RESUMO
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. NMR chemical shift, line width, and proton transverse relaxation rate measurements reveal that free and dendrimer-bound polyplex DNA exchange rapidly relative to the NMR time scale (Assuntos
DNA/química
, DNA/genética
, Vetores Genéticos/química
, Vetores Genéticos/genética
, Polímeros/química
, Dendrímeros/química
, Técnicas de Transferência de Genes
, Espectroscopia de Ressonância Magnética/métodos
, Polietilenoglicóis/química
RESUMO
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. As an intermediate phenotype, metabolite signatures capture a unique aspect of cellular dynamics that is not typically interrogated, providing a distinct perspective on cellular homeostasis. To date, there have been only a few metabolomics studies investigating cardiovascular diseases. In this review, we explore the principles of metabolomics and how it can provide further insight into the mechanisms of cardiovascular physiology and ultimately lead to improved diagnostic and therapeutic options for patients with cardiovascular disease.
Assuntos
Aterosclerose/metabolismo , Metabolômica , Doenças Cardiovasculares/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de MassasRESUMO
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. We also followed gene expression in cells sorted based on the retention of endogenous fluorescein. We have found that cell membrane porosity caused by polycationic vectors does not enhance internalization or gene expression. Based on this single-cell study, membrane permeability is found to be an unwanted side effect that limits transfection efficiency, possibly through leakage of the delivered nucleic acid through the pores prior to transcription and translation and/or activation of cell defense mechanisms that restrict transgene expression.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , DNA/genética , Poliaminas/farmacologia , Animais , Células COS , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , DNA/química , Dendrímeros/efeitos adversos , Dendrímeros/química , Citometria de Fluxo , Vetores Genéticos/efeitos adversos , Vetores Genéticos/química , Humanos , Modelos Teóricos , Polieletrólitos , TransfecçãoRESUMO
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. Herein, several analytical techniques such as dynamic light scattering, circular dichroism, thermal gravimetric analysis, isothermal titration calorimetry (ITC), and ethidium bromide exclusion assays were used to examine the pDNA binding strength of O4, S4, and A4, and the results are compared with the previous series of poly(glycoamidoamine)s. It was found that the length of the amide bond spacer in these nonhydroxylated analogs did affect the pDNA binding affinity to a small degree (binding affinity order A4 > S4 > O4). The increase in binding affinity with longer methylene spacer was not due to hydrophobic interactions but likely from optimization in electrostatic interactions and hydrogen bond formation. Even though O4 was revealed to have the lowest pDNA binding affinity of the nonhydroxylated series, this polymer yields the highest cellular transfection efficiency, which is likely an effect of the faster hydrolysis rate.
Assuntos
Amidas/química , Amidas/metabolismo , DNA/metabolismo , Plasmídeos/química , Poliaminas/química , Poliaminas/metabolismo , Linhagem Celular , DNA/genética , Plasmídeos/metabolismo , Ligação Proteica/genéticaRESUMO
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. Apocynin is a known antioxidant and inhibitor of NOX oxidase enzymes. A key phenol on the compound 1 controls ROS-initiated cyclization and makes 1 responsive to ROS with a EC50 comparable to common antioxidants in an ABTS assay. In an in vitro DNA nicking assay, the RIP reagent prevented DNA strand breaks. In cell-based assays, the reagent was not cytotoxic, apocynin was released only in cells treated with UVR, reduced UVR-induced cell death, and lowered DNA lesion formation. Finally, topical treatment of human skin explants with the RIP reagent reduced UV-induced DNA damage as monitored by quantification of cyclobutane dimer formation and DNA repair signaling via TP53. The reagent was more effective than administration of a catalase antioxidant on skin explants. This chemistry platform will expand the types of ROS-activated motifs and enable inhibitor release for potential use as a long-acting sunscreen.
Assuntos
Antioxidantes/administração & dosagem , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Estresse Oxidativo , Pele/efeitos dos fármacos , Raios Ultravioleta/efeitos adversos , Acetofenonas/administração & dosagem , Administração Tópica , Células Cultivadas , Ciclização , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Humanos , Queratinócitos/efeitos da radiação , Oxirredução , Espécies Reativas de Oxigênio , Pele/efeitos da radiaçãoRESUMO
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. Dynamic light scattering measurements revealed that the polyplexes formed with the longer polymers (n(w)=53, 75, or 100) inhibited flocculation in media containing serum, whereas the polyplexes formed with the shorter polymer (n(w)=35) aggregated rapidly. Similar results were observed via transmission electron microscopy studies, where the nanoparticles formed with the polymers having longer degrees of polymerization showed discrete particles in media containing 10% serum. Transfection experiments revealed that the polymers exhibited low cytotoxicity at low N/P ratios and could facilitate high cellular uptake and gene expression in HeLa and H9c2(2-1) cells, and the results were dependent on the degrees of polymerization (longer polymers yielded higher transfection and toxicity).
Assuntos
Nanopartículas/química , Plasmídeos/genética , Polímeros/química , Transfecção/métodos , Trealose/química , Ligação Competitiva , Linhagem Celular , DNA/química , DNA/genética , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão , Nanopartículas/ultraestrutura , Plasmídeos/química , Plasmídeos/metabolismo , Polímeros/síntese química , Polímeros/metabolismo , Reprodutibilidade dos Testes , Cloreto de Sódio/químicaRESUMO
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. Low conjugation ratios of 750Da PEG to PEI resulted in little decrease in DNA affinity, but a significant decrease-up to two orders of magnitude-was found for the other copolymers. We identified limitations in determination of affinity using indirect assays (electrophoretic mobility shift and ethidium bromide exclusion) commonly used in the field. Dynamic light scattering of the DNA complexes at physiological ionic strength showed that PEI modifications that did not reduce DNA affinity also did not confer significant colloidal stability, a finding that was supported by calorimetric data on the aggregation process. These results quantify the DNA interaction thermodynamics of PEGylated polycations for the first time and indicate that there is an optimum PEG chain length and degree of substitution in the design of agents that have desirable properties for effective in vivo gene delivery.
Assuntos
DNA/química , Polietilenoglicóis/química , Polietilenoimina/análogos & derivados , Termodinâmica , Calorimetria , Coloides/química , Estrutura Molecular , Peso Molecular , Polietilenoglicóis/síntese química , Polietilenoimina/síntese química , Polietilenoimina/químicaRESUMO
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. Detailed thermodynamic analysis has determined that CPC-GAG binding constants range from 8.7×10(3) to 2.4×10(6)M(-1) and that affinity is dependent upon GAG charge density and stereochemistry and CPC molecular weight. The effect of GAG composition on affinity is likely due to hydrogen bonding between CPC amines and amides and GAG hydroxyl and amine groups. These results were compared to the association of CPCs with lipid vesicles of varying composition as model plasma membranes to finally clarify the relative importance of each cell surface component in initial cell recognition. CPC-lipid affinity increases with anionic lipid content, but GAG affinity is higher for all cell-penetrating compounds, confirming the role these heterogeneous polysaccharides play in cellular association and clustering.
Assuntos
Glicosaminoglicanos/metabolismo , Bicamadas Lipídicas/metabolismo , Calorimetria , Dendrímeros/química , Glicosaminoglicanos/química , Bicamadas Lipídicas/química , Lipossomos/química , Lipossomos/metabolismo , TermodinâmicaRESUMO
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. The size of these pDNA-polymer complexes, or polyplexes, at various states of formation was determined through light scattering and zeta-potential measurements. Varying degrees of pDNA secondary structure change occurred upon interaction with the polymers, as evidenced by circular dichroism spectra through increasing molar ratios of polymer amine to DNA phosphate, and Fourier transform infrared (FT-IR) results demonstrated stronger electrostatic binding with the phosphate backbone with the least amine-dense of the series. It was concluded that, depending on the number of secondary amines in the repeat unit, these polymers interact with pDNA via different mechanisms with varying extents of electrostatic interaction and hydrogen bonding. These differing mechanisms may affect the ability of trehalose to serve as a deterrent against aggregation in serum conditions and lend insight into the roles of polymer-pDNA binding during the complex transfection process.
Assuntos
Aminas/química , DNA/química , Plasmídeos/química , Poliaminas/química , Trealose/química , Calorimetria , Dicroísmo Circular , Estrutura Molecular , Peso Molecular , Tamanho da Partícula , Polieletrólitos , Potenciometria , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , Termodinâmica , Titulometria , Água/químicaRESUMO
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). The size and charge of the polyplexes at various ITC injection points were then investigated by light scattering and zeta-potential measurements to provide comprehensive insight into the formation of these polyplexes. An analysis of the calorimetric data revealed a three-step process consisting of two different endothermic contributions followed by the condensation/aggregation of polyplexes. The strength of binding and the point of charge neutralization were found to be dependent upon the hydroxyl stereochemistry of the carbohydrate moiety within each polymer repeat unit. Circular dichroism spectra reveal that the PGAAs induce pDNA secondary structure changes upon binding, which suggest a direct interaction between the polymers and the DNA base pairs. Infrared spectroscopy experiments confirmed both base pair and phosphate group interactions and, more specifically, showed that the stronger-binding PGAAs had more pronounced interactions at both sites. Thus, we conclude that the mechanism of poly(glycoamidoamine)-pDNA binding is most likely a combination of electrostatics and hydrogen bonding in which long-range Coulombic forces initiate the attraction and hydroxyl groups in the carbohydrate comonomer, depending on their stereochemistry, further enhance the association through hydrogen bonding to the DNA base pairs.