Ambient synthesis of an iminium-linked covalent organic framework for synergetic RNA interference and metabolic therapy of fibrosarcoma

Small interfering RNA (siRNA)-mediated gene silencing is a promising therapeutic approach. Herein, we report the ambient synthesis of a positively charged iminium-linked covalent organic framework by a three-component one-pot reaction. Through anion exchange and siRNA adsorption, the resulting multifunctional siRNA@ABMBP-COF, which possesses both the HK2 inhibitor 3-bromopyruvate and SLC7A11 siRNA, exhibits powerful synergistic antitumor activity against fibrosarcoma via the ferroptosis and apoptosis pathways.

A reduced glutathione assay kit and haematoxylin-eosin (H&E) stain kit were purchased from Nanjing Jiancheng Bioengineering Institute (China).

Experimental instrumentation
Supercritical carbon dioxide drying was performed using a Tousimis Samdri-PVT-3D critical point dryer. Damp solids were contained in folded filter paper secured with a staple and dried with a 15 min purge time and 15 min equilibration time after heating.
Nitrogen-adsorption isotherms of samples that were degassed under vacuum at 120°C for 8 h were measured at 77 K using a Micromeritics ASAP2020 HD88 surface area and porosity analyser. The Brunauer-Emmett-Teller (BET) equation was used to calculate the specific surface areas.
Fourier transform infrared (FT-IR) spectra were obtained on a Thermo Scientific Nicolet iS50 FT-IR spectrometer equipped with a diamond attenuated total reflection (ATR) module between 4000-400 cm -1 . Each spectrum represented an average of 16 scans.
Thermogravimetric analysis (TGA) was performed with a Mettler Toledo TGA/DSC3+ thermogravimetric analyser. Approximately 5.0 mg of dried sample was analysed from room temperature to 900°C at a heating rate of 10°C/min under a N2 atmosphere. 13 C Cross-polarization/magic-angle spinning solid-state NMR ( 13 C CP/MAS ssNMR) spectra were recorded on a Bruker Advance III 400 MHz NMR spectrometer.
Ultraviolet-visible (UV-vis) absorption spectra were recorded on a Shimadzu UV-2700 double-beam UV-vis spectrophotometer using a 10 mm quartz cuvette.
Inductively coupled plasma-mass spectrometry (ICP-MS) measurements were carried out using a PerkinElmer NexION 300X ICP-MS.
The hydrodynamic particle size and zeta potential were measured using a Malvern Zetasizer Nano ZS90 system. Transmission electron microscopy (TEM) images were recorded on a Hitachi HT7700 120 kV compact-digital instrument. TEM samples were prepared in methanol by sonication for 5 min, followed by application on a carbon-coated copper TEM grid (200 mesh, Beijing Zhongxing Bairui Technology Co., Ltd.), and air drying at room temperature.
Scanning electron microscopy (SEM) images were recorded using a Hitachi SU8010 instrument. SEM samples were prepared by depositing a diluted suspension onto silicon wafers (approximately 3 × 3 mm, Beijing Zhongxing Bairui Technology Co., Ltd.), followed by air drying and coating with a thin layer of Pt to increase the contrast.
Microplate assays were conducted using a Molecular Devices SpectraMax i3x microplate detection system.
Western blot images were obtained using a GE Healthcare Amersham Imager 600 luminescent image analyser. Flow cytometric analysis was performed using a BD FACSCalibur flow cytometer.
In a black 96-well plate, ABMI-COF and ABMBP-COF (0-200 µg/mL, 100 µL) were mixed with siRNA-Cy3 (50 nM, 100 µL). The 96-well plate was shaken at room temperature for 10 min. Subsequently, the fluorescence intensity at 564 nm after excitation at 514 nm was measured using a multimode microplate detection system. The concentration of added ABMI-COF and ABMBP-COF was used as the horizontal axis, and the fluorescence intensity was used as the vertical axis to plot the fluorescence quenching curves.

Cell culture
The HT-1080 (human fibrosarcoma) cell line was provided by the Cell Bank, Chinese Academy of Sciences (Shanghai, China). HT-1080 cells were cultured in MEM supplemented with FBS (10 vol%) and normocin (100 μg/mL) at 37°C in a water-jacketed CO2 incubator with CO2 (5 vol%).

Clonogenic assays
HT-1080 cells were cultured in 6-well plates, treated with ABMI-COF, ABMBP-COF, siRNA@ABMI-COF, and siRNA@ABMBP-COF (2.0 mL, 40 μg/mL, COF equiv.) for 4 h in a CO2 incubator and washed twice with PBS. After approximately 7 days of incubation, the cells were fixed with paraformaldehyde (2.0 mL, 4 wt%) for 30 min and stained with fresh Giemsa staining solution for 1 h at room temperature. The plates were washed with water, air-dried, and photographed with a digital camera. Untreated wells were used as the control group, and wells without cells were used as the blank group.

Enzymatic activity assays
HT-1080 cells were cultured in 6-well plates, treated with ABMI-COF, ABMBP-COF, siRNA@ABMI-COF, and siRNA@ABMBP-COF (1.0 mL, 40 μg/mL, COF equiv.) for 4 h in a CO2 incubator and washed twice with PBS. After an additional 48 h of incubation, HK2 and GPX4 activity was analysed using commercially available kits. Untreated cells were used as the control group. HK2 and GPX4 activity was normalized to the total protein amount in the cell lysates from a parallel plate and are expressed as a percentage relative to the control group.

Intracellular GSH and malondialdehyde measurement
HT-1080 cells were cultured in 6-well plates, treated with ABMI-COF, ABMBP-COF, siRNA@ABMI-COF, and siRNA@ABMBP-COF (2.0 mL, 40 μg/mL, COF equiv.) for 4 h in a CO2 incubator and washed twice with PBS. After an additional 48 h of incubation, GSH and malondialdehyde measurements were performed using commercially available kits. Untreated cells were used as the control group. GSH and malondialdehyde levels were normalized to the total protein amount in the cell lysates from a parallel plate.

Experimental animals
On the 4 th day after nanomedicine injection, 4 mice in each group were randomly selected and euthanized. The harvested tumour tissues were washed with normal saline containing heparin (160 μg/mL), cut into small pieces (approximately 10 mg), and homogenized in Tris-HCl buffer (10 mM, pH=7.4) containing protease inhibitor cocktail (MedChemExpress, Cat# HY-K0010) and EDTA-2Na (0.1 mM) at 4°C. The tumour homogenates were centrifuged at 3500 rpm for 15 min at 4°C, and GSH levels, malondialdehyde levels, HK2 activity, and GPX4 activity in the supernatants were measured with assay kits according to the manufacturers' guidelines. In addition, the total protein level was measured in parallel using a BCA protein assay kit (Thermo Fisher, Cat# 23227). The data were normalized to total protein level and are expressed as an absolute value or a percentage value relative to the control group value.
The remaining mice were further reared. When the tumour size reached 15 mm in either dimension, the in vivo antitumor experiment was terminated, and all mice were euthanized. The tumour tissue and major organs were collected for H&E histological analysis, Ki67 Chem. Sci., 2022, DOI: 10.1039/D2SC02297D Electronic supplementary information S15 immunohistochemical staining, and TUNEL immunofluorescence staining.
Furthermore, to evaluate the biocompatibility of the nanodrugs, nude mice (aged 8 weeks, ♀) were randomly divided into 5 groups and intravenously injected with PBS, ABMI-COF, ABMBP-COF, siRNA@ABMI-COF, and siRNA@ABMBP-COF (80 μL, 0.4 mg/mL, COF equiv.). The nude mice were housed for 7 days, and the blood of each mouse was collected from the eyes for blood biochemical testing.