Ruby Bansal

Lipophilic and cationic triphenylphosphonium grafted linear polyethylenimine polymers for efficient gene delivery to mammalian cells

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.

Biodegradable and versatile polyethylenimine derivatives efficiently transfer DNA and siRNA into mammalian cells.

UNLABELLED Polyethylenimines (PEIs) are considered as the most promising vectors for non-viral gene delivery applications. Here, we report the synthesis and in vitro evaluation of two non-toxic and biodegradable polymers, TEPA@bPEI (TBP) and TEPA@lPEI (TLP), derived from low molecular weight branched and linear polyethylenimines by the stepwise reactions with methylacrylate (aza-Michael reaction) and amidation with tetraethylenepentamine (TEPA). These polymers not only showed their ability to bind and condense pDNA into nano-sized complexes but also provided protection against nucleases in cellular milieu. Both the polymers exhibited excellent buffering capacity and efficiently delivered nucleic acids (plasmid DNA and siRNA) across the mammalian cells (CHO and A549 cells) and outclassed native polymers and the commercial transfection reagents in terms of transfection efficiency and target gene silencing, and that too without compromising on biocompatibility i.e. TOXICITY The results advocate the promising potential of the PEI derivatives as safe and potent nucleic acid carriers for practical gene delivery applications.

Enhanced Antimicrobial Activity of Amine-Phosphonium (N-P) Hybrid Polymers Against Gram-Negative and Gram-Positive Bacteria

The authors report synthesis, characterization and evaluation of a series of linear polyethylenimine (lPEI)-grafted butyltriphenylphosphonium bromide (LBTP) polymers (N-P hybrid polymers) for their antimicrobial activity on various Gram-positive and Gram-negative bacteria. Polymers with ∼5.8–13.8% substitution of butyltriphenylphosphonium bromide (BTP) on the backbone of lPEI showed enhanced charge density as compared to native lPEI confirming the conjugation of BTP onto lPEI. These modified polymers displayed low hemolytic activity and excellent antimicrobial activity against these two types of bacteria with one of the modified polymers, LBTP-40, was found to exhibit high antimicrobial activity in all the strains. GRAPHICAL ABSTRACT

Galactomannan-PEI based non-viral vectors for targeted delivery of plasmid to macrophages and hepatocytes

Intracellular nature and diversified locations of infectious and parasitic diseases such as leishmaniasis, trypanosomiasis, tuberculosis and hepatitis B and C pose a significant global burden and challenge to the 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.

Synthesis, characterization and evaluation of diglycidyl-1,2-cyclohexanedicarboxylate crosslinked polyethylenimine nanoparticles as efficient carriers of DNA

Non-viral gene delivery vectors have shown promising potential to treat a variety of inherited and acquired disorders. Among various non-viral systems, cationic polymers have proved to be the most efficient gene carriers as they have the tendency to condense nucleic acids to nanosized particles and improve their transfer inside the cells. Polyethylenimine has been considered as a ‘gold standard’ in gene delivery applications. However, charge-associated toxicity has limited its clinical efficacy. Here, we have tried to address this concern by partially reducing the cationic charge density on branched polyethylenimines (PEIs, 10 and 25 kDa) and simultaneously converting these polymers into their respective nanoparticles using a commercially available reactive crosslinking reagent, diglycidyl-1,2-cyclohexanedicarboxylate (DCD). Varying the amounts of DCD during crosslinking reaction generated two small series of diglycidyl-1,2-cyclohexanedicarboxylate–PEI (DP10 and DP25) nanoparticles with the size ranging from 125–201 nm and zeta potential from +11–20 mV. Though these nanoparticles showed no difference in the nucleic acid condensing ability from their respective native polymers, a significant decrease in their buffering capacity was observed as determined by the acid–base titration method. Upon further evaluation, pDNA complexes of DP10 and DP25 nanoparticles were found to be non-toxic and exhibited several fold higher transfection efficiency than native polymers and the standard transfection reagent, Lipofectamine. Altogether, these results demonstrate that these nanoparticles can effectively be used for future gene delivery applications.

Oligoamine-tethered low generation polyamidoamine dendrimers as potential nucleic acid carriers

In recent years, dendrimers have emerged as the most widely explored materials for theranostics emphasizing their potential in therapeutic delivery and diagnostics as well as in pharmaceutical technology. Amongst them, PAMAM dendrimers have been extensively studied for their prospects in various biomedical applications due to their defined structures and distinctive features such as monodispersity, uniformity and amenability to functionalization. Here, low generation PAMAM dendrimers (G2-G4) have been modified via Michael addition reaction followed by amidation with an oligoamine linker, tetraethylenepentamine (TEPA). Subsequently, these modified dendrimers were characterized by physicochemical techniques and evaluated for their capability to transfer nucleic acids in vitro. The results displayed significant improvements in the transfection efficiency in both HeLa and A549 cells maintaining higher cell viability. Sequential delivery of GFP-specific siRNA resulted in ∼73% suppression of the target gene. Flow cytometry results revealed that one of the formulations, mG3-pDNA complex, exhibited the highest gene transfection (∼49-68%) outperforming pDNA complexes of native dendrimers and the standard transfection reagent, Superfect (∼32-36%). All these results ensure the potential of the modified dendrimers as effective vectors for future gene delivery applications.

Engineered polymeric amphiphiles self-assembling into nanostructures and acting as efficient gene and drug carriers

Nonviral gene delivery systems are finding widespread use due to their safety, rapid and economical production, and ease of modification. In this work, series of N-alkyl-substituted linear polyethylenimine (CP) polymers have been synthesized, characterized, and investigated about how degree of substitution (hydrophobic–hydrophilic balance) (i.e. N-alkylation) influenced the transfection efficiency. Mobility shift assay demonstrated efficient binding of plasmid DNA (pDNA). Transfection efficiency and cytotoxicity of CP polymers were assessed in vitro, which revealed that all the formulations exhibited higher transfection activity than linear polyethylenimine (lPEI) and commercial transfection reagents, Lipofectamine and Superfect, with negligible toxicity (MTT assay). In the projected series, one of the formulations, CP-3-pDNA complex, displayed the highest transfection efficiency (∼1.6–12 folds vs. lPEI and commercial transfection reagents) and effectively carried GFP-specific siRNA inside the cells as monitored by measuring the suppression in the gene expression of the target gene. Further, flow cytometry experiments confirmed that CP-3-pDNA complex transfected the highest number of cells. Besides, CP-3 was also evaluated in terms of its capability to entrap hydrophobic drug molecules. The results showed that it efficiently encapsulated an anti-cancer drug, etoposide, and released it in a controlled fashion over a period of time. Altogether, the data support that CP-3 is a promising vector for nucleic acid as well as hydrophobic drug delivery.