A carrier-free supramolecular nano-twin-drug for overcoming irinotecan-resistance and enhancing efficacy against colorectal cancer | Journal of Nanobiotechnology

Materials and reagents

Ir and Nir were purchased from Meilun Biotechnology Co. Ltd (Dalian, China). Dimethyl sulfoxide (DMSO) and annexin V-FITC/PI apoptosis detection kit were purchased from Sigma-Aldrich (Shanghai, China). Cyanine5.5 (Cy5.5) were purchased from MedChemExpress (NJ, USA). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), PBS, trypsin-EDTA and penicillin/streptomycin were purchased from Thermo Fisher Scientific (Waltham, USA). The CCK-8 kit was purchased from Beyotime Biotechnology (Shanghai, China). All the chemicals were used as supplied without further purification.

Cells and animals

Human HCT116, SW480, HCT8, HCT8/V authenticated colorectal cancer cell lines, and LO2 a normal cell line of human liver cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), with no mycoplasma contamination. All cells were cultured in recommended medium with 10% FBS at 37 ◦C in an incubator with 5% CO2. 5-week-old female BALB/c nude mice (18–22 g, SPF grade) were purchased from GemPharmatech (Nanjing, China). The animal protocols were approved by the Ethics Review Committee for Animal Experimentation for The Eighth Affiliated Hospital, Sun Yat-sen University (Shenzhen, China) (Approved number: 2022-009-01).

Preparation and characterization of the Nir-Ir supramolecular nanoparticles

Nir-Ir NPs were obtained via an amended nanoprecipitation method. Briefly, Ir and Nir were dissolved in dimethyl sulfoxide (DMSO) with appropriate ratio, and the mixture was added to deionized water (c = 1 mg/mL). The resultant solution was stirred slightly at room temperature for 30 min and stored at 4 °C for further use. The morphology of the resultant nanoparticles was studied using a transmission electron microscopy (TEM, JEOL JEM-2100 F, Japan). The hydrodynamic diameter and zeta potential of Nir-Ir NPs were measured by dynamic light scattering (DLS) using a NanoBrook 90Plus PALS. UV-Vis spectrophotometer was used to obtain the absorption spectra, and a Thermo Scientific Varioskan LUX was used to obtain the fluorescence emission spectra. The fourier transform infrared spectroscopy (FTIR) spectrum was scanned by the spectrometer (Nicolet 6700, thermo scientific, USA). Besides, to track Nir-Ir NPs, Cy5.5 was added to DMSO solution of irinotecan and niraparib (1% in total mole), and the other steps were the same as described above.

Stability test

To test colloidal stability, freshly prepared Nir-Ir NPs solution with a concentration of 1 mg/mL was stored at room temperature (RT) for 7 days, and the size change of NPs was recorded by DLS as described above. The colloidal stability of Nir-Ir NPs was also evaluated under incubation with normal saline and DMEM containing 10% FBS for 24 h. For low pH stability test, the solution of Nir-Ir NPs (1 mg/mL) was adjusted to pH 6.5. And the high redox condition was simulated by adding 10 µL H2O2 (30% v/v) into the Nir-Ir NPs solution.

Molecular dynamics (MD) simulation

The structures of Ir and Nir were optimized under B3LYP/6-31G* by Gaussian09 package. After that, the HF/6-31G* method and basis set were used to calculate the electrostatic potential (ESP) and then the result was employed to calculate the restricted ESP(RESP)2 charge. MMFF94x Force Field parameters were used for characterizing those two drugs. 24 Niraparib and 10 irinotecan molecules were initially packed randomly by PACKMOL in a cubic box with a length of 40 Å. Then the mixture was neutralized by adding sodium/chlorine counter ions and solvated in a cuboid box of TIP3P water molecules with solvent layers 10 Å between the box edges and solute surface. MD simulation was performed using AMBER18. The complex was centered in a box of 10 Å margin solvated by the TIP3P water model. Periodic boundary condition (PBC) was set to allow free motion along the 3D lattice. Nonbonded van der Waals interactions were calculated using the Lennard-Jones 12 − 6 potentials with a 10 Å cutoff, while long-range electrostatics were treated using the Particle Mesh Ewald (PME)algorithm [45]. The SHAKE algorithm was applied to constrain bonds involving hydrogen atoms [46]. To remove improper atom contacts, a steepest descent minimization of 500,000 steps was performed. And then the system was heated up to 300 K in 50 ps. Subsequently, a two-step equilibration phase was carried out to simulate constant volume (NVT) and constant pressure (NPT) ensembles, respectively. The phase was simulated for 100 ps at 300 K using the Langevin dynamics method to control the temperature with collision frequency of 1.0 ps-1. At last, a 50 ns MD simulation was conducted with the integration time step of 2.0 fs.

In vitro cytotoxicity study

Cytotoxicity was analyzed by the CCK-8 assay according to the manufacturer’s instructions. Briefly, for each cell line, 1–5 × 103 cells per well were seeded in a 96-well plate, and then incubated overnight. The cells were treated with different concentrations of Ir, Nir, Ir/Nir mixture or Nir-Ir NPs. After 72 h incubation, the medium was replaced, and the cell viability was detected using the CCK-8 kit. The absorbance at 450 nm of each well was recorded on a microplate reader. Untreated cells were used as controls. IC50 values were determined by CompuSyn 1.0 software. Colony formation assay was used to analyze the long-term proliferative potential of cell lines following treatments with Ir, Nir, Ir/Nir mixture and Nir-Ir NPs. 4–10 × 102 cells per well were seeded in 6-well plates and incubated with the drugs with the same Nir or Ir concentration (Ir, 0.2 µM and Nir, 0.4 µM) for 72 h. The medium was replaced every 3 days. After 2 weeks, cells were fixed with 4% paraformaldehyde for 20 min, and then stained with 0.1% crystal violet for 30 min.

Immunofluorescence and annexin-V FITC/PI assay

To study the DNA damage induced by Nir-Ir NPs, 5 × 104 cells/well were seeded on a confocal dish and treated with Ir, Nir, Ir/Nir mixture or Nir-Ir NPs with the same Nir or Ir concentration (Ir, 0.2 µM and Nir, 0.4 µM) for 24 h. Cells were washed in PBS, fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.2% Triton X-100/PBS solution for 10 min. Blocking was performed using 1% BSA for 30 min at room temperature. Cells were incubated with rabbit primary anti-phospho-Histone-H2AX antibody (Cell Signaling Technology Cat# 9718) and mouse anti-RAD51 antibody (Genetex Cat# GTX70230) in PBS overnight at 4 °C. Secondary goat anti-rabbit Alexa Fluor 488-conjugated (Thermo Fisher Scientific Cat# A-11,008) and goat anti-mouse Alexa Fluor 555-conjugated (Thermo Fisher Scientific Cat# A-21,424) antibodies were added for 1 h at RT after PBS wash once. Cells were then incubated with DAPI (Thermo Fisher Scientific, Cat# D1306) in PBS for 10 min in the dark. Images were collected under a Zeiss LSM 800 laser confocal scanning microscope. To analyze the cellular apoptosis induced by Nir-Ir NPs, 1*105 cells/well cells were plated in 6-well plates and cultured overnight. Then cells were incubated with the drugs as described above for 48 h. Afterwards, cells were washed with PBS and stained by annexin-V FITC and propidium iodide (PI) according to the manufacturer’s protocol. The fluorescence intensity of cells was measured by a BD LSRFortessa flow cytometry in green channel for annexin V-FITC and red channel for PI, respectively.

Western blotting and quantitative PCR

3 × 105 cells/well cells were seeded in 6-well plates and cultured overnight. Cells were treated with Ir, Nir, Nir/Ir mixture or Nir-Ir NPs for 48 h. Then the cells were washed with PBS and lysed by RIPA buffer containing protease/phosphatase inhibitor cocktails (Beyotime Cat# P1045). Cell lysates were centrifuged, and the supernatants were loaded on SDS-PAGE, followed by transferring to the PVDF membrane (BIORAD, Cat# 1704156). The blots ware blocked with TBST containing 5% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies against γH2Ax (Cell Signaling Technology Cat# 9718), Bax (Abcam Cat# ab182733), Bcl-2 (Cell Signaling Technology Cat# 3498), PARP-1 (Cell Signaling Technology Cat# 9532) and MRP-1 (Abcam Cat# ab233383) at 4 ◦C overnight. Then, membranes were washed with TBST and incubated with the HRP-linked antibody at RT for 1 h. A ChemiDoc Imager system (Bio-Rad, ChemiDoc Touch) was used to detect the bands of specific proteins. Total RNA was isolated from SW480 cells with Trizol (Invitrogen, USA). Reverse transcription was performed with a PrimeScript reverse transcription reagent kit (Takara, Japan). After cDNA was amplified in Thermal Cycler (Bio-Rad, C1000 Touch), quantitative PCR was performed with TB Green Premix Ex Taq (Takara, Japan) and a fluorescence quantitative real-time PCR machine (Roche, LightCyele480). GAPDH mRNA was used as a reference. Primers were: hCCL5: 5’ – CCTGCTGCTTTGCCTACATTGC-3’ (sense) and 5’ – ACACACTTGGCGGTTCTTTCGG-3’ (antisense); hCXCL10: 5’GTGGCATTCAAGGAGTACCTC-3’ (sense) and 5’ – TGATGGCCTTCGATTCTGGATT-3’(antisense); hIFNB1: 5’-CTGCATTACCTGAAGGCCAAG-3’ (sense) and 5’- TTGAAGCAATTGTCCAGTCCC-3’ (antisense); hGAPDH: 5’- GCACCGTCAAGGCTGAGAAC-3’ (sense) and 5’-TGGTGAAGACGCCAGTGGA-3’(antisense).

Cellular uptake and in vivo biodistribution of Nir-Ir NPs

To estimate the endocytosis of Nir-Ir NPs, cells were seeded in a 6-well plate at a density of 3 × 105 cells/well and incubated overnight. Then the cells were treated with Nir/Ir mixture or Nir-Ir NPs for another 2 to 12 h. After that, the fluorescence of Ir or Nir-Ir NPs in the cells were analyzed by flow cytometry using a specific channel (405 nm laser, 450 nm/40 nm filter). Fluorescence imaging were performed to study the in vivo biodistribution of Nir-Ir NPs. In brief, tumor-bearing mice were subcutaneously injected with 5 × 106 HCT8/V or HCT116 cells into the right flank of female BALB/c nude mice. When the tumor volume exceeded 100 mm3, Cy5.5-labelled Nir-Ir NPs or free Cy5.5, with an equivalent Cy5.5 dose of 0.2 mg/kg, were intravenously injected into the tumor-bearing mice (n = 3). Fluorescence signals were detected at 2 h, 4 h, 6 h, 12 h, 24 h post-intravenous injection by an in vivo fluorescence imaging system (Biolight Biotechnology, AniView100) with excitation at 630 nm and emission at 680 nm. Then the mice were sacrificed at 24 h post-injection to collect the tumors and major organs. The average fluorescence intensities from Cy5.5 in tumors and major organs were evaluated to reveal the in vivo biodistribution.

Hemocompatibility evaluation

The whole blood sample was collected from a BALB/c mice into an EDTA anti-coagulated tube, and then was supplemented with 1 mL PBS to wash once at 2000 rpm for 10 min. After removing the supernatant, 10 mL PBS was added to dilute the blood sample. Then 200 µL of the diluted blood cells were co-incubated with 1 mL PBS (negative control), deionized water (positive control), or various concentrations of Nir-Ir NPs diluted in PBS (3.8, 7.7, 12.5, 25, 50 and 100 µM) for 2 h at 37℃. Afterwards, samples were centrifuged at 12,000 rpm for 10 min, and the supernatant was added into a 96-well plate to detect the absorbance at 570 nm. The calculation method of hemolysis rate is hemolysis ratio (%) = (A (sample570 nm) – A (negative, 570 nm))/(mean value of A (positive, 570 nm)-A (negative, 570 nm)) × 100% .

In vivo therapeutic efficacy and biosafety

In vivo antitumor efficacy of Nir-Ir NPs was studied in HCT116 and HCT8/V tumor models. A total of 5 × 106 cells were resuspended in 200 µL PBS and implanted subcutaneously into the right flank of 20 mice for each cell line. The mice were randomly divided into five groups when the tumors reached a volume of 75–100 mm3, with 4 mice in each group, and were intravenously injected with: (i) PBS; (ii) Nir-Ir NPs (200 uL, 1 mg/mL); (iii) Ir, (iv) Nir, (v) Ir/Nir mixture (equivalent Ir or Nir dose) every three days. The volume of tumors was measured every other day and calculated by the following equation: V = L × W2/2. Mice were weighed every three days. When the tumor diameter reached 15 mm, mice were euthanized to collect whole blood, tumors, and major organs (liver, heart, kidney, lung, spleen) for further analysis. The tumors were weighed and photographed. The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CREA) were measured by serum biochemical analysis to reveal the long-term toxicity to the liver and kidney. The tissues were fixed with 4% paraformaldehyde solution and embedded in paraffin, followed by staining with hematoxylin and eosin (H&E) for further observation by optical microscopy. The tumor sections were also stained by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), γH2AX and MRP1 for histology studies.

Statistical analyses

Data were analyzed using GraphPad Prism 9.0 software. Statistical analysis was performed by Student’s t-test. The data were presented as means ± standard deviation (SD) unless otherwise indicated. Significant differences were considered if P values < 0.05; * for P < 0.05, ** for P < 0.01, *** for P < 0.001 and NS. for non-significant.