Precise delivery of doxorubicin and imiquimod through pH-responsive tumor microenvironment-active targeting micelles for chemo- and immunotherapy.

Recently, combining immunotherapy and chemotherapy has become a promising strategy to treat cancer. However, this therapeutic strategy still has its limitations because of the adverse effects caused by the simultaneous administration of multiple therapeutic agents. Using nanoparticles is an effective approach to successfully combine these therapies because they can reduce side effects, increase circulation time, and ensure the delivery of cytotoxic agents to tumor tissues. In this study, dual pH-sensitive and tumor microenvironment (TME)-active targeting micelles comprising poly(propyl methacrylate-co-glucosamine/histidine/doxorubicin) (P(PAA-co-GLU/HIS/DOX) and methoxy-poly(ethylene glycol)-block-poly(l-lysine) were prepared to encapsulate an immunomodulator, imiquimod (IMQ). Because these micelles can expose glucose targeting ligands at the TME and pH-dependently release IMQ and DOX, micelles effectively inhibit the growth of 4T1 cells selectively and highly accumulate in 4T1 cells as the pH decreased to 6.5. Moreover, in RAW 264.7 â€‹cells, these micelles prevent cell death and induce M1 macrophage polarization. In 4T1 orthotopic tumor-bearing mice, micelles not only exhibited high tumor accumulation, effective tumor inhibition, and fewer adverse effects, but also dramatically increased the number of mature dendritic cells, activate cytotoxic T cells, and polarize M1-like macrophages in tumor tissues. Overall, these micelles exhibit precise pH responsiveness and ideal drug delivery capabilities for combined chemo- and immunotherapy; these results significantly contribute to the future development of nanomedicines in cancer therapy.

Development of a Rapid-Onset, Acid-Labile Linkage Polyplex-Mixed Micellar System for Anticancer Therapy.

In the treatment of cancers, small interfering ribonucleic acids (siRNAs) are delivered into cells to inhibit the oncogenic protein's expression; however, polyanions, hydrophilicity, and rapid degradations in blood, endosomal or secondary lysosomal degradation hamper clinal applications. In this study, we first synthesized and characterized two copolymers: methoxy poly(ethylene glycol)-b-poly(2-hydroxy methacrylate-ketal-pyridoxal) and methoxy poly(ethylene glycol)-b-poly(methacrylic acid-co-histidine). Afterwards, we assembled two polymers with the focal adhesion kinase (FAK) siRNA, forming polyplex-mixed micelles for the treatment of the human colon cancer cell line HCT116. In terms of the physiological condition, the cationic pyridoxal molecules that were conjugated on the copolymer with ketal bonds could electrostatically attract the siRNA. Additionally, the pyridoxal could form a hydrophobic core together with the hydrophobic deprotonated histidine molecules in the other copolymer and the hydrophilic polyethylene glycol (PEG) shell to protect the siRNA. In an acidic condition, the pyridoxal would be cleaved from the polymers due to the breakage of the ketal bonds and the histidine molecules can simultaneously be protonated, resulting in the endosome/lysosome escape effect. On the basis of our results, the two copolymers were successfully prepared and the pyridoxal derivatives were identified to be able to carry the siRNA and be cleavable by the copolymers in an acidic solution. Polyplex-mixed micelles were prepared, and the micellar structures were identified. The endosome escape behavior was observed using a confocal laser scanning microscopy (CLSM). The FAK expression was therefore reduced, and the cytotoxicity of siRNA toward human colon cancer cells was exhibited, rapidly in 24 h. This exceptional anticancer efficiency suggests the potential of the pH-sensitive polyplex-mixed micellar system in siRNA delivery.

Preparation of Immunotherapy Liposomal-Loaded Thermal-Responsive Hydrogel Carrier in the Local Treatment of Breast Cancer.

To reduce the side effects of immune drugs and the sustainable release of immune drugs on local parts, we have designed an injectable thermal-sensitive hydrogel containing an imiquimod-loaded liposome system. In the extracellular environment of tumor tissues (pH 6.4), 50% of the drug was released from the carrier, which could be a result of the morphological changes of the liposomal microstructure in the acidic environment. According to the results in animals, the drug-containing liposomes combined with hydrogel can be effectively applied in breast cancer therapy to delay the growth of tumors as well as to dramatically reduce the death rate of mice.

Preparation and Characterization of Electrostatically Crosslinked Polymer⁻Liposomes in Anticancer Therapy.

pH-sensitive polymer⁻liposomes can rapidly release their payloads. However, it is difficult to simultaneously achieve stability and pH-responsiveness in the polymer⁻liposomes. In this study, stable and pH-sensitive crosslinked polymer⁻liposomes were fabricated through electrostatic interactions. The pH-sensitive copolymer methoxy poly(ethylene glycol)-block-poly(methacrylic acid)-cholesterol (mPEG-b-P(MAAc)-chol) and crosslinking reagent poly(ethylene glycol) with end-capped with lysine (PEG-Lys2) were synthesized and characterized. At physiological conditions, the pH-sensitive copolymers were anionic and interacted electrostatically with the cationic crosslinker PEG-Lys2, forming the electrostatically-crosslinked polymer⁻liposomes and stabilizing the liposomal structure. At pH 5.0, the carboxylic groups in mPEG-b-P(MAAc)-chol were neutralized, and the liposomal structure was destroyed. The particle size of the crosslinked polymer⁻liposomes was approximately 140 nm and the polymer⁻liposomes were loaded with the anticancer drug doxorubicin. At pH 7.4, the crosslinked polymer⁻liposomes exhibited good stability with steady particle size and low drug leakage, even in the presence of fetal bovine serum. At pH 5.0, the architecture of the crosslinked polymer⁻liposomes was damaged following rapid drug release, as observed by using transmission electron microscopy and their apparent size variation. The crosslinked polymer⁻liposomes were pH-sensitive within the endosome and in the human breast cancer cells MDA-MB-231, as determined by using confocal laser scanning microscopy. The intracellular drug release profiles indicated cytotoxicity in cancer cells. These results indicated that the highly-stable and pH-sensitive electrostatically-crosslinked polymer⁻liposomes offered a potent drug-delivery system for use in anticancer therapies.

A corn straw-based diet increases release of inflammatory cytokines in peripheral blood mononuclear cells of dairy cows / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Recent studies have shown that diet can affect the body's immunity. Roughage of dairy cows consists of a variety of plant materials which make different contributions to health. This study investigated the effect of different roughages on the immunity of dairy cows. Serum, peripheral blood mononuclear cells (PBMCs), and milk samples were collected from 20 multiparous mid-lactation cows fed mixed forage (MF)- or corn straw (CS)-based diets. Expression profile analysis was used to detect the differentially expressed genes (DEGs) from PBMCs. The results showed that milk protein in the MF group increased to 3.22 g/100 ml, while that of the CS group milk was 2.96 g/100 ml; by RNA sequencing, it was found that 1615 genes were differentially expressed between the CS group and the MF group among the 24 027 analyzed probes. Gene ontology (GO) and pathway analysis of DEGs suggested that these genes (especially genes coding cytokines, chemokine and its receptors) are involved in the immune response. Results were confirmed at the protein level via detecting the levels of interleukin-2 (IL-2), IL-6, IL-10, IL-12, leptin (LEP), interferon-γ (IFN-γ), transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) in peripheral blood by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay analysis. Our data supported the conclusions that the protein content in milk of the MF group was higher than that of the CS group, the CS-based diets induced more release of cytokines than the MF-based diets in dairy cows' PBMCs, and milk protein content may be affected by cytokines.

Multifunctional Polymer Nanoparticles for Dual Drug Release and Cancer Cell Targeting.

Multifunctional polymer nanoparticles have been developed for cancer treatment because they could be easily designed to target cancer cells and to enhance therapeutic efficacy according to cancer hallmarks. In this study, we synthesized a pH-sensitive polymer, poly(methacrylic acid-co-histidine/doxorubicin/biotin) (HBD) in which doxorubicin (DOX) was conjugated by a hydrazone bond to encapsulate an immunotherapy drug, imiquimod (IMQ), to form dual cancer-targeting and dual drug-loaded nanoparticles. At low pH, polymeric nanoparticles could disrupt and simultaneously release DOX and IMQ. Our experimental results show that the nanoparticles exhibited pH-dependent drug release behavior and had an ability to target cancer cells via biotin and protonated histidine.

Comparison of plaque-forming characteristics and morphogenetic changes of Newcastle disease virus isolated from chickens and geese on host cells / 病毒学报

The plaque-forming characteristics of Newcastle disease viruses of chickens and geese source were compared on various cells. The result showed that there were obvious differences of plaque formation between F48E9 and NA-1 on Vero cells, chicken embryo fibroblast cells (CEF) and goose embryo fibroblast cells (GEF). The plaque-forming ability of NA-1 was higher than F48E9 on GEF, but lower than F48E9 on CEF. On Vero cells, the plaque-forming ability of NA-1 was slightly stronger than F48E9. It demonstrated that the plaque-forming characteristics were consistent with host tropism of virus. The morphogenesis of F48E9 and NA-1 on Vero cells was observed with transmission electron microscope. There were different replication processes between F48E9 and NA-1 on cells at different stages. NA-1 had stronger adaptability to host than F48E9 according to budding processes and envelope integrity.

Comparison of effectiveness of whole viral, N and N199 proteins by ELISA for the rapid diagnosis of severe acute respiratory syndrome coronavirus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Although severe acute respiratory syndrome (SARS) has been controlled, the subsequently emerging sporadic cases in 2004 emphasize the necessity of developing a rapid diagnostic method, which would be of great help in clinical diagnosis and also wild host screening. This study aims to establish an effective and rapid serological tool for the diagnosis of SARS-CoV by comparison among whole viral, N and N199 proteins by ELISA.</p><p><b>METHODS</b>SARS-CoV N and N199 (a truncated nucleocapsid gene) genes were cloned, expressed, identified by Western blotting, and applied in screening of human and swine samples. Sera of SARS convalescent-phase patients, normal human sera, sera of patients with other respiratory diseases, and swine sera were screened by ELISA, with whole SARS-CoV F69, N and N199 proteins as antigens.</p><p><b>RESULTS</b>The sensitivity and specificity of N and N199 proteins in human sera diagnosis were approximate (P = 0.743), which was higher than whole viral protein but the difference was not significant (P = 0.234). The N199 protein proved to be more specific in swine sera screening than whole viral and N protein (P < 0.001).</p><p><b>CONCLUSION</b>N199 protein is feasible in both clinical diagnosis and SARS-CoV reservoir screening.</p>

Salmonella choleraesuis C500 delivering DNA immunization against classical swine fever virus / 生物工程学报

Classical Swine Fever Virus (CSFV) E2 protein eukaryotic expression plasmid pVAXE2 was constructed. The plasmid pVAXE2 was transformed into Salmonella choleraesuis C500 (S. C500) attenuated vaccine strain by electroporation to generate Salmonella choleraesuis engineering strain S. C500/pVAXE2. The characterization of S. C500/pVAXE2 in morphology, growth, biochemistry and serology indicated that it retained the same properties as its original strain S. C500 with exception of kanamycin resistance originated from the plasmid pVAXE2. The plasmid stable in the bacteria after 15 passages. Kunming mice and rabbits were vaccinated three times at two weeks interval with S. C500/pVAXE2 in oral and intramuscular routes at the dosage of 1 x 10(8) CFU for mice and 2 x 10(9) CFU for rabbits each time. The specific antibody response against CSFV and Salmonella choleraesuis was detected by ELISA. Two weeks after the third boost the immunized rabbits were challenged with 20 ID50 of hog cholera lapinized virus (HCLV), followed by a virulent strain of Salmonella choleraesuis two week later than HCLV challenge. The results showed that all immunized mice and rabbits produced significant antibodies against CSFV and Salmonella choleraesuis, and the immunized rabbits demonstrated the effective protection against the challenge of HCLV and virulent Salmonella choleraesuis. These results indicated the potential of developing multiplex swine DNA vaccine by using this bacteria as the vector.

Construction of recombinant fowlpox virus coexpressing gp120 of Chinese HIV-1 strain and IL-18 and its immunogenicity in mice / 生物工程学报

To screening out Chinese vaccine candidate against HIV-1, Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing gp120 of Chinese HIV-1 strain and IL-18, and the recombinant virus was indentified by PCR and Western blot. The specific DNA fragment could be amplified by PCR from the genome of rFPV. Western blot analysis showed that gp120 and IL-18 could be expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, the spleen specific CTL activities and serum antibodies in the immunized mice were detected, which demonstrated that the rFPV had good immunogenicity and could induce BALB/c mice to produce specific humoral and cellular immunity. IL-18 palyed the role of immunoadjuvant. The study lays the basis on the preparation of genetic engineering live vector vaccine against HIV-1.

Effects of oxLDL and simvastatin on PKC activity and level of cytosolic free Ca 2+ in cultured human umbilical vein endothelial cells / 第二军医大学学报

Objective: To investigate the effects of oxLDL and HMG-CoA reductase inhibitor simvastatin on PKC activity, and level of cytosol ic free Ca 2+ in cultured human umbilical vein endothelial cells. Methods: Th e activity of PKC was determined by its ability to transfer phosphate from 32P-ATP to lysine-rich histone and level of cytosolic free calcium［Ca2+ ］i was measured by flow cytometric analysis loading with the Ca2+ dye F luo-3/Am. Results: oxLDL increased PKC total activity in a dose-de pendent manner and peaked after 12 min, then decreased slowly and maintained for at least 30 min, while oxLDL induced biphasic ［Ca2+］i responses includ ing the rapid initial transient phase and the sustained phase. Removal of extrac ellular Ca2+ did not inhibit the rapid transient phase, but abolished the sustained phase. When simvastatin was added, the activity of PKC wasmarkedly dec reased with no impairment to the initial peak response, but significantly reduce d the sustained phase. Conclusion: oxLDL can induced dynamic changes of signal transduction of PKC and level of cytosolic free Ca2+ in HUVEC, these 2 events are closely linked. The change of rapid initial transient phase i s the result of mobilization of Ca2+ from intracellular pool and the chang e of sustained phase is from the influx of extracellular Ca2+. The inhibit ion of PKC activity induced by simvastatin may contribute to the changes of ［Ca 2+］i.

Effects of oxLDL and simvastatin on PKC activity and level of cytosolic free Ca 2+ in cultured human umbilical vein endothelial cells / 第二军医大学学报

Objective: To investigate the effects of oxLDL and HMG-CoA reductase inhibitor simvastatin on PKC activity, and level of cytosol ic free Ca 2+ in cultured human umbilical vein endothelial cells. Methods: Th e activity of PKC was determined by its ability to transfer phosphate from 32P-ATP to lysine-rich histone and level of cytosolic free calcium［Ca2+ ］i was measured by flow cytometric analysis loading with the Ca2+ dye F luo-3/Am. Results: oxLDL increased PKC total activity in a dose-de pendent manner and peaked after 12 min, then decreased slowly and maintained for at least 30 min, while oxLDL induced biphasic ［Ca2+］i responses includ ing the rapid initial transient phase and the sustained phase. Removal of extrac ellular Ca2+ did not inhibit the rapid transient phase, but abolished the sustained phase. When simvastatin was added, the activity of PKC wasmarkedly dec reased with no impairment to the initial peak response, but significantly reduce d the sustained phase. Conclusion: oxLDL can induced dynamic changes of signal transduction of PKC and level of cytosolic free Ca2+ in HUVEC, these 2 events are closely linked. The change of rapid initial transient phase i s the result of mobilization of Ca2+ from intracellular pool and the chang e of sustained phase is from the influx of extracellular Ca2+. The inhibit ion of PKC activity induced by simvastatin may contribute to the changes of ［Ca 2+］i.