Efficient ocular delivery of siRNA via pH-sensitive vehicles for corneal neovascularization inhibition

Abstract

Corneal neovascularization (CoNV)-induced blindness is an enduring and challenging condition with limited management options. Small interfering RNA (siRNA) is a promising strategy for preventing CoNV. This study reported a new strategy using siVEGFA to silence vascular endothelial growth factor A (VEGFA) for CoNV treatment. To improve the efficacy of siVEGFA delivery, a pH-sensitive polycationic mPEG_2k-PAMA₃₀-P(DEA₂₉-D5A₂₉) (TPPA) was fabricated. TPPA/siVEGFA polyplexes enter cells via clathrin-mediated endocytosis, resulting in higher cellular uptake efficiency and comparable silencing efficiency than that of Lipofectamine 2000 in vitro. Hemolytic assays verified that TPPA safe in normal physiological environments (pH 7.4) but can easily destroy membranes in acidic mature endosomes (pH 4.0). Studies on the distribution of TPPA in vivo showed that it could prolong the retention time of siVEGFA and promote its penetration in the cornea. In a mouse model induced by alkali burn, TPPA efficiently delivered siVEGFA to the lesion site and achieved VEGFA silencing efficiency. Importantly, the inhibitory effect of TPPA/siVEGFA on CoNV was comparable to that of the anti-VEGF drug ranibizumab. Delivering siRNA using pH-sensitive polycations to the ocular environment provides a new strategy to efficiently inhibit CoNV.

Keywords: Anti-angiogenesis; Cornea neovascularization; pH-sensitive polymer; siRNA delivery.

Graphical abstract

Fig. 1

Characterization of TPPA/siRNA complexes. (A) The pK_a determination of the pH-sensitive section by acid-base titration. (B) The particle size of TPPA. The curve is the hydrodynamic size detected by DLS, and the upper left corner is the morphology map taken by TEM under dry condition. (C) Surface potential (zeta potential) of TPPA detected by DLS. (D) The pH-sensitive assembly/disassembly behavior of TPPA NPs. Nile red dye was used as a fluorescent probe. (E) Agarose gel retardation and (F) Size and zeta potential of TPPA/siRNA complexes at different w/w ratios. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

In vitro performance of TPPA/siRNA polyplexes. (A) Cell viability of HCEC cells and (B) HUVEC cells evaluated by MTT assay 24 h after the transfection of TPPA/siRNA. Lipofectamine 2000 abbreviated Lipo 2000 was used as a positive control. (C) Relative VEGFA mRNA expression of HCEC cells and (D) HUVEC cells after TPPA/siRNA treatment. (E) VEGFA protein expression of HCEC cells and (F) HUVEC cells evaluated by western blot. The numbers in the figure represent the grayscale value. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

Endosomal escaping and cell internalization of TPPA/siRNA. (A) Flow cytometry histogram of HCEC cells after treatment with TPPA/Cy5-siRNA complexes. (B) Cellular uptake of TPPA/Cy5-siRNA represented by mean fluorescence intensity (MFI) of panel A. (C) Flow cytometry histogram of HCEC cells after treatment with TPPA/Cy5-siRNA and different endocytosis inhibitors. Amiloride inhibits macropinocytosis; chlorpromazine inhibits clathrin-mediated endocytosis; genistein inhibits caveolin-mediated endocytosis, and β-cyclodextrin disrupts lipid raft. (D) MFI analysis of panel D. (E) Confocal images of TPPA/Cy5- siRNA complexes in the HCEC cells with different inhibitors. Scale bar, 50 μm. (F) Fluorescence microscopy images of HCEC cells 24 h after transfecting Cy5-siRNA (red). LysoTracker (green) was used to label endosomes and lysosomes. Hoechst 33342 (blue) represents the cell nuclear. Scale bar, 50 μm. (G) Colocalization analysis of Cy5-siRNA (red) and LysoTracker (green) in panel F. (H) Hemolysis ratio of TPPA in different pH. Passive lysis buffer (PLB) was used as positive control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Biodistribution of TPPA/siRNA polyplexes with topical treatment (A-B) and subconjunctival injection (C-E). (A) IVIS imaging after topical treatment of TPPA/Cy5-siRNA. (B) Quantification of eyeballs in panel A. (C) IVIS images after subconjunctival injection. (D) Corneal sections 4 h, 24 h, and 72 h after subconjunctival injection. (E) Quantification of panel D. AU: Arbitrary Units.

Fig. 5

Anti-neovascular effect of TPPA/siVEGFA in alkali corneal burn model. (A1-D2”) Slit-lamp images three days after alkali burn. A1 and A2 are different samples treated with PBS, and A1, A1’, and A1” are different images of the same sample. The white dotted line represents the extent of the vascular. FS: fluorescein sodium staining, marking the corneal injury area. Ranibizumab was used as the positive control drug. (E) Clinical score and (F) injury area statistics three days after alkali burn. (G1-L) Slit-lamp images and analyses seven days after alkali burn.

Fig. 6

Anti-neovascular and biocompatibility assessment of TPPA/siVEGFA in mouse model. (A) Whole mount immunofluorescence pictures and HE sections of cornea. The first row is a picture of vascular staining of the whole cornea marked with CD31 (red); the second row is a magnified image of the outlined region in the first row; and the third row is a H&E staining of a paraffin section with blood vessels circled by black ovals. (B) Quantitative statistical results of panel A. (C) VEGFA mRNA expression detected by RT-qPCR. (D)WB and (E)grayscale analysis determined the protein level of cornea VEGFA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)