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Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

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Enhanced gene transfer with multilayered polyplexes assembled with layer-by-layer technique.

X Qu¹, P Li, D Liu

¹Shandong University, Institute of Pharmaceutics, School of Pharmaceutical Science, Ji'nan, People's Republic of China.

IET Nanobiotechnology

|August 17, 2012

Summary

A novel multilayered gene delivery system using DNA, protamine, and polyethylenimine offers enhanced DNA protection, nuclear transfer, and endosomal escape. This multifunctional vector shows lower cytotoxicity and higher transfection efficiency in vitro.

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Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

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Published on: March 1, 2013

Area of Science:

Biotechnology
Nanotechnology
Molecular Biology

Background:

Gene therapy requires efficient and safe DNA delivery vectors.
Current vectors often face challenges with DNA protection, nuclear entry, and endosomal escape.

Purpose of the Study:

To develop a novel multifunctional gene delivery system using a layer-by-layer (LbL) approach.
To enhance DNA protection, facilitate nuclear transfer, and improve endosomal escape for gene therapy.

Main Methods:

Constructed a quaternary polyplex system using DNA, protamine (Pro), and polyethylenimine (PEI) via the LbL technique.
Protamine condensed DNA and provided nuclear localization signals (NLS); additional DNA was layered, followed by a PEI outer layer for endosomal escape.
Characterized the polyplexes for surface charge, morphology, particle size distribution, and DNA protection capability.

Main Results:

The LbL technique successfully created quaternary polyplexes with high DNA loading capacity.
The novel vector demonstrated strong DNA protection, positive surface charge, spherical morphology, and narrow particle size distribution.
In vitro tests showed significantly lower cytotoxicity and higher transfection efficiency in HepG2 and HeLa cells compared to commercial PEI/DNA complexes.

Conclusions:

The developed multilayered vector is a promising multifunctional system for gene therapy.
The combination of protamine and PEI in an LbL structure enhances gene delivery efficiency and safety.
This approach offers a viable strategy for overcoming key barriers in gene delivery systems.