K C Gupta

Institute of Genomics and Integrative Biology, Old Delhi, NCT, India

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Publications (80)227.14 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The present study describes the formation of copolymers of linear and branched PEIs (25 kDa each). These polyethylenimines (bPEI and lPEI) were crosslinked with each other to obtain branched-linear (BL) PEI copolymers using epichlorohydrin as a crosslinker in two steps. First, lPEI was reacted with epichlorohydrin to form lPEI-chlorohydrin (CHL) and subsequently, bPEI was grafted onto CHL in basic medium by in situ generation of epoxy functionalities. The two PEIs were crosslinked by varying the weight ratio of bPEI while keeping the amount of lPEI fixed. The ratio of two PEIs (1:1, 2:1, 3:1, 4:1 and 5:1) and crosslinking percentage of epichlorohydrin (5, 10, 15 and 20%) appeared as the main parameters to have affected the transfection efficiency. The lead conjugate/DNA complex was tested for in vivo transgene expression in Balb/c mice and was found to show maximum expression in the spleen.
    Journal of Biomedical Nanotechnology 11/2014; 10(11). · 5.26 Impact Factor
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    ABSTRACT: Intracellular nature and diversified locations of infectious and parasitic diseases such as leishmaniasis, trypanosomiasis, tuberculosis, hepatitis B and C, etc. pose a significant global burden and challenge to scientists working in the area of drug discovery and drug delivery. The macrophages and hepatocytes are considered as potential target sites as they together play an important role in various infectious diseases. The present study scrutinizes the applicability of a natural biopolymer-based chemical vectors, capable of targeting both macrophages and hepatocytes, that can form a complex with plasmid and administer it into cells to produce a desired protein. The investigations were made to develop a novel series of gene carriers by conjugating depolymerized galactomannan (guar gum), a biocompatible polysaccharide with low molecular weight branched PEI (LMWP). A series of conjugates were developed and characterized using physicochemical techniques. All the GP/pDNA complexes showed significantly higher transfection efficiency with GP-3/pDNA, one of the best formulations, showed ∼2.0-7.7 folds higher transfection efficacy when compared with the standard transfection reagents. Further, GP-3/pDNA displayed significantly higher target specific transfection efficiency under both in vitro and in vivo conditions. The data demonstrate the potential of GP vectors to deliver nucleic acids simultaneously to macrophages and hepatocytes in gene delivery applications.
    European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 05/2014; · 3.15 Impact Factor
  • European Journal of Pharmaceutics and Biopharmaceutics. 01/2014;
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    ABSTRACT: Branched polyethylenimine was conjugated with hydrophobic pyridoxal phosphate (PLP) in the side chain via reaction with primary amines to obtain amphiphilic phosphopyridoxyl-polyethylenimine (PPyP) polymers. These polymeric amphiphiles with defined degree of hydrophobicity self-assembled into nanostructures, which were characterized by DLS and evaluated for their capability to condense nucleic acids and carry them into the cells. pDNA condensation further compacted the size of the self-assembled nanostructures from 421-559 nm to 134-210 nm with zeta potential from +20-32 mV to +18-28 mV. Conjugation of PLP with bPEI not only reduced the density of primary amines (i.e. charge density) but also improved the cell viability of the modified polymers considerably and weakened the binding of pDNA with these polymers. Efficient unpackaging of pDNA complexes inside the cells led to several folds enhancement in the transfection efficiency with one of the formulations, PPyP-3/pDNA complex, among the series, exhibited ~4.9 - 8.2 folds higher gene delivery activity than pDNA complexes of bPEI and LipofectamineTM in HeLa and MCF-7 cells. Flow cytometry analysis revealed very high percentage of transfected cells by PPyP/pDNA complexes compared to pDNA complexes of bPEI and LipofectamineTM. Further, GFP-specific siRNA delivery using PPyP-3 as a vector resulted in ~84% knockdown of the target gene expression (cf. ~54% by LipofectamineTM/pDNA/siRNA complex). Moreover, PPyP-3/pDNA complex displayed ~6.7 folds higher transfection efficiency than bPEI/pDNA complex in human peripheral blood dendritic cells. Intravenous administration of PPyP-3/pGL3 complex showed the highest gene expression in spleen tissues advocating the potential of these vectors in future gene delivery applications.
    J. Mater. Chem. B. 06/2013;
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    ABSTRACT: Conjugation through primary amines is one of the most commonly used methods to modify cationic vectors for efficient gene delivery. Here, dimethyl suberimidate, a commercially available homobifunctional reagent bearing imidoesters at the termini, has been used to crosslink branched polyethylenimine (bPEI) into its nanoparticles (crosslinked PEI nanoparticles, CLP NPs) specifically through primary amines without altering the total charge on the resulting NPs for interaction with biomolecules and cell membranes. By varying the degree of crosslinking, a small series of CLP NPs was prepared and evaluated for their capability to deliver nucleic acids in vitro and in vivo. Physico-chemical characterization revealed the size of the NPs in the range of 152 to 210 nm with zeta potential +35 to +38 mV. The plasmid DNA binding ability of these nanoparticles was examined by mobility shift assay, where the pDNA migration was found to be completely retarded by these NPs at an N/P ratio of 4 (cf. bPEI at N/P 3). In various mammalian cells, CLP/pDNA nanoplexes were not only found to be non-toxic but also exhibited significantly enhanced gene expression with one of the formulations, the CLP3/pDNA nanoplex, displaying the highest transfection efficiency, outperforming native bPEI and the selected commercial transfection reagents both in the presence and absence of serum. Further, the versatility of the vector, CLP3, was demonstrated by sequential delivery of GFP-specific siRNA to HEK293 cells, which resulted in 79% suppression of the target gene. Intracellular localization studies showed a significant population of the dual labeled nanoplex (CLP3/pDNA) in the nucleus in just 60 min of incubation. Luciferase reporter gene analysis in Balb/c mice post-intravenous administration of the CLP3/pDNA nanoplex showed the highest gene expression in their spleen. The study suggests that CLP NPs could be used as efficient gene delivery vectors for future gene therapy applications.
    J. Mater. Chem. B. 04/2013; 1(19):2515-2524.
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    R. Bansal, K.C. Gupta, P. Kumar
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    ABSTRACT: Gene therapy has inspired various researchers due to its potential of treating inherited disorders; however, many problems remain to be resolved such as development of a safe and efficient vector to overcome major hurdles of the gene transfer at the cellular level. To address these issues, various modifications have been incorporated in cationic polymers. Among these, hydrophobic modifications have also been considered; however, such modifications affect the solubility of the polymers post modification. In an attempt overcome this concern, we have deliberately selected a hydrophobic ligand bearing a charged moiety, i.e. Butyltriphenylphosphonium bromide (TPP+). A series of triphenylphosphonium bromide-grafted-linear polyethylenimine (BTP) polymers have been synthesized and characterized by physicochemical techniques prior to their evaluation for transfection efficacy and cytotoxicity. Subsequently, the BTP polymers were assessed for their buffering capacity, intracellular trafficking and transfection studies in mammalian cell lines and found to exhibit significantly higher transfection efficiency compared to the standard transfection reagents. The versatility of the projected vector was established by sequential delivery of GFP-specific siRNA, which resulted in efficient knockdown of the targeted gene expression. The study demonstrates that the BTP polymers can serve as efficient vectors for the delivery of nucleic acids in future gene therapy applications.
    Biomedical Engineering Conference (SBEC), 2013 29th Southern; 01/2013
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    ABSTRACT: Synthetic chemical vectors have recently provided a versatile and robust platform for the safe and efficient delivery of exogenous genes. Here, for the first time, a small series of N-butyltriphenylphosphonium bromide-grafted-linear polyethylenimine (BTP-g-lP) polymers (N–P hybrid polymers) have been evaluated for their ability to deliver genes into mammalian cell lines, viz., MCF-7 and A549 cells. Biophysical characterization revealed that the projected polymers efficiently interacted with plasmid DNA, and the resulting complexes displayed hydrodynamic diameters in the range of 249–307 nm with relatively higher zeta potential values of +31 to +34 mV (cf. lPEI, +26 mV). The tethering of lipophilic and cationic triphenylphosphonium moieties to linear PEI (lPEI) addressed several limitations associated with lPEI, such as solubility, the stability of the pDNA complexes and the timely release of pDNA for nuclear localization as assessed by protection and release assays. Also, the lipophilic interactions between cellular membranes and the pDNA complexes mediated the efficient cellular uptake and internalization of the pDNA complexes, resulting in significantly higher transfection efficiency in these cell lines, outperforming the GenePORTER 2™, Lipofectamine™ and Superfect™ used in the study for comparison purposes. Confocal studies using dual-labeled TMR-BTP-g-lP3/YOYO-1-pDNA complex in MCF-7 cells confirmed that the complex behaved more or less like native lPEI, as the substitution of the phosphonium moiety was too small to affect the intracellular trafficking. Furthermore, the versatility of the BTP-g-lP3 vector was established by GFP specific siRNA delivery, which resulted in 79% suppression of targeted gene expression (cf. Lipofectamine™, 55%). Altogether, the study demonstrates the potential of these hybrid polymers for the efficient delivery of nucleic acids for future gene therapy applications.
    Journal of Materials Chemistry 11/2012; 22(48):25427-25436. · 5.97 Impact Factor
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    ABSTRACT: An engineered polymer support 5 has been prepared for the solid-phase assembly of 3'-carboxyalkyl-modified oligonucleotides using commonly available reagents. A two-step deprotection procedure resulted in the quantitative cleavage of oligonucleotides from the support and removal of the protecting groups from phosphodiesters and exocyclic amino groups of the nucleic bases. The fully deprotected oligomers, obtained in high yield, were desalted and analyzed on RP-HPLC. After characterization by MALDI-TOF, these carboxyalkylated oligonucleotides were immobilized onto the epoxy-functionalized glass microslides to prepare biochips. The performance of these biochips was evaluated under different sets of conditions and then successfully validated by the detection of base mismatches and human infectious disease, bacterial meningitis, caused by N. meningitidis.
    Bioconjugate Chemistry 03/2012; 23(3):664-70. · 4.58 Impact Factor
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    ABSTRACT: A series of polyethylenimine (PEI) and γ-polyglutamic acid (PGA) nanocomposites (PPGA) was prepared and evaluated in terms of their cell viability and transfection efficiency in vitro and in vivo. On complexion with pDNA, the positively charged PPGA/DNA nanocomposites resulted in a higher level of in vitro reporter gene transfection (2.7-7.9-fold) as compared to native PEI, and selected commercial reagents and >95% cell viability in HEK293, HeLa and HepG2 cell lines. Further, PPGA-5 nanocomposite (the best working system in terms of transfection efficiency among the series) was found to efficiently transfect primary mouse keratinocytes up to 22% above the control level. PPGA-5, when tested for in vivo cytotoxicity in Drosophila, did not induce any stress in the exposed larvae in comparison with control. In vivo gene expression using PPGA-5 showed the highest transfection efficiency in spleen of mouse closely followed by heart tissues after intravenous injection through tail vein. Besides, these nanocomposites also delivered siRNA efficiently into mammalian cells, resulting in ∼ 80% suppression of EGFP expression. These results together demonstrated the potential of the projected nanocomposites for in vivo gene delivery.
    European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 07/2011; 79(3):473-84. · 3.15 Impact Factor
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    ABSTRACT: In an attempt to circumvent toxic effects of branched polyethylenimine (bPEI, 25 kDa), it was crosslinked with varying proportions of a novel linker, PEG600-bis(aminoethylphosphate) (PaP), which resulted in the formation of nanoparticles (PPaP) in the range of 61–99 nm. These nanoparticles were found to have significantly lower toxicity in vitro than the native PEI. GFP expression in cells mediated by PPaP (8.1%)/DNA complex was found to be 1.1–4.8 folds higher compared to GenePORTER 2™, Lipofectamine™, Superfect™ and native PEI in HeLa, HEK293 and CHOcells. FACS analysis on HeLa cells revealed 62% transfected cells, whereas, in the case of the GenePORTER 2™ transfection reagent, transfected cells were found to be 36%. Intracellular trafficking in HeLa cells showed a significant population of PPaP (8.1%) nanoparticles and their DNA complex in nucleus after 1 h of treatment. Also, efficient delivery of GFP specific siRNA resulted in 71% suppression of the target gene. DNase protection assay revealed that 78% of complexed DNA was protected by PPaP(8.1%) nanoparticles even after 2 h of treatment. In vivo transgene expression studies in Balb/c mice showed significantly higher expression in the spleen. The results advocate the potential of PPaPnanoparticles as efficient carriers of nucleic acidsin vivo.
    Soft Matter 06/2011; 7(13):6103-6112. · 3.91 Impact Factor
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    ABSTRACT: bPEI (polyethylenimine, 25 kDa, gold standard) is highly effective in transfection efficiency owing to its high buffering capacity, however, cytotoxicity limits its use in in vivo applications. We hypothesized that partial conversion of secondary amines in IPEI to tertiary amines, while preserving the overall number of amines, would result in improved buffering capacity, which may, in turn, improve transfection efficiency of the resulting nanoparticles with cell viability comparable to that of native IPEI. IPEI was crosslinked with BDE to obtain a series of IPEI nanoparticles (LPN-1 to LPN-8) which were obtained in approximately 80-85% yield. These particles were relatively non-toxic in vitro and in vivo. In vivo gene expression studies using LPN-5 in Balb/c mice through i.v. injection showed maximum expression of the reporter gene in the spleen. These results demonstrate the potential of these particles as efficient transfection reagents.
    Journal of Biomedical Nanotechnology 02/2011; 7(1):52-3. · 5.26 Impact Factor
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    ABSTRACT: Of the non-viral vectors, a cationic polymer like PEI is an attractive candidate which however, has been negatively impacted due to its marked toxicity. An anionic sugar polymer gelan gum (GG) has been introduced into PEI system to increase transfection efficiency with minimal toxicity. We showed that one of the synthesized (GP1-GP6) GG-PEI nanocomposites (NCs), GP3, exhibited negligible toxicity in in vitro (primary keratinocytes, HEK293, HeLa and HepG2 cells) and in vivo (Drosophila melanogaster) as compared to PEI or lipofectamin. GP3-pDNA complex was found to be transfected efficiently in the above cells as confirmed by FACS analysis (72.0 + 5.5%) while lipofectamine showed only 12.4 + 3.5% efficiency. GP3 mediated GFP specific siRNA delivery resulted in the knockdown of the GFP expression by approximately 77% and JNK (60%). In vivo gene expression studies in mice revealed reporter gene expression in spleen. The study demonstrates that GG blended PEI NCs hold promise for future applications in gene delivery both in vitro and in vivo.
    Journal of Biomedical Nanotechnology 02/2011; 7(1):38-9. · 5.26 Impact Factor
  • Soft Matter 01/2011; · 3.91 Impact Factor
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    ABSTRACT: 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.
    European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 01/2011; 79(1):3-14. · 3.15 Impact Factor
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    ABSTRACT: Microarray technology has become an important tool for detection and analysis of nucleic acid targets. Immobilization of modified and unmodified oligonucleotides on epoxy-functionalized glass surfaces is often used in microarray fabrication. Here, we demonstrate a protocol that employs coating of SU-8 (glycidyl ether of bisphenol A) onto glass microslides to obtain high density of epoxy functions for efficient immobilization of aminoalkyl-, thiophosphoryl-, and phosphorylated oligonucleotides with uniform spot morphology. The resulting microarrays exhibited high immobilization (∼65%) and hybridization efficiency (30-36%) and were sufficiently stable over a range of temperature and pH conditions. The prominent feature of the protocol is that spots can be visualized distinctly at 0.05 μM probe (a 20-mer oligonucleotide) concentration. The constructed microarrays were subsequently used for detection of base mismatches and bacterial diseases (meningitis and typhoid).
    Bioconjugate Chemistry 09/2010; 21(9):1703-8. · 4.58 Impact Factor
  • P. KUMAR, K. C. GUPTA
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 01/2010; 27(52).
  • P. KUMAR, A. K. SHARMA, K. C. GUPTA
    ChemInform 01/2010; 27(47).
  • [Show abstract] [Hide abstract]
    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 01/2010; 33(49).
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    ABSTRACT: 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.
    International Journal of Pharmaceutics 10/2009; 385(1-2):194-202. · 3.99 Impact Factor
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    ABSTRACT: Oligonucleotide-based arrays are increasingly becoming useful tools for the analysis of gene expression and single-nucleotide polymorphism. Here, we report a method that allows the direct immobilization of thiolated oligonucleotides onto an epoxy-activated glass surface via a stable thioether linkage under microwaves. The described chemistry efficiently immobilizes the probes via terminal thiol groups with uniform spot morphology. The thioether linkage could endure repeated PCR-like heat cycling with only 2.5% loss after 20 cycles, indicating that the chemistry can be used in integrated PCR/microarray devices. The highlighting feature of the proposed method is that the detection limit for the probe concentration can be reduced to 0.25 microM with 20-mer oligonucleotides. The efficiency of the projected method (approximately 33%) indicates its advantage over the existing standard methods, viz., NTMTA (approximately 9.8%), epoxide-amine (approximately 9.8%) and disulfide (approximately 1.7%). The constructed microarrays were validated through the detection of base mismatches and bacterial meningitis. These features make the projected strategy ideal for manufacturing oligonucleotide arrays and detection of mismatches and bacterial diseases.
    Bioconjugate Chemistry 09/2009; 20(9):1703-10. · 4.58 Impact Factor

Publication Stats

414 Citations
227.14 Total Impact Points

Institutions

  • 2003–2013
    • Institute of Genomics and Integrative Biology
      Old Delhi, NCT, India
  • 1993–2012
    • University of Delhi
      • • Institute of Genomics and Integrative Biology (CSIR)
      • • Centre of Biochemical Technology
      Delhi, NCT, India
    • Universität Ulm
      • Institute for Laser Technologies in Medicine & Metrology
      Ulm, Baden-Wuerttemberg, Germany
  • 2010–2011
    • Indian Institute of Toxicology Research
      Lakhnau, Uttar Pradesh, India
  • 2007
    • Banaras Hindu University
      • Department of Chemistry
      Benares, Uttar Pradesh, India
  • 1996
    • Università Telematica "E-Campus"
      Campobasso, Molise, Italy