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

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

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

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