PTX-resistant ovarian cancer cell line A2780/PTX and its sensitive counterpart A2780 were purchased from Keygen Biotech (Nanjing, Jiangsu, China). PTX-resistant lung carcinoma cancer cell line A549/PTX was purchased from Keygen Biotech and PTX sensitive cell line A549 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). A2780/PTX and A2780 were cultured in DMEM, while A549/PTX and A549 were cultured in RPMI-1640 medium (Gibco, Invitrogen, Carlsbad, CA, USA), supplemented with 10% FBS (Invitrogen).
Tissue microarray and immunohistochemical analysis of multiple organs
The tissue microarray was obtained from Shanghai Outdo Biotech Company (Cat# HOrgC120PG05, Shanghai, China). The ethical approval of using human multiple organs was approved by the Ethics Committee of Shanghai Outdo Biotech Company (No. SHYJS-CP-1904010). Immunohistochemical analysis was performed in Shanghai Outdo Biotech Company (Shanghai, China). The immunohistochemical tissue microarray was imaged by using an Aperio ImageScope system (v12.4.35008).
Construction and characterization of LANP-PTX-siSRI
The lipid modification method was the same as the previous report . In brief, 50 mg of DOPE and 50 mg of DOTAP were dissolved in 4 mL of chloroform. The solvent was then moved by rotary evaporation at room temperature. Lipid suspension was obtained by hydration with 5 mL ultrapure water, followed by mixing with 20 mg/mL of albumin-PTX (Jiangsu Hengrui Pharmaceuticals Co., Ltd., Jiangsu, China) at a weight ratio of 1:1. The lipid-coated albumin-PTX nanoparticles (LANP-PTX) were obtained after ultrasonic dispersion and 1% (w/w) dextran nanogels, as described in the previous report , were added to reduce precipitation generation. LANP-PTX-siSRI and LANP-PTX-siNC were prepared by mixing LANP-PTX (16 mg/mL) either with siSRI (20 μM) or with siNC (20 μM) in an equal volume and vortexing for 20 s at room temperature. The control LANP (without PTX) was prepared by mixing 20 mg/mL of albumin (Sigma-Aldrich, USA) with lipid suspension at a weight ratio of 1:1 and then, underwent ultrasonic dispersion. LANP-siSRI and LANP-siNC were prepared by mixing LANP (16 mg/mL) either with siSRI (20 μM) or siNC (20 μM) in an equal volume and vortexing for 20 s at room temperature.
Diameter and Zeta potential were measured using a Zetasizer Nano instrument (ZS90, Malvern, U.K.). Images were recorded using a transmission electron microscope (TEM, 120 kV, FEI Tecnai G2 Spirit BioTwin, FEI, U.S.A.).
Release of siRNA
A 250 µL of LANP-PTX-siSRI (16 mg/mL) was added to a microdialysis tube (4000 MW, Sangon Biotech, Shanghai, China). Then, the microdialysis tube was put into a microdialysis cup (Sangon Biotechnology, Shanghai, China) with 25 mL of pure water. The dialysate was taken at various times and the siRNA release was determined by measuring siRNA concentration in dialysates using Nanodrop (Thermo Fisher, USA).
Flow cytometry analysis
The cells were cultured in 6-well plates at a density of 2 × 105 cells/well for 24 h. Then, cells were trypsinized, centrifuged, and stained with a calcium ion assay kit (Beyotime Biotechnology, Shanghai, China) according to the product instructions. The cells were resuspended with 500 µL PBS and detected by flow cytometry (Gallios, Beckman Coulter, CA, USA).
A 5 μL of Fam-siRNA (20 μM) was loaded on 10 μL of LANP (8 mg/mL) and added to 6-well plates. After 4 h, cells were trypsinized, centrifuged, and resuspended with 500 µL PBS, and then detected by flow cytometry. Flow cytometry data were analyzed using FlowJo (V10).
Laser confocal microscopy assays
The cells were cultured in a confocal petri dish with a 35 mm diameter and 20 mm glass bottom. After the confluence reached 60–80%, cells were stained with a calcium ion assay kit according to the product instructions. The nuclei were further stained with Hoechst 33,342 (Beyotime Biotechnology, Shanghai, China). After washing with PBS 3 times, cells were photographed by a laser confocal microscope (SP8 STED 3X, Leica, Germany).
Green fluorescent Fam-labeled siRNA (20 μM in 2.5 μL, Fam-siRNA) was loaded into 5 μL of LANP (8 mg/mL). Electrophoretic separation was performed using a 2.5% (w/v) agarose gel containing SYBR Green Nucleic Acid Gel Stain. Electrophoresis was conducted in tris–acetate (TAE) running buffer at a voltage of 140 V for 20 min. Then, the gel was visualized and recorded using ultraviolet transmittance (Bio-Rad GelDoc EZ, USA).
RNA extraction and qRT-PCR
Total RNA was extracted using an RNA Rapid Purification kit (ES Science, Shanghai, China) according to the manufacturer’s instructions. PCR was performed using a qPCR reverse transcription kit (Mei5 Biotechnology, Beijing, China). The sequence of PCR primers was shown in Additional file 1: Table S1. A 7300 real-time PCR system (V1.4, Applied Biosystems, USA) was used to measure threshold circulation (Ct) and target gene expression was normalized to endogenous gene GAPDH.
RNA sequencing analysis
Total RNA was extracted using an RNA Rapid Purification kit (ES Science, Shanghai, China) according to the manufacturer's instructions. mRNA sequencing was conducted by Shanghai Biochip Co. Ltd. (Shanghai, China).
Protein extraction and Western blot
Cells were lysed with a sodium dodecyl sulfate lysate (SDS) containing benzyl sulfonyl fluoride (1%, w/v) and phosphatase inhibitor (1%, w/v). After ultrasonic lysis, the total protein was subjected to SDS–polyacrylamide gel electrophoresis. The primary antibodies were rabbit anti-P-gp (1:5000 diluent, CST, USA), rabbit anti-SRI (1:1000 diluent, CST, USA), mouse Smad2 (1:1000 diluent, CST, USA), rabbit P-Smad2 (1:1000 diluent, CST, USA), and mouse anti-β-actin (1:5000 diluent, CST, USA). The secondary antibodies were goat anti-rabbit IgG (1:10,000 diluent, Proteintech, USA) and anti-mouse IgG (1:10,000 diluent, Proteintech, USA) labeled with horseradish peroxidase. A chemiluminescence imaging system (Tanon Science & Technology, China) was used to photograph protein bands.
Cell viability and cell toxicity
Cells were seeded into 96-well plates at a density of 1 × 104. After 24 h, the medium was replaced by the fresh medium containing different concentrations of PTX ranging from 0.001–0.313 μM for sensitive cells and 0.625–40 μM for resistant cells. Each sample was performed with at least 4 parallel repetitions. After 48 h, a CCK-8 assay (Dojindo, Japan) was used to detect cell cytotoxicity by measuring the optical density (OD) of each well at 450 nm. The cell cytotoxicity calculation formula was used as indicated: cell cytotoxicity rate (%) = OD test /OD control × 100%.
In vitro therapy
The cells were cultured into 96-well plates at a density of 1 × 104/well. After cultivation for 24 h, the supernatant was removed and a fresh medium containing LANP-PTX, siRNA control (LANP-PTX-siNC), or LANP-PTX-siSRI was added to each well. The concentration of PTX was 0–20 μM and the corresponding siRNA concentration is in the range of 0–0.43 μM. After 48 h, cell viability was detected by CCK-8.
RBC hemolysis study
A hemolysis test was performed by using rabbit blood. The 5% erythrocyte suspension was mixed with a four-time volume of LANP-PTX-siSRI solution (test group), PBS (negative group), and water (control), respectively. After incubation at 37 ℃ for 4 h, the supernatant was collected by centrifugation. The optical density (OD) of the supernatant was measured at 577 nm. The hemolysis rate was calculated by the following formula: Hemolysis rate (%) = (ODtest—ODnegative)/(ODcontrol—ODnegative) × 100%.
Generation of tumor-bearing mice
The ethics approval of animal experiments was approved by the Ethics Committee of Shanghai Public Health Clinical Center (No. 2020-A027-01). About 3–4 weeks old female BALB/c nude mice (B&K Laboratory Animal Co., LTD., Shanghai, China) were raised under standard feeding conditions with adequate food and water. After 3 days, 3 × 106 A549/PTX cells in 100 µL of RPMI-1640 medium without FBS were injected into the right side of each mouse. After 3 weeks, the tumor-bearing mice were randomly assigned to various treatment groups.
Fluorescence imaging of tumor-bearing mice
Fluorescent-labeled LANP were fabricated by replacing DOPE with DOPE-Cy5.5 during the LANP preparation process. DOPE-Cy5.5 was synthesized as described previously . The mice were intravenous by 100 μL of Cy5.5-labeled LANP solution with 0.25 mg of PTX and 6.25 nM of SRI-siRNA. In vivo fluorescence imaging was performed at 0.5, 1.5, and 5 h after intravenous injection. The ex vivo fluorescence of major organs and tumors was imaged.
In vivo therapy
The tumor-bearing mice were divided into 3 groups (n = 8 in each group), including the normal saline group, the albumin-PTX group, and the LANP-PTX-siSRI group. The mice were intravenous and intratumor administration once every 2 days a total of 4 times. The corresponding solution is 100 μL (50 μL of intravenous + 50 μL of intratumor) each time. The dose of PTX was maintained at 12.5 mg/kg and the dose of siRNA was maintained at 312.5 nM/kg. The tumor volume and body weight were monitored every 2 days for 2 weeks. Tumor volume was calculated by V = ab2/2, where V is the tumor volume, a is the length of the tumor, and b is the width of the tumor. At the end of the experiment, the mice were anesthetized and sacrificed. The excised tumors were weighed and photographed.
The tumors harvested from various treated groups were fixed with formalin and embedded in paraffin. A pathological examination was performed by Shenghua Biological Technology Co., LTD (Shanghai, China). Tumor tissue sections were stained with H&E, PCNA, and TUNEL and were photographed under a microscope.
TGF-β treatment was carried out by seeding the cells into a 6-well plate at a density of 2 × 105 cells/well. After incubation for 24 h, a fresh medium containing TGF-β1 (R&D Systems, MN, USA) was added to wells at concentrations of TGF-β1 at 0, 1, and 10 ng/mL. The cells were collected for subsequent experiments after 48 h.
UCSC XENA (https://xenabrowser.net/datapages/) was used for the analysis of SRI expression levels in human serous ovarian cancer and lung adenocarcinoma. Data of lung adenocarcinoma and serous ovarian cancer were extracted from TCGA, and corresponding normal tissue data were extracted from GTEx. The prognostic value of SRI was assessed by Kaplan–Meier Plotter (www.kmplot.com) as described in our previous report .
The qRT-PCR data are presented as mean ± standard error (SE). The cell viability, IC50, particle size, Zeta potential, tumor volume, tumor weight, and mice body weight are presented as the mean ± standard deviation (SD). Results were performed for assays at least 3 times. Graphs were drawn using Origin (V5.0, OriginLab, USA) or GraphPad (V5.0, GraphPad Software, USA) software. The student's t-test was used for statistical analysis of differences between the 2 groups. A p < 0.05 was considered statistically significant.