Targeted dexamethasone nano-prodrug for corneal neovascularization management

Abstract

Background: To overcome the drawbacks of traditional therapy for corneal neovascularization (CNV), we evaluated the efficacy of polyethylene glycol (PEG)-conjugated Ala-Pro-Arg-Pro-Gly (APRPG) peptide modified dexamethasone (Dex), a novel nano-prodrug (Dex-PEG-APRPG, DPA).

Methods: Characterization of DPA nano-prodrug were measured with transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses. Cytotoxicity and effects on cell migration and tube formation of DPA were evaluated in vitro. A murine CNV model was established by cornea alkali burn. The injured corneas were given eye drops of DPA (0.2 mM), Dex solution (0.2 mM), Dexp (2 mM), or normal saline three times a day. After two weeks, eyes were obtained for the analysis of histopathology, immunostaining, and mRNA expression.

Results: DPA with an average diameter of 30 nm, presented little cytotoxicity and had good ocular biocompatibility. More importantly, DPA showed specific targeting to vascular endothelial cells with efficient inhibition on cell migration and tube formation. In a mouse CNV model, clinical, histological, and immunohistochemical examination results revealed DPA had a much stronger angiogenesis suppression than Dex, resembling a clinical drug with an order of magnitude higher concentration. This was ascribed to the significant downregulations in the expression of pro-angiogenic and pro-inflammatory factors in the corneas. In vivo imaging results also demonstrated that APRPG could prolong ocular retention time.

Conclusions: This study suggests that DPA nano-prodrug occupies advantages of specific targeting ability and improved bioavailability over conventional therapy, and holds great potential for safe and efficient CNV therapy.

Keywords: Angiogenic vessel-homing peptide; Corneal neovascularization; Dexamethasone; Nano-prodrug; Targeted drug delivery.

Scheme 1

Schematic representation of the preparation of DPA and this targeted delivery in the efficient therapeutic process for CNV.

Fig. 1

Characterization of Dex-SA and DPA. (A) ¹H NMR spectrum and (B) MS spectrum of Dex-SA. (C) HPLC spectrum and (D) MS spectrum of DPA. (E) DLS results and TEM image of DPA.

Fig. 2

Evaluation on the cytocompatibility, specific cell-targeted capacity and anti-angiogenic effects of DPA. (A) Cell viability of HCECs which were exposed to diverse concentrations of DPA tested by the CCK-8 assay. (B) Images of HCECs treated with diverse concentrations of DPA. The cells were co-stained by calcein-AM (live cells, green) and PI (dead cells, red). Scale bar = 500 μm. Cellular target of RhB and APRPG-RhB in (C) HCECs and (D) HUVECs. Cell nuclei were stained with Hoechst (blue), endo/lysosomes were stained with LysoTracker (green) and RhB was red. Scale bar = 50 μm. Semiquantitative analyses on fluorescence intensity of RhB in (G) HCECs and (H) HUVECs. In HUVECs-GFP, six groups were created: control (PBS only); Dex (20 μM); DPA (20 μM); VEGF (100 ng/mL); VEGF (100 ng/mL) combined with Dex (20 μM); and VEGF (100 ng/mL) combined with DPA (20 μM). (E) Cell migration was imaged at 0 h and 12 h and (F) tube formation was imaged at 2 h. Scale bar = 500 μm. Quantitative analyses of (I) cell migration rate and (J) total length of master segments (normalized to the value of the control). Results are presented as the mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 between the indicated groups.

Fig. 3

Clinical assessment of the efficacy of DPA on alkali burn-induced CNV in mice. (A) Representative ocular surface images on days 3, 7, 14. Scale bar = 1 mm. Measurements of (B) vessel length and (C) CNV area on day 14. Results are presented as the mean ± SEM (n = 10). ∗p < 0.05, ∗∗∗p < 0.001 between the indicated groups, and ns denotes no significance. (D) Anterior segment OCT imaging of the cornea. Scale bar = 0.5 mm.

Fig. 4

Inhibitory effect of DPA on CNV. (A) Histological examinations of the cornea with H&E staining. The black arrows indicate the neovascular section. Scale bar = 50 μm. (B) Immunofluorescent staining of corneal flat mounts with CD31 (blood vessels), LYVE1 (lymphatic vessels) and DAPI. Scale bar = 500 μm. Quantitative analyses of (C) corneal thickness, (D) neovascularization area and (E) lymphangiogenesis area on day 14. Results are expressed as % vascularized areas of the cornea. Results are presented as the mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01 between the indicated groups.

Fig. 5

Measurement of mRNA expression levels, including VEGF-A, -C, -D, MMP-2, -9 and IL-6 on day 14. Results are presented as the mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 between the indicated groups, and ns denotes no significance.

Fig. 6

Precorneal retention evaluation. (A) In vivo fluorescence images and (B) quantitative analyses of fluorescein intensity of RhB and APRPG-RhB instilled into the eye at 10 s, 1 min, 5 min, 10 min, and 30 min. Relative mean fluorescence intensity (MFI) is calculated as the MFI at observation time/MFI at 10 s. Results are presented as the mean ± SEM (n = 3).

Fig. 7

Biocompatibility evaluation of DPA. (A) Slit lamp observation and corneal fluorescein staining. (B) Change in mouse body weight after different treatments. Results are presented as the mean ± SEM (n = 10). (C) H&E staining of major organs, including the heart, liver, spleen, lung and kidney. Scale bar = 100 μm.