A mitochondria-targeting anti-tumor nano self-assembled particle and a preparation method and application thereof

Abstract

The application discloses an anti-tumor nano self-assembled particle targeting mitochondria and a preparation method and application thereof. The nano self-assembled particle takes a photothermal agent as a core and takes an active oxygen donor reagent coated on the surface of the photothermal agent as a shell. The application utilizes the photothermal agent and the active oxygen donor to prepare the nano particle through self-assembly, realizes photodynamic therapy excited by near-infrared laser, has a deeper tissue penetration depth and causes less damage to normal tissues; the active oxygen is produced by breaking a peroxide bridge in artemether, and the limitation of traditional photodynamic therapy on anoxic conditions in a tumor microenvironment is overcome; the active oxygen is produced on a subcellular level by targeting mitochondria of tumor cells; and the nano particle has self-reporting fluorescence and can realize real-time monitoring of the in-vivo distribution of the nano particle.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a mitochondria-targeting anti-tumor nano self-assembled particle and a preparation method and application thereof. BACKGROUND

[0002] At present, existing cancer treatment technologies such as surgical treatment and chemotherapy methods have certain limitations, and therefore, a laser photothermal treatment method having the advantages of non-invasiveness / minimal invasiveness and being capable of greatly reducing the pain of patients gradually enters the field of vision of people. Photodynamic therapy relies on a photosensitizer, under laser irradiation, the photosensitizer delivers energy to surrounding oxygen to generate highly active singlet oxygen, which oxidizes surrounding biological macromolecules to produce cytotoxicity and kill tumor cells. Compared with traditional treatment methods, photodynamic therapy has the advantage of accurately performing effective treatment with minimal side effects.

[0003] For example, CN113321644A discloses a supramolecular photothermal agent compound having a stimulus response, a composition and application thereof, which can self-assemble into a micro-nano structure in an aqueous solution, can be passively and actively double-targeted and enriched in tumor tissues, and can release the photothermal agent under pathological stimulus response of the tumor microenvironment, and exhibits higher photothermal conversion efficiency, excellent photothermal stability and the advantages of fluorescence imaging. However, the excitation of the traditional photosensitizer used in the photodynamic therapy usually requires visible light or ultraviolet light, however, long-term exposure of biological tissues to shorter wavelengths will cause DNA damage and normal cell death. Therefore, short-wavelength excitation of the photosensitizer molecule has obvious disadvantages. In addition, the tissue penetration ability in the ultraviolet and visible light regions is weak, and is only suitable for surface treatment, and is difficult to be applied to deep tumor treatment research.

[0004] In summary, it is of great significance to develop a new and effective photodynamic drug preparation for the field of tumor photodynamic therapy. SUMMARY

[0005] In view of the deficiencies of the prior art and actual needs, the application provides a mitochondria-targeting anti-tumor nano self-assembled particle and a preparation method and application thereof. The photothermal material having near-infrared absorption and the active oxygen donor reagent are combined to self-assemble into a nano particle in an aqueous solution, so as to realize mitochondria-targeting photodynamic therapy under near-infrared excitation independent of the oxygen content in the tumor environment, and to be used for efficient, safe and deep tumor treatment.

[0006] To achieve the above purpose, the application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a mitochondria-targeted anti-tumor nano self-assembled particle, which has a photo-thermal agent as a core and an active oxygen donor agent coated on the surface of the photo-thermal agent as a shell, and the structure of the active oxygen donor agent (artemether-PEG-Cy5 phospholipid chain) is shown in Formula I, wherein n is 50-100, including but not limited to 51, 52, 53, 54, 55, 60, 70, 80, 90, 95, 96, 97, 98 or 99.

[0008]

[0009] In the present application, the photo-thermal material with near-infrared absorption and the active oxygen donor agent (such as artemether-PEG-Cy5 phospholipid chain) are combined to self-assemble into nanoparticles in an aqueous solution, the photo-thermal agent and artemether are combined, the high photo-thermal conversion capability of the photo-thermal agent is utilized to promote the thermal decomposition of artemether to produce active oxygen (ROS), the near-infrared laser excited photodynamic therapy is realized, the tissue penetration depth is deeper, and the damage to normal tissues is smaller; the peroxide bridge in artemether is broken to produce active oxygen, which overcomes the limitation of traditional photodynamic therapy under the hypoxic condition in the tumor microenvironment; the mitochondria of tumor cells are targeted, and active oxygen is produced at the subcellular level; in addition, the nanoparticles have self-reporting fluorescence, and the in vivo distribution of the nanoparticles can be monitored in real time.

[0010] Optionally, the photo-thermal agent includes indocyanine green (ICG) and / or benzodithiabazole (BPBBT).

[0011] Optionally, the mass ratio of the photo-thermal agent and the active oxygen donor agent in the nano self-assembled particle is 1:(2-5), including but not limited to 1:3, 1:4 or 1:5.

[0012] Optionally, the particle size of the nano self-assembled particle is 100-400 nm, including but not limited to 101 nm, 102 nm, 110 nm, 120 nm, 130 nm, 140 nm, 145 nm, 148 nm, 150 nm, 200 nm, 260 nm, 300 nm, 350 nm, 390 nm, 450 nm, 460 nm, 480 nm, 490 nm or 498 nm.

[0013] In a second aspect, the present application provides a preparation method of the mitochondria-targeted anti-tumor nano self-assembled particle of the first aspect, and the preparation method comprises:

[0014] The photo-thermal agent, the active oxygen donor agent and the solvent are mixed to obtain a mixed solution, and the mixed solution is mixed with water to obtain the mitochondria-targeted anti-tumor nano self-assembled particle.

[0015] Optionally, the preparation method of the active oxygen donor agent comprises:

[0016] The artemether 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are mixed with a solvent, NH2-PEG 2000-Cy5 is added, stirring in the dark, and a condensation reaction is carried out to obtain a phospholipid skeleton modified with artemether and a Cy5 fluorescent group, i.e., an active oxygen donor reagent.

[0017] In a specific embodiment of the present application, 100 mg of artemether is dissolved in 2 mL of dimethyl sulfoxide, 10 equivalents of EDCl and NHS are added, stirring in the dark for 2 h, 100 mg of NH2-PEG(2000)-Cy5 is added, stirring in the dark for 12 h, and a condensation reaction is carried out to obtain a phospholipid skeleton modified with artemether and a Cy5 fluorescent group.

[0018] In a third aspect, the present application provides use of the mitochondria-targeting anti-tumor nano-self-assembling particle of the first aspect in the preparation of an anti-tumor drug.

[0019] Optionally, the tumor comprises pancreatic cancer.

[0020] In a fourth aspect, the present application provides a pharmaceutical composition comprising the mitochondria-targeting anti-tumor nano-self-assembling particle of the first aspect.

[0021] Preferably, the pharmaceutical composition further comprises an excipient.

[0022] Preferably, the excipient comprises any one or a combination of at least two of a pharmaceutically acceptable wetting agent, a solubilizing agent, an osmotic pressure regulator, a coating material, a coloring agent, a pH regulator, an antioxidant, or a buffer.

[0023] In a fifth aspect, the present application provides a method for killing tumor cells in vitro, the method comprising:

[0024] The target sample is mixed with the mitochondria-targeting anti-tumor nano-self-assembling particle of the first aspect, and the target sample is irradiated with near-infrared laser light, the target sample being an ex vivo tissue or a cell suspension, the tissue or cell suspension containing tumor cells and normal cells.

[0025] Preferably, the tumor cells comprise pancreatic cancer cells.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] The application uses a photo-thermal agent and an active oxygen donor to prepare nanoparticles through self-assembly, realizes photodynamic therapy excited by near-infrared laser, has a deeper tissue penetration depth, and causes less damage to normal tissues; the active oxygen is produced by breaking the peroxide bridge in artemether, which overcomes the limitation of traditional photodynamic therapy under the hypoxic condition in the tumor microenvironment; the active oxygen is produced at the subcellular level by realizing the targeting of tumor cell mitochondria; the nanoparticles have self-reporting fluorescence, and the in-vivo distribution of the nanoparticles can be monitored in real time. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The figure of artemether-PEG-Cy5 phospholipid skeleton nucleic acid verification results prepared for example 1 is shown in the figure;

[0029] Figure 2 The figure of ultraviolet absorption results of the mitochondria-targeted anti-tumor nano self-assembled particles prepared for example 1 is shown in the figure;

[0030] Figure 3 The figure of fluorescence emission results of the mitochondria-targeted anti-tumor nano self-assembled particles prepared for example 1 is shown in the figure;

[0031] Figure 4 The figure of particle size results of the mitochondria-targeted anti-tumor nano self-assembled particles prepared for example 1 is shown in the figure;

[0032] Figure 5 The figure of fluorescence of Art-PEG-Cy5@PTA in mice is shown in the figure;

[0033] Figure 6 The figure of tumor volume change of mice in test example 3 is shown in the figure;

[0034] Figure 7 The figure of body weight change of mice in test example 3 is shown in the figure. DETAILED DESCRIPTION

[0035] In order to further illustrate the technical means adopted by the application and its effects, the application will be further described below in conjunction with the embodiments and the drawings. It can be understood that the specific embodiments described herein are only used to explain the application, and not to limit the application.

[0036] If a specific technology or condition is not specified in the embodiments, the technology or condition described in the literature in the art or according to the product instruction is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be purchased through a regular channel.

[0037] Example 1

[0038] In this embodiment, the preparation and characterization of mitochondria-targeted anti-tumor nano self-assembled particles are carried out.

[0039] 1 mg near-infrared photothermal agent (ICG, purchased from Sigma, item number 1340009) and 2 mg artemether-PEG-Cy5 phospholipid skeleton were dissolved in 1 mL of tetrahydrofuran, slowly dropped into 5 mL of deionized water under ultrasonic conditions, and the organic solvent was blown dry with nitrogen to obtain mitochondria-targeted anti-tumor nano self-assembled particles (named Art-PEG-Cy5@PTA).

[0040] The preparation method of the artemether-PEG-Cy5 phospholipid skeleton is specifically as follows:

[0041] 100 mg of artemether was dissolved in 2 mL of dimethyl sulfoxide, 10 times the amount of EDCl and NHS was added, and stirred in the dark for 2 h, 100 mg of NH2-PEG(2000)-Cy5 (purchased from Xi'an Ruishi Biological Technology Co., Ltd., model R-C-27) was added, and stirred in the dark for 12 h to undergo condensation reaction to obtain a phospholipid skeleton modified with artemether and a Cy5 fluorescent group. The nuclear magnetic resonance verification result is shown in Figure 1 , and the specific molecular structure formula is shown below, n = 54.

[0042]

[0043] The ultraviolet absorption, fluorescence emission and particle size of the sample were determined. The specific method includes: using an ultraviolet visible spectrophotometer to determine the absorption of the sample in the range of 200-1100 nm, using a fluorescence spectrometer to determine the emission spectrum of the sample, and using a Malvern particle size detector to determine the particle size of the sample.

[0044] The ultraviolet absorption result is shown in Figure 2 , the absorption spectrum shows that the sample has two absorption peaks, which are the absorption peak of Cy5 at 630 nm and the absorption peak of the photothermal agent at 860 nm; the fluorescence emission result is shown in Figure 3 , the emission spectrum shows that the sample emits in the range of 640-750 nm; the particle size result is shown in Figure 4 , the particle size result shows that the particle size of the sample is mostly distributed at 140 nm.

[0045] Example 2

[0046] In this example, mitochondria-targeted anti-tumor nano self-assembled particles were prepared and characterized.

[0047] 1 mg near-infrared photothermal agent (ICG, purchased from Sigma, item number 1340009) and 2 mg artemether-PEG-Cy5 phospholipid skeleton were dissolved in 1 mL of tetrahydrofuran, slowly dropped into 5 mL of deionized water under ultrasonic conditions, and the organic solvent was blown dry with nitrogen to obtain mitochondria-targeted anti-tumor nano self-assembled particles.

[0048] The preparation method of the artemether-PEG-Cy5 phospholipid skeleton is referred to Example 1.

[0049] Test Example 1

[0050] This test example tests the reactive oxygen generation capability of the mitochondria-targeted anti-tumor nano self-assembled particles prepared in Examples 1 and 2 at the subcellular level.

[0051] The mitochondria-targeted anti-tumor nano self-assembled particles prepared in Examples 1 and 2, respectively, are incubated with pancreatic cancer cells (PANC-1) purchased from Biyun Tian with the product number C6725 for 4 hours to ensure sufficient intracellular entry, and then the reactive oxygen monitoring probe (2,7-dichlorodihydrofluorescein diacetate DCFH-DA probe, purchased from Biyun Tian with the product number S0033S) is added and incubated with the cells for 30 min, and then irradiated with near-infrared laser for 10 min, and then the probe is removed, and the fluorescence signal of the probe is detected under a confocal fluorescence microscope.

[0052] Test Example 2

[0053] This test example tests the enrichment capability of the mitochondria-targeted anti-tumor nano self-assembled particles prepared in Example 1 at the tumor site of a mouse.

[0054] The mitochondria-targeted anti-tumor nano self-assembled particles prepared in Example 1 are injected into the body of a tumor-bearing mouse (Balb / c immunodeficient mouse, purchased from Guangdong Yaoke, 100 μL (40 μg / mL) is injected) via the tail vein, and the fluorescence signal intensity in the mouse body at different time points is detected using an IVIS small animal imaging system.

[0055] The results are shown in Figure 5 After the self-assembled nanoparticles prepared in the present application are injected into the mouse body, strong fluorescence signals can be observed within 2 h, the nanoparticles are enriched at the tumor site, and obvious fluorescence signals can still be observed at the tumor site of the mouse after 24 h.

[0056] Test Example 3

[0057] This test example tests the tumor treatment effect of the mitochondria-targeted anti-tumor nano self-assembled particles prepared in Example 1 in a mouse body.

[0058] The mitochondria-targeted anti-tumor nano self-assembled particles prepared in Example 1 are injected into the body of a tumor-bearing mouse (Balb / c immunodeficient mouse, purchased from Guangdong Yaoke Biotechnology Co., Ltd., 100 μL (40 μg / mL) is injected) via the tail vein, and an equal amount of PBS buffer is injected as a control, and then the tumor site of the mouse is irradiated with near-infrared laser for 10 min at 12 h after injection, and the change in tumor volume and body weight of the mouse is detected.

[0059] The tumor volume changes of mice after injection of PBS and Art-PEG-Cy5@PTA nanoparticles, respectively, are shown in Figure 6 As shown in the figure, four experimental groups are divided, Blank is a blank control group (injection of PBS), without light treatment; Art-PEG-Cy5@PTA (NIR+) is injection of Art-PEG-Cy5@PTA and light treatment; Blank (NIR-) is a blank control group, light treatment; Art-PEG-Cy5@PTA (NIR-) is injection of Art-PEG-Cy5@PTA, without light treatment; the tumor volume of the control group (PBS) continues to grow, in contrast, the tumor growth of the treatment group is inhibited. The body weight changes of mice are shown in Figure 7 As shown in the figure, the body weight of the four groups of mice has no obvious change during the treatment, which proves that the material has good biological safety.

[0060] In summary, the nanoparticles are prepared by self-assembly of the photothermal agent and the active oxygen donor, the photodynamic therapy excited by near-infrared laser is realized, the tissue penetration depth is deeper, and the damage to normal tissues is smaller; the active oxygen is produced by the peroxo bridge rupture in artemether, which overcomes the limitation of traditional photodynamic therapy under the hypoxic condition in the tumor microenvironment; the mitochondria of tumor cells are targeted, and the active oxygen is produced at the subcellular level; the nanoparticles have self-reporting fluorescence, and the in-vivo distribution of the nanoparticles can be monitored in real time.

[0061] The applicant declares that the detailed method of the present application is illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, that is, it does not mean that the present application must rely on the above-mentioned detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A mitochondrial-targeted anti-tumor nanoparticle self-assembled particle, characterized in that: The nano self-assembled particles have a photothermal agent as a core and an active oxygen donor agent coated on the surface of the photothermal agent as a shell; The structural formula of the active oxygen donor reagent is shown in Formula I, wherein n is 50 to 100; ； Formula I The photothermal agent includes indocyanine green and / or benzobisthiadiazole; The mass ratio of the photothermal agent to the active oxygen donor agent in the nano self-assembled particles is 1:(2-5).

2. The mitochondria-targeting anti-tumor nano self-assembled particles according to claim 1, characterized in that: The particle size of the nano self-assembled particles is 100-400 nm.

3. The method for preparing the mitochondria-targeting anti-tumor nano self-assembled particles according to claim 1 or 2, characterized in that: The preparation method comprises: The photothermal agent, active oxygen donor agent and solvent are mixed to obtain a mixed solution, and the mixed solution is mixed with water to obtain the mitochondria-targeted anti-tumor nano self-assembled particles.

4. The method for preparing mitochondria-targeted anti-tumor nano self-assembled particles according to claim 3, characterized in that: The preparation method of the active oxygen donor reagent comprises: Artesunate 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are mixed with a solvent, NH2-PEG 2000-Cy5 is added, and the mixture is stirred in the dark to carry out a condensation reaction to obtain a phospholipid skeleton modified with artesunate and Cy5 fluorescent groups, which is a reactive oxygen donor reagent.

5. Use of the mitochondria-targeted anti-tumor nano self-assembled particles according to claim 1 or 2 in the preparation of anti-tumor drugs.

6. The use according to claim 5, characterized in that The tumor includes pancreatic cancer.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the mitochondria-targeted anti-tumor nano self-assembled particles according to claim 1 or 2.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition further contains excipients.

9. The pharmaceutical composition according to claim 8, characterized in that The excipients include any one or a combination of at least two of pharmaceutically acceptable wetting agents, solubilizers, osmotic pressure regulators, coating materials, colorants, pH regulators, antioxidants or buffers.

10. A method for killing tumor cells in vitro for non-therapeutic purposes, characterized in that: The method comprises: Mixing a target sample with the mitochondria-targeting anti-tumor nano self-assembled particles according to claim 1 or 2, and irradiating the target sample with a near-infrared laser; The target sample is an in vitro tissue or cell suspension, and the tissue or cell suspension contains tumor cells and normal cells.

11. The method for killing tumor cells in vitro for non-therapeutic purposes according to claim 10, characterized in that: The tumor cells include pancreatic cancer cells.

Images