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R Langer

Publications and source records attributed to R Langer.

At least 235 records · Page 13Linked to original sources
PubMed1995

Changes in the passive electrical properties of human stratum corneum due to electroporation.

The stratum corneum (SC) is the main barrier to molecular and ionic transport across mammalian skin and has been extensively studied by others at low voltages (U(skin)(t) < 10 V) in order to partially characterize the skin. Here we use one or more exponential pulses (tau pulse = 1 ms) and a temperature of 25 +/- 2 degrees C and found that the low voltage passive electrical properties (impedance) change rapidly and significantly if these pulse result in U(skin),0 > 40 V. In contrast, the dynamic resistance (describing passive electrical behavior in a nonlinear range) changes dramatically by application of pulses between 40 V and 80 V and then it settles at levels between 50 omega and 100 omega. We also found that recovery of the low voltage electrical parameters after pulsing depends mainly on the voltage, and, for multiple pulse protocols, on the number of pulses. For single pulses of U(skin),0 approximately 90 V or less the electrical recovery was almost complete, returning to within 0.90 of the pre-pulse value. In contrast, larger pulses result progressively in decreased recovery. The recovery for pulses > 90 V revealed several characteristic times, suggesting the involvement of different processes. For multiple pulses with U(skin),0 > 130 V almost no recovery of the transdermal resistance, R(skin), was evident (returning to < 0.10 of pre-pulse values), i.e., essentially permanent changes in the stratum corneum occurred. This is similar to that of single bilayer membrane electroporation, for which a transition from reversible to irreversible behavior occurs as transmembrane voltage is increased. Thus, these results are consistent with the hypothesis that 'high-voltage' pulses cause electroporation within the SC, i.e., that elevated transmembrane voltage result in creation of new aqueous pathways ('pores') across SC lipid regions.

Electric Conductivity↗
PubMed1995

Ultrasound-mediated transdermal protein delivery.

Transdermal drug delivery offers a potential method of drug administration. However, its application has been limited to a few low molecular weight compounds because of the extremely low permeability of human skin. Low-frequency ultrasound was shown to increase the permeability of human skin to many drugs, including high molecular weight proteins, by several orders of magnitude, thus making transdermal administration of these molecules potentially feasible. It was possible to deliver and control therapeutic doses of proteins such as insulin, interferon gamma, and erythropoeitin across human skin. Low-frequency ultrasound is thus a potential noninvasive substitute for traditional methods of drug delivery, such as injections.

Administration, Cutaneous↗
PubMed1995

Transdermal transport of DNA antisense oligonucleotides by electroporation.

Fluorescein-labeled antisense oligodeoxynucleotides (ODNs) corresponding to the promoters of the protooncogene c-myb (24-mer) and the oncogene c-myc (15-mer) were transported through the human skin in vitro by electroporation. Fluxes of 6.4 +/- 2.1 pM/cm2* hr and 11.5 +/- 3.5 pM/cm2* hr, respectively, were achieved during exponential pulsing [tau pulse = 1.1ms, transdermal voltage (Uskin) = 80V] every five seconds. The flux for Uskin < 70V was undetectable, rapidly increased at 80V, but plateaued at higher values. Fluorescence imaging demonstrated that transport of the ODNs is concentrated in localized transport regions (LTRs)1 approximately 30 microns in diameter.

Base Sequence↗
PubMed1995

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.

Amino Acid Sequence↗
PubMed1995

Biodegradable sponges for hepatocyte transplantation.

Liver cell transplantation may provide a means to replace lost or deficient liver tissue, but devices capable of delivering hepatocytes to a desirable anatomic location and guiding the development of a new tissue from these cells and the host tissue are needed. We have investigated whether sponges fabricated from poly-L-lactic acid (PLA) infiltrated with polyvinyl alcohol (PVA) would meet these requirements. Highly porous sponges (porosity = 90-95%) were fabricated from PLA using a particulate leaching technique. To enable even and efficient cell seeding, the devices were infiltrated with the hydrophilic polymer polyvinyl alcohol (PVA). This reduced their contact angle with water from 79 to 23 degrees, but did not inhibit the ability of hepatocytes to adhere to the polymer. Porous sponges of PLA infiltrated with PVA readily absorbed aqueous solutions into 98% of their pore volume, and could be evenly seeded with high densities (5 x 10(7) cells/mL) of hepatocytes. Hepatocyte-seeded devices were implanted into the mesentery of laboratory rats, and 6 +/- 2 x 10(5) of the hepatocytes engrafted per sponge. Fibrovascular tissue invaded through the devices' pores, leading to a composite tissue consisting of hepatocytes, blood vessels and fibrous tissue, and the polymer sponge.

Animals↗
PubMed1995

Cytotoxicity testing of poly(anhydride-co-imides) for orthopedic applications.

The cytotoxicity of a series of poly(anhydride-co-imides) with osteoblast-like cells (MC3T3-E1) was evaluated. The imide component of the copolymers was based on trimellitylimidoglycine (TMA-gly), and the anhydride component was based on either sebacic acid (SA) or 1,6-bis(carboxyphenoxy)hexane (CPH). Cell adhesion and proliferation on surfaces of the polymer discs were observed by environmental scanning electron microscopy (ESEM). During the first 24 h of attachment, cells showed normal morphology when cultured on the various copolymers of CPH. Concurrently, the effects of polymer breakdown products on osteoblast-like cells were evaluated by studying their proliferation (cell numbers), viability (dye exclusion), and morphology (light microscopy). Cell cultures in the presence of these breakdown products resulted in normal morphologies and reached confluency after 7 days. This initial in vitro evaluation with osteoblast-like cells suggests that the poly(anhydride-co-imides) may be viable carriers for osteoblasts.

Anhydrides↗
PubMed1995

Development of biomechanical properties and morphogenesis of in vitro tissue engineered cartilage.

Neocartilage was engineered by culturing bovine chondrocytes on poly(glycolic acid) (PGA) fibrous nonwoven scaffolds. The biomechanical properties and morphologies of the PGA-chondrocyte constructs were studied over 12 weeks of in vitro culture. PGA scaffolds without cells lost their mechanical strength and structural integrity between week 2 and week 3 in culture. The thickness of the PGA-chondrocyte constructs decreased by 35% during the first 3 weeks, but the thickness increased from week 3 to week 9 to a thickness 42% higher than that of the starting scaffolds, which was then maintained. Safranin O staining of PGA-chondrocyte constructs revealed increasing proteoglycan formation over time. The compressive modules of PGA-chondrocyte constructs increased with in vitro culture time, and reached the same order of magnitude as that of normal bovine cartilage at week 9. The aggregate modulus of the PGA-chondrocyte constructs decreased by 57% over the first 2 weeks but then increased, reaching the same order of magnitude as normal bovine cartilage at week 12. The apparent permeability of the PGA-chondrocyte constructs, which was initially four orders of magnitude above that of normal cartilage, decreased between weeks 1 and 3 and thereafter remained the same order of magnitude as that measured for normal cartilage.

Animals↗
PubMed1995

A mechanistic study of ultrasonically-enhanced transdermal drug delivery.

Although ultrasound has been shown to enhance the transdermal transport of a variety of drugs, the mechanisms underlying this phenomenon are not clearly understood. In this paper, we evaluate the roles played by various ultrasound-related phenomena, including cavitation, thermal effects, generation of convective velocities, and mechanical effects, in the ultrasonic enhancement of transdermal drug delivery (sonophoresis). Our experimental findings suggest that among all the ultrasound-related phenomena evaluated, cavitation plays the dominant role in sonophoresis using therapeutic ultrasound (frequency range, 1-3 MHz; intensity range, 0-2 W/cm2). Furthermore, confocal microscopy results indicate that cavitation occurs in the keratinocytes of the stratum corneum upon ultrasound exposure. It is hypothesized that oscillations of the cavitation bubbles induce disorder in the stratum corneum lipid bilayers, thereby enhancing transdermal transport. Evidence supporting this hypothesis is presented using skin electrical resistance measurements. Finally, a theoretical model is developed to predict the effect of ultrasound on the transdermal transport of drugs. The model predicts that sonophoretic enhancement depends most directly on the passive permeant diffusion coefficient, rather than on the permeability coefficient through the skin. Specifically, permeants passively diffusing through the skin at a relatively slow rate are expected to be preferentially enhanced by ultrasound. The experimentally measured sonophoretic transdermal transport enhancement for seven permeants, including estradiol, testosterone, progesterone, corticosterone, benzene, butanol, and caffeine, agree quantitatively with the model predictions. These experimental and theoretical findings provide quantitative guidelines for estimating the efficacy of sonophoresis in enhancing transdermal drug delivery.

Administration, Cutaneous↗
PubMed1995

Permeation of steroids through human skin.

The permeabilities of many steroids through human skin have been previously measured and reported in the literature. Analysis of these data reveals that significant discrepancies exist between the measurements of Scheuplein et al. and those of other groups. Six of the 14 steroids which were examined by Scheuplein et al., aldosterone, corticosterone, estradiol, hydrocortisone, progesterone, and testosterone, have also been examined by other groups. For each of these steroids, the permeability measurements of Scheuplein et al. are lower than those reported by other groups by factors of between 5.0 and 77. Eight independent measurements of the permeability of estradiol are in good agreement with one another, but are greater than the value reported by Scheuplein et al. by factors of between 11 and 20. Several possible sources of experimental error, including the variability of the skin samples, the differences in the experimental temperature, the establishment of steady-state conditions, the use of radiolabeled drugs, and the skin preparation technique, have been considered and do not appear to account for the magnitude of the observed discrepancies nor for the fact that the data of Scheuplein et al. are consistently lower than those reported by other groups.

Humans↗
PubMed1995

Tissue engineering heart valves: valve leaflet replacement study in a lamb model.

BACKGROUND: Valve replacements using either bioprosthetic or mechanical valves have the disadvantage that these structures are unable to grow, repair, or remodel and are both thrombogenic and susceptible to infection. These characteristics have significantly limited their durability and longevity. In an attempt to begin to overcome these shortcomings, we have tested the feasibility of constructing heart valve leaflets in lambs by seeding a synthetic polyglycolic acid fiber matrix in vitro with fibroblasts and endothelial cells. METHODS: Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted ovine arteries. Endothelial cells were selectively labeled with an acetylated low-density lipoprotein marker and separated from the fibroblasts using a fluorescent activated cell sorter. A synthetic biodegradable scaffold constructed from polyglycolic acid fibers was seeded with fibroblasts, which grew to form a tissue-like sheet. This tissue was subsequently seeded with endothelial cells, which formed a cellular monolayer coating around the leaflet. Using these constructs, autologous (n = 3) and allogenic (n = 4) tissue engineered leaflets were implanted in 7 animals. In each animal the right posterior leaflet of the pulmonary valve was resected and replaced with an engineered valve leaflet. RESULTS: All animals survived the procedure. Postoperative echocardiography demonstrated no evidence of stenosis and trivial pulmonary regurgitation in the autografts and moderate regurgitation in the allogenic valves. Collagen analysis of the constructs showed development of an extracellular matrix. Histologic evaluation of the constructs demonstrated appropriate cellular architecture. CONCLUSIONS: This preliminary experiment showed that a tissue engineered valve leaflet constructed from its cellular components can function in the pulmonary valve position. Tissue engineering of a heart valve leaflet is feasible, and these preliminary studies suggest that autograft tissue will probably be superior to allogenic tissue.

Animals↗
PubMed1995

Millisecond measurement of transport during and after an electroporation pulse.

Electroporation involves the application of an electric field pulse that creates transient aqueous pathways in lipid bilayer membranes. Transport through these pathways can occur by different mechanisms during and after a pulse. To determine the time scale of transport and the mechanism(s) by which it occurs, efflux of a fluorescent molecule, calcein, across erythrocyte ghost membranes was measured with a fluorescence microscope photometer with millisecond time resolution during and after electroporation pulses several milliseconds in duration. One of four outcomes was typically observed. Ghosts were: (1) partially emptied of calcein, involving efflux primarily after the pulse; (2) completely emptied of calcein, involving efflux primarily after the pulse; (3) completely emptied of calcein, involving efflux both during and after the pulse; or (4) completely emptied of calcein, involving efflux primarily during the pulse. Partial emptying, involving significant efflux during the pulse, was generally not observed. We conclude that under some conditions transport caused by electroporation occurs predominantly by electrophoresis and/or electroosmosis during a pulse, although under other conditions transport occurs in part or almost completely by diffusion within milliseconds to seconds after a pulse.

Biological Transport↗
PubMed1995

In vitro evaluation of polymerized liposomes as an oral drug delivery system.

The physical characteristics of polymerized liposomes for potential use as an oral drug delivery system were examined in vitro. The trap efficiency in monomeric liposomes composed of 1,2-di (2,4-octadecadienoyl) phosphatidylcholine was increased from 3% for original multilamellar vesicles to 35% for freeze-thaw treated liposomes. Polymerized liposomes with azobis (isobutyronitrile) and azobis (2-amidinopropane) hydrochloride as radical initiators showed complete stability against solubilization by Triton X-100, a detergent chosen to mimic bile salts. Release rates of 14C-BSA and 14C-sucrose in media simulating the gastro-intestinal fluids was 50% less than from regular liposomes composed of hydrogenated egg phosphatidylcholine mixed with cholesterol (molar ratio 1:1), which can be regarded as one of the most stable types of regular liposomes. It was estimated that, when administered orally, polymerized liposomes can reach the intestine while maintaining their vesicle structure and keeping at least 75% of their original content.

Administration, Oral↗
PubMed1995

Aggregation of a lyophilized pharmaceutical protein, recombinant human albumin: effect of moisture and stabilization by excipients.

In the presence of water vapor at 37 degrees C, lyophilized recombinant human albumin (rHA) undergoes intermolecular thiol-disulfide interchange, eventually forming high-molecular-weight, water-insoluble aggregates. The relationship between the extent of aggregation and the water content of the lyophilized protein was bell-shaped, with maximum aggregation (over 80% after one day) at approximately 50 g water per 100 g dry protein, corresponding to incubation at 96% relative humidity. Nineteen different excipients were co-lyophilized with rHA to test their ability to inhibit aggregation under these conditions. These compounds included low- and high-molecular-weight sugars, as well as various organic acids (amino, hydroxy, and aliphatic), and the simple inorganic salt sodium chloride. Seven of them afforded complete stabilization of rHA against moisture-induced aggregation. The stabilizing potency of the excipients correlated with their water-sorbing capability, presumably due to increasing the moisture level in the vicinity of rHA.

Albumins↗
PubMed1995

Transdermal delivery of heparin by skin electroporation.

Therapeutic uses of compounds produced by biotechnology are presently limited by the lack of noninvasive methods for continuous administration of biologically-active macromolecules. Transdermal delivery would be an attractive solution, except macromolecules have not previously been delivered clinically across human skin at therapeutic rates. To increase transport of a highly-charged macromolecule (heparin), high-voltage pulses believed to cause electroporation were applied to skin. Using this approach, transdermal heparin transport across human skin in vitro occurred at therapeutic rates (100-500 micrograms/cm2h), reported to be sufficient for systemic anticoagulation. In contrast, fluxes caused by low-voltage iontophoresis having the same time-averaged current were an order of magnitude lower. Heparin transported across the skin was biologically active, but with only one eighth the anticoagulant activity of heparin in the donor compartment due to preferential transport of small (less active) heparin molecules. Flux, activity, and transport number data together suggest that high-voltage pulsing creates transient changes in skin microstructure which do not occur during iontophoresis. Safety issues are discussed.

Administration, Cutaneous↗
PubMed1995

Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix.

This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In contrast, cell spreading could be completely inhibited by combining suboptimal doses of cytochalasin D and nocodazole, suggesting that intact microtubules can stabilize cell form when the microfilament lattice is partially compromised. The physiological relevance of the cytoskeleton and cell shape in hepatocyte physiology was highlighted by the finding that a short exposure (6 hour) of cells to nocodazole resulted in production of smaller cells 42 hours later that exhibited enhanced production of a liver-specific product (albumin). These data demonstrate that spreading and flattening of the entire cell body is not driven directly by net polymerization of either microfilaments or microtubules.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Enzymatic degradation of glycosaminoglycans.

Glycosaminoglycans (GAGs) play an intricate role in the extracellular matrix (ECM), not only as soluble components and polyelectrolytes, but also by specific interactions with growth factors and other transient components of the ECM. Modifications of GAG chains, such as isomerization, sulfation, and acetylation, generate the chemical specificity of GAGs. GAGs can be depolymerized enzymatically either by eliminative cleavage with lyases (EC 4.2.2.-) or by hydrolytic cleavage with hydrolases (EC 3.2.1.-). Often, these enzymes are specific for residues in the polysaccharide chain with certain modifications. As such, the enzymes can serve as tools for studying the physiological effect of residue modifications and as models at the molecular level of protein-GAG recognition. This review examines the structure of the substrates, the properties of enzymatic degradation, and the enzyme substrate-interactions at a molecular level. The primary structure of several GAGs is organized macroscopically by segregation into alternating blocks of specific sulfation patterns and microscopically by formation of oligosaccharide sequences with specific binding functions. Among GAGs, considerable dermatan sulfate, heparin and heparan sulfate show conformational flexibility in solution. They elicit sequence-specific interactions with enzymes that degrade them, as well as with other proteins, however, the effect of conformational flexibility on protein-GAG interactions is not clear. Recent findings have established empirical rules of substrate specificity and elucidated molecular mechanisms of enzyme-substrate interactions for enzymes that degrade GAGs. Here we propose that local formation of polysaccharide secondary structure is determined by the immediate sequence environment within the GAG polymer, and that this secondary structure, in turn, governs the binding and catalytic interactions between proteins and GAGs.

Animals↗