K.C. Gupta

Imidazolyl-PEI modified nanoparticles for enhanced gene delivery.

The derivatives of polyethylenimine (PEI 25 and 750kDa) were synthesized by partially substituting their amino groups with imidazolyl moieties. The series of imidazolyl-PEIs thus obtained were cross-linked with polyethylene glycol (PEG) to get imidazolyl-PEI-PEG nanoparticles (IPP). The component of hydrophobicity was introduced by grafting the lauryl groups in the maximal substituted IPP nanoparticles (IPPL). The nanoparticles were characterized with respect to DNA interaction, hydrodynamic diameter, zeta potential, in vitro cytotoxicity and transfection efficiency on model cell lines. The IPP and IPPL nanoparticles formed a loose complex with DNA compared to the corresponding native PEI, leading to more efficient unpackaging of DNA. The DNA loading capacity of IPP and IPPL nanoparticles was also lower compared to PEI. The imidazolyl substitution improved the gene delivery efficiency of PEI (750kDa) by nine- to ten-fold and PEI (25kDa) by three- to four-fold. At maximum transfection efficiency, the zeta potential of nanoparticles was positive after forming a complex with DNA. The maximum level of reporter gene expression was mediated by IPPL nanoparticles in both the series. The cytotoxicity, another pertinent problem with cationic polymers, was also negligible in case of IPP and IPPL nanoparticles.

Gellan gum blended PEI nanocomposites as gene delivery agents: Evidences from in vitro and in vivo studies

Branched Polyethylenimine, 25 kDa (PEI), was blended with gellan gum, an anionic heteropolysaccharide, for partial neutralization of its excess positive charge to form gellan gum-polyethylenimine (GP) nanocomposites (NCs). Subsequently, we manipulated the amount of gellan gum for obtaining a series of NCs and characterized them for their size, charge and morphology. Among all the NCs, one member, named GP3, showed the best transfection efficiency in tested cell lines in comparison with the rest of the series, PEI, Lipofectamine and other commercial transfection agents and also exhibited minimum cytotoxicity. It was found to transfect primary cells of mouse skin with better efficiency than PEI and Lipofectamine and was able to protect the plasmid DNA from nucleases and serum proteins present in the blood. GP3 exhibited efficient intracellular delivery of plasmid as revealed by confocal studies while its intracellular presence was also confirmed by the knockdown of GFP expression (using GFP specific siRNA) and JNKII by quantifying proteins in cell lysates and by western blotting and hybridization, respectively. In vivo cytotoxicity studies in Drosophila showed lack of induction of stress response in the exposed organisms. Further, exposed organisms did not show any developmental delay or mortality and no morphological defects were observed in the emerged flies. In vivo gene expression studies in Balb/c mice revealed maximum expression of luciferase enzyme in spleen. The study suggests that GP3 may act as an efficient non-viral gene carrier with diverse biomedical applications.

Express Protocol for Functionalization of Polymer Supports for Oligonucleotide Synthesis

Abstract A rapid and general one-pot method is described for the attachment of the leader nucleoside onto the polymer supports, suitable for polymer supported oligonucleotide synthesis following oxidation-reduction condensation. The method can also be used for support functionalisation in fully automated DNA synthesizer prior to oligonucleotide synthesis.

PEI-alginate nanocomposites: Efficient non-viral vectors for nucleic acids

Branched polyethylenimine (PEI, 25 kDa) was ionically interacted with varying amount of alginic acid to block different proportion (2.6-5.7%) of amines in PEI to form a series of nanocomposites, PEI-Al. These nanocomposites, upon interaction with DNA, protected it against DNase I. Among various complexes evaluated, PEI-Al(4.8%)/DNA displayed the highest transfection efficiency in HEK293, COS-1 and HeLa cells that was approximately 2-8-folds higher than Superfect, Fugene, PEI (750 kDa)-Al(6.26%) and PEI alone. The projected nanocomposites were nearly non-toxic to cells in vitro. Furthermore, the concentration of PEI-Al(4.8%) needed to deliver GFP-specific siRNA in COS-1 cells was 20 times lower than PEI (750 kDa)-Al(6.26%). Intracellular trafficking of PEI-Al(4.8%) with or without complexed DNA in HeLa cells shows that both appear in the nucleus after 1 h.

Solid phase synthesis and purification of 5′-mercaptoalkylated oligonucleotides

Abstract A novel strategy for the synthesis of thiol link phosphoramidites having base labile S-acyl type protecting groups is described. The method involves the reaction of 6-bromohexanol with potassium thioacetate or thiobenzoate in the presence of 18-crown-6 as a phase transfer catalyst to obtain the compounds 3(a,b) , which were then converted to their corresponding phosphoramidites 4(a,b) and used for solid phase synthesis of 5′-mercaptoalkylated oligonucleotides. The thiolated oligomers were purified by single step covalent chromatography using an activated sulfhydryl group containing support.

A RAPID METHOD FOR THE FUNCTIONALISATION OF POLYMER SUPPORTS FOR SOLID PHASE OLIGONUCLEOTIDE SYNTHESIS

Abstract A rapid method is described for the covalent anchoring of appropriately protected 2′-deoxyribonucleoside-3′-O-succinates to LCAA-CPG, widely used support for solid phase oligonucleotide synthesis. The method involves the reaction of nucleoside-3′-O-succinates with aminoalkyl functions of the support in the presence of improved and commercially available condensing reagent, TBTU or TPP-DTNP to generate fully functionalised polymer supports with excellent nucleoside loadings.

Microwave assisted high yielding preparation of N-protected 2'-deoxyribonucleosides useful for oligonucleotide synthesis

ABSTRACT A rapid and high yielding method for the synthesis of precursors of synthons for DNA synthesis, N-protected 2′-deoxyribonucleosides is described, which occur under mild conditions using microwave irradiation. The desired material, N-protected nucleosides, was obtained in 93–96% yield in few minutes. The final products were then characterized by 1H-NMR and MALDI-TOF and compared with the standard samples. The method is amenable to small to moderate scale of synthesis.

Microwave-assisted spectrophotometric estimation of polymer-supported functional groups using a universal reagent

A rapid method has been developed for the estimation of polymer-supported functionalities under microwave irradiation. The method involves the use of a novel universal reagent, S-(4,4′-dimethoxytrityl)-3-mercaptopropionic acid (DMPA) for the estimation of polymer-supported hydroxyalkyl, aminoalkyl and mercaptoalkyl functionalities in the presence of triphenylphosphine–bromotrichloromethane (TPP–BTCM) as an oxidation-reduction coupling reagent. The loadings obtained on the supports following the proposed method were found to be comparable with those obtained with the standard, 4,4′-dimethoxytrityl chloride (DMTr-Cl), method. The usefulness of the method was further demonstrated by monitoring the functionalization of polymer supports, suitable for solid-phase peptide and oligonucleotide synthesis.

An improved method for synthesis of biotin phosphoramidites for solid phase biotinylation of oligonucleotides

Abstract Biotin (potassium salt) 2 was selectively and quantitatively N1-tritylated to 3 in just 2 h in the presence of PEG-dimethyl ether. 3 was converted to its benzotriazolide active ester in 2h at room temperature, which in the subsequent reaction in situ with aminoalkanol resulted in 4(a,b) . The compounds 4(a,b) were converted to the corresponding phosphoramidite reagents 5(a,b) and were used for solid phase 5′-biotinylation of oligonucleotides in automated DNA synthesizer.

Tagging requirements for web application

Tagging systems exist that support tagging of resources, like, web pages, web links, videos, images and blogs. In web applications, use of tag facilitates categorization of related content and improves search within a resource, like, searching a blog or a page. During the development of web application, there is a need for eliciting the requirements for the tagging functionality in the web application. In this paper, we present a requirement checklist for tagging, from the viewpoint of incorporating the tagging functionality in a web application. A use-case based approach has been followed to identify the requirements for tagging. We see that the requirements for tagging vary, depending on the kind of permission provided to the user for the purposes of tagging. Also, there is a need to include resource-specific features. Accordingly, we categorize user requirements based on user-permissions for tagging, and also list the resource-specific features. Our checklist is beneficial both for the web application and the developer during the requirement specification process. During testing, the requirement checklist for tagging is useful for the validation and verification purposes.