Preparation and application of a diagnostic and therapeutic integrated nanocapsule based on pH reversibly activated photosensitizer

Abstract

The present invention discloses the preparation and application of an integrated diagnosis and treatment nanocapsule based on a pH reversibly activated photosensitizer, which belongs to the field of advanced functional materials and biomedicine technology. The method for preparing an integrated diagnosis and treatment nanocapsule described in the present invention comprises the following steps: (1) synthesizing a pH reversibly activated photosensitizer BAC808 by chemical reaction design; (2) selecting amphiphilic materials DSPE‑PEG‑S‑S‑PEG‑DMMA and DSPE‑PEG‑cRGD as carriers to prepare nanocapsules loaded with BAC808. The integrated diagnosis and treatment nanocapsules prepared by the present invention have intelligent tumor targeting, microenvironment-responsive precise imaging-guided photodynamic / photothermal therapy effects, as well as good water dispersibility and biocompatibility, and have great application potential.

Description

Technical Field

[0001] The invention relates to the preparation and application of a diagnosis and treatment integrated nanocapsule based on a pH reversibly activated photosensitizer, and belongs to the technical field of advanced functional materials and biomedicine. Background Art

[0002] Malignant tumors have always been one of the major diseases that threaten human life, health and safety. The diagnosis rate of early cancer in my country is low. As the tumor progresses, the treatment effect becomes worse and worse, and the patient's survival rate decreases. Therefore, developing a simple, efficient and safe method for early diagnosis and detection of tumors, and then achieving treatment for various types of tumors, is crucial to saving lives.

[0003] Phototherapy has been considered as a promising tumor treatment method because of its excellent characteristics such as high spatiotemporal precision, invasiveness and low side effects. They usually rely on excitable photoactive fluorophores, such as photosensitizers triggered by light of a specific wavelength. Therefore, designing emissive photosensitizers is of practical significance for realizing imaging-guided phototherapy, because it can provide diagnosis and treatment at the same time and monitor the therapeutic effect in real time during tumor treatment. In addition, compared with traditional "always on" probes, reversibly activated photosensitizers are particularly attractive because they can achieve specific imaging treatment of tumors while reducing toxic side effects. However, individual photosensitizers usually have poor water dispersibility, biocompatibility and tumor targeting, which hinders their development in the field of biotherapy. With the development of nanotechnology, the diagnosis and treatment system based on nanoplatforms has opened up new ideas for solving this problem. Therefore, designing and synthesizing a new nanomaterial that combines excellent biocompatibility, ideal tumor targeting, and activatable fluorescence imaging and treatment capabilities has become a new challenge in the field of tumor diagnosis and treatment. Summary of the invention

[0004] [Technical issues]

[0005] Traditional photosensitizers are mostly "always on" probes, which have strong background interference and nonspecific damage to normal cells. In addition, most individual photosensitizers have poor water dispersibility, biocompatibility and tumor targeting, which seriously affects their imaging and therapeutic effects in vivo.

[0006] [Technical solution]

[0007] In order to solve the above problems, the present invention designed and synthesized an integrated diagnosis and treatment nanocapsule loaded with a pH-reversibly activated photosensitizer, and used it for the photothermal / photodynamic combined therapy guided by precise fluorescence imaging of tumors, which overcomes and solves the problems of unsatisfactory treatment effects caused by traditional photosensitizers such as high background interference, large nonspecific damage and poor tumor targeting.

[0008] The first object of the present invention is a method for preparing a photothermal / photodynamic / fluorescence diagnosis and treatment integrated nanocapsule based on a pH-reversibly activated photosensitizer, comprising the following steps:

[0009] pH reversibly activated photosensitizer BAC808, DSPE-PEG-SS-PEG-DMMA (polyethylene glycol functionalized with amphiphilic distearyl phosphatidylethanolamine (DSPE) and 2,3-dimethylmaleic anhydride (DMMA) containing disulfide bonds) and DSPE-PEG-cRGD (polyethylene glycol functionalized with DSPE and cyclic arginine-glycine-aspartic acid (cRGD) polypeptide) were dissolved in an organic solvent, mixed and then slowly added to ultrapure water, and the organic solvent was removed after a period of time to obtain photothermal / photodynamic / fluorescence diagnosis and treatment integrated nanocapsules, recorded as DMMA@cRGD@BAC808;

[0010] Among them, the structure of pH reversibly activated photosensitizer BAC808 is shown in Formula I:

[0011]

[0012] In one embodiment of the present invention, the mass ratio of BAC808, DSPE-PEG-SS-PEG-DMMA and DSPE-PEG-cRGD is 1:(1-2):(1-2). Specifically, it can be 1:1.5:1.5.

[0013] In one embodiment of the present invention, the organic solvent may be tetrahydrofuran.

[0014] In one embodiment of the present invention, the volume ratio of the organic solvent to water is 1:(8-10), and can be specifically 1:9.

[0015] In one embodiment of the present invention, after removing the organic solvent, the mixture is filtered through a filter membrane to obtain DMMA@cRGD@BAC808. The diameter of the corresponding filter membrane is 0.22 μm.

[0016] In one embodiment of the present invention, the synthesis method of the pH reversibly activated photosensitizer comprises:

[0017] (a) using compound 1 and 3-methyl-2-butanone as starting materials to react to obtain compound 2; (b) using compound 2 and compound 3 as reaction substrates to react to obtain compound 4; (c) adding an alkali reagent to the ethanol solution of compound 4 and mixing for reaction; (d) after the reaction, removing ethanol and re-dissolving the compound in water, acidifying with an acid reagent, centrifuging, collecting the solid, washing, and drying to obtain a compound with a structure shown in formula I, recorded as BAC808;

[0018] Among them, compound 1: Compound 2: Compound 3: Compound 4: Compounds of formula I:

[0019] In one embodiment of the present invention, in step (a), the molar ratio of compound 1 to 3-methyl-2-butanone is 1:(1-2); the reaction temperature is 80-120° C., and the reaction time is 12 h.

[0020] In one embodiment of the present invention, in step (b), the molar ratio of compound 2 to compound 3 is 1:(1-2); the reaction temperature is 80-120° C., and the reaction time is 12 h.

[0021] In one embodiment of the present invention, in step (c), the alkaline reagent is a saturated solution of sodium hydroxide; the volume ratio in the solvent is 10%-20%; the reaction temperature is 50° C., and the reaction time is 6 hours.

[0022] In one embodiment of the present invention, in step (d), the method for removing ethanol is negative pressure evaporation at a temperature of 40-50°C.

[0023] In one embodiment of the present invention, in step (d), the acid reagent is a 1M hydrochloric acid solution acidified to pH 4-5.

[0024] In one embodiment of the present invention, in step (d), the centrifugal speed is 6000-8000 rpm, and the time is 5 min-10 min.

[0025] In one embodiment of the present invention, in step (d), the precipitate is washed 2-4 times; and the washing solvent is ultrapure water.

[0026] In one embodiment of the present invention, in step (d), the drying method is freeze-drying.

[0027] The second purpose of the present invention is to prepare a diagnostic and therapeutic integrated nanocapsule DMMA@cRGD@BAC808 based on a pH reversibly activated photosensitizer using the above method.

[0028] The third object of the present invention is to provide an application of the above-mentioned integrated diagnosis and treatment nanocapsule in the preparation of biological imaging reagents.

[0029] The fourth object of the present invention is to provide a method for live cell imaging or in vivo imaging, wherein the method uses the above-mentioned integrated diagnosis and treatment nanocapsule as a probe. The cells may specifically include mouse squamous cell carcinoma cell SCC-7.

[0030] The fifth objective of the present invention is to use the above-mentioned integrated diagnosis and treatment nanocapsules in the preparation of drugs for treating tumors.

[0031] [Beneficial Effects]

[0032] (1) The DMMA@cRGD@BAC808 prepared in the present invention has pH responsiveness, and the near-infrared absorption emission activated by the weakly acidic microenvironment enables it to effectively avoid the interference of biological autofluorescence and endogenous substances in cells, showing the characteristics of strong specificity, high sensitivity and low biological toxicity and side effects.

[0033] (2) The DMMA@cRGD@BAC808 prepared in the present invention can be used as an integrated diagnosis and treatment probe to achieve near-infrared imaging-guided synergistic photothermal / photodynamic therapy of tumors under the excitation of 808nm laser, overcoming the defects of single treatment and greatly improving the treatment effect.

[0034] (3) The DMMA@cRGD@BAC808 prepared by the present invention not only has excellent water dispersibility and biocompatibility, but also the DMMA and cRGD in DMMA@cRGD@BAC808 give it a longer blood circulation time and ideal tumor intelligent targeting properties, making it possible to be used as a tumor-targeted responsive diagnosis and treatment integrated nanomaterial to realize the diagnosis and treatment of tumors in living bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the NMR image of the pH reversibly activated photosensitizer BAC808 prepared in Example 1.

[0036] Figure 2 This is the mass spectrum of the pH reversibly activated photosensitizer BAC808 prepared in Example 1.

[0037] Figure 3 Infrared spectra of compound 4, BAC808 and DMMA@cRGD@BAC808 prepared in Example 1.

[0038] Figure 4 This is the transmission electron microscopy image of DMMA@cRGD@BAC808 prepared in Example 1.

[0039] Figure 5 The ultraviolet absorption spectrum and fluorescence emission spectrum of DMMA@cRGD@BAC808 prepared in Example 1.

[0040] Figure 6Graph showing the singlet oxygen generation capability of DMMA@cRGD@BAC808 prepared in Example 1, wherein A0 and A are the ultraviolet absorption intensity of the DPBF solution at 410 nm before irradiation and the ultraviolet absorption intensity of the DPBF solution at 410 nm after irradiation, respectively.

[0041] Figure 7 This is the photothermal effect diagram of DMMA@cRGD@BAC808 prepared in Example 1.

[0042] Figure 8 This is the dark cytotoxicity graph of BAC808 and DMMA@cRGD@BAC808 prepared in Example 1.

[0043] Fig. 9 This is a diagram showing the in vitro cancer cell killing effect of DMMA@cRGD@BAC808 prepared in Example 1.

[0044] Fig.10 This is the in vivo fluorescence imaging of DMMA@cRGD@BAC808 prepared in Example 1.

[0045] Fig.11 This is a graph showing the relative volume changes of tumors in different groups of mice during the treatment with DMMA@cRGD@BAC808 prepared in Example 1; V0 and V are the tumor volumes before treatment and on a certain day after treatment, respectively. DETAILED DESCRIPTION

[0046] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0047] The DSPE-PEG-SS-PEG-DMMA and DSPE-PEG-cRGD involved in the present invention are purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0048] Example 1

[0049] A method for preparing a diagnosis and treatment integrated nanocapsule based on a pH-reversibly activated photosensitizer, specifically comprising the following steps:

[0050] (1) Synthesis of BAC808:

[0051] The molar ratio of compound 1 to 3-methyl-2-butanone is 1:1.2. The mixture is heated to 100°C and reacted for 2 hours. The mixture is cooled to room temperature, extracted with dichloromethane, and concentrated. 20 mL of glacial acetic acid is added to the resulting red liquid, and the mixture is refluxed for 12 hours. The mixture is cooled to room temperature, 50 mL of dichloromethane is added, and the glacial acetic acid is neutralized with a saturated aqueous sodium carbonate solution. The mixture is extracted with dichloromethane, concentrated, and separated by column chromatography to obtain compound 2 with a yield of 74%.

[0052] The prepared compound 2 and compound 3 were dispersed in CH3CN at a molar ratio of 1:1, and stirred vigorously at 80°C under reflux overnight. After the mixture was cooled to room temperature, it was extracted with dichloromethane, washed with saturated potassium carbonate aqueous solution and ultrapure water until neutral, and concentrated by rotary evaporation. Compound 4 was obtained by column chromatography with the eluent firstly being petroleum ether: ethyl acetate = 10:1 and then petroleum ether: ethyl acetate = 3:1 (V / V) with a yield of 42%.

[0053] Compound 4 was dissolved in 30 mL of ethanol, and then 2 mL of saturated NaOH solution was added. After stirring at 50°C for 6 h, the ethanol was concentrated to remove the ethanol, 30 mL of purified water was added, and the solution was adjusted to acidity (pH 4-5) with 1 M hydrochloric acid. The precipitate was centrifuged at 8000 rpm for 5 min in a high-speed centrifuge, and the precipitate was placed in a freeze dryer to remove moisture to obtain BAC808.

[0054] The structure of synthesized BAC808 was characterized by NMR, mass spectrometry and Fourier transform infrared spectroscopy. Figure 1 ) and mass spectrum ( Figure 2 ) is the same as the theoretical value. In the Fourier transform infrared (FT-IR) spectrum, compound 4 has a peak at 1742 cm -1 The characteristic peak of the ester group disappeared, and the peak of the product at 1715 cm -1 A new absorption peak appeared at the position, which is the characteristic peak of -COOH, proving the successful preparation of BAC808 ( Figure 3 ).

[0055] (2) Preparation of DMMA@cRGD@BAC808:

[0056] 1.5 mg DSPE-PEG-SS-PEG-DMMA, 1.5 mg DSPE-PEG-cRGD and 1 mg BAC808 were dissolved in 1 mL tetrahydrofuran, and then the solution was dripped dropwise into 9 mL pure water with a 1 mL syringe under stirring conditions, and the tetrahydrofuran was removed by stirring at room temperature in a fume hood. After 12 h, the solution was filtered with a 0.22 μm filter membrane and fixed to volume in a 10 mL volumetric flask.

[0057] The absorbance of BAC808 precipitated on the bottle wall, syringe and filter membrane was measured by UV-visible spectrophotometer, and the content of BAC808 in DMMA@cRGD@BAC808 was calculated with reference to the standard curve of BAC808. The synthesized DMMA@cRGD@BAC808 was characterized by Fourier transform infrared spectroscopy. -1 (C=C) and 1643cm -1 The appearance of the (-CONH-) characteristic absorption peak confirmed the successful synthesis of DMMA@cRGD@BAC808 ( Figure 3 ). Figure 4 From its transmission electron microscopy image, it can be seen that the prepared DMMA@cRGD@BAC808 is spherical with an average diameter of about 131.0±8.9nm.

[0058] Comparative Example 1

[0059] Referring to Example 1, only the amounts of DSPE-PEG-SS-PEG-DMMA, DSPE-PEG-cRGD and BAC808 in step (2) were changed to 1.5 mg, 1.5 mg and 0.3 mg, and the other amounts remained unchanged. The results showed that the amount of probes encapsulated in the synthesized nanocapsules was too small.

[0060] Referring to Example 1, only the amounts of DSPE-PEG-SS-PEG-DMMA, DSPE-PEG-cRGD and BAC808 in step (2) were changed to 0.2 mg, 0.2 mg and 1 mg, and the other contents remained unchanged. The results showed that nanocapsules with uniform particle size could not be synthesized.

[0061] Referring to Example 1, only the amounts of DSPE-PEG-SS-PEG-DMMA, DSPE-PEG-cRGD and BAC808 in step (2) were changed to 0.2 mg, 1.5 mg and 1 mg, respectively, while the other contents remained unchanged. The results showed that the synthesized nanocapsules could not achieve charge reversal under acidic conditions and were not suitable for in vivo circulation and metabolism.

[0062] Example 2

[0063] The optical properties of DMMA@cRGD@BAC808 obtained in Example 1 were investigated:

[0064] DMMA@cRGD@BAC808 (expressed as BAC808 concentration, 1×10 -5 mol L -1 ), and adjusted to acidic and alkaline with 0.1M hydrochloric acid and 0.1M sodium hydroxide solution respectively. The absorption spectrum was measured by UV-visible near-infrared spectrophotometer and the emission spectrum was scanned by fluorescence spectrometer (the excitation wavelength was the maximum absorption wavelength).

[0065] The results are as follows Figure 5As shown, DMMA@cRGD@BAC808 exhibits a characteristic absorption peak at 518nm under alkaline conditions, and there is no fluorescence emission. When the solution becomes acidic, the characteristic absorption peak at 518nm disappears, and a strong characteristic absorption appears at 808nm, indicating that the excitation wavelength of DMMA@cRGD@BAC808 is 808nm, and it can have excellent excitation efficiency at 808nm. In addition, the fluorescence of DMMA@cRGD@BAC808 is also specifically activated under acidic conditions, and a strong near-infrared fluorescence emission appears near 830nm. More importantly, the reverse process of the above process occurs when the probe solution changes from acidic to alkaline. This pH-specific reversible response characteristic makes it possible for the obtained probe to achieve high signal-to-noise ratio near-infrared fluorescence imaging that is specifically activated in the weakly acidic microenvironment of the tumor environment, and also provides the possibility for tumor-specific treatment.

[0066] Meanwhile, the present invention also compares other photosensitizers (structures as shown in Formula II), and prepares corresponding nanocapsules by the same method as in Example 1.

[0067]

[0068] The optical properties were measured and it was found that the excitation wavelength of the nanocapsule was 780 nm, and the ultraviolet absorption at 808 nm was significantly weaker than that of DMMA@cRGD@BAC808, that is, the excitation efficiency of DMMA@cRGD@BAC808 at 808 nm was better than that of the nanocapsule prepared by the photosensitizer shown in Formula II.

[0069] Example 3

[0070] The in vitro photodynamic / photothermal therapy effect of DMMA@cRGD@BAC808 obtained in Example 1 was investigated:

[0071] (1) Singlet oxygen production performance evaluation:

[0072] Take 1,3-diphenylisobenzofuran (DPBF) as the indicator and take 18 mL of a 6×10 -5 M of DPBF solution and 2 mL of DMMA@cRGD@BAC808 with different concentrations were mixed in a 50 mL centrifuge tube. The concentrations of the obtained solutions were 2.5×10 -6 M, 5.0×10 -6 M and 1.0×10 -5 M and adjust its pH to acidic. Then, the power density was 0.6 W cm -2The 808nm laser was irradiated for 5min. At the same time, the UV absorption spectra of each solution were measured at different time points (0s, 10s, 20s, 30s, 40s, 50s, 60s, 75s, 1.5min, 2min, 3min, 5min) using a UV-visible near-infrared spectrophotometer. -5 M) and DPBF alone were used as the control group and blank group.

[0073] The results are as follows Figure 6 As shown in the figure, under acidic conditions, the corresponding UV absorption of DPBF gradually decreases with the extension of illumination time, indicating that singlet oxygen is produced in the process. Under alkaline conditions, no change in the fluorescence intensity of DPBF was found during the entire irradiation process, indicating that no singlet oxygen is produced under alkaline conditions. The experimental results confirm that DMMA@cRGD@BAC808 has the ability to produce singlet oxygen only under acidic conditions, ensuring tumor-specific photodynamic therapy without causing damage to adjacent normal tissues.

[0074] (2) Photothermal performance evaluation:

[0075] 1 mL of acidic DMMA@cRGD@BAC808 with different concentrations (expressed in terms of BAC808 concentration, 2.5×10 -6 M, 5.0×10 -6 M and 1.0×10 -5 M) were placed in a 48-well plate. The power density was 0.6 W cm -2 The 808 nm laser was irradiated for 10 min. The temperature of each solution was recorded by infrared thermal imager every 1 min. -5 M) and phosphate buffer solution were used as the control group and blank group.

[0076] The results are as follows Figure 7 As shown in the figure, under acidic conditions, the probe solution showed a significant temperature increase effect, which was enhanced with the extension of irradiation time and the increase of solution concentration. In contrast, under alkaline conditions, no obvious temperature change was shown during the entire irradiation process. It can be seen that the probe has a strong photothermal conversion effect only under acidic conditions, ensuring the possibility of tumor-specific treatment.

[0077] Example 4

[0078] The dark cytotoxicity and photothermal / photodynamic killing effects of DMMA@cRGD@BAC808 obtained in Example 1 were evaluated.

[0079] (1) Cytotoxicity:

[0080] Taking 3T3 as an example, 1×10 5 cell / mL cell suspension (100 μL / well) and incubate overnight. Then remove the culture medium and add 100 μL of fresh culture medium containing different concentrations of probe (0, 2.5, 5, 10, 20, 50 μM), repeat 5 times for each group, and continue incubation for 24 hours. Remove the culture medium, wash with PBS, and add 100 μL of 0.5 mg mL -1 The fresh medium of MTT was continued to be cultured for 4 hours. The culture medium was discarded, PBS was washed, 100 μL of DMSO was added, and the cells were placed in a 37°C constant temperature shaker in the dark for 30 minutes, and then the absorbance at a wavelength of 570 nm was measured with an enzyme marker.

[0081] The results are as follows Figure 8 As shown in the figure, the relative survival rate of 3T3 cells was still higher than 90% until the probe concentration reached 50 μM, which means that the probe had no obvious cytotoxicity at this concentration. In addition, compared with BAC808 alone, the integrated diagnosis and treatment nanocapsule has lower cytotoxicity and can be used for cell imaging studies.

[0082] (2) Evaluation of photothermal / photodynamic killing effects at the cellular level:

[0083] Cell culture and incubation were the same as the above cytotoxicity test. -5 M) After incubation, the pH of the culture system was adjusted to 6.5 and the 808nm 0.6W cm -2 The laser was irradiated for 3, 5 and 10 min, and then incubated for another 12 h. After 12 h, the culture medium was discarded, the cells were washed with PBS, and 100 μL of 0.5 mg mL -1 The cells were cultured in fresh MTT medium for 4 h. The liquid in the wells was removed, the wells were washed with PBS, 100 μL of DMSO was added, and the wells were placed in a 37°C constant temperature shaker in the dark for 30 min. The absorbance at a wavelength of 570 nm was measured using an enzyme marker.

[0084] The results are as follows Fig. 9 As shown, the probe concentration was 1×10 -5 When the pH value is 6.5, irradiation for 5 minutes can kill about 90% of tumor cells, while it has no killing effect on normal cells under normal body fluid conditions. This proves that the probe can only work in weakly acidic tumor sites.

[0085] Example 5

[0086] The in vivo imaging performance and in vivo therapeutic effect of DMMA@cRGD@BAC808 obtained in Example 1 were evaluated.

[0087] (1) In vivo imaging performance:

[0088] To explore the imaging effect of DMMA@cRGD@BAC808 on tumors, the SCC-7 tumor-bearing mice were randomly divided into three groups and injected with the same concentration of BAC808, DMMA@BAC808 and DMMA@cRGD@BAC808 (1×10 -4 M, 150 μL). Fluorescent images of mice were collected at different time points (0 h, 4 h, 6 h, 8 h, 10 h, 12 h and 24 h) by IVIS imaging system.

[0089] DMMA@BAC808 was prepared by the following method: referring to Example 1, except that DSPE-PEG-cRGD was not added in step (2), and the rest remained unchanged.

[0090] The results are as follows Fig.10 As shown in the figure, no fluorescence signal was detected in the tumor site of SCC-7 tumor-bearing mice injected with BAC808, which is because BAC808 alone has no targeting and is easily excreted from the body through blood circulation metabolism. In contrast, after tail vein injection of DMMA@BAC808, a strong fluorescence signal appeared in the tumor site of the mouse. The reason for this phenomenon is that compared with organic small molecule fluorescent groups, the enhanced penetration and retention effect of nanocapsules and the charge reversal property of DMMA on their surface simultaneously give DMMA@BAC808 an extended blood circulation time and ideal tumor targeting ability. In addition, compared with the DMMA@BAC808 experimental group, the fluorescence intensity of the tumor site of SCC-7 tumor-bearing mice injected with DMMA@cRGD@BAC808 nanocapsules was higher, because the cRGD exposed only in the tumor microenvironment enhanced the binding ability of the nanocapsule with tumor cells, proving that DMMA@cRGD@BAC808 can be used for high-sensitivity near-infrared fluorescence imaging of living tumors.

[0091] (2) Evaluation of in vivo treatment effects

[0092] The SCC-7 tumor-bearing mice were divided into 5 groups (n=3): (1) no treatment, (2) phosphate buffer solution + 808 nm laser (0.6 W cm -2 , 5 min), (3) DMMA@cRGD@BAC808, (4) DMMA@BAC808+808 nm laser (0.6 W cm -2 , 5 min), (5) DMMA@cRGD@BAC808+808 nm laser (0.6 W cm -2 , 5min). The tumor size was measured with a vernier caliper every day, and the changes in body weight and tumor volume were calculated.

[0093] The results are as follows Fig.11As shown, the tumor tissue volume of mice in groups (4) and (5) was significantly suppressed. Among them, the tumor in the DMMA@BAC808+808nm laser treatment group showed a recurrence trend within two weeks, while the tumor in the DMMA@cRGD@BAC808+808nm laser treatment group was completely eliminated, indicating that DMMA@cRGD@BAC808 has a more excellent in vivo photodynamic / photothermal treatment effect.

[0094] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a photothermal / photodynamic / fluorescence diagnosis and treatment integrated nanocapsule based on pH reversibly activated photosensitizer, comprising the following steps: The pH-reversibly activated photosensitizer BAC808, DSPE-PEG-SS-PEG-DMMA and DSPE-PEG-cRGD were dissolved in an organic solvent, mixed and slowly added to ultrapure water. After a period of time, the organic solvent was removed to obtain a photothermal / photodynamic / fluorescence diagnosis and treatment integrated nanocapsule, recorded as DMMA@cRGD@BAC808; The organic solvent is tetrahydrofuran; in, The structure of pH reversibly activated photosensitizer BAC808 is shown in Formula I: Formula I.

2. The method according to claim 1, characterized in that The mass ratio of BAC808, DSPE-PEG-SS-PEG-DMMA and DSPE-PEG-cRGD is 1:(1-2):(1-2).

3. The method according to claim 1, characterized in that The volume ratio of organic solvent to water is 1:(8-10).

4. The method according to any one of claims 1 to 3, characterized in that: The synthesis method of the pH reversibly activated photosensitizer comprises: (a) Compound 1 and 3-methyl-2-butanone are used as starting materials to react to obtain compound 2; (b) Compound 2 and compound 3 are used as reaction substrates to react to obtain compound 4; (c) an alkali reagent is added to the ethanol solution of compound 4, and the mixture is mixed for reaction; (d) after the reaction, the ethanol is removed and the compound is redissolved in water, acidified with an acid reagent, centrifuged, the solid is collected, washed, and dried to obtain a compound with a structure shown in formula I, which is recorded as BAC808; Among them, compound 1: ; Compound 2: ; Compound 3: ; Compound 4: ; Compound of formula I: ; In step (c), the alkaline reagent is a saturated sodium hydroxide solution with a volume fraction of 10%-20% in the solvent, the reaction temperature is 50°C, and the reaction time is 6 h; in step (d), the acid reagent is a 1 M hydrochloric acid solution acidified to pH 4-5.

5. The method according to claim 4, characterized in that In step (a), the molar ratio of compound 1 to 3-methyl 2-butanone is 1:(1-2), the reaction temperature is 80-120°C, and the reaction time is 12 h; in step (b), the molar ratio of compound 2 to 3 is 1:(1-2), the reaction temperature is 80-120°C, and the reaction time is 12 h.

6. The photothermal / photodynamic / fluorescence diagnosis and treatment integrated nanocapsule prepared by the method according to any one of claims 1 to 5.

7. Use of the photothermal / photodynamic / fluorescence diagnosis and treatment integrated nanocapsule according to claim 6 in the preparation of biological imaging agents.

8. Use of the photothermal / photodynamic / fluorescence diagnosis and treatment integrated nanocapsule according to claim 6 in the preparation of drugs for treating tumors.

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