Investigation on factors affecting drug delivery using polymers and phospholipids 1

i
ACKNOWLEDGMENTS
At the end of four years of research, I would like to thank all the people that have
contributed to the realization of my thesis.
I wish to acknowledge with gratitude the contribution of my supervisor A/P Chan Sui
Yung for providing an excellent environment for research. Her availability, and
numerous interesting discussions I have got with her, has been a powerful stimulant
during these years.
Financial assistance from the Agency for Science Technology and Research (A Star)
and the National University of Singapore is gratefully acknowledged.
I am grateful to my numerous friends and my colleagues at NUS who helped to make
this endeavor an enriching and enjoyable experience.
I wish to acknowledge my parents and my brother. Their love and support through the
years have been invaluable to me and I dedicate this thesis to them.
Finally, I wish to thank God for his blessings.
ii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS i
TABLE OF CONTENTS ii
SUMMARY vi
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF ABBREVIATIONS xiv
CHAPTER 1 General Introduction 1
1.1 Skin Structure 1
1.2 Topical and Transdermal Drug Delivery 3
1.3 Skin Permeation Models 6
1.4 Recent Formulation Developments 7
1.5 Hypothesis and Objective 8
CHAPTER 2 Effect of Non-Ionic Surfactants in Proniosomes on the
Permeation of Drugs Across Human Skin

28
2.1 Introduction 28
2.2 Materials and Methods 29
2.2.1 Materials 29
2.2.2 HPLC Analysis 30
2.2.3 Phase Solubility and Surface Tension Studies 30
2.2.4 Proniosome Formulations 31
2.2.5 Encapsulation Efficiency and Stability of
Proniosomes
33
2.2.6 Scanning Electron Microscopy (SEM) 33
2.2.7 Preparation of Human Epidermis 34
2.2.8 In vitro Skin Permeation Studies 34
2.2.9 Confocal Laser Scanning Microscopy (CLSM) 36
2.2.10 Statistics 36
2.3 Results and Discussion 36
2.3.1 Solubility and Surface Tension Studies 36
2.3.2 Encapsulation Efficiency and Vesicle Stability 37
2.3.3 SEM Imaging 38
2.3.4 In vitro Skin Permeation Studies 40
2.4 Conclusion 46
CHAPTER 3 Effect of Drug Complexation and Drug Ionization on the
Permeation of Haloperidol Across Human Skin
47
3.1 Introduction 47
3.2 Materials and Methods 49
3.2.1 Materials 49
3.2.2 HPLC Analysis 50
3.2.3 Molecular Modeling 50
iii

3.2.4 Phase Solubility Studies 51
3.2.5 Surface Tension and Contact Angle Measurements 51
3.2.6 In vitro Skin Permeation Studies 52
3.3 Results and Discussion 53
3.3.1 Molecular Modeling 53
3.3.2 Solubility Studies 54
3.3.3 Surface Tension and Contact Angle Measurements 55
3.3.4 In vitro Skin Permeation Studies 58
3.4 Conclusion 65
CHAPTER 4 Development of a Multilayered/Multicomponent Fiber
Mat for Improved Topical Delivery of L-Ascorbic Acid
and Retinoic Acid
66
4.1 Introduction 66
4.2 Materials and Methods 68
4.2.1 Materials 68
4.2.2 Electrospinning 68
4.2.3 Field Emission Scanning Electron Microscopy
(FESEM)
70
4.2.4 Fourier Transform Infrared Measurements (FTIR) 70
4.2.5 HPLC Analysis 70
4.2.6 Drug Release Profile 70
4.2.7 In vitro Skin Permeation Studies 71
4.3 Results and Discussion 71
4.3.1 Characterization of Nanofiber 71
4.3.2 FTIR Studies 72
4.3.3 In vitro Drug Release Studies 74
4.3.4 In vitro Skin Permeation Studies 76
4.4 Conclusion 77

CHAPTER 5 Development of a Nutrient-Rich Facial Mask for the
Topical Delivery of Ascorbic Acid and Retinoic Acid
79
5.1 Introduction 79
5.2 Materials and Methods 81
5.2.1 Materials 81
5.2.2 Electrospinning 81
5.2.3 FESEM and Energy Dispersive X-Ray Spectroscopy
(EDS) Analysis of the Fiber Mat
82
5.2.4 UV Spectroscopy 83
5.2.5 In vitro Skin Permeation Studies 83
5.2.6 Skin Histology 83
5.2.7 Statistical Analysis 84
5.3 Results 84
5.3.1 Fiber Morphology and EDS Analysis 84
5.3.3 In vitro Skin Permeation Studies 87
5.4 Conclusion 89
iv
CHAPTER 6 Development of a Thermosensitive Mat for Sustained
Topical Delivery of Levothyroxine
90
6.1 Introduction 90
6.2 Materials and Methods 91
6.2.1 Materials 91
6.2.2 HPLC Analysis 91
6.2.3 Electrospinning of PVA/PNIPAM Nanofibers 92
6.2.4 FTIR Studies of the Nanofibers 92
6.2.5 FESEM and Fluorescence Microscopy of the
Nanofibers

93
6.2.6 In vitro Drug Release Studies 93
6.2.7 In vitro Skin Permeation Studies 93
6.2.9 Confocal Laser Scanning Microscopy (CLSM) 94
6.3 Results and Discussion 94
6.3.1 FTIR Measurements of the Drug-loaded Nanofibers 94
6.3.2 FESEM and Florescence Image of Nanofibers 95
6.3.3 In vitro Drug Release Studies 98
6.3.4 In vitro Skin Permeation Studies 100
6.4 Conclusion 102
CHAPTER 7 Effect of Polymer Transition on the Topical Delivery of
Levothyroxine
104
7.1 Introduction 104
7.2 Materials and Methods 106
7.2.1 Materials 106
7.2.2 Preparation of Microparticles 106
7.2.3 Encapsulation Efficacy and Stability Studies 106
7.2.4 Microparticle Characterization 107
7.2.5 HPLC Analysis 107
7.2.6 In vitro Drug Release Studies 107
7.2.7 In vitro Skin Permeation Studies 107
7.2.8 FTIR of Skin Sample 108
7.2.9 Confocal Studies of the Treated Skin 108
7.3 Results and Discussion 108
7.3.1 FESEM Characterization of Microparticles 108
7.3.2 Determination of Encapsulation Efficiency 110
7.3.3 In vitro Release of Levothyroxine 111
7.3.4 In vitro Skin Permeation Studies 112
7.3.5 FTIR of Human Skin Samples 117

7.4 Conclusion 118
CHAPTER 8 Effect of Skin Lipid Fluidization and Drug Encapsulation
on the Transdermal Delivery of Diclofenac
120
8.1 Introduction 120
8.2 Materials and Methods 122
8.2.1 Materials 122
8.2.2 Preparation of Diclofenac Sodium-Loaded Vesicles 122
v
8.2.3 Determination of Encapsulation Efficiency 125
8.2.4 Storage Stability of Vesicles 125
8.2.5 In vitro Skin Permeation Studies 125
8.2.6 FTIR Studies of the Human Skin 126
8.2.7 HPLC Assay of Diclofenac Sodium 126
8.3 Results and Discussion 126
8.3.1 Vesicle Size Measurement 126
8.3.2 Determination of Encapsulation Efficiency 128
8.3.3 In Vitro Drug Permeation Studies 128
8.3.4 FTIR Studies of the Human Skin 131
8.4 Conclusion 134
CHAPTER 9 Conclusion and direction for future work 136
REFERENCES 142
LIST OF PUBLICATIONS 167
vi
SUMMARY
Topical drug delivery helps to achieve therapeutic concentrations at the site of
application to achieve a localized effect, while transdermal delivery is defined as
delivery of the drug through intact skin so that it reaches the systemic circulation at
sufficient concentrations to attain therapeutic levels. The objective of this thesis was
to study the effect of some physicochemical factors of the drug molecule and the

carrier, including their solubility, hydrophilicity, contact angle and surface area on the
human skin permeation or accumulation of drug molecules. In vitro flow-through
diffusion cells were employed to explore the skin permeation of hydrophilic (ascorbic
acid, diclofenac) and lipophilic molecules (levothyroxine, haloperidol, retinoic acid)
from various lipid vesicles namely, liposome, niosome, proniosome, transferosome,
ethosome, cerosome, or polymeric, poly N-isopropylacrylamide (PNIPAM), poly
vinylalcohol (PVA), randomly methylated β- cyclodextrin (RM β-CD), poly lactide
(PLA), poly lactide co glycolide (PLGA), ethyl cellulose (EC), microparticles and
nanofibers.
Proniosomal formulations with non-ionic surfactant, Spans and Tweens, were
studied. The effect of hydrophilic-lipophilic balance (HLB value) of one or two
surfactants on drug solubility, proniosome surface structure and stability and skin
permeation of haloperidol from different formulations were investigated. It was
found that a balance of hydrophilicity is needed for an efficient drug release from
proniosomes and a high diffusion rate to the skin. Formulations with single
surfactants were found to increase the permeation of HP more than mixtures of
surfactants. Mixtures of surfactants may form a new type of mixed micelle that
vii
could behave differently than the two single surfactants and thus decrease the skin
permeation rate. Surfactant characteristics such as HLB value, surface tension,
number of carbons in the alkyl chain influence the skin permeation of the drug
molecule.
The effect of RM β-CD on the solubility and complexation energy of
haloperidol was studied. Highest increase in drug solubility was observed when the
drug was in its degree of ionized form in RM β-CD, resulting in a 128-fold increase in
the intrinsic solubility of the drug. Interfacial tension of various concentrations of this
CD derivative was explored and it was found that controversial results regarding the
role of CD as a penetration enhancer reported by various scientists may be related to
the surface active behavior and the CMC value of the CD. It was found that contact
angle of the vehicle influences the extent of drug permeation across the skin layers.

Mixture of hydrophilic, (poly vinyl alcohol, PVA, and randomly methylated β-
cyclodextrin, RM β-CD), and hydrophobic (poly d,l-lactide, PLA, and poly d,l-
lactide-co-glycolide, PLGA) polymers were electrospun to make a
multilayered/multicomponent nanofiber mat. The release characteristic of the drug
was modified using the layer by layer approach to help compensate the limitation of
the individual materials. Incorporation of RM β-CD to the PVA solution significantly
decreased the degradation rate of the resulting fiber mat from a few weeks to a few
seconds. Polyesters, PLA and PLGA, releases drug via hydrolysis of the polymer and
could provide sustained and controlled release rate of the drug. Blends of these
hydrophilic and hydrophobic polymers could effectively prolong drug release and
decrease physiological toxicity resulting from fast release of drugs.
A novel anti-wrinkle polymeric nanofiber of PVA and RM β-CD face mask
containing ascorbic acid, retinoic acid, collagen and gold nanoparticles was
viii
developed. The formulation is dry in nature and would become wetted only when
applied on the skin. This would maintain the chemical stability of the ascorbic acid
and thus the shelf life of the product compared to the pre-moistened commercially
available facial masks. RM β-CD could help to increase the solubility of the low
water soluble compounds such as retinoic acid. The high surface area-to-volume ratio
and porosity of the fibers will ensure maximum contact with the skin surface and
enhance the permeation rate of the active compounds when compared to cotton facial
masks available in the market.
Polymeric nanofibers of poly (N-isopropylacrylamide) (PNIPAM) and PVA
and blends of the two polymers were developed to modify the drug release patterns.
PNIPAM is a thermosensitive polymer with low critical solution temperature (LCST)
of around 32
o
C in aqueous solution. The release of T
4
from mixed polymer mat was

found to be a function of PNIPAM concentration used. PNIPAM nanofibers
sustained the permeation of levothyroxine to the skin and therefore maintained the
effective drug concentration in the skin layers for a longer period.
Polymeric microparticles of PLA, PLGA, PNIPAM and EC were used as
carriers for the skin delivery of levothyroxine. These polymeric microparticles varied
in their surface morphology, drug encapsulation efficacy and stability profiles. The
low transition temperature, T
g
, of PLA and PLGA (~ 37
o
C), and low LCST of
PNIPAM (~ 32
o
C) in aqueous solutions caused precipitation of the rubber-like
polymer on the skin surface which created an impermeable barrier to prevent drug
penetration across the epidermis. Skin permeation observed from EC microparticles
was due to the high T
g
of this polymer.
Diclofenac-loaded conventional liposomes, ethosomes, transferosomes,
niosomes, and PEG-PPG-PEG niosomes were studied for their effects on the skin
ix
lipid fluidization and skin permeation. The lipid structure of the skin was modified,
however the skin permeation of diclofenac was not significantly enhanced. This
suggests that there may be no correlation between drug encapsulation and skin lipid
fluidization with skin permeation of hydrophilic drug.
It can be concluded that modification of the characteristics of the drug and the carrier
can help to increase the skin permeation/accumulation of drugs.
x
LIST OF TABLES

Table 1.1
Overview of lipid vesicle research in transdermal drug
delivery.
14
Table 1.2
Overview of cyclodextrin-drug inclusion research in
transdermal drug delivery.
19
Table 1.3
Overview of polyester and thermosensitive polymer research in
transdermal drug delivery.
25
Table 1.4
Scope of investigation of this thesis. 27
Table 2.1
Composition and appearance of proniosomal formulations. 32
Table 2.2
Properties of the surfactants incorporated in proniosomes. 45
Table 2.3
Permeation profiles of different proniosomal formulations
(n=3).
45
Table 3.1
Surface tension and contact angle values of the solutions (n=3). 58
Table 3.2
Flux value of HP across human epidermis (n=3). 60
Table 4.1
Details of the nanofiber formulations. 69
Table 5.1
Details and the composition of the face masks. 82

Table 6.1
Details of the nanofiber formulations. 92
Table 8.1
Composition of lipid suspensions. 124
Table 9.1
Scope of investigation and findings of this thesis. 141
xi
LIST OF FIGURES
Fig. 1.1
Image of the human epidermis, (a) Binary image of the human
epidermis and localization of green fluorescence, staining of cell
nuclei with DAPI is shown as blue signal. Slice view of stratum
granulosum is shown in red fluorescence. (b) Cross-section of
human epidermis. Details on the method of sample preparation are
mentioned in section 2.2.9 and 5.2.6.
2
Fig. 2.1
Solubility and surface tension measurements of HP solutions (n=3). 37
Fig. 2.2
Encapsulation efficiencies (%) of proniosomal formulations (n=3). 38
Fig. 2.3
Scanning electron microscopy images of proniosome formulations,
(a) HLB 1.8, (b) HLB 2, (c) HLB 6, (d) HLB 6.7, (e) HLB 10, (f)
HLB 16 and (g) HLB 16.7.
39
Fig. 2.4
Permeation profile of HP across human epidermis (n=3). 41
Fig. 2.5
(a) Image of the epidermis and localization of red fluorescence
incorporated in to the proniosomes as a function of depth into the

skin. (b) For better visualization, skin samples where stained with
fluorescein prior skin permeation studies. The image depths (from
left to right) are 0, 4, 8, 12, 16, 20 and 24 µm.
44
Fig. 3.1
(a) Hypothetical structure of the haloperidol-DM β-CD complex,
and (b) haloperidol-HP β-CD complex. (1) Side view; (2) Side
view with electron surface; (3) Top view; and (4) Top view with
electron surface.
54
Fig. 3.2
Phase solubility of haloperidol in CD solutions (n=3). 55
Fig. 3.3
Surface tension of RM β-CD and HP β-CD (n=3). 56
Fig. 3.4
Schematic aggregation of CD. 57
Fig. 3.5
Permeation profile of haloperidol across human epidermis.
Influence of (a) different RM β-CD concentrations, (b) limonene
and RM β-CD, (c) ionization and RM β-CD, (d) RM β-CD and sink
condition in receptor compartment, R and D denote receptor and
donor compartment of the flow through diffusion cells, respectively
(n=3).
63
Fig. 4.1
Schematic presentation of single layered and multilayered
nanofibers.
69
Fig. 4.2
FESEM images of single-layered haloperidol-loaded nanofibers. 72

xii
Fig. 4.3
FTIR spectra of (a) PLA, (b)PLGA 48:52 , (c)PLGA 73:27, (d)
PVA-RM β-CD, (e) PVA-RM β-CD and PLGA 48:52 (f) PVA-RM
β-CD and PLGA 73:27 (g) PVA-RM β-CD and PLA and (h) PVA
nanofibers.
73
Fig. 4.4
In vitro release profile of haloperidol from electrospun fiber mat in
phosphate buffer saline (pH 7.4) at body temperature (37
o
C), n=3.
75
Fig. 4.5
Cumulative HP permeation across human skin (n=3). 77
Fig. 5.1
FESEM morphology of the electrospun fiber mats and digital
image of the fiber mat.
85
Fig. 5.2
X-ray energy spectrum of nanofiber face mask, demonstrating the
presence of the gold element signals using area analysis and spot
analysis.
86
Fig. 5.3
Cumulative AA and RA across human epidermis (n=3). 88
Fig. 5.4
Morphology of human epidermis, before and after skin permeation
of gold nanoparticle-loaded nanofibers. The nucleated cells of the
epidermis have been stained blue, unsaturated lipids, including

fatty acids and esters have been stained red.
89
Fig. 6.1
FTIR spectra of nanofiber mats of (a) 10% w/v PVA - No drug, (b)
10% w/v PVA, (c) 10% w/v PVA - 5% w/v PNIPAM, (d) 10% w/v
PVA - 10% w/v PNIPAM, (e) 10% w/v PNIPAM and (f) 10% w/v
PNIPAM - No drug. All formulations contain drug unless
otherwise mentioned.
95
Fig. 6.2
FESEM images of T
4
-loaded nanofibers of (a) 10% w/v PVA, (b)
10% w/v PNIPAM in ethanol, (c) 10% w/v PNIPAM in water, (d)
10% w/v PVA - 5% w/v PNIPAM, (e) 10% w/v PVA - 10% w/v
PNIPAM, (f) fluorescein -loaded 10% w/v PNIPAM.
97
Fig. 6.3
In vitro release profile of T
4
from electrospun mat in phosphate
buffer (pH 7.4) at body temperature (37
o
C), n=3.
98
Fig. 6.4
In vitro release profile of T
4
from electrospun mat in phosphate
buffer (pH 7.4) at room temperature (20

o
C), n=3.
99
Fig. 6. 5
Cumulative T
4
permeation across human epidermis (n=3). 100
Fig. 6.6
(a) Image of the epidermis and localization of green fluorescence
incorporated in to the PNIPAM nanofibers as a function of depth
into the skin. The image depths (from left to right) are 0, 8, 16 and
24 µm. (b) Binary image of the skin.
102
Fig. 7.1
FESEM images of T
4
loaded (a) PLA, (b) PLGA, (c) EC and (d)
PNIPAM microparticles.
110
xiii
Fig. 7.2
Stability of lipid suspensions: Encapsulation efficacy of the
vesicles over time at room temperature (20
o
C) and fridge
temperature (4
o
C), n=3.
111
Fig. 7.3

In vitro release profile of T
4
from microparticles in phosphate
buffer (pH 7.4) at body temperature (37
o
C), n = 3.
112
Fig. 7.4
Permeation profile of T
4
across human epidermis (n=6-8). 113
Fig. 7.5
Binary image of the epidermis and localization of green
fluorescence on the skin after treatment with the polymeric
particles. For better visualization cell nuclei were counter stained
with DAPI.
116
Fig. 7.6
FTIR spectrum of (a) untreated human epidermis, and skin treated
with (b) 10% w/v PVA mat, (c) 10% w/v PNIPAM mat and (d)
PBS.
118
Fig. 8.1
Stability of lipid formulations: Mean diameter of vesicle
formulations (nm) with time, (n=3).
127
Fig. 8.2
Stability of lipid suspensions: Encapsulation efficacy of the
vesicles with time, (n=3).
129

Fig. 8.3
Cumulative concentrations of diclofenac sodium across human
epidermis (n=3).
130
Fig. 8.4
Representative FTIR spectra of (a) untreated human epidermis, and
skin in the presence of (b) PBS, (c) Ethosome, (d) transferosome,
(e) proniosome, (f) niosome, (g) PEG-PPG-PEG niosome and (h)
cerosome.
134
xiv
LIST OF ABBREVIATIONS
A Area
AA L-ascorbic acid
ANOVA One- way analysis of variance
Au Gold
AZT Azidothymidine
C Carbon
C
o
Initial drug concentration in the donor cell
C
f
Concentration of the free drug
C
ion
Concentration of the ionized drug
C
max
Maximum plasma concentration

C
t
Concentration of the total drug
C
union
Concentration of the unionized drug
CD Cyclodextrin
CLSM Confocal laser scanning microscopy
CM β-CD Carboxymethyl-β-cyclodextrin
CMC Critical micelle concentration
CP Capsaicin
D/L
2
Drug diffusion parameter
DAPI 4’, 6-diamidino-2-phenylindole
DM β-CD Dimethyl- β –cyclodextrin
DMSO Dimethyl sulfoxide
DSC Differential thermal analysis
EC Ethyl cellulose
EDS Energy Dispersive X-Ray Spectroscopy
EI Enhancement index
EE Encapsulation efficacy
FDA Food and drug administration
FESEM Field emission scanning electron microscopy
FTIR Fourier transform infrared spectroscopy
G Gauge
GJP Gap junction protein
HBsAg Hepatitis B surface antigen
HLB Hydrophilic-lipophilic balance
HP Haloperidol

HP β-CD Hydroxypropyl β-CD
HPMC Hydroxypropyl methyl cellulose
HPLC High-performance liquid chromatography
IPA Isopropyl alcohol
IV
Intravenous
J
ss
Steady state flux
J
tot
Total flux
K
pion
Drug permeability of the ionized drug
K
punion
Drug permeability of the unionized drug
KD/L Drug permeability
xv
KL Partition parameter
kV Kilo volt
LCST Lower critical solution temperature
M Molar
MPZ Metopimazine
Na Sodium
O Oxygen
PBS Phosphate buffer saline
PEG Polyethylene glycol
PEG-PPG-PEG Polyethylene glycol-block-polypropylene glycol-block-polyethylene glycol

PG Propylene glycol
PLA Poly D,L lactide
PLGA Poly D,L lactide co glycolide
PM β-CD Partialy methylated β-CD
PNIPAM Poly (N-isopropylacrylamide)
PVA Poly vinyl alcohol
PVP Poly vinyl pyrrolidone
Q Cumulative amount of released drug
RA 13-cis retinoic acid
RM β-CD Randomly methylated β-CD
SB Stratum basale
SC Stratum corneum
SD Standard deviation
SEM Scanning electron microscopy
SG Stratum granulosum
SS Stratum spinosum
T
4
Levothyroxine
T
g
Glass transition temperature
t
L
Lag time
TDD Transdermal drug delivery
TEWL Transepidermal water loss
TRA Tretinoin
UV Ultra violet