Regulation of the Contribution of Integrin to Cell Attachment on Poly(2-Methoxyethyl Acrylate) (PMEA) Analogous Polymers for Attachment-Based Cell Enrichment

Cell enrichment is currently in high demand in medical engineering. We have reported that non-blood cells can attach to a blood-compatible poly(2-methoxyethyl acrylate) (PMEA) substrate through integrin-dependent and integrin-independent mechanisms because the PMEA substrate suppresses protein adsorption. Therefore, we assumed that PMEA analogous polymers can change the contribution of integrin to cell attachment through the regulation of protein adsorption. In the present study, we investigated protein adsorption, cell attachment profiles, and attachment mechanisms on PMEA analogous polymer substrates. Additionally, we demonstrated the possibility of attachment-based cell enrichment on PMEA analogous polymer substrates. HT-1080 and MDA-MB-231 cells started to attach to poly(butyl acrylate) (PBA) and poly(tetrahydrofurfuryl acrylate) (PTHFA), on which proteins could adsorb well, within 1 h. HepG2 cells started to attach after 1 h. HT-1080, MDA-MB-231, and HepG2 cells started to attach within 30 min to PMEA, poly(2-(2-methoxyethoxy) ethyl acrylate-co-butyl acrylate) (30:70 mol%, PMe2A) and poly(2-(2-methoxyethoxy) ethoxy ethyl acrylate-co-butyl acrylate) (30:70 mol%, PMe3A), which suppress protein adsorption. Moreover, the ratio of attached cells from a cell mixture can be changed on PMEA analogous polymers. These findings suggested that PMEA analogous polymers can be used for attachment-based cell enrichment.     

A molecular simulation of the compatibility of chitosan and poly(vinyl pyrrolidone)

The compatibility of chitosan (CS) and poly(vinyl pyrrolidone) was investigated by molecular dynamic (MD) simulations using the Flory–Huggins theory. The specific interactions in blends were studied by the radial distribution function (RDF). The Flory–Huggins interaction parameter, χ, was calculated at 298 K to assess the blend compatibility at different component ratios in the polymers. Miscibility was observed for blends with more than 50% of CS in the molar fraction, while immiscibility was prevalent at the molar fraction of CS between 10 and 50% of CS. Miscibility between poly(N-vinyl-2-pyrrolidone) (PVP) and CS polymers is attributed to the hydrogen bond formation of the –C = O group of PVP and the –CH₂OH groups of CS. This was further confirmed by MD simulations of RDFs for groups or atoms that are involved in interactions. These results are correlated well to obtain more realistic information on interactions involved as a function of blend composition.     

Biosynthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) polymers

In support of programs to identify polyhydroxyalkanoates with improved materials properties, we report on our efforts to characterize the mechanical and thermal properties of copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). The copolyesters, having molar fraction of 3HHx ranging from 2.5 to 35 mol % and average molecular weights ranging from 1.15 x 10(5) to 6.65 x 10(5), were produced by fermentation using Aeromonas hydrophila and a recombinant strain of Pseudomonas putida GPp104. The polymers were chloroform extracted and characterized by solution-state and solid-state nuclear magnetic resonance (NMR) spectroscopy and a variety of mechanical and thermal tests. Solution-state (1)H NMR data were used to determine polymer composition-of-matter, while solution-state (13)C NMR data provided polymer-sequence information. Solvent fractionation and NMR spectroscopic characterization of these polymers showed that polymers containing up to 9.5 mol % 3HHx had a Bernoullian compositional distribution. By contrast, polymers containing more than 9.5 mol % 3HHx had a bimodal polymer composition. Solvent fractionation of these 3HHx-rich polyesters produced two polymer fractions, each of which was again consistent with Bernoullian polymerization statistics. Solid-state NMR relaxation experiments provided insight into aging in poly(3HB-co-3HHx) copolymers, demonstrating increased polymer-chain motion with increasing 3HHx content. The elongation-to-break ratio in the polyesters increased with increasing molar fraction of 3HHx monomers. Aging properties of the poly(3HB-co-3HHx) copolymers were very similar to copolymers of 3HB and 3-hydroxyvalerate (3HV). However, poly(3HB-co-3HHx) exhibited increased activation energy to thermal degradation with increasing 3HHx content.     

Semi-interpenetrating polymer network hydrogels based on water-soluble N-carboxylethyl chitosan and photopolymerized poly (2-hydroxyethyl methacrylate)

Semi-interpenetrating polymer network (semi-IPN) hydrogels were prepared by UV irradiation of water-soluble N-carboxylethyl chitosan (CECS) and 2-hydroxyethyl methacrylate (HEMA) aqueous solutions in the presence of D-2959 as photoinitiator. Hydrogels were characterized by using scanning electron microscopy (SEM), thermal gravimetric analysis (TGA) and X-ray diffractometry (XRD). SEM showed that semi-IPN hydrogels displayed porous surface and therefore had high surface area. XRD indicated that CECS/poly (HEMA) semi-IPN hydrogels had amorphous structure. The thermal stability and equilibrium degree of swelling improved obviously with increase of CECS content. Differential scanning calorimetry (DSC) indicated that free water content increased with increase of CECS content while bonded water content decreased. Cytotoxicity results suggested that semi-IPN hydrogels had good biocompatibility. From these preliminary evaluations, it is possible to conclude that these materials have potential applications in the biomedical field.     

Rapid deswelling response of poly(N-isopropylacrylamide)/poly(2-alkyl-2-oxazoline)/poly(2-hydroxyethyl methacrylate) hydrogels

Ternary poly( N-isopropylacrylamide)/poly(2-alkyl-2-oxazoline)/poly(2-hydroxyethyl methacrylate) (PNIPAAm/PROZO/PHEMA) hydrogels were prepared by the free-radical copolymerization of N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), and poly(2-alkyl-2-oxazoline) (PROZO) multifunctional macromonomers. The resulting polymeric materials were characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM), as well as by equilibrium swelling experiments. All synthesized hydrogels display temperature sensitivity in the 28-38 degrees C range. A high rate of response was registered as compared to that of materials based only on PNIPAAm. The swelling-deswelling peculiar behavior was related to the chemical composition (hydrophile/hydrophobe balance), the length of the inserted PROZO sequence, and inner morphology, an aspect which points on its possible control by synthesis. It was evidenced that the architecture of the resulting porous materials has a high order degree, emerging from the self-assembling of the microgel particles, which provided numerous, nearly uniform, large water release channels.     

PFG-NMR measurements of the self-diffusion coefficients of water in equilibrium poly(HEMA-co-THFMA) hydrogels

The self-diffusion coefficients for water in a series of copolymers of 2-hydroxyethyl methacrylate, HEMA, and tetrahydrofurfuryl methacrylate, THFMA, swollen with water to their equilibrium states have been studied at 310 K using PFG-NMR. The self-diffusion coefficients calculated from the Stejskal-Tanner equation, D(obs), for all of the hydrated polymers were found to be dependent on the NMR storage time, as a result of spin exchange between the proton reservoirs of the water and the polymers, reaching an equilibrium plateau value at long storage times. The true values of the diffusion coefficients were calculated from the values of D(obs) in the plateau regions by applying a correction for the fraction of water protons present, obtained from the equilibrium water contents of the gels. The true self-diffusion coefficient for water in polyHEMA obtained at 310 K by this method was 5.5 x 10(-10) m(2)s-1. For the copolymers containing 20% HEMA or more a single value of the self-diffusion coefficient was found, which was somewhat larger than the corresponding values obtained for the macroscopic diffusion coefficient from sorption measurements. For polyTHFMA and copolymers containing less than 20% HEMA, the PFG-NMR stimulated echo attenuation decay curves and the log-attenuation plots were characteristic of the presence of two diffusing water species. The self-diffusion coefficients of water in the equilibrium-hydrated copolymers were found to be dependent on the copolymer composition, decreasing with increasing THFMA content.     

Hemocompatibility of hydrophilic antimicrobial copolymers of alkylated 4-vinylpyridine

Quaternized poly(vinylpyridine) (PVP) is a polymer with inherent antimicrobial properties that is effective against Gram-positive bacteria, Gram-negative bacteria, viruses, and yeast cells. However, quaternized PVP has poor biocompatibility, which prevents its use in biomaterial applications. Copolymerization was examined as a method of modifying the structure to incorporate biocompatibility. Polyethyleneglycol methyl ether methacrylate (PEGMA) and hydroxyethyl methacrylate (HEMA) are polymers generally known to be biocompatible and thus were chosen as comonomers. Random copolymers of 4-vinylpyridine and PEGMA or HEMA were synthesized via free radical polymerization and quaternized with bromohexane. Copolymer biocompatibility was characterized by interaction with human red blood cells to analyze hemolysis. Hemolysis of human red blood cells was conducted on insoluble films and on water-soluble polymers in a serial dilution study. Hemolysis results demonstrated that blood compatibility does not depend on PEG chain length in PEGMA incorporated copolymers. Results indicate a critical weight ratio of PEGMA to VP in copolymers separating the no-hemolysis regime from 100% hemolysis.     

Synthesis and self-assembly of well-defined poly(amino acid) end-capped poly(ethylene glycol) and poly(2-methyl-2-oxazoline)

Poly(ethylene glycol) (PEG) and poly(2-methyl-2-oxazoline) (PMOx) are water-soluble, biocompatible polymers with stealth hemolytic activities. Poly(amino acid) (PAA) end-capped PEG and PMOx were prepared using amino-terminated derivatives of PEG and PMOx as macroinitiators for the ring-opening polymerization of γ-benzyl protected l-glutamate N-carboxyanhydride and S-benzyloxycarbonyl protected l-cysteine N-carboxyanhydride, respectively, in the presence of urea, at room temperature. The molecular weight of the PAA moiety was kept between M(n) = 2200 and 3000 g mol(-1). PMOx was polymerized by cationic ring-opening polymerization resulting in molecular weights of M(n) = 5000 and 10,000 g mol(-1), and PEG was a commercial product with M(n) = 5000 g mol(-1). Here, we investigate the self-assembly of the resulting amphiphilic block copolymers in water and the effect of the chemical structure of the block copolymers on the solution properties of self-assembled nanostructures. The PEG-block-poly(amino acid), PEG-b-PAA, and PMOx-block-poly(amino acid), PMOx-b-PAA, block copolymers have a narrow and monomodal molecular weight distribution (PDI < 1.3). Their self-assembly in water was studied by dynamic light scattering and fluorescence spectroscopy. In aqueous solution, the block copolymers associate into particles with hydrodynamic radii (R(H)) ranging in size from R(H) 70 to 130 nm, depending on the block copolymer architecture and the polymer molecular weight. Larger R(H) and critical association concentration values were obtained for copolymers containing poly(S-benzyloxycarbonyl-l-cysteine) compared to their poly(γ-benzyl-L-glutamate) analogue. FTIR investigations revealed that the poly(γ-benzyl-L-glutamate) block adopts a helical conformation, while the poly(S-benzyloxycarbonyl-L-cysteine) block exists as β-sheet.     

(1)H NMR study of the states of water in equilibrium poly(HEMA-co-THFMA) hydrogels

The spin-spin relaxation times, T(2), of hydrated samples of poly(hydroxymethyl methacrylate), PHEMA, poly(tetrahydrofurfuryl methacrylate), PTHFMA, and the corresponding HEMA-THFMA copolymers have been examined to probe the states of the imbibed water in these polymers. The decay in the transverse magnetization of water in fully hydrated samples of PHEMA, PTHFMA, and copolymers of HEMA and THFMA was described by a multiexponential function. The short component of T(2) was interpreted as water molecules that were strongly interacting with the polymer chains. The intermediate component of T(2) was assigned to water residing in the porous structure of the samples. The long component of T(2) was believed to arise from water residing in the remnants of cracks formed in the polymer network during water sorption.     

Poly(ethylene glycol)-grafted poly(3-hydroxyundecenoate) networks for enhanced blood compatibility

Poly(ethylene glycol)-grafted poly(3-hydroxyundecenoate) (PEG-g-PHU) networks were prepared by irradiating homogeneous solutions of poly(3-hydroxyundecenoate) (PHU) and the monoacrylate of poly(ethylene glycol) (PEG) with UV light. The resulting polymer networks were characterized by measuring the water contact angle, water uptake, and mechanical properties and by performing attenuated total reflectance infrared spectroscopy and scanning electron microscopy. These measurements showed that the PEG chains were present in polymer networks. Adsorption of blood proteins and platelets on cross-linked PHU (CLPHU) and PEG-g-PHU were examined using poly(L-lactide) (PLLA) surfaces as control. Blood proteins and platelets had significantly lower tendency of adhesion to surfaces composed of CLPHU and PEG-g-PHU networks than to PLLA. Blood compatibility of polymer networks increased as the fraction of grafted PEG increased. The results of this study suggest that PEG-g-PHU networks might be useful for blood-compatible biomedical applications.     

Synthesis of multiarm star poly(glycerol)-block-poly(2-hydroxyethyl methacrylate)

Well-defined multiarm star block copolymers poly(glycerol)-b-poly(2-hydroxyethyl methacrylate) (PG-b-PHEMA) with an average of 56, 66, and 90 PHEMA arms, respectively, have been prepared by atom transfer radical polymerization (ATRP) of HEMA in methanol by a core-first strategy. The hyperbranched macroinitiators employed were prepared on the basis of well-defined hyperbranched polyglycerol by esterification with 2-bromoisobutyryl bromide. Polydispersites M(w)/M(n) of the new multiarm stars were in the range of 1.11-1.82. Unexpectedly, with the combination of CuCl/CuBr(2)/2,2'-bipyridyl as catalyst, the polymerization conversion can be driven to maximum values of 79%. The control of CuCl catalyst concentration is also very important to achieve high conversion and narrow polydispersity. The absolute M(n) values of the obtained multiarm star polymers were in good agreement with the calculated ones, and the highest M(n) values of the multiarm star copolymer is around 10(6) g/mol. Kinetic analysis shows that an induction period exists in the polymerization of HEMA. After this induction period, a linear dependence of ln ([M](0)/[M](t)()) on time was observed. Due to the star architecture, the viscosity of the obtained multiarm star PHEMA is much lower than that of linear PHEMA.     

Complexation of DNA with poly(methacryl oxyethyl trimethylammonium chloride) and Its poly(oxyethylene) grafted analogue

Intermolecular complexes of genomic polydisperse DNA with synthetic polycations have been studied. Two cationic polymers have been used, a homopolymer poly(methacryl oxyethyl trimethylammonium chloride) (PMOTAC) and its analogue grafted with poly(oxyethylene). The amount of poly(oxyethylene) grafts in the copolymer was 15 mol % and Mw of the graft was 200 g/mol. Salmon DNA (sodium salt) was used. The average molecular weight (Mw) of DNA was 10.4 x 10(6) g/mol. Conductivity, pH, and dynamic light scattering studies were used to characterize the complexes. The size and shape of the polyelectrolyte complex particles have been studied as a function of the cation-to-anion ratio in aqueous solutions of varying ionic strengths. The polyelectrolyte complexes have extremely narrow size distributions taking into account the polydispersity of the polyelectrolytes studied. The poly(oxyethylene) grafts on PMOTAC promote the formation of small colloidally stabile complex particles. Addition of salt shifts the macroscopic phase separation toward lower polycation content; that is, complexes partly phase separate with the mixing ratios far from 1:1. Further addition of salt to the turbid, partly phase separated solution results in the dissociation of complexes and the polycation and DNA dissolve as individual chains.     

聚氨酯／液晶（PU／EBBA）复合膜的血液相容性研究

以聚氨酯弹性体为基质材料,与液晶化合物EBBA共混后,由溶剂蒸发法浇铸成膜。偏光显微镜观察证实了复合膜中液晶相的存在。用动态凝血实验、血小板粘附实验和扫描电镜观察的方法研究了复合膜中液晶含量对材料抗凝血性能的影响。结果表明,只有当液晶含量达到30%（wt）时,复合膜的血液相容性随着液晶含量的增加有明显改善,同时发现复合膜表面吸附的血小板随着液晶含量的增加而明显减少。     

Enhancement of blood compatibility of poly(urethane) substrates by mussel-inspired adhesive heparin coating

Heparin immobilization on surfaces has drawn a great deal of attention because of its potential application in enhancing blood compatibility of various biomedical devices such as catheters, grafts, and stents. Existing methods for the heparin immobilization are based on covalent linkage formation and electrostatic interaction between substrates and heparin molecules. However, complicated multistep procedures and uncontrolled desorption of heparin are limitations of these methods. In this work, we report a new heparin derivative that exhibits robust adhesion on surfaces. The derivative, called hepamine, was prepared via conjugation of dopamine, a mussel-inspired adhesive moiety, onto a heparin backbone. Immersion of poly(urethane) substrates into an aqueous solution of hepamine resulted in robust heparin coating of the poly(urethane), the most widely used polymeric material for blood-contacting medical devices. The hepamine-coated poly(urethane) substrate showed significant inhibition of blood coagulation and platelet adhesion. The use of hepamine for surface modification is advantageous for several reasons: for example, no chemical pretreatment of the substrates is necessary, and surface functionalization is a simple, one-step procedure. Thus, the heparin immobilization method described herein is an excellent alternative approach for the introduction of heparin molecules onto surfaces.     

Poly(N-(2-hydroxypropyl) methacrylamide mono/di lactate): a new class of biodegradable polymers with tuneable thermosensitivity

A novel class of thermosensitive and biodegradable polymers, poly(N-(2-hydroxypropyl) methacrylamide mono/di lactate) (poly(HPMAm-mono/di lactate)), was synthesized. The cloud points (CP) of poly(HPMAm-monolactate) and poly(HPMAm-dilactate) in water were 65 and 13 degrees C, respectively. The lower CP for poly(HPMAm-dilactate) is likely due the greater hydrophobicity of the dilactate side group over the monolactate side group. The CP of poly(HPMAm-monolactate-co-HPMAm-dilactate) increased linearly with mol % of HPMA-monolactate, which demonstrates that the CP is tuneable by the copolymer composition.     

Facile synthesis of multiamino vinyl poly(amino acid)s for promising bioapplications

We presented a general and facile strategy to prepare biocompatible multiamino polymers. Series of new monomers were synthesized by esterification of 2-hydroxyethyl methacrylate (HEMA) and Boc-amino acids, such as Boc-l-phenylalanine, Boc-glycine, Boc-l-alanine, Boc-l-valine, and Boc-l-lysine. Subsequent vinyl polymerization of monomers gave rise to vinyl poly(amino acid)s with a side primary amino group at each unit if deprotected. Both atom transfer radical polymerization (ATRP) and conventional free radical polymerization (FRP) were employed to prepare the multiamino polymers. A well controlled effect upon molecular weight with the standard first-order kinetics was achieved in cases of ATRP, and high molecular weight polymers were obtained via FRP. MTT assay showed that cell survival rates for the multiamino polymers were almost maintained above 90% and that their cytotoxicities were much lower than that of linear PEI (PEI 25000). Zeta potential measurements demonstrated that the vinyl poly(amino acid)s are electropositive, and AFM measurements showed that the vinyl poly(amino acid)s could tightly condense DNA into granular structures at a suitable concentration. The combination of facile availability, controlled productivity, low cytotoxicity and strong binding ability with DNA promises the great potential of the novel multiamino polymers in bioapplications.     

New sorbent for bilirubin removal from human plasma: Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads

Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads were investigated as a specific sorbent for bilirubin removal from human plasma. The poly(EGDMA–HEMA) microbeads were prepared by a modified suspension copolymerization technique. Cibacron Blue F3GA was covalently coupled to the poly(EGDMA–HEMA) microbeads via the nucleophilic reaction between the chloride of its triazine ring and the hydroxyl groups of the HEMA molecule, under alkaline conditions. Bilirubin adsorption was investigated from hyperbilirubinemic human plasma on the poly(EGDMA–HEMA) microbeads containing different amounts of immobilized Cibacron Blue F3GA, (between 5.0–16.5 μmol/g). The non-specific bilirubin adsorption on the unmodified poly(EGDMA–HEMA) microbeads were 0.32 mg/g from human plasma. Higher bilirubin adsorption values, up to 14.8 mg/g, were obtained with the Cibacron Blue F3GA-immobilized microbeads. Bilirubin molecules interacted with these sorbents directly. Contribution of albumin adsorption on the bilirubin adsorption was pronounced. Bilirubin adsorption increased with increasing temperature.     

Binding of streptavidin with biotinylated thermosensitive nanospheres based on poly(N,N-diethylacrylamide-co-2-hydroxyethyl methacrylate)

Thermosensitive polymer nanospheres based on N,N-diethylacrylamide and 2-hydroxyethyl methacrylate (HEMA) have been prepared, characterized, and conjugated with biotin. The thermosensitivity of poly(N,N-diethylacrylamide) was enhanced by the incorporation of HEMA up to about 40 mol %. Atomic force microscopic images show that these particles can be closely packed even without the surface charges as in the latex particles. Biotinylation reduces the thermosensitivity of the copolymer nanospheres. The biotinylated hydrogel nanospheres showed a reduction in size upon binding with streptavidin, indicating the formation of a less hydrophilic conjugate. No aggregation of the biotinylated particles due to the cross-linking effect of streptavidin was observed. This size change could be reversed by the addition of free biotin to the system. The interaction is specific, and no such changes were observed when streptavidin was replaced by bovine serum albumin.     

Factors affecting the production of prednisolone by immobilization of Bacillus pumilus E601 cells in poly(vinyl alcohol) cryogels produced by radiation polymerization

To increase the yield percent of prednisolone from hydrocortisone (cortisol), Bacillus pumilus E601 (a radioresistant microorganism) was incorporated into poly(vinyl alcohol) (PVA) cryogel grafted with hydroxyethyl-methacrylate (HEMA) as a crosslinking agent. The polymer was prepared by a radiation polymerization technique at 20 kGy from Co-60 source. The optimum temperature for the biotransformation of hydrocortisone by free cells, poly(PVA)/HEMA, and poly(PVA)/HEMA /N-isopropylacrylamide (N-IPAAm) was 30 °C. The highest yield % of prednisolone was obtained by immobilization of the cells on poly(PVA/HEMA), the addition of N-IPAAm to poly(PVA/HEMA) protected the immobilized cells from temperatures above 35 °C during the fermentation process. The optimal pH (buffered pH) of the biotransformation of hydrocortisone by immobilized and free cells was 7.0, but the maximum yield of prednisolone (60%) was obtained by immobilized cells in comparison with free cells (42%) also at pH 7.0. The prednisolone yield reached 60–65% with 1,2-propanediol cosolvent containing media and 60–62% in the case of ethanediol cosolvent containing media at 1% (v/v) of both cosolvents. 10 mg/50 ml Tween 80 the medium increased the prednisolone yield by only 1.1-fold compared with the control. The maximum bioconversion efficiency was obtained at a substrate concentration of 20 mg/50 ml medium. Stability studies showed that the immobilized cells can be used for seven times without any significant decrease in activity.     

Production of prednisolone by Bacillus pumilus E601 cells incorporated in radiation induced poly(vinyl alcohol g-2 hydroxyethylmethacrylate) cryogels

Immobilization of Bacillus pumilus E601 in poly(vinyl alcohol) (PVA) cryogels at a concentration of 10% grafted with hydroxy-ethylmethacrylate (HEMA) at a concentration of 0.4% using a radiation polymerization technique lead to an increase in the prednisolone yield (46%) compared with the prednisolone yield (38%) produced by immobilized B. pumilus E601 carrier on the surface of the polymer at the same concentrations. The Δ¹-dehydrogenase of B. pumilus E601 was affected by the molecular weight, the irradiation dose and the diameter of the polymer. The storage of immobilized B. pumilus E601 in poly(PVA)/HEMA at −4 °C for 30 days shows a higher yield of prednisolone (80%) as compared with prednisolone yield (75%) at 25 °C at the time of storage.