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1.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 49(1): 20-34, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32621413

RESUMO

Mesenchymal stem cells (MSCs) have the inherent tumor-homing ability with the attraction of multiple chemokines released by tumor tissues or tumor microenvironments, which can be utilized as promising cellular carriers for targeted delivery of anti-tumor drugs and genes. In most circumstances, large amount of systemicly administrated MSCs will be firstly trapped by lungs, following with re-distribution and homing to tumor tissues after lung clearance. Several approaches like enhanced interactions between chemokines and receptors on MSCs or reducing the retention of MSCs by changes of administration methods are firstly reviewed for improving the homing of MSCs towards tumor tissues. Additionally, the potentials and gains of utilizing MSCs to carry several chemotherapeutics, such as doxorubicin, paclitaxel and gemcitabine are summarized, showing the advantages of overcoming the short half-life and poor tumor targeting of these chemotherapeutics. Moreover, the applications of MSCs to protect and deliver therapeutic genes to tumor sites for selectively tumor cells eliminating or promoting immune system are highlighted. In addition, the potentials of using MSCs for tumor-targeting delivery of diagnostic and therapeutic agents are addressed. We believed that the continuous improvement and optimization of this stem cells-based cellular delivery system will provide a novel delivery strategy and option for tumor treatment.


Assuntos
Antineoplásicos , Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , Células-Tronco Mesenquimais , Neoplasias , Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Humanos , Neoplasias/terapia , Paclitaxel/administração & dosagem , Pesquisa/tendências
2.
Int J Nanomedicine ; 15: 3639-3647, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32547019

RESUMO

Purpose: Astrocyte dysfunction is a hallmark of central nervous system injury or infection. As a primary contributor to neurodegeneration, astrocytes are an ideal therapeutic target to combat neurodegenerative conditions. Gene therapy has arisen as an innovative technique that provides excellent prospect for disease intervention. Poly (lactide-co-glycolide) (PLGA) and polyethylenimine (PEI) are polymeric nanoparticles commonly used in gene delivery, each manifesting their own set of advantages and disadvantages. As a clinically approved polymer by the Federal Drug Administration, well characterized for its biodegradability and biocompatibility, PLGA-based nanoparticles (PLGA-NPs) are appealing for translational gene delivery systems. However, our investigations revealed PLGA-NPs were ineffective at facilitating exogenous gene expression in primary human astrocytes, despite their success in other cell lines. Furthermore, PEI polymers illustrate high delivery efficiency but induce cytotoxicity. The purpose of this study is to develop viable and biocompatible NPsystem for astrocyte-targeted gene therapy. Materials and Methods: Successful gene expression by PLGA-NPs alone or in combination with arginine-modified PEI polymers (AnPn) was assessed by a luciferase reporter gene encapsulated in PLGA-NPs. Cytoplasmic release and nuclear localization of DNA were investigated using fluorescent confocal imaging with YOYO-labeled plasmid DNA (pDNA). NP-mediated cytotoxicity was assessed via lactate dehydrogenase in primary human astrocytes and neurons. Results: Confocal imaging of YOYO-labeled pDNA confirmed PLGA-NPs delivered pDNA to the cytoplasm in a dose and time-dependent manner. However, co-staining revealed pDNA delivered by PLGA-NPs did not localize to the nucleus. The addition of AnPn significantly improved nuclear localization of pDNA and successfully achieved gene expression in primary human astrocytes. Moreover, these formulations were biocompatible with both astrocytes and neurons. Conclusion: By co-transfecting two polymeric NPs, we developed an improved system for gene delivery and expression in primary human astrocytes. These findings provide a basis for a biocompatible and clinically translatable method to regulate astrocyte function during neurodegenerative diseases and disorders.


Assuntos
Arginina/química , Astrócitos/metabolismo , Técnicas de Transferência de Genes , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , DNA/genética , Células HEK293 , Humanos , Tamanho da Partícula , Plasmídeos/genética , Polietilenoimina , Transfecção
4.
Nano Lett ; 20(6): 4543-4549, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32375002

RESUMO

Lipid nanoparticle (LNP) packaged mRNA vaccines have been deployed against infectious diseases such as COVID-19, yet their structural features remain unclear. Cholesterol, a major constituent within LNPs, contributes to their morphology that influences gene delivery. Herein, we examine the structure of LNPs containing cholesterol derivatives using electron microscopy, differential scanning calorimetry, and membrane fluidity assays. LNPs formulated with C24 alkyl derivatives of cholesterol show a polymorphic shape and various degrees of multilamellarity and lipid partitioning, likely due to phase separation. The addition of methyl and ethyl groups to the C24 alkyl tail of the cholesterol backbone induces multilamellarity (>50% increase compared to cholesterol), while the addition of a double bond induces lipid partitioning (>90% increase compared to cholesterol). LNPs with multilamellar and faceted structures, as well as a lamellar lipid phase, showed higher gene transfection. Unraveling the structure of mRNA-LNPs can enable their rational design toward enhanced gene delivery.


Assuntos
Colesterol/análogos & derivados , Infecções por Coronavirus/prevenção & controle , Portadores de Fármacos/química , Nanopartículas/química , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , RNA Mensageiro/administração & dosagem , Vacinas Virais/administração & dosagem , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/genética , Técnicas de Transferência de Genes , Células HeLa , Humanos , Lipídeos/química , Nanopartículas/ultraestrutura , Transição de Fase , Fitosteróis/química , RNA Mensageiro/genética , Transfecção , Vacinas Virais/genética
5.
Int J Nanomedicine ; 15: 2809-2828, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32368056

RESUMO

Introduction: Glioblastoma (GBM) is the most common and lethal of the central nervous system (CNS) malignancies. The initiation, progression, and infiltration ability of GBMs are attributed in part to the dysregulation of microRNAs (miRNAs). Thus, targeting dysregulated miRNAs with RNA oligonucleotides (RNA interference, RNAi) has been proposed for GBM treatment. Despite promising results in the laboratory, RNA oligonucleotides have clinical limitations that include poor RNA stability and off-target effects. RNAi therapies against GBM confront an additional obstacle, as they need to cross the blood-brain barrier (BBB). Methods: Here, we developed gold-liposome nanoparticles conjugated with the brain targeting peptides apolipoprotein E (ApoE) and rabies virus glycoprotein (RVG). First, we functionalized gold nanoparticles with oligonucleotide miRNA inhibitors (OMIs), creating spherical nucleic acids (SNAs). Next, we encapsulated SNAs into ApoE, or RVG-conjugated liposomes, to obtain SNA-Liposome-ApoE and SNA-Liposome-RVG, respectively. We characterized each nanoparticle in terms of their size, charge, encapsulation efficiency, and delivery efficiency into U87 GBM cells in vitro. Then, they were administered intravenously (iv) in GBM syngeneic mice to evaluate their delivery efficiency to brain tumor tissue. Results: SNA-Liposomes of about 30-50 nm in diameter internalized U87 GBM cells and inhibited the expression of miRNA-92b, an aberrantly overexpressed miRNA in GBM cell lines and GBM tumors. Conjugating SNA-Liposomes with ApoE or RVG peptides increased their systemic delivery to the brain tumors of GBM syngeneic mice. SNA-Liposome-ApoE demonstrated to accumulate at higher extension in brain tumor tissues, when compared with non-treated controls, SNA-Liposomes, or SNA-Liposome-RVG. Discussion: SNA-Liposome-ApoE has the potential to advance the translation of miRNA-based therapies for GBM as well as other CNS disorders.


Assuntos
Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Lipossomos/administração & dosagem , Interferência de RNA , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Técnicas de Transferência de Genes , Glioblastoma/genética , Glioblastoma/patologia , Ouro/química , Humanos , Lipossomos/química , Masculino , Nanopartículas Metálicas/química , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Ácidos Nucleicos/química , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/farmacocinética , Proteínas do Envelope Viral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Gene ; 747: 144677, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32304785

RESUMO

The progressive, late-onset, nonsyndromic, sensorineural hearing loss (PNSHL) is the most common cause of sensory impairment globally, with presbycusis affecting greater than a third of individuals over the age of 65. The etiology underlying PNSHL include presbycusis, noise-induced hearing loss, drug ototoxicity, and delayed-onset autosomal dominant hearing loss (AD PNSHL). The objective of this article is to discuss the potential diagnostic and therapeutic applications of genomic medicine in PNSHL. Genomic factors contribute greatly to PNSHL. The heritability of presbycusis ranges from 25 to 75%. Current therapies for PNSHL range from sound amplification to cochlear implantation (CI). PNSHL is an excellent candidate for genomic medicine approaches as it is common, has well-described pathophysiology, has a wide time window for treatment, and is amenable to local gene therapy by currently utilized procedural approaches. AD PNSHL is especially suited to genomic medicine approaches that can disrupt the expression of an aberrant protein product. Gene therapy is emerging as a potential therapeutic strategy for the treatment of PNSHL. Viral gene delivery approaches have demonstrated promising results in human clinical trials for two inherited causes of blindness and are being used for PNSHL in animal models and a human trial. Non-viral gene therapy approaches are useful in situations where a transient biologic effect is needed or for delivery of genome editing reagents (such as CRISPR/Cas9) into the inner ear. Many gene therapy modalities that have proven efficacious in animal trials have potential to delay or prevent PNSHL in humans. The development of new treatment modalities for PNSHL will lead to improved quality of life of many affected individuals and their families.


Assuntos
Terapia Genética , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/terapia , Análise Custo-Benefício , Epigênese Genética , Técnicas de Transferência de Genes , Terapia Genética/economia , Perda Auditiva Neurossensorial/genética , Humanos
7.
Int J Nanomedicine ; 15: 2131-2150, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32280211

RESUMO

Gene-based therapies have emerged as a new modality for combating a myriad of currently incurable diseases. However, the fragile nature of gene therapeutics has significantly hampered their biomedical applications. Correspondingly, the development of gene-delivery vectors is of critical importance for gene-based therapies. To date, a variety of gene-delivery vectors have been created and utilized for gene delivery. In general, they can be categorized into viral- and non-viral vectors. Due to safety issues associated with viral vectors, non-viral vectors have recently attracted much more research focus. Of these non-viral vectors, polymeric vectors, which have been preferred due to their low immunogenicity, ease of production, controlled chemical composition and high chemical versatility, have constituted an ideal alternative to viral vectors. In particular, biodegradable polymers, which possess advantageous biocompatibility and biosafety, have been considered to have great potential in clinical applications. In this context, the aim of this review is to introduce the recent development and progress of biodegradable polymers for gene delivery applications, especially for their chemical structure design, gene delivery capacity and additional biological functions. Accordingly, we first define and categorize biodegradable polymers, followed by describing their corresponding degradation mechanisms. Various types of biodegradable polymers resulting from natural and synthetic polymers will be introduced and their applications in gene delivery will be examined. Finally, a future perspective regarding the development of biodegradable polymer vectors will be given.


Assuntos
Materiais Biocompatíveis/química , Técnicas de Transferência de Genes , Polímeros/química , Cátions , Terapia Genética/métodos , Humanos , Polímeros/síntese química
8.
Science ; 368(6487): 181-186, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32273467

RESUMO

Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.


Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário , Microscopia Intravital/métodos , Crista Neural , Animais , Encéfalo/embriologia , Encéfalo/fisiologia , Divisão Celular , Movimento Celular , Quimera/embriologia , Quimera/fisiologia , Eletroporação , Feminino , Técnicas de Transferência de Genes , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica , Crista Neural/irrigação sanguínea , Crista Neural/citologia , Crista Neural/embriologia , Placenta/fisiologia , Gravidez , Retina/embriologia , Retina/fisiologia , Transmissão Sináptica , Útero
9.
Proc Natl Acad Sci U S A ; 117(16): 8845-8849, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32253306

RESUMO

The genetic incorporation of noncanonical amino acids (ncAAs) into proteins has been realized in bacteria, yeast, and mammalian cells, and recently, in multicellular organisms including plants and animals. However, the addition of new building blocks to the genetic code of tissues from human origin has not yet been achieved. To this end, we report a self-replicating Epstein-Barr virus-based episomal vector for the long-term encoding of ncAAs in human hematopoietic stem cells and reconstitution of this genetically engineered hematopoietic system in mice.


Assuntos
Aminoácidos/genética , Diferenciação Celular/genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/fisiologia , Engenharia de Proteínas/métodos , Animais , Sangue Fetal/citologia , Técnicas de Transferência de Genes , Código Genético , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Herpesvirus Humano 4/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Plasmídeos/genética , Cultura Primária de Células/métodos , Transfecção/métodos , Quimeras de Transplante , Transplante Heterólogo/métodos
10.
Int J Nanomedicine ; 15: 483-495, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32158206

RESUMO

Background: The complex preparation procedures and severe toxicities are two major obstacles facing the wide use of chimeric antigen receptor-modified T (CAR-T) cells in clinical cancer immunotherapy. The nanotechnology-based T cell temporary CAR modification may be a potential approach to solve these problems and make the CAR-T cell-based tumor therapy feasible and broadly applicable. Methods: A series of plasmid DNA-loaded self-assembled nanoparticles (pDNA@SNPsx/y) prepared from adamantane-grafted polyamidoamine (Ad-PAMAM) dendrimers of different generations (G1 or G5) and cyclodextrin-grafted branched polyethylenimine (CD-PEI) of different molecular weights (800, 2000, or 25,000 Da) were characterized and evaluated. The detailed physicochemical properties, cellular interaction, and cytotoxicity of selected pDNA@SNPG1/800 were systematically investigated. Thereafter, the epidermal growth factor receptor variant III (EGFRvIII) CAR-expression plasmid vector (pEGFRvIII-CAR) was constructed and encapsulated into SNPG1/800. The resulting pEGFRvIII-CAR@SNPG1/800 was used for Jurkat cell transient transfection, and the EGFRvIII-CAR expressed in transfected cells was measured by flow cytometry and Western blot. Finally, the response of EGFRvIII CAR-positive Jurkat T cell to target tumor cell was evaluated. Results: The pDNA@SNPG1/800 showed the highest efficacy in Jurkat cell gene transfection and exhibited low cytotoxicity. pEGFRvIII-CAR@SNPG1/800 can efficiently deliver pEGFRvIII-CAR into Jurkat T cells, thereby resulting in transient EGFRvIII-CAR expression in transfected cells. EGFRvIII-CAR that is present on the cell membrane enabled Jurkat T cells to recognize and bind specifically with EGFRvIII-positive tumor cells. Conclusion: These results indicated that pEGFRvIII-CAR@SNPG1/800 can effectively achieve T-cell transient CAR modification, thereby demonstrating considerable potential in CAR-T cancer therapy.


Assuntos
Receptores ErbB/genética , Técnicas de Transferência de Genes , Imunoterapia Adotiva/métodos , Nanopartículas/química , Linfócitos T/fisiologia , Linhagem Celular Tumoral , Dendrímeros/química , Vetores Genéticos , Humanos , Iminas/química , Imunoterapia , Células Jurkat , Peso Molecular , Polietilenos/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/genética , Transfecção/métodos
11.
Int J Nanomedicine ; 15: 497-511, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32158207

RESUMO

Introduction: RNA-based therapy for bone repair and regeneration is a highly safe and effective approach, which has been extensively investigated in recent years. However, the molecular stability of RNA agents still remains insufficient for clinical application. High porosity, tunable size, and ideal biodegradability and biosafety are a few of the characters of mesoporous silicon nanoparticles (MSNs) that render them a promising biomaterial carrier for RNA treatment. Materials and Methods: In this study, a novel miR-26a delivery system was constructed based on MSNs. Next, we assessed the miRNA protection of the delivery vehicles. Then, rat bone marrow mesenchymal stem cells (rBMSCs) were incubated with the vectors, and the transfection efficiency, cellular uptake, and effects on cell viability and osteogenic differentiation were evaluated. Results: The results demonstrated that the vectors protected miR-26a from degradation in vitro and delivered it into the cytoplasm. A relatively low concentration of the delivery systems significantly increased osteogenic differentiation of rBMSCs. Conclusion: The vectors constructed in our study provide new methods and strategies for the delivery of microRNAs in bone tissue engineering.


Assuntos
Diferenciação Celular , Técnicas de Transferência de Genes , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Nanopartículas/química , Osteogênese/genética , Animais , Materiais Biocompatíveis/química , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Iminas/química , Células-Tronco Mesenquimais/fisiologia , Peptídeos/química , Polietilenos/química , Porosidade , Ratos Sprague-Dawley , Dióxido de Silício/química , Transfecção
12.
Radiology ; 295(2): 254-272, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32208094

RESUMO

This review summarizes the use of high-voltage electrical pulses (HVEPs) in clinical oncology to treat solid tumors with irreversible electroporation (IRE) and electrochemotherapy (ECT). HVEPs increase the membrane permeability of cells, a phenomenon known as electroporation. Unlike alternative ablative therapies, electroporation does not affect the structural integrity of surrounding tissue, thereby enabling tumors in the vicinity of vital structures to be treated. IRE uses HVEPs to cause cell death by inducing membrane disruption, and it is primarily used as a radical ablative therapy in the treatment of soft-tissue tumors in the liver, kidney, prostate, and pancreas. ECT uses HVEPs to transiently increase membrane permeability, enhancing cellular cytotoxic drug uptake in tumors. IRE and ECT show immunogenic effects that could be augmented when combined with immunomodulatory drugs, a combination therapy the authors term electroimmunotherapy. Additional electroporation-based technologies that may reach clinical importance, such as gene electrotransfer, electrofusion, and electroimmunotherapy, are concisely reviewed. HVEPs represent a substantial advancement in cancer research, and continued improvement and implementation of these presented technologies will require close collaboration between engineers, interventional radiologists, medical oncologists, and immuno-oncologists.


Assuntos
Eletroporação/métodos , Oncologia/métodos , Neoplasias/terapia , Antineoplásicos/administração & dosagem , Fusão Celular/métodos , Terapia por Estimulação Elétrica/métodos , Eletroquimioterapia/métodos , Técnicas de Transferência de Genes , Humanos , Imunoterapia/métodos
13.
Life Sci ; 250: 117550, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32179071

RESUMO

Breast cancer is the frequently diagnosed cancer among women and it is the most lethal malignancy in women globally. With one million cases every year, breast cancer is the fast-growing cancer type that has a high prevalence rate in young women. The limitations and undesirable side effects of conventional therapies like chemotherapy and radiotherapy on malignant tumors necessitate the development of alternative therapeutic approaches. Gene therapy has emerged as a promising approach to cure a variety of malignant cancer types which involves the delivery of functional gene directly into the target tumor tissue. Efficient gene therapy approach relies on the effective delivery of therapeutic genes to the desired cell type. In this regard, biological and non-biological gene delivery vectors are used to protect the naked foreign DNA to mediate effective tissue entry of the desired gene of interest. In this review, the use of bacterial and viral vectors for breast cancer gene therapy was summarized.


Assuntos
Bactérias , Neoplasias da Mama/prevenção & controle , Neoplasias da Mama/terapia , Vacinas Anticâncer/administração & dosagem , Vetores Genéticos , Vírus , Animais , Feminino , Técnicas de Transferência de Genes , Terapia Genética/métodos , Humanos , Neoplasias Mamárias Experimentais/terapia , Transplante de Neoplasias , Vírus Oncolíticos , Prognóstico
14.
Int J Nanomedicine ; 15: 1397-1408, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32184594

RESUMO

Background: siRNA-mediated polo-like kinase 1 (PLK1) silencing has been proposed as a promising therapeutic method for multiple cancers. However, the clinic application of this method is still hindered by the low specific delivery of siPLK1 to desired tumor lesions. Herein, folate (FA)-modified and leucine-bearing polyethylenimine was successfully synthesized and showed excellent targeted silencing to folate receptor overexpressed cells. Materials and Methods: The condensation of siPLK1 by FA-N-Ac-L-Leu-PEI (NPF) was detected by the gel retardation assay. The targeted and silencing efficiency was evaluated by flow cytometry and confocal laser scanning microscope. The PLK1 expressions at gene or protein levels were detected by quantitative real-time PCR and Western blotting assay. Further impacts of the PLK1 silencing on cell viability, cell cycle, migration, and invasion were studied by MTT, colony formation, wound healing and transwell assays. Results: The NPF and siPLK1 could efficiently assemble to stable nanoparticles at a weight ratio of 3.0 and showed excellent condensation and protection effect. Owing to the FA-mediated targeted delivery, the uptake and silencing efficiency of NPF/siPLK1 to SGC-7901 cells was higher than that without FA modification. Moreover, NPF-mediated PLK1 silencing showed significant antitumor activity in vitro. The anti-proliferation effect of PLK1 silencing was induced via the mitochondrial-dependent apoptosis pathway with the cell cycle arrest of 45% at G2 phase and the apoptotic ratio of 28.3%. Conclusion: FA-N-Ac-L-Leu-PEI (NPF) could generate targeted delivery siPLK1 to FA receptor overexpressed cells and dramatically downregulate the expression of PLK1 expression.


Assuntos
Proteínas de Ciclo Celular/genética , Ácido Fólico/química , Técnicas de Transferência de Genes , Polietilenoimina/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Neoplasias Gástricas/terapia , Células A549 , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Ácido Fólico/farmacologia , Inativação Gênica , Terapia Genética/métodos , Humanos , Leucina/química , Nanopartículas/química , RNA Interferente Pequeno/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
15.
Chem Pharm Bull (Tokyo) ; 68(2): 133-139, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32009080

RESUMO

C1q/tumor necrosis factor (TNF)-related protein 12 (CTRP12) is a secretory protein that participates in the regulation of glucose and lipid metabolism in obesity and diabetes. Its role in cardiovascular disease, particularly sepsis-induced cardiac injury, is unclear. Here, we stimulated cardiomyocytes with lipopolysaccharide (LPS) to establish an in vitro cardiomyocyte injury model and CTRP12 was overexpressed with an adenovirus delivery system. Overexpression of CTRP12 reduced the transcription and release of pro-inflammatory cytokines from LPS-stimulated cardiomyocytes, including TNFα, interleukin-1 (IL-1), and IL-6. Reactive oxygen species (ROS) level increased and the oxidation/redox system was disturbed in LPS-stimulated cardiomyocytes, as evident from the decrease in superoxide dismutase activity and an increase in reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and malondialdehyde level. CTRP12 overexpression decreased the increasing level of ROS and ameliorated the unbalance in the oxidation/redox system in LPS-stimulated cardiomyocytes. The viability of cardiomyocytes decreased after LPS stimulation, and the cells underwent apoptosis. CTRP12-overexpressing cardiomyocytes showed a decrease in the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells, and the ratio of B cell lymphoma (Bcl)-1/Bax in these cells was recovered. In comparison with the control group, LPS-stimulated cardiomyocytes showed reduced expression of nuclear factor E2-related factor 2 (NRF2), while CTRP12-overexpressing cardiomyocytes showed elevated NRF2 expression. Small-interfering RNA-mediated silencing of NRF2 expression in cardiomyocytes resulted in the inhibition of the protective effects of CTRP12. Thus, CTRP12 ameliorated injury in LPS-stimulated cardiomyocytes in an NRF2-dependent manner.


Assuntos
Inflamação/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Lipopolissacarídeos/imunologia , Miócitos Cardíacos/imunologia , Regulação para Cima , Adenoviridae/genética , Animais , Linhagem Celular , Células Cultivadas , Regulação para Baixo , Técnicas de Transferência de Genes , Inflamação/imunologia , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Interleucina-1/genética , Interleucina-1/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
16.
Mol Biotechnol ; 62(4): 240-251, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32108286

RESUMO

In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.


Assuntos
Imunoglobulina G/biossíntese , Agricultura Molecular/métodos , Mucuna/genética , Agrobacterium tumefaciens/genética , Expressão Gênica , Técnicas de Transferência de Genes/instrumentação , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Mucuna/metabolismo , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Toxoplasma/imunologia , Transformação Bacteriana
17.
Gene ; 730: 144318, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-31917231

RESUMO

Although the chicken embryo has been a classical model for developmental studies, the lack of straightforward technologies for chicken transgenesis limited the usefulness of this animal model. Here, we assessed electroporation and lipofection approaches for in ovo transfection of Sleeping Beauty transposon system in stage X-XII chicken embryos. Electroporation of chicken embryos could transfect the trophectodermal cells. Then, a mixture of transposon lipoplexes and high concentrated carboxymethylcellulose (HCC) solution was injected into the subgerminal cavity of day 0 embryos. The lipoplex-HCC mixture substantially increased the number of trophectodermal cells expressing the reporter. Importantly, the fluorescent reporter was detected in cells inside of the embryos as well as circulation cells in the bloodstream during days 3-4 of incubation. This study provided evidence for direct in ovo transfection of early chicken embryos, though the long-term outcome of this approach warrants further studies.


Assuntos
Eletroporação/métodos , Transfecção/métodos , Transposases/genética , Animais , Animais Geneticamente Modificados , Carboximetilcelulose Sódica , Embrião de Galinha , Galinhas/genética , Elementos de DNA Transponíveis/genética , Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Transferência de Genes
18.
DNA Cell Biol ; 39(3): 451-458, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31910350

RESUMO

Gene delivery from tissue engineering scaffold is a novel strategy in regulating long-term growth and function of cells in vitro culture. In this study, a hepatocyte growth factor plasmid/polyetherimide (pHGF/PEI) polyplex delivering alginate (AL)/galactosylated chitosan (GC) (pHGF/PEI-AL/GC) sponge scaffold was prepared for the in vitro coculture of hepatocytes/3T3 cells. The pHGF/PEI polyplex released for 6 days in the sponge scaffold with weight ratio of AL/GC being 3:1 and fixed amount of pHGF being 40 µg (24-well scaffold). In addition, the 3T3 cells culturing in the pHGF/PEI-AL/GC sponge scaffold could be continually transfected and expressed the exogenous HGF for 6 days. Furthermore, the albumin secretion and urea synthesis of hepatocytes were significantly enhanced when cocultured with 3T3 cells in the pHGF/PEI-AL/GC sponge scaffold compared with that in the AL/GC sponge without pHGF. In summary, the preparation of AL/GC sponge scaffold delivering pHGF/PEI polyplex is a critical significance for maintaining the long-term survival and function of primary hepatocytes in vitro.


Assuntos
Quitosana/análogos & derivados , Técnicas de Transferência de Genes , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/metabolismo , Tecidos Suporte/química , Células 3T3 , Alginatos/química , Animais , Células Cultivadas , Técnicas de Cocultura/métodos , Galactose/análogos & derivados , Fator de Crescimento de Hepatócito/genética , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Masculino , Camundongos , Polímeros/química , Ratos , Ratos Wistar , Tecidos Suporte/efeitos adversos
19.
PLoS Biol ; 18(1): e3000589, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31922526

RESUMO

Electroporation is a basic yet powerful method for delivering small molecules (RNA, DNA, drugs) across cell membranes by application of an electrical field. It is used for many diverse applications, from genetically engineering cells to drug- and DNA-based vaccine delivery. Despite this broad utility, the high cost of electroporators can keep this approach out of reach for many budget-conscious laboratories. To address this need, we develop a simple, inexpensive, and handheld electroporator inspired by and derived from a common household piezoelectric stove lighter. The proposed "ElectroPen" device can cost as little as 23 cents (US dollars) to manufacture, is portable (weighs 13 g and requires no electricity), can be easily fabricated using 3D printing, and delivers repeatable exponentially decaying pulses of about 2,000 V in 5 ms. We provide a proof-of-concept demonstration by genetically transforming plasmids into Escherichia coli cells, showing transformation efficiency comparable to commercial devices, but at a fraction of the cost. We also demonstrate the potential for rapid dissemination of this approach, with multiple research groups across the globe validating the ease of construction and functionality of our device, supporting the potential for democratization of science through frugal tools. Thus, the simplicity, accessibility, and affordability of our device holds potential for making modern synthetic biology accessible in high school, community, and resource-poor laboratories.


Assuntos
Eletroporação/instrumentação , Técnicas de Transferência de Genes/instrumentação , Análise Custo-Benefício , Eletricidade , Eletroporação/economia , Desenho de Equipamento/economia , Escherichia coli , Técnicas de Transferência de Genes/economia , Humanos , Laboratórios/economia , Manufaturas/economia , Áreas de Pobreza , Impressão Tridimensional , Transformação Bacteriana , Transportes
20.
J Nanobiotechnology ; 18(1): 15, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31952530

RESUMO

BACKGROUND: The successful deliveries of siRNA depend on their stabilities under physiological conditions because greater in vivo stability enhances cellular uptake and enables endosomal escape. Viral-based systems appears as most efficient approaches for gene delivery but often compromised in terms of biocompatibility, patient safety and high cost scale up process. Here we describe a novel platform of gene delivery by elastin-like polypeptide (ELP) based targeting biopolymers. RESULTS: For better tumor targeting and membrane penetrating characteristics, we designed various chimeric ELP-based carriers containing a cell penetrating peptide (Tat), single or multiple copies of AP1 an IL-4 receptor targeting peptide along with coding sequence of ELP and referred as Tat-A1E28 or Tat-A4V48. These targeted polypeptides were further analyzed for its ability to deliver siRNA (Luciferase gene) in tumor cells in comparison with non-targeted controls (Tat-E28 or E28). The positively charged amino acids of these polypeptides enabled them to readily complex with negatively charged nucleic acids. The complexation of nucleic acid with respective polypeptides facilitated its transfection efficiency as well as stability. The targeted polypeptides (Tat-A1E28 or Tat-A4V48) selectively delivered siRNA into tumor cells in a receptor-specific fashion, achieved endosomal and lysosomal escape, and released gene into cytosol. The target specific delivery of siRNA by Tat-A1E28 or Tat-A4V48 was further validated in murine breast carcinoma 4T1 allograft mice model. CONCLUSION: The designed delivery systems efficiently delivered siRNA to the target site of action thereby inducing significant gene silencing activity. The study shows Tat and AP1 functionalized ELPs constitute a novel gene delivery system with potential therapeutic applications.


Assuntos
Peptídeos Penetradores de Células/química , Elastina/química , Peptídeos/química , RNA Interferente Pequeno/química , Animais , Biopolímeros , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Feminino , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Luciferases/genética , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Imagem Óptica , RNA Interferente Pequeno/administração & dosagem , Receptores de Interleucina-4/metabolismo , Transfecção
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