利用pH敏感性之弱電解質-磺基甜菜鹼共聚物 製備生物惰性表面

Abstract

Nonfouling表面已經被廣泛使用在生醫材料和生物晶片，由於能夠抵抗非專一性的吸附。本研究選用nonfouling材料製備生物惰性表面，將具有雙離子性的磺基甜菜鹼單體(sulfobetaine mathacrylate)分別與兩種pH敏感性弱電解質單體丙烯酸(acrylic acid)和2-氨乙基甲基丙烯酸酯鹽酸鹽(2-aminoethyl methacrylate hydrochloride)，利用自由基聚合法合成無規則性共聚物，藉以靜電作用力吸附於聚電解質修飾的表面，達到抗生物性貼附，而研究的目的是利用此機制去應用其他基材。本研究合成不同比例的磺基甜菜鹼和弱電解質共聚物，分別是0%(聚電解質)、10%、25%、50%、75%、100% 磺基甜菜鹼，主要探討共聚物吸附環境,例如pH和鹽濃度，藉由石英微量天平測量其吸附量的變化，進而影響血清蛋白吸附和纖維母細胞(L929)貼附。由於之前文獻已提出幾乎相同結構的磺基甜菜鹼和丙烯酸共聚物，利用靜電作用與聚丙烯胺鹽酸鹽堆疊共四層，具有抗蛋白質吸附和細胞貼附的能力，但並未細部討論吸附環境對生物性貼附的影響，因此，本研究將進一步探究其中原因。 對於不同比例之磺基甜菜鹼和丙烯酸共聚物，利用共聚物帶負電的特性吸附於三層PEI/PAA (Poly(ethylene imine) /Poly(acrylic acid)之聚電解質表面，當共聚物在酸性環境下吸附，能有效抑制細胞貼附，隨著磺基甜菜鹼比例增加，在50%磺基甜菜鹼之共聚物修飾的表面，最有效抑制蛋白質吸附和細胞貼附。當50%磺基甜菜鹼之共聚物在酸性環境且不同鹽濃度下吸附，皆能抑制細胞貼附，但在高鹽濃度，具有最佳抑制細胞貼附的能力。 對於另一種帶正電之磺基甜菜鹼和2-氨乙基甲基丙烯酸酯鹽酸鹽共聚物，其不同比例共聚物吸附於四層PEI/PSS聚電解質表面，只有75%磺基甜菜鹼之共聚物修飾的表面能有40%抑制細胞貼附，發現75%磺基甜菜鹼之共聚物直接吸附於未經聚電解質修飾的基材表面， 反而更有效減少細胞貼附。此外，當75%磺基甜菜鹼之共聚物在中性環境吸附於低解離度之PAA表面，也能有效減少細胞貼附。因此，本研究利用兩種具有相反電性之弱電解質和磺基甜菜鹼之共聚物，成功製備生物惰性表面。

Nonfouling surface has been widely used in both biomaterials and biosensor, due to its resistancy in non-specific adsorption. In our study, non-fouling materials were used to fabricate bioinert surfaces, where effect on the interface with different pH values and salt concentration was investigated. Sulfobetaine methacrylate (SBMA), with its zwitterionic character, was polymerized into two oppositely charged random copolymers. It was either polymerized with a weak electrolytic acrylic acid, or 2-aminoethyl methacrylate by a conventional free radical polymerization. Through this method, electrostatic interaction may aid in adsorption of the copolymers on the polyelectrolyte-modified surface for obtaining the ideal resistance in biological adsorption. The objective is that the use of polyelectrolytic mechanism is applied on the all substrates. Examinations were done on the copolymers with different content of SBMA at 0 % (polyelectrolyte), 25%, 50%, 75% and 100 %, mainly investigating the effect of pH and salt content on the adsorption of the surface. Methods such as Quartz Crystal Microbalance (QCM) was used to examine the changes on surface adsorption in order to correlate it with the protein adsorption and L929 cell adhesion on the different surfaces through cell culture experiments. From previous studies, 4 layer of P(SBAA-co-AA) with PAH were examined to show resistancy in protein adsorption and cell adhesion, though the influence of surrounding factor on the biological adhesion was not further discussed. Thus, the goal of this study is to explore environmental factors on its influence for cell adhesion. Using the negatively charged character of copolymer, copolymer with different ratio of SBMA and acrylic acid were adsorbed to form surfaces with 3 layers of PEI/PAA. As result, resistancy on cell adhesion was observed under acidic environment, where the resistance increases with increment in SBMA ratio, and the surface with 50% SBMA copolymer showed most resistancy in protein adsorption and cell adhesion without addition of salt. Inhibition of cell adhesion was also demonstrated in acidic environment under different salt concentration, where best cell adhesion resistancy was observed under high salt content. As for the other copolymer of positive-charged Poly(SBMA-co-AEMA) (poly(sulfobetaine methacrylate-co-2-aminoethyl methacrylate)), copolymer with different composition of SBMA were adsorbed to 4 layers of PEI/PSS surface. As result, only 75% SBMA copolymer showed 40% resistancy for cell adhesion, suggesting better resistance effect by the direct adsorption of the copolymer to the TCPS without polyelectrolyte modification as indicated on 75% SBMA copolymer. Nevertheless, great efficiency in decrement of cell adhesion was also observed under neutral environment for the 75% SBMA copolymer to low ionized PAA surface. Thus, through this research, bioinert surface were successfully created by electrostatic interaction to immobilize two types of opposite-charged poly(electrolyte-co-SBMA) copolymer.