Traditional chinese medicine self-assembled nanomaterial, preparation method therefor, and use thereof

Abstract

The present invention belongs to the technical field of biomedical engineering, and relates to a traditional Chinese medicine self-assembled nanomaterial, a preparation method therefor, and use thereof. The present invention uses a mineral traditional Chinese medicine and other traditional Chinese medicines as raw materials to prepare a traditional Chinese medicine self-assembled nanomaterial having a D50 particle size of 30-450 nm. The traditional Chinese medicine self-assembled nanomaterial has a T1-weighted nuclear magnetic resonance imaging function, a photoacoustic imaging function, and a photothermal therapy function.

Description

A self-assembled nanomaterial of traditional Chinese medicine, its preparation method and application Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, and relates to a self-assembled nanomaterial of traditional Chinese medicine, its preparation method and application. Background Technology

[0002] Magnetic resonance imaging (MRI), a non-invasive and high-resolution medical imaging technique, plays a crucial role in modern medical diagnosis. Contrast agents, in particular, enhance image contrast and highlight lesions, greatly assisting doctors in accurately diagnosing diseases. Currently, most mainstream contrast agents on the market are chemically synthesized products. For example, gadolinium-based contrast agents, due to their unique magnetic properties, are widely used in T1-weighted imaging; superparamagnetic iron oxide nanomaterials also have specific imaging applications for certain tissues or diseases, such as breast cancer, gastric cancer, and liver cancer. However, these chemically synthesized contrast agents have several significant drawbacks. In terms of stability, they require stringent storage conditions, typically involving low temperatures, which undoubtedly increases storage and transportation costs. Even at suitable low temperatures, their shelf life is relatively short, and they frequently encounter degradation and aggregation problems, significantly reducing the imaging effect. From a biosafety perspective, some chemical contrast agents can induce serious adverse reactions such as renal systemic fibrosis, limiting their use in special groups such as patients with renal insufficiency.

[0003] Traditional Chinese medicine (TCM), a treasure of the People's Republic of China, boasts a history of application spanning thousands of years. It contains a rich variety of natural components and exhibits complex and sophisticated pharmacological effects. TCM is derived from natural plants, animals, and minerals, resulting in a diverse and abundant array of substances with unique physiological activities. Furthermore, its safety has been proven through long-term clinical trials. For a long time, the application of TCM has been largely confined to traditional dosage forms such as oral decoctions, pills, and powders to regulate the functions of the body's internal organs. Its development in the field of modern cutting-edge medical imaging, particularly in contrast agents, is still in its early stages, with a wealth of potential value yet to be explored. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, the present invention aims to provide a self-assembled nanomaterial of traditional Chinese medicine, its preparation method, and its application, thereby overcoming the shortcomings of the existing technology.

[0005] The present invention employs the following technical solutions to achieve its objective:

[0006] One aspect of the present invention provides a self-assembled nanomaterial of traditional Chinese medicine, wherein the raw materials for preparing the self-assembled nanomaterial of traditional Chinese medicine include mineral traditional Chinese medicine and other traditional Chinese medicine, wherein the other traditional Chinese medicine is one or two of plant-derived traditional Chinese medicine and animal-derived traditional Chinese medicine;

[0007] The D50 particle size of the self-assembled nanomaterials of traditional Chinese medicine is 30-450 nm.

[0008] Preferably, the D50 particle size of the self-assembled nanomaterial of traditional Chinese medicine is 50-450 nm.

[0009] Preferably, the raw materials for preparing the self-assembled nanomaterials of traditional Chinese medicine further include a targeting agent, wherein the targeting agent is a coupling targeting agent with a carboxyl or amino group at the end.

[0010] Preferably, the mineral-based traditional Chinese medicine is one or more of the following: natural copper, ochre, hematite, magnetite, ferrous sulfate, gypsum, stachyite, and calamine.

[0011] Preferably, the plant-derived traditional Chinese medicine is one or more of the following: Cornus officinalis, Crataegus pinnatifida, Lycium barbarum, Jujube, Codonopsis pilosula, Cistanche deserticola, Ganoderma lucidum, Saffron, Amomum tsao-ko, Curcuma longa, Piper longum, Rehmannia glutinosa, Ophiopogon japonicus, Asparagus cochinchinensis, Citrus reticulata peel, Taxillus chinensis, Eucommia ulmoides, Dipsacus asper, Platycladus orientalis leaf, Clematis chinensis, Anemarrhena asphodeloides, Phellodendron chinense, Ligustrum lucidum, Angelica sinensis, Salvia miltiorrhiza, Citrus reticulata peel, Astragalus membranaceus, and Artemisia argyi.

[0012] Preferably, the animal-derived traditional Chinese medicine is one or more of the following: *Eupolyphaga sinensis*, *Eupolyphaga sinensis*, donkey-hide gelatin, chicken gizzard lining, centipede, bezoar, musk, leech, and earthworm.

[0013] Preferably, the conjugable target agent with a carboxyl or amino group at the end is one or more of folic acid and its derivatives, hyaluronic acid and its derivatives, arginine-glycine-aspartic acid tripeptide and its derivatives.

[0014] Preferably, the self-assembled nanomaterials of traditional Chinese medicine have one or more of the following functions: T1-weighted magnetic resonance imaging, photoacoustic imaging, and photothermal therapy.

[0015] The self-assembled nanomaterials made from traditional Chinese medicine are used for photoacoustic imaging and photothermal therapy.

[0016] Preferably, the photoacoustic imaging includes: the self-assembled nanomaterials of traditional Chinese medicine being enriched in tumor tissue and imaged at 808 nm.

[0017] Preferably, the photothermal therapy includes: the traditional Chinese medicine self-assembled nanomaterials targeting tumor tissue at 808 nm and heating to 30-60°C, shrinking the tumor tissue to 1-30% of its initial volume.

[0018] Preferably, when the self-assembled nanomaterials of the traditional Chinese medicine at a concentration of 1–20 μM are applied to tumor cells under the action of an 808 nm laser, the cell survival rate is <10%.

[0019] Further optimization, at 808nm, 1W / cm 2Under the action of laser light, the self-assembled nanomaterials of the traditional Chinese medicine at a concentration of 20 μM acted on tumor cells, and the cell survival rate was <5%.

[0020] Preferably, under the action of an 808nm laser, the self-assembled nanomaterials of the traditional Chinese medicine at a concentration of 0.1-2mM act on the tumor tissue, reducing the weight of the tumor tissue to 1-30% of its initial volume.

[0021] Preferably, at 808nm, 1W / cm 2 The traditional Chinese medicine self-assembled nanomaterials were subjected to laser treatment for 6 minutes, and the concentration of 1 mM was raised to 50-60℃.

[0022] A second aspect of this invention provides a method for preparing self-assembled nanomaterials of traditional Chinese medicine, the method comprising the following steps:

[0023] The mineral-based traditional Chinese medicine, other traditional Chinese medicines, and water are mixed, then heated to react, and finally filtered, centrifuged, dialyzed, and dried to obtain the self-assembled nanomaterials of the traditional Chinese medicine.

[0024] The other traditional Chinese medicines mentioned are one or two of the following: plant-derived traditional Chinese medicines and animal-derived traditional Chinese medicines.

[0025] The third aspect of this invention provides another method for preparing self-assembled nanomaterials of traditional Chinese medicine, in which the raw materials also include a targeting agent. The preparation method includes the following steps: mixing mineral-based traditional Chinese medicine, other traditional Chinese medicine, and water, then heating and reacting the mixture, followed by filtration, centrifugation, dialysis, and drying to obtain the self-assembled nanomaterials; wherein the other traditional Chinese medicine is one or two of plant-derived traditional Chinese medicine and animal-derived traditional Chinese medicine.

[0026] Self-assembled nanomaterials are coupled with targeting agents through chemical reactions to obtain self-assembled nanomaterials of traditional Chinese medicine.

[0027] Preferably, the mineral-based Chinese medicine and other Chinese medicines are pulverized before use, with the pulverized material having a size ≤1.5cm.

[0028] After the heating reaction is completed, filtration is performed. The filter paper used for filtration has a pore size ≥1μm, which is smaller than the size of the pulverized mineral Chinese medicine and other Chinese medicines.

[0029] Preferably, the mass ratio of the mineral-based traditional Chinese medicine to other traditional Chinese medicines is 1:0.1 to 1:10.

[0030] Preferably, the ratio of the mass of water to the total mass of all Chinese medicinal herbs is 1 to 100:1.

[0031] Preferably, the preparation method further includes the following step before heating the reaction: standing for 1 to 60 minutes.

[0032] Preferably, the temperature of the heating reaction is 70–120°C, and the heating reaction time is 10–200 min.

[0033] Preferably, the centrifugation step includes: performing 2 to 8 centrifugations, with the first centrifugation speed being 1000 to 7000 rpm, the subsequent centrifugation speed being higher than the previous centrifugation speed, and the last centrifugation speed being 8000 to 15000 rpm; each centrifugation time being 5 to 50 minutes.

[0034] Preferably, the difference between the centrifugation speed of the subsequent centrifugation and that of the previous centrifugation is 500 to 6000 rpm.

[0035] Preferably, the centrifugation step includes: performing 3 to 5 centrifugations, with the first centrifugation speed being 2000 to 5000 rpm, the difference between the centrifugation speed of each subsequent centrifugation and that of the previous centrifugation being 1000 to 5000 rpm, and the final centrifugation speed being 9000 to 12000 rpm.

[0036] Preferably, the supernatant obtained by centrifugation is placed in a dialysis bag for dialysis, the molecular weight cutoff of the dialysis bag is 1000-10000 Da, and the dialysis time is 10-100 h.

[0037] Preferably, the drying is freeze-drying or vacuum drying;

[0038] The freeze-drying temperature is -40 to -100℃, the vacuum degree is 1 to 100 Pa, and the time is 12 to 100 h.

[0039] Preferably, the chemical reaction is an activation coupling method of EDC and NHS.

[0040] Preferably, the coupling includes: dispersing the self-assembled nanomaterials in 2-(N-morpholino)ethanesulfonic acid buffer, sequentially adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for activation; then adding a coupling target agent with a carboxyl or amino group at the end, reacting at room temperature, and then purifying by centrifugation to obtain the self-assembled nanomaterials of traditional Chinese medicine.

[0041] Preferably, the mass ratio of the self-assembled nanomaterial to the coupled target agent with a carboxyl or amino group at the end is 1:0.1 to 10.

[0042] A fourth aspect of the present invention provides the application of the aforementioned self-assembled nanomaterials of traditional Chinese medicine, wherein the application is selected from one or more of the following:

[0043] Used in the preparation of nuclear magnetic resonance contrast agents;

[0044] Used in the preparation of photoacoustic imaging contrast agents;

[0045] Used to prepare drugs for the diagnosis and / or treatment of cancer diseases.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1. This invention uses mineral-based traditional Chinese medicine and other traditional Chinese medicines as raw materials to prepare self-assembled nanomaterials of traditional Chinese medicine with a D50 particle size of 30-450 nm. Because the source is traditional Chinese medicine, it has good biocompatibility and few side effects on the matrix.

[0048] 2. The self-assembled nanomaterials of traditional Chinese medicine provided by this invention can be used as nuclear magnetic resonance contrast agents, which can accumulate at the tumor tissue, thereby significantly enhancing the T1-weighted MRI signal of the tumor tissue; they can also be used as photoacoustic imaging contrast agents, which can accumulate at the tumor tissue, thereby significantly enhancing the photoacoustic signal of the tumor tissue; it is expected to accurately distinguish the tumor boundary from normal tissue.

[0049] 3. The self-assembled nanomaterials of traditional Chinese medicine provided by this invention have a D50 particle size of 30-450 nm. The small particle size of the contrast agent makes it easier to enter the tumor tissue, thereby realizing MRI imaging and photoacoustic imaging of the internal structure of the tumor.

[0050] 4. The self-assembled nanomaterials of traditional Chinese medicine provided by this invention have the function of generating reactive oxygen free radicals under acidic conditions and exhibit photothermal effects. Therefore, they have a killing effect on tumors. Under the action of laser, the photothermal killing effect is further enhanced. Thus, they can be used as drugs for the diagnosis and / or treatment of tumor diseases in the treatment of solid tumors.

[0051] 5. The self-assembled nanomaterials of traditional Chinese medicine of the present invention can be used as nuclear magnetic resonance contrast agents and photoacoustic imaging contrast agents, and can also have a killing effect on tumors, thus realizing the integrated diagnosis and treatment of malignant tumors.

[0052] 6. The method for preparing self-assembled nanomaterials of traditional Chinese medicine provided by the present invention uses mineral-based traditional Chinese medicines and other traditional Chinese medicines as raw materials. The raw materials are safe, the synthesis process is simple, and the cost can be effectively reduced. Furthermore, the self-assembled nanomaterials of traditional Chinese medicine prepared by this method can be stored at room temperature for up to 3 months and have high storage stability. Attached Figure Description

[0053] Figure 1 shows the particle size distribution and dispersion of the self-assembled nanomaterials of traditional Chinese medicine prepared in Examples 1-6 of this invention, and their appearance in water.

[0054] Figure 2 is a SEM image of the self-assembled nanomaterials of traditional Chinese medicine prepared in Example 3 of the present invention.

[0055] Figure 3 shows the room temperature storage stability and batch stability of the self-assembled nanomaterials of traditional Chinese medicine prepared in Example 3 of the present invention.

[0056] Figure 4 shows the CCK-8 experimental results of the self-assembled nanomaterials of traditional Chinese medicine prepared in Example 3 of the present invention in cells.

[0057] Figure 5 shows the H&E staining of organ tissue sections of the self-assembled nanomaterials of traditional Chinese medicine prepared in Example 3 of the present invention after an animal model experiment.

[0058] Figure 6 shows the MRI and relaxation properties of the self-assembled nanomaterials of traditional Chinese medicine prepared in Example 3 of this invention.

[0059] Figures 7 and 8 are MRI images of the self-assembled nanomaterials of traditional Chinese medicine prepared in Example 3 of the present invention in animal models.

[0060] Figure 9 shows the tumor image of the self-assembled nanomaterials of traditional Chinese medicine prepared in Example 3 of the present invention after treatment in an in situ osteosarcoma animal model.

[0061] Figure 10 shows the particle size distribution of the materials prepared in Comparative Examples 1 and 2 of the present invention, and their appearance in water.

[0062] Figure 11 is an MRI image of the Chinese medicine granules prepared in Comparative Example 2 of the present invention dissolved in water.

[0063] Figure 12 shows the appearance of the material prepared in Comparative Example 3 of the present invention after 48 hours at room temperature.

[0064] Figure 13 is a transmission electron microscope image of the self-assembled nanomaterials of traditional Chinese medicine in Examples 11-14 of this invention.

[0065] Figure 14 shows the hydration particle size distribution (a) and zeta potential (b) of the self-assembled nanomaterials of traditional Chinese medicine in Examples 11-14 of this invention.

[0066] Figure 15 shows the iron content of the self-assembled nanomaterials of traditional Chinese medicine in Examples 11-14 of this invention, as measured by inductively coupled plasma atomic emission spectrometry.

[0067] Figure 16 is an XPS image of the self-assembled nanomaterials of traditional Chinese medicine in Example 14 of the present invention.

[0068] Figure 17 shows the UV-Vis spectra of the self-assembled nanomaterials of traditional Chinese medicine in Example 14 of this invention at different concentrations.

[0069] Figure 18 shows the UV-Vis spectra of the self-assembled nanomaterials of traditional Chinese medicine in Example 14 and Comparative Example 4 of the present invention at the same concentration.

[0070] Figure 19 shows the photothermal performance and photothermal conversion efficiency of the self-assembled nanomaterials of traditional Chinese medicine in Example 14 of the present invention under different concentrations (a), different laser powers (b), 5 cycles (c), and different concentrations and times (e).

[0071] Figure 20 shows photoacoustic imaging (a) and quantitative results (b) of photoacoustic signals at different concentrations of the photothermal self-assembled nanomaterials of traditional Chinese medicine in Example 14 of Application Example 2 of the present invention.

[0072] Figure 21 shows the cell survival rate of normal cells after the self-assembled nanomaterials of traditional Chinese medicine in Examples 11-14 of Application Example 3 of this invention were applied.

[0073] Figure 22 shows the photothermal killing test results of each group on tumor cells in Application Example 4 of the present invention.

[0074] Figure 23 shows the tumor (a) and weight change (b) of tumor-bearing mice after photothermal treatment with the self-assembled nanomaterials of traditional Chinese medicine in Example 14 of Example 5 of this invention.

[0075] Figure 24 shows the photoacoustic imaging results of tumor-bearing mice at different times using the self-assembled nanomaterials of traditional Chinese medicine in Example 14 of Application Example 6 of the present invention. Detailed Implementation

[0076] In the following description, embodiments of the self-assembled nanomaterials of traditional Chinese medicine of the present invention, their preparation methods and applications will be described in detail. However, these embodiments are exemplary and the disclosure of the present invention is not limited thereto.

[0077] One aspect of the present invention provides a self-assembled nanomaterial of traditional Chinese medicine, the raw materials for which include mineral-derived traditional Chinese medicine and other traditional Chinese medicine, wherein the other traditional Chinese medicine is one or two of plant-derived traditional Chinese medicine and animal-derived traditional Chinese medicine;

[0078] The D50 particle size of the self-assembled nanomaterials of traditional Chinese medicine is 30-450 nm.

[0079] The raw materials for preparing the self-assembled nanomaterials of traditional Chinese medicine are one or two of plant-derived traditional Chinese medicines, animal-derived traditional Chinese medicines, and mineral-derived traditional Chinese medicines.

[0080] Plant-derived traditional Chinese medicine refers to medicines derived from natural plants, primarily consisting of the roots, stems, leaves, flowers, fruits, and seeds of natural plants. Preferably, the plant-derived traditional Chinese medicine includes one or more of the following: Cornus officinalis, Crataegus pinnatifida, Lycium barbarum, Ziziphus jujuba, Codonopsis pilosula, Cistanche deserticola, Ganoderma lucidum, Saffron, Amomum tsao-ko, Curcuma longa, Piper longum, Rehmannia glutinosa, Ophiopogon japonicus, Asparagus cochinchinensis, Citrus reticulata peel, Taxillus chinensis, Eucommia ulmoides, Dipsacus asper, Platycladus orientalis leaves, Clematis chinensis, Anemarrhena asphodeloides, Phellodendron chinense, Ligustrum lucidum, Angelica sinensis, Salvia miltiorrhiza, Citrus reticulata peel, Astragalus membranaceus, and Artemisia argyi.

[0081] Animal-derived traditional Chinese medicines are drugs obtained from animals or their secretions. These medicines typically consist of the whole animal or certain organs or tissues of a specific species, or animal excrement and secretions. Preferably, the animal-derived traditional Chinese medicines include, but are not limited to, one or more of the following: *Eupolyphaga sinensis*, *Eupolyphaga sinensis*, donkey-hide gelatin, chicken gizzard lining, centipede, bezoar, musk, leech, and earthworm.

[0082] Mineral-based traditional Chinese medicines are medicines made from natural minerals or rocks that have been processed for medical purposes. Preferably, the mineral-based traditional Chinese medicines include one or more of the following: natural copper, ochre, halloysite, magnetite, ferrous sulfate, gypsum, chalcedony, and calamine.

[0083] The D50 particle size of the self-assembled nanomaterials of traditional Chinese medicine is 30–450 nm. The D50 particle size represents the particle size corresponding to a cumulative particle size distribution percentage of 50%, and is determined using dynamic light scattering (DLS) method. The determination method mainly includes: firstly, uniformly dispersing the nanomaterials in a solvent (such as deionized water, anhydrous ethanol, etc.); placing the dispersed sample in a dedicated DLS measurement cell; performing multiple measurements on the sample; and then analyzing and processing the data using built-in software to obtain the D50 particle size.

[0084] Preferably, the D50 particle size of the self-assembled nanomaterial of traditional Chinese medicine is 50-450 nm. For example, it can be any value among 50, 60, 70, 80, 100, 120, 150, 180, 200, 220, 250, 270, 300, 330, 350, 380, 400, and 450 nm, but is not limited to the values ​​listed above.

[0085] The second aspect of this invention provides another self-assembled nanomaterial of traditional Chinese medicine, wherein the raw materials for preparing the self-assembled nanomaterial of traditional Chinese medicine include: mineral traditional Chinese medicine, other traditional Chinese medicine and a targeting agent, wherein the other traditional Chinese medicine is one or two of plant-derived traditional Chinese medicine and animal-derived traditional Chinese medicine, and the targeting agent is a coupling targeting agent with a carboxyl or amino group at the end;

[0086] The D50 particle size of the self-assembled nanomaterials of traditional Chinese medicine is 2–450 nm.

[0087] A terminal carboxyl or amino group is a conjugable targeting agent that can covalently couple with the surface functional groups of self-assembled nanomaterials of traditional Chinese medicine. The molecule has a terminal carboxyl or amino group. Preferably, the terminal carboxyl or amino group conjugable targeting agent is one or more of folic acid and its derivatives, hyaluronic acid and its derivatives, and arginine-glycine-aspartic acid tripeptide and its derivatives. Specifically, the core target site of folic acid is the folic acid receptor α, hyaluronic acid mainly targets the CD44 receptor highly expressed on the surface of tumor cells, and the arginine-glycine-aspartic acid tripeptide mainly targets the integrin receptor family on the surface of tumor cells and tumor vascular endothelial cells.

[0088] Preferably, the D50 particle size of the self-assembled nanomaterial of traditional Chinese medicine is 50-450 nm.

[0089] The self-assembled nanomaterials of traditional Chinese medicine have one or more of the following functions: T1-weighted magnetic resonance imaging, photoacoustic imaging, and photothermal therapy. Furthermore, the self-assembled nanomaterials of traditional Chinese medicine have T1-weighted magnetic resonance imaging, photoacoustic imaging, or photothermal therapy functions targeting tumor tissue.

[0090] The type of tumor is not limited in any way; any tumor in the common sense is within the scope of protection of this invention, including both benign and malignant tumors. Malignant tumors are preferred, including but not limited to osteosarcoma, liver cancer, gastric cancer, breast cancer, prostate cancer, kidney cancer, bladder cancer, lung cancer, colorectal cancer, pancreatic cancer, and thyroid cancer. The self-assembled nanomaterials of the traditional Chinese medicine can aggregate at the tumor tissue, significantly enhancing the T1-weighted MRI signal and photoacoustic signal of the tumor tissue. These self-assembled nanomaterials of the traditional Chinese medicine can be used as contrast agents for MRI and / or photoacoustic imaging, significantly enhancing the photoacoustic and / or T1-weighted MRI signals of the tumor tissue, accurately distinguishing the tumor boundary from normal tissue. Furthermore, these self-assembled nanomaterials of the traditional Chinese medicine have a photothermal effect, exhibiting a killing effect on tumors, and can be used as drugs for the diagnosis and / or treatment of solid tumors.

[0091] The third aspect of this invention provides a method for preparing a first type of self-assembled nanomaterial of traditional Chinese medicine, comprising the following steps:

[0092] The mineral-based traditional Chinese medicine, other traditional Chinese medicines, and water are mixed, then heated to react, and finally filtered, centrifuged, dialyzed, and dried to obtain the self-assembled nanomaterials of the traditional Chinese medicine.

[0093] The other traditional Chinese medicines mentioned are one or two of the following: plant-derived traditional Chinese medicines and animal-derived traditional Chinese medicines.

[0094] The fourth aspect of this invention provides a second method for preparing self-assembled nanomaterials of traditional Chinese medicine, comprising the following steps:

[0095] The process involves mixing mineral-based traditional Chinese medicine, other traditional Chinese medicines, and water, then heating the mixture to react with the water. After filtration, centrifugation, dialysis, and drying, self-assembled nanomaterials are obtained. The other traditional Chinese medicines are one or two of plant-derived and animal-derived traditional Chinese medicines.

[0096] The self-assembled nanomaterials are coupled with a targeting agent through a chemical reaction to obtain self-assembled nanomaterials of traditional Chinese medicine.

[0097] The preparation methods for the third and fourth aspects are described in detail below:

[0098] The mineral-containing Chinese medicinal herbs and other Chinese medicinal herbs mentioned are all commercially available dried medicinal materials. They can be commercially available prepared slices of Chinese medicinal herbs, or commercially available granular or block-shaped Chinese medicinal herbs. Preferably, they are Chinese medicinal herbs that have passed the testing of the Chinese Pharmacopoeia. Before use, the mineral-containing Chinese medicinal herbs and other Chinese medicinal herbs can be pulverized. There are no particular restrictions on the pulverized size; generally speaking, the smaller the pulverized size within a certain range, the more beneficial it is to the reaction. For example, pulverizing to a size ≤ 1.5 cm is acceptable.

[0099] Preferably, the mass ratio of the mineral-based traditional Chinese medicine to other traditional Chinese medicines is 1:0.1 to 1:10. More preferably, it is 1:0.5 to 1:5. For example, it can be 1:0.5, 1:0.7, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.

[0100] Preferably, the ratio of water to the total mass of all Chinese medicinal herbs is 1 to 100:1. More preferably, it is 2 to 80:1, and even more preferably, it is 5 to 50:1. For example, it can be 5:1, 8:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1.

[0101] Preferably, the preparation method further includes the following step before the heating reaction: standing for 1 to 120 minutes. Specifically, the mineral-based Chinese medicine, other Chinese medicines, and water are mixed, left to stand for 1 to 120 minutes, and then the heating reaction is carried out.

[0102] Soaking mineral-containing Chinese medicinal herbs and other Chinese medicinal herbs in water and allowing them to stand for a period of time helps to promote the dissolution of active ingredients, saves decoction time, and ensures balanced efficacy. The preferred standing time is 1–60 minutes, more preferably 15–60 minutes. For example, it can be 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes.

[0103] Preferably, the heating reaction temperature is 70–120°C, for example, 70, 80, 85, 88, 90, 93, 95, 97, 100, 110, or 120°C. The heating reaction can be carried out under pressure or atmospheric pressure, such as by pressurizing to achieve a reaction temperature exceeding 100°C. Preferably, the heating reaction is carried out at atmospheric pressure at 80–100°C. Preferably, the heating reaction time is 10–200 min, more preferably 20–120 min, for example, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 min.

[0104] After the heating reaction is complete, filtration is performed. The filter paper used for filtration should have a pore size ≥1μm, further ≥1.5μm, and further ≥2μm. The pore size of the filter paper should be smaller than the size of the pulverized mineral-based Chinese medicinal herbs and other Chinese medicinal herbs. For example, the pore size of the filter paper can be 1–200μm, further 1–100μm. Filtration effectively removes the medicinal residue, yielding a filtrate containing the active ingredients.

[0105] The filtrate obtained from filtration is further centrifuged, preferably using gradient centrifugation.

[0106] Preferably, the centrifugation process includes: performing 2 to 8 centrifugations, with the first centrifugation at a speed of 1000 to 7000 rpm, each subsequent centrifugation at a higher speed than the previous one, and the final centrifugation at a speed of 8000 to 15000 rpm; each centrifugation lasting 5 to 50 minutes. The centrifugation time can be the same or different for each centrifugation. After each centrifugation, the supernatant is collected for further processing.

[0107] Multiple gradient centrifugation can reduce the damage to the activity of the active ingredients in traditional Chinese medicine, improve the purity of the active ingredients, and remove minute impurities.

[0108] For example, perform four centrifugations: the first centrifugation, the second centrifugation, the third centrifugation, and the last centrifugation. The centrifugation speed in each subsequent centrifugation should be greater than the centrifugation speed in the previous centrifugation. That is, the centrifugation speed in the second centrifugation should be greater than the centrifugation speed in the first centrifugation, the centrifugation speed in the third centrifugation should be greater than the centrifugation speed in the last centrifugation, and the centrifugation speed in the last centrifugation should be greater than the centrifugation speed in the third centrifugation.

[0109] Further optimization is that the difference between the centrifugation speed of the subsequent centrifugation and that of the previous centrifugation is 500 to 6000 rpm.

[0110] Further preferred, the centrifugation step includes: performing 3 to 5 centrifugations, with the first centrifugation speed being 2000 to 5000 rpm, the difference between the centrifugation speed of each subsequent centrifugation and that of the previous centrifugation being 1000 to 5000 rpm, and the final centrifugation speed being 9000 to 12000 rpm.

[0111] The supernatant obtained from the final centrifugation is placed in a dialysis bag for dialysis in water. The molecular weight cutoff of the dialysis bag is preferably 1000-10000 Da, and more preferably 1500-8000 Da.

[0112] Preferably, the dialysis time is 10 to 100 hours, and more preferably 12 to 72 hours.

[0113] After dialysis, the solution in the dialysis bag is dried or concentrated and then dried.

[0114] Further preferably, the concentration is carried out by vacuum evaporation, with a temperature of 20–80°C, a system pressure of 5–80 kPa, and a time of 1–1200 min.

[0115] Further preferably, the reduced pressure evaporation method is rotary evaporation, with a rotation speed of 50-300 rpm, a temperature of 20-80℃, a system pressure of 5-80 kPa, and a time of 1-1200 min.

[0116] The drying process can be freeze-drying or vacuum drying. Freeze-drying is preferred. The freeze-drying temperature is -40 to -100°C, the vacuum degree is 1 to 100 Pa, and the time is 12 to 100 hours.

[0117] In the second method for preparing self-assembled nanomaterials of traditional Chinese medicine, the self-assembled nanomaterials and the targeting agent are coupled through a chemical reaction, wherein the chemical reaction is an EDC and NHS activation coupling method.

[0118] Preferably, the coupling includes: dispersing the self-assembled nanomaterials in 2-(N-morpholino)ethanesulfonic acid buffer, sequentially adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for activation; then adding a coupling target agent with a carboxyl or amino group at the end, reacting at room temperature, and then purifying by centrifugation to obtain the self-assembled nanomaterials of traditional Chinese medicine.

[0119] The mass ratio of the self-assembled nanomaterial to the coupling target agent with a carboxyl or amino group at the end is preferably 1:0.1 to 10. More preferably, it is 1:0.1 to 5.

[0120] In the coupling step, preferably, 1-10 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.1-1 mL of N-hydroxysuccinimide are added per 10 mg of self-assembled nanomaterial for activation. The pH of the 2-(N-morpholino)ethanesulfonic acid buffer is 5.5-7.4. The reaction time at room temperature is 0.5-24 h.

[0121] The fifth aspect of this invention provides the application of the aforementioned self-assembled nanomaterials of traditional Chinese medicine, wherein the application is selected from one or more of the following:

[0122] Used in the preparation of nuclear magnetic resonance contrast agents;

[0123] Used in the preparation of photoacoustic imaging contrast agents;

[0124] Used to prepare drugs for the diagnosis and / or treatment of cancer diseases.

[0125] The self-assembled nanomaterials made from traditional Chinese medicine possess T1-weighted magnetic resonance imaging (MRI) capabilities and can be used as MRI contrast agents. They also exhibit photoacoustic imaging capabilities, significantly enhancing the photoacoustic signal in tumor tissues, and can be used as photoacoustic imaging contrast agents. Furthermore, these nanomaterials can generate reactive oxygen species under acidic conditions and possess photothermal effects, exhibiting a tumor-killing effect, and can be used as drugs for the diagnosis and / or treatment of solid tumors.

[0126] Preferably, the photoacoustic imaging contrast agent comprises 0.1–100 wt% of the self-assembled nanomaterials of the traditional Chinese medicine.

[0127] Preferably, the drug for diagnosing and / or treating tumor diseases comprises 0.1 to 100 wt% of the self-assembled nanomaterials of the traditional Chinese medicine.

[0128] Therefore, the self-assembled nanomaterials of traditional Chinese medicine can realize the integrated diagnosis and treatment of tumors.

[0129] The technical solution of the present invention will be further described and illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the specific scope of the present invention. Furthermore, the accompanying drawings used herein are merely for better illustrating the content disclosed in the present invention and do not limit the scope of protection. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used in the art, and the methods used in the embodiments are all conventional methods in the art.

[0130] The Chinese medicinal herbs used in the following examples and comparative examples are all commercially available processed Chinese medicinal herbs, and have passed the testing of the Chinese Pharmacopoeia. Before use, all herbs were pulverized to a size of 0.3–1 mm, and the filter paper pore size was 2 μm.

[0131] Example 1

[0132] The preparation method of the self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0133] (1) Add 12g of natural copper and 15g of Cornus officinalis to the reaction vessel, then add 150mL of water to soak and let stand for 30 minutes;

[0134] (2) Heat the solution in (1) to 100 degrees Celsius under normal pressure and let it boil gently for 20 minutes;

[0135] (3) After filtering the residue from the solution in (2), take the filtrate and perform gradient centrifugation. Centrifuge at 4000 rpm, 6000 rpm, 8000 rpm and 10000 rpm for 30 minutes each time. Take the supernatant after each centrifugation for subsequent centrifugation.

[0136] (4) Transfer the supernatant obtained by centrifugation in (3) into a 3500 Da dialysis bag and dialyze in water for 48 hours.

[0137] (5) The solution obtained in (4) was freeze-dried (temperature -80℃, vacuum degree 10Pa) for 48 hours to obtain the self-assembled nanomaterial of traditional Chinese medicine.

[0138] Example 2

[0139] The preparation method of the self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0140] (1) Add 12g of natural copper and 15g of Cornus officinalis to the reaction vessel, then add 150mL of water to soak and let stand for 30 minutes;

[0141] (2) Heat the solution in (1) to 100 degrees Celsius under normal pressure and let it boil gently for 40 minutes;

[0142] (3) After filtering the residue from the solution in (2), take the filtrate and perform gradient centrifugation. Centrifuge at 4000 rpm, 6000 rpm, 8000 rpm and 10000 rpm for 30 minutes each time. Take the supernatant after each centrifugation for subsequent centrifugation.

[0143] (4) Transfer the supernatant obtained by centrifugation in (3) into a 3500 Da dialysis bag and dialyze in water for 48 hours.

[0144] (5) The solution obtained in (4) was freeze-dried (temperature -80℃, vacuum degree 10Pa) for 48 hours to obtain the self-assembled nanomaterial of traditional Chinese medicine.

[0145] Example 3

[0146] The preparation method of the self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0147] (1) Add 12g of natural copper and 15g of Cornus officinalis to the reaction vessel, then add 150mL of water to soak and let stand for 30 minutes;

[0148] (2) Heat the solution in (1) to 100 degrees Celsius under normal pressure and let it boil gently for 60 minutes;

[0149] (3) After filtering the residue from the solution in (2), take the filtrate and perform gradient centrifugation. Centrifuge at 4000 rpm, 6000 rpm, 8000 rpm and 10000 rpm for 30 minutes each time. Take the supernatant after each centrifugation for subsequent centrifugation.

[0150] (4) Transfer the supernatant obtained by centrifugation in (3) into a 3500 Da dialysis bag and dialyze in water for 48 hours.

[0151] (5) The solution obtained in (4) was freeze-dried (temperature -80℃, vacuum degree 10Pa) for 48 hours to obtain the self-assembled nanomaterial of traditional Chinese medicine.

[0152] Example 4

[0153] The preparation method of the self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0154] (1) Add 12g of natural copper and 15g of Cornus officinalis to the reaction vessel, then add 150mL of water to soak and let stand for 30 minutes;

[0155] (2) Heat the solution in (1) to 100 degrees Celsius under normal pressure and let it boil gently for 80 minutes;

[0156] (3) After filtering the residue from the solution in (2), take the filtrate and perform gradient centrifugation. Centrifuge at 4000 rpm, 6000 rpm, 8000 rpm and 10000 rpm for 30 minutes each time. Take the supernatant after each centrifugation for subsequent centrifugation.

[0157] (4) Transfer the supernatant obtained by centrifugation in (3) into a 3500 Da dialysis bag and dialyze in water for 48 hours.

[0158] (5) The solution obtained in (4) was freeze-dried (temperature -80℃, vacuum degree 10Pa) for 48 hours to obtain the self-assembled nanomaterial of traditional Chinese medicine.

[0159] Example 5

[0160] The preparation method of the self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0161] (1) Add 12g of natural copper and 15g of Cornus officinalis to the reaction vessel, then add 150mL of water to soak and let stand for 30 minutes;

[0162] (2) Heat the solution in (1) to 100 degrees Celsius under normal pressure and let it boil gently for 100 minutes;

[0163] (3) After filtering the residue from the solution in (2), take the filtrate and perform gradient centrifugation. Centrifuge at 4000 rpm, 6000 rpm, 8000 rpm and 10000 rpm for 30 minutes each time. Take the supernatant after each centrifugation for subsequent centrifugation.

[0164] (4) Transfer the supernatant obtained by centrifugation in (3) into a 3500 Da dialysis bag and dialyze in water for 48 hours.

[0165] (5) The solution obtained in (4) was freeze-dried (temperature -80℃, vacuum degree 10Pa) for 48 hours to obtain the self-assembled nanomaterial of traditional Chinese medicine.

[0166] Example 6

[0167] The preparation method of the self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0168] (1) Add 12g of natural copper and 15g of Cornus officinalis to the reaction vessel, then add 150mL of water to soak and let stand for 30 minutes;

[0169] (2) Heat the solution in (1) to 100 degrees Celsius under normal pressure and let it boil gently for 120 minutes;

[0170] (3) After filtering the residue from the solution in (2), take the filtrate and perform gradient centrifugation. Centrifuge at 4000 rpm, 6000 rpm, 8000 rpm and 10000 rpm for 30 minutes each time. Take the supernatant after each centrifugation for subsequent centrifugation.

[0171] (4) Transfer the supernatant obtained by centrifugation in (3) into a 3500 Da dialysis bag and dialyze in water for 48 hours.

[0172] (5) The solution obtained in (4) was freeze-dried (temperature -80℃, vacuum degree 10Pa) for 48 hours to obtain the self-assembled nanomaterial of traditional Chinese medicine.

[0173] Example 7

[0174] The preparation method of the self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0175] (1) Add 12g of natural copper, 5g of gentian and 24g of cornus officinalis to the reaction vessel, then add 200mL of water to soak and let stand for 50 minutes.

[0176] (2) Heat the solution in (1) to 100 degrees Celsius under normal pressure and let it boil gently for 60 minutes;

[0177] (3) After filtering the residue from the solution in (2), take the filtrate and perform gradient centrifugation. Centrifuge at 4000 rpm, 6000 rpm, 8000 rpm and 10000 rpm for 30 minutes each time. Take the supernatant after each centrifugation for subsequent centrifugation.

[0178] (4) Transfer the supernatant obtained by centrifugation in (3) into a 3500 Da dialysis bag and dialyze in water for 70 hours.

[0179] (5) The solution obtained in (4) was freeze-dried (temperature -80℃, vacuum degree 10Pa) for 48 hours to obtain the self-assembled nanomaterial of traditional Chinese medicine.

[0180] Example 8

[0181] The preparation method of the self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0182] (1) Add 12g of natural copper, 10g of Cornus officinalis, 14g of Ligustrum lucidum and 6g of Taxillus chinensis to the reaction vessel, then add 220mL of water to soak and let stand for 30 minutes;

[0183] (2) Heat the solution in (1) to 100 degrees Celsius under normal pressure and let it boil gently for 60 minutes;

[0184] (3) After filtering the residue from the solution in (2), take the filtrate and perform gradient centrifugation. Centrifuge at 3000 rpm, 5000 rpm, 8000 rpm and 10000 rpm for 20 minutes each time. Take the supernatant after each centrifugation for subsequent centrifugation.

[0185] (4) Transfer the supernatant obtained by centrifugation in (3) into a 3500 Da dialysis bag and dialyze in water for 30 hours.

[0186] (5) The solution obtained in (4) is freeze-dried (temperature -80℃, vacuum degree 10Pa) for 72 hours to obtain the self-assembled nanomaterial of traditional Chinese medicine.

[0187] Example 9

[0188] The preparation method of the self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0189] (1) Add 12g of natural copper, 10g of Cornus officinalis, 5g of centipede and 5g of Astragalus membranaceus to the reaction vessel, then add 180mL of water to soak and let stand for 30 minutes.

[0190] (2) Heat the solution in (1) to 100 degrees Celsius under normal pressure and let it boil gently for 60 minutes;

[0191] (3) After filtering the residue from the solution in (2), take the filtrate and perform gradient centrifugation. Centrifuge at 4000 rpm, 6000 rpm, 8000 rpm and 10000 rpm for 30 minutes each time. Take the supernatant after each centrifugation for subsequent centrifugation.

[0192] (4) Transfer the supernatant obtained by centrifugation in (3) into a 3500 Da dialysis bag and dialyze in water for 48 hours.

[0193] (5) The solution obtained in (4) was freeze-dried (temperature -80℃, vacuum degree 10Pa) for 48 hours to obtain the self-assembled nanomaterial of traditional Chinese medicine.

[0194] Example 10

[0195] The preparation method of the self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0196] (1) Add 6g of natural copper, 6g of ferrous sulfate, 6g of cornus officinalis, 5g of centipede and 6g of mugwort to the reaction vessel, then add 200mL of water to soak and let stand for 20 minutes.

[0197] (2) Heat the solution in (1) to 90 degrees Celsius under normal pressure and let it boil gently for 60 minutes;

[0198] (3) After filtering the residue from the solution in (2), take the filtrate and perform gradient centrifugation. Centrifuge at 4000 rpm, 8000 rpm and 10000 rpm for 40 minutes each time. Take the supernatant after each centrifugation for subsequent centrifugation.

[0199] (4) Transfer the supernatant obtained by centrifugation in (3) into a 2000 Da dialysis bag and dialyze in water for 48 hours.

[0200] (5) The solution obtained in (4) is freeze-dried (temperature -80℃, vacuum degree 10Pa) for 72 hours to obtain the self-assembled nanomaterial of traditional Chinese medicine.

[0201] Figure 1 shows the particle size distribution, dispersion, and appearance in water of the self-assembled nanomaterials of traditional Chinese medicine obtained in Examples 1-6. It can be seen that the self-assembled nanomaterials of traditional Chinese medicine have good dispersion in water, and the D50 particle size is between 150 and 350 nm.

[0202] The microstructure of the self-assembled nanomaterials of traditional Chinese medicine obtained in Example 3 is shown in Figure 2. The D50 particle size is about 190 nm.

[0203] Figure 3a) is a comparison of the particle size distribution and dispersity of the self-assembled nanomaterials of traditional Chinese medicine synthesized five times using the method of Example 3. It can be seen that the batch stability of the multiple syntheses is high, and the D50 particle size is stable at around 190 nm. Figure 3b) is a comparison of the particle size distribution and dispersity of the self-assembled nanomaterials of traditional Chinese medicine in Example 3 after storage at room temperature for 0, 1, 2, and 3 months. It can be seen that the D50 particle size of the self-assembled nanomaterials of traditional Chinese medicine is still stable at around 190 nm after 3 months of storage at room temperature. The self-assembled nanomaterials of traditional Chinese medicine have high stability at room temperature, and can be stored at room temperature for more than 3 months.

[0204] The self-assembled nanomaterials of traditional Chinese medicine obtained in Example 3 were dissolved in a complete culture medium to prepare solutions of different concentrations. The toxicity results on normal cell line L929 and cancer cell lines U2OS and 143B are shown in Figure 4. Even at a high concentration of 400 μg / mL, the cell viability was above 80%.

[0205] The self-assembled nanomaterials of traditional Chinese medicine obtained in Example 3 were dissolved in PBS to prepare a solution. 10 mg / kg of the material was injected into the tail vein of 6-week-old Balb / C female mouse models, while the control group was directly injected with PBS. After 14 days of normal feeding, the mice were sacrificed, and their internal organs (heart, liver, spleen, lungs, and kidneys) were collected. The tissue sections were observed after HE staining. The results are shown in Figure 5. Compared with normal mice (control group), there were no lesions in the internal organs, indicating that the contrast agent has extremely high biocompatibility.

[0206] The self-assembled nanomaterials of traditional Chinese medicine obtained in Example 3 were dissolved in water to prepare solutions with concentrations of 4, 8, 12, 16, and 20 mg / mL. The relaxation properties and MRI imaging properties of the materials were characterized using an MRI imaging system. The results are shown in Figure 6. The obtained self-assembled nanomaterials of traditional Chinese medicine have good MRI imaging effects and T1 relaxation properties.

[0207] The self-assembled nanomaterials of traditional Chinese medicine obtained in Example 3 were dissolved in PBS to prepare a solution (concentration of 100 mg / mL). 200 μL of the material was injected into a 6-week-old female Balb / CNu nude mouse model of subcutaneous osteosarcoma via the tail vein. The MRI and metabolic status of the animals were observed and recorded within 2 hours after administration. The results are shown in Figure 7. Comparison of solid tumor images and their MRI images shows that the material accumulates significantly at the tumor site, resulting in a significant enhancement of the tumor MRI signal. It can also be metabolized by the kidneys, showing potential for injection as an MRI contrast agent.

[0208] The self-assembled nanomaterials of traditional Chinese medicine obtained in Example 3 were dissolved in water to prepare a solution (concentration of 100 mg / mL). 200 μL of the material was orally administered to a normal 6-week-old Balb / CNu female nude mouse model via gavage. MRI scans of the animals were observed and recorded within 30 minutes after administration. The results are shown in Figure 8. Compared with the same concentration of the Example 3 solution placed in vitro, the MRI signal in the gastrointestinal tract of the nude mice increased after gavage, indicating that Example 3 is rapidly distributed in vivo and has the potential to be used as an oral MRI contrast agent.

[0209] The self-assembled nanomaterials of traditional Chinese medicine obtained in Example 3 were dissolved in PBS to prepare a solution. The solution was injected into a 6-week-old female Balb / CNu nude mouse model of osteosarcoma in situ via the tail vein at a dose of 5 mg / kg. The control group was directly injected with PBS. The tumor size of the animals was observed and recorded 2 weeks after administration. The results are shown in Figure 9. The comparison of the images shows that tumor growth was inhibited.

[0210] Comparative Example 1

[0211] The difference between Comparative Example 1 and Example 3 is that no other Chinese medicine was added to Comparative Example 1. That is, step (1) of Comparative Example 1 is: 12g of natural copper is added to the reaction vessel, 150mL of water is added to soak and stand for 30 minutes; subsequent steps (2)-(5) are the same as in Example 3, and Chinese medicine powder particles are obtained.

[0212] As shown in Figure 10, the Chinese medicine powder particles prepared in this comparative example have no particle size after dissolving in water.

[0213] Comparative Example 2

[0214] The difference between Comparative Example 2 and Example 3 is that Comparative Example 2 did not add mineral Chinese medicine. That is, step (1) of Comparative Example 2 is: add 15g of Cornus officinalis to the reaction vessel, add 150mL of water to soak and let stand for 30 minutes; subsequent steps (2)-(5) are the same as in Example 3, and Chinese medicine powder particles are obtained.

[0215] Figure 10 shows the appearance and particle size distribution of the prepared herbal powder particles dissolved in water. The herbal powder particles are nearly micrometer-sized. The absence of mineral-rich herbal ingredients meant there was no ion source to serve as a "seed" for the assembly of other herbal components, thus preventing the observation of self-assembling nanomaterials. The nearly micrometer-sized substances detected here are not small-molecule self-assembled nanomaterials precipitated from the herbal powder, but may simply be fine particles from the decomposition of Cornus officinalis itself.

[0216] The Chinese medicine powder particles obtained in Comparative Example 2 were dissolved in water to prepare solutions with concentrations of 4, 8, 12, 16, and 20 mg / mL. The MRI imaging performance of the materials was characterized using an MRI imaging system. The results are shown in Figure 11. The signal was similar to that of water, with almost no MRI imaging effect.

[0217] Comparative Example 3

[0218] The difference between Comparative Example 2 and Example 3 is that Comparative Example 3 did not undergo any subsequent processing. The specific preparation method of Comparative Example 3 is as follows:

[0219] (1) Add 12g of natural copper and 15g of Cornus officinalis to the reaction vessel, then add 150mL of water to soak and let stand for 30 minutes;

[0220] (2) Heat the solution in (1) to 100 degrees Celsius under normal pressure and let it boil gently for 60 minutes;

[0221] (3) After filtering the dregs from the solution in (2), the filtrate obtained is the Chinese medicine decoction.

[0222] The traditional Chinese medicine decoction prepared in this comparative example deteriorated and became smelly after only 48 hours at room temperature, as shown in Figure 12.

[0223] Example 11

[0224] The preparation method of the self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0225] (1) Add 8g of hematite and 10g of cornus officinalis to the reaction vessel, then add 100mL of water to soak and let stand for 30 minutes;

[0226] (2) Heat the solution in (1) to 100℃ under normal pressure and let it boil gently for 60 minutes;

[0227] (3) After filtering the residue from the solution in (2), take the filtrate and perform gradient centrifugation. Centrifuge at 4000 rpm, 6000 rpm, 8000 rpm and 10000 rpm for 30 minutes each time. Take the supernatant after each centrifugation for subsequent centrifugation.

[0228] (4) Transfer the supernatant obtained from the last centrifugation in (3) to a 3500 Da dialysis bag and dialyze in water for 48 hours to obtain dialysate;

[0229] (5) The dialysate obtained in (4) was concentrated by rotary evaporation for 30 minutes at a speed of 100 rpm, a temperature of 55 ℃, and a pressure of 12 kPa.

[0230] (6) The concentrated solution obtained in (5) was freeze-dried at a temperature of -80℃, a vacuum of 10Pa, and a time of 48h to obtain self-assembled nanomaterials.

[0231] (7) Disperse 10 mg of the self-assembled nanomaterial obtained in (6) in 5 mL of 2-(N-morpholino) ethanesulfonic acid buffer (pH 6.5), and then add 0.2 mL of 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.2 mL of 10 mg / mL N-hydroxysuccinimide sequentially for activation;

[0232] (8) Add 5 mg of folic acid to the activated self-assembled nanomaterials obtained in (7) and react at room temperature for 4 h; then centrifuge and purify to obtain targeted photoacoustic / photothermal self-assembled nanomaterials of traditional Chinese medicine.

[0233] Example 12

[0234] The method for preparing self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0235] (1) Add 8g of natural copper and 10g of Cornus officinalis to the reaction vessel, then add 100mL of water to soak and let stand for 30 minutes;

[0236] (2-8) Proceed according to steps (2-8) in Example 11.

[0237] Example 13

[0238] The method for preparing self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0239] (1) Add 8g of Yu Yu Liang and 10g of Shan Zhu Yu to the reaction vessel, then add 100mL of water to soak and let stand for 30 minutes;

[0240] (2-8) Proceed according to steps (2-8) in Example 11.

[0241] Example 14

[0242] The method for preparing self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0243] (1) Add 8g of magnetite and 10g of cornus officinalis to the reaction vessel, then add 100mL of water to soak and let stand for 30 minutes;

[0244] (2-8) Proceed according to steps (2-8) in Example 11.

[0245] Example 15

[0246] The method for preparing self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0247] (1-7) Proceed according to steps (1-7) in Example 14.

[0248] (8) Add 10 mg of hyaluronic acid to the activated Chinese medicine self-assembled nanomaterials obtained in (7) and react at room temperature for 8 h; then centrifuge and purify to obtain targeted photoacoustic / photothermal Chinese medicine self-assembled nanomaterials.

[0249] Example 16

[0250] The method for preparing self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0251] (1-7) Proceed according to steps (1-7) in Example 14.

[0252] (8) Add 12 mg of arginine-glycine-aspartic acid tripeptide to the activated Chinese medicine self-assembled nanomaterial obtained in (7) and react at room temperature for 2 h; then perform centrifugation purification to obtain the targeted photoacoustic / photothermal Chinese medicine self-assembled nanomaterial.

[0253] Example 17

[0254] The method for preparing self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0255] (1) Add 8g magnetite, 8g ochre, 10g cornus officinalis, 10g hawthorn and 5g bezoar to the reaction vessel, then add 150mL of water to soak and let stand for 30 minutes.

[0256] (2-7) Proceed according to steps (2-7) in Example 11.

[0257] (8) Add 5 mg of folic acid-PEG-OH to the activated Chinese medicine self-assembled nanomaterials obtained in (7) and react at room temperature for 4 h; then perform centrifugation purification to obtain targeted photoacoustic / photothermal Chinese medicine self-assembled nanomaterials.

[0258] Example 18

[0259] The method for preparing self-assembled nanomaterials of traditional Chinese medicine in this embodiment includes the following steps:

[0260] (1) Add 8g of ochre, 8g of natural copper, 10g of cornus officinalis, 10g of hawthorn and 10g of wolfberry to the reaction vessel, then add 150mL of water to soak and let stand for 30 minutes.

[0261] (2-7) Proceed according to steps (2-7) in Example 11.

[0262] (8) Add 1 mg of folic acid to the activated Chinese medicine self-assembled nanomaterial obtained in (7) and react at room temperature for 4 h; then perform centrifugation purification to obtain the targeted photoacoustic / photothermal Chinese medicine self-assembled nanomaterial.

[0263] Comparative Example 4

[0264] The preparation method of this comparative example of self-assembled nanomaterials from traditional Chinese medicine includes the following steps:

[0265] (1) Add 8g of magnetite and 1g of cornus officinalis to the reaction vessel, then add 100mL of water to soak and let stand for 30 minutes;

[0266] (2-8) Proceed according to steps (2-8) in Example 11.

[0267] As shown in Figure 13, the D50 particle size of the targeted photoacoustic / photothermal self-assembled nanomaterials of traditional Chinese medicine in Examples 11-14 is ~100 nm. As shown in Figure 14, they exhibit good dispersibility in water, with a hydrated particle size of 200-260 nm and a negative surface charge ranging from -20 to -40 mV. As shown in Figure 15, the self-assembled nanomaterials of traditional Chinese medicine in Examples 11-14 contain iron, with a content of 5-280 mg / L. As shown in Figure 16, the self-assembled nanomaterials of traditional Chinese medicine in Example 14 are mainly composed of carbon, oxygen, nitrogen, and iron.

[0268] Application Example 1

[0269] Near-infrared absorption performance test:

[0270] The self-assembled nanomaterials of traditional Chinese medicine in Example 14 and the self-assembled nanomaterials of traditional Chinese medicine in Comparative Example 4 were dissolved in water and prepared into solutions of different concentrations (0.2 mM, 0.4 mM, 0.6 mM and 0.8 mM, respectively, based on the iron content).

[0271] As shown in Figure 17, the self-assembled nanomaterials of traditional Chinese medicine in Example 14 at different concentrations showed absorption in the near-infrared region (e.g., at 808 nm), indicating that the self-assembled nanomaterials of traditional Chinese medicine have potential applications in the fields of photoacoustic and photothermal.

[0272] As shown in Figure 18, at the same concentration (0.2 mM), the self-assembled nanomaterials of traditional Chinese medicine in Comparative Example 2 exhibit virtually no absorption in the near-infrared region. This indicates that the ratio of mineral-based traditional Chinese medicines to other traditional Chinese medicines is a crucial factor affecting their performance.

[0273] Application Example 2

[0274] Thermal imaging test:

[0275] The self-assembled nanomaterials of the traditional Chinese medicine from Example 14 were dissolved in water and prepared into solutions of different concentrations (0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM, respectively, based on iron content for quantification); and 808 nm nanometers were applied to each solution (power 0.5 W / cm²). 2 1W / cm 2 1.5W / cm 2 2.0W / cm 2 ) irradiation.

[0276] As shown in Figure 19, Figure 19(a) shows solutions of different concentrations at 808 nm (1 W / cm²). 2 Figure 19(b) shows the temperature change of a 0.4 mM solution at 808 nm (0.5 W / cm²). 2 1W / cm 2 1.5W / cm2 2.0W / cm 2 Figure 19(c) shows the temperature change of the 0.8 mM solution under five cycles of switching laser irradiation. Figure 19(d) shows the temperature change of the 0.8 mM solution at 808 nm (1 W / cm²). 2 The photothermal conversion efficiency under irradiation was measured to be 36.73%. Figure 19(e) shows the thermal imaging of solutions of different concentrations over time. These test results demonstrate that the self-assembled nanomaterials of the traditional Chinese medicine in Example 14 possess excellent photothermal conversion efficiency and photothermal stability.

[0277] As shown in Figure 20, the photoacoustic signal at 808 nm increases with increasing concentration and exhibits a linear relationship, indicating that the self-assembled nanomaterials of traditional Chinese medicine in Example 14 have excellent photoacoustic imaging performance.

[0278] Under the same conditions as in Example 14, thermal imaging of other embodiments was performed, and the self-assembled nanomaterials of traditional Chinese medicine in Examples 1-13 and 15-18 of the present invention also exhibited excellent photoacoustic imaging performance.

[0279] Application Example 3

[0280] Cytotoxicity test:

[0281] The self-assembled nanomaterials of traditional Chinese medicine from Examples 11-14 were dissolved in culture medium solution and prepared into solutions of different concentrations (10 μM, 25 μM, 50 μM, 100 μM, 150 μM, and 200 μM, respectively, based on iron content for quantification). These solutions were then added to the normal cell line L929 and incubated in a CO2 incubator at 37°C for 24 h. Cell viability was then measured using the CCK-8 reagent.

[0282] As shown in Figure 21, cell viability was above 80% at concentrations below 100 μM.

[0283] Application Example 4

[0284] Photothermal killing test of cancer cells:

[0285] The same number of human renal cell carcinoma cell line 786-0 cells were dispersed in culture medium solution and seeded onto four confocal dishes. The dishes were incubated in a CO2 incubator at 37°C for 24 hours. After cell adhesion, the cells were divided into groups for subsequent experiments: control group, laser irradiation only group, Example 14 only group, and Example 14 + laser irradiation group.

[0286] Control group: The old culture medium was discarded, and after being replaced with fresh culture medium solution, it was placed in a CO2 incubator at 37°C for 24 hours.

[0287] Laser irradiation group only: After discarding the old culture medium and replacing it with fresh culture medium solution, 808nm (1W / cm) was applied. 2 Irradiate the sample with a laser for 6 minutes. Then, incubate it in a CO2 incubator at 37°C for 24 hours.

[0288] Example 14 only: The self-assembled nanomaterials of traditional Chinese medicine from Example 14 were dissolved in a culture medium solution and prepared into a 20 μM solution (based on iron content for quantification). The old culture medium was discarded, and the solution containing the culture medium from Example 14 was replaced. The solution was then placed in a CO2 incubator at 37°C for 24 hours.

[0289] Example 14 + Laser Irradiation Group: The self-assembled nanomaterials of traditional Chinese medicine from Example 14 were dissolved in a culture medium solution and prepared into a 20 μM solution (based on iron content for quantification). The old culture medium was discarded, and the solution containing the one from Example 14 was replaced. Then, an 808 nm (1 W / cm²) laser irradiation was applied. 2 Irradiate the sample with a laser for 6 minutes. Then, incubate it in a CO2 incubator at 37°C for 24 hours.

[0290] The above four groups of cells were stained with Calcein-AM / PI working solution, and cell viability was assessed by laser confocal microscopy.

[0291] As shown in Figure 22, laser irradiation alone has virtually no effect on human renal cell carcinoma cell line 786-0; however, the self-assembled nanomaterials of traditional Chinese medicine in Example 14 have a certain photothermal killing effect on human renal cell carcinoma cell line 786-0. Under the action of laser, the photothermal killing effect is significant, and the cell survival rate is ~5%.

[0292] Under the same conditions as in Example 14+ laser irradiation group, the traditional Chinese medicine self-assembled nanomaterials in Examples 1-13 and 15-18 of this invention also showed good photothermal killing effects on renal cancer cells.

[0293] In the absence of laser irradiation, the self-assembled nanomaterials of traditional Chinese medicine in Comparative Example 4 showed an effect on renal cell carcinoma cells with a cell survival rate of ~95%.

[0294] Under laser irradiation, the self-assembled nanomaterials of traditional Chinese medicine in Comparative Example 4 showed an effect on renal cell carcinoma cells, with a cell survival rate of ~95%. It can be concluded that the self-assembled nanomaterials of traditional Chinese medicine in Comparative Example 4 do not possess photothermal or photoacoustic properties.

[0295] Application Example 5

[0296] Animal model test 1:

[0297] (1) Animal model construction process: 100 μL (5 × 10 6786-0 cells were injected into the back (near the right upper or lower limb) of female BALB / c nude mice (16–19 g, 6 weeks old) and into the tumor when it reached ~100 mm. 3 Animal experiments were conducted at a time when the tumor volume was calculated as (tumor volume = width × width × length / 2).

[0298] (2) Dissolve the self-assembled nanomaterials of traditional Chinese medicine in physiological saline to prepare a 2mM solution (based on the iron content).

[0299] Control group: 100 μL of physiological saline was injected into tumor-bearing mice via the tail vein. The mice were sacrificed on day 14 and the tumors were removed for observation.

[0300] Laser irradiation group only: Six hours after injecting 100 μL of physiological saline into tumor-bearing mice via the tail vein, 808 nm (1 W / cm²) laser irradiation was applied to the tumor site on days 1 and 4. 2 The mice were irradiated with a laser for 6 minutes each time; on the 14th day, the mice were sacrificed and the tumors were removed for observation.

[0301] Example 14 only: 100 μL of the above solution was injected into tumor-bearing mice via the tail vein. The mice were sacrificed on day 14 and the tumors were removed for observation.

[0302] Example 14 + Laser Irradiation Group: 100 μL of the above solution was injected into tumor-bearing mice via the tail vein. Six hours later, on days 1 and 4, 808 nm (1 W / cm²) laser irradiation was applied to the tumor site. 2 The mice were irradiated with a laser for 6 minutes each time; on the 14th day, the mice were sacrificed and the tumors were removed for observation.

[0303] As shown in Figure 23, Example 14 can inhibit tumor growth; and under the action of laser, Example 14 + laser irradiation group has excellent photothermal killing effect on tumor.

[0304] Under the same conditions as in Example 14 + laser irradiation group, the self-assembled traditional Chinese medicine nanomaterials in Examples 1-13 and 15-18 of this invention also showed good photothermal killing effects on cancer cells. The self-assembled traditional Chinese medicine nanomaterials in Comparative Example 4, lacking photothermal and photoacoustic properties, exhibited almost zero tumor weight reduction.

[0305] Application Example 6

[0306] Animal model test 2:

[0307] (1) The process of constructing the animal model is the same as that in application example 5.

[0308] (2) The self-assembled nanomaterials of traditional Chinese medicine in Example 14 were dissolved in physiological saline to prepare a 2mM solution (based on iron content). 100μL of the solution was injected into tumor-bearing mice via the tail vein.

[0309] After injection, the changes in the tumor site in the tumor-bearing mice of Example 14 were observed at 0h, 1h, 2h, 4h, 6h, and 8h, resulting in the photoacoustic imaging in Figure 24. It can be seen that after injection, with the extension of time, the photoacoustic signal at the tumor site increased, and the vascular details at the tumor site increased, which can help to accurately distinguish the tumor boundary from normal tissue.

[0310] In summary, the small D50 particle size of the self-assembled nanomaterials of traditional Chinese medicine in this invention makes it easier to enter the tumor tissue and accumulate there, resulting in a significant enhancement of the MRI and photoacoustic signals of the tumor tissue, accurately distinguishing the tumor boundary from normal tissue. At the same time, it has a good photothermal killing effect on the tumor. Under the action of laser, the photothermal killing effect is further enhanced, effectively realizing MRI, photoacoustic, and photothermal effects.

[0311] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0312] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0313] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A self-assembled nanomaterial of traditional Chinese medicine, characterized in that, The raw materials for preparing the self-assembled nanomaterials of traditional Chinese medicine include mineral-based traditional Chinese medicine and other traditional Chinese medicines, wherein the other traditional Chinese medicines are one or two of plant-derived traditional Chinese medicines and animal-derived traditional Chinese medicines; The D50 particle size of the self-assembled nanomaterials of traditional Chinese medicine is 30-450 nm.

2. The self-assembled nanomaterials of traditional Chinese medicine according to claim 1, characterized in that, The D50 particle size of the self-assembled nanomaterials of traditional Chinese medicine is 50–450 nm.

3. The self-assembled nanomaterials of traditional Chinese medicine according to claim 2, characterized in that, The raw materials for preparing the self-assembled nanomaterials of traditional Chinese medicine also include a targeting agent, which is a coupling targeting agent with a carboxyl or amino group at the end.

4. The self-assembled nanomaterials of traditional Chinese medicine according to any one of claims 1 to 3, characterized in that, The mineral-based traditional Chinese medicine is one or more of the following: natural copper, ochre, hematite, magnetite, ferrous sulfate, gypsum, stachyite, and calamine.

5. The self-assembled nanomaterials of traditional Chinese medicine according to any one of claims 1 to 3, characterized in that, The plant-derived Chinese medicinal herbs are one or more of the following: Cornus officinalis, Crataegus pinnatifida, Lycium barbarum, Ziziphus jujuba, Codonopsis pilosula, Cistanche deserticola, Ganoderma lucidum, Saffron, Amomum tsao-ko, Curcuma longa, Piper longum, Rehmannia glutinosa, Ophiopogon japonicus, Asparagus cochinchinensis, Citrus reticulata peel, Taxillus chinensis, Eucommia ulmoides, Dipsacus asper, Platycladus orientalis leaf, Clematis chinensis, Anemarrhena asphodeloides, Phellodendron chinense, Ligustrum lucidum, Angelica sinensis, Salvia miltiorrhiza, Citrus reticulata peel, Astragalus membranaceus, and Artemisia argyi. The animal-derived traditional Chinese medicine is one or more of the following: *Eupolyphaga sinensis*, *Eupolyphaga sinensis*, donkey-hide gelatin, chicken gizzard lining, centipede, bezoar, musk, leech, and earthworm.

6. The self-assembled nanomaterial of traditional Chinese medicine according to claim 3, characterized in that, The conjugable target agent with a carboxyl or amino group at the end is one or more of folic acid and its derivatives, hyaluronic acid and its derivatives, arginine-glycine-aspartic acid tripeptide and its derivatives.

7. The self-assembled nanomaterials of traditional Chinese medicine according to any one of claims 1 to 3, characterized in that, The self-assembled nanomaterials of traditional Chinese medicine have one or more of the following functions: T1-weighted nuclear magnetic resonance imaging, photoacoustic imaging, and photothermal therapy.

8. The method for preparing self-assembled nanomaterials of traditional Chinese medicine as described in claim 1, characterized in that, The preparation method includes the following steps: The mineral-based traditional Chinese medicine, other traditional Chinese medicines, and water are mixed, then heated to react, and finally filtered, centrifuged, dialyzed, and dried to obtain the self-assembled nanomaterials of the traditional Chinese medicine. The other traditional Chinese medicines mentioned are one or two of the following: plant-derived traditional Chinese medicines and animal-derived traditional Chinese medicines.

9. The method for preparing self-assembled nanomaterials of traditional Chinese medicine as described in claim 3, characterized in that, The preparation method includes the following steps: mixing mineral-based traditional Chinese medicine, other traditional Chinese medicine, and water, then heating and reacting the mixture, followed by filtration, centrifugation, dialysis, and drying to obtain self-assembled nanomaterials; the other traditional Chinese medicine is one or two of plant-derived and animal-derived traditional Chinese medicine. The self-assembled nanomaterials and the targeting agent are coupled through a chemical reaction to obtain the self-assembled nanomaterials of traditional Chinese medicine.

10. The preparation method according to claim 8 or 9, characterized in that, Before use, the mineral-based Chinese medicine and other Chinese medicines shall be pulverized to a size ≤1.5cm. After the heating reaction is completed, filtration is performed. The filter paper used for filtration has a pore size ≥1μm, which is smaller than the size of the pulverized mineral Chinese medicine and other Chinese medicines.

11. The preparation method according to claim 8 or 9, characterized in that, The mass ratio of the mineral-based Chinese medicine to other Chinese medicines is 1:0.1 to 1:10; And / or, the ratio of the mass of water to the total mass of all Chinese medicinal herbs is 1 to 100:1; And / or, the preparation method further includes, before the heating reaction, Includes the following steps: Let stand for 1 to 60 minutes; And / or, the temperature of the heating reaction is 70–120°C, and the heating reaction time is 10–200 min.

12. The preparation method according to claim 8 or 9, characterized in that, The centrifugation process includes: performing 2 to 8 centrifugations, with the first centrifugation speed being 1000 to 7000 rpm, each subsequent centrifugation speed being higher than the previous one, and the last centrifugation speed being 8000 to 15000 rpm; each centrifugation time being 5 to 50 minutes.

13. The preparation method according to claim 12, characterized in that, The difference between the centrifugation speed of the subsequent centrifugation and that of the previous centrifugation is 500 to 6000 rpm.

14. The preparation method according to claim 8 or 9, characterized in that, The centrifugation process includes: performing 3 to 5 centrifugations. The first centrifugation speed is 2000 to 5000 rpm, the difference between the centrifugation speed of each subsequent centrifugation and the previous centrifugation speed is 1000 to 5000 rpm, and the final centrifugation speed is 9000 to 12000 rpm.

15. The preparation method according to claim 8 or 9, characterized in that, The supernatant obtained by centrifugation was placed in a dialysis bag for dialysis. The molecular weight cutoff of the dialysis bag was 1000-10000 Da, and the dialysis time was 10-100 h.

16. The preparation method according to claim 8 or 9, characterized in that, The drying process is either freeze-drying or vacuum drying. The freeze-drying temperature is -40 to -100℃, the vacuum degree is 1 to 100 Pa, and the time is 12 to 100 h.

17. The preparation method according to claim 9, characterized in that, The chemical reaction is an activation coupling method of EDC and NHS; The coupling process includes: dispersing the self-assembled nanomaterials in 2-(N-morpholino)ethanesulfonic acid buffer, sequentially adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for activation; then adding a coupling target agent with a terminal carboxyl or amino group, reacting at room temperature, and finally purifying by centrifugation to obtain the self-assembled nanomaterials of traditional Chinese medicine.

18. The preparation method according to claim 9, characterized in that, The mass ratio of the self-assembled nanomaterial to the coupled target agent with a carboxyl or amino group at the end is 1:0.1 to 10.

19. The application of the self-assembled nanomaterials of traditional Chinese medicine as described in any one of claims 1 to 7, characterized in that, The application is selected from one or more of the following: Used in the preparation of nuclear magnetic resonance contrast agents; Used in the preparation of photoacoustic imaging contrast agents; Used to prepare drugs for the diagnosis and / or treatment of cancer diseases.

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