Sushil K. Tripathi

Linear polyethylenimine-graft-chitosan copolymers as efficient DNA/siRNA delivery vectors in vitro and in vivo

Chitosan was partially converted to its chlorohydrin derivative by the reaction with epichlohydrin, which was subsequently reacted with varying amounts of lPEI(2.5 kD) to obtain a series of chitosan-lPEI(2.5 kD) copolymers (CP). These copolymers were then characterized and evaluated in terms of transfection efficiency (in vitro and in vivo), cell viability, DNA release and buffering capacity. The CP-4 copolymer (the best among the CP series) showed enhanced transfection (-2 - 24 folds) in comparison with chitosan, lPEI(2.5 kD), bPEI(25 kD) and Lipofectamine in HEK293, HeLa and CHO cells. The buffering capacity (in the pH range of 3 - 7.5), as shown by confocal microscopy, and DNA-release capability of the CP copolymers, was found to be significantly enhanced over chitosan. Intravenous administration of CP-4/DNA polyplex in mice followed by the reporter gene analysis showed the highest gene expression in spleen. Collectively, these results demonstrate the potential of CP-4 copolymer as a safe and efficient nonviral vector. From the Clinical Editor: Chitosan -PEI (2.5 kD) copolymers (CP) were characterized and their transfection efficiency, DNA release and buffering capacity were studied. The CP-4 copolymer significantly enhanced buffering capacity and provided the highest gene expression levels. The method may be used to enhance DNA transfection.

Depolymerized chitosans functionalized with bPEI as carriers of nucleic acids and tuftsin-tethered conjugate for macrophage targeting.

Development of efficient and safe nucleic acid carriers (vectors) is one of the essential requirements for the success of gene therapy. Here, we have evaluated the gene transfer capability of chitosan-PEI (CP) conjugates prepared by conjugating low molecular weight branched polyethylenimine (LMWP) with depolymerized chitosans (7 and 10 kDa) via their terminal aldehyde/keto groups. The CP conjugates interacted efficiently with nucleic acids and also showed higher cellular uptake. These conjugates on complexation with DNA yielded nanoparticles in the size range of 100-130 nm (in case of C7P) and 115-160 nm (in case of C10P), which exhibited significantly higher transfection efficiency (~2-42 folds) in vitro compared to chitosans (high and low mol. wt.) and the commercially available transfection reagents retaining cell viability almost comparable to the native chitosan. Of the two CP conjugates, chitosan 7 kDa-LMWP (C7P) displayed higher gene transfer ability in the presence and absence of serum. Luciferase reporter gene analysis in male Balb/c mice receiving intravenous administration of C7P3/DNA polyplex showed the maximum expression in their spleen. Further, tuftsin, a known macrophage targeting molecule, was tethered to C7P3 and the resulting complex, i.e., C7P3-T/DNA, exhibited significantly higher gene expression in cultured mouse peritoneal macrophages as compared to unmodified C7P3/DNA complex without any cytotoxicity demonstrating the suitability of the conjugate for targeted applications. Conclusively, the study demonstrates the potential of the projected conjugates for gene delivery for wider biomedical applications.

Selective blocking of primary amines in branched polyethylenimine with biocompatible ligand alleviates cytotoxicity and augments gene delivery efficacy in mammalian cells

Recently, polyethylenimines (PEIs) have emerged as efficient vectors for nucleic acids delivery. However, inherent cytotoxicity has limited their in vivo applications. To address this concern as well as to incorporate hydrophobic domains for improving interactions with the lipid bilayers in the cell membranes, we have tethered varying amounts of amphiphilic pyridoxyl moieties onto bPEI to generate a small series of pyridoxyl-PEI (PyP) polymers. Spectroscopic characterization confirms the formation of PyP polymers, which subsequently form stable complexes with pDNA in nanometric range with positive surface charge. The projected modification not only accounts for a decrease in the density of 1° amines but also allows formation of relatively loose complexes with pDNA (cf. bPEI). Alleviation of the cytotoxicity, efficient interaction with cell membranes and easy disassembly of the pDNA complexes have led to the remarkable enhancement in the transfection efficiency of PyP/pDNA complexes in mammalian cells with one of the formulations, PyP-3/pDNA complex, showing transfection in ∼68% cells compared to ∼16% cells by Lipofectamine/pDNA complex. Further, the efficacy of PyP-3 vector has been established by delivering GFP-specific siRNA resulting in ∼88% suppression of the target gene expression. These results demonstrate the efficacy of the projected carriers that can be used in future gene therapy applications.

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.

Hydrophobic and membrane permeable polyethylenimine nanoparticles efficiently deliver nucleic acids in vitro and in vivo

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.

Surface modification of crosslinked dextran nanoparticles influences transfection efficiency of dextran–polyethylenimine nanocomposites

Dextran–PEI grafted nanoparticles as transfection agents have been reported, however, with limited success due to their large size, poor buffering capacity and inadequate availability of charge for competent binding to DNA. To address these concerns, dextran was first crosslinked with 1,4-butanediol diglycidyl ether (BDE), a commercially available homobifunctional crosslinker, to form its nanoparticles (DN), which were then partially oxidized with sodium periodate to generate aldehyde functionalities on them. The resultant nanoparticles were grafted with branched polyethylenimine (bPEI, 25 kDa) to obtain a series of DN–PEI nanocomposites (DP), having comparatively smaller size, enhanced buffering capacity and competent binding ability with DNA. Subsequent to biophysical characterization, these nanocomposites were evaluated for their transfection efficiency in serum and serum-free environments and toxicity in various mammalian cell lines. A significantly (p < 0.05) improved transfection efficiency of these nanocomposites concurrent with their minimal cytotoxicity as compared to the selected commercial transfection agents and native PEI was observed. These results were further validated by intracellular trafficking, wherein DP4 (the best working system in the series) was able to carry the DNA inside the nucleus after 1 h of the addition of the complex. The in vivo gene expression profile of the DP4/DNA complex in male Balb/c mice exhibited maximum expression in their spleen tissue raising promise of using these nanocomposites as improved non-viral transfection agents.

Polyglutamic acid-based nanocomposites as efficient non-viral gene carriers in vitro and in vivo

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.

Self-assembled amphiphilic phosphopyridoxyl-polyethylenimine polymers exhibit high cell viability and gene transfection efficiency in vitro and in vivo

Branched polyethylenimine (bPEI) 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 a 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 cells. Further condensation of pDNA compacted the size of the self-assembled nanostructures from 421-559 nm to 134-210 nm with zeta potentials from +20-32 mV to +18-28 mV. Conjugation of PLP with bPEI not only reduced the density of the 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 the pDNA complexes inside the cells led to a several fold enhancement in the transfection efficiency with one of the formulations, PPyP-3/pDNA complex, among the series, exhibiting ∼4.9 to 8.2 folds higher gene delivery activity than pDNA complexes of bPEI and Lipofectamine™ in HeLa and MCF-7 cells. Flow cytometry analysis revealed a very high percentage of transfected cells by PPyP/pDNA complexes compared to pDNA complexes of bPEI and Lipofectamine™. Further, GFP-specific siRNA delivery using PPyP-3 as a vector resulted in ∼84% knockdown of the target gene expression (cf.∼54% by Lipofectamine™/pDNA/siRNA complex). Moreover, the PPyP-3/pDNA complex displayed ∼6.7 fold higher transfection efficiency than the bPEI/pDNA complex in human peripheral blood dendritic cells. Intravenous administration of PPyP-3/pGL3 complex showed the highest gene expression in spleen tissue, advocating the potential of these vectors in future gene delivery applications.

Synthesis and evaluation of N-(2,3-dihydroxypropyl)-PEIs as efficient vectors for nucleic acids

Branched polyethylenimine (bPEI, 25 kDa) has been widely used as an efficient delivery vector for nucleic acids in vitro. However, its charge-associated toxicity has limited its in vivo applications. In an attempt to control its toxicity, it was reacted with varying amounts of glycidol (2,3-epoxy-1-propanol) to obtain a small series of hydrophilic polymers, 2,3-dihydroxypropyl-grafted-polyethylenimines (DHP-g-P). The resulting polymers were characterized by (1)H-NMR and subjected to interaction with negatively charged pDNA, which yielded complexes in the size range of ~171-190 nm with a zeta potential of ∼+33-39 mV. Acid-base titration revealed no effect of substitution on the buffering capacity of the modified polymers. Grafting of 2,3-dihydroxypropyl groups on bPEI significantly improved the cell viability (i.e. almost non-toxic) as well as the DNA release properties of these modified polymers compared to native bPEI. Formation of a relatively loose DHP-g-P25/pDNA complex (the best working system in terms of transfection efficiency) resulted in the efficient nuclear release of pDNA for transcription, a prerequisite for efficient transfection. Subsequently, upon evaluation of their ability to transfer nucleic acids in vitro, the DHP-g-P/pDNA complexes exhibited higher gene transfection efficiency with one of the formulations, DHP-g-P25/DNA complex, displaying ~2.7 folds higher GFP expression than bPEI and ~2.3-3.5 folds higher than the selected commercial transfection reagents used in this study. Further to quantify the extent of GFP positive cells, FACS analysis was performed, which revealed DHP-g-P25/DNA mediated gene expression in ~51% cells outcompeting bPEI, Superfect™, Fugene™ and Lipofectamine™. Sequential delivery of GFP-specific siRNA resulted in ~78% suppression of the target gene compared to ~49% achieved by Fugene™. All these results demonstrate the potential of these polymers for in vivo gene delivery.

Engineered PEI-piperazinyl nanoparticles as efficient gene delivery vectors: evidence from both in vitro and in vivo studies

A small library of polyethylenimine (PEI) nanoparticles (NPs), wherein PEI was crosslinked with piperazinyl linkers, viz., piperazine-N,N′-dibutyric acid and piperazine-N,N′-dipropanal, yielding piperazine-N,N′-dibutyramide-PEI (PBAP) and piperazine-N,N′-dipropyl-PEI (PPP) nanoparticles, was designed and synthesized. The NPs were in the size range 103–216 nm. DNA complexes of all the NPs had low toxicity and exhibited significant enhancement in transfection efficiency compared to parent PEI and commercial transfection agents. Importantly, the transfection activity of NP/DNA complexes was preserved in the presence of serum. Amongst all the formulations, the PBAP4/DNA complex exhibited the highest transfection efficiency in all the cell lines tested. Also, PBAP4 NPs efficiently protected the complexed DNA against DNase in vitro. Intravenous delivery of PBAP4/DNA complex to male Balb/c mice showed the highest gene expression in spleen cells. The results indicate that PBAP nanoparticles may be used for tissue-specific gene delivery in vivo.