Formulation and Evaluation of Polymeric Nanoparticles of Itraconazole for Antifungal Therapy

Mechanisms of Drug Release

The drug from the polymeric drug carriers deliver at the site of the tissue by anyone of the three general physico- chemical mechanisms which are explained below:

• By hydration which causes the swelling of the polymer nanoparticles followed by release through diffusion.
• By an enzymatic reaction that leads to rupture or degradation or cleavage of the polymer at the site of delivery and results in the release of the drug from the entrapped inner core.
• Dissociation of the drug from the polymer and it does de- adsorption/release from the swelled nanoparticles

Analytical Method Development

Determination of Absorption Maxima: Absorption maxima are the wavelength at which maximum absorption takes place. For accurate analytical work, it is important to determine the absorption maxima of the substance under study [3]. Procedure: For the preparation of calibration curve stock solution was prepared by dissolving 100 mg of accurately weighed drug in 100ml of methanol (1mg/ml). Further 1ml of the stock solution was pipette out into a 100 ml volumetric flask and volume was made up with phosphate buffer (5.5pH). From this stock solution pipette out 1ml and dilute to 10 ml with phosphate buffer and subject for UV scanning in the range of 200-400 nm using double beam UV spectrophotometer. The absorption maxima were obtained at 260 nm with a characteristic peak. Preparation of Calibration Curve: It is soluble in Methanol; hence Methanol was used for solubilizing the drug. Stock solution (1 mg/mL) of Itraconazole was prepared in Dichloromethane and subsequent working standards (2, 4, 6, 8 and 10 mg/mL) were prepared by dilution with phosphate buffer of pH-5.5. These solutions were used for the estimation Itraconazole by UV method. The whole procedure was repeated three times and average peak area was calculated. Calibration plot was drawn between concentrations and peak area. Calibration equation and R2 value are reported.

Preparation of nanoparticles

Preparation of Itraconazole loaded nanoparticles: Chitosan was dissolved in aqueous solution of acetic acid (2%) in 50 ml of distilled water. Under magnetic stirring at room temperature, tween 80 was add above solution and 10 ml of (w/v) solution was added drop wise using syringe needle into 100 ml Chitosan solution containing respective mg of Itraconazole. The stirring was continued for about 2.30 hrs [4]. The resultant nanoparticles suspensions were centrifuged at 12000 × g for 15 minutes using C24 centrifuge. Samples were washed with water and dried. The formation of the particles was a result of the interaction between the negative groups of the TPP and the positively charged amino groups of chitosan (ionic gelation) (Table 1).

Table1: Composition of nanoparticles formulations (F1 to F12).

In Vitro Release Studies

Drug release was determined by dialysis method; two ml of each formulation (test and control) were poured into dialysis bags and put into 25 ml phosphate buffer (pH 6.8) and stirred (100 rpm, room temperature). At predetermined time intervals, 2 ml of phosphate buffer was taken and then substituted by fresh phosphate buffer. Finally, the amounts of released Simvastatin in phosphate buffer were measured by spectrophotometer at 235 nm. Aliquots withdrawn were assayed at each time interval for the drug released at λ max of 235 nm using UV‐Visible [5].

Spectrophotometer by keeping phosphate buffer pH 6.8 as blank and the amount of released drug was estimated by the standard curve.

Calibration Plot of Itraconazole Phosphate Buffer of Ph -5.5

A standard graph of Itraconazole in phosphate buffer of pH-5.5 was plotted using Absorbance and concentration as shown in Tables 2-5 and Figures 1 & 2. Equation for linearity curve and R2 were calculated as Y=0.076X+0.007 and R2 =0.999. Itraconazole showed maximum absorbance in phosphate buffer (pH 5.5) at 260 nm. The solution obeyed Beer-Lambert’s law for concentration range of 2 to 10μg/ mL with regression coefficient of 0.999. Standard curve of prepared Itraconazole in phosphate buffer pH 5.5 is shown below [6].

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Characterization of Nanoparticles

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SEM studies showed that the Itraconazole - loaded nanoparticles had a spherical shape with a smooth surface as shown in Figure 3 & 4.

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Release Kinetics

To analyze the drug release mechanism the in vitro release was fitted into various release equations and kinetic models first order, zero order, Higuchi and Korsmeyer- peppas. The release kinetics of optimized formulation F6 (Chitosan) is shown in Table 6 and in following Figures 5-8.

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The prepared F6 optimised Chitosan nanoparticles were subjected to the drug release kinetics and release mechanism [7, 8, 9]. The formulations were studied by fitting the drug release time profile with the various equations such as Zero order, First order, Higuchi and Korsmeyer pappas. The optimised formulation F6 optimised Chitosan a Nanoparticle was analyzed for the drug release mechanism. The best correlation coefficient value (0.983) indicates the best release mechanism (First order).

FTIR

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Infrared studies were carried out to confirm the compatibility between the polymer, drug, and selected excipients. From the spectra it was observed that there was no major shifting, as well as, no loss of functional peaks between the spectra of the drug and drug-loaded nanoparticles. This indicated no interaction between the drug and other excipients [10].