cRGD-Conjugated Bilirubin Nanoparticles Alleviate Dry Eye Disease Via Activating the PINK1-Mediated Mitophagy

Abstract

Purpose: The purpose of this study was to evaluate the cytoprotective effect and the mechanism of cRGD-conjugated bilirubin nanoparticles (cNPs@BR) in dry eye disease (DED).

Methods: The binding capacity and cellular uptake of cNPs@BR in human corneal epithelial cells (HCECs) were assessed by immunofluorescence. The anti-inflammation and anti-oxidative stress effects of cNPs@BR were determined by flow cytometry, immunofluorescence, Western blot, chromatin immunoprecipitation (ChIP), and ELISA assay in LPS-stimulated RAW264.7 cells and hypertonic HCECs. The function of ocular surface barrier, tear production, and the number of goblet cells after cNPs@BR treatment were further assessed by fluorescein sodium staining, phenol red cotton threads, quantitative PCR (qPCR), hematoxylin and eosin (H&E) staining, and Periodic Acid-Schiff (PAS) staining in a 0.2% BAC-induced DED mouse model. Furthermore, the mechanism of cNPs@BR in treating DED was explored by RNA sequencing and RNA interference.

Results: The cRGD peptide prolonged the retention time of nanoparticles on HCECs and enhanced the cellular uptake efficiency. In both cell models, 20 µM cNPs@BR pretreatment ameliorated oxidative stress by decreasing the intracellular reactive oxygen species (ROS) levels and the expression of NOX4 and 4-HNE, while promoting HO-1 and nuclear Nrf2 levels. Moreover, cNPs@BR alleviated the inflammatory response by inhibiting NF-κB p65 nuclear translocation and decreasing the expression of iNOS and the secretion of IL-1β, IL-6, and TNF-α. In addition, cNPs@BR protected ocular surface epithelium against oxidative stress and inflammation and restored conjunctival goblet cells in the mouse model of DED by activating PINK1-mediated mitophagy.

Conclusions: The cNPs@BR suppressed oxidative stress and inflammatory response in the ocular surface epithelium and restored goblet cells by activating PINK1-mediated mitophagy.

Dry eye disease (DED) is a common ocular disorder that disrupts the tear film and ocular surface.¹ The prevalence of DED has risen sharply, reaching 5% to 50% worldwide.² Although the etiology of DED has not yet been elucidated, tear deficiency and excessive evaporation can lead to tear film instability and chronic ocular surface inflammation, which in turn results in the loss of conjunctival goblet cells and the apoptosis of corneal and conjunctival epithelial cells.³ Therefore, anti-inflammatory and artificial tear supplementation are currently the main therapeutic strategies for DED.⁴ 

Currently, the primary cause of DED is the immuno-inflammatory process occurring on the surface of the eye.⁵ The hyperosmolar environment of the ocular surface triggers the release of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) from the stressed epithelium into tear films.⁶ The inflammatory cytokines are secreted by promoting Toll-like receptors (TLRs)/NF-κB/inducible nitric oxide synthase (iNOS) and mitogen-activated protein kinase (MAPK) pathway, which in turn recruit immune cells.⁷ Thereafter, activated immune cells further express pro-inflammatory mediators and matrix metalloproteinases (MMPs) to amplify the inflammatory cascade and generate a vicious cycle of DED.^8,9 On the other hand, DED is also accompanied by oxidative stress of the ocular surface barrier, which is characterized by increased intracellular reactive oxygen species (ROS) levels and inactivation of antioxidant enzymes.¹⁰ ROS production is often induced by the activation of NADPH oxidase 4 (NOX4).¹¹ Oxidative damage promotes the release of inflammatory cytokines and cell apoptosis. Several studies confirmed that the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) induces the expression of heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO-1), and protects against corneal epithelium damage and DED.¹² In addition, inflammation and oxidative stress disrupt the ocular surface epithelial barrier by modulating the autophagy and mitophagy pathways.^13,14 All the abovementioned evidence suggests that the crosstalk between immune-inflammation and oxidative stress participates in the initiation and development of DED. 

Bilirubin (BR), one of the metabolic byproducts of HO-1 degradation of heme, forms redox pairs with biliverdin to exert biological functions.¹⁵ Multiple studies have found that physiological BR has significant antioxidant, anti-inflammatory, and anti-apoptotic properties.¹⁶ Generally, BR has been regarded as a potential candidate to overcome inflammatory bowel disease, pulmonary fibrosis, liver ischemia-reperfusion injury, and type 2 diabetes by scavenging excessive intracellular ROS, suppressing inflammatory cytokines’ secretion, and modulating immune cells activity.^17–19 In the field of ocular disorders, serum BR levels have been found to be negatively correlated to the severity of retinopathy and corneal nerve fiber pathology in patients with type 2 diabetes.^20,21 Our previous works also depicted that the HO-1/BR signaling pathway inhibits hydrogen peroxide (H₂O₂)-induced oxidative damage in lens epithelial cells.^22–25 However, the protective effect of BR on DED remains unclear. 

In the present study, BR was encapsulated into PEG_2k/PLGA_5k nanoparticles (NPs@BR), a US Food and Drug Administration (FDA)-approved biomaterial with minimal ocular toxicity,²⁶ which was further conjugated with cyclic Arg-Gly-Asp (cRGD) peptide (cNPs@BR). Then, the antioxidative and anti-inflammatory effect of cNPs@BR eyedrops was assessed in 2 different cells models and in a 0.2% benzalkonium chloride (BAC)-induced murine model of DED. Lifitegrast (5%, Xiidra), an FDA-approved commercial eye drop for DED, serves as a positive control.²⁷ Concurrently, the mechanism was explored via RNA sequencing and RNA interference. 

Materials and Methods

Reagents

BR (B8431) and lipopolysaccharide (LPS, L8880) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Xi'an Ruixi Biological Technology Co., Ltd. (Shanxi, China) provided PEG_2k/PLGA_5k (R-PL1001-7KD) and cRGD-PEG_2k/PLGA_5k (R-RG0219). Sigma-Aldrich (St. Louis, MO, USA) provided Benzalkonium chloride (BAC, B6295). DCFH-DA probe (S0033S), MitoSOX red probe (S0061M), JC-1 probe (C2005), One Step TUNEL Apoptosis Assay Kit (C1088), Lyso-Tracker green (C1047S), Cell counting kit-8 (C0038), Hoechst (C1027), DAPI (C1002), Cell Mitochondria Isolation Kit (C3601), and Nuclear and Cytoplasmic Protein Extraction Kit (P0027) were purchased from Beyotime (Shanghai, China). The mouse IL-1β ELISA kit (BMS6002-2), IL-1β human high sensitivity ELISA kit (BMS224-2HS), mouse IL-6 ELISA kit (BMS603-2), human IL-6 ELISA kit (BMS-213-2), mouse TNF-α ELISA kit (BMS607-2HS), and human TNF-α ELISA kit (88-7346-22) were purchased from Thermo Fisher Scientific, Inc. (Shanghai, China). Abcam (Shanghai, China) provided the primary antibodies against HO-1 (ab189491), Iκkα (ab32041), NF-κB p65 (ab32536), Iκbα (ab32518), iNOS (ab178945), PGC1α (ab191838), NRF1 (ab175932), mtTFA (ab176558), LC3B (ab48394), p62 (ab109012), E-cadherin (ab40772), TOMM20 (ab56783), 4-HNE (ab48506), phospho-Iκkα (ab38515), phospho-Iκbα (ab92700), phospho-NF-κB p65 (ab76302), GAPDH (ab8245), COX IV (ab202554), β-actin (ab8226), Histon H3 (ab1791), goat anti-rabbit IgG H&L (ab7090), and goat anti-mouse IgG H&L (ab7063). The primary antibodies against Nrf2 (80592-1-RR) and NOX4 (14347-1-AP) were purchased from Proteintech Group, Inc. (Hubei, China). Pink1 small interfering RNA (siRNA; sc-44598) and scramble siRNA (sc-37007) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). 

Preparation of Bilirubin Loaded Nanoparticles

BR was encapsulated into PLGA_5K-PEG_2K (NPs) and PLGA_5K-PEG_2K-cRGD (cNPs) using nanoprecipitation method. NPs or cNPs (1 mg) and BR (0.25 mg) were dissolved in 0.1 mL of DMSO. After mixing the solutions, 0.9 mL of PBS was added and stirred at room temperature for 2 hours. The BR loaded nanoparticles (NPs@BR and cNPs@BR) were obtained through lyophilization. 

Characterization of Bilirubin Loaded Nanoparticles

The nanoparticles were observed for microstructural analysis using a transmission electron microscope (TEM; JEOL-2100F) after drying. Additionally, the dynamic light scattering (DLS) was used to determine the hydrated particle sizes of NPs and cNPs. The surface charge was assessed via detecting zeta potential of NPs and cNPs in water. The chemical structures of the materials were analyzed using 1H-nuclear magnetic resonance spectroscopy (¹H-NMR) on a Bruker Biospin Inc., AVANCE III HD system. 

The released BR from NPs@BR and cNPs@BR (3 mg each) was extracted using cryogenic ultracentrifugation. The drug load (W1) of the different materials was determined by UV spectrophotometry, and the cumulative release was calculated. Equations 1 and 2 were used to evaluate encapsulation efficiency and drug loading capacity:  

\begin{eqnarray} {\rm{Encapsulation}}\,{\rm{efficiency}} = \left( {{\rm{3}} - {\rm{W1}}} \right){\rm{/3}} \times {\rm{100\% }}\quad \end{eqnarray}

(1)

 

\begin{eqnarray} {\rm{Drug}}\,{\rm{loading}}\,{\rm{capacity}} = \left( {3 - {\rm{W}}1} \right)/10 \times 100\%\quad \end{eqnarray}

(2)

 

These equations allowed for the quantification of the percentage of encapsulated BR and the drug loading capacity, respectively. 

Cell Culture and Cell Models

Human corneal epithelial cells (HCECs; CL-0743) and RAW264.7 cells (CL-0190) were purchased from Pricella Biotechnology Co., Ltd. (Wuhan, China). LPS-stimulated RAW264.7 cells was established for assessing inflammation and oxidative stress. In brief, 2 × 10⁶ cells were seeded in 6-well plates and incubated for 24 hours to allow the cells grow to approximately 60%. Then, the different BR nanoparticles (20 µM) were pretreated with cells for 2 hours, followed by incubating with 10 µg/mL LPS for 24 hours. 

The culture medium was supplemented with sodium chloride to establish a hypertonic model of HCECs. The initial osmolarity of serum-free medium is 320 mOsm. To establish a hyperosmolar environment, 90 mM sodium chloride was added to serum-free medium, leading to the increased osmolarity to 450 mOsm. Then, the different BR nanoparticles (20 µM) were pretreated with cells for 2 hours, followed by incubating in hyperosmolar environment for 24 hours. 

Flow Cytometry

The total intracellular ROS levels were assessed via DCFH-DA probe. After treatment in each group, the cells were incubated with 10 µM DCFH-DA probe at 37°C for 30 minutes. Then, the cells were washed twice with PBS and detected by flow cytometry. 

The mitochondrial membrane potential was assessed by JC-1 probe. After treatment in each group, the cells were incubated with JC-1 probe at 37°C for 30 minutes. The fluorescence intensity was detected at 488 nm and 575 nm wavelength. 

ELISA Assay

In each group, the cell supernatants were harvested by high-speed centrifugation. The levels of IL-1β, IL-6, and TNF-α in cell culture supernatants were quantified by ELISA kits according to the manufacturer's protocols. 

Small Interfering RNA Transfection

The cells at 50% confluence were transfected with siRNA targeting Pink1 (siPINK1) within Lipofectamine 3000 for 6 hours. Then, the cells were incubated in hyperosmolar environment for 24 hours. The cells were harvested for immunofluorescence staining, Seahorse assay, mitochondrial ROS detection, and inflammatory cytokines measurement. 

Cellular Uptake Assay

The HCECs were incubated with Cy5.5-labeled nanoparticles for 5 minutes, 15 minutes, and 30 minutes at 37°C. To explore the mechanism of cellular uptake, the HCECs were pretreated with different endocytosis inhibitors, including 10 µM chlorpromazine, 5 µM filipin, 5 µM wortmannin, or 10 µM cytochalasin D for 2 hours. Then, the cells were incubated with Cy5.5-labeled nanoparticles for 30 minutes at 37°C. Cells were observed via fluorescence microscope. Fluorescence intensity was calculated by ImageJ software. 

Binding Capacity Assay

The binding capacity of cNPs on corneal epithelium were assessed. To observe the co-localization of the nanoparticles and cell surface membrane, the HCECs were incubated with different Cy5.5-labeled nanoparticles for 5 minutes. Then, the cells were incubated with primary antibody against E-cadherin (1:200). Finally, the DAPI was stained for 10 minutes. The cells were observed via a confocal fluorescence microscope. 

To evaluate the effect of cNPs in vivo, 5 µL of different Cy5.5 labeled nanoparticles were instilled into the mouse conjunctival sac. At predetermined time points (1, 6, 12, and 24 hours), the eyeballs were harvested and placed in optimal cutting temperature (OCT) for frozen section. After staining with DAPI, the mouse cornea was then observed to assess the retention time of nanoparticles on the ocular surface. 

Experimental Dry Eye Murine Model and Therapeutic Strategy

The experimental protocol was approved by the Animal Ethics Committee of Shanghai East Hospital, Tongji University School of Medicine. All procedures conducted in this study adhere to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. One hundred sixty male C57BL/6J mice (19–22 g) aged 4 weeks were purchased from Beijing Longan Animal Center. According to the guidelines, the mice were housed under specific pathogen-free (SPF) conditions at the center. 

Here, 0.2% BAC eye drops were used to induce experimental dry eye murine model. In brief, the right eyes were topically administered with 5 µL of 0.2% BAC twice daily (at 10:00 AM and 8:00 PM) for 7 consecutive days. Commencing from day 7, the 5 µL of different nanoparticles containing BR (20 µM) were topically administered twice daily (at 10:00 AM and 8:00 PM) for another 7 consecutive days. Mice were randomly divided into six groups: (1) the control group; (2) the BAC+PBS group; (3) the BAC+BR group; (4) the BAC+NPs@BR group; (5) the BAC+cNPs@BR group; and (6) the BAC+Xiidra group (n = 15 per group). 

Corneal Fluorescein Sodium Staining

Corneal fluorescein sodium staining was conducted on days 1, 3, 5, and 7 at the same time of treatment (at 11:00 AM). Each eye image was analyzed to quantify the fluorescein score as follows: 0 = no green color, 0.5 = microgreen spot staining, 1 = diffuse spot green color, 2 = less than one-third of green stained area, 3 = more than one-third of green stained area, and 4 = more than two-thirds of green stained area. Each corneal score is the sum of the 4 quadrant scores and the final score ranges from 0 to 16. 

Detection of Tear Production

Tear volume was measured using phenol red cotton threads on days 1, 3, 5, and 7 at the same time of treatment (at 12:00 AM). The cotton threads were put into the lower conjunctival fornix at around one third of the outer eyelid. Then, the wetted length of the threads was recorded after 15 seconds. 

Immunofluorescence Staining

In cell models, cells in each group were fixed with 4% paraformaldehyde, added with 0.3% Triton X-100 and sealed with 5% BSA. After that, the cells were incubated with primary antibody against 4-HNE (1:200), NOX4 (1:200), or p65 (1:200) followed by secondary antibody. Finally, DAPI was stained for 10 minutes. To assess the intracellular ROS and mitochondrial ROS levels, the cells were incubated with DCFH-DA probe and MitoSOX probe, respectively. Then, the cells were stained with Hoechst for 30 minutes. 

For cornea fluorescence staining, mouse eyeballs were excised, fixed, and subsequently paraffin-embedded on day 7 of treatment. Sections were then deparaffinized with xylene and underwent high-pressure antigen retrieval. To assess the oxidative stress of corneal epithelium, the samples were fixed and incubated with a primary antibody against 4-HNE (1:200) followed by a secondary antibody (1:500). To assess the apoptotic rate of corneal epithelium, the samples were incubated with TUNEL apoptosis assay kit. Cells and cornea sections were visualized by immunofluorescence microscopy and fluorescence intensity was quantified using ImageJ software. 

Hematoxylin and Eosin Staining, Periodic Acid-Schiff Staining, and Immunohistochemistry

Immunohistochemistry (IHC) was conducted to evaluate corneal epithelial tissue. Excised tissue was fixed, paraffin-embedded, and stained with hematoxylin and eosin (H&E) and Periodic Acid-Schiff (PAS). The slides were detected using a primary antibody against Pink1 (1:200) and analyzed by microscopy. Quantitative analysis of mouse corneas was carried out from five random fields of view per section. 

RNA Sequencing

On day 7 of treatment, corneas from BAC+PBS mice and BAC+cNPs@BR were collected for transcriptome sequencing. Mouse corneal RNA was extracted according to the instructions of the total RNA extraction reagent, and cDNA libraries were created using the Novitec mRNA Seq V3 Library Prep Kit. The cDNA library was sequenced using NextSeq 550 sequencer and NextSeq 500/550 High Output Kit V2 Sequencing Kit, with the parameter setting of 100 bp for single-end, and the sequencing volume of 40 to 50 M reads. Differentially expressed genes (DEGs) were analyzed using STRING, and the interactions between the genes and the proteins encoded were predicted. STRING was used to analyze the DEGs and predict the interactions between the proteins encoded by the genes. DEGs were defined as those with an adjusted P value (P adjust) < 0.05 and a fold change in gene expression ≥ 2 (|log₂fold change| ≥ 1) between samples. The STRING results were imported into Cytoscape version 3.9.1 software, and the degree of each node (Degree) was calculated by Cytohubba plug-in, and the network composed of genes with the top 10 Degree scores could be regarded as the core sub-network. Gene set enrichment analysis (GSEA) was subsequently performed on mitophagy-related genes to explore the potential role of mitophagy in ocular surface recovery. 

Western Blot Analysis

The mitochondrial, cytoplasmic, and nuclear extractions were performed as previously described.^23,28 The protein concentrations in mitochondria, cytoplasm, and nucleus were determined via the BCA kit. The protein samples were loaded into the wells of the SDS-PAGE gel for electrophoresis. Then, the proteins were transferred to PVDF membranes. The membranes were then blocked and incubated with primary antibodies against Nrf2 (1:1000), HO-1 (1:1000), NOX4 (1:1000), p65 (1:1000), Iκkα (1:1000), Iκbα (1:1000), PGC1α (1:1000), NRF1 (1:1000), mtTFA (1:1000), phospho-Iκkα (1:1000), phospho-Iκbα (1:1000), phospho-NF-κB p65 (1:1000), iNOS (1:1000), LC3B (1:1000), p62 (1:1000), Pink1 (1:1000), Parkin (1:1000), β-actin (1:5000), Histon H3 (1:5000), and GAPDH (1:5000), followed by treatment with secondary antibodies. The protein bands were visualized using ECL detection system, and the density of target bands was analyzed using Image J software. 

Quantitative PCR Assay

On day 7 of treatment, mouse corneas were collected for quantitative polymerase chain reaction (qPCR) assays targeting inflammatory cytokines (IL-1β, IL-4, IL-6, and MMP-9) and mitophagy markers (Pink1, Atg5, Mfn2, Map1lc3b, Binp3, and Sqstm1). Total RNA was extracted from the corneas and cDNA was synthesized followed by SYBR Green Supermix. The internal reference gene gapdh was used for normalization. The primer sequences used are provided in Supplementary Table S1. 

Seahorse Assay

The oxygen consumption rates (OCRs) of corneal epithelial cells in different groups were detected via the Seahorse Bioscience XF24 Analyzer. In brief, after 10 minutes of detecting the basal respiration, 2.5 µM oligomycin was added followed by 1 µM FCCP at 25 minutes, 2.5 µM rotenone and 2.5 µM antimycin A at 45 minutes. OCR was recorded as pMols per minute. ATP turnover and respiratory capacity were calculated as previously described.²⁹ 

Chromatin Immunoprecipitation Assay

The chromatin immunoprecipitation (ChIP) assay was performed as previously described.²⁴ In brief, the RAW264.7 cells were fixed at room temperature with 1% paraformaldehyde for 10 minutes. Followed with ultrasonic treatment to shear the genomic DNA into mostly 400 to 800 bp in size. The samples were added with Protein A/G Agarose beads and centrifuged at 1000 g for 1 minute at 4°C. The supernatant was transferred and incubated with p65 antibody, H2A.X antibody, and anti-rabbit IgG antibody at 4°C. Thereafter, 60 µL of protein A/G agarose beads were gently mixed and rotated at 4°C for 60 minutes. The supernatant was carefully removed, the pellet was retained, and placed at room temperature for 15 minutes. Then, the DNA was de-crosslinked and purified. The fragment region of the predicative binding sites on the iNOS promoter was amplified by PCR with the DNA extracted from NF-κB p65 antibody-immunoprecipitated chromatin fragments. Primers targeting promoter sequences were as follows: 5′- GAA GAT GAG TGG ACC CTG GC -3′ (forward) and 5′- GAT GGG CTG TGC TGA GTG AA -3′ (backward). The product size was about 499 bp. 

Statistical Analysis

Statistical analyses were performed using GraphPad Prism (GraphPad, San Diego, CA, USA), with data presented as mean ± standard error of the mean. Student's t-test or 1-way ANOVA with post hoc least significant difference test were applied for statistical comparisons, with P values < 0.05 as being statistically significant. 

Results

Characterization of cNPs@BR

TEM images depicted intact spherical structures for both NPs and cNPs. The particle sizes of NPs and cNPs, as determined by TEM and DLS, were approximately 100 nm (Figs. 1A, 1B). Furthermore, the zeta potentials of NPs and cNPs in water were measured as –13.8 mV and –16.7 mV, respectively (Fig. 1C). Distinct characteristic peaks corresponding to cRGD were observed via ¹H-NMR analysis, with the short peptide of cRGD clearly discernible. Additionally, a prominent signal consistent with the PEG methylene proton at 3.7 ppm was evident, further affirming the successful synthesis of cNPs (Fig. 1D). The encapsulation efficiency and loading capacity of NPs and cNPs were evaluated at various weight ratios of BR. The findings showed a gradual decrease in the encapsulation efficiencies of NPs and cNPs with increasing BR ratio, whereas the loading capacities exhibited the opposite trend (Fig. 1E). After 48 hours of incubation, both NPs and cNPs released approximately 60% of the drug (Fig. 1F). 

Figure 1.

 

Characterization of cNPs@BR. (A) Transmission electron microscopy (TEM) images showing the morphology of NPs and cNPs. Bar = 200 nm. (B) Dynamic light scattering (DLS) analysis reveals the size distribution of both NP formulations. (C) Zeta potential measurements illustrate the surface charge characteristics of NPs and cNPs in water. (D) Nuclear magnetic resonance (NMR) spectra demonstrate the chemical composition, particularly the presence of cRGD in the nanoparticles. (E) Encapsulation efficiency (EE) and loading capacity (LC) of bilirubin (BR) within NPs and cNPs. (F) Cumulative release profiles of BR from NPs and cNPs over 48 hours. Data are presented as mean ± SEM; n = 3.

cRGD Modification Enhanced Retention Time of Nanodrugs on the Ocular Surface

In the present study, we designed cRGD modified nanodrugs to address the issue of rapid drug clearance from the ocular surface. Because the HCECs and their tight junction mainly compose the integrity of ocular surface, we first assessed the binding capacity of cNPs on cell membrane of HCECs in vitro. Cy5.5, a red fluorescent probe, was used to replace BR. Additionally, endocytosis inhibitors, including 10 µM chlorpromazine, 5 µM wortmannin, and 10 µM cytochalasin D, were used to pretreat with HCECs for 2 hours to reduce the cellular uptake of nanoparticles. Thereafter, HCECs were incubated with Cy5.5-labeled nanoparticles for 5 minutes to mimic the rapid clearance of eye drops on the ocular surface. The fluorescent images depicted that cNPs exhibited the stronger binding capacity to the cell membrane (green fluorescence stained by E-cadherin antibody) compared to NPs (Fig. 2A). 

Figure 2.

 

The cRGD modification promoted corneal adhesive capacity of NPs. (A) In vitro binding assay of various NPs with HCECs. Representative confocal images illustrating the co-localization of various Cy5.5-labeled nanoparticles (red channel) and E-cadherin (epithelial cell surface marker, green channel) in HCECs. Bar = 25 µm. (B) Left: Representative fluorescent images depicting the retention of various Cy5.5-labeled NPs on the mouse cornea at different time points after topical administration. Right: Semi-quantification of the red fluorescence retained in the corneal epithelium at each time point. Bar = 25 µm; n = 4.

We further evaluated the retention time of NPs on the ocular surface of mice. After the initial instillation, the red signal of free Cy5.5 in the corneal epithelium vanished within 1 hour. The red signal of NPs@Cy5.5 in the corneal epithelium lasted for 6 hours. Intriguingly, cNPs@Cy5.5 persisted for 12 hours, displaying the most extended retention time in the corneal epithelium (Fig. 2B). 

cRGD Modification Enhanced Cellular Uptake of Nanodrugs

The uptake of Cy5.5-labeled nanoparticles by HCECs was studied by fluorescent microscope at 5 minutes, 15 minutes, and 30 minutes. After incubating with different nanoparticles for 30 minutes, more red dots were seen in cNPs@Cy5.5 treated cells compared to those in free Cy5.5 and NPs@Cy5.5 treated cells, which indicated that cNPs@Cy5.5 exhibited rapid endocytosis (Figs. 3A, 3B). To investigate the mechanism of endocytosis, we pretreated HCECs with different endocytosis inhibitors before incubating with NPs@BR or cNPs@BR. The endocytosis inhibition assay revealed that pretreatment with chlorpromazine, wortmannin, and cytochalasin D inhibited the cellular uptake of Cy3 probe, indicating that the endocytosis of NPs and cNPs by HCECs was mediated via lattice protein and giant vesicle phagocytosis pathways (Fig. 3C, Supplementary Fig. S1). 

Figure 3.

 

The cRGD modification enhanced cellular uptake of NPs. (A) Fluorescent images of uptake characteristics of various Cy5.5-labeled nanoparticles in HCECs. Bar = 25 µm. (B) The relative fluorescence intensity of various Cy5.5-labeled nanoparticles following incubation with HCECs for 30 minutes. All data are mean ± SEM (n = 4). ***P < 0.001, versus the Cy5.5 group; and #P < 0.05, versus the NPs@Cy5.5 group. (C) The relative fluorescence intensity of various Cy5.5-labeled NPs in HCECs following pretreatment with different endocytosis inhibitors. All data are mean ± SEM (n = 4). ***P < 0.001, versus the control group. (D) Representative confocal images demonstrating the co-localization of Cy5.5-labeled NPs and lysosome (green channel) in HCECs. Bar = 25 µm.

Furthermore, we investigated the intracellular migration of cNPs within HCECs using confocal microscopy. As illustrated in Figure 3D, both NPs and cNPs demonstrated the capability to achieve endosomal escape. Taken together, these findings underscore the rapid and efficient endocytosis of cNPs by HCECs. 

cNPs@BR Alleviated Oxidative Stress and Inflammation Response Via Modulating NOX4/Nrf2/HO-1 and NF-κB /iNOS Pathway In Vitro

As oxidative stress and inflammation were identified as the pivotal contributors to initiate the vicious cycle of DED, we evaluated the anti-inflammatory and anti-oxidative stress effects of BR nanodrugs in LPS-induced cell model and hypertonic cell model, respectively. 

The optimal concentration of BR nanodrugs on RAW264.7 cells was determined based on the result of relative cell viability. The cells were treated with 10 µg/mL LPS for 24 hours followed by the pretreatment of cNPs@BR ranging from 2 µM to 50 µM for 2 hours. The data revealed that pretreatment with 20 µM cNPs@BR showed relatively higher viability compared with LPS group. Thus, we selected 20 µM cNPs@BR before exposure to LPS in RAW264.7 cells (Supplementary Fig. S2). Upon LPS stimulation, the intracellular ROS levels in RAW264.7 cells was detected with DCFH-DA probe by flow cytometry. The data revealed that LPS increased intracellular ROS levels, whereas cNPs@BR effectively suppressed ROS production, with the better effect than that of the BR and NPs@BR (Fig. 4A). Then, the levels of ROS markers, 4-HNE and NOX4, were detected via fluorescence staining. It was obvious that BR, NPs@BR, and cNPs@BR could effectively reduce 4-HNE and NOX4 expression, with cNPs@BR having the most remarkable effect (Fig. 4B). Furthermore, cNPs@BR inhibited the expression of NOX4 and promoted the expression of Nrf2 and HO-1 (Fig. 4C). To further investigate the nuclear translocation of Nrf2 upon LPS stimulation, we conducted immunofluorescence assay. As shown in Figure 5A, LPS activation remarkably decreased the fluorescence intensity of nuclear Nrf2 (red channel), whereas cNPs@BR pretreatment restored the fluorescence intensity of nuclear Nrf2. Meanwhile, cNPs@BR significantly enhanced the protein level of nuclear Nrf2 without affecting the cytoplasmic Nrf2 content by Western blot assay (Fig. 5B). All the data suggested that cNPs@BR promoted nuclear translocation of Nrf2 in LPS-stimulated RAW264.7 cells. 

Figure 4.

 

The cNPs@BR attenuated oxidative stress in LPS-stimulated RAW264.7 cells. (A) Determination of intracellular ROS levels (labelled by DCFH-DA probe) by flow cytometry. (B) Representative confocal images and semi-quantification of 4-HNE (red channel) and NOX4 (green channel). (C) Relative protein levels of Nrf2, HO-1, and NOX4 in RAW264.7 cells detected by Western blot assay. All data are mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, versus the control group; #P < 0.05, versus the LPS group; &P < 0.05, versus the LPS+cNPs@BR group. Bar = 25 µm.

Figure 5.

 

The cNPs@BR promoted nuclear translocation of Nrf2 in LPS-stimulated RAW264.7 cells. (A) Fluorescent images of Nrf2 nuclear translocation in different groups. Arrow: Nrf2 translocated into the nuclei. (B) Relative protein levels of cytosolic and nuclear Nrf2 in RAW264.7 cells detected by Western blot assay. All data are mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, versus the control group; #P < 0.05, versus the LPS group; &P < 0.05, versus the LPS+cNPs@BR group. Bar = 25 µm.

Under stimulation of stressors, NF-κB is the key mediator of inflammation. Therefore, the effect of BR nanodrugs was further evaluated on the NF-κB pathway in the LPS-stimulated cells. In the present study, LPS promoted the nuclear translocation of NF-κB subunit p65, whereas cNPs@BR notably decreased the nuclear p65 level (Fig. 6A). After LPS treatment, the protein levels of p-Iκkα, p-Iκbα, p-p65, nuclear p65, and iNOS were remarkably upregulated, whereas cNPs@BR pretreatment inhibited their upregulation (Fig. 6B). Furthermore, we assessed the effect of the nuclear translocated p65 on the transcription of iNOS gene. It was predicted that transcriptional factor p65 binds to the promoter region of iNOS gene (binding sequence: GGGGATTTTC) by Jaspar online website. The ChIP assay found the enrichment level of iNOS promoter pulled by p65 antibody, which demonstrated the binding of p65 and iNOS promoter. Intriguingly, LPS enhanced the binding capacity between p65 and iNOS promoter, whereas cNPs@BR partially decreased the effect (Fig. 6C). Additionally, cNPs@BR exhibited a robust ability on decreasing the secretion of IL-1β, IL-6, and TNF-α and was more effective than BR and NPs@BR (Figs. 6D–F). All the results confirmed that cNPs@BR inhibited the activation of NF-κB pathway and its downstream inflammatory response. 

Figure 6.

 

The cNPs@BR ameliorated inflammatory response in LPS-stimulated RAW264.7 cells. (A) Fluorescent images of NF-κB p65 nuclear translocation in different groups. Arrow: NF-κB p65 translocated into the nuclei. (B) Relative protein levels of phospho-Iκkα, phospho-Iκbα, phospho-NF-κB p65, and iNOS in RAW264.7 cells detected by Western blot assay. (C) ChIP assay for the binding of NF-κB p65 to the promoter region of iNOS genes. IgG and H2A.X were set as the negative and positive control, respectively. (D–F) The levels of IL-1β, IL-6, and TNF-α in the supernatants of different groups of cells were detected via ELISA assay. All data are mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, versus the control group; #P < 0.05, versus the LPS group; &P < 0.05, versus the LPS+cNPs@BR group. Bar = 25 µm.

The optimal concentration of BR nanodrugs on HCECs was determined along with the levels of IL-1β. The HCECs were exposed to hyperosmolar condition for 24 hours followed by the pretreatment of cNPs@BR ranging from 2 µM to 50 µM for 2 hours. The data revealed that pretreatment with 20 µM cNPs@BR showed relatively lower IL-1β level compared with the hyperosmolar group. Thus, we selected 20 µM cNPs@BR before exposure to hyperosmolar conditions in HCECs (Supplementary Fig. S3). Under a hyperosmolar environment, we found that cNPs@BR effectively attenuated intracellular ROS levels in HCECs (Figs. 7A, 7B). Hypertonicity also promotes the secretion of inflammatory cytokines. On the contrary, cNPs@BR suppressed the levels of inflammatory cytokines, showing superior effect compared to BR and NPs@BR (Figs. 7C–E). All these findings suggested that cNPs@BR alleviated inflammation and oxidative stress via modulating NOX4/Nrf2/HO-1 and NF-κB pathways. 

Figure 7.

 

The cNPs@BR suppressed intracellular ROS levels and inflammatory cytokines in a hypertonic model of HCECs. (A) Representative confocal images of intracellular ROS (labeled by DCFH-DA probe, green channel). (B) Semi-quantification of ROS fluorescent intensity. (C–E) The levels of IL-1β, IL-6, and TNF-α in the supernatants of different groups of cells were detected via ELISA assay. All data are mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, versus the control group; #P < 0.05, versus the hyperosmolar group; & P < 0.05, versus the hyperosmolar+cNPs@BR group. Bar = 25 µm.

cNPs@BR Protected Ocular Surface in a Mouse Model of Dry Eye by Activating PINK1-Mediated Mitophagy

The optimal concentration of BR nanodrugs on the ocular surface of mice was determined along with the relative mRNA levels of IL-1β and MMP-9 in the cornea by qPCR. The cNPs@BR eye drops, ranging from 5 µM to 100 µM, were topically instilled in 0.2% BAC-induced mouse model of DED for 7 consecutive days. The data revealed that instillation with 20 µM cNPs@BR showed relatively lower IL-1β and MMP-9 mRNA levels compared with the DED group. Thus, 20 µM cNPs@BR eye drops was selected for the next step (Supplementary Fig. S4). In the mouse model of DED, the corneal fluorescent intensity was consistently scored > 12.0 after PBS treatment. In contrast, after 7 days of cNPs@BR treatment, the corneal fluorescence reduced to a score of 1.5, indicating a significant therapeutic effect of cNPs@BR (Figs. 8A, 8B). Additionally, cNPs@BR demonstrated an optimal therapeutic effect with a tear volume of 6.62 ± 0.69 mm, surpassing the efficacy of the commercial Xiidra (5.35 ± 0.51 mm) and approaching normal tear volume (Fig. 8C). 

Figure 8.

 

The cNPs@BR mitigated ocular surface damages in 0.1% BAC-induced DED mice. (A) Corneal sodium fluorescein staining images in mouse models of DEDs after different treatment for 7 days. (B) Quantitative analysis of sodium fluorescein staining scores. (C) Tear production in mice was measured by phenol red cotton threads. (D–G) The qPCR assays of mouse corneas were performed to evaluate the relative mRNA levels of inflammatory cytokines (IL-1β, IL-4, IL-6, and MMP-9) after treatment. (H) The images of goblet cells in the upper conjunctiva via PAS staining and the analysis of goblet cell counts (the arrow represents PAS positive goblet cell). (I) H&E staining of mouse corneas along with quantitative analysis of central and peripheral corneal epithelial thickness. Bar = 200 µm. (J) Immunofluorescence staining of 4-HNE in mouse corneas along with quantitative analysis of fluorescence intensity. All data are mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, versus the control group; #P < 0.05, versus the BAC+PBS group; &P < 0.05, versus the BAC+cNPs@BR group. Bar = 50 µm.

Then, we detected the relative mRNA levels of pro-inflammatory cytokines in mouse cornea tissue. Following 7 days of treatment, the BR, NPs@BR, and cNPs@BR groups exhibited effective inhibition of IL-1β, IL-4, IL-6, and MMP-9, whereas cNPs@BR showed the most significant suppression (Figs. 8D–G). PAS staining revealed that the goblet cell number was reduced to 8.2 ± 3.3 after PBS treatment. However, cNPs@BR and Xiidra treatment could both effectively restore goblet cell number to 47.7 ± 2.5 and 43.2 ± 2.9, respectively (Fig. 8H). H&E staining revealed that the central corneal epithelial thickness was reduced to 18.2 ± 0.46 µm after PBS treatment. However, cNPs@BR and Xiidra treatment could both effectively restore central corneal epithelial thickness to 37.6 ± 0.58 µm and 38.1 ± 0.28 µm, respectively. Meanwhile, the peripheral corneal epithelial thickness was reduced to 12.1 ± 0.65 µm after PBS treatment. However, cNPs@BR and Xiidra treatment could both effectively restore peripheral corneal epithelial thickness to 22.5 ± 0.47 µm and 21.6 ± 0.58 µm, respectively (Fig. 8I). Additionally, immunofluorescent images showed significant 4-HNE expression in the cornea of the PBS groups. However, treatment with cNPs@BR and Xiidra led to a substantial reduction in corneal 4-HNE expression (Fig. 8J). All these findings revealed that cNPs@BR effectively restored conjunctival goblet cells and inhibited inflammatory response and oxidative damage in the ocular surface of DED. 

To elucidate the molecular mechanisms of cNPs@BR in treating DED, the transcriptomic profiles of corneal tissues in DED mice with PBS and cNPs@BR treatment were analyzed at day 7. The data revealed that 16,270 shared genes between BAC+PBS group and BAC+cNPs@BR group. The volcano plot indicated 309 DEGs downregulated and 452 DEGs upregulated (Supplementary Fig. S5). GO functional enrichment analysis unveiled that differential genes associated with the mitochondrial respiratory chain and mitochondrial membrane protein expression were enriched among the top 10 cellular components, whereas genes related to inflammatory responses and oxidative stress responses were enriched among the top 10 biological processes (Supplementary Fig. S6). KEGG pathway analysis and GSEA revealed mitophagy (listed 5) among the top 20 pathways was enriched between the BAC+PBS group and the BAC+cNPs@BP treated group. Moreover, the significantly dysregulated genes in mitophagy pathway with fold changes ≥ 2 were presented as the heatmap (Figs. 9A–C). 

Figure 9.

 

The cNPs@BR promoted mitophagy in DED mice. (A) KEGG pathway enrichment analysis highlighting the significant enrichment of mitophagy pathway in mouse corneas of the BAC group and the cNPs@BR+BAC group. (B) Heatmap depicting differentially expressed genes in the mitophagy pathway. (C) GSEA map illustrating the enrichment of mitophagy genes. (D) The relative mRNA levels of mitophagy-related genes in mouse corneas. All data are mean ± SEM (n = 4). ***P < 0.001, versus the BAC group. (E) Relative expression of key mitophagy proteins (LC3, P62, Pink1, and Parkin) in mouse corneas. (F) Immunohistochemical (IHC) staining depicting PINK1 positive cell rate in corneal epithelium. All data are mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, versus the control group; #P < 0.05, versus the BAC+PBS group; &P < 0.05, versus the BAC+cNPs@BR group. Bar = 50 µm. (G) Confocal images of the co-localization of LC3B (red channel) and TOMM20 (mitochondria marker, green channel) in HCECs exposed to 450 mOsm medium for 24 hours. Cells were pretreated with 20 µM cNPs@BR for 2 hours. All data are mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, versus the control group; #P < 0.05, versus the hyperosmolar group; &P < 0.05, versus the hyperosmolar+cNPs@BR group. Bar = 25 µm.

As illustrated in Figure 9D, the relative mRNA levels of mitophagy-related genes, including Pink1, Atg5, Mnf2, and Map1lc3b, in the cornea tissue of BAC+cNPs@BR group were significantly upregulated compared to that of the BAC+PBS group, whereas Bnip3 and Sqstm1 were downregulated. The mitophagy pathway was further activated by cNPs@BR instillation in the cornea, evidenced by the higher expression of LC3B, PINK1, and Parkin with the lower level of P62 when compared to the cornea with PBS treatment (Fig. 9E). As shown by IHC data, the PINK1 intensity in the corneal epithelium in BAC+PBS mice was higher than that of the normal mice, whereas cNPs@BR further enhanced PINK1 positivity (Fig. 9F). Furthermore, the LC3B fluorescence intensity in HCECs upon hyperosmolar environment was higher than that of the control group, whereas cNPs@BR further augmented LC3B fluorescence intensity and was co-localized with TOMM20 (the marker of mitochondria; Fig. 9G). All these findings indicated that cNPs@BR ameliorated ocular surface damage induced by 0.2% BAC in mice by activating the mitophagy pathway. In contrast, Xiidra did not influence mitophagy activity. 

Because Pink1, the classic gene of mitophagy, was significantly enriched in the cornea tissue of the BAC+cNPs@BR group and the BAC+PBS group, we further investigated whether cNPs@BR protects the ocular surface against mitochondrial damage in a PINK1-dependent manner. The data revealed that cNPs@BR protected HCECs against mitochondrial dysfunction by decreasing mitochondrial ROS levels (Fig. 10A), increasing resting OCR, ATP turnover, and respiratory capacity (Fig. 10B), and restoring mitochondrial membrane potential (Fig. 10C) under hyperosmolarity. Moreover, cNPs@BR promoted mitochondrial biogenesis by increasing the protein levels of NRF1, PGC1α, and mtTFA and modulated mitochondrial dynamics by decreasing mitochondrial Drp1 content and increasing mitochondrial Mfn2 content (Fig. 10D). However, PINK1 knockdown largely abolished the protective effects of cNPs@BR. 

Figure 10.

 

The cNPs@BR restored mitochondrial homeostasis by activating PINK1-dependent mitophagy. HCECs were transfected with PINK1 siRNA or scramble siRNA. (A) Representative confocal images of mitochondrial ROS (labeled by MitoSOX probe, red channel). (B) Mitochondrial oxygen consumption rates (OCRs) were detected via Seahorse mitochondrial stress test. Respiration graphs were calculated by response to oligomycin, FCCP, and Rotenone/Antimycin A. (C) The representative diagram of the mitochondrial membrane potential determined by JC-1 staining. (D) Relative expression levels of proteins involved in mitochondrial biogenesis (NRF1, PGC1α, and mtTFA) and mitochondrial dynamics (Drp1 and Mfn2) detected by Western blot assay. All data are mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, versus the control group; #P < 0.05, versus the hyperosmolar group; &P < 0.05, versus the hyperosmolar+siPINK1+cNPs@BR group. Bar = 10 µm.

In addition, cNPs@BR inhibited the expression of 4-HNE and NOX4 (Fig. 11A) and intracellular ROS levels (Fig. 11B) and suppressing the levels of IL-1β, IL-6, and TNF-α (Figs. 11C–E) in hypertonicity-damaged cells. However, knockdown of Pink1 canceled out the anti-oxidative stress and anti-inflammatory effects of cNPs@BR. All the abovementioned findings suggested that cNPs@BR protected ocular surface against oxidative stress and inflammation via PINK1-mediated mitophagy. 

Figure 11.

 

The cNPs@BR protected against oxidative stress and inflammation by activating PINK1-dependent mitophagy. HCECs were transfected with PINK1 siRNA or scramble siRNA. (A) Representative confocal images and fluorescence quantification of 4-HNE (red channel) and NOX4 (green channel). (B) Determination of intracellular ROS levels by flow cytometry. (C–E) The levels of IL-1β, IL-6, and TNF-α in the supernatants of different groups of cells were detected via ELISA assay. All data are mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, versus the control group; #P < 0.05, versus the hyperosmolar group; &P < 0.05, versus the hyperosmolar+siPINK1+cNPs@BR group. Bar = 25 µm.

Biocompatibility Assessment of BR Nanodrugs

In this study, we assessed the biocompatibility of BR nanodrugs. The BR NPs exhibited no apparent cytotoxicity within the concentration of 500 µM in both RAW264.7 cells and HCECs (Supplementary Fig. S7). After instilling various BR nanodrugs (20 µM) twice daily for 28 consecutive days, the H&E and TUNEL immunofluorescence staining assay revealed no discernible pathological alterations in the eyeballs and multiple organs, including the lungs, liver, kidneys, and heart, among the mice in each group (Supplementary Fig. S8, Figs. 12A, 12B). Furthermore, key parameters of hepatic and renal functions in mouse blood samples, including ALT, total bilirubin, BUN, Cr, and glucose levels, showed no significant difference between each group (Fig. 12C). 

Figure 12.

 

The biocompatibility of different BR nanodrugs. Eye drops were topically instilled twice daily for 28 consecutive days in mouse eyes. (A) H&E staining of cornea, iris, ciliary body, and retina. (B) Terminal deoxynucleotidyl transferase (TdT)-dUTP nick-end labeling (TUNEL) immunofluorescence staining of eyeballs. (C) The parameters of hepatic and renal function in blood samples. Bar = 200 µm.

Discussion

In the present work, we prepared the cRGD-conjugated PEG-PLGA nano-platform (cNPs) to deliver BR via topical administration. The cNPs prolonged drug retention time on the ocular surface and improved the cellular uptake efficiency. Further, cNPs@BR decreased intracellular ROS levels and inflammatory cytokines through NOX4/Nrf2/HO-1 and NF-κB/iNOS pathways in both LPS-stimulated RAW264.7 cell model and hypertonic HCECs model. Consequently, cNPs@BR effectively ameliorated oxidative stress and inflammatory response in the ocular surface epithelium and restored conjunctival goblet cells by activating PINK1-mediated mitophagy in a 0.2% BAC-induced mouse model of DED. Therefore, cNPs@BR may be a potential candidate for the treatment of DED (Fig. 13). 

Figure 13.

 

Illustration of the preparation of cNPs@BR for alleviating DED. The anti-oxidative and anti-inflammatory cNPs@BR with prolonged ocular surface retention time and its therapeutic effect and mechanism by activating PINK1-mediated mitophagy and breaking the vicious cycle in DED.

Upon physiological conditions, the NOX4/Nrf-2/HO-1 axis plays a crucial role in regulating cellular redox homeostasis.³⁰ Yu et al. confirmed that the Nrf2 and HO-1 expression levels in the cornea of a PM2.5 exposure-induced DED mouse model were decreased compared to those in the healthy group. Conversely, overexpression of Nrf2 in corneal epithelial cells significantly attenuates PM2.5-induced ROS production.³¹ Additionally, various antioxidants can mitigate DED by increasing Nrf2 and HO-1 expression levels.^12,32,33 In our data, LPS promotes NOX4 expression in RAW264.7 cells while inhibiting Nrf2 nuclear translocation and HO-1 expression. In contrast, cNPs@BR restores NOX4, nuclear Nrf2, and HO-1 expression levels in RAW264.7 cells and exhibits remarkable antioxidant effects. 

In response to multiple stimuli, Iκkα and Iκbα were phosphorylated, which in turn promotes NF-κB subunit p65 phosphorylation and nuclear translocation.^34,35 Thereafter, p65 binds to the promoter of several pro-inflammatory genes, including iNOS. Multiple studies have confirmed that in DED models, NF-κB p65 nuclear translocation promotes the secretion of inflammatory cytokines and activates iNOS expression, leading to the loss of conjunctival goblet cells.^36,37 In the present study, our findings revealed that LPS stimulation induced the phosphorylation of Iκkα, Iκbα, and p65 and nuclear translocation of p65 in RAW264.7 cells, which in turn directly activated iNOS expression by binding p65 to the iNOS promoter region and induced the secretion of inflammatory cytokines. However, cNPs@BR inhibited the activation of the NF-κB/iNOS pathway and the secretion of inflammatory factors. Additionally, cNPs@BR further inhibited inflammation in corneal epithelial cells and restored the number of conjunctival goblet cells in the hypertonic HCECs model and the mouse model of DED. 

In BAC-induced mouse DED, we further confirm the protective effect of cNPs@BR on mitigating ocular surface damage by activating mitophagy pathway in corneal epithelium through RNA sequencing and RNAi techniques. Mitophagy is critical for regulating mitochondrial quality control in multiple cells.³⁸ Recently, several reports have found a correlation between the mitophagy and DED, whereas the results are controversial. Peng et al. found that the AMPK/MFF pathway promotes inflammatory responses by activating mitophagy.¹⁴ In contrast, Xu et al. confirmed that the activation of mitophagy protects corneal epithelial cells from inflammation and oxidative stress.³⁹ The differences in these results may be related to different cell models and the dual role of mitophagy in diseases. Our study found that mitophagy was activated in the BAC-damaged cornea, but the autophagic flux was blocked. However, cNPs@BR promoted the expression of mitophagy markers (LC3B, PINK1, and Parkin) in the cornea and facilitated the autophagic flux, thereby protecting the ocular surface epithelium against inflammation and oxidative stress. Additionally, gene silencing of PINK1 ameliorated the protective effect of cNPs@BR in LPS-damaged RAW264.7 cells. These results indicated that cNPs@BR alleviated DED by PINK1-mediated mitophagy. 

Despite of the significant anti-inflammatory and antioxidant effects, BR solution is difficult to use clinically. This is because BR is poorly soluble in water and is unstable when exposed to light.⁴⁰ Additionally, conventional eye drops are limited by tear washing, drug dilution, and metabolic degradation, resulting in low bioavailability of the drug on the ocular surface.⁴¹ However, breakthroughs in bionanotechnology offer a new direction for the treatment of DED. Currently, 0.09% CsA nanomicelle eye drops (Cequa) and 0.05% CsA nanoemulsion eye drops (TJ Cyporin) have been approved for clinical use.^42,43 In the present work, cRGD peptide was conjugated to PEG-PLGA nanoparticle to increase the retention time of the nanodrug on the ocular surface and enhance cellular uptake through receptor-mediated endocytosis.^44,45 Notably, we also compared the therapeutic effect of cNPs@BR with Xiidra in the murine model of DED. Several animal studies and clinical trials confirmed that Xiidra inhibits the binding of T cell and ICAM-1, decreases the release of pro-inflammatory cytokines, thus showing significant improvement in the ocular surface disease index (OSDI) and dry eye symptoms.^46–48 In the present study, we found no significant difference between cNPs@BR and Xiidra on the treatment of murine model of DED, which underscores the clinical potential of cNPs@BR. Although nano-delivery platform overcomes many drawbacks of conventional eye drops, further analysis of pharmacokinetic parameters of various candidates are acquired, including particle size, charge, composition, degradation rate, and cumulative release rate of nanodrugs. Moreover, large-scale manufacturing and refining clinical trial data are an urgent need. In addition, long-term biocompatibility, including systemic and ocular tissue toxicity, needs further confirmation. 

In conclusion, this study identified that the cRGD peptide prolonged the retention time and enhanced cellular uptake of cNPs@BR on ocular surface. Moreover, cNPs@BR effectively alleviated oxidative stress and inflammatory response by modulating NOX4/Nrf2/HO-1 and NF-κB /iNOS pathway in both LPS-stimulated RAW264.7 cell model and hypertonic HCECs model. In addition, cNPs@BR effectively ameliorated oxidative stress and inflammatory response in the ocular surface epithelium and restored conjunctival goblet cells by activating PINK1-mediated mitophagy in a 0.2% BAC-induced mouse model of DED. 

Acknowledgments

Supported by Natural Science Foundation of Shanghai (24ZR1459200). 

All data utilized and/or analyzed in this work can be obtained from the corresponding author. 

Disclosure: Y. Huang, None; L. Wang, None; H. Jin, None