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Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery
Published on: May 2, 2019
Stable and efficient transfection of siRNA for mutated KRAS silencing using novel hybrid nanoparticles
A Lakshmikuttyamma1, Y Sun, B Lu
1Department of Pharmaceutical Sciences, School of Pharmacy, and ‡Division of Molecular Radiation Biology, Department of Radiation Oncology, Thomas Jefferson University , Philadelphia, Pennsylvania 19107, United States.
Abstract:
siRNA is currently the most widely studied form of RNAi, and it has promising therapeutic potential not just in cancer but also in other diseases such as autoimmune and infectious diseases. However, efficient delivery of siRNA to target cells is being limited by lack of an effective delivery system that ensures efficient transfection into cells while protecting the encapsulated siRNA from nuclease. We hypothesized that a hybrid nanoparticle system composed of human IgG and poloxamer-188, a stealth polymer, will efficiently deliver mutated KRAS siRNA to A549 cells, leading to an efficient knockdown of mutated siRNA while protecting the siRNA from serum nuclease. We also hypothesized that the nanoparticles will not elicit an immunostimulatory effect in murine macrophages and also avoid clearance by macrophages. These nanoparticles were found to efficiently deliver siRNA to the cytoplasm and nuclease of A549 cells in a controlled and sustained manner while avoiding recycling by endosomes. An effective knockdown of mutated KRAS was achieved, which subsequently led to an increased sensitivity to erlotinib. These nanoparticles successfully avoided uptake by murine macrophages and reduced immune responses normally associated with siRNA/nanoparticle therapy. These results demonstrate that the novel hybrid nanoparticles could potentially serve as a platform for efficient delivery of siRNA to cells for stable gene knockdown.
Insights
This study introduces a novel hybrid nanoparticle system for delivering small interfering RNA (siRNA) effectively to cancer cells. The nanoparticles protect siRNA from degradation and reduce immune responses, enabling stable gene knockdown for potential therapeutic applications.
Area of Science:
- Biotechnology
- Nanomedicine
- Molecular Biology
Background:
- Small interfering RNA (siRNA) holds therapeutic promise for various diseases, including cancer.
- Efficient delivery and protection of siRNA from nucleases remain significant challenges in its application.
- Current delivery systems often face limitations in cellular transfection and immune evasion.
Purpose of the Study:
- To develop and evaluate a hybrid nanoparticle system for efficient delivery of mutated KRAS siRNA to A549 cells.
- To assess the nanoparticle's ability to protect siRNA from serum nucleases and ensure targeted delivery.
- To investigate the nanoparticles' immunomodulatory effects and macrophage interaction for therapeutic safety.
Main Methods:
- Formulation of a hybrid nanoparticle system using human IgG and poloxamer-188.
- Transfection of A549 cells with mutated KRAS siRNA encapsulated in the nanoparticles.
- Assessment of siRNA delivery, endosomal escape, and gene knockdown efficacy.
- Evaluation of nanoparticle interaction with murine macrophages and associated immune responses.
Main Results:
- The hybrid nanoparticles demonstrated efficient, sustained, and controlled delivery of siRNA to the cytoplasm of A549 cells, bypassing endosomal recycling.
- Effective knockdown of mutated KRAS was achieved, leading to increased sensitivity to erlotinib.
- Nanoparticles successfully avoided uptake by murine macrophages and mitigated associated immune responses.
Conclusions:
- The novel hybrid nanoparticle system shows potential as an effective platform for stable siRNA delivery and gene knockdown.
- This system offers a promising approach for overcoming current limitations in siRNA-based therapeutics.
- The nanoparticles exhibit favorable biocompatibility and immune evasion properties for clinical translation.
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