1. Formulation and Characterization of DTX-sHDL

The docetaxel-loaded sHDL (DTX-sHDL) was prepared as described previously ^[1]. Briefly, 22A, egg sphingomyelin (eSM), and DTX were dissolved in acetic acid. The acetic acid solutions of 22A, eSM, and DTX were mixed and freeze-dried for 24 h (mass ratio of 22A: eSM: DTX = 1:2:0.05). The lyophilized powder was rehydrated by PBS (pH 7.4). Three heat-cooling cycles (50 °C, 5 min followed by room temperature for 5 min) were performed to form DTX-sHDL. DTX-sHDL/CpG particles were prepared by incubating DTX-sHDL with CpG-Cholesterol in 10 mM phosphate buffer at room temperature for 2 h. The particle size of DTX-sHDL was analyzed by dynamic laser scattering (DLS). The purity of the DTX-sHDL nanoparticles was evaluated by gel permeation chromatography (GPC) at 220 nm using Tosoh TSK gel G3000SWx 7.8 mm × 30 cm column (Tosoh Bioscience, King of Prussia, PA, USA).

For the block lipid transport-1 (BLT-1) inhibition experiment, cells were pretreated with SR-B1 inhibitor BLT-1 with different concentrations for 1 h. Then, DiD-sHDL was added to each well (final 22A concentration = 10 mcg/mL). The cells were further incubated for 3 h followed by FACS analysis.

MC-38 cells were cultured in RPMI medium supplemented with 10% Fetal Bovine Serum and 1% Penicillin/Streptomycin antibiotics. When cells reached their exponential growth phase, they were trypsinized and plated on 96-well tissue culture plates at 10,000 cells per well to be incubated overnight at 37 °C to allow adherence. Cells were dosed with six different doses of DTX in either free drug form or encapsulated in HDL to test the effect of increasing dose on cell death. Following dosing, cells were incubated at 37 °C for 48 h before analysis using the CellTiter 96^® AQ_ueous Non-Radioactive Cell Proliferation Assay (MTS) from Promega, Madison, WI, USA. Absorbance at 490 nm was quantified using a BioTek SynergyNEO spectrophotometer. Negative control wells (without treatment) were considered to have the maximum absorbance at 100% viability, and the viability of other wells was calculated as the ratio of treated well absorbance to untreated well absorbance.

Thirty-five female C57BL/6 mice aged 7–8 weeks (Charles River Laboratories, Wilmington, MA, USA) were inoculated with 1 million MC-38 cells at a concentration of 10 million cells/mL subcutaneously superior to the right flank. On day 8 after tumor inoculation, mice were split into four groups of seven for treatments. Mice were injected intratumorally with (1) PBS, (2) DTX, (3) DTX-HDL, or (4) DTX-HDL/CpG twice a week at 1 mg/kg DTX and 15 µg CpG for five treatments. Mice were euthanized when tumors surpassed 15 mm in one dimension or ulcerated extensively. Blood samples were taken four days after the final treatments were administered for serum isolation, and analysis of toxicity markers was performed by the In-Vivo Animal Core Animal Diagnostic Laboratory.