UV cell stress induces oxidative cyclization of a protective reagent for DNA damage reduction in skin explants.

UV irradiation is a major driver of DNA damage and ultimately skin cancer. UV exposure leads to persistent radicals that generate ROS over prolonged periods of time. Toward the goal of developing long-lasting antioxidants that can penetrate skin, we have designed a ROS-initiated protective (RIP) reagent that, upon reaction with ROS (antioxidant activity), self-cyclizes and then releases the natural product apocynin.

Cell-penetrating compounds preferentially bind glycosaminoglycans over plasma membrane lipids in a charge density- and stereochemistry-dependent manner.

Cell-penetrating compounds (CPCs) are often conjugated to drugs and genes to facilitate cellular uptake. We hypothesize that the electrostatic interaction between the positively charged amines of the cell-penetrating compounds and the negatively charged glycosaminoglycans (GAGs) extending from cell surfaces is the initiating step in the internalization process. The interactions of generation 5 PAMAM dendrimer, Tat peptide and 25 kDa linear PEI with four different GAGs have been studied using isothermal titration calorimetry to elucidate structure-function relationships that could lead to improved drug and gene delivery methods to a wide variety of cell types.

Impact of molecular weight and degree of conjugation on the thermodynamics of DNA complexation and stability of polyethylenimine-graft-poly(ethylene glycol) copolymers.

Poly(ethylene glycol) (PEG) is often conjugated to polyethylenimine (PEI) to provide colloidal stability to PEI-DNA polyplexes and shield charge leading to toxicity. Here, a library of nine cationic copolymers was synthesized by grafting three molecular weights (750, 2000, 5000Da) of PEG to linear PEI at three conjugation ratios. Using isothermal titration calorimetry, we have quantified the thermodynamics of the associations between the copolymers and DNA and determined the extent to which binding is hindered as a function of PEG molecular weight and conjugation ratio.

Intrinsic dynamics of DNA-polymer complexes: a mechanism for DNA release.

The transfer of genetic material into cells using nonviral vectors offers unique potential for therapeutics; however, the efficacy of delivery depends upon a poorly understood, multistep pathway, limiting the prospects for successful gene delivery. Mechanistic insight into DNA association and release has been hampered by a lack of atomic resolution structural and dynamic information for DNA-polymer complexes (polyplexes). Here, we report a dendrimer-based polyplex system containing poly(ethyleneglycol) (PEG) arms that is suitable for atomic-level characterization by solution NMR spectroscopy.

Metabolomics and atherosclerosis.

Metabolites reflect the dynamic processes underlying cellular homeostasis. Recent advances in analytical chemistry and molecular biology have set the stage for metabolite profiling to help us understand complex molecular processes and physiology. Metabolomics is the comparative analysis of metabolite flux and how it relates to biological phenotypes.

Polycation-induced cell membrane permeability does not enhance cellular uptake or expression efficiency of delivered DNA.

Polycationic materials commonly used to delivery DNA to cells are known to induce cell membrane porosity in a charge-density dependent manner. It has been suggested that these pores may provide a mode of entry of the polymer-DNA complexes (polyplexes) into cells. To examine the correlation between membrane permeability and biological activity, we used two-color flow cytometry on two mammalian cell lines to simultaneously measure gene expression of a plasmid DNA delivered with four common nonviral vectors and cellular uptake of normally excluded fluorescent dye molecules of two different sizes, 668 Da and 2 MDa.

Amide spacing influences pDNA binding of poly(amidoamine)s.

Previously, a series of three poly(amidoamine)s was designed and synthesized by polymerizing oxylate, succinate, or adipate groups with pentaethylenehexamine. These resulting polymers (named O4, S4, and A4, respectively) were created as models to poly(glycoamidoamine) nucleic acid delivery agents to understand how the absence of hydroxyl groups and changes in the amide bond spacing affect polymer degradation, plasmid DNA (pDNA) encapsulation, toxicity, and transfection efficiency in vitro. To understand differences in the biological properties quantitatively, we investigated the mechanism of interaction between these macromolecules and pDNA to reveal differences in pDNA binding affinity and complexation as a function of structure.

Correlation of amine number and pDNA binding mechanism for trehalose-based polycations.

Glycopolymers with repeat units comprised of the disaccharide trehalose and an oligoamine of increasing amine have been previously synthesized by our group and shown to efficiently deliver pDNA (plasmid DNA) to HeLa cells while remaining relatively nontoxic. Complexes formed between the most amine-dense of these polycations and pDNA were also found to be relatively stable in serum and have low aggregation, which is desirable for in vivo gene delivery. To lend insight into these interesting results, this study was aimed at investigating the binding strength and mechanism of interaction between these macromolecules, via isothermal titration calorimetry (ITC) and ethidium bromide exclusion assays.

Deciphering the role of hydrogen bonding in enhancing pDNA-polycation interactions.

There is considerable interest in the binding and condensation of DNA with polycations to form polyplexes because of their possible application to cellular nucleic acid delivery. This work focuses on studying the binding of plasmid DNA (pDNA) with a series of poly(glycoamidoamine)s (PGAAs) that have previously been shown to deliver pDNA in vitro in an efficient and nontoxic manner. Herein, we examine the PGAA-pDNA binding energetics, binding-linked protonation, and electrostatic contribution to the free energy with isothermal titration calorimetry (ITC).

Effects of trehalose click polymer length on pDNA complex stability and delivery efficacy.

Cationic polymers are currently being studied as non-viral vectors to deliver therapeutic DNA into cells. In this study, a series of trehalose-based glycopolymers containing four secondary amines in the repeat unit were synthesized via the 'click reaction' [degrees of polymerization (n(w))=35, 53, 75, or 100] to elucidate how the polymer length affects the bioactivity. The four structures bound and charge-neutralized pDNA with similar affinity that was independent of the length, as determined through gel electrophoresis, heparin competitive displacement, and isothermal titration calorimetric assays.

Trehalose click polymers inhibit nanoparticle aggregation and promote pDNA delivery in serum.

Herein, three new glycopolymers have been synthesized via "click polymerization" to promote nucleic acid delivery in the presence of biological media containing serum. These structures were designed to contain a trehalose moiety to promote biocompatibility, water solubility, and stability against aggregation, amide-triazole groups to enhance DNA binding affinity, and an oligoamine unit to facilitate DNA encapsulation, phosphate neutralization, and interactions with cell surfaces. A 2,3,4,2',3',4'-hexa-O-acetyl-6,6'-diazido-6,6'-dideoxy-D-trehalose (4) monomer was polymerized via copper(I)-catalyzed azide-alkyne cycloaddition with a series of dialkyne-amide comonomers that contain either one, two, or three Boc-protected secondary amines (7a, 7b, or 7c, respectively).