The administration of gene-editing tools has been proposed as a promising therapeutic approach for correcting mutations that cause diseases. Gene-editing tools, composed of relatively large plasmid DNA constructs that often need to be co-delivered with a guiding protein, are unable to spontaneously penetrate mammalian cells. Although viral vectors facilitate DNA delivery, they are restricted by the size of the plasmid to carry. In this work, we describe a strategy for the stable encapsulation of the gene-editing tool piggyBac transposon into Poly (β-amino ester) nanoparticles (NPs). We propose a non-covalent and a covalent strategy for stabilization of the nanoformulation to slow down release kinetics and enhance intracellular delivery. We found that the formulation prepared by covalently crosslinking Poly (β-amino ester) NPs are capable to translocate into the cytoplasm and nuclei of human glioblastoma (U87MG) cells within 1 h of co-culturing, without the need of a targeting moiety. Once internalized, the nanoformulation dissociates, delivering the plasmid presumably as a response to the intracellular acidic pH. Transfection efficiency is confirmed by green fluorescence protein (GFP) expression in U87MG cells. Covalently stabilized Poly (β-amino ester) NPs are able to transfect ~55% of cells causing non-cytotoxic effects. The strategy described in this work may serve for the efficient non-viral delivery of other gene-editing tools.
Gene editing has emerged as a therapeutic approach to manipulate the genome for killing cancer cells, protecting healthy tissues, and improving immune response to a tumor. The gene editing tool achaete-scute family bHLH transcription factor 1 CRISPR guide RNA (ASCL1-gRNA) is known to restore neuronal lineage potential, promote terminal differentiation, and attenuate tumorigenicity in glioblastoma tumors. Here, we fabricated a polymeric nonviral carrier to encapsulate ASCL1-gRNA by electrostatic interactions and deliver it into glioblastoma cells across a 3D in vitro model of the blood−brain barrier (BBB). To mimic rabies virus (RV) neurotropism, gene-loaded poly(β-amino ester) nanoparticles are surface functionalized with a peptide derivative of rabies virus glycoprotein (RVG29). The capability of the obtained NPs, hereinafter referred to as RV-like NPs, to travel across the BBB, internalize into glioblastoma cells, and deliver ASCL1-gRNA is investigated in a 3D BBB in vitro model through flow cytometry and CLSM microscopy. The formation of nicotinic acetylcholine receptors in the 3D BBB in vitro model is confirmed by immunochemistry. These receptors are known to bind to RVG29. Unlike Lipofectamine which primarily internalizes and transfects endothelial cells, RV-like NPs are capable to travel across the 3D BBB in vitro model, preferentially internalizing glioblastoma cells, and delivering ASCL1-gRNA at an efficiency of 10%, causing noncytotoxic effects.
The administration of exogenous DNA has been proposed as a promising therapeutic approach for a variety of diseases. Unfortunately, exogenous DNA is unable to spontaneously penetrate mammalian cells. Although viral vectors facilitate DNA delivery at high transfection efficiency, they are restricted for in vivo applications as they could potentially induce immunogenicity and mutagenesis. To overcome the clinical challenge of viral delivery, a strategy for the encapsulation of plasmid DNA on the surface of poly(lactide-co-glycolide) nanoparticles (PLGA NPs) is shown. Plasmid green fluorescence protein (pEF-GFP) or piggybac transposon (PBCAG-eGFP) are assembled on the surface of PLGA NPs through layer by layer technique. The assembly of pEF-GFP with biopolyelectrolytes is monitored on a planar support using a quartz crystal microbalance with dissipation. The assembly of the biopolymer multilayers on PLGA NPs is followed by ζ-potential measurements. Encapsulation of plasmid DNA within the multilayers coating is confirmed by gel electrophoresis. Cellular uptake studies on HEK293 cells revealed that PLGA NPs are taken up by cells within the first 5 hr of co-culturing. Intracellular release of cargo is confirmed by GFP expression in HEK293 cells. PLGA NPs encapsulating pEF-GFP on their surface are able to transfect~20% of HEK293 cells, while those encapsulating PBCAG-eGFP can transfect up to 75% of cells after 72 hr, causing minimum to non-cytotoxic effects. K E Y W O R D Sbiomedical applications, drug delivery systems, polyelectrolytes, self-assembly, surfaces and interfaces
Noninvasive manipulation of cell signaling is critical in basic neuroscience research and in developing therapies for neurological disorders and tissue engineering and regenerative medicine approaches. In this work, biomimetic synthesized conductive copolymer 3,4-ethylenedioxythiophene (EDOT)-Pyrrole nanoparticles (RB02 NPs) were used for wireless and localized stimulation of neurons. 1 H nuclear magnetic resonance was used to monitor the polymerization. RB02 NPs were characterized by Raman spectroscopy, Fourier transform infrared spectroscopy, and dynamic light scattering. The electrochemical properties were characterized by galvanostatic charge−discharge, voltammetry, and electrochemical impedance spectroscopy. For electrical stimulation of neurons, RB02 NPs were charged by applying 1 V to a NP suspension using platinum electrodes. The effect of NPs on ND7/23 neuron hybrid cell line viability was assessed by live/dead staining using flow cytometry. ND7/23 differentiation was evaluated by cell cytoskeleton staining and quantification of morphological parameters such as the dendrite number and length. Primary cortex neuron stimulation was studied by calcium ion influx detectable through the dynamic fluorescence changes of Fluo-4. RB02 NPs presented no toxicity toward ND7/23 cells. Furthermore, charged NPs enhanced cell differentiation at short times after addition (<6 h). Charged RB02 NPs largely increased the cortex neuronal activity. Altogether, biocompatible copolymer EDOT-Pyrrole nanoparticles present great potential for remote control of neural activities.
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