Zhanguo Yue

Surface Charge Affects Cellular Uptake and Intracellular Trafficking of Chitosan-Based Nanoparticles

Chitosan-based nanoparticles (NPs) are widely used in drug delivery, device-based therapy, tissue engineering, and medical imaging. In this aspect, a clear understanding of how physicochemical properties of these NPs affect the cytological response is in high demand. The objective of this study is to evaluate the effect of surface charge on cellular uptake profiles (rate and amount) and intracellular trafficking. We fabricate three kinds of NPs (∼ 215 nm) with different surface charge via SPG membrane emulsification technique and deposition method. They possess uniform size as well as identical other physicochemical properties, minimizing any differences between the NPs except for surface charge. Moreover, we extend our research to eight cell lines, which could help to obtain a representative conclusion. Results show that the cellular uptake rate and amount are both positively correlated with the surface charge in all cell line. Subsequent intracellular trafficking indicates that some of positively charged NPs could escape from lysosome after being internalized and exhibit perinuclear localization, whereas the negatively and neutrally charged NPs prefer to colocalize with lysosome. These results are critical in building the knowledge base required to design chitosan-based NPs to be used efficiently and specifically.

The role of the lateral dimension of graphene oxide in the regulation of cellular responses

The nanomaterial graphene oxide (GO) has attracted explosive interests in various areas. However, its performance in biological environments is still largely unknown, particularly with regard to cellular response to GO. Here we separated the GO sheets in different size and systematically investigated size effect of the GO in response to different types of cells. In terms of abilities to internalize GO, enormous discrepancies were observed in the six cell types, with only two phagocytes were found to be capable of internalizing GO. The 2 μm and 350 nm GO greatly differed in lateral dimensions, but equally contributed to the uptake amount in macrophages. Similar amounts of antibody opsonization and active Fcγ receptor-mediated phagocytosis were demonstrated the cause of this behavior. In comparison with the nanosized GO, the GO in micro-size showed divergent intracellular locations and induced much stronger inflammation responses. Present study provided insight into selective internalization, size-independent uptake, and several other biological behaviors undergone by GO. These findings might help build necessary knowledge for potential incorporation of the unique two-dimensional nanomaterial as a biomedical tool, and for avoiding potential hazards.

Particle size affects the cellular response in macrophages

A deeper understanding of how the physical properties of particles regulate specific biological responses is becoming a crucial requirement for their successful biomedical application. To provide insights on their design and application, J774A.1 cells are exposed to particles with different diameters (430 nm, 1.9 μm and 4.8 μm), and the size effects on a series of cellular responses in macrophages are evaluated. Cellular uptake study demonstrates that nanosized particles accumulate in the cells at a faster rate, and with a higher surface area. Once the data are converted into the expression of particle volume, the maximum value is found with 1.9 μm particles instead of nanoparticles. Moreover, the uptake intermediates are also trapped, and the steps of particle internalization include filopodia sensing, skeleton rearrangement, and morphology change. Subsequent cellular trafficking reveals that only nanosized particles transport via lysosomal pathway, which is consistent with their uptake mechanisms. Furthermore, nanosized particles prefer to promote the secretion of Th1-specific molecule signals (e.g. IFN, IL-12) rather than immune suppressors. All these results, along with a couple of surprises, are discussed in the view of clinical practice. They are expected, in principle, to establish the basis of new design concepts for particle-based biomedical applications.

Galactosylated nanocrystallites of insoluble anticancer drug for liver-targeting therapy: an in vitro evaluation

AIM Low solubility in water has become an intrinsic property of many anticancer drugs, which poses a hurdle in the translation from the bench to the clinic. In this study, we developed a facile method to prepare 10-hydroxycamptothecin (HCPT) nanocrystallites and testified their feasibility for liver-targeting therapy. MATERIALS & METHODS HCPT nanocrystallites were prepared under the soft template effect of galactosylated chitosan. The internalization profile, intracellular trafficking, drug activity and cell viability were evaluated by exposing these nanocrystallites to human hepatocellular carcinoma HepG2 cells. RESULTS Galactosylated chitosan located on the HCPT nanocrystallites not only stabilized the formulation in aqueous medium, but also enhanced the cellular internalization through an asialoglycoprotein receptor-mediated pathway. These nanocrystallites also exhibited the advantages of nuclear entry and active HCPT delivery, and consequently better anticancer cytotoxicity could be achieved. CONCLUSION These data strongly support the superior properties of galactosylated HCPT nanocrystallites on liver-targeting therapy.

Nanoparticles-based multi-adjuvant whole cell tumor vaccine for cancer immunotherapy

Whole cell tumor vaccine (WCTV), as a potential treatment modality, elicits limited immune responses because of the poor immunogenicity. To address this issue, researchers have attempted to transduce a cytokine adjuvant into tumor cells, but these single-adjuvant WCTVs curtail the high expectations. In present study, we constructed a multi-adjuvant WCTV based on the nanoparticles modified with cell penetrating peptide, which could facilitate the transportation of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 2 (IL-2) into tumor cells. After inactivation, as-designed multi-adjuvant WCTV exhibited programmed promotions on DC recruitment, antigen presentation, and T-cell activation. In vivo evaluations demonstrated the satisfactory effects on tumor growth suppression, metastasis inhibition, and recurrence prevention. Therefore, the nanoparticles-based multi-adjuvant WCTV may serve as a high-performance treatment for anti-tumor immunotherapy.

The orchestration of cellular and humoral responses is facilitated by divergent intracellular antigen trafficking in nanoparticle-based therapeutic vaccine

Therapeutic vaccination for the treatment of chronic hepatitis B is promising but has so far shown limited clinical efficacy. Herein, we employ polylactide nanoparticles (NPs) as the vaccine adjuvant and systematically explore their effect on activation of specific immunity and the underlying theoretical mechanisms. In vitro studies show that hepatitis B surface antigen (HBsAg) accumulates in antigen-presenting cells (APCs) to a larger content (270%) with the assistant of NP in comparison with the pure-antigen group. Besides the elevated costimulators (CD80/86) and increased major histocompatibility complex (MHC) II molecules, the MHC I molecules are also found upregulated. This result is mostly owing to the divergent antigen trafficking ways of NP-antigen in APCs, especially for the escape of exogenous HBsAg from the lysosomes to the cytosol. Interestingly, the MHC I level is downregulated in alum-antigen group, indicating a possible reason for its inefficiency in priming cellular response. Further in vivo experiments establish that NP-antigen group indeed enhances the CD8(+) CTL cytotoxicity and IFN-γ cytokine secretion. Meanwhile, specific antibody titer is also upregulated, and even surpasses that of the commercialized alum-antigen. All these results strongly support that NP-based antigen promotes an orchestration of cellular and humoral immune response, exhibiting favorable intrinsic properties to be applied in therapeutic vaccines.

Novel vaccine delivery system induces robust humoral and cellular immune responses based on multiple mechanisms.

Aiming to enhance the immunogenicity of H5N1 split vaccine, the development of a novel antigen delivery system based on quaternized chitosan hydrogel microparticles (Gel MPs) with multiple mechanisms of immunity enhancement is attempted. Gel MPs based on ionic cross-linking are prepared in a simple and mild way. Gel MPs are superior as a vaccine delivery system due to their ability to: 1) enhance cellular uptake and endosomal escape of antigens in dendritic cells (DCs); 2) significantly activate DCs; 3) form an antigen depot and recruit immunity cells to improve antigen capture. Further in vivo investigation shows that Gel MPs, in comparison to aluminum salts (Alum), LPS, and covalent cross-linking quaternized chitosan MPs (GC MPs), induce higher humoral and cellular immune responses with a mixed Th1/Th2 immunity. In conclusion, these results demonstrate that Gel MPs are efficient antigen delivery vehicles based on multiple mechanisms to enhance both humoral and cellular immune responses against H5N1 split antigen.

Molecular structure matters: PEG-b-PLA nanoparticles with hydrophilicity and deformability demonstrate their advantages for high-performance delivery of anti-cancer drugs

Various carriers are being advanced for anti-cancer therapy, which can protect drugs and ferry them to the target site. However, little understanding exists regarding the effect of molecular structure on anti-cancer drug delivery efficiency. To fill this knowledge gap, we take poly(lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA), and poly-ethylene glycol-co-poly-lactide (PEG-b-PLA) polymers as prototype materials and comparatively explore the inherent relationship between the molecular structure and the delivery ability. Compared with PLA and PLGA NPs, PEG-b-PLA ones possess the advantages of longer blood circulation time, more tumor accumulation, and better intratumoral delivery ability. Subsequent mechanism investigations reveal that the molecular structure will regulate the polymer arrangement and render NPs different hydrophilicity/deformability, which dictate the distinct delivery performances. Finally, the superior PEG-b-PLA NPs are further loaded with the anti-cancer drug paclitaxel (PTX) and functionalized with magnetic (M) Fe3O4 nanocrystals. As-designed PTX/M PEG-b-PLA NPs show much better tumor inhibition efficacy and fewer side effects than the commercialized Taxol® formulation, strongly supporting their use as high-performance carriers for anti-cancer therapy.