CN106975078B - Application of nano material containing gadolinium polytungstate as sensitizer - Google Patents

Application of nano material containing gadolinium polytungstate as sensitizer Download PDF

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CN106975078B
CN106975078B CN201710210288.9A CN201710210288A CN106975078B CN 106975078 B CN106975078 B CN 106975078B CN 201710210288 A CN201710210288 A CN 201710210288A CN 106975078 B CN106975078 B CN 106975078B
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赵宇亮
雍媛
杜江锋
谷战军
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National Center for Nanosccience and Technology China
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Abstract

The invention provides an application of a nano material containing gadolinium polytungstate as a sensitizer, wherein the gadolinium polytungstate in the nano material containing gadolinium polytungstate can generate an oxidation-reduction reaction with glutathione, so that the content of the glutathione is reduced, and the internal sensitization is further realized. The invention also provides a gadolinium polytungstate composite nanomaterial consisting of gadolinium polytungstate, chitosan and small interfering RNA (ribonucleic acid), the gadolinium polytungstate composite nanomaterial has excellent radiosensitization performance, can absorb a large amount of X rays to generate Reactive Oxygen Species (ROS), and can also reduce the expression of HIF-1 alpha at a tumor part and inhibit the DNA self-repair of double-strand break; and most importantly, the gadolinium polytungstate can perform redox reaction with Glutathione (GSH) in vivo to reduce the content of glutathione in vivo, so that more effective active oxygen substances are generated, and effective combination of internal sensitization and external sensitization is realized to achieve an ideal radiosensitization effect.

Description

Application of nano material containing gadolinium polytungstate as sensitizer
Technical Field
The invention belongs to the technical field of nano materials and radiosensitizing drugs, relates to an application of a nano material containing gadolinium polytungstate as a sensitizer, and particularly relates to an application of a chitosan modification-based gadolinium polytungstate composite nano material as a sensitizer.
Background
Radiotherapy (RT), one of the most widespread methods for treating cancer in the clinic, is based on the principle that high-intensity ionizing radiation (X-rays or gamma-rays) with very strong penetrating power is used to inhibit tumor proliferation. During radiation therapy, ionizing radiation interacts with water or oxygen inside the tissue to generate large amounts of Reactive Oxygen Species (ROS) that are cytotoxic to induce damage to cellular DNA duplexes. Therefore, in order to enhance ionizing radiation-induced cellular damage during radiation therapy, it is necessary that sufficient reactive oxygen species be produced to cause DNA double strand damage by reacting with DNA double strands, thereby greatly preventing DNA self-repair where the double strands have been damaged. However, radiation therapy still faces the problem of not being able to effectively eradicate hypoxic tumors. One of the key difficulties is the presence of large amounts of reduced Glutathione (GSH) in the cell, significantly reducing the effective production of reactive oxygen species and impairing the effectiveness of radiotherapy. Yet another key limitation is that depletion of oxygen during rapid proliferation of the tumor leads to local hypoxia of the tumor, resulting in radioresistance of hypoxic cells, increasing the chance of DNA self-repair and significantly reducing the effect of radiotherapy. Also, high energy ionizing radiation (X-rays or gamma rays) is used in the radiation therapy process, which may exceed the tolerance of normal cells, resulting in inevitable damaging effects on normal tissues. Therefore, in order to effectively improve the radiation therapy efficiency and maximize tumor elimination, it is very attractive to develop an ideal and novel radiation sensitization system that can simultaneously absorb a large radiation dose and consume glutathione in a local tumor in a concentrated manner, and effectively inhibit DNA self-repair through other means to jointly enhance the radiation therapy effect of hypoxic tumors, and achieve good treatment effect through multiple sensitization means.
In recent years, a great deal of research effort has been devoted to the development of radiosensitizers based on traditional nano-drugs as external radiosensitization means, such as: metal-based nanoparticles, quantum dots, superparamagnetic iron oxide, and non-metal-based nanoparticles, among others. These conventional nano-drug radiosensitizers can generate radiochemistry (radical or ionization) for the treatment of hypoxic tumors by scattering out X-rays/photons, compton electrons, positive and negative electron pairs, auger electrons, etc. under high-energy radiation in order to concentrate the radiation locally to the tumor and effectively improve the therapeutic effect of radiotherapy. However, the low renal clearance and biodegradability of these conventional radiosensitizers results in a long-term accumulation of the radiosensitizer in the body, thereby creating potentially unavoidable toxic side effects in the body. Also, these radiosensitizers are not able to eliminate intracellular glutathione, resulting in a substantial reduction in the production of ROS.
In addition to addressing many extrinsic sensitization regimes, a number of intrinsic sensitization strategies have been developed in recent years, derived from the intrinsic properties of the organism, to enhance the effectiveness of radiotherapy for hypoxic tumors. According to previous reports, hypoxia inducible factor-1 a (HIF-1a), a key transcription factor in hypoxic cells, is thought to interact with polymerase-1 (PARP-1) under hypoxic conditions, as a potent regulator of the major adaptive stress to hypoxic conditions during tumor growth. At the same time, previous related studies have also demonstrated that: decreasing HIF-1a expression in hypoxic cells promotes the degradation of polymerase, inhibits the self-reconstitution of damaged double-stranded DNA (DSB), and upregulates caspase-3 expression, thereby promoting apoptosis. Thus, HIF-1a siRNA has been widely used to mediate targeting of hypoxic tumors, to down-regulate HIF-1a expression in hypoxic cells, to overcome the radiotolerance of hypoxic tumors and to inhibit self-repair of double stranded DNA that has been broken during radiotherapy. However, to date, there are few reports on whether the nano-materials can be used cooperatively by an external sensitization mode and an internal sensitization mode to achieve the effect of improving radiosensitization simultaneously, and thus the research on the aspect is of great significance.
Disclosure of Invention
In view of the above problems in the prior art, the present invention aims to provide a use of a nano material containing gadolinium polytungstate as a sensitizer. The invention discovers a new characteristic of the poly-gadolinium tungstate as a sensitizer, thereby realizing the cooperative coordination of external sensitization and internal sensitization and improving the radiosensitization effect in practical application.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of a nano material containing gadolinium polytungstate as a sensitizer, wherein the gadolinium polytungstate in the nano material and glutathione are subjected to oxidation-reduction reaction, so that the content of glutathione is reduced.
The gadolinium polytungstate in the gadolinium polytungstate-containing nano material can not only reduce the level of glutathione, but also well realize the radiosensitization effect.
According to the invention, the in-situ injection mode is adopted for the tumor part of the mouse, and then the local X-ray irradiation is carried out on the tumor for detection, so that the tumor has a certain inhibiting effect.
The invention creatively discovers that the gadolinium polytungstate can generate oxidation-reduction reaction with glutathione (such as in human bodies) to reduce the content and the level of the glutathione, so that when the gadolinium polytungstate is used as a sensitizer for treating diseases, the consumption of the glutathione to active oxygen substances can be reduced by reducing the level of the glutathione in cells, more effective active oxygen substances are generated, the DNA double-strand damage is more serious, and the internal sensitization effect is generated. In addition, the ray killing effect can be improved under the action of X rays, and the effect of external sensitization is achieved, so that the synergistic effect of internal sensitization and external sensitization is achieved.
The invention discloses a nano material containing gadolinium polytungstate, which is characterized in that: can be a poly-gadolinium tungstate nano material and can also be a poly-gadolinium tungstate composite nano material.
Preferably, the nano material is a gadolinium polytungstate nano material.
In the invention, the gadolinium polytungstate belongs to a crystal substance in polyoxometallate.
As a preferable technical scheme of the nano material containing the gadolinium polytungstate, the nano material is a gadolinium polytungstate composite nano material.
Preferably, the gadolinium polytungstate composite nanomaterial comprises gadolinium polytungstate and a modifier for modifying the gadolinium polytungstate.
Preferably, the modifier includes any one or a combination of at least two of Chitosan (Chitosan), liposome or aqueous solution positively charged high molecular polymer, but is not limited to the above-listed modifiers, and other positively charged modifiers commonly used in the art can also be used in the present invention.
Preferably, the gadolinium polytungstate composite nanomaterial comprises gadolinium polytungstate and chitosan modified with the gadolinium polytungstate, and the gadolinium polytungstate is used as an inner core, and the biopolymer chitosan with good biocompatibility and degradability is modified on the outer layer of the gadolinium polytungstate.
As a preferred technical scheme of the gadolinium polytungstate composite nanomaterial, the gadolinium polytungstate composite nanomaterial further comprises a substance with negative electricity, wherein the substance with negative electricity is preferably Small interfering RNA (siRNA), and is further preferably Small interfering RNA expressed by hypoxia inducible factor-1 alpha (HIF-1 alpha for short).
In the invention, the small interfering RNA expressed by hypoxia inducible factor-1 alpha is abbreviated as HIF-1 alpha siRNA.
The small interfering RNA of the invention is a small interfering RNA which can regulate a certain protein in tumor cells. The HIF-1 alpha siRNA of the invention mainly aims at the regulation of hypoxia inducible factor 1 alpha (HIF-1 alpha) in hypoxia tumor cells, and can down regulate the expression of the HIF-1 alpha in the hypoxia cells.
Preferably, the small interfering RNA is loaded on the surface of chitosan-modified gadolinium polytungstate.
Preferably, the particle size of the gadolinium polytungstate composite nano material is 30nm to 40nm, such as 30nm, 33nm, 35nm, 36nm, 38nm or 40nm and the like.
Preferably, the hydrated particle size of the gadolinium polytungstate composite nanomaterial is 40nm to 60nm, such as 40nm, 42nm, 43nm, 45nm, 48nm, 50nm, 51nm, 53nm, 55nm, 58nm or 60 nm.
Preferably, the potential of the water system of the gadolinium polytungstate composite nano material consisting of gadolinium polytungstate and chitosan is +30mV to +60mV, such as +30mV, +32mV, +34mV, +35mV, +37mV, +38mV, +39mV, +41mV, +43mV, +45mV, +47mV, +50mV, +53mV, +55mV, +58mV or +60 mV.
Preferably, the potential of the water system of the gadolinium polytungstate composite nanomaterial consisting of gadolinium polytungstate, chitosan and small interfering RNA is +10mV to +30mV, such as +10mV, +12mV, +14mV, +15mV, +17mV, +18mV, +20mV, +22mV, +23mV, +25mV, +27mV, +28mV or +30 mV.
Preferably, the chitosan has an average molecular weight of 100 KD-300 KD (i.e. 100000-300000), such as 100KD,120KD,150KD,180KD,200KD,210KD,220KD,250KD,255KD,260KD,270KD,280KD or 300KD, etc., considering the problem of nano-size of the composite material obtained after the chitosan is compounded with the gadolinium polytungstate crystal, the average molecular weight is preferably 100KD-200KD, so that the size of the synthesized composite nano-material is not particularly large and the problem of difficult cell uptake is not caused.
The invention further introduces the small interfering RNA into the composite material, and then carries out X-ray irradiation on the tumor part of the mouse, so that the growth inhibition of the tumor is more obvious, and the tumor does not relapse, which shows that compared with single radiotherapy or single gene therapy, the composite material obtained after introducing the RNA can achieve better synergistic treatment effect of radiotherapy and gene therapy as a radiation sensitizer.
The invention also provides a preparation method of the gadolinium polytungstate composite nano material, which comprises the following steps:
(1) reacting poly gadolinium tungstate with chitosan by using an ion gel method to prepare chitosan-modified poly gadolinium tungstate, namely a poly gadolinium tungstate composite nano material consisting of poly gadolinium tungstate and chitosan.
In the invention, the gadolinium polytungstate and the chitosan are used for carrying out the ionic gel reaction to realize the compounding, thereby obtaining the nano-grade gadolinium polytungstate composite nano-material.
Preferably, the process for preparing the chitosan-modified gadolinium polytungstate in the step (1) comprises the following steps:
(A) dissolving chitosan in acetic acid to obtain an acetic acid solution of chitosan;
(B) preparing aqueous solution of gadolinium polytungstate by using gadolinium polytungstate crystal and water;
(C) mixing aqueous solution of gadolinium polytungstate with acetic acid solution of chitosan to form suspension, and removing impurities to obtain the chitosan-modified gadolinium polytungstate.
Preferably, the mass concentration of acetic acid in step (a) is 0.1% to 5%, such as 0.1%, 0.5%, 0.8%, 1.0%, 1.3%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.7%, 3.0%, 3.2%, 3.5%, 3.7%, 3.9%, 4.2%, 4.4%, 4.7%, or 5.0%, etc., in order to completely dissolve chitosan and not to excessively increase the acetic acid, preferably 0.1% to 3%.
Preferably, the mass ratio of the chitosan in the acetic acid solution of the chitosan in the step (C) to the gadolinium polytungstate in the aqueous solution of gadolinium polytungstate is 5-20, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, and in order to enable the chitosan-modified gadolinium polytungstate to form a composite nanomaterial with uniform morphology and appropriate size, which is beneficial to the uptake of cells, the mass ratio is preferably 7-15.
Preferably, the mixing in step (C) is performed by: dropwise adding aqueous solution of gadolinium polytungstate into acetic acid solution of chitosan.
Preferably, the mixing of step (C) is accompanied by stirring.
Preferably, the impurity removal process in the step (C) is as follows: the suspension was centrifuged, and the centrifuged product was dispersed in ultrapure water and dialyzed in a dialysis bag.
Preferably, the rotation speed of the centrifugation is 12000rpm, and the time of the centrifugation is preferably 30 min.
Preferably, the molecular weight of the dialysis bag is 2.0Dka, and the dialysis time is preferably 1 day.
As a preferred technical scheme of the method, the gadolinium polytungstate crystal in the step (B) is synthesized by a simple and environment-friendly recrystallization method, and is more preferably prepared by the following method:
dissolving tungstate in ultrapure water to form a uniform and transparent aqueous solution A;
adjusting the pH value of the aqueous solution A to 6.50-8.50 by using an acidic compound under the stirring action to obtain a solution B;
adding gadolinium salt aqueous solution into the solution B under the stirring action to form aqueous solution C;
IV, heating and stirring the aqueous solution C until a uniform and transparent aqueous solution D is obtained;
and V, cooling to precipitate a gadolinium polytungstate crystal.
In this preferred embodiment, the stirring in step II and step III may be magnetic stirring, independently.
In the preferred embodiment, the stirring in step II and step III is carried out at room temperature (15-35%).
The invention provides a method for synthesizing gadolinium polytungstate by adopting a classical simple and environment-friendly recrystallization method, and the solution of gadolinium polytungstate prepared by the method not only contains sodium tungstate (Na)2WO4·2H2O) and also contains a small amount of NaCl, and more pure and larger gadolinium polytungstate crystals can be obtained by a recrystallization method.
Preferably, the tungstate salt in step I is sodium tungstate.
Preferably, the chemical composition of the sodium tungstate is Na2WO4·2H2O。
Preferably, the concentration of tungstate in the aqueous solution A in the step I is 300 mg/mL-500 mg/mL, for example: 300mg/mL, 310mg/mL, 320mg/mL, 330mg/mL, 350mg/mL, 370mg/mL, 390mg/mL, 410mg/mL, 430mg/mL, 450mg/mL, 480mg/mL, or 500mg/mL, etc.
Preferably, the acidic compound in step II comprises hydrochloric acid, acetic acid, nitric acid and the like, and glacial acetic acid is preferred.
In this preferred embodiment, step ii adjusts the pH of the aqueous solution a to 6.50-8.50, such as 6.50, 6.55, 6.60, 6.65, 6.70, 6.75, 6.80, 6.85, 6.90, 6.95, 7.00, 7.05, 7.10, 7.15, 7.20, 7.25, 7.30, 7.35, 7.40, 7.45, 7.50, 7.55, 7.60, 7.65, 7.70, 7.75, 7.80, 7.85, 7.90, 7.95, 8.00, 8.05, 8.10, 8.15, 8.20, 8.25, 8.30, 8.35, 8.40, 8.45, or 8.50.
Preferably, the gadolinium salt in the gadolinium salt aqueous solution in the step III is gadolinium chloride.
Preferably, the chemical composition of the gadolinium chloride is GdCl6·6H2O。
Preferably, the concentration of gadolinium salt in the gadolinium salt aqueous solution in step III is 300g/mol to 600g/mol, such as 300g/mol, 320g/mol, 330g/mol, 350g/mol, 360g/mol, 380g/mol, 400g/mol, 420g/mol, 440g/mol, 460g/mol, 490g/mol, 510g/mol, 530g/mol, 550g/mol, 570g/mol, 590g/mol or 600g/mol, etc.
Preferably, the heating temperature in step IV is 70-90 deg.C, such as 70 deg.C, 72 deg.C, 75 deg.C, 77 deg.C, 79 deg.C, 80 deg.C, 82 deg.C, 84 deg.C, 85 deg.C, 87 deg.C or 90 deg.C.
As a preferable technical scheme of the method, the method further comprises the following step (2) after the chitosan-modified gadolinium polytungstate is obtained in the step (1):
and loading a substance with negative electricity on the surface of the chitosan-modified gadolinium polytungstate by utilizing the interaction of positive charges and negative charges, thereby obtaining the gadolinium polytungstate composite nano material consisting of the gadolinium polytungstate, the chitosan and the substance with negative electricity.
As the preferred technical scheme of the method of the invention, the negatively charged substance comprises small interfering RNA, preferably small interfering RNA expressed by hypoxia inducible factor-1 alpha. The small interfering RNA of the invention is a small interfering RNA which can regulate a certain protein in tumor cells. The HIF-1 alpha siRNA of the invention mainly aims at the regulation of hypoxia inducible factor 1 alpha (HIF-1 alpha) in hypoxia tumor cells, and can down regulate the expression of the HIF-1 alpha in the hypoxia cells.
Preferably, the loading process in step (2) is as follows: preparing aqueous solution of chitosan modified gadolinium polytungstate, mixing with negatively charged substances, shaking, and standing.
The surface of the chitosan-modified gadolinium polytungstate can be loaded with a large amount of negatively charged substances (such as negatively charged small interfering RNA), and the mass ratio of the chitosan-modified gadolinium polytungstate to the negatively charged substances in the aqueous solution of the chitosan-modified gadolinium polytungstate is preferably 0.1-500, such as 0.1, 1, 10, 30, 50, 80, 100, 120, 150, 180, 210, 230, 240, 260, 280, 300, 310, 330, 350, 370, 390, 400, 420, 450, 460, 480, 490, or 500.
Preferably, the mass ratio of the chitosan-modified gadolinium polytungstate to the small interfering RNA in the aqueous solution of the chitosan-modified gadolinium polytungstate is preferably 0.1-500, and in order to enable the small interfering RNA with negative charges to be completely loaded and successfully released, the mass ratio is preferably 0.1-300, and further preferably 1-210.
Preferably, the mixing of the aqueous solution of chitosan-modified gadolinium polytungstate with the negatively charged substance is carried out in a nuclease-free aqueous solution.
As a further preferable embodiment of the method of the present invention, the method comprises the steps of:
(1) preparing a gadolinium polytungstate crystal:
i, mixing Na2WO4·2H2Dissolving O in ultrapure water to form a uniform and transparent aqueous solution A;
regulating the pH value of the aqueous solution A to 6.50-8.50 by using glacial acetic acid under the action of magnetic stirring at the temperature of 15-35 ℃ to obtain a solution B;
III, under the magnetic stirring action at 15-35 ℃, GdCl6·6H2Adding the O aqueous solution into the solution B to form an aqueous solution C;
finally cooling to precipitate a gadolinium polytungstate crystal;
(2) preparing chitosan modified gadolinium polytungstate:
(A) dissolving chitosan in glacial acetic acid to obtain a glacial acetic acid solution of chitosan;
(B) preparing aqueous solution of the gadolinium polytungstate by using the gadolinium polytungstate crystal obtained in the step (1) and water;
(C) mixing aqueous solution of gadolinium polytungstate with glacial acetic acid solution of chitosan to form suspension, centrifuging the suspension at 12000rpm for 30min, and dialyzing the centrifuged product in a dialysis bag with molecular weight of 2.0Dka for 1 day to obtain gadolinium polytungstate modified by chitosan;
(3) uniformly mixing small interfering RNA and aqueous solution of chitosan-modified gadolinium polytungstate in aqueous solution without nuclease, oscillating, and standing for 30min at the temperature of 15-35 ℃ to obtain the gadolinium polytungstate composite nano material.
The invention also provides the application of the nano material containing the gadolinium polytungstate as a carrier.
The invention also provides the application of the nano material containing the gadolinium polytungstate in tumor treatment.
Furthermore, the invention also provides the application of the poly-gadolinium tungstate composite nano material as a radiosensitizer in the cooperative treatment of tumor radiotherapy and gene therapy.
The gadolinium polytungstate composite nanomaterial has the functions of tumor multimode imaging (MRI/CT), radiotherapy and gene therapy as a radiosensitizer, and the functions can realize a synergistic treatment effect.
The "sensitizer" of the invention is also called radiosensitizer or radiosensitizer.
Compared with the existing radiosensitizer, the invention has the following beneficial effects:
(1) in the invention, in the nano material containing the gadolinium polytungstate, on one hand, the gadolinium polytungstate can absorb a large amount of high-energy X rays to generate Auger electrons or Compton electrons, and the electrons can react with water or oxygen in the organism environment (such as organism cells) to generate a large amount of Reactive Oxygen Species (ROS), thereby generating a damage effect on a cell DNA double chain and achieving the effect of radiotherapy; on the other hand, the gadolinium polytungstate can also perform an oxidation-reduction reaction with glutathione in cells to reduce the content level of the glutathione in the cells, thereby reducing the consumption of the glutathione on active oxygen substances, generating more effective active oxygen substances, causing the DNA double-strand damage to be more serious and achieving the enhanced radiotherapy effect.
(2) According to the invention, chitosan is further adopted to modify gadolinium polytungstate, small interfering RNA is loaded, and the expression of HIF-1 alpha in hypoxic tumor cells can be reduced by mediating the small interfering RNA to enter tumor parts, so that the self-repair of double-chain damaged DNA is prevented, and the synergistic treatment effects of the three effects of gadolinium polytungstate radiotherapy effect, gadolinium polytungstate gene treatment effect and reduction of intracellular HIF-1 alpha expression are finally achieved, so that the effects of internal sensitization and external sensitization are realized, and a very good radiotherapy effect is achieved.
(3) In the invention, a macromolecular compound chitosan is modified on the surface of the gadolinium polytungstate crystal, so that a composite nano material based on gadolinium polytungstate with uniform appearance can be formed, the biocompatibility of the material is improved, and the surface of a composite nano carrier formed by modifying gadolinium polytungstate with chitosan has very many positive charges, which is beneficial to the load of small interfering RNA with negative charges and successfully realizes the transfection and release of the small interfering RNA in hypoxic tumor cells.
(4) In the invention, the adopted macromolecular compound chitosan is a macromolecular compound, has good biodegradability, can be degraded in vivo, and reduces toxic and side effects on organisms, and the size of the residual gadolinium polytungstate crystal after degradation is very small, and can be quickly discharged out of the body through the kidney, thereby reducing the potential toxic and side effects caused by long-term accumulation of the nano material in the organisms.
Drawings
FIG. 1 is a schematic structural diagram of a radiosensitizer, gadolinium polytungstate composite nanomaterial of example 1;
FIG. 2 is a scanning electron microscope image of the chitosan-modified gadolinium polytungstate composite nanocarrier of step 2) of example 1;
FIG. 3 is a transmission electron microscope image of the chitosan-modified gadolinium polytungstate composite nanocarrier of step 2) of example 1;
FIG. 4 is an X-ray energy spectrum of the chitosan-modified gadolinium polytungstate composite nanocarrier obtained in step 2) of example 1;
FIG. 5 is an infrared spectrum of the chitosan-modified gadolinium polytungstate composite nanocarrier of step 2) of example 1, wherein GdW10@ CS represents a chitosan-modified gadolinium polytungstate composite nano-carrier;
FIG. 6 is a thermogravimetric analysis of chitosan, gadolinium polytungstate crystals and chitosan-modified gadolinium polytungstate composite nanocarriers of example 1, wherein CS represents chitosan; GdW10Represents a gadolinium polytungstate crystal; GdW10@ CS represents a chitosan-modified gadolinium polytungstate composite nano-carrier;
FIG. 7 is an agarose gel electrophoresis chart before and after loading small interfering RNA on the chitosan-modified gadolinium polytungstate composite nanocarrier prepared in example 1, wherein siRNA represents small interfering RNA; GdW10@ CS-siRNA represents the product after loading, and the mass ratios of the composite nanocarrier to the small interfering RNA are 1:5, 1:10, 1:20, 1:40, 1:50 and 1:100, respectively;
FIG. 8 shows blank, X-ray, gadolinium polytungstate GdW10Contrast of fluorescence enhancement patterns of gadolinium polytungstate plus X-ray, gadolinium polytungstate plus X-ray and glutathione under X-ray irradiation, wherein GSH represents glutathione, GdW10Represents a gadolinium polytungstate crystal;
FIG. 9 shows the glutathione content of pure hydrogen peroxide, pure gadolinium polytungstate, pure X-ray, gadolinium polytungstate plus X-rayWherein GSH represents glutathione, GdW10Represents a gadolinium polytungstate crystal;
FIG. 10 is a graph showing the effect of chitosan-modified gadolinium polytungstate composite nanocarriers prepared in example 1 on the cell viability of human hepatoma carcinoma cells BEL-7402 under hypoxic conditions at different concentrations, the concentrations being 0 and 6.25. mu.m
g/mL, 12.5. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, and 100. mu.g/mL;
FIG. 11A is a graph showing the killing effect of hypoxic BEL-7402 cells in a control group without radiosensitizer-gadolinium polytungstate composite nanomaterial; FIG. 11B is a graph showing the killing effect of hypoxic BEL-7402 cells on the treatment group of radiotherapy and gene therapy in combination with the addition of the radiosensitizer-gadolinium polytungstate composite nanomaterial of example 1;
FIGS. 12A to 12F are graphs showing the results of tumor suppression in combination with radiotherapy and gene therapy after various treatments were injected into tumors of a mouse bearing a BEL-7402 tumor, in which FIGS. 12A to 12F represent groups i to vi, respectively, and i corresponds to a PBS buffer solution; ii, corresponding to a chitosan-modified gadolinium polytungstate composite nano-carrier; iii corresponds to X-rays; iv corresponding to radiosensitizer gadolinium polytungstate composite nanomaterial; v adding X-rays to the chitosan-modified gadolinium polytungstate composite nano-carrier; vi corresponding to radiosensitizer gadolinium polytungstate composite nano material plus X-ray;
FIGS. 13A and 13B are pathological section images of the liver of mice in a control group without adding a radiosensitizer, gadolinium polytungstate composite nanomaterial at different magnifications, two arrows in FIG. 13A indicate cancer cells metastasized to the liver, and a dotted circle in FIG. 13B indicates cancer cells metastasized to the lung;
FIGS. 13C and 13D are pathological section views of the liver of mice at different magnifications after treatment with the radiosensitizer, gadolinium polytungstate composite nanomaterial of example 1 and radiotherapy;
FIG. 14 is a magnetic resonance imaging and computed tomography dual-mode imaging graph obtained by testing using chitosan-modified gadolinium polytungstate composite nanocarriers obtained in example 1 at different concentrations, which are 0,6.25 mg/mL, 12.5mg/mL, 25mg/mL and 50mg/mL, respectively.
Detailed Description
The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings.
The invention measures the capacity of the chitosan modified gadolinium polytungstate loaded with small interfering RNA, and the adopted measuring method is a well-known agarose gel electrophoresis method.
Example 1
The embodiment provides a gadolinium polytungstate composite nanomaterial, which is composed of gadolinium polytungstate, chitosan and small interfering RNA.
The preparation method comprises the following steps:
1) gadolinium polytungstate crystal (named GdW)10) Synthesis of (2)
In a typical experimental procedure, first 8.3g Na was weighed2WO4·2H2Dissolving O in 20mL of ultrapure water to form a uniform and transparent aqueous solution, and then slowly dropping acetic acid into the solution under the action of magnetic stirring to adjust the pH value of the solution to 7.40-7.50. Then 2mL of 477.95g mol were added under magnetic stirring at room temperature-1GdCl of6·6H2O was added dropwise to the above solution, and the whole solution system was heated to 85 ℃ on a heating table, and after the solution became completely clear and transparent, it was allowed to cool to room temperature, and then crystals were precipitated. In order to ensure that the obtained gadolinium polytungstate crystal has higher purity, a recrystallization method is adopted, the supernatant is discarded, the precipitated crystal is redissolved in ultrapure water with the same volume, the solution is heated to be completely dissolved to form clear and transparent aqueous solution, and then the solution is cooled to the room temperature until the crystal is precipitated again.
2) Preparation of macromolecular compound chitosan modified gadolinium polytungstate
Firstly, Chitosan (Chitosan, abbreviated as CS) is dissolved in1 wt% acetic acid solution to form acetic acid solution of Chitosan with mass concentration of 1%, and then 10mg of the prepared gadolinium polytungstate crystal is weighed and dissolved in10 mL of ultrapure water to form gadolinium polytungstate aqueous solution with concentration of 1 mg/mL. Then dropwise adding aqueous solution of gadolinium polytungstate into the solution under the action of continuous magnetic stirring at room temperatureIn acetic acid solution of chitosan to form stable chitosan modified gadolinium polytungstate composite nano-carrier (named GdW)10@ CS) suspension. Wherein the ratio of the amount of the substances added with the chitosan to the amount of the gadolinium polytungstate is 10. Subsequently, in order to obtain a chitosan-modified gadolinium polytungstate composite nano-carrier with higher purity, the formed chitosan-modified gadolinium polytungstate composite nano-carrier is centrifuged for 30min at the rotating speed of 12000rpm to remove unreacted free chitosan aqueous solution. Finally, the obtained chitosan-modified gadolinium polytungstate composite nano-carrier is re-dispersed in ultrapure water, placed in a dialysis bag with the molecular weight of 2.0Dka to be dialyzed in the ultrapure water for 1 day so as to remove impurity ions, and finally placed in a refrigerator at 4 ℃ for subsequent experiments.
The prepared macromolecular compound chitosan modified gadolinium polytungstate can be used as a carrier.
The obtained macromolecular compound chitosan-modified gadolinium polytungstate can also be called as a chitosan-modified gadolinium polytungstate composite nano-carrier.
3) Preparation of gadolinium polytungstate composite nano material
In order to obtain a gadolinium polytungstate composite nano material (named GdW) consisting of gadolinium polytungstate, chitosan and small interfering RNA (RNA)10@CSsiRNA) Firstly, uniformly mixing HIF-1 alpha siRNA with GdW10 aqueous solutions with different concentrations in a nuclease-free aqueous solution according to a preset amount, fully shaking, and standing for 30 minutes at room temperature to form the radiosensitizer gadolinium polytungstate composite nanomaterial.
The obtained gadolinium polytungstate composite nano material is composed of gadolinium polytungstate, chitosan and small interfering RNA.
The obtained gadolinium polytungstate composite nano material can be used as a radiosensitizer.
The obtained gadolinium polytungstate composite nano material can also be called as a radiosensitizer gadolinium polytungstate composite nano material.
The structural schematic diagram of the radiosensitizer gadolinium polytungstate composite nanomaterial obtained in the embodiment is shown in fig. 1, and the radiosensitizer gadolinium polytungstate composite nanomaterial comprises gadolinium polytungstate, chitosan and small interfering RNA loaded on the surface of a chitosan-modified gadolinium polytungstate composite nanomaterial.
The chitosan-modified gadolinium polytungstate composite nano-carrier obtained in step 2) of this example was characterized by using a scanning electron microscope (High _ technologies, Japan, S-4800), and as a result, as shown in fig. 2, it can be seen that the chitosan-modified gadolinium polytungstate composite nano-carrier is a spherical nano-particle with uniform morphology.
The transmission electron microscope (FEI Tecnai G2F20) is used to characterize the chitosan-modified gadolinium polytungstate composite nano-carrier obtained in step 2) of this embodiment, and as a result, as shown in fig. 3, it can be seen that the chitosan-modified gadolinium polytungstate composite nano-carrier has a uniform morphology and a particle size of 30-40 nm.
The chitosan-modified gadolinium polytungstate composite nano-carrier obtained in step 2) of this example was characterized by using X-ray energy spectrum (High _ technologies, Japan, S-4800), and the result is shown in fig. 4, from which it can be seen that the chitosan-modified gadolinium polytungstate composite nano-carrier contains a large amount of C, N, W, O, Gd elements, which indicates that chitosan is successfully modified on gadolinium polytungstate compound.
The chitosan-modified gadolinium polytungstate composite nano-carrier obtained iN step 2) of this example was characterized by an infrared spectrometer (Nicolet iN10, Thermo Fisher), and the result is 1639cm as shown iN fig. 5-1And 1423cm-1Is the characteristic absorption peak of chitosan; 1076cm-1、931cm-1、842cm-1And 783cm-1Is a characteristic absorption peak of the gadolinium polytungstate, and can be seen that characteristic peaks of chitosan and gadolinium polytungstate appear in an infrared spectrogram of the chitosan-modified gadolinium polytungstate composite nano-carrier.
Therefore, the prepared macromolecular compound chitosan-modified gadolinium polytungstate composite nano-carrier comprises macromolecular compound chitosan and gadolinium polytungstate crystals.
The chitosan, the gadolinium polytungstate crystal obtained in the step 2) of this example and the chitosan-modified gadolinium polytungstate composite nano-carrier obtained in the step 2) were characterized by using a thermogravimetric analyzer (Perkin, Elmer, Diamond TG/DTA), and the results are shown in fig. 6, where the mass fraction of the macromolecular compound chitosan in the whole chitosan-modified gadolinium polytungstate composite nano-carrier is 14.3%, and the mass fraction of the gadolinium polytungstate in the whole chitosan-modified gadolinium polytungstate composite nano-carrier is 85.7%.
Evaluating the small interfering RNA loading capacity of the chitosan-modified gadolinium polytungstate composite nano-carrier:
in step 3) of this embodiment, it can be shown that the polymeric compound chitosan-modified gadolinium polytungstate composite nano-carrier can successfully load the small interfering RNA through the interaction between positive and negative charges, and the load capacity of the composite nano-carrier for loading the small interfering RNA can be determined through an agarose gel electrophoresis experiment.
More specifically, in order to load small interfering RNA, the chitosan-modified gadolinium polytungstate composite nanocarrier obtained in example 1 was complexed with small interfering RNA (the mass ratio of the two was 100,50,40,20,10,5), and the radiosensitizer gadolinium polytungstate composite nanomaterial obtained was gently shaken on an oscillator and allowed to stand at room temperature for 15 minutes. In order to evaluate the ability of small interfering RNA to release from radiosensitizer gadolinium polytungstate composite nanomaterial, heparin and radiosensitizer gadolinium polytungstate composite nanomaterial were added and incubated at room temperature for 20 minutes, wherein the ratio of the amount of heparin to small interfering RNA species was 5: 1. In addition, in order to determine the protection effect of the chitosan modified gadolinium polytungstate composite nano-carrier on small interfering RNA, 2 μ L of RNase (RNase,1 μ g/μ L) and radiosensitizer gadolinium polytungstate composite nano-material were added and incubated for 1 hour at 37 ℃ to degrade 1 μ g of small interfering RNA. Wherein, free small interfering RNA as blank control. Finally, the small interfering RNA load capacity, the release amount and the protection capacity of the chitosan modified gadolinium polytungstate composite nano-carrier on the small interfering RNA are detected by 1% agarose gel electrophoresis under the voltage of 120V for 15 minutes.
The ability of the chitosan-modified gadolinium polytungstate composite nanocarrier obtained in example 1 to load small interfering RNA was characterized by using an agarose gel electrophoresis apparatus (C300, USA, azure biosystems), and the result is shown in fig. 7, when the mass ratio of the composite nanocarrier to the small interfering RNA was 50:1, the small interfering RNA was all successfully loaded on the composite nanocarrier, and a good loading effect was achieved.
Evaluation of sensitization effect:
this example illustrates that gadolinium polytungstate can produce radiosensitization effect under X-ray irradiation, and glutathione can scavenge reactive oxygen species.
The specific detection process is as follows: first, 0.5mL of DCFH-DA dissolved in DMSO and 2.0mL of NaOH with a concentration of 0.01M were subjected to hydrolysis reaction at room temperature in the dark to finally produce the probe DCFH. After the reaction for 30 minutes, 10mL of PBS (25mM, pH 7.2) was added to the above reaction system to terminate the reaction. The formed aqueous solution of DCFH was then covered with a layer of tinfoil paper on the outside and placed on ice for subsequent testing experiments. Next, the chitosan-modified gadolinium polytungstate composite nanocarrier (0.10mg/mL) obtained in example 1 was mixed with the above-generated DCFH aqueous solution (10. mu.M) in the absence or presence of GSH (1mM), and immediately irradiated with X-ray for 10 minutes. Immediately after irradiation, the solution was placed under a fluorescence spectrometer to measure the fluorescence spectra of the samples for the detection of ROS production in each sample.
Each sample (blank, X-ray, gadolinium polytungstate GdW) in this example was analyzed by fluorescence spectroscopy (Jobin Yvon FluoroLog3, Horiba, Japan)10Gadolinium polytungstate plus X-ray, gadolinium polytungstate plus X-ray and glutathione) at the characteristic absorption peak of the probe 530nm, as shown in fig. 8, the blank or the probe has very low fluorescence intensity under the irradiation of the X-ray, but the gadolinium polytungstate has very high fluorescence intensity under the irradiation of the X-ray, and once the Glutathione (GSH) is added, the fluorescence intensity is reduced again, which indicates that the gadolinium polytungstate can generate active oxygen substances under the irradiation of the X-ray, so that the radiosensitization effect is achieved, and the glutathione has the capability of removing the active oxygen substances.
This example demonstrates that gadolinium polytungstate can undergo a redox reaction with glutathione, reducing the level of glutathione, thereby achieving the production of more effective active oxygen species.
Dissolving chitosan modified gadolinium polytungstate composite nano-carrier in carbonate buffer solution (PBS, PH 8.6,50mM) to prepare 100 mug/mL, and then dissolving the chitosan modified gadolinium polytungstate composite nano-carrier in the concentrationThe carrier solution and glutathione aqueous solution (0.8mM) are placed in a centrifugal tube according to the volume ratio of 1:1 for oxidation reaction, and meanwhile, a layer of tin foil paper covers the outer wall of the centrifugal tube to prevent the interference of external visible light to the reaction. Subsequently, the mixed system was placed on a shaker and shaken at 150rpm for 4 hours at room temperature. After the reaction was completed, 785. mu.L of Tris-HCl (0.05M) and 15. mu.L of DTNB (100mM) were added to the reaction system, respectively, and the shaking was continued at the same speed for 5 minutes. Finally, the reaction product is placed under a microplate reader, and the ultraviolet absorption of the sample at 412nm is determined. Wherein PBS (pH 8.6,50mM) and pure H2O2(1.0mM) was used as negative and positive controls, respectively, throughout the experiment.
Ultraviolet absorption of a sample is measured at a 412nm characteristic absorption peak of a probe by using a fluorescence inverted microscope (Olympus, IX71, JAPAN), and an influence diagram of pure hydrogen peroxide, pure gadolinium polytungstate, pure X-ray, gadolinium polytungstate and X-ray on glutathione content is detected, as shown in figure 9, the pure glutathione with a negative control has strong absorption at 412nm, the glutathione is completely reacted by the hydrogen peroxide with a positive control, and almost no absorption is generated at 412nm, but the content of the glutathione is reduced after the gadolinium polytungstate is added, which indicates that the gadolinium polytungstate can generate an oxidation reduction reaction with the glutathione, the level of the glutathione is reduced, and the opportunity of glutathione for removing active oxygen substances is reduced.
Evaluation of the influence of human hepatoma carcinoma cell BEL-7402 on cell viability under hypoxic conditions:
the following example is used to illustrate the effect of the chitosan-modified gadolinium polytungstate composite nanocarrier obtained in example 1 on the cell viability of human hepatoma cells BEL-7402 under hypoxic conditions.
(1) Culture of BEL-7402 hypoxic cells
First, BEL-7402 cells were cultured in DMEM fresh medium containing 10% fetal bovine serum and placed at 37 ℃ in 5% CO2Is incubated in a cell incubator.
(2) Determination of cell viability
After the cells had grown to a certain number, the cells were transferred to a 96-well plate and incubated for a further 24 hours,wherein the cell density is 4 × 103A hole. After the cells were spread over substantially the entire well plate, various concentrations of chitosan-modified gadolinium polytungstate composite nanocarriers (6.25,12.5,25.0,50.0,100 μ g/mL) obtained in example 1 were dispersed in CoCl containing 100 μ M simulated hypoxic environment2Fresh medium. After 24 hours of co-incubation, 10 mu L of fresh CCK-8 is added into each well for further co-incubation for 1 hour, finally, the 96-well plate is placed under a microplate reader, the absorbance of each well cell is measured at 450nm, and the survival rate of the cells under different concentration conditions is calculated according to a cell survival rate calculation formula.
The effect of the chitosan-modified gadolinium polytungstate composite nanocarrier obtained in example 1 at different concentrations on the cell viability of human hepatoma carcinoma cells BEL-7402 under hypoxic conditions was determined by using a microplate reader (Thermo scientific, MULTISCAN MNK3), as shown in fig. 10, when the concentration of the composite nanocarrier was as high as 100 μ g/mL, the cell viability was still greater than 90%, which indicates that the obtained chitosan-modified gadolinium polytungstate composite nanocarrier did not produce significant cytotoxicity to human hepatoma carcinoma cells BEL-7402 under hypoxic conditions, and can be further applied to the evaluation of anti-tumor effect.
Evaluation of the synergistic therapeutic effect with radiotherapy and gene therapy in hypoxic tumor cells:
the following example is used to illustrate the synergistic therapeutic effect of radiosensitizer gadolinium polytungstate composite nanomaterial in example 1 in hypoxic tumor cells for radiotherapy and gene therapy.
(1) Culture of BEL-7402 hypoxic cells
First, BEL-7402 cells were cultured in DMEM fresh medium containing 10% fetal bovine serum and placed at 37 ℃ in 5% CO2Is incubated in a cell incubator.
(2) Evaluation of synergistic therapeutic Effect
For cloning experiments, BEL-7402 cells with a cell number of 2000 were dispersed in CoCl containing 100. mu.M of simulated hypoxic environment2In culture, the suspension cells were then incubated in 24-well plates for 48 hours. After the cells are attached to the wall, the radiosensitizer gadolinium polytungstate composite nano material for the cells is simultaneously treated with X rays. The blank control group is under the same other conditions except that no radiosensitizer-gadolinium polytungstate composite nanomaterial is added, after the two methods are used for treatment, the cells are continuously incubated for 10 days, then the cells are stained by Giemsa staining solution, and finally the inhibition effect of the two methods on the cell viability is evaluated by measuring the number of the formed cells. As shown in fig. 11A and fig. 11B, after the radiosensitizer gadolinium polytungstate composite nanomaterial is treated with X-rays, the survival rate of the cells (fig. 11B) is much lower than that of the control group (fig. 11A), which indicates that the radiosensitizer gadolinium polytungstate composite nanomaterial produces obvious synergistic therapeutic effect of radiotherapy and gene therapy on human liver cancer cell BEL-7402 under hypoxic conditions.
Evaluation of synergistic therapeutic effects with radiotherapy and gene therapy in vivo tumor models:
this example serves to illustrate that the radiosensitizer, gadolinium polytungstate composite nanomaterial in example 1 has a synergistic therapeutic effect of radiotherapy and gene therapy in a living tumor model.
(1) Construction of tumor model
First, 30 BALB/c nude mice were purchased from Experimental animals technology, Inc. of Wei Tongli, Beijing, and then 100. mu.L of nude mice containing cells at a density of 1.0X 10 were subcutaneously implanted6BEL-7402 cell suspension. After 10 days, the tumor volume of the mice reached about 100mm3At the time, the rats were randomly assigned according to the experimental design.
(2) Evaluation of synergistic therapeutic effects in vivo tumor models
Tumor-bearing nude mice were randomly assigned to 6 groups: wherein, (i) a PBS buffer solution set (PBS); (ii) single chitosan modified gadolinium polytungstate composite nano-carrier (GdW)10@ CS); (iii) a separate set of X-rays (X-ray); (iv) radiosensitizer gadolinium polytungstate composite nanomaterial (GdW)10@CSsiRNA) (ii) a (v) Composite nanomaterials and X-ray groups (GdW)10@ CS + RT); (vi) radiosensitizer gadolinium polytungstate composite nanomaterial and X-ray set (GdW10@ CS)siRNA+ RT). Injecting 20 μ L PBS solution into tumor of i groups of tumor-bearing mice with BEL-7402 tumorLiquid; injecting 20 mu L of chitosan modified gadolinium polytungstate composite nano-carrier aqueous solution into the tumor of the mice in the groups ii and v; for group iv and vi mice, 20 μ L of radiosensitizer gadolinium polytungstate composite nanomaterial aqueous solution was injected intratumorally. After injection, tumor-bearing mice bearing BEL-7402 tumors from groups i, v and vi were irradiated with X-ray (10Gy) for 10 minutes. Meanwhile, in order to ensure the activity of HIF-1 alpha siRNA in the tumor to ensure that higher treatment effect is achieved, a freshly prepared radiosensitizer gadolinium polytungstate composite nano-material aqueous solution is injected into the mouse body again every 2 to 3 days. Subsequently, changes in mouse tumor volume were measured and photographed using a vernier caliper or a camera, respectively. And the calculation formula of the mouse tumor volume is as follows: tumor volume V ═ ab2(ii)/2, wherein, a is tumor length; b-tumor width.
After the experiment, the mice tumors treated by various methods in this example were photographed by using a camera, as shown in fig. 12A-12F, which shows that the radiosensitizer gadolinium polytungstate composite nanomaterial also produces significant synergistic therapeutic effects of radiotherapy and gene therapy in the living tumor model, and compared with other groups, the tumor growth of the synergistic therapeutic group is maximally inhibited.
The following example is used to illustrate that the radiosensitizer, gadolinium polytungstate composite nanomaterial of example 1 also does not produce significant toxicity to living bodies.
(1) Construction of tumor model
First, 30 BALB/c nude mice were purchased from Experimental animals technology, Inc. of Wei Tongli, Beijing, and then 100. mu.L of nude mice containing cells at a density of 1.0X 10 were subcutaneously implanted6BEL-7402 cell suspension. After 10 days, the tumor volume of the mice reached about 100mm3At the time, the rats were randomly assigned according to the experimental design.
(2) Evaluation of toxicity of radiosensitizer-gadolinium polytungstate composite nano material on living body
After the experiment is finished, an eyeball blood sampling mode is adopted for each group of mice, collected whole blood is placed in a 1.5mL centrifuge tube and stands for 2-3 hours at room temperature, and then a centrifuge is adopted to centrifuge for 5 minutes at the rotating speed of 1500rpm so as to obtain serum for detecting biochemical indexes of blood and judging whether the liver function and the kidney function of each group of mice are normal or not.
The biochemical indexes of the blood of each group of mice obtained in the embodiment, such as the content of the liver function index, the content of the kidney function index and the like, are measured by using a blood biochemical analyzer, the result is shown in table 1, and the biochemical indexes of the blood of each group of mice are as follows: alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Creatinine (CREA) and UREA (UREA). The liver function index and the kidney function index are both in a normal range, and no obvious abnormal phenomenon occurs, which indicates that the radiosensitizer gadolinium polytungstate composite nano material does not generate obvious toxicity to living bodies.
Table 1
Figure BDA0001260832260000151
This example illustrates that the radiosensitizer, gadolinium polytungstate composite nanomaterial in example 1 has some inhibitory effect on mouse tumor metastasis.
(1) Construction of tumor model
First, 30 BALB/c nude mice were purchased from Experimental animals technology, Inc. of Wei Tongli, Beijing, and then 100. mu.L of nude mice containing cells at a density of 1.0X 10 were subcutaneously implanted6BEL-7402 cell suspension. After 10 days, the tumor volume of the mice reached about 100mm3At the time, the rats were randomly assigned according to the experimental design.
(2) Evaluation of mouse tumor metastasis suppressing Effect
After the experiment was completed, after the mice were sacrificed, the heart, liver, spleen, lung and kidney of each mouse were collected, and the obtained mouse organs were fixed with 4% paraformaldehyde for two days, followed by paraffin sectioning and staining with hematoxylin and eosin (H & E). Finally, each tissue section was analyzed pathologically using a fluorescence inverted microscope.
The case sections collected in the examples were analyzed by fluorescence inverted microscope (Olympus, IX71, JAPAN), and as shown in fig. 13A, 13B, 13C and 13D, the tumors of mice treated with radiosensitizer gadolinium polytungstate composite nanomaterial and X-ray did not develop any metastasis as compared with the control group (fig. 13C and 13D), while the tumors of the mice of the control group were found to have significant metastasis in both liver and lung (fig. 13A and 13B), indicating that the radiosensitizer gadolinium polytungstate composite nanomaterial has significant inhibitory effect on the tumors of mice and can also inhibit metastasis of tumors.
The following example is presented to illustrate that chitosan-modified gadolinium polytungstate composite nanocarriers can also be used for multimodal imaging.
Firstly, chitosan-modified gadolinium polytungstate composite nano-carriers (0,6.25,12.5,25.0,50mg/mL) with different concentrations are dissolved in ultrapure water, and then a sample is placed under a magnetic resonance imager for detecting the magnetic resonance signal intensity (MRI) of the sample. Also, in order to determine the signal intensity of CT under different concentration conditions, different concentrations of chitosan-modified gadolinium polytungstate composite nanocarriers (0,6.25,12.5,25.0,50mg/mL) were dissolved in 0.5% agarose aqueous solution, and then the samples were placed in 1.5mL centrifuge tubes for determination of the CT signal intensity of the samples. For comparison, the same concentration of the commercial CT contrast agent Iopromide (Iopromide) was used as a control experiment. Finally, each sample was placed under a computed tomography scanner for CT imaging.
Magnetic resonance imaging (Biospec; Bruker; Ettlingen; Germany) and computed tomography (MSOT observation 128, iTheramedical, Germany) are respectively adopted to measure the imaging effect of the chitosan-modified gadolinium polytungstate composite nanocarrier with different concentrations, as shown in FIG. 14, the magnetic resonance signal intensity and the CT signal intensity are greatly increased along with the increase of the concentration, which indicates that the chitosan-modified gadolinium polytungstate composite nanocarrier can also be used for multi-mode (MR/CT) imaging.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims (26)

1. The application of the gadolinium polytungstate composite nanomaterial for preparing the sensitizer is characterized in that gadolinium polytungstate in the nanomaterial is subjected to oxidation-reduction reaction with glutathione, so that the content of glutathione is reduced, and intrinsic sensitization is realized; the gadolinium polytungstate composite nanomaterial comprises gadolinium polytungstate, chitosan and small interfering RNA, wherein the small interfering RNA is loaded on the surface of modifier chitosan; the particle size of the gadolinium polytungstate composite nano material is 30-40nm, and the small interfering RNA is HIF-1 alpha siRNA;
the gadolinium polytungstate composite nano material is prepared by the following method:
(1) chitosan-modified gadolinium polytungstate: (A) dissolving chitosan in acetic acid to obtain an acetic acid solution of chitosan; (B) preparing aqueous solution of gadolinium polytungstate by using gadolinium polytungstate crystal and water; (C) mixing aqueous solution of gadolinium polytungstate with acetic acid solution of chitosan to form suspension, and removing impurities to obtain chitosan-modified gadolinium polytungstate;
(2) preparing aqueous solution of chitosan-modified gadolinium polytungstate, mixing the aqueous solution with small interfering RNA in a nuclease-free aqueous solution, oscillating, and standing to obtain a gadolinium polytungstate composite nano material consisting of gadolinium polytungstate, chitosan and small interfering RNA;
the mass ratio of the chitosan in the acetic acid solution of the chitosan to the gadolinium polytungstate in the aqueous solution of the gadolinium polytungstate in the step (C) is 5-20; and (3) the mass ratio of the chitosan-modified gadolinium polytungstate to the small interfering RNA in the chitosan-modified gadolinium polytungstate aqueous solution in the step (2) is 1-50.
2. The use according to claim 1, wherein the small interfering RNA is expressed by hypoxia inducible factor-1 alpha and is loaded on the surface of gadolinium polytungstate modified with chitosan.
3. Use according to claim 1, wherein the hydrated particle size of the gadolinium polytungstate composite nanomaterial is 40nm to 60 nm.
4. The use according to claim 1, wherein the potential of a water system of the gadolinium polytungstate composite nanomaterial consisting of gadolinium polytungstate and chitosan is +30mV to +60 mV.
5. The use according to claim 1, wherein the potential of a water system of the gadolinium polytungstate composite nanomaterial consisting of gadolinium polytungstate, chitosan and small interfering RNA is +10mV to +30 mV.
6. The use according to claim 1, wherein the chitosan has an average molecular weight of 100-300 KD.
7. The use according to claim 6, wherein the chitosan has an average molecular weight of 100-200 kD.
8. The use according to claim 1, wherein the mass concentration of the acetic acid in the step (A) is 0.1-5%.
9. The use according to claim 8, wherein the mass concentration of the acetic acid in the step (A) is 0.1-3%.
10. The use according to claim 1, wherein the mass ratio of chitosan in the acetic acid solution of chitosan in the step (C) to gadolinium polytungstate in the aqueous solution of gadolinium polytungstate is 7-15.
11. Use according to claim 1, wherein the mixing of step (C) is carried out by: dropwise adding aqueous solution of gadolinium polytungstate into acetic acid solution of chitosan.
12. Use according to claim 1, wherein the mixing of step (C) is accompanied by stirring.
13. The use according to claim 1, wherein the impurity removal process of step (C) is: the suspension was centrifuged, and the centrifuged product was dispersed in ultrapure water and dialyzed in a dialysis bag.
14. Use according to claim 13, wherein the rotation speed of the centrifugation is 12000rpm and the time of the centrifugation is 30 min.
15. Use according to claim 13, wherein the dialysis bag has a molecular weight of 2.0Dka and the dialysis time is 1 day.
16. The use according to claim 1, wherein the gadolinium polytungstate crystal of step (B) is prepared by the following method:
dissolving tungstate in ultrapure water to form a uniform and transparent aqueous solution A;
adjusting the pH value of the aqueous solution A to 6.50-8.50 by using an acidic compound under the stirring action to obtain a solution B;
adding gadolinium salt aqueous solution into the solution B under the stirring action to form aqueous solution C;
IV, heating and stirring the aqueous solution C until a uniform and transparent aqueous solution D is obtained;
and V, cooling to precipitate a gadolinium polytungstate crystal.
17. Use according to claim 16, wherein the tungstate salt of step i is sodium tungstate, and the chemical composition of the sodium tungstate is Na2WO4·2H2O。
18. The use as claimed in claim 16, wherein the concentration of tungstate in said aqueous solution a in step i is 300mg/mL to 500 mg/mL.
19. The use according to claim 16, wherein the acidic compounds of step ii comprise hydrochloric acid, acetic acid and nitric acid.
20. Use according to claim 19, wherein the acidic compound of step ii is glacial acetic acid.
21. Use according to claim 16, wherein the gadolinium salt in the aqueous gadolinium salt solution of step iii is gadolinium chloride, the chemical composition of which is GdCl6·6H2O。
22. Use according to claim 16, wherein the concentration of gadolinium salt in said aqueous gadolinium salt solution of step iii is 300g/mol to 600 g/mol.
23. Use according to claim 16, wherein the heating temperature in step iv is from 70 ℃ to 90 ℃.
24. The use as claimed in claim 1, wherein the gadolinium polytungstate composite nanomaterial is prepared by the following method:
(1) preparing a gadolinium polytungstate crystal:
i, mixing Na2WO4·2H2Dissolving O in ultrapure water to form a uniform and transparent aqueous solution A;
regulating the pH value of the aqueous solution A to 6.50-8.50 by using glacial acetic acid under the action of magnetic stirring at the temperature of 15-35 ℃ to obtain a solution B;
III, under the magnetic stirring action at 15-35 ℃, GdCl6·6H2Adding the O aqueous solution into the solution B to form an aqueous solution C;
finally cooling to precipitate a gadolinium polytungstate crystal;
(2) preparing chitosan modified gadolinium polytungstate:
(A) dissolving chitosan in glacial acetic acid to obtain a glacial acetic acid solution of chitosan;
(B) preparing aqueous solution of the gadolinium polytungstate by using the gadolinium polytungstate crystal obtained in the step (1) and water;
(C) mixing aqueous solution of gadolinium polytungstate with glacial acetic acid solution of chitosan to form suspension, centrifuging the suspension at 12000rpm for 30min, and dialyzing the centrifuged product in a dialysis bag with molecular weight of 2.0Dka for 1 day to obtain gadolinium polytungstate modified by chitosan;
(3) uniformly mixing small interfering RNA and aqueous solution of chitosan-modified gadolinium polytungstate in aqueous solution without nuclease, oscillating, and standing for 30min at the temperature of 15-35 ℃ to obtain the gadolinium polytungstate composite nano material.
25. Use according to any one of claims 1 to 24, wherein the gadolinium polytungstate composite nanomaterial is also used as a support.
26. Use according to any one of claims 1 to 24, wherein the gadolinium polytungstate composite nanomaterial is for use in tumour therapy.
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