Poly(ethylenimine) (PEI) and PEI-based systems have been widely studied for use as nucleic acid delivery vehicles. However, many of these vehicles display high cytotoxicity, rendering them unfit for ...therapeutic use. By exploring the mechanisms that cause cytotoxicity, and through understanding structure–function relationships between polymers and intracellular interactions, nucleic acid delivery vehicles with precise intracellular properties can be tailored for specific function. Previous research has shown that PEI is able to depolarize mitochondria, but the exact mechanism as to how depolarization is induced remains elusive and therefore is the focus of the current study. Potential mechanisms for mitochondrial depolarization include direct mitochondrial membrane permeabilization by PEI or PEI polyplexes, activation of the mitochondrial permeability transition pore, and interference with mitochondrial membrane proton pumps, specifically Complex I of the electron transport chain and F0F1-ATPase. Herein, confocal microscopy and live cell imaging showed that PEI polyplexes do colocalize to some degree with mitochondria early in transfection, and the degree of colocalization increases over time. Cyclosporin a was used to prevent activation of the mitochondrial membrane permeability transition pore, and it was found that early in transfection cyclosporin a was unable to prevent the loss of mitochondrial membrane potential. Further studies done using rotenone and oligomycin to inhibit Complex I of the electron transport chain and F0F1-ATPase, respectively, indicate that both of these mitochondrial proton pumps are functioning during PEI transfection. Overall, we conclude that direct interaction between polyplexes and mitochondria may be the reason why mitochondrial function is impaired during PEI transfection.
Bicontinuous thermotropic liquid crystal (LC) materials, e.g., double gyroid (DG) phases, have garnered significant attention due to the potential utility of their 3D network structures in ...wide-ranging applications. However, the utility of these materials is significantly constrained by the lack of robust molecular design rules for shape-filling amphiphiles that spontaneously adopt the saddle curvatures required to access these useful supramolecular assemblies. Toward this aim, we synthesized anomerically pure Guerbet-type glycolipids bearing cellobiose head groups and branched alkyl tails and studied their thermotropic LC self-assembly. Using a combination of differential scanning calorimetry, polarized optical microscopy, and small-angle X-ray scattering, our studies demonstrate that Guerbet cellobiosides exhibit a strong propensity to self-assemble into DG morphologies over wide thermotropic phase windows. The stabilities of these assemblies sensitively depend on the branched alkyl tail structure and the anomeric configuration of the glycolipid in a previously unrecognized manner. Complementary molecular simulations furnish detailed insights into the observed self-assembly characteristics, thus unveiling molecular motifs that foster network phase self-assembly that will enable future designs and applications of network LC materials.
Abstract Progenitor and pluripotent cell types offer promise as regenerative therapies but transfecting these sensitive cells has proven difficult. Herein, a series of linear ...trehalose-oligoethyleneamine “click” copolymers were synthesized and examined for their ability to deliver plasmid DNA ( p DNA) to two progenitor cell types, human dermal fibroblasts (HDFn) and rat mesenchymal stem cells (RMSC). Seven polymer vehicle analogs were synthesized in which three parameters were systematically varied: the number of secondary amines (4–6) within the polymer repeat unit ( Tr433 , Tr530 , and Tr632 ), the end group functionalities PEG ( Tr4128 PEG-a, Tr4118 PEG-b ), triphenyl ( Tr4107 -c ), or azido ( Tr499 -d ), and the molecular weight (degree of polymerization of about 30 or about 100) and the biological efficacy of these vehicles was compared to three controls: Lipofectamine 2000, JetPEI, and Glycofect. The trehalose polymers were all able to bind and compact p DNA polyplexes, and promote p DNA uptake and gene expression luciferase and enhanced green fluorescent protein (EGFP) with these primary cell types and the results varied significantly depending on the polymer structure. Interestingly, in both cell types, Tr433 and Tr530 yielded the highest luciferase gene expression. However, when comparing the number of cells transfected with a reporter plasmid encoding enhanced green fluorescent protein, Tr433 and Tr4107 -c yielded the highest number of HDFn cells positive for EGFP. Interestingly, with RMSCs, all of the higher molecular weight analogs ( Tr4128 PEG-a, Tr4118 PEG-b, Tr4107 -c, Tr499 -d ) yielded high percentages of cells positive for EGFP (30–40%).
Improving the delivery of nucleic acids to diverse tissue types in culture is important for translating genome editing for regenerative cell therapies. Herein, we examine the effect of transfection ...media additives, such as the sulfated glycosaminoglycan heparin, in dramatically increasing pDNA delivery efficiency and transgene expression in a wide variety of cell types. Polyplexes formed by combining pDNA and Tr4, a cationic glycopolymer containing repeated trehalose and pentaethylenetetramine groups, were treated with low concentrations of heparin prior to in vitro transfection with plasmid DNA. Polyplex formulations were found to be stable and form ternary complexes upon heparin addition according to dynamic light scattering and ethidium bromide dye exclusion assays. Heparin-coated polyplexes offer significant increases (approximately 4-fold) in GFP expression compared to polyplexes prepared with Tr4 only in primary fibroblasts, U87, and HepG2 cells. Heparin was also shown to increase GFP expression in a linear, dose-dependent manner. The heparin-treated Tr4 polyplexes exhibited more than 50% higher cellular internalization with HepG2 cells while showing minimal increases with U87 and primary fibroblasts. Pharmacological inhibition was used to further understand the endocytic pathways taken during transfection in the presence and absence of heparin. It was found that heparin-treated polyplexes are endocytosed primarily through macropinocytosis and clathrin-mediated pathways, while Tr4 polyplexes without heparin appear to be internalized primarily via caveolae. Heparin appears to also modify the nuclear localization behavior of Tr4 polyplexes, which likely contributes to increased efficiency and transgene expression.
A class of cationic poly(alkylamidoamine)s (PAAAs) containing lipophilic methylene linkers were designed and examined as in vitro plasmid DNA (pDNA) delivery agents. The PAAAs were synthesized via ...step-growth polymerization between a diamine monomer and each of four different diacid chloride monomers with varying methylene linker lengths, including glutaryl chloride, adipoyl chloride, pimeloyl chloride, and suberoyl chloride, which served to systematically increase the lipophilicity of the polymers. The synthesized polymers successfully complexed with pDNA in reduced serum medium at N/P ratios of 5 and greater, resulting in polyplexes with hydrodynamic diameters of approximately 1 μm. These polyplexes were tested for in vitro transgene expression and cytotoxicity using HDFa (human dermal fibroblast), HeLa (human cervical carcinoma), HMEC (human mammary epithelial), and HUVEC (human umbilical vein endothelial) cells. Interestingly, select PAAA polyplex formulations were found to be more effective than Lipofectamine 2000 at promoting transgene expression (GFP) while maintaining comparable or higher cell viability. Transgene expression was highest in HeLa cells (∼90% for most formulations) and lowest in HDFa cells (up to ∼20%) as measured by GFP fluorescence. In addition, the cytotoxicity of PAAA polyplex formulations was significantly increased as the molecular weight, N/P ratio, and methylene linker length were increased. The PAAA vehicles developed herein provide a new delivery vehicle design strategy of displaying attributes of both polycations and lipids, which show promise as a tunable scaffold for refining the structure-activity-toxicity profiles for future genome editing studies.
We report the facile synthesis and characterization of 1,6-α linked functional stereoregular polysaccharides from biomass-derived levoglucosan
cationic ring-opening polymerization (cROP). ...Levoglucosan is a bicyclic acetal with rich hydroxyl functionality, which can be synthetically modified to install a variety of pendant groups for tailored properties. We have employed biocompatible and recyclable metal triflate catalysts - scandium and bismuth triflate - for green cROP of levoglucosan derivatives, even at very low catalyst loadings of 0.5 mol%. Combined experimental and computational studies provided key kinetic, thermodynamic, and mechanistic insights into the cROP of these derivatives with metal triflates. Computational studies reveal that ring-opening of levoglucosan derivatives is preferred at the 1,6 anhydro linkage and cROP proceeds in a regio- and stereo-specific manner to form 1,6-α glycosidic linkages. DFT calculations also show that biocompatible metal triflates efficiently coordinate with levoglucosan derivatives as compared to the highly toxic PF
used previously. Post-polymerization modification of levoglucosan-based polysaccharides is readily performed
UV-initiated thiol-ene click reactions. The reported levoglucosan based polymers exhibit good thermal stability (
> 250 °C) and a wide glass transition temperature (
) window (<-150 °C to 32 °C) that is accessible with thioglycerol and lauryl mercaptan pendant groups. This work demonstrates the utility of levoglucosan as a renewably-derived scaffold, enabling facile access to tailored polysaccharides that could be important in many applications ranging from sustainable materials to biologically active polymers.
We examine connections among polycation composition, DNA-polycation binding thermodynamics, binding strength, and resulting complex properties, for circular and linear DNA and hydrophilic diblock ...copolymers possessing cationic blocks. Two poly(2-deoxy-2-methacrylamido glucopyranose)-block-poly(N-(2-aminoethyl) methacrylamide) (PMAG-b-PAEMA), with block degrees of polymerization of PMAG56-b-PAEMA30 and PMAG52-b-PAEMA63, are employed. DNA binding behavior of these diblocks is also compared with that of a PAEMA homopolymer, in order to evaluate the role of the hydrophilic, charge-neutral PMAG block. In addition, DNA structure was varied, utilizing both circular and linear DNA with the same contour length. The enthalpy change due to DNA-polycation interactions (ΔH
) is observed via isothermal titration calorimetry (ITC) during titrations of DNA with the polycations. With circular DNA, a higher cationic content is found to result in a completion of binding with a smaller amount of polycation, as well as a larger initial ΔH
. In contrast to the common understanding that a neutral block simply provides colloidal stability, the PMAG block turns out to significantly impact both the extent of the binding and the size and dispersity of the final complexes. With a lower cationic content, the complex is less compact, but both the size and dispersity are more stable. Changes in circular dichroism spectra of DNA are shown to be correlated with PMAG-to-PAEMA block length ratio. PMAG52-b-PAEMA63 leads to stronger binding with DNA, compared to PMAG56-b-PAEMA30. Better-defined polyplexes and more disruption in the DNA helices are observed when the PMAG-to-PAEMA ratio is lower. All in all, while PMAG itself does not directly interact with DNA, the DNA-polycation binding turns out to be sensitive to the balance between the DNA-PAEMA attraction and PMAG solvation. In addition, it is confirmed that polyelectrolyte complexation is favored both entropically and enthalpically when the ionic strength of the solution is low. While only endothermic interactions occur in the buffered systems, exothermic initial interactions are observed in low-salt, unbuffered cases. Finally, complexes formed with linear DNA show clear bimodal size distributions, distinct from those formed with circular DNA. Collectively, these data provide insights into the controllable parameters in DNA-polycation complexation, which may advance the development of polymeric vehicles for large biomolecules such as nucleic acids.
Bottlebrush polymers have great potential as vehicles to noncovalently sequester, stabilize, and deliver hydrophobic small molecule actives. To this end, we synthesized a poly(
-isopropylacrylamide-
...-
,
-dimethylacrylamide) bottlebrush copolymer using ring-opening metathesis polymerization and developed a facile method to control the thermoresponsive properties using postpolymerization modification. Six increasingly hydrophilic end-groups were installed, yielding cloud point temperature control over a range of 22-42 °C. Solubility enhancement of the antiseizure medication, phenytoin, increased significantly with the hydrophilicity of the end-group moiety. Notably, carboxylated bottlebrush copolymers solubilized formulations with higher drug loadings than linear copolymers because they exist as unimolecular nanoparticles with a synthetically defined density of polymer chains that are more stable in solution. This work provides the first investigation of bottlebrush polymers for hydrophobic noncovalent sequestration and solubilization of pharmaceuticals.
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