Hexadecylated linear PEI self-assembled nanostructures as efficient vectors for neuronal gene delivery

Area of Science:

• Biotechnology and Biomedical Engineering
• Gene Therapy and Drug Delivery
• Polymer Chemistry

Background:

• Efficient and safe nucleic acid carriers are crucial for advancing gene therapy.
• Linear polyethylenimine (lPEI) is a common gene delivery vector, but its efficiency and safety can be improved.
• Developing novel materials for self-assembled nanoparticles (SN) is key to overcoming current gene delivery challenges.

Purpose of the Study:

• To synthesize and characterize novel HD-lPEI polymers for self-assembled nanoparticle formation.
• To evaluate the transfection efficiency, gene knockdown capability, and cytotoxicity of these nanoparticles in vitro.
• To assess the in vivo gene delivery potential and neuronal delivery applicability of the optimized formulation.

Main Methods:

• Synthesis of 3-(hexadecyloxy)-1-chloropropan-2-ol (HD) linker and grafting onto linear polyethylenimine (lPEI) to create HD-lPEI polymers.
• Characterization of HD-lPEI polymers using 1H-NMR spectrometry to determine the extent of HD grafting.
• Formation of self-assembled nanoparticles (SN) and complexation with plasmid DNA (pDNA) and siRNA; evaluation of size, zeta potential, transfection efficiency, gene knockdown, and cytotoxicity in MCF-7 cells and in vivo studies in Balb/c mice.

Main Results:

• HD-lPEI polymers formed self-assembled nanoparticles (SN) with varying degrees of HD substitution.
• SN of HD-lPEI-3 (ca. 15% substitution) exhibited the highest in vitro transfection efficiency (~91%) with minimal cytotoxicity.
• HD-lPEI-3/pDNA/siRNA complexes achieved ~80% GFP gene knockdown in vitro, significantly higher than Lipofectamine™ (~48%).
• In vivo studies in Balb/c mice demonstrated enhanced luciferase gene expression, and safe stereotaxic delivery of FITC-labeled siRNA against α-synuclein gene.
• HD-lPEI-3 SN showed significant potential for both general gene delivery and targeted neuronal siRNA delivery.

Conclusions:

• The developed HD-lPEI polymers effectively form self-assembled nanoparticles suitable for gene delivery.
• HD-lPEI-3 nanoparticles represent a highly efficient and safe non-viral vector for nucleic acid delivery, outperforming commercial reagents in vitro.
• The study highlights the potential of HD-lPEI-3 SN for in vivo gene delivery and specifically for neuronal siRNA delivery, paving the way for future therapeutic applications.