To systematically elucidate the effect of surface charge on the cellular uptake and in vivo fate of PEG-oligocholic acid based micellar nanoparticles (NPs), the distal PEG termini of monomeric PEG-oligocholic acid dendrimers (telodendrimers) are each derivatized with different number (n = 0, 1, 3 and 6) of anionic aspartic acids (negative charge) or cationic lysines (positive charge). Under aqueous condition, these telodendrimers self-assemble to form a series of micellar NPs with various surface charges, but with similar particle sizes. NPs with high surface charge, either positive or negative, were taken up more efficiently by RAW 264.7 murine macrophages after opsonization in fresh mouse serum. Mechanistic studies of cellular uptake of NPs indicated that several distinct endocytic pathways (e.g., clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis) were involved in the cellular uptake process. After their cellular uptake, the majority of NPs were found to localize in the lysosome. Positively charged NPs exhibited dose-dependent hemolytic activities and cytotoxicities against RAW 264.7 cells proportional to the positive surface charge densities; whereas negatively charged NPs did not show obvious hemolytic and cytotoxic properties. In vivo biodistribution studies demonstrated that undesirable liver uptake was very high for highly positively or negatively charged NPs, which is likely due to active phagocytosis by macrophages (Kupffer cells) in the liver. In contrast, liver uptake was very low but tumor uptake was very high when the surface charge of NPs was slightly negative. Based on these studies, we can conclude that slightly negative charge may be introduced

Iron oxide nanoparticles (IONPs) have been increasingly used in biomedical applications, but the comprehensive understanding of their interactions with biological systems is relatively limited. In this study, we systematically investigated the in vitro cell uptake, cytotoxicity, in vivo distribution, clearance and toxicity of commercially available and well-characterized IONPs with different sizes and coatings. Polyethylenimine (PEI)-coated IONPs exhibited significantly higher uptake than PEGylated ones in both macrophages and cancer cells, and caused severe cytotoxicity through multiple mechanisms such as ROS production and apoptosis. 10 nm PEGylated IONPs showed higher cellular uptake than 30 nm ones, and were slightly cytotoxic only at high concentrations. Interestingly, PEGylated IONPs but not PEI-coated IONPs were able to induce autophagy, which may play a protective role against the cytotoxicity of IONPs. Biodistribution studies demonstrated that all the IONPs tended to distribute in the liver and spleen, and the biodegradation and clearance of PEGylated IONPs in these tissues were relatively slow (>2 weeks). Among them, 10 nm PEGylated IONPs achieved the highest tumor uptake. No obvious toxicity was found for PEGylated IONPs in BALB/c mice, whereas PEI-coated IONPs exhibited dose-dependent lethal toxicity. Therefore, it is crucial to consider the size and coating properties of IONPs in their applications.

To minimize premature release of drugs from their carriers during circulation in the blood stream, we have recently developed reversible disulfide cross-linked micelles (DCMs) that can be triggered to release drug at the tumor site or in cancer cells. We designed and synthesized thiolated linear-dendritic polymers (telodendrimers) by introducing cysteines to the dendritic oligo-lysine backbone of our previously reported telodendrimers comprised of linear polyethylene glycol (PEG) and a dendritic cluster of cholic acids. Reversibly cross-linked micelles were then prepared by the oxidization of thiol groups to disulfide bond in the core of micelles after the self-assembly of thiolated telodendrimers. The DCMs were spherical with a uniform size of 28 nm, and were able to load paclitaxel (PTX) in the core with superior loading capacity up to 35.5% (w/w, drug/micelle). Cross-linking of the micelles within the core reduced their apparent critical micelle concentration and greatly enhanced their stability in non-reductive physiological conditions as well as severe micelle-disrupting conditions. The release of PTX from the DCMs was significantly slower than that from non-cross-linked micelles (NCMs), but can be gradually facilitated by increasing the concentration of reducing agent (glutathione) to an intracellular reductive level. The DCMs demonstrated a longer in vivo blood circulation time, less hemolytic activities, and superior toxicity profiles in nude mice, when compared to NCMs. DCMs were found to be able to preferentially accumulate at the tumor site in nude mice bearing SKOV-3 ovarian cancer xenograft. We also demonstrated that the disulfide cross-linked micellar formulation of PTX (PTX-DCMs) was more efficacious than both free drug and the non-cross-linked formulation of PTX at equivalent doses of PTX in the ovarian cancer xenograft mouse model. The anti-tumor effect of PTX-DCMs can be further enhanced by triggering the release of PTX on-demand by the administration of the FDA approved reducing agent, N-acetylcysteine, after PTX-DCMs have reached the tumor site.