Carboxymethyl Chitosan-poly(amidoamine) Dendrimer Core-shell Nanoparticles For Intracellular Protein Delivery

Nowadays, advanced biotechnology promote the production of myriad proteinsand peptides which could be used as highly specific and effective therapeutics agents,such as vaccines, hormones, growth factors and enzymes. Particularly, enzymesrepresent an irreplaceable group of protein drugs to treat cancers, chronic disease,metabolic disorders and autoimmune disease. These protein drugs are acting throughvarious mechanisms. In some of them, passing through the cell in a cell-type ortissue-specific manner is needed, especially when the relevant pharmacological targetis located intracellularly. However, intracellular delivery of native, active proteins ischallenging due to the fragility of most proteins. Although many nanoscale vehiclesfor intracellular protein delivery have been reported to date, they may exhibit lowdelivery effciency and can disturb protein folding and impair biological activity.Nanoscale polyion complex (PIC) resulting from the self-assembly of proteins withnatural or synthetic polymers could overcome the defects of other nanoscale vehiclesand has drawn increasing attention especially for application in therapeutic proteindelivery.In order to develop a novel improved nanocarrier for intracellular protein delivery,a water-soluble amphiphilic dendronized chitosan derivative carbohydratechitosan-poly(amidoamine)(CMCS-PAMAM) was synthesized herein via thecondensation reaction among amino groups in CMCS and carboxyl groups inPAMAM. The novel dendronized chitosan derivative CMCS-PAMAM couldself-assemble into nanoparticles with core-shell structure via hydrophobic interactionand electrostatic interaction, and encapsulate the positively charged lysozyme viaelectrostatic attraction and hydrogen bond interaction at physiological pH. However,the novel core-shell nanoparticles will swell and release their cargo efficiently atendosomal pH, because of the electrostatic repulsion between lysozyme andnanoparticles.In the following research，we investigated the effect of feeding ratios on the sizeand structure of CMCS-PAMAM and find the optimum feeding ratios to CMCS-0.5GPAMAM, CMCS-1.5GPAMAM and CMCS-2.5GPAMAM respectively.The mechanism of nanoparticles formation for CMCS-PAMAM was proposed. Thesize of nanoparticles with optimum feeding ratios, which were used in the followingresearch, is about40-60nm. Characterization, including1H NMR and TEM wereperformed to investigate CMCS-PAMAM structure and morphology. Moreover, theCMCS-PAMAM dendrimer nanoparticles did not exhibit significant cytotoxicity inthe range of concentrations below3.16mg/ml.The process of lysozyme encapsulated by the CMCS-PAMAM nanoparticleswas followed by ζ-potential and Bradford analysis and the optimum weight ratio wasdetermined. The results of dynamic light scattering and TEM explored thatCMCS-PAMAM exerted much higher encapsulating efficiency for lysozyme ascompared to CMCS. The enzymic activity of the released lysozyme was determinedand the results show that only a fraction of lysozyme released from CMCS-PAMAMdendrimer nanoparticles and the activity of lysozyme was completely inhibited atphysiological pH, whereas the lysozyme was released into the medium and exhibitedalmost the full enzymatic activity in an acidic intracellular environment.