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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.

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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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References

  1. Babar, I. A. et al. Nanoparticle-based therapy in an in vivo microRNA-155 (miR-155)-dependent mouse model of lymphoma. Proc. Natl Acad. Sci. USA 109, E1695–E1704 (2012).

    CAS  Google Scholar 

  2. Lorenzo-Redondo, R. et al. Persistent HIV-1 replication maintains the tissue reservoir during therapy. Nature 530, 51–56 (2016).

    CAS  Google Scholar 

  3. Kuerer, H. M. et al. Residual metastatic axillary lymph nodes following neoadjuvant chemotherapy predict disease-free survival in patients with locally advanced breast cancer. Am. J. Surg. 176, 502–509 (1998).

    CAS  Google Scholar 

  4. Liu, H. et al. Structure-based programming of lymph node targeting in molecular vaccines. Nature 507, 519–522 (2014).

    CAS  Google Scholar 

  5. Lee, J.-W. et al. Peripheral antigen display by lymph node stroma promotes T cell tolerance to intestinal self. Nat. Immunol. 8, 181–190 (2006).

    Google Scholar 

  6. Kreiter, S. et al. Intranodal vaccination with naked antigen-encoding RNA elicits potent prophylactic and therapeutic antitumoral immunity. Cancer Res. 70, 9031–9040 (2010).

    CAS  Google Scholar 

  7. Komori, J., Boone, L., DeWard, A., Hoppo, T. & Lagasse, E. The mouse lymph node as an ectopic transplantation site for multiple tissues. Nat. Biotechnol. 30, 976–983 (2012).

    CAS  Google Scholar 

  8. Thomas, S. N. & Schudel, A. Overcoming transport barriers for interstitial-, lymphatic- and lymph node-targeted drug delivery. Curr. Opin. Chem. Eng. 7, 65–74 (2015).

    Google Scholar 

  9. Qi, H., Kastenmuller, W. & Germain, R. N. Spatiotemporal basis of innate and adaptive immunity in secondary lymphoid tissue. Annu. Rev. Cell Dev. Biol. 30, 141–167 (2014).

    CAS  Google Scholar 

  10. Chang, J. E. & Turley, S. J. Stromal infrastructure of the lymph node and coordination of immunity. Trends Immunol. 36, 30–39 (2015).

    CAS  Google Scholar 

  11. Kastenmuller, W., Torabi-Parizi, P., Subramanian, N., Lammermann, T. & Germain, R. N. A spatially-organized multicellular innate immune response in lymph nodes limits systemic pathogen spread. Cell 150, 1235–1248 (2012).

    CAS  Google Scholar 

  12. Sixt, M. et al. The conduit system transports soluble antigens from the afferent lymph to resident dendritic cells in the T cell area of the lymph node. Immunity 22, 19–29 (2005).

    CAS  Google Scholar 

  13. Gerner, M. Y., Torabi-Parizi, P. & Germain, R. N. Strategically localized dendritic cells promote rapid T cell responses to lymph-borne particulate antigens. Immunity 42, 172–185 (2015).

    CAS  Google Scholar 

  14. Junt, T. et al. Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells. Nature 450, 110–114 (2007).

    CAS  Google Scholar 

  15. Gretz, J. E., Kaldjian, E. P., Anderson, A. O. & Shaw, S. Sophisticated strategies for information encounter in the lymph node: the reticular network as a conduit of soluble information and a highway for cell traffic. J. Immunol. 157, 495–499 (1996).

    CAS  Google Scholar 

  16. Gretz, J. E., Norbury, C. C., Anderson, A. O., Proudfoot, A. E. & Shaw, S. Lymph-borne chemokines and other low molecular weight molecules reach high endothelial venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex. J. Exp. Med. 192, 1425–1440 (2000).

    CAS  Google Scholar 

  17. Wong, C. et al. Multistage nanoparticle delivery system for deep penetration into tumor tissue. Proc. Natl Acad. Sci. USA 108, 2426–2431 (2011).

    CAS  Google Scholar 

  18. Pridgen, E. M., Langer, R. & Farokhzad, O. C. Biodegradable, polymeric nanoparticle delivery systems for cancer therapy. Nanomedicine 2, 669–680 (2007).

    CAS  Google Scholar 

  19. Colson, Y. L. & Grinstaff, M. W. Biologically responsive polymeric nanoparticles for drug delivery. Adv. Mater. 24, 3878–3886 (2012).

    CAS  Google Scholar 

  20. Reddy, S. T., Berk, D. A., Jain, R. K. & Swartz, M. A. A sensitive in vivo model for quantifying interstitial convective transport of injected macromolecules and nanoparticles. J. Appl. Physiol. 101, 1162–1169 (2006).

    CAS  Google Scholar 

  21. Porter, C. J. H. & Charman, S. A. Lymphatic transport of proteins after subcutaneous administration. J. Pharm. Sci. 89, 297–310 (2000).

    CAS  Google Scholar 

  22. Nuhn, L. et al. pH-degradable imidazoquinoline-ligated nanogels for lymph node-focused immune activation. Proc. Natl Acad. Sci. USA 113, 8098–8103 (2016).

    CAS  Google Scholar 

  23. de Titta, A. et al. Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory recall at low dose. Proc. Natl Acad. Sci. USA 110, 19902–19907 (2013).

    Google Scholar 

  24. Olson, E. S. et al. Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases. Proc. Natl Acad. Sci. USA 107, 4311–4316 (2010).

    CAS  Google Scholar 

  25. Kwon, Y. J., James, E., Shastri, N. & Frechet, J. M. In vivo targeting of dendritic cells for activation of cellular immunity using vaccine carriers based on pH-responsive microparticles. Proc. Natl Acad. Sci. USA 102, 18264–18268 (2005).

    CAS  Google Scholar 

  26. Rehor, A., Hubbell, J. A. & Tirelli, N. Oxidation-sensitive polymeric nanoparticles. Langmuir 21, 411–417 (2005).

    CAS  Google Scholar 

  27. Schudel, A., Kassis, T., Dixon, J. B. & Thomas, S. N. S-nitrosated polypropylene sulfide nanoparticles for thiol-dependent transnitrosation and toxicity against adult female filarial worms. Adv. Healthc. Mater. 4, 1484–1490 (2015) .

    CAS  Google Scholar 

  28. Schudel, A., Sestito, L. F. & Thomas, S. N. S-nitrosated poly(propylene sulfide) nanoparticles for enhanced nitric oxide delivery to lymphatic tissues. J. Biomed. Mater. Res. A 106, 1463–1475 (2018).

    CAS  Google Scholar 

  29. Thomas, S. N., Vokali, E., Lund, A. W., Hubbell, J. A. & Swartz, M. A. Targeting the tumor-draining lymph node with adjuvanted nanoparticles reshapes the anti-tumor immune response. Biomaterials 35, 814–824 (2014).

    CAS  Google Scholar 

  30. Higginson, C. J., Eno, M. R., Khan, S., Cameron, M. D. & Finn, M. G. Albumin-oxanorbornadiene conjugates formed ex vivo for the extended circulation of hydrophilic cargo. ACS Chem. Biol. 11, 2320–2327 (2016).

    CAS  Google Scholar 

  31. Higginson, C. J., Kim, S. Y., Pelaez-Fernandez, M., Fernandez-Nieves, A. & Finn, M. G. Modular degradable hydrogels based on thiol-reactive oxanorbornadiene linkers. J. Am. Chem. Soc. 137, 4984–4987 (2015).

    CAS  Google Scholar 

  32. Kislukhin, A. A., Higginson, C. J., Hong, V. P. & Finn, M. G. Degradable conjugates from oxanorbornadiene reagents. J. Am. Chem. Soc. 134, 6491–6497 (2012).

    CAS  Google Scholar 

  33. van der Vlies, A. J., O’Neil, C. P., Hasegawa, U., Hammond, N. & Hubbell, J. A. Synthesis of pyridyl disulfide-functionalized nanoparticles for conjugating thiol-containing small molecules, peptides and proteins. Bioconjug. Chem. 21, 653–662 (2010).

    Google Scholar 

  34. Hirosue, S., Kourtis, I. C., van der Vlies, A. J., Hubbell, J. A. & Swartz, M. A. Antigen delivery to dendritic cells by poly(propylene sulfide) nanoparticles with disulfide conjugated peptides: cross-presentation and T cell activation. Vaccine 28, 7897–7906 (2010).

    CAS  Google Scholar 

  35. Hong, V., Presolski, S. I., Ma, C. & Finn, M. G. Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation. Angew. Chem. Int. Ed. 48, 9879–9883 (2009).

    CAS  Google Scholar 

  36. Reddy, S. T. et al. Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nat. Biotechnol. 25, 1159–1164 (2007).

    CAS  Google Scholar 

  37. Kourtis, I. C. et al. Peripherally administered nanoparticles target monocytic myeloid cells, secondary lymphoid organs and tumors in mice. PLoS ONE 8, e61646 (2013).

    CAS  Google Scholar 

  38. Nathanson, S. D., Nelson, L. & Karvelis, K. C. Rates of flow of technetium 99m-labeled human serum albumin from peripheral injection sites to sentinel lymph nodes. Ann. Surg. Oncol. 3, 329–335 (1996).

    CAS  Google Scholar 

  39. Charman, W. N. & Stella, V. J. Lymphatic Transport of Drugs (Taylor & Francis, 1992).

  40. Hettiaratchi, M. H. et al. A rapid method for determining protein diffusion through hydrogels for regenerative medicine applications. APL Bioeng. 2, 026110 (2018).

    Google Scholar 

  41. Gerner, M. Y., Casey, K. A., Kastenmuller, W. & Germain, R. N. Dendritic cell and antigen dispersal landscapes regulate T cell immunity. J. Exp. Med. 214, 3105–3122 (2017).

    CAS  Google Scholar 

  42. Roozendaal, R. et al. Conduits mediate transport of low molecular weight antigen to lymph node follicles. Immunity 30, 264–276 (2009).

    CAS  Google Scholar 

  43. Fiedler, J. D. et al. Engineered mutations change the structure and stability of a virus-like particle. Biomacromolecules 13, 2339–2348 (2012).

    CAS  Google Scholar 

  44. Golmohammadi, R., Fridborg, K., Bundule, M., Valegard, K. & Liljas, L. The crystal structure of bacteriophage Qβ at 3.5 Å resolution. Structure 4, 543–554 (1996).

    CAS  Google Scholar 

  45. Beaudette, T. T. et al. In vivo studies on the effect of co-encapsulation of CpG DNA and antigen in acid-degradable microparticle vaccines. Mol. Pharm. 6, 1160–1169 (2009).

    CAS  Google Scholar 

  46. Jeanbart, L. et al. Enhancing efficacy of anticancer vaccines by targeted delivery to tumor-draining lymph nodes. Cancer Immunol. Res. 2, 436–447 (2014).

    CAS  Google Scholar 

  47. Goldstein, M. J. et al. A CpG-loaded tumor cell vaccine induces antitumor CD4+ T cells that are effective in adoptive therapy for large and established tumors. Blood 117, 118–127 (2011).

    CAS  Google Scholar 

  48. Qi, X.-F. et al. CpG oligodeoxynucleotide induces apoptosis and cell cycle arrest in A20 lymphoma cells via TLR9-mediated pathways. Mol. Immunol. 54, 327–337 (2013).

    CAS  Google Scholar 

  49. Drobits, B. et al. Imiquimod clears tumors in mice independent of adaptive immunity by converting pDCs into tumor-killing effector cells. J. Clin. Invest. 122, 575–585 (2012).

    CAS  Google Scholar 

  50. Liu, Y. et al. Biphasic rapamycin effects in lymphoma and carcinoma treatment. Cancer Res. 77, 520–531 (2017).

    CAS  Google Scholar 

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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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