Formulation, characterization and cellular toxicity assessment of a novel bee-venom microsphere in prostate cancer treatment

Collection of BV

BV was collected from healthy workers of the honey bee, Apis mellifera (L.) according to Ref.20 using the electro-stimulation method. Briefly, the electric shock device (VC-6F model from Apitronic Services, 9611, No. 4 Road, Richmond, BC, Canada) comprises a frame with wire electrodes installed in parallel to each other. The frames were mounted on the top or under the hive and then connected to an electro-stimulator. The electrical impulses stimulated the bee workers to sting through latex, which was placed on a glass plate. We transferred the glass plate carefully to the laboratory, in which the venom was dried at an ambient temperature. Then, we used a sharp scraper to scrape off the dry venom. After that, fresh bee venom was stored in dark glass tubes at a temperature of – 4 °C until needed. 1 mg of BV was diluted in 1 mL of distilled water to prepare a stock solution of the venom. Centrifugation (15,000g, 5 min.) was conducted at 25 °C after vertex. The supernatant was filtered using a 0.2 membrane filter and kept at − 40 °C in the dark.

Three formulations of BV loaded cross-linked chitosan microspheres with different BV: polymer ratios were prepared (Table 1). Firstly, chitosan was added to 1% aqueous glacial acetic acid with continuous stirring overnight by a magnetic stirrer. Then BV was added to the prepared solution while mixing. The formed mixture was injected after that into liquid paraffin containing span 80 using a syringe with mechanical stirring for 30 min. to form w/o emulsion. Glutaraldehyde (5%) was then added dropwise and the mixture was left for 7 h to allow cross-linking21. 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 solvent evaporation method with ES 100. The prepared microspheres were initially dispersed in ES 100 solution containing ethanol and acetone. Then it was poured in a mixture of span 80 and liquid paraffin with subsequent agitation for 3 h at room temperature. The mixture was then filtered, washed with n-hexane, and finally overnight freeze-dried19.

Percentage yield (%)

BV loaded cross-linked chitosan-coated microspheres are weighed and the % yield is calculated using the following equation19:

$$% {text{Yield}} = Actual;weight;of;the;product times 100/{text{Total}};{text{weight}};{text {of}};{text{excipient}};{text{and}};{text{drug}}$$

The test was done in triplicate and the results are represented as percentage yield mean ± SD (n = 3).

Entrapment efficiency (% EE)

To calculate the amount of BV entrapped inside the prepared coated microspheres. Phosphate buffer saline (PBS) pH 7.4 was added to a known amount of BV loaded cross-linked chitosan-coated microspheres. The formed mixture was vigorously stirred with a mechanical stirrer for 24 h. Centrifugation was then applied and the supernatant was collected to determine BV content. Finally, the amount of BV was successfully measured spectrophotometrically at ʎmax595 using 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 carried out in triplicate, data were represented as mean ± SD (n = 3).

Degree of swelling

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

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

where SR is the swelling ratio, Wo is the initial weight, Wg is the Final weight.

The test was done in triplicate and the results are represented as mean ± SD (n = 3).

Scanning electron microscopy (SEM)

SEM (JSM 5300, JOEL, Japan) was used to detect the morphological structure of the prepared cross-linked chitosan-coated microspheres. Firstly, the microspheres were coated using a sputter coater with gold and then dried using an ion beam-based system with a single vacuum. For imaging by SEM, computer i-scan 2000 software was used23.

In vitro drug release study

An accurately weighed amount of BV loaded cross-linked chitosan-coated microspheres from each formulation were placed in tea bags and immersed in a pH progressive media of 37 °C ± 0.5 °C, 100 rpm. The study was done using dissolution test apparatus paddle type. The tea bag tying has been assisted by the stringed 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 another 3 h. After that, the pH was adjusted to pH 6.8 and continued for 24 h19. Finally, the samples were taken from the dissolution medium at different time intervals and the drug release rate was effectively measured spectrophotometrically at ʎmax595 using Bradford protein assay method. Each formula was estimated in triplicate and the results are represented as mean ± SD (n = 3).

Kinetic study

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

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

Cell culture

Human Prostate adenocarcinoma (PC3) has been used as a cancer cell line while oral epithelial cells (OEC) was used as a normal cell line during this investigation. 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 test to determine the effects of free BV and BV loaded cross-linked chitosan-coated microspheres as well as doxorubicin as positive control on PC3 and OEC cells. In brief, cells (1 x 105 cells/mL) were seeded in 96 well micro-titer plates (Nunc-Denmark) at a concentration of 1 x 105 cells/mL (100 µL/well) and incubated until a complete monolayer sheet developed . After the monolayer sheet of cells was formed, the growth media was decanted and the cells were treated with (1.93, 3.87, 7.75, 15.5, 31, and 62 µg/mL) of both BV & doxorubicin and 100 mg/mL of BV loaded cross-linked chitosan coated microspheres in the volume of 100 μL ⁄well. The control was added to saline of equal volume. Plates were incubated at 37 °C and 5% CO2 atmospheric conditions for 24 h. After, that the media were removed, plates were washed with phosphate-buffered saline (PBS), and the cells were incubated 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 was measured at 570 nm wavelength using an ELIZA reader.

The viability percent was calculated as follows:

$$mathrm{Viability ; %}=frac{mathrm{Mean ; OD; Treated }}{mathrm{ Mean ; OD ; Control}}times 100,$$

where, OD is optical density.

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

Morphological analysis

Cancer PC3 cells were seeded in 12-well plates containing RPMI-1640 supplemented with 10% fetal calf serum (FCS) at a density of 5 × 105 cells/well and incubated for 24 h. Then the media were removed and the cells were treated with (1.93, 3.87, 7.75, 15.5, 31, and 62 µg/mL) of both BV & doxorubicin and 100 mg/mL of BV loaded cross-linked chitosan coated microspheres and incubated for 24 h. After that, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet at room temperature, decolorized 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

In order to examine the type of cell death induced by 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) according to the manufacturer’s protocol. PC3 cells were treated with IC50 concentration of BV loaded cross-linked chitosan coated microspheres and incubated for 24 h. The treated and untreated cells as control were trypsinized and pelleted down, centrifuged (1000g5 min, 24 °C), washed with cold PBS, and centrifuged (1000g, 5 min, 24 °C). Then, 5 µL of Annexin-V-FITC and 5 µL of propidium iodide were added and maintained in the dark for15 minutes. Finally, the samples were analyzed using a flow cytometer (CyFlow SL, Partec-Germany) at 488 nm to quantify the proportion of live, dead, apoptotic and necrotic cells. The Navios software (Beckman Coulter) was used to analyze flow cytometry data. Experiments were performed independently in triplicates.

Statistical analysis

The examinations were performed in three replicates and the data were represented 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 experiment group and the control group in the flow cytometry assay. P < 0.05 was considered to indicate a statistically significant difference.