RESUMO
Crohn's disease (CD) is a refractory chronic inflammatory bowel disease (IBD) with unknown etiology. Transmural inflammation, involving the intestine and mesentery, represents a characteristic pathological feature of CD and serves as a critical contributor to its intractability. Here, this study describes an oral pyroptosis nanoinhibitor loaded with tumor necrosis factor-α (TNF-α) deoxyribozymes (DNAzymes) (DNAzymes@degradable silicon nanoparticles@Mannose, Dz@MDSN), which can target macrophages at the site of inflammation and respond to reactive oxygen species (ROS) to release drugs. Dz@MDSN can not only break the inflammatory cycle in macrophages by degrading TNF-α mRNA but also reduce the production of ROS mainly from macrophages. Moreover, Dz@MDSN inhibits excessive pyroptosis in epithelial cells through ROS clearance, thereby repairing the intestinal barrier and reducing the translocation of intestinal bacteria to the mesentery. Consequently, these combined actions synergistically contribute to the suppression of inflammation within both the intestine and the mesentery. This study likely represents the first successful attempt in the field of utilizing nanomaterials to achieve transmural healing for CD, which also provides a promising treatment strategy for CD patients.
Assuntos
Doença de Crohn , DNA Catalítico , Piroptose , Fator de Necrose Tumoral alfa , Doença de Crohn/tratamento farmacológico , Doença de Crohn/patologia , Doença de Crohn/metabolismo , Piroptose/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Humanos , Animais , Administração Oral , Camundongos , DNA Catalítico/química , DNA Catalítico/metabolismo , DNA Catalítico/farmacologia , Nanopartículas/química , Espécies Reativas de Oxigênio/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Silício/química , Silício/farmacologia , Manose/química , Manose/farmacologia , Células RAW 264.7 , MasculinoRESUMO
Influenza A viruses (IAV) are a high threat to humanity because of a lack of proper effective antiviral drugs and resistance of viruses to existing vaccines. We describe the sufficient anti-IAV effect of Ans/PL-Dz nanocomposites that contain deoxyribozymes (Dz) immobilized on anatase TiO2 nanoparticles (Ans) through polylysine linker (PL). The Dz-containing nanocomposites appear to be more efficient than the Ans/PL-ODN nanocomposites that contain common oligodeoxyribonucleotides (ODN) targeted to the same RNA regions of the viral genome. The simultaneous use of nanocomposites that contain Dz and ODN, which are targeted to different sites of viral RNA provides a higher overall effect than the independent action of each of them (synergism). The inhibition of IAV with the proposed nanocomposites was shown to be effective, sequence-specific, and dose-dependent. The most efficient Ans/PL-Dz nanocomposite exhibited a high antiviral effect in vivo on mice models. The efficiency of IAV inhibition with this nanocomposite in vitro and in vivo is higher than that for the approved antiflu drug oseltamivir. The results open the prospect of creating a unique antiviral agent suitable for IAV suppression.
Assuntos
DNA Catalítico , Vírus da Influenza A , Influenza Humana , Nanopartículas , Titânio , Cães , Animais , Camundongos , Humanos , Vírus da Influenza A/genética , Antivirais/farmacologia , DNA Catalítico/farmacologia , DNA Catalítico/uso terapêutico , Células Madin Darby de Rim Canino , Influenza Humana/tratamento farmacológicoRESUMO
Although DNAzymes have been found to reduce injury after myocardial ischemia/reperfusion (MI/R), their efficiency have been limited due to rapid degradation in vivo. Thus, this study was conducted to extend their half-life by encapsulation into nanoniosomes and examine their cardioprotective effects in a rat model of myocardial infarction (MI). In order to synthesize nanoniosomes, surface active agent film hydration method was used. Characterization of nanoniosomes was performed using the atomic force microscopy (AFM). In order to establish MI/R model in rats, left anterior descending coronary artery (LAD) was ligated for 30 min. A single dose (150µL) of drug formulations was injected into the infarcted region. The cardiac function was evaluated using echocardiography. The expression of pro-inflammatory cytokines, apoptotic factors, and nuclear factor-κB (NF-κB) were evaluated using Western blot and immunohistochemistry, respectively. Particle size of only nano-niosomes was in the range of 60-90 nm, while a shift to 70-110 nm was seen after DNAzyme encapsulation. MI rats treated with DNAzymeloaded nanoniosomes could markedly reduce Bax, caspase3, TNF-α, IL-1ß, and NF-κB as well as increase Bcl-2 compared to only MI/R group. Collectively, our finding show that nanoniosomes can be considered excellent drug delivery platforms to extend half-life and stability of DNAzyme, when it is used to reduce myocardial I/R injury.
Assuntos
DNA Catalítico , Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica , Ratos , Animais , NF-kappa B/metabolismo , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , DNA Catalítico/uso terapêutico , DNA Catalítico/farmacologia , Lipossomos , Ratos Sprague-Dawley , Inflamação , ApoptoseRESUMO
BACKGROUND: RNA-cleaving deoxyribozymes (DNAzymes) are catalytic deoxyribonucleic acid molecules that have become a promising new class of gene suppressors by binding and cleaving target mRNA. This study investigated whether DNAzymes targeting Bcl-xL enhanced the effectiveness of radiotherapy and chemotherapy in colorectal cancer (CRC) cells. METHODS: Two types of CRC cells, SW480 and SW837, were transfected with five DNAzymes. Cell viability, Bcl-xL expression and apoptosis were examined. SW480 xenograft model was used to examine the combined effects of Bcl-xL DNAzymes and 5-FU (or X-rays) on tumor growth. RESULTS: Three Bcl-xL DNAzymes, DT882, DT883, and DT884 were identified to be effective in suppressing Bcl-xL expression and causing cell apoptosis. Furthermore, DT882 combined with 5-FU or radiotherapy addictively promoted cell apoptosis and significantly inhibited the growth of SW480 xenografts in vivo. CONCLUSIONS: These results suggest that Bcl-xL DNAzymes can enhance the radiosensitivity and chemosensitivity in CRC cells via inducing apoptosis.
Assuntos
Apoptose , Neoplasias Colorretais , DNA Catalítico , Proteína bcl-X , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/radioterapia , DNA Catalítico/farmacologia , Humanos , Tolerância a Radiação , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/genéticaRESUMO
The development of nucleic-acid-based antimicrobials such as RNA-cleaving DNAzyme (RCD), a short catalytically active nucleic acid, is a promising alternative to the current antibiotics. The current rapid spread of antimicrobial resistance (AMR) in bacteria renders some antibiotics useless against bacterial infection, thus creating the need for alternative antimicrobials such as DNAzymes. This review summarizes recent advances in the use of RCD as a diagnostic and therapeutic agent against AMR. Firstly, the recent diagnostic application of RCD for the detection of bacterial cells and the associated resistant gene(s) is discussed. The next section summarises the therapeutic application of RCD in AMR bacterial infections which includes direct targeting of the resistant genes and indirect targeting of AMR-associated genes. Finally, this review extends the discussion to challenges of utilizing RCD in real-life applications, and the potential of combining both diagnostic and therapeutic applications of RCD into a single agent as a theranostic agent.
Assuntos
Antibacterianos , DNA Catalítico , Farmacorresistência Bacteriana , Antibacterianos/farmacologia , Bactérias/genética , DNA Catalítico/farmacologia , Farmacorresistência Bacteriana/genética , RNARESUMO
The RNA-cleaving DNAzyme (DZ) holds promising potential for RNA interference (RNAi) applications and is favored over siRNA owing to its high chemical stability, biocompatibility, predictable activity, and substrate versatility. However, its pharmaceutical applications for disease treatment are limited by the requirement of metal cofactor for activation, as well as the lack of effective co-delivery systems to combine with other therapeutic modalities. Herein, we designed and constructed metal organic framework (MOF) coated MnO2 nanosheets to realize the co-delivery of a survivin inhibiting DZ and doxorubicin (DOX) for chemo-gene combinatorial treatment of cancer. In our design, the DOX was adsorbed on MnO2 planar surface, and the DZ was loaded into the MOF shell layer through the coordination between Mn2+ and tannic acid. The nano-system could stably encapsulate the payloads under physiological condition, but rapidly degraded after endocytose into tumor cells in response to intracellular stimuli, resulting in triggered drugs release. Notably, the coreleased Mn2+ could act as metal cofactor for effective DZ activation. Both in vitro and in vivo studies have demonstrated the enhanced anti-tumor efficacy of the nanosystem, with co-contributions from anti-neoplastic DOX, survivin silencing effect of DZ, and to some extent, ROS generation by Mn2+. This work provides an ingenious strategy to address the key limitation of DZ for RNAi applications and realize the combination of DZ with other therapeutic modalities, in which the DZ can be in-situ activated for target gene silencing.
Assuntos
Antineoplásicos/administração & dosagem , DNA Catalítico/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Estruturas Metalorgânicas/química , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , DNA Catalítico/farmacologia , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Feminino , Humanos , Compostos de Manganês/química , Camundongos , Camundongos Nus , Nanopartículas/química , Terapêutica com RNAi/métodos , Espécies Reativas de Oxigênio/metabolismo , Tecnologia Farmacêutica/métodosRESUMO
The hyperphosphorylation of the microtubule-associated protein tau (MAPT) has been implicated in various neurological diseases, including Alzheimer's disease. It has been hypothesized that the reduction of MAPT would result in depolymerizing neurofibrillary tangles and could be a potential strategy for the treatment of Alzheimer's disease and other tauopathies. In this study, we report the development of novel DNAzymes and splice-modulating antisense oligonucleotides (AOs) for the efficient inhibition of MAPT. We designed and synthesized a range of DNAzymes and 2'-O-methyl (2'-OMe)-modified AOs on a phosphorothioate (PS) backbone targeting various exons across the MAPT gene transcript. Our results demonstrated that RNV563, an arm-loop-arm-type DNAzyme targeting exon 13, and an AO candidate AO4, targeting exon 4, efficiently downregulated MAPT RNA expression by 58% and 96%, respectively. In addition, AO4 also reduced the MAPT protein level by 74%. In line with our results, we believe that AO4 could be used as a potential therapeutic molecule for Alzheimer's disease and other tauopathies.
Assuntos
Doença de Alzheimer/tratamento farmacológico , DNA Catalítico/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Proteínas tau/genética , Processamento Alternativo/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Linhagem Celular , Éxons/genética , Expressão Gênica/efeitos dos fármacos , Humanos , RNA Mensageiro/genética , Tauopatias/tratamento farmacológico , Tauopatias/genética , Tauopatias/patologia , Proteínas tau/antagonistas & inibidoresRESUMO
DNAzymes with nucleic acid-cleaving catalytic activity are increasing in versatility through concerted efforts to discover new sequences with unique functions, and they are generating excitement in the sensing community as cheap, stable, amplifiable detection elements. This review provides a comprehensive list and detailed descriptions of the DNAzymes identified to date, classified by their associated small molecule or ion needed for catalysis; of note, this classification clarifies conserved regions of various DNAzymes that are not obvious in the literature. Furthermore, we detail the breadth of functionality of these DNA sequences as well as the range of reaction conditions under which they are useful. In addition, the utility of the DNAzymes in a variety of sensing and therapeutic applications is presented, detailing both their advantages and disadvantages.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , DNA Catalítico/química , DNA Catalítico/farmacologia , DNA Catalítico/uso terapêutico , Humanos , Oligonucleotídeos/químicaRESUMO
BACKGROUND: The phenylboronic acid-functionalized polyamidoamine (PP) was employed as a gene carrier for Dz13 delivery, inducing an obvious anticancer response. MATERIALS AND METHODS: The Dz13 condensation ability of PP was evaluated through gel retardation assay. The cellular uptake mechanism of PP/Dz13 nanoparticles was studied using confocal laser scanning microscope and flow cytometer. The inhibition ability of cell proliferation, migration and invasion was investigated through MTT assay, flow cytometry, wound healing and Transwell migration assays, using hepatocarcinoma cell line HepG2 as a model. Finally, Western blotting analysis was used to detect the signaling pathway associated with the inhibition of cell apoptosis and migration induced by Dz13 delivery. RESULTS: The carrier PP could efficiently condense Dz13 into stable nanoparticles at mass ratios of >1.5. The hydrodynamic diameter and zeta potential of PP/Dz13 nanoparticles were measured to be 204.77 nm and +22.00 mV at a mass ratio of 10.0, respectively. The nanoparticles could realize an efficient cellular uptake in sialic acid-dependent endocytosis manner. Moreover, the nanoparticles exhibited an obvious antiproliferation effect through the induction of cell apoptosis and cell cycle arrest due to the cleavage of c-Jun mRNA. Besides, the suppression of cell migration and invasion could be achieved after the PP/Dz13 transfection, attributing to the decreased expression level of MMP-2 and MMP-9. CONCLUSION: The PP provided a potential delivery system to achieve the tumor-targeting gene therapy.
Assuntos
Ácidos Borônicos/química , Movimento Celular , DNA Catalítico/administração & dosagem , DNA Catalítico/farmacologia , Poliaminas/química , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Células Hep G2 , Humanos , Nanopartículas/química , Nanopartículas/ultraestrutura , Espectroscopia de Prótons por Ressonância Magnética , RNA Mensageiro/genéticaRESUMO
RNA-cleaving deoxyribozymes (DNAzymes) are synthetic single-stranded DNA-based catalytic molecules that can be engineered to bind to and cleave target mRNA at predetermined sites. These have been used as therapeutic agents in a range of preclinical cancer models and have entered clinical trials in Europe, China, and Australia. This review surveys regulatory insights into mechanisms of disease brought about by use of catalytic DNA in vitro and in vivo, including recent uses as nanosensors, nanoflowers, and nanosponges, and the emerging role of adaptive immunity underlying DNAzyme inhibition of cancer growth. DNAzymes represent a promising new class of nucleic acid-based therapeutics in cancer. This article discusses mechanistic and therapeutic insights brought about by DNAzyme use as nanotools and reagents in a range of basic science, experimental therapeutic and clinical applications. Current limitations and future perspectives are also discussed.
Assuntos
DNA Catalítico/farmacologia , Animais , Antineoplásicos/farmacologia , Catálise , HumanosRESUMO
DNAzymes are synthetic oligonucleotides that are capable of cleavaging target mRNA to exert gene-silencing activity and are considered as promising therapeutic agents. Dz13 is a DNAzyme that cleaves the mRNA of c-Jun and suppresses the growth of squamous cell carcinomas. However, DNAzymes exhibit low cellular uptake efficacy and require a suitable drug delivery system. In this study, we directly added the Dz13 sequence to the 5'-end of single-stranded DNA to form modified tetrahedral DNA nanostructures (TDN-Dz13). The TDNs were used to deliver the single-stranded DNAzyme Dz13 into cells. Dz13 delivered by the TDNs showed high cellular uptake efficiency and still maintained intracellular gene-silencing activity to cleave the target c-Jun mRNA, which reduced cell proliferation. This study may help find a convenient approach for the delivery of DNAzymes to regulate target genes.
Assuntos
Carcinoma de Células Escamosas , DNA Catalítico , Sistemas de Liberação de Medicamentos , Inativação Gênica , Nanoestruturas , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , DNA Catalítico/química , DNA Catalítico/farmacologia , Humanos , Nanoestruturas/química , Nanoestruturas/uso terapêutico , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Proto-Oncogênicas c-jun/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismoRESUMO
BACKGROUND: Infections with human rhinoviruses (RVs) are responsible for millions of common cold episodes and the majority of asthma exacerbations, especially in childhood. No drugs specifically targeting RVs are available. OBJECTIVE: We sought to identify specific anti-RV molecules based on DNAzyme technology as candidates to a clinical study. METHODS: A total of 226 candidate DNAzymes were designed against 2 regions of RV RNA genome identified to be sufficiently highly conserved between virus strains (ie, the 5'-untranslated region and cis-acting replication element) by using 3 test strains: RVA1, RVA16, and RVA29. All DNAzymes were screened for their cleavage efficiency against in vitro-expressed viral RNA. Those showing any catalytic activity were subjected to bioinformatic analysis of their reverse complementarity to 322 published RV genomic sequences. Further molecular optimization was conducted for the most promising candidates. Cytotoxic and off-target effects were excluded in HEK293 cell-based systems. Antiviral efficiency was analyzed in infected human bronchial BEAS-2B cells and ex vivo-cultured human sinonasal tissue. RESULTS: Screening phase-generated DNAzymes characterized by either good catalytic activity or by high RV strain coverage but no single molecule represented a satisfactory combination of those 2 features. Modifications in length of the binding domains of 2 lead candidates, Dua-01(-L12R9) and Dua-02(-L10R11), improved their cleavage efficiency to an excellent level, with no loss in eminent strain coverage (about 98%). Both DNAzymes showed highly favorable cytotoxic/off-target profiles. Subsequent testing of Dua-01-L12R9 in BEAS-2B cells and sinonasal tissue demonstrated its significant antiviral efficiency. CONCLUSIONS: Effective and specific management of RV infections with Dua-01-L12R9 might be useful in preventing asthma exacerbations, which should be verified by clinical trials.
Assuntos
Antivirais/farmacologia , DNA Catalítico/farmacologia , RNA Viral/efeitos dos fármacos , Rhinovirus , Replicação Viral/efeitos dos fármacos , Resfriado Comum/prevenção & controle , Descoberta de Drogas , HumanosRESUMO
The catalytic DNA circuits play a critical role in engineered biological systems and molecular information processing. Actually, some of the natural or synthetic DNA circuits were triggered by covalent modifications, where conformational changes were induced to facilitate complex DNA engineering functions and signal transmissions. However, most of the reported artificial catalytic DNA circuits were regulated by the toehold-mediated reaction. Therefore, it is significant to propose a strategy to regulate the catalytic DNA circuit not only by the toehold-mediated mechanism, but also by involving the conformational changes induced by the covalent modification. In this study, we developed the catalytic DNA logic circuits regulated by DNAzyme. Here, a regulation strategy based on the covalent modification was proposed to control the DNA circuit, combing two reaction mechanisms: DNAzyme digestion and entropy-driven strand displacement. The DNAzyme and DNA catalyst can participate into the reactions alternatively, thus realizing the cascading catalytic circuits. Using the DNAzyme regulation, a series of logic gates (YES, OR and AND) were constructed. In addition, a two-layer cascading circuit and a feedback self-catalysis circuit were also established. The proposed DNAzyme-regulated strategy shows great potentials as a reliable and feasible method for constructing more complex catalytic DNA circuits.
Assuntos
Computadores Moleculares , DNA Catalítico/farmacologia , Entropia , Catálise , Eletroforese em Gel de Poliacrilamida , Fluorometria , Lógica , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The highly efficient cancer cell targeted delivery plays an important role in precise targeted therapies. Herein, a multifunctional DNA nano-scorpion nanostructure (termed AptDzy-DNS) functioned with aptamers and DNAzyme is developed for highly efficient targeted delivery of mRNA therapeutics in gene therapy. The designed AptDzy-DNS is self-assembled with specific aptamers as "scorpion stingers" for targeting tumor cell and DNAzymes as "scorpion pincers" for targeted gene therapy by cleaving mRNA into fragments. The as-prepared AptDzy-DNS can effectively distinguish cancer cells from normal cells by specific cross-talking between aptamers on AptDzy-DNS and overexpressed cell-surface receptors. In the process of gene therapy, by reacting with Mg2+-dependent DNAzyme on AptDzy-DNS, the mRNA oligonucleotide in cancer cell is auto-cleaved into broken strand, failing to be translated into corresponding protein. Following, the downregulation protein can block cancer cell growth and realize highly efficient targeted therapies. The results demonstrate that the multifunctional AptDzy-DNS shows promise for targeted cancer cell discrimination, highly efficient targeted delivery of mRNA therapeutics in gene therapy. Thus, this developed strategy provides impressive improvement on gene targeted therapy and paves the way for application of AptDzy-DNS in human cancer targeted therapies.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA Catalítico/química , Terapia Genética/métodos , RNA Mensageiro/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Linhagem Celular Tumoral , DNA Catalítico/farmacologia , Sistemas de Liberação de Medicamentos , Humanos , Conformação Molecular , NanoestruturasRESUMO
Non-small-cell lung cancer (NSCLC) is commonly caused by a mutation in the epidermal growth factor receptor (EGFR) and subsequent aberrant EGFR signaling with uncontrolled kinase activity. A deletion mutation in EGFR exon 19 is frequently observed in EGFR gene mutations. We designed a DNAzyme to suppress the expression of mutant EGFR by cleaving the mutant EGFR mRNA. The DNAzyme (named Ex19del Dz) specifically cleaved target RNA and decreased cancer cell viability when transfected into gefitinib-resistant lung cancer cells harboring EGFR exon 19 deletions. The DNAzyme decreased EGFR expression and inhibited its downstream signaling pathway. In addition to EGFR downregulation, Ex19del Dz containing CpG sites activated Toll-like receptor 9 (TLR9) and its downstream signaling pathway via p38 kinase, causing an immunostimulatory effect on EGFR-mutated NSCLC cells. Thus, dual effects of this DNAzyme harboring the CpG site, such as TLR9 activation and EGFR downregulation, leads to apoptosis of EGFR-mutated NSCLC cells. [BMB Reports 2018; 51(1): 27-32].
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ilhas de CpG , DNA Catalítico/farmacologia , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Receptor Toll-Like 9/metabolismo , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/genética , DNA Catalítico/genética , DNA Catalítico/metabolismo , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Éxons , Gefitinibe , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutação , Quinazolinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Toll-Like 9/biossíntese , Receptor Toll-Like 9/genéticaRESUMO
Nucleic-acid-based drugs are a promising class of novel therapeutics; however, their use in medicine is widely limited because of insufficient delivery into cells. This article proposes a new delivery strategy of nucleic acid fragments into cells as components of TiO2-based nanocomposites. For the first time, unmodified Dz molecules were non-covalently immobilized on TiO2 nanoparticles precovered with polylysine (TiO2â¢PL) with the formation of (TiO2â¢PL)â¢Dz nanocomposites. DNAzymes in the proposed nanocomposites were shown to retain their ability to cleave the RNA target in a cell-free system with the same selectivity as unbound Dz molecules. It was shown by confocal laser microscopy that the fluorescein-labelled (TiO2â¢PL)â¢DzFlu nanocomposites penetrate into eukaryotic cells, where DzFlu is internalized in the cytoplasm and predominantly in nuclei. Delivery of deoxyribozymes into cells in the proposed nanocomposites permits very efficient interactions with RNA targets inside cells. This was demonstrated by an example of inhibition of H5N1 influenza A virus replication (inhibition by a factor of ca. 3000). This effect was one order of magnitude higher than with using lipofectamine as the transfection agent. The proposed (TiO2â¢PL)â¢Dz nanocomposites demonstrated high antiviral activity and are thus potent as nucleic-acid-based drugs.
Assuntos
Antivirais/farmacologia , DNA Catalítico/farmacologia , Portadores de Fármacos/metabolismo , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Nanopartículas Metálicas , Nanocompostos , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/metabolismo , DNA Catalítico/metabolismo , Cães , Células HeLa , Humanos , Virus da Influenza A Subtipo H5N1/fisiologia , Células Madin Darby de Rim CaninoRESUMO
Apoptosis pathway has become one of the important targets for therapeutic exploration for cancer therapy. The increased Bcl-2 protein level and phosphorylation is implicated in a decreased chemotherapeutic response in many cancers. BCL-2 inhibitors have been developed as direct inducers of apoptosis. However, resistance to BCL2 inhibitors has been emerging and thus considerable effort has been made to seek novel approaches to BCL2 suppression. In this report we describe an in vitro DNAzyme selection strategy resulting in molecules that are effective in suppressing expression of the target gene BCL-2 in vitro. A 3'-inverted modification was shown to significantly increase the DNAzyme stability in serum and the modified DNAzyme delivered by an osmotic pump chemosensitized human prostate cancer to Taxol in vivo. Thus this study provides an alternative strategy for potential BCL-2-targetd therapy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , DNA Catalítico/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Humanos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oligonucleotídeos/genética , Paclitaxel/farmacologia , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Herein, a polyethylenimine derivative N-acetyl-L-leucine-polyethylenimine (N-Ac-L-Leu-PEI) was employed as a carrier to achieve the delivery of DNAzyme targeting aurora kinase A using PC-3 cell as a model. Flow cytometry and confocal laser scanning microscopy demonstrated that the derivative could realize the cellular uptake of nanoparticles in an energy-dependent and clathrin-mediated pathway and obtain a high DNAzyme concentration in the cytoplasm through further endosomal escape. After DNAzyme transfection, expression level of aurora kinase A would be downregulated at the protein level. Meanwhile, the inhibition of cell proliferation was observed through 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cell colony formation assay, attributing to the activation of apoptosis and cell cycle arrest. Through flow cytometric analysis, an early apoptotic ratio of 25.93% and G2 phase of 22.58% has been detected after N-Ac-L-Leu-PEI-mediated DNAzyme transfection. Finally, wound healing and Transwell migration assay showed that DNAzyme transfection could efficiently inhibit the cell migration. These results demonstrated that N-Ac-L-Leu-PEI could successfully mediate the DNAzyme delivery and downregulate the expression level of aurora kinase A, triggering a significant inhibitory effect of excessive proliferation and migration of tumor cells.
Assuntos
Aurora Quinase A/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA Catalítico/farmacologia , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Neoplasias da Próstata/tratamento farmacológico , Apoptose/efeitos dos fármacos , Western Blotting , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Masculino , Polietilenoimina/química , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Células Tumorais Cultivadas , CicatrizaçãoAssuntos
Descoberta de Drogas/métodos , Ácidos Nucleicos/uso terapêutico , Animais , DNA Catalítico/farmacologia , DNA Catalítico/uso terapêutico , Genoma Humano , Humanos , MicroRNAs/farmacologia , MicroRNAs/uso terapêutico , Ácidos Nucleicos/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , RNA Catalítico/farmacologia , RNA Catalítico/uso terapêutico , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêuticoRESUMO
Although activity has been reported in vivo, free nucleic acid-based drugs are rapidly degraded and cleared following systemic administration. To address these challenges and improve the potency and bioavailability of genetic drugs, significant efforts have been made to develop effective delivery systems of which lipid nanoparticles (LNP) represent the most advanced technology currently available. In this review, we will describe and discuss the improvements to the pharmacokinetic and pharmacodynamic properties of nucleic acid-based drugs mediated by LNP delivery. It is envisioned that the significant improvements in potency and safety, largely driven by the development of LNP encapsulated siRNA drugs, will be translatable to other types of genetic drugs and enable the rapid development of potent molecular tools and drugs.