1

2

THESIS/DISSERTATION APPROVED BY

Alekha K. Dash, Ph.D.

3

i

PREPARATION, CHARACTERIZATION, AND IN VITRO EVALUATION OF

MULTIPLE EMULSION FOR TOPICAL APPLICATION OF LAWSONE AND

DIHYDROXYACETONE

___________________________________

By

Anne Grana

___________________________________

A THESIS

Submitted to the faculty of the Graduate School of the Creighton University in Partial

Fulfillment of the Requirements for the degree of Master of Science in the Department of

Pharmaceutical Sciences

_________________________________

Omaha, NE

(November 25, 2014)

ii

© Anne Grana, 2014

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ABSTRACT

Dihydroxyacetone (DHA) is the primary ingredient in most sunless tanners.

Lawsone is a primary component of red henna, used in henna tattooing and reversed

tattooing. DHA initiates a reaction called the Maillard reaction which results in the

browning of the skin. Application of lawsone results in the production of colored

keratin-bound fluorescent polymers called melanoidins. DHA alone gives some

protection in the UVA spectrum, with minimal UVB. When lawsone is applied after the

application of DHA, a different set of melanoidins are produced, giving UVB and even

greater UVA protection.

In this study, a multiple emulsion (W1/O/W2) containing 0.035% (w/w) lawsone

and 1% (w/w) DHA were prepared using the two step process by Matsumoto et al. The

first step was the production of the primary (W1/O) emulsion. The primary emulsion,

making up 40% of the final volume of the emulsion, was then added to the secondary

aqueous phase (W2). A combination emulsion was prepared by combining doubled

concentrations of lawsone and DHA multiple emulsions (W1/O/W2) in a 1:1 ratio through

trituration. The structure of the multiple emulsions was determined by light and

fluorescent microscopy. The stability of the prepared emulsions was characterized after

storage at 25˚C, 32˚C, and 40˚C and over a period of 28 days. DHA and DHA in

combination showed a significant decrease in % entrapment efficiency to 37.41 ± 6.03%

and 5.41 ± 3.82% after 28 at 40˚C, showing physical/ chemical instability of emulsions at

increased temperature. Chemical instability of the emulsions was confirmed by varying

decrease in pH at 32˚C and 40˚C of DHA emulsion and combination emulsion. Structural

instability of emulsions at 40˚C was confirmed by increase of zeta potential and

iv

rheological tests using flow and oscillatory stress/time sweeps. No significant changes in

particle size suggest no coalescence occurring. Spreadability remained within the range

of ≈ 20 cm²/g to 35cm²/g respectively. Characterization testing suggest the stability of

lawsone and DHA emulsions were best when kept separate at 25˚C. In vitro surface

release and Franz diffusion studies verified and confirmed drug release were more

effective when lawsone and DHA emulsions were kept separate.

v

Dedicated to my family

vi

ACKNOWLEDGEMENTS

I would first and foremost like to express my immense gratitude to my advisor

and mentor Dr. Alekha Dash for his encouragement, guidance, patience, and support

throughout the duration of the program. I am thankful to him for providing me the

opportunities and skills in lab to think critically and scientifically in order to better myself

professionally and personally. I extend my gratitude to my committee members, Dr.

Somnath Singh and Dr. Justin Tolman for their valuable suggestions in my project and

manuscript.

I would also like to thank my lab members: Sneha Dhapare, Shantanu Chandratre,

Swasti Pandey, Dr. Igor Meerovich for their help and scientific input during lab meetings.

I especially would like to thank Dan Munt for his knowledge, guidance, and

encouragement on this project, of which he initiated. I extend my gratitude to Taunya

Plater, Dawn Trojanowski, and the entire Graduate School and Department of

Pharmaceutical Sciences for their support throughout the past two years. A special

thanks goes to all my fellow graduate students, both past and present, who have been my

family and moral support here in Omaha. You have made my years here memorable.

My never ending gratitude goes to my parents, Mr. Salvador Grana and Mrs. Eden

Grana, for their immense love and constant support. I would especially like to thank my

brothers, Albert and Alvin Grana, for their support and inspiration throughout the

duration of my program. A special thanks goes to all my friends and family members at

home who have been my source of encouragement from afar. Finally, I would like to

thank God for all the blessing he has bestowed upon me to get me to this point in my life.

vii

TABLE OF CONTENTS

Page Number

Abstract iii

Table of Contents vi

List of Figures xi

List of Tables xiv

List of Equations xv

CHAPTER 1: Introduction 1

1.1 Topical application of sunscreen 2

1.2 UV and Skin Pigmentation 4

1.3 Maillard Reaction 7

1.4 DHA and Lawsone 10

1.5 Mulitple Emulsion 12

1.6 Objective, Hypothesis, and Specifications 13

CHAPTER 2: Formulation of Multiple Emulsion System Incorporating

Lawsone and DHA 15

2.1 Introduction 16

2.2 Materials 18

2.3 Methods 18

viii

2.3.1 Formulation of water-in-oil-in-water multiple emulsion 18

2.3.2 Verification of W1/O/W2 multiple emulsion 20

2.3.2.1 Light Microscopy 20

2.3.2.2 Fluorescence Microscopy 21

2.3.3 % Entrapment Efficiency 21

2.3.3.1 Chromatography 22

2.4 Calculations 22

2.5 Statistical data analysis 23

2.6 Results and Discussion 23

2.6.1 Fluorescence Microscopy 23

2.6.2 Light Microscopy 24

2.6.3 % Entrapment Efficiency 26

2.7 Conclusion 29

CHAPTER 3: Characterization of Multiple Emulsion Incorporating Lawsone

and DHA 30

3.1 Introduction 31

3.2 Methods 36

3.2.1 Particle Size and Zeta Potential Analysis 36

ix

3.2.2 Determination of pH 36

3.2.3 Spreadability 37

3.2.4 Rheology 38

3.2.5 Statistical Data Analysis 39

3.3 Results 40

3.3.1 Particle Size and Zeta Potential 40

3.3.2 Spreadability 42

3.3.3 pH 43

3.3.4 Rheology 45

3.4 Discussion 52

3.5 Conclusion 56

CHAPTER 4: In vitro Evaluation of Multiple Emulsion Incorporating

Lawsone and DHA 58

4.1 Introduction 59

4.2 Materials 61

4.3 Methods 62

4.3.1 In vitro surface release studies 62

4.3.2 Franz diffusion studies using snake skin 64

x

4.3.3 Statistical Analysis of Data 66

4.4 Results and Discussion 66

4.4.1 In vitro surface release studies 66

4.4.2 Franz diffusion using snake skin 68

4.5 Conclusion 70

CHAPTER 5: Summary and Future Directions 73

5.1 Summary 74

5.2 Future Directions 78

References 80

xi

LIST OF FIGURES

Page number

Figure 1. Anatomy of the skin showing the epidermis, dermis, and

subcutaneous tissue; melanocytes are found within the basal

cells which are in the deepest part of the epidermis 5

Figure 2. Generalized reaction forming a Schiff base 8

Figure 3. Chemical structure of dihydroxyacetone 9

Figure 4. Chemical structure of lawsone 9

Figure 5. Illustration of 3 phases of multiple emulsion droplet: internal water

droplets (W1) dispersed within the oil phase which is also dispersed

into a continuous water (W2) phase 16

Figure 6. Fluorescence microscopy photographs (a and b) of W1/O/W2

emulsion containing rhodamine, diluted 1:100 with deionized

filtered 0.2 µm filtered water, and magnified at 500x 23

Figure 7. Light microscopy photographs (a and b) of W1/O/W2 emulsion

containing DHA, diluted 1:100 with deionized filtered 0.2 µm filtered

water and magnified at 500x 25

Figure 8. Light microscopy photographs of W1/O/W2 emulsion containing

lawsone, diluted 1:100 with deionized filtered 0.2 µm filtered water,

and magnified at a) 200x and b) 500x 26

Figure 9. Entrapment efficiency for lawsone, DHA, and combination

lawsone/DHA at 25˚C, 32˚C, and 40˚C 26

Figure 10. Particle size analysis for lawsone, DHA, and the combination of

lawsone/DHA emulsions at a) 25˚, b) 32˚C, and c) 40˚C after 28 days 40

xii

Figure 11. Zeta potential for lawsone, DHA, and the combination of

lawsone/DHA emulsions at a) 25˚C, b) 32˚C, and c) 40˚C

after 28 days 41

Figure 12. Spreadability (cm²/g) for lawsone, DHA, and the combination

lawsone/DHA emulsions at a) 25˚C, b) 32˚C, and c) 40˚C

after 28 days 43

Figure 13. pH readings for lawsone, DHA, and combination lawsone/DHA

emulsions at a) 25˚C, b) 32˚C, and c) 40˚C after 28 days 44

Figure 14. DHA rheograms for OSS of G’ vs oscillatory stress at a) 25˚C,

b) 32˚C, and c) 40˚C 46

Figure 15. DHA rheogram of flow curves plotting viscosity vs shear rate at

a) 25˚C, b) 32˚C, and c) 40˚C. 48

Figure 16. DHA rheogram flow curves plotting shear stress vs shear rate at

a) 25˚C, b) 32˚C, and c) 40˚C. 49

Figure 17. OTS rheograms for lawsone at a) 25˚C, b) 32˚C, and c) 40˚C, G’ vs

Time(s) 51

Figure 18. OTS rheograms for DHA at a) 25˚C, b) 32˚C, and c) 40˚C, G’

vs Time (s) 52

Figure 19. Schematic of USP Apparatus 5 (Paddle Over Disk) Assembly 62

Figure 20. Picture representation of altered version of USP Apparatus 5

(sample and vessel) and schematic of sampling process/ analysis 63

Figure 21. Illustrated representation of multi-station Franz Diffusion cell system 64

Figure 22. Picture representation of Franz diffusion cell 65

xiii

Figure 23. The in vitro release profiles of a) lawsone and b) DHA multiple

emulsions in comparison with the combination lawsone/DHA

multiple emulsion 66

Figure 24. In vitro release profile from franz diffusion testing as a function

of the square root of time for a) lawsone and b) DHA multiple

emulsions in comparison with the combination of lawsone/DHA

multiple emulsion 69

xiv

LIST OF TABLES

Page number

Table 1. Table of volume percentages of chemicals used in formulation of

W1/O/W2 multiple emulsion and corresponding HLB values 19

Table 2. Parameters for OSS step for analysis of lawsone/ DHA emulsions 38

Table 3. Parameters for SF step for analysis of lawsone/DHA emulsions 39

Table 4. Parameters for OTS for analysis of lawsone/DHA emulsions 39

Table 5. Storage modulus (G’), loss modulus (G”), and tan δ of lawsone,

DHA, and combination lawsone/DHA at temperatures 25˚C,

32˚C, and 40˚C 47

Table 6. Summary of yield stress values and maximum viscosity values

at 25˚C, 32˚C, and 40˚C for lawsone, DHA, and combination

lawsone/ DHA emulsions 50

Table 7. Comparison of thickness, lipid content, and water evaporation

rate between human stratum corneum and shed snake skin 60

xv

LIST OF EQUATIONS

Equation 1. % Entrapment Efficiency 22

Equation 2. Stokes Law 31

Equation 3. Laplace Equation 33

Equation 4. Dampening factor (tan δ ) 35

Equation 5. Area of circle 37

Equation 6. Spreadability 37

Equation 7. Average cumulative amount of drug released (Q) 61

xvi

1

CHAPTER 1

Introduction

2

1.1.Topical applications of sunscreens

Sunscreens are protective agents applied to the skin to reduce the harmful effects of

solar radiation. Solar radiation produced by the sun can be categorized into ultraviolet

radiation (UVR) or visible light. The ultraviolet (UV) spectrum, which lies within

200nm to 400nm, can further be categorized according to its wavelength: UVA (320-

400nm), UVB (280-320nm), and UVC (200-280nm)1. UV radiation from the sun is

strongest between daylight hours of 10:00am to 4:00pm2. The UVR that causes damage

to the skin are mostly UVA and UVB. UVC is not considered as harmful for it is mostly

absorbed and filtered by the ozone layer and is only transmitted artificially through

germicidal or mercury lamps3. UVA and UVB are considered the most common

radiation that can pose a threat when overexposed. UVA delivers less energy to the skin,

therefore is not as considered as effective as UVB. UVA is mainly responsible for

producing suntan and not sunburn3,4

and is considered the cause of photoaging5. UVA

overexposure is more apparent because of its consistency throughout the day, reaching

the earth’s surface twenty times more than UVB3. Photoaging results in extracellular

matrix degradation and dysregulation of collagen metabolism5. The change in

extracellular matrix affects the elasticity of skin and consequently results in the formation

of wrinkles6. UVB, on the other hand, is considered to be the most erythrogenic and

melanogenic because it delivers such a high amount of energy to the stratum corneum

and superficial layers of the skin1,7

. UVB is the main cause of sunburn, suntan and skin

cancers3. In a study linking UVB with skin cancer, it was found that UVB energy was

directly absorbed by DNA, forming photoproducts which block replication and

transcription8. The resulting mutations specifically occur in the tumor suppressor gene

3

p53. P53 mutations have been linked to squamous cell carcinoma (SCC)9. Because of

the potential immediate (i.e. erythema) and long-term (i.e. photoaging,

immunosuppression, and carcinogenicity) harm that both UVA and UVB can cause, the

stress on application of sunscreens has become more apparent2.

The most common sunscreens on the market can be separated into two types: physical

and chemical sunscreens. Physical sunscreens are also referred to either inorganic or

reflectant sunscreens because they tend to use UV filters that act as a physical barrier that

can block transmission of UVR to the skin10

. Some of the most commonly used physical

sunscreens are those containing titanium dioxide, zinc oxide, kaolin, and ferric chloride.

Physical sunscreens are generally stable, but are sometimes cosmetically unappealing

because of their often thick and opaque consistency can be difficult to apply.11

Because

of its grainy texture, application can be sandy and quite messy. Despite their unappealing

texture, physical sunscreens tend to not be easily washed off, appealing to those who

have laborious jobs that cause them to sweat or for those who work in the water such as

lifeguards3. In order to provide superior UV protection and improve on cosmetic

appearance, UV filters like zinc oxide and titanium dioxide have been incorporated into

nanoparticle delivery systems12

. Toxicity and percutaneous penetration of the nano-

materials through the skin are still debated subjects of concern.

The second type of sunscreen available on the market is chemical sunscreens.

Chemical sunscreens are commonly referred to as organic or absorbent sunscreens

because they reduce the amount of UVR that reaches the stratum corneum by absorbing

the harmful radiation1. Chemical sunscreens have been mainly UVB absorbers. They

have shown to a selectively absorb the photons of sunburn-producing (UVB) radiation

4

while still allowing for the tanning reaction to occur because they don’t completely block

the transmission of UVA radiation1. Some of the common chemical sunscreen active

ingredients include: octocrylene, avobenzene, cinnamates, octinoxate, octisalate,

oxybenzone, homosalate, helioplex, mexoryl SX and XL, salicylates, benzophenomes,

and uvinul T150 and A Plus. Chemical sunscreens are colorless and more cosmetically

accepted, often found in moisturizers or mineral makeup. After application of chemical

sunscreens, irritation to the skin or eyes may occur. Chemicals such as salicylates,

cinnamates, anthranilates, and benzophenones tend to be surface agents and do not adhere

to the skin13

. Therefore, reapplication becomes necessary in order to provide adequate

and effective protection.

1.2.UV and Skin pigmentation

When it comes to sun protection, it is important to understand the basics of what

happens to the skin when exposed to UV radiation. UV exposure on the skin results in

two defense mechanisms produced from the skin. The skin first undergoes an epidermal

thickening and then increases in production of melanin, the polymer responsible for

pigmentation of the skin14

. In Figure below, melanocytes can be seen within the basal

layer of the epidermis of the skin. The melanocytes then transfer melanosomes,

organelles that contain melanin, to surrounding keratinocytes which would result in the

various tanned color of the skin after sun exposure15

Melanin has two types of chemical

compositions: eumelanin and pheomelanin. Eumelanin is a brown polymer and

pheomelanin is a yellow-reddish pigment. Whether eumelanin or pheomelanin

predominates is usually dependent on polygenic inheritance15

.

5

Figure 1. Anatomy of the skin showing the epidermis, dermis, and subcutaneous tissue;

melanocytes are found within the basal cells which are in the deepest part of the

epidermis16

Most Caucasians of European decent are thought to have less melanin content in

their skin, as seen in a high frequency of skin cancer occurring within that population.

On the other hand individuals with darkly pigmented skin are thought to have

significantly more epidermal melanin content, of which protects the skin by acting as free

radical scavengers, preventing from the incidence of cancer.17

When the skin is exposed to UV radiation, increased pigmentation from melanin

occurs in a three stages:

1. IPD- Immediate pigment darkening

2. PPD- Persistent pigment darkening

3. DT- Delayed tanning

6

IPD occurs within minutes after exposure to the sun. The skin appears grayish in color

and gradually fades to a brown over minutes or days, depending on the amount of UV

exposure and the skin color of the individual18

. IPD is not caused by the synthesis of new

melanin, but results from the photoxidation of preexisting melanin and the movement of

melanosomes to a peripheral dendritic location19

. The second phase is the PPD which

occurs after UV exposure and persists at least 3 to 5 days20,21

. This phase results in a tan

to brown color which is a result of oxidation of melanin and with exposure to UVA more

so than UVB20

. The final stage is the DT phase, which results from an increase of

melanocyte activity and new melanin formation in response to UVB8. The DT effect is

considered to be mildly photoprotective, with a sun protective factor (SPF) of about 318

.

According to Jimbow et al, melanin pigmentation can be associated with the following14

.

1. Resistance to sunburn, solar degradation, and skin cancer

2. Thermoregulation by enhancement of absorption of solar radiation

3. Regulation of vitamin D3 biosynthesis by influences on penetration of UV light

into skin

4. Protection of vital metabolites from photodestruction

Although melanin can aid in prevention of UV damage, there are still plenty of fair

complexioned people who lack the necessary amounts of melanin for its benefits.

Therefore, another viable option for UV protection is the use of keratin-bound

melanoidins. In this form of photoprotection, the application of dihydroxyacetone

(DHA) initiates a reaction called the Maillard reaction which results in the browning of

the skin from its combination with keratin amines (filaggrin). This reaction is followed

by the addition of lawsone, also known as henna, resulting in the production of brown

7

colored keratin-bound fluorescent polymers called melanoidins.17

Melanoidins produced

from DHA alone gives some photoprotection in the long UVA and Soret band of the

electromagnetic spectrum with minimal UVB protection17,22

. In a study by Fusaro et al,

it was shown that when lawsone is applied after the application of DHA, a different set of

melanoidins are produced, giving high UVB SPF protection and even greater UVA

protection than when DHA is used alone17,23

. The Maillard reaction and the properties of

Lawsone and DHA will be further explained in the following sections.

1.3. Maillard Reaction

The Maillard reaction was named after its founder Louis-Camille Maillard in France

in 1912. Maillard’s goal was to explain the reaction when amino acids react with sugars

at elevated temperatures during biological protein synthesis. Instead, his explanation was

used in food science to explain the browning process which occurs in food when being

cooked (i.e. in baked bread or barbequed meat). In 1953, John E Hodge, a chemist who

worked for the U.S. Department of Agriculture, took Maillard’s concepts and further

established a chemical mechanism of the reaction. Hodge broke down the reaction in the

following parts24

:

1. Initial Stage: (products colorless, without absorption in the ultraviolet (≈ 280

nm))

a. Reaction A: Sugar-amine condensation

b. Reaction B: Amadori rearrangement

2. Intermediate Stage: (products colorless or yellow, with strong absorption in

the ultraviolet)

8

a. Reaction C: Sugar Dehydration

b. Reaction D: Sugar fragmentation

c. Reaction E: Amino acid degradation (Strecker degradation)

3. Final Stage: (products highly colored)

a. Reaction F: Aldol Condensation

b. Reaction G: Aldehyde- amine condensation and formation of

heterocyclic nitrogen compounds

Hodge’s mechanism of explaining Maillard’s reaction showed how truly complex

the process of browning can be. To summarize, the Maillard reaction starts off with the

condensation reaction between a sugar and amine forming a Schiff base and liberating a

water molecule, which can be seen in the Figure below:

Figure 2. Generalized reaction forming a Schiff base25

The Schiff base then undergoes the Amadori rearrangement generating a

ketoamine product also known as a Heyns product. The Heyns product undergoes a

series of dehydration, degradation, and condensation reactions to form the final products

of the non-enzymatic browning called melanoidins. Melanoidins are different form the

melanin that is produced from enzymatic browning24

. Maillard’s reaction is not only

used in food chemistry, but also has implications in the field of medicine, especially in

the skin. For the past 50 years, dihydroxyacetone (DHA) has been used as sunless tanner.

9

Figure 3. Chemical structure of dihydroxyacetone 26

The corneocytes in the upper layers of the stratum corneum contain a large concentration

of amino acids that are produced from the degradation of filaggrin, the filament

associated protein which binds to the keratin fibers in epithelial cells27

. Filaggrin in the

stratum corneum is responsible for the skin barrier function28

. When DHA is applied to

the skin, the carbonyl group on the DHA (Figure 3) reacts with the amines in the keratin

layer, resulting in water and the Schiff base17

. Because the water content of the

epidermis increases the deeper it goes, the production of free radicals from this part of the

Maillard reaction ceases at the lower edge of the keratin layer as the law of mass action

reverses the equilibrium of the chemical reaction 17,27

. Therefore, the free radicals

produced by the Maillard reaction stops at the keratin layer of the skin. The Schiff base

and the intermediate products of the Maillard reaction progress to form non-enzymatic

covalent bonding into the skin protein, forming the keratin bound browning polymer

known as melanoidins17,29

. Like in food, melanoidins on the skin produces a brown tone

that stains the skin.

Figure 4. Chemical structure of lawsone30

10

Lawsone (Figure 4), the principle coloring agent of henna dye, also is said to produce

melanoidins on the skin. This can be especially evident after applying henna as a

temporary tattoo where the skin is designed and dyed with a brown stain. According to

Forestier et al, lawsone reacts with keratin through either the hydrosulfide group on

keratin or by forming a Schiff base by binding the keto-carbon on lawsone with the free

amine group on keratin31,32

. The formation of a Schiff base is said to be more likely to

occur, resembling the same type of reaction as DHA32

. The melanoidins produced by

DHA alone are said to give some photoprotection in the long UVA and Soret band of the

electromagnetic spectrum with minimal UVB protection. The melanoidins induced from

lawsone, on the other hand, are thought to produce a high UVB SPF protection and an

even greater UVA photoprotection than DHA17

.

1.4.DHA and Lawsone

DHA is used in cosmetic products to darken the skin chemically, providing sunless

tanning 33

. Store bought sunless tanners containing DHA are usually within the range of

3% to 5% in concentration, with professional products usually ranging between 5% and

15% depending on extent of tanning needed by the consumer. DHA based sunless

tanning has been recommended as a safer alternative to sun exposed tanning by the Skin

Cancer Foundation, the American Academy of Dermatology Association, the Canadian

Dermatology Association, and the American Medical Association34

. In the 1970s, DHA

was added to the United States Food and Drug Administration’s (FDA) list of approved

cosmetic ingredients. Studies by Faurschou et al demonstrated that the use of DHA

provides modest SPF in humans depending on the concentration used. DHA was shown

to shield against longwave UVA, visible (blue) light, and short wave UVB, delaying

11

photocarcinogenesis in hairless mice35,36

. In addition to its versatility and photoprotective

benefits, DHA binds covalently to the amines in the keratin layer, thus is not easily

washed off after application. Even after harsh physical activity, the melanoidins formed

from DHA will remain on the skin for 1 to 2 weeks or until the stratum corneum sloughs.

Henna is a member of the Lythraceae family that is cultivated in warm dry climates.

The dried leaves of the plant are usually crushed to a powder to produce the pigment

called lawsone37

. Lawsone has been used for centuries as hair dye or an expression of

traditional decorative body art in many Arab or Hindu cultures. Some consumer products

that currently contain henna include shampoos, conditioners, hair dyes, and body washes.

The safety of use for lawsone has always been a constant debate. The Scientific

Committee for Consumer Products (SCCP) of the European Union evaluated the use of

lawsone in hair dyes in 2002 and claimed that use of lawsone caused mutagenic and

clastogenic results in both in vitro and in vivo experiments. It was then concluded that

lawsone was not suitable for use as hair dye. Then in 2005, the SCCP concluded that the

mutagenicity data was insufficient to assess the safe usage of lawsone in hair dye. A

study by Kraeling et al initiated studies of lawsone related shampoos and pastes in order

to determine the extent of absorption when applied to human skin38

. In determining the

amount of lawsone that has possibly been absorbed, an important factor to analyze was

the amount of lawsone still remaining on the skin after diffusion studies were conducted.

Kraeling determined that the amount of lawsone still remaining was isolated within the

skin reservoir, or the stratum corneum, and was not available for systemic skin

absorption. Extended studies, spanning 48 and 72 hours, were also performed to verify

the lack of systemic absorption of lawsone. Although an increase of lawsone was seen

12

within the receptor fluid after the 72 hour time span for 3 out of 4 of the products, the

amount would still not be considered absorbed.

When lawsone and DHA are combined together forming the keratin-bound

melanoidin sunscreen, they work symbiotically to protect the skin from both UVA and

UVB regions. In the initial patent produced by Fusaro et al, the application of the

mixture of lawsone and DHA would be done separately39

. First the carbonyl compound

(DHA) would be applied to prepare the skin for reception of the subsequently applied

quinone (lawsone). Then there is a15 minute wait time before application of lawsone and

the two step application is repeated in order to enhance efficacy. In preliminary data

produced in by Fusaro et al in 1966, it was shown that the combination of lawsone and

DHA within the same formulation caused decomposition of the drugs within one to

several days when stored at temperatures of 25˚C or above23

. Combination of the two

drugs just prior to application was the most effective way in inducing the sunscreen filters

within the skin. Because of the melanoidin’s ability to combine to the keratin of the skin,

constant reapplication after every 3 hours or after sweating/ swimming is unnecessary

with lawsone/DHA sunscreens. Application of DHA followed by lawsone at bedtime

would suffice without any reapplication during the daytime.

1.5.Multiple Emulsion

In the current study, lawsone and DHA were both formulated into a multiple

emulsion water-in oil-in-water (W/O/W) system in an attempt to create a formulation

containing both drugs. A W/O/W system is composed of initial emulsion (W/O)

emulsified into an addition water (W) phase. According to DeLuca et al, a three phase

13

emulsion is recommended for the extended delivery of an active material because the

material would have to pass through two interfaces than an single interface of a two-

emulsion system40,41

. This was the intention when formulating a multiple emulsion for

lawsone and DHA. The various phases of the multiple emulsion would act as inherent

barrier between drugs, therefore protecting the drugs from possible decomposition as

seen from Fusaro et al. A benefit in formulating a three phase W/O/W emulsion is its

ability to create creams and lotions with the desired consistency of the external water

phase42

. A W/O/W emulsion also is able to enhance the solubility of compounds which

otherwise are only slightly soluble in hydrocarbons or water and the slow release of

active substance from the emulsion droplet40

. Because of the two water phases within the

system, another advantage includes the ability of placing two incompatible hydrophilic

materials in the same emulsion, one in the internal aqueous phase and the other in the

external phase40

. With these integral benefits of a multiple emulsion system in mind, a

formulation incorporating both lawsone, DHA, and combination of lawsone/DHA were

created and characterized in the following study.

1.6.Objective, hypothesis, and specific aims

The objective of this study was to formulate and characterize a multiple emulsion

for topical delivery of both lawsone and dihydroxyacetone and to evaluate its

compatibility under in vivo conditions. The underlying hypothesis of this investigation

was that a stable multiple W/O/W emulsion containing both lawsone and

dihydroxyacetone (DHA) can be prepared and characterized and can be effective as a

topical application. The specific aims were the following:

14

I. Development of multiple emulsion system incorporating both lawsone and

DHA

II. Characterization of the lawsone/ DHA multiple emulsion under various

conditions to determine its stability through time and temperature change

III. Evaluation of lawsone/ DHA multiple emulsion through in vitro studies to

verify compatibility under in vivo conditions

15

CHAPTER 2

Formulation of Multiple Emulsion System Incorporating Lawsone and DHA

16

2.1. Introduction

A multiple emulsion is a delivery system that incorporates both water-in-oil

(W/O) and oil-in-water (O/W) emulsions simultaneously. Multiple emulsions can also be

seen as a heterogeneous system of one immiscible liquid dispersed in another in the form

of droplets, which usually have diameters greater than 1µm43

. Figure 5 is an illustration

representing the phases of a multiple emulsion system.

Figure 5. Illustration of 3 phases of multiple emulsion droplet: internal water droplets

(W1) dispersed within the oil phase which is also dispersed into a continuous water (W2)

phase44

A major issue concerning multiple emulsions is the stability of the two

thermodynamically unstable interfaces (i.e. W/O interface of the primary emulsion and

O/W interface of multiple emulsion)45,46

. Therefore, when formulating a W1/O/W2

emulsion, it is necessary to utilize two emulsifiers within the formulation. When choosing

17

an emulsifier, it is necessary to consider its hydrophilic-lipophilic balance (HLB) value.

According to Griffin¸ a low HLB value is attributed to lipophilic surfactants, whereas

hydrophilic surfactants are thought to possess higher HLB values47

. In a study done by

Matsumoto et al, the emulsion process occurs in two steps48

. The first step is the

production of the primary W1/O emulsion, of which utilizes an emulsifier between the

range of 3 and 8. After proper homogenization of the primary emulsion, it is then added

to the secondary aqueous phase (W2), making up 40% of the final volume of the emulsion

while the remaining volume would be the continuous phase. A hydrophilic surfactant

within the range of 9 and 12 would then be incorporated into the external aqueous phase.

In addition to choosing the right hydrophilic and lipophilic emulsifiers, other

factors that are imperative to formulating a stable multiple emulsion are the type of

equipment used for mixing, the nature of the oil phase, and the phase volume ratio. In

mixing the primary emulsion, a homogenizer would allow the droplets to properly

disperse into the continuous phase. On the other hand, the second emulsification stage

would require gentler mixing, for high shear rates would cause the primary emulsion

droplets to rupture. Generally, the oil phase chosen for a multiple emulsion should be

nontoxic. The use of vegetable oils (soybean, sesame, peanut, etc.), refined hydrocarbons

(i.e. liquid paraffin, squalene, etc.) and mineral oils have often been used in multiple

emulsions. In comparison, mineral oil based multiple emulsions were said to form more

stable emulsions than those produced with vegetable oil49

. The phase volume is the ratio

of aqueous to oil phase which influence the stability of a multiple emulsion. The phase

volume of the primary emulsion doesn’t hold as much importance as the phase volume

ratio of the secondary phase50

. According to Matsumoto et al, stable multiple droplets

18

are produced at low volume fractions only48

. However, De Luca et al reported that stable

multiple emulsions can be obtained with a high volume ratio of 70% to 90% 41

.

The present study utilized all the aforementioned considerations to develop a

multiple W1/O/W2 emulsion incorporating lawsone, DHA, and combination of

lawsone/DHA. In this chapter, the formulation and development of the multiple

emulsions, as well as the drug entrapment of lawsone, DHA, and combination of the two

drugs will be addressed and analyzed.

2.2. Materials

Lawsone was obtained from Sigma Aldrich (St. Louis, MO). Dihydroxyacetone

was manufactured by Pfaltz & Bauer (Waterbury, Ct) and purchased through Fisher

Scientific (Pittsburg, PA). Sorbitan monooleate (Span 80) and polyoxyethylene sorbitan

monooleate 80 (Tween 80) were provided by Spectrum (New Brunswick, NJ). Mineral

oil was obtained from PCCA (Houston, TX). Coconut oil was provided by Carrington

Farms (Closter, NJ). Beeswax was obtained from Acros (New Jersey) and stearic acid

was obtained from Fisher Scientific (Pittsburg, PA). Soy lecithin was provided by a The

Herbarie (Prosperity, SC).

2.3. Methods

2.3.1. Formulation of water-in-oil-in-water multiple emulsion

A multiple emulsion was prepared using a two-step procedure as stated by

Matsumoto et al.48

The first step is the preparation of the primary (W1/O) emulsion. The

second step is obtained by dispersing 40% of the primary emulsion into the final aqueous

19

solution containing a hydrophilic emulsifier. The mass (g) of the each chemical in Table

1 was determined using their densities, which was then used to accurately weigh the

volume percentages of each phase.

Table 1. Table of volume percentages of chemicals used in formulation of W1/O/W2

multiple emulsion, and corresponding HLB values

% (w/w) Material HLB Value

Water (W1)

14.10 Phosphate Buffer pH

7.4/ Deionized Water -

0.035% / 1% Lawsone/ DHA -

Oil (O)

13.20 Mineral oil 10.5

6.00 Beeswax 12.0

4.32 Coconut oil 8.0

0.48 Stearic Acid 15.0

1.10 Soy Lecithin 4.0

0.80 Span 80 4.3

Water (W2) 0.25 Tween 80 15.0

59.75 Deionized Water -

Water (W1) and oil (O) phases were weighed in separate beakers and were heated

at the same time until both simultaneously reached 65˚C to 70˚C, or until the oil phase

has melted in the oil phase. While remaining heated at 70˚C, a hand homogenizer (Omni

TH) was used to homogenize the mixture as W1 phase was incorporated drop by drop

into the oil phase. The emulsion was kept under the highest speed of the homogenizer for

15 minutes. Meanwhile, the final water phase (W2) was weighed and heated on a

separate hot plate. After homogenization, 40% (≈40 g) W1/O was weighed and returned

to the hot plate to remain in liquid state for easy pouring (≈ 65˚C to 70˚C). Using an

overhead mechanical stirrer (Caframo RZR1) with a paddle attachment (speed setting 2),

W1/O was added drop by drop into the external aqueous phase (W2). Once incorporated,

20

temperature was decreased until hot plate was turned off. Vigorous mixing was

continued until water-in –oil-in –water (W1/O/W2) multiple emulsion achieved right

consistency and was fully cooled.

In the internal aqueous (W1) phase of the multiple emulsion, DHA was combined

with deionized water which was filtered using a 0.2µm filter. Lawsone multiple

emulsion’s internal aqueous phase (W1) was comprised of the lawsone mixed with

phosphate buffer pH 7.4 (PB). Deionized water in the laboratory was within the range of

pH 5 to 5.5, decreasing the solubility of lawsone. Therefore, a slightly more alkaline PB

was used in replacement of the deionized water. Combination of lawsone/DHA multiple

emulsion was comprised of a doubled concentration of lawsone (0.07% w/w) and DHA

(2% w/w) multiple emulsions combined in a 1:1 ratio (w/w) through trituration until

homogenous.

2.3.2. Verification of W1/O/W2 multiple emulsion

2.3.2.1 Light microscopy

In order to inspect the dispersion state of the inner W1/O emulsion containing

both lawsone and DHA, the W1/O/W2 emulsion was diluted (1:100) by weight with 0.2

µm filtered deionized water and sonicated in a bath sonicator for 5 minutes until

emulsion is fully in the continuous phase. The dispersion was placed onto a microscope

slide and visualized with light microscopy, using Leica DM2500M at 200x and 500x

settings. Photographs were taken to record results.

21

2.3.2.2 Fluorescence microscopy

To confirm the structure of the internal W1/O of the W1/O/W2 emulsion,

rhodamine was incorporated into the internal water (W1) phase. The oil (O) and

secondary water phase (W2) was prepared and incorporated as previously stated into the

multiple emulsion. The emulsion was diluted (1:100) by weight with 0.2 µm filtered

deionized water and sonicated in a bath sonicator for 5 minutes until emulsion was fully

in the continuous phase. The dispersion was placed onto a microscope slide and

visualized with fluorescence setting on Leica DM2500M. Photographs were taken to

record results.

2.3.3. % Entrapment Efficiency

Lawsone (0.035% w/w) was added into the internal water (W1) phase of a

multiple emulsion. DHA (1% w/w) was added to the internal water (W1) of a separate

emulsion. A combination of lawsone (0.07% w/w) and DHA (2% w/w) was prepared by

forming two separate W1/O/W2 emulsions and combining the two separate emulsions by

triturating both emulsions until homogenous in a 1:1 ratio (by weight). To determine

drug content, a biphasic liquid-liquid extraction was performed for all three emulsions

(lawsone, DHA, and combination lawsone/DHA). The emulsion (0.2g) was weighed in

an Eppendorff tube. At a ratio of 1:1, 1.5 mL of methylene chloride and sodium

phosphate buffer (pH 7.4) was added to Eppendorff tube. The samples were vortexed

(Vortex Genie 2) at speed 8 for approximately 10 seconds, until emulsion was in solution.

Samples were centrifuged (Accu-spin MicroR, Fisher Scientific) for 5 minutes at 13,000

rpm, until emulsion was separated into biphasic layers. Because both lawsone and DHA

22

are miscible in sodium phosphate pH 7.4, the aqueous layer was extracted and filtered

with 0.2 µm syringe filter. Samples were analyzed using UPLC method in the following

section. Entrapment efficiency was calculated using Equation 1:

% Entrapment Efficiency =DrugTotal−DrugFree

DrugTotal× 100 (1)

2.3.3.1. Chromatography

A Ultra Performance Liquid Chromatography (UPLC) method was performed on

the lawsone and DHA samples using a reversed phase Waters Acquity system (Waters,

Milford, MA) equipped with a quaternary solvent pump, auto sampler, and photodiode

array detector. The chromatographic separation of lawsone and DHA was obtained by

isocratic elution on a Waters Acquity BEH C18 (1.7 µm, 2.1 ×50 mm) column. The

mobile phase consisted of 0.2 µm filtered 0.1M acetic acid was mixed with methanol in a

ratio of 70:30 (v/v). Prior to usage on UPLC, 0.1M acetic acid was degassed in a bath

sonicator for 5 minutes. The flow rate was maintained at 0.5 mL/minute and the column

effluents were monitored at the detector wavelength range of (250 - 400 nm). The

injection volume was 10 µL, with a total run time of 6 minutes.

2.4. Calculations

The standard solution for lawsone (0.05% (w/v)) was prepared by dissolving

25mg of lawsone in 50 mL of sodium phosphate buffer pH 7.4. DHA (5% (w/v))

standard solution was prepared by dissolving 0.5g of DHA with 10 mL of sodium

phosphate buffer pH 7.4. A series of 15 serial dilutions were made for both lawsone and

DHA standard solution. The standard curve was obtained by plotting the peak height of

23

the standards to their corresponding concentrations. The unknown concentrations of

lawsone, DHA, and the combination of lawsone/DHA were determined by interpolating

from the regression equation relating to the peak height obtained from the standard curve.

2.5. Statistical data analysis

The statistical analysis of this experimental data for the purpose of comparison

was performed using a 2 tailed Student’s T-Test. Data was considered statistically

significant if p<0.05.

2.6 Results and Discussion

2.6.1. Fluorescence Microscopy

Figure 6. Fluorescence microscopy photographs (a and b) of W1/O/W2 emulsion

containing rhodamine, diluted 1:100 with deionized filtered 0.2 µm filtered water, and

magnified at 500x

In order to visualize and observe if the W/O/W formulation was able to

incorporate a hydrophilic drug within the internal (W1) phase without secretion into the

surrounding (O) oil and (W2) external aqueous phase, rhodamine was incorporated into

a)

) 0102030405060708090100

020406080100120140

% T

b)

24

the W1 phase of the multiple emulsion. Rhodamine is a water soluble fluorescent dye and

therefore is able to dissolve into the (W1) initial aqueous phase quite easily. After

completing the emulsion preparation process, the final emulsion was perceived to be a

deep pink color. To determine whether or not rhodamine was able to remain in the

internal (W1) phase, the emulsion was diluted 1:100 and dispersed into the continuous

phase (W2) with deionized water. By doing so, it was easier to observe the internal

(W1/O) phase. In Figure 6a and 6b, the rhodamine dye was able to fluoresce under the

fluorescent microscope, of which it can be discerned that the dye was able to remain

within the internal aqueous phase (W1) of the multiple emulsion. It can also be observed

that the rhodamine dispersion was shaped in a spherical form which seemed uniform in

size and structure. This also verifies that no other phase of the multiple emulsion, (O) oil

or (W2) external aqueous phase, contained any of the rhodamine dye. Figure 6a and 6b

is a visual confirmation of the feasibility of incorporating a hydrophilic drug into the

internal (W1) aqueous phase of a multiple W1/O/W2 emulsion.

2.6.2. Light Microscopy

The emulsion containing DHA which was used in Figure 7was diluted (1:100)

and dispersed into the continuous aqueous (W2) phase, aiding in visualizing the internal

(W1/O) phase. This can be observed in both Figure 7a and 7b. With light microscopy

and magnification at 500x, the internal W1 phase containing DHA can be seen within the

oil globules, more so observed in Figure 7a. Assuming that DHA was fully dispersed

into W1 phase during the formulation and preparation process, this can be regarded as a

confirmation that DHA was able to be incorporated into a W1/O/W2 multiple emulsion.

25

Figure 7. Light microscopy photographs (a and b) of W1/O/W2 emulsion containing

DHA, diluted 1:100 with deionized filtered 0.2 µm filtered water and magnified at 500x

The same can be said about the Figure 8a and 8b. Because lawsone is a pigment

and naturally colored, the internal W1/O phase can be clearly observed. In the

photographs above, the lawsone emulsion was also diluted (1:100) and dispersed into the

continuous aqueous (W2) phase, making it easier to envision the internal W1/O phase. In

Figure 8a, the internal W1/O particles can be seen to be far less than 100 µm,

encompassing a spherical, tinted internal (W1) phase within the oil (O) globules. In

Figure 8b, the dispersion was magnified to 500x. The internal (W1) phase can be

observed even clearer, also confirming that lawsone was fully dispersed only into the

internal phase and not into the other (O) oil or (W2) external aqueous phases. The light

microscopy analysis of lawsone at both 200x and 500x magnification validates and

confirms that lawsone was able to be incorporated into W1/O/W2 multiple emulsion.

a)

)

) 0102030405060708090100

020406080100120140

% T

b)

26

Figure 8. Light microscopy photographs of W1/O/W2 emulsion containing lawsone,

diluted 1:100 with deionized filtered 0.2 µm filtered water, and magnified at a) 200x and

b) 500x

2.6.3. % Entrapment Efficiency

Figure 9. Entrapment efficiency for lawsone, DHA, and combination lawsone/DHA at

25˚C, 32˚C, and 40˚C

a)

) 0102030405060708090100

020406080100120140

% T

b)

a)

) 0102030405060708090100

020406080100120140

% T

c)

) 0102030405060708090100

020406080100120140

% T

b)

) 0102030405060708090100

020406080100120140

% T

27

Figure 9 is a graphical representation of the % entrapment efficiency of lawsone,

DHA, and both the drugs in combination. The entrapment efficiency determines the

amount of hydrophilic drug that was entrapped within the internal water phase.

Therefore, the lesser amount of drug detected by the UPLC, the greater the entrapment

within the inner phase of the multiple emulsion. In Figure 9a, the amount of entrapped

lawsone, DHA, and combined drugs were calculated after the emulsions were kept at a

storage temperature of 25˚C. Although there were significant changes observed within

lawsone, DHA, and DHA in combination emulsions, the entrapment efficiency remained

within the range of ≈71.1% to 83%, respectively. The emulsion containing lawsone in

combination showed a significant increase from 93.38% on day 0 and to approximately

99% on day 7 through 28. An important observation to note is that all values remained

within a consistent range within this storage temperature, which means that no drug

leakage is occurring from internal phase. In Figure 9b, the storage temperature

increased to 32˚C. The lawsone emulsion showed a significant decrease starting on day

21 to about 78.65 ± 2.43% entrapment efficiency on day 28. A significant decrease in

entrapment was also observed in DHA and DHA in combination emulsions starting on

day 21, but still remained within a range of ≈ 76% to 65% throughout the full 28 day

duration. On the other hand, lawsone in combination emulsion showed a significant

increase from day 7 to 28, which is similar to the pattern observed at 25˚C where the

entrapment efficiency increased to ≈ 99%. The pattern of consistency observed at 32˚C

was similar to that observed at 25˚C. Figure 9c displays the entrapment efficiency for

the emulsions stored at 40˚C. At day 0, all emulsions started with drug entrapment

efficiency within the range of ≈ 69% to 78%. Lawsone emulsion remained consistent

28

with an entrapment of 69.83 ± 0.53% on day 0 to 70.64 ± 3.02% by day 28. In the

lawsone in combination emulsion, the same pattern as the other storage temperatures was

observed, showing a significant increase in entrapment from ≈ 78.45% at day 0 to

99.58% respectively by day 28. On the other hand, a significant decrease in entrapment

was observed for both DHA and DHA in combination. For the DHA only emulsion, a

significant decrease in entrapment was seen from 69.13% on day 0 to 37.41 ± 6.03 % by

day 28. The DHA in combination showed a significant decrease in entrapment starting

from day 14 through day 28, resulting in a final entrapment of 5.41 ± 3.82 %.

The decrease in entrapment efficiency is correlated to the increase of free drug

detected by the UPLC. A possible explanation for decrease in entrapment may be the

chemical instability of DHA. As reported by Fusaro et al, preliminary stability studies

with DHA and lawsone showed that stock solutions of both drugs kept separately at room

temperature or 4˚C showed more stability as when stored at higher temperatures or in

combination23

. It can be suggested that DHA’s chemical instability in an environment

with increased temperature might have been the reason for its possible diffusion out of

the inner phase to the continuous phase and increased detection of free drug in both DHA

and DHA in combination emulsion. Another possible explanation for the increased

detection of DHA in both emulsions is that the structure of the emulsion at 40˚C might

have been compromised, resulting in complete delivery of the drug into the external

aqueous phase which would cause more of the free drug to be detected. The structural

integrity of the emulsion can only be determined through further characterization, which

can be found in the following chapter.

29

It should be noted that the entrapment efficiency for all emulsions were calculated

using UPLC analysis of drug content by biphasic extraction. After conducting the

extraction process using methylene chloride and phosphate buffer, centrifugation, and

removal of the aqueous phase, it was determined that not all of the hydrophilic drugs

(lawsone and DHA) were fully removed. Instead, the foam layer after centrifugation

housed the residual drugs. Therefore, in order to achieve 100% entrapment efficiency at

day 0, the samples needed a second extraction to remove any remaining drug. It can be

noted that the values presented were an underrepresentation of the true % entrapment

efficiency of the emulsion samples.

2.7 Conclusion

Using light microscopy and fluorescence microscopy, it was visually verified that

a multiple emulsion formulation was successfully produced. By conducting the

entrapment efficiency, the presence of both lawsone, DHA, and a combination mixture of

both drugs was validated. Stability studies and characterization of the multiple emulsion

system were conducted, after storage at 25˚C, 32˚C, and 40˚C, in the following chapter

for further validation.

30

CHAPTER 3

Characterization of Multiple Emulsion Incorporating Lawsone and DHA

31

3.1 Introduction

The stability of pharmaceutical emulsions is characterized by the absence of

coalescence of the internal phases, absence of creaming, and maintenance of elegance

with respect to appearance, odor, color, and other physical properties51

. According to

Martin, problems that may occur in formulating emulsions can be the following51

:

(1) Flocculation and creaming

(2) Coalescence and breaking

(3) Miscellaneous physical and chemical changes

(4) Phase inversion

Flocculation occurs when an attractive force between the internal droplets brings

them together, forming an aggregation of droplets, or clusters, without any increase in

droplet size. Monitoring the electrostatic repulsion of the internal droplet surface

by measuring the zeta potential can therefore help determine if flocculation may be

present. Hence, the greater the electrostatic charges between droplets, the less

likely flocculation would occur.

Creaming is a property related to the Stokes law:

ν =d2(ρs−ρ0)g

18η (2)

In Equation 2, the rate of creaming (𝜈) is proportional to the diameter (d) of the

droplet in cm, the densities of both the dispersed phase (𝜌s) and continuous phase

(𝜌o), the acceleration due to gravity (g), and inversely proportional to the viscosity

of the dispersion medium (𝜂). In a multiple emulsion, the dispersed phase is the

32

internal W1/O phase, which would be relatively lighter in weight, due to the

presence of oils, than the external (W2) water phase. Therefore, an upward or a

negative rate of creaming would be observed in an unstable multiple W/O/W

emulsion. From the Stokes law in Equation 3, the rate of creaming is shown to be

dependent upon the droplet or globule size. By doubling the globule size, the rate of

creaming can increase by a factor of 451

. Also droplet size measurements are a good

indicator of the formulation’s stability. A fast droplet size increase indicates low system

stability52

.

According to Ficheux et al, multiple emulsions instability usually follows two

possible mechanisms 53,54:

Coalescence of the small inner droplets with the globule interface

Coalescence of the inner droplets within the globule

Coalescence usually occurs when the particles of the dispersed phase comes

together to form larger particles40. As larger particles begin to accumulate, breaking

or phase separation usually occurs. Disproportionation, also known as Ostwald

ripening, is a process dependent on the diffusion of disperse phase molecules, or the

chemical components, from smaller to larger droplets to the continuous phase55. It

is a result of the pressure inside a droplet being higher than the pressure outside the

droplet40. The pressure of dispersed material is greater for smaller droplets as

shown by the Laplace equation (Equation 3), where P is the Laplace pressures, 𝛾 is

the surface tension, and r is the droplet radius. Consequently, monitoring the particle

33

size of the internal W/O droplets of a multiple emulsion can help the formulators

determine the stability of the formulation over a period of time.

𝑃 = 2γ

r⁄ (3)

Chemical reactions within the emulsion samples may also occur after being stored

at various temperatures and with an elongated time span. Chemical reactions that may

occur within the emulsion can be relatively determined by monitoring its pH during its

storage time range. By doing so, the emulsion’s stability and any potential chemical

reactions that may compromise the quality of the product could be determined. The pH of

the skin has always been thought to be around pH 5.5 to 6. According to Lambers et al,

the pH of healthy skin is actually closer to 4.7, preserving any resident bacterial flora 56

.

Therefore, by monitoring the pH, it can be determined whether the emulsion is

compatible with the skin or may cause any irritation. Any changes in batches and the

quality of emulsion could also be checked by detecting any variations in pH.

Phase inversion is another obstacle that may occur when formulating an emulsion.

Phase inversion involves the change of emulsion type from O/W to W/O or vice versa51

.

In the phase inversion phenomenon, the phase-volume ratio of the emulsion plays an

important part in its stability. Depending on what type of emulsion (W/O or O/W), the

phase-volume ratio is a term that refers to the volume of water and oil that is added to

emulsion. Theoretically, from the study done by Ostwald and Kolloid, the phase-volume

ratio should not exceed 74% of the total volume of the emulsion. Ostwald and Kolloid

showed that an increase in the phase-volume ratio of oil to water in an O/W emulsion to

above 74% resulted in the oil globules to coalesce and break the emulsion. This limit

34

reached is called the critical point, which can be defined as the concentration of the

internal phase above which the emulsifying agent cannot produce a stable emulsion51

.

When the volume concentration reaches the 74% limit, a change in viscosity

occurs within the emulsion. Viscosity and flow properties of the formulation may be

monitored and assessed through spreadability testing and rheological analysis of the

product. Characterizing the spreadability of the emulsion is important in order to

determine if an even and efficacious dose is being delivered to the target site.

Spreadability, in principle, is related to the contact angle of the drop of the liquid or a

semisolid preparation on a standardized substrate and is a measure of the lubricity, which

is directly related to the coefficient of friction57,58

. Related to spreadability, other factors

such as viscosity, elasticity, and rheology are also important to consider when creating a

consistent formulation. Rheology is the science that studies how materials deform and

flow under the influence of external forces59

. Characterizing the rheological properties of

a system is not only important in the design and application, but also during its

processing and to ensure a long shelf life59

. By undergoing rheological analysis, one can

determine the changes that the formulation experiences when subjected to external forces,

measure the deformation and flow of the system, and learn how to improve the

application properties of the product. In order to fully assess the rheological properties of

the sample after application of stress, the following test could be used:

(1) Oscillatory Stress Sweep

(2) Stepped Flow

(3) Oscillatory Time Sweep

(4) Creep and Recovery

35

Oscillatory stress sweep measures the viscoelastic properties of a material. As

stress is applied during the amplitude sweep, the linear viscoelastic region (LVR) is

determined. The LVR is a measure of the inherent strength of the structure of the

emulsion40

. The storage/elastic modulus (G’) is a measure of the extent of the elastic

component (i.e. crosslinking, aggregation, etc) within the emulsion. The loss modulus

(G”) measures the extent of the viscous component of the emulsion. The strength of the

interaction in the formulation would be calculated by the ratio of G’ and G” (Equation 4),

resulting in the dampening factor (tan δ), where δ is the phase angle. The smaller the

value of tan δ, the more pronounced the elastic character of the formulation.

G"/G′ = tan δ (4)

In the experiments performed, the G’ is plotted as a function of oscillatory stress,

which measures and determines the product’s ability to lose its elasticity after applied

stress. Therefore, the more defined and longer the LVR, the product would be considered

to have retained its elasticity longer. The experimental parameters used on the ARG2

Rheometer for the following experiments are listed in Table 2.

Stepped flow (SF) is used to determine the type of flow (Newtonian or non-Newtonian)

and the viscosity profile of the formulation. Viscosity (Pa/s) is determined solely by the

relationship of two parameters, the shear stress (Pa) and the shear rate (1/s)40

. By

measuring the shear stress and shear rate, an understanding of whether force or

displacement is responsible for the flow of the formulation is determined. The parameters

used in the following experiments for SF can be seen in Table 3.

36

Oscillatory time sweep (OTS) is important when testing materials such as dispersions and

polymers, which may undergo macro-micro structural rearrangements with time60

. OTS

directly provides the necessary information about how a material changes with time, by

plotting the G’ as a function of time when the applied stress is held constant. Thus, any

changes to the structure (i.e. chemical structure, chemical reaction, change in

temperature, or curing) can be determined through OTS40

. The parameters used for

analysis of the emulsion samples can be seen in Table 4.

3.2 Methods

3.2.1. Particle Size and Zeta Potential Analysis

The particle size (PS) and zeta potential (ZP) of the formulation were determined

using the zetameter (ZetaPlus, Brookhaven Instruments Corporation, Holtsville, NY).

The PS and ZP of lawsone, DHA, and the combination lawsone/DHA W1/O/W2

emulsions were determined at various temperatures and time points by diluting the

emulsion sample by a ratio of 1:100 with filtered 0.2µm deionized water within a

scintillation vial. The solution was sonicated in a bath sonicator for 5 minutes and shaken

before use. The PS measurements were measured in a series of 10 readings, reporting the

effective diameter (nm) of the particle. The ZP measurements were also measured in a

series of 10 measurements, reporting the mean charge (mV). PS and ZP were of all three

emulsions were taken in triplicate weekly for a total of 28 days at 25˚C, 32˚C, and 40˚C.

3.2.2. Determination of pH

The pH of lawsone, DHA, and combination lawsone/DHA emulsions were

obtained by using Fisher Scientific ™ Accumet™ Basic AB 15 with a glass body

37

combination double junction electrode. The pH was obtained by dipping the electrode in

each emulsion, recording the pH in triplicate weekly for a total of 28 days at 25˚C, 32˚C,

and 40˚C.

3.2.3. Spreadability

Spreadability of each lawsone, DHA, and combination lawsone/DHA was

determined through the parallel plate method. The emulsion was first placed within a 1

cm diameter circle within the center of a glass plate (75 × 50mm) and weighed (g). A

second glass plate was placed on top of the first glass plate, with the addition of a 500 g

weight for a total of 15 seconds. The final diameter was determined (cm) on an x, y, and

z axis. The average diameter (cm) is used to determine the area of the circle by using the

following equation:

𝐴 = 𝜋𝑟2 (cm²) (5)

Spreadability was then calculated using Equation 7:

S500 =A (cm2)

Weight of Emulsion (g) (6)

Spreadability was performed in triplicate weekly for a total of 28 days at 25˚C, 32˚C, and

40˚C

3.2.4 Rheology

Rheology was conducted using ARG2 Rheometer (TA Instruments, LTD, New

Castle, DE). Geometry attachment for rheometer was chosen to be 40mm, 2˚degree

stainless steel cone due to its common use with thicker dispersions such as emulsions or

38

gels. Prior to starting rheological analysis, calibration of inertia and bearing friction was

conducted to determine if rheometer was working at optimal function. Once the

geometry was attached, the gap was zeroed and then raised for application of sample.

The emulsion sample (≈1 to 2 g) was added onto the center of the Peltier plate and

lowered. Excess emulsion was strategically cleaned off at a 90˚ angle so a straight edge

was obtained. The geometry was then lowered further to the zero gap distance (53).

Temperature was sustained at 25˚C. Protocols for oscillatory stress sweep (OSS),

stepped flow test (SF), and oscillating time sweep (OTS) were obtained from TA

Instruments (New Castle, DE) for determination and analysis of rheologically unknown

material. Rheograms for OSS was viewed as elastic modulus G’ (Pa) vs oscillation stress

(Pa). Rheograms for SF was viewed both as viscosity (Pa.s) vs shear stress (Pa) and

shear rate (1/s) vs shear stress (Pa). Rheograms for OTS was viewed as G’ (Pa) vs time

(s). Protocols for each test were as follows:

Table 2. Parameters for OSS step for analysis of lawsone/ DHA emulsions

Oscillatory Stress Sweep

Conditioning Step

Temperature 25˚C

Equilibration time 8 minutes

Deformation Step

Stress Sweep Broad torque range (1-10,000 mN.m)

Frequency 1 Hz

Points per decade 10

Mode Log

39

Table 3. Parameters for SF step for analysis of lawsone/DHA emulsions

Stepped Flow Step

Conditioning Step

Temperature 25˚C

Equilibration time 8 minutes

Stepped Flow Step

Stress Broad torque range (1-10,000 mN.m)

Points per decade 5

Mode Log

Constant time 10 seconds, average last 5 seconds

Table 4. Parameters for OTS for analysis of lawsone/DHA emulsions

Oscillatory Time Sweep

Conditioning Step 1

Temperature 25˚C

Equilibration time 3 minutes

Conditioning Step 2

Pre-shear Value of shear rate when viscosity is

reduced in SF step

Equilibration time 0

Time Sweep Step

Time duration 15 minutes

Frequency 1 Hz

Control Variable 0.5968 Pa

Sampling time 5 seconds

3.2.5. Statistical data analysis

The statistical analysis of this experimental data for the purpose of comparison

was performed using a 2 tailed Student’s T-Test. Data was considered statistically

significant if p<0.05.

3.3 Results

3.3.1. Particle Size and Zeta Potential

40

Figure 10. Particle size analysis for lawsone, DHA, and the combination of

lawsone/DHA emulsions at a)25˚, b)32˚C, and c)40˚C after 28 days

Lawsone, DHA, and combination emulsions were prepared fresh before placing

into the stability chamber at each temperature. By diluting the emulsions with water at a

ratio of 1:100, the internal W1/O droplet was dispersed into the continuous phase of the

multiple emulsion, which was measured by the ZetaPlus particle analyzer. In Figure 10a

and 10c, no significant changes in particle size was seen between all three emulsions at

temperatures of 25˚C and 40˚C. A significant increase in particle size was observed in

the combination lawsone/DHA emulsion (Figure 10b) by day 21 at 32˚C, where the

particle size enlarged from 414.7 ± 13.5nm to 475.97 ± 43nm. Despite the significant

increase at that point, the overall particle size of all emulsions, despite change in

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41

temperature, remained within a diameter size of between 300 nm and 500 nm

respectively.

Figure 11. Zeta potential for lawsone, DHA, and the combination of lawsone/DHA

emulsions at a)25˚C, b)32˚C, and c)40˚C after 28 days

After dispersion into the continuous phase, the zeta potential of the internal W/O

droplets can determine the potential stability of the colloidal system61

. By having an

overly negative or positive zeta potential, the particles will tend to repel each with no

tendency for aggregation or flocculation, remaining generally stable61

. In Figure11a

below, no significant differences were seen from each emulsion after 28 days at 25˚C. At

32˚C (Figure 11b), however, there was a significant increase in zeta potential for DHA

after 28 days. The most significant changes, on the other hand, occurred at 40˚C (Figure

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42

11c) for DHA at 14 and 28 days respectively, where the zeta potential showed an increase

from -20.83 ± 0.99 mV to -14.93 ± 0.97 mV. Combination lawsone /DHA had an

increase in zeta potential to -19.3 mV by day 28. In the lawsone emulsion, an increase

was observed after 7 and 14 days, from -33.7 ±3.10 mV on day 0 to -25.4 ± 0.87 mV by

day 14, but returned back to baseline by day 21. Overall, variations in zeta potential for

all emulsions were seen as the temperature increased to 40˚C, showing relative instability

3.3.2. Spreadability

Spreadability was obtained by measuring the displacement in diameter of the

emulsion after application of a 500g weight when placed between two glass slides. The

area of resulting circular spread (cm²) was then measured and divided by the initial

weight of emulsion used (g). The measured spreadability of each emulsion varied with

temperature and time. At 25˚C (Figure 12a), lawsone exhibited a significant increase in

spreadability after 21 days to 21.6 ± 1.1 cm²/g, and combination lawsone/DHA showed

an increase at day 7 and 28 to about 24.1 ± 1.77 cm²/g. Combination lawsone/DHA also

showed an increase at 21 days at 32˚C (Figure 12b) to a spreadability of 29.5 ± 0.6

cm²/g. In Figure 12c, DHA showed a significant increase in spreadability at day 7 to

about 34.0 ± 1.1 cm²/g, and lawsone showed a significant decrease in spreadabiltiy at day

21, but both returned to baseline. In summary, no obvious spreadability patterns within

the 28 days were observed at each temperature. Although variance in results occurred,

the spreadability values remained within the range of ≈ 20 cm²/g to35cm²/g.

43

Figure 12. Spreadability (cm²/g) for lawsone, DHA, and the combination lawsone/DHA

emulsions at a) 25˚C, b) 32˚C, and c) 40˚C after 28 days

3.3.3. pH

The pH values of the multiple emulsions kept at different storage conditions

(25˚C, 32˚C, and 40˚C) are shown below in Figure 13a, 13b, and 13c. Lawsone and

DHA emulsions were separately prepared, as well as a combination containing both

drugs. They were all stored separately and kept at the various temperatures. For the

freshly prepared lawsone and combination lawsone/DHA emulsions, the pH value ranged

between 5 and 6 respectively. DHA emulsions that were freshly prepared were roughly

within the range of pH of 4 and 5. Values of pH at time zero were used as the standard

and significance level was calculated using Student’s T-Test (p < 0.05). Although the

lawsone emulsion showed a significant decrease at day 7 to pH 5.85 ± 0.12 from 6.27 ±

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0.18 at 25˚C, it remained within a range of its original value of ≈ 6.27 after 28 days. A

significant increase was observed in DHA within days 14 to 28, from a pH of 4.41 ± 0.33

at time zero to a pH of 5.15 ± 0.05. Combination lawsone/DHA showed a fluctuating

increase and decrease within the 28 day time span, with a final pH of ≈ 5.1.

Figure 13. pH readings for lawsone, DHA, and combination lawsone/DHA emulsions at

a)25˚C, b)32˚C, and c)40˚C after 28 days

At 32C, changes in pH were observed for the lawsone emulsion, with a significant

increase to on day 28 to pH ≈ 6.05 after 28 days in the stability chamber. DHA emulsion,

with a pH of 6.03 ± 0.18 after being freshly prepared, showed a significant decrease in

pH weekly throughout the 28 day time span to a final pH of 4.92 ±0.08. The same

pattern was seen with combination lawsone/DHA. At the start of the stability test at time

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45

zero, the emulsion was measured to be a pH of 5.21 ± 0.10, but throughout the 28 days a

significant drop in pH to an average of ≈ 3.88 ensued. This suggests that some chemical

degradation is happening in DHA and more so when both lawsone and DHA is

combined.

Finally at 40C, lawsone emulsions exhibited a significant decrease in pH from

6.83 ± 0.11 to 5.85 ± 0.04 after 28 days. The same pattern was also obvious in the

combination lawsone/DHA where a significant drop in pH from 6.64 ± 0.22 to 3.71± 0.25

occurred. DHA emulsion remained consistent and without any significant changes until

day 28, when the average pH dropped from ≈ 5 to 4.15. The change in pH levels as

temperature increase suggests chemical degradation of the drugs and/or instability of

chemical components of the emulsion.

3.3.4. Rheology

Linear viscoelastic region (LVR) was determined by conducting the oscillatory

stress sweep, which graphed the (elastic/storage modulus) G’ versus the oscillatory stress

(Pa) that was applied to the sample emulsions. Figures 14a, 14b, and 14c below are

examples of rheograms of weekly stability analysis of DHA at 25˚C, 32˚C, and 40˚C.

The LVR region is a measure of the inherent strength of the structure of the emulsion40

.

The elasticity (G’) of the emulsion samples was measured as a function of increasing

stress, until a point at the end of the LVR. By continuing the oscillatory stress applied on

the sample, the structure eventually becomes destroyed and therefore a downward curve

was observed. In Figure 14a and 14b, the LVR for DHA remained consistent at both

25˚C and 32˚C, which can be validated in Table 6 below. The G’ values showed

46

minimal decrease within the 28 day span throughout both temperatures. As the

temperature increased to 40˚C, the G’ values decreased significantly within each week

from 1516 Pa at day 0 to 497.8 Pa by day 28. Also in Figure 14c, the LVR for DHA

emulsion gradually becomes shorter until it underwent a quick decline at day 28. This

pattern suggests that the elasticity of the emulsion decreased, showing a decline in

structure with the increase of temperature. Lawsone and combination lawsone/DHA

emulsions also exemplified the same decline in elasticity at 40˚C. The resulting graphs

could be viewed in the Appendix.

Figure 14. DHA rheograms for OSS of G’ vs oscillatory stress at a) 25˚C, b)32˚C, and

c)40˚C

0 100.0 200.0 300.0 400.0 500.0 600.0

osc. stress (Pa)

0.1000

1.000

10.00

100.0

1000

10000

G'

(P

a)

Osc Stress Sweep DHA 25C OSS DHA DAY 0

OSS DHA DAY 7

OSS DHA DAY 14

OSS DHA DAY 21

OSS DHA DAY 28

0 100.0 200.0 300.0 400.0 500.0 600.0

osc. stress (Pa)

0.1000

1.000

10.00

100.0

1000

10000G

' (

Pa

)

OSS DHA DAY 0

OSS DHA DAY 7

OSS DHA DAY 14

OSS DHA DAY 21

OSS DHA DAY 28

Osc Stress Sweep DHA 32C

0 100.0 200.0 300.0 400.0 500.0 600.0

osc. stress (Pa)

1.000E-3

0.01000

0.1000

1.000

10.00

100.0

1000

10000

G'

(P

a)

OSS DHA DAY 0

OSS DHA DAY 7

OSS DHA DAY 14

OSS DHA DAY 21

OSS DHA DAY 28

Osc Stress Sweep DHA 40C

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LVR

LVR

LVR

47

Table 5 is a summary of results of OSS in terms of tan δ. Tan δ is the value that

measures the overall strength and interaction of the emulsion. Generally, the smaller

value of tan δ would suggest that the emulsion would exhibit more of an elastic character.

The values of G’, G” and tan δ which are represented were taken at time zero and day 28.

The values of tan δ at day 28 that are highlighted in orange displayed a decrease in value,

signifying an increase in elasticity which might have occurred from possible swelling.

The tan δ values at day 28 that are highlighted in yellow showed an opposite increase in

the value, signifying a decrease in elasticity of the emulsion. This may have occurred

from possible expulsion of the internal water droplets into the continuous medium after

swelling occurred or after having applied stress onto the samples during the oscillatory

stress sweep.

Table 5. Storage modulus (G’), loss modulus (G”), and tan δ of lawsone, DHA, and

combination lawsone/DHA at temperatures 25˚C, 32˚C, and 40˚C

Lawsone DHA Combination

Law/DHA

˚C Day G’ G” Tan δ G’ G” Tan δ G’ G” Tan δ

25 0 2060 157.8 0.077 1447 164.4 0.114 2040 104.6 0.051

28 1576 186.7 0.118 1311 60.3 0.046 1107 166.9 0.151

32 0 3510 707 0.201 1516 175.9 0.116 1905 97.4 0.051

28 1878 212.1 0.113 1408 157.8 0.112 1245 127.7 0.102

40 0 3510 707 0.201 1516 175.9 0.116 1905 97.4 0.051

28 2408 307.1 0.127 497.8 91.6 0.184 1437 208.8 0.145

During the SF tests, all emulsions were evaluated by two sets of rheograms. One

set of rheograms was presented as viscosity vs shear rate, and the other presented as shear

stress vs shear rate. In Figures 15a, 15b and 15c below, the non-Newtonian, shear

thinning behavior, or pseudoplastic flow, of the DHA emulsion can be observed. As

48

stress rates increased, viscosity of the emulsions decreased almost instantaneously. The

yield stress, or the minimum shear stress required to initiate flow, was also determined.

By the presence of yield stress values and its shear thinning behavior, it can be suggested

that the emulsion can be considered viscoelastic, containing both viscous properties of a

liquid and elastic properties of a solid. Viscoelasticity is especially common for cosmetic

semisolids such as lotions and creams.

Figure 15. DHA rheogram of flow curves plotting viscosity vs shear rate at a) 25˚C, b)

32˚C, and c) 40˚C.

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49

Figure 16. DHA rheogram flow curves plotting shear stress vs shear rate at a)25˚C,

b)32˚C, and c) 40˚C.

The shear stress (Pa) vs shear rate (1/s) curves, such as those found in Figure

16a,16b, and 16c above, was used to determine the inherent flow of the formulation. By

increasing the shear rate (i.e. the consumer started to rub onto the skin), the DHA

emulsion’s shear stress gradually increased as well, meaning minimal structural loss was

seen at 25˚C and 32˚C. The viscosity remained relatively stable even during stress. On

the other hand, at 40˚C, a decrease in viscosity was observed throughout each week until

day 28. The increase in temperature could have resulted in structural breakdown as the

weeks passed until it reached its lowest viscosity at 406.4Pa.s on day 28. In Table 6

below, a decrease in viscosity was observed for lawsone emulsions stored at higher

temperatures of 32˚C and 40˚C, which may be attributed to structural breakdown or phase

0 25.00 50.00 75.00 100.0 125.0 150.0 175.0 200.0 225.0

shear rate (1/s)

0

100.0

200.0

300.0

400.0

500.0

600.0

sh

ea

r s

tr

es

s (

Pa

)

Flow DHA 25C Stepped Flow DHA DAY 0

Stepped Flow DHA DAY 7

Stepped Flow DHA DAY 14

Stepped Flow DHA DAY 21

Stepped Flow DHA DAY 28

0 25.00 50.00 75.00 100.0 125.0 150.0 175.0 200.0 225.0

shear rate (1/s)

0

100.0

200.0

300.0

400.0

500.0

600.0

sh

ea

r s

tr

es

s (

Pa

)

Flow DHA 32C Stepped Flow DHA DAY 0

Stepped Flow DHA DAY 7

Stepped Flow DHA DAY 14

Stepped Flow DHA DAY 21

Stepped Flow DHA DAY 28

0 25.00 50.00 75.00 100.0 125.0 150.0 175.0 200.0

shear rate (1/s)

0

100.0

200.0

300.0

400.0

500.0

600.0

sh

ea

r s

tr

es

s (

Pa

)

Flow DHA 40C Stepped Flow DHA DAY 0

Stepped Flow DHA DAY 7

Stepped Flow DHA DAY 14

Stepped Flow DHA DAY 21

Stepped Flow DHA DAY 28

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separation may have occurred after 28 days. For combination lawsone/DHA an increase

in viscosity was detected. This may suggest swelling may have occurred within the

emulsion, increasing the rigidity of the second interface by progressive migration of the

lipophilic surfactant, which corroborated with the data observed during the OSS testing

for the same samples62

.

Table 6. Summary of yield stress values and maximum viscosity values at 25˚C, 32˚C,

and 40˚C for lawsone, DHA, and combination lawsone/ DHA emulsions

Lawsone DHA Lawsone/DHA

˚C Day

Yield

Stress

(Pa)

Viscosity

(Pa.s)

Yield

Stress

(Pa)

Viscosity

(Pa.s)

Yield

Stress

(Pa)

Viscosity

(Pa.s)

25 0 37.7 381.2 94.6 373.2 59.7 622.9

28 37.7 1353.0 94.6 298.1 37.7 2839.0

32 0 59.7 3111.0 37.7 2946.0 59.7 872.2

28 37.7 496.2 37.7 3061.0 37.7 3138.0

40 0 59.7 3111.0 37.7 2946.0 59.7 872.2

28 94.6 552.7 37.7 406.4 37.7 4272.0

The OTS rheograms for lawsone and DHA can be seen below in Figures 17a,

17b, and 17c. In the following rheograms, G’, or the elasticity modulus, is plotted along

with time in order to determine the extent of elasticity change after 15 minutes at each

weekly time point at 25˚C, 32˚C, and 40˚C. The rheogram for lawsone showed a

decrease in elasticity (G’) on day 28 at 25˚C and day 14 at 40˚C. The decrease in

elasticity may have resulted in a change or a loss of structure at that time point, which

may be due to a number of reasons such as a chemical reaction or just a change in

temperature. In Figure 18a and 18b, the G’ for DHA emulsion remained consistent at

both 25˚C and 32˚C storage temperatures, showing a minimal change in G’ within the 28

51

days span. In Figure 18c, there was an observed variance in G’ values at 40˚C, more

specifically a drastic decrease was detected on day 28. A change in elasticity and

structure for DHA may have been due to an increase in temperature for a prolonged

period of time, which is also consistent with the results obtained during the OSS tests

above. The results from OTS tests for combination lawsone/DHA showed minimal

changed throughout the 28 day span at all 3 temperatures, of which can be seen in the

Appendix.

Figure 17. OTS rheograms for lawsone at a) 25˚C, b) 32˚C, and c) 40˚C, G’ vs Time (s)

0 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.0

time (s)

100.0

1000

10000

G'

(P

a)

Osc Time Sweep LAWSONE 25C OTS LAW DAY 0

OTS LAW DAY 7

OTS LAW DAY 14

OTS LAW DAY 21

OTS LAW DAY 28

0 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.0

time (s)

100.0

1000

10000

G'

(P

a)

Osc Time Sweep LAW at 32C OTS LAW DAY 0 32C

OTS LAW DAY 7 32C

OTS LAW DAY 14 32C

OTS LAW DAY 21 32C

OTS LAW DAY 28 32C

0 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.0

time (s)

100.0

1000

10000

G'

(P

a)

Osc Time Sweep LAWSONE 40C OTS LAW DAY 0

OTS LAW DAY 7

OTS LAW DAY 14

OTS LAW DAY 21

OTS LAW DAY 28

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52

Figure 18. OTS rheograms for DHA at a)25˚C, b)32˚C, and c) 40˚C, G’ vs Time (s)

3.4 Discussion

Measuring the zeta potential of the internal droplets of a multiple emulsion is a

possible way of determining the attractive forces that may cause aggregation or

flocculation to occur. During flocculation, the droplets tend to aggregate without the

potential increase in droplet size. Zeta potential results showed an increase in zeta

potential for DHA at day 28. Despite that observation, lawsone, DHA, and combination

lawsone/DHA emulsions exhibited overall stability at 25˚C and 32˚C. At 40˚C, all

emulsions showed varying values of zeta potential. Significant increases in zeta potential

for all emulsions were observed. Therefore, the electrostatic repulsion of the internal

0 943.32time (s)

100.0

1000

10000

G'

(P

a)

Osc Time Sweep DHA 25C OTS DHA DAY 0

OTS DHA DAY 7

OTS DHA DAY 14

OTS DHA DAY 21

OTS DHA DAY 28

0 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.0

time (s)

100.0

1000

10000

G'

(P

a)

Osc Time Sweep DHA 32C OTS DHA DAY 0

OTS DHA DAY 7

OTS DHA DAY14

OTS DHA DAY 21

OTS DHA DAY 28

0 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.0

time (s)

100.0

1000

10000

G'

(P

a)

Osc Time Sweep DHA 40C OTS DHA DAY 0

OTS DHA DAY 7

OTS DHA DAY 14

OTS DHA DAY 21

OTS DHA DAY 28

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53

droplets’ surface may be less, which may lead to a greater chance of flocculation to

occur.

Monitoring particle size within the multiple emulsion may determine the presence

of creaming and coalescence. The results for particle size showed an increase for at 32˚C

for combination of lawsone/DHA. Despite this one increase, the particle size remained

consistent throughout all temperatures and days, lying within the range of 300nm to 500

nm. Therefore, in terms of creaming or coalescence, the emulsions were stable at varying

times and temperatures.

Determination of varying pH at various times and temperature can help determine

any possible chemical reactions that may occur with a multiple emulsion. At 25˚C,

lawsone experienced a pH drop on day 7 but remained within the pH range of its original

value about 6.22 after 28 days. A significant increase was observed within the DHA

emulsion from day 14 to day 28 to 5.1. As for combination lawsone/DHA emulsion, it

showed a fluctuating increase and decrease within the 28 time span, resulting in a final

pH of 5.1. At 32˚ C, lawsone showed a significant increase to a pH of 6.05 by day 28. A

drop in pH was observed for DHA emulsion from 6.03 at day 0 to 4.92 by day 28, and in

combination of lawsone/DHA to a pH of 3.88 from 5.2 at day 0. The same drop in pH

was also observed at 40˚C for combination lawsone/DHA, with a change in pH from 6.64

to 3.71. Drastic changes in pH of DHA and combination of lawsone/DHA suggest

chemical interactions may be occurring. This notion corroborates with the data from the

entrapment efficiency studies presented in the previous chapter and observations by

Fusaro et al, where DHA emulsions showed chemical instability when stored in elevated

temperatures and when in combination with lawsone23

. Hence, it was thought best if both

54

drugs were kept separate and stored at either room temperature or 4˚C, only combining

the two drugs before use.

In order to monitor any changes in viscosity, elasticity, and structure, measuring

spreadability and conducting rheological analysis may be necessary. Spreadability

results showed a significant increase in both lawsone and combination lawsone/DHA

multiple emulsions at 25˚C. At 32˚C, combination lawsone/DHA showed an increase to

29.5cm²/g on day 21. While at a stability temperature of 40˚C, a slight variance was seen

in the DHA emulsion with an increase in spreadability to 34cm²/g. Despite these varying

values, no obvious patterns were observed within each batch or temperature. Generally,

the spreadability of each emulsion remained rather consistent, remaining within the range

of 20cm²/g to 35cm²/g.

Rheological assessment of each emulsion is pertinent in knowing the structural

integrity and flow of the emulsions. The decrease in the LVR region in Figure 14c

suggests that at higher storage temperatures the elasticity of the emulsion decreases,

compromising its overall structure. In Table 5, the orange boxes showed a decrease in

value of tan δ for lawsone at 32˚C and 40˚C and for DHA at 25˚C and 32˚C suggesting an

increase in elasticity which may have happened from possible swelling. According to

Geiger et al, the swelling of the oil globules would lead to the increase in elasticity of the

system62

. Concurrently, the yellow boxes in Table 5 showed an increase in tan δ,

signifying a decrease in elasticity after 28 days. The decrease in elasticity can somewhat

be expected at elevated storage temperatures of 40˚C with any emulsion due to its use of

lipids or waxes. The decrease in elasticity may have occurred from possible expulsion of

55

the internal water droplets into the continuous medium after swelling or after having

applied stress onto the samples during the stress sweep.

Stepped flow curves showed all emulsion displayed a non-Newtonian,

pseudoplastic flow behavior. The presence of yield stress values, as seen in Table 6, and

shear thinning behavior of emulsions suggest viscoelastic flow properties. In Table 6,

the values in orange (lawsone and DHA emulsions) showed a decrease in viscosity

suggesting phase separation may have occurred for lawsone and DHA emulsions left at a

storage temperature of 32˚C and 40˚C after 28 days. This observation validated the

observed results of the orange boxes in Table 5. Since swelling of the oil globules may

have occurred, it may have eventually progressed to phase separation. The yellow boxes

in Table 6, primarily all of combination lawsone/ DHA emulsions left at 25˚C, 32˚C, and

40˚C, showed an increase in viscosity after 28 days, suggesting swelling may have

occurred. Swelling may have led to an increase in the rigidity of the second interface by

the progressive migration of the lipophilic surfactant. In the second step of the of the

multiple emulsion preparation, lipophilic surfactant molecules can diffuse from the first

to the second interface, where they produce a synergistic effect resulting in membrane

strengthening62

. This therefore explains how and why the viscosity possibly increased,

also validating the results found in the yellow boxes in Table 5.

By plotting G’ as a function of time as in the OTS tests, any structural changes of

the multiple emulsion can be monitored. OTS determines if and how the material

properties change after being loaded onto the rheometer, by monitoring certain

viscoelastic parameters as time advances. In this case, the control variable was 0.5968Pa

which was within the LVR of all the emulsions. Therefore, at this control variable and a

56

constant frequency of 1Hz, evaluation of the multiple emulsion’s behavior with time was

monitored directly. The OTS results showed a decrease in G’ for lawsone emulsion at

25˚C after 28 days (398 Pa). Also, a gradual decrease in G’ was observed at 40˚C for

DHA after a span of 28 days to 490.4Pa, which also exhibited a decrease in emulsion

elasticity through time. This observation validates the previous speculation made when

measuring the entrapment efficiency of DHA emulsions. In previous observations, the

DHA emulsion stored at 40˚C exhibited a decrease in entrapment efficiency, correlating

to the increase of free drug detected by the UPLC. It was previously speculated that the

structure of the emulsion at 40˚C might have been compromised, resulting in the

complete delivery of the drug into the external aqueous phase and consequently more free

DHA to be detected. The results from the OTS tests confirmed this speculation that the

structural integrity and loss of elasticity did occur over a period of time and increased

temperature, which may have been the reason for the decreased entrapment of DHA

within the internal aqueous phase.

3.5 Conclusion

In summary, the overall integrity of lawsone and DHA emulsions remained

generally stable within the storage temperatures of 25˚C for all characterization tests.

With increased storage temperatures of 32˚C and 40˚C, the emulsions have a greater

chance in experiencing flocculation, chemical instability, decrease in structural elasticity,

and possible swelling of the internal globule of the multiple emulsion. As for the

multiple emulsion combining both lawsone and DHA, pH issues suggest a chemical

degradation may be occurring at elevated temperatures which is in agreement with

previous stability studies conducted by Fusaro et al23

. Also, because the combination

57

emulsion was a physical mixture of two multiple emulsions combined in 1:1 ratio,

structural integrity and elasticity was compromised. When combining the two drugs in

that manner, an increased incidence of swelling was observed at all storage temperatures.

In conclusion, optimum stability was observed when lawsone and DHA emulsions were

kept at a storage temperature of 25˚C as separate entities and not in combination.

58

CHAPTER 4

In vitro Evalution of Multiple Emulsion Incorporating Lawsone and DHA

59

4.1. Introduction

In vitro drug release testing is a measure of the active pharmaceutical ingredient

(API) from the drug delivery system, which in this case is a multiple emulsion.

Evaluation of drug release profiles is pertinent to drug development and quality control.

It involves subjecting the dosage form to a set of conditions that will induce a drug

release and the measuring or quantifying of the amount of drug released under those

conditions. By simulating in vitro conditions to that of in vivo conditions, a better

understanding is gained of how the drug product will react within the body’s

environment. Typically, release profiles are determined using a Franz diffusion

apparatus, where the topical drug would spread over a suitable membrane and placed

application side up onto a diffusion cell. In the following experiments, the release profile

of lawsone, DHA, and combination lawsone DHA were determined conventionally by

use of the Franz diffusion apparatus using a snake skin membrane, as well as determined

without any interposed membrane. Because the emulsions were comprised of multiple

interphases with the drug embedded within internal most phase, preliminary release tests

were conducted in order to determine the amount of drug that actually diffused through

the system without the additional barrier of a membrane. By using a modified version of

USP Apparatus 5, the amount of drug released through the multiple emulsion system was

then determined.

Additional release studies were conducted using the vertical diffusion cell (VDC)

method, also known as the Franz diffusion cell. Diffusive communication between the

delivery system and the reservoir takes place through an inert, highly permeable support

membrane63

. In this study, the membrane used was shed snake skin. Shed snake skin is a

60

nonliving pure stratum corneum with no hair follicles64,65

. Snakes shed their skin

periodically, leaving their old stratum corneum behind, which makes it possible to obtain

multiple shed skins from the same individual snake65

. Unlike human stratum corneum

which is made up of 10 to 20 layers of α-keratin-rich intracellular layer and a lipid-rich

intracellular layer, shed snake skin consists of 3 distinct layers66,67

. The three layers are a

β-keratin-rich outermost beta layer, α-keratin and lipid rich intermediate mesos layer, and

α-kertain-rich innermost alpha layer68,69

. The meso layers shows three to five layers of

multilayer structure with cornified cells surrounded by intercellular lipids, which is

similar to the human stratum corneum. Further comparisons between human stratum

corneum and shed snake skin from Itoh et al can be seen below:

Table 7. Comparison of thickness, lipid content, and water evaporation rate

between human stratum corneum and shed snake skin

Human Stratum Corneum Shed Snake Skin

(Elaphe absoleta)

Thickness

13-15µm66,67

, 10µm66

10-20µm

Lipid Content 2.0-6.5%

3.0-6.8%70

6.0%

Water Evaporation Rate

0.1-0.8mg/cm²hr

0.34mg/cm²/hr

Ca. 0.2 mg/cm²hr

0.15-0.22 mg/cm²hr

With the similarities to human stratum cornuem listed above, shed snake skin was

thought to be a viable option as a substitute for actual human cadaver skin. Permeation

of a chemical through the stratum corneum is a process where active transport does not

apply. The layer with the highest resistance to diffusion is the rate-limiting membrane.

61

For many compounds, the lipophilic stratum corneum is the primary rate limiting

barrier71

. On the other hand, the rate limiting barrier for topically applied lipophilic

compounds would be the hydrophilic epidermis and dermis. When conducting in vitro

release testing (IVRT), the formulation is applied to the donor chamber with full contact

with the membrane that is in contact with the receiver liquid, in this case phosphate

buffer pH 7.4. The receiving medium should provide a “diffusional sink” for the active

ingredient released from the semisolid formulation72

. The receiving medium is sampled

as a function of time, and the API is obtained quantitatively to determine permeation

/flux profile. The cumulative amount (Q) release per surface was calculated by

Equation 7 below72

. The relationship of Q (cumulative amount released) versus √T

(square root of time) is derived from the Higuchi model with the assumption that there is

a reservoir of the drug always available to diffuse through72,73

.

Q =CnV+ ∑ Ci

n−1i=1 S

A (7)

The average cumulative amount of drug released was determined from Equation

7 where Cn is the concentration (mg/mL) determined at nth sampling interval, V is the

volume of individual Franz diffusion cell, ∑ 𝐶𝑖𝑛−1𝑖=1 is the sum of concentrations (mg/mL)

determined at sampling intervals 1 through n-1, S is the volume sampling aliquots

(0.1ml), and A is the surface area of the sample well (0.64cm²).

4.2. Materials

Python snake skin used for the Franz diffusion studies was provided by the

generous donations of Henry Doorly Zoo (Omaha, NE). Snake skin was obtained after

62

ecdysis, or sloughing of the skin, which is dependent on various aspects such as species,

age, nutrition etc.

4.3. Methods

4.3.1. In vitro Surface Release Studies

Figure 19. Schematic of USP Apparatus 5 (Paddle Over Disk) Assembly63

Under USP guidelines for measuring drug release from topical and transdermal

products, Apparatus 5 (Paddle Over Disk) assembly, which can be seen in Figure 19,

was to be used. Although the laboratory was equipped with a dissolution apparatus, it

was not equipped with the disk assembly necessary for the release study. Therefore, to

remedy the lack of supplies, an altered version of USP Apparatus 5 was assembled.

Instead of using a large volume dissolution vessel, either 150 mL or 1000 mL, a 20 mL

glass scintillation vial was instead used. Emulsion samples (lawsone, DHA, and

combination lawsone/DHA) were carefully filled into HPLC vial caps, which were used

as a replacement version of the disk assembly of USP Apparatus 5.

63

Figure 20. Picture representation of altered version of USP Apparatus 5 (sample and

vessel) and schematic of sampling process/ analysis

Three sets of each emulsion were weighed and excess emulsion was removed and

smoothened to provide a uniform release surface. The caps filled with emulsions were

carefully maneuvered into the glass scintillation vials. Approximately 10 mL of sodium

phosphate buffer pH 7.4 was used as dissolution medium and was placed into the

scintillation vial with the samples. The release assemblies were placed into an orbital

shaking incubator for a total of 120 hours at a speed of 100 rpm and kept at a temperature

of 32˚C. Samples, about 2% (0.2 mL) of the total volume of dissolution medium, were

removed at various time points using a needle and syringe and analyzed using the UPLC

analysis method previously stated. Time points for sampling were taken in hours (0, 1, 2,

3, 6, 12, 24, 48, 72, 96, and 120). Sodium phosphate buffer pH 7.4 (0.2mL) was used to

replace the volume that was extracted. Figure 20 is a picture representation of the altered

USP Apparatus 5 assembly that was created, which also displays the sampling process

and analysis through UPLC. Release studies were performed in triplicate (n=3).

64

4.3.2. Franz Diffusion using Snake Skin

The Franz diffusion apparatus, also known to the USP as the vertical diffusion

cell (VDC), was used for measuring the drug release from lawsone, DHA, and a

combination of lawsone/DHA multiple emulsions. An illustrated representation of the

multi-station Franz diffusion cell system which was used in this experiment can be seen

in the Figure 2174

.

Figure 21. Illustrated representation of multi-station Franz Diffusion cell system

The Franz diffusion cells, as represented in Figure 22, used for this experiment

were the standard jacketed cells with a flat ground joint, an internal 9 mm (≈ 5 mL)

receptor chamber, and a detachable donor assembly. The glass jacket surrounding the

cells allowed it to be connected to a circulator/heater that kept the temperature consistent

at 32˚C. A stir bar was placed within each cell and kept at a speed of 600 rpm. Before

use in the diffusion apparatus, snake skin was first prepared. The skin used was a large

portion, often over 3 feet long, of sloughed snake skin. Initially, the skin was soaked in

water to be cleaned. It was then dried overnight. The dried skin was cut according to

either the dorsal or ventral regions of the snake. In this particular experiment, dorsal

samples which were color and scale matched were utilized. Lastly, the skin was cut into

65

small (≈ 1 inch²) pieces prior to use. For ease of application, a small circle with the

diameter of the donor chamber was drawn as a reference for applying the emulsions.

The prepared skin squares where then further soaked in sodium phosphate buffer pH 7.4

overnight. The skin was weighed before and after application of the emulsions. Three

sets of approximately 0.2g to 0.4g of lawsone, DHA, and combination of lawsone/DHA

emulsions were weighed on top of the snake skins. To ensure full contact with the skin,

the emulsions were carefully rubbed onto the skin with a cotton tip applicator. Sodium

phosphate buffer pH 7.4 was placed into the receptor chamber and used as the diffusion

medium. The skin/emulsion samples were then positioned on top of the opening of the

receptor chamber. The donor assembly was maneuvered on top of the skin/emulsion

samples, ensuring the excess emulsions were kept within the donor chamber. The sample

(≈ 0.1 mL) aliquots were removed through the sampling port by needle and syringe.

Time points for sampling were taken in hours (0, 1, 2, 3, 6, 12, 24, 48, 72, 96, and 120).

Samples were analyzed using the UPLC method. Diffusion experiments were done in

triplicate.

Figure 22. Picture representation of Franz diffusion cell

66

4.3.3. Statistical Analysis of Data

The statistical analysis of this experimental data for the purpose of comparison

was performed using a 2 tailed Student’s T-Test. Data was considered statistically

significant if p<0.05.

4.4. Results and Discussion

4.4.1. In vitro Surface Release Studies

Figure 23. The in vitro release profiles of a) lawsone and b) DHA multiple emulsions in

comparison with the combination lawsone/DHA multiple emulsion

a) b)

67

Multiple emulsions are composed of two interphases. In this experiment, lawsone

and DHA are incorporated into the internal W1/O phase, while the external phase is the

continuous aqueous (W2) phase. Initial in vitro surface release studies were conducted to

determine the amount of drug that was able to release from the internal phase by

exposing only one portion of the emulsion to the phosphate buffer pH 7.4 medium.

Phosphate buffer pH 7.4 was used as well as kept at a temperature of 32 ͦC to mimic in

vivo skin conditions. In Figure 23 a and b, the graphs show comparative representation

between release profiles of multiple emulsions containing lawsone and DHA alone versus

in a combination multiple emulsion containing both drugs. The combination

lawsone/DHA multiple emulsion was comprised by combining two separate emulsions

double in concentration in a 1:1 ratio and triturated until homogenous. In Figure 23a¸ the

lawsone multiple emulsion obtained a final release of 85.3 ± 3.95%, while the

combination emulsion only obtained 56.4%, with significant differences (p <0.05)

starting from 72 hours until 120 hours. In Figure 23b, DHA only emulsion resulted in a

final release of 49.9 ± 2.08% while the combination lawsone/DHA emulsion had a final

release of about 40.7 ± 2.57%. Multiple emulsions have previously been considered a

controlled release drug delivery system, with the internal phase holding the hydrophilic

drug content. When incorporating two emulsions together as in the combination

lawsone/DHA emulsion, there are not only two interphases, but multiple interphases in

which the drug has to release from since two emulsions were combined to make one.

Therefore, this may be the reasoning behind the decreased release in the combination

lawsone/DHA emulsions for Figures 23a and 23b. The lawsone multiple emulsion’s

internal (W1) aqueous phase was comprised of phosphate buffer pH 7.4 instead of

68

deionized water which was used for the DHA emulsion. The literature value of the

distribution coefficient (log Doct/wat) for lawsone at pH 7.4 is -1.39 75

, while for DHA had

a log D at pH 7.4 of -1.0976

. The log D generally is a measurement of the hydrophobicity

of the drug, which affects how easily the drug can reach its intended target in the body,

how strong an effect it will have once it reaches the target, and how long it will remain in

the body in an active form77

. A higher value of log D would result in a more

lipophilic/hydrophobic drug, and vice versa. Therefore, at pH 7.4, lawsone’s low value

of log D of -1.39 would make it more hydrophilic which would allow more partition

through the oil layer and into the continuous aqueous (W2) phase, explaining the greater

release of lawsone into the distribution medium as opposed to DHA.

4.4.2 Franz Diffusion using Snake Skin

a)

69

Figure 24. In vitro release profile from franz diffusion testing as a function of the square

root of time for a) lawsone and b) DHA multiple emulsions in comparison with the

combination of lawsone/DHA multiple emulsion

The average rate of release is calculated by determining the relationship between

the cumulative amount released (Q) (mg/cm²) versus the square root of time (√T) (hr½

).

The average flux of release is obtained by the slope of the regression line where √T is the

x-axis and Q is the y-axis. In Figure 24a and 24b, the graphs presented compared the

lawsone and DHA multiple emulsions respective to the amount of drug released when

combined into one emulsion. Figure 24a shows a significant increase (p < 0.05) in the

average flux of release of lawsone (0.015 mg/cm²/hr½)

) when incorporated into a solo

emulsion as opposed to when in a combined multiple emulsion with both lawsone and

DHA (0.0014 mg/cm²/hr½). The opposite was observed in Figure 24b, displaying an

increased average flux of release of DHA from the combination multiple emulsion

(1.3326 mg/cm²/hr½) as opposed to the multiple emulsion with just DHA alone (0.7807

mg/cm²/hr½). As established prior, the combination lawsone/DHA multiple emulsion

was composed of two separate lawsone and DHA emulsions, triturated and homogenized

b)

70

to make one final emulsion. Hence, it was expected to see a slower rate of release from

the combined emulsion due to the increase in viscosity and the presence of multiple

interfaces that would act as barriers for release. An opposite reaction was observed for

DHA and its rate of release from the combined emulsion. As established in the previous

chapter in Table 6, combination lawsone/DHA multiple emulsions was shown to have a

tendency to increase in viscosity when stored at 32 ͦC which may be attributed to a

swelling reaction which may occur. Generally, an increase in viscosity would cause a

decrease in average flux release rate as seen in Thakker et al72

. But as discussed

previously, swelling had a possibility of occurring within combination lawsone/DHA

emulsion at temperatures of 32 ͦC and higher, which may be due to the migration of the

lipophilic surfactant to the second interface during the initial preparation process. In this

case, the lipophilic surfactant surrounding the external continuous phase would be the

cause for an increase in attraction to the lipophilic nature of the snake skin. Since the

combination lawsone/DHA emulsion is composed of two multiple emulsions together,

the amount of lipophilic surfactant would be double the amount of a single multiple

emulsion. Also, as discussed in the previous section, the distribution coefficient of DHA

(-1.09) is slightly greater than lawsone (-1.39) at a pH 7.4 resulting in a less hydrophilic

nature and may also be causing an affinity to the lipophilic nature of the snake skin more

so than lawsone. In order to further assess the reasoning behind the increased rate of

flux for DHA in combination, additional studies need to be conducted.

4.5. Conclusion

Lawsone, DHA, and combination lawsone/DHA multiple emulsions were

evaluated to determine their compatability in in vivo conditions. A preliminary release

71

study was conducted to determine if and how much of the drugs were able to release

without the addition of diffusion membrane. By utilizing an altered version of USP

Apparatus 5, the surface release profiles for lawsone, DHA, and combination

lawsone/DHA multiple emulsions were obtained. DHA and DHA in combination with

lawsone both showed comparable release profiles. Lawsone, on the other hand, showed a

cumulative % release of 85.3 ± 3.95% after 120 days as opposed to only 56.4 ± 0.45% for

lawsone in combination. This was mainly due to its inherent log D when at a pH 7.4.

Because lawsone’s log D was lesser (-1.39) than DHA (-1.09), it allowed more partition

through the oil layer to the external aqueous buffer medium and resulting in a greater

release of drug. The multiple interphases, which formed a barrier of sorts for the

combination emulsions, allowed no more than 40.7 ± 2.57% to 56.4 ± 0.45% release of

DHA and lawsone.

The IVRT for the emulsions using snake skin showed a significant increase in the

rate of release for lawsone when compared to the combination multiple emulsion. This

also correlates with the results obtained in the surface release studies. A greater

cumulative amount released (Q) was observed in DHA in combination than when DHA is

alone in the multiple emulsion. This may be due to the fact that at temperatures greater

than 32˚ C, combination lawsone/DHA multiple emulsions was previously shown to

swell, causing the lipophilic surfactant to migrate to the second interface during the

preparation process. Also, the distribution coefficient (log D) of DHA was slightly

higher than that of lawsone. Because of this and the lipophilic nature of the external

phase of the combination multiple emulsion after swelling, we can assume that a greater

affinity to the lipophilic nature of the snake skin was established. Consequently, a greater

72

cumulative amount of drug released would be observed for DHA in combination. In

order to confirm these results, further studies of the partitioning behavior of the multiple

emulsions need to be conducted.

73

CHAPTER 5

Summary and Future Directions

74

5.1. Summary

A multiple emulsions containing 0.035% lawsone (w/w) and 1% DHA (w/w)

were prepared using a two-step emulsion procedure provided by Matsumoto et al. The

first step of emulsion was process was the preparation of the primary (W1/O) phase where

the hydrophilic drugs were incorporated into the internal (W1) phase and was slowly

incorporated into the oil (O) phase through by hand homogenizer. The second step was

the addition of the primary (W1/O) phase to the external aqueous phase (W2) by over-

head stirrer with a paddle attachment. The combination lawsone/DHA emulsions were

prepared by formulating the lawsone and DHA emulsions separately with double the

concentration and triturating both in a 1:1 ratio until homogenous. The existence of all

interphases within the multiple emulsion was verified and visualized using light and

fluorescence microscopy. Entrapment efficiencies of lawsone and DHA within each

emulsion and in combination were determined through a biphasic extraction using a 1:1

ratio of methylene chloride and phosphate buffer (PB) pH 7.4. Entrapped drugs were

analyzed using a UPLC method. The entrapment efficiency at 25˚C showed a consistent

percent entrapment for lawsone, DHA, and DHA in combination multiple emulsions,

lying within the range of ≈ 71.1% to 83% entrapment within the internal (W1) water

phase after 28 days of storage. Meanwhile, the lawsone in combination multiple

emulsion showed an entrapment of ≈ 99%. At 32˚C, about the same patterns were

observed, except a slight decrease in entrapment efficiency for DHA and DHA in

combination emulsion to about ≈ 76% to 65% after 28 days. At 40˚C, lawsone emulsion

remained consistent at 70.64 ± 3.02% after 28 days and lawsone in combination

displayed a drastic increase in entrapment to 99.58 ± 0.07% by day 28. DHA emulsion

75

decreased by day to 37.41 ± 6.04 % entrapment efficiency and DHA in combination

emulsion showed a drastic decrease in entrapment to about 5.41 ± 3.82% after 28 days.

Therefore, less entrapment for DHA and DHA in combination was a clear indication of

the emulsion’s instability at 40˚C, which were later confirmed by characterization and

stability studies.

Lawsone, DHA, and a combination of lawsone/DHA were all stored at 25˚C,

32˚C, and 40˚C and characterized at various time points throughout a 28 day time span to

determine the emulsion’s chemical and structural stability. Particle size for all three

emulsions remained with a consistent range of 300 to 500nm suggesting no drastic

creaming or coalescence occurred despite temperature and time change. Regardless of

the varying values obtained through spreadability testing, the emulsions remained rather

consistent and remained within the range of ≈ 20cm²/g and 35cm²/g with time and

temperature change, suggesting no noticeable change in viscosity or elasticity would be

observed after application of the emulsions. Zeta potential displayed overall stability at

25˚C and 32˚C without any significant changes occurring. However at 40˚C, zeta

potential for DHA emulsion showed a significant increase (p<0.05) from -20.80 ±

0.98mV on day 14 to -14.93 ± 0.97 mV by day 28 and combination lawsone/DHA also

showed a significant increase to -19.3 ± 3.68mV. The variance in zeta potential at this

temperature implies that a greater chance of flocculation may happen at an increased

temperature of 40˚C. Chemical instability was determined by monitoring pH changes in

the emulsions. At increased temperatures of 32˚C and 40˚C, drops in pH were observed

for both DHA and combination lawsone/DHA multiple emulsions. At 32˚C, DHA

observed a significant decrease in pH from 6.03 ± 0.18to 4.92 ± 0.08 and combination

76

lawsone/DHA with a drop of ≈5.2 to 3.88 ± 0.05 after 28 days in the stability chamber.

At 40˚C, pH drops drastically from pH 6.64 ± 0.22 to 3.71 ± 0.26 after 28 days for

combination lawsone/DHA was observed. Drastic changes in pH of DHA and

combination of lawsone/DHA suggest chemical interactions occurred. This notion

corroborates with the data from the entrapment efficiency studies and observations by

Fusaro et al, where DHA emulsions showed chemical instability when stored in elevated

temperatures and when in combination with lawsone23

. Hence, it was thought best if both

drugs were kept separate and stored at either room temperature or 4˚C, only combining

the two drugs before use. This notion was later confirmed by rheological assessment of

the structure of the emulsions. Rheological studies, specifically the OTS tests, suggest

that structural integrity of the multiple emulsion containing DHA and combination DHA

may also have been the reason for the complete delivery of the DHA to the external (W2)

phase, hence leading to the decrease in entrapment efficiency. Stepped flow step showed

an increased in viscosity of combination lawsone/DHA when stored at all temperatures

after 28 days, which may indicate that swelling may have occurred. Swelling would have

occurred when the lipophilic surfactant molecules may have diffused form the first to the

second interface, which would have led to membrane strengthening and an increase in

viscosity of the emulsion. Therefore, in accordance with Fusaro et al, the individual

lawsone and DHA multiple emulsions may be best stored as separate emulsions until

application, with an optimum storage temperature of 25˚C or cooler.

Evaluation of the in vitro surface release profiles of lawsone, DHA, and both

lawsone/DHA in combination multiple emulsions was used to determine if and how

much of the drugs were able to release out of the emulsion without the added barrier of a

77

diffusion membrane. DHA and DHA in combination with lawsone emulsions had

comparable release profiles with a cumulative % release after 120 hours. DHA emulsion

had a cumulative drug release of about 49.9 ± 2.08% after 120 days while, DHA in

combination had a drug release of 40.7 ± 2.57% with no significant difference (p<0.05)

between the two formulations. However, lawsone multiple emulsion showed an increase

in cumulative release (85.3%) as opposed to lawsone in combination, the reason for this

being the distribution coefficient (log D) of lawsone at a pH of 7.4. Because the log D

for lawsone (-1.39) was less than DHA (-1.09), lawsone was allowed more partition

through the oil layer to the external aqueous buffer medium, therefore resulting in a

greater release of the drug. In the diffusion release studies using snake skin, a significant

increase in the average flux of release was observed in lawsone (0.015 mg/cm²/hr½)

multiple emulsion when compared to lawsone in combination (0.0014 mg/cm²/hr½).

Because the combination emulsion was composed of two emulsions integrated together

to make a homogenous formulation, it was expected to see a slower flux of release

especially from the increase in viscosity and the presence of multiple interfaces which

would act as barriers for release. The opposite reaction was seen when comparing the

flux release of DHA in combination and DHA multiple emulsions. The emulsion

containing DHA in combination indicated a higher average flux (1.3326 mg/cm²/hr½)

than DHA alone (0.7807 mg/cm²/hr½). Although the reasons for this reaction can be

speculated, further studies are needed in order to do a full evaluation of diffusion kinetics

of the multiple emulsion. In summary, evaluation of the multiple emulsions through in

vitro studies showed that the multiple emulsions containing lawsone, DHA and a

combination of both lawsone/DHA are capable to diffuse through the interphases of the

78

emulsion itself. Also, the studies exemplified how multiple emulsions can be a viable

drug delivery system for topical application of lawsone, DHA, and/or a combination of

both lawsone and DHA.

5.2. Future Directions

The present study elucidated the formulation and stability characterization of

multiple emulsions containing lawsone, DHA and a combination of lawsone and DHA.

Due to the variance in viscosity and decreased structural elasticity over a period of time

as storage temperatures increased as seen from the rheological assessment of the

emulsions, further fine tuning of the formulation is needed to ensure thermodynamic

stability. One approach to improving stabilization of a multiple emulsion is by stabilizing

the inner (W1/O) phase by addition of various emulsifier combinations78

. Also,

stabilization of the oil phase can be enhanced by choosing a suitable oil type and addition

of proper carriers, complexants, and viscosity builders may be another approach. By

doing so, the solubility and polarity of the oil phase would be modified, making it less

water soluble79

. Finally, stabilization of the external aqueous phase by increasing the

viscosity of the outer aqueous layer may be another way to enhance stability80

(tedajo).

After a new formulation is established, full extraction of the drug by possibly a double

extraction method would be needed in order to not under estimate the full drug load of

the multiple emulsions. In addition, further in vitro studies are needed to determine the

permeability and diffusion kinetics of the multiple emulsions. Concurrently, human

cadaver skin may also be used instead of snake skin in order to determine a truer

representation of skin diffusion. Finally, in vivo studies can be conducted to correlate the

results obtained from the in vitro studies by using actual human volunteers to determine

79

the efficacy of using lawsone and DHA in a multiple emulsion drug delivery system as a

form of sun protection.

80

References

81

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