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1.
Br J Anaesth ; 123(4): 439-449, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31383364

RESUMO

BACKGROUND: Nerve growth factor (NGF) has been implicated in hyperalgesia by sensitising nociceptors. A role for NGF in modulating myocardial injury through ischaemic nociceptive signalling is plausible. We examined whether inhibition of spinal NGF attenuates myocardial ischaemia-reperfusion injury and explored the underlying mechanisms. METHODS: In adult rats, lentivirus-mediated short-hairpin RNA targeted at reducing NGF gene expression (NGF-shRNA) or a transient receptor potential vanilloid 1 (TRPV1) antagonist (capsazepine) was injected intrathecally before myocardial ischaemia-reperfusion. Infarct size (expressed as the ratio of area at risk) and risk of arrhythmias were quantified. Whole-cell clamp patch electrophysiology was used to record capsaicin currents in primary dorsal root ganglion neurones. The co-expression of substance P (SP) and calcitonin gene-related peptide (CGRP), plus activation of TRPV1, protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) were also quantified. RESULTS: NGF levels increased by 2.95 (0.34)-fold in dorsal root ganglion and 2.12 (0.27)-fold in spinal cord after myocardial ischaemia-reperfusion injury. Intrathecal injection of NGF-shRNA reduced infarct area at risk from 0.58 (0.02) to 0.37 (0.02) (P<0.01) and reduced arrhythmia score from 3.67 (0.33) to 1.67 (0.33) (P<0.01). Intrathecal capsazepine was similarly cardioprotective. NGF-shRNA suppressed expression of SP/CGRP and activation of Akt/ERK and TRPV1 in spinal cord. NGF increased capsaicin current amplitude from 144 (42) to 840 (132) pA (P<0.05), which was blocked by the TRPV1 antagonist 5'-iodoresiniferatoxin. Exogenous NGF enhanced capsaicin-induced Akt/ERK and TRPV1 activation in PC12 neuroendocrine tumour cells in culture. CONCLUSIONS: Spinal NGF contributes to myocardial ischaemia-reperfusion injury by mediating nociceptive signal transmission.


Assuntos
Terapia Genética/métodos , Lentivirus/genética , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fator de Crescimento Neural/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , Animais , Arritmias Cardíacas/prevenção & controle , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Cardiotônicos/administração & dosagem , Cardiotônicos/uso terapêutico , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Injeções Espinhais , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/prevenção & controle , Fator de Crescimento Neural/biossíntese , Células PC12 , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo
2.
Pharm Res ; 36(10): 142, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31376020

RESUMO

BACKGROUND: With the recent approval of the first small interfering RNA (siRNA) therapeutic formulated as nanoparticles, there is increased incentive for establishing the factors of importance for the design of stable solid dosage forms of such complex nanomedicines. METHODS: The aims of this study were: (i) to identify factors of importance for the design of spray-dried siRNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles (LPNs), and (ii) to evaluate their influence on the resulting powders by using a quality-by-design approach. Critical formulation and process parameters were linked to critical quality attributes (CQAs) using design of experiments, and an optimal operating space (OOS) was identified. RESULTS: A series of CQAs were identified based on the quality target product profile. The loading (ratio of LPNs to the total solid content) and the feedstock concentration were determined as critical parameters, which were optimized systematically. Mannitol was chosen as stabilizing excipient due to the low water content of the resulting powders. The loading negatively affected the colloidal stability of the LPNs, whereas feedstock concentration correlated positively with the powder particle size. The optimal mannitol-based solid formulation, defined from the OOS, displayed a loading of 5% (w/w), mass median aerodynamic diameter of 3.3 ± 0.2 µm, yield of 60.6 ± 6.6%, and a size ratio of 1.15 ± 0.03. Dispersed micro-embedded LPNs had preserved physicochemical characteristics as well as in vitro siRNA release profile and gene silencing, as compared to non-spray-dried LPNs. CONCLUSION: The optimal solid dosage forms represent robust formulations suitable for higher scale-up manufacturing.


Assuntos
Dessecação/métodos , Lipídeos/química , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , RNA Interferente Pequeno/química , Administração por Inalação , Animais , Composição de Medicamentos , Excipientes/química , Inativação Gênica , Técnicas de Transferência de Genes , Manitol/química , Camundongos , Nanomedicina , Tamanho da Partícula , Pós , Células RAW 264.7 , RNA Interferente Pequeno/administração & dosagem , Solubilidade , Solventes/química
3.
Int J Nanomedicine ; 14: 5287-5301, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31406460

RESUMO

Purpose: Nanoparticle (NP)-mediated targeted delivery of therapeutic genes or siRNAs to tumors has potential advantages. In this study, hyaluronic acid (HA)-modified chitosan nanoparticles (CS NPs-HA) loaded with cyanine 3 (Cy3)-labeled siRNA (sCS NPs-HA) were prepared and characterized. Methods: Human non-small cell lung cancer (NSCLC) A549 cells expressing receptor CD44 and tumor-bearing mice were used to evaluate the cytotoxic and antitumor effects of sCS NPs-HA in vitro and in vivo. Results: The results showed that noncytotoxic CS NPs-HA of small size (100-200 nm) effectively delivered the Cy3-labeled siRNA to A549 cells via receptor CD44 and inhibited cell proliferation by downregulating the target gene BCL2. In vivo experiment results revealed that sCS NPs-HA directly delivered greater amounts of Cy3-labeled siRNA to the tumor sites, resulting in the inhibition of tumor growth by downregulating BCL2, as compared to unmodified NPs loaded with siRNA (sCS NPs) and to naked Cy3-labeled siRNA. Conclusion: The HA-modified NPs based on chitosan could serve as a promising carrier for siRNA delivery and targeted therapy for NSCLC expressing CD44.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/terapia , Quitosana/química , Ácido Hialurônico/química , Neoplasias Pulmonares/terapia , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Células A549 , Animais , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Fluorescência , Inativação Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem
4.
Life Sci ; 232: 116635, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31283925

RESUMO

AIMS: The pathological cardiac hypertrophy will develop into heart failure, which has no effective treatment currently. Previous studies have proved that microRNAs (miRNAs) participate in the development of cardiac hypertrophy and regulate the pathological progress. In this study, we want to investigate the role of microRNA-92b-3p (miR-92b-3p) in cardiomyocyte hypertrophy and the mechanisms involved. MATERIALS AND METHODS: Neonatal mouse ventricular cells (NMVCs) were isolated from the hearts of 1-3-d-old newborn C57BL6 mice. The isolated NMVCs were induced hypertrophic phenotype by Angiotensin-II (Ang-II) and the cell size was examined by FITC-phalloidin staining assay. The expression of miR-92b-3p was determined by quantitative real-time PCR (qRT-qPCR). MRNA and protein level of ß-MHC, ACTA1 and HAND2 in NMVCs transfected with miR-92b-3p mimic and inhibition were assessed by RT-qPCR assay and western blot assay, respectively. Dual luciferase assay was used to verify the interaction between miR-92b-3p and the 3'-untranslated region (UTR) of HAND2 gene. KEY FINDINGS: MiR-92b-3p and HAND2 were significantly increased in Ang-II-induced NMVCs. Overexpression of miR-92b-3p can ameliorate Ang-II-induced cardiomyocyte hypertrophy. MiR-92b-3p negatively regulated HAND2 expression at the transcriptional level. Both miR-92b-3p mimic and HAND2 siRNA could efficiently inhibit Ang-II-induced hypertrophy in mouse cardiomyocytes. SIGNIFICANCE: MiR-92b-3p inhibits Ang-II-induced cardiomyocyte hypertrophy via targeting HAND2.


Assuntos
Angiotensina II/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiomiopatia Hipertrófica/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/patologia , Regiões 3' não Traduzidas , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Modelos Animais de Doenças , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Ventrículos do Coração/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima
5.
Chem Commun (Camb) ; 55(56): 8170-8173, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31241120

RESUMO

Lipid-complexed small interfering RNA (siRNA) nanoparticles are promising gene regulation materials with excellent genetic, but little cellular, selectivity. Herein, we report a chemical strategy to enhance the gene silencing selectivity of these nanoparticles against cancer cells through the covalent integration of a reactive oxygen species (ROS)-degradable thioketal into the lipid nanoparticles. These lipid nanoparticles can efficiently deliver siRNA into cells, and selectively silence cancer cell gene expression in response to the high levels of intracellular ROS in cancer cells.


Assuntos
Inativação Gênica , Lipídeos/química , Nanopartículas/química , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética
6.
Int J Nanomedicine ; 14: 3557-3569, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31190812

RESUMO

Purpose: Combining siRNA and other chemotherapeutic agents into one nanocarrier can overcome the multidrug resistance (MDR) phenomenon by synergistically MDR relative genes silencing and elevated chemotherapeutic activity. Most of these systems are typically fabricated through complicated procedures, which involves materials preparation, drug loading and modifications. Herein, the purpose of this study is to develop a new and fast co-delivery system of siRNA and doxorubicin for potentially synergistic cancer treatment. Methods: The co-delivery system is constructed conveniently by a stable complex consisting of doxorubicin bound to siRNA via intercalation firstly, followed by interacting with (3-Aminopropyl)triethoxysilane (APTES) electrostatically and Tetraethyl orthosilicate (TEOS) co-condensed, and the characterizations of the resultant nanocarrier are also investigated. Furthermore, this study evaluates the synergistic anti-cancer efficacy in MCF-7/MDR cells after treatment of siRNA and doxorubicin 'two in one' nanocarriers. Results: We establish a new and fast method to craft a co-delivery system of siRNA and doxorubicin with controllable and nearly uniform size, and the entire fabrication process only costs in about 10 minutes. The resultant co-delivery system presents high loading capacities of siRNA and doxorubicin, and the encapsulated doxorubicin plays a pH-responsive control release. Further, biological functionality tests of the synthesized co-delivery nanocarriers show high inhibition of P-gp protein encoded by MDR-1 gene in MCF-7/MDR cells (a variant of human breast cancer cell line with drug resistance) after transfection of these nanocarriers carrying MDR-1 siRNA and doxorubicin simultaneously, which sensitize the MCF-7/MDR cells to doxorubicin, overall leading to improved cell suppression. Conclusion: Collectively, this co-delivery system not only serves as potent therapeutics for synergistic cancer therapy, it also may facilitate the bench-to-bedside translation of combinatorial delivery system as a robust drug nanocarrier by allowing for fabricating a simply and fast nanocarrier for co-delivery of siRNA and doxorubicin with predictable high production rate.


Assuntos
Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , RNA Interferente Pequeno/administração & dosagem , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Inativação Gênica , Humanos , Células MCF-7 , Nanopartículas/administração & dosagem , Nanopartículas/ultraestrutura , RNA Interferente Pequeno/genética , Dióxido de Silício/química
7.
Int J Oncol ; 55(1): 93-102, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31180520

RESUMO

Neuroblastoma (NB) is one of the most common extracranial solid tumors in children, which has complex molecular mechanisms. Increasing evidence has suggested that long noncoding RNAs (lncRNAs) account for NB pathogenesis. However, the function of small nucleolar RNA host gene 16 (SNHG16) in NB is currently unclear. In the present study, publically available data and clinical specimens were employed to verify the expression of SNHG16 in NB. Colony formation, real­time cell proliferation and migration assays were performed to demonstrate the status of cellular proliferation and migration. Flow cytometry was used to examine cell cycle progression in SH­SY5Y cells, and acridine orange/ethidium bromide staining and caspase­3/7 activity measurements were applied to study cell apoptosis. To explore the underlying mechanism of SNHG16 function, an online database was used to identify potential RNA­binding proteins that bind SNHG16. The expression of SNHG16 was revealed to be in line with the clinical staging of NB, and high SNHG16 expression was positively associated with poor clinical outcome. Furthermore, SNHG16 silencing inhibited cell proliferation, repressed migration, and induced cell cycle arrest at the G0/G1 phase in SH­SY5Y cells. Additionally, apoptosis was undetectable in SH­SY5Y cells following SNHG16 silencing. Bioinformatics analysis revealed that SNHG16 regulated cell proliferation in NB through transcriptional and translational pathways. These results suggested that SNHG16 may serve important roles in the development and progression of NB, and could represent a potential target for NB therapy.


Assuntos
Neuroblastoma/genética , RNA Longo não Codificante/genética , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Criança , Pré-Escolar , Inativação Gênica , Humanos , Lactente , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Oncogenes , RNA Longo não Codificante/biossíntese , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção
8.
Int J Nanomedicine ; 14: 4229-4245, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31239677

RESUMO

Purpose: Gene therapies via Noggin small interfering (si)RNA (siNoggin) and bone morphogenetic protein (BMP)-2 plasmid DNA (pBMP-2) may be promising strategies for bone repair/regeneration, but their ideal delivery vectors, efficacy difference, and underlying mechanisms have not been explored, so these issues were probed here. Methods: This study used lipopolysaccharide-amine nanopolymersomes (LNPs), an efficient cytosolic delivery vector developed by the research team, to mediate siNoggin and pBMP-2 to transfect MC3T3-E1 cells, respectively. The cytotoxicity, cell uptake, and gene knockdown efficiency of siNoggin-loaded LNPs (LNPs/siNoggin) were studied, then the osteogenic-differentiation efficacy of MC3T3-E1 cells treated by LNPs/pBMP-2 and LNPs/siNoggin, respectively, were compared by measuring the expression of osteogenesis-related genes and proteins, alkaline phosphatase (ALP) activity, and mineralization of the extracellular matrix at all osteogenic stages. Finally, the possible signaling pathways of the two treatments were explored. Results: LNPs delivered siNoggin into cells efficiently to silence 50% of Noggin expression without obvious cytotoxicity. LNPs/siNoggin and LNPs/pBMP-2 enhanced the osteogenic differentiation of MC3T3 E1 cells, but LNPs/siNoggin was better than LNPs/pBMP-2. BMP/Mothers against decapentaplegic homolog (Smad) and glycogen synthase kinase (GSK)-3ß/ß-catenin signaling pathways appeared to be involved in osteogenic differentiation induced by LNPs/siNoggin, but GSK-3ß/ß-catenin was not stimulated upon LNPs/pBMP-2 treatment. Conclusion: LNPs are safe and efficient delivery vectors for DNA and RNA, which may find wide applications in gene therapy. siNoggin treatment may be a more efficient strategy to enhance osteogenic differentiation than pBMP-2 treatment. LNPs loaded with siNoggin and/or pBMP-2 may provide new opportunities for the repair and regeneration of bone.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular , Lipopolissacarídeos/farmacologia , Nanopartículas/química , Osteogênese , Polímeros/química , RNA Interferente Pequeno/administração & dosagem , Fosfatase Alcalina/metabolismo , Aminas/química , Animais , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Matriz Extracelular/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Minerais/química , Nanopartículas/toxicidade , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Plasmídeos/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Transfecção , beta Catenina/metabolismo
9.
Med Oncol ; 36(8): 66, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31183633

RESUMO

Nuclear receptor subfamily 4, group A, member 3 (NR4A3) is a member of the NR4A subgroup of orphan nuclear receptors, implicated in the regulation of diverse biological functions, including metabolism, angiogenesis, inflammation, cell proliferation, and apoptosis. Although many reports have suggested the involvement of NR4A3 in the development and/or progression of tumors, its role varies among tumor types. Previously, we reported that DNA hypomethylation at NR4A3 exon 3 is associated with lower survival rate of neuroblastoma (NB) patients. As hypomethylation of this region results in reduced expression of NR4A3, our observations suggested that NR4A3 functions as a tumor suppressor in NB. However, the exact mechanisms underlying its functions have not been clarified. In the present study, we analyzed public databases and showed that reduced NR4A3 expression was associated with shorter survival period of NB in two out of three datasets. An in vitro study revealed that forced expression of NR4A3 in human NB-derived cell line NB1 resulted in elongation of neurites along with overexpression of GAP43, one of the differentiation markers of NB. On the other hand, siRNA-mediated knockdown of NR4A3 suppressed the expression level of GAP43. Interestingly, the forced expression of NR4A3 induced only the GAP43 but not the other molecules involved in NB cell differentiation, such as MYCN, TRKA, and PHOX2B. These results indicated that NR4A3 directly activates the expression of GAP43 and induces differentiated phenotypes of NB cells, without affecting the upstream signals regulating GAP43 expression and NB differentiation.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Neuroblastoma/metabolismo , Receptores de Esteroides/biossíntese , Receptores dos Hormônios Tireóideos/biossíntese , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Progressão da Doença , Proteína GAP-43/biossíntese , Técnicas de Silenciamento de Genes , Humanos , Neuritos/metabolismo , Neuritos/patologia , Neuroblastoma/genética , Neuroblastoma/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Regulação para Cima
12.
Nat Commun ; 10(1): 2705, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221969

RESUMO

Folded single chain polymeric nano-objects are the molecular level soft material with ultra-small size. Here, we report an easy and scalable method for preparing single-chain nanogels (SCNGs) with improved efficiency. We further investigate the impact of the dynamic molecular conformational change of SCNGs on cellular interactions from molecular to bulk scale. First, the supramolecular unfoldable SCNGs efficiently deliver siRNAs into stem cells as a molecular drug carrier in a conformation-dependent manner. Furthermore, the conformation changes of SCNGs enable dynamic and precise manipulation of ligand tether structure on 2D biomaterial interfaces to regulate the ligand-receptor ligation and mechanosensing of cells. Lastly, the dynamic SCNGs as the building blocks provide effective energy dissipation to bulk biomaterials such as hydrogels, thereby protecting the encapsulated stem cells from deleterious mechanical shocks in 3D matrix. Such a bottom-up molecular tailoring strategy will inspire further applications of single-chain nano-objects in the biomedical area.


Assuntos
Engenharia Celular/métodos , Portadores de Fármacos/química , Hidrogéis/química , Nanopartículas/química , Polímeros/química , Materiais Biocompatíveis/química , Diferenciação Celular/genética , Linhagem Celular , Humanos , Células-Tronco Mesenquimais/fisiologia , Conformação Molecular , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/metabolismo
13.
Nat Commun ; 10(1): 2702, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221991

RESUMO

Most cationic vectors are difficult to avoid the fate of small interfering RNA (siRNA) degradation following the endosome-lysosome pathway during siRNA transfection. In this study, the endoplasmic reticulum (ER) membrane isolated from cancer cells was used to fabricate an integrative hybrid nanoplexes (EhCv/siRNA NPs) for improving siRNA transfection. Compared to the undecorated Cv/siEGFR NPs, the ER membrane-decorated EhCv/siRNA NPs exhibits a significantly higher gene silencing effect of siRNA in vitro and a better antitumor activity in nude mice bearing MCF-7 human breast tumor in vivo. Further mechanistic studies demonstrate that functional proteins on the ER membrane plays important roles on improving cellular uptake and altering intracellular trafficking pathway of siRNA. It is worth to believe that the ER membrane decoration on nanoplexes can effectively transport siRNA through the endosome-Golgi-ER pathway to evade lysosomal degradation and enhance the silencing effects of siRNA.


Assuntos
Portadores de Fármacos/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Transfecção/métodos , Animais , Linhagem Celular Tumoral , Membrana Celular , Portadores de Fármacos/efeitos adversos , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Receptores ErbB/genética , Feminino , Terapia Genética/métodos , Complexo de Golgi/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Nanopartículas/química , Neoplasias/genética , Neoplasias/terapia , RNA Interferente Pequeno/efeitos adversos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Carbohydr Polym ; 216: 332-342, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31047074

RESUMO

Chitosan has received a lot of attention as a carrier for small interfering RNA (siRNA), due to its capacity for complexation and intracellular release of these molecules. However, one of its limitations is its insolubility at neutral pH and the tendency towards aggregation of its nanoparticles in isotonic ionic strength. In this study, a series of amphipathic chitosans were synthesized by varying the degree of acetylation (DA) from ˜2 to ˜30 mol% and the degree of substitution (DS) from 5 to 25%. by tertiary amino groups (DEAE) The results showed that the adjustment of these parameters decreases the interparticle interactions mediated by hydrogen bonding to obtain nanoparticles with improved colloidal stability. siRNA-containing nanoparticles of 100 to 150 nm with low polydispersities (0.15-0.2) and slightly positive zeta potentials (˜+ 5 mV) were resistant to aggregation at pH 7.4 and ionic strength of 150 mM. This resistance to aggregation is provided by changes on the nanoparticle surface and highlights the importance of more organized self-assembly in providing colloidal stability at physiological conditions. Additionally, the PEGylation of the most promising vectors conferred favorable physicochemical properties to nanoparticles. The chitosans and their nanoparticles exhibited low toxicity and an efficient cell uptake, as probed by confocal microscopy of rhodamine labeled vectors. The results provide a new approach to overcome the limited stability of chitosan nanoparticles at physiological conditions and show the potential of these amphipathic chitosans as siRNA carriers.


Assuntos
Quitosana/análogos & derivados , Portadores de Fármacos/química , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Tensoativos/química , Anidridos Acéticos/química , Acetilação , Animais , Quitosana/síntese química , Quitosana/metabolismo , Quitosana/toxicidade , Dietilaminas/química , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/toxicidade , Fluorescência , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Camundongos , Nanopartículas/metabolismo , Nanopartículas/toxicidade , Tamanho da Partícula , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoglicóis/toxicidade , Células RAW 264.7 , RNA Interferente Pequeno/química , Rodaminas/química , Tensoativos/síntese química , Tensoativos/metabolismo , Tensoativos/toxicidade
15.
Parasitol Res ; 118(7): 2247-2255, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31081529

RESUMO

In a previous study, immunoproteomics was used to identify a serine protease inhibitor (TsSPI) of T. spiralis excretory/secretory (ES) proteins that exhibited an inhibitory effect on trypsin enzymatic activity, but the precise role of TsSPI on worm infection and development in its host is not well understood. The objective of the present study was to use RNA interference to ascertain the function of TsSPI in larval invasion and growth. TsSPI-specific small interference RNAs (siRNAs) were delivered to muscle larvae (ML) to silence TsSPI expression by electroporation. Four days after electroporation, the ML transfected with 2 µM siRNA-653 exhibited a 75.75% decrease in TsSPI transcription and a 69.23% decrease in TsSPI expression compared with control ML. Although the silencing of TsSPI expression did not decrease worm viability, it significantly suppressed the larval invasion of intestinal epithelium cells (IEC) (P < 0.01), and the suppression was siRNA dose-dependent (r = 0.981). The infection of mice with siRNA-653-treated ML produced a 63.71% reduction of adult worms and a 72.38% reduction of muscle larvae. In addition, the length of the adults, newborn larvae, and ML and the fecundity of female T. spiralis from mice infected with siRNA-treated ML were obviously reduced relative to those in the control siRNA or PBS groups. These results indicated that the silencing of TsSPI by RNAi suppressed larval invasion and development and decreased female fecundity, further confirming that TsSPI plays a crucial role during the T. spiralis lifecycle and is a promising molecular target for anti-Trichinella vaccines.


Assuntos
Doenças Transmitidas por Alimentos/prevenção & controle , RNA Interferente Pequeno/administração & dosagem , Inibidores de Serino Proteinase/genética , Trichinella spiralis/genética , Triquinelose/prevenção & controle , Animais , Feminino , Fertilidade , Doenças Transmitidas por Alimentos/imunologia , Doenças Transmitidas por Alimentos/parasitologia , Humanos , Mucosa Intestinal/imunologia , Larva , Camundongos , Camundongos Endogâmicos BALB C , Músculos/parasitologia , Proteômica , Interferência de RNA , Inibidores de Serino Proteinase/metabolismo , Trichinella spiralis/crescimento & desenvolvimento , Trichinella spiralis/imunologia , Trichinella spiralis/patogenicidade , Triquinelose/imunologia , Triquinelose/parasitologia
16.
Nanomedicine (Lond) ; 14(9): 1153-1171, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31050581

RESUMO

Aim: To develop a peptide/phospholipid hybrid system for gastrin-releasing peptide receptor (GRPR)-targeted delivery of pDNA or siRNA. Materials & methods: A multifunctional GRPR-targeted peptide R9-K(GALA)-BBN(6-14) was combined with a phospholipid oligonucleotide delivery system (1:1 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and 1,2-dioleoyl-3-trimethylammonium-propane) and evaluated for pDNA and siRNA delivery in terms of complex size, toxicity, receptor-targeted delivery and gene expression or knockdown efficiency. Results: By combining peptide and phospholipid delivery systems, synergistic improvements in gene expression and knockdown were observed when compared with either system alone. The optimized formulation demonstrated high levels of EGFP expression and EGFP knockdown, GRPR-targeted delivery, enhanced endosomal release and minimal toxicity. Conclusion: The peptide/phospholipid hybrid system provides efficient GRPR-targeted DNA/siRNA delivery.


Assuntos
DNA/administração & dosagem , Ácidos Graxos Monoinsaturados/química , Peptídeos/química , Fosfatidiletanolaminas/química , Compostos de Amônio Quaternário/química , RNA Interferente Pequeno/administração & dosagem , Receptores da Bombesina/metabolismo , Sobrevivência Celular , Técnicas de Silenciamento de Genes , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Humanos , Terapia de Alvo Molecular , Células PC-3 , Plasmídeos , Transfecção
17.
Prep Biochem Biotechnol ; 49(7): 659-670, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31066619

RESUMO

Small interfering RNA (siRNA)-based gene silencing strategy has high potential on suppressing specific molecular targets, involved in cancer progression. However, the lack of an effective nanocarrier system that safely delivers siRNA to its target still limits the clinical applications of siRNA. This study aimed to develop albumin-sericin nanoparticles (Alb-Ser NPs) as a novel siRNA delivery system for laryngeal cancer treatment. Nanoparticle formulations composed of albumin and sericin at different ratios (1:1, 2:1, 1:2 w/w) were synthesized by desolvation method. The nanoparticles were modified with poly-L-lysine (PLL) for siRNA binding and decorated with hyaluronic acid (HA) to target laryngeal cancer cell line, Hep-2. HA/PLL/Alb-Ser NPs were individually loaded with siRNAs for casein kinase 2 (CK2), Absent, Small, or Homeotic-Like (ASH2L), and Cyclin D1 genes, which are overexpressed in Hep-2 cells. Downregulation of genes was confirmed by real-time PCR (RT-PCR). Size, morphological, and thermogravimetric characterizations revealed that Alb-Ser NPs having 2:1 (w/w) ratio are the most optimized formulation. Between 36.8 and 61.3% of siRNA entrapment efficiencies were achieved. HA/PLL-siRNA/Alb-Ser (2:1) NPs-mediated gene silencing resulted in a significant inhibition of cell growth and induction of apoptosis in cells. Our findings showed that HA/PLL/Alb-Ser (2:1) NPs were promising as a siRNA carrier.


Assuntos
Técnicas de Transferência de Genes , Neoplasias Laríngeas/terapia , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Terapêutica com RNAi , Sericinas/química , Albumina Sérica Humana/química , Caseína Quinase II/genética , Linhagem Celular Tumoral , Ciclina D1/genética , Proteínas de Ligação a DNA/genética , Portadores de Fármacos/química , Humanos , Neoplasias Laríngeas/genética , Nanopartículas/ultraestrutura , Proteínas Nucleares/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Terapêutica com RNAi/métodos , Fatores de Transcrição/genética
18.
Int J Nanomedicine ; 14: 2637-2653, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31043779

RESUMO

Background: For the past few years, gene-therapy has recently shown considerable clinical benefit in cancer therapy, and the applications of gene therapies in cancer treatments continue to increase perennially. EZH2, an ideal candidate for tumor gene therapy, plays an important role in the tumorigenesis. Methods: In this study, we developed a novel gene delivery system with a self-assembly method by Methoxy polyethylene glycol-polycaprolactone (MPEG-PCL) and DOTAP(DMC). And EZH2si-DMC was used to research anti-glioma both in vitro and in vivo. Results: DMC with zeta-potential value of 36.7 mV and size of 35.6 nm showed good performance in the delivery siRNA to glioma cell in vitro with high 98% transfection efficiency. EZH2si-DMC showed good anti-glioma effect in vitro through inducing cell apoptosis and inhibiting cell growth. What's more, treatment of tumor-bearing mice with DMC-EZH2si complex had significantly inhibited tumor growth at the subcutaneous model in vivo by inhibiting EZH2 protein expression, promoting apoptosis and reducing proliferation. Conclusion: The EZH2 siRNA and DMC complex may be used to treat the glioma in clinical as a new drug.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Glioma/tratamento farmacológico , Terapia de Alvo Molecular , Nanopartículas/química , RNA Interferente Pequeno/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Ácidos Graxos Monoinsaturados/síntese química , Ácidos Graxos Monoinsaturados/química , Feminino , Glioma/patologia , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Micelas , Nanopartículas/ultraestrutura , Poliésteres/síntese química , Poliésteres/química , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Compostos de Amônio Quaternário/síntese química , Compostos de Amônio Quaternário/química , RNA Interferente Pequeno/administração & dosagem
19.
Int J Mol Sci ; 20(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991632

RESUMO

The development of effective nanosystems for drug delivery represents a key challenge for the improvement of most current anticancer therapies. Recent progress in the understanding of structure and function of extracellular vesicles (EVs)-specialized membrane-bound nanocarriers for intercellular communication-suggests that they might also serve as optimal delivery systems of therapeutics. In addition to carrying proteins, lipids, DNA and different forms of RNAs, EVs can be engineered to deliver specific bioactive molecules to target cells. Exploitation of their molecular composition and physical properties, together with improvement in bio-techniques to modify their content are critical issues to target them to specific cells/tissues/organs. Here, we will discuss the current developments in the field of animal and plant-derived EVs toward their potential use for delivery of therapeutic agents in different pathological conditions, with a special focus on cancer.


Assuntos
Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Vesículas Extracelulares/química , Preparações Farmacêuticas/administração & dosagem , Animais , Portadores de Fármacos/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Lipossomos/química , Lipossomos/metabolismo , Plantas/química , Plantas/metabolismo , RNA Interferente Pequeno/administração & dosagem
20.
Med Sci Monit ; 25: 3090-3099, 2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31026254

RESUMO

BACKGROUND In the pathogenesis and progression of prostate cancer, cell proliferation and cell migration results in tumor invasion and metastasis that is associated with patient morbidity and mortality. Rho-associated protein kinase (ROCK) has previously been shown to be upregulated in prostate cancer, but its biological role remains poorly understood. This study aimed to investigate the role of ROCK in the proliferation and migration of PC-3 and DU145 prostate cancer cells and to identify the possible targets involved by knockdown of ROCK1 and ROCK2 RNA expression. MATERIAL AND METHODS An RNA interference (RNAi) assay was performed to silence the expression of ROCK1 and ROCK2 in the PC-3 and DU145 human prostate cancer cell lines. Cells were also treated with a specific ROCK inhibitor, Y27632. A cell counting kit-8 (CCK-8) assay was used to determine the proliferation rate of prostate cancer cells, and cell migration and invasion assays were performed. Western blot and polymerase chain reaction were used to measure protein and RNA expression levels. RESULTS In PC-3 and DU145 prostate cancer cells, knockdown of ROCK1 and ROCK2 reduced cell migration and invasion. ROCK1 and ROCK2 regulated cell proliferation in PC-3 and DU145 prostate cancer cells. Protein levels of phosphorylated LIM kinase 1 (p-LIMK1) and matrix metalloproteinase-2 (MMP-2) were reduced in ROCK1 and ROCK2 siRNA transfected cells. CONCLUSIONS In PC-3 and DU145 human prostate cancer cells, ROCK promoted cell proliferation and migration by targeting LIMK1 and MMP-2.


Assuntos
Quinases Lim/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Neoplasias da Próstata/enzimologia , Quinases Associadas a rho/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Nus , Células PC-3 , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais , Quinases Associadas a rho/genética
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