Skip to main content

Efficient delivery of clay-based nanovaccines to the mouse spleen promotes potent anti-tumor immunity for both prevention and treatment of lymphoma


Cancer therapeutic nanovaccines are ideal tools to inhibit tumor growth and provide the body with continuous protecting immune surveillance. However, the conventional subcutaneous (SC) vaccination normally induces limited anti-tumor immune responses with low therapeutic efficacy. Herein, we devised clay-based nanovaccines and directly delivered them to the spleen via intravenous (IV) injection to induce the stronger anti-tumor immunity with higher efficacy for tumor prevention and treatment. The clay, i.e., layered double hydroxide (LDH) was prepared as nanoadjuvant with the average size from 77 to 285 nm and co-loaded with the model antigen ovalbumin (OVA) and bioadjuvant CpG to form CpG/OVA-LDH (CO-LDH) nanovaccines. We found that CO-LDH-215 (the size of LDH was 215 nm) promoted dendritic cells to present the most antigen, and moreover showed the highest spleen enrichment (~ 1.67% of CO-LDH-215 enriched in the spleen at 24 h post IV injection). The in vivo immunologic data showed that CO-LDH-215 induced the most potent anti-tumor immune responses and completely prevented the growth of E.G7-OVA tumor in the mouse model. Furthermore, IV injected CO-LDH-215 nanovaccine more effectively delayed tumor growth than that SC injected, largely due to the direct and quick delivery of more nanovaccines to the spleen. This study demonstrates that the therapeutic efficacy of nanovaccines can be greatly enhanced by targeted delivery of nanovaccines to the spleen via the proper vaccination route.

This is a preview of subscription content, access via your institution.


  1. [1]

    Melief, C. J. M.; van Hall, T.; Arens, R.; Ossendorp, F.; van Der Burg, S. H. Therapeutic cancer vaccines. J. Clin. Invest. 2015, 125, 3401–3412.

    Google Scholar 

  2. [2]

    Melero, I.; Gaudernack, G.; Gerritsen, W.; Huber, C.; Parmiani, G.; Scholl, S.; Thatcher, N.; Wagstaff, J.; Zielinski, C.; Faulkner, I. et al. Therapeutic vaccines for cancer: An overview of clinical trials. Nat. Rev. Clin. Oncol. 2014, 11, 509–524.

    CAS  Google Scholar 

  3. [3]

    Wang, H.; Mooney, D. J. Biomaterial-assisted targeted modulation of immune cells in cancer treatment. Nat. Mater. 2018, 17, 761–772.

    CAS  Google Scholar 

  4. [4]

    Chen, W. Y.; Zuo, H. L.; Li, B.; Duan, C. C.; Rolfe, B.; Zhang, B.; Mahony, T. J.; Xu, Z. P. Clay nanoparticles elicit long-term immune responses by forming biodegradable depots for sustained antigen stimulation. Small 2018, 14, 1704465.

    Google Scholar 

  5. [5]

    Li, A. W.; Sobral, M. C.; Badrinath, S.; Choi, Y.; Graveline, A.; Stafford, A. G.; Weaver, J. C.; Dellacherie, M. O.; Shih, T. Y.; Ali, O. A. et al. A facile approach to enhance antigen response for personalized cancer vaccination. Nat. Mater. 2018, 17, 528–534.

    CAS  Google Scholar 

  6. [6]

    Xia, Y. F.; Wu, J.; Wei, W.; Du, Y. Q.; Wan, T.; Ma, X. W.; An, W. Q.; Guo, A. Y.; Miao, C. Y.; Yue, H. et al. Exploiting the pliability and lateral mobility of Pickering emulsion for enhanced vaccination. Nat. Mater. 2018, 17, 187–194.

    CAS  Google Scholar 

  7. [7]

    Itano, A. A.; Jenkins, M. K. Antigen presentation to naive CD4 T cells in the lymph node. Nat. Immunol. 2003, 4, 733–739.

    CAS  Google Scholar 

  8. [8]

    Von Andrian, U. H.; Mempel, T. R. Homing and cellular traffic in lymph nodes. Nat. Rev. Immunol 2003, 3, 867–878.

    Google Scholar 

  9. [9]

    Cyster, J. G. Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs. J. Exp. Med. 1999, 189, 447–450.

    CAS  Google Scholar 

  10. [10]

    Bachmann, M. F.; Jennings, G. T. Vaccine delivery: A matter of size, geometry, kinetics and molecular patterns. Nat. Rev. Immunol. 2010, 10, 787–796.

    CAS  Google Scholar 

  11. [11]

    Liu, J.; Li, H. J.; Luo, Y. L.; Xu, C. F.; Du, X. J.; Du, J. Z.; Wang, J. Enhanced primary tumor penetration facilitates nanoparticle draining into lymph nodes after systemic injection for tumor metastasis inhibition. ACS Nano 2019, 13, 8648–8658.

    CAS  Google Scholar 

  12. [12]

    Zhang, L. X.; Xie, X. X.; Liu, D. Q.; Xu, Z. P.; Liu, R. T. Efficient co-delivery of neo-epitopes using dispersion-stable layered double hydroxide nanoparticles for enhanced melanoma immunotherapy. Biomaterials 2018, 174, 54–66.

    CAS  Google Scholar 

  13. [13]

    Sultan, H.; Kumai, T.; Nagato, T.; Wu, J.; Salazar, A. M.; Celis, E. The route of administration dictates the immunogenicity of peptidebased cancer vaccines in mice. Cancer Immunol. Immunother. 2019, 68, 455–466.

    CAS  Google Scholar 

  14. [14]

    Han, X.; Shen, S. F.; Fan, Q.; Chen, G. J.; Archibong, E.; Dotti, G.; Liu, Z.; Gu, Z.; Wang, C. Red blood cell-derived nanoerythrosome for antigen delivery with enhanced cancer immunotherapy. Sci. Adv. 2019, 5, eaaw6870.

  15. [15]

    Bronte, V.; Pittet, M. J. The spleen in local and systemic regulation of immunity. Immunity 2013, 39, 806–818.

    CAS  Google Scholar 

  16. [16]

    Kranz, L. M.; Diken, M.; Haas, H.; Kreiter, S.; Loquai, C.; Reuter, K. C.; Meng, M.; Fritz, D.; Vascotto, F.; Hefesha, H. et al. Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature 2016, 534, 396–401.

    Google Scholar 

  17. [17]

    Liu, J. P.; Zhang, R.; Xu, Z. P. Nanoparticle-based nanomedicines to promote cancer immunotherapy: Recent advances and future directions. Small 2019, 15, 1900262.

    Google Scholar 

  18. [18]

    Zhang, L. X.; Liu, D. Q.; Wang, S. W.; Yu, X. L.; Ji, M.; Xie, X. X.; Liu, S. Y.; Liu, R. T. MgAl-layered double hydroxide nanoparticles co-delivering siIDO and Trp2 peptide effectively reduce IDO expression and induce cytotoxic T-lymphocyte responses against melanoma tumor in mice. J. Mater. Chem. B 2017, 5, 6266–6276.

    CAS  Google Scholar 

  19. [19]

    Chen, W. Y.; Zhang, B.; Mahony, T.; Gu, W. Y.; Rolfe, B.; Xu, Z. P. Efficient and durable vaccine against intimin β of diarrheagenic E. Coli induced by clay nanoparticles. Small 2016, 12, 1627–1639.

    CAS  Google Scholar 

  20. [20]

    Yan, S. Y.; Rolfe, B. E.; Zhang, B.; Mohammed, Y. H.; Gu, W. Y.; Xu, Z. P. Polarized immune responses modulated by layered double hydroxides nanoparticle conjugated with CpG. Biomaterials 2014, 35, 9508–9516.

    CAS  Google Scholar 

  21. [21]

    Yan, S. Y.; Gu, W. Y.; Zhang, B.; Rolfe, B. E.; Xu, Z. P. High adjuvant activity of layered double hydroxide nanoparticles and nanosheets in anti-tumour vaccine formulations. Dalton Trans. 2018, 47, 2956–2964.

    CAS  Google Scholar 

  22. [22]

    Xu, Z. P.; Stevenson, G. S.; Lu, C. Q.; Lu, G. Q.; Bartlett, P. F.; Gray, P. P. Stable suspension of layered double hydroxide nanoparticles in aqueous solution. J. Am. Chem. Soc. 2006, 128, 36–37.

    CAS  Google Scholar 

  23. [23]

    Gu, Z.; Zuo, H. L.; Li, L.; Wu, A. H.; Xu, Z. P. P. Pre-coating layered double hydroxide nanoparticles with albumin to improve colloidal stability and cellular uptake. J. Mater. Chem. B 2015, 3, 3331–3339.

    CAS  Google Scholar 

  24. [24]

    Wei, P. R.; Cheng, S. H.; Liao, W. N.; Kao, K. C.; Weng, C. F.; Lee, C. H. Synthesis of chitosan-coated near-infrared layered double hydroxide nanoparticles for in vivo optical imaging. J. Mater. Chem. 2012, 22, 5503–5513.

    CAS  Google Scholar 

  25. [25]

    Zhang, L. X.; Sun, X. M.; Xu, Z. P.; Liu, R. T. Development of multifunctional clay-based nanomedicine for elimination of primary invasive breast cancer and prevention of its lung metastasis and distant inoculation. ACS Appl. Mater. Interfaces 2019, 11, 35566–35576.

    CAS  Google Scholar 

  26. [26]

    Raeesi, V.; Chou, L. Y. T.; Chan, W. C. W. Tuning the drug loading and release of DNA-assembled gold-nanorod superstructures. Adv. Mater. 2016, 28, 8511–8518.

    CAS  Google Scholar 

  27. [27]

    Zhang, Z. P.; Tongchusak, S.; Mizukami, Y.; Kang, Y. J.; Ioji, T.; Touma, M.; Reinhold, B.; Keskin, D. B.; Reinherz, E. L.; Sasada, T. Induction of anti-tumor cytotoxic T cell responses through PLGAnanoparticle mediated antigen delivery. Biomaterials 2011, 32, 3666–3678.

    CAS  Google Scholar 

  28. [28]

    Zeng, Q.; Jiang, H.; Wang, T.; Zhang, Z. R.; Gong, T.; Sun, X. Cationic micelle delivery of Trp2 peptide for efficient lymphatic draining and enhanced cytotoxic T-lymphocyte responses. J. Controlled Release 2015, 200, 1–12.

    CAS  Google Scholar 

  29. [29]

    Liu, D. Q.; Lu, S.; Zhang, L. X.; Ji, M.; Liu, S. Y.; Wang, S. W.; Liu, R. T. An indoleamine 2, 3-dioxygenase siRNA nanoparticle-coated and Trp2-displayed recombinant yeast vaccine inhibits melanoma tumor growth in mice. J. Controlled Release 2018, 273, 1–12.

    CAS  Google Scholar 

  30. [30]

    Xu, Z. P.; Niebert, M.; Porazik, K.; Walker, T. L.; Cooper, H. M.; Middelberg, A. P. J.; Gray, P. P.; Bartlett, P. F.; Lu, G. Q. Subcellular compartment targeting of layered double hydroxide nanoparticles. J. Controlled Release 2008, 130, 86–94.

    CAS  Google Scholar 

  31. [31]

    Liu, D. Q.; Lu, S.; Zhang, L.; Zhang, L. X.; Ji, M.; Liu, X. G.; Yu, Z.; Liu, R. T. A biomimetic yeast shell vaccine coated with layered double hydroxides induces a robust humoral and cellular immune response against tumors. Nanoscale Adv. 2020, 2, 3494–3506.

    CAS  Google Scholar 

  32. [32]

    Choi, S. J.; Choy, J. H. Layered double hydroxide nanoparticles as target-specific delivery carriers: Uptake mechanism and toxicity. Nanomedicine 2011, 6, 803–814.

    CAS  Google Scholar 

  33. [33]

    Oh, J. M.; Choi, S. J.; Lee, G. E.; Kim, J. E.; Choy, J. H. Inorganic metal hydroxide nanoparticles for targeted cellular uptake through clathrin-mediated endocytosis. Chem. Asian J. 2009, 4, 67–73.

    CAS  Google Scholar 

  34. [34]

    Oh, J. M.; Choi, S. J.; Kim, S. T.; Choy, J. H. Cellular uptake mechanism of an inorganic nanovehicle and its drug conjugates: Enhanced efficacy due to clathrin-mediated endocytosis. Bioconjugate Chem. 2006, 17, 1411–1417.

    CAS  Google Scholar 

  35. [35]

    Dong, H. Y.; Parekh, H. S.; Xu, Z. P. Particle size- and numberdependent delivery to cells by layered double hydroxide nanoparticles. J. Colloid Interface Sci. 2015, 437, 10–16.

    CAS  Google Scholar 

  36. [36]

    Li, B.; Tang, J.; Chen, W. Y.; Hao, G. Y.; Kurniawan, N.; Gu, Z.; Xu, Z. P. Novel theranostic nanoplatform for complete mice tumor elimination via MR imaging-guided acid-enhanced photothermo-/chemo-therapy. Biomaterials 2018, 177, 40–51.

    CAS  Google Scholar 

  37. [37]

    Moghimi, S. M.; Hedeman, H.; Muir, I. S.; Illum, L.; Davis, S. S. An investigation of the filtration capacity and the fate of large filtered sterically-stabilized microspheres in rat spleen. Biochi. Biophys. Acta (BBA) - Gen. Subj. 1993, 1157, 233–240.

    CAS  Google Scholar 

  38. [38]

    Moghimi, S. M.; Hunter, A. C.; Andresen, T. L. Factors controlling nanoparticle pharmacokinetics: An integrated analysis and perspective. Annu. Rev. Pharmacol. Toxicol. 2012, 52, 481–503.

    CAS  Google Scholar 

  39. [39]

    Cataldi, M.; Vigliotti, C.; Mosca, T.; Cammarota, M.; Capone, D. Emerging role of the spleen in the pharmacokinetics of monoclonal antibodies, nanoparticles and exosomes. Int. J. Mol. Sci. 2017, 18, 1249.

    Google Scholar 

  40. [40]

    Demoy, M.; Andreux, J. P.; Weingarten, C.; Gouritin, B.; Guilloux, V.; Couvreur, P. In vitro evaluation of nanoparticles spleen capture. Life Sci. 1999, 64, 1329–1337.

    CAS  Google Scholar 

  41. [41]

    Ernsting, M. J.; Murakami, M.; Roy, A.; Li, S. D. Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles. J. Controlled Release 2013, 172, 782–794.

    CAS  Google Scholar 

  42. [42]

    Zhang, L. X.; Sun, X. M.; Jia, Y. B.; Liu, X. G.; Dong, M. D.; Xu, Z. P.; Liu, R. T. Nanovaccine’s rapid induction of anti-tumor immunity significantly improves malignant cancer immunotherapy. Nano Today 2020, 35, 100923.

    CAS  Google Scholar 

  43. [43]

    Schroeder, A.; Heller, D. A.; Winslow, M. M.; Dahlman, J. E.; Pratt, G. W.; Langer, R.; Jacks, T.; Anderson, D. G. Treating metastatic cancer with nanotechnology. Nat. Rev. Cancer 2012, 12, 39–50.

    CAS  Google Scholar 

  44. [44]

    Li, F.; Chen, Y.; Liu, S. B.; Pan, X.; Liu, Y. L.; Zhao, H. T.; Yin, X. J.; Yu, C. L.; Kong, W.; Zhang, Y. The effect of size, dose, and administration route on zein nanoparticle immunogenicity in BALB/c mice. Int. J. Nanomedicine 2019, 14, 9917–9928.

    CAS  Google Scholar 

Download references


This work was financially supported by the International Partnership Program of Chinese Academy of Sciences (No. 122111KYSB20180005), the Australian Research Council (ARC) Discovery Project (No. DP190103486), Zhejiang Provincial Natural Science Foundation of China (No. LY19H160011), and Ningbo Digestive System Clinical Medicine Research Center (No. 2019A21003). We also thank the support from the Key Laboratory of Diagnosis and Treatment of Digestive System Tumors of Zhejiang Province.

Author information



Corresponding authors

Correspondence to Ting Cai, Rui-Tian Liu or Zhi Ping Xu.

Electronic Supplementary Material


Efficient delivery of clay-based nanovaccines to the mouse spleen promotes potent anti-tumor immunity for both prevention and treatment of lymphoma

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, LX., Jia, YB., Huang, YR. et al. Efficient delivery of clay-based nanovaccines to the mouse spleen promotes potent anti-tumor immunity for both prevention and treatment of lymphoma. Nano Res. 14, 1326–1334 (2021).

Download citation


  • cancer immunotherapy
  • nanovaccine
  • layered double hydroxide
  • targeted delivery to the spleen
  • lymphoma