CNT fabrication
Sizes of CNT were purchased from SES Research. CNTs were heated at 300 ℃ for 2 h to evaporate the water vapor. Next, 400 mg CNT were added to 180 ml sulfuric acid and 60 ml nitric acid and reacted for 99 min under sonication. After 99 min, the reaction was conducted at 55 degrees/200 RPM for 48 h. After 48 h, 20 ml 240 ml CNT was mixed with 2 L of DW and diluted. Subsequently, 300 ml of diluted CNT was added and filtered using a 0.1 ~ 0.2 μm PTFE membrane filter purchased from Merck Millipore. DW (25 ml) was added to the filtered CNTs and washed once. After removing the filtered CNT filter paper, it was placed on a 150 dish, stored for 2 d at 60 ℃/0.07 vacuum, dried, collected, transferred to a 2 ml piece of e-tube, and stored at -20 ℃ until use.
Covalently conjugated CNT with DOX
CNT (40 mg) was placed in 10 ml DW and sonicated for 10 min. Centrifugation was performed at 4000 RPM for 10 min, and DW washing was performed 3 times in total. After washing, 40 mg CNT and 20 ml pH 6 MES buffer were warmed to 50 ℃, and 920 mg N-hydroxysuccinimide (NHS) and 10 mL pH 6 MES buffer were added; next, CNT and NHS were mixed, followed by sonication for 5 min. First, 900 mg 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was reacted by rotation at room temperature for 1 h. After the reaction was completed, centrifugation was performed at 4000 RPM for 15 min, and washing was performed three times using 10 ml MES (pH 6). For DOX, when washing CNTs with 10 ml pH 6 MES, 40 mg CNTs was pre-dissolved in a weight ratio of 1:1 in 5 ml pH 6 MES, stored at 4 ℃ after washing, released in 5 ml pH 6 MES, and mixed with DOX at 4 ℃ overnight. After the overnight reaction, washing was performed three times using 10 ml MES buffer (pH 6 MES buffer was warmed to 50 ℃ to remove DOX that did not adhere to CNTs; next, CNT DOX was released with 2 ml DW and collected in a 2 ml e-tube. CNT-DOX was diluted 50-fold in DW in a UV–vis cuvette, and the total volume was 2 ml. In the measured CNT DOX, DOX was measured at 480 nm and CNT at 700 nm. DOX loading (%) = weight of DOX attached to the CNT / Weight of CNT × 100.
Cell cultures
H69AR (non-SCLC, CRL-11351™) cells supplemented with 10% FBS (16000-044, Gibco) and 1% penicillin streptomycin (15140-163, Gibco) were maintained in RPMI 1640 medium (11875-093, Gibco) and were cultured in a humidified incubator with 5% CO2 at 37 ℃.
Uptake analysis of DOX resistance cancer cells
Poly D lysine coating was conducted by placing a cover glass in a 150 dish, putting 70% ethanol, and irradiating via UV for 1 d. After 1 d, using DW, washing was performed four times for 5 min each. After washing, the cover glass was transferred to a 24-well plate. The transferred cover glass was irradiated with UV until the phosphate-buffered saline (PBS) disappeared in 24 wells; when PBS disappeared, it was stored at 4 ℃ MV. Poly D lysine-coated cover glass was placed in a 24-well plate, and RPMI-1640 + 10% FBS + 1% P/S was added and preincubated for 30 min. After seeding 1 × 105 cells, the next-day inhibitors (macropinocytosis: EIPA, clathrin: CPZ, caveolin: GEN) were treated with 20 μM, 25 μM, and 200 μM at each concentration for 1 h before drug treatment. After treating CNT DOX 500 ng/ml, incubation was conducted for each hour; after removing the media, it was washed three times with PBS, 500 μl of 4% paraformaldehyde (PFA) was added, and this fixed for 1 d. After fixation, 5 µL mounting solution was placed on a glass slide, and the cover glass with cells was turned upside down for mounting. After incubation at room temperature for 30 min, the RFP fluorescence intensity was checked using EVOS.
Viability and apoptosis analysis
Next, 3 × 104 cells of H69AR were dispensed into a 96-well plate (100 ul each). The next day, GEN, a caveolin inhibitor, was treated with 200 μM 1 h before the CNT DOX treatment. CNT DOX was treated with the concentrations of 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, and 1 μg/ml and incubated at 36 ℃ / 5% CO2 for 48 h. After 48 h, 100 of 2 mg/ml MTT solution was put into each well and incubated at 36 ℃ and 5% CO2 for 2 h. After 2 h of reaction, the solution was removed, and 100 μl dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals, which were then measured at 570 nm.
For the investigation of the effect of covalent CNT DOX on the apoptosis of H69AR, the cells were aliquoted in 2 ml 8 × 105 cells in a 6-well plate. The next day, GEN, a caveolin inhibitor, was treated with 200 μM 1 h before CNT DOX treatment. After 1 h, 0.5 μg/ml CNT DOX was treated and incubated at 36 ℃ / 5% CO2 for 24 h. The medium was transferred to a 15 ml conical tube, and after washing once, 200 μl 1X TE was added, and the cells were removed by reacting for 5 min. The removed cells were placed in a 15 ml conical tube with medium and centrifuged at 1500 RPM for 5 min; next, the supernatant was removed, and the cells were washed once with PBS. Subsequently, 105 μl fluorescence-activated cell sorting (FACS) buffer (1X Annexin V binding buffer 100 μl + Annexin V 5 μl) was added, the pellet was released, and the reaction was conducted at room temperature for 20 min. After 20 min, 400 μl annexin V binding buffer was added to stop the reaction. After centrifugation at 1500 RPM for 5 min, the supernatant was removed, 500 μl was added to release the cells, the cells were transferred to a FACS tube, and the results were confirmed.
MRP-1 expression
H69AR cells were seeded in 2 ml of 8 × 105 cells in a 6-well plate. The next day, GEN, a caveolin inhibitor, was treated with 200 μM for 1 h before the CNT DOX treatment. CNT DOX was treated with 1 μg/ml and incubated at 36 ℃ / 5% CO2 for 2–24 h. After removing the media after 2–24 h, 1X TE treatment was performed to remove the cells, which were then centrifuged at 1500 RPM for 5 min. After removing the supernatant, 100 μl protein lysis buffer (RIPA buffer 100 μl + 1% protein inhibitor 10 μl) was added and transferred to 1.5 ml e-tube. The mixture was then placed on ice and incubated for 30 min. After 30 min, the mixture was centrifuged at 12,000 RPM at 4 ℃ for 30 min, and the supernatant was transferred to a new 1.5 ml e-tube. The extracted protein was quantified using a Bradford (Coomassie) protein assay kit. After protein quantification, 20 μl of the sample was prepared by adding protein at an appropriate concentration, 4X sample buffer and DW, and boiling at 100 ℃ for 5 min. For SDS PAGE gel, 8% was used, and each prepared sample was placed on the gel wall and loaded at 85 V. A semi-dry transfer was used to transfer the loaded protein to the membrane at 25 V for 35 min. Using 5% skim milk in TBS-T, blocking was performed at room temperature for 1 h, and mrp-1 and beta actin antibodies were conjugated at 4 ℃ / overnight. The next day, after removing the 1st antibody, the membrane was washed with TBS-T, incubated with the 2nd antibody (rabbit, mouse) at room temperature for 1 h, washed again with TBS-T, and detected using ECL. Immunodetection was performed using an enhanced chemiluminescence detection kit (34080, Thermo Fisher Scientific) and protein bands were photographed with a LAS4000 Chemi-Doc imager (Fuji-film, Japan). The full-length stain is represented in Figure S5.
ATP analysis
Total ATP inside the cells was measured using an ATP assay kit (Invitrogen, A22066). Viewing the inside of the cell is performed as follows: the cell is lysed with RIPA buffer to obtain the protein, and subsequently, the protein is quantified using the BCA assay. In a state containing 1 µg of protein in a volume of 10 μl, it was placed into a 96-well plate and mixed with 90 μl reaction buffer in the absence of light, and the result was checked using a VICTOR Nivo Multimode Microplate Reader (PerkinElmer). At this time, the ATP concentration used in the standard curve was 0.0625–2 μM; next, 10 μl was added in the same manner as the protein, and 90 μl reaction buffer was added to construct a calibration curve, to calculate the value of ATP measured in cells.
Mitochondrial membrane potential changes
The poly D lysine-coated cover glass was treated in RPMI-1640 medium before cells were dispensed; next, 1 × 105 cells of H69AR cells were dispensed (500 μl each). The next day, CNT-DOX was treated with 1 μg/ml and incubated at 37 °C for 24 h. After 20 h, 4 mM H2O2 as a positive control was treated for 4 h, and the cells were washed twice with PBS. Subsequently, 1 μM JC-1 was dissolved in PBS, dispense 500 μl to each well, and incubated at 37 °C for 30 min. It was removed after 30 min, washed twice with PBS, and 500 μl 4% PFA was added; next, it was fix for 1 d. For the fixed samples, 5 μl mounting solution was dispensed on a glass slide, and the cover glass with cells was turned over for mounting. Mitochondrial damage was confirmed by measuring the fluorescence intensities of RFP and GFP by using an EVOS7000.
Intracellular trafficking
The poly-D lysine-coated cover glass was transferred to a 24-well plate, and RPMI-1640 + 10% FBS + 1% P/S was added; it was preincubated for 30 min, and subsequently, 5 × 104 cells were aliquoted. The next day, 0.5 µg/ml CNT-DOX (60–100 nm) and DOX were treated for 6, 12, and 24 h. For each time-treated group, the medium was removed, washed with PBS, and 4% PFA was added, and the medium was fixed at 4 °C for 1 d. The fixed sample was washed three times for 10 min with PBS, and 500 μl of 0.5 Triton x-100 was added and treated for 10 min. Next, we added 500 µl 100 mM glycine, react at 4 °C for 10 min, add 500 µl of blocking buffer, and incubate at room temperature for 1 h. After blocking, the 1st antibody EEA-1 and mannose 6-phosphate receptor (M6PR) were incubated at 4 ℃ for 1 d. After washing 3 times with PBS for 10 min, the 2nd antibody (Alexa 488 goat anti-rabbit, Alexa 488 goat anti-mouse) was incubated at room temperature for 1 h. After washing it with PBS three times, mounting was performed to confirm the fluorescence wavelength in EVOS7000.
Intracellular pH change
The Poly-D lysine-coated cover glass was seeded 5 × 104. The next day, the drug was administered according to time, and the medium was removed the next day and washed with live cell imaging solution (LCIS). The pH rodo AM solution was then added, and this was incubated at 37 °C for 30 min. After 30 min, they were washed with LCIS and fixed with 4% PFA. In the case of a calibration curve, it was diluted with 10 mM nigericin 5 μl and 10 mM valinomycin 5 μl in 10 ml of pH calibration buffer (pH 4.5, 5.5, 6.5, 7.5) before fixing, placed on each wall, incubated at 37 °C for 5 min, washed with LCIS, and washed with 4% PFA fix it.
H69ARflu-GFP production
H69AR cells were seeded 1 × 105 in a 6-well plate. The next day, 1 ml lentiviral supernatant containing luciferase and GFP, and 2 ml RPMI-1640 without penicillin were exchanged. After 24 h, the medium containing the lentiviral vector was removed, replaced with RPMI-1640 containing penicillin, and cultured for 24 h. The next day, 2 ml RPMI-1640 was added to the appropriate concentration of puromycin, and the cells were cultured. Next, the puromycin of and the appropriate concentration were continuously added for 1–3 weeks and cultured, and it was observed.
H69ARflu-GFP Xenograft model
Female BALB/c nude-Foxn-1-nu mice were purchased from Orient Bio (Seoul, Korea). After shipment, five mice were housed per cage in a laminar air flow room maintained at a temperature of 22 ± 2 °C with a relative humidity of 55 ± 5%. In addition, all mice were cared in specific-pathogen-free environment with a 12-h light/dark cycle (lights on at 7:00 AM) for 7–14 days, to acclimatize before the experiment. All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of Gachon University (LCDI-2019-0092). H69ARFluc-GFP, a SCLC cell line, was dissolved in 1 × 107 cells in 100 μl PBS and injected between the dorsal endothelium and integument of mice. We confirmed whether the H69ARFluc-GFP tumor was established in the mice: 150 mg/kg D-luciferin was injected, and the size of the tumor was confirmed by fluorescence intensity measurement and imaging for 1 min by using IVIS.
In vivo antitumor efficacy
The tumors of the mice were larger than 100 mm3 and were divided into five groups (n = 5)—saline, CNT, free DOX, CNT DOX, and CNT DOX + inhibitor (in 100 μl of PBS). They were injected intratumorally (5 mg/kg) once per week. The size of the tumor was checked once per week by IVIS fluorescence intensity.
Bioluminescence imaging using GFP animal model
Bioluminescence imaging (BLI) was confirmed by measuring the luciferase activity with an in vivo imaging system (IVIS). BLI was checked once per week. Before imaging, 100 μl luciferin (150 mg/kg) was injected into the mouse, and anesthetized with isoflurane (approximately 3% in air). Anesthetized mice were placed in a chamber protected from the light of the IVIS, and measurements were collected for 1 min. Images were captured using Living Image software. BLI averaged the fluorescence intensity on the animal surface.
H69ARflu-GFP production
H69AR cells were seeded 1 × 105 in a 6-well plate. The next day, 1 ml lentiviral supernatant containing luciferase and GFP and 2 ml RPMI-1640 without penicillin were exchanged. After 24 h, the medium containing the lentiviral vector was removed, replaced with RPMI-1640 containing penicillin, and cultured for 24 h. The next day, 2 ml RPMI-1640 was added to the appropriate concentration of puromycin, and the cells were cultured. Next, the puromycin of and the appropriate concentration were continuously added for 1–3 weeks and cultured, and this was observed.
Immuno-histological analysis
The H69ARFluc-GFP xenograft model was sacrificed. After fixing the cancer tissue with 10% neutral buffered formalin, the cancer tissue was dehydrated and embedded in paraffin. Embedding tissue was sectioned at a size of 5 μm, rehydration was conducted, and the hematoxylin and eosin (H&E) and tunnel assays were performed.
Toxicity analysis
For the analysis of peripheral circulating blood cells, blood was placed in a vial containing heparin (5 unit/ml) and transferred to ice for analysis, and blood cells were automatically counted. In the case of serum, the blood was allowed to coagulate at room temperature without being disturbed and was centrifuged at 2000 × g at 4 ℃ for 15 min to remove the clot by transferring the supernatant to a new tube. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and blood urea nitrogen (BUN) were measured using clinical chemistry reagent kits.