Photothermal and cascade ROS generative multilayer as intraocular lens surface coating for effective posterior capsular opacification inhibition
Renjie Zhang, Qingqing Jia, Jiahao Wang, Wei Li, Jiang Chen, Wenxin Hong, Huiying Huang, Quankui Lin
TL;DR
This paper introduces a new intraocular lens coating that uses gold nanoparticles and metal-organic frameworks to prevent a common post-surgery complication by generating reactive oxygen species and heat.
Contribution
A novel multilayer coating using AuNPs@MIL for IOLs that combines photothermal therapy and ROS generation to inhibit posterior capsular opacification.
Findings
AuNPs@MIL coating effectively inhibits HLEC proliferation and migration in vitro and in vivo.
The coating demonstrates good biocompatibility and optical properties suitable for intraocular use.
NIR-induced photothermal therapy enhances cell apoptosis for long-term PCO prevention.
Abstract
Cataract is the leading cause for blindness. While the intraocular lens (IOL) implantation is the only effective treatment in cataract surgery. Posterior capsule opacification (PCO) is the most frequent complication after cataract surgery, mainly resulting from the proliferation and migration of residual human lens epithelial cells (HLECs) within the capsular bag. While surface modification of IOL has helped slow its progression, long-term effectiveness and biocompatibility remain major challenges. Supraphysiological concentration of reactive oxygen species (ROS) induced by natural enzyme stimulation can effectively inhibit proliferation and migration of HLECs, but the effect is limited by enzyme instability. Gold nanoparticles (AuNPs), a promising candidate with enzyme-mimicking activity and desirable photothermal effects, can promote ROS generation and enhance cell apoptosis. To…
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10- —Zhejiang Provincial Natural Science Foundation10.13039/501100004731
- —Science & Technology Program of Wenzhou
Peer Reviews
No public reviews on file for this paper yet. If you reviewed it on a platform where reviews are public (OpenReview, ICLR, NeurIPS, ICML), you can paste yours below so the community can read it here.
Videos
No videos yet. Explain this paper in a talk, walkthrough, or lecture? Add one.
Taxonomy
TopicsIntraocular Surgery and Lenses · Nanoplatforms for cancer theranostics · Advanced Nanomaterials in Catalysis
Introduction
Cataract is a common blindness causing disease characterized by lens opaque [1]. The only effective clinical treatment is phacoemulsification combined with intraocular lens (IOL) implantation [2, 3]. However, one of the most frequent postoperative complications, posterior capsule opacification (PCO) has a high incidence, which may impact the postoperative visual quality significantly [4–6]. PCO occurs when lens epithelial cells within the capsular bag proliferate, migrate and adhere to the IOL and posterior capsule. As a result, patients not only experience recurrent vision deterioration but also require additional treatment, such as Nd:YAG laser capsulotomy, increasing the medical burden [7–10]. More importantly, surgical intervention is not suitable for all patient groups. For children and individuals with partial or complete lens dislocation, surgery may introduce new complications [11–13]. There is a growing population of patients with heightened expectations for their visual quality [14]. Therefore, the development of effective PCO prevention strategies is critical for enhancing surgical efficacy following cataract surgery.
Surface modification of IOL has emerged as a promising strategy to reduce the incidence of PCO [15, 16]. A systematic investigation of surface modification techniques, such as plasma treatment, chemical grafting, layer by layer (LbL) self-assembly and surface coating, has been carried out in many studies [6, 17, 18]. Among these approaches, the LbL self-assembly technique, driven by electrostatic adsorption, has demonstrated the ability to preserve biomolecular activity and exhibits strong potential for long-term therapeutic applications [19–22]. Our previous work showed that coating IOL with anti-proliferative drugs via LbL self-assembly technique has prevented the development of PCO more effectively. However, nonspecific drug distribution caused unavoidable off-target toxicity, restricting therapeutic application [23]. Recently, the mechanism of reactive oxygen species (ROS)-induced apoptosis has offered a safer approach for PCO prevention. Supraphysiological ROS concentrations can induce cytotoxic effects via mitochondrial dysfunction, genomic instability and membrane peroxidation, culminating in cell apoptosis [24–26]. Our preliminary study has already verified that stimulation-triggered excess ROS can effectively inhibit abnormal proliferation and migration of human lens epithelial cells (HLECs) within the capsular bag. However, the construction and implementation of robust cascade catalytic nanoplatforms in biomedicine are often limited by the inherent instability of natural enzymes or the complex assembly processes between different nanozymes [27–29]. Therefore, developing new strategies that leverage ROS-induced apoptosis to prevent PCO holds great potential and significance.
Inspired by natural cascade processes, biomimetic cascade catalytic strategies based on nanozymes offer a simple and effective way to achieve optimal multimodal synergistic treatment [30]. In this context, gold nanoparticles (AuNPs) have emerged as a promising candidate for promoting apoptosis by producing excess ROS through cascade catalysis. Recent studies have demonstrated that AuNPs exhibit excellent enzyme-mimicking activities under specific conditions, similar to peroxidase, oxidase, catalase, superoxide dismutase and reductase, allowing them to autonomously drive ROS-producing cascade catalysis. Compared to biological enzymes, AuNPs as artificial enzymes offer several advantages, including easy synthesis, tunability, good biocompatibility and low cost, making them have highly promising applications in biomedical and biochemical analyses [31–33]. In addition, AuNPs have been extensively studied as the photothermal agents due to the unique surface plasmon resonance (SPR) properties and enhanced radiation absorption and scattering [34–36]. These advantages make them highly effective in photothermal therapy (PTT), where light can be converted into localized heat through photothermal agents, leading to targeted cellular destruction [37–41]. Moreover, AuNPs, stimulated by near-infrared light for PTT, offer much higher safety than UV light-based PTT. The above studies showed that the cascade catalytic generation of ROS based on AuNPs, together with their inherent photothermal properties, provide potential approaches for safer and more effective PCO prevention.
These IOL surface modification strategies effectively address the challenges of drug release from matrix IOL material. AuNPs possess dual enzyme-mimicking activities, including glucose oxidase-like and peroxidase-like functions. Under aerobic conditions, AuNPs can catalyze the oxidation of glucose to generate hydrogen peroxide (H_2_O_2_), which can be further decomposed into ROS via subsequent catalytic reactions mediated by AuNPs themselves. Notably, in acidic environments, the AuNPs can cooperate with Fe^3+^ to catalytically transform H_2_O_2_ into ·OH via combined oxidase-like nanocatalysis and Fenton-type reactions [42, 43]. However, AuNPs tend to aggregate in solution, which significantly diminishes their catalytic performance and hampers the localized diffusion of reactants and intermediate products. Immobilizing AuNPs onto suitable supports can effectively improve the coupling efficiency among sequential enzyme-mimicking catalytic steps [44, 45]. Metal-organic frameworks (MOFs), characterized by their periodic network structures, high porosity and low density, serve as excellent scaffold materials. Anchoring AuNPs onto the MOF surface markedly enhances the dispersion of AuNPs, thereby facilitating the construction of a stable and efficient cascade catalytic system [46–49]. Furthermore, to improve the stability and immobilization amount, the AuNPs-loaded MIL (AuNPs@MIL) was modified onto IOL through electrostatically driven LbL self-assembly technique. In this process, the biocompatible and positively charged chitosan (CHI) will be chosen as one of the driving forces for LbL self-assembly, which also endows the IOL with good hydrophilic properties, thereby offering additional anti-cell adhesion properties of IOL [50].
In this study, a cascade catalytic platform was constructed by immobilizing AuNPs onto MIL, which was subsequently modified onto IOL via LbL self-assembly technique to achieve desired stability and loading amount. After implantation into the capsular bag, the enzymatic activity of AuNPs within the AuNPs@MIL initiates a cascade catalytic reaction by consuming intra-capsular oxygen and glucose, generating ROS that promote cell apoptosis. The competitive consumption of oxygen and glucose also inhibits cell proliferation. Additionally, because the development of PCO is a dynamic process, PTT can be applied on demand during treatment to further eliminate residual HLECs surrounding the IOL, thereby providing more comprehensive prevention of PCO. The on-demand PTT based on AuNPs further enhances the effectiveness of PCO prevention, providing a feasible approach for long-term, safer and more efficient prevention of PCO (Figure 1). This study offers a novel strategy for the safe, efficient and long-term PCO prevention, with significant implications for clinical treatment.
Preparation of AuNPs@MIL and (AuNPs@MIL/CHI) n-IOL, along with a schematic diagram of cascade catalytic reactions and PTT after intraocular implantation.
Materials and methods
Materials
CHI (with an approximate molecular weight of 300 kDa and 92% deacetylation), branched polyethyleneimine (PEI), sodium borohydride (NaBH_4_), D-(+)-glucose and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich Co., Ltd. (USA). N, N-Dimethyl-formamide (DMF), 2-aminoterephthalic acid (NH_2_-BDC), iron chloride hexahydrate (FeCl_3_·6H_2_O), ascorbic acid, 1,10-phenanthroline and gold chloride trihydrate (HAuCl_4_·3H_2_O) were obtained from Aladdin Co., Ltd. Polyethylene terephthalate (PET) and human lens epithelial cell line (HLEC B3, CRL-11421TM) were sourced from American Type Culture Collection (ATCC). Cell culture medium DMEM/F12 (1:1), fetal bovine serum (FBS), trypsin, penicillin and streptomycin were acquired from Gibco Co., Ltd. Cell counting kit-8 (CCK-8) and phosphate buffer saline (PBS) were bought from Invitrogen Co., Ltd. Calcein/PI cell viability/cytotoxicity assay kit, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), BCA protein assay (BCA) kit, RIPA lysis buffer, Dihydroethidium (DHE), Hoechst 33342 staining solution for live cells and mitochondrial membrane potential detection kit (JC-1) were obtained from Beyotime Biotechnology Co., Ltd. Commercial hydrophobic acrylate foldable IOLs were bought from Suzhou 66 Vision Tech Co., Ltd (66VT^®^, FV- 60A, the diameter of the optical region was 6 mm). Clinical postoperative administration was provided by Zhejiang Eye Hospital (Wenzhou, China). All other chemicals used were of analytical grade and required no further purification.
Characterization
Scanning electron microscopy (SEM) images were acquired using an FEI Quanta 650 at an accelerating voltage of 20 kV. Transmission electron microscopy (TEM) images were obtained using the FEI Talos F200 instrument under 200 kV. X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) patterns were obtained using an X-ray photoelectron spectrometer (Thermo Scientific, K-Alpha) and an X-ray diffractometer (Panalytical, X’Pert PRO MPD), respectively. The hydrodynamic diameter of the samples was measured using dynamic light scattering (DLS) on a Malvern Zetasizer (Nano-ZS90, UK). The LbL assembly was characterized using a quartz crystal microbalance (QSense Explorer, Sweden). Surface wettability analysis was conducted using the dataphysics OCA20 instrument (Germany). Refractive index measurements of the IOL material were performed using an Abbe refractometer (NAR-1T SOLID, ATAGO Corporation). The transmittance of the IOL material was determined using a UV-visible spectrophotometer (UV-1780, Shimadzu, Japan). Optical density (OD) values were recorded using a microplate reader (SpectraMax 190, MD, USA). Microscopic images were obtained using an inverted fluorescence microscope (DMi8, Leica) and upright fluorescence microscope (DM4b, Leica). Cataract surgery was performed using a surgical microscope (OPMI VISU 160, Germany) and an ultrasonic phacoemulsification system (Infiniti, Alcon, USA). Postoperative observations and photography were conducted using a slit lamp microscope (SLM-9E, Chongqing Kanghua Co., Ltd., China). The surface morphology of the IOL was observed using a stereomicroscope (SMZ1500, Nikon, Japan). Retinal electrophysiology was recorded using a retinal electrophysiology system (Reti-Port21 system, Roland Consult, Germany).
Preparation and characterization of AuNPs@MIL nanoparticles
MIL can be synthesized via hydrothermal reaction. In brief, 600 mg FeCl_3_·6H_2_O and 800 mg NH_2_-BDC were first weighed and mixed with 15 mL DMF and sonicated until complete dissolution. The mixture was then poured into a 30 mL Teflon-lined stainless-steel autoclave and reacted in a vacuum oven at 110°C for 24 h. After the reaction, the mixture was centrifuged at 12 000 rpm for 5 min and the supernatant was removed. DMF and anhydrous ethanol were added to the brown precipitate, which was then washed twice and centrifuged to remove the supernatant. Finally, the sediment was dried in a vacuum oven at 70°C for 24 h to obtain MIL-101-NH_2_ (Fe). To obtain AuNPs@MIL, 200 μL 10 mM HAuCl_4_ was added to 30 mL 1 mg/mL MIL aqueous solution, stirred in an ice bath for 2 h for full reaction, centrifuged and washed twice with ultra-pure water, then completely dispersed in 30 mL water. 30 μL 0.1 M NaBH_4_ was added, stirred vigorously for 5 min, centrifuged and washed with ultra-pure water twice, and vacuum dried at 70°C for 12 h to collect the product AuNPs@MIL. The morphology and element distribution of nanoparticles were observed by SEM and TEM; the hydrodynamic diameters and Zeta potentials of MIL and AuNPs@MIL were analyzed by DLS; and the successful loading of AuNPs was characterized by XPS and XRD.
Preparation and characterization of AuNPs@MIL/CHI coating
The prepared AuNPs@MIL carried a negative charge, while the CHI naturally carried a positive charge, enabling their LbL adsorption onto the surface of the amino-functionalized IOL by electrostatic interaction. Briefly, clean PET film with a diameter of 6 mm was soaked in 5 mg/mL PEI solution for 24 h to make it have an amination surface, followed by blown dry with nitrogen and spread on the bottom of the 96-well plate. AuNPs@MIL aqueous solution with a certain concentration was added in and soaked for 15 min. Then the PET film was washed with PBS to remove surface unstable components and dried with nitrogen. Next, 100 μL of the same concentration of CHI solution was added in and soaked for 15 min. Then the PET film was washed with PBS, and dried with nitrogen. These steps were repeated n times to obtain n layers of AuNPs@MIL/CHI surface modified IOL material.
The self-assembly process of AuNPs@MIL and CHI layers was characterized by QCM. Specifically, PBS was firstly perfused for 10 min to achieve baseline stabilization. The gold film surface was then aminated by PEI solution treatment, followed by PBS rinsing. Finally, AuNPs@MIL solution was introduced and allowed to incubate for 30 min. After washing again with PBS, the CHI solution was perfused for 15 min. This process was repeated n times, and the frequency change was monitored by instruments to analyze the thickness of the layer. The changes in surface wetting properties of modified IOL materials were measured using a water contact angle (WCA) instrument. The Abbe refractometer was used to measure the refractive index of modified IOL materials. The absorption value of AuNPs@MIL solution in the visible range was measured by UV-visible spectrophotometer (UV-1780, Shimadzu, Japan), and the characteristic absorption peak was observed. The successful loading of AuNPs@MIL was characterized by measuring the absorption value of the modified IOL materials with different layers of AuNPs@MIL at the same concentration, and the transmittance of IOL materials was observed. The resolution of modified IOL materials was observed by stereomicroscope (SMZ1500, Nikon, Japan).
Cascade catalytic activities evaluation
The cascade catalytic mechanism of AuNPs@MIL proceeds through the following steps. The cascade catalytic process initiates with AuNPs exhibiting glucose oxidase-like activity to catalyze the oxidation of glucose and oxygen, yielding gluconic acid and H_2_O_2_ (Equation 1). This reaction competitively suppresses HLECs proliferation while inducing lactic acid production under the subsequently established hypoxic microenvironment. The resultant acidic byproducts (gluconic acid and lactic acid) collectively acidify the cellular microenvironment. Subsequently, the acidic conditions promote the disintegration of the labile coordination bonds of MIL, releasing Fe^3+^ ions (Equation 2). The Fe^3+^ released from MIL decomposition synergizes with AuNPs to convert H_2_O_2_ into ·OH through a dual mechanism involving the peroxidase-mimicking activity of AuNPs and the Fe^3+^-mediated Fenton-like reaction (Equation 3).
To verify the catalytic cascade activity of the coating, different layers of IOL material modified with varying concentrations of AuNPs@MIL/CHI were placed at the bottom of 24-well plates, and 2 mL of 50 mM glucose solution was added to each well. The plates were incubated at 37°C for 24 h under shaking, and pH changes were monitored using a pH meter. Then, different concentration of FeCl_3_·6H_2_O were reacted with ascorbic acid to obtain the standard curve of Fe^3+^ concentration versus absorbance. The release concentration of Fe^3+^ from the reaction mixture was detected according to the standard. The production of ROS was assessed by TMB colorimetry. IOL materials modified with different concentrations of AuNPs@MIL/CHI and various numbers of layers were placed at the bottom of 96-well plates, and 200 μL of the reaction mixture, including 50 mM glucose and 80 μM TMB, was added to each well. The plates were incubated at 37°C for 2 h under shaking, and absorbance was measured using a UV-visible spectrophotometer after the reaction.
Photothermal performance
The IOL material modified with different concentrations of AuNPs@MIL/CHI and different numbers of layers was placed at the bottom of each well of a 48-well plate, and 200 μL PBS was added to each well to immerse the modified IOL material. The thermal effect of the material was excited using an 808 nm laser transmitter at 3.5 W/cm^2^. The thermal image of each time point was captured and recorded in real-time using an infrared thermal imager (FOTRIC, 220 s). The 808 nm laser transmitter was used to stimulate the thermal effect for 3 min of the material, followed by cooling for 2 min. This process was repeated n times and the thermal image of each time point was captured and recorded in real-time using an infrared thermal imager (FOTRIC, 220 s) to observe the thermal cycling performance of the material.
Cell proliferation suppression assay
The inhibition of cell proliferation of IOL materials coated with different concentrations of AuNPs@MIL/CHI and different layers was evaluated using the CCK-8 reagent. The modified IOL material was first placed on the bottom of a 96-well plate, then HLECs with a density of 8 × 10^3^ cells/well were inoculated on the surface of the material and incubated in a CO_2_ incubator (5% CO_2_, 37°C) for 48 h. The old medium was discarded, the wells were washed with PBS once, and 100 μL of CCK-8 staining working solution was added to each well. The plate was then incubated away from light for 2 h. After the incubation, the IOL material at the bottom of the well was removed, and the absorbance value at 450 nm was detected with a microplate reader. By comparing with the control group, the appropriate concentration and number of layers of AuNPs@MIL/CHI modified IOL materials were screened for subsequent in vivo and in vitro assays.
The modified IOL material was placed at the bottom of a 96-well plate, with HLECs inoculated on its surface at a density of 8 × 10^3^ cells/well. The cells were incubated in a CO_2_ incubator (5% CO_2_, 37°C) for 48 h. Subsequently, part of the modified IOL material was excited with an 808 nm laser transmitter at 3.5 W/cm^2^ for 10 min, followed by further incubation for 2 h. The old culture medium was discarded and 100 μL Calcein/PI Cell Viability/Cytotoxicity staining working solution was added to each well, followed by being incubated away from light for 30 min. Cell images were obtained under an inverted fluorescence microscope after the incubation. Meanwhile, to exclude any cytotoxic effects arising from MIL alone, IOLs modified with MIL at different concentrations were co-incubated with HLECs for 48 h following the same experimental procedures described above. Subsequently, live/dead staining was performed, and representative fluorescence images of the cells were acquired.
Pro-apoptotic effects evaluation
Hoechst 33342 staining working solution and DCFH-DA reagent were used to observe apoptosis. Modified IOL material was placed at the bottom of 96-well plates, with HLECs inoculated on the material at a density of 8 × 10^3^ cells/well and incubated in a CO_2_ incubator (5% CO_2_, 37°C) for 48 h. Afterward, part of the modified IOL material was excited with an 808 nm laser transmitter at 3.5 W/cm^2^ for 10 min, followed by incubation for 2 h. The old culture medium was removed and the wells were washed once with PBS, after which 100 μL of Hoechst 33342 staining working solution was added to each well and incubated for 30 min. Images were then taken with an inverted fluorescence microscope. Similar to the above, when cell confluence reached 90%, the partially modified IOL material was excited with the same power for 10 min and ROS initiator was added to the positive control group, followed by 2 h of incubation. The old culture medium was removed and the wells were washed once with PBS, after which 1 mL of FBS-free culture medium containing 1 μL of DCFH-DA was added to each well. The plate was placed back in the incubator for 30 min. After the incubation, the old medium was discarded and the wells were washed once with PBS, followed by the addition of 100 μL of PBS. Fluorescence microscopy images were obtained in the same manner. Following the same procedure described above, when the cells reached approximately 90% confluence, both groups of IOLs were subjected to NIR irradiation at the same power for 10 min, followed by an additional 4 h of incubation. The culture medium was then removed, and the wells were washed once with PBS. Subsequently, 1 mL of serum-free medium containing 1 μL of JC-1 dye was added. The plate was returned to the incubator and incubated for 30 min. After incubation, the dye-containing medium was discarded, the wells were washed three times with PBS, and finally 100 μL of PBS was added for fluorescence microscopy imaging.
To determine whether ROS generated on the IOL surface could elicit a specific inflammatory response, a Transwell co-culture system was established. MIL- or AuNPs@MIL-modified IOLs were placed in the upper chamber, onto which 5 × 10^4^ HLECs were seeded, while glass coverslips were placed in the lower chamber and seeded with 1 × 10^5^ RAW cells. The co-culture was maintained for 48 h, followed by irradiation with an 808 nm NIR laser at a power density of 3.5 W/cm^2^ for 10 min. The cells were then further incubated for 2 h. After incubation, RAW cells on the coverslips were fixed with 4% paraformaldehyde for 10 min and washed three times with PBS (5 min each). The samples were blocked with 5% BSA for 1 h, and then incubated with a primary antibody against NF-κB p65 (1:500 dilution in PBS containing 3% BSA and 0.1% Triton X-100) at 4°C overnight. After three PBS washes (5 min each), the coverslips were incubated with the corresponding secondary antibody (1:1000 dilution, 300 μL) at room temperature for 1.5 h. Following removal of the secondary antibody and three PBS washes, the samples were further incubated with phalloidin (1:1000 dilution) at room temperature for 1.5 h to stain the actin cytoskeleton. After three additional PBS washes, the nuclei were counterstained with DAPI (20 μL), mounted, and subsequently observed using a confocal laser scanning microscope.
Animal experiments
Animal experiments were carried out under the approval of Animal Protection and Utilization Ethics Committee of Eye Hospital of Wenzhou Medical University (The issue number is YSG24112209). A preliminary examination of rabbit eyes was performed before surgery to rule out eye disease. Nine 4-week-old New Zealand white rabbits were randomly divided into three groups (n = 3). Group A received commercial IOL (unmodified), and group B and C received AuNPs@MIL/CHI modified IOLs. All rabbits underwent combined phacoemulsification and IOL implantation surgery in the right eye. Preoperatively, proparacaine eye drops were used for ocular surface anesthesia, and tropicamide eye drops were administered to induce mydriasis. Postoperatively, compound tobramycin-dexamethasone and levofloxacin eye drops were applied three to five times daily during the first week to prevent intraocular inflammation. The progression of PCO was observed by slit-lamp microscopy on days 1, 7, 14, 21 and 28. The rabbits were humanely euthanized by intravenous injection of pentobarbital sodium at the fifth postoperative week, followed by extraction of the eyeball tissue for fixation. Subsequently, the eyeball was dissected to isolate the cornea, iris, retina and capsular bag. The progress of PCO was evaluated by the images of each group of capsular bags taken by a stereo microscopy. The partial tissue was ground to evaluate the cascade catalytic activity of the modified IOL in vivo. Frozen and paraffin sections of the remaining tissue, including the cornea, iris, retina and capsular bag, were made to evaluate intraocular biocompatibility and the progression of PCO.
Statistical analysis
Each sample was repeated three times independently and all results were recorded as mean ± standard deviation. The data were analyzed using the Origin 2021 software’s independent samples test and one way ANOVA results. P > 0.05 was considered as no significant difference (P > 0.05, ns), P < 0.05 was considered as a significant difference (P < 0.05, *), P < 0.01 was considered as a significant difference (P < 0.01, **) and P < 0.001 was considered as a significant difference (P < 0.001, ***).
Results
Preparation and characterization of AuNPs@MIL
MIL was synthesized according to the literature method, and AuNPs@MIL was prepared by in-situ reduction of HAuCl_4_ [51, 52]. As shown in Figure 2A, the MIL could be observed under TEM with a typical regular octahedral structure. The morphology and particle size of nanoparticles did not change after in-situ reduction of HAuCl_4_, with only a marginal increase in surface roughness observed. This was also illustrated by the hydrodynamic diameter changes measured by DLS, as shown in Figure 2C. These results demonstrate that the in situ chemical deposition strategy serves as a robust synthetic approach for achieving well-dispersed and highly stable MIL structures. Element mapping of AuNPs@MIL in Figure 2B showed even distribution of C, N, O, Fe and Au elements, preliminarily illustrating the successful loading of AuNPs. As depicted in Figure 2D, Malvern Zetasizer measurements revealed that the MIL originally possessed a positive charge, whereas AuNPs@MIL exhibited a negative charge, indicating charge reversal may be due to the incorporation of negatively charged AuNPs. In addition, as shown in the XPS survey spectrum in Figure S3A, the characteristic peaks of both Fe and Au can be reliably detected. The Fe 2p_1/2_ and Fe 2p_3/2_ signals in Figure S3B further confirm the presence of Fe³^+^ within the MIL framework. Moreover, the XPS spectrum presented in Figure 2E displays Au 4f_7/2_ and Au 4f_5/2_ peaks at 84.1 eV and 87.9 eV, respectively, verifying the successful incorporation of AuNPs and the in situ reduction of Au^3+^ to Au^0^ by NaBH_4_. The XRD pattern in Figure 2F shows a noticeable attenuation of the characteristic MIL peaks in AuNPs@MIL, accompanied by the emergence of diffraction peaks corresponding to the (111) and (200) planes of AuNPs. These features collectively confirm the successful loading of AuNPs onto the MIL framework.
Preparation and characterization of AuNPs@MIL. (A) TEM images of MIL and AuNPs@MIL. (B) The element mapping images of C, N, O, P, Fe and Au. (C) Hydrodynamic diameters of MIL and AuNPs@MIL measured by DLS. (D) Zeta potential of MIL and AuNPs@MIL measured by DLS. (E) The XPS spectra of AuNPs@MIL. (F) XRD patterns of MIL and AuNPs@MIL.
Preparation and characterization of AuNPs@MIL/CHI coating
The surface assembly process of materials was characterized using quartz crystal microbalance with dissipation (QCM-D). The results in Figure 3A demonstrated a progressive decrease in resonance frequency with sequential deposition of AuNPs@MIL and CHI layers, confirming the successful formation of multilayer films. With the increase of the number of assembly layers, the UV absorption peak of the AuNPs@MIL/CHI modified IOL material also increased (Figure 3B), preliminarily indicating the successful modification of AuNPs@MIL/CHI onto IOL material via LbL technology. Figure 3C and D showed that the surface WCA of the modified IOL material progressively decreases with an increase of AuNPs@MIL concentration and the number of assembled layers, further confirming the successful modification of AuNPs@MIL/CHI. When the assembly reached five layers, the IOL surface exhibited enhanced hydrophilicity, which may facilitate its anti-proliferative properties. All these results show the successful modification of the AuNPs@MIL/CHI multilayer films.
Preparation and characterization of AuNPs@MIL/CHI coating. (A) The assembly process of AuNPs@MIL/CHI multilayer films characterized by QCM (‘V’ denotes the overtone number). (B) The changes in absorbance of IOL material with different bilayers of AuNPs@MIL/CHI multilayer film modification. (C) The changes in WCA of IOL material with different bilayers of AuNPs@MIL/CHI multilayer film modification. (D) The changes in WCA of IOL material after modification with five layers containing different concentrations of AuNPs.
Given the critical importance of optical properties for IOL, the comprehensive characterization of its optical performance was conducted in this study. As shown in Figure 4A, the refractive index of the IOL material remains constant at approximately 1.495 after modification with different concentrations of AuNPs@MIL/CHI multilayer films. Figure 4B and C showed the optical transmittance of IOL materials modified with AuNPs@MIL/CHI exhibited a slight decrease, which progressively declined with increasing layer number and AuNPs@MIL/CHI concentration, yet remained above 90% in all groups. Figure 4D showed that the IOL material modified with AuNPs@MIL/CHI multilayer film exhibited high resolution, similar to the unmodified IOL material. All the above results indicate that the IOL materials modified with AuNPs@MIL/CHI possess satisfactory optical properties and exhibit potential for intraocular implantation applications.
Optical properties of AuNPs@MIL multilayer film. (A) The changes in refractive index of IOL material with AuNPs@MIL/CHI multilayer film modification. (B) Optical transmittance variations of IOL materials modified with five AuNPs@MIL/CHI layers at different concentrations. (C) Optical transmittance variations of IOL materials modified with different numbers of AuNPs@MIL/CHI layers at the same concentration (200 μg/mL). (D) The resolution of IOL material with AuNPs@MIL/CHI multilayer film modification.
Catalytic performance in vitro
To confirm the occurrence of the cascade catalytic reaction, we monitored its key products, including H^+^, ROS and the release of Fe^3+^. The modified IOL material was immersed in glucose solution and co-incubated for 24 h. As shown in Figure 5A, a transition from neutral to acidic pH was observed in the glucose solution, with the pH decreasing more significantly as both the coating concentration and the number of layers increased. This result preliminarily shows the activation of the cascade catalytic reaction and suggests that the cascade catalytic capacity of the coating is enhanced with both the increased concentration of AuNPs@MIL and the number of assembled layers. The production of ROS was further assessed by TMB colorimetry, which can be oxidized from colorless to yellow. As shown in Figure 5B, AuNPs@MIL catalyzed the generation of ROS from glucose via a cascade reaction, and the produced ROS efficiently facilitated the oxidation of TMB and a colorimetric change, further demonstrating the successful implementation of the cascade catalytic process. More importantly, as shown in Figure S5A and B, ROS generation exhibited a clear dependence on both the concentration of AuNPs@MIL and the number of modification layers. Fe^3+^ is a key ion in the cascade catalytic process, further promoting the reaction through the Fenton reaction. To further confirm the cascade catalytic reaction, the released Fe^3+^ was detected. As shown in Figure 5C and D, when the number of layers of the coating was kept constant, the release of Fe^3+^ increased with the concentration of AuNPs@MIL. Similarly, when the concentration of AuNPs@MIL was fixed, the release of Fe^3+^ increased with the number of assembled layers. These results confirm the occurrence of the cascade catalytic reaction of AuNPs@MIL-modified IOL material, with the AuNPs@MIL loading capacity being a key factor in enhancing the cascade catalytic activity.
In vitro cascade catalytic activity evaluation of AuNPs@MIL coatings. (A) The changes in pH of modified IOL material after reaction with glucose solutions. (B) The generation of ROS evaluation based on TMB oxidation assay. (C and D) The releases of Fe3+ from modified IOL material after reaction with glucose solutions.
In vitro photothermal properties studies
Photothermal performances of the modified IOL material were investigated upon 808 nm laser irradiation. As shown in Figure 6A and Figure S4, when the IOLs were uniformly modified with AuNPs@MIL at a concentration of 200 μg/mL, the photothermal performance of the engineered IOLs exhibited a clear layer-dependent behavior. Notably, the IOL modified with five layers of 200 μg/mL AuNPs@MIL reached a surface temperature of approximately 45°C after 5 min of NIR irradiation, which is sufficient to effectively induce apoptosis in HLECs. Figure 6B shows that when the number of modified layers was fixed at 5 layers, the temperature of the modified IOL material increased in a concentration-dependent manner, suggesting that AuNPs@MIL retained excellent photothermal conversion ability during LbL self-assembly. To more intuitively observe the temperature changes, the temperature in each group was recorded. As shown in Figure 6C, the temperature of IOL material modified with 200 μg/mL and five bilayers AuNPs@MIL/CHI could rise to about 42°C within 5 min under NIR irradiation. The photothermal recirculation stability was also investigated. The result in Figure 6D showed that during three laser-on/off cycles of irradiation, there was no obvious change of the maximum temperature, indicating the AuNPs@MIL/CHI possess good reusability. All these results demonstrate the AuNPs@MIL/CHI multilayer modified IOL material has good and recyclable photothermal effect, which holds promise for the long-term treatment of PCO.
In vitro photothermal properties of AuNPs@MIL coatings. (A) The photothermal effect with increasing layers of AuNPs@MIL while keeping the concentration of AuNPs@MIL constant. (B) The photothermal effect with varying concentrations of AuNPs@MIL while keeping the layer number of AuNPs@MIL constant. (C) The temperature values and statistical analysis of modified IOL materials after NIR irradiation in (B). (D) Thermal stability and recyclability of the modified IOL materials measurement undergoing continuous three laser on/off cycles (808 nm, 3.5 W/cm2).
In vitro cell suppression evaluation
After cataract surgery, residual HLECs within the capsular bag proliferate, migrate and differentiate on the IOL surface, which constitutes the primary driving force for the onset and progression of PCO. To rigorously evaluate the HLECs‐clearing capability of AuNPs@MIL/CHI‐modified IOLs, it was first necessary to exclude any effects attributable to MIL alone. As shown in Figure S8, IOLs modified with MIL at concentrations ranging from 100 to 500 µg/mL exhibit negligible cytotoxicity. Furthermore, Figure S7A and B demonstrate that the ROS generated by the cascade catalytic and photothermal processes do not induce macrophage polarization, indicating favorable biosafety of the system. A significant decrease in HLECs viability (to below 50%) was observed when the AuNPs@MIL/CHI concentration exceeded 200 μg/mL and the number of assembled layers was greater than five (Figure 7A). Under 200 μg/mL AuNPs@MIL/CHI and (AuNPs@MIL/CHI)5, the inhibition effect of cell proliferation was further evaluated using Calcein/PI cell viability/cytotoxicity assay kit. As shown in Figure 7B and C, there was rare cell apoptosis in control group and partial cell apoptosis in AuNPs@MIL/CHI multilayer modified group (without 808 nm laser irradiation), with a survival less than 60%. Whereas, there was an obvious cell apoptosis with 808 nm laser irradiation, with a survival rate less than 10% and almost no living cells. These results indicate that IOL modified with AuNPs@MIL/CHI multilayer possess a good ability to inhibit cell proliferation, showing great potential for enhanced PCO prevention.
Suppression cell proliferation effect in vitro. (A) The cell viabilities of HLECs seeded on the different layers of IOL material modified with different concentrations of AuNPs@MIL/CHI. (B) Statistical analysis of cell viabilities in (C). (C) Fluorescence images of HLECs stained with calcein/PI cell viability/cytotoxicity assay kit after incubation on the surfaces of modified IOL material.
In vitro cell apoptosis assessment
Hoechst staining was utilized to examine the role of AuNPs@MIL/CHI-modified IOL material in the process of apoptosis. As shown in Figure 8A and C, the nuclei of HLECs in control group were stained with light blue, indicating that the HLECs were not undergoing apoptosis. However, half of the HLECs nuclei in the modified IOL material group showed a blue and dense staining, indicating that these cells were in the apoptotic state. After excitation with 808 nm laser, all cells showed a blue and dense staining, suggesting they had entered the apoptotic cycle. This phenomenon confirms the occurrence of cascade catalytic reactions and photothermal effects, where the generated ROS and photothermal effects promote cell apoptosis. Figure 8B and D further illustrates this phenomenon. As can be seen, negligible green fluorescence from DCF was observed in the control group, whereas a pronounced fluorescence was observed in the experimental group, indicating significantly higher level of ROS was generated within HLECs in the experimental group. Furthermore, the ROS content in the HLECs was significantly increased after excitation by 808 nm laser. The excellent pro-apoptotic effect of AuNPs@MIL multilayer modified IOL material can be attributed to the combination of cascade catalysis and localized hyperthermia effects. Similarly, as shown in Figure S6A and B, NIR irradiation of the AuNPs@MIL-modified IOLs led to a pronounced depolarization of the mitochondrial membrane potential in HLECs. After an additional 4 h of incubation, the number of viable HLECs was markedly reduced, indicating that the combined photothermal treatment can rapidly induce HLEC apoptosis. All these results indicate that AuNPs@MIL/CHI multilayer modified IOL material can effectively induce apoptosis, providing strong evidence for enhanced PCO prevention.
Evaluation of apoptosis induced by modified IOL materials. (A) Hoechst staining images and partial enlarged drawing of HLECs seeded on modified IOL material and pristine IOL material. (B) DCFH-DA staining images of HLECs after incubation on the surfaces of modified IOL material and pristine IOL material. (C) Hoechst fluorescence staining statistics of HLECs after incubation on modified IOL material and pristine IOL material measured by fluorescence spectrophotometer. (D) Statistical analysis of fluorescence intensity of (B).
In vivo cascade catalytic activity assessment
Encouraged by the remarkable in vitro cascade catalytic capabilities of AuNPs@MIL, the in vivo cascade catalytic activities were further investigated. As shown in Figure 9A and B, there was a significant reduction in the number of HLECs within the capsular bag in five-bilayer AuNPs@MIL/CHI modified IOL ((AuNPs@MIL/CHI)5-IOL). Moreover, a high level of ROS was observed in the HLECs of the (AuNPs@MIL/CHI)5-IOL group. To further clarify the details of the cascade catalytic reaction process in vivo, the capsular bag was completely disrupted using a lysate after grinding, and the protein content and Fe^3+^ concentration were measured. The results shown in Figure 9C indicated that the protein concentration in the pristine IOL group was significantly higher than in the (AuNPs@MIL/CHI)5-IOLs group. More importantly, the combination of the cascade catalytic effect and photothermal treatment resulted in the most significant reduction in both HLEC density and protein concentration within the capsular bag. The statistical analysis of Fe^3+^ content in Figure 9D and E showed that pristine IOL group exhibited only a minimal amount of Fe^3+^, while the (AuNPs@MIL/CHI)5-IOLs group exhibited the high Fe^3+^ concentration, further confirming the occurrence of the cascade catalytic reaction. These results suggest that the (AuNPs@MIL/CHI)5-IOLs retains its excellent cascade catalytic activity and PTT after implantation, and its combination enhanced the cell clearance effect, thereby effectively preventing the development of PCO.
In vivo cascade catalytic processes analysis. (A) Representative images of DHE fluorescent staining of capsular bag sections. (B) Statistical analysis of fluorescence intensity of (A). (C) The protein concentration of capsular bag. (D) Fe3+ concentration of capsular bag. (E) The content of capsular bag.
In vivo PCO prevention behavior
The development of PCO was monitored using a slit-lamp microscope at postoperative days 1, 7, 14, 21 and 28. As shown in Figure 10A, the Pristine IOL group experienced mild corneal edema with slight exudation on the first postoperative day, which resolved within 7 days. In contrast, the (AuNPs@MIL/CHI)5-IOL group showed much milder symptoms, preliminarily indicating good intraocular biocompatibility for the (AuNPs@MIL/CHI)5-IOL. At 14 days postop, all groups exhibited high light transmission and clarity, without iris adhesion or corneal opacity. But the pristine IOL group showed obvious adhesion of fibrous tissue near the capsular notch, which progressively extended to the central optical zone and increased over time. By day 28, numerous cells appeared in the central optical zone of the Pristine IOL group, accompanied by significant capsular wrinkling, and the capsular bag became virtually opaque, indicating there was a severe PCO. In contrast, although a small amount of fiber tissue adhesion was also observed on day 14 in the (AuNPs@MIL/CHI)5-IOL group, it did not progress over time. By day 28, fiber tissue adhesion was barely detectable, and the central optical zone remained transparent. Encouragingly, the central optical region of the (AuNPs@MIL/CHI)5-IOL+NIR group was clean and transparent throughout the observation time, with well-defined edges and no signs of dragging or distortion. All these results demonstrate that the (AuNPs@MIL/CHI)5-IOL exhibits desirable anti-PCO effects and good intraocular compatibility.
Assessment of the PCO development in vivo. (A) Representative images captured by slit-lamp microscope at different time after surgery. (B) Representative images of capsular bag by a stereomicroscope. (C) Representative H&E staining images of capsular bag sections. (D) Intraocular PCO scores at 28 days after operation.
The stereomicroscopic results of capsular bags in Figure 10B were consistent with the slit-lamp microscope results. Obvious capsular wrinkles were observed in the Pristine IOL group, and the proliferation of the posterior capsule had covered the entire central optical zone. In contrast, the (AuNPs@MIL/CHI)5-IOL group showed no capsular wrinkles, and the central optical zone remained relatively clear, with only slight subcapsular opacification around the capsular notch. In the (AuNPs@MIL/CHI)5-IOL+NIR group, almost no subcapsular opacification was observed, and the central optical region was very clear and transparent. Additionally, the development of PCO was evaluated according to capsular bag slices. The results in Figure 10C revealed that there was a significant cell proliferation within the capsular bag in control group, while there was a significant decrease in cell proliferation, confirming that (AuNPs@MIL/CHI)5-IOL implantation greatly suppressed the development of PCO. Finally, to visually assess PCO severity, the PCO score was determined based on the areas and locations of PCO: (i) Central PCO (CPCO), within a 3 mm diameter of the central optical zone; (ii) Peripheral PCO (PPCO), the annular region between 3 mm and 6 mm from the center and (iii) Soemmerring’s ring (SR), the portion outside the optical zone of the IOL. The slit-lamp microscope images taken on the 28th day after surgery were scored according to the PCO grading criteria [27]. The detailed regional delineation of the lens and the corresponding PCO grading criteria are presented in Figure S2, and the results of the scoring are shown in Figure 10D. The control group had the highest PCO score, while the (AuNPs@MIL/CHI)5-IOL group showed a significant decrease in PCO score, and the combination with photothermal treatment further decreased in PCO score. These results indicate that the (AuNPs@MIL/CHI)5-IOL can effectively harness the cascade catalytic and photothermal properties of AuNPs@MIL to inhibit PCO development after ocular implantation. Notably, the H&E staining results of corneal, iridal and retinal sections (Figure S1) indicate that all groups of IOLs exhibit favorable intraocular biocompatibility. The ocular tissues maintain normal morphology, and no inflammatory infiltration or pathological alterations were observed.
Discussion
Cataract remains the leading cause of reversible blindness worldwide. PCO is the most common complication following cataract surgery and is primarily driven by the proliferation, migration and differentiation of residual HLECs on the capsular surface. Although recent advances in IOL surface-modification technologies have partially mitigated PCO progression, substantial challenges persist regarding long-term efficacy and intraocular biocompatibility. A variety of studies have explored hydrophilic and hydrophobic surface coatings to inhibit cellular adhesion and proliferation, offering promising strategies for PCO prevention [53, 54]. Meanwhile, because the eye is an optical organ, photodynamic therapy has also been widely investigated as an adjunctive approach against PCO [55, 56]. In this context, our study proposes a surface-engineered IOL that integrates hydrophilicity, PTT and cascade catalytic functions to achieve triple-mode protection against PCO. Despite demonstrating the potential of AuNPs@MIL-modified IOLs in preventing PCO, several limitations remain. At present, aside from slit-lamp examination, there are no more direct modalities available to dynamically quantify PCO progression, such as real-time monitoring of proliferating HLECs. In addition, once an IOL is implanted, it is not feasible to continuously monitor the in vivo release kinetics or stability of the surface coating. These important aspects warrant deeper investigation in future studies.
Conclusions
In this study, we developed an AuNPs@MIL-modified IOL that directly targets the pathogenic mechanisms of PCO-including the proliferation, migration and differentiation of HLECs. The engineered IOL integrates three synergistic functionalities: a hydrophilic surface interface, a glucose and O_2_ driven cascade catalytic system, and a NIR-mediated PTT platform. Following implantation, the hydrophilic interface provides intrinsic anti-adhesive properties, thereby reducing HLECs attachment and proliferation on the IOL surface. Meanwhile, the AuNPs@MIL coating continuously consumes glucose and O_2_, competitively suppressing HLECs proliferation while generating ROS capable of efficiently inducing HLECs apoptosis. During postoperative monitoring, once early signs of PCO development are detected, NIR-triggered PTT can be applied as an adjunct therapy to further eliminate residual or proliferating HLECs. Both in vitro and in vivo studies demonstrated that AuNPs@MIL-modified IOLs exhibit excellent preventive efficacy against PCO, favorable biocompatibility and outstanding optical performance. Collectively, these findings provide a promising pathway toward long-term, safer and more effective PCO prevention.
Supplementary Material
rbag020_Supplementary_Data
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1Cicinelli MV , Buchan JC, Nicholson M, Varadaraj V, Khanna RC. Cataracts. Lancet 2023;401:377–89.36565712 10.1016/S 0140-6736(22)01839-6 · doi ↗ · pubmed ↗
- 2Auffarth GU , Gerl M, Tsai L, Janakiraman DP, Jackson B, Alarcon A, Dick HB, Quantum Study Group. Clinical evaluation of a new monofocal IOL with enhanced intermediate function in patients with cataract. J Cataract Refract Surg 2021;47:184–91.32932369 10.1097/j.jcrs.0000000000000399 · doi ↗ · pubmed ↗
- 3Chen X , Xu J, Chen X, Yao K. Cataract: advances in surgery and whether surgery remains the only treatment in future. Adv Ophthalmol Pract Res 2021;1:100008.37846393 10.1016/j.aopr.2021.100008 PMC 10577864 · doi ↗ · pubmed ↗
- 4Zhu SQ , Huang HY, Liu D, Wen SM, Shen LL, Lin QK. Augmented cellular uptake and homologous targeting of exosome-based drug loaded IOL for posterior capsular opacification prevention and biosafety improvement. Bioact Mater 2022;15:469–81.35386342 10.1016/j.bioactmat.2022.02.019PMC 8958386 · doi ↗ · pubmed ↗
- 5Xia JY , Han YM, Shen LL, Wang R, Wen SM, Zhu SQ, Lin QK. Photo-responsive intraocular lens with on demand drug release for posterior capsule opacification prevention and improved biosafety. Chem Eng J 2022;430:132716.
- 6Lu DD , Han YM, Liu D, Chen SQ, Qie JQ, Qu J, Lin QK. Centrifugally concentric ring-patterned drug-loaded polymeric coating as an intraocular lens surface modification for efficient prevention of posterior capsular opacification. Acta Biomater 2022;138:327–41.34800717 10.1016/j.actbio.2021.11.018 · doi ↗ · pubmed ↗
- 7Luo C , Wang H, Chen X, Xu J, Yin H, Yao K. Recent advances of intraocular lens materials and surface modification in cataract surgery. Front Bioeng Biotechnol 2022;10:913383.35757812 10.3389/fbioe.2022.913383 PMC 9213654 · doi ↗ · pubmed ↗
- 8Sun Y , Xiong L, Wang X, Wang L, Chen B, Huang J, Huang M, Chen J, Wu J, Huang S, Liu Y. Autophagy inhibition attenuates TGF-β2-induced epithelial-mesenchymal transition in lens epithelial cells. Life Sci 2021;265:118741.33181173 10.1016/j.lfs.2020.118741 · doi ↗ · pubmed ↗