Biomedical subjects

C Russell Middaugh

Publications and source records attributed to C Russell Middaugh.

At least 55 records · Page 3Linked to original sources

PubMed2003

An infrared spectroscopic study of the effect of hydration on cationic lipid/DNA complexes.

Infrared spectroscopy was used to examine the effect of dehydration on the structure of DNA and cationic lipid/DNA complexes (CLDCs). Information regarding the effect of hydration on the interface between the cationic lipids and DNA was obtained by following subtle but reproducible changes in vibrational bands arising from the DNA bases and phosphate backbone as well as bands from the lipid ester groups within the interfacial region of the bilayer. Dehydration of supercoiled plasmid DNA induces a transition from a B-conformation in solution to a mixed conformation in the dried state. Changes in vibrations of the bases upon drying suggest a change to an A-conformation whereas vibrations from the phosphate moieties suggest A- or C-forms. Vibrational changes in the ribose ring suggest adoption of a C-conformation. When CLDCs composed of either DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) or DDAB (dioctadecyldimethylammonium bromide) cationic lipids with or without equimolar amounts of the helper lipids cholesterol or DOPE (1,2-dioleoylphosphatidylethanolamin) are dried, the DNA is still able to undergo these structural transitions suggesting a nonrigid CLDC structure. The effect of dehydration on these interfacial interactions was found to be dependent on the type of cationic lipid used as well as the type of helper lipid. In addition, this work provides a simple spectroscopic analytical approach that can be used for the characterization of nonviral vectors that has potential pharmaceutical utility.

Cations↗

PubMed2003

Barriers to nonviral gene delivery.

The use of various synthetic lipids and polymers to deliver DNA for gene therapy applications has been the subject of intense examination for the last 15 years. Our understanding of the processes involved in the delivery of DNA, although still limited, can be described in terms of specific physical and chemical barriers encountered along the delivery pathway. Successful engagement of this pathway involves avoiding inactivation in the extracellular compartment and initial favorable interactions with the cell surface. Internalization of the delivery system by endocytosis results in a poorly defined endosomal trafficking process which, if not escaped, leads to degradation of the therapeutic DNA in lysosomes. For the small fraction of material that is able to escape this vesicular trafficking pathway, the cytosol provides additional physical and metabolic barriers to further trafficking to the nucleus. Finally, nuclear uptake has been demonstrated to be a significant barrier to gene delivery. In this review, we outline in greater detail the various processes involved in each step and describe various formulation variables that have been explored to overcome these delivery barriers to nonviral gene delivery.

Animals↗

PubMed2003

Thermal stability of vaccines.

Worldwide vaccination programs against infectious diseases and toxins are estimated to save approximately 3 million lives yearly. Tragically, however, another 3 million individuals (primarily children) die of vaccine-preventable diseases. A significant portion of this problem results from the thermal instability of many of the currently used vaccines. This review argues that modern methods of physical and chemical analysis permit for the first time characterization of the degradative pathways of thermally labile vaccines. A rigorous description of these pathways permit a more rational and systematic approach to the stabilization of vaccines. A direct result of the replacement of currently employed, primarily empirical, approaches to vaccine stabilization with a more molecular-based methodology should be the development of more universally available vaccinations against life-threatening diseases. This has the potential to have a dramatic impact on world health.

Animals↗

PubMed2003

Structural stability of adenovirus type 5.

Thermally induced structural changes in adenovirus type 5 (Ad) in the presence of either 2 or 10% sucrose were investigated using a variety of biophysical techniques. In solutions containing 2% sucrose, a highly cooperative transition in the structure of the virus was observed at 45 degrees C as detected by tryptophan fluorescence, derivative UV absorption spectroscopy, circular dichroism (CD), and dynamic and static light scattering. This transition resulted in (at least partial) disassembly of the virus and a concomitant increase in the accessibility of the viral DNA to the fluorescent dye, TOTO-1. Capsid disassembly was verified by transmission electron microscopy, which showed ruptured icosahedral vertices near 45 degrees C followed by complete capsid disassembly at higher temperatures. SDS-PAGE of thermally treated Ad suggests that the penton base (protein III) and protein IIIa (located in the peripentonal region) are significantly more labile than other capsid proteins and may be the initial instigators in capsid disassembly. Additional discrete structural transitions were observed in viral proteins using the aforementioned spectroscopic techniques. Thermally induced rearrangements of the condensed DNA at higher temperatures were also detected by the appearance of "psi"-like features in the CD spectra as well as a dramatic decrease in accessibility of DNA to TOTO-1. These transitions corresponded to discrete endothermic events that are also detected by differential scanning calorimetry. By increasing the concentration of sucrose to 10%, secondary and tertiary structural features of adenoviral proteins were significantly stabilized, although loss of quaternary structure at 45 degrees C was still observed.

Adenoviridae↗

PubMed2003

A fluorescence study of the structure and accessibility of plasmid DNA condensed with cationic gene delivery vehicles.

The cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dimethyldioctadecylammonium bromide, with or without the helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or cholesterol, and the cationic polymer polyethyleneimine, were compared for their ability to displace fluorescent dyes from DNA. Differences in displacement of the intercalating dyes ethidium bromide and ethidium homodimer correlate with their relative affinities with DNA, with the extent of ethidium homodimer displacement significantly less. Differences in ethidium homodimer and ethidium bromide displacement as a function of the ratio of polycation to DNA and the charge density of the polycation suggest a greater sensitivity of the former to topological changes in condensed DNA. Marked differences in the ability of these cationic delivery systems to displace the minor groove binding dyes 4',6-diamidino-2-phenylindole and Hoechst 33258 upon interaction with DNA are also apparent, with the majority of Hoechst 33258 remaining bound to DNA. Changes in the spectral properties of Hoechst 33258 were further used to characterize polycation-induced changes in solvent accessibility of the DNA minor groove. Taken together, these studies demonstrate differences in the interaction of various cationic lipids and polyethyleneimine in terms of regional displacement of dyes, polycation-induced structural changes in DNA, as well as polycation-mediated changes in solvent accessibility of the minor groove. The relevance of these studies to current models of the structure and assembly of polycation/DNA complexes are discussed.

Bisbenzimidazole↗

PubMed2003

Biophysical characterization of PEI/DNA complexes.

The main goal of this study was to determine the effects of polyethylenimine (PEI) molecular weight and structure (750 kDa, 25 kDa, 2 kDa branched, and 25 kDa linear PEI) and the nitrogen/phosphate (N/P) molar ratio on the physical properties and transfection efficiencies of PEI/DNA complexes. Fourier transform infrared spectroscopy revealed that DNA remained in the B conformation when complexed to all PEIs. Unique alterations in the circular dichroism spectra of DNA were observed in the presence of each PEI, whereas differential scanning calorimetry measurements showed that all PEIs examined destabilized supercoiled DNA at N/P < 3/1, but not at higher ratios. Isothermal titration calorimetry revealed the existence of protonation changes at low ionic strength due to possible shifts in pK(a) of the ionizable groups of PEI during complex formation. Twenty-five kilodalton branched and 25 kDa linear PEI complexes showed the highest transfection efficiencies at an N/P ratio of 6:1 in COS-7 and CHO-K1 cells, respectively. These investigations have detected alterations in the physical and colloidal properties of the complexes that were sensitive to polymer structure, molecular weight, and polymer/DNA ratio, but these properties did not directly correlate with their transfection efficiencies. To further probe any possible relationship between these parameters and activity, a more refined biophysical analysis of any subpopulations in these samples that may differ in transfection activity is suggested, although the existence of such species remains unknown.

Animals↗

PubMed2003

Derivative absorbance spectroscopy and protein phase diagrams as tools for comprehensive protein characterization: a bGCSF case study.

In protein and macromolecule pharmaceutical formulation development, the amount of information initially gathered about a drug's physical and chemical properties under different conditions is often quite limited. This generally requires more intensive studies using a variety of techniques if problems arise later in the development process. We propose a supplementary approach involving a comprehensive examination of a protein by derivative absorbance spectroscopy in conjunction with other methods and the subsequent construction of a phase diagram that permits the determination of optimal formulation conditions. Using bovine granulocyte colony stimulating factor (bGCSF) as a model protein, a thorough characterization is performed using high-resolution second-derivative absorbance spectroscopy. Derivative ultraviolet absorbance data are used to construct an empirical phase diagram for bGCSF using a multidimensional phase space approach. Between pH 2 and 7, and from 10 degrees to 90 degrees C, bGCSF is found to adopt more than six distinct structural phases. Surprisingly, the combination of the phase diagram approach with derivative absorbance data identifies phase boundaries that are not apparent upon initial examination of complementary biophysical data (previous article in this issue). The simplicity and pharmaceutical utility of this approach are discussed.

Algorithms↗

PubMed2003

Structural characterization of bovine granulocyte colony stimulating factor: effect of temperature and pH.

The protein bovine granulocyte colony stimulating factor (bGCSF) was studied in solution as a function of pH (2-7) and temperature (10 degrees -90 degrees C) using fluorescence, circular dichroism, and Fourier transform infrared spectroscopies, as well as differential scanning calorimetry and optical density as a measurement of aggregation. bGCSF possesses significant conformational lability under the solution conditions examined. Under all pH conditions examined, a major conformational change is observed as a function of temperature at 50 degrees -60 degrees C, although the magnitude and precise temperature at which this occurs varies with pH. Three major conformations are adopted with changing pH. One is observed at pH 2 and 3, a second at pH 4, and a third at pH 5-7. At low pH (2-3), bGCSF adopts a molten globule-like conformation at moderate temperatures (25 degrees -45 degrees C), whereas at pH 4 the protein appears to form a non-molten globule extended conformation. The use of this type of study as complementary data for protein phase diagram development as well as the relationship between the conformational lability demonstrated by bGCSF and that observed for recombinant human granulocyte colony stimulating factor and other similar cytokines is discussed.

Animals↗

PubMed2003

Nonclassical transport proteins and peptides: an alternative to classical macromolecule delivery systems.

The number of peptides and proteins known to exhibit nonclassical transport activity has increased significantly in recent years. In most cases, these entities have been studied in relation to their ability to deliver high molecular weight compounds, including proteins and DNA, for the ultimate purpose of developing new drug delivery strategies. In this review, an overview of the various types of vectors is presented. The in vitro and in vivo delivery successes of this technology, as well as preliminary therapeutic efforts, are described. Although a comprehensive mechanism of nonclassical transport has not yet been clearly established, we propose a straightforward model based on the cationic nature of the vectors and the need for lack of highly organized structure. In this hypothesis we suggest that the movement of polycations is mediated by a network of extra- and intracellular polyanions while transport across the bilayer is facilitated by cation-pi interactions between the vectors' basic groups and aromatic amino acid side chains in the bilayer spanning helices of membrane proteins.

Animals↗

PubMed2003

Structure/function analysis of peptoid/lipitoid:DNA complexes.

Previous transfection studies of cationic peptoid polymers (N-substituted polyglycines) and cationic lipitoid polymers (peptoid-phospholipid conjugates) have shown that only the polymers which possessed a repeating (cationic, hydrophobic, hydrophobic) substituent sequence are efficient in gene transfer in vitro. To determine if there is a physical attribute of peptoid and lipitoid complexes that correlates with efficient gene transfection, biophysical, and transfection measurements were performed with polymer:DNA complexes containing each of seven structurally diverse peptoid polymers and two lipitoids that possess different hydrophobic substituents. These measurements revealed that the biophysical properties of these complexes (size, zeta-potential, ethidium bromide exclusion) varied with polymer structure and complex (+/-) charge ratio but were not directly predictive of transfection efficiency. Unique alterations in the circular dichroism spectra of DNA were observed in complexes containing several of the peptoids and both lipitoids, although FTIR spectroscopy demonstrated that the DNA remained in the B-form. The lack of correlations between the physical properties and the transfection activities of these polyplexes suggests that a further subpopulation examination of these complexes by these methods may reveal hidden structure-activity relationships.

Animals↗

PubMed2003

The structure of DNA within cationic lipid/DNA complexes.

The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction with either the cationic lipid dimethyldioctadecylammonium bromide or 1,2-dioleoyltrimethylammonium propane. This loss of rotational strength is shown, however, by both FTIR and Raman spectroscopy to occur within the parameters of the B-type conformation. Contributions of absorption flattening and differential scattering to the CD spectra of complexes are unable to account for the observed spectra. Model studies of the CD of complexes prepared from synthetic oligonucleotides of varying length suggest that significant reductions in rotational strength can occur within short stretches of DNA. Furthermore, some alteration in the hydrogen bonding of bases within CLDCs is indicated in the FTIR and Raman spectroscopy results. In addition, alterations in base stacking interactions as well as hydrogen bonding are suggested by molecular dynamics simulations. A global interpretation of all of the data suggests the DNA component of CLDCs remains in a variant B form in which base/base interactions are perturbed.

Cations↗

PubMed2003

Formulation and characterization of DNA-polyethylenimine-dextran sulfate nanoparticles.

Polyethylenimine (PEI) is a promising non-viral gene delivery polymer that produces high transfection efficiency both in vitro and in vivo. The use of PEI, however, is hindered by its toxicity, reflecting its polycationic nature. In an attempt to decrease this charge-dependent cytotoxicity, a polyanionic polymer, dextran sulfate (DS), has been incorporated into self-assembling PEI-DNA complexes with zinc as stabilizing agent. Spherical particles with a mean particle size of approximately 200 nm and a polydispersity index of 0.2 were achieved using the following optimal conditions: PEI solutions at pH 8, PEI/DS mass ratios of >or=2, and 25 microM zinc sulfate. Plasmid DNA was completely condensed within the nanoparticles as confirmed by an ethidium bromide accessibility assay. This result correlates well with DNase protection studies which find partial protection of the DNA nanoparticles from degradation by the enzyme. The DNA was incorporated into the PEI-DS particles with a high efficiency (>95%) and maintained a primarily supercoiled B-form as determined by gel electrophoresis and circular dichroism. The cytotoxicity of the DNA nanoparticles appeared to decrease as the amount of DS in the formulation was increased and they produced moderate transfection activities that were only modestly inhibited by the presence of serum.

Circular Dichroism↗

PubMed2002

Structure-function analysis of invasion plasmid antigen C (IpaC) from Shigella flexneri.

Shigella flexneri causes a self-limiting gastroenteritis in humans, characterized by severe localized inflammation and ulceration of the colonic mucosa. Shigellosis most often targets young children in underdeveloped countries. Invasion plasmid antigen C (IpaC) has been identified as the primary effector protein for Shigella invasion of epithelial cells. Although an initial model of IpaC function has been developed, no detailed structural information is available that could assist in a better understanding of the molecular basis for its interactions with the host cytoskeleton and phospholipid membrane. We have therefore initiated structural studies of IpaC, IpaC I', (residues 101-363 deleted), and IpaC Delta H (residues 63-170 deleted). The secondary and tertiary structure of the protein was examined as a function of temperature, employing circular dichroism and high resolution derivative absorbance techniques. ANS (8-anilino-1-napthalene sulfonic acid) was used to probe the exposure of the hydrophobic surfaces under different conditions. The interaction of IpaC and these mutants with a liposome model (liposomes with entrapped fluorescein) was also examined. Domain III (residues 261-363) was studied using linker-scanning mutagenesis. It was shown that domain III contains periodic, sequence-dependent activity, suggesting helical structure in this section of the protein. In addition to these structural studies, investigation into the actin nucleation properties of IpaC was conducted, and actin nucleation by IpaC and some of the mutants was exhibited. Structure-function relationships of IpaC are discussed.

Actins↗

PubMed2002

The structural organization of cationic lipid-DNA complexes.

The interaction of cationic liposomes with supercoiled plasmid DNA results in a major rearrangement of each component to form compact multilamellar structures comprised of alternating layers of two-dimensional arrays of DNA sandwiched between lipid bilayers. Fluorescence resonance energy transfer was used to estimate the distance of closest approach of DNA to the lipid bilayers in these complexes. The effect of several compositional variables on this distance, including the ratio of cationic lipid to DNA, and the charge density, intrinsic curvature, and fluidity of the lipid bilayer were examined. Additionally, the effect of ionic strength was studied. For complexes prepared at or above a 3:1 charge ratio (+/-), the observed distance of closest approach was found to be in agreement with the intercalation of DNA between lipid bilayers. As the charge ratio was decreased, a monotonic increase in the distance was observed with a maximum observed at 0.5:1. Correlations between differences in the proximity of DNA to the lipid bilayer and the hydrodynamic size of the complexes were also found. A model based on these observations and previous reports suggests the formation of discrete populations of complexes below a charge ratio of 0.5:1 and above 3:1. The structure of the negatively charged complexes is consistent with DNA extending from the surface of the particles, whereas those possessing excess positive charge were multilamellar aggregates with the DNA effectively condensed between lipid bilayers. Complexes between these two states consist of weighted fractions of these two species.

Boron Compounds↗

PubMed2002

Investigation of protein/carbohydrate interactions in the dried state. 2. Diffuse reflectance FTIR studies.

Upon freeze-drying in the absence of lyoprotectants, Fourier transform infrared (FTIR) spectroscopy has detected changes in the secondary structures of proteins. Such FTIR studies have been typically conducted using protein/KBr pellets, where additional protein degradation could potentially occur due to pressure effects and partial dissolution of the chaotropic KBr. Diffuse reflectance FTIR spectroscopy, in which no sample preparation is necessary, was evaluated as an alternative spectroscopic method to examine protein structure upon freeze-drying. The therapeutic proteins recombinant human deoxyribonuclease I (rh-DNase) and recombinant human insulin like growth factor I (rh-IGF-I) were freeze-dried with mannitol, sucrose, trehalose, and two molecular weight dextrans (69 and 503 kDa) separately, at concentrations ranging from 0 to 100% (w/w). Upon freeze-drying, rh-DNase and rh-IGF-I underwent significant changes in their secondary structure. For both proteins, the presence of intermolecular beta-sheets due to aggregation was detected and the alpha-helix content decreased significantly. The addition of carbohydrates to the formulations inhibited the protein secondary structure rearrangement in a concentration-dependent manner. Sucrose and trehalose appeared to be the most efficient excipients in preventing secondary structure changes. The conformational changes observed for both proteins appeared to be reversible upon rehydration.

Carbohydrates↗

PubMed2002

The inhibitory action of phospholamban involves stabilization of alpha-helices within the Ca-ATPase.

We have used attenuated total reflection Fourier transform infrared (ATR-FTIR) and circular dichroism (CD) spectroscopies to identify secondary and dynamic structural changes within the Ca-ATPase that result from the functional inhibition of transport activity by phospholamban (PLB). Isotopically labeled [(13)C]PLB was expressed and purified from Escherichia coli and was functionally reconstituted with unlabeled Ca-ATPase, permitting the resolution of the amide I and II absorbance bands of the Ca-ATPase from those of [(13)C]PLB. Upon co-reconstitution of the Ca-ATPase with PLB, spectral shifts are observed in both the CD spectra and the amide I and II bands associated with the Ca-ATPase, which are indicative of increased alpha-helical stability. Corresponding changes in the kinetics of H/D exchange occur upon association with PLB, indicating that 100 +/- 20 residues in the Ca-ATPase that normally undergo rapid amide H/D exchange become exchange resistant. There are no corresponding large changes in the secondary structure of PLB. The affinity of the structural interaction between PLB and the Ca-ATPase is virtually identical to that associated with functional inhibition (K(d) = 140 +/- 30 microM), confirming that the inhibitory regulation of the Ca-ATPase by PLB involves the stabilization of alpha-helices within the Ca-ATPase.

Animals↗

PubMed2002

Investigation of protein/carbohydrate interactions in the dried state. 1. Calorimetric studies.

Isoperibol calorimetry was used to evaluate protein/carbohydrate interactions after freeze drying. rh-DNase, rh-GH, rh-MetGH, and rh-IGF-I were freeze dried with either mannitol, sucrose, trehalose, or dextran at concentrations ranging from 0% to 100% (w/w). Enthalpies of solution for both freeze-dried and physical mixtures were measured in water at 25 degrees C. Differential scanning calorimetry was used to monitor changes in the melting or crystallization temperatures of the lyoprotectants. Linear relationships between enthalpies of solution and the percentage of protein in the formulations were observed for all physical mixtures. In contrast, nonlinear relationships between the enthalpies of solution and protein content were observed for the freeze-dried mixtures. Mannitol-containing mixtures were characterized by negative deviation from linearity, while positive deviations were detected for mixtures containing sucrose or trehalose. Using DSC, sucrose was found to be amorphous at low and not detected at high protein content in the freeze-dried mixtures. Melting of mannitol was observed through almost all of the protein concentration range examined. Two melting endotherms, however, were observed for mannitol at most protein/mannitol ratios, indicating the presence of protein/mannitol interactions. This work suggests that direct interactions occur between proteins and carbohydrates in lyophilized mixtures.

Calorimetry, Differential Scanning↗

PubMed2002

Differential scanning calorimetric studies of the thermal stability of plasmid DNA complexed with cationic lipids and polymers.

The thermal stabilities of supercoiled (SC) and linear/open circular (LIN/OC) forms of plasmid DNA when complexed with cationic lipids or cationic polymers used for cellular transfection were assessed using differential scanning calorimetry. Differences in the stability of SC DNA produced by the cationic lipids DOTAP (1,2-dioleoyltrimethyl ammoniumpropane chloride), DSTAP (1,2-distearyltrimethyl ammoniumpropane chloride), and DDAB (dimethyldioctadecylammonium bromide) upon complexation suggest possible effects of headgroup structure on the stability of SC DNA and minimal effects of lipid acyl chain saturation/unsaturation. Complexation of DNA with the cationic polymers polyethylenimine (PEI) or poly-L-lysine (PLL) (but not poly-L-arginine) resulted in a decreased stability of SC DNA when the DNA was in charge excess, although all polymers stabilized SC DNA when the polymer was in charge excess. The effects of these cationic polymers on the stability of SC DNA can be explained by changes produced in the tertiary structure of SC DNA upon binding and may reflect the importance of the topological constraint of supercoiling upon the stability of the resulting complexes.

Calorimetry, Differential Scanning↗