Yonghyun Choi,Wooic Son,Youngpin Han,Jayoung Chae,Chul-Su Yang,Jonghoon Choi
Glycan targeting nanoparticle for photodynamic immunotherapy of melanoma
Immunotherapy,Macrophages,Phagocytosis,Lectin,Melanoma cancer,Photothermal nanoparticles,Immune checkpoint,Glycan
Interaction between tumour cells and macrophages enables cancer cells to evade immune detection and clearance by interfering with macrophage phagocytosis. The anti-phagocytic signals regulated by anti-phagocytic proteins are termed “don't eat me” signals; these signals include sialic acid-binding immunoglobulin-type lectin-10 (Siglec-10) and the recently revealed CD24 immune checkpoint (ICP). In this study, we demonstrate that targeting a specific glycan on CD24 exhibits the potential to inhibit ICP. Sambucus nigra agglutinin (SNA), a sialic acid-binding lectin, was employed to block CD24 and to enhance phagocytosis in melanoma tumours. In addition, we prepared SNA-conjugated hollow gold-iron oxide nanoparticles for photothermal therapy of tumours. Our findings show that the combination treatment of SNA-conjugated photothermal nanoparticles and near-infrared exposure successfully augments tumour cell phagocytosis both in vitro and in vivo models.
Glycan targeting nanoparticle for photodynamic immunotherapy of melanoma

Interaction between tumour cells and macrophages enables cancer cells to evade immune detection and clearance by interfering with macrophage phagocytosis. The anti-phagocytic signals regulated by anti-phagocytic proteins are termed “don't eat me” signals; these signals include sialic acid-binding immunoglobulin-type lectin-10 (Siglec-10) and the recently revealed CD24 immune checkpoint (ICP). In this study, we demonstrate that targeting a specific glycan on CD24 exhibits the potential to inhibit ICP. Sambucus nigra agglutinin (SNA), a sialic acid-binding lectin, was employed to block CD24 and to enhance phagocytosis in melanoma tumours. In addition, we prepared SNA-conjugated hollow gold-iron oxide nanoparticles for photothermal therapy of tumours. Our findings show that the combination treatment of SNA-conjugated photothermal nanoparticles and near-infrared exposure successfully augments tumour cell phagocytosis both in vitro and in vivo models.

Among the strategies used for tumour treatment, most of the anti-tumour reagents used in chemotherapy modulate tumour proliferation and induce tumoricidal effects1, 2, 3. However, as cancer cells are influenced by the tumour microenvironment, neighbouring cells, and angiogenesis within tumour tissue, conventional anti-tumour reagents tend to decrease toxicity rather than enhance therapeutic efficacy,. To overcome these obstacles, recent studies have actively identified the tumour microenvironment and the mechanism of anti-tumour treatment. In particular, understanding the interactions between tumour cells and neighbouring cells is pivotal for manipulating the tumour-inhibitory or tumour-promoting effects in anti-tumour therapies,. Abnormal cells, such as tumour cells, are recognised as antigens by immune cells within the body and are scavenged by the immune system9, 10, 11. However, tumour cells can escape from immune surveillance, a process that functions to detect and eliminate neoplastically-transformed cells. This immune evasion process interferes with immune recognition by immune cells, such as macrophages and natural killer (NK) cells,. The M2 phenotype—the most common phenotype of tumour-associated macrophages (TAMs)—promotes tumour progression and reduces the efficacy of chemotherapy. Additionally, during migration and invasion of tumour cells, TAMs can be polarised to the M2 phenotype (M2-TAMs) by interleukin-4 (IL-4), which is secreted from CD4+ T cells, and this plays a role in assisting the migration and metastasis of tumour cells. Moreover, tumour cells that interact with macrophages can activate immune checkpoints (ICPs) to evade immune surveillance,. Macrophage phagocytosis is a crucial component of the innate immune system response to tumour cells; however, activation of ICPs (representative ICPs were CD47 and SIRP α) as a result of interactions between tumour cells and macrophages inhibits macrophage-mediated phagocytosis, thus serving as a “don't eat me” signal. The interaction between sialic acid-binding immunoglobulin-type lectin-10 (Siglec-10) and CD24 is a representative mechanism related to the anti-phagocytic effect,. CD24 is a tumour cell surface marker that exists as a sialylated glycoprotein that binds to Siglec-10 on the surface of macrophages in proximity to tumour tissue. The majority of ICP proteins are glycoproteins. In particular, sialoglycan located on the surface of tumour cells interacts with Siglec protein groups on immune cells, and this protein is primarily involved in the activation or suppression of immune surveillance. Therefore, sialic acid-targeting could be considered a significant factor in tumour immunotherapy and possesses the potential for use as an immune checkpoint inhibitor (ICI). Previous studies have reported that sialic acid expression is suppressed by the delivery of sialyltransferase inhibitors into tumour cells, thereby increasing immune cell activity,. Moreover, sialidase conjugation to antibodies targeting tumour-specific proteins was introduced to suppress tumour growth. Our study introduces a lectin protein that exhibits a specific glycan preference for targeting the sialic acid moiety in glycoproteins of tumour cells. Lectin-conjugated photothermal nanoparticles (NPs) were introduced simultaneously for their anti-phagocytic inhibition and photothermal tumour destruction by blocking the sialic acid moiety attached to CD24 (Fig. 1). Sialic acid present on CD24 molecules could be blocked by Sambucus nigra agglutinin (SNA), and the phagocytic efficacy of melanoma cells was increased. Furthermore, synthesised hollow-structure photothermal nanoparticles (hSPION-Au) that are composed of superparamagnetic iron oxide nanoparticles (SPIONs) and gold (Au) induced the inactivation of ICPs and caused tumour death triggered by near-infrared (NIR) irradiation. We demonstrated that when SNA-conjugated hSPION-Au (SNA/hSPION-Au) was used to treat melanoma tumour cells, the sialic acid on CD24 was blocked by SNA, ultimately augmenting phagocytosis. Furthermore, the NIR irradiation-induced synergy with SNA/hSPION-Au in tumour death increased phagocytic activity compared to treatment with lectin alone. Photothermal tumour ablation in the presence of glycan-targeting NPs could demonstrate ICI function (an ICP blockade), followed by significant anti-tumour immune responses to promote effective tumour immunotherapy.

Carboxylated polystyrene (PS) fluorescent NPs (L5530) possessing a mean size of 500 nm (Ex: 520 nm, Em: 540 nm), 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC; E6383), iron(II) chloride tetrahydrate (44939), iron(III) chloride hexahydrate (31232), BSA (A9418), (hydroxypropyl)methylcellulose (H7509), PMA (P8139), lipopolysaccharide (LPS; L2880), gold(III) chloride trihydrate (520918), silver nitrate (S6506), l-ascorbic acid (A5960), KF (402931), Triton X-100 (T9284), 4-morpholineethanesulphonic acid (MES; M3671), phosphatase inhibitor cocktail 2 (P5726) and cocktail 3 (P0044) were all obtained from Sigma–Aldrich (St. Louis, MO, USA). Heterobifunctional polyethylene glycol (SH–PEG–COOH, used as a surface modifier) was obtained from Biochempeg (Watertown, MA, USA). The cell-mounting medium SNA used for cell fixation was purchased from Vector Laboratories (Burlingame, CA, USA). Alexa 488 anti-mouse CD24 antibody, Alexa 647 anti-CD11b antibody, PE anti-CD11b, Pacific blue anti-CD14, FITC anti-CD80 antibody, APC anti-CD206 and PE anti-Siglec-10 antibody were all purchased from BioLegend (San Diego, CA, USA). The bicinchoninic acid (BCA) protein assay kit for protein quantification, rhodamine‒phalloidin (R415), 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI; D1306), calcein-AM (C3100MP), and the LIVE/DEAD® Viability/Cytotoxicity Assay kit (L3224) were all obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). RIPA buffer (5X) with a protease inhibitor cocktail ( × 100) was obtained from Cell Biolabs (San Diego, CA, USA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from VWR Life Sciences (Radnor, PA, USA). Apoptosis and necrosis assay kits were purchased from Invitrogen (Carlsbad, CA, USA). Cytokines, including interferon-gamma (INF-γ), IL-4 and interleukin-13 (IL-13), used for macrophage differentiation and polarisation, were obtained from PeproTech (Cranbury, NJ, USA). Neuraminidase was purchased from Roche (Little Falls, NJ, USA). Water ammonia and N-hydroxysulphosuccinimide were obtained from TCI (Tokyo, Japan).

hSPION-Au was synthesised using the seed growth method. Core−shell NPs (SPION@Au) that consist of SPIONs for the core material and Au for the shell structure were utilised as seed particles to synthesise hSPION-Au. First, SPIONs were synthesised by following a previously reported method. Iron(II) chloride tetrahydrate (FeCl2·4H2O) and iron(III) chloride hexahydrate (FeCl3·6H2O) at a ratio of 2.5:1.55 g, respectively, were dissolved in 100 mL of deionised water (DW), and 12.5 mL of ammonia solution (28%, w/v) was then applied to the reaction batch. After 10 min, sodium citrate (11 g) was added, and then the reaction temperature was raised to 90 °C with continuous stirring (350 rpm, DMSD 633, Misung Scientific Co., Republic of Korea) for 30 min. After cooling the batch, the SPIONs were rinsed twice with DW to remove excess free citrate and ammonia. During rinsing, a sample was separated from the supernatant using a permanent magnet. Finally, the sample was resuspended in 100 mL of DW. For the synthesis of SPION@Au, 20 mL of HAuCl4 (1 mmol/L) was added to the SPION solution (1.5 mL) with 13.5 mL of DW. The reaction batch was then sonicated for 60 min. Sodium borohydride (NaBH4, 10 mmol/L, 500 μL) was added to the reaction batch every 15 min during sonication. After the completion of the reaction, SPION@Au was rinsed twice with DW to remove excess free chemicals and then the synthesised SPION@Au was resuspended in 5 mL of DW. Finally, seed-mediated and surfactant-assisted syntheses were used to synthesise hSPION-Au. The seed SPION@Au was formed using the process described above. The growth solution was prepared with 95 mL of Triton X-100 (100 mmol/L), 100 μL of KF (10 mmol/L), 1 mL of silver nitrate (20 mmol/L), and 5.4 mL of gold(III) chloride trihydrate (10 mmol/L). Then, 1.4 mL of the SPION@Au seed solution was added to the mixture solution. This mixture solution was stored overnight at 24 °C.

For the conjugation of SNA to hSPION-Au, hSPION-Au was functionalised with a carboxyl group. First, hSPION-Au was functionalised with heterobifunctional polyethylene glycol (SH−PEG−COOH) for carboxyl modification. The amine group of the lectin protein and the carboxyl group of nanoparticles were activated by EDC/NHS chemistry. For conjugation, hSPION-Au was resuspended in MES buffer (50 mmol/L, pH 6.0), and SNA was dissolved in MES buffer at 1 mg/mL. The EDC/NHS activation of the carboxyl groups of hSPION to an NHS ester was performed with final concentrations of 322 mmol/L EDC and 579 mmol/L NHS at room temperature for 20 min. Afterwards, the dissolved lectin protein was mixed with activated hSPION-Au under gentle stirring for 3 h and then washed twice in cold DW by centrifugation (12,000×g, 15 min). The concentration of conjugated lectins on hSPION-Au was measured using the BCA protein assay kit.

To characterise the morphology of hSPION-Au, transmission electron microscopy (TEM) was performed using a Carl Zeiss EF-TEM LIBRA 120 (Oberkochen, Germany). The samples were placed on a copper grid for TEM measurements. Furthermore, the magnetisation of the particles was assessed using a vibrating sample VSM-7410 magnetometer (Lake Shore, OH, USA) at room temperature within the range of −150 to +150 emu/g. Dynamic light scattering and electrophoretic light scattering (DLS/ELS) were measured using a Malvern Zetasizer Nano ZSU5800 (Malvern, UK) to evaluate the size distribution and zeta potential. Then, we used a NanoSight NS300 (Malvern, UK) to assess the particle size distributions and concentrations. All samples were diluted to appropriate volumes with phosphate-buffered saline (PBS). Tracking videos were recorded in triplicate for 30 s each and analysed using NTA software (version 2.3; NanoSight, Malvern, UK).

Murine melanoma cell lines (B16F1 and B16F10), a murine fibroblast cell line (L929) and immortalised human monocytes (THP-1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The melanoma cell lines were cultured in high-glucose Dulbecco's modified Eagle medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.). The L929 and THP-1 cell lines were cultured in ATCC-modified RPMI medium (Gibco; Thermo Fisher Scientific, Inc.). All culture media were supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotics–antimycotics (Hyclone, Logan, UT, USA) and then incubated at 37 °C in 5% CO2 and 90%–95% humidified air. To differentiate and polarise THP-1 cells into macrophages, THP-1 cells were incubated with PMA, LPS, IFN-γ and IL-4 or IL-13. First, THP-1 cells were differentiated into M0 macrophages by treatment with 320 nmol/L PMA and incubated for 24 h. M0 macrophages were then polarised by incubation with 20 ng/mL IFN-γ and 100 ng/mL LPS for 24 h. M2 macrophages were polarised by incubating M0 macrophages with 20 ng/mL IL-4 and IL-13, respectively, for 24 h.

To investigate the binding affinity of SNA onto the cell lines, the fluorescence of the SNA-PS beads was measured according to the types of lectins and time points. To measure the fluorescence intensity, cell lines were grown on an 18-mm coverslip in 12-well plates for 24 h after seeding with 4 × 104 cells and then treated with SNA-PS at a protein concentration of 5 μg/mL. To further evaluate the interactions between cells and lectins, SNA-PS-treated cells were incubated for 1, 2, 4 or 6 h. After incubation, the cells were washed three times with PBS, and the fluorescence intensity was measured using a confocal microscope (LSM 700, Zeiss Microscopy, Jena, Germany). FACS was performed to compare the SNA-positive population in each cell line. For FACS measurement, the SNA-PS-treated cells were rapidly detached from the 6-well plate using TryPLE™ Express Enzyme (Gibco) and resuspended in PBS containing 5% FBS. The samples were analysed using a 488-nm laser. Flow cytometry data were analysed using FlowJo™ v10.8.0 software (BD Life Sciences, Franklin Lakes, NJ, USA). Moreover, to evaluate the blockade effect on anti-CD24 antibody, SNA/hSPION-Au (SNA: 10 μg/mL, particles: 1.75 × 108) or hSPION-Au (particles: 1.75 × 108) were treated and incubated for 6 h, followed by Alexa 488 anti-CD24 antibody treatment. Then, FACS was performed to compare the blockade efficacy of Alexa 488 anti-CD24 antibody.

ELISA-like solid-phase binding assays were performed to evaluate the interaction between CD24 and Siglec-10. First, melanoma and macrophage cells were lysed in RIPA buffer containing phosphatase inhibitor cocktail 2 and cocktail 3 and protease inhibitor cocktail. Then, Nunc MaxiSorp™ flat-bottom ELISA plates (Thermo Fisher Scientific, Inc.) were coated with melanoma cell lysate (10 μg/mL) and stored overnight at 4 °C. The melanoma cell lysate was pre-incubated with BSA (10 μg/mL), SNA (10 μg/mL) for 2 h or neuraminidase (100 U/mL) for 1 h before interaction with the macrophage lysate-coated on an ELISA plate. Next, a blocking buffer containing 1% BSA in Tris-buffered saline (TBS) (20 mmol/L Tris, pH 7.4; 150 mmol/L NaCl) was applied to the ELISA plate for 30 min. Melanoma cell lysates were then applied to macrophage lysate-coated ELISA plates to induce interactions between CD24 and Siglec-10 in the lysates, followed by incubation with secondary antibody (Alexa 488 anti-CD24 antibody, 5 μg/mL) for 2 h. After final incubation with the secondary antibody, the plate was washed twice with ice-cold PBS. Fluorescence intensity was measured (Ex: 485 nm, Em: 528 nm) using a BioTek microplate reader (Synergy H1, BioTek Instruments, Inc., Winooski, VT, USA). The result was normalised based on control group of B16F1.

Flow cytometry measurements of THP-1-derived macrophages and melanoma cell lines were performed using a FACS Aria II (BD Biosciences, Franklin Lakes, NJ, USA) to evaluate the expressions of Siglec-10 and CD24, respectively. For FACS sampling, the cells were cultured in a 6-well plate at a cell density of 2 × 106 cells/well. Cells were then incubated at 37 °C for 24 h. THP-1 cells were polarised and differentiated into M0, M1 and M2 macrophage phenotypes. Then, the cells were rapidly detached from the plate using TryPLE™ Express Enzyme (Gibco) and resuspended in PBS containing 5% FBS. Samples were analysed by flow cytometry using 405-, 488- and 633-nm lasers after antibody labelling (Alexa 488 anti-CD24 antibody for melanoma cell lines, Alexa 647 anti-CD11b-antibody for macrophages, Pacific blue anti-CD14 antibody for M0 macrophages, Alexa 488 anti-CD80 antibody for M1 macrophages, Alexa 647 anti-CD206 antibody for M2 macrophages). Flow cytometry data were analysed using FlowJo™ v10.8.0 software (BD Life Sciences).

The tumoricidal effect of hSPION-Au and SNA/hSPION-Au was assessed using the MTT assay. The cell lines described above were cultured in DMEM or RPMI supplemented with 10% FBS and 1% antibiotics−antimycotics at 37 °C with 5% CO2 air. Cells were seeded at the same density of 5 × 103 cells/well in a 96-well plate and allowed to attach and grow for 18 h before sample treatment. Either hSPION-Au or SNA/hSPION-Au were treated with various concentrations of particles and lectin protein with or without NIR irradiation. Cytotoxicity assays were performed using MTT (Amresco, Solon, OH, USA) after 24 h of treatment. MTT (2 mg/mL) was added to each well, and the samples were incubated for 2 h. Dimethyl sulphoxide (DMSO) was added to dissolve the formazan crystals within the cells. Finally, the absorbance of formazan was read at 560 nm and expressed as a percentage of the control value. Furthermore, Apoptosis and necrosis assay was performed using the Annexin V-FITC Apoptosis kit (Invitrogen, V13242). Briefly, cells (1 × 106/well) were cultured for 24 h, and then samples were treated on each group, followed by 6-h incubation with or without NIR irradiation. The Annexin V-FITC and PE-stained cells were analysed by flow cytometry (SNA/hSPION-Au, SNA: 10 μg/mL, particles: 1.75 × 108; hSPION-Au, particles: 1.75 × 108; SNA: 10 μg/mL).

Melanoma cells (red fluorescence) and THP-1-derived macrophages (green fluorescence) were labelled with Alexa Fluor 633 and Calcein-Am, respectively. Cell suspensions consisting of 25,000 macrophages and 50,000 melanoma cells that were reacted with hSPION-Au (1.75 × 108) or SNA/hSPION-Au (SNA: 10 μg/mL, particles: 1.75 × 108) for 2 h were placed into a non-treated 18-mm coverslip in a 12-well plate to allow for the adherence of THP-1-derived macrophages while preventing melanoma cell adherence. Samples were incubated for 6 h in an incubator at 37 °C. After incubation, cell-attached coverslips were washed vigorously three times with serum-free RPMI to wash away free melanoma cells, and the coverslips were then mounted onto microscope glass slides. Melanoma phagocytosis was evaluated using an LSM 700 confocal microscope (Zeiss Microscopy). All images were processed using the Zeiss CLSM software (ZEN2009 Light Edition) and the National Institutes of Health (NIH) ImageJ software (Bethesda, MD, USA). The result was normalised based on hSPION-Au treatment group of B16F10 interacted with M2 macrophage.

The in vitro phagocytosis assay using flow cytometry was performed by co-culturing the melanoma and macrophage cells at a ratio of 700,000 melanoma cells to 350,000 macrophages for 2 h in an incubator at 37 °C with 5% CO2 air in 96-well U-bottom Cell Floater Plates (SPL, South Korea) in serum-free RPMI. Melanoma cells were treated with hSPION-Au (1.75 × 108) or SNA/hSPION-Au (SNA: 10 μg/mL, particles: 1.75 × 108) for 2 h before co-culture. Cells were collected from a culture plate using the TrypLE™ Express Enzyme (Gibco). Prior to co-culturing, the cells were fluorescently labelled with Alexa 647 anti-CD11b antibody (for macrophages) and Alexa 488 anti-CD24 antibody (for melanoma). Alexa 647 anti-CD11b and Alexa 488 anti-CD24 antibodies (5 μg/mL) were incubated with cells for 1 h on ice, followed by two washes with ice-cold PBS. After co-culturing, phagocytosis assays were terminated by placing the plates on ice. Following centrifugation at 350×g for 5 min at 4 °C, staining with DAPI was performed to identify the dead cell population. Flow cytometry was undertaken using a FACS Aria II (BD Biosciences), and the data were analysed using FlowJo™ v10.8.0 software (BD Life Sciences). The result was normalised based on hSPION-Au treatment group of B16F1 interacted with M0 macrophage.

To form melanoma spheroids, melanoma cells were harvested at a density of 1 × 106 cells/mL. Then, the cells were placed in a three-dimensional (3D) cell culture dish (StemFIT 3D, MicroFIT, Republic of Korea). An anti-adherence rinsing solution (STEMCELL™; Vancouver, Canada) was applied to the StemFIT culture dish overnight before plating the cells. Then, the 3D culture dish was filled with the cell suspension and incubated for 30 min under cell culture conditions. After incubation, the cell culture medium was aspirated carefully using a pipette to remove the remaining cells from outside of the well. Subsequently, 1 mL of methylcellulose solution at a final concentration of 4% was added to the 3D culture dish. The result was normalised based on phagocytosis of SNA/hSPION-Au-treated B16F10 by M2 macrophage. To evaluate the PTT-triggered tumoricidal effect of the melanoma spheroids that were prepared as described above, the cells were transferred to an ultra-low adherent 6-well plate containing 4% methylcellulose solution; the same number of melanoma spheroids were placed into each well (n = 10). Next, the spheroids were treated with hSPION-Au or SNA/hSPION-Au, followed by NIR irradiation treatment. A spheroid-based migration assay was performed to evaluate the migration inhibition triggered by SNA/hSPION-Au with NIR irradiation. Melanoma spheroids were placed in a 24-well culture plate with a culture medium containing supplements. Spheroid migration was monitored for several days. After 7 days, the plates were observed by an optical microscopy, and the diameters of the migration zone and initial spheroids were calculated using ImageJ (NIH). Spheroid migration was assessed using the following Eq. (1):

Female C57BL/6 mice (4–6 weeks old) were purchased from Samtako Bio Korea (Gyeonggi-do, Republic of Korea). To create melanoma tumour-bearing mice, melanoma cell lines (B16F1 and B16F10) were inoculated with 1 × 106 cells/100 μL suspended in PBS. The cells were then subcutaneously injected into the left flank of each female C57BL/6 mouse. When the tumour volume reached 100 mm3, the mice were injected intratumorally with a single dose of SNA/hSPION-Au (2 mg/kg SNA). Two hours after injection, all tumour-bearing mice were exposed to 850 nm IR LED light (PRIME-100™, Skycares, Gyeonggi-do, Republic of Korea) for 8 min, and IR irradiation was administered every 3 days. The tumour volume was recorded daily. Tumour volume was calculated using the following Eq. (2): The mice were sacrificed after 3 weeks. All animals were maintained in a pathogen-free environment, and all animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Hanyang University (protocol 2021-0224). Histological examination was performed by using paraffin-embedded hematoxylin-eosin (H&E) or 3,3′-diaminobenzidine (DAB)-stained histology slides. All histological examination was outsourced to KP&T, Korea Pathology Technical Center (Chungcheongbuk-do, Republic of Korea).

In accordance with the sample distribution and variance, all data and graphs were processed using OriginPro 2015 software (OriginLab, Northampton, MA, USA). Data were analysed by one-way ANOVA, followed by Tukey's multiple-comparison test (OriginPro 2015 software). Each experiment was performed in duplicate, and error bars indicate standard deviations. Statistical significance was set at P < 0.05. All significant values provided in the figures are indicated as follows: ∗P < 0.10, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001 vs. the control.

Au-modified NPs can act as photothermal reagents for photodynamic therapy. We selected iron oxide NPs for Au modification to optimise the NIR therapeutic window. hSPION-Au was synthesised using iron oxide and Au-based core−shell nanoparticles (SPION@Au) as seed NPs (Fig. 2A). The Au coating on the SPION serves as a strong surface plasmon resonance (SPR) peak for photothermal therapy (PTT). To optimise PTT within a NIR excitation wavelength window, the absorption wavelength of SPION@Au was modified by inducing the anisotropic growth of SPION@Au exposed to halide ions. Fluorine ion from potassium fluoride (KF) was applied to synthesize the hollow structured nanoparticles which have strong absorption in NIR. As a result, the SPION@Au particles agglomerated to form a conglomerate possessing a porous surface (hSPION-Au) (Fig. 2B). The synthesised hSPION-Au was then applied to deliver lectin, followed by NIR PTT. The optimal NIR wavelength range for tissue penetration by PTT is 700–1100 nm. To optimise the SPR peak of hSPION-Au, the fluorine concentration was adjusted using KF. The shape of the particles according to the concentration control of KF was different. Also, there was no trend in the absorbance spectrum change according to the KF concentration. However, it was shown that uniform particles were synthesized at a KF concentration of 0.01 mmol/L. The optimal concentration of KF was 0.01 mmol/L with a strong SPR peak of hSPION-Au at 846 nm (Fig. 2C). Fig. 2C depicts the room-temperature magnetisation versus a magnetic field and DLS/ELS characterisation of particles. All samples exhibited representative superparamagnetic behaviour, thus implying that SPIONs coated with Au shells can conserve their superparamagnetic properties. The bare SPION exhibited the highest values of magnetisation (145 emu/g), while the saturation magnetisation was determined as 65 and 8.7 emu/g at 150,000 Oe for the SPION@Au and hSPION-Au, respectively. After SNA binding of hSPION-Au, the average particle size increased by 42 nm. The zeta potential showed a negative charge. The negative zeta potential after protein binding is presumed to be due to the isoelectric point (PI) of SNA at 5. Furthermore, the photothermal properties of hSPION-Au were verified (Fig. 2D–F). The temperature increased to 55 °C for the hSPION-Au suspension, while the SPION@Au and SPION suspensions exhibited only a slight increase to 30 °C in response to the same laser exposure (Fig. 2D and E). Moreover, hSPION-Au reached an equilibrium temperature of approximately 57 °C after three heating and cooling cycles under 850-nm NIR LED light (Fig. 2F). Thus, the absorbance of hSPION-Au is suitable for effective PTT that reportedly occurs within the wavelength range of 780–940 nm. Numerous sialoglycans are reportedly expressed on the surface of tumour cells. We used the melanoma cell lines B16F1 and B16F10 as a proof-of-concept model to confirm the reaction between the surface of tumour cells and the SNA protein, a sialic acid-specific binding protein. L929 cells were used as a control. The interaction between cell lines and lectin was evaluated by treating the cell lines with SNA-bound fluorescent polystyrene (SNA-PS) beads. Melanoma cells reportedly show CD24 overexpression. The tendency of CD24 sialylation in each cell line can be confirmed by evaluating the binding rate of SNA to sialic acid, as CD24 is a highly sialylated glycoprotein. SNA PS was added to each cell line to monitor the fluorescence intensity due to SNA binding over time. We verified the SNA-PS distribution in the cell lines using confocal laser scanning microscopy (CLSM; Fig. 3A). Representative fluorescence images were obtained according to the incubation time after SNA-PS treatment, and then the distribution of SNA-PS on the cells was analysed. Our results revealed that the binding of SNA-PS was almost undetectable in the L929 cells used as a control cell line against melanoma. By contrast, the melanoma cell lines (B16F1 and B16F10) exhibited time-dependent SNA–PS binding. Both melanoma cell lines exhibited an increase in the bound SNA-PS on the cell surface over time, suggesting the presence of an increased amount of sialic acid moiety that is capable of binding to SNA. Furthermore, fluorescence-activated cell sorting (FACS) analysis was performed to compare the fluorescence intensities according to the reaction between the cell surface and SNA (Fig. 3B). In Fig. 3, the SNA-PS bound to the cells is almost undetectable in the representative image of the L929 cells. L929 cells incubated for 12 h with SNA-PS showed a low proportion of the sample population with positive fluorescence (6.6%) by FACS analysis. Melanoma cells displayed comparatively higher percentages of fluorescent-positive cells in the sample population, 62.3% in B16F1 cells and 92.1% in B16F10 cells (Fig. 3B). The results of fluorescence analysis suggest that the binding rate of SNA-PS differs depending upon the cell line, and more SNA could become attached to the tumour cells than to the normal ones. Although the tumour cell lines were derived from melanoma tissue, B16F1 and B16F10 exhibited different results. The fluorescence intensity indicates that the differences in the tumour cell-binding efficacy of lectin are due to the diversity of tumour cells. We hypothesised that these variations in fluorescence intensities are because the degree of glycosylation of tumour cells affects the binding affinity of lectins. In particular, in the B16 melanoma model, B16F1 and B16F10 reportedly possess low and high metastatic potential, respectively. We speculate that different metastatic characteristics may contribute to the glycosylation tendency of tumour cells. In particular, high sialylation and overexpression of CD24 reportedly occur in highly metastatic tumour cells. To verify these results, the expression level of CD24 in each melanoma cell line was evaluated (Fig. 4A). Furthermore, we analysed whether treatment with lectin-conjugated hollow gold-iron oxide nanoparticles (SNA/hSPION-Au) could block the interaction between melanoma cells and CD24 antibodies. The labelled CD24 antibody on the surface of B16F10 cells was significantly more abundant (>10%) than that on B16F1, supporting previous results. When melanoma cell lines were treated with hSPION-Au alone, the labelled CD24 antibody was reduced because the reaction between the CD24 antibody and melanoma cells was disturbed by the NPs. It can be assumed that NPs non-specifically bound to the cell surface induced the blockade of membrane proteins in tumour cells. Furthermore, we demonstrated that SNA/hSPION-Au significantly reduced the binding of the CD24 antibody to the melanoma cells. These results indicate that the SNA-mediated binding of SNA/hSPION-Au to CD24 interrupts CD24 antibody labelling. CD24 in melanoma tumour cells primarily induces immunosuppression via interaction with Siglec-10 on macrophages (Supporting Information Fig. S1). The sialic acid moiety triggered the binding of Siglec-10 to CD24. When Siglec-10 recognises CD24, anti-phagocytosis signalling is induced by the phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) following the binding of phosphatase. We speculated that anti-phagocytic signalling could be inhibited if sialic acid that functions as a cue for the interaction between Siglec-10 and CD24 was blocked by lectin. THP-1 human monocytes were introduced, and FACS analysis confirmed the expression of macrophage-related markers according to macrophage phenotypes (Fig. 4B). The THP-1 monocytes present before differentiation into M0 macrophages were confirmed through the CD11b-positive population, and the CD11b+Siglec-10+ population was observed to be 25.7%. Phorbol 12-myristate 13-acetate (PMA)-induced M0 macrophages were confirmed based on the expression of CD14, and the CD14+Siglec-10+ population was observed to be 20.1%. Furthermore, to confirm Siglec-10 expression according to functional phenotype, M0 macrophages were polarised into M1 and M2 macrophages by specific cytokines. Polarisation of M1 macrophages was confirmed in the CD80-positive population, and the CD80+Siglec-10+ population was observed to be 29.8%. M2 macrophages were confirmed in the CD206-positive population, and the CD206+Siglec-10+ population was observed to be 43.0%, thus exhibiting the highest Siglec-10 expression among the macrophage phenotypes. These results confirmed that macrophages derived from THP-1 cells possessed high Siglec-10 expression, and the highest expression was observed in M2 macrophages. After confirming the expression of CD24 and Siglec-10 in the melanoma cell lines and the THP-1-derived macrophages, lysates from each cell line were prepared to evaluate the interaction between CD24 and Siglec-10. The CD24 antibody was introduced into the melanoma lysate to monitor solid-phase binding with macrophage lysate by providing Siglec-10 proteins. To determine the sialic acid preference of Siglec-10, the sialic acid moiety was eliminated by sialidase or blocked by SNA, and the interaction between CD24 and Siglec-10 (depending on treatment with SNA, sialidase or bovine serum albumin [BSA] as a negative control) was evaluated according to the fluorescence intensity (Fig. 4C). The sialidase-treated group could not bind to Siglec-10 in the macrophage lysate due to the removal of the sialic acid moiety that functions as the binding site of Siglec-10; thus, the fluorescence intensity was insignificant. Similarly, the SNA-treated group exhibited a decrease in fluorescence. This implies that SNA treatment inhibited the CD24−Siglec-10 interaction by blocking sialic acid. Conversely, the BSA-treated negative control group exhibited little decrease in fluorescence compared to the enzyme- and lectin-treated groups. Our results suggest that sialic acid-preferring lectins can block CD24 interactions mediated by sialic acid. Considering the critical role of CD24 and Siglec-10 in the immune response, CD24 blockade after treatment with SNA-conjugated NPs may be a key for suppressing ICPs.

The hSPION-Au photothermal NP was introduced to induce the effective delivery of SNA to block CD24 and increase tumoricidal efficacy. To confirm the effect of PTT on melanoma tumour cells using SNA/hSPION-Au, the viability of melanoma cells was evaluated according to the concentration of SNA/hSPION-Au and NIR irradiation time (Supporting Information Fig. S2). At low concentrations (SNA: 5 μg/mL, NPs: 8.75 × 107) of SNA/hSPION-Au for 12-h incubation, the viability of B16F1 melanoma cells decreased with NIR irradiation time, while the efficacy of cell death was insignificant in L929 cells (Fig. 5A). Additionally, the tumoricidal effect on melanoma cells that depended upon the NIR irradiation time for the double concentrations of SNA and NPs was significantly different from the results at lower concentrations (Fig. 5B). Moreover, the tumoricidal efficacy was evaluated at a fixed NIR irradiation time (10 min) with SNA-conjugated or non-conjugated hSPION-Au (Fig. 5C and Supporting Information Fig. S3). Under a low concentration of NPs (8.75 × 107 NPs/mL), the cell viabilities differed depending on SNA conjugation for each cell line. There was no significant decrease in the cell viability of L929 cells treated with SNA/hSPION-Au (SNA, 5 μg/mL), while B16F1 and B16F10 cells exhibited 2.5% and 5.2% decreases in cell death, respectively (Fig. 5D). By contrast, the cell death in each group treated with a double concentration of NPs without lectin conjugation (1.75 × 108 NPs/mL, SNA: 0 μg/mL) in combination with NIR irradiation was 0% (L929), 16% (B16F1) and 15% (B16F10). However, the double-concentration SNA/hSPION-Au-treated cells exhibited cell death percentages of 9% (L929), 30% (B16F1) and 39% (B16F10). These results revealed that the tumoricidal efficacy of doubling the concentration of SNA/hSPION-Au was up to 24% higher than for doubling the concentration of hSPION-Au in the B16F10 cells. These results demonstrate that the combination treatment of hSPION-Au and NIR irradiation can induce melanoma cell death via the photothermal effect. Additionally, we demonstrated that the binding of SNA can increase the efficiency of photothermal treatment. Next, we performed apoptosis and necrosis rate profiling to elucidate the tumour death process triggered by photothermal treatment using hSPION-Au and SNA. Reportedly, inducing apoptosis may increase macrophage activity against tumour cells through processes, such as efferocytosis and phagocytosis, and may enhance the efficiency of anti-tumour immunotherapy. Therefore, an apoptosis/necrosis assay was performed on both B16F1 and B16F10 cells to confirm the apoptosis rate resulting from SNA/hSPION-Au treatment with or without NIR irradiation. When cells were treated with SNA, hSPION-Au or SNA/hSPION-Au—and depending on NIR irradiation—an increase in early apoptosis was observed. In the B16F1 group, when SNA and hSPION-Au were treated without NIR irradiation, each result exhibited 0.67% and 26.3% rates of early apoptosis, respectively (Supporting Information Fig. S4A). By contrast, when NIR irradiation was performed for 10 min under the above conditions, the SNA-treated group exhibited an increase in early apoptosis of <2%, while the hSPION-Au-treated group exhibited a 5% increase in early apoptosis. Moreover, B16F1 cells treated with SNA/hSPION-Au exhibited 30.2% early apoptosis without NIR and an 8.2% increase in early apoptosis with NIR irradiation (Fig. 5E). The B16F10 group without NIR possessed a higher early apoptosis percentage (2.24%) than the B16F1 group when only SNA was used, and the group treated with hSPION-Au exhibited higher early apoptosis (29%) than that of B16F1 (26.3%) (Fig. S4B). In particular, NIR irradiation of B16F10 cells demonstrated that the rate of early apoptosis was significantly increased by hSPION-Au treatment. B16F10 cells exhibited an increase of 10.6% in early apoptosis compared to a 5% increase in B16F1 cells triggered by NIR. Moreover, the B16F10 cells treated with SNA/hSPION-Au exhibited 48.9% early apoptosis with NIR irradiation. These results indicate that the combination treatment (SNA/hSPION-Au and NIR) increased the apoptosis rate more significantly in B16F10 cells than in B16F1 cells (Fig. 5F). Collectively, SNA/hSPION-Au exerted a promising tumoricidal effect upon NIR irradiation. The effect on tumour death differed according to the bound SNA on hSPION-Au, and it was confirmed that the photothermal tumour cell death from SNA/hSPION-Au was the highest in B16F10 cells with a highly induced early apoptosis rate. These results imply that B16F10 cells (a highly metastatic tumour cell line) may possess high glycosylation and increased CD24 expression; this can influence the tumoricidal effect of the combination of SNA/hSPION-Au treatment and NIR irradiation due to the relatively overexpressed CD24 that possesses an abundance of sialic acid moieties. Specifically, our results suggest that PTT using SNA/hSPION-Au possesses clinical potential for non-metastatic and metastatic melanoma tumours.

Anti-phagocytosis signalling in tumour cells is induced by interactions between ICPs on both tumour and immune cells. A representative model of this process is the interaction between Siglec-10 on macrophages and CD24 on tumour cells. Siglec-10 and CD24 are known immune inhibitors that can trigger an immune evasion cascade mediated by the sialic acid moiety terminalised on CD24 (Fig. S1). Therefore, we hypothesised that immune evasion signalling could be suppressed by blocking CD24 on melanomas by recognising the terminal sialic acid of CD24 using sialic acid-affinity lectins. Thus, we introduced an experimental model that involved sialic acid blockade by treatment with SNA/hSPION-Au (Fig. 6A) and evaluated the efficacy of CD24 blockade by the combination treatment of SNA/hSPION-Au and NIR irradiation for the inhibition of anti-phagocytic activity. Melanoma phagocytosis was observed by CLSM analysis upon treatment with PE anti-CD24 monoclonal antibodies (mAb) on the melanoma cell lines and upon treatment with calcein-AM on the macrophages. Subsequent co-culturing was conducted with the above cells. Macrophages were assumed to phagocytose melanoma cells because they demonstrated engulfment of melanoma cells, and we confirmed the co-localisation of melanoma and macrophages (Supporting Information Fig. S5). After 12 h of co-culturing, the blockade of CD24 using SNA/hSPION-Au augmented the phagocytic ability of macrophages, thereby confirming the fluorescence intensity of the co-localised melanoma cells (Fig. 6B). Furthermore, it was confirmed that the augmented phagocytic ability was more pronounced in high metastatic melanoma (B16F10) than in B16F1. In particular, the phagocytic effect of M2 macrophages following treatment with SNA/hSPION-Au was more significant in B16F10 cells than in low metastatic melanoma. Due to the overexpression of CD24 in the high metastatic tumours, they exhibited more significant anti-phagocytic activity than low metastatic or non-metastatic tumour cells. These results suggest that SNA/hSPION-Au plays a distinct role as an ICI. This may increase the possibility of immunotherapy by activating the phagocytosis of M1-and M2-like macrophages. Additionally, phagocytic activity was quantified by flow cytometry-based measurement of SNA/SPION-Au-induced phagocytosis of melanoma cells with and without NIR irradiation. The augmentation of phagocytic activity, indicative of the inhibition of ICPs, confirmed that treatment with SNA/hSPION-Au was more significant in this group than in the untreated group, in agreement with the results of the microscopic analysis. The gating strategy for flow cytometry was assessed by selecting the population where the co-localised fluorescence signals of Alexa 488 anti-CD24 mAb-labelled melanoma and Alexa 647 anti-CD11b mAb-labelled macrophages were detected in living cells (Supporting Information Fig. S6). Phagocytosis activity, as judged by the SNA bound to hSPION-Au, differed depending on the type of melanoma and macrophage. The hSPION-Au treatment of B16F1 cells co-cultured with M0 macrophages resulted in 21.5% phagocytosis, and SNA/hSPION-Au treatment augmented phagocytosis (30.9%) in the hSPION-Au-treated group (Supporting Information Fig. S7). Similarly, SNA/hSPION-Au treatment of B16F1 cells co-cultured with M1 macrophages showed an 8% increased phagocytic rate compared to the individual hSPION-Au treatment group. By contrast, the SNA/hSPION-Au treatment of B16F10 cells increased the phagocytic activity by more than two-fold that of B16F1 cells (Fig. S7). In particular, it was observed that M2 macrophages exhibited the highest rate of phagocytosis activation in high metastatic melanoma cells after treatment with SNA/hSPION-Au (Fig. 6C). Consequently, the CD24 blockade induced by SNA/hSPION-Au treatment augmented phagocytosis in melanoma cells. The fluorescence intensity values were normalised to compare each group (Fig. 6D); it was demonstrated that a 1.3–1.5-fold increase in phagocytosis was observed in low metastatic melanoma (B16F1), and a 1.75–2.07-fold increase in phagocytic activity was observed in high metastatic melanoma (B16F10). FACS results indicated that when SNA/hSPION-Au treatment was applied to B16F10 cells, the phagocytic activity of macrophages was the highest. We hypothesised that if SNA/hSPION-Au could effectively block CD24, it would inhibit the ICP more strongly when followed by NIR irradiation. Upon NIR irradiation, increased phagocytic activity was induced in both melanoma cell lines (Supporting Information Fig. S8), and a higher rate of phagocytosis was observed in B16F10 cells than in B16F1 cells. These results suggest that SNA/hSPION-Au treatment with PTT application can simultaneously suppress the specific ICP response and inhibit immune evasion. Moreover, macrophages, which showed high expression of Siglec-10, demonstrated significantly increased phagocytosis on melanoma cells mediated by CD24 blockade.

Removing tumour tissue is crucial for tumour treatment; however, it is also an important factor for preventing tumour metastasis and increasing the patient survival rate. Metastatic tumour cells are difficult to treat using conventional chemotherapy or surgery due to their anatomical infiltration into various organs, thus indicating the importance of early prevention. Therefore, we evaluated the tumoricidal effect and metastasis inhibition in a 3D culture tumour model under in vitro conditions that included treatment with SNA/hSPION-Au, followed by PTT. Tumour spheroids were constructed within the range of 100–150 μm as an ex vivo model to evaluate tumour death and metastasis inhibition. Then, we monitored changes in the size of the tumour spheroids treated with SNA/hSPION-Au, followed by NIR irradiation. After spheroid formation, monitoring was conducted for 7 days, and SNA/hSPION-Au and NIR irradiation were applied after 4 days. Tumour growth in both spheroid models was observed from Day 1 to Day 3, and particle treatment and PTT application were both performed on Day 4. The B16F1 spheroids were 150 μm in size before therapy, and changes in the spheroid size varied depending on the treatments with hSPION-Au or SNA/hSPION-Au, followed by NIR irradiation. hSPION-Au, SNA/hSPION-Au treatment and NIR application inhibited the growth of B16F1 spheroids (Fig. 7A). By contrast, the untreated group exhibited continuous tumour growth over time. Moreover, the untreated B16F10 spheroid group exhibited a slower growth curve than the untreated B16F1 spheroid group. The B16F1 spheroids exhibited a tumour growth inhibitory effect after hSPION-Au treatment with PTT, while the B16F10 spheroid underwent a slight decrease in size after the same treatment. We noted that the B16F10 spheroid quickly recovered in size after treatment; consequently, hSPION-Au treatment alone did not significantly induce therapeutic efficacy in the spheroid models (Fig. 7B). However, the combination of SNA/hSPION-Au and PTT demonstrated significant tumour reduction in B16F10 cells and resulted in the elevated incorporation of SNA/hSPION-Au and NIR irradiation. It was confirmed that the tumoricidal effect of SNA/hSPION-Au was more pronounced on the spheroids of B16F10 high metastatic tumours, thus demonstrating that the efficiency of PTT may be increased by introducing SNA to block the sialic acid moiety. A tumour spheroid invasion assay was performed to evaluate the suppression of metastasis in melanoma cells treated with SNA/hSPION-Au (Supporting Information Figs. S9 and S10). The spheroid invasion rate of B16F10 cells was higher than that of B16F1 cells, thus indicating that highly metastatic tumours possessed a rapid invasion tendency. It was observed that photothermal application after hSPION-Au treatment decreased but could not prevent the invasion of both spheroids. By contrast, SNA/hSPION-Au inhibited invasion through PTT. These results suggest that PTT mediated by SNA/hSPION-Au induces tumour death and suppresses tumour invasion by metastatic tumour cells. The distribution of macrophage infiltration into a melanoma spheroid model was confirmed to evaluate the performance of PTT using SNA/hSPION-Au as an immunotherapeutic agent. Spheroids cultured to 100 μm in diameter were treated with hSPION-Au or SNA/hSPION-Au and co-cultured with macrophages for each phenotype. The distribution of macrophages within the spheroids was confirmed by fluorescence imaging (Fig. 7C). Melanoma spheroids and macrophages were labelled with calcein-AM and Alexa 647 anti-CD24 antibodies, respectively. It was confirmed that macrophages penetrated the spheroids, and treatment with SNA/hSPION-Au augmented the infiltration of macrophages into the tumour. In B16F1 spheroids, M1 macrophages exhibited the best accumulation in tumour spheroids, and treatment with SNA/hSPION-Au augmented the infiltration of macrophages up to three-fold (Fig. 7D). The above results suggest that the introduction of SNA/hSPION-Au not only serves as a photothermal treatment agent but also possesses therapeutic potential as an immunotherapeutic agent that can activate the phagocytic activity of macrophages through mechanisms that include the inhibition of ICPs. The dual treatments involving blockading the sialic moiety on tumour cells in combination with NIR treatment may result in excellent anti-tumour efficacy and provide a promising new route for tumour immunotherapy. To evaluate the anti-tumour efficacy of sialic acid blockade with PTT using SNA/hSPION-Au, we performed an in vivo study using tumour-bearing C57BL/6 mice. For the in vivo study, a subcutaneous syngeneic mouse model was created to monitor the combinational tumour treatment using SNA/hSPION-Au (2 mg/kg of SNA concentration) with NIR irradiation (Fig. 8A). The dose concentration for the treatment was fixed because a dose concentration more than 2 mg/kg influenced the survival rate of mice. B16F1 and B16F10 cells were subcutaneously injected into the left flanks of the experimental mice. The subcutaneous tumours in the B16F1 group were eliminated after SNA/hSPION injection, followed by NIR irradiation (Fig. 8B). However, the B16F10-inoculated group exhibited mild tumour growth inhibition compared to the B16F1 group (Fig. 8C); this reveals that low metastatic tumours were significantly affected by applying photothermal after SNA/hSPION-Au treatment. Moreover, macrophage infiltration was found in tumour tissue (Supporting Information Fig. S11). In the treated group, the result showed an augmented macrophage infiltration into tumour tissue. This macrophage infiltration can promote the destruction of cancer cells. In addition, the histological information of major organs including liver, spleen, stomach, colon, lung and kidney were also shown in Supporting Information Fig. S12. The tissue structures of liver, spleen, stomach, colon, lung and kidney did not demonstrate significant changes, indicating that the treatment rarely had effect on major organs, further imply that SNA/hSPION-Au had good biosafety. Collectively, glycan-targeting anti-cancer treatment exhibited a synergistic effect in vivo when applying photothermal treatment after SNA/hSPION-Au. The above results demonstrate that SNA/hSPION-Au can act as an effective photothermal agent for tumour ablation following a single intratumoral injection and one-time laser irradiation with no noticeable side effects on a low metastatic tumour.

In conclusion, we suggest that the immune response of macrophages can be activated through ICP inhibition mediated by lectin protein. The sialic acid-preferable lectin SNA induced a blockade of the terminal sialic acid of CD24, leading to the inhibition of the interaction between CD24 and Siglec-10. SNA/hSPION-Au with NIR irradiation augmented the phagocytosis of macrophages for melanoma treatment. SNA/hSPION-Au introduced into melanoma cell lines resulted in the successful phagocytic activation of macrophages in both low and high metastatic melanoma cells. Furthermore, treatment with SNA/hSPION-Au-mediated combinational NIR irradiation of melanoma tumours resulted in enhanced anti-tumour effects and the significant inhibition of anti-phagocytic signalling, while simultaneous NIR irradiation with CD24 blockade by SNA/hSPION-AU induced the progression of apoptosis in tumour cells and augmented the phagocytic effect of macrophages. This study simultaneously performed ICP inhibition and PTT—thus suggesting a new therapeutic strategy for anti-tumour immunotherapy—and demonstrated metastasis inhibition in the context of melanoma. Lectin-based photo-immune therapy could provide an excellent anti-tumour strategy and clinical solution to overcome the limitations of conventional tumour therapies.

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (10.13039/501100014188MSIT) (Nos. 2020R1A5A1018052, 2017M3A7B8061942, 2019R1A2C1006018, 2021R1A4A5032463 and 2021M3H4A4079629, Republic of Korea).

Jonghoon Choi supervised the study. Yonghyun Choi designed the study and performed the experiments. Jayoung Chae participated in the experiments. Wooic Son, Youngpin Han and Chul-Su Yang supported the in vivo experiments. Yonghyun Choi performed data analyses. Jonghoon Choi and Yonghyun Choi wrote the manuscript.

The authors declare no conflicts of interest.