CN110075296B - Gold nanoflowers with liver cancer targeting and radiotherapy sensitization characteristics and preparation and application thereof - Google Patents
Gold nanoflowers with liver cancer targeting and radiotherapy sensitization characteristics and preparation and application thereof Download PDFInfo
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0038—Radiosensitizing, i.e. administration of pharmaceutical agents that enhance the effect of radiotherapy
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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Abstract
The invention relates to gold nanoflowers with liver cancer targeting and radiotherapy sensitivity enhancing characteristics, and a preparation method and application thereof. The invention constructs a multifunctional responsive surface on the gold nanoflowers, thereby realizing liver cancer enrichment and radiotherapy sensitization. The gold nanoflowers have specific morphology and surface plasmon resonance property, are beneficial to endocytosis of cells, and have higher X-ray absorption efficiency. The multifunctional responsive surface is stable under normal physiological conditions, and internal cell-penetrating peptide molecules are exposed after the multifunctional responsive surface responds to a tumor microenvironment, so that the multifunctional responsive surface is efficiently endocytosed by liver tumor cells, and the enrichment degree and retention time of gold nanoflowers in tumors are improved. Under the irradiation of radioactive rays, a better radiotherapy sensitization effect can be achieved.
Description
(I) technical field
The invention relates to gold nanoflowers with liver cancer targeting and radiotherapy sensitization characteristics, and a preparation method and application thereof.
(II) background of the invention
Tumor radiotherapy is a local treatment for tumors using radiation. The radiation includes alpha, beta and gamma rays generated by radioactive isotopes, and x-rays, electron beams, proton beams and other particle beams generated by various x-ray therapeutic machines or accelerators. About 70% of cancer patients require radiation therapy in the course of cancer treatment, and about 40% of cancers can be cured by radiation therapy. The role and position of radiotherapy in tumor treatment are increasingly prominent, and the radiotherapy has become one of the main means for treating malignant tumors. Because of the large side effects of radiation therapy, there is an urgent need to enhance the sensitivity of tumor tissue to radiation therapy to increase the efficacy without increasing the radiation dose (side effects).
Theoretically, high atomic number substances (such as heavy metals like gold) enter tumor tissues and can generate stronger photoelectric absorption in the tumor tissues than surrounding normal tissues, so that more radioactive energy can be transmitted to the tumor tissues, and the effect of radiotherapy sensitization is achieved. The albumin modified nano-gold has obvious sensitization effect on X-ray radiotherapy of liver cancer tumor-bearing mice: under the irradiation dose of 5Gy, the tumor inhibition rate can be improved to 58% from 28% of simple radiotherapy by injecting gold nanospheres with the particle size of 30-60 nm into tail veins, and the sensitization coefficient reaches 2.07.
Although the gold nanoparticles have good biocompatibility, the gold nanoparticles are easy to be identified and removed by the immune system of a human body in vivo, and have the defect of weak capability of targeting cancer tissues and cells; meanwhile, the conventional gold nanospheres have low absorption efficiency on X-rays and are not beneficial to being endocytosed by tumor cells; these disadvantages limit their popularization and application.
The nano material with the tumor microenvironment responsiveness can maintain the surface hydrophilicity and the electrical neutrality in a normal physiological environment, and is beneficial to the in vivo circulation; under the action of specific enzyme in the tumor microenvironment, the hydrophilic molecules on the surface are removed to expose the cell affinity molecules inside, so that the tumor cells can swallow the hydrophilic molecules, and the concentration of the nano material in the tumor tissues is increased. The cell-penetrating peptide is a common cell affinity molecule, is modified on the surface of a nano material, and can mediate cell endocytosis. GRKKRRQRRRPQ this sequence is derived from the Tat protein in the HIV virus and has been shown to be effective in a number of studies. The GPLG sequence can be cleaved by MMP-9, thereby removing the hydrophilic molecules from the surface. Polysarcosine is a hydrophilic polymer formed by polymerizing sarcosine, and previous researches show that the modification of the polymyosine on the surface of a nano material can reduce the effects of protein adsorption and cell interaction and obviously prolong the in vivo circulation time of the nano material.
The gold nanoflowers have special appearance and surface plasmon resonance property, and can absorb X rays more efficiently than gold nanospheres. And the rough surface of the nano-particle is more beneficial to being endocytosed by tumor cells, so that the enrichment degree in tumor tissues and the radiotherapy sensitization effect are improved.
Disclosure of the invention
The invention aims to provide gold nanoflowers with liver cancer targeting and radiotherapy sensitization characteristics, and a preparation method and application thereof.
The technical scheme adopted by the invention is as follows:
gold nanoflowers with liver cancer targeting and radiotherapy sensitivity enhancing characteristics are 50-200 nm in diameter, 4-10 protruding thorns are provided, and the gold nanoflowers are prepared by the following method:
(1) adding a glutathione solution into a gold nanorod solution, violently stirring at room temperature for 1-2 hours, and then sequentially adding a hexadecyl trimethyl ammonium chloride solution and HAuCl4Solution, AgNO3The solution and an ascorbic acid solution are stirred vigorously for 0.5-1 hour at room temperature, and are kept stand overnight in a constant-temperature water bath at the temperature of 26-28 ℃ to obtain monodisperse gold nanoflowers;
(2) centrifuging gold nanoflowers to remove excessive CTAC ligand, dispersing the gold nanoflowers in a polypeptide-polymyosin solution, placing the polypeptide-polymyosin solution in a constant-temperature water bath at the temperature of 28-30 ℃ to vibrate for 18-24 h, centrifuging to remove supernate, continuously dispersing the supernate in the polypeptide-polymyosin solution, placing the polypeptide-polymyosin solution in a constant-temperature water bath at the temperature of 28-30 ℃ to vibrate for 18-24 h, centrifuging, and collecting the gold nanoflowers with the characteristics of liver cancer targeting and radiotherapy sensitization.
The invention constructs a multifunctional responsive surface on the gold nanoflowers, thereby realizing liver cancer enrichment and radiotherapy sensitization.
The invention also relates to a method for preparing gold nanoflowers, comprising:
(A) adding a glutathione solution into a gold nanorod solution, violently stirring at room temperature for 1-2 hours, and then sequentially adding a hexadecyl trimethyl ammonium chloride solution and HAuCl4Solution, AgNO3The solution and an ascorbic acid solution are stirred vigorously for 0.5-1 hour at room temperature, and are kept stand overnight in a constant-temperature water bath at the temperature of 26-28 ℃ to obtain monodisperse gold nanoflowers; the gold nanorods: glutathione: hexadecyltrimethylammonium chloride: HAuCl4：AgNO3: the ratio of the amounts of ascorbic acid in the substance was 0.1 mmol: 1-5 mmol: 0.05-0.3 mmol: 5-15 mmol: 0.01-0.05 mmol: 1-10 mmol;
(B) centrifuging gold nanoflowers to remove excessive CTAC ligand, dispersing the gold nanoflowers in a 3-8 mg/mL polypeptide-polymyosin solution, placing the gold nanoflowers in a constant-temperature water bath at 28-30 ℃ to vibrate for 18-24 h, centrifuging to remove supernate, continuously dispersing the gold nanoflowers in a polypeptide-polymyosin solution, placing the gold nanoflowers in a constant-temperature water bath at 28-30 ℃ to vibrate for 18-24 h, centrifuging, and collecting the gold nanoflowers with the characteristics of liver cancer targeting and radiotherapy sensitization.
The polypeptide-poly-sarcosine has the molecular sequence of CCV GRKKRRQRRRPQGGPLGV-poly-sarcosine, wherein the molecular weight of poly-sarcosine is about 2 kD. The GRKKRRQRRRPQ sequence in the polypeptide plays a role of cell-penetrating peptide, the GPLG sequence plays a role of MMP-9 response, and the poly-sarcosine plays a role of stabilizing nano materials and resisting phagocytosis of cells.
Specifically, the method comprises the following steps:
(A) 1mL of glutathione solution was added to 1mL (0.1mM) of gold nanorod solution, and after vigorously stirring at room temperature for 2 hours, 5.81mL of cetyltrimethylammonium chloride (0.033 mM) and 40. mu.L of HAuCl (25 mM) were added in this order4Solution, 30. mu.L of 1mM AgNO3The solution and 60 mu L of ascorbic acid solution with the concentration of 100mM are stirred vigorously for 0.5 to 1 hour at room temperature, and are kept stand overnight in a constant-temperature water bath at the temperature of 26 to 28 ℃ to obtain monodisperse gold nanoflowers;
(B) centrifuging gold nanoflowers to remove excessive CTAC ligand, dispersing the gold nanoflowers in 5mg/mL polypeptide-polymyosin solution, placing the gold nanoflowers in 30 ℃ constant temperature water bath to shake for 24 hours, centrifuging to remove supernate, continuously dispersing the gold nanoflowers in polypeptide-polymyosin solution, placing the gold nanoflowers in 30 ℃ constant temperature water bath to shake for 24 hours, centrifuging, and collecting the gold nanoflowers with the characteristics of liver cancer targeting and radiotherapy sensitization.
The gold nanoflowers obtained by the method are stable under normal physiological conditions and can circulate in blood for a long time; when the nano gold flowers reach a liver tumor microenvironment, the inner cell-penetrating peptide molecules are exposed under the action of high-concentration MMP-2, so that the nano gold flowers are efficiently endocytosed by liver tumor cells, and the enrichment degree and retention time of the nano gold flowers in tumors are improved. The gold nanoflowers have specific morphology and surface plasmon resonance property, are beneficial to endocytosis of cells and have higher X-ray absorption efficiency. Under the irradiation of radioactive rays, active oxygen is generated on the surface of the gold nano material, so that the effect of radiotherapy can be improved, and the effect of radiotherapy sensitization is achieved.
The invention also relates to application of the gold nanoflowers with liver cancer targeting and radiotherapy sensitization characteristics in preparation of a medicine for treating liver cancer.
Specifically, the medicine is a radiotherapy medicine.
The invention has the following beneficial effects: the gold nanoflowers have specific morphology and surface plasmon resonance property, are beneficial to endocytosis of cells, and have higher X-ray absorption efficiency; the multifunctional responsive surface is stable under normal physiological conditions, and internal cell-penetrating peptide molecules are exposed after the multifunctional responsive surface responds to a tumor microenvironment, so that the multifunctional responsive surface is efficiently endocytosed by liver tumor cells, and the enrichment degree and retention time of gold nanoflowers in tumors are improved; under the irradiation of radioactive rays, a better radiotherapy sensitization effect can be achieved.
(IV) description of the drawings
FIG. 1 is a transmission electron micrograph of gold nanoflowers according to the present invention.
FIG. 2 is the relationship between the surface functional molecule mass and the charge amount of gold nanometer particles.
FIG. 3 is a graph of particle size (left) and potential (right) of gold nanoflowers with responsive surfaces before and after treatment in MMP-9 solution.
FIG. 4 is a graph showing the relationship between the time of incubation and the endocytosis of gold nanoflowers with responsive surfaces by hepatoma cells.
FIG. 5 is a tissue distribution of gold nanoflowers with responsive surfaces injected into tumor-bearing mice.
FIG. 6 shows the radiosensitizing effect of gold nanoflowers with responsive surfaces injected into tumor-bearing mice.
FIG. 7 shows the radiosensitization effect of gold nanospheres with responsive surfaces injected into tumor-bearing mice.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1:
1) to a solution of 1mL (0.1mM) gold nanorods (self-prepared, 14 × 50nm in size, method see below) was added 360. mu.lL glutathione solution with the concentration of 0.01M is vigorously stirred for 2 hours at room temperature; then 5.81mL of 0.033mM cetyltrimethylammonium chloride (CTAC) and 40. mu.L of 25mM HAuCl were added in succession4Solution, 30. mu.L of 1mM AgNO3Solution, 60. mu.L ascorbic acid solution with a concentration of 100 mM. Stirring vigorously for half an hour at room temperature, and standing overnight in a constant-temperature water bath at 28 deg.C to obtain monodisperse gold nanoflowers. The transmission electron micrograph is shown in FIG. 1.
Preparing gold nanorods by adopting a seed growth method:
preparing gold seeds: 0.25mL of 10mM HAuCl4Added to 10mL of a 0.1M CTAB solution, followed by 0.6mL of 0.01M NaBH under vigorous stirring4Continuously and violently stirring for 2min, standing in a water bath at 28 ℃ for 2h, and aging for later use.
Seed growth: HAuCl was added to 100mL of 0.1M CTAB aqueous solution in sequence with magnetic stirring4(5mL，0.01M)、AgNO3(2.2mL, 0.01M), HCl (0.8mL, 1M), adding AA (0.7mL, 78mM) after the solution is fully stirred, observing that the solution gradually turns colorless from orange yellow, then adding 120 μ L of seed solution, stirring, and standing at 28 deg.C for 12 h. And finally, centrifuging (12000rpm for 12min), discarding the supernatant, and washing twice to remove free CTAB, thereby obtaining the gold nanorods with the resonance absorption wavelength of 808 nm.
2) Centrifuging 10mg of gold nanoflowers obtained in the step 1) to remove excessive CTAC ligand, dispersing in 5mg/mL polypeptide-poly-sarcosine solution, and placing in a constant temperature water bath at 30 ℃ for shaking. After one day the supernatant was removed by centrifugation and the procedure repeated again. The functionalized gold nanoflowers were then collected by centrifugation. The polypeptide molecule content on the surface of the nanomaterial is analyzed by the BCA kit, and the result is shown in FIG. 2, which shows that the polypeptide accounts for 6.3% of the nanomaterial by mass.
3) 1mg of the gold nanoflowers obtained in step 2) were mixed with 5. mu.g/mL of MMP-9 physiological saline solution (containing 0.9% NaCl) and allowed to stand at 37 ℃ for 4 hours. The particle size and surface potential of the gold nanoflowers before and after mixing with MMP-9 were measured. The results are shown in FIG. 3, which shows that the size of gold nanoparticles is increased significantly by MMP-9 enzyme treatment, and the surface is changed from neutral to positive charge, indicating that the surface molecules are changed.
4) Co-culturing the gold nanoflowers (Au @ Res, 100 mu g/mL) obtained in the step 2) with the liver cancer cell HepG2 for 24 hours. MMP-9 was optionally added at 5. mu.g/mL to the cell culture medium, and gold nanoparticles (Au @ NoR) with surface-modified non-responsive polypeptide-polymyosine molecules served as controls. And detecting the concentration of the gold nanoflowers in the liver cancer cells. The results, see figure 4, show that nanoflowers with responsive surfaces have the highest amount of cellular phagocytosis in the presence of MMP-9 enzyme.
5) Injecting the gold nanoflower particles (Au @ Res, 5mg/kg) obtained in the step 2) into tumor-bearing mice, and detecting the content of the gold nanoflowers in main organs and tumors after 24 hours. Results referring to fig. 5, it is shown that a responsive surface can increase the degree of gold nanoflowers enrichment within the tumor.
6) Injecting gold nanoflowers (Au @ Res, 5mg/kg) obtained in the step 2) into tumor-bearing mice, and performing 6Gy irradiation treatment after 24 hours. Results referring to fig. 6, it is shown that gold nanoflowers with responsive surfaces can achieve the best radiosensitizing effect.
Example 2:
the difference from example 1 is step 2): centrifuging 10mg of gold nanoflowers obtained in the step 1) to remove excessive CTAC ligand, dispersing in 20mg/mL polypeptide-poly-sarcosine solution, and placing in a constant temperature water bath at 30 ℃ for shaking. After one day the supernatant was removed by centrifugation and the procedure repeated again. The functionalized gold nanoflowers were then collected by centrifugation. The polypeptide molecule content on the surface of the nanomaterial is analyzed by the BCA kit, and the result is shown in FIG. 2, which shows that the polypeptide accounts for 7.9% of the nanomaterial by mass.
Example 3:
the difference from example 1 is step 2): dispersing 10mg of citric acid-protected spherical gold nanoparticles (with the particle size of 5-20 nm) in 5mg/mL polypeptide-poly-sarcosine solution, and placing in a constant-temperature water bath at 30 ℃ for shaking. After one day the supernatant was removed by centrifugation and the procedure repeated again. And then collecting the functionalized gold nanospheres by centrifugation. The same dose of gold nanospheres (calculated according to the gold content) is injected into tumor-bearing nude mice, and radiation therapy is performed. The results are shown in fig. 7, which shows that the radiosensitization effect of the gold nanospheres is worse than that of the gold nanoflowers with the same responsive surface, and thus the gold nanoflowers have better radiosensitization effect.
Claims (5)
1. Gold nanoflowers with liver cancer targeting and radiotherapy sensitivity enhancing characteristics are 50-200 nm in diameter, 4-10 protruding thorns are provided, and the gold nanoflowers are prepared by the following method:
(1) adding a glutathione solution into a gold nanorod solution, violently stirring at room temperature for 1-2 hours, and then sequentially adding a hexadecyl trimethyl ammonium chloride solution and HAuCl4Solution, AgNO3The solution and the ascorbic acid solution are stirred vigorously for 0.5-1 hour at room temperature, and then are kept stand overnight in a constant-temperature water bath at the temperature of 26-28 ℃ to obtain monodisperse gold nanoflowers;
(2) centrifuging gold nanoflowers to remove excessive CTAC ligand, dispersing the gold nanoflowers in a polypeptide-polymyosin solution, placing the polypeptide-polymyosin solution in a constant-temperature water bath at the temperature of 28-30 ℃ to vibrate for 18-24 h, centrifuging to remove supernate, continuously dispersing the supernate in the polypeptide-polymyosin solution, placing the polypeptide-polymyosin solution in a constant-temperature water bath at the temperature of 28-30 ℃ to vibrate for 18-24 h, centrifuging, and collecting gold nanoflowers with liver cancer targeting and radiotherapy sensitization characteristics; the polypeptide-poly-sarcosine has the molecular sequence of CCV GRKKRRQRRRPQGGPLGV-poly-sarcosine, wherein the molecular weight of poly-sarcosine is about 2 kD.
2. A method of making gold nanoflowers according to claim 1, comprising:
(A) adding a glutathione solution into a gold nanorod solution, violently stirring at room temperature for 1-2 hours, and then sequentially adding a hexadecyl trimethyl ammonium chloride solution and HAuCl4Solution, AgNO3The solution and the ascorbic acid solution are stirred vigorously for 0.5-1 hour at room temperature, and then are kept stand overnight in a constant-temperature water bath at the temperature of 26-28 ℃ to obtain monodisperse gold nanoflowers; the gold nanorods: glutathione: hexadecyltrimethylammonium chloride: HAuCl4：AgNO3: the ratio of the amounts of ascorbic acid in the substance was 0.1 mmol: 1-5 mmol: 0.05-0.3 mmol: 5-15 mmol:0.01～0.05mmol：1～10mmol；
(B) centrifuging gold nanoflowers to remove excessive CTAC ligand, dispersing the gold nanoflowers into 3-8 mg/mL polypeptide-polymyosin solution, placing the gold nanoflowers into a constant-temperature water bath at 28-30 ℃ to vibrate for 18-24 h, centrifuging to remove supernatant, continuously dispersing the gold nanoflowers into polypeptide-polymyosin solution, placing the gold nanoflowers into a constant-temperature water bath at 28-30 ℃ to vibrate for 18-24 h, centrifuging, and collecting the gold nanoflowers with the characteristics of liver cancer targeting and radiotherapy sensitization; the polypeptide-poly-sarcosine has the molecular sequence of CCVGRKKRRQRRRPQGGPLGV-poly-sarcosine, wherein the molecular weight of poly-sarcosine is about 2 kD.
3. The method of claim 2, wherein the method is performed as follows:
(A) 1mL of glutathione solution was added to 1mL of 0.1mM gold nanorod solution, and after vigorous stirring at room temperature for 2 hours, 5.81mL of 0.033mM cetyltrimethylammonium chloride and 40. mu.L of 25mM HAuCl were added in this order4Solution, 30. mu.L of 1mM AgNO3The solution and 60 mu L of ascorbic acid solution with the concentration of 100mM are stirred vigorously for 0.5 to 1 hour at room temperature, and then are kept stand overnight in a constant-temperature water bath at the temperature of 26 to 28 ℃ to obtain monodisperse gold nanoflowers;
(B) centrifuging gold nanoflowers to remove excessive CTAC ligand, dispersing the gold nanoflowers in 5mg/mL polypeptide-polymyosin solution, placing the gold nanoflowers in 30 ℃ constant temperature water bath to shake for 24 hours, centrifuging to remove supernate, continuously dispersing the gold nanoflowers in polypeptide-polymyosin solution, placing the gold nanoflowers in 30 ℃ constant temperature water bath to shake for 24 hours, centrifuging, and collecting the gold nanoflowers with the characteristics of liver cancer targeting and radiotherapy sensitization.
4. The use of gold nanoflowers with liver cancer targeting and radiosensitizing properties according to claim 1 for the preparation of a medicament for the treatment of liver cancer.
5. The use of claim 4, wherein said medicament is a radiotherapeutic agent.
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