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Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

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Silk-based gene carriers with cell membrane destabilizing peptides.

Keiji Numata¹, David L Kaplan

¹Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, United States.

Biomacromolecules

|October 15, 2010

Summary

Engineered silk proteins with cell-destabilizing peptides show high gene transfer efficiency in human cells. These silk-based carriers offer controlled release and reduced toxicity, rivaling commercial reagents.

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Area of Science:

Biomaterials Science
Gene Therapy
Nanotechnology

Background:

Developing non-viral gene delivery vectors with high efficiency and low toxicity is crucial for gene therapy.
Silk proteins offer biocompatibility, but require functionalization for effective gene delivery.
Cell membrane destabilizing peptides can enhance cellular uptake and gene transfer.

Purpose of the Study:

To engineer recombinant silk-polylysine proteins incorporating ppTG1 peptides for enhanced gene delivery.
To evaluate the transfection efficiency and characteristics of these novel silk-based gene carriers.
To investigate the role of silk secondary structure in controlling gene release kinetics.

Main Methods:

Synthesis of silk-polylysine-ppTG1 dimer recombinant proteins in Escherichia coli.
Complexation with plasmid DNA (pDNA) at various N/P ratios.
Transfection of human embryonic kidney (HEK) cells and assessment of efficiency.
Characterization of complex morphology, size, and DNAse resistance.
Enzymatic degradation studies to determine pDNA release profiles.

Main Results:

Silk-polylysine-ppTG1 dimer complexes at N/P 2 exhibited transfection efficiency comparable to Lipofectamine 2000 in HEK cells.
The complexes displayed globular morphology (99 nm diameter) with minimal β-sheet structure.
Silk-based complexes demonstrated excellent DNAse resistance and tunable pDNA release via silk-degrading enzymes, influenced by β-sheet content.

Conclusions:

Bioengineered silk-based gene delivery vehicles incorporating cell membrane destabilizing peptides show significant potential.
These systems offer a less-toxic and controlled-release alternative for gene delivery applications.
The β-sheet structure of silk can be modulated to regulate gene release profiles.