NSC 167409

Glycyrrhizin micelle as a genistein nanocarrier: Synergistically promoting corneal epithelial wound healing through blockage of the HMGB1 signaling pathway in diabetic mice

Yuzhen Hou a, 1, Meng Xin a, b, 1, Qiqi Li a, Xianggen Wu a, c,*


The purpose of this study was to explore the feasibility of targeting the HMGB1 signaling pathway to treat diabetic keratopathy with a dipotassium glycyrrhizinate-based micelle ophthalmic solution encapsulating gen- istein (DG-Gen), and to evaluate whether these dipotassium glycyrrhizinate (DG) micelles could synergistically enhance the therapeutic effect of encapsulated genistein (Gen). An optimized DG-Gen ophthalmic solution was fabricated with a Gen/DG weight of ratio 1:15, and this formulation featured an encapsulation efficiency of 98.96 ± 0.82%, and an average particle size of 29.50 ± 2.05 nm. The DG-Gen ophthalmic solution was observed to have good in vivo ocular tolerance and excellent in vivo corneal permeation, and to remarkably improve in vitro antioXidant activity. Ocular topical application of the DG-Gen ophthalmic solution significantly prompted corneal re-epithelialization and nerve regeneration in diabetic mice, and this efficacy might be due to the inhibition of HMGB1 signaling through down-regulation of HMGB1 and its receptors RAGE and TLR4, as well as inflammatory factor interleukin (IL)-6 and IL-1β. In conclusion, these data showed that HMGB1 signaling is a potential regulation target for the treatment of diabetic keratopathy, and novel DG-micelle formulation encapsulating active agents such as Gen could synergistically cause blockage of HMGB1 signaling to prompt diabetic corneal and nerve wound healing.

Corneal epithelial wound healing Nerve regeneration
High-mobility group boX 1 Dipotassium glycyrrhizinate Micelle

1. Introduction

High mobility group boX 1 (HMGB1), as a highly conserved non- histone DNA-binding protein, participates in various physiological and pathophysiological processes (Liu et al., 2018; Nebbioso et al., 2020). As a damage-associated molecular pattern, HMGB1 can be released into extracellular fluid actively or passively after stress. The released HMGB1 leads to a pro-inflammatory cycle, causes an immune response, and plays a key role in tissue damage through the signaling of receptors for advanced glycation end products (RAGEs) and toll-like receptors (TLRs) (Paudel et al., 2019).
Reports have confirmed that HMGB1 is associated with many in- flammatory and sterile-inflammatory diseases, such as sepsis, hyper- tension, tumors, and diabetes and diabetic complications, though some of the underlying mechanisms still remain unclear (Yang et al., 2020). HMGB1 is a therapeutic target in both infectious and sterile-inflammatory conditions. It has also been revealed that HMGB1 is involved in many eye diseases, and that HMGB1 is an important thera- peutic target for improving the efficacy of treatments for these eye diseases (Liu et al., 2018).
Diabetic keratopathy (DK) is a severe complication with a high incidence in diabetes patients (Lutty, 2013). It is characterized by some typical symptoms including an intractable epithelial defect, corneal ulcer, impaired corneal sensitivity, and even blindness after ocular trauma and ophthalmic surgery. The current treatment of DK mainly focuses on prompting corneal re-epithelialization and nerve regenera- tion with clinical practices including antibiotics (e.g., levofloXacin eye drops), autologous serum, lubricants (e.g., sodium hyaluronate eye drops), growth factors (e.g., basic fibroblast growth factor eye drops) and other handles such as bandage contact lenses and tarsorrhaphy (Abdelkader et al., 2011; Priyadarsini et al., 2020). However, current clinical practices are not usually effective or adequate for the purpose of improving re-epithelialization/nerve regeneration in diabetes patients, and these insufficient treatments usually provide opportunities for infection and irreparable vision problems. Moreover, the aforemen- tioned clinical practices do not address the fundamental pathological mechanisms of delayed corneal healing in diabetes patients. Therefore, new potential treatment targets and novel practices are needed to intervene in this severe diabetic complication.
Our previous work revealed that the diabetic corneas of mice had significantly increased protein expression levels of HMGB1 and its re- ceptors, RAGE and TLR4 (Hou et al., 2020). Corneal HMGB1 levels significantly increased during the corneal epithelium/nerve wound Co., Ltd. (Qingdao, China). The present animal study was approved by the Qingdao University of Science and Technology Ethics Committee for Animal EXperimentation (approval document no. 2017–1, Qingdao, China). In this study, animal experiments were conducted according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

2. Animals and methods

2.1. Animals

C57BL/6 J mice (male, eight weeks old) were obtained from Jinan Pengyue EXperimental Animal Breeding Co., Ltd. (Jinan, China). New Zealand white rabbits were obtained from Qingdao Kangda Foodstuffs to determine the Gen concentration in filtrate and its encapsulation ef- ficiency. The Gen concentration in filtrate was then diluted to 5.0 mg/ml with PBS. Afterward, this solution was filtered a second time through a 0.22-μm filter to obtain a sterile DG-Gen ophthalmic solution. The DG- Gen ophthalmic solution was placed in 10-ml colorless glass vials under a sterile procedure and protected from light with aluminum foil. Then, it was stored at 4 ◦C for further use.
To the encapsulation efficiency determination, 100 μl unfiltered so- lution and filtrate solution was diluted into 10 ml methanol. Then, they were determined by UV at 260 nm (Infinite 200 Pro, Austria) with quartz cell with lid. The absorption of control sample (DG solution without Gen) was subtracted from the respective absorption values of samples and then the concentration of Gen was calculated according to a stan- dard curve of Gen methanol solution. The encapsulation efficiency was calculated as the ratio of the detected amount to the added Gen amount.

2.2. Preparation and characterization of DG-Gen

Gen (50 mg) and DG with different Gen/DG weight ratios (1:6, 1:9, 1:12, 1:15, and 1:18) were dissolved into ethanol. Then, ethanol was completely volatilized with a vacuum rotary evaporator at 40 ◦C. The dried powder obtained was dissolved into 9.0 ml of PBS. The solution healing process of the diabetic mice. EXogenous HMGB1 peptide was filtered through a 0.22 μm membrane filter to remove free Gen and significantly retarded corneal wound and nerve healing, while glycyr- rhizin (an HMGB1 inhibitor) significantly prompted corneal wound and nerve healing in diabetic mice. All of this shows that HMGB1 and its related receptors are highly involved in the development of diabetic keratopathy, and the blockage of HMGB1 might serve as a strategy to prompt diabetic corneal and nerve wound healing (Hou et al., 2020).
Glycyrrhizin is a triterpenoid saponin mainly isolated from Glycyr- rhiza glabra L. Glycyrrhizin is widely used as a food additive and is listed as a Generally Recognized as Safe (GRAS) agent (2006). Glycyrrhizin is also widely used in clinical practices due to its strong pharmacological activities, including those with antimicrobial, antioXidative, and anti- inflammatory properties (Pastorino et al., 2018). As glycyrrhizin has some unsuitable physicochemical features, such as poor water solubility and instability, dipotassium glycyrrhizinate (DG), a derivative salt of glycyrrhizin with similar pharmacological properties, is mainly used in clinic with approval of authority (http://app1.nmpa.gov.cn/data_nmp a/face3/dir.html). As it has an amphiphilic structure, glycyrrhizin can self-assemble into micelles in an aqueous solution and thus attracts additional attention as a novel multifunctional nanocarrier. The first thing our research group did was demonstrated that a DG micelle ophthalmic solution encapsulating thymol may be a promising novel ophthalmic formulation for the treatment of eye diseases, especially oXidative stress and inflammation-related eye diseases (Song et al., 2020b).
Glycyrrhizin is an HMGB1 inhibitor, and inhibition of HMGB1 by glycyrrhizin is a potential therapeutic regimen for treating many HMGB1-involved diseases (Burillon et al., 2018; Musumeci et al., 2014). Glycyrrhizin has attracted additional attention as a novel multifunc- tional drug-delivery system (Selyutina and Polyakov, 2019; Song et al., 2020b). This knowledge inspired the notion that the combination of glycyrrhizin as a nanocarrier encapsulating a variety of drugs with regulating HMGB1 signaling activities could be beneficial. Genistein (Gen), as a well-known isoflavone, exhibited protective effect on dia- betic wound healing (Tie et al., 2013), and it could be effectively used for the treatment of some ocular disorders as its reliable antioXidant and anti-inflammatory properties (Kim et al., 2008; Sulaiman et al., 2014). However, poor solubility and low bioavailability necessitate drug de- livery of Gen using nanotechnology. So, Gen was used as the model drug in this manuscript.
This work’s primary aim is to reveal the feasibility of combining glycyrrhizin as a nanocarrier encapsulating Gen with strengthened regulating HMGB1 signal pathway activities. This work also aims to explore the potential that blockage of the HMGB1 signal pathway could improve treatment of diabetic keratopathy.

2.3. Physicochemical characterization of DG-Gen

Micelle size and polydispersity index (PdI) were detected with OP- 90S nanoparticle sizer (Optek Instrument, Inc. [Zibo, Shandong, China]). The zeta potential of DG-Gen was detected with a Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK). The surface morphology of the micelles was observed with transmission electron microscopy (TEM, JEM-1200EX, JEOL Ltd., Tokyo, Japan). All these tests were conducted according to our group established method (Song et al., 2020b).

2.4. Antioxidant activities

Both a free radical scavenging assay of 2,2′-Azino-bis(3-ethyl- benzothiazoline-6-sulfonic acid) (ABTS) and a ferric-reducing antioXi- dant power (FRAP) assay were performed to explore the antioXidant activity profiles of DG-Gen. The FRAP assay was determined according to our group established method (Song et al., 2020a, 2020b). Briefly, free Gen aqueous suspension solution and DG-Gen solution with concentrations from 250 to 500 μg/ml Gen were prepared respectively, and blank DG micelle solution was used as control. The total antioXidant capacity of DG-Gen micelles was determined at 15, 30, 60, 90 and 120 min respectively according to the instruction of FRAP detection kit (Product ID: S0116, Beyotime Biotechnology, Shanghai, China). The ABTS radical scavenging assay was tested with a kit as previ- ously reported from our group (Song et al., 2020a, 2020b), and the concentration setting was consistent with the FRAP method.

2.5. In vivo ocular tolerance evaluation

An exaggerated ocular dosing regimen was performed in this test (Enriquez de Salamanca et al., 2006; Esteban-Perez et al., 2020). In summary, 50 μl/drop of DG-Gen ophthalmic solution was instilled top- ically into the rabbits’ eyes 13 times at 30-min intervals, while a PBS solution, a benzalkonium chloride (BAC, 0.1 mg/ml) in PBS solution, and a sodium lauryl sulfate (SLS, 5 mg/ml) in PBS solution were also applied as the control groups (Pauloin et al., 2009). Three rabbits (siX eyes) were used to each tested solution. The rabbit eyes were examined for any damages with a slit lamp microscope. The rabbits were then sacrificed and a histopathological observation was performed on the corneas, according to the methods reported by our group (Song et al., 2020a, 2020b).

2.6. In vivo corneal penetration test

In vivo corneal penetration characterizations of DG-Gen were visu- alized in mouse corneas with Coumarin 6 (Cou6) labeled DG-Gen (Cou6- DG-Gen). The preparation procedures of Cou6-DG-Gen were the same as for DG-Gen, except 50 mg of Gen was replaced with 49.5 mg Gen and 0.5 mg Cou6. The free Cou6 solution was prepared by a reported method and used as a control in the corneal permeability study of mouse eyes (Guo et al., 2015). Healthy C57BL/6 mice were randomly divided into siX groups with 2 mice (4 eyes) in each group. Three groups were given free Cou6 solution, and the other three groups were given Cou6-DG-Gen solution. Four drops of test solution (5 μl/drop, 4 drops, 10-min intervals) were instilled to the eye. Randomly, one group of mice for each test solution was sacrificed 30, 60, and 90 min after the last administration, and the whole corneas were dissected with surgical scissors, fiXation at 4 ◦C for 40 min with 4% paraformaldehyde, and then observed under fluores- cence microscope (Song et al., 2020a, 2020b).

2.7. Diabetic animal model and corneal epithelial wound healing protocol

Mouse models of type 1 diabetes were induced with intraperitoneal injections of streptozocin (STZ; 50 mg/kg; Solarbio). Mice with fasting blood-glucose levels above 16.7 mM by the tenth day following the last STZ injection were considered diabetic models; these diabetic mice were later rechecked, and only those with blood-glucose levels above 16.7 mM at 9 weeks after the successful establishment of them as diabetic models were included in experiments. Diabetic mice that had hyperglycemia for 9 weeks underwent both systemic anesthesia and local anesthesia of the eye. The central corneal epithelium, with a diameter of 2.0 mm, was uniformly debrided with an Algerbrush II Corneal Rust Ring Remover (Alger Equipment Co., Inc., Lago Vista, TX, USA). The corneal epithelium debrided mice were divided into 5 groups, with 8–10 mice in each group. The mice received ocular topical treatments of PBS, DG in PBS solution (75 mg/ml), Gen suspension solution in PBS (5 mg/ml), DG&Gen physical miXture solu- tion in PBS (DG 75 mg/ml and Gen 5 mg/ml), and DG-Gen ophthalmic solution (micelle formulation containing DG 75 mg/ml and Gen 5 mg/ml). The administered regimen was 5 μl/eye, 6 times/day. The wounded corneal epithelium healing profiles were observed under a slit lamp at 24, 48 and 72 h after debridement following a 0.25% fluorescein sodium staining. The defect area of each cornea was measured with Image J software to express the percentage of area of the residual epithelial defect (Hou et al., 2020).

2.8. Corneal mechanical sensitivity and corneal nerve staining

Seven days after debridement, mice were examined for corneal me- chanical sensitivity, first using a Cochet-Bonnet esthesiometer (Luneau Ophtalmologie, Chartres Cedex, France) in unanesthetized state, and then, after they were sacrificed, the whole corneas from each group were carefully separated, and the corneal nerve was stained with an Anti-Beta III Tubulin Alexa Fluor® 488 Conjugate (AB15708A4, Millipore, Merck KGaA, Darmstadt, Germany), as described in the procedures of previous studies (Guo et al., 2016; Hou et al., 2020). One image field was sampled in each quadrant of the whole-mount corneal image, and corneal sub- basal nerve fiber density was expressed as a percentage of the positive area of green fluorescence counted using Image J software (Hou et al., 2020; Li et al., 2020b).

2.9. Western blot determination

Protein expression levels of HMGB1, RAGE, and TLR4 in mouse corneas from these 5 experimental groups were determined with West- ern blot staining according to the standard procedures used in our pre- vious reports (Hou et al., 2020). The collected antibody information is listed in Table S1.

2.10. Immunohistochemical staining

Corneas from the 5 experimental groups underwent immunohisto- chemical staining procedures according to routine protocols (Hou et al., 2020). The antibody information for this portion of the study is listed in Table S1.

2.11. Enzyme-linked immunosorbent assay (ELISA)

Corneas from these 5 experimental groups were harvested for the quantitative measurement of inflammatory cytokines Interleukin 1β (IL- 1β) and Interleukin 6 (IL-6) with enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Enzyme-linked Biotechnology Co., Ltd.).

2.12. Data analysis

The data comparisons depicted in Figs. 3–5 and 7 were analyzed with multiple comparisons in an ANOVA. SPSS 11.5 software (SPSS Inc., Chicago, IL) was used for all analyses, and p < 0.05 was considered significant. 3. Results 3.1. Preparation and characterization of DG-Gen Gen could be encapsulated into DG micelles, but the encapsulation efficiency was highly dependent on the Gen/DG weight ratio, as the encapsulation efficiency was just 56.44% for a Gen/DG weight ratio of 1:6, but soared to 98.96% with a Gen/DG weight ratio of 1:15. However, further increasing the Gen/DG weight ratio to 1:18 did not lead to a further increase of encapsulation efficiency (Fig. 1A). The DG-Gen ophthalmic solution with a Gen/DG weight ratio of 1:15 was set as the optimized formulation and was used in the subsequent experiments. The DG-Gen ophthalmic solution was a light-yellow transparent solution displayed a small nanosize of 29.50 ± 2.05 nm, with a polydispersity index of 0.525 0.140, and a zeta potential of –(0.0360 0.0359) mV (Fig. 1B and C). The DG-Gen micelles were in sphere or nearly sphere with good dispersion under TEM observation, and the size observed from TEM was consistent with the dynamic light scattering determina- tion results (Fig. 1D). 3.2. Determination of antioxidant activity OXidative stress plays important roles in the development of DK (Wang et al., 2014). Although Gen has various pharmacological appli- cations, the main and important biological activity to Gen is the anti- oXidative activity, and the antioXidative activity exhibited a positive correlation to other biological activities, including the anti-inflammatory and neuroprotective activities. In this study, FRAP and ABTS were used to compare the antioXidant capacity of the DG-Gen and free-Gen suspensions. The antioXidant activities of the DG-Gen, free-Gen suspension, and DG solutions were all time and concentra- tion dependent (Figure S1). For each of the formulations with the same level of concentration, the antioXidant capacity of DG-Gen was signifi- cantly higher than that of free-Gen solution and DG solution. For example, in an ABTS assay (Figure S1A-B), when the concentration of DG-Gen was 300 μg/ml and incubation duration was 15 min, the TroloX-Equivalent antioXidant activity of DG-Gen was 0.5279, while the Gen solution was 0.0773 at 300 μg/ml and 15 min, and 0.1032 for the DG solution at 0.75 mg/ml and 15 min. When the incubation duration was increased to 120 min, the antioXidant activity of 300 μg/ml DG-Gen was 0.7717, while it was 0.3128 for the Gen solution and 0.2657 for the DG solution. But when the DG-Gen was increased to 500 μg/ml, the antioXidant activity was 1.6544 with a 15 min incubation, and this antioXidant activity increased to 1.7992 with a 30 min incubation; however, no further increase could be observed with a further increase of incubation time. The results to FRAP (Figure S1C-D) were similar to ABTS. Both the FRAP and ABTS experiments confirmed that the anti- oXidant capacity of Gen in DG-Gen was significantly improved when compared to the free-Gen suspension. 3.3. In vivo ocular tolerance study in rabbits SLS, set as a positive irritation agent in this test, caused severe ocular irritation symptoms, including sustained closure of eyes; severe discharge, even wetting the whole orbit; severe conjunctival congestion and edema; and iris vessel engorgement. With the DG-Gen ophthalmic solution, none of the rabbits developed signs of ocular irritation. The PBS and BAC groups also displayed no obvious irritation symptoms (Fig. 2A). Histopathological observation also revealed that SLS caused severe histopathological changes, including thinning of the corneal epithelium, the corneal stroma becoming disordered and thin, and inflammatory cell infiltration. Normal corneal structures were observed with the DG-Gen ophthalmic solution, PBS, and BAC groups (Fig. 2B). Together, the re- sults strongly suggest that the DG-Gen ophthalmic solution is well tolerated by rabbit eyes. 3.4. In vivo corneal permeation visualization After administration, corneas from the Cou6-DG-Gen groups dis- played strong green fluorescence at 30 min, with the fluorescence uni- formly distributed over the whole cornea. However, the fluorescence became diminished over time, and only weak fluorescence could be observed at 90 min. Corneas from free-Cou6 groups displayed a lot of weak fluorescence, and no obvious fluorescence could be observed at 30 min with the same fluorescence observation parameters that were applied to the Cou6-DG-Gen groups (Figure S2). 3.5. Corneal wound healing evaluation The DG-Gen ophthalmic solution effectively accelerated diabetic corneal epithelial repair, and the wounds were completely resolved after 48 h (Fig. 3); however, the free-Gen suspension solution failed to accelerate corneal epithelial repair. It is worth mentioning that the DG solution, as well as the DG&Gen physical miXture solution, were also shown to promote corneal epithelial repair efficacies, but not as strongly as the DG-Gen ophthalmic solution (p < 0.05 for the DG-Gen ophthalmic solution group). Corneal subbasal nerve fiber densities were further evaluated with whole-mount corneal nerve staining 7 days after the wounds were created (shown in Fig. 4). The DG-Gen ophthalmic solution effectively promoted diabetic corneal nerve regeneration, and the density of sub- basal nerves reached 22.50 2.09%; meanwhile, the density of subbasal nerves was just 11.42 1.72% for PBS treated corneas (p < 0.05). The DG solution, as well as the DG&Gen physical miXture solution, had a positive effect on nerve regeneration (p < 0.05 for the PBS group), though their efficacies were lower than the DG-Gen ophthalmic solution (p < 0.05). However, the free-Gen suspension solution failed to promote corneal nerve regeneration. Simultaneously, the corneal sensitivities of diabetic mice treated with DG-Gen exhibited a marked increase in comparison to the PBS treated group (p < 0.05). The DG solution and the DG&Gen physical miXture solution showed a positive effect on corneal sensitivity when compared to PBS treated corneas (p < 0.05 for the PBS group), but they were weaker than DG-Gen treated corneas, while the free-Gen suspension solution failed to improve corneal sensitivity. The corneal sensitivity results were consistent with the corneal nerve regeneration results. As shown in Fig. 5, the Western blot revealed that DG-Gen ophthalmic solution treated corneas demonstrated decreased HMGB1 expression levels when compared to the diabetic corneas treated with PBS, and their protein level was just 0.47-fold that of PBS treated corneas (p < 0.05). Compared to the PBS treatment, the DG solution and DG&Gen physical miXture solution showed a positive effect on down expression of HMGB1 protein levels (p < 0.05 vs. PBS group), though their efficacies were lower than the DG-Gen ophthalmic solution (p < 0.05). The free-Gen suspension solution failed to down regulate HMGB1 expression. Protein expression regulation profiles of RAGE and TLR4 after different group treatments were similar to HMGB1. As for the immunohistochemical staining evaluation results shown in Fig. 6, a massive distribution of extracellular HMGB1 in the corneal epithelium was detected in PBS-treated corneas. EXtracellular HMGB1 levels were reduced with the application of the DG-Gen ophthalmic solution. The DG solution and the DG&Gen physical miXture solution treatments also reduced extracellular HMGB1 levels, but were weaker than the DG-Gen treatment. However, the free-Gen suspension solution showed no obvious effect as its extracellular HMGB1 level was similar to the PBS treatment group. RAGE and TLR4 also exhibited massive expression in PBS-treated corneas, and their expression regulation pro- files of different treatment groups were similar to those of HMGB1. Correspondingly, the ELISA results (Fig. 7) showed that there were reduced IL-1β and IL-6 levels in the diabetic corneas after treatment with the DG-Gen ophthalmic solution (p < 0.05 vs. the PBS treatment group). The DG and DG&Gen physical miXture solutions also displayed the effect of reducing IL-1β and IL-6 levels (p < 0.05 vs. the PBS treatment group), but more weakly than the DG-Gen ophthalmic solution (p < 0.05 vs. the DG-Gen treatment group). The free-Gen suspension solution treatment failed to reduce IL-1β and IL-6 levels in corneas. 4. Discussion In clinical reports, the HMGB1 signal pathway has been confirmed to be involved in many eye diseases. Increased HMGB1 and RAGE were detected in the vitreous cavity of eyes with proliferative diabetic reti- nopathy (PDR) and proliferative vitreoretinopathy (PVR); HMGB1 also presented in the epiretinal membranes (ERMs) of eyes with these pro- liferative retinal diseases (Pachydaki et al., 2006); Similar results of HMGB1 were reported in PDR patients with active neovascularization and in PDR patients with hemorrhaging; HMGB1 levels were signifi- cantly related to levels of inflammatory biomarkers soluble intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 (El-Asrar et al., 2011). Upregulated expression of HMGB1 contributed to early retinal neuropathy of diabetes and could be a target to ameliorate this disease (Abu El-Asrar et al., 2014). The vitreous level of HMGB1 is elevated in ocular sarcoidosis (Takeuchi et al., 2017). For patients with endophthalmitis, HMGB1 is released in the vitreous of eyes and related to the progression of endophthalmitis (Arimura et al., 2008); HMGB1 is highly related to inflammatory diseases of the external eye, and 18β-glycyrrhetinic acid might treat these diseases by inhibiting HMGB1 (Cavone et al., 2011); HMGB1 and its soluble receptor RAGE could play a role in vernal keratoconjunctivitis (VKC) (Zicari et al., 2014); these results are similar to those of Roberto Caputo et al. (2019) HMGB1 is involved in dry-eye disease (DED), and glycyrrhizin 2.5% eye drops have good ocular tolerance and good treatment results for patients with moderate DED (Burillon et al., 2018). There are also several experimental reports confirming that HMGB1 plays a key role in some eye diseases, including corneal wounds (Zhou et al., 2020), fungal keratitis (Li et al., 2020a), and cataracts (Xu et al., 2018). Our recent work provides evidence that HMGB1 and its related receptors are involved in the development of diabetic keratopathy in mice, and that the blockage of HMGB1 could serve as a strategy to prompt diabetic corneal and nerve wound healing (Hou et al., 2020). As HMGB1 is involved in many eye diseases, a glycyrrhizin eye drop could have good clinical values, and an eye drop combination of gly- cyrrhizin with other active agents could have even better clinical values in treating these stubborn diseases. One reason for this might be that the HMGB1 signal pathway has many signal molecules, and a part of these signal molecules is also involved with other signal pathways. Blockage of HMGB1 might trigger a compensatory activation of other signal pathways. The other reason might be that many of these stubborn dis- eases have complicated mechanisms, and just a glycyrrhizin eye drop might not be expected to achieve satisfying treatment results. For example, an antifungal drug is needed to effectively kill fungi while glycyrrhizin relieves the inflammatory reaction to secretions from fungi or dead fungi. If glycyrrhizin is designed as a nanocarrier to be formu- lated with many active molecules, a synergistic therapeutic effect and even an improved therapeutic effect could be anticipated, as many pharmacological active agents have limitations such as low aqueous solubility, low stability, and low permeability, while nanoformulations of these agents can regulate their pharmacokinetic and pharmacody- namic properties. Glycyrrhizin can provide not only the drug delivery nanocarrier, but the drug itself. Inspired by this expectation, a DG-Gen ophthalmic solution was designed and evaluated in vitro/in vivo for its efficacy in the regulation of the HMGB1 signal pathway. Gen has strong pharmacological activ- ities, including anti-inflammatory, antioXidative, and neuroprotective, and it has the confirmed activity of inhibiting development of diabetic complications including cataracts and diabetic retinopathy (Park and Surh, 2004; Weng et al., 2019). However, Gen is insoluble in water; a solubilizing agent is needed to form an ophthalmic solution. In this study, Gen was encapsulated into DG micelles with the proper DG/Gen weight ratio. The Gen concentration of this optimized DG-Gen ophthalmic solution, with a DG/Gen weight ratio of 15:1, was set as 5 mg/ml, though the apparent solubility could be obtained at a higher concentration than this, and the apparent solubility of Gen was improved 2644-fold (the solubility of Gen in water was 1.89 μg/ml at 30 ◦C (Chen et al., 2013)). Gen could successfully be formulated into an ophthalmic solution with solubilizing DG micelles. DG-Gen had nano- scale micelle size, though with some wide size distribution (PdI>0.3). It has been widely accepted that the smaller size allows nanoparticles to traverse the corneal epithelium and enhances corneal permeation (Niamprem et al., 2019; Sharma et al., 2016). The improved apparent solubility of Gen and the nanoscale micelle formulation of Gen indicated a good in vivo corneal permeation characterization. And the in vivo corneal permeation results confirmed this expectation. The Cou6-DG-Gen group displayed a much stronger green fluorescence than the free-Cou6 group.
The improved apparent solubility of Gen and a better in vivo corneal permeation characterization of DG-Gen also anticipated a strong in vitro/in vivo pharmacological characterization. Both FRAP and ABTS evaluations confirmed great improvement of in-vitro antioXidant ac- tivities of DG-Gen compared to the free-Gen suspension solution. It is worth mentioning that DG also displayed antioXidant activity and that this result was consistent with other reports (Jiang et al., 2020; Kwon et al., 2020; Paudel et al., 2020). The observed antioXidant activity of DG verified our proposal that DG was not only a delivery nanocarrier, but the drug itself.
Diabetic keratopathy is a severe diabetic complication in the eye without effective treatment practices, therefore, there is an unmet medical need to identify new potential treatment targets and novel practices to intervene in this severe diabetic complication. Our previous work had identified that the HMGB1 signaling pathway played a vital role in the development of diabetic keratopathy; thus, blockage of this pathway could improve treatment of diabetic keratopathy. Therefore, the feasibility of targeting the HMGB1 signaling pathway with a DG-Gen ophthalmic solution to treat diabetic keratopathy was explored in this manuscript. This evaluation of efficacy also provides feedback on whether DG micelles can synergistically enhance the therapeutic effects of encapsulated Gen.
Diabetic mice with corneal epithelial debridement wounds are frequently used as animal models in evaluating treatment efficacies for and revealing the mechanisms of diabetic keratopathy (Di et al., 2017; Lu et al., 2020; Yang et al., 2014). Corneal re-epithelialization and nerve regeneration profiles were the two main parameters in evaluating treatment efficacies (Li et al., 2020b). The DG-Gen ophthalmic solution significantly accelerated corneal re-epithelialization and nerve regen- eration, while the free-Gen suspension solution was negative in the evaluation of these two parameters. The DG solution was also observed to have strong treatment efficacies, and the DG&Gen physical miXture solution displayed similar treatment efficacies to the DG solution. This result also confirmed the negative treatment efficacies of free Gen. The strong contribution of nanoformulation itself can safely be proven, as the DG-Gen ophthalmic solution displayed much stronger efficacies than the DG&Gen physical miXture solution, though the same concentration of DG and Gen was used in both these solutions.
Responses to corneal epithelium damage treated with the DG-Gen ophthalmic solution appeared to involve HMGB1. The DG-Gen ophthalmic solution treatment led to significantly lower expression levels of HMGB1, as well as the protein levels of its receptors RAGE and TLR4, in wounded diabetic corneas. The DG solution and the DG&Gen physical miXture solution also down-regulated these protein levels, while the free-Gen suspension solution failed to do this. These protein- level expression profiles were consistent with the treatment efficacies.
As the HMGB1 signal pathway is involved in inflammatory responses, IL- 1β and IL-6, two inflammatory factors widely detected in experiments on the HMGB1 signal pathway, were detected and found in low levels in the DG-Gen ophthalmic solution treated groups. The DG solution and the DG&Gen physical miXture solution also down-regulated these two in- flammatory factors levels, while the free-Gen suspension solution failed to do this. Our findings include the fact that DG-Gen prompted corneal wound healing and nerve regeneration involving the regulation of the HMGB1 signal pathway, including suppression of the level of HMGB1, as well as its receptors RAGE and TLR4, and the down-regulation of IL-1β and IL-6. Thus, it seems reasonable to conclude that during treatment with DG-Gen, the mechanism that inhibits inflammation through the HMGB1 signal pathway was involved in prompting corneal epithelial wound healing and nerve regeneration in diabetic mice.
In conclusion, this study developed a novel DG-Gen ophthalmic so- lution with DG as a nanocarrier. The DG-Gen solution exhibited nano- spherical particles with nanosacle size dispersion and good ocular tolerance. The in-vivo permeation results revealed that DG-Gen dis- played an excellent corneal permeation profile. The DG-Gen ophthalmic solution was identified to have a strong efficacy in prompting epithelial wound healing and nerve regeneration in diabetic corneas. This activity appears to involve regulation of the HMGB1 signal pathway, since the promotion of activity by DG-Gen on epithelial and nerve repair was involved with suppressing expression levels of HMGB1 and its receptors RAGE and TLR4, as well as inflammatory factors IL-1β and IL-6. These findings provide new insights into the mechanism underlying HMGB1 signaling effects on diabetic corneal wound healing, as well as an effective drug therapy intervention based on DG micelles, a nanocarrier designed as an HMGB1 inhibitor.


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