STAR Protocols
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2021
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IF 0
Lidia Wrobel,Farah H. Siddiqi,David C. Rubinsztein
Summary We present a protocol for in vivo siRNA-mediated knockdown of a gene of interest in mouse liver using systemic delivery via intravenous injection. We describe a step-by-step protocol for delivery of siRNA particles, with tips on how to optimize dosage. We detail steps for feeding/starving cycles as well as for liver tissue isolation, followed by gene expression analysis, measured at the mRNA and protein levels. For complete information on the generation and use of this protocol, please refer to Wrobel et al. (2020) .
研究领域:
分子生物学
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遗传学
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DOI
PubMed
RSC Advances
·
2017
·Vol. 7
·期 15
·8823-8831
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IF 3.108
Tsogzolmaa Ganbold,Gerile Gerile,Hai Xiao,Huricha Baigude
RNA interference (RNAi) has shown great potential for clinical treatment of a variety of diseases. As an effective post-transcriptional regulation, RNAi induces degradation of specific target mRNA, leading to a decreased level of disease related gene product, which could be resistant to conventional small molecule therapeutics. Because of the relatively small size of short interfering RNA (siRNA) as well as it's robustness in the design of efficient 20–22 nucleotide sequences targeting any known gene, delivery of siRNA in vitro and in vivo has achieved tremendous success by the design and fabrication of various carrier materials including peptides, lipids, and polymers. While siRNA delivery efficiency of these materials proved to be appreciable, their tissue specificity, biocompatibility and cytotoxicity still need much improvement. We report a novel siRNA delivery material designed by optimization of our previously reported peptidomimetic based lipoplex. The novel lipoplex, which bears a D-amino acid based dipeptide head group, possesses an ideal balance of increased stability in a tissue environment, and enhanced efficiency of tissue specific delivery as well as minimized cytotoxicity. The novel lipoplex carrying siRNA efficiently targeted mouse liver, initiated RNAi and knocked down the apoB dose dependently, which is confirmed by both RT-qPCR and Western blotting. Moreover, the novel lipoplex does not induce any apparent cytotoxicity, as confirmed by the measurement of liver enzyme levels in serum.
研究领域:
分子生物学
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药学
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生物技术
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DOI
高影响因子
POLYMER INTERNATIONAL
·
2022
·Vol. 71
·期 8
·1022-1029
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IF 3.213
Mei Lin,Aizhu Lin,Sheng Huang,Tianhui Liu,Fang Ke,Deshun Qiu,Xuefeng Lin,Daoshu Luo
An ideal carrier that delivers small interfering RNA (siRNA) should be designed based on two criteria: high gene transfection efficiency and low cytotoxicity. In this work, arginine was used to modify polyvinylamine (PVAm) to form a novel PVAm derivative (PVAm-Arg) for siRNA delivery and the derivative was characterized using Fourier transform infrared spectroscopy. PVAm-Arg/siRNA complexes were formed by the self-assembly method. The zeta potential and size of the complexes were measured using electrophoretic light scattering and dynamic light scattering, respectively. The siRNA binding capacity of PVAm-Arg was assessed using gel retardation assay. The results showed that PVAm-Arg derivatives can bind siRNA effectively and form complexes with sizes of about 72 ± 2 nm. What is more, the ability of PVAm-Arg to condense siRNA was better than that of PVAm. Cytotoxicity assay with RSC96 cells also showed that PVAm-Arg had lower cytotoxicity compared with PVAm. In in vitro cellular uptake assay, PVAm-Arg showed good transfection efficiency. Therefore, we concluded that PVAm-Arg would be a promising candidate as a gene delivery vector. © 2022 Society of Industrial Chemistry.
研究领域:
生物技术
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药学
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分子生物学
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DOI
高影响因子
International Journal of Nanomedicine
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2025
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IF 6.5
Li Gao,Li-Hua Yang,Fang-Cheng Jiang,Wang-Yang Ye,Xin Chen,Zhao-Ji Liu,Rong-Quan He,Wan-Ying Huang,Zhen-Bo Feng,Jin-Liang Kong,Ze-Feng Lai,Gang Chen
Background Although immune checkpoint blocking (ICB) therapeutic agents have significantly improved the survival for many cancer patients, the efficacy of ICB therapy for non-small cell lung cancer (NSCLC) patients remains limited. Combining ICB therapy with other strategies that is able to stimulate tumor immunogenicity or increase infiltration of T cells aroused great concern in cancer therapy.Results Herein, we constructed a kind of AuNP@NH2-PEG-SH/PD-L1 siRNA nanoparticle complex which showed prominent photothermal therapeutic (PTT) effects triggered by NIR laser and could efficiently deliver PD-L1 siRNA into NSCLC cells (knock-down efficiency were 75.8% and 83% in HCC827 and A549 cells). During the process of photothermal stimulation, discrepancies in heat shock protein expression were observed in NSCLC cells. Knocking down PD-L1 expression in NSCLC cells such as A549 and HCC827 cells activated the co-culturing Jurkat cells and enhanced their tumor-killing effect in vitro (cell inhibition rate was 62.65% for HCC827 cells and 57.03% for A549 cells). The gold nanoparticle complexes exhibited remarkable PTT effect for the engrafted NSCLC cells in zebrafish larvas. Furthermore, the nano complexes could promote the activation of the xenografted human peripheral blood mononuclear cells (PBMCs) and enhance the killing of the NSCLC cells in the larvas.Conclusion It is well known that gold nanoparticle is one of the several metal nanoparticles approved for clinical trial by American FDA. This work has demonstrated the outstanding PTT and immunotherapeutic effects of gold nanoparticle complexes on NSCLC, indicating great potential in clinical regiment of lung cancers.
研究领域:
肿瘤学
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免疫学
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生物医学工程
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分子生物学
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转化医学
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
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2022
·Vol. 23
·期 15
·8380
·
IF 6.208
Zhipeng Dong,Yunxue Yin,Jun Luo,Bingxia Li,Fangning Lou,Qiyan Wang,Qingfa Zhou,Baofen Ye,Yue Wang
Liposome modification by targeting ligands has been used to mediate specific interactions and drug delivery to target cells. In this study, a new peptide ligand, CP7, was found to be able to effectively bind to FGFR1 through reverse molecular docking and could cooperate with VEGFR3 to achieve targeting of A549 cells. CP7 was modified on the surface of the liposome to construct a targeted and safe nanovehicle for the delivery of a therapeutic gene, Mcl-1 siRNA. Due to the specific binding between CP7 and A549 cells, siRNA-loaded liposome-PEG-CP7 showed increased cellular uptake in vitro, resulting in significant apoptosis of tumor cells through silencing of the Mcl-1 gene, which is associated with apoptosis and angiogenesis. This gene delivery system also showed significantly better antitumor activity in tumor-bearing mice in vivo. All of these suggested that siRNA-loaded liposome-PEG-CP7 could be a promising gene drug delivery system with good bioavailability and minimal side effects for treatment.Keywords:CP7;liposome;target delivery;FGFR1;gene drug
研究领域:
药学
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癌症研究
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分子生物学
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药物递送系统
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产品关联
DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
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2018
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IF 8.097
Tsogzolmaa Ganbold,Huricha Baigude
6-Amino-6-deoxy-curdlan is a promising nucleic acid carrier that efficiently delivers plasmid DNA as well as short interfering RNA (siRNA) to various cell lines. The highly reactive C6-NH2 groups of 6-amino-6-deoxy-curdlan prompt conjugation of various side groups including tissue-targeting ligands to enhance cell-type-specific nucleic acid delivery to specific cell lines. Herein, to test the primary-cell-targeting efficiency of the curdlan derivative, we chemically conjugated a macrophage-targeting ligand, mannose, to 6-amino-6-deoxy-curdlan. The resulting curdlan derivative (denoted CMI) readily complexed with siRNA and formed nanoparticles with a diameter of 50–80 nm. The CMI nanoparticles successfully delivered a dye-labeled siRNA to mouse peritoneal macrophages. The delivery efficiency was blocked by mannan, a natural ligand for a macrophage surface mannose receptor (CD206), but not by zymosan, a ligand for the dectin-1 receptor, which is also present on the surface of macrophages. Moreover, CMI nanoparticles were internalized by macrophages only at 37 °C, suggesting that the cellular uptake of CMI nanoparticles was energy-dependent. Furthermore, CMI nanoparticle efficiently delivered siRNA against tumor necrosis factor α (TNFα) to lipopolysaccharide-stimulated primary mouse peritoneal macrophages. In vivo experiments demonstrated that CMI nanoparticles successfully delivered siTNFα to mouse peritoneal macrophages, liver, and lung and induced significant knockdown of the TNFα expression at both messenger RNA and protein levels. Therefore, our design of CMI may be a promising siRNA carrier for targeting CD206-expressing primary cells such as macrophage and dendritic cells.
研究领域:
药学
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分子生物学
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纳米医学
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免疫学
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产品关联
DOI
PubMed
高影响因子
Frontiers in Immunology
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2022
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IF 8.786
Lixia Gao,Jie Yang,Jutao Feng,Ziying Liu,Ying Dong,Jiangyan Luo,Liangzhentian Yu,Jiamei Wang,Hongying Fan,Weifeng Ma,Tiancai Liu
A viable therapy is needed to overcome the deadlock of the incurable chronic hepatitis B (CHB). The prolonged existence of covalently closed circular DNA (cccDNA) and integrated HBV DNA in the nucleus of hepatocytes is the root cause of CHB. As a result, it is critical to successfully suppress HBV DNA replication and eliminate cccDNA. RNA interference has been proven in recent research to silence the expression of target genes and thereby decrease HBV replication. However, siRNA is susceptible to be degraded by RNA enzymes in vivo , making it difficult to deliver successfully and lacking of tissue targeting. To exploit the advantages of siRNA technology while also overcoming its limitations, we designed a new strategy and prepared biomimetic nanoparticles that were directed by PreS/2-21 peptides and precisely loaded HBV siRNA. Experiments on these nanoparticles in vitro and in vivo revealed that they are tiny, stable, safe and highly targetable, with high inhibitory effects on HBV DNA, pgRNA, cccDNA, HBeAg and HBsAg. PreS/2-21-directed nanoparticles loaded with HBV gene therapy drugs are expected to be promising for the treatment of CHB.
研究领域:
分子生物学
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病毒学
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药学
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治疗学
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产品关联
DOI
PubMed
高影响因子
International Journal of Nanomedicine
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2015
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IF 4.383
Zhenzhen Liu,Haijun Gong,Rui Zeng,Xuan Liang,Li-Ming Zhang,Liqun Yang,Yuqing Lan
A hyperbranched cationic polysaccharide derivative-mediated small interfering (si)RNA interference strategy was proposed to inhibit nuclear transcription factor-kappa B (NF-κB) activation in human retinal pigment epithelial (hRPE) cells for the gene therapy of diabetic retinopathy. Two hyperbranched cationic polysaccharide derivatives containing the same amount of cationic residues, but with different branching structures and molecular weights, including 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) and amylopectin (DMAPA-Amp) derivatives, were developed for the efficient delivery of NF-κB siRNA into hRPE cells. The DMAPA-Glyp derivative showed lower toxicity against hRPE cells. Furthermore, the DMAPA-Glyp derivative more readily condensed siRNA and then formed the nanoparticles attributed to its higher branching architecture when compared to the DMAPA-Amp derivative. Both DMAPA-Glyp/siRNA and DMAPA-Amp/siRNA nanoparticles were able to protect siRNA from degradation by nuclease in 25% fetal bovine serum. The particle sizes of the DMAPA-Glyp/siRNA nanoparticles (70–120 nm) were smaller than those of the DMAPA-Amp/siRNA nanoparticles (130–180 nm) due to the higher branching architecture and lower molecular weight of the DMAPA-Glyp derivative. In addition, the zeta potentials of the DMAPA-Glyp/siRNA nanoparticles were higher than those of the DMAPA-Glyp/siRNA nanoparticles. As a result, siRNA was much more efficiently transferred into hRPE cells using the DMAPA-Glyp/siRNA nanoparticles rather than the DMAPA-Amp/siRNA nanoparticles. This led to significantly high levels of suppression on the expression levels of NF-κB p65 messenger RNA and protein in the cells transfected with DMAPA-Glyp/siRNA nanoparticles. This work provides a potential approach to promote hyperbranched polysaccharide derivatives as nonviral siRNA vectors for the inhibition of NF-κB activation in hRPE cells.
研究领域:
药理学
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分子生物学
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眼科学
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基因治疗
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产品关联
DOI
PubMed
高影响因子
DRUG METABOLISM AND DISPOSITION
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2025
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IF 4
Hong Zhang,Gengyao Qin,Liqi Shi,Ruixing Li,Liang Shen,Weiqun Cao,Yi Tao
GalNAc-conjugated small interfering RNAs (siRNAs) have emerged as a promising new class of therapeutics, demonstrating potential across various disease areas. Metabolic stability is a critical factor in the development of siRNA therapies, necessitating the selection of suitable in vitro metabolism models for comprehensive studies. However, many existing in vitro models often fail to accurately predict in vivo metabolism. Therefore, our objective was to systematically identify the optimal models and conditions that can simulate in vivo metabolism. Inclisiran, a representative GalNAc-conjugated siRNA, was chosen as the model compound for this investigation. A thorough comparison of in vitro metabolism was conducted to identify optimal models and corresponding conditions that could predict Inclisiran's metabolic profiles in rat plasma and liver in vivo. The models tested included serum, plasma, plated hepatocytes, liver homogenate, S9 fractions, tritosomes, lysosomes, and cytosol, reflecting Inclisiran's trajectory from injection to RNA-induced silencing complex (RISC) in hepatocytes. The results indicated that both rat serum and heparin-anticoagulant plasma are suitable models to mimic the nucleolytic metabolism of Inclisiran in in vivo blood. For hepatic metabolism, liver homogenate in phosphate buffer (pH 6.0), liver tritosomes in acetate buffer (pH 5.0), and plated hepatocytes were suitable models. Liver homogenate and cytosol in Tris-HCl buffer (pH 7.4) were effective for studying nuclease-mediated metabolism of Inclisiran's antisense strand under neutral conditions, though they did not fully simulate in vivo metabolism of the sense strand. These findings underscore the importance of selecting appropriate in vitro models and conditions to accurately simulate in vivo metabolism of Inclisiran.
研究领域:
药理学
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Acta Pharmaceutica Sinica B
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2025
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IF 14.6
Yunfei Yi,Zhangwen Peng,Yuanqi Liu,Huisong Hao,Liu Yu,Simin Wen,Shengjie Sun,Jianlin Shi,Meiying Wu,Lin Mei
Among tumor microenvironment (TME), the entire metabolic characteristics of tumor-resident cells are reprogrammed to benefit the expansion of tumor cells, which count on glutamine in large part to fuel the tricarboxylic acid cycle for energy generation and anabolic metabolism support. Endothelial cells that are abducted by tumor cells to form a pathological tumor vascular network for constructing the hypoxic immunosuppressive TME, also rely on glutaminolysis as the “engine” of angiogenesis. Additionally, the glutamine metabolic preference benefits the polarization of TAMs towards pro-tumoral M2 phenotype as well. Herein, we developed a type of siRNA micelleplexes (MH@siGLS1) to reverse immunosuppressive TME by targeting glutaminolysis within tumor-resident cells for tumor vasculature normalization- and TAMs repolarization-enhanced photo-immunotherapy. Tumor cell starvation and antioxidant system destruction achieved by MH@siGLS1-mediated glutaminolysis inhibition could promote photodynamic therapy efficacy, which was available to trigger immunogenic cell death for adaptive antitumor immune responses. Meanwhile, glutaminolysis inhibition of tumor endothelial cells and TAMs could realize tumor vascular normalization and TAMs repolarization for antitumor immunity amplification. This study provides a unique perspective on cancer treatments by focusing on the interrelations of metabolic characteristics and the biofunctions of various cell types within TME.
研究领域:
肿瘤学
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生物化学
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免疫学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
·
2016
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IF 7.145
Hai Xiao,Altanzul Altangerel,Gerile Gerile,Yinga Wu,Huricha Baigude
RNA interference (RNAi) is a highly efficient approach for gene silencing. Regulation of gene expression at post-transcriptional level provides great potential for curing diseases caused by abnormal overexpression of disease-related genes. However, the application of RNAi in the clinic has been hindered by the lack of efficient and biocompatible delivery systems. Therefore, the development of a safe and tissue-targeted double-stranded interfering RNA (siRNA) carrier for clinical application is urgently needed. Here we report the discovery of a highly efficient liposomal siRNA delivery agent based on a novel peptidomimetic built from natural amino acids. Fine tuning of the composition of amino acids, the type of amide linkage in the peptidomimetic, as well as the formulation and the physicochemical parameters of the novel lipoplex resulted in a lipid nanoparticle (LNP) that efficiently encapsulates and carries siRNA to mouse liver. In vivo experiments showed that a single injection of unmodified siRNA complexed to one of the peptidomimetics at a clinically feasible dose induced significant RNAi in mouse liver, resulting in a 90% decrease in apolipoprotein B (ApoB) mRNA level, as well as a 60% decrease in serum ApoB protein level. Analysis of mouse serum by ELISA indicated that the novel peptidomimetic based lipoplex did not elevate the level of liver enzymes (ALT, AST) in the serum. Our novel peptidomimetic-based lipoplex demonstrated great potential for the development of a safe and efficient siRNA delivery agent for clinical applications.
研究领域:
药理学
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分子生物学
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治疗学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Science China-Chemistry
·
2025
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IF 9.7
Jiang Yifan,Li Lisha,Fang Xiao,Zeng Tao,Su Lichao,Liu Yichang,Wu Ying,Li Jinyu,Lyu Peng,Lu Zhonglei,Lu Chunhua,Yang Huanghao
The development of RNA interference (RNAi) therapy offers a potential solution for Alzheimer’s disease (AD). However, the brain-blood barrier (BBB) with its selective permeability and pharmacokinetic-related challenges poses restrictions on the delivery of small interfering RNA (siRNA) to the central nervous system (CNS). In this study, we demonstrate that the incorporation of 2′-fluoro (2′-F) substitutions and L-carnitine modification facilitates the self-assembly of siRNA through triple interaction, leading to the formation of nanorings, called LCSF-NR. Based on the enhanced cellular uptake and lysosomal escape by 2′-F substitution and the transport across the BBB promoted by L-carnitine, the nanorings realized the improved brain-targeted delivery of siRNA, both in zebrafish and mice models. Moreover, our findings highlight the therapeutic potential of LCSF-NR formulation in an AD zebrafish model through a synergistic effect of downregulating the β-site APP cleavage enzyme 1 (BACE1) gene and L-carnitine-mediated neuroprotection, effectively inhibiting pathological processes. Overall, these results suggest that the chemical modification-based siRNA self-assembly strategy enables trans-BBB delivery and presents a concise approach for synergistic therapy of AD.
研究领域:
分子生物学
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神经科学
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药物递送系统
✓ 已验证
产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）