Yijuan Zhang

Polypeptide cationic micelles mediated co-delivery of docetaxel and siRNA for synergistic tumor therapy

Combination of two or more therapeutic strategies with different mechanisms can cooperatively impede tumor growth. Co-delivery of chemotherapeutic drug and small interfering RNA (siRNA) within a single nanoparticle (NP) provides a rational strategy for combined cancer therapy. Here, we prepared polypeptide micelle nanoparticles (NPs) of a triblock copolymer poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-leucine) (PEG-PLL-PLLeu) to systemically codeliver docetaxel (DTX) and siRNA-Bcl-2 for an effective drug/gene vector. The hydrophobic PLLeu core entrapped with anticancer drugs, while the PLL polypeptide cationic backbone allowed for electrostatic interaction with the negatively charged siRNA. The resulting micelle NP exhibited very stable, good biocompatible and excellent passive targeted properties. The micelle complexes with siRNA-Bcl-2 effectively knocked down the expression of Bcl-2 mRNA and protein. Moreover, the co-delivery system of DTX and siRNA-Bcl-2 (DTX-siRNA-NPs) obviously down-regulation of the anti-apoptotic Bcl-2 gene and enhanced antitumor activity with a smaller dose of DTX, resulting the significantly inhibited tumor growth of MCF-7 xenograft murine model as compared to the individual siRNA and only DTX treatments. Our results demonstrated well-defined PEG-PLL-PLLeu polypeptide cationic micelles with the excellent synergistic effect of DTX and siRNA-Bcl-2 in combined cancer therapy.

PEGylated cationic liposomes robustly augment vaccine-induced immune responses: Role of lymphatic trafficking and biodistribution

Lymph nodes (LNs) are peripheral lymphoid organs essential for vaccine-induced immune responses. Although cationic liposomes have been documented as a novel adjuvant and vaccine delivery system, whether enhancing LN targeting would improve the efficiency of cationic liposome-formulated vaccines has not been elucidated yet. In the present study we investigated the effect of PEGylation on LN targeting and the immunogenicity of cationic liposome-formulated vaccines. DOTAP cationic liposomes were incorporated with 1 or 5mol% of DSPE-PEG2000 and labeled with near infrared fluorescent dyes. The lymphatic trafficking and biodistribution of different liposomes after subcutaneous (s.c.) injection were recorded using an in-vivo imaging system. The results showed that incorporation of 1mol% DSPE-PEG2000 not only accelerated the drainage of DOTAP liposomes into draining LNs, but also prolonged their LN retention and enhanced liposome uptake by resident antigen-presenting cells. On the other hand, although incorporating 5mol% of DSPE-PEG2000 into DOTAP liposomes enhanced their LN retention and uptake to a lesser extent, it prolonged blood circulation of DOTAP liposomes and increased their splenic accumulation. In addition, PEGylated DOTAP liposomes augmented primary and secondary anti-OVA antibody responses more potently than nonPEGylated DOTAP liposomes did. Hence, incorporating a small amount of DSPE-PEG2000 into DOTAP liposomes not only increased the passive LN targeting of DOTAP-formulated vaccines but also modulated their biodistribution in vivo, which consequently improved the efficiency of cationic liposome-formulated vaccines.

Bioreducible alginate-poly(ethylenimine) nanogels as an antigen-delivery system robustly enhance vaccine-elicited humoral and cellular immune responses

Although polysaccharide nanogels have emerged as a novel antigen delivery system for vaccine development, whether modulating the redox sensitivity of nanogels could improve vaccine efficacy remains unclear. In the present study, we generated bioreducible cationic alginate-polyethylenimine (PEI) nanogels as a novel vaccine delivery system. Briefly, nanogels were prepared by the electrostatic interaction of negatively charged alginate sodium with branched PEI2k, followed by disulfide cross-linking to generate bioreducible nanogels (AP-SS). The AP-SS nanogels demonstrated great antigen-loading capacity and minimal cytotoxicity. The in vitro study showed that reducible AP-SS nanogels not only facilitated antigen uptake by mouse bone marrow dendritic cells (BMDCs), but also promoted intracellular antigen degradation and cytosolic release. Moreover, AP-SS nanogels significantly enhanced both MHC class I and II antigen presentation by BMDCs. Compared with the non-reducible nanogels, AP-SS nanogels more potently enhanced vaccine-induced antibody production and CD8+ T cell-mediated tumor cell lysis. Hence, the bioreducible alginate-PEI nanogels could serve as a potent adjuvant to improve vaccine-elicited humoral and cellular immune responses.

Cationic polypeptide micelle-based antigen delivery system: A simple and robust adjuvant to improve vaccine efficacy

Modern subunit vaccines with purified or recombinant antigens are important alternatives to the traditional vaccines. However, there remains a big challenge to elicit potent antibody production and CD8 T cell response. Nanoparticle-based antigen delivery systems have emerged as an innovative strategy to improve the efficacy of subunit vaccines. The present study reported self-assembled cationic micelles based on poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-leucine) (PEG-PLL-PLLeu) hybrid polypeptides as a simple and potent vaccine delivery system. The results showed that the PEG-PLL-PLLeu micelles spontaneously encapsulated OVA antigens with great loading capacity (LC=55%) and stability. More importantly, the polypeptide micelle formulations robustly enhanced vaccine-induced antibody production by 70-90 fold, which could be due to their capability of inducing dendritic cell maturation, enhancing antigen uptake and presentation, as well as promoting germinal center formation. Furthermore, the polypeptide micelles could simultaneously encapsulate OVA and polyriboinosinic: polyribocytidylic acid (PIC), a TLR3 agonist, to synergistically augment tumor specific cytotoxic-T-lymphocyte (CTL) response. Hence, the polypeptide micelle-based antigen delivery system could be a robust adjuvant to enhance vaccine-induced immune responses.

Toll-like receptor 3 agonist complexed with cationic liposome augments vaccine-elicited antitumor immunity by enhancing TLR3-IRF3 signaling and type I interferons in dendritic cells.

Cancer vaccine-based immunotherapy is emerging as a novel therapeutic strategy for cancer treatment. However, its antitumor effect remains unsatisfied due to the poor immunogenicity of tumor antigens (Ags). Although polyriboinosinic: polyribocytidylic acid (PIC), a TLR3 agonist, has been reported as a promising adjuvant for cancer vaccines, its immunopotency may be limited by insufficient cellular penetration. In the present study, we incorporated PIC into DOTAP cationic liposome to generate PIC-DOTAP Liposome Complex (PDLC) nanoparticles. The results showed that PDLC was more potent than DOTAP or PIC to enhance vaccine-induced tumor-specific cytotoxic T lymphocyte (CTL) response and IFN-γ production. Moreover, two doses of PDLC vaccines remarkably suppressed tumor growth in mice, which involved the participance of CD8(+) T cells and depended on the presence of Ag. The superior antitumor effect of PDLC vaccines could be attributable to enhanced maturation of mouse bone-marrow dendritic cells (BMDCs) and increased production of type I IFNs. More importantly, PDLC strengthened the TLR3 signaling in BMDCs by enhancing the interaction of PIC with TLR3 and augmenting downstream IRF-3 phosphorylation, as well as elevating IRF-3/IRF-7 mRNA transcription. Taking together, the complex of PIC and DOTAP liposomes enhanced PIC uptake and consequential TLR3 signaling in BMDCs, which in turn promoted DC maturation and type I IFN production, thereby augmenting the antitumor effect of cancer vaccines.

Autophagy mediates avian influenza H5N1 pseudotyped particle-induced lung inflammation through NF-κB and p38 MAPK signaling pathways

Since avian influenza virus H5N1-induced hypercytokemia plays a key role in acute lung injury, understanding its molecular mechanism is highly desirable for discovering therapeutic targets against H5N1 infection. In the present study, we investigated the role of autophagy in H5N1-induced lung inflammation by using H5N1 pseudotyped viral particles (H5N1pps). The results showed that H5N1pps significantly induced autophagy both in A549 human lung epithelial cells and in mouse lung tissues, which was primarily due to hemagglutinin (HA) of H5N1 virus. Blocking autophagy with 3-methyladenine (an autophagy inhibitor) or siRNA knockdown of autophagy-related genes (beclin1 and atg5) dramatically attenuated H5N1pp-induced proinflammatory cytokines and chemokines, such as IL-1β, TNF-α, IL-6, CCL2, and CCL5, both in vitro and in vivo. Autophagy-mediated inflammatory responses involved the activation of NF-κB and p38 MAPK signaling pathways, which required the presence of clathrin but did not rely on p62 or autophagosome-lysosome fusion. On the other hand, the activation of NF-κB also promoted H5N1pp-induced autophagosome formation. These data indicated a positive feedback loop between autophagy and NF-κB signaling cascade, which could exacerbate H5N1pp-induced lung inflammation. Our data demonstrated an essential role of autophagy in H5N1pp-triggered inflammatory responses, and targeting the autophagic pathway could be a promising strategy to treat H5N1 virus-caused lung inflammation.

Dual effects of interleukin-18: inhibiting hepatitis B virus replication in HepG2.2.15 cells and promoting hepatoma cells metastasis

Interleukin-18 (IL-18) has been reported to inhibit hepatitis B virus (HBV) replication in the liver of HBV transgenic mice; however, the molecular mechanism of its antiviral effect has not been fully understood. In the present study, it was shown that IL-18 and its receptors (IL-18R) were constitutively expressed in hepatoma cell lines HepG2 and HepG2.2.15 as well as normal liver cell line HL-7702. We demonstrated that IL-18 directly inhibited HBV replication in HepG2.2.15 cells via downregulating the activities of HBV core and X gene promoters. The suppressed HBV replication by IL-18 could be rescued by the administration of BAY11-7082, an inhibitor of transcription factor NF-κB. On the other hand, it was of interest that IL-18 promoted HepG2 cell metastasis and migration dose dependently in both wound-healing assays and Transwell assays. The underlying mechanism could be partially attributable to the increased activities of extracellular matrix metalloproteinase (MMP)-9, MMP-3, and MMP-2 by IL-18, which upregulated the mRNA levels of MMP-3 and MMP-9 in a NF-κB-dependent manner. Furthermore, it was confirmed that expression of IL-18/IL-18R and most MMPs were remarkably upregulated in hepatocellular carcinoma (HCC) liver cancer tissue specimens, suggesting that IL-18/IL-18R-triggered signaling pathway was closely related to HCC metastasis in vivo. Therefore, we revealed the dual effects of IL-18 in human hepatocytes: it not only inhibited HBV replication but also promoted hepatoma cells metastasis and migration. NF-κB played a critical role in both effects. Our work contributed to a deeper understanding of the biological function of IL-18 in human hepatocytes.

Noninvasive visualization of respiratory viral infection using bioorthogonal conjugated near-infrared-emitting quantum dots.

Highly pathogenic avian influenza A viruses are emerging pandemic threats in human beings. Monitoring the in vivo dynamics of avian influenza viruses is extremely important for understanding viral pathogenesis and developing antiviral drugs. Although a number of technologies have been applied for tracking viral infection in vivo, most of them are laborious with unsatisfactory detection sensitivity. Herein we labeled avian influenza H5N1 pseudotype virus (H5N1p) with near-infrared (NIR)-emitting QDs by bioorthogonal chemistry. The conjugation of QDs onto H5N1p was highly efficient with superior stability both in vitro and in vivo. Furthermore, QD-labeled H5N1p (QD-H5N1p) demonstrated bright and sustained fluorescent signals in mouse lung tissues, allowing us to visualize respiratory viral infection in a noninvasive and real-time manner. The fluorescence signals of QD-H5N1p in lung were correlated with the severity of virus infection and significantly attenuated by antiviral agents, such as oseltamivir carboxylate and mouse antiserum against H5N1p. The biodistribution of QD-H5N1p in lungs and other organs could be easily quantified by measuring fluorescent signals and cadmium concentration of virus-conjugated QDs in tissues. Hence, virus labeling with NIR QDs provides a simple, reliable, and quantitative strategy for tracking respiratory viral infection and for antiviral drug screening.

Retinal-conjugated pH-sensitive micelles induce tumor senescence for boosting breast cancer chemotherapy

Evoking tumor cellular senescence, an irreversible status of cell growth quiescence, has been recently proposed as a potential strategy to improve the efficacy of cancer treatment. In the current study, all-trans retinal, the precursor of all-trans retinoic acid, was conjugated to dextran via hydrazone bond to generate amphiphilic dextran-retinal (DR) conjugates, which self-assembled into pH-sensitive DR micelles. Our results showed that DR micelles moderately inhibited MCF-7 breast cancer cell growth through inducing p21-associated cellular senescence, which relied on retinoic acid receptors (RARs) and was accompanied by significant G0/G1 cell cycle arrest. Moreover, DR micelles were capable of encapsulating doxorubicin (DOX) to generate DOX-loaded DD micelles, facilitating the uptake and release of DOX in cancer cells. Compared with free DOX, DD micelles more effectively suppressed tumor growth and prolonged survival time of mouse xenograft model through inducing tumor apoptosis and cellular senescence. However, blocking cellular senescence diminished DD-caused apoptosis in MCF-7 cells by 40-50%. Therefore, pH-sensitive DR micelles not only served as a potent platform for DOX delivery, but also enhanced the anti-tumor effect of DOX by inducing tumor cellular senescence. These data reveal a great potential of evoking tumor senescence with retinal-conjugated micelles for boosting breast cancer chemotherapy.

Fluorophore Analyses of Vulnerable Atherosclerotic Plaque and Its Early Detection

The diagnosis of atherosclerotic plaque based on time-resolved laser-induced fluorescence spectroscopy and the relationship of the endogenous fluorophores in lesions with the wavelength-resolved and especially the time-resolved data were investigated. The major fluorophores in arterial tissues are elastin and collagen (type I and III), and lipid components such as free cholesterol, cholesterol esters and LDLs. Elastin which has broad spectral emission is the primary fluorophore in normal artery, whereas collagen which has narrow band and blue-shifted peak emission and the highest lifetime is the mainly fluorophore in collagen-rich plaques. Lipid components that have the fastest fluorescence dynamics mainly exist in lipid-rich lesions which are always instability. At last a dual-modality system combined two complementary techniques was also introduced to diagnose vulnerable plaques.