Proniosomal Gel as a Carrier for Improved Transdermal Delivery of Griseofulvin:
Preparation and In-Vitro Characterization
Sandeep Gupta*, Dheeraj Ahirwar, Neeraj K Sharma, and Deenanath Jhade
School
of
ABSTRACT
The
present investigation aimed at formulation, and performance evaluation of
vesicular drug carrier system proniosomal gel for transdermal delivery of antifungal agent, griseofulvin. Proniosomal gel
(PNG) formulations of griseofulvin were prepared, and
characterized for vesicles shape, size, entrapment efficiency, and drug
permeation across pig ear skin. The effects of different non-ionic surfactants
on transdermal permeability profile were assessed.
The optimized PNG formulation showed enhanced in vitro skin permeation flux of
3.682±0.186 µg/cm2/hr as
compared to 0.028 ± 0.02 µg/cm2/hr for plain drug solution in water.
Results indicated that the optimized PNG formulation of griseofulvin
had better skin permeation potential than plain drug solution in water.
KEYWORDS:- Transdermal
delivery, Griseofulvin (GF), Proniosomal
gel (PNG), Non-ionic surfactants
INTRODUCTION
Superficial
fungal infections affect millions of people throughout the world. Among them, tinea represents cutaneous
infections by dermatophytes. Dermatophytes cause fungal infections of keratinised
tissues, e.g. skin, hair and nails. Topical antifungals
remain the most commonly recommended treatment for many superficial dermatophytoses 1. Topical
preparation of 1% GF is effective and safe in treating tinea
corporis and interdigital
dermatophyte infections 2.
Despite
decades of research, the barrier function of the stratum corneum
still remains a problem, which makes the development of new transdermal
drug delivery systems an interesting challenge. Vesicular systems have been
widely studied as vehicles for dermal and transdermal
drug delivery. Their benefits in enhancing drug permeation have been well
established 3. Vesicular
system, both liposomes and niosomes
are uni- or multilamellar spheroidal structures composed of amphiphilic
molecules assembled into bilayers. They are
considered primitive cell models, cell-like bioreactors and matrices for bioencapsulation. In the recent years, nonionic surfactant
vesicles known as niosomes received great attention
as an alternative potential drug delivery system to conventional liposomes. Moreover, compared to liposomes,
niosomes offer higher chemical and physical stability
4 with lower cost and greater
availability of surfactant classes 5. Niosomal vesicles can
encapsulate both lipophilic and hydrophilic drugs 6.
It has been reported to enhance the
residence time of drugs in the stratum corneum and
epidermis 7, while reducing the systemic absorption of the drug and
improve penetration of the trapped substances across the skin. In addition,
these systems have been reported to decrease side effects and to give a
considerable drug release 3. They
are thought to improve the horny layer properties
both by reducing transepidermal water loss and by
increasing smoothness via replenishing lost skin lipids8. Moreover, it has been
reported in several studies that compared to conventional dosage forms,
vesicular formulations exhibited an enhanced cutaneous
drug bioavailability 5,9. However,
there may be problems of physical instability in niosome
dispersions during storage like vesicles aggregation, fusion, leaking or
hydrolysis of encapsulated drugs, which affected the shelf life of the
dispersion 10.
Proniosomes; semisolid liquid crystal (gel)
products of nonionic surfactants easily prepared by dissolving the surfactant
in a minimal amount of an acceptable solvent namely ethanol and the least
amount of water could solve the problem 4. The proniosomal
structure was liquid crystalline-compact niosomes
hybrid that was converted into niosomes immediately
upon hydration 11,12,13. Lecithin still acts as a component of the proniosomal structure as they reported. Stimulated
by these findings it was envisaged to extend the concept of proniosomes
to the delivery of GF across the skin.
The aim of
present work was to enhance the absorption of GF across the skin by formulating
it with appropriate nonionic surfactant in the form of proniosomal
gel, a precursor dosage form of niosomes. In the
present study the provesicular approach has been
extended to the liquid crystalline proniosomes, which
are reported to have superior skin penetration ability. Liquid crystalline proniosomal gel (PNG) will be converted into the niosomes, in situ by absorbing water from the skin 4.
MATERIALS AND
METHODS
Materials: Span-20,40,60,80
(
Preparation
of Formulations: Proniosomes were
prepared by a method modified from Perrett et al.,
199114. Two mg of Griseofulvin with
surfactant, lecithin, and cholesterol were mixed with 0.125 ml absolute Ethanol
in a wide mouth glass tube. The composition of additives is listed in Table-1.
Then the open end of the glass tube was covered with aluminium
foil and warmed in a water bath at 65 ± 3°C for 5 min. A 0.08
ml; pH 7.4 phosphate buffer was added and still warmed on the water bath for
about 2 min till the clear solution was observed. The mixture was allowed to
cool down at room temperature till the dispersion was converted to proniosomal gel. In case of formulations in which drug was
not properly dissolved, the drug and formulation surfactants were dissolved in
chloroform, followed by evaporation of solvent.
Figure-1.
Photomicrograph of proniosomal gel under cross
polarizer (X 40).
In Vitro Characterizations:
(1).
Visualization of Vesicles and Size Determination: A thin layer of PNG was
spread
on a slide
and a drop of phosphate buffer was added through the side of the cover slip
into the cavity slide and again observed. Photomicrographs were taken at
suitable Magnifications before and after addition of water (Leica,
DMLB,
(2). Encapsulation Percentage Measurement: PNG
in the glass tube was reconstituted with 10 ml; pH 7.4 phosphate buffer. The
GF-containing niosomes were separated from untrapped drug by centrifuging at 20,000 rpm at 20°C for 30 min 11.
The supernatant was taken and diluted with phosphate buffer (pH 7.4). The GF
concentration in the resulting solution was assayed by UV method at 291.0 nm 15.
The percentage of drug encapsulation was calculated by the following equation:
EP (%) = [(Ct - Cf)/ Ct] ´ 100. Where Ct is
the concentration of total GF, and Cf is the concentration of free GF 11.
Figure-2.
Photomicrograph of niosomes formed after shaking proniosomal gel with water (X 40).
In Vitro Skin Permeation Studies: The in
vitro release of GF from different PNG formulations was studied using locally
fabricated diffusion cell through the excised full-thickness pig ear skin. Skin
was prepared for this study by the method reported by Trotta
et al., 200416. Capacity of receptor compartment was 50
ml and area of the donor compartment was 45.87 cm2. The prepared
formulation with 1% drug was applied to the stratum corneum
side of the skin surface, which had an available diffusion area of 4.17 cm2.
This gel-applied skin was mounted and clamped between the donor and receptor
compartment with stratum corneum side facing donor
compartment. A 20 ml aliquot of 40%: 60% (v/v) ethanol/pH 7.4-phosphate buffer was
used as receptor medium to maintain a sink condition. GF aqueous solution was
used as control formulation. The temperature of receptor compartment was
maintained at 37ºC using a thermostatic hot plate temperature controller
available on magnetic stirrer. The receptor fluid was stirred at 600 rpm by
magnetic bead on a magnetic stirrer. The top of the donor compartment was open
for air circulation. At appropriate intervals, 0.2 ml aliquots of the receptor
medium were withdrawn and immediately replaced by an equal volume of fresh
receptor solution. Samples withdrawn were analyzed at
Figure-3. Effect of
amount of drug on permeation profile across pig ear skin (Formulation PNG 40).
Mean ± S.D. (n = 3).
Figure-4.
Effect of Different Spans on Drug Permeation Profile Across Pig Ear Skin. Mean
± S.D. (n = 3).
Table-1:
Composition of Various Proniosomal Gel Formulations
in (mg).
|
Formulation
code |
GF |
Span 20 |
Span 40 |
Span 60 |
Span 80 |
Lecithin |
Cholesterol |
|
PNG 20 PNG 40 PNG 60 PNG 80 |
2 2 2 2 |
90 – – – |
– 90 – – |
– – 90 – |
– – – 90 |
90 90 90 90 |
10 10 10 10 |
Table-2:
Characterization of niosomes formed from proniosomal gel after dilution with phosphate buffer (pH
7.4).
|
Formulation
Code |
Size in
µm |
Rate of Hydration |
Encapsulation
(%) |
|
|
VS ± SD |
PI |
|||
|
PNG 20 PNG 40 PNG 60 PNG 80 |
0.782±0.016 0.881±0.014 0.783±0.011 1.001±0.019 |
0.020 0.016 0.014 0.018 |
8.06×104 7.96×103 7.38×103 9.16×104 |
86.67±0.21 87.98±0.18 87.54±0.44 87.46±0.88 |
Values are
represented as mean ± S.D. (n = 3); SD = Standard Deviation; VS = Vesicle Size;
PI = Polydispersity Index obtained as PI = SD/VS
291.0 nm
using UV-VIS Double Beam Spectrophotometer (SYSTRONICS
2101). In-vitro
release rate studies were done for different formulations and effect of
variation in composition, alcohols and amount of drug on release rate were
studied. Cartesian plots of cumulative amount of drug present in receptor
compartment versus time were plotted. Steady State Transdermal
Flux (Jss, µg/cm2/ hr) was calculated from
the slop of the steady state portion of these graphs.
RESULTS AND DISCUSSION
Preparation and In Vitro Characterization of Formulations: The method
of preparation involves the principle of coacervation
phase separation 17. The method is based on the simple idea, that
the mixture of surfactants: alcohol: aqueous phase can be used to form the
concentrated proniosomal gel, which can be converted
to stable niosomal dispersion, respectively, by
dilution with excess aqueous phase. The composition of formulations was taken
according to the previous report by 14 and optimized for PNG formulation using the principle of
three-phase diagram. Different formulations using different surfactants were
prepared. Span-40 was used as representative of non-ionic surfactants because
it gives the vesicles of larger size with higher entrapment of drug 18.
Further, the drug leaching from the vesicles composed of Span-40 is low, due to
its high phase transition temperature and low permeability.
Figure-5. Steady state
transdermal flux of different formulations for the
transport of GF across pig ear skin.
Morphological characterization of the proniosomal gel as well as Existence of vesicular structure
after hydration of PNG was confirmed by photomicrograph. The PNG when observed
under cross polarizer showed birefringent streaks
lamellar structures in liquid crystalline form (Figure-1). When the prepared
gel was hydrated, niosomes formed from it were multivesicular, multilamellar,
spherical and somewhat elongated in shape (Figure-2). For the topical
administration of vesicles, size and size distribution studies are important 19.
Size of the vesicles was measured by dynamic light scattering method in two
conditions: a). without agitation b). with agitation and the size of vesicles was
found to be in the order of hydration: Without agitation > With agitation.
Hydration without agitation results in largest vesicle size. While the
application of energy i.e. hydration with agitation results in the breakage of
vesicles into smaller size. The permeability index (PI), which is the ratio of
standard deviation (SD) to vesicle size (VS) was also low, which indicates that
this method of PNG formation results in vesicles of uniform size. The vesicles
formed from PNG were more uniform. Encapsulation efficiency of the PNG
formulation was high (maximum
87.98±0.18 for S-40) (Table-2). Highly lipophilic
drugs like GF are intercalated almost completely within the lipid bilayer of liposomes and niosomes 20. Hence, maximum drug molecules seem
to be intercalated within the lipid bilayer forming a
part of the bilayer. This result was consistent with
the entrapment efficiency of levonorgestrel in proniosomes incorporated with Span 40 4. The
mean vesicle size of niosomes formed from PNG
formulations is given in (Table-2). Vesicle size of PNG 40 and 60 was larger
than PNG 20 and 80. It may be due to their high HLB values (Span-40:6.7;
Span-60:4.7) which results in reduction in surface free energy and allows to
form vesicles of larger size 18.
In Vitro
Skin Permeation Study: In vitro permeation studies give us valuable
information about the product behaviour in vivo. The
drug permeated dictates the amount of drug available for absorption. A 20 ml
aliquot of 40%: 60% (v/v) ethanol/pH 7.4-phosphate buffer was used as receptor
fluid for the in-vitro drug permeation studies based on the solubility
consideration of GF. For the different PNG formulations, drug release profiles
were studied in triplicate and standard deviation, transdermal
flux, permeation coefficient, regression coefficient and enhancement ratio (ER)
were calculated from the data (Table-3). The sampling time is one hour so no
lag phase could be detected and the release of GF from PNG formulations through
the pig ear skin was found to be constant slow (zero order) release i.e. near
linear. Data of each permeation profile of GF through excised pig ear skin were
linearly regressed and fitted into the straight-line equation to get the slop
values. These slop values (n) were calculated from Log Q = n log t. The slop
values for the formulations were very close to one showing the zero order
release profile. The no lag phase or small lag time with our formulation was
perhaps due to penetration enhancing properties of the alcohols and surfactants
and increase in solubility of free drug in stratum corneum
lipid. The conversion of PNG to noisome in situ releases the partition of free
drug, which in the presence of alcohol and surfactants might penetrate faster. Junginger et al., 19918, reported the molecular mixing of soya lecithin
lipids with stratum corneum lipids, which might
enhance in the presence of alcohols. The amount of drug released from different
PNG formulation was found to be in the following order: PNG 80 > PNG 20 >
PNG 40 > PNG 60. Effect of different amount of the drug, present in the PNG
formulation, on the skin permeation profile are shown in (Figure-3) for
formulation PNG 40. Almost linear correlation was observed between
concentration of drug and transdermal flux i.e. as
the concentration of drug increases, the steady state transdermal
flux also increases (Table-3). Effect of different Spans (Figure-4) on drug
permeation profile showed that flux value was highest for Span-80 and lowest
for Span-60. No significant difference was observed in skin permeation profile
of formulations containing Span-40 and Span-60 due to their higher phase
transition temperature which is responsible for their less permeable nature18.
Moreover, their high HLB values (Span-40:6.7; Span-60:4.7) results in reduction
in surface free energy which allows to form vesicles of larger size and hence
small area exposed to the dissolution medium and skin. Span-20 has highest HLB
value of 8.6 and vesicles of largest size but faster drug release was obtained
perhaps due to its low transition temperature. With the Span-80 formulation,
(HLB 4.3), highest transdermal flux value was
obtained probably due to smallest size of vesicles and its low transition
temperature 18. The intrinsic unsaturation
of oleate in Span-80 responsible for low transition
temperature might have better penetration enhancing ability than laurate in Span-20 21.
Table-3. Steady State Transdermal Flux, and Enhancement Ratio for the Transport
of GF Across Pig Ear Skin.
|
Formulation Code |
Steady State Transdermal Fluxa
(µg/cm2 hr.) |
ERb |
|
PNG 80 (0.5mg) PNG 80 (1.0mg) PNG 80 (1.5mg) PNG 80 (2.0mg) PNG 20 PNG 40 PNG 60 PNG 80 CF |
1.517±0.061 5.547±0.036 14.695±0.074 17.113±0.047 3.441±0.103 2.879±0.197 2.716±0.128 3.682±0.186 0.028±0.02 |
54.18 198.1 524.8 611.1 122.9 102.8 97 131.5 - |
Values are represented
as mean ± S.D. (n = 3); (a) =>Amount of Drug/Time × Area of the Skin: Q/T×A ;
(b) => Enhancement Ratio (ratio of transdermal
flux from prepared formulation to control formulation)
The flux value obtained from PNG 80 (3.682±0.186
µg/cm2hr.) is 1.07-fold, 1.28-fold, and 1.36-fold higher than that
of PNG 20, PNG 40, and PNG 60 formulations (3.441±0.103, 2.879±0.197,
2.716±0.128 µg/cm2hr.) respectively and 131.5-fold higher than that
attained by the control formulation (0.028±0.02) (Table-3). The very low skin
permeability of plain drug solution is due to extreme hydrophobicity
and low solubility of GF in water (1.27±0.1µg/ml). Better transdermal
flux and no lag phase with PNG formulations was perhaps a result of the
combination of one or more of following mechanism: (1). Increased solubility of
GF, (2). High association of drug with vesicle bilayer,
(3). Increased partitioning of vesicles into the stratum corneum,
(4). Penetration enhancement effect of the short chain alkanols
and nonionic surfactants.
CONCLUSION
In the present study, in vitro permeation of Griseofulvin from proniosomal
formulations with various types and contents of nonionic surfactant was
evaluated. Griseofulvin included in proniosomes was entrapped within the lipid bilayers formed by this technique with very high
efficiency. Griseofulvin from proniosomes
seems to pass through the skin with comparable facility to free drug. The
experimental result and supportive theoretical analysis suggest either direct
transfer of drug from vesicles to the skin or the penetration enhancer effect
by non-ionic surfactant may contribute to the mechanism of Griseofulvin
permeation from proniosomal formulations. Hence, the
PNG formulation developed for transdermal
administration of Griseofulvin possessed better skin
permeation potential and high entrapment efficiency. A 131.5-fold increase in transdermal flux of Griseofulvin
as compared to plain drug solution suggested that PNG formulation provide a
better in vitro skin delivery of Griseofulvin.
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Received on
06.06.2009
Accepted on
10.06.2009
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Journal . of Pharmaceutical Dosage Forms and Technology. 1(1): July.-Aug. 2009, 33-37