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Programmable multistage drug delivery to lymph nodes

A Publisher Correction to this article was published on 06 July 2020

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Abstract

Therapeutic delivery selectively to lymph nodes has the potential to address a variety of unmet clinical needs. However, owing to the unique structure of the lymphatics and the size-restrictive nature of the lymph node reticular network, delivering cargo to specific cells in the lymph node cortex and paracortex is difficult. Here, we describe a delivery system to overcome lymphatic and intra-lymph node transport barriers by combining nanoparticles that are rapidly conveyed to draining lymph nodes after administration in peripheral tissues with programmable degradable linkers. This platform enables the controlled release of intra-lymph-mobile small-molecular cargo, which can reach vastly more immune cells throughout the lymph node than either the particles or free compounds alone. The release rate can be programmed, allowing access to different lymph node structures and therefore specific lymphocyte subpopulations. We are thus able to alter the subtypes of drugged lymph node cells to improve immunotherapeutic effects.

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Fig. 1: NP-OND preparation and properties.
Fig. 2: Altered access by NP-OND to dLN-resident cell populations.
Fig. 3: Increased cargo delivered by NP-OND to LN-resident cells.
Fig. 4: Temporal effects of multistage NP-OND delivery.
Fig. 5: Augmented immunotherapeutic effects of adjuvant delivery with the NP-OND system.

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request.

Code availability

The custom code used in analysis of the fluorescence distribution in LN immunohistochemistry images is available from the corresponding authors upon reasonable request.

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Acknowledgements

This work was supported by the National Institutes of Health (R01CA247484, R01CA207619, S10OD016264), the National Science Foundation (CHE 1011796), grants from the Parker H. Petit Institute for Bioengineering and Bioscience at the Georgia Institute of Technology and Georgia CORE/It’s the Journey and by National Institutes of Health training grants T32EB021962 and T32GM008433. A.S. was an American Heart Association Predoctoral Fellow. C.J.H. and M.P.M. gratefully acknowledge support by the National Science Foundation Graduate Research Fellowship Program.

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S.N.T. and M.G.F. conceived the project and, with A.S., designed the experiments. A.S., A.P.C., D.M.F. and M.P.M. carried out the experiments. M.-K.Y. and C.J.H. performed all of the linker chemistry and analysis. N.A.R. and A.R.C.A. performed immunohistochemistry. N.A.R. wrote the image analysis script. A.S., A.P.C., S.N.T. and M.G.F. analysed the data. S.N.T. and M.G.F. supervised the project. S.N.T., M.G.F. and A.S. wrote the manuscript and all parties reviewed the manuscript.

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Correspondence to M. G. Finn or Susan Napier Thomas.

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Extended data

Extended Data Fig. 1 Compound structures.

OND (1-4 Dn) and epoxyoxanorbornene (5-Dn) electrophiles, model fluorophore (Fluor-Mal, Rhod-Mal, Cy5.5-COOH, 1-Coum, Rhod-F, 3-Rhod, 4-Rhod) and drug cargos (Irino-F, 3-Irino, OND-CpG) presented in this study.

Extended Data Fig. 2 Spatiotemporal effects of NP encapsulation-and-release delivery platform.

a, In vitro extended release of encapsulated Cy5.5 Cargo from NP (teal; half-life = 10.9 h) versus free dye (dark blue; half-life = 1.2 h). n = 6 samples per group. Fraction b and percentage of total c dLN cells of various types positive for encapsulated Cy5.5 cargo (blue) and AF488-NPs (orange) measured by flow cytometry 6, 12, 24, and 48 h after i.d. administration Cy5.5-encapsulating NPs. n = 6 biological replicates per group. For all graphs, the columns/points and error bars represent the mean + SEM.

Extended Data Fig. 3 Delivery of OND cargo using virus like particle carriers.

a, Schematic representation of the preparation of VLPs conjugated to 3-Rhod. b, Liquid chromatography time-of-flight mass spectrometry (C3 column) of VLP-OND conjugates. Blue trace represents VLP coat protein associated with AF647-NHS via lysine residues, red trace represents species associated with 3-Rhod. This experiment was repeated once with similar results. c, VLPs covalently labeled with NIR-NHS ester were injected i.d. into mice, followed by organ extraction and IVIS imaging after 6 and 24 h. This experiment was repeated once with similar results. d, dLN size after i.d. injection of VLP. Axial and brachial LN from draining and contralateral (non-draining) sides were photographed and measured using ImageJ. n = 6 biological replicates per group. e, Total numbers of AF647 positive and Rhodamine positive dLN cells 24 h after i.d. treatment with VLPs labelled with AF647-NHS (VLP, non-cleavable linker) and 3-Rhod (OND). Saline injected and naïve (uninjected) animals served as Rhodamine channel negative controls. n = 6 biological replicates per group. f Percent of Rhodamine positive cells in dLN 4 h (teal), 6 h (black), 16 h (green), or 24 h (red) after i.d. injection with either VLP (VLP) or pyridyl disulfide PPS NP (PDS) conjugated with 3-Rhod measured by flow cytometry. n = 3 biological replicates per group. g) Percent of positive lymphocytes and barrier cells in dLN 6 h (black) or 24 h (red) after i.d. injection of VLP labeled with both Alexa647-NIH (VLP, non-cleavable linker) and 3-Rhod (OND) measured by flow cytometry, normalized to average uptake at 6 h. n = 5 biological replicates per group. For all graphs, the columns/points and error bars represent the mean + SEM. Statistics were performed by ordinary one-way ANOVA with Tukey’s multiple comparisons test. ****=p < .0001, ***=p < .005, *=p < .05.

Extended Data Fig. 4 Toxicity of OND derivatives of irinotecan.

a, Log dose testing of 3-Irino (OND), Irino-F (Furan), and unmodified irinotecan against C57Bl6 splenocytes after 24 h in IMDM at 37 °C. n = 5 samples per group. b, Log IC50 values derived from panel a. c, Effects of cargo delivered to lymph node-resident cells by lymph-accessing NP-OND compared to free drug. Number of dead LN cells normalized to saline-treated animals 24 h after i.d. administration of (NP-OND) NPs labeled with 3-Irino (4 mg/kg) or i.p. administration of free Irino-F at the indicated doses. n = 5 biological replicates per group. For all graphs, the columns/points and error bars represent the mean + SEM. Statistics were performed by two-way ANOVA with Tukey’s multiple comparisons test. ****=p < .0001, ***=p < .005, *=p < .05 relative to each dose of free Irino-F. For all graphs, the columns/points and error bars represent the mean + SEM.

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Supplementary methods, Figs. 1–16, Table 1, synthesis schemes 1–10, discussion, additional data figures and all associated labels.

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Schudel, A., Chapman, A.P., Yau, MK. et al. Programmable multistage drug delivery to lymph nodes. Nat. Nanotechnol. 15, 491–499 (2020). https://doi.org/10.1038/s41565-020-0679-4

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