Formulation, characterization and evaluation of the cellular toxicity of a new bee venom microsphere in the treatment of prostate cancer


BV collection

BV was collected from healthy workers of the honey bee, Apis mellifera (L.) according to ref.20 using the method of electro-stimulation. Briefly, the electric shock device (model VC-6F from Apitronic Services, 9611, No. 4 Road, Richmond, BC, Canada) comprises a frame with electrode wires installed parallel to each other. The frames were mounted on top or under the hive and then connected to an electro-stimulator. The electrical pulses stimulated the bee workers to sting through the latex, which was placed on a glass plate. We carefully transferred the glass plate to the laboratory, where the venom was dried at room 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 used. 1mg of BV was diluted in 1ml of distilled water to prepare a stock solution of the venom. Centrifugation (15,000g, 5 min.) was performed at 25°C after the vertex. The supernatant was filtered using a 0.2 membrane filter and kept at -40°C in the dark.

Preparation of BV loaded cross-linked chitosan microspheres

Three BV-loaded cross-linked chitosan microsphere formulations with different BV:polymer ratios were prepared (Table 1). First, 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 mixture formed was then injected into liquid paraffin containing span 80 using a syringe with mechanical stirring for 30 min. to form a w/o emulsion. Glutaraldehyde (5%) was then added dropwise and the mixture was left for 7 h to allow crosslinking21. The microspheres formed were collected by centrifugation, washed with acetone and finally dried at 50° C. in a hot air oven.

Table 1 Composition of various cross-linked chitosan microspheres loaded with BV.

Coating of BV-loaded cross-linked chitosan microspheres

The coating process was carried out using the solvent evaporation method with ES 100. The prepared microspheres 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 with subsequent stirring for 3 h at room temperature. The mixture was then filtered, washed with n-hexane and finally lyophilized overnight.19.

Characterization of the microsphere coated with cross-linked BV-loaded chitosan

Percent yield (%)

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

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

The assay was performed in triplicate and the results are represented as mean percent yield ± SD (n=3).

Trapping efficiency (%EE)

To calculate the amount of BV trapped inside the prepared coated microspheres. Phosphate buffered saline (PBS) pH 7.4 was added to a known amount of cross-linked chitosan-coated microspheres loaded with BV. The mixture formed was stirred vigorously with a mechanical stirrer for 24 h. Centrifugation was then applied and the supernatant was collected to determine the BV content. Finally, the amount of BV was successfully measured by spectrophotometry at ʎmaximum595 using the Bradford protein assay method22. Bovine serum albumin (BSA) was used as a protein concentration standard. The trapping efficiency is calculated using the following equation19:

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

%EE was performed in triplicate, data were represented as mean ± SD (n = 3).

Degree of swelling

Place the weighed amount of different BV-loaded cross-linked chitosan-coated microspheres in simulated intestinal fluid without enzyme pH 7.4, leave until swollen in the dissolving apparatus at 37°C ± 0.5°C. Then, the treated microspheres were dried between filter paper and then weighed. Weight change is always measured until equilibrium is reached. The following equation is used to calculate the swelling rate19:

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

where SR is the swelling rate, Wo is the initial weight, Wg is the final weight.

The assay was performed in triplicate and 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 prepared microspheres coated with cross-linked chitosan. First, the microspheres were coated with gold using a spray coater and then dried using an ion beam system with a single vacuum. For SEM imaging, i-scan 2000 computer software was used23.

In vitro drug release study

A precisely weighed amount of BV-loaded cross-linked chitosan-coated microspheres of each formulation were placed in tea bags and immersed in a gradual pH medium of 37°C ± 0.5°C, 100 rpm. The study was carried out using a paddle-type dissolution tester. Tying the tea bags was assisted by the string 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, samples were taken from the dissolution medium at different time intervals and the drug release rate was effectively measured spectrophotometrically at ʎmax595 using the Bradford protein assay method. Each formula was estimated in triplicate and the results are represented as the mean ± SD (n = 3).

Kinetic study

In vitro release data were fitted to first-order, zero-order kinetics, and Higuchi equations, as well as the general exponential function: Myou/M=ktnotwhere Myou /Mrepresents solute release relative to equilibrium conditions; the diffusion exponent (n) is the characteristics of the release mechanism and k is used for the properties of the drug and the polymer23.

In vitro cytotoxic effect of free BV, cross-linked chitosan coated microspheres loaded with BV 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 during this investigation. Both cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM I-glutamine, 1 mM sodium pyruvate and penicillin/streptomycin (100 U/mL). Cell lines were maintained at 37°C and 5% CO2 .

MTT test

Cell viability was assessed using the MTT reduction assay to determine the effects of cross-linked chitosan-coated microspheres loaded with BV and BV as well as doxorubicin as a positive control on PC3 and OEC cells. Briefly, cells (1 × 105 cells/mL) were seeded into 96-well microtiter plates (Nunc-Denmark) at a concentration of 1 × 105 cells/mL (100 µL/well) and incubated until what a full monolayer sheet develops. . After formation of the cell monolayer sheet, 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 and doxorubicin and 100 mg/mL of BV loaded microspheres coated with cross-linked chitosan in a volume of 100 μL⁄well. The control was added to a saline solution of equal volume. Plates were incubated at 37°C and 5% CO2 atmospheric conditions for 24 hours. After removing the medium, the 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 at 0.05 ml/well Finally, the absorbance of each well was measured at a wavelength of 570 nm using an ELIZA reader.

Percent viability was calculated as follows:

$$mathrm{Viability; %}=frac{mathrm{Average; OD; Processed }}{mathrm{ Average; DO; Control}}times 100,$$

where OD is the optical density.

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

Morphological analysis

PC3 cancer cells were seeded in 12-well plates containing RPMI-1640 supplemented with 10% fetal bovine 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 BV and doxorubicin and 100 mg/mL of microspheres coated with cross-linked chitosan loaded with BV and incubated for 24 h. After that, the 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 cytometry test

In order to examine the type of cell death induced by the tested formula (cross-linked chitosan-coated microspheres loaded with BV) in PC3 cells, flow cytometry analysis was performed using the Annexin Apoptosis Detection Kit V-FITC I (BD Biosciences) according to manufacturer’s instructions. protocol. PC3 cells were treated with an IC50 concentration of BV-loaded cross-linked chitosan-coated microspheres and incubated for 24 h. Treated and untreated control cells were trypsinized and pelleted, 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 kept in the dark for 15 minutes. Finally, the samples were analyzed using 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 analyzes

Examinations were performed in three replicates and data was plotted 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 group experimental and the control group in the flow cytometry test. P


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