(291f) pH-Responsive, Cell-Penetrating, Magnetite-Silver Nanoparticles for Delivery of Plasmid-Based Gene Therapies: Preparation, Characterization, and in Vitro Evaluation
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Here, we propose to develop a nanostructured core-shell cell-penetrating vehicle composed of magnetite at the core and surrounded by a silver shell. A subsequent superficial conjugation of a pH-responsive polymer (poly(2-dimethylamino) ethyl methacrylate, pDMAEMA) was proposed to assure that the vehicle is capable of carrying and releasing circular DNA. Finally, Buforin II was conjugated to the vehicle to facilitate translocation and endosomal escape as well as reaching the nucleus5. The prepared core-shell system exhibited colloidal stability such that agglomerate formation and thereby precipitation was avoided. Besides the role of DNA carrier, the pDMAEMA was conjugated to attempt to avoid endosome formation, which is a cell internalization mechanism that traps exogenous material and consequently might lead to a significant reduction in transfection rate4. Buforin II was immobilized via the organosilane molecule aminopropyl 3-ethoxy silane and the surface spacer polyether amine (PEA) (Jeffamine 600) by following a protocol described by Cuellar et al5. Finally, a 10kb circular DNA vector was loaded on the modified nanoparticles at pH values in the range of 6 to 8.5 to estimate the loading efficiency and the loading maximum capacity. The designed vector contained information for a Cas9 enzyme, a CRISPR gRNA, and a mCherry reporter. This is an attempt to explore the suitability of the vehicle in the delivery of gene editing systems. The release of DNA was also tested in vitro, by exposing the loaded systems to a medium with pH values in the range of 8.5 to 6. The obtained nanobioconjugates were characterized by UV-Vis spectrophotometer, dynamic light scattering (DLS), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), scanning electron microscope equipped with energy dispersive spectroscopy (SEM+EDS), Transmission Electron Microscopy (TEM), and DLS. Moreover, the conjugates were tested in neuroblastoma (SH-SY5Y) and Vero cells to evaluate endosomal escape and cytotoxicity via LDH. Finally, hemolytic tendency and platelet aggregation assays were conducted to complete the biocompatibility analysis and therefore estimate the clinical potential of the vehicle.
The results indicate that the average hydrodynamic diameter of the obtained nanobioconjugate is 478 nm. SEM and TEM images revealed some unevenness of the Silver coating on nanoparticles but the crystalline structure corresponding to magnetite at the core. According to the TEM images, individual nanobioconjugates approached 10.4 nm in diameter. TGA analysis confirmed a conjugation efficiency of pDMAEMA of about 8.8% and of about 3% for Buforin II. The FTIR confirmed the characteristic bands of the polymer as well as those of Buforin II. The vehicle was capable of loading 35 ng of DNA per microgram of nanoparticles and it loads almost 16% of the added DNA while releasing around 8% of that amount. Figure 1 shows that cell viability remains above 95% for the pDMAEMA conjugated to the core@shell system (magnetite@silver-pDMAEMA) as well as for the vehicle with the additional conjugation of Buforin II (magnetite@silver-pDMAEMA-BUFII). This was also the case for the hemolysis assay where the release of hemoglobin was below 5% in both cases. To evaluate endosomal escape, the nanobioconjugates were fluorescently-labeled with Rhodamine B (magnetite@silver-pDMAEMA-BUFII-RhodB), delivered to SH-SY5Y, and subsequently incubated for 30 minutes and 4 hours. Pearsonâs correlation coefficients were calculated to evaluate the colocalization of the nanobioconjugates with the endosomes (labeled with Lysotracker Green). The results suggest an endosomal escape of about 80% for SH-SY5Y and 60% for Vero cells. Further evidence of escape and even cytosol distribution was provided by a calculated surface area coverage of about 50% for both cell lines.
Our findings regarding the possibilities of the vehicle to load and release of DNA, cell-penetrating and endosomal escape capacity as well as high biocompatibility, confers it with important chances for further in vivo testing. Also, it highlights a route to enable gene therapy applications that require large-size plasmid vectors such as those involved in CRISPR/Cas9 editing systems.
- Dai, W.-J. et al. CRISPR-Cas9 for in vivo Gene Therapy: Promise and Hurdles. Mol. Ther. - Nucleic Acids 5, e349 (2016).
- Ho, B. X., Loh, S. J. H., Chan, W. K. & Soh, B. S. In vivo genome editing as a therapeutic approach. International Journal of Molecular Sciences 19, (2018).
- Jin, S. & Ye, K. Nanoparticle-mediated drug delivery and gene therapy. in Biotechnology Progress 23, 32â41 (2007).
- Varadan, V. K. Nanomedicine : design and applications of magnetic nanomaterials, nanosensors, and nanosystems LK - https://univdelosandes.on.worldcat.org/oclc/916159742. TA - TT - (Wiley, 2008).
- Cuellar, M. et al. Novel BUF2-magnetite nanobioconjugates with cell-penetrating abilities. Int. J. Nanomedicine Volume 13, 8087â8094 (2018).
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