Platinum-Metformin Conjugates Acting as Promising PD-L1 Inhibitors through the AMP-Activated Protein Kinase Mediated Lysosomal Degradation Pathway.

With the development of metalloimmunology, the potential of platinum drugs in cancer immunotherapy has attracted extensive attention. Although immunochemotherapy combining PD-1/PD-L1 antibodies with platinum drugs has achieved great success in the clinic, combination therapy commonly brings new problems. Herein, we have developed a platinum-metformin conjugate as a promising alternative to antibody-based PD-L1 inhibitors, not only disrupting PD-1/PD-L1 axis on cell surface but also down-regulating the total PD-L1 levels in non-small cell lung cancer (NSCLC) cells comprehensively, thus achieving highly efficient immunochemotherapy by a single small molecule. Mechanism studies demonstrate that Pt-metformin conjugate can selectively accumulate in lysosomes, promote lysosomal-dependent PD-L1 degradation via the AMPK-TFEB pathway, and modulate the upstream regulatory proteins related to PD-L1 expression (e.g. HIF-1α and NF-κB), eventually decreasing the total abundance of PD-L1 in NSCLC, overcoming tumor hypoxia, and activating anti-tumor immunity in vivo. This work suggests an AMPK-mediated lysosomal degradation pathway of PD-L1 for the first time and provides a unique design perspective for the development of novel platinum drugs for immunochemotherapy.

Influences of repeated exposure to low levels of TiO2 nanoparticles on Kruppel-like factors in 3D Caco-2 spheroids and mouse intestines.

Kruppel-like factors (KLFs) are a set of transcription factors (TFs) involved in the regulation of many basic biological processes, and recent studies suggested that nanoparticles (NPs) were capable to change KLFs in different models even at non-cytotoxic concentrations. In this study, we repeatedly exposed 3D Caco-2 spheroids and mice to TiO2 NPs, one of the most frequently used metal oxide NPs, and investigated the changes of KLF-signaling pathways based on RNA-sequencing. Although the internalization of TiO2 NPs did not induce cytotoxicity in vitro, repeated exposure (three times within 7 days) to 15.7 ng/ml TiO2 NPs increased KLF4 but decreased KLF6. Consistently, KLF4/KLF6-regulated gene ontology terms were altered, including those involved in the regulation of gene expression. We further verified that repeated exposure to 15.7 ng/ml TiO2 NPs increased the expression of KLF4 and proto-oncogene, bHLH transcription factor (MYC), but decreased the expression of KLF6 and activating transcription factor 3 (ATF3). But with the increase of NP concentrations, the expression of these genes was decreased. In mice following intragastrical exposure to 4.39 and 43.9 mg/kg TiO2 NPs (once a day for 5 continuous days), we observed increased expression of klf4, klf6, myc, and atf3, along with morphological changes of intestines. We concluded that repeated exposure to low levels of TiO2 NPs altered KLF-signaling pathways in intestinal cells both in vitro and in vivo.

Nano vaccines for T. gondii Ribosomal P2 Protein With Nanomaterials as a Promising DNA Vaccine Against Toxoplasmosis.

Caused by Toxoplasma gondii, toxoplasmosis has aroused great threats to public health around the world. So far, no effective vaccine or drug is commercially available, and the demands for a safe and effective therapeutic strategy have become more and more urgent. In the current study, we constructed a DNA vaccine encoding T. gondii ribosomal P2 protein (TgP2) and denoted as TgP2-pVAX1 plasmid. To improve the immunoprotection, nanomaterial poly-lactic-co-glycolic acid (PLGA) and chitosan were used as the delivery vehicle to construct TgP2-pVAX1/PLGA and TgP2-pVAX1/CS nanospheres. Before vaccinations in BALB/c mice, TgP2-pVAX1 plasmids were transiently transfected into Human Embryonic Kidney (HEK) 293-T cells, and the expression of the eukaryotic plasmids was detected by laser confocal microscopy and Western blotting. Then the immunoprotection of naked DNA plasmids and their two nano-encapsulations were evaluated in the laboratory animal model. According to the investigations of antibody, cytokine, dendritic cell (DC) maturation, molecule expression, splenocyte proliferation, and T lymphocyte proportion, TgP2-pVAX1 plasmid delivered by two types of nanospheres could elicit a mixed Th1/Th2 immune response and Th1 immunity as the dominant. In addition, TgP2-pVAX1/PLGA and TgP2-pVAX1/CS nanospheres have great advantages in enhancing immunity against a lethal dose of T. gondii RH strain challenge. All these results suggested that TgP2-pVAX1 plasmids delivered by PLGA or chitosan nanomaterial could be promising vaccines in resisting toxoplasmosis and deserve further investigations and applications.

Comparison of P25 and nanobelts on Kruppel-like factor-mediated nitric oxide pathways in human umbilical vein endothelial cells.

Recently, we reported that titanium dioxide (TiO2 ) materials activated endothelial cells via Kruppel-like factor (KLF)-mediated nitric oxide (NO) dysfunction, but the roles of physical properties of materials are not clear. In this study, we prepared nanobelts from P25 particles and compared their adverse effects to human umbilical vein endothelial cells (HUVECs). TiO2 nanobelts had belt-like morphology but comparable surface areas as P25 particles. When applied to HUVECs, P25 particles or nanobelts did not induce cytotoxicity, although nanobelts were much more effective to increase intracellular Ti element concentrations compared the same amounts of P25 particles. Only nanobelts significantly induced THP-1 adhesion onto HUVECs. Consistently, nanobelts were more significant to induce the expression of intracellular adhesion molecule-1 (ICAM1) and the release of soluble ICAM-1 (sICAM-1), indicating that nanobelts were more potent to induce endothelial activation in vitro. As the mechanisms for endothelial activation, both P25 and nanobelts reduced the generation of intracellular NO as well as the expression of NO regulators KLF2 and KLF4. Combined, the results from this study indicated that the different morphologies of P25 particles and nanobelts only changed their internalization into HUVECs but showed minimal impact on KLF-mediated NO signaling pathways.

Effects of epigallocatechin gallate on the stability, dissolution and toxicology of ZnO nanoparticles.

Previously we reported the cytoprotective effects of polyphenols rich in hydroxyl groups against ZnO nanoparticles (NPs). This study used RNA-sequencing to evaluate the toxicity of ZnO NPs and epigallocatechin gallate (EGCG) to 3D Caco-2 spheroids. EGCG altered the colloidal stability of ZnO NPs, shown as the changes of atomic force microscopic height, solubility in cell culture medium, and hydrodynamic sizes. EGCG almost completely reversed ZnO NP-induced cytotoxicity, and consistently, alleviated ZnO NP-induced gene ontology (GO) terms and genes related with apoptosis. EGCG also modestly decreased intracellular Zn ions and changed GO terms and genes related with endocytosis/exocytosis in ZnO NP-exposed spheroids. Meanwhile, EGCG changed ZnO NP-induced alteration of GO terms and genes related with the functions of mitochondria, endoplasmic reticulum and lysosomes. We concluded that EGCG alleviated the cytotoxicity of ZnO NPs to 3D Caco-2 spheroids by altering NPs' colloidal properties and the pathways related with internalization and organelle dysfunction.

Nano DNA Vaccine Encoding Toxoplasma gondii Histone Deacetylase SIR2 Enhanced Protective Immunity in Mice.

The pathogen of toxoplasmosis, Toxoplasma gondii (T. gondii), is a zoonotic protozoon that can affect the health of warm-blooded animals including humans. Up to now, an effective vaccine with completely protection is still inaccessible. In this study, the DNA vaccine encoding T. gondii histone deacetylase SIR2 (pVAX1-SIR2) was constructed. To enhance the efficacy, chitosan and poly (d, l-lactic-co-glycolic)-acid (PLGA) were employed to design nanospheres loaded with the DNA vaccine, denoted as pVAX1-SIR2/CS and pVAX1-SIR2/PLGA nanospheres. The pVAX1-SIR2 plasmids were transfected into HEK 293-T cells, and the expression was evaluated by a laser scanning confocal microscopy. Then, the immune protections of pVAX1-SIR2 plasmid, pVAX1-SIR2/CS nanospheres, and pVAX1-SIR2/PLGA nanospheres were evaluated in a laboratory animal model. The in vivo findings indicated that pVAX1-SIR2/CS and pVAX1-SIR2/PLGA nanospheres could generate a mixed Th1/Th2 immune response, as indicated by the regulated production of antibodies and cytokines, the enhanced maturation and major histocompatibility complex (MHC) expression of dendritic cells (DCs), the induced splenocyte proliferation, and the increased percentages of CD4+ and CD8+ T lymphocytes. Furthermore, this enhanced immunity could obviously reduce the parasite burden in immunized animals through a lethal dose of T. gondii RH strain challenge. All these results propose that pVAX1-SIR2 plasmids entrapped in chitosan or PLGA nanospheres could be the promising vaccines against acute T. gondii infections and deserve further investigations.

With Chitosan and PLGA as the Delivery Vehicle, Toxoplasma gondii Oxidoreductase-Based DNA Vaccines Decrease Parasite Burdens in Mice.

Toxoplasma gondii (T. gondii) is an intracellular parasitic protozoan that can cause serious public health problems. However, there is no effectively preventive or therapeutic strategy available for human and animals. In the present study, we developed a DNA vaccine encoding T. gondii oxidoreductase from short-chain dehydrogenase/reductase family (TgSDRO-pVAX1) and then entrapped in chitosan and poly lactic-co-glycolic acid (PLGA) to improve the efficacy. When encapsulated in chitosan (TgSDRO-pVAX1/CS nanospheres) and PLGA (TgSDRO-pVAX1/PLGA nanospheres), adequate plasmids were loaded and released stably. Before animal immunizations, the DNA vaccine was transfected into HEK 293-T cells and examined by western blotting and laser confocal microscopy. Th1/Th2 cellular and humoral immunity was induced in immunized mice, accompanied by modulated secretion of antibodies and cytokines, promoted the maturation and MHC expression of dendritic cells, and enhanced the percentages of CD4+ and CD8+ T lymphocytes. Immunization with TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres conferred significant immunity with lower parasite burden in the mice model of acute toxoplasmosis. Furthermore, our results also lent credit to the idea that TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres are substitutes for each other. In general, the current study proposed that TgSDRO-pVAX1 with chitosan or PLGA as the delivery vehicle is a promising vaccine candidate against acute toxoplasmosis.