A near-infrared two-region chemiluminescence resonance energy transfer nanosystem, a preparation method and application thereof

Abstract

The application provides a near-infrared two-region (NIR-II, 1000-1700 nm) chemiluminescence resonance energy transfer nanosystem and a preparation method and application thereof; the near-infrared two-region chemiluminescence resonance energy transfer nanosystem is constructed by multi-component synergistic self-assembly of a pH-responsive amphiphilic short peptide, metal ions, a NIR-II CRET system and a photosensitizer, has a NIR-II CRET signal activated by double responses of pH and MPO enzymes in a microenvironment, can be used as a diagnostic reagent to realize accurate tracking and positioning of breast cancer sentinel lymph nodes through a near-infrared two-region contrast agent, realizes the qualitative determination of sentinel lymph nodes through the double-response activated NIR-II CRET signal, and thus overcomes the technical difficulties that the traditional sentinel lymph node diagnostic method cannot avoid false negative and false positive.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical detection technology, in particular to a near-infrared two-region chemiluminescence resonance energy transfer nanosystem and a preparation method and application thereof. BACKGROUND

[0002] Breast cancer is one of the most important public health problems endangering women's health. Metastasis of breast cancer is the main cause of death in clinical patients, therefore, early detection and effective intervention of breast cancer is the key link to improve the efficacy and improve the quality of life of patients. Clinical studies have shown that breast cancer cells will preferentially spread through the lymphatic metastasis pathway. Among them, the sentinel lymph node is considered to be the first site reached by the spread of breast cancer cells. Therefore, accurate identification of metastatic sentinel lymph nodes is helpful for clinical breast cancer typing and staging, and has important clinical significance for guiding the treatment of patients.

[0003] The sentinel lymph node biopsy technique for breast cancer is the most common method for diagnosing sentinel lymph nodes at present, mainly including dye tracing method, radioisotope labeling method represented by 99mTc, ultrasonic contrast and fluorescent tracer method, etc. The above methods achieve accurate sentinel lymph node positioning effect and benefit many patients, but they still have some problems that cannot be ignored: first, part of the tissue extraction is difficult to obtain complete information of the metastatic sentinel lymph node; second, invasive tissue extraction increases the risk of metastasis; in addition, the identification result needs a long pathological evaluation process. Therefore, how to realize the rapid and accurate diagnosis of the sentinel lymph node of breast cancer is the urgent clinical demand to be solved.

[0004] Fluorescent molecular imaging technology has a series of advantages such as high sensitivity, high specificity, intuitive visualization, green and convenient, among which, near-infrared two-region (NIR-II, 1000-1700nm) imaging technology shows excellent tissue penetration ability, spatial resolution and lower tissue autofluorescence, significantly improving the accuracy and sensitivity of cancer diagnosis. The pathological characteristics of tumor microenvironment have been applied to develop various activated nano-fluorescent probes in tumor tissues, however, a single detection index cannot ensure the accuracy of the result.

[0005] Studies have shown that tumor-associated macrophages (TAM) and neutrophil (TAN) cells account for a significant proportion in metastatic breast cancer; among them, myeloperoxidase (MPO enzyme) is an important component of TAM and TAN, which is closely related to tumor metastasis, and specific recognition of MPO enzyme activity signal has become a new strategy for detecting metastatic lymph nodes.

[0006] In view of the problems in the prior art, the present application provides a near-infrared two-zone CRET technology method based on microenvironment dual-response activation, and applies it to a drug preparation for positioning and qualitative integrated diagnosis and treatment of breast cancer sentinel lymph nodes. SUMMARY

[0007] Therefore, the present application provides a near-infrared two-zone chemiluminescence resonance energy transfer nanosystem, a preparation method and application thereof, and realizes rapid and accurate diagnosis and treatment of breast cancer sentinel lymph nodes.

[0008] To achieve the above purpose, the present application provides a near-infrared two-zone (NIR-II, 1000-1700 nm) chemiluminescence resonance energy transfer nanosystem, which is constructed by multi-component synergistic self-assembly of a pH-responsive amphiphilic short peptide, a metal ion, a NIR-II CRET system and a photosensitizer; the near-infrared two-zone chemiluminescence resonance energy transfer nanosystem has a dual-response activation function of pH and MPO enzyme in the microenvironment to enhance the NIR-II CRET signal.

[0009] Preferably, the pH-responsive amphiphilic short peptide is any one of N-fluorenylmethyloxycarbonyl-L-histidine (Fmoc-His), N-fluorenylmethyloxycarbonyl-L-lysine (Fmoc-Lys), N-fluorenylmethyloxycarbonyl-L-arginine (Fmoc-Arg), N-fluorenylmethyloxycarbonyl-L-cysteine (Fmoc-Cys), and any combination of two or more thereof.

[0010] Preferably, 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+ ).

[0011] Preferably, the NIR-II CRET system comprises a near-infrared two-zone contrast agent and a compound having chemiluminescence ability.

[0012] More preferably, the compound having chemiluminescence ability is luminol or a luminol derivative; the luminol derivative is selected from 4-amino hexyl-N-ethyl iso-luminol (ABEI) and / or ethyl luminol (AHEI).

[0013] Preferably, the near-infrared two-region contrast agent at least comprises near-infrared quantum dots.

[0014] Preferably, the near-infrared quantum dots are near-infrared quantum dots modified by one or two or more of any combination of mercaptopropionic acid, undecylic acid, glutathione, and thioctic acid.

[0015] More preferably, the near-infrared quantum dots are one or two or more of any combination of Ag2S, Ag2Se, AgTe, Au:Ag2Te, and AgAuSe.

[0016] Preferably, the photosensitizer is one or two or more of any combination of chlorin e6 (Ce6), benzoporphyrin derivative (BPD), 2-(1-hexyloxyethyl)-2-devinyl pyrophosphate (HPPH), and benzophthalocyanine monocyclic acid A (BPD-MA).

[0017] As another object, the present application also provides a preparation method of the above-mentioned near-infrared two-region chemiluminescence resonance energy transfer nanosystem, comprising mixing a pH-responsive amphiphilic short peptide, metal ions, a NIR-II CRET system, and a photosensitizer, and then performing multi-component synergistic self-assembly to obtain the near-infrared two-region chemiluminescence resonance energy transfer nanosystem; the multi-component synergistic self-assembly comprises electrostatic adsorption, metal coordination, hydrophobic interaction, and π-π stacking interaction between the pH-responsive amphiphilic short peptide, the metal ions, the NIR-II CRET system, and the photosensitizer.

[0018] Specifically, the preparation method comprises the following steps:

[0019] (1) providing a NIR-II CRET system solution;

[0020] dissolving the near-infrared two-region contrast agent and the chemiluminescence compound in water respectively to obtain a contrast agent solution and a chemiluminescence compound solution; mixing the contrast agent solution and the chemiluminescence compound solution, and continuously stirring for 30-120 min before purification, to obtain the preparation method of the NIR-II CRET system, characterized in that the NIR-II CRET system solution;

[0021] (2) providing an amphiphilic short peptide solution, a metal ion solution, and a photosensitizer solution;

[0022] dissolving the pH-responsive amphiphilic short peptide in water to obtain the amphiphilic short peptide solution;

[0023] dissolving the metal ions in water to obtain the metal ion solution;

[0024] dissolving the photosensitizer in an organic solvent to obtain the photosensitizer solution;

[0025] (3) preparing the NIR-II CRET nanosystem;

[0026] The purified NIR-II CRET system solution in step (1) is mixed with the amphiphilic short peptide solution and the metal ion solution in step (2), and stirring is continued for 10-40 min; then, the photosensitizer solution prepared in step (2) is added, and after mixing, the pH is adjusted to neutral, and after purification and concentration, the NIR-II CRET nanosystem is obtained.

[0027] Preferably, in steps (1) and (4), the purification is ultrafiltration purification.

[0028] Preferably, 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.

[0029] Preferably, the mass ratio of the near-infrared two-region contrast agent to the chemiluminescent compound is 1-10:0.1-1.

[0030] Preferably, in step (2), the organic solvent is dimethyl sulfoxide.

[0031] Preferably, the concentration of the amphiphilic short peptide solution is 1-5 mg / mL.

[0032] Preferably, the concentration of the metal ion solution is 5-20 mmol / mL.

[0033] Preferably, the concentration of the photosensitizer solution is 1-10 mg / mL.

[0034] Preferably, in step (3), the mass addition ratio of the near-infrared two-region contrast agent, the chemiluminescent compound, the amphiphilic short peptide, the metal ion, and the photosensitizer in the near-infrared two-region CRET nanosystem is 1-10:50-100:5-50:50-200:1-5.

[0035] The pH value of the near-infrared two-region chemiluminescence resonance energy transfer nanosystem is neutral; under neutral conditions, the average size of the NIR-II CRET nanosystem is 20-500 nm.

[0036] Based on the above technical scheme, the near-infrared two-region chemiluminescence resonance energy transfer nano system is constructed by mixing the pH-responsive amphiphilic short peptide, metal ions, the NIR-II CRET system and the photosensitizer through multi-component synergistic self-assembly, and the multi-component synergistic self-assembly includes electrostatic adsorption, metal coordination, hydrophobic interaction and pi-pi stacking effect between the pH-responsive amphiphilic short peptide, metal ions, the NIR-II CRET system and the photosensitizer. The near-infrared two-region chemiluminescence resonance energy transfer nano system is used as a diagnostic reagent to establish a general breast cancer sentinel lymph node positioning and qualitative integrated diagnosis and treatment technology, and a new method for applying the near-infrared two-region contrast agent and the CRET optical system to clinical breast cancer sentinel lymph node diagnosis and treatment is provided.

[0037] The beneficial technical effects obtained by the present application are as follows:

[0038] (1) The present application integrates a dual-mode optical and pH / MPO synergistic activation strategy to realize accurate positioning and diagnosis of metastatic sentinel lymph nodes, and effectively avoids "false negative" and "false positive" results.

[0039] (2) Benefiting from the high tissue penetration, high spatiotemporal resolution and high signal-to-noise ratio imaging characteristics in the NIR-II window, the present application helps to detect deeper and earlier metastatic lesions. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is a transmission electron microscope image of the microenvironment dual-response activated NIR-II CRET nano system prepared by the present application in different pH environments.

[0041] Figure 2 is a transmission electron microscope image of the microenvironment dual-response activated NIR-II CRET nano system prepared by the present application in different pH environments.

[0042] Figure 3a and Figure 3b are the CRET spectrum and the fluorescence quantitative curve of the NIR-II CRET nano system of the present application in vitro through MPO enzyme catalytic reaction, respectively.

[0043] Figure 4 is an effect diagram of the microenvironment dual-response activated NIR-II CRET nano system prepared by the present application for in vivo breast cancer sentinel lymph node detection.

[0044] Figure 5 is an effect diagram of the metastatic sentinel lymph node tissue and the negative sentinel lymph node tissue removed by CRET signal guidance and hematoxylin-eosin staining (H&E). DETAILED DESCRIPTION

[0045] So that the purposes, technical solutions and advantages of the embodiments of the present application are more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some embodiments of the present application, rather than all the embodiments of the present application.

[0046] The present application provides a microenvironment dual-responsive activated NIR-II CRET nanosystem, which comprises a pH-responsive amphiphilic short peptide, a metal ion, a CRET system and a photosensitizer. The nanosystem is prepared by synergistic self-assembly of electrostatic adsorption, metal coordination, hydrophobic interaction, π-π stacking and the like between the above-mentioned multiple components.

[0047] The present application combines the high tissue penetration, high spatiotemporal resolution optical properties of near-infrared two-region imaging technology and the pathological characteristics of breast cancer tumor microenvironment, and prepares a microenvironment dual-responsive activated NIR-II CRET nanosystem through a multi-component synergistic self-assembly process. The nanosystem can realize the positioning of sentinel lymph nodes through near-infrared fluorescence signals, and realize the qualitative analysis of sentinel lymph nodes through the pH and MPO enzyme programmed activation of NIR-II CRET signals, overcoming the difficulty of realizing the integration of in vivo positioning and qualitative analysis in traditional methods, and providing a new method for realizing the precise diagnosis and treatment of breast cancer sentinel lymph nodes.

[0048] In some specific embodiments, the pH-responsive amphiphilic short peptide is any one of N-fluorenylmethyloxycarbonyl-L-histidine (Fmoc-His), N-fluorenylmethyloxycarbonyl-L-lysine (Fmoc-Lys), N-fluorenylmethyloxycarbonyl-L-arginine (Fmoc-Arg), N-fluorenylmethyloxycarbonyl-L-cysteine (Fmoc-Cys) and any combination thereof.

[0049] In some specific embodiments, the metal ion is one or any combination of gadolinium ion (Gd 3+ ), erbium ion (Er 3+ ), iron ion (Fe 3 + ), zinc ion (Zn 2+ ).

[0050] In some specific embodiments, the NIR-II CRET system loads any combination of a near-infrared two-region contrast agent and a compound with chemiluminescence capability.

[0051] In some embodiments, the photosensitizer is one of chlorin e6 (Ce6), benzoporphyrin derivative (BPD), 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH), benzoporphyrin monomethyl ether A (BPD-MA), or any combination thereof.

[0052] In some embodiments, the nanosystem, CRET system, and NIR-II contrast agent comprise near-infrared quantum dots.

[0053] In some embodiments, the near-infrared quantum dots are one of Ag2S, Ag2Se, AgTe, Au:Ag2Te, AgAuSe, or any combination thereof.

[0054] In some embodiments, the near-infrared quantum dots are one of mercaptopropionic acid, undecylenic acid, glutathione, thioctic acid modified near-infrared quantum dots, or any combination thereof.

[0055] In some embodiments, the chemiluminescent compound is one of luminol or its derivatives 4-aminohexyl-N-ethylisoluminol (ABEI), ethyl luminol (AHEI), or any combination thereof.

[0056] In another aspect, the present application provides a method for preparing a microenvironment dual-responsive activated NIR-II CRET nanosystem as described above, characterized in that the method comprises the following steps:

[0057] (1) Dissolve the near-infrared two-region contrast agent and the chemiluminescent compound in water separately to obtain two solutions;

[0058] (2) Mix the two solutions obtained in (1) and continue stirring for one hour, and purify to remove excess chemiluminescent compound.

[0059] (3) Dissolve the amphiphilic short peptide and the metal ion in water separately, and dissolve the photosensitizer in an organic solvent to obtain a photosensitizer solution by ultrasonication.

[0060] (4) Mix the CRET system solution obtained after purification in (2) and the amphiphilic short peptide solution in (3) with the metal ion solution in (3) at the same time, continue stirring for 30 minutes, then add the photosensitizer solution in (3), adjust the pH of the above mixed solution to neutral, and purify and concentrate to obtain the pH and MPO enzyme dual-responsive activated NIR-II CRET nanosystem.

[0061] The application provides application of a microenvironment dual-response activated NIR-II CRET nanosystem in breast cancer sentinel lymph node positioning and qualitative integrated diagnosis and treatment.

[0062] It should be particularly pointed out that, unless otherwise specified, the raw materials and chemical reagents used in the application are commercially available.

[0063] The technical solutions in the embodiments of the application will be clearly and completely described below with specific examples and drawings. Obviously, the described embodiments are only some of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the application.

[0064] Embodiment 1

[0065] The embodiment provides a preparation method of a microenvironment dual-response activated NIR-II CRET nanosystem, and the specific steps include:

[0066] (1) 10 mg of 4-(N-ethyl-N-aminobutylamino) phthalhydrazide (ABEI), 0.5 g of sodium bicarbonate and 200 μL of concentrated ammonia water are dissolved in water to obtain an ABEI solution;

[0067] (2) 1 mg of undecyl acid modified Ag2S quantum dots is dissolved in water to obtain a quantum dot solution in water phase (average concentration is 0.5 mg / mL);

[0068] (3) The ABEI solution obtained in (1) and the quantum dot solution obtained in (2) are mixed in a mass ratio of 100:1, and continuously stirred at a stirring speed of 300 rpm for 1 hour;

[0069] (4) The solution obtained in (3) is purified by ultrafiltration for three times, 10 minutes each time, to remove excess ABEI, and a purified CRET system solution is obtained;

[0070] (5) 1 mg of fluorenylmethoxycarbonyl lysine (Fmoc-Lys) and 5 mmol of ErCl3 are respectively dissolved in 1 mL of water to obtain Fmoc-Lys solution and ErCl3 solution; 1 mg of phenyl dichlorophosphine (BPD) is dissolved in 1 mL of DMSO to obtain BPD solution.

[0071] (6) The NIR-II CRET system solution obtained in step (4) is mixed with the Fmoc-Lys solution and the ErCl3 solution obtained in (5) at the same time, and continuously stirred at a stirring speed of 300 rpm for 30 minutes, and finally a mixed solution is obtained;

[0072] (7) The BPD solution prepared in step (5) is mixed with the mixed solution prepared in step (6) to obtain a solution, which is divided into two parts, and the pH of each part is adjusted to pH = 7.4 and pH = 5.5, respectively. After stirring for 1 hour, the purified NIR-II CRET nanosystem is obtained by ultrafiltration and concentration.

[0073] As shown in FIG. 1, the morphology characterization diagram of the NIR-II CRET nanosystem provided in the embodiment is shown. As can be seen from the transmission electron microscopy diagram, the size of the nanosystem is about 120 nm under the condition of pH 7.4, and the size of the nanosystem is about 30 nm under the condition of pH 5.5. It is illustrated that the NIR-II CRET nanosystem is successfully prepared under the condition of pH 7.4, and is depolymerized under the condition of pH 5.5. Figure 1

[0074] Embodiment 2

[0075] The embodiment provides a preparation method of a NIR-II CRET nanosystem based on a microenvironment dual-response activation, and the specific steps include:

[0076] (1) 20 mg luminol, 1 g sodium bicarbonate and 100 μL concentrated ammonia water are dissolved in water to obtain a 10 mM luminol solution;

[0077] (2) 1 mg of thiol propionic acid modified AgAuSe quantum dots is dissolved in water to obtain a quantum dot solution of 2 mg / mL;

[0078] (3) The luminol solution obtained in step (1) is mixed with the quantum dot solution obtained in step (2) (according to an average mass ratio of 50:1), and stirring is continuously performed at a stirring speed of 300 rpm for 1 hour.

[0079] (4) The solution obtained in step (3) is purified by ultrafiltration for three times, each time for 10 minutes, to remove excess luminol, and a purified NIR-II CRET system solution is obtained.

[0080] (5) 1 mg of fluorenylmethoxycarbonyl histidine (Fmoc-His) and 2 mmol of GdCl3 are respectively dissolved in 1 mL of water to obtain a Fmoc-His solution and a GdCl3 solution, and 2 mg of chlorin e6 (Ce6) is dissolved in 1 mL of DMSO.

[0081] (6) The NIR-II CRET solution obtained in step (4) is mixed with the Fmoc-His solution and the GdCl3 solution obtained in step (5), and stirring is continuously performed at a stirring speed of 300 rpm for 30 minutes.

[0082] ​(7) The whole Ce6 solution obtained in step (5) is mixed with the whole mixed solution in step (6), then divided into two parts, and the pH of each part is adjusted to pH = 7.4 and pH = 5.5 respectively, and after continuous stirring for 1 hour, the purified NIR-II CRET nanosystem is obtained by ultrafiltration and concentration.

[0083] As shown in FIG. 1, the morphology characterization diagram of the NIR-II CRET nanosystem provided in the embodiment is shown, and the characterization of the transmission electron microscope diagram shows that the size of the nanosystem is about 80 nm under the condition of pH 7.4, and the size of the nanosystem is about 27 nm under the condition of pH 5.5; further, as can be seen from the diagram, the NIR-II CRET nanosystem is successfully prepared in the pH 7.4 environment, and depolymerization occurs in the pH 5.5 environment. Figure 2 As shown in FIG. 2, the CRET spectrum and fluorescence quantification curve of the NIR-II CRET nanosystem of the embodiment in vitro through MPO enzyme catalytic reaction are shown. As can be seen from FIG. 2, the CRET signal can be generated in vitro through MPO enzyme catalytic reaction; referring to FIG. 3, the quantitative curve of the CRET signal changing with time (CRET signal-time quantitative curve) under different pH environments shows that the CRET signal under the condition of pH 5.5 is stronger than that under the condition of pH 7.4, proving that the nanosystem has a significant pH-dependent MPO enzyme response function.

[0084] Figure 3a As shown in FIG. 4, the CRET spectrum and fluorescence quantification curve of the NIR-II CRET nanosystem of the embodiment in vitro through MPO enzyme catalytic reaction are shown. As can be seen from FIG. 4, the CRET signal can be generated in vitro through MPO enzyme catalytic reaction; referring to FIG. 5, the quantitative curve of the CRET signal changing with time (CRET signal-time quantitative curve) under different pH environments shows that the CRET signal under the condition of pH 5.5 is stronger than that under the condition of pH 7.4, proving that the nanosystem has a significant pH-dependent MPO enzyme response function. Figure 3b Figure 3a Figure 3b

[0085] Example 3

[0086] The NIR-II CRET nanosystem obtained in Example 2 of the application is selected as a diagnosis and treatment reagent and applied to in vivo evaluation of breast cancer metastatic sentinel lymph node evaluation / diagnosis, and the specific steps include:

[0087] (1) A breast cancer lymph metastasis mouse model is constructed, and the model mice and normal mice are subcutaneously injected with the optimal dose of nanosystem solution through the mouse paw pad; real-time imaging of the above mice is performed using a near-infrared in vivo imaging instrument; the changes in the fluorescence signals of the sentinel lymph nodes of the two groups of mice are observed, and real-time fluorescence images are collected; after imaging is completed, the sentinel lymph node tissues of the mice are taken for histopathological staining analysis.

[0088] (2) Postoperative histopathological staining analysis: the above removed lymph node tissue sections are subjected to immunofluorescence staining and H&E staining. The inverted fluorescence microscope is used to collect images of the stained sections, and pathological analysis is performed. The above in vivo fluorescence images and histopathological staining results are compared, and the consistency of the near-infrared fluorescence signals, NIR-II CRET signals and histopathological diagnosis of the metastatic lymph nodes and normal lymph nodes is analyzed.​​​​

[0089] The analysis methods of the above steps are all general technical means in the prior art.

[0090] As shown in the formula (I), the nano system can accurately locate the breast cancer sentinel lymph node under 808nm laser irradiation, and the nano system successfully realizes the identification of metastatic sentinel lymph node through NIR-II CRET signal without laser, and no obvious NIR-II CRET signal is observed at the negative sentinel lymph node; it is proved that the metastatic sentinel lymph node can be accurately detected through NIR-II CRET signal under the dual response activation of pH and MPO enzyme. Figure 4 As shown in the formula (I), the nano system can accurately locate the breast cancer sentinel lymph node under 808nm laser irradiation, and the nano system successfully realizes the identification of metastatic sentinel lymph node through NIR-II CRET signal without laser, and no obvious NIR-II CRET signal is observed at the negative sentinel lymph node; it is proved that the metastatic sentinel lymph node can be accurately detected through NIR-II CRET signal under the dual response activation of pH and MPO enzyme.

[0091] As shown in the formula (I), the nano system can accurately locate the breast cancer sentinel lymph node under 808nm laser irradiation, and the nano system successfully realizes the identification of metastatic sentinel lymph node through NIR-II CRET signal without laser, and no obvious NIR-II CRET signal is observed at the negative sentinel lymph node; it is proved that the metastatic sentinel lymph node can be accurately detected through NIR-II CRET signal under the dual response activation of pH and MPO enzyme. Figure 5 As shown in the formula (I), the nano system can accurately locate the breast cancer sentinel lymph node under 808nm laser irradiation, and the nano system successfully realizes the identification of metastatic sentinel lymph node through NIR-II CRET signal without laser, and no obvious NIR-II CRET signal is observed at the negative sentinel lymph node; it is proved that the metastatic sentinel lymph node can be accurately detected through NIR-II CRET signal under the dual response activation of pH and MPO enzyme.

[0092] Therefore, the above results show that the near-infrared two-zone chemiluminescence resonance energy transfer nano system provided by the technical scheme of the present application can be used for the diagnosis and treatment reagent of breast cancer sentinel lymph node, has pH dependence and MPO enzyme response function, occurs depolymerization reaction under acidic conditions, can enhance the NIR-II CRET signal, so as to realize the positioning and qualitative integration diagnosis and treatment of breast cancer sentinel lymph node at the in-vivo level.

[0093] Aspects, embodiments, features, and examples of the present application are to be considered in all respects as illustrative only and not restrictive, and the scope of the present application is defined only by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed application.

[0094] The use of headings and description contents in the present application scheme does not mean the limitation of the present application; each part can be applied to any aspect, embodiment or feature of the present application.

[0095] Throughout this present application scheme, where compositions are described as having, containing, or including certain components, or where processes are described as having, containing, or including certain process steps, it is contemplated that the present teachings also consist essentially of and can consist of the recited components and the recited process steps, respectively.

[0096] The use of the terms "include," "includes," "including," "have," "has," or "having," to describe the presence of a feature, object, or action, etc., are intended to be construed to be open-ended, and not transitive, unless specifically stated otherwise.

[0097] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present teachings remain operable. Moreover, two or more steps or actions can be conducted simultaneously or concurrently.

[0098] In addition, the inventors have found that the aforementioned embodiments, as well as other raw materials, process operations, process conditions, etc., described herein, have been tested and have resulted in desirable results.

[0099] While the present application has been described with reference to the illustrative embodiments, those with ordinary skill in the art will appreciate that various modifications, omissions, and / or additions can be made without departing from the spirit and scope of the application. Additionally, many modifications can be made to adapt a particular situation or material to the teachings of the present application without departing from its scope. Therefore, the present application is not intended to be limited to the disclosed embodiments associated with performing the present application, but rather, the scope of the present application is to be accorded the broadest interpretation so as to encompass all embodiments falling within the scope of the appended claims. Furthermore, to the extent that the term "includes" is used in either the detailed description or the claims, such term is intended to be inclusive in a manner similar to the term "comprising" as "comprising" is interpreted when employed as a transitional word in the introductory clauses of the claims.

Claims

1. A near-infrared II chemiluminescence resonance energy transfer nanosystem, characterized in that, It was constructed by multi-component synergistic self-assembly of pH-responsive amphiphilic short peptides, metal ions, NIR-IICRET system and photosensitizer; The near-infrared II chemiluminescent resonance energy transfer nanosystem exhibits a NIR-IICRET signal activated by both pH and MPO enzymes in the microenvironment. The pH-responsive amphiphilic short peptide is any one or any combination of two or more of N-fluorenmethoxycarbonyl-L-histidine, N-fluorenmethoxycarbonyl-L-lysine, N-fluorenmethoxycarbonyl-L-arginine, and N-fluorenmethoxycarbonyl-L-cysteine. The metal ion is one or any combination of two or more of the following: gadolinium ion, erbium ion, iron ion, calcium ion, copper ion, magnesium ion, zinc ion, and manganese ion. The NIR-IICRET system contains a near-infrared II contrast agent and a compound with chemiluminescence capabilities; The compound with chemiluminescent ability is luminol or a luminol derivative; The luminol derivatives are selected from 4-aminohexyl-N-ethyl isorhuno and / or ethyl luminol; The near-infrared II contrast agent includes at least one type of contrast agent, including near-infrared quantum dots. The near-infrared quantum dots are one or any combination of two or more of Ag2S, Ag2Se, AgTe, Au:Ag2Te, and AgAuSe.

2. The near-infrared II chemiluminescence resonance energy transfer nanosystem according to claim 1, characterized in that, The photosensitizer is one or any combination of two or more of dihydroporphyrin E6, benzoporphyrin derivatives, 2-(1-hexaoxyethyl)-2-devinyl pyrophosphate, and phenylporphyrin monocyclic acid A.

3. A method for preparing a near-infrared II chemiluminescence resonance energy transfer nanosystem as described in any one of claims 1-2, characterized in that, A near-infrared II region chemiluminescent resonance energy transfer nanosystem was prepared by mixing pH-responsive amphiphilic short peptides, metal ions, an NIR-IICRET system, and a photosensitizer and then performing multi-component synergistic self-assembly. The multi-component synergistic self-assembly includes electrostatic adsorption, metal coordination, hydrophobic interactions, and π-π stacking interactions between pH-responsive amphiphilic short peptides, metal ions, the NIR-IICRET system, and photosensitizers.

4. The preparation method according to claim 3, characterized in that, Includes the following steps: (1) Provide NIR-IICRET system solution; The near-infrared II contrast agent and the chemiluminescent compound were dissolved in water to obtain a contrast agent solution and a chemiluminescent compound solution, respectively. The contrast agent solution and the chemiluminescent compound solution were mixed and stirred continuously for 30-120 minutes before purification to obtain the NIR-IICRET system solution. (2) Provide amphiphilic short peptide solution, metal ion solution and photosensitizer solution; The pH-responsive amphiphilic short peptide was dissolved in water to obtain the amphiphilic short peptide solution. The metal ion solution is obtained by dissolving the metal ions in water; The photosensitizer is dissolved in an organic solvent to obtain the photosensitizer solution; (3) Preparation of NIR-IICRET nanosystem: The NIR-IICRET system solution purified in step (1) is mixed with the amphiphilic short peptide solution and the metal ion solution in 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 purified and concentrated to obtain NIR-IICRET nanosystem.

5. The preparation method according to claim 4, characterized in that, In steps (1) and (4), the purification is ultrafiltration purification.

6. The preparation method according to claim 4, characterized in that, 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.

7. The preparation method according to claim 4, characterized in that, The mass ratio of the near-infrared II contrast agent to the chemiluminescent compound is 1~10:50~100.

8. The preparation method according to claim 4, characterized in that, In step (2), the organic solvent is dimethyl sulfoxide.

9. The preparation method according to claim 4, characterized in that, The concentration of the amphiphilic short peptide solution is 1~5 mg / mL.

10. The preparation method according to claim 4, characterized in that, The concentration of the metal ion solution is 5~20 mmol / mL.

11. The preparation method according to claim 4, characterized in that, The concentration of the photosensitizer solution is 1~10 mg / mL.

12. The preparation method according to claim 4, characterized in that, In step (3), the NIR-IICRET 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.

13. The preparation method according to claim 4, characterized in that, Under neutral conditions, the average size of the NIR-IICRET nanosystem is 20~500 nm.

14. The application of the near-infrared II chemiluminescence resonance energy transfer nanosystem as described in any one of claims 1-2 in the preparation of a diagnostic reagent for sentinel lymph nodes in breast cancer.

15. A diagnostic reagent for sentinel lymph nodes in breast cancer, comprising at least the near-infrared II chemiluminescent resonance energy transfer nanosystem as described in any one of claims 1-2, wherein the diagnostic reagent is pH-dependent and MPO enzyme responsive, undergoes a depolymerization reaction under acidic conditions, and can enhance the NIR-IICRET signal.

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