RESUMEN
CRISPR-Cas9 is a revolutionary genome-editing technology that has enormous potential for the treatment of genetic diseases. However, the lack of efficient and safe, non-viral delivery systems has hindered its clinical application. Here, we report on the application of polymeric and hybrid microcarriers, made of degradable polymers such as polypeptides and polysaccharides and modified by silica shell, for delivery of all CRISPR-Cas9 components. We found that these microcarriers mediate more efficient transfection than a commercially available liposome-based transfection reagent (>70% vs. <50% for mRNA, >40% vs. 20% for plasmid DNA). For proof-of-concept, we delivered CRISPR-Cas9 components using our capsules to dTomato-expressing HEK293T cells-a model, in which loss of red fluorescence indicates successful gene editing. Notably, transfection of indicator cells translated in high-level dTomato knockout in approx. 70% of transfected cells. In conclusion, we have provided proof-of-principle that our micro-sized containers represent promising non-viral platforms for efficient and safe gene editing.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Polímeros/química , Solanum lycopersicum/metabolismo , Portadores de Fármacos , Fluorescencia , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/antagonistas & inhibidores , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Solanum lycopersicum/genética , Dióxido de Silicio/químicaRESUMEN
Engineered calcium carbonate (CaCO3) particles are extensively used as drug delivery systems due to their availability, biological compatibility, biodegradability, and cost-effective production. The synthesis procedure of CaCO3 particles, however, suffers from poor reproducibility. Furthermore, reducing the size of CaCO3 particles to <100 nm requires the use of additives in the reaction, which increases the total reaction time. Here we propose on-chip synthesis and loading of nanoscaled CaCO3 particles using microfluidics. After the development and fabrication of a microfluidic device, we optimized the synthesis of CaCO3 NPs by varying different parameters such as flow rates in the microfluidic channels, concentration of reagents, and the reaction time. To prove the versatility of the used synthesis route, we performed single and double loading of CaCO3 NPs with various compounds (Doxorubicin, Cy5 or FITC conjugated with BSA, and DNA) using the same microfluidic device. Further, the on-chip loaded CaCO3 NPs were used as carriers to transfer compounds to model cells. We have developed a microfluidic synthesis method that opens up a new pathway for easy on-chip fabrication of functional nanoparticles for clinical use.
Asunto(s)
Carbonato de Calcio , Dispositivos Laboratorio en un Chip , Nanopartículas , Carbonato de Calcio/química , Nanopartículas/química , Doxorrubicina/química , Doxorrubicina/farmacología , Doxorrubicina/administración & dosificación , Humanos , Microfluídica/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Portadores de Fármacos/química , Tamaño de la Partícula , ADN/química , ADN/administración & dosificaciónRESUMEN
An important area in modern malignant tumor therapy is the optimization of antitumor drugs pharmacokinetics. The use of some antitumor drugs is limited in clinical practice due to their high toxicity. Therefore, the strategy for optimizing the drug pharmacokinetics focuses on the generation of high local concentrations of these drugs in the tumor area with minimal systemic and tissue-specific toxicity. This can be achieved by encapsulation of highly toxic antitumor drug (vincristine (VCR) that is 20-50 times more toxic than widely used the antitumor drug doxorubicin) into nano- and microcarriers with their further association into therapeutically relevant cells that possess the ability to migrate to sites of tumor. Here, we fundamentally examine the effect of drug carrier size on the behavior of human mesenchymal stem cells (hMSCs), including internalization efficiency, cytotoxicity, cell movement, to optimize the conditions for the development of carrier-hMSCs drug delivery platform. Using the malignant tumors derived from patients, we evaluated the capability of hMSCs associated with VCR-loaded carriers to target tumors using a three-dimensional spheroid model in collagen gel. Compared to free VCR, the developed hMSC-based drug delivery platform showed enhanced antitumor activity regarding those tumors that express CXCL12 (stromal cell-derived factor-1 (SDF-1)) gene, inducing directed migration of hMSCs via CXCL12 (SDF-1)/CXCR4 pathway. These results show that the combination of encapsulated antitumor drugs and hMSCs, which possess the properties of active migration into tumors, is therapeutically beneficial and demonstrated high efficiency and low systematic toxicity, revealing novel strategies for chemotherapy in the future.
Asunto(s)
Sistemas de Liberación de Medicamentos , Células Madre Mesenquimatosas/química , Neoplasias/tratamiento farmacológico , Vincristina/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimiocina CXCL12/genética , Colágeno/química , Colágeno/farmacología , Portadores de Fármacos/química , Portadores de Fármacos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Neoplasias/patología , Cultivo Primario de Células , Receptores CXCR4/genética , Transducción de Señal/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Vincristina/químicaRESUMEN
Stem cell engineering-the manipulation and functionalization of stem cells involving genetic modification-can significantly expand their applicability for cell therapy in humans. Toward this aim, reliable, standardized, and cost-effective methods for cell manipulation are required. Here we explore the potential of magnetic multilayer capsules to serve as a universal platform for nonviral gene transfer, stem cell magnetization, and magnetic cell separation to improve gene transfer efficiency. In particular, the following experiments were performed: (i) a study of the process of internalization of magnetic capsules into stem cells, including capsule co-localization with established markers of endo-lysosomal pathway; (ii) characterization and quantification of capsule uptake with confocal microscopy, electron microscopy, and flow cytometry; (iii) intracellular delivery of messenger RNA and separation of gene-modified cells by magnetic cell sorting (MACS); and (iv) analysis of the influence of capsules on cell proliferation potential. Importantly, based on the internalization of magnetic capsules, transfected cells became susceptible to external magnetic fields, which made it easy to enrich gene-modified cells using MACS (purity â¼95%), and also to influence their migration behavior. In summary, our results underline the high potential of magnetic capsules in stem cell functionalization, namely (i) to increase gene-transfer efficiency and (ii) to facilitate enrichment and targeting of transfected cells. Finally, we did not observe a negative impact of the capsules used on the proliferative capacity of stem cells, proving their high biocompatibility.