Peripherally Administered Nanoparticles Target Monocytic Myeloid Cells, Secondary Lymphoid Organs and Tumors in Mice

Nanoparticles have been extensively developed for therapeutic and diagnostic applications. While the focus of nanoparticle trafficking in vivo has traditionally been on drug delivery and organ-level biodistribution and clearance, recent work in cancer biology and infectious disease suggests that targeting different cells within a given organ can substantially affect the quality of the immunological response. Here, we examine the cell-level biodistribution kinetics after administering ultrasmall Pluronic-stabilized poly(propylene sulfide) nanoparticles in the mouse. These nanoparticles depend on lymphatic drainage to reach the lymph nodes and blood, and then enter the spleen rather than the liver, where they interact with monocytes, macrophages and myeloid dendritic cells. They were more readily taken up into lymphatics after intradermal (i.d.) compared to intramuscular administration, leading to similar to 50% increased bioavailability in blood. When administered i.d., their distribution favored antigen-presenting cells, with especially strong targeting to myeloid cells. In tumor-bearing mice, the monocytic and the polymorphonuclear myeloid-derived suppressor cell compartments were efficiently and preferentially targeted, rendering this nanoparticulate formulation potentially useful for reversing the highly suppressive activity of these cells in the tumor stroma

Engineering antigens for in situ erythrocyte binding induces T-cell deletion

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens

6-Thioguanine-loaded polymeric micelles deplete myeloid-derived suppressor cells and enhance the efficacy of T cell immunotherapy in tumor-bearing mice

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress effector T cell responses and can reduce the efficacy of cancer immunotherapies. We previously showed that ultra-small polymer nanoparticles efficiently drain to the lymphatics after intradermal injection and target antigen-presenting cells, including Ly6c(hi) Ly6g(-) monocytic MDSCs (Mo-MDSCs), in skin-draining lymph nodes (LNs) and spleen. Here, we developed ultra-small polymer micelles loaded with 6-thioguanine (MC-TG), a cytotoxic drug used in the treatment of myelogenous leukemia, with the aim of killing Mo-MDSCs in tumor-bearing mice and thus enhancing T cell-mediated anti-tumor responses. We found that 2 days post-injection in tumor-bearing mice (B16-F10 melanoma or E.G7-OVA thymoma), MC-TG depleted Mo-MDSCs in the spleen, Ly6c(lo) Ly6g(+) granulocytic MDSCs (G-MDSCs) in the draining LNs, and Gr1(int) Mo-MDSCs in the tumor. In both tumor models, MC-TG decreased the numbers of circulating Mo- and G-MDSCs, as well as of Ly6c(hi) macrophages, for up to 7 days following a single administration. MDSC depletion was dose dependent and more effective with MC-TG than with equal doses of free TG. Finally, we tested whether this MDSC-depleting strategy might enhance cancer immunotherapies in the B16-F10 melanoma model. We found that MC-TG significantly improved the efficacy of adoptively transferred, OVA-specific CD8(+) T cells in melanoma cells expressing OVA. These findings highlight the capacity of MC-TG in depleting MDSCs in the tumor microenvironment and show promise in promoting anti-tumor immunity when used in combination with T cell immunotherapies

Monocytes internalize nanoparticles via macropinocytosis while B and T cell associate externally.

<p>(<b>a</b>) Representative flow cytometry plots of <i>in vivo</i> NP-Dy649⁺ uptake kinetics after intradermal administration: monocytes (CD11b⁺GR1^midSSC^lowF4/80⁺) and B cells (B220⁺) in the spleen. (<b>b</b>) Characteristic flow cytometry plots of biotinylated nanoparticle (NP-biotin) association with splenic (B, CD4, and CD8 cells) and bone marrow (CD11b⁺Ly6c⁺ and CD11b⁺Ly6g⁺) cells after 12 h incubation <i>in vitro</i>. To distinguish surface-associated- from internalized-NPs, cells were incubated before permeabilization with streptavidin-A488 (for extracellular association) and after permeabilization with streptavidin-A647 (for intracellular uptake). (<b>c</b>) Percentage of fluorescently labeled NPs (NP-Dy649) taken up by bone marrow cells as a function of the PI3K inhibitor (Ly294002) concentration. Bone marrow cells were incubated with increasing concentrations of Ly294002 (maximum 50 µM) for 45′ prior to the addition of NP-Dy649 for 12 h. Cells were subsequently stained and analyzed by flow cytometry. Open circles: CD11b⁺Ly6c⁺, filled squares: CD11b⁺Ly6g⁺, continuous line: vehicle control (VH, DMSO) for CD11b⁺Ly6c⁺, dashed line: VH for CD11b⁺Ly6g⁺.</p

Nanoparticles target lymph node dendritic cells better after i.d. vs. i.m. delivery.

<p>(<b>a</b>) Blood concentrations of Dy649-labeled NPs after i.v., i.m. and i.d. administration. (<b>b</b>) Heat maps representing the median percentage of NP⁺ cells for indicated cell populations. Note that maxima vary from 10% in total leukocytes to 100% in monocytes. <i>P</i> values were computed by comparing the adjusted means of each organ between i.d. and i.m. for each cell type with a two-tailed Student's t-test. (<b>c</b>) Importance of route of administration for each cellular subtype. The log-likelihood ratio represents the likelihood of the alternate model, i.e. the model without taking account the route of administration, over the likelihood of the full factorial model. <i>P</i> values were computed using the <i>Chi</i> Square test between the alternate model and the full model for each population. For 144 h, <i>n</i> = 2, for all else, <i>n</i>≥4. Leukocytes: CD45⁺, mature myeloid DCs: CD11c⁺CD11b⁺I/A^b+, cross-presenting DCs: CD11c⁺CD8α⁺ I/A^b+, immature myeloid DCs: CD11c⁺CD11b⁺I/A^b−, immature lymphoid DCs: CD11c⁺CD11b⁻I/A^b−, medullary/red pulp (RP) macrophages (MØ): CD11b⁺F4/80⁺, monocytes: CD11b⁺GR1^midSSC^lowF4/80⁺, granulocytes: CD11b⁺GR1^highSSC^high, T cells: CD3ε⁺, B cells: B220⁺. Draining lymph nodes are indicated by Ax: axillary, Br: brachial, In: inguinal, Po: popliteal; Sp: spleen. *<i>p</i>≤0.05, **<i>p</i><0.01, ***<i>p</i><0.005.</p

Nanoparticles are taken up by MDSCs in draining nodes, spleen and tumor.

<p>Mice were inoculated subcutaneously with 10⁶ E.G7-OVA thymoma cells underneath the left shoulder blade (dorsoanterior left lateral side). After tumors reached 100 mm³, mice were injected with Dy649-labeled nanoparticles (NPs). Flow cytometry plots illustrating targeting of (<b>a</b>) monocytic (MO) MDSCs and (<b>b</b>) polymorphonuclear (PMN) MDSCs in the tumor draining lymph node (TDLN), the spleen and the tumors. (<b>c</b>) Three-dimensional flow-cytometry representation of the MDSC compartment (MO and PMN) of the tumor. Comparison between different organs of interest of the (<b>d</b>) MO-MDSCs and (<b>e</b>) PMN-MDSCs subpopulation accumulating NPs. One-way ANOVA followed by Bonferroni post test. n = 3 *<i>p</i>≤0.05, **<i>p</i>≤0.01. Tu: Tumor; Sp: Spleen.</p

Lymphatic drainage is required for nanoparticle targeting of the lymph node and spleen after i.d. administration.

<p>(<b>a</b>) Bioavailability of Dy649-nanoparticles (NPs) in the blood compartment after i.d. administration in mice that lack peripheral lymphatics (K14-VEGR-3-Ig) and their wild type littermates. VH: vehicle control (non-fluorescently labeled NPs). Two-way repeated measures ANOVA followed by Bonferroni post test. (<b>b</b>) Comparison of NP⁺ association as assessed by flow cytometry in the brachial lymph node (LN) and the spleen 24 h post-i.d. administration. n = 4 *<i>p</i>≤0.05, ***<i>p</i>≤0.005. (<b>c</b>) 9 µm thick section of a Dy649-NP (red) draining wild type popliteal LN stained with nuclei (DAPI, blue). Scale bar, 200 µm. (<b>d</b>) 9 µm thick section of the NP-Dy649 (red) draining wild type brachial LN 12 h after i.d. administration, stained for lymphatic endothelium (LYVE-1, green), the T cell zone stroma (ERTR7, white). Scale bar, 40 µm. (<b>e</b>) 40 µm section of the wild type anterior spleen stained with DAPI (blue) and NP-Dy649 (red) shows NP accumulation in the red pulp (RP) and the marginal zone (MZ), as well as surrounding the B cell follicles (FO). Scale bar, 300 µm. (<b>f</b>) Enlarged region of the central arteriole of the spleen (white filled arrow) (<b><i>i</i></b>) immunofluorescence image (NP-Dy649, red and DAPI, blue). (<b><i>ii</i></b>) Hematoxylin & eosin staining of the same section of the spleen; dark blue FO, purple red pulp and pink blood vessels. Scale bar, 100 µm.</p

Nanoparticle biodistribution in tissues and cells show secondary lymphoid organ accumulation.

<p>Heat maps show nanoparticle (NP)-positive percentages of each indicated cell type in lymph nodes (LN) or blood-filtering organs 12 h after i.d. injection as analyzed by flow cytometry. (<b>a</b>) Overall leukocyte (CD45+) in different tissues. leukocyte subpopulations with (<b>b</b>) low to medium levels (0–15%) or (<b>c</b>) high levels (up to 98%) of NP accumulation. B cells: B220⁺, T cells: (CD3ε⁺ then CD4⁺CD25⁺, CD4⁺CD25⁻, CD8⁺), TCRγδ: CD3ε⁺CD4⁻CD8⁻ TCRγδ⁺, immature myeloid dendritic cells (DCs): CD11c⁺CD11b⁺I/A^b−, immature lymphoid DCs: CD11c⁺CD11b⁻I/A^b−. (<b>c</b>) Granulocytes: CD11b⁺GR1^highSSC^high, monocytes: CD11b⁺GR1^lowSSC^lowF4/80⁺, mature myeloid DCs: CD11c⁺CD11b⁺I/A^b+, CD11c⁺CD8α⁺I/A^b+, CD11c⁺CD11b⁻I/A^b+, medullary macrophages (MØ): CD11b⁺F4/80⁺. Draining LNs are indicated by Ax: axillary, Br: brachial, In: inguinal, Po: popliteal; Sp: spleen, Bl: blood, Kd: kidneys, Li: liver, Lu: lungs. Heatmap color scales indicated on the right. Refer to gating strategies in Figures S2 and S3.</p