Near-infrared-ii chemiluminescence resonance energy transfer nanosystem, preparation method therefor, and use thereof

Abstract

The present invention provides a near-infrared-II (NIR-II, 1000-1700 nm) chemiluminescence resonance energy transfer (CRET) nanosystem, a preparation method therefor, and a use thereof. The NIR-II CRET nanosystem is constructed by means of multi-component synergistic self-assembly of a pH-responsive amphiphilic short peptide, a metal ion, an NIR-II CRET functional unit, and a photosensitizer, and has an NIR-II CRET signal activated by responding to both pH and MPO enzyme in a microenvironment. The NIR-II CRET nanosystem can be used as a diagnostic reagent to achieve accurate tracing and localization of a sentinel lymph node of breast cancer by means of an NIR-II contrast agent. The nature of the sentinel lymph node is determined by means of the NIR-II CRET signal activated by responding to both pH and MPO enzyme, thereby overcoming the technical problem that it is difficult for conventional sentinel lymph node diagnosis methods to avoid false negatives and false positives, and achieving in-vitro localization and qualitative integrated diagnosis and treatment of a metastatic sentinel lymph node of breast cancer.

Description

A near-infrared II chemiluminescence resonance energy transfer nanosystem, its preparation method and application

[0001] This application claims priority to Chinese invention patent application No. CN 202410948618.4, filed on July 16, 2024, entitled “A near-infrared II region chemiluminescence resonance energy transfer nanosystem and its preparation method and application”, which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of optical detection technology, specifically to a near-infrared II chemiluminescence resonance energy transfer nanosystem, its preparation method, and its application. Background Technology

[0003] Breast cancer is one of the most significant public health problems threatening women's health. Metastasis is a leading cause of death in clinical patients with breast cancer; therefore, early detection and effective intervention are crucial for improving treatment outcomes and enhancing patients' quality of life. Clinical studies have shown that breast cancer cells preferentially spread via the lymphatic system. Sentinel lymph nodes are considered the first sites reached by breast cancer cells after metastasis. Therefore, accurate identification of metastatic sentinel lymph nodes is essential for clinical breast cancer classification and staging, and is of significant clinical importance in guiding patient treatment.

[0004] Sentinel lymph node biopsy is currently the most common method for diagnosing sentinel lymph nodes in breast cancer. It involves obtaining a solid tumor tissue sample through surgery or puncture, followed by in vitro processing and pathological staining evaluation. While this method has benefited many patients, it still has limitations: firstly, partial tissue extraction makes it difficult to obtain complete information on sentinel lymph node metastasis; secondly, invasive tissue extraction increases the risk of metastasis; and thirdly, the identification results require a lengthy pathological evaluation process. Therefore, achieving rapid and accurate diagnosis of sentinel lymph nodes in breast cancer is a pressing clinical need.

[0005] Fluorescent molecular imaging technology boasts a series of advantages, including high sensitivity, high specificity, intuitive visualization, and ease of use. Among these, near-infrared II (NIR-II, 1000–1700 nm) fluorescence imaging technology exhibits excellent tissue penetration, spatial resolution, and low tissue autofluorescence, significantly improving the accuracy and sensitivity of cancer diagnosis. The pathological characteristics of the tumor microenvironment have been applied to the development of various tumor tissue-activated fluorescent nanoprobes; however, relying on a single detection indicator is insufficient to ensure accurate results. A multi-indicator synergistic diagnostic strategy is an effective way to improve accuracy.

[0006] Studies have shown that, including breast cancer, a slightly acidic environment is a typical characteristic of solid tumors, regardless of their stage of development. Furthermore, metastatic breast cancer is characterized by a significant proportion of tumor-associated macrophages (TAMs) and neutrophils (TANs); myeloperoxidase (MPO) is an important component of both TAMs and TANs and is closely related to tumor metastasis. Specific recognition of MPO activity signals has become a potential new target for detecting metastatic lymph nodes.

[0007] To address the shortcomings of existing lymph node metastasis identification technologies, this invention provides a near-infrared two-zone CRET technology method based on pH and MPO dual-response activation, and applies it to drug formulations for integrated diagnosis and treatment of sentinel lymph nodes in breast cancer.

[0008] Summary of the Invention

[0009] This invention provides a technical method, namely a near-infrared II chemiluminescence resonance energy transfer nanosystem and its preparation method and application, which aims to achieve rapid and accurate diagnosis and treatment of sentinel lymph nodes in breast cancer.

[0010] To achieve the above objectives, this invention provides a near-infrared II (NIR-II, 1000–1700 nm) chemiluminescent resonance energy transfer nanosystem, which is constructed by multi-component synergistic self-assembly of a pH-responsive amphiphilic short peptide, metal ions, NIR-II CRET functional units, and a photosensitizer; the near-infrared II chemiluminescent resonance energy transfer nanosystem possesses a NIR-II CRET signal activated by both pH and MPO enzymes in the microenvironment.

[0011] Preferably, the pH-responsive amphiphilic short peptide is any one of N-fluorenmethoxycarbonyl-L-histidine (Fmoc-His), N-fluorenmethoxycarbonyl-L-lysine (Fmoc-Lys), N-fluorenmethoxycarbonyl-L-arginine (Fmoc-Arg), N-fluorenmethoxycarbonyl-L-cysteine ​​(Fmoc-Cys), or any combination of two or more of them.

[0012] Preferably, the metal ion is gadolinium ion (Gd). 3+ ), Erbium ions (Er) 3+ ), iron ions (Fe) 3+ ), calcium ions (Ca 2+ ), copper ions (Cu) 2+ ), magnesium ions (Mg 2+ ), zinc ions (Zn) 2+ ), manganese ions (Mn) 2+ One or any combination of two or more of the following.

[0013] Preferably, the NIR-IICRET functional unit comprises a near-infrared II contrast agent and a compound with chemiluminescence capability.

[0014] More preferably, the compound with chemiluminescence capability is luminol or a luminol derivative; the luminol derivative is selected from 4-aminohexyl-N-ethylisoluminol (ABEI) and / or ethylluminol (AHEI).

[0015] Preferably, the near-infrared II contrast agent includes at least one type of contrast agent, including near-infrared quantum dots.

[0016] Preferably, the near-infrared quantum dots are near-infrared quantum dots modified by one or more of mercaptopropionic acid, undecyl acid, glutathione, and lipoic acid.

[0017] More preferably, the near-infrared quantum dot is one or any combination of two or more of Ag2S, Ag2Se, AgTe, Au:Ag2Te, and AgAuSe.

[0018] Preferably, the photosensitizer is one or any combination of two or more of dihydroporphyrin e6 (Ce6), benzoporphyrin derivative (BPD), 2-(1-hexaoxyethyl)-2-devinyl pyrophosphate (HPPH), and phenylporphyrin monocyclic acid A (BPD-MA).

[0019] As another objective, the present invention also provides a method for preparing the aforementioned near-infrared II chemiluminescence resonance energy transfer nanosystem, comprising mixing a pH-responsive amphiphilic short peptide, metal ions, NIR-II CRET functional units and a photosensitizer and then performing multi-component synergistic self-assembly to prepare the near-infrared II chemiluminescence resonance energy transfer nanosystem; the multi-component synergistic self-assembly mechanism includes electrostatic adsorption, metal coordination, hydrophobic interaction, π-π stacking interaction, etc., between the pH-responsive amphiphilic short peptide, metal ions, NIR-II CRET functional units and photosensitizer.

[0020] Specifically, the preparation method includes the following steps:

[0021] (1) Provide NIR-II CRET functional unit solution;

[0022] A near-infrared II contrast agent and a chemiluminescent compound are dissolved in water to obtain a contrast agent solution and a chemiluminescent compound solution, respectively. The contrast agent solution and the chemiluminescent compound solution are mixed and stirred continuously for 30-120 minutes before purification to obtain the preparation method of the NIR-II CRET functional unit.

[0023] (2) Provide amphiphilic short peptide solution, metal ion solution and photosensitizer solution;

[0024] The pH-responsive amphiphilic short peptide was dissolved in water to obtain the amphiphilic short peptide solution.

[0025] The metal ion solution is obtained by dissolving the metal ions in water;

[0026] The photosensitizer is dissolved in an organic solvent to obtain the photosensitizer solution;

[0027] (3) Preparation of NIR-II CRET nanosystem;

[0028] The purified NIR-II CRET functional unit solution from step (1) is mixed with the amphiphilic short peptide solution and the metal ion solution from step (2) and stirred continuously for 10–40 min. Then, the photosensitizer solution prepared in step (2) is added, mixed evenly, the pH is adjusted to neutral, and the mixture is purified and concentrated to obtain the NIR-II CRET nanosystem.

[0029] Preferably, in steps (1) and (4), the purification is ultrafiltration purification.

[0030] Preferably, the method for preparing the chemiluminescent compound solution includes dissolving the chemiluminescent compound in an alkaline solution; the pH of the alkaline solution is 8.0 to 9.0.

[0031] Preferably, the mass ratio of the near-infrared II contrast agent to the chemiluminescent compound is in the range of 1–10:0.1–1.

[0032] Preferably, in step (2), the organic solvent is dimethyl sulfoxide.

[0033] Preferably, the concentration of the amphiphilic short peptide solution is 1–5 mg / mL.

[0034] Preferably, the concentration of the metal ion solution is 5–20 mmol / mL.

[0035] Preferably, the concentration of the photosensitizer solution is 1–10 mg / mL.

[0036] Preferably, in step (3), the NIR-II CRET nanosystem contains the near-infrared II contrast agent, the chemiluminescent compound, the amphiphilic short peptide, the metal ion and the photosensitizer in a mass addition ratio of 1-10:50-100:5-50:50-200:1-5.

[0037] The NIR-IICRET nanosystem has a neutral pH value; under neutral conditions, the average size of the NIR-IICRET nanosystem is 20–500 nm.

[0038] Based on the above technical solutions, this invention constructs an NIR-II CRET nanosystem by mixing a pH-responsive amphiphilic short peptide, metal ions, NIR-II CRET functional units, and a photosensitizer, and then constructing it through multi-component synergistic self-assembly. The multi-component synergistic self-assembly mechanism includes electrostatic adsorption, metal coordination, hydrophobic interactions, and π-π stacking interactions between the pH-responsive amphiphilic short peptide, metal ions, the NIR-II CRET system, and the photosensitizer. Using this as a diagnostic reagent, a universal integrated diagnostic and therapeutic technique for the localization and characterization of sentinel lymph nodes in breast cancer is established, providing a new method for the clinical application of the NIR-II CRET nanosystem in the diagnosis and treatment of sentinel lymph nodes in breast cancer.

[0039] The beneficial technical effects obtained by this invention are as follows:

[0040] (1) This invention integrates dual-mode optics and pH / MPO synergistic activation strategy to achieve accurate localization and diagnosis of metastatic sentinel lymph nodes, effectively avoiding false negative and false positive results.

[0041] (2) Benefiting from the high tissue penetration, high spatiotemporal resolution and high signal-to-noise ratio imaging characteristics of the NIR-II window, the invention of this technology can help to quickly detect metastatic lesions at deeper and earlier levels. Attached Figure Description

[0042] Figure 1 shows transmission electron microscopy images of the NIR-IICRET nanosystem activated by both pH and MPO enzymes prepared in Example 1 of this invention under different pH conditions.

[0043] Figure 2 shows transmission electron microscopy images of the pH and MPO enzyme dual-responsive NIR-IICRET nanosystem prepared in Example 2 of the present invention under different pH environments.

[0044] Figures 3a and 3b show the CRET spectrum and fluorescence quantitative curve of the NIR-IICRET nanosystem of Example 2 of the present invention, respectively, generated in vitro by MPO enzyme catalysis.

[0045] Figure 4 shows the effect of the pH and MPO enzyme dual-responsive activated NIR-IICRET nanosystem prepared in Example 1 of the present invention on in vivo diagnosis of metastatic sentinel lymph nodes in breast cancer.

[0046] Figure 5 shows the results of immunofluorescence staining and hematoxylin-eosin (H&E) staining of metastatic sentinel lymph node tissue and negative sentinel lymph node tissue removed under the guidance of CRET signal. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of this application, but not all embodiments.

[0048] This invention provides a pH- and MPO enzyme-responsive NIR-II CRET nanosystem, comprising a pH-responsive amphiphilic short peptide, a metal ion, an NIR-II CRET functional unit, and a photosensitizer. The nanosystem is prepared through synergistic self-assembly of the aforementioned components via electrostatic adsorption, metal coordination, hydrophobic interactions, and π-π stacking.

[0049] The nanosystem can locate sentinel lymph nodes through near-infrared II fluorescence signals and characterize sentinel lymph nodes by programmed activation of NIR-IICRET signals in response to the pH and MPO enzymes of the breast cancer tumor microenvironment. This overcomes the difficulty of traditional methods in achieving integrated in-situ diagnosis of sentinel lymph node location and characterization, and provides a new method for precise diagnosis and treatment of metastatic sentinel lymph nodes in breast cancer.

[0050] In some specific embodiments, the pH-responsive amphiphilic short peptide is any one of N-fluorenmethoxycarbonyl-L-histidine (Fmoc-His), N-fluorenmethoxycarbonyl-L-lysine (Fmoc-Lys), N-fluorenmethoxycarbonyl-L-arginine (Fmoc-Arg), N-fluorenmethoxycarbonyl-L-cysteine ​​(Fmoc-Cys), and any combination thereof.

[0051] In some specific embodiments, the metal ion is gadolinium ion (Gd). 3+ ), Erbium ions (Er) 3+ ), iron ions (Fe) 3+ ), zinc ions (Zn) 2+ One of or any combination thereof.

[0052] In some specific embodiments, the NIR-IICRET functional unit is loaded with any combination of near-infrared II contrast agents and compounds with chemiluminescence capabilities.

[0053] In some specific embodiments, the photosensitizer is one of dihydroporphyrin e6 (Ce6), benzoporphyrin derivative (BPD), 2-(1-hexaoxyethyl)-2-devinyl pyrophosphate (HPPH), benzoporphyrin monocyclic acid A (BPD-MA), or any combination thereof.

[0054] In some specific embodiments, the nanosystem includes at least one type of contrast agent, including near-infrared quantum dots.

[0055] In some specific embodiments, the near-infrared quantum dot is one of Ag2S, Ag2Se, AgTe, Au:Ag2Te, AgAuSe, or any combination thereof.

[0056] In some specific embodiments, the near-infrared quantum dots are near-infrared quantum dots modified with one or any combination of mercaptopropionic acid, undecyl acid, glutathione, lipoic acid, or mercaptopropionic acid.

[0057] In some specific embodiments, the chemiluminescent compound is one of luminol or its derivatives 4-aminohexyl-N-ethylisoluminol (ABEI) and ethylluminol (AHEI), or any combination thereof.

[0058] On the other hand, the present invention provides a method for preparing a near-infrared II chemiluminescence resonance energy transfer nanosystem activated by both pH and MPO enzymes as described above, comprising the following steps:

[0059] (1) Dissolve the near-infrared II contrast agent and the chemiluminescent compound in water to obtain two solutions;

[0060] (2) Mix the two solutions obtained in (1) and stir continuously for one hour, and purify to remove excess chemiluminescent compounds.

[0061] (3) Dissolve the amphiphilic short peptide and metal ions in water respectively, dissolve the photosensitizer in an organic solvent, and obtain a photosensitizer solution by ultrasound.

[0062] (4) The NIR-IICRET functional unit solution obtained after purification in (2) and the amphiphilic short peptide solution in (3) are mixed with the metal ion solution in (3) and stirred continuously for 30 minutes. Then, the photosensitizer solution in (3) is added, the pH of the above mixed solution is adjusted to neutral, and after purification and concentration, the pH and MPO enzyme dual-response activated nanosystem solution is obtained.

[0063] This invention provides a novel method for integrated diagnosis and treatment of sentinel lymph node localization and characterization in breast cancer using a near-infrared two-zone chemiluminescence resonance energy transfer nanosystem activated by both pH and MPO enzyme responses.

[0064] It should be noted that, unless otherwise specified, all raw materials and chemical reagents used in this invention are commercially available.

[0065] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific embodiments and 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0066] Example 1

[0067] This embodiment provides a method for preparing a near-infrared II chemiluminescence resonance energy transfer nanosystem based on pH and MPO enzyme dual-response activation, the specific steps of which include:

[0068] (1) Dissolve 10 mg of 4-(N-ethyl-N-aminobutylamino)phthalohydrazide (ABEI), 0.5 g of sodium bicarbonate and 200 μL of concentrated ammonia in water to obtain an ABEI solution;

[0069] (2) Dissolve 1 mg of undecyl acid-modified Ag2S quantum dots in water to obtain an aqueous quantum dot solution (average concentration of 0.5 mg / mL);

[0070] (3) Mix the ABEI solution obtained in (1) with the quantum dot solution obtained in (2) at a mass ratio of 100:1 and stir continuously for 1 hour at a stirring speed of 300 rpm.

[0071] (4) The solution obtained in (3) was purified by ultrafiltration three times for 10 minutes each time to remove excess ABEI and obtain purified NIR-II CRET functional unit solution.

[0072] (5) Dissolve 1 mg of fluorenylmethoxycarbonyl lysine (Fmoc-Lys) and 5 mmol of ErCl3 in 1 mL of water to obtain Fmoc-Lys solution and ErCl3 solution respectively; dissolve 1 mg of phenyl phosphorus dichloride (BPD) in 1 mL of DMSO to obtain BPD solution.

[0073] (6) The NIR-II CRET functional unit solution obtained in step (4) and the Fmoc-Lys solution obtained in (5) are mixed with ErCl3 solution at the same time and stirred continuously for 30 minutes at a stirring speed of 300 rpm to obtain a mixed solution.

[0074] (7) Mix the BPD solution prepared in step (5) with the mixed solution prepared in step (6) evenly, divide the solution into two parts, and adjust the pH to pH=7.4 and pH=5.5 respectively. After stirring for 1 hour, ultrafiltration concentration is used to obtain the purified NIR-II CRET nanosystem solution.

[0075] Figure 1 shows the morphological characterization image of the near-infrared II chemiluminescence resonance energy transfer nanosystem provided in this embodiment. The transmission electron microscope image shows that the size of the nanosystem is about 120 nm under pH 7.4 conditions and about 30 nm under pH 5.5 conditions. This indicates that the NIR-II CRET nanosystem was successfully prepared in pH 7.4 environment and depolymerized in pH 5.5 environment.

[0076] Example 2

[0077] This embodiment provides a method for preparing a near-infrared II chemiluminescence resonance energy transfer nanosystem based on pH and MPO enzyme dual-response activation, the specific steps of which include:

[0078] (1) Dissolve 20 mg luminol, 1 g sodium bicarbonate and 100 μL concentrated ammonia in water to obtain a 10 mM luminol solution;

[0079] (2) Dissolve 1 mg of mercaptopropionic acid-modified AgAuSe quantum dots in water to obtain a quantum dot solution of 2 mg / mL;

[0080] (3) Mix the luminol solution obtained in step (1) with the quantum dot solution obtained in step (2) (at an average mass ratio of 50:1) and stir continuously for 1 hour at a stirring speed of 300 rpm.

[0081] (4) The solution obtained in step (3) is purified by ultrafiltration three times for 10 minutes each time to remove excess luminol and obtain purified NIR-II CRET functional unit solution.

[0082] (5) Dissolve 1 mg of fluorenylmethoxycarbonylhistidine (Fmoc-His) and 2 mmol of GdCl3 in 1 mL of water to obtain Fmoc-His solution and GdCl3 solution, respectively. Dissolve 2 mg of dihydroporphyrin e6 (Ce6) in 1 mL of DMSO to obtain the solution.

[0083] (6) Mix the NIR-II CRET functional unit obtained in step (4) and the Fmoc-His solution obtained in step (5) with the GdCl3 solution at the same time, and stir continuously for 30 minutes at a stirring speed of 300 rpm.

[0084] (7) After mixing all the Ce6 solution obtained in step (5) with all the mixed solution in step (6), the mixture is divided into two parts, and the pH is adjusted to pH=7.4 and pH=5.5 respectively. After stirring for 1 hour, the solution is concentrated by ultrafiltration to obtain the purified nanosystem solution.

[0085] Figure 2 shows the morphological characterization image of the near-infrared II region chemiluminescence resonance energy transfer nanosystem provided in this embodiment. The transmission electron microscope image shows that the size of the nanosystem is about 80 nm under pH 7.4 conditions and about 27 nm under pH 5.5 conditions. Furthermore, as shown in the figure, the NIR-IICRET nanosystem was successfully prepared in a pH 7.4 environment and underwent depolymerization in a pH 5.5 environment.

[0086] As shown in Figures 3a and 3b, the CRET spectrum and fluorescence quantitative curve of the near-infrared II chemiluminescence resonance energy transfer nanosystem of this embodiment were plotted in vitro via MPO enzyme catalysis. Figure 3a shows that CRET signals can be generated in vitro via a dual-response catalytic reaction of pH and MPO enzyme. Referring to Figure 3b, the quantitative curves of CRET signals over time under different pH conditions (CRET signal-time quantitative curves) show that a stronger CRET signal is generated at pH 5.5 than at pH 7.4, demonstrating that the nanosystem has a significant pH-dependent MPO enzyme response function.

[0087] Example 3

[0088] The near-infrared II chemiluminescence resonance energy transfer nanosystem obtained in Example 2 of this invention was selected as a diagnostic reagent and applied to the integrated diagnosis and treatment of metastatic sentinel lymph nodes in in vivo breast cancer. The specific steps include:

[0089] (1) A breast cancer lymph node metastasis model was constructed. The nanosystem solution was injected subcutaneously into the paw pads of mice with tumors and normal mice. The mice were imaged in real time using a near-infrared two-zone fluorescence in vivo imager. The changes in the fluorescence signal of the sentinel lymph nodes of the two groups of mice were observed and real-time fluorescence images were acquired. After the imaging was completed, the sentinel lymph node tissue of the mice was taken for histopathological staining analysis.

[0090] (2) Postoperative histopathological staining analysis: The lymph node tissue sections obtained above were subjected to immunofluorescence staining and H&E staining. Images of the stained sections were acquired using an inverted fluorescence microscope and pathological analysis was performed. The above in vivo fluorescence images and histopathological staining results were compared to analyze the consistency between the near-infrared fluorescence signal and NIR-II CRET signal of metastatic lymph nodes and normal lymph nodes and the histopathological diagnosis.

[0091] The analytical methods used in the above steps are all conventional techniques commonly used in existing technologies.

[0092] As shown in Figure 4, the nanosystem can accurately locate sentinel lymph nodes in breast cancer under 808nm laser irradiation. Without laser, the nanosystem successfully identified metastatic sentinel lymph nodes through NIR-II CRET signal, while no obvious NIR-II CRET signal was observed at negative sentinel lymph nodes. This indicates that metastatic sentinel lymph nodes can be accurately detected by NIR-II CRET signal under the dual activation of pH and MPO enzyme.

[0093] As shown in Figure 5, the histological H&E staining results of metastatic sentinel lymph nodes indicate that TAM and TAN fully infiltrate tumor cells, and the immunofluorescence staining images show that MPO enzyme is highly expressed in metastatic sentinel lymph nodes. The results indicate that the nanosystem can achieve the qualitative identification of metastatic sentinel lymph nodes through the CRET signal generated by the dual response catalysis of pH and MPO enzyme.

[0094] Therefore, the above results indicate that the near-infrared II chemiluminescence resonance energy transfer nanosystem provided by the present invention can be used for integrated diagnosis and treatment of sentinel lymph nodes in breast cancer metastasis. It has dual pH and MPO enzyme response functions. After depolymerization under acidic conditions, it outputs NIR-IICRET signals through MPO enzyme catalysis, thereby realizing integrated diagnosis and treatment of localization and characterization of sentinel lymph nodes in breast cancer metastasis at the in vivo level.

[0095] All aspects, embodiments, features, and examples of this invention are to be regarded as illustrative in all respects and are not intended to limit the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0096] The use of titles and descriptions in this invention does not imply limitation of the invention; each part can be applied to any aspect, embodiment, or feature of the invention.

[0097] Throughout this invention, wherever a composition is described as having, containing, or including specific components, or wherever a process is described as having, containing, or including specific process steps, it is contemplated that the compositions taught in this invention are also substantially composed of or comprised of the described components, and that the processes taught in this invention are also substantially composed of or comprised of the described process steps.

[0098] Unless otherwise specifically stated, the use of the terms “include, include, including” or “have, has, or having” should generally be understood as open-ended and non-restrictive.

[0099] It should be understood that the order of the steps or the order in which specific actions are performed is not particularly important, as long as the teachings of this invention remain operable. Furthermore, two or more steps or actions can be performed simultaneously.

[0100] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0101] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated otherwise, any use of the terms first, second, etc., does not indicate any order or importance, but is used to distinguish one element from another.

Claims

1. A near-infrared two-region chemiluminescence resonance energy transfer nanosystem, characterized in that, The pH-responsive amphiphilic short peptide, metal ions, NIR-II CRET functional units and photosensitizers are constructed by multi-component synergistic self-assembly to obtain; The near-infrared two-region chemiluminescence resonance energy transfer nano system has a NIR-II CRET signal activated by a microenvironment pH and MPO enzyme dual-response.

2. The near-infrared two-region chemiluminescence resonance energy transfer nanosystem of claim 1, wherein, The pH-responsive amphiphilic short peptide is any one of N-fluorenylmethoxycarbonyl-L-histidine (Fmoc-His), N-fluorenylmethoxycarbonyl-L-lysine (Fmoc-Lys), N-fluorenylmethoxycarbonyl-L-arginine (Fmoc-Arg), N-fluorenylmethoxycarbonyl-L-cysteine (Fmoc-Cys), and any combination of two or more thereof.

3. The near-infrared two-region chemiluminescence resonance energy transfer nano system of claim 1, wherein The metal ion is one or any combination of two or more of gadolinium ion (Gd 3+ ), erbium ion (Er 3+ ), iron ion (Fe 3+ ), calcium ion (Ca 2+ ), copper ion (Cu 2+ ), magnesium ion (Mg 2+ ), zinc ion (Zn 2+ ), and manganese ion (Mn 2+ ).

4. The near-infrared two-region chemiluminescence resonance energy transfer nanosystem of claim 1, wherein, The NIR-II CRET functional unit comprises a near-infrared two-region contrast agent and a chemiluminescence compound; The chemiluminescence compound is luminol or a luminol derivative; the luminol derivative is selected from 4-aminoheptyl-N-ethylisoluminol (ABEI) and / or ethyl luminol (AHEI).

5. The near-infrared two-region chemiluminescence resonance energy transfer nanosystem of claim 1, wherein, The near-infrared two-region contrast agent includes at least one of a contrast agent including a near-infrared quantum dot.

6. The near-infrared two-region chemiluminescence resonance energy transfer nanosystem of claim 5, wherein, The near-infrared quantum dot is a near-infrared quantum dot modified by one or any combination of two or more of mercaptopropionic acid, undecylenic acid, glutathione, and thioctic acid.

7. The near-infrared two-region chemiluminescence resonance energy transfer nanosystem of claim 5, wherein, The near-infrared quantum dot is one or any combination of two or more of Ag2S, Ag2Se, AgTe, Au:Ag2Te, and AgAuSe.

8. The near-infrared two-region chemiluminescence resonance energy transfer nano system of claim 1, wherein The photosensitizer is one or any combination of two or more of chlorin e6 (Ce6), benzoporphyrin derivative (BPD), 2-(1-hexyloxyethyl)-2-devinyl pyrophosphate (HPPH), and benzoporphyrin monomethyl ether A (BPD-MA).

9. A method of preparing the near-infrared two-region chemiluminescence resonance energy transfer nanosystem according to any one of claims 1-8, characterized in that, The near-infrared two-region chemiluminescence resonance energy transfer nano system is prepared by mixing the pH-responsive amphiphilic short peptide, metal ions, NIR-II CRET functional units, and photosensitizers and then performing multi-component synergistic self-assembly. The multi-component synergistic self-assembly mechanism includes electrostatic adsorption, metal coordination, hydrophobic interaction, and π-π stacking interaction between the pH-responsive amphiphilic short peptide, metal ions, NIR-II CRET functional units, and photosensitizers.

10. The production method according to claim 9, wherein The method comprises the following steps: (1) providing a NIR-II CRET functional unit solution; The near-infrared two-region contrast agent and the chemiluminescence compound are dissolved in water to obtain a contrast agent solution and a chemiluminescence compound solution; The contrast agent solution and the chemiluminescence compound solution are mixed, continuously stirred for 30-120 min, and then purified to obtain the NIR-II CRET functional unit solution; (2) providing an amphiphilic short peptide solution, a metal ion solution, and a photosensitizer solution; dissolving the pH-responsive amphiphilic short peptide in water to obtain the amphiphilic short peptide solution; dissolving the metal ion in water to obtain the metal ion solution; dissolving the photosensitizer in the organic solvent to obtain the photosensitizer solution; (3) preparing the NIR-II CRET nanosystem; mixing the purified NIR-II CRET functional unit solution in step (1) with the amphiphilic short peptide solution and the metal ion solution in step (2), and continuously stirring for 10-40 min; then adding the photosensitizer solution prepared in step (2), mixing uniformly, adjusting the pH to neutral, and purifying and concentrating to obtain the NIR-II CRET nanosystem.

11. The preparation method of claim 10, wherein, in steps (1) and (4), the purification is ultrafiltration purification; the preparation method of the chemiluminescent compound solution comprises dissolving the chemiluminescent compound in an alkaline solution; the pH of the alkaline solution is 8.0-9.0; the mass ratio of the near-infrared second region contrast agent to the chemiluminescent compound is 1-10:0.1-1; in step (2), the organic solvent is dimethyl sulfoxide; the concentration of the amphiphilic short peptide solution is 1-5 mg / mL; the concentration of the metal ion solution is 5-20 mmol / mL; the concentration of the photosensitizer solution is 1-10 mg / mL; in step (3), the NIR-II CRET nanosystem contains the near-infrared second region contrast agent, the chemiluminescent compound, the amphiphilic short peptide, the metal ion, and the photosensitizer, and the mass addition ratio is 1-10:50-100:5-50:50-200:1-5.

12. The method of claim 10, wherein, The average size of the near-infrared second region chemiluminescence resonance energy transfer nanosystem under neutral conditions is 20-500 nm.

13. Use of the near-infrared second region chemiluminescence resonance energy transfer nanosystem of any one of claims 1-8 in the preparation of a breast cancer sentinel lymph node diagnosis and treatment reagent.

14. A diagnosis and treatment reagent for breast cancer sentinel lymph nodes, comprising at least the near-infrared second region chemiluminescence resonance energy transfer nanosystem of any one of claims 1-8, the diagnostic reagent having pH-dependent and MPO enzyme dual response functions, and after depolymerization under acidic conditions, outputting a NIR-II CRET signal through MPO enzyme catalysis to realize integrated diagnosis and treatment of breast cancer metastatic sentinel lymph nodes at the body level.

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