WO2016015173A1 - Tumor treatment method for blocking tumor vasculature by means of nanomaterial and external radiation source - Google Patents

Abstract

Provided is an application of a nanomaterial to the preparation of a tumor vasculature blocking agent, said nanomaterial having all of the following properties: (1) the surface is hydrophilic; (2) the surface has electronegativity; (3) the size of the nanomaterial is 50-250 nm, and the material is rigid, such that it is not prone to passing through the hole in the wall of a tumor vessel and becoming stuck in said hole; (4) the nanomaterial can absorb external radiation energy, and internally convert same to thermal energy, and accumulate said thermal energy, causing the morphological structure of said nanomaterial to change dramatically. During treatment, the described nanomaterial is injected into a tumor-bearing organism, then radiation is applied to the tumor section, causing the nanomaterial to absorb the radiation energy and its internal temperature to rise rapidly; when the temperature has risen to a certain level, the morphological structure of the nanomaterial changes (explosion or dramatic volume expansion), thereby causing the morphological structure or function of the tumor vascular endothelial cells to change, destroying the tumor vasculature.

Description

 Tumor treatment method for tumor vascular occlusion by nano material and external radiation source

 The invention belongs to the field of medicine, and particularly relates to a tumor treatment method for realizing tumor vascular blockage by using nano materials and an external radiation source.

Background technique

 In recent years, the incidence and mortality of cancer have been on the rise. There are as many as 15 million new cancer patients worldwide each year, which seriously threatens human health. The traditional three major tumor treatment methods, namely, surgery, radiotherapy and chemotherapy, are aimed at killing tumor cells. However, with the progress of cancer research, people gradually realize that there are not only tumor cells in the tumor tissue, but also abundant tumor neovascularization. Tumor tissue is actually a complete ecosystem formed by tumor cells and tumor neovascularization. If a tumor tumor grows up rapidly, it must rely on the formation of tumor neovascularization, and the tumor blood vessel is the main transfer channel of cancer cells. Therefore, blocking or destroying the tumor blood vessels can cut off the nutrient supply of tumor tissue. Dead "tumors, on the other hand, will block the spread and metastasis of cancer cells to the greatest extent.

 There are large differences in anatomy and pathology between solid tumors and normal tissues. The normal vascular growth cycle is one year, usually consisting of a three-layer membrane of intima, media, and adventitia. The growth cycle of tumor blood vessels is 4 days, and there is only a thin inner membrane in the structure. Due to the large gap and incomplete structure of the endothelial cells, the tumor blood vessels contain a large number of nanometer-sized pores, and the plasma can pass through. Out. In addition, the tumor is rich in blood supply, and the tumor blood vessels also have blood flow and high blood pressure, which is three times the normal blood pressure.

Based on the differences between normal blood vessels and tumor blood vessels, tumor treatment methods that specifically destroy tumor blood vessels can be designed for tumor blood vessel specificity, similar to tumor vascular disrupting agents (VDAs). VDAs are drugs for the purpose of treating cancer by selectively destroying neovascularization of malignant tumors. They target the tumor blood vessels that have been formed, and cause apoptosis of endothelial cells by rapidly changing the shape of endothelial cells or damaging endothelial cells, exposing the basement membrane, and significantly affecting Capillary blood flow, thereby disintegrating the vascular system inside the solid tumor, leading to tumor ischemia and extensive necrosis. The most studied of these drugs are small molecule VDAs, and a variety of small molecule VDAs have been introduced into preclinical experiments. However, most VDAs candidates have been found to have more serious side effects, such as ibsstatin, CA-1P, and MPC-6827, etc. Cardiovascular adverse reactions such as hypertension, tachycardia, bradyarrhythmia, atrial fibrillation, myocardial infarction have been reported and are dose-related, and these adverse reactions have limited their widespread clinical use. Invention disclosure

 One of the objects of the present invention is to provide a new use of nanomaterials.

 The new use of the nanomaterial provided by the present invention is as a tumor vascular blocker drug, combined with a matching radiation source for the purpose of treating tumors.

 The nanomaterial of the present invention has all of the following properties: (1) the surface is hydrophilic so that it can be injected into the living body via the vein and carried to the tumor blood vessel; (2) the surface is electrically negative, so that It does not produce non-specific adsorption with normal cells and the inner wall of blood vessels of normal tissues, on the one hand, avoids aggregation of the nanomaterials in blood vessels, and on the other hand, it is not easy to adsorb to normal tissues or blood vessel walls, and avoids normal cells. And tissue damage; (3) nanomaterials have a particle size range of 50-250 nm, and the material is rigid (ie, not easily deformable), allowing it to become stuck when it is intended to pass through the pores of the tumor vessel wall; 4) The nanomaterial is capable of absorbing external radiant energy and internally converting it into thermal energy, and accumulating the thermal energy, causing the morphological structure of the nano material to change drastically, thereby causing a change in the morphological structure or function of the tumor vascular endothelial cell. , eventually destroying the tumor blood vessels.

 Possible ways for the morphological structure of the nanomaterial to change are as follows: For a nanomaterial having a single crystal/polycrystalline structure, when the accumulated temperature exceeds the phase transition temperature point of the material (40-150 ° C), the material volume is rapidly Expansion occurs; for nano-materials that are loose inside and contain bound water, the accumulated temperature exceeds 10 CTC. The water inside the evaporation produces water vapor. When the vapor pressure accumulates to a certain extent, the micro-explosion occurs beyond the binding force of the material; or the material sudden heat occurs directly. Explosion; all of the possible pathways can act on tumor blood vessels, and ultimately achieve the effect of highly effective treatment of tumors.

The nanomaterial of the present invention is obtained by modifying the water-soluble and electronegative properties of the host material. The host material is a nano material having the ability to absorb external radiation (infrared, visible light, X-ray, alternating magnetic field, radio frequency, etc.) and accumulating heat, and may be a single metal or a non-metal material, or a composite material, typically Materials such as: fullerene nanoparticles, embedded metal fullerene nanoparticles, noble metal nanoclusters (including gold nanoclusters, silver nanoclusters, platinum nanoclusters), ferroferric oxide nanomaterials, zinc oxide nanomaterials Materials, zinc sulfide nanomaterials, Cadmium selenide nanomaterials, cadmium telluride nanomaterials, rare earth oxide nanomaterials, carbon quantum dots, carbon nanohorns, carbon nanotubes, graphene, and the like, and any combination thereof.

The nano material according to the present invention may specifically be Au@S i0 ₂ nano particles (ie, a negatively charged Si ₂ layer coated with Au nanoparticles, a Zeta potential of -34±0. 4 eV), and a water-soluble rich Receptor nanoparticles, water-soluble metal fullerene nanoparticles, Fe ₃ 0 ₄ nanoparticles coated with a hydrophilic layer, and the like.

 The externally radiated energy sources of the present invention include radio frequency (also known as radio waves), microwaves, infrared light, visible light, laser light, X-rays, alternating magnetic fields, and the like, and any combination thereof. The energy form is as short as possible short-wave pulse radiation, such as laser pulse, electromagnetic pulse (including radio frequency pulse), etc., in order to facilitate the rapid absorption of the radiant energy by the nano material to make the temperature rise rapidly, and then before the energy is lost through heat exchange or the like. Deformation (expansion or explosion) occurs quickly, releasing energy. The pulse energy source can also reduce the damage of the external radiation to the living body.

 The nanomaterial used in the present invention needs to be water-soluble, can be injected into a living body via a vein, and exerts a therapeutic effect as blood is circulated to the tumor blood vessel. To achieve this, the host material described above is typically water soluble to make it more biocompatible. For example, the surface of the host material is modified with one or more hydrophilic chemical groups, such as: hydroxyl group, amino group, sulfhydryl group, carboxyl group, etc., and any combination thereof; and a hydrophilic inorganic substance such as dioxide may be coated on the surface of the host material. Silicon or directly encapsulating the host material with hydrophilic biological small molecules such as amino acids, peptide chains, etc., or supporting the host material, such as liposome, cell membrane, etc., as a carrier by means of a biocompatible carrier material, or The assembly forms a form of a water-soluble supramolecular system. All of the above modification methods can be modified in accordance with the methods disclosed in the prior art.

 The nanomaterial used in the present invention should have a certain electronegativity, and the electronegativity modification method includes directly introducing an electronegative functional group such as a hydroxyl group, a carboxyl group and an amino acid through a covalent bond on the surface of the host material, or The electronegative carrier is non-covalently coated, such as a hydroxylated silica layer, a carboxylated carbon film, a peptide chain, and the like. The material is not easily phagocytosed by cells or adsorbed by normal tissues and blood vessels, and even if it is contacted, it is quickly bounced off, which can reduce damage to normal cells or biological tissues when the nanomaterial is deformed (expanded or exploded).

 Of course, in order to simplify the modification step, water-solubilization modification and negative-electrode modification of the host material can also be simultaneously achieved by selecting an appropriate modification method.

The size distribution of the nanomaterials used in the present invention should generally be in the range of 50-250 nm. Moreover, the material needs to have a certain rigidity (not easily deformed), so that it cannot pass through the nanopores between the tumor vascular endothelial cells, and can be closely adhered to the pores of the blood vessel wall by the pressure difference between the blood vessels inside and outside the tumor, so as to facilitate Nanomaterials are more effective in damaging tumor vascular endothelial cells when they are rapidly deformed (expanded or exploded).

 Another object of the present invention is to provide a pharmaceutical kit for treating a tumor.

 The kit of parts provided by the present invention consists of the above-described nanomaterials and means for providing a source of radiant energy matched thereto.

 The drug kit can be specifically gold nanoclusters + pulsed laser; fullerene nanoparticles + pulsed laser; metal fullerene nanoclusters + radio frequency; ferroferric oxide nanoparticles + alternating magnetic field.

 Regarding the above energy source, those skilled in the art can make a reasonable choice according to the teachings of the prior art, depending on the absorption properties of the host material in the nanomaterial for different radiant energies.

 A third object of the present invention is to provide a method for treating tumors based on the above-described nanomaterials which can specifically block tumor blood vessels.

 The method for treating tumors provided by the invention comprises the following steps:

 1) administering an effective dose of nanomaterial to a tumor-bearing organism in need of treatment;

 2) Irradiating the tumor site of the organism with a source of radiant energy that matches the nanomaterial.

 The "effective dose" as used in the present invention means an amount sufficient to efficiently deliver an active ingredient for treating a disease to an organism when the biological nanomaterial is administered by the method of the present invention.

 The organism described in the present invention refers to a mammal including a human.

 The irradiation according to the present invention is irradiated with nanomaterials for 0-1 h and irradiated for a period of time, for example, 10 min-1 h.

 The preferred method of injection of the above tumor treatment method is intravenous injection, which acts directly in the blood without osmosis, and the amount of the medicament used is small and the curative effect is high.

The nanomaterial used in the present invention treats tumor by specifically blocking tumor blood vessels, and utilizes the difference between blood vessels in the tumor tissue and normal blood vessels, and thus has broad spectrum and is applicable to all solid tumors, including: liver cancer, lung cancer, colorectal Cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, laryngeal cancer, liver cancer, cholangiocarcinoma, cervical cancer, uterine cancer, testicular cancer, meningioma , skin cancer, melanoma, sarcoma (such as fiber Uterine sarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, etc.

 The nanomaterial used in the present invention is a specific nanoparticle having a size of 50-250 nm, which can be deformed after absorbing a specific wavelength of radiant energy (infrared, visible light, X-ray, alternating magnetic field, radio frequency, etc.). Or explosion). When used, the nanomaterial is intravenously injected into the tumor-bearing organism and transported to the tumor via the blood. Due to the large amount of nanoporosity between the tumor vascular wall cells, the smaller size nanomaterials will enter the tumor through these voids, but the nanomaterials of suitable size and rigidity (ie, nanomaterials having the properties described in the present invention) It is trapped by these nanopores and is retained in the gaps of these tumor vascular cells for a long time due to the pressure difference between the blood vessels and the inside. At this time, a suitable wavelength of radiation is applied to the vicinity of the tumor, so that the nanomaterial absorbs the radiant energy and the internal temperature rapidly rises. When the temperature accumulates to a certain extent, the morphology and/or structure of the nanomaterial changes (such as an explosion or The volume is exploding). Because nanomaterials are closely contacted with blood vessel pores by intravascular and extravascular blood pressure, the rapid deformation of the nanomaterials causes damage and apoptosis of peripheral tumor vascular endothelial cells, exposing the basement membrane, thereby significantly affecting capillary blood flow and destroying the transport of tumor blood vessels. The blood function causes the tumor cells to lose their nutrient supply and starve to death.

 The nanomaterials used in the present invention are rapidly deformed (rapidly expanding or exploding) when absorbed, and if they are located in normal blood vessels or organs, since the surface electronegativity of the nanomaterials repels them away from these blood vessels and organ surfaces, Normal blood vessels or biological organs have little damage; if nanomaterials pass through the interstitial cell space located in tumor blood vessels, when the absorbed radiant energy rapidly deforms (rapid expansion or explosion), it will seriously damage nearby endothelial cells and change the morphology of peripheral tumor vascular endothelial cells. And function, leading to cell apoptosis, exposure of the basement membrane, and finally the loss of nutrients in the tumor vascular damage, rapid starvation of tumor cells.

 The nanomaterial used in the present invention treats a tumor by specifically blocking tumor blood vessels, causing a large number of tumor cells to starve or be damaged, and the tumor cells after death are absorbed by the body, which will stimulate the body's immune response and further produce an anti-tumor effect. .

The nanomaterial used in the invention treats the tumor by specifically blocking the tumor blood vessel, and the administration method is intravenous injection, the medicine directly functions in the blood, does not need to penetrate, and the dosage is small, the curative effect is high, and the medicine can be delivered to the living body. The whole body, therefore, can not only treat primary tumors, but also treat late tumors that have spread throughout the body. Since the above treatment method is purely physical damage to the tumor, there is no problem of drug resistance;

 The nanomaterial used in the present invention treats tumor by specifically blocking tumor vasculature, rapidly damages tumor vascular endothelial cells, induces apoptosis of tumor vascular endothelial cells, exposes basement membrane, vascular damage, and then tumor cells die due to lack of nutrition, the whole The process takes place within 2-5 hours and thus has the effect of rapidly treating the tumor.

 The tumor treatment method provided by the invention utilizes nano materials to block tumor blood vessels with high selectivity, has strong specificity, large power, direct and rapid, and is safe and efficient, and has little damage to normal tissues, and is a promising new type of tumor treatment. method.

In summary, the method for treating tumors provided by the present invention has the following advantages: (1) has broad spectrum and is effective for any solid tumor; (2) water-soluble nanomaterial directly acts on tumor blood vessel wall (endothelial cells) through blood circulation The drug delivery method is simple and the targeting effect is strong; (3) The therapeutic effect has a magnifying effect. Since one vascular endothelial cell supports the growth of 50-100 tumor cells, the effect of inhibiting the tumor will be multiplied after destroying the tumor blood vessel; 4) The side effects are small, only for tumor blood vessels with pores, and the blood vessels of normal tissues are not easily damaged due to different structures; (5) Physical destruction of tumor blood vessels, no drug resistance problem; (6) Tumor metastasis is also dependent on blood vessels. Therefore, it can inhibit tumor metastasis and spread; (7) The action time is short. Once the tumor blood vessel bleeds or blocks the nutrient supply, the tumor necrosis occurs for less than 2 hours _. (8) The treatment is not limited to the location of the tumor. And depth, and also have a therapeutic effect on advanced tumors; (9) protection and activation of immunity; (10) stronger town Effect. DRAWINGS

 1 is a schematic diagram of nanomaterial-specific destruction of tumor vascular endothelial cells, in which black particles refer to nanomaterials that satisfy the properties of the present invention, that is, can undergo deformation (rapid expansion or explosion); green particles refer to those that do not satisfy the present invention. Nanoparticles of the nature; the purple fraction refers to the action of the nanomaterials leading to apoptosis of tumor vascular endothelial cells.

Figure 2 is a transmission electron micrograph of water-soluble Au@SiO ₂ nanoparticles.

Figure 3 shows that the water-soluble Au@SiO ₂ solution rapidly generates a large number of bubbles under the action of 760 nm laser, indicating that some nanoparticles have exploded.

Figure 4 shows the treatment of tumors after 24 cycles of laser pulse using Au@SiO ₂ nanoparticles. Effect chart.

Figure 5 is an environmental scanning electron micrograph of tumor blood vessels that have been pulsed with Au@SiO ₂ nanoparticles and laser pulses (the yellow arrow in the figure is where the tumor is damaged). The best way to implement the invention

 The invention will now be described by way of specific examples, but the invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.

The nanomaterials having all of the properties described in the present invention used in the following examples are water-soluble Au@^-based SiO ₂ nanoparticles, and the preparation method thereof is referred to the literature (J. Am. Chem. Soc. 2014, 136, 7317). ; Angew. Chem. Int. Ed. 2010, 49, 3777 )

2 is a transmission photograph of the above water-soluble Au@SiO ₂ nanoparticles, the size of the particles is about 80-100 nm, and the thickness of the hydroxylated SiO ₂ coating layer is about 7 nm. Since the surface of the modified SiO ₂ has a hydroxyl group, the surface of the Au@SiO ₂ nanoparticle is electronegative, and the Zeta potential value is determined to be -34 ± 0.4 eV. Example 1: Au@SiO ₂ aqueous solution produced bubbles under the action of laser

As shown in Figure 3, Au@SiO ₂ aqueous solution (concentration 25 mg/mL) produced a large number of bubbles under the action of 760 nm laser pulse (average power 500 mW, 16 W/cm ² ), indicating that the laser pulse was passed through the gold nanoparticles. Under the action, heat can be generated. Because the outer layer is covered with a layer of heat-insulating SiO ₂ , it does not exchange energy with the outside world. When the internal heat accumulates to a certain extent, an explosion occurs. The laser-treated sample no longer explodes. Example 2: Tumor treatment and effect verification of Au@SiO ₂ nanomaterials under laser irradiation

1. Establish a tumor-bearing mouse model:

The ascites of mice inoculated with H22 hepatoma cell line were intraperitoneally, and the supernatant was counted by centrifugation, and a cell suspension having a cell concentration of 10 ⁷ /mL was inoculated to the right thigh of each mouse. After 5-7 days of growth, the tumor size was about 5 mm.

 2, animal experiments in vivo magnetic resonance imaging and tumor treatment experiments:

An aqueous solution of Au@SiO _{2 was} injected through the tail vein (concentration 25 mg/mL, where Au concentration was 10 Mg/kg) 200 μL in tumor-bearing mice. The 760 nm laser pulse was applied to the tumor site for 30 min, and the imaging of tumor tissue, kidney and liver at each time point was collected.

 Fig. 4 is a photograph of a tumor after 24 hours of treatment using the method of the present invention. After treatment, the inside of the tumor is hollow, external scab, and most of the tumor tissue is digested by the body.

Figure 5 shows an environmental scanning electron micrograph of tumor blood vessels after radiofrequency exposure for 2 days after treatment with Au@SiO ₂ . It can be observed that after exposure to Au@SiO ₂ nanomaterials and laser pulses, tumor angiogenesis has a large range of endothelial cell necrosis. The exposed vascular basement membrane indicates that the explosion impact of the Au@SiO^ft rice material under the action of laser can quickly and effectively destroy the tumor blood vessels, and finally "starved" the tumor tissue to achieve efficient and rapid treatment. Industrial application

 The tumor treatment method provided by the invention utilizes nano materials to block tumor blood vessels with high selectivity, has strong specificity, large power, direct and rapid, and is safe and efficient, and has little damage to normal tissues, and is a promising new type of tumor treatment. method.

Claims

Rights request  1. The use of a nanomaterial in the preparation of a tumor vascular blocker;  The nanomaterial has all of the following properties: (1) the surface is hydrophilic so that it can be injected into the living body via the vein and carried to the tumor blood vessel; (2) the surface is electronegative, making it and normal cells And the normal tissue of the inner wall of the blood vessel is repulsive; (3) the nanomaterial has a particle size ranging from 50 to 250 nm, and the material is rigid so that it is stuck when passing through the pore of the tumor blood vessel wall, due to the tumor The pressure inside and outside the blood vessel is poor and stays there for a long time; (4) the nanomaterial is capable of absorbing external radiant energy and internally converting it into thermal energy, and simultaneously accumulating the thermal energy, causing the morphological structure of the nano material to change drastically, thereby causing the morphological structure or function of the tumor vascular endothelial cell. change.  2. The use according to claim 1, wherein: the nanomaterial is obtained by water-soluble and electronegative modification of at least one of the following materials: fullerene nanoparticles, embedded metal fullerene nanoparticles , precious metal nanoclusters, ferroferric oxide nanomaterials, zinc oxide nanomaterials, zinc sulfide nanomaterials, cadmium selenide nanomaterials, cadmium telluride nanomaterials, rare earth oxide nanomaterials, carbon quantum dots, carbon nanohorns, carbon Nanotubes and graphene.  3. The use according to claim 1 or 2, characterized in that the energy source for providing the external radiant energy is selected from at least one of the following: radio frequency, microwave, infrared light, visible light, laser light, X-ray and alternating magnetic field.  4. Use according to any one of claims 1-3, characterized in that the energy form of the external radiated energy is ultrashort time pulsed radiation, the energy source of which is preferably: a laser pulse, an electromagnetic pulse. The use according to any one of claims 1 to 4, wherein the nano material is a composite nanoparticle formed by coating a Au nanoparticle with a negatively charged SiO ₂ layer, that is, Au@SiO^ft rice particles. , water-soluble fullerene nanoparticles, water-soluble metal fullerene nanoparticles or Fe ₃ O ₄ nanoparticles coated with a hydrophilic layer.  6. A pharmaceutical kit for treating a tumor, comprising the nanomaterial of any of claims 1-5 and means for providing a source of radiant energy that matches the nanomaterial. The drug kit according to claim 6, wherein: the nano material is Au@SiO ₂ nanoparticles, and the radiant energy source matched thereto is a pulsed laser; the nano material is water-soluble fullerene nanometer. a particle, the radiant energy source matched thereto is a pulsed laser; The rice material is a water-soluble metal fullerene nano-cluster, and the matching radiant energy is radio frequency; the nano-material is Fe ₃ O ₄ nano-particle coated with a hydrophilic layer, and the matching radiation source is alternating magnetic field.  8. A method of treating a tumor comprising the steps of:  1) administering to the tumor-bearing organism in need of treatment an effective amount of the nanomaterial of any one of claims 1-5;  2) Irradiating the tumor site of the organism with a source of radiant energy that matches the nanomaterial.  9. The method according to claim 8, wherein: said organism is a mammal including a human; said injection is by intravenous injection;  The irradiation is for a period of time after intravenous injection of the nano material for 0-1 h, for example 10 min- 1 h.  The application according to any one of claims 1 to 5, the pharmaceutical kit according to any one of claims 6 to 7, and the treatment method according to claims 8-9, wherein: the tumor is a solid tumor; The solid tumor includes: liver cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, laryngeal cancer, liver cancer, bile duct Cancer, cervical cancer, uterine cancer, testicular cancer, meningioma, skin cancer, melanoma, sarcoma.