Preparation and cellular uptake study of anemoside B4 and PD-L1 siRNA co-delivered cRGD-modified targeting liposomes / 中国药房

OBJECTIVE To prepare anemoside B4 （AB4） and programmed cell death ligand 1 （PDL1） siRNA （siP） co- delivered cRGD-modified targeting liposomes （AB4/siP-c-L）， and to study the cellular uptake in vitro. METHODS The cRGD- modified AB4-loaded targeted liposomes （AB4-c-L） were prepared by ethanol injection. AB4-c-L was mixed with 20 nmol/L siP in the same volume and AB4/siP-c-L was obtained through electrostatic adsorption. The particle size， Zeta potential， morphology， encapsulation efficiency and drug content， in vitro release behavior and serum stability of AB4/siP-c-L were investigated by laser scattering particle size tester， transmission electron microscopy， ultrafiltration centrifugation， dialysis and agar-gel electrophoresis block test. Cellular uptake of AB4/siP-c-L by Lewis lung cancer cells LLC and its intracellular localization were evaluated by flow cytometry and confocal laser scan technique. RESULTS The average particle size of AB4/siP-c-L was （187.4±3.1） nm， and the Zeta potential was （33.5±1.4） mV. AB4/siP-c-L was spheroidal in shape. The encapsulation efficiency and content of AB4 were （95.2±0.4） % and （1.0±0.2） mg/mL， respectively. AB4/siP-c-L could better package siP， and exhibited good serum stability， obvious pH sensitivity and sustained release property. The uptake rate of AB4/siP-c-L by LLC cells was significantly higher than that of free drug， and was able to accumulate in cytoplasm. CONCLUSIONS AB4/siP-c-L can effectively realize the co-loading of AB4 and gene drug siP， which has certain in vitro targeting to LLC cells.

Establishment an aggregation-induced emission vesicle material based on supramolecular host-guest chemical assembly for siRNA delivery and study on its toxicity to tumor cells / 国际生物医学工程杂志

Objective:To establish an aggregation-induced emission vesicle material based on supramolecular host-guest chemical assembly (AIE-HG-Vesicle) for siRNA delivery and fluorescence imaging, and to explore its uptake effect by tumor cells and siRNA-based cell killing effect.Methods:By synthesizing β-cyclodextrin modified with polyethyleneimine dendrimer (H-β-CD-dendrimer) as a host compound and a Bola type adamantane containing tetrastyrene AIE group (G-Ada-AIE) as a guest compound, the nanovesicle material was prepared by a supramolecular host-guest self-assembly process for loading siRNA. The morphology and size of the materials were tested by transmission electron microscopy and the dynamic light scattering method. The aggregation-induced luminescence properties of the materials were investigated by fluorescence spectrophotometry. The loading effect of the material on siRNA was investigated by gel retardation experiments. The delivery effect of siRNA-loaded AIE-HG-Vesicle vesicles in tumor cells was observed by a confocal laser scanning microscope. The killing effect of siRNA-loaded AIE-HG-Vesicle vesicles on tumor cells was tested by an MTT assay.Results:The prepared host-guest compounds can be assembled into vesicles with a size of about 100 nm and wall thickness of 9 nm in solution, and the positively charged vesicles on the surface can efficiently load siRNA. The siRNA-loaded AIE-HG-Vesicle vesicles can deliver siRNA into HeLa tumor cells and can be observed through aggregation-induced luminescence. The siRNA-loaded vesicles have an obvious killing effect on HeLa tumor cells.Conclusions:A vesicle material with aggregation-induced luminescence properties was prepared by a method based on supramolecular host-guest chemical assembly, which can be used to deliver siRNA. The material has fluorescence imaging and siRNA-based tumor cytotoxic effects and is expected to be applied to tumor treatment in vivo.

Doxorubicin-conjugated siRNA lipid nanoparticles for combination cancer therapy

Evasion of apoptosis is a hallmark of cancer, attributed in part to overexpression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2). In a variety of cancer types, including lymphoma, Bcl-2 is overexpressed. Therapeutic targeting of Bcl-2 has demonstrated efficacy in the clinic and is the subject of extensive clinical testing in combination with chemotherapy. Therefore, the development of co-delivery systems for Bcl-2 targeting agents, such as small interfering RNA (siRNA), and chemotherapeutics, such as doxorubicin (DOX), holds promise for enabling combination cancer therapies. Lipid nanoparticles (LNPs) are a clinically advanced nucleic acid delivery system with a compact structure suitable for siRNA encapsulation and delivery. Inspired by ongoing clinical trials of albumin-hitchhiking doxorubicin prodrugs, here we developed a DOX-siRNA co-delivery strategy via conjugation of doxorubicin to the surface of siRNA-loaded LNPs. Our optimized LNPs enabled potent knockdown of Bcl-2 and efficient delivery of DOX into the nucleus of Burkitts' lymphoma (Raji) cells, leading to effective inhibition of tumor growth in a mouse model of lymphoma. Based on these results, our LNPs may provide a platform for the co-delivery of various nucleic acids and DOX for the development of new combination cancer therapies.

Progress in non-viral localized delivery of siRNA therapeutics for pulmonary diseases

Emerging therapies based on localized delivery of siRNA to lungs have opened up exciting possibilities for treatment of different lung diseases. Localized delivery of siRNA to lungs has shown to result in severalfold higher lung accumulation than systemic route, while minimizing non-specific distribution in other organs. However, to date, only 2 clinical trials have explored localized delivery of siRNA for pulmonary diseases. Here we systematically reviewed recent advances in the field of pulmonary delivery of siRNA using non-viral approaches. We firstly introduce the routes of local administration and analyze the anatomical and physiological barriers towards effective local delivery of siRNA in lungs. We then discuss current progress in pulmonary delivery of siRNA for respiratory tract infections, chronic obstructive pulmonary diseases, acute lung injury, and lung cancer, list outstanding questions, and highlight directions for future research. We expect this review to provide a comprehensive understanding of current advances in pulmonary delivery of siRNA.

Effects of Chitosan Nanoparticles with Long Synthetic siRNAs Targeting VEGF in Triple-Negative Breast Cancer Cells

Abstract Vascular endothelial growth factor (VEGF) is an essential angiogenic factor in breast cancer development and metastasis. Small interfering RNAs (siRNAs) can specifically silence genes via the RNA interference pathway, therefore were investigated as cancer therapeutics. In this study, we investigated the effects of siRNAs longer than 30 base pairs (bp) loaded into chitosan nanoparticles in triple-negative breast cancer cells, compared with conventional siRNAs. 35 bp long synthetic siRNAs inhibited VEGF gene expression by 51.2% and increased apoptosis level by 1.75-fold in MDA-MB-231 cell lines. Furthermore, blank and siRNA-loaded chitosan nanoparticles induced expression of IFN-γ in breast cancer cells. These results suggest that long synthetic siRNAs can be as effective as conventional siRNAs, when introduced into cells with chitosan nanoparticles

Swertiamarin attenuates paraquat-induced pulmonary epithelial-like cell apoptosis via NOX4-mediated regulation of redox and mitochondrial function

Abstract The aim of the present study was to investigate the effect of swertiamarin (STM) in attenuating paraquat (PQ)-induced human lung alveolar epithelial-like cell (A549) apoptosis and the underlying mechanisms. A549 cells were pretreated with different concentrations of STM for 2 hr and then cultured with or without PQ (700 µM) for 24 hr. Cell survival was determined using the CCK8 assay. Morphological changes, MDA content, inflammatory factors, fibrogenesis parameters, apoptosis rates, redox status and mitochondrial membrane potential (MMP) were evaluated. The expression of several genes involved in the modulation of redox status was measured by Western blotting. Cell viability and MMP were decreased, but the apoptosis rate and DCFH oxidation were elevated by PQ exposure. STM pretreatment notably increased cell viability and MMP and reduced the apoptosis rate and DCFH oxidation. Furthermore, TLR4- NOX4 signaling was significantly inhibited by STM. The downregulation of NOX4 by siRNA exerted the same protective effects as STM. This study provides the first evidence that STM attenuates PQ-induced pulmonary epithelial-like cell apoptosis via NOX4-mediated regulation of redox and mitochondrial function

Research progress on sRNA regulation of mosquito metabolic detoxification enzyme gene expression / 中国热带医学

@#Abstract: With the development of molecular biology, non-coding sRNA has been found to play an important regulatory role in gene expression and protein activity, affecting various biological pathways including mosquito resistance against insecticides. Understanding the molecular regulatory mechanisms of drug resistance is essential for controlling mosquitoes, , of which metabolic resistance being the most critical mechanism, mainly referring to the high expression of metabolic detoxification enzyme-related genes (especially the cytochrome P450 enzyme system) in mosquitoes. On the basis of verification of insecticide resistance-related genes, further research on the correlation between sRNA and mosquito resistance-related genes provides new ideas and directions for further exploring the mechanism of mosquito resistance. The study of mosquito metabolic resistance mechanism is of great significance for the control of vector mosquitoes, drug resistance monitoring and novel insecticide development. This article reviews the progress of research on the resistance genes, sRNAs biosynthesis, genes involved in regulating mosquito metabolic detoxification enzymes and their applications.

Synthesis and anti-HCC activity of full 2ʹ-F/OMe-siRNA encapsulated with neutral cytidinyl/cationic lipid / 药学学报

A variety of full 2ʹ-F/OMe-modified siRNAs were designed and synthesized, and the activity against hepatocellular carcinoma Huh-7 and HepG2 cells was evaluated. K&A DNA/RNA H-8 synthesizer was used to synthesize siRNAs, and neutral cytidinyl lipid DNCA mixed with cationic lipid CLD were used to transfect siRNA. By RT-qPCR and CCK-8 assay, the target gene silence and the proliferation of Huh-7 and HepG2 cells were detected. The siRNAs loading into Ago2 protein was detected by RNA-binding protein immunoprecipitation. Drug uptake and cell apoptosis were detected by flow cytometry, and the expression of PLK1 protein was detected by Western blot. Partial full 2ʹ-F/OMe modified siRNAs, especial siPLK1A3, increased the uptake of Huh-7 cells, enhanced their binding to Ago2 and gene silencing activity, down-regulated PLK1 protein, as well as induced more Huh-7 cell apoptosis and proliferation inhibition activity. It provides important data for the development of novel siRNA modification patterns and anti-HCC formulations.

Progress in RNA-targeting and Gene Editing Therapies for Transthyretin Amyloidosis Cardiomyopathy / 罕见病研究

Transthyretin(TTR) protein is a tetramer protein, synthesized mainly by the liver. TTR can be misfolded and deposited as amyloid fibrilae and deposited in the myocardial interstroma leading to transthyroxin amyloidosis cardiomyopathy (ATTR-CM). ATTR-CM was included in China's First List of Rare Diseases. Therapeutic strategies for ATTR-CM include blocking TTR synthesis in the liver, stabilizing TTR tetramers and destroying TTR fibra. Small molecule drugs such as tafamidis and diflunisal offer new treatment options for patients. Chlorobenzolic acid became the first drug approved by the U.S. Food and Drug Administration for the treatment of ATTR-CM. Small interfering RNA(siRNA)patisiran and antisense oligonucleotide (ASO)inotersen block TTR expression in the liver and have been approved for the treatment of ATTR variant polyneuropathy (ATTRv-PN)and are in phase Ⅲ trials for the treatment of ATTR-CM. Other siRNA drugs, vutrisiran, and ASO, eplontersen, are being evaluated for clinical efficacy. This article reviews the development of RNA-targeted therapeutics and gene-editing drugs using CRISPR-Cas9.

Lipid nanomaterials-based RNA therapy and cancer treatment

We summarize the most important advances in RNA delivery and nanomedicine. We describe lipid nanoparticle-based RNA therapeutics and the impacts on the development of novel drugs. The fundamental properties of the key RNA members are described. We introduced recent advances in the nanoparticles to deliver RNA to defined targets, with a focus on lipid nanoparticles (LNPs). We review recent advances in biomedical therapy based on RNA drug delivery and state-of-the-art RNA application platforms, including the treatment of different types of cancer. This review presents an overview of current LNPs based RNA therapies in cancer treatment and provides deep insight into the development of future nanomedicines sophisticatedly combining the unparalleled functions of RNA therapeutics and nanotechnology.

Concurrent silencing of TBCE and drug delivery to overcome platinum-based resistance in liver cancer

Platinum-based chemotherapy resistance is a key factor of poor prognosis and recurrence in hepatocellular carcinoma (HCC). Herein, RNAseq analysis revealed that elevated tubulin folding cofactor E (TBCE) expression is associated with platinum-based chemotherapy resistance. High expression of TBCE contributes to worse prognoses and earlier recurrence among liver cancer patients. Mechanistically, TBCE silencing significantly affects cytoskeleton rearrangement, which in turn increases cisplatin-induced cycle arrest and apoptosis. To develop these findings into potential therapeutic drugs, endosomal pH-responsive nanoparticles (NPs) were developed to simultaneously encapsulate TBCE siRNA and cisplatin (DDP) to reverse this phenomena. NPs (siTBCE + DDP) concurrently silenced TBCE expression, increased cell sensitivity to platinum treatment, and subsequently resulted in superior anti-tumor effects both in vitro and in vivo in orthotopic and patient-derived xenograft (PDX) models. Taken together, NP-mediated delivery and the co-treatment of siTBCE + DDP proved to be effective in reversing chemotherapy resistance of DDP in multiple tumor models.

Reprogrammed siTNFα/neutrophil cytopharmaceuticals targeting inflamed joints for rheumatoid arthritis therapy

Rheumatoid arthritis (RA) is an autoimmune disease characterized by severe synovial inflammation and cartilage damage. Despite great progress in RA therapy, there still lacks the drugs to completely cure RA patients. Herein, we propose a reprogrammed neutrophil cytopharmaceuticals loading with TNFα-targeting-siRNA (siTNFα) as an alternative anti-inflammatory approach for RA treatment. The loaded siTNFα act as not only the gene therapeutics to inhibit TNFα production by macrophages in inflamed synovium, but also the editors to reprogram neutrophils to anti-inflammatory phenotypes. Leveraging the active tendency of neutrophils to inflammation, the reprogrammed siTNFα/neutrophil cytopharmaceuticals (siTNFα/TP/NEs) can rapidly migrate to the inflamed synovium, transfer the loaded siTNFα to macrophages followed by the significant reduction of TNFα expression, and circumvent the pro-inflammatory activity of neutrophils, thus leading to the alleviated synovial inflammation and improved cartilage protection. Our work provides a promising cytopharmaceutical for RA treatment, and puts forward a living neutrophil-based gene delivery platform.

Highly penetrable nanoparticles co-loading doxorubicin and IDO1 siRNA enhance tumor immunotherapy / 药学学报

There are two serious obstacles to tumor immunotherapy. Firstly, the immune response of the tumor is seriously reduced due to immunosuppressive tumor microenvironment (ITM) and low immunogenicity of tumor. The second obstacle is the dense and complex heterogeneous structures, which seriously prevent the nanoparticles (NPs) from penetrating deeper into tumor tissue. Immunogenic cell death (ICD) induced by doxorubicin (DOX) is an effective method to enhance tumor immune activity. However, interferon-γ (IFN-γ) secreted by cytotoxic T lymphocytes (CTL) after ICD induction would increase the expression of indoleamine 2,3-dioxygenase 1 (IDO1) and enhance ITM. IDO1 siRNA would reduce the expression of IDO1 protein, regulate the tumor immunosuppressive microenvironment and regulate ITM, so as to enhance the ICD effect of DOX. In this paper, a novel charge conversional, particle size reduction and highly penetrable NPs based on a pH sensitive copolymer poly(ethylene glycol)-poly-L-lysine-2,3-dimethylmaleic anhydride (mPEG-PLL-DMA, PLD) and polyamidoamine (PAMAM) dendrimers to achieve deep delivery of tumor tissue. DOX and IDO1 siRNA were encapsulated to achieve efficient tumor immunotherapy. Preparation and cell level experiments showed that PLD material had significant pH sensitivity. Results of 3D tumor penetrable experiment in vitro showed that adding the pH sensitive material PLD significantly improved the permeability of the preparation. In addition, 4T1 tumor model was established for BALB/c mice and all animal experiments were displayed in according with the requirements of the Animal Experiment Ethics Committee of Shenyang Pharmaceutical University. The results of in vivo efficacy experiments and tissue experiments evaluated that IDO1 siRNA significantly improved the ICD effect owing to DOX, so as to significantly inhibit tumor growth.

Expression and regulation of keratin 6B in CD44+ bladder cancer stem cells / 中国组织工程研究

BACKGROUND: Bladder cancer stem cells could promote the recurrence and drug resistance of bladder cancer. Numerous studies have shown that keratin 6B (KRT6B) is involved in the production and progression of tumors, and is closely related to the prognosis of tumors. OBJECTIVE: To observe the expression of keratin 6B in CD44+ bladder cancer stem cells and to show the influence of keratin 6B on proliferation, migration, and self-renewal of bladder cancer stem cells, and to further explore the effect of keratin 6B expression on the prognosis of bladder cancer patients. METHODS: (1) CD44+ 5637 bladder cancer stem cells were isolated by magnetic active cell sorting. Cancer stem cell-related gene expression of SOX2, OCT4, and NANOG was detected via real-time polymerase chain reaction. The spheroid formation assay was used to detect the ability of self-renewal of cancer stem cells in CD44+ cells. Keratin 6B expression was detected in CD44+ bladder cancer stem cells by real-time polymerase chain reaction. (2) The CD44+5637 bladder cancer stem cells were divided into two groups. In the keratin 6B siRNA group, keratin 6B small interfering RNA was transfected into CD44+ bladder cancer stem cells. Untransfected CD44+ bladder cancer stem cells were used as the black control group. Cells were collected at 2 days post-transfection. The proliferation, migration, and self-renewal capacity of keratin 6B siRNA CD44+ bladder cancer stem cells were detected by the colony and wound healing assay and spheroid formation respectively. (3) Totally 24 bladder cancer tissues were used by immunohistochemistry to analyze the expression of CD44v6 and keratin 6B. (4) ONCOMINE database was used to analyze the effect of keratin 6B expression on the overall survival of bladder cancer. RESULTS AND CONCLUSION: (1) Cancer stem cell-related genes (SOX2, OCT4, NANOG) and keratin 6B expression was higher in CD44+ cells isolated by magnetic active cell sorting compared with CD44- cells (P < 0.05). Cell proliferation, migration, and in vitro spheroid formation were significantly increased (P < 0.05). Keratin 6B small interfering RNA down-regulated the expression of keratin 6B in CD44+ bladder cancer stem cells (P < 0.05). (2) Compared with the blank control group, the proliferation and migration of CD44+ bladder cancer stem cells after transfection of keratin 6B small interfering RNA (P < 0.05), and the number of tumorsphere significantly diminished (P < 0.05); the expression of Notch1 and Hes1 mRNA increased (P < 0.05). (3) Keratin 6B and CD44v6 were significantly different in bladder cancer tissue (P=0.006). The overall survival rate of bladder cancer patients with high expression of keratin 6B was lower than that of patients with low expression of keratin 6B. (4) The results showed that keratin 6B was highly expressed in CD44+ bladder cancer stem cells, and could promote the proliferation, migration, and self-renewal capacity of bladder cancer stem cells. The high expression of keratin 6B contributes to improving the survival of bladder cancer patients.

Expression of integrin-lined kinase in esophageal squamous cell carcinoma tissues and its effect on proliferation and apoptosis of KYSE-150 cells and the growth of xenografts in nude mice / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：分析整合素连接激酶（ILK）基因在食管鳞状细胞癌（ESCC）组织中的表达水平及其与患者临床病理特征之间的关系，探讨其对KYSE-150细胞增殖、凋亡和裸鼠皮下移植瘤生长的影响。方法:选取2012年1月至2014年12月手术切除并经病理证实的75例ESCC患者的癌组织和其配对的癌旁组织标本，用组织芯片技术及免疫组织化学染色法检测ESCC组织和癌旁组织中ILK的表达情况；qPCR法检测ESCC细胞ECA109、TE-1、EC9706、KYSE-150中ILK mRNA的表达，选用ILK表达最高的KYSE-150细胞进行后续细胞功能学研究。使用ILK干扰慢病毒感染KYSE-150细胞下调ILK的表达，qPCR和WB法检测ILK基因敲降效率；MTT实验、克隆形成实验和FACS检测干扰ILK表达对KYSE-150细胞增殖能力和凋亡水平的影响；裸鼠皮下成瘤实验检测干扰ILK对KYSE-150细胞移植瘤生长的影响。结果:ESCC组织中ILK蛋白阳性表达率高于癌旁组织（P<0.05），且ILK高表达与淋巴结转移有关联（P<0.05）。ILK干扰慢病毒感染的KYSE-150细胞中ILK mRNA表达明显受到抑制（P<0.05），ILK蛋白水平表达下调，以上结果提示ILK敲降成功。与感染阴性对照病毒的KYSE-150细胞相比，ILK干扰慢病毒感染的KYSE-150细胞的增殖能力、克隆形成数均显著降低（均P<0.05），但细胞凋亡率升高（P<0.05）。与对照组相比，干预组裸鼠移植瘤生长缓慢，移植瘤的质量及体积均较小（均P<0.05）。结论:ESCC组织中ILK的表达高于癌旁组织，且ILK高表达与患者发生淋巴结转移有关联；抑制ILK基因可导致KYSE-150细胞增殖能力降低，促进细胞凋亡而抑制裸鼠移植瘤生长。

Effects of RhoA silencing on the migration and invasion of salivary adenoid cystic carcinoma / 口腔疾病防治

Objective@# To explore whether RhoA plays a role in the migration and invasion of the salivary adenoid cystic carcinoma cell lines SACC-LM and SACC-83.@*Methods@#Total RNA and total protein were extracted from 20 salivary adenoid cystic carcinoma (SACC) and normal adjacent tissues frozen in liquid nitrogen to detect RhoA expression. RhoA-siRNA was constructed to transfect two cell lines (SACC-LM and SACC-83) for cytological experiments. The research included an experimental group (RhoA-siRNA transfection), negative control group (siRNA-NC transfection) and blank group by transient transfection with liposomes. Expression of RhoA mRNA and protein as well as the protein expression of biomarkers of epithelial-mesenchymal transition (EMT) were analyzed, including E-cadherin, N-cadherin, and Vimentin. Furthermore, the changes in invasion and migration of cells in each group were analyzed by comparing the number of transmembrane cells in the Transwell assay and the results of the scratch test.@*Results@#Compared with normal adjacent tissues, RhoA protein and mRNA levels increased in SACC tissues. Compared with the control group, the relative expression levels of RhoA mRNA and protein decreased (P < 0.01), the relative expression levels of E-cadherin protein increased, and the relative expression levels of N-cadherin and vimentin protein increased in the experimental group (P < 0.01). Additionally, the trial results revealed that RhoA knockdown restrained cell migration and invasion (P < 0.01).@*Conclusion @#RhoA expression increased in SACC tissue. Silencing RhoA in vitro could effectively restrain cell migration and invasion in SACC-LM and SACC-83 cells through the regulation of EMT signaling pathways.

Construction and application of gene delivery systems for primary dendritic cells / 中国药科大学学报

@#Nowadays, there is still no mature gene delivery system for safe and effective transfection on primary dendritic cells (DC). Herein, we constructed a liposome-based gene delivery system for primary DCs and optimized the preparation method to improve the transfection efficiency of siRNA on primary DCs. In this study, different methods, including co-incubation method, ethanol injection method, and protamine compound method, were used to prepare liposome/siRNA complexes based on different cationic lipids. Moreover, particle size, zeta potential, siRNA loading capacity, safety, stability, uptake efficiency and gene silencing efficiency of various liposome/siRNA complexes were detected to screen the optimal cationic lipid as well as its preparation method. We demonstrated that the OA2/siRNA delivery system prepared by the co-incubation method exhibited the best safety, uptake efficiency and gene silencing effect, compared to other siRNA delivery systems including the commercial Lipo2000. In summary, we provide a safe and effective gene delivery vector for primary DC cells through simple preparation method, which could also offer a gene delivery platform for other immune cells.

Design and preparation of a new multi-targeted drug delivery system using multifunctional nanoparticles for co-delivery of siRNA and paclitaxel / 药物分析学报

Drug resistance is a great challenge in cancer therapy using chemotherapeutic agents.Administration of these drugs with siRNA is an efficacious strategy in this battle.Here,the present study tried to incor-porate siRNA and paclitaxel(PTX)simultaneously into a novel nanocarrier.The selectivity of carrier to target cancer tissues was optimized through conjugation of folic acid(FA)and glucose(Glu)onto its surface.The structure of nanocarrier was formed from ternary magnetic copolymers based on FeCo-polyethyleneimine(FeCo-PEI)nanoparticles and polylactic acid-polyethylene glycol(PLA-PEG)gene delivery system.Biocompatibility of FeCo-PEI-PLA-PEG-FA(NPsA),FeCo-PEI-PLA-PEG-Glu(NPsB)and FeCo-PEI-PLA-PEG-FA/Glu(NPsAB)nanoparticles and also influence of PTX-loaded nanoparticles on in vitro cytotoxicity were examined using MTT assay.Besides,siRNA-FAM internalization was investi-gated by fluorescence microscopy.The results showed the blank nanoparticles were significantly less cytotoxic at various concentrations.Meanwhile,siRNA-FAM/PTX encapsulated nanoparticles exhibited significant anticancer activity against MCF-7 and BT-474cell lines.NPsAB/siRNA/PTX nanoparticles showed greater effects on MCF-7 and BT-474 cells viability than NPsA/siRNA/PTX and NPsB/siRNA/PTX.Also,they induced significantly higher anticancer effects on cancer cells compared with NPsA/siRNA/PTX and NPsB/siRNA/PTX due to their multi-targeted properties using FA and Glu.We concluded that NPsAB nanoparticles have a great potential for co-delivery of both drugs and genes for use in gene therapy and chemotherapy.

The effect of silencing the endoplasmic reticulum stress-related protein calnexin on the proliferation, invasion, and migration of tongue squamous cell carcinoma cells / 口腔疾病防治

Objective@#To investigate the effect of silencing the endoplasmic reticulum stress-related protein calnexin on the proliferation, invasion, and migration of tongue squamous cell carcinoma cells. @* Methods @#Calnexin siRNA was transfected into SCC-9 and SCC-25 tongue squamous cell carcinoma cells, and the expression of calnexin was detected by qRT-PCR. The silencing effect of calnexin siRNA was further verified by Western blotting. CCK-8 assay was applied to detect the effect of silencing calnexin on the proliferation of tongue squamous cell carcinoma cells; Transwell assay was used to detect the effect of silencing calnexin on the invasion and migration of tongue squamous cell carcinoma cells.@* Results @#qRT-PCR showed that calnexin siRNA could effectively downregulate the expression of calnexin. Western blot analysis further confirmed the silencing effect of calnexin siRNA on calnexin. The CCK-8 assay showed that silencing calnexin expression on the 4th and 5th days could inhibit the proliferation of tongue squamous cell carcinoma cells, and the difference was statistically significant (P < 0.01). The Transwell assay showed that knockdown of calnexin could inhibit the invasion and migration of tongue squamous cell carcinoma cells (P < 0.001).@*Conclusion@#Knockdown of calnexin can inhibit the proliferation, invasion, and migration of tongue squamous cell carcinoma cells.

Construction of a curcumin-siRNA co-delivery system based on mesoporous silica and its effect on M2-type polarization of macrophages / 口腔疾病防治

Objective @#To fabricate a co-delivery system of curcumin (CUR) and siRNA based on mesoporous silica nanoparticles (MSN) and investigate its potential application in inducing macrophage M2 polarization.@*Methods@# MSNs were synthesized using the conventional sol-gel method. The interior mesochannels were occupied by small-molecule CUR, while the exterior surface was adsorbed by cationic polymeric polyethyleneimine (PEI) to link the negatively charged siRNA molecules to formulate the (CUR@MSN)PEI/siRNA co-delivery system. The formulation process was monitored by transmission electron microscopy(TEM). The MTT assay was used to evaluate the cytotoxicity in RAW264.7 cells under various concentrations of nanoparticles. Confocal laser scanning microscopy and TEM were used to observe cell internalization using FAM-labeled siRNA. GAPDH-targeting siRNA was used to prepare nanoparticles and then was transfected into RAW264.7 cells to observe the silencing efficiency of target genes. The knockdown efficiency was examined by real-time quantitative PCR. The related control groups were untreated cells, CUR delivery only and the co-delivery of CUR and siRNA negative control. By loading miRNA-130a-3p antisense oligonucleotide (ASO) to transfect RAW264.7 cells, the effects on the polarization of macrophages were observed. The M2 polarization marker arginase 1 (Arg-1) was measured by western blotting. The related control groups were untreated cells, CUR delivery only and co-delivery of CUR and miRNA negative control. @* Results @# The (CUR@MSN)PEI/siRNA co-delivery system was successfully formulated. The nanoparticles exhibited dose-dependent cytotoxicity, and the cell viability was maintained over 90% when the nanoparticle concentration was less than 10 μg/mL. A high cell uptake efficiency was observed, and the target gene knockdown efficiency was greater than 80% (P < 0.05 vs. all the other groups). The CUR delivery-only group and co-delivery of the CUR-and miRNA-negative control group improved Arg-1 expression ~ 3-fold (P < 0.05 vs. untreated cells). Using the co-delivery of CUR and ASO, synergistic effects were obtained, and Arg-1 expression was increased ~ 8-fold (P < 0.05 vs. all the other groups).@*Conclusion @#The CUR-siRNA co-delivery system can effectively transfect macrophages and synergistically induce M2 polarization.