Inhalable hybrid nanovaccines with virus-biomimetic structure boost protective immune responses against SARS-CoV-2 variants.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with different antigenic variants, has posed a significant threat to public health. It is urgent to develop inhalable vaccines, instead of injectable vaccines, to elicit mucosal immunity against respiratory viral infections. METHODS: We reported an inhalable hybrid nanovaccine (NVRBD-MLipo) to boost protective immunity against SARS-CoV-2 infection. Nanovesicles derived from genetically engineered 293T cells expressing RBD (NVRBD) were fused with pulmonary surfactant (PS)-biomimetic liposomes containing MPLA (MLipo) to yield NVRBD-MLipo, which possessed virus-biomimetic structure, inherited RBD expression and versatile properties. RESULTS: In contrast to subcutaneous vaccination, NVRBD-MLipo, via inhalable vaccination, could efficiently enter the alveolar macrophages (AMs) to elicit AMs activation through MPLA-activated TLR4/NF-κB signaling pathway. Moreover, NVRBD-MLipo induced T and B cells activation, and high level of RBD-specific IgG and secretory IgA (sIgA), thus elevating protective mucosal and systemic immune responses, while reducing side effects. NVRBD-MLipo also demonstrated broad-spectrum neutralization activity against SARS-CoV-2 (WT, Delta, Omicron) pseudovirus, and protected immunized mice against WT pseudovirus infection. CONCLUSIONS: This inhalable NVRBD-MLipo, as an effective and safe nanovaccine, holds huge potential to provoke robust mucosal immunity, and might be a promising vaccine candidate to combat respiratory infectious diseases, including COVID-19 and influenza.

Splenic-targeting biomimetic nanovaccine for elevating protective immunity against virus infection.

BACKGROUND: The prevalence of viral infectious diseases has become a serious threat to public safety, economic and social development. Vaccines have been served as the most effective platform to prevent virus transmission via the activation of host immune responses, while the low immunogenicity or safety, the high cost of production, storage, transport limit their effective clinical application. Therefore, there is a need to develop a promising strategy to improve the immunogenicity and safety of vaccines. METHODS: We developed a splenic-targeting biomimetic nanovaccine (NV) that can boost protective humoral and cellular immunity against african swine fever virus (ASFV) infection. The universal PLGA nanoparticles (CMR-PLGA/p54 NPs) coated with mannose and CpG (TLR9 agonist) co-modified red blood cell (RBC) membrane were prepared, which comprised a viral antigen (p54) and can be served as a versatile nanovaccine for elevating protective immunity. RESULTS: CMR-PLGA/p54 NVs could be effectively uptaken by BMDC and promoted BMDC maturation in vitro. After subcutaneous immunization, antigen could be effectively delivered to the splenic dendritic cells (DCs) due to the splenic homing ability of RBC and DC targeting capacity of mannose, which promoted antigen presentation and DCs maturation, and further elicited higher levels of cytokines secretion and specific IgG titers, CD4+ and CD8+ T cells activation and B maturation. Moreover, NVs demonstrated notable safety during the immunization period. CONCLUSIONS: This study demonstrates the high potential of CMR-PLGA NPs as vaccine delivery carriers to promote humoral and cellular immune responses, and it provides a promising strategy to develop safe and effective vaccines against viral infectious diseases.

Wnt8B, transcriptionally regulated by ZNF191, promotes cell proliferation of hepatocellular carcinoma via Wnt signaling.

Dysregulation of wingless-type (Wnt) signaling is implicated in hepatocellular carcinoma (HCC). Wnt family member 8B (Wnt8B), one of the canonical Wnt ligands, is implicated in oncogenesis. However, the role of Wnt8B in human HCCs and its transcriptional regulation mechanism are presently unknown . Here, we report that Wnt8B expression was frequently increased in HCCs and was significantly associated with poorer patient prognosis. Wnt8B knockdown suppresses HCC cell growth both in vitro and in vivo via inhibiting the canonical Wnt signaling. Zinc finger transcription factor 191 (ZNF191) can positively regulate Wnt8B mRNA and protein expression, and promoter luciferase assay indicated that ZNF191 can increase the transcription activity of the 2-Kbps WNT8B promoter. Chromatin immunoprecipitation-qPCR and electrophoretic mobility shift assay showed that ZNF191 protein directly binds to the WNT8B promoter, and the binding sites are at nt-1491(ATTAATT) and nt-1178(ATTCATT). Moreover, Wnt8B contributes to the effect of ZNF191 on cell proliferation, and Wnt8B expression correlates positively with ZNF191 in human HCCs. Our findings suggested that Wnt8B, directly transcriptionally regulated by ZNF191, plays a pivotal role in HCC proliferation via the canonical Wnt pathway and may serve as a new prognostic biomarker and a potential therapeutic target for HCC patients.

Platelet sonicates activate hair follicle stem cells and mediate enhanced hair follicle regeneration.

An increasing number of studies show that platelet-rich plasma (PRP) is effective for androgenic alopecia (AGA). However, the underlying cellular and molecular mechanisms along with its effect on hair follicle stem cells are poorly understood. In this study, we designed to induce platelets in PRP to release factors by calcium chloride (PC) or by sonication where platelet lysates (PS) or the supernatants of platelet lysate (PSS) were used to evaluate their effect on the hair follicle activation and regeneration. We found that PSS and PS exhibited a superior effect in activating telogen hair follicles than PC. In addition, PSS injection into the skin activated quiescent hair follicles and induced K15+ hair follicle stem cell proliferation in K14-H2B-GFP mice. Moreover, PSS promoted skin-derived precursor (SKP) survival in vitro and enhanced hair follicle formation in vivo. In consistence, protein array analysis of different PRP preparations revealed that PSS contained higher levels of 16 growth factors (out of 41 factors analysed) than PC, many of them have been known to promote hair follicle regeneration. Thus, our data indicate that sonicated PRP promotes hair follicle stem cell activation and de novo hair follicle regeneration.

Cyclin Y binds and activates CDK4 to promote the G1/S phase transition in hepatocellular carcinoma cells via Rb signaling.

Inactivation of Rb is a major event in the development of hepatocellular carcinoma (HCC). The activity of CDK4, determined by T172 phosphorylation, correlates with the onset of RB phosphorylation and G1/S cell cycle transition. However, the regulation of CDK4 activation and of the Rb pathway in HCC remain unclear. Here, we report that cyclin Y, a novel member of the cyclin family, is a potential regulator of the Rb pathway. We demonstrate that the Cyclin Y protein was overexpressed in human HCC tissues and that it was associated with poor patient prognosis. Cyclin Y could regulate the G1/S phase transition in human HCC cell lines. We found that CDK4 can bind to Cyclin Y in vitro. Furthermore, the accumulation of Cyclin Y could activate CDK4 through T172 phosphorylation of CDK4, inactivate Rb with increasing Rb phosphorylation, and enable the expression of E2F target genes such as CDK2 and Cyclin A. Thus, our findings suggest that Cyclin Y plays a role in the G1/S phase transition of HCC cells via Cyclin Y/CDK4/Rb signaling and that Cyclin Y could be used as a potential prognostic biomarker in HCC.

Multi-Site Attack, Neutrophil Membrane-Camouflaged Nanomedicine with High Drug Loading for Enhanced Cancer Therapy and Metastasis Inhibition.

Background: Advanced breast cancer is a highly metastatic tumor with high mortality. Simultaneous elimination of primary tumor and inhibition of neutrophil-circulation tumor cells (CTCs) cluster formation are urgent issues for cancer therapy. Unfortunately, the drug delivery efficiency to tumors and anti-metastasis efficacy of nanomedicine are far from satisfactory. Methods: To address these problems, we designed a multi-site attack, neutrophil membrane-camouflaged nanoplatform encapsulating hypoxia-responsive dimeric prodrug hQ-MMAE2 (hQNM-PLGA) for enhanced cancer and anti-metastasis therapy. Results: Encouraged by the natural tendency of neutrophils to inflammatory tumor sites, hQNM-PLGA nanoparticles (NPs) could target delivery of drug to tumor, and the acute hypoxic environment of advanced 4T1 breast tumor promoted hQ-MMAE2 degradation to release MMAE, thus eliminating the primary tumor cells to achieve remarkable anticancer efficacy. Alternatively, NM-PLGA NPs inherited the similar adhesion proteins of neutrophils so that NPs could compete with neutrophils to interrupt the formation of neutrophil-CTC clusters, leading to a reduction in extravasation of CTCs and inhibition of tumor metastasis. The in vivo results further revealed that hQNM-PLGA NPs possessed a perfect safety and ability to inhibit tumor growth and spontaneous lung metastasis. Conclusion: This study demonstrates the multi-site attack strategy provides a prospective avenue with the potential to improve anticancer and anti-metastasis therapeutic efficacy.

Self-Rectifiable and Hypoxia-Assisted Chemo-Photodynamic Nanoinhibitor for Synergistic Cancer Therapy.

Photodynamic therapy (PDT) can eradicate cancer cells under light irradiation, mainly because of reactive singlet oxygen (1O2) being transformed from intratumoral oxygen. Nonetheless, the consumption of oxygen during PDT results in serious hypoxic conditions and an elevated hypoxia-inducing factor-1α (HIF-1α) level that hamper further photodynamic efficacy and induce tumor metastasis. To address this problem, we developed hypoxia-assisted NP-co-encapsulating Ce6 (photosensitizer) and YC-1 (HIF-1α inhibitor) as a self-rectifiable nanoinhibitor for synergistic antitumor treatment. PDT-aggravated intracellular hypoxic stress facilitated NP dissociation to release the drug (YC-1), which achieved tumor killing and HIF-1α inhibition to further enhance the therapeutic effect of PDT and prevent tumor metastasis. Besides, in vivo studies revealed that the HC/PI@YC-1 NPs afforded synergistic anticancer efficacy with minimal toxicity. Therefore, this study provides a prospective approach against PDT drawbacks and combination cancer therapy.

ZNF191 alters DNA methylation and activates the PI3K-AKT pathway in hepatoma cells via transcriptional regulation of DNMT1.

BACKGROUND: Alteration of DNA methylation is an important event in pathogenesis and progression of hepatocellular carcinoma (HCC). DNA methyltransferase (DNMT) 1, the foremost contributor in DNA methylation machinery, was revealed elevated in HCC and significantly correlates with poor prognosis. However, the transcriptional regulation of DNMT1 in HCC remains unknown. METHODS: Real-time PCR and immunohistochemistry were performed to detect DNMT1 and zinc finger transcription factor 191 (ZNF191) expressions in HCCs. Transcription activity of DNMT1promoter was analyzed with Luciferase reporter activity assay. The binding capacity of ZNF191 protein to DNMT1 promoter was examined with chromatin immunoprecipitation-qPCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA). DNA methylation level of hepatoma cells was detected with Methylation array. RESULTS: ZNF191 can regulate DNMT1 mRNA and protein expression positively, and increase the transcription activity of the DNMT1 promoter. ChIP-qPCR and EMSA revealed that ZNF191 protein directly binds to the DNMT1 promoter at nt-240 AT(TCAT)3 TC. Moreover, DNMT1 and ZNF191 expression correlate positively in human HCCs. With methylation array, DNA methylation alteration was observed in hepatoma cells with ZNF191 knockdown, and the differential methylation sites are enriched in the PI3K-AKT pathway. Furthermore, we proved DNMT1 contributes the effect of ZNF191 on hepatoma cell growth via the PI3K-AKT pathway. CONCLUSION: ZNF191 is a novel transcription regulator for DNMT1, and the pro-proliferation effect of ZNF191/DNMT1/p-AKT axis in hepatoma cells implies that ZNF191 status in HCCs may affect the therapeutic effect of DNMTs inhibitors and PI3K inhibitors for precise treatment of the disease.