RESUMO
Effective intracellular DNA transfection is imperative for cell-based therapy and gene therapy. Conventional gene transfection methods, including biochemical carriers, physical electroporation and microinjection, face challenges such as cell type dependency, low efficiency, safety concerns, and technical complexity. Nanoneedle arrays have emerged as a promising avenue for improving cellular nucleic acid delivery through direct penetration of the cell membrane, bypassing endocytosis and endosome escape processes. Nanostraws (NS), characterized by their hollow tubular structure, offer the advantage of flexible solution delivery compared to solid nanoneedles. However, NS struggle to stably self-penetrate the cell membrane, resulting in limited delivery efficiency. Coupling with extra physiochemical perforation strategies is a viable approach to improve their performance. This study systematically compared the efficiency of NS coupled with polyethylenimine (PEI) chemical modification, mechanical force, photothermal effect, and electric field on cell membrane perforation and DNA transfection. The results indicate that coupling NS with PEI modification, mechanical force, photothermal effects provide limited enhancement effects. In contrast, NS-electric field coupling significantly improves intracellular DNA transfection efficiency. This work demonstrates that NS serve as a versatile platform capable of integrating various physicochemical strategies, while electric field coupling stands out as a form worthy of primary consideration for efficient DNA transfection.
Assuntos
DNA , Eletroporação , Transfecção , Membrana Celular , Terapia Genética , Polietilenoimina/químicaRESUMO
In vitro, drug assessment holds tremendous potential to success in novel drug development and precision medicine. Traditional techniques for drug assessment, however, face remarkable challenges to achieve high speed, as limited by incubation-based drug delivery (>several hours) and cell viability measurements (>1 d), which significantly compromise the efficacy in clinical trials. In this work, a nano-electroporation-DNA tensioner platform is reported that shortens the time of drug delivery to less than 3 s, and that of cellular mechanical force analysis to 30 min. The platform adopts a nanochannel structure to localize a safe electric field for cell perforation, while enhancing delivery speed by 103 times for intracellular delivery, as compared to molecular diffusion in coculture methods. The platform is further equipped with a DNA tensioner to detect cellular mechanical force for quantifying cell viability after drug treatment. Systematic head-to-head comparison, by analyzing FDA (food and drug administration)-approved drugs (paclitaxel, doxorubicin), demonstrated the platform with high speed, efficiency, and safety, showing a simple yet powerful tool for clinical drug screening and development.
Assuntos
Sistemas de Liberação de Medicamentos , Eletroporação , Estados Unidos , Eletroporação/métodos , Terapia com Eletroporação , Difusão , DNARESUMO
Chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising cell-based immunotherapy approach for treating blood disorders and cancers, but genetically engineering CAR-T cells is challenging due to primary T cells' sensitivity to conventional gene delivery approaches. The current viral-based method can typically involve significant operating costs and biosafety hurdles, while bulk electroporation (BEP) can lead to poor cell viability and functionality. Here, a non-viral electroactive nanoinjection (ENI) platform is developed to efficiently negotiate the plasma membrane of primary human T cells via vertically configured electroactive nanotubes, enabling efficient delivery (68.7%) and expression (43.3%) of CAR genes in the T cells, with minimal cellular perturbation (>90% cell viability). Compared to conventional BEP, the ENI platform achieves an almost threefold higher CAR transfection efficiency, indicated by the significantly higher reporter GFP expression (43.3% compared to 16.3%). By co-culturing with target lymphoma Raji cells, the ENI-transfected CAR-T cells' ability to effectively suppress lymphoma cell growth (86.9% cytotoxicity) is proved. Taken together, the results demonstrate the platform's remarkable capacity to generate functional and effective anti-lymphoma CAR-T cells. Given the growing potential of cell-based immunotherapies, such a platform holds great promise for ex vivo cell engineering, especially in CAR-T cell therapy.
Assuntos
Linfoma , Receptores de Antígenos de Linfócitos T , Humanos , Linfócitos T , Transfecção , Eletroporação , Linfoma/metabolismoRESUMO
Cell perforation is a critical step for intracellular drug delivery and real-time biosensing of intracellular signals. In recent years, the nanostraws system has been developed to achieve intracellular drug delivery with minimal invasiveness to the cells. Repeated cell perforation via nano-system could allow delivery of multiple drugs into cells for cell editing, but the biosafety is rarely explored. In this work, a nanostraw-mediated nano-electroporation system was developed, which allowed repeated perforation of the same set of cells in a minimally invasive manner, while the biosafety aspect of this system was investigated. Highly controllable fabrication of Al2O3 nanostraw arrays based on a porous polyethylene terephthalate (PET) membrane was integrated with a microfluidic device to construct the nanostraw-electroporation system. The pulse conditions and intervals of nano-electroporation were systematically optimized to achieve efficient cells perforation and maintain the viability of the cells. The cells proliferation, the early apoptosis activities after nanostraw-electroporation and the changes of gene functions and gene pathways of cells after repeated nano-electroporation were comprehensively analyzed. These results revealed that the repeated nanostraw-electroporation did not induce obvious negative effects on the cells. This work demonstrates the feasibility of repeated nano-electroporation on cells and provides a promising strategy for future biomedical applications.
Assuntos
Nanoestruturas , Contenção de Riscos Biológicos , Eletroporação/métodos , Dispositivos Lab-On-A-Chip , Preparações FarmacêuticasRESUMO
Probing nuclear protein expression while correlating cellular behavior is crucial for deciphering underlying causes of cellular disorders, such as tumor metastasis. Despite efforts to access nuclear proteins by trafficking the double barriers of cell membrane and nuclear membrane, they mostly fall short of the capacity for analyzing various proteins in different cells. Herein, we introduce a Companion-Probe & Race (CPR) platform that enables interrogating nuclear proteins in living cells, while guiding and tracking cellular behaviors (e.g., migration) in real time. The Companion-Probe consists of two polypeptide complexes that were structured with nuclear localization signal (NLS) for entering nucleus, recognition polypeptide for targeting different sites of nuclear proteins, and fragments of green fluorescent protein (GFP) that can recover a whole fluorescent GFP once the two polypeptide complexes combine with a same target protein. The two polypeptide complexes were expressed by two plasmids (named "probe plasmids") that were uniformly and efficiently delivered into cells by nano-electroporation (NEP), a high-performance delivery method for cell focal-poration and accelerated intracellular delivery. To track cell migration, multiple radial microchannels were designed with micro-landmarks on the platform to serve as addressable runways for cells. The proof-of-concept of CPR platform was validated with clinical primary cells that indicated the positive-correlation between nuclear protein murine double minute 2 (MDM2) expression level and cell migration velocity. This platform shows great promises to interrogate nuclear proteins in live cells, and to decode their roles in determining cellular behaviors on a chip.
Assuntos
Técnicas Biossensoriais , Proteínas Nucleares , Animais , Núcleo Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/metabolismoRESUMO
Transfection is a critical step for gene editing and cell-based therapies. Nanoscale technologies have shown great promise to provide higher transfection efficiency and lower cell perturbation than conventional viral, biochemical and electroporation techniques due to their small size and localized effect. Although this has significant implications for using cells post-transfection, it has not been thoroughly studied. Here, we developed the nano-electro-injection (NEI) platform which makes use of localized electric fields to transiently open pores on cell membrane followed by electrophoretic delivery of DNA into cells. NEI provided two-folds higher net transfection efficiency than biochemicals and electroporation in Jurkat cells. Analysis of cell doubling time, intracellular calcium levels and mRNA expression changes after these gene delivery methods revealed that viruses and electroporation adversely affected cell behavior. Cell doubling times increased by more than 40% using virus and electroporation methods indicative of higher levels of cell stress, unlike NEI which only minimally affected cell division. Finally, electroporation, but not NEI, greatly altered the expression of immune-associated genes related to immune cell activation and trafficking. These results highlight that nanoscale delivery tools can have significant advantages from a cell health perspective for cell-based research and therapeutic applications.