Fine tuning neuronal targeting of nanoparticles by adjusting the ligand grafting density and combining PEG spacers of different length

Abstract

Poly(ethylene glycol) (PEG) has been extensively used to coat the surface of nanocarriers to improve their physicochemical properties and allow the grafting of targeting moieties. Still, to date there is no common agreement on the ideal PEG coverage-density or length to be used for optimum vector performance. In this study, we aimed to investigate the impact of both PEG density and length on the vectoring capacity of neuron-targeted gene-carrying trimethyl chitosan nanoparticles. The non-toxic fragment from the tetanus toxin (HC) was coupled to a 5 kDa heterobifunctional PEG (HC-PEG_5k) reactive for the thiol groups inserted into the polymer backbone and grafted at different densities onto the nanoparticles. Internalization and transfection studies on neuronal versus non-neuronal cell lines allowed to determine the PEG density of 2 mol% of PEG chains per mol of primary amine groups as the one with superior biological performance. To enhance HC exposure and maximize cell-nanoparticle specific interaction, NPs containing different ratios of HC-PEG_5k and 2 kDa methoxy-PEG at the same grafting density were produced. By intercalating HC-PEG_5k with methoxy-PEG_2k we attained the best performance in terms of internalization (higher payload delivery into cells) and transfection efficiency, using twice lower amount of HC. This outcome highlights the need for fine-tuning of PEG-modified nanoparticles towards the achievement of optimal targeting.

Statement of significance: The amount and exposure of targeting moieties at a nanoparticle surface are critical parameters regarding the targeting potential of nanosized delivery vectors. However, to date, few studies have considered fundamental aspects impacting the ligand-receptor pair interaction, such as the effect of spacer chain length, flexibility or conformation. By optimizing the PEG spacer density and chain length grafted into nanoparticles, we were able to establish the formulation that maximizes cell-nanoparticle specific interaction and has superior biological performance. Our work shows that the precise adjustment of the PEG coverage-density presents a significant impact on the selectivity and bioactivity of the developed formulation, emphasizing the need for the fine-tuning of PEG-modified nanoparticles for the successful development of the next-generation nanomedicines.

Keywords: Cellular uptake; Nanoparticle surface modification/functionalization; PEGylation; Spacer length; Targeted nanoparticles.