title: Preparation of Concentrated Chitosan/DNA Nanoparticle Formulations by Lyophilization for Gene Delivery at Clinically Relevant Dosages.
abstract: Chitosan/DNA polyplexes have been optimized for efficient and safe in vitro and in vivo gene delivery. Clinical application of this technology requires the development of formulations with higher concentrations to reach therapeutic dosages. Polyplexes were prepared using chitosan and EGFPLuc plasmids. Freeze-thawing and freeze-drying studies were performed to identify and optimize lyoprotectant and buffer contents in formulations. Freeze-dried samples were rehydrated in reduced volumes to increase their final DNA dose. Nanoparticle physicochemical properties were analyzed, and their transfection efficiency and cytotoxicity were measured in human embryonic kidney 293 cells. Data showed that 3.5 mM histidine buffer (pH 6.5) combined with one of 0.5% wt/vol sucrose, dextran 5 kDa, or trehalose was required to prevent polyplex aggregation during freeze-drying. Optimal formulations could be concentrated 20-fold, to a clinically desired ∼1 mg of DNA/mL, while maintaining near physiological pH and tonicity. Polyplexes were predominantly spherical, with diameters below 200 nm, polydispersity indexes below 0.32, and zeta potentials above +19 mV. Rehydrated formulations had transfection efficiencies no less than 65% of fresh polyplexes without excipients and had no effect on viability and metabolic activity of human embryonic kidney 293 cells. These concentrated formulations represent an important step toward clinical use of chitosan-based gene delivery systems.
authors: Institute of Biomedical Engineering, Polytechnique Montreal, Montreal, Quebec, Canada.
authors: Daniel
authors: D
authors: Veilleux
authors: Department of Chemical Engineering, Polytechnique Montreal, Montreal, Quebec, Canada.
authors: Monica
authors: M
authors: Nelea
authors: Department of Scientific Affairs, ANRis Pharmaceuticals Inc., Kirkland, Quebec, Canada.
authors: Kristof
authors: K
authors: Biniecki
authors: Department of Chemical Engineering, Polytechnique Montreal, Montreal, Quebec, Canada.
authors: Marc
authors: M
authors: Lavertu
authors: Institute of Biomedical Engineering, Polytechnique Montreal, Montreal, Quebec, Canada; Department of Chemical Engineering, Polytechnique Montreal, Montreal, Quebec, Canada. Electronic address: michael.buschmann@polymtl.ca.
authors: Michael D
authors: MD
authors: Buschmann
mesh_terms: D002470:Cell Survival / Q000187:drug effects; D048271:Chitosan / Q000737:chemistry* / Q000633:toxicity; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D004337:Drug Carriers / Q000737:chemistry* / Q000633:toxicity; D005612:Freeze Drying; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D053758:Nanoparticles / Q000737:chemistry* / Q000633:toxicity; D010957:Plasmids; D011094:Polyethyleneimine / Q000737:chemistry / Q000633:toxicity; D013499:Surface Properties
chemical_list: D004337:Drug Carriers; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; D004247:DNA; D048271:Chitosan; D008156:Luciferases
keywords: DNA/oligonucleotide delivery; chitosan; formulation; freeze-drying/lyophilization; nanoparticles; nonviral gene delivery; toxicity