Methods of delivering anionic agents in vivo using non-viral nanoparticle-based delivery systems

A method for in vivo targeted delivery of a nucleic acid or an anionic agent to an individual in need thereof, said method comprising:

• (a) providing a polymeric delivery system comprising the nucleic acid or the anionic agent,
• wherein the polymeric delivery system comprises non-viral nanoparticles comprising:
• a plurality of cationic chitosan polymers, polyethylene glycol and a peptide sequence,
• and wherein the non-viral nanoparticles comprise an internal core and the nucleic acid or the anionic agent are placed in the inner core,
• and wherein the peptide sequence comprises a cell penetrating peptide, a protein transduction domain, and a cell surface receptor targeting moiety,
• and the polymeric delivery system is made by a method comprising:
• a) reacting amine groups of chitosan with phthalic anhydride as a protection step;
• b) activating monomethoxy polyethylene glycol (mPEG-OH) with succinic anhydride to form mPEG-COOH;
• c) activating mPEG-COOH with thionyl chloride to form mPEG-COCl to form an activated PEG;
• d) reacting hydroxyl groups of chitosan with the activated mPEG-COCl to form PEGylated chitosan;
• e) reacting the monomethoxy group of the PEGylated chitosan with aluminum chloride in ethane thiol to convert the monomethoxy group to a hydroxyl group to form hydroxyl terminated PEGylated chitosan;
• f) reacting the hydroxyl terminated PEGylated chitosan with succinic anhydride to form an activated chitosan-PEG-COOH;
• g) reacting the activated chitosan-PEG-COOH with a peptide sequence comprising a cell penetrating peptide, a protein transduction domain, and a cell surface receptor targeting moiety, wherein said peptide sequence comprises a terminal amine, and wherein this reaction is done using EDC and DMAP as a catalyst to form chitosan-PEG-peptide;
• and
• h) reacting the chitosan-PEG-peptide with hydrazine monohydrate to deprotect amine groups of chitosan,
• and wherein the plurality of cationic chitosan polymers are cross linked to each other to form the outer layer of the nanoparticle to generate an internal core;
• and the nanoparticles have an average diameter of between 5 to 300 nm;
• and (b) administering the non-viral nanoparticle to the individual.