Biomedical subjects

Swaroop Mishra

Publications and source records attributed to Swaroop Mishra.

6 recordsLinked to original sources

PubMed2006

Structure-function correlation of chloroquine and analogues as transgene expression enhancers in nonviral gene delivery.

To understand how chloroquine (CQ) enhances transgene expression in polycation-based, nonviral gene delivery systems, a number of CQ analogues with variations in the aliphatic amino side chain or in the aromatic ring are synthesized and investigated. Our studies indicate that the aliphatic amino moiety of CQ is essential to provide increased gene expression. Further, the enhancements are more dramatically affected by changes to the aromatic ring and are positively correlated to the strength of intercalation between DNA and the CQ analogues. Quinacrine (QC), a CQ analogue with a fused acridinyl structure that can strongly intercalate DNA, enhances transfection similarly to CQ at a concentration 10 times lower, while N(4)-(4-pyridinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CP), a CQ analogue that has a weakly intercalating pyridinyl ring, shows no effect on gene expression. Subtle change on the 7-substituent of the chloroquine aromatic structure can also greatly affect the ability of the CQ analogues to enhance transgene expression. Transfection in the presence of N(4)-(7-trifluoromethyl-4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamin e (CQ7a) shows expression efficiency 10 times higher than in the presence of CQ at same concentration, while transfection in the presence of N(4)-(4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CQ7b) does not reveal any enhancing effects on expression. Through a number of comparative studies with CQ and its analogues, we conclude that there are at least three mechanistic features of CQ that lead to the enhancement in gene expression: (i) pH buffering in endocytic vesicles, (ii) displacement of polycations from the nucleic acids in polyplexes, and (iii) alteration of the biophysical properties of the released nucleic acid.

Cell Line, Tumor↗

PubMed2006

Imidazole groups on a linear, cyclodextrin-containing polycation produce enhanced gene delivery via multiple processes.

The linear, cyclodextrin-containing polycation (CDP) is one of many non-viral gene delivery vectors that show improved transfection efficiency when modified to have pH-buffering capacity. The buffering activity is presumed to confer enhanced ability to escape the endocytic pathway. Here, the differences in delivery behavior between CDP and its pH-buffering, imidazole-containing variant (CDPim) are investigated in order to elucidate the mechanism(s) by which these related materials exhibit differences in gene delivery. In cell-free assays that include dye exclusion and heparan sulfate displacement, CDP appears to have weaker binding strength with nucleic acids than CDPim. Numerous analyses involving transfected cells, however, indicate that CDPim more readily releases nucleic acids in the intracellular setting. Together, these data suggest that differences in transfection efficiency between CDP and CDPim result from factors beyond buffering activity and endosomal escape.

Buffers↗

PubMed2005

Molecular conjugates.

Molecular conjugates are nanometer-sized entities consisting of synthetic materials (lipids, polycations, targeting agents, and so on) and nucleic acids. These composites are delivery vehicles that function to provide the transport of nucleic acids to sites of action. Recently, great progress has been made in the construction of these nonviral delivery vehicles and the understanding of how they function in cells and animals. Here, we review some of the important issues in assembling molecular conjugates and understanding their behavior in biological fluids, cells, and animals. One of the largest challenges in the field of molecular conjugates is how to integrate the components into a workable system that exploits the combined attributes of the components without suffering losses due to the assembly of the system. We discuss some of the difficulties involved in the assembly of a functioning delivery system for in vivo use.

Biocompatible Materials↗

PubMed2004

PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles.

In vitro studies of non-viral gene delivery vectors are typically not performed at physiological conditions, and thus may not provide meaningful results for in vivo investigations. We determine if polycation-plasmid DNA complexes (polyplexes) exploited for in vitro studies behave similarly to variants more applicable to in vivo use by examining their cellular uptake and trafficking. Branched polyethylenimine (25 kDa) or a linear beta-cyclodextrin-containing polymer are each used to formulate polyplexes, which can be PEGylated (PEG: poly(ethylene glycol)) to create particles stable in physiological salt concentrations. Particle size, cellular uptake, intracellular trafficking, and reporter gene expression are reported for polyplexes and for their PEGylated variants. PEGylation confers salt stability to particles but produced a reduction in luciferase expression. Examination of in vitro particle internalization by transmission electron microscopy shows unmodified polyplexes entering cells as large aggregates while PEGylated particles remain small and discrete, both outside and within cells. Unmodified and PEGylated particles enter cells through the endocytic pathway and accumulate in a perinuclear region. Immunolabeling reveals unpackaged exogenous DNA in the cytoplasm and nuclei. It appears all particle types traffic towards the nucleus within vesicles and undergo degradation in vesicles and/or cytoplasm, and eventually some exogenous DNA enters the nucleus, where it is transcribed. In comparing polyplexes and their PEGylated variants, significant differences in particle morphology, cellular uptake, and resultant expression suggest that in vitro studies should be conducted with particles prepared for physiological conditions if the results are to be relevant to in vivo performance.

Animals↗

PubMedDate not supplied

Structural effects of carbohydrate-containing polycations on gene delivery. 3. Cyclodextrin type and functionalization.

Linear cationic beta-cyclodextrin (beta-CD)-based polymers can form polyplexes with plasmid DNA and transfect cultured cells. The effectiveness of the gene delivery and the cellular toxicity has been related to structural features in these polycations. Previous beta-CD polycations were prepared from the cocondensation of 6(A),6(D)-dideoxy-6(A),6(D)-diamino-beta-CD monomers with other difunctionalized monomers such as dimethyl suberimidate (DMS). Here, the type of CD and its functionalization are varied by synthesizing numerous 3(A),3(B)-dideoxy-3(A),3(B)-diamino-beta- and gamma-CD monomers. Both alkyl- and alkoxydiamines are prepared in order to vary the nature of the spacing between the CD and the primary amines in the monomers. These diamino-CD-monomers are polymerized with DMS to yield amidine-based polycations. The nature of the spacer between the CD-ring and the primary amines of each monomer is found to influence both molecular weight and polydispersity of the polycations. When these polycations are used to form polyplexes with plasmid DNA, longer alkyl regions between the CD and the charge centers in the polycation backbone increase transfection efficiency and toxicity in BHK-21 cells, while increasing hydrophilicity of the spacer (alkoxy versus alkyl) provides for lower toxicity. Further, gamma-CD-based polycations are shown to be less toxic than otherwise identical beta-CD-based polycations.

Animals↗

PubMedDate not supplied

Single cell kinetics of intracellular, nonviral, nucleic acid delivery vehicle acidification and trafficking.

Mechanistic understanding of the intracellular trafficking of nonviral nucleic acid delivery vehicles remains elusive. A live, single cell-based assay is described here that is used to investigate and quantitate the spatiotemporal, intracellular pH microenvironment of polymeric-based nucleic acid delivery vehicles. Polycations such as polyethylenimine (PEI), poly-l-lysine (PLL), beta-cyclodextrin-containing polymers lacking or possessing imidazole termini (CDP or CDP-imid), and cyclodextrin-grafted PEI (CD-PEI) are used to deliver an oligonucleotide containing a single fluorophore with two emission lines that can be employed to measure the pH. Delivery vehicles were also sterically stabilized by addition of poly(ethylene glycol) (PEG) and investigated. The intracellular trafficking data obtained via this new methodology show that vectors such as PEI and CDP-imid can buffer the endocytic vesicles while PLL and CDP do not. Additionally, the PEGylated vectors reveal the same buffering capacity as their unstabilized variants. Here, the live cell, spatiotemporal mapping of these behaviors is demonstrated and, when combined with cell uptake and luciferase expression data, shows that there is not a correlation between buffering capacity and gene expression.

Base Sequence↗