Sarah Alexandra Pais Pereira

Development of automatic systems

to study the loading and release of

anticancer drugs from mesoporous

silica nanoparticles

Dissertation of the 2nd Cycle of Studies

Degree of Master in Quality Control in the Specialty of Drug Substances and

Medicinal Plants.

Supervisors: Professor Maria Lúcia Marques Ferreira de Sousa Saraiva and

Professor André Ricardo dos Santos Araújo Pereira

March, 2018

ii

THE COMPREHENSIVE REPRODUCTION OF THIS DISSERTATION

IS AUTHORIZED ONLY FOR RESEARCH EFFECTS, THROUGH A

WRITTEN STATEMENT BY THE INTERESTED PARTY THAT IT IS

COMMITTED TO THAT.

iii

Acknowledgments

This work was carried out at the Division of Applied Chemistry, Faculty of Pharmacy,

University of Porto during the years 2017-2018.

I am extremely grateful to my supervisor, Professor Lúcia Saraiva for supporting,

encouraging and believing in my abilities from the beginning and at every step. Her positive

attitude, enthusiasm, patience and generosity, both personally and scientifically, have

always inspired me and have paved the way of my accomplishments.

I would like to express my gratitude to my co-supervisor, Professor André Pereira for

supporting and encouraging me in the development of this work. Without him, I would not

have had the opportunity to meet all the people who are currently part of my day-to-day in

laboratory.

A special acknowledgement to Marieta who besides being my laboratory colleague, I

consider her a great friend and an example for me. Thank you for your patience, support

and thoughtfulness, both personally and scientifically and for sharing with me the best and

the worst of the last years, always cheering-me up.

I wish to thank to Mafalda and Jorge for being an example for me as a couple and for

their supporting and encouraging in the last years of my life.

I am very grateful to all my colleagues from the Divison of Appllied Chemistry for their

friendship and pleasant working atmosphere.

I would also like to express my sincere gratitude to Dr. Hélder A. Santos and

Alexandra Correia for helping me in the development of this work.

I wish to thank my parents Cristina and João for their love and unconditional support

with every decisions I have made, always encouraging me to pursue my dreams.

I would like to dedicate this work to my sister Ana Luísa for lighting up my life, for her

positive wishes, inspiration and love.

I wish to also thank my grandparents Emília and Elisio for their unlimited love and

support.

Last but not the least, I want to thank to my boyfriend António for his unconditional

love, constant motivation, patience and support. Thank you for always being there for me,

in good and bad times. You’re the pillar of my strength and nothing would have been

possible without you.

iv

Resumo

O cancro é uma das principais causas de morte em todo o mundo e representa um

grupo de doenças heterogéneas caracterizadas pelo crescimento descontrolado, divisão e

disseminação de células anormais.

Uma vez que as terapias convencionais, usadas atualmente, são limitadas e têm

demonstrado uma variedade de efeitos adversos, o desenvolvimento da nanotecnologia

baseada na medicina (nano medicina), permite um avanço na eficácia terapêutica de um

grande número de fármacos anticancerígenos bem como na redução do seu perfil de

toxicidade.

As nanopartículas de sílica mesoporosa (MSNs, do inglês “Mesoporous sílica

nanoparticles”) são materiais sólidos que contêm centenas de mesoporos vazios dispostos

numa rede bidimensional. Esta rede de poros com homogeneidade no tamanho, permite

uma elevada capacidade de encapsulação e a libertação controlada de fármacos

anticancerígenos.

Neste trabalho, foram desenvolvidos dois sistemas automáticos baseados na análise

por injeção sequencial (SIA, do inglês “Sequential Injection Analysis”), um para o ensaio de

encapsulação e o outro para o ensaio de libertação. O fármaco anticancerígeno usado foi

o 5-fluorouracilo (5-FU) que é um análogo da pirimidina e cuja atividade baseia-se na

inibição irreversível da enzima timidilato sintase. As MSNs foram usadas para os ensaios

de encapsulação e libertação deste fármaco.

No sistema SIA em que se realizaram os ensaios de encapsulação, foram otimizadas

condições, como por exemplo: o volume de 5-FU e de nanopartículas (NPs) usados, o

número de alíquotas e a sua ordem de aspiração, o caudal de aspiração da solução de 5-

FU e de NPs bem como o caudal de propulsão das NPs encapsuladas para o frasco. No

sistema SIA em que se realizaram os ensaios de libertação foram também otimizadas

condições como: o volume de 5-FU aspirado e o volume de reposição para o frasco no final

do ciclo analítico., bem como o caudal de propulsão para o detetor de fluorescência

Comparando os resultados obtidos nos sistemas SIA e os obtidos em modo discreto

provou-se que as metodologias automáticas podem ser usadas na encapsulação e no

traçado dos perfis de libertação do fármaco 5-FU a partir de MSNs.

Palavras-chave: cancro, nanopartículas de sílica mesoporosa, 5-fluorouracilo,

análise por injeção sequencial, encapsulação, libertação.

v

Abstract

Cancer is one of the leading cause of death worldwide and represent a group of

heterogeneous diseases characterized by uncontrolled growth, division and spread of

abnormal cells.

Since the conventional therapies currently used are limited and have shown a variety

of adverse effects, the development of nanotechnology based medicine (nanomedicine)

provides a great breakthrough in the therapeutic efficacy of a large number of anticancer

drugs as well as reduce their toxicity profile.

Mesoporous silica nanoparticles (MSNs) are solid materials which contain hundreds

of empty mesopores arranged in a two-dimensional network of porous structure. This

ordered pore network with size homogeneity, allows a high loading capacity and enables

the controlled-release of anticancer drugs.

In this work, it was developed two automatic sequential injection analysis (SIA)

systems, one to perform the loading assays and another one to the release assays. The

chemotherapeutic agent used was 5-fluorouracil (5-FU), a pyrimidine analogue, whose

activity is based on the irreversible inhibition of thymidylate synthase. MSNs were used to

load and release this drug.

In SIA developed for loading assays, optimized conditions were studied, such as: the

volume of 5-FU and of nanoparticles (NPs) suspension used, the number of the aliquots

and their order of aspiration, the aspiration flow rate of the 5-FU solution and NPs as well

as the flow rate to propel the loaded nanoparticles to the vessel. In SIA for release assays,

optimized conditions were also studied such as: the volume of 5-FU solution aspirated to

the system and replaced to the vessel of the NPs at the end of analytical cycle, the

propulsion flow rates to the fluorescence detector.

The results obtained in SIA systems compared with the ones obtained using batch

procedures proved that the automatic methodologies can be used to load and to plot the

release profiles of 5-FU from MSNs.

Keywords: cancer, mesoporous silica nanoparticles, 5-fluorouracil, sequential

injection analysis, loading, release.

vi

Table of Contents

Acknowledgments ...................................................................................................... iii

Resumo ....................................................................................................................... iv

Abstract ........................................................................................................................ v

Table of Contents ....................................................................................................... vi

List of Figures ........................................................................................................... viii

List of tables ................................................................................................................ x

List of abbreviations ................................................................................................... xi

Chapter 1 - Introduction .................................................................................................. 1

1. Flow techniques and sequential injection analysis (SIA) .................................. 2

1.1. Determination of pharmaceuticals in SIA systems 6

1.2. Nanoparticles and SIA systems 13

1.3. Mesoporous silica nanoparticles (MSNs) and SIA systems 15

2. Mesoporous silica nanoparticles (MSNs) synthesis and surface chemistry

modifications ............................................................................................................. 17

3. Chemotherapeutic agents used in treatment of cancer ................................... 18

4. Biomedical applications of MSNs: drug delivery ............................................. 21

5. Aims of the study ................................................................................................ 22

Chapter 2 - Materials and Methods .............................................................................. 24

1. Introduction ......................................................................................................... 25

2. Reagents and solutions ..................................................................................... 25

3. Instrumentation .................................................................................................. 25

3.1. Auxiliary equipment 25

3.2. Flow Apparatus 26

3.2.1. Aspiration/ propulsion device 28

3.2.2. Fluid selector valve 30

3.2.3. Tubing and other manifold components 31

3.2.4. Informatics’ control 32

3.2.5. Detection device 33

4. Experimental procedure ..................................................................................... 34

4.1. Loading batch procedure 34

4.2. Loading sequential injection procedure 35

vii

4.3. Release batch procedure 35

4.4. Release sequential injection procedure 35

Chapter 3 - Results and Discussion............................................................................. 37

1. Introduction ......................................................................................................... 38

2. Optimization of sequential injection procedure for the loading of NPs .......... 38

3. Optimization of release sequential injection procedure .................................. 42

4. Determination of % loading using SIA system and batch procedure ............. 45

5. 5-FU release profiles obtained in batch procedure and SIA system ............... 46

6. 5-FU release profiles using different loading methodologies ......................... 49

Chapter 4 - Conclusions ............................................................................................... 52

Chapter 5 - Bibliography ............................................................................................... 54

viii

List of Figures

Figure 1 - Basic schematic of an FIA assembly, where CS: carrier solution; R: reagent; P:

pump; IV: sample injection valve (sampling); RT: reaction tube; D: detector; W: waste ..... 2

Figure 2 - Schematic sequential aspiration and propulsion of aliquots of sample and

reagent, where S, P, and R are sample, product, and reagent, respectively. ..................... 3

Figure 3 - Schematic representation of axial and radial diffusion in SIA system. ............... 4

Figure 4 - Basic schematic of a SIA assembly where CS: carrier solution; PD: peristaltic

pump or syringe pump; HC: holding coil; SV: selection valve; R: reagent; S: sample; RT:

reaction tube; D: detector and W: waste. ........................................................................... 4

Figure 5 - Chemical structures of THCPSi, TCPSi, UnTHCPSi and APTES NPs. ........... 18

Figure 6 - Chemical structure of 5-FU. ............................................................................ 23

Figure 7 - (a) Schematic representation of the SIA manifold used in loading assays. C:

carrier (phosphate buffer 0.2 mol L-1 pH 7.4), S: syringe, HC: holding coil (2m), SV: selection

valve, 5-FU: 5-fluorouracil, NPs: nanoparticles, M: loaded NPs, W: waste. (b) Sequence of

aspiration of solutions into the holding coil of the flow system: NPs and 5-FU, repeated ten

times. .............................................................................................................................. 27

Figure 8 - Schematic representation of the SIA manifold used in release assays. C: carrier

(phosphate buffer 0.2 mol L-1 pH 7.4), PP: peristaltic pump, HC: holding coil, SV: selection

valve, F: filter, S: sample, D: detector, W: waste. ............................................................ 28

Figure 9 - Syringe pump module Bu1S from Crison Instruments S.A. ............................. 29

Figure 10 - Peristaltic pump Miniplus 3 from Gilson. ....................................................... 30

Figure 11 - Selector valve with 10-port multiposition from Valco®. .................................. 31

Figure 12 - PTFE tubing used in SIA systems (1) and PTFE tubing in an eight configuration

(2). .................................................................................................................................. 31

Figure 13 - Computer software in the SIA system where it was performed loading assays.

........................................................................................................................................ 32

Figure 14 - Computer software in the SIA system where it was performed release assays.

........................................................................................................................................ 33

Figure 15 - Photography of fluorescence detector device. .............................................. 33

Figure 16 - Calibration curve obtained in HPLC to determine the concentration of 5-FU in

loading assays. ............................................................................................................... 39

Figure 17 – Volume sample and flow rate optimization in SIA system for release studies.

........................................................................................................................................ 43

Figure 18 - Calibration curve used to determine the concentration values of 5-FU obtained

in release assays. ........................................................................................................... 44

ix

Figure 19 - Release profiles using SIA system for loading, where A are APTES NPs, B are

TCPSi NPs, C are THCPSi NPs, D are UnTHCPSi NPs, 1 corresponding to the release

using batch procedure and 2 corresponding to the release using SIA system. ................ 49

Figure 20 - Release profiles using different loading methodologies and batch release,

where A are APTES NPs, B are UnTHCPSi NPs, 1 corresponding to the loading and

release using batch methods and 2 corresponding to the loading in SIA system and release

in batch method. .............................................................................................................. 50

Figure 21 - Release profiles using different loading methodologies and SIA system release,

where A are TCPSi NPs, B are THCPSi NPs, 1 corresponding to the loading and release

using SIA methods and 2 corresponding to the loading in batch method and release in SIA

system. ............................................................................................................................ 51

x

List of tables

Table 1 - Basic comparisons between FIA and SIA. Adapted from (Santos & Masini, 2010)

.......................................................................................................................................... 6

Table 2 - Main anticancer drugs used in conventional therapy ........................................ 19

Table 3 - Results of the optimization of loading assay in the flow system. ....................... 39

Table 4 – Tested conditions and respective results of the optimization of loading assay in

SIA system. ..................................................................................................................... 40

Table 5 - Analytical cycle to perform loading assay in SIA system .................................. 41

Table 6 – Analytical cycle to the implementation of release assays in SIA system. ......... 44

Table 7 - Results of % loading obtained with both loading methodologies ...................... 45

xi

List of abbreviations

A – Area

ABC – ATP-binding cassette

APTES – 3-aminopropyltriethoxysilane

ATP – Adenosine triphosphate

C – Concentration

CS – Carrier solution

CTC – Chlortetracycline

D – Detector

DDS – Drug delivery systems

DMSO – Dimethyl sulfoxide

DNA – Deoxyribonucleic acid

EPR – Enhanced permeability and retention

FdUMP – 5-fluorodeoxyuridine monophosphate

FI – Fluorescence intensity

FIA – Flow injection analysis

H2 – Hydrogen

HC – Holding coil

HF – Hydrofluoric acid

HPLC – High performance liquid chromatography

IV – Injection valve

MDR – Multidrug resistance

MSN – Mesoporous Silica Nanoparticles

NPs – Nanoparticles

OTC – Oxytetracycline

P – Product

PBS – Phosphate buffer solution

PD – Propulsion device

POEGMA – poly (oligoethylene glycol) monomethyl ether methacrylate

PP – Peristaltic pump

PTFE – Polytetrafluoroethylene

PVC – Polyvinyl chloride

R – Reagent

RSD – Relative Standard Deviation

RT – Reaction tube

S – Sample

xii

Si – Silicon

SIA – Sequential Injection Analysis

SPE – Solid phase extraction

SPS – Solid phase spectroscopy

TC – Tetracycline

TCPSi – Thermally carbonized porous silicon

THCPSi – Thermally hydrocarbonized porous silicon

TS – Thymidylate synthase

UnTHCPSi – Undecylenic acid modified THCPSi

W – Waste

5-FU – 5-fluorouracil

Chapter 1

Introduction

2

1. Flow techniques and sequential injection analysis (SIA)

It is easy to see the increase of the analytical efficiency translated by the automation

of the different stages of an analytical process by means of continuous flow techniques.

Flow techniques rely on the introduction of an aliquot in a flow sample, where contact

occurs with one or more reagents. From this process, a reaction zone will be sent to a

detection system, giving rise to an analytical signal, whose intensity is related to the physical

and chemical properties of the analysed solution.

Flow injection analysis (FIA) was the first flow technique that emerged in 1975

(Ṙuz̆ic̆ka & Hansen, 1975) and has as its operating principle the injection of an aliquot of

liquid into a non-segmented, continuous flowing carrier fluid. FIA is a general solution-

handling technique, applicable to a variety of tasks ranging from pH or conductivity

measurements to colorimetry and enzymatic assays. The injected sample is transported

towards a detector. The sample and the reagent are mixed with the flowing stream mainly

by diffusion controlled processes, with a chemical reaction occurring. The detector

continuously records the absorbance, fluorescence, electrode potential or other physical

parameter, as it changes, as a result of the passage of the reaction product through the flow

cell. Despite the simplest FIA system has only a single-flow line, FIA systems may have a

variety of manifold configurations, by reconfiguration, to allow application to nearly any

chemical system (Christian, 2004).

A FIA system typically consists of a propulsion device (PD), an injection valve (IV),

whose loop dimensions determines the volume of sample to be injected, a portion of the

reaction tube (RT) where the chemical reaction occurs and a detector (D) device where the

product formed is measured, as shown in figure 1.

On the other hand, sequential injection analysis (SIA) emerged in 1990 (Jaromir

Ruzicka & Marshall, 1990) and, according to its authors, can be considered as the second

generation of non-segmented stream injection techniques. Its proposal was primarily aimed

.

Figure 1 - Basic schematic of an FIA assembly, where CS: carrier solution; R: reagent;

P: pump; IV: sample injection valve (sampling); RT: reaction tube; D: detector; W: waste

3

at satisfying certain requirements imposed on the implementation of routine analysis of

procedures based on continuous flow namely, versatility, robustness, and simplicity.

SIA is based on the sequential aspiration of accurate sample and reagent volumes

from a selection valve to a holding tube which, after reversal of the flow direction, are

propelled to the reaction tube and then monitored in the detector. In the SIA system, the

reproducible overlap of sample zones and reagents is checked by the combination of

stoppages and flow direction changes (Jaromir Ruzicka & Marshall, 1990), as can be seen

in Figure 2.

Figure 2 - Schematic sequential aspiration and propulsion of aliquots of sample and

reagent, where S, P, and R are sample, product, and reagent, respectively.

The mixing of the different zones aspirated sequentially depends on the operational

parameters of the assembly itself and affects the extent of the dispersion suffered by the

reaction zone. In this type of systems, the dispersion is also related with convex transport

processes that occur with the flow and molecular diffusion in the contact interfaces of the

adjacent zones that affect its interpenetration (Staden; & Botha, 1998)

Considering that the flow is predominantly laminar, a segment inserted in the system

will initially be subjected to a parabolic velocity profile, favouring the concentration gradients

responsible for radial and axial diffusion (Figure 3).

4

The reversal of the flow direction causes a decrease in the axial dispersion of the

segment, resulting in a relatively symmetrical concentration profile, which leads to an

analytical signal of approximately Gaussian shape, by passage through the detector

(Staden & Botha, 1998).

Contrary to the FIA system, in the SIA assembly, after the reversal of the flow

direction, the overlapping of adjacent sample zones and reagents is only partial. This fact

is actually one of the limitations of this type of technique since, despite the possibility of

sequentially aspirating a greater number of reagents, there is a limitation as to the number

of zones that can be mixed by reversing the flow in a tubular reactor.

However, the SIA technique, due to its enormous versatility and computer control

mode, has features that facilitate the implementation of complex analytical procedures

without the physical reconfiguration of the assembly (T. Gübeli, G.D. Christian, & Ruzicka,

1991).

A SIA system consists of a propulsion device (PD) that can be a peristaltic pump or a

syringe pump; a fluid selector valve, a detector system, a holding coil, which connects the

propellant device to the fluid selection valve (SV) and a reaction tube (RT), which connects

the fluid selector valve to the detector (D) system, as shown in Figure 4.

Figure 3 - Schematic representation of axial and radial diffusion in SIA system.

Figure 4 - Basic schematic of a SIA assembly where CS: carrier solution; PD: peristaltic

pump or syringe pump; HC: holding coil; SV: selection valve; R: reagent; S: sample; RT:

reaction tube; D: detector and W: waste.

PD

5

SIA is a computer-controlled, single-line, injection technique that simplifies the

manifold and is more robust than FIA. Once computer-controlled, the propulsion device and

the selection valve operate synchronously, allowing the definition of the volume, direction

and flow velocity of the various solutions.

SIA uses a multiport selection valve as shown in Figure 4. The circulation of the fluids

occurs from the central orifice of the selection valve which can access any of the other ports,

including to the holding coil, by electrical rotation of the valve.

The holding coil is placed between the propulsion device and the selection valve and

it is essential to ensure that the length of the holding coil is sufficient to prevent

contamination of the carrier solution with the aspirated solutions (J. Ruzicka & Gubeli,

1991). The different ports of the selection valve are connected to different solutions such

as sample, reagents, standards, waste (W) reservoir and detector.

In operation, all the tubes in the SIA system are first filled with carrier solution. The

selection valve is switched to the port of detector and the propulsion device propels the

carrier solution through the system until it exits at the waste end. The selection valve is then

switched to the sample position and a few microliters of sample are drawn in by reversing

the flow of the propulsion device using precision time (Christian, 2004)

The volumes are defined by the flow rate and time of aspiration or propulsion, whereby

the propulsion device must provide precise movements of start, stop and reverse direction

of the flow, which ensures that the aspiration and propulsion of the solutions are

reproducible (T. Gübeli et al., 1991).

The propulsion device is stopped during the rotation of the selection valve to avoid

pressure surges. After the introduction of the sample in holding coil, the reagent is aspirated.

Thus, the sample and reagent solutions are sequentially injected into the holding coil and,

finally, the selection valve is switched to the detector port. The propulsion device is changed

to forward flow and the aspirated solutions are propelled through the reaction coil to the

detector flow cell. The solutions merge via diffusion and secondary forces and react to form

a product that is detected, resulting in a transient signal as in conventional FIA (Christian,

2004)

The SIA system is a solution to the need of simplifying systems and to increase their

analytical versatility while allowing independence of the empirical knowledge of the user. Its

mode of operation, based on reversals of the flow direction, coupled with the operation of

the fluid selector valve, enables not only different chemical reactions but also all types of

sample pre-treatment to be performed online (Jaromir Ruzicka & Marshall, 1990).

SIA systems have some advantages comparing with FIA methodologies since SIA

readily allows a significant reduction in the solvents, samples, and reagents consumed and

6

in waste produced. SIA allows different methodologies to be implemented without manifold

modifications (Ṙuz̆ic̆ka & Hansen, 1975). Other characteristics can be analysed in the

following table (Table 1).

Table 1 - Basic comparisons between FIA and SIA. Adapted from (Santos & Masini, 2010)

Characteristics FIA SIA

Complexity of the system Bigger Smaller

Programming knowledge Optional Necessary

Reagent consumption Higher Lower

Computer control Optional Present

Purchase cost Smaller Bigger

Addition of reagent by confluence Present Absent

Frequency of sampling

(typical values)

>100 analysis per hour < 60 analysis per hour

Operating time without parameter

adjustment (robustness)

Smaller Bigger

In addition to the above, SIA presents more characteristics as robustness, reliability,

long-term stability and the maintenance routine that allow an increase in automation of the

analytical modules considered important attributes of the methodology to be implemented.

1.1. Determination of pharmaceuticals in SIA systems

SIA system, with its advantages, have been used in the determination of different

pharmaceuticals through release, permeation and dissolution studies. In this kind of studies,

different analytical methodologies can be used with the detection of pharmaceuticals or their

metabolites, recurring to spectrophotometry, fluorimetry, chemiluminescence,

electrochemistry and so on. (Al-Kindy, Suliman, Al-Wishahi, Al-Lawati, & Aoudia, 2007;

Fraihat, 2014; Molina-Garcia, Llorent-Martinez, Fernandez-de Cordova, & Ruiz-Medina,

2012; Tzanavaras, 2011).

The majority of the determinations of active ingredients in pharmaceutical

formulations are spectrophotometric-based methods and within this group, the most of

those described are based on chromogenic reactions or on the specific characteristics of

light absorption by analytes. On the other hand, fluorimetric determinations are the most

used widespread, since the sensitivity is higher than in the spectrophotometric

determinations both by the lower interference of the matrix and also due to the wide linear

range between the intensity of the radiation source and the analytical signal measured.

7

However, in this type of determinations, it was necessary to perform an appropriate

derivatization reaction of the desired compound, since many compounds are not

fluorescence. Chemiluminescent detection can be classified into two groups. In the first

group, the analyte is the emission specie through the direct action of common strong

oxidants and in another group the analyte interferes in some way in the oxidation reaction

of the chemiluminescent precursors. (Pimenta, Montenegro, Araujo, & Calatayud, 2006).

Numerous determination studies in several SIA system configurations have been

developed using different groups of pharmaceuticals.

Pinto et al. developed a pulsed flow sequential injection analysis (SIA) system to

determinate indomethacin in pharmaceutical preparations upon alkaline hydrolysis in

micellar medium and detection of the fluorescent formed products. This new methodology

took advantageous of the enhance mixture of solutions due to the use of solenoid micro-

pumps as impulsion devices and was favourably compared with the reference method being

though considered a good alternative to other already available proposals (Pinto, Saraiva,

Santos, & Lima, 2005).

Indomethacin was also assayed with another SIA methodology coupled with solid

phase spectroscopy (SPS). SPS is an alternative to the limitation of the spectroscopic

methods since their applicability to complex matrixes, such as biological samples, is difficult.

SPS is based on the direct measurement of the degree of light absorption or emission

shown by an active solid support on which the analyte or its derivative compound is retained,

thereby achieving an important improvement with respect to conventional solution

spectrophotometry in both sensitivity and selectivity. The method proposed by Molina-

Garcia et al. is suitable for the analysis of indomethacin not only in pharmaceuticals but also

in human urine at levels usually found in patients after administration. Hence, the method

could be useful for easy, rapid, selective, and highly sensitive analysis of indomethacin

using a low-cost analytical instrumentation (Molina-Garca, Crdova, & Ruiz-Medina, 2010).

Silva et al. developed a SIA spectrophotometric methodology for the determination of

metoclopramide in pharmaceutical preparations (de Souza Silva, Saraiva, Santos, & Lima,

2007). The approach was based on the reaction of metoclopramide with Folin–Ciocalteu

reagent. In this SIA system, it was incorporated an in-line mixing chamber to promote

reaction zone homogenization, to increase reaction time, and to attenuate the high

refractive index gradient generated by the utilization of concentrated solutions, thus

enabling the minimization of the Schlieren effect. In a conventional sequential injection

system, the reaction coil length and flow rate have a profound effect on sample/reagent

mixing and reaction time, therefore on sensitivity and sampling rate. However, their impact

was minimized due to the introduction of the mixing chamber. With this configuration, it was

8

possible to enhance the reaction zone, both by increasing sample and Folin–Ciocalteu

reagent volumes, while ensuring an adequate homogenization. So, the inclusion of a mixing

chamber in an SIA manifold was revealed to be an expeditious alternative to the commonly

used reaction coil, as it ensures enhanced sample/reagents mixing and provides the means

for increased reaction development when a stopped-flow approach is implemented (de

Souza Silva et al., 2007). Comparing the linear range and the detection limit of this study

and another one (Guo, Feng, & Fan, 2009), the results are different. In the first study, the

linear range obtained was of up to 100 mg L-1 and the detection limit was 2.0 mg L-1 while

in the second study, the linear range obtained was 20–250 μg mL-1 for metoclopramide with

a detection limit of 0.034 μg mL-1.

In another perspective, Llorent-Marínez et al. developed a SIA methodology coupled

with SPS as an alternative to conventional optosensors, that is, the coupling of FIA and

SPS. This methodology was used for the determination of paracetamol after appropriate

derivatization reaction. The fluorimetric SIA optosensor used in this study was based on the

continuous measurement of the fluorescence signal from the oxidation product of the

analyte directly retained on the solid sensing phase. The method has been satisfactorily

applied to the determination of paracetamol in pharmaceuticals, and an additional recovery

study had been also performed, obtaining good results (Llorent-Martínez, Šatínský, Solich,

Ortega-Barrales, & Molina-Díaz, 2007).

On the other hand, Passos et al. developed a SIA methodology with a

spectrophotometric detector capable of determining three pharmaceuticals: metoprolol,

acebutolol, and propranolol. Comparing this methodology with British Pharmacopeia, SIA

allowed a drastic reduction in the time necessary for each determination (10-90 minutes

using BP procedures to 5 minutes in the SIA procedure). This fact is a significant

disadvantage in the pharmaceutical quality control, which advocates the use of the SIA

methodology that consumes a small sample and the characteristics present to be used for

the on-line pre-treatment of the sample and an almost real-time monitoring of the processes

of dissolution (M. L. C. Passos, Saraiva, Lima, & Korn, 2008).

In the next study, a SIA spectrophotometric method was described for the

determination of tetracycline (TC), chlortetracycline (CTC) and oxytetracycline (OTC) in

different sample matrices. The proposed method has been satisfactorily applied for the

determination of tetracycline and its derivatives in pharmaceutical preparations together

with their residues in milk and honey samples collected in Chiang Mai Province. The method

was rapid with a sampling rate of over 60 samples h−1 for the three drugs. The described

SIA system proved to be accomplished to sensitivity and detection limit, simplicity,

reproducibility, and rapidity. The advantages of the proposed SIA method are cost-

9

effectiveness and less operator input requirements system that can be controlled by means

of the laboratory-made software which is relatively inexpensive. Therefore, this method is

suitable for routine analysis (Thanasarakhan et al., 2011).

Naheid et al. reported the optimization and validation of a new, rapid, reagent-saving

and environmentally-safe method for chlorpromazine assay in pharmaceutical formulations

utilizing a SIA technique. This new SIA system was compared with another SIA technique

and the authors concluded that the sampling frequency of the new method was 51.4

samples/h while in the previous method was 22 samples/h. Furthermore, the total disposed

volume in the new SIA method, including water as a propeller solution, was 1260 mL while

that of the previous method was 2290 mL. The drastic reduction of the consumed volumes

of reagents and samples does not only render analytical method reagent-saving but also

offers better safety for the environment (Naheid et al., 2013).

In another study, Šrámková et al. developed a SIA methodology allowing the

determination of propofol without the requirement of sample pre-treatment. The method was

based on spectrophotometric detection after the enzymatic oxidation catalysed by

horseradish peroxidase and subsequent coupling with 4-aminoantipyrine leading to a

coloured product with an absorbance maximum at 485 nm. This procedure was compared

with a simple fluorimetric method, which was based on the direct selective fluorescence

emission of propofol in ethanol at 347 nm. In the spectrophotometric method, the time of

analysis per run was longer which included the enzymatic reaction. However, this did not

lead to higher solvent and reagent consumption, compared to other flow methods.

Furthermore, comparing both methods, it can be seen that the spectrophotometric provided

a sensitivity comparable to the simple fluorimetric detection and higher selectivity is

expected due to the enzymatic reaction (Šrámková et al., 2014).

Vakh et al. developed a SIA system based on the sandwich technique for the

simultaneous separate determination of iron (II) and ascorbic acid in pharmaceuticals (Vakh

et al., 2015). The implementation of a sandwich technique assumed the strict order

aspiration of sample solution between two selective reagents and allowed the carrying out

in reaction coil two chemical reactions simultaneously: iron (II) with 1,10-phenanthroline and

ascorbic acid with sodium 2,6-dichlorophenolindophenol. Despite the need to use the

relatively large sample volume for preventing the overlapping of the coloured zones, the

sandwich technique is characterized by satisfactory sampling frequency because of the high

use. Moreover, due to the optimization of physical parameters of the flow systems, it

becomes possible to achieve the excellent repeatability of analysis and considerable

reagent saving. So, the implementation of this flow system based on sandwich technique,

10

allow the determination of the iron (II) and ascorbic acid giving rise to two analytical signals

at the wavelength 510 nm for iron (II) and 512 nm for ascorbic acid (Vakh et al., 2015).

Regarding permeation studies, Franz diffusion cells can be used for monitoring the

release of pharmaceuticals. In a few words, Franz diffusion cells consist of two

compartments, a donor compartment and an acceptor compartment, with a barrier between

them. The donor compartment maintains the preparation of the drug; the acceptor

compartment contains an acceptor medium, the composition of which is determined by the

solubility of the drug. When performing the experiments, samples from the acceptor medium

are drawn through the sampling port of the Franz cell. The entire apparatus is maintained

at constant temperature by a water bath. The receiver fluid is mixed by a magnetic stir bar

(Solich, Sklenarova, Huclova, Satinsky, & Schaefer, 2003).

The apparatus was used in a SIA system to monitoring release profiles of

Indomethacin 1% gel. In this study, the authors compared the release rates of gels and

ointment prepared by different technological procedures since they used membranes of

different materials (poly-carbonate, mixed esters of cellulose, teflon, silicon, poly-vinylidene

fluoride) and different pore diameters. After this studies, a poly-carbonate membrane with

a pore diameter of 0.4 µm was selected to compare the release rates of indomethacin. In

SIA system, the release profile of the three different pharmaceutical formulations tested was

monitored without any necessity of human control during the whole 6 hours per cycle. This

enables the measurement of a high number of experimental points of the profile in shorter

time periods and therefore allows calculating the release rate after 2 h of the liberation

experiment. Moreover, the results obtained with the SIA system were confirmed by

conventional batch UV measurement (Solich et al., 2003).

On the other hand, Klimundová et al. performed a SIA system for carrying out

simultaneous release tests with three or more Franz cells to examine Belosalic® ointment

containing 3% salicylic acid as the active substance. This SIA system did not require any

human control during the experiments and allowed simultaneous monitoring of the release

tests for up to six Franz diffusion cells, making this system a useful tool for the release tests

used during manufacturing process control, monitoring pre- and post-changes in product

properties, monitoring batch uniformity as well as pre-formulation screening and product

development (Klimundova, Mervartova, Sklenarova, Solich, & Polasek, 2006).

In a different way, a SIA system could be used in pharmaceuticals dissolution assays,

since this methodology enables the analysis of samples within a wide range of

concentrations without manifold reconfigurations and with the consumption of a low sample

volume. The dissolution assays are important for quality control of pharmaceuticals

formulations. These tests supply detailed information about the dynamic characteristics of

11

the dissolution process and they are also useful for the development of new dosage forms

(Pimenta et al., 2006).

The first SIA system developed for dissolution assays was described for Liu and Fang

in 1998 (Liu & Fang, 1998). This study evaluated the dissolution profiles of ibuprofen tablets

sustained-release capsules and controlled-release matrix tablets. In this system, the

samples were collected after on-line filtration and the ibuprofen determined by monitoring

at 222 nm. Every hour, seven samples run in hexa-replicates were processed with a total

of 42 measurements. The system was continuously run for 12 h with an overall relative

standard deviation of absorbance 2.18%. The excellent long-term precision demonstrated

the stability and robustness of the automated system (Liu & Fang, 1998).

Subsequently, the same authors taking the advantage of the versatility and flexibility

of the system developed, performed the simultaneous monitoring aspirin, phenacetin, and

caffeine in compound aspirin tablets. For the simultaneous determination of the three active

substances, a partial least squares calibration technique was used. Using this system, it

was possible to analyse a total of 45 measurements (15 samples h-1) and the sample

consumption per determination was 0.2 ml. This system did not require the separation of

the analytes, since was quick and easily adapted to monitoring various dissolution vessels

and allowed the attainment of results in real time, with an elevated resolution of the

dissolution kinetics in the three analytes (Liu, Liu, Wu, & Fang, 1999).

Paseková et al. developed an automated procedure for formulation assays and

dissolution tests based on a SIA system involving an ion-selective electrode as a sensing

device. A tubular salicylate selective electrode was constructed for potentiometric

determination of acetylsalicylic acid in pharmaceutical formulations. The electrode was used

for sensing acetylsalicylic acid after its on-line chemical hydrolysis to salicylate in the SIA

system. The sampling rate was 6 h-1 but for the dissolution tests, the frequency was

increased to 20 h-1. The SIA set-up was employed for the assay of acetylsalicylic acid in

plain tablets, composed tablets, and effervescent tablets and for performing dissolution

tests of normal and sustained-release tablets. The results obtained in the acetylsalicylic

acid dosage assays in various pharmaceutical formulations, including composed and

effervescent tablets, were in compliance with the reference HPLC methodology, described

in the American Pharmacopeia. These two methodologies selectively determined the

acetylsalicylic acid even in the presence of other active substances (Pasekova, Sales,

Montenegro, Araujo, & Polasek, 2001).

In another study, Legnerová et al. monitored the dissolution assay of ergotamine

tartare in pharmaceutical formulations using a SIA system (Legnerova, Sklenarova, &

Solich, 2002). The samples were filtered and aspirated by the system and then directed

12

towards the fluorescent detector. These dissolution assays had a duration of 20 min, a

sampling rate of 120 samples h-1 and a sample consumption per measurement of 50 µL.

Various dissolution tests of ergotamine tablets were carried out and the results obtained

were in compliance with the British Pharmacopeia reference methodology (Legnerova et

al., 2002). These authors also proposed the fully automated approach for monitoring the

profiles of prazosin hydrochloride by fluorescence. In this study, the dissolution tests had a

duration of 60 min, a sampling rate of 70 samples h-1 and a sample consumption per

measurement of 20 µL. The results obtained by SIA technique was compared well with

HPLC standard method (Legnerova, Huclova, Thun, & Solich, 2004).

In addition to the previous studies, Legnerová et al. developed a SIA system for the

separation and simultaneous determination of two active substances in combined

pharmaceutical formulation, composed of ascorbic acid and rutin trihydrate, using a solid

phase extraction (SPE) microcolumn coupled to the SIA system (Legnerova, Satinsky, &

Solich, 2003). The SPE microcolumn was used for retention of rutin trihydrate, while the

ascorbic acid was eluted with the solvent front phase. The proposed methodology based

on SIA system with spectrophotometric detection was successfully applied to the assays

and dissolution studies of these active pharmacological compounds. The SIA–SPE method

exhibited similar or slightly lower sensitivity than conventional separation techniques (liquid

chromatography and capillary electrophoresis). These two methods were reliable and

sensitive, but required expensive instrumentation, involving lengthy sample preparation

procedures, making the whole analysis relatively slow and complicated. On the other hand,

SIA–SPE was much more rapid (the frequency of injections of samples was 26 h−1) and

allowed on-line treatment of the samples without time consumption possible. Furthermore,

this new system exhibited the advantages of both methods with minimum drawbacks:

analytes separation, robustness, full automation and online performance of dissolution

studies, high sample throughput and non-continuous flow and a drastic decrease in the

organic waste generation (Legnerova et al., 2003).

Pinto et al. developed a SIA methodology with a fluorimetric detector for the

determination of aminocaproic acid in pharmaceutical formulations. Since aminocaproic

acid does not emit fluorescence, it was necessary to perform a derivatization reaction.

Comparing the results obtained with the recommended method (USP 24), the results were

in agreement with the imposed criteria by USP 24, which refers that not less than 75% of

the amount of aminocaproic acid should be dissolved in 45 minutes (Pinto, Saraiva, Santos,

& Lima, 2006).

13

1.2. Nanoparticles and SIA systems

Nowadays, nanoparticles (NPs) associated with analytical methodologies are the

most extensively exploited area of nanotechnology. The fact that NPs have peculiar

characteristics allow the improvement of well established analytical methods or the

development of new methodologies for new analytes or matrices (Kaur & Gupta, 2009).

Currently, automated flow analytical methods enable the incorporation of all types of

NPs for varied applications. The automation of all stages of the analytical process allows

the implementation of effective in-line pre-treatments of samples, guaranteeing the

reproducibility during the insertion of solutions and the implementation of the reaction and

transport zone for detection. Beyond this, the automation provides conditions to perform the

assays in a closed environment, with inherent advantages in terms of reaction control,

reagent consumption and analysis time, allowing the implementation of more complex

reaction schemes or multiparameter determinations (Marieta L. C. Passos, Pinto, Santos,

Saraiva, & Araujo, 2015).

Flow-based techniques present also adequate characteristics for the synthesis of

nanomaterials since they provide large surface areas and reduced diffusion resulting in fast

mass transport due to the downsized dimensions of tubing or channels, covering a wide

range of compositions and tunable sizes. These characteristics facilitate the attainment of

high synthesis yields by means of a precise control of the reaction environment while

reducing the consumption of expensive or toxic reactants as well as waste production

(Zhao, He, Qiao, & Middelberg, 2011).

Thus, the association of flow systems with NPs exhibits enormous potential due to

the combination of different factors such as the inherent advantageous properties of NPs,

the precise and reproducible control of NPs / chemical analyte detection mechanisms and

the simplicity, versatility and easy techniques of flow-based techniques.

In the last years, flow researchers have informed the scientific community about the

implementation of NPs-based assays in SIA systems, mainly for analytical purposes,

exploring the versatility of SIA systems that allows the incorporation of additional devices

such as sensors, reactors or pre-treatment devices of samples allowing the accomplishment

of several concomitant actions. In this way, the automation of NPs-based assays using the

SIA systems mainly explores the versatility of the selection valve and the computer-

controlled mode of operation which, in addition to allowing the implementation of parallel

events at the valve's lateral inlets, also allows the strict control of conditions of reaction in

terms of space and time. All these possibilities make this technique very suitable not only

for analytical applications but also for the implementation of synthetic processes in which

14

the assays conditions determine the properties of the resulting NPs (Marieta L. C. Passos

et al., 2015).

Alarfaj and El-Tohany determined naloxone and raloxifene, an opioid antagonist and

anticancer respectively, through two different SIA methodologies, both with

chemiluminescence detector. In the determination of naloxone, the authors used the gold

nanoparticles-luminol-ferricyanide system, while in the determination of raloxifene it also

included the synthesis gelatin-capped bimetallic Au–Ag nanoparticles in the SIA system

and determinate raloxifene. So, despite the different configurations of SIA systems, this

proves the versatility of SIA methodology (N. A. Alarfaj & M. F. El-Tohamy, 2015; Alarfaj &

El-Tohamy, 2016).

The same authors developed another study in which they performed two novel

sensitive sequential injection chemiluminescence analysis and fluorescence methods for

the antibiotic trovafloxacin mesylate detection. The methods were based on the

enhancement effect of gold nanoparticles on luminol–ferricyanide–trovafloxacin and

europium(III)–trovafloxacin complex systems. The fluorescence method gave a wide linear

concentration range, but use one reagent was more favorable than using three reagents in

fluorescence detection. Based to its characteristic advantages, for example, robustness,

versatility, automated sampling and analytical procedures, low consumption of sample and

reagents, short analysis time, and computer compatibility, SIA with chemiluminescence

detection is more sensitive than the fluorescence method and facilitates precise fluidic

handling of sample and reagents (Nawal A. Alarfaj & Maha F. El-Tohamy, 2015).

In addition to previous studies, Alarfaj et al. developed SIA chemiluminescence

detection method for the determination of cephalosporin antibiotic cefditoren pivoxil. In this

case, the developed method was based on the enhancement effect of silver nanoparticles

on the chemiluminescence signal arising from a luminol–potassium ferricyanide reaction in

the presence of cefditoren pivoxil. According to the results of this study, the method was

found to be sensitive, reproducible and accurate for the determination of the drug in its bulk

powder, dosage forms, and biological fluids. The enhancement effect of cefditoren pivoxil

on the employed system was proportional to its concentration, which showed good results

relevant to the linear concentration range 0.001–5000 ng mL-1. The proposed method has

been proved to be fast, inexpensive, highly sensitive and precise (Alarfaj, Aly, & El-Tohamy,

2015).

In another perspective, the synthesize of NPs could be performed inside a SIA

system. Passos et al. developed the first successful attempt to synthesis biosilica NPs. In

this study, there was synthesis the NPs and also the immobilization of an enzyme, laccase,

on the nanostructures. The results obtained showed some advantages, namely,

15

reproducibility between all the samples used when compared with the small-scale batch-

based process, and the absence of clogging due to the operational characteristics of the

SIA technique. Besides the good reaction conditions, such as ambient temperatures,

physiological pH range, and aqueous solvents, this SIA procedure was shown to be a rapid,

simple, and more sensitive alternative method for the enzyme immobilization that results in

the physical entrapment of enzymes within silica nanospheres as they are formed (M. L. C.

Passos, Lima, & Saraiva, 2013).

1.3. Mesoporous silica nanoparticles (MSNs) and SIA systems

One of the types of nanoparticles that have been associated with flow systems is

mesoporous silica nanoparticles (MSNs). MSNs are solid materials which contain hundreds

of empty mesopores arranged in a two-dimensional network of a porous structure. This

ordered pore network, with size homogeneity, allows a high loading capacity and enables

the controlled-release of anticancer drugs (Alvarez-Berrios, Sosa-Cintron, Rodriguez-Lugo,

Juneja, & Vivero-Escoto, 2016).

MSNs exhibit the typical characteristics of inorganic nano-biomaterials such as high

thermal/chemical stability, tunable biocompatibility/biodegradability and resistance to

corrosion under extreme conditions (Chen, Chen, & Shi, 2014b).

MSNs present a variety of successful characteristics that potentiate the application on

drug delivery such as: high drug loading efficiency, high degree of tunability regarding size,

controlling drug release, protecting cargoes and cell and tissue targeting to prevent the

premature release of anticancer drugs, biodistribution, biodegradation and excretion (Hu,

Xiao, & Zhang, 2016; Martinez-Carmona, Colilla, & Vallet-Regi, 2015).

Furthermore, MSNs have unique tunable properties such as high surface area (> 700

m2 g-1), large pore volume (> 0.9 cm3 g-1) and surface area (2-10 nm and recently until 50

nm). MSNs also present a stable and rigid framework with great chemical, mechanical and

thermal stability. Although they have demonstrated good properties, it is also possible to

easily perform surface functionalization on the internal or external pore surface, for the site-

specific delivery (Alvarez-Berrios et al., 2016; Beltran-Osuna & Perilla, 2016; Douroumis,

Onyesom, Maniruzzaman, & Mitchell, 2013).

The structural properties of MSNs enable the encapsulation of hydrophobic and

hydrophilic drugs, making MSNs a promising drug delivery carrier for cancer therapy

(Alvarez-Berrios et al., 2016).

MSNs can be synthesized by different methods such as modified Stöber method,

aerosol-assisted synthesis or spray drying method, soft and hard templating, and dissolving

reconstruction (S. H. Wu, Mou, & Lin, 2013)

16

In an ideal synthesis of MSNs, it is necessary that the nanoparticles present several

good characteristics to be desirable for versatile applications, such as well-suspended

stable solution, controllable pore size, controllable uniform particle size and large pore

volume (S. H. Wu et al., 2013).

A larger pore volume and surface area enable a higher amount of drug encapsulation

within them (Rosenholm, Zhang, Linden, & Sahlgren, 2016). The different MSN pore sizes

allow to effectively control the anticancer drug release inside the tumor (Huang, Cole, Cai,

& Cai, 2016).

Although these properties can be tunable for the controlled drug delivery and

multifunctional in cancer treatment, the cytotoxicity and the biocompatibility have been

studied in vitro and in vivo. The results of these studies demonstrated that MSNs are very

biocompatible and non-cytotoxic (Douroumis et al., 2013).

In addition, MSNs can be also used in catalysis (Dickschat et al., 2013; Fu, Li, Han,

Liu, & Yang, 2014; Lin, Ren, & Qu, 2014; Rimoldi, Fodor, van Bokhoven, & Mezzetti, 2013),

dye-doped imaging and sensing (Miletto et al., 2014; Montalti, Prodi, Rampazzo, &

Zaccheroni, 2014; Wang, Sun, Wang, Bai, & Wu, 2014), adsorption (Gibson, 2014; Rimola,

Costa, Sodupe, Lambert, & Ugliengo, 2013), detection (Panda, Dhar, Malvi, Bhattacharjee,

& Sen Gupta, 2013; Zhou, Zheng, & Wang, 2014) and biomedical and theranostic systems

(Chen, Chen, & Shi, 2014a; Q. J. He & Shi, 2014; Z. X. Li, Barnes, Bosoy, Stoddart, & Zink,

2012).

Furthermore, the surface of MSNs has been considered a key factor directly affecting

the interaction of the MSNs with cells and it has been widely studied in biomedical and

pharmaceutical fields (Shahabi et al., 2015).

MSNs have high amounts of silanol (Si-OH) on their surface that provide covalent

conjugations with different types of functionalization, mainly polar molecules, such as:

carboxylate, amine, amine/phosphonate, polyethylene glycol, octadecyl and

carboxylate/octadecyl groups (Bagwe, Hilliard, & Tan, 2006; Dou, Hu, Li, Qiao, & Hao,

2011).

Other functional groups such as aromatic rings induce hydrophobicity on the surface

of MSNs, thereby preventing the loading with hydrophilic compounds into the mesopores

(Manzano & Vallet-Regi, 2010).

Since these nanoparticles have all these advantageous characteristics, they have

been associated with other types of nanoparticles aiming at mainly the development of

sensing devices. Pan and Yang developed the first FIA assay based on NPs resorting to

MSNs associated to magnetic NPs for the immobilization of an anti-carcinoembryonic

antibody. This study demonstrated that the implementation of this flow system provides also

17

appropriate conditions for the automation of the steps of antigen-antibody interaction and

sensor regeneration (J. Pan & Yang, 2007).

2. Mesoporous silica nanoparticles (MSNs) synthesis and surface

chemistry modifications

The synthesis of MSNs is commonly carried out by the electrochemical dissolution of

bulk silicon (Si) in a hydrofluoric acid (HF) based electrolyte. For this, a Si wafer is placed

between two HF resistant electrolyte cells in which platinum electrodes are located on both

sides of the Si wafer and an electrical current is applied between them. The upper side of

the Si substrates acts as the anode, where the oxidation and chemical etching of the Si

surface takes place with the subsequent dissolution of Si and formation of a Si layer. The

lower side of the wafer is the cathode, which is in contact with a conductive metal. Here,

the proton reduction takes place, leading to the formation and evacuation of hydrogen (H2)

(J. Salonen, A. M. Kaukonen, J. Hirvonen, & V.-P. Lehto, 2008).

Usually, the surface of MSNs is stabilized through different methods such as

oxidation, hydrosilylation or carbonization (Salonen & Lehto, 2008).

The surface of the UnTHCPSi, THCPSi, and TCPSi NPs (Figure 5), used herein, are

stabilized by carbonization. Carbonization of the surface of this NPs refers to the

substitution of the Si-H bonds for Si-C bonds, providing to this NPs stability towards harsh

chemical conditions and oxidation. The most common method that is used is the thermal

carbonization of Si in the presence of acetylene. Since the small molecules of acetylene

rapid diffuse through the pores, a complete carbonization can be achieved. Two types of

surface chemistries are obtained, depending on the temperature used. So, these are, Si-

CH, when the treatment temperature is below 650 ºC, rendering thermally hydrocarbonized

Si (THCPSi) and hydrogen-free Si-C species, and when the treatment temperature is above

700 ºC, thermally carbonized MSNs (TCPSi) (Bjorkqvist, Paski, Salonen, & Lehto, 2006;

Salonen, Björkqvist, Laine, & Niinistö, 2004; Sarparanta et al., 2011) are generated.

Similarly to the hydrosilylation of native MSNs, the surface of THCPSi NPs has been

functionalized by thermal treatment of the particles in undecylenic acid for 16h at 120 ºC to

obtain undecylenic acid modified THCPSi (UnTHCPSi). The presence of –COOH groups

allows easy covalent attachment of other biopolymers, macromolecules, or fluorescent dyes

having a great impact on drug delivery applications (Kovalainen et al., 2012).

APTES NPs are 3-aminopropyltriethoxysilane functionalized TCPSi NPs, in order to

covalently attach a –NH2 termination on the surface. This NPs provide to TCPSi NPs a new

secondary functionalization with amine group termination (-NH2). In drug delivery, a

positively charged surface enables the adhesion of the particles onto the cell walls where

18

they may more effectively release the drug payload (Makila et al., 2012; Slowing, G. Trewyn,

& S.-Y. Lin, 2006). Moreover, the amine termination, is common in immobilization of

peptides and specific cells, making this property surface very beneficial for biosensing

purpose (Kim, Cho, Seidler, Kurland, & Yadavalli, 2010).

APTES NPs improve drug delivery and present, also, other advantages since there

are many studies that provide evidence that APTES can reduce the toxicity of MSNs both

in vitro and in vivo. It is believed that the amine groups on the silica surface help to decrease

the surface reactivity of exposed surface silanol groups on the NPs. In other words, linking

APTES to MSNs can increase its overall biocompatibility by effectively reducing the

production of damage levels of intracellular reactive oxygen species (ROS) (Lehman et al.,

2016).

3. Chemotherapeutic agents used in treatment of cancer

An important aspect of cancer therapy is the selection of the best chemotherapeutic

agent for the specific type of cancer.

Chemotherapy proposes to silence the cancerous cells in order to completely

eradicate cancer, promoting a healthy change of life.

A variety of anticancer drugs that have been used to induce apoptosis, interfere with

the cell cycle and gene transcription and inhibit angiogenesis and, are summarized in Table

2.

Figure 5 - Chemical structures of THCPSi, TCPSi, UnTHCPSi and APTES NPs.

19

Table 2 - Main anticancer drugs used in conventional therapy

Drug Mechanism of Action Side Effects References

Doxorubicin

Intercalates with DNA.

Disrupts the topoisomerase-II-

mediated DNA repairing

mechanism, which results in cell

death.

Generates free radicals that

lead to the rupture of DNA

strands and damage of cellular

membranes and proteins.

Cardiotoxicity: iron-

related free radicals and

formation of a doxorubicinol

metabolite, and

mitochondrial disruption

that leads to apoptosis.

(Clementi,

Giardina, Di

Stasio, Mordente,

& Misiti, 2003;

Pang et al., 2013)

Paclitaxel

A mitotic inhibitor that blocks

the cells G2/M phase of the cell

cycle.

Enhances the polymerization

of tubulin to stable microtubules.

Stabilizes microtubules and

prevents their depolymerization

by cold and calcium.

Causes oxidative stress

and mitochondrial damage

after accumulation in

neuronal tissues, leading to

neuropathic pain.

(Jordan & Wilson,

2004)

Cisplatin

This platinum compound

reacts with water molecules to

obtain an unstable intermediate

that quickly interacts with DNA to

prevent cell division, resulting in

the cellular apoptosis.

Nephrotoxicity and

neurotoxicity.

Associated with cells

resistant to the drug.

(Bugarcic,

Bogojeski,

Petrovic,

Hochreuther, &

van Eldik, 2012;

Florea &

Büsselberg, 2011)

Methotrexate

The folic acid antagonist that

acts on the S phase of the cell

cycle, inhibiting the cell division.

Hepatotoxicity caused by

oxidative stress in liver

tissue.

Nephrotoxicity due to the

precipitation of the drug and

its metabolites in the renal

tubules.

(Hagag, Elgamsy,

El-Asy, &

Mabrouk, 2016;

Sayilmaz,

Karabulut, &

Ozgorgulu, 2016)

5-Fluorouracil

Pyrimidine analog that causes

an irreversible inhibition of

thymidylate synthase.

Renal toxicity.

Production of the

reactive oxygen species

(ROS) that damages the

(Longley, Harkin,

& Johnston, 2003;

Xiong et al., 2016)

20

Into tumor cells, 5-FU is

converted into fluorodeoxyuridine

monophosphate which can mis-

incorporate into DNA and RNA

chains and ultimately lead to cell

apoptosis.

kidney and induces toxic

effects such as necrosis

and apoptosis.

However, the conventional therapies are limited and have shown a variety of adverse

effects, such as high toxicity, poor tumor-specific delivery and nonspecific distribution, low

water solubility and therapeutic index, possibility to induce multidrug resistance (MDR), as

well as nausea and low counts of white blood cells are still the predominant used one (Lim,

Phua, Xu, Sreejith, & Zhao, 2016; Masood, 2016; G. B. Yang, Liu, Wu, Feng, & Liu, 2016).

The MDR is a phenotype acquired by cancer cells that confers resistance to certain

chemotherapeutic drugs, being responsible for 90% of the chemotherapeutic failure in

patients with metastatic cancer (L. Z. He et al., 2014; Huang et al., 2016).

Usually, the MDR of cancer cells is associated with the overexpression of ATP-binding

cassette (ABC) transporters, but it can also be due to other mechanisms, including

decreased drug influx, increased drug efflux, tumor proliferation driven by a small population

of self-renewing cancer cells, activation of detoxifying systems, activation of DNA repair

mechanism, avoidance of drug-induced apoptosis or activation of the immune response

(Chen, Chen, & Shi, 2014c).

Taking all of these into account, it is imperative to develop new approaches for

targeted drug delivery, in order to precisely release the chemotherapeutics in the tumor site,

and therefore, reduce the tumor recurrence, metastasis, MDR and side effects caused by

the conventional therapies (Feng et al., 2016; Y. N. Yang & Yu, 2016). In this way,

nanotechnology has emerged as an effective tool for the development of targeted drug

delivery systems employed for diagnosis, monitoring, and treatment of cancer (L. L. Li et

al., 2014).

These nanomedicines promoted an increase in the therapeutic efficacy of a large

number of anticancer drugs and reduce their toxicity profile (Lim et al., 2016; Y. N. Yang &

Yu, 2016).

In general, drug delivery systems (DDS) should be biocompatible, present high

loading/encapsulation of drug molecules without leaking the drug molecules, specificity to

a certain cell or tissue and controlled release of drug molecules that allows an effective

concentration at the target site. Furthermore, DDS can improve the solubility of poorly

water-soluble drugs, enhance the bioavailability and pharmacokinetic profiles of anticancer

21

drugs and increase the drug's half-life by reducing immunogenicity, while minimizing their

side effects (Dianzani et al., 2014).

The nanoscale dimensions of DDS can alter the drug biodistribution by accumulating

the drug at the tumor site as a result of the enhanced permeability and retention (EPR)

effect (Mo & Gu, 2016). This effect is a unique anatomical and pathological characteristic

of solid tumors, triggered by an extensive vascular permeability and insufficient lymphatic

drainage (Mo & Gu, 2016).

Other important features of a DDS rely on the ability to modify the surface of the

nanocarriers with targeting ligands for active targeting of tumor cells, as well as to achieve

a stimuli-responsive drug release through the coating with microenvironment-sensitive

molecules of the surface of the DDS (Q. J. He, Guo, Qian, & Chen, 2015).

Therefore, targeted nanomedicine therapeutics that improve the pharmacokinetic

profile and decrease the toxicity of chemotherapeutic agents can decrease the resistance

of tumors against anticancer drugs to overcome the MDR problems (Dianzani et al., 2014;

Zhu & Liao, 2015).

4. Biomedical applications of MSNs: drug delivery

The beneficial biocompatibility and biodegradability of MSNs along with the vast

surface area-to-volume ratio and pore volume makes them ideal candidates as drug

delivery vehicles (Anglin, Cheng, Freeman, & Sailor, 2008; Bimbo et al., 2010; Godin et al.,

2010; Low, Voelcker, Canham, & Williams, 2009).

The drug loading capacity has been one of the key parameters for an efficient drug

delivery system (Couvreur, 2013).

The loading and adsorption of drugs into the pores of MSNs and their release is

controlled by many factors that can be tuned in order to successfully load and release a

different kind of cargos. Among these factors are the surface area and the pore volume,

which determine the amount of drug that can potentially be loaded; and the degradation

rate of MSNs itself. Moreover, the surface chemistry which determines the physicochemical

properties of the surface of the pores and the interactions between the pore wall, to load

the drug are also important. Beyond this, there are other parameters that have an impact

on the loading of drugs into the pores of MSNs that are related to the solvent used and the

properties of the drug. The surface properties of the pores, the surface tension, and the

viscosity of the drug solution interfere with the wettability of the pores. So, low viscosity and

surface tension allow better filtration of the solvent into the pores improving the drug loading.

Furthermore, the physicochemical properties and concentration of the drugs also play a role

in drug loading into MSNs (Lehto, Riikonen, & Santos, 2014; E. C. Wu et al., 2011).

22

Drug loading within MSNs can be achieved by immersion, impregnation, or covalent

attachment, however, the most common loading method is immersion method (Salonen et

al., 2005). This method consists of the immersion of MSNs in a drug solution for a certain

period of time, usually in the range of hours, to allow the solvent to diffuse into the pores

carrying the drug which is adsorbed on the pore walls driven by geometrical and chemical

interactions. The solvent is then removed and MSNs can be carefully washed with an

appropriate solvent to remove the drug bound to the external surface. In this method, it is

required a relatively high concentration of drug solutions, being immersion method not

optimal for the loading of valuable drugs (Salonen et al., 2005).

To overcome this disadvantage, other methods can be used to incorporate drugs into

MSNs, such as impregnation or covalent attachment. In the impregnation method, the drug

solution is mixed with MSNs and the solvent is dried, forcing the diffusion of the drug into

the pores (Limnell et al., 2011; Nieto et al., 2015). Thereby, this valuable drugs may be

efficiently loaded, although the adsorption of the drug on the surface is difficult to control (J.

Salonen, A. M. Kaukonen, J. Hirvonen, & V. P. Lehto, 2008)

The drug loading can be also affected by several factors involved in the process.

These include pH dependency, temperature and time but the most important factor is the

possible chemical reactivity of the drug loading solution with the MSNs surface

(Chakraborty, Dhakshinamurthy, & Misra, 2017).

The loading solvent is chosen according to the solubility of the drug and is in several

cases water, ethanol or dimethylsulfoxide (DMSO) solutions. The solvent has the function

to drive drug molecules into the pores (Kovalainen et al., 2012).

The release assays are usually performed in buffered salt solution with fixed pH in

order to simulate the stability of the delivery system in the different stages of the oral

administration route. For targeted drug delivery it is extremely important to always test the

release with other simulating mediums, especially in plasma in order to get a wider insight

of the drug release in the complex intravenous environment (Tay et al., 2004).

5. Aims of the study

In this study, it is our intention to shift the focus of the application of the SIA

methodology to assays using MSNs and to exploit the potential of this assembly to perform

drug loading and drug release studies. While for drug loading it will be explored the

reproducible characteristics of flow systems, in the second its versatility in accommodating

different dispositives around the valve will be tested.

23

As a chemotherapeutic agent it was used 5-fluorouracil, represented in figure 6,

because is one of the most frequently used chemotherapeutic drugs in the treatment of

cancer.

5-FU acts by blocking key enzymes in nucleotide synthesis and works during the S-

phase of cell division. This chemotherapeutic drug is metabolized to 5-fluorodeoxyuridine

monophosphate (FdUMP) which, in the presence of 5, 10-methylenetetrahydrofolate,

irreversibly inhibits thymidylate synthase (TS). TS inhibition results in nucleotide pool

imbalances, impaired DNA synthesis and a reduction in DNA repair (Eynali, Khoei, Khoee,

& Esmaelbeygi, 2017; Loc et al., 2017).

Despite 5-FU potency in treating cancers, its clinical applications are limited due to its

short half-life, disease resistance, and severe side effects such as myelosuppression,

dermatitis, cardiotoxicity, neurotoxicity, nausea and vomiting which is associated with its

high non-specific in vivo distribution (Tawfik, Ahamed, Almalik, Alfaqeeh, & Alshamsan,

2017). Since 5-FU presents severe systemic assays, and a poor accumulation in tumor

tissues with a low therapeutic response rate of cancer tissues with it is administrated in high

dosage for expected efficacy (Pico, Avila-Garavito, & Naccache, 1998; Viele, 2003).

So, it is advantageous for this chemotherapeutic drug to be loaded in a NP to

overcome these drawbacks. Different functionalized MSNs were used to evaluate the

differences in their behaviour. Batch and SIA procedures were tested and compared for the

different behaviours of the MSNs towards both methodologies.

Figure 6 - Chemical structure of 5-FU.

Chapter 2

Materials and Methods

25

1. Introduction

In this chapter, it will be reported all techniques and experimental procedures applied

in this work, including the preparation of the solutions as well as the instrumentation and

the materials.

The schematic representation of SIA systems manifold will be also exposed.

Moreover, the apparatus used in SIA systems and their mode of operation and control will

be explained.

It will be also describe the preparation of the solutions and formula of the experimental

calculations used.

2. Reagents and solutions

All solutions were prepared using ultrapure water with a specific conductance less

than 0.1 µS cm-1 and chemicals of analytical grade.

5-fluorouracil (5-FU) and methanol were purchase to Sigma-Aldrich and Chem-Lab,

respectively.

A phosphate buffer solution (PBS) 0.2 mol L-1 (pH 7.4) was used as carrier solution of

both sequential injection procedures.

The mobile phase used in HPLC consists in PBS 0.2 mol L-1 (pH 6) and methanol

(90:10, v/v).

The mobile phase of column storage consists of water mixed and methanol (50:50,

v/v).

A solution of 5-fluorouracil (5-FU) 5mg mL-1 was daily prepared through the dissolution

of 5-FU (Sigma-Aldrich) in PBS 0.2 mol L-1 (pH 7.4).

The four types of MSNs used in this study were: 3-aminopropyltriethoxysilane

(APTES), thermally hydrocarbonized porous silicon (THCPSi), thermally carbonized porous

silicon (TCPSi) and undecylenic acid functionalized thermally hydrocarbonized porous

silicon nanoparticles (UnTHCPSi).

3. Instrumentation

3.1. Auxiliary equipment

According to the required precision, the reagents were weighed in Kern (Balingen,

Germany) ABT 120-5 DM analytical balance (precision of 1×10-6 g).

26

The solutions were stirred by Labbox MAGM-005-010 magnetic stirring bar. To degas

the mobile phase solution used in HPLC a VWR® USC 100T5 (VWR International, Radnor

USA) ultrasonic bath was used.

The pH of buffer solutions was measured using a Crison® model GLP 22 milivoltimeter

(Crison Instruments, Allela, Spain) coupled to a combined Ag/AgCl glass electrode (Crison®

52-02). The calibration of the combined electrode was daily performed with commercial

standards of pH = 4.00 (Riedel-de Haën, 33543), pH = 7.00 (Riedel-de Haën, 33546) and

pH = 9.00 (Riedel-de Haën, 9889).

The temperature of release assays was controlled by a stirrer with heating IKA® C-

MAG HS7 equipped with the sensor IKA® ETS-D5 (IKA-Werke GmbH & Co., Staufen,

Germany) was employed to control temperature and continuous stirring of the 5-FU

solution.

Automatic pipettes DiscoveryPro® with maximum capacities of 100, 1000 and 5000

μL were used, and calibrated regularly. All solutions were prepared in class A glassware,

suitably washed.

In loading and release batch procedure, a stirring plate (Velp Scientifica) an HPLC

equipment (Jasco PU-4180) with a C18 column (Grace Smart RP C18 50mm x 4.6mm), a

100µL syringe (Microsyringes borosilicate glass 3.3) and a photodiode array detector

(Jasco MD-4010) were used to determine the concentration of 5-FU.

3.2. Flow Apparatus

The SIA system used in loading assays (Figure 7) consisted of a syringe module Bu1S

from Crison Instruments S.A. (Allela, Barcelona, Spain) and a 10-port multiposition

CheminertTM selection valve. A glass syringe of 5 mL total dispense volume (Hamilton

Bonaduz AG, Switzerland) was coupled to the syringe equipment and driven by a stepper

motor. Solenoid head-valves allowed the commutation of the syringe either to the manifold

or to the carrier. The communication between the computer and the modules was done

resorting to a RS232C serial protocol. The software used for the instrumental control was

developed using visual basic and communication with instruments was accomplished by

means of RS-232C asynchronous protocols, using embedded dynamic libraries. A

sequential output of the commands and evaluation of the equipment status was performed

through the implementation of the control algorithm based on the use of a set of

interdependent timers. Computer control enabled the control of flow rate, flow direction,

valve position, stop flow duration, sample, enzyme and substrate volumes, as well as data

acquisition and processing. Manifold components were connected by means of 0.8 mm i.d.

PTFE tubing, which was also used for the holding coil (2m).

27

Figure 7 - (a) Schematic representation of the SIA manifold used in loading assays. C:

carrier (phosphate buffer 0.2 mol L-1 pH 7.4), S: syringe, HC: holding coil (2m), SV: selection

valve, 5-FU: 5-fluorouracil, NPs: nanoparticles, M: loaded NPs, W: waste. (b) Sequence of

aspiration of solutions into the holding coil of the flow system: NPs and 5-FU, repeated ten

times.

The SIA system used in the release assays (Figure 8) consisted of a Crison® module

that incorporates an 8-port selection valve and a Gilson® Minipuls 3 peristaltic pump

equipped with polyvinyl chloride pumping tube of 1.30 mm i.d. The pump responsible for

the propulsion of the carrier was connected to the holding coil and through this to the central

port of the SV.

Fluorescent measurements were performed in a Jasco® FP-2020 Plus fluorescence

detector, incorporating a 16 µL flow cell that was connected to the RC2.

The excitation and emission wavelengths were set at 301 and 345 nm, respectively.

A Microsoft QuickBasic 4.5 software was applied in the control of the flow system.

The analytical signals were recorded on a Kipp & Zonen BD 111 strip chart recorder

or acquired via computer.

28

3.2.1. Aspiration/ propulsion device

The aspiration/ propulsion device used in loading SIA system was a syringe pump

module Bu1S from Crison Instruments S.A. (Allela, Barcelona, Spain) (Figure 9). The

equipment included a glass syringe, a piston and connectors of polytetrafluoroethylene

(PTFE). The device was equipped with a syringe of 5mL total dispense volume (Hamilton

Bonaduz AG, Bonaduz, Switzerland), driven to a stepper motor. Solenoid head-valves

allowed the commutation of the syringe either to the manifold tubes or to the buffer

container, depending on the position “OUT” or “IN”, respectively, independently of the

movement assumed by the piston (aspiration or propulsion). The propulsion/aspiration

operation was controlled by computer, namely the beginning and the end of the operation,

velocity and direction of solutions movement. The complete movement of the piston

corresponded to 40,000 steps. The volume corresponding to one step depended on the

capacity of the selected syringe.

Figure 8 - Schematic representation of the SIA manifold used in release assays. C: carrier

(phosphate buffer 0.2 mol L-1 pH 7.4), PP: peristaltic pump, HC: holding coil, SV: selection

valve, F: filter, S: sample, D: detector, W: waste.

29

The aspiration/ propulsion device used in release SIA system was a peristaltic pump

Miniplus 3 from Gilson® with 4 channels (Figure 10). This device is controlled by a computer

taking into account the aspiration/ propulsion time, direction and rotation speed. In this work,

it was used polyvinyl chloride (PVC) impulsion tubes (Gilson®) with an internal diameter of

1.3 mm. A proper calibration of PVC tubes was carried out before starting the experimental

work, in order to confirm the flow rates to be used in the aspiration/ propulsion of the

solutions volumes. Basically, the mass of water aspirated or propelled in a determined

period of time was weighed, at different speeds of rotation of the peristaltic pump. So, a

relation was established between the speed of rotation of the pump and the time and,

inherently, between the flow rate and the time. This relation allowed to define the volumes

to be aspirated or propelled in terms of time.

These tubes substitution was accomplished when there were alterations of the flow

rate, loss of elasticity and increase of the number of pulses.

Figure 9 - Syringe pump module Bu1S from Crison Instruments S.A.

30

3.2.2. Fluid selector valve

In loading SIA system, a 10-port multiposition CheminertTM selection valve (Valco®

Instruments, Houston, EUA) was used as fluid selection valve device to perform the loading

assay of 5-FU into the NPs. The rotor moved in a two-way mode, which enables the access

to side port by shorter route.

The selection valve had a central channel which was connected to one of the ten side

ports, managing the fluids direction. The reagents and samples were aspirated by side ports

to system (holding coil) and one of the side ports was used to connect to detector.

In release SIA system, a 8-port multiposition from Valco® (Figure 11) was used as

fluid selection valve device to perform the release assay of 5-FU. As in previous described

selection valve, the rotor and the operation of the apparatus is similar.

The rotor in both selection valves are two-way mode. It is necessary to take into

account some features of these devices, such as: the number of peripheral ports and the

number of operations to be performed, the direction of valve rotation being selected to

correspond to the smallest path between two sequential ports, as well as consider that the

presence of some ports with no purpose can decrease the sampling rate.

Figure 10 - Peristaltic pump Miniplus 3 from Gilson.

31

3.2.3. Tubing and other manifold components

The different components of the SIA system were connected with PTFE tubing

(Omnifit), with 0.8 mm internal diameter and 1.6 mm external diameter. The holding coil and

reaction tube used in the SIA systems were made with the same type of PTFE tubing in an

eight configuration to promote radial diffusion and reduce laminar diffusion of solutions

throughout the systems (Figure 12). The holding coil length ensured, simultaneously, a

minimal dispersion of solutions and no contamination of buffer solution present in the

aspiration/propulsion device.

Although all holding coils used in both SIA systems, the eight configuration used in

loading SIA system was applied to promote the efficiency of the NPs and 5-FU solution

mixture.

2 1

Figure 11 - Selector valve with 10-port multiposition from Valco®.

Figure 12 - PTFE tubing used in SIA systems (1) and PTFE tubing in an eight

configuration (2).

32

3.2.4. Informatics’ control

SIA systems require an informatics control to enable the

synchronization between the aspiration/ propulsion device and the fluids selector valve, and

thus the automatization of the system. In this work, it was used two different informatics

control.

In the SIA system used to perform the loading assays the control by computer was

based in Visual Basic software that enabled the control of analytical parameters that may

influence the performance of the flow system. The experimental conditions, previously

established in a multi column table (Figure 13), describe the valve position, speed of the

piston movement (according the time and steps selected), the time to be spent in each step

and the possibility to repeat any step previously done. The computer program also allowed

the simultaneous control of piston movement (aspiration or propulsion) and commutation of

the syringe to the manifold or to the carrier container. The commutation to the manifold or

to the carrier container was represented as “OUT” or “IN”, respectively. The direction of the

piston movement was commanded by positive steps to propel and negative steps to

aspirate.

On the other hand, SIA system where it was performed the release assays was

controlled by a specific software, named Microsoft Quick-Basic which commanded the

analytical cycle according to the data entered. With the informatic programme used it was

selected (Figure 14) the valve position, the time and flow rate that determines the volume

Figure 13 - Computer software in the SIA system where it was performed loading

assays.

33

spent, the direction of the selector valve (aspiration or propulsion) and the length of stay in

this position.

3.2.5. Detection device

In automatic loading assays it wasn’t used a detection device, however, in automatic

release assays a FP-2020 Plus fluorescence detector, from Jasco® was used (Figure 15).

This device is equipped with a 16 µL internal volume flow cell and covers a wide

wavelength range both for excitation and emission from 220 to 700 nm with proven stability.

Figure 14 - Computer software in the SIA system where it was performed release assays.

Figure 15 - Photography of fluorescence detector device.

34

4. Experimental procedure

4.1. Loading batch procedure

The drug loading is affected by several factors involved in the process. These include

pH dependency, temperature, and time, but the most vital factor is the possible chemical

reactivity of the drug loading solution with the MSNs surface. The possible chemical

reactivity of the drug loading solution with MSNs surface can change the physicochemical

properties of MSNs materials.

The loading batch procedure of the model anticancer drug, 5-FU, into the NPs, was

performed by the immersion method using concentrated aqueous solutions of the drug.

The immersion method is the most commonly loading technique used since it is a

simple process and has the possibility to be performed at room temperature. This method

follows the principle of physical adsorption of the drug molecule onto the surface of the

pores of MSNs. The charge and hydrophobicity along with the concentration of the drug

loading solution, loading time and temperature are factors that influence the drug adsorption

onto MSNs (Haidary, Corcoles, & Ali, 2012). Basically, the MSNs is dispersed into the drug

solution at a suitable concentration in a chosen solvent. After reaching equilibrium, meaning

that the loading agent fills the pores' volume, the drug-loaded MSNs can be obtained by

filtration or centrifugation. The amount of drug molecules loaded is largely determined by

the pore volume and the concentration of the drug-loading solution that can also affect the

drug-loading degree in MSNs (Jarvis, Barnes, & Prestidge, 2012).

About the procedure, firstly 5 mg of 5-FU was dissolved in 1 mL of PBS (pH 7.4).

Then, 5-FU was added to the 200 µg of NPs. After 120 min stirring (300 rpm) at room

temperature (RT) 5-FU-loaded NPs were separated from the supernatant by centrifugation

at 13,500 rpm for 5 min (supernatant 1). To remove drug molecules loosely adsorbed on

the surface of the NPs, the NPs were gently washed with 500 µL of PBS pH 7.4 (supernatant

2).

Prior to HPLC analyses, the 5-FU-loaded NPs were re-suspended in 2 mL of PBS at

pH 7.4 and stirred (300 rpm) at RT for 60 min. The suspensions were then centrifuged at

13,500 rpm for 5 min and the supernatant was used for the detection of 5-FU using an

HPLC analysis (supernatant 3).

The calculation of percentage (%) of loading of 5-FU within each NPs was made

through the following equation:

%loading= HPLC conc. (mg mL−1) × final volume

weight NPs + ( HPLC conc. × final volume) (equation 1)

35

where HPLC conc. (mg mL -1) was the concentration of the supernatant 3, the final volume

was the last volume added to 5-FU loaded NPs (2mL) and the weight NPs was the weight

of NPs used in the assay (200 µg).

Each MSN was evaluated in triplicate.

4.2. Loading sequential injection procedure

In this assay, 5mg of 5-FU was dissolved in 450 µL of PBS pH 7.4. Also, it was

withdrawn from the suspension of NPs 85 µL which were placed in 250 µL of PBS pH 7.4.

The loading sequential injection procedure of 5-FU into the NPs was performed by the

sequential aspiration of 10 aliquots of 5-FU and NPs within the flow system. After that, the

aliquots are propelled to a glass bottle, resulting in a suspension of loaded NPs in a solution

of 5-FU not loaded.

Then, 5-FU-loaded NPs were separated from the supernatant by centrifugation at

13,500 rpm for 5 min (supernatant 1). To remove 5-FU molecules loosely adsorbed on the

surface of the NPs, these were gently washed with 500 µL of PBS pH 7.4 (supernatant 2).

Prior to HPLC analyses, the 5-FU-loaded NPs were re-suspended in 2 mL of PBS at

pH 7.4 and stirred (300 rpm) at RT for 60 min. The suspensions were then centrifuged at

13,500 rpm for 5 min and the supernatant was used for the detection of 5-FU using an

HPLC analysis (supernatant 3).

The calculation of percentage (%) of loading of 5-FU within each NPs was through

the equation 1 previously defined.

Each MSN was evaluated in triplicate.

4.3. Release batch procedure

In order to perform the 5-FU release assay of the NPs, it was necessary initially to

load the drug into the NPs, using the same assay described in points 4.1 or in 4.2. However,

instead of the NPs being re-suspended in 2 ml of PBS pH 7.4 in the last step, they are re-

suspended in 5 mL. Then 100 µL aliquots of the supernatant are withdrawn from the solution

at different time points: 5, 10, 15, 20, 25, 30, 60, 120, 180 minutes.

Each MSN was evaluated in triplicate.

4.4. Release sequential injection procedure

To perform the 5-FU release assay of the NPs, it was necessary initially to load the

drug into the NPs, using the same assay described in points 4.1 or 4.2. The only difference

is that after loading NPs, they were re-suspended in 10 mL. Then, before doing the analysis,

36

it was necessary to fill the tube where the NPs were aspirated in the different time points:

5, 10, 15, 20, 25, 30, 60, 120 and 180 minutes.

Each MSN was evaluate in duplicate.

Chapter 3

Results and Discussion

38

1. Introduction

MSNs have been established as versatile platforms for drug delivery assays. The

loading of drugs into the porous structures of these NPs is a possibility to control drug

release or deliver the ideal concentration of therapeutic molecules to the suitable location

in a controlled manner (Makila et al., 2014). The implementation of the assays in the SIA

systems was accomplished through the optimization of several physical and chemical

parameters by the univariate method, where only one parameter is changed while others

are kept constant. The optimization of the process of loading of these kind of NPs with drugs

and at the same time the monitoring of the release of the same drug is detailed and

discussed.

2. Optimization of sequential injection procedure for the loading of NPs

The mesoporous particles may present different sizes and so the first stage involved

the selection of those that could be used in order to avoid the obstruction of the flow system

tubes.

Subsequently, the optimization of the loading assay was conducted in strictly aqueous

media with the purpose of reducing the time to load 5-FU into NPs, not affecting the % of

loading normally obtained in batch assays.

Previously, it was defined the total volume of the 5-FU solution and NPs that should

be aspirated to the system so as to have a final volume of loaded NPs of 1 mL inside the

glass bottle. This guaranteed a similar final volume used in the batch studies. So, a volume

of 450 µL of 5-FU and 67 µL of NPs were aspirated to the system and then propelled to the

glass bottle with PBS pH 7.4 until the volume of 1 mL.

All the optimization of this SIA system was done with the TCPSi NPs. The loading

percentage evaluation was carried on using the batch procedure referred in point 4.3 of

Materials and Methods – Release batch procedure. The concentration values used were

obtained by the conversion of the areas obtained in HPLC using the next calibration curve

(Figure 16).

39

The calibration curve was A=5×107 (±1.2×106) C + 4×104 (± 6×104); R2= 0.9987,

where A is the area obtained in HPLC and C is the concentration of 5-FU in mg mL-1,

respectively, with 95% confidence limits for the intercept and slope.

The optimized parameters comprised the study of aspiration order of the reagents,

aspiration, and propulsion flow rate as well as the aliquots number, presented in Table 3.

Table 3 - Results of the optimization of loading assay in the flow system.

Parameter Range Selected value

Aspiration order NPs-5-FU; 5-FU-NPs NPs-5-FU

NPs aspiration flow rate 0.075 mL/min ; 0.15 mL/min 0.15 mL/min

5-FU aspiration flow rate 0.5 mL/min ; 1 mL/min 1 mL/min

Propulsion flow rate 0.5 mL/min ; 1 mL/min 0.5 mL/min

Aliquots number 5 ; 10 10

For the choice of the better conditions, it was considered into account the values of

% loading obtained in the assays and the RSD obtained. So, in the next table (Table 4) it

was described the different conditions tested and the results obtained with the average

value, standard deviation and RSD (relative standard deviation). Each condition tested was

evaluated in triplicate.

As it was previously referred, an important feature affecting system performance is

the aliquots mixing and zone homogenisation. In a SIA system these aspects are, to a great

extent, determined by the sequence of insertion (or aspiration) which dictates how the

0

1000000

2000000

3000000

4000000

5000000

6000000

0 0.02 0.04 0.06 0.08 0.1 0.12

Are

a

concentration 5-FU (mg mL -1)

Figure 16 - Calibration curve obtained in HPLC to determine the concentration of 5-FU

in loading assays.

40

different zones will mutually inter-disperse or which zone will endure a higher degree of

dispersion.

The finest results, in terms of mixing efficiency were obtained when 10 aliquots of 6.7

µL of NPs and 45 µL of 5-FU were used being the first one of the mixture of reagents was

first introduced in the holding coil followed by the sample plug, which could be explained by

the lower sample dispersion and the greater reagents zone penetration.

Table 4 – Tested conditions and respective results of the optimization of loading assay in SIA system.

Tested conditions Results (average ± standard deviation and RSD)

NPs – 5- FU 0.15 mL/min aspiration NPs 1 mL/min aspiration 5-FU 1 mL/min propulsion 10 aliquots (6.7µL and 45 µL)

3.28 ± 0.90 ; 27.55 %

5- FU - NPs 0.15 mL/min aspiration NPs 1 mL/min aspiration 5-FU 1 mL/min propulsion 10 aliquots (45 µL and 6.7 µL)

4.61 ± 2.85 ; 61.88 %

NPs – 5- FU 0.075 mL/min aspiration NPs 0.5 mL/min aspiration 5-FU 1 mL/min propulsion 10 aliquots (6.7µL and 45 µL)

5.02 ± 5.62 ; 112 %

5- FU - NPs 0.075 mL/min aspiration NPs 0.5 mL/min aspiration 5-FU 1 mL/min propulsion 10 aliquots (45 µL and 6.7 µL)

4.68 ± 2.33 ; 50 %

NPs – 5- FU 0.15 mL/min aspiration NPs 1 mL/min aspiration 5-FU 1 mL/min propulsion 5 aliquots (13.4 µL and 90 µL)

9.73 ± 2.36 ; 24.23 %

NPs – 5- FU 0.15 mL/min aspiration NPs 1 mL/min aspiration 5-FU 0.5 mL/min propulsion 10 aliquots (6.7 µL and 40 µL)

7.88 ± 0.24 ; 3.11 %

NPs – 5- FU 2.11 ± 1.11 ; 52 %

41

0.15 mL/min aspiration NPs 1 mL/min aspiration 5-FU 0.5 mL/min propulsion 5 aliquots (13.4 µL and 90 µL)

The aspiration flow rate was fixed at 1.0 ml min-1 for the 5-FU solution and 0.15 mL/min

for the NPs suspension. The propulsion flow rate towards the detector was fixed at 0.5 ml

min-1 avoiding the dilution of the final compound.

The choice of the parameters previously described has been done accordingly not

only to the % loading obtained but also to the RSD obtained. So, although the % loading

obtained with the selected parameters wasn’t the % loading best result (7.88 ± 0.24; 3.11%),

the RSD is less than the others. This is important as it assures reproducibility between the

different loadings, with non-significant differences in the three assays done.

The analytical cycle established for the optimization and implementation of loading

assay in the SIA system is presented in Table 5.

Table 5 - Analytical cycle to perform loading assay in SIA system

Step Position valve

IN/OUT Volume (mL)

Time (s)

Flow rate (mL min-1)

Repeat Step Event

1 4 i 5 22 13.6 0 0 Filling the syringe

2 4 i 1.0625 21.25 3 0 0 Partial emptying of the syringe

3 9 o 6.7×10−3 2.7 0.15 0 0 Aspiration of NPs

4 8 o 4.5×10−2 2.5 1 9 3 Aspiration of 5-FU

5 10 o 1 120 0.5 0 0 Propulsion the loaded NPs to the glass bottle

6 4 o 0 2 0 0 0 End of the analytical cycle

In each cycle, the syringe was filled completely and next it was necessary to partial

emptying the syringe to enable the aspiration of the aliquots of NPs and 5-FU afterwards.

Then, it was aspirated sequentially 10 aliquots of 6.7 µL of NPs and 45 µL of 5-FU,

respectively. After this, the flow was reversed and the loaded NPs was propelled by the

42

carrier solution to the glass bottle. The following steps are explained in point 4.2. of Materials

and Methods - Loading sequential injection procedure .

For each NP, the assay was performed in triplicate.

3. Optimization of release sequential injection procedure

To obtain the release profile using the SIA system it was used a fluorescence detector

to monitorize the 5-FU released. For that it was necessary to evaluate first the conditions

that would give a linear interval range for the drug accordingly to the concentrations

expected. So, it were selected the volume of standard solution and the flow rate used

towards the detector. Then after its connection to the release vessel apparatus and the filter

support it was necessary to evaluate again the performance of the developed flow manifold.

The sample feeding tubing was immersed into the release medium in such a way as to

restrain the occurrence of hydrodynamic disturbances and the influence of the filter support

was also studied since it could impose a resistance to the flowing stream affecting the

aspiration flow rate.

It was observed that the performance of the developed system, including the

aspiration and propelling flow rates, was unaltered and that all of the previously optimised

analytical parameters were adequate and did not require any adjustment. The release

profiles resulting from periodic measurements showed that the release of 5-FU is very fast,

requiring no more than 3 h to obtain the release profile. Sample aliquots were collected at

pre-set time intervals through an in-line filter and analysed without further treatment.

The first stage of development the automated procedure involved the adaptation of

previously described batch release assays to the SIA format. Subsequently, the

optimization of the assay was conducted in strictly aqueous media with the purpose of

simulating physiological human conditions (pH 7.4 and 37 ºC) to further apply to the release

assay of 5-FU of the NPs.

The volume necessary to fill the tube between the filter and the valve was determined

using the blue dye of bromothymol. With this dye, it was possible to acknowledge the

volume for the complete filling of the tube, since the filter holder used had a considerable

dead volume.

The optimized parameters comprised the study of the 5-FU volume aspirated to the

system, the replacement volume to the release vessel and the flow rate to the detector. The

aspiration flow rate of the 5-FU solution from the tube with the filter was stablished as 0.5

mL min -1 and the flow rate to clean the holding coil was defined as 2 mL min -1, both as a

compromise between sensitivity and sampling rate.

43

5-FU aspirated volume was studied between 100 - 300 µL and the results showed

that there is an increase of sensibility with a volume of 300 µL, as it can be seen in Figure

17.

From the graphics, it is possible to verify the relationship between the volume

aspirated and the propulsion flow rate to the detector with an increase of fluorescence

signal. Although the better combination is a volume of 300 µL with a flow rate to the detector

of 1 mL min-1, the 5-FU aspirated volume in the release assays was 200 µL as a compromise

between the sensitivity and the replacement volume of fresh solution at the end of the assay

into the release vessel.

The replacement volume was also studied. It was tested an interval between 417 µL

– 750 µL and it was selected the volume of 500 µL as it was the one that didn´t originated

a dilution in the release vessel, confirmed by the maintenance of the fluorescence signal

along the successive aliquots analysed.

Figure 17 – Volume sample and flow rate optimization in SIA system for release studies.

44

The calibration curve used to determine the concentration values of 5-FU solution is

represented in the figure 18.

Figure 18 - Calibration curve used to determine the concentration values of 5-FU obtained

in release assays.

The calibration curve was FI = 69.146 (± 8.17) C + 0.0294 (± 0.041); R2 = 0.9959,

where FI is the fluorescence intensity and C is the concentration of 5-FU in mg mL-1,

respectively, with 95% confidence limits for the intercept and slope.

So, the analytical cycle established for the implementation of release assay in the SIA

system is presented in Table 6.

Table 6 – Analytical cycle to the implementation of release assays in SIA system.

SIA valve position (1-8)

Time (s) Flow rate (mL min -1)

Direction (a/b)

Event

5 90 0.5 a Filling the tube with the filter

6 10 2 b

Cleaning holding coil 7 10 2 b 8 10 2 b

1 10 2 b

5 24 0.5 a Aspiration of 5-FU solution

4 100 1 b Propulsion to the detector

5 30 1 b Replacing clean solution to the vessel

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.002 0.004 0.006 0.008 0.01 0.012

FI

concentration 5-FU (mg mL -1)

45

a- aspiration and b- propulsion

In each cycle, the tube with the filter (valve position number 5) is filled with a new 5-

FU solution, corresponding to the quantity released by NPs at that time. After this, the

holding coil is cleaned during 4 steps as it was used to relieve the back pressure created in

the SIA system due to the filter. Next, 200 µL of 5-FU solution was aspirated to the holding

coil and then it was propelled to the detector and the fluorescence signal is obtained.

The last step aims not only to return the solution that was aspirated to the filling tube

between the filter and the valve to the releasing vessel and at the same time fill this tube

with fresh carrier assuring that in the next time point, there is no 5-FU solution of the

previous time point.

For each NP, the assay is performed in duplicate.

4. Determination of % loading using SIA system and batch procedure

As previously mentioned, the drug loading degree of MSNs is affected by several

properties of the material, such as hydrophilicity/hydrophobicity, pore size, surface

chemistry, load drug (charge, chemical structure, shape and molecular size) and drug

loading method. Furthermore, the drug loading is affected also by several factors involved

in the process. These include pH dependency, temperature and time. However, the most

vital factor is the possible chemical reactivity of the drug loading solution with the MSNs

surface. So, the surface properties of the NPs, the chemical nature of the drug and the drug

loading solution determine the compound affinity towards the MSNs pores (Chakraborty et

al., 2017).

Taking into account the previously mentioned, in the next table (Table 7) it is

represented the % loading values obtained with both loading methodologies.

Table 7 - Results of % loading obtained with both loading methodologies

Nanoparticles % loading

batch % loading SIA RSD (%)

APTES 11.1mg/mL 3.03 ± 0.21 2.93 ± 1.40 3.3%

UnTHCPSi 17.3mg/mL 7.13 ± 4.13 6.13 ± 2.50 14.0%

THCPSi 13.8mg/mL 2.87 ± 1.38 2.78 ± 0.56 3.1%

TCPSi 20.1mg/mL 4.55 ± 1.55 3.95 ± 2.47 13.2%

46

Considering table 7, it was possible to verify that each MSNs studied presents

different % of loading and this results can be justified taking into account the characteristics

of these NPs.

The final characteristics of MSNs obtained such as pore size shape and porosity is

directly dependent on the conditions of the synthesis. Voltage and density of the electrical

current applied to the electrodes, the concentration of HF in the electrolyte solution, the

type, doping, resistivity, and crystallographic orientation of the Si wafer, the temperature

and the time are some conditions of synthesis that affect the final characteristics of this NPs

(Salonen & Lehto, 2008).

So, taking into account the temperature used in the synthesis, UnTHCPSi, THCPSi

and TCPSi NPs have different surface properties. THCPSi NPs have hydrophobic surface

properties and TCPSi NPs have hydrophilic surface properties. UnTHCPSi and APTES NPs

have also hydrophilic surface properties (Salonen et al., 2004).

UnTHCPSi, THCPSi, and TCPSi NPs have a negative charge while APTES NPs have

a positive charge (Makila et al., 2012) and this can be justified through the chemical groups

presented in the surface of each type of MSNs used.

From the results, the differences in % of loading for this NPs can be partially ascribed

to the different chemical structures and the solubility of the 5-FU. So, the % of loading of 5-

FU is better in UnTHCPSi and TCPSi NPs because they present hydrophilic surface

properties and negative cargo, improving the affinity of 5-FU to this type of functionalized

NPs.

It was possible to observe that the results obtained by both loading methodologies

are very similar between them. Agreement between both methods was evaluated using the

t-test, carried out as a bilateral coupled test (J.C. Miller & Miller, 1993). The tabulated t value

of 3.18 when compared to the calculated t value of 2.04 showed the absence of statistical

differences for those results obtained by the methodologies at the 95% confidence level.

SIA system presents some advantages comparing with the batch procedure adopted,

being the more significant the time spent in both methodologies, since the time spent in

batch procedure was 2 h and in SIA system was about 4 minutes. These differences in time

are justified by the efficiency in mixture of NPs and 5-FU solution inside the 0.8 mm internal

diameter of PTFE tubes used in SIA system. It was also avoided the use of a magnetic

stirrer as the mixture is performed inside the system with a decrease of costs.

5. 5-FU release profiles obtained in batch procedure and SIA system

47

A major drawback limitation of the application of MSNs for the delivery of drugs is the

uncontrolled release of the loaded cargo. The release of drugs from these type of NPs is

mediated by the cleavage of the covalent bond between the MSN and the drug or by the

degradation of the Si structure (Lehto et al., 2014).

In this subchapter it will be presented the 5-FU release profiles obtained in batch

procedure and SIA system. The release profile of the 5-FU loaded in the four different MSNs

used were studied in buffer with pH value of 7.4, for simulating the physiological human

conditions.

The next figure (Figure 19) represents the release profiles in batch procedure and SIA

system when NPs are loaded using the SIA system.

From the results, it was possible to compare all the release profiles obtained using

batch procedure, all the release profiles using SIA system and to compare the release

profiles of both methodologies used.

The release profiles obtained using batch procedure (A1, B1, C1 and D1) are very

similar to each other, since the total release of 5-FU from any of the four studied

nanoparticles occurs at the end of the first 5 minutes. It was also possible to verify that at

the end of the three hours of the release assays, a stabilization of the curve of the release

profile was achieved. However, in charts B1, C1 and D1, 1 h after the start of the assay the

release curve begins to decline, perhaps due to the dilution effect of the aliquots of clean

solution added in each time point.

Now, comparing the release profiles obtained using SIA system, some differences in

release profiles arise. In the four charts (A2, B2, C2, and D2), all 5-FU loaded in the NPs

were released in the first hour of the assays, but on the chart C2 the % of release decreased

between 10 minutes and 30 minutes of the begin of the assay. On all charts except A2, 1 h

after the start of the assay the % release slightly rise. The presence of the filter may justify

these differences in profiles as there are adsorbed NPs at the filter compared to batch

procedure.

Comparing the results obtained using batch procedure and SIA system, some

differences arise when the release profiles are analysed. While in the release profiles

obtained using batch procedure (A1, B1, C1 and D1), 5-FU release is immediate (after 5

minutes), when carrying out the release assay on the SIA system, the charts B2 and C2

give the 100% release at the end of 1 h and in the charts A2 and D2 are obtained to the

end of 10 minutes. This can be justified by the use of a filter in the SIA system to retain NPs

throughout the assay that might release the 5-FU later on but also by the detection method

used in the two methodologies (HPLC in batch procedure and fluorescence detector in SIA

system).

48

Another difference, previously justified, is the behaviour of the release curve at the

end of the release assay in both methodologies. While in release profiles obtained using

batch procedure, at the end of the assay the curves begin to decline, the release profiles

obtained in SIA system, at the end of the assay the curves begin to rise.

In general and taking into account a clinical approach, this four functionalization type

and also this type of MSNs are not the best way to use in therapy because the release of

the drug occurs very quickly. A controlled drug delivery system should ensure that the drug

is gradually released over time in order to reduce the drug-related adverse effects and

decrease the number of drug administrations.

There is only one article that associates 5-FU with this type of NPs in which Pan et al.

used MSNs anchored to a hydrophilic and biocompatible poly(oligo( ethylene glycol)

monomethyl ether methacrylate) (POEGMA) and tumor targeting peptide (RGD) onto the

MSNs to load 5-FU. With this functionalization and under simulated physiological conditions

(PBS, pH 7.4, 37 ºC), ~90 % cumulative 5-FU release was observed for free 5-FU in the

first 6 h. However, only ~55 % cumulative 5-FU release was observed for 5-FU@MSN-RGD

in the same period, exhibiting controlled release characteristics. At extended time period,

the release of 5-FU gradually increased and finally reached to ~94 % at 24 h (G. Pan et al.,

2017).

So, with the different features described in the paper by Pan et al., the release profile

of 5-FU is very different from that obtained in this work. This may be justified by the different

functionalities used in both studies, where in Pan et al., this functionalization allows the

controlled release of 5-FU over time, and in our case the functionalization didn´t give the

best results proven that functionalization influences the release of 5-FU over time.

49

Figure 19 - Release profiles using SIA system for loading, where A are APTES NPs, B are

TCPSi NPs, C are THCPSi NPs, D are UnTHCPSi NPs, 1 corresponding to the release

using batch procedure and 2 corresponding to the release using SIA system.

6. 5-FU release profiles using different loading methodologies

In this subchapter, it was analysed if using different loading methods (batch or SIA)

this affect the release profile of 5-FU.

50

Initially, it was used two different NPs (APTES and UnTHCPSi NPs) which were

tested in two different conditions: in the first (1), the loading and release assays were doing

in batch and in the second one (2), the loading was doing in SIA system and the release

using batch procedure.

Looking for Figure 20, it is possible to verify that with different loading, the release

profiles obtained using batch procedure presented few differences. In the first case (A1 and

A2), the only difference is that in the first time point in A1, the % of release is close to 85%

and in A2 is close to 95%. In the second case, the release profiles are very similar to each

other.

After this comparison, it was decided to do the same assay described below but with

other NPs and other conditions.

So, it was used the other two types of NPs (TCPSi and THCPSi NPs) but here it were

tested two different conditions: in the first (1), the loading and release assays were done in

SIA system and in the second (2), the loading was done using batch procedure and the

release was done in the SIA system.

Considering the figure 21, it is possible to verify that in the first case (A1 and A2),

although the behaviour of the curves are not similar to each other, the % of release in the

first time point is close to 80%. In the second case (B1 and B2), although in B1 the third and

Figure 20 - Release profiles using different loading methodologies and batch release, where

A are APTES NPs, B are UnTHCPSi NPs, 1 corresponding to the loading and release using

batch methods and 2 corresponding to the loading in SIA system and release in batch

method.

51

fourth time points will fall slightly in comparison with the first two time points, the % of release

in the first time point in both release curves are close than 90%.

Figure 21 - Release profiles using different loading methodologies and SIA system release,

where A are TCPSi NPs, B are THCPSi NPs, 1 corresponding to the loading and release

using SIA methods and 2 corresponding to the loading in batch method and release in SIA

system.

Chapter 4

Conclusions

53

In this work, two SIA methodologies were developed to study two different

approaches. An SIA system for use in loading assays was developed to improve the loading

batch procedure, with an effective decrease of time spent while getting similar results.

Another perspective was the development of an SIA system to evaluate the release profiles

of a drug from MSNs and comparing if the release profiles obtained in batch procedure are

similar to the release profiles obtained using SIA system.

The % of loading obtained in both methodologies (batch and SIA) were compared

through Student's t-test, for a 95% confidence interval, showing that there were no

significant differences between them, attesting the agreement between the two

methodologies and, consequently, confirming the use of SIA system as a valid alternative

method. The use of SIA compared to batch for loading assays led to the decrease of time

spent to 4 minutes in comparison to 2 h of batch procedure.

Concerning the release assays, and comparing the release profiles obtained in both

methodologies, when the NPs are loaded using the SIA system for loading assays, some

differences arise mainly in the % of release obtained in the first time point as well as in the

behaviour of the curve at the end of the test. However, when the NPs are loaded by different

loading methods (batch and SIA) and the release assay was done using SIA system or

batch procedure, there are no such differences in release profiles.

The fact is that this functionalized NPs aren’t the better nanosystems to use as

controlled drug delivery of 5-FU, since this chemotherapeutic agent present hydrophilic

characteristics and has more affinity for the aqueous solution in which it is dissolved than

for loading this type of NPs. However, a change in the functionalization of this MSNs with

more hydrophobic characteristics probably it would enable the use of this type of

nanosystems in the delivery of this drug, and so can be used in the clinic in order to reduce

the effects of conventional chemotherapy.

When comparing SIA methodology used in loading assays with batch procedure, this

enabling the results in a short period (4 minutes in opposite to 2 h followed by batch

procedure) and in a reproducible manner, besides its environment friendly nature due to the

low volumes spent and effluents produced. With automation, there is also the prevention of

errors associated to human manipulation and an increase of cost-effectiveness. The SIA

methodology used in release assays present all the advantages described above.

Chapter 5

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