Characterization of chitosan microsphere

2.4.1. Percentage yield of spray dried samples.

The yield of the process was determined by weighing the spray dried particles using Precisa analytical balance (405M-200A Swiss quality Switzerland) by using following equation

𝑃𝑜𝑤𝑑𝑒𝑟 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑒𝑑

𝑇𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑎𝑛𝑑 𝑐ℎ𝑖𝑡𝑜𝑠𝑎𝑛 X 100---Equation. 1 2.4.2. Drug Content determination

The powder collected contains drug and polymer was dissolved in specified amount of distilled water to make suspension. 1ml of suspension was centrifuged. Acetonitrile and acetic acid was added to pellets to dissolve the drug and chitosan and then injected 0.5ml into HPLC. The concentration was calculated from AUC by using standard calibration curve of furazolidone.

2.4.3. Particle Size Analysis

Particle size was determined by Izon qNano particle sizer (Izon Science Ltd. NewZealand) by using tris buffer. Reference calibration particles (CPC) were selected on the basis of expected size of formulation particles and then suspended in tris buffer provided by Izon company which was filtered through syringe by using syringe filter 0.2μm and put into Izon qnano by using micro pipette. By maintaining the steady stable current flow on screen CPC particles were replaced with particulate formulations in the same tris buffer and particles size was measured in terms of current block that is proportional to the diameter of particle size. Two

55 different nanopores (1000 and 2000) were selected for each formulation for the measurement of microparticles. However, for mucoadhesive liposomes nanopores 100, 200 and 400 nm were used for each formulation to get more reliable data. For mucopenetration liposomes calibration particles CPC 500 and nanopore 400 nm were used. Selection of nanopores for each formula was based on the expected size and particle size distribution.

2.4.4. Muco-adsorption of spray dried microparticles

For mucin adsorption calorimetric method was used in which periodic acid and Schiff reagent were used for the determination of remaining free mucin after its adsorption on the chitosan micro-particles. 0.1 g of Sodium metabisulphate was added to every 6ml of Schiff reagent and incubated at 37 °C till it turned into pale yellow color. 10µl of 50% periodic acid was added to 7 ml of 7% acetic acid to make periodic acid reagent. Periodic acid reagent (0.2ml) was added to sample and incubated for 2 hours followed by addition of 0.2ml of Schiff reagent room temperature and kept it for 30 minutes. Absorbance was measured at 555nm by UV spectrophotometer (Talaei et al., 2011).

Mucin solution was prepared and 2 ml of micro-particle suspensions (F1 to F3) con- taining different amount of chitosan and crosslinking agent were centrifuged. Super- natant was discarded and pellets were dispersed into standard mucin solution sep- arately and vortexed for 5 minutes and analyzed for free mucin concertation at pre- determined time intervals (1.5, 3, 4.5 and 6 hours). Separate Eppendorf with mucin and micro-particles formulation was used for each time interval. The dispersion was centrifuged at 4000 rpm for 5minutes, supernatant was used for the measurement of free mucin by using the method stated above.

56 The concentration of free mucin was determined by using standard calibration curve of mucin in section 2.2.2. (Type-I with bound salicylic acid 9-17% from sigma Al- drich).

2.4.5. In-vitro drug release

Modified dispersion method stated by (Shazly et al., 2008) was used to perform in vitro dissolution. Release was determined for all formulations (spray dried) (0, 0.5, 1, 1.5, 2, 3, 4 and 5 hours) at pH 1.3 and pH 4.5. However, in vitro release of freeze dried microparticles was determined at pH 4.5 only. In this method spray dried mi- croparticles equal to 2 mg of drug was centrifuged at 4000 rpm for 5min. However, in case of freeze dried microparticles 2ml of suspension was used that contained different amount of drug corresponding to each formulation. Supernatant was dis- carded and pellets were transferred to new vials without disturbing the yellow col- ored drug at the bottom. Pellets were washed with water three times and suspended in 5ml of SGF at desired pH. After specified time 0.5ml of sample was withdrawn from and replaced by equal volume of fresh SGF which was filtered by 0.2 µm filter and then analyzed by HPLC analysis.

2.4.6. Particle morphology

Scanning electron photomicrographs of all microspheres were taken by microscope (Hitachi S3000N, Hitachi High-Technologies UK-Electron Microscopes, Wokingham Berkshire, UK). Small amount of each sample was attached to a 15mmdiameter aluminum specimen stub using double sided carbon adhesive tabs (Mikrostik adhe- sive, Agar Scientific), and the powder samples were sputter-coated with a thin layer of gold/palladium mixture to allow them to be electrically conductive. This was car- ried out using a Quorum Technology (Polaron range) SC760, whereby the samples are exposed to argon atmosphere at 10 Pa. The samples are coated at a process current of 18–20mAfor 2 × 105 s,with a turning through 180° in between.

57 2.5. Preparation of liposomes.

Liposomes were prepared by thin film hydration method using different ratio of cholesterol. Accurately weighed phosphatidylcholine, cholesterol (table 2.3) and cremophore ELP as co-surfactant were dissolved in 8 ml of chloroform. However, furazolidone (table. 2.3) was dissolved in 3ml of acetonitrile. After dissolving, chloroform-acetonitrile mixture containing drug and other constituents was transferred in 100 round bottom flask and organic solvents were evaporated by rotary evaporator (Buchi RE 121 Switzerland) at 60°C. After evaporation thin film on inner surface of flask was flushed with nitrogen gas for 10 min to remove the traces of organic solvent followed by rehydration of film by 5 ml tris buffer pH 7.4 at 53°C for half an hour.

Table 2.3. Composition of mucoadhesive liposomal formulations

Formulation Drug (mg) Lipid: Cholesterol (weight/mg)

L1 4 106:53 L2 5 106:53 L3 8 106:53 L4 4 106:10.6 L5 5 106:10.6 L6 8 106:10.6

2.5.1. Preparation of Mucoadhesive liposomes

Liposomes were prepared by conventional film hydration method previous section with coumarin-6 as fluorescent dye instead of furazolidone and then coated by chitosan for mucoadhesion. Equal volumes of liposomal suspension and 0.6% w/v solution of chitosan in 0.1% v/v glacial acetic acid were mixed at rate of 1 ml per

58 minute by continuous stirring at 25°C as shown in table 2.4. Resulting suspension was kept in a refrigerator overnight.

Table 2.4. Composition of fluorescence labelled liposomes Formulation Composition

Coumarin-6: Lipid: Cholesterol

Mucoadhesive liposomes

Chitosan

LC1 2.5 µg:26.5mg: 2.5mg 0.6% (W/V)

NLC1 2.5 µg:26.5mg: 2.5mg 0

LC1: Liposomes containing coumarin-6 with chitosan; NLC1: Liposomes containing coumarin-6

without chitosan.