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Long-acting IL-2 release from pressure-fused biomineral tablets promotes antitumor immune response

Nature Cancer volume 6, pages 1384–1399 (2025)Cite this article

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Abstract

Long-acting controlled drug release formulations are highly desired for potentiating efficacy and reducing administration frequency. Here we present a kinetically controllable long-term interleukin-2 (IL-2) release platform by the fusion and boundary elimination of calcium carbonate and calcium phosphate amorphous phases. Unlike mixtures, a group of hybrid biominerals with the chemical formula Ca(CO3)x(PO4)2(1−x)/3 (CaCPs, 0 < x < 1) was fabricated under high pressure (2 GPa), and the CaCPs showed crystallization-driven release behaviors to optimize the in vivo fate of IL-2. Ca(CO3)1/2(PO4)1/3 dynamically remodeled immunosuppressive tumor microenvironments, preferentially activated cytotoxic and memory T cells by improving IL-2 redistribution and achieved weeks-long IL-2 retention in tumors with high tolerance and biosafety. In a melanoma model in female mice, Ca(CO3)1/2(PO4)1/3 revealed superior antitumor effects to inhibit local tumor recurrence, hinder the growth of distant untreated tumors and maintain long-term T cell responses against the rechallenged metastatic tumors.

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Fig. 1: Schematic and characterization of pressure-fused IL-2@CaCPs.
Fig. 2: IL-2 retention and biosafety of IL-2@Ca(CO3)1/2(PO4)1/3 (30 µg IL-2) through i.t. administration.
Fig. 3: Local IL-2@CaCPs therapy for preventing the recurrence of B16F10 melanoma tumors after surgery.
Fig. 4: Local IL-2@CaCPs therapy for systemic antitumor immunotherapy and long-term T cell memory responses.
Fig. 5: Local IL-2@Ca(CO3)1/2(PO4)1/3 in combination with anti-PD-1 checkpoint therapy for growth suppression of orthotopic and metastatic pancreatic tumors.

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Data availability

The data that support the findings of this study are available within the Article and its source data files, and/or from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Key R&D Program of China (grant 2021YFA0909900), the National Natural Science Foundation of China (grant 52233013), the Startup Packages of Zhejiang University to Z.G., the National Natural Science Foundation of China (grants 22435006 and 22275161), the Fundamental Research Funds for the Central Universities (grant 2024FZZX02-01-04) to Z.L., the Fundamental Research Funds for the Central Universities (grant 2022ZJJH02-01) to R.T., the National Natural Science Foundation of China (grant 223B2504) to W.F. and the China Postdoctoral Science Foundation (grant 2022M712716) and the National Natural Science Foundation of China (grant 52303208) to J.H. We appreciate the help from Z. Xing for the calculation and analysis of the crystallinity of CaCPs. We thank Q. Lin for her technical assistance on TEM characterization.

Author information

Author notes

  1. These authors contributed equally: Jinpeng Han, Shenqiang Wang, Weifeng Fang.

  2. These authors jointly supervised this work: Ruikang Tang, Zhaoming Liu, Zhen Gu.

Authors and Affiliations

  1. State Key Laboratory of Advanced Drug Delivery and Release Systems, Key Laboratory of Advanced Drug Delivery Systems of Zhejiang Province, School of Pharmacy, Zhejiang University, Hangzhou, China

    Jinpeng Han, Shenqiang Wang, Yinxian Yang, Ruyi Zhou, Yuqi Zhang, Jicheng Yu & Zhen Gu

  2. College of Pharmaceutical Sciences, Soochow University, Suzhou, China

    Shenqiang Wang

  3. Department of Chemistry, Zhejiang University, Hangzhou, China

    Weifeng Fang, Ruikang Tang & Zhaoming Liu

  4. Department of Burns and Wound Care Center, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China

    Yuqi Zhang

  5. Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, China

    Jicheng Yu & Zhen Gu

  6. Jinhua Institute of Zhejiang University, Jinhua, China

    Jicheng Yu & Zhen Gu

  7. Department of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China

    Jicheng Yu & Zhen Gu

  8. Institute of Fundamental and Transdisciplinary Research Zhejiang University, Zhejiang University, Hangzhou, China

    Ruikang Tang & Zhen Gu

  9. MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, China

    Zhen Gu

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Contributions

Z.G., R.T., Z.L. and J.H. initiated and supported the project. Z.G. and J.H. conceived and designed the project. Z.L. and R.T. provided important scientific suggestions on the project. J.H., S.W., W.F., Y.Y. and R.Z. performed the experiments. J.H. drafted the manuscript. Z.G., Z.L., R.T., S.W., W.F., J.H., Y.Z. and J.Y. analyzed the data and revised the manuscript. All authors reviewed and approved the manuscript.

Corresponding authors

Correspondence to Ruikang Tang, Zhaoming Liu or Zhen Gu.

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Competing interests

Z.G. is the co-founder of Zenomics Inc., Zcapsule Inc. and μZen Inc. The other authors declare no competing interests.

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Nature Cancer thanks Luiz Bertassoni, Xiaoyuan Chen and Honggang Cui for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Characterization of the pressure-fused IL-2@Ca(CO3)1/2(PO4)1/3.

a,d, TEM images of CaC (a) and CaP (d) nanoparticles. Scale bar, 250 nm. b,e, Size distributions of CaC (b) and CaP (e) nanoparticles. c,f, XRD patterns of CaC (c) and CaP (f) tablets. g,h, XRD patterns of calcite (g) and hydroxyapatite (h) with Ca(CO3)1/2(PO4)1/3, the right figures are the magnified regions. i, SEM images of the cross section of the pressure-fused IL-2@Ca(CO3)1/2(PO4)1/3. Scale bar, 10 µm. j, SEM-EDS mapping results of the enlarged cross-section image. The dots corresponded to the elemental N from IL-2. Scale bar, 200 nm. k, SEM images of the cross section of the crystallized IL-2@Ca(CO3)1/2(PO4)1/3 tablet after 2 months. The enlarged images represented the amorphous cross-section (upper right) and the crystallized cross-section (lower right). The images indicated that the crystallized thickness of the tablet was about 90 µm. Scale bar, 30 µm (left) and 3 µm (right). l, Measurement of IL-2 contents in amorphous and crystalline phases collected from the used tablets; n = 3 samples from three independent experiments. Statistical significance was calculated via two-tailed unpaired t-test. m, Changes in the transparency of IL-2@Ca(CO3)1/2(PO4)1/3 tablet as IL-2 contents increased; n = 3 samples from three independent experiments. The total mass of IL-2@Ca(CO3)1/2(PO4)1/3 tablet was 10 mg. The loading capacity of IL-2 in the tablet was at least 3 wt.%. n, SEM image of the pressure-fused IL-2@Ca(CO3)1/2(PO4)1/3 containing 300 µg IL-2. Scale bar, 10 µm. In l,m, data are presented as mean ± s.d. In a,d, i-k, and n, experiments were repeated three times.

Source data

Extended Data Fig. 2 Bioactivity of protein drugs and mechanical properties of the pressure-fused IL-2@Ca(CO3)1/2(PO4)1/3.

a,b, XRD patterns of calcite (a) and hydroxyapatite (b) with IL-2@Ca(CO3)1/2(PO4)1/3 containing 300 µg IL-2. c, Bioactivity of lyophilized IL-2 with the protection of sucrose; n = 3 samples from three independent experiments. d, Relative bioactivity of glucose oxidase (GOX, 160 kDa) released from GOX@Ca(CO3)1/2(PO4)1/3 tablet; n = 4 samples from four independent experiments. Although the tablet was stored at 4 °C for half a year, the released GOX still remained 68.83% activity of the native GOX. e, Relative bioactivity of insulin (5.8 kDa) released from insulin@ Ca(CO3)1/2(PO4)1/3 tablet; n = 3 samples from three independent experiments. Statistical significance was calculated via two-tailed unpaired t-test. f, AFM images to reveal the surface topography changes of IL-2@Ca(CO3)1/2(PO4)1/3 tablet by maintaining the applied pressure (2 GPa) for different times. The blue dashed line corresponded to the directions of the residual height variation curves. Experiments were repeated three times. Scale bar, 500 nm. g, Residual height variation curves obtained from the corresponding AFM images in f. h, Representative loading–unloading nano-indentation curve of IL-2@Ca(CO3)1/2(PO4)1/3 tablet. i, Young’s modulus of the tablet obtained from the nano-indentation curves; n = 3 samples from three independent experiments. j, Digital photo of the tablet subcutaneously implanted in mice for three months. The experiments were repeated three times from n = 3 independent mice in one experiment. Scale bar, 4 mm. In c-e, and i, data are presented as mean ± s.d. In a,b,j, experiments were repeated three times.

Source data

Extended Data Fig. 3 Crystallization kinetics and in vitro accumulated protein drug release of pressure-fused CaC and CaP tablets.

a,b, In situ XRD patterns of CaP (a) and CaC (b) tablets immersed in PBS for different time. c,d, The curves of crystallinity as a function of time from pure Ca3(PO4)2 (c) and CaCO3 (d) tablets. The crystallization kinetics were characterized by XRD by incubating the tablets in PBS for different times and the crystallinity was calculated from XRD results by the modified Rietveld method. e, Digital photos of pressure-fused CaP and CaC tablets exposed to air for different time. f, SEM images of pressure-fused CaP and CaC tablets immersed in PBS for different time. Experiments were repeated three times. Scale bar, 5 μm (upper) and 4 μm (below). g, Long-term in vitro accumulated IL-2 release (%) from IL-2@CaCPs with different fused compositional ratios; n = 3 samples from three independent experiments. The average release efficiency of IL-2@CaCPs ranged from 32.7% to 36.5%. h,i, In vitro accumulated protein drug release from the pressure-fused CaC (h) and CaP (i) tablets under different pressures; n = 4 samples from four independent experiments. We employed bovine serum albumin (BSA) as a model protein drug to investigate the roles of different applied pressures on in vitro release behaviours. In g-i, Data are presented as mean ± s.d. In a-d, experiments were repeated three times.

Source data

Extended Data Fig. 4 Modulation of TMEs and Ca2+-induced mitochondrial dysfunctions of IL-2-free CaCPs.

a, Time-dependent pH value changes of PBS containing pressure-fused CaCPs; n = 3 samples from three independent experiments. b, In vitro accumulated Ca2+ release from CaCPs; n = 3 samples from three independent experiments. c, Confocal fluorescence images of intracellular Ca2+ concentrations in B16F10 tumour cells. Scale bar, 200 μm. d, Relative quantification of Ca2+ concentrations in B16F10 tumour cells; n = 3 cell cultures from three independent experiments. e, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide) staining of B16F10 tumour cells treated with CaCPs. The red aggregates and green monomers represented high mitochondrial membrane potentials (normal mitochondrial functions) and low mitochondrial membrane potentials (abnormal mitochondrial functions), respectively. Scale bar, 50 μm. f, Relative quantification of the fluorescence intensity ratios of red and green channels in B16F10 tumour cells; n = 3 cell cultures from three independent experiments. g, Relative ATP levels of B16F10 tumour cells treated with different CaCPs; n = 3 cell cultures from three independent experiments. h-j, Relative quantification of CD45+ cells in lymphocytes (h), CD4+ cells in CD45+ cells (i), and CD8+ cells in CD45+ cells (j); n = 4 independent tumour samples from one experiment. k,l, Representative flow cytometric analysis images (k) and relative quantification (l) of MDSCs (CD11b+Gr-1+, myeloid-derived suppressor cells) gating on CD45+ cells; n = 4 independent tumour samples from one experiment. m,n, Representative flow cytometric analysis images (m) and relative quantification (n) of M1-like macrophages (CD80hi) gating on F4/80+CD11b+CD45+ cells; n = 4 independent tumour samples from one experiment. o,p, Representative flow cytometric analysis images (o) and relative quantification (p) of M2-like macrophages (CD206hi) gating on F4/80+CD11b+CD45+ cells; n = 4 independent tumour samples from one experiment. Statistical significance was calculated via one-way ANOVA with a Dunnett’s multiple comparisons test. In a,b,d,f,g, h-j, and l,n,p, data are presented as mean ± s.d.

Source data

Extended Data Fig. 5 Biosafety assessment of IL-2@Ca(CO3)1/2(PO4)1/3 tablet.

a, H&E staining images of the skin excised from IL-2@Ca(CO3)1/2(PO4)1/3 tablet-embedded mice after one month, n = 3 mice in each group. Scale bar, 100 μm. b,c, Representative immunohistochemical staining images of TNFα, IL-6, IL-10, IL-12, and IL-17 in the skin tissues subcutaneously injected of IL-2 (b) or implanted with IL-2@Ca(CO3)1/2(PO4)1/3 tablets (c) after 7 days, n = 3 mice in each group. Scale bars, 200 μm. Restricted by the high modulus of tablets, the tablets were removed before the slicing process. d, Blood routine and serum biochemistry analysis of the mice intratumorally implanted with IL-2@Ca(CO3)1/2(PO4)1/3 tablet for two weeks; n = 3 mice in each group. Light blue area indicated the normal reference range of blood routine data. Statistical significance was calculated via two-tailed unpaired t-test. In d, data are presented as mean ± s.d. In a-c, experiments were repeated three times.

Source data

Extended Data Fig. 6 Collagen deposition and the spatial distribution of immune cells in tumours induced by IL-2@Ca(CO3)1/2(PO4)1/3 tablets.

a, Representative Masson’s trichrome staining of the mice skin tissues implanted with IL-2@Ca(CO3)1/2(PO4)1/3 tablets for different time (1, 2, 4, and 8 weeks). Scale bar, 500 µm. b, The corresponding enlarged Masson’s trichrome staining images. Scale bar, 100 µm. c, Relative collagen areas calculated from the corresponding enlarged Masson’s trichrome staining images; n = 3 independent skin tissue samples from n = 3 independent mice in one experiment. Statistical significance was calculated via one-way ANOVA with a Dunnett’s multiple comparisons test. d-f, Representative immunofluorescence staining images showing co-localization and distribution of CD4+ T cells (pink), CD8+ T cells (green), and DC cells (CD11c-red) in B16F10 tumour tissues implanted with IL-2@Ca(CO3)1/2(PO4)1/3 tablets for 1 day (d), 3 days (e), and 7 days (f), n = 3 mice in each group. Cell nuclei were stained blue with DAPI. The location of tablets was marked by dashed area. Scale bars, 500 μm. In c, data are presented as mean ± s.d. In d-f, experiments were repeated three times.

Source data

Extended Data Fig. 7 Local IL-2@CaP-tablet therapy for preventing recurrence of B16F10 tumours after surgery.

a, IVIS images showing the growth of subcutaneous B16F10 tumours after primary resection. Five representative mice per group were shown. IL-2 with different doses (20, 40, 80 and 160 µg) were used. b,c, Individual (b) and average (c) tumour growth curves in different groups; n = 9 mice in each group. d, Overall survival over time for mice treated with different formulations; n = 9 mice in each group. Statistical significance was calculated via the log-rank (Mantel–Cox) test. e, Changes in the body weight over time; n = 9 mice in each group. f, Relative quantification of local CD8+ T cells as frequency of CD3+ T cells; n = 4 independent tumour samples from one experiment. g, Relative quantification of local Tregs (CD4+CD25+Foxp3+) as frequency of CD4+ T cells; n = 4 independent tumour samples from one experiment. Statistical significance was calculated via one-way ANOVA with a Dunnett’s multiple comparisons test unless otherwise specified. In c, and e-g, data are presented as mean ± s.d.

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Extended Data Fig. 8 Representative flow cytometric analysis images and the relative quantifications of IL-2@CaCPs.

a, Changes in the body weight over time; n = 9 mice in each group. b, Representative flow cytometric analysis images of CD103+ dendritic cells (DCs) gating on CD45+CD11c+ cells in the treated tumour. c,d, Representative flow cytometric analysis images (c) and relative quantification (d) of CD45+ cells in lymphocytes; n = 4 independent spleen samples from one experiment. e,f, Representative flow cytometric analysis images (e) and relative quantification (f) of CD3+ cells in CD45+ cells; n = 4 independent spleen samples from one experiment. g,h, Representative flow cytometric analysis images (CD8+ cells in CD3+ cells) (g) and relative quantification (h) of CD8+ cells in CD45+ cells; n = 4 independent spleen samples from one experiment. i, Representative flow cytometric analysis images of effector memory CD8+ T cells (CD8+CD62L−CD44+) and central memory CD8+ T cells (CD8+CD62L+CD44+). j, Degradation rate curve of IL-2@Ca(CO3)1/2(PO4)1/3 tablet within two months; n = 3 mice in each group. k, Data fitting of the degradation rate curve to estimate the degradation timeline of the crystallized tablet; n = 3 mice in each group. Statistical significance was calculated via one-way ANOVA with a Dunnett’s multiple comparisons test. In a,d,f,h,j,k, data are presented as mean ± s.d.

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Extended Data Fig. 9 Long-term biosafety assessment of IL-2@Ca(CO3)1/2(PO4)1/3 tablet.

a, Blood routine and serum biochemistry analysis of mice subcutaneously implanted with IL-2@Ca(CO3)1/2(PO4)1/3 tablet for three months; n = 3 mice in each group. Light blue area indicated the normal reference range of blood routine data. b, Blood routine and serum biochemistry analysis of mice implanted with IL-2@Ca(CO3)1/2(PO4)1/3 tablet under pancreatic capsule fixed by biocompatible 3 M BioGlue for three months; n = 3 mice in each group. Light blue area indicated the normal reference range of blood routine data. Statistical significance was calculated via two-tailed unpaired t-test. Data are presented as mean ± s.d.

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Extended Data Fig. 10 Representative sequential gating strategies to identify specific immune cells by flow cytometry.

a, CD8+ T cells and CD4+ T cells as displayed in Fig. 4g, h and Extended Data Fig. 4h–j. b, MDSCs, M1-like macrophages, and M2-like macrophages as displayed in Fig. 5k–m and Extended Data Fig. 4k–p. c, Local CD4+ and CD8+ T cell subsets including Tregs, pSTAT5+Tregs, pSTAT5+CD8+ T cells, CD25+CD8+ T cells, and IFN-γ+CD8+ T cells as displayed in Fig. 3f–k, Fig. 5f–j, and Extended Data Fig. 7f, j. d, DC cells as displayed in Fig. 4f and Extended Data Fig. 8b. e, Memory CD8+ T cells including effector memory CD8+ T cells (CD8+CD62L−CD44+) and central memory CD8+ T cells (CD8+CD62L+CD44+) as displayed in Fig. 4i, j and Extended Data Fig. 8c–i.

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Han, J., Wang, S., Fang, W. et al. Long-acting IL-2 release from pressure-fused biomineral tablets promotes antitumor immune response. Nat Cancer 6, 1384–1399 (2025). https://doi.org/10.1038/s43018-025-00993-4

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