Preparation and drug release effect evaluation of drug-loaded cross-linked decellularized corneal stromal lenticules in vitro

Objective

To prepare a drug release system of drug-loaded cross-linked decellularized corneal stromal lenticules and evaluate its drug release characteristics in vitro.

Methods

Lenticules were obtained during femtosecond laser-assisted small incision lenticule extraction (SMILE) surgery in Chongqing Aier Ophthalmology Hospital.Decellularized corneal stromal lenticules were prepared using high concentration sodium chloride (NaCl) combining nuclease.The decellularized corneal stromal lenticules were randomly divided into normal group, 0.5% levofloxacin group, 3% levofloxacin group and 5% levofloxacin group, with 4 lenticules in each group.The lenticules did not receive any treatment in the normal group, and drug-loading those were soaked in different doses of levofloxacin solution for three hours according to grouping.In the crosslinking test, 12 decellularized corneal stromal lenticules were randomly divided into non-crosslinking group, 0.01 mmol 1-(3-dimethylamino) propylimine (EDC) group, 0.05 mmol EDC group and 0.25 mmol EDC group.The lenticules for cross-linking were soaked in different contents of mixed solution of EDC with N-hydroxysuccinyl (NHS) for four hours respectively according to grouping, and then in 3% levofloxacin solution for three hours.Only 3% levofloxacin solution soaking was carried in the non-crosslinking group.High performance liquid chromatography (HPLC) was employed to detect the drug release concentration of the lenticules, and spectral scanning method was performed to measure light transittance of the lenticules.The surface ultrastructure of the decellularized lenticules among different cross-linking groups was examined and compared with scanning electron microscope.The use of the human corneal lenticules was approved by an Ethics Committee of Chongqing Aier Ophthalmology Hospital (No.2019012). Written informed consent was obtained from each patient before surgery.

Results

The release concentrations of decellularized corneal stroma lenticules were significantly different at 1 day, 7, 14, and 21 days among 0.5%, 3%, and 5% levofloxacin group (P<0.05) or also among the 0.01 mmol EDC, 0.05 mmol EDC, and 0.25 mmol EDC cross-linked groups (P<0.01). The drug release concentrations in 0.05 mmol EDC group were the highest at various time points, and the release time of the three cross-linked groups lasted until 21 days after release concentrations of decellularized corneal stroma lenticules.The drug release concentrations in cross-linked groups and non-crosslinking group were gradually declined with the prolong of drug-loading time, showing a significant difference at different time points (P<0.05). The transmittance of the lenticules was (88.68±1.19)% and (91.55±1.16)% in the non-crosslinking group and normal group, respectively, with no significant difference (P>0.05). The average transmittance of the lenticules was significantly reduced in the drug-loaded groups compared with the normal group (P<0.05). The smaller collagen fiber voids and closely arranged collagen fibers were displayed in the cross-linking groups under the scanning electron microscope with the best effect in the 0.25 mmol EDC group.

Conclusions

EDC/NHS cross-linking can improve the drug-loading effect of decellularized corneal stromal lenticules probably by lessening collagen fiber voids.The drug-loaded cross-linked decellularized corneal stromal lenticules have a good drug release effect in vitro.