INVESTIGATION OF PRONIOSOMES GEL AS A PROMISING CARRIER FOR TRANSDERMAL DELIVERY OF GLIMEPIRIDE.

: The aim of the study was to develop a proniosomal carrier system that is capable of efficiently delivering entrapped glimepiride over an extended period of time for the treatment of type 2 diabetes. Proniosomal gels were developed based on Span 60 with and without cholesterol. The entrapment efficiency ofdrug inside niosomes developed from hydration of the proniosomes gel was also characterized. The in vitro release and skin permeation of glimepiride from various proniosome gel formulations were investigated. The stability studies were performed at 4°C and at room temperature. The maximum entrapment efficiency was obtained when the cholesterol concentration was 10% of total lipid (90.02%). In vitro release through Mixed Cellulose Estermembrane showed sustained release of drug from proniosomes gels. In vitro drug permeation across rabbit skin revealed improved drug permeation and higher transdermal flux with proniosomes gels compared to hydro-alcoholic gel of drug. Also, good physical stability was also achieved with proniosomes gels. Kinetics of in vitro skin permeation showed diffusion model of drug release from formulations. The study proved that the concentration of cholesterol had great influences on the properties of proniosomes gels. Hence, preparation containing 10% cholesterol can significantly increase trans-epidermal flux and prolong the release of glimepiride.

proniosomal gel (Table 1). The obtained formulations were kept in the same closed glass vials in dark for further characterization 15 . Drug loading into proniosomesgels GMD was added as (5, 10, 12, 15, 17and 20 mg) to the nonionic surfactant/ cholesterol mixture (400mg) and dissolved by the aid of absolute ethanol while warming at 50-60°C in water bath. It was noted that the addition of the drug didn't show turbidity or precipitated crystals in preparations. * Each result is the mean ± SD. (n = 3)

Hydration step and formation of niosomes
About 7 ml of phosphate buffer (pH 7.4) were added into each vial followed by heating for 10 minat a temperature 60°C±3 in a water bath while vortexing. The final volume was adjusted to 10 ml by the same buffer 15 .

Preparation of 50%alcoholic HPMC gels containing GMD
Weighted amount of HPMC-4000 cP were dispersed in distilled water containing 50% ethyl alcohol and stirring sufficient time (about 30min) was allowed for complete hydration and gel formation, then weighted amount of GMD was added and dispersed thoroughly. The concentration of HPMC-4000 cp was 2% and the final concentration of GMD was adjusted to be 15mg/gm. Characterization of proniosomes gel bases Total drug content 1 ml of aqueous dispersionwas disrupted using sufficient quantity of methyl alcohol 16 and the absorbance was recorded at 228 nm.

Microscopic examination Light microscopy
A thin layer of proniosomes gel were spread on a glass slide, and examined for the gel structure and the presence of insoluble drug crystals using ordinary light microscope with magnification powers (40 X). Photomicrographs were taken for either proniosomal gel or niosomes 12 .

Transmission electron microscopy (TEM)
Transmission electron microscopy (JEOL-JEM-2100, Tokyo, Japan) was performed to investigate the morphology of niosomes.A drop of the diluted dispersion was applied to a carbon-coated 300 mesh copper grid and left for 1min to allow some of the niosomes to adhere to the carbon substrate. The remaining dispersion was removed by absorbing the drop with the corner of a piece of filter paper and the sample was air dried 17 .

Fourier transform-infrared spectroscopy (FT-IR) studies
The FT-IR spectral analysis was conducted to verify the possible chemical intermolecular interactions between GMD, Span 60 and cholesterol. The samples were scanned using FT-IR spectrophotometer, Nicolet 200 FT-IR, USA in the spectral region between 4000 and 400 cm -1 . The smoothing of the spectra and the baseline correlation procedures were applied. The detector was purged with dry nitrogen gas to reduce moisture and to increase the signal level.

Differential scanning calorimetry (DSC)
The thermotropic properties of the samples were explored to assess the degree of crystallinity and the presence of possible interactions between GMD, Span 60 and cholesterol. DSC was performed with a DSC Q2000 V24.4 Build 116. The calorimeter was calibrated for temperature and heat flow accuracy using the melting of pure indium (mp156.6°C and ΔH of 25.45 J gm −1 ). The temperature range was from 0 to 300°C with a heating rate of 10°C/min. The gas used was nitrogen with a purging rate of 50 ml/min. The weight of each sample was 5-9 mg. Determination of entrapment efficiency of GMD in niosomes by Freez-thawing/ Centrifugation method 1 ml Samples of niosomes dispersion were frozen for 24 h at 20°C in Eppendorf tubes. The frozen samples were removed from the freezer and let to thaw at room temperature, then centrifuged at 20000 rpm for 60 min at 4°C. Two times washings with phosphate buffer (pH 7.4) were done for complete removal of drug adsorbed on the surface of niosomal vesicles. The supernatant was separated each time from niosomal pellets and prepared for the assay of free drug 15 . Each result was the mean of three determinations (±SD).The drug content was determined spectrophotometrically at 228nm against phosphate buffer (pH 7.4) as blank. The %of GMD entrapped was calculated by subtracting amount of un-entrapped drug from the total drug incorporated 18 .

Assessment of GMD release rates from proniosomesacross mixed cellulose ester (MCE)membrane
The release of GMD from different proniosomes gelswere determined using modified Franz cell fabricated locally with 6.6 cm 2 of diffusion area. MCE membrane (0.45μm pore size), previously soaked in phosphate buffer (pH 7.4),was mounted between the donor and receptor compartments of the diffusion cells. The proniosomes gels were placed on MCE membrane in the donor chamber whilst phosphate buffer containing 10% methanol (pH 7.4) was used as a receiver medium in the receptor chamber. The temperature was kept at 32± 0.5 °C and the stirring rate was 100 rpm in Kottermann shaker (D3165 Hangisen, Germany). About two milliliter samples were withdrawn at specified time intervals (0.5, 1, 2, 3, 4, 6, and 8 h) and replaced with equal volumes of fresh receiver medium to keep the volume constant during the experiment. Samples were analyzed spectrophotometrically at 228 nm against samples collected from diffusion of drug free systems as blank.

Assessment of GMD release rates from niosomes
The niosomal pellets were resuspended in 50 ml of phosphate buffer (pH 7.4). The suspension was placed in a stoppered glass flask in a shaking water bath at 37°C. 1 ml samples were withdrawn at the following intervals; 0, 1, 2, 3, 4, 6 and 8 h after incubation. Samples were centrifuged as before and supernatants assayed at 228nm. The percentage of drug release was plotted as a function of time 15 .

Ex vivo skin permeation study
Abdominal skin of male rabbits (2-2.5 kg) was used in the study. Hair was removed from the abdominal skin with the aid of an electric animal clipper and shaver. Care was taken not to damage the skin surface. Rabbits were sacrificed and the abdominal skin of the rabbit was separated. Sodium azide (0.02%) is applied to the skin as preservative and the skin was stored at -20°C and used within three days for the permeation study. The permeation experiments were run by using the same diffusion cells previously described in the release through MCE membrane. However, skin membranes were mounted, with the stratum corneum side towards the donor (drug loaded system) and the dermal side facing and in contact the receptor compartment which contain phosphate buffer containing 10% methanol (pH 7.4) maintained at 32±0.5°C 19 and the stirring rate was 100 rpm in Kottermann shaker.
Proniosomes gels of Span 60 and alcoholic HPMC were applied to the stratum corneum side. 2 ml aliquots were sampled at 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 hr after starting the experiment from the receptor medium and replaced with equal volume of the fresh receiver medium. Samples were analyzed spectrophotometrically at 228 nm using samples collected from permeation of drug free systems as a blank. Each experiment was carried out in triplicate.
All skin permeation parameters such as steady state transdermal flux (SSTF), permeability coefficient (PC), maximum diffusion (D max ) and diffusion coefficient (D) of GMD across rabbit skin were estimated for different formulations. Permeability parameters of GMD were constructed by plotting the cumulative amount of the drug permeated per unit area (Q) as a function of time. The steady state flux (J SS ) was calculated from the slope. The permeability coefficient (P C ) was calculated by dividing the flux by the initial drug load (C o ).The diffusion coefficient (D) was calculated by plotting the cumulative amount of drug permeated versus square root of time according to Equation 13 .

Assessment of physical stability for proniosomes gels
Proniosomes gels stability and aggregation of niosomes dispersions as a function of temperature were determined as the change in total drug content and entrapment efficiency after storage. The proniosomal gels and niosomes dispersions were stored in glass vials at room temperature or kept in refrigerator (4°C) for 3 months 17 .

Statistical analysis
The data were reported as mean ± SD (n = 3) and statistical analysis of the data were carried out using one-way ANOVA at a level of significant of P< 0.05.

RESULTS AND DISCUSSION: Formation of proniosomes gels
A sol phase of Proniosomes gelswere formed when distilled water (about 30-40% w/w of total lipids) at 60°C is added in drops to the nonionic surfactant dissolved into hot absolute ethanol while vortexing, where the temperature was kept at 60°C. Since the solvent (absolute ethanol) amount is small and the formation of micelles is not possible into this solvent, it is expected that the addition of small amount of water favors the selfassembly of the surfactant into w/o microemulsion sol phase, where the aqueous droplets bound by surfactant interfacial films which are dispersed in the continuous solvent phase 20 .
Cooling the sol phase results in a decrease in the solubility of Span 60 and cholesterol in the solvent and consequently, lower solvent-cholesterol affinities due to the limited solvent system present. The formed gel structure proposed to be amphiphilic system comprising double layers of oriented molecules placed head to head and tail to tail with water present as droplets or sheets of water molecules between the hydrophilic residues of the surfactant layers. Span 60 (Tc=53°C) produced white creamy gel in the presence or absence of cholesterol as it has the greatest transition temperature and is solid at room temperature, so it acts as gelator by itself. The formed proniosomes gels is thermo-reversible as an increase in temperature results in the solvation of the gelator into the solvent and returns again to the gel structure upon cooling 15 .

Hydration of proniosomes gels
Niosomes are formed only when the proniosomes gels comes into contact with an aqueous environment such as moisture found on the skin or mucosal surfaces. The contact of proniosomes gels with water leads to swelling of the lamellar liquid crystal bilayers and vesicles due to the interaction of the hydrophilic head groups of the surfactant with water molecules. The surfactant tubules serve as conduits for water penetration into the gel network and the gel fragments 21 .
The complete hydration of proniosomes gels was found to take long time ranging from 1h to 2h at room temperature with continuous stirring or vortexing. So, warming the gel with excess water above the transition temperature of Span 60 (>60°C) accelerates the transformation to niosomal structures within 5-10 min only. Drug loading in proniosomes gels 15 mg of GMD was found to be completely dissolved into proniosomes gels. This is perhaps due to high solubility of the drug in surfactant/cholesterol/ethanol mixture. Microscopic examination showed no drug crystals precipitated.

Drug content
The drug content of the developed formulations was not found to be significantly different (P< 0.001) from the added amount.

Gel structure
The gel structures were formed fromfloccules of small tubular and vesculating particles which have creamy opaque appearance and also no drug precipitates (Figure 1). The units of the gel are often bound together by van der Waals forces so as to form crystalline regions throughout the entire system. The dominant coherent gel phase is built up by surfactant/cholesterol lamellae with water phase mainly bound interlamellarly to Comment [kc18]: The manuscript lacking size assessment data for the formed niosomal vesicles which is a basic evaluation technique for nano-drug delivery systems to clearly prove its nano-size.

Comment [kc19]:
What is the viscosity of the formed gel? Numerical drug content data is missing. Niosomes appeared under ordinary microscope as multilamellar vesicles while under TEM as unilamellar vesicles, how does the author explain this contradictory? the hydrophilic head groups of surfactant/ cholesterol lamellae. This resulted in an interconnected network of a mixture of lamellar liquid crystals resembling palisades or tubular aggregates and vesculating lamellae 12 .

FT-IR studies
The FT-IR spectra of GMD, Span 60, cholesterol and physical mixture are displayed in Figure 4. The GMD showed strong absorption peak at 1159 cm -1 corresponding to the sulphonamide group (S=O) and peaks at 1696 cm -1 corresponding to carbonyl group (C=O). The broad bands at 3374 cm -1 and 3377cm -1 could be assigned to O-H stretching vibrations of Span 60 and cholesterol, respectively. The FT-IR spectrum of the physical mixture revealed that the characteristic bands of GMD did not disappear or exhibit major shifts. Furthermore, no new bands were formed. These findings pointed out the lack of considerable intermolecular interactions between GMD, Span 60 and cholesterol.

Differential scanning calorimetry (DSC)
DSC is a fast and reliable method to screen drug-excipient interactions as indicated by appearance of a new peak(s), change in the peak shape and its onset, peak temperature/ melting point and relative peak area or enthalpy. Figure 5 depicts various DSC thermograms obtained during the study. Pure GMD showed a sharp endothermic peak at 205.72°C. Thermogram of Span 60 exhibits an endothermic peak with onset at 44.62°C and maximum occurrence at 52.24°C. Cholesterol is reported to show an endothermic peak at 148-150°C. It is evident that the original peaks of GMD disappear from the thermogram of proniosomal gel (containing Span 60, Cholesteroland GMD or Span 60 and GMD). These observations confirm incorporation of GMD into proniosomal gel proving complete entrapping of drug into the vesicles.

Effect of cholesterol
Cholesterol is one of the common and essential additives in niosomesdispersions in the present study. Incorporation of cholesterol was known to influence vesicle stability and permeability 22 . The effect of cholesterol on GMD entrapment was varied according to its concentration. A significant increase (P < 0.05) in the entrapment efficiency of GMD was obtained when 10% of cholesterol was incorporated into niosomes prepared from Span 60 (P6A) followed by a decrease in entrapment efficiency of the drug upon further increase in cholesterol content ( Table 2).
Reason revealed for this type of behavior is cholesterol molecules accommodate itself as ''vesicular cement'' in the molecular cavities formed when surfactant monomers are assembled into bilayers to form niosomal membranes and this space filling action results in the increased rigidity and the improved entrapment efficiency. On further increase of cholesterol beyond certain concentration it compete with the drug for the space within the bilayers, hence excluding the drug and can disrupt the regular linear structure of vesicular membranes 23 . In vitro release studies Figure 6 showed the percentage GMD released from proniosomes gels over 8 h. From the obtained results it is clear that P6 showed the lowest release rates among all proniosomal preparations. Addition of 10% cholesterol (P6A) gave greater release rates than those without cholesterol. Increasing cholesterol content up to 50% was accompanied by gradual decrease in the release rate, however, remained significantly higher than that of cholesterol free systems (P< 0.05). This could be due to the fact that the addition of cholesterol appeared to disrupt the ordered array of the hydrocarbon chains in the gel phase 15,24 . Below the transition temperature, addition of cholesterol made the membrane less ordered, while above the transition temperature made the membrane more ordered 25,26 . At 37°C P6 gave rise to highly ordered gel phase than those containing cholesterol, consequently, they produced lower release amounts of GMD.

Kinetic analysis of the release data of GMD Proniosomal systems
The kinetic analysis of GMD release data from different Proniosomes gels were tested according to zero, first order kinetic and diffusion controlled model. The results clearly revealed that all proniosomal systems fitted better with Higuchi diffusion model.

Glimepiride release rates from niosomes
The release profiles of GMD from niosomes of different cholesterol contents is an apparently biphasic release process. Rapid drug leakage was observed during the initial phase where about 30-35% of the entrapped drug was released from various formulations in the first hour. However, during the following 8 ha slow release occurred in which only further 3% to5% of GMD was lost from different niosomal preparations (Figure 7). This could be explained on the basis that the drug is mainly incorporated between the fatty acid chains in the lipid bilayers of niosomal vesicles. This leads to rapid ionization and release upon dispersing niosomes in increased buffer (pH 7.4) volumes until reaching equilibrium 27 . Figure 7 showed that P6A displayed the lowest extent of drug release after 8h. The percent GMD released from P6A & P6Cafter 8 h was 34.54% and 39.08%, respectively. The increase in release rates of GMD from P6C formulation was statistically significant (P< 0.05) compared to P6 and P6A formulation. This result attributed to the cholesterol concentration.10% cholesterol produced an optimum hydrophobicity that decreased the formation of the transient hydrophilic holes, responsible for drug release through liposomal layers 28 . On the other hand, further increase in cholesterol amounts into niosomes formulations could increase the release of GMD 23 .

Comment [kc25]:
The effect of 50% cholesterol versus 10% cholesterol on the in vitro release rates needs to be more clarified.

Comment [kc26]:
The effect of 50% cholesterol on the in vitro release rates from niosomes needs further explanation (50% cholesterol showed the highest release rate which was not the case in proniosomes gel).
The reduced permeation of GMD from proniosomal gels of Span 60 is primarily attributed to its high transition temperatures which made them in a highly ordered gel state at the permeation temperature (32°C) 19 .

Kinetic analysis of the permeation data of GMD Proniosomal systems
The kinetic analysis of the permeation data of GMD from different proniosomal systems were tested according to zero, first order kinetic and diffusion controlled model. The results clearly revealed that all proniosomes gels fitted better with the diffusion controlled model.

Physical stability
The results of stability studies were compiled in Figures 9-12. The effects of storage temperature on entrapment efficiency were expressed as % of GMD retained entrapped according to the following equation.
Generally, drug leakiness from niosomal dispersions upon storage in refrigerator was significantly low; while at room temperature there was an appreciable drug loss and decreased in entrapment efficiency. Hence the niosomes dispersions needs to be refrigerated for use as is the case with all other vesicular systems 17 .
Proniosomes gels were more stable than niosomal vesicles under refrigerated and room temperature condition. This suggests that proniosomes offered a more stable system that could minimize the problems associated with conventionally prepared niosomes like degradation by hydrolysis, or oxidation, sedimentation, aggregation and fusion during storage. P6A (1.86% loss in drug content and 5.89% decrease in entrapment efficiency) were more stable than other formulations at 3 months.

CONCLUSION:
Concluding the above mentioned results; Proniosomes gels could enhance the solubility of certain poorly soluble drugs but to a maximum limit after which any increase in the drug concentration may lead to drug precipitation.The EE% of GMD (as a model for poor soluble drugs) into niosomes prepared by the proniosomal method was a function of cholesterol content.Cholesterol content affected the drug release and permeation properties of proniosomal system.All proniosomal gels fitted to diffusion model when using MCE membranes and when using rabbit skin for release and permeation studies, respectively.It is clear that niosomal formulations containing 10% cholesterol are the most stable among other tested formulations.

Comment [kc28]:
The author needs to clarify the reasons why P6A (10 % cholesterol) showed the highest stability.