RESUMO
Multidrug resistance (MDR) is a major factor in the failure of many forms of tumor chemotherapy. Development of a specific ligand for MDR-reversal would enhance the intracellular accumulation of therapeutic agents and effectively improve the tumor treatments. Here, an aptamer was screened against a doxorubicin (DOX)-resistant human hepatocellular carcinoma cell line (HepG2/DOX) via cell-based systematic evolution of ligands by exponential enrichment. A 50 nt truncated sequence termed d3 was obtained with high affinity and specificity for HepG2/DOX cells. Multidrug resistance protein 1 (MDR1) is determined to be a possible recognition target of the selected aptamer. Aptamer d3 binding was revealed to block the MDR of the tumor cells and increase the accumulation of intracellular anticancer drugs, including DOX, vincristine, and paclitaxel, which led to a boost to the cell killing of the anticancer drugs and lowering their survival of the tumor cells. The aptamer d3-mediated MDR-reversal for effective chemotherapy was further verified in an in vivo animal model, and combination of aptamer d3 with DOX significantly improved the suppression of tumor growth by treating a xenograft HepG2/DOX tumor in vivo. This work demonstrates the feasibility of a therapeutic DNA aptamer as a tumor MDR-reversal agent, and combination of the selected aptamer with chemotherapeutic drugs shows great potential for liver cancer treatments.
Assuntos
Antineoplásicos , Resistencia a Medicamentos Antineoplásicos , Animais , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Resistência a Múltiplos Medicamentos , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Quimioterapia Combinada , Linhagem Celular TumoralRESUMO
Overexpression of receptor tyrosine kinases (RTKs) or binding to ligands can lead to the formation of specific unliganded and liganded RTK dimers, and these two RTK dimers are potential targets for preventing tumor metastasis. Traditional RTK dimer inhibitor analysis was mostly based on end point assays, which required cumbersome cell handling and behavior monitoring. There are still challenges in developing intuitive process-based analytical methods to study RTK dimer inhibitors, especially those used to visually distinguish between unliganded and liganded RTK dimer inhibitors. Herein, taking the mesenchymal-epithelial transition factor (MET) receptor, an intuitive method for evaluating MET inhibitors has been developed based on atomic force microscopy (AFM) lifetime analysis. The time interval between the start of the force and the bond break point was regarded as the bond lifetime, which could reflect the stability of the MET dimer. The results showed that there was a significant difference in the lifetime (τ) of unliganded MET dimers (τ1 = 207.87 ± 4.69 ms) and liganded MET dimers (τ2 = 330.58 ± 15.60 ms) induced by the hepatocyte growth factor, and aptamer SL1 could decrease τ1 and τ2, suggesting that SL1 could inhibit both unliganded and liganded MET dimers. However, heparin only decreased τ2, suggesting that it could inhibit only the liganded MET dimer. AFM-based lifetime analysis methods could monitor RTK dimer status rather than provide overall average results, allowing for intuitive process-based analysis and evaluation of RTK dimers and related inhibitors at the single-molecule level. This study provides a novel complementary strategy for simple and intuitive RTK inhibitor research.
Assuntos
Microscopia de Força Atômica , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-met , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Humanos , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Multimerização Proteica/efeitos dos fármacos , Ligantes , Fator de Crescimento de Hepatócito/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismoRESUMO
Synthetic cells function as biological mimics of natural cells by mimicking salient features of cells such as metabolism, response to stimuli, gene expression, direct metabolism, and high stability. Droplet-based microfluidic technology presents the opportunity for encapsulating biological functional components in uni-lamellar liposome or polymer droplets. Verified by its success in the fabrication of synthetic cells, microfluidic technology is widely replacing conventional labor-intensive, expensive, and sophisticated techniques justified by its ability to miniaturize and perform batch production operations. In this review, an overview of recent research on the preparation of synthetic cells through droplet-based microfluidics is provided. Different synthetic cells including lipid vesicles (liposome), polymer vesicles (polymersome), coacervate microdroplets, and colloidosomes, are systematically discussed. Efforts are then made to discuss the design of a variety of microfluidic chips for synthetic cell preparation since the combination of microfluidics with bottom-up synthetic biology allows for reproductive and tunable construction of batches of synthetic cell models from simple structures to higher hierarchical structures. The recent advances aimed at exploiting them in biosensors and other biomedical applications are then discussed. Finally, some perspectives on the challenges and future developments of synthetic cell research with microfluidics for biomimetic science and biomedical applications are provided.
RESUMO
BACKGROUND: Minimally invasive glaucoma surgery (MIGS) has experienced a surge in popularity in recent years. Glaucoma micro-stents serve as the foundation for these minimally invasive procedures. Nevertheless, the utilization of these stents still presents certain short-term and long-term complications. This study aims to elucidate the creation of a novel drainage stent implant featuring a diverging channel, produced through microfluidic template processing technology. Additionally, an analysis of the mechanical properties, biocompatibility, and feasibility of implantation is conducted. RESULTS: The stress concentration value of the proposed stent is significantly lower, approximately two to three times smaller, compared to the currently available commercial XEN gel stent. This indicates a stronger resistance to bending in theory. Theoretical calculations further reveal that the initial drainage efficiency of the gradient diverging drainage stent is approximately 5.76 times higher than that of XEN stents. Notably, in vivo experiments conducted at the third month demonstrate a favorable biocompatibility profile without any observed cytotoxicity. Additionally, the drainage stent exhibits excellent material stability in an in vitro simulation environment. CONCLUSIONS: In summary, the diverging drainage stent presents a novel approach to the cost-effective and efficient preparation process of minimally invasive glaucoma surgery (MIGS) devices, offering additional filtering treatment options for glaucoma.
Assuntos
Glaucoma , Stents , Glaucoma/cirurgia , Animais , Microfluídica/instrumentação , Teste de Materiais , Humanos , Procedimentos Cirúrgicos Minimamente Invasivos/instrumentação , Fenômenos Mecânicos , Desenho de Equipamento , CoelhosRESUMO
There is considerable interest in creating a precise and sensitive strategy for in situ visualizing and profiling intracellular miRNA. Present here is a novel photocaged amplified FRET nanoflare (PAFN), which spatiotemporal controls of mRNA-powered nanomachine for precise and sensitive miRNA imaging in live cells. The PAFN could be activated remotely by light, be triggered by specific low-abundance miRNA and fueled by high-abundance mRNA. It offers high spatiotemporal control over the initial activity of nanomachine at desirable time and site, and a 'one-to-more' ratiometric signal amplification model. The PAFN, an unprecedented design, is quiescent during the delivery process. However, upon reaching the interest tumor site, it can be selectively activated by light, and then be triggered by specific miRNA, avoiding undesirable early activation and reducing nonspecific signals, allowing precise and sensitive detection of specific miRNA in live cells. This strategy may open new avenues for creating spatiotemporally controllable and endogenous molecule-powered nanomachine, facilitating application at biological and medical imaging.
Assuntos
Técnicas Biossensoriais , MicroRNAs , Diagnóstico por Imagem , Transferência Ressonante de Energia de Fluorescência , MicroRNAs/genética , RNA Mensageiro/genéticaRESUMO
The reprogramming of cell signaling and behavior through the artificial control of cell surface receptor oligomerization shows great promise in biomedical research and cell-based therapy. However, it remains challenging to achieve combinatorial recognition in a complicated environment and logical regulation of receptors for desirable cellular behavior. Herein, we develop a logic-gated DNA nanodevice with responsiveness to multiple environmental inputs for logically controlled assembly of heterogeneous receptors to modulate signaling. The "AND" gate nanodevice uses an i-motif and an ATP-binding aptamer as environmental cue-responsive units, which can successfully implement a logic operation to manipulate receptors on the cell surface. In the presence of both protons and ATP, the DNA nanodevice is activated to selectively assemble MET and CD71, which modulate the HGF/MET signaling, resulting in cytoskeletal reorganization to inhibit cancer cell motility in a tumor-like microenvironment. Our strategy would be highly promising for precision therapeutics, including controlled drug release and cancer treatment.
Assuntos
DNA , Neoplasias , Humanos , DNA/genética , Oligonucleotídeos , Transdução de Sinais , Neoplasias/tratamento farmacológico , Trifosfato de Adenosina , Microambiente TumoralRESUMO
The design and construction of synthetic protocells capable of stimuli response and homeostatic regulation is an important challenge for synthetic protobiology. Here, we develop a step toward the construction of model protocells capable of a hypotonic stress-induced volume response that facilitates an increase in membrane permeability and the triggering of endogenous enzyme reactions. We describe a facile self-transformation process for constructing single- or multichambered molecularly crowded protocells based on the osmotic reconfiguration of lipid-coated coacervate droplets into multicompartmentalized coacervate vesicles. Hypotonic swelling broadens membrane permeability and increases transmembrane transport such that protease-based hydrolysis and enzyme cascades can be triggered and enhanced within the protocells by osmotically induced expansion. Specifically, we demonstrate how the enhanced production of nitric oxide (NO) within the swollen coacervate vesicles can be used to induce in vitro blood vessel vasodilation in thoracic artery rings. Our approach provides opportunities for designing reconfigurable model protocells capable of homeostatic volume regulation, dynamic structural reorganization, and adaptive functionality in response to changes in environment osmolarity, and could find applications in biomedicine, cellular diagnostics, and bioengineering.
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Células Artificiais , Células Artificiais/química , BioengenhariaRESUMO
Sensitive detection of miRNA targets in complex biological samples possesses great value in biopsy analysis and disease diagnosis but is still challenging because of low abundance and nonspecific interferences. In this work, self-primer DNA polymerization-propelled stochastic walkers (SWs) were proposed to detect miRNA-24 by combining magnetic microbeads (MMBs) and flow cytometry. The MMBs not only provide a three-dimensional interface for DNA walkers but also facilitate the enrichment and isolation of RNA targets from complex biological samples such as serum. The SWs can be initiated to walk through the entire surface of MMBs and transduce RNA walking into amplified fluorescence signals, with the detection limit of miRNA-24 at 0.95 pM. Moreover, this strategy integrating with flow cytometry was demonstrated to have good specificity with other homologous miRNAs. This platform offers promising applications in RNA biosensing and biomedical diagnostics.
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Técnicas Biossensoriais , MicroRNAs , MicroRNAs/análise , Microesferas , Polimerização , Limite de Detecção , DNA/análise , Fenômenos MagnéticosRESUMO
Rational regulation of nanozyme activity can promote biochemical sensing by expanding sensing strategies and improving sensing performance, but the design of effective regulatory strategies remains a challenge. Herein, a rapid DNA-encoded strategy was developed for the efficient regulation of Pt nanozyme activity. Interestingly, we found that the catalytic activity of Pt nanozymes was sequence-dependent, and its peroxidase activity was significantly enhanced only in the presence of T-rich sequences. Thus, different DNA sequences realized bidirectional regulation of Pt nanozyme peroxidase activity. Furthermore, the DNA-encoded strategy can effectively enhance the stability of Pt nanozymes at high temperatures, freezing, and long-term storage. Meanwhile, a series of studies demonstrated that the presence of DNA influenced the reduction degree of H2PtCl6 precursors, which in turn affected the peroxidase activity of Pt nanozymes. As a proof of application, the sensor array based on the Pt nanozyme system showed superior performance in the accurate discrimination of antioxidants. This study obtained the regulation rules of DNA on Pt nanozymes, which provided theoretical guidance for the development of new sensing platforms and new ideas for the regulation of other nanozyme activities.
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Antioxidantes , DNA , Peroxidases , Peroxidase , Peróxido de Hidrogênio/análiseRESUMO
Usually, different assays and instrumentation are required for different types of targets, e.g., nucleic acids, proteins, small molecules, etc., because of significant differences in their structures and sizes. To increase efficiency and reduce costs, a desirable solution is to develop a versatile platform suitable for diverse objectives. Here, we established a versatile detection technique: first, target separation and enrichment were carried out using magnetic beads (MBs); then, different targets were converted to same barcoded DNA strands (BDs) released from gold nanoparticles; finally, sensitive detection of three different targets (miRNA-21, digoxigenin antibody, and aflatoxin B1) was achieved through exonuclease III (Exo III) cyclic cleavage-assisted signal amplification. To simplify the operation, we integrated this technique into a microfluidic chip with multiple chambers in which the requisite reagents were prestored. Just by moving the MBs through different chambers with a magnet, multiple steps can be completed. Due to the limited space in microfluidic chips, the full mixing of MBs and solution is a key point to improve reaction efficiency. The mixing can be achieved by acoustic vibration generated by a small, portable sonic toothbrush. Based on the microfluidic chip, the detection limits of the above three targets were 0.76 pM, 0.16 ng/mL, and 0.56 nM, respectively. Furthermore, miRNA-21 and Digoxigenin antibody (Dig-Ab) in serum and AFB1 in corn powder were also used to demonstrate the performance of this chip. Our versatile platform is easy to operate and is expected to develop into an automatic "sample-to-answer" device.
Assuntos
Nanopartículas Metálicas , MicroRNAs , Técnicas Analíticas Microfluídicas , Microfluídica , Ouro/química , Digoxigenina , Nanopartículas Metálicas/química , AnticorposRESUMO
DNA cascaded circuits have great potential in detecting low abundance molecules in complex biological environment due to their powerful signal amplification capability and nonenzymatic feature. However, the problem of the cascaded circuits is that the design is relatively complex and the kinetics is slow. Herein, a new design paradigm called catalyst-accelerated circular cascaded circuits is proposed, where the catalyst inlet is implanted and the reaction speed can be adjusted by the catalyst concentration. This new design is very simple and only requires three hairpin probes. Meanwhile, the results of a series of studies demonstrate that the reaction speed can be accelerated and the sensitivity can be also improved. Moreover, endogenous mRNA can also be used as a catalyst to drive the circuits to amplify the detection of target miRNA in live cells and in mice. These catalyst-accelerated circular cascaded circuits can substantially expand the toolbox for intracellular low abundance molecular detection.
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Técnicas Biossensoriais , MicroRNAs , Animais , Camundongos , DNA Circular , DNA , MicroRNAs/genética , RNA Mensageiro , Cinética , Técnicas Biossensoriais/métodos , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
A ß-amyloid (Aß) aggregation process is a spontaneous process where the original random coil or helical structure changes into a regularly arranged ß-sheet structure. The development of inhibitors with the features of low cost, high efficiency, and biosafety by targeting Aß self-aggregation is significant for Alzheimer's disease treatment. However, the issues of low inhibition efficiency under low concentrations of inhibitors and biological toxicity are currently to be addressed. To resolve the above problems, a DNA nanoassembly (HCR-Apt) based on spatially ordered recognition elements was constructed by targeted disruption of Aß ordered arrangement. It was discovered that HCR-Apt could inhibit effectively the fibrillation of Aß40 monomers and oligomers at substoichiometric ratios. This may be due to orderly arrangement of aptamers in rigid nanoskeletons for enhancing the recognition interaction between aptamers and Aß40. The strong interaction between HCR-Apt and Aß40 limited the flexible conformational conversion of Aß40 molecules, thereby inhibiting their self-assembly. Computational simulations and experimental analysis revealed the interactions of Apt42 with Aß40, which explained different inhibition effects on the fibrillation of Aß40 monomers and oligomers. Furthermore, the analysis of tyrosine intrinsic fluorescence spectra and surface plasmon resonance imaging showed that the interaction of HCR-Apt and Aß40 was stronger than that of Apt42 and Aß40. These findings contributed to establishing a promising method of boosting the recognition interaction by orderly arrangement of recognition elements. Taken together, this work is expected to provide a simple and efficient strategy for inhibiting Aß aggregation, expanding aptamer's application potential in neurodegenerative diseases.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/química , Amiloide/química , Oligonucleotídeos , DNA , Fragmentos de Peptídeos/químicaRESUMO
A simple and wash-free POCT platform based on microcapillary was developed, using breast cancer cell-derived exosomes as a model. This method adopted the "one suction and one extrusion" mode. The hybridized complex of epithelial cell adhesion molecule (EpCAM) aptamer and complementary DNA-horseradish peroxidase conjugate (CDNA-HRP) was pre-modified on the microcapillary's inner surface. "One suction" meant inhaling the sample into the functionalized microcapillary. The exosomes could specifically bind with the EpCAM aptamer on the microcapillary's inner wall, and then the CDNA-HRP complex was released. "One extrusion" referred to squeezing the shedding CDNA-HRP into the 3,3',5,5'-tetramethylbenzidine (TMB)/H2O2 solution, and then the enzyme-catalyzed reaction would occur to make the solution yellow using sulfuric acid as the terminator. Therefore, exosome detection could be realized. The limit of detection was 2.69 × 104 particles mL-1 and the signal value had excellent linearity in the concentration range from 2.75 × 104 to 2.75 × 108 particlesâ mL-1 exosomes. In addition, the wash-free POCT platform also displayed a favorable reproducibility (RSD = 2.9%) in exosome detection. This method could effectively differentiate breast cancer patients from healthy donors. This work provided an easy-to-operate method for detecting cancer-derived exosomes without complex cleaning steps, which is expected to be applied to breast cancer screening.
Assuntos
Neoplasias da Mama , Exossomos , Humanos , Feminino , Neoplasias da Mama/diagnóstico , DNA Complementar/metabolismo , Exossomos/metabolismo , Peróxido de Hidrogênio/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Reprodutibilidade dos Testes , Sucção , Peroxidase do Rábano Silvestre/metabolismoRESUMO
Visualizing intracellular microRNA (miRNA) is of great importance for revealing its roles in the development of disease. However, cell membrane barrier, complex intracellular environment and low abundance of target miRNA are three main challenges for efficient imaging of intracellular miRNA. Here, we report a size-controllable and self-assembled DNA nanosphere with ATP-fueled dissociation property for amplified miRNA imaging in live cells and mice. The DNA nanosphere was self-assembled from Y-shaped DNA (Y-DNA) monomers through predesigned base pair hybridization, and the size could be easily controlled by varying the concentration of Y-DNA. Once the nanosphere was internalized into cells, the intracellular specific target miRNA would trigger the cyclic dissociation of the DNA nanosphere driven by ATP, resulting in amplified FRET signal. The programmable DNA nanosphere has been proven to work well for detecting the expression of miRNA in cancer cells and in mice, which demonstrates its fairish cell penetration, stability and sensitivity.
Assuntos
Técnicas Biossensoriais , MicroRNAs , Nanosferas , Camundongos , Animais , DNA/genética , Hibridização de Ácido Nucleico , Trifosfato de AdenosinaRESUMO
The ability to reproduce signal transduction and cellular communication in artificial cell systems is significant in synthetic protobiology. Here, we describe an artificial transmembrane signal transduction through low pH-mediated formation of the i-motif and dimerization of DNA-based artificial membrane receptors, which is coupled to the occurrence of fluorescence resonance energy transfer and the activation of G-quadruplex/hemin-mediated fluorescence amplification inside giant unilamellar vesicles. Moreover, an intercellular signal communication model is established when the extravesicular H+ input is replaced by coacervate microdroplets, which activate the dimerization of the artificial receptors, and subsequent fluorescence production or polymerization in giant unilamellar vesicles. This study represents a crucial step towards designing artificial signalling systems with environmental response, and provides an opportunity to establish signalling networks in protocell colonies.
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Células Artificiais , Receptores Artificiais , Lipossomas Unilamelares , Transdução de Sinais , DNA , Comunicação , Células Artificiais/metabolismoRESUMO
DNA logic gates, as a class of smart molecular devices with excellent biocompatibility and convenient information processing mode, have been widely used for identification of cancer cells based on logic analysis of cancer biomarkers. However, most of the developed DNA logic gates for identification of cancer cells are mainly driven by homogeneous biomarkers such as membrane proteins or RNAs, which may suffer from insufficient accuracy. Herein, we reported a membrane protein and extracellular acid heterogeneity-driven amplified DNA logic gate (HDLG) for accurate and sensitive identification of cancer cells by combining the superior signal amplification characteristics of the hybridization chain reaction (HCR) and the precise computation ability of the logic operation. In this strategy, a DNA aptamer was employed for membrane protein recognition, and a split i-motif was used for the response of the extracellular acid. Only when the two heterogeneous biomarkers existed simultaneously, the DNA logic gate could be driven to perform the "AND" logic operation and induce the formation of an intact trigger to initiate a HCR process on the cell surface, generating an amplified "ON" fluorescence signal. Benefiting from the design of heterogeneity-driven and signal amplification, this DNA logic gate could not only autonomously perform high-resolution fluorescence imaging on the surface of target cancer cells, but also perform sensitive analysis of target cancer cells with a cell number of 70 detected in 200 µL of buffer and desirable accuracy in differentiating target cancer cells from complicated cell mixtures. We anticipate that this novel HDLG is expected to be applied in precise disease diagnosis.
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Aptâmeros de Nucleotídeos , Computadores Moleculares , DNA , Proteínas de Membrana , Neoplasias , Aptâmeros de Nucleotídeos/metabolismo , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , DNA/genética , DNA/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neoplasias/diagnóstico , Neoplasias/genéticaRESUMO
The desire for a cancer theranostic system with simultaneously accurate diagnosis and efficient therapy is undeniably interminable. Heretofore, theranostic systems with simple components were designed for cancer theranostics but with confined accuracy of diagnosis and side effects of administered drugs. Here, we report an activatable theranostic system for simultaneously imaging dual cancer-related RNAs, mRNA Bcl-2 and piRNA-36026, and combined gene-chemotherapy through the target-induced intracellular disassembly of DNA tetrahedron. Briefly, five customized oligonucleotides are used to assemble the functionalized DNA tetrahedron. The relevant functional nucleic acids, including the antisequence of mRNA Bcl-2, the antisequence of piRNA-36026, and aptamer AS1411, are designed in the customized oligonucleotides with the signal reporters Cy3 and Cy5. Doxorubicin (DOX) is loaded in the functionalized DNA tetrahedron by inlaying between cytosine and guanine to form the activatable cancer theranostic system. The activatable cancer theranostic system is able to recognize MCF-7 cells by aptamer AS1411 and then enter the cells. In the presence of targets, the antisequences in the activatable cancer theranostic system hybridize with intracellular mRNA Bcl-2 and piRNA-36026, leading to the fluorescence signal recovery of Cy3 and Cy5 and the downregulation of two targets in the cytoplasm as well as the consequent apoptosis of MCF-7 cells in the form of gene therapy. Interestingly, as the antisequences are designed in the assembly strands, the hybridization between targets and the antisequences results in the disassembly of the activatable cancer theranostic system and the release of DOX as well as sequential chemotherapy. Advantageously, the activatable cancer theranostic system can achieve imaging of dual cancer-related RNAs with an imaging time window as long as 15 h and exhibit an obvious therapeutic effect in vivo. Therefore, this work is in furtherance of exploration for activatable cancer theranostic systems with high accuracy and efficiency and sheds new light on the development of precision medicine.
Assuntos
Neoplasias , Nanomedicina Teranóstica , DNA , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro , RNA Interferente Pequeno , Nanomedicina Teranóstica/métodosRESUMO
Mesenchymal stem cells (MSCs) mainly found in the bone marrow of adult mammals demonstrate unique capacities of differentiating into multiple cell lineages and undifferentiated MSCs are considered an ideal source of seed cells for cell therapy and tissue engineering. However, MSCs are heterogeneous and not abundant in bone marrow, and there are few specific markers for these cells currently. Therefore, new methods to isolate and characterize MSCs are urgently required. To address the problem, we successfully developed a high-specificity aptamer, called Apt-W2, to specifically recognize mouse bone marrow mesenchymal stem cells (mBMSCs). We synthesized Apt-W2 modified magnetic beads (Apt-W2-MBs) and used them as bait to fish out the MSCs from mouse bone marrow accurately by magnetic-activated cell sorting (MACS). Next, the sorted cells could break free from the Apt-W2-MBs by the competition of C-W2 (complementary strands of Apt-W2). As a result, the sorted cells were intact, and maintained the stem cell phenotype and good proliferative ability. Simultaneously, the sorted cells showed high pluripotency to differentiate into osteoblasts, chondrocytes, and adipocytes. More importantly, the Apt-W2-MB cocktail showed a fine capture performance for MSCs (â¼88.33%). This new methodological approach can greatly facilitate MSC isolation efficiently and intactly, thereby enhancing the rate of in vitro differentiation of MSC-derived cells for the emerging field of tissue engineering and regenerative medicine. This new instrumental application of aptamers is an important innovation that achieved both high efficiency and nondestructive cell sorting, opening the door to novel cell sorting approaches.
Assuntos
Aptâmeros de Nucleotídeos , Células-Tronco Mesenquimais , Camundongos , Animais , Medula Óssea , Diferenciação Celular , Células da Medula Óssea , Células Cultivadas , Proliferação de Células , MamíferosRESUMO
Sensitive and accurate analysis of low-concentration of tumor-derived exosomes (Exos) in biofluids is essential for noninvasive cancer diagnosis but is still challenging due to the lack of high-sensitive methods with low-cost and easy-operation. Herein, exploiting target Exos as a three-dimensional (3D) track for the first time, we developed a self-serviced-track DNA walker (STDW) for wash-free detection of tumor Exos using exosomal glycoprotein, which was enabled by split aptamer-recognition-initiated autonomous running powered by a catalytic hairpin assembly (CHA). Benefiting from high selectivity and sensitivity of the STDW assay, direct detection of tumor Exos in cell culture medium and serum could also be realized. Furthermore, this method exhibited high accuracy in clinical sample analysis, offering the potential for early cancer diagnosis and postoperative response prediction.
Assuntos
Técnicas Biossensoriais , Exossomos , Neoplasias , DNA/genética , Glicoproteínas , Humanos , Neoplasias/diagnósticoRESUMO
Although giant unilamellar vesicles (GUVs) have been extensively studied as synthetic cell-like microcompartments, their applicability as cytomimetic models is severely compromised by low levels of membrane permeability, low encapsulation efficiencies, and high physicochemical instability. Here, we develop an integrated cytomimetic model comprising a macromolecularly crowded interior with high sequestration efficiency and enclosed within a phospholipid membrane that is permeable to molecules below a molecular weight cutoff of ca. 4 kDa. The protocells are readily prepared by spontaneous assembly of a phospholipid membrane on the surface of preformed polynucleotide/polysaccharide coacervate microdroplets and are designated as giant coacervate vesicles (GCVs). Partial anchoring of the GCV membrane to the underlying coacervate phase results in increased robustness, lower membrane fluidity, and increased permeability compared with GUV counterparts. As a consequence, enzyme and ribozyme catalysis can be triggered in the molecularly crowded interior of the GCV but not inside the GUVs when small molecule substrates or inducers are present in the external environment. By integrating processes of membrane-mediated compartmentalization and liquid-liquid microphase separation, GCVs could offer substantial advantages as cytomimetic models, synthetic protocells, and artificial biomolecular microreactors.