A large arsenal of nanoparticles (NPs) have been developed for several biomedical applications ranging from property enhancers of polymeric scaffolds, gene therapy strategies to drug delivery vehicles for highly targeted therapies. However, to improve therapeutic efficacy, cell penetration and drug leakage from intracellular endosomal compartments are, thus far, some of the most difficult challenges to tackle. A family of NPs of interest is the polymeric NPs due to their protection upon labile molecules, biodegradability and biocompatibility. To improve cell penetration and prevent endosomal entrapment after crossing the cell membrane, polymeric NPs can be superficially modified with translocating agents. Therefore, this study focuses on developing low and high molecular weight chitosan (LMW/HMW) and type B gelatin NPs, conjugated with the translocating peptide Buforin II (BUF-II). Prior to modification, the chitosan NPs were synthesized by the ionic gelation method while the gelatin ones by the desolvation method. The obtained chitosan- and type B gelatin-BUF-II nanobioconjugates’ size and zeta potential were characterized via DLS, and then the synthesis was analyzed by Fourier transform infrared spectroscopy (FTIR). The NPs FTIR showed peaks resembling those of pure polymers and BUF-II after conjugation. The average NP diameters were approximately 500 nm, chitosan NPs zeta potential was close to +30 mV, and the one of gelatin NPs was -13 mV, which is slightly above what is required for colloidal stability (±10 mV). SEM microscopy of LMW and HMW chitosan NPs showed round and oval-shaped morphology, respectively, while gelatin NPs were more filamentous. TEM microscopy showed that the nominal size of the NPs was about 10 nm. Rhodamine B was also conjugated to the immobilized BUF-II to track their internalization and endosomal escape via confocal microscopy. Our results indicate that all the synthesized nanobioconjugates successfully penetrated the monocytic THP-1 cell line. Hemolysis and platelet aggregation assays demonstrated their high biocompatibility. We are currently exploring the potential of the nanobioconjugates to penetrate other cell lines, including Vero, Neuroblastoma, and HaCaT. Also, we are investigating in detail the intracellular trafficking routes via early and late endosome labeling.

Chitosan and type B gelatin nanoparticles functionalized with BUF-II for highly biocompatible cell-penetrating vehicles

ACS Fall 2021

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