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. 2025 Jun 6;33:101954. doi: 10.1016/j.mtbio.2025.101954

Extracellular matrix-degradable polymer nanostimulants elicit potent immune responses in orthotopic pancreatic cancer via sono-activatable dual-drug synergism

Meng Li a,1, Danling Cheng a,1, Yue Wang b, Chongwen Xuan a, Viktar Abashkin c,⁎, Jindong Xia b,⁎⁎, Ling Ding a,⁎⁎⁎, Jingchao Li a,⁎⁎⁎⁎
PMCID: PMC12178713  PMID: 40538751

Abstract

Pancreatic cancer is a highly aggressive malignancy with a poor prognosis due to its complex tumor microenvironment (TME), which includes a dense extracellular matrix (ECM) and immunosuppressive pathways. Nanomedicine capable of achieving profound tumor penetration and modulating the tumor immune microenvironment is urgently needed to enhance the efficacy of cancer therapy. Herein, we introduce ECM-degradable semiconducting polymer nanostimulants (SPNs) as a novel nanostimulant for deep tumor penetration and multifaceted remodeling of the tumor microenvironment. The SPNs were constructed by loading two immune drugs: toll-like receptor 7/8 agonist (R848) and indoleamine 2,3-dioxygenase inhibitor (NLG919), onto singlet oxygen (1O2)-responsive SPNs, and modifying their surface with hyaluronidase (HAase). Upon accumulation at orthotopic pancreatic tumor sites, HAase-mediated degradation of the ECM significantly enhances the penetration of nanomedicine into the tumor and facilitates the infiltration of immune cells. Upon sono-activation, the SPNs produce 1O2, which is not only used for sonodynamic therapy of deep-seated pancreatic tumors and, but also induces immunogenic cell death (ICD) in tumor cells. Simultaneously, the generated 1O2 can be cleaved by 1O2-responsive fragments, disrupting the nanoparticle structure and enabling controlled and precise release of the two immune drugs at the tumor site, thus minimizing off-target effects. Through such a multifaceted remodeling mechanism, SPN-based treatment triggers a potent antitumor immunological response. Consequently, the growth of orthotopic pancreatic tumors in mouse models is nearly inhibited, and tumor metastases are effectively suppressed. This study presents an ECM-degradable semiconducting polymer nanostimulant for multifaceted remodeling of the tumor microenvironment, enabling effective and precise immunotherapy of deep-seated orthotopic tumors.

Keywords: Polymer nanoparticles, Sonodynamic therapy, Immunotherapy, Orthotopic pancreatic cancer, Tumor microenvironment

Graphical abstract

Image 1

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Highlights

  • •

    Extracellular matrix-degradable nanostimulants enable deep tumor penetration and tumor microenvironment remodeling.

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    Precise drug release at the tumor site can be achieved by nanostimulants through sono-activation.

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    Sono-activatable dual-drug synergy elicits potent immune responses in orthotopic pancreatic cancer.

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    This strategy enables effective and precise immunotherapy of deep-seated orthotopic tumors.

1. Introduction

Pancreatic cancer stands as a paradigm of therapeutic resistance among malignancies, with a 5-year survival rate of less than 13 %, which drops to a mere 3 % when distant metastasis occurs [1]. A hallmark of the pancreatic cancer microenvironment is its densely packed extracellular matrix (ECM), characterized by elevated concentrations of hyaluronic acid (HA) and collagen fibers [2]. This ECM not only drives tumor initiation, progression, invasion, and immune evasion but also acts as a physical barricade, severely impeding the penetration of therapeutic agents into the tumor parenchyma [[2], [3], [4]]. Despite multimodal therapies (surgery, chemotherapy, and radiotherapy), outcomes remain dismal due to late diagnosis and inherent treatment resistance [[5], [6], [7], [8], [9]]. A key obstacle is the ECM dual role as a physical barrier and an immunosuppressive modulator within the tumor microenvironment (TME) [10,11]. HA, a dominant ECM component, is strongly associated with tumor invasiveness and metastasis [[11], [12], [13]]. Notably, enzymatic HA degradation using hyaluronidase (HAase) improves chemotherapeutic efficacy by enhancing drug penetration and alleviating interstitial pressure [[14], [15], [16]]. Thus, targeting the ECM while reprogramming the immunosuppressive TME represents a promising strategy to overcome therapeutic resistance and improve survival.

Sonodynamic therapy (SDT) has emerged as a highly promising modality in recent years, leveraging sono-activatable nanoparticles to generate reactive oxygen species (ROS) under ultrasound (US) irradiation [[17], [18], [19], [20]]. Particularly, SDT enables deep tissue penetration and precisely focuses energy on targeted tumor sites, thereby triggering immunogenic cell death (ICD) in tumor cells [[21], [22], [23], [24], [25]]. This cascade of events not only stimulates robust antitumor immune responses but also minimizes collateral damage to adjacent normal tissues, enhancing the overall therapeutic efficacy and safety profile [26,27]. However, the therapeutic outcomes of SDT are often compromised by the complex immunosuppressive TME and apoptosis-resistant mechanisms inherent to cancer cells [23,24]. These factors can significantly undermine the effectiveness of SDT, highlighting the need for complementary strategies to enhance its therapeutic potential.

Immunotherapy has fundamentally reshaped cancer treatment by harnessing and enhancing the body's own immune cells to elicit a powerful systemic anti-tumor response [[28], [29], [30]]. This innovative approach not only effectively eradicates primary tumors but also targets distant metastases, offering comprehensive therapeutic benefits [[31], [32], [33]]. Moreover, immunotherapy holds the promise of establishing long-term immune memory, which can help to suppress the recurrence of cancer [5,34,35]. Despite these advancements, the benefits of immunotherapy for pancreatic cancer remain limited due to its highly immunosuppressive TME [10,35,36]. In addition, systemic biodistribution of these drugs poses safety issues, which might lead to the use of less-than-ideal doses or, in some cases, prevent their use in treating pancreatic cancer altogether [37]. Advancements in nanoparticle-based delivery systems have demonstrated potential to improve cancer immunotherapy through the effective transport of immune drugs to tumor sites, T cells, and circulating dendritic cells (DCs) [28,34,[38], [39], [40], [41], [42]]. Given these, the integration of SDT with nanoparticle-based immunotherapy emerges as a compelling strategy, which not only enhances the delivery of immune drugs but also leverages the deep tissue penetration and precise targeting capabilities of SDT, thereby overcoming the limitations posed by the immunosuppressive TME.

In this study, we designed singlet oxygen (1O2)-responsive semiconducting polymer-based nanostimulants (SPNs) to achieve multifaceted remodeling of the TME (Fig. 1). We incorporated the immunomodulatory agents NLG919, an indoleamine 2,3-dioxygenase 1 (IDO-1) inhibitor [43] and R848, a TLR7/8 agonist [44], into the SPNs, which are encapsulated with the semiconducting polymer poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7bis(thiophen-2-yl)benzo-2,1,3-thiadiazole] (PFODBT) functioning as a sonosensitizer. PFODBT exhibits stable fluorescence properties and can generate 1O2 upon stimulation by US radiation, which is crucial for the therapeutic mechanism of SPNs [10]. To further improve therapeutic efficacy, the surface of the SPNs is functionalized with HAase (Fig. 1a). As shown in Fig. 1b, degradation of HA in the tumor microenvironment by HAase boosts the accumulation of nanostimulants and facilitates the infiltration of T cells into orthotopic pancreatic tumor sites. R848 is a potent TLR7/8 agonist that activates DCs and T cells, thereby promoting an immune response and enhancing antitumor immunity [[45], [46], [47]]. NLG919 inhibits the expression of IDO-1, which converts tryptophan (Trp) into kynurenine (Kyn), thus modulating the immunosuppressive TME in pancreatic cancer by increasing the proportion of DCs and T cells while reducing the proportion of regulatory T cells (Tregs) [43,48]. Upon sono-activation, the SPNNRH nanostimulants produce 1O2, which not only facilitates SDT of deep-seated pancreatic tumors and induces ICD in tumor cells but also triggers the controlled release of R848 and NLG919, thereby activating DCs and inhibiting IDO. This comprehensive strategy effectively remodels the TME and elicits a robust antitumor immune response, resulting in near-complete inhibition of orthotopic pancreatic tumor growth and metastasis.

Fig. 1.

Fig. 1

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Schematic of sono-activated immunotherapy for pancreatic cancer with ECM-degrading nanostimulants. (a) Design and fabrication of SPNNRH nanostimulants employing film-dispersion, hydration, and modification. (b) A schematic illustration clarifies the multifaceted mechanistic pathways of SPNNRH in orthotopic pancreatic cancer therapy. This includes degradation of the tumor ECM, enhanced SDT, potentiated ICD, stimulation of antitumor immunity, and regulation of immune suppression, collectively achieving comprehensive remodeling of the tumor microenvironment.

2. Materials and methods

2.1. Materials

The PFODBT was sourced from Sigma-Aldrich (USA). All ELISA kits were purchased from Solarbio (Beijing, China). All solvents were supplied by Sinopharm (China). The TLR7/8 agonist R848 and IDO inhibitor NLG919 was procured from MedChemExpress (USA). The culture media were purchased from Procell (China)

2.2. Synthesis of SPNNRH, SPNNR and SPNNH

The 1O2-responsive polymer 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- thioketal-(polyethylene glycol) (DSPE-TK-PEG) was synthesized following a previously reported method [49]. PFODBT, DSPE-TK-PEG, DSPE-PEG-COOH, R848, and NLG919 were dissolved together in chloroform at a mass ratio of 0.25:10:20:0.25:0.25. The mixture was then sonicated to achieve uniform dispersion. Subsequently, the chloroform was removed via rotary evaporation to form a thin film containing PFODBT and the drugs. This film was hydrated in ultrapure water at 55 °C with stirring for 1 h. The hydrated solution was then extruded through 200 and 100 nm membranes using a liposome extruder. After purification by ultrafiltration and washing with water, SPNNR was obtained. To obtain the final product SPNNRH, SPNNR was reacted with HAase overnight at 4 °C. The preparation method for SPNNH is similar to that of SPNNRH, except that R848 is omitted in the initial step.

2.3. Characterization of nanostimulants

The particle size and surface charge of SPNNR, SPNNH, and SPNNRH were measured by a Malvern Zetasizer (Nano-ZS90). A UV–Vis spectrophotometer was used to measure their UV–Vis absorption spectra, and a fluorescence spectrophotometer was employed to evaluate the fluorescence properties of the samples.

2.4. 1O2 generation assessment

The production of 1O2 was evaluated using singlet oxygen sensor green (SOSG). The production of 1O2 by SPNNR, SPNNH, and SPNNRH (concentration = 20 μg/mL) was measured after US irradiation for 2, 4, 6, 8, and 10 min.

2.5. Evaluation of R848 and NLG919 release

To evaluate the US-activated release of R848 and NLG919 by SPNNRH, PBS solutions of SPNNRH were treated with US for 0, 5, 10, and 15 min. The release of R848 and NLG919 before and after laser irradiation was investigated using the LC-16 SHIMADZU high-performance liquid chromatography (HPLC) method.

2.6. In vitro cytotoxicity analysis

Panc02 cells were incubated with varying concentrations of SPNNR, SPNNH, and SPNNRH for 24 h, after which cell viability was evaluated using the CCK-8 assay. In a subsequent experiment, Panc02 cells were incubated with SPNNR, SPNNH, and SPNNRH at a concentration of 35 μg/mL for 12 h, followed by US treatment (1 W/cm2, 5 min). Cell viability was then assessed using CCK-8 kit.

2.7. In vitro cellular uptake analysis

Panc02 cells were treated with SPNNR, SPNNH, and SPNNRH for 12 h. Subsequently, the cells were trypsinized, washed three times with PBS, and then resuspended in 500 μL of PBS for flow cytometer analysis.

2.8. Evaluation of intracellular ROS generation

Panc02 cells were treated with SPNNR, SPNNH, and SPNNRH for 12 h, followed by incubation with H2DCFH-DA for 20 min. Afterward, the cells were subjected to US irradiation. Finally, the cells were washed three times with PBS and visualized using a fluorescence microscope.

2.9. In vitro spheroid penetration assessment

To evaluate the penetration of nanostimulants, multicellular Panc02 spheroids were constructed and exposed to SPNNR, SPNNH, and SPNNRH (50 μg/mL). Confocal fluorescence microscopy was employed to capture images of these spheroids at various depths (30, 60, 90, 120, 150 and 180 μm).

2.10. In vitro Trp metabolism

The methods reported in previous studies were employed to evaluate the Trp metabolism and spheroid penetration of Panc02 cells [49]. Panc02 cells were first treated with interferon-γ (IFN-γ) for 24 h. Subsequently, PBS, SPNNR, SPNNH, and SPNNRH were introduced into the cell culture medium at a final concentration of 20 μg/mL, followed by an additional 24-h incubation period. For the US irradiation group, cells were treated with US (1 W/cm2, 5 min) and then cultured for another 24 h. After these treatments, the supernatant from each well was collected, and the concentrations of Trp and Kyn were determined using HPLC.

2.11. In vitro ICD characterization and assessment

Panc02 cells were treated with SPNNR, SPNNH, or SPNNRH for 12 h and then subjected to US irradiation treatment. The cells were subsequently placed in an incubator for overnight culture. The following day, the levels of Adenosine Triphosphate (ATP) and high-mobility group box 1 (HMGB1) in the cell supernatant were detected using ELISA kits, respectively. Calreticulin (CRT) expression was assessed by immunofluorescence staining, and the corresponding fluorescence images were obtained with a confocal microscope.

2.12. Intratumoral HA level detection

To detect the HA content in orthotopic pancreatic Panc02 tumors, the tumors were removed from mice and then injected intravenously with PBS, SPNNR, SPNNH, or SPNNRH (200 μg/mL, 0.2 mL) through the tail vein. Subsequently, the tumor tissues were placed in a PBS solution with pH = 7.4, ground into a cell suspension, and the HA content was measured using a mouse HA ELISA kit.

2.13. In vivo fluorescence imaging and biodistribution study

A solution of SPNNR, SPNNH, or SPNNRH (200 μg/mL, 0.2 mL) was injected intravenously into orthotopic pancreatic cancer mouse models via the tail vein. Using the IVIS fluorescence imaging system, the mice were imaged at 0, 6, 12, 24, and 36 h post-injection (excitation wavelength 520 nm, emission wavelength 700 nm) to observe the dynamic accumulation of nanostimulants in the orthotopic pancreatic tumor. At 36 h post-injection, the C57BL/6 mice bearing tumors were euthanized, and the corresponding tissues were removed and further analyzed for biodistribution using the IVIS fluorescence imaging system.

2.14. Detection of intratumoral ROS generation in vivo

To detect the generation of ROS in pancreatic tumors, PBS, SPNNR, SPNNH, or SPNNRH were intravenously injected into orthotopic pancreatic cancer mouse models via the tail vein. After 24 h, the mice were intraperitoneally injected with H2DCFH-DA solution (5 μM, 100 μL), and the tumor sites were irradiated with US for 10 min. Subsequently, the ROS levels within the tumors were assessed using fluorescence imaging techniques.

2.15. In vivo assessment of antitumor and antimetastatic efficacy

Orthotopic pancreatic cancer mice were intravenously injected with PBS, SPNNR, SPNNH, or SPNNRH (concentration 200 μg/mL, 200 μL) via the tail vein. At 24 h post-injection, the orthotopic pancreatic tumors were treated with US for 10 min. On days 0, 7, and 14, the mice were intraperitoneally injected with D-luciferin potassium salt solution (concentration 20 mg/mL, 150 μL). To monitor tumor growth in real time, bioluminescence (BL) images were acquired using the IVIS imaging system, and the images were analyzed using Living Image software. Subsequently, the tissues from the mice were harvested for ex vivo bioluminescence imaging to evaluate the antimetastatic effect. Meanwhile, the orthotopic pancreatic tumor tissues were collected, weighed, and photographed for documentation.

2.16. Monitoring of mouse survival rate

After different treatments, the survival of orthotopic pancreatic cancer mice (n = 5 per group) was monitored daily for 25 days.

2.17. In vivo evaluation of immune response

After subjecting the mice to various treatment regimens, they were humanely euthanized. Subsequently, the tumor-draining lymph nodes and orthotopic pancreatic tumors were harvested to generate single-cell suspensions. These suspensions were then stained with fluorescently labeled antibodies. Flow cytometry was employed to analyze the stained cells, enabling the assessment of the immune response.

2.18. Statistical analysis

Data were presented as mean ± SD, with sample sizes (n) specified. Significant differences were denoted by ∗(p < 0.05), ∗∗(p < 0.01), and ∗∗∗(p < 0.001). Statistical significance was assessed using a two-tailed unpaired t-test, and analyses were conducted with GraphPad Prism 8.0.

3. Results and discussion

3.1. Sonodynamic and drug release properties of nanostimulants

The SPNNR nanostimulants were synthesized by encapsulating semiconducting polymer (PFODBT), R848 and NLG919 within a 1O2-responsive shell using thin film dispersion method. Subsequently, HAase was conjugated onto the surface of SPNNR to form the nanostimulants (SPNNRH). The zeta potential measurements revealed values of −33.6 mV for SPNNR, −27.3 mV for SPNNH, and −28.3 mV for SPNNRH (Fig. 2a). The protein content in SPNNH and SPNNRH was significantly higher compared to that in SPNNR (Fig. S1), confirming the successful surface modification with HAase. The nanostimulants (SPNNR, SPNNH and SPNNRH) exhibited a spherical shape and a consistent size distribution (Fig. 2b). The hydrodynamic diameters were 61.31 nm for SPNNR, 71.43 nm for SPNNH and 72.35 nm for SPNNRH (Fig. 2c), indicating that HAase successfully modified the surface of the nanostimulants and that all nanostimulators exhibited good colloidal behavior potential. The optical properties of the nanostimulants were then characterized using UV–vis absorption and fluorescence emission spectra. Similar absorbance peaks at 398 nm and 554 nm, characteristic of PFODBT, were observed for SPNNR, SPNNH and SPNNRH (Fig. 2d). Additional absorbance peaks at around 320 nm were detected for SPNNH and SPNNRH, attributed to the surface-modified HAase. As shown in Fig. 2e, all nanostimulants exhibited a fluorescence emission peak at 690 nm. These results confirmed that the nanostimulants retained comparable absorbance and fluorescence characteristics, which confirmed successful encapsulation and surface modification.

Fig. 2.

Fig. 2

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Characterization of the sonodynamic and drug release properties of nanostimulants. (a) Zeta potential measurements of SPNNR, SPNNH, and SPNNRH in aqueous solutions. b) Morphology characterization of SPNNR, SPNNH, and SPNNRH using TEM. (c) DLS size of SPNNR, SPNNH, and SPNNRH. (d) Absorbance spectra of SPNNR, SPNNH, and SPNNRH. (e) Fluorescence emission spectra of SPNNR, SPNNH, and SPNNRH. (f) The 1O2 generation efficacies of SPNNR, SPNNH, and SPNNRH (n = 3). (g) Release profiles of R848 inhibitors from SPNNRH after US irradiation at different time points (n = 3). (h) Release percentages of NLG919 from SPNNRH after US irradiation at various time points (n = 3). Data are expressed as mean ± SD.

Further exploration was conducted into the sonodynamic effect and the drug release characteristics of SPNNRH. SOSG was employed as an indicator of 1O2. Solutions containing SPNNR, SPNNH, and SPNNRH exhibited a time-dependent increase in fluorescence intensities (Fig. S2), verifying the production of 1O2 through the sonodynamic effect of the semiconducting polymer under US irradiation. Following 2, 4, 6, 8, and 10 min of US irradiation, the fluorescence intensities increased by roughly 1.5-, 2.1-, 2.7-, 3.5-, and 4.0-fold, respectively (Fig. 2f). The 1O2-responsive shells, containing thioketal bond (TK) linked PEG, were designed to facilitate drug release on demand upon US irradiation. In the absence of US irradiation, the release percentage of NLG919 from SPNNRH was minimal but increased to 31.1 % after 5 min, 41.6 % after 10 min, and 76.7 % after 15 min of US irradiation (Fig. 2g). Likewise, without US irradiation, negligible release of R848 from SPNNRH was observed. In contrast, significant release of R848 was observed after 5, 10, and 15 min of US irradiation (Fig. 2h). These findings indicated that US irradiation activates the release of R848 and NLG919 from SPNNRH. Specifically, when subjected to US irradiation, SPNNRH produces 1O2, which degrades the 1O2-responsive shells, leading to the controlled release of the drugs. Importantly, minimal hemolysis of erythrocytes was observed after incubation with SPNNR, SPNNH, and SPNNRH (Fig. S3), confirming their biocompatibility and suitability for systemic administration.

3.2. In vitro anticancer activity and penetrating capability evaluation

The cytotoxicity of SPN nanostimulants was first assessed to determine their intrinsic effects on cancer cells. Panc02 cancer cells treated with SPNNR, SPNNH, and SPNNRH at various concentrations exhibited cell viabilities similar to those of untreated control cells (Fig. 3a), indicating minimal cytotoxicity of the nanostimulants themselves. To evaluate in vitro anticancer efficacy, cells treated with the SPN nanostimulants were subjected to US irradiation. Following US exposure, the viability of cells treated with SPNNR, SPNNH, and SPNNRH decreased, with a more pronounced reduction observed at higher SPNs concentrations (Fig. 3b). At a concentration of 35 μg/mL, cell viability was significantly reduced to 78.7 %, 62.9 %, and 59.3 % for SPNNR, SPNNH, and SPNNRH group, respectively. Flow cytometry was employed to investigate the cellular internalization of the nanostimulants. Panc02 cells treated with SPNNH, and SPNNRH exhibited stronger fluorescence signals compared to those treated with SPNNR and the PBS group (Fig. 3c). These findings indicated that the HAase modification enhances the cellular internalization of the SPN nanostimulants.

Fig. 3.

Fig. 3

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Evaluation of anticancer efficacy and penetrating capability. (a) Cell viability of Panc02 cells treated with SPNNR, SPNNH, and SPNNRH (n = 3). (b) Cell viability of Panc02 cells treated with SPNNR, SPNNH, and SPNNRH under US irradiation (n = 3). (c) Cellular internalization of SPNNR, SPNNH, and SPNNRH via a flow cytometer in Panc02 cells analyzed by flow cytometry. (d) Fluorescence images of 3D multicellular tumor spheroids treated with fluorescence-labeled SPNNR, SPNNH, and SPNNRH at different depths. (e) Fluorescence intensity in 3D multicellular tumor spheroids after treatment at various depths (n = 3). (f) Fluorescence images of Panc02 cells with generation of ROS. (g) Fluorescence intensity of ROS signals in treated Panc02 cells (n = 5). Data are expressed as mean ± SD. Statistical significance was determined using a two-tailed unpaired t-test, ∗p < 0.05 and ∗∗∗ (p < 0.001).

To investigate the degradation of the ECM and the subsequent enhancement of nanostimulants penetration, three-dimensional (3D) multicellular tumor spheroids were constructed. At equivalent depths, the red fluorescence signals of nanostimulants in the SPNNH, and SPNNRH-treated groups were significantly stronger than those in the SPNNR-treated group (Fig. 3d). Specifically, at depths of 90, 120, 150, and 180 μm, the fluorescence intensity in the SPNNH, and SPNNRH groups was approximately 2.5-, 2.7-, 3.9-, and 10.3-fold higher, respectively, compared to the SPNNR group (Fig. 3e). These findings indicated that the surface modification of SPNs with HAase significantly improves nanostimulants penetration within the cell spheroids.

The production of 1O2 via SDT was verified by fluorescence imaging using DCFH-DA as a probe. Green fluorescence, indicative of ROS signals, was exclusively detected in groups treated with SPNNR + US, SPNNH + US, and SPNNRH + US -treated groups (Fig. 3f), thereby confirming the production of 1O2. Notably, the ROS fluorescence intensity was higher in the SPNNH + US, and SPNNRH + US groups than in the SPNNR + US group. Relative to the PBS control group, the ROS signal intensity increased by 104.0-, 133.6-, and 134.2-fold for the SPNNR + US, SPNNH + US, and SPNNRH + US groups, respectively (Fig. 3g). These findings confirmed 1O2 generation via the SDT effect.

3.3. In vitro ICD effect evaluation

The in vitro ICD effect induced by nanostimulants treatment and US irradiation was investigated. The ability of SPNNRH to inhibit IDO activity was assessed by measuring the extracellular levels of Kyn and Trp. The Kyn/Trp ratio was significantly reduced in the SPNNR + US, SPNNH + US, and SPNNRH + US groups, whereas no significant changes were observed in the groups treated with nanostimulants alone (SPNNR, SPNNH, and SPNNRH) (Fig. 4a). These results indicated that sono-activation of SPNNR, SPNNH, and SPNNRH effectively inhibits IDO activity, thereby modulating the levels of Kyn and Trp.

Fig. 4.

Fig. 4

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In vitro ICD effect evaluation. (a) Kyn/Trp ratio in panc02 cells treated with SPNNR, SPNNH, and SPNNRH. (n = 5). (b) HMGB1 secretion levels in panc02 cells treated with SPNNR, SPNNH, and SPNNRH. (n = 5). (c) ATP secretion levels in panc02 cells treated with SPNNR, SPNNH, and SPNNRH. (n = 5). (d) Fluorescence intensity of CRT staining signals in panc02 cells treated with SPNNR, SPNNH, and SPNNRH. (n = 5). (e) Confocal fluorescence images of CRT staining in panc02 cells treated with SPNNR, SPNNH, and SPNNRH. Data are expressed as mean ± SD. Statistical significance was determined using a two-tailed unpaired t-test, ∗ (p < 0.05), ∗∗ (p < 0.01), and ∗∗∗ (p < 0.001).

The secretion levels of HMGB1 were significantly increased in the SPNNR + US, SPNNH + US, and SPNNRH + US groups (Fig. 4b). Similarly, the secretion levels of ATP were increased by 5.1-, 5.6-, and 6.7-fold, respectively, in these groups (Fig. 4c). No significant changes in HMGB1 and ATP secretion levels were observed in cells treated with nanostimulants alone, without US irradiation. Confocal microscopy images showed strong green fluorescence signals indicative of CRT exposure on the cell surface in the SPNNR + US, SPNNH + US, and SPNNRH + US groups, which were markedly distinct from those in the groups treated with nanostimulants alone (Fig. 4e). Quantitative analysis revealed a 4.3-, 7.7-, and 9.4-fold increase in CRT fluorescence intensity in the SPNNR + US, SPNNH + US, and SPNNRH + US groups, respectively (Fig. 4d). These results demonstrate that SPNNH and SPNNRH-mediated SDT effectively induces ICD, as evidenced by increased secretion levels of HMGB1 and ATP, and enhanced CRT exposure. These findings confirmed the excellent therapeutic efficacy of SDT in inducing ICD and highlight its potential for in vivo applications.

3.4. In vivo TME modulation evaluation

The ability of the SPNNRH in degrading the tumor ECM and modulating the TME was initially confirmed by evaluating the levels of HA and the accumulation of nanostimulants at the tumor sites. Specifically, the HA levels in orthotopic pancreatic tumor tissues were significantly lower in the SPNNH, SPNNRH, SPNNH + US, and SPNNRH + US groups compared to the PBS, PBS + US, SPNNR, SPNNR + US groups (Fig. 5a), confirming that the surface-conjugated HAase effectively degraded HA within the tumor site. Additionally, the fluorescence intensity in tumors was higher for SPNNH and SPNNRH compared to SPNNR (Fig. 5b), with the fluorescence intensity in the SPNNH, and SPNNRH groups was approximately 1.2-fold higher than that in the SPNNR group (Fig. 5c). These results indicated that SPNNR, SPNNH, and SPNNRH were effectively delivered to orthotopic pancreatic tumor tissues, likely through in situ tumor-generated extracellular vesicle-mediated transport and the enhanced permeability and retention (EPR) effect [[50], [51], [52], [53]]. Furthermore, SPNNH and SPNNRH demonstrated improved tumor accumulation efficacy due to the surface-conjugated HAase, which degraded the dense ECM, thereby promoting nanostimulants diffusion and penetration.

Fig. 5.

Fig. 5

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In vivo tumor microenvironment modulation evaluation. (a) Content of HA levels in tumors across different treatment groups. (b) In vivo fluorescence images of Panc02 orthotopic pancreatic cancer bearing mice following intravenous (i.v.) injection of SPNNR, SPNNH, and SPNNRH. (c) Quantitative analysis of fluorescence intensity in orthotopic pancreatic tumors for each group at various time points (n = 3). (d) Confocal fluorescence images of the generated 1O2 in orthotopic pancreatic tumors for each treatment group. (e) Fluorescence intensity of generated ROS signals in orthotopic pancreatic tumors (n = 5). Data are expressed as mean ± SD. Statistical significance was determined using a two-tailed unpaired t-test, ∗∗ (p < 0.01).

Subsequently, SDT-mediated ROS generation in the context of HA degradation was investigated. Consistent with the above findings, ROS were detected exclusively in the SPNNR + US, SPNNH + US, and SPNNRH + US treated groups, with SPNNH + US and SPNNRH + US exhibiting superior ROS production compared to SPNNR + US (Fig. 5d and e). These results collectively demonstrate that the SPNNRH nanostimulants effectively degrade the tumor ECM through HAase-mediated HA degradation, enhancing tumor accumulation and ROS generation upon US activation, thus highlighting their potential for modulating the TME and improving antitumor outcomes.

3.5. Deep-tissue orthotopic pancreatic cancer therapeutic and anti-metastasis efficacy evaluation

The therapeutic outcomes of deep-tissue orthotopic pancreatic cancer were evaluated following i.v. injection of SPNNR, SPNNH, and SPNNRH, combined with US irradiation (Fig. 6a). The antitumor and anti-metastasis efficacies were assessed using Panc02-Luc orthotopic pancreatic cancer mouse models the measurement of bioluminescence (BL) signals. D-Luciferin potassium salt, which is oxidized by luciferase in Panc02-Luc cells to generate BL signals, was used to monitor tumor growth and metastasis. After treatment with SPNNH + US, and SPNNRH + US, the BL signals were significantly reduced and nearly undetectable by day 14 (Fig. 6b). While the BL signals were also reduced in the SPNNR + US group, which remained detectable after 14 days of treatment. In contrast, the BL signals increased in the other groups due to tumor growth. The lowest BL signal intensities were consistently observed in the SPNNRH + US group (Fig. 6c). On day 14, the BL signal intensity in the SPNNRH + US group was 7.0- and 6.1-fold lower than that in the SPNNR + US and SPNNH + US groups, respectively.

Fig. 6.

Fig. 6

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Deep-tissue orthotopic pancreatic cancer therapeutic and anti-metastasis efficacy evaluation. (a) Illustration of the therapeutic efficacy evaluation for deep-tissue orthotopic pancreatic cancer via i.v. injection of SPNNR, SPNNH, and SPNNRH and US followed by US irradiation. (b) In vivo BL imaging analysis of Panc02-Luc orthotopic pancreatic cancer-bearing mice on day 0, 7, and 14 (n = 5). (c) Quantification of BL intensity at tumor sites in Panc02-Luc orthotopic pancreatic cancer-bearing mice (n = 5). (d) H&E staining images of orthotopic pancreatic tumors from mice following various treatments. (e) Photograph of tumors from orthotopic pancreatic cancer-bearing mice (n = 5). (f) Tumor weights of collected tumors (n = 5). (g) Survival curves of Panc02-Luc orthotopic pancreatic cancer-bearing mice following various treatments (n = 10). (h) BL imaging analysis of tumor metastasis in different organs for Panc02-Luc orthotopic pancreatic cancer-bearing mice. (i) Quantification of BL signal intensity in the intestines, stomach, kidney, lung, spleen, heart, and liver from mice in each group (n = 5). Data are expressed as mean ± SD. Statistical significance was determined using a two-tailed unpaired t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗ (p < 0.001).

Histological analysis revealed significant cell apoptosis in the SPNNR + US, SPNNH + US, and SPNNRH + US groups (Fig. 6d), with the most pronounced apoptosis observed in the SPNNRH + US group. Photographs of excised tumors showed that the tumors in the SPNNR + US, SPNNH + US, and SPNNRH + US groups were smaller than those in the PBS group (Fig. 6e). Notably, all tumors in the SPNNRH + US group were completely eradicated in five individual mice, while only one tumor and three tumors with smaller sizes were observed in the SPNNR + US and SPNNH + US groups, respectively. The average tumor weight in the SPNNH + US group was as low as 0.03 g, significantly lower than that in the SPNNR + US (0.14 g) and PBS (0.54 g) groups (Fig. 6f). The survival rate of mice in the SPNNRH + US group remained at 100 % after 25 days of treatment, compared to less than 70 % in the other groups (Fig. 6g). These results demonstrated the strong tissue penetration capability of US and highlight the superior antitumor therapeutic efficacy of SPNNRH + US.

Furthermore, the anti-metastasis efficacy was explored using BL imaging. Minimal BL signals were detected in the intestines, stomach, kidney, lung, spleen, heart, and liver in the SPNNRH + US group, whereas BL signals were observed in the liver, stomach, and intestines in the SPNNR, SPNNH, SPNNRH, PBS + US, SPNNR + US, and SPNNH + US groups (Fig. 6h). The BL intensity in the heart, stomach, and intestines of the SPNNRH + US group was at least 3.2-, 5.6-, and 1.4-fold lower than that in the corresponding organs of the other groups, respectively (Fig. 6i). Furthermore, the BL signals and intensities in these tissues in the SPNNRH + US group were similar to those in healthy mice, indicating complete suppression of tumor metastasis.

3.6. In vivo ICD effect and immunological effect evaluation

To further elucidate the immune-activating potential of SPNNRH, which has demonstrated remarkable antitumor efficacy, the alterations in the tumor immune microenvironment were meticulously assessed. The initial focus was on evaluating the ICD effect in orthotopic pancreatic tumors through immunofluorescence analysis. The findings revealed that pronounced fluorescence signals for CRT and HMGB1 were evident in the SPNNR + US, SPNNH + US, and SPNNRH + US groups. In contrast, these signals were virtually undetectable in the remaining groups (Fig. 7a). Notably, the SPNNRH + US group exhibited the strongest CRT and HMGB1 staining signals. Specifically, the intensity of CRT fluorescence signals in the SPNNRH + US group was approximately 65.9 times higher than that in the non-US irradiation treatment group, 9.4 times higher than that in the PBS + US group, 2.2 times higher than that in the SPNNR + US group, and 1.2 times higher than that in the SPNNH + US group (Fig. S4). Similarly, the highest HMGB1 fluorescence intensity was observed in the SPNNRH + US group (Fig. S5). Additionally, ATP levels in orthotopic pancreatic tumors were measured. While SPNNR, SPNNH, SPNNRH treatments alone did not significantly increase ATP levels, the combination of these nanostimulants with US irradiation led to a 5.1-fold increase in ATP levels for SPNNR + US, a 5.6-fold increase for SPNNH + US, and a 6.6-fold increase for SPNNRH + US (Fig. 7b). These findings confirmed the potent ICD effect in the SPNNRH + US group.

Fig. 7.

Fig. 7

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In vivo ICD effect and immunological effect evaluation. (a) Confocal fluorescence images of CRT and HMGB1 staining in orthotopic pancreatic tumors. (b) Quantification of ATP levels in each treatment group (n = 5). (c) Quantification of Kyn/Trp ratio in each treatment group (n = 5). (d) Quantification of matured DCs in each treatment group (n = 5). (e) Quantification of intratumoral CD4+T cells in each group (n = 5). (f) Quantification of intratumoral CD8+T cells in each treatment group (n = 5). (g) Quantification of intratumoral Treg cells in each treatment group (n = 5). Data are expressed as mean ± SD. Statistical significance was determined using a two-tailed unpaired t-test, ∗ (p < 0.05) and ∗∗ (p < 0.01).

To evaluate the modulation of the IDO immunosuppressive tumor microenvironment, the Kyn/Trp ratio in orthotopic pancreatic tumors was analyzed. The Kyn/Trp ratios in the US irradiation treatment groups were almost identical, whereas the ratios in the SPNNH + US and SPNNRH + US groups were significantly reduced (Fig. 7c). This indicates that following US irradiation, the SPN-based nanostimulants effectively degraded to release NLG919, thereby efficiently inhibiting IDO activity.

DCs play pivotal roles in cancer immunotherapy by facilitating antigen delivery and presentation, as well as activating T cells [32]. The content of mature DCs was only increased in the SPNNR + US, SPNNH + US, and SPNNRH + US groups (Fig. S6). The mature DCs levels in these groups were 41.4 %, 38.8 %, and 57.5 %, respectively, compared to approximately 19.0 % in the remaining groups (Fig. 7d). To confirm the activation of antitumor immune responses, the intratumoral levels of T cells were then evaluated. while CD4+ T cell levels in SPNs groups did not significantly affect, all SPNs + US groups led to a noticeable increase (Fig. S6). Specifically, the CD4+ cell level in the SPNNRH + US group reached 23.6 %, higher than that in the SPNNR + US (18.7 %) and SPNNH + US (20.3 %) groups (Fig. 7e). Similarly, the levels of CD8+ T cells within primary tumors were found to rise following SPNs + US treatments (Fig. S7). Notably, the CD8+ T cell number in SPNNRH + US group reached 33.2 %, which was higher than those in all other groups (Fig. 7f). Treg cells play a crucial role in suppressing antitumor immune responses [54]. To further elucidate the immunological landscape within the tumor microenvironment, the intratumoral levels of Treg cells were also analyzed. The treatments involving SPNs + US were found to down-regulate the levels of Treg cells (Fig. S8). The lowest percentage of Treg cells in tumor tissues was observed in the SPNNRH + US group (13.5 %), which was reduced by 2.3-fold (Fig. 7g). Consequently, the SPNNRH + US group exhibited significant suppression of Treg cells, which in turn enhanced the functionality and activity of effector T cells.

3.7. Side effect evaluation

The safety profile of SPNNRH + US treatment was assessed in orthotopic pancreatic tumor-bearing mice. No significant changes in body weight were observed across various treatment groups (Fig. S9). Additionally, histological analysis via H&E staining revealed no evident damage to major organs between the SPNNRH + US group and the PBS control group (Fig. S10). Collectively, these results indicated that the SPNNRH + US combination treatment is both safe and effective for their in vivo anticancer applications.

4. Conclusion

In summary, we have developed ECM-degradable SPNs that facilitate precise drug delivery and extensive remodeling of the TME, thereby augmenting antitumor immunological effects in deep-seated orthotopic tumors. These SPNs incorporate HAase to degrade HA within the tumor ECM, diminishing the dense stroma and enhancing nanostimulants accumulation in tumors. Under US irradiation, the SPNs effectively generate 1O2 through the SDT effect of the PFODBT. The generated 1O2 not only induces ICD but also triggers the release of R848 and NLG919 from the 1O2-responsive nanostimulants, ensuring targeted delivery to tumor tissues. Additionally, ECM degradation improves immune cell infiltration into tumors. This multifaceted remodeling strategy using SPNs elicits a robust antitumor immunological response, leading to significant growth inhibition of deep-tissue orthotopic pancreatic tumors and resistance to tumor metastasis in mouse models. Our findings suggest that these ECM-degradable SPNs hold great promise for developing next-generation antitumor drugs, offering a novel approach to comprehensively remodel the TME for the immunotherapy of deep-seated orthotopic tumors.

CRediT authorship contribution statement

Meng Li: Writing – original draft, Methodology, Investigation, Data curation. Danling Cheng: Writing – original draft, Methodology, Investigation, Data curation. Yue Wang: Methodology, Investigation. Chongwen Xuan: Methodology, Investigation. Viktar Abashkin: Writing – review & editing, Resources, Funding acquisition. Jindong Xia: Writing – review & editing, Resources, Funding acquisition. Ling Ding: Writing – review & editing, Supervision, Resources, Conceptualization. Jingchao Li: Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (52311530771), Science and Technology Commission of Shanghai Municipality (23ZR1457400) and Donghua University 2024 Cultivation 798 Project of Discipline Innovation.

Footnotes

This article is part of a special issue entitled: Multiscale Composites published in Materials Today Bio.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtbio.2025.101954.

Contributor Information

Viktar Abashkin, Email: viktar.abashkin@gmail.com.

Jindong Xia, Email: xiajd_21@163.com.

Ling Ding, Email: ling_ding@dhu.edu.cn.

Jingchao Li, Email: jcli@dhu.edu.cn.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (4.4MB, docx)

Data availability

Data will be made available on request.

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Data Availability Statement

Data will be made available on request.

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