Biomedical subjects

David Putnam

Publications and source records attributed to David Putnam.

At least 19 recordsLinked to original sources

PubMed2006

Characterizing the structure/function parameter space of hydrocarbon-conjugated branched polyethylenimine for DNA delivery in vitro.

Polyethylenimine is a popular DNA transfection reagent, and many approaches have been explored to further enhance its transfection efficiency. Substitution of branched polyethylenimine's primary amine groups is an attractive approach because it is amenable to a variety of chemistries and is also implicated as a primary factor in its cytotoxicity. The purpose of this work was to serially substitute saturated hydrocarbons to branched polyethylenimine and determine what structure/function relationships exist between the hydrocarbon length and its degree of substitution, relative to transfection efficiency in multiple cell lines. Specifically, acetate, butanoate and hexanoate were conjugated to branched polyethylenimine (M(w) = 25,000) using an aqueous condensation protocol. Transfections were performed in culture using HeLa, NIH/3T3 and Clone 9 cell lines. Biophysical characteristics of the polyelectrolyte complexes were also measured (hydrodynamic diameter, relative binding affinity) and correlated to transfection efficiency. The results show that substitution of the primary amines generally increases transfection efficiency relative to unconjugated polyethylenimine, but increasing the degree of substitution beyond approximately 25 mol% generally decreases transfection efficiency from the optimum. Additionally, increasing hydrocarbon length generally decreased transfection efficiency. There was little correlation between particle size and binding efficiency to transfection efficiency.

Acylation↗

PubMed2006

Biophysical and structural characterization of polyethylenimine-mediated siRNA delivery in vitro.

PURPOSE: The goals of this study were as follows: 1) to evaluate the efficacy of different polyethylenimine (PEI) structures for siRNA delivery in a model system, and 2) to determine the biophysical and structural characteristics of PEI that relate to siRNA delivery. MATERIALS AND METHODS: Biophysical characterization (effective diameter and zeta potential), cytotoxicities, relative binding affinities and in vitro transfection efficiencies were determined using nano-complexes formed from PEI's of 800, 25,000, (both branched) and 22,000 (linear) molecular weights at varying N:P ratios and siRNA concentrations. The HR5-CL11 cell line stably expressing luciferase was used as a model system in vitro. RESULTS: Successful siRNA delivery was observed within a very narrow window of conditions, and only with the 25,000 branched PEI at an N:P ratio of 6:1 and 8:1 and with 200 nM siRNA. While the zeta potential and size of PEI:siRNA complexes correlated to transfection efficacy in some cases, complex stability may also affect transfection efficacy. CONCLUSIONS: The ability of PEI to transfer functionally active siRNA to cells in culture is surprisingly dependent on its biophysical and structural characteristics when compared to its relative success and ease of use for DNA delivery.

Biophysical Phenomena↗

PubMed2006

A functionalizable biomaterial based on dihydroxyacetone, an intermediate of glucose metabolism.

A biomaterial and its potential degradation products should be biocompatible, nontoxic, and removed by the body upon expiration of its functional lifetime. One historically successful approach is to create new materials from biomolecules that naturally occur in the human body. Herein, we report the synthesis and characterization of a polycarbonate based on dihydroxyacetone, a 3-carbon ketose, and an intermediate in the glucose metabolic pathway. The polymer was synthesized in a range of molecular weights ( approximately 8000 to approximately 37,500) by ring-opening polymerization. The C2 carbonyl of dihydroxyacetone is reactive to amines, and this reactivity was used to functionalize the polymer's surface in a one-step reaction by reductive amination. Additionally, contact angle measurements show the surface of poly(2-oxypropylene carbonate) is hydrophilic even though it is insoluble in water. Mechanical analysis of the polymer revealed it is exceptionally strong for an aliphatic polycarbonate. Specifically, poly(2-oxypropylene carbonate), M(w) 37 500, yielded a Young's modulus of 0.5 GPa and a compressive yield stress of 50 MPa. These values equal or exceed those of cancellous bone with similar dimensions.

Biocompatible Materials↗

PubMed2006

Diblock copolymers based on dihydroxyacetone and ethylene glycol: synthesis, characterization, and nanoparticle formulation.

Polymeric biomaterials have played an integral role in tissue engineering, biomedical devices, and targeted drug delivery. Block copolymers are especially important because their physical and chemical properties can be controlled by adjusting the ratio, size, and type of constituting blocks. Herein, the synthesis and characterization of diblock copolymers composed of poly(ethylene glycol) and a polycarbonate based on the metabolic intermediate, dihydroxyacetone, are reported. The length of the dihydroxyacetone-based block was controlled by adjusting the reactant feed ratios and initiator injection conditions. Intermediates and final products were characterized via (1)H NMR, GPC, DSC, TGA, and diffusion-ordered NMR spectroscopy. The dihydroxyacetone-based hompolymer is insoluble in water and most organic solvents, but is hydrophilic in nature. This, coupled with poly(ethylene glycol)'s solubility characteristics, allows the block copolymer to form nanoparticles in aqueous and organic anti-solvents. Dynamic light scattering and TEM results indicated the formation of spherical nanoparticles.

Calorimetry, Differential Scanning↗

PubMed2006

Polymers for gene delivery across length scales.

A number of human diseases stem from defective genes. One approach to treating such diseases is to replace, or override, the defective genes with normal genes, an approach called 'gene therapy'. However, the introduction of correctly functioning DNA into cells is a non-trivial matter, and cells must be coaxed to internalize, and then use, the DNA in the desired manner. A number of polymer-based synthetic systems, or 'vectors', have been developed to entice cells to use exogenous DNA. These systems work across the nano, micro and macro length scales, and have been under continuous development for two decades, with varying degrees of success. The design criteria for the construction of more-effective delivery vectors at each length scale are continually evolving. This review focuses on the most recent developments in polymer-based vector design at each length scale.

DNA↗

PubMed2006

Identification of compartments involved in mammalian subcellular trafficking pathways by indirect immunofluorescence.

A characteristic of a successful DNA vaccine is its trafficking to the nucleus where it can be transcribed. Plasmid DNA coupled to a delivery vector must enter the cell, navigate its way through endocytic compartments, and ultimately reach the nucleus. Currently, the precise pathway taken by plasmid DNA is not clear. Understanding how plasmid DNA interacts with the cell and which path it follows to reach the nucleus will aid in the rational design of improved delivery vectors. Achieving this goal requires a means by which to monitor the subcellular trafficking of plasmid DNA and delivery vectors. Presented here are methods for identifying various endocytic compartments involved in mammalian subcellular trafficking pathways using indirect immunofluorescence. Together with labeled delivery vectors and/or plasmid DNA, these methods can aid in the understanding of the trafficking pathways involved in DNA delivery, and contribute to the rational design of more efficient delivery vectors.

Active Transport, Cell Nucleus↗

PubMed2005

Structure-function relationships of gene delivery vectors in a limited polycation library.

A library of 13 polylysine-graft-imidazoleacetic acid conjugates was synthesized to examine the collective effects of polymer molecular weight, side chain substitution, and DNA:polymer ratio on cytotoxicity, transfection efficiency, and polycation-DNA interaction. In general, the relationships between the physicochemical characteristics and the gene transfer capabilities of these polycations appear nonlinear. The in vitro cytotoxicity of these polymers decreased, while total protein expression increased, with decreasing molecular weight and increasing imidazole content. Flow cytometry experiments indicated, however, that an increase in marker gene expression does not always correlate with the total number of cells transfected, even when similar polymer structures are used for transfection. The maximum level of luciferase gene expression was mediated by transfection with a low molecular weight, high imidazole content (9400 Mw, 95 mol% imidazole) polymer. The extent of DNA condensation, as determined by ethidium bromide fluorescence quenching, also decreased with decreasing polymer molecular weight and increasing imidazole content. Relative binding affinity between DNA and the polycations, measured via competitive binding in the presence of a synthetic polyanion, decreased with decreasing polymer molecular weight; however, the relative affinity also appeared to increase with increasing imidazole, suggesting that electrostatic contributions are not solely responsible for DNA-polycation binding interactions. This limited library and corresponding structure/function analysis forms the foundation upon which larger, more comprehensive polycationic libraries can be designed and evaluated to further understand how polycation transfection reagents function.

Animals↗

PubMed2005

Biomaterial microarrays: rapid, microscale screening of polymer-cell interaction.

The identification of biomaterials that induce optimal gene expression patterns and allow for appropriate levels of cellular attachment is of central importance in tissue engineering and cell therapy. Herein, we describe the creation of cell-compatible, biomaterial microarrays, that allow rapid, microscale testing of biomaterial interactions with cells. As proof of principle, we simultaneously characterized over 3456 human mesenchymal stem cell (hMSC)-biomaterial composite interactions, and describe preliminary studies on the utility of these arrays with a neural stem cell line (NSC), and primary articular chondrocytes.

Biocompatible Materials↗

PubMed2005

Breath ammonia testing for diagnosis of hepatic encephalopathy.

Measurement of arterial ammonia has been used as a diagnostic test for hepatic encephalopathy, but obtaining an arterial specimen is an invasive procedure. The aim of this study was to evaluate the ability of a minimally invasive, highly sensitive optical sensing device to detect ammonia in the breath of patients with end-stage liver disease and to evaluate the correlation of breath ammonia levels, arterial ammonia levels, and psychometric testing. Fifteen subjects with liver cirrhosis and clinical evidence of hepatic encephalopathy underwent mini-mental status examination, number connection test, focused neurological examination, and arterial ammonia testing. On the same day, breath ammonia testing was performed using an apparatus that consists of a sensor (a thin membrane embedded with a pH-sensitive dye) attached to a fiberoptic apparatus that detects optical absorption. Helicobacter pylori testing was performed using the 14C urea breath test. A positive correlation was found between arterial ammonia level and time to complete the number connection test (r = 0.31, P = 0.03). However, a negative correlation was found between breath ammonia level and number connection testing (r = -0.55, P = 0.03). Furthermore, no correlation was found between breath and arterial ammonia levels (r = -0.005, P = 0.98). There is a significant correlation between the trailmaking test and arterial ammonia levels in patients with cirrhosis. However, no correlation was found between breath and arterial ammonia levels using the fiberoptic ammonia sensor apparatus in this small study.

Ammonia↗

PubMed2004

Molecularly engineered poly(ortho ester) microspheres for enhanced delivery of DNA vaccines.

Genetic vaccination using plasmid DNA presents a unique opportunity for achieving potent immune responses without the potential limitations of many conventional vaccines. Here we report the design of synthetic biodegradable polymers specifically for enhancing DNA vaccine efficacy in vivo. We molecularly engineered poly(ortho ester) microspheres that are non-toxic to cells, protect DNA from degradation, enable uptake by antigen-presenting cells, and release DNA rapidly in response to phagosomal pH. One type of microsphere of poly(ortho esters) that releases DNA vaccines in synchrony with the natural development of adaptive immunity, elicited distinct primary and secondary humoral and cellular immune responses in mice, and suppressed the growth of tumour cells bearing a model antigen. This polymer microparticulate system could, with further study, have implications for advancing the clinical utility of DNA vaccines as well as other nucleic-acid-based therapeutics against viral infections and cancer.

Adjuvants, Immunologic↗

PubMed2004

Determination of P-glycoprotein inhibition by excipients and their combinations using an integrated high-throughput process.

Excipients often used in pharmaceutical formulations have been reported to have inhibitory effects on P-glycoprotein, an important membrane-associated transport protein. Because inhibition of efflux transporters can have an effect on drug bioavailability, identification of these excipients and their extent of inhibition are therefore important for pharmaceutical development. We have developed an automated and integrated high-throughput process for identifying these excipients and their combinations. Common excipients containing polyethylene glycol (PEG) in the chemical structure were screened using a cytotoxic cell growth assay, and excipients giving inhibition were further combined to identify synergistic effects. Our screens identified excipients previously reported to inhibit P-glycoprotein, such as PEG stearates, PEG fatty acid esters, polysorbates, and poloxamers. We also found new excipients, such as those in the PEG glyceryl fatty acid family, which were among the best inhibitors identified. Dose-response studies of these compounds and of cyclosporin A indicated that the extent of inhibition depended logarithmically on the concentration. This suggests a similar mechanism by which inhibition is obtained, despite widely varying chemical structures. In the particular set of combinatorial studies performed, which involved >20,000 samples, we found that inhibitory effects in binary combinations followed the single-excipient logarithmic trend, rather than being synergistic. These experiments showcased the potential for integrated high-throughput processes that enable combinatorial screens which would otherwise be difficult to perform manually.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Enhancement of a human immunodeficiency virus env DNA vaccine using a novel polycationic nanoparticle formulation.

In an effort to develop a more effective DNA immunization strategy for HIV, we synthesized an HIV-2 env DNA vaccine and delivered it in a novel polycationic adjuvant formulation that forms nanoparticles in solution and enhances protein expression. The polycationic adjuvant contained imidazole moieties to facilitate endosomal escape. Nanoparticles containing the DNA vaccine plasmid were formed by electrostatic condensation with the polycationic adjuvant. We hypothesized that this formulation would improve immune responses to the gp140 env gene from HIV-2(UC2) by increasing the level of expressed antigen. We found that the nanoparticles were superior at inducing high levels of systemic antibody responses compared to naked DNA when delivered by the intradermal route in BALB/c mice. In addition, the nanoparticles induced higher levels of IgM, IgG, and IgA antibodies. These results suggest that nanoparticles may be an important adjuvant formulation to improve the effectiveness of genetic immunization and rationalize its use in the evaluation of vaccine candidates in non-human primate models for AIDS.

AIDS Vaccines↗

PubMed2003

Competitive reactions in solutions of poly-L-histidine, calf thymus DNA, and synthetic polyanions: determining the binding constants of polyelectrolytes.

The physicochemical characteristics of a nonviral gene delivery system will govern its functional bioactivity; however, empiricism dominates the literature in this field, and a significant deficiency of quantitative investigation and evaluation of nonviral gene delivery vehicles remains. Herein, we derive a physical model and experimental method to quantitatively determine the binding constants between a model polycationic nonviral gene delivery vehicle poly-L-histidine (PLH) and calf thymus DNA. The approach has utility to a variety of systems and is not limited to the described polymer model. The interaction of PLH with DNA was monitored by fluorescence quenching of an ethidium bromide probe in the pH range 4 to 8. The interaction increased with pH decrease with the most pronounced change between pH 6 and 7. The obtained pH-dependence of fraction of salt bonds formed between PLH and DNA was used to estimate pK(a) of PLH in the presence of DNA, which equaled 6.24. The interaction of PLH with DNA in the presence of added synthetic polyanions was studied by the same approach and found to be controlled by pH, nature of the charge groups of the polyanion, and its degree of polymerization. In the mixture with sodium poly(styrenesulfonate) the interaction was negligible in the whole studied pH range, whereas in the mixtures with sodium poly(acrylate) (PA) or sodium poly(methacrylate), DNA was able to compete effectively for the binding with PLH. For PA samples with degree of polymerization higher than degree of polymerization of PLH, DP(PA) > DP(PLH), the fraction of polycation bound to DNA was constant regardless of DP(PA.) In contrast, at DP(PA) < DP(PLH), a pronounced increase in the bound fraction was observed. It substantiates the notion that the binding energy of two polymers is mainly controlled by the DP of the shorter component of polyelectrolyte complex. The data on PLH distribution between DNA and added polyanion with different values of DP were treated according to the developed procedure to yield the effective binding constants of PLH with DNA and polyanion-competitor, calculated both per mole of interacting units K(1) and mole of interacting chains K(n). In all cases, K(1) had similar numerical values reflecting common type of interaction stabilizing the complexes, i.e., electrostatics. Slight variation of K(1) yielded in drastic changes in K(n) and alteration of dominance of PLH interaction with DNA or synthetic polyanion. The results of the study can have a high impact in deriving the correlation between the binding constant of a polycation to DNA and its ability to serve as gene delivery vehicle.

Acrylic Resins↗

PubMed2003

Polyhistidine-PEG:DNA nanocomposites for gene delivery.

Complexation of plasmid DNA with polycations is a popular method by which to transfer therapeutic nucleic acid sequences to cells. One caveat of the approach is that the positive zeta potential of the complexes facilitates interaction with blood constituents, leading to serum protein adsorption and complement activation. As a countermeasure, investigators have developed polycations combined with polyethylene glycol (PEG) to create complexes with reduced protein adsorption potential. We have designed and synthesized PEG-polyhistidine conjugates to evaluate the material class as potential gene delivery vehicles. Two conjugate architectures (comb-shaped and linear A-B block copolymers) were synthesized and formulated with plasmid DNA. The complexes were characterized with respect to DNA complexation capacity, hydrodynamic diameter, zeta potential, in vitro cytotoxicity and transfection capacity in a model cell line. PEG content of the conjugate significantly influenced the hydrodynamic diameter of the DNA:conjugate composite in aqueous solution. For comb-shaped conjugates steric hindrance attributed to PEG led to a direct relationship between the PEG content and the complex size. Both architectures could condensed plasmid DNA into complexes with hydrodynamic diameters <150 nm. Complexation of DNA with the polyhistidine-PEG conjugates resulted in nanocomposites with negative zeta potentials that retarded DNase I-mediated hydrolysis, and all conjugates showed low cytotoxicity to macrophages cultured in vitro. The transfection efficiency was approximately equivalent to DNA:polylysine complexes. The formulation characteristics and low cytotoxicity suggest that polyhistidine-PEG conjugates may be useful for gene delivery.

Animals↗

PubMed2002

A retrospective electronic chart review of blood pressure changes in elderly patients treated with amlodipine or an angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker.

BACKGROUND: Despite the high costs of managing hypertension, pharmacologic intervention is cost-effective, particularly in patients at highest risk for cardiovascular events. The prevalence of hypertension in the elderly and the age-associated risks of coronary artery disease and stroke suggest that early identification and aggressive treatment should be priorities in this population. OBJECTIVE: The aim of this study was to compare the effect of amlodipine and angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs) in the treatment of essential hypertension in elderly patients (>60 years) in an actual practice setting. METHODS: This was a retrospective cohort analysis using electronic medical records stored in the Physicians Data Corporation cardiology database. Patients aged >60 years who received care from a cardiologist and who had a recorded diagnosis of hypertension during 1997 or 1998 were identified. For inclusion, patients had to have received an initial prescription for amlodipine, an ACE inhibitor, or an ARB at the index visit. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) readings from the index visit and > or =1 subsequent visit (<180 days after the index visit) were assessed. RESULTS: A total of 192 patients (56.3% male; mean age, 71.9 years) met the inclusion criteria. Amlodipine-treated patients experienced a mean decrease in SBP of 26.7 mm Hg, compared with 18.8 mm Hg in patients receiving an ARB and 15.8 mm Hg for patients receiving an ACE inhibitor (P = 0.008, amlodipine vs ACE inhibitor). DBP decreased 8.8 mm Hg with amlodipine, 8.7 mm Hg with an ARB, and 6.2 mm Hg with an ACE inhibitor. After adjusting for age, sex, and disease severity, amlodipine-treated patients were -4 times as likely to move to a better blood pressure stage than patients treated with an ARB or an ACE inhibitor (odds ratio, ARB vs amlodipine: 0.245; 95% CI, 0.080-0.753; odds ratio, ACE inhibitor vs amlodipine: 0.234; 95% CI, 0.072-0.761). CONCLUSION: Results of this study indicate that in patients aged >60 years, amlodipine may be an effective therapy for hypertension.

Aged↗

PubMed2002

Breath ammonia measurement in Helicobacter pylori infection.

Our aim was to define the utility of breath ammonia measurement in assessing Helicobacter pylori infection. Volunteers breathed into a device containing three fiberoptic NH3 sensors at baseline and after ingesting 300 mg of urea. Breath ammonia levels were compared to the [14C]urea breath test. Thirteen subjects were tested. Before urea ingestion, H. pylori-positive subjects had significantly lower breath ammonia levels than negative subjects (mean +/- SD, 0.04 ppm +/- 0.09 vs 0.49 ppm +/- 0.24, P = 0.002) and had a significantly greater increases in breath ammonia after urea ingestion (range 198-1,494% vs 6-98%). One H. pylori-positive subject underwent treatment and breath ammonia levels shifted from the pattern seen in positive subjects to that seen in negative subjects. In conclusion, breath ammonia measurement for H. Pylori-positive and negative subjects showed distinct patterns. Breath ammonia measurement may be feasible as a diagnostic test for H. pylori.

Adult↗

PubMedDate not supplied

Aliphatic ionenes as gene delivery agents: elucidation of structure-function relationship through modification of charge density and polymer length.

Polycation-based gene delivery agents are generally polydisperse populations whose properties are averaged among the different molecular weight species. Therefore, to understand the physicochemical properties of polycations and their relationships to cellular gene transfer, one needs to control the molecular weight of the polymer as well as its cationic charge density. To investigate the structure-function correlation of polycations with respect to the degree of polymerization (DP) and charge density, a series of model materials based on aliphatic ionenes was synthesized and fractionated into distinct molecular weight fractions with DP range from 14 to 32. The aliphatic ionene fractions and their polyelectrolyte complexes (PEC) with DNA were studied using physicochemical and biological methods. Ionene polymers were shown to possess low cytotoxicity (minimal viability of the P388D1 murine macrophage cells 80%). DP and charge density of the ionenes were shown to be the factors of effective control of PEC dissociation in water-salt solutions, with a diminished role of charge density upon lengthening the ionene chain. These polymer characteristics were also important for DNA-ionene PEC resistivity to DNase activity and the ability of ionenes to serve as gene delivery vectors in vitro and exhibited good correlation with the results of salt-induced dissociation of PEC. These data may be useful for developing correlations and mathematical models to predict synthetic gene delivery vector efficiency.

Animals↗

PubMedDate not supplied

Kinetic and efficacy analysis of RNA interference in stably and transiently expressing cell lines.

RNA interference, particularly through the use of small interfering RNA (siRNA), has become an important laboratory tool for both fundamental and applied investigations. However, it is currently unknown whether siRNA-mediated knockdown of transiently expressed proteins is an acceptable quantitative surrogate for stably expressed proteins. Further, the best means by which to transfect cells with functionally active siRNA are poorly defined, and determination of the best reagent and transfection conditions for a particular cell line is a burdensome prerequisite for RNA interference studies. We therefore established the optimal transfection conditions for six commercial siRNA delivery reagents in three cell lines (HR5-CL11, HeLa, and NIH/3T3) transiently or stably expressing the firefly luciferase gene. The delivery efficiency, knockdown kinetics, and cytotoxicity of the reagents were evaluated. siPORT Amine, X-tremeGENE, and TransIT-siQUEST achieved the best knockdown and consistency of performance among the three cell lines. Delivery efficiency varied and was cell line dependent in some cases. The knockdown kinetics were reagent-dependent, and knockdown was generally more rapid in the stably transfected cells. Cytotoxicity of the reagents was variable. GeneSilencer was the least cytotoxic reagent for all three cell lines, and TransIT-siQUEST was the most cytotoxic to the HeLa and HR5-CL11 cell lines. These comparative results provide an initial basis for reagent selection and experimental design for RNA interference studies in HeLa, NIH/3T3, and their respective derivative cell lines.

Animals↗