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Updated: Sep 7, 2025
Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform
Published on: February 25, 2021
Multifunctional Lipid Nanoparticles for Protein Kinase N3 shRNA Delivery and Prostate Cancer Therapy
Ji Wang1, Yanhao Zhang1, Chao Liu1
1School of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu 214122, PR China.
Abstract:
Protein kinase N3 (PKN3), by virtue of its abnormal expression in prostate cells, has been widely used as a target of RNAi (shRNA, siRNA, miRNA) therapy. The major challenges of PKN3 RNAi therapy lie in how to design effective interference sequences and delivery systems. Herein, new PKN3 shRNA sequences (shPKN3-2459 and shPKN3-3357) were designed, and bioreducible, biodegradable, ionizable lipid-based nanoparticles were developed for shPKN3 delivery. First, an ionizable lipid (DDA-SS-DMA) bridged with disulfide bond and ester bonds was synthesized by a three-step reaction and confirmed by MS, 1H NMR, and 13C NMR. The ionizable lipid was mixed with cholesterol, DSPC, PEG-lipid, and shPKN3 by a microfluidic mixer to prepare lipid nanoparticles (LNP-shPKN3) which were characterized by DLS and TEM. Afterward, the pH and glutathione (GSH)-responsiveness of the DDA-SS-DMA based LNP delivery system were investigated by lysosome escape and gel electrophoresis assays. Compared with the commercial transfection reagent Lipo2000, the DDA-SS-DMA based delivery system showed higher transfection efficiency and lower toxicity. Western blot analysis, invasion tests, and migration assays were performed to evaluate the silencing effect of shPKN3 in vitro. In in vivo studies, high tumor suppression (65.8%) and treatment safety were evident in the LNP-shPKN3-2459 treatment group. Taken together, the DDA-SS-DMA based delivery system encapsulating shPKN3-2459 showed significant antitumor efficacy and might be a promising formulation for the treatment of prostate cancer.
Insights
New lipid nanoparticles effectively deliver shRNA targeting PKN3 for prostate cancer therapy, demonstrating significant tumor suppression and safety in vivo.
Area of Science:
- Biotechnology
- Nanomedicine
- Oncology
Background:
- Abnormal expression of Protein kinase N3 (PKN3) in prostate cells makes it a target for RNA interference (RNAi) therapy.
- Challenges in PKN3 RNAi therapy include designing effective interference sequences and delivery systems.
Purpose of the Study:
- To design novel PKN3 shRNA sequences (shPKN3-2459 and shPKN3-3357).
- To develop bioreducible, biodegradable, ionizable lipid-based nanoparticles for efficient shPKN3 delivery.
- To evaluate the antitumor efficacy and safety of the developed delivery system for prostate cancer treatment.
Main Methods:
- Synthesis and characterization of a novel ionizable lipid (DDA-SS-DMA) using MS, 1H NMR, and 13C NMR.
- Preparation and characterization (DLS, TEM) of lipid nanoparticles (LNP-shPKN3) encapsulating shPKN3.
- Assessment of nanoparticle pH and glutathione (GSH)-responsiveness, transfection efficiency, toxicity, in vitro gene silencing, invasion, and migration assays.
- In vivo studies to evaluate tumor suppression and treatment safety.
Main Results:
- The DDA-SS-DMA based delivery system exhibited higher transfection efficiency and lower toxicity compared to Lipo2000.
- In vitro studies confirmed the gene silencing effect of shPKN3.
- In vivo studies showed significant tumor suppression (65.8%) and good safety in the LNP-shPKN3-2459 treatment group.
Conclusions:
- The developed DDA-SS-DMA based ionizable lipid nanoparticles are effective for shPKN3 delivery.
- The LNP-shPKN3-2459 formulation demonstrated significant antitumor efficacy in vivo.
- This delivery system represents a promising therapeutic strategy for prostate cancer treatment.
