A medical multifunctional optical probe, a preparation method thereof and application thereof in surgical navigation

Abstract

The application discloses a kind of medical multifunctional optical probes, belong to tumor operation navigation material technical field.The multifunctional optical probe is the gold nanostar embedding doped Gd 3+ Of magnetic silica, and further modified with polyethylene glycol on the surface of the magnetic silica shell Au@Cu 2‑x Se core-shell nanoparticle modification with near-infrared two-zone plasmonic effect.The medical multifunctional optical probe provided by the application can be applied to tumor operation navigation, and the operation resection can be guided by preoperative MR / SERS imaging and intraoperative SERS using the MATRA probe, which can significantly improve the operation effect.Immunotherapy can further remove local residual microtumor and distant metastatic tumor, thereby effectively inhibiting the recurrence and metastasis of tumor, and significantly improving the anticancer effect of 4T1 breast cancer mouse model.

Description

Technical Field

[0001] This invention relates to the field of tumor surgical navigation materials technology, specifically to a medical multifunctional optical probe, its preparation method, and its application in surgical navigation. Background Technology

[0002] Surgical resection is currently the primary means of cancer treatment in clinical practice and remains the only treatment option for most malignant solid tumors. However, local recurrence and distant metastasis occur after surgery, which is a significant reason for surgical failure in cancer treatment. Studies have shown that cancer recurrence and metastasis are closely related to residual microtumors on the operating table, surgical tissue damage, and local inflammation. The ideal approach to cancer treatment in clinical practice is to completely surgically remove the entire tumor mass without adversely affecting surrounding blood vessels and vital organs, without removing adjacent healthy tissue, and to further implement effective perioperative treatments (such as chemotherapy, radiotherapy, thermal ablation, and immunotherapy) to remove microscopic residual lesions and unresectable distant metastases that are not visible to the naked eye. Accurate preoperative and intraoperative detection provides surgeons with crucial information on the presence, location, size, number, and stage of a tumor for their clinical decision-making. In clinical treatment, the surgical workflow typically begins with preoperative medical imaging to precisely locate the tumor and delineate its boundaries to formulate a surgical plan. Currently, commonly used methods for preoperative non-invasive detection and guidance of tumor surgery in clinical practice include computed tomography (CT), positron emission tomography (PET), magnetic resonance (MR) imaging, and ultrasound imaging. However, these methods have drawbacks such as high cost, large instrument footprint, limited spatial resolution, poor sensitivity, long operation time, and inconsistencies between the preoperatively determined tumor location and the actual intraoperative location due to soft tissue displacement during surgery. Furthermore, these imaging techniques face challenges in detecting residual microscopic lesions (typically dozens of tumor cells, <1 mm in size) and accurately determining the complete tumor boundary contour after tumor cells infiltrate adjacent normal tissue. Recently, optical imaging techniques such as photoacoustic imaging and fluorescence imaging have been used for surgical navigation to guide tumor resection. Although photoacoustic imaging has advantages such as large penetration depth and high spatial resolution, its limited sensitivity prevents the detection of microscopic lesions and real-time guidance for tumor resection. Near-infrared fluorescence imaging (NIFI) offers advantages such as high sensitivity and good molecular specificity, enabling surgical navigation without ionizing radiation. It provides real-time guidance for precise localization, demarcation, and surgical resection of solid tumors. However, its clinical translation is hampered by factors such as the inability to visualize invasive lesions at tumor boundaries, photobleaching effects, strong interference from tissue autofluorescence, and long surgical procedures. Therefore, there is an urgent need to develop effective surgical navigation strategies to achieve preoperative tumor localization, delineation of tumor margins, and real-time guidance for complete resection of microtumors.

[0003] Recently, surface-enhanced Raman spectroscopy (SERS) nanoparticles (NPs) have attracted considerable interest as a next-generation multifunctional optical contrast agent for in vivo biosensing and bioimaging. They can integrate traditional imaging techniques such as CT and MRI, as well as multiple functions including photothermal ablation and chemotherapy, with SERS detection to achieve multimodal imaging and combined therapy. SERS possesses high sensitivity for single-molecule detection, excellent molecular specificity, powerful multiplexing capabilities, negligible photobleaching properties, and excellent resistance to interference from complex biological matrices, overcoming common problems in traditional clinical medical technologies and optical imaging technologies (such as photoacoustic and fluorescence imaging). Given these superior properties, SERS NPs can serve as an ideal "all-in-one" multifunctional imaging contrast agent for multimodal imaging, for example, accurately delineating tumor boundaries and providing real-time intraoperative SERS guidance for surgical resection of tumors, including microtumors and invasive tumor cells at the tumor boundary that lead to poor surgical outcomes. The significant efforts invested in reforming high-level SERS platforms for biosensing and bioimaging over the past few decades demonstrate the enormous application potential of SERS imaging in biomedical imaging. In addition, our research group and other research teams have recently reported many results that use in vivo tumor SERS imaging to achieve precise preoperative planning and intraoperative detection, and to eliminate tiny lesions on the operating table in real time, ultimately improving surgical outcomes.

[0004] Following surgical resection, adjuvant therapy is crucial for cancer patients because surgery typically cannot treat metastatic tumors and residual microtumors invisible to the naked eye, which often lead to local recurrence and distant metastasis. Currently, clinical postoperative cancer treatments mainly include adjuvant radiotherapy, oral chemotherapy, or thermal ablation; however, due to their poor clinical efficacy and severe side effects, they are rarely used to treat metastatic tumors. Furthermore, to allow patients sufficient time to recover from the initial resection, these adjuvant therapies are often administered several weeks later. This prolonged recovery period may miss the optimal window for treating microtumors on the operating table, potentially leading to local recurrence and distant metastasis. Recent research indicates that cancer immunotherapy can stimulate both innate and adaptive immunity, inhibiting local recurrence and treating distant metastasis, making it a superior alternative to traditional postoperative adjuvant therapy. Immunotherapy has proven effective in treating malignant tumors such as breast cancer, brain cancer, lung cancer, and melanoma. Therefore, we infer that postoperative immunotherapy is an effective adjuvant therapy strategy for treating residual microtumors and distant metastases after surgery. Summary of the Invention

[0005] In view of the above-mentioned shortcomings, this invention provides a medical multifunctional optical probe, its preparation method, and its application in surgical navigation. This invention utilizes the MATRA probe for preoperative MR / SERS imaging and intraoperative SERS-guided surgical resection, which can significantly improve surgical outcomes. Subsequent immunotherapy further eliminates local residual microtumors and distant metastatic tumors, thereby effectively inhibiting tumor recurrence and metastasis, and significantly improving the anti-cancer effect in the 4T1 breast cancer mouse model.

[0006] To achieve the above objectives, the present invention provides a medical multifunctional optical probe, wherein the multifunctional optical probe is a Raman-encoded gold nanostar with Gd intercalation. 3+ Au@Cu with near-infrared II plasmon resonance effect, further modified with polyethylene glycol on the surface of the magnetic silica shell. 2-x Se core-shell nanoparticle modification.

[0007] Among them: the AuSt (gold nanostar) core is used to amplify the Raman signal of p-NTP molecules adsorbed on its surface for SERS imaging; Gd 3+ For MR imaging; Au@Cu 2-x The high photothermal conversion efficiency of Se core-shell nanoparticles makes them suitable for photothermal tumor ablation; while the outermost layer of polyethylene glycol is modified to enhance the overall biocompatibility of the MATRA probe.

[0008] Based on the same inventive concept, the present invention also provides a method for preparing the above-mentioned medical multifunctional optical probe, comprising the following steps:

[0009] Step 1: React HAuCl4 aqueous solution with NaBH4 in a system with surfactant in a redox reaction and let stand to obtain gold nano-seed solution;

[0010] Step 2: Mix the HAuCl4 aqueous solution with the PVP and DMF solutions evenly, add the gold nanoseed solution, and after purification, obtain the gold nanostar solution;

[0011] Step 3: Add specific Raman molecules to the gold nanostar solution and add MPTMS under alkaline conditions to hydrolyze it, grow a SiO2 shell on the surface of the gold nanostar, and after purification, obtain Raman molecule labeled AuSt@SiO2 nanoparticles.

[0012] Step 4: Add an alkaline mixed solution of C6H5Na7·2H2O and GdCl3·6H2O to the Raman-labeled AuSt@SiO2 nanoparticles to form a Gd-doped Gd complex. 3+ -SERS nanoparticles;

[0013] Step 5: Reconcile HAuCl4 aqueous solution with NaBH4 in Gd 3+ -SERS nanoparticles undergo redox reactions on their surface to obtain Au@Gd 3+ -SERS nanoparticles;

[0014] Step 6: Add SeO2 solution and reducing agent to Au@Gd 3+ Elemental Se was deposited on the surface of gold spheres containing SERS nanoparticles, followed by the addition of CuSO4·5H2O and a reducing agent to obtain Au@Cu. 2-x Se@Gd 3+ -SERS nanoparticles;

[0015] Step 7: Add Au@Cu 2-x Se@Gd 3+ -SERS nanoparticles were dispersed in a solvent, and then methoxy polyethylene glycol-mercapto groups were added. After stirring, centrifugation, and purification, polyethylene glycol-alcoholized Au@Cu was obtained. 2-x Se@Gd 3+ -SERS nanoparticles, also known as multifunctional optical probes.

[0016] According to one aspect of the invention, in step 1, the surfactant is polyvinylpyrrolidone.

[0017] According to one aspect of the invention, in step 3, the specific Raman molecule is p-nitrothiophenol.

[0018] According to one aspect of the invention, in step 6, the reducing agent is ascorbic acid.

[0019] Based on the same inventive concept, the present invention also provides the application of the above-mentioned medical multifunctional optical probe or the medical multifunctional optical probe prepared by the above-mentioned preparation method in tumor surgical navigation.

[0020] According to one aspect of the present invention, the tumor surgical navigation includes: preoperative planning using MR / SERS imaging of the probe to determine the size, location, and boundaries of the tumor; intraoperative SERS imaging of the probe to detect and locate the tumor boundaries and microtumor lesions on the operating table in real time; and postoperative immunotherapy combining the near-infrared II photothermal effect of the probe and programmed death-1 antibody to eliminate residual microtumors and distant metastatic tumors.

[0021] The beneficial effects of this invention are:

[0022] The GCS@Gd of the present invention 3+ -SERS NPs exhibit a distinct core-shell structure; and the GCS@Gd of this invention... 3+The AuSt on -SERS NPs has a plasmonic resonance band (plasmonic resonance wavelength of 742nm), which makes Gd 3+ -SERS NPs are redshifted to 776 nm; Au NPs are deposited on the silica surface, making Au@Gd 3+ -SERS NPs showed a novel plasmon absorption band at 559 nm; GCS@Gd synthesized from 1.0 mM SeO2 3+ In the extinction spectrum of SERS NPs, a strong near-infrared II plasmon resonance band can be clearly seen at 1051 nm, which is caused by the plasmon resonance absorption of GCS NPs. As the SeO2 concentration increases from 0.5 mM to 2.0 mM, the plasmon resonance absorption of the near-infrared II plasmon resonance band is enhanced. This invention prepares a multifunctional optical probe (MATRA probe) for tumor surgical navigation. The probe's MR / SERS imaging is used for preoperative planning to determine the size, location, and boundaries of the tumor; intraoperative SERS imaging detects and locates the tumor boundaries and micro-tumor lesions on the operating table in real time; postoperatively, the near-infrared II photothermal effect of the MATRA probe and programmed death-1 antibody are combined for adjuvant immunotherapy to eliminate residual micro-tumors and distant metastases. This multifunctional optical probe first uses routine clinical MR imaging for preoperative planning to determine tumor size and location, avoiding the time-consuming drawbacks of SERS macroscopic imaging. Secondly, during surgery, the ultra-high sensitivity of SERS imaging allows for real-time localization of tumor boundaries and micro-tumor lesions, resolving issues such as the low sensitivity of MR imaging and the discrepancy between the preoperatively determined tumor location and the actual intraoperative location due to soft tissue displacement during surgery. Finally, the near-infrared II photothermal effect of the MATRA probe, combined with programmed death-1 antibody, further improves the efficacy of conventional immune checkpoint blockade immunotherapy through postoperative adjuvant immunotherapy.

[0023] Similarly, this method can also be applied to surgical navigation in HER2+ breast cancer mouse models. First, a HER2-targeting multifunctional optical probe (SERS probe) is prepared. This probe can specifically target the tumor location in the HER2+ breast cancer mouse model. Preoperative SERS imaging completely outlines the tumor contour. During the operation, the ultra-high sensitivity of SERS is used to detect residual micro-tumor lesions. Postoperatively, photothermal ablation removes the residual micro-tumors. Attached Figure Description

[0024] Figure 1A schematic diagram of a design strategy to improve cancer treatment by combining SERS-guided cancer surgery with postoperative immunotherapy to eliminate local and distant metastatic microtumors; A is a three-dimensional schematic diagram of the MATRA probe structure, in which the MATRA probe is embedded in a Gd3+-doped silica shell of Au nanostars (AuSt) encoded by Raman molecules (p-NTP), then modified with Au@Cu2-xSe (GCS) core-shell nanoparticles, and subsequently modified with polyethylene glycol; B is a schematic diagram of surgical resection guided by the MATRA probe, in which the MATRA probe is injected intravenously and accumulates in the tumor through enhanced permeability and retention (EPR) effect; C is a whole-body MR imaging of 4T1 breast tumor-bearing mice before surgery for tumor localization; D is a flowchart of the workflow of SERS-guided surgical resection, including (i) intraoperative in vivo SERS imaging to accurately delineate the tumor boundary, (ii) real-time detection of residual microtumors on the operating table during surgery, and (iii) near-infrared II laser irradiation / PD-1 antibody-induced immunotherapy for local residual and distant metastatic microtumors;

[0025] Figure 2 This is a schematic diagram illustrating the synthesis of the MATRA probe of this invention;

[0026] Figure 3 This is a TEM image of the MATRA probe synthesized using different concentrations of SeO2 (0.5 (left), 1.0 (mM), and 2.0 (right) mM) in Example 2 of the present invention.

[0027] Figure 4 The STEM image of SeO2 at a concentration of 1.0 mM and the elemental distribution diagrams of Au, Si, Cu and Se are shown in Example 2 of this invention.

[0028] Figure 5 AuSt and Gd are from Embodiment 2 of the present invention. 3+ -SERS NPs、Au@Gd 3+ - Optical extinction spectra of SERS NPs;

[0029] Figure 6 GCS@Gd synthesized using different concentrations of SeO2 (0.5, 1.0, and 2.0 mM) in Example 2 of this invention. 3+ - Optical extinction spectra of SERSNPs;

[0030] Figure 7 For Au and Cu 2-x The standard XRD data for SE are JCPDS numbers 01-1172 and 06-0680, respectively.

[0031] Figure 8 GCS@Gd synthesized with different concentrations of SeO2 (0.5, 1.0, and 2.0 mM) in Example 2 of this invention. 3+- Powder XRD patterns of SERSNPs;

[0032] Figure 9 AuSt and Gd are from Embodiment 2 of the present invention. 3+ -SERS NPs、Au@Gd 3+ GCS@Gd synthesized from -SERS NPs and different concentrations of SeO2 (0.5, 1.0 and 2.0 mM) 3+ -Hydrated particle size of SERS NPs;

[0033] Figure 10 For 1064nm laser (1.0W / cm) 2 (10 min) Continuous irradiation with different concentrations of GCS@Gd 3+ Temperature distribution of SERS NPs (PBS, 25, 50, 100, 200 and 400 μg / mL);

[0034] Figure 11 Near-infrared photothermal properties of GCS@Gd3+-SERS NPs prepared in Example 2 of this invention; In A: (i) under 1064nm laser (1.0W / cm 2 GCS@Gd synthesized under irradiation with different SeO2 concentrations (0.5, 1.0, and 2.0 mM) of 200 μg / mL. 3+ -SERS NPs aqueous solution after 0-10 min infrared thermal imaging, and under 1064nm laser (1.0W / cm²) 2 Under irradiation, (ii) 0.5 mM and (iii) 2.0 mM SeO2 were used to synthesize different concentrations (PBS, 25, 50, 100, 200 and 400 μg / mL) of GCS@Gd. 3+ -Temperature distribution of SERS NPs in aqueous solution; In B, (i) is GCS@Gd synthesized with 200 μg / mL of 0.5 mM SeO2. 3+ -SERS NPs in 1064nm laser (1.0W / cm) 2 (ii) The photostability test was performed on GCS@Gd synthesized with 200 μg / mL of 1.0 mM SeO2. 3+ -SERS NPs in 1064nm laser (1.0W / cm) 2 (iii) The photostability test was performed on GCS@Gd synthesized with 200 μg / mL SeO2 at a concentration of 2.0 mM. 3+ -SERS NPs in 1064nm laser (1.0W / cm) 2 Light stability test under )

[0035] Figure 12 For GCS@Gd 3+ Calculation of near-infrared II photothermal conversion efficiency of SERS NPs. In AC, (i) under 1064nm laser (1.0W / cm²) 2 GCS@Gd was prepared at a SeO2 concentration of 200 μg / mL, with A at 0.5 mM, B at 1.0 mM, and C at 2.0 mM. 3+ - Temperature curve of SERS NPs aqueous solution and (ii) Change of cooling time and cooling portion -Ln(θ) in photothermal curve;

[0036] Figure 13 For products containing different concentrations of GCS@Gd 3+ (i) SERS images and (ii) average SERS spectra of agarose models of -SERS NPs (6.25-400 μg / mL), and (iii) 1340 cm⁻¹ SERS spectra. -1 SERS intensity at GCS@Gd 3+ - Relationship between SERS NP concentrations; where the SERS image is composed of 1340 cm⁻¹ -1 Peak intensity values ​​are created at the location;

[0037] Figure 14 For GCS@Gd 3+ - Near-infrared SERS performance of SERS NPs; A represents the near-infrared SERS performance of NPs containing different concentrations of GCS@Gd. 3+ (i) Representative SERS spectra and (ii) SERS images of agarose models of SERS NPs synthesized from different concentrations of SeO2 (0.5, 1.0, and 2.0 mM); in BC, (i) average SERS spectra of agarose models at different concentrations (6.25–400 μg / mL), in B, (ii) 0.5 mM SeO2, and in C, (ii) 2.0 mM SeO2 synthesized from GCS@Gd 3+ -SERS NPs at 1340cm -1 A graph showing the changes in SERS intensity and concentration at a given location;

[0038] Figure 15 The photostability and colloidal stability of the MATRA probe are shown; A represents the MATRA probe at 1340 cm⁻¹ under 785 nm laser (9.2 mW) irradiation. -1 Figure B shows the change of SERS intensity with laser irradiation time; Figure B shows the change of MATRA probe hydrated particle size with culture time in (i) 10% fetal bovine serum and (ii) DMEM medium.

[0039] Figure 16T1-weighted MR images of MATRA probe aqueous solutions and graphs showing the variation of relaxation rate (1 / T1) with MATRA probe concentration; (i) T1-weighted MR images of MATRA probe aqueous solutions at different concentrations (0, 0.0156, 0.0313, 0.0625 and 0.125 mM, with Gd concentration as standard); (ii) Graphs showing the variation of relaxation rate (1 / T1) with MATRA probe concentration.

[0040] Figure 17 (i) After treating 4T1 cells with different concentrations of MATRA probe (0 (PBS, control), 25, 50, 100, 200, and 400 μg / mL), the cells were irradiated with a 1064 nm laser (+ laser, 1.0 W / cm²). 2 (ii) Cell viability images under 1064nm laser irradiation (5 min) or no laser irradiation (-laser), and fluorescence images of 4T1 cells treated with PBS and different concentrations of MATRA probes, then incubated with calcein-AM (live cells, green) and PI (dead cells, red) after staining.

[0041] Figure 18 The assays were performed using PBS + laser, 120 μg / mL MATRA probe, and 120 μg / mL MATRA probe + laser (1064 nm laser, 1.0 W / cm²). 2 4T1 cells treated for 5 min were then co-incubated with Hoechst 33342 (nucleus, blue) and goat anti-rabbit secondary antibody conjugated with Alexa Fluor488 (green) to express CRT; (i) Immunofluorescence image after staining; (ii) Fluorescence intensity spectrum along the direction of the arrow;

[0042] Figure 19 HMGB1 expression in 4T1 cells treated with PBS + laser, 120 μg / mL MATRA probe, and 120 μg / mL MATRA probe + laser (1064 nm laser, 1.0 W / cm2, 5 min) followed by co-incubation with Hoechst 33342 (nucleus, blue) and Alexa Fluor488 conjugated goat anti-rabbit secondary antibody (green); (i) Immunofluorescence image after staining; (ii) Fluorescence intensity spectrum along the direction of the arrow;

[0043] Figure 20 (i) Optical photographs and (ii) corresponding SERS images of 4T1 live cells incubated with 120 μg / mL MATRA probe; (iii) SERS spectra acquired from points I, II, and III, as shown in Figure (ii); the SERS images were obtained from a 1340 cm⁻¹ plate. -1 Peak intensity was created at the location;

[0044] Figure 21 This is a schematic diagram of the surgical procedure guided by MR imaging / SERS. SERS detection was performed on micro-tumors on the operating table during the operation, and the surgical results were examined after the operation.

[0045] Figure 22 MR images of 4T1 tumor-bearing mice before (without MATRA probe injection) and before and after MATRA probe injection.

[0046] Figure 23 (i) H&E stained images, (ii) SERS images, and (iii) threshold segmentation integration maps (scale: 2.0 mm) of the operating table before and after surgical resection of the primary tumor. A small bottle of water was placed in the upper left corner as a reference during MRI; A is before resection of the primary tumor; B is after resection of the primary tumor.

[0047] Figure 24 The contrast signal-to-noise ratio (CNR) of the primary tumor location before (before) injection of the MATRA probe and 24 h after (after) injection of the MATRA probe, and the percentage of microtumors (total microtumor area / operating bed area) calculated based on the segmentation map after processing of (ii) H&E stained images and (iii) SERS images (n=22).

[0048] Figure 25 SERS-guided microtumor resection on an operating table in a 4T1 breast cancer mouse model after intravenous injection of MATRA probe; (i) Raman device for SERS-guided surgery, (ii) 4T1 breast cancer mouse, (iii) optical photograph of the primary tumor, and (iv) superposition of in vivo SERS image of the primary tumor with its optical photograph.

[0049] Figure 26 SERS-guided stepwise surgical resection of primary tumors; A shows (i) optical photographs of the operating table before and (ii) after the first resection, (iii) H&E staining image (left) and SERS image (right) of the resected tumor, (iv) magnified H&E staining image of the resected tumor region shown in (iii), and (v) SERS image of the operating table after the first resection and superimposed image of the first resected tumor; B shows (i) optical photographs of the operating table after the second resection, (ii) H&E staining image (left) and SERS image (right) of the second resected tumor, (iii) magnified H&E staining image of the second resected tumor region shown in (ii), (iv) SERS image of the operating table after the second resection and superimposed image of the second resected tumor, and (v) optical photographs of mice after surgery;

[0050] Figure 27H&E staining images of the entire tissue (left), internal boundary region I (middle), and region II (right) on the operating table after surgery;

[0051] Figure 28 Following intravenous injection of the MATRA probe and SERS-guided surgical resection of the primary tumor, the tumor was irradiated with a 1064nm laser (1.0W / cm²). 2 (i) Infrared thermographic image and (ii) temperature change image of the operating table position of 4T1 breast cancer-bearing mice at 5 min;

[0052] Figure 29 The study aimed to determine the biodistribution of the MATRA probe in the major organs (heart, liver, spleen, lung, and kidney) and tumors of 4T1 tumor-bearing mice after intravenous administration.

[0053] Figure 30 Optical photographs of 4T1 tumor-bearing mice after receiving various treatments (#1: control group; #2: surgery; #3: surgery + PD-1; #4: surgery + laser; #5: surgery + laser + PD-1);

[0054] Figure 31 The average tumor volume of the primary and distal tumors in each group of mice;

[0055] Figure 32 The recurrence rate of primary tumors in each treatment group;

[0056] Figure 33 Kaplan-Meier survival curves of 4T1 tumor-bearing mice after different treatments;

[0057] Figure 34 Tumor growth curves for (i) primary tumors and (ii) distant metastases in mice after different treatments;

[0058] Figure 35 The curves showing the changes in body weight of 4T1 tumor-bearing mice after different treatments;

[0059] Figure 36 H&E stained images of tissue sections from major organs of 4T1 tumor-bearing mice after different treatments;

[0060] Figure 37 Serum biochemical analysis of 4T1 tumor-bearing mice treated with PBS and MATRA probe, respectively; A: ALB; B: ALP; C: ALT; D: AST; E: BUN; F: CREA;

[0061] Figure 38(i) Flow cytometry analysis of CD4+ T cells and CD8+ T cells in spleen tissue (n=4); (ii) Immunofluorescence analysis of distant metastases in 4T1 tumor-bearing mice after different treatments; (iii) Quantitative results of CD4+ T cells and CD8+ T cells in spleen tissue and distant metastases. Figure 38 B represents the serum levels of (i) TNF-α and (ii) IFN-γ in 4T1 tumor-bearing mice that received different treatments (n=3);

[0062] Figure 39 For 4T1 tumor-bearing mice 31 days after treatment, the following images were obtained: (i) optical photographs of lung tissue stained with Indian ink, (ii) H&E-stained images of corresponding lung sections, and (iii) quantitative statistics of lung metastatic nodules (n=3). Detailed Implementation

[0063] To make the present invention easier to understand, specific embodiments are described below to further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. 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. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art; unless otherwise specified, the raw materials and reagents involved herein can be purchased commercially or obtained by known methods.

[0064] It should be noted that the experimental materials for this invention were sourced from the following sources:

[0065] Chloroauric acid (HAuCl4-4H2O, 99% Au) was purchased from Shanghai Siyu Chemical Co., Ltd.

[0066] Polyvinylpyrrolidone (PVP, average MW≈10kg / mol) was purchased from Sigma-Aldrich Trading Ltd.

[0067] (3-Mercaptopropyl)trimethoxysilane (MPTMS, 97.0%) and copper sulfate pentahydrate (CuSO4·5H2O, 99%) were purchased from Aladdin Reagent Co., Ltd.

[0068] N,N-Dimethylformamide (DMF, 99.8%), trisodium citrate dihydrate (C6H5Na7·2H2O), L-ascorbic acid (H2Asc, ≥99.7%), Triton X-100, paraformaldehyde, and sodium borohydride (NaBH4, >98.0%) were obtained from Sinopharm Chemical Reagent Co., Ltd.

[0069] Selenium dioxide (SeO2, 99.99%) was ordered from Shandong Xiya Chemical Co., Ltd.

[0070] Ammonia (NH3·H2O, 25-28%, v / v) and p-nitrobenzenethiophenol were ordered from Shanghai Maclean Biochemical Co., Ltd.

[0071] Gadolinium chloride hexahydrate (GdCl3·6H2O, 99.99%) was purchased from Shanghai Xianding Biotechnology Co., Ltd.

[0072] Methoxy polyethylene glycol mercapto (mPEG-SH, average MW≈2kg / mol) was purchased from Hunan Huateng Pharmaceutical Co., Ltd.

[0073] The anti-mouse PD-1 antibody (CD279) was purchased from Bioxcell, Inc., USA.

[0074] The tumor necrosis factor-α (TNF-α) ELISA kit and the interleukin-γ (IFN-γ) ELISA kit were developed by Wuhan Saiweier Biotechnology Co., Ltd.

[0075] CD4 monoclonal antibody (GK1.5), FITC, CD3e monoclonal antibody (145-2C11), APC, CD8a monoclonal antibody (53-6.7), and PE were all purchased from Thermo Fisher Scientific, Inc., USA.

[0076] All chemicals and solvents were used as is, without further purification.

[0077] All experiments used deionized (DI) water (Milli-Q grade, 18.2 MΩ-cm, 25°C).

[0078] It should be noted that the characterization and instrumentation of this invention are as follows:

[0079] The optical extinction spectrum was measured on an Agilent Cary 5000 UV-Vis-NIR spectrophotometer.

[0080] Hydrated particle size and zeta potential were measured using a Malvern Zetasizer Nano ZSE instrument.

[0081] Transmission electron microscopy (TEM) was performed using a FEI Tecnai G2 transmission electron microscope with an accelerating voltage of 200 kV.

[0082] Scanning electron microscopy (SEM) was performed on a Hitachi Regulus 8230 field emission scanning electron microscope.

[0083] Quantitative analysis of gold, copper, and selenium was performed using an Agilent 5100 inductively coupled plasma optical emission spectrometer (ICP-OES) or an Agilent 7900 inductively coupled plasma mass spectrometer (ICP-MS).

[0084] Infrared thermal imaging analysis was performed on a FLIR A65 system.

[0085] Magnetic resonance (MR) imaging analysis was performed on a clinical 3.0-T Siemens Magnetom Skyra scanner.

[0086] Fluorescence images and laser confocal images were acquired using a Zeiss Axio vert. A1 fluorescence microscope and an LSM880 Airyscan T laser confocal microscope, respectively.

[0087] Flow cytometry sorting analysis was performed on an Agilent NovoCyte 2040 flow cytometer.

[0088] All SERS measurements were performed on a Renishaw inViaQontor confocal Raman microscope equipped with a Leica DM2700M Ren RL / TL microscope. The excitation source was a 785nm diode laser (maximum output: 300mW), the detector was an infrared-enhanced CCD, and the grating was a high-resolution grating (1200 lines / mm). All SERS spectra were processed and background corrected using Renishaw WiRE 5.3 software, and all SERS images were generated using direct classical least squares calculations within the software.

[0089] It should be noted that the specific surgical procedure mainly includes: firstly, using the MATRA probe for preoperative MR imaging for surgical planning and SERS detection to identify the tumor; then, utilizing the high sensitivity of SERS for real-time detection, which can display the tumor boundary and micro-tumor lesions on the operating table in real time; and finally, combining the near-infrared II photothermal effect of the MATRA probe with programmed death-1 antibody for adjuvant immunotherapy after surgery. Figure 1 BD)

[0090] Example 1

[0091] A medical multifunctional optical probe

[0092] The MATRA probe is primarily composed of gold nanostars (AuSts) intercalated with Gd encoded by p-nitrothiophenol (p-NTP). 3+ In the magnetic silica shell, the shell is further modified with polyethylene glycol to form Au@Cu exhibiting near-infrared II plasmon resonance. 2-x Se(GCS) core-shell NPs modification ( Figure 1 A).

[0093] Example 2

[0094] A method for preparing a medical multifunctional optical probe:

[0095] Preparation of AuSts: First, PVP-modified gold seeds were synthesized. 2 mL of 38.8 mM C6H5Na7·2H2O (stabilizer) solution was rapidly added to 90 mL of 0.27 mM HAuCl4·4H2O solution. Then, 1 mL of freshly prepared NaBH4 (0.075 wt%) solution was added dropwise to the mixture, and the reaction was continued for 12 h. 0.94 mM PVP (K30) was added to the solution, and the mixture was stirred continuously for 24 h to obtain the PVP-modified gold seed solution. Then, 1 mM HAuCl4·4H2O was added to a DMF solution containing 10 mM PVP (K15), followed by 1.66 pM of gold seeds. After reacting at room temperature for 3 h, a deep blue solution was obtained, indicating successful preparation of AuSts. The AuSts were washed at least twice consecutively with ultrapure water and ethanol, and finally stored in ultrapure water at a concentration of 1.2 nM for later use.

[0096] Preparation of AuSt@p-NTP@SiO2 nanoparticles: Freshly prepared p-NTP solution (10 μM) was added to 0.8 nM AuSts and stirred at room temperature for 30 min. Then, 20 μL MPTMS and 140 μL NH3·H2O were added to grow the SiO2 shell. The reaction mixture was stirred at high speed for 3 min and then allowed to stand at 30 °C for 12 h. Finally, purified AuSt@p-NTP@SiO2 nanoparticles were obtained by centrifugation at 4500 rpm and washing with ultrapure water.

[0097] Gd 3+ Preparation of -SERS nanoparticles: First, a storage solution was prepared by uniformly mixing 100 μL of C6H5Na7·2H2O solution (1M), 50 μL of GdCl3·6H2O solution (1M), and 150 μL of NH3·H2O solution (1.5M). Then, 40 μL of the storage solution was added to 10 mL of AuSt@p-NTP@SiO2 nanoparticle solution (200 pM). After continuous stirring in a water bath at 40 °C for 24 h, the mixture was further purified by washing twice with ethanol and once with water to obtain Gd-doped Gd composites. 3+ -SERS nanoparticles.

[0098] Au@Gd 3+ Preparation of -SERS nanoparticles: 100 pM Gd 3+The SERS nanoparticle solution was mixed with 5 mL of HAuCl4 solution (0.1 mM), and the pH of the mixture was adjusted to 9.0 with 0.01 M NaOH. After stirring for 15 min, 600 μL of NaBH4 solution (10 mM) was added. The color of the mixture rapidly changed from pale blue to purple. Stirring was continued for 6 h, and the mixture was centrifuged at 4500 rpm for 10 min. After washing three times, Au@Gd was obtained. 3+ -SERS nanoparticles.

[0099] GCS@Gd 3+ Preparation of -SERS nanoparticles: First, 150 pM Au@Gd 3+ -SERS nanoparticles were dispersed in 5 mL of PVP (K15) solution (5 mg / mL), followed by the addition of SeO2 solutions of different concentrations (0.5 mM, 1 mM, and 2 mM). After stirring for 10 min, 250 μL of ascorbic acid solution (0.1 M) was added. After reacting for 15 min, a mixture of CuSO4·5H2O solution of corresponding concentrations (1 mM, 2 mM, and 4 mM) and 500 μL of ascorbic acid solution (0.1 M) was added sequentially, and the reaction was continued for 24 h. Finally, purified GCS@Gd was obtained by centrifugation at 4000 rpm for 10 min and washing three times with water. 3+ -SERS NPs. GCS@Gd 3+ -SERSNPs were dispersed in 1 mL of 95% ethanol, and then 10 mg of mPEG-SH was added. After stirring continuously for 24 h, the mixture was centrifuged and washed three times with ultrapure water to obtain polyethylene glycol-alcoholized GCS@Gd. 3+ -SERS nanoparticles (hereinafter referred to as MATRA probes) were synthesized, and the resulting 1 mg / mL MATRA probe was stored in ultrapure water for subsequent experiments. The synthesis process of the MATRA probe is as follows: Figure 2 As shown.

[0100] The prepared MATRA probe was subjected to microscopic detection. For example... Figure 3 and Figure 4 TEM and STEM images show that GCS@Gd 3+ -SERS NPs consist of a GCS-modified silica shell enclosing an AuSt core, indicating a distinct core-shell structure. Energy-dispersive X-ray spectroscopy (EDX) Figure 4 This further validates GCS@Gd 3+ -SERS NPs have a core-shell structure. GCS@Gd 3+ The synthesis of -SERS NPs was further verified by extinction spectroscopy. The synthesized AuSt plasmon resonance wavelength was 742 nm, while Gd 3+-SERS NPs redshifted to 776nm ( Figure 5 In addition to the AuSt-related plasmon resonance bands, Au@Gd is also present due to the deposition of Au NPs on the silica surface. 3+ -SERS NPs exhibited a novel plasmon absorption band at 559 nm. This was observed in GCS@Gd synthesized from 1.0 mM SeO2. 3+ In the extinction spectrum of -SERS NPs, a strong near-infrared II plasmon resonance band is clearly visible at 1051 nm, which is due to the plasmon resonance absorption of GCS NPs. We also observed that as the SeO2 concentration increased from 0.5 mM to 2.0 mM, the plasmon resonance absorption of the near-infrared II plasmon resonance band increased. Figure 6 GCS@Gd 3+ Powder X-ray diffraction (XRD) patterns of GCS NPs showed that the 2θ peaks of the cubic structure of GdSeCo (JCPDS no. 1) were located at 26.7°, 44.6°, 52.9°, and 65.0°, while the 2θ peaks of the gold nanocubic structure were located at 38.3° and 77.5° (JCPDS no. 01-1172), proving that GCS NPs were successfully modified on GdSeCo. 3+ -SERS NPs on ( Figure 7 In MATRA probes, as the loading of GCS NPs increases, the characteristic XRD peaks of the GCS component become more dominant than those of the Au component. Figure 8 The changes in hydration particle size and zeta potential measurements further support the successful preparation of the MATRA probe. Figure 9 ).

[0101] Due to GCS@Gd 3+ -SERS NPs exhibited strong near-infrared II plasmon absorption, and we further investigated their near-infrared II photothermal conversion capability. Under 1064nm laser irradiation (1.0W / cm²), [the following was observed]: 2 Below, we can clearly see that all GCS@Gd 3+ -SERS NPs all exhibited a concentration-dependent increase in temperature (ΔT) over 10 minutes. Figure 10 and Figure 11 It is worth noting that GCS@Gd synthesized at a concentration of 400 μg / mL using 0.5, 1.0, and 2.0 mM SeO2 3+ The maximum ΔT values ​​for the aqueous solutions of -SERS NPs were 42.5, 43.5, and 44.5 °C, respectively, while the ΔT value for the PBS buffer solution (approximately 2 °C) was negligible. GCS@Gd synthesized with 0.5 mM, 1.0 mM, and 2.0 mM SeO2... 3+The near-infrared II photothermal conversion efficiencies (η) of the -SERS NPs were 55.2%, 64.7%, and 68.4%, respectively. Figure 12 Furthermore, during five consecutive on / off laser cycles, all GCS@Gd 3+ -SERS NPs steady-state temperature changes are negligible. Figure 11 These results demonstrate that GCS@Gd 3+ -SERS NPs have good near-infrared II photothermal conversion capability and photostability.

[0102] Then, we tested GCS@Gd synthesized with different SeO2 concentrations (0.5, 1.0, and 2.0 mM). 3+ Near-infrared SERS properties of -SERS NPs. Preparation of GCS@Gd NPs with different concentrations. 3+ - An agarose model of SERS NPs (6.25–400 μg / mL) was used for SERS testing under near-infrared 785 nm laser light. Figure 13 and Figure 14 These agarose models all exhibited characteristic p-NTP Raman peaks, and their SERS images showed changes with GCS@Gd. 3+ - The concentration of SERS NPs increased, resulting in brighter colors. Notably, we found that GCS@Gd... 3+ In -SERS NPs, the SERS intensity decreases with increasing GCS load. This is due to the optical shielding effect caused by the strong optical absorption of the GCS component at the laser wavelength. Figure 13 and Figure 14 Additionally, GCS@Gd synthesized with 0.5, 1.0, and 2.0 mM SeO2 3+ The limits of detection for -SERS NPs were 2.39, 4.26, and 6.84 μg / mL, respectively. Therefore, GCS@Gd synthesized using 1.0 mM SeO2... 3+ -SERS NPs were used as MATRA probes for subsequent experiments, and their Gd content was determined by inductively coupled plasma optical emission spectrometry (ICP-OES). 3+ The loading was 20%. We observed that after culturing in 10% fetal bovine serum or cell culture medium (Dulbecco modified Eagle's medium, DMEM), the hydration size of the MATRA probe did not change significantly, and the SERS intensity did not decrease significantly after continuous irradiation with a 785 nm laser (9.2 mW power) for 1 h. Figure 15 These results further demonstrate that the probe possesses good colloidal and photostability. Due to Gd... 3+It is the most widely used T1-weighted MR imaging contrast agent in clinical practice, and we evaluated the MR performance of the MATRA probe. For example... Figure 16 As shown, the T1-weighted MR image becomes brighter with increasing MATRA probe concentration. This is further demonstrated by adjusting the relaxation time and Gd... 3+ Concentration ([Gd) 3+ The relaxation efficiency r1 is calculated using linear regression of [Gd], and the formula is: 1 / T1=r1×[Gd] 3+ ]+b, where T1 is the relaxation time, r1 is the relaxation efficiency, and b is the intercept. The measured relaxation rate r1 is 11.02 mM. -1 ·s -1 It is significantly higher than the commercial Gd-DOTA by approximately 3.11mM. -1 ·s -1 (1.41T). In summary, these results indicate that the MATRA probe is a multifunctional contrast agent that can be used for MR imaging, SERS ultrasensitive detection, and near-infrared II photothermal ablation.

[0103] In vitro immunostimulation and SERS delimitation of live cells. Given the excellent near-infrared II photothermal conversion capability of our MATRA probe, we performed a CCK-8 assay to evaluate its cytotoxicity and photothermal killing ability against 4T1 cells. Results are as follows: Figure 17 As shown in (i), 4T1 cells were incubated for 12 hours at a MATRA probe concentration as high as 400 μg / mL, and the cell viability remained above 92%. However, when the MATRA probe was irradiated with a 1064 nm laser (1.0 W / cm²), the cell viability decreased. 2 At 5 min, the cell viability of 4T1 cells decreased in a dose-dependent manner. When the MATRA probe concentration was 400 μg / mL, the survival rate of the treated 4T1 cells was only 24%, indicating that the MATRA probe has excellent photothermal killing ability. Similar results were also observed in the live / dead cell co-staining experiment. Figure 17 (ii)).

[0104] Photothermal treatment can induce immunogenic cell death (ICD) in cancer cells and release damage-associated molecular patterns (DAMPs), subsequently activating the immune system. Therefore, we then used a MATRA probe combined with 1064nm laser irradiation (1.0W / cm²). 2 4T1 cells were treated with PBS (1064 nm laser irradiation) for 5 min to assess the expression of two key DAMPs: calreticulin (CRT) and high-mobility group box 1 (HMGB1). PBS with 1064 nm laser irradiation or MATRA probe treatment served as controls. Immunofluorescence images showed that CRT expression was highest in the plasma membrane of 4T1 cells treated with MATRA probe and laser, while CRT expression was negligible in the control group. Figure 18 Similarly, we can clearly see that after combined treatment with MATRA probe and laser irradiation, the expression of HMGB1 in the nuclei of 4T1 cells was the lowest, indicating that the secretion of HMGB1 was the highest in the MATRA probe + laser group. Figure 19 Compared with PBS+laser or MATRA probe therapy, the above results indicate that MATRA probe combined therapy can induce a stronger ICD effect in 4T1 cells.

[0105] To investigate the targeted SERS imaging capability of the MATRA probe on live tumor cells, 4T1 cells were incubated with 120 μg / mL MATRA probe for 12 h, then excess MATRA probe was washed away with PBS, followed by SERS testing. Figure 20 As shown, SERS images of live cells clearly depict the cell's outline. Furthermore, strong p-NTP-characteristic SERS signals are observed intracellularly, while extracellular SERS signals are negligible. Therefore, MATRA probes can detect cancer cells with high sensitivity and specificity, demonstrating their strong potential for accurately delineating tumor boundaries and identifying microtumors on the operating table.

[0106] Image-guided surgical resection of the primary tumor. After validating the excellent SERS detection performance of the MATRA probe at the cellular level, we further explored its potential to guide surgical resection of the primary tumor in a 4T1 breast cancer mouse model. Figure 21 We injected the MATRA probe into 4T1 tumor-bearing mice via the tail vein, and performed whole-body MR imaging and in vivo SERS imaging one day after injection. For ease of comparison, we also captured MR images of 4T1 tumor-bearing mice before MATRA probe injection. Figure 22 As shown, compared with the group without MATRA probe injection, the tumor was more clearly displayed in the MR images after MATRA probe injection, indicating enhanced tumor localization ability after MATRA probe treatment. Further, after visually guided surgical resection, no obvious tumor lesions were shown in the operating table MR images. To verify whether the tumor lesions were completely removed surgically, we performed H&E staining and SERS analysis on the operating table tissues of 4T1 tumor-bearing mice (n=22). Figure 24 Representative operating table H&E stained images and operating table SERS images after resection of the primary tumor. Figure 23 (A) Multiple micro-tumor foci can be clearly seen within the operating table, and Figure 23 (B) and Figure 26 , 27 No microtumor lesions were observed within the operating table. We observed that after treatment with the MATRA probe, the MR contrast signal-to-noise ratio (CNR) at the tumor site increased by 2.5 times. Figure 25(i)). H&E staining analysis showed that 77% of mice had tumor-positive tissue after surgery, with a mean microtumor area percentage of 4.3% (±3.5%), defined as the tumor-positive area divided by the total operating table area. Figure 24 (ii) Strong MATRA probe SERS signal characteristics were clearly observed in these tumor-positive sites, while no obvious signal was observed in tumor-negative sites on the operating table, which is consistent with the H&E analysis results. Threshold segmentation was used to distinguish the SERS characteristics of tumor-positive and tumor-negative sites, i.e., a SERS peak intensity of 1340 cm⁻¹ was established. -1 (υ(NO2)) segmentation diagram ( Figure 23 (A) and (B)). Quantitative analysis of the segmentation map showed that approximately 6.7% (±5.0)% of the total operating table area was tumor-positive. Figure 24 (iii) Compared with H&E analysis, SERS measured a much higher percentage of tumor-positive area, thanks to the high sensitivity of MATRA probe SERS detection. Therefore, we demonstrate that SERS detection using MATRA probes can more sensitively detect microtumors on the operating table, thereby improving surgical outcomes through more complete removal of microtumors.

[0107] Next, the primary tumor is surgically removed under image guidance, such as... Figure 25 As shown. First, 24 hours after intravenous injection of the MATRA probe, 4T1 tumor-bearing mice were anesthetized, and whole-body MR imaging was performed to locate the tumor, followed by in vivo SERS imaging to delineate the tumor boundaries. Figure 25 As shown, the SERS images clearly depicted the contour of the primary tumor, highlighting its edges. Strong SERS signals were observed within the tumor, while the signals outside the tumor were weak and negligible. Guided by the SERS images, the primary tumor was then surgically removed. Intraoperatively, we acquired SERS images of the operating table and the removed tumor tissue after each step of the resection, and correlated them with the optical images. Figure 26 After each resection, the SERS images from the operating table clearly depicted the outline of the residual tumor, and the SERS images of the resected tumor also matched its visual shape. Further H&E staining analysis of the resected tissue confirmed that it was cancerous tissue with no obvious normal tissue present. Macroscopic observation of the operating table revealed no residual tumor lesions, but the SERS images from the operating table showed obvious microtumor lesions. Furthermore, histological analysis also confirmed the presence of microtumors at the tumor boundary due to tumor cell infiltration into adjacent normal tissue. Figure 27 Therefore, we demonstrate that intraoperative SERS detection using the MATRA probe can guide real-time surgical resection and identify micro-tumor lesions invisible to the naked eye and clinical MR imaging.

[0108] Image-guided surgical resection and postoperative immunotherapy were evaluated in vivo. The results clearly indicate that multiple residual microtumor lesions remained on the operating table after SERS-guided surgical resection, a fact confirmed by intraoperative real-time SERS monitoring. However, surgical resection did not treat distant metastases. In recent years, tumor immunotherapy has demonstrated significant potential in cancer treatment. Therefore, our next step is to investigate whether combining image-guided surgery with postoperative adjuvant immunotherapy can improve treatment outcomes and eliminate local residual microtumors and distant metastases. Anti-PD-1 immunotherapy has proven to be a novel and promising strategy for treating various cancers, significantly improving the treatment outcomes of metastatic cancers. To evaluate the in vivo therapeutic effect, 4T1 tumor-bearing mice were randomly divided into 5 groups (n=6) and intravenously injected with: (1) 200 μL PBS (control group), (2) SERS-guided cancer surgery (surgery group), (3) SERS-guided cancer surgery plus PD-1 antibody immunotherapy (surgery + PD-1 group), (4) SERS-guided cancer surgery plus laser irradiation (surgery + laser group), and (5) SERS-guided cancer surgery plus laser irradiation plus PD-1 antibody immunotherapy (surgery + laser + PD-1 group). 4T1 tumor-bearing mice were intravenously injected with PBS (200 μL) or MATRA probe (200 μL, 2.4 mg / mL). Whole-body magnetic resonance imaging and in vivo SERS imaging were performed 24 h after injection (day 0) to guide surgical resection of the primary tumor. After surgical resection, the operating table was continuously exposed to a 1064 nm laser at 1.0 W / cm² for 5 min. As can be seen, the operating table temperature in the MATRA probe + laser group gradually increased to ~42.5℃ within 5 minutes, which is much higher than the 38.6℃ in the PBS + laser group. Figure 28 This demonstrates the excellent in vivo photothermal effect of the MATRA probe. Subsequently, 4T1 cells were inoculated into the right abdomen of treated mice to establish a mouse model of distant metastatic tumors, and PD-1 antibody was injected three times consecutively on days 1, 4, and 8 postoperatively for adjuvant immunotherapy. Biodistribution analysis of major organs (heart, liver, spleen, lung, and kidney) and tumors showed that the injected MATRA probe had a high accumulation rate in tumor tissue, approximately 5.3%, only lower than that in the liver and spleen, indicating that it can effectively accumulate in tumors. Figure 29 ).

[0109] According to the results ( Figure 30 and Figure 34Surgical treatment can effectively remove the primary tumor, although minor recurrence may occur over time. Combined treatment with surgical resection, laser irradiation, and PD-1 antibody immunotherapy can completely remove the primary tumor, inhibit the growth of distant metastases, and result in no significant local recurrence post-surgery. Its therapeutic effect is superior to other groups. The inhibition rates of the surgery + laser + PD-1 group on the primary tumor and distant metastases were 100% and 80%, respectively, while the inhibition rates of the surgery + PD-1 group on the primary tumor and distant metastases were only 50% and 0%, respectively, and the surgery + laser group was 33.2% and 0%, respectively. Figure 32 Kaplan-Meier survival curves showed that all mice in the surgery + laser + PD-1 group survived the 31-day treatment period, while the average survival rates of the control group, surgery group, surgery + PD-1 group, and surgery + laser group during this period were approximately 0%, 50%, 66.7%, and 66.7%, respectively. Figure 33 The above results indicate that anti-PD-1 adjuvant immunotherapy and laser irradiation play an important role in inhibiting local tumor recurrence and distant metastatic tumor growth. Furthermore, the body weight of mice in each treatment group did not change significantly, confirming the good biocompatibility of the MATRA probe. Figure 35 H&E histological analysis of major organ sections from mice in each treatment group showed virtually no histological abnormalities, indicating that the MATRA probe had no significant systemic toxicity. Figure 36 Serum biochemical analysis showed no significant difference between mice treated with PBS and mice treated with the MATRA probe (5 mg / kg). Figure 37 The results indicate that it has no significant adverse effects on liver and kidney function or immune response.

[0110] The impact of immunosuppressive effects on postoperative local residual microtumors and lung metastases. Subsequent studies used flow cytometry to analyze the immune response in spleen tissue. Specifically, on day 15 after various treatments, spleen tissue was collected from 4T1 tumor-bearing mice in each group and analyzed using flow cytometry. Local presentation of tumor antigens in dendritic cells (DCs) triggered an increase in cytotoxic CD8+ T cells and CD4+ T cells. As shown in 38(A), compared with other groups, flow cytometry analysis showed a significant increase in CD8+ T cells and CD4+ T cells in the spleen tissue of the surgery + laser + PD-1 group. Furthermore, immunofluorescence staining also showed a significant increase in CD8+ T cells and CD4+ T cells in the distant metastatic tumor tissue of the surgery + laser + PD-1 group, significantly higher than in other groups (38(A)). These results confirm that injection of the MATRA probe followed by near-infrared II laser irradiation and PD-1 immunotherapy can effectively trigger an immune response, thereby effectively inhibiting postoperative local tumor recurrence and distant metastasis. We further found that the serum levels of multiple cytokines, including tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), were significantly increased in the surgery + laser, surgery + PD-1, and surgery + laser + PD-1 groups of 4T1 tumor-bearing mice (39(B)). The surgery + laser + PD-1 group showed the highest expression of TNF-α and IFN-γ, which is attributed to the more effective activation of the anti-cancer immune system by the combined treatment of surgical resection, near-infrared photothermal ablation, and anti-PD-1 immunotherapy. In addition, we observed lung metastasis in 4T1 tumor-bearing mice after different treatments. Lung tissue was collected on day 31 after treatment and stained with Indian ink. The number of metastatic nodules was as follows: control group > surgery > surgery + laser > surgery + PD-1 > surgery + laser + PD-1. No obvious metastatic nodules were observed in the surgery + laser + PD-1 group, which was further confirmed by H&E histological analysis. Figure 39 Therefore, we believe that combined treatment with surgical resection, near-infrared photothermal ablation, and anti-PD-1 immunotherapy can effectively inhibit postoperative lung metastasis of tumors.

[0111] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A medical multifunctional optical probe, characterized in that, The multifunctional optical probe is a Raman-encoded gold nanostar with Gd-doped core. 3+ Au@Cu with near-infrared II plasmon resonance effect, further modified with polyethylene glycol on the surface of the magnetic silica shell. 2-x Se core-shell nanoparticle modification; The preparation method of the medical multifunctional optical probe includes the following steps: Step 1: React HAuCl4 aqueous solution with NaBH4 in a system with surfactant in a redox reaction and let stand to obtain gold nano-seed solution; Step 2: Mix the HAuCl4 aqueous solution with the PVP and DMF solutions evenly, add the gold nanoseed solution, and after purification, obtain the gold nanostar solution; Step 3: Add specific Raman molecules to the gold nanostar solution and add MPTMS under alkaline conditions to hydrolyze it, grow a SiO2 shell on the surface of the gold nanostar, and after purification, obtain Raman molecule labeled AuSt@SiO2 nanoparticles. Step 4: Add an alkaline mixed solution of trisodium citrate dihydrate and GdCl3·6H2O to the Raman-labeled AuSt@SiO2 nanoparticles to form a Gd-doped complex. 3+ -SERS nanoparticles; Step 5: Reconcile HAuCl4 aqueous solution with NaBH4 in Gd 3+ -SERS nanoparticles undergo redox reactions on their surface to obtain Au@Gd 3+ -SERS nanoparticles; Step 6: Add SeO2 solution and reducing agent to Au@Gd 3+ Elemental Se was deposited on the surface of gold spheres containing SERS nanoparticles, followed by the addition of CuSO4·5H2O and a reducing agent to obtain Au@Cu. 2-x Se@Gd 3+ -SERS nanoparticles; Step 7: Add Au@Cu 2-x Se@Gd 3+ -SERS nanoparticles were dispersed in a solvent, and then methoxy polyethylene glycol-mercapto groups were added. After stirring, centrifugation, and purification, polyethylene glycol-alcoholized Au@Cu was obtained. 2-x Se@Gd 3+ -SERS nanoparticles, also known as multifunctional optical probes.

2. A method for preparing a medical multifunctional optical probe as described in claim 1, characterized in that, Includes the following steps: Step 1: React HAuCl4 aqueous solution with NaBH4 in a system with surfactant in a redox reaction and let stand to obtain gold nano-seed solution; Step 2: Mix the HAuCl4 aqueous solution with the PVP and DMF solutions evenly, add the gold nanoseed solution, and after purification, obtain the gold nanostar solution; Step 3: Add specific Raman molecules to the gold nanostar solution and add MPTMS under alkaline conditions to hydrolyze it, grow a SiO2 shell on the surface of the gold nanostar, and after purification, obtain Raman molecule labeled AuSt@SiO2 nanoparticles. Step 4: Add an alkaline mixed solution of C6H5Na7·2H2O and GdCl3·6H2O to the Raman-labeled AuSt@SiO2 nanoparticles to form a Gd-doped Gd complex. 3+ -SERS nanoparticles; Step 5: Reconcile HAuCl4 aqueous solution with NaBH4 in Gd 3+ -SERS nanoparticles undergo redox reactions on their surface to obtain Au@Gd 3+ -SERS nanoparticles; Step 6: Add SeO2 solution and reducing agent to Au@Gd 3+ Elemental Se was deposited on the surface of gold spheres containing SERS nanoparticles, followed by the addition of CuSO4·5H2O and a reducing agent to obtain Au@Cu. 2-x Se@Gd 3+ -SERS nanoparticles; Step 7: Add Au@Cu 2-x Se@Gd 3+ -SERS nanoparticles were dispersed in a solvent, and then methoxy polyethylene glycol-mercapto groups were added. After stirring, centrifugation, and purification, polyethylene glycol-alcoholized Au@Cu was obtained. 2-x Se@Gd 3+ -SERS nanoparticles, also known as multifunctional optical probes.

3. The method for preparing the medical multifunctional optical probe according to claim 2, characterized in that, In step 1, the surfactant is polyvinylpyrrolidone.

4. The method for preparing the medical multifunctional optical probe according to claim 2, characterized in that, In step 3, the specific Raman molecule is p-nitrothiophenol.

5. The method for preparing the medical multifunctional optical probe according to claim 2, characterized in that, In step 6, the reducing agent is ascorbic acid.

6. The application of a medical multifunctional optical probe as described in claim 1 or a medical multifunctional optical probe prepared by any of the preparation methods described in claims 2-5 in the preparation of multifunctional imaging contrast agents for tumor surgical navigation.

7. The application according to claim 6, characterized in that, The tumor surgical navigation includes: preoperative planning using probe MR / SERS imaging to determine the size, location, and boundaries of the tumor; intraoperative SERS imaging using probe to detect and locate the tumor boundaries and micro-tumor lesions on the operating table in real time; and postoperative immunotherapy combining the near-infrared II photothermal effect of probe and programmed death-1 antibody to eliminate residual micro-tumors and distant metastatic tumors.

Images