC, the remainder of the epidermis is viable tissue called viable

epidermis. It has a thickness of 50-100 µm and contains nucleated cells called

keratinocytes. It is a region for drug binding, metabolism, active transport, and

surveillance. As the water content of epidermis is about 90%, the density of this region is

not much different than water. The epidermis is avascular (without blood vessels) and it

6

depends on blood vessels of the dermis for oxygenation, metabolic provision and removal

of metabolic waste products. The epidermis is made up of a number of layers-

➢ Stratum basale- is the nearest layer to the dermis and is made up of a single row of

columnar keratinocytes. It is the only layer within the epidermis that consists of

cells capable of division. Keratinocytes in the stratum basale undergo mitosis and

produce two daughter cells. One remains in this layer while the other migrates up

through the other layers to the surface of the epidermis. As the daughter cells move

away from the stratum basale, they receive less nutrition and the cells die. This

whole process takes approximately 28 days. In healthy skin there is a balance

between the formation of new keratinocytes in the stratum basale and the shedding

of dead keratinocytes from the stratum corneum. The stratum basale also contains

melanocytes, which produce skin pigment called melanin that protects the skin

from the harmful effects of ultraviolet (UV) light. Merkel cells are also found in

the stratum basale. They make contact with the flattened process of a sensory

neuron called a Merkel disc. Merkel cells and their discs together detect the

sensation of touch.

➢ Stratum spinosum- as the daughter cells move to the stratum spinosum they lose

their ability to divide. This layer is 5 to 12 cells thick. Langerhans cells are found

in this layer. They are produced in the red bone marrow and then migrate to the

stratum spinosum where they become involved in immune responses against

microorganisms. They are also important for antigen presentation and for the

activation of T lymphocytes to destroy the appropriate cells.

7

➢ Stratum granulosum- is composed of 3 to 5 layers of flattened keratinocytes. In this

layer the cells go through a process of programmed cells death known as apoptosis.

➢ Stratum lucidum- is only found in areas where the skin is thick, such as the palms

of the hands and soles of the feet. It contains 3 to 5 layers of flattened, dead

keratinocytes and provides some degree of waterproofing to the skin.

Viable dermis

A dermal-epidermal junction separates the viable epidermis from the dermis. The

dermal-epidermal junction is not flat and distinct papillae or rete pegs can be observed and

it plays a role in the permeation of large molecular weight proteins and peptides. The

dermis is 3 to 5 mm thick and is composed of connective tissue containing collagen and

elastic fibers. This layer plays an important role in the regulation of body temperature, it

delivers nutrients and oxygen to the skin while removing waste products and toxins.

Subcutaneous connective tissue (hypodermis)

It is the deepest layer of the skin and is formed from loose connective tissue and fat

(50% of the body fat). The dermis and subcutaneous layers contain blood vessels,

lymphatics, nerve cells and skin appendages. It may also contain sensory pressure organ.

It provides thermal insulation, mechanical protection, and an energy reserve.

8

1.2. Artificial membrane

Although human skin is the most relevant membrane to evaluate permeation of a

molecule [14], there are different factors which can also affect the penetration through the

skin, such as- skin age and site, skin temperature, state of the skin (normal, diseased or

abraded), degree of hydration of the skin, pretreatment of the skin etc [6]. As the use of

human skin in research encounters ethical, health, and supply problems there is a need for

the development of skin parallel artificial membrane [15]. Table 2 shows the advantages

and disadvantages of various skin model.

Table 1.2. A list of various skin models and membranes used for skin research [5].

Artificial skin models are convenient and reproducible alternatives to in vivo and

ex vivo tests with human and animal skins. The artificial skin models range from simple

homogeneous polymer materials to lipid-based parallel artificial membrane-permeability

assay (PAMPA) [16].The synthetic membranes can be classified into two groups: group 1-

consisting of polysulfone, acrylic polymer, glass fiber, silicone, and mixed cellulose ester,

9

showed higher drug permeation compared to group 2, which included

polytetrafluoroethylene–polyethylene, mixed cellulose ester (of greater thickness), and

polypropylene [5]. Although they are not capable of mimicking the complexity of

biological skin properties, they are relatively reproducible due to their simple standardized

construction. As they eliminate the complexity of human skin, their simple structure is

particularly advantageous for understanding the basic mechanisms controlling passive

transport through a membrane [17].

Strat-M™ (Merck Millipore, USA) is a commercially available skin-mimic

artificial membrane without lot-to-lot variability, safety and storage limitations. This

membrane is composed of multiple layers of polyester sulfone (two layers of

polyethersulfone and one layer of polyolefin) with a very tight top layer creating

morphology similar to human skin [18] (Figure 1.4). These layers are increasingly porous

and larger in thickness. The polyolefin and polyethersulfone present in this membrane

represent the epidermis and dermis layer of the skin. It is generally known that SC is the

Figure 1.4. Scanning electron microscopic image of a cross-section of Strat-M™. The

first (i), second (ii), and third layer (iii) of Strat-M™ [19].

10

rate limiting barrier in the passive transport of most molecules across the skin due to the

presence of protein rich corneocytes and intercellular lipids [20]. Therefore, an artificial

membrane should have a blend of lipids in order to mimic skin barrier properties. To

provide the Strat-M™ membrane with its lipophilic quality the polymeric layers are

impregnated with a proprietary blend of synthetic lipids (Figure 1.5) that finally imparts a

skin-like properties to the synthetic membrane. Artificial membrane can be potentially used

as a screening tool to narrow the selection of formulations to be evaluated with a more

biologically relevant model. Additionally, they can be easily manufactured, easy to handle,

doesn’t require membrane preparation time and more reproducible.

Figure 1.5. Transmission scanning electron microscopic observation of a cross-section of

Strat-M™. The first (i), second (ii), and third layer (iii) of Strat-M™. Lipids in the layer

were stained with a black color. Arrows show the lipid region [19].

11

1.3. Skin penetration enhancement

The use of chemical penetration enhancers is the most widely used approach to skin

penetration enhancement across the SC barrier. There are a variety of mechanisms for

penetration enhancement by these chemical enhancers [21]:

• Lipid-fluidization of the SC leads to decreased barrier function;

• Influence the thermodynamic activity of the drug in the vehicle and the skin;

• Affect the partition coefficient of the drug in order to increase its release from the

formulation into the upper layers of the skin;

• Increase drug diffusivity in the skin;

• Create drug reservoir within the skin.

Penetration enhancers may act by one or more of these mechanisms. Figure 6 shows

the influence of enhancers on the lipophilic and hydrophilic pathway of drug penetration

[22]. Based on their mechanism of permeation enhancement more than 300 penetration

enhancers can be classified into three groups [23]:

• Group 1- enhancers that weaken the barrier by extracting skin lipids (e.g., ethanol)

• Group 2- enhancers increase drug solubility within the skin (e.g., propylene glycol)

• Group 3- enhancers disorder intercellular lipids (e.g., Azone (laurocapram))

12

Figure 1.6. Lipophilic and hydrophilic pathways of drug penetration and mode of action

of penetration enhancers [22].

Table 1.3 shows the principal classes of penetration enhancers. For the better

selection of enhancers its physicochemical properties should be compared with the drug

(e.g, the solubility parameter of the enhancer should be similar to the skin solubility, that

is 10 (cal/cm3)1/2) [21]. Penetration enhancers should contain the following properties [24]:

• It should be non-toxic, non-irritant and non-allergen

• It should be pharmacologically inert

• It should be unidirectional- should allow therapeutic agents into the body whilst

preventing the loss of endogenous material from the body

• It should be compatible with both excipients and drugs

• It should work rapidly, and the activity and duration of effect should be both

predictable and reproducible

13

• Its action should be reversible, barrier properties should return both rapidly and

fully.

Table 1.3. Examples of chemical penetration enhancers on the bases of their structure [25].

The simplest way to model the process of skin penetration is to consider the skin as

a passive membrane through which the drug has to pass. With in-vitro skin permeation

studies, a membrane is clamped between two compartments (Figure 1.7). The donor

compartment contains a drug formulation and the receiver compartment holding a receptor

solution provides a sink condition (essentially zero concentration).

14

Figure 1.7. Franz diffusion cell.

The diffusion of the compound through the skin is described by Fick’s First Law:

𝐽 = −𝐴𝐷 (𝑑𝑐

𝑑𝑥) (1)

This equation describes the rate of transfer or flux (J) of the diffusing substance through

unit area A of the membrane as being proportional to the velocity of molecular movement

through the diffusional medium or diffusion coefficient D and to the differential

concentration change 𝑑𝑐 over the differential distance 𝑑𝑥 (concentration gradient

measured across the membrane (𝑑𝑐

𝑑𝑥)). The negative sign indicates the diffusion process

occurs in the opposite direction to increased concentration or the flow is in the direction of

decreasing thermodynamic activity. Fick’s First Law is combined with the differential

15

mass balance existing in a membrane and resulted in Fick’s Second Law. This second law

of diffusion is also based on some assumptions like, the compound is not metabolized, it

does not bind with the membrane and its diffusion coefficient does not vary with position

or composition.

𝜕𝐶

𝜕𝑡= 𝐷

𝜕2𝐶

𝜕𝑥2 (2)

Equation 2 may be written as Equation 3, given that the drug is applied to the membrane

at a maximum fixed concentration in the donor compartment and maintaining sink

conditions in the receptor compartment.

𝐽 = 𝐴𝐷𝐶𝑚

ℎ (3)

Where Cm is the concentration of the compound at the donor-membrane interface and h is

the effective diffusional pathlength of the membrane. The vehicle-membrane partition

coefficient (k) can be defined as the ratio between the concentrations of the permeant in

the membrane at the donor-membrane interface and the vehicle in which it is applied (CV).

By replacing Cm in Equation 3 a modified form of Fick’s first law of diffusion can be

obtained-

𝐽𝑠𝑠 =𝐴𝐷𝐾𝐶𝑣

ℎ (4)

Equation 4 for passive drug permeation enhancement indicates that increased drug flux

should be achieved by a change in D, K and C and it can be rewritten in terms of

thermodynamic activities:

16

𝐽 =𝛼𝐷

𝛾ℎ (5)

𝛼 = 𝑡ℎ𝑒𝑟𝑚𝑜𝑑𝑦𝑛𝑎𝑚𝑖𝑐 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑤𝑖𝑡ℎ𝑖𝑛 𝑓𝑜𝑟𝑚𝑢𝑙𝑎𝑡𝑖𝑜𝑛

𝛾 = 𝑡ℎ𝑒𝑟𝑚𝑜𝑑𝑦𝑛𝑎𝑚𝑖𝑐 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑤𝑖𝑡ℎ𝑖𝑛 𝑚𝑒𝑚𝑏𝑟𝑎𝑛𝑒

Equation 2 can also be simplified as-

𝑑𝑚

𝑑𝑡=

𝐷𝐶𝑜

ℎ (6)

Where m is the cumulative mass of diffusant that passes per unit area through the

membrane in time t, Co is the concentration of diffusant in the first layer of the membrane

at the skin surface contacting the source of the penetrant, and h is the membrane thickness.

In most diffusion experiments it is difficult to measure Co, but Cʹo (the concentration of

diffusant in the donor phase bathing the membrane) is usually known. Both Co and Cʹo are

related by:

𝐶𝑜 = 𝑃𝐶ʹ𝑜 (7)

Where P is the partition coefficient of the diffusant between the membrane and the bathing

solution. Substituting Equation 7 into Equation 6 gives:

𝑑𝑚

𝑑𝑡=

𝐷𝐶ʹ𝑜𝑃

ℎ (8)

The cumulative amount of drug crossing a unit area of skin against time gives a

permeation profile of the drug through the membrane (Figure 1.8). Extrapolation of the

17

pseudo steady-state portion of the graph to the intercept on the time axis gives the lag time

(L). This is the period during which the rate of diffusion across the membrane is increasing

and the steady-state conditions prevail after approximately 2.7 times the lag time [23]. The

lag time L is related to the diffusion coefficient D and D may be obtained by measuring L:

𝐿 =ℎ2

6𝐷 (9)

Figure 1.8. A typical plot of permeation study [25].

From the above equation the ideal characteristics of a molecule that would penetrate SC

well can be deduced. These are [26]:

18

• Adequate solubility in oil and water to ensure high membrane concentration

gradient

• Low molecular mass, less than 600 Da is preferred when diffusion coefficient is

high

• Optimal partition coefficient (1-3)

• Low melting point (<200°F), correlating with good solubility as predicted by ideal

solubility theory

• It should not metabolize in skin.

1.4.Topical and transdermal drug delivery

Skin formulation can be divided into three depending on different functions that

can be achieved when applying drugs to the human skin. 1) Epidermal formulation- when

the active is desirable to remain on the surface of the skin, e.g. insect repellent, sunscreen.

2) Dermal formulation- delivery of the active into the skin to target the pathological sites

within the skin, e.g. skin cancer, psoriasis, eczema and microbial infections while ensuring

minimal systemic absorption. 3) Transdermal formulation- by using skin as the application

site the active diffuses through the various layers of the skin and into the systemic

circulation to exert a therapeutic effect.

Once applied to the skin initially, the drug must be released from the

topical/transdermal formulation followed by partitioning into the SC. As a result of

concentration gradient, drug molecules will subsequently diffuse through the SC before a

further partitioning process into the viable epidermis, and further diffusion through the

viable epidermis towards the dermis. Finally, the vasculature and lymphatic vessels in the

19

dermis will clear the drug from the skin and will make it available into the systemic

circulation. The process involved in drug transport from topical or transdermal formulation

are illustrated in Figure 1.9.

Figure 1.9. Schematic representation of the process involved in drug transport from topical

or transdermal formulation [27].

20

Topical and transdermal route gives an alternative to oral and intravenous delivery.

There are several advantages of cutaneous delivery [1, 13]:

1. The avoidance of first pass metabolism and stomach environment where the drug

can be degraded,

2. Sustained and controlled delivery over a prolonged period of time,

3. Improves bioavailability,

4. Reduction in side effects associated with systemic toxicity,

5. Improved patient compliance,

6. Ease of dose termination,

7. Convenient and painless administration,

8. Suitable route for unconscious or vomiting patient,

9. Best route for pediatric patients.

There are also several limitations associated with cutaneous delivery [13]-

1. A molecular weight less than 500 Da is essential to ensure ease of diffusion across

the SC,

2. Required enough aqueous and lipid solubility,

3. Variability associated with the different skin types,

4. Pre-systemic metabolism due to the presence of skin enzymes that might

metabolize the drug into a therapeutically inactive form,

5. Skin irritation and sensitization.

21

1.5.Thymoquinone

Thymoquinone (TQ) (2- isopropyl- 5- methyl- 1,4- benzoquinone) is the main

constituent of Nigella sativa (Black cumin) seeds [28]. This herb has been popularly called

as the “seed of blessing” by the Arab population [29]. Black seed contains up to 30-48%

of TQ along with 15 amino acids, proteins, carbohydrates, fixed oils, volatile oils,

alkaloids, saponins, crude fiber, minerals such as, calcium, iron, sodium and potassium

[30]. TQ is a yellow crystalline molecule and has a basic quinone structure consisting of a

para substituted dione conjugated to a benzene ring to which a methyl and an isopropyl

side chain groups are added in positions 2 and 5, respectively (Figure 1.10). TQ has many

pharmacological properties such as anticancer, anti-inflammatory, antioxidant,

antiasthmatic and immunomodulatory effect [31].

22

Figure 1.10. Nigella sativa, black cumin seed and chemical structure of thymoquinone

[32].

The ability to formulate TQ is hindered by various factors. It is a lipophilic

molecule (log P = 2.54) and poorly soluble in aqueous medium that causes bioavailability

issues. Hence, it shows poor formulation characteristics into a conventional dosage forms

such as tablets or capsules. Additionally, its thermo-labile nature limits the application of

nano-formulation techniques to enhance its bioavailability. On the other hand, it’s low

molecular weight (164.2 gmol-1), low melting point (44-45°C), and it’s lipophilic

characteristic can be useful to formulate it in a topical or transdermal delivery system.

1.6.Specific aims

Skin has significant barrier properties that inhibit the passive transport of many

active molecules. Different strategies are developed to overcome this skin barrier such as,

chemical enhancement techniques using penetration enhancers and targeted drug delivery

using various topical or transdermal delivery system. Usually these approaches are tested

using human or animal skin. Human skin is not easily accessible and animal skin has

significant biological and barrier differences when compared with human skin. Due to

these issues the possibility of having a synthetic skin membrane is an attractive option. The

goal of this research was to further investigate the effectiveness of a synthetic membrane

compared with human skin samples for drug permeability testing, to propose for the first

time a Solubility-Physicochemical-Thermodynamic theory to define the action of

penetration enhancers and to investigate the feasibility of transdermal delivery of

23

thymoquinone (TQ) using different topical/transdermal formulation approaches and to

show its effectiveness in the treatment of wound healing and bacterial skin infections. To

achieve this goal following specific aims were identified for this research:

Specific Aim 1: Evaluating Strat-M® synthetic membrane as a screening tool for

topical/transdermal formulation.

To provide data on applicability of a synthetic membrane for in-vitro diffusion

studies in transdermal arena in place of human or animal skin as a model. To compare

different formulations containing various enhancers regarding their ability to enhance or

reduce the delivery of nicotine as a model drug through human cadaver skin and to correlate

that to a novel synthetic membrane (Strat-M® EMD Millipore, MA) to examine the

usefulness of this membrane as a convenient screening tool to investigate topically applied

formulations and TDDS (Transdermal Delivery System). To obtain the aforementioned

goal we aimed to investigate the correlation of permeation behavior of transdermal

formulations through Strat-M® membrane and human cadaver skin. Strat-M® membranes

were designed with the intent to share similar structural and chemical characteristics found

in the human skin however, omitting any biological behavior due to the absence of viable

cells. Both human skin and the membrane display a layered structure with a very tight top

layer. Additionally, the Strat-M® membrane contains a combination of lipids in a specific

ratio similar to what is found in the human stratum corneum (SC). Formulations containing

nicotine and a chemical penetration enhancer (CPE) were used for evaluating drug

penetration to understand how each enhancer impacts the permeability of nicotine as a

model compound.

24

Specific Aim 2: Solubility-physicochemical-thermodynamic theory of penetration

enhancer mechanism of action.

To date, there is limited research that demonstrate the underlying mechanism of

action of penetration enhancer. Thus, it is very important to clearly define their actions in

the formulation with the drug. The objective of this study was to propose for the first time

a Solubility-Physicochemical-Thermodynamic (SPT) theory to define the action of

penetration enhancers. The hypothesis for this investigation was that the overall

mechanism of action of skin penetration enhancers is best explained by the SPT theory.

The SPT theory puts forward the concept that the mode of action of enhancers is related to

solubility parameters, physicochemical interactions and thermodynamic activity. This

study discusses these concepts by using experimentally derived permeation data, various

physicochemical and solubility parameters (ingredient active gap (IAG), ingredient skin

gap (ISG), solubility of active in the formulation (SolV) and the formulation solubility in

the skin (SolS)) generated by using FFE (Formulating for EfficacyTM - ACT Solutions

Corp) software. This work will connect the solubility and physicochemical properties of

the active and enhancers/ingredients with the thermodynamic activity of the model drug

used in order to explain the mode of action of enhancers in a given formulation with that

specific drug.

Specific Aim 3: Effects of solvents and penetration enhancers on transdermal

delivery of thymoquinone: permeability and skin deposition study.

Thymoquinone (TQ) is a quinone-based phytochemical and was first identified in

1963 in Nigella sativa (black cumin seed) by El-Dakhakhany. Based on the ideal

characteristics of transdermal delivery, TQ can be an attractive candidate for TDDS

25

(transdermal drug delivery system). The aim of this study was to investigate for the first

time the feasibility of transdermal delivery of thymoquinone (TQ) and to assess the effect

of ethanol and propylene glycol (PG) as solvents together with the effects of selected

chemical penetration enhancers on the in vitro human skin deposition and permeation of

TQ. To investigate both transdermal flux and skin deposition of thymoquinone and the

various conditions that may influence these including various vehicle/solvents, receiver

composition and permeation enhancers. The effects of penetration enhancers on the in vitro

skin permeation and TQ skin absorption were studied using human cadaver skin and Franz

diffusion cell method. The permeation of saturated solutions of TQ was investigated with

5% concentrations of each of the following enhancers: Azone (laurocapram), Transcutol®

P (Tc), oleic acid, ethanol, Polysorbate 80 (Tween 80), and N-methyl-pyrrolidone (NMP).

Specific Aim 4: Thymoquinone loaded polymeric films and hydrogels for wound

healing and the treatment of bacterial skin infections.

The purpose of this study was to synthesize and characterize a biocompatible novel

topical polymeric film and hydrogel system that has the potential to deliver

antibacterial/anti-inflammatory agent thymoquinone (TQ) directly to the skin target site

and that may be useful for the treatment and management of skin wound infections. TQ

loaded polyvinyl pyrrolidone (PVP) matrix-type films and hydrogels with different

polymers were prepared. The developed films were evaluated for thickness, drug content

uniformity, weight variation, flatness, folding endurance, percentage of moisture content

and uptake. The surface morphology of the film was recorded with a Zeiss field emission

scanning electron microscopy (FESEM) and in vitro skin permeation studies were

performed on human cadaver skin by using Franz diffusion cells (FDC). Human dermal

26

fibroblasts (HDFs) and Human keratinocytes (HaCaT) cell lines were used for wound

healing scratch assay. The proliferative effect of different concentration of TQ (1-1000 ng)

were also investigated on HDF and HaCat cell lines. Antibacterial activity of TQ loaded

films and hydrogels against Staphylococcus aureus were assessed using the disc diffusion

method (in vitro) and an ex vivo human cadaver skin explant. To evaluate the TQ film’s

preclinical and in vivo efficacy wound infection animal model was used. This animal

model was made by inoculating Staphylococcus aureus at a concentration of 108 CFU/mL

to create an in vivo bacterial wound infection at their dorsal side. Bacterial samples were

taken from the animal site at pre-determined time points and were analyzed for the bacterial

numbers. At the end of the study, all the animals were sacrificed, and the histology of the

skin samples were examined. Efficacy was determined by the % of wound closure and log

of bacterial reduction of the wound site.

27

Chapter 2. Evaluating strat-M® synthetic membrane as a screening tool for

topical/transdermal formulation

2.1. Introduction

Synthetic membranes for in vitro permeation studies were originally developed to

be used as an alternative to using a human skin models [33]. Determination of drug

permeation of formulations using ex vivo human skin methods possesses several

drawbacks which hinder reproducibility data of drug candidate screening including:

variations of skin thickness from skin donors, diseased skin states, skin storage conditions,

membrane preparation complexity, density of hair follicles, age of donor, and high

laboratory costs [34, 35]. Some advantages of using a synthetic membrane are: controlled

membrane thickness, faster membrane preparation time, low storage space, and relatively

low cost. The human stratum corneum (SC) is commonly the rate limiting step for

successful API (active pharmaceutical ingredient) delivery [36, 37]. It consists of 10-15

parallel layers of corneocytes embedded in an intercellular lipid matrix of mainly

ceramides (50%), cholesterol (25%) and free fatty acids (15%), in a bricks and mortar

arrangement [22]. There are several techniques such as chemical enhancement, physical

enhancement, and drug modification that have been employed to change the barrier

properties of stratum corneum [26]. Among these using chemical penetration enhancers is

the most widely used technique since these compounds can reversibly alter the stratum

corneum’s barrier function [38]. Usually chemical enhancers act by lipid disruption and at

acceptable concentrations they interact and affect the stratum corneum intercellular lipid

domain or organization and make the stratum corenum more permeable [39].

28

Understanding the physiochemical relationship of API/vehicle interactions through a

membrane barrier is critical for selection of optimal formulation penetration enhancement

efficacy [40]. In this regard, we report here on the development of nicotine solutions

containing penetration enhancers to evaluate the permeability correlations of Strat-M®

(EMD Millipore, MA) synthetic membrane with human cadaver skin. Nicotine is

commonly used for nicotine replacement therapy (NRT) to encourage successful smoking

cessation from tobacco products [41]. Particular chemical properties such as: low

molecular weight (162.23 g/mol), logP (1.2) and its wide-spread use make this compound

an ideal candidate for testing. It is commercially available as a TDDS (Transdermal

Delivery System).

Most commonly used synthetic membranes models lack the biological composition

of a highly structured stratum corneum, metabolic processes, and interactions of proteins

found in the human epidermis. Strat-M® membranes were designed with the intent to share

similar structural and chemical characteristics found in the human epidermis. The multiple

layers of stratum corneum undergo a process called keratinization. In this process as the

cells formed and migrated upwards to the skin surface from the basal layer stem cells the

concentration of oxygen and nutrients decrease, and the cells become flatter and

accumulation of keratin and lipids occurs. Strat-M® membrane was engineered to mimic

the layered structure and lipid chemistry of human skin (Figure 2.1). The thickness of each

Strat-M® membrane is approximately 300 µm; comprising a top layer supported by two

layers of porous polyether sulfone (PES) on top of one single layer of polyolefin non-

woven fabric support. Membrane layers are increasingly more porous and open and also

increasingly larger in thickness to mimic different layers of human skin (epidermis, dermis

29

and subcutaneous tissue). These multiple layers of the membrane create a morphology

similar to that of human skin. Both human skin and the membrane display a layered

structure with a very tight top layer. The porous membrane was treated with a proprietary

blend of synthetic lipids. Skin contains various lipids, such as phospholipids and ceramides,

which impart hydrophobic character to skin. This synthetic membrane contains a

combination of lipids (ceramides, cholesterol, free fatty acids, and other components) in a

specific ratio similar to what is found in the human SC.

Figure 2.1. Multilayered structure of Strat-M® membrane.

Strat-M® serves a purpose to be a cost-effective membrane for testing and

optimizing pharmaceutical formulations with good reproducibility to increase confidence

during early stage drug/formulation development. This synthetic membrane can be used

30

for high throughput formulation screening during the early stages of formulation

development to test for API’s, personal care products, pesticides, cosmetic actives, and

chemical warfare protective formulations. Furthermore, a need for high quality methods

that help determine safety and bioequivalence for formulations including those containing

CPE’s are sought by regulatory agencies to speed up the long approval times needed in

order for generic drug clearance and approvals [42]. Bioequivalence studies are typically

conducted using human cadaver skin or animal models. Unfortunately these models

experience a number of drawbacks that make them not very suitable for development

including: complex sample preparation, strict sample storage requirements, biohazard

issues and expensive study costs [43].

The objective of the present study was to compare different formulations containing

various enhancers regarding their ability to enhance or reduce the delivery of nicotine

through human cadaver skin and to correlate that to Strat-M® synthetic membrane to

examine the usefulness of this membrane as a convenient screening tool to investigate

topically applied formulations and TDDS.

2.2.Materials and Methods

2.2.1. Materials

Polysorbate 80 (Tween 80), eucalyptol, N-methyl-2-pyrrolidone (NMP), propylene

glycol, sodium phosphate monobasic were purchased from Sigma-Aldrich Co. (St. Louis,

MO, USA). Nicotine was purchased from Alfa Aesar (Haverhill, MA, USA) and

Laurocapram (Azone) was purchased from BOC Sciences (Shirley, NY, USA). Phosphate-

buffered saline tablets (PBS, pH 7.4) was purchased from MP Biomedicals, LLC (Solon,

31

OH, USA) and o-Phosphoric Acid 85% was purchased from Fisher Scientific (Hampton,

NH, USA). Dermatomed human cadaver skin from the posterior torso of three different

donors were obtained from New York Firefighter Skin Bank (NY, USA). Strat-M®, high-

performance liquid chromatography (HPLC) grade water and acetonitrile was a gift from

EMD Millipore (Danvers, MA, USA).

2.2.2. Preparation of formulations

Five formulations were prepared (10 mL) containing 1% nicotine, with or without

5% of enhancer (Azone, Tween 80, eucalyptol, or N-methyl-2-pyrrolidone) in propylene

glycol.

2.2.3. In vitro skin permeation test (IVPT) studies

Each of the five formulations (as described above) were applied to dermatomed

human cadaver skin with the dermal side in contact with filtered PBS (pH 7.4) and Strat-

M® membrane with the shiny side in contact with the donor compartment, both mounted

on Franz diffusion cells with a donor area of 0.64 cm2 and a receptor volume of 5.0 mL

(Permegear Inc., Hellertown, PA). Dermatomed human cadaver skin samples (~500 μm)

from the posterior torso of three different donors (2 white males at the age of 68, 45 and

one white female at the age of 34) obtained from New York Firefighters Skin Bank (New

York, NY) were used for skin permeation study. Prior to using the skin, the samples were

slowly thawed, cut into appropriate pieces and then soaked in filtered PBS (pH 7.4) for 15

minutes. Strat-M® membrane (EMD Millipore, MA, USA) does not require any

32

pretreatment, thus was used immediately after removing from the packaging. The skin and

the Strat-M® membrane was not occluded in the Franz cells and the receptor compartment

of each cell was filled with filtered PBS (pH 7.4) and maintained at 37oC under

synchronous continuous stirring using magnetic stirrers at 600 rpm. The diffusion cells

were allowed to equilibrate at 37oC for 15 minutes. Then at time zero 200 µL of formulation

was added to the donor compartment of each Franz diffusion cell using a positive

displacement pipette set and the dose was spread through the surface with the tip of pipette.

At each time point (1, 2, 3, 4, 5, 6, 7 and 8 hours) 300 µL of receptor were withdrawn from

the sampling port. At the end of 8 hours, all receptor samples were analyzed using a valid

HPLC method described below.

2.2.4. High-performance liquid chromatography (HPLC)

Nicotine was quantified using a validated HPLC method and an Agilent 1100 series

HPLC (Agilent Technologies, CA, USA) coupled with UV (259 nm) and a diode array

detector (DAD). A mobile phase of 65% sodium phosphate buffer (adjusted to pH 3.2 with

85% orthophosphoric acid) and 35% acetonitrile was pumped at a flow rate of 1.0 mL/min

through a Phenomenex Luna® 5 µm C18(2) 100 Å Column 250 X 4.6 mm (ambient

temperature). The retention time for nicotine was 2.5 minutes. The method was linear at a

concentration range 4 -500 µg/mL with R2 of 1. The limit of quantification is 1.2 µg/mL

and the limit of detection is 0.35 µg/mL.

33

2.2.5. Data analysis

Penetration parameters were obtained from the cumulative amount of nicotine

permeated per unit skin surface area (µg/cm2) versus time (hours) plot. The effectiveness

of the penetration enhancers (EF= enhancement factor) was determined using Equation 1.

EF = Flux with the enhancer/Flux without the enhancer (1)

2.2.6. Statistical analysis

Results are reported as mean ± SD (n=6). The statistical analysis of the data was

performed by using one-way Anova and Student’s t-test, and p-values < 0.05 were

considered significant.

2.3. Results and discussion

Penetration enhancers are employed across various areas of drug delivery and are

classified into separate classes depending on their chemical composition [44]. In this

experiment 5% of different enhancers were selected to represent enhancer groups (amide,

surfactant, pyrrolidone, glycol, terpene) due to the fact that each class of enhancers

modifies skin lipids in different ways and the mechanism of action is linked to their ability

to interact with skin lipids [27]. The results showed that the rank order for nicotine flux for

each enhancer for both Strat-M® and human cadaver skin are: Azone > eucalyptol + Tween

80> control> N-methyl pyrrolidone. Effect of each enhancer is discussed below.

34

2.3.1. Effect of Azone

Azone (1-dodecylazacycloheptan-2-one) was the first compound specifically

designed as a chemical penetration enhancer [45]. It is a colorless and odorless liquid and

has a melting point of -7°C. Structurally, Azone comprises a polar headgroup attached to

a C12 chain and is a highly lipophilic material with a logPoctanol/water value around 6.2. Due

to its non-polar nature Azone dramatically affected the lipid structure and showed no

protein interaction. It further suggests that at a low concentration Azone does not enter the

cells in significant amount. Azone is known to significantly enhance penetration of API’s

in transdermal formulations by disrupting the normal lipid bilayer packing structure

arrangement in the SC [26, 46]. It is highly effective when used in conjunction with

propylene glycol. Our study also showed its effectiveness with propylene glycol. Tables

2.1 and 2.2 and Figure 2.2 show the amount of nicotine permeated per square cm from

formulation 4 (Nicotine 1% in propylene glycol + 5% Azone) in both Strat-M® and human

cadaver skin after 8 hours (Q8) was significantly greater when compared to control and all

other formulations (p<0.05). This behavior can be explained by the fact that Azone

enhances intercellular drug diffusion only and cannot affect intracellular protein contents.

On the other hand, propylene glycol enhances intracellular transport. So, the combination

of propylene glycol and Azone is more effective. The mechanism of perturbation is

suggested to be caused by Azone attaching and separating the junctions between polar

heads and lipids tails of the SC causing fluidization of the intercellular lipids which result

in a more fluid-like structure (Figure 2.3) [47]. The above mechanism of action was

supported by a ‘soup spoon’ model for Azone’s conformation within stratum corneum

lipids [48].

35

Table 2.1 Penetration parameters of nicotine through Strat-M® membrane after 8 hours.

Formulation Q at 8 hours (µg/cm2) J (µg/cm²/hr) EF

through Strat-M

Control 1220 ± 64 163 -

NMP 1181 ± 73 157 0.96

Tween 80 1802 ± 135 250 1.53

Azone 2606 ± 302 338 2.07

Eucalyptol 1845 ± 278 264 1.61

Q, cumulative amount of nicotine penetrated per cm2 at 8 hours (mean ± SD, n=6); J is

flux determined from slope of the cumulative amounts of nicotine permeated versus time

profiles; EF, flux of nicotine from the formulation containing an enhancer divided by the

flux of nicotine from the control formulation without an enhancer.

Table 2.2 Penetration parameters of nicotine through human cadaver skin after 8 hours.

Formulation Q at 8 hours (µg/cm2) J (µg/cm²/hr) EF

through Skin

Control 87 ± 8 7 -

NMP 81 ± 17 6 0.85

Tween 80 167 ± 43 14 2

Azone 484 ± 86 21 3

Eucalyptol 168 ± 41 16 2.28

Q, cumulative amount of nicotine penetrated per cm2 at 8 hours (mean ± SD, n=6); J

is flux determined from slope of the cumulative amounts of nicotine permeated versus

time profiles; EF, flux of nicotine from the formulation containing an enhancer

divided by the flux of nicotine from the control formulation without an enhancer.

36

Figure 2.2. Cumulative amounts of nicotine per cm2 of membrane/skin permeated after 8

hours through Strat-M® membrane (n=6) and human cadaver skin (n=6) samples.

2.3.2. Effect of Propylene Glycol

Propylene glycol is considered to be part of the polyols class which has superior

solubilizing capabilities in both water and oil. Since 1932 propylene glycol has been used

either as a co-solvent and/or to enhance drug permeation through skin. This co solvent was

selected due formulation compatibility to be used with CPE’s for increasing the effects of

solubility [49-51]. Bouwstra et al. (1989) suggested that propylene glycol decreased the

hydration of skin [52]. It might be incorporated in the head group regions of the lipids and

0

500

1000

1500

2000

2500

3000

Control N-Methyl

Pyrrolidone

Tween 80 Azone Eucalyptol

Cu

mu

lati

ve

am

ou

nt

of

nic

oti

ne

per

mea

ted

(ug

/cm

2)

Strat-M

Skin

37

probably acts by solvating alpha-keratin and occupying hydrogen-bonding sites. Evidence

suggests a very mild enhancement effect of propylene glycol for estradiol and 5-

fluorouracil [24].

Figure 2.3. Schematic representation of Azone disrupting the stratum corneum

intercellular lipids.

2.3.3. Effect of Eucalyptol and Tween 80

In this study, eucalyptol and Tween 80 shared similar trends by providing lower

nicotine flux in comparison to the formulation containing 5% Azone and higher flux of

nicotine when compared to control and N-methyl pyrollidone (5%). Tween 80 is a non-

ionic surfactant widely used to make emulsion formulations. The mechanism of enhancing

drug delivery is different than that of Azone and this surfactant works by allowing polar

molecules to partition across the barrier more easily by the incorporation of micelles [53].

38

Micelles may use some SC barrier lipids to favor permeation of these vehicles containing

hydrophilic API’s. Eucalyptol was selected from the class of terpenes and can be rapidly

metabolized by the skin making them a useful penetration enhancer for transdermal drug

delivery systems [54]. The mechanisms of enhancement include the modification of the

structure of the SC lipids to increase diffusivity of hydrophilic compounds [55].

2.3.4. Effect of N-methyl pyrrolidone

The formulation with 5% N-methyl pyrrolidone did not show any enhancement

effect on nicotine permeation through Strat-M® membrane and human cadaver skin. The

mechanism of enhancement is different than others mentioned in which reservoirs or

clusters containing drug in vehicle are formed between the lipids of the SC and are more

soluble providing a prolonged sustained release of drug from vehicle [56, 57].

2.3.5. Enhancement factor

Tables 2.1 and 2.2 and Figure 2.4 shows the enhancement factor for a given

enhancer of nicotine permeation for Strat-M® and human cadaver skin. Although, Strat-M®

lacks the highly organized SC intercellular structure and provided higher nicotine flux

compared to human cadaver skin where the highly structured SC significantly reduced

nicotine permeability, both membranes still provided similar enhancement factors for a

given enhancer. These studies suggest that the Strat-M® synthetic membrane lipid

composition probably closely mimics that of human cadaver skin based on the data

obtained.

39

Figure 2.4. Human cadaver skin and Strat-M® membrane enhancement factors for different

formulation enhancers.

2.3.6. Correlation of Strat-M® membrane to human cadaver skin

Several studies have investigated the correlation of different synthetic membrane

to human cadaver skin. Shah et al., prepared hydrocortisone creams and tested using pure

cellulose acetate, cellulose and polysulfone synthetic membranes [58]. These membranes

had good reproducibility and were eventually used for quality control assurance for batch

uniformity, but studies concluded that no correlations existed between the synthetic

membranes and ex vivo human skin models. Another study performed by Ng et al., used

an ibuprofen formulation to test drug penetration using various polymeric membranes [59].

This work concluded that no correlations with human skin existed with membrane pore

0

2

4

N-Methyl Pyrrolidone Tween 80 Azone Eucalyptol

En

ha

nce

men

t F

act

or

Strat-M

Skin

40

size, thickness, and polymer molecular weight and that only the polymer matrices

themselves were limiting drug vehicle interactions. Additionally, no correlations were

drawn comparing the flux of drug with the membrane and human skin. In a recent study

conducted by Simon et al., synthetic membranes were used to compare with an ex vivo pig

ear skin model for transdermal drug permeation of rivastigmine [60]. Results concluded

that most polymeric membranes tested had low correlation factors (R2 = < 0.90). In this

study, Strat-M® synthetic membrane demonstrated a relatively high correlation to human

skin for nicotine flux with a R2 value of 0.99 (Figure 2.6). Moreover, the time point

correlation between Strat-M® and human cadaver skin were in the range 0.90-0.99 (Figure

2.5C). This work suggests that some of the main transport mechanisms for drug diffusion

and permeation could be similar to an ex vivo human skin model. But more studies needed

to be performed with different hydrophilic and lipophilic compounds to further confirm

these suggestions.

2.4. Conclusions

In summary, this study has demonstrated that a novel Strat-M® synthetic membrane

has potential to be used as an early screening model to select the best performing CPE for

transdermal formulations containing nicotine. Our findings suggest that Strat-M® can

provide useful predictions based on the major role partitioning plays in passive drug

diffusion. The synthetic membrane also offers an additional value by providing rank

ordering of penetration enhancers from enhancement effectiveness standpoint. Although

41

Fig. 6.

(C)

0

25

50

75

100

0 2 4 6 8

Per

mea

tio

n (

ug/c

m-2

)

Time (h)

Control

0

1000

2000

3000

0 2 4 6 8

Per

mea

tio

n (

ug/c

m-2

)

Time (h)

Tween 80

0

1000

2000

3000

0 2 4 6 8

Per

mea

tio

n (

ug/c

m-2

)

Time (h)

Eucalyptol

0

500

1000

1500

0 2 4 6 8

Per

mea

tio

n (

ug/c

m-2

)

Time (h)

N-Methyl Pyrrolidone

0

100

200

300

0 2 4 6 8

Per

mea

tio

n (

ug/c

m-2

)

Time (h)

Tween 80

0

25

50

75

100

0 500 1000 1500

Q (

Skin

, ug/c

m-2

)

Q (Strat-M, ug/cm-2)

Control

0

25

50

75

100

0 2 4 6 8

Per

mea

tio

n (

ug/c

m-2

)

Time (h)


0

25

50

75

100

0 500 1000 1500

Q (

Skin

, ug/c

m-2

)



0

50

100

150

200

0 1000 2000

Q (

Skin

, ug/c

m-2

)


Tween-80

0

50

100

150

200

0 1000 2000Q (

Skin

, ug/c

m-2

)


Eucalyptol

0

100

200

300

0 2 4 6 8

Per

mea

tio

n (

ug/c

m-2

)

Time (h)

Eucalyptol

0

200

400

600

0 2 4 6 8

Per

mea

tio

n (

ug/c

m-2

)

Time (h)

Azone

0

1000

2000

3000

4000

0 2 4 6 8

Per

mea

tio

n (

ug/c

m-2

)

Time (h)

Azone

0

1

2

3

0 1000 2000 3000

Q (

Skin

, ug/c

m-2

)


Azone

0

500

1000

1500

0 2 4 6 8Per

mea

tio

n (

ug/c

m-2

)

Time (h)

Control

(A) (B)

R²=0.98

R²=0.99

R²=0.99

R²=0.98

R²=0.90

42

Figure 2.5. Nicotine penetration profiles of transdermal formulations (A) human cadaver

skin, (B) Strat-M® membrane, and (C) time point correlations between the amounts of drug

penetrated through human cadaver skin and Strat-M® membrane. Means plus minus S.D.

and correlation coefficients.

Figure 2.6. Correlation of flux between Strat-M® synthetic membrane and human cadaver

skin.

Strat-M® will only provide information on trends and correlations and not match the

absolute permeability values for human skin, still ranking the order of penetration

enhancement efficiency is helpful for understanding which modes are the most effective

for developing an optimized formulation. This however, is information that can be used in

preliminary screening efforts in the pharmaceutical, cosmetic and personal care industries

to select formulations or penetration enhancers or vehicles for drugs/actives. Moreover, the

synthetic membranes are a convenient tool to investigate and to determine the most

effective chemical penetration to be used in vivo with human skin. The Strat-M® synthetic

R² = 0.99

0

5

10

15

20

25

0 50 100 150 200 250 300 350 400

Hu

ma

n C

ad

av

er S

kin

Flu

x (

µg

/cm

²)

Strat-M Synthetic Membrane Flux (µg/cm²)

43

membrane serves as a rapid, cost-effective, easy and ready to use method with no solvent

activation requirements. Thus, synthetic membranes can be successfully used as a

screening tool in order to choose the best performing chemical enhancers and to evaluate

the transdermal formulations through the human cadaver skin.

The Strat-M® membranes share similar drug permeability behavior of nicotine

permeation to an ex vivo human skin model; which is mainly driven by drug partitioning.

Major structure organizational differences between both models; limiting the applicability

to be used as a direct correlation prediction. Enhancement of physiochemical behavioral

properties in formulations show similar trends between both models. Future work in this

area is still needed to understand the impact of CPE’s from other classes on other drug

candidates including ones with lipophilic properties.

44

Chapter 3. Solubility-physicochemical-thermodynamic theory of penetration

enhancers mechanism of action

3.1.Introduction

The passage of the drugs through skin mainly follows laws of passive diffusion and

can be described by Fick’s first law [27]. According to this law a permeant will move down

a concentration gradient [60] from a region of high concentration to a region of lower

concentration. Mathematical models derived by Higuchi explained this passive diffusion

process in terms of percutaneous absorption [61]. Moreover, Higuchi used

physicochemical principles in describing the importance of the thermodynamic activity of

the permeating molecule [62]. The thermodynamic driving force for the partitioning of

drug depends on the difference between the chemical potential of drug in a particular

vehicle and skin [63]. From the thermodynamic point of view, the steady state flux (J) can

be expressed by [61] -

J = αD/ϒ L (1)

where α is the thermodynamic activity of the drug in its vehicle, ϒ is the activity

coefficient of the drug in the skin, D and L are diffusion coefficient of the drug in the skin

and skin thickness respectively.

The extent of the percutaneous drug absorption from a topically applied formulation

is greatly influenced by the physicochemical properties of the drug. Due to the dependency

on physicochemical properties there is only a very small group of drugs that can be

45

delivered through the transdermal route utilizing passive diffusion alone. There are several

techniques among which is the use of chemical enhancers – a most convenient and popular

technique to modify the permeation through the skin. By using different penetration

enhancers, we can modify the thermodynamic activity of the drug in the formulation and

as a result can manipulate permeant flux. In this way we can also increase the range of

drug candidates that can be effectively delivered through transdermal route.

To date, there is limited research that demonstrate the underlying mechanism of

action of these enhancers [64]. Thus, it is very important to clearly define their actions in

the formulation with the drug. Lipid-Protein-Partitioning theory [65] is based on the fact

that enhancers usually work by one or more of the following three mechanisms-

1) The disruption of the lipid domains of the stratum corneum;

2) Interaction with intracellular protein; and

3) Increasing partitioning of a drug, co-enhancer or co-solvent into the tissue.

These are all broadly described mechanisms that do not provide details why a given

enhancer will not increase the permeation of all types of drugs and why the permeation

ability of enhancers depends on their concentration and why some of them are most

effective at lower rather than higher concentrations. In this manuscript we provide evidence

that the mechanism of action of enhancers is related to solubility parameters,

physicochemical interactions as well as thermodynamic activity. The objective of the

present investigation was to propose for the first time a Solubility-Physicochemical-

Thermodynamic theory to define the action of penetration enhancers. While developing

this approach, the following important factors had to be considered-

46

1) The potency of penetration enhancers appears to be drug specific due to the

importance of physicochemical interactions between drug and enhancers and thus

should be compared. Here we describe this interaction as the “ingredient active

gap” (IAG).

2) The effectiveness of most penetration enhancers is concentration dependent and it

must be remembered that penetration enhancers also permeate through the skin with

the vehicle and active molecule.

3) The affinity of the enhancers to the skin. There is a link between the mechanism of

action of enhancers and their affinity to skin. Here we describe this affinity as the

“ingredient skin gap” (ISG).

4) The thermodynamic activity effect provided by the addition of an enhancer.

Enhancers can increase or optimize the thermodynamic activity of the drug in the

formulation as well as the effects in the skin.

5) The solvent properties of the skin that affect the permeants. Thus, solubility of

active in the formulation (SolV) and the formulation solubility in the skin (SolS)

should also be considered as both influence drug permeation.

This work is the missing piece of the puzzle that will connect the physicochemical

properties of the active and enhancers/ingredients with the thermodynamic activity of the

drug in order to explain the mode of action of enhancers in a given formulation with a

specific drug. There is definitely a need for theoretical bases that will allow prediction of

the effect of enhancers on skin flux of drugs. Here we propose the basis of this theory that

47

allows the prediction of outcomes and to correlate these with in vitro experimental

diffusion cell data.

3.1.1.Theoretical background

The cohesive energy density which is equal to the energy of vaporization divided

by the molar volume reflects the degree of attractive forces holding the molecules together

[66]. In 1950 Hildebrand introduced solubility parameter δt, the square root of the cohesive

energy density (ced) [67, 68].

δt = (ced)1/2 = (𝛥𝐸𝑣

𝑉)1/2 (2)

where 𝛥𝐸𝑣 is the latent energy of vaporization, V is the molar volume. The latent energy

of vaporization is usually calculated by-

𝛥𝐸𝑣 = 𝛥𝐻𝑣 -RT (3)

where 𝛥𝐻𝑣 is the latent heat of vaporization, 𝑅 is the universal gas constant, and 𝑇 is the

absolute temperature.

It was realized by Charles M. Hansen in the mid-1960s that in the evaporation

process all of the cohesive bonds holding the liquid together were broken and led to an

expansion of the Hildebrand solubility parameters δt into three dimensions [69] : dispersive

interactions (δD) – the dispersion or van der Waals properties of a molecule, or the amount

of polarizable electrons. It has a strong correlation with refractive index which is, at a

deeper level, a correlation with the polarizability of a molecule. Those molecules with more

electrons able to move freely at the surface have a higher polarizability, a higher refractive

48

index, stronger van der Waals interactions and a higher δD. polar interactions (δP) – the

polar contribution – related to (and calculable from) dipole moment. This is generally

thought of as classic polarity. Something with a large dipole moment will have a large δP.

and hydrogen bonding (δH) – the hydrogen bonding contribution is usually found by

subtracting the polar and dispersion parameters from the total parameter, or in terms of

energies, by subtracting the dispersion and polar energies from the cohesive energy. This

is important for a number of the solvents and solutes in the study and should not be ignored.

(δt)2 = (δD)2 + (δP)2 + (δH)2 (4)

These three solubility parameters can be visualized using FFE (Formulating for

EfficacyTM - ACT Solutions Corp) Software with a three-dimensional coordinate system,

the so-called Hansen space with axes δD, δP and δH. The Hansen Solubility Parameter (HSP)

methodology is based on the Hansen Solubility Parameters in Practice (HSPiP) program

from Steven Abbott, Charles Hansen and Hiroshi Yamamoto. It incorporates the Y-MB

(Yamamoto Molecular Breaking) methodology for calculating HSP from SMILES

developed by Hiroshi Yamamoto. The Formulating for Efficacy (FFE) software program

was written for Dr. Johann Wiechers, a noted expert in active delivery by Professor Steven

Abbott and based on the HSPiP software with additions and adaptations for utility in

pharmaceutical and cosmetic applications. Among the additions is the Diffusion Modeler

which mimics the dynamics of a Franz diffusion cell, using accepted principles of diffusion

and HSP of the stratum corneum.

The closer the HSP (Hansen solubility parameters) values of two substances or

smaller the distance between the coordinates of two substances in the 3D HSP space the

49

higher is their similarity and the stronger is their affinity [70]. The distance between the

materials (Ra) on such plots is calculated by the following equation [69]:

Ra2 = 4(δD1 - δD2)2 + (δP1 – δP2)

2 + (δH1 – δH2)2 (5)

In this equation subscript 1 and 2 refer to material 1 and 2. For high affinity between

two materials Ra must be less than the R0 (radius of a Hansen solubility parameter sphere)

of the test material. R0 describes how large or small the interaction range is. Relative

Energy Difference (RED) value is used to quantify relative distances between Ra and R0

and RED < 1 represents high affinity whereas RED > 1 represents low affinity [71]

RED = Ra/ R0 (6)

The FFE software plots the δD, δP and δH of each compound along the three axes of

a 3D graph after which the program calculates a sphere whose center coordinates give the

δD, δP and δH of the skin. This software was chosen as it has an integrated suite of programs

that allow calculation of various physicochemical and solubility properties including

ingredient active gap (IAG), ingredient skin gap (ISG), solubility of active in the

formulation (SolV) and the formulation solubility in the skin (SolS) [72]. IAG; this is the

difference in polarity between an Ingredient and an Active, expressed in this work through

comparison of the bonding energies of each – Dispersion, Dipolar, and Hydrogen Bonding

forces of the molecules. In another sentence, the IAG is the sum of the square root of the

difference of the squares between the HSP’s of the Ingredient and the Active. An Active

with a high IAG between it and a specific Ingredient indicates that there are vast differences

in the polarity between the molecules and likely not a good match for solvency or delivery,

but possibly could be used to induce a driving force in a mixture with an Ingredient with a

50

low IAG. The smaller the IAG, the more alike are the Active and the Ingredient and as

“like dissolves like” the solubility will be higher. ISG; analogous to the Ingredient Active

Gap. This is the difference in polarity between an Ingredient and the skin; expressed in

this work through comparison of the bonding energies of each – Dispersion, Dipolar, and

Hydrogen Bonding forces of the molecules; an Ingredient with a low ISG may be more apt

to penetrate the skin and carry an Active, but other such factors as SolS, IAG, MVol (molar

volume), and SolV should be taken into account. The smaller it is, the more compatible the

ingredient is with the stratum corneum. This means that it will also ‘mix’ better with the

skin. Skin penetration can be seen as a way of ‘mixing’ the skin and the ingredient. So, the

smaller the ISG, the more the ingredient will penetrate the skin. This ISG was formerly

called the PPG, the Penetrant Polarity Gap. SolV; predicted solubility of an Active in a

specific Ingredient, expressed as a percentage; uses IAG and comparisons of MVol, and

Melting Point (MPt. ⁰C) to predict the percentage of Active that will remain soluble in an

Ingredient at room temperature; this Hansen model has been used for more than 50 years,

mainly in industrial applications, to predict solubility with excellent reliability. SolS;

solubility in the skin; whether an Ingredient has low or high solubility in the barrier is a

factor to consider when evaluating an Ingredient for use in delivery enhancement;

expressed as a percentage.

The aim of the present study was to investigate whether various physicochemical

properties derived from Hansen solubility parameters of actives, excipients, and the skin

and FFE software can be used to describe the ability of drugs and enhancers/excipients to

cross the biological membrane. The purpose of these studies was to develop a model to

explain the enhancers mode of actions in drug permeation. The overall concept may be

51

referred as the Solubility-Physicochemical-Thermodynamic (SPT) theory so that this

designation may facilitate our consideration of enhancer action based on solubility

parameters and physicochemical interactions governing the thermodynamic activity of

drug.

3.2.Materials and Methods

3.2.1. Materials

Polysorbate 80 (Tween 80), eucalyptol, N-methyl-2-pyrrolidone, propylene glycol,

sodium phosphate monobasic, thymoquinone (TQ), High-performance liquid

chromatography (HPLC) grade water and acetonitrile were purchased from Sigma-Aldrich

Co. (St. Louis, MO, USA). Nicotine was purchased from Alfa Aesar (Haverhill, MA, USA)

and laurocapram (Azone) was purchased from BOC Sciences (Shirley, NY, USA).

Phosphate-buffered saline tablets (PBS, pH 7.4) was purchased from MP Biomedicals,

LLC (Solon, OH, USA), ethanol was purchased from Decon Labs, Inc. (King of Prussia,

PA, USA) and O-phosphoric Acid 85% was purchased from Fisher Scientific (Hampton,

NH, USA). Dermatomed human cadaver skin from the posterior torso (2 Females at the

age of 34, 69 and 2 males at the age of 68, 45) were obtained from New York Firefighter

Skin Bank (NY, USA). Oleic acid and Transcutol® P was a gift from Croda (Edison, NJ,

USA) and Gattefosse (Paramus, NJ, USA) respectively.

52

3.2.2. Preparation of formulation and solubility determination

10 mL of five different formulations containing 1% nicotine, with or without 5%

of one type of enhancer (Azone, oleic acid, Tween 80, eucalyptol, and N-Methyl-2-

pyrrolidone) in propylene glycol (as vehicle) were prepared. Saturated solutions of TQ

were prepared by adding an excess amount of TQ and 250 µL of the enhancer to 5 mL of

propylene glycol in a well-closed container. Using Julabo SW22 shaker (Julabo USA Inc.,

Allentown, PA) all the amber containers with TQ in their respective solvents were agitated

at 37°C for 48 hours. After 48 hours all the samples were filtered through a 0.2 µm PES

syringe filter media with polypropylene housing. All experiments were performed in

triplicate and the drug content was measured by HPLC after appropriate dilution.

3.2.3. Permeation procedure for enhancer studies

Franz diffusion cells (FDC) with a donor area of 0.64 cm2 and a receptor volume

of 5.0 mL (Permegear Inc., Hellertown, PA) were used throughout the study. The samples

of dermatomed human cadaver skin were slowly thawed, cut into appropriate pieces to fit

the Franz cells and then soaked in filtered PBS (pH 7.4) for 15 minutes. After that they

were mounted on FDC with the epidermal side in contact with the formulation or donor

compartment. The receptor compartment of each cell was filled with filtered PBS (pH 7.4)

and was maintained at 37oC under synchronous continuous stirring using a magnetic stirrer

at 600 rpm. The diffusional membranes were left to equilibrate at 37oC for 15 minutes.

Once reached equilibrium, at time zero 200 µL of nicotine formulation and 500 µL of

thymoquinone formulation was added to the donor compartment of each Franz diffusion

53

cell. At each time point (1, 2, 3, 4, 5, 6, 7 and 8 hours for nicotine and 3, 4, 6, 8, 10, 12 and

24 hours for thymoquinone) 300 µL of receptor samples were withdrawn from the sampling

port. At the end of experimental hours, receptor aliquots of 300 µL were then analyzed

using a valid HPLC method described below.

3.2.4. Skin deposition study

At the end of permeation study, the skin was removed from the diffusion cell and

was cut around the diffusional area, air dried, and accurately weighed. The samples were

then placed into bead bug tubes and using a scissor they were cut into very small pieces. 1

mL ethanol was added to each tube and they were homogenized for 9 minutes (3 min of 3

cycles) by using BeadBugTM Microtube homogenizer, D1030 (Benchmark Scientific,

Sayreville, NJ). All the skin samples were then placed in a Julabo SW22 shaker (Julabo

USA Inc., Allentown, PA) and were agitated at 37 °C for 24 hours. After that all the skin

samples were centrifuged at 1200 rpm for 5 minutes and were filtered through a 0.45 μm

polypropylene filter media with polypropylene housing. TQ concentrations were expressed

as ng of TQ per skin weight in mg.


A validated HPLC method was used for this study. The HPLC instrument used

was Agilent 1100 series instrumentation (Agilent Technologies, CA, USA) coupled with

54

UV detection (DAD) and HP Chemstation software V. 32. The HPLC method of analysis

for each compound is provided below:

3.2.5.1. Nicotine

For the analysis of nicotine, a mobile phase of 65% sodium phosphate buffer

(adjusted to pH 3.2 with 85% orthophosphoric acid) and 35% acetonitrile was pumped

through a Phenomenex Luna® 5 µm C18(2) 100 Å Column 250 X 4.6 mm. Injection

volumes of 10 uL with a flow rate of 1.0 mL/minute set to 25°C with UV detection of 259

nm were used with retention time of 2.5 minutes. The method was linear at a concentration

range 0.9 -500 µg/mL with R2 of 0.999.

3.2.5.2. Thymoquinone

For the analysis of TQ, a mobile phase of 80% acetonitrile and 20% water was

pumped through an Agilent Eclipse XDB-C18 5 µm, 250 X 4.6 mm column. Injection

volumes of 20 uL with a flow rate of 1.0 mL/minute was set to 23°C with UV detection of

250 nm were used with retention time of 4.2 minutes. The method was linear at a

concentration of 0.39-100 µg/mL with R2 value of 0.998.

3.2.6. Calculated solubility and physicochemical parameters and permeation data

The FFE Software can be used to derive the Hansen Solubility Parameters of actives

and excipients with the input of a linear chemical structure expression called SMILES;

from there, solubility profiles, permeation and different physicochemical properties of drug

55

actives and excipient ingredients are calculated automatically. Measured drug permeation

data were compared with the calculated permeation data and solubility parameters of the

drugs. Eight-hour diffusion studies for nicotine and twenty-four-hour diffusion studies for

TQ were modeled for each formulation were run using the software.

3.2.7. Data and statistical analysis

The cumulative amounts of nicotine and TQ permeated per unit area were plotted

against time. The flux was calculated by determination of the slope of the linear portion of

the permeation profile. Results are reported as mean ± SD (n=6). The statistical analysis of

the data was performed by using one-way Anova and Student’s-t test, and p-values < 0.05

were considered significant.

3.3.Results and discussion

3.3.1. Nicotine

If a penetration enhancer is added into the formulation, then its affinity to the skin

must be considered. For example, if a penetration enhancer has lower ISG indicating it has

higher affinity to the skin that will further increase the formulation solubility in the skin

(SolS) (Figure 3.3A and B). The addition of certain penetration enhancers can also increase

the solubility of active in the formulation (SolV). The more extreme the difference in

solubility between the formulation (SolV) and the skin (SolS) the greater this driving force

for partitioning into the stratum corneum. In another scenario, if a penetration enhancer has

56

lower skin affinity (increased ISG) that will lead to the reduction of formulation solubility

in the skin and can further increase the permeability of the active across the skin given that

there is an increased active solubility in the formulation and an increased physicochemical

interaction between the enhancer and active (Figure 3.3A and B). If there is an increase in

the thermodynamic activity of the drug in the formulation, then an increase in rate of

delivery/flux of the drug can be achieved which will improve the clinical effect as well.

The desired modification of the thermodynamic activity of the drug can be achieved by

thoughtfully selecting the penetration enhancers for the formulation or by changing the

concentration or relative composition of the enhancers. Such modifications can benefit

both topical and transdermal delivery of either pharmaceutical or cosmetic active

ingredients.

In the present investigation the three-dimensional Hansen solubility parameters

were used to correlate the experimental permeation data of drugs. It was demonstrated that

the permeation of drug can be predicted by analyzing different physicochemical (Table

3.2) and solubility parameters (Table 3.1) of drug and ingredients that were assessed using

FFE Software. The three solubility parameters (δD, δP and δH) from Table 3.1 can be further

visualized using FFE ((Formulating for EfficacyTM - ACT Solutions Corp) Software with

a three-dimensional coordinate system, the so-called Hansen space with axes δD, δP and δH

(Figure 3.2). The closer the HSP (Hansen solubility parameters) values of two substances

in the 3D HSP space the higher is their similarity and the stronger is their affinity. Figure

3.1A represents the measured permeation data from the Franz diffusion cell method using

human cadaver skin and Figure 3.1B represents the correlations between the calculated and

measured permeation of nicotine, where X axis represent calculated permeation data from

57

the software model (µg/cm2) and Y axis represent measured permeation data from the

Franz diffusion cell study (µg/cm2). Through this graph we have also found out the

theoretical value of the correlation of permeation that exist between the measured

permeation data and the calculated permeation data. It is represented by R2 value, the

coefficient of multiple determination for multiple regression. Figure 3.1B shows a linear

correlation between measured and calculated permeation of nicotine from various

formulation using different ingredients. Among other ingredients Azone showed lower

regression coefficient value of R2 = 0.62. This result supports our previous study of

comparing the permeability of different formulations using various enhancers to human

cadaver skin and Strat-M® synthetic membrane [73].

Table 3.1. Hansen solubility parameters and molar volume for nicotine and different

solvents/enhancers.

1

2

Solvent Diffusion Coefficient δD δP δH Mvol 3 (m2/s) 4 5 Nicotine 5.49E-10 18.6 5.1 5 159.9 6 Propylene Glycol 1.30E-09 16.8 10.4 21.3 73.7 7 Tween 80 8.72E-15 16.2 6.6 9.6 1265 8 Eucalyptol 5.09E-10 16.6 2.7 2.5 167.5 9 N-Methyl Pyrrolidone 1.02E-09 18.1 10.3 6.6 98.1 10 Azone 1.20E-10 17 1.6 3.2 311.7 11 Oleic Acid 1.14E-10 16.5 3.2 5.7 317.5 12 13

14

58

Table 3.2. Physicochemical parameters of nicotine and different enhancers.

This study has also shown lower R2 value while comparing the permeation of drug

using Azone with human skin and synthetic membrane. This is probably due to the fact

that both synthetic membrane and software-based model are unable to account for the more

biological interaction of penetration enhancer and skin. In case of Azone the biological

interaction with the skin is more prominent as it causes the fluidization of the intercellular

lipids by creating a ‘soup spoon’ model within stratum corneum lipids [47, 48]. This unique

characteristic of Azone must have played a significant role while driving formulation

through skin. On the other hand, this biological influence was absent while running

diffusion using FFE software and this same effect was also noticeable while performing

permeation study using synthetic membrane. Moreover, Azone is associated with removal

of skin lipids and provide additional effects on the membrane components by acting as

1 2

Penetration Enhancers ASG IAG ISG SolV SolS 3

Nicotine 5.26 - - 100 100 4 Propylene Glycol - 17.51 9.96 0.9 100 5 Tween 80 - 6.82 33.66 100 0.6 6 Eucalyptol - 5.29 12.86 100 3.4 7 N-Methyl Pyrrolidone - 5.53 3.41 100 100 8 Azone - 5.07 24.94 100 0.2 9 Oleic Acid - 4.66 17.19 100 0.2 10 11 ASG – Active skin gap; IAG – Ingredient active gap; ISG – Ingredient skin gap; SolV – 12

Solubility of active in the formulation; SolS – The formulation solubility in the skin 13

59

A B

Figure 3.1. Nicotine permeation profiles of transdermal formulations (A) with the Franz

diffusion cell method using human cadaver skin, (B) the correlations between the

calculated and measured permeation of nicotine.

60

Figure 3.2. Position of the active nicotine and penetration enhancers/ingredients in 3D

Hansen Space.

surfactants as well as solvents [74]. The amount of nicotine permeated per square cm from

formulation 4 (nicotine 1% in propylene glycol + 5% Azone) and from formulation 6

(nicotine 1% in propylene glycol + 5% oleic acid) after 8 hours (Q8) were significantly

greater when compared to control and all other formulations (p<0.05) (Table 3.3). There is

synergy between Azone and propylene glycol since propylene glycol assists Azone

penetration into the stratum corneum and Azone also promotes the flux of propylene

glycol. Barry, B.W. has demonstrated the importance of the correct choice of vehicle or

co-solvent for Azone and oleic acid. For such enhancers to reach the polar surface of the

lipid bilayer in sufficient amounts, they may require a co-solvent such as propylene glycol

or ethanol [65]. The study conducted by Wotton, P.K. et al. has demonstrated that

61

metronidazole penetration is enhanced both by 1% Azone and by propylene glycol alone

and together they produced a synergistic effect by giving highest permeation of the

compound [75]. In another study Squillante, E., et al. have identified positive synergistic

interactions among the following formulation components; propylene glycol, cis-oleic

acid, and dimethyl isosorbide—which strongly affected nifedipine permeation [76]. The

synergistic action of propylene glycol with oleic acid may be due to the co-solvent’s ability

to reduce the polarity of the aqueous regions of the stratum corneum, so increasing the

ability of the stratum corneum to solubilize oleic acid [77]. Our previous study showed

highest permeation of active using 5% of Azone and oleic acid from propylene glycol and

ethanol vehicle compared to other enhancers [78]. It may be deduced from this study that

there must be other factors such as the change in the barrier properties by these solvents,

that have played an additional role besides the synergy present between the solvent and

enhancer to increase the flux of same active from two different solvents. The formulation

with 5% N-Methyl Pyrrolidone did not show any enhancement effect on nicotine

permeation through human cadaver skin. Eucalyptol and Tween 80 shared similar trends

by providing lower nicotine flux in comparison to the formulation containing 5% oleic

acid, 5% Azone, and higher flux of nicotine when compared to N-Methyl Pyrrolidone

(5%). The rank order of each enhancer/ingredient for the enhancement of nicotine skin

permeation was as follows: Oleic acid>Azone>Eucalyptol + Tween 80>N-Methyl

Pyrrolidone. This permeation ranking can be further understood by analyzing the ratio of

SolV and SolS, IAG and ISG values showed on Table 3.2 and illustrated in Figure 3.3A

and B. By using FFE Software it was found that the SolV and SolS ratio of NMP is 100:100,

which is the same as nicotine. For this reason, in spite of having lowest ISG (3.41) value

62

among all other ingredients, most likely the N-Methyl Pyrrolidone did not achieve high

enough thermodynamic activity to drive the active from the formulation into the skin

efficiently (Figure 3.3B). On the other hand, oleic acid, with SolV and SolS ratio (100:0.2)

and low IAG (4.66) value compared to all other ingredients used in this experiment, was

found to be best in providing maximum nicotine flux. Additionally, Azone shares the same

SolV and SolS values with oleic acid but showed lower nicotine flux than oleic acid due

likely to higher IAG and ISG values (Figure 3.3A and B).

There appears to be an inverse relationship between measured flux and IAG values

given that there is an optimum ingredient skin gap, SolV and SolS ratio. The sensitivity of

the Hansen solubility and physicochemical parameters can be further understood by

analyzing the behavior of eucalyptol and Tween 80. From the experimental study, Q at 8

hours (µg/cm2) for nicotine permeation using eucalyptol was 168 ± 41 and Q at 8 hours

(µg/cm2) for nicotine permeation using Tween 80 was 167 ± 43. The experimental data

showed that in spite of having different functional group they influenced the

thermodynamic activity of the nicotine in the formulation at a same rate. Interestingly, we

can explain this behavior using SPT theory. According to this theory, after analyzing their

physicochemical characteristics it was found that eucalyptol has higher affinity to the skin

with a SolS value 3.4 compare to Tween 80 (SolS value 0.6). Additionally, IAG value of

eucalyptol is 5.29 and for Tween 80 is 6.82. It can be assumed that in spite of having higher

skin solubility (SolS) this small fraction of reduction of IAG values of eucalyptol may

influenced the thermodynamic activity of nicotine in a better way by giving slightly

increased flux compared to Tween 80 (Figure 3.3A). It is to be remembered that what is

being evaluated here, especially regarding Tween 80 systems, are SPT effects and not

63

[A]

Penetration enhancers Action 1

Oleic acid Increased physicochemical interaction with nicotine due to small IAG value (4.66) 2

and optimum SolV (100) and SolS (0.2) values. Increased thermodynamic activity 3

drove nicotine from the formulation efficiently for the drug to enter and pass through 4

the skin. Higher skin permeability and flux was observed. 5

Azone Physicochemical interaction between Azone and nicotine was reduced compared to 6

Oleic acid due to slightly increased IAG value (5.07). Azone showed optimum SolV 7

(100) and SolS (0.2) values. Due to lower physicochemical interaction Azone 8

provided lower skin permeability of nicotine compared to oleic acid. 9

Eucalyptol Increased IAG value (5.29) reduced physicochemical interaction of eucalyptol and 10

nicotine. Eucalyptol has higher affinity to the skin due to lower ISG value (12.86) 11

that produced improved/increased formulation solubility in the skin (SolS value 3.4). 12

Provided slightly increased flux of nicotine compared to Tween 80 due to small 13

reduction in IAG value. 14

Tween 80 Physicochemical interaction was reduced with increasing IAG value (6.82). Tween 15

80 has lower affinity to the skin compared to eucalyptol due to increased ISG 16

(33.66) that further reduced the formulation solubility in the skin as indicated by 17

SolS value of 0.6. 18

NMP Reduction in physicochemical interaction due to increased IAG (5.53). Produced 19

highest SolV and SolS ratio (100:100). Higher affinity to the skin compared to 20

other ingredients as indicated by lower ISG (3.41). Due to the same SolV:SolS 21

ratio like nicotine NMP has provided lower skin permeability of nicotine thus 22

lower flux. 23

24

64

[B]

Figure 3.3. The influence of physicochemical interactions (IAG, SolV) between

penetration enhancer and active (Nicotine) on the driving force for diffusion and, the

influence of various physicochemical and solubility parameters (ISG, SolS) of the

formulation on the skin affinity of the penetrant is illustrated. (A) The possible mechanism

of action of skin penetration enhancers; (B) a representation of the active-enhancer and

stratum corneum interactions promoting partitioning into the stratum corneum.

Vehicle Stratum Corneum Lipid

Physicochemical interaction

between nicotine and Oleic

acid

IAG 4.66

Increased thermodynamic

activity drove nicotine from

the formulation efficiently to

enter and pass through the

skin. Higher skin permeability

and flux was observed.

ISG 17.19

SolS 0.2


between nicotine and NMP

IAG 5.53

Due to the same SolV:SolS

(100:100) ratio like nicotine

NMP has provided lower

skin permeability of

nicotine thus lower flux.

Increasing affinity for stratum

corneum ISG 3.41

65

colloidal effects. Further enhancement based on such colloidal structures as

microemulsions or lamellar gel networks can be overlain on the SPT approach.

Table 3.3. Penetration parameters of nicotine through human cadaver skin after 8 hours.

3.3.2.Thymoquinone

After analyzing the applicability of Solubility-Physicochemical-Thermodynamic

(SPT) theory using 1% nicotine and 5% of each enhancer from five different groups

(Amides-Azone; fatty acids- oleic acid; surfactants- Tween 80; pyrrolidones- N-Methyl

Pyrrolidone, terpenes- eucalyptol) now we want to use another drug of a solid form

(thymoquinone) to measure their experimental solubility in different enhancers to see

whether we can correlate their solubility, permeability and skin deposition experimental

66

data with the measured permeability, solubility and physicochemical parameters to

understand the influence of enhancers on thermodynamic activity of thymoquinone.

Tables 3.4 and 3.5 represent the Hansen solubility and physicochemical parameters

of thymoquinone and different solvents/enhancers. Penetration parameters of

thymoquinone through human cadaver skin (N=5) after 24 hours have been provided in

Table 3.6. The rank order of each enhancer/ingredient for the enhancement of

thymoquinone skin permeation was as follows: Azone + Oleic Acid>Transcutol®

P>Control + Tween 80>Ethanol>NMP. Figure 3.4A shows the measured permeation data

from the Franz diffusion cell method using human cadaver skin and Figure 3.4B shows the

correlations between the calculated and measured permeation of TQ, where X axis

represent calculated permeation data from the software model (µg/cm2) and Y axis

represent measured permeation data from the Franz diffusion cell study (µg/cm2). Figure

3.4B shows a linear correlation between measured and calculated flux of TQ from various

formulation using different enhancers and Figure 3.5 shows the position of thymoquinone

and penetration enhancers in 3D Hansen Space.

67

Table 3.4. Hansen solubility parameters and molar volume of thymoquinone and different

solvents/enhancers.

Table 3.5. Physicochemical parameters of thymoquinone and different enhancers.

1

2

Solvent Diffusion Coefficient δD δP δH Mvol 3 (m2/s) 4 5 Thymoquinone 5.72E-10 18.3 9.2 5.1 159.9 6 Propylene Glycol 1.30E-09 16.8 10.4 21.3 73.7 7 Tween 80 8.72E-15 16.2 6.6 9.6 1265 8 N-Methyl Pyrrolidone 1.02E-09 18.1 10.3 6.6 98.1 9 Azone 1.20E-10 17 1.6 3.2 311.7 10 Oleic Acid 1.14E-10 16.5 3.2 5.7 317.5 11 Ethanol 1.51E-09 15.4 9.2 19.6 58.7 12 Transcutol® P 7.03E-10 16.3 7.1 11.9 135.2 13

14

1 2

Penetration Enhancers ASG IAG ISG SolV SolS 3

Thymoquinone 4.08 - - 73.4 100 4 Propylene Glycol - 16.52 9.96 1.3 100 5 Tween 80 - 6.68 33.66 21.7 0.6 6 N-Methyl Pyrrolidone - 1.9 3.41 81.6 100 7 Azone - 8.25 24.94 22.4 0.2 8 Oleic Acid - 7.02 17.19 35.3 0.2 9 Ethanol - 15.62 7.1 4.9 100 10 Transcutol® P - 8.16 5.73 67 100 11

ASG – Active skin gap; IAG – Ingredient active gap; ISG – Ingredient skin gap; SolV – 12

Solubility of active in the formulation; SolS – The formulation solubility in the skin 13

68

A B

Figure 3.4. Thymoquinone permeation profiles of transdermal formulations (A) with the

Franz diffusion cell method using human cadaver skin, (B) the correlations between the

calculated and measured permeation of Thymoquinone.

69

Figure 3.5. Position of the active Thymoquinone and penetration enhancers/ingredients in

3D Hansen Space.

In spite of having lowest IAG (1.9) value compared to all other enhancers NMP

was not able to provide better TQ permeation due to its high solubility in the skin and in

the formulation (Figure 3.6A). From the solubility data (Table 3.7) it was found that

thymoquinone has highest solubility in ethanol. On the other hand, the permeation data

showed that thymoquinone flux was lower than the control formulation with 5% of ethanol

(Table 3.6). It shows that the flux is actually proportional to a gradient of thermodynamic

activity rather than the concentration. The thermodynamic activity of thymoquinone was

70

reduced as ethanol has highest IAG value and it is also very soluble in the skin (Figure

3.6A).

Table 3.6. Penetration parameters of thymoquinone through human cadaver skin (N=5)

after 24 hours [78].

TQ Flux TQ Q 24 ER

Formulation (µg/cm²/h) (µg/cm²)

Control 11.02±1.2 208±23

Tween 80 11.09±1.5 208±16 1

NMP 9±1.5b 167±38 0.81

Azone 49.3±5.6a 854±93 4.47

Ethanol 10.59±1 180±55 0.96

Oleic Acid 46.3±4.5a 865±113 4.2

Transcutol® P 14.23±1.4a 247±26 1.29

ER= Enhancement Ratio

a, significant increase in TQ flux (p<0.05)

b, significant reduction in TQ flux (p<0.05)

71

Table 3.7. Summary of the solubility study results showing the effect of 5% penetration

enhancers on the solubility of TQ using propylene glycol. The values represent the

concentration of TQ ± SD (N=3) in mg/mL at 48 hours [78].

Enhancers Solubility (mg/mL) ± SD

Propylene glycol 8.6 ± 0.3

Tween 80 9.4 ± 1.1

N-Methyl Pyrrolidone 8.5 ± 0.2

Azone 15.0 ± 1.4

Ethanol 15.7 ± 0.5

Oleic Acid 13.6 ± 2.1

Transcutol® P 11.1 ± 0.7

Table 3.4 and 3.5 and Figure 3.6A and B demonstrated that although Tween 80 has lower

IAG value compared to Azone and oleic acid it showed lower permeability due to its

highest ISG (33.66) and Mvol (1265). Transcutol® P has lower IAG value but does not

possess an optimum SolV : SolS ratio, thus did not provide better skin flux of TQ (Figure

3.6B). Oleic acid has a smaller IAG value than Azone (Table 3.5). Additionally, both

compounds have similar SolS values, but oleic acid has a higher SolV value than Azone.

It can be stated that, the more extreme the difference in solubility between the formulation

and the skin the greater the driving force for partitioning of the active into the stratum

corneum. With thymoquinone Azone and oleic acid showed similar trends in terms of

permeability. Whereas there was significant increase (p<0.05) of nicotine flux with Azone

compared to oleic acid. It is possible that this reversed behavior of Azone and oleic acid

72

[A]

Penetration enhancers Action 1

NMP Increased physicochemical interaction with TQ due to small IAG value (1.9). 2

Due to increased solubility of active in the formulation (SolV 81.6) and formulation 3

solubility in the skin (SolS 100) the driving force for partitioning into the stratum 4

corneum was reduced. Thus, lower skin permeability and flux was observed. 5

Ethanol Physicochemical interaction between TQ and ethanol was reduced with increasing 6

IAG value (15.62). It has higher affinity to the skin as indicated by the lower ISG 7

value (7.1). Most probably due to the higher IAG and lower ISG value there was 8

lower SolV (4.9) and higher SolS (100) value. This further reduced the flux of TQ. 9

Tween 80 Increased IAG value (6.68 ) reduced the physicochemical interaction of Tween 80 10

and TQ. Has lower affinity to the skin due to higher ISG value (33.66) that further 11

reduced the formulation solubility in the skin (SolS value 0.6). Provided lower skin 12

permeability and flux. 13

Transcutol Physicochemical interaction was reduced with increasing IAG value (8.16). It has 14

higher affinity to the skin (ISG 5.73). No optimum SolV, SolS ratio 15

(67:100) resulting in lower flux of TQ. 16

Oleic acid Reduction in physicochemical interaction due to increased IAG (7.02). Higher 17

affinity to the skin compared to Azone due to lower ISG (17.19). SolV, SolS ratio 18

was found to be (35.3:0.2) and provided higher flux of TQ. 19

Azone Increased IAG value (8.25) reduced the physicochemical interaction between 20

Azone and TQ. Lower affinity to the skin compared to oleic acid due to increased 21

ISG (24.94). Optimum SolV:SolS ratio (22.4:0.2) resulted in higher flux of TQ. 22

23

73

[B]

Figure 3.6. The influence of physicochemical interactions (IAG, SolV) between

penetration enhancer and active (Thymoquinone) on the driving force for diffusion and,

the influence of various physicochemical and solubility parameters (ISG, SolS) of the

formulation on the skin affinity of the penetrant is illustrated. (A) The possible mechanism

of action of skin penetration enhancers; (B) a representation of the active-enhancer and

stratum corneum interactions promoting partitioning into the stratum corneum.

Vehicle Stratum Corneum Lipid


between TQ and Azone

IAG 8.25

The more extreme difference in

solubility between the formulation

(SolV) and the skin (SolS) provided the

greater driving force for partitioning of

the active into the stratum corneum and

increased flux.

ISG 24.94

SolS 0.2


between TQ and Transcutol

IAG 8.16

No optimum SolV:SolS

ratio (67:100) resulting in

lower skin permeability and

lower flux of TQ.

Increasing affinity for stratum

corneum ISG 5.73

74

with the two different drugs is due to the complex concentration dependency of oleic acid

since oleic acid is more effective at lower concentration. We should also take in account

that penetration enhancers also permeate through the skin together with the vehicle and

active molecule.

When experiments were carried out for 8 hours with nicotine 5% oleic acid showed

highest permeability of the nicotine. On the other hand, use of 5% oleic acid did not result

in better permeability of thymoquinone compared to Azone when the experiment was

continued for 24 hours. It can be assumed that oleic acid was permeating together with the

active and at some point, there was not enough oleic acid to drive the thermodynamic

activity of the drug to influence its permeation. To understand the concentration

dependency of oleic acid we performed another experiment with 3% and 10% of oleic acid.

Figure 7 shows the amount of thymoquinone detected at 24 hours in human cadaver skin.

Since Azone and oleic acid showed the highest skin deposition of TQ it can be stated that

the maximum skin penetration and deposition was achieved when the drug is at its highest

thermodynamic activity.

3.3.3.Concentration dependency of oleic acid

Thymoquinone permeation profiles at different oleic acid concentrations (Figure

3.8) clearly show that thymoquinone permeated optimally with 3% oleic acid. In addition,

as the enhancer concentration increased the TQ permeation decreased. At the beginning,

for first 6 hours of experiment TQ permeated at a similar rate from each of the formulation

containing different concentrations (3%, 5% and 10%) of oleic acid (Figure 3.8). Then

gradually with increasing time the permeation become influenced by the varied

75

concentration of oleic acid. This may be due to the fact that with increasing concentration

and with increasing time higher amount of oleic acid was also leaving the formulation with

active that in terms reduced the thermodynamic activity of TQ. In this case, the

thermodynamic activity of the drug which is affected by the vehicle composition

determines the drug permeation [79].

Figure 3.7. Amount of Thymoquinone detected at 24 hours in human cadaver skin.

76

Figure 3.8. Thymoquinone permeation profile in propylene glycol vehicle containing

different concentration of oleic acid. Time points were measured at 3, 4, 6, 8, 10, 12 and

24 hours. Each point represents the mean ± S.D. of five experiments. ***p<0.02.

The concurrent use of FFE Software and FDC (Franz diffusion cell) methodologies

has demonstrated some interesting aspects of how physicochemical interaction of drug and

enhancers regulate the thermodynamic activity of the system. It was shown that the model

developed in the present study to describe the permeation of drugs and enhancers action

enables prediction of permeation behavior of active compounds through the skin.

Although, differential scanning calorimetry (DSC), X-ray diffraction, infrared (IR) and

confocal Raman spectroscopy offer some possibilities to shed light on the mechanism of

action of enhancers in terms of the interactions of the stratum corneum lipid systems with

***

77

the enhancers, none of these methods provide important information about the

thermodynamic activity regulated by the physicochemical properties of both drug and

enhancers that further governs the permeation of drug.

3.4. Conclusions

Better understanding of the physicochemical properties and solubility parameters

of the active and enhancers, interaction of enhancers with the drug and skin will aid in to

simplify our concept of enhancers behavior. By considering thermodynamic activity

previously described simple generalized enhancers mechanism of action can be improved

significantly to the point where one can explain the mode of action of enhancers in terms

of the specific experimental set-up (drug, vehicle, enhancers and its composition and

concentration differences, experimental duration). The knowledge of physicochemical

interactions and thermodynamic influence of enhancers on the permeant flux can not only

enable us to modify permeation but can also help in designing new and exciting chemical

enhancers. The solubility and physicochemical parameter is a fundamental thermodynamic

property. So, understanding Solubility-Physicochemical-Thermodynamic (SPT) theory

can open scope to design new chemical enhancers and can answer regulatory related

inquiry in the process of approval of the upcoming new chemical enhancers. Moreover, the

adoption of this SPT theory can save countless hours of trial and error in drug/active

formulation and delivery and can benefit pharmaceutical, cosmetic and chemical industry

and the related entity.

78

Chapter 4. Effects of solvents and penetration enhancers on transdermal delivery of

thymoquinone: permeability and skin deposition study

4.1. Introduction

The alternative route of oral drug administration is achieving growing interest. The

experts predict that the transdermal and intradermal drug delivery system market will

exceed $25 billion by 2018. It was found that approximately 74% of orally administered

drugs failed to exert desired pharmacological effectiveness [80]. In such case transdermal

delivery should be considered as an attractive alternative route to oral delivery. There are

many important advantages associated with transdermal drug delivery system (TDDS).

This delivery system can be very beneficial in avoiding hepatic first pass effect [81] and

stomach environment; a potential site of drug degradation [82]. It can also provide steady

state plasma level, improved bioavailability, decreased side effects and ultimately can

improve patient compliance [80, 83]. In 1979, FDA first approved scopolamine for

transdermal delivery system [83]. Until the present only nicotine, lidocaine, estradiol,

testosterone, fentanyl, nitroglycerine etc. represent this novel group of TDDS [39]. It

indicates that there are very small group of drugs that are able to cross the skin barrier in

an amount that will be sufficient to give desired therapeutic concentrations and

effectiveness. One should consider several physicochemical properties of active molecules

while choosing them for transdermal delivery. An ideal candidate for transdermal delivery

should a) have low molecular weight (<500 gmol-1) b) Optimum lipophilicity (log P = 1-

3) and c) low melting point (<200°F).

79

There is also an increasing interest in the development of bioactive compounds

isolated from natural products as a potential drug [84]. Poor oral bioavailability has

diminished the potential of using drugs of natural origin [85], thus there is a scope for

alternative route of drug administration for natural drugs. Thymoquinone (TQ) is a

quinone-based phytochemical and was first identified in 1963 in Nigella sativa (black

cumin seed) by El-Dakhakhany [86]. Black seed has been listed by US-FDA on its GRAS

(Generally recognized as safe) list [87]. This is one of the most widely studied plants and

for more than 2000 years the seed has been used to treat various maladies [88]. The seeds

contain volatile oils, proteins, alkaloids, saponins, crude fiber, minerals and vitamins

(calcium, potassium, iron, sodium, zinc, vitamin A, vitamin B, vitamin B2, vitamin C etc.),

linoleic acid, oleic acid etc.. [88, 89]. TQ is a yellow crystalline molecule and is the main

constituents of (30-48%) Nigella sativa extract [87]. It exhibits anti-oxidant, anti-

inflammatory and anti-neoplastic properties and has been studied for the treatment of

cancer and various neurodegenerative diseases e.g., Alzheimer’s and Parkinson’s disease

[90]. Based on the ideal characteristics of transdermal delivery TQ can be an attractive

candidate for TDDS. It is lipophilic (log P = 2.54), has low molecular weight (164.2 gmol-

1) and low melting point (44-45°C). On the other hand, due to its lipophilic character

thymoquinone is not an ideal candidate for tablet or capsule formulations. So, one approach

that may be successful is to develop a transdermal formulation of thymoquinone.

Overcoming skin barrier properties that is mainly regulated by stratum corneum, is

primarily composed of multiple layers of keratin-rich corneocytes (the bricks) surrounded

with lipid lamellae (the mortar) in a bilayer form is the major challenge while delivering a

drug through the skin [62, 91]. There are several strategies to overcome skin barrier to

80

successfully deliver active molecules. They include- chemical permeation enhancers,

iontophoresis, electroporation, microneedle, ultrasound, magnetophoresis,

photomechanical waves etc. [92]. Using chemical enhancers is the commonly used

technique that reversibly decrease skin barrier function by disrupting intercellular stratum

corneum lipids [38, 93, 94]. Many chemicals have been investigated as penetration

enhancers, e.g., surfactants, n-methyl-2-pyrrolidone, terpenes, transcutol, azone, oleic acid

etc. [64].

The objective of this study was to investigate the feasibility of transdermal delivery

of thymoquinone. To the best of our knowledge, this is the first study to investigate both

transdermal flux and skin deposition of thymoquinone and the various conditions that may

influence these including various vehicle/solvents, receiver composition and permeation

enhancers. This study would be further useful for the development of novel thymoquinone

transdermal formulation.

4.2. Materials and Methods

4.2.1. Materials

Polysorbate 80 (Tween 80), N-Methyl-Pyrrolidone (NMP), propylene glycol (PG),

thymoquinone (TQ), High-performance liquid chromatography (HPLC) grade water and

acetonitrile were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Ethanol was

purchased from Decon Labs, Inc. (King of Prussia, PA, USA) and Azone (laurocapram)

was purchased from BOC Sciences (Shirley, NY, USA). Phosphate-buffered saline tablets

(PBS, pH 7.4) was purchased from MP Biomedicals, LLC (Solon, OH, USA). Dermatomed

81

human cadaver skin from the posterior torso was obtained from New York Firefighter Skin

Bank (NY, USA). Oleic Acid and Transcutol® P was a gift from Croda (Edison, NJ, USA)

and Gattefosse (Paramus, NJ, USA) respectively.

4.2.2. Solubility determination

Saturated solutions of TQ were prepared by adding an excess amount of TQ in 5

mL of series of different solvents in well closed amber containers. Using Julabo SW22

shaker all the amber containers with TQ in their respective solvents were agitated at 37°C

for 48 hours. After 48 hours all the samples were filtered through a 0.2 µm PES syringe

filter media with polypropylene housing. All experiments were done in triplicate and the

drug content was measured by HPLC after appropriate dilution.

4.2.3. In vitro skin permeation test (IVPT) studies

The skin permeability of TQ was studied in vitro by using Franz diffusion cells

(Permegear Inc., Hellertown, PA). Dermatomed human cadaver skin with the dermal side

in contact with receptor compartment was mounted on Franz diffusion cells. The

donor/diffusion area was 0.64 cm2 and the receptor compartment was filled with PBS (pH

7.4). The precise volume of PBS that was needed to fill the receptor compartment was

measured for each cell and was included into the calculations. Before using skin, they were

slowly thawed, cut into appropriate pieces and then soaked in filtered PBS (pH 7.4) for 15

minutes. The diffusion cells were allowed to equilibrate at 37oC for 15 minutes. Once

reached equilibrium, at time zero the donor compartment was filled with 0.5 mL of

saturated solutions of TQ in PG/ethanol vehicle with or without 5% of each enhancer used

82

in this study. The skin was occluded with parafilm and the receptor compartment of each

cell was maintained at 37oC under synchronous continuous stirring using a magnetic stirrer

at 600rpm. At each time point (3,4,6,8,10,12 and 24 hours) 300 µL of receptor samples

were withdrawn from the sampling port and were immediately replaced with an equal

volume of PBS (pH 7.4). At the end of 24 hours, receptor aliquots of 300 µL were then

analyzed using a validated HPLC method described below.

4.2.4. High-performance liquid chromatography (HPLC) method development and

validation for TQ

4.2.4.1. Method characteristics

TQ was quantified using a validated HPLC method by Agilent 1100 series HPLC

instrumentation (Agilent Technologies, CA, USA) coupled with UV detection (DAD). A

mobile phase of 80% Acetonitrile and 20% water was pumped through an Agilent Eclipse

XDB-C18 5 µm, 250 X 4.6 mm column. Injection volumes of 20 uL with a flow rate of

1.0mL/minute was set to 23°C with UV detection of 250 nm were used with retention time

of 4.2 minutes. The method was linear at a concentration of 0.39-100 µg/mL with R2 value

of 0.99.

4.2.4.2. Standard solutions and calibration curve

Thymoquinone standard stock solution of 200 µg/mL was prepared by dissolving

2 mg of TQ into 10 mL of mobile phase (80 Acetonitrile : 20 Water). 1 mL of the standard

stock solution was transferred to a test tube and was mixed with 1mL of mobile phase to

83

give a concentration of 100 µg/mL. Process was repeated to produce calibration standards

of 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.39 µg/mL.

4.2.4.3. Method validation

The precision of an analytical method is the closeness of a series of individual

analyte measurements applied repeatedly to multiple aliquots of the same sample and can

be calculated as a Relative Standard Deviation (RSD).

%RSD=(SD/mean)×100

The method was validated for linearity of the calibration curve. Intra and inter-day

variability was determined by running HPLC for the standard solutions three times per day

and on two different days respectively.

4.2.5. Determination of TQ concentration in the skin

At the end of the 24 hours of permeation study the left-over donor solutions and

donor compartments were carefully transferred into a 50 mL centrifuge tube. Skin surface

was then patted dry with a cotton swab. Franz diffusion cell with still skin mounted on it

was held in slightly tilted position over the 50 mL centrifuge tube and 1mL of ethanol was

placed dropwise to wash the skin surface and again few cotton swab was used to patted dry

the skin. All the cotton swab with additional 4 mL of ethanol were placed in 50 mL

centrifuge tube to further analyze the concentration of donor. The skin was then removed

from the diffusion cell and was cut around the diffusional area, air dried and precisely

84

weighed. Then it was placed into a bead bug tube, using a scissor the skin was cut into very

small pieces. The tube was then filled with 1 mL ethanol and was homogenized for 9

minutes (3 min of 3 cycles) by using bead bug homogenizer. All the skin and donor samples

were then placed in a Julabo SW22 shaker and were agitated at 37°C for 24 hours. After

24 hours all the skin samples were centrifuged at 1200 rpm for 5 minutes and were filtered

through a 0.45 µm polypropylene filter media with polypropylene Housing. To further

calculate TQ skin concentration we have divided TQ skin concentration in ng by the

respective skin weight in mg.


The flux of TQ was determined from the slope of the linear portion of the

cumulative amount of TQ permeated per unit skin surface area (µg/cm2) versus time

(hours) plot. Individual permeation profile was generated to calculate average TQ flux with

their respective standard deviations. Permeability coefficient (Kp) and the effectiveness of

the penetration enhancers (ER= enhancement ratio) were determined using equation 1 and

2 respectively,

Kp = Flux/solubility………………………………………...Equation 1 [95]

ER = Flux with the enhancer/Flux without the enhancer……………Equation 2

The lag time was calculated from extrapolation of the linear portion to the x-axis intercept

of the permeation profile. Results are reported as mean ± SD (n=5). The statistical analysis

85

of the data was performed by using one-way Anova and Student’s t - test, and p-values <

0.05 were considered significant.

4.3. Results and discussion

4.3.1. HPLC method validation

Using the current validated HPLC method the TQ chromatogram peak appeared

around 4.2 min and showed in Figure 4.1. The peak’s shape passed the requirement for

symmetry and sharpness.

Figure 4.1. Thymoquinone chromatogram peak at retention time of 4.2 min.

86

The average standard calibration curve obtained from three separate HPLC runs for TQ

solutions ranging from 0.39-100 μg/mL was shown in Figure 4.2. The method was linear

with an R2 value of 0.99.

Figure 4.2. Thymoquinone standard curve for HPLC assay.

The results of intra-day and inter-day variability assessment are provided in Table 4.1 and

4.2 respectively. For inter-day and intra-day precision the %RSD for the slope of the best

fit line was calculated as 0.11% and 0.01% respectively. These values are lower than the

requirement %RSD value of 2%.

0

2000

4000

6000

8000

10000

12000

14000

0 20 40 60 80 100 120

Response A

rea (

mA

U*s

)

Concentration of Thymoquinone (µg/mL)

y = 130.02x + 119.31R² = 0.9985

87

Table 4.1. Intra-day variability of TQ standard solutions of three separate runs in one

day.

Conc (µg/mL) Average AUC SD %RSD

100 12910.5 44.7 0.34

50 6941.7 19.9 0.28

25 3561.3 11.5 0.32

12.5 1814.2 6.3 0.34

6.25 921.9 0.1 0.01

3.125 465 2.1 0.46

1.56 236 1 0.43

0.78 119.9 0.46 0.38

0.39 60.7 0.25 0.41

Table 4.2. Inter-day variability of TQ standard solutions of two separate runs in two

days.

Conc (µg/mL) Average AUC SD %RSD

100 12902.1 54.1 0.41

50 6937.8 26.9 0.38

25 3559.3 15.5 0.43

12.5 1811.9 6.8 0.37

6.25 921.2 1 0.11

3.125 465.1 2.8 0.62

1.56 235.4 2 0.87

0.78 119.5 0.98 0.82

0.39 60.5 0.35 0.58

88

4.3.2. Thymoquinone solubility study

There is not enough literature available on TQ solubility data in commonly used

vehicle [96]. The saturation solubility of TQ in several commonly used solvents is shown

in Table 4.3. The results show that highest TQ solubility can be achieved with ethanol

solvent (19 ± 2.6 mg/ml). Adding water to ethanol in 1:1 ratio significantly decreased

(p<0.0002) the solubility of TQ (0.42 ± 0.06 mg/ml). Although TQ has good solubility in

PBS pH 7.4 (0.66 ± 0.01 mg/ml), interestingly it can be further increased to 0.79 ± 0.03

mg/ml by adding ethanol in 1:1 ratio. Thus, we can depict that ethanol synergistically

increase TQ solubility when used with another vehicle. On the other hand, water reduced

TQ solubility when used with another solvent like, ethanol and methanol.

Table 4.3. Summary of the Solubility Study Results. The values represent the

concentration of TQ ± SD (N=3) in mg/mL at 48 hours.

Solvents Solubility (mg/mL) ± SD

Methanol 0.4 ± 0.02

Ethanol 19 ± 2.6

Ethanol : Water (1:1) 0.42 ± 0.06

Methanol : Water (1:1) 0.34 ± 0.03

Propylene Glycol 9.7 ± 0.16

PBS pH 7.4 0.66 ± 0.01

Ethanol : PBS pH 7.4 (1:1) 0.79 ± 0.03

Polyethylene Glycol 2.9 ± 0.2

89

4.3.3. Effect of propylene glycol and ethanol donor solvent

Ethanol and propylene glycol are commonly used solvents for lipophilic drug [79].

Both of them are important solvents and/or co-solvents for topical and transdermal

formulation. Penetration parameters of thymoquinone using PG vehicle are summarized in

Table 4.4 and Penetration parameters of thymoquinone using ethanol vehicle are

summarized in Table 4.5. For both of the solvents the formulations were applied on the

skin as saturated suspensions with 5% of each enhancer and the study was continued for

24 hours. Figure 4.3 and 4.4 shows that 24 hours study was sufficient to reach steady state

plasma concentrations. It was found that there are significant differences in the penetration

parameters of thymoquinone in presence of various chemical enhancers and vehicles

(ethanol and PG). The rank order for the TQ flux of each enhancers from the PG vehicle

was as follows: Azone + oleic acid>Tc>control + Tween 80>ethanol>NMP. On the other

hand, the rank order for the TQ flux of each enhancers from the ethanol vehicle was as

follows: Tc>oleic acid>Azone>control>Tween 80 + NMP. Cumulative amount of TQ

penetrated per cm2 from both Azone and oleic acid formulation after 24 hours through

human cadaver skin was 1.8 folds reduced in ethanol vehicle compare to PG vehicle. In

this study, TQ solubility in PG vehicle was found to be 9.7 ± 0.16 mg/ml (Table 4.3), which

is significantly lower (p<0.0035) than the solubility of TQ in ethanol vehicle (19 ± 2.6

mg/ml). The solubility and penetration parameters data showed that although ethanol was

able to increase the solubility of TQ it was not able to increase the flux of permeant from

the saturated solutions of TQ. From the above findings, it can be postulated that

90

Figure 4.3. Thymoquinone permeation profile in propylene glycol vehicle. Time points

were measured at 3,4,6,8,10,12 and 24 hours. Each point represents the mean ± S.D. of five

experiments.

thermodynamic activity of TQ in the formulation was modified most probably by the rapid

permeation and evaporation of ethanol vehicle. Due to the varying results between PG and

ethanol vehicle we cannot suggest that only the permeation and evaporative effect of

ethanol either reduced or increased the thermodynamic activity of TQ. Moreover, different

penetration enhancers and vehicles act differently due to their varying mechanism of

action. It is believed that ethanol may disrupts cutaneous barrier function by removing

intercellular material [97]. Ethanol is also involved in lipid fluidization and extraction [98,

99]. On the contrary, PG might increase drug permeability by solvating the α-keratin

structures of the cells [79].

91

Figure 4.4. Thymoquinone permeation profile in ethanol vehicle. Time points were

measured at 3,4,6,8,10,12 and 24 hours. Each point represents the mean ± S.D. of five

experiments.


after 24 hours using propylene glycol vehicle.

TQ Flux TQ Q 24 Lag time Px10-3 ER

Formulation (µg/cm²/hr) (µg/cm²) (Hr) (cm/h)±SD

Control 11.02±1.2 208±23 3.17±0.07 1.25±0.13

Tween 80 11.09±1.5 208±16 3.25±0.4 1.03±0.14 1

NMP 9±1.5b 167±38 3.9±0.3 1.07±0.19 0.81

Azone 49.3±5.6a 854±93 4.2±0.3 3.59±0.36 4.47

Ethanol 10.59±1 180±55 2.34±0.2 0.69±0.06 0.96

Oleic Acid 46.3±4.5a 865±113 3.2±0.3 3.03±0.26 4.2

Transcutol P 14.23±1.4a 247±26 3.7±0.2 1.37±0.12 1.29



92

b, significant reduction in TQ flux (p<0.05)


after 24 hours using ethanol vehicle.



Control 21.28±1.8 347±18 4.21±0.8 0.90±0.07

Tween 80 20.68±2.8 325±43 4.8±0.2 0.85±0.11 0.97

NMP 20.68±2.8 382±62 4.30±0.2 0.90±0.12 0.97

Azone 27.97±5.8a 468±98 4.38±0.3 0.97±0.20 1.31

Oleic Acid 28.25±10a 466±146 4.31±0.6 0.97±0.35 1.32

Transcutol P 29.53±3a 483±48 4.93±0.2 0.96±0.09 1.38



Comparing the penetration parameters of two vehicle it was found that there was significant

increase (p<0.05) in TQ flux of control, Tween 80 and NMP formulation from ethanol

vehicle. On the other hand, there was significant increase (p<0.05) in TQ flux of Azone

and oleic acid formulation from PG vehicle compare to ethanol vehicle. Such behavior of

Azone and oleic acid in PG vehicle can be explained by the fact that there might be a

synergistic effect between Azone and oleic acid with PG vehicle [64]. Azone is a highly

lipophilic compound (log P = 6.2) and is the first molecule to be specifically designed as a

skin penetration enhancers [45]. Although efficacy of Azone is influenced by the vehicle

choice, it mostly shows its ability to permeate an active molecule by interacting with the

stratum corneum lipid domains [64]. Oleic acid was also found to exerts its effectiveness

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by either modifying or by interacting with the stratum corneum lipid domains [26].

Interestingly, 5% Tc performed better in ethanol vehicle by showing two folds increase in

TQ flux compare to PG vehicle. It is reported that Transcutol increase the solubility of

drugs in the skin, its exact mechanism of action is still unexplored [100]. By analyzing

penetration parameters of TQ in PG and ethanol vehicle it was found that 5% Azone in PG

vehicle showed highest permeation of TQ. Additionally, 5% NMP in PG vehicle showed

lowest permeation of TQ. Although there was significant improvement of TQ permeation

with 5% NMP in ethanol vehicle (p<0.05) compare to PG vehicle, the permeation was

lower when comparing with ethanol control and was equal with Tween 80 formulation in

ethanol vehicle. This result is not surprising as NMP acts well with hydrophilic molecule

Figure 4.5. Thymoquinone permeation profile in ethanol vehicle and ethanol:PBS (pH 7.4)

receptor. Time points were measured at 3,4,6,8,10,12 and 24 hours. Each point represents

the mean ± S.D. of five experiments.

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by providing higher flux and TQ is a highly lipophilic molecule [64]. Additionally, Tween-

80 as a non-ionic surfactant has a minor penetration enhancement effect in human skin and

can lower thermodynamic activity of permeants [27]. Moreover, there was reduction of lag

time when 5% ethanol was used in PG vehicle (Table 4.4), although this formulation was

not able to increase TQ flux compare to all other formulations except NMP in PG vehicle.

This result support the study conducted by Rao (2015) and also confirmed that ethanol can

reduce the time needed for a drug to reach the steady state [101].

4.3.4. Effect of receiver solvent composition

As from solubility study we have found adding ethanol with PBS pH 7.4 has

increased the amount of thymoquinone that can be detected, we choose to change the

receiver solvent composition to 60:40 (Ethanol : PBS pH 7.4) to further run the permeation

study. Table 4.6 summarizes the penetration parameters of thymoquinone using ethanol

vehicle and ethanol : PBS pH 7.4 (60:40) receptor solvents. The rank order for the TQ flux

of each enhancers from the ethanol vehicle and ethanol : PBS receptor was as follows:

Azone>Tc >oleic acid>Tween 80 >control> NMP. In all three different permeation studies

NMP showed lowest permeation ability of TQ. On the other hand, Azone came out as a

better penetration enhancer by providing highest flux in PG vehicle and in ethanol : PBS

receptor solvent study. Additionally, Tc was a better enhancer in ethanol vehicle and PBS

receptor. It was interesting to find out that Tween-80 acted as a control in PG vehicle and

it acted as a NMP in ethanol vehicle. By changing the receptor composition, we were able

to increase the permeability capacity of Tween-80 compare to control and NMP. May be

this is due to the phenomenon that receptor composition improved the thermodynamic

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activity of TQ in Tween-80 formulation as the flux is proportional to a gradient of

thermodynamic activity and also due to the improvement of TQ skin reservoir (Figure 4.8).


after 24 hours using ethanol vehicle and ethanol : PBS pH 7.4 (60:40) receptor solvents.



Control 160±5 3140±270 3.6±0.5 5.54±0.2

Tween 80 168±11 2966±188 4.3±0.1 5.74±0.38 1.05

NMP 155±15 2918±250 3.8±0.5 5.30±0.53 0.96

Azone 206±18a 3885±202 3.8±0.4 7.26±0.64 1.28

Oleic Acid 171±8a 3839±316 2.7±0.8 6.04±0.30 1.06

Transcutol P 177±19a 3504±353 3.4±0.2 6.22±0.66 1.11



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Figure 4.6. Amount of TQ (PG vehicle) detected after 24 hours in human cadaver skin

(N=5, mean ± SD).

4.3.5. Effect (pull or drag) of permeation enhancers and vehicle on TQ skin

deposition

Both donor and receptor solvent composition influence the permeation enhancers

and vehicle capability to either increase or decrease TQ skin deposition. Figure 4.6 shows

the amount of TQ in ng/mg (PG vehicle) detected after 24 hours in human cadaver skin.

The rank order for TQ skin deposition of PG vehicle using different enhancers are as

follows: Azone>oleic acid> ethanol>control>Tc>NMP>Tween 80. This result shows both

Azone and oleic acid was able to provide reservoir for TQ skin deposition. Moreover, with

PG vehicle Azone showed almost two folds increase in TQ skin deposition compare to

oleic acid. Figure 5 shows the amount of TQ in ng/mg (ethanol vehicle) detected after 24

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hours in human cadaver skin and the rank order are as follows: oleic

acid>Azone>control>Tc>Tween 80> NMP. Although oleic acid, Azone and Tc in ethanol

vehicle were not as efficient as oleic acid, Azone and Tc in PG vehicle in terms of TQ flux,

but they were able to provide highest TQ skin deposition. So, with the ethanol vehicle all

the enhancers were able to create reservoirs within the skin membrane and more TQ was

deposited in the skin rather than migrating to the receptor compartment of Franz diffusion

cell. In another words, ethanol increases the capacity of the stratum corneum for drug

uptake. As both vehicle and enhancer also penetrate through the skin with the active

molecule there might be some pull or drag effect associated with this varying results by

using two different vehicle. We can further understand the pull/drag effect of ethanol

vehicle on TQ skin deposition by analyzing the skin deposition study (Figure 4.8) after

changing the receptor composition to 60:40 (Ethanol : PBS). The rank order are as follows:

control>Tc>NMP>Tween 80>Azone>oleic acid. This skin distribution ranking profile is

somewhat reversed comparing the skin permeation ranking profile. This time ethanol

control was able to provide highest skin reservoir compare to other formulation with

different enhancers and Azone and oleic acid showed lowest TQ skin deposition. From the

above result, it can be depicted that ethanol control was able to pull more drug in the skin

and as the control formulation didn’t contain any enhancer the ethanol vehicle was not

further able to enhance the drug permeability. On the other hand, all the enhancers used in

this study showed low pulling effect as they were not as efficient like control to pull more

drug in skin membrane. They rather showed enhanced permeation as the enhancers has

permeation enhancing effect. It must be noted here, that we have analyzed the whole skin

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for TQ skin deposition/concentration study and the skin samples were analyzed after 24

hours of permeation study. However, further study of TQ skin absorption is needed.

Figure 4.7. Amount of TQ (ethanol vehicle) detected after 24 hours in human cadaver

skin (N=5, mean ± SD).

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Figure 4.8. Amount of TQ (ethanol : PBS receptor) detected after 24 hours in human

cadaver skin (N=5, mean ± SD).

4.4. Conclusions

Skin penetration of TQ was influenced not only by the physicochemical properties

of the vehicle but also by the other experimental parameters such as, receiver composition

and permeation enhancers. This study showed donor-receiver inter-relationships governing

TQ penetration and skin absorption along with effects of penetration enhancers. It can be

concluded that transdermal permeation and skin deposition of TQ can be obtained by using

penetration enhancers and different vehicles. Azone, oleic acid and Tc at a concentration

of 5% was able to provide measurable TQ flux and can be the choice of penetration

enhancer to further develop a novel transdermal formulation of TQ. These penetration

enhancers were also able to generate TQ reservoirs in the skin that might be useful to exert

sustained release of TQ from the stratum corneum over longer period of time.

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Chapter 5. Thymoquinone loaded polymeric films and hydrogels for the treatment

of wound healing and bacterial skin infections

5.1.Introduction

Skin is the largest organ in the body and occupies about 16% of the total body

weight of an adult and has a surface area of about 2 m2 [2]. It is a complex arrangement of

structures and has a multifunctional role- provides a physical barrier to the environment by

acting as a protective barrier against the ingress of foreign material, maintains homeostasis

and thermoregulation by limiting the loss of water, electrolytes, and heat and prevents

microbial colonization [1, 102]. Although skin act as a shield against bacterial invasion,

bacteria can still invade the epidermis and dermis to produce localized infection and cause

a variety of pathologic changes in the skin (impetigo, furuncles, subcutaneous abscesses)

[103]. Microbial infections of the skin and underlying tissues are among the most frequent

conditions found in ambulatory care patients [104]. Staphylococcus aureus is one of the

most important human and veterinary pathogens and is the causative agent for the majority

of primary skin infections [105]. It causes infections ranging from benign to life threatening

diseases [106]. Skin and soft tissue infections (SSTIs) encompass a wide variety of clinical

outcomes, ranging from mild cases of cellulitis, erysipelas, trauma, subcutaneous tissue

infections, wound related infections to complicated deep-seated infections with systemic

sign of sepsis [107]. SSTIs may lead to severe complications and hospital admission when

associated with co-morbidities and/or bacteraemia. The most commonly reported cause of

SSTIs is Staphylococcus aureus followed by β-haemolytic streptococci (BHS) [108, 109].

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Staphylococcus aureus can internalize by a variety of nonphagocytic host cells and can

contribute to the development of persistent or chronic infections and may lead to deeper

tissue infections or dissemination [110-112].

The skin of patients with atopic dermatitis (AD), eczema and psoriasis show a

striking susceptibility to colonization with Staphylococcus aureus. About 7% of patients

with psoriasis have bacterial skin infections [113] and 80-100% of patients with AD and

eczema are colonized with Staphylococcus aureus [114, 115]. On the other hand,

Staphylococcus aureus can be isolated from the skin of only 5-30% of normal individuals,

mainly from intertriginous areas of the body (axilla of the arm, the anogenital region

between digits etc.). There is a relationship between disease severity, extent and

Staphylococcus aureus colonization of lesional and non-lesional skin and the density of

Staphylococcus aureus has been shown to correlate with cutaneous inflammation [116].

The colonization density of Staphylococcus aureus can reach up to 107 colony-forming

units cm-2 without clinical signs of infection in patients with AD. Bacterial infection can

often lead to a chronic wound [117]. Wound management is a prevalent clinical problem

as wound healing involves a series of complex process including inflammation phase,

proliferative phase (formation of granulation tissue, reepithelialization and matrix

formation) and remodeling phase [118]. Each phase of wound healing is well defined,

although they overlap with next [119]. The process of wound healing becomes delayed

when wounds are colonized and the colonizing agent is sustained [120]. In patient with

weaken immune system bacterial contamination can prolong wound healing [121, 122] and

colonization of bacteria in wounds is a serious threat. Open wounds are also at high risk of

invasive wound infections, which can further lead to amputation and disability [123].

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SSTIs including atopic dermatitis (AD), eczema, psoriasis and wound healing all rely on

efficient antibiotic therapies. As temporary eradication of Staphylococcus aureus with

antibiotics often leads to clinical improvement of AD, data from clinical studies suggest

that antimicrobial treatments should be applied in AD patients with apparent or recurrent

skin infections [124]. Antimicrobial resistance is one of the biggest challenges in the global

health sector [125]. The high incidence of methicillin resistance in hospitals complicated

the prevention and treatment of serious infections due to staphylococci [126]. As infections

due to multi-resistant Gram-positive organisms are increasing day by day their early

recognition, treatment and proper management are greatly required. Several antimicrobial

agents in different dosage forms are available for the treatment of SSTIs. Topical

application of antibiotic agents have several benefits over oral and systemic therapy [127]-

localized and targeted delivery can provide required concentration for antibiotic activity

more efficiently at the skin target site, can avoid an unnecessary exposure of gut flora that

may exert selection for resistance, can avoid side-effect and allergic reactions associated

with systemic antibiotic treatment. Therefore, topical application may highly influence the

treatment efficiency and can increase the patient compliance.

In such scenario, development of new treatment strategy is crucial to deal with the

emerging issues of skin infections. Topical delivery of antibacterial agent of medicinal

plants can be considered as a source for new therapeutic agents aimed at the treatment and

management of skin infections. Thymoquinone (TQ) (2- isopropyl- 5- methyl- 1,4-

benzoquinone) is the main constituent of Nigella sativa (Black cumin) seeds [28]. TQ is a

yellow crystalline molecule and has a basic quinone structure consisting of a para

substituted dione conjugated to a benzene ring to which a methyl and an isopropyl side

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chain groups are added in positions 2 and 5, respectively. TQ has many pharmacological

properties such as anticancer, antimicrobial, anti-inflammatory, antioxidant, antiasthmatic

and immunomodulatory effect [31]. Thus far, several experiments have explored its

anticancer and brain targeting properties - Odeh et al. loaded TQ in a liposome system and

tested that on breast cancer cell lines (MCF-7 and T47D) to evaluate its anticancer

properties [28], Ng et al. prepared TQ loaded nanostructured lipid carrier and showed its

effectiveness towards breast cancer and cervical cancer cell lines [128], Ahmad et al.

evaluated TQ loaded mucoadhesive nanoemulsion for the treatment of cerebral ischemia

[129]. However, there has no report on its delivery from a topical delivery system and its

effectiveness for the treatment of wound healing and Staphylococcus aureus associated

bacterial skin infections.

The objective of this study was to synthesize and characterize a biocompatible

novel topical polymeric film and hydrogel system that has the potential to deliver

antibacterial TQ agent directly at the skin target site that may be useful for the treatment

and management of Staphylococcus aureus related bacterial skin infections and for the

wound management. To achieve this objective, TQ loaded polyvinyl pyrrolidone (PVP)

films were prepared using solvent casting method and TQ wound hydrogels were prepared

using different polymers. The prepared films and hydrogels were characterized for physical

parameters, permeability and stability studies. Its biocompatibility was assessed, and the

antibacterial efficacy of films and hydrogels were evaluated in vitro and ex vivo on selected

strains of Staphylococcus aureus (ATCC 49230). Further, in vitro scratch assay models

using HDF (fibroblast) and HaCat (keratinocytes) cell lines were used to demonstrate its

wound healing properties. To evaluate its preclinical and in vivo efficacy biopsy punch

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wound infection animal model was used. This work demonstrates that PVP/TQ film is

effective in facilitating wound healing. Both the film and wound hydrogel can be useful in

the treatment of bacterial skin or wound infections and TQ can be a promising candidate.

5.2. Materials and Methods

5.2.1. Materials

Thymoquinone (TQ), polyvinylpyrrolidone (PVP), dibutyl phthalate (DBP),

hydroxy propyl methyl cellulose (HPMC), potassium chloride, benzoic acid, Gentamicin

solution, formalin solution (10%), triethanolamine, propylene glycol, dipropylene glycol,

alamarBlue® (resazurin) assay kit, High-performance liquid chromatography (HPLC)

grade water and acetonitrile were purchased from Sigma-Aldrich Co. (St. Louis, MO,

USA). Laurocapram (Azone) was purchased from BOC Sciences (Shirley, NY, USA).

Phosphate-buffered saline tablets (PBS, pH 7.4) was purchased from MP Biomedicals,

LLC (Solon, OH, USA), ethanol was purchased from Decon Labs, Inc. (King of Prussia,

PA, USA), xanthan gum was purchased from Spectrum Chemical (New Brunswick, NJ,

USA), hydroxy propyl cellulose (HPC) was purchased from Ashland (Wilmington, DE,

USA). Carbopol 980 and ultrez 10 were purchased from Lubrizol (Cleveland, OH, USA)

and drierite was purchased from Acros organics (Morris Plains, NJ, USA). Bacto tryptic

soy broth, bacto agar, Dulbecco's Modified Eagle's Medium (DMEM) and Dulbecco's

phosphate-buffered saline (DPBS) were purchased from Fisher Scientific (Hampton, NH,

USA). Human epidermal keratinocytes (HaCat) and Human dermal fibroblasts (HDF) cell

lines and Pen/strep were purchased from Life Technologies (Carlsbad, CA, USA).

Staphylococcus aureus (ATCC 49230) was purchased from ATCC (Manassas, VA, USA).

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Isoflurane was purchased from Henry Schein (Dublin, OH, USA). Fetal bovine serum

(FBS) was purchased from Atlanta Biologicals (Minneapolis, MN, USA). CellTiter 96®

was purchased from Promega (Madison, WI, USA). Gentamicin sulfate cream USP, 0.1%

was purchased from Perrigo (Allegan, MI, USA). Dermatomed human cadaver skin from

the posterior torso were obtained from New York Firefighter Skin Bank (NY, USA). Nine

to ten week old male BALB/c mice were purchased from Charles River (Wilmington, MA,

USA).

5.2.2. Fourier Transform Infrared (FTIR) analysis

FTIR spectra of samples were taken on Thermo Scientific (model Nicolet iS10)

instrument to investigate the possible interaction between the drug and polymer. FTIR

spectra of pure drug, polymer, physical mixture of drug and polymer in ratio of 1:1, films

with TQ and films without TQ were scanned in the ranged between 4000-400 cm¯¹.

5.2.3. Fabrication of films

The matrix-type polymeric films containing TQ were prepared by solvent casting

method. Accurately weighted TQ were dissolved in ethanol and were sonicated for 30

minutes to ensure solubilization. DBP was used as a plasticizer and Azone was used as a

penetration enhancer. The weighted amount of PVP, DBP (4%, v/v) and Azone (5%, v/v)

was added in the drug solution. The mixture was stirred at 200 rpm at 25°C for 20 minutes.

The solution was poured on Teflon dish (15 cm2) and placed in an oven maintained at 60°C

± 5°C. To allow complete evaporation the system was left undisturbed for 3 hour 20

minutes. The formed films were completely removed from the Teflon dish and punched

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out into 0.64 cm2 pieces. Control films without drug containing only PVP, plasticizer and

penetration enhancer were also prepared.

5.2.4. Preparation of TQ hydrogel formulations

Drug-loaded hydrogels were prepared using gelling agents, preservatives,

penetration enhancer and vehicles. Different concentrations of various polymers (gelling

agents) with or without xanthan gum were dispersed slowly in an aqueous-based solution

containing TQ (0.2% w/w), 1:1 concentration of propylene glycol and dipropylene glycol

(20% w/w, as a vehicle), benzoic acid (0.1% w/w, as a preservative), ethanol (5% w/w, as

a penetration enhancer), using an overhead mechanical stirrer at a moderate speed.

Triethanolamine was used to adjust the pH of Carbopol and Ultrez 10. The prepared

hydrogels were packed in wide mouth jar covered with screw capped plastic lid and kept

in dark and at laboratory ambient temperature. The composition of different prepared TQ

hydrogel formulations is given in Table 5.1.

5.2.5. Field Emission Scanning Electron Microscopic (FESEM) studies

The surface morphology of the film was recorded with a Zeiss field emission

scanning electron microscopy (FESEM) (FSD PRE-AMP 4CH, Germany). The film

sample was mounted on an aluminium stub with double-sided adhesive band then gold was

sputtered on the specimen (20 nm) to ensure sufficient electrical conductivity. An

accelerating voltage of 5 kV was applied and the image was photographed by secondary

electron detector.

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Table 5.1. Composition of TQ topical hydrogels (% w/w).

5.2.6. Physicochemical characterization of films

5.2.6.1. Film thickness

Film thickness was measured using Digital Caliper (Fisher Scientific, New

Hampshire, USA) at three different places, and mean value was calculated.

5.2.6.2. Drug content uniformity

A prepared film was dissolved in 10mL ethanol and stirred continuously for 24

hour. The drug content was analyzed using HPLC method.

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5.2.6.3. Weight variation

Weight variation was studied by individually weighing 5 randomly selected films.

5.2.6.4. Flatness

Three longitudinal strips were cut out from each film (one from the center, one from

the left side, and one from the right side). The length of each strip was measured and the

variation in length because of nonuniformity in flatness was measured by determining

percent constriction, with 0% constriction equivalent to 100% flatness.

% Constriction = L1- L2/L2 x 100

where L1= initial length of each strip and L2 = final length of each strip

5.2.6.5. Folding endurance

Folding endurance was determined by repeatedly folding the film at the same place

until it broke. The number of times the film could be folded at the same place without

breaking was the folding endurance value.

5.2.6.6. Percentage of moisture content

The prepared films were marked, then weighed individually and kept in a desiccator

containing drierite at room temperature for 24h. The films were weighed again and again

individually until it showed a constant weight. The percentage of moisture content was

calculated as the difference between initial and final weight with respect to final weight.

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5.2.6.7. Percentage of moisture uptake

The films were weighed and kept in a desiccator at room temperature for 24 hour.

Films were taken out and placed in a desiccator containing 100mL of saturated solution of

potassium chloride to maintain 84% relative humidity until a constant weight for the films

were obtained. The percentage of moisture uptake was calculated as the difference between

final and initial weight with respect to initial weight.

5.2.7. Physicochemical characterization of the prepared hydrogels

5.2.7.1. Visual inspection

TQ hydrogels were examined visually for their color and homogeneity (appearance

and presence of any aggregates).

5.2.7.2. pH determination

The pH of various TQ hydrogel formulations was determined using pH meter

(VWR pH meter symphony B10P, Radnor, PA, U.S.A). 1 gm of TQ hydrogel was

dissolved in 10 gm of DI water. After 2 hours pH was determined at room temperature.

5.2.7.3. Spreadability test

Spreadability was determined in mm. A 10 mg sample was placed on top of a

microscopic slide and covered with another slide, 50 gm of standardized weight was put

on it and after 1 minute the diameter of the sample was taken in mm.

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5.2.7.4. Drug content uniformity

Three samples of a specific quantity (100 µL) of each prepared hydrogel was taken

and dissolved in 10 mL of ethanol solvent. To ensure drug solubility the 20 mL glass vial

containing the gel solution was put on a magnetic stirrer at 600 rpm at 25°C for overnight.

The drug content was then determined using HPLC. The variability of TQ content in

hydrogels was reported as % RSD,

% RSD = (standard deviation/mean drug content) × 100.

5.2.8. Rheological characterization of hydrogel formulation

Rheological characterization was performed on rheometer (Kinexus Ultra +,

Malvern, UK) equipped with a 25 mm flat stainless-steel plate. All tests were done at 32ºC

and a gap of 1 mm. Following tests were carried out-

5.2.8.1. Oscillation stress sweep

The samples were subjected to increasing stress (0.1 - 500 Pa) at a constant

frequency of 1 Hz. This test allows determination of the liner viscoelastic region (LVR) of

the sample, and therefore the consequent choice of the stress value to use in the subsequent

oscillation test.

5.2.8.2. Frequency sweep

All the prepared samples were subjected to increasing frequency of 0.1–50 rad/sec

at a constant stress (5 Pa) obtained from LVR. Effect of stress on elastic modulus (G′) and

viscous modulus (G″) was monitored.

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5.2.9. In Vitro skin permeation studies

In vitro skin permeation studies were performed by using a Franz diffusion cells

(FDC) with a donor area of 0.64 cm2 and a receptor volume of 5.0 mL (Permegear Inc.,

Hellertown, PA). The samples of dermatomed human cadaver skin were slowly thawed at

room temperature, cut into appropriate pieces and then soaked in filtered PBS (pH 7.4) for

15 minutes. After that they were mounted on FDC with the epidermal side in contact with

the formulation or donor compartment. The receptor compartment of each cell was filled

with filtered PBS (pH 7.4) and was maintained at 37oC under synchronous continuous

stirring using a magnetic stirrer at 600 rpm. The diffusional membranes were left to

equilibrate at 37oC for 15 minutes. Once reached equilibrium, at time zero formulated films

and 100 µL of hydrogels were placed over the skin to the donor compartment of each Franz

diffusion cell. At each time point 300 µL of receptor samples were withdrawn from the

sampling port. At the end of experimental hours, receptor aliquots of 300 µL were then

analyzed using a valid HPLC method described below.


A validated HPLC method was used for this study. The HPLC instrument used was

Agilent 1100 series instrumentation (Agilent Technologies, CA, USA) coupled with UV

detection (DAD) and HP Chemstation software V. 32. For the analysis of TQ, a mobile

phase of 80% acetonitrile and 20% water was pumped through an Agilent Eclipse XDB-

C18 5 µm, 250 X 4.6 mm column. Injection volumes of 20 uL with a flow rate of 1.0

mL/minute was set to 23°C with UV detection of 250 nm were used with retention time of

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4.2 minutes. The method was linear at a concentration of 0.39-100 µg/mL with R2 value of

0.99.

5.2.11. Skin deposition study

At the end of permeation study, the skin was removed from the diffusion cell and

was cut around the diffusional area, air dried, and accurately weighed. The samples were

then placed into bead bug tubes and using a scissor they were cut into very small pieces. 1

mL ethanol was added to each tube and they were homogenized for 9 minutes (3 min of 3

cycles) by using BeadBugTM Microtube homogenizer, D1030 (Benchmark Scientific,

Sayreville, NJ). All the skin samples were then placed in a Julabo SW22 shaker (Julabo

USA Inc., Allentown, PA) and were agitated at 37 °C for 24 hours. After that all the skin

samples were centrifuged at 1200 rpm for 5 minutes and were filtered through a 0.45 μm

polypropylene filter media with polypropylene housing. TQ concentrations were expressed

as ng of TQ per skin weight in mg.

5.2.12. Stability study

The films were put in a petri dish and the dish was wrapped by aluminium foil and

stored at 20 °C for 60 days. The samples were analyzed for physical changes such as color,

texture and other physical parameters. The FTIR spectra of stored films were compared

with the freshly prepared films. The films were also analyzed for drug content. On the other

hand, all the hydrogel formulations were filled in glass wares and covered with aluminum

foil and were kept at laboratory ambient temperature for 8 months. The physical stability

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of the formulation was examined visually for appearance, color and odor in every two

weeks. After 8 months an antibacterial efficacy study was performed to confirm the

formulation stability of hydrogel formulation.

5.2.13. In vitro antibacterial activity of TQ films and hydrogels

The prepared control films and TQ loaded films were tested for their antibacterial

activity against Staphylococcus aureus (ATCC 49230) using disc diffusion method.

Briefly, Muller Hinton agar (MHA) plates were used for screening, prepared by pouring

15 mL of molten media into sterile petri dishes. Then 150 µL of overnight cultured bacteria

adjusted to OD concentration of 0.602 (OD 1 = 1X109/mL of bacteria) in sterile TSB

(Tryptic soy broth) was spread on the surface of MHA agar plates with the help of sterile

spreader. The disc shaped polymer film of 0.64 cm2 and 100 µL of TQ gel were then placed

on the surface of the medium and incubated at 37 °C for 24 hours. Gentamicin 500 µg/mL

and 50 ug/mL was used as a positive control, UV irradiated filter paper was used as a

negative control and control film without TQ was used as a control. At the end of

incubation, the inhibition zones were examined around the polymer disc films. The study

was performed in triplicate.

5.2.14. Ex vivo antibacterial activity of TQ films and hydrogels using human cadaver

skin explants

Human cadaver skin was thoroughly washed three times using sterile PBS. Using

surgical gloves and sterile scissors they were cut into pieces (2 cm x 2 cm) and two skin

114

samples were placed on each agar plate. 5 µL of 1 X 10⁶ CFU/mL was put onto each skin

pieces followed by the application of treatment. After overnight incubation at 37°C bacteria

were extracted for counting using sterile PBS and 10 seconds of vortex. Serial dilutions of

bacteria were prepared and were plated on TSB agar plates. Bacteria were counted after

overnight incubation at 37°C.

5.2.15. Cyto-compatibility study

alamarBlue® (resazurin) assay was used to evaluate the cyto compatibility of the

TQ film using two cell lines, HaCat (Human epidermal keratinocytes, passage 8) and HDF

(Human dermal fibroblasts, passage 5). The cells were counted and were seeded into the

6 well plates at a density of 200000 cells/cm². After reaching confluency, the cells were

treated with the samples for 24 h. 1% Triton treated cells served as positive control and

cells in media without any treatment acted as negative control. After 24 h the cells were

treated with alamarBlue® and incubated for 4 h. The optical density was measured at

excitation-emission wavelength of 560-590 nm using Spark 10M multimode microplate

reader (Tecan, Switzerland). The percentage of cell viability was calculated using the

formula given below,

% Cell viability = [ [Fluorescent intensity] test/[Fluorescent intensity] control] X 100

5.2.16. Scratch assay for wound closure activity

HDF (Passages 2-4) cells were counted and were seeded into the 24 well plates at

a density of 50000 cells/well. After reaching confluency, the culture media was replaced

with sterile base media (DMEM with 1% P/S) and scratch wounds were created in the cell

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monolayer using a 200 µL sterile pipette tip [130]. TQ in DMEM at different concentration

(1 ng- 1000 ng) was put into the culture media and the experiment was continued for 24

hours. Base media was used as a control and 100 ng FGF 2 (Fibroblast Growth Factor) was

used as a positive control. Images were taken at 0, 4, 8, 12 and 24 hours. HaCat (Passage

37-39) cells were counted and were seeded into the 24 well plates at a density of 250k

cells/well. After reaching confluency, the culture media was replaced with sterile base

media (DMEM with 1% FBS+ 1% P/S) and scratch wounds were created in the cell

monolayer using a sterile pipette tip. TQ in DMEM at different concentration (1 ng- 1000

ng) was put into the culture media and the experiment was continued for six days. Base

media was used as a control and 10% DMEM was used as a positive control. Images were

taken at 0, 24, 48, 72 and 144 hours.

5.2.17. In vivo bacterial skin infection study

Animal infection experiments were performed at the Nelson Biological

Laboratories, Rutgers University (Piscataway, NJ, USA) in accordance with a protocol

approved by the Rutgers University Institutional Animal Care and Facilities Committee

(ACFC). Mice were housed under standard conditions of light and temperature and were

fed standard diet and water ad libitum. Adult male mice (BALB/c, 10 weeks) were used

for all experiments [131, 132]. Prior to the experiment day, the mice were anesthetized

using an inhaler beginning with 5% isoflurane, and then decrease to 2-3% to maintain

sedation for the remainder of the procedures. The hair over the dorsum (head to tail) were

shaved with an electric clipper. To remove the remaining hair the depilatory cream was

applied for 3 min. Finally, the shaved area was washed with wet scrub, and the animals

116

were returned to their cages. On next day, 10 mm biopsy punch was used to create the

wound on the dorsum of the animals. A bacterial infection at the wound site was initiated

by placing on the skin a 10 µL droplet containing 108 CFU/mL cells of concentrated

Staphylococcus aureus from an overnight bacterial culture in stationary phase. Mice were

divided into following groups namely, Control wound (10 mm biopsy skin wound),

Bacterial wound (skin wound infected with bacteria), Control film (wound infected with

bacteria and then treated with control film without TQ), TQ film (wound infected with

bacteria and then treated with TQ loaded film), Gentamicin (wound infected with bacteria

and then treated with gentamicin marketed cream formulation)

Film with or without TQ were applied at the wound infection site on Day 0, 1, 2

and 3. Gentamicin was applied similarly as TQ film. Wounds were covered using Tegaderm

film (3M, Saint Paul, MN, USA). At each time point (Day 1, 2, 3 and 7) bacterial samples

were taken by taking out the Tegaderm film from the wound site and collected in 2 mL

microtube containing PBS. The tubes were then vortexed for 10 sec to extract the bacteria.

Different bacterial dilutions were made by adding 10 µL of bacterial solution to the 990 µL

of TSB solution and were plated on TSB agar plates. After overnight incubation at 37°C

bacteria were counted. The experiments were continued for 21 days. Wounds were visually

monitored for local inflammatory reaction and photographed at Day 0, 3, 7, 10, 14 and 21

days. All the animals were euthanized at the end of day 21.

5.2.18. Behavioral response of mice

The mice were observed at least once each day for signs of fatigue, stress, and

aggressiveness. The mice were weighed at each time point.

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5.2.19. Histopathological examinations

Immediately after the animals were killed, wound skin samples were collected

and immediately fixed in phosphate-buffered (pH 7.4) formalin (10%) for 48-72 hours at

room temperature and then switched into 70% ethanol and stored at 4°C until process.

Skin tissues including wound scar area were sectioned (5um thickness) and stained with

Masson’s trichrome.


The cumulative amounts of TQ permeated per unit area were plotted against time.

The flux was calculated by determination of the slope of the linear portion of the

permeation profile. The % wound closure for each time interval was determined by the

following formula and were calculated using ImageJ software-

𝑤𝑜𝑢𝑛𝑑 𝑐𝑙𝑜𝑠𝑢𝑟𝑒 (%) =𝑤𝑜𝑢𝑛𝑑 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑛 𝑑𝑎𝑦 0 − 𝑤𝑜𝑢𝑛𝑑 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟𝑜𝑛 𝑜𝑛 𝑑𝑎𝑦 𝑛

𝑤𝑜𝑢𝑛𝑑 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑛 𝑑𝑎𝑦 0 𝑋 100 (1)

Results are reported as mean ± SD. The statistical analysis of the data was performed by

using one-way Anova, the Tukey post-hoc tests and Student’s-t test, and p-values < 0.05

were considered significant.

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5.3.Results and discussion

5.3.1. Fourier Transform Infrared (FTIR) Spectroscopic studies

The FTIR analysis was employed to study the compatibility of the drug with the

polymer used (Figure 5.1A). The samples were scanned in the region of 4000-400 cm -1.

The IR spectral analysis of pure TQ showed that the major peaks were observed at

wavenumbers 2967.30 (C-H stretching of aliphatic group), 1640.54 (C = C stretching),

1462.23, 1358.15 (C-H methyl rock), 1246.90, 1133.03, 1023.62 (C-H in-plane bend),

1006.33, and 933.08, confirming the purity of the drug (Figure 5.1A). A weaker band

observed at a higher wavenumber (3253.95) corresponds to the stretching observed in the

vinylic C-H in the C = C-H groups [133]. In the IR spectra of the physical mixture of TQ

and PVP (Figure 5.1A) the major peaks of TQ were observed at wavenumbers 2966.73,

1644.49, 1461.38, 1374.84, 1248.05, 1133.32, 1023.30, 1006.10 and 933.43. Infrared

spectra of physical mixture of TQ and polymer showed all the characteristic peaks

indicating the absence of any possible interaction between the drug and polymer.

Therefore, it can be stated that the drug and polymer are compatible and can be formulated

into films. The characteristic peaks of the drug can also be seen in TQ films of both freshly

prepared and stored films (Figure 5.1A).

5.3.2. Physicochemical characterization of films

The results of the physicochemical studies are summarized in Table 5.2. The drug

content in the prepared films was found to be 100% with a low standard deviation,

indicating good uniformity in drug content. The thickness and weight variation of films are

associated with the uniformity and accuracy of dosing [134]. Uniformity of thickness of

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Figure 5.1. Physicochemical characterization of TQ films. (A) FTIR spectrum of TQ pure

drug, PVP, physical mixture of drug and polymer, freshly prepared films containing drug

and polymer, stored films containing drug and polymer; (B) control films (i); Field

emission scanning electron microscopic (FESEM) images showing surface morphology of

control film (ii-iii) at different magnifications; and (C) TQ films (i); FESEM images

showing surface morphology of TQ films (ii-iii) at different magnifications.

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each film and minimal weight variation was ensured as low standard deviation values were

observed in the thickness of films and weight variation studies. The flatness study showed

that the films had the same strip length before and after cutting, indicating 100% flatness.

These data also indicate 0% constriction in the films meaning they could maintain a smooth

surface when applied onto the skin. In other words, the films provided intimate contact

with skin and hence better drug permeation. Folding endurance test results indicated that

the films would not break and would maintain their integrity when folded. The low

moisture uptake at laboratory ambient condition protects the material from microbial

contamination and avoids extra bulkiness of the films. The moderate moisture content of

the prepared films could assist the formulation stability by preventing drying and

brittleness. These results indicated that the polymeric combinations showed good film-

forming properties and the process employed to prepare films in this study could produce

films with uniform drug content and minimal film variability.

The surface morphology of the drug loaded film was assessed using field emission

scanning electron microscopy (FESEM) and shown in Figure 5.1C. FESEM images were

taken at different magnifications 100X, 500X and 1000X to investigate the surface of films.

At all magnifications the film surface appeared smooth and compact. FESEM photograph

of TQ film shows polymer networks inside the film and homogeneous dispersion of drug

inside the polymer networks (Figure 5.1C).

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Table 5.2. Physicochemical properties of TQ films (data shows mean of five

determinations with ± standard deviation).

Physicochemical TQ polymeric

parameters films

Drug content (%) 100 ± 6.4

Thickness (mm) 1.17 ± 0.04

Weight variation (mg) 82.04 ± 1.9

Flatness (%) 100

Folding endurance 68 ± 2.38

Moisture content (%) 14.12 ± 0.42

Moisture uptake (%) 2.26 ± 0.47

5.3.3. Characterization of the TQ hydrogels

Table 5.3 shows the results of physicochemical properties of prepared TQ hydrogel

formulations (F1-F10). All the prepared hydrogels were light yellow in color and either

opaque or clear. Most of the formulations showed good homogeneity with no lumps and

smooth homogeneous texture. pH values of the formulations were found in the range of

3.91-4.86. The relative standard deviation (% RSD) of prepared hydrogel formulations

ranged from 0.12 to 0.34%. Good spread ability is one of the criteria for gel as it shows the

behavior of the gel when it comes out form the tube. It is the term that is used to indicate

the extent of area to which gel readily spreads on application. It was observed that spread

ability of TQ hydrogels decreased by increasing the polymer concentration and the values

were in the range of 12-21 mm.

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Table 5.3. Physicochemical properties of TQ topical hydrogel formulations (F1- F10).

+: not good; ++: good; +++: very good

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Rheological properties of gel formulation provide important information regarding

physical form, appearance, texture and flow behavior [135]. The determination of linear

viscoelastic region (LVR) is used to understand the physical form or microstructure of the

gel formulation. In this study, oscillation stress sweep was used to obtain the LVR. The

range of stress over which the elastic modulus G′ is independent of the applied stress

amplitude is called the LVR. So, it presents a critical stress beyond which the sample may

show significant structural breakdown. The mean stress value of 5 Pa obtained from LVR

was used for other oscillation tests such as frequency sweep.

To obtain information about viscoelastic behavior of the prepared samples

oscillation frequency sweep test was performed to measure the response of a system as a

function of frequency at constant stress amplitude (within LVR). Elastic modulus (G′) and

viscous modulus (G″) were determined as a function of frequency. G″ is a measure of the

energy lost per cycle and reflects the fluid-like component whereas G′ is a measure of

energy stored per cycle and reflects the solid like component of the viscoelastic material.

The G′ will be large if a material is predominantly elastic or highly structured. In this

experiment, the addition of Carbopol and Ultrez 10 showed drastic increase in G′, whereas

addition of HPMC and HPC in formulation reduced G′. The order of increment in G′ was

F10 > F9 > F4 > F3>F1 (Figure 5.2A).

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Figure 5.2. Rheological characterization of TQ hydrogel formulations (F1-F10). (A)

Oscillation frequency sweep data. The elastic modulus (i); The viscous modulus (ii) were

plotted against angular frequency. TQ permeation and skin deposition from film and gel

formulations (B). TQ permeation profile for different hydrogel formulations (i). Time

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points were measured at 1, 2, 3, 4, 5, 6 and 8 hours. Each point represents the mean ± S.D.

of five experiments; TQ permeation from film formulation across human cadaver skin

(mean ± S.D., n=5) (ii); Amount of TQ detected after 8 hours in human cadaver skin (N=5,

mean ± SD) using different TQ hydrogel formulations (iii).

5.3.4. In Vitro skin permeation and deposition studies

Penetration parameters of thymoquinone are summarized in Table 5.4. The results

showed that the rank order for thymoquinone flux from each formulation are: F 2 > F 5 >

F 3 > F 7 > F 10 > F 9 > F 6 > F 8 > F 4 > F 1. Figure 5.2B shows the thymoquinone

permeation profile and amount of TQ detected after 8 hours in human cadaver skin. It was

observed that formulation 7 and 9 was able to retain more drugs in human cadaver skin

compared to other formulations that might be useful in the treatment and management of

wound infections.

5.3.5. Stability study

After storage, no significant changes in color and texture of the film were observed.

The drug content of the stored films was comparable and were within limits. Additionally,

FTIR spectra of stored film and freshly prepared films can be superimposed, indicating the

stability of the films (Figure 5.1A). Hence, the film can be used after storage of two months

without any loss of physical and chemical attributes. After storage for 8 months, hydrogels

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after 8 hours.

Formulation Q at 8 hours (µg/cm2) TQ Flux

(µg/cm²/hr)

1 62 ± 9 5.38 ± 0.3

2 77 ± 13 9.56 ± 1.0

3 73 ± 11 7.87 ± 0.9

4 76 ± 16 5.59 ± 0.7

5 67 ± 15 9.19 ± 2.0

6 36 ± 7 6.70 ± 1.8

7 99 ± 17 7.11 ± 1.6

8 52 ± 9 6.24 ± 1.1

9 48 ± 10 6.89 ± 1.4

10 40 ± 9 7.1 ± 2.3

Q, cumulative amount of thymoquinone penetrated per cm2 at 8

hours (mean ± SD, n=5)

did not show any change in color and odor. Additionally, no phase separation occurred.

The antibacterial study with the stored hydrogel showed similar efficacy like the marketed

gentamicin cream against Staphylococcus aureus. This indicated that the drug was stable

in gels even after 8 months of storage and the gel formulations were physically and

chemically stable.

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5.3.6. Cyto-compatibility study

The viability of HDF and HaCat cells at the presence of TQ film was analyzed

using alamarBlue® assay- is a cell viability assay reagent which contains the cell

permeable, non-toxic and weakly fluorescent blue indicator dye called resazurin. Resazurin

is used as an oxidation-reduction (REDOX) indicator that undergoes colorimetric change

in response to cellular metabolic reduction. The reduced form resorufin is pink and highly

fluorescent. As the intensity of fluorescence produced is proportional to the number of

living cells respiring, the viable cells continuously convert resazurin to resorufin,

increasing the overall fluorescence and color of the media surrounding cells. Through

detecting the level of oxidation during respiration alamarBlue® acts as a direct indicator to

quantitatively measure cell viability and cytotoxicity. The results of the test are shown in

Figure 5.3. As can be seen from the graph, the cell viability is not affected by the addition

of TQ film in the media. TQ film showed 90% cell viability on HaCat and 96% cell viability

on HDF cell lines after 24 h of incubation. According to ISO 10993–5: 2009 [136]

standards, it can be confirmed that the prepared samples were nontoxic in nature, because

the cell viability is > 80%

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Figure 5.3. Cytocompatibility study of TQ film. Cell viability of TQ film with HDF and

HaCat cells using alamarBlue® assay.

5.3.7. In vitro and Ex vivo bacterial inhibition study

Prior to in vivo experiments, the in vitro antibacterial efficacy of TQ film was first

validated along with ex vivo antibacterial activity. For this, the materials were inoculated

with Staphylococcus aureus and their antibacterial efficacies against bacterial growth were

assessed after 24 h incubation. Results from in vitro study showed that the presence of

gentamicin generated a zone of growth inhibition of Staphylococcus aureus on the plate in

a dosage dependent manner (Figure 5.4A). However, there was no growth of bacteria in

the presence of TQ films and hydrogels (Figure 5.4A) and suggested that a complete

inhibition of Staphylococcus aureus. The completely absence of bacteria in the presence

of TQ films was verified by three independent experiments. Furthermore, the results from

0

20

40

60

80

100

120

Media TQ Film Control Film Stored Film Triton

Cell

via

bili

ty (

%)

HaCat

HDF

129

ex vivo antibacterial study showed complete eradication of bacteria using TQ film from

human cadaver skin. Whereas, gentamicin cream and TQ hydrogel showed 5 and 4 log

reduction of bacteria respectively (Figure 5.4B).

Figure 5.4. Bacterial inhibition study. (A) Inhibition of bacterial growth on agar plate by

Control negative (i); Gentamicin positive control 50 µg/mL (ii) right upper and 500 µg/mL

(ii) right lower; Control film (iii); TQ hydrogel (iv) and TQ film (v) against Staphylococcus

a b

c

130

aureus; (B) Ex vivo antibacterial activity by Control (i); Control film (ii); Gentamicin

sulfate USP, 0.1% marketed cream (iii); TQ hydrogel (iv); TQ film (v) and Log of bacterial

reduction with different treatment groups (vi). Data represent mean ± SD of four replicates.

***p = < 0.001 and ^^^p = < 0.05.

5.3.8. Scratch assay for wound closure activity

In vitro scratch wound healing assay has been proven as a simple, valuable and

inexpensive experiment to obtain first insights into how plant preparations or their isolated

compounds can positively influence formation of new tissue [137]. This assay has

commonly been applied to measure cell migration, cell proliferation and wound closure in

response to test components. In this experiment a “wound gap” in a cell monolayer of both

HDF and HaCat is created by scratching, and the ability to migrate cells to repopulate the

scratch area over time is monitored. The results of these experiments are shown in Figure

5.5 (A-E). It was found that TQ showed significant positive effects on wound healing

activities of HDF and HaCaT cell lines. TQ (100 ng) showed significant wound closure

activity with HDF compared to control (p<0.05) (Figure 5.5D). 77% of wounds were

closed with 100 ng TQ at 12 hours followed by 100% wound closure at 24 hours. On the

other hand, 43% wounds were closed with control at 12 hours (Figure 5.5C and D).

Additionally, at 4 h, 8 h and 12 h all the different concentration of TQ (1-1000 ng) showed

increased wound closure activity compared to control, suggest the cell migrates faster in

presence of TQ. The number of fibroblast cells in the scratched area was found to

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Figure 5.5. Effect of different treatment groups on the wound healing of keratinocytes and

fibroblasts. (A) Representative micrographs from control, 1 ng/mL and 100 ng/mL of TQ,

showing the original wound and the wound after 6 days; (B) Quantitative analysis of wound

closure as a function of time. The wound area was determined as the wound area at a given

time relative to the original wound area. Data are presented as the means ± SD (n=5-6).

***p<0.001 (control vs 100 ng/mL) and ^p<0.05 (Control vs 1 ng/mL). (C) Representative

micrographs from control, 1 ng/mL and 100 ng/mL of TQ, showing the original wound

and the wound after 24 hour; (D) Quantitative analysis of wound closure as a function of

time. The wound area was determined as the wound area at a given time relative to the

original wound area. Data are presented as the means ± SD (n=6). **p<0.01 and

***p<0.001 (control vs 100 ng) and ^p<0.05 (Control vs 1 ng); (E) Quantitative

measurement of cells number migrating in the corresponding scratched wound areas at

different treatment groups. The values plotted were means of 6 determinations (𝑛 = 6).

***p<0.001 (100 ng/mL vs control/1 ng/mL/10 ng/ml).

increase between 10 ng to 200 ng TQ and then decrease with the increased concentration

of TQ. 100 ng TQ showed a significant increase in the number of fibroblast cells compared

to all other experimental conditions (Figure 5.5E). Using the HaCaT cell line 100 ng TQ

showed 85% wound closure activity at day six which is significantly higher (p = 0.0001)

than the experimental control (Figure 5.5A and B). At all time points (Day 1, Day 2, Day

3 and Day 6) 1 ng, 10 ng, 100 ng, 200 ng and 1000 ng TQ showed increased wound closure

activity compared to the control. Rapid wound closure activity was observed between day

three and day six at all TQ concentrations (1-1000 ng).

5.3.9. Wound healing and anti-bacterial activity of TQ film in vivo

The photographs of the wounds at 0, 3, 7, 14, and 21 days post-wounding are shown

in Figure 5.6A. Almost all of the wound areas were re-epithelialized in different

experimental groups. The percentage of wound closure were determined and compared

among all experimental groups (Figure 5.6C). At day 7 and 10, 25% and 42% wounds

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were closed respectively using TQ film. Whereas, applying gentamicin marketed cream

12% and 39% wound closure activity were observed at day 7 and 10 respectively. Based

on this observation, it can be stated that an accelerated but not statistically significant

wound healing was observed using TQ film at day 7 and 10 compared to the gentamicin

marketed formulation (Figure 5.6C). Additionally, at day 14 similar wound closure activity

was obtained using both TQ film (62%) and gentamicin marketed formulation (63%). At

all time points both TQ film and gentamicin showed higher wound closure activity

compared to the control film experimental group. A higher wound closure activity (though

not statistically significant) was observed at all time points with the bacterial wound

experimental group compared to the TQ film, gentamicin cream and control film. One

possibility may be fewer handling or disturbance of the wounds in this group helped wound

closure. In this case, the bacterial wound with the vehicle control serves a more appropriate

control for the study. As the vehicle control would provide the same wound environment

as the treatment group whereas the bacterial wound without any vehicle would not provide

the same microenvironment of the wound.

Bacterial sample analysis of the wound site at day 1, day 2, day 3 and day 7 showed

significant (p<0.001) bacterial reduction using both TQ film and gentamicin compared to

the bacterial wound and control film (Figure 5.6B). Significant bacterial reduction coupled

with wound closure activity of TQ film that is similar to the gentamicin marketed cream

may be considered as a promising aspect of TQ’s usefulness in treating infected wounds.

Further studies should investigate in more details the benefits of using TQ in the treatment

and management of wound infection.

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Figure 5.6. Macroscopic observations, wound closure and bacterial reduction. (A)

Photographs of wounds in BALB/c mice in which the wounds received TQ loaded film

and Gentamicin. The animal with bacterial wounds and wound with no bacterial served as

control group and animal with control film served as a vehicle control. Representative

photographs of the wounds were taken at 0, 3, 7, 10, 14, and 21 days post-wounding; (B)

Log of bacterial reduction at each time point (Day 1, 2, 3 and 7) using different

136

experimental groups. Data are presented as the means ± SD (n=2-4). ***p<0.001 (Bacterial

wound vs TQ Film) and ^^^p<0.001 (Bacterial wound vs Gentamicin); (C) Percentage of

wound closure in all experimental groups at 0, 3, 7, 10, and 14 days post-wounding.

5.3.10 . Histological examination

Histopathological study was performed by taking 1 cm2 of animal tissue at the end

of the study. The tissues were stained with Masson’s trichrome. Re-epithelization was

observed in experimental groups of control wound, bacterial wound, TQ film and

gentamicin formulation (Figure 5.7). A complete re-epithelization was observed with the

animal received one extra dose of TQ film (+ dose) compared to the group which received

four doses of TQ film (Figure 5.7v and vi). Therefore, future research should be conducted

to find out the appropriate dosage regimen (formulation type, TQ concentration in the

formulation, dosing interval, length of treatment) that would be needed to bring in the

beneficial effect of TQ in wound re-epithelization. We also predict that wound remodeling

was still in progress in animals with TQ film treatment. Whereas, wound remodeling was

may be completed with other animal groups. More studies need to be conducted to confirm

these findings and to understand the wound environments under different conditions in the

future. Future research should also examine the usefulness of other dosage form of TQ like,

cream, gel, topical spray formulation in wound closure activity.

137

Figure 5.7. Masson’s trichrome staining of the different samples at day 21 post-wounding

(control wound (i); Gentamicin sulfate USP, 0.1% marketed cream (ii); bacterial wound

(iii); control film (iv); TQ film (v); TQ film + dose (vi)) indicates epidermis and

indicates dermis.

5.4. Conclusions

Open wounds are prone to bacterial infection and if not treated at earlier time point

might also provide an entry point for microbes that cause systemic infections. Additionally,

infected wounds heal less rapidly as infection at the wound site can produce toxins that can

further kill the regenerating cells and can delay the progression of wound healing. Several

topical and oral antibiotics are presently being used to treat wound infections in humans.

However, due to their adverse effects and the presence of antibiotic-resistant organisms,

researchers are now investigating different bioactive compounds of plant origin for their

antibacterial activity to offer an innovative treatment strategy. The use of TQ to treat

138

infected wound is justified by this work, as TQ exhibited commendable activity against

Staphylococcus aureus.

In this study, novel TQ loaded polymeric films and hydrogels were developed using

different polymers. The application of these TQ containing films and hydrogels showed in

vitro skin permeation and a strong antibacterial activity against Staphylococcus aureus

since no bacterial growth was observed with TQ loaded film and hydrogel formulations. In

vitro scratch wound healing assay revealed wound closure activity of TQ. Moreover,

preclinical mice model of Staphylococcus aureus wound infection demonstrates a

significant reduction in bacterial population using TQ film and wound closure activity.

Based on this result it can be stated that, significant bacterial reduction coupled with wound

closure activity of TQ film might be able to provide a new treatment strategy specially for

those who has developed resistance towards the commonly used antibacterial agents. In

summary, TQ has potential to be used as an agent for wound healing and to treat bacterial

skin infections. TQ/PVP films and hydrogels developed in this study have potential for the

treatment and management of wound and Staphylococcus aureus related bacterial skin or

wound infections. This study would provide the supplementary evidence of TQ’s

significant potential in the area of microbial control and wound healing. But further

research is needed to confirm this novel finding.

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Appendix A. Development of lidocaine loaded EUDRAGIT® RLPO transdermal

patch application

A.1. Introduction

Transdermal route has attracted most of the formulation scientists owing to its

advantages such as prevention of hepatic first pass metabolism, avoidance of

gastrointestinal degradation, higher bioavailability at lower dose, direct transport into

systemic circulation, non-invasive, easy administration, higher patience compliance, lower

risk of overdose etc. [13, 138, 139]. Transdermal drug delivery systems in the form of

patches have been available on the market for several decades. Patches can be classified as

matrix (drug-in-adhesive) systems, or reservoir, or membrane-controlled systems [140].

PSAs (Pressure-Sensitive Adhesives) are traditionally used in patch production. The main

families of PSAs present several drawbacks related to their chemical structures. Therefore,

nowadays acrylics and silicone-based PSAs have been largely replaced by

polyisobutylenes (PIBs) due to the reduced allergenicity. Poly(methyl methacrylate)s are

commonly used in various pharmaceutical preparations including in transdermal patches

because of their well-recognized biocompatibility and safety even if additives or chemical

cross-linking agents are required to provide them with required adhesive properties [141-

143].

EUDRAGIT® RL is referred to as ammoniomethacrylate copolymers in the

USP/NF [144]. This copolymer is synthesized from acrylic acid and methacrylic acid esters

[145]. Unlike natural cellulose derivatives the synthesis of methacrylic copolymers via

140

free-radical polymerization is highly reproducible. Firstly, using various acrylic acid or

methacrylic acid derivatives long polymer chains are formed by chain growth reactions. In

a subsequent step, the functional properties of the final (meth)acrylic copolymers can be

adjusted by selection from a variety of monomers. The functional co-monomers are

responsible for adjusting the solubility profile. Whereas, the non-functional co-monomers

are responsible for steering the polymer properties. Various grades of EUDRAGIT® can

be obtained by varying the chain length through various termination and transfer reactions.

Different grades of EUDRAGIT® has different physicochemical properties and their

applicability in pharmaceutical industries also differ. EUDRAGIT® RLPO is insoluble at

physiological pH and able to swell to form permeable films as it contains a higher number

of functional quaternary ammonium groups (10%). This ammonium groups are present as

salts which allow water molecules to penetrate freely into EUDRAGIT® RLPO and can

further influence the drug flux. Additionally, EUDRAGIT® RLPO is a polymer with

moderate glass transition temperature of 63°C and it has a pH of 6 that is close to the human

skin pH. Moreover, this polymer is stable, possess good film making characters and act as

crystallization inhibitors [146]. All of these properties can be useful to solve some of the

existing issues with transdermal patch delivery, such as; 1) a pronounced tendency to drug

crystallization with silicon-based PSAs (Pressure-Sensitive Adhesives) [147]; 2) the

disadvantage of using (PIBs) in place of PSAs are related to their easy oxidation and low

air and water vapor permeability that can interfere with drug flux through the skin and can

cause skin maceration.

Hydrophobic polymers of EUDRAGIT® classes including EUDRAGIT® RLPO,

EUDRAGIT® RSPO, EUDRAGIT® NE 30D, EUDRAGIT® EPO have been widely used

141

in pharmaceutical formulations. The high release and flux of lipophilic drugs can be

achieved by combining the hydrophobic and hydrophilic polymers in a suitable ratio. The

previous study showed the use of EUDRAGIT® RL 100, EUDRAGIT® RS 100 and

HPMC (Hydroxy propyl methylcellulose) in the preparation of matrix patches of lipophilic

drugs [148]. HPMC is a water-soluble cellulose ether. It has been widely used in

pharmaceutical products due to its hydration and gel forming abilities. Therefore,

EUDRAGIT® RLPO and HPMC were chosen to develop lidocaine transdermal patches.

In the current study, lidocaine was chosen as a model drug due to its lipophilic

characteristic (log P = 2.3) and its applicability as a topical/transdermal anesthetic. It was

also used in this study because of its tendency to become crystalline in PSAs. The aim of

this work was to develop and characterize the prolonged release transdermal patch using

EUDRAGIT® RLPO and lidocaine as a model drug to improve its adhesive and cohesive

strength, to study the drug release of lipophilic drug using the hydrophobic polymer, to

examine the lidocaine crystallization, to evaluate the water vapor transmission rate

(WVTR) of the developed transdermal patch system and to compare the rheological

properties of the formulation.

A.2.Materials and Methods

A.2.1. Materials

Lidocaine was purchased from MP Biomedicals (Solon, OH, USA). Chitosan was

purchased from Alfa Aesar (Haverhill, MA, USA), Hydroxy propyl methylcellulose

(HPMC) was purchased from Ashland (Parlin, NJ, USA), Triethyl Citrate (TEC) was

142

purchased from Jungbunzlauer (Newton Centre, MA, USA) and Acetone was purchased

from J.T. Baker (Phillipsburg, NJ, USA). Eudragit® RLPO was a gift from Evonik

(Piscataway, NJ, USA). Drierite was purchased from W.A. Hammond Drierite Company

Ltd (Xenia, OH, USA). Glacial acetic acid, 1N sodium hydroxide, Acetonitrile, Monobasic

potassium phosphate were purchased from Fisher Scientific (Bridgewater, NJ, USA).

Nonwoven polyester layer CoTran™ 9695 and fluorosilicone coated polyester film

Scotchpak™ 9709 were gifts from 3M Drug Delivery Systems (St. Paul, MN, USA).

A.2.2. Preparation of lidocaine-loaded transdermal patches

Transdermal patches containing lidocaine were prepared by solvent evaporation

techniques (Figure A.1) using composition given in Table A.1. Lidocaine and Eudragit®

RLPO polymer were first dissolved in an acetone solvent then HPMC and Chitosan were

incorporated into the lidocaine containing Eudragit® RLPO dispersion with constant

stirring. The obtained uniform dispersion was casted on nonwoven polyester layer coated

with a hypoallergenic acrylate adhesive (3M CoTran™ 9695; 3M Drug Delivery Systems,

St. Paul, MN) and allowed for air drying at laboratory ambient temperature for overnight.

The patches were then covered with Fluorosilicone Coated Polyester film (3M

Scotchpak™ 9709), cut into appropriate sizes and stored at room temperature.

143

Figure A.1. Schematic representation of solvent evaporation method.

Table A.1. Lidocaine-loaded patch composition % (w/w) at different drug loading ranging

from 4% to 20% (Formulation A-D).

Formulation

Code

Eudragit®

RLPO

HPMC Chitosan TEC Lidocaine Acetone

A 10 7 7 16 4 56

B 10 7 7 16 5 55

C 10 7 7 16 10 50

D 10 7 7 16 20 40

Solvent evaporation

144

A.2.3. Patch characterization

A.2.3.1 Thickness

Patch thickness was determined using a digital caliper (Mitutoyo America

Corporation, Aurora, IL, USA) and three measurements were performed on each patch to

obtain the average thickness.

A.2.3.2 Weight variation

To ensure the weight uniformity, 3 randomly selected patches from each

formulation were subjected to individual weighing.

A.2.3.3. Content uniformity

The lidocaine-loaded patch was cut into 7 x 5 inch2 pieces and accurately weighted.

The patch was then dissolved in 500 mL of 50:50 acetonitrile and water solvent and filtered

through a syringe filter (0.45 µm). Drug concentration was quantified using HPLC method.

A.2.4. High Performance Liquid Chromatography (HPLC)

Lidocaine was quantified using a Shimadzu Prominence PDA HPLC system

(Empower 3 software). A C18 column (Waters, Xbridge) with dimensions of 250 X 4.6

mm and 5 µm packing was used. Acetonitrile and Solution A (20:80) was used as a mobile

phase. Solution A was prepared by mixing 50 mL of glacial acetic acid with 930 mL of DI

water and finally adjusted the pH to 3.4 with 1N sodium hydroxide. The column

temperature was set to 25 °C. The flow rate used was 1.5 mL/min with injection volume of

145

20 µL and run time for 15 minutes. Lidocaine peak was detected at 254 nm, 4 min retention

time. The method was linear at a concentration of 0.17–1.7 mg/mL with R2 value of 0.99.

A.2.5. Mechanical properties

Mechanical properties in term of tensile strength (TS) and percent elongation at

break (E/B) were determined using Tinius Olsen Material testing equipment (H50KT,

Horsham, PA, USA) working with a 100 N loaded cell. The patch strip between two

clamps was positioned at a distance of 50 mm and the test speed was set to 5 mm/s. Tensile

stress (N/m²) is the force applied to induce film deformation. Tensile stress was calculated

using the following equation-

Stress = Force applied (N)/Area (m²) (1)

The elongation at break is the distance traveled by the upper plate up to the point where

the film separates compared to the origin and was calculated as follows-

% 𝐸𝑙𝑜𝑛𝑔𝑎𝑡𝑖𝑜𝑛=(𝐿𝑒𝑛𝑔𝑡ℎ 𝑎𝑡 𝑏𝑟𝑒𝑎𝑘𝑖𝑛𝑔 𝑝𝑜𝑖𝑛𝑡−𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑙𝑒𝑛𝑔𝑡ℎ 𝑜𝑓 𝑓𝑖𝑙𝑚)/(𝑂𝑟𝑖𝑔𝑖𝑛𝑎𝑙

𝑙𝑒𝑛𝑔𝑡ℎ 𝑜𝑓 𝑓𝑖𝑙𝑚) X 100 (2)

A.2.6. Loop tack test or adhesive strength study

Tinius Olsen Material testing equipment (H50KT, Horsham, PA, USA) equipped

with software (Horizon) was used to study the adhesive properties of the transdermal patch.

Stainless steel plate was used due to its low surface energy like the human epidermis.

146

Adhesive strength in terms of peak adhesive force (N) was determined. The force required

to detach the film on the upper platen from the stainless-steel plate known as the adhesive

force. The upper platen with a 1-inch width patch attached was lowered to the surface of

the stainless-steel, allowed to secure contact with a load of approximately 100 N for 10 s

and then raised at a constant rate of 300 mm/min.

A.2.7. Rheology

Rheological characterization was performed on rheometer (Anton Paar, Graz,

Austria) equipped with a 25 mm flat stainless-steel plate. All tests were done at 32ºC and

a gap of 1 mm. Following tests were carried out-

A.2.7.1. Oscillation stress sweep

The samples were subjected to increasing stress (0.1 - 100% strain) at a constant

frequency of 1 Hz. This test allows determination of the liner viscoelastic region (LVR) of

the sample, and therefore the consequent choice of the stress value to use in the subsequent

oscillation test.

A.2.7.2. Frequency sweep

Frequency sweeps were done by oscillating the samples at angular velocity (ω)

range of 0.1 – 100 rad/sec at a constant stress obtained from LVR. Effect of stress on elastic

modulus (G') and viscous modulus (G'') were recorded.

147

A.2.8. In vitro release study

Vankel VK 7000 dissolution system was used to obtain the release profile of

Eudragit patches. The release studies were performed according to USP apparatus 5, paddle

over disc method. One liter of phosphate buffer pH 7.4 at 32ºC was used as the dissolution

medium. The paddle rotation speed was adjusted to 50 rpm. Two milliliter samples were

withdrawn at predetermined time intervals. At 24 hours the paddle rotation speed was set

to 100 rpm for an hour for infinity sample. At the end of the study the samples were

analyzed using a UV-Visible spectrophotometer at 254 nm.

A.2.9. Water vapor transmission of transdermal patch system

The water vapor transmission rate (WVTR) is the amount of moisture transmitted

through a unit area of film in a given duration and at specified temperature. All tests were

done at 32ºC and 90% RH chamber. Water vapor cups were filled to within 3 mm of the

opening with Drierite and was assured that it will not make contact with the adhesive of

the test specimen. The test specimens were applied adhesive face down over the opening.

The test adhesives were brought into intimate contact with the flange using pressure. The

assembly was placed in the humidity cabinet for 24 hours conditioning period. After 24

hour it was removed from the cabinet, cooled for 15 minutes at standard conditions (24ºC

and 50% RH) and weighted on an analytical balance. This weight is the W1. After the

initial weighing the assembly was returned to the humidity cabinet for 72 hours. Removed,

conditioned for 15 minutes and weighed. This weight was used as W2. Finally, the WVTR

for the prepared patches was calculated using the following equation-

148

WVTR = W2- W1 X 2400/T X A (1)

Where,

W1 = weight (in grams) before exposure period

W2 = weight (in grams) after exposure period

T = exposure time (in hours)

A = area (in square inches) of opening in dish

A.2.10. Shower resistance study

A 10 minutes shower test was performed on the marketed and experiments patches.

Both of the patches were cut into same sizes (10 X 7 cm) and were brought into intimate

contact with the stainless-steel plate using pressure. A water flow of 40 psi was applied on

the patches for 10 minutes. This cycle was repeated until the sign of detachment was

observed.

A.2.11. SEM-EDS (Scanning Electron Microscopy-Energy Dispersive Spectroscopy)

The surface morphology of the transdermal patch was examined using a scanning

electron microscope (TM3000, Hitachi, USA) at 500 magnification.

149

A.2.12. Differential scanning calorimetry studies

Patches used for studying the crystallization of the drug were kept at laboratory

ambient temperature in a closed container for 30 days. A Perkin-Elmer Pyris-6 differential

scanning calorimeter (DSC) was used to study the crystallinity of the drug in the

transdermal patches and the physical mixtures of the polymers used in this study.

Approximately 6-8 mg of each sample of Ex patch 10%, Ex patch 20%, pure drug, pure

polymers (Eudragit® RLPO, HPMC, Chitosan), 1:1 physical mixtures of polymers and 1:1

physical mixtures of polymers, drug and TEC were hermetically sealed in a flat bottomed

aluminum pan with aluminum cover and heated over a temperature range of -30ºC to 150ºC

at a linear heating rate of 10ºC/min.

A.2.13. Data and statistical analysis

Results are reported as mean ± SD (n=3). The statistical analysis of the data was

performed by using one-way Anova, the Tukey post-hoc tests and Student’s-t test, and p-

values < 0.05 were considered significant.

A.3. Results and discussion

The transdermal patches of lidocaine were prepared according to Table A.1. The

patches were the combination of hydrophilic polymer (HPMC and Chitosan) and

hydrophobic polymer (EUDRAGIT®) with varying amounts of lidocaine. Solvent casting

150

technique used to prepare the patches was satisfactory. The developed patches were thin,

flexible and smooth.

A.3.1. Appearance and patch thickness

A matrix dispersion transdermal patch was prepared. Lidocaine-loaded patch had a

smooth surface with transparent color. The thickness, weight and drug content assay of

lidocaine patches were shown in Table A.2. Thickness of all five experimental patches was

found to be in the range of 0.85±0.02 to 1.31±0.005 mm. The marketed lidocaine patch

thickness was 1.17±0.04 mm. Each formulated patch provided the low value of SD,

indicating the uniformity of its thickness. The average weights of these formulations ranged

from 2.03±0.05 to 3.80±0.13 gm. Each formulation showed the low value of SD, indicating

the uniformity of the developed patch.

A.3.2. Content uniformity

To determine the content uniformity other parameters were kept constant

(EUDRAGIT® RLPO, HPMC, Chitosan). Drug loading was varied from 4% to 20%

(Table A.1). Drug content (%) of formulations varied from 94±1 to 107±3.5 indicating

drug was dispersed uniformly throughout the patches. The small SD also indicates that the

solvent evaporation method was efficient to fabricate lidocaine-loaded patches with good

uniformity of dosage unit and minimal variation.

151

Table A.2. Physical and mechanical properties of transdermal patches containing

lidocaine. Data represents N=3, mean ± SD.

A.3.3. The effect of EUDRAGIT® RLPO in adhesive and cohesive strength

The adhesion of patches to the skin is a prerequisite for maintaining drug release.

The loop tack tests define tack as the force required to separate a loop made by clamping

the ends of a patch strip at a specified time that has been brought in contact with a specified

area of a defined surface. Formulation B and C showed significantly higher adhesive

strength compared to the other formulations possibly due to the varying concentration of

drugs (Figure A.2). All the formulations have the same concentration of HPMC, Chitosan,

EUDRAGIT® RLPO and TEC. The control positive formulation doesn’t contain any

EUDRAGIT® RLPO. The presence of a large number of hydroxyl groups in HPMC and

152

Chitosan may have been played a role in adhesion [149]. Though control formulation

without EUDRAGIT® RLPO showed adhesive properties but the adhesive layer of the

control patch did not strip cleanly from the plate, leaving noticeable residues, which was

an evidence of cohesive failure (Figure A.3). This could be due to the observed lower

cohesive properties of the patches made of just hydrophilic polymer. EUDRAGIT® RLPO

may have function as an adjunct adhesive in the patch containing hydrophilic polymers.

That further may have been contributed in improving the cohesive properties of the

patches.

Figure A.2. Peak adhesive force of different formulations containing Eudragit® RLPO

and different concentration of Lidocaine. Data represents mean ± SD (n=3).

0

1

2

3

4

5

6

Controlnegative

From A Form B Form C Form D MarketedPatch

Controlpositive

Pe

ak a

dh

esiv

e f

orc

e (

N/c

m²)

***

***

153

Figure A.3. Cohesive properties (a) formulation with EUDRAGIT® RLPO, (b)

formulation without EUDRAGIT® RLPO.

A.3.4. The effect of drug loading and EUDRAGIT® RLPO on the mechanical

properties of transdermal patches

The mechanical properties are useful indications of patch strength that might be

useful to hold up to the rigorous friction from day to day patch use. The results of

mechanical properties in terms of tensile strength and percentage of elongation at break are

shown in Table A.2, Figure A.4 and Figure A.5. All formulations showed tensile strength

with the range of 131- 143 N/m2 and provided percentage of elongation at break in the

range of 107-139%. Formulation A, B, C and D has varying amounts of lidocaine ranging

from 4% to 20%. There was almost no difference in the tensile strength and % elongation

at break values among different drug loaded patches. These results revealed that the

presence of hydrophobic polymer EUDRAGIT® RLPO in formulations may delivered a

patch with higher tensile strength compared to the marketed lidocaine patch (Figure A.4).

a b

154

On the other hand, the % elongation at break was higher with the marketed patch compared

to the all other experimental patches (Figure A.5). This is may be the effect of different

liners used in marketed and experimental patches.

Figure A.4. The effect of different formulations on tensile stress. Data represents mean ±

SD (n=3), ***p < 0.005.

Figure A.5. The effect of different formulations on % Elongation. Data represents mean

± SD (n=3), ***p < 0.005.

0

20

40

60

80

100

120

140

160

Controlnegative

Form A Form B Form C Form D Marketedpatch

Controlpositive

Te

nsile

str

ess (

N/m

²)

0

50

100

150

200

250

Controlnegative

Form A Form B Form C Form D Marketedpatch

Controlpositive

Str

ain

(%

Elo

nga

tio

n)

***

155

A.3.5. The effect of drug loading on rheological behavior of the formulations

The adhesive properties of PSAs are strictly related to the solid and liquid like

behaviors and are dependent on frequency of the applied stress at a given temperature. In

this study, the rheological analysis of the different formulations was used to determine the

viscoelastic parameters such as elastic modulus (G´) and viscous modulus (G´´). If G´ >

G´´, then the material is more solid than liquid as G´ value is the representative to the solid

like behavior. The converse is also true. Initial bonding with the skin usually occurs at low

frequencies and the PSA liquid like nature predominates since it must wet the substrate and

therefore low values of G´ is desirable [150]. Debonding process requires the formulation

to behave like solid and occurs at high frequencies. It also requires high cohesive strength

and is associated with a larger G´´. Figure A.6 shows the rheological behavior of different

drug loaded patch formulations. The result demonstrates that there is a decrease in G´ and

G´´ values with the increase of drug concentrations. Comparing the G´ and G´´ values of

different formulations it is observed that G´ ´> G´. That might be useful in bonding and

debonding process.

156

[A]

[B]

Figure A.6. Rheological behavior of different drug loaded patch formulations in terms of

oscillation frequency sweep data (A) the elastic or storage modulus and (B) the viscous or

loss modulus were plotted against angular frequency.

157

A.3.6. In Vitro release study

All the experimental EUDRAGIT® RLPO patch formulations provided the most

controlled release of lidocaine with 7% being released after 2 h and 100% after 24 h (Figure

A.7). On the other hand, 36% lidocaine was released from the marketed patch after 2 h and

65% drug was released at 4 h (Figure A.7). This result indicated that, due to the presence

of EUDRAGIT® RLPO there was more sustain and controlled release of lidocaine from

the experimental patch compared to the marketed and control patch without EUDRAGIT®

RLPO. Experimental control patch showed 53% drug release at 2 h followed by 84% drug

release at 4 h. This result demonstrated the usefulness of EUDRAGIT® RLPO in providing

a sustain and controlled drug release over a longer period of time in the transdermal patch

formulations.

Figure A.7. In vitro release profile of lidocaine from experimental and marketed patch

formulations in phosphate buffer at pH 7.4 (N=3).

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12 18 24 25

% R

ele

ase

Time (h)

Marketed Patch

Ex Patch 4%

Ex Patch 5%

Ex Patch 10%

Ex Control Patch

Ex Patch 20%

158

The zero-order model, the first-order model and the Higuchi square root law model has

been used to describe the drug release kinetics from the polymer matrix systems [151]. The

zero-order, first-order and Higuchi models were applied to the release data. The R2 value

of both zero-order and first-order model indicated less fit. Whereas, the drug release kinetic

data fitted the Higuchi model indicating the release of lidocaine from the experimental

patch was controlled by the diffusion process (Figurer A.8). This study is in agreement

with the study conducted by Doungdaw et al. where the piroxicam release kinetic data from

the EUDRAGIT® RL100 and EUDRAGIT® RS100 fitted with the Higuchi model [145].

In another study, Mohamed et al. also demonstrated that the release of tizanidine

hydrochloride from a EUDRAGIT® RL100 and EUDRAGIT® RS100 bioadhesive buccal

patch occurred by diffusion through the film matrix [152]. Therefore, it can be stated that

the drug molecules are released from the polymer metrices by the following mechanisms:

(a) the presence of higher amounts of quaternary ammonium groups in the EUDRAGIT®

RLPO influenced the water to enter more easily into the polymer matrix and provided

hydration, (b) diffusion of drug molecules through the hydrated polymer layer. It should

be also noted that the significance of these mechanisms will depend on the drug

characteristics and the polymer combinations.

159

Figure A.8. Higuchi release kinetics, in phosphate buffer at pH 7.4 after 25 h (N=3).

A.3.7. Scanning electron microscopy

Drug crystallization is a critical issue in transdermal patch formulations as it is an

indication of formulation instability and can also impact the drug delivery [153]. The

prepared patches were evaluated for their surface morphology and for the indication of

drug crystallization. At the end of 30 days the prepared patches were examined using SEM.

Patches containing higher concentrations of lidocaine (10%) did not show any signs of

crystallization (Figure A.9B) after 30 days of storage at ambient laboratory temperature

suggesting that 10% (w/w) lidocaine was under saturation level as crystallization is likely

to initiate in supersaturated systems with the formation of a nucleation of drug molecules

that is difficult to re-dissolve [154].

0

10

20

30

40

50

60

70

80

2 2.4 2.8 3.1

Cum

ula

tive %

dru

g r

ele

ased

Time½ (h1/2)

Ex Patch 4% Ex Patch 5% Ex Patch 10% Ex Patch 20%

R² = 0.9974

R²= 0.9913

R² = 0.9915

R²= 0.9968

160

[A]

[B]

161

[C]

Figure A.9. SEM-EDS photographs of (A) lidocaine 4% transdermal patch (B) lidocaine

10% transdermal patch after 30 days and (C) marketed 5% lidocaine patch.

A.3.8. Differential scanning calorimetry

Differential scanning calorimetry (DSC) studies of the pure drug produced an

endotherm representing its melting point at 69ºC indicating that lidocaine is the crystalline

drug (Figure A.10A). In the physical mixtures- EUDRAGIT® RLPO, HPMC and chitosan

produced a single melting peak at 60ºC. The melting peak was shifted to 46ºC with the

addition of lidocaine in the 1:1 physical mixture of polymers (Fig. A.10C). No peak

representing lidocaine was observed in the experimental lidocaine 10% and 20% patch

stored for 30 days. This may be explained by the fact that lidocaine is still being solubilized

162

[A]

[B]

163

[C]

[D]

164

[E]

Figure A.10. Differential scanning calorimetry profiles of different components in

transdermal patches: (A) Pure lidocaine; (B) 1:1:1 physical mixture of EUDRAGIT®

RLPO, HPMC and chitosan; (C) 1:1:1:1:1 physical mixture of EUDRAGIT® RLPO,

HPMC, chitosan, TEC and lidocaine; (D) Ex lidocaine patch 10% and [E] Ex lidocaine

patch 20%.

in the patch and even after 30 days of storage lidocaine didn’t change its physical state to

appear as crystalline form.

A.3.9. WVP evaluation

The WVP of marketed patch was 2.21 ± 0.19 g/100 sq inches/24h, whereas the

WVP measured for experimental 4% patch was 0.98 ± 0.04 g/100 sq inches/24h. The

differences in backing liner and polymer composition might have played a role in WVP.

165

The marketed and experimental patch has different backing liner, additionally, the

marketed patch has hydrophilic polymer whereas the experimental patch has hydrophobic

polymer that could have influenced the WVP of both patches.

A.3.10. Evaluation of shower effect on the patches

As transdermal patches are intended to be used for longer periods of time,

evaluating their ability to withstand water force equivalent to usual shower time is logical.

The marketed lidocaine patch was able to stick on the stainless-steel plate for 10 minutes

under 40 psi water pressure but showed complete detachment from the plate at 15 minutes

(Figure A.11). On the other hand, experimental patch was able to withstand the continuous

flow of 40 psi water pressure for 60 minutes (Figure A.11). This is may be due to the reason

that lidocaine marketed patch is composed of hydrophilic polymer like polyvinyl alcohol,

carboxymethylcellulose that tends to swell in water. This water uptake and swelling

tendency reduced the bonding between the patch and stainless-steel plate. Whereas, the

experimental patch has a combination of hydrophilic and hydrophobic polymer that might

have reduced the water uptake of the polymer during the shower test. Which further played

a role in maintaining the bond between the patch and stainless-steel plate.

166

Figure A.11. Effect of shower or 40 psi water pressure on marketed (A-C) and

experimental patches (D-F).

A.4. Conclusions

Lidocaine-loaded transdermal adhesive patch was successfully developed using

EUDRAGIT® RLPO, 3M CoTran™ 9695 nonwoven layer and Fluorosilicone Coated

167

Polyester film (3M Scotchpak™ 9709) as a backing liner. DSC and SEM studies

demonstrated that lidocaine even in higher concentration (20%) is in amorphous or

dissolved state in the patch formulation with EUDRAGIT® RLPO. All experimental

patches showed sustained release of lidocaine over time and the release kinetics followed

the Higuchi model. All the patch formulations showed higher adhesive strength and tensile

strength compared to the marketed patch that might be useful to hold up to rigorous friction

from day to day patch use. Additionally, the experimental patch was able to withstand 40

psi shower pressure till 60 minutes and did not show any sign of detachment from stainless

steel plate. The development of these patches with EUDRAGIT® RLPO would be relevant

as a potential dosage form for potent drug with crystalline tendency to deliver through

transdermal route. Further, investigation should be conducted to confirm the results.

168

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Thesis summary and future perspectives

The scope of this thesis was the development of a novel topically applied polymeric

film system to deliver the bioactive compound thymoquinone (TQ) to the targeted dermal

site for the treatment and management of wound infections. Firstly, this research aimed to

identify different physicochemical and solubility parameters such as, ingredient active gap

(IAG), ingredient skin gap (ISG), solubility of active in the formulation (SolV) and the

formulation solubility in the skin (SolS) to understand the physicochemical interaction of

the active and the penetration enhancer. Based on the study results, we proposed for the

first time a Solubility-Physicochemical-Thermodynamic (SPT) theory to define the action

of penetration enhancers in a given formulation with a specific drug. These studies suggest

that - there is an inverse relationship between measured flux and IAG values given that

there is an optimum ISG, SolV and SolS ratio; The larger the difference in solubility

between the formulation and the skin the greater the driving force for partitioning of the

active into the stratum corneum; the flux is actually proportional to a gradient of

thermodynamic activity rather than the concentration and maximum skin penetration and

deposition can be achieved when the drug is at its highest thermodynamic activity.

Secondly, we have applied the knowledge of SPT theory to evaluate the interaction of our

experimental drug TQ with the skin and various penetration enhancers. The study

concluded that transdermal permeation and adequate skin deposition of TQ can be obtained

by using penetration enhancers and different vehicles. Azone, oleic acid and Transcutol®

P (Tc) at a concentration of 5% was able to provide measurable TQ flux. Additionally,

these penetration enhancers were also able to generate TQ reservoirs in the skin that may

be useful to exert sustained release of TQ from the stratum corneum over longer period of

178

time. This fact provided information to deduce that, Azone, oleic acid or Tc can be the

penetration enhancer of choice to further develop a novel transdermal formulation of TQ.

Thirdly, we have used TQ to synthesize and characterize a biocompatible novel topical

polymeric film and hydrogel system. The developed system showed to be very useful and

efficient in controlling Staphylococcus aureus infection and promoting wound closure. The

presence of TQ-containing films and hydrogels completely wiped out Staphylococcus

aureus in a 10 cm in diameter TSA (Tryptic Soy Agar) plates while 500 µg/mL gentamicin

containing filters gave 10 mm of ZOI. In an ex vivo model, the presence of TQ-film

eradicated the bacterial colonization on human cadaver skin. Furthermore, in the BALB/c

mice wound model, TQ-films showed significant activity in controlling Staphylococcus

aureus wound infection without compromising the healing process.

This research indicates that, TQ has the potential to become an active of interest for

use in both the pharmaceutical prescription/OTC area but also for personal care and

cosmetic uses. It’s antioxidant, anti-inflammatory and anti-neoplastic properties can be

applied together to treat various diseases like breast cancer, cervical cancer etc. and

neurodegenerative diseases e.g., Alzheimer’s and Parkinson’s disease. Since TQ has not

been commercially used before as an antibacterial agent, it has the potential to offer a new

treatment strategy specially for those who has developed resistance towards the commonly

used antibacterial agents. In future work, investigating different concentrations of TQ to

find the minimum inhibitory concentration that might exert efficacy against both gram-

positive and gram-negative microorganisms might expand the scope of this research. It will

be important that future research investigate the combination therapy of TQ with other

known antibacterial agents like, gentamicin, fusidic acid, metronidazole etc. to evaluate the

179

potential benefit of combination therapy in different disease conditions such as, rosacea,

psoriasis, skin and soft tissue infection etc.. Further studies could investigate the different

strategies to improve TQ’s photostability and different delivery systems (antimicrobial

topical spray, microbial sealant, wound dressing etc.) for successful delivery of TQ. The

information obtained in this study for the thesis leaves further questions to be answered

such as how TQ promotes wound healing; which immune cells are recruited in the process

of the antibacterial effect of TQ in wound infections; is there any effect of TQ in promoting

angiogenesis during wound healing? Therefore, further studies are needed to provide TQ

related topical drug delivery system in the treatment and management of wound infections.

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