Hybrid LNP Prime Dendritic Cells for Nucleotide Delivery

Abstract The efficient activation of professional antigen‐presenting cells—such as dendritic cells (DC)—in tumors and lymph nodes is critical for the design of next‐generation cancer vaccines and may be able to provide anti‐tumor effects by itself through immune stimulation. The challenge is to stimulate these cells without causing excessive toxicity. It is hypothesized that a multi‐pronged combinatorial approach to DC stimulation would allow dose reductions of innate immune receptor‐stimulating TLR3 agonists while enhancing drug efficacy. Here, a hybrid lipid nanoparticle (LNP) platform is developed and tested for double‐stranded RNA (polyinosinic:polycytidylic acid for TLR3 agonism) and immune modulator (L‐CANDI) delivery. This study shows that the ≈120 nm hybrid nanoparticles‐in‐nanoparticles effectively eradicate tumors by themselves and generate long‐lasting, durable anti‐tumor immunity in mouse models.

Fig. S1: L-CANDI Characterization. A. Three-step synthesis to loaded L-CANDI particles starting from 1) cross-linking of succinyl-beta-cyclodextrin (s--CD) and L-Lysine with EDC/NHS, 2) surface functionalization of nucleophilic groups with palmitic acid NHS ester in carbonate buffer and 3) payload (LCL-161) adsorption on particle's surface via inclusioncomplexation with CD units.B. Plots showing the size distribution and surface charge of all particles obtained from dynamic light scattering (DLS) and zeta measurements, respectively (Zetaziser, Malvern).All experiments were performed in triplicates (N = 3).Note that LNP17 exhibited long-term stability with no evidence of aggregation in the presence of serum.

Fig. S6: Comparison of efficacy (IL-12 induction in BMDM) and cellular toxicity (iMAC). A.
A. IL-12 induction measured in BMDM.B. Cellular toxicity in iMAC as a function of dose.The shaded area in green represents the therapeutic window.The in vivo dose (5 µg poly I:C/mouse in 0.774 mL of plasma corresponds to a vascular concentration of 6.5 µg/mL(1)) is denoted by the red dotted line.There is cellular toxicity at very high doses (> 100x used therapeutically) due to the combination of LCL-161 and poly:IC.The LNP preparation is not toxic at these doses.

Fig. S2 :
Fig. S2: C12-200 Characterization. A. 1 H-NMR of C12-200 in CDCl3 showing the structure, chemical formula, and exact mass (ChemDraw 20, Perkin Elmer).B. HPL chromatograph with (ELDS source) of the purified product shows a single peak with a retention time of t = 3.65 min and ionizes with an m/z of ES + = 1137.24{M +} .

Fig. S5 :
Fig. S5: Stability of LNP stored at 4°C A. Size as a function of time.B. Polydispersity was measured by DLS C. Poly I:C encapsulation efficiency was determined by the RiboGreen assay.D. stability of LNP17 in the presence of 0, 10, 25% fetal bovine serum measured by DLS.Note that LNP17 exhibited long-term stability with no evidence of aggregation in the presence of serum.

Fig. S7 :
Fig. S7: Blood half-life of LNP. A. LNP17Fl or PBS control was injected into BL6 mice and serial imaging of the ear vasculature was performed.Blood half-life (T1/2) was determined to be 30 min.B. Time-series of images depicts the fluorescent LNP17Fl in the ear microvasculature.

Fig. S8 :
Fig. S8: Tissue distribution of LNP. A. LNP17Fl or PBS control was injected into tumorbearing mice (MC38; N = 6 mice), and tissues were surgically removed 24 hr later.The graph shows the relative distribution of LNP to different organs.As expected, liver and spleen tumor lymph nodes showed the highest concentrations.B. Tissues were imaged using an OV100 epifluorescence system.Bright signal (647 nm) corresponds to fluorescent LNP17 accumulation.C. Flow cytometry data.LNP17Fl was injected into tumor-bearing mice (MC38; N = 6 mice), and tissues were surgically removed 24 hr later.The graph shows the relative distribution of LNP to different organ cells as measured by flow cytometry, confirming the fluorescence imaging experiments.

Fig. S9 :
Fig. S9: Tumoral accumulation.A. LNP17Fl was injected into tumor-bearing mice (MC38; N = 3 mice), and tumors were imaged serially over 24 hr.The LNP is initially seen in tumor microvasculature but accumulates in non-tumor cells at 24 hr.Scale bar = 100 µm.Fig. S10: Dynamic changes in the tumor immune microenvironment induced by fluorescent LNP17 .A. Representative images of MC38 tumors with a fluorescent LNP17Fl

Fig. S11 :
Fig. S11: Spatial analysis of immune cell subsets in LNP17 treated (A) or untreated (C) tumors.For each specimen, 12 representative fields of views are shown.Gray marks, CD45+ cells and blue dots represent superimposed, dendritic cells.Note the higher DC infiltration in