Gene transfection is an effective therapeutic avenue to target many kinds of eye diseases.Non-viral vectors with high transfection efficiency, long-term expression, low toxicity and high expression levels are pivotal in gene therapy of corneal disease.
This study was to evaluate and compare the safety and efficiency between Entranster™ and liposome vectors for transfer of CD25 siRNA in rat cornea.
Eighty male SPF SD rats were randomly divided into Entranster™-CD25 siRNA group, liposome-CD25 siRNA group, simple CD25 siRNA group and normal saline solution (NSS) group with the right eye as experimental eyes.Corneal epithelia of the rats were completely removed after ocular surficial anesthesia, and 50 μl Entranster™-CD25 siRNA, liposome-CD25 siRNA, CD25 siRNA solution and NSS were topical administered in the eyes respectively.Ocular response and green fluorescence number on the corneas were examined under the slit lamp assisted microscope 12 hours, 24 hours, 3 days and 7 days after use of the drugs.The rats were sacrificed and the corneas were obtained, and corneal histopathological examination was performed by using hematoxylin eosin stain.The gene transferred efficiency in the corneas was evaluated by fluorescence technology, and the safety of Entranster™ and liposome carriers was assessed using TUNEL stain.The expression and location of CD11b in the corneas were detected by immunofluorescence technology.The use and care of the experimental animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee.
The quantity and intensity of fluorescence staining in the corneas were significantly increased in the Entranster™-CD25 siRNA group in comparison with the liposome-CD25 siRNA group, and the corneal fluorescence appeared earlier in the simple CD25 siRNA group, but it disappeared in 24 hours after transfection.Corneal histopathological examination revealed that the corneal edema and inflammatory cell infiltration in corneal epithelium after gene transfection were more dominant in the liposome-CD25 siRNA group than those in the Entranster™-CD25 siRNA group, simple CD25 siRNA group and NSS group, and no abnormality was seen in the stroma and endothelium.The number of inflammatory cells was more in the liposome-CD25 siRNA group than that in the Entranster™-CD25 siRNA group, simple CD25 siRNA group and NSS group (all at P=0.00). The number of apoptosis cells was significantly more in the liposome-CD25 siRNA group than that in the Entranster™-CD25 siRNA group, simple CD25 siRNA group and NSS group in 12 hours and 3 days after transfection (all at P=0.00). Immunofluorescence assay showed the expression of CD11b primarily located in the corneal epithelial and stromal layers.The expression of CD11b was gradually enhanced over time in the liposome-CD25 siRNA group and peaked in 24 hours after transfection.However, the expression was absent in the Entranster™-CD25 siRNA group, simple CD25 siRNA group and NSS group.
Entranster™ nanometer material-mediated transfection of CD25 siRNA in corneas of normal SD rats appears to have high transfection efficiency, low toxicity and slight irritating response to corneas, and Entranster™ vector is currently available for the gene therapy of corneal disease.