Fabrication Of TGF-β2multilayers On Intraocular Iens Surfaces And Its Effect On Preventing Posterior Capsule Opacification

Part I Fabrication of TGF-β2antibody multilayers on acrylic intraocular lens surfaces by layer-by-layer self assembly techniqueObjective:To fabricate TGF-β2antibody (anti-TGF-β2) multilayers on the surfaces of hydrophobic acrylic intraocular lens (10L) by layer-by-layer self assembly (LBL) technique, and test the physico-chemical and molecular biological properties of the surface modified IOL.Methods:Hydrophobic acrylic IOL was pretreated by atmospheric pressure glow discharge plasma, then polyethylenimine (PEI), anti-TGF-(32and poly (L-lysine)(PLL) were sequentially deposited onto IOL surfaces by LBL technique until multilayers of PEI-(anti-TGF-β2/PLL)4-(anti-TGF-β2) was completed. Quartz Crystal Microbalance (QCM) was used to monitor assembly process of anti-TGF-β2multilayers, and test the immunological activity of the deposited anti-TGF-β2on IOL surfaces, as well as evaluate the orientational aspect of anti-TGF-β2immobilized onto IOL. Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS) were used to test the hydrophilicity and surface element changes of the surface modified IOL. Atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM) were used to characterize the surface roughness of the surface modified IOL. The optical and mechanical properties of surface modified IOL were tested on the basis of National Quality Test Standard of IOL (YY-0290). Immunofluorescence combined with laser scanning confocal microscopy (LSCM) was used to test the long-term stability of anti-TGF-β2multilayers and immunological activity of the immobilized anti-TGF-(32on IOL surfaces.Results:QCM showed linear growth of the multilayers when PEI, anti-TGF-(32and PLL sequentially deposited onto IOL surfaces, and confirmed good immunological activity of the immobilized anti-TGF-β2on IOL surfaces. QCM also revealed that mean mass, thickness, and density of anti-TGF-β2monolayer was (77.45±8.00) ng,(1.42±0.15) nm, and (0.48±0.05)μg/cm2, respectively, which confirmed that anti-TGF-β2was deposited on the IOL surfaces with a side-on orientation in the multilayers. WCA revealed an improved hydrophilicity of surface modified IOL. XPS revealed an significantly elevated percentage of Oxygen and Nitrogen element on the surface of surface modified IOL. AFM revealed a slightly elevated roughness of surface modified IOL. FESEM showed no difference in smoothness between surface modified10and control IOL. The optical and mechanical properties of surface modified IOL were proved to be qualified by National Quality Test Standard of IOL. Immunofluorescence combined with LSCM revealed that anti-TGF-β2multilayers could keep stable on IOL surfaces for at least3months, and that the immobilized anti-TGF-β2on surface modified IOL kept good immunological activity, based on the fact that it could combine with applied TGF-β2factor and form immunoflurescence deposite particles.Conclusions:Anti-TGF-β2multilayers was firstly fabricated onto IOL surfaces via atmospheric pressure glow discharge plasma pretreatment and LBL technique in this study. Anti-TGF-β2multilayers showed long-term stability and good immunological activity of antibody on IOL surfaces, while had no side effects on the smoothness, optical and mechanical properties of IOL. Part Ⅱ Prevention of posterior capsule opacification by surface modified acrylic intraocular lens with TGF-β2antibody multilayersObjective:To evaluate the efficacy and safety of surface modified acrylic intraocular lens (IOL) with TGF-β2antibody (anti-TGF-β2) multilayers in preventing posterior capsule opacification (PCO).Methods:Human lens epithelial cells (LECs) were cultured onto surface modified acrylic IOL with anti-TGF-β2multilayers and untreated acrylic IOL, inverted phase contrast microscope was used to compare LECs adhesion and proliferation on IOL surfaces at6h,24h and48h after LECs culture. Scratch test was used to compare LECs migration between surface modified IOL and untreated IOL group. α-SMA was uesed as an epithelial-mysenchymal transition indicator, and tested by immunoflurorescence microscope to compare LECs transition on IOL surfaces between surface modified IOL and untreated IOL group.20coloured rabbits were randomly devided into test group or control group, each group had10rabbits. The right eye of each rabbit was performed phacoemusification combined with implantation of a surface midified IOL (test group) or untreated IOL (control group). Slitlamp microscope was used to examine the anterior chamber inflammation including aqueous flare, cells, and iris synechia on lday,3days, lweek, lmonth and3months after operation. Scores of anterior chamber inflammation were calculated and compared between the test and control group. Standardized digital retroilluminated slitlamp images of posterior capsule was taken and PCO areas were calculated by software designed by ourselves. PCO scores were then compared between between the test and control group. All rabbits were killed and right eyes were enucleated3months after operation, Miyake-Apple posterior photography was used to evaluate PCO severity between the test and control group. IOLs were then extracted from rabbits eyes, tested by immunofluorescence and laser scanning confocal microscope to check the integrity of anti-TGF-β2multilayers on IOL surfaces. Lens capsule was embedded by paraffin and paraffin-cut sections were then dyed by hematoxylin-eosin (HE) staining, LECs proliferation and extracellular matrix deposition above posterior capsule were examined. Immunohistochemistry was used to test a-SMA, Collagen Ⅰ and Collagen Ⅲ expression above posterior capsule.Results:Short-terminhibition of adhesion to LECs on surface modified IOL was found at6h after LECs culture, while no difference in adhesion and proliferation of LECs between surface modified IOL and untreated IOL at24h and48h. LECs migration was dramatically decreased in surface modified IOL group, compared to untreated IOL group, immunoflurorescence microscopy showed much less a-SMA expression in surface modified IOL group, compared to untreated IOL group. In vivo PCO model showed no difference in anterior chamber inflammation was found between the test and control group except for less aqueous flare and cells in the anterior chamber in the test group at1w and1mon follow-up. Both retroillumination slitlamp photography and Miyake-Apple posterior photography revealed less PCO occurred in test group. HE staining revealed a thinner posterior capsule and less LECs above posterior capsule in test group, compared to the control group. Immunohistochemistry revealed less a-SMA and Collagen Ⅰ expression above posterior capsule in test group.Conclusions:Surface modified acrylic IOL with TGF-β2antibody multilayers is effective and safe in the prevetion of PCO.