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. 2022 Jul 12;13(1):4032.
doi: 10.1038/s41467-022-31551-6.

Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

Jingchao Li #  1 , Yu Luo #  2 , Ziling Zeng  1 , Dong Cui  1 , Jiaguo Huang  1 , Chenjie Xu  1 , Liping Li  3 , Kanyi Pu  4 , Ruiping Zhang  5
Affiliations

Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

Jingchao Li et al. Nat Commun. .

Abstract

Nanomedicine holds promise to enhance cancer immunotherapy; however, its potential to elicit highly specific anti-tumor immunity without compromising immune tolerance has yet to be fully unlocked. This study develops deep-tissue activatable cancer sono-immunotherapy based on the discovery of a semiconducting polymer that generates sonodynamic singlet oxygen (1O2) substantially higher than other sonosensitizers. Conjugation of two immunomodulators via 1O2-cleavable linkers onto this polymer affords semiconducting polymer immunomodulatory nanoparticles (SPINs) whose immunotherapeutic actions are largely inhibited. Under ultrasound irradiation, SPINs generate 1O2 not only to directly debulk tumors and reprogram tumor microenvironment to enhance tumor immunogenicity, but also to remotely release the immunomodulators specifically at tumor site. Such a precision sono-immunotherapy eliminates tumors and prevents relapse in pancreatic mouse tumor model. SPINs show effective antitumor efficacy even in a rabbit tumor model. Moreover, the sonodynamic activation of SPINs confines immunotherapeutic action primarily to tumors, reducing the sign of immune-related adverse events.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Design and mechanism of SPINs for deep-tissue activatable sono-immunotherapy.
a Schematic illustration of US-triggered deep-tissue activation of SPINs to release immunomodulators. b Schematic illustration of sonodynamic activation of SPINs to debulk tumor, enhance tumor immunogenicity, and release immunomodulators in situ as well as synergetic action of IDO inhibition and PD-L1 blocking on enhancing antitumor immunity with alleviated irAEs relative to free-drug administration.
Fig. 2
Fig. 2. Screening of SPNs for sonodynamic therapy.
a Chemical structures of SPs and small molecular sonosensitizers. b Schematic illustration of the synthesis of SPNs via nanoprecipitation. c Representative TEM image of SPN7. The experiment was repeated independently three times with similar results. d UV–vis absorption spectra of SPNs in 1× phosphate-buffered saline (PBS) solution (pH = 7.4). e ESR spectra of SPN7, PpIX and TiO2 nanoparticles (20 µg/mL) after US irradiation using TEMP as the trap. f Quantification of sonodynamic 1O2 generation for each sample (20 µg/mL) (n = 3). g ESR spectra of SPN7 (20 µg/mL) after 1, 2, 3, and 4 cycles of US irradiation with TEMP as the trap. h Relative 1O2 generation for SPN7, ICG, PpIX and AO (20 µg/mL) after different cycles of US irradiation (n = 3). US-irradiation conditions: 1.0 MHz, 1.2 W/cm2, 50% duty cycle, 5 min for each cycle. Data are presented as mean values ± SD. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Synthesis, characterization, and sonodynamic activation of SPINs.
a Chemical structures of amphiphilic semiconducting polymeric modulators and schematic illustration of their self-assembly and surface modification to form SPINs. b The molar ratios of each component in different SPINs. c Zeta potentials and hydrodynamic sizes of different SPINs in 1× PBS buffer (pH = 7.4) (n = 4). d Photographs of erythrocytes after incubation with 1× PBS buffer (negative control), 1% Triton X-100 (positive control), and 1× PBS buffer containing SPINs at the concentration of 100 µg/mL for 2 h, followed by centrifugation. e Hemolysis percentages of erythrocytes after incubation with SPINs at different concentrations for 2 h (n = 4). f Schematic illustration of US irradiation of SPIND2 solutions covered with a pork tissue. g ESR spectra of 1O2 for SPIND2 (20 µg/mL) after US irradiation (1.2 W/cm2, 3 min) without or with coverage of pork tissues at different thicknesses. h Release profiles of aPD-L1 and NLG919 from SPIND2 (40 µg/mL) after US irradiation for different time (n = 4). i PD-L1/PD-1 binding activity assay after treatment with free aPD-L1 or SPIND2 (40 µg/mL) with or without US irradiation (n = 4). SPIND2 – US versus SPIND2 + US: P < 0.0001. Statistical significance was calculated via a two-tailed Student’s t test. ***P < 0.001. In (g–i), the power intensity of US irradiation was 1.2 W/cm2 (1.0 MHz, 50% duty cycle). Data are presented as mean values ± SD. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Evaluation of SPIN-mediated sono-immunotherapy in Panc02 tumor mouse model.
a Schedule for the establishment of primary and distant tumors, triple systemic injection of SPINs (0.2 mL, 0.6 mg/mL) via tail vein, US irradiation (1.0 MHz, 1.2 W/cm2, 50% duty cycle, 10 min), and analysis of immune responses. b, c Relative tumor volumes of primary (b) and distant (c) tumors of Panc02 tumor-bearing C57BL/6 mice (n = 6) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), or SPIND2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm2, 50% duty cycle, 10 min). SPIND2 + US versus drug + US: P < 0.0001 for primary tumors (b); SPIND2 + US versus drug: P < 0.0001 for distant tumors (c). d Survival curves of Panc02 tumor-bearing C57BL/6 mice (n = 10) receiving different treatments as indicated. e Schematic illustration of treatment of rechallenged tumor mouse models using SPINs. f Growths of rechallenged tumors in Panc02 tumor-bearing mice after injection of saline or SPIND2 (0.2 mL, 0.6 mg/mL) with US irradiation (1.0 MHz, 1.2 W/cm2, 50% duty cycle, 10 min) (n = 5). Saline versus SPIND2 + US: P < 0.0001. g The survival curves of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge (n = 10). h Flow cytometry analysis of populations of effector memory T cells in the spleen of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge (n = 4). Saline versus SPIND2 + US: P = 0.0059. i Differentially expressed gene numbers in tumor tissues of mice after different treatments. j Relative expression of Carl, Hmgb1-ps1, Hmgb1-ps2, Cd80, Cd86, Cd40, Pdcd1, Cd3e, Cd8a, Ifng, Gzmb, Cxcl1, Cxcl2, Cxcl9, Cxcl10, Cxcl11, Ccl4, Ccl5, Il1b, Il2, Il6, Il7, Il15, Ido1, and Cd274 in tumors of Panc02 tumor-bearing mice after different treatments (the experiment was repeated independently five times with similar results). k Unsupervised hierarchical clustering of relative gene expression in tumors of Panc02 tumor-bearing C57BL/6 mice after different treatments (n = 5). Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Deep-tissue therapy of tumor models using SPINs.
a Schematic of sono-immunotherapy of subcutaneous pancreatic mouse tumors covered with 5-cm tissue. b, c Relative tumor volumes of primary (b) and distant (c) tumors of Panc02 tumor-bearing C57BL/6 mice (n = 5) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), SPIN0 or SPIND2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm2, 50% duty cycle, 10 min). The primary tumors were covered with 5-cm tissue under US irradiation. SPIND2 + US versus SPIN0 + US: P < 0.0001 for primary tumors (b); SPIND2 + US versus SPIN0 + US: P < 0.0001 for distant tumors (c). d Survival curves of Panc02 tumor-bearing C57BL/6 mice (n = 10) after different treatments for 60 days. e Schematic of US-mediated deep-tissue sonodynamic therapy of orthotopic pancreatic rabbit tumors. f Radiolabeling stability of 131I-SPIN0 after storage in saline or 50% serum at 37 °C for different time (n = 3). g, h SPECT imaging (g) and signal intensity (h) of orthotopic pancreatic rabbit tumors after systemic injection of 131I-SPIN0 (1.0 mL, 1.5 mg/mL) for different time (n = 4). The white dotted circle indicated tumors. i Computed tomography (CT) imaging of orthotopic pancreatic rabbit tumors after systemic injection of saline or SPIN0 (1.0 mL, 1.5 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm2, 50% duty cycle, 30 min). The white dotted circle indicated tumors. j Tumor volume of orthotopic pancreatic rabbit tumors (n = 3) after treatments as indicated for different days. Saline + US versus SPIN0 + US: P = 0.0108. k H&E staining images of orthotopic pancreatic rabbit tumors after different treatments. The experiment was repeated independently three times with similar results. l Survival curves of orthotopic pancreatic tumor-bearing rabbits (n = 4) after different treatments for 20 days. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; *P < 0.05, ***P  <  0.001. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. Preliminary evaluation of minimized irAEs for SPIND2-mediated sono-immunotherapy.
a, b Flow cytometry analysis of percentages of CD3+CD4+ Th cells (a), and CD3+CD8+ CTLs (b) in blood of mice (n = 4) at 30 day after systemic administrations of saline, SPIN0, SPIND2 (0.2 mL, 1.2 mg/mL) or free-drug mixture (8 mg/kg body weight for NLG919 and aPD-L1) with or without US irradiation (1.0 MHz, 1.2 W/cm2, 50% duty cycle, 10 min). Saline – US versus drug − US: P = 0.0023; saline − US versus drug + US: P = 0.0006; drug + US versus SPIND2 + US: P = 0.0071 for CD3+CD4+ Th cells (a); saline − US versus drug − US: P = 0.0004; saline − US versus drug + US: P = 0.0001; drug + US versus SPIND2 + US: P = 0.0093 for CD3+CD8+ CTLs (b). c, d Flow cytometry analysis of percentages of CD3+CD4+ Th cells (c), and CD3+CD8+ CTLs (d) in spleen of mice (n = 4) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0008; saline − US versus drug + US: P = 0.0005; drug + US versus SPIND2 + US: P = 0.0015 for CD3+CD4+ Th cells (c); saline − US versus drug − US: P = 0.0001; saline  − US versus drug + US: P = 0.0002; drug + US versus SPIND2 + US: P = 0.0049 for CD3+CD8+ CTLs (d). e Representative H&E staining images of liver after 30 days of treatments in different groups (white arrows indicate the infiltrated lymphocytes). The experiments were repeated independently three times with similar results. f Heatmap to show relative fold of cytokine levels in serum of mice after different treatments for 30 days relative to those in saline control group. g, h Serum levels of ALT (g) and AST (h) in mice (n = 5) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0010; saline − US versus drug + US: P = 0.0020; drug + US versus SPIND2 + US: P = 0.0054 for ALT (g); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P < 0.0001; drug + US versus SPIND2 + US: P = 0.0013 for AST (h). i Summary comparison of the antitumor immunity and irAEs between SPIND2-mediated sono-immunotherapy and free-drug treatment. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.
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