Cellular internalization of poly(ethylene oxide)-b-poly(epsilon-caprolactone) diblock copolymer micelles

Poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PEO-b-PCL) block copolymers self-assemble into micelles in aqueous solution. We have examined whether these micelles can internalize into P19 cells in vitro. Fluorescently labeled PEO(45)-b-PCL(23) block copolymer was prepared by conjugating a tetramethylrhodamine molecule to the end of the hydrophobic PCL block. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) studies yielded 24 +/- 2 and 25 +/- 2 nm, respectively, for the diameters of the micelles. The studies also showed that chemical labeling did not effect the morphology or size. When the rhodamine-labeled PEO(45)-b-PCL(23) block copolymer micelles were tested in vitro, time-, concentration-, and pH-dependence of the internalization process suggested that internalization proceeded by endocytosis. The results from these studies provide the first direct evidence for the internalization of PEO(45)-b-PCL(23) micelles. Future studies will utilize multiple labeling of these micelles, allowing questions to be addressed related to the fate of internalized micelles as drug carriers, the destination of the incorporated drugs or fluorescent probes released from micelles, and the identification of the subcellular localization of the whole drug-carrier system within cells, both in vitro and in vivo.     

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.     

Composition dependence of the crystallization behavior and morphology of the poly(ethylene oxide)-poly(epsilon-caprolactone) diblock copolymer

The crystallization behavior and morphology of the crystalline-crystalline poly(ethylene oxide)-poly(epsilon-caprolactone) diblock copolymer (PEO-b-PCL) was studied by differential scanning calorimetry (DSC), wide-angle X-ray diffraction (WAXD), Fourier transform infrared spectroscopy (FTIR), small-angle X-ray scattering (SAXS), and hot-stage polarized optical microscope (POM). The mutual effects between the PEO and PCL blocks were significant, leading to the obvious composition dependence of the crystallization behavior and morphology of PEO-b-PCL. In this study, the PEO block length was fixed (Mn = 5000) and the weight ratio of PCL/PEO was tailored by changing the PCL block length. Both blocks could crystallize in PEO-b-PCL with the PCL weight fraction (WFPCL) of 0.23-0.87. For the sample with the WFPCL of 0.36 or less, the PEO block crystallized first, resulting in the obvious confinement of the PCL block and vice versa for the sample with WFPCL of 0.43 or more. With increasing WFPCL, the crystallinity of PEO reduced continuously while the variation of the PCL crystallinity exhibited a maximum. The long period of PEO-b-PCL increased with increasing WFPCL from 0.16 to 0.50 but then decreased with the further increase of WFPCL due to the interaction of the respective variation of the thicknesses of the PEO and PCL crystalline lamellae. Only the PEO spherulites were observed in samples with WFPCL of 0.16-0.36 by POM, in contrast to only the PCL spherulites in samples with WFPCL of 0.56-0.87. For samples with WFPCL of 0.43 and 0.50, a unique concentric spherulite was observed. The morphology of the inner and outer portions of the concentric spherulites was determined by the PCL and PEO spherulites, respectively. The growth rate of the PEO spherulites reduced rapidly with increasing WFPCL from 0 to 0.50. However, when increasing WFPCL from 0.43 to 0.87, the variation of the growth rate of the PCL spherulites exhibited a maximum rather than a monotonic change.     

Degradation behavior of poly(epsilon-caprolactone)-b-poly(ethylene glycol)-b-poly(epsilon-caprolactone) micelles in aqueous solution

Poly(epsilon-caprolactone)-b-poly(ethylene glycol)-b-poly(epsilon-caprolactone) triblock copolymers were synthesized by the ring-opening polymerization of epsilon-caprolactone in the presence of hydroxyl-terminated poly(ethylene glycol) with different molecular weights, using stannous octoate catalyst. Micelles prepared by the precipitation method with these triblock copolymers exhibit a core-shell structure. The degradation behaviors of these core-shell micelles in aqueous solution were investigated by FT-IR, 1H NMR, GPC, DLS, TEM, and AFM. It was found that the degradation behavior of micelles in aqueous solution was quite different from that of bulk materials. The size of the micelles increased in the initial degradation stages and decreased gradually when the degradation period was extended. The caprolactone/ethylene oxide (CL/EO) ratio in micelles measured by NMR also shows an increase at the initial degradation stage and a decrease at later stages. The morphology of these micelles became more and more irregular during the degradation period. We explain the observed behavior by a two-stage degradation mechanism with interfacial erosion between the cores and the shells followed by core erosion.     

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.     

Compatibilization effect of poly(epsilon-caprolactone)-b-poly(ethylene glycol) block copolymers and phase morphology analysis in immiscible poly(lactide)/poly(epsilon-caprolactone) blends

The miscibility and phase behavior of two stereoisomer forms of poly(lactide) (PLA: poly (L-lactide) (PLLA) and poly(DL-lactide) (PDLLA)) blends with poly(epsilon-caprolactone)-b-poly(ethylene glycol) (PCL-b-PEG) and PCL-b-monomethoxy-PEG (PCL-b-MPEG) block copolymers have been investigated by differential scanning calorimetry (DSC). The DSC thermal behavior of both the blend systems revealed that PLA is miscible with the PEG segment phase of PCL-b-(M)PEG but is still immiscible with its PCL segment phase although PCL was block-copolymerized with PEG. On the basis of these results, PCL-b-PEG was added as a compatibilizer to PLA/PCL binary blends. The improvement in mechanical properties of PLA/PCL blends was achieved as anticipated upon the addition of PCL-b-PEG. In addition, atomic force microscopy (AFM) measurements have been performed in order to study the compositional synergism to be observed in mechanical tests. AFM observations of the morphological dependency on blend composition indicate that PLA/PCL blends are immiscible but compatible to some extent and that synergism of compatibilizing may be maximized in the compositional blend ratio before apparent phase separation and coarsening.     

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.     

Enzymatic degradation of block copolymers prepared from epsilon-caprolactone and poly(ethylene glycol)

Block copolymers were prepared by ring-opening polymerization of epsilon-caprolactone in the presence of monohydroxyl or dihydroxyl poly(ethylene glycol) (PEG), using Zn powder as catalyst. The resulting poly(epsilon-caprolactone) (PCL)-PEG diblock and PCL-PEG-PCL triblock copolymers were characterized by various analytical techniques such as NMR, size-exclusion chromatography, differential scanning calorimetry, and X-ray diffraction. Both copolymers were semicrystalline polymers, the crystalline structure being of the PCL type. Films were prepared by casting dichloromethane solutions of the polymers on a glass plate. Square samples with dimensions of 10 x 10 mm were allowed to degrade in a pH = 7.0 phosphate buffer solution containing Pseudomonas lipase. Data showed that the introduction of PEG blocks did not decrease the degradation rate of poly(epsilon-caprolactone).     

One-pot synthesis of star-shaped macromolecules containing polyglycidol and poly(ethylene oxide) arms

Synthesis of fully hydrophilic star-shaped macromolecules with different kinds of arms (A(x)B(y)C(z)) based on polyglycidol (PGL, A(x)) and poly(ethylene oxide) (PEO, C(z)) arms and diepoxy compounds (diglycidyl ethers of ethylene glycol (DGEG) or neopentyl glycol (DGNG) in the core, B(y)) forming the core is described. Precursors of arms were prepared by polymerization of glycidol with protected -OH groups. The first-generation stars were formed in the series of consecutive-parallel reactions of arms A(x) with diepoxy compounds (B). These first-generation stars (A(x)B(y)), having approximately O-, Mt+ groups on the cores, were used as multianionic initiators for the second generation of arms (C(z)) built by polymerization of ethylene oxide. The products with M(n) up to 10(5) and having up to approximately 40 arms were obtained. The number of arms (f) was determined by direct measurements of M(n) of the first-generation stars (M(n) of arms A(x) is known), compared with f calculated from the branching index g, determined from R(g) measured with size-exclusion chromatography (SEC) triple detection with TriSEC software. The progress of the star formation was monitored by 1H NMR and SEC. These novel water-soluble stars, having a large number of hydroxyl groups, both at the ends of PEO arms as well as within the PGL arms, can be functionalized and further used for attaching compounds of interest. This approach opens, therefore, a new way of "multiPEGylation".     

Formation of a unique crystal morphology for the poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymer

The crystallization behaviors of the poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymer with the PEG weight fraction of 0.50 (PEG(50)-PCL(50)) was studied by DSC, WAXD, SAXS, and FTIR. A superposed melting point at 58.5 degrees C and a superposed crystallization temperature at 35.4 degrees C were obtained from the DSC profiles running at 10 degrees C/min, whereas the temperature-dependent FTIR measurements during cooling from the melt at 0.2 degrees C/min showed that the PCL crystals formed starting at 48 degrees C while the PEG crystals started at 45 degrees C. The PEG and PCL blocks of the copolymer crystallized separately and formed alternating lamella regions according to the WAXD and SAXS results. The crystal growth of the diblock copolymer was observed by polarized optical microscope (POM). An interesting morphology of the concentric spherulites developed through a unique crystallization behavior. The concentric spherulites were analyzed by in situ microbeam FTIR, and it was determined that the morphologies of the inner and outer portions were mainly determined by the PCL and PEG spherulites, respectively. However, the compositions of the inner and outer portions were equal in the analysis by microbeam FTIR.     

A convenient method for the synthesis of 2-(beta-D-glycopyranosylthio) pyridines

Treatment of piperidinium salts of dihydropyridinethiolates 3 with glycosyl bromides 4 in dry acetone provides a convenient and high yielding synthesis of 1,4-dihydro-3-cyanopyridine thioglycosides 5. The structures of 5 were confirmed by oxidation as well as by 1H NMR and 13C NMR spectral analysis.     

Application of molecular dynamics simulation to predict the compatability between water-insoluble drugs and self-associating poly(ethylene oxide)-b-poly(epsilon-caprolactone) block copolymers

In the present work, molecular dynamics (MD) simulation was applied to study the solubility of two water-insoluble drugs, fenofibrate and nimodipine, in a series of micelle-forming PEO-b-PCL block copolymers with combinations of blocks having different molecular weights. The solubility predictions based on the MD results were then compared with those obtained from solubility experiments and by the commonly used group contribution method (GCM). The results showed that Flory-Huggins interaction parameters computed by the MD simulations are consistent with the solubility data of the drug/PEO-b-PCL systems, whereas those calculated by the GCM significantly deviate from the experimental observation. We have also accounted for the possibility of drug solubilization in the PEO block of PEO-b-PCL.     

Conjugation of arginine-glycine-aspartic acid peptides to poly(ethylene oxide)-b-poly(epsilon-caprolactone) micelles for enhanced intracellular drug delivery to metastatic tumor cells

An arginine-glycine-aspartic acid (RGD) containing model peptide was conjugated to the surface of poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) micelles as a ligand that can recognize adhesion molecules overexpressed on the surface of metastatic cancer cells, that is, integrins, and that can enhance the micellar delivery of encapsulated hydrophobic drug into a tumor cell. Toward this goal, PEO-b-PCL copolymers bearing acetal groups on the PEO end were synthesized, characterized, and assembled to polymeric micelles. The acetal group on the surface of the PEO-b-PCL micelles was converted to reactive aldehyde under acidic condition at room temperature. An RGD-containing linear peptide, GRGDS, was conjugated on the surface of the aldehyde-decorated PEO-b-PCL micelles by incubation at room temperature. A hydrophobic fluorescent probe, that is, DiI, was physically loaded in prepared polymeric micelles to imitate hydrophobic drugs loaded in micellar carrier. The cellular uptake of DiI loaded GRGDS-modified micelles by melanoma B16-F10 cells was investigated at 4 and 37 degrees C by fluorescent spectroscopy and confocal microscopy techniques and was compared to the uptake of DiI loaded valine-PEO-b-PCL micelles (as the irrelevant ligand decorated micelles) and free DiI. GRGDS conjugation to polymeric micelles significantly facilitated the cellular uptake of encapsulated hydrophobic DiI most probably by intergrin-mediated cell attachment and endocytosis. The results indicate that acetal-terminated PEO-b-PCL micelles are amenable for introducing targeting moieties on the surface of polymeric micelles and that RGD-peptide conjugated PEO-b-PCL micelles are promising ligand-targeted carriers for enhanced drug delivery to metastatic tumor cells.     

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.     

Morphological control of poly(ethylene glycol)-block-poly(epsilon-caprolactone) copolymer aggregates in aqueous solution

In aqueous solution, it was found that the amphiphilic copolymer poly(ethylene glycol)-b-poly(caprolactone) (PEG(5000)-b-PCL(4100)) formed different morphologies, including long rod-like, short rod-like, or spherical aggregates, when the copolymer concentration was increased. Nearly identical morphologies were observed with the addition of increasing amounts of PEG(2000)-distearoylphosphoethanolamine (PEG(2000)-DSPE) to the copolymer. The morphologies of the aggregates in solution were confirmed by negative stain transmission electron microscopy (TEM) and cryogenic-TEM (cryo-TEM). The critical micelle concentrations of the PEG(5000)-b-PCL(4100) copolymer, PEG(2000)-DSPE and a mixture of the two materials (PEG(5000)-b-PCL 4100/PEG(2000)-DSPE) were evaluated to determine the thermodynamic stability of the aggregates. Differential scanning calorimetry was performed to gain insight into the degree of mixing of PEG(5000)-b-PCL(4100) and PEG(2000)-DSPE. Overall, combining PEG(5000)-b-PCL(4100) and PEG(2000)-DSPE produced a single population of mixed micelles with rod-like or spherical morphologies depending on the material composition and concentration.     

Convenient synthesis of heterobifunctional poly(ethylene glycol) suitable for the functionalization of iron oxide nanoparticles for biomedical applications

A straightforward route is proposed for the multi-gram scale synthesis of heterobifunctional poly(ethylene glycol) (PEG) oligomers containing combination of triethyloxysilane extremity for surface modification of metal oxides and amino or azido active end groups for further functionalization. The suitability of these PEG derivatives to be conjugated to nanomaterials was shown by pegylation of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (NPs), followed by functionalization with small peptide ligands for biomedical applications.     

Tracking the physical aging of poly(ethylene oxide): A technical note

Physical aging of 2 types of PEO could be tracked by the combination of PALS, DSC, and SEM methods. After storing the samples at 40°C±2°C and 75%±5% RH, a decrease in the o-Ps lifetime values and an increase in the melting enthalpies as a function of storage time indicated a reorientation of the polymer chains. The limitations of monitoring enthalpy relaxation confirm the importance of methods that track volume relaxation, such as PALS. Structural changes could be observed even after a short storage time (4 weeks), which highlights the effect of further investigations of the influence of physical aging on the properties of PEO-containing dosage forms.     

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.     

Electrospinning Bombyx mori silk with poly(ethylene oxide)

Electrospinning for the formation of nanoscale diameter fibers has been explored for high-performance filters and biomaterial scaffolds for vascular grafts or wound dressings. Fibers with nanoscale diameters provide benefits due to high surface area. In the present study we explore electrospinning for protein-based biomaterials to fabricate scaffolds and membranes from regenerated silkworm silk, Bombyx mori, solutions. To improve processability of the protein solution, poly(ethylene oxide) (PEO) with molecular weight of 900,000 was blended with the silk fibroin. A variety of compositions of the silk/PEO aqueous blends were successfully electrospun. The morphology of the fibers was characterized using high-resolution scanning electron microscopy. Fiber diameters were uniform and less than 800 nm. The composition was estimated by X-ray photoelectron spectroscopy to characterize silk/PEO surface content. Aqueous-based electrospining of silk and silk/PEO blends provides potentially useful options for the fabrication of biomaterial scaffolds based on this unique fibrous protein.     

Heterobifunctional poly(ethylene oxide) oligomers containing carboxylic acids

Syntheses of vinylsilyl alcohols having one to three vinyl moieties and their use as initiators for ethylene oxide polymerizations are discussed. Poly(ethylene oxide) oligomers with vinylsilanes at one end and a hydroxyl group at the other were prepared in base-catalyzed reactions. Molecular weights determined from 1H NMR and gel permeation chromatography were close to the targeted values. Carboxylic acid functional poly(ethylene oxide) oligomers were prepared from ene-thiol addition reactions of mercaptoacetic acid across the vinylsilane terminus. It is anticipated that these carboxylic acid functional oligomers will complex to magnetite nanoparticles to afford complexes that can be dispersed in aqueous media.