Micromolding of PDMS scaffolds and microwells for tissue culture and cell patterning: A new method of microfabrication by the self-assembled micropatterns of diblock copolymer micelles

We introduce an innovative fabrication of the polymer scaffolds for tissue culture by utilizing the evaporation induced self-assembled micropatterns of polystyrene-block-poly(acrylic acid) (PS-b-PAA) diblock copolymer micelles. The microstructures were used as templates for micromolding a silicon elastomer, poly(dimethylsiloxane) (PDMS), into tissue scaffolds and microwells for cell patterning purpose. Cultivation of human epithelial cells (Calu-3 cell line) on the PDMS scaffolds demonstrates potential applications in tissue engineering and cell-based biosensors. The reported method is rapid, simple, economical, and versatile comparing with the existing microfabrication techniques.     

Block copolymer micelles as carriers of transition metal ions Y(III) and Cu(II) and gelation thereof

Poly(ethylene oxide)-block-poly(2-(diethylamino)ethyl methacrylate) (PEO-b-PDEAEMA) diblock copolymer was synthesized by anionic polymerization, whose molecular structure was characterized by ¹H NMR and size exclusion chromatography (SEC). The diblock copolymer self-assembled into micelles in nonacid aqueous solution with PEO and PDEAEMA as corona and core respectively. By virtue of the coordinating property of PDEAEMA block to metal ions, the resultant micelles were then used as carriers to load metal ions Y(III) and Cu(II) in the micellar core. The morphology and stability of the metal loaded micelles were characterized by dynamic light scattering (DLS), atomic force microscopy (AFM) and transmission electron microscopy (TEM). The metal loading amounts were determined by elemental analyses, UV spectrometry and titrimetric analysis. In addition, the Y(III) loaded micelles were demonstrated to complex with α-cyclodextrins and form supramolecular hydrogels in-situ. The metal loaded micelles and the resultant supramolecular hydrogels will have potential application for cancer internal radiotherapy.     

A new approach in the study of tethered diblock copolymer surface morphology and its tethering density dependence

A poly(l-lactic acid)-block-polystyrene-block-poly(methyl methacrylate) (PLLA-b-PS-b-PMMA) triblock copolymer was synthesized with a crystalline PLLA end block. Single crystals of this triblock copolymer grown in dilute solution could generate uniformly tethered diblock copolymer brushes, PS-b-PMMA, on the PLLA single crystal substrate. The diblock copolymer brushes exhibited responsive, characteristic surface structures after solvent treatment depending upon the quality of the solvent in relation to each block. The chemical compositions of these surface structures were detected via the surface enhanced Raman scattering technique. Using atomic force microscopy, the physical morphologies of these surface structures were identified as micelles in cyclohexane and “onion”-like morphologies in 2-methoxyethanol, especially when the PS-b-PMMA tethered chains were at low tethering density.     

Functionalized organic nanoparticles from core-crosslinked poly(4-vinylbenzocyclobutene-b-butadiene) diblock copolymer micelles

Surface-functionalized polymeric nanoparticles were prepared by: a) self-assembly of poly(4-vinylbenzocyclobutene-b-butadiene) diblock copolymer (PVBCB-b-PB) to form spherical micelles (diameter: 15-48 nm) in decane, a selective solvent for PB, b) crosslinking of the PVBCB core through thermal dimerization at 200-240 °C, and c) cleavage of the PB corona via ozonolysis and addition of dimethyl sulfide to afford aldehyde-functionalized nanoparticles (diameter: ∼16-20 nm), along with agglomerated nanoparticles ranging from ∼30 to ∼100 nm in diameter. The characterization of the diblock copolymer precursors, the intermediate micelles and the final surface-functionalized crosslinked nanoparticles was carried out by a combination of size exclusion chromatography, static and dynamic light scattering, viscometry, thermogravimetric analysis, ¹H NMR and FTIR spectroscopy and transmission electron microscopy.     

Doubly thermo-responsive brush-linear diblock copolymers and formation of core-shell-corona micelles

Doubly thermo-responsive brush-linear diblock copolymer of poly[poly(ethylene glycol) methyl ether vinylphenyl]-block-poly(N-isopropylacrylamide) (PmPEGV-b-PNIPAM) is prepared by RAFT polymerization. The obtained brush-linear diblock copolymer exhibits two lower critical solution temperatures (LCSTs) corresponding to the linear poly(N-isopropylacrylamide) (PNIPAM) block and the brush poly[poly(ethylene glycol) methyl ether vinylphenyl] (PmPEGV) block in water. This brush-linear diblock copolymer undergoes a two-step temperature sensitive micellization. At temperature above the first LCST, the brush-linear diblock copolymer self-assembles into core-corona micelles with the dehydrated PNIPAM block forming the core and the solvated brush PmPEGV block forming the corona. When temperature increases above the second LCST, the polystyrene backbone in the brush PmPEGV block collapses onto the dehydrated PNIPAM core to form core-shell-corona micelles, in which the dehydrated PNIPAM block forms the core, the collapsed polystyrene backbone in the brush PmPEGV block forms the shell and the solvated poly(ethylene glycol) side-chains forms the corona. The effect of the length of the PNIPAM block and the length of the poly(ethylene glycol) side-chains on the thermo-responsive micellization and the size of core-shell-corona micelles is investigated.     

Fluorescence studies of pyrene-labelled, pH-responsive diblock copolymer micelles in aqueous solution

Fluorescence spectroscopic techniques, and time-resolved anisotropy measurements (TRAMS) in particular, have provided valuable information regarding micelle formation in luminescently labelled pH-responsive diblock copolymers of 2-(diethylamino)ethyl methacrylate (DEA) and 2-(dimethylamino)ethyl methacrylate (DMA). A pyrenyl derivative, located at the DEA block, allowed motion of this site to be monitored via TRAMS in aqueous solution: a significant reduction in the mobility of this label was apparent at concentrations in excess of the critical micelle concentration, CMC, of the diblock copolymer. This is consistent with the labelled DEA block being located in the core of the micelles. At concentrations below the CMC, unimers were detected in solution. The micelle size estimated from TRAMS is approximately half of that determined from dynamic light scattering measurements. This suggests that the chain ends of the block copolymer are not “frozen” into position but that limited motion may occur due to fluidity within the micelle core. This is reasonable given the low T_g of the DEA block. Alternatively, a model is proposed which suggests that the interior of the micelle is a hard sphere, surrounded by flexible, fast-moving corona, which imparts little viscous drag on the core.     

Regular and irregular micelles formed by A LEL triblock copolymer in aqueous solution

Laser light scattering (LLS) techniques were used to characterize the micellization of poly(d,l-lactide)-poly(ethylene glycol)-poly(d,l-lactide) (LEL) triblock copolymer (MW 1K-2K-1K) in aqueous solution. We observed the existence of both thermodynamically stable flower-like micelles (regular micelles) and large, less soluble nanoparticles (irregular micelles) in dilute aqueous solutions with the same preparation procedure. Both kinds of micelles were found to co-exist with single copolymer chains. The initial copolymer concentration determines the nature of the micelles. The regular core-shell micelle formation follows a closed association mechanism, resulting in flower-like micelles. The hydrophobicity of a L unit is estimated as ∼0.5-0.6 B (polyoxybutylene) units from the micellization parameters, which is quite consistent with earlier estimations obtained from EL diblock copolymers.     

Dissipative particle dynamics study on the multicompartment micelles self-assembled from the mixture of diblock copolymer poly(ethyl ethylene)-block-poly(ethylene oxide) and homopolymer poly(propylene oxide) in aqueous solution

Dissipative particle dynamics (DPD) method is applied to model the self-assembly of diblock copolymer poly(ethyl ethylene)-block-poly(ethylene oxide) (PEE-b-PEO) and homopolymer poly(propylene oxide) (PPO) in aqueous solution. In this study, several segments are coarse-grained into a single simulation bead based on the experimental density. For the self-assembly of pure diblock copolymer PEE-b-PEO in dilute solution, the DPD simulation results are in good agreement with experimental data of micelle morphologies and sizes. The chain lengths of the block copolymers and the volume ratios between PPO and PEE-b-PEO are varied to find the conditions of forming multicompartment micelles. The micelles with core-shell-corona structure and the micelles with two compartments are both formed from the mixture of PEE-b-PEO and PPO in aqueous solution.     

Clustered assembly of Au nanoparticles from spherical diblock copolymer micelles encapsulating Au nanoparticle

Superstructures composed of diblock copolymer micelles and inorganic nanoparticles are quite interesting because the specific arrangement of inorganic nanoparticles within the micellar structure can reveal interesting opportunities in many field of science. In this perspective, we report a simple method to produce clustered assembly of Au nanoparticles in the micelles in attempt to induce plasmonic coupling among multiple Au nanoparticles in the assembled structures. Here, we utilized polystyrene‐block‐poly(acrylic acid), PS‐PAA, micelles containing single Au nanoparticle in the core (Au@PS‐PAA micelles) as building materials to initiate next‐level assembling process. In particular, the addition of HCl to the solution of Au@PS‐PAA micelles affected the overall equilibrium condition as well as kinetic process in the micellar solution. As a result, individual Au@PS‐PAA micelles could be merged together to form more large micelles with inclusion of multiple nanoparticles in the core, the process of which was accompanied with plasmonic coupling of Au nanoparticles. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 44693.     

Phase behaviors of bidisperse nanoparticle/block copolymer mixtures in dilute solutions

The complex microstructures of bidisperse nanoparticles/diblock copolymer mixtures in dilute solutions have been investigated by a theoretical approach which combines the self-consistent field theory (SCFT) and the density functional theory (DFT). Special attention is payed to the role played by the block ratio and the interaction parameters between each component in the mixture. It is shown that the conformational entropy of the polymer chains, the block ratio of the diblock copolymer, the chemical difference between two kinds of particles and the steric packing effect of the particles play important roles in determining the morphologies of the systems. It is found that with the increase of the block ratio, the mixture undergoes a morphological transition from compound micelles to spherelike micelles. The increase of chemical difference between the two kinds of particles can promote the formation of “a jujube set in a cake”. When the selectivity of the particles is changed, another type of micelle emerges. Specifically, in the case where the particles are nonselective to the A- and B-blocks, ordered structures from the phase separation between the two types of particles emerge inside the micelles formed by the amphiphilic diblock copolymers in solutions.     

Synthesis, characterization, and drug delivery research of an amphiphilic biodegradable star-shaped block copolymer

An amphiphilic biodegradable three-arm star-shaped diblock copolymer containing poly(ε-caprolactone) (PCL) and poly(N-vinylpyrrolidone) (PVP) (TEA(PCL-b-PVP)₃) has been successfully synthesized by the ring-opening polymerization of ε-caprolactone (ε-CL), RAFT polymerization of N-vinylpyrrolidone and a coupling reaction of PCL with carboxyl-terminated PVP (PVP-COOH). In aqueous media, the star-shaped copolymer self-assembled into spherical micelles with diameters of near 106 nm. The critical micelle concentration of TEA(PCL-b-PVP)₃ copolymer was determined to be 5.96 × 10^?3 mg/mL. Folic acid was then used as a model drug to incorporate into TEA(PCL-b-PVP)₃ micelles, the drug loading content and encapsulation efficiency is 16.36 and 49.08 %, respectively. In vitro release experiments of the drug-loaded micelles exhibited sustained release behavior and it was affected by the pH of release media. These results indicate that the copolymer may serve as a promising “intelligent” drug delivery alternative.     

Controlled organization of self-assembled rod-coil block copolymer micelles

Spherical micelles of a series of poly(styrene-block-(2,5-bis[4-methoxyphenyl]oxycarbonyl)styrene) (PS-b-PMPCS) rod-coil diblock copolymers in a selective solvent can organize into large mono-layered films with a well-ordered hexagonal packing of the spheres after solvent evaporation. Organized domains in the spherical micelle film were observed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The core-shell structure of the spherical micelle remained after solvent evaporation. The micelle diameter in the ordered film as observed by TEM and AFM agree. The size of the spherical micelles can be controlled by the length of PMPCS when the length of the PS is fixed. The sphere diameters were varied from several tens of nanometers to more than one hundred nanometers. Solutions of smaller micelle spheres formed less ordered films than those from larger micelle particles. Additionally, monolayer films of cylindrical worm-like micelles were also prepared. Those cylindrical micelles were observed to be end-capped by spherical micelles. The monolayer micelle film from the largest spherical micelles appeared red when observed in optical microscopy in the reflection mode. A broad adsorption peak with a maximum adsorption wavelength of 545 nm was observed via UV-Vis spectroscopy.     

New folate-functionalized biocompatible block copolymer micelles as potential anti-cancer drug delivery systems

The main objective of this study was to synthesize novel folic acid-functionalized diblock copolymer micelles and evaluate their solubilization of two poorly water-soluble anti-tumor drugs, tamoxifen and paclitaxel, which suffer from low water solubility and/or poor hydrolytic stability. The diblock copolymer consisted of a permanently hydrophilic block comprising 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) residues and a pH-sensitive hydrophobic block comprising 2-(diisopropylamino)ethyl methacrylate (DPA) residues. Folic acid (FA) was conjugated to the end of the MPC block so that this group was located on the micelle periphery. Tamoxifen- and paclitaxel-loaded micelles were prepared from FA-MPC-DPA copolymers prepared with two different block compositions that were designed to produce optimal solubilization of each drug. Their drug-loading capacities and aqueous stabilities were determined by high performance liquid chromatography. The hydrodynamic diameters of tamoxifen- and paclitaxel-loaded FA-MPC-DPA micelles ranged from 30 to 60 nm, as judged by dynamic light scattering (DLS) and transmission electron microscopy (TEM) studies. Finally, tamoxifen and paclitaxel release profiles were evaluated in phosphate buffer solution at pH 7.4 and 5. These studies demonstrated that FA-MPC-DPA micelles acted as useful drug carriers, leading to relatively slow release of both tamoxifen and paclitaxel into aqueous solution over a period of 7 days. In addition, rapid release can be triggered by lowering the solution pH to 5, which leads to protonation of the DPA block and hence rapid micellar dissociation.     

Temperature-composition phase diagrams of binary blends of block copolymer and homopolymer

The temperature-composition phase diagrams for six pairs of diblock copolymer and homopolymer are presented, putting emphasis on the effects of block copolymer composition and the molecular weight of added homopolymers. For the study, two polystyrene-block-polyisoprene (SI diblock) copolymers having lamellar or spherical microdomains, a polystyrene-block-polybutadiene (SB diblock) copolymer having lamellar microdomains, and a series of polystyrene (PS), polyisoprene (PI), and polybutadiene (PB) were used to prepare SI/PS, SI/PI, SB/PS, and SB/PB binary blends, via solvent casting, over a wide range of compositions. The shape of temperature-composition phase diagram of block copolymer/homopolymer blend is greatly affected by a small change in the ratio of the molecular weight of added homopolymer to the molecular weight of corresponding block (M_H,A/M_C,A or M_H,B/M_C,B) when the block copolymer is highly asymmetric in composition but only moderately even for a large change in M_H,A/M_C,A ratio when the block copolymer is symmetric or nearly symmetric in composition. The boundary between the mesophase (M₁) of block copolymer and the homogeneous phase (H) of block copolymer/homopolymer blend was determined using oscillatory shear rheometry, and the boundary between the homogeneous phase (H) and two-phase liquid mixture (L₁+L₂) with L₁ being disordered block copolymer and L₂ being macrophase-separated homopolymer was determined using cloud point measurement. It is found that the addition of PI to a lamella-forming SI diblock copolymer or the addition of PB to a lamella-forming SB diblock copolymer gives rise to disordered micelles (DM) having no long-range order, while the addition of PS to a lamella-forming SB diblock copolymer retains lamellar microdomain structure until microdomains disappear completely. Thus, the phase diagram of SI/PI or SB/PB blends looks more complicated than that of SI/PS or SB/PS blends.     

Design and stabilization of block copolymer micelles via phenol-pyridine hydrogen-bonding interactions

A new approach for the preparation of block copolymer micelles in non-selective solvent is introduced. Phenol-pyridine hydrogen-bonding interactions are used for the first time to prepare core-shell micelles in non-selective solvents using block copolymers and bifunctional low-molecular-weight hydrogen-bonding crosslinkers. Poly(styrene-b-4-vinylphenol)/Bis-pyridyl ethane and poly(styrene-b-4-vinylpyridine)/Bisphenol A were investigated as micelle formation due to phenol-pyridine hydrogen bond crosslinking. The influence of several factors such as temperature, concentration, solvent and pH in micellization-demicellization process was analyzed by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), dynamic light scattering (DLS) and atomic force microscopy (AFM). This method opens new possibilities to the generation of block copolymer micelles in non-selective solvents.     

Re-assembly behaviors of polystyrene-b-poly(acrylic acid) micelles

The re-assembly behaviors of spherical micelles of the polystyrene-b-poly(acrylic acid) (PS-b-PAA) diblock copolymer in different solvent mixtures were investigated using dynamic light scattering, transmission electron microscopy and atomic force microscopy. Depending on the nature of the solvent, PS-b-PAA micelles re-assembled from spheres to nanorings in toluene or to necklace-like aggregates in water induced by solvent evaporation. Systematic studies suggested that the re-assembly behaviors on a neutral surface are strongly correlated with the micellar surface components, the solvent polarity and the chain length of the micelle corona of the solvated blocks. We proposed that the formation of nanorings from PS-b-PAA micelles in toluene is mainly induced by the dewetting process of the solvent, while the necklace-like structure arises from the hydrogen bonding interactions among the partially dissociated PAA units.     

Thermo-responsive behavior of hybrid core cross-linked polymer micelles with biocompatible shells

Poly(2-(methacryloyloxy)ethyl phosphorylcholine)-b-poly(N-isopropylacrylamide-co-2-(N,N-dimethylamino)ethyl methacrylate) (pMPC-b-p(NIPAM/DMA)) was synthesized via reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization. Below the critical aggregation temperature (CAT), i.e., about 40 °C, the diblock copolymer dissolved in water as a unimer with a hydrodynamic radius (R_h) of ca. 10 nm. Above CAT the diblock copolymers formed polymer micelles with an R_h of ca. 40 nm, composed of a p(NIPAM/DMA) core and biocompatible pMPC shell due to hydrophobic self-aggregation of the thermo-responsive p(NIPAM/DMA) block. The pendent 2-(N,N-dimethylamino)ethyl group of DMA in pMPC-b-p(NIPAM/DMA) reduced HAuCl₄ to form gold nanoparticles (AuNPs) and could attach to their surfaces. The cores of these polymer micelles could be cross-linked above CAT by HAuCl₄, which upon being reduced generated AuNPs as cross-linking points to form core cross-linked (CCL) polymer micelles, as confirmed by UV-vis absorption and dynamic light scattering measurements. The CCL polymer micelles absorbed visible light at 532 nm because of surface plasmon resonances of the AuNPs. The R_h of the CCL polymer micelles remained at ca. 40 nm regardless of temperature.     

Crystallization and coalescence of block copolymer micelles in semicrystalline block copolymer/amorphous homopolymer blends

Crystallization of two oxyethylene/oxybutylene block copolymers (E₇₆B₃₈ and E₁₅₅B₇₆) from micelles in block copolymer/amorphous homopolymer blends was studied by differential scanning calorimetry (DSC) and time-resolved small angle X-ray scattering (SAXS). Unlike the simultaneous crystallization and formation of superstructure in crystallization from an ordered structure, crystallization of block copolymer from micelles can be divided into two steps. The core of the micelles firstly crystallizes individually, with first-order crystallization kinetics and homogeneous nucleation mechanism. The SAXS revealed that crystallization-induced deformation occurs for the micelles, which strongly depends on microstructure of the block copolymers. For the shorter block copolymer E₇₆B₃₈, larger deformation induced by crystallization was observed, leading to coalescence of the micelles after crystallization, while for the longer block copolymer E₁₅₅B₇₆ the micelles show little deformation and the morphology of micelle is retained after crystallization.     

Synthesis and micelle behavior of (PNIPAm-PtBA-PNIPAm)m amphiphilic multiblock copolymer

A linear amphiphilic multiblock copolymer (PNIPAm-PtBA-PNIPAm)_m was successfully synthesized by a two-step reversible addition-fragmentation transfer (RAFT) polymerization in the presence of a cyclic trithiocarbonate as RAFT agent. The micelle behavior of (PNIPAm-PtBA-PNIPAm)_m multiblock copolymer in aqueous solution was then investigated by means of normal TEM, cryo-TEM, static and dynamic light scattering. The morphology, size, and size distribution of (PNIPAm-PtBA-PNIPAm)_m micelles were found to be dependent on the initial concentration of multiblock copolymer in THF. Spherical micelles, associated aggregates of spherical micelles, cage-like micelles, layered structures, and vesicular micelles were experimentally observed, which were in good agreement with the prediction of theory and simulations on linear amphiphilic multiblock copolymer in selective solvent. The (PNIPAm-PtBA-PNIPAm)_m micelles also exhibit thermo-sensitive behavior in aqueous solution because of the PNIPAm blocks.     

Enhanced loading of doxorubicin into polymeric micelles by a combination of ionic bonding and hydrophobic effect,and the pH-sensitive and ligand-mediated delivery of loaded drug

Folate-conjugated micelles were fabricated from amphiphilic diblock copolymers with poly(ethylene glycol) as the hydrophilic block and a random copolymer of n-butyl methacrylate and methacrylic acid as the hydrophobic block. Doxorubicin (DOX), a model drug that contains an amine group and hydrophobic moiety, was loaded with a high loading capacity into micelles by a combination of ionic bonding and hydrophobic effect. The combined interactions imparted a pH-sensitive delivery property to the system. The release rate of loaded DOX was slow at pH 7.4 (i.e., mimicking the plasma environment) but increased significantly at acidic pH (i.e., mimicking endosome/lysosome conditions). Acid-triggered drug release resulted from the carboxylate protonation of poly(methacrylic acid), which dissociated the ionic bonding between the micelles and DOX. Cellular uptake by folate receptor-overexpressing HeLa cells of the DOX-loaded folate-conjugated micelles was higher than that of micelles without folate conjugation. Thus, the DOX-loaded folate-conjugated micelles displayed higher cytotoxicity to HeLa cells.