Chitosan Nanoparticles for Nuclear Targeting: The Effect of Nanoparticle Size and Nuclear Localization Sequence Density

Abstract

Many recently discovered therapeutic proteins exert their main function in the nucleus, thus requiring both efficient uptake and correct intracellular targeting. Chitosan nanoparticles (NPs) have attracted interest as protein delivery vehicles due to their biocompatibility and ability to escape the endosomes offering high potential for nuclear delivery. Molecular entry into the nucleus occurs through the nuclear pore complexes, the efficiency of which is dependent on NP size and the presence of nuclear localization sequence (NLS). Chitosan nanoparticles of different sizes (S-NPs ≈ 25 nm; L-NP ≈ 150 nm) were formulated, and they were modified with different densities of the octapeptide NLS CPKKKRKV (S-NPs, 0.25, 0.5, 2.0 NLS/nm(2); L-NPs, 0.6, 0.9, 2 NLS/nm(2)). Unmodified and NLS-tagged NPs were evaluated for their protein loading capacity, extent of cell association, cell uptake, cell surface binding, and finally nuclear delivery efficiency in L929 fibroblasts. To avoid errors generated with cell fractionation and nuclear isolation protocols, nuclear delivery was assessed in intact cells utilizing Förster resonance energy transfer (FRET) fluorometry and microscopy. Although L-NPs showed ≈10-fold increase in protein loading per NP when compared to S-NPs, due to higher cell association and uptake S-NPs showed superior protein delivery. NLS exerts a size and density dependent effect on nanoparticle uptake and surface binding, with a general reduction in NP cell surface binding and an increase in cell uptake with the increase in NLS density (up to 8.4-fold increase in uptake of High-NLS-L-NPs (2 NLS/nm(2)) compared to unmodified L-NPs). However, for nuclear delivery, unmodified S-NPs show higher nuclear localization rates when compared to NLS modified NPs (up to 5-fold by FRET microscopy). For L-NPs an intermediate NLS density (0.9 NLS/nm(2)) seems to provide highest nuclear localization (3.7-fold increase in nuclear delivery compared to High-NLS-L-NPs). Results indicate that a higher NLS density does not result in maximum protein nuclear localization and that a universal optimal density for NPs of different sizes does not exist.

Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells

“…Small and large chitosan NPs were formulated by the inotropic gelation method using low-molecular weight chitosan (50–190 kDa; Sigma-Aldrich, St. Louis, MO) and tripolyphosphate (TPP; Mistral Chemicals, Antrim, UK) [28]. NP average hydrodynamic diameter and zeta potential were determined using a Zetasizer Nano ZS90 device (Malvern Instruments, Herrenberg, Germany).…”

“…NP encapsulation of BSA-RITC was performed as described [28]. NPs were purified and protein content was determined by fluorometry (FLUOstar Optima, BMG Labtech, Ortenberg, Germany; λex 540 nm; λem 625 nm).…”

“…S-NPs with low (L-NLS; 0.25 NLS/nm 2 ), intermediate (I-NLS; 0.5 NLS/nm 2 ) and high (H-NLS; 2 NLS/nm 2 ) NLS density were synthesized. NLS tagging and characterization of the modified NPs was performed as detailed previously [28]. …”

“…After 24 h, culture supernatants were removed and cells were washed twice with PBS. The amount of cell-associated NPs was calculated by fluorometry from standard curves as described [28, 44]. …”

“…The presence of primary amine groups on the NP surface facilitates endosomal escape via the proton sponge effect. Moreover, tagging of nuclear localization sequences (NLS) to the chitosan NPs allows directing NPs to the cell nucleus [28]. …”