Carrier-free self-assembled nano-drug responding to tumor microenvironment as well as preparation method and application of carrier-free self-assembled nano-drug

Abstract

The invention belongs to the technical field of biological medicine preparation, and discloses a carrier-free self-assembled nano-drug responding to a tumor microenvironment as well as a preparation method and application of the carrier-free self-assembled nano-drug. According to the present invention, self-assembly is performed based on ENBS-C6, DOX and DSPE-MPEG2000, such that the nanoparticle ENBS / DOX NPs is constructed, and the nanoparticle ENBS / DOX NPs is used for preparing the tumor chemotherapy-photodynamic combined treatment drug; due to enhanced permeability and retention, the drug can be self-delivered to a tumor site without a carrier. And in an acidic tumor microenvironment, interaction of multiple supermolecules is destroyed, and release of free ENBS and DOX is accelerated. Moreover, after in-vitro chemotherapy and photodynamic combined treatment, the growth of tumor cells is almost completely inhibited. Due to no carrier, near-infrared imaging and effective treatment effect, the nano-drug provided by the invention can provide a promising strategy for combined treatment of cancers.

Description

Technical Field

[0001] This invention relates to the field of biomedical preparation technology, and in particular to a carrier-free self-assembled nanomedicine that responds to the tumor microenvironment, its preparation method, and its application. Background Technology

[0002] Cancer has become one of the most serious diseases worldwide. Many treatments for cancer, such as surgery, chemotherapy, photodynamic therapy, photothermal therapy, radiotherapy, and immunotherapy, have been developed. Chemotherapy is considered a primary treatment due to its high efficacy compared to other methods. Although each individual treatment has some efficacy, monotherapy remains unsatisfactory in eradicating tumors. After decades of experimental and clinical research, combination therapy has proven to be an effective and superior treatment approach. Utilizing the advantages of different drugs' combined therapies and anti-cancer mechanisms while mitigating the disadvantages of each individual approach can synergistically inhibit tumor development. Furthermore, to guide the treatment process and track drug delivery, a common and effective strategy is to incorporate therapeutic agents and imaging agents into nanoplatforms. In recent years, increasing attention has been paid to bioimaging techniques, particularly the absorption and emission of organic photosensitizer dyes in the near-infrared (NIR) window, which not only enhances the depth of penetration into biological tissues but also allows for phototherapy. In combination therapy options, "chemotherapy + photodynamic therapy" has become a promising approach. On the one hand, it enhances the cytotoxicity of drugs through a combined approach, and on the other hand, it utilizes the fluorescent properties of photosensitizers for drug tracking. With the rapid development of nanotechnology and fluorescent nanomaterials, carrier-free nanomedicines with good bioactivity have been rapidly developed and have attracted widespread attention. Compared with traditional carrier-based nanomedicines, carrier-free self-release nanomedicines, as a new generation of drug delivery systems, not only avoid the significant safety issues associated with nanocarriers but also simultaneously achieve the advantages of the nanoscale and the effective drug payload, showing broad application prospects.

[0003] Photodynamic therapy (PDT) is a widely accepted clinical cancer treatment strategy with unique advantages over chemotherapy, including less invasiveness, good spatial and temporal selectivity, and minimal damage to normal tissues, making it a promising alternative treatment. As an externally triggered therapy, PDT typically consists of three separate components: a light source of appropriate wavelength, oxygen (O2), and a photosensitizer (PS). The photosensitizer is activated by laser light of a specific wavelength, generating superoxide anions (O2) via a type I process. -· ), and part of the O2 -· It can be further converted into H2O2 and its downstream highly toxic hydroxyl radicals through a cascade reaction mediated by superoxide dismutase (SOD) and the Fenton reaction. ·OH); or produced through a type II process. 1 O2 induces apoptosis and leads to tumor ablation. Most photodynamic therapy (PDT) is considered to have undergone a type II process. Due to the high O2 dependence of most available photosensitizers, type II PDT still faces significant challenges in preclinical and clinical progression because of the pervasive extreme hypoxia in the harsh microenvironment of solid tumors. A previous research group reported a near-infrared light-activated photostimulant, thionilo blue (ENBS), which can generate reactive oxygen species (O2) through a type I photodynamic reaction. -· H2O2 and · OH, thus effectively killing cancer cells. Furthermore, the type I photosensitizer-induced tumor (PDT) process can compensate for O2 depletion through disproportionation, the Haber-Weiss reaction, or the Fenton reaction, thereby significantly improving the therapeutic effect on hypoxic tumors. Given that nanomedicine has become a common and effective strategy for achieving multimodal tumor therapy, and there are currently no reports on the construction of nanomedicines based on ENBS, the development of carrier-free nanoparticles with low oxygen dependence and type I photosensitizers is of great significance.

[0004] Doxorubicin (DOX), an anthracycline antibiotic, disrupts or blocks cancer cell replication by intercalating into DNA and inhibiting macromolecular biosynthesis. However, the use of DOX is limited by its side effects, such as severe cardiotoxicity. To reduce side effects and improve therapeutic efficacy, researchers often use different carriers to prepare nanoparticles for targeted drug delivery. Nanoparticles, due to their sufficient stability, suitable size, and excellent surface properties, can effectively prevent drug release via vascular convection before reaching the tumor site, avoid rapid filtration by the kidneys and clearance by the reticuloendothelial system, and accelerate drug accumulation at the tumor site through enhanced permeability and retention (EPR) effects, thus effectively reducing drug toxicity. They hold great promise for selectively delivering therapeutic drugs to tumors. Although doxorubicin has been widely used in the construction of nanomedicines, reports on carrier-free nanomedicines based on self-assembly are currently limited. Summary of the Invention

[0005] The purpose of this invention is to provide a carrier-free self-assembled nanomedicine that responds to the tumor microenvironment, its preparation method, and its application. The aim is to achieve the synergistic construction of carrier-free nanomedicine by the type I photosensitizer ENBS and the chemotherapeutic drug DOX, providing a novel drug system for chemotherapy-photodynamic therapy.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing carrier-free self-assembled nanomedicines that respond to the tumor microenvironment, comprising the following steps:

[0008] (1) Preparation of ENBS-C6:

[0009] (1.1) Aluminum sulfate, N,N-diethyl-p-phenylenediamine, sodium thiosulfate, zinc chloride, potassium dichromate and water are mixed and reacted to obtain the first intermediate;

[0010] (1.2) Mix 1-naphthylamine, the first intermediate, silver carbonate and methanol, and react to obtain the second intermediate;

[0011] (1.3) The second intermediate, K2CO3, KI, 6-bromohexanol and N,N-dimethylformamide were mixed and reacted to obtain ENBS-C6;

[0012] (2) Preparation of ENBS / DOX NPs:

[0013] Doxorubicin solution, methanol solution of ENBS-C6, DSPE-MPEG2000 solution and water were mixed and solvent exchanged to obtain ENBS / DOX NPs, which are carrier-free self-assembled nanomedicines that respond to the tumor microenvironment.

[0014] Furthermore, in the preparation method, the mass ratio of aluminum sulfate, N,N-diethyl-p-phenylenediamine, sodium thiosulfate, zinc chloride, and potassium dichromate in step (1.1) is 7.5–9.2:3–5:13–15:3–5:1–2.5.

[0015] Furthermore, in the preparation method, the reaction conditions in step (1.1) include: being carried out in an ice bath for 1.5 to 2.5 hours.

[0016] Furthermore, in the preparation method, the mass ratio of 1-naphthylamine, the first intermediate, and the silver carbonate in step (1.2) is 1.6–2.5: 7.5–8: 7.5–8;

[0017] The mass ratio of 1-naphthylamine to the volume of methanol in step (1.2) is 1.8 to 2.2 g: 40 mL.

[0018] Furthermore, in the preparation method, the reaction conditions in step (1.2) include: a temperature of 60–80°C and a time of 0.5–1 h.

[0019] Furthermore, in the preparation method, the mass ratio of the second intermediate, the K2CO3, the KI and the 6-bromohexanol in step (1.3) is 190-210:120-130:15-25:120-140;

[0020] In step (1.3), the mass ratio of the second intermediate to the volume of the N,N-dimethylformamide is 190-210 mg: 10 mL.

[0021] Furthermore, in the preparation method, the reaction conditions in step (1.3) include: a temperature of 50–70°C and a time of 5–7 h.

[0022] Furthermore, in the preparation method, in the doxorubicin solution of step (2), the ratio of the mass of doxorubicin, the volume of triethylamine, and the volume of dimethyl sulfoxide is 9-10.5 mg: 19-20.5 μL: 0.8-1 mL;

[0023] In step (2), the concentration of ENBS-C6 in the methanol solution is 8-10 mg / mL.

[0024] In step (2), the concentration of DSPE-MPEG2000 in the DSPE-MPEG2000 solution is 8-10 mg / mL;

[0025] In step (2), the volume ratio of the doxorubicin solution, the methanol solution of ENBS-C6, the DSPE-MPEG2000 solution, and the water is 550-650 μL: 0.5-1.5 mL: 350-450 μL: 8-10 mL;

[0026] The conditions for solvent exchange in step (2) include: room temperature, stirring speed of 800-1000 rpm, and time of 1-3 hours.

[0027] This invention also provides a carrier-free self-assembled nanomedicine that responds to the tumor microenvironment.

[0028] This invention also provides the application of a carrier-free self-assembled nanomedicine responsive to the tumor microenvironment in the preparation of drugs for combined chemotherapy-photodynamic therapy for tumors.

[0029] In this invention, the synthetic route for preparing ENBS-C6 in step (1) is shown below:

[0030]

[0031] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0032] like Figure 1As shown, this invention constructs ENBS / DOX nanoparticles (NPs) through self-assembly of thioniloblue derivatives ENBS-C6, DOX, and DSPE-MPEG2000, for the preparation of chemotherapy-photodynamic therapy drugs for tumors. DOX is one of the most widely used anticancer chemotherapy drugs in clinical practice. ENBS, as a type I photosensitizer, can still efficiently generate O2 even under severe hypoxia conditions. ·- and · OH reactive oxygen species enable simultaneous near-infrared imaging and photodynamic therapy (PDT). DOX possesses a hydrophobic anthraquinone ring and a hydrophilic amino sugar. ENBS-C6, with its conjugated benzene ring backbone and alkyl chain, imparts hydrophobicity, while its positively charged tertiary amine structure imparts hydrophilicity. Therefore, ENBS-C6 and DOX can be used to achieve nanoparticle self-assembly based on π-π stacking, hydrophobic interactions, and hydrogen bonding. DSPE-MPEG2000 is used to modify the nanoparticles and facilitate their formation, ensuring a long cycle time for ENBS / DOX NPs nanoparticles. Due to enhanced permeability and retention (EPR), these nanoparticles can be spontaneously delivered to tumor sites without a carrier. In the acidic tumor microenvironment, various supramolecular interactions are disrupted, accelerating the release of free ENBS and DOX. Furthermore, after in vitro chemotherapy and photodynamic therapy combined with other treatments, tumor cell growth was almost completely inhibited. Due to their carrier-free nature, near-infrared imaging capabilities, and effective therapeutic effects, the nanomedicines self-assembled by small molecules described in this application offer a promising strategy for combination therapy of cancer. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0034] Figure 1 The study focuses on carrier-free ENBS / DOX nanoparticles self-assembled from ENBS-C6 and DOX, and their application in the preparation of drugs for chemotherapy-photodynamic therapy of tumors.

[0035] Figure 2 (A) in the figure represents the TEM characterization of ENBS / DOXNPs in Example 1; Figure 2 (B) in the figure represents the DLS characterization of the ENBS / DOX NPs in Example 1; Figure 2 (C) represents the hydration particle size of ENBS / DOX NPs in Example 1 at different times in PBS and DMEM;

[0036] Figure 3(A) shows the UV-Vis-NIR absorption spectra of DOX, ENBS-C6, and ENBS / DOXNPs from Example 1 in PBS. Figure 3 (B) shows the fluorescence emission spectra of ENBS-C6 and ENBS / DOX NPs (pH 7.4, pH 5.5) from Example 1 in PBS; Figure 3 In the diagram (C), the infrared spectra of DOX, ENBS-C6, and ENBS / DOX NPs of Example 1 are shown, where a is DOX, b is ENBS-C6, and c is ENBS / DOX NPs.

[0037] Figure 4 (A) in the figure represents the fluorescence emission spectra of different concentrations of DOX; Figure 4 (B) in the figure represents the linear relationship between DOX concentration and fluorescence intensity; Figure 4 (C) in the figure represents the DOX release curves of ENBS / DOX NPs in Example 1 in 1×PBS at different pH and at 37°C for different incubation times; Figure 4 (D) in the figure represents the fluorescence emission spectra of ENBS-C6 at different concentrations; Figure 4 In the figure, (E) represents the linear relationship between the concentration of ENBS-C6 and the fluorescence intensity. Figure 4 (F) in the figure represents the ENBS-C6 release curves of ENBS / DOX NPs in Example 1 in 1×PBS at different pH and at 37°C for different incubation times;

[0038] Figure 5 (A) shows the use of DHR123 as a fluorescent probe to detect O2 production in different groups under both light and non-light conditions. -· Ability; Figure 5 (B) in the figure represents the ENBS / DOX NPs of Example 1 detected by DHR123 under illumination (660nm, 100mW / cm). 2 Fluorescence emission spectra at different times; Figure 5 (C) represents the detection of different groups under light or no light conditions using HPF as a fluorescent probe. · The ability of OH; Figure 5 In the figure (D), the ENBS / DOX NPs of Example 1 were detected by HPF under illumination (660nm, 100mW / cm). 2 Fluorescence emission spectra at different times;

[0039] Figure 6 HepG-2 cells after different treatments were subjected to light (660nm, 100mW / cm²). 2 Cytotoxicity under conditions of 5 min or no light exposure;

[0040] Figure 7 Fluorescence imaging of live and dead cells in HepG-2 cells treated with different groups under light or no light conditions. Detailed Implementation

[0041] This invention provides a method for preparing carrier-free self-assembled nanomedicines that respond to the tumor microenvironment, comprising the following steps:

[0042] (1) Preparation of ENBS-C6:

[0043] (1.1) Aluminum sulfate, N,N-diethyl-p-phenylenediamine, sodium thiosulfate, zinc chloride, potassium dichromate and water are mixed and reacted to obtain the first intermediate;

[0044] (1.2) Mix 1-naphthylamine, the first intermediate, silver carbonate and methanol, and react to obtain the second intermediate;

[0045] (1.3) The second intermediate, K2CO3, KI, 6-bromohexanol and N,N-dimethylformamide were mixed and reacted to obtain ENBS-C6;

[0046] (2) Preparation of ENBS / DOX NPs:

[0047] Doxorubicin solution, methanol solution of ENBS-C6, DSPE-MPEG2000 solution and water were mixed and solvent exchanged to obtain ENBS / DOX NPs, which are carrier-free self-assembled nanomedicines that respond to the tumor microenvironment.

[0048] In this invention, the preferred method for mixing aluminum sulfate, N,N-diethyl-p-phenylenediamine, sodium thiosulfate, zinc chloride, potassium dichromate, and water in step (1.1) is as follows:

[0049] Aluminum sulfate was mixed with water to obtain an aqueous solution of aluminum sulfate; N,N-diethyl-p-phenylenediamine was added to the solution, followed by sodium thiosulfate and zinc chloride in sequence, and the reaction mixture was cooled; then an aqueous solution of potassium dichromate was added.

[0050] In this invention, the mass ratio of aluminum sulfate to the volume of water is preferably 8.5-9.5:100mL, more preferably 9-9.2:100mL, and even more preferably 9.2g:100mL.

[0051] In this invention, the concentration of potassium dichromate in the aqueous solution is preferably 0.03-0.05 g / mL, more preferably 0.035-0.045 g / mL, and even more preferably 0.04 g / mL.

[0052] In this invention, the cooling is preferably carried out in an ice bath.

[0053] In this invention, the mass ratio of aluminum sulfate, N,N-diethyl-p-phenylenediamine, sodium thiosulfate, zinc chloride, and potassium dichromate in step (1.1) is preferably 7.5–9.2:3–5:13–15:3–5:1–2.5, more preferably 8.5–9.2:4–5:14–14.5:3.5–4:1.5–2, and even more preferably 9.2:5:14.2:3.6:2.

[0054] In this invention, the reaction conditions in step (1.1) include: the temperature is preferably carried out in an ice bath; the time is preferably 1.5 to 2.5 h, more preferably 1.8 to 2.2 h, and even more preferably 2 h.

[0055] In this invention, the reaction described in step (1.1) is preferably followed by post-processing, including: filtering and washing the precipitate generated by the reaction; refluxing the obtained filter residue in methanol, cooling it to room temperature, and then filtering it.

[0056] In this invention, the washing reagent is preferably acetone.

[0057] In this invention, the reflux conditions include: a temperature preferably of 60-80°C, more preferably of 65-75°C, and even more preferably of 70°C; and a time preferably of 0.5-2 hours, more preferably of 1-1.5 hours, and even more preferably of 1 hour.

[0058] In this invention, the preferred method for mixing 1-naphthylamine, the first intermediate, silver carbonate, and methanol in step (1.2) is as follows:

[0059] 1-Naphthylamine and the first intermediate were refluxed in methanol; then silver carbonate was added to the aforementioned reaction mixture.

[0060] In this invention, the reflux conditions include: a temperature preferably of 70-80°C, more preferably of 75-80°C, and even more preferably of 80°C; and a time preferably of 3-5 hours, more preferably of 3.5-4.5 hours, and even more preferably of 4 hours.

[0061] In this invention, the mass ratio of 1-naphthylamine, the first intermediate and the silver carbonate in step (1.2) is preferably 1.6-2.5:7.5-8:7.5-8, more preferably 1.8-2.2:7.6-7.8:7.6-7.8, and even more preferably 2:7.8:7.7.

[0062] In this invention, the mass ratio of 1-naphthylamine to the volume of methanol in step (1.2) is preferably 1.8-2.2 g: 40 mL, more preferably 1.9-2.1 g: 40 mL, and even more preferably 2 g: 40 mL.

[0063] In this invention, the reaction conditions in step (1.2) include: the temperature is preferably 60-80°C, more preferably 65-75°C, and even more preferably 70°C; the time is preferably 0.5-1h, more preferably 0.5-0.8h, and even more preferably 0.5h.

[0064] In this invention, the preferred method for mixing the second intermediate, K2CO3, KI, 6-bromohexanol, and N,N-dimethylformamide in step (1.3) is as follows:

[0065] The second intermediate, K2CO3, and KI were mixed with N,N-dimethylformamide, and 6-bromohexanol was added to the mixture.

[0066] In this invention, the mass ratio of the second intermediate, the K2CO3, the KI and the 6-bromohexanol in step (1.3) is preferably 190-210:120-130:15-25:120-140, more preferably 195-205:122-128:18-22:125-135, and even more preferably 200:125:20:130.

[0067] In this invention, the mass ratio of the second intermediate to the volume of the N,N-dimethylformamide in step (1.3) is preferably 190-210 mg:10 mL, more preferably 195-205 mg:10 mL, and even more preferably 200 mg:10 mL.

[0068] In this invention, the reaction conditions in step (1.3) include: a temperature preferably of 50-70°C, more preferably of 55-65°C, and even more preferably of 60°C; and a time preferably of 5-7 h, more preferably of 5.5-6.5 h, and even more preferably of 6 h.

[0069] In this invention, the preferred method for mixing the doxorubicin solution, the methanol solution of ENBS-C6, the DSPE-MPEG2000 solution, and water in step (2) is as follows:

[0070] Mix the doxorubicin solution with the methanol solution of ENBS-C6, then add the DSPE-MPEG2000 solution and mix thoroughly. Add the mixture to water under stirring at room temperature.

[0071] In this invention, the ratio of the mass of doxorubicin, the volume of triethylamine, and the volume of dimethyl sulfoxide in the doxorubicin solution in step (2) is preferably 9-10.5 mg: 19-20.5 μL: 0.8-1 mL, more preferably 9.5-10 mg: 19.5-20 μL: 0.9-1 mL, and even more preferably 10 mg: 20 μL: 1 mL.

[0072] In this invention, the mixing conditions for preparing the doxorubicin solution in step (2) include: the temperature is preferably room temperature; the time is preferably 40-50 h, more preferably 45-50 h, and even more preferably 48 h.

[0073] In this invention, the concentration of ENBS-C6 in the methanol solution of ENBS-C6 in step (2) is preferably 8 to 10 mg / mL, more preferably 9 to 10 mg / mL, and even more preferably 10 mg / mL.

[0074] In this invention, the concentration of DSPE-MPEG2000 in the DSPE-MPEG2000 solution in step (2) is preferably 8 to 10 mg / mL, more preferably 9 to 10 mg / mL, and even more preferably 10 mg / mL.

[0075] In this invention, the solvent in the DSPE-MPEG2000 solution in step (2) is preferably dimethyl sulfoxide.

[0076] In this invention, the volume ratio of the doxorubicin solution, the methanol solution of ENBS-C6, the DSPE-MPEG2000 solution and the water in step (2) is preferably 550-650 μL: 0.5-1.5 mL: 350-450 μL: 8-10 mL, more preferably 580-620 μL: 0.8-1.2 mL: 380-420 μL: 9-10 mL, and even more preferably 600 μL: 1 mL: 400 μL: 10 mL.

[0077] In this invention, the conditions for solvent exchange in step (2) include: the temperature is preferably room temperature; the stirring speed is preferably 800-1000 rpm, more preferably 900-1000 rpm, and even more preferably 1000 rpm; the time is preferably 1-3 h, more preferably 1.5-2.5 h, and even more preferably 2 h.

[0078] In this invention, after solvent exchange in step (2), post-processing is preferably performed, including: dialysis, centrifugation to remove the supernatant, dispersing the product in water and storing it at 4°C.

[0079] In this invention, the dialysis conditions include: the molecular weight cutoff of the dialysis bag is preferably 3500 Da; the time is preferably 12-24 h, more preferably 18-24 h, and even more preferably 24 h.

[0080] In this invention, the centrifugation conditions include: a rotation speed preferably of 8000-10000 rpm, more preferably of 9000-10000 rpm, and even more preferably of 10000 rpm; and a centrifugation time preferably of 10-20 min, more preferably of 12-18 min, and even more preferably of 15 min.

[0081] This invention also provides a carrier-free self-assembled nanomedicine that responds to the tumor microenvironment.

[0082] This invention also provides the application of a carrier-free self-assembled nanomedicine responsive to the tumor microenvironment in the preparation of drugs for combined chemotherapy-photodynamic therapy for tumors.

[0083] In this invention, the method of application is not limited, and any method well known in the art can be used.

[0084] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0085] Example 1

[0086] This embodiment provides a method for preparing carrier-free self-assembled nanomedicines that respond to the tumor microenvironment, including the following steps:

[0087] (1) Preparation of ENBS-C6:

[0088] (1.1) N,N-diethyl-p-phenylenediamine (5.0 g, 30.4 mmol) was added to an aqueous solution of aluminum sulfate (9.2 g, 26.9 mmol) in 100 mL. Then sodium thiosulfate (14.2 g, 57.3 mmol) and zinc chloride (3.6 g, 25.7 mmol) were added sequentially. The reaction mixture was cooled in an ice bath. Then potassium dichromate (2.0 g, 6.8 mmol) aqueous solution (concentration 0.04 g / mL) was slowly added. The reaction mixture was then stirred in an ice bath for 2 h. After precipitation, the mixture was washed with acetone. The residue was refluxed in methanol (80 mL) at 70 °C for 1 h. After cooling to room temperature, the mixture was filtered to obtain a gray solid first intermediate with a yield of 93%. This product was used directly in the next step.

[0089] (1.2) A mixture of 1-naphthylamine (2.0 g, 14 mmol) and intermediate 5a (7.8 g, 25 mmol) was refluxed in 40 mL of methanol for 4 h at 80 °C. Then, silver carbonate (7.7 g, 28 mmol) was slowly added to the reaction mixture, and the mixture was refluxed at 70 °C for 30 min. After cooling, the mixture was filtered and evaporated to obtain a dark blue solid. Then, dichloromethane (30 mL) was added for extraction, and the mixture was washed with saturated sodium carbonate solution. The organic layer was dried with anhydrous MgSO4, filtered, and acidified with 0.4 mL of concentrated hydrochloric acid. The product was purified by silica gel column purification (CH2Cl2 / methanol) to obtain a dark blue solid second intermediate with a yield of 18%.

[0090] (1.3) Weigh the second intermediate (200 mg, 0.6 mmol), K2CO3 (125 mg, 0.9 mmol) and KI (20 mg) in sequence and place them in a 100 mL round-bottom flask. Dissolve them in 10 mL of DMF. Then add 6-bromohexanol (130 mg, 0.7 mmol) to the reaction system and react at 60 °C for 6 h. The reaction was confirmed to be complete by TLC. Add 100 mL of ice water to the reaction solution and extract with dichloromethane (20 mL × 3) in sequence. Wash with saturated brine (20 mL × 3), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain a blue crude product, and purify by column chromatography to obtain a blue solid ENBS-C6 with a yield of 43%.

[0091] (2) Preparation of ENBS / DOX NPs:

[0092] 10 mg DOX was dissolved in 1 mL of dimethyl sulfoxide (DMSO) solution, and 20 μL of triethylamine solution was added. The mixture was stirred at room temperature for 48 h. 600 μL of DOX in DMSO solution was mixed with 1.0 mL of 10 mg / mL ENBS-C6 methanol solution, and then 400 μL of 10 mg / mL DSPE-MPEG2000 DMSO solution was added and mixed thoroughly. The mixture was added to 10 mL of deionized water under vigorous stirring (1000 rpm) at room temperature. After stirring for 2 h, the mixture was dialyzed for 24 h (dialysis band cutoff 3500 Da). The solution was then centrifuged (10000 rpm, 15 min), and the supernatant was removed to obtain carrier-free self-assembled nanomedicine nanoparticles responsive to the tumor microenvironment, namely NBS / DOX NPs. These nanoparticles were dispersed in 1 mL of ultrapure water and stored at 4 °C.

[0093] Test 1: Preparation and characterization of ENBS / DOX NPs

[0094] In Example 1, ENBS / DOX NPs were prepared by removing hydrochloric acid from DOX·HCl in an alkaline environment to convert it into hydrophobic DOX molecules. Then, an organic solvent of ENBS-C6, DOX, and DSPE-MPEG2000 was dropwise into a deionized aqueous solution under stirring to prepare carrier-free ENBS / DOX NP nanoparticles. A trace amount of amphiphilic polyethylene glycol-modified lipid (DSPE-MPEG2000) can improve the stability, biocompatibility, and long-term cycling ability of the nanoparticles in a biological environment. The feed ratio of ENBS-C6 to DOX was 5:3 (m / m), and the drug loading of DOX and ENBS-C6 in the obtained ENBS / DOX NPs was 31.04% and 60.80%, respectively.

[0095] The 1H and 1C NMR spectra of the second intermediate obtained in step (1.2) of Example 1 are as follows:

[0096] 1 H-NMR (400MHz, DMSO-d6): δ9.53(br,2H),8.91(s,1H),8.43(s,1H),7.92(br,2H),7 .82(s,2H),7.28(br,2H),6.99(s,2H),3.61(q,J=8.0Hz,4H),1.19(t,J=8.0Hz,6H).

[0097] 13 C-NMR (101MHz, DMSO-d6): δ156.53,151.12,138.95,137.08,134.05,133.57,132.11,132.04 ,130.99,129.77,124.98,124.22,123.72,117.41,106.56,105.64,53.36,45.61,13.13(2C).

[0098] The proton and carbon NMR spectra of ENBS-C6 obtained in step (1.3) of Example 1 are as follows:

[0099] 1H-NMR (500MHz, MeOD): δ8.63(d,J=5Hz,1H),8.05(d,J=10Hz,1H),7.69(m,2H),7.63(t,J=10Hz,1H),7.20(d,J=10Hz,1H),6.96(s,1 H), 6.93 (s, 1H), 3.62 (m, 6H), 3.48 (t, J = 10Hz, 2H), 1.79 (t, J = 5Hz, 2H), 1.61 (t, J = 5Hz, 2H), 1.50 (d, J = 5Hz, 4H), 1.32 (t, J = 5Hz, 6H).

[0100] 13 C-NMR (126MHz, MeOD): δ152.86,151.14,139.74,136.91,133.50,133.15,132.46,131.66,130.69,129.30,124.7 6,123.91,121.99,117.14,104.64,101.69,61.39,53.76,45.48,44.10,32.12,28.45,26.57,25.35,11.80(2C).

[0101] Example 1, step (1.3), ESI-HRMS data for ENBS-C6: ESI-HRMS Calc.for C 26 H 32 N3OS + [M] + ,434.2261,found:434.2255.

[0102] The size and morphology of ENBS / DOX NPs nanoparticles were evaluated using transmission electron microscopy (TEM), revealing that the ENBS / DOX NPs particles were monodisperse nanospheres with a size of approximately 80–110 nm. Figure 2 In (A), due to hydration in aqueous solution, the nanoparticles are smaller than dynamic light scattering (DLS). Figure 2 The hydrodynamic diameter (~122 nm) obtained in (B) was also observed. Furthermore, DLS measurements of ENBS / DOX NPs in different solutions were performed at predetermined times (days 1, 2, 3, 4, and 5), as shown in Figure 1. Figure 2 As shown in (C), the nanoparticles maintained stability for at least 5 days in PBS and cell culture medium (DMEM).

[0103] The UV-Vis-NIR absorption spectra of DOX, ENBS-C6, and ENBS / DOX NPs are as follows: Figure 3As shown in (A) of the diagram, a redshift and broader absorption band of the ENBS-C6 absorption peak were observed in ENBS / DOX NPs, while a redshift also occurred in the DOX absorption band, suggesting that DOX may interact with ENBS-C6 through π-π stacking. Furthermore, from... Figure 3 As shown in (B), the fluorescence emission intensity of ENBS-C6 in ENBS / DOX NPs is significantly lower than that of free ENBS-C6, indicating that energy migration due to molecular stacking quenches the fluorescence of ENBS-C6. Under acidic conditions, the fluorescence of ENBS-C6 is released, indicating that the nanoparticles can release ENBS-C6 in an acidic environment. Furthermore, the Fourier transform infrared spectrum of ENBS / DOX NPs was recorded. Figure 3 (C) in the image shows that after the formation of nanoparticles, the C=O in DOX is at 1731 cm⁻¹. -1 The location has moved to 1705cm. -1 The -OH group of ENBS-C6 is at 3465 cm⁻¹ -1 The location has moved to 3395cm. -1 The presence of a broadened spectral band at the [location] indicates an interaction between ENBS-C6 and the DOX molecule. These results demonstrate the successful construction of ENBS / DOX NPs.

[0104] Test 2: Drug Release

[0105] The standard curves of DOX and ENBS-C6 were determined by fluorescence spectrophotometry. Figure 4 (A), (B), (D), and (E) in the example). The ENBS / DOX NPs from Example 1 were dialyzed at 37°C for 48 h in 1×PBS solution at pH 5.5, 6.0, and 7.4, respectively. Figure 4 As shown in (C) and (F), ENBS / DOX NPs exhibit a significant pH-dependent release characteristic. At pH 7.4, the nanoparticles are relatively stable, indicating a tight binding between ENBS-C6 and DOX. However, under acidic conditions at pH 5.5, the release rates of DOX and ENBS-C6 reach as high as 76.48% and 80.87%, respectively. This is because in an acidic environment, hydrogen ions interfere with the π-π stacking of DOX and ENBS-C6, disrupting the stability of π-electron stacking. Furthermore, the protonation of nitrogen atoms within the molecule makes it hydrophilic, thus weakening the hydrophobic interactions between molecules. In addition, the hydrogen bonds between molecules can also be dissociated in an acidic environment, leading to the dissociation of ENBS / DOX NPs and the release of DOX and ENBS-C6. The accelerated release at lower pH also indicates that the release of nanomedicines has a certain selectivity in tumor cells, as the pH of the tumor microenvironment is lower than that of normal tissues.

[0106] Test 3: The ability of ENBS / DOX NPs in Example 1 to generate reactive oxygen species

[0107] ENBS has been proven to be an excellent photosensitizer with highly efficient O2... -· O2 generation capacity. The O2 generation capacity of ENBS-C6 was further evaluated using a dihydrorhodamine 123 (DHR123) probe. -· The ability of DHR123 as O2. -· The probe itself is non-fluorescent, but reacts with O2. -· A green fluorescence was produced at 525 nm after the reaction. For example... Figure 5 As shown in (A), under laser irradiation, except for the free ENBS-C6 group and the ENBS / DOX NPs group, the O2 under illumination... -· The generation efficiency was similar, and the fluorescence intensity of the other groups did not change significantly under illumination. Furthermore, the fluorescence intensity of DHR123 at 525 nm increased with prolonged illumination time. Figure 5 (B) indicates that ENBS / DOX NPs have a high O2 generation rate. -· The ability to form nanoparticles also demonstrates that the photodynamic effect of ENBS-C6 is not affected. Furthermore, hydroxyphenylfluorescein (HPF) was used as a detection... · OH fluorescent probes are themselves non-fluorescent and... · After the OH reaction, a strong green fluorescence is produced at 515 nm, such as Figure 5 As shown in (C), no significant changes in fluorescence intensity were observed in different groups under both light and non-light conditions, and the fluorescence intensity remained unchanged with prolonged light exposure. Figure 5 (D) indicates that ENBS / DOX NPs cannot be generated in solution. · OH. The above results indicate that ENBS / DOX NPs can effectively generate O2 in solution under light irradiation. -· It has the potential to be used in PDT applications.

[0108] Test 4: Evaluation of ENBS / DOX NPs cytotoxicity in Example 1

[0109] The MTT assay was used to detect the cytotoxicity of different groups on HepG-2 cells. Figure 6As shown, DOX, DOX+laser, ENBS-C6, ENBS-C6+laser, ENBS / DOX NPs, and ENBS / DOX NPs+laser groups exhibited dose-dependent cytotoxicity against HepG-2 cells. Compared with chemotherapy with free DOX or free ENBS-C6 alone, the ENBS / DOX NPs group showed higher cytotoxicity, with tumor cells treated with ENBS / DOX NPs showing a survival rate of 53.5%, lower than that of free DOX (64.6%) and ENBS-C6 (90.4%). This may be because the internalization of ENBS / DOX NPs is more efficient than that of free molecules. Under light irradiation, the cell survival rate of cells treated with ENBS / DOX NPs was only 12.6%, indicating that chemotherapy combined with photodynamic therapy significantly increased the mortality rate of tumor cells.

[0110] To further evaluate the antitumor effect of ENBS / DOX NPs in Example 1, Calcein-AM / PI was used to visualize the live and dead cells of HepG-2 cells. The results are as follows: Figure 7 As shown, compared with other groups, the ENBS / DOX NPs + laser group had a significantly higher rate of cell death, indicating that ENBS / DOX NPs have excellent combined chemotherapy and photodynamic therapy effects.

[0111] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing carrier-free self-assembled nanomedicines responsive to the tumor microenvironment, characterized in that, Includes the following steps: (1) Preparation of ENBS-C6: (1.1) Aluminum sulfate, N,N-diethyl-p-phenylenediamine, sodium thiosulfate, zinc chloride, potassium dichromate and water are mixed and reacted to obtain the first intermediate; (1.2) Mix 1-naphthylamine, the first intermediate, silver carbonate and methanol, and react to obtain the second intermediate; (1.3) The second intermediate, K2CO3, KI, 6-bromohexanol and N,N-dimethylformamide were mixed and reacted to obtain ENBS-C6; (2) Preparation of ENBS / DOX NPs: Doxorubicin solution, methanol solution of ENBS-C6, DSPE-MPEG2000 solution and water were mixed and solvent exchanged to obtain ENBS / DOX NPs, which are carrier-free self-assembled nanomedicines that respond to the tumor microenvironment.

2. The preparation method according to claim 1, characterized in that, The mass ratio of aluminum sulfate, N,N-diethyl-p-phenylenediamine, sodium thiosulfate, zinc chloride, and potassium dichromate in step (1.1) is 7.5–9.2:3–5:13–15:3–5:1–2.

5.

3. The preparation method according to claim 1 or 2, characterized in that, The reaction conditions described in step (1.1) include: being carried out in an ice bath for 1.5 to 2.5 hours.

4. The preparation method according to claim 1, characterized in that, The mass ratio of 1-naphthylamine, the first intermediate, and the silver carbonate in step (1.2) is 1.6–2.5: 7.5–8: 7.5–8; The mass ratio of 1-naphthylamine to the volume of methanol in step (1.2) is 1.8 to 2.2 g: 40 mL.

5. The preparation method according to claim 1 or 4, characterized in that, The reaction conditions described in step (1.2) include: a temperature of 60–80°C and a time of 0.5–1 h.

6. The preparation method according to claim 1, characterized in that, In step (1.3), the mass ratio of the second intermediate, the K2CO3, the KI, and the 6-bromohexanol is 190–210: 120–130: 15–25: 120–140; In step (1.3), the mass ratio of the second intermediate to the volume of the N,N-dimethylformamide is 190-210 mg: 10 mL.

7. The preparation method according to claim 1 or 6, characterized in that, The reaction conditions described in step (1.3) include: a temperature of 50–70°C and a time of 5–7 h.

8. The preparation method according to claim 1, characterized in that, In the doxorubicin solution described in step (2), the ratio of the mass of doxorubicin, the volume of triethylamine, and the volume of dimethyl sulfoxide is 9–10.5 mg: 19–20.5 μL: 0.8–1 mL. In step (2), the concentration of ENBS-C6 in the methanol solution is 8-10 mg / mL. In step (2), the concentration of DSPE-MPEG2000 in the DSPE-MPEG2000 solution is 8-10 mg / mL; In step (2), the volume ratio of the doxorubicin solution, the methanol solution of ENBS-C6, the DSPE-MPEG2000 solution, and the water is 550-650 μL: 0.5-1.5 mL: 350-450 μL: 8-10 mL; The conditions for solvent exchange in step (2) include: room temperature, stirring speed of 800-1000 rpm, and time of 1-3 hours.

9. A carrier-free self-assembled nanomedicine responsive to the tumor microenvironment, prepared by the method according to any one of claims 1 to 8.

10. The application of the carrier-free self-assembled nanomedicine responsive to the tumor microenvironment as described in claim 9 in the preparation of a drug for combined chemotherapy-photodynamic therapy of tumors.

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