In this manuscript we have designed a synthetic approach for the preparation of a series of chitosan-graft-poly(L-glutamate) copolymers with different length poly(L-glutamate) grafts. First, organosulfonic chitosan salt, soluble in DMSO, was prepared in order to effectively initiate ring-opening polymerization of γ-benzyl-L-glutamate N-carboxyanhydride. The chitosan-graft-poly(γ-benzyl-L-glutamate) copolymers were fully deprotected by applying tetrabutylammonium hydroxide. The molar mass characteristics and chemical composition of graft copolymers with various lengths of polypeptide grafts were determined by SEC-MALS and various NMR spectroscopic techniques. The synthesized chitosan-graft-poly(sodium-L-glutamate) copolymers were used in combination with trimethyl chitosan for the preparation of nanoparticles of recombinant granulocyte colony-stimulating factor (GCSF). The most efficient polymer in the preparation of the complexes with GCSF was chitosan with the longest polyglutamate grafts. The formed complexes of GCSF with this particular chitosan-graft-poly(γ-benzyl-L-glutamate) and trimethyl chitosan show average particle size of 224 nm and narrow size distribution. These nanoparticles exhibit very high association efficiency (82 %) as well as final GCSF loading (42 %).
The negatively charged, water-soluble, hydrophobically modified poly(sodium glutamate)s which contain different amounts of randomly distributed alkyl grafts are synthesized. First, poly(γ-benzyl-L-glutamate) is prepared by ring-opening polymerization of the corresponding N-carboxyanhydride, which is in the next step aminolysed with octylamine. After removal of the remaining benzyl protected groups, the alkyl modified poly(sodium glutamates) (P(Glu-oa)) are obtained and, together with oppositely charged N,N,N-trimethyl chitosan, used for the preparation of nanoparticles of recombinant granulocyte colony-stimulating factor (GCSF) protein by polyelectrolyte complexation method. It is observed that, beside electrostatic interaction, the hydrophobic grafts on poly(sodium glutamate) polymers significantly contribute to association efficiency of P(Glu-oa) with GCSF protein. The addition of N,N,N-trimethyl chitosan solution to the dispersion of GCSF/P(Glu-oa) complexes results in formation of well-defined nanoparticles with very high association efficiency and high final protein loading.
A glycosylated 2,2-bis(methylol)propionic acid (bisMPA) derivative was synthesized as a versatile glycodendritic building block, which was, in the next step, applied for modification of appropriately protected chitosan to yield a protected glycodendritic chitosan derivative. Two ortogonal protecting groups, enabling additional flexibility for further derivatization, were removed in two separate deprotection steps. A 6-O-trityl protection group on chitosan was removed under acidic conditions, whereas an acetyl protection group of D-glucose under alkaline conditions. The structure and molar mass characteristics of the intermediates and final product were determined by NMR and FTIR spectroscopic methods, MALDI-TOF mass spectrometry and SEC-MALS, respectively. The synthesized glycodendritic chitosan is water-soluble at physiological conditions and, thus, suitable for further investigation as a potential carrier for drug delivery.