Materials & Methodology

Venlafaxine HCl was received as a gift sample from Dr.Reddys Lab, Hyderabad; Chitosan powder was gifted from India Sea Foods, Cochin, Kerala; Sodium alginate procured from Fluka Chem, Buchs from Sigma, USA; Lactic acid from Merck Limited, Mumbai; and all other chemicals and solvents were of analytical grade satisfying pharmacopoeia specifications.

Preparation of chitosan coated alginate beads:

Sodium alginate was dissolved in distilled water at various concentrations ranging from 1% to 4% (w/v), as indicated in Table 1. Venlafaxine hydrochloride was pre-dissolved in double distilled water. The solution was slowly added to the sodium alginate solution while maintaining constant stirring. To eliminate any air bubbles that might have formed during mixing, the solution was subjected to sonication for 30 minutes. The gelation medium was prepared by thoroughly mixing equal proportions of calcium chloride solution (0.5% to 2% w/v) with different concentrations of chitosan solution (0.5% to 2%). The chitosan solution had previously been prepared with 2.4% lactic acid, and the pH of the medium was adjusted to 4.5 ± 0.1. The mixture was stirred continuously for 2 hours and kept at room temperature (25ºC) until further use. Using a 5 ml hypodermic syringe equipped with a #21 needle, the homogenous mixture of sodium alginate drug solution was added drop-wise into the gelation medium. The addition was performed under constant stirring at room temperature (25ºC). The beads formed in the gelation medium were left to cure for 4 hours. Subsequently, they were washed with distilled water to remove any excess gelation medium. The beads were then air-dried at room temperature (25ºC) in a dust-free chamber until they reached a constant weight. The prepared beads were carefully stored in an airtight container at room temperature (25ºC) to ensure their stability and integrity for further analysis and evaluation 6.

IR Spectral and DSC Study

To investigate and predict any physiochemical interaction between different excipients, I.R spectroscopy was utilized. Spectra matching approach was employed to detect potential chemical interactions between the drugs and polymer. A physical mixture consisting of the drug, polymer, and other excipients was prepared. The mixture was then combined with an appropriate quantity of potassium bromide. This mixture was compressed under 15 tons of pressure using a hydraulic press to form a transparent pellet. The resulting pellet was scanned in the range of 4000 to 400 cm-1 using an FTIR spectrophotometer (FTIR 8400 S, Shimadzu). The obtained IR spectrum of the physical mixture was compared with those of the pure drug and polymers. Peak matching analysis was performed to identify any appearance or disappearance of peaks, indicating potential chemical interactions between the components as well as other excipients 7. The thermal behaviors of the pure drug(s), polymer(s), physical mixture of drug(s) and polymer(s), as well as the drug-loaded beads, were studied using a Pyris Diamond TG/DTA model no. instrument from PerkinElmer, Singapore. The analysis was carried out in a nitrogen atmosphere with a flow rate of 150 ml/min and a platinum crucible was used, and alpha alumina powder served as the reference material. Thermograms were recorded with a scanning speed of 10ºC/min, covering a temperature range from 30ºC to 300ºC.

X-Ray Powder Diffractometry [X-RD] Analysis

In order to investigate the physical state of the drug within the formulations, X-ray diffraction patterns of the pure drug, polymer(s), physical mixture of drug and polymer(s), blank microspheres, and drug-loaded microspheres were recorded using a Miniflex goniometer operating at a scanning speed of 1º/min. The analysis covered a 2θ angle range of 10-70 8.

Surface Morphology Investigations and Particle Size Analyses

Particle size analysis of the microspheres was conducted using optical microscopy. The instrument was calibrated, with one unit of the eyepiece micrometer equivalent to 17.5 μm. Approximately 50 microspheres were measured for each sample to determine their sizes. The shape and surface morphology of the drug-loaded microspheres were examined using Scanning Electron Microscopy (SEM) with Vega © Tescan, USA 9.

Determination of Drug loading and Entrapment efficiency

For each batch, a total of fifty milligrams of venlafaxine hydrochloride loaded chitosan-coated alginate beads were placed in 100 ml of distilled water. The mixture was allowed to stand for 24 hours with intermittent shaking to ensure complete dissolution of the drug from the beads. Following the dissolution period, the solution was filtered through Whatman filter paper to remove any undissolved particles or impurities. An aliquot of the filtered solution, appropriately diluted, was subjected to spectrophotometric analysis using a UV-Visible spectrophotometer (Shimadzu 1800, Japan). The spectrophotometric assay was conducted at a wavelength of 274 nm, which corresponds to the specific absorption peak of venlafaxine hydrochloride. The absorbance of the aliquot was measured and recorded, allowing for the quantification of the released drug concentration.

Drug Loading was calculated using the formula in Equation 1 10.

Drug Loading in % = W/W_t x 100 …..…… (Equation 1)

Where,

W = Drug content of the beads

W_t = Weight of the beads

Theoretical drug loading was determined by calculation assuming that the entire drug present in the polymer solution gets entrapped in beads and no loss occurs at any stage of preparation of the beads. The unloaded beads did not interfere with the spectrophotometric determination of drugs, which was checked before performing the drug loading studies.

Entrapment efficiency was determined by using the following relationship:

$\begin{array}{l}Entrapment Efficiency=\\ (Experimental Drug Content/\\ Theoretical Drug Content)\\ \times 100\end{array}$ ... (Eqn 2)

Invitro Drug Release and Its Release Behavior Studies

In vitro drug release studies were conducted using drug-loaded beads weighing 100 mg from each batch. The studies were performed using a USP XXI dissolution rate test apparatus, in 350 ml of double distilled water at a temperature of 37 ± 0.5ºC. The apparatus was set to operate at a stirring speed of 50 rpm.At predetermined time intervals, 3 ml of the dissolution medium was sampled, and an equal volume of fresh dissolution medium was replenished to maintain a constant volume. The collected samples were filtered through Whatman No. 1 qualitative filter paper and analyzed for drug content at a wavelength of 274 nm using a Shimadzu 1800 UV spectrophotometer from Japan.Statistical analysis was carried out using GraphPad Prism 5 software. Each parameter was repeated three times (n=3) to ensure the reliability and reproducibility of the results. To analyze the release kinetics of the drug, data obtained from in vitro drug release studies were fitted into three different kinetic models: zero order, first order 11, and Higuchi's model 12, 13. The zero-order model involved plotting the cumulative amount of drug released against time. The first-order model involved plotting the logarithm of the cumulative percentage of drug remaining against time. Higuchi's model involved plotting the cumulative percentage of drug released against the square root of time. By fitting the release data to these kinetic models, valuable insights into the release behavior and kinetics of the drug from the formulation were obtained.

Stability Studies

The prepared microspheres were packed securely in aluminum foil and subjected to stability studies for a period of three months, following the guidelines provided by the International Council for Harmonisation (ICH). Stability testing was conducted at two different temperature and relative humidity (RH) conditions 14:

• 25ºC and 65% RH

• 40ºC and 75% RH

To maintain the desired humidity levels, a saturated solution of sodium chloride was used. By subjecting the microspheres to these temperature and humidity conditions, their stability and potential changes over time were evaluated in accordance with ICH guidelines.