Investigation on factors affecting drug delivery using polymers and phospholipids 3

47
CHAPTER 3
Effect of Cyclodextrin-Drug Complexation and Drug Ionization on
the Permeation of Haloperidol Across Human Skin
3.1 Introduction
According to Fick’s law of diffusion, the delivery rate of molecules across the skin is
dependent on their physicochemical properties, partitioning coefficients and
solubilities. Higher drug solubility, increases the drug concentration in the donor
phase resulting in increased permeability (Ceschel et al., 2005; Wang et al., 2005).
Haloperidol (HP) is practically insoluble in water and has a basic pK of 8.3 (Lim et
al., 2006). To increase the solubility, pH of 2.5-3.8 and 2.5-4.5 are used for injection
and oral dosage forms respectively. However such acidic solutions can cause
irritation in the site of injection (Loukas et al., 1997).
Current approaches to soubilize water-insoluble drugs are complex formation with
cyclodextrins, liposomes, microemulsion-based drug delivery systems and
supersaturation. Cyclodextrins (CDs) are attractive candidates for increasing the
aqueous solubilities of lipophilic drugs. They are cyclic oligosaccharides of D-
glucopyranose units in the shape of cones, each with an outer hydrophilic surface and
an inner hydrophobic cavity. The solubilization effect of CDs is due to the formation
of a non-covalent water soluble inclusion complex, therefore drug-CD complexes are
48
easily dissociated and in equilibrium with free drug (Loukas et al., 1997; Liu et al.,
2003). Due to the solubility, hygroscopicity and toxicity concerns of CDs, they were
modified and examples are hydroxypropyl β-CDs (HP β-CDs) and randomly
methylated β-CD (RM β-CDs) (Liu et al., 2003; Gibaud et al., 2005; Murthy et al.,
2004).
CD derivatives can influence the solubilities of drugs (Loukas et al., 1997; Liu et al.,
2003; Sigurðardóttir and Loftsson 1995). They were also reported to decrease local
irritation (Amdidouche et al., 1994; Hoshino et al., 1989; Ventura et al., 2006) as well
as stabilize photosensitive drugs (Godwin et al., 2006). Some investigators reported
that CDs increased the skin permeation rates of drugs by extracting the lipid from the

skin (Bently et al., 1997; Okamoto et al., 1986; Uekama et al., 1982; Vianna et al.,
1998) while others reported that CDs did not show any enhancing effect on the flux
rates of drugs through the skin (Larrucea et al., 2001; Shaker et al., 2003; Williams et
al., 1998).
The pH of the vehicle influences the solubility and partitioning of the drug into the
skin, implying that the ionized and unionized moieties of a drug influence its
solubility and partitioning through stratum corneum and hence affect the skin
permeation (Hadgraft and Valenta 2000; Sridevi and Diwan 2000a and b). Wagner’s
group reported that pH values of donor and receptor compartments influence skin pH
and change the permeability of the drug (Wagner et al., 2003). On the contrary,
Sznitowska’s team reported that there were no significant differences in permeability
49
of hydrocortisone in the pH range of 1-10, and only extreme pH values affected
permeability (Sznitowska et al., 2001; Thune et al., 1988).
To further understand the effect of CD and pH, the aim of the present work is to
investigate the solubility and permeation of a drug from CD inclusions. For this
purpose the complexation of haloperidol with two derivatives of β-CD (RM β-CD and
HP β-CD) at pH 5 were studied by the phase solubility method. Molecular modeling
was conducted using DM β-CD (Dimethyl--cyclodextrin) and HP β-CD. Surface
tension and contact angle measurements were carried out to further elucidate the
effect of CDs on the permeability of HP though human epidermis. The effect of
concentrations of RM β-CD alone and then combined with limonene on the skin
permeation were studied. To elucidate the influence of pH of the donor phase on skin
permeability, further experiments using phosphate buffer at pH 5 in the donor
compartment alone and also in combination with RM β-CD were carried out. Then,
RM β-CD was added to the receptor solution to maintain a sink condition, while the
donor compartment consisted of solutions of HP in RM β-CD or propylene glycol.
3.2 Materials and Methods
3.2.1 Materials
Haloperidol (HP) was purchased from Sigma, Singapore. 2-hydroxypropyl-β-

cyclodextrin (HP β-CD) (degree of substitution of about 0.6) and randomly
methylated-β-cyclodextrin (RM β-CD) (degree of substitution of about 1.8) were kind
gifts from Roquette (Lestrem, France) and Wacker (Burghausen, Germany),
50
respectively.
3.2.2 HPLC Analysis
HP concentration was quantified by HPLC from Shimadzu (Kyoto, Japan) 2010A.
The analysis was carried out using a reversed-phase Waters Symmetry Shield column
(3.5 m, 3.0 mm  100 mm). Mobile phase was a 55:45 volume ratio of acetonitrile
and 0.05M phosphate buffer adjusted to pH 3 using phosphoric acid, flowing at a rate
of 0.4 ml/min. UV detection at wave length 254 nm, injection volume 100 μL gave a
retention time of 5 min. Standard solutions of HP (0.05 - 2 μg/ml) were prepared in
0.03% v/v lactic acid (Lim et al., 2006).
3.2.3 Molecular Modeling
Molecular modeling was carried out to elaborate the complexation modes. Dimethyl-
-cyclodextrin (DM β-CD) was adopted as a substitution for RM β-CD (degree of
substitution = 1.8) to facilitate the determination of the stable structure of the
haloperidol-RM β-CD complex, for RM β-CD is a mixture of different structures.
HP β-CD, with a degree of substitution of 0.6 was used for the experimental study;
four 2-hydroxypropyl groups were added on the primary hydroxyl groups of -
cyclodextrin (Mura et al., 1995). The structures of haloperidol, DM β-CD and HP β-
CD were individually minimized by MMFF94s force field using software SYBYL
version 7.2 (Tripos Co., USA). The structures of haloperidol, DM β-CD and HP β-
CD were individually minimized by MMFF94s force field using software SYBYL
version 7.2 The HP molecule, in its favorable conformation was introduced into the
51
respective DM β-CD and HP β-CD cavities and the interaction energies were
computed. The most likely conformation of each complex was the one with the
lowest interaction energy.
3.2.4 Phase Solubility Studies

Drug-CD inclusion complexes were prepared by adding an excess concentration of
HP (15 mg/ml), dissolved in water or buffer phosphate (pH 5), using RM β-CD and
HP β-CD solutions of different concentrations (0, 0.01, 0.05, 0.1, 0.2, 0.3 M). The
suspensions were shaken on a horizontal rotary shaker in the absence of light for 7
days and finally filtered through a membrane filter (Millipore filters
®
, 0.45 μm pore
size, 25 mm diameter) to obtain clear solutions. All samples were prepared in
triplicates. The concentrations of HP in the inclusion complexes were determined by
the HPLC assay.
3.2.5 Surface Tension and Contact Angle Measurements
Surface tensions of RM β-CD and HP β-CD solutions and each formulation used in
the permeation study were measured using the method described previously in section
2.2.3. The wettability of the excised human skin sample was determined by sessile
drop contact angle using a Rame-Hart 100 goniometer (USA). Drops were placed on
the surface using a micrometer with a flat tip needle. For contact angle measurements
excised human skin similar to that used in permeation studies was employed. This
was done because of its availability and its potential for elucidation of the mechanism
of drug permeation studies. Skin samples were prepared as those for the permeation
52
studies.
3.2.6 In vitro Skin Permeation Studies
Permeation studies of drug alone or as complexes with RM β-CD were performed
using a flow-through diffusion cell apparatus described earlier in section 2.2.8. The
donor compartment was filled with 1 ml of formulations containing 2 mg/ml drug.
The first receptor phase was isotonic phosphate buffer saline pH 7.4 (PBS). Samples
from the receptor phase were collected every 6 hour over a 30-h period, and the
amount of HP permeated was analyzed by HPLC. The steady state flux (J) was
estimated from the slope of the straight line portion of the cumulative HP absorbed
against time profile. Experiments were carried out in triplicates.

The effect of RM β-CD on the skin permeation of HP was studied using two sets of
experiments. First a concentration dependent effect of RM β-CD (0, 0.01, 0.05, 0.1
M) was studied. Further, synergistic effect of RM β-CD in combination with
limonene 0.1% v/v in propylene glycol (PG) solution was investigated. The effect of
pH on the permeability of the drug was studied at pH 5. The combined effects of
ionization and 0.01 M RM β-CD were also observed. In another set of experiments,
PBS in the receiver solution was replaced by 0.01% w/v RM β-CD, while the donor
compartment consisted of HP in 0.01 M RM β-CD or PG solutions.
53
3.3 Results and Discussion
3.3.1 Molecular Modeling
The hypothetical structures of the complexes formed by haloperidol and cyclodextrins
are presented in Fig. 3.1. For the haloperidol-DM β-CD complex, the computed total
energy is -40.7 kcals/mol and the steric energy is -30.587 kcals/mol. For the
haloperidol-HP β-CD complex, total energy is -39.6 kcals/mol and the steric energy is
-29.848 kcals/mol. The differences in energy values indicate that the interaction
between haloperidol and DM β-CD might be stronger than that of haloperidol and HP
β-CD.
54
a (1) a (2)
a (3) a (4)
b (1) b (2)
b (3) b (4)
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.
3.3.2 Solubility Studies
The solubilities of HP in phosphate buffer of pH 5 solutions with and without RM β-
CD or HP β-CD are presented in Fig. 3.2. This pH was selected as it is the same pH
of the skin and may therefore minimize skin irritation. The highest increase in drug

solubility occurred for RM β-CD, indicating that this oligosaccharide complexed
55
more of the drug than HP β-CD. Molecular modeling supports the results obtained
for solubility profile such that the increase in solubility was due to greater interaction
of HP with DM β-CD. The solubilization profile in Fig. 3.2 is linear for all
formulations indicating the formation of a 1:1 complex irrespective of the ionization
of the drug (Loukas et al., 1997). More solubilization was achieved when the drug
was in its degree of ionized form in RM β-CD, resulting in a 128-fold increase of the
intrinsic solubility of the drug. When phase solubility experiments were performed
with CD in the presence of buffer, the change in solubility was higher than in the
presence of CD alone, indicating a synergistic effect. Methylated CDs have been
observed to have larger cavity volumes than HP β-CD. Consequently, RM β-CD can
easily accommodate the hydrophobic drugs such as HP (Torque et al., 2004).
R
2
= 0.9909
R
2
= 0.9895
R
2
= 0.9624
R
2
= 0.9698
0
3
6
9
12

0 0.1 0.2 0.3
RM β-CD and HP β-CD concentration (M)
Concentration of HP (mg/ ml)
RM β-CD buffer pH 5
HP β-CD buffer pH 5
RM β-CD
HP β-CD
Fig. 3.2 Phase solubility of haloperidol in CD solutions (n=3).
3.3.3 Surface Tension and Contact Angle Measurments
The surface tensions of aqueous solutions of different concentrations of RM β-CD
and HP β-CD are shown in Fig. 3.3. A remarkable change in the surface tension of
56
pure water occurred when RM β-CD or HP β-CD was added, indicating that these
systems have effect on the surface tensions of pure water. The methylated
compounds showed the most reduction (Evrard et al., 2004; Thompson 1997). From
Fig. 4.3 it is evident that surface tension reached a constant value after certain
concentration suggesting the formation of super molecular aggregates of RM β-CD
and HP β-CD (Leclercq et al., 2007; Binkowski-Machut et al., 2006). Critical micelle
concentration values were determined from the sharp changes in the slope of the
surface tension versus log [CD] plot. CMC values for RM β-CD and HP β-CD were
4.4 mM and 3.2 mM respectively. The aqueous solution of naturally occurring β-CD
does not have any surface activity (Leclercq et al., 2007; Lu et al., 1997).
45
55
65
75
-8 -7 -6 -5 -4 -3 -2 -1 0
log [β-CD] (M)
Surface Tension (mN/m)
RM β-CD

HP β-CD
Fig. 3.3 Surface tension of RM β-CD and HP β-CD (n=3).
Based on above results, a possible mechanism for the formation of large micelle
assemblies was deduced as shown in Fig. 3.4. Particle size analysis, as observed by
the light scattering, supported this hypothesis (Binkowski-Machut et al., 2006).
However as compared with conventional surfactants these aggregates do not behave
57
as micellar systems. This maybe due to the short length of the hydrophobic chain in
the CD structure (Leclercq et al., 2007; Lombardo et al., 2004).
Fig. 3.4 Schematic aggregation of CD.
Surface tension and contact angle values of the solutions used are stated in Table 3.1.
It was observed that surface tension of water (70.3 ± 0.25 mN/m) decreases with
increase in the concentration of RM β-CD. The interfacial tension of RM β-CD 0.05
and 0.1 M were of similar values, 54.8 ± 0.31 and 54.1 ± 0.22 (mN/m), respectively,
however surface tension of RM β-CD 0.01M was 57.5 ± 0.68 mN/m. Phosphate
buffer solutions had lower surface tension of 59.2 ± 0.42 mN /m, addition of RM β-
CD further decreased the surface tension to 56.5 ± 0.03 mN/m. Addition of limonene
did not demonstrate any decrease in interfacial tension of the PG (36.2 ± 0.06 mN/m)
when compared to pure PG solutions (36.2 ± 0.21 mN/m), indicating that limonene
does not possess any surface active effect (see Table 3.1).
58
Table 3.1 Surface tension and contact angle values of the solutions (n=3).
Formulations Surface Tension (mN/m) ± SD Contact angle ± SD
water 70.3 ± 0.25 91.6 ± 3.13
RM β-CD 0.01M 57.5 ± 0.68 −
RM β-CD 0.05M 54.8 ± 0.31 −
RM β-CD 0.1M 54.1 ± 0.22 52.0 ± 3.82
PG 36.2 ± 0.21 40.0 ± 4.54
PG-Limonene 36.2 ± 0.06 −
PG-Limonene-RM β-CD 36.2 ± 0.05 −

buffer pH 5 59.2 ± 0.42 54.11 ± 6.58
buffer pH 5- RM β-CD 56.5 ± 0.03 −
From Table 3.1, water had the highest contact angle of 91.6
o
± 3.13
o
, indicating its
low wettability on the skin surface, whereas contact angle for propylene glycol was
found to be 40
o
± 4.54
o
, which resulted in higher wettability of the skin surface. The
contact angle for RM β-CD 0.1 M and buffer pH 5 were 52
o
± 3.82
o
and 54.11
o
±
6.58
o
, respectively. The contact angle for RM β-CD 0.05 M and 0.01 M, PG-
limonene, PG-limonene-RM β-CD and buffer pH 5-RM β-CD were not measured and
were thought to be similar to RM β-CD 0.1M, PG and buffer solutions respectively,
as their interfacial values did not differ much.
3.3.4 In vitro Skin Permeation Studies
RM β-CDs were used in permeation studies due to their significant effect on the
solubility of HP compared with HP β-CDs. Fig. 3.5a shows the effect of different
molar ratios of RM β-CD on the permeation of HP. RM β-CD concentrations used

for skin permeation were in the ranges of below (0.05 and 0.1 M) and above the CMC
(0.1 M) value. The cumulative HP concentrations decreased with increasing RM β-
59
CD concentrations. The in vitro permeation of HP through human stratum corneum
showed a similar trend in the presence of both RM β-CD 0.1 M and 0.05 M, with a
low drug penetration through the skin (p > 0.05). This is not surprising as it could be
due to the super molecular arrangement and of high concentrations of RM β-CD; the
aggregations produced were too large to increase drug permeability. However at
lower concentrations of RM β-CD (0.01 M), an increase in permeation was observed
(p < 0.01) and the flux rate was 2.7-fold higher than that of control (Table 3.2). The
flux is due to HP molecules which have not formed complexes with RM β-CD. An
increase in RM β-CD concentrations caused a decrease in dissociated HP molecules,
therefore the amount of free drug available for permeation decreased (Shaker et al.,
2003; Dias et al., 2003). From the phase solubility diagram, it is evident that the
CDs are potent solubilizers, however it is important to use just enough CD to dissolve
the drug; addition of too much CD will decrease drug partitioning into the skin
(Loftsson et al., 1991 and 1994; Felton 2002). It can be seen that the controvercial
results regarding the role of CD derivatives as penetration enhancers reported by
other scientists may be related to their surface active behavior and depends on the
concentration of the CD. At concentration above the CMC values the skin penetration
of the durg molecule may not be affected where as at lower concentrations the skin
permeation of the active compound may be significantly increased.
CD has been used in combination with chemical enhancers (Larrucea et al., 2001;
Maestrelli et al, 2005 and 2006; Zerrouk et al., 2006) and electroporation (Murthy et
al., 2004) to increase the skin permeation rate of drugs. Previous studies from our
laboratories showed that limonene acts as a good penetration enhancer for the
60
delivery of HP (Lim et al., 2006). To investigate the possible use of CD as a
candidate co-enhancer, additional tests were carried out by combining limonene with
0.01 M RM β-CD. As shown in Fig. 3.5b and Table 3.2, the combination of 0.01 M

RM β-CD with 0.1% v/v limonene in PG solution improved percutaneous absorption
by 1.4 fold compared to the control, but not significantly (p > 0.05). However, the
drug permeation profiles were completely different and the lack of significant
difference between the flux in limonene and limonene RM β-CD solutions suggests
high permeation enhancing effect of limonene that masks the effect of RM β-CD.
Table 3.2 Flux value of HP across human epidermis (n=3).
Formulation Mean Flux ± SD (μg/cm
2
/h)
a Water ( control) 0.18 ± 0.01
RM β-CD 0.1 M 0.04 ± 0.01
RM β-CD 0.05 M 0.11 ± 0.03
RM β-CD 0.01 M 0.49 ± 0.12
Phosphate buffer pH 5 0.90 ± 0.14
Phosphate buffer pH 5, RM β-CD 0.01 M 1.71 ± 0.22
b PG (control) 0.12 ± 0.01
PG-Limonene 0.1% v/v 1.98 ± 0.35
PG-Limonene 0.1% v/v, RM β-CD 0.01 M 2.74 ± 0.14
c PG 0.09 ± 0.01
RM β-CD 0.01 M 0.77 ± 0.13
* In a and b, receiver solution is PBS
* In c, receiver solution is RM β-CD 0.01% w/v
Fig. 3.5c shows the effect of pH alone and in combination with RM β-CD on
permeation rate of HP. Ionization of the donor solution with pH 5 increased the flux
rate but did not result in any significant difference compared with the control (p >
0.05). However the flux of the ionized drug at pH 5 and RM β-CD 0.01 M
concentration was enhanced to an order of approximately 1.9 times compared to that
61
when the buffer was used alone (p < 0.05). The higher flux is a result of increased
solubility of the drug. Synergistic effects can be achieved by adjusting the pH and

concentration of RM β-CD to obtain improved solubility and therefore skin
permeability of the drug (Sridevi and Diwan 2002 a and b; Singh et al., 2005). Both
ionized and unionized moieties of drug molecules contributed to the total flux, (J
tot
)
which can be calculated using the following equation:
ionpionunionpuniontot
CKCKJ ** 
(3-1)
Where K
punion
and K
pion
are the dependent drug permeabilities and C
union
and C
ion
are
the dependent concentrations of ionized and unionized moieties, respectively
(Hadgraft and Valenta 2000). The results from the drug solubility profiles showed,
that the ionized moieties have higher solubilities and therefore greater impact on the
total flux of the drug. Results from our studies suggested that the simultaneous
presence of both RM β-CD and phosphate buffer pH 5 at suitable concentrations
could be exploited to improve drug solubility and permeability resulting in enhanced
permeation of the drug though the skin.
Physiological buffer solutions with pH 7.4 are normally placed in the receptor of the
flow-though cell. However for a less water-soluble drug, sink conditions and
therefore total flux of the drug may increase with changes in the receptor phase. CD
molecule is not expected to cross the skin when used in the receptor compartment.
The purpose of adding CD is to increase the solubility of the haloperidol that will be

getting absorbed from the donor compartment to the receptor compartment.
Maintaining sink condition will help increase the rate of drug permeation across the
62
skin. Our recommendation to add CD to the receptor compartment would better
mimic the sink condition across in vitro skin delivery.
Our next approach was to study the possible promoting effect of the CDs in receptor
solutions. Sclafani and co-workers reported that γ-CD in receiver solutions
influenced the permeation rate of progesterone (Sclafani et al., 1995). In our study,
RM β-CD (0.1% w/v) was chosen because of its significant increase on the solubility
of HP. Formulations containing PG in donor compartments did not give significantly
different drug flux compared to control (p > 0.05). A substantial increase in HP
permeability was obtained by using 0.1% w/v RM β-CD as the receptor solution and
RM β-CD 0.01 M in donor compartment (Fig 3.5d). The flux was enhanced by a 1.6-
fold increase compared to that of the control (p < 0.05).
It was found that the critical contact angle value, which distinguishes between
penetration and non-penetration, helped to optimize the drug permeation rate. By
comparing Fig. 3.5a and b, PG with lower contact angle, resulted in higher drug
permeation rate, compared with water. Before any molecule can penetrate through
the skin, it has to first adhere to the skin surface, thus the wetting rates and the
viscosities of these formulations influence the penetration rate of the drug. Therefore
the physicochemical characteristics of the vehicle and other ingredients in
formulation have great impact on skin permeation rate (Venkatraman and Gale 1998;
Wokovich et al., 2006).
63
a)
0
2
4
0 5 10 15 20 25 30
Time (h)

Cumulative HP μg\cm
2
Water
RM β-CD 0.1 M
RM β-CD 0.05 M
RM β-CD 0.01 M
b)
0
5
10
15
20
0 5 10 15 20 25 30
Time (h)
Cumulative HP μg\cm
2
PG
PG-Limonene-RM β-CD 0.01M
PG-Limonene
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
compartments of the flow-through diffusion cells, respectively (n=3).
64
c)
0
2
4
6
8

10
12
14
0 5 10 15 20 25 30
Time (h)
Cumulative HP μg\cm
2
Buffer pH 5
Water
RM β-CD 0.01M-Buffer pH 5
d)
0
2
4
6
8
10
0 5 10 15 20 25 30
Time (h)
Cumulative HP μg\cm
2
D: PG, R: PBS
D: PG, R: RM β-CD
D: RM β-CD 0.01M, R: PBS
D: RM β-CD 0.01M, R: RM β-CD
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
compartments of the flow-through diffusion cells, respectively (n=3).
65

3.4 Conclusion
The effects of surface tension, contact angle and solubility on the skin permeation
of haloperidol were investigated using randomly methylated β-cyclodextrin (RM
β-CD) and hydroxypropyl β-cyclodextrin (HP β-CD). Molecular modeling was
carried out to explain the complexation modes and the solubility data. 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. Surface tension measurements indicate a surface active
effect for RM β-CD and HP β-CD. Contact angle measurements showed that
vehicles with higher skin wettability increased the contact of the drug with skin
surface and therefore resulted in significantly higher drug permeation across
human epidermis (p<0.05). Skin permeation was significantly increased when the
concentration of RM β-CD was below its CMC point (p<0.01) when compared to
formulations with concentrations of RM β-CD above the CMC point. Ionization
and RM β-CD synergistically increased the skin permeation of haloperidol
(p<0.05). It is concluded that transdermal flux of a drug through the skin may be
optimized by controlling surface tension, drug solubility and skin wettability.
66
CHAPTER 4
Development of a Multilayered/ Multicomponent Fiber Mat for
Improved Topical Delivery of Haloperidol
4.1 Introduction
Biodegradable polymeric nanofibers have been used as carriers for drug delivery
and may offer advantages such as reduced toxicity and increased therapeutic level
and biocompatibility (Yang et al., 2007; Jia et al., 2007; Huang et al., 2004). Poly
vinyl alcohol (PVA) is a biodegradable, hydrophilic polymer with distinct
properties such as high degree of swelling, inherent non-toxicity and good
biocompatibility (Kenawy et al., 2007). Nanofibers of PVA alone (Kenawy et al.,
2007; Tao and Shivkumar 2007) or blended with other polymers such as chitosan
(Jia et al., 2007; Li and Hsieh 2006; Mincheva et al., 2007) and gelatin (Yang et

al., 2007) have been studied. Blends of polymers have been widely used to
enhance physical properties and physiological functionality of fiber mats (Jia et
al., 2007; Mincheva et al., 2007; Liang et al., 2007).
Aliphatic polyesters such as poly D,L lactide (PLA) and its copolymers with
glycolic acid (PLGA) are remarkable for sustained drug delivery and targeting
drugs to specific sites. PLA and PLGA are biocompatible and biodegradable
polymers, and undergo bulk hydrolysis thereby providing sustained delivery of the
67
therapeutic agent (Basarkar et al., 2007; Guo et al., 2007; Budhian et al., 2007;
Blanco et al., 2006).
Electrospinning utilizes high voltage source to produce nanoscale and microscale
polymeric fibers with high surface area-to-volume ratio and porosity (Maretschek
et al., 2008; Siepmann et al., 2008; Verreck et al., 2003). When the electrostatic
charge exceeds the surface tension of the solution, the fiber jet travel from the
syringe nozzle to the electrically charged ground collector and allows the solvent
to evaporate, thus leading to the deposition of the non-woven solid polymer fiber
on the surface of the target metallic collector. When many fibers are collected on
the same collector, they produce a mat of fibers. Multilayered nanofiber mat is
obtained by sequentially electrospinning the second polymer solution on the same
target collector as the first polymer. This strategy helps to compensate the
limitation of the individual polymers while the inherent advantage of both the
polymers can be obtained in a single fiber mat (Kidoaki et al., 2005; Sill and von
Recum 2008; Kim et al., 2007; Pham et al., 2006).
One of the approaches for modulating the release of drugs from a delivery system
and changing the release kinetics over a period of time is to use blends of
hydrophilic and hydrophobic polymers (Siepmann et al., 2008). The aim of this
work is the preparation of haloperidol-loaded nanofiber via electrospinning of
hydrophobic, PLA (poly d,l lactide) and PLGA (poly d,l lactide coglycolide), and
hydrophilic polymers, PVA (poly vinylalcohol) and RM β-CD. We intend to
demonstrate that multi-component/multilayered polymeric mixture influence the

biological properties of electrospun fibers. The nanofiber morphology and
structure were investigated by field emission scanning electron microscopy
(FESEM) and fourier transform infrared (FTIR) measurements. The release
68
characteristics of haloperidol from the drug-loaded fiber mats were investigated.
In vitro skin permeation was carried out.
4.2 Materials and Methods
4.2.1 Materials
PLA (R 203H with inherent viscosity of 0.34 dl/g), PLGA (73:27 and 48:52) RG
503, RG 755S with inherent viscosity of 0.63 dl/g and 0.52 dl/g were kind gifts
from Boehringer Ingelheim (Ingelheim, Germany). PVA Mw 70 000-100 000 and
haloperidol were purchased from Sigma. Randomly methylated β-CD (RM β-CD)
(degree of substitution of about 1.8) was a gift from Wacker (Burghausen,
Germany). All other reagents used were of analytical grade.
4.2.2 Electrospinning
A weighed amount of PVA was dissolved in water to obtain 10% w/w solution,
RM β-CD powder was added to the solution to obtain 0.5M concentration. PLA,
PLGA (48:52) and PLGA (72:28) solutions were prepared by dissolving the
polymers in acetone at concentration of 35 % w/w, 30 % w/w and 30 % w/w
respectively. Haloperidol was 2 mg/ml for all formulations as shown in Table 4.1.
The electrostatic spinner used for the experiments was equipped with an
adjustable DC power supply (RP50-1.25 R/230 DDPM, Gamma high voltage
Research, USA), a syringe pump (KD-100, KD scientific, Inc., USA) on which a
3-ml syringe was connected to a blunt 27 G stainless steel needle. The applied
voltage was set at 25 kV. The distance between the syringe and the fiber collector
was kept constant at 13 cm for all experiments. A constant flow rate of 2 ml/h was
applied to all formulations. The depositions were performed at room temperature.
69
Table 4.1 Composition of nanofiber formulations.
Double-layered fiber mats were obtained by fabricating each polymer to form its

individual layer and were deposited on the same target collector (Fig. 4.1). The
non-woven fabric was removed from the collector and was vacuum dried for a
week at room temperature to remove residual solvent prior to usage.
Fig. 4.1 Schematic presentation of single layered and multilayered nanofibers.
4.2.3 Field Emission Scanning Electron Microscopy (FESEM)
Morphology of the obtained fibers was studied using FESEM. The fiber mats were
coated by a sputtering unit (Jeol JFC-1600 auto fine coater; Japan) under vacuum.
The coated samples were then examined using a field emission scanning electron
microscope (Jeol JSM-6701F; Japan) at an accelerating voltage of 5 kV.
Ingredients (% w/w)Formulation
PVA RM β-CD PLA PLGA 48:52 PLGA 73:27
PVA-RM β-CD 10 50 - - -
PLA - - 35 - -
PLGA 48:52 - - - 30 -
PLGA 73:27 - - - - 30
PVA-RM β-CD + PLA 10 50 35 - -
PVA-RM β-CD + PLGA 48:52 10 50 - 30 -
PVA-RM β-CD + PLGA 73:27 10 50 - - 30
Single-layered
Multi-layered
70
4.2.4 Fourier Transform Infrared Measurements (FTIR)
Interaction between polymers and their functional groups were studied using
FTIR. The spectra of the polymeric nanofiber mats were taken using FTIR
spectroscopy (Perkin Elmer Spectrum 100) at wavelengths of 500 - 4000 cm
-1
at
room temperature.
4.2.5 HPLC Analysis
Haloperidol concentrations were quantified by HPLC from Agilent HP. The

analysis was carried out using an Agilent column (5 µm, 4.6 mm × 150 mm).
Mobile phase was a 50:50 volume ratio of acetonitrile and 0.05M phosphate
buffer adjusted to pH 3 using phosphoric acid, flowing at a rate of 1 ml/min. UV
detection at wave length 254 nm, injection volume 20 μL gave a retention time of
6 min. Standard solutions of (0.05 - 2 μg/ml) were prepared in aqueous solutions
containing 0.03 % v/v lactic acid.
4.2.6 Drug Release Profile
Release studies were carried out in phosphate buffer saline pH 7.4 (PBS)
solutions. Samples of the non-woven nanofiber mat (10 mg) were put in 15 ml
plastic tubes and 10 ml release medium was added. The tubes were constantly
moved at a speed of 75 rpm at 37
o
C. At particular time interval, 1 ml samples
were removed and an equal volume of fresh PBS was immediately added after
each sampling. The amount of released HP was determined via HPLC method.
71
4.2.7 In vitro Skin Permeation Studies
Skin samples were prepared according to the method in section 2.2.7. Permeation
studies of drug-loaded 30% w/v PLA and 10% w/v PVA-RM β-CD nanofibers
were performed (see section 2.2.8). The donor compartment was filled with 60
mg of fiber mat or 250 mg of control solution, all having an equal amount of drug.
The amount of HP permeated was analyzed by HPLC.
4.3 Results and Discussion
4.3.1 Characterization of Nanofiber
Fig. 4.2 shows the FESEM image of the electrospun polymer fibers. The
influence of the polymer type on the morphology of the resulting fibers was
examined. All formulations presented a bead-free nanofibrous structure with wide
ranges of fiber diameters. Haloperidol was encapsulated within the entire
nonwoven fiber mat and the FESEM images did not observe any drug crystals.
The large surface area of the fibers results in fast evaporation of the organic

solvent which minimized the possibility of drug crystallization (Verreck et al.,
2003).