1: Formulating PESP: Encapsulating Purified Compound in Submicron Particles:
The process for formulating Polymer Encapsulated Submicron Particles (PESP) has been detailed in Indian patent file no: 544591. Briefly, Polyvinyl alcohol (PVA) was utilized as the primary component. PVA powder (Loba-Chemie) with a molecular weight of 115,000 and a polymerization degree below 2000 was dispersed in distilled water at 70°C. The mixture was stirred at 650 rpm for 1.5 hours to obtain a 5% clear, homogeneous aqueous solution. The purified compound, bis(isothiocyanato methyl)benzene, was extracted from Moringa oleifera leaf extract [8], serving as the drug for enhancing bioavailability and retention in targeted tissues.
The formulation employed a modified binary non-solvent conservation method, using the Flory-Huggins Interaction parameter to assess drug-polymer compatibility. The PVA solution was dispersed into the drug solution in predetermined volumetric ratios (Figure 1).
These formulations were tested on HeLa cell lines to determine the optimal ratio, which was found to be 7:3 (v/v) for the purified compound to polymer solution. Maleic acid (10% by weight) was added, with the mixture heated to 120°C to form a cross-linked polymer.
Chloroform and acetone were chosen as non-solvents based on their interaction parameters and hydrogen bonding capabilities. Adding the drug-loaded polymeric solution to chloroform initiated the formation of fine particles. Acetone, due to its dual miscibility with alcohol and strong non-solvent properties, further compacted the polymeric structure, minimizing particle size. The resultant PESP was filtered through a 0.2µm membrane and lyophilized under reduced pressure for four hours.
2: Morphology and Physio-chemical characterization of PESP through SEM, TEM, FTIR and UV-Vis spectroscopic analysis:
High-resolution field emission Scanning Electron Microscopy (S-4800, HI-9155-0005) and Transmission Electron Microscopy (JEOL JEM 2100 HR) were used for the morphological observation of the delivery system. The newly formed PESP was dispersed in double-distilled water, with drops of the dispersion air-dried on a glass slide and carbon coated copper grid. The Micro-photographs provided detailed morphological observations of PESP [9]. Additionally, FTIR spectroscopy was conducted to analyze the chemical composition of the PESP, with observations across wavelengths ranging from 4500 cm⁻¹ to 500 cm⁻¹.
3: Evaluating Drug Loading Efficiency and Release:
The effect of pH on the newly formed PESP was evaluated in buffer solutions at acidic, alkaline, and neutral pH levels. The drug-loaded PESP was incubated at 37°C to simulate human body conditions. Methanol was added to prevent polymer dissolution. Samples were centrifuged, collected at specific intervals, and analyzed using UV-spectroscopy [10,11]. The concentration of the purified compound (PC) released from the PESP was determined using a standard calibration curve with isothiocyanate methyl benzene. This pH-dependent release study quantified the PC loading efficiency using the following formula [12].
4: Cancer cell culture
Authenticated MCF-7 (epithelial breast cancer), MDA-MB-231 (mesenchymal breast cancer), and MC3T3-E1 (non-cancerous osteoblast) cell lines were purchased from NCCS Pune, India. MCF-7 and MDA-MB-231 were used to evaluate anti-metastatic activity, with MC3T3-E1 serving as a control in the soft agar assay. The cells were cultured per standard protocols, with the growth medium supplemented with 10% FBS and 2% L-Glutamine-Penicillin-Streptomycin, maintained at 37°C in a humidified CO2 incubator [8].
5: Isolation and Culture of PBMCs:
Peripheral blood was collected from healthy donors of any gender, with their consent and ethical approval for the same was taken from the Institutional Bioethics Committee, Bioequivalence Study Center, Jadavpur University. A modified density gradient separation method was used. Blood (3 mL) was collected in an EDTA vial and was diluted 1:3 with chilled saline followed by layered over 2.5 mL of Lymph Prep density gradient solution. After centrifugation at 5000 rpm for 30 minutes, the mononuclear cell layer was aspirated, washed it in PBS twice, and then resuspended in complete media (RPMI-1640, FBS, and L-Glutamine-Penicillin-Streptomycin). The cells were maintained at 37°C in a humidified incubator with 5% CO2 [13,14].
6: PBMC Proliferation and Growth Kinetic:
Once the cells reached confluence, PBMCs were plated in 12-well plates with serial dilutions ranging from 1x104 to 0.1x104cells per well, each with 1.5 mL of complete media. After 24 hours, an MTT assay was conducted to measure absorbance at 590 nm, which was used to create a standard calibration curve.
In a separate experiment, PBMCs were seeded at 0.2x104cells per well in 12-well plates. At predefined intervals (24 to 120 hours), cells were harvested and analyzed for viability using the MTT assay. Cell numbers were determined from the standard calibration curve, and proliferative activity and cellular growth kinetics were assessed over time. Population doubling time (PDT) was calculated using cell counts from a hemocytometer, with T and T0 representing the times of cell harvesting and seeding, respectively, and N and N0 denoting the cell counts at these times [15]
7: Cell viability and cytotoxicity assay:
Cell viability and cytotoxicity were assessed in MCF-7, MDA-MB-231, and PBMCs by treating them with PC-loaded PESP and performing an MTT assay. The IC50 value and treatment dose were evaluated, comparing cancer cells to PBMCs. MTT (Thiazol Blue Tetrazolium Bromide, Sigma-Aldrich) was added, and absorbance was measured at 590 nm after 3.5 hours. Viable cell percentages were calculated against a blank.
8: Cell based adhesion assay:
12-well plates were coated with 500 µL of diluted gelatin (250 µg/mL) and incubated at 37°C for 1.5 hours to allow polymerization. Excess gelatin was removed, and the plates were washed with PBS. The polymerized gelatin surfaces were then treated with a diluted fibronectin solution (2 µg/mL in PBS). MCF-7 and MDA-MB-231 cells were seeded onto the gelatin-fibronectin (GF) coated plates at 10x104 cells per well along with 1 mL of complete media. After 6 hours, the media was removed, and non-adherent cells were washed off. MTT assay was performed to determine the percentage of cell adhesion, calculated using the formula-
Cell numbers were determined using a standard curve based on OD values. The same procedure was applied to the treatment group after 24 hours of PC-loaded PESP administration to assess cell adhesion, cell death, and anti-metastatic activity. Additionally, MCF-7 and MDA-MB-231 cells were cultured and analyzed for proliferation up to 120 hours [16].
In separate 25 mm² dishes, cells adhering to gelatin-fibronectin coated coverslips were fixed with chilled ethanol, washed with FBS in PBS (1:1, v/v), stained with DAPI (HI media), and incubated in the dark at 4°C for 10 minutes. Observations were made using a fluorescence microscope with a DAPI filter at 360 nm [17,18].
9: Soft agar colony forming assay:
The soft agar colony formation assay is a well-established method for assessing the anchorage-independent growth of cells, which is indicative of tumor malignancy. The procedure was modified as follows [18]:
- Plate Preparation: Six-well plates and a 25 mm² dish were labeled as
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Control
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Treatment With
Purified compound (PC)
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Treatment With PC-loaded-PESP
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MCF-7
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MDA-MB-231
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MC3T3-E1 (non cancerous osteoblast cell line). It was taken as another control for inspecting non cancerous cell growth formation (or not). It was cultured in 25mm2 single dish as per appropriate protocol but no treatment was given to it.
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- Bottom Layer Production: A mixture of 2 mL complete media and 2 mL 0.7% agarose solution was prepared. After thorough mixing, 0.5 mL of this mixture was pipetted into each well and dish, allowed to cool, and solidified at room temperature.
- Top Layer Production: Cells were harvested and resuspended in 1 mL of 0.3% agarose solution (liquid at 25°C ± 2°C) at a 1:1 ratio. 0.5 mL of this cell-agarose mixture was layered on top of the solidified bottom layer in the wells. The plates were then allowed to solidify at 4°C. 800 µL of complete media was added on top to prevent agar drying, and the plates were incubated at 37°C in a humidified incubator with 95% air and 5% CO2, with media replenished as needed.
After 18 days of incubation, the MC3T3-E1, MCF-7, and MDA-MB-231 cells from the control plate were fixed with chilled ethanol, stained with Giemsa, and colony formation was observed and counted under a light microscope with microphotographs taken. On the same day the cells were treated appropriately as per mention in the well plate and incubated for 24h. On the 19th day, cells from the treatment groups were similarly fixed, stained, and analyzed. The experiment was repeated three times, and the average number of colonies from the replicates was used for analysis.
10: Flow Cytometric investigation for Cellular Death:
Flow cytometry dot plot analysis was performed using a BD FACS Aria flow cytometer to detect treatment-induced cellular apoptosis and necrosis. The Annexin V/PI apoptosis kit (Biovision Inc; K101-25) was used according to its protocol. Adherent control and treatment-induced cells were harvested, washed with ice-cold PBS containing 2% FBS, and fixed with 80% chilled ethanol while vortexing, followed by 30 minutes of incubation [19]. After staining with Annexin V/PI, cells were incubated in the dark for 20 minutes. Fluorescence emission was analyzed at 530 nm and 575 nm with 488 nm excitation.
11: Immunoblotting scrutinized pathways for cellular death:
Caspase3 Immunoblotting assay was executed using ELISA kit (Elabscience; cat no: E-EL-H0017). It was employed for the purpose of exploring the positive pathways for cellular death. Sandwich model ELISA was performed as per provided protocol.
12: 3D Molecular Docking of LR-simulation:
In-silico Ligand-Receptor (LR) interactions were conducted to assess complex stability and reactivity using UCSF Chimera, Biovia Discovery Studio, Ligplot+, and PubChem Sketcher V2.4. Structural data from the Protein Data Bank, including unique PDB IDs, was processed. Ligand structures were sketched in PubChem, and molecular docking was performed with UCSF Chimera and Autodock Vina, visualized using Biovia Discovery Studio 2017R2 [20]. Standard inhibitors, 17β-estradiol for the human estrogen α receptor (1ERE) and 4-anilinoquinazoline for the human epidermal growth factor receptor (1M17), were used for validation.
13: Data archiving & analysis
All experiments were carried out three times and the data were documented in MS-Excel 2007. One way ANOVA between control and treated groups was performed using statistical software origin 6.1. Data was reported either by line diagram or by bar with mean ± Standard Deviation (SD). The significant difference having value p<0.05 was calculated at 95% confidence interval level and represented alphabetically.