Materials
Chitosan (LMW with degree of deacetylation 75–85%) (BDH, England), Thioglycolic Acid (TGA) 99%, 5,5-dithiobis (2-nitrobenzoic acid) or Ellman’s reagent, 1-ethyl-3-(3- Dimethylaminopropyl) Carbodiimide (EDAC), hydroxyl- amine, sodium hydroxide, disodium di hydrogen phosphate, sodium dihydrogen phosphate, and dialysis membrane (MW 12–14 kD) were purchased from Sigma-Aldrich, Germany. PDMS moulds were imported from micro point Technology, Singapore, acetic acid (Riedel-de Haën, Germany), acetonitrile (Merck, Germany), ethanol (BDH, England). Raloxifene was received as a gift from Vaylor Pharmaceutical Industry, Pakistan.
Methods
Synthesis of thiolated chitosan: To synthesize thiolated chitosan 1 gm chitosan was dissolved in 100 ml 0.5% acetic acid solution, and then 1 gm TGA was added in it on stirring. After that EDAC was dissolved in it and and pH was adjusted to 5 using HCl 1 M solution. Continue stirring for 3 hrs, finally the mixture was dialyzed for three days at 10°C. The purified thiolated chitosan was lyophilized and stored at 4°C [11].
Fabrication of micro needle patch: Three formulations containing mixture of 1%, 2% and 3% thiolated chitosan solution and Raloxifene (3 mg dissolved in methanol) were prepared and loaded on micromoles (Micro point Technology, Singapore) using casting method to develop micro needle patches. Afterward, filled mould was dried at room temperature for 24 hrs and dried patches were removed from mould by using adhesive backing tape.
Characterization of micro needle patch
SEM studies: Differential scanning was performed by using Scanning Electron Microscope (SEM) in which the dimensional analysis of micro needle patches was performed i.e height of needle, length and width of patch and needle pitch etc [12]. In this procedure the micro needle patch was attached on copper grid that was coated with carbon and a drop of ammonium molbydate solution was poured on it [13].
FTIR analysis: Fourier transform infrared studies were performed using FTIR (Agilant technology) to check the compatibility studies of thiolated chitosan, Raloxifene and their micro needle patch [14]. In this study spectras of all the ingredients and in patch form were studied and compared.
Moisture contents: The moisture contents of micro needle patches were determined by using moisture analyser (Sartorious, Germany) by following the reported method [15].
Thickness: The thickness of patches was observed by using vernier caliper. The average thickness of patch should be between 50–250 µm. Patch thickness was observed from all the four sides and from center and average of all should be mentioned [16].
Tensile strength: Tensile strength of micro needle patches was observed by using auto tensile tester (Shimatzu). The patch was hold in the jaws of tensile tester and force was applied until patch torn in to two pieces. The displayed force on tester would be noted as a result [17].
The formula to calculate tensile strength is as below:
Tensile strength = Load at failure x 100 /Patch thickness x Patch width
Percent elongation: Percent elongation of patch was also measured by tensile tester. Patch was placed in the jaws of tester and force was applied. The increase in length of patch after applying load was noted and results were calculated by comparing it with initial length [18, 19].
Formula for result calculation is:
Percent Elongation = Final Length of Patch/Initial length x 100 Penetration study: Skin penetration study of micro needle patch
was performed by using para film by following reported method [20]. The Para film was folded into eight folds. The para film was placed on smooth surface and micro needle patch was placed over it, then force was applied manually for 30 seconds. Rhodamine B was used over the surface; it will help to clarify the holes formed by the needles of patch and also any needle damaged on patch after penetration [21]. Finally the patch was removed and both patch and the film were observed microscopically for piercing and any damaged tip of needles [22].
The percentage compression was calculated using formula:
%compression = Hbc-Hac/Hbc x 100.
In-Vitro drug release study: In-vitro drug release study was performed by using Franz Diffusion cell. The micro needle patch with para film was placed between sealed chambers of the cell (donor and recipient) [23, 24]. The compartment was filled with buffer solution with pH 7.4, sample was drawn at predetermined intervals for
48 hrs and analyzed by HPLC method [25]. Comparative study was performed by Raloxifene ointment as control.
HPLC method validation: Quantitative study was performed using HPLC (Shimadzu). The reported method was first validated using linear equation. The C-18 column (5 µ × 4.6 mm × 150 mm) was used for the analysis, and the mobile phase comprised of Acetonitrile (ACN)/Trifluoroacetic Acid (TFA) (70:30) with a flow rate 1.2 mL/min. The run time was set at 10 min with injection volume 20
µL. The absorbance was measured at 214 nm using UV-visible detector [26].