Doxorubicin loaded pH-sensitive micelle targeting acidic extracellular pH of human ovarian A2780 tumor in mice.

The purpose of this study was to examine the efficacy of a chemotherapeutic drug, doxorubicin (DOX), loaded in pH-sensitive micelles poly(l-histidine) (M(n):5K)-b-PEG (M(n):5K) micelles. The micelles were designed to target the acidic extracellular pH of solid tumors. Studies of pH-dependent cytotoxicity, growth rate of the tumor, pharmacokinetics and biodistribution were conducted. In vitro DOX uptake upon A2780 cells by incubating the cells in a pH 6.8 complete medium at a concentration of 20 microg DOX/ml in the micelle formulation was more than five times that of pH 7.4 condition for initial 20 min. In vivo pharmacokinetic data showed that AUC (area under concentration curve) and half life time (t(1/2)) (plasma half life) of DOX in the pH sensitive micelles increased about 5.8- and 5.2-fold of free DOX in phosphate buffered saline (PBS), respectively. It appeared that DOX in the pH-sensitive micelles preferentially accumulated in the tumor site. The distributions at 12 h post injection in other organs including liver, kidney, spleen, lung and heart were not significantly different from those of DOX in PBS at a 6 mg DOX/kg dose. The in vivo test of anti-tumor activity was performed with human ovarian carcinoma A2780 which was subcutaneously xenografted in female nu/nu athymic mice. The pH-sensitive micelle formulation significantly retarded tumor growth rate without serious body weight loss. The triggered drug release by the reduced tumor pH is believed to be a major mechanism of the observed efficacy after passive accumulation of the micelles by EPR effect. This may have resulted in a local high dose of drug in the tested solid tumor.

Cell transplantation for endocrine disorders.

Current hormonal replacement therapy for endocrine disorders cannot, unfortunately, reproduce the complex metabolic interactions of hormones. The organ or cell transplantation would be a more physiological approach to the treatment of endocrine disorders. For decades, remarkable progress in organ or cell transplantation in endocrine disorders has been made, especially in recent years. But there are many limitations in the widespread application of allotransplantation because of rejection. Various methods of immunomanipulations designed to overcome rejection have been proposed, which include immunosuppression, immunomodulation and immunoisolation. The transplantation of immunoisolated cells and some clinical results of the transplants were reviewed. Also a perspective for future directions on endocrine cell transplantation was provided in this review. Human islet cell transplantation for the cure of diabetes was emphasized in this chapter and other cell transplantation for endocrine disorders was also discussed briefly, including parathyroid tissue transplantation, bioartificial thyroid transplantation and adrenal cell transplantation.

Phase-transition polymers for drug delivery.

Phase-transition polymers show changes in response to external stimuli, such as pH, temperature, light, metabolite, and electric current. Based on the stimuli-induced phase transition, many applications have been developed to improve drug delivery. This paper summarizes various phase-transition polymers and their applications relevant to modulated-drug delivery.

PDMS-based polyurethanes with MPEG grafts: synthesis, characterization and platelet adhesion study.

Polyurethane (PU), based on poly(dimethyl siloxane) (PDMS) as a soft segment, with monomethoxy poly(ethylene glycol) (MPEG) grafted onto it, was synthesized as a new polymeric biomaterial for coating PDMS-based biomedical devices. Two different chain extenders, ethylene glycol (EG) and diethyl bis(hydroxymethyl) malonate (DBM), were used for the synthesis of PDMS-based PUs and then MPEG was grafted onto them by allophanate and esterification reactions, respectively. Their molecular structures were confirmed qualitatively and quantitatively using FT-IR and 1H-NMR measurements. PDMS-based PU was more hydrophobic than Pellethane, which is a commercial biomedical-grade poly(ether urethane), due to the PDMS-rich phase at the polymeric surface. However, the incorporation of MPEG in PDMS-based PU induced an increase in hydrophilicity. Analyses of their morphology using dynamic mechanical analysis and differential scanning calorimetry showed that the degree of phase separation increased with the content of hard segments. It also showed that MPEG is compatible with a hard segment consisting of 4,4'-diphenylmethane diisocyanate (MDI) and DBM, while being incompatible with one consisting of MDI and EG. Platelet adhesions with PDMS-based PUs were significantly reduced when compared with Pellethane. It was also observed from a platelet adhesion experiment that the incorportion of MPEG further reduced platelet adhesion. PDMS-based PUs with MPEG grafts, which have few hard segments and a distinct PEG phase, exhibited the least platelet adhesion among the polymer samples tested.

Synthesis of sulfonylurea conjugated copolymer via PEO spacer and its in vitro short-term bioactivity in insulin secretion from islets of Langerhans.

In order to reduce the number of immunoprotected islets required in xeno- or allogenic transplants for reversing diabetes, analogues of glyburide (a sulfonylurea), an extremely hydrophobic insulin secretagogue, were synthesized and used in an attempt to produce water soluble sulfonylurea (SU) grafted polymers. After synthesizing various polymers containing glyburide analogues, a poly(N-vinyl-2-pyrrolidone-co-sulfonylurea succinyl PEO (Mw = 3000) acrylate) was found to be soluble in a cell culture medium at pH 7.4. However, solubility was only obtained by decreasing solution pH from 11 to 7.4. When the copolymer was added to the islet cell culture media at a concentration of 5 microg ml(-1) (based on the theoretical SU content of the copolymer), insulin secretion was enhanced by about 30% at low glucose concentrations of 50 and 100 mg dl(-1) compared to the control. This is equivalent to 40-60% bioactivity of glyburide. The polymer's effect on insulin secretion at a higher glucose concentration of 200 mg dl(-1) was not significant. Considering the previous results where a similar but insoluble polymer without a PEO spacer was used and the polymer showed SU bioactivity only at a glucose concentration of 50 mg dl(-1), the observations from this study indicates that the solubility of SU-grafted polymers may affect the binding of SU groups to SU receptors on the pancreatic beta-cells, resulting in improved pharmacodynamic effect of SU.

Preparation and characterization of albumin-heparin microspheres.

Albumin-heparin microspheres were prepared by a two-step process which involved the preparation of a soluble albumin-heparin conjugate, followed by formation of microspheres from this conjugate or by a double cross-linking technique involving both coupling of soluble albumin and heparin and microsphere stabilization in one step. The first technique was superior since it allowed better control over the composition and the homogeneity of the microspheres. Microspheres could be prepared with a diameter of 5-35 microns. The size could be controlled by adjusting the emulsification conditions. The degree of swelling of the microspheres was sensitive to external stimuli, and increased with increasing pH and decreasing ionic strength of the medium.

Degradation and intrahepatic compatibility of albumin-heparin conjugate microspheres.

The in vitro degradation properties of glutaraldehyde cross-linked albumin and albumin-heparin conjugate microspheres (AMS and AHCMS respectively) were evaluated using light microscopy, turbidity measurements and heparin release determinations, showing that the microspheres are degraded by proteolytic enzymes such as trypsin, proteinase K and lysosomal enzymes. The degradation rate was inversely related to the cross-link density of the microspheres. After intrahepatic administration of AHCMS, cross-linked with 0.5% glutaraldehyde, to male Wag/Rij rats by injection into a mesenteric vein (intravenoportal: i.v.p.), the microspheres were entrapped in the hepatic vascular system. The AHCMS were entrapped within terminal portal veins predominantly at the periphery of the liver. The AHCMS were degraded by cellular enzymatic processes within 2 wk after injection, with a half life of approximately 1 d. Biocompatibility of AHCMS and adriamycin-loaded AHCMS was evaluated by histological assessment of the mitotic activity of liver parenchyma and inflammatory response, and by determination of liver damage marker enzymes during 4 wk after administration. Liver damage marker enzymes were not increased compared with controls, nor were adverse effects observed upon histological examination. There was no difference in response between empty and adriamycin-loaded AHCMS.

The effect of zinc-crystallized glucagon-like peptide-1 on insulin secretion of macroencapsulated pancreatic islets.

This research investigates the use of an insulinotropic factor, glucagon-like peptide-1 (GLP-1), to enhance insulin secretion from islets within a macrocapsule. A zinc-crystallized form of GLP-1 was added to the macrocapsule device to have a longer and more controlled release of the bioactive monomer GLP-1. The type of macrocapsule device used for this study consisted of a hollow fiber (MWCO 100,000 and 1 mm inner diameter) containing rat islets and GLP-1 crystals within a poly(N-isopropylacrylamide-co-acrylic acid) (2 mol% acrylic acid) matrix. When incubating the system in media with a high glucose concentration (300 mg/dL), insulin secretion was enhanced with a >85% increase after an induction period. When the same type of system was used in a dynamic perfusion experiment, similar results were obtained. GLP-1 crystals can be an effective form to be entrapped in a bioartificial pancreas to enhance insulin secretion function, especially at high glucose concentrations.

Drug release from biodegradable injectable thermosensitive hydrogel of PEG-PLGA-PEG triblock copolymers.

An aqueous solution of newly developed low-molecular-weight PEG-PLGA-PEG triblock copolymers with a specific composition is a free flowing sol at room temperature but becomes a gel at body temperature. Two model drugs, ketoprofen and spironolatone, which have different hydrophobicities, were released from the PEG-PLGA-PEG triblock copolymer hydrogel formed in situ by injecting the solutions into a 37 degrees C aqueous environment. Ketoprofen (a model hydrophilic drug) was released over 2 weeks with a first-order release profile, while spironolactone (a model hydrophobic drug) was released over 2 months with an S-shaped release profile. The release profiles were simulated by models considering degradation and diffusion, and were better described by a model assuming a core-shell structure of the gel.

Biodegradable amphiphilic multiblock copolymers and their implications for biomedical applications.

Alternating multiblock copolymers composed of short blocks of poly(ethylene oxide) (PEO) and poly(epsilon-caprolactone) (PCL) or poly(L-lactic acid) (PLLA) were synthesized by a coupling reaction. The block copolymers of relatively high molecular weights (M(n)20,000) formed a physically crosslinked thermoplastic network, while low molecular weight polymers were water-soluble. The block copolymers demonstrated solubility in a variety of solvents including acetone, tetrahydrofuran, methylene chloride, dioxane, water/acetone mixtures, and water/ethanol mixtures. The degree of swelling, optical transparency, and mechanical property of the films, prepared by a solvent casting method, were affected by the nature of the hydrophobic block used, polymer composition, temperature, and thermal history. The crystalline melting temperatures of PCL and PLLA in the block copolymers were significantly lowered due to the chemical structure of difunctional PCL and PLLA, and partial phase mixing with PEO segments. The properties of the block copolymers may be useful for biomedical applications as well as controlled drug release formulations. When PEO/PLLA multiblock copolymers were applied as a wound healing material loaded with basic fibroblast growth factor (bFGF), the feasibility study showed improved wound healing when compared to controls of no treatment and the same wound covering without bFGF, indicating that a certain degree of the bioactivity of bFGF is preserved.

Extracellular matrix for a rechargeable cell delivery system.

Above a critical concentration, aqueous polymer solutions of N-isopropylacrylamide copolymers with small amounts of acrylic acid, synthesized in benzene by radical polymerization, exhibited four distinct phases as the temperature increased; clear solution, opaque solution, gel and shrunken gel. The transition between the opaque solution phase and the gel phase was in the range of 30-34 degrees C and was reversible without syneresis and noticeable hysteresis under the experimental conditions used in this study. Islets of Langerhans, isolated from Sprague-Dawley rat pancreata and entrapped in the gel matrix, remained viable, with no significant decrease in insulin secretion function in vitro for one month. When islets were encapsulated with the gel matrix in hollow fibers [molecular weight cut-off (MWCO)= approximately 400000] and were exposed to dynamic changes in glucose and theophylline concentrations, their insulin secretion patterns demonstrated a smaller lag time and higher amplitude in insulin release than islets entrapped in a conventional alginate matrix under the same experimental conditions. From these two observations, i.e. gel reversibility and islet functionality in the matrix observed in in vitro experiments, the N-isopropylacrylamide copolymers with acrylic acid synthesized in this study are optimum candidates for the extracellular matrix in a diffusion chamber-type cell delivery system in order to recharge the entrapped cells when cell functionality in the system decreases.

Self-assembled hydrogel nanoparticles from curdlan derivatives: characterization, anti-cancer drug release and interaction with a hepatoma cell line (HepG2).

Self-assembled hydrogel nanoparticles were synthesized from carboxymethylated (CM)-curdlan, substituted with a sulfonylurea (SU) as a hydrophobic moiety for self-assembly. The degree of SU substitution was 2.4, 5.6, or 7.2 SU groups per hundred anhydroglucose units of curdlan. The physicochemical properties of the self-assembled hydrogel nanoparticles (DS 2.4, DS 5.6, and DS 7.2) in aqueous media were characterized by dynamic light scattering, transmission electron microscopy, and fluorescence spectroscopy. The mean diameter of all samples was less than 300 nm with a unimodal size distribution. The critical aggregation concentrations (CAC) of self-assembled hydrogel nanoparticles in distilled water were 4.2 x 10(-2), 3.1 x 10(-2) and 1.9 x 10(-2) mg/ml for DS 2.4, 5.6 and 7.2, respectively. The loading and release of all-trans retinoic acid (ATRA) was studied. The ATRA loading efficiencies and loading contents of CM-curdlan/SU nanoparticles increased as the degree of SU substitution increased. The ATRA release rate was controlled by the degree of substitution and drug-loading. For specific interaction with a hepatic carcinoma cell line (HepG2), CM-curdlan was additionally conjugated with lactobionic acid (LBA; galactose moiety) (5.5 LBA molecules per hundred glucose units). HepG2 was strongly luminated by ligand-receptor interactions with fluorescence-labeled LBA/CM-curdlan/SU hydrogel nanoparticles. The luminescence was not observed for other control cases. It is concluded that LBA/CM-curdlan/SU hydrogel nanoparticles are a useful drug carrier for the treatment of liver cancer, because of the potential immunological enhancement activities of CM-curdlan in the body, the ligand-receptor mediated specific interactions, and the controlled release of the anti-cancer drug.

New biodegradable polymers for injectable drug delivery systems.

Many biodegradable polymers were used for drug delivery and some are successful for human application. There remains fabrication problems, such as difficult processability and limited organic solvent and irreproducible drug release kinetics. New star-shaped block copolymers, of which the typical molecular architecture is presented, results from their distinct solution properties, thermal properties and morphology. Their unique physical properties are due to the three-dimensional, hyperbranched molecular architecture and influence microsphere fabrication, drug release and degradation profiles. We recently synthesized thermosensitive biodegradable hydrogel consisting of polyethylene oxide and poly(L-lactic acid). Aqueous solution of these copolymers with proper combination of molecular weights exhibit temperature-dependent reversible sol-gel transition. Desired molecular arrangements provide unique behavior that sol (at low temperature) form gel (at body temperature). The use of these two biodegradable polymers have great advantages for sustained injectable drug delivery systems. The formulation is simple, which is totally free of organic solvent. In sol or aqueous solution state of this polymer solubilized hydrophobic drugs prior to form gel matrix.

Bioactive polymers for biohybrid artificial pancreas.

To address the solution for some of the obstacles, such as low insulin secretion, limited lifespan and aggregation of transplanted islets, encountered in developing a biohybrid artificial pancreas (BAP), polymeric materials including a reversible polymeric extracellular matrix (ECM), crystallized glucagon-like peptide-1, and oxygen carrying polymers, were prepared and their potential utilities in designing a compact and rechargeable BAP were investigated. For a synthetic, reversible ECM, high molecular weight N-isopropylacrylamide copolymer with a small amount of acrylic acid (2 mole%) was synthesized by conventional radical polymerization in benzene, and its aqueous solution above a critical polymer concentration displayed a sol-gel transition temperature near physiological temperature (33-35 degrees C) without noticeable hysteresis. The physicochemical properties of the gel with islet compatibility proved that the synthetic ECM is an appropriate matrix which can make a BAP rechargeable. Glucagon-like peptide-1 (GLP-1, 7-37) is known to have a strong stimulatory effect on insulin secretion, particularly at high glucose concentrations. When zinc-crystallized GLP-1 was entrapped along with islets in a hollow fiber macrocapsule device, insulin secretion was enhanced at a high glucose concentration (300 mg/dl) with a >85% increase in insulin secretion after an induction period. The cross-linked hemoglobin with difunctional PEO (Hb-C) was prepared to increase the high molecular weight of Hb. This prevents diffusional loss when enclosed in an immunoprotecting membrane. The Hb-C, entrapped in microcapsules, enhanced insulin secretion and improved the viability of microencapsulated islets by promoting oxygen supply to islets. The introduction of the synthetic ECM, crystallized GLP-1, and Hb-C into a BAP may provide a basis for designing a compact and rechargeable BAP (macrocapsule).

Concanavalin A microspheres for a self-regulating insulin delivery system.

A self-regulating insulin delivery system based on the competitive binding of glucose and p-succinylamidophenyl-alpha-D-glycopyranoside-insulin (SAPG-insulin) to crosslinked concanavalin A (Con A) microspheres (MSs) has been investigated. Con A MSs prepared by a water-in-oil emulsion technique demonstrated properties of binding to glucose and SAPG-insulin (binding constants of 1.25 x 10(3) and 1.28 x 10(4) M-1, respectively) that are similar to the literature values of these properties for unmodified Con A. The retained binding capacity (number of binding sites per molecule) of Con A MSs, produced with 20% of the epsilon-amino groups crosslinked, was approximately 28% that of intact Con A. As a new device, Con A MSs loaded with SAPG-insulin was enclosed in a heat-sealed, surface-modified, porous poly(vinylidene difluoride) membrane pouch. This system showed a pulsatile release pattern for insulin with a short lag time in response to glucose challenges of 50-500 mg/dL. The pattern of release of SAPG-insulin from the devices was studied with varying design parameters, such as surface area, membrane pore size, and loading content of SAPG-insulin.

Innovative ambulatory drug delivery system using an electrolytic hydrogel infusion pump.

This report describes an ambulatory infusion device developed to provide parenteral drug delivery at a precisely controlled rate. The device is based on the innovative and unique concept of utilizing electrohydrolysis of a negatively charged hydrogel. The system consists of two modules: a pump unit and an electronic control unit. The pump module, which can be a disposable unit, contains medication separated by a flexible membrane from a gas generating chamber; this latter is an electrolytic cell comprising a hydrogel block and two platinum electrodes. The microcontroller-based control module is constructed with a user interface which includes input keys and a liquid crystal display, as well as a control to alter driving current level, depending on the infusion rate required. A microprocessor instantaneously calculates the current level required; this is based on operator-selected infusion rate, ambient pressure, and temperature sensor output. The accuracy and precision of the device were verified for all flow rates and for different environmental conditions; in vitro test results showed acceptable accuracy with less than +/- 5% error over the whole operating range of 0.1-100 [ml/h]. The device is small, lightweight, simple and easy to manufacture, and is also designed to be comfortably and conveniently worn by patients. It can be used for a variety of regimens including, for example, chemotherapy, insulin delivery, and pain management, antibiotic and AIDS therapy.

Pure or predominant sensory stroke due to brain stem lesion.

BACKGROUND AND PURPOSE: Pure or predominant hemisensory symptoms can be seen in patients with brain stem stroke. However, there have been no reports in which sufficient numbers of patients were studied with detailed descriptions on the sensory patterns and imaging findings. METHODS: We describe 17 patients presenting with pure or predominant hemisensory symptoms due to brain stem stroke in whom CT scan and/or MRI identified appropriate lesions. RESULTS: Eleven patients had an infarct and 6 had a hemorrhage. Aside from sensory deficit, the majority had dizziness and gait ataxia. Fifteen patients had paramedian dorsal pontine lesions associated with pure or predominant lemniscal sensory involvement, often in the cheiro-oral (n = 4) or leg dominance (n = 4) patterns. The lesions of the former group tended to be located more medially compared with those of other patients, which is in agreement with the sensory topography of the pontine lemniscal sensory tract. Bilateral facial or perioral sensory symptoms were noted in 6 patients. One patient with a dorsolateral pontine lesion had selective spinothalamic modality impairment, while one with a lateral midbrain infarct had sensory deficit of all modalities. CONCLUSIONS: Pure or predominant brain stem sensory stroke is most often produced by small infarcts or hemorrhages in the paramedian dorsal pontine area and may be differentiated from thalamic pure sensory stroke by the following characteristics: frequent association of dizziness/gait ataxia, predominant lemniscal sensory symptoms, occasional leg dominance or cheiro-oral pattern, and frequent bilateral perioral involvement.

A glucose oxidase electrode based on electropolymerized conducting polymer with polyanion-enzyme conjugated dopant.

An enzyme immobilization method has been developed by electropolymerization chemistry of conducting polymer which results in a more effective and reproducible enzyme electrode. As a model system, in this study, glucose oxidase (GOD) was conjugated with a polyanion, poly(2-acrylamido-2-methylpropane sulfonic acid), via a poly(ethylene oxide) spacer to improve the efficiency of enzyme immobilization into a conducting polymer. GOD was successfully conjugated with a high conjugation yield of more than 90%, and its bioactivity was preserved. The resulting polyanion-GOD conjugate was used as a dopant for the electrochemical polymerization of pyrrole. Polypyrrole was effectively deposited on a Pt wire working electrode with the polyanion-GOD conjugate. The enzyme electrode responded to glucose concentrations of up to 20 mM with a sensitivity of 40 nA/mM at an applied potential of 0.4 V within a response time of 30 s. Although the response signal decreased at the low applied potential of 0.3 V, the enzyme electrode showed sensitive response signals of about 16 nA/mM up to 20 mM in glucose concentration. Under the deoxygenated condition, reduced but clear response current signal was obtained. The results show that the current signal response of the enzyme electrode to glucose concentration may be produced by mixed mechanisms.

"On-off" thermocontrol of solute transport. II. Solute release from thermosensitive hydrogels.

Poly(N-isopropylacrylamide) (NIPAAm)/polytetramethylene ether glycol (PTMEG) interpenetrating polymer networks (IPNs) were synthesized and their feasibility as thermosensitive hydrogels for drug release was investigated. The release of indomethacin incorporated into these matrices showed pulsatile patterns in response to temperature changes and was sensitive to a few degrees of temperature fluctuation. The temperature inducing on-off release deviated from the gel collapse temperature of unloaded gel, possibly because of solute effects on network properties. The lag time and release profile of indomethacin in the low-temperature region (on process) of each temperature cycle were affected by the gel composition and applied temperature. The results of this study demonstrate that solute release can be regulated by rapid deswelling of the surface of the gels in response to temperature.

"On-off" thermocontrol of solute transport. I. Temperature dependence of swelling of N-isopropylacrylamide networks modified with hydrophobic components in water.

The swelling in water, as a function of temperature, of two series of N-isopropylacrylamide (NIPAAm) polymer networks was studied. In the first series, n-butylmethacrylate (BMA) was copolymerized with NIPAAm, and in the second, polytetramethylene ether glycol (PTMEG) was incorporated into NIPAAm network as a chemically independent interpenetrating network. With increasing BMA content in the poly(NIPAAm-co-BMA) network, the gel collapse point was lowered and the gels deswelled in a more gradual manner with increasing temperature. In the interpenetrating polymer networks (IPN) system, the gel collapse point was not significantly changed by the amount of incorporated PTMEG. In DSC thermograms of swollen samples, the shape and onset temperature of the endothermic peak corresponded to the gel deswelling behavior and gel collapse point. The temperature dependence of equilibrium swelling in water was shown to be a function of the gel composition in both network series. The synthesized networks formed a dense surface layer as the temperature increased past the gel collapse point. This dense layer retarded water efflux and thereby resulted in water pockets at the membrane surface.