Dexamethasone acetate loaded poly(ε-caprolactone) nanofibers for the treatment of rat corneal chemical burn

doi:10.21203/rs.3.rs-3769368/v1

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Dexamethasone acetate loaded poly(ε-caprolactone) nanofibers for the treatment of rat corneal chemical burn

https://doi.org/10.21203/rs.3.rs-3769368/v1

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published 18 Sep, 2024

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Nanofibers serve to overcome the limitation of the short duration of action of topical eye drops used against ocular inflammation in dry eyes. Several attempts to develop suitable nanofibers have been made; however, there is no ideal solution. The purpose of this study was to determine the effect of poly(ε-caprolactone) (PCL) nanofibers with encapsulated dexamethasone on alkali-injured cornea in rats. Thirty-nine Sprague Dawley (SD) rats (7 weeks old males) were divided into four treatment groups after alkaline burns of the cornea; negative control (no treatment group); dexamethasone eyedrops (DEX group); PCL fiber (PCL group); dexamethasone loaded PCL (PCL/DEX group). Corneal neovascularization was lower in the group treated with PCL + DEX than in that treated with DEX eyedrops, while PCL alone did not cause corneal neovascularization by irritation or inflammation. Histological analysis revealed lower infiltration of stromal inflammatory cells in corneas treated with PCL + DEX than in those treated with DEX eyedrops. Proinflammatory cytokines, IL-1β, MK2, TGFβ1, TGFβ2, and VEGF-A showed lower expression in corneas treated with PCL + DEX than in those treated with DEX eyedrops. Based on the results, PCL + DEX may be a promising approach to effective drug delivery in corneal burn injuries. In particular, anti-inflammatory function of DEX observed in the acute phase of ocular inflammation appears to be significantly improved on the first day in the PCL + DEX group.

Biological sciences/Drug discovery/Drug delivery

Health sciences/Diseases/Eye diseases/Corneal diseases

dry eye

dexamethasone eye drops

nanofibers

rats

The ocular surface is constantly exposed to the external environment, making it the most vulnerable tissue to external stimuli. As a result, continuous inflammation and immune responses at the surface of cornea and conjunctiva occur more often than in other body parts. If inflammation or wound healing due to trauma, surgery, infection, or burns, are not properly treated, corneal neovascularization and scar may cause irreversible fibrosis and vascularization which result in ocular pain and vision loss ^1,2. Topical steroids and cyclosporine A (CsA) are the most common treatment strategies used to control inflammation ^2,3. Moreover, in acute inflammatory conditions such as chemical or thermal burn, Stevens-Johnson syndrome and ocular graft-versus-host disease, intensive treatment with steroid instillation every few minutes or hours are often mandatory ^4–6. However, the effect of the topical drugs on the ocular surface is limited in time due to tear dilution, lacrimal turnover, blinking reflex, nasolacrimal drainage or ocular barriers ^7,8. Low ocular bioavailability of topically applied drug molecules can considerably limit their efficacy ⁹. Therefore, there is an urgent need for a superior treatment delivery method that can provide more stable and continuous effect on the ocular surface.

Electrospun nanofibers have been extensively used in regenerative medicine for efficient drug delivery due to their rapid and easy fabrication and controllability of density and thickness by factors such as polymer solution concentration, flow rate, and voltage ^10,11. Electrospun nanofibers can be used as a drug delivery system to treat local lesions because of its advantage of high drug loading capacity, high drug encapsulation efficiency, and simultaneous delivery of diverse therapeutic agents ¹². Nanofibers loaded with triamcinolone acetonide, an anti-inflammatory drug, reduced inflammation in rat experimental autoimmune uveitis ¹³. Furthermore, previous study reported that CsA-loaded nanofibers transferred onto the ocular surface injured with alkali could represent an effective alternative mode of therapy for reducing inflammation and further damage ¹⁴. Nanofibers made of poly (ε-caprolactone) (PCL) and dexamethasone (DEX) acetate were developed to deliver corticosteroids to the back of the eye during the post-operative period of any vitreoretinal disease ¹². Electrospun fiber with gelatin and PCL mixture in a rabbit organ culture model showed that the fiber was able to support wound healing in the cornea ¹⁵. Although there have been cases where DEX has been incorporated into electrospun fibers and used in drug delivery systems, there has been no study of alkali burn treatment. Therefore, we aimed to evaluate the usefulness of PCL nanofibers with encapsulated DEX for the treatment of alkali-injured cornea in rats.

Morphological characterization

Morphology analysis of the electrospun nanofibers was performed using SEM images. Figure 1A and Fig. 1B show the morphology of the electrospun PCL nanofibers prepared with 13 wt% PCL and the PCL:DEX blend (13:5 wt%), respectively. The average diameters of the nanofibers were measured to be 1.79 ± 0.14 µm for the 13 wt% PCL fibers and 1.76 ± 0.13 µm for the PCL:DEX (13:5 wt%) fibers.(Table 1.)

Table 1

Diameter of PCL and PCL + DEX fiber
	PCL (13wt%)	PCL (13wt%) + DEX (5wt%)
Average (n = 10)	1.79 ± 0.14 µm	1.76 ± 0.13 µm

In vitro drug release studies

The drug release patterns of Rhodamine 6G from the PCL fibers are shown in Fig. 2. Macroscopic observations (Fig. 2A) revealed a noticeable change in the appearance of the fibers. The initial intense dark red color attributed to Rhodamine 6G gradually changed to a pale pink color within the first hour after Rhodamine 6G release. In addition, the color became significantly lighter after 6 hours. On quantitative analysis, the PCL fibers loaded with Rhodamine 6G exhibited a rapid release profile with over 80% of the drug released within the first 20 minutes (Fig. 2B).

Corneal re-epithelialization

Facilitating re-epithelialization is of paramount importance in corneal wound healing. Here, we examined the extent of corneal erosion after chemical injury for 14 days. While the extent of corneal erosion was decreased gradually over time in all experimental groups, significant differences in the extent of corneal erosion were not observed among groups on days 1, 2, 3, 4, 7, and 14 post-injury (Fig. 3). Representative images of corneas in each group on days 0, 1, 2, 3, 4, 7, and 14 after alkali burn are shown in Fig. 3.

Evaluation of corneal opacity and neovascularization

The mean values of corneal opacity grading on day 7 after alkali burn in the untreated, DEX eyedrops, PCL, and PCL+DEX treated groups were 0, 1.5 ± 0.5, 1.5 ± 0.5, 2, and 2 ± 0.5, respectively (p = 0.3502) (Figure 4). No significant differences were observed between the groups. Subsequently, the mean values of corneal neovascularization grading in the untreated, DEX eyedrop, PCL, and PCL+DEX treated groups were 0, 1.5 ± 0.5, 1.5 ± 0.5, 0.5 ± 0.5, and 0.3 ± 0.5, respectively (p = 0.2358) (Figure 4). There were no significant differences between the groups.

Histology

H-E staining of untreated corneas revealed greater stromal thickness and looser stromal collagen structure compared to that in the DEX eyedrop, PCL, and DEX + PCL fiber-applied groups (Fig. 5).

In IHC staining, the expression of α-SMA was most prominent in the untreated cornea, and cornea treated with the DEX eyedrop. In addition, MMP9 and IL1-β were not expressed in the cornea treated with the PCL or PCL + DEX, whereas weak staining with the antibodies was observed in the untreated cornea and those treated with DEX eyedrops (Fig. 5).

Western blot and qPCR

In the western blot analysis, we found that IL-1β, MK2, TGFβ1, TGFβ2, and VEGF-A were expressed much stronger in the negative control and DEX-treated corneas than in the cornea treated with PCL + DEX fiber (Fig. 6).

In the short-term qPCR, IL-6 showed significantly lower expression in DEX + PCL compared to DEX group on the first day after injury. In the case of IL-1β, the expression was much lower in PCL + DEX in the first day after injury, but over time, it was relatively higher than in the DEX group. (Fig. 7)

Multiple studies have shown that PCL fibers were promising ocular delivery platforms due to their outstanding properties, including high kinetic and thermodynamic stability, biocompatibility, and biodegradability ^16–20. Various drug-loaded PCL fibers prepared from solution, suspension, and emulsion showed that PCL fibers were a potential drug delivery option for any kind of drug ^21–23. These fibers can also be prepared after co-polymerization with other polymers such as PEG, chitosan, or chlorophyllin, and some researchers have investigated polymer combinations in recent years ^24–27. However, most of the previous studies have been in vitro or ex vivo studies introducing characteristics of the materials or evaluating drug release profiles. There have never been in vivo studies evaluating the therapeutic effects of drug-loaded PCL fibers using rat models with corneal chemical burns. Accordingly, in this study, we prepared a PCL fiber mixed with dexamethasone acetate by electrospinning technique and attempted to study the therapeutic effects of the fibers on a rat model with corneal chemical burns.

For the re-epithelialization, no significant differences were observed between the groups on 1, 2, 3, 4, 7, and 14 days after burns. Steroids are known to inhibit the re-epithelialization of the cornea ^28–30. Nevertheless, our results showed that DEX eyedrops or PCL + DEX did not inhibit the re-epithelialization of the cornea. In addition, PCL itself did not have a negative effect on the re-epithelialization of the cornea, suggesting the capability of PCL fibers to deliver the drug to the cornea.

Although the mean grading value of neovascularization was lower in the group treated with PCL + DEX than in that treated with DEX eyedrop, there was no statistical significance. We identified that PCL + DEX had a tendency for the better corneal neovascular inhibition effect than DEX eyedrops. In the group treated with PCL, the mean grading value of the neovascularization was similar to that of PCL + DEX, suggesting that PCL itself did not cause corneal neovascularization by irritation or inflammation. Meanwhile, there were no significant differences in corneal opacity. In the process of wound healing and tissue repair, mature myofibroblasts secrete collagens and extracellular matrix materials that form fibrotic scars ³¹ Since the process takes time over weeks to years, 7 days may have been too short to observe the differences between groups. In addition, the therapeutic effect of PCL + DEX may have been insufficient to suppress corneal opacity due to the short release time of the drug as seen in vitro drug release study results.

In H-E staining, the infiltration of inflammatory cells into the corneal stroma was more pronounced in the corneas treated with DEX eyedrops than in those treated with PCL + DEX. Consistent with H-E staining, greater expression of α-SMA was identified in the corneas treated with DEX eyedrops compared to those treated with PCL + DEX in IHC staining. Although it was less prominent, MMP9 and IL1-β showed greater expression in DEX eyedrop-treated corneas compared to the PCL + DEX-treated corneas as well. α-SMA is used as a marker of activated myofibroblasts, and α-SMA expression implies activated fibrosis in the corneal stroma ³¹. MMP9 production is stimulated by the pro-inflammatory cytokine IL1-β, and these factors play a role in corneal matrix degradation ³². Hence, the expression of MMP9 and IL1-β in the anterior stroma is associated with inflammation. These results imply that PCL + DEX is superior to DEX eyedrops in controlling the fibrosis and inflammation of the cornea. Meanwhile, corneas treated with PCL showed a compact collagen structure similar to corneas treated with PCL + DEX fiber, and no definite expression of the antibodies was observed, suggesting that PCL fiber did not show negative effects related to fibrosis or inflammation in the process of wound healing and tissue repair for corneal alkali burns.

In western blot analysis, there were no statistically significant differences between the groups. However, IL-1β, MK2, TGFβ1, TGFβ2, and VEGF-A showed lower expression levels in corneas treated with PCL + DEX than in those treated with DEX eyedrops, and both values were lower than those in the negative control. We identified that PCL + DEX was superior to DEX eyedrops in terms of anti-inflammatory and neovascular suppression. Meanwhile, these factors were randomly expressed in the corneas treated with PCL fiber without a specific trend. We assumed that PCL fibers did not show a positive or negative effect consistently in terms of inflammation or neovascularization in the cornea.

In order to confirm a more immediate anti-inflammatory response and changes over time after the injury, qPCR was additionally performed on days 1, 3, and 7. IL-1β and IL-6 are known to act as proinflammatory cytokines, especially as angiogenic activities in the acute phase of ocular inflammation ^33,34. DEX regulates inflammation by inhibiting the action of these cytokines ³⁵. In our study, the anti-inflammatory function of DEX seen in the acute phase of ocular inflammation appears to be significantly improved on the first day in the PCL + DEX group.

There are several limitations to our study. The first limitation is that the number of animals was small. Even in short-term, qPCR assessments were performed on only one eye in each treatment. We could not observe statistical significance in all the results obtained through this study. Instead, we estimated the relative superiority in therapeutic effects by analyzing trends between groups. For this reason, it is necessary to conduct additional experiments under the same conditions in future studies to confirm the reproducibility and consistency of the results and to examine statistical significance. Second, the fibers might unexpectedly fall off early due to eyelid tension or blinking rate which can act as variables on the residence time of the fibers. Although the fibers were injected into the fornix and lateral 1/3 of the eyelid was sutured, we were unable to identify PCL fibers in all PCL and PCL + DEX groups when we checked the day after the injury. It was not clear whether the fiber was melted and its detection was no possible or not; however, we were not able to rule out the possibility of its early falling off. However, since nearly 80% of the drug is released from the fibers within 20 minutes, it is unlikely that it would have affected the experimental results even if it did fall off in the middle of the study.

Third, the observation period was short. Since complications, such as corneal opacity or neovascularization in corneal alkali burns progress over several weeks to months, 14 days might be too short to cause the differences between groups. In the future, it is necessary to conduct studies by increasing the observation period to compare the long-term therapeutic effects in the process of tissue repair. Fourth, the action time of the drug was too short. Nearly 100% of the drugs were released within 1 hour after showing an initial burst for 20 minutes. Despite the short action time, PCL + DEX showed superiority in inhibiting corneal neovascularization compared to DEX eyedrops on day 14 after burns. However, since corneal remodeling takes place over several weeks to months, we can expect a better long-term prognosis if we use a drug deliverer with a longer action time. Typically, a matrix type of drug delivery system exhibits a high initial drug release, followed by a decreased release rate due to the increasing diffusion distance from the drug molecules inside to the surface ³⁶. The drug release rate can be modulated by altering fabrication parameters such as the concentration of polymer and drug, type of solvent, or electrospinning conditions. The concentration of polymer and drug affects the fiber diameter, drug encapsulation, and tensile strength ^26,37,38. When the drug amount is increased, drug molecules in the polymer solution may form drug crystals on the fiber surface due to the high ionic strength of the fiber, leading to a faster drug release rate. In addition, a more sustained release is shown when the molecular weight of the drug is high ³⁹. Therefore, in future studies, PCL fibers with a longer residence time on the ocular surface can be prepared by mixing different ratios of PCL and DEX or loading other steroids such as loteprednol etabonate, fluocinolone acetonide, or difluprednate with a higher molecular weight than dexamethasone acetate. However, it will be necessary to identify a ratio of PCL and steroids with optimal residence time, considering that steroids can cause infection or inhibit corneal re-epithelialization.

Nevertheless, this study is valuable in that it is the first in vivo study to examine the therapeutic effects of steroids-loaded PCL fibers in various clinical aspects such as corneal re-epithelialization, opacity, and neovascularization and ascertain the possibility of PCL fibers as a drug deliverer on corneal alkali burns using a rat model. In addition, placing the fibers in the fornix has advantages in that it is less invasive and easier to apply in clinical practice compared to injecting fiber into the subconjunctival area ^18,19, anterior chamber ⁴⁰, or vitreous cavity ^12,41. However, it can easily fall off because it is easy to apply. To increase the bioavailability of the sutureless drug-loaded PCL fibers, it is necessary to develop strategies to ensure that the fibers stay inside the cornea during the drug action time. The fiber can be fabricated to have cations or a similar structure with the corneal epithelial cell’s microvilli. These strategies take advantage of the negatively charged ocular surface to increase their precorneal residence time through electrostatic interactions, and strengthen the adhesion with ocular surface by increasing the friction force, respectively. In addition, the fibers can be fabricated in a ring shape which has biocompatible metallic ring on a rim to preserve visual axis. It has the advantage of securing its position by placing the upper and lower parts in fornix without interfering with the vision.

Although there were no statistical differences, we identified that PCL + DEX had a greater therapeutic effect than DEX eyedrop in terms of anti-inflammation in the initial treatment of corneal alkali burns within 14 days. In particular, it was shown to maximize the anti-inflammatory function of DEX in acute inflammation. Based on these results, we concluded that rapid control of inflammation with an initial application of drugs within 20 minutes before the chemicals penetrate the corneal stroma and cause inflammation throughout the cornea is a key aspect that determines the prognosis of the disease.

Fabrication of PCL fiber

Unless otherwise noted, all chemicals and solvents were used as provided by the manufacturers. PCL (MW 80,000), 2,2,2-trifluoroethanol (TFE), Rhodamine 6G, and DEX were acquired from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS; pH 7.4) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

To fabricate PCL fiber, PCL was dissolved in TFE as 13 wt%, and DEX was added to 5 wt% of the total solution. The polymer solution was filled into a syringe and ejected through a 23G needle. The flow rate, voltage, and run time of electrospinning were 1.5 mL/h, 16.0 kV, and 1 h, respectively. The distance between the needle tip and the collector was 10 cm (Fig. 8).

Scanning electron microscope

Scanning electron microscope (SEM) photographs were obtained using an SEM (SU8600, Hitachi, Japan) at the Smart Materials Research Center for IoT, Gachon University, Korea. All samples were attached to a brass sample holder with double-sided adhesive tape and sputter-coated with gold to make them electrically conductive.

In vitro release profile

Rhodamine 6G was added to quantify the release profile from PCL electrospun fibers. Rhodamine 6G was added to 5 wt% of the total solution, and the electrospinning was performed in the same condition. Rhodamine 6G release was carried out in 24-well plates by immersing each scaffold in 2 mL of PBS. At each point, the entire solution of each well was removed and stored, and then PBS was added to each well. The fluorescence intensity of the stored solution was measured using Nanodrop (BioTek, Winooski, VT, YSA) and the intensity was converted to concentration via the fluorescence intensity standard curve.

Animals and corneal alkali burn model

Experimental protocols and animal care were performed according to the guidelines for the care and use of animals established by Yeouido St. Mary’s Hospital and complied with the ARRIVE guidelines. The Animal Experimentation Ethics Committee of Yeouido St. Mary’s Hospital approved the experimental protocols (approval number: YEO-2021-012FA, YEO-2022-008FA). A total of 33 male Sprague Dawley (SD) rats (7 weeks old, 200–300 g) were purchased from Saeronbio Inc. Anesthesia was achieved by intraperitoneal injection of ketamine hydrochloride (30 mg/kg). Alkali burn injury was performed on the right eye and was induced by pressing a qualitative filter paper disc (3.0 mm in diameter, Hyundal micro) containing 1 N NaOH onto the central cornea for 30 s. After removal of the filter paper disc, the corneal surface was carefully rinsed with 20 mL of physiological saline solution for 5 min. Study protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea (IRB No: YEO-2020-004FA), and this study was conducted in compliance with Animal Welfare Regulations. The animals were randomly divided into four groups (n = 5–9 per group). Sample size requirement estimates were based on clinical examination scores and testing at the study endpoint. In the first group of animals, no further treatment was applied to the injured eyes (control group). In the second group, DEX eyedrops were applied onto the injured corneal surface (DEX group). In the third group, PCL fibers were cut into about 1×1 mm sizes and implanted into the lower fornix (PCL group). In the fourth group, DEX/PCL fibers were cut into about 1×1 mm sizes and implanted into the lower fornix (Fig. 9) (DEX/PCL gorup). In all animals, the outer third of the upper and lower eyelids were sutured (Fig. 9). DEX eyedrops were applied once daily to all experimental groups except for a group of the normal and negative control. Photographs of the corneas were taken on days 0, 1, 2, 3, 4, 7, and 14 post-injury. Animals were sacrificed on day 7 or 14 after alkali burn. The experimental eyes were enucleated and embedded within a mold. Healthy corneas served as controls.

Six other male SD rats of the same model were prepared for a short-term effect evolution. Anesthesia and alkaline burns were performed in the same manner as described above, except for those performed on both eyes. The eyes were randomly divided into four groups (n = 3 per group). In the first group of eyes, no further treatment was applied to the injured eyes. In the second group, DEX eyedrops were applied onto the injured corneal surface. In the third group, PCL fibers were cut into about 1×1 mm sizes and implanted into the lower fornix. In the fourth group, DEX/PCL fibers were cut into about 1×1 mm sizes and implanted into the lower fornix (Fig. 9). In all animals, the outer third of the upper and lower eyelids were sutured (Fig. 9). DEX eyedrops were applied once daily in all experimental groups except for a group of the normal and negative control. Animals were sacrificed on day 1, 3, or 7 after alkali burn. The experimental eyes were enucleated and embedded within a mold.

Evaluation of the corneal re-epithelialization

The extent of the corneal chemical injury was calculated using the following equation and expressed as a percentage (%):

E (%) = W_s / W_t

Where W_s is the extent of the cornea stained with fluorescence dye, W_t is the extent of the whole cornea, and E represents the ratio indicating the extent of the corneal injury relative to the whole cornea. Denuded epithelium was stained with fluorescein dye, and it was visible under blue LED light. Blue LED light photos of rat corneas following alkali burn were taken on days 0, 1, 2, 3, 4, 7, and 14 after alkali burn. Measurements were processed using NIH ImageJ software (National Institutes of Health, Bethesda, MD, USA). The extent of corneal chemical injury at baseline was assumed to be identical in all three experimental groups and was presented as a percentage (%).

Evaluation of corneal opacity and neovascularization

The degree of corneal opacity and neovascularization (NV) was measured after administration of 0.1% HA eye drops, crosslinked HA hydrogels, or no treatment to evaluate the efficacy of corresponding treatments. Two researchers (K.S.N and S.K.P.) assessed the high-resolution photographs of the cornea and scored the degree of relevant parameters on day 7 post-injury using grading measfures previously described by Larkin et al ³¹. The mean value of two measurements was obtained as a representative value. All data analyses were performed in a manner blinded to treatment groups. Corneal opacity was graded as follows: 0, completely transparent; 1, minimal loss of transparency; 2, moderate loss of transparency but iris vessels visible on retroillumination; 3, iris vessels not visible but pupil outline visible; 4, pupil outline not visible. Corneal neovascularization was graded as follows: 0, no vascularization of graft; 1, vessel growth to 25% of graft radius in any quadrant; 2, vessel growth to 50% of graft radius; 3, vessel growth to 75% of graft radius; 4, vessel growth to center of graft.

Histological examinations

After completion of the study (on day 7), tissues were isolated. The cornea was carefully separated from the eye while avoiding damage and washed in 1×PBS. Tissues were fixed using 4% paraformaldehyde (PFA) solution overnight at 4°C and then washed in 1×PBS. Tissue segments were dehydrated in graded ethanol, embedded in paraffin, and cut into 3- to 4-µm-thick sections using a microtome. To study postoperative inflammatory cell infiltration, tissue sections were stained with hematoxylin-eosin (H-E). For H-E staining, tissue sections (3–4 µm) were rehydrated by immersing in xylene (3×5 min) followed by 100% (twice), 95, 85, and 70% ethanol for 1 min each, deionized water for 1 min, and hematoxylin (BBC Biochemical) and eosin (BBC Biochemical) solutions for 10–20 s before clearing with pure xylene.

Immunochemistry

For immunohistochemistry, tissue sections (3–4 µm) were rehydrated by immersing in xylene (3x5 min), 100% (twice), 95, 85, and 70% ethanol for 5 min each, and deionized water for 5 min. Antigen retrieval buffer (citrate buffer, pH 6.0) was preheated to 92–95°C. Slides were immersed in the pre heated solution for 5–10 min. Tissues were encircled with a hydrophobic barrier using a barrier pen. Tissues were further blocked with 2.5% normal horse serum (Vector) for 30 min-1 h. The slides were washed and incubated with primary antibodies: mouse anti-CK3 monoclonal antibody (ab68260, Abcam, Cambridge, UK; 1:50 dilution), mouse anti-alpha smooth muscle actin monoclonal antibody (ab7817, Abcam, Cambridge, UK; 1:100 dilution), rabbit IL-1 beta polyclonal antibody (ab9722, Abcam, Cambridge, UK; 1:1000 dilution), and mouse anti-MMP9 monoclonal antibody (ab58803, Abcam, Cambridge, UK; 1:1000 dilution) at 4°C overnight. The next day, sections were washed three times in 1×PBS followed by incubation with anti-mouse/rabbit IgG secondary antibodies (MP-7500, Vector, Burlingame, CA, USA) at room temperature (20–22°C) for 1 h in the dark. For the negative controls, nonimmune serum at 1:100 dilution was used instead of the specific primary antibody. After three washes in 1×PBS, visualization was performed using a freshly prepared DAB (K5007, DAKO, USA) substrate-chromogen solution for 30–40 s, followed by three washes in 1×PBS. Contrast staining was performed using hematoxylin (BBC Biochemical). Sections were observed and imaged with a Trinocular Microscope (Olympus BH2 BHS312, Shinjuku, Tokyo, Japan).

Western blot analysis

Rat corneal proteins were extracted with cold RIPA buffer (#89900, Thermo Fisher Scientific, USA) with Halt™ Protease and Phosphatase Inhibitor Cocktail (#78441, Thermo Fisher Scientific, USA) and the protein concentration was determined using a protein assay kit (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, USA). Aliquots having equal protein content were subjected to electrophoresis on 10%, 12% Tricine gels and then electrophorectially transferred to PVDF membranes (IPVH00010, Millipore, USA). After 1h blocking in 5% Skim milk, the blots were incubated with primary antibodies for: Anti-alpha smooth muscle Actin (ab7817, Abcam, UK; 1:1000 dilution), Anti-IL-1 beta (ab9722, Abcam, UK; 1:1000 dilution), Anti-VEGFA (ab1316, Abcam, UK; 1:1000 dilution), VEGF (MA1-16629, Invitrogen, USA; 1:1000 dilution), Anti-Vimentin (ab45939, Abcam, UK; 1:1000 dilution), TGFβ1 (sc-130348, Santa cruz, USA; 1:200 dilution), TGF-βRII (sc-17792, Santa cruz, USA; 1:500 dilution), MK2 (#12155S, Cell Signaling, USA; 1:1000 dilution ), p-MK2 (#3316S, Cell Signaling, USA; 1:1000 dilution ), p38 (#9212S, Cell Signaling, USA; 1:1000 dilution), p-p38(#9211S, Cell Signaling, USA; 1:1000 dilution), PEDF (PA5-75359, Invitrogen, USA; 1:1000 dilution) and Anti-β-Actin (A5441, Sigma, USA; 1:5000 dilution) as a loading control. After washing each membrane three times with Tris-buffered saline containing 1% Tween 20 for 10 min and subsequently reacted with HRP-conjugated secondary antibodies: anti-rabbit IgG (#7074S, Cell Signaling, USA; 1:3000), anti-mouse IgG (#7076S, Cell Signaling, USA; 1:3000) for 1h at room temperature, respectively. The specific bands were visualized by an enhanced chemiluminescence reagent (#32132, ECL, Thermo Fisher Scientific, USA) and exposed to Hyperfilm™ ECL ™ film (#28906838, Cytiva, USA). The film was developed and the band intensities quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Quantitative PCR (qPCR)

The corneas were cut into small pieces and homogenized in TRIzol™ (Invitrogen, #15596018, USA). In brief, each cornea was immersed in 1 mL of ice-chilled TRIzol and ground with a disposable homogenizer (Biomasher II, #890864; JPN). The concentration of RNA was measured with NanoDrop™ 2000 (Thermo Fisher Scientific, ND-2000, USA), and 1 µg of RNA templates were used for cDNA synthesis. Generation of cDNA was performed with the reverse transcription reaction kit (PrimeScript™ RT Master Mix, TaKaRa, RR036A; JPN). To determine the gene expression levels, we analyzed 100ng cDNA with quantitative PCR (qPCR) using the TB Green® Fast qPCR Mix (TaKaRa, RR430A; JPN) based on real-time detection of accumulated fluorescence, in accordance with the manufacturer’s instructions (Thermal Cycler Dice Real Time System,Applied, TaKaRa, TP800, JPN). The sequence of primers are as follows: IL-1β (5′-CTGTGACTCGTGGGATGATG-3′ and 5′-GGGATTTTGTCGTTGCTTGT-3’), IL-6 (5′-CACAAGTCCGGAGAGGAGAC-3′ and 5′-ACAGTGCATCATCGCTGTTC-3′), GAPDH (5′-GCAAGTTCAACGGCACAG-3′ and 5′-GCCAGTAGACTCCACGACAT-3’). The amplification program included an initial denaturation step at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, and 60°C for 10 s, dissociation step 95°C for 15 s, 60°C for 30 s, 95°C for 15 s after which a melt curve analysis was conducted to verify amplification specificity. Results were analyzed by the comparative threshold cycle (Ct) method, normalized with GAPDH as an endogenous reference and calibrated against the normal control group.

Statistical analysis

One-way ANOVA followed by post hoc analysis was used to analyze the differences in corneal re-epithelialization between untreated corneas, and corneas treated with DEX eye drops, PCL fibers, and DEX/PCL fibers. P-values < 0.05 were considered statistically significant. Data were analyzed using SPSS statistical software, version 22, (SPSS Inc., USA).

Acknowledgements

This study was supported by a grant from the National Research Foundation of Korea (NRF) (2022R1A2C2006109). The sponsor or funding organization had no role in the design or conduct of this research.

Author contribution

Study concept and design: D.R.K., S.K.P., E.J.K., K.S.N Acquisition, analysis, or interpretation of data: D.R.K., S.K.P., E.J.K., D.K.K., K.S.N Drafting of the manuscript: D.R.K., S.K.P., K.S.N Critical revision of the manuscript for important intellectual content: D.R.K., S.K.P., E.J.K., D.K.K., Y.C.Y, K.S.N, D.M., H.J.L Administrative, technical or material support : E.J.K., D.M., H.J.L Study supervision: K.S.N Funding acquisition : K.S.N

Data availability

All data are available from the corresponding author upon reasonable request.

Declaration of competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Dexamethasone acetate loaded poly(ε-caprolactone) nanofibers for the treatment of rat corneal chemical burn

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Abstract

Figures

Introduction

Results

Morphological characterization

In vitro drug release studies

Corneal re-epithelialization

Evaluation of corneal opacity and neovascularization

Histology

Western blot and qPCR

Discussion

Conclusions

Materials and methods

Fabrication of PCL fiber

Scanning electron microscope

In vitro release profile

Animals and corneal alkali burn model

Evaluation of the corneal re-epithelialization

Evaluation of corneal opacity and neovascularization

Histological examinations

Immunochemistry

Western blot analysis

Quantitative PCR (qPCR)

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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