# Formulation, characterization and cytotoxicity assessment of a novel bee venom microsphere for the treatment of prostate cancer

### Collection of BV

BV was isolated from healthy workers of the honey bee Apis mellifera (L.) according to Ref.20 with the electrostimulation method. Briefly, the stun gun (Model VC-6F from Apitronic Services, 9611, No. 4 Road, Richmond, BC, Canada) comprises a frame with wire electrodes installed parallel to one another. The frames were mounted on or under the hive and then connected to an electrostimulator. The electrical impulses stimulated the worker bees to sting through latex placed on a glass plate. We carefully transferred the glass slide to the lab, where the venom was dried at ambient temperature. Then we used a sharp scraper to scrape off the dry poison. Fresh bee venom was then stored in dark glass tubes at a temperature of -4 °C until use. 1 mg of BV was diluted in 1 ml of distilled water to make a stock solution of the venom. Centrifugation (15,000G, 5 min) was performed at 25 °C post-peak. The supernatant was filtered with a 0.2 membrane filter and stored in the dark at −40°C.

Three formulations of BV-loaded crosslinked chitosan microspheres with different BV:polymer ratios were prepared (Table 1). First, chitosan was added to 1% aqueous glacial acetic acid with continuous stirring overnight with a magnetic stirrer. BV was then added to the prepared solution with mixing. The resulting mixture was then injected into liquid paraffin containing Span 80 using a syringe with mechanical agitation for 30 min. to form a W/O emulsion. Glutaraldehyde (5%) was then added dropwise and the mixture was left for 7 hours to allow crosslinking21. The formed microspheres were collected by centrifugation, washed with acetone and finally dried at 50°C in a hot air oven.

The coating process was performed using the solvent evaporation method with ES 100. The microspheres produced were initially dispersed in an ES 100 solution containing ethanol and acetone. Then it was poured into a mixture of Span 80 and liquid paraffin followed by stirring for 3 h at room temperature. The mixture was then filtered, washed with n-hexane and finally freeze-dried overnight19.

#### Percent Yield (%)

BV-loaded, crosslinked, chitosan-coated microspheres are weighed and percent yield calculated using the following equation19:

$$% {text{Yield}} = actual;weight;of;product times 100/{text{total}};{text{weight}};{text{of} };{text{excipients}};{text{and}};{text{drugs}}$$

The test was performed in triplicate and the results are presented as percent yield mean ± standard deviation (n=3).

### Containment Efficiency (%EE)

Calculation of the amount of BV entrapped in the coated microspheres produced. Phosphate-buffered saline (PBS), pH 7.4, was added to a known amount of BV-loaded, cross-linked, chitosan-coated microspheres. The resulting mixture was stirred vigorously with a mechanical stirrer for 24 hours. Then it was centrifuged and the supernatant collected to determine the BV content. Finally, the amount of BV was successfully measured spectrophotometrically at ʎMax595 using the Bradford protein assay method22. Bovine serum albumin (BSA) was used as a protein concentration standard. The entrapment efficiency is calculated using the following equation19:

(% {text{EE}} = {text{Practical}};{text{Drug}};{text{Content}} times 100/{text{Theoretical}}; {text{drug}};{text{content}}.)

EE% was performed in triplicate, data presented as mean ± standard deviation (n=3).

### degree of swelling

Place a weighed amount of different BV-loaded, cross-linked chitosan-coated microspheres into enzyme-free simulated intestinal fluid, pH 7.4 and leave in the dissolution apparatus at 37°C ± 0.5°C until swelling. Then the treated microspheres were dried between filter paper and then weighed. The change in weight continues to be measured until equilibrium is reached. The following equation is used to calculate the swell ratio19:

$$mathbf{S}mathbf{R}=frac{mathrm{Wg}-mathrm{Wo}}{mathrm{Wo}},$$

where SR is the swelling ratio, Where is the initial weight, Wg is the final weight.

The test was performed in triplicate and the results are presented as mean ± standard deviation (n=3).

### Scanning Electron Microscopy (SEM)

SEM (JSM 5300, JOEL, Japan) was used to demonstrate the morphological structure of the prepared crosslinked chitosan-coated microspheres. First, the microspheres were coated with gold using a sputter coater and then dried using a single vacuum ion beam based system. i-scan 2000 computer software was used for imaging by SEM23.

### In vitro drug release study

An accurately weighed amount of BV-loaded, crosslinked chitosan-coated microspheres from each formulation was placed in tea bags and immersed in a pH-progressive medium at 37°C ± 0.5°C, 100 rpm. The study was conducted using a paddle type dissolution tester. The tying of the tea bags was aided by the cord paddle. Gastrointestinal transit conditions can be simulated by changing the pH of the dissolution medium at different time intervals. The pH of the dissolution medium was maintained at 1.2 with 0.1 N HCl for 2 h. By adjusting the pH to 7.4, the release study was observed and continued for a further 3 hours. Thereafter the pH was adjusted to pH 6.8 and continued for 24 h19. Finally, samples were removed from the dissolution medium at different time intervals and the drug release rate was measured effectively spectrophotometrically at ʎmax595 using the Bradford protein assay method. Each formula was estimated in triplicate and the results are presented as mean ± standard deviation (n=3).

### kinetic study

The in vitro release data were fitted to first-order, zero-order kinetics and Higuchi equations, and to the general exponential function: Mt/M= ctnwhere mt /Mrepresents solute release relative to equilibrium conditions; the diffusion exponent (n) is the characteristic of the release mechanism and k is used for drug and polymer properties23.

### In vitro cytotoxic effects of free BV, BV-loaded cross-linked chitosan-coated microspheres and doxorubicin

#### cell culture

Human prostate adenocarcinoma (PC3) was used as the cancer cell line while oral epithelial cells (OEC) were used as the normal cell line throughout this study. Both cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM sodium pyruvate and penicillin/streptomycin (100 U/ml). Cell lines were maintained at 37°C and 5% CO2 .

### MTT assay

Cell viability was assessed using the MTT reduction assay to determine the effects of free BV and BV-loaded cross-linked chitosan-coated microspheres and doxorubicin as a positive control on PC3 and OEC cells. Briefly, cells (1×10 5 cells/ml) were seeded in 96-well microtiter plates (Nunc-Denmark) at a concentration of 1×10 5 cells/ml (100 μl/well) and incubated until a complete monolayer formed developed . After the cell monolayer was formed, the growth medium was decanted and the cells were treated with (1.93, 3.87, 7.75, 15.5, 31 and 62 µg/ml) BV & doxorubicin and 100 mg/ml BV loaded cross-linked chitosan-coated microspheres in a volume of 100 μl/well. The control was added to an equal volume of saline. The plates were incubated at 37°C and 5% CO22 atmospheric conditions for 24 h. After the media was removed, the plates were washed with phosphate buffered saline (PBS) and the cells were treated with 50 µl/well of (3-(4,5-dimethylthiazol-2-yl)-2,5-ditetrazolium bromide (MTT ) solution for 4 h, then DMSO solution was added as 0.05 ml/well. Finally, the absorbance of each well at 570 nm wavelength was measured using an ELIZA reader.

The viability percentage was calculated as follows:

$$mathrm{viability ; %}=frac{mathrm{mean value ; OD; Handles }}{mathrm{ mean ; OD; Control}}times 100,$$

where OD is the optical density.

The IC50is the concentration of tested material required to inhibit 50% cell growth and the value was calculated using an online tool24.

### Morphological Analysis

PC3 cancer cells were seeded in 12-well plates with RPMI-1640 supplemented with 10% fetal calf serum (FCS) at a density of 5 x 10 5 cells/well and incubated for 24 h. Then the media was removed and the cells were treated with (1.93, 3.87, 7.75, 15.5, 31 and 62 µg/ml) BV & doxorubicin and 100 mg/ml BV-loaded, cross-linked, treated with chitosan-coated microbeads and incubated for 24 h. Thereafter, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet at room temperature, destained with 33% acetic acid. Morphological changes in treated cells were observed and compared to untreated cells using an inverted phase contrast microscope (Helmut Hund GmbH, Wetzlar, Germany).

### Detection of apoptosis by flow cytometric assay

To investigate the mode of cell death induced by the tested formula (BV-loaded, cross-linked, chitosan-coated microspheres) in PC3 cells, flow cytometric analysis was performed using the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences ) Protocol performed according to the manufacturer’s specifications. PC3 cells were treated with an IC50 concentration of BV-loaded, cross-linked, chitosan-coated microspheres and incubated for 24 h. The treated and untreated control cells were trypsinized and peeled off, centrifuged (1000G5 min, 24 °C), washed with cold PBS and centrifuged (1000G, 5 minutes, 24 °C). Then 5 µl of annexin-V-FITC and 5 µl of propidium iodide were added and kept in the dark for 15 minutes. Finally, the samples were analyzed with a flow cytometer (CyFlow SL, Partec-Germany) at 488 nm to quantify the proportion of living, dead, apoptotic and necrotic cells. Navios software (Beckman Coulter) was used to analyze flow cytometry data. Experiments were performed independently in triplicate.

### Statistical analysis

Examinations were performed in triplicate and data were presented as mean ± standard deviation using Sigma Plot 12.5 and Microsoft Office 365. Student’s t-test was used to analyze the difference between the experimental group and the control group in the flow cytometry assay. P