Improvement of Uveal and Capsular Biocompatibility of Hydrophobic Acrylic Intraocular Lens by Surface Grafting with 2-Methacryloyloxyethyl Phosphorylcholine-Methacrylic Acid Copolymer

Biocompatibility of intraocular lens (IOL) is critical to vision reconstruction after cataract surgery. Foldable hydrophobic acrylic IOL is vulnerable to the adhesion of extracellular matrix proteins and cells, leading to increased incidence of postoperative inflammation and capsule opacification. To increase IOL biocompatibility, we synthesized a hydrophilic copolymer P(MPC-MAA) and grafted the copolymer onto the surface of IOL through air plasma treatment. X-ray photoelectron spectroscopy, atomic force microscopy and static water contact angle were used to characterize chemical changes, topography and hydrophilicity of the IOL surface, respectively. Quartz crystal microbalance with dissipation (QCM-D) showed that P(MPC-MAA) modified IOLs were resistant to protein adsorption. Moreover, P(MPC-MAA) modification inhibited adhesion and proliferation of lens epithelial cells (LECs) in vitro. To analyze uveal and capsular biocompatibility in vivo, we implanted the P(MPC-MAA) modified IOLs into rabbits after phacoemulsification. P(MPC-MAA) modification significantly reduced postoperative inflammation and anterior capsule opacification (ACO), and did not affect posterior capsule opacification (PCO). Collectively, our study suggests that surface modification by P(MPC-MAA) can significantly improve uveal and capsular biocompatibility of hydrophobic acrylic IOL, which could potentially benefit patients with blood-aqueous barrier damage.


Results
P(MPC-MAA) was synthesized and grafted onto the IOL surface. P(MPC-MAA) copolymer was synthetized via free radical polymerization. Fourier transform infrared (FT-IR) spectroscopy and proton nuclear magnetic resonance ( 1 H NMR) spectroscopy of P(MPC-MAA) are shown in Fig. 1. A transmission absorption peak was observed at 1,720 cm −1 for all of the samples (Fig. 1a), which corresponded to the carbonyl group (C= O) in the PMAA and P(MPC-MAA). However, an absorption peak at 1,080 cm −1 was observed only in the spectra for P(MPC-MAA), which corresponded to the phosphate group (P-O) in the MPC unit 30,31 . The proton signals at 3.2 ppm were observed in the 1 H NMR spectroscopy of P(MPC-MAA) (Fig. 1b), which was attributed to -N + (CH 3 ) 3 of the MPC units 30,32,33 . Collectively, these results demonstrated that the P(MPC-MAA) copolymer was successfully synthesized. The molar fractions of MPC and MAA were 5.7:4.3 calculated from 1 H NMR spectroscopy. The Molecular weight (Mw) and polydispersity index (PDI, Mw/Mn) of P(MPC-MAA) copolymer were 2.3 × 10 5 and 3.02, respectively ( Supplementary Fig. S2).
To construct a protein-resistant IOL surface, P(MPC-MAA) copolymer was grafted onto the IOL surface via plasma technology. Untreated hydrophobic IOL, IOL treated by plasma alone, and P(MPC-MAA) modified IOL are abbreviated as IOL, IOL-Plasma and IOL-P(MPC-MAA), respectively. X-ray photoelectron spectroscopy (XPS) spectra of the binding energy regions of the nitrogen (N) and phosphorous (P) electrons of IOL, IOL-Plasma and IOL-P(MPC-MAA) are shown in Fig. 1c, d. Relative intensities of nitrogen element are listed in Supplementary Table S1. Compared to IOL, a strong and broad N1s peak at approximately 400 eV appeared on IOL-Plasma, indicating that plasma treatment was achieved successfully [32][33][34] . After P(MPC-MAA) grafting, the peaks at 401.96 and 134 eV appeared on IOL-P(MPC-MAA). These peaks corresponded to the -N-(CH 3 ) 3 and phosphate groups attributed to the MPC unit. Meanwhile, the peak at 400.04 eV corresponded to -NH-C(= O). These data indicate that P(MPC-MAA) was successfully grafted onto the IOL surface via the amidation reaction.
Surface characterization of the IOLs. Surface topography affects protein adsorption and subsequent cell behaviors 35 . We first characterized the surface morphology of the samples by atomic force microscopy (AFM) (Fig. 2). IOL had a surface roughness of 0.787 nm, exhibiting a relatively even morphology with a few particles and shallow grooves (Fig. 2a,b). IOL-Plasma had many deep grooves appeared on the surface, and the roughness Scientific RepoRts | 7:40462 | DOI: 10.1038/srep40462 was increased to 4.818 nm (Fig. 2c,d). IOL-P (MPC-MAA) exhibited many wave-like clusters of polymer chains, and the surface roughness was 3.469 nm (Fig. 2e,f).
Next, we measured the water contact angles (WCAs) to characterize the hydrophilicity of the IOL surface ( Fig. 3a,b). The WCA of IOL was 78.9 ± 2.2°, suggesting a hydrophobic surface property. Plasma treatment introduced amino groups onto the IOL surface, and the WCA of IOL-Plasma decreased to 21.8 ± 5.0°, indicating increased surface hydrophilicity. The WCA of IOL-P(MPC-MAA) also decreased to 24.5 ± 3.1°.
To investigate the electrokinetic properties of the samples, we measured the zeta potential of the samples (Fig. 3c). At pH 7.2, the zeta potential of IOL was − 13.6 mv. The average zeta potential of IOL-Plasma increased to − 11.5 mv due to the introduction of positively charged amino groups, while the average zeta potential of IOL-P(MPC-MAA) decreased to − 16.4 mv due to the introduction of the negatively charged carboxylic acid groups.
The optical characteristics of the samples, such as diopter, resolution and transmission properties, demonstrated no significant differences between IOL-P(MPC-MAA) and IOL. The haptics of all groups could endure bending and stretching 2.5 million times with a compression amplitude of + /− 0.25 mm. These optical and physical properties meet the standards of State Food and Drug Administration (SFDA) in China.

IOL-P(MPC-MAA) inhibits protein adsorption.
Protein adsorption is the first phenomenon observed after IOL implantation, and will affect subsequent cell interaction in the material-tissue interface in the following minutes or hours 8 . We used bovine serum albumin (BSA) to monitor protein adsorption on the IOL surface by quartz crystal microbalance with dissipation (QCM-D) analysis (Fig. 3d). BSA adsorption on IOL was 130.8 ± 9.9 ng/cm 2 , which was similar to that on other hydrophobic IOL surfaces we previously reported 36 . Compared to IOL, BSA adsorption on IOL-Plasma decreased to 43.1 ± 8.2 ng/cm 2 , and BSA adsorption on IOL-P(MPC-MAA) further decreased to 14.5 ± 3.1 ng/cm 2 . These data were consistent with the previous reports that increased surface hydrophilicity and introduction of negative charges onto the material surface can significantly decrease protein adsorption 16,17 .

IOL-P(MPC-MAA) inhibits the adhesion and proliferation of lens epithelial cells in vitro.
Cell interaction in the material-tissue interface include an initial phase of cell adhesion followed by subsequent cell proliferation and migration 12 . We used human lens epithelial cell (LEC) line SRA01/04 to evaluate cell behaviors on modified IOLs in vitro. Adhesion of LECs on IOL-P(MPC-MAA) (107.1 ± 5.1/mm 2 ) was significantly decreased compared to that on IOL (201.7 ± 8.1/mm 2 ) and IOL-Plasma (176.7 ± 8.9/mm 2 ) (Fig. 4a,b). However, there was no significant difference between cell adhesion on IOL and IOL-Plasma (P = 0.174). In order to characterize cell proliferation on the IOL surfaces, we incubated LECs on the IOL for 24 and 48 hours and performed a cell viability assay. Compared to IOL and IOL-Plasma, IOL-P(MPC-MAA) significantly decreased cell proliferation after 24 and 48 hours of incubation (Fig. 4c). Collectively, these results demonstrate that P(MPC-MAA) modification significantly increases cell repellency of the IOL surface.

IOL-P(MPC-MAA) reduces postoperative inflammation.
Uveal biocompatibility of the IOL can be assessed by the severity of postoperative inflammation 8 . Breakdown of the blood-aqueous barrier and the foreign body reaction to the IOL implant results in release of protein and cells into the anterior chamber, which can be manifested as anterior chamber flare (ACF) and anterior chamber cell (ACC) respectively 37 . Therefore, we first evaluated ACF and ACC scores as indicators of inflammation (Fig. 5a,b). Similar to the inflammatory responses in human patients after IOL implantation 4,38 , both ACF and ACC scores peaked 1 day postoperatively, and then decreased to the baseline after 4 weeks. Rabbit eyes with implantation of IOL-P(MPC-MAA) had significantly lower ACF and ACC scores 1 day, 4 days, and 1 week after surgery. Persistent inflammation may cause IPS, which refers to the adhesion of the iris to the anterior surface of the IOL or lens capsule. Eight weeks after surgery, slit lamp examination showed that IOL-P(MPC-MAA) implantation group had a significantly lower IPS score than IOL implantation group (Fig. 5c, Supplementary Fig. S3a). We also noticed other postoperative complications occurred in IOL, IOL-Plasma groups, including pupil capture (1 eye in IOL group and 1 eye in IOL-Plasma group), IOL displacement (1 eye in IOL group and 1 eye in IOL-Plasma group) and severe cortical proliferation (1 eye in IOL group) ( Supplementary Fig. S3b). However, no obvious postoperative complication due to inflammation was found in IOL-P(MPC-MAA) implantation group. Intraocular pressure (IOP) values were within normal range in all the groups ( Supplementary Fig. S4).
To further characterize the cellular response to the IOL implants, we extracted the IOLs 8 weeks after surgery and performed scanning electron microscopy (SEM). A large number of amorphous debris and polygonal cells were found adhered to the surfaces of IOL (Fig. 5d,e). However, only a few debris and small round cells were   found on the surfaces of IOL-P(MPC-MAA) (Fig. 5f,g). Collectively, these results indicate that P(MPC-MAA) modification greatly improves uveal biocompatibility of hydrophobic acrylic IOLs in vivo.

IOL-P(MPC-MAA) inhibits anterior capsule opacification. ACO is caused by proliferation and
epithelial-mesenchymal transition (EMT) of the remnant LECs between the inner surface of the anterior capsule and IOL implant 39 . In our study, IOL and IOL-plasma implantation groups developed ACO 2 weeks after surgery (Fig. 6a). After 4 weeks, severe fibrosis occurred on the anterior capsule covering IOL and IOL-plasma optics, leading to anterior capsule shrinkage (black arrows). However, in IOL-P(MPC-MAA) implantation group, ACO developed slowly, and the anterior capsule was relatively transparent 6 weeks after surgery. The ACO score of IOL-P(MPC-MAA) group was significantly lower than that of the IOL and IOL-plasma groups 6 weeks postoperatively (Fig. 6b). Also, histopathological examination showed that multilayer LECs presented underneath the anterior capsule in IOL implantation group, while LECs were arranged regularly in a single layer in IOL-P(MPC-MAA) group (Fig. 6c, black arrowheads). The expression levels of EMT markers fibronectin (Fn) and α -smooth muscle actin (α -SMA) were lower in IOL-P(MPC-MAA) group compared to those in IOL group ( Supplementary Fig. S5). TEM showed that in IOL group, LECs underneath the anterior capsule presented an elongated fibroblast-like appearance with massive ECM deposition ( Supplementary Fig. S6a). However, in IOL-P(MPC-MAA) group, LECs maintained epithelial morphology with a few ECM depositions ( Supplementary Fig. S6b). These results indicate that IOL-P(MPC-MAA) significantly suppressed LECs proliferation and EMT under the anterior capsule and thus inhibited ACO formation.

IOL-P(MPC-MAA) does not affect posterior capsule opacification. PCO is caused by migration of
remnant LECs to the posterior capsule 39 . In our study, slit lamp examination showed that all three groups developed moderate PCO 8 weeks after surgery (Fig. 7a). Fundus examination showed that the optic disk, retinal vessels and choroid vessels could not be clearly seen 8 weeks postoperatively (Supplementary Fig. S7). EPCO 2000 analysis showed no significant difference in all the groups, and Miyake-Apple view analysis showed no difference of CPCO, PPCO and Soemmering's area in all the groups (Fig. 7b). In both IOL and IOL-P(MPC-MAA) groups, LECs exhibited fibroblast-like morphology and were arranged irregularly underneath the posterior capsule (Fig. 7c, black arrowheads) with massive ECM surrounded ( Supplementary Fig. S6c,d). These results suggest that MPC-MAA surface grafting does not affect PCO formation.

Discussion
Optimization of IOL biocompatibility is critical for vision reconstruction after cataract surgery. Uveal biocompatibility is typically important for hydrophobic acrylic IOL because many studies have shown that hydrophobic IOL will cause more inflammatory responses than hydrophilic IOL after implantation 3,40,41 . Both the cataract surgery and the IOL implant trigger the release and adhesion of inflammatory cells, including macrophages and giant cells, onto the IOL surface 8 , leading to a high incidence of IPS and ACO, especially in patients with blood-aqueous barrier damage. MPC has excellent biocompatibility since the phosphorylcholine group on MPC mimics the neutral phospholipids of the cell membrane 14 . Previous studies have shown that grafting MPC onto silicone IOL surface can reduce adhesion of macrophages 20 . However, grafting MPC monomers does not reduce aqueous flare in vivo, possibly due to inadequate negative charges on the material surface 14 . In this study, we synthesized the copolymer P(MPC-MAA) and covalently grafted this copolymer onto the surface of hydrophobic acrylic IOLs. Compared to MPC monomer, P(MPC-MAA) has two advantages. First, P(MPC-MAA) is heavily negatively charged (Fig. 3c). The introduction of negative charges by MAA resulted in a significant reduction of protein adsorption (Fig. 3d) and cell adhesion (Fig. 4). Second, the intermolecular repulsion between MAA in the copolymer could make more MPC diffuse into the aqueous humor, so that the IOL surface was more inert to the surrounding biological system. Therefore, P(MPC-MAA) modification significantly reduced post-operative inflammation after IOL implantation (Fig. 5a-c) and showed excellent biocompatibility in vivo. The remaining anterior LECs (A cells) following cataract surgery have the potential to form fibrous tissue and cause capsular opacification around the capsulorhexis margin, resulting in ACO. Formation of ACO includes two stages: an early stage of LEC proliferation and a late stage involving EMT and ECM production. The LEC proliferation process is regulated by various cytokines and growth factors, such as IL-1, IL-6, transforming growth factor (TGF) and fibroblast growth factor (FGF), which are secreted by residual LECs and inflammatory cells [42][43][44] . Hydrophobic IOL has a higher incidence rate of ACO than hydrophilic IOL, because hydrophobic surfaces tend to attract more remnant LECs and inflammatory cells to adhere and proliferate 5,6,45 . Our results showed that surface modification by P(MPC-MAA) significantly suppressed ACO formation, which could be a direct consequence of decreased LECs adhesion and proliferation. Also suppression LECs and inflammatory cells adhesion may lead to less secretion of cytokines, contributing to a relatively mild inflammatory response in IOL-P(MPC-MAA) group than IOL and IOL-Plasma groups. This is consistent with other studies that hydrophilic surface modifications such as HSM coating 38 or PEG-grafting 15,46 could significantly reduce LECs adhesion and postoperative foreign-body reaction of hydrophobic IOL.
Posterior capsule opacification (PCO), also known as secondary cataract, results from proliferation, migration and EMT of residual LECs across the posterior capsule. In clinical application, hydrophobic IOL has a relatively low PCO rate compared to hydrophilic IOL, as the rapid adhesion of IOL to the posterior capsule can effectively inhibit the migration of LECs 12,47 . In this study, we did not observe a difference in PCO severity between IOL, IOL plasma and IOL-P(MPC-MAA) groups. Similarly, Xiaodan et al. 14 also did not find a change in PCO incidence after grafting MPC on silicone IOL. It is possible that the surface property of IOL may be not as important as the optic configuration in the prevention of PCO. Many studies have shown that a sharp optic edge is the key factor for preventing LECs migration from anterior to the posterior capsule [48][49][50] . Although all the IOLs we used in this study had sharp optic edges, we still observed PCO formation in all the groups 8 weeks after surgery, possibly because rabbit LECs have higher proliferation and migration capacity than human LECs.
Interestingly, we noticed that introduction of amino groups by ammonia plasma treatment alone could also increase surface hydrophilicity, decrease protein adsorption and cell proliferation. However, our previous study showed that the increased hydrophilicity of IOL after plasma treatment can only last for 14 days 51 . On the contrary, covalent immobilization of hydrophilic molecules onto the material surface can greatly weaken the hydrophobic recovery process 20,46 . Here, we showed that although the hydrophilicities of IOL-Plasma and IOL-P(MPC-MAA) were comparable after modification, IOL-P(MPC-MAA) exhibited more protein resistance. Moreover, only IOL-P(MPC-MAA) showed decreased postoperative inflammation and ACO formation in vivo. Therefore, modification by plasma treatment alone is insufficient for the improvement of IOL biocompatibility.
In conclusion, we synthesized a new copolymer P(MPC-MAA) and successfully grafted the copolymer onto the surface of hydrophobic acrylic IOL by plasma technology. IOL-P(MPC-MAA) showed increased surface hydrophilicity and reduced protein adsorption while maintaining the bulk optical and physical properties. IOL-P(MPC-MAA) significantly inhibited LECs adhesion and proliferation in vitro. and suppressed postoperative inflammation and ACO formation in vivo. Overall, these results suggest that P(MPC-MAA) modification improved uveal and capsular biocompatibility of hydrophilic acrylic IOLs. More studies need to be carried out to assess the long-term biocompatibility of IOL-P(MPC-MAA).

Methods
Synthesis and purification of P(MPC-MAA). 0.01 mol MPC (Nanjing Institute of Natural Science and Technology Development, Nanjing, China) and 0.01 mol MAA (Kemiou Chemical Reagent Co., Ltd, Tianjing, China) were dissolved in 20 g of ultrapure water (monomer concentration: 1 mol/L). After argon was introduced for 30 minutes, a sodium sulfite/ammonium sulfate initiator system (ammonium persulfate, sodium sulfite = 1:1.5) was added at a concentration of 0.03 mol/L. The reaction was carried out at 37 °C for 24 hours and was stopped with liquid nitrogen. After introducing anhydrous ethanol, sedimentation was carried out and collected by a suction filter. The sediment was again dissolved in water and dialyzed for 3 days. Then, the sample was freeze-dried for 2 days, and P(MPC-MAA) was obtained. FT-IR spectra was obtained using an FT-IR analyzer (VECTOR-22, Bruker, Germany) with the potassium bromide pressed-disk technique for 32 scans over the 500-4,000 cm −1 range at a resolution of 4.0 cm −1 . The composition of the polymers was determined by 1 H NMR (AVANCE 300, Bruker, Germany) spectral measurements at 400 MHz. Static water contact angle (WCA) measurement. WCA was characterized with a contact angle goniometer (OCA15, Dataphysics, Germany) at 25 °C using distilled water as a reference liquid. A total of 1.00 μ L of reference liquid was pumped onto the surface through a stainless steel needle at a rate of 1.0 μ L/s. The results are mean values calculated from five independent measurements on different points of the films. Zeta potential measurement. Zeta potential was obtained using an electrokinetic analyzer (SurPASS, Anton Paar Surpass, Austria). For the determination of zeta potential, streaming current measurements were performed using an Adjustable Gap Cell (SurPASS, Anton Paar Surpass, Austria). 1.0 mM potassium chloride (KCl) solution was used as the background electrolyte, and 0.1 M potassium hydroxide as well as 0.1 M hydrochloric acid solutions were used to adjust the pH value to 7.2.
Optical and physical characteristics. Diopter, resolution, transmission properties and anti-fatigue resistance of the IOL haptics were assessed according to the standards of State Food and Drug Administration (SFDA) by Medical Equipment Quality Supervision and Inspection Center of Zhejiang Province in China.
BSA adsorption assay. BSA adsorption of the surface was measured by quartz crystal microbalance with dissipation (QCM-D, E4, Q-Sense, Sweden). Briefly, the BSA solution (dissolved in PBS buffer at a concentration of 50 μ g/mL) was introduced onto the samples. After balancing, PBS was introduced again to wash off the non-adsorbed protein. Then, BSA adsorption was obtained from Q-Tools.
Cell adhesion assay. IOLs were placed into a 48-well plate. 300 μ L SRA01/04 cell (human lens epithelial cell) suspension at a concentration of 1 × 10 4 /mL was loaded onto the IOL surface. After incubation for 12 hours, the IOLs were stained with hematoxylin and eosin (HE) and examined with an inverted phase contrast microscope (CKX41, Olympus, Japan). Five fields were selected with one in the central and four in peripheral quadrants at random. Image-Pro Plus 6.0 was used to quantify the number of LECs in each field. At least 5 IOLs in each group were tested.
Cell viability assay. The seeding procedure was the same as described above. After incubation for 24 or 48 hours, 200 μ L Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 20 μ L Cell Counting Kit-8 (CCK-8) reagent were added to IOLs. Wells without IOLs were used as controls. After incubation for 1 hour, the OD values at 450 nm were measured with a microplate reader. The assay was repeated 3 times.
Phacoemulsification and IOL implantation. Twenty-four 1.5 kg male New Zealand albino rabbits were divided into three groups at random. Phacoemulsification was performed on the left eye using the Legacy 20000 System (Alcon Laboratories, Fort Worth, TX, USA). Briefly, a 3.2-mm corneal limbus tunnel incision was made at the 12 o' clock position, followed by a central continuous curvilinear capsulorhexis with 5.5 mm in diameter. Then, the lens materials were extracted and IOL was implanted into the capsule. The tunnel incision was closed with interrupted 10-0 nylon sutures. All surgeries were performed by one surgeon (M.X.W.), who was blind to the group assignment. All experiments were conducted in accordance with the ethical guidelines set forth by the Laboratory Animal Care and Use Committee of the Association for Research in Vision and Ophthalmology (ARVO). The study protocol was reviewed and approved by the Animal Ethics Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University, China.
Follow-up ophthalmic examinations. Digital slit lamp photos were taken by the SL-D7 anterior eye segment analysis system (Topcon Medical Systems, Inc., Tokyo, Japan) at indicated times postoperatively. Intraocular pressure (IOP) was measured by a Tono-Pen tonometer (Reichert Inc., Seefeld, Germany). Fundus images were acquired by a fundus camera (Topcon Medical Systems, Inc., Tokyo, Japan). All examinations were conducted by two researchers who were blind to the group assignment. Serious PCO usually occurred at 8 weeks due to the strong proliferative ability of rabbit LECs, so we defined 8 weeks postoperatively as the endpoint of the ophthalmic examinations.
Inflammation evaluation. Anterior chamber flare (ACF), anterior chamber cell (ACC), iris posterior synechiae (IPS) were scored to evaluate uveal biocompatibility of the IOL as previously described 52 . The grading is summarized in Supplementary Table S2. Postoperative complications, such as corneal edema, glaucoma, IOL displacement, pupil capture, and cortical proliferation, were also recorded. ACO scoring. Six weeks after surgery, ACO was scored from grade 0 to IV based on the severity of the anterior capsule opacity and the contraction of the anterior capsulorhexis opening: Grade 0: clear (transparent) anterior capsule; Grade I: opacification localized at the edge of the capsulorhexis; Grade II: moderate and diffuse opacification, in some cases with areas of capsular folding; Grade III: intense opacification, with areas of capsular folding; Grade IV: constriction (phimosis) of the capsulorhexis opening 45 .
PCO scoring. PCO was quantified by Evaluation of Posterior Capsule Opacification (EPCO) 2000 software or Miyake-Apple view analysis. Standard retroillumination pictures were taken 6 and 8 weeks postoperatively, imported into EPCO 2000 and processed as previously described 53 .
Eight weeks after surgery, the rabbits were euthanatized and the eye balls were enucleated for Miyake-Apple view analysis. Eye balls were sectioned at the equator and gross examinations were performed from the posterior aspect. Miyake-Apple view analysis of PCO was conducted as previously described 54 .