Solubilization of docetaxel in poly(ethylene oxide)-block-poly(butylene/styrene oxide) micelles

Poly(ethylene oxide)-block-poly(styrene oxide) (PEO-b-PSO) and PEO-b-poly(butylene oxide) (PEO-b-PBO) of different chain lengths were synthesized and characterized for their self-assembling properties in water by dynamic/static light scattering, spectrofluorimetry, and transmission electron microscopy. The resulting polymeric micelles were evaluated for their ability to solubilize and protect the anticancer drug docetaxel (DCTX) from degradation. The drug release kinetics as well as the cytotoxicity of the loaded micelles were assessed in vitro. All polymers formed micelles with a highly viscous core at low critical association concentrations (<10 mg/L). Micelle morphology depended on the nature of the hydrophobic block, with PBO- and PSO-based micelles yielding monodisperse spherical and cylindrical nanosized aggregates, respectively. The maximum solubilization capacity for DCTX ranged from 0.7 to 4.2% and was the highest for PSO micelles exhibiting the longest hydrophobic segment. Despite their high affinity for DCTX, PEO-b-PSO micelles were not able to efficiently protect DCTX against hydrolysis under accelerated stability testing conditions. Only PEO-b-PBO bearing 24 BO units afforded significant protection against degradation. In vitro, DCTX was released slower from the latter micelles, but all formulations possessed a similar cytotoxic effect against PC-3 prostate cancer cells. These data suggest that PEO-b-P(SO/BO) micelles could be used as alternatives to conventional surfactants for the solubilization of taxanes.     

Polycationic block copolymers of poly(ethylene oxide) and poly(propylene oxide) for cell transfection

A facile, one-step synthesis of cationic block copolymers of poly(2-N-(dimethylaminoethyl) methacrylate) (pDMAEMA) and copolymers of poly(propylene oxide) (PPO) and poly(ethylene oxide) (PEO) has been developed. The PEO-PPO-PEO-pDMAEMA (L92-pDMAEMA) and PEO-pDMAEMA copolymers were obtained via free radical polymerization of DMAEMA initiated by polyether radicals generated by cerium(IV). Over 95% of the copolymer fraction was of molecular mass ranging from 6.9 to 7.1 kDa in size, indicating the prevalence of the polyether-monoradical initiation mechanism. The L92-pDMAEMA copolymers possess parent surfactant-like surface activity. In contrast, the PEO-pDMAEMA copolymers lack significant surface activity. Both copolymers can complex with DNA. Hydrodynamic radii of the complexes of the L92-pDMAEMA and PEO-pDMAEMA with plasmid DNA ranged in size from 60 to 400 nm, depending on the copolymer/DNA ratio. Addition of Pluronic P123 to the L92-pDMAEMA complexes with DNA masked charges and decreased the tendency of the complex to aggregate, even at stoichiometric polycation/DNA ratios. The transfection efficiency of the L92-pDMAEMA copolymer was by far greater than that of the PEO-pDMAEMA copolymer. An extra added Pluronic P123 further increased the transfecton efficacy of L92-pDMAEMA, but did not affect that of PEO-pDMAEMA.     

Enhanced growth of canine bone marrow stromal cell cultures in the presence of acidic fibroblast growth factor and heparin

Summary The ex vivo establishment, expansion, transduction, and reintroduction of autologous bone marrow stromal cells offers a potential efficacious system for somatic cell gene therapy. It is likely that any ex vivo system will require the use of large numbers of cells which express high levels of transgene products. We present a method for routine expansion of canine bone marrow stromal cells, established from initial 10–20 ml marrow aspirates, to greater than 10⁹ cells. This high level expansion of cell cultures uses the stimulatory effect of acidic fibroblast growth factor (aFGF) and heparin. In the absence of these factors, stromal cell cultures grow actively for only 1 to 2 passages, become flattened in morphology, and expand to only 10⁸ cells. In the presence of heparin (5 U/ml), aFGF exerts its effect over a wide range of concentrations (0.1–10 ng/ml) in a dose-dependent manner. The stimulatory effect is dependent on the presence of both aFGF and heparin. Immunocytochemical and cytochemical analyses phenotypically characterize these stromal cells as bone marrow stromal myofibroblasts. Stromal cells grown in the presence of aFGF and heparin grow actively and maintain a fibroblast-like morphology for a number of passages, transduce efficiently with a human growth hormone (hGH) expression vector, and express and secrete high levels of hGH. Human marrow stromal cells were also established and expanded by the same culture method. This culture method should be of great value in somatic cell gene therapy for the delivery of secreted gene products to the plasma of large mammals.     

Synthesis, characterization, and biodegradation of novel poly(ether ester amide)s based on L-phenylalanine and oligoethylene glycol

A new family of novel biodegradable poly(ether ester amide)s (PEEAs) consisting of three building blocks (L-phenylalanine, oligoethylene glycol, and aliphatic acid dichloride) were synthesized by solution polycondensation. Using N,N-dimethylacetamide as the solvent, these PEEA polymers were obtained with fairly good yields with reduced viscosity (eta(red)) ranging from 0.13 to 0.61 dL/g. The chemical structures of the PEEAs were confirmed by IR, NMR spectra, and elemental analysis. The PEEAs had Tg values lower than that of the saturated poly(ester amide)s (PEAs) of similar structures due to the incorporation of ether bonds in the backbones. An increase in the number of ether bonds in PEEA resulted in a lower Tg value. The solubility of the PEEA polymers in a wide range of common organic solvents was significantly improved when compared with unsaturated PEAs. The preliminary in vitro biodegradation behaviors of PEEA polymers were investigated in both pure PBS buffer and alpha-chymotrypsin solution of different concentrations. The polymers showed a significantly faster weight loss in an enzyme solution (alpha-chymotrypsin) but a very slow biodegradation rate in pure PBS buffer. The enzymatic hydrolysis rates of PEEAs (in terms of weight loss) were found to be much faster than those of saturated and unsaturated polyesteramides reported in previous studies. The zero-order-like biodegradation kinetics and molecular weight data also suggested surface erosion biodegradation mechanisms for these PEEAs.     

Material properties and cytocompatibility of injectable MMP degradable poly(lactide ethylene oxide fumarate) hydrogel as a carrier for marrow stromal cells

Injectable in situ crosslinkable biomaterials seeded with multipotent progenitor cells and coupled with minimally invasive arthroscopic techniques are an attractive alternative for treating irregularly shaped osteochondral defects. An in situ crosslinkable poly(lactide-co-ethylene oxide-co-fumarate) (PLEOF) macromer has been developed with ultralow molecular weight poly(L-lactide) and poly(ethylene glycol) (PEG) units linked by fumaryl unit. The PLEOF macromer was crosslinked with the MMP-13 degradable peptide sequence QPQGLAK with acrylate end-groups or the methylene bisacrylamide (BISAM) crosslinker to form enzymatically or hydrolytically degradable hydrogels, respectively. Cell viability of the peptide crosslinker was significantly higher than that of BISAM. The relatively higher molecular weight peptide crosslinker significantly affected the water content and the rate of crosslinking (e.g., sol vs gel fraction). The addition of a small fraction of a highly reactive BISAM crosslinker to the PLEOF/peptide mixture reduced the gelation time and increased the elastic modulus while retaining enzymatic degradability of the hydrogel. Bone marrow stromal (BMS) cells were encapsulated in the peptide crosslinked PLEOF hydrogel; 84% of the encapsulated cells was viable after 1 week of incubation in osteogenic media. The encapsulated BMS cells differentiated to osteoblasts and produced a mineralized matrix, as measured by ALPase activity and calcium content. The degradation rate of the hydrogel depended on the ratio of the peptide to the BISAM crosslinker, MMP-13 concentration, and incubation time. The results demonstrate that the peptide crosslinked PLEOF hydrogel with tunable degradation characteristics is potentially useful as an injectable in situ crosslinkable carrier for bone marrow stromal cells.     

Bouquet-type dendrimerlike poly(ethylene oxide)s with a focal aldehyde and peripheral hydroxyls

Doubly functionalized dendrimerlike poly(ethylene oxide)s (PEOs) carrying 16 hydroxyl groups at their periphery and one aldehyde group at their focal point were synthesized up to the fourth generation through an iterative divergent approach. First, a protected aldehyde dihydroxyl compound, namely, 3,3-diethoxy-1,2-propanediol, was used as initiator for the anionic ring-opening polymerization (AROP) of ethylene oxide after partial deprotonation (30%) in dimethyl sulfoxide. The two hydroxyls carried by the PEO chain ends of the first generation were subsequently derivatized so as to generate four hydroxyls via a two-step reaction (allylation and osmylation). The next generations of such dendrimerlike PEOs were grown upon repeating the same cycle of AROP and chain-end modification. At the completion of these reactions, the acetal group present at the core was deprotected under acidic conditions to afford the targeted dendrimerlike PEO of fourth generation with a central aldehyde group. The reactivity and accessibility of the latter function was demonstrated upon its conjugation with aniline used as a model compound.     

Effects of growth factors on growth of stromal CFU-f in mouse bone marrow cell cultures]

Purified mouse IL-1 at doses 15-100 mu/ml inhibits the growth of stromal clonogenic cells /CFU-f/ both in full bone marrow cell cultures /F-cultures/ and in adherent bone marrow cell cultures /A-cultures/. Rec. human TNF-alpha inhibits growth of these cells at doses greater than 50 u/ml, but stimulates it /in 1.5 fold increase/at low doses /0.1-20 u/ml/ in cultures of both types. Rec. mouse IL-3 at doses 0.8-50 mu/ml slightly increases/in 1.6 fold increase/the in vitro growth of CFU-f and inhibits it at low doses in F-cultures. In A-cultures this factor stimulates CFU-f growth at all doses tested, but this stimulating effect takes place if only explantation density of mouse bone marrow cells in sufficiently high.     

Evaluation of human bone marrow stromal cell growth on biodegradable polymer/bioglass composites

Bone tissue engineering using human bone marrow mesenchymal stem cells (HBMCs) and biocompatible materials provides an attractive approach to regenerate bone tissue to meet the major clinical need. The aim of this study was to examine the effects of novel porous biodegradable composite materials consisting of a bioactive phase (45S5 Bioglass, 0, 5, and 40 wt%) incorporated within a biodegradable poly(dl-lactic acid) matrix, on HBMCs growth. Cell adhesion, spreading, and viability was examined using Cell Tracker Green/Ethidium Homodimer-1. Bone formation was assessed using scaffolds seeded with stro-1 positive HBMCs in nude mice. In vitro biochemistry indicated that with minimal scaffold pre-treatment osteoblast activity falls with increasing Bioglass content. However, 24h scaffold pre-treatment with serum resulted in a significant increase in alkaline phosphatase specific activity in 5 wt% Bioglass composites relative to the 0 and 40 wt% Bioglass groups. In vivo studies indicate significant new bone formation throughout all the scaffolds, as evidenced by immunohistochemistry.     

Thermally cross-linked oligo(poly(ethylene glycol) fumarate) hydrogels support osteogenic differentiation of encapsulated marrow stromal cells in vitro

A novel polymer, oligo(poly(ethylene glycol) fumarate) (OPF), cross-linked with a thermal radical initiation system has recently been developed in our laboratory as an injectable, biodegradable cell carrier for regeneration of orthopaedic tissues. The cross-linking, swelling, and degradative properties of hydrogels prepared from OPF with poly(ethylene glycol) of two different chain lengths were assessed. The two OPF types had similar gelation onset times ( approximately 3.6 min) but, when cross-linked for 8 min at 37 degrees C, exhibited significantly different swelling characteristics (fold swelling: 17.5 +/- 0.2 vs 13.4 +/- 0.4). Rat marrow stromal cells (MSCs) were then directly combined with the hydrogel precursors and encapsulated in a model OPF formulation at approximately 14 million cells/mL, cultured in vitro in the presence of osteogenic supplements (dexamethasone), and monitored over 28 days via histology. MSC differentiation in these samples (6 mm diameter x 0.5 mm thick before swelling), as determined by Von Kossa staining for calcified matrix, was apparent by day 21. At day 28, mineralized matrix could be seen throughout the samples, many microns away from the cells. These experiments strongly support the usefulness of thermally cross-linked OPF hydrogels as injectable cell carriers for bone regeneration.     

Tumour necrosis factor-alpha (TNFalpha) stimulates the growth of human bone marrow stromal cells

This study reports that TNF-alpha is a potent mitogen for human bone marrow sternal cells in vitro (assessed by [(3)H]-thymidine incorporation into DNA and cell counts). In contrast, cytokines such as IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-6, LIF, SCF, M-CSF, G-CSF and GM-CSF had no effect. The effect of TNF-alpha on the growth of human bone marrow stromal cells could be of importance during inflammatory processes which take place in the marrow, for example marrow fibrosis.     

Biodegradable polymeric nanospheres formed by temperature-induced phase transition in a mixture of poly(lactide-co-glycolide) and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer

The mixture of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer(F-127) and PLGA (poly(lactide-co-gycolide)) forms a liquid state above their phase transition temperatures, and the phase-separated state is induced by decreasing the temperature below the phase transition temperature. On the basis of the temperature-induced phase transition behavior in the mixture of F-127 and PLGA, a novel method for the preparation of drug-loaded PLGA nanospheres was designed and characterized by measuring the loading amount, the encapsulation efficiency, and the drug release pattern. Paclitaxel, used as a potent anticancer drug, was selected as a model drug.     

A convenient method for the synthesis of amine-terminated poly(ethylene oxide) and poly(epsilon-caprolactone)

A convenient synthetic route to prepare amine-terminated poly(ethylene oxide) (PEO) and poly(epsilon-caprolactone) (PCL) was described. The strategy involved two-step reactions, the condensation of hydroxyl-terminated PEO and PCL with N-benzyloxycarbonyl amino acid followed by the catalytic hydrogenation under mild conditions. NMR and GPC measurements indicated that the reactions proceeded nearly quantitatively. Amine-terminated PEO thus prepared was used to initiate the polymerization of alpha-(N(epsilon)-benzyloxycarbonyl-L-lysine) N-carboxy anhydride [lys(Z)-NCA], and the results confirmed that the reactivity of the amino group was high.     

Crystalline/amorphous phase structure and molecular mobility of biodegradable poly(butylene adipate-co-butylene terephthalate) and related polyesters

Differential scanning calorimetry (DSC), atomic force microscopy (AFM), wide-angle X-ray scattering (WAXD), and solid-state (13)C NMR have been used to investigate the crystalline/amorphous structure and molecular mobility of biodegradable poly(butylene adipate-co-44 mol % butylene terephthalate) [P(BA-co-44 mol % BT)] copolyester sample crystallized from the melt. The DSC endothermic peak, which is ascribed to the melting of the crystalline region, was broad relative to those reported for conventional partially crystalline polyesters. In AFM observation, spherulitic morphology was not observed while small particles with a size of about 100 nm were detected. The WAXD pattern of the sample was very broad. These results have indicated that a melt-crystallized P(BA-co-44 mol % BT) sample contains small crystals with a wide distribution in size. A solid-state (13)C NMR technique was also used to perform molecular-level and selective analyses for both butylene terephthalate and butylene adipate units. For the butylene terephthalate units, the existence of two components with different microstructure and molecular mobility was detected: one component was assigned to the alpha-form crystal of poly(butylene terephthalate) homopolymer (PBT) and the other was in amorphous regions. In contrast, all of butylene adipate units were located in amorphous regions. Solid-state NMR data have suggested that sizes of crystalline regions are less than 3 nm.     

In situ immobilization of proteins and RGD peptide on polyurethane surfaces via poly(ethylene oxide) coupling polymers for human endothelial cell growth

A "CBABC"-type pentablock coupling polymer, mesylMPEO, was designed and synthesized to promote human endothelial cell growth on the surfaces of polyurethane biomaterials. The polymer was composed of a central 4,4'-methylenediphenyl diisocyanate (MDI) coupling unit and poly(ethylene oxide) (PEO) spacer arms with methanesulfonyl (mesyl) end groups pendent on both ends. As the presurface modifying additive (pre-SMA), the mesylMPEO was noncovalently introduced onto the poly(ether urethane) (PEU) surfaces by dip coating, upon which the protein/peptide factors (gelatin, albumin, and arginine-glycine-aspartic acid tripeptide [RGD]) were covalently immobilized in situ by cleavage of the original mesyl end groups. The pre-SMA synthesis and PEU surface modification were characterized using nuclear magnetic resonance spectroscopy ((1)H NMR), attenuated total reflection infrared spectroscopy (ATR-FTIR), and X-ray photoelectron spectroscopy (XPS). Human umbilical vein endothelial cells (HUVEC) were harvested manually by collagenase digestion and seeded on the modified PEU surfaces. Cell adhesion ratios (CAR) and cell proliferation ratios (CPR) were measured using flow cytometry, and the individual cell viability (ICV) was determined by MTT assay. The cell morphologies were investigated by optical inverted microscopy (OIM) and scanning electrical microscopy (SEM). The gelatin- and RGD-modified surfaces were HUVEC-compatible and promoted HUVEC growth. The albumin-modified surfaces were compatible but inhibited cell adhesion. The results also indicated that, for HUVEC in vitro cultivation, the cell adhesion stage was of particular importance and had a significant impact on the cell responses to the modified surfaces.     

Poly(ester urethane)s consisting of poly[(R)-3-hydroxybutyrate] and poly(ethylene glycol) as candidate biomaterials: characterization and mechanical property study

Poly(ester urethane)s with poly[(R)-3-hydroxybutyrate] (PHB) as the hard and hydrophobic segment and poly(ethylene glycol) (PEG) as the soft and hydrophilic segment were synthesized from telechelic hydroxylated PHB (PHB-diol) and PEG using 1,6-hexamethylene diisocyanate as a nontoxic coupling reagent. Their chemical structures and molecular characteristics were studied by gel permeation chromatography, 1H NMR, and Fourier transform infrared spectroscopy. Results of differential scanning calorimetry and X-ray diffraction indicated that the PHB segment and PEG segment in the poly(ester urethane)s formed separate crystalline phases with lower crystallinity and a lower melting point than those of their corresponding precursors, except no PHB crystalline phase was observed in those with a relatively low PHB fraction. Thermogravimetric analysis showed that the poly(ester urethane)s had better thermal stability than their precursors. The segment compositions were calculated from the two-step thermal decomposition profiles, which were in good agreement with those obtained from 1H NMR. Water contact angle measurement and water swelling analysis revealed that both surface hydrophilicity and bulk hydrophilicity of the poly(ester urethane)s were enhanced by incorporating the PEG segment into PHB polymer chains. The mechanical properties of the poly(ester urethane)s were also assessed by tensile strength measurement. It was found that the poly(ester urethane)s were ductile, while natural source PHB is brittle. Young's modulus and the stress at break increased with increasing PHB segment length or PEG segment length, whereas the strain at break increased with increasing PEG segment length or decreasing PHB segment length.     

Enzymatic surface hydrolysis of poly(ethylene terephthalate) and bis(benzoyloxyethyl) terephthalate by lipase and cutinase in the presence of surface active molecules

A lipase from Thermomyces lanuginosus and cutinases from Thermobifida fusca and Fusarium solani hydrolysed poly(ethylene terephthalate) (PET) fabrics and films and bis(benzoyloxyethyl) terephthalate (3PET) endo-wise as shown by MALDI-Tof-MS, LC–UVD/MS, cationic dyeing and XPS analysis. Due to interfacial activation of the lipase in the presence of Triton X-100, a seven-fold increase of hydrolysis products released from 3PET was measured. In the presence of the plasticizer N,N-diethyl-2-phenylacetamide (DEPA), increased hydrolysis rates of semi-crystalline PET films and fabrics were measured both for lipase and cutinase. The formation of novel polar groups resulted in enhanced dye ability with additional increase in colour depth by 130% and 300% for cutinase and lipase, respectively, in the presence of plasticizer.     

Studies on the activity and stability of immobilized horseradish peroxidase on poly(ethylene terephthalate) grafted acrylamide fiber

Having been activated with glutaraldehyde, modified poly(ethylene terephthalate) grafted acrylamide fiber was used for the immobilization of horseradish peroxidase (HRP). Both the free HRP and the immobilized HRP were characterized by determining the activity profile as a function of pH, temperature, thermal stability, effect of organic solvent and storage stability. The optimum pH values of the enzyme activity were found as 8 and 7 for the free HRP and the immobilized HRP respectively. The temperature profile of the free HRP and the immobilized HRP revealed a similar behaviour, although the immobilized HRP exhibited higher relative activity in the range from 50 to 60 °C. The immobilized HRP showed higher storage stability than the free HRP.     

Detection and characterization of a B cell stimulatory factor (BSF-TC) derived from a bone marrow stromal cell line

Stromal cell lines derived from murine bone marrow support the growth of immature pre-B cells and produce cytokines that affect the growth and differentiation of other hematopoietic precursors. Conditioned medium (CM) from one such line (TC-1) stimulated marked proliferation of B cells previously activated by anti-Ig (anti-Ig blasts). Proliferation of anti-Ig blasts was not induced by purified cytokines known to be produced by TC-1 (CSF-1, GM-CSF, or G-CSF) or by IL-1, IL-2, IL-3, IL-4, IL-5, or IL-6. Furthermore, IL-2, IL-4, and IL-5, alone or in combination, failed to support proliferation or differentiation of anti-Ig blasts. TC-1 CM enhanced proliferation of B cells that were co-cultured with LPS, anti-Ig, or dextran sulfate; co-stimulation with anti-Ig was unaffected by the presence of monoclonal anti-IL-4. Proliferation of low, but not high, density B cells isolated from spleen was directly stimulated by TC-1 CM. These results suggest that bone marrow stromal cells produce a novel B cell stimulatory factor (BSF-TC) that induces proliferation of activated B cells.     

Chemical modification of wheat protein-based natural polymers: grafting and cross-linking reactions with poly(ethylene oxide) diglycidyl ether and ethyl diamine

Mobile poly(ethylene oxide) diglycidyl ether (PEODGE) segments were chemically grafted onto a soluble wheat protein (WP), and different network structures were formed via coupling reactions with ethyl diamine (EDA) in different PEODGE/EDA (PE) ratios. When the PE ratio was 1:1, linear PEs were the predominant segments grafted onto WP chains and the whole WP-PEODGE-EDA (WPE) system was still soluble with an increased molecular weight. Reducing the amount of EDA in the systems produced insoluble cross-linked WPE networks. The broad distribution of network structures and chain mobility resulted in a broad glass transition for the WPE materials. However, the glass transition started at lower temperatures, and the materials became flexible at room temperature. The PE segments were present in all rigid, intermediate, and mobile phases in WPE networks, while the proportion of mobile WP chains was increased as a result of the plasticization effect from the mobile PE segments. The mobility of the most mobile component lipid was also restricted to some extent when forming the cross-linked WPE networks. The study demonstrated that the formation of different network structures with PE segments could significantly improve the flexibility of WP materials, vary the solubility, and modify the mechanical performance of WP-based natural polymer materials.     

Human bone marrow stromal cell lines from myeloma and rheumatoid arthritis that can support murine pre-B cell growth.

In order to elucidate the pathologic significance of the bone marrow (BM) microenvironment in multiple myeloma (MM) and rheumatoid arthritis (RA), we established patient- or healthy donor (HD)-derived BM stromal cell lines by transfecting the plasmid for expression of SV40 large T Ag and examined their ability to support the stromal cell-dependent growth of a pre-B cell line, DW34. The means of recovered cell numbers of DW34 co-cultured with MM- and RA-derived BM stromal cell lines ranged from 6- to 10-fold more than those with HD-derived ones. Their enhanced ability to support DW34 cell growth was not caused by cytokines, including IL-6, IL-7, and c-kit ligand, although exogenous IL-7 could augment the growth-supporting ability. DW34 cell growth on the stromal cell lines was abolished by inhibiting cell-to-cell interaction with a membrane filter. FACS analysis revealed that the stromal cell lines did not express LFA-1 alpha, beta, NCAM, or ELAM-1. Both patient and HD BM stromal cell lines variably expressed ICAM-1, VCAM-1, and CD44. However, surface expression levels of these molecules did not correlate with the ability of the stromal cell lines to support DW34 cell growth. Taken together, these results suggested that BM microenvironment might play important roles in the pathogenesis of MM and RA.