RESUMO
Pulmonary delivery of small interfering RNA (siRNA) is a promising therapeutic strategy for treating various respiratory diseases but an effective carrier for the delivery of siRNA into the cells of the lungs and a robust gene-silencing effect is still lacking. Previously, we reported that the KL4 peptide, a synthetic cationic peptide with a repeating KLLLL sequence, can mediate effective siRNA transfection in lung epithelial cells but its high hydrophobic leucine content, and hence poor water solubility, limits its application as a delivery vector. Here, we show that the covalent attachment of monodisperse poly(ethylene glycol) (PEG) improves the solubility of KL4 and the uptake of its complex with siRNA into lung epithelial cells, such that very robust silencing is produced. All PEGylated KL4 peptides, with PEG length varying between 6 and 24 monomers, could bind and form nanosized complexes with siRNA, but the interaction between siRNA and peptides became weaker as the PEG chain length increased. All PEGylated KL4 peptides exhibited satisfactory siRNA transfection efficiency on three human lung epithelial cell lines, including A549 cells, Calu-3 cells, and BEAS-2B cells. The PEG12KL4 peptide, which contains 12 monomers of PEG, was optimal for siRNA delivery and also demonstrated a low risk of inflammatory response and toxicity in vivo following pulmonary administration.
Assuntos
Portadores de Fármacos/química , Pulmão/metabolismo , Peptídeos/química , RNA Interferente Pequeno/administração & dosagem , Doenças Respiratórias/terapia , Células A549 , Inativação Gênica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Polietilenoglicóis/química , RNA Interferente Pequeno/genética , Doenças Respiratórias/genética , Solubilidade , Transfecção/métodosRESUMO
Therapeutic gene silencing by RNA interference relies on the safe and efficient in vivo delivery of small interfering RNAs (siRNAs). Polyethylenimines are among the most studied cationic polymers for gene delivery. For several reasons including superior tolerability, small linear PEIs would be preferable over branched PEIs, but they show poor siRNA complexation. Their chemical modification for siRNA formulation has not been extensively explored so far. We generated a set of small linear PEIs bearing tyrosine modifications (LPxY), leading to substantially enhanced siRNA delivery and knockdown efficacy in vitro in various cell lines, including hard-to-transfect cells. The tyrosine-modified linear 10 kDa PEI (LP10Y) is particularly powerful, associated with favorable physicochemical properties and very high biocompatibility. Systemically administered LP10Y/siRNA complexes reveal antitumor effects in mouse xenograft and patient-derived xenograft (PDX) models, and their direct application into the brain achieves therapeutic inhibition of orthotopic glioma xenografts. LP10Y is particularly interesting for therapeutic siRNA delivery.
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Terapia Genética , Neoplasias Experimentais , Polietilenoimina , RNA Interferente Pequeno , Transfecção , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Neoplasias Experimentais/genética , Neoplasias Experimentais/terapia , Polietilenoimina/química , Polietilenoimina/farmacologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Small interfering RNA (siRNA) is a critical loss-of-function tool for elucidating the role of genes in biomedical studies. The effective use of siRNA needs transfection technology that delivers siRNA into the correct location of target cells, especially those which are extremely difficult to transfect. Macrophages, which play an important role in the pathogenesis of many diseases, are known to be extremely hard to transfect. Thus, to elucidate the functions of genes in human macrophage biology, it is essential to devise technology for efficient siRNA transfection. However, a fast and efficient method for siRNA transfection in primary human macrophages has not been reported. The siRNA transfection is a tug-of-war between transfection rate and cytotoxicity. A higher transfection rate is generally accompanied with increased cytotoxicity, therefore, choosing a transfection reagent that limits cell death while maintain a desirable transfection rate is important. In this study, we employed auto-analysis function of the IncuCyte® to devise a fast and cost-saving technology for efficient transfection of adherent cells and particularly human macrophages. We show that DharmaFECT3 transfection reagent from Dharmacon was the most efficient in transfecting primary human monocyte-derived macrophages and PMA-differentiated U937 cells, whereas other transfection reagents tested were cytotoxic. This method exhibited approximately 85% transfection efficiency in human macrophages. Moreover, siRNA silencing of Bax with this technique effectively protected primary human macrophages and PMA-differentiated U937 cells against Resveratrol-induced cell death. In addition, this method inherently takes the balance between transfection rate and cytotoxicity of siRNA transfection reagents into consideration.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , RNA Interferente Pequeno/genética , Resveratrol/farmacologia , Proteína X Associada a bcl-2/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Dosagem de Genes , Expressão Gênica , Humanos , Macrófagos/citologia , TransfecçãoRESUMO
Although gene therapy offers hope against incurable diseases, nonreplicating transduction vectors remain lacking. We have previously characterized a cell-penetrating peptide hPP10 for the delivery of various cargoes; however, whether hPP10 can mediate nucleic acid delivery is still unknown. Here, examining via different ways, we demonstrate that hPP10 stably complexes with plasmid DNA (pDNA) and safely mediates nucleic acid transfection. hPP10 can mediate GFP-, dsRed-, and luciferase-expressing plasmids into cells with nearly the same efficiency as commercial transfection reagents Turbofectin or Lipofect. Furthermore, hPP10 can mediate Cre fusion protein delivery and pDNA transfection simultaneously in the Cre/loxp system in vitro. In addition, hPP10 fused with an RNA-binding domain can mediate delivery of small interfering RNA into cells to silence the reporter gene expression. Collectively, our results suggest that hPP10 is an option for nucleic acid delivery with efficiencies similar to that of commercial reagents.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Técnicas de Transferência de Genes , Espaço Intracelular/metabolismo , Ácidos Nucleicos/administração & dosagem , Linhagem Celular Tumoral , DNA/administração & dosagem , Proteínas de Fluorescência Verde/metabolismo , Humanos , Plasmídeos/administração & dosagem , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
CD133, putative cancer stem cell marker, deemed to aid chemoresistance. However, this claim has been challenged recently and we previously reported that patients with CD133(+) colon cancer have benefit from 5-fluorouracil (5-FU) chemotherapy incontrast to no benefit in patients with CD133(-) cancer. To elucidate the role of CD133 expression in chemoresistance, we silenced the CD133 expression in a colon cancer cell line and determined its effect on the biological characteristics downstream. We comparatively analyzed the sequential changes of MDR1, ABCG2, AKT1 and survivin expression and the result of proliferation assay (WST-1 assay) with 5-FU treatment in CD133(+) and siRNA-induced CD133(-) cells, derived from Caco-2 colon cancer cell line. 5-FU treatment induced significantly increase of the mRNA expression of MDR1, ABCG2 and AKT1genes, but not protein level. CD133 had little to no effect on the mRNA and protein expression of these genes. However, survivin expression at mRNA and protein level were significantly increased in CD133(+) cells compared with siRNA-induced CD133-cells and Mock (not sorted CD133(+) cells) at 96 h after siRNA transfection. The cytotoxicity assay demonstrated notable increase of chemoresistance to 5-FU treatment (10 µM) in CD133(+) cells at 96 h after siRNA transfection. From this study, we conclude that CD133(+) cells may have chemoresistance to 5-FU through the mechanism which is related with survivin expression, instead of MDR1, ABCG2 and AKT1 expression. Therefore a survivin inhibitor can be a new target for effective treatment of CD133(+) colon cancer.
Assuntos
Antígenos CD/genética , Antimetabólitos Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Glicoproteínas/genética , Proteínas Inibidoras de Apoptose/genética , Peptídeos/genética , Antígeno AC133 , Células CACO-2 , Colo/efeitos dos fármacos , Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Interferência de RNA , RNA Interferente Pequeno/genética , Survivina , Regulação para CimaRESUMO
Glycol chitosan nanogels have been widely used in gene, drug, and contrast agent delivery in an effort to improve disease diagnosis and treatment. Herein, we evaluate the internalization mechanisms and intracellular fate of previously described glycol chitosan nanogels decorated with folate to target the folate receptor. Uptake of the folate-decorated nanogel was impaired by free folate, suggesting competitive inhibition and shared internalization mechanisms via the folate receptor. Nanogel uptake was shown to occur mainly through flotillin-1 and Cdc42-dependent endocytosis. This was determined by inhibition of uptake reduction observed upon siRNA depletion of these two proteins and the pathways that they regulate. The data also suggest the involvement of the actin cytoskeleton in nanogel uptake via macropinocytosis. After 7 h of incubation with HeLa cells, approximately half of the nanogel population was localized in endolysosomal compartments, whereas the remaining 50% of the material was in undefined regions of the cytoplasm. Glycol chitosan nanogels may thus have potential as drug delivery vectors for targeting different intracellular compartments.
Assuntos
Quitosana/química , Ácido Fólico/química , Polietilenoglicóis/química , Polietilenoimina/química , RNA Interferente Pequeno/genética , Western Blotting , Eletroforese em Gel de Poliacrilamida , Endocitose/fisiologia , Citometria de Fluxo , Células HeLa , Humanos , NanogéisRESUMO
Tumor associated macrophages (TAMs) are the most abundant immune cell type in the tissue microenvironment, affecting tumor progression, metastasis and therapeutic response. Different macrophage activation ("polarization") states can be distinguished: resting (M0; non-activated), pro-inflammatory/anti-tumorigenic (M1) and anti-inflammatory/pro-tumorigenic (M2). When exploring macrophages as targets in novel cancer immunotherapy approaches, TAM repolarization from the M2 into the M1 phenotype is an intriguing strategy to block their pro-tumoral and enhance their anti-tumoral properties. In the context of RNAi-based gene knockdown of M2 promoting genes, major bottlenecks include cellular siRNA delivery and correct intracellular processing. This is particularly true in case of macrophages as a cell type well-known to be notoriously hard-to-transfect. Among polymeric nanocarriers, the cationic polymer polyethylenimine (PEI) is widely explored for delivering nucleic acids. Further advanced nanocarriers are tyrosine-modified polymers based on PEI or polypropylenimine dendrimers (PPI) for highly efficient siRNA delivery in vitro and in vivo. In this paper, we explored a panel of PEI- or PPI-based nanoparticle systems for siRNA-mediated gene knockdown efficacy in macrophages and subsequent TAM repolarization. The tyrosine-modified linear 10 kDa PEI (LP10Y) or branched 5 kDa PEI (P5Y) as well as a tyrosine-modified PPI (PPI-Y) were found most efficient for gene knockdown in macrophage cell lines or primary macrophages, independent of their polarization. Knockdown of STAT6 or STAT3 led to repolarization of M2 macrophages, as indicated by alterations in various M2 and M1 marker levels. This highly specific approach also demonstrated non-redundant functions of STAT3 and STAT6. Importantly, macrophage re-polarization from M2 to M1 upon PPI-Y/siRNA-mediated STAT6 knockdown increased tumor cell phagocytosis in a co-culture model. In conclusion, we identify certain tyrosine-modified PEI- or PPI-based nanoparticles as particularly efficient for macrophage transfection, and the specific, siRNA-mediated STAT6 knockdown as a promising approach for macrophage repolarization and enhancement of their tumor cell suppressive role.
Assuntos
Macrófagos , Nanopartículas , Interferência de RNA , Linhagem Celular Tumoral , Macrófagos/metabolismo , RNA Interferente Pequeno/metabolismo , TirosinaRESUMO
Genome-wide association study (GWAS) analysis has exposed that genetic factors play important roles in COVID-19. Whereas a deeper understanding of the underlying mechanism of COVID-19 was hindered by the lack of expression of quantitative trait loci (eQTL) data specific for disease. To this end, we identified COVID-19-specific cis-eQTLs by integrating nucleotide sequence variations and RNA-Seq data from COVID-19 samples. These identified eQTLs have different regulatory effect on genes between patients and controls, indicating that SARS-CoV-2 infection may cause alterations in the human body's internal environment. Individuals with the TT genotype in the rs1128320 region seemed more susceptible to SARS-CoV-2 infection and developed into severe COVID-19 due to the abnormal expression of IFITM1. We subsequently discovered potential causal genes, of the result, a total of 48 genes from six tissues were identified. siRNA-mediated depletion assays in SARS-CoV-2 infection proved that 14 causal genes were directly associated with SARS-CoV-2 infection. These results enriched existing research on COVID-19 causal genes and provided a new sight in the mechanism exploration for COVID-19.
Assuntos
COVID-19 , Estudo de Associação Genômica Ampla , Humanos , SARS-CoV-2/genética , RNA Interferente Pequeno , RNA-SeqRESUMO
FOXM1, a proto-oncogenic transcription factor, plays a critical role in cancer development and treatment resistance in cancers, particularly in breast cancer. Thus, this study aimed to identify potential FOXM1 inhibitors through computational screening of drug databases, followed by in vitro validation of their inhibitory activity against breast cancer cells. In silico studies involved pharmacophore modeling using the FOXM1 inhibitor, FDI-6, followed by virtual screening of DrugBank and Selleckchem databases. The selected drugs were prepared for molecular docking, and the crystal structure of FOXM1 was pre-processed for docking simulations. In vitro studies included MTT assays to assess cytotoxicity, and Western blot analysis to evaluate protein expression levels. Our study identified Pantoprazole and Rabeprazole as potential FOXM1 inhibitors through in silico screening and molecular docking. Molecular dynamics simulations confirmed stable interactions of these drugs with FOXM1. In vitro experiments showed both Pantoprazole and Rabeprazole exhibited strong FOXM1 inhibition at effective concentrations and that showed inhibition of cell proliferation. Rabeprazole showed the inhibitor activity at 10 µM in BT-20 and MCF-7 cell lines. Pantoprazole exhibited FOXM1 inhibition at 30 µM and in BT-20 cells and at 70 µM in MCF-7 cells, respectively. Our current study provides the first evidence that Rabeprazole and Pantoprazole can bind to FOXM1 and inhibit its activity and downstream signaling, including eEF2K and pEF2, in breast cancer cells. These findings indicate that rabeprazole and pantoprazole inhibit FOXM1 and breast cancer cell proliferation, and they can be used for FOXM1-targeted therapy in breast or other cancers driven by FOXM1.
Assuntos
Neoplasias da Mama , Proliferação de Células , Reposicionamento de Medicamentos , Proteína Forkhead Box M1 , Simulação de Acoplamento Molecular , Rabeprazol , Humanos , Proteína Forkhead Box M1/antagonistas & inibidores , Proteína Forkhead Box M1/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Feminino , Rabeprazol/farmacologia , Células MCF-7 , Proliferação de Células/efeitos dos fármacos , Simulação de Dinâmica Molecular , Antineoplásicos/farmacologia , Antineoplásicos/química , Pantoprazol/farmacologia , Linhagem Celular Tumoral , Piridinas , TiofenosRESUMO
Despite the wide variety of available cationic lipid platforms for the delivery of nucleic acids into cells, the optimization of their composition has not lost its relevance. The purpose of this work was to develop multi-component cationic lipid nanoparticles (LNPs) with or without a hydrophobic core from natural lipids in order to evaluate the efficiency of LNPs with the widely used cationic lipoid DOTAP (1,2-dioleoyloxy-3-[trimethylammonium]-propane) and the previously unstudied oleoylcholine (Ol-Ch), as well as the ability of LNPs containing GM3 gangliosides to transfect cells with mRNA and siRNA. LNPs containing cationic lipids, phospholipids and cholesterol, and surfactants were prepared according to a three-stage procedure. The average size of the resulting LNPs was 176 nm (PDI 0.18). LNPs with DOTAP mesylate were more effective than those with Ol-Ch. Core LNPs demonstrated low transfection activity compared with bilayer LNPs. The type of phospholipid in LNPs was significant for the transfection of MDA-MB-231 and SW 620 cancer cells but not HEK 293T cells. LNPs with GM3 gangliosides were the most efficient for the delivery of mRNA to MDA-MB-231 cells and siRNA to SW620 cells. Thus, we developed a new lipid platform for the efficient delivery of RNA of various sizes to mammalian cells.
RESUMO
Background: The poor response of metastatic osteo- and chondrosarcomas to chemotherapy could be the result of multidrug resistance (MDR), which may be overcome through the use of small interfering RNA (siRNA). However, several methodologic questions remain unresolved. Aims: To test the toxicity of three commonly used siRNA transfection reagents and apply the least toxic reagent to investigate the siRNA-induced MDR1 mRNA knockdown. Methods: The toxicity of TransIT-TKO, Lipofectamine 2000, and X-tremeGENE siRNA transfection reagents was investigated on osteosarcoma (MG-63) and chondrosarcoma (SW1353) cell lines. The toxicity was measured at 4 and 24 hours using a MTT toxicity assay. The least toxic transfection reagent was applied to investigate the siRNA-induced MDR1 mRNA knockdown effect using qRT-PCR. Furthermore, five housekeeping genes were assessed in the BestKeeper software to obtain mRNA expression normalization. Results: Lipofectamine 2000 was the least toxic transfection reagent, reducing the cell viability only in chondrosarcoma 24 hours following exposure to the highest dose. In contrast, TransIT-TKO and X-tremeGENE transfection reagents displayed a significant reduction in cell viability in both chondrosarcoma after 4 hours and in osteosarcoma after 24 hours. Significant MDR1 mRNA silencing of over 80% was achieved in osteo- and chondrosarcoma using Lipofectamine at a final siRNA concentration of 25 nM. No significant dose response was observed in knockdown efficiency in either Lipofectamine or siRNA concentration. Conclusion: Lipofectamine 2000 was the least toxic transfection reagent in osteo- and chondrosarcoma. Successful siRNA-induced MDR1 mRNA silencing of over 80% was achieved.
Assuntos
Neoplasias Ósseas , Condrossarcoma , Osteossarcoma , Humanos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Osteossarcoma/genética , Osteossarcoma/patologia , RNA Interferente Pequeno/genética , Transfecção , Linhagem Celular Tumoral , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Condrossarcoma/tratamento farmacológico , Condrossarcoma/genéticaRESUMO
The concept of using two-photon excitation in the NIR for the spatiotemporal control of biological processes holds great promise. However, its use for the delivery of nucleic acids has been very scarcely described and the reported procedures are not optimal as they often involve potentially toxic materials and irradiation conditions. This work prepares a simple system made of biocompatible porous silicon nanoparticles (pSiNP) for the safe siRNA photocontrolled delivery and gene silencing in cells upon two-photon excitation. PSiNP are linked to an azobenzene moiety, which possesses a lysine group (pSiNP@ICPES-azo@Lys) to efficiently complex siRNA. Non-linear excitation of the two-photon absorber system (pSiNP) followed by intermolecular energy transfer (FRET) to trans azobenzene moiety, result in the photoisomerization of the azobenzene from trans to cis and in the destabilization of the azobenzene-siRNA complex, thus inducing the delivery of the cargo siRNA to the cytoplasm of cells. Efficient silencing in MCF-7 expressing stable firefly luciferase with siRNAluc against luciferase is observed. Furthermore, siRNA against inhibitory apoptotic protein (IAP) leads to over 70% of MCF-7 cancer cell death. The developed technique using two-photon light allows a unique high spatiotemporally controlled and safe siRNA delivery in cells in few seconds of irradiation.
Assuntos
Nanopartículas , Neoplasias , Humanos , RNA Interferente Pequeno/genética , Silício , Porosidade , Transfecção , Linhagem Celular TumoralRESUMO
Mesenchymal stromal cells (MSCs) are multipotent cells derived from different sources and able to differentiate into distinct cell lineages. For their possible biomedical application, the "tuning" of MSCs also involves the specific knockdown of defined target genes. A major limitation, however, is the notoriously low transfection efficacy especially of primary MSCs. In this paper, we systemically analyze a large set of tyrosine-modified linear or branched low molecular weight polyethylenimines (PEIs) of different sizes, as well as the tyrosine-modified polypropylenimine dendrimer PPI-G4, for their capacity of non-viral siRNA transfection into umbilical cord-derived MSCs from two different donors. Knockdown efficacies are determined on the molecular level and confirmed in functional assays. Beyond the determination of cell viabilities, acute cytotoxicity, induction of apoptosis/necrosis and mitochondrial membrane alterations are also studied. On the molecular level, caspase activation, ROS induction and genotoxic effects are analyzed. Major differences are observed between the various tyrosine-modified PEIs, with some candidates showing high knockdown efficacy and biocompatibility. PPI-G4-Y dendrimers, however, are identified as most efficient for siRNA transfection into MSCs. PPI-G4-Y/siRNA nanoparticles lead to particularly high gene knockdown, without cytotoxic and genotoxic effects on the cellular and molecular level, and are thus particularly well-suited for the tuning of MSCs.
Assuntos
Células-Tronco Mesenquimais , Tirosina , Polietilenoimina , RNA Interferente Pequeno , TransfecçãoRESUMO
Annexin A1 (ANXA1) is an endogenous protein involved in the control of proliferation, cell cycle, phagocytosis, and apoptosis in several types of cancer. To investigate the effects of ANXA1 knockdown in leukemia cells, transfection with specific ANXA1 siRNA was performed. Cell cycle and apoptosis were analyzed using flow cytometry and a mechanism involving caspases and Bcl-2 was quantified using Western blotting. Phagocytosis activity was evaluated using hematoxylin & eosin staining. The ANXA1 expression was significantly downregulated after the knockdown and apoptosis was induced in tested cells. The expression of caspase-9 and -3 increased in U937 and Jurkat cells respectively. Bcl-2 expression was downregulated in K562 and Jurkat cells while upregulated in U937. The number of leukemic cells arrested at the G2/M phase and the phagocytosis index were significantly increased in transfected cells. This suggests that ANXA1 knockdown might be a potential approach in the therapeutic strategy for leukemia.
Assuntos
Anexina A1 , Leucemia , Humanos , Anexina A1/genética , Anexina A1/metabolismo , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Pontos de Checagem da Fase G2 do Ciclo Celular , Leucemia/genética , Fagocitose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Differentiation of macrophages toward osteoclasts is crucial for bone homeostasis but can be detrimental in disease states, including osteoporosis and cancer. Therefore, understanding the osteoclast differentiation process and the underlying regulatory mechanisms may facilitate the identification of new therapeutic targets. Hereby, we tried to reveal new miRNAs potentially involved in the regulation of early steps of osteoclastogenesis, with a particular focus on those possibly correlated with NFATc1 expression, by studying miRNAs profiling. During the first 24 h of osteoclastogenesis, 38 miRNAs were differentially expressed between undifferentiated and RANKL-stimulated RAW264.7 cells, while 10 miRNAs were differentially expressed between RANKL-stimulated cells transfected with negative control or NFATc1-siRNAs. Among others, the expression levels of miR-411, miR-144 and members of miR-29, miR-30, and miR-23 families changed after RANKL stimulation. Moreover, the potential role of miR-124 during osteoclastogenesis was explored by transient cell transfection with anti-miR-124 or miR-124-mimic. Two relatively unknown miRNAs, miR-880-3p and miR-295-3p, were differentially expressed between RANKL-stimulated/wild-type and RANKL-stimulated/NFATc1-silenced cells, suggesting their possible correlation with NFATc1. KEGG enrichment analyses showed that kinase and phosphatase enzymes were among the predicted targets for many of the studied miRNAs. In conclusion, our study provides new data on the potential role and possible targets of new miRNAs during osteoclastogenesis.
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Extracellular vesicles (ECVs) are secreted cell-derived membrane particles involved in intercellular signaling and cell-cell communication. By transporting various bio-macromolecules, ECVs and in particular exosomes are relevant in various (patho-) physiological processes. ECVs are also released by cancer cells and can confer pro-tumorigenic effects. Their target cell tropism, effects on proliferation rates, natural stability in blood and immunotolerance makes ECVs particularly interesting as delivery vehicles. Polyethylenimines (PEIs) are linear or branched polymers which are capable of forming non-covalent complexes with small RNA molecules including siRNAs or antimiRs, for their delivery in vitro and in vivo. This study explores for the first time the combination of PEI-based nanoparticles with naturally occurring ECVs from different cell lines, for the delivery of small RNAs. ECV-modified PEI/siRNA complexes are analyzed by electron microscopy vs. ECV or complex alone. On the functional side, we demonstrate increased knockdown efficacy and storage stability of PEI/siRNA complexes upon their modification with ECVs. This is paralleled by enhanced tumor cell-inhibition by ECV-modified PEI/siRNA complexes targeting Survivin. Pre-treatment with various inhibitors of cellular internalization reveals alterations in cellular uptake mechanisms and biological activities of PEI/siRNA complexes upon their ECV modification. Extending our studies towards PEI-complexed antimiRs against miR-155 or miR-1246, dose-dependent cellular and molecular effects are enhanced in ECV-modified complexes, based on the de-repression of direct miRNA target genes. Differences between ECVs from different cell lines are observed regarding their capacity of enhancing PEI/siRNA efficacies, independent of the target cell line for transfection. Finally, an in vivo therapy study in mice bearing s.c. PC3 prostate carcinoma xenografts reveals marked inhibition of tumor growth upon treatment with ECVPC3-modified PEI/siSurvivin complexes, based on profound target gene knockdown. We conclude that ECV-modification enhances the activity of PEI-based complexes, by altering pivotal physicochemical and biological nanoparticle properties.
Assuntos
Vesículas Extracelulares , Polietilenoimina , Animais , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Masculino , Camundongos , RNA Interferente Pequeno , TransfecçãoRESUMO
The delivery of small interfering RNAs (siRNA) is an efficient method for gene silencing through the induction of RNA interference (RNAi). It critically relies, however, on efficient vehicles for siRNA formulation, for transfection in vitro as well as for their potential use in vivo. While polyethylenimines (PEIs) are among the most studied cationic polymers for nucleic acid delivery including small RNA molecules, polypropylenimines (PPIs) have been explored to a lesser extent. Previous studies have shown the benefit of the modification of small PEIs by tyrosine grafting which are featured in this paper. Additionally, we have now extended this approach towards PPIs, presenting tyrosine-modified PPIs (named PPI-Y) for the first time. In this study, we describe the marked improvement of PPI upon its tyrosine modification, leading to enhanced siRNA complexation, complex stability, siRNA delivery, knockdown efficacy and biocompatibility. Results of PPI-Y/siRNA complexes are also compared with data based on tyrosine-modified linear or branched PEIs (LPxY or PxY). Taken together, this establishes tyrosine-modified PPIs or PEIs as particularly promising polymeric systems for siRNA formulation and delivery.
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The existence of tightly integrated cross talk through multiple signaling and effector pathways has been appreciated in malignant cells. The realization of the plasticity of such networks is stimulating the development of combinational therapy to overcome the limitations of one-dimensional therapies. Synergistic pairs of siRNAs or siRNA and drug combinations are the new frontiers in identifying effective therapeutic combinations. To elucidate effective combinations, we developed a versatile protocol to screen siRNA libraries in triple-negative breast cancer cell models. This protocol outlines the steps to identify synergistic combinations of siRNA-siRNA or siRNA-drug combinations using siRNA libraries via a robotic screen. By focusing on smaller functional siRNA libraries, we present methodologies to identify synergistic siRNA pairings against cancerous cell growth and molecular targets to augment the activity of pro-apoptotic TRAIL protein. Here, we summarize the critical steps to undertake such combinational target identification, emphasizing critical factors that affect the outcome of the screens. Our experience suggests that siRNA library screening is an efficient protocol to identify complementary therapeutic pairs of new or already-existing drugs. This protocol is simple, robust and can be completed within a 1-week working period.
Assuntos
Detecção Precoce de Câncer/métodos , Ensaios de Triagem em Larga Escala/métodos , RNA Interferente Pequeno/genética , Neoplasias de Mama Triplo Negativas/genética , Proliferação de Células/genética , Biblioteca Gênica , Humanos , RNA Interferente Pequeno/isolamento & purificação , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/terapiaRESUMO
Recently, our group has contrasted an endothelial cell-smooth muscle cell (EC-SMC) co-culture model with 3D-cultured SMCs and found that SMCs could respond to high shear stress (SS), which has not been explored before. SMCs were not directly exposed to the flow but were under an EC monolayer; therefore, it is necessary to explore the influence of EC on SMC behaviors under high SS for understanding the mechanism of SMC response to various magnitudes of SS. In the present study, TGF-ß1 expression in ECs in an EC-SMC co-culture model was suppressed by an siRNA transfection method. Next, phenotypic changes were observed and MMP-2 and -9 productions were measured in SMCs in the co-culture model after 72-h flow exposure to different SS levels. We confirmed that TGF-ß1 expression in ECs could influence SMC phenotypic change under SS conditions and that TGF-ß1 expression in ECs could also change MMP-2 production but not MMP-9 production in SMCs under SS conditions in the co-culture model. These results could be useful for understanding the mechanisms of SMC response to SS, particularly for understanding signal transduction emanating from ECs.
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BACKGROUND: Human embryonic stem (hES) cells serve as an invaluable tool for research and future medicine, but their transfection often leads to unwanted side effects as the method itself may induce differentiation. On the other hand, RNA interference (RNAi)-based targeted gene silencing is a quick, cost-effective, and easy-to-perform method to address questions regarding the function of genes, especially when hypomorphic knockdowns are needed. Therefore, effective transfection method with minimal side effects is essential for applying RNAi to hES cells. Here, we report a highly promising approach for targeted gene silencing in hES cells with siRNA complexed with cell-penetrating peptide PepFect 14 (PF14). This strategy provides researchers with efficient tool for unraveling the functions of genes or addressing the differentiation of pluripotent stem cells. METHODS: We present a method for delivery of siRNA into hES cells with cell-penetrating peptide PF14. Accordingly, hES cells were transfected in ROCK inhibitor containing medium for 24 h right after EDTA passaging as small cell clumps. Fluorescently labeled siRNA and siRNAs targeting OCT4 or beta-2-microglobulin (B2M) mRNA sequences were used to evaluate the efficiency of transfection and silencing. Analyses were performed at various time points by flow cytometry, RT-qPCR, and immunofluorescence microscopy. RESULTS: Effective downregulation of OCT4 in 70% of treated hES cells at protein level was achieved, along with 90% reduction at mRNA level in bulk population of cells. The applicability of this low-cost and easy-to-perform method was confirmed by inducing silencing of another target not associated with hES cell pluripotency (B2M). Furthermore, we discovered that downregulation of OCT4 induces neuroectodermal differentiation accompanied by reduced expression of B2M during early stage of this lineage. CONCLUSIONS: The results demonstrate PF14 as a promising tool for studying gene function and regulatory networks in hES cells by using RNAi.