Ginseng-derived nanoparticles alter macrophage polarization to inhibit melanoma growth

All human experimental protocols were approved by the Ethics Committee for Human Studies of Affiliated Hospital of Integrated Traditional Chinese and Western Medicine (2018LWKYZ010).

Wild-type (WT) C57BL/6 mice were purchased from the Comparative Medicine Center, Yangzhou University (Yangzhou, Jiangsu, China). MyD88-, TLR4- and TLR2-deficient C57/BL6 mice were gifts from Dr. Lixin Wang (Department of Microbiology and Immunology, Medical School of Southeast University, Nanjing, China) [21]. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine.

The murine melanoma cell line (B16F10), breast cancer cell line (4T1) and human embryonic kidney cell line (HEK293T) were purchased from the Institute of Biochemistry and Cell Biology, Academy of Science (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) or RPMI 1640, supplemented with 10% foetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin (all from Thermo Fisher Scientific, USA). All cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂.

For isolation of GDNPs, fresh ginseng roots were purchased from Panax ginseng Base (Wanshan, Jilin, China) and washed with deionized water three times at room temperature (20 °C). After the final wash, ginseng roots were ground in a slow juicer to obtain ginseng fluid. Then, the juice was sequentially centrifuged at 200×g for 10 min, 2000×g for 20 min and 10,000×g for 30 min to remove large particles and fibres. The final supernatant was ultracentrifuged at 100,000×g for 60 min (Beckman Optima XE-100, Beckman, USA), and the pellets were resuspended in PBS, transferred to a gradient sucrose solution (15, 30, 45 and 60%) and ultracentrifuged at 150,000×g for another 60 min. The band at the 45% sucrose layer was collected and defined as GDNPs according to TEM (transmission electron microscopy) examination [22]. Finally, the GDNPs were diluted in PBS and ultracentrifuged at 100,000×g for 60 min, then the pellets were resuspended in sterile PBS. The resuspension was filtered (0.45 μm) and used freshly or stored at − 80 °C until further use.

The size and zeta potential of GDNPs were measured by dynamic light scattering using a Zetasizer nano ZS Zen3600 (Malvern, UK). For TEM imaging, a drop of purified GDNPs was deposited onto the surface of formvar-coated copper grids, followed by incubation with 1% uranyl acetate for 15 s. The samples were left to dry at room temperature and observed using a HITACHI H-7650 electron microscope operated at 200 kV at a magnification of 38,000×. The protein concentration in the GDNPs was quantified using a BCA protein assay kit (Beyotime Biotechnology, China) following the manufacturer’s instructions.

Human peripheral blood mononuclear cells (PBMCs) were collected from venous blood of healthy volunteers, and in leukocyte reduction chambers, diluted 2x with PBS and separated via Ficoll density gradient (Serumwerk Bernburg AG, Norway). CD14⁺ monocytes were positively selected to > 95% purity by MACS using anti-CD14 microbeads (Miltenyi, USA). Mouse bone marrow was collected by flushing the femurs of C57BL/6 mice (8~10 weeks old) with cold PBS. After collection, red blood cells were lysed with RBC lysis buffer (Thermo Fisher Scientific, USA), and the remaining cells were washed twice with PBS. For induction of macrophage differentiation, sorted monocytes or bone-marrow cells were cultured in RPMI 1640 or DMEM supplemented with 10% FBS and 20 ng/ml human or mouse macrophage colony-stimulating factor (M-CSF) (R&D Systems, USA). Fresh medium with M-CSF was added every 3 days. On day 7, M2-like polarization was achieved by treatment with human/mouse 20 ng/ml IL-4 and 20 ng/ml IL-13 (R&D Systems, USA) for 2 days. For M0, only DMEM-10% FBS was added. After polarization, the cells were phenotyped and used in different assays. The media from M0, M2 and GDNP-treated M2 macrophages was collected for ELISA, cytokines array and apoptosis assays.

For analysing the role of TLR signalling pathways in macrophage polarization induced by GDNPs, M2-like macrophages were purified from wild type, MyD88-, TLR4- and TLR2-deficient C57/BL6 mice and incubated with GDNPs for 72 h. The supernatants were collected for detection of IL-6 and tumor-necrosis factor-α (TNF-α) using ELISA kits (R&D Systems, USA). Then, the cells were harvested for detection of associated macrophage surface markers by flow cytometry analysis.

Cells (M0 macrophage, B16F10, 4T1 and HEK293T) were seeded on 12-chamber slides (Thermo Fisher Scientific, USA) at a density of 5 × 10⁵/well and cultured overnight at 37 °C. The media was then replaced with fresh culture media containing GDNPs (10 μg/ml) previously labelled with DiI (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. After 12 h incubation, the cells were fixed with 4% paraformaldehyde for 10 min and then dehydrated with acetone at − 20 °C for 5 min. After the cells were blocked with anti-CD16/32 (Fc block, BioLegend, USA) in PBS for 30 min, antifade mountant with 4′,6-Diamidino-2-Phenylindole (DAPI) (Thermo Fisher Scientific, USA) was added, and the mixture was incubated for an additional 30 min. Finally, cells were coverslip-mounted with mounting medium and imaged using an Olympus FV10i confocal microscope with Olympus Fluoview software version 4.0b (Olympus, Japan).

To determine the efficiency of GDNP uptake by macrophages, cells (1 × 10⁶/well) were co-incubated with DiI-labelled GDNPs (10 μg/ml) for 12 h or 24 h. The cells were harvested and single cell suspensions were prepared and analysed using the FACSAria II system (BD Biosciences, USA). Data analysis was performed using FlowJo Version7.6 (BD Biosciences, USA).

For analysis of biodistribution of GDNPs in vivo, healthy male C57BL/6 mice (6–8 weeks old) were intraperitoneally (i.p.), intragastrically (i.g.), intravenously (i.v.) and subcutaneously (s.c.) administrated DiR-labeled GDNPs. Mice were sacrificed and different organs were collected at 72 h after the injection. The intensity of DiR signal from different samples was then measured using IVIS series in vivo imaging systems (PerKinElmer, USA). In vivo stability of DiR dye-labelled GDNPs was determined by scanning (IVIS series) mice that received an i.p. injection of DiR-GDNPs for 7 days.

To study GDNPs taken up by macrophages in vivo, healthy male C57BL/6 mice (6–8 weeks old) were received i.p. injection with clodronate liposomes (CL, 200 μg per mouse) [23], which deplete macrophages (n = 3). After 3 days, mice were i.p. administrated with DiR-labeled GDNPs in the presence or absence of CL. Mice were sacrificed and organs were collected at 72 h after the injection. The intensity of DiR signal of livers and spleens from mice treated with DiR-labeled GDNPs was then measured using IVIS series. In addition, splenocytes from mice treated with DiI-labeled GDNPs for 72 h were obtained by gently pressing the spleens between two sterile glass slides followed by washing the lymphocytes with PBS. Single cell suspensions were prepared by filtering through a 100-μm nylon filter strainer and washed thoroughly in PBS. Nonspecific labelling was blocked with anti-CD16/32 followed by staining with the following mouse monoclonal antibodies (BioLegend, USA and Thermo Fisher Scientific, USA) to detect splenocytes surface markers: anti-CD45 APC; anti-CD45 Brilliant Violet 510; anti-CD3 FITC; anti-CD4 PE/Cy7; anti-CD335 PerCP/Cy5.5; anti-CD11b APC/Cy7; anti-F4/80 PE/Cy7; anti-Ly6C/6G APC; anti CD11c FITC; and anti-CD45R/B220 Brilliant Violet 510, according to the manufacturer’s instruction (Additional file 1: Table S1). The stained cells were analysed on a FACSAria II Flow Cytometer using BD FACSDiva software (BD Biosciences, USA), and the data were processed using FlowJo Version 7.6 (BD Biosciences, USA).

M2-like macrophages (1 × 10⁶/well) were incubated with or without GDNPs (10 μg/ml). At 48 h, supernatants were collected for detection of M1-associated cytokines, including IL-6 and TNF-α, using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocol (R&D Systems, USA). Then, the cells were harvested for measurement of gene expression and detection of surface markers as described below.

Total RNA was isolated from treated macrophages using TRIzol reagent (TaKaRa, Japan) and reverse transcribed into cDNA using a cDNA Synthesis Kit (TaKaRa, Japan) according to the manufacturer’s protocol. Then, RT-PCR was performed using SYBR Green Mastermix (Toyobo, Japan) following the manufacturer’s instructions and run on an ABI Prism 7500 Sequence Detection System (Applied Biosystems, USA). The primer sequences are shown in Additional file 1: Table S2. The 2^ΔΔCt method was used to calculate fold changes in gene expression normalized to the housekeeping gene Gapdh (Additional file 1: Table S2).

Single-cell suspensions of treated macrophages were prepared in PBS. Nonspecific labelling was blocked with anti-CD16/32 followed by staining with the following mouse monoclonal antibodies (BioLegend, USA and Thermo Fisher Scientific, USA) to detect macrophage surface markers: anti-CD206 APC; anti-CD80 APC; anti-CD86 PE; anti-MHC-II FITC; anti-CD11b APC; anti-F4/80 PE; anti-TLR2 FITC; and anti-TLR4 PE/Cy7, according to the manufacturer’s instruction. For each sample at least 2 × 10⁴ cells were analysed by flow cytometry. Data analysis was performed using FlowJo software (BD Biosciences, USA).

For lipidomic analysis, lipids from GDNPs were submitted to the APTBIO Co. Ltd. (Shanghai, China). Briefly, high-throughput identification and relative quantification of lipids was performed separately for positive and negative ionization mode data using LipidSearch software (Thermo Fisher Scientific, USA) using the default parameters for QExactive as previously described [24]. Data for each lipid molecular species were presented as mol % of the total lipids analyzed.

GDNPs were digested with proteinase K or DNase I/RNase I according to the manufacturer’s instructions (Beyotime Institute of Biotechnology, China). After digestion with proteinase K or DNase I/RNase I, proteins or DNA in supernatant were analyzed via 2% SDS polyacrylamide gel electrophoresis or agarose gel electrophoresis. The effect of protein- and nucleic acid-depleted GDNPs on macrophage polarization was analyzed as described above.

On day 0 of the experiments, sixteen C57BL/6 male mice (6–8 weeks old) were subcutaneously inoculated with 2 × 10⁵ B16F10 cells on their right flanks. The tumor size was measured every 2 days using digital calipers and tumor volume was calculated using the following equation: V = (length×width²)/2. Body weight was also monitored every two days. On day 7 post-tumor implantation, tumor-bearing mice were randomly separated into two groups (8 mice per group) that received four intraperitoneal injections (100 μl per mouse) in total, administered every four days, containing the following formulations: PBS and GDNPs (250 μg per mouse). Tumor growth was monitored for up to 21 days after implantation, at which point animals were euthanized in a CO₂ chamber and tumors were removed for further analysis. All tumors were divided in two pieces, weighed and processed for both flow cytometry and histopathology as described below.

To investigate tumor growth inhibition in our mouse melanoma model was driven by GDNP-mediated TAMs polarization, B16F10-bearing C57BL/6 mice were treated with GDNPs in the presence or absence of clodronate liposomes. CL treatment (200 μg per mouse) was repeated every 4 days by i.p. injection. Mice in the control group (n = 5) were treated with the same dose of liposomes containing PBS at the same time. The tumor size was measured and mice were treated with GDNPs as described above. Twenty-one days after implantation, tumors were processed for IF.

Tumor samples were minced with scissors before incubation with 66 μg/ml Liberase and 0.2 mg/ml DNase (Roche, Switzerland) in DMEM for 30 min at 37 °C. Single cell suspensions were prepared by filtering through a 100-μm nylon filter strainer and washed thoroughly in Hank’s balanced salt solution (HBSS) buffer supplemented with 2% FBS, 20 mM HEPES, and 5 mM EDTA. Fixable Viability Dye (Thermo Fisher Scientific, USA) was applied to eliminate dead cells in combination with anti-CD16/32 monoclonal antibody (BioLegend, USA) for 15 min on ice in the dark. Then, cells were stained for 30 min in PBS with appropriate dilutions of various combinations of the following fluorochrome-conjugated antibodies: anti-CD206 PE; anti-CD86 APC/Cy7; anti-CD45 APC; anti-CD11b FITC; and anti-F4/80 PE/Cy7; anti-CD4 PE/Cy7; anti-CD3 APC/Cy7; anti-CD25 APC; anti-CD8 FITC; anti-NK1.1 PE; anti-CD45 Brilliant Violet 510; anti-CD45R Brilliant Violet 510; anti-CD335 PerCP/Cy5.5; anti-CD45 Brilliant Violet 421; anti-Ly6G Alexa Fluor® 647; anti-CD11c FITC. For intracellular staining, cells were further permeabilized using a FoxP3 Fixation and Permeabilization Kit (Thermo Fisher Scientific, USA) and stained with anti-FoxP3 PE antibody (Thermo Fisher Scientific, USA). All flow cytometry data was acquired and analyzed as mentioned above.

The results are expressed as the mean ± standard error (S.E.M). All data were analyzed using GraphPad Prism 6.0 (GraphPad Software, USA) by unpaired Student’s t-test and two-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

To isolate EVs from ginseng, ginseng roots were harvested, followed by a combination of extraction, filtration and differential centrifugation. Four bands were formed after sucrose gradient ultracentrifugation. Transmission electron microscopy (TEM) examination indicated the majority of the GDNPs accumulated at the 45% interface (band 3), generally spherical in shape (Fig. 1a and b). Purified GDNPs were identified with an average diameter (as determined by dynamic light scattering) of ~ 344.8 nm (band 3) and a low polydispersity (Fig. 1c). Zeta potential analysis indicated that GDNPs had a negative zeta potential value of − 25.4 mV (Fig. 1d). The GDNPs were quantified by protein concentration using a micro BCA protein assay kit. Extracts from ginseng roots are enriched for nanoparticles (approximately 500 mg/kg ginseng), suggesting that ginseng could produce a large amount of GDNPs. In addition, the ginsenoside Rg3 were detected highly concentrated in the GDNPs by electrospray spray ionization (ESI) scanning. Our data indicated that the concentrations of the ginsenoside Rg3 were similar within different batches (Fig. 1e). It is a potential component to control quantities of GDNPs within different batches.

Next, we analysed the composition of the purified GDNPs using mass spectrometry (MS) in duplicate. Metabolic analysis revealed that GDNPs contained amino acids (~ 25%), nucleotides (~ 13%), lipids/fatty acids (10%) and organic acids (8%) (Fig. 1f). In addition, we identified 3129 proteins that were classified using Gene Ontology (GO) analysis into three categories: biological process, cellular compartment and molecular function (Additional file 2: Figure S1a-c).

Previous reports have shown that ginseng polysaccharide extracts stimulate the activity of macrophages and enhance the production of different mediators or active components [4, 5]. Since macrophages exhibited a high uptake potential for nanoparticles, we tested whether GDNPs are taken up by macrophages in vitro. BMDM, B16F10, 4T1 and HEK293T were incubated with GDNPs labelled with DiI, a lipophilic fluorescent dye, for 12 h. Compared to other cells, we found that GDNPs (red) were taken up more effectively by BMDMs and preferentially localized in the cytoplasm of the cells (Fig. 2a). Flow cytometry showed that the percentage of cells containing GDNPs increased with time from 41.3% at 12 h to 57.4% at 24 h (Fig. 2b and c).

To determine in vivo biodistribution of GDNPs, we first evaluated the effect of different routes of administration of DiR-labelled GDNPs. At 72 h following i. p. and i.v. injection, the majority of the DiR-labelled GDNPs were located in the liver and spleen, whereas i.g. administration of DiR-labelled GDNPs were predominantly localized in stomach and gut when compared with PBS-treated control mice. However, no signal was detected in lung, heart, kidney and brain (Fig. 2d; Additional file 2: Figure S2a and b). In vivo imaging to continuously track the stability of injected DiR-GDNPs further revealed that fluorescent signals remained strong in the liver and spleen at day 7 (Additional file 2: Figure S2c). Our findings suggested that the size and structure of nanoparticulate increased the stability and retention of GDNPs in the circulation.

FACS analysis was done on splenic cells from mice received i.p. injection of DiI- GDNPs. The results indicated that 72 h after GDNPs were i.p. administration, they were easily taken up by macrophages (13.7%) (Fig. 2e). Upon analysis of the biodistribution of DiR-GDNPs with i.p. injection in the presence of CL, we found that the DiR fluorescent signals decreased significantly in the liver and spleen (Additional file 2: Figure S3a and b). Our findings showed the cellular tropism of GDNPs to macrophages in vitro and in vivo.

To assess the biocompatibility of GDNPs in vitro, cell viability assays were performed. The results of cell viability assays revealed that GDNPs exhibited no cytotoxicity on cells for 72 h, even at the high concentration of 30 μg/mL (Additional file: Figure S4a). To further evaluate the biosafety of GDNPs in vivo, mice were treated with GDNPs via i.p. injection. Sex-, age- and weight-matched healthy mice were used as controls. The body weights of the mice from GDNPs treated groups did not differ significantly from those of the control group (Additional file: Figure S4b). Two weeks after injection, all mice were euthanized for blood biochemistry and hematology analyses as well as histological examinations. As shown in Additional file: Figure S4c and d, i.p. injection of GDNPs did not lead to any changes in blood cells, hemoglobin, and platelets. No statistically significant differences were detected by evaluating liver enzymes, kidney function and hematologic toxicity. In addition, the heart, lungs, liver, spleen and kidneys were collected for haematoxylin & eosin (H&E) staining (Additional file: Figure S4e). No apparent organ or tissue damages in the brain, heart, kidney, liver, lungs or spleen were observed in GDNP-administrated mice, compared with those in control group. Thus, these results indicate that GDNPs show no significant toxic effects in the range of administration in vitro and in vivo. 2 2 2 2

M2-like macrophages account for the majority of TAMs. Thus, inhibiting or altering M2-like cells is considered to be an effective therapeutic strategy in cancer therapy. Next, we determined whether GDNPs can alter M2-like polarization of macrophages. To this end, we incubated BMDMs with IL-4 and IL-13 for 24 h to polarize the cells to an M2-like phenotype, and then added GDNPs (10 μg/ml) for 48 h. Polarization corroboration by flow cytometric analysis was performed to examine the levels of polarization-related surface markers CD80^mid, CD86^mid, MHC-II(I-A^b)^mid, TLR2/4^mid, and CD206^high, characteristic of M1/M2 macrophages [25–27]. Treatment with GDNPs resulted in significantly reduced levels of CD206 in M2-like macrophages, whereas the expression of CD80, CD86, MHC-II and TLR2/4 was up-regulated (Fig. 3a).

To further confirm that treatment with GDNPs alter M2-like polarization, we prepared RNA samples from M1 and M2 macrophages and measured the expression of M1- and M2-associated genes using quantitative real-time polymerase chain reaction (RT-PCR). Transcriptional profiling revealed that GDNP exposure significantly induced M1-related markers while M2-associated markers were down-regulated (Fig. 3b). Increased production of the M1 markers, IL-6 and TNF-α, in the medium of GDNP-treated macrophages was further verified by ELISA (Fig. 3c).

Moreover, production of inflammatory cytokine by M2-like macrophages before and after GDNP treatment was quantified in the macrophage medium using a cytokine array. The results showed that treatment with GDNPs resulted in a dramatic increase in the production of M1-related cytokines and chemokines, such as CCL5, IL-6, MCP-1, TNF-α IL-1α and IL-12 (Fig. 3d). As shown in the heat maps of human cytokines microarray analysis, the similar results were observed (Fig. 3e). The proinflammatory cytokine IL-12 and TNF-α are known to promote cell-mediated immunity via stimulation of Th1 immune response. Thus, we tested the suppressive function of M2 macrophages with GDNPs treatment on naive CD8⁺ T cell proliferation. The results showed that suppression of CD8⁺ T cells was mitigated when GDNPs were added to the M2 macrophages (Fig. 3f). Collectively, these data revealed that GDNPs effectively inhibit M2-like polarization of macrophages in vitro.

In innate immunity, macrophages produce proinflammatory mediators upon activation of several receptors that recognize pathogens, including the family of Toll-like receptors (TLRs) [28–30]. Based on cytokine array examination, the signalling pathway analysis suggested that the response induced by GDNPs on macrophages is similar in TLRs/MyD88 (myeloid differentiation antigen 88) signalling pathway induced by pathogen-associated molecular patterns (PAMPs) (Fig. 3g). We hypothesized that the immunomodulatory effect of GDNPs might occur via a similar signalling pathway. To examine this hypothesis, we generated M2-like macrophages from mice deficient in MyD88, a common signalling adaptor for different TLRs [31]. Up-regulation of M1-related surface markers and production of the cytokines IL-6 or TNF-α did not occur when MyD88^−/− M2-like macrophages were incubated with GDNPs (10 μg/ml) for 48 h (Fig. 3h). To determine which TLRs were specifically responsible for the GDNP-induced M1-like macrophages associated cytokines production, the response to GDNP treatment was analysed in M2-like macrophages derived from mice lacking TLR2 or TLR4. We found that TLR2^−/− M2-like macrophages produced those cytokines in response to GDNPs but TLR4^−/− M2-like macrophages failed to do so (Fig. 3i). These findings suggest that TLR4 on macrophages may interact with ligands on GDNPs, resulting in macrophage polarization.

For evaluating whether the EVs-like nanoparticles from non-medicinal plant have the similar effect of macrophages polarization, EVs-like nanoparticles from cucumber (Cucumis sativus L.) and kiwi (Actinidia chinensis) were isolated. BMDMs were incubated with nanoparticles (10 μg/ml) derived from ginseng (GDNPs), cucumber (CDNPs) and kiwi fruit (KDNPs) for 48 h. Polarization corroboration by FACS analysis was performed to examine the levels of M1-related surface markers. These results showed that nanoparticles from cucumber and kiwi fruit could not polarize macrophages to M1 type (Additional file 2: Figure S5).

Since M1-like macrophages are actually capable of killing tumor cells by producing proinflammatory cytokines, promoting T helper type 1 cell response and releasing ROS [26, 32]. We investigated the effect of GDNPs on the macrophage-tumor cell interaction. M2-like macrophages were treated with or without GDNPs (10 μg/ml), and the culture medium was replaced with fresh medium. After further 48 h, the supernatant medium was collected as conditioned medium (CM). To quantitatively assess the apoptotic effects of different CM, we treated B16F10 melanoma cells with the different CM for 24 h and stained them with an annexin V-PE/7-AAD apoptosis assay detection kit. We found that treatment with CM from GDNP-stimulated macrophages significantly increased apoptosis of B16F10 melanoma cells compared to treatment with CM from unstimulated macrophages as measured by increased Annexin V binding (Fig. 4a) and increased caspase 3/7 expression (Fig. 4b).

Previous reports have demonstrated that production of total ROS and superoxide levels is increased in tumoricidal M1-like macrophages, resulting in production of highly toxic hydrogen peroxide through TLR-mediated signalling [33]. Therefore, we measured total ROS production in M2-like macrophages treated with or without GDNPs. We found that total ROS production in GDNP-treated M2-like macrophages was higher than in untreated macrophages (Fig. 4c). We further investigated whether hydrogen peroxide was induced by GDNPs. As shown in Fig. 4d, treatment with GDNPs resulted in a 14-fold increase in hydrogen peroxide production in M2-like macrophages. Thus, treatment of macrophages with GDNPs increases the production of ROS, which is known to contribute to the tumoricidal function of M1-like macrophages.

To explore which components in GDNPs mediate the polarization of macrophages. We assessed comparative lipid profiles generated from lipidomic analysis (Additional file 1: Table S3). The results revealed that GDNPs were enriched with digalactosyl monoacylglycerol (DGMG, 59.4%), phosphatidyl ethanolamine (PE, 16.8%) and ceramide (Cer, 13.8%) (Fig. 5a). In contrast, the majorities of the lipid in other plants derived nanoparticles were phosphatidylcholine (PC) and glycerophosphate (PA), whereas DGMG and Cer were not detected [34].

Next, we digested GDNPs with proteinase K or DNase I/RNase I or ultrasonic and used these protein- and nucleic acid-depleted GDNPs to treat M2-like macrophages (Fig. 5b-d). Proteinase K treatment of EVs was shown to significantly reduce their uptake by ovarian cancer cells which strongly supports the role of proteins in the EV uptake pathway [35]. Many EV proteins have been shown to interact with membrane receptors on target cells [36, 37]. We found that DNase I/RNase I treatment did not affect GDNP-induced up-regulation of M1-related surface markers (Fig. 5e), suggesting that GDNP-associated nucleic acids were not involved in macrophage polarization. By contrast, up-regulation of these surface markers did not occur in macrophages stimulated with proteinase K-digested GDNPs, indicating that GDNP proteins may participate in the effect of these particles on macrophages polarization.

In addition, no significant differences in IL-6 and TNF-α secretion were observed in macrophages exposed to DNase I/RNase I-digested GDNPs compared to macrophages exposed to undigested GDNPs. However, only small amounts of IL-6 and TNF-α were detected when macrophages were treated with proteinase-digested GDNPs (Fig. 5f). Interestingly, treatment with sonicated GDNPs resulted in both down-regulation of M1-associated surface markers and a significant reduction in IL-6 and TNF-α secretion by M2-like macrophages compared with treatment with unsonicated GDNPs, indicating that the intact structure of GDNPs is necessary for macrophage polarization.

To further evaluate the routes of uptake of GDNP by macrophages, the percentage of DiI-GDNPs in cells was examined by using confocal microscopy (Additional file 2: Figure S6a) and FACS analysis, and determined by quantitative analysis of DiI-GDNPs⁺ cells (Additional file 2: Figure S6b and c). Our results indicated that uptake of GDNPs was remarkably inhibited by LY294002, but treatment with 5-(N,N-Dimethyl) amiloride hydrochloride (EIPA), the macropinocytosis inhibitor, resulted in no reduction of GDNPs uptake, suggesting that macropinocytosis is not main route of macrophages internalizing GDNPs. By FACS analysis, we further demonstrated that macropinocytosis inhibition had no apparent effect on the polarization of macrophages with GDNPs treatment, compared with phagocytosis inhibition (Additional file 2: Figure S6d). These findings suggested that the polarization of macrophages depended on GDNPs internalization.

Our in vitro results showed that GDNP treatment of macrophages results in altering M2-like polarization, increased production of proinflammatory cytokines and ROS, and induction of apoptosis of melanoma cells. Next we investigated whether treatment with GDNPs had a similar effect in vivo and resulted in a change in TAM polarization towards a favourable antitumor profile. To this end, we established a tumor-bearing mouse model by subcutaneously inoculating B16F10 cancer cells into the right flanks of male C57BL/6 mice as described previously [21]. After 7 days, mice were treated with PBS (control) or GDNPs every four days and the experiment was ended on day 21 post-tumor implantation (Fig. 6a). Treatment with GDNPs significantly suppressed tumor growth as measured by tumor volume from day 14 of treatment (Fig. 6b). In addition, at day the end of the experiment, tumor weight in GDNP-treated mice was reduced by 53% (Fig. 6c; Additional file 2: Figure S7a), and these mice had gained more body weight than control mice (Fig. 6d).

To better understand the antitumor mechanism induced by GDNPs, we purified the tumor-infiltrating leukocytes (TILs) using specific anti-CD45 antibodies and analysed the different cell populations by multicolour flow cytometry and immunofluorescence (IF). Treatment of B16F10-allografted mice with GDNPs for 21 days resulted in a significantly higher quantity of M1 macrophages in the TIL population (Fig. 6e) than in the PBS treated control mice. The ratio of M1 cell counts (CD86⁺/total macrophages) was significantly higher in GDNP-treated mice than in control mice. In addition, the median fluorescence intensity (MFI) of CD86, a marker of M1-like macrophages, was significantly elevated in GDNP-treated mice. Meanwhile, the proportion of CD206-positive cells, which indicate M2-like macrophages, decreased among all examined macrophages in GDNP-treated mice. These results were further confirmed by IF staining (Fig. 6f and g). We evaluated major immune cell in TME by FACS analyses at day 21 after B16F10 cancer cells implantation. The proportion of T cells and natural killer (NK) cells in the TILs population were increased in the tumors of GDNP-treated mice (Fig. 6h). And the GDNPs treatment correlates with increased T cell infiltrates and a higher CD8⁺/regulatory T cell ratio at day 21 in tumors (Fig. 6i). In comparison, there are no remarkable differences in the quantity of DCs, B cells and granulocytes in the TME (Additional file 2: Figure S7b). FACS and IF images analysis was also done on main sets of TILs from tumor-bearing mice received i.p. injection of DiI-labelled GDNPs. The results indicated that 14 days after GDNPs were i.p. administration, the majority of them taken up by macrophages (23.2%) in the TME, with minority populations of DCs (3.92%), granulocytes (3.42%) and CD8⁺ T cells (2.51%) (Additional file 2: Figure S7c and d). Confocal fluorescence analysis further indicated GDNPs co-localized mainly with macrophages signal in liver, spleen and tumor (Additional file 2: Figure S7e). Moreover, the impact of GDNPs was compromised in mice lacking T cells (after anti-CD8⁺ T cell depletion) (Additional file 2: Figure S8). Therefore, our results showed that GDNP-treatment in vivo alters M2-like polarization to M1-like polarization with the consequent increase in T cells in the TME.

Finally, we studied whether treatment with GDNPs induces tumor cell apoptosis in vivo by histopathology analysis. Similarly to what we had observed in vitro, we found that treatment with GDNPs induced death of tumor cells in TME (Fig. 6j). Overall these results indicate that GDNP treatment polarizes M1 macrophages, represses M2 macrophages and subsequently induces tumor cell death, thus inhibiting tumor growth.

To further determine whether macrophage was responsible for the GDNP-mediated tumor growth inhibition, we compared tumor growth and M1 polarization in tumor-bearing mice treated with either GDNPs or GDNPs plus clodronate liposomes, which deplete TAMs. Control mice inoculated with melanoma cells were treated with control liposomes that were coated in PBS. As expected, macrophages were depleted efficiently with clodronate liposomes treatment (Fig. 6k). We found that no significant tumor growth inhibition in mice treated with GDNPs plus clodronate liposomes (Fig. 6l). Control mice also showed no significant tumor growth inhibition. These results confirmed that GDNPs inhibit tumor growth through a TAM-dependent mechanism.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.