Figure 1.
Nanoparticle biodistribution in tissues and cells show secondary lymphoid organ accumulation.
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. (a) Overall leukocyte (CD45+) in different tissues. leukocyte subpopulations with (b) low to medium levels (0–15%) or (c) 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/Ab−, immature lymphoid DCs: CD11c+CD11b−I/Ab−. (c) Granulocytes: CD11b+GR1highSSChigh, monocytes: CD11b+GR1lowSSClowF4/80+, mature myeloid DCs: CD11c+CD11b+I/Ab+, CD11c+CD8α+I/Ab+, CD11c+CD11b−I/Ab+, 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.
Figure 2.
Nanoparticles target lymph node dendritic cells better after i.d. vs. i.m. delivery.
(a) Blood concentrations of Dy649-labeled NPs after i.v., i.m. and i.d. administration. (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. P 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. (c) 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. P values were computed using the Chi Square test between the alternate model and the full model for each population. For 144 h, n = 2, for all else, n≥4. Leukocytes: CD45+, mature myeloid DCs: CD11c+CD11b+I/Ab+, cross-presenting DCs: CD11c+CD8α+ I/Ab+, immature myeloid DCs: CD11c+CD11b+I/Ab−, immature lymphoid DCs: CD11c+CD11b−I/Ab−, medullary/red pulp (RP) macrophages (MØ): CD11b+F4/80+, monocytes: CD11b+GR1midSSClowF4/80+, granulocytes: CD11b+GR1highSSChigh, T cells: CD3ε+, B cells: B220+. Draining lymph nodes are indicated by Ax: axillary, Br: brachial, In: inguinal, Po: popliteal; Sp: spleen. *p≤0.05, **p<0.01, ***p<0.005.
Figure 3.
Lymphatic drainage is required for nanoparticle targeting of the lymph node and spleen after i.d. administration.
(a) 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) 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 *p≤0.05, ***p≤0.005. (c) 9 µm thick section of a Dy649-NP (red) draining wild type popliteal LN stained with nuclei (DAPI, blue). Scale bar, 200 µm. (d) 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. (e) 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. (f) Enlarged region of the central arteriole of the spleen (white filled arrow) (i) immunofluorescence image (NP-Dy649, red and DAPI, blue). (ii) Hematoxylin & eosin staining of the same section of the spleen; dark blue FO, purple red pulp and pink blood vessels. Scale bar, 100 µm.
Figure 4.
Monocytes internalize nanoparticles via macropinocytosis while B and T cell associate externally.
(a) Representative flow cytometry plots of in vivo NP-Dy649+ uptake kinetics after intradermal administration: monocytes (CD11b+GR1midSSClowF4/80+) and B cells (B220+) in the spleen. (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 in vitro. 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). (c) 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+.
Figure 5.
Nanoparticles are taken up by MDSCs in draining nodes, spleen and tumor.
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 (a) monocytic (MO) MDSCs and (b) polymorphonuclear (PMN) MDSCs in the tumor draining lymph node (TDLN), the spleen and the tumors. (c) Three-dimensional flow-cytometry representation of the MDSC compartment (MO and PMN) of the tumor. Comparison between different organs of interest of the (d) MO-MDSCs and (e) PMN-MDSCs subpopulation accumulating NPs. One-way ANOVA followed by Bonferroni post test. n = 3 *p≤0.05, **p≤0.01. Tu: Tumor; Sp: Spleen.