1. Pharmaceutical nanotechnology
Liposomal buccal mucoadhesive film for improved delivery and
permeation of water-soluble vitamins
Heba Abd El Azim a,b
, Noha Nafee a,
*, Alyaa Ramadan a
, Nawal Khalafallah a
a
Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
b
Department of Pharmaceutics, Faculty of Pharmacy, Damanhour University, Damanhour, Egypt
A R T I C L E I N F O
Article history:
Received 6 February 2015
Received in revised form 11 April 2015
Accepted 16 April 2015
Available online 18 April 2015
Keywords:
Liposomes
Mucoadhesive film
Water-soluble vitamins
Vitamin B6
Buccal permeation
A B S T R A C T
This study aims at improving the buccal delivery of vitamin B6 (VB6) as a model highly water-soluble, low
permeable vitamin. Two main strategies were combined; first VB6 was entrapped in liposomes, which
were then formulated as mucoadhesive film. Both plain and VB6-loaded liposomes (LPs) containing
Lipoid S100 and propylene glycol ($200 nm) were then incorporated into mucoadhesive film composed
of SCMC and HPMC. Results showed prolonged release of VB6 (72.65%, T50% diss 105 min) after 6 h from
LP-film compared to control film containing free VB6 (96.37%, T50% diss 30 min). Mucoadhesion was
assessed both ex vivo on chicken pouch and in vivo in human. Mucoadhesive force of 0.2 N and residence
time of 4.4 h were recorded. Ex vivo permeation of VB6, across chicken pouch mucosa indicated increased
permeation from LP-systems compared to corresponding controls. Interestingly, incorporation of the
vesicles in mucoadhesive film reduced the flux by 36.89% relative to LP-dispersion. Meanwhile, both films
provided faster initial permeation than the liquid forms. Correlating the cumulative percent permeated
ex vivo with the cumulative percent released in vitro indicated that LPs retarded VB6 release but improved
permeation. These promising results represent a step forward in the field of buccal delivery of water-
soluble vitamins.
ã 2015 Elsevier B.V. All rights reserved.
1. Introduction
A great deal of interest has focused on the implementation of
buccal mucosa for systemic delivery of various drugs and dietary
supplements including vitamins (Puratchikody and Mathew,
2011). Drug delivery across buccal mucosa offers a variety of
advantages such as providing direct entry into the systemic
circulation through the jugular vein, thus bypassing the stomach
environment and first-pass liver metabolism leading possibly to
increased bioavailability of drugs suffering from gut or first pass
liver metabolism. Ease of drug administration, accessibility and
improved patient compliance all provide acceptable alternative for
pediatrics, geriatric and nauseous patients. The buccal route allows
for both prolonged and rapid drug release for local or systemic
action, in addition to versatility in designing uni- or multi-
directional release systems (Gilhotra et al., 2014).
Among the buccal dosage forms, caffeine chewing gum, (Stay
Alert1
) and nicotine chewing gums (e.g., Nicorette1
and Nic-
otinell1
) have been marketed for systemic drug delivery (Kami-
mori et al., 2002; Patel et al., 2011). A novel liquid aerosol insulin
formulation (Oralin1
, Generex Biotechnology) was developed
allowing pain-free, user-friendly administration as well as precise
insulin dose delivery into the mouth (comparable to injection)
(Modi et al., 2002).
Attempts for prolonged and improved drug contact with the
mucosa have lead to the development of mucoadhesive delivery
systems (e.g., patches, flexible films and wafers) as extensively
reported (Kianfar et al., 2012; Patel et al., 2011; Sharma et al., 2012).
Intraoral polymeric films have gained a wide range of applications
from breath fresheners to local anesthetics, vitamin supplements,
vaccines, in allergic remedies, emetic condition and in chronic
attacks of some diseases (Chaturvedi et al., 2011). Both the
controlled release and fast dissolving films can be suitable for
young children and for patients with difficulty in swallowing
(dysphagic patients) (Boateng et al., 2013).
* Corresponding author at: Department of Pharmaceutics, Faculty of Pharmacy,
Alexandria University, 21521-El Khartoom Square, Alexandria, Egypt. Tel.: +20
34868482; fax: +20 34871668.
E-mail addresses: n.nafe3@gmail.com, noha.nafee@pharmacy.alexu.edu.eg
(N. Nafee).
http://dx.doi.org/10.1016/j.ijpharm.2015.04.052
0378-5173/ã 2015 Elsevier B.V. All rights reserved.
International Journal of Pharmaceutics 488 (2015) 78–85
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm

2. Meanwhile, colloidal systems such as micelles, liposomes,
nanoemulsions and polymeric nanoparticles showed great prom-
ise in drug delivery by improving the therapeutic index of drugs,
modifying their distribution, thus increasing their efficacy and/or
reducing their toxicity (Tan and Liu, 2011). Among colloidal
systems, liposomes (LPs) have gained considerable attention due to
their ability to entrap both lipophilic (within the phospholipids
bilayer) and hydrophilic drugs (in the inner aqueous compartment)
(Kumar et al., 2010; Xu et al., 2012). Various buccal liposomal
dosage forms reported in the literature include silymarin
liposomes for improved buccal permeation through chicken pouch
(El-Samaligy et al., 2006), buccal micellar insulin spray and buccal
deformable liposomes to deliver insulin with significantly greater
bioavailability than conventional LPs and subcutaneous insulin
solution in rabbits (Yang et al., 2002). Nevertheless, drug leakage
and colloidal instability upon storage are reported as the main
drawbacks of liposomal carriers.
Multiple-unit mucoadhesive carriers combine the abilities of
the mucoadhesive dosage forms with the advantageous features
of the multiparticulate delivery systems (e.g., uniform dispersion
in the targeting site, more reproducible drug absorption and
reduced local irritation) (Albertini et al., 2009; Nitin et al., 2010).
Mucoadhesive liposomal ointment for the buccal delivery of
peptides increased the intimate contact with mucosa and
accordingly local drug concentration (Veuillez et al., 2001).
As well, liposome encapsulated corticosteroids paste was devel-
oped for the treatment of oral lichen planus (Hearnden et al.,
2012). In another study, mucoadhesive ointment containing
liposomal triamcinolone acetonide was found to be well
tolerated with no local irritation (Sveinsson and Peter Holbrook,
1993).
Water-soluble vitamins represent a structurally and function-
ally diverse set of organic molecules that play a crucial role in
maintaining the metabolic, energy, differentiation and prolifera-
tion status of cells (Said and Seetharam, 2006). Vitamin B6 (VB6)
as member of the vitamin B complex group is involved in the
metabolism of carbohydrates, amino acids, lipids and neuro-
transmitters (Ball, 2004). It also regulates the expression factors
responsible for efficient absorption of microelements, e.g.,
calcium or transport drugs or antigens (Zielinska-Dawidziak
et al., 2008). VB6 deficiency in humans leads to a variety of
clinical abnormalities that include sideroblastic anemia, weakness,
insomnia, and neurologic disorders (Kevadiya et al., 2010). One of
the applications of interest of VB6 is as an antiemetic. Many oral
and parenteral products are already on the market, yet a buccal
prolonged release product if developed, could be less nauseating
than oral product to be swallowed and less painful compared to
parenteral VB6.
Therefore, the aim of this study was to develop a prolonged
release buccal formulation of VB6 as a model highly water-soluble,
low permeable vitamin. In this context, two main strategies were
combined; first VB6 was entrapped in liposomal carrier in an
attempt to improve permeability, and second, this liposomal
dispersion was formulated as mucoadhesive buccal film in order to
prolong residence time and release profile. Loaded liposomes,
control drug solution and control drug film were tested in parallel
for drug release and permeation for comparison.
2. Materials and methods
2.1. Materials
Lipoid S100 (LS100) also known as phosphatidylcholine (PC)
from soybean lecithin, containing not less than 94% PC, was a kind
gift from Lipoid GmbH (Ludwigshafen, Germany). Vitamin B6 was a
kind gift from European Egyptian Pharmaceuticals (Alexandria,
Egypt). Propylene glycol (PG), anhydrous ethanol were purchased
from ADWIC, El-Nasr Pharmaceutical Chemicals Co. (Abu Zaabal,
Egypt). Hydroxypropyl methyl cellulose (HPMC, molecular weight
4 kDa) and carboxymethyl cellulose sodium (SCMC) were from
Prolabo Pharmaceutical Chemicals Co. (Cairo, Egypt).
2.2. Methods
2.2.1. Preparation of plain and VB6-loaded liposomes
The formulation prepared contained 2, 5 and 0.6% w/v of LS100,
PG and VB6, respectively, (calculated with respect to final LP-
dispersion). A minimum amount of ethanol was used to dissolve
VB6. LS100 was dissolved in the ethanolic VB6 solution. Ethanol
was slowly removed at reduced pressure, using a rotary evaporator
(Rotavapor, Büchi, Germany) above the lipid transition tempera-
ture. The lipid film was hydrated with deionized water containing
the PG.
The resulting vesicle dispersion was subjected to sonication in
ice bath (30 min intermittent) and to extrusion cycles through
membrane filters of descending pore size (once through nylon
filter 0.45 mm and twice through cellulose acetate filter 0.20 mm).
The final LP-dispersion were stored in refrigerator at 4
C. Plain
(VB6-free) LPs were similarly prepared.
2.2.2. Colloidal characterization of liposomes
Vesicle size, polydispersity index (PdI) and zeta potential for
plain and loaded LPs were determined using Malvern Zetasizer
Nano ZS (Malvern Instruments, Malvern, UK). Deionized water was
used as dilution medium (20 fold dilution). For morphological
characterization, LPs were examined by TEM (Jeol-100CX, Japan)
after negative staining using uranyl acetate.
2.2.3. Determination of drug entrapment efficiency by dialysis
A volume of LP-dispersion (0.2 ml equivalent to 1.2 mg VB6) was
filled in dialysis bags (Carolina1
dialysis tubing, 12–14 kDa
molecular weight cut off, North Carolina, USA) and suspended in
deionized water (pH 6.5 Æ 0.05) for 2 h at 4
C, to ensure an
internal: external volume ratio of 1:300. VB6 in dialysate was
measured spectrophotometrically at 292 nm (Thermospectronic,
Helios alpha, NC 9423 UVA 1002E, UK) a wavelength corresponding
to maximum UV absorption of VB6, as pyridoxine HCl, in water
(Moffat et al., 2004; Ristilä et al., 2006).
Entrapment efficiency (EE) was calculated using the following
equation:
EE ¼
Total VB6 added À VB6 in dialysate
Total VB6 added
 100
2.2.4. Preparation of mucoadhesive buccal liposomal film
Solvent casting method was used to prepare buccal mucoad-
hesive films containing VB6-loaded LPs. HPMC and SCMC (1% w/v
each, calculated with respect to LP-dispersion volume) were added
to the LP dispersion while stirring till complete dissolution of the
powders. The gels formed were left overnight at room temperature
in a dessicator to ensure clear, bubble-free gel. PG already present
in the LPs formula served as plasticizer. The gel was cast into glass
Petri dishes and allowed to dry at 40
C. Film discs (1 cm2
,
containing 1.4 mg VB6) were wrapped in aluminum foil, and stored
in glass containers at 4
C. A control film containing free VB6 was
similarly prepared.
H. Abd El Azim et al. / International Journal of Pharmaceutics 488 (2015) 78–85 79

3. 2.3. Characterization of liposomal mucoadhesive films (LP-film)
2.3.1. Physicochemical characterization
Film thickness, weight uniformity, surface pH, swelling and
endurance were assessed (n = 3) as detailed in Supplementary
materials as previously reported (Mohamed et al., 2011).
2.3.2. Integrity of the liposomes in mucoadhesive films
Films were dissolved in deionized water. Vesicle size, PdI and
surface charge of restored LPs in the film solution were measured.
Morphology was examined by TEM.
2.3.3. In vitro release of VB6 from LP-films
VB6 release from the buccal film was assessed by dialysis at
37
C. A film disc (equivalent to 1.4 mg drug) was placed in the
dialysis bag and immersed in 20 ml simulated saliva (pH
6.75 Æ 0.05), prepared by dissolving 2.38 g Na2HPO4, 0.19 g KH2PO4
and 8.0 g NaCl in 1000 ml of distilled water (Wong et al., 1999). At
predetermined time intervals, samples (0.5 ml) were withdrawn
and replaced with fresh release medium. VB6 released was
analyzed at 254 nm, a wavelength corresponding to maximum
absorption of VB6 at pH 6.75 (Moffat et al., 2004; Ristilä et al.,
2006). A calibration curve in simulated saliva was used.
2.3.4. Stability on storage
LP-film was stored for three months at 4
C. Vesicle size, PdI and
VB6 release were measured. Morphology was examined by TEM.
Films were dissolved in deionized water before size measurements
and morphological examination. Release was tested by dialysis of
the films (Section 2.2.3). Data for LP-dispersion stability were also
generated under the same storage conditions for comparison.
2.4. Mucoadhesion
2.4.1. Ex vivo determination of mucoadhesive strength
The mucoadhesion strength was determined using an in house
balance method based on the representative sketch illustrated in
Fig. S1 (Supplementary materials) (Nafee et al., 2003, 2004). The
chicken pouch membrane was used as model mucosa for these
studies. The film (attached to one arm of the balance) was brought
into contact with the chicken pouch fitted on a rubber cork and
kept moistened with simulated saliva (pH 6.75, 37
C). The weight
of water, in grams (poured into a beaker hanging from the other
arm of the balance), required to detach the film from the mucosal
surface gave the measure of mucoadhesive strength. Average
values were recorded (n = 3). The force of adhesion was calculated
using the equation (Hamzah et al., 2010):
Force of adhesionðNÞ ¼
Bioadhesive strength
1000
 9:81
2.4.2. In vivo testing of mucoadhesion in human
Mucoadhesion was also assessed in vivo in three volunteers. The
study followed the rules approved by the Faculty of Pharmacy
(Alexandria University, Alexandria, Egypt) ethics committee for
research involving volunteers. Volunteers were asked to rinse their
mouth with distilled water before a unit of the film was placed
between the cheek and gingiva in the region of the upper canine
and gently pressed onto the mucosa for about 30 s. The time for
complete erosion or detachment of the film from the buccal
mucosa was recorded. Volunteers were allowed to eat and drink
during the testing period. Possible irritation, bad taste, swelling,
dry mouth or increase in salivary flow were monitored (Nafee et al.,
2003).
2.5. Ex vivo permeation using chicken pouch mucosa
2.5.1. Tissue preparation
Excised chicken pouch mucosa was carefully cleaned and kept
frozen at À20
C in saline, thawed to room temperature directly
before use and washed with simulated saliva (pH 6.75 Æ 0.05).
Tissue integrity was confirmed histologically by HE staining.
2.5.2. Ex vivo permeation testing
LPs, LP-film, control VB6 solution and control VB6 film were
tested for VB6 permeation through chicken pouch mucosa using
the QuixSep Micro Dializer (Roth, Karlsruhe, Germany). The unit is
a two-piece capsule that securely locks with a push fit. The donor
chamber was filled with 230 ml LP-dispersion or control solution.
In case of mucoadhesive films, the donor chamber was filled with
230 ml simulated saliva (pH 6.75 Æ 0.05) and the film was pressed
on the mucosal side of chicken pouch for 30 s.
In all cases, the mucosal side of chicken pouch was stretched
over the capsule open end towards the donor compartment and
covered tightly with the lid collar. A beaker containing phosphate
buffer (15 ml, pH 7.4 Æ 0.05) was used as the recipient solution. The
whole set was put in thermostatically controlled shaking water
bath at 50 rpm and 37 Æ 0.5
C for 6 h. Samples were withdrawn
over 6 h and compensated with fresh buffer. The concentrations of
permeated drug were calculated from the absorbance measured at
254 nm, a wave length corresponding to maximum absorption of
VB6 at pH 7.4. For blank readings, microdialyser units fitted with
chicken pouch membranes, containing donor and recipient
solutions, were placed in the shaking water bath; samples from
Fig. 1. TEM micrographs of VB6-loaded LPs, (A) freshly prepared; (B) after six months storage at 4
C.
80 H. Abd El Azim et al. / International Journal of Pharmaceutics 488 (2015) 78–85

4. the recipient compartment were used as blanks. Experiments were
performed in triplicate and mean value was used to calculate the
flux and permeability coefficient (Hamzah et al., 2010).
2.5.3. Calculation of permeation parameters
Cumulative amount of permeated drug per square centimeter
was plotted versus time, and steady-state flux was estimated from
the slope of the linear portion of the plot using the following
equation:
Flux ¼ Jss ¼
dQ=dt
A
where, Jss is the steady-state flux (mg cmÀ2
hÀ1
); dQ/dt is the
permeation rate (mg cmÀ2
); A is the active diffusion area (cm2
).
The VB6 permeability coefficient (P) across chicken pouch
mucosa was calculated using the relation derived from Fick’s first
law of diffusion as follows:
P ¼
Jss
Cd
where, P is the permeability coefficient (cm sÀ1
) and Cd is the donor
drug concentration (mg mlÀ1
).
2.6. Statistical analysis
Results are expressed as mean Æ standard deviation. Statistical
analysis was performed using Student t-test or One-way ANOVA.
Differences were considered significant at a level of p 0.05.
3. Results and discussion
3.1. Colloidal characteristics of VB6-loaded LPs
VB6-loaded LPs were 207.4 Æ17.1 nm in diameter (PdI
0.228 Æ 0.03). Zeta potential of loaded LPs was À1.98 mV compared
to an average value of À15.5 mV for corresponding plain LPs
indicating possible surface adsorption of the positively-charged
vitamin on the phospholipid bilayer. The weak surface potential of
the loaded vesicles suggests stabilization mechanisms other than
electrostatic repulsion.
TEM micrographs of VB6-loaded LPs (freshly-prepared and
6-months stored LPs) showed spherical, evenly distributed vesicles
with smooth surface (Fig. 1A and B). Estimated TEM size was in the
nano-range in accordance with size measurement by photon
correlation spectroscopy. However, LPs appear relatively smaller;
this might be attributed to drying and staining processes prior to
TEM measurement.
3.2. Entrapment efficiency
Percent entrapment efficiency value was 45.99 Æ1.75. Deter-
mination of EE by dialysis was favored over ultracentrifugation as
the strong energy transmitted to the samples disrupted the
vesicles in spite of cooling (Lopez-Pinto et al., 2005). Preliminary
trials indicated that 2 h was enough period for complete dialysis of
the free drug.
Generally speaking, the entrapment efficiency of drugs in
liposomes is a function of various factors such as the preparation
technique, the geometry of the prepared vesicles (size and
lamellarity), the lipid concentration, drug physicochemical prop-
erties, possible drug–lipid interactions, aqueous phase composi-
tion among others (Adrian and Huang,1979; Dominak and Keating,
2007; Nii and Ishii, 2005; Pidgeon et al.,1987; Sun and Chiu, 2005).
Passive encapsulation of water-soluble drugs depends on the
ability of liposomes to trap aqueous medium containing the
dissolved drug during vesicle formation. Trapping effectiveness is
limited by the trapped volume in the liposomes and drug solubility
(Akbarzadeh et al., 2013).
3.3. Characteristics of LP-film
The mucoadhesive film was 0.1 mm thick, with an average
weight of 15.46 mg cmÀ2
and a surface pH of 5.93. The films
showed good flexibility; no cracks appeared when folded more
than 250 times (Table S1, Supplementary materials). Swelling
assessment indicated that the film weight was 5 times the initial
weight in 5 min and 25 times in 120 min (Fig. S2, Supplementary
materials). The film remained intact after 2 h immersion in
deionized water in spite of the increase in weight.
3.4. In vitro VB6 release from LP-film
The in vitro release profiles of VB6 from control solution, LP-film
and control film (Fig. 2) indicated that the films provided slower
VB6 release from the dialysis bag compared to VB6 solution.
Hydrophilic polymers in water produce a water-swollen gel-like
state that reduces the penetration of the dissolution medium into
the films and lengthens the diffusion path length resulting in
retarded drug release (Sekhar et al., 2008; Vishnu et al., 2007).
Apart from the impact of hydrophilic polymers, entrapment of
VB6 in the vesicles contributed to the controlled release. After 6 h,
LP-film achieved slower release (72.65 %, T50% diss 105 min) than
the control film (96.37 %, T50% diss 30 min). Initial amount of VB6
released from LP-film (after 30 min) was significantly lower (t-test,
p 0.05) than from control film. The slower release profile from
the LP-film compared to the control film (Fig. 2) also suggested that
gelling and film casting process did not compromise the integrity
of the incorporated vesicles.
Release kinetics followed the Higuchi model; Korsmeyer–
Peppas n value (0.321) indicated diffusion release mechanism.
3.5. Stability attributes
3.5.1. Integrity of liposomes in the film before and after storage
An important prerequisite for the effective use of liposomes as a
drug carrier is demonstration of physical and performance stability
in the final dosage form (Kirby et al., 1980). The data generated in
this study suggested maintenance of vesicle integrity after
incorporation in the film and also after 3 months-storage of the
film (in both cases, the film was dissolved in deionized water to
permit measuring vesicle size (Fig. 3A) and obtaining TEM
micrographs) (Fig. 3B and C, respectively).
Fig. 2. In vitro release profiles of VB6 from LP-film, control film and control solution.
H. Abd El Azim et al. / International Journal of Pharmaceutics 488 (2015) 78–85 81

5. Concerning size measurements, insignificant fluctuation in the
mean size of LPs and polydispersity was evident before and after LP
incorporation in the film (Fig. 3A). TEM micrographs indicated that
LP retained their morphology (spherical shape and smooth, intact
surface) before and after incorporation in the film (Figs.1A and 3B),
respectively.
3.5.2. Effect of storage on the in vitro release of VB6 from LP-film
No change in VB6 release profiles was observed after three
months of storage of the LP-film at 4
C (Fig. 3D). Korsmeyer–
Peppas kinetics (n = 0.321) revealed the persistence of diffusion
release mechanism with ageing of the film.
Release profiles also lent further evidence; superimposed
release profiles (dialysis of films before and after storage,
Fig. 3D) is less likely to occur from destroyed and reformed LPs
compared to stable LPs. Based on these data, the suggestion that
LPs remained stable and preserved their integrity during film
formation appears better evidenced.
The stability attributes of LP-film were in contrast to LP
dispersion; the latter showed gradual drug leakage on storage at
4
C (EE data, Table 1), while no change in vesicle size and PdI was
recorded (Table 1).
In the present study, the film provided a possible patient-
friendly buccal dosage form and improved stability against leakage
of the LPs by immobilizing them in the film, as evident in the lack of
Fig. 3. Stability on storage at 4
C: (A) particle size and PdI of LPs in fresh and stored films, TEM micrograph of: (B) VB6-loaded LP-film, (C) same film after storage, (D) in vitro
release of VB6 from fresh and stored mucoadhesive films.
Table 1
Stability data at 4
C of VB6-loaded LPs.
Storage time (month) Z-average (nm) PdI Zeta potential
(mV)
EE%
0 185.4 Æ 1.14 0.245 Æ 0.01 À1.98 45.99 Æ 1.75
1 223.1 Æ 2.57 0.242 Æ 0.01 3.18 45.98 Æ 1.04
2 208.4 Æ 1.64 0.174 Æ 0.01 4.52 34.55 Æ 0.62
3 211.9 Æ 2.92 0.262 Æ 0.03 1.18 29.60 Æ 0.99
6 174.3 Æ 1.00 0.234 Æ 0.01 À1.52 10.83 Æ 0.39
82 H. Abd El Azim et al. / International Journal of Pharmaceutics 488 (2015) 78–85

6. change in VB6 release profiles from the film before and after
storage.
3.6. Ex vivo and in vivo mucoadhesion
The mucoadhesive strength determined ex vivo was
20.55 Æ 0.2 g corresponding to a force of adhesion of 0.2 N. These
mucoadhesive properties resulted in an in vivo duration of
attachment of LP-film to the buccal mucosa of 4.43 Æ 0.07 h in
three volunteers (Table S2, Supplementary materials). Incorpo-
ration of LPs in the film had no remarkable apparent impact on
mucoadhesion. None of the volunteers complained of irritation,
bad taste, dry mouth or increase in salivary flow during the in vivo
study.
3.7. Ex vivo permeation through chicken pouch membrane
Histological examination of the isolated chicken pouch
confirmed membrane integrity (Fig. 4A).
3.7.1. LP systems versus controls
Ex vivo permeation of VB6, across chicken pouch mucosa, from
control solution, LP-dispersion, control film and LP-film (Fig. 4B
and C), indicated significant increase in permeation from LP
systems compared to corresponding controls (One-way ANOVA:
p = 0.0005 for LP-film versus control film, p = 0.014 for LP-dispersion
versus control solution of free VB6).
Flux values (Table 2), confirmed the increased VB6 flux from LP
systems (dispersion and film) by 13.06 and 35.21%, respectively,
compared to their controls.
The data indicate the permeation enhancing potential of LPs. In
accordance with our findings, a novel post-expansile hydrogel
foam containing PG-liposomes ensured drug permeation through
porcine vaginal mucosa higher than hydrogel foam aerosol, PG-
liposome aerosol and hydrogel (Li et al., 2012). The penetration
enhancing effect of phospholipids and propylene glycol was
previously reported (Elsayed et al., 2007; Manconi et al., 2009).
However, the data generated in the present study, did not allow
concluding whether the vesicles themselves permeate or whether
they release the drug at the permeation surface, while the
phospholipid solely acts as permeation enhancer.
3.7.2. Mucoadhesive films versus liquid systems
Comparing LP-film with LP-dispersion as well as control film
with control solution, mucoadhesive films reduced the cumulative
permeated amount of VB6 relative to the corresponding disper-
sion, Fig. 4B and C. Fig. 5A shows that, at 6 h, the amount of VB6
permeated from control VB6 solution was 1.5 times that permeated
from control film. Similarly, VB6 permeated from LP-dispersion
was 1.2 times that permeated from LP-film. In terms of flux values,
incorporation of LPs in mucoadhesive film reduced the flux by
36.89% relative to LP-dispersion (113.10 and 179.20 mg cmÀ2
hÀ1
,
respectively, Table 2). Similar results were previously recorded for
mucoadhesive-coated curcumin-loaded liposomes for vaginal
delivery (Berginc et al., 2014).
However, looking at the rate of permeation and judging by the
amounts of VB6 permeated across the chicken pouch membrane in
30 min (Fig. 5A), both films provided faster initial permeation than
the liquid forms. The films, being mucoadhesive, presumably
allowed better drug contact with the permeating membrane. By
time, swelling of the polymeric film increased the diffusion path
length of VB6 leading to reduced permeation in case of LP-film
compared to the dispersion.
The permeability coefficient calculated from flux value of the
control VB6 solution (7.3 Â10À6
cm sÀ1
,Table 2) was of the same
magnitude reported for other hydrophilic drugs such as caffeine
Fig. 4. (A) Chicken pouch membrane (H E staining), (B) permeation of VB6 control
solution and VB6-loaded LPs across chicken pouch membrane, (C) permeation of
VB6 from control film and VB6-loaded LP-film across chicken pouch membrane.
Table 2
Ex vivo permeation parameters of VB6 across chicken pouch mucosa determined at
37
C.
Parameter LP-dispersion Control
solution
LP-film Control
film
Ja
(mg cmÀ2
hÀ1
) 179.20 158.50 113.10 83.65
Pb
(cm sÀ1
) Â 10À6
8.18 7.23 5.16 3.82
R2
0.983 0.993 0.999 0.965
a
Steady state flux obtained from the linear portion of the cumulative amount
permeated (mg) plotted against time (h).
b
Permeability coefficient.
H. Abd El Azim et al. / International Journal of Pharmaceutics 488 (2015) 78–85 83

7. (8.14 Â10À6
cm sÀ1
) determined across porcine mucosa (Kulkarni
et al., 2011) but lower than that recorded for diltiazem
hydrochloride from HPC/SCMC mucoadhesive film across chicken
pouch mucosa (25.7 Â10À6
cm sÀ1
) (Mohamed et al., 2011). In
comparison, the permeability coefficient of PEG hydrophilic
molecules was in the range 1–3 Â10À6
cm sÀ1
according to
molecular weight (Goswami et al., 2009).
Relevant permeability studies necessitate careful choice of the
mucosal membrane; the oral epithelia of a number of experimental
animals such as rats and rabbits are entirely keratinised (Harris and
Robinson, 1992), with a very thick keratinised buccal mucosa
(Shojaei, 1998). In contrast, chicken pouch represents a better
alternative as it resembles the human thin and non-keratinised
oral lining mucosa (Erjavec et al., 2006; Hamzah et al., 2010).
Concerning the mechanism of VB6 transport across mucosal
membranes, Zielinska-Dawidziak et al. (2008) suggested a dual
mechanism of VB6 transport; at low concentrations, the uptake
process was concentration dependant via simple diffusion, while
at high concentrations, a concentration independent, carrier-
mediated mechanism was recognized. Heard and Annison (1986)
reported that VB6 absorption through chicken intestinal epitheli-
um occurred by simple diffusion.
3.8. Diffusion-permeation relation
Seeking to assess possible relation between diffusion (dialysis)
data and permeation (chicken pouch) data generated for the film
systems, the percent cumulative amount permeated ex vivo across
chicken pouch was plotted against the percent cumulative amount
diffused in vitro through dialysis membrane. Regression analysis
was performed (Fig. 5B and C). Both correlations were impressive,
with a higher correlation coefficient for the LP-film (R2
= 0.99) than
for the control film (R2
= 0.95) inspite of the difference in study
conditions. The higher than unity regression line slope (1.37,
Fig. 5B) for the LP-film indicated that percent increment in
permeation exceeded corresponding increments in diffusion
during a time interval which suggested that permeation of some
vesicles containing entrapped VB6 through the chicken pouch was
likely, but was ruled out in the dialysis study.
The slope of the regression line for the control film (no
liposomes) had a value close to unity (1.058, Fig. 5C), indicating,
comparable increments in percent diffusion and permeation.
4. Conclusions
In summary, results of this study demonstrated that liposomes
represent a suitable carrier for water-soluble vitamins. Incorpo-
ration of VB6-loaded liposomes into buccal mucoadhesive film
provided a possible patient-friendly dosage form, ensured slower
release and higher stability upon storage. Meanwhile, the LP
carrier coupled with the mucoadhesive film improved the
permeability through chicken pouch membrane relative to
controls. The results encourage future investigations of the
behavior of other water soluble vitamins as well as water-insoluble
ones in such systems.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
ijpharm.2015.04.052.
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