Photo-thermal immunoregulation nanofiber scaffold as well as preparation method and application thereof

Abstract

The invention discloses a photo-thermal immunoregulation nanofiber scaffold and a preparation method and application thereof.The photo-thermal immunoregulation nanofiber comprises nanofiber with polylactic acid as a base material, and black phosphorus nanosheets and an HSP90 inhibitor 17-DMAG are jointly loaded in the nanofiber through a high-voltage electrostatic spinning technology; the photo-thermal immunoregulation nanofiber scaffold is applied to liver cancer postoperative immunotherapy through a photo-thermal therapy method. According to the photo-thermal immune regulation and control nanofiber scaffold, a PTT effect is locally generated under near-infrared irradiation, and a strong ISR-ICD cascade reaction is driven. Meanwhile, the exposed DAMPs enhance tumor immunogenicity, recruit cytotoxic T lymphocytes and reprogram an immunosuppressive cell population, so that an immunosilence microenvironment is remodeled into a responsive tumor microenvironment and cooperates with anti-PD-L1, lasting immune memory is established, and finally recurrence and metastasis of postoperative liver cancer are effectively inhibited.

Description

Technical Field

[0001] This invention belongs to the technical field of postoperative medication in medicine, and relates to a photothermal immunomodulatory nanofiber scaffold, its preparation method and application. Background Technology

[0002] Postoperative recurrence of liver cancer is a formidable challenge in modern oncology. Although immune checkpoint inhibitors (ICIs) targeting programmed death receptor 1 / programmed death ligand 1 (PD-1 / PD-L1) have been widely used to assist in recurrence prevention, the results have not been ideal. In recent years, photothermal therapy (PTT), as a local physical therapy method, has provided a new approach to improve this situation. By irradiating photothermal agents, such as two-dimensional nanomaterials, with near-infrared (NIR) light, high temperatures can be generated locally in the tumor, directly killing tumor cells. Moreover, photothermal therapy can induce a special death mechanism called immunogenic cell death (ICD). During immunogenic cell death, dying tumor cells release or expose a series of damage-associated molecular patterns (DAMPs), such as the membrane translocation of calreticulin (CRT), the release of high-mobility group box 1 (HMGB1), and the secretion of adenosine triphosphate (ATP). These signals can effectively promote the maturation and antigen presentation of dendritic cells (DCs), thereby initiating a tumor-specific T-cell immune response, theoretically transforming "cold" tumors into "hot" tumors.

[0003] However, when faced with protein denaturation and misfolding induced by photothermal therapy (collectively known as proteotoxicity), tumor cells initiate a self-protective mechanism called the integrated stress response (ISR). The core of the ISR is the PERK-eIF2α-ATF4 signaling axis, whose moderate activation upregulates the expression of molecular chaperones such as heat shock protein 90 (HSP90), helping to restore intracellular protein homeostasis, establish heat tolerance, thereby limiting the killing effect of photothermal therapy and weakening the intensity of immunogenic cell death. This cellular-level adaptive response is a significant reason why traditional photothermal therapy has limited immune activation effects and why tumors are prone to recurrence.

[0004] Therefore, developing a treatment method that can synergistically activate the ISR-ICD cascade reaction through photothermal therapy is of vital significance and application value for overcoming the current bottleneck in postoperative immunotherapy for liver cancer. Summary of the Invention

[0005] To address the aforementioned problems, this invention proposes a photothermal immunomodulatory nanofiber scaffold and its application, which effectively solves the problems in the prior art.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A photothermal immunomodulatory nanofiber scaffold comprises nanofibers based on polylactic acid, wherein black phosphorus nanosheets and the heat shock protein 90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygerdom are co-loaded in the nanofibers by high voltage electrospinning technology.

[0008] A preparation method, wherein the preparation method is the above-described method for preparing a photothermal immunomodulatory nanofiber scaffold, comprising:

[0009] A suitable amount of black phosphorus crystals were ground into a fine powder in an argon-filled glove box. The fine powder was dispersed in N-methyl-2-pyrrolidone at an initial concentration of 0.5-1.5 mg / mL. The dispersion was placed in an ice bath and sonicated at 250-350 W for 10-14 hours. The brown suspension formed after sonication was centrifuged at 3500-4500 rpm for 15-30 minutes. The supernatant was collected and centrifuged at 12000-15000 rpm for 10-20 minutes to remove N-methyl-2-pyrrolidone. The precipitate was collected to obtain black phosphorus nanosheets.

[0010] Polylactic acid was dissolved in hexafluoroisopropanol to prepare a PLA solution with a mass-volume fraction of 10-14% w / v. 0.04-0.06 g of 17-(dimethylaminoethylamino)-17-demethoxygerdromycin and 0.01-0.03 g of black phosphorus nanosheet solution were dissolved in 10 mL of the above PLA solution to form an electrospinning solution.

[0011] The electrospinning solution was loaded into a 5 ml syringe with a 22 gauge needle and placed on a syringe pump for electrospinning. The electrode voltage was set to 10-20 kV, the electrode spacing to 10-15 cm, and the flow rate to 0.3-1 ml / h.

[0012] One application is the use of the aforementioned photothermal immunomodulatory nanofiber scaffold combined with photothermal therapy in postoperative immunotherapy for liver cancer.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. A self-enhancing therapeutic system was constructed by integrating the HSP90 inhibitor 17-DMAG and the photothermal agent BPNSs into an implantable PLA nanofiber scaffold. Utilizing the protein toxicity induced by PTT itself as a "primary attack," and simultaneously using NIR-triggered 17-DMAG release to inhibit the cell's own repair mechanism (HSP90), the system forcibly transforms moderate, survival-promoting ISR into strong, apoptosis-promoting, and ICD-promoting ISR, achieving an endogenous amplification of the therapeutic effect.

[0015] 2. The application of photothermal immunomodulatory nanofiber scaffolds can reprogram the immunodeficient tumor microenvironment (TME) into an acceptable immunosuppressive tumor microenvironment (TME), effectively modulating multiple nodes in the cancer immune cycle, promoting the cyclic amplification and expansion of the anti-tumor immune response, and ultimately linking local therapeutic intervention with sustained systemic immunity.

[0016] 3. In the nanofiber scaffold, the PLA and BPNSs used are both biocompatible and biodegradable raw materials. These raw materials are widely available, green and environmentally friendly, and have simple preparation processes and low costs. At the same time, by adjusting the dosage ratio of 17-DMAG / BPNSs, electrospinning parameters and irradiation parameters during use, the anti-tumor effect can be adapted according to the needs of the tumor microenvironment after surgery, which helps to achieve the best balance between tumor treatment effect and patient safety in precise intervention. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the preparation process of a photothermal immunomodulatory nanofiber scaffold disclosed in an embodiment of the present invention;

[0018] Figure 2 EDS energy dispersive spectroscopy analysis of PBD nanofiber scaffold;

[0019] Figure 3 The images show the combined Fourier transform infrared spectrum and the Raman spectrum.

[0020] Figure 4 The graph shows the experimental analysis of photothermal performance.

[0021] Figure 5 The image shows the CCK-8 results of H22 cells. The groups from left to right are Control group, PB group, PBD group, PB+NIR group, and PBD+NIR group.

[0022] Figure 6 The Transwell results and HSP90 immunofluorescence staining images of H22 cells are shown. G1 is the Control group, G2 is the PB group, G3 is the PBD group, G4 is the PB+NIR group, and G5 is the PBD+NIR group.

[0023] Figure 7 This is a transmission electron microscope scan image;

[0024] Figure 8 The images show the Western blot results and related statistical analysis of PERK, p-PERK, elF2α, p-elF2α and ATF4 proteins in H22 cells. G1 is the control group, G2 is the PB group, G3 is the PBD group, G4 is the PB+NIR group, and G5 is the PBD+NIR group.

[0025] Figure 9 The image shows the immunofluorescence staining results of p-eIF2α and CHOP in H22 cells;

[0026] Figure 10 The image shows the immunofluorescence staining results of CRT and HMGB1 in H22 cells. G1 is the Control group, G2 is the PB group, G3 is the PBD group, G4 is the PB+NIR group, and G5 is the PBD+NIR group.

[0027] Figure 11 The results of ELISA quantification of HMGB1 concentration and ATP content in H22 cells are shown in the figure. Among them, G1 is the control group, G2 is the PB group, G3 is the PBD group, G4 is the PB+NIR group, and G5 is the PBD+NIR group.

[0028] Figure 12 The bar chart shows the levels of IL-12p70, IL-1β, IL-6 and TNF-α cytokines secreted by mature bone marrow-derived dendritic cells (BMDCs) using ELISA. G1 represents the Control group, G2 the PBD group, G3 the PB+NIR group, and G4 the PBD+NIR group.

[0029] Figure 13 Images and quantitative analysis of MHCⅡ and IL-1β double immunofluorescence staining of BMDCs are shown. Among them, G1 is the control group, G2 is the PBD group, G3 is the PB+NIR group, and G4 is the PBD+NIR group.

[0030] Figure 14 Flow cytometry images and quantitative analysis diagrams of BMDCs (CD11c+CD80+CD86+), where G1 is the control group, G2 is the PBD group, G3 is the PB+NIR group, and G4 is the PBD+NIR group.

[0031] Figure 15 The images, in order, are thermal imaging images of H22 tumor-bearing BALB / c mice undergoing incomplete resection model treatment, temperature-time curves, and curves showing mouse body weight and tumor volume during treatment.

[0032] Figure 16 Figure 1 shows the experimental results verifying the inhibitory effect of NIR-assisted PBD stent single strategy on postoperative recurrence of liver cancer and the existence of the molecular mechanism of "ISR-ICD cascade activation". In the figure, G1 is the control group, G2 is the PB group, G3 is the PBD group, G4 is the PB+NIR group, and G5 is the PBD+NIR group.

[0033] Figure 17The figure shows the results of the experiment on the prevention of postoperative tumor recurrence and recruitment of cytotoxic T lymphocytes by NIR-assisted PBD scaffold and anti-PD-L1. Among them, G1 is the control group, G2 is the anti-PD-L1 group, G3 is the PBD+NIR group, and G4 is the PBD+NIR+anti-PD-L1 group.

[0034] Figure 18 The figure shows the results of the experimental verification of NIR-assisted PBD scaffold synergistic anti-PD-L1 inhibition of distant tumor metastasis and establishment of immune memory. Among them, G1 is the control group, G2 is the anti-PD-L1 group, G3 is the PBD+NIR group, and G4 is the PBD+NIR+anti-PD-L1 group. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This invention discloses a photothermal immunomodulatory nanofiber scaffold (PBD nanofiber scaffold), comprising nanofibers based on polylactic acid (PLA), wherein black phosphorus nanosheets (BPNSs) and heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygerdin (17-DMAG) are co-loaded in the nanofibers by high voltage electrospinning technology.

[0037] Specifically, HSP90 is not only a major molecular chaperone during high temperatures but also a key downstream regulator of mild heat shock responses. It stabilizes denatured and unfolded proteins to mitigate protein toxicity and fine-tunes the activation threshold of the heat shock response, thereby preventing the stress response from escalating uncontrollably. Targeted inhibition of HSP90 may disrupt this adaptive balance, prompting PTT-induced protein toxicity-driven activation of the decisive heat shock response-immune cell death cascade, while simultaneously sensitizing the tumor microenvironment to immune checkpoint inhibitors. Traditional systemic HSP90 inhibitors face conversion barriers, including low tumor bioavailability and dose-limiting off-target effects.

[0038] In this application, BPNSs and the HSP90 inhibitor 17-DMAG are integrated into a PLA nanofiber scaffold using electrospinning technology. This PBD nanofiber scaffold forms a self-enhanced photothermal immunomodulatory strategy. Under near-infrared (NIR) irradiation, the PBD nanofiber scaffold locally generates a PTT effect and photothermally releases 17-DMAG to specifically inhibit HSP90-mediated protein homeostasis. This, in turn, activates a strong PERK-eIF2α-ATF4 signaling axis, synergistically driving a robust immunostimulatory response-immune cell death (ISR-ICD) cascade. Simultaneously, the exposed damage-associated molecular patterns (DAMPs) enhance tumor immunogenicity, recruit cytotoxic T lymphocytes (CTLs), and reprogram immunosuppressive cell populations, thereby reshaping the immune-silenced microenvironment into a responsive tumor microenvironment (TME). Furthermore, the PBD nanofiber scaffold further synergistically combats PD-L1 and establishes durable immune memory, ultimately effectively inhibiting postoperative recurrence and metastasis of liver cancer.

[0039] Please see Figure 1 The present application describes a process for preparing a photothermal immunomodulatory nanofiber scaffold. Black phosphorus crystals are prepared into black phosphorus nanosheets (BPNSs) through liquid phase exfoliation. Polylactic acid (PLA) is dissolved in hexafluoroisopropanol (HFIP) and stirred to form a solution. BPNSs, 17-DMAG and PLA solution are then mixed. The above mixture is then prepared into a nanofiber scaffold through an electrospinning process.

[0040] To facilitate the implementation of this application, the specific preparation process is also disclosed as follows:

[0041] A suitable amount of black phosphorus crystals were ground into a fine powder in an argon-filled glove box. The fine powder was dispersed in N-methyl-2-pyrrolidone at an initial concentration of 0.5-1.5 mg / mL. The dispersion was placed in an ice bath and sonicated at 250-350 W for 10-14 hours. The brown suspension formed after sonication was centrifuged at 3500-4500 rpm for 15-30 minutes. The supernatant was collected and centrifuged at 12000-15000 rpm for 10-20 minutes to remove N-methyl-2-pyrrolidone. The precipitate was collected to obtain black phosphorus nanosheets.

[0042] Polylactic acid was dissolved in hexafluoroisopropanol to prepare a PLA solution with a mass-volume fraction of 10-14% w / v. 0.04-0.06 g of 17-(dimethylaminoethylamino)-17-demethoxygerdromycin and 0.01-0.03 g of black phosphorus nanosheet solution were dissolved in 10 mL of the above PLA solution to form an electrospinning solution.

[0043] The electrospinning solution was loaded into a 5 ml syringe with a 22 gauge needle and placed on a syringe pump for electrospinning. The electrode voltage was set to 10-20 kV, the electrode spacing to 10-15 cm, and the flow rate to 0.3-1 ml / h.

[0044] To facilitate verification of the performance of the photothermal immunomodulatory nanofiber scaffold of this application, the following experimental results are also disclosed in this embodiment:

[0045] 1. Preparation of Black Phosphorus Nanosheets (BPNSs)

[0046] A suitable amount of black phosphorus crystals were ground in an argon-filled glove box for 2 hours until a fine powder was formed. The powder was then dispersed in N-methyl-2-pyrrolidone (NMP) at an initial concentration of 1 mg / mL. The dispersion was placed in an ice bath and sonicated at 300 W for 12 hours. Subsequently, the sonicated brown suspension was centrifuged at 4000 rpm for 20 minutes to remove unpeeled black phosphorus, and the supernatant was collected. The NMP was removed by centrifugation at 13000 rpm for 15 minutes, and the precipitate was collected to obtain black phosphorus nanosheets.

[0047] 2. Synthesis of electrospun nanofiber scaffolds

[0048] 1.25 g of polylactic acid (PLA) was dissolved in 10 mL of hexafluoroisopropanol (HFIP) to prepare a PLA solution with a mass fraction of 12.5%. Three electrospinning solutions were prepared by dissolving 17-DMAG solution, BPNSs solution, and 17-DMAG / BPNSs solution in three separate portions of the above PLA solution.

[0049] The electrospinning solution was loaded into a 5 mL syringe with a 22-gauge needle and placed on a syringe pump for electrospinning. The electrode voltage was set to 15 kV, the electrode spacing to 12 cm, and the flow rate to 0.5 mL / h. A series of nanofiber scaffolds were obtained and named P, PD, PB, and PBD, respectively, where P represents PLA nanofiber scaffold, PD represents 17-DMAG / PLA nanofiber scaffold, PB represents BPNSs / PLA nanofiber scaffold, and PBD represents 17-DMAG / BPNSs / PLA nanofiber scaffold. The sample codes and compositions are shown in Table S1.

[0050] Table S1 Sample Code and Ingredient List

[0051] Sample number PLA(g) 17-DMAG(g) BPNSs(g) HFIP(g) PLA 1.25 0 0 15.96 17-DMAG / PLA(PD) 1.25 0.05 0 15.96 BPNSs / PLA(PB) 1.25 0 0.02 15.96 17-DMAG / BPNSs / PLA(PBD) 1.25 0.05 0.02 15.96

[0052] Please see Figure 2 Energy dispersive spectroscopy (EDS) mapping showed that carbon, nitrogen, oxygen and phosphorus were spatially uniformly distributed throughout the PBD nanofiber scaffold, confirming the uniform loading of 17-DMAG and BPNSs.

[0053] Please see Figure 3 Please see Figure 3 Images a and b in the image are Fourier transform infrared (FTIR) spectra. The analysis shows that the typical peak of PLA particles is at 1747 cm⁻¹. -1 Stretched to C=O, 1453cm -1 CH bend, 1180cm -1 For COC stretching, 1128 and 1079 cm -1 For CO stretching, and 1041cm -1 The OH-bend is observed. The FTIR spectrum of 17-DMAG shows a depth of 3435 cm⁻¹. -1 The characteristic absorption band at 3727 cm⁻¹ is related to the stretching of -OH. -1 For NH bending, 2967 and 2932 cm -1 For CH bending, 1631 and 1608 cm -1 For C=O stretching, and 1254 and 1059 cm -1 For use in CO stretching. Additionally, BPNS is available in 1608, 1102, and 961 cm. -1 The absorption peaks observed at these locations originate from the vibrational stretching of P=O, PPO, and PO, respectively, and are attributed to the slight oxidation of the BPNS surface to produce PO. x 3- Although PD, PB, and PBD nanofiber scaffolds exhibit typical characteristic peaks of PLA, there are slight shifts, while the characteristic peaks of 17-DMAG and BPNS are masked and undetectable due to PLA dominance.

[0054] Please see Figure 3 Images c and d in the image show Raman spectroscopy used for chemical group analysis of the raw material and nanofiber scaffold: the characteristic peak of 17-DMAG powder appears at 1279 cm⁻¹. -1 This is attributed to the coupled CN stretching vibration and NH plane bending vibration in the cycloamide III band, 1367 cm. -1 Belongs to CH3 symmetric bending vibration, 1637 cm -1 This reflects the C=C and C=O stretching vibrations of benzoquinone. Furthermore, BPNSs exhibit vibrations at 362, 440, and 467 cm⁻¹. -1 The display shows three characteristic peaks, corresponding to the out-of-plane phonon mode (A1g) and two in-plane modes (B1g). 2g and A 2 g The P nanofiber scaffold exhibits the same spectral characteristics as PLA particles, showing a characteristic peak of CCO bending at 407 cm⁻¹. -1 The characteristic peak of C-COO stretching vibration is at 876 cm⁻¹. -1The characteristic peak of the asymmetric stretching vibration of COC is at 1131 cm⁻¹. -1 The characteristic peak of CH3 symmetric bending is at 1385 cm⁻¹. -1 The characteristic peak of CH3 bending vibration is at 1459 cm⁻¹. -1 The characteristic peak of C=O stretching vibration is at 1775 cm⁻¹. -1 The characteristic peak of the symmetric stretching vibration of CH3 is at 2880-2900 cm⁻¹. -1 The characteristic peaks of CH3 asymmetric stretching vibration are located at 2947-3003 cm⁻¹. -1 The characteristic peak of the PD nanofiber scaffold is at 1643 cm⁻¹. -1 The key changes are observed at 3342 cm⁻¹, corresponding to the C=C and C=O stretching vibrations of benzoquinone. -1 The peaks are mainly attributed to the hydrogen bond stretching vibrations of the lactam NH and the vibrations of the side-chain secondary amide NH (mainly derived from 17-DMAG). The PB nanofiber scaffold additionally exhibits three characteristic peaks of BPNSs superimposed on the signal of the P nanofiber scaffold.

[0055] The PBD nanofiber scaffold exhibited characteristic peaks of BPNSs and enhanced hydrogen bonding interactions. All observed significant peak shifts were attributed to tensile stress, chemical bonding, and π-π stacking of the PLA chains after loading with 17-DMAG and BPNSs. Overall, this demonstrates that the two functional components, 17-DMAG and BPNSs, are not simply mixed, and these spectroscopic analyses confirm the successful integration of 17-DMAG and / or BPNSs into the PLA nanofiber scaffold.

[0056] Please see Figure 4 At times including 0, 10, 15, 20, 60, and 120 s, the light was emitted by 808 nm near-infrared (NIR) at a wavelength of 0.8 W / cm². 2 Infrared thermal imaging of the power irradiated; at 0.8 W / cm 2 Temperature-time curves of P, PD, PB, and PBD nanofiber scaffolds under NIR irradiation; at 0.8 W / cm 2 The temperature changes of the PBD nanofiber scaffold during five switching cycles under NIR irradiation; the 17-DMAG release curves of the PD and PBD nanofiber scaffolds under NIR irradiation of different powers; and the 17-DMAG release curves of the PBD nanofiber scaffold in response to multiple stimulations under intermittent NIR irradiation of different powers. The images demonstrate the photothermal performance of the photothermal immunomodulatory nanofiber scaffold of this application, at a relatively high 0.8 W / cm². 2At high intensity, PB and PBD nanofiber scaffolds reached cytotoxic equilibrium temperatures of 49.4 ± 1.7 °C and 48.8 ± 1.5 °C, respectively, within 60 s, a stark contrast to the negligible heating in the BPNS-free control. This superior efficiency stems from the inherent semiconductor properties of BPNSs, where photon absorption at the band gap generates electron-hole pairs, which undergo nonradiative recombination via the Shockley-Read-Hall or Auger mechanism to achieve efficient photothermal conversion. The PBD nanofiber scaffold, at 0.8 W / cm², showed improved efficiency after five consecutive cycles. 2 The NIR maintained sustained photothermal stability during on / off cycling, repeatedly reaching 55.0°C without degradation, demonstrating that structural integrity and resistance to photodegradation are fundamental standards for clinical photothermal nanoplatforms.

[0057] Please see Figure 4 To evaluate near-infrared triggered drug release behavior, at variable near-infrared intensities (0, 0.5, and 0.8 W / cm²), 2 The cumulative 17-DMAG release profiles of PD and PBD nanofiber scaffolds over 14 days were quantified. In the absence of near-infrared spectroscopy, both PD and PBD nanofiber scaffolds showed similar baseline release profiles, reaching only about 50% of the cumulative release after 14 days, indicating that temperature-independent diffusion is the dominant mechanism. Crucially, near-infrared stimulation induced a power-dependent release enhancement only in the PBD nanofiber scaffold, with 0.5 W / cm²... 2 The cumulative release increased to 76.0 ± 4.1% and 0.8 W / cm³. 2 Further scaling up to 87.8 ± 4.7% indicates that photothermally enhanced drug release is proportional to heat input. Conversely, the PD stent exhibits minimal responsiveness at 0.5 and 0.8 W / cm². 2 The increases were only 8% and 14% at the time, clarifying that BPNSs-mediated photothermal transduction is crucial for NIR response release. Further analysis showed that the PBD nanofiber scaffold achieved on-demand release regulation, from no irradiation to intermittent 0.5 and 0.8 W / cm². 2 Under NIR irradiation, the baseline increased from 14.1±1.3% to 33.5±2.3% and 46.8±4.0% within 24 hours. This spatiotemporally controlled release paradigm, achieved through remote drive and sensitive near-infrared response, demonstrates targeted drug delivery capability while reducing the risk of systemic toxicity.

[0058] Please see Figure 5Three-day Cell Count Kit-8 (CCK-8) assays showed that the cytotoxic effect of the nanofiber scaffold on H22 cells was time- and treatment-dependent. On day three, H22 cell viability in the PB group was close to baseline, while H22 cell viability in the PBD group was moderately reduced to 62.03±4.89%, attributed to the sustained release of 17-DMAG. Near-infrared irradiation further amplified this effect, with cell viability in the PBD+NIR group significantly decreasing to 20.74±3.16%, a 2.3-fold decrease compared to the PB+NIR group's 47.0±2.3%, confirming the crucial role of 17-DMAG in enhancing photothermal damage sensitivity.

[0059] Please see Figure 6 Transwell assays showed that the control group and PB group had ≥95% coverage of the migrated H22 cells, while the PBD group had the fewest migrated cells, indicating that the near-infrared assisted PBD nanofiber scaffold has the strongest anti-metastasis effect.

[0060] To elucidate the effects of HSP90-targeted inhibition, immunofluorescence staining assessed the influence of nanofiber scaffolds on HSP90 expression. In the Control group, H22 cells exhibited constitutive HSP90 expression, consistent with its crucial role in tumor proliferation and protein homeostasis. The relative mean fluorescence intensity (MFI) of HSP90 in the PBD group was effectively reduced to 0.72 ± 0.10, mechanistically explaining the observed viability inhibition and apoptosis. Notably, the PB+NIR group, representing PTT alone, triggered a compensatory 1.9-fold upregulation of HSP90, confirming an adaptive survival response activated under thermotherapy-induced protein toxicity. Conversely, the NIR-assisted PBD nanofiber scaffold counteracted this recovery response, reducing the relative HSP90 MFI to near baseline levels and establishing a self-enhancing therapeutic effect: local 17-DMAG delivery blocked the HSP90-mediated protein quiescence buffer, transforming transient heat stress into end-stage tumor eradication, thereby overcoming the limitations of conventional PTT.

[0061] Please see Figure 7 A strong ISR-ICD axis represents a transformative strategy to reprogram the immunosuppressive postoperative resection microenvironment into an immunogenic one. Since the endoplasmic reticulum (ER) coordinates the synthesis, folding, and transport of cellular proteins, thermotherapy-induced protein toxicity leads to ER dysregulation, directly initiating ISR activation. Transmission electron microscopy (TEM) ultrastructural observations revealed that NIR-assisted PBD nanofiber scaffolds induced significant ER swelling in tumor cells, indicating a breakdown in protein homeostasis.

[0062] Please see Figure 8Protein toxicity stress triggered phosphorylation of PERK on the endoplasmic reticulum membrane, activating the downstream PERK-eIF2α-ATF4 signaling cascade-mediated ISR. In Western blot analysis of PERK, p-PERK, eIF2α, p-eIF2α, and ATF4 protein expression, the phosphorylation levels of PERK and eIF2α (p-PERK and p-eIF2α) in the PBD and PBNIR groups were 1.56-fold and 1.80-fold higher than those in the control group, and 1.99-fold and 2.37-fold higher, respectively. This was attributed to 17-DMAG-mediated HSP90 inhibition and PTT-induced protein toxicity.

[0063] Please see Figure 9 In the PBD+NIR group, the levels of p-eIF2a and CHOP were significantly increased, which significantly promoted cell apoptosis.

[0064] Please see Figure 10 Immunofluorescence staining and quantification results of CRT and HMGB1 showed that when evaluating nanofiber scaffold-induced ICD, immunofluorescence revealed CRT translocation and HMGB1 release in the PBD and PB+NIR groups, with the most significant CRT membrane exposure and HMGB1 release in the PBD+NIR group.

[0065] Please see Figure 11 The concentrations of HMGB1 and ATP in the supernatant were further quantified, with the highest concentrations found in the PBD+NIR group, at 3.83±0.16 ng / mL and 27.86±2.36 nM, respectively. These results indicate that NIR-assisted PBD nanofiber scaffolds effectively activate the ISR-ICD cascade.

[0066] Please see Figure 12 Using the corresponding enzyme-linked immunosorbent assay (ELISA) kit, it was strongly associated with the secretion of immune-related cytokines, including IL-12p70 (T cell initialization), IL-1β (T cell activation), IL-6 (pro-inflammatory signaling), and TNF-α (direct tumor killing activity).

[0067] Please see Figure 13 Immunofluorescence co-staining of MHC-II and IL-1 confirmed that BMDCs had enhanced antigen presentation capabilities, and the relative MFI of the PBD+NIR group was 3.5 times higher than that of the control group.

[0068] Please see Figure 14Dendritic cells (DCs), acting as the "shepherds" of T-cell immunity in cancer, serve as a hub connecting innate and adaptive immune responses, achieving this function by initiating T-cell activation. Flow cytometry revealed an increase in the number of CD11c+CD80+CD86+ mature bone marrow-derived dendritic cells (BMDCs) in both the PBD and PB+NIR groups. This was attributed to heat shock protein 90 (HSP90) inhibition and the release of sublethal damage-associated molecular patterns (DAMPs) from photothermal therapy (PTT), respectively. Compared to the PB+NIR group and the control group, the near-infrared (NIR)-assisted PBD nanofiber scaffold significantly enhanced the maturity of BMDCs, increasing it by 2.0-fold and 4.2-fold, respectively.

[0069] To further elucidate the inhibitory effect of NIR-assisted PBD stent (PLA+BPNSs+17-DMAG) single strategy on postoperative recurrence of liver cancer and the molecular mechanism of "ISR-ICD cascade activation," and to demonstrate that it can effectively inhibit postoperative recurrence of liver cancer by inhibiting HSP90 and enhancing the ISR-ICD cascade (such as promoting CRT exposure and HMGB1 release), without significant systemic toxicity, the following experiments were conducted:

[0070] Please see Figure 15 The in vivo therapeutic effect was evaluated using an incomplete resection model in H22 tumor-bearing BALB / c mice. Mice were randomly divided into five groups: control group, PB group, PBD group, PB+NIR group, and PBD+NIR group. 5 × 10⁶ mice were used as the in vivo treatment model. 6 / 100μL of H22 cells were subcutaneously injected into the right anterior dorsal side of BALB / c mice. When the tumor volume approached approximately 150 mm³, the tumor was excised, leaving approximately 1% residual tumor tissue at the surgical site to stimulate residual microtumors. After implanting PB or PBD nanofiber scaffolds at the excision site, near-infrared irradiation was performed on tumor-bearing mice at 0.8 W / cm² at 5, 10, 15, 20, 30, 60, and 90 seconds postoperatively. 2 Near-infrared irradiation increased the temperature at the tumor resection site to 48.7°C and 47.8°C in the PB+NIR and PBD+NIR groups, respectively, with no statistically significant difference. On day 10, mice were sacrificed for endpoint analysis; weight trajectory analysis showed no significant difference between groups, indicating negligible systemic toxicity. Furthermore, tumor weight analysis showed that while both the PBD and PB+NIR groups delayed recurrence, the PBD+NIR group most effectively inhibited tumor recurrence.

[0071] Please see Figure 16Images a and b, along with flow cytometry analysis of lymph nodes, revealed that the PBD+NIR group had the highest proportion of mature CD11c+CD80+CD86+ dendritic cells (47.27±3.05%), which was superior to the PB+NIR group (25.47±1.93%) and the PBD group (19.33±1.17%). Please refer to [link to relevant documentation]. Figure 16 Images c and d show that in recurrent tumor tissue, the proportions of CD3+CD4+ and CD3+CD8+ T cells in the PBD+NIR group were significantly increased to 23.83±0.75% and 11.33±1.06%, respectively. Please refer to [link to relevant image]. Figure 16 In the MRI images, tumor-associated macrophage analysis showed that the recurrent tumor tissue in the control group was predominantly composed of M2 macrophages, while the PBD group and PB+NIR group increased the proportion of M1 macrophages to 15.13±0.40% and 18.40±1.14%, respectively, while decreasing the proportion of M2 macrophages to 16.27±0.50% and 14.13±0.81%, respectively. The PBD+NIR group achieved the greatest M1 polarization, with the proportion of M1 macrophages at 25.63%±1.32, while inhibiting the presence of M2 macrophages, whose proportion was 10.37%±0.67, and the M1 / M2 ratio was 2.48±0.15. Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) are two important immunosuppressive cell types in the tumor microenvironment (TME). They secrete immunosuppressive factors such as IL-10 and TGF-β and suppress T cell-mediated immune responses. See also... Figure 16 Further analysis of the f and g images showed that in the PBD+NIR group, CD4+FOXP3+ regulatory T cells and CD11b+Gr-1+ myeloid-derived suppressor cells were significantly reduced to 4.02±0.91% and 7.79±1.21%, respectively, which were significantly lower than those in the PB+NIR group and the control group. Please refer to [link to relevant documentation]. Figure 16 Mid-h images and quantitative enzyme-linked immunosorbent assay confirmed the immunomodulatory efficacy of the PBD+NIR group, showing elevated levels of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6, and IL-12p70, while significantly reduced immunosuppressive IL-10 and TGF-β, compared with monotherapy.

[0072] These results demonstrate that near-infrared-assisted PBD nanofiber scaffolds coordinate a multi-pronged immunomodulatory cascade: first, enhanced dendritic cell maturation promotes antigen-specific T cell responses; second, M1 macrophage polarization reprograms the inflammatory environment; and third, depletion of regulatory T cells and myeloid-derived suppressor cells alleviates immunosuppression. By amplifying the ISR-ICD axis, this self-reinforcing photothermal immunomodulatory strategy transforms the immunosuppressive tumor microenvironment into an immunogenic, active niche.

[0073] To further elucidate the clinical translational value of the photothermal immunomodulatory nanofiber scaffold proposed in this application, based on the above experiments, commonly used clinical ICIs (anti-PD-L1) were introduced to verify the synergistic effect of "PBD+NIR+ICIs". The following experiments were conducted to better align with actual clinical treatment needs:

[0074] Please see Figure 17 Mice were randomly divided into four groups: control group, anti-PD-L1 group, PBD+NIR group, and PBD+NIR+anti-PD-L1 group, thus re-establishing the incomplete resection model. Please refer to... Figure 17 Image a shows mice receiving near-infrared light irradiation on days 1, 4, and 7 post-surgery after implantation of a PBD nanofiber scaffold at the resection site, followed by intravenous injection of anti-PD-L1 drugs on days 2, 5, and 8 post-surgery. Tumor weight quantification analysis indicated a gradual decrease in tumor weight. Please refer to [link to relevant documentation]. Figure 17 Image b in the diagram shows that tumor recurrence and progression were delayed in all treatment groups, with the PBD+NIR+anti-PD-L1 group exhibiting the most significant inhibitory effect. Please refer to [link to image]. Figure 17 The c-images and survival analysis (follow-up for 50 days) showed that the local near-infrared light-assisted PBD nanofiber scaffold and the anti-PD-L1 drug synergistically prolonged postoperative survival.

[0075] Histological evaluation by Ki67 immunohistochemistry showed that the cell nuclei in the control group were densely packed and deeply stained, while the tumor proliferation activity in the treatment group was significantly reduced. Please refer to [link to relevant documentation]. Figure 17 The images in the d image show that the reduction in Ki67+ cell nuclei was most pronounced in the PBD+NIR+ anti-PD-L1 group. (See also...) Figure 17 The e-image in the image, further immunospectral analysis showed that in the PBD+NIR+anti-PD-L1 group, please refer to [link to relevant documentation]. Figure 17 In the f to g images, CD3+CD4+ and CD3+CD8+ T cell infiltration surged to 36.80±1.23% and 18.00±1.68%, respectively. Additionally, please refer to... Figure 17 In the h-j images, macrophage polarization significantly shifts towards a pro-inflammatory M1 phenotype, while the effector function of cytotoxic T lymphocytes (CTLs) is significantly enhanced. Please refer to [link to relevant documentation]. Figure 17 In the k-to-l image, the CD3+CD8+IFN-γ+ and granzyme B+ cell populations reached 29.85±2.02% and 17.33±1.02%, respectively. Please refer to [link to image]. Figure 17The m-images, analyzed by enzyme-linked immunosorbent assay (ELISA) for cytokine profiling, confirmed that TNF-α peaked at 821.53±17.61 pg / mL in the PBD+NIR+anti-PD-L1 group, IFN-γ reached 593.12±8.30 pg / mL, while TGF-β decreased to 126.57±19.76 pg / mL, and the M2 marker arginase-1 decreased to 20.89±6.89 pg / mL. Compared with other groups, this multi-pronged immunomodulation—enhanced cytotoxic T lymphocyte (CTL) cytotoxicity, M1 macrophage polarization, and depletion of regulatory T cells (Tregs)—indicates that the combination of a locally self-reinforcing photothermal immunomodulatory strategy and anti-PD-L1 can overcome adaptive PD-L1 upregulation, reactivate CTLs, achieve durable relapse suppression, and promote the cancer immune cycle.

[0076] To further elucidate the effect of this photothermal immunomodulatory nanofiber scaffold in inhibiting distant metastasis and establishing immune memory function, a re-attack model was established to evaluate the inhibitory effect on distant metastasis, and the following experiments were conducted:

[0077] Please see Figure 18 The experimental mice were randomly divided into four groups: control group, anti-PD-L1 group, PBD+NIR group, and PBD+NIR+anti-PD-L1 group. Please refer to [link to relevant documentation]. Figure 18 Image a shows mice that, after implantation of a PBD nanofiber scaffold at the resection site, received near-infrared irradiation on days 1, 4, and 7 post-surgery, and were intravenously injected with anti-PD-L1 on days 2, 5, and 8. On day 10 post-treatment, H22 cells were injected into the contralateral groin to establish a re-attack model to evaluate the inhibitory effect on distant metastasis. Please refer to [link to relevant documentation]. Figure 18 In the middle group (b), the PBD+NIR+anti-PD-L1 group showed the most significant inhibitory effect on distant tumor growth, indicating that systemic immunity was activated. (See also...) Figure 18 Image C, contralateral tumor tissue collected on day 21. For flow cytometry analysis of the contralateral tumor, please refer to [link to relevant documentation]. Figure 18 Images d through e show that the proportions of CD3+CD4+ and CD3+CD8+ T cells in the PBD+NIR group were 11.97±1.08% and 7.67±0.61%, respectively. This indicates that this self-reinforcing photothermal immunomodulatory strategy can, to some extent, further influence the systemic anti-tumor immune response by reshaping the immune microenvironment. It is worth noting that... (See also...) Figure 18From f to g images, cytokine profile analysis of the contralateral tumor showed a pro-inflammatory shift. In the PBD+NIR+anti-PD-L1 group, TNF-α and IFN-γ reached peak values ​​of 350.01±18.29 pg / mL and 245.21±14.76 pg / mL, respectively, while in the control group, they were 128.53±19.17 pg / mL and 95.81±7.22 pg / mL, respectively.

[0078] In addition, please see Figure 18 Mid-to-high frequency (h-I) images and spleen memory T cell analysis confirmed the establishment of durable systemic immunity. The number of central memory T cells (TCM) and effector memory T cells (TEM) was significantly increased in the PBD+NIR+anti-PD-L1 group, at 13.50±1.30% and 25.58±0.95%, respectively. These findings collectively indicate that the combined use of local near-infrared assisted PBD nanofiber scaffold and anti-PD-L1 can remodel the primary and systemic immune environment—enhancing CTL migration to distant sites, maintaining pro-inflammatory cytokine cascade responses, and forming immune memory responses, thereby establishing a systemic immune barrier to resist tumor metastasis.

[0079] This invention discloses a photothermal immunomodulatory nanofiber scaffold that utilizes a bidirectional ISR mechanism mediated by the PERK-eIF2α-ATF4 signaling axis. Locally released 17-DMAG specifically antagonizes upregulated HSP90, forcibly recalibrating the ISR activation threshold. This molecular intervention not only disrupts adaptive protein homeostasis mechanisms but also redirects cell fate to terminal pro-apoptotic pathways by upregulating GADD34 and CHOP. Simultaneously, it induces strong ICD (immunogenic cell death) through CRT exposure and HMGB1 release. Comparative analysis with photothermal monotherapy (NIR-assisted PB nanofiber scaffold) shows that the NIR-assisted PBD nanofiber scaffold significantly enhances the immunogenicity of the postoperative microenvironment, accelerates dendritic cell (DC) maturation, and increases the infiltration density of cytotoxic T lymphocytes (CTLs) while completely eliminating residual tumor. By precisely regulating the ISR-ICD cascade reaction, it amplifies the high-temperature damage and immune activation effects induced by photothermal therapy (PTT).

[0080] Strategic remodeling of the immunosuppressive tumor microenvironment (TME) is a key prerequisite for optimizing the efficacy of adjuvant immunotherapy. This invention utilizes a photothermal immunomodulatory nanofiber scaffold to reprogram an immune-unfavorable TME into an acceptable one. Through strategic regulation of regulatory T cell (Tregs) and myeloid-derived suppressor cell (MDSC) populations, and recalibration of immunomodulatory cytokine levels, a synergistic effect is established between eliminating ICD suppression and enhancing the efficacy of helper immune checkpoint inhibitors (ICIs). Near-infrared (NIR) assistance demonstrates a significant ability to regulate PD-L1 expression dynamics, thereby creating a time-optimized window for adjuvant ICIs to revitalize cytotoxic T lymphocytes (CTLs) and restore their cytolytic capacity. This hierarchical immune regulation effectively modulates multiple nodes in the cancer immune cycle, promoting the cyclic amplification and expansion of the anti-tumor immune response, ultimately linking local therapeutic intervention with durable systemic immunity.

[0081] The photothermal immunomodulatory nanofiber scaffold of this invention also exhibits favorable toxicological properties and excellent biocompatibility. Its local 17-DMAG dissolution mechanism avoids the systemic toxicity risks inherent in traditional drug delivery methods, while maintaining therapeutic concentrations at the target site for sustained efficacy. By adaptively adjusting the 17-DMAG / BPNSs dosage ratio, electrospinning parameters, and irradiation parameters, release can be precisely controlled, allowing for the selection of appropriate antitumor effects based on the postoperative tumor microenvironment. The mutual control of release kinetics and biological responsiveness offers personalized potential for postoperative local treatment, achieving an optimal balance between tumor therapeutic efficacy and patient safety through precise intervention.

[0082] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A photothermal immunomodulatory nanofiber scaffold, comprising nanofibers based on polylactic acid, characterized in that: Black phosphorus nanosheets and the heat shock protein 90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygerdomyl were co-loaded in the nanofibers using high-voltage electrospinning technology.

2. A preparation method, wherein the preparation method is the preparation method of the photothermal immunomodulatory nanofiber scaffold according to claim 1, characterized in that, include: A suitable amount of black phosphorus crystals were ground into a fine powder in an argon-filled glove box. The fine powder was dispersed in N-methyl-2-pyrrolidone at an initial concentration of 0.5-1.5 mg / mL. The dispersion was placed in an ice bath and sonicated at 250-350 W for 10-14 hours. The brown suspension formed after sonication was centrifuged at 3500-4500 rpm for 15-30 minutes. The supernatant was collected and centrifuged at 12000-15000 rpm for 10-20 minutes to remove N-methyl-2-pyrrolidone. The precipitate was collected to obtain black phosphorus nanosheets. Polylactic acid was dissolved in hexafluoroisopropanol to prepare a PLA solution with a mass-volume fraction of 10-14% w / v. 0.04-0.06 g of 17-(dimethylaminoethylamino)-17-demethoxygerdromycin and 0.01-0.03 g of black phosphorus nanosheet solution were dissolved in 10 mL of the above PLA solution to form an electrospinning solution. The electrospinning solution was loaded into a 5 ml syringe with a 22 gauge needle and placed on a syringe pump for electrospinning. The electrode voltage was set to 10-20 kV, the electrode spacing to 10-15 cm, and the flow rate to 0.3-1 ml / h.

3. An application, characterized in that: The application is the use of the photothermal immunomodulatory nanofiber scaffold synergistic with photothermal therapy as described in claim 1 for postoperative immunotherapy of liver cancer.

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