DOTAP, DOPE, cholesterol, 2,7-dichlorodi -hydrofluorescein diacetate (DCFH-DA), fluorescein isothiocyanate (FITC), 4’,6-diamidino-2-phenylindole (DAPI), and benzalkonium chloride were purchased from Sigma. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly(ethylene glycol))2000] (DSPE-PEG2000) and FITC- DSPE-PEG2000 were purchased from Yuan Ye Biological Company. FK506 was purchased from MedChemExpress. Sodium dodecyl sulfate was purchased from Sangon Biotech. 1X phosphate buffer solution (PBS) was purchased from Biosharp. Commercial FK506 eye drops were purchased from SENJU (FK506 concentration: .1%), and it was a white turbid liquid. Hylo-COMOD was a commercial artificial tear whose first ingredient listed .1% sodium hyaluronate, and it was purchased from URSA PHARM Arzneimittel DmbH. The chemicals and solvents used for mass spectrum–high-performance liquid chromatography (MS-HPLC) and HPLC were of HPLC grade, while the others were of analytical grade.

The FK506 liposomes were prepared using the thin film hydrated dispersion method. Five mg DOTAP, 5.3 mg DOPE (DOTAP: DOPE = 1:1 in molar rate), 5 mg DSPE-PEG2000, 2.77 mg cholesterol, and 2 mg FK506 were dissolved in 6 ml of trichloromethane in a round-bottom flask. A rotary evaporator was used to remove the organic solvent in a 40°C water bath under a .06–.08 MPa vacuum; the rotation speed was kept at a rate of 100 rpm. Then, solvent traces were removed by keeping the solution in a 0.1-MPa vacuum for 1 h at 100 rpm. The resultant dried lipid film was hydrated with 10 ml 1X PBS at 40°C for 20 min until it formed a homogenous emulsion. Last, the final product was obtained by squeezing the liposomes through a 0.4-μm pore-size polycarbonate film (Whatman, Life Science) to decrease the particle size at room temperature. The liposomes were stored at 4°C until use.

The FITC-labeled FK506 liposome was synthesized following the abovementioned steps, except using FITC-labeled DSPE-PEG2000 to replace DSPE-PEG2000.

Particle size, zeta potential, and polydispersity index (PDI) of the liposomes were measured by dynamic light scattering (DLS) conducted on a particle size analyzer (Zetasizer nano-ZSE, Malvern). The liposomes were diluted with ultrapure water at a ratio of 1:100 in advance. Every sample was tested three times.

The morphology of the liposomes was analyzed using negative staining TEM (Tecnai G2 spirit, Thermo FEI). Ten μl of FK506 liposomes was dropped onto a 300-mesh carbon-coated copper grid. Excess liposome solution was stained before 1 μl 2% uranium acetate was added for 60s. The uranium acetate was then blotted, and another 1 μl drop of 2% uranium acetate was added; this process was repeated three times. The samples were then dried and examined at 120 kV.

The entrapment efficiency was determined by ultrafiltration. Freshly prepared FK506 liposomes were added to the ultrafiltration centrifugal tube (Merck Millipore, 10,000 NMWL) and centrifuged at 3,000 g at 4°C for 60 min. The filtered liquid, which contained free FK506, was collected, then lyophilized for 24 h, and redissolved with .6 ml methyl alcohol. The concentration of FK506 in the filtered liquid was determined by HPLC. Entrapment efficiency was calculated by the following formula: Entrapment   efficiency ( % ) = total   mass   of   added   FK506−the   mass   of   free   FK506  total   mass   of   added   FK506 × 100 % . (1)

Product loss was not considered in the preparation process, where the mass of free FK506 stands for the amount of FK506 in the filtered liquid, and the total mass of added FK506 is the initial mass of FK506 added into trichloromethane.

The FK506 loading level was calculated by the following formula: FK506   loading   level ( % ) = total   mass   of   FK506   encapsulated   in   liposomes total   mass   of   FK506   liposomes ×100% . (2)

Total mass of FK506 encapsulated in liposomes equals total mass of added FK506 minus the mass of free FK506, and the total mass of the FK506 liposome equals 20.07 mg minus the mass of free FK506.

The release profile of the FK506 liposome was confirmed by dynamic dialysis. First, FK506 liposomes were freshly synthesized in accordance with the procedures mentioned above; following this, PBS containing .1% sodium dodecyl sulfate (SDS) was prepared to serve as a release medium under sink condition (Dai et al., 2013). Ten ml of the FK506 liposomes was pipetted into a preprocessed dialysis bag (Biosharp, 3,500 MW); this was placed in 30 ml of the release medium and stirred at 200 rpm at 37°C. At the pre-established time points (.5, 1, 2, 4, 6, 9, 12, 24, 48, 72, 120, and 168 h), 1 ml of the release medium was taken out before 1 ml of the new release medium was added to maintain the volume of the release medium. All the samples were collected in centrifuge tubes and lyophilized for 24 h. The resulting powders were dissolved in .4 ml of methyl alcohol and centrifuged at 10,000 g for 10 min. The supernatant was then collected, and the final concentration of FK506 was measured by HPLC.

The quantitative analysis of FK506 in entrapment efficiency was determined by HPLC (Shimadzu, Japan), while the corneal concentration was determined using MS-HPLC (Agilent Technologies, USA). The experimental conditions of HPLC were as follows: The mobile phase comprised acetonitrile/10 mM ammonium formate solution (72/28, v/v). The flow rate was kept at 1.2 ml/min, and 20 μl of the samples was injected; the absorption was monitored at 210 nm. Chromatographic separation was carried out on an InfinityLab Poroshell 120 SB-C18 column (50 × 4.6 mm, 2.7 µm), and the column temperature was maintained at 60°C. The retention time of FK506 on this column was approximately 1.6 min; the peak area was calculated using the least square method, and the standard curve of the FK506 concentration peak was pictured. The regression equation was used to calculate the corresponding FK506 concentrations when the areas were known.

HCECs were purchased from ATCC and cultivated at an atmosphere of 95% air/5% CO₂ at 37°C. The culture medium used was Dulbecco’s modified Eagle medium (DMEM) (Solarbio); this was supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY), 4.5 mg/ml glucose, 100 U/ml penicillin, and 100 ug/ml streptomycin.

Cytotoxicity was measured using a cell counting kit-8 (CCK-8) assay (Dojindo, Japan) according to the manufacturer’s protocol. HCECs were seeded into 96-well plates at a ratio of 5,000 cells per well with 100 μl culture medium. The plates were placed into a cell incubator to allow them to adhere for 24 h. The cell culture medium was removed, and the cells were washed three times. The cells were incubated with 100 μl DMEM, blank liposomes, FK506 liposomes, and commercial FK506 eye drops for 2, 4, and 6 h. FK506 concentration of FK506 liposomes was .2 mg/ml, as well as that of commercial FK506 eye drops. Subsequently, 10 μl of the CCK-8 solution was added into each well, and then the cells were re-incubated for 2 h. The optical density values were measured at 450 nm using a microplate reader (SpectraMax M5, Molecular Device), and cell viability was calculated. The optimum concentration of FK506 liposomes was also determined using CCK-8 assay. FK506 liposomes with a series of drug concentration gradients (.0025 mg/ml, .005 mg/ml, .01 mg/ml, .02 mg/ml, .05 mg/ml, .1 mg/ml, and .02 mg/ml) were tested, and cell viability was calculated.

1*10⁵ HCECs were seeded into confocal dishes and adhered to for 24 h, the culture medium was then removed, and the cells were washed three times. Hundred μl of FK506 liposome labeled with FITC was co-incubated with cells for 2 h with 100 μl DMEM in the cell incubator. The excess liposomes and DMEM were removed, and the cells were washed three times. The nuclei of the HCECs were stained by DAPI. Confocal microscopy (A1 Ti, Nikon) was used to show the position of FK506 liposomes in cells.

The cell models of dry eye were established as follows: HCECs were seeded into 12-well plates at a density of 10⁵ per well and cultivated in the conditions mentioned above. After 24 h, the culture medium was removed, and 200 μM of hydrogen peroxide (H₂O₂) was added, and the cells were incubated for 1 h (Li et al., 2019).

The HCECs were seeded at a density of 1.0 × 10⁵ cells per well with 100 μl culture medium. After 200 μM H₂O₂ treatment was carried out for 1 h, FK506 liposomes and commercial FK506 were added into each well. After co-incubation for 2 h, the cells were washed three times with PBS and stained with DCFH-DA for 30 min. The fluorescence intensity of intracellular ROS was measured by flow cytometry at 484 nm (excitation) and 520 nm (emission).

The processed HCECs were treated with 1 ml of TRIzol and collected. MRNA was collected, quantified, and reverse-transcribed in turn. Reverse transcription was performed using the PrimeScript™ RT-PCR Kit, followed by quantitative real-time polymerase chain reaction (qRT-PCR) using the SYBR® Premix Ex Taq™ Kit. All the experimental procedures were in accordance with the manufacturer’s protocols. The primer pairs were as follows: human GAPDH, 5′-ACA​ACT​TTG​GTA​TCG​TGG​AAG​G-3′ (forward) and 5′-GCC​ATC​ACG​CCA​CAG​TTT​C-3′ (reverse); human interleukin-1β (IL-1β), 5′-AGC​TAC​GAA​TCT​CCG​ACC​AC -3′ (forward) and 5′-CGT​TAT​CCC​ATG​TGT​CGA​AGA​A -3′ (reverse); and human interleukin-6 (IL-6), 5′- ACT​CAC​CTC​TTC​AGA​ACG​AAT​TG -3′ (forward) and 5′-CCA​TCT​TTG​GAA​GGT​TCA​GGT​TG -3′ (reverse).

Animal experiments were carried out after ethical approval (No. 2021057) and conformed to the ethical standards of The Second Affiliated Hospital of Zhejiang University. The animal models of dry eye were established as follows: New Zealand rabbits (2–2.5 kg, male) were obtained from the Zhejiang province medical science institute. They were housed in their own cages individually in an environment where the temperature was 20–26°C and the humidity was around 40%–70%. The rabbits were treated with 25 μl of .2% benzalkonium chloride twice a day in the interval of 12 h for 7 days according to a previously reported study (Tan et al., 2019). C57BL/6 mice (20–25 g, male) were purchased from Shanghai SLAC Animal Co., Ltd. The mice were housed in pathogen-free conditions as stipulated.

The biocompatibility of FK506 liposomes was evaluated in the aspect of central corneal thickness (TOMEY anterior segment optical coherence tomography, Japan), endothelial cell density (TOMEY specular microscope EM-3000, Japan), corneal edema and conjunctival edema, and hyperemia (slit lamp microscope). Eighteen normal New Zealand rabbits were randomly put into three groups: the PBS group, the FK506 liposome group, and the artificial tear group. Each group had six rabbits. The rabbits in the PBS group were given PBS, those in the FK506 liposome group were treated with fresh FK506 liposome, and those in the artificial tear group were given Hylo-COMOD eye drops. All of them were administered twice a day (8 a.m. and 8 p.m.) at 50 μl. The abovementioned indicators were rigorously observed at day 0, day 7, and day 28. The anatomical structure of the cornea and conjunctiva was observed by H and E staining.

The ocular irritation of FK506 liposomes was evaluated via the modified Draize test (Bhatta et al., 2012). The normal rabbits were treated with 20 μl of FK506 liposomes directly on the cornea in the right eye and 20 μl of PBS in the left eye. All of them were installed at 30-min interval for 6 h. Then, ophthalmological tests were performed to observe the level of irritation, including secretions, redness of conjunctiva, and corneal opacity and swelling. The observations were conducted at a 12-hour interval for 72 h.

The dry eye rabbits were treated with 50 μl of FK506 liposomes in the right eye and 50 μl of the commercial FK506 eye drops in the left eye. After 5, 30 min, 1, and 2 h, the rabbits were euthanized. The cornea was excised wholly and washed gently three times with normal saline. All samples were stored at −20°C. In addition, the amount of FK506 in the cornea was determined by MS-HPLC.

The experimental conditions of MS-HPLC were as follows: The cornea was cut into pieces and placed in centrifuge tubes. One ml of methyl alcohol was added along with 2 mm ceramic beads to prepare the cornea homogenate. All the samples were centrifuged at 10,000 g for 10 min, and the liquid supernatant was removed. The FK506 concentrations were measured utilizing MS-HPLC. The mobile phase comprised 0.1% formic acid/5 mM ammonium acetate in water (Solvent A) and acetonitrile (Solvent B), the flow rate was kept at 0.3 ml/min, and 20 μl of the samples was injected. Chromatographic separation was carried out on a Zorbax SB C18 column (150 × 2.1 mm, 3.5 µm). The gradient program was as follows: 80% of B for .5 min; kept at 95% of B for 4.5 min and finally, remained at 100% of B. MS was performed under 5 L/min gas flow at 325°C and 11 L/min sheath gas flow at 350°C. The ion spray voltages were 3,000 V and −3,500 V in the positive and negative ionization modes, respectively. The integral peak area of m/z 821.5>786.4 was used for quantitative analysis.

The pre-ocular retention time was assessed by in vivo fluorescence imaging. The C57BL/6 mice were anesthetized, and a drop of FITC-labeled FK506 liposomes and commercial FK506 eye drops mixed with FITC and FITC solutions (nearly 5 μl) was added to their eyes according to the group. The excitation wavelength was set at 488 nm, and the emission wavelength was 520 nm. After 5, 10, and 30 min, the fluorescence signal was collected and analyzed.

The 16 dry eye rabbits were assigned into four groups using a random number table, and they were as follows: the negative control group, positive control group, commercial FK506 eye drop group, and FK506 liposome group. Each group had four rabbits. The rabbits in the negative control group had no treatment or intervention. The rabbits in the positive group were given PBS after modeling of dry eye, commercial FK506 eye drop in the commercial FK506 eye drop group, whereas FK506 liposome in the FK506 liposome group. All of them were administrated twice a day (8 a.m. and 8 p.m.) at 50 μl.

To evaluate the clinical signs of dry eye, two ophthalmological tests were carried out on day 0, day 7, and day 9, including corneal fluorescein staining and TFBUT. Corneal fluorescein staining was carried out as follows: sodium fluorescein strips were wetted with a drop of normal saline and used to dye the conjunctiva of the lower eyelid; the rabbits blinked, and then corneal staining was observed and graded according to the SICCA OSS Group Scale. TFBUT was measured as follows: the rabbits were helped to blink several times to form a tear film in the ocular surface, and it started to time until the first dark point or crack appeared, and the abovementioned steps were repeated three times, and the average value was counted (Valim et al., 2015).

The corneal epithelial layer thickness was measured using ImageJ software. The H and E–stained slides were photographed using a fluorescence microscope (Nikon DM4000) at 400 times magnification, and ten points were randomly chosen to measure the thickness. The mean value was calculated, and the difference was analyzed.

The cornea and conjunctiva of rabbits were collected following euthanasia on day 9. Part of these was fixed by 4% paraformaldehyde, embedded, and sectioned to observe corneal epithelial regeneration, conjunctival goblet cell density, and inflammation cell infiltration via H and E staining. The remaining tissue was kept in liquid nitrogen before being used to determine the level of inflammatory cytokines by ELISA. Inflammatory cytokines in the cornea and conjunctiva were extracted. The tissue was cut into pieces and further broken up using ultrasonication at 0°C. The resulting homogenate was centrifuged at 2,000 rpm for 20 min to collect the protein. The ELISA procedures were conducted according to the manufacturer’s protocol (Jiancheng Bioengineering institute, Nanjing, China).

All the data in this article were reported as the mean ± standard deviation (SD). The statistical analyses performed were one-way ANOVA to calculate the significance of experimental data, except the analysis of FK506 concentration in the cornea and the comparison of relative corneal fluorescein score change between day 7 and day 9 using unpaired t-test (p < .05).

The FK506 liposomes were synthesized using the classical thin-film hydration dispersion method (Wang et al., 2021; Wu et al., 2021; Zhong et al., 2021), as described in the Materials and Methods. The physical and chemical properties of the liposomes were investigated. The particle size of the FK506 liposomes was around 292 ± 7 nm, while the surface zeta potential was 31.1 ± 0.83 mV (Table 1). TEM showed that the morphology of the FK506 liposomes was spherical (Figure 1A), and the size distribution of the FK506-loaded liposomes was a little smaller in TEM than DLS. It might be attributed to the fact that DLS determined the hydrodynamic radius of nanoparticles. The entrapment efficiency of the FK506 liposomes was 90.11 ± 2.87%, and the FK506 loading level was around 9.07 ± 0.03%.

The in vitro release profile of the FK506 liposome showed that FK506 liposome released FK506 slowly overall (Figure 1C). This formulation released the drug slowly in the first 48 h, and nearly 5.13 ± 0.92% FK506 was released in this period. There was an accumulative concentration of 6.97 ± 1.08% of total FK506 released until day 7.

The positive charge of the liposome surface played a significant role in adhesion; however, it was also responsible for the cytotoxicity of cationic liposomes (Kim et al., 2013). Therefore, to evaluate the cytocompatibility of FK506 liposomes, the cell viability was measured using CCK-8. As shown in Figure 2A, the blank liposome group showed 85.54 ± 18.31% cell viability after 2-hour co-incubation with HCECs. In addition, there was no significant difference in viability between the blank liposome group and the negative control group. It suggested that the cationic liposomes did not exhibit apparent cytotoxicity. Furthermore, at the same FK506 concentration of 0.2 mg/ml, FK506-loaded liposomes were less detrimental to HCECs than commercial FK506 eye drops after 2 h. The cell viability was 67.17 ± 0.14% in the FK506-loaded liposome group and 4.38 ± 1.21% in the commercial FK506 eye drop group, and there was a statistically significant difference (p < .001). This result inferred that using liposomes as the drug delivery vehicle decreased drug toxicity.

To reduce the cytotoxicity further, a gradient of concentration test was then performed to determine the safe concentrations of the formulation in vitro. After 24-hour co-incubation, FK506 liposomes with a drug concentration below 0.02 mg/ml showed good cell viability (Figure 2B). Based on this result, .02 mg/ml FK506 concentration was chosen in the subsequent experiments in vitro.

The ability to enter cells was a prominent characteristic of cationic liposomes. It was important to deliver drugs (Takikawa et al., 2020). To investigate whether FK506 liposomes could enter cells, confocal microscopy was used. The nucleus was stained blue using DAPI, while FK506-loaded liposomes were stained green using FITC-labeled DSPE-PEG2000 (Figure 2E). After a 2-hour co-incubation with HCECs, FITC-labeled FK506 liposomes were shown to adhere to HCECs and were observed in the cytoplasm. This result implied that HCECs will uptake FK506 liposomes, facilitating the transfer of FK506 into cells.

Oxidative stress was pathogenesis of dry eye and was associated with its progression (Perez-Garmendia et al., 2020). ROS levels were evaluated using flow cytometry via the measurement of the mean intracellular fluorescence intensity. In Figure 2D, the mean ROS fluorescence intensity of the FK506 liposome group was 11.14 ± .65*10⁴; this result was significantly lower than that of the positive control group, which scored 16.07 ± .94*10⁴. It indicated that FK506 liposomes might reduce ROS generation following H₂O₂ stimulation. In addition, the FK506 liposome group had significantly lower ROS levels than the commercial FK506 eye drops group, which scored 14.52 ± 1.16*10⁴ (p < .001).

In terms of the dry eye–related expression of inflammatory factors, the levels of IL-1β and IL-6 were assessed using qRT-PCR. Compared to the positive control group, FK506 liposomes and commercial FK506 eye drops both downregulated the expression of IL-1β and IL-6. FK506 liposomes exhibited a more significant inhibitory effect on IL-1β (p < .05) and IL-6 (p < .01) expression than commercial eye drops. It was attributed to the fact that the liposomal FK506 dosing method allowed the drug to enter the cell easily, making it more efficient to decrease the expression of inflammatory factors, which was in accordance with previous studies (Nattika et al., 2014).

The cornea is an extremely sensitive tissue due to dense coverage of nerve endings within the corneal epithelium, which protects the eyes from harmful substances and foreign objects (Lum et al., 2019). Once stimulated, tearing, blinking, and foreign body sensation in the eye occur; this progresses into pain, edema, optical congestion, and increased secretions. To assess the biocompatibility of FK506 liposomes, central corneal thickness and endothelial cell density were observed in vivo. The FK506 concentration in the FK506 liposome was 0.2 mg/ml. Both of the central corneal thickness and endothelial cell density remained stable after 28-day administration; there was no significant difference among the PBS group, commercial eye drop group, and FK506 liposome eye drops group. In addition to this, none of the groups presented obvious clinical signs of irritation. The cornea remained transparent throughout; there was no evidence of conjunctival congestion, edema, or a change to eye secretions during the observation period (Figure 3A). Moreover, H and E staining of the cornea and conjunctiva showed that there were no corneal epithelial defects, no corneal stromal edema, and no evidence of inflammatory cell infiltration (Figure 3B). Overall, it was concluded that FK506 liposome eye drops (FK506 concentration was 0.2 mg/ml) were compatible with the ocular surface and were safe for ophthalmic use.

It was necessary to evaluate the ocular irritation in vivo because the surfactant is one of the materials to prepare FK506 liposomes. To study the ocular irritation of FK506 liposomes, the ocular irritation test was carried out. During the 72-hour observation, there were no signs of irritation after installing FK506 liposomes and PBS (Figure 3C). Specifically, there was no conjunctival redness, no secretions, and no corneal swelling. Thus, it was concluded that FK506 liposomes have no ocular irritation.

To investigate whether the use of FK506 liposomes would prolong retention time in the ocular surface, the fluorescence intensity following eye drop treatment was compared. Ten minutes after administration, there were similar fluorescence intensity levels among the three following groups, which are the FITC solution group, commercial FK506 eye drops mixed with FITC group, and FITC-labeled FK506 liposomes group (Figure 4A). However, only the group treated with FITC-labeled FK506 liposomes showed an intense signal after 30 min. Following this experiment, it can be concluded that FK506 liposomes can extend the ocular retention time.

In addition, the group treated with FK506 liposomes had a significantly higher FK506 concentration in the corneal tissue 1 h after administration than the group treated with commercial FK506 eye drops (Figure 4B (p < .01)). The FK506 concentration of the cornea in the FK506 liposome group was 3.59 ± 1.96 ng/ml, 2.79 ± 1.07 ng/ml, and 5.26 ± 1.75 ng/ml after 5, 30 min, and 1 h, respectively, while it was 1.20 ± .43 ng/ml, .57 ± .17 ng/ml, and .93 ± .43 ng/ml at the corresponding time points in the commercial FK506 eye drop group. It was in accordance with the ocular retention time, suggesting that the liposome was a promising drug delivery vehicle to facilitate FK506 penetration into the cornea.

As well as being a kind of macrolide antibiotic, FK506 is more commonly used in the suppression of the immune system. In ophthalmology, the primary use for FK506 eye drops was conjunctivitis catarrhalis aestiva. In addition, clinicians also prescribe FK506 for patients with severe dry eye or following corneal transplantation surgery.

The scheme of the evaluation of the effect on alleviating signs of dry eye in vivo is shown in Figure 5A. As shown in Figure 5D, there was a sharp improvement to the tear film break up time (TFBUT) of the FK506 liposome group. Dry eye would interfere with the stability of the tear film and influence the health state of the ocular surface. TFBUT is an indicator of the stability of the tear film. The long TFBUT implies the stable tear film and mild dry eye. On the contrary, the short TFBUT hints at a brittle tear film and severe dry eye. Corneal fluorescein staining score could evaluate the health state of the ocular surface, especially the cornea. The appearance of fluorescein staining suggests that the integrity of the cell is compromised. The high corneal fluorescein score means corneal epithelial layer defect and severe dry eye (Workshop, 2007; Wolffsohn et al., 2017). Hence, TFBUT and corneal fluorescein staining score are indicators used to assess the severity of dry eye.

Compared with baseline (day 7), TFBUT increased by 3.57 ± 1.50 s when using FK506 liposomes; this was statistically significantly different to the 1.50 ± 1.29 s when using the commercial FK506 eye drops (p < .05). Notably, TFBUT was notably longer in the FK506 liposome group than in the positive control group at day 9 (p < .01). It showed that liposomes prepared as given above promote tear film stability. Similarly, corneal fluorescein staining levels were lower at day 9 in all groups than at day 7; the lowest levels were found in the FK506 liposome eye drop group. Statistical analysis showed a significant difference between day 7 and 9 (p < .01). By contrast, the commercial FK506 eye drop group showed no statistical difference between day 7 and 9 (Figures 5B,C). It indicated that there was a more rapid recovery of the corneal epithelial cell layer in the FK506 liposome eye drop group. FK506 liposomes not only cure epithelial cell injury but also prompt clinical sign alleviation.

Severe dry eye always has severe punctate erosions on the cornea, and fluorescein staining could help evaluate the range of corneal damage (Workshop, 2007). To assess the depth of corneal injury, the thickness of the corneal epithelial layer was measured. As is shown in Figure 6A, the reconstruction of the corneal epithelial cell layer varied widely from group to group at day 9. The commercial FK506 eye drop group and the FK506 liposome group had a more significant increase in thickness than the positive control group; in addition, the FK506 liposome group had a more considerable increase than the commercial FK506 eye drop group. The mean thickness of the epithelial layer was 22.31 ± 1.35 μm in the positive control group, 26.69 ± 2.18 μm in the commercial FK506 group, 29.14 ± 1.89 μm in the FK506 liposome group, and 35.28 ± 1.51 μm in the negative control group (Figure 6B). There was a significant difference between the positive control group and the FK506 liposome group (p < .0001), as well as the commercial FK506 eye drop group (p < .0001). Moreover, the difference between the FK506 liposome group and the commercial FK506 eye drop group was significant (p < .05).

To determine the effect of FK506 liposome eye drops on dry eye, the expression levels of dry eye–related inflammatory factors were determined via ELISA. As shown in Figure 6C, after treatment with .2% benzalkonium chloride, matrix metalloproteinase-9 (MMP-9), IL-1β, and IL-6 levels markedly increased in the cornea and conjunctiva. Following administration with FK506 liposomes, the concentration of IL-1β decreased to 99.91 ± 9.68 pg/ml, while the level in the commercial eye drops was 138.98 ± 4.32 pg/ml. This difference was statistically significant (p < .05). The level in the positive control group was 228.99 ± 25.59 pg/ml. Likewise, the expression levels of IL-6 and MMP-9 were downregulated in the FK506 liposome group at 22.58 ± 4.87 pg/ml and 40.26 ± 11.83 pg/ml, respectively. The concentration of IL-6 in the positive control group was 64.38 ± 17.35 pg/ml (p < .01) and the MMP-9 concentration was 95.95 ± 4.23 pg/ml (p < .001).