A nanotherapeutic agent based on near-infrared photosensitizer and its preparation method and application

Abstract

The present invention discloses a near-infrared photosensitizer-based nanotherapeutic agent, its preparation method, and application. The near-infrared photosensitizer-based nanotherapeutic agent comprises a protein and a near-infrared prodrug bound to a hydrophobic cavity of the protein. The near-infrared prodrug comprises a near-infrared photosensitizer connected to a cupric sulfide bond and a chemotherapy drug bound to the near-infrared photosensitizer connected to the cupric sulfide bond via a thioketone bond. The near-infrared photosensitizer-based nanotherapeutic agent provided by the present invention significantly improves the solubility and bioavailability of the chemotherapy drug and the near-infrared photosensitizer, achieves tumor targeting and selectivity, thereby increasing the drug accumulation at the tumor site, improving the therapeutic effect, and reducing toxic side effects. This solves the problems of existing near-infrared fluorescent dyes, which have limited targeting and selectivity and low therapeutic effect.

Description

Technical Field

[0001] The present invention relates to the field of medical nanomaterials, and in particular to a nano therapeutic agent based on a near-infrared photosensitizer, and a preparation method and application thereof. Background Art

[0002] Near-infrared fluorescent dyes (near-infrared photosensitizers) can reduce background absorption and improve the sensitivity of fluorescent probes and chemical sensors, and their development is very rapid. In addition, light in the near-infrared region (650-900nm) has the ability to penetrate deep tissues, allowing for in vivo optical imaging and photodynamic therapy. Therefore, the synthesis and design of near-infrared fluorescent dyes have attracted increasing interest. However, the complex physiological environment of cancer and tumor heterogeneity have made the targeting, selectivity, and sensitivity of near-infrared fluorescent dyes face major challenges.

[0003] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a nanotherapeutic agent based on a near-infrared photosensitizer, and a preparation method and application thereof, aiming to solve the problems of limited targeting and selectivity and low therapeutic effect of existing near-infrared fluorescent dyes.

[0005] The technical solutions of the present invention are as follows:

[0006] In a first aspect of the present invention, a nanotherapeutic agent based on a near-infrared photosensitizer is provided, wherein the nanotherapeutic agent based on a near-infrared photosensitizer comprises a protein and a near-infrared prodrug bound to a hydrophobic cavity of the protein, the near-infrared prodrug comprises a near-infrared photosensitizer connected to a thioketal (TK) bond and a chemotherapy drug bound to the near-infrared photosensitizer connected to the TK bond via the TK bond.

[0007] Optionally, the mass ratio of the near-infrared photosensitizer connected with a TK bond to the chemotherapy drug is 2:1 to 3:1.

[0008] Optionally, the mass ratio of the near-infrared prodrug to the protein is 1:25 to 1:40.

[0009] Optionally, the near-infrared photosensitizer-based nanotherapeutic agent is a nanoparticle, and the particle size of the nanoparticle is 80 to 120 nm.

[0010] Optionally, the protein is selected from one or more of glucose oxidase (GOx), albumin (ALB), ferritin (SF), whey protein (WPI), collagen (COL), silk fibroin (SF), lipoprotein (Lp), and recombinant protein.

[0011] Optionally, the chemotherapy drug is selected from one or more of paclitaxel (PTX), camptothecin (CPT), doxorubicin (ADM), bicoumarol (BHC), apigenin (API), tirapazamine (TPZ), vincristine (VCR), and thiotepa (TSPA).

[0012] The second aspect of the present invention provides a method for preparing the nanotherapeutic agent based on the near-infrared photosensitizer as described above, which comprises the steps of:

[0013] Provided are near-infrared prodrugs and proteins, wherein the near-infrared prodrugs include a near-infrared photosensitizer connected to a TK bond and a chemotherapy drug bound to the near-infrared photosensitizer connected to the TK bond via a TK bond;

[0014] The near-infrared prodrug is mixed with protein to obtain the near-infrared photosensitizer-based nanotherapeutic agent.

[0015] Optionally, the preparation method of the near-infrared prodrug comprises the steps of:

[0016] Providing near-infrared photosensitizers and chemotherapy drugs connected with TK bonds;

[0017] The near-infrared photosensitizer connected with a TK bond and the chemotherapy drug are dissolved in an organic solvent, 4-dimethylaminopyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride are added, and after reaction, the near-infrared prodrug is obtained.

[0018] Optionally, the preparation method of the near-infrared photosensitizer connected with a TK bond comprises the steps of:

[0019] 1-Bronaphthalene and 1,4-diaminohexane were added to 2-methoxyethanol, and then CuI and CsCO3 were added to reflux;

[0020] The obtained product is mixed with alkyl thiosulfate and potassium dichromate, and methanol and hydrochloric acid are added to react to obtain a near-infrared photosensitizer;

[0021] Copper sulfide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride are added to dimethylformamide, and then the near-infrared photosensitizer is added. After reaction, the near-infrared photosensitizer connected with a TK bond is obtained.

[0022] The third aspect of the present invention provides an application of the nanotherapeutic agent based on the near-infrared photosensitizer as described above in the preparation of a tumor treatment preparation, and / or provides an application of the nanotherapeutic agent based on the near-infrared photosensitizer prepared by the preparation method as described above in the preparation of a tumor treatment preparation.

[0023] Beneficial Effects: The present invention combines a near-infrared photosensitizer with a TK bond, which is then combined with a hydrophobic chemotherapy drug via the TK bond to obtain a hydrophobic near-infrared prodrug. The hydrophobic near-infrared prodrug is then combined with the protein via a hydrophobic interaction using a protein as a carrier, so that the near-infrared prodrug is bound to the protein cavity and thus encapsulated by the protein, thereby obtaining a near-infrared photosensitizer-based nanotherapeutic agent. When the near-infrared photosensitizer-based nanotherapeutic agent is targeted to a tumor, the near-infrared prodrug is released. Hydrogen peroxide in the tumor environment cleaves the TK bond between the near-infrared photosensitizer and the chemotherapy drug in the near-infrared prodrug, breaking the TK bond and releasing the chemotherapy drug and the near-infrared photosensitizer, thereby simultaneously achieving synergistic treatment of tumors with near-infrared imaging-guided chemotherapy and photodynamic therapy. The nanotherapeutic agent based on near-infrared photosensitizer provided by the present invention greatly improves the solubility and bioavailability of chemotherapy drugs and near-infrared photosensitizers, can achieve tumor targeting and selectivity, thereby increasing the accumulation of drugs in the tumor site, improving the therapeutic effect, and reducing toxic side effects, solving the problems of limited targeting and selectivity and low therapeutic effect of existing near-infrared fluorescent dyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the synthetic route of the nanotherapeutic agent based on near-infrared photosensitizer in Example 1 of the present invention.

[0025] Figure 2 This is a high-resolution mass spectrum of the NTP prepared in Example 1 of the present invention.

[0026] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of NTP@G prepared in Example 1 of the present invention.

[0027] Figure 4 This is a TEM image of NTP@G prepared in Example 1 of the present invention.

[0028] Figure 5 This is a diagram showing the killing effect of NTP@G on 4T1 tumor cells in Example 3 of the present invention.

[0029] Figure 6 4 is a comparison diagram of the intracellular superoxide radical content of different treatment groups in Example 4 of the present invention, wherein (a) is a fluorescence confocal comparison diagram of different treatment groups, and (b) is a relative fluorescence intensity comparison diagram of different treatment groups.

[0030] Figure 7 (a) is a tumor-targeted fluorescence imaging image of mice injected with NTP and NTP@G, respectively, in Example 5 of the present invention; (b) is a comparison of the corresponding fluorescence intensities of tumor targeting of mice injected with NTP and NTP@G, respectively, in Example 5 of the present invention.

[0031] Figure 8Graphs illustrating the inhibitory effect of starvation / chemotherapy / photodynamic therapy on 4T1 tumor growth in Example 6 of the present invention, wherein (a) shows the change in tumor volume of nude mice in different treatment groups over time, and (b) shows the tumor weights of different treatment groups after dissection 15 days after treatment. DETAILED DESCRIPTION

[0032] The present invention provides a nanotherapeutic agent based on a near-infrared photosensitizer, a preparation method thereof, and an application thereof. To make the objectives, technical solutions, and effects of the present invention more clear and explicit, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0033] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0034] Prodrugs are compounds that have low or even no in vitro activity but can release their active form in vivo through the action of enzymes or non-enzymes to exert their pharmacological effects. Prodrugs have the potential to increase drug stability and selectivity, reduce drug toxicity, and improve drug bioavailability. With the continuous advancement of chemical and biotechnological technologies, prodrug design has garnered increasing attention in new drug research. Combining prodrugs with cancer therapy has enabled the design of a large number of anticancer prodrugs. While currently developed nanodrug delivery systems can deliver prodrugs to tumors for therapeutic efficacy, challenges such as low drug loading, poor stability, early drug leakage, and low specific drug release efficiency remain to be addressed. Furthermore, while near-infrared fluorescent dyes can reduce background absorption and enhance the sensitivity of fluorescent probes and chemical sensors, their development has been rapid. However, the complex physiological environment of cancer and the heterogeneity of tumors pose significant challenges to the targeting, selectivity, and sensitivity of these dyes. Based on this, an embodiment of the present invention provides a near-infrared photosensitizer-based nanotherapeutic agent, wherein the near-infrared photosensitizer-based nanotherapeutic agent includes a protein and a near-infrared prodrug bound to the hydrophobic cavity of the protein, the near-infrared prodrug includes a near-infrared photosensitizer connected with a TK bond and a chemotherapy drug bound to the near-infrared photosensitizer connected with a TK bond through a TK bond.

[0035] In this embodiment, a near-infrared photosensitizer is combined with a TK bond to obtain a near-infrared photosensitizer connected with a TK bond, and then a hydrophobic chemotherapy drug is combined with the near-infrared photosensitizer connected with a TK bond (i.e., the chemotherapy drug and the near-infrared photosensitizer are combined through the TK bond) to obtain a hydrophobic near-infrared prodrug; using protein as a carrier, the hydrophobic near-infrared prodrug is combined with the protein through hydrophobic interaction, so that the near-infrared prodrug is combined in the hydrophobic cavity of the protein, thereby being encapsulated by the protein, to obtain a nanotherapeutic agent based on the near-infrared photosensitizer.

[0036] In this embodiment, a hydrophobic chemotherapy drug is combined with a near-infrared photosensitizer connected with a TK bond (having reactive oxygen species response characteristics) to obtain a hydrophobic near-infrared prodrug, which is then combined with the hydrophobic near-infrared prodrug in the hydrophobic cavity of the protein, thereby improving the water solubility of the chemotherapy drug and the near-infrared photosensitizer, increasing the bioavailability of the chemotherapy drug and the near-infrared photosensitizer, and thus increasing the bioavailability of the near-infrared prodrug. When the nanotherapeutic agent based on the near-infrared photosensitizer is targeted to the tumor, the protein microstructure changes in the acidic environment of the tumor, releasing the near-infrared prodrug. The hydrogen peroxide in the tumor environment cleaves the TK bond between the near-infrared photosensitizer and the chemotherapy drug in the near-infrared prodrug, breaking the TK bond, thereby releasing the chemotherapy drug and the near-infrared photosensitizer, further achieving tumor targeting and selectivity, thereby increasing the accumulation of the drug in the tumor site, improving the therapeutic effect, reducing toxic side effects, and simultaneously achieving synergistic treatment of tumor near-infrared imaging-guided chemotherapy and photodynamic therapy, solving the problems of limited targeting and selectivity and low therapeutic effect of existing near-infrared fluorescent dyes. Furthermore, in the near-infrared photosensitizer-based nanotherapeutic agent described in this embodiment, the near-infrared prodrug is stably bound to the hydrophobic cavity of the protein, the encapsulation efficiency of the near-infrared prodrug is as high as 57.4%, and the encapsulation efficiency of the protein is as high as 31.5%. Moreover, only in the tumor environment can the near-infrared photosensitizer-based nanotherapeutic agent release the chemotherapy drug and the near-infrared photosensitizer. That is, the near-infrared photosensitizer-based nanotherapeutic agent has a high drug loading capacity, bioavailability and selectivity, and can realize the synergistic treatment of chemotherapy and photodynamic therapy guided by near-infrared imaging in the tumor site, solving the problems of low drug loading capacity, poor stability and low specific drug release efficiency of existing nanodrug delivery systems, and will have good application prospects in the field of tumor diagnosis and treatment.

[0037] In one embodiment, the mass ratio of the near-infrared photosensitizer connected with a TK bond to the chemotherapy drug is 2:1 to 3:1. This ratio can maximize the connection rate between the near-infrared photosensitizer and the chemotherapy drug and reduce loss.

[0038] In one embodiment, the mass ratio of the near-infrared prodrug to the protein is 1:25 to 1:40. This ratio allows the near-infrared photosensitizer-based nanotherapeutic agent to have an appropriate particle size and maximize the encapsulation efficiency, with the encapsulation efficiency of the near-infrared prodrug reaching 57.4% and the encapsulation efficiency of the protein reaching 31.5%.

[0039] In one embodiment, the near-infrared photosensitizer-based nanotherapeutic agent is a nanoparticle having a particle size of 80 to 120 nm. Within this particle size range, the near-infrared photosensitizer-based nanotherapeutic agent can better achieve targeted accumulation in tumors and synergistic treatment combining chemotherapy and photodynamic therapy for tumors.

[0040] In one embodiment, the protein is selected from one or more of GOx, ALB, SF, WPI, COL, SF, Lp, and recombinant proteins, but is not limited thereto. In this embodiment, the protein not only has the function of a carrier, but can also achieve tumor treatment. For example, when the protein is selected from GOx, the GOx in the near-infrared photosensitizer-based nanotherapeutic agent exerts a starvation therapy effect in the tumor microenvironment and can generate a large amount of reactive oxygen species. Specifically, when the near-infrared photosensitizer-based nanotherapeutic agent is targeted to the tumor, GOx oxidizes glucose in the tumor site to produce a large amount of hydrogen peroxide, which can achieve starvation therapy. The hydrogen peroxide produced at the same time can destroy the TK bond between the chemotherapeutic drug and the near-infrared photosensitizer in the near-infrared prodrug, breaking it to generate a thiol and acetone. The thiol end then undergoes intramolecular cyclization to form a sulfur-containing ring that falls off the ester bond, thereby releasing the chemotherapeutic drug (such as PTX, etc.) and the near-infrared photosensitizer. The released chemotherapeutic drug (such as PTX, etc.) achieves chemotherapy treatment, while the released near-infrared photosensitizer can achieve photodynamic therapy through infrared light irradiation. The nanotherapeutic agent based on near-infrared photosensitizer has high drug loading, bioavailability and selectivity, and can simultaneously achieve synergistic treatment of tumor starvation therapy guided by near-infrared imaging, chemotherapy and photodynamic therapy.

[0041] In one embodiment, the chemotherapeutic drug is selected from one or more of PTX, CPT, ADM, BHC, API, TPZ, VCR, and SPA, but is not limited thereto. PTX is a natural anticancer drug widely used clinically, mainly used for the treatment of ovarian cancer and breast cancer, and also has certain therapeutic effects on lung cancer, melanoma, brain tumors, and colon cancer. However, PTX has poor water solubility, weak absorption, low bioavailability, and high toxic and side effects, which limit its application in the field of anti-tumor treatment. The present invention combines PTX with a near-infrared photosensitizer and, through hydrophobic interaction, encapsulates it in a protein, effectively solving the problem of poor water solubility of PTX.

[0042] In one embodiment, the structural formula of the near-infrared photosensitizer is The near-infrared photosensitizer with the structure has deep tissue penetration ability under 660nm light irradiation and can generate highly toxic superoxide free radicals, thereby realizing in vivo optical imaging and photodynamic therapy of cancer.

[0043] In one embodiment, the structural formula of the near-infrared photosensitizer connected with a TK bond is:

[0044]

[0045] The present invention also provides a method for preparing the nanotherapeutic agent based on the near-infrared photosensitizer as described above in the embodiment of the present invention, which comprises the steps of:

[0046] S1. Providing a near-infrared prodrug and a protein, wherein the near-infrared prodrug includes a near-infrared photosensitizer connected to a TK bond and a chemotherapy drug bound to the near-infrared photosensitizer connected to the TK bond via the TK bond;

[0047] S2. Mixing the near-infrared prodrug with protein to obtain the near-infrared photosensitizer-based nanotherapeutic agent.

[0048] The preparation method provided by the embodiments of the present invention is simple and easy to operate, does not require complex and expensive equipment, and is easily scalable for industrial production. The resulting near-infrared photosensitizer-based nanotherapeutic agent can simultaneously achieve synergistic treatment of tumors through near-infrared imaging-guided starvation therapy and chemotherapy combined with photodynamic therapy.

[0049] In step S1, in one embodiment, the method for preparing the near-infrared prodrug comprises the steps of:

[0050] S11, providing a near-infrared photosensitizer and a chemotherapy drug connected with a TK bond;

[0051] S12. Dissolve the near-infrared photosensitizer connected with a TK bond and the chemotherapy drug in an organic solvent, add 4-dimethylaminopyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and react to obtain the near-infrared prodrug.

[0052] In step S11, in one embodiment, the method for preparing the near-infrared photosensitizer connected with a TK bond comprises the steps of:

[0053] S111, adding 1-bromonaphthalene and 1,4-diaminohexane to 2-methoxyethanol, and then adding CuI and CsCO3 to reflux;

[0054] S112, mixing the obtained product with alkyl thiosulfate and potassium dichromate, adding methanol and hydrochloric acid, and reacting to obtain a near-infrared photosensitizer;

[0055] S113, adding copper sulfide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to dimethylformamide, and then adding the near-infrared photosensitizer, and after reaction, obtaining the near-infrared photosensitizer connected with a TK bond.

[0056] In step S11, in one embodiment, the chemotherapy drug is selected from one or more of PTX, CPT, ADM, BHC, API, TPZ, VCR and SPA, but is not limited thereto.

[0057] In one embodiment, the protein is selected from one or more of GOx, ALB, SF, WPI, COL, SF, Lp and recombinant protein, but is not limited thereto.

[0058] In step S12, in one embodiment, the organic solvent is selected from dimethylformamide, but is not limited thereto.

[0059] In step S2, in one embodiment, the step of mixing the near-infrared prodrug with a protein to obtain the near-infrared photosensitizer-based nanotherapeutic agent specifically comprises:

[0060] S21, adding β-mercaptoethanol and protein to a Tris buffer solution and mixing, then adding the near-infrared prodrug while stirring to obtain the near-infrared photosensitizer-based nanotherapeutic agent.

[0061] The β-mercaptoethanol is used to break the disulfide bonds of the protein and open its structure, making it easier for the protein to hydrophobically bind to the near-infrared prodrug, so that the near-infrared prodrug is bound to the hydrophobic cavity of the protein.

[0062] In step S21, in one embodiment, the solvent in the Tris buffer solution is ultrapure water, which has almost no impurities and is more conducive to forming a pure biomimetic nanoemulsion.

[0063] The present invention also provides an embodiment of the present invention, wherein the nanotherapeutic agent based on a near-infrared photosensitizer is used in the preparation of a tumor treatment preparation. The present invention also provides an embodiment of the present invention, wherein the nanotherapeutic agent based on a near-infrared photosensitizer is used in the preparation of a tumor treatment preparation. Specifically, the nanotherapeutic agent is used for simultaneous starvation therapy, chemotherapy, and photodynamic therapy.

[0064] The following describes it in detail through specific examples.

[0065] Example 1

[0066] Preparation of nanotherapeutic agents based on near-infrared photosensitizers, the synthetic route is as follows Figure 1 shown.

[0067] 4 g of 1-bromonaphthalene and 4.65 g of 1,4-diaminohexane were dissolved in 30 mL of 2-methoxyethanol. 3.69 g of CuI and 3.74 g of CsCO₃ were then added and refluxed at 125°C for 24 h. The product was then extracted with ethyl acetate, dried over Na₂SO₄, and the solvent evaporated. Finally, it was purified on a silica gel column (CH₂Cl₂:CH₃OH, volume ratio 20:1) to obtain the initial near-infrared photosensitizer, designated NB.

[0068] Dissolve 1.2 g of alkyl thiosulfate and 1 g of NB in ​​20 mL of DMSO, then add 1.2 g of potassium dichromate. After stirring at room temperature for 15-20 minutes, add 150 mL of methanol and 15 mL of hydrochloric acid (2 mol / L). The mixture is then stirred at room temperature for an additional 30 minutes. After completion of the reaction, remove the solvent by rotary evaporation and purify on a silica gel column (CH2Cl2:methanol volume ratio 8:3) to obtain a near-infrared photosensitizer, designated NBS.

[0069] Under nitrogen, 300 mg of TK was dissolved in 10 mL of dimethylformamide, and 47 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 100 mg of 4-dimethylaminopyridine were added. After stirring for 20 minutes, 242.74 mg of NBS was added dropwise to the reaction mixture and stirred at room temperature for 24 hours. The reaction mixture was purified by silica gel column (CH2Cl2:CH3OH, volume ratio 5:1) to obtain a near-infrared photosensitizer with a TK bond, designated NT.

[0070] 70 mg of PTX and 150 mg of NT were added to 10 mL of dimethylformamide (mass ratio of NT to PTX: 2.14:1). Then, 54 mg of 4-dimethylaminopyridine and 90 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added and stirred at room temperature for 48 hours. The mixture was then purified on a silica gel column (CH2Cl2:CH3OH, volume ratio: 10:1) to obtain the near-infrared prodrug, designated NTP.

[0071] 5 mM β-mercaptoethanol and 20 mg of GOx were added to 10 mL of Tris buffer (pH 7.4) and stirred for 10 minutes. NTP was slowly added to the reaction mixture to a final concentration of 0.05 mg / mL (NTP:GOx mass ratio of 1:40). The mixture was then stirred at 37°C for 10 minutes. Finally, the solution was centrifuged at 9000 rpm for 5 minutes to obtain a near-infrared photosensitizer-based nanotherapeutic agent, designated NTP@G.

[0072] The high resolution mass spectrum of NTP prepared in Example 1 is as follows Figure 2As shown in the figure, the results show that its molecular weight is 1474.57; the nuclear magnetic resonance hydrogen spectrum of NTP@G is as follows Figure 3 As shown, the structure of the nanotherapeutic agent based on near-infrared photosensitizer is demonstrated; the TEM imaging results of NTP@G are shown in Figure 4 As shown, from Figure 4 It can be seen that NTP@G is a spherical particle with a diameter of 80 to 120 nm.

[0073] Example 2

[0074] Preparation of nanotherapeutic agents based on near-infrared photosensitizers, the synthetic route is as follows Figure 1 shown.

[0075] 4 g of 1-bromonaphthalene and 4.65 g of 1,4-diaminohexane were dissolved in 30 mL of 2-methoxyethanol. 3.69 g of CuI and 3.74 g of CsCO₃ were then added and refluxed at 125°C for 24 h. The product was then extracted with ethyl acetate, dried over Na₂SO₄, and the solvent evaporated. Finally, it was purified on a silica gel column (CH₂Cl₂:CH₃OH, volume ratio 20:1) to obtain the initial near-infrared photosensitizer, designated NB.

[0076] Dissolve 1.2 g of alkyl thiosulfate and 1 g of NB in ​​20 mL of DMSO, then add 1.2 g of potassium dichromate. After stirring at room temperature for 15-20 minutes, add 150 mL of methanol and 15 mL of hydrochloric acid (2 mol / L). The mixture is then stirred at room temperature for an additional 30 minutes. After completion of the reaction, remove the solvent by rotary evaporation and purify on a silica gel column (CH2Cl2:methanol volume ratio 8:3) to obtain a near-infrared photosensitizer, designated NBS.

[0077] Under nitrogen, 300 mg of TK was dissolved in 10 mL of dimethylformamide, and 47 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 100 mg of 4-dimethylaminopyridine were added. After stirring for 20 minutes, 242.74 mg of NBS was added dropwise to the reaction mixture and stirred at room temperature for 24 hours. The reaction mixture was purified by silica gel column (CH2Cl2:CH3OH, volume ratio 5:1) to obtain a near-infrared photosensitizer with a TK bond, designated NT.

[0078] 70 mg of PTX and 210 mg of NT were added to 10 mL of dimethylformamide (NT:PTX mass ratio of 3:1), followed by 54 mg of 4-dimethylaminopyridine and 90 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The mixture was stirred at room temperature for 48 hours. The product was then purified on a silica gel column (CH2Cl2:CH3OH volume ratio of 10:1) to obtain the near-infrared prodrug, designated NTP.

[0079] 5 mM β-mercaptoethanol and 20 mg of GOx were added to 10 mL of Tris buffer (pH 7.4) and stirred for 10 minutes. NTP was slowly added to the reaction mixture to a final concentration of 0.05 mg / mL (NTP:GOx mass ratio of 1:40). The mixture was then stirred at 37°C for 10 minutes. Finally, the near-infrared photosensitizer-based nanotherapeutic, designated NTP@G, was obtained after centrifugation at 9000 rpm for 5 minutes.

[0080] Example 3

[0081] Evaluation of the cytotoxicity of NTP@G to 4T1 tumor cells

[0082] The effect of NTP@G on the viability of 4T1 cells was evaluated using the standard MTT assay. 4T1 cells were cultured at 37°C and 5% CO2 at a density of 5 × 10 cells per well. 3 The cells were seeded into a 96-well plate at a density of 100 μg / mL. After 24 h, the old culture medium in the 96-well plate was aspirated and DMEM culture medium containing 0, 12.5, 25, 50, and 100 ng / mL of the NTP described in Example 1 was added. After 4 h of continuous culture, the DMEM culture medium containing NTP in the 96-well plate was washed away and replaced with fresh DMEM culture medium. Each well was illuminated with a 660 nm laser at 0.2 W / cm 2 Irradiate with a power of 100 nm for 5 minutes. After incubating the cells for another 20 hours, aspirate the old medium from the 96-well plate and add 100 μL of a medium solution containing 5 mg / mL MTT to each well. Incubate for another 4 hours. Finally, replace the medium with 150 μL of DMSO per well and measure the OD value of each well (at a wavelength of 490 nm). This is referred to as the NTP group.

[0083] The effect of NTP@G on the viability of 4T1 cells was evaluated using the standard MTT assay. 4T1 cells were cultured at 37°C and 5% CO2 at a density of 5 × 10 cells per well. 3 The cells were seeded into a 96-well plate at a high density. After 24 h, the old culture medium in the 96-well plate was aspirated and DMEM culture medium containing 0, 12.5, 25, 50, and 100 ng / mL NTP@G from Example 1 was added. After 4 h of continuous culture, the DMEM culture medium containing NTP@G in the 96-well plate was washed away and replaced with fresh DMEM culture medium. Each well was illuminated with a 660 nm laser at 0.2 W / cm 2Irradiate with a power of 500 nm for 5 minutes. After incubating the cells for another 20 hours, aspirate the old medium from the 96-well plate and add 100 μL of a medium solution containing 5 mg / mL MTT to each well. Incubate for another 4 hours. Finally, replace the medium with 150 μL of DMSO per well and measure the OD value of each well (detection wavelength: 490 nm). This is referred to as the NTP@G group.

[0084] The cell viability was calculated using the following formula: Cell viability (%) = (OD490 value of sample / OD490 value of blank) × 100%. Figure 5 As shown, the NTP group exhibited minimal toxicity to 4T1 cells within the 0-100 ng / mL concentration range, while the NTP@G group exhibited significant toxicity. At a concentration of 50 ng / mL, cell survival was less than 50%, while at the same concentration, the NTP group achieved a 90% cell survival rate. Under illumination at a concentration of 100 ng / mL, cell survival in the NTP@G group fell below 10%, while that in the NTP group reached 80%. These results suggest that GOx-mediated starvation therapy enhances NTP-mediated chemotherapy and photodynamic therapy of tumor cells.

[0085] The toxicity evaluation results of NTP@G on 4T1 tumor cells in Example 2 are similar to those of NTP@G in Example 1 and are not repeated here.

[0086] Example 4

[0087] Evaluation of the effects of different treatments on intracellular superoxide radical levels

[0088] 4T1 cells were cultured at 37°C and 5% CO2 at a density of 5 × 10 cells per well. 3 Cells were seeded into 96-well plates at a high density. After 24 hours, the old culture medium in the 96-well plates was aspirated and DMEM medium containing 50 ng / mL of the NTPs described in Example 1 was added. After 1 hour of incubation, 5 μM dihydroethidium (a superoxide radical indicator, DHE) was added and incubated for a further 30 minutes. Finally, the fluorescence change of DHE was measured and quantitatively analyzed using a PerkinElme high-content screening instrument, which was recorded as the NTP group.

[0089] 4T1 cells were cultured at 37°C and 5% CO2 at a density of 5 × 10 cells per well. 3 The cells were seeded into a 96-well plate at a density of 100 μg / mL. After 24 h, the old culture medium in the 96-well plate was aspirated and sugar-free DMEM medium containing 50 ng / mL of the NTPs described in Example 1 was added. After 1 h of incubation, 5 μM dihydroethidium (superoxide radical indicator, abbreviated as DHE) was added and incubated for another 30 min. Next, each well was illuminated with a 660 nm laser at 0.2 W / cm 2Finally, the fluorescence changes of DHE were measured using a PerkinElme high-content screening instrument and quantitatively analyzed, which was recorded as the NTP+illumination group.

[0090] 4T1 cells were cultured at 37°C and 5% CO2 at a density of 5 × 10 cells per well. 3 The cells were seeded into a 96-well plate at a high density. After 24 hours, the old culture medium in the 96-well plate was aspirated and sugar-free DMEM containing 50 ng / mL of NTP@G from Example 1 was added. After 1 hour of incubation, 5 μM dihydroethidium (a superoxide radical indicator, DHE) was added and incubated for a further 30 minutes. Finally, the fluorescence change of DHE was measured and quantitatively analyzed using a PerkinElme high-content screening instrument, which was recorded as the NTP@G group.

[0091] 4T1 cells were cultured at 37°C and 5% CO2 at a density of 5 × 10 cells per well. 3 The cells were seeded into a 96-well plate at a high density. After 24 hours, the old culture medium in the 96-well plate was aspirated and sugar-free DMEM medium containing 50 ng / mL of NTP@G in Example 1 was added. After 1 hour of incubation, 5 μM dihydroethidium (superoxide radical indicator, abbreviated as DHE) was added and incubated for another 30 minutes. Next, each well was illuminated with a 660 nm laser at 0.2 W / cm 2 Finally, the fluorescence changes of DHE were measured using a PerkinElme high-content screening instrument and quantitatively analyzed, which was recorded as the NTP@G+ illumination group.

[0092] 4T1 cells were cultured at 37°C and 5% CO2 at a density of 5 × 10 cells per well. 3 The cells were seeded into a 96-well plate at a high density. After 24 hours, the old culture medium in the 96-well plate was aspirated and DMEM medium containing 50 ng / mL of the NTP@G described in Example 1 was added, wherein the DMEM medium contained 2 mM glucose. After 1 hour of incubation, 5 μM dihydroethidium (a superoxide radical indicator, abbreviated as DHE) was added and incubated for a further 30 minutes. Finally, the fluorescence change of DHE was measured and quantitatively analyzed using a PerkinElme high-content screening instrument, which was recorded as the NTP@G + glucose group.

[0093] 4T1 cells were cultured at 37°C and 5% CO2 at a density of 5 × 10 cells per well. 3The cells were seeded into a 96-well plate at a high density. After 24 hours, the old culture medium in the 96-well plate was aspirated and a DMEM medium containing 50 ng / mL of NTP@G in Example 1 was added, wherein the DMEM medium contained 2 mM glucose. After 1 hour of incubation, 5 μM dihydroethidium (superoxide radical indicator, abbreviated as DHE) was added and incubated for another 30 minutes. Next, each well was illuminated with a 660 nm laser at 0.2 W / cm 2 Finally, the fluorescence changes of DHE were measured using a PerkinElme high-content screening instrument and quantitatively analyzed, which was recorded as the NTP@G+glucose+light group.

[0094] 4T1 cells were cultured at 37°C and 5% CO2 at a density of 5 × 10 cells per well. 3 The cells were seeded into a 96-well plate at a density of 100 μg / mL. After 24 h, the old culture medium in the 96-well plate was aspirated and a DMEM medium containing 50 ng / mL of NTP@G in Example 1 was added, wherein the DMEM medium contained 2 mM glucose and 50 μM ascorbic acid. After culturing for 1 h, 5 μM dihydroethidium (superoxide radical indicator, abbreviated as DHE) was added and incubated for another 30 min. Next, each well was illuminated with a 660 nm laser at 0.2 W / cm 2 Finally, the fluorescence changes of DHE were measured using a PerkinElme high-content screening instrument and quantitatively analyzed, which was recorded as the NTP@G+glucose+ascorbic acid+light group.

[0095] The results are as follows Figure 6 As shown. Among them, the DHE excitation wavelength is fixed at 488nm, and the emission wavelength is between 570nm and 630nm. Figure 6 As shown in (a), after 4T1 cells were treated with NTP or NTP@G, 0.2W / cm 2 The relative fluorescence intensity of DHE in the light-treated group was significantly higher than that in the non-light-treated group. Interestingly, as shown in Figure (b) (where ★★★ The results indicate statistically significant differences (p ≤ 0.001). The horizontal axes 1, 2, 3, 4, 5, 6, and 7 correspond to the NTP group, NTP + light group, NTP@G group, NTP@G + light group, NTP@G + glucose group, NTP@G + glucose + light group, and NTP@G + glucose + ascorbic acid light group, respectively. The NTP@G + glucose + light group exhibited the highest relative fluorescence intensity, with a 6-fold increase in fluorescence. DHE fluorescence was higher in the NTP@G + glucose + light group than in the NTP + light group, indicating that NTP@G can induce the production of more superoxide radicals. These results demonstrate that NTP@G can induce the production of more superoxide radicals during GOx-mediated starvation therapy, potentially enabling multimodal synergistic cancer therapy.

[0096] The same experiment was conducted on NTP and NTP@G in Example 2. The results were basically the same as those of NTP and NTP@G in Example 1, and are not described again here.

[0097] Example 5

[0098] Evaluation of the effect of NTP@G on the accumulation of 4T1 subcutaneous tumors in mice

[0099] Female Balb / c nude mice (6 weeks, 15-20 g) were injected subcutaneously in the right hind leg with 1×10 6 4T1 tumor cells were used to establish a mouse subcutaneous tumor model. When the subcutaneous tumor volume exceeded 100 mm 3 In vivo fluorescence imaging experiments were performed on mice injected with NTP and NTP@G in Example 1. Under 660nm light excitation, NBP@G has a fluorescence signal in the 680-720nm band. Therefore, the changes in tumor accumulation were observed by the small animal fluorescence effect system. The results are shown in Figure 2. Figure 7 .

[0100] like Figure 7 As shown in (a) and (b), NTP accumulation in the tumor reaches its maximum 12 hours after caudal vein administration, after which it begins to be gradually metabolized. In contrast, NTP@G in vivo accumulation reaches its maximum 24 hours after caudal vein administration, and its metabolism is relatively slow, indicating that NTP@G can be targeted to the tumor site with higher targeting efficiency than NTP. Therefore, NTP@G demonstrates good tumor targeting and high tumor accumulation.

[0101] The results of NTP and NTP@G accumulation in mouse 4T1 subcutaneous tumors in Example 2 are similar to those of NTP and NTP@G in Example 1 and are not described again here.

[0102] Example 6

[0103] Evaluation of the inhibitory effect of starvation / chemotherapy / photodynamic therapy on 4T1 tumor growth

[0104] Female Balb / c nude mice (6 weeks, 15-20 g) were injected subcutaneously in the right hind leg with 1×10 6 4T1 tumor cells were used to establish a mouse subcutaneous tumor model. When the tumor volume reached 60 mm 3 In the tumor model, the tumor-bearing mice were randomly divided into six groups: (1) Control group (blank control); (2) Control light group; (3) NTP injection group; (4) NTP injection light group; (5) NTP@G injection group; (6) NTP@G injection light group. The tumor volume was measured with a vernier caliper every other day, and the weight of the mice was monitored at the same time. The tumor volume was calculated according to the formula V = AB 2 / 2 to calculate the tumor volume, where A is the long diameter of the tumor (mm) and B is the short diameter of the tumor (mm). Each measurement result was normalized by the initial tumor volume before treatment. The experimental results are shown in Figure 8 .

[0105] Figure 8 Figure (a) shows the change in tumor volume over time in nude mice treated in different groups in the tumor model, while figure (b) shows the weight of the dissected tumors in the different treatment groups after 15 days of treatment (the horizontal axes 1, 2, 3, 4, 5, and 6 represent the control group, control illumination group, NTP group, NTP illumination group, NTP@G group, and NTP@G illumination group, respectively). As shown in Figure (a), the NTP@G illumination group significantly inhibited the growth of subcutaneous 4T1 breast cancer tumors and had the best tumor inhibition effect compared to the other treatment groups. As shown in Figure (b), the weight of the dissected tumor was the lightest 15 days after NTP@G injection and illumination treatment, further indicating that NTP@G illumination treatment almost completely inhibited tumor growth.

[0106] The same experiment was conducted on NTP and NTP@G in Example 2. The results were basically the same as those of NTP and NTP@G in Example 1, and are not described again here.

[0107] In summary, the present invention provides a nanotherapeutic agent based on a near-infrared photosensitizer, and its preparation method and application. The present invention combines a near-infrared photosensitizer with a TK bond, and then combines the near-infrared photosensitizer with a hydrophobic chemotherapy drug through the TK bond to obtain a hydrophobic near-infrared prodrug; then, using a protein as a carrier, the hydrophobic near-infrared prodrug is combined with the protein through a hydrophobic effect, so that the near-infrared prodrug is bound to the hydrophobic cavity of the protein, thereby being encapsulated by the protein to obtain a nanotherapeutic agent based on a near-infrared photosensitizer. When the nanotherapeutic agent based on a near-infrared photosensitizer is targeted to a tumor, the near-infrared prodrug is released, and the hydrogen peroxide in the tumor environment cleaves the TK bond between the near-infrared photosensitizer and the chemotherapy drug in the near-infrared prodrug, breaking the TK bond, thereby releasing the chemotherapy drug and the near-infrared photosensitizer, thereby simultaneously achieving synergistic treatment of tumor near-infrared imaging-guided chemotherapy and photodynamic therapy. The nanotherapeutic agent based on near-infrared photosensitizer provided by the present invention greatly improves the solubility and bioavailability of chemotherapy drugs and near-infrared photosensitizers, can achieve tumor targeting and selectivity, thereby increasing the accumulation of drugs in the tumor site, improving the therapeutic effect, and reducing toxic side effects, solving the problems of limited targeting and selectivity and low therapeutic effect of existing near-infrared fluorescent dyes.

[0108] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A nanotherapeutic agent based on a near-infrared photosensitizer, characterized in that: The nanotherapeutic agent based on the near-infrared photosensitizer includes a protein and a near-infrared prodrug bound to the hydrophobic cavity of the protein through a hydrophobic interaction. The structural formula of the near-infrared prodrug is: The mass ratio of the near-infrared prodrug to the protein is 1:25 to 1:40; The protein is glucose oxidase; The nano therapeutic agent based on near-infrared photosensitizer is nanoparticles, and the particle size of the nanoparticles is 80-120 nm.

2. A method for preparing a nanotherapeutic agent based on a near-infrared photosensitizer according to claim 1, characterized in that: Including steps: Provided are near-infrared prodrugs and proteins, wherein the structural formula of the near-infrared prodrug is: The protein is glucose oxidase; mixing the near-infrared prodrug with a protein to obtain the near-infrared photosensitizer-based nanotherapeutic agent; The preparation method of the near-infrared prodrug comprises the steps of: Will and paclitaxel are dissolved in an organic solvent, 4-dimethylaminopyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride are added, and after reaction, the near-infrared prodrug is obtained; The preparation method comprises the steps of: 1-Bronaphthalene and 1,4-diaminohexane were added to 2-methoxyethanol, and then CuI and CsCO3 were added to reflux; The obtained product is mixed with alkyl thiosulfate and potassium dichromate, and methanol and hydrochloric acid are added to react to obtain a near-infrared photosensitizer; Copper sulfide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride are added to dimethylformamide, and then the near-infrared photosensitizer is added. After reaction, the product is obtained.

3. Use of a near-infrared photosensitizer-based nanotherapeutic agent according to claim 1 or a near-infrared photosensitizer-based nanotherapeutic agent prepared by the preparation method according to claim 2 in the preparation of a preparation for treating tumors, wherein the tumor is breast cancer.

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