Dimethylaminoethyl methacrylate copolymer-siRNA nanoparticles for silencing a therapeutically relevant gene in macrophages

Therapeutic gene silencing using small-interfering RNA (siRNA) for treatment of bacterial infections has been neglected in comparison with cancer and viral infections. The aim of our investigation was to formulate siRNA-loaded nanoparticles, using an established cationic polymethacrylate polymer, to enhance the delivery of siRNA into the cytoplasm of macrophages that host many pathogenic bacterial species, including tuberculosis. Nanoparticles of cationic dimethylaminoethyl methacrylate copolymer (Eudragit[registered sign] E 100) were successfully formulated and were found to efficiently bind the siRNA molecules (Cy3-siRNA, Bfl1/A1 siRNA). The efficiency of nanoparticles in overcoming cellular barriers to intracellular siRNA delivery and the precise pathway of endocytosis of nanoparticles were both confirmed using confocal microscopy. Through efficient siRNA release into the cytoplasm, the siRNA-loaded nanoparticles enabled a five-fold enhancement in the knockdown efficiency of the endogenous host gene Bfl1/A1, when the formulation was compared with free siRNA. Persistence of Bfl1/A1 is useful for phagolysosomal survival of tuberculosis bacteria in macrophages, and the nanoparticles offer a promising concept for exploitation as an anti-tuberculosis therapy

Alternative Approach to Synthesize Methylated Chitosan Using Deep Eutectic Solvents, Biocatalyst and “Green” Methylating Agents

Conventional synthesis of N-methylated chitosan involves the use of organic solvents in alkaline conditions, using methyl iodide as the methylating agent. However, the method does not result in selective N-methylation and is known to cause heavy polymer scission. In this investigation, we have reported alternative “green” approaches for methylated chitosan synthesis. Two types of deep eutectic solvents (DESs), viz., DES­(Urea) and DES­(Gly), either alone or in combination with other solvents, were screened as media for facilitating the methylation of chitosan. Our results indicated that DESs mediated selective N-methylation in the absence of NaOH, with no polymer scission, when compared with the reported methods. ¹H NMR spectra confirmed selective N-methylation in the case of products obtained using DES­(Urea), while that obtained using DES­(Gly) demonstrated some O-methylation. Another green method investigated the unexplored property of biocatalyst lipase for methylating chitosan in the presence of “green” methylating agents in DESs systems. Furthermore, lipase from <i>Burkhorlderia</i> species exhibited ability to methylate the chitosan polymer, while the enzyme from <i>Candida Antartica</i> failed to methylate the polymer. Our investigation also confirmed the possibility of using dimethyl carbonate as a benign methylating agent

Three-dimensional spheroids of choroid-retinal vascular endothelial cells as an in-vitro model for diabetic retinopathy: Proof-of-concept investigation

Diabetic retinopathy (DR) is a primary microvascular complication of diabetes mellitus and a vision-threatening condition. Vascular endothelial growth factor (VEGF) induces neovascularization and causes metabolic damage to the retinal and choroidal vasculature in diabetic patients. Existing drug screening models and treatment strategies for DR need to be refined through the establishment of relevant pre-clinical models, which may enable development of effective and safe therapies. The present study discusses the development of an in-vitro three-dimensional (3D) spheroid model, using RF/6A choroid-retinal vascular endothelial cells, to closely mimic the in-vivo disease condition. Compact, reproducibly-sized, viable and proliferating RF/6A spheroids were fabricated, as confirmed by microscopy, live/dead assay, cell proliferation assay and histological staining. In-vitro angiogenesis was studied by evaluating individual effects of VEGF and an anti-VEGF monoclonal antibody, Bevacizumab, and their combination on cellular proliferation and 3D endothelial sprout formation. VEGF stimulated angiogenic sprouting while Bevacizumab demonstrated a dose-dependent anti-angiogenic effect, as determined from the cellular proliferation observed and extent and length of sprouting. These investigations validated the potential of RF/6A spheroids in providing an alternative-to-animal, pathophysiologically-relevant model to facilitate pre-clinical and biomedical research related to DR

Starch based nanofibrous scaffolds for wound healing applications

Starch is an attractive polymer for wound healing applications because of its wide availability, low cost, biocompatibility, biodegradability and wound-healing property. Here, we have fabricated starch-based nanofibrous scaffolds by electrospinning for wound healing applications. The diameter of the optimized nanofibers was determined by field emission scanning electron microscopy (FE-SEM) and was found to be in the range of 110–300 nm. The mechanical strength (0.5–0.8 MPa) of the nanofibrous scaffolds was attuned using polyvinyl alcohol (plasticizer) and glutaraldehyde (crosslinking agent), to impart them with sufficient durability for skin tissue engineering. Absence of negative interactions between the polymers was confirmed by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), differential scanning microscopy (DSC) and thermal gravimetric analysis (TGA). Cellular assays with L929 mouse fibroblast cells indicated the ability of the scaffolds to promote cellular proliferation, without exhibiting any toxic effect to the cells. Thus, the nanofibrous scaffolds demonstrated potential for wound healing applications. Keywords: Starch, Nanofibers, Electrospinning, Scaffold, Wound healin

Curcumin-loaded hydrogel nanoparticles: application in anti-malarial therapy and toxicological evaluation

The present investigation involved preparation of hydrogel nanoparticles using a combination of hydroxyl propyl methyl cellulose and polyvinyl pyrrolidone. The objective was to exploit the size and hydrophilic nature of the formulated nanocarriers to enhance absorption and prolong the rapid clearance of curcumin due to possible evasion of the reticulo-endothelial system. Reproducible nanoparticles of size around 100nm, a fairly narrow distribution and encapsulation efficiency of 72%, were produced by the solvent emulsion-evaporation technique. This optimized system was further subjected to freeze-drying. The freeze-dried product was readily reconstituted with distilled water. The reconstituted product exhibited a size and distribution similar to that before freeze-drying, drug content of greater than 99% and presence of amorphous drug when analyzed by differential scanning calorimetry (DSC) which may result in possible improved absorption of curcumin. In vivo anti-malarial studies revealed significant superior action of nanoparticles over curcumin control suggesting the possibility of the formulation being employed as an adjunct anti-malarial therapy along with the standard therapy. Acute and subacute toxicity studies confirmed the oral safety of the formulation. A battery of genotoxicity studies was conducted to evaluate the nongenotoxic potential of the developed formulation thus indicating the possibility of the formulation being employed for prolonged duration

Cellular delivery of polynucleotides by cationic cyclodextrin polyrotaxanes.

Cationic polyrotaxanes, obtained by temperature activated threading of cationic cyclodextrin derivatives onto water-soluble cationic polymers (ionenes), form metastable nanometric polyplexes with pDNA and combinations of siRNA with pDNA. Because of their low toxicity, the polyrotaxane polyplexes constitute a very interesting system for the transfection of polynucleotides into mammalian cells. The complexation of Cy3-labeled siRNA within the polyplexes was demonstrated by fluorescence correlation spectroscopy. The uptake of the polyplexes (red) was imaged by confocal fluorescence microscopy using the A549 cell line as a model (blue: nuclei, green: membranes). The results prove the potential of polyrotaxanes for further investigations involving knocking down genes of therapeutic interest