Larry Brown

Cellular Response to Transforming Growth Factor-β1 and Basic Fibroblast Growth Factor Depends on Release Kinetics and Extracellular Matrix Interactions

The extracellular matrix plays an important role in growth factor biology, serving as a potential platform for rapid growth factor mobilization or a sink for concentrated sequestration. We now demonstrate that when a growth factor binds reversibly to the matrix, its effects are augmented by this interaction, and when the factor is absorbed irreversibly to the extracellular matrix, it becomes sequestered. These findings call into question the notion that all growth factors are best presented to cells and tissues in a sustained and controlled fashion. In our studies, we examined basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) release kinetics from synthetically fabricated microsphere devices and naturally synthesized extracellular matrix. While the sustained release of bFGF was up to 3.0-fold more potent at increasing vascular endothelial and smooth muscle cell proliferation than bolus administration, the reverse was true for TGF-β1. A bolus of TGF-β1 inhibited vascular cells up to 3.8-fold more efficiently than the same amount of TGF-β1 if control-released. Both growth factors bound to the extracellular matrix, but only bFGF was released in a controlled fashion (2.8%/day). Contact with the extracellular matrix and subsequent release enhanced bFGF activity such that it was 86% more effective at increasing smooth muscle cell numbers than equal amounts of growth factor diluted from frozen stock. TGF-β1 remained tightly adherent. The small amount of TGF-β1 released from the extracellular matrix was ∼30% less effective than bolus administration at inhibiting vascular endothelial and smooth muscle cell growth. Sustained growth factor release may be the preferable mode of administration, but only when a similar mode of metabolism is utilized endogenously.

A MICRO SUSTAINED RELEASE SYSTEM FOR EPIDERMAL GROWTH FACTOR

SummaryA technique for ensuring the controlled release of microgram and smaller amounts of biologically active epidermal growth factor (EGF) from polymeric delivery systems is described. We show that albumin in milligram quantities can facilitate the sustained release of picogram amounts of EGF for at least 3 wk. The EGF-containing polymer matrix can be placed directly into cell culture and will increase the proliferation rate of serum-starved cells. The method reported here should be suited particularly to the delivery of biologically active growth factors that are obtainable in only microgram or smaller amounts.

Controlled Release of Insulin From Polymer Matrices: Control of Diabetes in Rats

The controlled release of insulin from ethylene-vinyl acetate copolymer matrices wasdemonstrated for over 100 days in vivo. The matrices were designed to release sodium insulin at near-constant rates. These 0.06-cm3 implants were coated completely with an impermeable layer of polymer. An aperture was drilled in the center of one face of the matrix, restricting release through this opening. These devices were implanted into 13 streptozocin-induced diabetic rats. Plasma glucose concentrations fell from 386 ± 18 to 119 ± 35 mg/dl (mean ± SEM), and urinary glucose was eliminated. Thee parameters were controlled for up to 105 days by a single implant. Glycosylated hemoglobin concentrations measured 90 days after implantation were 3.86 ± 0.11% for the polymer-treated rats, 3.10 ± 0.18% for the normal controls, and 5.42 ± 0.33% for the diabetic controls. The average weight gain of the treated rats was similar to that of the controls, whereas the diabetic controls failed to thrive. In addition, all of the diabetic controls developed cataracts 1 mo after diabetes induction, whereas none of the treated rats developed cataracts.

A Microsphere-Based Vaccine Prevents and Reverses New-Onset Autoimmune Diabetes

OBJECTIVE—This study was aimed at ascertaining the efficacy of antisense oligonucleotide-formulated microspheres to prevent type 1 diabetes and to reverse new-onset disease. RESEARCH DESIGN AND METHODS—Microspheres carrying antisense oligonucleotides to CD40, CD80, and CD86 were delivered into NOD mice. Glycemia was monitored to determine disease prevention and reversal. In recipients that remained and/or became diabetes free, spleen and lymph node T-cells were enriched to determine the prevalence of Foxp3+ putative regulatory T-cells (Treg cells). Splenocytes from diabetes-free microsphere-treated recipients were adoptively cotransferred with splenocytes from diabetic NOD mice into NOD-scid recipients. Live-animal in vivo imaging measured the microsphere accumulation pattern. To rule out nonspecific systemic immunosuppression, splenocytes from successfully treated recipients were pulsed with β-cell antigen or ovalbumin or cocultured with allogeneic splenocytes. RESULTS—The microspheres prevented type 1 diabetes and, most importantly, exhibited a capacity to reverse clinical hyperglycemia, suggesting reversal of new-onset disease. The microspheres augmented Foxp3+ Treg cells and induced hyporesponsiveness to NOD-derived pancreatic β-cell antigen, without compromising global immune responses to alloantigens and nominal antigens. T-cells from successfully treated mice suppressed adoptive transfer of disease by diabetogenic splenocytes into secondary immunodeficient recipients. Finally, microspheres accumulated within the pancreas and the spleen after either intraperitoneal or subcutaneous injection. Dendritic cells from spleen of the microsphere-treated mice exhibit decreased cell surface CD40, CD80, and CD86. CONCLUSIONS—This novel microsphere formulation represents the first diabetes-suppressive and reversing nucleic acid vaccine that confers an immunoregulatory phenotype to endogenous dendritic cells.

Controlled Release of Insulin From Polymer Matrices: In Vitro Kinetics

A biocompatible system was developed that permits continuous release of biologicallyactive insulin from small polymer matrices. Powdered insulin particles were incorporated into an ethylene-vinyl acetate copolymer matrix. The presence of particulate insulin resulted in a matrixcomposed of tortuous channels and constricted pores through which release occurred. When aqueous release media permeated the matrix, the insulin dissolved and diffused slowly through this tortuous network. The large concentration of insulin within the matrix provided the driving force for release. Release kinetics from these insulin polymer matrices were enhanced by increasing the insulin solubility, the insulin powder particle size, the loading of insulin within the matrix, and the porosity of the matrix. Appropriate geometric design of the polymer matrix resulted in near-constant insulin release rates.

[30] Controlled release and magnetically modulated release systems for macromolecules

Publisher Summary This chapter presents the methodology of formulating of polymeric delivery systems. The methods of preparing polymeric delivery systems, methods of regulating the release kinetics of these systems, and a discussion of the advantages and limitations of the systems as well as potential directions for future research in this area are presented. The basis for the slow release of macromolecules through these polymers appears to be diffusion through a series of interconnecting macrochannels in the polymer matrix. These channels do not normally exist within the polymer but are caused by the incorporation of solid or liquid in the matrix during the casting procedure. Thus, factors that increase the size of these channels or provide simpler pathways (lower tortuosity) for diffusion out of the matrix would be expected to increase release rates. Modulated delivery systems controlled by external means may ultimately improve the release pattern of certain drugs such as insulin. Modulated release systems triggered by magnetism or other mean may also be useful for delivering hormones for birth control and other therapies.

Drug Clearance and Arterial Uptake After Local Perivascular Delivery to the Rat Carotid Artery

OBJECTIVES We attempted to characterize how drug released into the perivascular space enters the arterial wall and how it is cleared from the local environment. BACKGROUND Drug released into the perivascular space can enter the artery either from the adventitial aspect or from the lumen after absorption by the extraarterial capillaries and mixing within the systemic circulation. Some investigators suggest that this latter mechanism dominates, and they question whether local drug release is synonymous with local deposition. METHODS We investigated both the pathways by which adventitially released drug is cleared from the perivascular space and those by which drug enters the blood vessel wall. Inulin was used to follow drug release from implanted devices and subsequent entry to the circulation, because of its first-pass urinary excretion. Heparin was used to follow arterial deposition because of its vasoactivity and tissue-binding properties. The different potential pathways of drug entry and egress were systematically removed and the effects on metabolism and deposition determined. RESULTS Ligature occlusion of the artery did not decrease inulin excretion or heparin deposition. Extravascular wraps designed to shield the device from extramural capillaries reduced inulin excretion rates 10-fold but did not alter heparin deposition into the vessel wall. The deposition of drug after perivascular delivery was 500 times higher than after intraperitoneal administration. CONCLUSIONS Although almost all the drug released into the perivascular space is cleared through the extravascular capillaries, virtually all the deposited drug diffuses directly from the perivascular space, and little arrives from the endovascular aspect. These data support the view that local drug release leads directly to increased local drug concentration.

Optimal Control of Blood Glucose: The Diabetic Patient or the Machine?

Computer-controlled infusion of multiple hormones might more precisely control blood glucose, changing the insulin-centric treatment of diabetes, but perhaps creating new issues in the face of residual challenges. In this issue of Science Translational Medicine, El-Khatib et al. describe a “closed-loop” bihormonal artificial pancreas, designed to avert episodes of low blood sugar in patients with insulin-dependent diabetes. We discuss the benefits and challenges of therapy directed at tight control of blood glucose and ask whether this and similar technological breakthroughs can address as yet unanswered questions in the biology of diabetes.

Sintered Polymers for Sustained Macromolecular Drug Release

Polymeric devices for the sustained release of macromolecules have been in use since 1976 (1). Although the primary application thus far has been for bioassays (2, 3), there is great promise for the use of these devices for pharmaceutical purposes (4). In particular, many polypeptides, which are now available due to genetic engineering, are difficult to administer either orally (due to attack by the digestive system), or intravenously (due to short half-lives in the circulation) (5). Moreover, many of these drugs must be administered on a continuous basis in order to be effective. It is our belief that polymeric devices provide one solution to the problem of delivery of such unstable drugs.

Controlled Release and Magnetically Modulated Systems for Macromolecules: Recent Advances

Since the initial report that inert biocompatible polymers such as ethylene-vinyl acetate copolymer could be used for the controlled release of macromolecules (M.W.> 1000) (1), these systems have been used by different investigators in many areas of research (2–21). Macromolecules such as enzymes (22), antigens (23) and insulin (24) have been released in biologically active form for up to six months in vivo. Extensive studies in vitro have demonstrated that the release rates of drugs from these devices can be adjusted over a 2000-fold range by simple alterations in the fabrication procedures of those macromolecule-polymer matrices (25). Constant release rates have been achieved using appropriate geometric design of these matrices (26) and a method of externally regulating the release rates of these systems using magnetism has been developed (27).