探讨钛表面儿茶酚化聚电解质多层膜对蛋白质的吸附行为，为钛种植体表面改性提供参考。

根据本课题组前期建立的方法，采用脂多糖胺纳米囊泡（NPs）和3，4-二羟苯基丙酸反应制备儿茶酚接枝率为40%的儿茶酚化NPs（cNPs）；采用透明质酸（HA）和多巴胺反应制备儿茶酚接枝率为10%的儿茶酚化透明质酸（cHA）。利用层层自组装技术，以cNPs为引发层、cHA/NPs为阴、阳离子聚电解质，在钛或石英表面构建含3个（cHA/NPs）双层的儿茶酚化聚电解质膜[（基底-cNPs-（cHA/NPs）₃]，记为cPEM。同时以NPs为引发层，构建含（HA/NPs）₃的未儿茶酚化聚电解质膜（PEM）。采用红外光谱分析膜表面化学组成、激光扫描共聚焦显微镜（LSCM）检测膜表面粗糙度，Zeta电位分析仪记录膜表面Zeta电位。选取4种等电点（pI）分别小于、等于、大于生理pH 7.4的蛋白质：牛血清白蛋白（BSA，pI = 4.7）、纤连蛋白（Fn，pI = 5.8）、牛血红蛋白（BHb，pI = 6.8 ~ 7.0）、多聚赖氨酸（PLL，pI = 9.74），以其为模型蛋白，用0.15 mol/L的NaCl配制成1 mg/mL的水溶液。采用石英晶体微天平（QCM）实时动态监测膜表面蛋白吸附情况、原子力显微镜观察样品蛋白吸附前后形貌，LSCM、荧光酶标仪分别分析荧光标记蛋白在膜表面吸附情况，并测试荧光标记蛋白的吸附量。使用SPSS 20.0对数据进行单因素方差分析、SNK和LSD法进行比较，P<0.05认为差异有统计学意义。

LSCM结果表明，石英表面粗糙度为（301 ± 12）nm，组装cPEM、PEM后，表面粗糙度增加，分别为（656 ± 88）、（446 ± 25）nm，组间差异具有统计学意义（F = 66.974，P<0.001）。cPEM组的红外谱图中出现儿茶酚中的苯环（ν_{C = C}）、PEM组的胺基和烷基、多糖中的糖醛酸环等特征峰，证实钛表面引入cPEM和PEM。组装过程中Zeta电位呈锯齿状交替上升，cPEM组表面电位为+22.53 mV，PEM组的表面电位为+17.36 mV。QCM结果表明，生理pH下，所有表面均基本不吸附PLL。在不同表面，BSA和BHb的吸附量cPEM组>PEM组>Ti组。原子力显微镜下可见cPEM、PEM组表面为分布均匀的水滴形海岛状结构，吸附BSA后，表面可见圆盘状结构，且cPEM组量大于PEM组，说明可能BSA在cPEM组表面的吸附量大于PEM组。采用LSCM和荧光酶标仪分析绿色荧光标记蛋白在不同表面的吸附情况，发现在不同种膜表面，同一蛋白吸附量cPEM组>PEM组>Ti组；在同一种膜表面，不同蛋白吸附量BSA>Fn>BHb。

本实验研发的聚电解质多层膜对钛表面进行改性后，能提高蛋白在表面的吸附，儿茶酚化改性则进一步促进这种吸附。蛋白吸附的驱动力可能主要源于静电相互作用和儿茶酚基团对蛋白偶联捕捉作用。

To provide a theoretical basis for surface modification of titanium implants, the protein adsorption of the catechol functionalized polyelectrolyte multilayer film on titanium surface was explored.

According to the methods established in our previous study, catechol functionalized lipopolysaccharide-amine nanopolymersomes (cNPs) grafting rate of 40% was prepared by the reaction of lipopolysaccharide-amine amino nanopolymersomes (NPs) and 3, 4-dihydroxy phenyl propionic acid. The catechol functionalized hyaluronic acid (cHA) grafting rate of 10% was prepared by the reaction of hyaluronic acid (HA) and dopamine. Via layer-by-layer technique and initiated by cNPs, catechol functionalized polyelectrolyte multilayer film (cPEM) was constructed on titanium or quartz surface by using cHA and NPs, containing 3 (cHA/NPs) catechol double polyelectrolyte membrane. Similarly, initiated by NPs layer, polyelectrolyte multilayer film (PEM) without catechol was constructed on titanium or quartz surface by using hyaluronic acid (HA) and NPs. The chemical composition of the film surface was analyzed by infrared spectrum, and the surface roughness was measured by laser scanning confocal microscope (LSCM) . Zeta potential of film surface was recorded with a Zeta potential analyzer. Four kinds of proteins, namely Bovine serum albumin (BSA, pI = 4.7) , Fibronectin (Fn, pI = 5.8) , Bovine hemoglobin (BHb, pI = 6.8-7.0) and Polylysine (PLL, pI = 9.74) , were selected, as their isoelectric points (pI) were less than, equal to and greater than physiological pH 7.4, respectively. Solutions with proteins above (1 mg/mL) were prepared with 0.15 mol/L NaCl. The real-time protein adsorption process was monitored by using quartz crystal microbalance (QCM) . Atomic force microscopy was used to observe the morphology of protein before and after adsorption. The adsorption of fluorescently labeled proteins on the membrane surface was analyzed by LSCM and fluorescence microplate reader, respectively. The data were analyzed by One-Way ANOVA, SNK and LSD tests with SPSS 20.0 software package. The difference was statistically significant with P<0.05.

The results of LSCM showed that the surface roughness of quartz was (301 ± 12) nm, and the surface roughness increased after assembling into cPEM and PEM, which were (656 ± 88) nm and (446 ± 25) nm, respectively. The three groups showed statistical difference (F = 66.974, P<0.001) . The infrared spectrum showed the characteristic peaks of benzene ring (ν_C=C) of cPEM, amino group and alkyl group in PEM, uronic acid ring in polysaccharide. It was proved that cPEM and PEM were introduced into the surface. When the Zeta potential increased in zigzag shape during the film assembly, the surface potential of cPEM was about +22.53 mV and the PEM was about +17.36 mV. QCM showed that under physiological pH, PLL was hardly adsorbed onto all surfaces. The adsorption amount of BSA and BHb on different surfaces was cPEM > PEM > Ti. Atomic force microscopy showed that the surface of cPEM and PEM groups was uniform distribution of water-drop and island-like structure. After adsorption of BSA, the disk structure was observed on the surface, and the amount of cPEM was larger than that of PEM, suggesting that the adsorption amount of BSA on cPEM surface was more than that on PEM. Observation under LSCM and the results from fluorescence microplate reader showed that the adsorption amount of protein on quartz surface increased after being catechol functionalized. On the surface of the same kind of membrane, different proteins showed the capacity of BSA > Fn > BHb.

Protein adsorption on titanium surface can be improved by the modification of polyelectrolyte multilayer film, which can be further promoted by catechol functionalized modification. The driving force of protein adsorption may be mainly due to electrostatic interaction and the catechol catching influence on protein coupling.