Reagents
Celastrol was bought from Chengdu Zhibiaohuachun Biotechnology (Chengdu, China); betulinic acid, cholesterol and coumarin-6, from J&K Scientific (Beijing, China); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] -folic acid and soybean phospholipid (SPC), from Xi’an Ruixi Biotechnology (Xi’an, China); fetal bovine serum (FBS), calf serum, RPMI-1640 medium, and Dulbecco’s modified Eagle medium (DMEM), from GIBCO (Thermo Fisher Scientific, (Carlsbad, USA); 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), from Beyotime Biotechnology; and DAPI, from Solarbio Life Sciences (Beijing, China).
The Cell Counting Kit-8 (CCK-8) was obtained from APExBIO Technology (Houston, TX, USA). 2' ,7'-Dichlorodihydrofluorescein diacetate and JC-1 mitochondrial membrane potential fluorescent probe was obtained from Solarbio (Beijing, China). Antibodies were obtained from Abcam (Cambridge, UK) against α-SMA (catalog no. ab124964), Bcl-2 (ab196495) or collagen I (ab260043). Antibody against P-glycoprotein 1 (catalog no. AF5185) was obtained from Affnity Biosciences (Jiangsu, China).
Cell lines
Mouse mammary breast tumor cell line 4T1 and mouse embryonic fibroblast cell line NIH 3T3 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cultures were maintained in RPMI-1640 medium supplemented with 10% FBS or DMEM supplemented with 10% FBS in a standard incubator at 37 ºC in an atmosphere of 5% CO2.
Animals
All procedures and experiments were carried out in accordance with the guidelines of the Laboratory Animal Ethics Committee at Southwest Medical University. Six-week-old female BALB/c mice (16 ± 2 g) were purchased from Chengdu Yaokang Biotechnology (Chengdu, China) and housed individually in cages with ad libitum access to food and water. Animals were co-injected subcutaneously into the fourth mammary pad with 4T1 cells (1 × 105) and NIH 3T3 cells (5 × 104).
Tumor volumes were regularly determined according to the formula [length × (width)2] / 2.
Preparation of BM and F/CL@BM
To prepare BM, we mixed TPGS and betulinic acid in a weight atio of 10:1 in ethanol (5 mL), The mixed solution was evaporated on a rotary evaporator at 50°C, forming a lipid film, which was rehydrated in 10 min in PBS (5 mL).
To prepare CL@BM, we mixed SPC and cholesterol in a weight ratio of 10:2:1 in methylene dichloride (5 mL), celastrol (1mg) was added, and the mixture was evaporated on a rotary evaporator at 35°C to form a lipid film. The film was rehydrated for 30 min with the above-prepared BM (10 mL), followed by probe sonication at 65 W for 10 min in ice water.
To prepare F/CL@BM, we incubated CL@BM and DSPE-PEG2000-folic acid (2 mg) at 37°C for 60 min.
Before use in experiments, all these preparations were passed through a 220-nm filter to remove unencapsulated drugs.
Characterization of formulations
Particle size and zeta potential were assessed using dynamic light scattering (NanoZS 90, Malvern, Malvern city, UK). Morphology was assessed using transmission electron microscopy (JEM-200CX, JEOL, Tokyo, Japan) after diluting the samples to 2 mg mL− 1 with PBS, dispersing the dilution onto copper grids, staining with 10 µL of 1% (v/v) phosphotungstic acid in PBS, and drying in air.
Loading capacity and encapsulation efficiency for betulinic acid and celastrol were measured using high-performance liquid chromatography at, respectively, 220 and 425 nm. Encapsulation efficiency (EE) was calculated as follows:
EE (%) = (weight of the drug in F/CL@BM / weight of the feeding drug) × 100%.
Co-loading of betulinic acid and celastrol was confirmed using FRET experiments, in which celastrol was replaced with DiO to serve as energy donor and betulinic acid was replaced with DiI to serve as energy acceptor. For comparison, liposomes containing DiO (L-DiO) and micelles containing DiI (M-DiI) were combined in a mass ratio of 1:1 to form M@DiI@L-DiO. Fluorescence emission spectra were recorded after excitation at 470 nm using a Synergy H1 Microplate Reader (BioTek Instruments, Winooski, VT, USA). The FRET ratio was quantified as the ratio of fluorescence emission at 568 nm to the sum of the emission at 568 and 503 nm.
A dialysis method was used to measure the release of betulinic acid and celastrol from BM@CL in vitro. BM@CL in PBS (1 mL, 1 mg/mL for both drugs) was placed in a dialysis bag with a molecular weight cut-off of 10 kDa (Shanghai Titan Technology), which was immersed in 20 mL of PBS (pH adjusted to 6.5) containing 0.5% Tween 80 with stirring (100 rpm). At the indicated times, an aliquot (1 mL) was withdrawn and replaced with the same volume of fresh PBS. The aliquot was assayed for betulinic acid and celastrol using high-performance liquid chromatography.
The stability of F/CL@BM in vitro was assessed in PBS or DMEM by measuring size and polydispersity index every other day for two weeks by dynamic light scattering.
Effect of formulations on cell viability in vitro
4T1 cells alone or together with NIH 3T3 fibroblasts (1 × 104 cells per well, 5:1 ratio of 3T3:4T1 cells) were seeded into 96-well plates and incubated for 48 h. Then cells were treated for 24 h with F/CL@BM at concentrations (µg/ml) of CEL and BA of 0, 0.025, 0.125, 0.25, 0.5 and 1. CCK-8 reagent (10 µl) was added to each well, and the plates were incubated at 37°C for 30 min. Optical density was measured at 450 nm.
As a complementary assessment of cell viability, 4T1 cells were seeded into 6-well plates (5 × 105 cells per well), incubated for 24 h, then exposed for 4 h to F/CL@BM containing CEL and BA at concentrations of 0.1 µg/L. Cells were washed with PBS, stained with calcein-AM and propidium iodide, and analyzed under a fluorescence microscope (Leica, Germany).
Cellular uptake of formulations
4T1 and NIH 3T3 cells were seeded into 12-well plates (4×104 cells per well, 5:1 ratio of 3T3:4T1 cells), incubated overnight, then the medium was replaced with 1 mL of fresh medium containing free coumarin-6 (C6), micelles containing C6 (MC), liposomes containing C6 (LC), micelles-in-liposomes containing C6 (MC@LC) or folate-coated micelles-in-liposomes with C6 (F/MC@LC). In all cases, the C6 concentration was 1 µg/mL. After 2 h, the cells were fixed, stained with DAPI, and examined under a confocal laser scanning microscope (TCS SP2, Leica). In other experiments, the cells were harvested, resuspended in PBS, and analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) to quantitate cellular uptake.
In some experiments, cultures were pretreated for 30 min with 1 mg/mL free folic acid to saturate folate receptors on the surface of tumor cells. Then cultures were exposed to the different formulations as described above.
Effect of formulations on cell migration
4T1 cells alone or together with NIH 3T3 cells were seeded into six-well plates (1×105 cells per well, 5:1 ratio of 3T3:4T1 cells) and incubated for 24 h, then the monolayer was scratched with a sterile pipette tip. Scratched cells were washed away using PBS, and the adherent cells were treated with free betulinic acid, free celastrol, a physical mixture of the two drugs, CL, CL@BM or F/CL@BM. Wound width was measured using an Eclipse Ti-S microscope (Nikon) immediately after the scratch and at 24 h later.
Effect of formulations on intracellular production of reactive oxygen species
4T1 cells were seeded into 6-well plates (1 × 105 cells per well) or 96-well plates (1 × 104 cells per well), incubated for 24 h, washed three times with PBS, then treated for 24 h with free BA, free CEL, a physical mixture of the two drugs, BM, CL, CL@BM, or F/CL@BM. The cells were washed three times with PBS, incubated for 30 min with 2' ,7'-Dichlorodihydrofluorescein diacetate (DCFH-DA), and washed free DCFH-DA. Six-well plates were then examined for the presence of the fluorescent product ROS under a fluorescence microscope, while 96-well plates were analyzed on a microplate reader to quantitate levels of the fluorescent product ROS (excitation 488 nm, emission 525 nm).
Effect of formulations on mitochondrial membrane potential
4T1 cells were seeded into six-well plates (5 × 104), incubated for 24 h, then treated for 30 min with free betulinic acid, free celastrol, a physical mixture of the two drugs, CL, CL@BM, or F/CL@BM. The cultures were incubated for 20 min with 5 µg/mL JC-1 in RPMI 1640 medium, washed with PBS, then observed under a fluorescence microscope.
Western blotting
4T1 cells alone or together with NIH 3T3 cells were seeded into 6-well plates (1 × 105 cells per well, 5:1 ratio of 3T3:4T1 cells), incubated for 24 h, then treated for 24h with various drug formulations. As a control, 4T1 cells were cultured alone and treated the same way. Cells were harvested, lysed with radio-immunoprecipitation assay (RIPA) buffer, microcentrifuged to pellet debris, and the supernatant was fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were incubated 12h with primary rabbit antibodies against α-SMA, Bcl-2, P-gp or GAPDH, followed by incubation for 1 h at 25°C with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase. Antibody binding was detected using enhanced chemiluminescence (Image Quant LAS 4000 Mini; Fuji, Tokyo, Japan) and quantitated using Gel Image System 4.00 (Tanon, China).
Biodistribution of formulations in vivo
Mice bearing 4T1/TAF desmoplastic tumors with a volume of approximately 200 mm3 were injected through the tail vein with DiD-loaded micelles (BM), DiD-loaded micelles-in-liposomes (CL@BM), and DiD-loaded, folate-modified micelles-in-liposomes (F/CL@BM) (three animals per condition). In all cases, the dose of DiD was 0.1 mg/kg. At the indicated time points, mice were anesthetized and imaged (Caliper Life Sciences, Mountain View, CA, USA). At 24 h after injection, tumors and major organs were excised and imaged.
Antitumor efficacy of formulations in vivo
Mice bearing 4T1/TAF desmoplastic tumors with a volume of approximately 80 mm3 were injected through the tail vein with PBS (Control), free betulinic acid, free celastrol, a physical mixture of the two drugs, BM, CL, CL@BM or F/CL@BM). The doses of betulinic acid and celastrol were 2 mg/ kg. Tumor volume and body weight were measured throughout the experiment.
On day 14 after injection, mice were sacrificed, tumors and organs were excised, and blood was sampled. The excised tumors were weighed, photographed, and sectioned for TUNEL staining. In parallel, tumor samples were fixed in 10% formalin, embedded in paraffin, cut into sections, deparaffinized and subjected to antigen recovery. Sections were immunostained overnight at 4°C against α-SMA or collagen I, followed by incubation with a secondary antibody conjugated to horseradish peroxidase. Antibody binding was detected by incubating the sections in xxxx. Sections were also stained with hematoxylin.
Organs were sectioned for hematoxylin-eosin staining. Blood was analyzed for levels of creatinine, AST and ALT.
Statistical analysis
Data were reported as mean ± standard deviation (SD) and analyzed using GraphPadPrism 9.4.0. Pairwise differences were assessed for significance using the two-tailed Student’s t test, while differences among three or more groups were assessed using one-way analysis of variance.