Erosion of biodegradable block copolymers made of poly(D,L-lactic acid) and poly(ethylene glycol)

Biodegradable block copolymers made of poly(ethylene glycol) monomethylether (Me.PEG) and poly(D,L-lactic acid) (PLA) were investigated for their erosion properties. Wide angle X-ray diffraction (WAXD) and differential scanning calorimetry (DSC) investigations prior to erosion revealed that despite the low content of crystallizable Me.PEG of 10%, Me.PEG5-PLA45 is a partially crystalline polymer. The erosion of the polymer was investigated using cylindrical polymer matrix discs with a diameter of 8 mm and a height of 1.5 mm. WAXD and DSC spectra obtained from eroded polymer matrix discs suggest that both polymer blocks separate completely during erosion. The crystallinity of Me.PEG5-PLA45 was found to increase during erosion, which is probably due to the improved mobility of Me.PEG inside the polymer with a progressive degree of degradation. The erosion kinetics were found to be similar to that of PLA or poly(lactic-co-glycolic acid). During erosion the polymer matrix weight of dried samples remains constant for 11 weeks after which erosion sets in rapidly. From this observation one can conclude that the impact of the relatively small Me.PEG chains on Me.PEG5-PLA45 erosion is not pronounced. This is beneficial for all those applications that require the stability of the polymer matrix and in which the Me.PEG chain is intended to bring about other effects such as the modification of the surface properties of PLA polymers.     

Interfacial thickness of liposomes containing poly(ethylene glycol)-cholesterol from electrophoresis

The electrophoretic mobilities of liposomes incorporating a polyethylene glycol (PEG) headgroup coupled to cholesterol for PEG of average chain index 3.0, 13.2, and 22.3 have been determined as a function of PEG-cholesterol mole fraction between 5% and 40% and ionic strength between 2 and 200 mM. The liposome compositions were 40 mole % cholesterol plus PEG-cholesterol, 10 mole % 1,2-dipalmitoyl-sn-glyerco-3-phosphoglycerol, and 50 mole % egg phosphatidylcholine. The mobilities were fit to a model in which the PEG forms a surface layer of polymer subject to viscous drag arising from electroosmotic flow within this layer. The model provides estimates of the average layer thickness that are comparable to those determined from contemporary models of surface-attached polymer.     

Surface characteristics and biocompatibility of lactide-based poly(ethylene glycol) scaffolds for tissue engineering

Novel lactide-based poly(ethylene glycol) (PEG) polymer networks (GL9-PEGs) were prepared by UV copolymerization of a glycerol-lactide triacrylate (GL9-Ac) with PEG monoacrylate (PEG-Ac) to use as scaffolds in tissue engineering, and the surface properties and biocompatibility of these networks were investigated as a function of PEG molecular weight and content. Analysis by ATR-FTIR and ESCA revealed that PEG was incorporated well within the GL9-PEG polymer networks and was enriched at the surfaces. From the results of SEM, AFM, and contact angle analyses, GL9-PEG networks showed relatively rough and irregular surfaces compared to GL9 network, but the mobile PEG chains coupled at their termini were readily exposed toward the aqueous environment when contacting water such that the surfaces became smoother and more hydrophilic. This reorientation and increase in hydrophilicity were more extensive with increasing PEG molecular weight and content. As compared to GL9 network lacking PEG, protein adsorption as well as platelet and S. epidermidis adhesion to GL9-PEG networks were significantly reduced as the molecular weight and content of PEG was increased, indicating that GL9-PEG networks are more biocompatible than the GL9 network due to PEG's passivity. Based on the physical and biological characterization reported, the GL9-PEG materials would appear to be interesting candidates as matrices for tissue engineering.     

Interfacial tension of aqueous two-phase systems containing poly(ethylene glycol) and dipotassium hydrogenphosphate

Liquid-liquid equilibrium (LLE) compositions and interfacial tensions of the aqueous two-phase system containing poly(ethylene glycol) (PEG 4000, average Mr=3500; PEG 6000, average Mr=7500; and PEG 20000, average Mr =20000) and dipotassium hydrogenphosphate were experimentally determined by using a shaking flask method and a drop volume method at 288.15, 298.15 and 308.15 K, respectively.     

Water-soluble polyion complex associates of DNA and poly(ethylene glycol)-poly(L-lysine) block copolymer

Complex formation of poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) AB type block copolymer with salmon testes DNA or Col E1 plasmid DNA in aqueous milieu was studied. The PLL segment of PEG-PLL interacts with nucleic acid through an electrostatic force to form a water-soluble complex associate with a diameter of ca. 50 nm. PEG segments surrounding the core of the polyion complex prevented the complex from precipitation even under stoichiometric conditions, at which the unit ratio of L-lysine in PEG-PLL and phosphate in the DNA is equal. The profile of the thermal melting curve revealed a higher stabilization of DNA structure in PEG-PLL/DNA complexes compared to that in the complex made from DNA and PLL homopolymer with the same molecular weight as the PLL segment in PEG-PLL. This stabilizing effect on the DNA structure may be due to the compartmentalization of DNA into the microenvironment of PEG with low permittivity. The reversible nature of the PEG-PLL/DNA complex was further verified through the addition of polyanion [poly-(L-aspartic acid)]: Poly(L-aspartic acid) replaced DNA in the complex with PEG-PLL, resulting in the release of free DNA in the medium. Furthermore, the PEG-PLL/DNA complex showed high resistance against DNase I attack, suggesting DNA protection through the segregation into the core of the associate having PEG palisade.     

Molecular calculations of poly(ethylene glycol) transport across a swollen poly(acrylic acid)/mucin interface

The transport of poly(ethylene glycol) chains than can promote mucoadhesion across the interface between lightly cross-linked poly(acrylic acid) and mucin may be analyzed as a function of molecular characteristics using theories of chain penetration in a dilute network. The fracture energy for the ensuing adhesive bond is proportional to the number of polymer chains crossing the interface, which, in turn, is related to the polymer volume fraction, the chain diffusion coefficient, and the degree of polymerization. Relevant calculations were performed for a number of cross-linked poly(acrylic acid) gels and three different types of poly(ethylene glycol) chains.     

Hydrogels of poly(ethylene glycol): mechanical characterization and release of a model drug

Thermosensitive polymer networks were synthesized from poly(ethylene glycol), hexamethylene diisocyanate and 1,2,6-hexanetriol in stoichiometric proportions. By varying the amount of 1,2,6-hexanetriol and the molar mass of the poly(ethylene glycol), a wide range of networks with different crosslinking densities was prepared. The networks obtained were characterized by the temperature dependence of their degree of equilibrium swelling in water and by their Young's moduli. For each network, the molecular weight between crosslinks was estimated. The structure of the hydrogels was analysed with respect to scaling laws, and it was found that the results obtained with PEG 1500 and PEG 6000 hydrogels are in agreement with theoretical predictions, whereas those obtained with PEG 400 hydrogels are in disagreement. The release properties of PEG hydrogels were studied by the determination of the diffusion coefficient for acebutolol chlorhydrate and by an analysis of the effect of temperature on these coefficients. Finally, these release properties were correlated with the swelling and structural properties of the hydrogels.     

Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels

Hydrogel membranes formed by interfacially photopolymerizing poly(ethylene glycol) (PEG) diacrylate precursor solution were prepared from PEG diacrylate of molecular weights (MW) ranging from 2000 (2K) to 20000 (20K) with concentrations ranging from 10% to 30% w/w. The effects of PEG diacrylate MW and concentration in the membrane precursor solution upon the diffusivities of vitamin B12, myoglobin, ovalbumin, albumin, and IgG were determined. Regardless of the concentration of the PEG diacrylate in the precursor solution, hydrogels prepared with PEG 2K, 4K, and 8K diacrylate were impermeable to proteins with a size equal to or larger than myoglobin (22 kDa), while hydrogels prepared with PEG 20K diacrylate were impermeable to proteins with a size equal to or larger than ovalbumin (45 kDa). Similarities between hydrogels formed from PEG 2K, 4K, and 8K diacrylates were also seen in calculations of the molecular weight between crosslinks and the mesh size, with values in the range of 150-750 g/mol and 15-35 A, respectively, depending on PEG diacrylate concentration. In contrast, hydrogels formed from PEG 20K diacrylate had molecular weight between crosslinks ranging from 1150 to 2000 g/mol and mesh sizes ranging from 45-70 A, with larger values being observed in membranes polymerized from more dilute PEG diacrylate precursor.     

Exchange of monooleoylphosphatidylcholine as monomer and micelle with membranes containing poly(ethylene glycol)-lipid

Surface-grafted polymers, such as poly(ethylene glycol) (PEG), provide an effective steric barrier against surface-surface and surface-macromolecule interactions. In the present work, we have studied the exchange of monooleoylphosphatidylcholine (MOPC) with vesicle membranes containing 750 mol wt surface-grafted PEG (incorporated as PEG-lipid) from 0 to 20 mol % and have analyzed the experimental results in terms of thermodynamic and stationary equilibrium models. Micropipette manipulation was used to expose a single lipid vesicle to a flow of MOPC solution (0.025 microM to 500 microM). MOPC uptake was measured by a direct measure of the vesicle area change. The presence of PEG(750) lipid in the vesicle membrane inhibited the partitioning of MOPC micelles (and to some extent microaggregates) into the membrane, while even up to 20 mol % PEG-lipid, it did not affect the exchange of MOPC monomers both into and out of the membrane. The experimental data and theoretical models show that grafted PEG acts as a very effective molecular scale "filter" and prevents micelle-membrane contact, substantially decreasing the apparent rate and amount of MOPC taken up by the membrane, thereby stabilizing the membrane in a solution of MOPC that would otherwise dissolve it.     

Cholesterol derivative of poly(ethylene glycol) inhibits clathrin-independent, but not clathrin-dependent endocytosis

The effect of poly(ethylene glycol) cholesteryl ethers (PEG(n)-Chols) with two different numbers of units (n = 50 and 200) in the hydrophilic PEG moiety on cellular endocytic activity was studied on HT-1080 cells. The amphipathic molecules were soluble in aqueous solution. When fluorescein derivatives of PEG-Chols (one fluorescein at the distal end of PEG) were incubated with the cells in culture, the cellular fluorescence was localized at the plasma membrane level and in intracellular vesicles. Fluorescence quantification indicated that for the same external concentration, twice more FPEG(50)-Chol than FPEG(200)-Chol was associated with the cells under the same conditions. Regardless of the length of PEG moiety, PEG-Chols' interaction with cells reduced the endocytic internalization of a fluid phase marker, horseradish peroxidase (HRP) depending on the cell-associated amount. In contrast, internalization of 125I-labeled epidermal growth factor (EGF) through receptor-mediated endocytosis did not change upon incubation with PEG(50)-Chol. The effect of PEG(200)-Chol was also small, since EGF internalization showed a reduction of 10-20%, while at the same concentration as much as 80% of HRP uptake was inhibited. PEG(50)-Chol did not influence the internalization of a larger ligand, 125I-transferrin (Tfn). However, in the presence of PEG(200)-Chol, the uptake of 125I-Tfn decreased remarkably, and yet, PEG(200)-Chol has no influence on the binding and internalization of a monoclonal antibody directed toward the ectodomain of the Tfn-receptor. These results suggested that incorporation of PEG-Chols in the outer monolayer of the plasma membrane specifically inhibited clathrin-independent, but not clathrin-dependent endocytosis.     

In vivo and in vitro responses to poly(ethylene terephthalate-co-diethylene glycol terephthalate) and polyethylene oxide blends

Although biocompatible polymeric compounds are generally nontoxic, nonimmunogenic, and chemically inert, implants made from these materials may trigger acute and chronic inflammatory responses. These inflammatory reactions may induce degeneration of implanted biopolymer. Interactions between implanted biomaterial and inflammatory cells are mediated by many cellular events involving cellular adhesion and activation. We studied the inflammatory responses in vivo and in vitro to samples of biopolymers composed of poly(ethylene terephthalate-co-diethylene glycol terephthalate) plus 0, 5, 25% of polyethylene oxide. We observed that these biopolymers did not induce inflammatory responses when implanted in the peritoneal cavity of mice for 28 days. However we observed deposition of hyaluronic acid at the surface of implanted biomaterial, suggesting that tolerance to biomaterial occurred after surgical implantation. No significant adhesion of inflammatory cells such as mononuclear phagocytes and peripheral leukocytes were observed in vitro, when poly(ethylene terephthalate-co-diethylene glycol terephthalate) blends were used as substratum to cellular adhesion. These results suggest that blends composed of poly(ethylene terephthalate-co-diethylene glycol terephthalate) induce low inflammatory cell adhesion, since no rejection of biopolymer was observed when implanted in experimental animal models.     

Comparison of different hydrophobic anchors conjugated to poly(ethylene glycol): effects on the pharmacokinetics of liposomal vincristine

The cost-benefit ratio of tetravalent rhesus rotavirus vaccine (RRV-TV) in Finland for prevention of rotavirus gastroenteritis was assessed in a randomized, double-blind, placebo-controlled trial. Costs related to vaccination, side effects, and gastroenteritis were identified. Children received RRV-TV (n = 1,191) or placebo (n = 1,207) at 2, 3, and 5 months of age with other infant vaccinations. Prospective follow-up averaged 1.0 years per child. An intention-to-treat analysis was performed from the perspective of society. Nine cases of severe rotavirus gastroenteritis occurred in the RRV-TV group, versus 100 in the placebo group (P < .0001); mean cost per vaccinated child was 4 Finnish marks (FIM) in the RRV-TV group, versus 203 FIM in the placebo group. Side effects with related costs occurred after 11% and 7% of doses in the RRV-TV group and placebo group, respectively (P < .001); mean cost per child was 89 FIM vs. 75 FIM. The break-even cost (i.e., net benefit, excluding cost of vaccine) of RRV-TV in prevention of severe rotavirus gastroenteritis was 109 FIM (U.S. $19.60) per child.     

Preparation and characterization of poly(propylene fumarate-co-ethylene glycol) hydrogels

We describe the preparation and bulk characterization of a cross-linked poly(propylene fumarate-co-ethylene glycol), p(PF-co-EG), hydrogel. Eight block copolymer formulations were made varying four different design parameters including: poly(ethylene glycol) (PEG) molecular weight, poly(propylene fumarate) (PPF) molecular weight, copolymer molecular weight, and ratio of PEG to PPF. Two different cross-linking formulations were also tested, one with a cross-linking monomer and one without. The extent of the cross-linking reaction and the degree of swelling in aqueous solution were determined on copolymer formulations made without a cross-linking monomer. The values of molecular weight between cross-links, Mc ranged from 300 +/- 120 to 1190 +/- 320 as determined from swelling data (n = 3). The equilibrium volume swelling ratios, Q, varied from 1.5 +/- 0.1 to 3.0 +/- 0.1. This ratio was found to increase with increasing PEG content in the copolymer and decrease with increasing PPF molecular weight. The values for complex dynamic elastic moduli magnitudes of E*, ranged from 0.9 +/- 0.2 to 13.1 +/- 1.1 MPa for the formulations with the cross-linking monomer, N-vinyl pyrrolidinone (VP) (n = 3). The ultimate tensile stresses on the formulations made with VP ranged from 0.15 +/- 0.03 to 1.44 +/- 1.06 MPa, and tensile moduli ranged from 1.11 +/- 0.20 to 20.66 +/- 2.42 MPa (n = 5). All of the mechanical properties increased with increasing PPF molecular weight and decreased with increasing PEG content in the copolymer. These data show that the physical properties of p(PF-co-EG) hydrogels can be tailored for specific applications by altering the material composition.     

Variation of penicillin acylase partition coefficient with phase volume ratio in poly(ethylene glycol)-sodium citrate aqueous two-phase systems

The influence of phase volume ratio on partition and purification of penicillin acylase from Escherichia coli on poly(ethylene glycol)-sodium citrate aqueous two-phase systems was studied. In PEG 1000 systems both partition coefficients of the enzyme and total protein increased with decreasing phase volume ratio. However, in PEG 3350 containing NaCl, penicillin acylase follows a reverse trend, while total protein behaves in the same way. Implications for protein purification designs are discussed.     

Preparation and characterization of biodegradable nanospheres composed of methoxy poly(ethylene glycol) and DL-lactide block copolymer as novel drug carriers

We synthesized amphiphilic diblock copolymer based on methoxy poly(ethylene glycol) (MePEG) and dl-lactide with different molar composition in bulk without catalyst. Using the resulting amphiphilic diblock copolymers, we prepared drug-loaded polymeric nanospheres by micelle formation through solution behavior of amphiphilic copolymer in selective solvents. The structure of MePEG/dl-lactide diblock copolymers was identified by IR, WAXD, GPC, 1H-NMR. The size of nanosphere measured by dynamic light scattering showed a narrow monodisperse size distribution and average diameter less than 200 nm. From the surface chemical composition of nanosphere by ESCA, the presence of MePEG chains on the nanosphere layers was confirmed. The critical micelle concentration of ML50 sample investigated by fluorescence spectroscopy was 1.44x10-7 mol/l which is lower than common low molecular weight surfactants. In addition, we could obtain nanospheres having a relatively high drug-loading of about 33.0% when the feed weight ratio of indomethacin to polymer was 1:1. In vitro release experiments of the indomethacin-loaded MePEG/dl-lactide nanospheres exhibited sustained release behavior without any burst effects. The results of cytotoxicity tests showed that the MePEG/dl-lactide nanospheres didn't induce any relevant cell damage.     

Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine

In mice, the mdr1a and mdr1b genes encode drug-transporting proteins that can cause multidrug resistance in tumor cells by lowering intracellular drug levels. These P-glycoproteins are also found in various normal tissues such as the intestine. Because mdr1b P-glycoprotein is not detectable in the intestine, mice with a homozygously disrupted mdr1a gene [mdr1a(-/-) mice] do not contain functional P-glycoprotein in this organ. We have used these mdr1a(-/-) mice to study the effect of gut P-glycoprotein on the pharmacokinetics of paclitaxel. The area under the plasma concentration-time curves was 2- and 6-fold higher in mdr1a(-/-) mice than in wild-type (wt) mice after i.v. and oral drug administration, respectively. Consequently, the oral bioavailability in mice receiving 10 mg paclitaxel per kg body weight increased from only 11% in wt mice to 35% in mdr1a(-/-) mice. The cumulative fecal excretion (0-96 hr) was markedly reduced from 40% (after i.v. administration) and 87% (after oral administration) of the administered dose in wt mice to below 3% in mdr1a(-/-) mice. Biliary excretion was not significantly different in wt and mdr1a(-/-) mice. Interestingly, after i.v. drug administration of paclitaxel (10 mg/kg) to mice with a cannulated gall bladder, 11% of the dose was recovered within 90 min in the intestinal contents of wt mice vs. <3% in mdr1a(-/-) mice. We conclude that P-glycoprotein limits the oral uptake of paclitaxel and mediates direct excretion of the drug from the systemic circulation into the intestinal lumen.     

Biodegradation of poly(ethylene adipate) microcapsules in physiological media

Spherical reservoir-type microcapsules fabricated using a water/oil/water (W/O/W) double emulsion technique with solvent evaporation and composed of poly(ethylene adipate) (PEAD) blended with 20% poly-epsilon-caprolactone (PCL II) containing a range of bovine serum albumin (BSA) loadings were incubated in Hank's buffer, pH 7.4, newborn calf serum, 1.5% pancreatin and synthetic gastric juice containing 10% pepsin A over 30 days and their percentage weight loss (PWL) and changes in ultrastructural morphology monitored by gravimetry and stereoscan electron microscopy (SEM) respectively. The greatest PWL from microcapsules was observed after incubation in newborn calf serum (NCS) and pancreatin and decreased in the order NCS > pancreatin > synthetic gastric juice > Hank's buffer. Only microcapsules theoretically loaded with 5-20% BSA and incubated in synthetic gastric juice showed a significant increase in PWL with increasing percentage BSA loading. The structural biodegradation of PEAD microcapsules in both Hank's buffer and synthetic gastric juice was minimal whilst the morphological changes observed during incubation in NCS involved pitting of the membrane, some surface erosion and reduction in diameter, followed by microcapsule membrane disruption and loss of reservoir contents. Biodegradation in pancreatin was associated with surface flaking and loss of large fragments of the microcapsule membrane. Only in NCS and pancreatin, where one would expect to see the effects of enzyme activity in addition to simple ester hydrolysis, did biodegradation proceed to the stage where there was a loss of spherical shape and almost total disruption of the microcapsule structure within 30 days.     

High speed separation of DNA fragments by capillary electrophoresis in poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock polymer

The high speed separation of DNA fragments by using a triblock copolymer, 25% w/v F127 (PEO99PPO69PEO99 with PEO and PPO denoting polyethylene oxide and polypropylene oxide, respectively) which is easy to handle and does not need coating of the quartz capillary, has been investigated. Two ways to decrease the run time are presented: one is to shorten the effective capillary length and the other to increase the electric field strength. In a short capillary, the sieving ability of the separation medium versus the initial band width, and the band width spreading as a function of distance traveled dominate the resolution; at high electric field strength, Joule heating could deteriorate the separation. By taking both effects into account, the phi X174/HaeIII digest could be separated within 100 s by using an 8 mm effective length, 50 microns diameter capillary operating at 300 V/cm.     

Effects of inorganic salts on solubilization of estriol in an aqueous solution of poly(ethylene oxide)/poly(propylene oxide)/poly(ethylene oxide) triblock copolymer

Blood vessels originate as simple endothelial cell tubes. It has been proposed that platelet-derived growth factor B polypeptide (Pdgfb) secreted by these endothelial cells drives the formation of the surrounding muscular wall by recruiting nearby mesenchymal cells. However, targetted inactivation of the Pdgfb gene or the Pdgf receptor beta (Pdgfrb) gene, by homologous recombination, does not prevent the development of apparently normal large arteries and connective tissue. We have used an in vivo competition assay in which we prepared chimaeric blastocysts, composed of a mixture of wild-type (Pdgfrb[+/+]) and Pdgfrb(+/-) or wild-type and Pdgfrb(-/-) cells, and quantified the relative success of cells of the two component genotypes in competing for representation in different cell lineages as the chimaeric embryos developed. This study revealed that the participation of Pdgfrb(-/-) cells in all muscle lineages (smooth, cardiac, skeletal and pericyte) was reduced by eightfold compared with Pdgfrb(+/+) cells, and that participation of Pdgfrb(+/-) cells was reduced by twofold (eightfold for pericytes). Pdgfrb inactivation did not affect cell contribution to non-muscle mesodermal lineages, including fibroblasts and endothelial cells. Chimaera competition is therefore a sensitive, quantitative method for determining developmental roles of specific genes, even when those roles are not apparent from analysis of purebred mutants; most likely because they are masked by homeostatic mechanisms.