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.

PDMS-based polyurethanes with MPEG grafts: mechanical properties, bacterial repellency, and release behavior of rifampicin.

PDMS-based polyurethanes (PUs) grafted with monomethoxy poly(ethylene glycol) (MPEG) were synthesized to develop a coating material for urinary catheters with a silicone surface for minimizing urinary tract infections. MPEG was grafted on PDMS-based PUs by two methods depending on the PU synthetic routes: esterification and allophanate reactions. It was confirmed from mechanical characterization that an increase of the hard segment amount enhanced the ultimate strength and Young's modulus, while reducing elongation at the end-points. The incorporation of MPEG in PDMS-based PUs induced a decrease in tensile strength and Young's modulus, and increased elongation at the break point due to its high flexibility. When hydrated in distilled water, mechanical properties of all PUs synthesized in this study deteriorated due to water absorption. It was evident from the bacterial adhesion test that PDMS-based PUs showed moderate resistance to adhesion of E. coli on their surfaces compared to Pellethane, while the incorporation of MPEG significantly enhanced repellency to bacteria, including E. coli and S. epidermidis. We also studied the release behavior of an antibiotic drug, rifampicin, from the polymeric devices fabricated by solvent evaporation. Although rifampicin is hydrophilic and soluble in pH 7.4 phosphate buffer, it showed a sustained release over 45 days from PDMS-based PUs with MPEG that were grafted on ethylene glycol residues by allophanate reaction. This release characteristic was predominantly influenced by a hydrogen bond interaction between the polymers and rifampicin, which was confirmed through an ATR-IR study. This may imply that the specific interaction is responsible for the delayed release. Considering the mechanical properties, morphologies of drug-incorporated polymeric matrices, and drug release behaviors, PDMS-based PU with MPEG that were grafted on ethylene glycol (a chain extender) residues by allophanate reaction showed better material properties for uretharal catheter coating pusposes in order to minimize urinary tract infections.

Interaction of sulfonylurea-conjugated polymer with insulinoma cell line of MIN6 and its effect on insulin secretion.

A carboxylated derivative of sulfonylurea (SU), an insulinotropic agent, was synthesized and grafted onto a water-soluble polymer as a biospecific and stimulating polymer for insulin secretion. To evaluate the effect of the SU-conjugated polymer on insulin secretion, its solution in dimethyl sulfoxide was added to the culture of insulinoma cell line of MIN6 cells to make 10 nM of SU units in the medium and incubated for 3 h at 37 degrees C. The culture medium was conditioned with glucose concentration of 3.3 or 25 mM. To verify the specific interaction between the SU (K+ channel closer)-conjugated polymer and MIN6 cells, the cells were pretreated with diazoxide, an agonist of adenosine triphosphate-sensitive K+ channel (K+ channel opener), before adding the SU-conjugated polymer to the cell culture medium. This treatment suppressed the action of SUs on MIN6 cells. Fluorescence-labeled polymer with rodamine-B isothiocyanate was used to visualize the interactions, and we found that the labeled polymer strongly absorbed to MIN6 cells, probably owing to its specific interaction mediated by SU receptors on the cell membrane. The fluorescence intensity on the cells significantly increased with an increase in incubation time and polymer concentration. A confocal laser microscopic study further confirmed this interaction. The results from this study provided evidence that SU-conjugated copolymer stimulates insulin secretion by specific interactions of SU moieties in the polymer with MIN6 cells.

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.

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).

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.

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.

Anti-oxidative neuroprotection by estrogens in mouse cortical cultures.

Estrogen replacement therapy in postmenopausal women may reduce the risk of Alzheimer's disease, possibly by ameliorating neuronal degeneration. In the present study, we examined the neuroprotective spectrum of estrogen against excitotoxicity, oxidative stress, and serum-deprivation-induced apoptosis of neurons in mouse cortical cultures. 17beta-estradiol as well as 17alpha-estradiol and estrone attenuated oxidative neuronal death induced by 24 hr exposure to 100 microM FeCl2, excitotoxic neuronal death induced by 24 hr of exposure to 30 microM N-methyl-D-aspartate (NMDA) and serum-deprivation induced neuronal apoptosis. Furthermore, estradiol attenuated neuronal death induced by Abeta25-35. However, all these neuroprotective effects were mediated by the anti-oxidative action of estrogens. When oxidative stress was blocked by an antioxidant trolox, estrogens did not show any additional protection. Addition of a specific estrogen receptor antagonist ICI182,780 did not reverse the protection offered by estrogens. These findings suggest that high concentrations of estrogen protect against various neuronal injuries mainly by its anti-oxidative effects as previously shown by Behl et al. Our results do not support the view that classical estrogen receptors mediate neuroprotection.

Thermoreversible copolymer gels for extracellular matrix.

To improve the properties of a reversible synthetic extracellular matrix based on a thermally reversible polymer, copolymers of N-isopropylacrylamide and acrylic acid were prepared in benzene with varying contents of acrylic acid (0 to 3%) and the thermal properties were evaluated. The poly(N-isopropylacrylamide) and copolymers made with acrylic acid had molecular weights from 0.8 to 1.7 x10(6) D. Differential scanning calorimetry (DSC) showed the high-molecular-weight acrylic acid copolymers had similar onset temperatures to the homopolymers, but the peak width was considerably increased with increasing acrylic acid content. DSC and cloud point measurements showed that polymers with 0 to 3% acrylic acid exhibit a lower critical solution temperature (LCST) transition between 30 degrees and 37 degrees C. In swelling studies, the homopolymer showed significant syneresis at temperatures above 31 degrees C. Copolymers with 1 and 1.5% showed syneresis beginning at 32 degrees and 37 degrees C, respectively. At 37 degrees C the copolymers with 1.5-3% acrylic acid showed little or no syneresis. Due to the high water content and a transition near physiologic conditions (below 37 degrees C), the polymers with 1.5-2.0% acrylic acid exhibited properties that would be useful in the development of a refillable synthetic extracellular matrix. Such a matrix could be applied to several cell types, including islets of Langerhans, for a biohybrid artificial pancreas.

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.

In situ gelation of PEG-PLGA-PEG triblock copolymer aqueous solutions and degradation thereof.

Aqueous solutions of poly(ethylene glycol-b-[DL-lactic acid-co-glycolic acid]-b-ethylene glycol) (PEG-PLGA-PEG) triblock copolymers form a free-flowing sol at room temperature and become a gel at body temperature. In this study, in situ gel formation was investigated in rats. Upon subcutaneous injection of 33 wt % aqueous solutions of PEG-PLGA-PEG triblock copolymer into rats, transparent gels were observed. The gel showed good mechanical strength and the integrity of gels persisted longer than 1 month. The gel underwent degradation by hydrolysis and turned opaque. Degradation study showed preferential mass loss of PEG-rich segment from the in situ formed gel. Number average molecular weight determined by gel permeation chromatography decreased from 3300 to 1900 and 30% mass loss was observed over 1 month.

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.

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.

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.

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.

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.

Insulin release from islets of Langerhans entrapped in a poly(N-isopropylacrylamide-co-acrylic acid) polymer gel.

A copolymer of N-isopropylacrylamide (98 mol% in feed) and acrylic acid, poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAAm-co-AAc)), was prepared by free radical polymerization for development of a thermally reversible polymer to entrap islets of Langerhans for a refillable biohybrid artificial pancreas. A 5 wt% solution of the polymer in Hanks' balanced salt solution forms a gel at 37 degrees C that exhibits no syneresis. Diffusion of fluorescein isothiocyanate (FITC) dextrans having molecular weights of 4400 and 70000 were used to evaluate mass transport in the gel at 37 degrees C. Insulin secretion from islets in the polymer gel was also investigated in both static and dynamic systems. The polymer gel exhibited excellent diffusion of FITC dextran 4400 and FITC dextran 70000 with diffusion ratios, D/D0 (ratio of diffusion in the gel to diffusion in water), of 0.20+/-0.04 and 0.35+/-0.17, respectively. Human islets entrapped in the polymer gel showed prolonged insulin secretion in response to basal (5.5 mM) glucose concentration compared to free human islets. Rat islets showed prolonged insulin secretion in response to high (16.5 mM) glucose concentrations compared to free rat islets. Rat islets in the polymer gel maintained insulin secretion in response to the higher glucose concentration for over 26 days. Rat islets entrapped by the polymer also released higher quantities of insulin more rapidly in response to changes in concentrations of glucose and other stimulants than rat islets entrapped in an alginate control. These results suggest that this material would provide adequate diffusion for rapid insulin release in an application as a synthetic extracellular matrix for a biohybrid artificial pancreas.

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.