WO2022252012A1 - 肿瘤诊疗剂及其制备方法和应用 - Google Patents

肿瘤诊疗剂及其制备方法和应用 Download PDF

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WO2022252012A1
WO2022252012A1 PCT/CN2021/097236 CN2021097236W WO2022252012A1 WO 2022252012 A1 WO2022252012 A1 WO 2022252012A1 CN 2021097236 W CN2021097236 W CN 2021097236W WO 2022252012 A1 WO2022252012 A1 WO 2022252012A1
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albumin
tumor
treatment agent
solution
nanoparticles
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PCT/CN2021/097236
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English (en)
French (fr)
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郑海荣
胡德红
盛宗海
刘新
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深圳先进技术研究院
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Publication of WO2022252012A1 publication Critical patent/WO2022252012A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • 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
    • A61K47/51—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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the application relates to the field of biomedical technology, in particular to tumor diagnosis and treatment agents and their preparation methods and applications.
  • molecular imaging technology provides technical support for early diagnosis, treatment and prognosis evaluation of tumors.
  • molecular imaging probes such as magnetic, optical, acoustic, and nuclide, have been developed around the early diagnosis, treatment, and prognosis evaluation of tumors.
  • detection depth and resolution of molecular imaging probes still need to be improved, and the current molecular imaging probes are only used for imaging and have a single role.
  • the application provides a tumor diagnosis and treatment agent and its preparation method.
  • the tumor diagnosis and treatment agent can realize magneto-optic dual-mode imaging and drug treatment at the same time, achieve the integration of diagnosis and treatment, and has good targeting and biocompatibility properties and stability, and has broad application prospects in the field of biomedicine.
  • the present application provides a tumor diagnosis and treatment agent, including albumin, anti-tumor drugs and gold clusters dispersed in the albumin, and a magnetic resonance contrast agent and a Target penetrating peptides.
  • targeting membrane-penetrating peptides enables tumor diagnosis and treatment agents to penetrate the cell membrane into tumor cells; gold clusters can emit light in the second near-infrared region to achieve optical imaging, magnetic resonance contrast agents can achieve magnetic resonance imaging, and realize magneto-optical dual Simultaneous imaging; the tumor diagnosis and treatment agent carries anti-tumor drugs, so as to achieve precise drug treatment while imaging.
  • the particle size of the tumor diagnosis and treatment agent is 30nm-200nm.
  • the particle size of the gold clusters is less than 2nm.
  • the gold cluster includes 8-100 gold atoms.
  • the molar ratio of the targeting penetrating peptide to the albumin is 1-100.
  • the targeting penetrating peptide includes at least one of iRGD, iNGR, TAT and CTX.
  • the molar ratio of the magnetic resonance contrast agent to the albumin is 1-100.
  • the magnetic resonance contrast agent includes at least one of Gd-DTPA, Gd-DOTA, Gd-DTTA and Gd-HOPO.
  • the molar ratio of the anti-tumor drug to the albumin is 1-10000.
  • the antitumor drug includes at least one of paclitaxel, docetaxel, doxorubicin, cisplatin, irinotecan, camptothecin, carmustine and curcumin.
  • the tumor diagnosis and treatment agent provided in this application can realize magneto-optical dual-mode imaging and drug treatment at the same time, achieve the integration of diagnosis and treatment, and can meet the needs of clinical applications.
  • the present application provides a method for preparing a tumor diagnosis and treatment agent, comprising:
  • the solution containing the first nanoparticles Adding a reducing agent with a sulfhydryl group to the solution containing the first nanoparticles, and reacting to obtain a reaction solution, wherein the first nanoparticles include albumin and gold clusters dispersed in the albumin;
  • the reaction solution is ultrasonically treated, and a solution containing anti-tumor drugs is added to the reaction solution to react to obtain a solution containing second nanoparticles, the second nanoparticles include the albumin and are dispersed in the said gold clusters and said antineoplastic drug within albumin;
  • the tumor diagnosis and treatment agent includes the albumin, all the components dispersed in the albumin The anti-tumor drug and the gold cluster, as well as the magnetic resonance contrast agent and the targeting penetrating peptide linked on the surface of the albumin through a chemical bond.
  • the preparation of the first nanoparticles includes: mixing the chloroauric acid solution and the albumin solution, adding sodium hydroxide solution and sodium borohydride solution, reacting and filtering to obtain the first nanoparticles.
  • the reducing agent with a thiol group includes at least one of glutathione, cysteine, mercaptoethanol and dithiothreitol; the reducing agent with a thiol group and the first
  • the molar ratio of the albumin in the solution of nanoparticles is 5-30.
  • the solution containing the second nanoparticle reacts with the targeting penetrating peptide, comprising: adding N-hydroxysuccinimide and 1-(3-dimethyl After aminopropyl)-3-ethylcarbodiimide hydrochloride is reacted, the solution containing the second nanoparticles is added to react to obtain a solution containing the third nanoparticles, wherein the third nanoparticles include the The albumin, the anti-tumor drug and the gold cluster dispersed in the albumin, and the targeting penetrating peptide connected on the surface of the albumin through chemical bonds.
  • the preparation method of the tumor diagnosis and treatment agent provided by the application is simple and convenient to operate, can realize industrial production, and is beneficial to the wide use of the tumor diagnosis and treatment agent.
  • the present application provides the application of the tumor diagnosis and treatment agent prepared by the preparation method described in the first aspect or the second aspect in a drug for preventing, diagnosing or treating tumors.
  • Fig. 1 is a flowchart of a preparation method of a tumor diagnosis and treatment agent provided in an embodiment of the present application.
  • Fig. 2 is a schematic diagram of the preparation of a tumor diagnosis and treatment agent provided by an embodiment of the present application.
  • Example 3 is a transmission electron micrograph of the tumor diagnosis and treatment agent prepared in Example 1.
  • Figure 4 is a near-infrared two-zone fluorescence microscope image of the cells.
  • Figure 5 is the near-infrared second region optical and magnetic resonance imaging images of mice.
  • the application provides a tumor diagnosis and treatment agent, including albumin, anti-tumor drugs and gold clusters dispersed in the albumin, and a magnetic resonance contrast agent and a targeted transmembrane chemically bonded on the surface of the albumin peptide.
  • the tumor absorbs and dissipates incident light to varying degrees, resulting in attenuation of the incident light, reducing the imaging depth, signal-to-background ratio, and resolution, making the border of the tumor blurred Difficult to identify;
  • traditional fluorescence such as near-infrared first-range fluorescence
  • near-infrared second-range fluorescence 1000nm-1700nm
  • the tumor diagnostic and therapeutic agent provided by the application has gold clusters, which can emit light in the second near-infrared region, so that high-quality tumor imaging results can be obtained
  • the tumor has a magnetic resonance contrast agent, which can realize magnetic resonance imaging.
  • the tumor diagnosis and treatment agent carries anti-tumor drugs
  • tumor Cells contain a large amount of glutathione, which can open the disulfide bonds of tumor diagnostic and therapeutic agents to release anti-tumor drugs, and then realize the release and treatment of anti-tumor drugs while imaging, achieving the integration of diagnosis and treatment.
  • albumin is a biological endogenous protein, which has the advantages of biodegradability and non-toxicity.
  • albumin As a carrier of tumor diagnostic and therapeutic agents, albumin has high stability and water solubility, which can improve the performance of tumor diagnostic and therapeutic agents. stability and hydrophilicity.
  • the albumin includes at least one of human serum albumin, bovine serum albumin, porcine serum albumin, and recombinant serum albumin.
  • the above-mentioned albumin has good biocompatibility and is favorable for clinical use.
  • the albumin is bovine serum albumin.
  • the particle size of the albumin is 20nm-150nm. Further, the particle size of albumin is 25nm-100nm. Specifically, the particle size of the albumin can be, but not limited to, 20nm, 25nm, 40nm, 50nm, 80nm, 90nm, 100nm or 120nm. It can be understood that multiple albumins may be included in the tumor diagnosis and treatment agent provided in this application.
  • gold clusters are uniformly dispersed inside albumin, which can be imaged in the second near-infrared region, and high-quality imaging results can be obtained.
  • the particle size of the gold clusters is less than 2nm. Further, the particle size of the gold clusters is less than 1.5nm.
  • the gold cluster includes 8-100 gold atoms. In one embodiment, the gold cluster includes 8-20 gold atoms. In another embodiment, the gold cluster includes 30-50 gold atoms. In yet another embodiment, the gold cluster includes 80-95 gold atoms. In this application, the amount of gold clusters dispersed in albumin can be set according to need.
  • the molar ratio of gold atoms to albumin in the tumor diagnosis and treatment agent is 10-1000. Therefore, the tumor diagnosis and treatment agent can have a high luminous intensity in the second near-infrared region and a good imaging effect. Further, the molar ratio of gold atoms to albumin in the tumor diagnosis and treatment agent is 50-700. In the embodiment of the present application, there is an Au-S bond between the gold cluster and the albumin. Through the Au-S bond, the gold clusters can exist in the interior of albumin stably and for a long time.
  • the anti-tumor drugs are dispersed inside the albumin, targeting the penetrating peptide to allow the tumor diagnosis and treatment agents to enter the tumor cells, so that the anti-tumor drugs can enter the tumor cells, and at the same time, the water solubility of the hydrophobic anti-tumor drugs is improved. Therefore, anti-tumor drugs can precisely act on tumor cells and improve the therapeutic effect.
  • the molar ratio of the antitumor drug to the albumin is 1-10000. Therefore, the tumor diagnosis and treatment agent carries a sufficient amount of antitumor drugs, and the therapeutic effect of the tumor diagnosis and treatment agent is improved. Further, the molar ratio of the antitumor drug to the albumin is 20-5000.
  • the molar ratio of the antineoplastic drug to the albumin is 100-1000. Specifically, the molar ratio of the antineoplastic drug to the albumin can be, but not limited to, 20-100, 50-500, 100-2000, 150-4000 or 500-5000.
  • the antitumor drug includes at least one of paclitaxel, docetaxel, doxorubicin, cisplatin, irinotecan, camptothecin, carmustine and curcumin.
  • the above-mentioned anti-tumor drugs can form a bond energy with the albumin, so that they can be stably dispersed inside the albumin for a long time, and after entering the tumor cells, they can be released and act on the tumor cells.
  • the antineoplastic drug includes paclitaxel.
  • the magnetic resonance contrast agent is connected to the surface of albumin through a chemical bond, so that the magnetic resonance contrast agent can exist stably for a long time and ensure the progress of magnetic resonance imaging.
  • the magnetic resonance contrast agent is linked to the surface of the albumin through a covalent bond.
  • the magnetic resonance contrast agent is linked to the surface of the albumin through an amide bond.
  • the magnetic resonance contrast agent includes at least one of Gd-DTPA, Gd-DOTA, Gd-DTTA and Gd-HOPO.
  • the magnetic resonance contrast agent has stable performance, low toxicity, good water solubility and less required amount.
  • the molar ratio of the magnetic resonance contrast agent to the albumin is 1-100.
  • the molar ratio of the magnetic resonance contrast agent to the albumin is 10-80. Furthermore, the molar ratio of the magnetic resonance contrast agent to the albumin is 20-65. Specifically, the molar ratio of the magnetic resonance contrast agent to albumin may be, but not limited to, 15, 25, 30, 38, 45, 50, 60, 74 or 85, etc. Within the above molar ratio range, the magnetic resonance contrast agent has no effect on the structure of the tumor diagnosis and treatment agent, and can realize the magneto-optical dual-modal imaging function together with the gold cluster, improving the imaging quality.
  • the targeting penetrating peptide is linked to the surface of albumin through a chemical bond, so that the targeting penetrating peptide can exist stably for a long time, ensuring that tumor diagnostic and therapeutic agents can target and enter tumor cells for imaging and treatment.
  • the targeting penetrating peptide is linked to the surface of the albumin through a covalent bond.
  • the targeting penetrating peptide is linked to the surface of the albumin through an amide bond.
  • the molar ratio of the targeting penetrating peptide to the albumin is 1-100. Further, the molar ratio of the targeting penetrating peptide to the albumin is 15-80.
  • the molar ratio of the targeting penetrating peptide to the albumin is 20-70.
  • the molar ratio of the targeting penetrating peptide to albumin can be, but not limited to, 15, 20, 25, 30, 35, 42, 55, 60, 75 or 80, etc.
  • the tumor diagnosis and treatment agent can efficiently and rapidly target tumor cells and enter into tumor cells, which is beneficial to magneto-optical dual-mode imaging and drug release therapy.
  • the targeting penetrating peptide includes at least one of iRGD, iNGR, TAT and CTX.
  • targeting penetrating peptides can enhance the permeability of blood vessels by interacting with vascular permeability regulating molecules, thereby promoting the entry of tumor diagnostic and therapeutic agents into cells;
  • iRGD has an RGD domain
  • iNGR has an NGR domain, which can specifically bind to integrins ⁇ v ⁇ 3 and ⁇ v ⁇ 5 on the surface of tumor cells and blood vessels around the tumor, and target to tumor sites with high integrin expression.
  • the amino acid sequence of iRGD is CRGDKGPDC.
  • the amino acid sequence of iNGR is CRNGRGPDC.
  • the particle size of the tumor diagnosis and treatment agent is 30nm-200nm. Further, the particle size of the tumor diagnosis and treatment agent is 40nm-180nm. Furthermore, the particle size of the tumor diagnosis and treatment agent is 50nm-150nm. Specifically, the particle size of the tumor diagnosis and treatment agent can be, but not limited to, 40nm-60nm, 50nm-60nm, 70nm-90nm, 80nm-110nm, 100nm-150nm, 110nm-150nm, 130nm-160nm or 160nm-200nm. In this particle size range, the tumor diagnosis and treatment agent has better dispersion performance and stability, which is beneficial to its use.
  • Fig. 1 is a flow chart of the preparation method of the tumor diagnosis and treatment agent provided in one embodiment of the present application.
  • the method prepares the tumor diagnosis and treatment agent in any of the above embodiments, and the preparation method includes:
  • Operation 101 Add a reducing agent with a sulfhydryl group to the solution containing the first nanoparticles, and react to obtain a reaction solution, wherein the first nanoparticles include albumin and gold clusters dispersed in the albumin.
  • Operation 102 Ultrasonicize the reaction solution, and add a solution containing anti-tumor drugs to the reaction solution to react to obtain a solution containing second nanoparticles, the second nanoparticles include albumin and gold clusters dispersed in the albumin and anticancer drugs.
  • the tumor diagnosis and treatment agent After reacting the solution containing the second nanoparticle with the targeting penetrating peptide and the magnetic resonance contrast agent, the tumor diagnosis and treatment agent is obtained by drying, and the tumor diagnosis and treatment agent includes albumin, antitumor drugs dispersed in the albumin, and gold clusters Clusters, as well as magnetic resonance contrast agents and targeting penetrating peptides attached to the surface of albumin by chemical bonds.
  • the first nanoparticles are prepared by biomineralization.
  • the preparation of the first nanoparticles includes: mixing the chloroauric acid solution and the albumin solution, adding sodium hydroxide solution and sodium borohydride solution, reacting and filtering to obtain the first nanoparticles.
  • the solubility of the chloroauric acid solution is 1 mM-50 mM, and the volume of the chloroauric acid solution is 0.1 mL-3 mL.
  • the solubility of the albumin solution is 1 mg/mL-50 mg/mL, and the volume of the albumin solution is 0.1 mL-5 mL.
  • the solubility of the sodium hydroxide solution is 0.1M-2M, and the volume of the sodium hydroxide solution is 0.1mL-2mL.
  • the solubility of the sodium borohydride solution is 0.01M-0.5M, and the volume of the sodium borohydride solution is 0.01mL-0.5mL.
  • the chloroauric acid solution and the albumin solution are mixed at 15°C-30°C, and then sodium hydroxide solution and pre-cooled sodium borohydride solution are added in sequence, and the solution turns dark brown; then the solution is Incubate at 130rpm-200rpm, 25°C-37°C to prepare the first nanoparticles.
  • the first nanoparticle can be stored in a buffer solution for future use. Specifically, but not limited to, the first nanoparticle can be placed in a 0.1M phosphate buffer solution for dialysis and then stored at low temperature.
  • the reducing agent with mercapto group before adding the reducing agent with mercapto group, it also includes adjusting the pH of the solution containing the first nanoparticles to 7-12. This facilitates the subsequent opening of albumin disulfide bonds.
  • the pH of the solution containing the first nanoparticles is adjusted to 7-9.
  • the pH of the solution containing the first nanoparticles is adjusted to 9-12.
  • the pH of the solution containing the first nanoparticles can be adjusted to 7, 8, 9, 10, 11 or 12, but not limited to.
  • a sodium hydroxide solution is used to adjust the pH, for example, a 1 mol/L NaOH solution is used to adjust the pH of the solution containing the first nanoparticles.
  • the reducing agent with sulfhydryl group includes at least one of glutathione, cysteine, mercaptoethanol and dithiothreitol.
  • the disulfide bond of albumin can be opened by using a reducing agent with sulfhydryl group, so that the structure of albumin can be unfolded, which is beneficial to the entry and dispersion of subsequent anti-tumor drugs.
  • the molar ratio of the reducing agent with sulfhydryl group to the albumin in the solution containing the first nanoparticles is 5-30.
  • the molar ratio of the reducing agent with sulfhydryl group to the albumin in the solution containing the first nanoparticles is 10-25. Furthermore, the molar ratio of the reducing agent with sulfhydryl group to the albumin in the solution containing the first nanoparticle is 12-20. Specifically, the molar ratio of the reducing agent with sulfhydryl groups to the albumin in the solution containing the first nanoparticles may be, but not limited to, 10, 12, 15, 17, 20, 21, 24, 28 or 30, etc. The above-mentioned range is beneficial to the unfolding of the albumin structure without excessive damage to the albumin structure, so that the albumin can still load gold clusters and anti-tumor drugs.
  • the concentration of the reducing agent with sulfhydryl group is 0.01 mol/L-2 mol/L. Further, the concentration of the reducing agent with mercapto groups is 0.05mol/L-1.8mol/L. In the embodiment of the present application, the reaction is carried out at 30°C-60°C for 1min-5min. Further, the reaction is carried out at 35°C-60°C for 3min-5min. Under these reaction conditions, it is conducive to the rapid opening of albumin disulfide bonds.
  • anti-tumor drugs are added at the same time of ultrasonic treatment, so as to facilitate the entry of anti-tumor drugs into the unfolded albumin, increase the amount of anti-tumor drugs loaded on albumin, enhance the curative effect of tumor diagnosis and treatment agents, and at the same time prevent Aggregation between albumins affects the preparation of nano-diagnostics.
  • the power of ultrasound is 5W-20W. Further, the power of ultrasound is 10W-15W.
  • the reaction solution is processed in an ultrasonic cell breaker, so that a large amount of anti-tumor drugs can be further entered into the albumin, and the loading of anti-tumor drugs in the tumor diagnosis and treatment agent can be increased.
  • the injection rate of the solution containing antitumor drugs is 30ml/s-80ml/s.
  • the injection rate of the solution containing antitumor drugs may be, but not limited to, 30ml/s, 40ml/s, 50ml/s, 60ml/s, 70ml/s or 80ml/s.
  • the solvent of the antitumor drug-containing solution includes at least one of dimethyl sulfoxide, methanol, ethanol, propanol and tert-butanol.
  • the concentration of the solution containing the antitumor drug is 0.01M-2M.
  • the concentration of the solution containing the antitumor drug may be, but not limited to, 0.01M, 0.05M, 0.1M, 0.3M, 0.7M, 1M, 1.2M, 1.5M, 1.8M or 2M.
  • the molar ratio of the antitumor drug to the albumin is 1-10000.
  • the tumor diagnosis and treatment agent carries a sufficient amount of antitumor drugs, and the therapeutic effect of the tumor diagnosis and treatment agent is improved.
  • the molar ratio of the antineoplastic drug to the albumin can be, but not limited to, 20-100, 50-500, 100-2000, 150-4000 or 500-5000.
  • the reaction includes performing at 30°C-60°C for 10min-30min. Further, the reaction is carried out for 15min-25min. Under such reaction conditions, it is favorable for the full contact and mixing of antitumor drugs and albumin, so that the albumin can be loaded with a sufficient amount of antitumor drugs and improve the curative effect of tumor diagnosis and treatment agents.
  • antineoplastic drugs are dispersed inside albumin.
  • a reducing agent with a sulfhydryl group is used to open the disulfide bond of the albumin, so that the albumin structure unfolds, and after the antineoplastic drug is added, multiple albumins aggregate and encapsulate the antineoplastic drug.
  • the tumor diagnosis and treatment agent includes a plurality of albumins, the gold clusters are dispersed in the albumin, and the plurality of albumins aggregate and wrap the anti-tumor drug, and the magnetic resonance contrast agent and the targeting penetrating peptide are connected in the albumin through a chemical bond. protein surface. Further, antineoplastic drugs are encapsulated in the center of albumin.
  • the dialysis treatment is performed by placing the solution containing the second nanoparticles in a phosphate buffer solution with a pH of 7-12.
  • the solution containing the second nanoparticles is placed in a dialysis bag, and the dialysis bag is placed in 1 L, pH7-12 phosphate buffer solution for dialysis for 5h-10h, and then the dialysis bag is placed in water Dialysis 1h-3h can be.
  • the solution containing the second nanoparticle reacts with the targeting penetrating peptide, comprising: adding N-hydroxysuccinimide and 1-(3-dimethylamino to the solution containing the targeting penetrating peptide After the reaction of propyl)-3-ethylcarbodiimide hydrochloride, the solution containing the second nanoparticle is added to react to obtain the solution containing the third nanoparticle, wherein the third nanoparticle includes albumin, dispersed in white Antineoplastic drugs and gold clusters within the protein, and targeted penetrating peptides linked to the surface of albumin by chemical bonds.
  • N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added to the solution containing the targeting penetrating peptide at 25° C. React at -30°C for 2h-4h. In another embodiment, after adding the solution containing the second nanoparticles, react at 25°C-30°C for 12h-14h.
  • the solvent of the solution containing the penetrating peptide includes at least one of dimethyl sulfoxide, methanol, ethanol, propanol and tert-butanol.
  • the tumor diagnosis and treatment agent can be obtained by mixing the solution containing the third nanoparticle with the NHS-magnetic resonance contrast agent and reacting at 25°C-30°C for 2h-4h.
  • the targeting penetrating peptide and the magnetic resonance contrast agent form a chemical bond with albumin through a chemical reaction, so that they can be stably loaded on the surface of albumin for a long time.
  • dialysis treatment is also included before drying; through dialysis treatment, impurities in the solution can be removed.
  • dialysis is performed by placing the solution in a phosphate buffered saline solution with a pH of 7-12.
  • the solution is placed in a dialysis bag, and the dialysis bag is placed in 1L, pH 7-12 phosphate buffer solution for dialysis at 25°C-30°C for 5h-10h, and then the dialysis bag is placed in water Dialysis 1h-3h can be.
  • the drying includes pre-freezing at 0°C for 1h-3h, then transferring to -20°C for freezing for 2h-5h, and then freeze-drying for 8h-15h.
  • FIG. 2 is a schematic diagram of the preparation of a tumor diagnosis and treatment agent provided in an embodiment of the application.
  • albumin is mixed with a solution containing gold elements, and under the action of sodium hydroxide and sodium borohydride, a Albumin gold clusters, that is, the first nanoparticles, the first nanoparticles include albumin and gold clusters dispersed in albumin; through glutathione treatment, and adding paclitaxel solution, the albumin gold clusters are generated Cluster nanomedicine, that is, the second nanoparticle, the second nanoparticle includes albumin and gold clusters dispersed in albumin and anti-tumor drug paclitaxel; then react with NHS-iRGD and DTPA-Gd to prepare albumin Gold cluster-targeted nanomedicine, that is, a tumor diagnosis and treatment agent, the tumor diagnosis and treatment agent includes albumin, the antitumor drug paclitaxel and gold clusters dispersed in the albumin, and a magnetic resonance contrast agent linked to the surface
  • This application provides a magneto-optical dual-mode nano-therapeutic agent that uses albumin as a carrier, uses a targeting penetrating peptide as a targeting ligand, and is loaded with antitumor drugs; uses albumin as a carrier, and uses biomineralization to synthesize nearly Gold clusters that emit light in the second infrared region to obtain the first nanoparticles; then prepare water-soluble, monodisperse second nanoparticles by a one-step bioreduction method; finally, couple the targeting membrane-penetrating peptide on the second nanoparticles by chemical bonds and magnetic resonance contrast agents to achieve magneto-optic dual-mode imaging.
  • the preparation method of the tumor diagnosis and treatment agent provided by the application is simple and convenient to operate, can realize industrial production, and is beneficial to the wide use of the tumor diagnosis and treatment agent.
  • the present application provides the application of the above-mentioned tumor diagnosis and treatment agent in drugs for preventing, diagnosing or treating tumors.
  • the tumor can be but not limited to cancer, such as brain glioma and the like.
  • the tumor diagnosis and treatment agent provided by the present application can be applied to the prevention, diagnosis or treatment of glioma; the tissue boundary of glioma is unclear, and the blood-brain barrier hinders the entry of imaging materials and drugs,
  • the tumor diagnosis and treatment agent provided by this application has a targeted penetrating peptide, which can avoid the influence of the blood-brain barrier and enter the glioma cells. At the same time, it can effectively identify gliomas through near-infrared second-region optical imaging and magnetic resonance imaging.
  • the anti-tumor drugs carried at the same time can precisely target the brain glioma and play a therapeutic role.
  • Step 1 Mix chloroauric acid solution (10mM, 1.25mL) and albumin solution (50mg/mL, 2.5mL) at 25°C, then add sodium hydroxide solution (1M, 0.25mL) and pre-cooled Sodium borohydride solution (0.1M, 0.1mL), the solution turned dark brown; then the solution was incubated at 180rpm, 37°C to obtain the first nanoparticles; the first nanoparticles were placed in 0.1M phosphate buffer Store at 4°C after dialysis.
  • Step 2 Take 2mL of the first nanoparticle solution, and then use 1mol/L NaOH solution to adjust the pH value of the first nanoparticle solution to 7; add glutathione to it to obtain a reaction solution, and shake gently at 60°C After reacting for 0.05h, the mole number of glutathione was 10 times that of albumin.
  • Step 3 Treat the solution after the reaction in step 2 with an ultrasonic cell disruptor at 60°C.
  • the power of the ultrasonic cell disruptor is 10W, and at the same time inject 2 mL of paclitaxel dimethyl The sulfoxide solution, the solution was reacted at 60° C. for 20 minutes to obtain the second nanoparticle solution.
  • Step 4 Transfer the second nanoparticle solution obtained in step 3 into a dialysis bag, and keep the temperature at 30°C, place the dialysis bag in 1 L of PBS buffer solution with pH 7 for dialysis for 10 hours, and change the solution every 12 hours during this period. Each time, 1 L of PBS buffer solution with pH 7 was used, and then the dialysis bag was placed in 5 L of double distilled water for dialysis for 1 hour to obtain the second nanoparticles.
  • Step 5 Dissolve iRGD in DMSO solution, then add N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, in React at 25°C for 3h, then add the second nanoparticle, react at 25°C for 15h; then add NHS-DTPA-Gd to the above solution, react at 25°C for 3h, and obtain a solution containing the tumor diagnosis and treatment agent.
  • Step 6 Transfer the solution containing the tumor diagnosis and treatment agent obtained in step 5 into a dialysis bag, and keep the temperature at 30°C, place the dialysis bag in 1L, pH7 PBS buffer solution for dialysis for 10 hours, and change the solution every 12 hours during the period.
  • 1 L of PBS buffer solution with pH 7 was used, and then the dialysis bag was dialyzed in 5 L of double-distilled water for 1 h; then it was pre-frozen at -0°C for 1 h, then transferred to -20°C for 2 h, and then placed in Freeze-dry in a freeze dryer for 12 hours to obtain a tumor diagnosis and treatment agent.
  • the prepared tumor diagnosis and treatment agent was observed by transmission electron microscope, and the results are shown in Figure 3. It can be seen that the tumor diagnosis and treatment agent prepared by the present application is nano-scale, uniform in size, and the particles are relatively dispersed.
  • the tumor diagnosis and treatment agent prepared in Example 1 After incubating the tumor diagnosis and treatment agent prepared in Example 1 with C6 cells for 3 hours, the cells were observed by a near-infrared two-zone fluorescence microscope, and the results are shown in FIG. 4 . It can be seen that the tumor diagnosis and treatment agent provided by the present application can generate fluorescence signals in the second near-infrared region, which is beneficial for imaging.
  • the tumor diagnosis and treatment agent prepared in Example 1 was dissolved in PBS solution (concentration is 9 mg/ml), and injected into the mouse body through tail vein injection, and after 24 hours of injection, the mouse was observed by light in the second near-infrared region and nuclear magnetic resonance. The brain tumor site, the results are shown in Figure 5. It can be seen that the tumor diagnosis and treatment agent provided by the present application has the effect of magneto-optic dual-mode imaging.

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Abstract

提供了一种肿瘤诊疗剂，包括白蛋白、分散在所述白蛋白内的抗肿瘤药物和金团簇，以及通过化学键连接在所述白蛋白表面的磁共振造影剂和靶向穿膜肽。该肿瘤诊疗剂可同时实现磁光双模成像和药物治疗，达到诊疗一体化，并且具有良好的靶向性、生物相容性和稳定性，在生物医学领域有广泛的应用前景。还提供了肿瘤诊疗剂的制备方法和应用。

Description

肿瘤诊疗剂及其制备方法和应用 技术领域
本申请涉及生物医学技术领域，尤其涉及肿瘤诊疗剂及其制备方法和应用。
背景技术
分子影像学技术的发展为肿瘤的早期诊断、治疗及预后评价提供了技术支持。目前，围绕肿瘤的早期诊断、治疗及预后评价已经发展了磁、光、声、核素等多种分子影像探针。然而目前分子影像探针的检测深度和分辨率还有待提高，并且目前的分子影像探针仅仅用于成像，作用单一。
发明内容
有鉴于此，本申请提供了一种肿瘤诊疗剂及其制备方法，该肿瘤诊疗剂可同时实现磁光双模成像和药物治疗，达到诊疗一体化，并且具有良好的靶向性、生物相容性和稳定性，在生物医学领域有广泛的应用前景。
第一方面，本申请提供了一种肿瘤诊疗剂，包括白蛋白、分散在所述白蛋白内的抗肿瘤药物和金团簇，以及通过化学键连接在所述白蛋白表面的磁共振造影剂和靶向穿膜肽。
在本申请中，靶向穿膜肽使得肿瘤诊疗剂能够穿过细胞膜进行肿瘤细胞中；金团簇能够在近红外二区发光实现光学成像，磁共振造影剂实现磁共振成像，实现磁光双模成像；该肿瘤诊疗剂携带有抗肿瘤药物，从而在成像的同时实现药物的精准治疗。
可选的，所述肿瘤诊疗剂的粒径为30nm-200nm。
可选的，所述金团簇的粒径小于2nm。
可选的，所述金团簇包括8个-100个金原子。
可选的，所述靶向穿膜肽和所述白蛋白的摩尔量比值为1-100。
可选的，所述靶向穿膜肽包括iRGD、iNGR、TAT和CTX中的至少一种。
可选的，所述磁共振造影剂和所述白蛋白的摩尔量比值为1-100。
可选的，所述磁共振造影剂包括Gd-DTPA、Gd-DOTA、Gd-DTTA和Gd-HOPO中的至少一种。
可选的，所述抗肿瘤药物和所述白蛋白的摩尔量比值为1-10000。
可选的，所述抗肿瘤药物包括紫杉醇、多西紫杉醇、阿霉素、顺铂、伊立替康、喜树碱、卡莫司汀和姜黄素中的至少一种。
本申请提供的肿瘤诊疗剂可同时实现磁光双模成像和药物治疗，达到诊疗一体化，能够满足临床应用的需求。
第二方面，本申请提供了一种肿瘤诊疗剂的制备方法，包括：
向含第一纳米颗粒的溶液中加入带巯基的还原剂，反应后得到反应液，其中，所述第一纳米颗粒包括白蛋白以及分散在所述白蛋白内的金团簇；
将所述反应液进行超声处理，并向所述反应液中加入含抗肿瘤药物的溶液，反应得到 含第二纳米颗粒的溶液，所述第二纳米颗粒包括所述白蛋白以及分散在所述白蛋白内的所述金团簇和所述抗肿瘤药物；
所述含第二纳米颗粒的溶液与靶向穿膜肽和磁共振造影剂反应后，经干燥得到肿瘤诊疗剂，所述肿瘤诊疗剂包括所述白蛋白、分散在所述白蛋白内的所述抗肿瘤药物和所述金团簇，以及通过化学键连接在所述白蛋白表面的所述磁共振造影剂和所述靶向穿膜肽。
可选的，所述第一纳米颗粒的制备包括：氯金酸溶液与白蛋白溶液混合后，加入氢氧化钠溶液和硼氢化钠溶液，反应后过滤得到所述第一纳米颗粒。
可选的，所述带巯基的还原剂的包括谷胱甘肽、半胱氨酸、巯基乙醇和二硫苏糖醇中的至少一种；所述带巯基的还原剂与所述含第一纳米颗粒的溶液中的所述白蛋白的摩尔比为5-30。
可选的，所述含第二纳米颗粒的溶液与靶向穿膜肽反应，包括：含所述靶向穿膜肽的溶液中加入N-羟基琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐反应后，加入所述含第二纳米颗粒的溶液，反应得到含第三纳米颗粒的溶液，其中，所述第三纳米颗粒包括所述白蛋白、分散在所述白蛋白内的所述抗肿瘤药物和所述金团簇，以及通过化学键连接在所述白蛋白表面的所述靶向穿膜肽。
本申请提供的肿瘤诊疗剂的制备方法操作简单、方便，可以实现工业化生产，有利于肿瘤诊疗剂的广泛使用。
第三方面，本申请提供了第一方面所述的或第二方面所述的制备方法制得的肿瘤诊疗剂在预防、诊断或治疗肿瘤的药物中的应用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
图1为本申请一实施方式提供的肿瘤诊疗剂的制备方法流程图。
图2为本申请一实施方式提供的肿瘤诊疗剂的制备示意图。
图3为实施例1制得的肿瘤诊疗剂的透射电镜图。
图4为细胞的近红外二区荧光显微镜图。
图5为小鼠的近红外二区光和磁共振成像图。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
本申请提供了一种肿瘤诊疗剂，包括白蛋白、分散在所述白蛋白内的抗肿瘤药物和金团簇，以及通过化学键连接在所述白蛋白表面的磁共振造影剂和靶向穿膜肽。
在预防、诊断和治疗肿瘤过程中，首先需要对肿瘤进行成像，而肿瘤不同程度地吸收和散热入射光，造成入射光衰减，降低成像深度、信背比以及分辨率，使得肿瘤边界模糊不清难以辨认；相较于近红外一区荧光等传统荧光，近红外二区荧光(1000nm-1700nm)具有更纵深的穿透深度、更好的空间分辨率以及低的背景噪声，有利于实现深部组织的高分辨成像，并且对生物组织光损伤和毒性小；本申请提供的肿瘤诊疗剂中具有金团簇，其能够在近红外二区发光，从而可以获得高质量的肿瘤成像结果，并且该肿瘤诊疗剂具有磁共振造影剂，进而可以实现磁共振成像，具有磁光双模成像结果，大幅度提升了成像质量，为诊断提供强有力的技术支持；该肿瘤诊疗剂携带有抗肿瘤药物，肿瘤细胞含有大量谷胱甘肽，可以将肿瘤诊疗剂的二硫键打开，使得抗肿瘤药物释放出来，进而在成像的同时实现抗肿瘤药物的释放和治疗，达到诊疗一体化。
在本申请中，白蛋白是一种生物内源性蛋白，具有可生物降解、无毒等优点，白蛋白作为肿瘤诊疗剂的载体，具有高稳定性和水溶性的性能，可以提高肿瘤诊疗剂的稳定性以及亲水性。
在本申请实施方式中，白蛋白包括人血清白蛋白、牛血清白蛋白、猪血清白蛋白、重组血清白蛋白中的至少一种。上述白蛋白的生物相容性好，有利于在临床使用。在一实施例中，白蛋白为牛血清白蛋白。在本申请实施方式中，白蛋白的粒径为20nm-150nm。进一步的，白蛋白的粒径为25nm-100nm。具体的，白蛋白的粒径可以但不限于为20nm、25nm、40nm、50nm、80nm、90nm、100nm或120nm等。可以理解的，本申请提供的肿瘤诊疗剂中可以包括多个白蛋白。
在本申请中，金团簇均匀分散在白蛋白内部，其能够在近红外二区成像，进而可以获得高质量的成像结果。在本申请实施方式中，金团簇的粒径小于2nm。进一步的，金团簇的粒径小于1.5nm。在本申请实施方式中，金团簇包括8个-100个金原子。在一实施例中，金团簇包括8个-20个金原子。在另一实施例中，金团簇包括30个-50个金原子。在又一实施例中，金团簇包括80个-95个金原子。在本申请中，可以根据需要设置分散在白蛋白中金团簇的量。在本申请一实施例中，肿瘤诊疗剂中金原子与白蛋白的摩尔比为10-1000。从而可以使得肿瘤诊疗剂在近红外二区发光强度大，成像效果好。进一步的，肿瘤诊疗剂中金原子与白蛋白的摩尔比为50-700。在本申请实施方式中，金团簇和白蛋白之间具有Au-S键。通过Au-S键，使得金团簇可以稳定、长期的存在在白蛋白的内部。
在本申请中，抗肿瘤药物分散在白蛋白内部，靶向穿膜肽使得肿瘤诊疗剂进入肿瘤细胞中，从而使得抗肿瘤药物可以进入肿瘤细胞中，同时还提高了疏水性抗肿瘤药物的水溶性，进而使抗肿瘤药物可以精准作用肿瘤细胞，提高治疗效果。在本申请实施方式中，抗肿瘤药物和白蛋白的摩尔量比值为1-10000。从而使得肿瘤诊疗剂携带足够量的抗肿瘤药物，提高肿瘤诊疗剂的治疗效果。进一步的，抗肿瘤药物和白蛋白的摩尔量比值为20-5000。更进一步的，抗肿瘤药物和白蛋白的摩尔量比值为100-1000。具体的，抗肿瘤药物和白蛋白的摩尔量比值可以但不限于为20-100、50-500、100-2000、150-4000或500-5000等。在本申请实施方式中，抗肿瘤药物包括紫杉醇、多西紫杉醇、阿霉素、顺铂、伊立替康、喜树 碱、卡莫司汀和姜黄素中的至少一种。上述抗肿瘤药物与白蛋白之间可以形成键能，从而长期稳定分散在白蛋白内部，并且在进入肿瘤细胞后，可以释放并作用在肿瘤细胞中。在一实施例中，抗肿瘤药物包括紫杉醇。
在本申请中，磁共振造影剂通过化学键连接在白蛋白的表面，使得磁共振造影剂可以长期稳定存在，保证磁共振成像的进行。在本申请实施方式中，磁共振造影剂通过共价键连接在白蛋白的表面。进一步的，磁共振造影剂通过酰胺键连接在白蛋白的表面。在本申请实施方式中，磁共振造影剂包括Gd-DTPA、Gd-DOTA、Gd-DTTA和Gd-HOPO中的至少一种。上述磁共振造影剂性能稳定、毒性低、水溶性好，并且所需量少。在本申请实施方式中，磁共振造影剂和白蛋白的摩尔量比值为1-100。进一步的，磁共振造影剂和白蛋白的摩尔量比值为10-80。更进一步的，磁共振造影剂和白蛋白的摩尔量比值为20-65。具体的，磁共振造影剂和白蛋白的摩尔量比值可以但不限于为15、25、30、38、45、50、60、74或85等。在上述摩尔比范围内，磁共振造影剂对肿瘤诊疗剂的结构无影响，并且能够与金团簇共同实现磁光双模态成像功能，提高成像质量。
在本申请中，靶向穿膜肽通过化学键连接在白蛋白的表面，使得靶向穿膜肽可以长期稳定存在，保证肿瘤诊疗剂能够靶向并进入肿瘤细胞内，实现成像和治疗。在本申请实施方式中，靶向穿膜肽通过共价键连接在白蛋白的表面。进一步的，靶向穿膜肽通过酰胺键连接在白蛋白的表面。在本申请实施方式中，靶向穿膜肽和白蛋白的摩尔量比值为1-100。进一步的，靶向穿膜肽和白蛋白的摩尔量比值为15-80。更进一步的，靶向穿膜肽和白蛋白的摩尔量比值为20-70。具体的，靶向穿膜肽和白蛋白的摩尔量比值可以但不限于为15、20、25、30、35、42、55、60、75或80等。在上述摩尔比范围内，肿瘤诊疗剂能够高效、快速地靶向肿瘤细胞并进入肿瘤细胞内，有利于磁光双模成像和药物释放治疗。在本申请实施方式中，靶向穿膜肽包括iRGD、iNGR、TAT和CTX中的至少一种。在本申请中，靶向穿膜肽，如iRGD、iNGR等，能够通过与血管通透性调节分子作用，增强血管的通透性，从而促进肿瘤诊疗剂进入细胞内；iRGD具有RGD结构域，iNGR具有NGR结构域，能够与肿瘤细胞表面以及肿瘤周围血管上的整合素αvβ3、αvβ5特异性结合，靶向到整合素表达较高的肿瘤部位。在一实施例中，iRGD的氨基酸序列为CRGDKGPDC。在另一实施例中，iNGR的氨基酸序列为CRNGRGPDC。
本申请实施方式中，肿瘤诊疗剂的粒径为30nm-200nm。进一步的，肿瘤诊疗剂的粒径为40nm-180nm。更进一步的，肿瘤诊疗剂的粒径为50nm-150nm。具体的，肿瘤诊疗剂的粒径可以但不限于为40nm-60nm、50nm-60nm、70nm-90nm、80nm-110nm、100nm-150nm、110nm-150nm、130nm-160nm或160nm-200nm等。在该粒径范围下肿瘤诊疗剂具有较好的分散性能和稳定性，有利于其使用。
请参阅图1，为本申请一实施方式提供的肿瘤诊疗剂的制备方法流程图，该方法制得上述任一实施方式中的肿瘤诊疗剂，该制备方法包括：
操作101：向含第一纳米颗粒的溶液中加入带巯基的还原剂，反应后得到反应液，其中，第一纳米颗粒包括白蛋白以及分散在白蛋白内的金团簇。
操作102：将反应液进行超声处理，并向反应液中加入含抗肿瘤药物的溶液，反应得到含第二纳米颗粒的溶液，第二纳米颗粒包括白蛋白以及分散在白蛋白内的金团簇和抗肿瘤药物。
操作103：含第二纳米颗粒的溶液与靶向穿膜肽和磁共振造影剂反应后，经干燥得到肿瘤诊疗剂，肿瘤诊疗剂包括白蛋白、分散在白蛋白内的抗肿瘤药物和金团簇，以及通过化学键连接在白蛋白表面的磁共振造影剂和靶向穿膜肽。
在本申请实施方式中，采用生物矿化法制得第一纳米颗粒。在本申请一实施例中，第一纳米颗粒的制备包括：氯金酸溶液与白蛋白溶液混合后，加入氢氧化钠溶液和硼氢化钠溶液，反应后过滤得到第一纳米颗粒。在一实施例中，氯金酸溶液的溶度为1mM-50mM，氯金酸溶液的体积为0.1mL-3mL。在另一实施例中，白蛋白溶液的溶度为1mg/mL-50mg/mL，白蛋白溶液的体积为0.1mL-5mL。在又一实施例中，氢氧化钠溶液的溶度为0.1M-2M，氢氧化钠溶液的体积为0.1mL-2mL。在又一实施例中，硼氢化钠溶液的溶度为0.01M-0.5M，硼氢化钠溶液的体积为0.01mL-0.5mL。在一具体实施例中，将氯金酸溶液与白蛋白溶液在15℃-30℃下混合，随后依次加入氢氧化钠溶液和预冷的硼氢化钠溶液，溶液变为深棕色；然后将溶液在130rpm-200rpm、25℃-37℃下孵育，制得第一纳米颗粒。在本申请中，第一纳米颗粒可以置于缓冲液中保存备用，具体的，可以但不限于将第一纳米颗粒置于0.1M磷酸盐缓冲液中透析后低温保存。
在本申请实施方式中，在加入带巯基的还原剂之前，还包括将含第一纳米颗粒的溶液的pH调节至7-12。从而有利于后续白蛋白二硫键的打开。在一实施例中，将含第一纳米颗粒的溶液的pH调节至7-9。在另一实施例中，将含第一纳米颗粒的溶液的pH调节至9-12。具体的，可以但不限于将含第一纳米颗粒的溶液的pH调节至7、8、9、10、11或12等。在一具体实施例中，采用氢氧化钠溶液进行pH调节，如采用1mol/L的NaOH溶液调节含第一纳米颗粒的溶液的pH。
在本申请实施方式中，带巯基的还原剂的包括谷胱甘肽、半胱氨酸、巯基乙醇和二硫苏糖醇中的至少一种。通过采用带巯基的还原剂可以打开白蛋白的二硫键，使得白蛋白结构展开，有利于后续抗肿瘤药物的进入和分散。在本申请实施方式中，带巯基的还原剂与含第一纳米颗粒的溶液中的白蛋白的摩尔比为5-30。进一步的，带巯基的还原剂与含第一纳米颗粒的溶液中的白蛋白的摩尔比为10-25。更进一步的，带巯基的还原剂与含第一纳米颗粒的溶液中的白蛋白的摩尔比为12-20。具体的，带巯基的还原剂与含第一纳米颗粒的溶液中的白蛋白的摩尔比可以但不限于为10、12、15、17、20、21、24、28或30等。上述范围有利于白蛋白结构的展开，同时又不会过多破坏白蛋白的结构，使得白蛋白仍然可以负载金团簇以及抗肿瘤药物。在本申请实施方式中，带巯基的还原剂的浓度为0.01mol/L-2mol/L。进一步的，带巯基的还原剂的浓度为0.05mol/L-1.8mol/L。在本申请实施方式中，反应在30℃-60℃进行1min-5min。进一步的，反应在35℃-60℃进行3min-5min。在此反应条件下，有利于白蛋白二硫键的快速打开。
在本申请中，在超声处理的同时加入抗肿瘤药物，从而有利于抗肿瘤药物进入展开的 白蛋白的内部，提高白蛋白负载抗肿瘤药物的量，增强肿瘤诊疗剂的疗效，同时还可以防止白蛋白之间聚集，影响纳米诊疗剂的制备。在本申请实施方式中，超声的功率为5W-20W。进一步的，超声的功率为10W-15W。在一实施例中，将反应液在超声细胞破碎仪中进行处理，从而可以进一步使得抗肿瘤药物大量进入白蛋白内部，提高肿瘤诊疗剂中抗肿瘤药物的负载量。在本申请实施方式中，含抗肿瘤药物的溶液的注入速度为30ml/s-80ml/s。具体的，含抗肿瘤药物的溶液的注入速度可以但不限于为30ml/s、40ml/s、50ml/s、60ml/s、70ml/s或80ml/s等。在本申请实施方式中，含抗肿瘤药物的溶液的溶剂包括二甲基亚砜、甲醇、乙醇、丙醇和叔丁醇中的至少一种。采用上述溶剂有利于提高抗肿瘤药物的溶解度，使得抗肿瘤药物与白蛋白之间充分混合和接触。在本申请实施方式中，含抗肿瘤药物的溶液浓度为0.01M-2M。具体的，含抗肿瘤药物的溶液浓度可以但不限于为0.01M、0.05M、0.1M、0.3M、0.7M、1M、1.2M、1.5M、1.8M或2M等。在本申请实施方式中，抗肿瘤药物和白蛋白的摩尔量比值为1-10000。从而使得肿瘤诊疗剂携带足够量的抗肿瘤药物，提高肿瘤诊疗剂的治疗效果。具体的，抗肿瘤药物和白蛋白的摩尔量比值可以但不限于为20-100、50-500、100-2000、150-4000或500-5000等。在操作102中，反应包括在30℃-60℃进行10min-30min。进一步的，反应进行15min-25min。在此反应条件下，有利于抗肿瘤药物与白蛋白充分接触和混合，以使得白蛋白负载足量的抗肿瘤药物，提高肿瘤诊疗剂的疗效。
在本申请中，抗肿瘤药物分散在白蛋白内部。在本申请一实施方式中，采用带巯基的还原剂打开白蛋白的二硫键，使得白蛋白结构展开，抗肿瘤药物加入后，多个白蛋白团聚并包裹抗肿瘤药物。在一实施例中，肿瘤诊疗剂包括多个白蛋白，金团簇分散在白蛋白内，多个白蛋白团聚并包裹抗肿瘤药物，磁共振造影剂和靶向穿膜肽通过化学键连接在白蛋白的表面。进一步的，抗肿瘤药物被包裹在白蛋白的中心。
在操作102后，还包括将含第二纳米颗粒的溶液进行透析处理。通过透析处理可以去除未反应的带巯基的还原剂等杂质，同时也提高第二纳米颗粒的分散效果。在本申请实施方式中，通过将含第二纳米颗粒的溶液置于pH7-12的磷酸盐缓冲液进行透析处理。在一具体实施例中，将含第二纳米颗粒的溶液置于透析袋中，将透析袋置于1L、pH7-12的磷酸盐缓冲液内透析5h-10h，然后再将透析袋置于水中透析1h-3h即可。
在本申请实施方式中，含第二纳米颗粒的溶液与靶向穿膜肽反应，包括：含靶向穿膜肽的溶液中加入N-羟基琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐反应后，加入含第二纳米颗粒的溶液，反应得到含第三纳米颗粒的溶液，其中，第三纳米颗粒包括白蛋白、分散在白蛋白内的抗肿瘤药物和金团簇，以及通过化学键连接在白蛋白表面的靶向穿膜肽。在一实施例中，含靶向穿膜肽的溶液中加入N-羟基琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐在25℃-30℃反应2h-4h。在另一实施例中，加入含第二纳米颗粒的溶液后在25℃-30℃反应12h-14h。在另一实施例中，含靶向穿膜肽的溶液的溶剂包括二甲基亚砜、甲醇、乙醇、丙醇和叔丁醇中的至少一种。在本申请实施方式中，将含第三纳米颗粒的溶液与NHS-磁共振造影剂混合，在在25℃-30℃反应2h-4h，即可得到肿瘤诊疗剂。在本申请中，靶向穿膜肽和磁共振造影剂通过化学反应与白蛋白之间产生化学键合， 从而可以长期稳定地负载在白蛋白的表面。
在本申请实施方式中，在干燥之前还包括透析处理；通过透析处理，可以去除溶液中的杂质。在本申请实施方式中，通过将溶液置于pH 7-12的磷酸盐缓冲液进行透析处理。在一具体实施例中，将溶液置于透析袋中，将透析袋置于1L、pH 7-12的磷酸盐缓冲液内25℃-30℃透析5h-10h，然后再将透析袋置于水中透析1h-3h即可。在一实施例中，干燥包括在0℃下预冻1h-3h后转移至-20℃下冷冻2h-5h，然后冷冻干燥8h-15h。
请参阅图2，为本申请一实施方式提供的肿瘤诊疗剂的制备示意图，在本申请实施方式中，白蛋白和含金元素的溶液混合，在氢氧化钠和硼氢化钠的作用下，生成了白蛋白金簇，即第一纳米颗粒，所述第一纳米颗粒包括白蛋白以及分散在白蛋白内的金团簇；通过谷胱甘肽处理，并加入紫杉醇溶液，生成了白蛋白金团簇纳米药物，即第二纳米颗粒，所述第二纳米颗粒包括白蛋白以及分散在白蛋白内的金团簇和抗肿瘤药物紫杉醇；再与NHS-iRGD、DTPA-Gd反应，制得白蛋白金团簇靶向纳米药物，即肿瘤诊疗剂，所述肿瘤诊疗剂包括白蛋白、分散在白蛋白内的抗肿瘤药物紫杉醇和金团簇，以及通过化学键连接在白蛋白表面的磁共振造影剂DTPA-Gd和靶向穿膜肽iRGD。
本申请提供了一种以白蛋白为载体，以靶向穿膜肽为靶向配体并负载抗肿瘤药物的磁光双模纳米诊疗剂；通过白蛋白作为载体，采用生物矿化法合成近红外二区发光的金团簇，获得第一纳米颗粒；再通过一步生物还原法制备水溶性的、单分散的第二纳米颗粒；最后在第二纳米颗粒上通过化学键偶连靶向穿膜肽和磁共振造影剂，以实现磁光双模成像。本申请提供的肿瘤诊疗剂的制备方法操作简单、方便，可以实现工业化生产，有利于肿瘤诊疗剂的广泛使用。
本申请提供了上述肿瘤诊疗剂在预防、诊断或治疗肿瘤的药物中的应用。可以理解的，肿瘤可以但不限于为癌症，如脑胶质瘤等。在一实施例中，本申请提供的肿瘤诊疗剂可以应用于预防、诊断或治疗脑胶质瘤中；脑胶质瘤的组织边界不清，还有血脑屏障阻碍成像材料和药物的进入，本申请提供的肿瘤诊疗剂中具有靶向穿膜肽，可以避免血脑屏障的影响，进入脑胶质瘤细胞中，同时通过近红外二区光成像以及磁共振成像，有效识别脑胶质瘤区域，同时携带的抗肿瘤药物可以精准靶向脑胶质瘤，起到治疗作用。
实施例
步骤1：将氯金酸溶液(10mM，1.25mL)与白蛋白溶液(50mg/mL，2.5mL)在25℃下混合均匀，随后依次加入氢氧化钠溶液(1M，0.25mL)和预冷的硼氢化钠溶液(0.1M，0.1mL)，溶液变为深棕色；然后将溶液在180rpm、37℃下孵育，制得第一纳米颗粒；将第一纳米颗粒置于0.1M磷酸盐缓冲液中透析后置于4℃保存。
步骤2：取2mL第一纳米颗粒溶液，然后采用1mol/L的NaOH溶液调节第一纳米颗粒溶液的pH值到7；向其中加入谷胱甘肽获得反应液，然后在60℃下轻轻摇动反应0.05h，谷胱甘肽的摩尔数为白蛋白摩尔数的10倍。
步骤3：将步骤2反应后的溶液在60℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为10W，同时以50ml/s的速度向溶液中注入的2mL紫杉醇二甲基 亚砜溶液，溶液在60℃的条件下反应20min后得到第二纳米颗粒溶液。
步骤4：将步骤3所得的第二纳米颗粒溶液移入透析袋，保持温度为30℃的条件下，将透析袋置于1L、pH7的PBS缓冲液内透析10h，期间每12h换液1次，每次都采用1L、pH7的PBS缓冲液，然后再将透析袋置于5L双蒸水内透析1h即可获得第二纳米颗粒。
步骤5：将iRGD溶于二甲基亚砜溶液中，然后加入N-羟基琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，在25℃反应3h，再加入第二纳米颗粒，25℃反应15h；然后向上述溶液中加入NHS-DTPA-Gd，25℃反应3h，获得含肿瘤诊疗剂的溶液。
步骤6：将步骤5所得含肿瘤诊疗剂的溶液移入透析袋，保持温度为30℃的条件下，将透析袋置于1L、pH7的PBS缓冲液内透析10h，期间每12h换液1次，每次都采用1L、pH7的PBS缓冲液，然后再将透析袋置于5L双蒸水内透析1h；然后置于-0℃下预冻1h后，转移至-20℃下冷冻2h，再在冷冻干燥机中冷冻干燥12h，得到肿瘤诊疗剂。将制得的肿瘤诊疗剂进行透射电镜观测，结果如图3所示，可以看出，本申请制得的肿瘤诊疗剂为纳米级别，尺寸均一，颗粒较为分散。
效果实施例
将实施例1制得的肿瘤诊疗剂与C6细胞孵育3h后，通过近红外二区荧光显微镜观测细胞，结果如图4所示。可以看出，本申请提供的肿瘤诊疗剂能够在近红外二区产生荧光信号，有利于成像。将实施例1制得的肿瘤诊疗剂溶于PBS溶液中(浓度为9mg/ml)，并通过尾静脉注射注入小鼠体内，在注射24h后，通过近红外二区光以及核磁共振观测小鼠脑部肿瘤部位，结果如图5所示。可以看出，本申请提供的肿瘤诊疗剂具有磁光双模成像效果。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本申请专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种肿瘤诊疗剂，其特征在于，包括白蛋白、分散在所述白蛋白内的抗肿瘤药物和金团簇，以及通过化学键连接在所述白蛋白表面的磁共振造影剂和靶向穿膜肽。
  2. 如权利要求1所述的肿瘤诊疗剂，其特征在于，所述肿瘤诊疗剂的粒径为30nm-200nm。
  3. 如权利要求1所述的肿瘤诊疗剂，其特征在于，所述金团簇的粒径小于2nm，所述金团簇包括8个-100个金原子。
  4. 如权利要求1所述的肿瘤诊疗剂，其特征在于，所述靶向穿膜肽和所述白蛋白的摩尔量比值为1-100；所述靶向穿膜肽包括iRGD、iNGR、TAT和CTX中的至少一种；
    所述磁共振造影剂和所述白蛋白的摩尔量比值为1-100；所述磁共振造影剂包括Gd-DTPA、Gd-DOTA、Gd-DTTA和Gd-HOPO中的至少一种。
  5. 如权利要求1所述的肿瘤诊疗剂，其特征在于，所述抗肿瘤药物和所述白蛋白的摩尔量比值为1-10000，所述抗肿瘤药物包括紫杉醇、多西紫杉醇、阿霉素、顺铂、伊立替康、喜树碱、卡莫司汀和姜黄素中的至少一种。
  6. 一种肿瘤诊疗剂的制备方法，其特征在于，包括：
    向含第一纳米颗粒的溶液中加入带巯基的还原剂，反应后得到反应液，其中，所述第一纳米颗粒包括白蛋白以及分散在所述白蛋白内的金团簇；
    将所述反应液进行超声处理，并向所述反应液中加入含抗肿瘤药物的溶液，反应得到含第二纳米颗粒的溶液，所述第二纳米颗粒包括所述白蛋白以及分散在所述白蛋白内的所述金团簇和所述抗肿瘤药物；
    所述含第二纳米颗粒的溶液与靶向穿膜肽和磁共振造影剂反应后，经干燥得到肿瘤诊疗剂，所述肿瘤诊疗剂包括所述白蛋白、分散在所述白蛋白内的所述抗肿瘤药物和所述金团簇，以及通过化学键连接在所述白蛋白表面的所述磁共振造影剂和所述靶向穿膜肽。
  7. 如权利要求6所述的制备方法，其特征在于，所述第一纳米颗粒的制备包括：
    氯金酸溶液与白蛋白溶液混合后，加入氢氧化钠溶液和硼氢化钠溶液，反应后过滤得到所述第一纳米颗粒。
  8. 如权利要求6所述的制备方法，其特征在于，所述带巯基的还原剂的包括谷胱甘肽、半胱氨酸、巯基乙醇和二硫苏糖醇中的至少一种；所述带巯基的还原剂与所述含第一纳米颗粒的溶液中的所述白蛋白的摩尔比为5-30。
  9. 如权利要求6所述的制备方法，其特征在于，所述含第二纳米颗粒的溶液与靶向穿膜肽反应，包括：
    含所述靶向穿膜肽的溶液中加入N-羟基琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐反应后，加入所述含第二纳米颗粒的溶液，反应得到含第三纳米颗粒的溶液，其中，所述第三纳米颗粒包括所述白蛋白、分散在所述白蛋白内的所述抗肿瘤药物和所述 金团簇，以及通过化学键连接在所述白蛋白表面的所述靶向穿膜肽。
  10. 如权利要求1-5任一项所述的或权利要求6-9任一项所述的制备方法制得的肿瘤诊疗剂在预防、诊断或治疗肿瘤的药物中的应用。
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