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Background and objective: The combination of two or more different mechanisms of drugs in the treatment of cancer has become one of the effective methods. The purpose of this study was to successfully prepare a non-viral delivery system that could efficiently co-delivery siRNA and gambogenic acid (GNA) to improve the anti-cancer efficiency in HepG2 cells. Methods: The delivery system was prepared by a two-step method. First, the GNA-anionic liposome took shape by a solvent evaporation method, and then the liposome was bound to the PEI/siRNA complex by electrostatic interaction to form the final carrier system (lipopolyplexes). Agarose gel electrophoresis, MTT, particle size and zeta potential were detected to analyse the lipopolyplexes formation. The transfection efficiency of siRNA was determined by confocal laser scanning microscopy and flow cytometry. Western blotting was used to assess the VEGF protein expression levels of HepG2 cells. The cell apoptosis assay was used to assess the anti-tumour superiority of lipopolyplexes. Results: GNA-PEI/siRNA-liposome (lipopolyplexes) are significantly less cytotoxicity compared to PEI mediated carriers. Simultaneously, the results of flow cytometry and confocal laser scanning microscopy indicated that the lipopolyplexes could successfully carry siRNA into the cytoplasm, and the western-blot result evidence that the delivery system has a potential for VEGF to express down. Also compared with the control group, the results of apoptosis test suggest that the lipopolyplexes can significantly promote cell apoptosis. Conclusion: The delivery system has a potential in the combination of various drugs for cancer therapy in future.
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Antineoplásicos/química , Lipossomos/química , Polietilenoimina/química , RNA Interferente Pequeno/química , Fator A de Crescimento do Endotélio Vascular/genética , Xantenos/química , Ânions/química , Apoptose/efeitos dos fármacos , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada/métodos , Citoplasma/metabolismo , Liberação Controlada de Fármacos , Inativação Gênica/efeitos dos fármacos , Terapia Genética/métodos , Células Hep G2 , HumanosRESUMO
A successful siRNA delivery system is dependent on the development of a good siRNA carrier. Graphene oxide (GO) has gained great attention as a promising nanocarrier in recent years. It has been reported that GO could be used to deliver a series of drugs including synthetic compounds, proteins, antibodies, and genes. Our previous research indicated that functionalized GO could deliver siRNA into tumor cells and induce a gene silencing effect, to follow up the research, in this research, GO-R8/cRGDfV(GRcR) was designed and prepared for VEGF-siRNA delivery as a novel carrier. The Zeta potential and particle size of the new designed GRcR carrier was measured at (29.46 ± 5.32) mV and (135.7 ± 3.3) nm respectively, and after transfection, the VEGF mRNA level and protein expression level were down-regulated by 48.22% (p < 0.01) and 38.3% (p < 0.01) in HeLa cells, respectively. The fluorescent images of the treated BALB/c nude mice revealed that GRcR/VEGF-siRNA could conduct targeted delivery of VEGF-siRNA into tumor tissues and showed a gene silencing effect as well as a tumor growth inhibitory effect (p < 0.01) in vivo. Further studies showed that GRcR/VEGF-siRNA could effectively inhibit angiogenesis by suppressing VEGF expression. Histology and immunohistochemistry studies demonstrated that GRcR/VEGF-siRNA could inhibit tumor tissue growth effectively and have anti-angiogenesis activity, which was the result of VEGF protein downregulation. Both in vitro and in vivo results demonstrated that GRcR/VEGF-siRNA could be used as an ideal nonviral tumor-targeting vector for VEGF-siRNA delivery in gene therapy.
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Técnicas de Transferência de Genes , Grafite/química , RNA Interferente Pequeno/metabolismo , Animais , Varredura Diferencial de Calorimetria , Morte Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Inativação Gênica , Células HeLa , Humanos , Camundongos Nus , Neoplasias/patologia , Neovascularização Fisiológica , Oligopeptídeos/química , Tamanho da Partícula , Peptídeos Cíclicos/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , Termogravimetria , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Anti-tumor angiogenesis therapy, targeting the suppression of blood vessel growth in tumors, presents a potent approach in the battle against cancer. Traditional therapies have primarily concentrated on single-target techniques, with a specific emphasis on targeting the vascular endothelial growth factor, but have not reached ideal therapeutic efficacy. In response to this issue, our study introduced a novel nanoparticle system known as CS-siRNA/PEITC&L-cRGD NPs. These chitosan-based nanoparticles have been recognized for their excellent biocompatibility and ability to deliver genes. To enhance their targeted delivery capability, they were combined with a cyclic RGD peptide (cRGD). Targeted co-delivery of gene and chemotherapeutic agents was achieved through the use of a negatively charged lipid shell and cRGD, which possesses high affinity for integrin αvß3 overexpressed in tumor cells and neovasculature. In this multifaceted approach, co-delivery of VEGF siRNA and phenethyl isothiocyanate (PEITC) was employed to target both tumor vascular endothelial cells and tumor cells simultaneously. The co-delivery of VEGF siRNA and PEITC could achieve precise silencing of VEGF, inhibit the accumulation of HIF-1α under hypoxic conditions, and induce apoptosis in tumor cells. In summary, we have successfully developed a nanoparticle delivery platform that utilizes a dual mechanism of action of anti-tumor angiogenesis and pro-tumor apoptosis, which provides a robust and potent strategy for the delivery of anti-cancer therapeutics.
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Combinations of two different therapeutic modalities of VEGF inhibitors against angiogenesis can cooperatively impede breast cancer tumor growth and enhance therapeutic efficacy. Itraconazole (ITZ) is a conventional antifungal drug with high safety; however, it has been repurposed to be a multi target anti-angiogenesis agent for cancer therapy in recent years. In the present study, composite nanoparticles co-loaded with ITZ and VEGF siRNA were prepared in order to investigate their anti-angiogenesis efficacy and synergistic anticancer effect against breast cancer. The nanoparticles had a suitable particle size (117.9 ± 10.3 nm) and weak positive surface charge (6.69 ± 2.46 mV), as well as good stability and drug release profile in vitro. Moreover, the nanoparticles successfully escaped from endosomes and realized cell apoptosis and cell proliferation inhibition in vitro. In vitro and in vivo experiments showed that the nanoparticles could induce the silencing of VEGF-related expressions as well as anti-angiogenesis efficacy, and the co-loaded ITZ-VEGF siRNA NPs could inhibit tumor growth effectively with low toxicity and side effects. Taken together, the as-prepared delivery vehicles are a simple and safe nano-platform that improves the antitumor efficacy of VEGF siRNA and ITZ, which allows the repositioning of the generic drug ITZ as a great candidate for antitumor therapy.
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BACKGROUND: The dual-loaded nano-delivery system can realize chemotherapeutic drug and small interfering RNA (siRNA) co-loading as well as enhance the therapeutic effect of drugs on tumors through a synergistic effect, while reducing their toxic and side effects on normal tissues. METHODS: Previously, we developed layered smart nanoparticles (NPs) to co-deliver survivin siRNA as well as small molecule drugs for lung cancer. In this study, we used such smart NPs to co-deliver paclitaxel (PTX) and siRNA against vascular endothelial growth factor (VEGF) gene for breast cancer therapy in mice models. For the prepared NPs, characterizations such as particle size, zeta potential, gel electrophoresis imaging and in vitro stability were investigated. Then, 4T1 cells were used to evaluate the in vitro VEGF silencing capacity, tumor cell inhibitory and anti-apoptotic abilities. Finally, an orthotopic model of mouse breast cancer was established to evaluate the in vivo antitumor effects and safety properties of PTX-siRNAVEGF-NPs. RESULTS: We prepared PTX-siRNAVEGF-NPs with particle size of 85.25 nm, PDI of 0.261, and zeta potential of 5.25 mV. The NPs with VEGF siRNA effectively knocked down the expression of VEGF mRNA. Cell counting kit-8 (CCK-8) and apoptosis assays revealed that the PTX-siRNAVEGF-NPs exhibited antiproliferation effect of PTX on 4T1 cells. The in vivo anti-tumor study indicated that PTX-siRNAVEGF-NPs could exert an antitumor effect by inhibiting the formation and development of new blood vessels in tumor tissues, thereby cutting off nutrient and blood supplies required for tumor tissue growth. Both the anti-tumor efficacy and in vivo safety of the PTX-siRNAVEGF-NPs group were better than that of the PTX-NPs and siRNAVEGF-NPs groups. CONCLUSION: The combination of PTX and VEGF siRNA exerts good antitumor effect on 4T1 tumor cells. This study provides a theoretical and practical basis for breast cancer therapy.
Assuntos
Neoplasias da Mama , Nanopartículas , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Paclitaxel/uso terapêutico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/uso terapêuticoRESUMO
OBJECTIVE: Although great progress has been made in the field of siRNA gene therapy, safe, efficient, and targeted delivery of siRNA are still major challenges in siRNA therapeutics. METHODS: We developed an up-conversion nanoparticle-based nanocage system. This system protected the siRNA from being degraded by nucleases in organisms and selectively delivered the siRNAs to the tumor sites, due to modifications of targeted molecules on the surfaces of nanocages and local inhalation. RESULTS: The siRNAs delivered by the up-conversion nanoparticle nanocages were protected from degradation in transit to the tumor sites, where they accumulated. Compared with the passive target and control groups, the up-conversion nanoparticles based on the nanocage system showed a tumor suppressive effect after approximately 3 weeks of treatment. CONCLUSIONS: The up-conversion nanoparticle nanocages efficiently delivered vascular endothelial growth factor siRNAs to tumor sites. Mice with lung tumors treated with tumors targeting up-conversion nanoparticle nanocages showed steady body weight changes, high tumor inhibition ratios, and longer survival times.
RESUMO
The combination of chemotherapeutic drug paclitaxel (PTX) and VEGF siRNA could inhibit cancer development with synergistic efficacy. However, efficient and safe delivery systems with high encapsulation efficiency of PTX and a long-time release of drugs are urgently needed. In this study, novel nanoparticles (PTX/siRNA/FALS) were constructed by using tripeptide lipid (L), sucrose laurate (S), and folate-PEG2000-DSPE (FA) to co-deliver PTX and siRNA. The cancer cell targeting nanoparticle carrier (PTX/siRNA/FALS) showed anticipated PTX encapsulation efficiency, siRNA retardation ability, improved cell uptake and sustained and controlled drug release. It led to significant anti-tumor activity in vitro and in vivo by efficient inhibition of VEGF expression and induction of cancer cell apoptosis. Importantly, the biocompatibility of the carriers and low dosage of PTX required for effective therapy greatly reduced the toxicity to mice. The targeting nanoparticles show potential as an effective co-delivery platform for RNAi and chemotherapy drugs, aiming to improve the efficacy of cancer therapy.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas/administração & dosagem , Paclitaxel/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Lipídeos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Peptídeos/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The present work explores the ability of poly(1-vinylimidazole) (PVI) to complex small interfering RNA (siRNA) silencing vascular endothelial growth factor (VEGF) and the in vitro efficiency of the formed complexes in A549 lung cancer cells. The polyplex formed was found to exhibit 66% complexation efficiency. The complexation was confirmed by gel retardation assays, FTIR and thermal analysis. The blank PVI polymer was not toxic to cells. The polyplex was found to exhibit excellent internalization and escaped the endosome effectively. The polyplex was more effective than free siRNA in silencing VEGF in lung cancer cells. The silencing of VEGF was quantified using Western blot and was also reflected in the depletion of HIF-1α levels in the cells treated with the polyplex. VEGF silencing by the polyplex was found to augment the cytotoxic effects of the chemotherapeutic agent 5-fluorouracil. Microarray analysis of the mRNA isolated from cells treated with free siRNA and the polyplex reveal that the VEGF silencing by the polyplex also altered the expression levels of several other genes that have been connected to the proliferation and invasion of lung cancer cells. These results indicate that the PVI complexes can be an effective agent to counter lung cancer.
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Non-viral nanocarrier affords a platform for drug and siRNA combination, the focus of which is to load drug and siRNA into a single carrier, allowing for co-delivery and a synergistic effect at tumour site. In our previous study, pH-sensitive carboxymethyl chitosan-modified liposomes (CMCS-SiSf-CL) were assembled for sorafenib (Sf) and Cy3-siRNA co-loaded. The present study evaluated in vitro and in vivo co-delivery of the co-loaded liposomes. Further, in vitro inhibiting hepatocellular carcinoma of the pH-sensitive sorafenib (Sf) and VEGF-siRNA co-loaded liposomes was discussed. The experimental results demonstrated co-delivery and penetration into 2-dimensional (2D) cultured HepG2 cells, 3-dimensional (3D) cultured HepG2 tumour spheroids and tumour regions of H22 tumour-bearing mice. Compared with free siRNA and single loaded carrier, co-delivery liposomes exhibited enhanced VEGF downregulating effect, inducing cell early apoptosis. Therefore, the CMCS-SiSf-CL delivery system can lay the foundation for the co-delivery systems development and provide new area for HCC therapy.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Sorafenibe/administração & dosagem , Sorafenibe/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Inativação Gênica , Humanos , Concentração de Íons de Hidrogênio , Lipossomos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Camundongos , Sorafenibe/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/deficiênciaRESUMO
Targeting tumor angiogenesis pathway via VEGF siRNA (siVEGF) has shown great potential in treating highly malignant and metastatic non-small cell lung cancer (NSCLC). However, anti-angiogenic monotherapy lacked sufficient antitumor efficacy which suffered from malignant tumor proliferation. Therefore, the combined application of siVEGF and chemotherapeutic agents for simultaneous targeting of tumor proliferation and angiogenesis has been a research hotspot to explore a promising NSCLC therapy regimen. Methods: We designed, for the first time, a rational therapy strategy via intelligently co-delivering siVEGF and chemotherapeutics etoposide (ETO) by multi-functional nanoparticles (NPs) directed against the orthotopic NSCLC. These NPs consisted of cationic liposomes loaded with siVEGF and ETO and then coated with versatile polymer PEGylated histidine-grafted chitosan-lipoic acid (PHCL). We then comprehensively evaluated the anti-angiogenic and anti-proliferation efficiency in the in vitro tumor cell model and in bioluminescent orthotopic lung tumor bearing mice model. Results: The NPs co-delivering siVEGF and ETO exhibited tailor-made surface charge reversal features in mimicking tumor extracellular environment with improved internal tumor penetration capacity and higher cellular internalization. Furthermore, these NPs with flexible particles size triggered by intracellular acidic environment and redox environment showed pinpointed and sharp intracellular cargo release guaranteeing adequate active drug concentration in tumor cells. Enhanced VEGF gene expression silencing efficacy and improved tumor cell anti-proliferation effect were demonstrated in vitro. In addition, the PHCL layer improved the stability of these NPs in neutral environment allowing enhanced orthotopic lung tumor targeting efficiency in vivo. The combined therapy by siVEGF and ETO co-delivered NPs for orthotopic NSCLC simultaneously inhibited tumor proliferation and tumor angiogenesis resulting in more significant suppression of tumor growth and metastasis than monotherapy. Conclusion: Combined application of siVEGF and ETO by the multi-functional NPs with excellent and on-demand properties exhibited the desired antitumor effect on the orthotopic lung tumor. Our work has significant potential in promoting combined anti-angiogenesis therapy and chemotherapy regimen for clinical NSCLC treatment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Etoposídeo/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sistemas de Liberação de Medicamentos/métodos , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , Esferoides Celulares/citologia , Esferoides Celulares/metabolismoRESUMO
Cell-penetrating peptides (CPPs) have been widely used to deliver nucleic acid molecules. Generally, CPPs consisting of short amino acid sequences have a linear structure, resulting in a weak complexation and low transfection efficacy. To overcome these drawbacks, a novel type of CPP is required to enhance the delivery efficacy while maintaining its safe use at the same time. Herein, we report that a bioreducible branched poly-CPP structure capable of responding to reducing conditions attained both outstanding delivery effectiveness and selective gene release in carcinoma cells. Branched structures provide unusually strong electrostatic attraction between DNA and siRNA molecules, thereby improving the transfection capability through a tightly condensed form. We designed a modified type of nona-arginine (mR9) and synthesized a branched-mR9 (B-mR9) using disulfide bonds. A novel B-mR9/pDNA polyplex exhibited redox-cleavability and high transfection efficacy compared to conventional CPPs, with higher cell viability as well. B-mR9/VEGF siRNA polyplex exhibited significant serum stability and high gene-silencing effects in vitro. Furthermore, the B-mR9 polyplex showed outstanding tumor accumulation and inhibition ability in vivo. The results suggest that the bioreducible branched poly CPP has great potential as a gene delivery platform.
Assuntos
Arginina/análogos & derivados , Peptídeos Penetradores de Células/química , DNA/administração & dosagem , Técnicas de Transferência de Genes , Plasmídeos/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Animais , Sobrevivência Celular , DNA/genética , Feminino , Células HEK293 , Células HeLa , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Plasmídeos/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transfecção , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The development of an efficient colorectal cancer therapy is currently a public health priority. In the present work, we proposed a multifunctional theranostic micellar drug delivery system utilizing cationic PDMA-block-poly(ε-caprolactone) (PDMA-b-PCL) micelles as nanocarriers of SN-38 (7-ethyl-10-hydroxycamptothecin), ultra-small superparamagnetic iron oxide nanoparticles (USPIO), and small interfering RNA (siRNA) that targets human vascular endothelial growth factor (VEGF). The VEGF siRNA was conjugated to polyethylene glycol (PEG) (siRNA-PEG) before complexation with the micelles in order to improve the siRNA's stability and to prolong its retention time in the blood circulation. To further improve the in vivo biosafety, we prepared mixed micelles using mPEG-PCL together with PDMA-b-PCL copolymer. The SN-38/USPIO-loaded siRNA-PEG mixed micelleplexes passively targeted to tumor regions and synergistically facilitated VEGF silencing and chemotherapy, thus efficiently suppressing tumor growth via a multi-dose therapy regimen. Additionally, the SN-38/USPIO-loaded siRNA-PEG mixed micelleplexes acted as a negative magnetic resonance imaging (MRI) contrast agent in T2-weighted imaging, resulting in a powerful tool for the diagnosis and for tracking of the therapeutic outcomes. In summary, we established a theranostic micellar drug and gene delivery system that not only synergistically combined gene silencing and chemotherapy but also served as a negative MRI contrast agent, which reveal its potential as a novel colorectal cancer therapy.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Camptotecina/análogos & derivados , Neoplasias Colorretais/terapia , Portadores de Fármacos/química , RNA Interferente Pequeno/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Camptotecina/administração & dosagem , Camptotecina/uso terapêutico , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Dextranos/química , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Irinotecano , Nanopartículas de Magnetita/química , Metacrilatos/química , Camundongos Endogâmicos BALB C , Camundongos Nus , Micelas , Poliésteres/química , Polietilenoglicóis/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Terapêutica com RNAi/métodos , Reto/efeitos dos fármacos , Reto/metabolismo , Reto/patologia , Nanomedicina TeranósticaRESUMO
In this data article, carboxyfluorescein-loaded liposomes were prepared and purified from free carboxyfluorescein using gel filtration chromatography in the first part. In the next part, following preparation of anti-VEGF siRNA loaded liposomes incorporating hydrophobically modified G2 PAMAM dendrimer (G2-Chol40%) (Golkar et al., 2016) [1], the cell growth inhibition induced by the formulations (siRNA/Metafectene complexes and siRNA loaded liposomes incorporating hydrophobic G2) was evaluated at two exposure times through MTT assay in a breast cancer cell (SKBR-3) and compared by two-way ANOVA.
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A novel lipopolymer based system was designed and characterized for cellular delivery of anti-VEGF siRNA in SKBR-3 breast tumor cell line. Polyamidoamine (PAMAM) dendrimers of low generations (G1, G2 and G3) were incorporated into polyethylene glycol (PEG)-stabilized liposomes by following the consecutive steps: (a) synthesis of the cholesterol conjugates (40% molar ratio of cholesterol to primary amines of PAMAM), (b) incorporation of the conjugates in liposome by lipid mixing and (c) microencapsulation of the siRNA using the ethanol drop method. The cholesterol conjugates of PAMAM dendrimers (G1-Chol40%, G2-Chol40% and G3-Chol40%) formed self assembly with low CMC values (<11µg/ml). Not only did G2-Chol40% show the highest lipid mixing among the cholesterol conjugates, but also, had the lowest leakage of encapsulated carboxyfluorescein tracer. Various N(amine))/L(lipid)/P(phosphate) mole ratios were investigated for siRNA condensation by ethidium bromide dye exclusion assay. The optimum N/L/P ratio of 20:33:10 was chosen for microencapsulation of anti-VEGF siRNA by ethanol drop method, showing particle size of 130nm, zeta-potential of +4mV, siRNA loading efficiency and capacity of 96% and 13wt%, and high stability against heparin sulfate (extracellular matrix). TEM shows uniform and discrete oligo- or multi-lamellar vesicular structures. The liposome incorporating G2-Chol40% was successfully internalized into SKBR-3 cells mainly through clathrin-mediated endocytosis, which was able to escape from endosomes and showed a significantly higher sequence-specific inhibition of VEGF expression and cell growth than the respective G2-Chol40%/siRNA dendriplexes. Importantly, the cytotoxicity decreased with incorporation of G2-Chol40% in the liposomes.
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Dendrímeros/administração & dosagem , Portadores de Fármacos/administração & dosagem , Poliaminas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dendrímeros/química , Relação Dose-Resposta a Droga , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Lipossomos , Poliaminas/química , RNA Interferente Pequeno/química , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Galactose modified trimethyl chitosan-cysteine (GTC) conjugates with various galactose grafting densities were developed for oral delivery of Survivin shRNA-expression pDNA (iSur-pDNA) and vascular endothelial growth factor (VEGF) siRNA (siVEGF) in the synergistic and targeted treatment of hepatoma. iSur-pDNA and siVEGF loaded GTC nanoparticles (NPs) were prepared via electrostatic complexation and showed desirable stability in physiological fluids and improved intestinal permeation compared to naked genes. Galactose grafting density of GTC NPs significantly affected their in vitro and in vivo antitumor activities. GTC NPs with moderate galactose grafting density, termed GTC2 NPs, were superior in facilitating cellular uptake, promoting nuclear distribution, and silencing target genes, leading to notable inhibition of cell growth. In tumor-bearing mice, orally delivered GTC2 NPs could effectively accumulate in the tumor tissues and silence the expression of Survivin and VEGF, evoking increased apoptosis, inhibited angiogenesis, and thus the most efficient tumor regression. Moreover, compared with single gene delivery, co-delivery of iSur-pDNA and siVEGF showed synergistic effects on inhibiting in vitro cell proliferation and in vivo tumor growth. This study could serve as an effective approach for synergistic cancer therapy via oral gene delivery, and highlighted the importance of ligand grafting density in the rational design of targeted nanocarriers.
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Carcinoma Hepatocelular/terapia , Quitosana/análogos & derivados , Proteínas Inibidoras de Apoptose/genética , Neoplasias Hepáticas/terapia , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Feminino , Galactose/análogos & derivados , Terapia Genética , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , SurvivinaRESUMO
Targeted delivery of therapeutic genes to the tumor site is critical for successful and safe cancer gene therapy. The arginine grafted bio-reducible poly (cystamine bisacrylamide-diaminohexane, CBA-DAH) polymer (ABP) conjugated poly (amido amine) (PAMAM), PAM-ABP (PA) was designed previously as an efficient gene delivery carrier. To achieve high efficacy in cancer selective delivery, we developed the tumor targeting bio-reducible polymer, PA-PEG1k-RGD, by conjugating cyclic RGDfC (RGD) peptides, which bind αvß3/5 integrins, to the PAM-ABP using polyethylene glycol (PEG, 1 kDa) as a spacer. Physical characterization showed nanocomplex formation with bio-reducible properties between PA-PEG1k-RGD and plasmid DNA (pDNA). In transfection assays, PA-PEG1k-RGD showed significantly higher transfection efficiency in comparison with PAM-ABP or PA-PEG1k-RAD in αvß3/5 positive MCF7 breast cancer and PANC-1 pancreatic cancer cells. The targeting ability of PA-PEG1k-RGD was further established using a competition assay. To confirm the therapeutic effect, the VEGF siRNA expressing plasmid was constructed and then delivered into cancer cells using PA-PEG1k-RGD. PA-PEG1k-RGD showed 20-59% higher cellular uptake rate into MCF7 and PANC-1 than that of non-targeted polymers. In addition, MCF7 and PANC-1 cancer cells transfected with PA-PEG1k-RGD/pshVEGF complexes had significantly decreased VEGF gene expression (51-71%) and cancer cell viability (35-43%) compared with control. These results demonstrate that a tumor targeting bio-reducible polymer with an anti-angiogenic therapeutic gene could be used for efficient and safe cancer gene therapy.
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Técnicas de Transferência de Genes , Oligopeptídeos/química , Plasmídeos/administração & dosagem , Polímeros/química , RNA Interferente Pequeno/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/genética , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Neoplasias/genética , Neoplasias/terapia , Oxirredução , Plasmídeos/genética , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
To develop a gene carrier for cancer therapy by systemic injection, we synthesized methoxypolyethylene glycol-polycaprolactone (MPEG-PCL) diblock copolymers conjugated with a cytoplasm-responsive cell-penetrating peptide (CPP), CH2R4H2C (C, Cys; H, His; R, Arg). The carrier/small interfering RNA (siRNA) complexes (N/P ratio of 20) had a particle size of approximately 50 nm and stabilized the siRNA against RNase. The cellular uptake ability of the carrier/FAM-siRNA complexes with fetal bovine serum was significantly higher than that of naked FAM-siRNA. In addition, the carrier/anti-vascular endothelial growth factor siRNA (siVEGF) complexes attained a significantly greater silencing effect than naked siVEGF with low cytotoxicity, resulting from higher uptake, early endosomal escape, and efficient release from the complexes in the cytoplasm. Furthermore, intravenous injection of MPEG-PCL-CH2R4H2C/siVEGF complexes had a significantly higher anti-tumor effect in S-180 tumor-bearing mice, which could be attributed to the rigid compaction of siRNA by ionic interactions and disulfide linkages in the CPP polymer micelles in the blood, as well as higher release following cleavage of the disulfide bonds in the reductive cytosol. Taken together, our data demonstrated that these cytoplasm-responsive polymer micelles conjugated with multi-functional CPP, could facilitate siVEGF delivery to tumor tissues after systemic injection and could exert an extremely strong anti-tumor effect.
Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Portadores de Fármacos/administração & dosagem , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Citoplasma , Portadores de Fármacos/química , Masculino , Camundongos , Camundongos Endogâmicos ICR , Nanopartículas/química , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Poliésteres/química , Polietilenoglicóis/química , RNA Interferente Pequeno/química , Carga Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: Small interfering RNA (siRNA) can silence target genes in the cytoplasm and be a major tool in gene therapy. Vascular endothelial growth factor (VEGF), a potent regulator of angiogenesis, is overexpressed in most tumors and is closely associated with tumor growth and metastasis. It has been shown that inhibition of VEGF expression by siRNA is an effective and useful method for antiangiogenic tumor therapy. METHODS: In the present study, we synthesized a targeted delivery system of PEI-PEG-APRPG incorporating angiogenic vessel-homing Ala-Pro-Arg-Pro-Gly (APRPG) peptide into cationic polyethylenimine (PEI) via a hydrophilic poly(ethylene glycol) (PEG) spacer. RESULTS: PEI-PEG-APRPG effectively condensed siRNA into 20-50 nm nanoparticles with a positive surface charge using a suitable N/P ratio. The siRNA/PEI-PEG-APRPG complex effectively enhanced the stability of siRNA in RNase A, and improved the proliferation-inhibiting ability and transfection efficiency of siRNA in vitro and tumor accumulation in vivo. In addition, the siRNA/PEI-PEG-APRPG complex exhibited high efficiency as antitumor therapy with regard to tumor growth, microvessel density, and VEGF protein and mRNA levels. CONCLUSION: These findings suggest that PEI-PEG-APRPG effectively delivers siRNA to tumors overexpressing VEGF and thereby inhibits tumor growth.