DNA-based Hybrid Hydrogels Sustain Water-insoluble Ophthalmic

ABSTRACT: Clinical need for treating allergic conjunctivitis (AC) is rapidly ... conjunctivitis (AC) is one of the common allergic diseases that plagu...
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DNA-Based Hybrid Hydrogels Sustain Water-Insoluble Ophthalmic Therapeutic Delivery against Allergic Conjunctivitis Ning Ren,†,∥ Rui Sun,†,∥ Kai Xia,†,∥ Qi Zhang,† Wei Li,† Fei Wang,‡ Xueli Zhang,*,‡ Zhilei Ge,§ Lihua Wang,† Chunhai Fan,§ and Ying Zhu*,†

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†

Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 201800, China ‡ Joint Research Center for Precision Medicine, Shanghai Jiao Tong University & Affiliated Sixth People’s Hospital South Campus, Southern Medical University Affiliated Fengxian Hospital, Shanghai 201499, China § School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China S Supporting Information *

ABSTRACT: Clinical need for treating allergic conjunctivitis (AC) is rapidly increasing. However, AC-relevant anti-inflammatory compounds are generally difficult to solubilize in water, thus limiting their therapeutic potential. Solubility-improved eye drop formulations of these compounds have poor bioavailability and a short retention time in ophthalmic tissues. Herein, we report a DNA/poly(lactic-co-glycolicacid) (PLGA) hybrid hydrogel (HDNA) for water-insoluble ophthalmic therapeutic delivery. PLGA pre-encapsulation enables loading of water-insoluble therapeutics. HDNA’s porous structure is capable of sustained delivery of therapeutics. Dexamethasone (DEX), with demonstrated activities in attenuating inflammatory symptom in AC, was used as a model system. The designed HDNA hybrid hydrogels significantly improved the DEX accumulation and mediated the gradual DEX release in ophthalmic cells and tissues. Using the HDNA−DEX complexes, potent efficacy in two animal models of AC was acquired. Given this performance, demonstrable biocompatibility, and biodegradability of DNA hydrogel, the HDNA-based ophthalmic therapeutic delivery system enables novel treatment paradigms, which will have widespread applications in the treatment of various eye diseases. KEYWORDS: DNA hydrogels, poly(lactic-co-glycolicacid) (PLGA), ophthalmic drug, anti-inflammatory, allergic conjunctivitis (AC)

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INTRODUCTION

the functioning of these water-insoluble ophthalmic drugs for AC treatment. DNA hydrogels, owing to their excellent solubility, biocompatibility, biodegradability,13−16 and especially “spongelike” porous structure,15,17−19 have shown great potential as vehicles for sustained therapeutic delivery. However, current efforts on DNA hydrogel-based delivery are confined to watersoluble drugs such as model antigen OVA20,21 or DNA-binding agents such as chemotherapeutic drug doxorubicin hydrochloride (DOX).22,23 This hampers their translation into ophthalmic therapeutic delivery applications. In this work, we develop a hybrid of DNA hydrogel/ poly(lactic-co-glycolicacid) (PLGA) material (HDNA) for water-insoluble ophthalmic drug delivery (Figure 1a). Dexamethasone (DEX), with demonstrated activities in attenuating the clinical signs of AC, served as a model system.4,5 Biocompatible and biodegradable PLGA nanoparticles (NPs)24−26 are used to pre-encapsulate hydrophobic DEX to form microspheres. We found that the complexation of DEX

With the increasingly serious environmental pollution, chemical abuse, radiation, and various food additives, allergies have become a big killer of humans. Allergic conjunctivitis (AC) is one of the common allergic diseases that plagues people. The prevalence of AC has steadily risen, affecting up to 25% of the general population. It includes seasonal, perennial, and more severe cases such as atopic and vernal keratoconjunctivitis, which are prone to increased complications due to corneal damage, leading to permanent visual loss.1−3 The most characteristic symptom of AC is ocular itching. Common clinical signs of AC include conjunctival redness, tearing, and scratching behavior.4,5 Several anti-inflammatory compounds are used to manage these symptoms.6,7 Due to poor water solubility, they are generally modified with water-soluble chemicals and administered in the form of eye drops. However, the highly permi-selective corneal epithelium and the precorneal tear clearance lead to low bioavailability (less than 5%) and a short retention time in ocular tissues.8−10 It requires repeated administration every day, compromising patient obedience.11,12 Hence, it is highly demanding to develop a new topical formulation that can both increase the bioavailability and sustain © XXXX American Chemical Society

Received: May 18, 2019 Accepted: July 2, 2019 Published: July 2, 2019 A

DOI: 10.1021/acsami.9b08652 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 1. Preparation of HDEX. (a) Schematic showing the preparation of HDEX- and HDNA-mediated sustained DEX release in ophthalmic tissues. (b) Characterization of PLGA−DEX NPs. Left: scanning electron microscopy (SEM) image of PLGA−DEX NPs. Scale bar: 3 μm. Middle: plot of the w/w ratio of PLGA-to-DEX to the complexation ratios (DEX loaded in PLGA NPs). Right: UV−vis spectrum of PLGA−DEX, DEX, and PLGA. (c) The viscosity η* corresponding to HDNA consisting of various ratios between the Y-shaped monomers and the cross-linker monomers. (d) Characterization of HDEX. Left: SEM image of HDEX. Scale bar: 60 μm. Middle: plot of the molar ratio of HDNA-to-PLGA-DEX NPs to the complexation ratios (DEX loaded in HDEX) and formulation pH. Right: rheology analysis of HDEX complexes. (e) In vitro release of DEX from HDEX or PLGA−DEX formulation at 37 °C in phosphate-buffered saline (PBS). Data represented as mean ± standard deviation (SD) (n = 3).

surface (Figure 1b, left). UV−vis absorption spectrum analysis confirmed their encapsulation with DEX (Figure 1b, right). Then, we prepared the Y-shaped and cross-linker DNA monomers from individual single-stranded DNA strands with rationally designed, partially complementary sequences (Table S1).30,31 By polyacrylamide gel electrophoresis (PAGE), we confirmed the successful formation of these monomers (∼90% yield) (Figure S2). To prepare HDEX, we incubated these two DNA monomers with PLGA−DEX NPs (Figure 1a). It is worthy to note that when HDNA was prepared at a molar ratio of Y-shaped to crosslinker units of 2:3, it had the highest viscosity η* of 9.3 Pas (Figure 1c), which ensures a prolonged retention and thus improving the ocular bioavailability of DEX.32 We found it could not be successfully prepared when the molar ratio of HDNA/ PLGA−DEX NPs was less than 1:10 probably due to the low degree of cross-linking (Figure 1d, left, and Figures S3 and S4).23,33 For a fixed HDNA concentration, high-performance liquid chromatography (HPLC) analysis showed that the DEX concentration increased with increasing molar ratio of HDNA/ PLGA−DEX NPs. Considering that the optimum pH of an ocular formulation is 7.2 ± 0.2,34 we chose a 5:6 molar ratio of HDNA/PLGA−DEX NPs for the following experiments (Figure 1d, middle). Rheology analysis of the prepared HDEX revealed that the shear-storage modulus (G′) value was higher

with HDNA (HDEX) remarkably increased its cellular uptake by ∼8-fold. More importantly, HDEX exhibited a long retention time of 24 h at the ophthalmic cells and tissues, which arises from the sustained release properties mediated by HDNA’s sponge-like porous structure. We further topically applied HDEX into two animal models of acute AC, i.e., BALB/c mice and New Zealand white rabbits. Importantly, the efficacy of once daily treatment with this HDEX is much better than that of multiple treatments with commercial DEX drops. Given this performance, demonstrable biocompatibility, and convenient use, this HDNA hybrid hydrogel vector opens new opportunities for AC treatment.

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RESULTS AND DISCUSSION Preparation of DNA/PLGA Hybrid Hydrogel for DEX Delivery. To design a therapeutic delivery vector for DEX, we first loaded DEX in PLGA nanospheres using oil-in-water emulsion solvent evaporation technique (Figure 1a).27−29 Drugloading studies revealed that PLGA nanospheres were saturated with DEX when the w/w ratio of PLGA/DEX was 5:1 (Figure 1b, middle) and the DEX in PLGA nanospheres was 10%. After the formation of PLGA−DEX nanospheres, they had a mean diameter of 500 nm and a ζ-potential of −13 mV (Figure S1). Scanning electron microscopy (SEM) images showed that these nanospheres were homogeneous in size and had a smooth B

DOI: 10.1021/acsami.9b08652 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

using real-time live-cell imaging with a DV Elite microscope. We observed that green-colored particle slowly moved along the cell membrane for ∼60 s and suddenly crossed the membrane and rapidly came into the cytoplasm (Figures 2c, S5, and Video S1). Next, we investigate the clearance of HDNA−DEX in cells after the initial 6 h incubation. Following extensive washing of cells to remove noninternalized HDNA, we measured the intracellular DEX concentrations at 2, 4, 6, 12, and 24 h, respectively. We observed that it slowly decreased within 24 h after the clearance of drug treatment, indicating that DEX vectorized by the sustained release of HDNA from HDEX. However, in the case of initial 6 h incubation with DEX alone, intracellular DEX concentration dramatically decreased at 2 h after the clearance of drug treatment, and it was undetectable in the subsequent time intervals (Figure 2d). Interestingly, the PLGA nanosphere alone also showed a similar release trend, although the corresponding DEX concentrations were generally lower than those of HDNA (Figure 2d), suggesting that the use of DNA hydrogels slowed down the release of DEX. HDNA-Mediated Increased Accumulation and Sustained Release of Drug in Ophthalmic Tissues. Next, we investigated the effect of HDNA on DEX delivery and release in ophthalmic tissues in two animal models, i.e., BALB/c mice and New Zealand white rabbits. Animals topically treated with HDEX were analyzed by measuring the DEX concentration in ophthalmic tissues with HPLC. At 15 min after HDEX instillation, the mean drug concentration in conjunctivas and corneas of the mice was 37.8 and 33.3 μg/g, respectively, whereas that after PLGA−DEX/DEX instillation at the same amount was only 10.5/1.7 and 11.7/2.4 μg/g, respectively. Compared with the PLGA−DEX/DEX instillation group, HDEX dramatically enhanced the mean drug concentration in conjunctivas and corneas of the mice by 4/22 and 3/14 times, respectively. For the rabbits, at 15 min after HDEX instillation, the mean drug concentration in conjunctivas, corneas, and aqueous humor was 40.5, 13.1 μg/g, and 1.5 μg/mL, respectively, whereas that after PLGA−DEX/DEX instillation was only 14.1/6.0, 6.6/1.2 μg/g, and 0.7/0.2 μg/mL, respectively. HDEX enhanced the mean drug concentration in conjunctivas, cornea, and aqueous humor by 3/7, 2/11, and 2/8 times, respectively (Figure 3a). Further, we found after the HDEX treatment that the drug concentration in the ophthalmic tissues of the mice slowly decreased within 24 h. However, in the case of PLGA−DEX/DEX treatment, the drug concentration in the ophthalmic tissues drastically decreased to almost zero within 6/2 h. For the rabbits, at 24 h after HDEX instillation, we noticed that the amount of mean drug concentration in conjunctiva, cornea, and aqueous humor was 2 μg/g, 0.7 μg/g, and 52 ng/mL, respectively, while it decreased to the detection limit at 10/4 h after the PLGA−DEX/DEX treatment (Figure 3a). We labeled HDNA with nucleic acid-specific dye GelGreen.39 We found the green fluorescence signal on the rabbit eye surface slowly decreased along with time, and it was still prominent 16 h after instillation (Figure S6), indicating that it stayed on the eye for at least 16 h. Additionally, following the instillation of FluoSpheres with similar particle size to PLGA NPs, the green fluorescence signal on the eye surface dropped dramatically at 5 min and completely vanished at 10 min after dosing. In contrast, a bright fluorescent signal was observed throughout the surface of the eyes at 5−10 min after dosing when instilled with HDNA-FluoSphere complexes. Fluorescent signal on the surface of eyes slowly decreased along with time, and it was still prominent even 16 h after dosing (Figure 3b).

than the shear-loss modulus (G″), suggesting their typical hydrogel properties (Figure 1d, right). We then compared the release profile of DEX from HDNA−DEX with that from PLGA−DEX NPs. For both formulations, 50% DEX release occurred in a short duration (10 h for HDNA−DEX and 3.4 h for PLGA−DEX) possibly due to sudden release.35 The time taken for 100% DEX release from HDNA−DEX complexes was 7 days, which is much longer than 60 h for PLGA−DEX NPs (Figure 1e). HDNA-Mediated Increased Accumulation and Sustained Release of Drug in Ophthalmic Cells. We first investigated the effect of HDNA on DEX delivery and release (Figure 2a). Human corneal epithelial cells (HCE-T), which are

Figure 2. HDNA-mediated increased accumulation and sustained release of drug in ophthalmic cells. (a) General experimental design. (b) The intracellular concentrations of DEX were measured by HPLC. HCE-T cells were treated with indicated materials [DEX equivalent (32 μg/mL)] for 6 h. Data are expressed as means ± SD. (c) Cell uptake of HDNA-FluoSphere as visualized in real time with a DV Elite microscope. Upper: two stages (blue: staying in the membrane; red: inside the cytoplasm). Bottom: speed analysis of the representative trajectory. A representative trajectory of internalization of the HDNAFluoSphere, see Figure S4 and Video S1 in the Supporting Information. Scale bar: 10 μm. (d) The intracellular concentrations of DEX were measured at the intervals of 2, 4, 6, 12, and 24 h after the initial 6 h incubation by HPLC. Data are expressed as means ± SD.

widely used in eye disease research studies,36−38 were chosen as a cell model. DEX internalized by HCE-T cells were quantitatively measured by HPLC. After 6 h incubation, the amount of intracellular DEX reached 543 ± 32 ng after exposure to HDEX, whereas it was only 68 ± 18 ng after exposure to DEX alone (Figure 2b). Compared with DEX alone, HDNA−DEX dramatically increased the DEX concentration in cells by 8-fold. Control studies showed that the cellular uptake of DEX for HDNA was ∼2.0-fold higher than that for PLGA nanosphere alone, suggesting that the DNA hydrogel cross-linking is important for the delivery (Figure 2b). Having demonstrated the efficient cellular uptake of HDNA−DEX, we incubated FluoSpheres with similar particle size to PLGA NPs with two DNA monomers by the same method mentioned above. Also, we tracked the internalization of HDNA-FluoSphere in cells by C

DOI: 10.1021/acsami.9b08652 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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conjunctiva. As shown in the histology images and the corresponding statistical analysis, OVA-challenged mice showed significant eosinophils infiltration in the conjunctiva. After HDEX or DEX treatment, eosinophil counts reduced when compared with those of the untreated group. Of note, eosinophil counts of the HDEX-treated group were much lower than those of the DEX-treated group (22 ± 4 vs 42 ± 6; Figure 4c). As AC are closely associated with immunoglobulin E (IgE)-mediated immune responses,1,40,41 we then measured the secreted levels of IgE and OVA-specific IgE in animal serum. We found that local boosting with OVA significantly induced the production of IgE/OVA-specific IgE in serum. However, after HDEX treatment, the production of IgE/OVA-specific IgE in mouse serum after OVA challenge was greatly reduced by 91/54%, which was much lower than that of the DEX-treated group. In the rabbit model of AC, the IgE production in serum also showed a similar reducing trend (Figure 4d). In addition, control studies showed that PLGA−DEX had poor efficacy, similar to DEX alone treatment, in animal models of acute AC (Figure S7 and Table S2). Additionally, at the end of day 21, we observed no morphological/structural changes according to the histological examination of the cornea and retina of the animal eyes (Figure S8). This confirmed that HDEX did not induce any gross toxicity in the ocular tissues, which is consistent with the in vitro cytotoxicity results (Figure S9). All data suggest that this DNA/ PLGA hybrid hydrogel-based water-insoluble ophthalmic therapeutic delivery system is safe and effective. In future, using DNA assembly technology or chemical modification, we can incorporate more functionalities such as carrying antibodies or sensing into these DNA-based hybrid hydrogels.42−44

Figure 3. HDNA-mediated increased accumulation and sustained release of drug in ophthalmic tissues. (a) Mice (upper) or rabbits (lower) were topically treated with indicated materials [DEX equivalent: 20 μg/mouse (n = 4) or 200 μg/rabbit (n = 3)]. Ophthalmic samples were collected at 15 min, 40 min, 1, 2, 4, 6, 8, 12, and 24 h (for mice) or at 15 min, 1, 2, 4, 8, 10, and 24 h (for rabbits) after treatment. DEX concentrations were measured by HPLC. (b) Rabbits were topically treated with HDNA-FluoSpheres or FluoSpheres [FluoSphere equivalent: 1 mg/rabbit (n = 3)]. FluoSpheres were entrapped in DNA hydrogel or PBS instead of PLGA NPs. Representative fundus camera images of rabbits at 10 min, 1, 6, 10, and 16 h after instillation. Scale bars: 0.5 cm.

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CONCLUSIONS

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EXPERIMENTAL SECTION

In summary, we have fabricated DNA/PLGA hybrid hydrogels for water-insoluble ophthalmic therapeutic delivery. This hybrid DNA hydrodels show a porous structure, thus enabling sustained release of ophthalmic drug loaded on them. DEX was selected as a model ophthalmic drug. The efficacy of the prepared HDEX with once daily treatment was much better than that of commercial DEX drops with multiple treatments in animal models of AC. We expect that our design approach could facilitate the development of new topical formulation to advance the bioavailability and sustained functioning of a water-insoluble ophthalmic drug for eye disease treatment.

These results indicate that the NPs gained a significantly prolonged residence time with the use of DNA hydrogels. Taken together, we demonstrated that the drug retention of the DEX treatment was transient and HDNA significantly improved drug accumulation and mediated gradual drug release in ophthalmic cells and tissues. HDEX-Mediated Potent Efficacy in Animal Models of Acute AC. Having demonstrated the improved retention and slow clearance abilities of HDEX in ophthalmic cells and tissues, we next assessed its effect on AC in two animal models, i.e., mouse and rabbit. The animals were sensitized intraperitoneally with OVA and alum at days 0 and 7 and then challenged with OVA from day 15 to 20. HDEX complexes and commercial DEX drops were given topically in the conjunctival sac once and twice daily from day 15 to 20, respectively (Figure 4a). Compared with the control group, OVA-challenged BALB/c mice had severe clinical signs. HDEX or DEX treatment reduced the clinical scores when compared with the untreated group. Notably, clinical scores of the HDEX once daily treated group were much lower than those of the DEX twice daily treated group (3.0 ± 0.6 vs 8.0 ± 0.6; Figure 4b and Table S2). To assess the conjunctival inflammation severity after various treatments, we evaluated the infiltration of eosinophils in the

Preparation of Drug Formulations. PLGA NPs loaded with DEX (PLGA−DEX NPs) were prepared by an oil-in-water (O/W) emulsion-evaporation technique. Briefly, 100 mg of PLGA and 20 mg of DEX were dissolved in an organic solvent (1 mL of methylene chloride and 30 μL of dimethyl sulfoxide mixture). The organic solution was then pre-emulsified with 5 mL of a PVA aqueous solution on ice (2%, w/v) by sonication at 40 W for 3 min (SONICS&MATERIALS INC). The formed emulsion was added to 25 mL of 0.1% (w/v) PVA aqueous solution and stirred at 250 rpm for 6 h at room temperature. The resulting nanoparticles were collected by centrifugation at 14 000g for 30 min and washed three times to remove organic solvent residue and free DEX. To prepare the HDNA−DEX complexes (HDEX), Y-shaped and cross-linker DNA monomers were first prepared according to the literature.30 Briefly, Y-shaped strands (1A)/(2A)/(3A) and the crosslinker structure strands (4)/(5) with final concentrations of 1 mM were mixed in PBS separately (Table S1). Each toehold of the Y-shaped monomer can hybridize with each end of the cross-linker monomer.45 D

DOI: 10.1021/acsami.9b08652 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 4. Potent efficacy of HDEX in animal models of acute AC. (a) General experimental design. (b−d) Mice (b and d, left) or rabbits (d, right) were sensitized i.p. with albumin (OVA, 5 mg/kg for mice and 1.8 mg/kg for rabbits) and aluminum hydroxide (ALUM, 250 mg/kg for mice and 90 mg/kg for rabbits) on days 0 and 7. Then, they were challenged with OVA (4 mg/mL in PBS, 10 μL for mice and 100 μL for rabbits) via eye drops from day 15 to 20. Commercial DEX drops (twice per day) or HDEX (once per day) were topically administered to both eyes 1 h before the OVA challenge. (b) Representative eye appearance (left) and clinical scores of the AC mice at 20 min after challenge (n = 5). (c) Left: conjunctival tissues were obtained on the second day after the final instillation, and hematoxylin and eosin (H&E) histopathological sections (n = 3) were analyzed. Eosinophils are indicated with arrows. Scale bars: 40 μm. Right: corresponding eosinophil infiltration quantification (means ± SD). (d) Blood samples were obtained on the second day after the final instillation and serum IgE/OVA-specific IgE levels were analyzed by ELISA (n = 5 for mice n = 3 for rabbits). Data are represented as means ± SD. **P < 0.01, ***P < 0.001, significantly different from control; †P < 0.05; ††P < 0.01; †††P < 0.001, significantly different from model. #P < 0.05, ###P < 0.001, significantly different from DEX. The mixture solutions were heated to 90 °C for 5 min and then slowly cooled down to 4 °C for 1 h. The prepared monomers were characterized by nondenaturing polyacrylamide gel electrophoresis (PAGE). Then, these two DNA monomers were mixed (HDNA, the molar ratio between Y-shaped units and cross-linker units at a value of 2:3; the concentration of Y-shaped units is 250 μM) and incubated with PLGA−DEX NPs at the molar ratio of HDNA/PLGA−DEX NPs of 5:6. 10 or 100 μL of the hydrogels were put on the slide. The diameter is 0.5 or 1.2 cm, and the thickness is 0.09 or 0.17 cm, respectively (Figure S10). The percent porosity of HDNA−DEX is 51.2% (see details in “Supplementary Methods” section). After storage at 4 °C for 7 days, the morphology and rheology of the HDEX are unchanged (Figures S11 and S12). To prepare the HDNA-FluoSphere complexes (HS), the same HDNA as mentioned above was incubated with FluoSpheres carboxylate-modified nanospheres (500 nm, Invitrogen, Grand Island, NY) in a molar ratio of HDNA/nanosphere of 5:6. After storage at 4 °C for 7 days, the fluorescence intensity of HS also remained unchanged (Figure S13), which indicates no leakage of spheres from HS during storage. Cell Line and Treatment. Human corneal epithelial cells (HCE-T, RIKEN Cell Bank, Tsukuba, Japan) were grown in the Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum, and the resultant cell suspension (2.5 × 105 cells/mL) was dispensed into 6-well plates and incubated overnight to allow for cell adherence. After washing twice with phosphate-buffered saline (PBS), the cells were exposed to DEX, PLGA−DEX, or HDEX for required time and concentration.

Animals and Treatment. Both BALB/c mice (male, 18−22 g) and New Zealand white rabbits (male, 2.1−2.5 kg) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd., China, and kept in conventional conditions. All animal experiments were conducted in accordance with the Institute’s Guide for the Care and Use of Laboratory Animals and were approved by the ethical committee of Shanghai University of Traditional Chinese Medicine (Approval No. ACSHU-2014-200, approved in 16 July, 2014). For the ophthalmic tissue drug release experiments, 10 μL (for mice) or 100 μL (for rabbits) of commercial DEX drops, PLGA−DEX, or HDEX was instilled topically into both eyes. The concentration of DEX in these three formulations was 1 mg/mL. For mice, at 1/4, 2/3, 1, 2, 4, 6, 8, 12, and 24 h after instillation, the eyes were enucleated and frozen immediately on dry ice/isopentane bath. The eyes were dissected in frozen condition to isolate conjunctive and cornea (4 per group at each time point). For rabbits, at 1/4, 1, 2, 4, 8, 10, and 24 h after instillation, conjunctive, cornea, and aqueous humor were carefully dissected under the same conditions (3 per group at each time point). All samples were weighed before HPLC analysis. For the mouse AC model establishment, BALB/c mice were sensitized i.p. with 5 mg/kg of ovalbumin (OVA, Sigma-Aldrich Corp., St. Louis, MO) and 250 mg/kg of aluminum hydroxide (ALUM, SigmaAldrich Corp.) on days 0 and 7. Then, the mice were challenged with 10 μL of OVA (4 mg/mL in PBS) via eye drops from days 15 to 20. 10 μL of commercial DEX drops (twice per day), PLGA−DEX (once per day), or HDEX (once per day) was applied topically to both eyes 1 h before the OVA challenge. The concentration of DEX in these two formulations was the same as mentioned above. Meanwhile, 10 μL of normal saline was used as a placebo in the control and model groups. E

DOI: 10.1021/acsami.9b08652 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Allergic Conjunctivitis in A Mouse Model. Graefe’s Arch. Clin. Exp. Ophthalmol. 2013, 251, 1717−1721. (6) Normann, E. K. Conjunctivitis in Children. Lancet 2005, 366, 6− 7. (7) Leonardi, A.; Piliego, F.; Castegnaro, A.; Lazzarini, D.; Valerio, A. L.; Mattana, P.; Fregona, I. Allergic Conjunctivitis: A Cross-Sectional Study. Clin. Exp. Allergy 2015, 45, 1118−1125. (8) Than, A.; Liu, C. H.; Chang, H.; Duong, P. K.; Cheung, C. M. G.; Xu, C. J.; Wang, X. M.; Chen, P. Self-Implantable Double-Layered Micro-Drug-Reservoirs for Efficient and Controlled Ocular Drug Delivery. Nat. Commun. 2018, 9, No. 4433. (9) Yuan, X. Y.; Marcano, D. C.; Shin, C. S.; Hua, X.; Isenhart, L. C.; Pflugfelder, S. C.; Acharya, G. Ocular Drug Delivery Nanowafer with Enhanced Therapeutic Efficacy. ACS Nano 2015, 9, 1749−1758. (10) Huang, J. F.; Zhong, J.; Chen, G. P.; Lin, Z. T.; Deng, Y. Q.; Liu, Y. L.; Cao, P. Y.; Wang, B. W.; Wei, Y. T.; Wu, T. F.; Yuan, J.; Jiang, G. B. A Hydrogel-Based Hybrid Theranostic Contact Lens for Fungal Keratitis. ACS Nano 2016, 10, 6464−6473. (11) Tsai, J. C. Medication Adherence in Glaucoma: Approaches for Optimizing Patient Compliance. Curr. Opin. Ophthalmol. 2006, 17, 190−195. (12) Sverrisson, T.; Gross, R.; Pearson, J.; Rusk, C.; Adamsons, I. The Dorzolamide/Timolol Combination versus Timolol Plus Pilocarpine: Patient Preference and Impact on Daily Life. J. Glaucoma 1999, 8, 315− 324. (13) Song, J.; Lee, M.; Kim, T.; Na, J.; Jung, Y.; Jung, G. Y.; Kim, S.; Park, N. A RNA Producing DNA Hydrogel as A Platform for A High Performance RNA Interference System. Nat. Commun. 2018, 9, No. 4341. (14) Song, P.; Ye, D. K.; Zuo, X. L.; Li, J.; Wang, J. B.; Liu, H. J.; Hwang, M. T.; Chao, J.; Su, S.; Wang, L. H.; Shi, J. Y.; Wang, L. H.; Huang, W.; La, R.; Fan, C. H. DNA Hydrogel with Aptamer-ToeholdBased Recognition, Cloaking, and Decloaking of Circulating Tumor Cells for Live Cell Analysis. Nano Lett. 2017, 17, 5193−5198. (15) Li, C.; Faulkner-Jones, A.; Dun, A. R.; Jin, J.; Chen, P.; Xing, Y. Z.; Yang, Z. Q.; Li, Z. B.; Shu, W. M.; Liu, D. S.; Duncan, R. R. Rapid Formation of a Supramolecular Polypeptide-DNA Hydrogel for In Situ Three-Dimensional Multilayer Bioprinting. Angew. Chem., Int. Ed. 2015, 54, 3957−3961. (16) Jin, J.; Xing, Y. Z.; Xi, Y. L.; Liu, X. L.; Zhou, T.; Ma, X. X.; Yang, Z. Q.; Wang, S. T.; Liu, D. S. A Triggered DNA Hydrogel Cover to Envelop and Release Single Cells. Adv. Mater. 2013, 25, 4714−4717. (17) Um, S. H.; Lee, J. B.; Park, N.; Kwon, S. Y.; Umbach, C. C.; Luo, D. Enzyme-Catalysed Assembly of DNA Hydrogel. Nat. Mater. 2006, 5, 797−801. (18) Lee, J. B.; Peng, S. M.; Yang, D. Y.; Roh, Y. H.; Funabashi, H.; Park, N.; Rice, E. J.; Chen, L. W.; Long, R.; Wu, M. M.; Luo, D. A Mechanical Metamaterial Made from A DNA Hydrogel. Nat. Nanotechnol. 2012, 7, 816−820. (19) Wang, J. B.; Chao, J.; Liu, H. J.; Su, S.; Wang, L. H.; Huang, W.; Willner, I.; Fan, C. H. Clamped Hybridization Chain Reactions for the Self-Assembly of Patterned DNA Hydrogels. Angew. Chem., Int. Ed. 2017, 56, 2171−2175. (20) Umeki, Y.; Saito, M.; Takahashi, Y.; Takakura, Y.; Nishikawa, M. Retardation of Antigen Release from DNA Hydrogel Using Cholesterol-Modified DNA for Increased Antigen-Specific Immune Response. Adv. Healthcare Mater. 2017, 6, No. 1700355. (21) Nishikawa, M.; Ogawa, K.; Umeki, Y.; Mohri, K.; Kawasaki, Y.; Watanabe, H.; Takahashi, N.; Kusuki, E.; Takahashi, R.; Takahashi, Y.; Takakura, Y. Injectable, Self-gelling, Biodegradable, and Immunomodulatory DNA Hydrogel for Antigen Delivery. J. Controlled Release 2014, 180, 25−32. (22) Nishikawa, M.; Mizuno, Y.; Mohri, K.; Matsuoka, N.; Rattanakiat, S.; Takahashi, Y.; Funabashi, H.; Luo, D.; Takakura, Y. Biodegradable CpG DNA Hydrogels for Sustained Delivery of Doxorubicin and Immunostimulatory Signals in Tumor-Bearing Mice. Biomaterials 2011, 32, 488−494. (23) Kang, H.; Liu, H. P.; Zhang, X. L.; Yan, J. L.; Zhu, Z.; Peng, L.; Yang, H. H.; Kim, Y. M.; Tan, W. H. Photoresponsive DNA-Cross-

Similarly, for the rabbit AC model establishment, New Zealand white rabbits were sensitized i.p. with 1.8 mg/kg of ovalbumin (OVA, SigmaAldrich Corp., St. Louis, MO) and 90 mg/kg of aluminum hydroxide (ALUM, Sigma-Aldrich Corp.) on days 0 and 7. Then, the rabbits were challenged with 100 μL of OVA (4 mg/mL in PBS) via eye drops from days 15 to 20. 100 μL of commercial DEX drops (twice per day), PLGA−DEX (once per day), or HDEX (once per day) was applied topically to both eyes 1 h before the OVA challenge. The concentration of DEX in these two formulations was the same as mentioned above. Meanwhile, 100 μL of normal saline was used as a placebo in the control and model groups.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b08652. Supplementary methods; dynamic light scattering measurement statistics of PLGA−DEX NPs; native PAGE of DNA monomers and HDEX; photograph of HDEX complex; SEM images of HDEX; efficacy of PLGA−DEX in animal models of acute AC; DNA sequences; clinical scores of the AC mice (PDF) Real-time imaging of HDNA-FluoSphere entry into a HCE-T cell (AVI)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (X.Z.). *E-mail: [email protected] (Y.Z.). ORCID

Zhilei Ge: 0000-0001-7184-7565 Lihua Wang: 0000-0002-6198-7561 Chunhai Fan: 0000-0002-7171-7338 Ying Zhu: 0000-0003-0418-919X Author Contributions ∥

N.R., R.S., and K.X. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Program (2016YFA0400902), the Open Large Infrastructure Research of Chinese Academy of Sciences, the National Natural Science Foundation of China (11675251), and the LU JIAXI International team program supported by the K.C. Wong Education Foundation and CAS and the Youth Innovation Promotion Association of CAS (Grant No. 2016236).

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