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Published on: June 16, 2023
Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma
Anup K Kundu1,2, Swathi V Iyer3, Sruti Chandra2
1Center for Nanomedicine and Drug Delivery, Xavier University College of Pharmacy, New Orleans, Louisiana, United States of America.
Objectives:
The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318-1, a murine osteosarcoma cell line.
Methods:
The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP) and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency.
Key Findings:
Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent.
Conclusions:
This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.
Insights
Researchers developed optimized lipid-based nanoparticles to deliver siRNA for silencing mutant p53 in osteosarcoma cells. The best formulation showed high gene silencing efficiency and cell viability, but stability decreased with storage.
Area of Science:
- Biomedical Engineering
- Nanotechnology
- Cancer Research
Background:
- The tumor suppressor p53 is critical in osteosarcoma development.
- Targeting mutant p53 is a potential therapeutic strategy for osteosarcoma.
Purpose of the Study:
- To develop and optimize lipid-based nanoparticles for siRNA delivery.
- To achieve effective silencing of mutant p53 in a murine osteosarcoma cell line (318-1).
Main Methods:
- Nanoparticles were formulated using cholesterol, DOTAP, and either PLGA or PLGA-PEG.
- High-pressure homogenization was used for nanoparticle preparation.
- Optimization involved varying nanoparticle composition, siRNA concentration, cell density, incubation time, and storage conditions.
Main Results:
- PLGA or PLGA-PEG incorporation reduced particle size and cytotoxicity.
- Nanoparticles with 10% PLGA demonstrated the highest mutant p53 knockdown efficiency.
- Optimal silencing was achieved with a nanoparticle to siRNA ratio of 6.8:0.66 and 75 nM siRNA, maintaining high cell viability.
- Long-term storage led to a significant decrease in gene silencing efficiency.
Conclusions:
- The developed lipid-polymer nanoparticles show promise for targeting mutant p53 in osteosarcoma.
- Further evaluation in tissue culture and animal models is warranted for potential therapeutic applications.
- Formulation stability requires further investigation for clinical translation.
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