ROS-Responsive Nanoparticles with Antioxidative Effect for the treatment of Diabetic Retinopathy

doi:10.21203/rs.3.rs-4134272/v1

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ROS-Responsive Nanoparticles with Antioxidative Effect for the treatment of Diabetic Retinopathy

https://doi.org/10.21203/rs.3.rs-4134272/v1

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Diabetic retinopathy (DR) is a prevalent microvascular complication of diabetes. While current clinical treatments focus on later stages of the disease, early intervention is crucial to impede its progression. Essential oils derived from Fructus Alpiniae zerumbet (EOFAZ) have shown promise in protecting against high glucose (HG)-induced Müller cell activation and the development of DR. In this study, we designed a reactive oxidative species (ROS)-responsive drug delivery system (NPS_PHE@EOFAZ) to target early DR stages and combat oxidative stress. Our nanoparticles were engineered to detect and respond to elevated oxidative stress levels, effectively transporting EOFAZ into HG-exposed Müller cells. The NPS_PHE@EOFAZ formulation exhibited significant efficacy in inhibiting abnormal cell growth, reducing oxidative stress, and alleviating inflammation in these cells. Moreover, in vivo experiments on diabetic mice with DR demonstrated that NPS_PHE@EOFAZ mitigated early pathological changes by reducing oxidative stress and inflammation. Additionally, the NPS_PHE@EOFAZ formulation minimized pathological damage in vital organs such as the heart, liver, spleen, lung, and kidney. These results highlight the potential of NPS_PHE@EOFAZ as a promising antioxidant for early intervention in DR pathogenesis.

ROS-responsiveness

Diabetic retinopathy

Nanoparticle

Fructus Alpiniae zerumbet

Diabetic retinopathy (DR) is acknowledged as the primary ocular complication of diabetes mellitus, often leading to visual impairment among individuals of working age in developed countries.¹ Various treatment approaches, such as laser surgery and anti-vascular endothelial growth factor therapy, are utilized for DR management, focusing mainly on advanced disease stages and carrying notable adverse effects.^2–3 Nevertheless, as DR follows an irreversible course, it is crucial to implement an effective approach for early intervention in the pathological progression of DR.

Müller cells, as the predominant glial cell type in the retina, play a crucial role in the pathophysiology of Diabetic Retinopathy (DR), ultimately leading to visual impairment.^{1, 4–6} Under high-glucose (HG) conditions, Müller cells undergo activation, triggering excessive oxidative stress, which in turn leads to cellular inflammation, dysfunction, death, tissue damage, and inflammatory responses.^7–9 Excessive oxidative stress is a key mechanism in the development of DR, emphasizing the importance of reducing oxidative stress as a fundamental approach for early prevention and treatment of the disease. Antioxidants are commonly used to alleviate oxidative stress in various conditions due to their well-documented antioxidative properties.^10–11 For instance, Essential oil extracted from Fructus Alpiniae zerumbet (EOFAZ) demonstrates potent antioxidant effects.^12–13 Previous research has shown the protective effects of EOFAZ against HG-induced Müller cell activation and the pathogenesis of DR.^{5, 14} However, EOFAZ's poor water solubility hinders its distribution in the body and limits its clinical applicability.

With the advancements in the understanding of pathogenesis and molecular mechanisms underlying retinal diseases, various novel therapeutic approaches have been employed for their treatment, including topical administration, systemic delivery, periocular injection, and intravitreal injection.^15–16 Among these strategies, stimulus-responsive polymeric nanocarriers have emerged as versatile platforms to address retinal diseases.^{15, 17} A thermo-responsive polymeric nanocarrier has been developed to enable sustained release in the retina without inducing inflammation, gliosis or apoptosis.¹⁸ Lai et al. also developed a biodegradable drug delivery system that exhibited prolonged drug release and enhanced antioxidative effects in chronic ocular hypertension.¹⁹ Consequently, polymeric nanoparticles have been used to encapsulate poorly soluble drugs to enhance solubility, improve delivery efficiency, reduce drug toxicity, and prolong ocular residence time.^20–25

In response to the challenges presented by EOFAZ, we engineered ROS-responsive polymeric nanoparticles for the precise delivery of EOFAZ, examining their efficacy in mitigating the initial pathological mechanisms of DR. Comprehensive analyses were conducted on the dimensions, structure, and reactivity of these nanoparticles. Subsequently, EOFAZ was successfully encapsulated within the nanoparticles, and their antioxidative potential was assessed in vitro using HG-induced Müller cells. Ultimately, the therapeutic impact of these nanoparticles on early-stage DR was investigated.

2.1 Materials

2-Hydroxyethyl methacrylate (HEMA), poly(ethylene glycol) methacrylate (PEGMA, Mn ≈ 300), 4-(hydroxymethyl) phenylboronic acid pinacol ester, 4-cyanopentanoic acid dithiobenzoate (CPADB), 4-hydroxyphenylboronic acid pinacol ester, and acryloyl chloride were purchased from Innochem Technology Co., Ltd. HEMA and PEGMA were filtered using an alumina column prior to use. 2,2′-Azobis-(isobutyronitrile) (AIBN, 98%), 4’,6-diamidino-2-phenylindole (DAPI), and 3-[4,5-dime-thylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Heowns Biochem Technologies LLC (Tianjin, China). Malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) was bought from KeyGen Biotech Co. Ltd. (Jiangsu, China). ELISA kits of IL-6 and IL-1β were obtained from Shanghai Fanyin Biotechnology Co., Ltd. All other reagents used in this study were of analytical grade and used as received.

2.2 Synthesis of copolymer PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀

2.2.1 Synthesis of PBAE

4-Hydroxyphenylboronic acid pinacol ester (5.85 g, 25 mmol), triethylamine (TEA, 3.05 g, 30 mmol), and anhydrous dichloromethane (DCM, 30 mL) was added to a dry flask in an ice bath. Acryloyl chloride (2.7 g, 30 mmol) dissolved in anhydrous DCM (2.5 mL) was added dropwise to the flask. The mixture was reacted for 10 h at 25°C. Afterward, the mixture was filtered and concentrated using rotary evaporation. The resulting residue was extracted with diethyl acetate, washed with salt water, and dried with anhydrous magnesium sulfate. The crude product was then purified using silica gel column chromatography with a petroleum ether/diethyl acetate (30:1, v/v) eluent. The eluent was collected and concentrated to obtain the product, named as PBAE.

2.2.2 Synthesis of copolymer PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀

PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀ was synthesized via the reversible addition-fragmentation chain transfer (RAFT) method. The polymerization used PBE, HEMA, and PEGMA as the monomers, CPADB as a RAFT agent, and AIBN as an initiator. PBE (432.23 mg, 3 mmol), HEMA (130.14 mg, 1 mmol), PEGMA (0.41 mL, 3 mmol), CPADB (13.97 mg, 0.1 mmol), and AIBN (3.28 mg, 0.02 mmol) were dissolved in 1,4-dioxane (2.0 mL). The mixture was purged with argon for 30 min and then incubated at 70°C for 48 h. After the reaction was terminated in an ice bath, PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀ was obtained through precipitation in hexane, followed by washing and drying.

2.3 Preparation of nanoparticles

PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀ was dissolved in dimethyl sulfoxide (DMSO). Deionized water was added dropwise into the copolymer solution under ultrasound. The resulting suspension was dialyzed against deionized water for 24 h using a dialysis tube (MWCO = 3500). The resulting nanoparticles were named NPS_PHE.

A similar method was used to prepare EOFAZ-loaded nanoparticles. PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀ and EOFAZ were dissolved in DMSO. Deionized water was added dropwise into the mixed solution and stirred for 4 h. The suspension was dialyzed against deionized water for 24 h using dialysis tube (MWCO = 3500). As a result, EOFAZ-loaded nanoparticles were obtained and named NPS_PHE@EOFAZ.

2.4 Characterizations of copolymer and nanoparticles

The ¹HNMR spectra were measured using a Varian Unity-plus 400 NMR spectrometer at room temperature. Dynamic light scattering (DLS) with a Brookhaven 90Plus PALS instrument was used to measure the hydrodynamic diameter (D_H) of the nanoparticles. The morphology of the nanoparticles was observed using a transmission electron microscope (TEM, Philips EM400ST).

2.5 The stability of NPS_PHE and NPS_PHE@EOFAZ

The stable assays of NPS_PHE and NPS_PHE@EOFAZ were conducted by incubating them in water or DMEM with 10% fetal bovine serum (FBS) medium. The size measurements were performed using the DLS method. Each sample was measured in triplicate, and the results were presented as the mean ± standard deviation (n = 3).

2.6 Determination of critical micelle concentration (CMC).

Nile red fluorescent method was used to measure CMC value.²⁶ A series of PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀ solutions with different concentrations (ranging from 6.0 × 10^− 4 to 0.6 mg/mL) were prepared. Nile red (1 × 10^− 6 mol/L) was mixed with the polymer solutions. The mixture was treated under ultrasonic (10 min) and then kept in the dark for 2 h. Fluorescence spectra were measured using a microplate reader, with an emission spectrum recorded from 570 to 750 nm and an excitation wavelength at 550 nm. The CMC was determined by finding the intersection of tangents to the two linear portions of the graph depicting fluorescence intensity against polymer concentration.

2.7 ROS responsiveness of NPS_PHE

The ROS responsiveness of nanoparticles was tested using a UV spectrometer. NPS_PHE was dispersed in two colorimetric vials. One vial was supplemented with H₂O₂ (10 mM), while the other was mixed with PBS buffer. After thorough mixing, the absorbance of nanoparticles in the H₂O₂ solution was measured during 24 h using the UV spectrometer. Both samples were taken photos after the treatment for 24 h.

2.8 Cell viability

The cell viability of EOFAZ, NPS_PHE, and NPS_PHE@EOFAZ was investigated using Müller cells. Müller cells were cultured in DMEM containing 5.5 mM of glucose. The cells were seeded into a 96-well plate and cultured for 48 h at 37°C. A cell proliferation model was induced by treating the cells with a 30 mM high glucose (HG) solution for 48 h. EOFAZ, NPS_PHE, and NPS_PHE@EOFAZ were diluted with the HG medium to the predetermined concentrations and added to the wells. The cells were then incubated for an additional 24 h. After the incubation period, MTT solution (5 mg/mL, 10 µL) was added to each well and the plate was further incubated for 4 h. Subsequently, the medium was removed, and 150 µL of DMSO was added to dissolve the formazane crystals. The optical density was measured using a microplate reader at 492 nm. All samples were analyzed in quintuplicate, and the results were reported as the mean ± standard deviation (n = 5). The cell viability of normal Müller cells was also performed using the similar method, except that the Müller cells was treated without HG medium.

2.9 Cellular uptake

To investigate the cellular uptake of NPS_PHE, FITC was loaded into the nanoparticles, resulting in NPS_PHE@FITC. Müller cells were seeded into a 12-well plate and cultured for 48 h at 37°C. After treated with HG for 48 h, the Müller cells were treated with EOFAZ/FITC or NPS_PHE@EOFAZ/FITC for 4 h at 37°C. Then, the cells were stained with Mito-Tracker Orange for 30 min and DAPI for 10 min. The cellular uptake images were taken using a fluorescent microscope (Leica Dmi8).

2.10 Cellular ROS measurement

Müller cells were seeded into 12-well plates and cultured for 48 h. After treated with HG for 48 h, the Müller cells were treated with PBS, EOFAZ-L (0.25 µg/L), EOFAZ-M (0.5 µg/L), or NPS_PHE@EOFAZ-L (EOFAZ: 0.25 µg/L) for 4 h at 37°C. Then, the cells were stained with DCFH-DA dye for 30 min and observed using a fluorescent microscope. The cellular ROS in normal Müller cells was also performed using the similar method, except that the Müller cells was treated without HG medium.

2.11 The detection of SOD and MDA

Müller cells were cultured in 6-well plates and cultured for 48 h. After treated with HG for 48 h, the Müller cells were treated with PBS, EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L for 4 h at 37°C. Then, the cells were lysed and centrifuged at 10 000 rpm for 10 min. The supernatants were collected to measure the levels of MDA, GSH, and SOD according to the manufacturer’s instructions.

2.12 The inflammatory response

Müller cells were seeded into 6-well plates and cultured for 48 h. After treated with HG for 48 h, the Müller cells were treated with PBS, EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L for 4 h at 37°C. Total protein extraction from Müller cells was performed using RIPA lysis buffer according to the manufacturer's instructions. The resulting lysates were obtained through centrifugation, and protein concentrations were quantified using a BCA kit. Inflammatory cytokines of IL-6 and IL-1β were detected using ELISA Kits following the manufacturer's instructions.

2.13 In vivo studies

Six-week-old male C57BL/6J mice were purchased from Guizhou Medical University Laboratory Animal Center, and bred in a pathogen-free conditions at the central animal facility of animal experiment center in Guizhou Medical University. All protocols for animal experiments were performed according to the guidelines of Council for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Public Health, Government of China.

Type 2 diabetic model mice were purchased from Guizhou Medical University Laboratory Animal Co., Ltd. in Guizhou, China. The mice were raised for 8 weeks. The diabetic mice were randomly assigned to three groups: the HG group (n = 6), EOFAZ-L group (n = 6), EOFAZ-M group (n = 6), and NPS_PBE@EOFAZ-L group (n = 6). The three mice were administrated with sterile PBS, EOFAZ-L (0.15g/kg), EOFAZ-M (0.3g/kg), and NPS_PBE@EOFAZ-L (EOFAZ: 0.15g/kg), respectively. A control group of healthy mice was administrated with sterile PBS. All mice were given the corresponding agents once a day via intragastric administration. After two months, all mice were sacrificed. Their eyes, hearts, livers, spleens, lungs, and kidneys were collected. Eye samples were fixed with eyeball fixed liquid, while the other primary organs were fixed with 4% paraformaldehyde. Hematoxylin and eosin (H&E) staining was performed on all fixed organs for histological evaluation. Fresh eyeball tissues were homogenized and centrifuged to extract protein, which was then measured using a BCA protein assay kit. The levels of MDA, GSH, and SOD were examined according to the manufacturer's instructions. The levels of IL-1β and IL-6 were measured using an ELISA kit following the manufacturer's instructions.

3.1 Preparation and characterization of the copolymer and nanoparticles

The copolymer PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀ was synthesized via RAFT polymerization, as outlined in Scheme 1. The chemical structure of PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀ was elucidated using 1H NMR spectroscopy (Fig. 1A). Comparative analysis with the ¹H NMR spectra of the individual HEMA, PEGMA, and PBE monomers (Figures S1-S3) revealed the absence of double bond proton peaks, retention of characteristic peaks from the three monomers, and emergence of new proton peaks from the main chain at 0.8–2.5 ppm in the copolymer spectrum (Fig. 1A). Consequently, the ¹H NMR data validated the successful synthesis of PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀. Functioning as an amphiphilic copolymer, PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀ demonstrated the ability to self-assemble into nanoparticles using a nano-precipitation technique, denoted as NPS_PHE. The nanoparticles were composed of a hydrophilic PEGMA/HEMA shell and a hydrophobic PBE core. The PEGMA component improved water solubility and extended circulation time, while HEMA promoted cell adhesion. The PBE core was designed to degrade in the presence of excessive ROS, facilitating effective drug delivery in disease microenvironments.^27–31 The size and distribution of the nanoparticles (NPS_PHE) were determined using DLS, revealing an average size of 163.2 ± 4.87 nm (Fig. 1B). TEM analysis showed that NPS_PHE exhibited spherical structures (Fig. 1C). The CMC of NPS_PHE was measured using the Nile red fluorescent method, yielding a value of 0.039 mg/mL (Fig. 1D). To create drug-loaded nanoparticles, the PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀ copolymer was combined with EOFAZ to form self-assembled nanoparticles (NPS_PHE@EOFAZ) via the nano-precipitation method. The size of NPS_PHE@EOFAZ was 189 ± 0.54 nm (Fig. 1E), with a spherical morphology (Fig. 1F). The observed changes in size and morphology of NPS_PHE@EOFAZ compared to NPS_PHE were attributed to the incorporation of EOFAZ into the nanoparticles. Due to the hydrophobic nature of EOFAZ, its dialysis out of the aqueous solution during preparation was minimal, leading to the assumption that the final amount of EOFAZ in NPS_PHE@EOFAZ was the initially added quantity.³² Consequently, the drug loading and encapsulation efficiency of EOFAZ were not further investigated based on previous laboratory findings.

The stability of NPS_PHE and NPS_PHE@EOFAZ was assessed in various media, as depicted in Fig. 1G-H. Both NPS_PHE and NPS_PHE@EOFAZ were stored in PBS at 4°C, maintaining their size for 7 days. In DMEM (with 10% FBS) at 37°C, the particle size remained constant for 48 h. These findings demonstrate the robust stability of NPS_PHE and NPS_PHE@EOFAZ across different media. Gavage administration, known for its patient-friendly and long-term use benefits, facilitates drug absorption through the gastrointestinal tract into systemic circulation, resulting in reduced drug intake and bioavailability. For effective gastric medication, tolerance to the gastrointestinal tract's acidity and alkalinity is crucial. Therefore, we simulated strong acidic stomach conditions using a hydrochloric acid buffer (pH 1.5), and intestinal conditions with acetic acid buffer (pH 4.6) and phosphate buffer (pH 6.8) to evaluate NPS_PHE and NPS_PHE@EOFAZ stability. Both formulations remained stable for at least 18, 24, and 48 h in pH 1.5, 4.6, and 6.8 buffer solutions, respectively. Although stability in acidic environments was shorter compared to neutral conditions, the duration was adequate for gastrointestinal absorption. Consequently, the NPS_PHE@EOFAZ formulations developed are suitable for oral administration.

3.2 Cellular uptake

The biotoxicity of NPS_PHE@EOFAZ was assessed through experiments conducted on Müller cells using the MTT viability assay (Fig. 2A-B). Three concentrations of EOFAZ (0.25, 0.5, and 1 µg/L) were selected based on prior laboratory investigations. Initially, the cytotoxic effects of EOFAZ and NPS_PHE@EOFAZ on normal cells were examined across varying concentrations. Results depicted in Fig. 2A indicate that both EOFAZ and NPS_PHE@EOFAZ exhibited no toxicity within the tested range of 0.25 to 1 µg/L.⁵ Subsequently, a HG concentration of 30 mM, as identified in previous research, was employed to induce cellular damage. As illustrated in Fig. 2B, Müller cells treated with HG demonstrated aberrant proliferation, which was mitigated upon treatment with EOFAZ and NPS_PHE@EOFAZ. Notably, the cell viability at 1 µg/L EOFAZ was lower compared to 0.25 and 0.5 µg/L, leading to the selection of EOFAZ-L (0.25 µg/L), EOFAZ-M (0.5 µg/L), and NPS_PHE@EOFAZ-L (EOFAZ: 0.25 µg/L) for subsequent assays.

The impact of NPS_PHE@EOFAZ on HG-induced Müller cell morphology was assessed using the Giemsa assay (Fig. 2C). In the control group (Con group), cells exhibited elongated morphology with well-defined and evenly distributed intercellular spaces. Upon HG induction, cell proliferation increased, leading to blurred morphology and unclear intercellular spaces. Treatment with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L resulted in amelioration of abnormal proliferation and morphology, with NPS_PHE@EOFAZ-L demonstrating superior efficacy compared to EOFAZ-L in mitigating HG-induced Müller cell damage. These findings suggest that NPS_PHE@EOFAZ suppresses abnormal proliferation and enhances the morphology of HG-induced Müller cells.

The water solubility of EOFAZ is limited, prompting its encapsulation into nanoparticles to enhance its solubility. The delivery of EOFAZ via nanomaterials was investigated using normal and HG-treated Müller cells. To facilitate tracking, FITC, a green fluorescent isothiocyanate, was linked to EOFAZ and NPS_PHE@EOFAZ, yielding FITC-labeled EOFAZ and NPS_PHE@EOFAZ. These compounds were then co-cultured with normal (Con) and HG-induced cells for 4 h. Mitochondrial fluorescent probes and DAPI were employed for mitochondria and nuclear staining, respectively. The results depicted in Fig. 2D-F reveal that NPS_PHE@EOFAZ exhibited heightened green fluorescence in HG-treated cells, indicating enhanced drug uptake compared to other groups. Notably, NPS_PHE@EOFAZ demonstrated superior green fluorescence signals relative to the EOFAZ group in both normal and HG-induced cells, underscoring its targeted drug delivery capability. Furthermore, NPS_PHE@EOFAZ exhibited increased cellular uptake in HG-induced cells compared to normal cells. Under HG conditions, Müller cells exhibit elevated oxidative stress levels, leading to heightened cytomembrane permeability that facilitates cellular drug internalization.³³ Conversely, EOFAZ, being hydrophobic with poor solubility, hinders cellular uptake. In contrast, nanoparticles show promise in improving the cellular uptake of hydrophobic drugs.¹⁵ Consequently, the incorporation of EOFAZ into NPS_PHE resulted in enhanced cellular uptake owing to the superior drug delivery capacity of the nanoparticles.

3.3 ROS measurement

Müller cells, the primary glial cells spanning the entire retinal layer, normally play a role in glycogen synthesis and storage, providing nourishment to retinal neurons.^34–36 However, underHG conditions, Müller cells become activated, leading to the generation of oxidative stress and inflammation.^37–38 Initially, in the early stages of HG, Müller cells produce oxidative stress, triggering the expression of various inflammatory factors. Prolonged HG exposure results in the excessive buildup of ROS in the retinas of DR patients, causing damage to retinal endothelial cells and eventual vision loss.^39–40 The accumulation of ROS, coupled with their inadequate clearance, may represent an irreversible factor in DR progression.⁴¹ Therefore, reducing oxidative stress in HG-induced Müller cells is crucial. For instance, Tu et al. demonstrated that melatonin activates the Sirt1 pathway, mitigating oxidative stress and inflammation in Müller cells during DR, thereby safeguarding the retina against diabetes-induced harm.¹ To this end, NPS_PHE, comprising a ROS-responsive PBE core, is engineered to selectively release drugs within environments of heightened oxidative stress.

To assess the ROS responsiveness of NPS_PHE, it was co-incubated with an H₂O₂ medium, and the absorbance was measured at 600 nm using a UV spectrometer. The OD₆₀₀ value and turbidity of the NPS_PHE suspension decreased over time after H₂O₂ treatment, as depicted in Fig. 3A-B, indicating its sensitivity to ROS. ROS levels in HG-induced Müller cells were determined using the DCFH-DA probe. Following a 4-h co-culture of EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L with normal and HG-induced cells, DCFH-DA dye was utilized for cell staining, with results presented in Figs. 3C-D. In normal cells, no significant change in intracellular green fluorescence signal was observed with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L, suggesting no alteration in intracellular oxidative stress. Conversely, HG-induced cells exhibited elevated green fluorescence intensity, indicating increased ROS levels. Treatment with EOFAZ-L, EOFAZ-M, and NPS_PHE@EOFAZ-L led to decreased green fluorescence intensity in HG-induced cells, signifying reduced cellular ROS levels. NPS_PHE@EOFAZ-L demonstrated superior efficacy in alleviating oxidative stress compared to EOFAZ-L. The impact of EOFAZ and NPS_PHE@EOFAZ on HG-induced intracellular oxidative stress was further evaluated through MDA and SOD assays (Fig. 3E-F). HG-induced cells displayed elevated MDA and decreased SOD levels compared to normal cells, indicative of heightened oxidative stress. Treatment with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L resulted in decreased MDA levels and increased SOD levels in HG-induced cells, reflecting reduced oxidative stress. NPS_PHE@EOFAZ-L exhibited better performance in reducing oxidative stress compared to EOFAZ-L, albeit inferior to EOFAZ-M.

The excessive production of ROS triggers the generation of inflammatory mediators and causes cellular damage, establishing a detrimental cycle in the retinal vasculature. To explore the inflammatory response, cell culture media were collected post-treatment with EOFAZ and NPS_PHE@EOFAZ. Levels of IL-6 and IL-1β were quantified using ELISA kits as per the manufacturer's instructions. Results depicted in Fig. 3G-H illustrated elevated IL-6 and IL-1β levels in HG-induced Müller cells, indicating heightened inflammatory activity. Conversely, treatment with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L led to decreased IL-6 and IL-1β levels, signifying potent anti-inflammatory effects. Notably, NPS_PHE@EOFAZ-L exhibited superior efficacy in mitigating inflammatory responses compared to EOFAZ-L. In summary, NPS_PHE@EOFAZ-L demonstrated enhanced antioxidative and anti-inflammatory properties in HG-induced Müller cells relative to EOFAZ-L, likely attributed to improved bioavailability facilitated by nanoparticle-mediated delivery.

3.4 In vivo assays

Due to the toxicity of EOFAZ, mice were orally administered with EOFAZ and NPS_PHE@EOFAZ. Ocular tissues, comprising the cornea, conjunctiva, and retina, possess unique properties that pose challenges for the effective delivery of orally administered drugs. The presence of protective barriers in the eye, including the corneal epithelium and the blood-ocular barrier, presents a significant obstacle.⁴² These barriers function to impede the penetration of potentially harmful substances into the eye. The role of ocular barriers is essential for maintaining eye health, yet it poses challenges for drug delivery into ocular tissues. Oral administration primarily targets systemic drug distribution rather than specific delivery to the eyes. Upon ingestion, drugs are absorbed in the gastrointestinal tract, enter the bloodstream, and distribute throughout the body. Consequently, drug levels reaching the eyes are often limited. This study utilized small animal in vivo imaging to investigate the distribution of NPS_PHE@EOFAZ in mouse eyes. The results showed high concentrations of NPS_PHE@EOFAZ at 3 h post-administration, gradually decreasing over time in eye tissues. NPS_PHE@EOFAZ exhibited higher fluorescence intensity and longer retention compared to EOFAZ in the eyes at specified time points. Following oral administration for 6 h, the fluorescence intensity of EOFAZ-BODIPY in the eyeball decreased to 48% of the 3-h level, while NPS_PHE@EOFAZ-BODIPY maintained a high intensity at 87%. Even after 12 h, although both EOFAZ-BODIPY and NPS_PHE@EOFAZ-BODIPY fluorescence decreased significantly in ocular tissues, NPS_PHE@EOFAZ-BODIPY still showed stronger fluorescence than EOFAZ-BODIPY. These findings suggest that NPS_PHE@EOFAZ can be absorbed and circulated through the gastrointestinal tract, ultimately leading to increased drug accumulation and retention in eye tissues.

DR is a common microvascular complication associated with diabetes, characterized by the leakage and blockage of retinal micro-vessels due to the chronic and progressive nature of the disease. While patients may not initially experience visual impairment during the early stages of metabolic dysfunction and DR, the condition is irreversible, underscoring the critical need for effective treatments in its early phases. Therefore, research focusing on intervening in the early pathological progression of DR and assessing the efficacy of potential treatments is imperative. In this study, diabetic mice were raised for 8 weeks to induce early-stage DR. The investigation aimed to evaluate the impact of NPS_PHE@EOFAZ on the early pathological processes of DR. Analysis, as depicted in Fig. 4A, revealed that the control group displayed intact retinal cell layers with a well-organized structure. Conversely, the DR group exhibited disorganized outer nuclear layer (ONL) and inner nuclear layer (INL) cells, indicative of early-stage DR. Following treatment with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L, the ONL and INL cells demonstrated a more orderly arrangement, suggesting a mitigation of disease severity. Notably, NPS_PHE@EOFAZ-L exhibited superior efficacy compared to EOFAZ-L in ameliorating DR damage. These findings indicate that NPS_PHE@EOFAZ decelerated the pathological progression and improved the pathogenesis of early-stage DR.

The in vivo antioxidant capacity of NPS_PHE@EOFAZ was assessed through MDA, GSH, and SOD assays (Fig. 4B-D). DR mice exhibited elevated MDA levels and reduced GSH and SOD levels, indicating heightened oxidative stress. Treatment with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L resulted in decreased MDA levels and increased GSH and SOD levels, indicating a mitigation of oxidative stress. Levels of IL-6 and IL-1β, markers of inflammatory response, were measured using ELISA kits (Fig. 4E-F). DR mice displayed elevated IL-6 and IL-1β levels. Following treatment with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L, IL-6 and IL-1β levels decreased, suggesting a reduction in inflammatory response. NPS_PHE@EOFAZ-L demonstrated superior antioxidant and anti-inflammatory effects compared to EOFAZ. In summary, NPS_PHE@EOFAZ ameliorated the early pathological progression of DR by alleviating oxidative stress and inflammatory response.

Diabetes, a metabolic disorder marked by elevated blood sugar levels, can lead to chronic damage and dysfunction in multiple tissues, including the eyes, kidneys, heart, blood vessels, and nerves.⁴³ Fluorescence imaging in Figure S4 revealed significant accumulation of NPS_PHE@EOFAZ in the heart, liver, spleen, lungs, and kidneys. Subsequently, H&E staining was employed to examine the pathological alterations in these primary tissues. As depicted in Fig. 6, severe pathological changes were evident in all tissues of DR mice, which were mitigated following treatment with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L. In the cardiac tissue of DR mice, cardiac myocytes exhibited irregular and disordered arrangement, along with irregularly shaped nuclei and noticeable bleeding. Treatment with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L led to an improvement in the arrangement and morphology of myocardial cells. In the hepatic tissue of DR mice, hepatocytes appeared swollen, with large vacuoles in the cytoplasm displacing the nucleus to one side. Following treatment with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L, vacuoles decreased in size and lesion severity improved. In spleen images of DR mice, the distinction between white and red pulp was initially unclear. However, treatment with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L led to a clearer boundary between the white and red pulp, indicating a positive impact on spleen structure. Analysis of lung tissue in DR mice revealed inflammatory cell infiltration, alveolar cavity expansion, and alveolar septa rupture. Treatment with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L resulted in improved alveolar cavity expansion, reduced inflammatory cell infiltration, and significantly decreased alveolar septa rupture. Examination of kidney tissue in DR mice showed irregular and disordered arrangement of epithelial cells in renal tubules. Treatment with EOFAZ-L, EOFAZ-M, or NPS_PHE@EOFAZ-L improved renal lesions, indicating a beneficial effect on kidney tissue. Notably, NPS_PHE@EOFAZ-L exhibited superior improvement in primary tissue damage compared to EOFAZ-L, and comparable results to EOFAZ-M. In conclusion, NPS_PHE@EOFAZ demonstrated efficacy in reducing damage and inflammatory response in the primary organs of DR mice.

We synthesized ROS-responsive nanoparticles (NPS_PHE@EOFAZ) for early intervention in DR. NPS_PHE@EOFAZ demonstrated stability for 7 days in water and 48 h in DMEM with 10% FBS. In vitro studies using MTT and Giemsa assays revealed the efficacy of NPS_PHE@EOFAZ in inhibiting hyperglycemia-induced abnormal proliferation of Müller cells. Cellular internalization assays confirmed efficient delivery of EOFAZ by NPS_PHE@EOFAZ into Müller cells, enhancing cellular uptake. NPS_PHE@EOFAZ exhibited superior antioxidative and anti-inflammatory properties compared to EOFAZ in hyperglycemia-induced Müller cells. Moreover, NPS_PHE@EOFAZ facilitated enhanced delivery of EOFAZ in mice, leading to increased accumulation and retention in ocular tissues. In vivo experiments in DR mice demonstrated that NPS_PHE@EOFAZ attenuated DR progression, improved ocular pathology by reducing oxidative stress and inflammation, and minimized pathological damage in primary organs. These findings underscore the potential of NPS_PHE@EOFAZ in ameliorating early pathological processes associated with DR.

AUTHOR INFORMATION

Corresponding Authors

*Email address: [email protected]; ORCID: 0000-0002-4333-9106.

*Email address: [email protected]; ORCID: 0000-0001-8373-3530.

Conflict of Interest

There are no conflicts to declare.

Acknowledgments

The Guizhou Provincial Science and Technology Projects (Grant No. [2020]1Y210). The Guizhou Provincial Natural Science Foundation (Grant No. [2020]1Z069). The National Natural Science Foundation of China (Grant No. 52263014 and 82260827).

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Scheme 1 is available in the Supplementary Files section.

Scheme1.jpeg
Scheme 1. Synthetic route of copolymer PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀.
Supportinginformation.doc

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You are reading this latest preprint version

ROS-Responsive Nanoparticles with Antioxidative Effect for the treatment of Diabetic Retinopathy

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and experiments

2.1 Materials

2.2 Synthesis of copolymer PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀

2.2.1 Synthesis of PBAE

2.2.2 Synthesis of copolymer PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀

2.3 Preparation of nanoparticles

2.4 Characterizations of copolymer and nanoparticles

2.5 The stability of NPS_PHE and NPS_PHE@EOFAZ

2.6 Determination of critical micelle concentration (CMC).

2.7 ROS responsiveness of NPS_PHE

2.8 Cell viability

2.9 Cellular uptake

2.10 Cellular ROS measurement

2.11 The detection of SOD and MDA

2.12 The inflammatory response

2.13 In vivo studies

3. Results and discussion

3.1 Preparation and characterization of the copolymer and nanoparticles

3.2 Cellular uptake

3.3 ROS measurement

3.4 In vivo assays

4. Conclusion

Declarations

References

Scheme 1

Supplementary Files

Status:

Version 1

ROS-Responsive Nanoparticles with Antioxidative Effect for the treatment of Diabetic Retinopathy

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and experiments

2.1 Materials

2.2 Synthesis of copolymer PBE30-r-HEMA10-r-PEGMA30

2.2.1 Synthesis of PBAE

2.2.2 Synthesis of copolymer PBE30-r-HEMA10-r-PEGMA30

2.3 Preparation of nanoparticles

2.4 Characterizations of copolymer and nanoparticles

2.5 The stability of NPSPHE and NPSPHE@EOFAZ

2.6 Determination of critical micelle concentration (CMC).

2.7 ROS responsiveness of NPSPHE

2.8 Cell viability

2.9 Cellular uptake

2.10 Cellular ROS measurement

2.11 The detection of SOD and MDA

2.12 The inflammatory response

2.13 In vivo studies

3. Results and discussion

3.1 Preparation and characterization of the copolymer and nanoparticles

3.2 Cellular uptake

3.3 ROS measurement

3.4 In vivo assays

4. Conclusion

Declarations

References

Scheme 1

Supplementary Files

Status:

Version 1

2.2 Synthesis of copolymer PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀

2.2.2 Synthesis of copolymer PBE₃₀-r-HEMA₁₀-r-PEGMA₃₀

2.5 The stability of NPS_PHE and NPS_PHE@EOFAZ

2.7 ROS responsiveness of NPS_PHE