Silicone contact lenses (SCL), as an emerging ocular drug delivery system, achieve controlled drug release. However, the existing drug loading methods have limitations such as low drug uptake, complicated operation process, poor welling rate and transmittance of the lens after drug loading. In this study, an effective microemulsion soaking method was proposed to increase the drug-loading capacity of silicone contact lenses. Levofloxacin (LVF) was encapsulated into the microemulsion by direct agitation, then the microemulsion was loaded into silicone contact lenses using the immersion method. The adsorption capacity of levofloxacin and its effect on drug release kinetics were explored. The results showed that the particle size of the microemulsion was approximately 160 nm. The levofloxacin microemulsion soaking method (LVF-ME-SCL) significantly enhanced the drug loading of levofloxacin in the silicone contact lenses, achieving a maximum drug loading of 216.32 ± 1.15 μg/lens (p > 0.05). The total release rate of levofloxacin was 95.96% when the sustained release time was 10 h, and the drug leakage observed after 10 h was negligible. The survival rate of E. coli and S. aureus in LVF-ME-SCL-1 (LVF concentration was 4.8 mg/mL) group was 0 and 19.33 ± 0.02% (p < 0.0001), with a significant difference, indicating that the drug-loaded silicone contact lenses exhibited effective bactericidal properties against E. coli and S. aureus. Following the addition of maximum levofloxacin, the surface contact angle of silicone contact lenses decreased significantly to 32.88 ± 1.19° (p > 0.05), while the swelling, mechanical properties, and oxygen permeability remained relatively unchanged. There was no significant decrease in the transmittance of the contact lenses after the addition of levofloxacin, which remained above 95%. In conclusion, these results show that the microemulsion impregnation method effectively improves the drug loading and sustained release time of levofloxacin, and maintains lens performance stability before and after drug loading, so it is expected to be used in ophthalmic treatment.
The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5′-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 µg/mL) in the soaking buffer for 24 h in the dark at 25 °C.The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.
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