Linker-based Lecithin Oral Drug Delivery Systems · Linker-based Lecithin Oral Drug Delivery Systems Jacquelene Phia Chu Masters of Applied Science Department of Chemical Engineering

Linker-based Lecithin Oral Drug Delivery Systems

By

Jacquelene Phia Chu

A thesis submitted in conformity with the requirements

for the degree of Masters of Applied Science

Graduate Department of Chemical Engineering and Applied Chemistry

University of Toronto

© Copyright by Jacquelene Phia Chu 2010

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Linker-based Lecithin Oral Drug Delivery Systems

Jacquelene Phia Chu

Masters of Applied Science

Department of Chemical Engineering and Applied Chemistry

University of Toronto

2010

ABSTRACT

In this study, pharmaceutical-grade and food-grade linker-based lecithin self-emulsifying

delivery systems (SEDS) were developed with a combination of lipophilic and hydrophilic

linkers. These additives at suggested concentrations are safe for pharmaceutical and food

applications. The ratio of surfactant lecithin and linkers in these systems was optimized to

develop surfactant in oil preconcentrates. The preconcentrates containing different surfactant

concentrations and oil were diluted with fed state simulated intestinal fluid to produce pseudo-

ternary phase diagrams and to identify the formulations that produced self-emulsifying or self-

microemulsifying delivery systems. Optimal SEDS preconcentrates were evaluated using a

dialyzer model to simulate intestinal uptake. An uptake of 39.6 mg/cm2

for the pharmaceutical-

grade SEDS was obtained within 72 minutes, which promises substantial improvement in the

bioavailability of hydrophobic actives. The optimal uptake of 12.2 mg/cm2

for food-grade SEDS

suggests enhancement in the bioavailability of omega-3 fatty acids.

iii

ACKNOWLEDGMENTS

I would like to express my gratitude to:

• Professor Edgar Acosta, my supervisor, for his support and intellectual guidance

throughout this investigation.

• Professor Levente Diosady and Professor Yu-Ling Cheng, my committee members, for

their advice and constructive feedback throughout this research process.

• The Government of Ontario for Ontario Graduate Scholarship (OGS) 2009-2010, Natural

Science and Engineering Research Council of Canada (NSERC), University of Toronto

Fellowship, for their financial support.

• The American Oil Chemists' Society (AOCS) for providing the opportunities to present

this work and interact with people in the pharmaceutical industry.

• Fellow colleagues and researchers: Floryunuen Garcia Becerra, Sumit Kiran, Jessica

Yuan, Suniya Quraishi, Carol Xuan, Ziheng Wang, Americo Troncoso, Yi Guo, Oliver

Chung, Victor Castellino, Varun Maholtra, Matthew Baxter, Albert Lam for their

discussion, feedback and friendship.

• The Chemical Engineering and Applied Chemistry Department staff for their

administrative support.

• My family and my fiancée for their support and encouragement.

iv

TABLE OF CONTENTS

ABSTRACT ....................................................................................................................................... ii

ACKNOWLEDGMENTS .................................................................................................................... iii

TABLE OF CONTENTS ..................................................................................................................... iv

List of Tables ................................................................................................................................ vii

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

Chapter 1 Overview ....................................................................................................................... 1

1.1 Introduction ......................................................................................................................... 1

1.2 Hypothesis ........................................................................................................................... 3

1.3 Specific Objectives ............................................................................................................. 3

1.4 Thesis Outline ..................................................................................................................... 3

1.5 References ........................................................................................................................... 4

Chapter 2 Formulation of Pharmaceutical grade Linker-based Self-Emulsifying Delivery

Systems ...................................................................................................................................... 6

2.1 Introduction ......................................................................................................................... 6

2.2 Materials and Methods ...................................................................................................... 10

2.2.1 Materials ............................................................................................................... 10

2.2.2 Microemulsion Formulation ................................................................................. 11

2.2.3 Physicochemical Characterization ........................................................................ 12

2.2.4 Pseudo-ternary Phase Diagram ............................................................................. 13

2.2.5 Self-emulsification Studies ................................................................................... 14

2.2.6 Dialyzer Studies .................................................................................................... 14

2.3 Results ............................................................................................................................... 15

2.3.1 Phase Scans ........................................................................................................... 15

2.3.2 Pseudo- Ternary Phase Diagram ........................................................................... 17

2.3.3 Formulation of Preconcentrates ............................................................................ 21

v

2.3.4 In- Vitro Absorption- Dialyzer studies ................................................................. 24

2.4 Discussion ......................................................................................................................... 28


2.4.2 Formulation of Preconcentrate .............................................................................. 30

2.4.3 In-Vitro Absorption- Dialyzer Studies .................................................................. 31

2.5 Conclusion ........................................................................................................................ 32

2.6 References ......................................................................................................................... 33

Chapter 3 Formulation of Food- Grade Linker-based Self-emulsifying Delivery Systems ......... 38

3.1 Introduction ....................................................................................................................... 38

3.2 Materials and Methods ...................................................................................................... 42

3.2.1 Materials ............................................................................................................... 42

3.2.2 Microemulsion Formulation ................................................................................. 42

3.2.3 Physicochemical Characterization ........................................................................ 43


3.2.5 Self-emulsification Studies ................................................................................... 44


3.3 Results ............................................................................................................................... 46

3.3.1 Phase Scans ........................................................................................................... 46

3.3.2 Pseudo- Ternary Phase Diagram ........................................................................... 47

3.3.3 Formulation of Preconcentrates ............................................................................ 50

3.3.4 In- Vitro Absorption- Dialyzer Studies ................................................................. 52

3.4 Discussion ......................................................................................................................... 54

3.4.1 Optimal Surfactant-linkers ratio ........................................................................... 54


3.4.3 Formulation of Preconcentrate .............................................................................. 56


vi

3.5 Conclusion ........................................................................................................................ 58

3.6 References ......................................................................................................................... 58

Chapter 4 Conclusions and Future Recommendations ................................................................. 62

4.1 Conclusions ....................................................................................................................... 62

4.2 Recommendations for Future Work .................................................................................. 64

4.3 References ......................................................................................................................... 66

Appendix 1 ............................................................................................................................... 67

Appendix 2 ............................................................................................................................... 73

Appendix 3 ............................................................................................................................... 77

Appendix 4 ............................................................................................................................... 79

vii

List of Tables

Table 2.1 Composition of aqueous medium to simulate fed state intestinal conditions ............... 11

Table 2.2 Composition of S1 phase scan ...................................................................................... 12

Table 2.3 Particle size of Type IV vials in S1 series .................................................................... 16

Table 2.4 Physical conditions of an adult human and the uptake model ...................................... 26

Table 3.1 Composition of F1 phase scan ...................................................................................... 43

Table 3.2 Physical conditions of an adult human and the uptake model ...................................... 54

viii

List of Figures

Figure 2.1 Schematic of the linker effect with surfactant and linkers used at the oil/aqueous

interface ........................................................................................................................................... 9

Figure 2.2 Chemical structure of β-sitosterol (left) and β-carotene ............................................. 10

Figure 2.3 Flow-Thru Dialyzer method to measure uptake of β-carotene .................................... 15

Figure 2.4 S1 Hydrophilic linker scan (% w/w of PEG-6-caprylic/capric glycerides) ................ 16

Figure 2.5 S1 particle sizes after FeSSIF dilution factor of 500 ................................................... 17

Figure 2.6 Pseudo-ternary phase diagrams of the S1 formulation using the 13% PEG-6-

caprylic/capric glycerides surfactant ratio at 4 hours (a), and after 2 weeks (b), (c). Oil phase

contains ethyl caprate (EC) in (a) and (b), and 3.35% nutraceutical β-sitosterol (β-sito.) in (c). SL

represents surfactant-linker mixture, aqueous phase is FeSSIF (F). Surfactants and linker

mixture (lecithin, sorbitan monooleate, decaglyceryl monocaprylate/ caprate, PEG-6-

caprylic/capric glycerides,) are in the ratio of 4:7:3:13. The red triangles denote regions with

liquid crystals at specified preconcentrate dilutions. .................................................................... 20

Figure 2.7 T40 and T50 dilution line containing from the left of each preconcentrate: 17%, 33%,

50%, 67%, 83%, 91%, and 99% FeSSIF. ..................................................................................... 21

Figure 2.8 FeSSIF dilution (factor of 500) of surfactant in oil preconcentrates T10-T80. ........... 21

Figure 2.9 (a) Mean particle sizes of preconcentrate containing 10%-80% surfactant in oil, and

diluted with FeSSIF by a factor of 500. (b) Mean particle size of T50 preconcentrate dilution

factors of 100, 200, 500, and 1000. ............................................................................................... 22

Figure 2.10 Conductivity plots of T10-T80 dilution lines. Each point along the dilution line

reflects the same FeSSIF dilution increment used in the pseudo-ternary phase diagram. The

connected lines are for ease of visualization. Note that the maximum meter reading is 14.67 mS.

....................................................................................................................................................... 23

ix

Figure 2.11 Setup of the turbidity assessment consisting of the diluted oil (ethyl caprate) and

diluted T50 at time 0 in (a) and after 4 hours in (b). The FeSSIF dilution factor is 500. ............. 24

Figure 2.12 Turbidity Plot of (a) diluted oil (ethyl caprate) and surfactant in oil preconcentrate

T50 (b) Change in turbidity over time of diluted oil (measurement at 0% surfactant in oil) and

surfactant in oil preconcentrate T10-T80. The changes between 0 and 4 hours are measured at the

bottom of each sample. The dilution is with FeSSIF at a factor of 500. ...................................... 24

Figure 2.13 Absorbance and desorption profile of β-carotene in donor solution (0.11 % β-

carotene in SEDS) ......................................................................................................................... 25

Figure 2.14 Flow-Thru Dialyzer percentage uptake of SEDS dilution containing 0.23 % β-

carotene in oil. Results from three separate trials. ....................................................................... 26

Figure 2.15 (a) The donor side chamber interior showing oil phase separation after an oil dilution

uptake test. (b) FeSSIF dilution (factor 500) of T50 and ethyl caprate (both dosed with 0.23 % β-

carotene in ethyl caprate) mixed and left aside after two hours. .................................................. 27

Figure 2.16 T50 dilution line showing no change in phase behaviour after two hours at (a) 25°C,

(b)37 °C and with (c) β-carotene at 25°C. Dilution ratio of preconcentrate to FeSSIF are as

follows: 1:0.2, 1:0.5, 1:1, 1:2, 1:5, 1:10, 1:100. ............................................................................ 27

Figure 3.1 Structure of omega-3 fatty acids docosahexaenoic acid (top), and eicosapentaenoic

acid (bottom). ................................................................................................................................ 41


interface ......................................................................................................................................... 41

Figure 3.3 Flow-Thru Dialyzer method to measure uptake of β-carotene .................................... 45

Figure 3.4 F series particle sizes after FeSSIF dilution factor of 500 ........................................... 46

Figure 3.5 F1 Hydrophilic linker scan, % polyglyceryl-6 caprylate ............................................. 47

Figure 3.6 Pseudo-ternary phase diagrams of the F1 formulation using the 35% polyglyceryl-6

caprylate surfactant ratio at 4 hours (a), and after 2 weeks (b). SL represents the surfactant-linker

apex, F is FeSSIF, FO is fish oil ethyl esters. Surfactants and linker mixture (lecithin, glyceryl

x

monooleate, and polyglyceryl-6 caprylate) are in the ratio of 6:3:35. The red triangles denote

regions with liquid crystals at specified preconcentrate dilutions. *Liquid crystals (LC), liquid

crystals and excess oil (LC + oil), liquid crystals and FeSSIF lower phase (LC + F), 2 separate

phases of liquid crystals (LC1+LC2), liquid crystals and FeSSIF upper phase (F+LC),

microemulsion and excess oil (µE +oil), single phase microemulsion (µE),and surfactant, fish

oil, FeSSIF mixture (S+FO+F) .................................................................................................... 49

Figure 3.7 FeSSIF dilution of preconcentrates containing 10%-90% surfactant in oil. ............... 50

Figure 3.8 Average particle sizes of diluted preconcentrates containing 10%-90% surfactant in

oil. ................................................................................................................................................. 50

Figure 3.9 Turbidity Plot of (a) diluted oil and surfactant in oil preconcentrates T40,T70 and (b)

Change in turbidity of diluted oil (measurement at 0% surfactant in oil) and surfactant in oil

preconcentrates T10-T90. The changes between 0 and 4 hours are measured at the bottom of

each sample. The dilution is with FeSSIF at a factor of 500. ....................................................... 51

Figure 3.10 (a) The Flow-Thru Dialyzer donor chamber interior of T35 system revealing oil

phase separation, (b) FeSSIF dilution (factor 500) of T50 on the left and fish oil ethyl ester on

the right (both dosed with 0.23 % β-carotene in oil phase) after two hours. ............................... 52


carotene in oil phase). ................................................................................................................... 53

Figure 3.12 Flow-Thru Dialyzer percent uptake of T40 and T50 SEDS dilution containing 0.23

% β-carotene in oil. ....................................................................................................................... 54

1

Chapter 1

Overview

1.1 Introduction

The most common and preferred route of drug delivery is the oral route. However, an estimated

40%-70% of new drug candidates are insufficiently soluble in aqueous media which renders poor

absorption in the gastrointestinal tract. Other challenges posed by the gastrointestinal tract

include variations in pH, semi-permeability of membranes, high dilutions, rapid metabolism, and

enzyme interactions (Hauss, 2007). It is therefore desirable to use a suitable delivery vehicle to

enhance the absorption of the drug. Fortunately, applications in nanotechnology have been found

to improve drug delivery systems. Microemulsions and self-emulsifying delivery vehicles

(SEDS) in particular, have shown promising potential as nano-sized oral drug delivery vehicles

(Garti, 2008; Gursoy and Benita, 2004). The growing interest in these systems arises from their

physicochemical properties that confer many advantages including high solubilization capacity

of hydrophilic and lipophilic drugs, stability, and ease of preparation (Dungan, 1997). Currently,

there are commercial products on the market using SEDS and microemulsion technology such as

Neoral for the oral delivery of cyclosporine and Ritonavir for the delivery of HIV protease

inhibitor (Gursoy and Benita, 2004).

Compared to other types of oral delivery vehicles such as tablets, powders, suspensions, and

liposomes, SEDS and microemulsions have shown superior performance for oral delivery of

lipophilic drugs. Both systems are also suitable for oral delivery in gelatin capsules (Grove and

Mullertz, 2007). Microemulsions are mixtures of water, oil, and surfactant forming optically

isotropic and thermodynamically stable liquid solutions. These systems contain particle sizes of

less than 100 nm (Acosta et al., 2004; Kesisoglou et al., 2007, Rosen 2004). More importantly,

these systems enhance the bioavailability of hydrophobic drugs due to the presence of lipophilic

and hydrophilic solubilization sites (Solans and Kunieda, 1997; Lawrence and Rees, 2000;

Acosta, 2008). Three types of microemulsions can be produced: oil-in-water (o/w, Type I),

water-in-oil (w/o, Type II), and bicontinuous (Type III or IV).

2

SEDS preconcentrates are also mixtures of oils and surfactants that do not contain an aqueous

component. Upon aqueous dilution and gentle agitation, SEDS preconcentrates emulsify to form

fine oil in water emulsions (SEDS) or microemulsions (SMEDS). Following oral administration,

the gastrointestinal tract can provide sufficient dilution and agitation for the self-emulsification

process (Constantinides, 1995; Gursoy and Benita, 2004). SEDS form an emulsion of fine

particles that are typically between 100nm to 300 nm, while the particle sizes of SMEDS are less

than 100 nm (Charman et al., 1992; Khoo et al., 1998).

One critical aspect in oral drug delivery is the biocompatibility of the drug delivery vehicle. The

use of SEDS for safe and approved oral delivery is often dependent on the amount and type of

surfactant comprising the system. Although surfactants of natural origin (e.g. lecithin and

glyceryl monooleate) are generally safer and are thus preferred over synthetic surfactants, their

self-emulsification process is less efficient in comparison to synthetic surfactants (Constantinides,

1995). Non-ionic surfactants are often safer and preferred over anionic surfactants due to their

greater stability over a wider range of ionic strength and pH values (Tenjarla, 1999). However,

non-ionic surfactants may potentially lead to reversible changes in intestinal lumen permeability

(Swenson et al., 1994). In SEDS preconcentrates, surfactant concentrations usually range from

30%-60% of total volume, yet high surfactant concentrations may irritate the gastrointestinal

tract (Gursoy and Benita, 2004; Rosen 2004). Therefore, minimal surfactant concentrations

should be utilized to promote product safety and efficacy. Phospholipids, which are present in

cell membranes, are good surfactants for SEDS formulations as they are generally recognized as

safe (GRAS, FDA - 21 CFR 184.14) and have no limit on their concentration. Lecithin in

particular, has been used in microemulsions for oral and topical delivery (Rogerio, 2010; Yuan et

al., 2008). However, lecithin as a surfactant is too hydrophobic to form microemulsions. This can

be mitigated with the use of medium-chain alcohols (Tenjarla, 1999, Yuan et al., 2008; Gupta

and Moulik, 2008). However, alcohols of medium chain length pose toxicity issues (Solans and

Kunieda, 1997; De Villiers et al., 2009; Gursoy and Benita).

An alternative to alcohols in formulations is to use amphiphilic molecules known as linkers.

Linkers can be added to surfactant systems to enhance the interaction between the surfactant and

oil (lipophilic linkers) or surfactant and water (hydrophilic linkers). Linkers can increase the oil

solubilization capacity due to the modification of interfacial properties (Sabatini et al, 2003). The

3

linker technique to form microemulsions has been demonstrated for a wide variety of oils and

surfactants (Acosta et al, 2003). Yuan and Acosta (2009) have also developed and patented work

(U.S. Pub. No.20080139392) on the lecithin-linker system for transdermal drug delivery

applications. In their work, caprylic acid and sodium caprylate were used as hydrophilic linkers

and sorbitan monooleate as a lipophilic linker. However, the linkers used by Yuan and Acosta

(2009) are approved for topical and not oral applications. Prior to this investigation, no reports

existed on the use of linker-based lecithin microemulsions for oral drug delivery.

1.2 Hypothesis

Lecithin-based self-emulsifying delivery systems (SEDS) can be developed using alternative

GRAS or food additive status ingredients, and the systems can be used as potentially safe and

efficient oral delivery vehicles.

1.3 Specific Objectives

The main objective of this investigation is to optimize the formulation of linker-based lecithin

SEDS and to examine their potential on the oral delivery of drugs through understanding the

uptake mechanisms from the delivery system. The specific objectives in this study are as follows:

1. To formulate pharmaceutical and food grade, lecithin-based microemulsions with suitable

hydrophilic and lipophilic linkers and to use a pseudo-ternary phase diagram to guide the

formulation of SEDS preconcentrates;

2. To investigate the effectiveness of SEDS preconcentrates as oral drug delivery vehicles using

in-vitro dilution tests using simulated intestinal fluids;

3. To examine the effect of surfactant concentration in the formulated SEDS on their oral

potential and to understand the role of surfactants on intestinal uptake.

1.4 Thesis Outline

This thesis is organized into four chapters. Chapter 1 consists of an introduction of the research

topic, hypothesis, and objectives. Chapter 2 presents a proof of concept on the use of linkers in

4

oral drug delivery. A lecithin linker based SEDS formulation and uptake performance is

described. Chapter 3 reports on the formulation of food grade microemulsions and SEDS, and

uptake performance using linker-based lecithin systems as oral delivery vehicles for omega-3

fatty acid ethyl esters (fish oil esters). In addition, the impact of surfactant concentrations on

uptake performance of the developed lecithin-linker systems is discussed. Lastly, conclusions

from the investigation and recommendations are presented in Chapter 4. Appendices are also

included that present part of the experimental work in this study.

1.5 References

Acosta, E., Do Mai, P., Harwell, J.H., Sabatini, D.A., 2003. Linker-Modified Microemulsions for

a Variety of Oils and Surfactants. Journ. of Surfactants and Detergents. 6, 353-363.

Acosta, E.J., Nguyen, T., Witthayapanyanon, A., Harwell, J.H., Sabatini, D.A., 2004. Linker-

Based Bio-Compatible Microemulsions. Enviro. Sci. Tech. 39, 1275-1282.

Acosta, E., 2008. Testing the effectiveness of delivery systems. In: Delivery and Controlled

Release of Bioactives in Foods and Nutraceuticals, Nissim Garti (Ed), 53-106. Woodland

Publishing Co., Cambridge, England.

Charman, S., Charman, W., Rogge, M., Wilson, T., Dutko, F., Pouton, C., 1992. Self-

emulsifying drug delivery systems: formulation and biopharmaceutic evaluation of an

investigational lipophilic compound. Pharm Res. 9(1):87-93.

Constantinides, P., 1995. Lipid microemulsions for improving drug dissolution and oral

absorption: physical and biopharmaceutical aspects. Pharm Res., 12, 1561–72.

De Villiers, M., Aramwit, P., Kwon, G. 2009. Nanotechnology in drug delivery. Springer :

AAPS Press, New York .

Dungan, S. R., 1997. Microemulsions in foods: properties and applications. In Industrial

Applications of Microemulsions, Solans, C., Kunieda, H., Eds.; Dekker: New York; 66, 148-170.

Garti, N., 2008. Delivery and Controlled Release of Bioactives in Foods and Nutraceuticals.

Woodland Publishing Co., Cambridge, England.

Grove, M., Mullertz, A., 2007. Liquid Self-Microemulsifying Drug Delivery Systems. In Oral

Lipid-Based Formulations Enhancing the Bioavailability of Poorly Water-Soluble Drugs, Hauss,

D., Ed, Informa Healthcare, New York, 107-127.

Gupta, S., Moulik, S.P., 2008. Biocompatible microemulsions and their prospective uses in drug

delivery. Journ.Pharmaceutical Sci., 97, 22-45.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Wilson%20TD%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Dutko%20FJ%22%5BAuthor%5D

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Gursoy, R., Benita, S., 2004 . Self-emulsifying drug delivery systems (SEDS) for improved oral

delivery of lipophilic drugs. Biomedicine & Pharmacotherapy 58, 173–182.

Hauss, D., 2007. Oral Lipid-Based Formulations Enhancing the Bioavailability of Poorly Water-

Soluble Drugs. Informa Healthcare, New York.

Kesisoglou, F., Panmai, S., Wu, Y.H., 2007. Application of nanoparticles in oral delivery of

immediate release formulations. Current nanosci., 3, 183.

Khoo, S., Humberstone, A., Porter, C., Edwards, G., Charman, W., 1998, Formulation design

and bioavailability assessment of lipidic self-emulsifying formulations of halofantrine. Intern.

Journ. Of Pharma. 167 (1, 2), 155-164.

Lawrence, M.J., Rees, G. D., 2000. Microemulsion-based media as novel drug delivery systems.

Adv. Drug Delivery Rev., 45, 89-121.

Rogerio, A.P., Dora, C.L., Andrade, E.L., Chaves, J.S., Silva, L. Lemos-Senna, E., Calixto, J.B.,

2010. Anti-inflammatory effect of quercetin-loaded microemulsion in the airways allergic

inflammatory model in mice. Pharma. Research,61(4), 288-297.

Rosen, M., 2004. Surfactants and Interfacial Phenomena. 3rd

edition. John Wiley & Sons.

Hoboken, New Jersey.

Sabatini, D.A., Acosta, E., Harwell, J.H., 2003. Linker molecules in surfactant mixtures.Curr.

Opin. Colloid Interf. Sci. 8, 316–326.

Solans, C., Kunieda, H., 1997. Industrial Applications of Microemulsions. Marcel Dekker, New

York.

Swenson, E.S., Milisen, W.B., Curatolo, W., 1994. Intestinal permeability enhancement: efficacy,

acute local toxicity and reversibility. Pharm. Res.,11,1132–1142

Tenjarla, S., 1999. Microemulsions: an overview and pharmaceutical applications. Cri. Rev. Ther.

Drug Carrier Syst., 16 (5), 461-521.

Yuan, J.S., Ansari, M., Samaan, M., Acosta, E., 2008. Linker-based lecithin microemulsions for

transdermal delivery of lidocaine. Intern. Journ. Pharmaceutics, 349, 130-143.

Yuan, J.S., Acosta, E., 2009. Extended release of lidocaine from linker-based lecithin

microemulsions. Intern. Journ. Pharmaceutics, 368, 63-71.

http://search0.scholarsportal.info.myaccess.library.utoronto.ca/ids70/p_search_form.php?field=au&query=lemos+senna+e&log=literal&SID=91b35719d53a8e4d363396ea05a254f5

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2 Chapter 2

Formulation of Pharmaceutical grade Linker-based Self-

Emulsifying Delivery Systems

2.1 Introduction

Consumer demand for healthy food products (i.e. nutraceuticals) has been increasing due to

growing awareness of proper nutrition. However there are issues with respect to optimal delivery

of functional food ingredients. These issues include having sufficient oral bioavailability,

improved water solubility, and physiological stability. This has led researchers to use

nanotechnology to tackle issues pertaining to food and nutrition (Garti and Yuli-Amar, 2008).

Nanotechnology involves manipulating properties of matter with sizes between 1-100 nm with

the goal of developing systems with novel properties and functions (Farhand, 2007). The use of

nano-sized delivery systems can lead to enhanced drug solubility and targeted delivery (Acosta

2009; Ravichandran, 2009; Puneet and Sanjay, 2010). Nanoparticles are potentially more

bioactive than larger particles because of the greater surface area to volume ratio. These benefits

can thus address issues in oral drug delivery as there is a need to improve the bioavailability of

nutraceuticals and poorly soluble drugs. This is of great interest as an estimated 50% of orally

delivered drug compounds are highly hydrophobic in nature which result in limited

bioavailability and drug delivery efficiency (Gupta and Moulik, 2008; Kesisoglou et al., 2007;

Gursoy and Benita, 2004). Although mass transfer equations suggest that great enhancements in

bioavailability is expected with nanoparticles less than 100 nm, experimental data show that in

some cases 100 nm -500 nm nanoparticles can also result in improved bioavailability. The

enhanced bioavailability is seemingly correlated to direct nanoparticle uptake in the majority of

cases (Acosta, 2009). Bioavailability refers to the extent to which the active ingredient is

absorbed from a drug product and becomes available at the site of action (Van de Waterbeemd et

al., 2003; Mu, 2008). On the other hand, the term uptake refers to the extent of the drug that is

absorbed through the intestinal walls. The dosage that is taken up by the intestine may not

entirely be bioavailable because of various processes involved in nutrient absorption (Acosta

7

2009). In the past decade, nanotechnology has experienced rapid growth and has demonstrated

promising potential. According to research firm Cientifica, nanotechnology-enabled drug

delivery systems represented a $3.4 billion market in 2007 and is estimated to grow to $26

billion by 2012. The U.S. nanotechnology market alone is predicted to grow and reach $20

billion by 2013 (Huang et al., 2010).

Microemulsions are one type of nano-sized oral delivery vehicles capable of enhancing the

bioavailability of poorly soluble drugs. These systems enhance the bioavailability of

hydrophobic drugs by reducing drop size, increasing solubilization of active ingredient, and

increasing the residence time of the drug in the intestine through bioadhesion. Microemulsion

microstructures include oil-in-water (Type I) where water is the continuous phase; bicontinuous

(Type III, IV) where approximate equal amounts of oil and water are present; and water-in-oil

(Type II) where oil is the continuous phase. These systems are nano-sized particles that are less

than 100 nm and are smaller than the range (1 to 100 μm) for conventional emulsions

(Kesisoglou et al., 2007, Rosen 2004; Huang et al., 2010).

Another type of delivery vehicle is self-emulsifying delivery systems (SEDS). SEDS are ideally

isotropic, water-free mixtures of oils, surfactants, and or co-solvents/surfactants that emulsify to

form fine oil in water emulsions or microemulsions (SMEDS) upon aqueous dilution and gentle

agitation (Pouton, 1997; Gursoy and Benita, 2004). These systems have been used to enhance

lipophilic drug absorption (Narang et al., 2007; Pouton, 2000; Acosta, 2008).

In addition to sufficient drug absorption, oral delivery formulations should be safe for

consumption. Despite the limited selection of food-grade ingredients (Acosta, 2009; Garti et al.,

2008; Calderon et al., 2010), biocompatible microemulsions have been developed, some of

which have used plant oils such as corn oil, peppermint oil, and coconut oil (Gupta et al., 2006).

Triglycerides and esters of fatty acids such as ethyl caprate and ethyl oleate have also been

utilized in formulations (Von Corswant et al., 1997; Gupta and Moulik, 2008; Torchilln, 2008).

Some of the commonly used surfactants are polysorbates and sorbitan monoesters. Lecithin

based microemulsions are desirable as lecithin has GRAS status (21 CFR 184.14) although they

have a tendency to form liquid-crystal phases. These phases can be circumvented with the use of

medium-chain alcohols (Tenjarla, 1999, Yuan et al., 2008; Gupta and Moulik, 2008). However,

8

medium-chain alcohols pose a safety risk and may also evaporate into the shells of gelatin

capsules causing drug precipitation. Although alcohol-free formulations have been developed,

the oil solubilization capacity may be limited (Gursoy and Benita, 2004; Constantinides, 1995;

Solans and Kunieda, 1997).

Linker-based formulations have been developed and show promising potential as alternatives to

alcohol –based systems. At the oil-water interface, hydrophilic linkers lessen the rigidity of the

surfactant film (Acosta et al., 2003; Sabatini et al, 2003). The combination of lipophilic and

hydrophilic linkers results in a self-assembly that further enhances the system solubilization that

is proportional to the linker concentration (Acosta et al, 2005). Yuan and Acosta (2009) have

used the linker technique and developed linker-based lecithin microemulsions that (for

transdermal drug delivery) resolved toxicity issues present in alcohol-based microemulsion

systems.

In this work, we hypothesize that linker-based lecithin microemulsions can be formulated as

effective oral drug delivery vehicles for hydrophobic drugs using food and pharmaceutical grade

ingredients. To produce such microemulsions, the transdermal lecithin-based linker

microemulsion developed by Yuan and Acosta (2009) was used as the starting point. The

transdermal formula consisted of lecithin as the main surfactant, isopropyl myristate as the

carrier oil, sorbitan monooleate as the lipophilic linker, and caprylic acid and sodium caprylate as

the hydrophilic linkers. In order to avoid pH sensitivity, the caprylic acid and its salt were

substituted with a mixture of non-ionic hydrophilic linkers: decaglyceryl caprylate/caprate (food

additive) and PEG-6-caprylic/capric glycerides (pharmaceutical grade). The oil isopropyl

myristate (IPM) was replaced with ethyl caprate as IPM is not a food grade additive. Figure 2.1

shows a surfactant and linker-self assembly at the oil/aqueous interface. To forecast in-vivo drug

performance, Fed-State Simulated Intestinal Fluid (FeSSIF) developed by Dressman and

collaborators, was used as the aqueous dilution media (Dressman et al., 1998, Dressman et al.,

2000; Galia et al., 1998). The FeSSIF media simulates the small intestine fluids in terms of bile

salt, phospholipid levels, pH, osmolarity, and buffer capacity (Augustijns et al., 2007). The fed-

state media was used as the bioavailability of several hydrophobic drugs is known to improve

when administered with food. This observation of enhanced drug solubilization and absorption is

9

due to increased gastrointestinal secretions (i.e. bile salts emulsify fat) and residence time in the

small intestine (Patel and Brocks, 2009; Hauss, 2007).

To evaluate the linker-based SEDS as oral delivery vehicles, β-sitosterol and β-carotene, both

lipophilic nutraceuticals, were selected as model drugs. Their chemical structures are shown in

Figure 2.2. β-sitosterol is a plant sterol that slows or inhibits the incorporation of dietary and

biliary cholesterol into micelles leading to reduced cholesterol levels in the serum (Rozner et al.,

2007). However, phytosterols have low oil and water solubility and must be taken in high doses

to lower cholesterol levels (Rozner et al., 2006). β-carotene is also insoluble in water and

slightly soluble in oil. This nutraceutical is a carotenoid (yellow-orange colour) with antioxidant

properties and is affirmed as GRAS for use as a nutrient (21 CFR 184.1245) by the FDA. β-

carotene was also used as a lipophilic marker in the in-vitro uptake tests. In this investigation, the

initial objective consisted of constructing a pseudo-ternary phase diagram to examine phase

behaviour with the view of guiding the formulation of self-emulsification delivery systems

(SEDS). The phase behaviour containing a lipophilic active was also investigated by

incorporating β-sitosterol in the carrier oil. FeSSIF was used as the aqueous component and

dilution medium to further simulate in-vivo conditions. A new in-vitro method to assess active

ingredient uptake was developed with a Flow-Thru dialyzer.


interface

10

OH

CH3

CH3

CH3CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3 CH3

CH3

Figure 2.2 Chemical structure of β-sitosterol (left) and β-carotene

2.2 Materials and Methods

2.2.1 Materials

2.2.1.1 Chemicals

The following chemicals were purchased from Sigma-Aldrich Canada with purity levels shown

in parenthesis: Sorbitan monooleate or span 80 (99%+), taurocholic acid sodium salt hydrate

(95%+), ethyl caprate (99%+), Dulbecco's phosphate buffer saline (PBS), albumin from bovine

serum (BSA), β-carotene (95%), sodium chloride (99.5%+, Fluka brand), and β-sitosterol (assay

~ 60%, Fluka brand). Sodium hydroxide pellets were purchased from Caledon chemicals. Glacial

acetic acid (99.7%+) was purchased from EMD Chemicals Inc. Samples of decaglyceryl

caprylate/ caprate (drewpol 10-1-CC) and PEG-6-caprylic/capric glycerides (softigen 767) were

kindly donated by Stepan Company and Sasol respectively. Laboratory grade soybean lecithin

(99%+) was purchased from Fisher Scientific. Frozen sheep intestine was purchased from a local

market. The compositions listed are on a mass basis (wt. %) unless stated otherwise.

2.2.1.2 Tissue

Prior to use, the frozen sheep intestine was thawed by soaking it in warm water for 1 hour.

Afterwards, a three inch section of the sheep small intestine (jejunum) was excised and the

exterior impermeable membrane was removed. The excised section was rinsed 4 times with

distilled water and then soaked in PBS for 30 minutes to render it ready for use in the in-vitro

study.

11

2.2.2 Microemulsion Formulation

Phase behaviour studies were required to determine optimal surfactant and linker ratios for

microemulsion formation. Phase behaviour was investigated using equivalent masses of oil and

aqueous phases at room temperature (~ 25 °C) and pressure (1 atm). The oil and aqueous phases

were then combined and vortexed for two minutes at 3200 rpm. The formulations were set aside

to equilibrate (occurred within two weeks). Fed state simulated intestinal fluid (FeSSIF)

(Dressman et al., 2000) was used as the aqueous medium rather than water and the composition is

shown in Table 2.1.

Based on preliminary scans, it was determined that a minimum of 7% sorbitan monooleate was

required to prevent the formation of lecithin gels. A hydrophilic linker scan included 7% sorbitan

monooleate in oil while concentrations of PEG-6-caprylic/capric glycerides were varied 0% to

20% to obtain transparent isotropic regions. This scan will be referred to as S1 and the

composition is in Table 2.2. A nutraceutical was also incorporated into the S1 formulations.

When close to the maximum amount of β-sitosterol (~8% in ethyl caprate) was introduced into

Type IV microemulsions, the drug precipitated out of solution. By reducing the drug content to

3.35% in oil, isotropic single phases could still be formed at linker concentrations of > 13%

PEG-6-caprylic/capric glycerides.

Table 2.1 Composition of aqueous medium to simulate fed state intestinal conditions

Fed State Simulated Intestinal Fluid (FeSSIF)

Sodium taurocholate 15 mM

Lecithin 3.75 mM

Acetic Acid 8.65 g

NaCl 11.9 g

NaOH qs pH 5

Deionized water qs 1 liter

12

Table 2.2 Composition of S1 phase scan

Phase Component S1 Weight %

Lecithin 4%

Aqueous Decaglyceryl caprylate/ caprate 3%

PEG-6-caprylic/capric glycerides 0-40%

FeSSIF To 50%

Oil Sorbitan monooleate 7%

Ethyl Caprate To 50%

2.2.3 Physicochemical Characterization

2.2.3.1 Particle Size

The average particle diameters were determined using dynamic light scattering (DLS)

measurements from a 90Plus Particle Size Analyzer instrument (λ = 635 nm; Brookhaven

Instruments). The photomultiplier detector is positioned at a scattering angle of 90°. The

cuvettes were set aside for 2 minutes to equilibrate prior to the measurements. The data was

obtained by averaging five measurements per vial set at 1 minute per measurement. The

viscosities of the samples were measured in triplicate using a CV-2200 falling ball viscometer

(Gilmont Instruments, Barrington, IL, USA). These measurements were conducted at room

temperature ~ 25°C.

2.2.3.2 Conductivity

A VWR conductivity meter (model 21800-012) was used, which was equipped to measure in the

range of 0.001 µS-14.67 mS. Conductivity was measured at 25°C.

13

2.2.3.3 Turbidity

Turbidity can be examined by the changes in transmitted light intensity. It is represented

mathematically in Equation 2.1 where τ is turbidity having units of inverse length, L is the path

length of light, I is transmitted light intensity, and I0 is the transmitted light intensity of the

―clear‖ sample (Acosta et al, 2003), which in this case is distilled water. A red line drawn on

white paper and served as a reference point. The changes in the light intensity of the red line

through the sample served as a turbidity indicator. Since drug residence time in the small

intestine is ~ 3 hours (Fadda et al., 2009) images were captured over four hours to examine

stability. The light intensities were assessed via image analysis tools Corel PaintShop Photo Pro

X3 (RGB tool, blue channel) and Scion Image Beta 4.03 (Plot Profile tool).

τ

I

I

L

0ln1

(Equation 2.1)

2.2.4 Pseudo-ternary Phase Diagram

A pseudo-ternary phase diagram was generated to examine phase behaviour and identify the range

of isotropic regions with microemulsion structures. The diagram was created along aqueous

phase dilution lines where mixtures of surfactant, linkers and oil (designated as preconcentrates)

were diluted with FeSSIF at room temperature. Oil (ethyl caprate) dilution lines of surfactant,

linkers and fed state were also used to further assist in phase identification. The fed state medium

was used as the aqueous phase to generate the diagram rather than water to better simulate

biological conditions and thus forecast in-vivo phase behaviour upon dilution (Dressman et al.,

1998; Staggers et al., 1990; Wang et al., 1996).

The surfactant and linker mixture comprised of lecithin, sorbitan monooleate, decaglyceryl

monocaprylate/ caprate, and PEG-6-caprylic/capric glycerides, in the ratio of 4:7:3:13 as

explained in section 2.3.1. Preconcentrates containing 10%, 20%, 30%, 40%, 50%, 60%, 70%,

and 80% surfactant and linker mixture in oil were each diluted with FeSSIF at ratios of 1:0.2,

1:0.5, 1:1, 1:2, 1:5, 1:10, 1:100. Similarly, 10%-80% surfactant and linker in FeSSIF were

http://www.scioncorp.com/downloads/scion_image/SetupWinB403.exe

14

diluted with oil to characterize the preconcentrates and phases near the surfactant-FeSSIF axis.

Shorter notations will be used to denote specific preconcentrate dilutions, for instance T10

represents the preconcentrate containing 10% surfactant in oil, T10-1 represents the first dilution

(16.7% aqueous phase) of the T10 preconcentrate, and T60-SF represents the preconcentrate

containing 60% surfactant in FeSSIF.

Each dilution was vortexed thoroughly for one minute at 3200 rpm and kept in a separate vial.

Cross polarizers were used to identify liquid crystal regions. The formulations were set aside to

equilibrate (occurred within 2 weeks) for classification. Formulations that remained transparent

were categorized as monophasic and isotropic in the phase diagram. In addition, the effect of

phytosterols was investigated by incorporating 3.35% of β-sitosterol in oil (ethyl caprate).

2.2.5 Self-emulsification Studies

The self-emulsification studies consisted of mimicking in-vivo dilution with simulated intestinal

media and in-vitro evaluation of the uptake by the intestines. The procedure involves vortexing the

sample for two minutes prior to dilution with fed state simulated intestinal fluid (FeSSIF). This

method assists in assessing whether the microemulsion preconcentrate will produce a SMEDS,

SEDS, an insoluble phase or a conventional emulsion upon dilution. The criteria distinguishing

these systems will be based on the particle sizes discussed earlier, where SMEDS produce

particle sizes of less than 100 nm, SEDS produce particle sizes exceeding 100 nm, and

conventional emulsions are in the range of 1-100 µm.

2.2.6 Dialyzer Studies

The Flow-Thru Dialyzer (Harvard Apparatus models: 741202, 741105, 74104) setup shown in

Figure 2.3 shows the receiver chamber containing 4% bovine serum albumin in PBS (simulating

human plasma concentration). The SEDS (T50) dosed with β-carotene (0.23% in oil) was diluted

with FeSSIF and circulated through the donor chamber. The donor and receiver chambers were

separated by the excised intestine (6 mm diameter cross section). The FeSSIF dilution factor of

500 was used to approximate the dilution ratio in the small intestine for an ingested pill (assuming a

pill volume of 1mL) (Hauss, 2007). Both reservoirs were mixed at the lowest setting. An Ocean

Optics UV/VIS Spectrophotometer (HR2000) and software (OOIBase32) tracked changes in

15

absorbance of the active. After the two hour absorption simulation, the donor chamber was

rinsed with 6 mL of FeSSIF and this effluent was discharged. The loop of the donor side was

then closed to recirculate 15 mL of FeSSIF for one hour to simulate the desorption process.

Additional simulations consisted of diluted oil (0.23 % β-carotene in ethyl caprate diluted with

FeSSIF) as the donor solution to serve as a baseline comparison. The dialyzer fluids circulated at

a rate of 3 mL/min, which was consistent with the measured postprandial flow rate in the

jejunum in the first hour of a study (Fadda et al., 2009; Kerlin et al., 1982). The absorbance data

was obtained by subtracting the absorbance at 450 nm (signal detection wavelength) from the

baseline at 600 nm. The integration time, average, boxcar, and strobe frequency parameters were

set at 8, 10, 2, and 10 respectively. The volumes of simulated plasma and simulated intestinal

fluid after consumption of SEDS were 30mL and 10 mL. These volume ratios were designed

based on the ratios found in literature (Sherwood, 2004).

Figure 2.3 Flow-Thru Dialyzer method to measure uptake of β-carotene

2.3 Results

2.3.1 Phase Scans

The phase behaviour of the S1 scan is shown in Figure 2.4. For 0% and 2% PEG-6-

caprylic/capric glycerides (hydrophilic linker) milky emulsion phases are formed, and at 4%

hydrophilic linker a Type II system is formed. The concentrations of the hydrophilic linker in the

16

range of 6%-12%, liquid crystals (lower phase) are present. Higher concentrations consisting of

13%-20% PEG-6-caprylic/capric glycerides resulted in isotropic, bicontinuous Type IV systems

with no excess phases. These concentrations also have low viscosities between 15-30 cP. The

isotropic particle sizes are less than 12 nm as reported in Table 2.3. Figure 2.5 shows the S1

particle sizes after a fed state dilution ratio of 500. The dilution of 0%-6% PEG-6-caprylic/capric

glycerides resulted in emulsion particle sizes between 570 nm-830 nm. In comparison, the higher

amount of PEG-6-caprylic/capric glycerides in the range of 8% - 20% had led to nanoemulsions

with more uniform sizes (smaller deviation) of 105 nm - 175 nm. A 13% PEG-6-caprylic/capric

glycerides concentration was the least amount of surfactant that resulted in a transparent phase

with low particle size upon dilution. Thus the ratio of 4% lecithin/ 7% sorbitan monooleate/ 3%

decaglyceryl monocaprylate/caprate/ 13% PEG-6-caprylic/capric glycerides was used to generate

the pseudo-ternary phase diagram.

0% 2% 4% 6% 8% 10% 12% 13% 14% 15% 20%

Figure 2.4 S1 Hydrophilic linker scan (% w/w of PEG-6-caprylic/capric glycerides)

Table 2.3 Particle size of Type IV vials in S1 series

% PEG-6-caprylic/capric glycerides

w/w

Mean diameter (nm) + SD

Mean Viscosity (cP) +

SD

13% 8.8 + 1.3 16.4 + 0.5

14% 11.3 + 0.9 15.5 + 0.5

15% 8.9 + 0.2 18.8 + 0.5

20% 5.2+ 0.2 28.6 + 1.0

*SD- standard deviation

17

Figure 2.5 S1 particle sizes after FeSSIF dilution factor of 500

2.3.2 Pseudo- Ternary Phase Diagram

The pseudo- ternary phase diagrams for the oil –surfactant mixture–FeSSIF system are depicted

in Figure 2.6. By visual inspection, any changes in the mixtures occurred notably within the first

week. After a period of two weeks changes were slight or nonexistent. The blue lines illustrate

aqueous dilution lines for the T30 and T50 preconcentrates.

Figure 2.6.a shows the pseudo-ternary phase diagram for the system after 4 hours. A transparent,

single phase region was identified as 1Ø. The single phase regions were observed upon the initial

dilutions for systems T30, T40, and T50. For T30, the single phase region was observed within

the first dilution up to ~ 17% FeSSIF. The T40 and T50 dilution lines within the monophasic

area could incorporate about 33% and 50% aqueous phase respectively. The remainder of the

phase diagram designated by mØ represents a turbid area with multiphase systems.

Figure 2.6.b displays the phase behavior after two weeks where the single phase limits and

multiphase regions are identified. The monophasic transparent region comprised 14.2% of the

diagram. Preconcentrates containing low surfactant concentrations resulted in microemulsions

18

with excess oil (µE + oil) after FeSSIF dilutions, as observed for T10 and T20. A microemulsion

region with no excess phases (µE region) was formed upon initial dilutions of the T30, T40, and

T50 preconcentrates. These systems transitioned to another region (µE + oil) comprised of

microemulsions with excess oil at approximately 17%, 33%, and 50% aqueous content

respectively. For T30, dilutions greater than 50% FeSSIF led to phase separation. Systems T40

and T50 could be diluted with high FeSSIF content (~ 91% FeSSIF) while higher volumes of

aqueous content resulted in nanoemulsions that separated within a week. These transitions from

monophasic microemulsion to microemulsion plus excess oil region, and to phase separations are

illustrated by the blue dilution lines for the T30 and T50 systems. The dilutions of the

preconcentrates containing higher surfactant concentration led to liquid crystal formation. The

different regions containing liquid crystals exhibited birefringence under cross polarizers and are

denoted by red triangles in Figure 2.6.b. The initial dilutions of T60-T80 content led to two

phases where the lower and upper phase comprised of liquid crystal and microemulsion

respectively (LC+µE). An example of this phase under cross polarizers is denoted by the triangle

at T70-1 and contains 17% FeSSIF. Going further along this dilution line resulted in liquid

crystals with excess oil (LC+oil) as seen with the T70-3 (50% FeSSIF) image. The LC region

containing surfactant and FeSSIF with low oil content represent viscous lyotropic liquid crystals

as shown with T70-SF. Multiple phases were also present near the surfactant-oil and surfactant-

FeSSIF axis. The S+O+P region comprised of a mixture of surfactant, oil, and precipitate while

the S+F+P region comprised of a mixture of surfactant, fed state, and precipitate.

The changes in phase behaviour when incorporating a nutraceutical can be observed in Figure

2.6.c, where β-sitosterol (3.35%) is dissolved in oil. In comparison to Figure 2.6.b, the addition

of the plant sterol decreased the isotropic microemulsion areas. The microemulsion and liquid

crystal (LC+µE) region was also enlarged, and lower surfactant in oil regions of T20 to T50

formed liquid crystals prior to the formation of the microemulsion phases. Increased dilution of

T30, T40 and T50 led to a monophasic microemulsion region at 11%, 17% and 23% aqueous

content. Further dilution for these systems resulted in two phase areas consisting of

microemulsion with excess oil at 18%, 42% and 46% FeSSIF content. These microstructure

transitions are seen with the T30 and T50 blue dilution lines. Phase separation and β-sitosterol

19

precipitate was observed for systems at high dilution (greater than 91% FeSSIF). The S+F+P

region also extended from 20% to 30% surfactant in FeSSIF.

From the phase diagrams, we were able to identify microemulsion regions and the optimal range

of oil and surfactant required to form an effective self-emulsification formulation. T40 - T50

preconcentrates were the optimal systems as they formed stable microemulsion structures along

most of the dilution line. The T50 system was selected for in-vitro uptake evaluation as it was

able to dissolve more oil along the dilution line in comparison to T40 as shown in Figure 2.7. To

avoid precipitate or liquid crystal formation upon dilution, small volumes of water should be

added to the preconcentrates prior to oral delivery.

20

(a)

(b) (c)

Figure 2.6 Pseudo-ternary phase diagrams of the S1 formulation using the 13% PEG-6-

caprylic/capric glycerides surfactant ratio at 4 hours (a), and after 2 weeks (b), (c). Oil phase

contains ethyl caprate (EC) in (a) and (b), and 3.35% nutraceutical β-sitosterol (β-sito.) in (c). SL

represents surfactant-linker mixture, aqueous phase is FeSSIF (F). Surfactants and linker

mixture (lecithin, sorbitan monooleate, decaglyceryl monocaprylate/ caprate, PEG-6-

caprylic/capric glycerides,) are in the ratio of 4:7:3:13. The red triangles denote regions with

liquid crystals at specified preconcentrate dilutions.

* Liquid crystals (LC), liquid crystals and excess oil (LC + oil), microemulsion and excess oil

(µE +oil), single phase microemulsion (µE), microemulsion and liquid crystals (µE + LC),

surfactant, oil, precipitate (S+O+P), and surfactant, FeSSIF, precipitate (S+F+P).

T40

21

T40 T50

Figure 2.7 T40 and T50 dilution line containing from the left of each preconcentrate: 17%,

33%, 50%, 67%, 83%, 91%, and 99% FeSSIF.

2.3.3 Formulation of Preconcentrates

Particle size, conductivity and turbidity measurements were obtained to characterize and

examine the performance of surfactant-oil mixtures upon dilution. T10 - T80 were diluted by a

ratio of 500:1 with FeSSIF as shown in Figure 2.8. The preconcentrates T30 - T80 have particle

sizes under 500 nm, while T10 and T20 sizes are near a micron as reported in Figure 2.9.a. T40

and T50 preconcentrate produce the smallest sizes of 151 nm and 170 nm. These systems are

therefore SEDS. This was expected since the pseudo-ternary phase diagrams showed that

systems formed microemulsions along most of the dilution line. Figure 2.9.b shows the dilution

of the T50 preconcentrate at a ratio of 100-1000. The trend shows particle sizes decreased with

increasing dilution. However, the sizes remained between 120 nm-245 nm.

T10 T20 T30 T40 T50 T60 T70 T80

Figure 2.8 FeSSIF dilution (factor of 500) of surfactant in oil preconcentrates T10-T80.

22

(a) (b)

Figure 2.9 (a) Mean particle sizes of preconcentrate containing 10%-80% surfactant in oil, and

diluted with FeSSIF by a factor of 500. (b) Mean particle size of T50 preconcentrate dilution

factors of 100, 200, 500, and 1000.

The conductivity of the T10-T80 dilution lines were measured from the phases containing the

surfactants (not excess phase) and plotted in Figure 2.10. The T40 and T50 preconcentrates show

an exponential increase for the Type IV bicontinuous microemulsion region in the initial

dilutions; however after dilutions with 33% FeSSIF for T40 and 50% FeSSIF for T50 the

conductivity changed to a linear trend marking the transition to the bicontinuous microemulsion

and excess oil region. At higher dilutions, conductivity indicate that the regions approach Type I

behaviour. Both preconcentrates could be diluted to a content of 91% FeSSIF. The T60-T80

systems contained liquid crystals and exhibited mostly low conductivity until increased dilutions

that forced higher conductance. The remaining systems appear bicontinuous and approach Type I

behaviour upon higher FeSSIF dilution. The systems that contained bicontinuous regions were

not stable at high dilution ratios where oil-in-water continuity was forced. The best

preconcentrate formulations (i.e. T50) thus approached Type I continuity and this was observed

later on in the uptake evaluation.

23

Figure 2.10 Conductivity plots of T10-T80 dilution lines. Each point along the dilution line

reflects the same FeSSIF dilution increment used in the pseudo-ternary phase diagram. The

connected lines are for ease of visualization. Note that the maximum meter reading is 14.67 mS.

Another necessary feature of SMEDS and SEDS is being stable over the course of digestion

(Pouton, 1997; Gursoy and Benita, 2004). The stability of the diluted preconcentrates and diluted

oil (ethyl caprate) were assessed via turbidity measurements. Figure 2.11 shows the turbidity

measurement setup and compares the turbidity of diluted oil and diluted T50 SEDS at time 0 (a)

and after 4 hours (b). By visual inspection, the diluted oil has clearly separated in 4 hours. The

turbidity measurements of the T50 and oil dilutions are shown in Figure 2.12.a at times 0 and 4

hours with respect to sample height. The top of the sample is the initial sample height (0 cm).

Figure 2.12.b shows the change in turbidity after four hours of the diluted preconcentrates and

oil. The percent change was determined by taking measurements at the bottom of each vial

where the greatest separation would occur. The diluted oil has shown a significant reduction in

turbidity (44%) indicating separation within four hours. T10-T30 are also not stable and showed

changes in turbidity in the range of 12%-24%. T10 however, had more visible phase separation

in comparison to T20 and T30. Systems T40 and T50 were the most stable dilutions. Since T40

and T50 have smaller particle sizes it was expected that they would have the smallest turbidity at

24

time 0. However, having increasing surfactant in the diluted preconcentrates led to a slight

decrease in stability as T60- T80 had 5%-11% changes in turbidity readings.

Oil T50 Oil T50

(a) t = 0 hour (b) t = 4 hours

Figure 2.11 Setup of the turbidity assessment consisting of the diluted oil (ethyl caprate) and

diluted T50 at time 0 in (a) and after 4 hours in (b). The FeSSIF dilution factor is 500.

(a) (b)

Figure 2.12 Turbidity Plot of (a) diluted oil (ethyl caprate) and surfactant in oil preconcentrate

T50 (b) Change in turbidity over time of diluted oil (measurement at 0% surfactant in oil) and

surfactant in oil preconcentrate T10-T80. The changes between 0 and 4 hours are measured at the

bottom of each sample. The dilution is with FeSSIF at a factor of 500.

2.3.4 In- Vitro Absorption- Dialyzer studies

The optimal surfactant-linker ratio determined from the phase behaviour scans was 4:7:3:13 for

lecithin, sorbitan monooleate, decaglyceryl monocaprylate/ caprate, PEG-6-caprylic/capric

25

glycerides. Based on the dilution, conductivity and turbidity results using this ratio, the system

that contained 50% surfactant and 50% oil (T50) produced the most optimal and stable SEDS.

Therefore it was presumed that this particular formulation should enhance uptake, and thus in

this section we evaluate uptake using the in-vitro dialyzer.

The concentration profile of β-carotene is illustrated in Figure 2.13. The concentration reaches a

plateau in ~ 72 minutes. The process of desorption is also depicted (trial 1 desorption) and is shown

to have negligible release of β-carotene back to the donor solution. The transport of SEDS from the

donor solution to the intestinal tissue is therefore an irreversible process. Since the dye remained

solubilized in the donor solution during the simulation, we can assume that the uptake of the

SEDS is equivalent to the uptake of β-carotene. The uptake of the SEDS was 56.0 + 0.8% and is

shown in Figure 2.14. Table 2.4 reports the physical conditions of the human intestine and the

model. For the model the absorption capacity (maximum SEDS absorbed per unit intestinal

surface area) was 39.6 mg/cm2, based on the uptake of the initial dose (0.02g SEDS) divided by

the intestinal surface area. The tabular values show that the uptake model has a surface area to

volume ratio that is two orders of magnitude smaller than in humans. This suggests that for in-

vivo conditions, more surface area would come in contact with the donor solution thus

potentially improving the uptake beyond 56%. Refer to Appendix 1 for sample calculations and

absorbance spectra.


carotene in SEDS)

26

Figure 2.14 Flow-Thru Dialyzer percentage uptake of SEDS dilution containing 0.23 % β-

carotene in oil. Results from three separate trials.

Table 2.4 Physical conditions of an adult human and the uptake model

SA - surface area, * Grassi et al. (2007)

Dialyzer studies were also conducted by using only diluted oil as the donor solution, this served

as a baseline of comparison. The results showed negligible changes in absorbance and no uptake.

As seen in Figure 2.15.a, the lipophilic marker (yellow-orange colour) accumulated within the

dialyzer. Phase separation occurred and the dye did not permeate into the plasma reservoir but

coalesced in the chamber on the donor side. Figure 2.15.b shows a separate comparison of

diluted T50 and ethyl caprate (FeSSIF dilution factor 500) after two hours. The phase behaviour

was comparable to the uptake investigation as phase separation for the oil was evident while T50

had maintained dispersion and solubilized most of the oil.

Small intestine SA

(cm2)

SA: FeSSIF

cm2/L

Max SEDS absorbed (mg)/

Area (cm2)

Model 0.28 28 39.6

Human 9800* 9800*

27

(a) (b)

Figure 2.15 (a) The donor side chamber interior showing oil phase separation after an oil

dilution uptake test. (b) FeSSIF dilution (factor 500) of T50 and ethyl caprate (both dosed with

0.23 % β-carotene in ethyl caprate) mixed and left aside after two hours.

A scan of the T50 dilution line at 25°C, 37 °C and one containing β-carotene (0.23 % in ethyl

caprate) was conducted to illustrate that these conditions did not affect phase behaviour. In

Figure 2.16 the scans showed no significant difference in phase volumes and phase behaviour

after two hours. This was expected since β-carotene was dosed in a low concentration, and

lecithin is relatively insensitive of the change to body temperature. The temperature insensitivity

within the 25°C-37 °C range is desirable in drug delivery vehicles as we are able to better predict

phase behaviour at in vivo conditions.

(a)

(b)

(c)

1:0.2 1:0.5 1:1 1:2 1:5 1:10 1:100

Dilution ratio

Figure 2.16 T50 dilution line showing no change in phase behaviour after two hours at (a) 25°C,

(b)37 °C and with (c) β-carotene at 25°C. Dilution ratio of preconcentrate to FeSSIF are as

follows: 1:0.2, 1:0.5, 1:1, 1:2, 1:5, 1:10, 1:100.

28

2.4 Discussion


In this investigation, we established pseudo-ternary phase diagrams based on the optimal mixture

of oil, surfactants and aqueous phase from the S1 scan described in section 2.3.1. The diagrams

used fed state aqueous media to model the phase behaviour of postprandial conditions in the

small intestine. The multiple phases present in the pseudo-phase diagrams (Figure 2.6) were

comparable to studies examining phase behaviour of postprandial intestinal conditions. Studies

have shown that intestinal fluids are multiphase and comprise of an oil phase, aqueous micellar

phase, and a precipitate phase. The presence of dispersed liquid crystalline regions, emulsion

particles, and mixed micelles has also been observed (Stafford et al., 1981; Staggers et al., 1990;

Wang and Carey, 2002). The mixed bile salt micelles are desirable for optimal lipid uptake since

they solubilize lipids and are smaller than 100 nm in size. From Figure 2.6.b and c, 99% fed

state content led to dilute emulsions that separated in the first week. The mechanism of formation

of some of these nanoemulsions is possibly by swelling of mixed micelles by solubilized solvent

followed by breakdown to tiny droplets of less than 400 nm (Rosen, 2004). The partitioning of

the hydrophilic surfactants into the aqueous phase could also lead to phase separation and

reduced oil solubilization. At lower aqueous content (less than 91%) stable microemulsion

structures were formed along selected regions of the phase diagram. At low surfactant in oil

systems, T10- T30 contained insufficient concentrations of surfactant for self-emulsification at

high dilutions, hence the phase separation observed after FeSSIF dilutions (Grove and Mullertz,

2007). The surfactants thus partitioned into the aqueous phase.

Preconcentrates containing higher surfactant concentrations resulted in more stable dilutions

capable of solubilizing more oil/nutraceutical/FeSSIF at high aqueous content (i.e. 91%). This is

due to the self-emulsifying properties of SEDS formulations that require relatively large

quantities of surfactant in the formulation (Grove and Mullertz, 2007). T40 – T50 systems were

deemed optimal as bicontinuous microemulsions formed along the dilution line while avoiding

liquid crystal formation and phase separation (FeSSIF content less than 91%) as observed in

Figure 2.6. a, b. These preconcentrates are thus considered SEDS as they transitioned through a

microemulsion phase that promoted self-emulsification (Solans et al., 2005).

29

On the other hand, further increase of surfactant in oil (greater than ~ 60%) formed liquid

crystalline phases as the micelles arranged in ordered arrays. The presence of multiple phases

and precipitate near the surfactant-oil and surfactant-FeSSIF axis is explained by the surfactant

mixture having incomplete solubility in ethyl caprate and FeSSIF. The hydrophilic linkers

decaglyceryl monocaprylate/ caprate and PEG-6-caprylic/capric glycerides are water soluble but

are not soluble in oil. Conversely, the lipophilic linker sorbitan monooleate is soluble in oil but

exhibits poor solubility in water. This is consistent with the fact that the microemulsion was

optimized towards bicontinuous systems.

The addition of β-sitosterol (3.35% in oil) reduced the area of the o/w and bicontinuous

microemulsion region. Upon aqueous dilution, the solubility of the phytosterol was reduced and

led to a smaller microemulsion area and extended liquid crystal regions (LC+μE in Figure 2.6.c).

In another study (Spernath et al., 2003), this trend is observed where the phytosterol

solubilization capacity was reduced by 96% after diluting to 90% w/w aqueous content. In our

system, 8% β-sitosterol can be solubilized in ethyl caprate. However, a lower dose was

incorporated in the oil (3.35%) to circumvent phase separation and crystallization upon fed state

dilution. The decrease in solubility may be attributed to changes in microstructure and a shift in

the locus of solubilization upon dilution (Spernath et al., 2003). In the presence of water, β-

sitosterol tends to form crystals of β-sitosterol hydrates (Bonsdorff-Nikander et al., 2005).

T40 and T50 were optimal systems in all phase diagrams. It is desirable though, to attain a larger

isotropic region as it gives more flexibility in maintaining microemulsion structures under

varying environmental conditions and compositions. For this system and model nutraceutical it

seems that adding water to the preconcentrates (microemulsion vehicle) prior to ingestion is

necessary to avoid precipitation (S+O+P region) and liquid crystals (LC+µE) formation.

According to Spernath et al. (2003) phytosterols should be delivered as water-based

microemulsions to optimize competitive solubilization in bile salt micelles. Their studies

revealed that phytosterols exhibited higher solubilization capacity than cholesterol in

microemulsions comprising of more than 50% aqueous phase.

30

2.4.2 Formulation of Preconcentrate

The preconcentrates were diluted to simulate intestinal digestion. In the system, the increase in

conductivity as a function of FeSSIF was due to the increase in the fraction of FeSSIF ions that

were not contained in the microemulsion core (Fanun, 2008). Liquid crystals exhibited low

conductivity as they are weak electrolytes with a low degree of dissociation (Stegemeyer et al.,

1993). As seen in Figure 2.10 the optimal systems (T40, T50) transitioned from the phase with

low conductivity (resembled Type II), to a phase with intermediate conductivity (typical of Type

IV) and to a phase with high conductivity (typical of Type I), and resembled the Type II-IV-I

transition behaviour observed in state of the art U-type microemulsions reported by Garti et al.

(2004). The best preconcentrate formulations (e.g. T50) approached Type I continuity and this

was observed in the uptake evaluation.

Notable changes in turbidity of the systems indicate that they are not in thermodynamic

equilibrium after simulated digestion. However, T40 and T50 remain fairly stable over time. The

selection of T50 for uptake studies is justified as it had the least change in turbidity and has one

of the smallest particle sizes (171 nm). The other measured particle sizes were also greater than

100 nm and are thus classified as SEDS. T40 and T50 were the optimal SEDS having 2.4%and

2.1% change in turbidity and particle sizes of 152 nm and 171 nm respectively.

Although it is desirable to form microemulsions (i.e. mixed bile salt micelles less than 100 nm)

after dilution to enhance drug absorption, many studies exist in which drug absorption is not

dependent on size. Khoo et al. (1998) showed that the bioavailability of lipophilic, anti-malarial

drug halofantrine was similar between SEDS (average droplet size of 119 nm) and SMEDS

(average droplet size of 52 nm). Another investigation compared the bioavailability of

cyclosporine in humans from SEDS and SMEDS system. The administered SEDS (16-20 μm

droplet size) had similar absorption characteristics to the SMEDS vehicle (200 nm droplet size)

and were approximately bioequivalent (Odeberg et al., 2003). These findings however, are in

contrast to another human bioavailability study that compared the cyclosporine Sandimmune

SEDS formulation to the Neoral SMEDS formulation. In comparison to the SEDS, the SMEDS

formula showed improved rate and extent of absorption of cyclosporine (Mueller et al., 1994).

31

Based on these studies one concludes that while size may play a role on absorption there are

other factors such as the surface chemistry of the particle that also influence the overall uptake.

2.4.3 In-Vitro Absorption- Dialyzer Studies

The dialyzer study was conducted to evaluate the potential absorption of linker-based SEDS for oral

delivery. From the study approximately half of the dosage is absorbed within 30 minutes. The

uptake reaches a plateau in ~ 72 minutes and results in an uptake of 56.0 + 0.8%. This suggests

that there is sufficient time for optimal absorption. Appendix 1 also shows an absorption model

fitted to the data. The in-vitro experiments only accounts for passive absorption and droplet

entrapment in the mucin layer, but even with these conditions it was observed to have significant

improvement in performance than simply dosing an equivalent amount of oil (ie. oil capsule) in

the system. As seen in Figure 2.15 the oil separated from the donor solution and was not

absorbed by the intestinal section. Since studies have shown that polyethylene glycol chains

adsorb onto phospholipid membranes, the adsorption of PEG-6-caprylic/capric glycerides on the

intestinal section could be promoting further uptake (Liu et al., 2009; Wang et al., 2008;

Efremova et al., 2002). Further in-vivo studies will confirm whether the uptake is limited by

adsorption or absorption (slow diffusion) processes.

If we assume that the performance is the same for SEDS containing β-sitosterol (3.35% in ethyl

caprate), then the SEDS and β-sitosterol absorption capacities are 39.6 mg/cm2 and 0.66 mg/cm

2

respectively. For a recommended phytosterol intake of 10 g (Arnoldi, 2004; Kirk-Othmer,

2007) that has a bioavailability of 2% (Ostlund et al., 2002; Rozner and Garti, 2006), only 0.2 g

of phytosterol is absorbed. Based on a small intestinal surface area for an adult human (Grassi et

al., 2007) the resulting phytosterol absorption capacity is 0.02 mg/cm2. This result is an order of

magnitude lower than the model uptake of β-sitosterol and three orders of magnitude lower than

the model uptake of SEDS. The in-vivo uptake of SEDS could also be potentially greater since

the absorption surface area to fed state volume ratio is two orders of magnitude greater for a

human intestine in comparison to the uptake model. Therefore, having surfactants and linkers in

the SEDS enhances drug absorption and potentially bioavailability. Refer to Appendix 1 for

calculations.

32

2.5 Conclusion

In this investigation, alcohol-free SEDS have been developed using sorbitan monooleate as a

lipophilic linker and a combination of decaglyceryl caprylate/ caprate and PEG-6-caprylic/capric

glycerides as hydrophilic linkers. Increasing the concentration of PEG-6-caprylic/capric

glycerides in the systems increased the hydrophilicity of the formulations and produces Type IV

bicontinuous microemulsions upon FeSSIF dilutions. The surfactant-linker system is partly

insoluble in oil and FeSSIF as it contains hydrophilic and lipophilic linkers, yet it is capable of

co-solubilizing equal amounts of oil and FeSSIF.

Phase behaviour scans (S1) and physical properties of the resulting linker-based SEDS were used

to select the optimal surfactant and linker combination that resulted in the largest solubilization

of the oil while facilitating the emulsification process upon dilution. The S1 microemulsion

aggregates are small (less than 12 nm) suggesting a large surface area to volume ratio to enhance

mass transfer. Based on the optimal S1 surfactant ratio, preconcentrates (10% -80% surfactant in

oil) were formulated and their phase behaviour was examined with a pseudo- ternary phase

diagram. Measurements on particle size and turbidity of diluted preconcentrates were conducted

to assist in selecting the optimal preconcentrate. The phase behaviour from the pseudo-ternary

phase diagram showed a reduction in the microemulsion regions due to the poor solubility of the

phytosterol in oil and aqueous phases and its tendency to crystallize upon aqueous contact. T40

and T50 were optimal systems that formed microemulsion structures, even at high dilutions

(91% FeSSIF). Higher dilutions (dilution factor 500) resulted in nanoemulsions with particle

sizes of 151 nm for T40 and 170 nm for T50. However, T50 was selected for uptake evaluation

as it showed the greatest stability in the turbidity studies.

The SEDS performance was evaluated using a developed Flow-Thru Dialyzer scheme. The

baseline (oil + active) simulation showed negligible uptake thus a substantial improvement is

observed when using SEDS. The T50 preconcentrate (SEDS) resulted in an uptake of 39.6

mg/cm2. The enhanced uptake is explained by the presence of surfactants and linkers which

enhance the interaction between the surfactant and oil/aqueous phases. The surfactant linker

combination prevents phase separation and improves drug solubilization. In-vivo absorption

however, could potentially be greater since the model does not account for active absorption, and

33

the human fed state volume to surface area ratio differs by two orders of magnitude. The Flow-

Thru Dialyzer technique shows consistent results and is therefore recommended as a method to

evaluate uptake from various delivery vehicles to predict in vivo behaviour.

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38

3 Chapter 3

Formulation of Food- Grade Linker-based Self-emulsifying

Delivery Systems

3.1 Introduction

According to the World Health Organization (WHO), cardiovascular diseases are the leading

cause of death on a global scale. Cardiovascular diseases (CVD) are known as disorders of the

heart and blood vessels. Fortunately, extensive studies have shown that fish oils, a major

source of omega-3 fatty acids, have substantial potential to prevent the onset of CVD. The

consumption of fish oil would be of great benefit in not only maintaining one's health (normal

triglyceride levels < 150 mg/ dL), but facilitating rapid recovery for individuals with very high

levels of triglycerides (>500 mg/dL). In fact, fish oils even appear to have greater potential for

deterring CVDs than vegetable oils (Nettleton, 1995).

The main components of fish oil responsible for cardiovascular benefits are omega-3 fatty

acids (ω3FA). Two of the most common ω3FA are eicosapentaenoic acid (EPA) and

docosahexaenoic acid (DHA) shown in Figure 3.1 (Shahidi and Finley, 2001). The benefits of

ω3FA are not exclusive to maintaining cardiovascular health as they also play a role in normal

growth and development, brain function and lowers the incidence of cancer and diseases

(Raatz et al, 2009; Nettleton, 1995; Shahidi, 2001; Drevon et al, 1993). However, many

individuals consume less than the recommended ω3FA intake and thus, cannot attain the health

benefits (Raatz et al, 2009; Nettleton, 2005). According to a workshop on the "Essentiality of

and Recommended Dietary Intake for Omega-6 and Omega-3 Fatty Acids" held at the National

Institutes of Health of the United States, the recommended daily intake consists of 650 mg

EPA/DHA for the reduction in chronic disease. Individuals who do not consume sufficient

levels of omega-3 fatty acids from food sources (e.g. 2-4 servings of fish per week) can take

supplements as a convenient alternative (Raatz et al, 2009; Rupp, 2009). According to Ocean

Nutrition Canada (ONC), fish oil capsules (omega-3 EPA/DHA) is ranked as one of the top

five supplements and the omega-3 market is growing at an annual rate of about 30%.

http://www.amazon.ca/s/178-7499199-3784524?_encoding=UTF8&search-alias=books-ca&field-author=Fereidoon%20Shahidi

39

Fish oil supplements can actually be a better, long-term source of omega-3 as they are purified

(reduced toxins) and more concentrated. Formulations can contain as much as 90% ω3FA in

ethyl ester form whereas natural fish oil has at most, a modest concentration of ~19% to 30%

ω3FA (Rupp, 2009; Raatz et al, 2009; Farooqui, 2009). However, not every product contains

the same quantity or quality fish oil. The chemical form also affects the rate and extent of

absorption. Fish oil can be manufactured into several forms including capsules containing

triglycerides or ethyl ester forms, wax esters (semi-solid), emulsified (semi-liquid), and

microencapsulated, all with varying degrees of performance. The bioavailability of ω3FA from

fish oil varies and is based on a number of factors such as the chemical form and level of fat

intake consumed with the fish oil.

A study by Gorreta et al. (2002) showed that the extent of ω3FA absorption in rats was greatest

for fish oil, followed by wax esters. They proposed that in comparison to fish oils, wax esters

are more stable as they are less susceptible to oxidation. There exists though, contradicting

views as human studies have revealed that wax esters are not hydrolyzed but are eliminated

quickly from the digestive tract (James et al., 1986; Berman et al., 1981). Microencapsulated

fish oil can also be incorporated into foods and have bioavailability similar to fish oil capsules

(Higgins et al., 1999; Wallace et al., 2000).

Lawson and Hughes (1988) showed that after the intake of a low fat meal the absorption of

EPA and DHA from fish oil triglycerides and ethyl esters were 69%, 61% and 20%, 22%

respectively. These rates changed to 90%, 68% and 65%, 59% respectively after the intake of a

high fat meal. It was suggested that the ethyl ester form deterred pancreatic lipase enzyme

activity as its double bond was closer to the carboxyl end.

Other studies showed similar trends of fish oil triglycerides having greater uptake of ethyl

esters (El Boustani et al., 1987). Opposing investigations indicated that ester and triglyceride

forms of fish oil had nearly equivalent absorption in humans (Harris et al., 1988; Nordoy et al.,

1991). The difference in observations could be explained by the varying levels of fat and time

frame in which the measurements were taken. The studies suggest that the absorption of fish

oil ester forms is enhanced over time after consuming a high fat meal. Evidently, the use of

ethyl esters is likely the preferred form of fish oil delivery as they are less expensive than

40

purified free fatty acids, and are more stable (Nettleton, 1995). Currently Lovaza/ Omacor is

the only EU and FDA approved prescription that contains purified omega-3 ethyl esters.

According to Lovaza, up to 14 capsules/day of an omega-3 product may be required to provide

the equivalent active ingredient dosage in a Lovaza capsule.

Further improved absorption of ω3FA is observed with emulsified fish oil. Studies have

demonstrated that emulsified fish oil was better absorbed than fatty acid ethyl esters and

triglyceride fish oil (Garaiova et al., 2007; Raatz et al., 2009). From these studies it seems that

for maximum bioavailability of fish oil, one needs to consume a high fat meal with a high

concentration of ω3FA (i.e. fish oil ethyl esters) in an emulsified form. One method to

facilitate the formation of emulsified ω3FA delivery systems is to develop self-emulsifying

delivery systems (SEDS). However, the use of SEDS and microemulsions has been restricted

by the use of alcohols and ionic surfactants used in several formulations.

In this study, it was hypothesized that linker-based lecithin self-emulsifying delivery systems can

be formulated as effective oral drug delivery vehicles for ω3FA using food grade ingredients. To

develop such microemulsions, the transdermal lecithin-based linker microemulsion developed by

Yuan and Acosta (2009), was used as an initial template. The transdermal formula comprised of

lecithin as the main surfactant, sorbitan monooleate as the lipophilic linker, and caprylic acid and

sodium caprylate as the hydrophilic linkers. The hydrophobic drug lidocaine was dosed in carrier

oil isopropyl myristate. In order to avoid pH sensitivity and biocompatibility limitations, the

caprylic acid and its salt were substituted with polyglyceryl-6 caprylate (food additive

21CFR172.854, acceptable daily intake (ADI) of 25 mg/kg body weight), a non-ionic

hydrophilic linker. The oil isopropyl myristate (IPM) was replaced with fish oil omega-3 ethyl

esters. Limonene and vitamin E were added to the fish oil ester to mask the odour and to prevent

oxidation. The lipophilic linker sorbitan monooleate was replaced with glyceryl monooleate

which has GRAS status (21CFR184.1323). Figure 3.2 shows a surfactant and linker-self

assembly at the oil/aqueous interface. To model in vivo drug performance, Fed-State Simulated

Intestinal Fluid (FeSSIF), was used as the aqueous dilution media (Dressman et al., 1998,

Dressman et al., 2000; Galia et al., 1998). The goal of this work is to formulate food-grade

lecithin linker microemulsions for the delivery of polyunsaturated fatty acid ethyl esters (fish oil

esters). To this end, the first objective is to find the optimal ratio among lecithin, the lipophilic

41

linker glyceryl monooleate, and the hydrophilic linker polyglyceryl-6 caprylate. The second

objective is to generate a ternary phase diagram using this optimal lecithin-linkers ratio as the

surfactant mixture, fish oil ester, and FeSSIF as the aqueous phase to determine the optimal

surfactant to oil ratio that produces stable SEDS (greater than 100 nm) and SMEDS (less than

100nm) formulations. The third and final objective is to assess, in-vitro, the intestinal uptake of

these systems to identify the potential advantages, or limitations of these lecithin-linker

formulations.

Figure 3.1 Structure of omega-3 fatty acids docosahexaenoic acid (top), and eicosapentaenoic

acid (bottom).


interface

42

3.2 Materials and Methods

3.2.1 Materials

3.2.1.1 Chemicals

The following chemicals were purchased from Sigma-Aldrich Canada with concentrations shown

in parenthesis: taurocholic acid sodium salt hydrate (95%+), Dulbecco's phosphate buffer saline

(PBS), albumin from bovine serum (BSA) and β-carotene (95%), sodium chloride (99.5%+,

Fluka brand), tocopheryl acetate (1000 IU/g Jamieson brand), (+)- limonene (95%, Acros

Organics brand). Sodium hydroxide pellets were purchased from Caledon chemicals. Glacial

acetic acid (99.7%+) was purchased from EMD Chemicals Inc. Samples of glyceryl monooleate

from Gattefossé, fish oil ethyl esters (55% EPA, 13% DHA) from Ocean Nutrition Canada, and

polyglyceryl-6 caprylate from Dr. Straetmans were kindly donated. Laboratory grade soybean

lecithin (99%+) was purchased from Fisher Scientific. Frozen sheep intestine was purchased

from a local market. The compositions listed in this paper are on a mass basis (wt. %) unless

stated otherwise.

3.2.1.2 Tissue

The frozen sheep intestine soaked in warm water for 1 hour for thawing. A three inch section of

the sheep small intestine (jejunum) was then excised and the exterior impermeable membrane

was removed. The excised section was rinsed 4 times with distilled water and then soaked in

PBS for 30 minutes to render it ready for use in the in vitro study.

3.2.2 Microemulsion Formulation

Phase behavior studies were conducted to determine the optimal surfactant and linker ratios for

SEDS formation. Equivalent masses of oil and aqueous solutions were combined and vortexed for

two minutes at 3200 rpm and allowed to equilibrate (~ two weeks) at room temperature (~ 25 °C)

and pressure (1 atm). Fed state simulated intestinal fluid (FeSSIF) was used as the dilution

medium rather than water (Dressman et al., 2000). Refer to Chapter 2, Table 2.1 for composition.

Preliminary phase scans (F series) were conducted where the polyglyceryl-6 caprylate

concentration ranged from 1 to 40%. Each series was prepared using different ratios of glyceryl

43

monooleate: lecithin. The scans containing glyceryl monooleate: lecithin ratios, more

specifically, 0:1, 0.5:1, 1:1, 2:1, 3:1 correspond to series F0, F1, F2, F3, F4. Based on the scans,

it was determined that a ratio of 0.5:1 (F1) was optimal in producing transparent isotropic

regions with the smallest particle sizes upon FeSSIF dilution. The composition of F1 is shown in

Table 3.1. Since the scan was at room temperature, oxidation occurred within 24 hours due to

the presence of polyunsaturated fatty acids in fish oil. The rate of oxidation is substantially

reduced if the fish oil ethyl esters are stored at refrigeration temperature of ~9°C. 2% antioxidant

tocopherol acetate was added yet this did not prevent the onset of oxidation.

Table 3.1 Composition of F1 phase scan

Phase Component F1 Weight %

Aqueous Lecithin 6%

Polyglyceryl-6 caprylate 1-35%

FeSSIF To 50%

Oil Glyceryl monooleate 3%

Fish oil ethyl ester To 50%

3.2.3 Physicochemical Characterization

3.2.3.1 Particle Size

The average particle diameters were determined using dynamic light scattering measurements

from a 90Plus Particle Size Analyzer instrument (Brookhaven Instruments). The cuvettes were

set aside for 2 minutes to equilibrate prior to the measurements. The data was obtained by

averaging five measurements per vial set at 1 minute per measurement. The viscosity of the vials

was measured in triplicate using a CV-2200 falling ball viscometer (Gilmont Instruments,

Barrington, IL, USA). These measurements were conducted at room temperature ~ 25°C.

3.2.3.2 Turbidity

Turbidity measurements were calculated according to Equation 3.1 where τ is turbidity having

units of inverse length, L is the path length of light, I is the transmitted light intensity, and I0 is

44

the transmitted light intensity of the ―clear‖ sample (Acosta et al., 2003b), which in this case is

distilled water. A red line drawn on a white background served as a reference. Since drug

residence time in the small intestine is ~ 3 hours (Fadda et al., 2009), images were captured over

four hours to examine stability. The light intensities were assessed via image analysis tools Corel

PaintShop Photo Pro X3 and Scion Image Beta 4.03.

τ

I

I

L

0ln1

(Equation 3.1)


A pseudo-ternary phase diagram was generated to examine phase behaviour and identify the range

of isotropic microemulsion regions. Mixtures of surfactant, linkers and oil (preconcentrates)

were diluted with FeSSIF to produce the diagram. Oil (fish oil ethyl esters) dilution lines of

surfactant, linkers and fed state were also created to assist in phase characterization. FeSSIF was

used to mimic biological conditions and predict in-vivo phase behaviour upon dilution

(Dressman et al., 1998; Staggers et al., 1990; Wang et al., 1996). The surfactant -linker mixture

consisted of lecithin, glyceryl monooleate, polyglyceryl-6 caprylate, in the ratio of 6:3:35 as

explained in section 3.3.1. Surfactant-linker in oil preconcentrates containing 10%, 20%, 30%,

40%, 50%, 60%, 70%, 80%, and 90% surfactant were each diluted with FeSSIF at ratios of

1:0.2, 1:0.5, 1:1, 1:2, 1:5, 1:10, 1:100, 1:500. Similarly, 10% to 90% surfactant and linker

mixtures in FeSSIF were diluted with oil to determine phase behaviour around the surfactant-

FeSSIF axis. Short form notations will be used to identify preconcentrate dilutions, for example

T20 represents the preconcentrate containing 20% surfactant in oil, T20-1 (16.7% aqueous

phase) represents the first dilution of the T20 preconcentrate, and T60-SF is the 60% surfactant

in FeSSIF preconcentrate. Each sample was vortexed for 1 minute at 3200 rpm. The remaining

formulations were left to equilibrate (~2 weeks) for further classification.

3.2.5 Self-emulsification Studies

This portion of the study will involve mimicking intestinal conditions via dilution with FeSSIF and

measuring the SEDS uptake. The sample is vortexed for two minutes prior dilution with FeSSIF. A

dilution ratio of 500 was used to approximate the intestinal dilution (Hauss, 2007). The diluted

http://www.scioncorp.com/downloads/scion_image/SetupWinB403.exe

45

microemulsion preconcentrates will be classified based on the particle sizes discussed earlier,

where SMEDS are less than 100 nm, SEDS exceed 100 nm, and conventional emulsions are in

the range of 1-100 µm (Acosta et al., 2005; Kesisoglou et al., 2007, Rosen 2004; Huang et al.,

2010).


The Flow-Thru Dialyzer (Harvard Apparatus models: 741202, 741105, 74104) setup shown in

Figure 3.3 consisted of a receiver chamber containing 4% bovine serum albumin in PBS. The

SEDS (0.23% β-carotene in oil) was diluted with FeSSIF and pumped through the donor

chamber. The chambers are divided by a cleansed section of intestine. Both reservoirs were

mixed at the lowest setting. An Ocean Optics UV/VIS Spectrophotometer (HR2000) and

software (OOIBase32) tracked changes in absorbance of the β-carotene marker. The absorption

run was set for two hours, after the donor chamber was rinsed with 6 mL of FeSSIF and

discharged. The donor side then pumped 15 mL of fresh FeSSIF for one hour to simulate

desorption. The fluids flowed at 3 mL/min, which is in agreement with the postprandial flow rate

in the jejunum (Fadda et al., 2009; Kerlin et al., 1982). The absorbance data was obtained by

subtracting the absorbance at 450 nm (signal detection wavelength) from the baseline at 600 nm.

The integration time, average, boxcar, and strobe frequency parameters were set at 8, 10, 2, and

10 respectively. The volumes of simulated plasma and intestinal fluid after SEDS administration

were 30mL and 10 mL. These volume ratios were designed based on the ratios found in literature

(Sherwood, 2004).

Figure 3.3 Flow-Thru Dialyzer method to measure uptake of β-carotene

46

3.3 Results

3.3.1 Phase Scans

The preliminary scans from 1% to 40% polyglyceryl-6 caprylate for the F series (F0, F1, F2, F3,

F4) showed that most concentrations of 30% or less of polyglyceryl-6 caprylate produced liquid

crystal systems. A higher concentration of 35% polyglyceryl-6 caprylate was required to produce

isotropic microemulsions. The samples containing 35% polyglyceryl-6 caprylate in each of the F

series were diluted at ratios of 100 and 500, with the average particle size measurements reported

in Figure 3.4. The F1 dilution was the desired system as the dilution of 100 leads to a

microemulsion particle size of 68.8+3.5 while the F0, F2, F3, resulted in nanoemulsions (Rosen,

2004). F4 which contained more glyceryl monooleate (lipophilic linker) led to a turbid dilution

with average drop size exceeding 500 nm. At dilution 500, F1 had the smallest average particle

size and thus the F1 ratio of 6% lecithin/35% polyglyceryl-6 caprylate/3% glyceryl monooleate

was used to develop the pseudo-ternary phase diagram. The F1 phase behaviour scan is shown in

Figure 3.5 below where in the range of 1% - 25% polyglyceryl-6 caprylate, multiple phases were

formed along with liquid crystals (lower phase). Higher concentrations consisting of 30% and

35% polyglyceryl-6 caprylate resulted in isotropic microemulsion systems, where the latter

concentration solubilized all the oil.

Figure 3.4 F series particle sizes after FeSSIF dilution factor of 500

500

47

1% 5% 10% 15% 20% 25% 30% 35%

Figure 3.5 F1 Hydrophilic linker scan, % polyglyceryl-6 caprylate

3.3.2 Pseudo- Ternary Phase Diagram

Figure 3.6 shows the pseudo-ternary phase diagrams for the oil–surfactant mixture–FeSSIF

system at 4 hours and 2 weeks. Visible changes were either slight or negligible after two weeks

indicating equilibrium. Figure 3.6.a shows the phase behaviour of the system after 4 hours where

a clear, monophasic region was identified as 1Ø. T60 and T70 were monophasic along the

dilution lines up to a FeSSIF content of 83% and 91% respectively. At dilution 500, T80 and T90

formed clear isotropic systems. The surfactant-linker systems were also dissolved in the fish oil

leading to the single phase along the surfactant-oil axis. On the contrary, the surfactant-linker-oil

mixtures solubilized the aqueous phase at low and high concentrations. The areas labeled mØ

are turbid regions with multiphase systems. The blue and green lines are the T60 and T40

dilution lines. These preconcentrates were evaluated with the dialyzer uptake model. Upon

dilution T40 formed a turbid multiphase system however it resulted in the most uptake as

determined in 3.3.4.

Figure 3.6.b presents the phase diagram after two weeks where the various regions are identified.

Dilutions of the T10-T30 systems led to phase separation, yet higher surfactant concentrations

led to enhanced oil solubilization as observed with T40-T50. However, not all the oil was

solubilized which resulted in excess oil phases. Multiple phases were also present near the

surfactant-linker-oil axis. The S+FO+F region comprised of an upper phase mixture of

surfactant/linker, oil, FeSSIF and a separated lower phase. Liquid crystals were present in

systems containing high surfactant concentration. These regions exhibited birefringence under

cross polarizers and are marked by red triangles in Figure 3.6.b. The initial dilutions of T80 and

T90 led to single clear phases while further dilution resulted in liquid crystals (lower phase) with

an excess oil phase (upper phase ) in the LC+oil region. An example of this phase under cross

polarizers is denoted by the triangle at T80-2 and contains 33% FeSSIF. Increasing increments of

48

fed state for T80 formed structurally different phases of liquid crystals resulting in phase

separation. This region is the LC1+LC2 as seen with the T80-3 (50% FeSSIF) image. The LC

region containing surfactant and FeSSIF with low oil content represent lyotropic liquid crystals

as shown near the axis of 30% to 80% surfactant in FeSSIF. At very high dilutions of T80 and

T90, a liquid crystal area with excess aqueous phase (upper phase) is formed (F+LC) as observed

with the T80-6 triangle (91% FeSSIF). T90 went through these transitions with the exception of

an additional liquid crystal and upper phase excess FeSSIF (LC+F) region. The surfactant-linker

systems at T10-T40 (S+FO+F) resulted in phase separation where polyglyceryl-6 caprylate

separated over time from the oil-surfactant rich phase. From the phase diagrams at a dilution

factor of 500, T40 to T60 were found to be SEDS while T70 to T90 resulted in SMEDS as a

single phase was maintained.

49

Figure 3.6 Pseudo-ternary phase diagrams of the F1 formulation using the 35% polyglyceryl-6

caprylate surfactant ratio at 4 hours (a), and after 2 weeks (b). SL represents the surfactant-linker

apex, F is FeSSIF, FO is fish oil ethyl esters. Surfactants and linker mixture (lecithin, glyceryl

monooleate, and polyglyceryl-6 caprylate) are in the ratio of 6:3:35. The red triangles denote

regions with liquid crystals at specified preconcentrate dilutions. *Liquid crystals (LC), liquid

crystals and excess oil (LC + oil), liquid crystals and FeSSIF lower phase (LC + F), 2

separate phases of liquid crystals (LC1+LC2), liquid crystals and FeSSIF upper phase

(F+LC), microemulsion and excess oil (µE +oil), single phase microemulsion (µE),and

surfactant, fish oil, FeSSIF mixture (S+FO+F)

T40

50

3.3.3 Formulation of Preconcentrates

The average droplet sizes and turbidity measurements were obtained to characterize the

performance of the preconcentrates upon dilution. T10 - T90 were diluted by a factor of 500 with

FeSSIF as shown in Figure 3.7 and sizes are reported in Figure 3.8. The preconcentrates T70 -

T90 have particle sizes under 100 nm and are thus classified as SMEDS which confirms the

ternary phase dilutions at the ratio of 1:500. Systems T40-T60 are SEDS with particle sizes

between 150 nm -285 nm. T10-T30 have an average range from ~ 330 nm- 580 nm.

10% 20% 30% 40% 50% 60% 70% 80% 90%

Figure 3.7 FeSSIF dilution of preconcentrates containing 10%-90% surfactant in oil.

Figure 3.8 Average particle sizes of diluted preconcentrates containing 10%-90% surfactant in

oil.

A necessary feature of SMEDS and SEDS is being stable over the course of digestion (Hauss,

2007). The stability of the diluted preconcentrates and diluted fish oil ethyl esters were

51

determined via turbidity measurements. Figure 3.9.a shows the turbidity plot at times 0 and 4

hours with respect to sample height comparing the dilutions of the oil, T40 and T70 systems. The

top of the sample is the initial sample height (0 cm). Figure 3.9.b shows the change in turbidity

after four hours of the diluted preconcentrates and oil. The percent variations were determined by

measuring the bottom of each vial as this is where the greatest separation would occur. The

diluted oil has shown a significant reduction in turbidity (50%) indicating drastic separation. T10

and T30 are also not stable and showed visible changes in turbidity in the range of 12%-21%.

T20, T40, and T50 were more stable with values of 6%-9%. The most stable dilutions were T60-

T90 with changes in the range of 1% - 3%. Since T70-T90 had the smallest particle sizes and are

classified as SMEDS it was expected that they would have the negligible changes in turbidity as

they are thermodynamically stable.

(a) (b)

Figure 3.9 Turbidity Plot of (a) diluted oil and surfactant in oil preconcentrates T40,T70 and (b)

Change in turbidity of diluted oil (measurement at 0% surfactant in oil) and surfactant in oil

preconcentrates T10-T90. The changes between 0 and 4 hours are measured at the bottom of

each sample. The dilution is with FeSSIF at a factor of 500.

52

3.3.4 In- Vitro Absorption- Dialyzer Studies

From the phase behaviour scans, the optimal surfactant ratio was determined to be 6:3:35 for

lecithin, glyceryl monooleate, and polyglyceryl-6 caprylate respectively. Based on the dilution

and turbidity results of various preconcentrates using this surfactant ratio, systems containing at

least 60% surfactant produced the most stable SEDS. Although T90 is the most stable, it is not

very practical for oral delivery as it contains only 10% oil and 90% surfactant. Therefore T60 is

selected for uptake evaluation. We hypothesized that this formulation should enhance ω3FA

uptake, thus in this section we evaluate uptake using the in-vitro dialyzer.

Dialyzer runs showed that the 60% surfactant in oil preconcentrate resulted in negligible absorption.

A lower surfactant in oil concentration of 50% (T50) resulted in ~7% uptake as shown in Figure

3.12 . Preconcentrates containing lower surfactant concentrations were tested to determine whether

higher uptake could be obtained. Preconcentrates containing 25%, 30%, and 35% surfactant in oil

led to phase separation as the oil and β-carotene dye accumulated within the donor side chamber.

Figure 3.10.a shows the β-carotene and oil phase separation from the FeSSIF solution for the

preconcentrate containing 35% surfactant in oil. Figure 3.10.b shows a separate comparison of

diluted T50 and diluted fish oil (FeSSIF dilution factor 500), which were mixed and left aside for

two hours. T50 had remained stable over time while the diluted fish oil separated from the

aqueous phase. A surfactant concentration of 40% (T40) was determined as the optimal, stable

(no phase separation) formulation where an uptake of 17.2+ 0.2% was attained. Therefore the

T40 system was selected for further assessment of SEDS absorption.

(a) (b)

Figure 3.10 (a) The Flow-Thru Dialyzer donor chamber interior of T35 system revealing oil

phase separation, (b) FeSSIF dilution (factor 500) of T50 on the left and fish oil ethyl ester on

the right (both dosed with 0.23 % β-carotene in oil phase) after two hours.

53

The concentration profile of β-carotene is illustrated in Figure 3.11. The process of desorption is

depicted in black and shows that the in-vitro absorption is an irreversible process. During the two

hour simulation, the changes in concentration appeared as a linear trend. It is assumed that the

uptake of the SEDS is equivalent to the uptake of the dye as the solution remained dispersed over

the course of the simulation. SEDS uptake is shown in Figure 3.12. Table 3.2 lists the physical

conditions of the dialyzer model and human intestine. For the model the absorption capacity

(maximum SEDS absorbed per unit intestinal surface area) was 12.2 mg/cm2 after 2 hours, based

on the uptake of the initial dose (0.02g SEDS) divided by the intestinal surface area. From the

table, there exists a difference of two orders of magnitude in the model versus human surface

area to volume ratio. This suggests that the increase in surface area would potentially enhance

the uptake if ingested in-vivo. Refer to Appendix 2 for sample calculations and absorbance

spectra.


carotene in oil phase).

54

Figure 3.12 Flow-Thru Dialyzer percent uptake of T40 and T50 SEDS dilution containing 0.23 % β-

carotene in oil.

Table 3.2 Physical conditions of an adult human and the uptake model

Small intestine SA

(cm2) SA:FeSSIF

Max SEDS absorbed (g)/ Area

(cm2)

Model 0.28 28 0.012

Human 9800* 9800*

SA - surface area, *Grassi et al. (2007)

3.4 Discussion

3.4.1 Optimal Surfactant-linkers ratio

In this work polyglyceryl-6 caprylate has been used, for the first time, as a hydrophilic linker.

This amphiphile was classified as a hydrophilic linker because it is highly hydrophilic and has 8

carbons in its tail group (hydrophilic linkers have between 6 to 9 carbons in their hydrophobic

tail) (Acosta et al., 2002; Sabatini et al., 2003). One of the advantages of using hydrophilic

linkers is that they help prevent the formulation of liquid crystals (Yuan et al., 2008); however,

in this case, 35% of polyglyceryl-6 caprylate was needed to avoid liquid crystal formation in

systems containing 50% fish oil. This suggests that polygylceryl-6 caprylate is not an effective

hydrophilic linker but is the only food grade hydrophilic linker identified thus far. In general,

55

high concentrations of hydrophilic linkers also require high concentrations of lipophilic linkers to

compensate these changes (Acosta et al., 2003a; Sabatini et al., 2003). However, in this case, the

optimal formulation was produced with 35% polyglyceryl-6 caprylate and only 3% glycerol

monooleate. This observation further supports the hypothesis that polyglyceryl caprylate is a

relatively weak hydrophilic linker. Nevertheless, this optimal formulation was capable of

producing stable SEDS and SMEDS formulations.


In this study, a pseudo-ternary phase diagram was created to examine the phase behaviour of the

F1 system using simulated intestinal media. The types of phases (emulsions, dispersed liquid

crystalline regions and mixed micelles) present in the pseudo-ternary phase diagram are similar

to the kind of phases found in the small intestine (Stafford et al., 1981; Staggers et al., 1990).

From Figure 3.6, it is evident that more surfactant is required to maintain stability and solubility

upon FeSSIF dilution. T10-T30 had low surfactant combinations leading to less oil solubilization

and phase separation upon dilution. T40-T50 showed microemulsion phases capable of oil

solubilization. However, T60-T70 led to bicontinuous structures with the ability to integrate

nearly all the oil upon high dilutions. A high concentration of 70%-90% surfactant is required

though to produce microemulsions upon dilution factor of 500 (SMEDS). It is desirable to form

these types of bile salt micelles as this form enhances gastrointestinal absorption of lipids by

promoting diffusion. Micelle particles are also better able to penetrate the space between

microvilli thus facilitating uptake and transfer (Gajjar et al., 2007). However, if the surfactant

concentration is too high, it becomes impractical for therapeutic use as only a small amount of

oil is present in the self-emulsification system.

From the phase diagram, it is observed that the stability of the phases is dependent on the

surfactant and dilution volume. Higher FeSSIF dilutions promote instability and phase separation

with the exception of preconcentrates containing large amounts of surfactant. Bicontinuous

microemulsions seem to be unstable at high dilution ratios therefore the optimal formulations

would be the ones that approach Type I continuity (i.e. T40, T50). With this in mind, it could

explain the enhanced uptake obtained with T40 and T50 dilutions in comparison to the T60

system.

56

3.4.3 Formulation of Preconcentrate

Evaluating the dilutions of the fish oil preconcentrates is a necessary step to predict in-vivo

behaviour and stability. The variations in turbidity over time as well as particle sizes can serve as

indicators of stability. The systems that had the least change in turbidity also had the smallest

particle sizes. The SMEDS T70-T90 formed transparent solutions with sizes less than 100 nm

yet also remained solubilized and stable over time. However, even the SEDS would be sufficient

for oral delivery as systems T40 and T50 remained well dispersed in the uptake studies. The

general trend showed that the higher the polyglyceryl-6 caprylate concentration, the smaller the

particle size and the more stable the dilution. This observation can be explained by the increased

amounts of polyglyceryl-6 caprylate, which is a good oil solubilizer with good wetting

properties. Despite the enhanced stability and size reduction, one also has to consider the surface

chemistry and preferential affinity of surfactants as this property may ultimately decide the

viability of the formulation for oral delivery. For instance, from the dialyzer results, although

T60 was fairly stable and had small size it had very poor uptake due to the higher affinity for the

aqueous phase.


From the study, the dosage absorbed in two hours shows a linear uptake that has not yet attained

equilibrium. This suggests that more time is required for optimal absorption. Research has shown

that levels of plasma phospholipid EPA and DHA derived emulsified fish oil peaked at 8 and 24

hours respectively (Raatz et al., 2009). Harris and Williams (1989) reported that emulsified fish

oil was absorbed to a greater extent than triglyceride forms over 6 hours despite literature

reports of drug intestinal transit times of ~ 3 hours (Fadda et al., 2009). Based on in-vivo studies

the plasma EPA/DHA levels do not show a linear trend over several hours, after two hours the

EPA/DHA levels actually have an increased rate of absorption until approaching the peak levels,

after which the concentrations of omega-3 fatty acids decreased (Garaiova et al., 2007; Raatz et

al., 2009). Further in-vivo studies will confirm the actual extent and time frame of uptake for this

particular system.

57

Although the prescence of linkers and surfactants in the SEDS has promoted uptake, it is

necessary to aquire the right combination of these components. As reported earlier on, 25%-35%

surfactant concentrations in the preconcentrate lead to phase separation as a minimum of ~40%

surfactant is required to emulsify and maintain stability upon simulated intestinal dilution. On the

other hand, having too much surfactant was counterproductive as 50% surfactant in the

preconcentrate led to reduced uptake and 60% had negligible uptake. These findings suggest

that, at least for this formulation, the SMEDS produced at high surfactant content are well

dispersed in water and have a lesser affinity for the tissue when compared to SEDS.

Despite the model accounting only for passive transport and entrapment in the mucin layer, the

uptake of 17% from the T40 preconcentrate has been found to be comparable to other studies.

Ikeda et al. (2006) assessed intestinal uptake in rats of EPA and DHA after three hours and found

that 26% of EPA and 6.4% DHA were absorbed. The dialyzer uptake could potentially be higher

than 18% since the simulation was conducted over two and not three hours.

Based on the uptake model the maximum SEDS absorption capacity is 12.2 mg/cm2, which

translates to an omega-3 fatty acid absorption capacity of about 5 mg/cm2

. For an FDA approved

4 g dose of fish oil ethyl ester from Omacor (Rupp, 2009) that has a postprandial bioavailability

of 65% for EPA and 59% for DHA (Lawson and Hughes, 1988), an omega-3 fatty acid

absorption capacity of 0.2 mg/cm2 results based on human intestinal surface area (Table 3.2).

This value is an order of magnitude lower than the model uptake of omega-3 fatty acid and two

orders of magnitude lower than the model uptake of SEDS. The in-vivo uptake of SEDS may

also be much greater since the absorption surface area to fed state volume ratio is two orders of

magnitude greater for a human intestine in comparison to the in-vitro model. In addition, the

uptake from the model is likely incomplete and has not reached the optimal value as in-vivo

studies have demonstrated that the absorption of emulsified fish oil still occurs after 8 hours of

administration (Raatz et al., 2009). Therefore, the presence of surfactants and linkers in the

SEDS formulation enhances drug absorption and potentially bioavailability. Refer to Appendix 2

for calculations.

58

3.5 Conclusion

In this study, linker based alcohol-free SEDS were developed using food grade ingredients for

the delivery of omega-3 ethyl esters. The formulation comprised of using glyceryl monooleate as

a lipophilic linker and polyglyceryl-6 caprylate as a hydrophilic linker. The ratio of lecithin and

linkers was optimized to produce the smallest drop size upon dilution with fed state simulated

intestinal fluid. The preconcentrates containing different proportions of surfactants (optimal

ratio of lecithin and linkers) and fish oil were diluted with FeSSIF to produce pseudo-ternary

phase diagrams and to identify the formulations that produced SEDS and SMEDS. Particle size

and turbidity measurements of the diluted preconcentrates were also used to direct the selection

of the optimal SEDS preconcentrate ratio for the uptake studies. Preconcentrates containing 40%

to 60% surfactants produced SEDS while systems containing 70% to 90% produced SMEDS.

Surprisingly, SEDS systems formulated with preconcentrates containing 40% surfactant

produced the best uptake using an in-vitro model of intestinal sorption. Based on phase

behaviour, turbidity and particle size measurements, it seemed that the SMEDS were optimal for

uptake evaluation. However T60 resulted in negligible uptake while less stable systems T40 and

T50 (SEDS) were capable of producing 17.2% and 7% uptake (12.2 mg/cm2

and 5 mg/cm2

),

respectively. These results highlight that size and stability of the emulsions or microemulsions

are not the only defining factors for enhanced uptake, but also the affinity of the emulsion drops

for the tissue.

3.6 References

Acosta, E., Uchiyama, H., Sabatini, D., Harwell, J., 2002. The Role of Hydrophilic Linkers.

Journ. Surfact. Deterg. 5 (2), 151-157.

Acosta, E., Do Mai, P., Harwell, J.H., Sabatini, D.A., 2003a. Linker-Modified Microemulsions

for a Variety of Oils and Surfactants. Journ. of Surfactants and Detergents. 6, 353-363.

Acosta, E., Le, M., Harwell, J.H., Sabatini, D.A., 2003b. Coalescence and Solubilization

Kinetics in Linker-Modified Microemulsions and Related Systems. Langmuir. 19, 566-574.



59

Berman, P., Harley, E.H., Spark, A.A., 1981. The passage of oil per-rectum - after ingestion of

marine wax esters. South A. Med. Journ., 59 (22), 791-792.

Bryhn, M., Hansteen, H., Schanche, T., Aakre, S.E., 2006. The bioavailability and

pharmacodynamics of different concentrations of omega-3 acid ethyl esters. Prostag., Leuko.

and Ess. Fatty Acids, 75, 19–24.

Dressman, J. B., Amidon, G. L., Reppas, C., Shah, V. P., 1998. Dissolution testing as a

prognostic tool for oral drug absorption: immediate release dosage forms. Pharm. Res., 15, 11–

22.

Dressman, J. B., Reppas, C., 2000. In vitro-in vivo correlations for lipophilic, poorly water-

soluble drugs. Euro. Journ. Of Pharm. Sci., 11, 73-80.

Drevon, C. A., Baksaas, I., Krokan, H. E. 1993. Omega-3 fatty acids: metabolism and

biological effects. Germany. Birkhauser.

El Boustani, S., Colette, C., Monnier, L., Descomps, B., Crastes de Paulet, A., Mendy, F.,

1987. Enteral Absorption in Man of Eicosapentaenoic Acid in Different Chemical Forms.

Lipids. 22, 711-714.

Fadda, H., McConnell, E., Short, M., Basit, A., 2009. Meal Induced Acceleration of Tablet

Transit Through the Human Small Intestine. Pharmaceutical Research 26, 356-35.

Farooqui, A., 2009. Beneficial effects of fish oil on human brain. Springer. New York.

Gajjar, R., Lo, C., Tso, P., 2007. Physiological Processes Giverning the Gastrointestinal

Absorption of Lipids and Lipophilic Xenobiotics. Oral Lipid-Based Formulations Enhancing the

Bioavailability of Poorly Water-Soluble Drugs. Ed. David Hauss. Informa Healthcare, New

York.. 207-239.

Garaiova, I., Guschina, I., Plummer, S., Tang, J., Wang, D., Plummer, N., 2007. A randomised

cross-over trial in healthy adults indicating improved absorption of omega-3 fatty acids by pre-

emulsification. Nutrition Journal, 6:4.

Gorreta, F., Bernasconi, R., Galliani, G,; Salmona M., Tacconi, M.T., Bianchi. R., 2002. Wax

Esters of n -3 Polyunsaturated Fatty Acids: A New Stable Formulation as a Potential Food

Supplement. 1 — Digestion and Absorption in Rats. Lebens.-Wiss. u.-Technol., 35(5), 458-465.


Absorption Mechanisms: A Physical and Mathematical Approach . CRC Press, Fl, U.S.A.

Harris, W.S., Zucker, M.L., Dujovne, C.A., 1988. ω-3 Fatty acids in hypertriglyceridemic

patients: Triglycerides vs. Methyl esters. Am. J. Clin. Nutr.48, 992-997.

Harris, W.S., Williams, G.G. 1989. Emulsification enhances the absorption of fish oil in man.

In : Health Effects of Fish and Fish Oils, R.K. Chandra, ed. Newfoundland, Canada. Arts

Biomedical Publishers & Distributors, 211-218.

http://www.ingentaconnect.com/content/ap/fs

60

Higgins, S., Carroll, Y.L., O'Brien, N.M., Morrissey, P. A., 1999, Use of microencapsulated

fish oil as a means of increasing n-3 polyunsaturated fatty acid intake. Journ. of Human Nutr.

& Diet., 12, 265-271 .

Huang, Q., Yu, H., Ru., Q., 2010. Bioavailability and Delivery of Nutraceuticals Using

Nanotechnology. Journ. Food Sci., 75, R50-R57.

Ikeda, I., Imasato, Y., Nagao, H., et al., 1993. Lymphatic transport of eicosapentaenoic and

docosahexaenoic acids as triglyceride, ethyl ester and free acid, and their effect on cholesterol

transport in rats. Life Sci.,52,1371-1379.

James, K., Body, D., Smith, W., 1986. A nutritional evaluation of orange roughy (hoplostethus

atlanticus) using growing pigs. New Zeal. Journ. Tach.2 (4).219 -223.



Kerlin,P., Zinsmeister,A., Phillips. S., 1982.Relationship of motility to flow of contents in the

human small-intestine. Gastroenterology. 82,701–706.

Lawson, L.D., and Hughes, B. 1988. Absorption of eicosapentaenoic acid and docosahexaenoic

acid from fish oils triacylglycerols or fish oil ethyl esters co-ingested with a high-fat meal.

Biochem. Res. Commun. 156, 960-963.

Mu, H., 2008. Bioavailability of omega-3 long-chain polyunsaturated fatty acids from foods.

Agrofood industry hi-tech. 19, 24-26.

Nettleton, J. 1995. Omega-3 Fatty Acids and Health. Chapman & Hall. New York, NY.

Nordoy, A., Barstad, L., Connor, W., Hatcher, L., 1991. Absorption of the n-3 eicosapentaenoic

and docosahexaenoic acids as ethyl esters and triglycerides by humans. Am. J.Clin. Nutr.53,

1185-1190.

Raatz, S., Redmon, B., Wimmergren, N., Donadio, and J., Bibus, D. 2009. Enhanced

absorption of omega-3 Fatty Acids from Emulsified Compared with Encapsulated Fish Oil, J.

American Dietetic Assoc., 1076-1081.

Rupp, H., 2009. Omacor® (Prescription Omega-3-Acid Ethyl Esters 90): From Severe Rhythm

Disorders to Hypertriglyceridemia. Adv. Ther., 26(7), 675-690.

Shahidi, F., and Finley, J. 2001. Omega-3 Fatty Acids: Chemistry, Nutrition, and Health

Effects. An American Chemical Society.

Sherwood, L. 2004. Human Physiology: From Cells to Systems, Brooks Cole; 5th edition , 623-

632.

Stafford, R. J., Donovan, J. M. , Benedek, G. B. , Carey, M.C., 1981. Physical-chemical

characteristics of aqueous duodenal content after a fatty meal. Gastroenterology. 80 (5), 1291A.

Staggers, J., Hernell, O., Stafford, R., Carey, M., 1990. Phase – Chemical Behavior or Dietary

and Biliary Lipids during Intestinal Digestion and Absorption. 1. Phase Behavior and

http://www.amazon.ca/s/178-7499199-3784524?_encoding=UTF8&search-alias=books-ca&field-author=Fereidoon%20Shahidi

http://www.amazon.ca/s/178-7499199-3784524?_encoding=UTF8&search-alias=books-ca&field-author=John%20W.%20Finley

61

Aggregation States of Model Lipid Systems Patterned after Aqueous Duodenal Contents of

Healthy Adult Human Beings. Biochemistry. 29, 2028-2040.

Swahn, E., Olsson, A., 1993. Omega-3 ethyl ester concentrate. Fish oil or secondary

prevention of myocardial infarction? In Drevon, C. A., Baksaas, I., Krokan, H. E. (Eds.).

Omega-3 fatty acids: metabolism and biological effects (217 -222). Birkhauser. Germany.

Wang, D.Q.-H., Carey, M.C., 1996. Complete mapping of crystallization pathways during

cholesterol precipitation from model bile: Influence of physical-chemical variables of

pathophysiologic relevance and identification of a stable liquid crystalline state in cold, dilute

and hydrophilic bile salt-containing systems Journal of Lipid Research, 37 (3), 606-630.

Wallace, J.M., McCabe, A.J., Robson, P.J., Keogh, M.K., Murray, C.A., Kelly, P.M., Marquez-

Ruiz, G., McGlynn, H., Gilmore, W.S., Strain, J.J., 2000. Bioavailability of n-3 polyunsaturated

fatty acids (PUFA) in foods enriched with microencapsulated fish oil. Ann. Nutr. Metab.44,157-

162.



62

4 Chapter 4

Conclusions and Future Recommendations

4.1 Conclusions

Pharmaceutical and food-grade linker-based lecithin self-emulsifying delivery systems (SEDS)

have been formulated and show promising potential as effective oral drug delivery vehicles. The

formulations also pose less toxicity issues in comparison to alcohol based systems. It was found

that the optimal SEDS substantially improved intestinal uptake in comparison to oil dosage

forms. The uptake of SEDS preconcentrates were obtained for specific ranges of surfactant

concentrations. The results also indicate that size and stability of the emulsions or

microemulsions are not the only factors that influence uptake, but also the affinity of the

emulsion drops for the tissue. The difference in uptake between the pharmaceutical and food-

grade SEDS is attributed to the differences in chemical composition. The conclusions drawn rely

on the following results:

1. The formulation of alcohol-free lecithin SEDS can be developed by using a combination

of decaglyceryl caprylate/caprate and PEG-6-caprylic/capric glycerides as hydrophilic

linkers. Food grade alcohol-free lecithin SEDS can also be developed by using

polyglyceryl-6 caprylate as a hydrophilic linker. The optimal systems produced small

droplets at high FeSSIF dilution. The average size was less than 200 nm for

pharmaceutical SEDS and less than 300nm for food-grade SEDS. Preconcentrates in

food-grade SEDS also produced microemulsions (less than 100 nm) at high surfactant

concentrations.

2. Phase behaviour examined in pseudo-ternary phase diagrams and conductivity tests

indicate that the preconcentrates can produce bicontinuous microemulsions with FeSSIF

dilutions. However, these bicontinuous microemulsions become destabilized at higher

dilutions. The systems that approach Type I (o/w) microemulsion behaviour are optimal

63

for absorption at high aqueous dilutions as preconcentrates containing 40% and 50%

surfactant were selected for the pharmaceutical grade SEDS (SEDS-P) and food grade

SEDS (SEDS-F) respectively.

3. The intestinal uptake observed with the linker based SEDS could be explained by the

presence of hydrophilic linkers, which lower the interfacial rigidity of the systems, thus

enhancing the SEDS-intestine mass transfer. The combination of hydrophilic-lipophilic

linkers may also enhance the oil solubilization capacity of the systems.

4. The dialyzer studies reported an uptake of 56.0% and 17.2 % for SEDS-P and SEDS-F.

The systems that approached Type I behaviour (T40, T50) at high dilution were selected

for optimal absorption. At lower surfactant concentrations, microemulsion and excess oil

phases were produced. However, higher concentration of surfactants were required to

solubilize more oil phase (smaller volume of excess oil phases). At higher concentrations

the in-vitro absorption of SEDS-F started to substantially decrease as the lipophilic

linkers partitioned into the aqueous phase. For SEDS-P the uptake using T50 reached a

plateau after 72 minutes. After two hours the SEDS-F had yet to complete absorption.

Other in-vivo studies showed that absorption could occur even after 24 hours of

administration. Therefore, the uptake should be substantially greater.

5. The increased rate and extent in intestinal uptake of SEDS-P in comparison to SEDS-F

could be explained by the presence of hydrophilic linker PEG-6-caprylic/capric

glycerides in SEDS-P. Studies have shown that the use of PEG promotes mucoadhesive

properties involving interpenetration with the mucus fibers. PEG have hydrogen acceptor

groups that facilitate hydrogen bonding between hydrogen donor groups of mucin

thereby enhancing absorption (Shojaei et al., 1997; Liu et al., 2009; Wang et al., 2008;

Efremova et al., 2002). In the case of SEDS-F, the hydrophilic linker polyglyceryl-6

caprylate has hydrogen donor groups and cannot bond to the mucin layer.

6. For SEDS-F, systems developed with preconcentrates containing 40% surfactant

produced the best uptake. The SEDS-F formulations also produced SMEDS at higher

surfactant concentrations such as 70%. Based on the phase behaviour, turbidity and

particle size results, the SMEDS were initially preferred for uptake evaluation. However,

64

T60 showed poor uptake while larger, less stable SEDS systems (T40 and T50) were

capable of enhancing uptake. A recent study has also shown a similar result where large

nanoparticles (200 nm and 500 nm) diffused through human cervicovaginal mucus more

rapidly than 100 nm particles (Lai et al., 2007). This result may be partly explained by

size-exclusion chromatography principles where smaller particles can access a greater

number of pores leading to reduced transport rate because of increased path tortuosity.

Larger particles cannot diffuse through the smaller pores and thus travel more freely in

low-viscosity channels. In addition to size and stability, the surface chemistry and

preferential affinity of surfactants must also be considered. As observed with the food-

grade SEDS the latter factors ultimately played a larger role in determining the optimal

formulation for absorption.

It is important to note however, that the reported uptake results are not the maximum values. In-

vivo absorption could potentially be greater since the dialyzer model accounts only for passive

absorption and mucin layer entrapment of particles. As well, the intestinal absorption surface

area to fed state intestinal volume ratio is two orders of magnitude greater for an adult human in

comparison to the uptake model (Grassi et al., 2007). The dialyzer runs were also conducted for

2 hours while literature has shown that the absorption process in humans can be longer

depending on the type of drug and meal content administered. These factors could affect not

only the extent but rate of drug absorption.

4.2 Recommendations for Future Work

In this work we have demonstrated the development and characterization of the linker-based

lecithin SEDS, their phase behaviour, intestinal performance of model nutraceuticals in-vitro,

and the impact of surfactant concentration. These results show potential enhancement of the

bioavailability of hydrophobic actives. However, future research can address the questions that

may arise from this investigation. The recommendations are as follows:

1. The selection of oils is an important factor in the development of SEDS as it can serve as

a carrier for actives and is involved in the self-emulsification process. It is desirable to

65

select an oil that can solubilize a large amount of drug to promote increased drug

bioavailability. However, one must consider the molecular structure, polarity and

molecular interactions between oil and active. In Appendix 3, the solubility of β-sitosterol

in various polar oils was investigated. It was found that β-sitosterol had a greater

solubility in other polar oils in comparison to ethyl caprate. However, those oils are not

food-grade and thus oils with food status should be further investigated.

2. The effect of dilution ratios can affect SEDS phase behaviour and dialyzer performance.

This can be further investigated by measuring uptake of SEDS with dilutions at factors of

300 and 1000 for instance. However at dilutions of 300 the solution may be too turbid to

be tracked effectively by the UV/Vis spectrophotometer.

3. The effect of FeSSIF bile salt concentrations may influence intestinal absorption. Higher

fat content in meals may induce increased bile salt secretion thereby potentially

promoting more efficient emulsification and absorption. The bile salt concentrations

reflecting a meal containing high fat and low fat can be compared in dialyzer uptake

studies.

4. The length of the uptake simulation for the SEDS containing fish oil omega-3 should be

extended (e.g. 8 hours) as the uptake was not yet complete. For the pharmaceutical SEDS

the time frame can be reduced since equilibrium was reached in 72 minutes.

5. The phase behaviour of food grade SEDS showed rapid oxidation despite incorporation

of an antioxidant. Increased concentrations of antioxidants or a combination of

antioxidants should be added and examined in an attempt to prevent oxidation.

Encapsulation technology is also currently used to deliver fish oils in order to protect the

oil from heat, moisture, air and to extend shelf life. Over 90% of microencapsulated

omega-3 fish oils are created by spray drying (Lee et al., 2008). The process of spray

drying fish oils should be attempted along with analysis of omega-3 content prior and

after encapsulation. This can be in conducted in collaboration with Professor Diosady's

lab.

66

6. Since the optimal SEDS formulations contained at least 50% oil, it may be interesting to

incorporate an additional lipophilic active and examine the uptake of the system. This

could prove to be a delivery system with multiple nutritional and health benefits. It is

therefore important to test the versatility of the developed SEDS by incorporating various

nutraceuticals. Thus far, the delivery of lipophilic drugs β- sitosterol, β- carotene and

omega-3 ethyl esters has been demonstrated using the linker-based delivery systems.

Appendix 4 reports additional studies involving the development of food grade

formulations for the delivery of retinyl palmitate (vitamin A) and tocopheryl acetate

(vitamin E).

4.3 References

Efremova, N.V., Huang, Y., Peppas, N.A., Lecknamd, D.E., 2002. Direct Measurement of

Interactions between Tethered Poly(ethylene glycol) Chains and Adsorbed Mucin Layers.

Langmuir. 18 (3), 836–845.


Absorption Mechanisms: A Physical and Mathematical Approach . CRC Press, Florida.

Liu, G., Fu., L., Zhang., G., 2009, Role of hydrophobic interactions in the adsorption of

poly(ethylene glycol) chains on phospholipid membranes investigated with a quartz crystal

microbalance. J. Phys. Chem. B., 113 (11), 3365-9.

Lai, S., O’Hanlon, D.E., Harrold, S., Man, S. T., Wang, Y., Cone, R., Hanes, J., 2007. Rapid

transport of large polymeric nanoparticles in fresh undiluted human mucus. PNAS, 104 (5),

1482-1487.

Lee, S., Ying., D., 2008. Encapsulation of fish oils. In: Controlled Release Technologies for

Targeted Nutrition (Nissim Garti (Ed)), 370-403.Woodland Publishing Co., Cambridge,

England.

Shojaei, A., Li, X., 1997. Mechanisms of buccal mucoadhesion of novel copolymers of acrylic

acid and polyethylene glycol monomethylether monomethacrylate. Journ. Cont. Rel. 151-161.

Wang, Y., Lai, S., Suk, J.S., Pace, A., Cone, R., Hanes, J., 2008. Addressing the PEG

mucoadhesivity paradox to engineer nanoparticles that "slip" through the human mucus barrier.

Ang. Chem. 47, 9726-9729.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Liu%20G%22%5BAuthor%5D

67

5 Appendix 1

1 UV/Vis absorbance and Uptake Calculations

1.1 UV/VIS Absorbance

The absorbance spectra of T50 with respect to wavelengths at time 0 and after two hours are

shown in Figure 1 and Figure 2 respectively. The two hour profile also shows a set of curves on

the left that represent actual absorbance values over time for specified channels. The absorbance

values were acquired for channels at 450nm, 460 nm, 490 nm, 800 nm, 600nm, and 700 nm. The

shift in absorbance observed from time 0 to two hours indicated that that relative absorbance

values were required to determine uptake and concentration calculations. The relative absorbance

values are obtained by subtracting the absorbance values at 450nm from baseline values at

600nm.

Figure 1 Trial 1 absorbance spectrum at time 0 for T50.

68

Figure 2 Absorbance spectrum for trial 1 over the two hour simulation for T50.

The calibration curve is shown in Figure 3. Figure 4 shows the trial 1 relative absorbance profile

after two hours.

Figure 3 Calibration curve for dilution factors of 500, 600, 700, 800 and 1500.

69

Figure 4 Relative absorbance profile for trial 1 T50.

1.2 Modeled Uptake Calculations

Since the concentration curves were non-linear, a first-order model was developed and fitted to the

data. The concentration of the β-carotene in the donor solution can therefore be expressed in

Equation 1 where K is the mass transfer coefficient in the donor side liquid, A is the tissue

surface area, C is the β-carotene concentration in the donor solution, Ce is the β-carotene

concentration at equilibrium (after absorption has completed) and V is the volume of donor

solution. k is a constant and is shown in Equation 2.

eCCKAdt

dCV (Equation 1)

k =V

AK (Equation 2)

Substituting Equation 2 into Equation 1 to obtain

eCCkdt

dC (Equation 3)

Integrating between t = 0 at the beginning of the uptake and any 't':

C

C e

t

CCk

dCdt

00 (Equation 4)

70

where C0 is the initial β-carotene concentration at time t= 0.

From Equation 4, the β-carotene concentration and percentage uptake at time t is as follows:

e

kt

e CCeCC

0 (Equation 5)

% Uptake %100110

kte e

C

C (Equation 6)

The percentage uptake of the model shown in Equation 6, is solved through an iterative process

using Microsoft excel's solver tool. The equation was entered as shown in column 'L' of Figure 5.

The measured uptake was calculated based on changes in absorption. The error between the

measured uptake and modeled uptake is calculated in column 'M'. Using solver, the target cell is

M25 where the goal is to minimize the sum of all errors by determining the corresponding

constants k and Ce/C0 shown in cells M21 and M22. Since the fluids in the donor side were well

mixed (no phase separation), we can assume that the uptake of SEDS is equivalent to the uptake

of β-carotene. Table 1 shows the uptake and parameters obtained by the first-order model fitted

to the data. Figure 6 shows the actual and modeled uptake.

Figure 5 Microsoft excel solver solution spreadsheet of trial 1 uptake.

71

Figure 6 Flow-Thru Dialyzer percentage uptake of SEDS dilution containing 0.23 % β-carotene

in oil. Fitted uptake curves for each independent trial are shown in black.

Table 1 Percentage uptake of β-carotene and SEDS, and uptake parameters: constant (k, s-1

),

mass transfer coefficient (K, m/s) determined by Equation 6, β-carotene or SEDS fraction

remaining in the donor (Ce/C0 ), calculated by the first-order model fitted to the 2 h uptake data.

% Uptake Ce/C0 k ( x10-3

s-1

) K (cm/h)

55.1% + 0.9 0.45+ 0.009 1.1 + 0.02 135.2+ 3.0

Figure 7 shows the absorption capacities based on the physical values of the human and model.

The calculations were based on the assumption of equivalent uptake model performance for T50

SEDS containing β-sitosterol (3.35% in ethyl caprate). The absorbing surface areas were based

on the diameter and length of the small intestine for the human and cross sectional area for the

model. The uptake model has a SEDS absorption capacity of 39.6 mg/cm2 and β-sitosterol

absorption capacity of 0.66 mg/cm2. For an average measured human surface area of ~9800 cm

2

only 0.2 g of phytosterol is absorbed from a 10 g dose (2% bioavailability), this translates to a

phytosterol absorption capacity of 0.02 mg/cm2, which is approximately 30 folds smaller than

the β-sitosterol model absorption capacity.

Sample calculation of absorption capacity using SEDS example:

(Dose * % Uptake) / Model intestine area = 0.0200 g x 56.0 % / 0.280 cm2 = 39.6 mg/cm

2

72

Figure 7 Physical values of the human and model in rows 48-52 which served as the basis for

calculating T50 and β-sitosterol absorption capacities and in-vivo absorption capacities (rows 59

and 63). SI- small intestine, SA- intestinal absorbing surface area, B-sito. - B-sitosterol

73

6 Appendix 2

1 UV/Vis absorbance and Uptake Calculations

1.1 UV/VIS Absorbance

The absorbance spectra of T40 with respect to wavelengths at time 0 and after two hours are

shown in Figure 1 and Figure 2. The two hour profile shows a set of curves on the left that

represent actual absorbance values over time for specified channels. The absorbance values were

acquired for channels at 450nm, 460 nm, 490 nm, 800 nm, 600nm, and 700 nm. The shift in

absorbance observed at time 0 and two hours indicated that that relative absorbance values were

required for calculations. The relative absorbance values are obtained by subtracting the

absorbance values at 450nm from baseline values at 600nm.

Figure 1 Absorbance spectrum for trial 1 at time 0.

74

Figure 2 Absorbance spectrum for trial 1 over the two hour simulation.

Figure 3 shows the absolute absorbance profile for T35 over the two hour simulation after the

300 nm wavelength values. The set of curves on the left side represents the actual absorbance

values over time for each acquired channel. Substantial changes in absorbance is observed as the

oil and active separated from the donor solution and accumulated within the dialyzer chamber.

Figure 3 Relative absorbance profile for trial 1 with T35.

75

The calibration curve in Figure 4 was created by measuring absorbance at various dilutions. The

equation of the line and correlation coefficient are also shown. Figure 5 shows the trial 1 relative

absorbance profile after two hours.

Figure 4 Calibration curve for dilutions factors of 500, 700, 800, 1100 and 1500.

Figure 5 Relative absorbance profile for trial 1 with T40.

76

1.2 Modeled Uptake Calculations

Figure 6 shows the absorption capacities based on the physical values of the human and model.

The absorbing surface areas were based on the diameter and length of the small intestine for the

human and cross sectional area for the model. The uptake model has a SEDS absorption capacity

of 12.2 mg/cm2 and SEDS omega-3 EPA/DHA absorption capacity of 5.0 mg/cm

2. For an

average measured human surface area of ~9800 cm2, 2.09 g of omega-3 EPA/DHA is absorbed

from a 4 g dose (65% EPA and 59% DHA bioavailability); this translates to an absorption

capacity of 0.21 mg/cm2, which is approximately 20 folds smaller than the omega-3 EPA/DHA

model absorption capacity.

Sample calculation of absorption capacity using recommended omega-3 fatty acid dose example:

Dose x (% EPA x % Bioavailability + % DHA x % Bioavailability) / human intestine area

= (4g x 38% x 59% + 4g x 46% x 65%) / 9817 cm2 = 0.21 mg/cm

2

Figure 6 Physical values of the human and model in rows 48-52 which served as the basis for

calculating T40 and in-vivo absorption capacities (rows 58 and 62).

SI- small intestine, SA- intestinal absorbing surface area, B-sito. - B-sitosterol

77

7 Appendix 3

Ms. Elizabeth Wong, a recent chemical engineering graduate had investigated the solubility of β-

sitosterol in various oils and provided the data in Table 1. An increase in solubility was observed

when the active was dissolved in polar oils due to similar polarities and attractive molecular

interactions between the two components. However, similar to findings in chapter 2, Ms. Wong

discovered that incorporating these polar oils into microemulsions caused the solubility of the

drug in the oil to decrease. She had also used the Hildebrand solubility parameter (Equation 1) to

quantify the intermolecular interactions between the active and oils. Matching the type and

strength of the intermolecular interactions creates favourable thermodynamic conditions for drug

dissolution. The solubility parameter of β-sitosterol was found to be closest to decanoic acid as it

has highest solubility in this solvent. The solubility parameter of β-sitosterol is approximately in the

range of 20.7 - 22.7 based on Ms. Wong's findings. The determination of the solubility parameter

of β-sitosterol can thus assist future studies in finding oils that can maximize its solubility.

Equation 1 Scatchard-Hildebrand equation

Where ΔmH: enthalpy of mixing; V: total molar volume of the mixture;

Φ1: volume fraction of species 1; δ1: solubility parameter of species 1;

Φ2: volume fraction of species 2; δ2: solubility parameter of species 2

78

Table 1 Summary of β-Sitosterol solubility in various polar oils

Component Solubility wt%

Error Value

(mg/mL)

Octanoic Acid 256.12 21.96% 22.97

Decanoic Acid (70%) + EC 211.60 19.29% 8.76

Decanoic Acid (50%) + EC 210.11 19.20% 16.19

Decanoic Acid (60%) + EC 209.07 19.20% 11.93

Propylene glycol monolaurate 200.48 17.89% 18.31

Decanoic Acid (40%) + EC 189.31 17.17% 21.68

Propylene glycol monocaprylate 174.95 15.64% 5.98

Glyceryl monocaprylate + EC 163.20 14.81% 15.28

Oleic Acid (50%) + EC 113.37 11.39% 14.11

Oleic Acid 113.30 11.24% 14.20

Glyceryl monooleate + EC 79.75 8.24% 9.43

Ethyl Caprate 75.86 8.09% 12.06

Medium Chain Triglycerides 36.53 3.72% 4.00

79

8 Appendix 4

1 Food-Grade Formulations- Vitamins A and E

In Chapter 3, linker-based lecithin SEDS and SMEDS have been formulated with food-grade

ingredients. The system showed promising results as an effective vehicle for oral drug delivery.

Therefore this work involved examining whether other lipophilic nutraceuticals could be

incorporated into food grade formulations using the surfactant and linkers from Chapter 3. The

model nutraceuticals were lipophilic vitamins: vitamin E (DL-α-tocopheryl acetate) and vitamin

A (Retinyl palmitate). Their structures are shown in Figure 1. Vitamin E is an antioxidant that

must be obtained from the diet. In-vivo studies have shown that vitamin E prevents lipid

peroxidation which contributes to chronic diseases (i.e. atherosclerosis) and the aging process.

Vitamin A is also an antioxidant that maintains ocular and mucous membrane health (Arnoldi,

2004)

Figure 1 Molecular structure of retinyl palmitate (left) and DL-α-tocopheryl acetate

The materials and microemulsion/SEDS formulation methods used are the same as those

described in Chapter 3. DL-α-tocopheryl acetate (1000 IU/g, Jamieson brand) was purchased

from a local pharmacy while retinyl palmitate (1,600,000-1,800,000 IU per g) was purchased

from Sigma Aldrich. Ethyl oleate (98%, Fischer brand) was used as the food grade carrier oil

(21CFR172.515). The compositions are on a mass basis (wt. %) unless stated otherwise.

1.1 Microemulsion Formulation Studies

For the DL-α-tocopheryl acetate formulation, a hydrophilic linker (polyglyceryl-6 caprylate)

scan was conducted as shown in Figure 2, and the composition is shown in Table 1. For this

series, polyglyceryl-6 caprylate concentrations of less than 10% result in white emulsion phases

80

while above the concentration of 10%, Type II structures form and transition to Type IV

microemulsions after 15% hydrophilic linker concentration. The dilutions (FeSSIF dilution

factor 500) of 10% polyglyceryl-6 caprylate resulted in an average particle size of 393 nm, while

for concentrations at 20% and 30% hydrophilic linker, the average diluted particle sizes were

220nm and 221 nm respectively. Thus these systems are classified as SEDS. This scan shows a

desirable property of using less polyglyceryl-6 caprylate content to form Type IV. About double

(35%) the hydrophilic linker concentration is required to form Type IV for omega-3 fatty acid

microemulsions (F1 scan in Chapter 3).

Similarly, a hydrophilic linker (polyglyceryl-6 caprylate) scan was conducted for retinyl

palmitate microemulsion formulation with the same composition in Table 1 except the oil phase

is dosed with retinyl palmitate. The scan is shown in Figure 3 where a minimum of 30%

polyglyceryl-6 caprylate is required for microemulsion formation, which is comparable to the

35% required for the omega-3 fish oil microemulsion scan (Chapter 3). Polyglyceryl-6 caprylate

concentrations of 12% or less resulted in emulsion phases while the concentration between 18 to

24% led to phase separation. The FeSSIF dilution (dilution factor 500) of 30% polyglyceryl-6

caprylate resulted in an average particle size of 180 nm.

Table 1 Composition of Polyglyceryl-6 caprylate scan containing DL-α-tocopheryl acetate

Phase Component

Weight

%

Aqueous NaCl 0.45%

Polyglyceryl-6 caprylate 0-35%

Deionized H2O To 50%

Oil Glyceryl monooleate 10%

Lecithin 6%

Ethyl oleate + 20% Tocopheryl acetate To 50%

81

0 1 5 8 10 11 12 13 14 15 16 17 18 19 20 22 24 26 28 30 32 35

% Polyglyceryl-6 caprylate

Figure 2 The hydrophilic linker (polyglyceryl-6 caprylate) scan containing 20% DL-α-

tocopheryl acetate in ethyl oleate and equal amounts of oil and aqueous phases (1:1). Type II-IV

microemulsions are formed at concentrations between 10% to 35%.

1% 6% 12% 18% 24% 30%

% Polyglyceryl-6 caprylate

Figure 3 The hydrophilic linker (polyglyceryl-6 caprylate) scan containing 20% retinyl palmitate

in ethyl oleate and equal amounts of oil and aqueous phases (1:1).

1.2 SEDS Formulation Studies

SEDS preconcentrates were also formulated by keeping the glyceryl monooleate to lecithin ratio

constant (10%: 6%), using the vitamins as the oil phase (no ethyl oleate) and varying the

polyglyceryl-6 caprylate content at concentrations of 10%, 20%, 30%. These preconcentrates

were then diluted with FeSSIF at factors of 1, 10 and 100 to examine phase behaviour

performance. Based on these dilutions, 30% and 10% polyglyceryl-6 caprylate content was

optimal for DL-α-tocopheryl acetate and retinyl palmitate respectively. Figure 4 shows the

FeSSIF dilutions for the 30% polyglyceryl-6 caprylate content containing 30% and 40% DL-α-

tocopheryl acetate. The preconcentrates are clear single phases and form opaque mixtures upon

dilution, the diluted phase behaviour are similar between 30% and 40% DL-α-tocopheryl acetate.

Figure 5 shows the FeSSIF dilutions for 10% polyglyceryl-6 caprylate content containing 30%

and 40% retinyl palmitate. The preconcentrates are clear single phases and form opaque

mixtures upon dilution. However 40% retinyl palmitate is less stable as visible phase separation

82

occurred after four hours. However, both of the vitamin A and vitamin E formulations show

enhanced solubility compared to dilutions of the pure vitamin forms. Figure 6 shows the

dilutions of pure retinyl palmitate (a) and pure DL-α-tocopheryl acetate (b) with FeSSIF. Clearly,

minimal solubilization has occurred as all oil phases have separated from the aqueous media.

The results suggest that the SEDS preconcentrate would enhance lipophilic drug solubility and

absorption in the human digestive system.

1:1 1:10 1:100 1:1 1:10 1:100

30% DL-α-tocopheryl acetate 40% DL-α-tocopheryl acetate

Figure 4 The preconcentrate containing 30% DL-α-tocopheryl acetate and its FeSSIF dilutions

at factors of 1, 10, 100 (left) and 40% DL-α-tocopheryl acetate with FeSSIF dilutions at factors

of 1, 10, 100 after four hours (right).

1:1 1:10 1:100 1:1 1:10 1:100

30% retinyl palmitate 40% retinyl palmitate

Figure 5 The preconcentrate containing 30% retinyl palmitate and its FeSSIF dilutions at factors

of 1, 10, 100 (left) and 40% retinyl palmitate with FeSSIF dilutions at factors of 1, 10, 100 after

four hours (right).

1:1 1:10 1:100 1:1 1:10 1:100

(a) (b)

Figure 6 The Dilution of pure retinyl palmitate (a) and DL-α-tocopheryl acetate (b) with FeSSIF

at dilution factors of 1, 10, and 100.

83

The results in this study confirm that the food-grade linker-based lecithin microemulsions can be

potential oral vehicles for various oil-soluble drugs. Different types of microemulsions can be

formed including Type IV bicontinuous phases and Type II. Based on the microemulsion scans

and dilutions of SEDS preconcentrates, retinyl palmitate is observed to be more hydrophobic

than DL-α-tocopheryl acetate. For retinyl palmitate, more polyglyceryl-6 caprylate is required to

form Type IV bicontinuous microemulsions when compared to DL-α-tocopheryl acetate

formulations. In addition, dilutions of the preconcentrate containing 40% vitamin results in phase

separation for retinyl palmitate. Therefore it may be more feasible to formulate the food grade

system using DL-α-tocopheryl acetate. However, further investigation would be required to

compare the in-vitro uptake performance from the SEDS preconcentrates.

References

Arnoldi, A. 2004. Functional Foods, Cardiovascular Disease and Diabetes. CRC Press LLC,

Florida, U.S.A.

Documents

Linker-based Lecithin Oral Drug Delivery Systems · Linker-based Lecithin Oral Drug Delivery Systems Jacquelene Phia Chu Masters of Applied Science Department of Chemical Engineering