Synthesis and characterization of triblock copolymers of methoxy poly(ethylene glycol) and poly(propylene fumarate)

Amphiphilic block copolymers were synthesized by transesterification of hydrophilic methoxy poly(ethylene glycol) (mPEG) and hydrophobic poly(propylene fumarate) (PPF) and characterized. Four block copolymers were synthesized with a 2:1 mPEG:PPF molar ratio and mPEGs of molecular weights 570, 800, 1960, and 5190 and PPF of molecular weight 1570 as determined by NMR. The copolymers synthesized with mPEG of molecular weights 570 and 800 had 1.9 and 1.8 mPEG blocks per copolymer, respectively, as measured by NMR, representing an ABA-type block copolymer. The number of mPEG blocks of the copolymer decreased with increasing mPEG block length to as low as 1.5 mPEG blocks for copolymer synthesized with mPEG of molecular weight 5190. At a concentration range of 5-25 wt % in phosphate-buffered saline, copolymers synthesized with mPEG molecular weights of 570 and 800 possessed lower critical solution temperatures (LCST) between 40 and 45 degrees C and between 55 and 60 degrees C, respectively. Aqueous solutions of copolymer synthesized with mPEG 570 and 800 also experienced thermoreversible gelation. The sol-gel transition temperature was dependent on the sodium chloride concentration as well as the mPEG block length. The copolymer synthesized from mPEG 570 had a transition temperature between 40 and 20 degrees C with salt concentrations between 1 and 10 wt %, while the sol-gel transition temperatures of the copolymer synthesized from mPEG molecular weight 800 were higher in the range 75-30 degrees C with salt concentrations between 1 and 15 wt %. These novel thermoreversible copolymers are the first biodegradable copolymers with unsaturated double bonds along their macromolecular chain that can undergo both physical and chemical gelation and hold great promise for drug delivery and tissue engineering applications.     

PNA conjugated to high-molecular weight poly(ethylene glycol): synthesis and properties

The conjugation of a bioactive, fluorescent PNA sequence to high-molecular weight poly(ethylene glycol) (PEG) is described and the properties of the PEG-PNA conjugate are evaluated.     

Physiochemical properties of low and high molecular weight poly(ethylene glycol)-grafted poly(ethylene imine) copolymers and their complexes with oligonucleotides

Inefficient delivery of antisense oligonucleotides (AOs) to target cell nuclei remains as the foremost limitation to their usefulness. Copolymers of cationic poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) have been well-studied for delivery of plasmids. However, the properties of PEG-PEI-AO polyplexes have not been comprehensively investigated. Therefore, we synthesized a series of PEG-PEI copolymers and evaluated their physiochemical properties alone and when complexed with AO. The M(w) of PEG was found to be the main determinant of polyplex size, via its influence on particle aggregation. DLS measurements showed that when PEG5000 was grafted to PEI2K and PEI25K, polyplex diameters were extremely small (range 10-90 nm) with minimal aggregation. In contrast, when PEG550 was grafted to PEI2K and PEI25K, polyplexes appeared as much larger aggregates (approximately 250 nm). As expected, the surface charge (zeta potential) was higher for polyplexes containing PEI25K than those containing PEI2K, but decreased with increased levels of PEG grafting. Surprisingly, within the physiological range (pH 7.5-5), the buffering capacity of all copolymers was nearly equivalent to that of unsubstituted PEI2K or PEI25K, and was barely influenced by PEGylation. The stability of polyplexes was evaluated using a heparin polyanion competition assay. Unexpectedly, polyplexes containing PEI2K showed stability equal to or greater than that of PEI25K polyplexes. The level of PEG grafting also had a dramatic effect on polyplex stability. The relationships established between molecular formulations and polyplex size, aggregation, surface charge, and stability should provide a useful guide for future studies aimed at optimizing polymer-mediated AO delivery in cell and animal studies. A summary of the relationships between polyplex structures and recent studies of their transfection capacity is provided.     

Star-shaped poly(ethylene glycol)-block-polyethylenimine copolymers enhance DNA condensation of low molecular weight polyethylenimines

Star-shaped poly(ethylene glycol)-block-polyethylenimine [star-(PEG-b-PEI)] significantly enhance plasmid DNA condensation of low molecular weight (MW) PEIs. The star-block copolymers were prepared via a facile synthesis route using hexamethylene diisocyanate as linker between PEG and PEI blocks. NMR and FT-IR spectroscopy confirmed the structures of intermediately activated PEG and final products. Furthermore, the copolymers were characterized by size exclusion chromatography, static light scattering, and viscosimetry. Their molecular weights (M(w) 19-26 kDa) were similar to high MW PEI (25 kDa). Thermoanalytical investigations (thermogravimetric analysis, differential scanning calorimetry) were also performed and verified successful copolymer synthesis. DNA condensation with the low MW PEIs (800 and 2000 Da) and their 4- and 8-star-block copolymers was studied using atomic force microscopy, dynamic light scattering, zeta-potential measurements, and ethidium bromide (EtBr) exclusion assay. It was found that low MW PEIs formed huge aggregates (500 nm to 2 microm) in which DNA is only loosely condensed. By contrast, the star-block copolymers yielded small (80-110 nm), spherical and compact complexes that were stable against aggregation even at high ionic strength and charge neutrality. Furthermore, as revealed in the EtBr exclusion assay these star-block copolymers exhibited a DNA condensation potential as high as high MW PEI. Since these star-(PEG-block-PEI) copolymers are composed of relatively nontoxic low MW PEI and biocompatible PEG, their potential as gene delivery agents merits further investigations.     

Diblock copolymers based on dihydroxyacetone and ethylene glycol: synthesis, characterization, and nanoparticle formulation

Polymeric biomaterials have played an integral role in tissue engineering, biomedical devices, and targeted drug delivery. Block copolymers are especially important because their physical and chemical properties can be controlled by adjusting the ratio, size, and type of constituting blocks. Herein, the synthesis and characterization of diblock copolymers composed of poly(ethylene glycol) and a polycarbonate based on the metabolic intermediate, dihydroxyacetone, are reported. The length of the dihydroxyacetone-based block was controlled by adjusting the reactant feed ratios and initiator injection conditions. Intermediates and final products were characterized via (1)H NMR, GPC, DSC, TGA, and diffusion-ordered NMR spectroscopy. The dihydroxyacetone-based hompolymer is insoluble in water and most organic solvents, but is hydrophilic in nature. This, coupled with poly(ethylene glycol)'s solubility characteristics, allows the block copolymer to form nanoparticles in aqueous and organic anti-solvents. Dynamic light scattering and TEM results indicated the formation of spherical nanoparticles.     

Long functionalized poly(ethylene glycol)s of defined molecular weight: synthesis and application in solid-phase synthesis of conjugates

A concise synthesis of long-chain poly(ethylene glycol) (PEG) of defined molecular weight up to 29 ethyleneoxy units is described. These PEG diols were converted in a two-step synthesis into Fmoc-protected PEG amino acids, suitable as long linkers and compatible with solid-phase peptide synthesis. Long PEG chains (MW > 1000) can be readily synthesized with this method, which has the advantage of defined single molecular weight products over the comparable commercial polymers. The application of these PEG linkers to the synthesis of peptide-PEG-folate conjugates on a solid support was investigated. A method for the solid support synthesis of the targeting component of the conjugate, folic acid-cysteine, was developed, resulting in improved yields with respect to literature methods. The assembly of the peptide, PEG linker, and targeting group on solid support resulted in the synthesis of a conjugate of defined molecular weight and structure.     

Low molecular weight polyethylenimine-graft-Tween 85 for effective gene delivery: synthesis and in vitro characteristics

The development of safe and efficient gene delivery systems is still a challenge for successful gene therapy. In this work, low molecular weight polyethylenimine (PEI 2K) was modified by Tween 85, which bears three oleate chains. Tween 85 modified PEI 2K (TP) could condense DNA efficiently, and TP/DNA complexes (TPCs) showed high resistance to salt-induced aggregation and enzymatic degradation. In addition, TP did not show the obvious cytotoxicity. The introduction of Tween 85 led to a significant increase in the cellular uptake of complexes with higher transfection efficiency, which was strongly inhibited by the addition of free Tween 85 in MCF-7/ADR cells, but not in MCF-7 cells. These results indicated that TP could be a potentially safe and effective copolymer for gene delivery, and TPCs could be taken up mainly by Tween 85-mediated endocytosis in MCF-7/ADR cells.     

Galactose-poly(ethylene glycol)-polyethylenimine for improved lung gene transfer

Polyethylenimine (PEI) is a potential gene transfer agent, but is limited by its poor transfection efficiency in vivo due to poor solubility and stability, pronounced toxicity and non-specific interaction with target cells. To improve its pulmonary gene transfection property, galactose (whose binding lectins are abundantly expressed in the lung) was selected as a ligand to improve the binding and uptake of the modified PEI/pDNA (plasmid DNA) polyplexes into lung cells. A novel protocol was developed to synthesize galactose-polyethylenglycol (PEG)-PEI copolymers. The resulting galactose-PEG-PEI/pDNA polyplexes showed improved solubility, stability, and reduced toxicity. Compared with that obtained by PEI/pDNA at a N/P ratio of 6, the transfection efficiency of 1% galactose-PEG-PEI/pDNA polyplexes at the N/P ratio of 36 was 4.5- and 11.6-fold in the A549 cell line and in mice lung, respectively. These data taken suggest that galactose-PEG-PEI may be a promising pulmonary gene delivery system.     

Synthesis and characterization of star poly(epsilon-caprolactone)-b-poly(ethylene glycol) and poly(L-lactide)-b-poly(ethylene glycol) copolymers: evaluation as drug delivery carriers

Two types of 32 arm star polymers incorporating amphiphilic block copolymer arms have been synthesized and characterized. The first type, stPCL-PEG 32, is composed of a polyamidoamine (PAMAM) dendrimer as the core with radiating arms having poly(epsilon-caprolactone) (PCL) as an inner lipophilic block in the arm and poly(ethylene glycol) (PEG) as an outer hydrophilic block. The second type, stPLA-PEG 32, is similar but with poly(L-lactide) (PLA) as the inner lipophilic block. Characterization with SEC, (1)H NMR, FTIR, and DSC confirmed the structure of the polymers. Micelle formation by both star copolymers was studied by fluorescence spectroscopy. The stPCL-PEG 32 polymer exhibited unimolecular micelle behavior. It was capable of solubilizing hydrophobic molecules, such as pyrene, in aqueous solution, while not displaying a critical micelle concentration. In contrast, the association behavior of stPLA-PEG 32 in aqueous solution was characterized by an apparent critical micelle concentration of ca. 0.01 mg/mL. The hydrophobic anticancer drug etoposide can be encapsulated in the micelles formed from both polymers. Overall, the stPCL-PEG 32 polymer exhibited a higher etoposide loading capacity (up to 7.8 w/w % versus 4.3 w/w % for stPLA-PEG 32) as well as facile release kinetics and is more suitable as a potential drug delivery carrier.     

Synthesis and characterization of six-arm star poly(delta-valerolactone)-block-methoxy poly(ethylene glycol) copolymers

Novel amphiphilic six-arm star diblock copolymers based on biocompatible and biodegradable poly(delta-valerolactone) (PVL) and methoxy poly(ethylene glycol) (MePEG) were synthesized by a two-step process. First, the hydrophobic star-shaped PVL with hydroxyl terminated functional groups was synthesized using a multifunctional alcohol, dipentaerythritol (DPE), as the initiator and fumaric acid as the catalyst. The amphiphilic six-arm star copolymer of poly(delta-valerolactone)-b-methoxy poly(ethylene glycol), (PVL-b-MePEG)(6), was then synthesized by coupling the hydroxyl terminated six-arm PVL homopolymer with alpha-methoxy-omega-chloroformate-poly(ethylene glycol) (MePEG-COCl). (1)H NMR and GPC analyses confirmed the successful synthesis of star-shaped copolymers with predicted compositions and narrow molecular weight distributions. DSC analysis revealed that the glass transition temperatures of the star PVL homopolymers with M(n) between 5000 and 49 000 are not dependent on their molecular weights, whereas the melting temperatures of both the PVL homopolymers and the amphiphilic (PVL-b-MePEG)(6) copolymers increase with an increase in the PVL molecular weight. Micelles were prepared from the (PVL-b-MePEG)(6) copolymers via the dialysis method and found to have effective mean diameters ranging from 10 to 45 nm, depending on the copolymer composition. In addition, the (PVL-b-MePEG)(6) copolymers having lower PVL content were found to form micelles with a narrow monomodal size distribution, whereas the copolymers having higher PVL content tended to form aggregates with a bimodal size distribution. The noncytotoxicity of the copolymers was also confirmed in CHO-K1 fibroblast cells using a cell viability assay, indicating that the (PVL-b-MePEG)(6) copolymers are suitable for biomedical applications such as drug delivery.     

Study of the synthesis, crystallization, and morphology of poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymers

Poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymers PEG-PCL were synthesized by ring-opening polymerization of epsilon-caprolactone using monomethoxy poly(ethylene glycol) as the macroinitiator and calcium ammoniate as the catalyst. Obvious mutual influence between PEG and PCL crystallization was studied by altering the relative block length. Fixing the length of the PEG block (Mn = 5000) and increasing the length of the PCL block, the crystallization temperature of the PCL block rose gradually from 1 to about 35 degrees C while that of the PEG block dropped from 36 to -6.6 degrees C. Meanwhile, the melting temperature of the PCL block went up from 30 to 60 degrees C, while that of the PEG block declined from 60 to 41 degrees C. If the PCL block was longer than the PEG block, the former would crystallize first when cooling from a molten state and led to obviously imperfect crystallization of PEG and vice versa. And they both crystallized at the same temperature, if their weight fractions were equal. We found that the PEG block could still crystallize at -6.6 degrees C even when its weight fraction is only 14%. A unique morphology of concentric spherulites was observed for PEG5000-PCL5000. According to their morphology and real-time growth rates, it is concluded that the central and outer sections in the concentric spherulites were PCL and PEG, respectively, and during the formation of the concentric spherulite, the PEG crystallized quickly from the free space of the PCL crystal at the earlier stage, followed by outgrowing from the PCL spherulites in the direction of right angles to the circle boundaries until the entire area was occupied.     

Cationic amphiphilic star and linear block copolymers: synthesis, self-assembly, and in vitro gene transfection

A series of amphiphilic star and linear block copolymers were synthesized using ATRP. The core consisted of either polystyrene (PS) or poly(n-butyl acrylate) (PBuA), having different glass-transition (T(g)) values. These polymers were used as macroinitiators in the polymerization of the cationic 2-(dimethylamino)ethyl methacrylate (DMAEMA). The polymers were used to study the effects of polymer architecture and flexibility on the self-assembling properties, DNA complexation, and transfection. All polymers formed core-shell micelles in aqueous solutions and condensed plasmid DNA. Linear PDMAEMA-PBuA-PDMAEMA has transfection efficiency comparable to PEI25K in ARPE19 cell line. Glassy state of the micellar core and star-shaped architecture decreased the DNA transfection compared with the rubbery and linear polymer structures. The polymers showed low cellular toxicity at low nitrogen/phosphate (n/p) ratios.     

Poly(ethylene glycol)-carboxymethyl chitosan-based pH-responsive hydrogels: photo-induced synthesis, characterization, swelling, and in vitro evaluation as potential drug carriers

In this study, carboxymethyl chitosan was prepared, characterized, and then photo-induced graft copolymerized with poly(ethylene glycol) under a nitrogen atmosphere in aqueous solution using 2,2-dimethoxy-2-phenyl acetophenone (DMPA) as the photo-initiator. The grafting copolymerization process was confirmed and the resulting copolymers were characterized using differential scanning calorimetry (DSC), FTIR spectroscopy, 2D-X ray diffraction, and elemental analysis. The kinetics of the grafting reactions was also studied. Under the applied experimental conditions, the optimum grafting values were obtained at: CMCs = 0.2 g, PEGA = 249 mM, DMPA = 10.4 mM at a 2 h reaction time. Some of the resulting copolymers were selected and used in the presence of methylene bisacrylamide (MBA) as a crosslinking agent to develop pH-responsive hydrogel matrices. The swelling characteristics and the in vitro release profiles of 5-fluorouracil (5-FU), as a model drug, from the hydrogels were investigated. The results revealed that the hydrogel matrices developed in this study can be customized to act as good candidates in drug delivery systems.     

Low molecular weight polyethylenimine grafted N-maleated chitosan for gene delivery: properties and in vitro transfection studies

To develop chitosan-based efficient gene vectors, chitosans with different molecular weights were chemically modified with low molecular weight polyethylenimine. The molecular weight and composition of polyethylenimine grafted N-maleated chitosan (NMC-g-PEI) copolymers were characterized using gel permeation chromatography (GPC) and (1)H NMR, respectively. Agarose gel electrophoresis assay showed that NMC-g-PEI had good binding ability with DNA, and the particle size of the NMC-g-PEI/DNA complexes was 200-400 nm, as determined by a Zeta sizer. The nanosized complexes observed by scanning electron microscopy (SEM) exhibited a compact and spherical morphology. The NMC-g-PEI copolymers showed low cytotoxicity and good transfection activity, comparable to PEI (25 KDa) in both 293T and HeLa cell lines, except for NMC 50K-g-PEI. The results indicated that the molecular weight of NMC-g-PEI has an important effect on cytotoxicity and transfection activity, and low molecular weight NMC-g-PEI has a good potential as efficient nonviral gene vectors.     

Synthesis and characterization of RGD peptide grafted poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-glutamic acid) triblock copolymer

Advances in tissue engineering require biofunctional scaffolds that can provide not only physical support for cells but also chemical and biological cues needed in forming functional tissues. To achieve this goal, a novel RGD peptide grafted poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-glutamic acid) (PEG-PLA-PGL/RGD) was synthesized in four steps (1) to prepare diblock copolymer PEG-PLA-OH and to convert its -OH end group into -NH(2) (to obtain PEG-PLA-NH(2)), (2) to prepare triblock copolymer PEG-PLA-PBGL by ring-opening polymerization of NCA (N-carboxyanhydride) derived from benzyl glutamate with diblock copolymer PEG-PLA-NH(2) as macroinitiator, (3) to remove the protective benzyl groups by catalytic hydrogenation of PEG-PLA-PBGL to obtain PEG-PLA-PGL, and (4) to react RGD (arginine-glycine-(aspartic amide)) with the carboxyl groups of the PEG-PLA-PGL. The structures of PEG-PLA-PGL/RGD and its precursors were confirmed by (1)H NMR, FT-IR, amino acid analysis, and XPS analysis. Addition of 5 wt % PEG-PLA-PGL/RGD into a PLGA matrix significantly improved the surface wettability of the blend films and the adhesion and proliferation behavior of human chondrocytes and 3T3 cells on the blend films. Therefore, the novel RGD-grafted triblock copolymer is expected to find application in cell or tissue engineering.     

Synthesis and characterization of photo-cross-linked hydrogels based on biodegradable polyphosphoesters and poly(ethylene glycol) copolymers

Novel biodegradable hydrogels by photo-cross-linking macromers based on polyphosphoesters and poly(ethylene glycol) (PEG) are reported. Photo-cross-linkable macromers were synthesized by ring-opening polymerization of the cyclic phosphoester monomer 2-(2-oxo-1,3,2-dioxaphospholoyloxy) ethyl methacrylate (OPEMA) using PEG as the initiator and stannous octoate as the catalyst. The macromers were characterized by 1H NMR, Fourier transform infrared spectroscopy, and gel permeation chromatography measurements. The content of polyphosphoester in the macromer was controlled by varying the feed ratio of OPEMA to PEG. Hydrogels were fabricated by exposing aqueous solutions of macromers with 0.05% (w/w) photoinitiator to UV light irradiation, and their swelling kinetics as well as degradation behaviors were evaluated. The results demonstrated that cross-linking density and pH values strongly affected the degradation rates. The macromers was compatible to osteoblast cells, not exhibiting significant cytotoxicity up to 0.5 mg/mL. "Live/dead" cell staining assay also demonstrated that a large majority of the osteoblast cells remained viable after encapsulation into the hydrogel constructs, showing their potential as tissue engineering scaffolds.     

Adriamycin release from poly(lactide-coglycolide)-polyethylene glycol nanoparticles: synthesis, and in vitro characterization

The preparation, properties, and application in adriamycin delivery ofbiocompatible and biodegradable poly(lactide-co-glycolide)-polyethylene glycol (PLGA-PEG) nanoparticles are discussed. PLGA-PEG copolymers were synthesized by ring opening polymerization of the dl-lactide and glycolide in the presence of PEG1000. 1H-NMR and FT-IR spectrum were consistent with the structure of PLGA-PEG copolymers. The adriamycin-loaded nanoparticles could be prepared using a precipitation-solvent evaporation technique. The nanoparticles have been produced by a precipitation-solvent evaporation technique. The physical characteristics and drug loading efficiency of the PLGA-PEG nanoparticles were influenced by the composition of the PLGA-PEG copolymers used to prepare the nanoparticles. Particle sizes were between 65 and 100 nm for different compositions of PLGA-PEG copolymers. PLGA-PEG nanoparticles prepared from copolymers having relatively high PLGA/PEG ratios were smaller. Entrapment efficiency was 25%-33%. Adriamycin release from the nanoparticles at pH 7.4 showed an initial burst release and then sustained release phase. These results showed that PLGA-PEG nanoparticles could be an effective carrier for cancer therapy.     

Mannosylated poly(ethylene oxide)-b-poly(epsilon-caprolactone) diblock copolymers: synthesis, characterization, and interaction with a bacterial lectin

A novel bioeliminable amphiphilic poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PEO-b-PCL) diblock copolymer end-capped by a mannose residue was synthesized by sequential controlled polymerization of ethylene oxide and epsilon-caprolactone, followed by the coupling of a reactive mannose derivative to the PEO chain end. The anionic polymerization of ethylene oxide was first initiated by potassium 2-dimethylaminoethanolate. The ring-opening polymerization of epsilon-caprolactone was then initiated by the omega-hydroxy end-group of PEO previously converted into an Al alkoxide. Finally, the saccharidic end-group was attached by quaternization of the tertiary amine alpha-end-group of the PEO-b-PCL with a brominated mannose derivative. The copolymer was fully characterized in terms of chemical composition and purity by high-resolution NMR spectroscopy and size exclusion chromatography. Furthermore, measurements with a pendant drop tensiometer showed that both the mannosylated copolymer and the non-mannosylated counterpart significantly decreased the dichloromethane/water interfacial tension. Moreover, these amphiphilic copolymers formed monodisperse spherical micelles in water with an average diameter of approximately 11 nm as measured by dynamic light scattering and cryo-transmission electron microscopy. The availability of mannose as a specific recognition site at the surface of the micelles was proved by isothermal titration microcalorimetry (ITC), using the BclA lectin (from Burkholderia cenocepacia), which interacts selectively with alpha-D-mannopyranoside derivatives. The thermodynamic parameters of the lectin/mannose interaction were extracted from the ITC data. These colloidal systems have great potential for drug targeting and vaccine delivery systems.     

Synthesis, characterization, and in vitro evaluation of long-chain hyperbranched poly(ethylene glycol) as drug carrier

A series of novel long-chain hyperbranched poly(ethylene glycol)s (LHPEGs) with biodegradable connections were designed and synthesized in one pot through proton-transfer polymerization using PEG and commercial glycidyl methacrylate as monomers and potassium hydride as catalyst. The LHPEGs were hydrolyzed at neutral pH resulting in the decrease of molecular weights. In vitro evaluation demonstrated that LHPEGs were biocompatible and displayed negligible hemolytic activity. The efficient cellular uptake of LHPEGs was confirmed by flow cytometry and confocal laser scanning microscopy. Moreover, conjugation of a model hydrophobic anticancer drug methotrexate to LHPEGs inhibited the proliferation of a human cervical carcinoma Hela cell line. MTT assay indicated that the conjugated methotrexate dose required for 50% cellular growth inhibition against Hela cells was 20 μg/mL. By combining the advantages of long-chain hyperbranched structure and PEG, LHPEG provides a promising drug carrier for therapeutic fields.     

Poly(ethylene glycol)-poly(ester-carbonate) block copolymers carrying PEG-peptidyl-doxorubicin pendant side chains: synthesis and evaluation as anticancer conjugates

Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to <40,000 g/mol. The aim of this ambitious study was to synthesize and evaluate a novel, prototype biodegradable polymeric system based on high molecular weight, water-soluble functionalized polyesters. The main polymeric platform was prepared from bis(4-hydroxy)butyl maleate (DBM) and poly(ethylene glycol) (PEG4000) blocks to give the polymer DBM2-PEG4000 containing biodegradable carbonate bonds and having a M(w) of 100,000-190,000 g/mol; M(n) of 37,000-53,000 g/mol, and M(w)/M(n) of 3.0-3.7. Using thioether linkages, this polymer was then grafted with HS-PEG3000-Gly-Phe-Lue-Gly doxorubicin (HS-PEG3000-GFLG-Dox) pendant side chains ( approximately 30 per DBM2-PEG chain). The final construct, DBM2-PEG4000-S-PEG3000-GFLG-Dox had a total Dox content of 3-4 wt % and a free Dox content of < or = 0.7% total Dox. During incubation with isolated lysosomal enzymes, the rate of Dox release from the polymer backbone was relatively slow (<5% release over 5 h) compared to that seen for PEG5000-GFLG-Dox alone (>20% over 5 h). The in vitro cytotoxicity was assessed using B16F10 murine melanoma (MTT assay). DBM2-PEG4000-S-PEG3000-GFLG-Dox was 10-20-fold less toxic than free Dox. In vivo antitumor activity of the DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates was assessed using a subcutaneous (s.c.) B16F10 murine melanoma model, and an intraperitoneal (i.p.) L1210 leukaemia model. The increased toxicity (attributed to poor solubility) and low antitumor activity of DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates compared to PEG5000-GFLG-Dox and HPMA copolymer-Dox conjugates was attributed to the slow rate of Dox release. The DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were considered unfavorable as candidates for further development. However, the successful scale-up synthesis of DBM2-PEG4000-S-PEG3000 constructs suggest that they are worthy of further investigation as carriers for controlled release and targeting of less hydrophobic agents.