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.     

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.     

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.     

Rational design of antithrombotic peptides to target the von Willebrand Factor (vWf) - GPIb integrin interaction

Conventional antithrombotic drug discovery requires testing of large numbers of drug candidates. We used computer-aided macromolecular interaction assessment (MIAX) to select antithrombotic molecules that mimic and therefore block platelet GPIb’s binding to von Willebrand factor (vWf), an early step in thrombus formation. We screened a random array of 15-mer D-amino acid peptides for binding vWf. Structures of 4 candidate peptides were inferred by comparison to sequences in protein databases, conversion from the L to D conformations and molecular dynamics (MD) determinations of those most energetically stable. By MIAX, we deduced the amino acids and intermolecular hydrogen bonds contributing to the GPIb-vWf interaction interface. We docked the peptides onto vWf in silico to localize their binding sites and consequent potential for preventing GPIb-vWf binding. In vitro inhibition of ristocetin-initiated platelet agglutination confirmed peptide function and suitability for antithrombotic development, thereby validating this novel approach to drug discovery.     

Molecular dynamics simulation on miscibility of trans-1,4,5,8-tetranitro-1,4,5,8 -tetraazadecalin (TNAD) with some propellants

The solubility parameters of TNAD, HMX, RDX, DINA, DNP propellants were predicted by molecular dynamics (MD) simulation in order to evaluate the miscibility of TNAD and the other four propellants. The results show that the order of miscibility is TNAD/DINA > TNAD/DNP > TNAD/RDX > TNAD/HMX from the analysis of miscibility. The densities and binding energies of TNAD/propellants blends were further investigated. The results indicate that the better the miscibility between TNAD and the propellants, the smaller the variation of the density rate. The larger the intermolecular interaction, the better the miscibility between components. The analysis of radial distribution function shows that the main interaction ways between TNAD and other energetic components are short-range interactions.

Homology modeling of dopamine d(2) and d(3) receptors: molecular dynamics refinement and docking evaluation

Dopamine (DA) receptors, a class of G-protein coupled receptors (GPCRs), have been targeted for drug development for the treatment of neurological, psychiatric and ocular disorders. The lack of structural information about GPCRs and their ligand complexes has prompted the development of homology models of these proteins aimed at structure-based drug design. Crystal structure of human dopamine D(3) (hD(3)) receptor has been recently solved. Based on the hD(3) receptor crystal structure we generated dopamine D(2) and D(3) receptor models and refined them with molecular dynamics (MD) protocol. Refined structures, obtained from the MD simulations in membrane environment, were subsequently used in molecular docking studies in order to investigate potential sites of interaction. The structure of hD(3) and hD(2L) receptors was differentiated by means of MD simulations and D(3) selective ligands were discriminated, in terms of binding energy, by docking calculation. Robust correlation of computed and experimental K(i) was obtained for hD(3) and hD(2L) receptor ligands. In conclusion, the present computational approach seems suitable to build and refine structure models of homologous dopamine receptors that may be of value for structure-based drug discovery of selective dopaminergic ligands.     

Modeling the interaction between anti-cancer drug penicillamine and pristine and functionalized carbon nanotubes for medical applications: density functional theory investigation and a molecular dynamics simulation

Abstract

The present study focuses on the prediction and investigation of binding properties of penicillamine with pure (5,5) single-walled carbon nanotube (SWCNT) and functionalized SWCNT (f-SWCNT) through the B3LYP and M06-2X functionals using the 6-31G** basis set. The electronic and structural properties, adsorption energy and frontier molecular orbitals of various configurations are examined. Our theoretical results indicated that the interaction of the nanotubes with penicillamine molecule is weak so that the drug adsorption process is typically physisorption. Also, results of theoretical calculations show that the adsorption of the drug molecule on f-SWCNT is stronger with shorter intermolecular distances in comparison to pure SWCNT. The natural bond orbital (NBO) analysis of studied systems demonstrates that the charge is transferred from penicillamine molecule to the nanotubes. Furthermore, molecular dynamics (MD) simulation is employed to evaluate the dynamic and diffusion behavior of drug molecule on SWCNT and f-SWCNT. Energy results show that drug molecule spontaneously moves toward the carriers, and the van der Waals energy contributions in drug adsorption are more than electrostatic interaction. The obtained results from MD simulation confirm that the functionalization of SWCNT leads to increase in the solubility of the carrier in aqueous solution.

Communicated by Ramaswamy H. Sarma     

Preparation of micelle-forming polymer-drug conjugates.

Adriamycin, a hydrophobic anticancer drug, was conjugated with poly(ethylene oxide)-poly(aspartic acid) block copolymers composed of various lengths of each block copolymer segment ranging from 1000 to 12,000 in molecular weight and from 10 to 80 units, respectively. Conjugation was achieved without precipitation by adjusting the ratio of adriamycin to aspartic acid residues of the block copolymer and the quantity of DMF used for the reaction. Thus obtained conjugates showed high water solubility irrespective of a large amount of the conjugated adriamycin. Furthermore, these conjugates were found to form micellar structures with a hydrophobic inner core and a hydrophilic outer shell. This micellar architecture may be utilized for effective drug targeting.     

Hyperbranched double hydrophilic block copolymer micelles of poly(ethylene oxide) and polyglycerol for pH-responsive drug delivery

We report the synthesis of a well-defined hyperbranched double hydrophilic block copolymer of poly(ethylene oxide)-hyperbranched-polyglycerol (PEO-hb-PG) to develop an efficient drug delivery system. In specific, we demonstrate the hyperbranched PEO-hb-PG can form a self-assembled micellar structure on conjugation with the hydrophobic anticancer agent doxorubicin, which is linked to the polymer by pH-sensitive hydrazone bonds, resulting in a pH-responsive controlled release of doxorubicin. Dynamic light scattering, atomic force microscopy, and transmission electron microscopy demonstrated successful formation of the spherical core-shell type micelles with an average size of about 200 nm. Moreover, the pH-responsive release of doxorubicin and in vitro cytotoxicity studies revealed the controlled stimuli-responsive drug delivery system desirable for enhanced efficiency. Benefiting from many desirable features of hyperbranched double hydrophilic block copolymers such as enhanced biocompatibility, increased water solubility, and drug loading efficiency as well as improved clearance of the polymer after drug release, we believe that double hydrophilic block copolymer will provide a versatile platform to develop excellent drug delivery systems for effective treatment of cancer.     

Molecular dynamics simulation of fluorination effect for solvation of trifluoromethylbenzoic acid isomers in supercritical carbon dioxide

A molecular dynamics (MD) simulation was applied to carbon dioxide+trifluoromethylbenzoic acid isomer and carbon dioxide+methylbenzoic acid isomer systems to investigate the interactions between carbon dioxide and the solutes. The pair correlation functions between the carbon dioxide and trifluoromethyl group or methyl group in the solutes were calculated to study the fluorination effect of solvation. As a result, it was found that the interactions between carbon dioxide and trifluoromethyl group in trifluoromethylbenzoic acid isomers were stronger than those between carbon dioxide and the methyl group in methylbenzoic acid isomers. The simulation results had the same tendency as the experimental solubility enhancements and coincided with the trend of the interaction parameters of the Peng-Robinson equation of state that were determined from the solubility data.     

Theoretical study on the stability of insulin within poly-isobutyl cyanoacrylate (PIBCA) nanocapsule

To study the physical stability of insulin in drug delivery particles, we developed a coarse-grained (CG) model for insulin based on dissipative particle dynamics (DPD). Three insulin modelling schemes were considered: each amino acid as a bead (IM1), each amino acid being separated into one to three beads (IM2), and adding secondary structural information of insulin to IM2 (IM3). The best possible bead-bead interaction parameters were obtained from Hildebrand and Hansen solubility parameters by performing the constant-temperature DPD simulation with insulin models in 20% acetic acid solution. IM3 showed good results in terms of RMSF, RMSD and A1B30 distance compared to those of all-atom models from the literature. Then, the IM3 model was considered in an oil-filled poly (isobutyl cyanoacrylate) (PIBCA) nanocapsule. Two crucial factors were found that mainly influence the stability of insulin in oil: the PIBCA shell thickness and the amount of ethanol in the oil droplet. An appropriate PIBCA shell thickness is necessary to block the interaction between insulin and water outside, and ethanol could stabilise insulin with its good affinity for both insulin and oil.     

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.     

Identification of functional motions in the adenylate kinase (ADK) protein family by computational hybrid approaches

Based on integrative computational hybrid approaches that combined statistical coupling analysis (SCA), molecular dynamics (MD), and normal mode analysis (NMA), evolutionarily coupled residues involved in functionally relevant motion in the adenylate kinase protein family were identified. The hybrids identified four top-ranking site pairs that belong to a conserved hydrogen bond network that is involved in the enzyme's flexibility. A second group of top-ranking site pairs was identified in critical regions for functional dynamics, such as those related to enzymatic turnover. The high consistency of the results obtained by SCA with NMA (SCA.NMA) and by SCA.MD hybrid analyses suggests that suitable replacement of the matrix of cross-correlation analysis of atomic fluctuations (derived by using NMA) with those based on MD contributes to the identification of such sites by means of a fast computational calculation. The analysis presented here strongly supports the hypothesis that evolutionary forces, such as coevolution at the sequence level, have promoted functional dynamic properties of the adenylate kinase protein family. Finally, these hybrid approaches can be used to identify, at the residue level, protein motion coordination patterns not previously observed, such as in hinge regions.     

Synthesis and characterization of biodegradable poly(ethylene glycol)-block-poly(5-benzyloxy-trimethylene carbonate) copolymers for drug delivery

Amphiphilic diblock copolymers with various block compositions were synthesized with monomethoxy-terminated poly(ethylene glycol) (MePEG) as the hydrophilic block and poly(5-benzyloxy-trimethylene carbonate) (PBTMC) as the hydrophobic block. When the copolymerization was conducted using MePEG as a macroinitiator and stannous 2-ethylhexanoate (Sn(Oct)2) as a catalyst, the molecular weight of the second block was uncontrollable, and the method only afforded a mixture of homopolymer and copolymer with a broad molecular weight distribution. By contrast, the use of the triethylaluminum-MePEG initiator yielded block copolymers with controllable molecular weight and a more narrow molecular weight distribution than the copolymers obtained using Sn(Oct)2. GPC and 1H NMR studies confirmed that the macroinitiator was consumed and the copolymer composition was as predicted. Two of the newly synthesized MePEG-b-PBTMC copolymers were evaluated in terms of properties primarily relating to their use in micellar drug delivery. MePEG-b-PBTMC micelles with a narrow monomodal size distribution were prepared using a high-pressure extrusion technique. The MePEG-b-PBTMC copolymers were also confirmed to be biodegradable and noncytotoxic.     

HIV-1整合酶核心区野生型和F185K突变型活性和溶解性的比较及分子动力学模拟分析

表达纯化了野生型(WT)及F185K突变型HIV-1整合酶核心区蛋白(IN_C),并对二者的溶解性和活性进行了比较．实验结果表明：F185K 突变后IN_C溶解性显著提高,活性有一定程度降低．对WT和F185K IN_C体系进行了1800 ps的分子动力学模拟．模拟结果表明：F185K IN_C功能loop区柔性和蛋白质整体运动性降低,使蛋白质活性降低,F185K突变后盐桥网络的变化驱动了IN_C局部构象改变,引起IN_C表面的部分疏水残基被包埋,亲水残基暴露,相对亲水溶剂可接近面积增大,同时,突变后IN_C与水之间形成氢键的数量增加,与水之间作用加强,以上变化使IN_C溶解性提高．分子动力学模拟与实验结果相吻合．为理解蛋白质溶解性和对蛋白质进行可溶性改造提供了一定的理论依据．     

A coarse-grain MD (molecular dynamic) simulation of PCL–PEG and PLA–PEG aggregation as a computational model for prediction of the drug-loading efficacy of doxorubicin

Abstract

Formulating a hydrophobic drug that is water-soluble is a pharmaceutical challenge. One way is to incorporate the drug in an amphiphilic micelle composed from an aggregation of block copolymers. Design of a good nano-micelle requires many trial-and-error attempts. In this article, we developed a computational model based on a coarse-grained molecular dynamic (MD) simulation and correlated outputs with previous studies. A good correlation shows that this model reliably simulates poly-lactic acid–poly-ethylene glycol (PLA–PEG) and poly-caprolactone (PCL)–PEG aggregation in water with and without the presence of doxorubicin.

Communicated by Ramaswamy H. Sarma     

Calculating three-dimensional molecular structure from atom-atom distance information: cyclosporin A

In recent years methods for deriving spatial molecular structure from atom-atom distance information have gained in importance due to the emergence of two-dimensional nuclear magnetic resonance (n.m.r) techniques, which make it possible to obtain such distance information for polypeptides, small proteins, sugars, and DNA fragments in solution. Distance geometry (DG) and restrained molecular dynamics (MD) refinement are applied to a cyclic polypeptide, the immunosuppressive drug cyclosporin A, and the results are compared. Two different procedures, DG followed by restrained MD, and straightforward restrained MD starting from the X-ray structure, both lead to a unique conformation that satisfies the 58 experimentally determined distance constraints. The results nicely show the relative merits of DG and restrained MD techniques for determining spatial molecular structure from distance information.     

Photoresponsive poly(S-(o-nitrobenzyl)-L-cysteine)-b-PEO from a L-cysteine N-carboxyanhydride monomer: synthesis, self-assembly, and phototriggered drug release

A photoresponsive S-(o-nitrobenzyl)-l-cysteine N-carboxyanhydride (NBC-NCA) monomer was for the first time designed, and the related poly(S-(o-nitrobenzyl)-l-cysteine)-b-poly(ethylene glycol) (PNBC-b-PEO) block copolymers were synthesized from the ring-opening polymerization (ROP) of NBC-NCA in DMF solution at 25 °C. Their molecular structures, physical properties, photoresponsive self-assembly, and drug release of PNBC-b-PEO were thoroughly investigated. The β-sheet conformational PNBC block within copolymers presented a thermotropic liquid crystal phase behavior, and the crystallinity of PEO block was progressively suppressed over the PNBC composition. The characteristic absorption peaks of these copolymers at about 310 and 350 nm increased over UV irradiation time and then leveled off, indicating that the o-nitrobenzyl groups were gradually photocleaved from copolymers until the completion of photocleavage. The PNBC-b-PEO copolymers self-assembled into spherical nanoparticles in aqueous solution, presenting a photoresponsive self-assembly behavior, together with a size reduction of nanoparticles after irradiation. The anticancer drug doxorubicin can be released in a controlled manner by changing the light irradiation time, which was induced by gradually photocleaving the PNBC core of nanoparticles. This work provides a facile strategy not only for the synthesis of photoresponsive polypeptide-based block copolymers but also for the fabrication of photoresponsive nanomedicine potential for anticancer therapy.