Formulation and Evaluation of Nystatin Microparticles as a Sustained Release System

Nystatin is the drug of choice for treatment of cutaneous fungal infections with main disadvantage that is the need for multiple applications to achieve complete eradication which may reduce patient compliance. Microparticles offer a solution for such issue as they are one of sustained release preparations that achieve slow release of drug over an extended period of time. The objectives of this study were to fabricate nystatin-loaded chitosan microparticles with the ultimate goal of prolonging drug release and to analyze the influence of polymer concentration on various properties of microparticles. Microparticles were prepared by chemical cross-linking method using glutaraldehyde as cross-linking agent. Five formulas, namely N1C1, N1C2, N1C3, N1C4 and N1C5, were prepared and the effect of drug to polymer ratio was studied with respect to drug loading, encapsulation efficiency, particle size and morphology. Furthermore the prepared microparticles were subjected to various physico-chemical studies, such as drugpolymer compatibility by Fourier Transform Infrared Spectroscopy (FTIR) and invitro drug release characteristics. Microparticles obtained from N1C1, N1C2 and N1C3 formulas were regular in shape with mean particle size ranging between 1μm and 10μm. N1C5 formula was resulted in particles with irregular shape while N1C4 showed a blend of microparticles and deformed particles. The effect of chitosan concentration on drug loading and entrapment efficiency was studied. The results showed increment in these parameters that was directly proportional to the increment in polymer concentration. Percentage yield showed a significant increment which was related to the increment in the ratio of chitosan used during the study. FTIR results showed no interactions between nystatin and chitosan. DSC studies proved the crystalline nature of nystatin and chitosan. On other hand, the thermogram of loaded microparticles showed the absence of endothermic peak corresponding to nystatin which may indicate the loss of the crystalline nature of the drug presented inside the microparticles. Invitro release studies resulted in 95.6% release of nystatin for N1C1 after 15 hours. N1C1 appeared to be promising in formulating microparticles that provide nearly complete release of the drug within15 hours. This formula can be selected in future work to be formulated as topical gel that prolongs the release of nystatin.


Introduction
The development of new delivery systems for the controlled release of drugs is one of the most interesting fields of research in pharmaceutical sciences.Microparticles are one of sustained release preparations that achieve slow release of drug over an extended period of time.Microparticles are micrometric matrix systems essentially spherical in shape having size ranging between 1μm -1000μm (1) .Microparticles can be used for the controlled release of drugs, vaccines, antibiotics and hormones.
Microparticles have many advantages such as providing a larger surface area and possessing an easier estimation of diffusion and mass transfer behavior.Besides these, the encapsulated small molecules could diffuse out of the barrier with precise kinetics modeling and control-release of drugs to the body fluid (2) .Candida albicans infection is a problem of growing clinical importance worldwide.Literature data point out that about 96% of all opportunistic mycoses are caused by Candida species (3) .Nystatin is a polyene antifungal antibiotic characterized by a potent broad-spectrum antifungal action against a variety of fungal pathogens including Candida, Aspergillus, Histoplasma, and Coccidioides (4) .Successful eradication of cutaneous fungal infections requires 2-4 applications of all available topical dosage forms of nystatin (5) .Consequently, the aim of this study was to fabricate nystatin-loaded, sustained-release formulation that would prolong the release of the drug at the site of application, reduce the frequency of application, reduce the amount of drug administered and improve patient compliance and acceptance.Chitosan was selected as a release-retardant polymer and the chemical cross-linking was selected as the technique.To find the best formula (the one with the highest drug content, best encapsulation efficiency, optimum particle size and morphology), different formulas were prepared using different ratios of chitosan.

Materials
Nystatin was obtained as a gift from Ninavah Drug Industry (NDI).Chitosan was obtained from Johnson CAO, US.Span 80 is purchased from BDH ® (UK).Liquid paraffin, methanol and glutaraldehyde were obtained from TEDIA ® (USA).Glacial acetic acid was obtained from THOMAS BAKER (UK) while hexane obtained from POCH ® (Poland).

Preparation of microparticles
Glutaraldehyde cross-linking method was employed for preparation of microparticles (6) .Accurately weighed amounts of polymer and drug (see Table 1 for the different amounts of drug and polymer) were added step by step into 2% acetic acid solution.The resultant nystatin-chitosan dispersion was added drop wise by syringe with 23G needle into a continuous phase consisting of 125 mL of light liquid paraffin containing 0.5% of Span 80 as an emulsifying agent.The mixture was stirred for 30 minutes at 1500 rpm using a 4-blade mechanical stirrer to form a w/o emulsion.A drop-by-drop solution of a measured quantity of aqueous glutaraldehyde (25% v/v) was added to the prepared emulsion at 15, 30, 45, and 60 minutes.Stirring was continued for 2.5 hours to obtain microparticles which were washed first with hexane and then with distilled water to remove the adhered liquid paraffin and glutaraldehyde.
Finally the resultant microparticles were filtered, dried at room temperature and stored in a desiccator for further evaluation.

Physicochemical characterization Morphological characteristics and particle size analysis:
A microscopical image analysis technique was applied for determination of particle size.The morphology and particle sizes were determined with Motic ® digital microscope equipped with imaging accessory.The particle diameters of 200 microparticles were measured randomly by supplied software and the average particle size was determined (7) .In order to be able to define a size distribution or compare the characteristics of particles with many different diameters, the size distribution can be broken down into different size ranges, which can be presented in the form of a histogram.

Determination of drug loading and entrapment efficiency
A known quantity of microparticles (10 mg) was triturated by mortar and pestle and extracted with 10 ml methanol (containing 5% acetic acid).The resultant solution was centrifuged and the obtained supernatant was diluted appropriately with respective solvent system (8) .Absorbance was measured at 305 nm using LABOMED ® UV-VIS spectrophotometer and concentration of nystatin in microparticles was calculated according to the standard regression.Each determination was made in triplicate.The drug loading of prepared microsphere was determined by applying the following equation (9) : Drug loading (%) = Entrapment efficiency represents the proportion of the initial amount of drug incorporated/associated into/with the microparticles (10) .This parameter was determined by applying the following equation (11) : Entrapment efficiency (%) =

Determination of percentage yield
The relative yield was calculated based on the amount of microparticles of each formulation obtained relative to the amount of the total weight of the polymer and drug used in the dispersed phase.The percentage yield was calculated according to the following equation (12) .

Fourier transforms infrared spectroscopy (FTIR)
Drug-polymer interactions were studied by FTIR spectroscopy.The spectrum was recorded for chitosan, pure nystatin, blank chitosan microparticles, physical mixture of blank chitosan microparticles and nystatin and for nystatin loaded chitosan microparticles using BRUKER ® (Germany) infrared spectrophotometer (13) .

Differential Scanning Calorimetry (DSC)
Thermal analysis was performed using DSC (Shimadzu DSC plus, Japan).Approximately 10 mg of each sample (chitosan, pure nystatin, blank chitosan microparticles, physical mixture of blank chitosan microparticles and nystatin in the ratio of (1:1) and nystatin loaded chitosan microparticles) were used.Each sample was placed in a sealed aluminum pan and heated at a scanning rate of 10ºC/min from 30ºC to 250ºC (14) .

In-vitro drug release studies
Nystatin-loaded chitosan microparticles equivalent to (10) mg of nystatin were subjected to in-vitro drug release studies to ascertain the drug release pattern of nystatin from the prepared microparticles formulations.
Microparticles were introduced in a beaker containing (300) mL of phosphate buffer solution (pH 5.5), used as the release medium.The beaker was rotated at 100 rpm and maintained at 32±0.5°Cin a thermostat shaking water bath (15,16) .An aliquot of (5) mL was withdrawn periodically at 1, 2, 3, 4, 5, 6, 9, 12 and 15 hours and replaced with a same volume of fresh medium each time.The withdrawn samples were filtered, then diluted appropriately and analyzed with LABOMED ® UV-VIS spectrophotometer at 305 nm to determine the amount of drug released.

Drug release kinetics
To examine the drug release kinetics, the cumulative release data were fitted to models representing zero-order (Q v/s t), first-order (log (Q0-Q) v/s t) and Higuchi"s square root of time (Q v/s t 1/2 ), respectively, Where Q is the cumulative percentage of drug released at time t and (Q0-Q) is the cumulative percentage of drug remaining after time t (17) .To determine the mode of drug release, the initial 60% drug release values were fitted to the Korsmeyer-Peppas model: Mt/M∞ = Kt n , where Mt/M∞ is the fraction( ≤ 60%) of drug released at time t, K is the drug release rate constant, and n is the release exponent.The n value is employed for the characterization of the mechanism of solute release from formulation (18) .

Statistical analysis
All experiments were repeated at least three times.Results are expressed as means ± standard deviation (SD).Statistical analysis was carried out employing one-way ANOVA test.A p-value ≤ 0.05 was considered statistically significant.

Preparation of microparticles
Different formulas of nystatin microparticles were fabricated successfully using chemical cross-linking method (Table 1).The formula N1C5 has shown very high viscous dispersion that was difficult to be extruded from a 23G needle, so it was applied by a disposable syringe without needle.Chitosan has 1 primary amino and 2 free hydroxyl groups for each C6 building unit.Due to the availability of free amino groups in chitosan, it carries a positive charge and thus it reacts with many negatively charged compounds (6) .Chitosan could be covalently cross-linked with glutaraldehyde through its amino groups.The aldehyde groups of the glutaraldehyde formed covalent imine bonds with the amino groups of chitosan, due to the resonance established with the adjacent double ethylenic bonds via a Schiff reaction (19) .

Particle size analysis and morphological characteristics
Optical microscope images show that N1C1, N1C2 and N1C3 formulas have uniform spherical shaped microparticles while N1C5 microparticles were irregularly shaped and highly aggregated in nature and practically difficult to be distinguished as individual microparticles.In other hand, N1C4 showed a blend of microparticles and deformed particles (Figure .1).It was noted that as the concentration of chitosan increased, the color of microparticles changed from light yellow to dark brown.
The size distribution profiles of the microparticles are shown in Figure 2. The particle size of all formulas was ranged from 1µm to 10 µm.The results showed that particle size was affected significantly (p<0.05) by the polymer concentration, as the chitosan concentration increased a larger mean size of microparticles was obtained (Table .2).This effect may due to the increase in the viscosity of the droplets (due to the increase in concentration of polymer solution) (11) .The increase in solution viscosity led to a decrease in stirring efficiency hence result in difficulty in dispersion and subdivision of droplets and thus increased the resultant microsphere size (20) .

Determination of drug loading and entrapment efficiency
The concentration of chitosan had significant (p<0.05)effect on the drug loading and entrapment efficiency of nystatin-loaded microparticles which is reflected by the variations in the percentage of drug loading and entrapment efficiency that observed in all formulations as shown in Table 2. Better entrapment efficiency were achieved by increasing the ratio of chitosan which complies with Patel et al. who obtained the same results with Tramadol-loaded chitosan microspheres (21) .Senthil et al. revealed that entrapment efficiency was also increased with the higher polymer concentration (22) .Another study conducted by Palanisamy et al. also achieved highest drug entrapment with drug: polymer ratio of 1:3 indicating that polymer concentration has a great impact on this parameter (23) .Increase in the concentration of chitosan increases the yield of the prepared microparticles and thereby resulting in higher drug entrapment levels (24) .It was further observed that the drug entrapment was proportional to the size of the microparticles (21) .The contribution of a high polymer concentration to drug loading can be interpreted in two ways.First, the high viscosity of the polymer solution would be expected to decrease the diffusion of the drug into the external phase which would result in high entrapment efficiency (8,25) .Second, the higher polymer concentration produce large size microparticles in which the loss of drug from surface during washing process is lesser in comparing to small microparticles.Thus size of microparticles is also affecting the drug loading (26,27) .

Determination of percentage yield
It was observed that as the polymer ratio increased, the product yield percentage also significantly (p<0.05)increased (Table 2).The low percentage yield in N1C1 in relative to other formulas may be due to microparticles loss during washing process.

Fourier transforms infrared spectroscopy (FTIR)
Owing to that each specific chemical bond often has unique energy absorption; FTIR spectroscopy has been extensively applied to identify the presence of certain functional groups or chemical bonds in a molecule.Spectral data could confirm the stability of the drug during microparticles preparation and the absence of any drugpolymer interaction.FTIR spectra of pure nystatin, chitosan, blank chitosan microparticles, physical mixture of blank chitosan microparticles and nystatin and the spectrum of nystatin loaded chitosan microparticles are shown in Figure 3. Nystatin displays characteristic absorption bands at about 1702 cm −1 due to the stretching vibration of carbonyl group (28) .On the other hand, the characteristic absorption of the chitosan  (29) .Some changes can be observed after chitosan cross-linking with glutaraldehyde.The characteristic peak at 1642 cm -1 is due to Schiff base (C=N) formed by a cross linking reaction between the amino group and the aldehydic group of glutaraldehyde.Moreover, no absorption is observed at ~1715 cm -1 , related to the free aldehyde group which further confirms the cross-linking reaction (18) .FTIR spectra of nystatin loaded microparticles and blank polymer microparticles showed the same characteristic absorption peaks with the exception of characteristic band value at ~1700 cm -1 for loaded microparticles which can be assigned to the C=O group of the drug.This result clearly indicated the stability of the drug during the microparticles preparation and revealed the absence of any drug-polymer interaction.

Differential scanning calorimetry (DSC)
The thermograms of nystatin (a), chitosan (b), physical mixture of chitosan and nystatin (c), plain microparticles (d) and loaded microparticles (e) are shown in figure 4. Nystatin presented a single, well defined sharp endothermic peak at 165.5 • C which is corresponded to the melting point of nystatin.Chitosan showed initial endothermic peak at 75.15 • C which can be correlated with loss of water associated to hydrophilic groups of polymer (30,31) .Physical mixture thermogram showed an endothermic peak at 164 • C due to the presence of nystatin, which reveals the absence of interactions between the nystatin and chitosan.Thermogram of drug-loaded chitosan microparticles showed the same peaks observed in blank microparticles, but there was no peak corresponding to nystatin.This might be an indication of loss of crystallinity of nystatin presented within the prepared micoparticles (21,24,29) .

In-vitro drug release studies
The cumulative percentage of nystatin release from chitosan microparticles versus time was drawn and represented graphically (Figure 5).

Figure (5): Cumulative % of nystatin released versus time.
The in-vitro drug release plot exhibited a biphasic mode with an initial "burst" release for the different plots.Thereafter, a slow release phase was started.The initial rapid release of drug might due to fast dissolution of drug molecules attached to the surface of the microparticles while subsequent slow but steady release was related to diffusion of drug molecules present in the core of the microparticles (20) .This is in agreement with Kumar et al. who found that increasing the concentration of chitosan leads to decrease the release of Indomethacin from chitosan microparticles (29) .Khalandar et al. who prepared nasal microspheres of Sumatriptan using different concentrations of chitosan also concluded that the drug release is retarded by increasing polymer ratio (32) .The results show that an increase in polymer ratio reduced the first phase (burst phase) significantly (p< 0.05) as is evident from T 50% value (time required to release 50% of the loaded dose) as seen in table 3. Nystatin was released from microparticles in a controlled manner for about 15 hours.The particle size would be expected to influence the rate of drug release (29) .This can explain the higher initial burst release of drug from N1C1 which has a smaller particle size.The overall release of nystatin from chitosan microparticles (from N1C1 to N1C4) decreased as chitosan concentration increased, suggesting that drug release could be controlled by varying chitosan concentration.released the highest cumulative percentage of nystatin as 95.6% was released after 15hr.N1C2 did not show much variation.The remaining formulas (N1C3 and N1C4) exhibited less drug release.At the end of dissolution test , the release of nystatin was found to be incomplete in all formulas; this could be due to the relatively slow erosion of the polymeric substance under dissolution test conditions, with a consequential slow release of entrapped drug from the matrices (33) .

Drug release kinetics
The in-vitro release data were plotted according to different models, and these curves were used to draw some conclusions regarding the mode of drug release from the microparticles.The curve fitting and plotting was performed in Excel (Microsoft Software Inc., USA).Correlation coefficient (R 2 ) values were calculated for the curves obtained by the regression analysis of the plots.The model with the highest correlation coefficient was considered to be the best fitted model.As Table 3 shows, the kinetic model fits more appropriately with the Higuchi matrix model (linear nature of curve).This was confirmed by high values of regression coefficients obtained in all formulas.All other models produced curvilinear plots with lower values of regression coefficient.To find out the mechanism of drug release, the obtained drug release data were fitted in Korsmeyer-Peppas model.This equation was used to calculate the value of release point (n) which gave an additional evidence for the diffusion controlled mechanism.The values of (n) for all formulas suggested a non-Fickian (anomalous) diffusion mode of nystatin release from chitosan microparticles, for spheres, when n= 0.85 it indicates case-II (swellingcontrolled drug release).Case-II is the relaxational release drug transport mechanism that is associated with stresses and state-transition in hydrophilic glassy polymers which swell in water or biological fluids.This term also includes polymer disentanglement and erosion (33) .When n value is between 0.43 and 0.85 (0.43 < n < 0.85) it corresponds to non-Fickian (anomalous) model while if n≤ 0.43, it indicates Fickian diffusion mechanism (34) .Non-Fickian refers to a combination of both diffusion and erosion to obtain controlled dug release (10) .Typically, the diffusion process consists of an initial "burst" release of drug at or near the surface of the microsphere followed by the additional release of drug from the pores of the microsphere.Erosion occurs by hydrolysis of the polymer matrix that generate pores which expose interior pockets of Nystatin to the bathing liquid.For continuous release, the diffusion and erosion process must balance each other to allow the drug to diffuse out of the microsphere at a constant rate (35) .

Conclusion
In an attempt to formulate nystatin as sustained release microparticles by chemical cross-linking method, five different formulas were prepared and evaluated.Chitosan concentration had a substantial effect on the main parameters of microparticles (particle size, drug loading, entrapment efficiency and yield) as any increase in polymer ratio was accompanied by a similar increase in the aforementioned parameters.In vitro release studies of microparticles showed that N1C1 formula has resulted in higher release of nystatin from the loaded microparticles after 15 hours of study (95.6% release).The kinetic study demonstrated that nystatin release from the prepared microparticles is likely to follow Higuchi matrix model.From the aforementioned results, chitosan appears to be a good choice as a polymer for producing a modified release microparticles.The produced microparticles have been found to have a good potential for prolonged nystatin release and therefore can be beneficial for use in treatment of fungal infections.In the future, the optimized nystatin microspheres formula will be incorporated in a topical gel that will be evaluated for its possible utilization in the prolongation of nystatin release.