1,3-Dithioheterocyclic fused naphthalenetetracarboxylic acid diimide derivatives, near-infrared II photothermal molecules containing them, their preparation methods and applications

Abstract

This invention discloses a 1,3-dithioheterocyclic fused naphthalenetetracarboxylic acid diimide derivative, a near-infrared II photothermal molecule containing it, its preparation method, and its applications, belonging to the field of biomedical technology. The thioheterocyclic fused naphthalenetetracarboxylic acid diimide derivative provided by this invention has a donor-acceptor-donor configuration. The thioheterocyclic fused naphthalenetetracarboxylic acid diimide derivative and doxorubicin are co-encapsulated in a temperature / pH dual-responsive nanogel matrix to form a PND core. Nasopharyngeal carcinoma tumor cell membranes expressing growth arrest-specific gene 6 are coated on the PND surface to construct a nanodecoy with homologous targeting capability. This integrates four major functions—homological targeted delivery, dual-mode imaging guidance, local chemotherapy-photothermal synergistic therapy, and systemic metastasis inhibition—into a single platform, simultaneously tackling the dual challenges of in situ treatment of nasopharyngeal carcinoma and prevention and control of lung cancer cell metastasis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to 1,3-dithia-fused naphthalene tetracarboxylic diimide derivatives, near-infrared two-region photothermal molecules containing the same, and a preparation method and application thereof. BACKGROUND

[0002] Nasopharyngeal carcinoma (NPC) is a highly metastatic malignant tumor derived from nasopharyngeal epithelium, and its incidence is particularly significant in East Asia and Southeast Asia. Its occurrence is closely related to dietary habits, long-term smoking and chronic inflammatory stimulation. Early-stage NPC can cause distant metastasis to organs such as lung, bone and brain, leading to organ dysfunction and significantly increasing the risk of death. Current conventional radiotherapy and chemotherapy strategies face bottlenecks due to low treatment efficiency, high recurrence rate and poor prognosis, especially the lack of effective metastasis prevention means.

[0003] At the molecular mechanism level, the Gas6 / AXL signaling axis has been confirmed to be a key pathway driving nasopharyngeal carcinoma cell proliferation and migration. Current clinical interventions mainly rely on AXL inhibitors (such as Foretinib, Cabozantinib, Bemcentinib, etc.) to block this pathway, but there are limitations such as high off-target toxicity, low kinase selectivity, and unstable response rate of combined immunotherapy. Although nanodiagnosis and treatment science has attempted to develop new nanomaterials to improve the stability of AXL inhibitors or inhibit the Gas6 / AXL signaling axis, these designs still lack the ability to actively kill tumors. For example, covalent organic frameworks (COFs) carry osimertinib to improve drug resistance, and non-Aβ peptide derivative nanofibers inhibit Gas6 / AXL binding. Although these designs have improved the influence of the material on the Gas6 / AXL signaling axis, they do not have the ability to actively initiate tumor killing. SUMMARY

[0004] To solve the above technical problems, the present application provides 1,3-dithia-fused naphthalene tetracarboxylic diimide derivatives, near-infrared two-region photothermal molecules containing the same, and a preparation method and application thereof. The present application provides a four-function integrated (homologous targeting delivery, dual-mode imaging guidance, local chemotherapy-photothermal synergistic treatment, and systemic metastasis inhibition) dual-responsive near-infrared two-region (NIR-II) intelligent nanobait PND@Gas6(+): first, the cell membrane of nasopharyngeal carcinoma tumor cells (Gas6@5-8F) that highly express growth arrest-specific gene 6 is disguised to achieve homologous targeting delivery for precise accumulation of metastatic lesions; at the same time, NIR-II fluorescence / photothermal dual-mode imaging is integrated to monitor the lesions in real time; then, acid / pH dual-responsive release of chemotherapy drugs and activation of photothermal effect are used to synergistically kill primary tumors; finally, the "ligand decoy" strategy is used to competitively inhibit the Gas6 / AXL axis to block lung metastasis colonization, thereby simultaneously solving the dual problems of nasopharyngeal carcinoma in situ clearance and systemic metastasis prevention, and providing a revolutionary solution for cancer diagnosis and treatment integration.

[0005] To achieve the above object, the present application provides the following technical solutions.

[0006] The present application provides a 1,3-dithia ring-fused naphthalene tetracarboxylic diimide derivative, the structure general formula is shown as formula I:

[0007]

[0008] Formula I

[0009] Wherein, R1 is selected from C 14 Straight chain or C2-C 10 Branch chain; R1 is preferably , Or-C 14 H 29 .

[0010] The present application also provides a preparation method of the above-mentioned 1,3-dithia ring-fused naphthalene tetracarboxylic diimide derivative, comprising the following steps: reacting sodium hydride and compound A under ice bath condition, then adding carbon disulfide to continue the reaction at room temperature, then adding N-alkyl R1 substituted 2,3,6,7-tetrabromonaphthalene tetracarboxylic diimide to react, after the reaction is completed, using saturated ammonium chloride solution to end the reaction, using dichloromethane or ethyl acetate to extract, drying the organic phase with anhydrous sodium sulfate, then rotary evaporation, and separating the residue by column chromatography to obtain the 1,3-dithia ring-fused naphthalene tetracarboxylic diimide derivative;

[0011] The structural formula of the compound A is ;

[0012] The structural formula of the N-alkyl R1 substituted 2,3,6,7-tetrabromonaphthalene tetracarboxylic diimide is: , R1 is selected from C 14 Straight chain or C2-C 10 Branch chain; R1 is preferably , Or-C 14 H 29 .

[0013] Further, in the preparation method of the 1,3-dithia ring-fused naphthalene tetracarboxylic diimide derivative, the molar ratio of sodium hydride, compound A and carbon disulfide is 2: (1-2): (1-2);

[0014] The molar ratio of the N-alkyl R1 substituted 2,3,6,7-tetrabromonaphthalene tetracarboxylic diimide and compound A is 1: (8-12).

[0015] Further, the reaction time of sodium hydride and compound A under ice bath condition (0℃) is 1 hour; after adding N-alkyl R1 substituted 2,3,6,7-tetrabromonaphthalene tetracarboxylic acid diimide, the reaction time is 4 hours.

[0016] The application further provides a preparation method of the near-infrared two-region photothermal molecule, comprising the following steps: dissolving a 1,3-dithia annellated naphthalene tetracarboxylic acid diimide derivative, compound B, a deprotonating agent, a ligand and a catalyst in an organic solvent, performing a reflux reaction, and performing post-treatment after the reaction is completed to obtain the near-infrared two-region photothermal molecule.

[0017] The compound B has a structural formula as shown in the following formula: 、 or .

[0018] The near-infrared two-region photothermal molecule is prepared from the above-mentioned 1,3-dithia annellated naphthalene tetracarboxylic acid diimide derivative, and is a near-infrared two-region photothermal molecule based on a dithia annellated naphthalene tetracarboxylic acid diimide derivative. The near-infrared two-region photothermal molecule has a donor-acceptor-donor (D-A-D) configuration, uses a dithia naphthalene tetracarboxylic acid diimide containing a long alkyl chain as an electron acceptor, and has quinone electron delocalization characteristics in a large conjugated main chain, thereby significantly reducing a molecular band gap and realizing long-wave near-infrared light absorption capacity. Meanwhile, triphenylamine is used as an electron donor and a flexible molecular rotor, a strong twisted intramolecular charge transfer (TICT) effect is formed through electron-rich characteristics, and near-infrared two-region light-emitting efficiency and photothermal conversion performance are synergistically improved. A long alkyl chain at the electron acceptor end effectively blocks excessive accumulation of molecules, and completely eliminates problems of imaging signal-to-noise ratio reduction and light stability degradation caused by aggregation-induced quenching (ACQ). The alkyl chain at the donor end endows triphenylamine with greater rotational freedom, and inhibits intermolecular interaction in an aggregated state, so that near-infrared two-region fluorescence emission and efficient photothermal conversion are simultaneously realized under laser irradiation, and high light stability and anti-quenching characteristics are achieved, thereby providing key support for construction of a dual-response near-infrared two-region intelligent nanobait PND@Gas6(+).

[0019] Chinese patent CN119708007A discloses a near-infrared emitting aggregation-induced emission organic photothermal molecule and a preparation method thereof. The patent uses a three-step reaction to prepare the near-infrared emitting aggregation-induced emission organic photothermal molecule: first, a first step reaction is addition annelation, and is prepared; then, a second step reaction is: a reducing agent (iron powder) is added, and a nitro group (-NO2) is reduced into an amino group (-NH2), and is prepared; and a third step reaction is: a (R' is selected from one of H, methyl and methoxy), Buchwald-Hartwig carbon-nitrogen coupling reaction is carried out to generate the final near-infrared emission aggregation-induced emission organic photothermal molecule, which is an infrared two-region photothermal molecule based on sulfur heterocycle-fused naphthalene tetracarboxylic diimide derivative. Compared with the patent, the preparation method of the infrared two-region photothermal molecule based on sulfur heterocycle-fused naphthalene tetracarboxylic diimide derivative is reduced from three steps to two steps (first, 1,3-disulfur heterocycle-fused naphthalene tetracarboxylic diimide derivative is prepared, and then the infrared two-region photothermal molecule based on sulfur heterocycle-fused naphthalene tetracarboxylic diimide derivative is prepared by using the 1,3-disulfur heterocycle-fused naphthalene tetracarboxylic diimide derivative), and the reactant conditions are optimized, the reaction process is simplified, and the conversion rate of the reaction raw material to the final structure is improved.

[0020] Further, the structural formula of the near-infrared two-region photothermal molecule is shown as formula II:

[0021]

[0022] Formula II

[0023] wherein, R1 is selected from C 14 linear or C2-C 10 branched chain, R1 is preferably , or -C 14 H 29 ; R2 is selected from one of diphenylamine, 4,4'-dimethoxydiphenylamine and 4,4'-dimethyldiphenylamine.

[0024] Further, the near-infrared two-region photothermal molecule is selected from the following structures:

[0025] , , , or .

[0026] Further, in the preparation method of the near-infrared two-region photothermal molecule, the molar ratio of the 1,3-disulfur heterocycle-fused naphthalene tetracarboxylic diimide derivative, compound B, deprotonating agent, ligand and catalyst is 1: (3-4): (4-6): (0.3-0.8): (0.15-0.45).

[0027] Further, in the preparation method of the near-infrared two-region photothermal molecule, the deprotonating agent is selected from sodium tert-butoxide or potassium tert-butoxide;

[0028] the ligand is selected from tri-tert-butylphosphonium tetrafluoroborate, 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl or 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl;

[0029] the organic solvent is toluene;

[0030] the catalyst is selected from tris(dibenzylideneacetone)dipalladium(0) or palladium acetate.

[0031] The application further provides a use of the above-mentioned near-infrared two-region photothermal molecule in the preparation of a double-response near-infrared two-region intelligent nanobait, a preparation method of the double-response near-infrared two-region intelligent nanobait, comprising the following steps:

[0032] N-isopropyl acrylamide (NIPAM), acrylic acid, a crosslinking agent and a surfactant are mixed in water, heated under magnetic stirring to obtain a mixture, oxygen in the mixture is removed, an initiator is added, and a polymerization reaction is carried out, after the polymerization reaction, the temperature / pH double-response (PNA) nanogel is obtained through dialysis purification and drying;

[0033] The above-mentioned near-infrared two-region photothermal molecule is dissolved in tetrahydrofuran to obtain a solution;

[0034] The solution is added to a Tris buffer solution containing the temperature / pH double-response nanogel, and the mixture is left overnight to obtain a nanogel, which is denoted as PN nanogel;

[0035] An anti-tumor drug solution is mixed with a dispersion of the nanogel, stirred, ultrafiltered and washed with water to obtain an anti-tumor drug-loaded nanogel;

[0036] The anti-tumor drug-loaded nanogel is coated with a cell membrane of a nasopharyngeal carcinoma tumor (Gas6@5-8F) cell with high expression of growth arrest-specific gene 6 to obtain the double-response near-infrared two-region intelligent nanobait.

[0037] Further, in the preparation method of the double-response near-infrared two-region intelligent nanobait, the amount of acrylic acid added is 5% of the molar amount of N-isopropyl acrylamide, the amount of crosslinking agent added is 5% of the molar amount of NIPAM, and the amount of surfactant added is 1.5% of the molar amount of NIPAM.

[0038] Further, in the preparation method of the double-response near-infrared two-region intelligent nanobait, the crosslinking agent is N,N'-methylenebisacrylamide, and the surfactant is sodium dodecyl sulfate.

[0039] Further, in the preparation method of the dual-response near-infrared two-region intelligent nanobait, the antitumor drug is doxorubicin (Dox). When the antitumor drug is Dox, the nanogel loaded with the antitumor drug is denoted as PND nanogel.

[0040] The application also provides application of the above-mentioned dual-response near-infrared two-region intelligent nanobait in preparation of drugs for diagnosing or treating in-situ tumors and in-situ metastatic tumors and avoiding early tumor metastasis. That is, the dual-response near-infrared two-region intelligent nanobait of the application can diagnose or treat in-situ tumors and in-situ metastatic tumors and avoid early tumor metastasis.

[0041] Compared with the prior art, the application has the following advantages and technical effects:

[0042] The application provides a method for preparing an infrared two-region photothermal molecule based on a sulfur-heterocycle-fused naphthalene tetracarboxylic diimide derivative by a two-step method, which is simple and efficient, and the obtained novel infrared two-region photothermal molecule has near-infrared two-region luminescence performance and excellent photothermal performance. Meanwhile, the preparation process of the dual-response near-infrared two-region intelligent nanobait PND@Gas6(+) in the application is simple and has excellent performance. First, Gas6@5-8F cell membrane camouflage is used to realize homologous targeted delivery and precise accumulation of metastatic foci; meanwhile, NIR-II fluorescence / photothermal dual-mode imaging is integrated to realize real-time monitoring of the foci; then, acid / pH dual-response is used to release chemotherapeutic drugs and activate photothermal effects to cooperatively kill primary tumors; at the same time, the growth-arrest-specific gene 6 (Gas6) protein overexpressed on the cell membrane surface plays a "molecular bait" core function, competitively binds to AXL with high affinity in a lung metastasis microenvironment, strongly blocks the Gas6 / AXL signal axis activation pathway, and precisely inhibits the migration, invasion and colonization abilities of tumor cells from a molecular source, so that the four functions of homologous targeted delivery, dual-mode imaging guidance, local chemotherapy-photothermal cooperative treatment and systemic metastasis inhibition are integrated in a single platform, and the dual problems of nasopharyngeal carcinoma in-situ treatment and lung cancer cell metastasis prevention and control are simultaneously solved, thereby providing a solution for cancer diagnosis and treatment integration. BRIEF DESCRIPTION OF DRAWINGS

[0043] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and serve as an explanation of the illustrative embodiments of the present application, and do not constitute an improper limitation of the present application. In the drawings:

[0044] Figure 1 The proton nuclear magnetic resonance spectrum of the 1,3-dithiacycle-fused naphthalene tetracarboxylic diimide derivative 1 obtained in Example 1 is characterized;

[0045] Figure 2 The proton nuclear magnetic resonance spectrum of the near-infrared two-region photothermal molecule 4 of the sulfur-heterocycle-fused naphthalene tetracarboxylic diimide derivative obtained in Example 5 is characterized;

[0046] Figure 3 Carbon-13 nuclear magnetic resonance spectroscopic characterization of near-infrared two-region photothermal molecule 4 of the thia-fused naphthalene tetracarboxylic diimide derivative obtained in Example 5;

[0047] Figure 4 Mass spectroscopic characterization of near-infrared two-region photothermal molecule 4 of the thia-fused naphthalene tetracarboxylic diimide derivative obtained in Example 5;

[0048] Figure 5 Molecular absorption and fluorescence emission spectra of near-infrared two-region photothermal molecule 4 of the thia-fused naphthalene tetracarboxylic diimide derivative obtained in Example 5 in tetrahydrofuran solvent;

[0049] Figure 6 Scanning electron microscope image of the dual-responsive near-infrared two-region smart nanobait PND@Gas6(+) obtained in Example 11;

[0050] Figure 7 Photothermal heating and cooling test images of the dual-responsive near-infrared two-region smart nanobait PND@Gas6(+) obtained in Example 11 and its process product;

[0051] Figure 8 pH / temperature versus tumor drug Dox time-release rate curve of the dual-responsive near-infrared two-region smart nanobait PND@Gas6(+) obtained in Example 11;

[0052] Figure 9 Schematic diagram of the photothermal ability treatment (PTT) effect of the dual-responsive near-infrared two-region smart nanobait PND@Gas6(+) obtained in Example 11 in a subcutaneous tumor model;

[0053] Figure 10 Treatment effect diagram of the dual-responsive near-infrared two-region smart nanobait PND@Gas6(+) obtained in Example 11 in a subcutaneous tumor model;

[0054] Figure 11 Treatment effect diagram of the dual-responsive near-infrared two-region smart nanobait PND@Gas6(+) obtained in Example 11 in a lung metastatic tumor model. DETAILED DESCRIPTION

[0055] Various illustrative embodiments of the present application are now described in detail below. The following description includes specific details for the purpose of providing a thorough understanding of the present application. However, it will be apparent to those skilled in the art that the present application can be practiced without these specific details. In other instances, well-known structures and functions have not been described in detail in order to avoid obscuring the understanding of the present application.

[0056] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentrations, solvent amounts, and other compositional constraints, each intervening value of the category is also specifically included within the scope of the present application. The intervening values are each of

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the present specification will control.

[0058] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0059] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0060] Compared with patent CN119708007A, the preparation method of the near-infrared two-region photothermal molecule based on the sulfur-heterocycle-fused naphthalene tetracarboxylic diimide derivative in the present application is reduced from three steps to two steps (firstly preparing a 1,3-dithia-fused naphthalene tetracarboxylic diimide derivative, and then preparing an infrared two-region photothermal molecule based on the sulfur-heterocycle-fused naphthalene tetracarboxylic diimide derivative using the 1,3-dithia-fused naphthalene tetracarboxylic diimide derivative), and the reactant conditions are optimized, the reaction process is simplified, and the conversion rate (which can also be referred to as the synthesis yield) of the reaction raw material to the final structure is improved. The calculation method of the synthesis yield is as follows: the synthesis yield (%) of the two-step method = first-step yield A% x second-step yield B% x 100%, and the synthesis yield (%) of the three-step method = first-step yield A% x second-step yield B% x third-step yield C% x 100%. For example, the synthesis yield of NDA-1 ( ) in patent CN119708007A is 52% x 76% x 46% = 18.1%, while the synthesis yield of the same The synthesis yield of the structure (i.e. the structure of embodiment 10) is 49% x 66% = 32.3%, and the two-step method of the application improves the synthesis yield.

[0061] Compared with CN119708007A, the reaction conditions of the application are optimized as follows:

[0062] The method of CN119708007A:

[0063] ,

[0064] The method of the application:

[0065] .

[0066] In the following embodiments of the application, doxorubicin (Dox) is used as an antitumor drug to prepare a near-infrared second region smart nano-bait.

[0067] In the application, PNA is used as a temperature / pH dual-responsive nanogel matrix to provide a basic response framework for constructing an intelligent drug delivery system; PN nanogel introduces near-infrared second region photothermal molecules on the basis of PNA, endows the material with the dual functions of near-infrared second region imaging and photothermal conversion, and realizes the optical monitoring and energy conversion capabilities; PND (nanogel loaded with doxorubicin (Dox)) further integrates the chemotherapeutic drug doxorubicin (Dox) to form a trifunctional core, and simultaneously has the environment-responsive drug release, photothermal killing, and chemotherapy synergistic mechanism. By coating Gas6@5-8F cell membranes on the surface of PND, the nanosystem is endowed with key dual-core capabilities: on the one hand, based on the homologous recognition protein on the membrane surface, the active targeting and precise accumulation of in situ tumors are realized, which significantly improves the drug concentration at the tumor site; on the other hand, the membrane protein Gas6 is used as a “molecular decoy” to competitively bind to the metastatic Axl receptor, and the Gas6 / Axl signaling axis activation pathway is blocked, which inhibits tumor cell migration and colonization from the source, and finally solves the dual challenges of in situ clearance and metastasis prevention in a single platform.

[0068] In the following embodiments of the application, the preparation method of the doxorubicin solution is: dissolving doxorubicin (Dox) in PBS to obtain a solution with a concentration of 1 mg·mL -1 , which is the doxorubicin solution; the preparation method of the dispersion of PN nanogel is: dissolving PN nanogel in Tris buffer (15 mM, pH=8.5) and stirring uniformly.

[0069] In the following embodiments of the present application, the specific preparation process of Gas6@5-8F cell membrane membrane protein is as follows: the tumor cells with high expression of growth arrest-specific gene 6 are incubated in 1 M Tris-HCl buffer (pH=7.4) at 4℃ for 1 hour, then the cell lysate is collected and centrifuged at 500xg for 10 minutes, and then the supernatant is aspirated and ultrasonicated for 10 minutes; after ultrasonication, the sample is centrifuged at 10,000xg for 10 minutes, the supernatant is separated and collected, and then the component is subjected to ultracentrifugation at 100,000xg for 1 hour, and the precipitate is separated; after determining the protein concentration of the supernatant by using a BCA kit, the sample is resuspended with ultrapure water to a final concentration of 1 mg / mL, and finally the preparation of Gas6@5-8F cell membrane membrane protein is completed by ultrasonication for 30 seconds.

[0070] In the following embodiments of the present application, regarding the membrane camouflage of Gas6@5-8F cell membrane, the obtained Gas6@5-8F cell membrane membrane protein is ultrasonicated and dispersed in PBS, and then mixed with PND nanogel by ultrasonication for 3 minutes to obtain a mixture, and the mixture is subjected to at least five physical extrusion cycles of 400 nm polycarbonate membrane by Avatar extruder, and finally purified by ultrafiltration (molecular weight cut-off=100 kDa).

[0071] In the following embodiments of the present application, the compound is prepared according to the method disclosed in Chinese patent CN101885732A; the compound is prepared according to the method disclosed in Chinese patent CN101885732A; the compound is prepared according to the method disclosed in Chinese patent CN101885732A.

[0072] The present application firstly constructs a near-infrared two-zone photothermal molecule (which can also be called a near-infrared two-zone photothermal molecule) of a sulfur-heterocyclic fused naphthalene tetracarboxylic diimide derivative with a donor-acceptor-donor (D-A-D) configuration through molecular engineering technology, so as to realize significant near-infrared two-zone (NIR-II) fluorescence emission and excellent photothermal conversion efficiency by taking triphenylamine as a strong electron donor and a flexible rotor; then the near-infrared two-zone photothermal molecule is co-encapsulated with doxorubicin (Dox) in a temperature / pH dual-responsive PNA nanogel matrix to form a PND core, and then the membrane of nasopharyngeal carcinoma cells with high expression of growth arrest-specific gene 6 (Gas6) is coated on the surface of the PND to construct a nano-decoy with homologous targeting ability. The system is precisely enriched at the tumor site through homologous targeting in the nasopharyngeal carcinoma model, and the acidic microenvironment triggers the release of Dox to realize chemotherapy, while near-infrared irradiation activates the photothermal effect to directly kill the tumor and enhance drug release, forming a chemotherapy-photothermal synergistic treatment mechanism; further relying on the dual-mode functions of NIR-II fluorescence imaging and photothermal imaging to realize high-resolution real-time monitoring, and using the "decoy" strategy to make the nano-decoy competitively bind to AXL receptor tyrosine kinase (AXL), so as to block the Gas6 / Axl signal axis to prevent metastasis and proliferation. The present application integrates four functions of homologous targeting delivery, dual-mode imaging guidance, local chemotherapy-photothermal synergistic treatment and systemic metastasis inhibition into a single platform, simultaneously solves the dual problems of nasopharyngeal carcinoma in situ treatment and lung cancer cell metastasis prevention and control, and provides a breakthrough solution for cancer precision diagnosis and treatment integration.

[0073] Unless otherwise specified, the room temperature in the present application is 25±2℃.

[0074] The raw materials used in the embodiments of the present application are all commercially available.

[0075] The technical solutions of the present application are further described by the following examples. As an example, 5-8F tumor cells (human nasopharyngeal carcinoma cells) are purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing); Gas6 (high expression growth arrest-specific gene 6) is purchased from Wuhan Moli Biological Technology Co., Ltd.; nude mice are purchased from the Guangdong Provincial Animal Center; the BCA kit is purchased from Thermo Fisher Scientific; 4,5,9,10-tetrabromo-2,7-bis(2-octyldodecyl)benzo[lmn][3,8]phenalene-1,3,6,8(2H,7H)-tetraone is purchased from Guangzhou Yunsan Biochemical Technology Co., Ltd.; tris(dibenzylideneacetone)dipalladium(0) and palladium acetate are both purchased from Beijing Bailingwei Technology Co., Ltd.; toluene is purchased from Guangzhou Chemical Reagent Factory; 4-bromophenylacetonitrile, 4-iodophenylacetonitrile, diphenylamine, 4,4'-dimethoxydiphenylamine, 4,4'-dimethyldiphenylamine, sodium tert-butoxide and N,N-dimethylformamide are all purchased from Angene Chemicals.

[0076] It should be noted that the invention is not detailed in the place, which is the conventional operation means of the art, and is not the focus of the invention, for example, the method of transfecting luciferase into 5-8F cells is completed by using conventional method.

[0077] Example 1

[0078] The present embodiment provides a preparation method of 1,3-dithia-fused naphthalene tetracarboxylic diimide derivative 1, and the synthetic route is as follows:

[0079]

[0080] The specific steps are as follows:

[0081] Under the protection of nitrogen, sodium hydride (210 mg, 5.24 mmol) was added to 10 mL of redistilled N,N-dimethylformamide (DMF) and stirred under ice bath condition; then 2-(4-iodophenyl)acetonitrile (513 mg, 2.62 mmol) was added and reacted for 30 minutes, carbon disulfide (300 mg, 3.93 mmol) was added, the ice bath was removed and the reaction was continued at room temperature for 2 hours, the reaction solution changed from colorless to light green and gradually turned to brown; then 4,5,9,10-tetrabromo-2,7-bis(2-octyldodecyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetrone (300 mg, 0.262 mmol) was added at one time, the reaction solution was black purple after stirring at room temperature for 1 hour, the reaction was quenched with 20 mL of normal saline, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and rotary evaporated, the residue was separated by column chromatography with dichloromethane / petroleum ether (2:1, by volume, the same below), and finally the ink green solid product was obtained, which was 1,3-dithia-fused naphthalene tetracarboxylic diimide derivative 1, and the yield was 49%. 1 H NMR (500 MHz, CDCl3)7.88 (d, J = 8.0 Hz, 4H), 7.43 (d, J = 8.3 Hz, 4H), 4.21 – 4.11 (m, 4H), 2.01(s, 2H), 1.38 – 1.15 (m, 64H), 0.85 (d, J = 5.2 Hz, 12H). 13C NMR (126 MHz, CDC13) 162.40, 162.25, 148.13, 147.62, 147.46, 144.63, 143.90, 143.61, 143.38, 141.04, 140.49, 139.61, 132.45, 131.39, 128.08, 127.75, 127.67, 126.56, 126.46, 125.63, 125.35, 124.29, 121.88, 117.31, 115.56, 115.28, 101.64, 77.31, 77.05, 76.80, 46.05, 36.30, 31.98, 31.94, 31.90, 31.85, 31.57, 31.49, 31.40, 30.11, 29.74, 29.70, 29.67, 29.62, 29.56, 29.52, 29.39, 29.35, 29.31, 26.41, 22.72, 22.70, 22.68, 14.18, 0.04.

[0082] Example 2

[0083] This example provides a method for preparing 1,3-dithia-fused naphthalene tetracarboxylic diimide derivative 2, and the synthetic route is as follows:

[0084]

[0085] The specific steps are the same as those in Example 1, except that is replaced by an equal molar amount of The product 1,3-dithia-fused naphthalene tetracarboxylic diimide derivative 2 is prepared in this example with a yield of 31%.

[0086] Example 3

[0087] This example provides a method for preparing 1,3-dithia-fused naphthalene tetracarboxylic diimide derivative 3, and the synthetic route is as follows:

[0088]

[0089] The specific steps are the same as those in Example 1, except that is replaced by an equal molar amount of The product 1,3-dithia-fused naphthalene tetracarboxylic diimide derivative 3 is prepared in this example with a yield of 45%.

[0090] Example 4

[0091] The embodiment provides a preparation method of infrared two-region photothermal molecule 4 based on a sulfur-heterocycle-fused naphthalene tetracarboxylic diimide derivative, and a synthesis route is as follows:

[0092]

[0093] The specific steps are as follows:

[0094] Under nitrogen protection, compound (referred to as compound i, 100.0 mg, 0.07 mmol), 4,4'-dimethyldiphenylamine (41.4 mg, 0.21 mmol), sodium tert-butoxide (26.8 mg, 0.28 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropyl biphenyl (11.5 mg, 0.02 mmol) and tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3, 9.2 mg, 0.01 mmol) are dissolved in toluene, and reaction is carried out at 120 DEG C for 12 hours; after reaction is completed, cooling is carried out, extraction is carried out with dichloromethane, and washing is carried out with saturated brine for three times; after the organic phase is dried over anhydrous sodium sulfate, concentration is carried out under reduced pressure, and the residue is purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 2:1) to obtain green powder product, that is, infrared two-region photothermal molecule 4, and the yield is 48%. 1 H NMR (500 MHz, CDCl3) 7.40 (d, J = 8.4 Hz, 1H), 7.14(d, J = 8.2 Hz, 2H), 7.08 (d, J = 8.3 Hz, 2H), 7.01 (d, J = 8.7 Hz, 1H), 4.14(s, 1H), 2.35 (s, 3H), 2.03 (s, 1H), 0.82 (s, 4H). 13C NMR (126 MHz, CDCl3)162.27, 162.13, 152.63, 147.46, 146.30, 145.74, 132.16, 130.20, 129.81,128.01, 126.24, 125.83, 121.37, 115.46, 115.27, 115.14, 93.35, 46.02, 45.66,36.25, 32.82, 31.92, 31.87, 31.83, 31.50, 31.44, 31.09, 30.17, 30.11, 29.71,29.68, 29.64, 29.59, 29.56, 29.36, 29.33, 29.29, 28.46, 26.37, 23.91, 22.69,22.67, 22.17, 20.93, 15.18, 14.13, 12.66, -0.00, -9.57. MS (MALDI-TOF) [m / z]:calcd for C 72 H 94 N6O4S4, 1596.3220; found, 1596.0077.

[0095] Example 5

[0096]

[0097] The preparation method of the infrared II photothermal molecule 4 based on the thioheterocyclic fused naphthalenetetracarboxylic acid diimide derivative in this embodiment is the same as that in Example 4, the only difference being that compound i ( The following were replaced with an equimolar amount of 1,3-dithioheterocyclic fused naphthalenetetracarboxylic acid diimide derivative 1 (0.07 mmol), the 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl was replaced with 2.5 times the molar amount of tritert-butylphosphine tetrafluoroborate (0.05 mmol), and the tris(dibenzylideneacetone)dipalladium(0) was increased to twice the molar amount in Example 4 (0.02 mmol).

[0098] In this embodiment, the yield of near-infrared II photothermal molecule 4 was 54%.

[0099] Example 6

[0100] The preparation method of the infrared II photothermal molecule 4 based on the thioheterocyclic fused naphthalenetetracarboxylic acid diimide derivative in this embodiment is the same as that in Example 4, except that the deprotonating agent is potassium tert-butoxide (0.28 mmol) and the catalyst is palladium acetate (0.01 mmol).

[0101] The yield of the product near-infrared two-region photothermal molecule 4 prepared in this example is 35%.

[0102] Example 7

[0103] This example provides a preparation method of an infrared two-region photothermal molecule 5 based on a naphthalene tetracarboxylic acid diimide derivative fused with a sulfur heterocycle, and the synthetic route is as follows:

[0104]

[0105] The specific steps are the same as those in Example 4, except that compound i is replaced by an equal molar amount of compound ii ( ), and the yield of the product near-infrared two-region photothermal molecule 5 prepared in this example is 33%.

[0106] Example 8

[0107] This example provides a preparation method of an infrared two-region photothermal molecule 6 based on a naphthalene tetracarboxylic acid diimide derivative fused with a sulfur heterocycle, and the synthetic route is as follows:

[0108]

[0109] The specific steps are the same as those in Example 4, except that compound i is replaced by an equal molar amount of compound iii ( ), and the yield of the product near-infrared two-region photothermal molecule 6 prepared in this example is 21%.

[0110] Example 9

[0111] This example provides a preparation method of an infrared two-region photothermal molecule 7 based on a naphthalene tetracarboxylic acid diimide derivative fused with a sulfur heterocycle, and the synthetic route is as follows:

[0112]

[0113] The specific steps are the same as those in Example 5, except that is replaced by an equal molar amount of , and the yield of the product near-infrared two-region photothermal molecule 7 prepared in this example is 33%.

[0114] Example 10

[0115] This example provides a preparation method of an infrared two-region photothermal molecule 8 based on a naphthalene tetracarboxylic acid diimide derivative fused with a sulfur heterocycle, and the synthetic route is as follows:

[0116]

[0117] The specific steps are the same as those in Example 5, except that the compound i is replaced by an equal molar amount of the compound 1 prepared in Example 1 (0.07 mmol), and the is replaced by an equal molar amount of The product near-infrared two-region photothermal molecule 8 prepared in this example has a yield of 54%.

[0118] Figure 1 The proton nuclear magnetic resonance spectrum of the 1,3-dithia-fused naphthalene tetracarboxylic diimide derivative 1 obtained in Example 1 is characterized. Figure 2 The proton nuclear magnetic resonance spectrum of the near-infrared two-region photothermal molecule 4 of the thia-fused naphthalene tetracarboxylic diimide derivative obtained in Example 5 is characterized, Figure 3 The carbon-13 nuclear magnetic resonance spectrum of the near-infrared two-region photothermal molecule 4 of the thia-fused naphthalene tetracarboxylic diimide derivative obtained in Example 4 is characterized, Figure 4 The mass spectrum of the near-infrared two-region photothermal molecule 4 of the thia-fused naphthalene tetracarboxylic diimide derivative obtained in Example 4 is characterized, proving that the structure of the near-infrared two-region photothermal molecule is correct.

[0119] Figure 5 The molecular absorption and fluorescence emission spectra of the near-infrared two-region photothermal molecule 4 of the thia-fused naphthalene tetracarboxylic diimide derivative obtained in Example 5 are tested in tetrahydrofuran solvent, and it can be seen that the molecular absorption is located in the near-infrared region, and the absorption wavelength is longer; the molecular emission is in the near-infrared two-region, and it can be seen that the material has excellent near-infrared two-region imaging potential.

[0120] Example 11

[0121] A preparation method of a dual-response near-infrared two-region intelligent nano-bait PND@Gas6(+) includes the following steps:

[0122] N-isopropyl acrylamide (NIPAM, 0.02 M, monomer), acrylic acid (5% of the molar amount of NIPAM, monomer), N,N'-methylenebisacrylamide (5% of the molar amount of NIPAM, crosslinking agent) and sodium dodecyl sulfate (1.5% of the molar amount of NIPAM, surfactant) were dissolved in deionized water and heated to 70°C under magnetic stirring to obtain a mixture; nitrogen was bubbled into the mixture for 30 minutes to remove oxygen, and then an aqueous solution of potassium persulfate was quickly added to initiate the polymerization reaction, and the polymerization reaction was carried out for 4 hours; the obtained responsive PNA nanogel was purified by dialysis (molecular weight cut-off = 100 kDa) for one week, and then freeze-dried for later use; the near-infrared two-region photothermal molecule prepared in Example 5 was dissolved in tetrahydrofuran (THF) to obtain a solution, and then the solution was gradually added to the Tris buffer (15 mM, pH = 8.5) containing the responsive PNA nanogel under stirring, and the mixture was allowed to stand overnight to evaporate the organic solvent THF, thereby forming a PN nanogel; then, in order to load doxorubicin (Dox), a Dox solution (concentration of 1 mg·mL -1 ) was added to the dispersion of the obtained PN nanogel, and stirred at 25°C for 12 hours, and then the PND nanogel was separated by ultrafiltration (molecular weight cut-off = 100 kDa) and washed with deionized water for multiple times until the pH = 7.4. In order to realize the function of the nanobait, the 5-8F tumor cell membrane was broken and dispersed by ultrasonic treatment in PBS to obtain membrane proteins; then the membrane proteins were mixed with the PND nanogel by ultrasonic treatment for 3 minutes to obtain a mixture, and the mixture was subjected to five physical extrusion cycles through a 400 nm polycarbonate membrane using an Avatar extruder, and then purified by ultrafiltration (molecular weight cut-off = 100 kDa) to finally obtain the dual-responsive near-infrared two-region intelligent nanobait PND@Gas6(+). Similarly, the nanobait without Gas6 protein is denoted as PND@Gas6(-).

[0123] Figure 6 The scanning electron microscope image of the dual-responsive near-infrared two-region intelligent nanobait PND@Gas6(+) obtained in this example can be seen that the particle size and morphology of the dual-responsive near-infrared two-region intelligent nanobait PND@Gas6(+) are uniform, and the particle size is about 140 nanometers.

[0124] Performance test:

[0125] 1. Photothermal heating and cooling test of the dual-responsive near-infrared two-region intelligent nanobait PND@Gas6(+) and its process product

[0126] The photothermal performance of the dual-responsive NIR two-region smart nanobait PND@Gas6(+), PN, PND and PND@Gas6(-) was tested in aqueous solution (concentration was 100 μM), under the irradiation of 808 nm laser with a power density of 0.8 W / cm 2 The local temperature change was monitored using a thermal imaging camera.

[0127] Figure 7 The photothermal heating and cooling test diagram of the dual-responsive NIR two-region smart nanobait PND@Gas6(+), PN, PND and PND@Gas6(-) obtained in Example 11 shows that all the materials exhibit efficient photothermal conversion, the temperature rises rapidly, and reaches about 76℃ in 5 minutes. The rapid temperature rise is attributed to the free molecular motion of the NIR two-region photothermal molecules of the sulfur-heterocyclic-fused naphthalene tetracarboxylic diimide derivative in the nanogel matrix, which enhances the dissipation of photothermal energy.

[0128] 2. pH / temperature versus time-release rate curve test of the dual-responsive NIR two-region smart nanobait PND@Gas6(+)

[0129] To verify the pH / temperature dual-responsive performance of the dual-responsive NIR two-region smart nanobait PND@Gas6(+), the release kinetics of the dual-responsive NIR two-region smart nanobait PND@Gas6(+) was tested in PBS buffer with different pH (pH=5.0 or 7.4) and different temperature (0~90℃) under the conditions of no laser and laser (laser wavelength was 808 nm, power density was 0.3 W·cm -2 or 0.6 W·cm -2 ), by measuring the absorbance at 480 nm, using the standard concentration curve of Dox to quantify the content of Dox in the PND nanogel, and then using zero-order reaction, Higuchi model and Peppas-Sahlin model to analyze and fit the release curve of Dox.

[0130] Figure 8The pH / temperature-dependent release rate curves of the bi-responsive NIR dual-region smart nanobait PND@Gas6(+) for the tumor drug Dox obtained in Example 11 can be seen. The cumulative release of Dox at pH = 5.0 was significantly higher than that at pH = 7.4, whether in the presence or absence of a laser, indicating that the drug has a pH-responsive release characteristic. This effect is attributed to the enhanced protonation under acidic conditions, which weakens the electrostatic interaction between Dox and PNA, thereby promoting the dissociation of the drug. In addition, increasing the 808 nm laser power density further accelerates the release of Dox, mainly due to the destruction of hydrogen bonds between the nanogel and water molecules caused by the increase in temperature. This thermal effect causes the nanogel to shrink, thereby expelling Dox from the interior of the nanogel.

[0131] 3. Application of the bi-responsive NIR dual-region smart nanobait PND@Gas6(+) in the diagnosis and treatment of orthotopic tumors and orthotopic metastatic tumors

[0132] The therapeutic effect was evaluated in a mouse model of subcutaneous tumor transplantation on the back (5-8F tumor cells). The mice were randomly divided into six groups: PBS+L group, Dox+L group, PN+L group, PND+L group, PND@Gas6(-)+L group, and PND@Gas6(+)+L group, wherein the PBS+L group was the negative control group, the mice were injected intravenously with PBS and applied with 808 nm laser, the Dox+L group was the mice injected intravenously with the same dose of free Dox solution as in the treatment group and applied with 808 nm laser, the PN+L group was the mice injected intravenously with PN nanogel and applied with 808 nm laser, the PND+L group was the mice injected intravenously with PND nanogel and applied with 808 nm laser, the PND@Gas6(-)+L group was the mice injected intravenously with PND@Gas6(-) nanobait and applied with 808 nm laser, and the PND@Gas6(+)+L group was the mice injected intravenously with PND@Gas6(+) nanobait and applied with 808 nm laser. On the first day of the experiment, the drug was administered by intravenous injection at a concentration of 6 mg / mL and a volume of 200 μL. Twelve hours after injection, the tumor area was irradiated with 808 nm near-infrared laser (power density 0.8 W·cm -2 , for 12 minutes), and the local temperature change was monitored in real time using a thermal imager.

[0133] Figure 9 Schematic diagram of the photothermal ability treatment (PTT) effect of the bi-responsive NIR dual-region smart nanobait PND@Gas6(+) obtained in Example 11 in a subcutaneous tumor model, Figure 10For the treatment effect diagram of the dual-responsive near-infrared two-region intelligent nanobait PND@Gas6(+) obtained in Example 11 in the subcutaneous tumor model, it can be seen that after 808 nm laser irradiation for 12 minutes, the tumor temperature of the PBS+L group was only slightly increased to 36℃. In contrast, the tumor temperature of the PN+L group and the PND+L group increased to about 45℃ due to the high penetration and long retention effect. Benefiting from the targeting effect of the homologous tumor cell membrane, the tumor temperature of the PND@Gas6(-)+L group increased to 53℃. The highest temperature (60℃) appeared in the PND@Gas6(+)+L group, and the Gas6 protein overexpressed on the homologous tumor cell membrane significantly enhanced the targeting ability, thereby greatly improving the photothermal effect.

[0134] 4. Application of dual-responsive near-infrared two-region intelligent nanobait PND@Gas6(+) in avoiding early tumor migration

[0135] After one week of adaptive feeding, the nude mice were injected with luciferase transfected 5-8F tumor cells (5x10 6 per mouse) through the tail vein to establish a lung metastasis model of nasopharyngeal carcinoma. After 7 days of modeling, the mice were injected with luciferin substrate intraperitoneally, and the tumor formation was confirmed by whole-body bioluminescence imaging. Subsequently, the mice were randomly divided into five groups (n=5 / group): (a) PBS group, (b) Dox group, (c) PND group, (d) PND@Gas6(-) group, (e) PND@Gas6(+) group. On day 1, each group was injected with the corresponding drug intravenously, and the treatment was repeated once every other day. After 14 days of treatment, the mice were injected with luciferin substrate intraperitoneally and whole-body imaging (IVIS) was performed to analyze the fluorescence intensity and evaluate the tumor metastasis inhibition effect.

[0136] Figure 11 For the treatment effect diagram of the dual-responsive near-infrared two-region intelligent nanobait PND@Gas6(+) obtained in Example 11 in the lung metastasis tumor model, the results showed that the tumor fluorescence signal of the PBS group was the strongest, while the PND@Gas6(+) group had the most significant effect, and the tumors in five mice were basically completely eliminated. These results indicate that the dual-responsive near-infrared two-region intelligent nanobait PND@Gas6(+) has excellent anti-tumor effect in the lung metastasis model, providing a very promising treatment strategy for inhibiting tumor metastasis.

[0137] As can be seen from the above, the near-infrared two-zone photothermal molecules designed in the application all exhibit excellent 808 nm excitation photothermal performance and near-infrared two-zone imaging performance, and are particularly suitable for preparing nano-bait materials for homologous targeted delivery, dual-mode imaging guidance, localized chemotherapy-photothermal synergistic treatment and systemic metastasis inhibition, and the core is to introduce photothermally controllable drug release into the nano-treatment platform for nasopharyngeal carcinoma. The dual-response near-infrared two-zone intelligent nano-bait PND@Gas6(+) of the application exhibits excellent and efficient loading efficiency and stability for various photothermal molecules, and provides a solution for integrating the four functions of homologous targeted delivery, dual-mode imaging guidance, localized chemotherapy-photothermal synergistic treatment and systemic metastasis inhibition into a single platform, and simultaneously solves the dual problems of in-situ treatment of nasopharyngeal carcinoma and prevention and control of lung cancer cell metastasis.

[0138] The above merely illustrates the preferred embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which shall be covered within the protection scope of the present application.

Claims

1. The use of a near-infrared two-region photothermal molecule in the preparation of a dual-responsive near-infrared two-region intelligent nanobait, characterized in that, The preparation method of the dual-response near-infrared two-region intelligent nanobait comprises the following steps: N-isopropyl acrylamide, acrylic acid, a crosslinking agent and a surfactant are mixed in water, heated under magnetic stirring to obtain a mixture, oxygen in the mixture is removed, an initiator is added, a polymerization reaction is carried out, after the polymerization reaction is completed, the temperature / pH dual-response nanogel is obtained through dialysis purification and drying; The near-infrared two-region photothermal molecule is dissolved in tetrahydrofuran to obtain a solution; The solution is added to a tris-hydroxymethyl aminomethane buffer containing the temperature / pH dual-response nanogel, and left overnight to obtain a nanogel; An anti-tumor drug solution is mixed with a dispersion of the nanogel, stirred, ultrafiltrated, and washed with water to obtain an anti-tumor drug-loaded nanogel; The anti-tumor drug-loaded nanogel is coated with a nasopharyngeal carcinoma tumor cell membrane that highly expresses growth arrest-specific gene 6 to obtain the dual-response near-infrared two-region intelligent nanobait; The structure of the near-infrared two-region photothermal molecule is shown in formula II: Formula II wherein R1is selected from C 14 linear or C2-C 10 branched alkyl or R2is selected from one of diphenylamine, 4,4'-dimethoxydiphenylamine, and 4,4'-dimethyldiphenylamine.

2. Use according to claim 1, characterized in that, The near-infrared two-region photothermal molecule is selected from the following structures: , , , or . 3.The application of the dual-responsive near-infrared two-zone intelligent nanolure in the preparation of a drug for diagnosing or treating nasopharyngeal carcinoma in situ treatment and lung cancer cell metastasis, characterized in that, The dual-response near-infrared two-region intelligent nanobait is the dual-response near-infrared two-region intelligent nanobait in claim 1.

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