Preparation and characterization of CNSI-Fe
Carbon nanoparticle suspension injection (CNSI) and ferrous sulfate for injection were produced under GMP conditions according to the laboratory-scale preparation method we previously reported,33 with some necessary adjustments. Briefly, 1.00 kg of carbon nanoparticles, 0.06 kg of sodium citrate, 0.40 kg of sodium chloride, and 0.18 kg of poloxamer were added to water for injection to achieve a total weight of 20.52 kg, and a CNSI with a concentration of 50 mg/mL was prepared, with 2 mL filled in each vial. Using 2.24 kg of FeSO₄·7H₂O, followed by dilution with water for injection to achieve a total weight of 30.96 kg, ferrous sulfate was prepared for injection as a lyophilized powder with 30 mg of ferrous ions per vial. CNSI-Fe was freshly prepared by dissolving ferrous sulfate for injection with CNSI at the designated concentration immediately before clinical use.
To characterize the physicochemical properties of CNSI-Fe, we employed a comprehensive suite of analytical techniques. Field-emission transmission electron microscopy (TEM; Talos F200S, Thermo Fisher Scientific, USA) was utilized to analyze the morphological characteristics and dispersion state. The particle size distribution was determined by dynamic light scattering (DLS; Zetasizer Nano ZS90; Malvern Instruments, UK). Surface functional groups were identified using Fourier transform infrared spectroscopy (FTIR; Nicolet iS50, Thermo Fisher Scientific, USA). The characteristic peaks of the carbon materials were examined by confocal Raman spectroscopy (LabRAM HR Evolution, Horiba Scientific, France). The elemental composition was precisely analyzed through X-ray photoelectron spectroscopy (XPS; K-Alpha, Thermo Fisher Scientific, USA). We also measured the stability of CNSI-Fe within 6 hours of preparation, with evaluation indicators including appearance, pH, particle size and polydispersity index (PDI), zeta potential, and Fe2+ and Fe3+ contents.
Study design and participants
This first-in-human, single-arm, open-label, dose-escalation trial employed a 3 + 3 design to evaluate the safety, pharmacokinetics (PK), and preliminary efficacy of intratumorally administered CNSI-Fe in patients with refractory solid tumors. Conducted across three Chinese clinical centers (West China Hospital of Sichuan University, Shandong First Medical University Affiliated Cancer Hospital, and Enshi Tujia and Miao Autonomous Prefecture Central Hospital) from December 2022 to September 2024, the study protocol received approval from institutional ethics committees (Approval No.: 2022 Clinical Trial (Western Medicine) Review No. 238, SDZLEC2023-203-01, and 2023-003-01; ClinicalTrials.gov: NCT06048367; NMPA CTR: CTR20222235). All participants provided written informed consent prior to enrollment.
The key inclusion criteria were as follows: age 18–80 years; histologically/cytologically confirmed advanced solid tumor; disease progression after standard therapy or lack of effective standard treatment; ≥1 measurable lesion per RECIST v1.1; intratumoural injection (direct or imaging-guided) was possible; ECOG performance status ≤1; and adequate hematologic and organ function.
The exclusion criteria included iron metabolism disorders, hollow organ perforation risk at the injection site, and local skin abnormalities (ulceration, necrosis, hemorrhage) potentially interfering with drug administration.
Procedures
Dose escalation followed a 3 + 3 sequential cohort design with planned doses of 30, 60, and 90 mg according to the order in which informed consent was obtained. DLT criteria triggered protocol-specified adjustments: If ≥2 DLTs occurred at 90 mg, de-escalation to 75 mg would occur. If no MTD was identified at 90 mg, the Safety Review Committee (SRC) would evaluate escalation to 120 mg or 150 mg on the basis of cumulative safety, PK/pharmacodynamic data, and efficacy data. Treatment cycles spanned 21 days, with CNSI-Fe administered by imaging-guided (ultrasound/CT) intratumoral injection on day 1. The DLT observation window covered cycle 1. Patients deriving clinical benefit per the investigator’s judgment could receive a second injection in cycle 2.
Study assessments
Primary endpoints: Safety/tolerability, including DLT incidence and MTD determination. Secondary endpoints: PK profile of serum iron ions and preliminary evaluation of antitumour activity. Safety evaluations included the following: AE monitoring per CTCAE v5.0; physical examinations; vital signs; laboratory tests; ECOG performance status; and twelve-lead electrocardiogram (12-lead ECG) and echocardiography. DLTs were defined as follows: Grade ≥3 nonhaematologic toxicity; Grade 4 hematologic toxicity; and any AE leading to treatment discontinuation. MTD was defined as the highest dose at which ≤1/6 of the patients experienced a DLT.
PK analysis
Serum iron concentrations were quantified via inductively coupled plasma mass spectrometry (ICP-MS) at predefined intervals (before injection to 72 h after injection). Key PK parameters (Cmax, Tmax, AUC0-t, AUC0-∞, t1/2) were derived using noncompartmental analysis for cycle 1 and cycle 2.
Efficacy evaluation
Tumor response was assessed by using modified RECIST ver1.1 and RECIST ver1.1. Initial assessment: Weeks 3-4 of cycle 1. Follow-up assessment: Weeks 7–8 for cycle 2 recipients. The objective response rate (ORR; complete [CR] + partial response [PR]) and disease control rate (DCR; CR + PR + stable disease [SD]) were calculated with two-sided 95% CIs (Clopper–Pearson method).
Statistical analysis
The safety population included all patients who received ≥1 CNSI-Fe dose. The efficacy population was composed of patients with ≥1 posttreatment tumor assessment. AE incidence and drug-related adverse reactions (ADRs) were recorded. SAS® software version 9.4 or higher was used to perform the statistical analyses.
In vitro antitumour effects
To evaluate the antiproliferative effects of CNSI-Fe on cells and investigate the underlying mechanism, 4T1 TNBC cells were seeded in three 12-well plates (5 × 10⁴ cells/well), incubated for 24 h and then divided into nine groups (triplicate wells per group), namely, the control, CNSI control, and seven CNSI-Fe groups, with increasing concentrations of Fe2+ (6.25–400 μg/mL). After the culture medium was removed, fresh medium, CNSI-containing medium, and CNSI-Fe-containing medium ([Fe2+]: 6.25–400 μg/mL) were added to the corresponding plates, followed by a 48-h incubation. The cells were then collected and counted to calculate proliferation rates. For mechanistic studies, cells from the control, CNSI, and CNSI-Fe groups were subjected to comprehensive analyses.
Intracellular iron ions
To measure the amount of intracellular iron ions, the cells were placed in a microwave digestion tube and treated with 2 mL of nitric acid and 0.5 mL of hydrogen peroxide. The mixture was then subjected to microwave digestion at 240 °C for 30 min using an EXPEC 790S instrument (Expec Technology Development Co., Hangzhou, China). After cooling, the digested samples were transferred and diluted to a final volume of 50 mL. The iron ion concentration was subsequently determined using an Agilent 7850 ICP-MS (Agilent Technologies, USA).
Oxidative stress markers
To measure hydroxyl radicals, cells were lysed using an ultrasonic cell disruptor (SCIENTZ-IID, Scientz Biotechnology Co., Ningbo, China). After dimethyl-α-phenyl-N-tert-butylnitrone (DMPO) was added, electron spin resonance (ESR, JES-FA200, JEOL, Japan) analysis was performed.
Oxidative stress markers, including hydrogen peroxide (H₂O₂), peroxidase (POD), GSH, and MDA, were measured according to the instructions provided by the manufacturers of the respective assay kits (Solarbio Biotechnology Co., Beijing, China).
To measure GPX4 activity, the collected cells were completely lysed by using RIPA lysis buffer. The lysates were subsequently centrifuged, and the supernatant was retained. Protein samples were predenated and then loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. After separation by electrophoresis, the protein bands in the gels were electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 1 h, followed by incubation with primary antibodies against GPX4 (Product No. GB124327; Wuhan Servicebio Technology Co., Ltd., China) and β-actin (Product No. GB15001; Wuhan Servicebio Technology Co., Ltd) at 4 °C for 3 h. The membranes were subsequently incubated with a secondary antibody (Product No. GB23301; Wuhan Servicebio Technology Co., Ltd., China) for 30 min. After the membranes were washed, chemiluminescence detection and analysis were performed. Data are presented as the mean ± SD. Statistical significance was defined by using Student’s t test, and p < 0.05 was considered significant.
Biological safety
In the 4T1 TNBC tumor-bearing mouse model, CNSI-Fe was administered via intratumoural injection twice. The mice in the control and CNSI-Fe groups were euthanized 21 days after the first administration of CNSI-Fe. Whole blood was collected for hematological parameter analysis by using a fully automatic blood cell analyzer (BC-2800vet, Mindray, China). The collected serum samples were utilized for biochemical index analysis by using a fully automatic biochemical analyzer (Chemray 800, Rayto, China). Simultaneously, the livers, hearts, lungs, spleens, and kidneys of mice were harvested, fixed in 10% (v/v) formalin, embedded in paraffin, sectioned, and finally stained with hematoxylin and eosin (H&E). The sections were then examined and photographed by an Eclipse Ci-L optical microscope (Nikon, Japan) for histopathological analysis.
Antitumour effects of CNSI-Fe in vivo
CNSI-Fe treatment of COLO205 colon cancer, HCC1954 breast cancer and MDA-MB-231 triple-negative breast cancer
COLO205 colon cancer, HCC1954 breast cancer, and MDA-MB-231 TNBC cells were inoculated into the right upper limbs of BALB/c nude mice. When the tumor volume reached 100–150 mm³, the mice were divided randomly into 6 groups (10 each), namely, the control group, CNSI group, 5‑fluorouracil (5-Fu)- or paclitaxel (PTX)-positive control group, and low-, medium-, and high-dose CNSI-Fe groups. The tumors were intratumourally injected with 50 μL of normal saline, 50 μL of CNSI, or 50 μL of CNSI-Fe. The concentration of CNSI was 50 mg/mL, and the ferrous ion concentrations of CNSI-Fe were 3.75, 7.5, and 15 mg/mL. The injections were administered twice per week for a total of two doses. The 5-Fu group received intraperitoneal injections at a dosage of 15 mg/kg for 21 consecutive days. The PTX group received intravenous injections at a dosage of 15 mg/kg once per week for a total of three doses. The tumor volume was measured twice per week after treatment using the formula: tumor volume = length × width²/2. The data are presented as the means ± SEMs.
CNSI-Fe treatment of Mia PaCa-2, Capan-2 and AsPC-1 pancreatic cancer cells
Mia PaCa-2, Capan-2, and AsPC-1 pancreatic cancer cells were separately inoculated into the right upper limbs of BALB/c nude mice. When the tumor volume reached 100–150 mm³, the mice were divided randomly into 4 groups, namely, the control group, PTX plus gemcitabine (GEM)-positive control group, medium-dose group, and high-dose CNSI-Fe groups (8 mice each). The tumors were intratumourally injected with 50 μL of normal saline or 50 μL of CNSI-Fe. The ferrous ion concentrations of CNSI-Fe were 7.5 and 15 mg/mL. The injections were administered twice per week for a total of two doses. The PTX plus GEM group received intravenous injections of PTX at a dosage of 20 mg/kg and intraperitoneal injections of GEM at a dosage of 120 mg/kg once per week for a total of four doses. The tumor volume was measured twice per week after treatment using the formula: tumor volume = length × width²/2. The data are presented as the means ± SEMs.
Flow cytometry detection
CT26. WT colon cancer tumor-bearing mice were intratumourally injected with CNSI-Fe twice. On day 17 after the first administration of CNSI-Fe, the mice in the control and CNSI-Fe groups were euthanized. Tumors were collected and prepared into single-cell suspensions for flow cytometry staining with corresponding antibodies (as shown in Supplementary Table 6). After staining, the samples were analyzed on a flow cytometer (FACS LRSfortessa™, BD, USA). FACS data were evaluated using Kaluza 2.1 software to assess the levels of relevant immune cell infiltrates in the samples.
PK detection after administration of CNSI-Fe
CT26. WT colon cancer tumor-bearing mice were divided into three groups and intratumourally injected with CNSI-Fe at volumes of 30 μL, 50 μL, and 60 μL. The ferrous ion concentration of CNSI-Fe was 15 mg/mL. Serum samples were collected from the mice before administration and at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h after administration. The iron ion concentrations in the serum were detected using ICP-MS (8900; Agilent Technologies, USA), and drug concentration-time curves were plotted.
Animal experiment ethical approval
All animal experiments described in this study were conducted in accordance with the relevant institutional guidelines and regulations, and were formally approved by the Institutional Animal Care and Use Committees (IACUCs) of Shanghai Medicilon Inc. (Approval Nos.: YRY2101P, YRY2103P, YRY2104P, YRY2110P, YRY2111P, and YRY2112P), Crown Bioscience (Taicang) Co., Ltd. (Approval No.: AN-2204-05-726), West China-Frontier Pharma Tech Co., Ltd. (Approval No.: IACUC-A2019012-K001-03), and Sichuan Enray Pharmaceutical Sciences Company (Approval No.: YRLL-2022-A05).
