Targeted Delivery of Immunomodulators to Lymph Nodes

Abstract

Active-targeted delivery to lymph nodes represents a major advance toward more effective treatment of immune-mediated disease. The MECA79 antibody recognizes peripheral node addressin molecules expressed by high endothelial venules of lymph nodes. By mimicking lymphocyte trafficking to the lymph nodes, we have engineered MECA79-coated microparticles containing an immunosuppressive medication, tacrolimus. Following intravenous administration, MECA79-bearing particles showed marked accumulation in the draining lymph nodes of transplanted animals. Using an allograft heart transplant model, we show that targeted lymph node delivery of microparticles containing tacrolimus can prolong heart allograft survival with negligible changes in tacrolimus serum level. Using MECA79 conjugation, we have demonstrated targeted delivery of tacrolimus to the lymph nodes following systemic administration, with the capacity for immune modulation in vivo.

Figure 1. Preparation of TAC-PLA Polymer Conjugate and Formulation of PEGylated MP-TAC-MECA79 and MP-TAC

(A) Schematic of the synthesis of TAC-PLA polymer conjugate by means of TAC-initiated LA polymerization in the presence of (BDI-EI)ZnN(TMS)₂ catalyst and preparation of MP-TAC-MECA79 with the surface modified by the MECA79 antibody. (B) FT-IR spectra of LA (red) and TAC-PLA conjugates (blue). The complete conversion of LA was determined via FT-IR by monitoring the disappearance of the band at 1772 cm⁻¹). (C) MALDI-TOF analysis for TAC-PLA_n conjugates. The obtained mass to charge ratio (m/z) is identical to the calculated m/z of [TAC-PLA_n + Na]⁺ (804.0+143.9 × n + 23).

Figure 2. Characterization of PEGylated MP-TAC-MECA79 and MP-TAC

(A) DLS measurement of MP-TAC-MECA79. D, average diameter; PDI, polydispersity index. (B) A representative scanning electron microscope image of PEGylated MP-TAC-MECA79 (scale bar, 1 µm). (C) Formulation parameters and physicochemical properties of PEGylated MP-TAC-MECA79 and MP-TAC. M_w PEG, molecular weight of poly(ethylene glycol. (D) Particle sizes and polydispersities of MP-TAC-MECA79 and MP-TAC before and after lyophilization.

Figure 3. MP-TAC-MECA79 Suppresses T Cell Proliferation in a Dose-Dependent Fashion In Vitro and Suppresses Inflammatory Cytokine Production by T Cells

(A) C57BL/6 splenocytes were stimulated in vitro with anti-CD3 and anti-CD28 antibodies in a flat-bottom plate. MP-TAC-MECA79 was added in escalating doses to the T cell stimulation assay. MP-TAC-MECA79 suppressed T cell proliferation, as measured by thymidine incorporation, in a dose-dependent fashion, starting at 0.5 ng/ml TAC-equivalent concentration (*p < 0.05). Ctrl, control, CPM, count per minute. (B) Free TAC suppressed T cell proliferation, as measured by thymidine incorporation, in a dose-dependent fashion, starting at 0.5 ng/ml TAC-equivalent concentration (*p < 0.05). (C) The suppression of IL2 production by CD3/CD28-stimulated T cells by MP-TAC-MECA79 at a concentration of 1 ng/ml TAC equivalent (*p < 0.05). (D) The suppression of IFNγ production by CD3/CD28-stimulated T cells by MP-TAC-MECA79 at a concentration of 1 ng/ml TAC equivalent (*p < 0.05). (E) The suppression of IL6 production by CD3/CD28-stimulated T cells by MP-TAC-MECA79 at a concentration of 1 ng/ml TAC equivalent (*p < 0.05). (F) The suppression of IL17 production by CD3/CD28-stimulated T cells by MP-TAC-MECA79 at a concentration of 1 ng/ml TAC equivalent (*p < 0.05).

Figure 4. Increased Expression of PNAd in DLNs Facilitates Targeted Delivery of MP-MECA79 in a Transplant Mouse Model

(A) Western blot analysis of cell suspensions from the DLNs of mice transplanted with syngeneic or allogeneic skin grafts showed a 4-fold increase in PNAd expression in allogeneic compared with syngeneic transplants. Total ERK1 was used as a loading control. The fold change over the syngeneic sample was calculated after densitometric analysis and correction for ERK1 (***p < 0.001). (B) Western blot analysis of cell suspensions from the DLNs and non-DLNs of mice transplanted with allogeneic skin grafts showed significant increases in PNAd expression in DLNs compared with non-DLNs. The fold change over the non-DLN sample was calculated after densitometric analysis and correction for ERK1 (*p < 0.05). (C) DLNs of mice transplanted with allogeneic skin grafts were analyzed by immunohistochemistry after intravenous injection of rhodamine-labeled MP-TAC-MECA79 (MP-Rhd-MECA79) or MP-TAC (MP-Rhd). DLN showed greatly increased PNAd (arrows) compared with non-draining LNs (pancreatic LNs). A 20× objective was used. Further extension of PNAd-positive cells from much larger vessels in the DLNs was also observed (left). A 40× objective was used. (D) MECA79 particles but not control particles are localized to adjacent spaces of PNAd-positive endothelial cells in the DLNs. Many MECA79 particles are lined up by PNAd. The cell nuclei were visualized by DAPI. A 40× objective was used. (E) A higher number of particles was counted per high-power field (200×) in DLNs isolated from mice treated with MP-Rhd-MECA79 compared with MP-Rhd (*p < 0.05, n = 4 mice/group). (F) Two-photon imaging shows greatly increased PNAd expression (green) in DLNs compared with non-draining LNs. A higher number of MP-Rhd-MECA79 is observed in the DLNs compared with the non-draining LNs.

Figure 5. Blocking PNAd Reduces Targeted Delivery of MP-MECA79 in a Transplant Mouse Model

(A) FACS analysis of cell suspension from the inguinal DLNs of skin transplant recipients injected with either MP-Rhd-MECA79 or MP-Rhd, showing a greater accumulation of rhodamine-labeled MP-MECA79 compared with MPs. (B) FACS analysis of cell suspension from the DLNs of skin transplant recipients injected with blocking anti-PNAd antibody 1 hr before injection of rhodamine-labeled particles. (C) The absolute number of rhodamine-labeled particles in the four different groups of mice (n = 4 mice /group, *p < 0.05).

Figure 6. MP-TAC-MECA79 Prolongs Heart Allograft Survival in Mice with Lower Serum Peak and Trough TAC Levels Compared with the Free TAC Group

(A) Immunohistochemistry analysis of the DLN of mice transplanted with heart allografts showed an increase in PNAd expression compared with non-draining LNs (axillary LNs) on day 7 post-transplantation. A 20× objective was used. Further extension of PNAd-positive cells from much larger vessels in the DLNs was also observed, giving a mesh-like appearance (arrows). (B) C57BL/6 mouse recipients of fully mismatched BALB/c hearts were injected daily via the tail vein with either MP-TAC-MECA79, MP-TAC, MP-MECA79, or free TAC at 1 mg/kg TAC equivalent. The TAC level was measured in the peripheral blood of the transplanted mice on day 3 after transplantation. The graph shows that the peak TAC level 2 hr after injection of the compounds was significantly lower for MP-TAC-MECA79 compared with free TAC (*p < 0.05, n = 4 mice/group). (C) The TAC trough level 24 hr after injection was significantly lower for MP-TAC-MECA79 compared with free TAC (*p < 0.05). (D) Kaplan-Meier survival curve of heart allograft recipients treated with MP-TAC-MECA79. C57BL/6 mouse recipients of fully mismatched BALB/c hearts were injected daily via the tail vein with either MP-TAC-MECA79, MP-TAC, MP, MP-MECA79, or free TAC at 1 mg/kg TAC equivalent. Compared with the other groups, treating recipients with MP-TAC-MECA79 significantly increased heart allograft survival (median survival 7 versus 7 versus 7 versus 7 versus 11 days, respectively; n = 4/group; p < 0.05). (E) FACS analysis of DLNs from heart transplant recipients treated with either MP-TAC-MECA79, MP-TAC, or free TAC showing a significant reduction in the percentage of effector CD4 T cells (CD4⁺CD62L^lowCD44^high) in DLNs of mice treated with MP-TAC-MECA79 compared with the MP-TAC- or free TAC-treated groups. (F) The absolute count of CD4 effector T cells in the DLNs of the three different groups of transplanted mice shows a significantly lower number of CD4 effector T cells in the DLNs of mice treated with MP-TAC-MECA79 compared with mice treated with MP-TAC or free TAC (*p < 0.05, n = 3–4 mice/group).