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

Abstract

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-PEGmaleimide (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.

A Multiple siRNA-Based Anti-HIV/SHIV Microbicide Shows Protection in Both In Vitro and In Vivo Models

“…Preclinical safety and efficacy testing of microbicide candidates involves a large number of in vitro , ex vivo , and in vivo assays and models. Here we demonstrated that chitosan-based nanoparticles showed no significant toxicity in rhesus monkeys, which is consistent with other modified chitosan nanoparticles for siRNA delivery [ 55 , 56 ]. Thus, the siRNA-based chlipid microbicides described in this study were subjected to a very cautious and rigorous preclinical evaluation to assess their efficacy in models used for microbicide development.…”

Nanotoxicity: A Key Obstacle to Clinical Translation of siRNA-Based Nanomedicine

“…For certain diseases, such as cancer, passive targeting based on an enhanced permeation and retention effect can increase tumor drug accumulation [113], however, the improvement is often moderate. Therefore, siRNA nanocarriers developed in recent years have often been functionalized with active-targeting moieties, such as mono-clonal antibodies, receptor substrates and cell-penetrating peptides [90,114–116]. The advantage of this active-targeting strategy is illustrated in the study of asymmetric liposome particles, which were designed with the positive charges on the inner surface to minimize the nonspecific cellular uptake [114].…”

Nanoalginates via Inverse-Micelle Synthesis: Doxorubicin-Encapsulation and Breast Cancer Cytotoxicity

“…Biopolymers have been used to encapsulate therapeutics in part due to their ease of use and their biocompatible nature [ 30 ]. Polymers used include alginate [ 31 , 32 ], heparin [ 33 , 34 ], chitosan [ 35 , 36 ], and carrageenan [ 37 , 38 ], among others. Alginate, a naturally derived polymer, is composed of varied amounts of β- d -mannuronate and α- l -guluronate residues linked by 1,4-glycosidic linkages [ 39 ] (M and G blocks, respectively).…”