Development and characterization of chitosan-PEG-TAT nanoparticles for the intracellular delivery of siRNA

Meenakshi Malhotra,1 Catherine Tomaro-Duchesneau,1 Shyamali Saha,2 Imen Kahouli,3 Satya Prakash11Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, Faculty of Medicine, 2Faculty of Dentistry, 3Department of Experimental Medicine, McGill University, Montreal, QC, CanadaAbstract: Recently, cell-penetrating peptides have been proposed to translocate antibodies, proteins, and other molecules in targeted drug delivery. The proposed study presents the synthesis and characterization of a peptide-based chitosan nanoparticle for small interfering RNA (siRNA) delivery, in-vitro. Specifically, the synthesis included polyethylene glycol (PEG), a hydrophilic polymer, and trans-activated transcription (TAT) peptide, which were chemically conjugated on the chitosan polymer. The conjugation was achieved using N-Hydroxysuccinimide-PEG-maleimide (heterobifunctional PEG) as a cross-linker, with the bifunctional PEG facilitating the amidation reaction through its N-Hydroxysuccinimide group and reacting with the amines on chitosan. At the other end of PEG, the maleimide group was chemically conjugated with the cysteine-modified TAT peptide. The degree of substitution on chitosan with PEG and on PEG with TAT was confirmed using colorimetric assays. The resultant polymer was used to form nanoparticles complexing siRNA, which were then characterized for particle size, morphology, cellular uptake, and cytotoxicity. The nanoparticles were tested in-vitro on mouse neuroblastoma cells (Neuro2a). Particle size and surface charge were characterized and an optimal pH condition and PEG molecular weight were determined to form sterically stable nanoparticles. Results indicate 7.5% of the amines in chitosan polymer were conjugated to the PEG and complete conjugation of TAT peptide was observed on the synthesized PEGylated chitosan polymer. Compared with unmodified chitosan nanoparticles, the nanoparticles formed at pH 6 were monodispersed and of <100 nm in size, exhibiting maximum cell transfection ability and very low cytotoxicity. Thus, this research may be of significance in translocating biotherapeutic molecules for intracellular delivery applications.Keywords: chemical conjugation, peptide, transfection, polymer, synthesis, in-vitr

Use of Artificial Cell Microcapsule Containing Thalidomide for Treating TNBS-induced Crohn's Disease in Mice

In this study, we examined the in-vivo characteristics of a novel microencapsulated thalidomide formulation in a murine model of experimental Crohn's disease. Crohn's disease was induced with a single intra-colonic injection of 120 mg/kg of bodyweight of 2,5,6-trinitrobenzene sulfonic acid (TNBS) dissolved in 30% ethanol in Balb/c mice. Level of tumor necrosis factor alpha (TNF-), interleukin one beta (IL-1), interleukin 6 (IL-6) and nitric oxide (NO) were measured in tissue homogenate. Moreover, myeloperoxidase (MPO) activity was determined to assess the extent of neutrophil infiltration. Dose response study showed that treating the mice with microencapsulated thalidomide (100 mg/kg of bodyweight) for two weeks significantly decreased the degree of intestinal inflammation related to Crohn’s disease. Higher and lower doses (0, 25, 50 and 200 mg/kg of bodyweight) did not exhibit comparable effects. The present study validates the success of alginate-poly-L-lysine-alginate (APA) microcapsules containing thalidomide in reducing colonic inflammation, and proposes a potential remedy for Crohn’s disease

Superluminescence and macroscopic exciton propagation in freestanding ZnO thin films

Recently we have reported properties of ZnO semiconductor films attached to CaF2 substrate. Presently we characterized absorption, emission and superluminescence of freestanding ZnO films, comparing these to the attached films with the same thickness values. The absorption spectra of the freestanding films had resolved bands, with the band density increasing with the nanofilm thickness. Additionally, the spectral transitions in these films were blue-shifted as compared to attached films. The absorption and emission spectra of freestanding films were resolved better than those of attached films, with the difference traceable to the surface roughness of the substrate used for deposition. We also explored exciton dynamics and propagation over macroscopic distances in freestanding films. The excitons lived longer and propagated further in freestanding films as compared to attached films. The superluminescence yield in freestanding 9.3 nm film of 0.43 +/- 0.05 was significantly larger than 0.17 +/- 0.03 in an equivalent attached film. We provided a detailed analysis of the results obtained. The reported data are unique, demonstrating significant difference in the optical properties of attached and freestanding ZnO thin films. Freestanding ZnO films are promising for optoelectmnic applications.PR NASA EPSCoR (NASA)National Aeronautics & Space Administration (NASA) [80NSSC19M0049]PR Space Grant (NASA) [NNX15AI11H]http://creativecommons.org/licenses/by/4.0