Boronate affinity nanoparticles for RNA isolation

Macromolecular Bioscience, 2006

Enzyme and Microbial Technology, 2012

Boronate affinity chromatography involves the interaction between cis-diol containing molecules and the hydroxyl group of boronate. Boronate affinity based cryogel chromatography matrices have been developed and the ligands were immobilized by two methods i.e., grafting of the boronate ligand on to the matrix and by copolymerization of monomer containing boronate with other co-monomer. The boronate grafted cryogel column was used to capture adherent and non-adherent cells and the captured cells were recovered at different fructose concentrations as an eluting agent, in chromatography mode. It was found that the adherent cells could be recovered at relatively higher fructose concentration (0.5 M) than non-adherent cells which could be recovered by using low fructose concentration (0.1 M). This might be due to the difference in the content of glycoprotein in adherent and non-adherent cells. In this way a new separation method can be devised for the fractionation of adherent and non-adherent cells. In another study, a copolymerized boronate cryogel column was developed for the separation of RNA from the bacterial crude extract without any pre-processing. RNA molecules were specifically retained in the cryogel column due to interaction between 2,3 diol group of ribose sugar in RNA and the hydroxyl group of boronate. The DNA molecules were passed through the column uninteracted due to absence of 2hydroxyl group. Later, bound RNA molecules were recovered from the boronate affinity cryogel column.

Pharmaceutics

We report a physiologically stable and cytocompatible glucose-responsive nonviral gene delivery system made up of boronate functionalized polymeric material. Herein, we utilize boronate cis-diol interactions to develop a glucose-responsive submicron particle (SMP) system. The stability of the boronate interaction at a physiological pH was achieved by copolymerization of dimethyl aminoethyl methacrylate (DMAEMA) with acrylamidophenylboronic acid (AAPBA) and the formation of a complex with polyvinylalcohol (PVA) which is governed by cis-diol interactions. The shift in hydrodynamic diameter of SMPs was observed and correlated with increasing glucose concentrations at a physiological pH. Optimal transfection was observed for a 5 µg dose of the gaussia luciferase reporter gene in NIH3T3 cells without any adverse effect on cellular viability. The destabilization of the AAPBA–PVA complex by interacting with glucose allowed the release of encapsulated bovine serum albumin (BSA) in a glucose...

Reactive and Functional Polymers, 2010

The novel chelating adsorbent has been synthesized by functionalization of monodisperse-porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) particles with N-methylglucamine (NMDG) as a boron selective ligand. The resulting resin can adsorb boron in almost all pH range (pH 4-10) and its maximum capacity is ca. 12 mg/g. The adsorption behavior of poly(GMA-co-EDM)NMDG resin obeyed Langmuir isotherm well.

Polysaccharides are increasingly being used as biomaterials for tissue engineering and regenerative medicine. Quantitative analysis of gene expression from cells in three-dimensional (3D) scaffolds requires extraction of messenger RNA, which is complicated in polysaccharide materials by ionic com-plexing between nucleic acids and the matrix. We used a strongly cationic surfactant, cetyl-trimethylammonium bromide (CTAB), to extract RNA from human mesenchymal stem cells embedded in 3D chitosan, agarose and collagen matrices. CTAB extraction was compared to conventional guanidinium thiocyanate-based methods for RNA isolation by assessing RNA yield, purity (A260/A280 and A260/A230) and integrity (28S/18S and RIN). For polysaccharide-based matrices, CTAB extraction yielded significantly more RNA with higher purity than guanidinium thiocyanate-based methods alone. The extracted RNA was largely intact as indicated by 28S/18S ratios and RIN values, while these parameters could not be measured using conventional kits alone. For pure collagen matrices, the CTAB method was comparable or better than guanidinium thiocyanate-based methods in terms of RNA yield and quality. We further validated the CTAB protocol using semi-quantitative and quantitative RT-PCR to amplify both large and small amplicons. Our results show that the CTAB-based method is a facile and effective way to extract abundant, high quality RNA from polysaccharide and protein biomaterials.

International Journal of Molecular Sciences, 2021

Epidermal growth factor receptor (EGFR) is one of the most promising molecular targets for anticancer therapy. We used boron clusters as a platform for generation of new materials. For this, functional DNA constructs conjugated with boron clusters (B-ASOs) were developed. These B-ASOs, built from 1,2-dicarba-closo-dodecaborane linked with two anti-EGFR antisense oligonucleotides (ASOs), form with their complementary congeners torus-like nanostructures, as previously shown by atomic force microscope (AFM) and transmission electron cryo-microscopy (cryo-TEM) imaging. In the present work, deepened studies were carried out on B-ASO’s properties. In solution, B-ASOs formed four dominant complexes as confirmed by non-denaturing polyacrylamide gel electrophoresis (PAGE). These complexes exhibited increased stability in cell lysate comparing to the non-modified ASO. Fluorescently labeled B-ASOs localized mostly in the cytoplasm and decreased EGFR expression by activating RNase H. Moreover, ...

Journal of Materials Science

The recent outbreak of the novel corona virus disease 2019 (COVID-19) has been made a serious global impact due to its high infectivity and severe symptoms. The Severe Acute Respiratory Syndrome (SARS-CoV-2) RNA extraction is considered as one of the most important steps in COVID-19 detection. Several commercially available kits and techniques are currently being used for specific extraction of SARS-CoV-2 RNA. However, such methods are time consuming and expensive due to the requirement of trained labors, and several chemical reagents. To overcome the mentioned limitations, magnetic RNA adsorption methodology of glycine functionalized iron oxide nanoparticles (GNPs) was established. It showed an efficient potential in SARS-CoV-2 RNA extraction due to pH responsive nature of GNPs. The highly magnetic pH responsive GNPs were synthesized by one-pot co-precipitation method. Random morphology and average 20 nm size of GNPs were denoted by Transmission Electron Microscopy (TEM). X-ray diffractometer (XRD) showed the crystalline magnetite nature. Fourier transform infrared spectroscopy (FT-IR) and UV-visible spectrometry confirmed the presence of glycine on the surface of magnetic nanoparticles. Furthermore, the magnetic nature and thermal properties of GNPs were examined by vibrating sample magnetometer (VSM) and thermogravimetric analysis (TGA), respectively. In this study, glycine performed the role of RNA adsorbent. The adsorption of RNA onto the surface of GNPs was achieved in acidic medium (pH 6

AAPS PharmSciTech, 2010

Chemical coupling of polyethylene glycol (PEG) to proteins or particles (PEGylation), prolongs their circulation half-life by greater than 50-fold, reduces their immunogenicity, and also promotes their accumulation in tumors due to enhanced permeability and retention effect. Herein, phase separation method was used to prepare bovine serum albumin (BSA) nanoparticles. PEGylation of BSA nanoparticles was performed by SPA activated mPEG through their free amino groups. Effect of process variables on PEGylation efficiency of BSA nanoparticles was investigated and optimized through response surface methodology with the amount of free amino groups as response. Optimum conditions was found to be 32.5 g/l of PEG concentration, PEG-nanoparticle incubation time of 10 min, incubation temperature of 27°C, and pH of 7 for 5 mg of BSA nanoparticles in 1 mL phosphate buffer. Analysis of data showed that PEG concentration had the most noticeable effect on the amount of PEGylated amino groups, but pH had the least. Mean diameter and zeta potential of PEGylated nanoparticles under these conditions were 217 nm and −14 mV, respectively. In conclusion, PEGylated nanoparticles demonstrated reduction of the negative surface charge compared to the non modified particles with the zeta potential of −31.7 mV. Drug release from PEGylated nanoparticles was almost slower than non-PEGylated ones, probably due to existence of a PEG layer around PEGylated particles which makes an extra resistance in opposition to drug diffusion.

RNA interference (RNAi) technology provides a powerful, yet selective, molecular tool to reduce the expression of genes in eukaryotic cells. Despite the success associated with the effective use of siRNA duplexes for gene silencing, there is a need to improve their properties. These properties, related mainly to migration through the cell membranes, stability of siRNA in vivo, and specificity of their silencing activity, can be improved by chemical modifications of siRNA backbone. In this study, we examined the physicochemical and biological properties of siRNA duplexes targeted against BACE1 gene modified at various positions with a lipophilic boron cluster (C2B10H11, CB). The lipophilicity and resistance to enzymatic degradation of the modified oligomers was higher than the unmodified counterparts. As measured in a dual fluorescence assay (BACE1-GFP/RFP), the carboranyl siRNAs (CBsiRNAs) were as active as the parent nonmodified duplexes and their toxicity toward HeLa cells was also similar. The helical structure of CB-siRNAs remained unchanged upon boron cluster introduction, as determined by CD and UV melting experiments.

Journal of Pharmaceutical Sciences, 2016

Small-interfering RNA (siRNA)-mediated gene silencing with the aid of chitosan (CS)-based carriers has shown efficient and reliable outcome in vitro, but the gene silencing efficiency in vivo is still limited. It is of great importance to balance the protection and release of siRNA from nanoparticles (NPs) so as to achieve high efficiency. However, siRNA release profile from CS/siRNA NPs has been rarely concerned. Here, F€ orster resonance energy transfer technique was adopted for in vitro investigation of siRNA release from CS NPs in lysozyme-contained buffer. The results clearly showed that siRNA molecules experienced a fast and short release phase under lysozyme competition to both CS and siRNA, and then a slow and long release under lysozyme degradation on CS. Moreover, lysozyme competition played more important role than enzymolysis on trigging siRNA release. This preliminary study of siRNA release is the first step to get insight of in vivo siRNA release mechanism from CS/siRNA NPs, which will be helpful to adjust the design of CS/siRNA NPs for balancing the protection and release of siRNA molecules.