RESUMO
Isolation of specific rare cell subtypes from whole blood is critical in cellular analysis and important in basic and clinical research. Traditional immunomagnetic cell capture suffers from suboptimal sensitivity, specificity, and time- and cost-effectiveness. Mimicking the features of octopuses, a device termed a "NanoOctopus" was developed for cancer cell isolation in whole blood. The device consists of long multimerized aptamer DNA strands, or tentacle DNA, immobilized on magnetic microparticle surfaces. Their ultrahigh sensitivity and specificity are attributed to multivalent binding of the tentacle DNA to cell receptors without steric hindrance. The simple, quick, and noninvasive capture and release of the target cells allows for extensive downstream cellular and molecular analysis, and the time- and cost-effectiveness of fabrication and regeneration of the devices makes them attractive for industrial manufacture.
Assuntos
Aptâmeros de Nucleotídeos/química , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/metabolismo , Separação Celular/métodos , Nanotecnologia/métodos , Células Neoplásicas Circulantes/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Sanguíneas/análise , Estudos de Casos e Controles , Humanos , Fenômenos Magnéticos , Microesferas , Células Neoplásicas Circulantes/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaRESUMO
Self-assembled monolayers designed to immobilize capture antibodies are usually prepared using a mixture of functional and inactive linkers. Here, using low molar ratios (1:1 to 1:100) of the two linkers resulted in loss of binding capability of the anti-EGFR (epidermal growth factor receptor) antibody nimotuzumab, as assessed by surface plasmon resonance imaging. We then developed a simple theoretical model to predict the optimal surface density of the functional linker, taking into account the antibody size and linker diameter. A high (1:1000) dilution of the functional linker yielded the best results. As an advantage, this approach does not require chemical modification of the protein.
Assuntos
Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Receptores ErbB/imunologia , Ligantes , Modelos Moleculares , Propriedades de SuperfícieRESUMO
Surface plasmon resonance imaging (SPRi) has emerged as a versatile biosensor to detect a wide range of biomolecular interactions with divergent potential applications. However, the use of this advanced-level technology for stem cell lysate study is still not much explored. Cell lysates are significant biological analytes used for disease diagnostics and proteomic studies, but their complex nature limits their use as an analyte for SPRi biosensors. Here, we review the problems associated with the use of SPRi for stem cell lysate study and examine the role of surface chemistry, running buffer, and blocking solution in order to minimize nonspecific adsorption (NSA). We detect the expression of Oct4, Sox2, Nanog, Rex1, and Lin28 biomarkers present in mouse embryonic stem cell (mESC) lysate against their corresponding antibodies immobilized on the sensor surface with reduced NSA. The current study shows that the conjunction of SPRi and microarray can be used as a label-free, high-throughput, and rapid technique for detection of biomarkers and their relative abundance in stem cell lysate study.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Adsorção , Animais , Biomarcadores/metabolismo , Soluções Tampão , Morte Celular , Regulação da Expressão Gênica , Camundongos , Fatores de Tempo , Análise Serial de TecidosRESUMO
In recent years, in situ protein synthesis microarray technologies have enabled protein microarrays to be created on demand just before they are needed. In this paper, we utilized the TUS-TER immobilization technology to allow label-free detection with real-time kinetics of protein-protein interactions using surface plasmon resonance imaging (SPRi). We constructed an expression-ready plasmid DNA with a C-terminal TUS fusion tag to directionally immobilize the in situ synthesized recombinant proteins onto the surface of the biosensor. The expression plasmid was immobilized on the polyethylene imine-modified gold surface, which was then coupled with a cell-free expression system on the flow cell of the SPRi instrument. The expressed TUS fusion proteins bind on the surface via the immobilized TER DNA sequence with high affinity (â¼3-7×10(-13)M). The expression and immobilization of the recombinant in situ expressed proteins were confirmed by probing with specific antibodies. The present study shows a new low cost method for in situ protein expression microarrays that has the potential to study the kinetics of protein-protein interactions. These protein microarrays can be created on demand without the problems of stability associated with protein arrays used in the drug discovery and biomarker discovery fields.
Assuntos
Análise Serial de Proteínas , Proteínas/análise , Ressonância de Plasmônio de Superfície/métodos , Técnicas Biossensoriais , CinéticaRESUMO
To evaluate the transdermal delivery of six analgesic drugs (i.e., ketamine, gabapentin, clonidine, lidocaine, ketoprofen, and amitriptyline) that were compounded into three commercially available bases, Salt Stable LS Base, Transdermal Pain Base, and Lipoderm ActiveMax Base, the Franz finite dose model was used for an in vitro penetration study using porcine skin over 48 hours. Rapid penetration with a steady-state flux after the first 24 hours was detected in all the formulations. The present study demonstrates the successful delivery of six compounded analgesic drugs, using all of the noted bases. A high flux rate within 1 hour to 4 hours of application would correlate to effective pain relief, and the prolonged delivery over the first 24 hours would reduce the need for frequent reapplication. This can aid in pain management with the potential for enhanced pain control.
Assuntos
Analgésicos , Pele , Animais , Suínos , Administração Cutânea , Gabapentina , Dor/tratamento farmacológicoRESUMO
Simultaneous isolation of various circulating tumor cell (CTC) subtypes from whole blood is useful in cancer diagnosis and prognosis. Microfluidic affinity separation devices are promising for CTC separation because of their high throughput capacity and automatability. However, current affinity agents, such as antibodies (mAbs) and aptamers (Apts) alone, are still suboptimal for efficient, consistent, and versatile cell analysis. By introducing a hybrid affinity agent, i.e., an aptamer-antibody (Apt-mAb) conjugate, we developed a universal and regenerative microchip with high efficiency and non-invasiveness in the separation and profiling of various CTCs from blood. The Apt-mAb conjugate consists of a monoclonal antibody that specifically binds the target cell receptor and a surface-bound aptamer that recognizes the conserved Fc region of the mAb. The aptamer then indirectly links the surface functionalization of the microfluidic channels to the mAbs. This hybrid affinity agent and the microchip platform may be widely useful for various bio-particle separations in different biological matrices. Further, the regeneration capability of the microchip improves data consistency between multiple uses and minimizes plastic waste while promoting environmental sustainability. STATEMENT OF SIGNIFICANCE: A hybrid affinity agent, Apt-mAb, consisting of a universal aptamer (Apt) that binds the conserved Fc region of monoclonal antibodies (mAbs) was developed. The invented nano-biomaterial combines the strengths and overcomes the weakness of both Apts and mAbs, thus changing the paradigm of affinity separation of cell subtypes. When Apt-mAb was used to fabricate microfluidic chips using a "universal screwdriver" approach, the microchip could be easily tuned to bind any cell type, exhibiting great universality. Besides high sensitivity and selectivity, the superior regenerative capacity of the microchips makes them reusable, which provides improved consistency and repeatability in cell profiling and opens a new approach towards in vitro diagnostic point-of-care testing devices with environmental sustainability and cost-effectiveness.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes , Anticorpos Monoclonais , Linhagem Celular Tumoral , Separação Celular , Dimaprit/análogos & derivados , Humanos , Microfluídica , Células Neoplásicas Circulantes/patologia , PlásticosRESUMO
Androgenetic alopecia (AGA) remains an unsolved problem for the well-being of humankind, although multiple important involvements in hair growth have been discovered. Up until now, there is no ideal therapy in clinical practice in terms of efficacy and safety. Ultimately, there is a strong need for developing a feasible remedy for preventing and treating AGA. The Wnt/ß-catenin signaling pathway is critical in hair restoration. Thus, AGA treatment via modulating this pathway is rational, although challenging. Dickkopf-related protein 1 (DKK1) is distinctly identified as an inhibitor of canonical Wnt/ß-catenin signaling. Thus, in order to stimulate the Wnt/ß-catenin signaling pathway, inhibition of DKK1 is greatly demanding. Studying DKK1-targeting microRNAs (miRNAs) involved in the Wnt/ß-catenin signaling pathway may lay the groundwork for the promotion of hair growth. Bearing in mind that DKK1 inhibition in the balding scalp of AGA certainly makes sense, this review sheds light on the perspectives of miRNA-mediated hair growth for treating AGA via regulating DKK1 and, eventually, modulating Wnt/ß-catenin signaling. Consequently, certain miRNAs regulating the Wnt/ß-catenin signaling pathway via DKK1 inhibition might represent attractive candidates for further studies focusing on promoting hair growth and AGA therapy.
Assuntos
Cabelo/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , MicroRNAs/metabolismo , Regeneração/fisiologia , Alopecia/patologia , Alopecia/fisiopatologia , Cabelo/crescimento & desenvolvimento , Humanos , MicroRNAs/genética , Via de Sinalização WntRESUMO
ß-Cyclodextrin (ß-CD) was grafted onto hyaluronic acid (HA) in a single step to generate a supramolecular biopolymer (HA-ß-CD) that was explored for targeted drug delivery applications. Along with its excellent biocompatibility, the prepared HA-ß-CD exhibits not only exceptionally high loading capacity for the model drugs doxorubicin and Rhodamine B through the formation of inclusion complexes with the ß-CD component, but also the capability of targeted drug delivery to cancerous cells with a high level of expression of CD44 receptors, attributable to its HA component. The polymer can release the drug under slightly acidic conditions. With all its attributes, HA-ß-CD may be a promising cancer-cell-targeting drug carrier.
Assuntos
Ácido Hialurônico , beta-Ciclodextrinas , Doxorrubicina , Portadores de Fármacos , Sistemas de Liberação de MedicamentosRESUMO
Circulating tumor cells (CTCs) are indicative for cancer diagnosis and prognosis. However, conventional immuno-magnetic cell capture technologies using antibody- and aptamer-functionalized magnetic particles generate increased intracellular oxidative stress through endocytosis. Herein, we efficiently, selectively, and non-invasively isolate CTCs from whole blood by mimicking double-sided tape using DNA.
Assuntos
Aptâmeros de Nucleotídeos/química , Separação Celular/métodos , Células Neoplásicas Circulantes/metabolismo , Linhagem Celular Tumoral , DNA/química , Humanos , Nanopartículas de Magnetita/química , Microscopia Confocal , Células Neoplásicas Circulantes/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
The abilities of a drug delivery system to target and penetrate tumor masses are key factors in determining the system's chemotherapeutic efficacy. Here, we explored the utility of an anti-carbonic anhydrase IX (anti-CA IX) antibody and CPP33 dual-ligand modified triptolide-loaded liposomes (dl-TPL-lip) to simultaneously enhance the tumor-specific targeting and increase tumor cell penetration of TPL. In vitro, the dl-TPL-lip increased the cytotoxicity of TPL in CA IX-positive lung cancer cells, which showed tunable size (137.6 ± 0.8 nm), high-encapsulation efficiency (86.3 ± 2.6%) and sustained release. Dl-TPL-lip significantly improved the ability of liposomes to penetrate 3 D tumor spheroids and exhibited a superior inhibiting effect. Furthermore, pharmacokinetic studies in rats that received TPL liposomal formulations by endotracheal administration showed a reduced concentration of TPL (17.3%-30.6% compared to free TPL) in systemic circulation. After pulmonary administration in orthotopic lung tumor-bearing mice, dl-TPL-lip significantly enhanced TPL anti-cancer efficacy without apparent systemic toxicity. This dual-ligand modified liposomal vehicle presents a potential system for localized and targeted delivery of anti-cancer drugs to improve their efficacy.
Assuntos
Anticorpos/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Peptídeos Penetradores de Células/química , Lipossomos/química , Neoplasias Pulmonares/tratamento farmacológico , Células A549 , Animais , Antineoplásicos/farmacocinética , Anidrase Carbônica IX/metabolismo , Linhagem Celular Tumoral , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Humanos , Ligantes , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Tamanho da Partícula , Ratos , Ratos Sprague-Dawley , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismoRESUMO
Optical biosensors have great significance for rapid and sensitive detection and monitoring of biomolecular interactions. Here, recent advancement in the nano-functionalized long-period fiber grating (LPFG) is used to develop a cost-effective approach for label-free and real-time detection of the carbonic anhydrase-IX (CA9) monoclonal antibody, a diagnostic biomarker in hypoxia condition cultured carcinoma cells.
RESUMO
BACKGROUND: Repeated administrations of insulin injection on daily basis evoke pain and numerous complications with adverse effects on the diabetic patients' life quality. Moreover, wearing insulin pump is also associated with several problems of diabetic ketoacidosis, catheter site infection, contact dermatitis and high cost. METHOD: We have developed an in situ gel system, consisting of insulin-loaded liposomes dispersed within a thermoreversible gel (Pluronic® F127 gel), which increases the duration of insulin action for the treatment of diabetes. Vesicular phospholipid gel technique was used to encapsulate the insulin into liposomes. RESULTS: The resulting liposomal gel formulation had a longer drug-release period in vitro than a free insulin solution or liposomes and Pluronic® F127 gel individually. Furthermore, the addition of liposomes to the Pluronic® F127 gel improved the stability of the encapsulated insulin at a physiological temperature. In vivo study was performed to investigate the bioactivity and absorption of insulin released from the liposomal gel and other formulations. The liposomal gel released insulin into the bloodstream continuously for up to 7 days and significantly enhanced drug bioavailability compared to insulin released from liposomes or Pluronic® F127 gel individually. Blood glucose levels were reduced for up to 4 days. Histology data demonstrated excellent biocompatibility of the Pluronic® F127 gel-based delivery systems, with no observable inflammatory response in rat subcutaneous tissues. CONCLUSION: Obtained results show that the insulin-loaded liposomes dispersed within Pluronic® F127 gel can be used as a long-acting drug delivery system, and replacement for conventional insulin therapy.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Hidrogéis/química , Insulina/uso terapêutico , Poloxâmero/química , Temperatura , Animais , Temperatura Corporal , Diabetes Mellitus Experimental/induzido quimicamente , Modelos Animais de Doenças , Injeções Subcutâneas , Insulina/administração & dosagem , Lipossomos/administração & dosagem , Lipossomos/química , Masculino , Ratos , Ratos Sprague-Dawley , EstreptozocinaRESUMO
Antibody-decorated liposomes can facilitate the precise delivery of chemotherapeutic drugs to the lung by targeting a recognition factor present on the surface of lung tumor cells. Carbonic anhydrase IX (CA IX) is an enzyme expressed on the surface of lung cancer cells with a restricted expression in normal lungs. Here, we explored the utility of anti-carbonic anhydrase IX (CA IX) antibody, conjugated to the surface of triptolide (TPL)-loaded liposomes (CA IX-TPL-Lips), to promote the therapeutic effects for lung cancer via pulmonary administration. It was found that the CA IX-TPL-Lips significantly improved the cellular uptake efficiency in both CA IX-positive human non-small cell lung cancer cells (A549) and A549 tumor spheroids, resulting in the efficient cell killing compared with free TPL and non-targeted TPL-Lips. In vivo, CA IX-Lips via pulmonary delivery showed specificity and a sustained release property resided up to 96 h in the lung, both of which improved the efficiency of TPL formulations in restraining tumor growth and significantly prolonged the lifespan of mice with orthotopic lung tumors. The results suggest that CA IX-decorated liposomes can potentially be used as an effective therapeutic strategy for lung cancer.