Hemocompatible Control of Zwitterionic-bias Interfaces and Membranes

• Main Authors: Sheng-Han Chen, 陳聖翰
• Other Authors: Yung Chang
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/aptu69

博士 === 中原大學 === 化學工程研究所 === 103 === Zwitterionic polymers recently received medical attention to study and design the next generation of hemocompatible materials superior than the typical blood-contacting materials in the advanced healthcare development. Previous works have already demonstrated that the biomimetic inspiration of zwitterionic betaines from the cell membrane nature with original anti-fouling characteristic introducing for the resistance of protein, bacteria, cell, and algae. Importantly, zwitterionic polymers also performed the excellent antithrombogenic properties potentially used as a blood-inert surface. It is generally acknowledgement that the hemocompatibility of betaine polymers might be associated with the formation of bounded water layer on a highly hydrated zwitterionic structure. Zwitterionic polymers containing the pendant groups of phosphobetaine (PB), sulfobetaine (SB), and carboxybetaine (CB) have received growing attention for use in the new generation of blood inert materials because of their excellent inhibition in plasma protein adsorption. This research thesis systematically lays the focus on the study of bioadhesive control based on zwitterionic and zwitterionic-bias interfaces in human blood. Two case studies: (1) Zwitteironic interfaces - For the physical-immobilized surface zwitterionization, random and block zwitterionic copolymers of PB methacrylate or SB methacrylate combined with hydrophobic moieties were synthesized with a controlled polydispersity. Two types of zwitterionic copolymers were physically self-assembled coating onto the surface of polypropylene (PP) flat films for enhancing blood compatibility. For the chemical-immobilized surface zwitterionization, the hemocompatibility of zwitterionic PP fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. (2) Zwitterionic-bias interfaces - Herein, a zwitterionic structure bearing moieties with extra positive or negative charges is introduced to develop a potential biomaterial for blood cell selections. The charge balance and charge-bias level was effectively regulated through the control of the initial negatively and positively charged monomer ratio. The focus was then laid on the assessment of a variety of essential properties in human blood contacting, including plasma protein adsorption, blood cell attachment and related hemocompatibility. In the first part of this thesis, two surface zwitterionization processes are presented, aiming at improving the blood compatibility of PP flat substrate and fibrous membranes. For the physical-immobilized surface zwitterionization, zwitterionic diblock copolymers containing hydrophobic n-butyl methacrylate (BMA) and hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC),[3-(methacryloylamino)propyl] dimethyl(3-sulfopropyl)ammonium hydroxide inner salt (SBAA) and 2-hydroxyethyl methacrylate (HEMA) blocks were prepared with well-controlled molecular weight via the conventional free radical polymerization and addition-fragmentation chain transfer polymerization (RAFT). Poly(2-methacryloyloxyethyl phosphorylcholine)-co-poly(n-butyl methacrylate) (PMPC-co-PBMA), poly(sulfobetaine acrylamide)-co-poly(n-butyl methacrylate) (PSBAA-co-PBMA), and poly(2-hydroxyethyl methacrylate)-co-poly(n-butyl methacrylate) (PHEMA-co-PBMA) copolymers with varying hydrophilic blocks or segments were self-assembly coated onto the surface of PP flat substrate for comparing surface hydrophilicity and protein resistance. In this work, we report a systematic study of how different hydrophilic moieties of PMPC-co-PBMA, PSBAA-co-PBMA, and PHEMA-co-PBMA copolymers affect hemocompatibility of modified PP membranes in human blood solution. The coating layers of anchoring zwitterionic copolymers highly regulate the PP surfaces to resist the adsorption of plasma proteins, the adhesion of platelets, the attachment of leukocytes and whole blood cells. This work suggests that the hemocompatible nature of self-assembled zwitterionic brushes by controlling anchoring copolymer structures gives them great potential in the molecular design of antithrombogenic substrates for use in human blood. For the chemical-immobilized surface zwitterionization, the hemocompatibility of zwitterionic PP fibrous membranes with varying grafting coverage of PSBMA via plasma-induced surface polymerization were studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions were measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from electrical balance of positively- and negatively- charged moieties was well-controlled via atmospheric plasma-induced interfacial zwitterionization and further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrombogenic activities when comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood. In the second part of this thesis, the charge neutral structure of zwitterionic polymer was designed as the basis matrix component. Zwitterionic-bias hydrogels were prepared by the chemical cross-linking reaction with positively charged monomer [2-(Methacryloyloxy)ethyl] trimethylammonium chloride (TMA) and negatively charged monomer 3-sulfopropyl methacrylate potassium salt (SA). To further determine the quantitative analysis of charge distribution in the prepared zwitterionic-bias hydrogels, the degree of charge-bias level (bias) from -100 to +100% is defined as the percentage difference in the relative composition amount of each component (PSA or PTMA) compared to zwitterionic component (PSBMA). A systematic study of how charge-bias variations in poly(SBMA-co-SA) and poly(SBMA-co-TMA) hydrogels affect hemocompatibility in human blood plasma and whole blood was reported. It was found that the protein adsorption, blood cell attachment, and swelling capability of prepared hydrogels can be effectively controlled by regulating the charge balance of the SA/SBMA or TMA/SBMA compositions in the copolymeric gel networks. The results suggest that 0% bias of zwitterionic-bias hydrogels with overall charge neutrality has the best antifouling, anticoagulant, and antihemolytic activities while contacting with blood plasma in human body temperature. It was found that the swelling ratio of poly(SBMA-co-SA) and poly(SBMA-co-TMA) hydrogels increased with the increment of negative and positive bias. The relative fibrinogen adsorption on the zwitterionic-bias surfaces increased as the bias shifted from negatively to positively charged level. However, the observed protein adsorption was independent of bias in the negatively charged level. The hemocompatible test results of platelet, erythrocyte and leukocyte attachment showed that only few platelets and erythrocytes adhered on the negatively charged poly(SBMA-co-SA) surfaces. As the bias is controlled in a range between +8.7% and +18.6%, the poly(SBMA-co-TMA) surfaces showed the preferential attachment of leukocytes and the slight adhesion of platelets and erythrocytes. Furthermore, a maximum number of specific leukocytes attached on the poly(SBMA-co-TMA) surfaces was observed as the bias is controlled in a range between +26.3% to +39.2%. Then, the concept of the zwitterionic-bias formulation was further applied to incorporate with various amine monomers from acrylamide (AA, 1o N), N-isopropylacrylamide (NIPAAm, 2o N), 2-(dimethylamino)ethyl methacrylate (DMAEMA, 3o N) to TMA (4o N) combined with the zwitterionic SBMA grafted on the hydrophobic PP fibrous membranes. The degree of amine-bias level (bias) in a range of 30-40% is precisely controlled and defined as the percentage difference in the relative composition amount of each amine component (1o N, 2o N, 3o N or 4o N) compared to zwitterionic component (PSBMA). The results suggested that the poly(SBMA)-grafted fibrous membranes exhibited the ultrahigh resistance of human plasma protein adsorption and blood cell attachment. But the amount of protein adsorption, platelet adhesion and leukocyte attachment was gradually increased from primary amine to quaternary amine groups. Especially for the quaternary amine of poly(TMA), the positively charged surfaces attached high amount of blood cells to adhere than other amine-grafted surfaces including poly(AA), poly(NIPAAm) and poly(DMAEMA). After the zwitterionic SBMA added into the amine monomers, the poly(S65T35)-grafted surfaces adsorbed a most high number of leukocytes in the fixed bias in a range of 3040%, indication the preferential bioadhesion of specific leukocytes from human blood. The performance of platelet concentration (PC) filtration showed that the modified membranes of poly(S65T35)-grafted PP fibrous membranes depleted 99% leukocytes, but lost 99% of platelets due to the serious platelet adhesion and activation. In the consideration between leukocyte deletion and platelet recovery as a balance, the poly(S80N20)-grafted PP fibrous membranes supported the best leukocyte depletion in PC filtration. This work not only performed the excellent hemocompatibility of zwitterionic polymers, but also demonstrated the controllable charge-bias from zwitterionic neutrality to preferentially capture specific blood cells, which provide a potential possibility in the development of advanced blood filtration systems.