The LNC internalization by blood leukocyte subsets depends on particle size.
Given the importance of T cell activation and the negative role of myeloid cells in anti-tumor immune response, we rationally modified LNC formulations to target myeloid cells, while reducing the uptake by T cells. LNCs, prepared as previously described, were composed of FDA-approved excipients showing a good safety record and a tunable size (between 20-100 nm) [22, 23]. In this study, we tested LNCs with different physico-chemical properties, i.e. variable size and surface charge, to modulate cell uptake [4, 24, 25].
First, we tested the uptake of LNCs with 25 nm, 50 nm, and 100 nm size, neutrally charged (Table 1), and loaded with the fluorescent dye DiD (DiD-LNC). Incorporation by leukocyte subsets present in the peripheral blood of HDs was carried out by multicolor flow cytometry, with mAbs directed against markers present on the cell surface of different leukocyte cell subsets and by assessing the signal emitted by DiD in T cells (CD3+), monocytes (CD14+), B cells (CD19+), NK cells (CD56+), eosinophils (CD11b+/CD16-), and polymorphonuclear cells (PMN, CD11b+/CD16+). Blank-LNCs were used as control. As shown in Figure 1A, the incorporation of 25 nm LNCs was very low in all the considered leukocyte populations, while 50 nm LNCs showed the highest uptake in the analyzed subsets and in particular in monocytes (30.1±3.2%). The 100 nm LNCs did not reach the same uptake of 50 nm LNCs on monocytes (19.4±0.4%) but allowed the reduction in internalization by T lymphocytes (10.5±3.1% with 50 nm LNCs vs 3.3±0.9% with 100 nm LNCs). We excluded from the analysis the 25 nm LNCs and further investigated the internalization properties of neutral 50 nm and 100 nm LNCs, focusing on monocytes and T cells, and increasing the incubation time from 90 minutes to 3 hours in order to reach the highest LNC internalization (Figure 1B). Under these experimental conditions, both LNC formulations reached comparable high levels of internalization in monocytes, but the incorporation by T cells was significantly lower when 100 nm LNCs were used (Fig. 1B). By calculating the ratio between the signal of DiD in monocytes and T cells, we observed that 100 nm LNCs allowed increasing specificity of LNC targeting towards monocytes (mean ratio of 4.9±2.7 for 50 nm LNCs vs 11.2±3.8 for 100 nm LNCs) (Figure 1C). Therefore, neutral 100 nm LNC formulation was chosen for further experiments.
Effect of 100 nm LNC surface charge on the internalization ability of peripheral blood leukocytes (PBLs)
We next set out to assess the surface charge of 100 nm LNCs to increase the specific uptake by monocytes compared to all the other main leukocyte populations. To this aim, we compared 100 nm neutral LNCs (-3 mV) with a slightly positive surface charge. The loading of cationic surfactant DDAB in nanosystems did not alter the size of the systems, while it affected the surface properties of the LNCs. The physico-chemical characteristics are summarized in Table 2.
After 3h of incubation of PBLs with DiD-loaded LNCs (Figure 2A), the internalization by T cells was very low and comparable in all the tested LNC formulations, while in monocytes the incorporation was always significantly higher than that of T cells, and had a trend toward an increase as the positive charge augmented (98.6±1.2% of DiD+ monocytes using +31 mV LNCs vs 90.7±4.7% with neutral LNCs; 82.0±16.5% with +6 mV LNCs; 85.1±12.9% with +16 mV LNCs; 95.6±4.3% with +25 mV LNCs).
We therefore tested the internalization of 100 nm positive LNCs (+31 mV) by all leukocyte subsets present in the peripheral blood of HDs and compared the results to neutral LNC formulation (Figure 2B). Monocytes showed the highest incorporation of positive (+31 mV) LNCs (92.0±1.4%), but a high uptake was also noticed for B lymphocytes (43.7±19.2%), an effect that was not observed using neutral LNCs (2.1±0.3%). This could lead to a deleterious consequence on the patient’s immune response if LNCs loaded with a cytotoxic drug were used. We thus selected the 100 nm neutral surface charge formulation to target monocytes in the peripheral blood and avoid incorporation by B cells.
Targeting circulating immunosuppressive cells in GBM patients by 100 nm neutral LNCs
Data from literature  and our own results (Pinton et al., unpublished) indicate that GBM patients have a significant expansion in circulating MDSC populations. We thus tested the uptake of the 100 nm neutral LNC formulation on leukocyte subsets present in the peripheral blood of these patients, extending the analysis to three MDSC subsets: two monocytic subsets (identified as CD14+/IL4Rα+ and CD14+/HLA-DRlow cells) and one PMN type (CD15+/IL4Rα+) . Following PBL incubation with DiD-loaded LNCs, maximum LNC internalization was observed by total monocytes (CD14+ cells: 83.0±6.4%) and by monocyte subsets CD14+/HLA-DRlow (86.4±8,2%) and CD14+/IL4Rα+ (84.7±6,9%) cells, corresponding to monocytic MDSCs (Fig. 3A), thus highlighting that this nanocarrier system could efficiently target immunosuppressive myeloid cells in GBM patients, while sparing lymphocyte subsets that, instead, showed very low uptake. A lower level of incorporation was observed in PMN-MDSCs, defined as CD15+/IL4Rα+ cells, as compared to the two monocytic subsets; it should be noted that this immunosuppressive granulocytic population shows a significantly higher LNC uptake as compared to total PMNs (defined as CD15+ cells) (7.6±7% DiD+ cells in CD15+/IL4Rα+ vs 2.4±1.2% DiD+ cells in PMNs), thus reinforcing the efficacy of LNCs in targeting immunosuppressive cells. To confirm intracellular localization of LNCs, we performed confocal analysis in isolated peripheral blood mononuclear cells (PBMCs) and PMN fractions, and observed that DiD-loaded lipid nanoparticles are internalized by monocytes showing cytoplasmic localization, while no uptake by granulocytes was observed (Fig. 3B).
Mechanism of LNC internalization by circulating monocytes
To gain evidence about the mechanisms involved in 100 nm neutral LNC internalization by monocytes, PBLs from GBM patients were treated with inhibitors of different uptake mechanisms and their effect was verified on particle internalization. Colchicine was used to inhibit pinocytosis , cytochalasin B as inhibitor of phagocytosis , LY294002 and Wortmannin as inhibitors of fluid phase pinocytosis and FcR-mediated phagocytosis [27, 28], and nystatin as inhibitor of caveolae-mediated endocytosis . Results indicate that nystatin is the most effective inhibitor, causing a significant reduction in the uptake of monocytes (Figure 4A). Another inhibitor that showed a trend toward a reduced LNC internalization by monocytes is colchicine, although its effect did not reach the same level observed with nystatin, lacking statistical significance. Altogether, these results indicate that 100 nm neutral LNCs are mainly internalized by monocytes from GBM patients through caveolae-mediated endocytosis. We also visualized the effect of nystatin on LNC uptake by PBMCs from three glioma patients, by means of confocal microscopy. Monocytes were enriched by adhesion onto microscope slides and further selected by nuclear morphology; following nystatin treatment, we observed a remarkable reduction in the internalization of DiD-loaded LNCs (Fig. 4B), thus confirming the results obtained by flow cytometry.
Evaluation of LNC uptake by the cells present in GBM microenvironment
Since 100 nm neutral LNCs efficiently target myeloid cells in the blood of GBM patients, we evaluated whether the same nanosystem could be incorporated by different cell subsets present in the tumor microenvironment. In fact, our recent results highlight the presence of an abundant leukocyte infiltrate of myeloid origin, mainly constituted by macrophages, as a main characteristic in GBM. We demonstrated that only macrophages of bone marrow origin, and not the resident microglial cells (MG), were endowed with a strong immunosuppressive activity . In fact, during tumor growth, macrophages originating from bone-marrow (BMDM) are recruited to the tumor, and can be distinguished by tissue resident MG through a marker combination in multicolor flow cytometry (Fig. 5A upper panels) [17, 18]. We therefore tested the incorporation of different LNC formulations with a size of 100 nm, carrying overall positive, neutral or negative charge, in the cell suspension freshly obtained from tumor tissue and analyzed the incorporation by BMDM, MG, tumor cells, PMNs, and lymphocytes. The maximum LNC uptake was obtained for macrophages (BMDM and MG) and tumor cells when positively charged LNCs were used; the low level the incorporation by T cells was comparable across different formulations. Moreover, both BMDM and MG cells also reached an uptake significantly higher than PMNs and this LNC formulation also allowed an increased uptake by tumor cells, evaluated as CD45- cells (Fig. 5A and B).
Positively charged LNCs show a very high uptake by B cells from the peripheral blood (Fig.2B), but B lymphocytes are not present in GBM tumors (data not shown); therefore, these data indicate that 100 nm positively charged LNCs could be used as a drug-loaded nanosystem to target the main immune suppressive cell subset in these tumors. Moreover, tumor cells also show a significant uptake, thus reinforcing the possibility of using this nanosystem to target both tumor cells and tumor-promoting cells.
Mechanism of LNC internalization by the GBM microenvironment
Given the high ability of tumor macrophages to internalize positively charged LNCs, we analyzed the mechanism by which these nanoparticles were incorporated by the cells of the tumor microenvironment. We thus treated the cell suspension obtained after enzymatic digestion of GBM tumor tissue with uptake inhibitors and added DiD-loaded LNCs to cell suspensions for overnight incubation. As shown in Figure 6, internalization by both types of macrophages (BMDM and MG) was significantly reduced by the addition of nystatin, in line with the internalization observed by blood monocytes. A reduction in LNC uptake was also observed using colchicine in both BMDM and MG and cytochalasin B in BMDM, but without statistical significance.