Poster Y1 - Development of microvesicle-based novel drug depot

Introduction: Pharmaceutical research depends significantly on drug delivery system optimization to maximize therapeutic efficacy and minimize side effects. A promising path in this approach is the use of microvesicles-small, lipid bilayer-bound structures released by cells as carriers for drugs. Doxorubicin (Doxo) is one of the most effective chemotherapy drugs available and has a high probability of curing a variety of cancers. Researchers have been exploring novel ways to improve the delivery of doxorubicin. Bovine serum albumin (BSA) is well-known for improving drug stability and solubility and has also been shown to have a high affinity for doxorubicin. Using microvesicles encapsulated BSA-Doxo aggregates, we expect to create a unique drug delivery system that enhances therapeutic results. Microvesicles (MVs) were first isolated from RAW 264.7 cells and then encapsulated with BSA-Doxo. Thioflavin-T (ThT) and dynamic light scattering (DLS) were used to characterize the resulting formulation. Our initial studies reveal that the formulations induced significant cytotoxicity against lung cancer cells. The formulation will be further optimized and characterized, and additional studies on apoptosis, oxidative stress, and mechanisms of action will be carried out.

Materials and

Methods: All chemicals used were purchased from Fisher Scientific or Sigma-Aldrich. The RAW 264.7 and A549 GFP cell lines were currently cultured in the lab and used for the studies. Microvesicles were isolated from RAW 264.7 cells. A procedure consisting of continuous centrifugation steps was used to separate MVs from 1.8 mL of conditioned medium. The concentration of MVs were measured by BCA protein assay. A combination of 2 mg/mL Doxo and 40 mg/mL BSA was prepared. Next BSA was gently mixed with MVs isolated from RAW cells. Then 0.2% saponin was added to each formulation, with and without Doxo, and the reaction was allowed to progress for 20 minutes. The formulations were then subjected to purification using centrifugation at 15,000 × g for 30 minutes at 4°C, followed by 2 hours incubation at 37°C. The size of the formulations was determined using dynamic light scattering (DLS). Thioflavin-T (ThT) fluorescence and the release of Doxo were measured by spectroscopy at 440/482 nm and 470/595 nm, respectively. A549 GFP lung cancer cells were cultured in 96-well plate at a density of 10,000 cells per well to determine the toxicity of the cells. Following incubation for 24 hours, the cells were treated with doxorubicin alone and in combination with the prepared formulation with and without doxorubicin. Cell viability was measured using alamarBlue assays after a 48-hour incubation time. One-way ANOVA was used to determine statistical significance between the formulations: * p < 0.05, or ** p < 0.01, or *** p < 0.001.

Results, Conclusions, and Discussions:

Results:
The size of the BSA-Doxo-loaded MVs was found to be 388.67±25.33 nm, as determined by the dynamic light scattering (DLS) analysis.
Thioflavin-T (ThT) fluorescence measurements at 15 min and following a 2-hour incubation period at 37°C were used to confirm BSA-Doxo nanoaggregation formation. The findings obtained indicate that both the formulations with and without Doxo exhibited significant ThT fluorescence (Figure 1).
Further, initial studies indicate the MVs formulation causes significant cytotoxicity in lung cancer cells compared to the drug alone at a concentration of 1 µg/ml, as shown in Figure 2.
Conclusion/

Discussion:
We have formulated and characterized MV encapsulated BSA-Doxo nanoaggregates. Our initial studies show MV encapsulated BSA-Doxo aggregates induce significant toxicity compared to doxorubicin alone. Additional research is currently underway, including optimization of the formulation, detail characterization, apoptosis assay, and mechanism of action.
Improving therapeutic results in cancer treatment using MVs as carriers for BSA-Doxo drug delivery is a promising approach.

Acknowledgements (Optional): We would like to thank NIH R15GM135766 for funding and the University of Michigan-Dearborn Department of Mechanical Engineering for Funding.