Preparation of MT-BHC Microparticles and Solid Lipid Nanoparticles

Based on our previous studies (Tian et al., 2018 ), MT-BHC MPs were prepared by the emulsion–solvent evaporation method. Briefly, 200 mg of EUD RS PO, 200 mg of EUD RL PO, 50 mg of BHC, and 30 mg of MT-BHC were completely dissolved in an acetonitrile solution containing 80 mg of triethyl citrate, 80 mg of glycerol, and 124 mg of Tween 80 (O₁ phase). The O₁ phase was emulsified by slowly dropping into the O₂ phase consisting of 248 mg of spirulina 80 and 10 mL of liquid paraffin and mixed rapidly with a stirrer (XW-80A, Haimen Kylin-Bell Lab Instruments Co., Ltd.), then sonicated (JY 92-II, Ningbo Scientz Biotechnology Co., Ltd. China) for 15 min in an ice bath with a ultrasonic power of 53% at 5-s intervals and stirred at 800 rpm for 1 h in the ice bath and later at RT for 0.5 h, followed by adjusting the speed to 650 rpm until a clear mixture was obtained. The resulting MPs were washed with n-hexane to remove any excess oil content on the surface, recovered by centrifugal filtration, and air-dried at room temperature. The MT-BHC MPs eye drops were prepared by dispersing the MPs powder in a sarcosine phosphate buffer containing a co-suspension agent and adding 3.1% mannitol (mass/vol) and 0.1% (mass/vol) Tween 80 to adjust the osmotic pressure and viscosity.
MT-BHC SLNs were prepared by the melt-emulsion sonication and low temperature-solidification method in line with our previous research (Liu S et al., 2020 (link); Han et al., 2021 (link)). Briefly, the organic phase was composed of 30 mg of BHC, 30 mg of GMS, and 100 mg of SPL dissolved in 5 mL of ethanol under heating at 75 °C. The water phase was a mixture of 200 mg of Tween 80, 100 mg of PEG-400, and 5 mg of sodium deoxycholate. First, 5 mg of MT-BHC complex was added to the organic phase and then ice bath ultrasonication for 10 min, injected into the aqueous phase at 75 °C under stirring at 800 rpm. Next, the stirring speed was increased to 1000 rpm and the mixture was stirred until an initial emulsion was formed. To maintain the stable morphology of SLNs, the initial emulsion was quickly injected into a 3.6% (wt/vol) aqueous mannitol solution at 0 °C and stirred for 2 h. The obtained SLNs were stored in a refrigerator at 4 °C. Figure 1 shows a schematic of the two preparation methods.