2.1 Preparation of Ir-cR8-BSA-NP
Ir-cR8-BSA-NP was synthesised in a four-step process. First, the synthesis and quantification of oligoarginine peptide (cRn) was performed by GL Biochem (Shanghai) Ltd. and a purity of 97.52% was obtained. The resulting solid was thoroughly dissolved in sterile ultrapure water, then diluted 5, 10, 20, 40, 80, 160 and 320 times and measured using a microvolume spectrophotometer at an absorbance wavelength of 205 nm. The original solution concentration was calculated and samples were aliquoted for storage at -20°C. A pH 7.4 Tris-HCl solution was then used to dilute the cRn solution and the bis(2-phenylpyridinato) iridium (III) hexafluorophosphate [Ir(ppy)2(H2O)2]OTf storage solution. The two components were thoroughly mixed in a volume ratio of 1:1 and incubated at 37°C for 24 hours to obtain well-coordinated Ir-cRn.
pH 7.4 Tris-HCl buffer was purchased from Beyotime Biotechnology Co., Ltd., while [Ir(ppy)2(H2O)2]OTf was procured from SunaTech Inc. Subsequently, [Ir(ppy)2(H2O)2]OTf and imidazole (C3H4N2) were dissolved in a mixture of acetonitrile (CH3CN) and PBS (50 mL: 10 mL, v/v). The solution was refluxed for 24 hours, cooled to room temperature, and then rotary evaporated under reduced pressure. The resulting material was purified through column chromatography using a methanol/dichloromethane mixture (1:20, v/v) to obtain a brown solid, namely, Ir-diimidazole. Finally, Ir-cR8-BSA-NP was obtained by assembling Ir-cR8 with bovine serum albumin at 1:1 mole ratio under room temperature for 2 h.
2.3 In vitro experiments
The H22 murine hepatocellular carcinoma cell line was purchased from FuHeng Biology. The VX2 rabbit squamous cell carcinoma cell line was purchased from Shanghai Guandao Biological Engineering Co. Mouse bone marrow-derived dendritic cells (BMDC) and mouse bone marrow-derived macrophages (BMDM) were induced as previously reported.
MTT and CCK-8 assays were used to evaluate the toxicity of Ir-cR8, Ir-cR8-BSA-NP and oxaliplatin on H22 and VX2 cells. To investigate the selectivity of Ir-cR8-BSA-NP for normal and tumour cells, CCK-8 assays were used to evaluate the toxicity of Ir-cR8-BSA-NP to BMDCs and BMDMs.
H22 cells or induced BMDCs and BMDMs in logarithmic growth phase were suspended in 1× pH 7.4 PBS buffer. After cell counting, the cell density was adjusted to 10^5 cells/ml and the ζ-potential of these three cells was measured using a dynamic light scattering instrument (Malvern).
2.4 In vivo experiments
Orthotopic mouse model of hepatocellular carcinoma 6 to 8-week-old female BALB/c mice were obtained from Suzhou Jingweiyu Biotechnology Co., Ltd. Mice were prepared by creating a skin flap around the liver. H22 cells in the logarithmic growth phase were digested, centrifuged and adjusted to a density of 2 × 10^6 cells/ml. Each mouse was inoculated with 10^6 cells mixed with standard matrix gel. Surgical procedures included an anterior abdominal incision, removal of a liver lobe, injection of cells and closure of the incision. When the tumour size reached approximately 50 mm^3, the mice were divided into three groups (n = 5): the control group received an i.v. injection of 100 µL PBS; the Ir-cR8-BSA-NP group received an i.v. injection of 100 µL Ir-cR8-BSA-NP (500 mM) for 6 consecutive days; and the oxaliplatin group received an i.v. injection of oxaliplatin 5 mg-kg^-1 every two days for a total of three injections. Starting on the day of tumour inoculation, on days 5, 10 and 15, each mouse received an intraperitoneal injection of 200 µL of 15 mg/mL D-luciferin sodium salt. Tumour growth was monitored using IVIS Spectrum (IVIS Lumina XRMS Series III, PerkinElmer) and the weight of each mouse was recorded.
H&E staining of mouse tissues: Three days after injection, tissues (heart, liver, spleen, lung, kidney and tumour) were collected and fixed in 4% neutral paraformaldehyde. After 24 hours of fixation, the samples were paraffin-embedded and sections were stained with haematoxylin and eosin (HE). The stained tissue sections were observed under a fluorescence upright microscope (Axio Scope A1, Zeiss).
Rabbit VX2 orthotopic liver tumour experiment.
To further validate the efficacy of Ir-cR8-BSA-NP and oxaliplatin, a rabbit VX2 orthotopic liver cancer model was generated from 4-month-old New Zealand White rabbits (n = 4 per group). Rabbits were divided into three groups: control (PBS, arterial infusion of PBS), oxaliplatin (arterial infusion of oxaliplatin 5 mg-kg^-1) and Ir-cR8-BSA-NP (arterial infusion of Ir-cR8-BSA-NP 5 mg-kg^-1).
Under ultrasound guidance, 1 mm3 blocks of tumour tissue were injected into the left lobe of the rabbit liver using a 20-gauge needle. Postoperatively, the rabbits received intramuscular injections of 40 WU/d penicillin sodium for 3 consecutive days to prevent infection. Tumour size was measured by computed tomography (CT) after surgery.
The doses used for arterial infusion treatment with Ir-cR8-BSA-NP and oxaliplatin were calculated based on body surface area using the formula Dose(b) = Dose(a) × (kb/ka) × (Wb/Wa)^(2/3). In situ VX2 tumours were treated by arterial infusion under digital subtraction angiography (DSA) guidance. Rabbits in each group underwent PET/CT before treatment and 7 days after infusion. The standardised uptake values (SUVs) of the tumours for each group were compared before and after treatment to assess the anti-tumour effects.
At the end of the experiment, the rabbit livers were harvested, the tumour tissues were isolated and the tissues were immersed in 4% neutral paraformaldehyde. After 24 hours of fixation, paraffin embedding was performed and the sections were stained with HE to further compare the necrotic conditions of the tumours in each group. These HE staining images were obtained using a fluorescence upright microscope (Axio Scope A1, Zeiss).