title: 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.
authors.affiliation: Laboratory of Pharmaceutical Technology and Biopharmaceutics, University of Bonn , Bonn 53121, Germany.|Department of Chemistry, The American University in Cairo , Cairo 11835, Egypt.
authors.forename: Salma N
authors.initials: SN
authors.lastname: Tammam
authors.affiliation: Department of Chemistry, The American University in Cairo , Cairo 11835, Egypt.
authors.forename: Hassan M E
authors.initials: HM
authors.lastname: Azzazy
authors.affiliation: Department of Biochemistry, The German University in Cairo , Cairo 11432 Egypt.
authors.forename: Hans G
authors.initials: HG
authors.lastname: Breitinger
authors.affiliation: Laboratory of Pharmaceutical Technology and Biopharmaceutics, University of Bonn , Bonn 53121, Germany.|Laboratory of Pharmaceutical Engineering, University of Franche-Comté , Besançon 25000, France.
authors.forename: Alf
authors.initials: A
authors.lastname: Lamprecht
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D048271:Chitosan / Q000008:administration & dosage / Q000737:chemistry*; D004337:Drug Carriers / Q000008:administration & dosage / Q000737:chemistry; D011992:Endosomes / Q000378:metabolism; D051379:Mice; D053758:Nanoparticles / Q000008:administration & dosage / Q000737:chemistry*; D019913:Nuclear Localization Signals / Q000008:administration & dosage / Q000737:chemistry*; D010316:Particle Size
chemical_list: D004337:Drug Carriers; D019913:Nuclear Localization Signals; D048271:Chitosan
keywords: FRET; active targeting; chitosan nanoparticles; nuclear delivery; nuclear localization sequence; nuclear localization sequence density; nuclear targeting; protein delivery