Where a pill won't reach.
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Administration, Cutaneous↗
Biomedical subjects
Robert Langer
Publications and source records attributed to Robert Langer.
Visualization of oleic acid-induced transdermal diffusion pathways using two-photon fluorescence microscopy.
In a novel application of dual-channel high-speed two-photon fluorescence microscopy, the skin autofluores-cence and the transdermal fluorescent model drug spatial distributions were imaged simultaneously over precisely the same spatial coordinates. The dual channels enable the detection of the fluorescence emission wavelengths characteristic of the endogenous (intrinsic) skin fluorophores, as well as of the rhodamine-based model drug intensity emission at a different wavelength range of the fluorescence emission spectrum. These fluorescent model drugs delineate the oleic acid induced changes in permeant diffusion with respect to the skin structural features over the 0.3 mm by 0.3 mm skin area imaged per skin sample. The dual-channel high-speed two-photon fluorescence microscopy studies presented here provide evidence for the existence of intracorneocyte diffusion in addition to the commonly cited lipid multilamellar transdermal pathway. The image quantification analysis methodology introduced in this paper reveals that intracorneocyte diffusion exists for the hydrophobic (rhodamine B hexyl ester) and for the hydrophilic (sulforhodamine B) model drugs, in the absence of oleic acid chemical enhancer action. The mechanism of oleic acid chemical enhancer action, however, depends on the model drug physicochemical properties, where the oleic acid induces hydrophobic model drug localization to the lipid multilamellar region, while increasing the hydrophilic model drug lipid to corneocyte partitioning.
Biological Transport↗
Effect of implantation site on hepatocytes heterotopically transplanted on biodegradable polymer scaffolds.
We investigated the engraftment of heterotopically transplanted hepatocytes in three sites: the subcutaneous space, the small intestinal mesentery, and the omentum to determine the optimal location for tissue-engineered liver constructs. Hepatocytes were isolated from inbred Lewis rats and placed on polymer constructs. Cell-polymer constructs were implanted into the subcutaneous space of the abdominal wall, the small intestinal mesentery, and the omentum of Lewis rats. One group of rats had undergone previous portacaval shunt. Animals were killed 2 or 4 weeks after implantation and the constructs were analyzed for engraftment, using computer-assisted morphometric analysis. Engraftment was greatest in the omentum with less engraftment in the mesentery. There was minimal engraftment in the subcutaneous space in all specimens. Prior portacaval shunt increased engraftment in the mesentery and the omentum, but not the subcutaneous space. The omentum is the most favorable bed for engraftment of hepatocyte-polymer tissue-engineered constructs and the addition of a portacaval shunt increases survival of transplanted hepatocytes in the omentum and mesentery.
Abdominal Wall↗
Temporal characteristics of activation, deactivation, and restimulation of signal transduction following depolarization in the pheochromocytoma cell line PC12.
This study focuses on the transient and dynamic activation of intracellular signal transduction following different protocols of depolarization. During chronic depolarization, phosphorylation of extracellular signal-regulated kinases (ERKs) was observed to peak and subsequently fall to low levels within 10 min of depolarization. Short periods of depolarization, from 1 to 5 min in duration, also led to phosphorylation of ERK, and the rate of ERK dephosphorylation was not affected by the duration of depolarization. Phosphorylation of the cyclic AMP response element binding protein (CREB) also peaked as a result of chronic depolarization but decreased to intermediate levels that were maintained for more than 1 h. Pulsatile depolarization was explored as a means to circumvent the deactivation of intracellular signaling activity during chronic depolarization. Both ERK and CREB were rephosphorylated by a second period of depolarization that followed a recovery period of 10 min or more. The effects of the durations of depolarization and interpulse recovery on reactivation of ERK and CREB were characterized. Measurements of free cytoplasmic Ca(2+) confirmed the transient rise in the intracellular calcium concentration ([Ca(2+)](i)) during chronic depolarization and the pulsatile increase in [Ca(2+)](i) that can be achieved with short periods of depolarization. This study characterizes the dynamic activities of signal transduction following depolarization. Electrical stimulation of neurons induces many cellular changes that unfold over time, and the influx of Ca(2+) ions that mediate these events is transient. This study suggests that pulsatile activity may be a means of maintaining signaling activity over long periods of time.
Animals↗
Recent advances in the development of an inhaled insulin product.
Inhaled insulin first entered clinical human testing in the mid 1990s. Since then, the commercial potential and technical challenges of an inhaled insulin product have grown increasingly clear, with several pharmaceutical partnerships now targeting treatment of diabetes mellitus through inhalation products in clinical development. While clinical results to date show the therapy to be generally promising, recent data have raised questions related to human safety and slowed progress toward a commercial product. Answering these questions positively in the coming years will be critical to making inhalation therapy a practical diabetes-care reality.
Administration, Inhalation↗
Gene transfer of a human insulin-like growth factor I cDNA enhances tissue engineering of cartilage.
The repair of articular cartilage lesions remains a clinical problem. Two novel approaches to cartilage formation, gene transfer and tissue engineering, have been limited by short-term transgene expression in transplanted chondrocytes and inability to deliver regulatory signals to engineered tissues according to specific temporal and spatial patterns. We tested the hypothesis that the transfer of a cDNA encoding the human insulin-like growth factor I (IGF-I) can provide sustained gene expression in cell-polymer constructs in vitro and in vivo and enhance the structural and functional properties of tissue-engineered cartilage. Bovine articular chondrocytes genetically modified to overexpress human IGF-I were seeded into polymer scaffolds, cultured in bioreactors in serum-free medium, and implanted subcutaneously in nude mice; constructs based on nontransfected or lacZ-transfected chondrocytes served as controls. Transgene expression was maintained throughout the duration of the study, more than 4 weeks in vitro followed by an additional 10 days either in vitro or in vivo. Chondrogenesis progressed toward the formation of cartilaginous tissue that was characterized by the presence of glycosaminoglycans, aggrecan, and type II collagen, and the absence of type I collagen. IGF-I constructs contained increased amounts of glycosaminoglycans and collagen and confined-compression equilibrium moduli as compared with controls; all groups had subnormal cellularity. The amounts of glycosaminoglycans and collagen per unit DNA in IGF-I constructs were markedly higher than in constructs cultured in serum-supplemented medium or native cartilage. This enhancement of chondrogenesis by spatially defined overexpression of human IGF-I suggests that cartilage tissue engineering based on genetically modified chondrocytes may be advantageous as compared with either gene transfer or tissue engineering alone.
Animals↗
Lipid-sugar particles for intracranial drug delivery: safety and biocompatibility.
Controlled release of drugs to specific locales in the brain has engendered considerable interest. Here we evaluate the safety and biocompatibility of 6-microm diameter particles composed of dipalmitoylphosphatidylcholine and chondroitin sulfate A, when delivered into the cerebral parenchyma and ventricles, and in the case of intravascular injection. Some particles were loaded with fluorescein-labeled albumin to facilitate detection. Particles placed in medium with cultured murine primary cortical neurons did not increase cell death at concentrations as high as 4 mg/ml. When particles (100 microg in 2 microl) were placed stereotactically in the striatum and lateral ventricles, there was no histological evidence on hematoxylin-eosin stained sections of tissue injury outside of the needle track in any animal 3, 7, and 14 days after injection (n=6 each), and no inflammation. Ventricular size was not significantly different between animals given intraventricular injections of particles and albumin solution at those time points (n=4 each). Intracarotid injection of particles at concentrations of 0.2 and 1 mg/ml (n=4 each) did not affect relative cerebral blood flow, and there were no embolic events on histology. In one animal in the group injected with 5 mg/ml (n=3), there was a profound decrease in rCBF, with patchy emboli on histology. These novel biodegradable particles are biocompatible in and around the brain, and may be safe for intracranial sustained drug delivery either in the parenchyma or into the CSF.
1,2-Dipalmitoylphosphatidylcholine↗
Adipose tissue mass can be regulated through the vasculature.
Tumor growth is angiogenesis dependent. We hypothesized that nonneoplastic tissue growth also depends on neovascularization. We chose adipose tissue as an experimental system because of its remodeling capacity. Mice from different obesity models received anti-angiogenic agents. Treatment resulted in dose-dependent, reversible weight reduction and adipose tissue loss. Marked vascular remodeling was evident in adipose tissue sections, which revealed decreased endothelial proliferation and increased apoptosis in treated mice compared with controls. Continuous treatment maintained mice near normal body weights for age without adverse effects. Metabolic adaptations in food intake, metabolic rate, and energy substrate utilization were associated with anti-angiogenic weight loss. We conclude that adipose tissue mass is sensitive to angiogenesis inhibitors and can be regulated by its vasculature.
Adipose Tissue↗
Effects of oxygen on engineered cardiac muscle.
Concentration gradients associated with the in vitro cultivation of engineered tissues that are vascularized in vivo result in the formation of only a thin peripheral tissue-like region (e.g., approximately 100 microm for engineered cardiac muscle) around a relatively cell-free interior. We previously demonstrated that diffusional gradients within engineered cardiac constructs can be minimized by direct perfusion of culture medium through the construct. In the present study, we measured the effects of medium perfusion rate and local oxygen concentration (p(O2)) on the in vitro reconstruction of engineered cardiac muscle. Neonatal rat cardiomyocytes were seeded onto biodegradable polymer scaffolds (fibrous discs, 1.1 cm diameter x 2 mm thick, made of polyglycolic acid, 24 x 10(6) cells per scaffold). The resulting cell-polymer constructs were cultured for a total of 12 days in serially connected cartridges (n = 1-8), each containing one construct directly perfused with culture medium at a flow rate of 0.2-3.0 mL/min. In all groups, oxygen concentration decreased due to cell respiration, and depended on construct position in the series and medium flow rate. Higher perfusion rates and higher p(O2) correlated with more aerobic cell metabolism, and higher DNA and protein contents. Constructs cultured at p(O2) of 160 mm Hg had 50% higher DNA and protein contents, markedly higher expression of sarcomeric alpha-actin, better organized sarcomeres and cell junctions, and 4.5-fold higher rate of cell respiration as compared to constructs cultured at p(O2) of 60 mm Hg. Contraction rates of the corresponding cardiac cell monolayers were 40% higher at p(O2) of 160 than 60 mm Hg. The control of oxygen concentration in cell microenvironment can thus improve the structure and function of engineered cardiac muscle. Experiments of this kind can form a basis for controlled studies of the effects of oxygen on the in vitro development of engineered tissues.
Animals↗
Measuring the pH environment of DNA delivered using nonviral vectors: implications for lysosomal trafficking.
The degradation of DNA in lysosomes represents a major obstacle to efficient nonviral gene delivery. The rational design of vectors that overcome this obstacle requires a better understanding of the lysosomal barrier to gene delivery, which in turn requires a means to investigate this intermediate step. To this end, we developed a technique to measure the pH environment of delivered DNA, from which the degree to which vectors avoided trafficking to acidic Iysosomes could be determined. The measured average pH of DNA delivered using poly-L-lysine (PLL) polyplexes was 4.5, suggesting that PLL polyplexes were trafficked to acidic lysosomes. Other vectors could avoid or buffer the pH of Iysosomes as DNA delivered using Lipofectamine Plus, polyethylenimine (PEI), linear polyethylenimine (LPEI), and two degradable poly(beta-amino ester)s (poly-1 and poly-2) had average pH values of 7.1, 5.9, 5.0, 6.7, and 6.4, respectively.
3T3 Cells↗
Local delivery of basic fibroblast growth factor increases both angiogenesis and engraftment of hepatocytes in tissue-engineered polymer devices.
BACKGROUND: We investigated heterotopic hepatocyte transplantation on biodegradable polymers as a potential treatment for end-stage liver disease. The primary problem has been insufficient engraftment of transplanted cells partly because of insufficient vascularization. Increasing vascularization through locally delivered angiogenic factors may increase angiogenesis and hepatocyte engraftment. METHODS: We studied the effect of local delivery of basic fibroblast growth factor (bFGF) on angiogenesis and hepatocyte engraftment within tissue-engineered liver constructs. Poly-l-lactic acid discs were fabricated and coated with either a mixture of saline, sucralfate, and Hydron (control group) or bFGF, sucralfate, and Hydron (bFGF group). bFGF release from polymers in vitro was tested using an ELISA. Hepatocytes were isolated from Lewis rats, seeded on control (n=9) or bFGF (n=11) polymers, and implanted into the small bowel mesentery of syngeneic animals. Specimens were harvested after 2 weeks and analyzed for hepatocyte engraftment. Microvascular density was compared between control (n=6) and bFGF groups (n=5). RESULTS: Three hundred twenty-three thousandths of a microgram of bFGF were incorporated per polymer. Greater than 99% of the bFGF was released into solution by 72 hr in vitro. Two weeks after implantation, microvascular density, as measured by capillaries per high-powered field (c/hpf), was significantly greater in the bFGF group (43.8 c/hpf), compared with the control group (30.5 c/hpf; P<0.005). Specimens from the bFGF group (mean engraftment, 61,355 microm2) showed a 2.5-fold increase in hepatocyte engraftment as compared with control (24,197 microm2; P<0.002). CONCLUSIONS: The angiogenic growth factor bFGF can be incorporated into degradable polymers used as delivery devices for hepatocyte transplantation. Implantation of these devices increases angiogenesis into the device and increases hepatocyte engraftment.
Animals↗
Biodegradable, elastic shape-memory polymers for potential biomedical applications.
The introduction of biodegradable implant materials as well as minimally invasive surgical procedures in medicine has substantially improved health care within the past few decades. This report describes a group of degradable thermoplastic polymers that are able to change their shape after an increase in temperature. Their shape-memory capability enables bulky implants to be placed in the body through small incisions or to perform complex mechanical deformations automatically. A smart degradable suture was created to illustrate the potential of these shape-memory thermoplastics in biomedical applications.
Animals↗
Endothelial cells derived from human embryonic stem cells.
Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.
Cell Differentiation↗
Biocompatibility of lipid-protein-sugar particles containing bupivacaine in the epineurium.
Novel lipid-protein-sugar particles (LPSPs) are potentially biocompatible because they are composed of naturally occurring ingredients and their expected tissue dwell times are relatively short. In this research, we used histological sections to study tissue reaction to LPSPs (4.4-microm median diameter) when used for sciatic nerve block in the rat. As a reference, we compared LPSPs to 60-microm median diameter poly(lactic-co-glycolic) acid (PLGA) microspheres (110,000 MW PLGA, glycolic/lactic ratio 65:35). Four days after injection, both particle types produced acute inflammation within the confines of the injectate, inflammation in adjacent tissues, and myotoxicity. Bupivacaine-free particles did not display myotoxicity, and inflammation in adjacent tissues was reduced. At 2 weeks, inflammation from LPSPs had almost disappeared, whereas PLGA microspheres had a foreign-body giant cell reaction until at least 8 weeks after injection. In contrast, 3.6-microm median diameter, 20,000-MW PLGA microspheres produced a primarily histiocytic reaction 2 weeks after injection. In summary, the LPSPs and PLGA microspheres studied herein have excellent biocompatibility, but tissue reaction to the former is of much shorter duration. Myotoxicity and inflammation of surrounding tissue is largely attributed to bupivacaine. Foreign-body giant cells may be attributed to particle size rather than a specific reaction to PLGA.
1,2-Dipalmitoylphosphatidylcholine↗
Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells.
To better direct repair following spinal cord injury (SCI), we designed an implant modeled after the intact spinal cord consisting of a multicomponent polymer scaffold seeded with neural stem cells. Implantation of the scaffold-neural stem cells unit into an adult rat hemisection model of SCI promoted long-term improvement in function (persistent for 1 year in some animals) relative to a lesion-control group. At 70 days postinjury, animals implanted with scaffold-plus-cells exhibited coordinated, weight-bearing hindlimb stepping. Histology and immunocytochemical analysis suggested that this recovery might be attributable partly to a reduction in tissue loss from secondary injury processes as well as in diminished glial scarring. Tract tracing demonstrated corticospinal tract fibers passing through the injury epicenter to the caudal cord, a phenomenon not present in untreated groups. Together with evidence of enhanced local GAP-43 expression not seen in controls, these findings suggest a possible regeneration component. These results may suggest a new approach to SCI and, more broadly, may serve as a prototype for multidisciplinary strategies against complex neurological problems.
Animals↗
Erodible conducting polymers for potential biomedical applications.
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Biocompatible Materials↗
Porous resins as a cavitation enhancer for low-frequency sonophoresis.
The application of low-frequency ultrasound enhances drug transport through the skin, a phenomenon referred to as low-frequency sonophoresis. This enhancement is mediated through cavitation, the formation and collapse of gaseous bubbles. We hypothesized that the efficacy of low-frequency sonophoresis can be significantly enhanced by provision of nuclei for cavitation. In this study, we used two porous resins, Diaion HP20 and Diaion HP2MG (2MG), as cavitation nuclei. We measured the effect of these resins on cavitation using pitting of aluminum foil. 2MG showed a higher efficacy in enhancing cavitation compared with Diaion HP20. 2MG was also effective in enhancing transdermal mannitol transport. These results confirm that the addition of cavitation nuclei such as porous resins further increases the effect of low-frequency ultrasound on skin permeability.
Administration, Cutaneous↗
Effects of low-frequency ultrasound on the transdermal permeation of mannitol: comparative studies with in vivo and in vitro skin.
The in vivo and the in vitro correlation of the effects of low-frequency ultrasound (low-frequency sonophoresis, LFS) on the percutaneous penetration of mannitol, a model hydrophilic permeant, was investigated using three in vitro skin models (including full-thickness and split-thickness pig skin, and heat-stripped human cadaver skin) and in vivo pig as the animal model. The central objective of this article was to identify the relevant in vitro skin models and ultrasound conditions that may be used in in vitro LFS studies to predict the effects of LFS in vivo on the transdermal delivery of hydrophilic permeants. In this article, by conducting comparative studies of the in vivo pig skin and of the three in vitro skin models under two LFS protocols (a constant ultrasound energy dose protocol, and a constant skin electrical resistance protocol), we demonstrated that: (1) under a constant ultrasound energy dose protocol (protocol A, 5 min LFS), no good correlation was observed between the in vivo skin and the in vitro skin models in terms of the measured skin permeabilities to mannitol. Moreover, the effects of LFS on the barrier functions of the in vivo pig skin, as measured by the enhancement ratio of the skin permeation rate of mannitol and by the reduction of the skin electrical resistance, are much more pronounced than those observed with the excised skin models in vitro; (2) under a constant skin electrical resistance protocol (protocol B) of LFS, a good correlation was found between the skin permeability to mannitol measured using the three in vitro skin models and that of the in vivo pig skin. This result indicates that by utilizing the skin electrical resistance as a quick indicator of the skin permeabilization state due to LFS, the three in vitro skin models can be utilized to predict the transport rate of mannitol across the in vivo skin during LFS; (3) by applying a recently developed skin porous-pathway theory, we demonstrated that within the range of LFS conditions examined, the three in vitro skin models exhibit similar transport properties to mannitol and similar skin effective pore radius values, and hence, represent equivalent skin models for the in vitro LFS studies in the case of hydrophilic permeants; (4) histological studies revealed that the LFS protocol that was shown to be efficacious in enhancing the skin penetration rate of mannitol across the in vivo pig skin, and was also utilized for the in vivo/in vitro skin comparative studies, is safe for the living skin; and (5) through measuring the skin concentration of mannitol in the presence and in the absence of the LFS treatment, we found that the LFS-induced flux enhancement outweighs the enhancement of the skin concentration of mannitol during the LFS studies both in vivo and in vitro. This result suggests that LFS represents a good method of enhancing the systemic absorption of hydrophilic permeants, while it does not significantly alter the vehicle-to-skin partition coefficient for the same class of permeants.
Administration, Cutaneous↗