聚丙烯酸酯人工晶状体的表面改性及生物相容性研究
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摘要
目前白内障唯一有效的治愈手段是手术摘除混浊的晶状体并植入人工晶状体(IOL)。生物相容性是临床选择IOL的重要标准，随着现代小切口白内障手术的普及，可折叠型疏水性聚丙烯酸酯IOL成为应用最多的IOL之一。此种IOL由于后表面良好的生物粘性和方形边缘的设计，能有效抑制与后表面相关的并发症后囊膜混浊(PCO)的发生。但缺点是与前表面相关的并发症异物反应和前囊膜混浊(ACO)发生率明显高于亲水性聚丙烯酸酯IOL。为了提高其生物相容性，本课题率先将两种表面改性新技术应用于IOL表面，目的在于提高其表面亲水性和生物相容性，同时不影响IOL本体的光学和机械性能；并为以后的连续性工业化生产提供可行性依据。这两种技术包括大气压下介质阻挡放电(DBD)等离子体技术和静电层层自组装技术，均为近二十年来出现的表面改性新技术，工艺简单环保，价格低廉易于实现连续性工业化生产。
本研究利用X射线光电子能谱(XPS)进行IOL表面化学元素分析，场发射扫描电子显微镜(FESEM)和原子力显微镜(AFM)观察表面形貌，静态水接触角(WCA)评价表面亲水性，以考察改性后IOL表面理化性能的改变。同时利用血小板、巨噬细胞、晶状体上皮细胞(LECs)的体外粘附实验观察细胞粘附数量和形态变化，并进一步行吖啶橙(AO)/碘化丙啶(PI)双染色法分析LECs的凋亡和坏死的形态学变化，以考察改性后IOLs的生物相容性。
首先，本研究采用以氩气为工作气体的大气压下DBD等离子体对疏水性聚丙烯酸酯IOL进行表面改性。表面元素分析证实DBD等离子体处理在IOL表面成功引入了含氮/氧的亲水性极性基团。表面形貌观察显示等离子体的刻蚀作用使IOLs表面粗糙度在纳米级别上有一定程度的增加。等离子体处理过的IOL表面亲水性明显提高，但处理时间超过180 s后WCA趋向恒定，可能与极性基团的引入和表面粗糙度增加这两种作用的平衡有关。等离子体处理在IOL表面产生了极易被洗去的低分子量氧化物(LMWOM)。将处理后的IOL置于干燥空气中一月后发现WCA稳定在75°左右。等离子体处理能明显减少IOL表面血小板的粘附和活化，抑制巨噬细胞的黏附，处理时间大于180 s时能延迟LECs的伸展和增殖，同时保持其上皮细胞表型。
其次，本研究首次在大气压下DBD等离子体处理的基础上，通过静电层层自组装技术在疏水性聚丙烯酸酯IOL表面交替沉积带正电荷的聚赖氨酸(PLL)和带负电荷的透明质酸(HA)以构建聚电解质多层膜(PEMs)。表面化学元素分析提示改性后IOL表面氮/氧元素含量的明显增加，证实了PEMs在IOL表面的成功构建。表面形貌分析显示表面改性后IOL表面无明显损伤，AFM更进一步提示聚电解质均匀平滑地沉积在IOL表面，改性前后表面粗糙度亦无明显变化。PLL/HA多层膜的构建极大地提高了IOL表面亲水性，WCA随着组装层数的改性呈折线型变化证实了静电层层自组装过程的成功进行。PEMs的构建显著减少了血小板的粘附和活化、巨噬细胞的粘附和伸展。并显著抑制了LECs的粘附、伸展和增殖，同时AO/PI双染色试验证实PLL/HA多层膜对LECs曾殖的抑制并非细胞毒作用，多数细胞仍存活。
大气压下DBD等离子体技术和静电层层自组装技术均能有效地对疏水性聚丙烯酸酯IOL进行表面改性。相较于氩气大气压下DBD等离子体，PLL/HA多层膜的构建在提高亲水性、抑制细胞粘附、提高生物相容性方面更有优势。我们推测前表面经上述方法改性，后表面维持方形边缘设计的疏水性聚丙烯酸酯IOL有望减少术后异物反应和ACO发生率，同时保持低PCO发生率。
Up to now,it is well known that surgery with the extraction of natural opacifiedcrystalline lens and then the implantation of IOLs is the only proven therapy for cataract.Biocompatibility is a critical guideline for the selection of commercial IOLs in cataractsurgery.With the prevalence of the modern small incision cataract surgery,thefoldable hydrophobic acrylate IOLs have become the most preferred one by thesurgeons.In terms of biocompatibility,the greatest advantage of these IOLs is theirprominent performance in the PCO prevention,as the sharp-edged and the inherentbioadhesive posterior surface can effectively inhibit the migration and proliferation ofLECs.However,compared with the hydrophilic acrylate IOLs,higher rates of theforeign-body reaction and ACO were also reported for the hydrophobic acrylate IOLs,both of which are related to the anterior IOL surface.We propose that surfacemodification should be an avenue to improve the surface hydrophilicity and thebiocompatibility of the hydrophobic acrylate IOLs,without change in the optical andmechanical properties of the bulk.Two new techniques,which are dielectric barrierdischarge (DBD) plasma at atmospheric pressure and electrostatic layer-by-layerself-assembly,are used for the surface mofication of acrylate IOL.They can work in a flexible,reliable,environmentally friendly,less expensive and continuous way andfavor the industrial production.
To evaluate the physicochemical properties of plasma-treated IOL surfaces,changesin chemical composition,morphology and hydrophilicity were comprehensivelyevaluated by X-ray photoelectron spectroscopy (XPS),field emission scanning electronmicroscopy (FESEM),atomic force microscopy (AFM) and water contact angle (WCA)measurements.Meanwhile,the surface biocompatibility of the untreated andplasma-treated IOLs was compared by the adhesion behaviors ofplatelets,macrophagesand lens epithelial cells (LECs) in vitro.The density of adhered cells was countedunder the optical microscopy and the changes in cell morphology were also observed byenvironmental scanning electron microscope (ESEM).Further,LECs death andapoptosis were evaluated by acridine orange (AO) / propidium iodide (PI) staining.
First,DBD plasma at atmospheric pressure (argon as the discharge gas) was carriedout to modify the acrylate IOLs surfaces.After DBD plasma treatment,thehydrophilicity of the IOL surface is obviously improved.The changes in WCA withtreatment time can be attributed to both the introduction of oxygen-/nitrogen-containingpolar groups and the increase of surface roughness induced by plasma etching effect.The existence of low molecular weight oxidized material (LMWOM) is proved on theplasma-treated IOL which is caused by the chain scission effect of plasma treatment.The plasma-treated IOLs resist the adhesion of platelets and macrophages significantly.The LECs spreading and proliferation are postponed on the IOLs plasma-treated formore than 180 s,with a well maintained epithelial phenotype of LECs.The IOLbiocompatibility is improved after the DBD plasma treatment.
Second,based on the DBD plasma treatment,the polyelectrolyte multilayered films(PEMs) were constructed by the alternate deposition of poly(L-lysine) (PLL) andhyaluronic acid (HA) onto the surface of the acrylate IOLs.XPS analysis confirmedthe immobilization of polyelectrolyte multilayers (PEMs) on the IOLs surfaces. FESEM results showed no change on the IOLs surfaces after the modification.AFMshowed that the polyelectrolytes were uniformly and smoothly deposited,and noobvious changes in the surface roughness were observed.The hydrophilicity wassignificantly improved,and WCA oscillated between the PLL and HA layers,verifyingthe process of alternate deposition.Further,in vitro cell adhesion tests revealed thatthe adhesion and spreading of the platelets,macrophages on the PEMs-modified IOLwere both apparently suppressed.The adhesion,spreading and proliferation of LECswere also suppressed because of the cytostatic effect of PEMs rather than cytotoxicity.
Both techniques of DBD plasma at atmospheric pressure and electrostaticlayer-by-layer self-assembly are effective to modify the hydrophobic acrylate IOLsurfaces.The construction of PLL/HA PEMs seems to excel in the improvement ofsurface hydrophilicity and the suppression of cell adhesion.We speculate that lessforeign-body reaction and slower incidence of anterior capsule opacification (ACO)could be expected after the implantation of surface-mofied IOL.
本研究利用X射线光电子能谱(XPS)进行IOL表面化学元素分析，场发射扫描电子显微镜(FESEM)和原子力显微镜(AFM)观察表面形貌，静态水接触角(WCA)评价表面亲水性，以考察改性后IOL表面理化性能的改变。同时利用血小板、巨噬细胞、晶状体上皮细胞(LECs)的体外粘附实验观察细胞粘附数量和形态变化，并进一步行吖啶橙(AO)/碘化丙啶(PI)双染色法分析LECs的凋亡和坏死的形态学变化，以考察改性后IOLs的生物相容性。
首先，本研究采用以氩气为工作气体的大气压下DBD等离子体对疏水性聚丙烯酸酯IOL进行表面改性。表面元素分析证实DBD等离子体处理在IOL表面成功引入了含氮/氧的亲水性极性基团。表面形貌观察显示等离子体的刻蚀作用使IOLs表面粗糙度在纳米级别上有一定程度的增加。等离子体处理过的IOL表面亲水性明显提高，但处理时间超过180 s后WCA趋向恒定，可能与极性基团的引入和表面粗糙度增加这两种作用的平衡有关。等离子体处理在IOL表面产生了极易被洗去的低分子量氧化物(LMWOM)。将处理后的IOL置于干燥空气中一月后发现WCA稳定在75°左右。等离子体处理能明显减少IOL表面血小板的粘附和活化，抑制巨噬细胞的黏附，处理时间大于180 s时能延迟LECs的伸展和增殖，同时保持其上皮细胞表型。
其次，本研究首次在大气压下DBD等离子体处理的基础上，通过静电层层自组装技术在疏水性聚丙烯酸酯IOL表面交替沉积带正电荷的聚赖氨酸(PLL)和带负电荷的透明质酸(HA)以构建聚电解质多层膜(PEMs)。表面化学元素分析提示改性后IOL表面氮/氧元素含量的明显增加，证实了PEMs在IOL表面的成功构建。表面形貌分析显示表面改性后IOL表面无明显损伤，AFM更进一步提示聚电解质均匀平滑地沉积在IOL表面，改性前后表面粗糙度亦无明显变化。PLL/HA多层膜的构建极大地提高了IOL表面亲水性，WCA随着组装层数的改性呈折线型变化证实了静电层层自组装过程的成功进行。PEMs的构建显著减少了血小板的粘附和活化、巨噬细胞的粘附和伸展。并显著抑制了LECs的粘附、伸展和增殖，同时AO/PI双染色试验证实PLL/HA多层膜对LECs曾殖的抑制并非细胞毒作用，多数细胞仍存活。
大气压下DBD等离子体技术和静电层层自组装技术均能有效地对疏水性聚丙烯酸酯IOL进行表面改性。相较于氩气大气压下DBD等离子体，PLL/HA多层膜的构建在提高亲水性、抑制细胞粘附、提高生物相容性方面更有优势。我们推测前表面经上述方法改性，后表面维持方形边缘设计的疏水性聚丙烯酸酯IOL有望减少术后异物反应和ACO发生率，同时保持低PCO发生率。
Up to now,it is well known that surgery with the extraction of natural opacifiedcrystalline lens and then the implantation of IOLs is the only proven therapy for cataract.Biocompatibility is a critical guideline for the selection of commercial IOLs in cataractsurgery.With the prevalence of the modern small incision cataract surgery,thefoldable hydrophobic acrylate IOLs have become the most preferred one by thesurgeons.In terms of biocompatibility,the greatest advantage of these IOLs is theirprominent performance in the PCO prevention,as the sharp-edged and the inherentbioadhesive posterior surface can effectively inhibit the migration and proliferation ofLECs.However,compared with the hydrophilic acrylate IOLs,higher rates of theforeign-body reaction and ACO were also reported for the hydrophobic acrylate IOLs,both of which are related to the anterior IOL surface.We propose that surfacemodification should be an avenue to improve the surface hydrophilicity and thebiocompatibility of the hydrophobic acrylate IOLs,without change in the optical andmechanical properties of the bulk.Two new techniques,which are dielectric barrierdischarge (DBD) plasma at atmospheric pressure and electrostatic layer-by-layerself-assembly,are used for the surface mofication of acrylate IOL.They can work in a flexible,reliable,environmentally friendly,less expensive and continuous way andfavor the industrial production.
To evaluate the physicochemical properties of plasma-treated IOL surfaces,changesin chemical composition,morphology and hydrophilicity were comprehensivelyevaluated by X-ray photoelectron spectroscopy (XPS),field emission scanning electronmicroscopy (FESEM),atomic force microscopy (AFM) and water contact angle (WCA)measurements.Meanwhile,the surface biocompatibility of the untreated andplasma-treated IOLs was compared by the adhesion behaviors ofplatelets,macrophagesand lens epithelial cells (LECs) in vitro.The density of adhered cells was countedunder the optical microscopy and the changes in cell morphology were also observed byenvironmental scanning electron microscope (ESEM).Further,LECs death andapoptosis were evaluated by acridine orange (AO) / propidium iodide (PI) staining.
First,DBD plasma at atmospheric pressure (argon as the discharge gas) was carriedout to modify the acrylate IOLs surfaces.After DBD plasma treatment,thehydrophilicity of the IOL surface is obviously improved.The changes in WCA withtreatment time can be attributed to both the introduction of oxygen-/nitrogen-containingpolar groups and the increase of surface roughness induced by plasma etching effect.The existence of low molecular weight oxidized material (LMWOM) is proved on theplasma-treated IOL which is caused by the chain scission effect of plasma treatment.The plasma-treated IOLs resist the adhesion of platelets and macrophages significantly.The LECs spreading and proliferation are postponed on the IOLs plasma-treated formore than 180 s,with a well maintained epithelial phenotype of LECs.The IOLbiocompatibility is improved after the DBD plasma treatment.
Second,based on the DBD plasma treatment,the polyelectrolyte multilayered films(PEMs) were constructed by the alternate deposition of poly(L-lysine) (PLL) andhyaluronic acid (HA) onto the surface of the acrylate IOLs.XPS analysis confirmedthe immobilization of polyelectrolyte multilayers (PEMs) on the IOLs surfaces. FESEM results showed no change on the IOLs surfaces after the modification.AFMshowed that the polyelectrolytes were uniformly and smoothly deposited,and noobvious changes in the surface roughness were observed.The hydrophilicity wassignificantly improved,and WCA oscillated between the PLL and HA layers,verifyingthe process of alternate deposition.Further,in vitro cell adhesion tests revealed thatthe adhesion and spreading of the platelets,macrophages on the PEMs-modified IOLwere both apparently suppressed.The adhesion,spreading and proliferation of LECswere also suppressed because of the cytostatic effect of PEMs rather than cytotoxicity.
Both techniques of DBD plasma at atmospheric pressure and electrostaticlayer-by-layer self-assembly are effective to modify the hydrophobic acrylate IOLsurfaces.The construction of PLL/HA PEMs seems to excel in the improvement ofsurface hydrophilicity and the suppression of cell adhesion.We speculate that lessforeign-body reaction and slower incidence of anterior capsule opacification (ACO)could be expected after the implantation of surface-mofied IOL.
引文
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1.Abela-Formanek C,Anion M,Schild G,Schauersberger J,Heinze G,Kruger A.Uveal and capsular biocompatibility of hydrophilic acrylic,hydrophobic acrylic,and silicone intraocular lenses.J Cataract Refract Surg,2002,28(1):50-61
2.Amon M.Biocompatibility of intraocular lenses.J Cataract Refract Surg,2001,27(2):178-179
3.Werner L.Biocompatibility of intraocular lens materials.Curr Opin Ophthalmol,2008,19(1):41-49
4.Learning D V.Practice styles and preferences of ASCRS members-2003 survey.J Cataract Refract Surg,2004,30(4):892-900
5.Findl O,Buehl W,Bauer P,Sycha T.Interventions for preventing posterior capsule opacification.Cochrane Database Syst Rev,2007,(3):CD003738
6.Mullner-Eidenbock A,Amon M,Schauersberger J,Kruger A,Abela C,Petternel V,Zidek T.Cellular reaction on the anterior surface of 4 types of intraocular lenses.J Cataract Refract Surg,2001,27(5):734-740
7.Vock L,Georgopoulos M,Neumayer T,Buehl W,Findl O.Effect of the hydrophilicity of acrylic intraocular lens material and haptic angulation on anterior capsule opacification.Br J Ophthalmol,2007,91(4):476-480
8.Yuen C,Williams R,Batterbury M,Grierson I.Modification of the surface properties of a lens material to influence posterior capsular opacification.Clin Exp Ophthalmol,2006,34(6):568-574
9.Larsson R,Selen G,Bjorklund H,Fagerholm P.Intraocular pmma lenses modified with surface-immobilized heparin-evaluation of biocompatibility in vitro and in vivo.Biomaterials,1989,10(8):511-516
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