Successful large gene augmentation of USH2A with non-viral episomal vectors.

USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene augmentation is a valuable therapeutic strategy for treating many inherited retinal diseases; however, conventional adeno-associated virus (AAV) gene therapy cannot accommodate cDNAs exceeding 4.7 kb, such as the 15.6-kb-long USH2A coding sequence. In the present study, we adopted an alternative strategy to successfully generate scaffold/matrix attachment region (S/MAR) DNA plasmid vectors containing the full-length human USH2A coding sequence, a GFP reporter gene, and a ubiquitous promoter (CMV or CAG), reaching a size of approximately 23 kb. We assessed the vectors in transfected HEK293 cells and USH2A patient-derived dermal fibroblasts in addition to ush2au507 zebrafish microinjected with the vector at the one-cell stage. pS/MAR-USH2A vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Twelve months of GFP expression was detected in the photoreceptor cells, with rescue of Usher 2 complex localization in the photoreceptors of ush2au507 zebrafish retinas injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector that can be used to express full-length usherin with functional rescue. S/MAR DNA vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development.

A multifunctional nanocomplex for enhanced cell uptake, endosomal escape and improved cancer therapeutic effect.

AIM: To evaluate the chemotherapeutic potential of a novel multifunctional nanocomposite encapsulating both porous silicon (PSi) and gold (Au) nanoparticles in a polymeric nanocomplex. MATERIALS & METHODS: The nanocomposite was physicochemically characterized and evaluated in vitro for biocompatibility, cellular internalization, endosomolytic properties, cytoplasmatic drug delivery and chemotherapeutic efficacy. RESULTS: The nanocomposites were successfully produced and exhibited adequate physicochemical properties and superior in vitro cyto- and hemocompatibilities. The encapsulation of PSi nanoparticles in the nanocomplexes significantly enhanced their cellular internalization and enabled their endosomal escape, resulting in the efficient cytoplasmic delivery of these nanosystems. Sorafenib-loaded nanocomposites showed a potent in vitro antiproliferative effect on MDA-MB-231 breast cancer cells. CONCLUSION: The multifunctional nanocomposite herein presented exhibits great potential as a chemotherapeutic nanoplatform.

In vitro and in vivo assessment of heart-homing porous silicon nanoparticles.

Chronic heart failure, predominantly developed after myocardial infarction, is a leading cause of high mortality worldwide. As existing therapies have still limited success, natural and/or synthetic nanomaterials are emerging alternatives for the therapy of heart diseases. Therefore, we aimed to functionalize undecylenic acid thermally hydrocarbonized porous silicon nanoparticles (NPs) with different targeting peptides to improve the NP's accumulation in different cardiac cells (primary cardiomyocytes, non-myocytes, and H9c2 cardiomyoblasts), additionally to investigate the behavior of the heart-targeted NPs in vivo. The toxicity profiles of the NPs evaluated in the three heart-type cells showed low toxicity at concentrations up to 50 µg/mL. Qualitative and quantitative cellular uptake revealed a significant increase in the accumulation of atrial natriuretic peptide (ANP)-modified NPs in primary cardiomyocytes, non-myocytes and H9c2 cells, and in hypoxic primary cardiomyocytes and non-myocytes. Competitive uptake studies in primary cardiomyocytes showed the internalization of ANP-modified NPs takes place via the guanylate cyclase-A receptor. When a myocardial infarction rat model was induced by isoprenaline and the peptide-modified [(111)In]NPs administered intravenously, the targeting peptides, particularly peptide 2, improved the NPs' accumulation in the heart up to 3.0-fold, at 10 min. This study highlights the potential of these peptide-modified nanosystems for future applications in heart diseases.

In vitro assessment of biopolymer-modified porous silicon microparticles for wound healing applications.

The wound healing stands as very complex and dynamic process, aiming the re-establishment of the damaged tissue's integrity and functionality. Thus, there is an emerging need for developing biopolymer-based composites capable of actively promoting cellular proliferation and reconstituting the extracellular matrix. The aims of the present work were to prepare and characterize biopolymer-functionalized porous silicon (PSi) microparticles, resulting in the development of drug delivery microsystems for future applications in wound healing. Thermally hydrocarbonized PSi (THCPSi) microparticles were coated with both chitosan and a mixture of chondroitin sulfate/hyaluronic acid, and subsequently loaded with two antibacterial model drugs, vancomycin and resveratrol. The biopolymer coating, drug loading degree and drug release behavior of the modified PSi microparticles were evaluated in vitro. The results showed that both the biopolymer coating and drug loading of the THCPSi microparticles were successfully achieved. In addition, a sustained release was observed for both the drugs tested. The viability and proliferation profiles of a fibroblast cell line exposed to the modified THCPSi microparticles and the subsequent reactive oxygen species (ROS) production were also evaluated. The cytotoxicity and proliferation results demonstrated less toxicity for the biopolymer-coated THCPSi microparticles at different concentrations and time points comparatively to the uncoated counterparts. The ROS production by the fibroblasts exposed to both uncoated and biopolymer-coated PSi microparticles showed that the modified PSi microparticles did not induce significant ROS production at the concentrations tested. Overall, the biopolymer-based PSi microparticles developed in this study are promising platforms for wound healing applications.

Amine-modified hyaluronic acid-functionalized porous silicon nanoparticles for targeting breast cancer tumors.

Active targeting of nanoparticles to receptor-overexpressing cancer cells has great potential for enhancing the cellular uptake of nanoparticles and for reducing fast clearance of the nanoparticles from the body. Herein, we present a preparation method of a porous silicon (PSi)-based nanodelivery system for breast cancer targeting, by covalently conjugating a synthesized amide-modified hyaluronic acid (HA(+)) derived polymer on the surface of undecylenic acid-modified thermally hydrocarbonized PSi (UnTHCPSi) nanoparticles. The resulting UnTHCPSi-HA(+) nanoparticles showed relatively small size, reduced polydispersibility, high biocompatibility, improved colloidal and human plasma stability, as well as enhanced cellular interactions and internalization. Moreover, we demonstrated that the enhanced cellular association of UnTHCPSi-HA(+) relies on the capability of the conjugated HA(+) to bind and consequently target CD44 receptors expressed on the surface of breast cancer cells, thus making the HA(+)-functionalized UnTHCPSi nanoparticles a suitable and promising nanoplatform for the targeting of CD44-overexpressing breast tumors and for drug delivery.

Augmented cellular trafficking and endosomal escape of porous silicon nanoparticles via zwitterionic bilayer polymer surface engineering.

The development of a stable vehicle with low toxicity, high cellular internalization, efficient endosomal escape, and optimal drug release profile is a key bottleneck in nanomedicine. To overcome all these problems, we have developed a successful layer-by-layer method to covalently conjugate polyethyleneimine (PEI) and poly(methyl vinyl ether-co-maleic acid) (PMVE-MA) copolymer on the surface of undecylenic acid functionalized thermally hydrocarbonized porous silicon nanoparticles (UnTHCPSi NPs), forming a bilayer zwitterionic nanocomposite containing free positive charge groups of hyper-branched PEI disguised by the PMVE-MA polymer. The surface smoothness, charge and hydrophilicity of the developed NPs considerably improved the colloidal and plasma stabilities via enhanced suspensibility and charge repulsion. Furthermore, despite the surface negative charge of the bilayer polymer-conjugated NPs, the cellular trafficking and endosomal escape were significantly increased in both MDA-MB-231 and MCF-7 breast cancer cells. Remarkably, we also showed that the conjugation of surface free amine groups of the highly toxic UnTHCPSi-PEI (Un-P) NPs to the carboxylic groups of PMVE-MA renders acceptable safety features to the system and preserves the endosomal escape properties via proton sponge mechanism of the free available amine groups located inside the hyper-branched PEI layer. Moreover, the double layer protection not only controlled the aggregation of the NPs and reduced the toxicity, but also sustained the drug release of an anticancer drug, methotrexate, with further improved cytotoxicity profile of the drug-loaded particles. These results provide a proof-of-concept evidence that such zwitterionic polymer-based PSi nanocomposites can be extensively used as a promising candidate for cytosolic drug delivery.

Poly(methyl vinyl ether-alt-maleic acid)-functionalized porous silicon nanoparticles for enhanced stability and cellular internalization.

Currently, developing a stable nanocarrier with high cellular internalization and low toxicity is a key bottleneck in nanomedicine. Here, we have developed a successful method to covalently conjugate poly(methyl vinyl ether-co-maleic acid) (PMVE-MA) copolymer on the surface of (3-aminopropyl)triethoxysilane-functionalized thermally carbonized porous silicon nanoparticles (APSTCPSi NPs), forming a surface negatively charged nanovehicle with unique properties. This polymer conjugated NPs could modify surface smoothness, charge, and hydrophilicity of the developed NPs, leading to considerable improvement in the colloidal and plasma stabilities via enhanced suspensibility and charge repulsion. Furthermore, despite the surface negative charge of the polymer-conjugated NPs, the cellular internalization was increased in both MDA-MB-231 and MCF-7 breast cancer cells. These results provide a proof-of-concept evidence that such polymer-based PSi nanocomposite can be extensively used as a promising candidate for intracellular drug delivery.

The mechanisms of surface chemistry effects of mesoporous silicon nanoparticles on immunotoxicity and biocompatibility.

Despite steadily increasing insights on the biocompatibility of PSi nanoparticles (NPs), an extensive biosafety study on the immune and red blood cells (RBCs) is still lacking. Herein, we evaluated the impact of the PSi NPs' surface chemistry on immune cells and human RBCs both in vitro and in vivo. Negatively charged hydrophilic and hydrophobic PSi NPs caused less ATP depletion and genotoxicity than the positively charged amine modified hydrophilic PSi NPs, demonstrating the main role of PSi NPs' surface charge on the immunocompatibility profile. Furthermore, cells with lower metabolic activity, longer doubling time, and shorter half-life were more sensitive to the concentration- and time-dependent toxicity in the following order: T-cells ≈ monocytes > macrophages ≈ B-cells. RBC hemolysis and imaging assay revealed a significant correlation between the surface chemistry, the amount of the PSi NPs adsorbed on the cell surface and the extent of morphological changes. The in vivo results showed that despite mild renal steatosis, glomerular degeneration, hepatic central vein dilation and white pulp shrinkage in spleen, no notable changes were observed in the serum level of biochemical and hematological factors. This study is a comprehensive demonstration of the mechanistic direct and indirect genotoxicity effects of PSi NPs, elucidating the most influencing properties for the PSi NPs' design.