CN109806403B

CN109806403B - Nano sheet containing nitric oxide donor, and preparation method and application thereof

Info

Publication number: CN109806403B
Application number: CN201910126863.6A
Authority: CN
Inventors: 刘庄; 田龙龙
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2019-02-20
Filing date: 2019-02-20
Publication date: 2021-09-21
Anticipated expiration: 2039-02-20
Also published as: CN109806403A

Abstract

The invention belongs to the field of medicines, and discloses a nano-sheet containing a nitric oxide donor, and a preparation method and application thereof. The nano-sheet provided by the invention is composed of a nitric oxide donor drug and metal ions. The method for synthesizing the nano-sheets is simple and can prepare the nano-sheets in a large scale. The nano-sheet synthesized by the invention can directly mark nuclide without chelating agent. The nano sheet synthesized by the invention can release nitric oxide by utilizing the Cherokee fluorescence of nuclide without external stimulation. After the nano sheet is used for marking the radionuclide, nitric oxide can be released by utilizing nuclide-induced Cherotkoff fluorescence, the tumor microenvironment can be adjusted, and the tumor radiotherapy and immunotherapy effects can be enhanced, so that the nano sheet can be used for preparing a therapeutic agent for treating cancers.

Description

Nano sheet containing nitric oxide donor, and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a nanosheet and a preparation method and application thereof, in particular to application of the nanosheet in preparation of a cancer therapeutic agent.

Background

Cancer causes millions of deaths worldwide each year, and is one of the most dangerous diseases people are currently facing. The combination of traditional radiotherapy and emerging immunotherapy has great clinical prospects and applications in the field of tumor treatment. It was found that after radiation therapy, regression of the tumor also occurred in the unirradiated areas, which is called the distal effect. Further research shows that radiotherapy causes tumor cells to generate immunogenic apoptosis and activates T cell tumor immunity. Immune checkpoint inhibitor therapy also treats cancer by activating T cell immunity, so combining radiation therapy with immune checkpoint inhibitors has strong scientific implications.

The tumor microenvironment has a large impact on the efficacy of radioimmunotherapy. Rapidly growing tumor cells need to take up more oxygen and nutrients from the surrounding vascular network to meet their growth and proliferation needs. However, inside a solid tumor, the growth rate of tumor cells is much greater than the generation rate of blood vessels inside the tumor, and a non-vascular hypoxic region, namely hypoxic, is generated in the solid tumor. The factors that lead to the formation of tumor hypoxia mainly include: abnormal vascular structure and function inside solid tumors, increased inter-vascular substance transport distance (some blood vessels even do not carry oxygenated red blood cells), oxygen deprivation among rapidly proliferating tumor cells, anemia caused by disease or therapy, and decreased oxygen carrying capacity of blood. Because the rapid proliferation of tumor cancer cells leads to solid tumor hypoxia, free radicals generated by radiotherapy are quickly reduced in a hypoxic environment, and then the tumor generates resistance to radiotherapy. And a large number of immunosuppressive cells in the tumor microenvironment inhibit the tumor immunotherapy effect. Therefore, the regulation of the tumor microenvironment has great significance for tumor radioimmunotherapy.

Nitric oxide is a gas signaling molecule, and as a newly discovered biological messenger, it has important physiological functions in the transmission of neural information, regulation of blood vessels, and regulation of immune function. Recently, the relationship between NO and tumor-induced immunosuppression has attracted attention. The current research shows that the compound can be used for not only sensitizing radiotherapy but also regulating tumor immune microenvironment. The nitric oxide molecule is unstable, however, because efficient delivery of nitric oxide to tumors cannot be guaranteed.

Disclosure of Invention

In view of the above, the present invention aims to provide a nanomaterial capable of delivering nitric oxide to a tumor, which solves the problem in the prior art that effective delivery of nitric oxide to a tumor cannot be guaranteed.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

a nano-sheet comprises an ionic compound composed of a nitric oxide donor and metal ions.

Wherein, the nitric oxide donor in the nano sheet is sodium nitroprusside or a nitroso-iron complex; the metal ions are zinc ions, iron ions, copper ions, manganese ions, cobalt ions or cadmium ions.

In some embodiments, the nanoThe sheet consists of zinc, iron, oxygen, nitrogen and carbon elements, the structure of the nano sheet is orthorhombic ZnFe (CN) as shown in figure 5 by X-ray powder diffraction₅NO, ZnNO for short. The observation of an electron microscope shows that the nano sheet is in a two-dimensional nano sheet structure, and the thickness is 0.7-10 nanometers. In some embodiments the nanoplatelets have a thickness of 0.85 nanometers. The nanosheet (104) peak was reduced after milling, indicating that it was a preferentially growing curved surface. The atomic structural arrangement is shown in fig. 6.

The preparation method of the nano-sheet is synthesized by metal ions and nitric oxide donor drugs through a reverse microemulsion method.

In some embodiments, the preparation method of the nanosheet is to mix zinc ions and sodium nitroprusside by using a reverse microemulsion method, and precipitating with ethanol to obtain the nanosheet ZnNO.

The invention also provides a nano sheet for marking nuclide, which is marked with medical radionuclide emitting high-energy alpha and beta rays.

Nuclide therapy is one of the commonly used radiotherapy means, and the nuclide is used to emit rays to kill tumor cells, and simultaneously, the high-energy rays can excite water molecules of a medium to generate Cherotkoff fluorescence mainly comprising ultraviolet/blue light. The nano-sheet of the labeled nuclide can be stimulated by blue-violet light or Cherokee fluorescence to release nitric oxide, and the radioimmunotherapy is enhanced.

Preferably, the nuclide is³²P、⁹⁰Y、¹⁷⁷Lu、¹⁸⁸Re、¹⁸⁶Re or⁶⁴Cu。

In some embodiments, the nanoplatelet label³²P, abbreviated as ZnNO: (³²P)。

The invention also provides a preparation method of the nano-sheet for marking nuclide, wherein the nano-sheet is dispersed in water, and radionuclide is added and fully and uniformly mixed.

In some embodiments, the present invention contemplates nanoplatelet ZnNO(s) after labeling the radionuclide³²P) and the results show ZnNO: (³²P) can induce the release of nitric oxide, has strong cell killing effect, and can emit strong Cherotkoff fluorescenceLong term retention within the tumor. ZnNO (b) after labeling the nanoplatelets with a radionuclide according to the invention relative to a control group was discovered by means of intratumoral injection³²P) can completely remove the tumor, and obviously improves tumor hypoxia and immune microenvironment. Intratumoral injection of ZnNO: (³²P) can also significantly extend the survival of mice. ZnNO: (³²P) and PD-1 monoclonal antibody can obviously enhance infiltration of T cells in primary and remote tumors and obviously increase cytokine interferon gamma and tumor necrosis factor alpha secreted by the T cells. The nano-sheet marked with the radionuclide can release nitric oxide by utilizing nuclide-induced Cherotkoff fluorescence, adjust the tumor microenvironment and enhance the tumor radiotherapy and immunotherapy effects. The invention thus provides the use of said nanoplatelets and nanoplatelets of said labelled nuclide in the preparation of a therapeutic agent for the treatment of cancer.

According to the technical scheme, the invention provides the nanosheet and the preparation method and application thereof. The nano-sheet provided by the invention consists of a nitric oxide donor drug and metal ions, and compared with the prior art, the nano-sheet provided by the invention has at least one of the following advantages:

1. the method for synthesizing the nano-sheets is simple and can prepare the nano-sheets in a large scale.

2. The nano-sheet synthesized by the invention can directly mark nuclide without chelating agent.

3. The nano sheet synthesized by the invention can release nitric oxide by utilizing nuclearity-cutting continuous koff fluorescence of nuclide without external stimulation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a schematic diagram of synthesis of ZnNO nanosheets as synthesized in the first example;

FIG. 2 is an electron microscope image of the synthesized nanosheets of the first example;

FIG. 3 is an atomic force microscope image of the synthesized nanosheet of the first example;

FIG. 4 is an X-ray photoelectron spectrum of a nanosheet of the first example;

FIG. 5 is an X-ray diffraction spectrum of a nanosheet of the first example;

FIG. 6 is a diagram of the atomic structure of the nanosheet in one embodiment;

fig. 7 shows the nanosheet releasing nitric oxide under the induction of ultraviolet light in the first embodiment;

FIG. 8 shows the labeling of the radionuclide by the nanoplatelets in the second embodiment³²post-P label stability;

FIG. 9 shows nanosheet-labeled nuclides of the second embodiment³²P postdiclokoff fluorescence induces nitric oxide release;

FIG. 10 is the killing of colon cancer cells by different concentrations of radiolabeled nanoplatelets in example three;

FIG. 11 shows the concentration of nitric oxide in the supernatant of the final culture medium of the cell experiment of example three;

FIG. 12 is the intratumoral injection of ZnNO (B in example four)³²P) nuclide imaging pictures of the mouse at different time points after the nanosheet;

FIG. 13 shows the intratumoral injection of ZnNO (B in example four)³²P) tumor nuclide signal intensity curves of the mice at different time points after the nanosheets;

FIG. 14 is the intratumoral injection of ZnNO (B in EXAMPLE four)³²P) connecting koff fluorescence imaging pictures of the mouse at different time points after the nano-sheet;

FIG. 15 shows the intratumoral injection of ZnNO (B in EXAMPLE four)³²P) linking the fluorescence signal intensity of the mouse tumor at different time points after the nano-sheet;

FIG. 16 is the intratumoral injection of ZnNO (B in EXAMPLE V)³²P) mouse tumor growth curve after nanosheet;

FIG. 17 shows the intratumoral injection of ZnNO (B in EXAMPLE V)³²P) mouse survival curve after nanosheet;

FIG. 18 shows the intratumoral injection of ZnNO (B in EXAMPLE V)³²P) hematoxylin-eosin staining image of tumor 12 days after the nano-sheet;

FIG. 19 is the intratumoral injection of ZnNO (B in EXAMPLE six)³²P) tumor cryosection a after 12 days of nanosheetnti-HIF-alpha, anti-CD8 (white arrow), anti-PD-L1, anti-F4/80and anti-Foxp3 antibody immunofluorescent staining laser confocal images;

FIG. 20 shows the intratumoral injection of ZnNO (B in EXAMPLE six)³²P) percentage of immunofluorescence anti-HIF-alpha staining positive area of tumor cryosections 12 days after the nanosheets;

FIG. 21 is the intratumoral injection of ZnNO (B in EXAMPLE six)³²P) percentage of immunofluorescence anti-CD8 staining positive area of tumor cryosections 12 days after the nanoplatelets;

FIG. 22 is the intratumoral injection of ZnNO (B in EXAMPLE six)³²P) percentage of immunofluorescence anti-F4/80 staining positive area of tumor cryosections 12 days after the nanoplatelets;

FIG. 23 shows the intratumoral injection of ZnNO (B in EXAMPLE six)³²P) percentage of immunofluorescent anti-Foxp3 staining positive area of tumor cryosections 12 days after nanoplatelets;

FIG. 24 shows the intratumoral injection of ZnNO (B in EXAMPLE six)³²P) frozen tumor section immunofluorescence staining CD 8/T12 days after nanosheet_regA ratio;

FIG. 25 shows the combination of ZnNO (b) in example VII³²P) nanoplate therapy and immune checkpoint inhibitor PD-1 treatment of the mouse primary tumor growth curve;

FIG. 26 shows the combination of ZnNO (b) in example VII³²P) nanoplate therapy and immune checkpoint inhibitor PD-1 treatment distal tumor growth curve in mice;

FIG. 27 shows the combination of ZnNO (b) in example VII³²P) nanosheet therapy and immune checkpoint inhibitor PD-1 treatment mouse survival curves;

FIG. 28 shows the combination of ZnNO (b) in example eight³²P) original tumor and distal tumor cryosection immunofluorescence anti-CD8 staining laser confocal images at 12 days of nanoplate treatment and immune checkpoint inhibitor PD-1 treatment;

FIG. 29 shows the combination of ZnNO (b) in example eight³²P) percentage of immunofluorescent anti-CD8 staining positive areas of primary and distal tumor cryosections at 12 days of nanoplate treatment and immune checkpoint inhibitor PD-1 treatment;

FIG. 30 shows Zn combination in example eightNO(³²P) interferon gamma levels in mouse serum at 12 days of nanoplate treatment and immune checkpoint inhibitor PD-1 treatment;

FIG. 31 shows the combination of ZnNO (b) in example eight³²P) tumor necrosis factor alpha levels in mouse serum at 12 days of treatment with nanoplate therapy and immune checkpoint inhibitor PD-1.

Detailed Description

The invention discloses a nanosheet and a preparation method and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the invention can be implemented and applied by modifying or appropriately combining the methods described herein without departing from the spirit, scope and spirit of the invention.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

The first embodiment is as follows: synthesis and characterization of nanosheet ZnNO:

as shown in fig. 1, nanosheets were synthesized by the reverse microemulsion method: in the first step, surfactant Igepal CO-520 is added to cyclohexane (the volume ratio of Igepal CO-520 to cyclohexane is 2:5) and mixed uniformly to form a microemulsion. The anionic phase was composed by adding 0.4 mol/l sodium nitroprusside solution to the microemulsion (v/v. 1/50). 0.1-0.4 mol/l zinc ion solution is added to the microemulsion (v/v-1/50) to make up the cationic phase. The temperature was controlled at 25-35 deg.C and an equal volume of the anionic phase was added dropwise to the cationic phase, mixed and stirred overnight. And secondly, centrifugally precipitating the nano material, and washing the nano material for multiple times by using organic solvents such as ethanol and the like.

Then, the nano-sheets are characterized, and an electron microscope image shows as figure 2, and the nano-material is in a two-dimensional nano-sheet structure. Atomic force microscopy images as figure 3 shows that the thickness of the nanoplatelets is about 1 nanometer. An X-ray photoelectron spectrum as shown in FIG. 4 shows that the components of the nanosheet are zinc, iron, oxygen, nitrogen and carbon elements. X-ray powder diffraction Zn [ Fe (CN) ] with nanosheet structure in orthorhombic phase as shown in FIG. 5₅NO]. The nanosheet (104) peak was reduced after milling, indicating that it was a preferentially growing curved surface. The atomic structural arrangement is shown in fig. 6. The nanosheet is abbreviated as ZnNO.

The growth conditions of the material are adjusted, and nanospheres and nano-cubic blocks are prepared as comparison materials. The material was excited using a weak ultraviolet light source (365 nm wavelength, 0.16 mw power) to detect nitric oxide release. As a result, as shown in fig. 7, the nanoplatelets can release nitric oxide efficiently and continuously, whereas the comparative material cannot.

Example two: nano-sheet labeled nuclide and induced nitric oxide release

Dispersing the nano-sheet in water solution by ultrasonic wave, adding radionuclide³²P, oscillating for 1 hour in an oscillator at 50 ℃, centrifuging to remove free nuclide in supernatant to obtain ZnNO (B) ((B))³²P). The stability of the radiolabel was tested and the results are shown in FIG. 8, ZnNO (C: (N-N)) (³²P) has a higher radiolabel stability. Dispersing in water solution again, placing in dark condition, taking solution at different time points, centrifuging, and testing the concentration of nitric oxide in supernatant. As shown in fig. 9, nuclides induced nitric oxide release.

Example three: killing of cancer cells by nuclide-labeled nanosheets:

different concentrations and radioactive doses of ZnNO (B)³²P) were incubated with mouse colon cancer cells and cell viability was measured after 72 hours. The results are shown in fig. 10, with the blank nanoplatelets not being significantly cytotoxic, whereas ZnNO: (³²P) has a strong cell killing effect. Detecting the presence of oxygen in the supernatant of the last group of cell culture mediaNitrogen concentration as shown in FIG. 11, ZnNO: (³²Group P) has a high concentration of nitric oxide.

Example four: nanosheet ZnNO (³²P) retention imaging within the tumor:

ZnNO (B) by intratumoral injection³²P) were injected into mice in a subcutaneous colon cancer model, at different time points radionuclide imaging as shown in fig. 12 and 13 showed that free nuclides rapidly escaped from the tumor site, while the nuclides labeled on the nanomaterials remained in the tumor for a long time. Cherotkoff fluorescence imaging ZnNO (C) (15) is shown in FIGS. 14 and 15³²P) emits strong dicke fluorescence and remains in the tumor for a long time.

Example five: treatment of tumors with nanosheet ZnNO (32P)

PBS, ZnNO, free by intratumoral injection³²P、ZnCN(³²P) and ZnNO: (³²P) was injected into mice of a subcutaneous colon cancer model and the tumor volume of the mice was measured with a vernier caliper. The growth curves of the mouse tumors are shown in FIG. 16, in which ZnNO and free are present relative to the control group³²P has no obvious inhibition effect on tumors; ZnCN (b), (c), (d) and (d)³²P) treatment only partially inhibited tumor growth; ZnNO: (³²P) treatment can completely eliminate the tumor. The survival curve of the mouse is shown in FIG. 17, ZnNO: (³²P) can cure the tumor of the mouse without recurrence. HE staining of tumors 12 days after treatment in mice is shown in FIG. 18, ZnNO: (³²P) tumor cell nuclear condensation in mice after treatment indicated cancer cell apoptosis.

Example six: nanosheet ZnNO (³²P) modulation of the tumor microenvironment

PBS, ZnNO, free by intratumoral injection³²P、ZnCN(³²P) and ZnNO: (³²P) (30 μm, 125 μ g per mouse) was injected into mice of a subcutaneous colon cancer model, and 12 days later, the tumors of the mice were taken out and embedded in OCT gel, and stored in a refrigerator at-80 ℃ for two months. After the nuclide basically decays, the tumor tissue is sliced, stained by a fluorescence-labeled antibody, and an image is taken by a laser confocal microscope as shown in fig. 19. Software statistics of percent of positive area in image, HIF-alpha expression is shown in FIG. 20Is ZnNO: (³²P) treatment significantly inhibited HIF- α expression; FIG. 21 shows ZnNO: (³²P) treatment significantly enhanced the infiltration of cytotoxic T lymphocytes in the tumor; FIG. 22 shows ZnNO: (³²P) treatment slightly upregulated recruitment of tumor-associated macrophages; FIG. 23 shows ZnNO: (³²P) treatment pair T_regThe number was not significantly changed; FIG. 24 shows ZnNO: (³²P) treatment significantly increased CD8/T_regThe ratio of (a) to (b). These results indicate that ZnNO: (³²P) treatment significantly improved the tumor hypoxia and immune microenvironment.

Example seven: nanosheet ZnNO (³²P) treatment in combination with immune checkpoint inhibitor PD-1 monoclonal antibody treatment

Establishing an artificial metastatic tumor subcutaneous colon cancer model, inoculating subcutaneous colon cancer on the right side of the back of the mouse for five days, and then inoculating subcutaneous colon cancer on the left side of the back of the mouse again. Four days after the second inoculation, the right tumor was intratumorally injected with PBS or ZnNO (II)³²P) treatment, followed by tail vein injection of PBS or PD-1 mab at

days

1, 3 and 5 post treatment, and mice tumor growth was recorded. The right-hand primary tumor growth curve is shown in FIG. 25, in which ZnNO (B) is injected intratumorally³²P) can eliminate tumors. Left tumor growth curves are shown in FIG. 26, in combination with ZnNO: (³²The P) treatment and the PD-1 monoclonal antibody treatment can obviously inhibit the growth of the remote tumor. Mouse survival curves the combination ZnNO (b) (shown in FIG. 27)³²The survival period of the mice can be obviously prolonged by the P) treatment and the PD-1 monoclonal antibody treatment.

Example eight: nanosheet ZnNO (³²P) modulation of tumor immune microenvironment by treatment in combination with immune checkpoint inhibitor PD-1 monoclonal antibody

Establishing an artificial metastatic tumor subcutaneous colon cancer model, inoculating subcutaneous colon cancer on the right side of the back of the mouse for five days, and then inoculating subcutaneous colon cancer on the left side of the back of the mouse again. Four days after the second inoculation, the right tumor was intratumorally injected with PBS or ZnNO (II)³²P) treatment followed by tail vein injection of PBS or PD-1 mab on

days

1, 3 and 5 post treatment. On day 12 of treatment, mice were tumor-removed and embedded in OCT gel and stored in a-80 ℃ freezer for two months. After the nuclide basically decays, the tumor tissue section is cut, and the fluorescence is labeledThe body was stained and the confocal laser microscopy images were taken as shown in FIG. 28. Software statistics of percent of positive area in image, CD8 staining result as shown in FIG. 29 shows the combination of ZnNO: (B)³²P) treatment with PD-1 mab treatment significantly enhanced T cell infiltration in both primary and distal tumors. Sera from mice taken on

days

0, 7 and 12 of treatment and the results of the Elisa test are shown in FIGS. 30 and 31, in combination with ZnNO (R) ((R))³²P) treatment and PD-1 monoclonal antibody treatment can obviously increase cytokine interferon gamma and tumor necrosis factor alpha secreted by T cells.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A nuclide nanoplate, Zn [ Fe (CN) ], for the treatment of cancer₅NO]The nano-chip is marked with medical radioactive nuclide emitting high-energy beta rays³²P。

2. A process for producing nanoplatelets bearing a nuclide as defined in claim 1, which comprises reacting Zn [ Fe (CN)₅NO]Dispersing the nano-plate in the water solution, adding radionuclide, and fully and uniformly mixing.

3. Use of nanoplatelets of the labelled nuclide of claim 1 in the preparation of a therapeutic agent for the treatment of cancer.