Lacritin-mediated regeneration of the corneal epithelia by protein polymer nanoparticles

Abstract

The avascular corneal epithelium plays an important role in maintaining normal vision and protecting the corneal interior from environmental infections. Delayed recovery of ocular wounds caused by trauma or refractive surgery strengthens the need to accelerate corneal wound healing and better restore the ocular surface. To address this need, we fused elastin-like polypeptide (ELP) based nanoparticles SI with a model mitogenic protein called lacritin. Lacritin fused at the N-terminus of the SI diblock copolymer is called LSI. This LSI fusion protein undergoes thermo-responsive assembly of nanoparticles at physiologically relevant temperatures. In comparison to ELP nanoparticles without lacritin, LSI showed potent signs of lacritin specific effects on a human corneal epithelial cell line (HCE-T), which included enhancement of cellular uptake, calcium-mediated signaling, and closure of a scratch. In vivo, the corneas of non-obese diabetic mice (NOD) were found to be highly responsive to LSI. Fluorescein imaging and corneal histology suggested that topical administration of LSI onto the ocular surface significantly promoted corneal wound healing and epithelial integrity compared to mice treated with or without plain ELP. Most interestingly, it appears that ELP-mediated assembly of LSI is essential to produce this potent activity. This was confirmed by comparison to a control lacritin ELP fusion called LS96, which does not undergo thermally-mediated assembly at relevant temperatures. In summary, fusion of a mitogenic protein to ELP nanoparticles appears to be a promising new strategy to bioengineer more potent biopharmaceuticals with potential applications in corneal wound healing.

Keywords: Elastin-like polypeptide (ELPs); Lacritin; cornea; nanoparticle; wound healing.

Figure 1. Construction of a thermo-responsive nanoparticle decorated with a mitogenic protein

(A) A gene encoding lacritin was fused to an ELP diblock copolymer called SI and expressed as a protein called LSI. LSI undergoes temperature-dependent assembly of a nanoparticle above its phase transition temperature (T_t). (B) After purification, gel electrophoresis (SDS-PAGE) was used to confirm the molecular weight for expressed LSI and SI. (C) An optical density profile was obtained upon heating purified polymers in phosphate buffered saline (25 μM). SI shows two inflections, where the first represents the assembly of a nanoparticle (T_t1) and the second represents the bulk phase separation (T_t2) of that nanoparticle. In contrast, LSI shows only a single inflection and does not undergo bulk phase separation. (D) Optical density data was compiled to create a concentration-temperature phase diagram for LSI and SI. Dashed lines indicate the fit of T_t to the following equation: $T_t=m{log}_{10}[C_{\mathit{ELP}}]+b$ where C_ELP is the concentration, m is the slope, and b is the transition temperature at 1 μM. For T_t of LSI, b = 19.69 °C and m = −0.64 °C (Log 10[μM])⁻¹. For T_t1 of SI, b = 65.06 °C, m = −22.56 °C (Log 10[μM])⁻¹. For T_t2 of SI, b = 77.98 °C, m = −3.72 °C (Log 10[μM])⁻¹.

Figure 2. LSI and SI assemble nanoparticles at physiological temperatures

(A) Dynamic Light Scattering (DLS) was performed during heating, which shows that SI assemble nanoparticles with a R_h of 22.3 ± 1.1 nm at 37 °C. Below T_t1 LSI form 30–40 nm structures; however, above T_t these reconstitute into stable 147 ± 36 nm nanoparticles at 37 °C. (B,C) TEM images of (B) SI and (C) LSI nanoparticles, with average diameter of 36.5 ± 5.8 nm and 67.1 ± 11.5 nm accordingly. The scale bar represents 100 nm.

Figure 3. LSI nanoparticles stimulate Ca²⁺- mediated signaling in corneal epithelial cells

HCE-T cells were treated with Fluo-4AM to detect calcium-mediated signaling and imaged using live cell confocal microscopy. The upper images are representative of the peak intensity following the administration of either LSI or SI (40 μM). The lower plot presents the fluorescence intensity as a function of time in ten individual cells. The percentage change in fluorescence intensity, F_t, compared to the initial time point, F₀, was estimated as follows: F_t = (F_t-F₀)/F₀×100%. (A) LSI nanoparticles were administered twice, each time triggering a 3–6 fold increase in intracellular Ca²⁺. (B) In contrast, the same concentration of SI did not elicit a significant effect (****p<0.0001, n=10). The scale bar represents 20 μm. Maximum fluorescence intensity changes were analyzed using an un-paired t-test.

Figure 4. LSI nanoparticles mediate scratch closure in corneal epithelial cells

HCE-T cells were grown to confluence on tissue culture polystyrene, scratched, and imaged for 24 h to observe the rate of closure. (A) At low concentrations, 10 nM LSI nanoparticles completely closed the scratch. This finding was similar to that obtained by a positive control containing epidermal growth factor (EGF) (5 ng/ml) and bovine pituitary extract (BPE) (50 μg/ml). In contrast, the no treatment group (medium only) failed to close the scratch. The scale bar represents 100 μm. (B) The scratch closure at 24 h was quantified to show that LSI nanoparticles induce an effect similar to control EGF and BPE. The no treatment group retained more than 80 % of initial scratch width. Each treatment condition was performed in triplicate and four representative distances in each well were measured for statistical analysis (***p<0.001, n=12). Data were analyzed by a blind reviewer. Images were quantified using ImageJ and analyzed using one-way ANOVA followed by Tukey’s post-test.

Figure 5. Lacritin mediates nanoparticle uptake in corneal epithelial cells

HCE-T cells were incubated with rhodamine labeled LSI or SI nanoparticles and imaged using confocal microscopy. (A) Representative images show time dependent uptake of LSI while SI nanoparticles do not internalize to the same degree. Red: rhodamine labeled LSI and SI; Blue: DAPI staining of nuclei. The scale bar represents 10 μm. (B) For cell uptake quantification, each treatment was repeated three times and three representative cells on each plate were chosen for analysis purpose. Remarkably, LSI exhibits significantly higher uptake level than SI (****p<0.0001, n=9) at 60 m. Images were quantified using ImageJ and analyzed via two-way ANOVA followed by a Bonferroni post-hoc t-test.

Figure 6. Lacritin-decorated nanoparticles heal abrasions in the corneal epithelium of mice

An algerbrush II was used to create a 2 mm defect in the corneal epithelium of female non-obese diabetic (NOD) mice, which were monitored using fluorescein staining at 0, 12 and 24 h with or without treatment by LSI, SI, and a positive control EGF+BPE. (A) Representative images showing the time-lapse healing of the defect on the corneal epithelium. (B) The area of the wounds as a percent of the initial wound area (PctArea) was determined by a blind reviewer to ensure objectivity. A Kruskal-Wallis non-parametric test was used to compare groups. These revealed that LSI at both 12 and 24 h significantly (***p=0.001, n=4) decreased the percentage of initial wound area (PctArea) compared to SI, EGF+BPE, and no treatment groups. (C) After 24 h, corneas were fixed, sectioned across the defect, and stained by hematoxylin and eosin. The corneal epithelium of the LSI treatment group revealed normal pathology, absent of inflammation. Although reduced fluorescein staining was observed at late times in the SI group, the epithelium did not recover fully, as evidenced by its irregular surface (black arrows). EP: epithelium; BM: Bowman’s membrane; ST: Stroma; DM: Descenet’s membrane; EN: Endothelium.

Figure 7. ELP-mediated assembly is essential for the in vivo activity of LSI nanoparticles

To determine whether the potency of LSI nanoparticles depends on ELP-mediated assembly of SI, a control lacritin fusion called LS96 (Table 1) was expressed. (A) Optical density measurements confirmed that LS96 lacks any detectable phase transitions at 25 μM in phosphate buffer saline (PBS). (B) Under the same conditions, Dynamic Light Scattering (DLS) was performed at 37 °C, which confirmed that LSI assembles nanoparticles with a R_h of 147.0 ± 35.8 nm, while LS96 produced particles with a R_h of 37.0 ± 8.8 nm, which is similar to that observed for LSI below T_t (Fig. 2A). (C) A 2 mm corneal defect was induced in female NOD mice, treated with LSI or LS96, stained by fluorescein at 12 h, and quantified by a blind reviewer. A representative image shows superior integrity of the ocular surface treated with LSI after 12 h. (D) Comparison of the PctArea was made by the non-parametric Mann-Whitney U test, which confirms that LSI at 12 h significantly decreases the wound area compared to LS96 (*p<0.05, n=8).