Wayne Thomas Shier

Gene transfer efficiency of high primary amine content, hydrophobic, alkyl-oligoamine derivatives of polyethylenimine

In this study, a series of alkyl-oligoamine derivatives of low-toxicity 10 kDa polyethylenimine (PEI) were synthesized to enhance the hydrophobicity of PEI while preserving most of its primary amine content. PEI was reacted with a series of omega-bromoalkylcarboxylic acids with different chain lengths (2-bromoacetic, 6-bromohexanoic, 10-bromodecanoic and 16-bromohexadecanoic acids) to modify hydrophobicity followed by coupling to various oligoamines (spermine, spermidine, ethylendiamine or diethylentriamine) to partially restore primary amine density. These modifications were designed to influence hydrophobic-hydrophilic balance as well as maintain the proton sponge effect in order to create an efficient vector with low toxicity. Ethidium bromide exclusion assays and dynamic light scattering studies showed that the modified PEIs could bind to plasmid DNA and form nanoparticles in the range of 100 nm. The transfection efficiency of modified PEIs complexed with a luciferase reporter gene (pCMV-luc) in N2A murine neuroblastoma cells was increased to a level comparable to that of 25,000 Da PEI. These results indicate that hydrophobic modification of low-toxicity PEI without reduction in primary amine content is an effective strategy for improving transfection efficiency of polycation-based non-viral vectors while maintaining low toxicity.

Alkylcarboxylate grafting to polyethylenimine: a simple approach to producing a DNA nanocarrier with low toxicity

Various strategies have been examined to improve both transfection efficiency and cytotoxicity of polyethylenimine (PEI), a widely used polycationic nonviral gene vector. In the present study, we sought to improve PEI transfection efficiency by combining the osmotic burst mechanism for lysing endocytotic vesicles with the lipid depletion mechanism, which was accomplished by maintaining buffering capacity at the same time as adding a lipid‐absorbing hydrophobic shell.

Non-covalent functionalization of single-walled carbon nanotubes with modified polyethyleneimines for efficient gene delivery

Functionalized carbon nanotubes (CNTs) have been recently emerged as important class of vectors for delivery of DNA and other biomolecules into various cells. In this study, single-walled carbon nanotubes (SWNTs) were functionalized by non-covalent binding of hydrophobic moieties, which were covalently linked to polyethyleneimines (PEIs). PEIs of three molecular weights (25, 10 and 1.8kDa) were used. CNTs were functionalized with the PEI series either through phospholipid moiety (via a polyethyleneglycol linker) or through directly-attached long (18 carbons) or intermediate (10 carbons) hydrophobic alkyl moieties. All PEI-functionalized CNTs exhibited good stability and dispersibility in biological media. Visualizing of functionalized CNTs and lack of aggregation were confirmed by atomic force microscopy. The PEI derivatives bound to CNTs retained the ability to fully condense plasmid DNA at low N/P ratios and substantial buffering capacity in the endosomal pH range. PEI-functionalized CNTs exhibited increased transfection efficiency compared to underivatized PEIs up to 19-fold increase being observed in the functionalized CNT with the smallest PEI tested, the smallest hydrophobic attachment moiety tested and no linker. Also PEI-functionalized CNTs were effective gene delivery vectors in vivo following tail vein injection in mice with the largest expression occurring with the vector PEI-functionalized through a polyethyleneglycol linker.

Dendrimeric Alkylated Polyethylenimine Nano-carriers with Acid-Cleavable Outer Cationic Shells Mediate Improved Transfection Efficiency Without Increasing Toxicity

PurposeImproved polycation-based non-viral DNA vectors were sought by preparing dendrimers with polyethylenimine cores surrounded by various shells incorporating structural features intended to facilitate steps in transfection mechanisms. Dendrimeric vectors were designed with (a) an outer oligocation shell, intended to facilitate DNA release inside cells, (b) a hydrophobic C-16 alkyl shell, and (c) a polycationic core, the latter two intended to combine lipid-depletion and osmotic burst endosome escape mechanisms, respectively, and were (d) attached through an a acid-cleavable linker reported to hydrolyze at endosomal pH values.MethodsVectors and DNA complexes were characterized by dynamic and static light scattering. Flow cytometry was used to quantitate transfection activity and cytotoxicity in CHO–K1 cells.ResultsAbout 5-fold increased transfection activity was obtained for a vector constructed with an outer shell of oligocations attached through acid-cleavable linkers, relative to a control dendrimer with an acid-stable linker. The most effective oligocation component of outer shells tested was spermine. Neither modification was associated with increased cytotoxicity. This vector design did not permit an evaluation of the benefit of combining endosome release mechanisms.ConclusionUsing acid-cleavable linkers to attach an outer shell of short, highly-charged oligocations to a PEI-based dendrimeric vector substantially increased transfection efficiency without increasing cytotoxicity.

From rationally designed polymeric and peptidic systems to sophisticated gene delivery nano-vectors

Lack of safe, efficient and controllable methods for delivering therapeutic genes appears to be the most important factor preventing human gene therapy. Safety issues encountered with viral vectors have prompted substantial attention to in vivo investigations with non-viral vectors throughout the past decade. However, developing non-viral vectors with effectiveness comparable to viral ones has been a challenge. The strategy of designing multifunctional synthetic carriers targeting several extracellular and intracellular barriers in the gene transfer pathway has emerged as a promising approach to improving the efficacy of gene delivery systems. This review will explain how sophisticated synthetic vectors can be created by combining conventional polycationic vectors such as polyethylenimine and basic amino acid peptides with additional polymers and peptides that are designed to overcome potential barriers to the gene delivery process.

Arginine-rich hydrophobic polyethylenimine: Potent agent with simple components for nucleic acid delivery

Conjugation of various arginine-rich peptide sequences to vectors based on 10 kDa polyethylenimine (PEI) and its hydrophobic derivative (hexanoate-PEI) was investigated as a strategy for improving pDNA and siRNA transfection activities. Six different arginine-histidine (RH) sequences and two arginine-serine (RS) sequences with a range of R/H ratios were designed and coupled to PEI and hexanoate-PEI. All arginine-rich peptide derivatives of PEI significantly enhanced luciferase gene expression compared to PEI 10 kDa alone. Hexanoate-PEI derivatives exhibited higher transfection activity than underivatized PEI vectors. Improved transfection activity may have resulted at least in part from use of higher vector/DNA ratios made possible by reduced cytotoxicity of vectors, and to use of vectors with higher molecular weights. Vectors that were the most efficient in pDNA delivery and transfection were also the most effective in siRNA delivery and protein expression knock down.

Targeted gene delivery with noncovalent electrostatic conjugates of sgc-8c aptamer and polyethylenimine.

Several strategies have been shown to improve the transfection efficiency of polyethylenimine (PEI) as a nonviral gene delivery vector. In the present study, a nucleic acid aptamer specific for protein tyrosine kinase 7 (PTK7) surface marker, sgc‐8c, was conjugated electrostatically to pre‐formed 10‐kDa PEI/plasmid DNA polyplexes, and the ability of the conjugate to transfer genetic material was evaluated in MOLT‐4 human acute lymphoblastic leukemia T‐cells, which express PTK7 on their surface.

Heterocyclic amine-modified polyethylenimine as gene carriers for transfection of mammalian cells

Branched polyethylenimine (PEI) is extensively used as a polycationic non-viral vector for gene delivery. Polyplexes formed with PEI are believed to be released from endocytotic vesicles by the osmotic burst mechanism in the rate-limiting step in transfection. Increasing the buffering capacity of PEI derivatives in the endosomal pH range of 4.5-7.5 should enhance transfection efficiency. In this study, PEI was derivatized by covalently attaching heterocyclic amine moieties (piperazine, pyridine and imidazole rings with pKa values from 5.23 to 6.04) through amide bonds. PEI derivatives with 50% of the primary amines on PEI exhibited increased buffering capacity, increased transfection activity and decreased cytotoxicity in murine neuroblastoma (Neuro-2a) cells. The relative effectiveness in enhancing transfection efficiency was piperazine>pyridine>histidine, but each type of amine was the most effective under a particular set of conditions. Modified vectors could significantly improve transfection efficiency in murine mesenchymal stem cells. PEI25 derivatized at 50% with histidine administered as polyplexes in the tail veins of mice resulted in remarkably enhanced luciferase gene expression in the expected organ distribution and much lower toxicity than underivatized PEI25.

Molecular weight-dependent genetic information transfer with disulfide-linked polyethylenimine-based nonviral vectors

One strategy for improving gene vector properties of polyethylenimine is to facilitate individual transfection mechanism steps. This study investigates (i) improving transfection efficiency by attaching peptide nuclear localization signals (nuclear localization signals: SV40 large T antigen nuclear localization signal or C-terminus of histone H1) to polyethylenimine (10 kDa) and (ii) using disulfide linkages, which are expected to be stable during polyplex formation, but cleaved inside cells giving improved gene release. Nuclear localization signal-containing polyplexes exhibited low cytotoxicity, whereas transfection efficiency with high molecular weight plasmid DNA increased up to 3.6 times that of underivatized polyethylenimine in Neuro2A cells at higher molar ratio of polyethylenimine-nitrogen to DNA-phosphate (N/P) ratios. However, with luciferase-specific low molecular weight small interfering RNA in Neuro2A/EGFPLuc cells, nuclear localization signal-containing polyplexes with disulfide linkages caused substantial cytotoxicity at N/P ratios >15 and no consistent significant reduction in luciferase expression. Possible explanations for molecular weight-dependent differences in genetic information transfer by polyplexes containing disulfide-linked nuclear localization signals are discussed.

Efficient megalin targeted delivery to renal proximal tubular cells mediated by modified-polymyxin B-polyethylenimine based nano-gene-carriers.

Non-viral vectors have attracted great interest, as they are simple to prepare, easy to modify and relatively safe, compared to viral vectors. Kidney-targeted gene delivery systems depict a promising technology to improve drug efficacy in renal diseases treatments. In order to develop a novel kidney-targeted gene delivery system, we synthesized polyamine-PEI conjugates using polymyxin B as ligand and investigated their potential targeting efficiency. After grafting either PEI25 kDa or PEI10 kDa with polymyxin B through amidation reaction, the modified-polymyxin-PEI/DNA-nanoplexes were produced via electrostatic attraction between the cationic polymers and EGFP plasmid. The properties of modified polymers including size, surface charge density, DNA condensation ability, buffering capacity and cytotoxicity were evaluated. Results revealed that the average size of -modified-polymyxin- PEI25kDa was about 143-180nm and modified-polymyxin-PEI10kDa 115-194nm. The zeta potentials were in the range of 16.4±1.87 to 23.43±1.25mV and 11.3±1.4 to 19.3±2.1mV for conjugates based on PEI25 and PEI10 respectively. The AFM images revealed that the complexes were spherical and nano-sized at C/P=4. The buffering capacity of both PEI 10 and 25kDa increased as the percentage of polymyxin B grafting increased. In vitro study demonstrated that modified-polymyxin-PEI conjugates could remarkably improve the gene transfection efficiency to kidney cells. The transfection efficiency of the polyplexes was dependent on the weight ratio of ligand in the formulation (~12 and 8 fold increase for PEI25 and PEI10kDa, respectively) and was significantly higher than that of unmodified PEIs/DNA nanoparticles. These results suggest that modified-polymyxin-PEI /DNA nanoparticles can effectively target megalin-expressing kidney cells and show improved transfection efficiency and low cytotoxicity in In vitro and In vivo studies. Animal studies confirmed the in vivo study. Thus, these conjugates can be considered as a safe and efficient non-viral therapeutic therapy vector for kidney diseases.