RESUMEN
Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) is a promising gene editing tool to treat diseases at the genetic level. Nonetheless, the challenge of the safe and efficient delivery of CRISPR/Cas9 to host cells constrains its clinical applicability. In the current study, a facile, redox-responsive CRISPR/Cas9-Ribonucleoprotein (RNP) delivery system by combining iron-coordinated aggregation with liposomes (Fe-RNP@L) is reported. The Fe-RNP is formed by the coordination of Fe3+ with amino and carboxyl groups of Cas9, which modifies the lipophilicity and surface charge of RNP and alters cellular uptake from primary endocytosis to endocytosis and cholesterol-dependent membrane fusion. RNP can be rapidly and reversibly released from Fe-RNP in response to glutathione without loss of structural integrity and enzymatic activity. In addition, iron coordination also improves the stability of RNP and substantially mitigates cytotoxicity. This construct enabled highly efficient cytoplasmic/nuclear delivery (≈90%) and gene-editing efficiency (≈70%) even at low concentrations. The high payload content, high editing efficiency, good stability, low immunogenicity, and ease of production and storage, highlight its potential for diverse genome editing and clinical applications.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Hierro , Oxidación-Reducción , Ribonucleoproteínas , Edición Génica/métodos , Hierro/química , Humanos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Liposomas/química , Técnicas de Transferencia de Gen , Proteína 9 Asociada a CRISPR/metabolismoRESUMEN
Plastic particle pollution poses an emerging threat to ecological and human health. Laboratory animal studies have illustrated that nano-sized plastics can accumulate in the testis and cause testosterone deficiency and spermatogenic impairment. In this study, TM3 mouse Leydig cells were in vitro exposed to polystyrene nanoparticles (PS-NPs, size 20 nm) at dosages of 50, 100 and 150 µg/mL to investigate their cytotoxicity. Our results demonstrated that PS-NPs can be internalized into TM3 Leydig cells and led to a concentration-dependent decline in cell viability. Furthermore, PS-NPs stimulation amplified ROS generation and initiated cellular oxidative stress and apoptosis. Moreover, PS-NPs treatment affected the mitochondrial DNA copy number and collapsed the mitochondrial membrane potential, accompanied by a disrupted energy metabolism. The cells exposed to PS-NPs also displayed a down-regulated expression of steroidogenesis-related genes StAR, P450scc and 17ß-HSD, along with a decrease in testosterone secretion. In addition, treatment with PS-NPs destructed plasma membrane integrity, as presented by increase in lactate dehydrogenase release and depolarization of cell membrane potential. In summary, these data indicated that exposure to PS-NPs in vitro produced cytotoxic effect on Leydig cells by inducing oxidative injury, mitochondrial impairment, apoptosis, and cytomembrane destruction. Our results provide new insights into male reproductive toxicity caused by NPs.
Asunto(s)
Células Intersticiales del Testículo , Nanopartículas , Ratones , Animales , Masculino , Humanos , Células Intersticiales del Testículo/metabolismo , Microplásticos/metabolismo , Poliestirenos/toxicidad , Plásticos/metabolismo , Nanopartículas/toxicidad , Nanopartículas/metabolismo , Testosterona/metabolismoRESUMEN
Immobilized enzymes outperform free enzymes in many properties and are widely used in environmental monitoring, engineering applications, food and medical fields. Based on the developed immobilization techniques, the search for immobilization with wider applicability, lower cost and more stable enzyme properties is of significant importance. In this study, we reported a molecular imprinting strategy for immobilizing peptide mimics of DhHP-6 on mesoporous materials. The DhHP-6 molecularly imprinted polymer (MIP) showed much higher adsorption capacity than raw mesoporous silica toward DhHP-6. The DhHP-6 peptide mimics was immobilized on the surface of mesoporous silica for the fast detection of phenolic compounds, a widely spread pollutant with highly toxic and difficult in degradation. Immobilized enzyme of DhHP-6-MIP exhibited higher peroxidase activity, better stability, and recyclability than free peptide. Notably, DhHP-6-MIP showed excellent linearity for the detection of the two phenols with detection limits of 0.28 µM and 0.25 µM, respectively. In combination with the spectral analysis and PCA method, DhHP-6-MIP provided better discrimination between the six phenolic compounds (phenol, catechol, resorcinol, hydroquinone, 2-chlorophenol, 2, 4-dichlorophenol). Our study showed that immobilization of peptide mimics by the molecular imprinting strategy using mesoporous silica as carriers was a simple and effective approach. The DhHP-6-MIP has great potentiality for the monitoring and degradation of environmental pollutants.
Asunto(s)
Impresión Molecular , Nanopartículas , Polímeros/química , Fenoles/química , Impresión Molecular/métodos , Polímeros Impresos Molecularmente , Péptidos , Dióxido de Silicio/química , AdsorciónRESUMEN
We investigate the structure of gluten polymer-like gels in a binary mixture of water/ethanol, 50/50 v/v, a good solvent for gluten proteins. Gluten comprises two main families of proteins, monomeric gliadins and polymer glutenins. In the semi-dilute regime, scattering experiments highlight two classes of behavior, akin to standard polymer solution and polymer gel, depending on the protein composition. We demonstrate that these two classes are encoded in the structural features of the proteins in very dilute solution, and are correlated with the presence of proteins assemblies of typical size tens of nanometers. The assemblies only exist when the protein mixture is sufficiently enriched in glutenins. They are found directly associated to the presence in the gel of domains enriched in non-exchangeable H-bonds and of size comparable to that of the protein assemblies. The domains are probed in neutron scattering experiments thanks to their unique contrast. We show that the sample visco-elasticity is also directly correlated to the quantity of domains enriched in H-bonds, showing the key role of H-bonds in ruling the visco-elasticity of polymer gluten gels.
Asunto(s)
Glútenes , Polímeros , Geles/química , Gliadina/química , Glútenes/química , Polímeros/química , Proteínas , ViscosidadRESUMEN
Polypeptide carriers have a good cell compatibility, rich functionality, and facile synthesis and modification, make them promising materials as siRNA vectors. Phenylalanine dipeptide (FF) has been previously assessed as an siRNA vector and showed to have two major drawbacks, namely poor water solubility and poor serum stability. Herein, the FF backbone was modified by ligating a PEG-Arg-Ala (PEG-RA) sequence at the N-terminus to increase its hydrophilicity and serum stability. Arg is a typical amino acid in the cell penetrating peptide, which can increase the efficiency of cell internalization. Ala acts as a spacer to avoid steric hindrance. The target sequence PEG-RAFF was synthesized by a solid phase peptide synthesis. The morphology, particle size, and siRNA ratio were assessed by SEM, TEM, DLS, and gel electrophoresis. Further, MCF-7 cells were used as a model and survivin-siRNA as a passenger to assess cell internalization, inhibition of gene expression rate, and apoptosis rate using confocal microscopy, real-time PCR, and flow cytometry. At a concentration of 1â¯mg/mL, PEG-RAFF took the form of nanovesicles with a diameter of 154.74⯱â¯14.36â¯nm. The optimal PEG-RAFF to siRNA ratio was N/Pâ¯=â¯100:1. Compared with the control group, the red fluorescence of TAMRA(Carboxytetramethylrhodamine, Red fluorescence)-siRNA transfected into cells was clearly visible in the confocal microscope image. The inhibition rate of survivin was 67.99⯱â¯10.31%, and the apoptotic rate was 16.07%. Therefore, PEG-RAFF has potential as an siRNA carrier in cancer treatment.