Effect of surface modification on the distribution of magnetic nanorings in hepatocellular carcinoma and immune cells

Abstract

Magnetic nanomaterial-mediated magnetic hyperthermia is a localized heating treatment modality that has been applied to treat aggressive cancer in clinics. In addition to being taken up by tumor cells to function in cancer therapy, magnetic nanomaterials can also be internalized by immune cells in the tumor microenvironment, which may contribute to regulating the anti-tumor immune effects. However, there exists little studies on the distribution of magnetic nanomaterials in different types of cells within tumor tissue. Herein, ferrimagnetic vortex-domain iron oxide nanorings (FVIOs) with or without the liver-cancer-targeting peptide SP94 have been successfully synthesized as a model system to investigate the effect of surface modification of FVIOs (with or without SP94) on the distribution of tumor cells and different immune cells in hepatocellular carcinoma (HCC) microenvironment of a mouse. The distribution ratio of FVIO-SP94s in tumor cells was 1.3 times more than that of FVIOs. Immune cells in the liver tumor microenvironment took up fewer FVIO-SP94s than FVIOs. In addition, myeloid cells were found to be much more amenable than lymphoid cells in terms of their ability to phagocytose nanoparticles. Specifically, the distributions of FVIOs/FVIO-SP94s in tumor-associated macrophages, dendritic cells, and myeloid-derived suppressor cells were 13.8%/12%, 3.7%/0.9%, and 6.3%/1.2%, respectively. While the distributions of FVIOs/FVIO-SP94s in T cells, B cells, and natural killer cells were 5.5%/0.7%, 3.0%/0.7%, and 0.4%/0.3%, respectively. The results described in this article enhance our understanding of the distribution of nanomaterials in the tumor microenvironment and provide a strategy for rational design of magnetic hyperthermia agents that can effectively regulate anti-tumor immune effects.

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1. Introduction

Magnetic nanomaterial-mediated magnetic hyperthermia therapy (MHT) has emerged as a promising treatment modality for deep tumors. Specifically, under an alternating magnetic field (AMF), magnetic iron oxide nanoparticles can generate localized heat through magnetic hysteresis.^1–4 Compared with traditional thermal therapies, MHT has the unique advantages of deep tissue penetration, high selectivity, and minimal toxic effects.^5–7 The most attractive point is that MHT could effectively activate anti-tumor immunity,^8–12 which resulted from the dual action of magnetothermal and enhanced reactive oxygen species (ROS) effect.

At present, MHT focuses on (1) how to improve the magnetothermal conversion efficiency of magnetic nanomaterials,^13–16 (2) how to improve the internalization amounts of magnetic nanomaterials inside tumor cells,^17–20 and (3) how to improve the anti-tumor immune effects, in fighting tumor cells.^21–24 All these studies highlighted the tumoricidal roles of therapeutic heating located inside tumor cells and the induction of a strong immune response initiated by provoking immunogenic tumor cell death. For example, in our previous studies, ferrimagnetic vortex-domain iron oxide nanorings (FVIO)-mediated MHT enable the induction of immunogenic tumor cell death when FVIOs enter the tumor cells by endocytosis, further enhancing the infiltration of T lymphocytes to activate the adaptive immunity.^9,11,12,21 Emerging evidence has revealed that MHT is not solely dependent on its direct heating or bio-regulation effects on tumor cells. Zanganeh et al. discovered that ferumoxytol nanoparticles could be engulfed by tumor-associated macrophages (TAMs) and further induced a polarization from the anti-inflammatory M2 TAM to the pro-inflammatory M1-mode.²⁵ Inspired by this, we found that FVIOs after being internalized by TAMs in the tumor microenvironment (TME) could polarize M2 TAM into M1-mode macrophages under AMF exposure.^10,11 Indeed, TME is a sophisticated spatiotemporal interaction that occurs among heterogeneous cell types, including malignant and immune cells.^26–28 Park et al. also reported that internalized magnetic nanomaterials did not affect the maturation of dendritic cells, while expression of the CD40 protein was attenuated on the surface of dendritic cells.²⁹ These findings suggest that the internalization of magnetic nanoparticles by immune cells in the TME would affect their immune function. Thus, it is urgent to elucidate the cellular-level distribution of magnetic nanoparticles in vivo, particularly within tumor cells or immune cells in the TME. This is an essential precondition for further studying the induction effect of magnetic nanomaterial-mediated MHT on anti-tumor immunity towards various types of immune cells.

Herein, FVIO with a unique vortex-domain magnetic structure³⁰ was employed as a model magnetic hyperthermia agent, as it exhibited (1) higher magnetothermal efficiency and (2) stronger anti-tumor immunity compared to clinically used superparamagnetic iron oxide nanoparticles.³¹ SP94 is a 12-amino-acid peptide, which could specifically bind to the plasma membrane of HCC cells in vivo.^32–34 FVIO with or without SP94 was prepared to investigate the effect of surface coating on the distribution of the magnetic hyperthermia agent in tumor cells and immune cells, via a multiplex immunofluorescence technique. The goal of this study is to gain insight into the surface modification (with or without targeting peptide) of the magnetic hyperthermia agent and the effect of its distribution inside tumor cells, dendritic cells (DCs), macrophages, myeloid-derived suppressor cells (MDSCs), T cells, B cells, and natural killer (NK) cells, as well as provide a general strategy to optimize the surface coating of magnetic hyperthermia agent for enhancing the distribution amounts in immune cells and provoking strong anti-tumor immunity.

2. Materials and methods

2.1. Materials

Ferric chloride hexahydrate (>99.0%) and sodium chloride were purchased from Kemiou Chemical Reagent Co., Ltd (Tianjin, China). Trioctylamine, fluorescein isothiocyanate (FITC), dopamine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Ammonium dihydrogen phosphate and fluorescein isothiocyanate (FITC), hexane, oleic acid (90%), and phenanthroline were purchased from Aladdin Reagent Co., Ltd (Shanghai, China). Absolute ethanol, dimethylsulfoxide, and tetrahydrofuran were purchased from Damao Chemical Reagent Factory (Tianjin, China). Liver-cancer-targeting peptide SP94 (SFSIIHTPILPL) was purchased from Synpeptide Co., Ltd (Nanjing, China). Paraformaldehyde, phosphate-buffered saline (PBS), and Cell Counting Kit-8 (CCK-8) were purchased from Solarbio Biotechnology Co., Ltd (Shanghai, China). Heat-inactivated fetal bovine serum (FBS), trypsin, and penicillin–streptomycin solution were purchased from Thermo Fisher Scientific Inc. (MO, USA). Cell culture medium was purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China). Pentobarbital sodium was purchased from Servicebio Technology Co., Ltd (Wuhan, China). Anti-CD45 antibody, anti-CD3 antibody, anti-NKR-P1C antibody, anti-CD11b antibody, anti-CD11c antibody, anti-F4/80 antibody, and anti-(LY6G/C) antibody were purchased from Abcam (Cambridge, United Kingdom). Anti-B220 antibody was purchased from BioLegend (London, United Kingdom).

2.2. Synthesis of FVIOs

In accordance with a previous report,³⁰ α-Fe₂O₃ nanorings were prepared using a hydrothermal method. Briefly, 0.80 mL of ferric chloride hexahydrate (0.50 M), 0.72 mL of ammonium dihydrogen phosphate (0.02 M), and 0.72 mL of sodium chloride (0.02 M) were added into a high-temperature reaction kettle. Then, deionized water was added to form a solution with a total volume of 40.0 mL, and the mixture was stirred for 10 min. The reaction kettle was sealed, and the reaction was carried out at 220 °C for 2280 min. After the reaction kettle cooled naturally, the precipitate was collected and washed three times with water and ethanol, followed by drying to obtain α-Fe₂O₃ nanorings. The dried α-Fe₂O₃ powder was reduced at 450 °C in a tube furnace for 2 h under 5% H₂/95% Ar. The black powder obtained at the end of the reaction was FVIOs.

2.3. Synthesis of hydrophilic FVIOs

The FVIO powder (20 mg) and 10 g of trioctylamine were mixed by ultrasonication and added to a 50 mL three-necked bottle. Then, 0.4 mL of oleic acid (0.81 g) was added as a surfactant, the temperature was rapidly increased to 280 °C, and stirring was maintained under a constant flow of argon for 50 min. After the reaction mixture was cooled to room temperature, the products were separated by centrifugation and washed three times with hexane and absolute ethanol. The purified oleic acid-coated FVIOs were then redispersed in tetrahydrofuran. Dopamine (200 mg) dissolved in 1 mL of deionized water was mixed with 20 mL of tetrahydrofuran and added to a 50 mL three-necked bottle. Then, 1 mL of oleic-acid-coated FVIOs were added to the bottle dropwise under the protection of argon. The solution was stirred at 55 °C for 6 h. The products were washed three times and dispersed in 1 mL of deionized water.

2.4. Synthesis of FVIO-SP94s

FITC (10 mg) dissolved in 1 mL of DMSO was added to a 50 mL centrifuge tube, followed by 4 mL of deionized water. FVIOs (5 mg) in water solution with a concentration of 1 mg mL⁻¹ were mixed with the FITC solution. The pH of the mixed solution was adjusted to 8 with 10% ammonia water, and the reaction was carried out on a shaker for 12 h in a dark room at room temperature. After washing three times, the solution was dispersed in deionized water to obtain the FITC-labeled FVIO solution. Then, 0.1 mmol of EDC and 0.05 mmol of liver-cancer-targeting peptide SP94 were dissolved in 5 mL of PBS solution (pH 7.4) and mixed with 0.2 mmol of NHS at room temperature in a dark room. After 15 min, 0.5 mmol of FVIO-FITC solution was added to the peptide SP94 solution with stirring for 6 h. Finally, the resulting mixture was further purified using a 5000-dialysis membrane. The purified FVIO-SP94s were stored under 4 °C for further use.

2.5. Characterization techniques

The morphology of the samples was observed using a Talos F200X J transmission electron microscope (Thermo Fisher Scientific Inc., USA) operated at 200 kV and a Hitachi SU8010 scanning electron microscope (Hitachi Corp., Japan) operated at 3.0 kV. The hydrodynamic diameters and zeta potential were recorded using a Zetasizer Nano-ZS (Malvern Corp., UK). The surface chemical composition was examined using a Fourier transform infrared spectrometer Tensor 27 (Bruker, Germany). The crystal structure was identified using a D8 Advance powder X-ray diffraction (Bruker, Germany) with a 1.54 Å Cu kα source. The iron concentration of the synthetic solution was determined using inductively coupled plasma mass spectrometry using a 7700X spectrometer (Agilent Technologies Co., Ltd, USA).

2.6. Cell culture

Murine hepatocellular carcinoma Hepa1-6 cells and mouse normal hepatocyte AML12 cells were purchased from the American Type Culture Collection. Based on mycoplasma testing, no contamination was detected in these cell lines. Hepa1-6 and AML12 cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) penicillin–streptomycin in a humidified 5% CO₂ incubator at 37 °C. The cells were trypsinized at room temperature and passaged 2–3 times per week.

2.7. Animal models

All in vivo experimental operations were approved by the Animal Ethical and Welfare Committee of Northwest University (Xi’an, China) and performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals and the institutional ethical guidelines for animal experiments. C57BL/6 mice (6 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). To establish subcutaneous Hepa1-6 HCC tumor models in the mice, 1 × 10⁷ cells were injected into the right posterior flank of the mice using a syringe. All mice were housed under specific pathogen-free conditions (temperature ∼22 °C, humidity ∼50%) with a 12/12 h dark/light cycle.

2.8. In vivo biodistribution

The tumor growth and body weight of mice were recorded every 3 days. The tumor size was measured using a sliding caliper across its longest (L) and shortest (W) diameters, and tumor volume was calculated using the following formula: volume = 0.5 × L × W². When the tumor volume reached approximately 200 mm³, 10 mice were randomly divided into two groups. FVIOs or FVIO-SP94s labeled with FITC were injected intravenously at a dose of 5 mg kg⁻¹ body weight (5 mice per group). Then, 24 h after injection, the mice were sacrificed, and tumors were removed in a dark room.

2.9. Tumor tissue sectioning

The removed tumor tissue was rinsed in normal saline and placed in a brown plastic bottle. Paraformaldehyde (4%) was added to the bottle to fix the tissue. After 3 days, the tumor tissue was dehydrated using alcohol of progressively increasing concentration. The tumor tissue was then made transparent with xylene. The transparent tissue was soaked in melted paraffin at about 62 °C for embedding. After cooling and solidification, the tissue was cut into 5-μm thick sections, which were placed in hot water to flatten, pasted onto slides, and dried in a 45 °C incubator.

2.10. Multiplex immunofluorescence

For multiplex immunofluorescence staining, 5 μm sections of tumor tissue were stained using tyramide signal amplification (TSA). First, deparaffinization, rehydration, and permeabilization were performed on all slides, followed by 20 min of 10% formalin fixation and 15 min of Tris-EDTA antigen retrieval at high temperature and high pressure. Afterwards, the slides were incubated with primary antibodies, secondary-HRP antibodies, and TSA dyes for 16 h (4 °C), 10 min (25 °C), and 20 min (25 °C), respectively. DAPI was used for nuclear counterstaining after the antibody staining. The slides were finally mounted with an antifade reagent. For all markers in the experiment, it was necessary to prepare tumor sections for preliminary staining to establish a single-color spectral database. After antibody prestaining, new tumor sections were used for multiplex immunofluorescence staining. After staining with the first antibody, the second antibody was applied, and so on, until finally the nucleus was stained. Tissue FAXS imaging software (v7.134) was used to capture images and identify all markers of interest. Multispectral fluorescence images were selected and quantitatively analyzed using Strata Quest software (Tissue Gnostics, v7.0.0). Tumor and immune cells in the Hepa1-6 tumor tissue were stained for the cell markers shown in Table 1. We employed the CD45 marker to identify immune cells, the primary subject of our research. Notably, the CD45⁻ population may include not just tumor cells but also other types such as cancer-associated fibroblasts and tumor endothelial cells, which typically support tumor growth without being directly involved in immune responses. Therefore, for the scope of this research, we have tentatively categorized CD45⁻ cells as tumor cells to keep our focus on the dynamics of immune cells. Gating strategies for multiplex immunofluorescence analysis are shown in Fig. S1 (ESI†).

Table 1 Markers of different types of cells in the TME

Marker	Cell Type
CD45^+ CD3^+	T cells
CD45^+ B220^+	B cells
CD45^+ CD3^− NKR-P1C^+	NK cells
CD45^+ CD11b^+ F4/80^+	TAMs
CD45^+ CD11b^+ Ly6G/C^+	MDSCs
CD45^+ CD11c^+	DCs
CD45^−	Tumor cells

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2.11. Cell cytotoxicity assay

A CCK-8 assay was used to evaluate the cytotoxicity of the synthesized nanoparticles to Hepa1-6 cells and AML12 cells. Both types of cells were seeded into 96-well plates (5 × 10³ cells per well) and cultured overnight. The cells were then cultured for 8 h in media containing nanoparticles of the indicated iron concentration (0, 10, 25, 50, 75, and 100 g L⁻¹). Subsequently, 10 μL CCK-8 solution was added to each well and incubated for an additional 2 h. Cell viability was assessed by measuring the absorbance at 450 nm (Thermo Fisher Scientific, Wilmington, DE, USA). Data are expressed as a percentage of the survival of viable cells versus the survival of the control cells (untreated cells served as a control for 100% viability).

2.12. In vivo safety evaluation

Healthy Sprague-Dawley (SD) rats (6 weeks of age, 180–200 g), randomly divided into three groups (n = 3), received 200 μL of saline or saline containing FVIOs or FVIO-SP94s (5.0 mg Fe per kg body weight) intravenously. Serum samples were collected 1 day and 14 days after injection. Parameters for determining liver function (ALT: alanine aminotransferase, AST: aspartate aminotransferase) and kidney function (BUN: blood urea nitrogen and CREA: creatinine) were measured using a Hitachi 7100 blood biochemistry automatic analyzer 7100 (Hitachi Corp.). After 14 days of material injection, all rats were euthanized. The heart, kidneys, liver, lungs, and spleen were collected after 14 d for hematoxylin and eosin staining. The sections were observed for significant pathological changes using an optical microscope (AMG EVOS xl core, Life Technologies, USA).

2.13. Statistical analysis

Data are represented as the mean ± standard deviation (±sd), as indicated in the figure legends. One-way ANOVA with Tukey's multiple comparisons was used for multiple comparisons when more than two groups were compared, and the two-tailed Student's t-test was used for two-group comparisons.

The distribution of FVIOs in the different types of cells in the sections is evaluated by the calculation method listed below:

C_n is the count of a certain type of cells, F_n is the mean FITC fluorescence intensity in these cells. C is the total number of all cells, and F is the mean FITC fluorescence intensity in all cells.

3. Results and discussion

Transmission electron microscope (TEM) images (Fig. 2A) and scanning electron microscope (SEM) images (Fig. S2, ESI†) showed that the obtained FVIOs had a ring-like morphology. The FVIOs were 58.53 ± 7.87 nm in mean outer diameter based on Gaussian fitting analysis (Fig. 2B). The powder X-ray diffraction (XRD) patterns of as-prepared FVIOs (Fig. 2C) exhibited a typical inverse spinel pattern, consistent with the standard spinel Fe₃O₄ diffraction pattern card (JCPDS No. 19-0629).

To confirm the successful conjugation of SP94 on the surface of FVIOs, we performed FTIR spectroscopy analysis of the freeze-dried samples (Fig. 2D). Strong adsorption at 572 cm⁻¹ due to Fe–O stretching was observed in the spectra for both FVIOs and SP94-modified FVIOs. Peaks at 3442 cm⁻¹ (ascribed to the –N–H– stretching vibration of amides), 1640 cm⁻¹ (ascribed to the overlap of C=O stretching vibrations and amide N–H bending vibrations), 1081 cm⁻¹ and 1154 cm⁻¹ (ascribed to C–H bending vibrations) appeared on the spectrum for FVIO-SP94s. These were caused by reactions between the carboxyl group of SP94 and the amine group of dopamine (Fig. 1A), which demonstrated the successful addition of SP94 onto FVIOs.

Fig. 1 Schematic illustration of FVIOs and FVIO-SP94s preparation and distribution in mouse HCC tumor tissue. (A) FVIOs and FVIO-SP94s preparation process. (B) Distribution of FVIOs or FVIO-SP94s in mouse Hepa1-6 HCC tissues.

Similar results were observed for the variations in hydrodynamic size and zeta potential. The hydrodynamic size of FVIOs increased from 81.1 nm to 86.1 nm in deionized water after conjugation with SP94 (Fig. 2E). Simultaneously, the zeta potential of FVIO-SP94s samples changed from 28.5 ± 1.5 mV to 13.1 ± 1.2 mV (Fig. 2F). These results suggested that SP94 was successfully grafted onto the FVIOs.

Fig. 2 Characterization of FVIOs and FVIO-SP94s. (A) TEM image of FVIOs. Scale bar: 50 nm. (B) Size distribution of FVIOs. (C) XRD patterns of FVIOs. (D) FTIR spectra of FVIOs (black) or FVIO-SP94s (red). (E) Hydrodynamic size and (F) zeta-potential of FVIOs and FVIO-SP94s, respectively.

As-synthesized FVIOs or FVIO-SP94s labelled with FITC were injected into mice through the tail vein, then 24 h after injection, the mice were sacrificed, and tumors were removed in a dark room. The distribution of samples in different types of cells within the Hepa1-6 subcutaneous tumor of a mouse was studied via a multiplex immunofluorescence technique.

To analyze the distribution of FVIOs more accurately, the tumor sections were divided into two groups, one with six fluorescence colors and another with seven fluorescence colors. Six-color sections were used to identify tumor cells and lymphoid cell lineages, including B cells, T cells, and NK cells. Seven-color sections were used to identify tumor cells and myeloid cells, including DCs, TAMs, and MDSCs. FVIOs were labeled with green fluorescence by FITC (Fig. S3, ESI†). The proportion of different types of cells in Hepa1-6 tumor tissue was first studied. We used the software Strata Quest (Tissue Gnostics) for image analysis. In the mouse Hepa1-6 tumor model, cancer cells were the most abundant cell type in the TME. For three major lymphocytes in the Hepa1-6 tumor tissue section (Fig. 3A), the proportions of B cells, T cells, and NK cells were 0.28%, 0.12%, and 0.43%, respectively. The proportions of myeloid cells including TAMs, MDSCs, and DCs in the Hepa1-6 tumor tissues are shown in Fig. 3B. TAM (19.3%) was the most abundant of the three myeloid cell types. The proportion of DCs and MDSCs was 0.8% and 1.3%, respectively. Compared to different cell types, the counts of the major lymphocytes in the TME were less than major myeloid cells in Hepa1-6 tumor tissues.

Fig. 3 The proportions of different cell types in the Hepa1-6 tumor tissues. (A) The proportions of tumor cells (CD45⁻), B cells (CD45⁺B220⁺), T cells (CD45⁺CD3⁺), and NK cells (CD45⁺CD3⁻NKR-P1C⁺) in the six-color images. (B) The proportions of tumor cells (CD45⁻), DCs (CD45⁺ CD11c⁺), TAMs (CD45⁺CD11b⁺F4/80⁺), and MDCSs (CD45⁺CD11b⁺Ly6G/C⁺) in the seven-color images.

Next, we investigate the distribution of FVIOs with or without SP94 in different lymphocytes. Photographs of the six-color fluorescence sections showing the co-localization of B cells (CD45⁺B220⁺), T cells (CD45⁺CD3⁺), and NK cells (CD45⁺CD3⁻NKR-P1C⁺) with FVIOs or FVIO-SP94s (green fluorescence) in Hepa1-6 tumor tissue are shown in Fig. 4A and B, respectively. The proportion of FVIO-SP94s in tumor cells was 87.1%, which was 1.3 times higher than that of FVIOs (67.2%; p < 0.05) (Fig. 4C). In the FVIOs group, the highest percent of FITC events was within CD45⁻ tumor cells (67.2%), and this percentage was significantly higher than those in B cells (3.0%, p < 0.0001), T cells (5.5%, p < 0.0001), and NK cells (0.4%, p < 0.0001) (Fig. 4D). The distribution of nanomaterials exhibited the same trend in the FVIO-SP94s group; the highest percent of FITC events was found within CD45⁻ tumor cells (87.1%), and this percentage was significantly higher than that in B cells (0.7%, p < 0.0001), T cells (0.7%, p < 0.0001), and NK cells (0.3%, p < 0.0001) (Fig. 4E).

Fig. 4 Distribution of FVIOs and FVIO-SP94s in the TME of Hepa1-6 tumor tissues. (A) Fluorescence images of FVIOs (A) or FVIO-SP94s (B) with B cells, T cells, and NK cells, in six-color sections. Scale bar: 50 μm. (C) Distribution of the two nanoparticle types in tumor cells. Distributions of FVIOs (D) and FVIO-SP94s (E) in tumor cells and lymphoid cells. To determine the differences within the groups and between groups, statistical analysis was conducted via one-way ANOVA with Tukey's multiple comparison tests for multiple groups or the student's T test for two groups. To be considered statistically significant, the P value should be <0.05. The range of P values is indicated by the number of asterisks, i.e., *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001; ****P < 0.0001.

Previous studies have shown that nanoparticles can affect the proliferation and function of T cells in vitro, but only at high concentrations.³⁵ Our results demonstrated that the distribution of FVIOs in T cells was reduced by adding the SP94 peptide. After Parra et al. found that B cells from fish, amphibians, and reptiles have significant phagocytic ability, researchers realized that B cells may also phagocytose nanoparticles.^36,37 The distribution of FVIOs in B cells changed from 3% to 0.7% after modifying the FVIOs with the SP94 peptide. A likely explanation is that, as more SP94-labeled nanoparticles were endocytosed by tumor cells, the probability of B cells contacting nanoparticles declined. Once activated, NK cells can rapidly recognize and kill tumor cells.³⁸ The results showed that the distribution of both FVIOs and FVIO-SP94s in NK cells was less than 1%. FVIOs might be detrimental to the function of NK cells as cancer cell killers. However, to design nanoparticles for the regulation of lymphocyte function in tumors, especially NK cells, it is obviously critical to endow nanoparticles with the ability to elevate the accumulation level in the target lymphocytes.^39,40

Then, we investigate the distribution of FVIOs with or without SP94 in different myeloid cells. Photographs of seven-color fluorescence sections showing DCs, TAMs, MDSCs and their co-localization with FVIOs (FITC⁺) are shown in Fig. 5A and B, respectively. We evaluated the distribution of FITC⁺ events in the different types of myeloid cells in the seven-color sections. At 24 h post-injection, the distribution of FVIOs and FVIO-SP94s in tumor cells were 51.5% and 65.8%, respectively (Fig. 5C). The distribution of FVIO-SP94s in tumor cells was 1.3-fold higher than that of FVIOs. This higher proportion of FVIO-SP94s, as well as the results of the six-color sections (Fig. 4C), again suggested that most of the nanoparticles tended to be taken up by the tumor cells in Hepa1-6 tumor tissues. The data also indicated that the modification of FVIOs with SP94 enhanced the concentration of the nanoparticles in tumor cells.

Fig. 5 Distribution of FVIOs and FVIO-SP94s in TME of Hepa1-6 tumor tissues (A) Fluorescence images of FVIOs (A) or FVIO-SP94s (B) with DCs, TAMs, and MDSCs, in seven-color sections. Scale bar: 50 μm. (C) Distribution of the two nanoparticles in tumor cells. Distribution of FVIOs (D) and FVIO-SP94s (E) in tumor cells and myeloid cells. To determine the differences within the groups and between groups, statistical analysis was conducted via one-way ANOVA with Tukey's multiple comparison tests for multiple groups or a student's T test for two groups. To be considered statistically significant, the P value should be <0.05. The range of P values is indicated by the number of asterisks, i.e., “ns”, no significant difference, *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001; ****P < 0.0001.

The distributions of the two types of nanoparticles in myeloid immune cells are shown in Fig. 5D and E. TAMs were the myeloid cells with the highest uptake of nanomaterials, and they were also hardly affected by the addition of SP94 of all the above immune cells, which could be related to the large number and strong phagocytic ability of macrophages. Approximately 13.8% of FVIOs and 12.0% of FVIO-SP94s were distributed in TAMs, both above 10%. As the most abundant innate immune cell in the TME, TAMs are significantly associated with poor prognosis.^41,42 Taking advantage of the high phagocytic activity of macrophages, labeling macrophages with intravenously administered nanoparticles can be used to quantitatively monitor the TAM load in tumors.⁴³ In recent years, with ever deeper research into macrophages, it has been found that iron oxide nanoparticles can cause TAM polarization to the M1 type and promote the tumor immune response.¹¹ This suggests that TAMs are a potential target of FVIOs, and FVIO-mediated TAM repolarization should be conducive to tumor therapy.

Furthermore, 3.7% of FVIOs and 0.9% of FVIO-SP94s were detected in DCs. Although DCs have phagocytic functions, their more important role is as tumor-associated antigen-presenting cells.⁴⁴ The results of this experiment could potentially be attributed to the effect of the tumor-targeting peptide, which led to the material being more concentrated in tumor cells and in a significantly lower concentration in DCs.

The percentages of FVIOs and FVIO-SP94s in MDCSs were 6.3% and 1.2%, respectively. MDSCs have a strong immunosuppressive function, and their large-scale expansion occurs during tumor development.^45,46 Hence, targeting MDSCs with nanoparticles may have favorable effects during a period when MDSCs are more abundant in tumors.

Determining the fate of nanoparticles is important for regulatory purposes because of concerns about chronic accumulation and patient safety. We evaluated the potential cytotoxicity of FVIOs and FVIO-SP94s in vitro using CCK8 assay. The viability of mouse hepatoma (Hepa1-6) cells and mouse normal hepatocyte (AML12) cells was not significantly affected after incubation with different concentrations of FVIOs or FVIO-SP94s for 8 h (Fig. S4, ESI†).

We also investigated the in vivo biosafety of the two materials by injecting them into the tail veins of non-tumor-bearing healthy SD rats at a dose of 5 mg kg⁻¹ Fe. Rats injected with the same volume of saline were used as a control group. We additionally performed blood biochemical tests 1 day and 14 days after intravenous injection of the nanoparticles. Functional indicators for the liver and kidney, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), carbamide (UREA), and creatinine (CREA), showed no significant differences between the control and nanoparticle-injection groups (Fig. 6A–D).

Fig. 6 Biosafety of FVIOs and FVIO-SP94s. Results of blood biochemical assessments. (A) ALT, (B) AST, (C) UREA, (D) CREA in SD rats 1 day and 14 days after nanoparticle injection. (E) H&E staining of hearts, livers, spleens, lungs, and kidneys of treated and control rats, scale bar: 100 μm.

H&E staining of the hearts, livers, spleens, lungs, and kidneys from treated rats showed no abnormalities in cellular morphology compared with those from control rats (Fig. 6E). These results demonstrated that the well-designed FVIOs and FVIO-SP94s showed no observable cytotoxicity or in vivo toxicity.

4. Conclusion

This study elucidated the distribution of FVIOs and FVIO-SP94s in different cell types within Hepa1-6 tumor tissue of mouse subcutaneous tumor models. The results showed that the distribution of the different cell types in the tumor tissue was dependent on the surface modification of the FVIOs. In the Hepa1-6 subcutaneous tumor models, there are many more tumor cells than immune cells. Therefore, more than 50% of the nanoparticles were concentrated in tumor cells 24 h after injection, regardless of whether the FVIOs were modified with the HCC-specific targeting peptide SP94 or not. The concentration of FVIOs in the tumor cells was further enhanced after modification with SP94, which was 1.3 times than that of the FVIOs group. Specifically, six kinds of immune cells were studied, including B cells, T cells, NK cells, DCs, TAMs, and MDSCs. The number of nanoparticles endocytosed was related to the type of immune cell. The ability of myeloid cells to uptake nanoparticles is much stronger than that of lymphocytes. The proportion of FVIOs distributed in immune cells decreased after SP94 modification. This indicates that the conjugation of tumor-targeting peptides is a better choice in the formulation of nanoparticles designed to interact with tumor cells. A better understanding of the mechanistic basis for the distribution of nanoparticles will be helpful for the future surface modification design of magnetic hyperthermia agents to manipulate the function of immune cells. Ultimately, the FVIO distributions characterized here could provide essential information for future MHT or even other nanomaterial-related medical studies.

Author contributions

The contributions are presented in a non-sequential manner. Conceptualization (Xiaoli Liu and Nan Zhang), investigation (Wangbo Jiao, Nana Wen, Siyao Wang, and Guxiang Zhou), methodology (Wangbo Jiao, Nana Wen, Siyao Wang, Shuwei Hu and Qiaoyi Lu), visualization (Zijun Su and Xinxin Wang), writing – original draft (Wangbo Jiao, Nana Wen and Xiaoli Liu), writing – review and editing (Xiaoli Liu, Youbang Xie, and Nan Zhang), supervision (Xiaoli Liu and Nan Zhang).

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (Grant number: 2022YFC2408000), the National Natural Science Foundation of China (NSFC) for Excellent Young Scientists (Grant number: 82322039), NSFC projects (Grant numbers: 82072063, 32001005, 32101136, and 82202306), the Key Research and Development Program of Shaanxi Province (Grant number: 2023-YBSF-132), the Shaanxi Province Youth Science and Technology New Star (Grant number: 2022KJXX-09), the Natural Science Foundation of Shaanxi Province (Grant numbers: 2020JQ610), the Medical-Engineering Cross Project of the First Affiliated Hospital of Xi’an Jiaotong University (Grant number: QYJC02), and the Science Foundation of Nanjing Chia Tai Tianqing Project (Grant number: TQ202215).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3tb02560h

‡ These authors contributed equally.

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