Research ArticleHEALTH AND MEDICINE

A combinational chemo-immune therapy using an enzyme-sensitive nanoplatform for dual-drug delivery to specific sites by cascade targeting

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Science Advances  05 Feb 2021:
Vol. 7, no. 6, eaba0776
DOI: 10.1126/sciadv.aba0776
  • Fig. 1 Schematic diagram of dual-drug chemo- and immune-combinational therapy mechanism.

    (A) Schematic illustration for the self-assembly process of the cascade-targeting enzyme-sensitive hierarchical nanoplatform. (B) Schematic illustration of the combinational effects including chemotherapeutics in combination with anti–PD-L1 for activating the immune system to maximize the chemo-immune therapeutics. Cyto C, cytochrome C.

  • Fig. 2 Characterization the multifunctional properties of nanoparticles.

    (A) Particle sizes and (B) zeta potential of TPT, LTPT, DLTPT, and HAase + DLTPT. (C) Stability of DLTPT in pH 7.4 PBS and 10% serum-rich media. (D) Transmission electron microscopy (TEM) images and sizes of LTPT (1), DLTPT (2), and DLTPT preincubated with HAase at pH 5.0 buffer solution (3). Scale bars, 200 nm (left) and 100 nm (right). (E) Particle sizes and zeta potentials of DLTPT within HAase buffer solution (pH 5.0 and pH 6.8). (F) DOX release and (G) d-LND release profiles of DLTPT under different conditions.

  • Fig. 3 Cascade-targeting and apoptosis mechanism evaluations of DLTPT in vitro.

    (A) The CLSM images of DLTPT and HA + DLTPT cultured with cells for 2 hours and then incubated with fresh medium without nanoparticles for an additional 0, 2, and 4 hours, where the lysosomal staining channel is red and the DOX channel is green. Scale bars, 5 μm. (B) CLSM images and 2.5 D displays of 4T1 cells after treatment with coumarin-3 (cou)–labeled DLTPT for 2 hours and then incubated with fresh medium without nanoparticles for an additional 0, 2, and 4 hours, where the mitochondrial staining channel is red and cou representing TPT is blue. Scale bars, 10 μm. (C) CLSM images for mitochondrial membrane potentials in 4T1 cells after treatment with control, DOX, DTPT, DLTPT, and HA + DLTPT for 4 hours with JC-1–stained mitochondria channel (red, JC-1 aggregate). (D) Adenosine triphosphate (ATP) contents of control, d-LND, DOX, DTPT, DLTPT, and HA + DLTPT were detected after incubation with 4T1 cells for 4 hours. (E) Immunohistochemical staining images of cytochrome C. Scale bar, 25 μm.

  • Fig. 4 In vitro permeability and in vivo targeting ability of DLTPT.

    (A) CLSM images of CRT exposure secretion in 4T1 cells after treating with control, LTPT, DTPT, and DLTPT. Red and blue represent CRT signals and nucleus, respectively. Scale bar, 10 μm. (B) CLSM images, (C) regional coexistence fluorescence profiles (along with yellow arrow at 70 μm), and (D) quantitative analysis of DLTPT and HAase + DLTPT (incubation with HAase for 4 hours) incubated with 4T1 multicellular tumor spheroids (MTSs) for 6 hours (means ± SD, n = 3; scale bar, 100 μm). (E) Ex vivo fluorescence distribution of different tissues and (F) quantitative analysis of tumor tissues after administration with phosphate-buffered saline (PBS), TPT/DOX, and DLTPT for 24 and 36 hours in 4T1 tumor model mice. (G) Fluorescence intensity of frozen sections of tumor tissues detected by confocal laser, the core dyed by 4′,6-diamidino-2-phenylindole (DAPI; blue), the blood vessels stained with CD-31 mouse antibodies (green), and the nanoparticles represented with the red color (scale bar, 50 μm).

  • Fig. 5 In vivo antitumor efficiency in 4T1 tumor model and immune response induced by combinatorial therapy.

    (A) Schematic diagram of therapy timeline. i.p., intraperitoneal. (B) Relative tumor volume and (C) body weight of mice with PBS, DOX, DTPT, DLTPT, and DLTPT + anti–PD-L1 formulations at the end of each treatment (means ± SD, n = 6; symbol on each column represents statistical difference compared with DLTPT + anti–PD-L1; *P < 0.05, **P < 0.01, and ***P < 0.001). (D) Representative hematoxylin and eosin (H&E) for tumors; blue represents nuclei, and red are intercellular substance. Scale bar, 20 μm. (E) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining analysis for 4T1 tumors; normal and apoptosis cells are blue and yellow, respectively. Scale bar, 50 μm. (F) CD11c+ CD80+ cells in the lymph node, (G) CD11c+ CD86+ cells in the lymph node, (H) CD11c+ CD80+ cells in the spleen, (I) CD11c+ CD86+ cells in the spleen, (J) CD4+ T cells, (K) CD8+ T cells, (L) the ratio of (CD8+ T and CD4+ T cells) and Treg and (M) CD3+CD4+ Foxp3 (Treg) in tumor (means ± SD, n = 3; *P < 0.05 and **P < 0.01). (N) Immunohistochemical staining CD4, CD8, PD-L1, and interleukin-10 (IL-10) from 4T1 tumor sections. Blue represents nuclei, and feature expression cells are brown. Scale bar, 50 μm.

  • Fig. 6 Antimetastasis effect evaluation.

    (A) Schematic diagram of lung metastasis experiment timeline. (B) Inhibition of lung metastasis detected by bioluminescence imaging. (C) H&E staining of ex vivo lungs in lung metastasis 4T1 tumor–bearing mice. Scale bar, 100 μm.

Supplementary Materials

  • Supplementary Materials

    A combinational chemo-immune therapy using an enzyme-sensitive nanoplatform for dual-drug delivery to specific sites by cascade targeting

    Yanmei He, Lei Lei, Jun Cao, Xiaotong Yang, Shengsheng Cai, Fan Tong, Dennis Huang, Heng Mei, Kui Luo, Huile Gao, Bin He, Nicholas A. Peppas

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