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Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodul

Issuing time:2022-07-15 09:53

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.

Introduction

Cancer immunotherapy that targets the host immune system for anticancer therapeutic intervention has revolutionized the paradigm of oncology1,2,3. Particularly, checkpoint blockade immunotherapy that interferes inhibitory pathways in adaptive immunity has improved patient survival across a variety of cancer types4, as exemplified by the clinical practice of the U.S. Food and Drug Administration (FDA)-approved antibodies that antagonize cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1)5,6. However, as the therapeutic index largely depends on the conditions of pre-existing immunity, many non-immunogenic “cold” tumors (e.g., glioblastomas, pancreatic, breast, and colorectal carcinoma) often manifested with a low number of tumor-infiltrating lymphocytes are poorly responsive to immune checkpoint immunotherapy (~10–30% in clinical patients)7. To overcome this issue, other therapeutic modalities with the ability to elicit immunogenic cell death (ICD) (such as chemotherapy, phototherapy, and radiotherapy) can be combined with checkpoint blockade immunotherapy, which convert “cold” tumors into immunogenic “hot” phenotypes and sensitize tumors for improved immunotherapy8. However, these combinational immunotherapies inevitably encounter increased risk of immune-related adverse events (irAEs) due to enhanced disruption in homeostatic immune tolerance relative to monotherapy9,10.

Nanomedicine has the potential to enhance immunotherapy and simultaneously to reduce irAEs11. Local administration of immunotherapeutic hydrogels allows slow release of immune checkpoint blockers, cytokines or immune cells in the tumor microenvironment to induce regional responses12,13,14,15, which however is difficult to treat invisible or inaccessible tumors16. Alternatively, nanoparticles that release immunotherapeutic agents in response to the tumor biomarkers can be systemically administered, which still permits relatively controlled immunotherapy to tumor sites17,18,19,20. However, their strong reliance on the different levels of the selected biomarker between tumors and normal tissues limits the regional selectivity of immune action and constrains the design flexibility to the availability of tumor-specific biomarkers21. In contrast to endogenous biomarkers, exogenous stimuli bypass both limitations22, and can provide a more precise way to remotely control immune activation in tumors. Although immunotherapeutic nanoagents responsive to near-infrared (NIR) light have been developed for immunotherapy in a more safe and efficient manner23,24,25,26, light often shows shallow tissue penetration (<1 cm)27,28. By contrast, ultrasound (US) has deep-tissue penetration and the ability to induce the generation of reactive oxygen species (ROS) from sonosensitizers for cancer therapy29,30,31,32. However, such a sonodynamic process has not been integrated into nanomedicines as the exogenous stimulus to gain the spatiotemporal control of immunotherapy in preclinical settings.

We herein report the development of semiconducting polymer nanoparticles (SPNs) for deep-tissue activatable cancer sono-immunotherapy. By screening a library of agents, an ideal SPN is identified to have the highest sonodynamic singlet oxygen (1O2) generation efficiency. This SPN is used to further construct semiconducting polymer immunomodulatory nanoparticles (SPINs) by conjugating immunomodulators via a 1O2-cleavable linker to the backbone of SPNs (Fig. 1a). The antitumor immunity of SPINs is tested against the subcutaneous pancreatic mouse tumors, as the pancreatic tumor is one of the most aggressive tumors with low immunogenicity, high metastasis, and low survival rate33. SPINs can effectively accumulate into mouse subcutaneous pancreatic tumors and rabbit orthotopic pancreatic tumors after systemic administration. Under US irradiation directed to the tumor regions, SPINs generate 1O2 that can (i) debulk tumor tissues, (ii) reprogram the tumor microenvironment through upregulating the expression levels of PD-L1 and indoleamine 2,3-dioxygenase (IDO), and (iii) scissor the 1O2-cleavable linkers to remotely release the immunomodulators, NLG919 and anti-PD-L1 antibody (aPD-L1). NLG919 is a potent inhibitor for IDO and can promote the proliferation of cytotoxic T lymphocytes (CTLs) and inhibit regulatory T cells (Treg)34. The aPD-L1 can bind to PD-L1 on the surface of cancer cells to block its binding with PD-1 on the surface of PD-1+ T cells35, thereby inhibiting T-cell exhaustion and in turn promoting the tumoricidal functions of T-cell immunity36. In addition, NLG919 has shown a great potential to improve the efficacy of aPD-L1, achieving a synergistic effect37. In such a way, sonodynamic activation cascade precisely restricts the immunotherapeutic action to tumor regions. Thus, SPIN-mediated activatable sono-immunotherapy not only elicits high antitumor immunity to reject both primary and metastatic distant pancreatic tumors and prevent tumor recurrence, but also reduces the probability of irAEs (Fig. 1b). SPIN-mediated deep-tissue therapy of pancreatic tumors covered with 5-cm tissues and deep-seated rabbit orthotopic pancreatic tumors is also achieved.

Fig. 1: Design and mechanism of SPINs for deep-tissue activatable sono-immunotherapy.
figure 1

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.

Results

Screening of SPNs for sonodynamic 1O2 generation

A library of hydrophobic semiconducting polymers (SPs) (Fig. 2a) were transformed into water-soluble nanoparticles via nanoprecipitation with the assistance of an amphiphilic triblock copolymer, poly(ethylene glycol)-block-poly-(propylene glycol)-block-poly(ethylene glycol) (PEG-b-PPG-b-PEG) (Fig. 2b). As shown in the transmission electron microscopy (TEM) image, the prepared SPNs exhibited a spherical morphology with a uniform size distribution (Fig. 2c). Dynamic light scattering (DLS) measurements revealed that the hydrodynamic diameters of SPNs ranged from ~24 to 40 nm (Supplementary Fig. 1). Owing to the different molecular structures, these SPNs had the absorption maxima ranging from 320 to 680 nm (Fig. 2d), and fluorescence emission maxima ranging from 430 to 840 nm (Supplementary Fig. 2).

Fig. 2: Screening of SPNs for sonodynamic therapy.
figure 2

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.

The sonodynamic 1O2 generation properties of SPNs were investigated and compared with other commonly used sonosensitizers including silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NCBS), indocyanine green (ICG), protoporphyrin IX (PpIX), acridine orange (AO), curcumin (CUR), and inorganic titanium dioxide (TiO2) nanoparticles at the same mass concentration. Electron spin resonance (ESR) was applied to measure the generation of 1O2 under US irradiation using 2,2,6,6-tetramethylpiperidine (TEMP) as the radical trap. The characteristic ESR peak of 1O2 was observed for all samples (Fig. 2e and Supplementary Fig. 3); however, SPN7 generated the highest amount of 1O2 (34.5 nmol/μg), which was at least threefold higher relative to the others (Fig. 2f). The ranking was consistent with the 1O2 generation efficiencies under white light irradiation (Supplementary Fig. 4). This implied that sonoluminescence-induced 1O2 generation probably was the main mechanism involved for SPNs: US induced the sonoluminescence of water in the range of 350–650 nm38, which subsequently excited the agents to produce 1O2 in a way similar to photodynamic process.

In addition to the efficient 1O2 generation, the intensity of 1O2 peak for SPN7 remained nearly the same (Fig. 2g) after four cycles of US irradiation (5 min for each cycle); in contrast, the ability to generate 1O2 for ICG, PpIX, and AO was almost completely demolished (dropped by 95.8, 100, and 91.7%, respectively) (Fig. 2h and Supplementary Fig. 5). This was attributed to the high stability of SPN7 under US irradiation as witnessed by its unchanged absorption, while ICG, PpIX, and AO were molecularly damaged after US irradiation (Supplementary Fig. 6). These data confirmed that SPN7 is an optimal sonosensitizer for in vivo applications.

Construction of SPINs and deep-tissue sonodynamic activation

SPINs were constructed based on SP7 due to its highest 1O2 generation efficacy among tested SPNs and excellent sonodynamic stability of SPN7. SP7 with bromide groups on the side chains (SP7-Br) was synthesized via Suzuki polycondensation and then reacted with sodium azide to afford azide groups linked polymer (SP7-N3) (Supplementary Figs. 710). The molecular weight of the synthesized SP7-N3 was measured to be 7200 Da, and the maximum absorbance and fluorescence emission was observed at 574 and 770 nm, respectively (Supplementary Fig. 11). To construct SPINs, a 1O2-cleavable linker was synthesized and utilized to cage the hydroxyl group of NLG919, followed by covalent conjugation with the carboxyl group of alkynyl PEG (Mw = 2000), affording the pro-modulator PEG-NLG919 conjugate (defined as PEG-N) (Supplementary Figs. 1215). A 1O2-responsive linker (2,2’-(propane-2,2-diylbis(sulfanediyl)diacetic acid, PSDA)-conjugated PEG chain (defined as PEG-PSDA) was synthesized according to our previous report25. SP7-N3 was then grafted with mPEG-alkyne (Mw = 2000), PEG-N, PEG-PSDA or alkyne-PEG-COOH at different feeding molar ratios to obtain the amphiphilic semiconducting polymer conjugates (Supplementary Figs. 1620), which would self-assemble into nanoparticles (defined as SPIN0, SPINN, SPN-PEG1, SPN-PEG2, and SPN-PEG3, respectively) in aqueous solution (Fig. 3a). Further modifications of SPN-PEG1, SPN-PEG2 and SPN-PEG3 on their surface with aPD-L1 afforded SPINA, SPIND1, and SPIND2, respectively. The molar ratios of NLG919/aPD-L1 in different SPINs were shown in Fig. 3b.

Fig. 3: Synthesis, characterization, and sonodynamic activation of SPINs.
figure 3

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 (gi), 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.

A spherical morphology with uniform size distribution was observed for all SPINs (Supplementary Fig. 21). The hydrodynamic sizes of SPINA, SPIND1 and SPIND2 (from 62.9 to 75.0 nm) were larger than those of SPIN0 and SPINN (~25 nm), and all SPINs showed negative zeta potentials ranging from −14.3 to −4.9 mV (Fig. 3c). They showed excellent colloidal stability in saline buffer solution as witnessed by negligible size change after 21 days of storage (Supplementary Fig. 22). SPINs showed similar absorption profiles with two characteristic peaks of SP7 at ~360 and 553 nm, and almost identical fluorescence emission peaked at ~810 nm (Supplementary Fig. 23). The NIR fluorescence of SPINs allowed for in vivo fluorescence imaging and tracking their tumor accumulation. All SPINs exhibited negligible cytotoxicity even at the high incubation concentration of 100 µg/mL (Supplementary Fig. 24). More importantly, almost no hemolysis of erythrocytes was observed after incubation with SPINs (Fig. 3d-e), testifying their suitability for systemic administration.

The 1O2 generation properties of SPINs under US treatment were almost identical to that of SPN7 prepared via nanoprecipitation (Supplementary Fig. 25). To investigate the effect of tissue penetration depth on US-induced 1O2 generation of SPINs, SPIND2 solutions covered by a pork tissue were exposed under US irradiation for ESR measurement (Fig. 3f). The ESR characteristic peaks of 1O2 slightly decreased with the increasing of tissue thickness (0, 2, 4, 6, 8, and 10 cm), which was still 65% of its original at a thickness of 10 cm (Fig. 3g). This envisioned that SPINs should be able to efficiently generate 1O2 to break the 1O2-cleavable linkers to release the immunomodulators even in deep tissues.

Deep-tissue sonodynamic activation of SPINs without and with the coverage of 10-cm pork tissues was evaluated by quantifying the amounts of released immunomodulators under US treatment. Under US irradiation at a power intensity of 1.2 W/cm2, the release amount of aPD-L1 and NLG919 in SPIND2 containing solution gradually increased as a function of treatment time regardless of the coverage of tissues (Fig. 3h). After US treatment for 30 min, the release amount of aPD-L1 and NLG919 was measured to be 76.9% and 90.6%, respectively, which was slightly decreased to 70.9% and 84.8% after coverage with 10-cm tissues. Note that the releases of aPD-L1 and NLG919 were negligible under white light irradiation when covering with 10-cm tissues (Supplementary Fig. 26). These results suggested deep-tissue activation of SPINs to control the release of immunomodulators using US, which however was impossible for white light. The PD-L1/PD-1 binding activity was only slightly inhibited for SPIND2 treatment without US irradiation, while which was inhibited by 3.8-fold after US irradiation (Fig. 3i), further confirming the sonodynamic activation of SPIND2.

Activatable sono-immunotherapy of subcutaneous pancreatic tumors

The therapeutic efficacy of SPIN-mediated sono-immunotherapy was evaluated using a bilateral Panc02 tumor-bearing mouse model. In vivo NIR fluorescence imaging was used to confirm the accumulation of SPINs in tumors after administration through tail vein. The fluorescence signals of tumors gradually increased and similarly reached the maximum at 24 h post injection of SPINs, which was around 5.3–7.2-fold higher than the background signals of tumors (Supplementary Fig. 27). Moreover, intense red fluorescence signals of nanoparticles were observed in tumor tissues at 24 h post-injection of SPINs (Supplementary Fig. 28). The major accumulation of SPINs was found in tumors (15–17% ID/g) and livers (21–23% ID/g) and substantial lower accumulations were found in other organs such as the heart (<3% ID/g), spleen (<6% ID/g), lung (<4% ID/g), and kidney (<5% ID/g) (Supplementary Fig. 29). In contrast, free drug (NLG919) showed a very high accumulation percentage in liver (16% ID/g), but limited accumulation in tumors.

To evaluate ICD biomarkers after SPIN-mediated sonodynamic therapy, the expression levels of calreticulin (CRT) and high mobility group box 1 (HMGB1), and secretion of adenosine triphosphate (ATP) in tumors of mice after intravenous injection of SPINs or free-drug mixture of NLG919 and aPD-L1 at a similar dosage with or without US irradiation were investigated. Obvious fluorescence signals of singlet oxygen sensor green (SOSG) were observed in tumors of SPIN-injected and US-irradiated groups (Supplementary Fig. 30), verifying the generation of 1O2. As shown in the immunofluorescence staining images, the expression levels of CRT and HMGB1 were greatly increased after SPIN injection and US irradiation, which were at least 6.1- and 4.3-fold higher relative to those in the control groups, respectively (Supplementary Fig. 31a–c). The treatments of SPINs plus US irradiation also increased the ATP levels in tumors by more than 1.8-fold compared with the control groups (Supplementary Fig. 31d). In contrast, the levels of CRT, HMGB1 and ATP did not have remarkable changes in free-drug-injected groups regardless of US irradiation. Furthermore, the expression levels of PD-L1 in tumors after SPIN injection and US irradiation were at least 2.2-fold higher relative to that in the control group (Supplementary Fig. 32), while the treatment of SPIND2 and free drug without US irradiation did not obviously change the PD-L1 expression levels. Immunohistochemical staining images revealed that the saline-injected control group had few CD3+ and CD8+ tumor-infiltrating lymphocytes inside the tumor tissues (Supplementary Fig. 33), which should be due to the intrinsically low immunogenicity of Panc02 pancreatic tumors. The numbers of CD3+ and CD8+ tumor-infiltrating lymphocytes were obviously increased after SPIN injection with US irradiation, while which were not increased much for sole SPIND2 injection without US irradiation and free-drug injection regardless of US irradiation. The results indicated that SPIN-mediated treatment could effectively trigger ICD of tumor cells and thus enhance tumor immunogenicity in the tumor microenvironment.

To evaluate the therapeutic efficacy of SPIN-mediated sono-immunotherapy, bilateral Panc02 tumor-bearing mice were treated through a triple therapeutic injection (on days 0, 3, and 6) and US irradiation (on days 1, 4, and 7) (Fig. 4a). Without US irradiation, no obvious cell apoptosis/necrosis was observed in the H&E and immunofluorescence terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining images of tumors after free drug and SPIND2 injection (Supplementary Fig. 34), which was similar to that of the saline-injected control group. The growths of primary and distant tumors in the SPIND2-injected group without US irradiation did not have any changes, and those in free-drug-injected group without US irradiation were slightly inhibited compared to that in the control group (Fig. 4b, c). Without US irradiation, the median survival of free drug and SPIND2 injected mice was 36 and 35 days, respectively, which was slightly longer than that of the control mice (27 days) (Fig. 4d). With US irradiation, as shown in H&E and TUNEL staining images, cell apoptosis/necrosis could be observed in both primary and distant tumors of mice after injection of SPINs (Supplementary Fig. 34). Particularly the highest degree of cell apoptosis/necrosis of both primary and distant tumors was found in the SPIND2 injected and US-irradiated group. The growths of primary tumors in the free drug, SPIN0, SPINN, SPINA, SPIND1, and SPIND2 injected group with US irradiation was inhibited by 22.3%, 35.1%, 68.9%, 76.8%, 78.6%, and 98.9% compared to that in the control group, respectively (Fig. 4b and Supplementary Fig. 35a). For distant tumors, the inhibition ratio of tumor growth in these groups was 25.6%, 30.1%, 56.9%, 68.1%, 69.5%, and 97.1%, respectively (Fig. 4c and Supplementary Fig. 35b). The median survival of SPIN0, SPINN, SPINA, and SPIND1 injected and US-irradiated mice was 33, 35, 39, and 46 days, respectively (Fig. 4d and Supplementary Fig. 36). More importantly, the survival of mice was still 100% after SPIND2 injection and US irradiation for 80 days. These results suggested that SPIND2-mediated treatment showed the highest efficacy in inhibiting the growths of primary and distant metastatic tumors and prolonging the survival of mice.

Fig. 4: Evaluation of SPIN-mediated sono-immunotherapy in Panc02 tumor mouse model.
figure 4

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.

To further confirm the superior antitumor efficacy of SPIND2-mediated sono-immunotherapy, the tumor-bearing mice after treatment were rechallenged with Panc02 tumor cells on the opposite flank (Fig. 4e). Note that the volume of rechallenged tumors in the saline-injected control mice gradually increased, while no rechallenged tumors were observed for mice after SPIND2 injection with US irradiation (Fig. 4f). All saline-injected control mice after tumor rechallenge died within 40 days (Fig. 4g). In contrast, the SPIND2-injected and US-irradiated mice exhibited a 100% survival for 80 days even after tumor rechallenge. The population of effector memory T cells in spleen of SPIND2-injected and US-irradiated mice was 25.5%, which was 1.9-fold higher than that for the control mice (Fig. 4h and Supplementary Fig. 37). This verified that SPIND2-mediated sono-immunotherapy could build a long-term immunological memory to prevent tumor rechallenge.

We evaluated the gene expression profiles of tumor tissues after different treatments using multiplexed gene expression analysis. Without US irradiation, the number of differentially expressed genes was less than 180 in the saline control, free drug, and SPIND2 injection groups (Fig. 4i). The expression levels of 76 immune-related genes in free-drug and SPIND2-injected group did not have obvious changes compared to those in the control group (Fig. 4j, k). With US irradiation, 261 genes were found to be differentially expressed in SPIND2- injected groups (Fig. 4i). The expression levels of 76 immune-related genes in the SPIND2-injected and US-irradiated group were overall higher than those in the other groups (Fig. 4j, k). The upregulated genes were associated with several key immune pathways, such as MHC class II protein complex binding and lymphocyte costimulation (Supplementary Fig. 38). In the SPIND2-injected and US-irradiated group, the expression levels of ICD-related genes (Carl, Hmgb1-ps1, and Hmgb1-ps2, >5-fold), DC activation, and T-cell priming-related genes (Cd80, Cd86, Cd40, Pdcd1, Cd3e, Cd8a, Ifng, and Gzmb, >1.2-fold), chemokine-encoded genes (Cxcl1, Cxcl2, Cxcl9, Cxcl10, Cxcl11, Ccl4, and Ccl5, >2.5-fold), and cytokine-encoded genes (Il1b, Il2, Il6, Il7, and Il15, >2.4-fold) were obviously upregulated compared to the control group (Fig. 4j). More importantly, the expression levels of immunosuppressive genes (Ido1 and Cd274) were also increased by 1.6- and 4.0-fold, respectively. This further verified that SPIN-mediated US treatment could convert non-immunogenic “cold” tumor microenvironment into immunogenic “hot” one via upregulating the expression of IDO and PD-L1 in cancer cells.

SPIND2-mediated antitumor immune activation effect was then investigated using flow cytometry and immunofluorescence staining. The free drug and SPIND2 injection did not obviously increase the population of matured DCs (CD80+CD86+) in tumor-draining lymph nodes (TDLNs) compared to the saline control group (Supplementary Figs. 39, 40). The population of CTLs (CD3+CD8+) in primary and distant tumors of SPIND2 injected mice was slightly increased compared to that in the control group, which however was 1.2-fold lower relative to that of free-drug-injected mice (Supplementary Fig. 41). Free-drug injection reduced the population of intratumor Treg cells (CD25+Foxp3+) by 2.6-fold, but the treatment of SPIND2 did not have a remarkable influence on the population of Treg cells (Supplementary Fig. 42). The immunofluorescence staining images revealed that the expression of CD8, IFN-γ, and Granzyme B in tumor tissues of mice after free drug and SPIND2 injection without US irradiation was similar or slightly higher than those in the control group (Supplementary Figs. 43, 44). The treatments of SPINs with US irradiation overall increased the populations of matured DCs in TDLNs, and CTLs in both primary and distant tumors, but reduced the populations of Treg cells (Supplementary Figs. 4042). The highest populations of matured DCs (36.4%), and CTLs in primary (24.3%) and distant tumors (23.4%) were observed for SPIND2-injected and US-irradiated group, which was 2.4-, 1.6-, and 1.8-fold higher relative to that in free-drug-injected and US-irradiated group, respectively. The lowest population of intratumor Treg cells in primary tumors (2.8%) and distant tumors (4.2%) was also observed for the SPIND2-injected and US-irradiated group, which was 2.4- and 1.8-fold lower than that in free-drug injection plus US-irradiation group, respectively. Moreover, the expression levels of CD8, IFN-γ and Granzyme B in tumor tissues were overall increased after SPIN injection and US irradiation compared to those in the control group (Supplementary Figs. 43 and 44). Particularly, the SPIND2-injected and US-irradiated group showed the highest expression levels of intratumor CD8, IFN-γ, and Granzyme B. These results verified that SPIND2-mediated sono-immunotherapy triggered the highest antitumor efficacy.

SPIN-mediated deep-tissue therapy of tumor models

To verify the deep-tissue therapeutic efficacy mediated by SPINs, US irradiation (on days 1, 4, and 7) of tumors covered with 5-cm pork tissues was conducted after systemic injection of SPIND2 (on day 0, 3, and 6) into Panc02 tumor-bearing mice (Fig. 5a). Without US irradiation, the growth of primary and distant tumors for the SPIND2 injection group was similar as that of the saline control group, and free-drug treatment slightly reduced the growth of both tumors (Fig. 5b, c). The median survival of free drug and SPIND2-injected mice without US irradiation was 35 and 32 days, respectively, which was almost the same as that of the control group (34 days) (Fig. 5d). Although free-drug treatment slightly increased the population of CTLs in tumors by 1.9-fold, the sole SPIND2 injection did not obviously change the population of CTLs compared to saline control group (Supplementary Fig. 45). With US irradiation, the growths of both primary and distant tumors in free drug, SPIN0, and SPIND2 injection groups were inhibited (Fig. 5b, c). In particular, the tumor growth in SPIND2 injected and US-irradiated group was inhibited by 100% for primary tumors and 96.4% for distant tumors. In the SPIND2 injection and US-irradiation group, the animal survival was still 100% for 60 days, while the median survival of mice in free drug and SPIN0 injection and US-irradiation group was only 37 days (Fig. 5d). The population of intratumoral CTLs in the SPIND2-injected and US-irradiated group was 23.6%, which was 2.1- and 1.6-fold higher than that in free drug and SPIN0 injection with US-irradiation group, respectively (Supplementary Fig. 45). These results verified that SPIND2-mediated treatment showed an excellent therapeutic efficacy for tumors covered with 5-cm tissues.

Fig. 5: Deep-tissue therapy of tumor models using SPINs.
figure 5

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.

SPIN-mediated deep-tissue therapy was then evaluated using orthotopic pancreatic rabbit tumors (Fig. 5e). As the antibodies against rabbit PD-L1 have not been proven to be effective for in vivo immunotherapy, we only conducted sonodynamic therapy. To track the accumulation of SPINs in orthotopic pancreatic rabbit tumors, radionuclide 131I was used to label SPIN0 for single-photon emission computed tomography (SPECT) imaging, as iodine could be covalently immobilized onto aromatic backbones via electrophilic substitution of protons39,40. The obtained 131I-SPIN0 showed excellent radiolabeling stability in saline and 50% serum solutions at 37 °C (Fig. 5f). After systemic injection of 131I-SPIN0 into orthotopic pancreatic tumor-bearing rabbits, the signal intensity of tumors gradually increased and reached the maximum at 12 h post-injection time, as shown in the SPECT images (Fig. 5g, h), confirming effective accumulation of SPIN0 in orthotopic pancreatic rabbit tumors. The tumors of rabbits were treated via a triple therapeutic injection (on days 0, 3, and 6) and US irradiation (on days 1, 4, and 7) to evaluate the therapeutic efficacy. Without US irradiation, the growth of tumors for SPIN0 injected rabbits was similar as that for the saline-injected control group (Fig. 5i, j), and no apparent apoptosis/necrosis of tumor cells was observed from histological hematoxylin and eosin (H&E) and TUNEL staining images of tumor tissues (Fig. 5k and Supplementary Fig. 46). The median survival of rabbits after saline and SPIN0 injection without US irradiation was 12 and 14 days, respectively (Fig. 5l). With US irradiation, the tumor growth of SPIN0-injected rabbits was inhibited by 2.4-fold compared to the saline-injected group after treatment for 15 days (Fig. 5i, j). As shown in H&E and TUNEL staining images, obvious apoptosis/necrosis was observed for tumors in the SPIN0-injected and US-irradiated group (Fig. 5k and Supplementary Fig. 46). The median survival of rabbits in SPIN0-injected and US-irradiated group was 17 days, longer than that for the saline group (Fig. 5l). The results suggested that SPIN-mediated therapy could be used to treat orthotopic pancreatic rabbit tumors due to the deep-tissue penetration of US.

Preliminary evaluation of minimized irAEs for sono-immunotherapy

The minimized irAEs for SPIN-mediated activatable sono-immunotherapy relative to the free-drug treatment was evaluated. Without US irradiation, the percentages of immune cells in blood and spleen for free-drug treatment were observably increased, which was 1.7-fold (CD3+CD4+ Th cells in blood), 1.8-fold (CD3+CD8+ CTLs in blood), 1.6-fold (CD3+CD4+ Th cells in spleen), and 1.8-fold (CD3+CD8+ CTLs in spleen) higher relative to those for the saline-injected control group, respectively (Fig. 6a–d and Supplementary Figs. 47, 48). In contrast, the percentages of CD3+CD4+ Th cells and CD3+CD8+ CTLs in SPIND2-treated group were similar as those in the control group and were at least 1.6-fold lower than those for the free-drug treatment group, which should be attributed to the minimal pharmacokinetic property of SPIND2 without sonodynamic activation. As shown in the H&E staining images, obvious accumulation of lymphocytes in the liver (especially around central vein) was observed for the free-drug treatment (Fig. 6e and Supplementary Fig. 49), while which was hardly found in the control and SPIND2 treated groups. Moreover, the infiltrations of CD3+, CD4+, and CD8+ T cells in livers and spleens of mice after treatment of free drug were overall higher than those for the control and SPIND2 treatment groups (Supplementary Figs. 5055). The treatment of free-drug would cause severe cytokine storm as the serum levels of various cytokines including interleukin-23 (IL-23), IL-1α, interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), IL-1β, IL-10, IL-6, IL-27, IL-17A, IFN-β, and granulocyte–macrophage colony-stimulating factor (GM-CSF) in this group was increased by more than 2.3-fold compared to those in the control group (Fig. 6f and Supplementary Fig. 56). In contrast, the levels of these cytokines in the SPIND2 treatment group were similar as those in the control group. The free-drug treatment was also found to increase the serum levels of key hepatic enzymes, including alanine aminotransferase (ALT) (by 1.6-fold), aspartate aminotransferase (AST) (by 1.6-fold), and alkaline phosphatase (ALP) (by 1.4-fold) compared to the saline control group (Fig. 6g, h and Supplementary Fig. 57a). However, the serum levels of these hepatic enzymes in the SPIND2 treatment group were almost identical to those in the control group. These results suggested that free-drug treatment potentially caused hepatic irAEs, which was common for immune checkpoint inhibitor therapy41; while which was not observed for SPIND2 treatment because of minimized bioactivities of caged NLG919 and aPD-L1.

Fig. 6: Preliminary evaluation of minimized irAEs for SPIND2-mediated sono-immunotherapy.
figure 6

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.

With US irradiation, the percentages of CD3+CD4+ Th cells and CD3+CD8+ CTLs in the blood and spleen for the SPIND2-injected group were more than 1.2-fold lower relative to the free-drug-treated group (Fig. 6a–d). The treatment of free drug with US treatment caused the accumulation of a larger number of lymphocytes in liver tissues, while nearly no accumulation of lymphocytes in livers was observed for SPIND2 treatment with US irradiation (Fig. 6e). Moreover, the infiltration of CD3+, CD4+, and CD8+ T cells in the heart, liver, spleen, lung, and kidney for SPIND2-treated and US-irradiated group was overall lower than those after free-drug treatment with US irradiation (Supplementary Figs. 5055). Although obvious cytokine storm was observed for the mice after treatment with free drug and US irradiation, the serum levels of IL-23, IL-1α, IFN-γ, TNF-α, MCP-1, IL-1β, IL-10, IL-6, IL-27, IL-17A, IFN-β, and GM-CSF in SPIND2-treated and US-irradiated group was overall 1.6-fold lower than those in the free-drug-treated and US-irradiated group (Fig. 6f and Supplementary Fig. 56). The serum levels of ALT, AST, and ALP in the SPIND2-treated and US-irradiated group was only slightly increased compared to the control group, while they were still 1.5-, 1.3-, and 1.3-fold lower relative to those after free-drug treatment with US irradiation (Fig. 6g, h and Supplementary Fig. 57a). Furthermore, in the SPIND2-treated and US-irradiated group, the serum levels of creatinine (CRE), urea, and gamma-glutamyl transferase (GGT) (Supplementary Fig. 57b–d), several vital blood parameters (Supplementary Fig. 58), the body weight of mice (Supplementary Fig. 59), and histological morphologies of major organs (heart, liver, spleen, lung, and kidney) (Fig. 6e and Supplementary Fig. 49) did not change obviously. These results demonstrated that SPIND2 treatment and US irradiation had minimal impact on disrupting homeostatic immune tolerance in normal organs of mice, indicating the reduced probability of irAEs relative to free-drug treatment regardless of US irradiation (Fig. 6i).

Discussion

With deep-tissue penetration and safe nonionizing radiation, US serves as an ideal external stimulus to precisely activate cancer immunotherapy in situ in living subjects. Although sonodynamic process has been combined with chemotherapy, chemodynamic, or phototherapy to treat cancer in preclinical settings, it has been rarely exploited for immunotherapy with only several examples wherein nanosonosensitizers and immune checkpoint inhibitors were independently administrated into mice (Supplementary Table 1)42,43. To acquire the specificity of US-induced immune activation, we have synthesized semiconducting polymer immunomodulatory nanoparticles (SPINs) that underwent sonodynamic process to generate 1O2 to specifically cleave the 1O2-sensitive linkers and release the covalently linked immunomodulators. Such a sonodynamic activation mechanism is distinct from the conventional US-mediated cavitation used in drug release from nanoparticles or microbubbles44,45, wherein the physically loaded drug molecules still encounter the issue of nonspecific release in normal tissues.

Although SPNs have been used for optical imaging and phototherapy46,47,48,49, their sonodynamic properties have been unknown. Screening of SPNs led to the discovery of a robust sonosensitizer (SPN7), which had the sonodynamic ability to produced 1O2 at least 3.0-fold stronger than other commonly used sonosensitizers including small molecule dyes and TiO2 at the same mass concentration (Fig. 2f). More importantly, SPNs intrinsically possessed excellent sonodynamic stability (Fig. 2g), which was superior to small molecule sonosensitizers that are structurally fragile to US irradiation (Fig. 2h and Supplementary Fig. 6). Besides, the sonodynamic 1O2 generation ability of SPNs was retained even in the tissue depth of 10 cm (Fig. 3g), forming the basis of deep-tissue activatable sono-immunotherapy. This was also validated by sole sonodynamic therapy using SPIN0, which inhibited the growth of orthotopic pancreatic rabbit tumors by 2.4-fold (Fig. 5j, k).

Molecular reengineering of SPN7 allowed us to covalently conjugate immunomodulators (NLG919 and aPD-L1) to the backbone via a 1O2-cleavable linker to form pro-therapeutic systems (SPINs) wherein the bioactivities of immunomodulators are intrinsically silenced and only triggered upon US irradiation (Fig. 3a). Although pancreatic tumors have excessive extracellular matrix (ECM) proteins, SPINs passively accumulated in subcutaneous pancreatic mouse tumors after systematic administration (Supplementary Fig. 27), which should be due to effective enhanced permeability and retention (EPR) effect50,51. Under US irradiation, SPINs were able to efficiently generate 1O2 to ablate tumor cells and induce ICD (Supplementary Fig. 31), which led to the enhanced tumor infiltration of lymphocytes (Supplementary Fig. 33) and thus primed tumors for immunotherapy. Meanwhile, the generation of 1O2 triggered in situ release of immunomodulators from SPINs in the tumor microenvironment, remotely activating localized immunotherapy. Among five SPINs, SPIND2 had the highest sono-immunotherapeutic efficacy in inhibiting growth of primary and distant tumors, extending animal survival and preventing tumor recurrence (Fig. 4b–h). The therapeutic benefits were also proven to be obtainable for mouse tumors covered with 5-cm pork tissues, verifying the in vivo deep-tissue activatable sono-immunotherapy (Fig. 5b–d).

The underlying molecular mechanism study via multiplexed gene expression analysis showed that SPIND2 injection and US irradiation upregulated the gene expression level of Ido1 and Cd274 in tumors by 1.6- and 4.0-fold, respectively (Fig. 4j, k). To date, photothermal and photodynamic effects induced upregulated expressions of IDO and PD-L1 in tumors cells has been reported52,53,54, while the influence of sonodynamic effect on these immunosuppressive proteins has not been evaluated. This study disclosed that SPIN-mediated US treatment could reprogram tumor microenvironment to transform the non-immunogenic “cold” tumors into immunogenic “hot” ones via upregulating the expression of IDO and PD-L1. Therefore, targeting of the IDO and PD-L1 pathways followed by US treatment should be an effective strategy to strengthen the immune response. Note that even though the injected dosage of NLG919 and aPD-L1 was overall lower than those used for combinational immunotherapy in previous studies (Supplementary Table 2), SPIND2 almost completely eradicated both primary and distant tumors and fully inhibited rechallenged tumors. Such a high antitumor immune response elicited by SPIND2 should be attributed to the co-existence of two immunomodulators (NLG919 for IDO inhibition and aPD-L1 for PD-L1 blocking) which synergistically improved the tumoricidal activity of CTLs (Supplementary Figs. 4144).

The high specificity of sonodynamic activation mechanism of SPINs enabled precise restriction of immunotherapeutic action to the tumors of living mice, potentially reducing the incidence of irAEs. This was supported by multi-fold observations. Administration of SPIND2 alone without US irradiation even had poor antitumor efficacy than free-drug administration and did not significantly affect the populations of immune cells in normal organs and serum cytokine levels as free drug did (Fig. 6a–f). In contrast, directed US irradiation after SPIND2 accumulation in tumors led to at least twofold increase in the population of CTLs in the tumor tissues as compared with free-drug administration; whereas, the populations of effector T cells were only slightly higher than those in the saline group and 1.2-fold lower than free-drug administration in normal organs (Fig. 6a–d). This could be explained by the minimal bioactivity of NLG919 and aPD-L1 within SPIND2 without US irradiation.

In summary, we have developed deep-tissue activatable sono-immunotherapeutic nanoagents based on SPNs and validated their efficacy in the pancreatic tumor mouse model. SPIN-mediated sono-immunotherapy capitalizes on excellent sonodynamic properties of SPNs to noninvasively eradiate tumors and enhance the immunogenicity of pancreatic tumors via induction of ICD, and also remotely activate the therapeutic functions via scission of covalently conjugated immunomodulators in tumor in situ. Deep-tissue therapy was achieved using SPINs on subcutaneous pancreatic mouse tumors covered with 5-cm tissues and rabbit orthotopic pancreatic tumors. Thus, SPINs represent a type of precision immunotherapeutic agents which elicit high antitumor immunity without significantly breaking systemic immune tolerance. Furthermore, the flexibility in structural modification of SPINs allows to easily generalize sonodynamic activation approach beyond checkpoint blockade immunotherapy through conjugation other immunotherapeutic molecules. Thus, our study provides a precision nanoplatform to further equip nano-immunotherapy with temporospatial control over immunomodulation.


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