Zwitterionic Modifications for Enhancing the Antifouling Properties of Poly(vinylidene fluoride) Membranes.

The development of effective antibiofouling membranes is critical for many scientific interests and industrial applications. However, the existing available membranes often suffer from the lack of efficient, stable, and scalable antifouling modification strategy. Herein, we designed, synthesized, and characterized alternate copolymers of p(MAO-DMEA) (obtained by reaction between poly(maleic anhydride-alt-1-octadecene) and N,N-dimethylenediamine) and p(MAO-DMPA) (obtained by reaction between poly(maleic anhydride-alt-1-octadecene) and 3-(dimethylamino)-1-propylamine) of different carbon space length (CSL) using a ring-opening zwitterionization. We coated these copolymers on poly(vinylidene fluoride) (PVDF) membranes using a self-assembled anchoring method. Two important design parameters-the CSL of polymers and the coating density of polymers on membrane-were extensively examined for their effects on the antifouling performance of the modified membranes using a series of protein, cell, and bacterial assays. Both zwitterionic-modified membranes with different coating densities showed improved membrane hydrophilicity, increased resistance to protein, bacteria, blood cells, and platelet adsorption. However, while p(MAO-DMEA) with two CSLs and p(MAO-DMPA) with three CSLs only differ by one single carbon between the amino and ammonium groups, such subtle structural difference between the two polymers led to the fact that the membranes self-assembled with MAO-DMEA outperformed those modified with MAO-DMPA in all aspects of surface hydration, protein and bacteria resistance, and blood biocompatibility. This work provides an important structural-based design principle: a subtle change in the CSL of polymers affects the surface and antifouling properties of the membranes. It can help to achieve the design of more effective antifouling membranes for blood contacting applications.

Preparation of chitosan/hydroxyapatite substrates with controllable osteoconductivity tracked by AFM.

In this study, cell-material adhesive strength and cellular mechanical properties were measured using atomic force microscopy (AFM) to track cell attachment and osteogenic differentiation. First, chitosan substrates were treated with simulated body fluid (SBF) for various periods, resulting in substrates with different osteoconductivity. The X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS) and in vitro tests revealed that the biomimeticity and osteoconductivity of substrates increased with increasing time of SBF treatment. When the SBF immersion exceeded 14 days, the chitosan substrates exhibited their highest biocompatibility and osteoconductivity. AFM measurements indicated specifically high adhesive forces between SBF-treated chitosan and osteogenic cells, causing better cell attachment. The results demonstrate that cell adhesion was controlled by cell-material adhesive strength, which were in turn controlled via the SBF treatment time. The adhesive strength between cells and material also accounted for the chitosan substrates' specific selectivity toward osteogenic cells. A two-step increase in mechanical strength was observed for the nucleus and cytoplasm of osteogenic cells. The results indicate that through the use of AFM, the real-time cell-material interforce and cellular mechanics can be identified. The adhesive strength was positively correlated to the cell attachment, and the second increase in the Young's modulus of nucleus and cytoplasm was correlated to early osteogenic differentiation.

Bacterial resistance of self-assembled surfaces using PPOm-b-PSBMAn zwitterionic copolymer - concomitant effects of surface topography and surface chemistry on attachment of live bacteria.

Three well-defined diblock copolymers made of poly(sulfobetaine methacrylate) (poly(SBMA)) and poly(propylene oxide) (PPO) groups were synthesized by atom transfer radical polymerization (ATRP) method. They were physically adsorbed onto three types of surfaces having different topography, including smooth flat surface, convex surface, and indented surface. Chemical state of surfaces was characterized by XPS while the various topographies were examined by SEM and AFM. Hydrophilicity of surfaces was dependent on both the surface chemistry and the surface topography, suggesting that orientation of copolymer brushes can be tuned in the design of surfaces aimed at resisting bacterial attachment. Escherichia coli, Staphylococcus epidermidis, Streptococcus mutans and Escherichia coli with green fluorescent protein (E. coli GFP) were used in bacterial tests to assess the resistance to bacterial attachment of poly(SBMA)-covered surfaces. Results highlighted a drastic improvement of resistance to bacterial adhesion with the increasing of poly(SBMA) to PPO ratio, as well as an important effect of surface topography. The chemical effect was directly related to the length of the hydrophilic moieties. When longer, more water could be entrapped, leading to improved anti-bacterial properties. The physical effect impacted on the orientation of the copolymer brushes, as well as on the surface contact area available. Convex surfaces as well as indented surfaces wafer presented the best resistance to bacterial adhesion. Indeed, bacterial attachment was more importantly reduced on these surfaces compared with smooth surfaces. It was explained by the non-orthogonal orientation of copolymer brushes, resulting in a more efficient surface coverage of zwitterionic molecules. This work suggests that not only the control of surface chemistry is essential in the preparation of surfaces resisting bacterial attachment, but also the control of surface topography and orientation of antifouling moieties.

Immobilization of naringin onto chitosan substrates by using ozone activation.

Ozone oxidation can easily produce peroxides containing active free radicals that can be used for the surface modification of biomaterials. This process is highly efficient and nontoxic. In this research, naringin, an HMG-CoA reductase inhibitor that can promote bone formation, was immobilized onto a chitosan film using ozone activation. First, a chitosan film was treated by ozone to produce peroxides; these peroxides were then quantified and their amount was optimized by an iodide assay. For the in vitro delivery of naringin, a chitosan-naringin substrate was immersed in phosphate-buffered saline to quantify the released amount of naringin. It was found that the immobilized naringin was slowly released over the course of two weeks, where its concentration in the medium was controlled by this delivery process. The results of cell culture showed that cell viability and early osteogenic differentiation, as measured by alkaline phosphatase expression, were promoted with the immobilized naringin on chitosan substrates. The expression of osteogenic proteins, including type-I collagen, bone siloprotein, and osteocalcin, were also enhanced. According to the results of Smad1 and Smad6 phosphorylation, immobilized naringin on ozonated chitosan substrates would be able to initiate bone morphogenetic protein-Smad signaling by activating receptor Smad and by suppressing inhibitory Smad. The results in this research demonstrated that the naringin-chitosan substrate produced by biocompatible ozone activation was highly osteoconductive without cytotoxicity.

Surface self-assembled PEGylation of fluoro-based PVDF membranes via hydrophobic-driven copolymer anchoring for ultra-stable biofouling resistance.

Stable biofouling resistance is significant for general filtration requirements, especially for the improvement of membrane lifetime. A systematic group of hyper-brush PEGylated diblock copolymers containing poly(ethylene glycol) methacrylate (PEGMA) and polystyrene (PS) was synthesized using an atom transfer radical polymerization (ATRP) method and varying PEGMA lengths. This study demonstrates the antibiofouling membrane surfaces by self-assembled anchoring PEGylated diblock copolymers of PS-b-PEGMA on the microporous poly(vinylidene fluoride) (PVDF) membrane. Two types of copolymers are used to modify the PVDF surface, one with different PS/PEGMA molar ratios in a range from 0.3 to 2.7 but the same PS molecular weights (MWs, ∼5.7 kDa), the other with different copolymer MWs (∼11.4, 19.9, and 34.1 kDa) but the similar PS/PEGMA ratio (∼1.7 ± 0.2). It was found that the adsorption capacities of diblock copolymers on PVDF membranes decreased as molar mass ratios of PS/PEGMA ratio reduced or molecular weights of PS-b-PEGMA increased because of steric hindrance. The increase in styrene content in copolymer enhanced the stability of polymer anchoring on the membrane, and the increase in PEGMA content enhanced the protein resistance of membranes. The optimum PS/PEGMA ratio was found to be in the range between 1.5 and 2.0 with copolymer MWs above 20.0 kDa for the ultrastable resistance of protein adsorption on the PEGylated PVDF membranes. The PVDF membrane coated with such a diblock copolymer owned excellent biofouling resistance to proteins of BSA and lysozyme as well as bacterium of Escherichia coli and Staphylococcus epidermidis and high stable microfiltration operated with domestic wastewater solution in a membrane bioreactor.

Using crosslinkable diacetylene phospholipids to construct two-dimensional packed beds in supported lipid bilayer separation platforms.

Separating and purifying cell membrane-associated biomolecules has been a challenge owing to their amphiphilic property. Taking these species out of their native lipid membrane environment usually results in biomolecule degradation. One of the new directions is to use supported lipid bilayer (SLB) platforms to separate the membrane species while they are protected in their native environment. Here we used a type of crosslinkable diacetylene phospholipids, diynePC (1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine), as a packed material to create a 'two-dimensional (2D) packed bed' in a SLB platform. After the diynePC SLB is exposed to UV light, some of the diynePC lipids in the SLB can crosslink and the non-crosslinked monomer lipids can be washed away, leaving a 2D porous solid matrix. We incorporated the lipid vesicle deposition method with a microfluidic device to pattern the location of the packed-bed region and the feed region with species to be separated in a SLB platform. Our atomic force microscopy result shows that the nano-scaled structure density of the '2D packed bed' can be tuned by the UV dose applied to the diynePC membrane. When the model membrane biomolecules were forced to transport through the packed-bed region, their concentration front velocities were found to decrease linearly with the UV dose, indicating the successful creation of packed obstacles in these 2D lipid membrane separation platforms.

Interactions between chitosan and cells measured by AFM.

Chitosan, a biocompatible material that has been widely used in bone tissue engineering, is believed to have a high affinity to osteoblastic cells. This research is the first to prove this hypothesis. By using atomic force microscopy (AFM) with a chitosan-modified cantilever, quantitative evaluation of the interforce between chitosan and cells was carried out. A chitosan tip functionalized with Arg-Gly-Asp (RGD) was also used to measure the interforce between RGD-chitosan and osteoblastic cells. This research concluded by examining cell adhesion and spreading of chitosan substrates as further characterization of the interactions between cells and chitosan. The force measured by AFM showed that the interforce between chitosan and osteoblasts was the highest (209 nN). The smallest adhesion force (61.8 nN) appeared between chitosan and muscle fibroblasts, which did not demonstrate any osteoblastic properties. This result proved that there was a significant interaction between chitosan and bone cells, and correlated with the observations of cell attachment and spreading. The technique developed in this research directly quantified the adhesion between chitosan and cells. This is the first study to demonstrate that specific interaction exists between chitosan and osteoblasts.

Immobilization of peptides by ozone activation to promote the osteoconductivity of PLLA substrates.

In this study, ozone treatment was applied to modify poly(L-lactic acid) (PLLA) with the intermediate reagent acryl-N-succinimide (ASI). Then, P15, the peptide related to the attachment and differentiation of osteoblastic cells, was reacted with ASI. Ozone activation successfully created peroxides on the surface of PLLA, which was quantitatively determined by the iodide method. By changing the activation temperature, oxygen flow rate, reaction bath, reaction temperature or addition of ferrous ions, the amount of peroxides was controlled and the effects of these variables were explored in this research. The immobilizations of ASI and P15 were confirmed and quantitative analyzed by FT-IR spectroscopy, elementary analysis and amino-acid analysis. Also, the optimization for ozone activation and ASI grafting were performed. From in vitro experiments, the cultured ROS cells expressed significantly higher ALPase activity and calcium deposition after P15 immobilization. The results demonstrated that the ROS cells expressed osteoblastic phenotypes more significantly when cultured with the substrate modified with P15. In this study, PLLA was successfully modified with P15 by ozone activation and the modification promoted the osteoconductivity of PLLA substrates, which could be helpful in bone tissue engineering.

Monoclonal antibody-based quantitation of poly(ethylene glycol)-derivatized proteins, liposomes, and nanoparticles.

Covalent attachment of poly(ethylene glycol) (PEG) molecules to drugs, proteins, and liposomes is a proven technology for improving their bioavailability, safety, and efficacy. Qualitative and quantitative analysis of PEG-derivatized molecules is important for both drug development and clinical applications. We previously reported the development of a monoclonal IgM antibody (AGP3) to PEG. We now describe a new IgG1 monoclonal antibody (E11) to PEG and show that it can be used in combination with AGP3 to detect and quantify PEG-derivatized molecules. Both antibodies bound the repeating subunits of the PEG backbone and could detect free PEG and PEG-modified proteins by ELISA, immunoblotting, and flow cytometry. Detection sensitivity increased with the length and the number of PEG chains on pegylated molecules. Both antibodies also efficiently accelerated the clearance of a PEG-modified enzyme in vivo. A sandwich ELISA in which E11/AGP3 were employed as the capture/detection antibodies was developed to detect PEG-modified proteins at concentrations as low as 1.2 ng/mL. In addition, the ELISA could also quantify, in the presence of 10% fetal bovine serum, free methoxy-PEG20,000, PEG2,000-quantum dots, and PEG2,000-liposomes at concentrations as low as 20 ng/mL (1.0 nM), 1.4 ng/mL (3.1 pM), and 2.4 ng/mL (3.13 nM phospholipids), respectively. Finally, we show that the sandwich ELISA could accurately measured the in vivo half-life of a PEG-modified enzyme. These antibodies should be generally applicable to the qualitative and quantitative analysis of all PEG-derivatized molecules.