ABSTRACT
Title
Non-viral HPMA-oligolysine copolymers for improved siRNA delivery to neuron-like cells
Authors
G. Carbonari1, R. N. Johnson2, S. H. Pun2, S. Spampinato1
1 Dept of Pharmacology, University of Bologna, Bologna, Italy
2 Dept of Bioengineering, University of Washington, Seattle, WA, USA
1 Dept of Pharmacology, University of Bologna, Bologna, Italy
2 Dept of Bioengineering, University of Washington, Seattle, WA, USA
Abstract
Gene therapy can be defined as the treatment of human disease by the transfer of genetic material into specific cells. The first step is to identify the target gene; then it is necessary to choose the vehicle to get to the target cells. The lack of safe and efficient gene-delivery methods is one of the limiting obstacles to gene therapy.Recently different synthetic vectors have been designed for gene deliveryand they became a good alternative to viral vectors: synthetic agents, although safer than recombinant viruses, do not possess the required efficacy.
Polymer-based gene carriers are synthesized starting from materials that interact electrostatically with nucleic acids: when aqueous solutions of nucleic acids and cationic polymers are mixed they interact to form nanoparticles called polyplexes. Using an excess of cationic polymer it is possible to obtain particles with a positive surface charge. However, in delivering nucleic acids to the nucleus, polyplexes must overcome a series of obstacles, both extracellular and intracellular: to improve delivery efficiency bioactive peptides, as DNA-binding, endosomal release, and cell targeting peptides, can be integrated into synthetic gene carriers.
The aim of this research is to develop multifunctional, peptide-based polymers that incorporate motifs to condense DNA and facilitate sequential trafficking steps to neuron-like cells: of particular interest is the combination of a synthetic monomer, N-(2-hydroxypropyl)methacrylamide (HPMA), with peptides that enable DNA condensation. Previous studies have combined a cationic material, poly-L-lysine, with HPMA: the modified polyplexes had enhanced serum stability in vitro, but elevated cytotoxic effect [Oupicky D. et al., 2002]. In a recent study copolymerization of cationic oligolysine peptides (K12) with HPMA results in narrowly dispersed and highly controlled molecular weight and compositioncopolymers [Johnson RN et al., 2010]. HPMA-K12 was first tested for its ability to form polyplexes: it condensed effectively plasmid DNA into small particles, of less than 200 nm in diameter, both in water and in PBS.
In our study copolymers were tested for their ability to deliver plasmid DNA and siRNA to logarithmically growing or NGF-differentiated PC-12 cells: this is a particularly suitable model for our study because a 6-days exposure to NGF causes terminal differentiation to neuron-like cells. HPMA-K12 polymers demonstrated plasmid DNA delivery efficiencies in undifferentiated PC-12 cells that were comparable to polyethylenimine; on the contrary, the transfection efficiency in 2- and 6-days differentiated cells is reduced up to two order of magnitude. It has been shown that loss of transfection efficiency in differentiated cells is not related to their sensitivity to the copolymers: in fact differentiated PC-12 cells resulted to be more resistant to HPMA-K12 polymers than their undifferentiated counterpart.
In the second part of the study HPMA-K12 polymers have been tested for their ability to deliver a GAPDH-specific siRNA to PC-12 cells, and residual GAPDH enzymatic activity and mRNA levels have been evaluated. While in undifferentiated cells any alteration of GAPDH activity was observed, in 6-days differentiated cells HPMA-K12-mediated transfection of siGAPDH 100 nM caused about 50% reduction of the enzymatic activity, even in presence of serum. Real Time PCR analysis revealed that GAPDH mRNA levels were reduced up to 40% 18 hours post-transfection.
The synthesized copolymers were shown to possess important properties for gene delivery applications, including efficient DNA binding and condensation, the ability to stabilize particles and efficient delivery of plasmid and siRNA. With the growing understanding of gene-delivery mechanisms, polymer-based gene delivery could become an important tool for human gene therapy.
Oupicky et al. (2002). Molecular Therapy.4, 463-72
Johnson et al. (2010). Biomacromolecules. Epub ahead of print
Polymer-based gene carriers are synthesized starting from materials that interact electrostatically with nucleic acids: when aqueous solutions of nucleic acids and cationic polymers are mixed they interact to form nanoparticles called polyplexes. Using an excess of cationic polymer it is possible to obtain particles with a positive surface charge. However, in delivering nucleic acids to the nucleus, polyplexes must overcome a series of obstacles, both extracellular and intracellular: to improve delivery efficiency bioactive peptides, as DNA-binding, endosomal release, and cell targeting peptides, can be integrated into synthetic gene carriers.
The aim of this research is to develop multifunctional, peptide-based polymers that incorporate motifs to condense DNA and facilitate sequential trafficking steps to neuron-like cells: of particular interest is the combination of a synthetic monomer, N-(2-hydroxypropyl)methacrylamide (HPMA), with peptides that enable DNA condensation. Previous studies have combined a cationic material, poly-L-lysine, with HPMA: the modified polyplexes had enhanced serum stability in vitro, but elevated cytotoxic effect [Oupicky D. et al., 2002]. In a recent study copolymerization of cationic oligolysine peptides (K12) with HPMA results in narrowly dispersed and highly controlled molecular weight and compositioncopolymers [Johnson RN et al., 2010]. HPMA-K12 was first tested for its ability to form polyplexes: it condensed effectively plasmid DNA into small particles, of less than 200 nm in diameter, both in water and in PBS.
In our study copolymers were tested for their ability to deliver plasmid DNA and siRNA to logarithmically growing or NGF-differentiated PC-12 cells: this is a particularly suitable model for our study because a 6-days exposure to NGF causes terminal differentiation to neuron-like cells. HPMA-K12 polymers demonstrated plasmid DNA delivery efficiencies in undifferentiated PC-12 cells that were comparable to polyethylenimine; on the contrary, the transfection efficiency in 2- and 6-days differentiated cells is reduced up to two order of magnitude. It has been shown that loss of transfection efficiency in differentiated cells is not related to their sensitivity to the copolymers: in fact differentiated PC-12 cells resulted to be more resistant to HPMA-K12 polymers than their undifferentiated counterpart.
In the second part of the study HPMA-K12 polymers have been tested for their ability to deliver a GAPDH-specific siRNA to PC-12 cells, and residual GAPDH enzymatic activity and mRNA levels have been evaluated. While in undifferentiated cells any alteration of GAPDH activity was observed, in 6-days differentiated cells HPMA-K12-mediated transfection of siGAPDH 100 nM caused about 50% reduction of the enzymatic activity, even in presence of serum. Real Time PCR analysis revealed that GAPDH mRNA levels were reduced up to 40% 18 hours post-transfection.
The synthesized copolymers were shown to possess important properties for gene delivery applications, including efficient DNA binding and condensation, the ability to stabilize particles and efficient delivery of plasmid and siRNA. With the growing understanding of gene-delivery mechanisms, polymer-based gene delivery could become an important tool for human gene therapy.
Oupicky et al. (2002). Molecular Therapy.4, 463-72
Johnson et al. (2010). Biomacromolecules. Epub ahead of print