Self-assembled coating with a metal-polyphenolic network for intraocular lens modification to prevent posterior capsule opacification

Yunqing Wang; Chan Wen; Ruihua Jing; Yunfei Yang; Yazhou Qin; Tiantian Qi; Conghui Hu; Xinshan Bai; Changrui Wu; Cheng Pei

doi:10.1088/1748-605X/ad1c9e

1. Introduction

Cataracts are the most significant contributor to blindness worldwide [1–3], and surgery is the only effective curative method. During surgery, the blurred lens is removed and implanted with a new transparent intraocular lens (IOL) through the residual capsule bag to maintain the IOL in an appropriate position [4–6]. Posterior capsule opacification (PCO) is the most common complication after cataract surgery [7–9]. In the clinic, the only solution for developed PCO is neodymium:YAG (Nd:YAG) laser capsulotomy, which places an extra burden on the patients and the hospital. Additionally, visual acuity cannot be restored to 100% after YAG treatment and may cause other complications, such as ocular edema and iris hemorrhage, leading to internal damage [10–13].

Preventing PCO development is a major task for Ophthalmology researchers, as small incision cataract surgery techniques popularized, the IOL material has involved a few generations, from non-foldable polymethyl methacrylate material to foldable hydrophobic acrylic and hydrophilic acrylic lenses [14]. Research has manifested [15] that hydrophobic acrylic IOL is associated with PCO development in vitro for its greater surface-adhesion and greater lens epithelial cell (LEC) adhesion. On the contrary, in clinic observation, hydrophilic acrylic IOL has a shorter service life owing to the lenses calcification [14]. It is reported that the square-edge intraocular lenses could reduce PCO rates, and most IOL manufacturers adopted this design [16]. However, although the material renewal and shape design updated, the PCO incidence is still 7.1%–22.6%. Clearly, current IOL have a promising PCO inhibition effectiveness, which leaves us a room for improvement in IOL materials.

In recent years, IOL modification has received increasing attention, as IOL is a potential drug device [17, 18] that focuses on the posterior capsule directly and requires no extra procedure or implantation during surgery. Some researchers added a hydrophobic surface [19, 20], and some added a nanocurve to prevent PCO [21]. The most widely used modification of the IOL is fabricating a drug coating, such as straight dipping [22], layer-by-layer [23] and spray coating [24]. Nevertheless, the IOL as an optical lens used in worldwide, the transmittance and economy are the first things we should take into consideration. Therefore, the choice of IOL modification materials is still unsolved.

Tannic acid (TA), a plant polyphenol that is extracted from many plants, deposits many multifunctional coatings onto surfaces and was first illustrated with a simple experiment involving unadulterated tea. The presence of a thin polyphenol coating was revealed by immersing the tea cup in an AgNO3 solution [25]. The unique merit of TA-based coatings is that they combine the functions of TA and metal ions to prepare self-assembled multifunctional surfaces, which we called metal-polyphenolic networks (MPNs) [26]. Based on the hard–soft acid base theory [27], the deprotonated phenolic ligand behaves as a hard Lewis base, combined with a high value of metal iron, which functions as a hard Lewis acid. Taking this theory into consideration, we chose a high value of metal iron, which can increase the MPNs and drug loading on the IOL surface. Fe3+ is commonly used when forming MPNs, but Fe³⁺ is black, which violates the IOL fundamental use as a refracting media to maintain surface transparency; therefore, we chose AlCl₃ combined with TA to form our MPN coating.

The formation of PCO is a complicated process, and inflammatory factors [28] and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway [29, 30] play essential roles in this process. Pterostilbene (PTE) is a natural stilbenoid and a dimethylated analog of resveratrol that is found primarily in blueberries. PTE exhibits a range of pharmacological properties, particularly anti-inflammatory and anticancer effects [31, 32]. In terms of safety, PTE has few, if any, toxic side effects and is classified as low risk. It is safe for use at doses of up to 250 mg d⁻¹, according to human clinical trials [33, 34]. Therefore, we chose PTE loaded in the surface coating to prevent inflammatory cytokines.

ERK1/2 signaling cross-interacts with the canonical TGFβ/Smad and Jagged/Notch signaling pathways, thus regulating EMT in LECs [35], and our previous study showed that the ERK1/2 signaling pathway can be activated by connective tissue growth factor (CTGF), contributing to CTGF-induced EMT-specific protein and ECM synthesis in human LECs (HLECs). AZD0364 is a highly selective ERK1/2 pathway inhibitor that targets the p-P90RSK protein, which was discovered in 2019 [36]. AZD0364 was characterized as a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity and has shown encouraging antitumor activity in preclinical models [37]. Both of these compounds have potential as yet unknown uses in the ophthalmology field and in preventing PCO.

As shown in figure 1, based on the self-assembly technology of TA and AlCl₃ to build an MPN, a multifunctional IOL coating was fabricated on the IOL surface, which incorporates AZD0364 and PTE on the surface. A series of experiments were carried out to characterize the physical, chemical, and biological properties of modified IOL in vitro and in vivo. The purpose of this study is to develop a new type of IOL coating that can efficiently resist the adhesion and inflammation of HLECs on the IOL surface, thus preventing PCO formation.

2. Materials and methods

2.1. Materials

Phosphate-buffered saline (PBS), modified Eagle's medium (MEM), and Dulbecco's MEM (DMEM) were purchased from HyClone (USA), fetal bovine serum (FBS) was purchased from Biological Industries (Israel), and cell culture dishes and cell culture plates were purchased from NEST (China). TA and AlCl3 were purchased from Aladdin Chemistry Co. Ltd (China), PTE was purchased from Meilunbio (China), and AZD0364 was purchased from Selleck (USA). The MTT assay was purchased from Solarbio (China), lipopolysaccharide (LPS) was purchased from Sigma‒Aldrich (USA), the RNA easy animal RNA Isolation kit and Calcein-AM reagent were purchased from Beyotime Biotechnology (China), and CTGF was purchased from PeproTech (USA).

2.2. Cell culture and treatment

The HLEC line SRA01/04 was a gift from the Ophthalmology Institute of the Fourth Military Medical University. HLECs were cultured in MEM containing 10% FBS and 1% PS at 37 °C in a 5% CO₂ incubator to test the antimigratory and antiproliferative activities of different treatments. RAW 264.7 cells were purchased from ATCC (USA) to test PTE anti-inflammatory activity and were cultured in high-glucose DMEM containing 10% FBS and 1% PS as the complete medium at 37 °C in a humidified atmosphere containing 5% CO₂.

2.2.1. Cell viability experiments

An MTT assay was used to evaluate the effects of AZD0364 and PTE on HLEC viability using the SRA01/04 cell line. Briefly, HLECs were seeded into a 96-well cell culture plate at a density of 2 × 10⁴ cells in 100 μl of medium per well 24 h before the experiment. The cells were then treated with different concentrations of AZD0364 (0.25, 0.5, 0.75, 1.0, 2.0, 5.0, 10.0, or 25.0 nmol l⁻¹) and PTE (0, 2.5, 5.0, 12.5, 25.0, or 50.0 μg ml⁻¹). After incubation for an additional 24 h, 100 μl of MEM containing 0.5% MTT was added to each well and then incubated for an additional 4 h. Next, the solutions were removed, 150 μl of DMSO was added to each well, and the plates were shaken using a low-speed vibrator to completely dissolve the crystals. The absorbance was detected at 490 nm using a microplate reader.

MTT assays were performed to determine the effect of PTE on RAW264.7 cells. The cells were seeded into a 96-well cell culture plate at a density of 2 × 10⁴ cells in 100 μl of medium per well 24 h before the experiment and then treated with different concentrations of PTE (0, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, or 25.0 μg ml⁻¹). The operation protocol was the same as described previously.

2.2.2. Anti-inflammatory detection of PTE

A LPS-induced macrophage inflammation model was established to investigate the anti-inflammatory effect of PTE in vitro. LPS causes an acute inflammatory response by activating the TLR4/MyD88 signaling pathway to produce many proinflammatory cytokines [38, 39]. Different concentrations of PTE (8 μg ml⁻¹ and 16 μg ml⁻¹) were incubated with RAW264.7 cells stimulated with LPS (100 ng ml⁻¹) for 24 h. The control group included a blank control group and a positive control group (treated with LPS alone). Total RNA was extracted from the cells using an RNA easy Animal RNA Isolation Kit with a spin column, and the expression of proinflammatory cytokines (IL-6 and IL-1β) was evaluated using real-time PCR (CFX Connect Real-Time System PCR, Bio-Rad, USA). The primers are listed in table S1. The expression levels of the target genes were normalized to GAPDH levels and calculated using the delta-delta Ct method.

2.2.3. Cell migration assays

SRA01/04 cells were used in wound healing and Transwell assays to confirm the effects of AZD0364 on HLEC migration. As described in our previous study [29], CTGF was used to activate the ERK signaling pathway and the migration ability of HLECs. Here, the experimental group was divided into three groups: the control group, CTGF group and CTGF + AZD0364 group.

The wound healing assay used the culture-Insert 2 Well, which provides two cell culture reservoirs with a special sticky and biocompatible surface at the bottom side separated by a 500 μm thick wall. A suspension of 3 × 10⁵ HLECs in 70 μl was seeded. When cells had grown to 90% confluence, the Culture-Insert 2 Well was gently removed with sterile tweezers. The cells in the experimental group were then treated with serum-free medium containing 60 μg l⁻¹ CTGF or 60 μg l⁻¹ CTGF + 2 nmol l⁻¹ AZD0364 for 48 h. Images of migrating cells were captured at 0 h, 24 h and 48 h using an inverted microscope. More than three random fields were selected for assessment, and the wound area at the 0 h time point was used as a reference. Images of wound areas were quantified using ImageJ software (USA).

Transwell experiments were performed in 24-well plates with Corning^®-coated chambers (8 μm pore size). Cells were grown to 70% confluence and serum starved for 24 h. Following detachment with trypsin, cells were washed with PBS and resuspended in serum-free medium, and 2 × 10⁴ cells were added to the upper chamber. Serum-free medium containing 60 μg l⁻¹ CTGF or 60 μg l⁻¹ CTGF + 2 nmol l⁻¹ AZD0364 was added to the bottom wells of the chambers. After 48 h, the cells that had not migrated were removed from the upper surface of the filters using cotton swabs, and the cells that had migrated were fixed and stained with a crystal violet solution. Images were captured at 48 h with an inverted microscope, and more than three random fields were selected for assessment. The number of migrated cells was quantified using ImageJ software (USA).

2.2.4. Western blot

AZD0364, an inhibitor of the ERK/RSK/P90RSK pathway [37], decreases the level of the pP90RSK protein. Thus, Western blot analysis was used to determine the role of AZD0364 in the HLEC line SRA01/04. After two washes with ice-cold PBS, the cells were harvested with 2× Laemmli sample buffer (Bio-Rad, USA). Equal amounts of protein were separated on 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, US) that were blocked with Tris-buffered saline containing Tween^® 20 (TBST) and 1% bovine serum albumin (BSA). PVDF membranes were incubated overnight at 4 °C with the following primary antibodies: ERK1/2 (1:1000, CST, USA), p-ERK1/2 (1:1000, CST, USA), GAPDH (1:2000, Proteintech, China), RSK (1:1000, CST, USA) and p-P90RSK (1:1000, CST, USA). After washing with TBST, the membrane was incubated at room temperature for 1 h with appropriate horseradish peroxidase-conjugated secondary antibodies (1:3000, Bio-Rad, USA). The protein bands were visualized using enhanced chemiluminescence, and the expression levels were normalized to the GAPDH level.

2.3. Fabrication of the self-assembled MPN surface

The concentrations of TA and AlCl₃ are not certain, and confirming an exact concentration is the primary task. Referencing a previous study [40], we first optimized the TA concentration by mixing 2 mg ml⁻¹, 4 mg ml⁻¹, and 6 mg ml⁻¹ TA with 40 mg ml⁻¹ AlCl₃ dissolved in ddH₂O. The IOL was placed in the mixture and centrifuged at 350 rpm for 1 h at room temperature. Next, the AlCl₃ concentration was optimized, and 40 mg ml⁻¹ and 60 mg ml⁻¹ were chosen. Other reaction conditions were the same as described above. After selecting the optimum concentrations of TA and AlCl₃, certain concentrations of AZD0364 and PTE were added to ddH2O in one pot based on the drug release data, and the other reaction conditions were the same as those described above. The MPN based on the self-assembled coating-modified IOL was fabricated named TA (AZD0364/PTE) IOL.

2.4. Evaluation of drug release kinetics

We determined the drug release kinetics as a reference for the initial drug loading concentration. A UV‒vis spectrophotometer (Ultraviolet and Visible Spectrophotometer, UV‒vis Shimadzu, UV-2700, Japan) was used to test the kinetics of drug release from the modified IOLs. The samples were placed in a quartz cuvette with 2 ml of artificial aqueous fluid to simulate the environment of the human eye, and the formula is shown in table S2. Then, at predetermined time points (1, 4, 12, 24, 48, 76, and 144 h), 100 μl of medium was removed and replaced with fresh medium. The absorbance value of the released drugs was measured and used to calculate the corresponding drug concentration from the standard curve. Through dynamic detection and repeated experiments, the release kinetics were obtained.

2.5. Characterizations and optical performance of the IOL surface

The surface micromorphology of the TA (AZD0364/PTE) IOL was observed using a scanning electron microscope (SEM, Carl Zeiss AG, Germany). The chemical composition of IOL surfaces was analyzed using x-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific, Netherlands).

The IOL we use is the commonly used in clinic to prevent PCO [41], its hydrophobic surface has a longer life span than the hydrophilic surface. Herein, all the IOLs were divided into four groups: original IOL, TA IOL, TA (AZD0364) IOL and TA (AZD0364/PTE) IOL to test its wettability and optical performance. The wettability of the IOL surface was measured using a water contact angle analyzer (WCA, Data Physics, Germany) and reported as the value of the static WCA for a water droplet volume of 2 μl. The optical quality of the four groups was tested. Imaging quality was reported as Zernick Polynomias and modulation transfer function (MTF), which were tested using an IOL optical analyzer (NIMO TR0815, China). The transmittance was tested using UV‒vis spectroscopy. The tension resistance of the IOL was evaluated using an electric tension and compression tester (accurate to ±0.01 N), which stretched at a rate between 1 mm min⁻¹ and 6 mm min⁻¹. The machine pulled until the loop broke or separated from the IOL, at which point a value was recorded.

2.6. Anti-adhesive and anti-inflammatory activities of the modified IOL in vitro

The four groups were placed into separate wells of a 48-well plate with a 1 × 10⁶ SRA 01/04 cell suspension and incubated at 37 °C with 5% CO₂ for 24 h to estimate cell adhesion on different IOL surfaces. Afterwards, IOLs were stained with Calcein-AM reagent (green fluorescent dye for labeling live cells) after washing with PBS and photographed using a fluorescence microscope (Olympus, ORCA-03G). Relative fluorescence intensity was quantified using ImageJ.

We tested the anti-inflammatory effects of different IOL surfaces by first administering LPS (100 mg ml⁻¹) to activate RAW 264.7 cells, as described above. The four groups of IOLs were placed into separate wells of a 6-well plate and incubated at 37 °C with 5% CO₂ for 24 h. Then, total RNA was extracted, and proinflammatory cytokine (IL-6 and IL-1β) levels were measured as described previously.

2.7. In vivo evaluation of PCO prevention

All animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guidelines for the Care and Use of Laboratory Animals (NIH publication 85–23, revised 1985). Twelve female Chinese white rabbits (12 weeks old, body weight 2.0–2.5 kg) were used in the experiment, all protocols in this study were performed in accordance with the Ethics Committee of Health Science Center of Xi'an Jiaotong University Approval for Research Involving Animals (No. XJTUAE2023/448). Throughout the observation process, we excluded four rabbits for different reasons, and the specific reasons are shown in table S3. The experimental rabbits were randomly divided into two groups (n = 4) as follows: 1) the Original IOL group and 2) the TA (AZD0364/PTE) IOL group.

All animals underwent extracapsular cataract extraction (ECCE) and IOL implantation in the right eye only, as shown in figure S1. All surgeries were performed by the same doctor (Dr Changrui Wu), and the animals were anaesthetized with an IV injection of 1 ml kg⁻¹ body weight 3% pelltobarbitalum natricum. Pupillary dilation was induced by applying one drop of 0.5% tropicamide (Santen, Japan) three times at intervals of 15 mins. The experimental eyes were dilated and evaluated with a slit lamp photography system (66Vision-Tech Co. Ltd, Suzhou, China) on days 1, 7, 14 and 28 after the operation. For the evaluation of anterior chamber inflammation after surgery, due to the difficulty of counting the number of cells in the anterior chamber of the rabbit eye, we defined four grades to evaluate anterior chamber inflammation according to the number of inflammatory cells and fibrinous exudate, and the exact criteria are shown in table S3. PCO development was evaluated using photographs captured under retroillumination and the EPCO (Evaluation of PCO) system.

The rabbits were sacrificed by injecting an overdose of anesthesia on Day 30 after the operation. At the end of the experiment, the right eyes of the rabbits were enucleated to evaluate PCO development. To evaluate the TA (AZD0364/PTE) IOL biocompatibility, the ocular tissues, including the posterior capsules, corneas, irises, and retinas, were assessed by performing haematoxylin and eosin (H&E) staining using a standard protocol. Posterior capsule tissue slices were collected for immunohistochemical staining and immunofluorescence staining, including α-smooth muscle actin (α-SMA) (Proteintech, China), collagen I (Proteintech, China) and collagen III (Proteintech, China). Aqueous humor was also collected to quantify the levels of interleukin-6 (IL-6) using an ELISA kit (Abcam ab277389). All the tissues were rendered intact and neat structures with no significant damage.

2.8. Statistical analysis

At least three replicates were performed for each experiment, and the results are presented as the means ± standard errors of the means. All statistical analyses were performed with GraphPad Prism 8 software (GraphPad Software, USA). Significant differences were determined using a t test or ANOVA. A p value < 0.05 (*) indicated a statistically significant difference, and p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****) were considered very significant differences.

3. Results and discussions

3.1. In vitro analysis of cell behaviors

3.1.1. Drug concentration screening

To the best of our knowledge, AZD0364 and PTE have never been used in the HLEC line SRA01/04. An MTT kit was used to test the effects of the two drugs on the HLEC line SRA01/04. The results from HLECs shown in figures 2(A) and (B) indicated that cell viability was maintained above 90% after treatment with up to 10.0 nM AZD0364 for 24 h, and cell viability was maintained above 80% after treatment with up to 25 μg ml⁻¹ PTE for 24 h. According to previous studies, both AZD0364 and PTE [33, 37] are drugs with dose-dependent effects. In our study, the MTT assay illustrated that cell viability was reduced in a dose-dependent manner by both AZD0364 treatment and PTE treatment, which also provided a reference for the initial drug dose in the surface coating.

**Figure 2.** Cell viability results and anti-inflammatory effects *in vitro*. (A) Cell viability results of AZD0364 in the SRA01/04 cell line. (B) Cell viability results of pterostilbene in the SRA01/04 cell line. (C) mRNA levels of IL-6 in RAW264.7 cells treated with or without LPS at different doses of pterostilbene. (D) mRNA levels of IL-1β in RAW264.7 cells treated with or without LPS at different doses of pterostilbene.
Download figure:
Standard image High-resolution image

3.1.2. Anti-inflammatory effects of PTE

PTE, a ramification of resveratrol, exerts a strong anti-inflammatory effect [31]. Using a classic LPS-induced macrophage inflammation model, we determined the different concentrations of PTE that mediated the anti-inflammatory effect. PTE (8.0 μg ml⁻¹ and 16.0 μg ml⁻¹) were chosen as the experimental groups. As shown in figures 2(C) and (D), as the concentration of PTE increased, its anti-inflammatory effects increased substantially. The concentrations we used, 8.0 μg ml⁻¹ and 16.0 μg ml⁻¹, exerted significant anti-inflammatory effects compared with the blank control group and the positive control group. The results were consistent with a previous study [42] using PCR technology to quantify the anti-inflammatory effect of PTE and proved that IL-6 and IL1-β levels were decreased in a dose-dependent manner.

3.1.3. AZD0364 inhibits HLEC migration

AZD0364 is a new and highly selective drug that inhibits the ERK1/2 pathway [36, 37, 43] and has not been used in ophthalmology before. Since the ERK1/2 pathway plays an important role in PCO progression [35, 44] and cell migration contributes greatly to PCO progression [5, 6], we wondered whether AZD0364 can prevent HLEC migration by inhibiting the ERK1/2 pathway. Using 60 μg l⁻¹ [29] CTGF to activate the ERK1/2 pathway in the SRA01/04 cell line, we investigated the effect of AZD0364 on HLEC migration using Transwell migration and wound healing assays. The results obtained from the captured images and quantified data in figures 3(A)–(C) show that the number of migrated cells and the migration rate of the CTGF group were significantly higher than those of the control group, and cotreatment with CTGF and AZD0364 significantly reduced the number of migrated cells and the migration rate compared with CTGF treatment and the control group. Our results showed a wider wound in the wound-healing assay and fewer migrated cells in the Transwell assay in the AZD0364 group than in the other two groups, which was in accordance with our assumption, suggesting that AZD0364 significantly suppressed the CTGF-induced migration of HLECs without inducing morphological changes.

**Figure 3.** Effects of AZD0364 on cell migration and Western blot results. (A) Images of the wound healing assay and Transwell assay. (B) Quantitation of the wound healing assay. (C) Quantitation of the Transwell assay. (D) Protein expression of RSK, p-p90RSK, ERK1/2 and p-ERK1/2 in SRA01/04 cells treated with or without CTGF or AZD0364. (E) Quantitation of the Western blot results for p-p90RSK.
Download figure:
Standard image High-resolution image

3.1.4. Protein expression of the ERK signaling pathway in cells treated with AZD0364

AZD0364 restrained SRA01/04 cell migration in our previous experiment, and we wanted to determine the effect of AZD0364 on the levels of proteins in the ERK signaling pathway using Western blotting. As shown in figure 3(D), the p-p90RSK protein levels were decreased significantly after treatment with different concentrations of AZD0364 and 60 μg l⁻¹ CTGF compared with the control group and CTGF group. The quantified results in figure 3(E) indicated that there was a high statistical significance between these groups. The Western blot results were in accordance with a previous study [37], indicating that at the protein level, AZD0364 suppressed the ERK1/2 pathway in HLECs by reducing the level of p-P90RSK protein.

3.2. Optimization of the self-assembly MPN IOL surface

The modified IOL was fabricated to maintain a whole MPN with the minimum TA concentration and a higher iron concentration to increase the drug loading. For uncertain concentrations of TA and AlCl3, the MPN coating must be optimized. As shown in figures 4(A)–(D), we aimed to form a whole smooth MPN coating based on a self-assembled technique on the IOL surface with the minimum concentration of TA to ensure that the IOL transparency was not damaged. Apparently, 6 mg ml⁻¹ TA + 40 mg ml⁻¹ AlCl3 figure 4(C) was the minimum concentration of TA needed to achieve this goal. While maintaining TA at a concentration of 6 mg ml⁻¹, as shown in figure 4(D), an increase in the AlCl3 concentration to 60 mg ml⁻¹ resulted in a higher iron concentration that still generated a smooth surface and the interaction of as much Al3+ with TA as possible to increase the drug loading. To reach the goal of maintaining a whole MPN with the minimum TA concentration and a higher iron concentration to increase the drug loading, we chose 6 mg ml⁻¹ TA + 60 mg ml⁻¹ AlCl3 to fabricate our MPN self-assembled coating. The energy dispersive x-ray spectrometer analysis of the TA (AZD0364/PTE) IOL surface is shown in figure S2. The crevices in figures 4(C) and (D) were caused by the MPN coating covered with Pt. Because of the transparency of the IOL, the IOL surface must be sprayed with Pt when it is scanned by SEM.

**Figure 4.** SEM and drug release kinetics results. (A) SEM image in 2 mg ml⁻¹ TA and 40 mg ml⁻¹ AlCl3. (B) SEM image in 4 mg ml⁻¹ TA and 40 mg ml⁻¹ AlCl3. (C) SEM image in 6 mg ml⁻¹ TA and 40 mg ml⁻¹ AlCl3. (D) SEM image in 6 mg ml⁻¹ TA and 60 mg ml⁻¹ AlCl3. (E) Drug release kinetics of AZD0364. (F) Drug release kinetics of PTE.
Download figure:
Standard image High-resolution image

3.3. Drug release kinetics

The exact concentration of MPN is certain, but the initial drug doses are not certain. Analysis of the drug-release behaviors of the modified IOL can be a reference for the original drug dosage. The cumulative drug release profiles are shown in figures 4(E) and (F). In the first hour, approximately 30% of the two drugs were released; then, in the next 96 h, the drugs were constantly released. Approximately 50% of AZD0364 was released at the end of the experiment compared with the initial solution density, and approximately 40% of PTE was released at the end of the experiment compared with the initial solution density. According to the percentage of the initial drug dose and the final amount of effective drug release and the results of the MTT assay, we chose the original AZD0364 drug dose of 5 nmol l⁻¹ and the original PTE drug dose of 25 μg ml⁻¹ as the original drug doses for our IOL coating.

3.4. Characterization of modified IOL surfaces

Further evidence of TA (AZD0364/PTE) IOL was obtained using XPS, and the binding energy sites of chemical bonding of the Original IOL and TA (AZD0364/PTE) IOL are shown in figures 5(A) and (B). The high-resolution C1s XPS spectra of the TA (AZD0364/PTE) IOL exhibited peaks at 284.5 eV (C=O) that were higher than those of the Original IOL. The higher C=O peak was mainly due to the combined coating of AZD0364 and PTE on the IOL surface, indicating that the two drugs were loaded in the MPN on the IOL surface. In wide-scan spectra, as shown in figure 5(C), the characteristic signal peaks of C 1 s (288.2 eV), O 1 s (536.1 eV), Al 2p (73.8 eV) and Cl 2p (198.1 eV) were clearly observed, and the signals of Al 2p and Cl 2p were naturally attributed to AlCl3 compared with the unmodified group. Al was analyzed by XPS peak differentiation imitation, and the results are shown in figure S3. The XPS results exemplified that the MPN was successfully coated on the IOL surface, and AZD0364 and PTE were loaded onto the MPN.

The surface wettability before and after the modification was studied using WCA analysis. As shown in figure 5(D), the static WCA on the unmodified surface was 71.17 ± 2.94°. After the MPN coating was self-assembled on the surface, the WCA decreased to 59.0 ± 2°, and WCA analysis was conducted to test the surface of the TA (AZD0364) IOL and the TA (AZD0364/PTE) IOL. The WCA on the surface of the TA (AZD0364) IOL was 62.73 ± 3.32°, and the WCA on the surface of the TA (AZD0364/PTE) IOL was 20.8 ± 6.2°. In contrast to the Original IOL, the hydrophobic of TA (AZD0364/PTE) IOL surface increased significantly. The increase in the hydrophilic surface was explained by the formation of the MPN and the additional drugs that were added to the coating, which led to a significantly hydrophilic surface, as we mentioned that a hydrophilic surface has advantages in inhibits cell adhesion, thus a hydrophobic IOL has a hydrophilic surface, can prevent the calcification progress and inhibit cell adhesion to prevent PCO development at the same time.

Regarding the type of drug coating, the priority standard for a qualified IOL is its mechanical and optical performance to maintain its original and primary function as a lens, which refracts the outside images to the retina and retains steadily in the posterior capsule. The tension resistance and imaging quality of the four groups were tested. The image quality results are shown in figures 5(E) and (F). The MTFs of the four groups were not significantly different, and they were greater than 0.43 at 100 Ip mm⁻¹, which was considered within the standard range. The Zernick Polynomias data showed that the four groups were not significantly different, suggesting that the four kinds of IOLs showed no differences in higher-order aberrations, which cannot be rectified through surgery. Transparency, one of the most essential factors for intraocular optics, was quantified by the percentage of visible light transmittance, and the results are shown in figure 5(G). The four groups showed no significant differences in visual light transmittance at wavelengths ranging from 300–1000 nm, indicating the excellent transparencies of the four groups. Tension resistance is the criterion of IOL stability in the intraocular capsule. The results for the IOLs are shown in figure 5(H), and the values for the four IOLs were significantly higher than the standard value of 0.25 N, which shows the strong resistance and stability of the modified IOLs. In summary, the TA (AZD0364/PTE) IOL is a qualified IOL in the mechanical and optical fields.

3.5. Antiadhesive and anti-inflammatory effects of the modified IOL surface in vitro

Cataract surgery procedures may disrupt the blood-aqueous barrier, leading to the entry of a large number of proteins and cells into the anterior chamber [45–47]. This is associated with an inflammatory response; coincidentally, fibrotic disorders are also often associated with inflammatory responses. When protein adsorbs to the IOL surface, proteins can cause monocytes to invade, adhere and differentiate into macrophages, thus aggravating the inflammatory reaction and driving the proliferation and migration of peripheral residual LECs to form PCO [48, 49]. Residual cell adhesion and inflammation in the capsule bag are the main causes of IOL implantation-related postoperative complications. The modified coating was designed to prevent proteins and cells from adhering to the IOL surface. We verified the antiadhesive effect of our modified IOL using calcein-AM reagent to quantify living cells on the IOL surface, and the fluorescence microscopy image results are shown in figures 6(A)–(D). Figure 6(E) shows the scheme of the antiadhesive effect in the in vitro experiment. ImageJ software was used to quantify the images. The results are shown in figure 6(F). The TA (AZD0364) IOL group and TA (AZD0364/PTE) IOL group had fewer cells, and the difference was statistically significant. This result shows that the surface of the TA (AZD0364) IOL and TA (AZD0364/PTE) IOL benefits from the MPN coating to resist LEC attachment, which plays a significant role in triggering postoperative complications.

**Figure 6.** Anti-adhesive and anti-inflammatory activities of TA (AZD0364/PTE) IOL. (A) Calcein AM/PI results on Original IOL. (B) Calcein AM/PI results on TA IOL. (C) Calcein AM/PI results on TA (AZD0364) IOL. (D) Calcein AM/PI results on TA (AZD0364/PTE) IOL. (E) Scheme of cells incubated with modified IOL. (F) Quantification of cell adhesion. (G) Quantification of IL-6 cytokine expression in Raw264.7 cells incubated with TA (AZD0364/PTE) IOL. (H) Quantification of IL1-β cytokine expression on Raw264.7 cells incubated with TA (AZD0364/PTE) IOL.
Download figure:
Standard image High-resolution image

Anti-inflammatory activity can alleviate PCO after cataract surgery, and the classic acute inflammatory model was used to test whether the modified IOLs had the same function. The results in figures 6(G) and (H) show that IL-6 and IL-1β mRNA expression in the drug-coated IOL was significantly decreased, suggesting that TA (AZD0364/PTE) IOL exerted a valid anti-inflammatory effect in vitro. These two experiments proved that the TA (AZD0364/PTE) IOL reached our expectation in vitro.

3.6. Efficiency of PCO prevention in vivo

Importantly, ECCE combined with IOL implantation was performed in female Chinese white rabbit eyes, and several indicators, including inflammation grades and inflammatory factor expression levels, the degree of PCO, the modified surface biocompatibility and EMT labels on the posterior capsule, were assessed on day 30 after surgery. Figure 7(A) shows the image captured by a slit lamp under diffuse illumination. The anterior segment structures were clearly visible, and the inflammation grades were evaluated using these images. Table 1 and figure 7(B) show the quantified results for inflammation grades, which indicate that after 7 d, the Original IOL group had a significantly higher inflammatory reaction than the TA (AZD0364/PTE) IOL group. After 14 and 28 d, the inflammatory response decreased. Figure 7(C) shows the quantified results of IL-6 in rabbit-operated eyes, suggesting that in Original IOL group, the concentration of IL-6 cytokine was significantly higher than that in the TA (AZD0364/PTE) IOL group, indicating that the TA (AZD0364/PTE) IOL exerted a considerable anti-inflammatory effect.

**Figure 7.** (A) Diffuse illumination pictures in operated eyes. (B) Quantification of the anti-inflammatory effect in operated eyes. (C) Quantification of IL-6 in rabbit operated eyes.
Download figure:
Standard image High-resolution image

Table 1. Inflammation grades. A grade of 'none = 0' represented a clear aqueous humor or only a few floating cells in the anterior chamber; a grade of 'mild = 1' indicated that there were a few cells in the anterior chamber; a 'moderate = 2' grade was characterized by many cells and some fibrinous exudate in the anterior chamber; and a 'severe = 3' grade indicated the presence of a very large number of cells and a large amount of fibrinous exudate in the anterior chamber.

Grade	Control-IOL group				TA (AZD0364/PTE) IOL group
Grade	1 d	7 d	14 d	28 d	1 d	7 d	14 d	28 d
None = 0	0	0	2	4	0	0	3	4
Mild = 1	2	0	1	0	3	3	1	0
Moderate = 2	2	1	1	0	1	0	0	0
Severe = 3	0	3	0	0	0	1	0	0

Applying the EPCO system to quantify the degree of posterior capsule opacity, table 1 shows the specific EPCO grading standard. Figure 8(A) shows the image captured by the slit lamp using the back lighting method. The posterior capsule was clearly visible, and PCO development was evaluated using these images. Figure 8(B) shows the EPCO images based on the original images, and the specific evaluation criteria are shown in table 2. Figure 8(C) presents the quantified results for EPCO. After 14 d, the original IOL group developed an obvious PCO compared with the TA (AZD0364/PTE) IOL group, and after 28 d, the differences were more visible. The differences observed at 28 d were statistically significant, and TA (AZD0364/PTE) IOL exerted a substantial antimigration effect. In addition, previous [28, 50, 51] studies suggested that IL-6 contributes to PCO progression. In our study, the original IOL group had more severe inflammation and an obvious PCO, and the inflammatory cytokines and residual LECs both contributed greatly to this progression. TA (AZD0364/PTE) IOL had anti-inflammatory effects and prevented PCO progression. The two complement each other in rabbit eyes, and the final result manifested as clear refracting media.

Table 2. EPCO grading standard.

Grade	Description	Color
0 (none)	Transparent of capsule
1 (minimal)	Mild wrinkling, mild homogenous layers, or sheets of LECs
2 (mild)	Honeycomb patterns of PCO, thicker homogenous layers, and denser fibrosis
3 (moderate)	Classical Elschnigg pearls and of very thick homogenous layers
4 (severe)	Very thick Elschnigg pearls with 'darkening effect' or of any type of severe opacification

To observe the concrete form of PCO and the TA (AZD0364/PTE) IOL biocompatibility, the rabbit eye tissues were stained with hematoxylin and eosin (H&E), we added a normal rabbit ocular tissue in the experiment, to evaluate whether IOL implantation or AZD0364/PTE has any influence on ocular tissue. The results are shown in figure 9. The eye slide was placed in the same position, and the intraocular contents from outside to inside that we can see in the image are cornea anterior chamber iris posterior capsule and PCO. The location of each constructure is shown with arrows in figure 9. Under the inverted microscope, ×4 magnification was used to locate the location of the posterior capsule, and then ×10 magnification was used to observe the specific structures. Few cells were observed on the posterior capsules in the TA (AZD0364/PTE) IOL group; however, in the Original IOL group, a large number of cells accumulated on the capsules. The slide results are consistent with previous slit lamp results, and the TA (AZD0364/PTE) IOL manifests an outstanding effect on preventing PCO. In addition, in terms of biocompatibility, the corneas and retinas from three groups showed no significant differences. The cornea maintained all five full continuous layers without inflammation or oedema. Retinal tissues did not display any structural abnormalities in the two groups, suggesting that TA (AZD0364/PTE) IOLs have great biocompatibility.

**Figure 9.** H&E staining results: (a) H&E results in posterior capsule; (b) H&E results in cornea and retina.
Download figure:
Standard image High-resolution image

The assessment of PCO development in vivo includes subjective and objective assessments. The fibrotic marker α-SMA Collagen I and III are the common proteins we test in PCO research, and detecting their expression can quantify PCO development in the two groups. The immunohistochemical and immunofluorescence images are shown in figures 10 and S4. Figure 10(A) shows capsular wrinkling and Soemmerring's ring, as indicated by the arrow. In the Original IOL group, there was a wide range of brown labeling around the migrating LECs, especially around the capsular wrinkling. At the same time, the brown labeling scope was hardly observed in the TA (AZD0364/PTE) IOL group, likely due to less cell migration on posterior capsules. The immunofluorescence staining results in figure 10(B) show that α-SMA revealed strong positive reactions in the Original IOL where the capsular wrinkling was located and was significantly elevated, but there was almost no obvious expression in the TA (AZD0364/PTE) IOL group. A weak signal was observed where the posterior capsule was located. The same results can be seen in figure S4; because of the low reaction of the antigen product to the rabbit, the whole signal is low and just as a reference. Overall, the TA(AZD0364/PTE) IOL could significantly reduce PCO pathogenesis and the expression of related key proteins.

**Figure 10.** Immunohistochemical and immunofluorescence results: an immunohistochemical results in two groups under 4× magnification. (B) α-SMA expression by immunofluorescence in two groups under 10× magnification.
Download figure:
Standard image High-resolution image

PCO is the most common complication after cataract surgery. To date, laser treatment is the only effective treatment in the clinic, but the patients' acuity is not completely recovered to the same extent as before PCO. A modified IOL can be a perfect solution to this problem depending on its convenience and disposable treatment as a drug delivery device. In our study, the newest differences were due to the use of TA and AlCl3, which are easily obtained and are used for the first time on the IOL surface to fabricate an MPN, and we targeted the two aims, antimigration and anti-inflammation. A study [52] using a mouse cataract surgery model showed that circulating innate immune cells are recruited to the lens post wounding. The recruitment of these immune cells to wounded lens tissue occurred subsequent to the rapid induction of chemoattractant and proinflammatory cytokines [52], they also suggest a link between the inflammatory response to lens wounding and the mechanisms responsible for the induction of fibrosis. Applying PTE to decrease inflammatory cytokines after cataract surgery is a sufficient way to prevent PCO. Residual LECs can be activated by an acute immune response, and residual cells can migrate on the IOL surface. By endowing the IOL with antiadhesive ability, residual LECs can avoid cells crawling on the IOL surface, thus preventing PCO progression. Inactivation of ERK1/2 signaling completely inhibits TGFβ2-induced EMT in LECs [35]. Several studies [36, 37, 43] have shown that AZD0364 is a highly selective ERK1/2 pathway inhibitor that targets the pP90RSK protein, which was discovered in 2019 and has been proven to be highly efficient with a low predicted oral dose in humans. Using AZD0364 could inhibit ERK1/2 pathway signaling; thus, the minimal inflammatory cytokines and residual cells in the posterior capsule could contribute to PCO progression. With this triple safety, our TA (AZD0364/PTE) IOL has sufficient treatment achievement.

Admittedly, this study has limitations. Drug release from the modified IOL still exhibits burst release initially. PCO progression is a continuous process, and our IOL prevented PCO and inflammation at the beginning of the process. This result also reflects that the PCO does not entirely vanish in the rabbit eye. In the future, a constant and smooth drug release behavior is expected, and the relationship between the drug release behavior and therapeutic target should be further studied by slowing the release of the drug and changing the newest target.

4. Conclusions

Here, we developed a promising modified IOL as a new drug delivery system. We chose TA and AlCl₃ to fabricate the classic MPN through a self-assembly method loaded with AZD0364 and PTE using a spinning coating process to fabricate a TA (AZD0364/PTE) IOL. We successfully tested its effect on preventing cell adhesion and inflammation in vitro. Implantation in rabbit eyes revealed a valid effect on preventing PCO progression. In summary, TA (AZD0364/PTE) IOLs have future prospects in preventing PCO, and this technique might be a practical and effective approach in drug device applications. By improving the drug release behavior, we expect that our research will contribute to the development of a new generation of IOLs in the future.

Data availability statement

The data cannot be made publicly available upon publication because they are not available in a format that is sufficiently accessible or reusable by other researchers. The data that support the findings of this study are available upon reasonable request from the authors.

Ackowledgement

Yunqing Wang: generated the idea, writing original draft, designed and performed the experiments, data curation, and formal analysis. Chan Wen: performed the in vivo experiments. Ruihua Jing: generated the idea Yunfei Yang: performed the vivo experiments, Data curation. Yazhou Qin: performed the in vivo experiments. Tiantian Qi: editing the manuscript. Conghui Hu: performed the in vivo experiments. Xinshan Bai: performed the vitro experiments. Changrui Wu supervised the study, generated the idea and edited the manuscript. Cheng Pei: generated the idea, writing—review & editing, supervised the study, revised and finalized the manuscript.

This work was financially supported by Shaanxi Science and Technology Key Project Fund (2021GXLH-Z-027), Xi'an Science and Technology Project Fund (20KYPT0001-07), Shaanxi Provincial Key Research and Development Program （2017SF-236） and Shaanxi Provincial Science and Technology Plan （2023-JC-QN-0861）.

Conflict of interest

The authors declare no competing financial interests.

Self-assembled coating with a metal-polyphenolic network for intraocular lens modification to prevent posterior capsule opacification

Article metrics

Submit

Author e-mails

Author affiliations

Author notes

ORCID iDs

Dates

Peer review information

Abstract

1. Introduction