Chitosan-plasmid nanoparticle formulations for IM and SC delivery of recombinant FGF-2 and PDGF-BB or generation of antibodies.
Growth factor therapy is an emerging treatment modality that enhances tissue vascularization, promotes healing and regeneration and can treat a variety of inflammatory diseases. Both recombinant human growth factor proteins and their gene therapy are in human clinical trials to heal chronic wounds. As platelet-derived growth factor-bb (PDGF-BB) and fibroblast growth factor-2 (FGF-2) are known to induce chemotaxis, proliferation, differentiation, and matrix synthesis, we investigated a non-viral means for gene delivery of these factors using the cationic polysaccharide chitosan. Chitosan is a polymer of glucosamine and N-acetyl-glucosamine, in which the percentage of the residues that are glucosamine is called the degree of deacetylation (DDA). The purpose of this study was to express PDGF-BB and FGF-2 genes in mice using chitosan-plasmid DNA nanoparticles for the controlled delivery of genetic material in a specific, efficient, and safe manner. PDGF-BB and FGF-2 genes were amplified from human tissues by RT-PCR. To increase the secretion of FGF-2, a recombinant 4sFGF-2 was constructed bearing eight amino-acid residues of the signal peptide of FGF-4. PCR products were inserted into the expression vector pVax1 to produce recombinant plasmids pVax1-4sFGF2 and pVax1-PDGF-BB, which were then injected into BALB/C mice in the format of polyelectrolyte nanocomplexes with specific chitosans of controlled DDA and molecular weight, including 92-10, 80-10, and 80-80 (DDA-number average molecular weight or M(n) in kDa). ELISA assays on mice sera showed that recombinant FGF-2 and PDGF-BB proteins were efficiently expressed and specific antibodies to these proteins could be identified in sera of injected mice, but with levels that were clearly dependent on the specific chitosan used. We found high DDA low molecular weight chitosans to be efficient protein expressors with minimal or no generation of neutralizing antibodies, while lowering DDA resulted in greater antibody levels and correspondingly lower levels of detected recombinant protein. Histological analyses corroborated these results by revealing greater inflammatory infiltrates in lower DDA chitosans, which produced higher antibody titers. We found, in general, a more efficient delivery of the plasmids by subcutaneous than by intramuscular injection. Specific chitosan carriers were identified to be either efficient non-toxic therapeutic protein delivery systems or vectors for DNA vaccines.
Department of Chemical Engineering, Institute of Biomedical Engineering, Ecole Polytechnique, Montréal, Québec, Canada.
M
M
Jean
F
F
Smaoui
M
M
Lavertu
S
S
Méthot
L
L
Bouhdoud
M D
MD
Buschmann
A
A
Merzouki
D000818:Animals; D000917:Antibody Formation; D001483:Base Sequence; D000077214:Becaplermin; D048271:Chitosan; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D007273:Injections, Intramuscular; D007279:Injections, Subcutaneous; D051379:Mice; D008807:Mice, Inbred BALB C; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000473:pathology; D053758:Nanoparticles / Q000008:administration & dosage* / Q000648:ultrastructure; D010957:Plasmids / Q000008:administration & dosage; D010982:Platelet-Derived Growth Factor / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins / Q000235:genetics; D012867:Skin / Q000473:pathology
D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D000077214:Becaplermin; D048271:Chitosan