Multi-modal imaging nano diagnosis and treatment agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a multi-mode imaging nano diagnosis and treatment agent and a preparation method and application thereof. The preparation method comprises the following steps: S1, preparing a dimethyl sulfoxide solution of IR780; s2, preparing a tannic acid aqueous solution; s3, preparing a ferric trichloride hexahydrate aqueous solution; s4, dropwise adding ultrapure water into the dimethyl sulfoxide solution of IR780 in an ultrasonic state, and continuously performing ultrasonic treatment after dropwise adding is completed; s5, dropwise adding a tannic acid aqueous solution into the mixed solution in the step S4 in an ultrasonic state, and continuously performing ultrasonic treatment after dropwise adding is completed; and S6, dropwise adding a ferric trichloride aqueous solution into the mixed solution in the step S5 in an ultrasonic state, continuously carrying out ultrasonic treatment after dropwise adding, and carrying out centrifugal washing at the rotating speed of 8000-10000. The invention overcomes the defects of long treatment cycle and the like of the traditional diagnostic agent and therapeutic agent, and has the advantages of good biocompatibility, small toxic and side effects and strong light regulation and control capability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a multimodal imaging nano-diagnostic agent and a preparation method and application thereof. Background Art

[0002] Early screening of cancer and integrated precise diagnosis and treatment are the difficulties in the field of cancer prevention and treatment. Usually, the diagnosis and treatment of tumors are two relatively independent stages in clinical practice. The separate use of diagnostic agents and therapeutic agents not only prolongs the diagnosis / treatment cycle, but also greatly increases the risk of delaying the disease and increases the economic burden on patients. With the rapid development of nanotechnology, integrated diagnosis and treatment preparations have been cleverly designed and constructed, and have received widespread attention in the biomedical field. However, how to achieve accurate tumor diagnosis and efficient and controllable tumor treatment in vivo is a research difficulty in integrated diagnosis and treatment preparations.

[0003] Photothermal therapy uses photothermal agents to convert light energy into heat energy, and kills tumor cells by heating up the local tumor. It is an efficient, controllable, non-invasive tumor treatment technology. Most photothermal agents have photothermal imaging function, which can provide a certain reference for disease diagnosis. At present, photothermal agents mainly include inorganic photothermal agents and organic photothermal agents. Inorganic photothermal agents are mainly carbon materials, precious metal materials and transition metal materials, and have the advantages of strong photothermal conversion ability, stable photothermal performance and easy modification, but they generally have poor biosafety and non-degradability. Organic photothermal agents include polymer photothermal agents (such as polydopamine and polypyrrole, etc.) and organic small molecule photothermal agents (such as ICG and IR780, etc.). Organic small molecule photothermal agents have good biocompatibility and easy metabolism, but they have problems such as short circulation time in the body and poor photothermal cycle stability. In addition, organic small molecule photothermal agents not only have photothermal imaging function, but also often have fluorescence imaging function. The two types of imaging can make up for each other's shortcomings in depth and accuracy. Dual-modality imaging can improve the precise diagnosis and treatment of tumors. Therefore, how to improve the photothermal stability of organic small molecule photothermal agents and prolong their circulation time in the body is an urgent problem to be solved in order to promote the further application of organic small molecule photothermal agents in the field of integrated tumor diagnosis and treatment.

[0004] In addition, in order to effectively determine the location of the lesion and realize real-time monitoring and efficacy evaluation of tumor treatment, the contrast technology is combined with the photothermal agent. The advantage of the high resolution of the contrast agent is used to provide guidance for the irradiation of photothermal therapy, which can greatly improve the sensitivity of diagnosis and the efficiency of treatment.

[0005] As one of the most widely used imaging diagnostic methods in scientific research and medical applications, nuclear magnetic resonance has the advantages of high sensitivity and large spatial resolution. Combining it with photothermal agents can complement each other's advantages and achieve accurate diagnosis of tumors through photothermal / fluorescence / nuclear magnetic resonance multimodal imaging. By guiding the irradiation conditions of photothermal therapy (irradiation position, irradiation nodes and irradiation duration, etc.), it can effectively improve the treatment efficiency and avoid damage and destruction of normal tissues. Summary of the invention

[0006] To address the deficiencies of the prior art, the present invention provides a multimodal imaging nano-diagnostic agent and its preparation method and application. The multimodal imaging nano-diagnostic agent provided by the present invention is an integrated diagnosis and treatment preparation, specifically integrating photothermal, fluorescence and MRI imaging with photothermal therapy functions. It overcomes the shortcomings of traditional diagnostic agents and therapeutic agents such as long treatment cycles, and has the advantages of good biocompatibility, low toxic side effects, and strong light regulation ability.

[0007] The technical solution provided by the present invention is as follows:

[0008] A method for preparing a multimodal imaging nano-diagnostic agent comprises the following steps:

[0009] S1, prepare dimethyl sulfoxide solution of IR780;

[0010] S2, preparing tannic acid aqueous solution;

[0011] S3, preparing an aqueous solution of ferric chloride hexahydrate;

[0012] S4, add ultrapure water dropwise to the dimethyl sulfoxide solution of IR780 under ultrasonic condition, and continue ultrasonication after the addition is completed;

[0013] S5, adding tannic acid aqueous solution dropwise to the mixed solution in S4 under ultrasonic condition, and continuing ultrasonication after the addition is completed;

[0014] S6. Add ferric chloride aqueous solution drop by drop into the mixed solution in S5 under ultrasonic condition. Continue ultrasonication after the addition is completed. Centrifuge and wash with water at 8000-10000 rpm to obtain a nano-diagnostic agent for multimodal imaging.

[0015] In the above technical solution:

[0016] The photothermal agent IR780 is coated with a complex of tannic acid and iron ions. Specifically, the dimethyl sulfoxide solution of IR780 is slowly precipitated during the slow dripping of ultrapure water. At this time, the added tannic acid is adsorbed on the surface of IR780 by its own adhesion. In the further dripping of ferric chloride, a polyphenol network is formed to wrap IR780.

[0017] The problem of insufficient water solubility and aqueous solution stability of photothermal agent IR780 can be overcome by coating the photothermal agent IR780 with a complex of tannic acid and iron ions.

[0018] The photothermal capacity and photothermal cycle stability of the photothermal agent IR780 can be further increased by coating the photothermal agent IR780 with a complex of tannic acid and iron ions.

[0019] Specifically, in steps S4-S6: the molar ratio of tannic acid to ferric chloride hexahydrate is 1:(2-3).

[0020] Specifically, in steps S4-S6, the molar ratio of tannic acid to IR780 is (2-4):1.

[0021] Specifically, in steps S4-S6: the ultrasonic time in each step is 5 to 30 minutes respectively.

[0022] Preferably, the molar ratio of tannic acid, ferric chloride hexahydrate and IR780 in the reaction system is 4:(8-12):(1-2).

[0023] Specifically, the concentration of the dimethyl sulfoxide solution of IR780 is 0.4-0.5 mg / mL.

[0024] Specifically, the concentration of the tannic acid aqueous solution is 2 to 5 mg / mL.

[0025] Specifically, the concentration of the ferric chloride hexahydrate aqueous solution is 2 to 5 mg / mL.

[0026] Among them, too little tannic acid will result in the inability to effectively encapsulate IR780, while too much tannic acid will not result in a stable nano-diagnostic agent and flocs will precipitate.

[0027] The present invention also provides a multimodal imaging nano-diagnostic agent prepared by the above preparation method, with a hydrated particle size of 100 to 120 nm and a dry particle size of 20 to 30 nm.

[0028] This particle size range has the advantage of being able to be passively targeted to tumor tissue via the enhanced permeation and retention effect (EPR effect).

[0029] Specifically, the ultraviolet maximum absorption peak of the nano-diagnostic agent for multimodal imaging is in the range of 760 to 810 nm.

[0030] This range can be fully applicable to commonly used near-infrared light sources, thereby facilitating the application of photothermal therapy under near-infrared light irradiation conditions.

[0031] The present invention also provides an application of a multimodal imaging nano-diagnostic and therapeutic agent for preparing an anti-tumor diagnostic and therapeutic agent with multimodal imaging function and photothermal therapy function.

[0032] Specifically: the multimodal imaging function includes fluorescence imaging, MRI imaging and photothermal imaging.

[0033] Specifically: the photothermal therapy function is photothermal therapy under a light source of 760 to 810 nm.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1) The synthesis method of the present invention is simple, controllable, green and environmentally friendly.

[0036] 2) The nano-diagnostic and therapeutic agent prepared by the present invention is a highly efficient carrier system, and the photothermal agent treatment component accounts for 50% of the mass of the multifunctional nano-diagnostic and therapeutic agent.

[0037] 3) The nano-diagnostic and therapeutic agent prepared by the present invention has excellent photothermal conversion performance, can absorb 808nm near-infrared light to generate heat, effectively ablate tumor cells, and improve the efficiency of tumor treatment.

[0038] 4) The nano-diagnostic and therapeutic agent prepared by the present invention has excellent fluorescence imaging performance and photothermal imaging performance, and has certain MRI imaging capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is an optical photograph of the multimodal nano-diagnostic agent of Example 3.

[0040] Figure 2 This is the transmission electron microscopy image of the multimodal nanodiagnostic agent of Example 3.

[0041] Figure 3 This is the hydrated particle size diagram of the multimodal nano-diagnostic agent of Example 3.

[0042] Figure 4 This is the ultraviolet absorption spectrum of the multimodal nano-diagnostic agent of Example 3.

[0043] Figure 5 The photothermal curves of the multimodal nano-therapeutic agent of Example 3: (Part A) photothermal cycle stability, (Part B) photothermal imaging at different concentrations, and (Part C) photothermal imaging at different powers.

[0044] Figure 6 This is the T1 relaxation rate of the multimodal nanotherapeutic agent of Example 3.

[0045] Figure 7 This is the cell survival rate after co-culture of the multimodal nano-diagnostic and therapeutic agent of Example 3 with (Part A) tumor cells 4T1 or (Part B) normal cells 3T3.

[0046] Figure 8 This is the in vivo imaging image of the multimodal nano-diagnostic and therapeutic agent in mice of Example 3. DETAILED DESCRIPTION

[0047] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0048] Unless otherwise specified, the test methods used in the examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0049] IR780 is a kind of organic small molecule photothermal agent with excellent fluorescence performance and photothermal conversion performance, and efficient mitochondrial localization ability, but its defects such as poor water solubility, poor stability and high acute toxicity in vivo limit its further application in vivo. In order to improve the water solubility and stability of IR780 and reduce acute toxicity and side effects, the inventors used a polyphenol network formed by tannic acid and iron ions to coat IR780 and prepared a multimodal imaging nano-diagnostic agent (referred to as multimodal nano-diagnostic agent). Through the coating of the polyphenol network, the water solubility, stability and photothermal cycle stability of IR780 have been greatly improved. The prepared multimodal nano-diagnostic agent has spectral absorption at 760-810, can absorb 808nm near-infrared light to generate heat, has a photothermal effect, and has fluorescence / photothermal / MRI multimodal imaging functions.

[0050] The molecular formula of IR780 is C 36 H 44 ClIN2, CAS number is 207399-07-3.

[0051] Example 1

[0052] Preparation of multimodal nanotheranostics

[0053] Prepare 2 mL of dimethyl sulfoxide solution of IR780 with a concentration of 0.5 mg / mL, slowly add 7 mL of ultrapure water to the dimethyl sulfoxide solution of IR780 under ultrasound, and continue ultrasound for 5 minutes. Then slowly add 60 μL of tannic acid aqueous solution (5 mg / mL) to the above solution and ultrasound for 5 minutes. Finally, continue to add 60 μL of hexahydrate ferric chloride aqueous solution (2 mg / mL) and ultrasound for 5 minutes to obtain a multimodal nano-diagnostic agent solution. The resulting solution was centrifuged at 10000 rpm for 10 minutes, and the resulting solid product was repeatedly washed with ultrapure water, and dried to obtain a multimodal nano-diagnostic agent solid product. The maximum ultraviolet absorption peak of the obtained multimodal nano-diagnostic agent aqueous solution is at 780 nm, and the hydrated particle size is 100-110 nm.

[0054] Example 2

[0055] Preparation of multimodal nanotheranostics

[0056] Prepare 4 mL of dimethyl sulfoxide solution of IR780 with a concentration of 0.5 mg / mL, slowly add 14 mL of ultrapure water to the dimethyl sulfoxide solution of IR780 under ultrasound, and continue ultrasound for 5 minutes. Then slowly add 180 μL of tannic acid aqueous solution (5 mg / mL) to the above solution and ultrasound for 5 minutes. Finally, continue to add 180 μL of ferric chloride hexahydrate aqueous solution (2 mg / mL) and ultrasound for 5 minutes. The resulting solution was centrifuged at 8000 rpm for 10 minutes, and the resulting solid product was repeatedly washed with ultrapure water, and after drying, a multimodal nanodiagnostic agent solid product was obtained. The maximum ultraviolet absorption peak of the obtained multimodal nanodiagnostic agent aqueous solution is at 780 nm, and the hydrated particle size is 100-120 nm.

[0057] Example 3

[0058] Preparation of multimodal nanotheranostics

[0059] Prepare 2 mL of dimethyl sulfoxide solution of IR780 with a concentration of 0.5 mg / mL, slowly add 7 mL of ultrapure water to the dimethyl sulfoxide solution of IR780 under ultrasound, and continue ultrasound for 5 minutes. Then slowly add 120 μL of tannic acid aqueous solution (5 mg / mL) to the above solution and ultrasound for 5 minutes. Finally, continue to add 60 μL of hexahydrate ferric chloride aqueous solution (4 mg / mL) and ultrasound for 5 minutes. The resulting solution was centrifuged at 9000 rpm for 10 minutes, and the resulting solid product was repeatedly washed with ultrapure water, and after drying, a multimodal nanodiagnostic agent solid product was obtained. The maximum ultraviolet absorption peak of the obtained multimodal nanodiagnostic agent aqueous solution is at 780 nm, and the hydrated particle size is 100-120 nm.

[0060] Comparative Example 1

[0061] IR780 was solubilized by DMSO and then ultrasonically dispersed in water to prepare an IR780 aqueous solution of the same concentration. Within 30 minutes after the IR780 aqueous solution was prepared, free IR780 was precipitated and adsorbed on the tube wall (e.g. Figure 1 As shown, the multimodal nanodiagnostic agent prepared in Example 3 with the same concentration of IR780 can be stably dispersed without precipitation within one week, indicating that the water solubility and aqueous solution stability of the present invention are improved. Figure 1 As shown, TA-Fe is a metal-polyphenol network aqueous solution formed by tannic acid and ferric chloride hexahydrate.

[0062] Test Example 1

[0063] In vitro photothermal performance testing of multimodal nanodiagnostic agents

[0064] The multimodal nano-diagnostic and therapeutic agent of Example 3 was prepared into 0.5 mL aqueous solutions with concentrations of 10.58 mg / L, 14.10 mg / L and 21.15 mg / L, and the heating rates of the multimodal nano-diagnostic and therapeutic agents of different concentrations under irradiation of near-infrared light of 808 nm were tested.

[0065] like Figure 5 As shown, after testing, the multimodal nano-diagnostic agent prepared in Example 3 was irradiated with 808nm near-infrared light power of 1W at concentrations of 10.58mg / L, 14.10mg / L and 21.15mg / L, and the temperature increased by 21.0℃, 28.7℃ and 34.4℃ in 90s respectively. The multimodal nano-diagnostic agent of 14.10mg / L was irradiated with 808nm near-infrared light power of 0.75W, 1W and 1.5W in 90s and the temperature increased by 18.8℃, 24.9℃ and 33.2℃ respectively. It is proved that the multimodal nano-diagnostic agent of the present invention has excellent photothermal conversion efficiency and photothermal imaging capability.

[0066] Test Example 2

[0067] In vitro cell growth inhibition assay

[0068] Tumor cells 4T1 ( Figure 7 Part A) and normal cells 3T3 ( Figure 7 Part B) were inoculated into 96-well plates at a density of 6000 cells / well. After culturing for 24 hours, multimodal nanodiagnostic agents and free IR780 were added to the cells in different groups. The sample concentrations in each group were set at different gradients, containing IR780 concentrations of 0 mg / L, 0.2 mg / L, 0.3 mg / L, 0.7 mg / L, 1.3 mg / L, 2.6 mg / L, 5.3 mg / L, 10.5 mg / L and 21.0 mg / L, respectively. A blank control group was set. After 4 hours of co-culture, fresh culture medium was replaced and culture continued. The non-NIR group did not receive light (respectively recorded as multimodal nanodiagnostic agents and IR780), and the irradiated group received 808nm near-infrared light power 1W irradiation for 90 seconds (respectively recorded as multimodal nanodiagnostic agents + L and IR780 + L). After 44 hours, the relative survival rate was determined by MTT method. The final results are as follows Figure 7As shown in the figure, under near-infrared light irradiation, the cell growth inhibition ability of multimodal nanotheranostics + L is equivalent to that of IR780 + L, and can effectively inhibit the growth of tumor cells 4T1, while the inhibition ability of multimodal nanotheranostics without near-infrared radiation is significantly weakened. This result shows that multimodal nanotheranostics not only have the ability to effectively inhibit the growth of tumor cells, but also can achieve controllable cell growth inhibition through light regulation. In normal cells 3T3, the cell viability of the multimodal nanotheranostics group was significantly higher than that of free IR780. In addition, in normal cells, multimodal nanotheranostics + L had significant differences with multimodal nanotheranostics in a wide concentration range of 0.2 to 21.0 mg / L, while the cell viability of IR780 + L in 3T3 cells was only significantly different from IR780 in a narrow concentration range of 0.2 to 5.3 mg / L, and the dark toxicity of free IR780 was significantly stronger than that of multimodal nanotheranostics. The above results show that the toxic side effects of multimodal nanotheranostics on normal cells are much less than IR780, and multimodal nanotheranostics have better light regulation differences than IR780. Cytotoxicity data show that multimodal nanotheranostics can achieve efficient inhibition of tumor cells through light regulation, and the toxic side effects of multimodal nanotheranostics on normal cells are much lower than IR780.

[0069] Test Example 3

[0070] In vivo imaging tests

[0071] Since the ability of nanophotothermal agents to accumulate in tumor tissues through blood circulation directly affects the final therapeutic effect, the distribution of multimodal nanotheranostics in mice was evaluated by in vivo imaging. After the multimodal nanotheranostics were injected into tumor-bearing mice, in vivo fluorescence imaging was performed regularly. Figure 8 As shown, after injection, the multimodal nanotherapeutic agent accumulated rapidly and effectively in the tumor area. The tumor fluorescence of mice was strongest at 12 hours and could still be effectively retained in the tumor tissue after 24 hours. This shows that the multimodal nanotherapeutic agent has efficient tumor targeting ability and long-term tumor retention ability, as well as excellent in vivo imaging ability. It shows that the multimodal nanotherapeutic agent can accumulate in the tumor area and achieve excellent tumor fluorescence imaging diagnosis.

[0072] like Figure 2 The figure shows a transmission electron microscope image of the multimodal nano-diagnostic agent of Example 3. It can be seen that the multimodal nano-diagnostic agent has a uniform spherical morphology in a vacuum dry state, and the particle size is 20-30 nm.

[0073] like Figure 3 The figure shows the hydrated particle size of the multimodal nano-theranostic agent of Example 3. It can be seen that the average hydrated particle size of the multimodal nano-theranostic agent is between 100 and 120 nm, and can be passively targeted to tumor tissues through the EPR effect.

[0074] like Figure 4 As shown, it is the ultraviolet absorption spectrum of the multimodal nano-diagnostic agent of Example 3. It can be seen that the multimodal nano-diagnostic agent has a strong absorption peak at 780nm, indicating that IR780 has been successfully encapsulated. At the same time, the multimodal nano-diagnostic agent has a strong absorbance value in the range of 760-810nm, indicating that the multimodal nano-diagnostic agent has the potential to absorb near-infrared light in a wide range.

[0075] like Figure 6 , which is the T1 relaxation rate of the multimodal nanotheranostic agent of Example 3. It can be seen that the relaxation rate is linearly correlated with the concentration increase, proving that the multimodal nanotheranostic agent has MRI imaging potential.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a multimodal imaging nanodiagnostic agent, characterized in that: The following steps are involved: S1, prepare dimethyl sulfoxide solution of IR780; S2, preparing tannic acid aqueous solution; S3, preparing an aqueous solution of ferric chloride hexahydrate; S4, add ultrapure water dropwise to the dimethyl sulfoxide solution of IR780 under ultrasonic condition, and continue ultrasonication after the addition is completed; S5, adding tannic acid aqueous solution dropwise to the mixed solution in S4 under ultrasonic condition, and continuing ultrasonication after the addition is completed; S6. Add ferric chloride aqueous solution drop by drop into the mixed solution in S5 under ultrasonic condition. Continue ultrasonication after the addition is completed. Centrifuge and wash with water at 8000-10000 rpm to obtain a nano-diagnostic agent for multimodal imaging.

2. The method for preparing the multimodal imaging nano-diagnostic agent according to claim 1, characterized in that: In steps S4-S6: The molar ratio of tannic acid to ferric chloride hexahydrate is 1:(2-3); Alternatively, the molar ratio of tannic acid to IR780 is (2-4):1; Alternatively, the ultrasonic time in each step is 5 to 30 minutes.

3. The method for preparing the multimodal imaging nano-diagnostic agent according to claim 2, characterized in that: The molar ratio of tannic acid, ferric chloride hexahydrate and IR780 in the reaction system is 4:(8-12):(1-2).

4. The method for preparing the multimodal imaging nano-diagnostic agent according to claim 1, characterized in that: The concentration of DMSO solution of IR780 is 0.4-0.5 mg / mL; The concentration of tannic acid aqueous solution is 2 to 5 mg / mL; The concentration of the ferric chloride hexahydrate aqueous solution is 2-5 mg / mL.

5. A multimodal imaging nano-diagnostic agent prepared by the preparation method according to any one of claims 1 to 4, characterized in that: The hydrated particle size is between 100 and 120 nm; the dry particle size is between 20 and 30 nm.

6. The multimodal imaging nano-diagnostic agent according to claim 5, characterized in that: Its maximum ultraviolet absorption peak is in the range of 760 to 810 nm.

7. An application of the multimodal imaging nano-diagnostic agent according to claim 5 or 6, characterized in that: Used to prepare anti-tumor diagnostic and therapeutic agents with multimodal imaging and photothermal therapy functions.

8. The use according to claim 7, characterized in that: The multimodal imaging function includes fluorescence imaging, MRI imaging and photothermal imaging.

9. The use according to claim 7, characterized in that: The photothermal therapy function is photothermal therapy under a light source of 760 to 810 nm.

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