Sentinel lymph node nano tracer agent as well as preparation method and application of sentinel lymph node nano tracer agent

Abstract

The invention discloses a sentinel lymph node nano tracer and a preparation method and application thereof. The sentinel lymph node nano tracer is prepared from the following raw materials: a quantum dot material with near-infrared two-region luminescence characteristic, an amphiphilic polymer, a catalyst, a diamino polymer, a macrophage CD206 receptor targeting molecule and a temperature-sensitive polymer. Wherein the carboxyl group ratio of the macrophage CD206 receptor targeting molecule to the temperature sensitive polymer is (500 to 1000): (100 to 5000). The nano tracer integrates the high-resolution imaging advantage of quantum dots, the temperature response type aggregation characteristic and the receptor-mediated targeted delivery mechanism, can effectively prolong the developing time window while realizing accurate positioning of sentinel lymph nodes, and can provide a new design idea for developing intelligent response type surgical navigation probes; good application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a sentinel lymph node nano-tracer and a preparation method and application thereof. Background Art

[0002] Breast cancer is a malignant tumor that poses a serious threat to women's health. Currently, surgical resection is the primary treatment. With the advancement of precision medicine, axillary-sparing breast cancer surgeries have become increasingly important, with sentinel lymph node biopsy (SLNB) playing a crucial role. By detecting the metastatic status of the first lymphatic drainage node of the primary tumor, SLNB can avoid unnecessary axillary lymph node dissection, significantly reducing the incidence of postoperative complications such as upper limb lymphedema and paresthesia. It also provides critical pathological evidence for tumor staging, prognostic assessment, and the development of adjuvant treatment plans.

[0003] Among related technologies, the sentinel lymph node tracing technology system still faces multiple challenges. For example, although radionuclides (such as technetium-99m labeled colloids) are the clinical gold standard, they have the risk of radiation exposure, the need for nuclear medicine equipment support, and complex operation. Dyes such as methylene blue and indocyanine green are prone to rapid tissue diffusion, blurred imaging boundaries, and the risk of allergic reactions. Conventional near-infrared region I (NIR-I) fluorescent probes are limited by insufficient tissue penetration depth and background autofluorescence interference, resulting in reduced sensitivity for deep lymph node imaging. In addition, the above technologies generally have problems such as low lymph node targeting and enrichment efficiency, insufficient tissue penetration depth, short imaging duration, and non-specific uptake, which affect the accuracy and reliability of intraoperative real-time navigation and sentinel lymph node status assessment.

[0004] Based on this, there is an urgent need to develop a new tracer that can break through these clinical pain points to improve its targeting and imaging efficacy. Summary of the Invention

[0005] The present invention aims to address at least one of the technical problems existing in the prior art. To this end, the present invention proposes a sentinel lymph node nanotracer, its preparation method, and its application. This nanotracer integrates the high-resolution imaging advantages of quantum dots, their temperature-responsive aggregation properties, and a receptor-mediated targeted delivery mechanism. While achieving precise sentinel lymph node localization, it can effectively extend the imaging time window, providing new design ideas for the development of intelligent, responsive surgical navigation probes.

[0006] The first aspect of the present invention provides a sentinel lymph node nanotracer, the raw materials for its preparation include:

[0007] Quantum dot materials, amphiphilic polymers, catalysts, diamino polymers, macrophage CD206 receptor targeting molecules, and temperature-sensitive polymers with near-infrared second-region luminescence properties;

[0008] Wherein, the carboxyl ratio of the macrophage CD206 receptor targeting molecule to the temperature-sensitive polymer is 500-1000:100-5000.

[0009] In some embodiments of the present invention, the carboxyl ratio of the macrophage CD206 receptor targeting molecule to the temperature-sensitive polymer is 1000:300 to 3000. For example, the carboxyl ratio can be 1000:300, 1000:500, 1000:1000, 1000:1500, 1000:2000 or 1000:3000, etc.

[0010] In some embodiments of the present invention, the quantum dot material having near-infrared second-zone luminescence characteristics is selected from one or more combinations of lead sulfide quantum dots (PbS QDs), lead selenide quantum dots, indium arsenide quantum dots, silver sulfide quantum dots, and lead telluride quantum dots.

[0011] In some embodiments of the present invention, the quantum dot material having near-infrared second-region luminescence characteristics is lead sulfide quantum dots.

[0012] In some embodiments of the present invention, the amphiphilic polymer is selected from one or more combinations of octylamine grafted polyacrylic acid, polyacrylic acid-hyperbranched polyester complex, alkylamine grafted polycarboxylic acid polymer, polyethylene glycol-based amphiphilic copolymer, polyacrylic acid-poly N-isopropylacrylamide-octylamine graft.

[0013] In some embodiments of the present invention, the amphiphilic polymer is octylamine grafted polyacrylic acid.

[0014] In some embodiments of the present invention, the catalyst is selected from one or more combinations of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysulfosuccinimide (Sulfo-NHS), N-hydroxysuccinimide (NHS), N,N'-dicyclohexylcarbodiimide (DCC), and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM).

[0015] In some embodiments of the present invention, the catalyst is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or N-hydroxysulfosuccinimide.

[0016] In some embodiments of the present invention, the bisamino polymer is selected from one or more combinations of bisamino polyethylene glycol molecules, bisamino polylysine, bisamino polyethyleneimine, bisamino block copolymers, and bisamino polyacrylic acid grafts.

[0017] In some embodiments of the present invention, the bisamino polymer is a bisamino polyethylene glycol molecule.

[0018] In some embodiments of the present invention, the average molecular weight of the polyethylene glycol in the bisamino polyethylene glycol molecule is 1000 to 10000. Specifically, the molecular weight of the polyethylene glycol can be 1000, 1500, 2000, 2500, 3000, 5000, 8000 or 10000.

[0019] In some embodiments of the present invention, the macrophage CD206 receptor targeting molecule is selected from one or more combinations of polymannose, mannose-modified proteins, mannose-modified antibodies, mannose liposomes, thiomannose, CD206 aptamers, cysteine-containing targeting peptides, and CD206 targeting antibody fragments.

[0020] In some embodiments of the present invention, the macrophage CD206 receptor targeting molecule is polymannose (ie, polymannose chain).

[0021] In some embodiments of the present invention, the degree of polymerization of the polymannose is 20 to 100. Specifically, the degree of polymerization of the polymannose may be 20, 30, 40, 50, 60, 70, 80, 90 or 100.

[0022] In some embodiments of the present invention, the temperature-sensitive polymer is selected from one or more combinations of poly (N-isopropylacrylamide) (PNIPAM), polyvinyl methyl ether, polyethylene glycol-polypropylene glycol block copolymer, polydiethylacrylamide, and poly (N-vinylcaprolactam).

[0023] In some embodiments of the present invention, the temperature-sensitive polymer is poly (N-isopropylacrylamide).

[0024] Poly (N-isopropylacrylamide) has good biocompatibility, low toxicity, and unique temperature-responsive properties. When the temperature is below its critical solution temperature (approximately 32°C), poly (N-isopropylacrylamide) is water-soluble; when the temperature is above its critical solution temperature, poly (N-isopropylacrylamide) undergoes a phase transition and becomes insoluble in water (gelation or aggregation), forming hydrophobic aggregates. Using it as a temperature-sensitive polymer for sentinel lymph node nanotracers helps to extend the development time window.

[0025] In some embodiments of the present invention, the mass molar ratio of the amphiphilic polymer to the quantum dot material having near-infrared second-region luminescence characteristics is 0.1-5 mg: 0.1-10 nmol / L.

[0026] In some embodiments of the present invention, the mass molar ratio of the amphiphilic polymer to the quantum dot material having near-infrared second region luminescence characteristics is 0.1-1 mg:0.5-5 nmoL. For example, the mass molar ratio can be 0.1 mg:1 nmoL, 0.2 mg:1 nmoL, 0.3 mg:1 nmoL, 0.4 mg:1 nmoL, 0.5 mg:1 nmoL, 0.8 mg:1 nmoL, or 1 mg:1 nmoL.

[0027] The second aspect of the present invention provides a method for preparing the sentinel lymph node nanotracer according to the first aspect of the present invention, comprising the following steps:

[0028] S1, mixing the amphiphilic polymer with the quantum dot material having near-infrared second region luminescence characteristics, and reacting them to obtain an amphiphilic polymer-coated quantum dot material;

[0029] S2, mixing the amphiphilic polymer-coated quantum dot material with a diamino polymer, and performing a coupling reaction to obtain an amino-modified amphiphilic polymer-coated quantum dot material;

[0030] S3. Under the catalyst conditions, the quantum dot material coated with the amino-modified amphiphilic polymer, the macrophage CD206 receptor targeting molecule and the temperature-sensitive polymer are mixed, and after amide coupling reaction, purification is performed to obtain.

[0031] In some embodiments of the present invention, the quantum dot material having near-infrared second-region luminescence characteristics is lead sulfide quantum dots.

[0032] In some embodiments of the present invention, the preparation method of the lead sulfide quantum dots comprises: mixing a lead precursor and a sulfur precursor, reacting and quenching, and obtaining the lead sulfide quantum dots after graded purification.

[0033] In some embodiments of the present invention, the lead precursor comprises PbCl2 and oleylamine.

[0034] In some embodiments of the present invention, the ratio of PbCl2 to oleylamine is 1-5 mmoL:5-10 mL.

[0035] In some embodiments of the present invention, the sulfur precursor includes sulfur, oleylamine, and tri-n-octylphosphine.

[0036] In some embodiments of the present invention, the molar volume ratio of sulfur, oleylamine, and tri-n-octylphosphine is 0.5-1 mmoL: 1-3 mL: 2-20 μL.

[0037] In some embodiments of the present invention, the reaction temperature is 100-140° C., and the reaction time is 5-40 s.

[0038] In some embodiments of the present invention, the amphiphilic polymer is octylamine grafted polyacrylic acid.

[0039] In some embodiments of the present invention, the preparation method of the octylamine grafted polyacrylic acid comprises: acidifying sodium polyacrylate and then mixing it with octylamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, performing a coupling reaction, and purifying to obtain the product.

[0040] In some embodiments of the present invention, the catalyst is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS).

[0041] In some embodiments of the present invention, the macrophage CD206 receptor targeting molecule is polymannose, and the degree of polymerization of the polymannose is 20-100.

[0042] In some embodiments of the present invention, the degree of polymerization of the polymannose is 30 to 80. For example, it can be 30, 35, 40, 50, 60, 70 or 80.

[0043] In some embodiments of the present invention, the method for preparing polymannose comprises:

[0044] α-D-mannose pentaacetate, acrylamide, and metal salt are mixed and reacted to obtain mannose monomers (i.e., D-mannose acyloxyethyl acrylamide); then, a reversible addition-fragmentation chain transfer polymerization method is used to synthesize polymannose chains, which are obtained after purification.

[0045] In some embodiments of the present invention, the metal salt is selected from one or more combinations of sodium hydroxide, potassium hydroxide, and calcium hydroxide.

[0046] In some embodiments of the present invention, the mass volume ratio of the α-D-mannose pentaacetate, acrylamide, and metal salt is 2-8 g: 0.5-2.5 mL: 30-80 mg.

[0047] In some embodiments of the present invention, in step S3, in the coupling reaction system, the molar concentration ratio of the PbS quantum dots, EDC, and Sulfo-NHS is 1:500-3000:500-3000.

[0048] In some embodiments of the present invention, in step S3, in the coupling reaction system, the molar concentration ratio of the PbS quantum dots, EDC, and Sulfo-NHS is 1:1000-2000:1000-2000.

[0049] In some embodiments of the present invention, in step S3, in the amide coupling reaction system, n(-NH2):n(-COOH):n(EDC)=1:500-3000:1500-5000.

[0050] In some embodiments of the present invention, in step S3, in the amide coupling reaction system, n(-NH2):n(-COOH):n(EDC)=1:1000-2000:2000-3000.

[0051] The third aspect of the present invention provides the use of the method for preparing the sentinel lymph node nanotracer according to the first aspect of the present invention or the sentinel lymph node nanotracer according to the second aspect of the present invention in any of the following:

[0052] A) Preparation of products for sentinel lymph node detection;

[0053] B) Screening for drugs related to sentinel lymphoma treatment;

[0054] C) Preparation of a reagent for detecting axillary sentinel lymph nodes in breast cancer.

[0055] The sentinel lymph node nanotracer provided by the present invention has at least the following beneficial effects:

[0056] (1) The sentinel lymph node nanotracer of the present invention can significantly enhance enrichment efficiency. The targeting molecule designed in the present invention can specifically bind to the CD206 receptor on lymph node macrophages, significantly enhancing the nanotracer enrichment efficiency in lymph nodes. Its specific targeting mechanism can reduce non-lymph node uptake, providing a strong basis for accurately assessing the metastatic status of sentinel lymph nodes.

[0057] (2) The sentinel lymph node nanotracers of the present invention are capable of temperature-sensitive aggregation. By utilizing the phase transition properties of temperature-sensitive polymers, local temperature regulation enables controlled aggregation of nanotracers within the sentinel lymph node, while preventing spread to non-sentinel lymph nodes and increasing the duration of sentinel lymph node imaging.

[0058] (3) The sentinel lymph node nanotracer of the present invention has the advantage of near-infrared zone II imaging. The present invention selects quantum dot materials with near-infrared zone II luminescence characteristics, which can achieve deeper tissue penetration and high signal-to-noise ratio, surpassing conventional tracers and near-infrared zone I materials, and significantly increasing tissue penetration depth.

[0059] In summary, the sentinel lymph node nanotracer of the present invention has the advantages of simultaneously improving the problems of low lymph node targeting enrichment efficiency, insufficient tissue penetration depth, short development duration and nonspecific uptake of existing tracers. The use of the sentinel lymph node nanotracer of the present invention in clinical sentinel lymph node detection can significantly improve the accuracy and reliability of intraoperative real-time navigation and sentinel lymph node status assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0061] Figure 1 These are the relevant characterization diagrams of PbS QDs prepared in Example 1 of the present invention, where A and B are transmission electron microscope images at different magnifications (A scale: 50 nm; B scale: 20 nm), C is a statistical diagram of the PbS QDs particle size based on the TEM image, and D is a UV-visible absorption spectrum of PbS QDs.

[0062] Figure 2 Schematic diagram of the hydrophilic modification of PbS QDs prepared in Example 1 of the present invention.

[0063] Figure 3 The hydrated particle size and Zeta potential measurement diagram of the PbS QDs related products of the present invention, wherein A is the hydrated particle size detection result, and B is the Zeta potential detection result.

[0064] Figure 4 The PbS-PNIPAM / M 50 and indocyanine green (ICG) real-time imaging of the lymphatic system at different time points in vivo, where A is PbS-PNIPAM / M 50 , B is indocyanine green.

[0065] Figure 5 The right foot pad of the mice of the present invention was injected with ICG or PbS-PNIPAM / M 50 NIR-II imaging of relevant lymph nodes 15 minutes later, where A is the ICG injection group and B is the PbS-PNIPAM / M injection group 50 Group.

[0066] Figure 6 The nano-tracer PbS-PNIPAM / M prepared in Example 1 of the present invention 50 In vivo imaging of lymphatic system nodes.

[0067] Figure 7 The nano-tracer PbS-PNIPAM / M prepared in Example 2 of the present invention 50 In vivo imaging of lymphatic system nodes.

[0068] Figure 8 The nano-tracer PbS-PNIPAM / M prepared in Example 3 of the present invention 50 In vivo imaging of lymphatic system nodes.

[0069] Figure 9 The nano-tracer PbS-PNIPAM / M prepared in the embodiment of the present invention 50 Fluorescence intensity statistics of lymphatic system nodes in vivo.

[0070] Figure 10 The PbS-PNIPAM / M 50 NIR fluorescence imaging of ICG and fat emulsion at different depths, where A is indocyanine green and B is PbS-PNIPAM / M 50 .

[0071] Figure 11 The PbS-PNIPAM / M 50 The fluorescence intensity distribution of ICG in fat emulsions of different depths, where A is the fluorescence intensity distribution of ICG in fat emulsions of different depths, and B is the fluorescence intensity distribution of PbS-PNIPAM / M 50 Fluorescence intensity distribution at different depths of fat emulsion.

[0072] Figure 12 The PbS-PNIPAM / M 50 The fluorescence intensity histogram and signal-to-noise ratio comparison of ICG and PbS-PNIPAM / M at different depths of fat emulsion, where A is the fluorescence intensity histogram of ICG and PbS-PNIPAM / M 50 B is the fluorescence intensity histogram of ICG and PbS-PNIPAM / M 50 Signal-to-noise ratio comparison chart.

[0073] Figure 13 The present invention regulates the nano-tracer PbS-PNIPAM / M by temperature 50 Real-time imaging of the in vivo lymphatic system, where A is real-time NIR-II imaging without continuous ice compress, and B is real-time NIR-II imaging under ice compress and rewarming. DETAILED DESCRIPTION

[0074] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0075] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0076] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0077] In the description of the present invention, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the exemplary expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0078] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0079] Example 1: Nanotracer

[0080] 1. Sentinel lymph node nanotracer

[0081] This embodiment provides a nano-tracer for tracing the axillary sentinel lymph nodes of breast cancer. The raw materials for its preparation include:

[0082] PbS quantum dots (i.e., PbS QDs), amphiphilic polymers, catalysts, diamino polymers, macrophage CD206 receptor targeting molecules and temperature-sensitive polymers.

[0083] Wherein, the above-mentioned amphiphilic polymer is octylamine grafted polyacrylic acid (abbreviated as OPA);

[0084] The catalysts are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS).

[0085] The bisamino polymer is a bisamino polyethylene glycol molecule (NH2-PEG2000-NH2);

[0086] The macrophage CD206 receptor targeting molecule is a polymannose chain (degree of polymerization 50);

[0087] The temperature-sensitive polymer is poly (N-isopropylacrylamide) (PNIPAM for short).

[0088] 2. Preparation method of sentinel lymph node nanotracer

[0089] This embodiment also provides a method for preparing a nano-tracer for tracing axillary sentinel lymph nodes of breast cancer, which specifically comprises the following steps:

[0090] (1) PbS quantum dot synthesis:

[0091] PbS quantum dots (PbS QDs) were synthesized using a hot injection method with oleylamine as a ligand. Specifically, a lead precursor (3 mmol / L PbCl2, 7.5 ml oleylamine) and a sulfur precursor (0.75 mmol / L sulfur / 2.25 mL oleylamine / 15 μL tri-n-octylphosphine) were rapidly reacted at 120°C for 20 seconds. After quenching, the mixture was purified by acetone fractionation, resuspended in toluene, and reacted with oleic acid (oleic acid: toluene = 1.5:10) for 30 seconds to perform ligand exchange and enhance stability, thus obtaining PbS QDs.

[0092] in, Figure 1 1 is a characterization diagram of the PbS QDs synthesized in this embodiment, wherein A and B are transmission electron microscope (TEM) images at different magnifications, C is a statistical diagram of the PbS QDs particle size based on the TEM image, and D is a UV-visible absorption spectrum of the PbS QDs.

[0093] (2) Amphiphilic polymer OPA@PbS modification:

[0094] First, amphiphilic octylamine-grafted polyacrylic acid (OPA) was synthesized by acidifying 0.03 mol sodium polyacrylate, mixing it with 0.0096 mol octylamine and 0.015 mol 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, coupling it through an amide reaction, and purifying it to obtain the amphiphilic OPA.

[0095] Then, 2.5 mg of OPA chloroform solution and 1 nmol / L of PbS QDs chloroform solution were mixed by a thin film method, and the solvent was removed by vacuum rotary evaporation at room temperature. BR buffer was sonicated for 30 s to allow OPA to coat PbS QDs, and hydrophilic modification was achieved through hydrophobic interaction to obtain an OPA@PbS aqueous dispersion system, namely, OPA@PbS.

[0096] in, Figure 2 This is a schematic diagram of obtaining OPA@PbS after hydrophilic modification of PbS QDs in this example.

[0097] (3) Aminated PEG modification:

[0098] 10g of PEG2000 was activated with 17.2g of sulfonyl chloride, followed by aminolysis with 300mL of ammonia to prepare NH2-PEG2000-NH2. EDC / Sulfo-NHS and OPA@PbS were stirred in PBS at room temperature for 15min. The EDC / Sulfo-NHS system was used to activate the carboxyl groups on the surface of OPA@PbS (nQDs:nEDC:nSulfo-NHS = 1:1500:1500). NH2-PEG2000-NH2 was then added and reacted for 10h for covalent coupling (nQDs:nNH2-PEG2000-NH2 = 1:2000). Aminated OPA@PbS (containing PEG2000), namely PbS-NH2, was obtained after ultrafiltration purification.

[0099] (3) Polymannose chain M 50 synthesis:

[0100] Mannose monomer (D-mannose acyloxyethyl acrylamide) was prepared from 5 g of α-D-mannose pentaacetate by functionalization with 1.595 mL of acrylamide and deprotection with 42.87 mg of KOH. Subsequently, polymannose chains M were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. 50 (degree of polymerization 50), and purified by dialysis to obtain.

[0101] (4) Nanotracer assembly:

[0102] The synthesized PbS-NH2 and polymannose chain M 50 , thermosensitive poly N-isopropylacrylamide (PNIPAM) was coupled via EDC / Sulfo-NHS-mediated amide reaction, and EDC / Sulfo-NHS, M 50 Stir with PNIPAM in PBS solution at room temperature for 15 minutes, activate the carboxyl group using EDC / Sulfo-NHS system, and then add PbS-NH2 to react for 10 hours for covalent coupling. Where n(-NH2):n(-COOH):n(EDC)=1:1500:2500, polymannose chain M 50 The ratio of carboxyl groups to PNIPAM was 500:1000, and the sentinel lymph node nanotracer, namely PbS-PNIPAM / M 50 .

[0103] Example 2: Nanotracer

[0104] This embodiment provides a nano-tracer for tracing the sentinel lymph nodes in the axillary region of breast cancer and a preparation method thereof. The difference between this embodiment and embodiment 1 is that during the assembly of the nano-tracer, the polymannose chain M 50 The ratio of carboxyl groups to PNIPAM was 750:750, and the rest of the steps were the same.

[0105] Example 3: Nanotracer

[0106] This embodiment provides a nano-tracer for tracing the sentinel lymph nodes in the axillary region of breast cancer and a preparation method thereof. The difference between this embodiment and embodiment 1 is that during the assembly of the nano-tracer, the polymannose chain M 50 The ratio of carboxyl groups to PNIPAM was 1000:500, and the rest of the steps were the same.

[0107] Comparative Example 1: Indocyanine Green

[0108] This comparative example provides a sentinel lymph node tracer indocyanine green (ICG), which was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0109] Test Example 1: Hydration Particle Size and Zeta Potential Measurement

[0110] This test example is to test the PbS QDs, OPA@PbS, PbS-NH2 and PbS-PNIPAM / M prepared in Example 1. 50 The hydrated particle size of OPA@PbS, PbS-NH2 and PbS-PNIPAM / M 50 The zeta potential was detected.

[0111] Test results such as Figure 3 As shown, A is the hydrated particle size test result, B is the Zeta potential test result, the above results show that with the physical and chemical modification of PbS (wrapping OPA, grafting NH2-PEG-NH2, grafting PNIPAM and M 50 ), the particle size and distribution changed, indicating that the presence of the modification layer increased the particle size and changed the particle surface charge.

[0112] Test Example 2: In Vivo Lymphatic System Imaging

[0113] This test example tests the in vivo lymphatic system imaging effect of the sentinel lymph node tracer of Examples 1 to 3 and Comparative Example 1, as follows:

[0114] In this case, the lower limb lymphatic system of 6-week-old female nude normal mice was used as an evaluation model. After the sentinel lymph node tracer is injected into the mouse footpad, it will first enter the popliteal lymph nodes through lymphatic drainage, thus the popliteal lymph nodes are the sentinel lymph nodes (SLN) in the footpad area. The tracer will then further enter the sacral lymph nodes, the next level of lymph nodes, and the iliac lymph nodes, the third level of lymph nodes. Ultimately, the lymph fluid containing the tracer will flow into the mouse lumbar trunk. Specifically, the in vivo lymphatic system imaging method includes: fixing an injection needle connected to a catheter to the right footpad of the mouse, while the other end of the catheter is connected to a microsyringe, and injecting 20μL of the sentinel lymph node tracer into the right footpad of the mouse through the microsyringe, and imaging in real time, while recording images at different time points.

[0115] Figure 4 PbS-PNIPAM / M prepared in Example 1 50 Real-time imaging of the lymphatic system of indocyanine green (ICG) at different in vivo time points (0, 15, 30, 45, 60, 75, and 90 min) compared to Comparative Example 1. The upper part shows the whole-body fluorescence imaging of the mouse, and the lower part shows the magnified fluorescence imaging of the mouse lymphatic system. Figure 5 Inject ICG or PbS-PNIPAM / M into the right footpad of mice 50 After 15 minutes, lymph nodes related to the lower limb lymphatic system were obtained for NIR-II imaging (a, b, and c in the figure are popliteal lymph nodes, sacral lymph nodes, and iliac lymph nodes, respectively).

[0116] The above imaging results show that ICG and nano-tracer PbS-PNIPAM / M 50 After the footpad injection, they quickly converged in the popliteal lymph nodes, and both showed the brightest fluorescence signals at about 15 minutes. Among them, within 15 to 90 minutes after the injection of ICG, the lymphatic vessel imaging between the popliteal lymph nodes and the sacral lymph nodes can be observed. The fluorescence imaging of the sacral lymph nodes is more obvious, and the fluorescence signal of the popliteal lymph nodes gradually weakened. The fluorescence signal of the sacral lymph nodes also weakened synchronously, indicating that ICG did not stay in the sentinel lymph nodes but continued to drain to the secondary lymph nodes. At the same time, in the fluorescence images at 30 minutes and 45 minutes, fluorescence signals can be observed in the mouse liver, indicating that ICG is quickly injected into the circulatory system through lymphatic drainage and metabolized after being enriched in the liver, and its stability is relatively poor. After the injection of the nanotracer PbS-PNIPAM / M 50 At 15 minutes, the drainage lymphatic vessels between the foot pad and the popliteal lymph nodes can be clearly observed. 50Within 15 to 90 minutes, the fluorescence signal of the popliteal lymph node tended to be stable without significant changes; and the fluorescence signal of the sacral lymph node was weaker than that of ICG imaging and also tended to be stable, indicating that PbS-PNIPAM / M 50 Most of it can accumulate in the sentinel lymph nodes, and only a small amount enters the secondary lymph nodes, indicating that it has good targeting specificity and stability.

[0117] Figure 6 、 Figure 7 and Figure 8 The nano-tracers PbS-PNIPAM / M containing different ratios of PNIPAM and polymannan chains prepared in Examples 1 to 3 are respectively 50 In vivo lymphatic system node imaging (excitation light λex = 750 nm, laser power 5 W, exposure time 5000 ms, long-pass filter 1200 nm), the time points are (1, 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 60, 72, 84, 96, 108 and 120 h), Figure 9 The imaging results show that at a relatively high PNIPAM ratio (1000:500), the tracer tends to slowly accumulate and accumulate in the lymph nodes, where it remains for a long time, exhibiting a bright fluorescence signal and a long fluorescence window. At a relatively low PNIPAM ratio (500:1000), the tracer does not show up in the draining lymph nodes. At a ratio of 750:750, the tracer's accumulation is intermediate between the two, with weak visualization in the draining lymph nodes but still maintaining a long retention time.

[0118] Test Example 3: Fluorescence Penetration Depth and Quality Testing

[0119] This test example is to test the PbS-PNIPAM / M prepared in Example 1 above. 50 The fluorescence penetration depth and quality of indocyanine green (ICG) of comparative example 1 were tested, and the specific method is as follows:

[0120] In order to intuitively 50 The fluorescence penetration depth was compared with that of ICG. In this case, fat emulsion was used to simulate the in vivo environment, and capillary pipette was used to simulate the in vivo pipeline. 50 Capillary pipettes containing TNF-α and ICG were placed in fat emulsions at different depths (0 mm, 1 mm, 2 mm, 3 mm, 4 mm, and 5 mm), and then near-infrared fluorescence imaging was performed.

[0121] Among them, PbS-PNIPAM / M 50The peak wavelength range of the fluorescence emission spectrum is between 1200-1450nm, and the maximum emission wavelength is 1345nm; ICG is a commonly used near-infrared fluorescent dye in clinical practice, and its emission light is in the NIR-I region.

[0122] Figure 10 for ICG and PbS-PNIPAM / M 50 The results of NIR fluorescence imaging at different depths of fat emulsion show that ICG presents a clearer fluorescence signal at 0mm and 1mm depth, which appears as a bright line. However, as the depth increases to 2mm and 3mm, the fluorescence signal is significantly weakened and becomes blurred, and the contrast and clarity of the image decrease (e.g. Figure 10 This indicates that the fluorescence signal of ICG decays faster when propagating in deeper tissues or media. 50 In the depth range of 0mm-3mm, its fluorescence signal is brighter and clearer than that of ICG at the same depth, and relatively obvious fluorescence signals can still be observed at the depth of 4mm and 5mm (such as Figure 10 As shown in B in the figure, although the intensity has weakened, the image is still highly recognizable, indicating that PbS-PNIPAM / M 50 The ability of fluorescence signal propagation in deep tissues is better than that of ICG.

[0123] Figure 11 for ICG and PbS-PNIPAM / M 50 Fluorescence intensity distribution diagram at different depths of fat emulsion, where the excitation light λex = 750nm, laser power 5W, exposure time 1000ms, long pass filter 1200nm. The results show that in the ICG group, as the depth increases from 0mm to 3mm, the peak intensity of the curve gradually decreases, and the shape of the curve becomes flatter (e.g. Figure 11 As shown in A in the figure, this means that the fluorescence intensity of ICG decays rapidly with increasing depth, and the fluorescence intensity becomes weaker at deeper depths, which is not conducive to deep imaging. 50 The fluorescence intensity of the group is at a depth of 0 mm to 3 mm, and the peak intensity of the curve is high and the change is relatively small (e.g. Figure 11 The fluorescence intensity is relatively stable within this depth range. At the depths of 4 mm and 5 mm, although the peak intensity of the curve decreases slightly, it is still higher than that of ICG at the same depth, further indicating that PbS-PNIPAM / M 50 A certain fluorescence intensity can still be maintained in deep layers, resulting in better imaging effects.

[0124] Figure 12 for ICG and PbS-PNIPAM / M 50Fluorescence intensity distribution histogram and signal-to-noise ratio comparison diagram under different depths of fat emulsion, where A is the fluorescence intensity distribution histogram of ICG and PbS-PNIPAM / M 50 B is the fluorescence intensity histogram of ICG and PbS-PNIPAM / M 50 The results show that at each depth point, PbS-PNIPAM / M 50 The fluorescence intensity of PbS-PNIPAM / M 50 The fluorescence intensity of PbS-PNIPAM / M is several times that of ICG, and the difference in fluorescence intensity between the two still exists as the depth increases, which directly reflects the 50 The advantage in fluorescence signal intensity helps to obtain more effective signals in deep imaging (such as Figure 12 Figure 3 (a) shows ICG and PbS-PNIPAM / M 50 The SNR comparison chart shows that with the increase of depth, both curves show a downward trend, indicating that the imaging SNR of the two materials will decrease with the increase of depth. However, within the depth range of detection, PbS-PNIPAM / M 50 The curves of PbS-PNIPAM / M 50 The signal-to-noise ratio of PbS-PNIPAM / M is increased compared with that of ICG. 50 It can provide clearer images with less noise interference, and its imaging quality is superior to ICG.

[0125] Test Example 4: Testing the Effect of Temperature on Fluorescence Imaging Time and Quality

[0126] In order to clarify the nanotracer PbS-PNIPAM / M 50 Thermosensitive properties, and the application of its thermosensitive properties in SLN NIR-II imaging was tested. In this example, the PbS-PNIPAM / M prepared in Example 1 was used. 50 The lower limb lymphatic system of 6-week-old female normal SD rats was used as the evaluation model. The rats were divided into two groups to test the effect of temperature on the nanotracer PbS-PNIPAM / M 50 The impact of NIR-II fluorescence imaging time and quality. The specific method is as follows:

[0127] Group 1: First, the left lower limb of anesthetized SD rats was locally iced (about 10 minutes). After the ice pack was removed, 100 μL of nanotracer PbS-PNIPAM / M 50The nanoparticles were injected into the rat's left footpad and subjected to real-time NIR-II fluorescence imaging after rewarming. To more intuitively demonstrate the transport status of the nanotracer in the lower limb lymphatic system as temperature changes, the fluorescence signal was adjusted to a black signal in this example. The detection parameters were set to excitation light λex = 750nm, laser power 5W, exposure time 5000ms, long-pass filter 1200nm, and frame rate 0.198fps.

[0128] Group 2: First, the left lower limb of anesthetized SD rats was locally iced (about 10 minutes). After the ice pack was removed, 100 μL of nanotracer PbS-PNIPAM / M 50 The nanoparticles were injected into the rat's left paw pad and then iced for 30 minutes before the ice pack was removed. The rats were then rewarmed to 36°C using a heating pad while undergoing real-time NIR-II fluorescence imaging. To more intuitively demonstrate the transport status of the nanotracer in the lower limb lymphatic system as temperature changes, the fluorescence signal was adjusted to a black signal in this example. The detection parameters were set to excitation light λex = 750nm, laser power 5W, exposure time 5000ms, long-pass filter 1200nm, and frame rate 0.198fps.

[0129] Figure 13 Demonstrated the temperature-controlled nanotracer PbS-PNIPAM / M 50 Real-time imaging of the lymphatic system in vivo, where Figure 13 Figure A shows the injection of nanotracer PbS-PNIPAM / M 50 NIR-II fluorescence imaging images at different time points (0-1500s) show that the black arrow points to the drainage lymphatic vessels between the foot pad and the popliteal lymph node. Starting from 150s, a clear black fluorescent signal can be seen in the drainage lymphatic vessels. As time goes by, during the period of 150-450s, the intensity and distribution range of the fluorescence signal of the drainage lymphatic vessels change relatively little, indicating that within 0-450s, the nanotracer is rapidly diffused and transported to the surrounding lymphatic tissue. During the period of 600-1050s, the fluorescence signal of the drainage lymphatic vessels decreases rapidly; at 1050-1200s, the black fluorescent signal of the drainage lymphatic vessels is almost no longer observable. A faint black fluorescent signal can be seen in the popliteal lymph nodes at 450s; and as time goes by, its fluorescent signal value increases rapidly, indicating that the nanotracer accumulates rapidly in the popliteal lymph nodes; at 1200-1500s, its fluorescent signal value remains stable, indicating that the nanotracer in the popliteal lymph nodes accumulates at the SLN and does not spread to the next level of lymph nodes.

[0130] Figure 13 B is the low-temperature nano-tracer PbS-PNIPAM / M 50Real-time NIR-II imaging of the left lower limb of SD rats injected with the drug, followed by ice application and rewarming, revealed a distinct black fluorescent signal in the draining lymphatic vessels at 150 seconds. This signal rapidly weakened between 300 and 600 seconds, and remained extremely weak in the draining lymphatic vessels between 600 and 900 seconds. At subsequent observation time points, no significant fluorescent signal was observed in the draining lymphatic vessels. Furthermore, the fluorescent signal in the popliteal lymph nodes remained relatively strong.

[0131] The above test results show that the nano-tracer PbS-PNIPAM / M 50 In the low temperature state, it helps to drain the lymph nodes quickly and continuously. As the temperature gradually rises to around body temperature (including self-warming and external heating pad), the drainage of the nanotracer gradually slows down. The external heating pad can quickly stop the drainage, indicating that the nanotracer PbS-PNIPAM / M 50 The rate of drainage slowing down is related to the speed of rewarming. Therefore, in practical applications, the nano-tracer PbS-PNIPAM / M 50 The effect and accuracy of lymphatic imaging can be improved by controlling temperature conditions (local ice compress, hot compress).

[0132] In summary, the present invention provides a sentinel lymph node nanotracer, its preparation method, and application. This nanotracer combines the high-resolution imaging advantages of quantum dots with their temperature-responsive aggregation properties and receptor-mediated targeted delivery mechanism. On the one hand, it can significantly enhance the efficiency of nanotracer enrichment in lymph nodes, reduce non-lymph node uptake, and achieve deeper tissue penetration and a high signal-to-noise ratio. On the other hand, through local temperature regulation, it can controllably aggregate the nanotracer within the sentinel lymph node, preventing non-sentinel lymph node spread and increasing the duration of sentinel lymph node imaging. Thus, the sentinel lymph node nanotracer of the present invention effectively extends the imaging time window while achieving precise sentinel lymph node localization, providing a new design approach for the development of intelligent, responsive surgical navigation probes.

[0133] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A sentinel lymph node nanotracer, characterized in that: The raw materials for preparation include: Quantum dot materials, amphiphilic polymers, catalysts, diamino polymers, macrophage CD206 receptor targeting molecules, and temperature-sensitive polymers with near-infrared second-region luminescence properties; Wherein, the carboxyl ratio of the macrophage CD206 receptor targeting molecule to the temperature-sensitive polymer is 500-1000:100-5000.

2. The sentinel lymph node nanotracer according to claim 1, characterized in that The quantum dot material having near-infrared second-region luminescence characteristics is selected from one or more combinations of lead sulfide quantum dots, lead selenide quantum dots, indium arsenide quantum dots, silver sulfide quantum dots, and lead telluride quantum dots; And / or, the amphiphilic polymer is selected from one or more combinations of octylamine grafted polyacrylic acid, polyacrylic acid-hyperbranched polyester complex, alkylamine grafted polycarboxylic acid polymer, polyethylene glycol-based amphiphilic copolymer, polyacrylic acid-poly N-isopropylacrylamide-octylamine graft; And / or, the catalyst is selected from one or more combinations of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysulfosuccinimide, N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; And / or, the bisamino polymer is selected from one or more combinations of bisamino polyethylene glycol molecules, bisamino polylysine, bisamino polyethyleneimine, bisamino block copolymers, and bisamino polyacrylic acid grafts; And / or, the macrophage CD206 receptor targeting molecule is selected from one or more combinations of polymannose, mannose-modified protein, mannose-modified antibody, mannose liposome, thiomannose, CD206 aptamer, cysteine-containing targeting peptide, and CD206 targeting antibody fragment; And / or, the temperature-sensitive polymer is selected from one or more combinations of poly (N-isopropylacrylamide), vinyl methyl ether, polyethylene glycol-polypropylene glycol block copolymer, polydiethylacrylamide, and poly (N-vinylcaprolactam).

3. The sentinel lymph node nanotracer according to claim 1 or 2, characterized in that The mass molar ratio of the amphiphilic polymer to the quantum dot material having near-infrared second-region luminescence characteristics is 0.1-5 mg: 0.1-10 nmol / L.

4. A method for preparing the sentinel lymph node nanotracer according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, mixing the amphiphilic polymer with the quantum dot material having near-infrared second region luminescence characteristics, and reacting them to obtain an amphiphilic polymer-coated quantum dot material; S2, mixing the amphiphilic polymer-coated quantum dot material with a diamino polymer, and performing a coupling reaction to obtain an amino-modified amphiphilic polymer-coated quantum dot material; S3. Under the catalyst conditions, the quantum dot material coated with the amino-modified amphiphilic polymer, the macrophage CD206 receptor targeting molecule and the temperature-sensitive polymer are mixed, and after amide coupling reaction, purification is performed to obtain.

5. The preparation method according to claim 4, characterized in that The quantum dot material having near-infrared second-region luminescence characteristics is lead sulfide quantum dots; The preparation method of the lead sulfide quantum dots comprises: mixing a lead precursor and a sulfur precursor, reacting and quenching, and obtaining the lead sulfide quantum dots after graded purification.

6. The preparation method according to claim 4, characterized in that The amphiphilic polymer is octylamine grafted polyacrylic acid; The preparation method of the octylamine grafted polyacrylic acid comprises: acidifying sodium polyacrylate, mixing it with octylamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, performing coupling reaction, and purifying to obtain the product.

7. The preparation method according to claim 4, characterized in that The catalyst is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide.

8. The preparation method according to claim 4, characterized in that The macrophage CD206 receptor targeting molecule is polymannose, and the polymerization degree of the polymannose is 20-100.

9. The preparation method according to any one of claims 4 to 8, characterized in that In step S2, the catalysts used in the coupling reaction are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide.

10. Use of the sentinel lymph node nanotracer according to any one of claims 1 to 3 or the preparation method of the sentinel lymph node nanotracer according to any one of claims 4 to 9 in any of the following: A) Preparation of products for sentinel lymph node detection; B) Screening for drugs related to sentinel lymphoma treatment; C) Preparation of a reagent for detecting axillary sentinel lymph nodes in breast cancer.

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