Efficient Cytosolic Delivery of siRNA Using Lyophilized and Reconstituted Polymer-siRNA Polyplexes

Area of Science:

• Biotechnology
• Nanomedicine
• Polymer Chemistry

Background:

• Small interfering RNA (siRNA) offers targeted gene silencing for disease treatment.
• Current siRNA delivery methods often fail due to endosomal entrapment and degradation.
• Direct cytosolic delivery is crucial for siRNA efficacy.

Purpose of the Study:

• To develop a stable formulation for direct cytosolic siRNA delivery using guanidinium functionalized poly(oxanorbornene)imide (PONI) polymers.
• To evaluate the efficacy of lyophilized PONI-Guan/siRNA polyplexes for gene knockdown and therapeutic applications.
• To demonstrate the potential of lyophilization as a translation tool for siRNA therapeutics.

Main Methods:

• Formation of PONI-Guan/siRNA polyplexes.
• Lyophilization of polyplexes and storage under various temperature conditions for 4 weeks.
• Assessment of cytosolic siRNA delivery via confocal microscopy.
• Evaluation of gene knockdown efficacy in GFP-expressing cells using flow cytometry.
• Testing therapeutic efficacy in 4T1 breast cancer cells via qRT-PCR and Alamar blue assay.

Main Results:

• Lyophilized PONI-Guan/siRNA polyplexes retained functional integrity after reconstitution.
• Effective cytosolic delivery of siRNA was achieved with reconstituted polyplexes.
• Significant STAT-3 gene knockdown (~80%) and cell growth inhibition (~70%) were observed in 4T1 cells using lyophilized polyplexes.

Conclusions:

• Lyophilization provides a stable and effective method for storing siRNA delivery systems.
• This strategy offers a transferable and versatile approach for the translation of siRNA therapeutics.
• Direct cytosolic delivery via lyophilized polyplexes enhances gene silencing efficacy.