WO2023041005A1 - Virus-like hollow oxide loaded near-infrared two-b region excited rare earth nanocrystal, preparation method therefor and application thereof - Google Patents

Virus-like hollow oxide loaded near-infrared two-b region excited rare earth nanocrystal, preparation method therefor and application thereof Download PDF

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WO2023041005A1
WO2023041005A1 PCT/CN2022/119211 CN2022119211W WO2023041005A1 WO 2023041005 A1 WO2023041005 A1 WO 2023041005A1 CN 2022119211 W CN2022119211 W CN 2022119211W WO 2023041005 A1 WO2023041005 A1 WO 2023041005A1
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rare earth
nanoparticles
core
preparation
virus
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French (fr)
Chinese (zh)
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刘小龙
王培园
吴名
李佳琦
李阳
蔺金燕
罗强
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福建医科大学孟超肝胆医院(福州市传染病医院)
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching

Definitions

  • the invention belongs to the field of biomedical nanomaterials, and in particular relates to a rare earth nanocrystal excited by a near-infrared 2b region supported by a virus-like hollow oxide, a preparation method thereof, and an application for surgical navigation and postoperative chemodynamic therapy.
  • NIR I near-infrared region
  • NIR II fluorescence imaging can be used to precisely locate the tumor location, clearly observe the tumor margin and perform efficient resection.
  • Manganese oxide is widely used in the treatment of tumors. The manganese ions released by its degradation can generate singlet oxygen in the acidic environment of tumors, perform chemodynamic therapy, and can also be used for nuclear magnetic resonance imaging.
  • virus-like silica nanoparticles with rough surfaces can greatly increase the uptake rate by cells due to cell adhesion. Therefore, it is urgent to develop a new method that can be used to construct virus-like metal mesoporous oxides with simple methods to improve tumor cell uptake and biosafety, which is of great significance for biological applications and clinical transformation.
  • the present invention provides a rare earth nanocrystal, which includes a carrier and rare earth core-shell nanoparticles loaded on the surface of the carrier.
  • the mass ratio of the carrier to the rare earth core-shell nanoparticles is 1:(1-2), preferably the mass ratio is 1:(1-1.5), exemplarily 1: 1, 1:1.2, 1:1.5, 1:2.
  • the carrier is connected to the rare earth core-shell nanoparticles through a valence bond, for example, through an amide bond.
  • the carrier has amino groups that form amide bonds with carboxyl groups in the rare earth core-shell nanoparticles.
  • the rare earth nanocrystals can be selectively degraded in the weakly acidic microenvironment of the tumor, and preferably can be selectively degraded to release manganese ions (Mn 2+ ), iron ions ( Fe 2+ ).
  • the carrier is a hollow sphere whose surface is modified with an amino functional group compound.
  • the carrier is aminated hollow manganese oxide and/or hollow iron oxide.
  • the compound with amino functional group may be aminosilane APTES ((3-Aminopropyl)-triethoxysilane).
  • fluorescence quenching molecules are loaded in the cavity of the carrier.
  • the fluorescence quencher molecule can be a near-infrared absorber, such as IR1064.
  • the loading amount of the fluorescence quencher molecule is 10-15% of the total weight of the carrier, exemplarily 10%, 12%, and 15%.
  • the hollow manganese oxide is nanoparticles, which have a virus-like shape and can be quickly phagocytosed by tumor cells to achieve precise imaging and treatment of tumors.
  • the virus-like hollow manganese oxide of the present invention will degrade and collapse in the acidic microenvironment of the tumor, and the released manganese ions can be used for chemodynamic therapy of postoperative tumor metastases or for nuclear magnetic resonance imaging of tumors; it releases fluorescence quenching After the molecule IR1064, the fluorescence of rare earth nanoparticles is restored, so it can be used for surgically guided resection of tumors with fluorescence imaging.
  • the rare earth core-shell nanoparticles include core nanoparticles, at least one shell covering the outside of the core nanoparticles, and a carboxyl functional group-containing substance modified on the outside of the shell,
  • the core nanoparticle and the shell layer are independently selected from AREF 4 , wherein: A is Na or K, RE is at least one of Er, Ho, Gd and Y.
  • the carboxyl functional group-containing substance may be a carboxyl functional group-containing polymer.
  • A is Na.
  • the RE elements in the core nanoparticles and the shell are different.
  • RE is two of Er and Ho; in the shell nanoparticles, RE is Y.
  • the doping amount of Ho is 1-5%, exemplarily 1%, 2%, 5%.
  • the particle size of the rare earth core-shell nanoparticles is 24-25.5nm, exemplarily 24nm, 24.13nm, 25nm, 25.25nm, 25.5nm.
  • the ratio of the particle size of the core nanoparticles to the thickness of the shell layer is 1:(1 ⁇ 1.5), exemplarily 1:1, 1:1.18, 1:1.5.
  • the particle diameter of the core nanoparticles is 17-18nm, exemplarily 17.09nm, 17.5nm, 17.95nm.
  • the carboxyl functional group-containing substance can be selected from PEG polymers, small molecular acids or carboxyl-containing polymers, such as phospholipid polyethylene glycol (DSEP-PEG 2000 -COOH), Malay At least one of amic acid and polyacrylic acid, etc.
  • PEG polymers small molecular acids or carboxyl-containing polymers, such as phospholipid polyethylene glycol (DSEP-PEG 2000 -COOH), Malay At least one of amic acid and polyacrylic acid, etc.
  • the polymer containing carboxyl functional group of the present invention can enhance the biocompatibility of materials and reduce cytotoxicity.
  • the rare earth core-shell nanoparticles have optical imaging functions in the first near-infrared region (650-900nm) and the second near-infrared region (1000-1700nm).
  • the rare earth nanocrystals are monodisperse and uniform in size.
  • the average particle size of the rare earth nanocrystals is about 24-26 nm, exemplarily 24.13 nm, 24.69 nm, 25 nm, 25.25 nm.
  • the rare earth nanocrystals can be excited by the near-infrared region 2b to generate fluorescence emission in the near-infrared region 1 and near-infrared region 2 .
  • the rare earth nanocrystals can emit red light in the near-infrared region of 660 nm when excited at 1532 nm. It can be used for fluorescence imaging in the first near-infrared region, and can be used as a reference for imaging in the second near-infrared region.
  • the rare earth nanocrystals can emit fluorescence at 1180 nm when excited at 1532 nm. It can be used for surgical resection of tumors guided by near-infrared two-zone fluorescence imaging, which solves the problems of low resolution and shallow penetration in current surgical navigation, and can be used for background-free fluorescence imaging of deep tissues.
  • the rare earth core-shell nanoparticles may be ⁇ -NaErF 4 :Ho@NaYF 4 .
  • the rare earth nanocrystalline particles of the present invention can realize fluorescence imaging navigation tumor resection, fluorescence imaging guidance of postoperative metastatic lesions, tumor photodynamic enhancement, and tumor nuclear magnetic resonance imaging, greatly improving the removal efficiency of tumors, and providing clinical treatment for malignant tumors. guide.
  • the present invention also provides a method for preparing the above-mentioned rare earth nanocrystals, which includes reacting a carrier with rare earth core-shell nanoparticles to prepare the rare earth nanocrystals.
  • the mass ratio of the rare earth core-shell nanoparticles to the carrier is 2-5:1, exemplarily 2:1, 3:1, 4:1, 5:1.
  • the preparation method of the rare earth core-shell nanoparticles comprises the following steps:
  • step (2) is performed at least once to obtain nanoparticles coated with at least one shell layer.
  • step (2) can be carried out 1 time, 2 times, 3 times or more;
  • the alkali metal fluoride refers to an alkali metal ion or a compound formed of a cation similar to an alkali metal ion and a F ion.
  • the alkali metal ions are Na + , K + , and the cations with properties similar to alkali metal ions are NH 4 + .
  • the alkali metal fluoride solution is one of NaF, NH 4 F and KF solutions, preferably NH 4 F solution.
  • the molar ratio of the rare earth ion and the alkali metal fluoride in the rare earth salt is 1:(2-5), exemplarily 1:4.
  • the rare earth ion in the rare earth salt is at least one of Er ion, Ho ion and Y ion, preferably two of Er ion and Ho ion.
  • the molar ratio of the Er ions and Ho ions used is (48-49.5):(0.5-2), exemplarily 49.5:0.5, 49:1, 48:2.
  • step (1) it also includes dispersing the nuclear nanoparticles in a solvent to obtain a nuclear nanoparticle dispersion;
  • the solvent is cyclohexane.
  • concentration of the nuclear nanoparticle dispersion liquid may be 0.05 ⁇ 0.2 mol/L, exemplarily 0.1 mol/L.
  • the rare earth salt is rare earth chloride, rare earth nitrate or rare earth acetate, preferably rare earth acetate.
  • the rare earth chloride is at least one of HoCl 3 , ErCl 3 and YCl 3 .
  • the rare earth nitrate is at least one of Ho(NO 3 ) 3 , Er(NO 3 ) 3 and Y(NO 3 ) 3 .
  • the rare earth acetate is at least one of Ho(CH 3 COO) 3 , Er(CH 3 COO) 3 and Y(CH 3 COO) 3 .
  • the alkaline solution is sodium hydroxide solution or potassium hydroxide solution.
  • the solvent used in the alkali metal fluoride solution and the alkaline solution is methanol.
  • the total amount of the rare earth salt is 0.5-2 mmol, exemplarily 0.5 mmol, 1 mmol, 2 mmol.
  • the volume ratio of oleic acid to octadecene is 1:(2-3), exemplarily 1:2, 1:2.5, 1:3.
  • heating and stirring the mixed solution of oleic acid and octadecene after adding the rare earth salt is also included, so as to remove water and oxygen in the system.
  • the temperature of the heating and stirring is 130-150°C, exemplarily 130°C, 140°C, 150°C.
  • the heating and stirring time is 20-40 minutes, exemplarily 20 minutes, 30 minutes, 40 minutes. Further, the heating and stirring are carried out under vacuum conditions.
  • step (1) it also includes stirring the mixed solution after adding the alkali metal fluoride solution and the alkaline solution to nucleate.
  • the stirring nucleation adopts a two-stage heating method.
  • the secondary heating method includes:
  • the temperature of the first stage heating is 40-60°C, exemplarily 40°C, 50°C, 60°C; the time of the first stage heating is 0.5-2h, exemplarily 0.5h, 1h, 2h;
  • the temperature of the second stage heating is 60-80°C, exemplarily 60°C, 70°C, 80°C; the time of the second stage heating is 0.5-2h, exemplarily 0.5h, 1h, 2h.
  • the preparation method further includes a process of solid-liquid separation of the nuclear nanoparticle mixture prepared in step (1) to obtain a reaction product.
  • the solid-liquid separation method is to add a precipitating agent to the mixture of nuclear nanoparticles for precipitation, and centrifuge to obtain a solid product.
  • the precipitating agent may be ethanol.
  • the preparation method further includes washing the reaction product obtained from the solid-liquid separation in step (1).
  • the washing solvent can be absolute ethanol.
  • step (2) before adding RE-OA and A-TFA-OA precursor solution to the product of step (1), it also includes adding oleic acid (OA) and mixed solution of octadecene.
  • OA oleic acid
  • the volume ratio of the nuclear nanoparticle dispersion to the mixed solution of oleic acid and octadecene is 1:(1-3), exemplarily 1:2.
  • the volume ratio of oleic acid and octadecene is 1:(1-2), exemplarily 1:1.5.
  • the concentration of the RE-OA precursor solution is 0.05-0.2 mol/L, exemplarily 0.05 mol/L, 0.1 mol/L, 0.2 mol/L.
  • the RE-OA precursor solution is prepared by dissolving a salt containing RE elements in a mixed solvent of oleic acid and octadecene.
  • a mixed solvent Preferably, in the mixed solvent, the volume ratio of oleic acid to octadecene is 1:(1-2), exemplarily 1:1, 1:1.5, 1:2.
  • heating and stirring the mixed solution is also included.
  • the heating temperature may be 130-150°C, exemplarily 130°C, 140°C, 150°C; the heating time is 0.5-2h, exemplarily 0.5h, 1h, 2h.
  • the salt containing RE element is rare earth chloride, rare earth nitrate or rare earth acetate, preferably chloride.
  • the concentration of the A-TFA-OA precursor solution is 0.3-0.5 mol/L, exemplarily 0.3 mol/L, 0.4 mol/L, 0.5 mol/L.
  • the A-TFA-OA precursor solution is prepared by dissolving trifluoroacetate salt containing A element (ie, A-TFA) in oleic acid.
  • the preparation of the A-TFA-OA precursor solution also includes stirring the mixed solution.
  • the RE-OA precursor solution and the A-TFA-OA precursor solution are added to the nuclear nanoparticle dispersion prepared in step (1) at alternating intervals.
  • the number of alternate additions of the RE-OA precursor solution and the A-TFA-OA precursor solution is at least one.
  • each is added alternately 1 time, 2 times or more; preferably alternately added three times.
  • the time interval may be 10-20 minutes, exemplarily 10 minutes, 15 minutes, 20 minutes.
  • the molar ratio of the rare earth ion in the rare earth salt to the rare earth ion (RE) in the RE-OA precursor solution in step (2) is 1:(1 ⁇ 10), preferably 1:(6 ⁇ 8), exemplarily 1:1, 1:2, 1:5, 1:6, 1:7.5, 1:8, 1:10.
  • the reactions of step (1) and step (2) are carried out under an inert atmosphere.
  • the inert atmosphere is nitrogen, argon.
  • the reaction temperatures are the same or different, and are independently 200-400°C, exemplarily 200°C, 280°C, 300°C, 400°C.
  • the reaction time is 40-80 min, exemplarily 40 min, 60 min, 80 min.
  • the heating rate of the reaction is 5-20°C/min, exemplarily 5°C/min, 10°C/min, 20°C/min.
  • the preparation method further includes step (3): mixing the nanoparticles coated with at least one shell layer obtained in step (2) with a substance (such as a polymer) containing a carboxyl functional group in an organic solvent, and statically Place until the organic solvent volatilizes, then add water to disperse, and centrifuge to separate the carboxyl-modified rare earth core-shell nanoparticles.
  • a substance such as a polymer
  • the organic solvent is chloroform, cyclohexane, n-hexane, tetrahydrofuran, preferably chloroform.
  • the rotational speed of centrifugation is 8000-20000rpm, exemplarily 8000rpm, 10000rpm, 15000rpm, 17500rpm, 20000rpm.
  • the centrifugation time is 20-40 minutes, for example, 20 minutes, 30 minutes, 40 minutes.
  • the dosage ratio of the nanoparticles coated with at least one shell layer and the substance (such as a polymer) containing carboxyl functional groups is 0.1 mmol: (20-30) mg, exemplarily as 0.1mmol: 20mg, 0.1mmol: 25mg, 0.1mmol: 30mg.
  • the preparation method of the rare earth nanocrystals includes the following steps:
  • Synthesis of Y-OA precursor Dissolve YCl 3 in a mixed solvent of oleic acid and octadecene, heat and stir under vacuum conditions to obtain a clear and transparent Y-OA precursor solution;
  • ⁇ -NaErF 4 2% Ho core nanoparticle dispersion is added to the mixed solution of oleic acid and octadecene, under vacuum, cyclohexane is removed from the system, and then argon is introduced, the temperature is raised, and then alternately Adding Y-OA precursor solution and Na-TFA-OA precursor solution to prepare the rare earth nanocrystal.
  • the preparation method of the carrier includes using the virus-like silicon mesoporous nanoparticle as a template, reacting with manganese salt and urotropine, and removing the virus-like silicon mesoporous nanoparticle template through alkali treatment, That is, the virus-like hollow manganese oxide (ie, it is mesoporous manganese oxide) is obtained.
  • the viroid silicon mesoporous nanoparticles are prepared by reacting raw materials including tetraethylorthosilicate, cetyltrimethylammonium bromide (CTAB) and triethylamine.
  • raw materials including tetraethylorthosilicate, cetyltrimethylammonium bromide (CTAB) and triethylamine.
  • both the tetraethyl orthosilicate and cetyltrimethylammonium bromide are added to the reaction system in the form of a solution.
  • a cyclohexane solution containing tetraethylorthosilicate and an aqueous solution of cetyltrimethylammonium bromide (CTAB) are first prepared respectively.
  • an aqueous solution of cetyltrimethylammonium bromide (CTAB) is mixed with triethylamine, and then mixed with a cyclohexane solution of tetraethyl orthosilicate to react to obtain the viroid silicon mesoporous nanoparticles.
  • the dosage ratio of tetraethyl orthosilicate to cetyltrimethylammonium bromide (CTAB) and triethylamine is 5.3mL:2g:1mL.
  • the concentration of the triethylamine may be 10-30%, exemplarily 10%, 25%, 30%.
  • the preparation method of the virus-like silicon mesoporous nanoparticles further includes a step of solid-liquid separation of the prepared virus-like silicon mesoporous nanoparticles.
  • the solid-liquid separation can be performed by means known in the art, such as filtration and centrifugation.
  • the preparation method further includes washing the reaction product obtained by solid-liquid separation.
  • the solvent used for washing can be water, ethanol.
  • the number of times of washing may be one, two, or three times.
  • the reaction of the viroid silicon mesoporous nanoparticles with manganese salt and hexatropine is carried out in a solvent system.
  • the virus-like silicon mesoporous nanoparticles are first dispersed in water, and then manganese salt and urotropine are added thereto.
  • the concentration of the viroid silicon mesoporous nanoparticle dispersion is 1-3 mg/mL, exemplarily 1 mg/mL, 2 mg/mL, 3 mg/mL.
  • the mass ratio of the viroid silicon mesoporous nanoparticles to the manganese salt and hexatropine is 10:9:9.
  • the manganese salt may be Mn(NO 3 ) 2 ⁇ 6H 2 O.
  • the reaction temperature of the virus-like silicon mesoporous nanoparticles with the manganese salt and urotropine is 80-100°C, exemplarily 80°C, 90°C, 100°C.
  • the reaction time is 3-5 hours, exemplarily 3 hours, 4 hours, 5 hours.
  • the alkali solution used in the alkali treatment is NaOH aqueous solution.
  • the temperature of the alkali treatment is 60-80°C, exemplarily 60°C, 75°C, 80°C.
  • the concentration of the lye is 1-3 mol/L, exemplarily 1 mol/L, 2 mol/L, 3 mol/L.
  • the preparation method of the carrier further includes a step of solid-liquid separation of the prepared viroid mesoporous manganese oxide reaction liquid.
  • the solid-liquid separation can be performed by means known in the art, such as filtration and centrifugation.
  • the preparation method of the carrier further includes washing the reaction product obtained by solid-liquid separation.
  • the solvent used for washing can be water, ethanol.
  • the number of times of washing may be one, two, or three times.
  • the preparation method of the carrier further includes reacting the washed reaction product with a compound containing an amino functional group, so as to prepare a virus-like hollow manganese oxide whose surface is modified with an amino functional group.
  • the preparation method of the carrier also includes reacting the above-mentioned virus-like hollow manganese oxide with amino functional groups on its surface and fluorescent quencher molecules to prepare virus-like hollow manganese oxides loaded with fluorescent quencher molecules and modified with amino functional groups on the surface.
  • manganese oxide for example, the mass ratio of the viroid hollow manganese oxide surface-modified with amino functional groups to the fluorescence quencher molecule is (3-5):1, exemplarily 3:1, 4:1, 5:1.
  • the reaction time is not less than 12 hours, exemplarily 24 hours.
  • the preparation method of the carrier further includes the step of solid-liquid separation of the above-mentioned virus-like hollow manganese oxide loaded with fluorescence quenching molecules and modified with amino functional groups on the surface.
  • the solid-liquid separation can be performed by means known in the art, such as centrifugation.
  • the preparation method of the carrier includes the following steps:
  • Cetyltrimethylammonium bromide (CTAB) is dissolved in water, then a mixed solution of triethylamine, cyclohexane and tetraethylorthosilicate is added, and the virus-like silicon mesoporous nanoparticles are prepared by reaction ;
  • step (S2) Dissolving the viroid hollow manganese oxide particles prepared in step (S2) in water, then adding IR1064, stirring the reaction at room temperature, and then centrifuging to remove excess IR1064, so as to prepare the viroid hollow manganese oxide particles loaded with IR1064.
  • the reaction between the carrier and the rare earth core-shell nanoparticle further includes adding an activator.
  • the activator can be at least one of carbodiimide (EDC), N-hydroxysuccinimide (NHS) and dimethylacetamide (DMAC); Exemplarily, the activator is EDC and NHS.
  • the mass ratio of the rare earth core-shell nanoparticles to the activator is 1:3-5.
  • the mass ratio of the rare earth core-shell nanoparticles to the carrier is 2-5:1.
  • the present invention also provides a composite probe, which contains the above-mentioned rare earth nanocrystals.
  • the present invention also provides the preparation and/or preparation of the above-mentioned rare earth nanocrystals or composite probes in the fields of surgical navigation (such as fluorescence imaging navigation tumor resection), postoperative chemodynamic therapy, fluorescence guidance of postoperative metastases, and nuclear magnetic resonance imaging of tumors.
  • surgical navigation such as fluorescence imaging navigation tumor resection
  • postoperative chemodynamic therapy fluorescence guidance of postoperative metastases
  • nuclear magnetic resonance imaging of tumors Application as a contrast agent.
  • the preparation and/or application of the rare earth nanocrystal as an optical contrast agent in the second near-infrared region/nuclear magnetic resonance contrast agent for example, the preparation and/or application of the rare earth nanocrystal as an optical contrast agent in the second near-infrared region/nuclear magnetic resonance contrast agent.
  • the optical contrast agent in the second near-infrared region is used in blood vessel imaging and lymph node imaging.
  • its emission wavelength is 1000-1700 nm
  • its excitation wavelength is 700-1100 nm
  • the present invention also provides a contrast agent, which contains the above-mentioned rare earth nanocrystals.
  • the present invention utilizes the hydrothermal solvothermal method to synthesize rare earth nanocrystals excited in the near-infrared 2b region and emitted in the near-infrared 2 region; manganese; and further load the fluorescence quenching molecule IR1064 in the cavity of the viroid hollow manganese oxide; then modify the rare earth nanocrystals by carboxylation reaction, modify the viroid hollow manganese oxide by amination reaction, and finally pass the condensation between the amino-carboxyl groups Reaction, modifying rare earth nanocrystals to the surface of virus-like hollow manganese oxide to construct a composite probe that can be used for surgical navigation and postoperative photodynamic therapy.
  • manganese oxide is responsive to acidic environments, chemokinetic therapy of metastases can be achieved, and manganese ions can be used for nuclear magnetic resonance imaging and restore the fluorescence of the released rare earth nanocrystals.
  • the material of the present invention has the advantages of high tissue penetration, no background autofluorescence interference, and high resolution, so it can be used for early diagnosis of clinical tumors without background interference, high resolution, and strong tissue penetration. Fluorescent imaging of areas, navigating tumor resection, and guiding chemodynamic therapy of metastases.
  • the present invention utilizes the hydrothermal solvothermal method to synthesize rare earth nanocrystals excited by the near-infrared 2b region, near-infrared 2 region, red light emitting, monodisperse and uniform in size through doping adjustment, thereby realizing tumor tissue High-penetration, high-resolution imaging and photodynamic therapy of tumor metastases.
  • the virus-like hollow manganese oxide nanoparticles synthesized by the present invention can be phagocytized by tumor cells in large quantities, and degrade in the slightly acidic environment of tumor cells to release singlet oxygen and manganese ions, so they can be used for the chemical kinetics of tumors. Therapy and MRI for precise tumor treatment.
  • the virus-like hollow manganese oxide-loaded rare earth nanocrystal composite probe excited by the near-infrared 2b region can realize fluorescence imaging of tumor microenvironment response, navigation tumor resection and photodynamic therapy of metastases, so as to The tumor is completely removed, thereby improving the survival rate of patients after surgery, and providing reference for the clinical treatment of malignant tumors.
  • a and B in Fig. 1 are the nuclei ( ⁇ -NaErF 4 : 2% Ho) and ⁇ -NaErF 4 : 2% Ho@NaYF 4 nanocrystals of the near-infrared two-b region excited rare earth nanocrystals prepared in Example 1, respectively TEM image of the core-shell structure.
  • a and B in FIG. 2 are the transmission electron micrographs of the virus-like silicon mesoporous nanoparticles and the virus-like hollow manganese oxide prepared in Example 2, respectively.
  • a and B in Fig. 3 are the rare earth nanocrystals (MnO 2 -IR1064@ ⁇ -NaErF 4 : 2%Ho@NaYF 4 ), the transmission electron microscope image, and the rare earth nanocrystal ( ⁇ -NaErF 4 :2%Ho@NaYF 4 nanocrystal core-shell structure and composite probe (MnO 2 -IR1064@ ⁇ -NaErF 4 : Fluorescence spectrum of 2%Ho@NaYF 4 ).
  • Figure 5 shows the rare earth nanocrystals (MnO 2 -IR1064@ ⁇ -NaErF 4 : 2%Ho@NaYF 4 ) and clinical approval of the surface-modified IR1064-loaded hollow manganese oxide prepared in Example 3 excited by the near-infrared 2b region Comparison of fluorescence imaging of ICG penetration depth under different laser irradiation.
  • ⁇ -NaErF 4 2%Ho@NaYF 4 core-shell structure nanocrystals were prepared by one-pot successive layer-by-layer protocol:
  • Y-OA precursor yttrium-oleic acid (Y-OA) precursor: the YCl 3 of 5.0mmol, oleic acid (20.0mL), octadecene (30.0mL) are added in the 100mL three-necked flask and mixed, and the temperature is raised to 140°C, keep stirring for 60 minutes, and keep the system in a highly anhydrous and oxygen-free environment. Subsequently, the synthesized complex solution was cooled down to obtain a clear and transparent Y-OA precursor solution (0.1mol/L);
  • Na-TFA-OA sodium trifluoroacetate-oleic acid
  • ⁇ -NaErF 4 2%Ho@NaYF 4 : First, add 4.0mL oleic acid and 6mL octadecene into a 50mL three-necked flask and mix well, and then add 2.5mL of the above-synthesized ⁇ -NaErF 4 : 2%Ho nanoparticle dispersion (0.25mmol); Under vacuum, cyclohexane is removed from the system, and then argon is fed into the there-necked flask, and the temperature is raised to 280°C (the heating rate is 20 °C/min), then alternately add Y-OA precursor solution (0.10mol/L, 1.0mL) and Na-TFA-OA (0.40mol/L, 0.50mL) precursor solution, between the two precursor solutions The dripping time interval is 15min.
  • the nanoparticles coated with a shell layer are obtained; a total of three layers are coated (that is, repeated and alternately added Y-OA precursor solution (0.10 mol/L, 1.0mL) and Na-TFA-OA (0.40M, 0.50mL) precursor solution three times), the coating time interval between each layer is 15min, and the thickness of the shell layer is controlled by the number of groups.
  • the reaction mixed solution was cooled to room temperature, and the obtained ⁇ -NaErF 4 : 2% Ho@NaYF 4 rare earth nanocrystals were washed and separated by centrifugation at 3000 rpm with absolute ethanol for 10 min, and the separated ⁇ -NaErF 4 : The 2% Ho@NaYF 4 rare earth nanocrystalline solid product was dispersed in cyclohexane and stored frozen until use.
  • a and B are respectively the nucleus of the rare earth nanocrystal excited by the near-infrared 2b region prepared in this embodiment (that is, the core layer nanocrystal ⁇ -NaErF 4 :2%Ho in the figure A) and the core-shell structure nanocrystal TEM image of the crystal (namely the core-shell structure nanocrystal ⁇ -NaErF 4 :2%Ho@NaYF 4 in Figure B).
  • both the nanocrystals of the core layer and the core-shell layer have a uniform ⁇ -shaped structure; and the particle size is uniform.
  • the particle size of the nanocrystal is about 24.69 ⁇ 0.52nm.
  • virus-like silicon mesoporous nanoparticles prepared in step (1) were ultrasonically dispersed in 50 mL of deionized water, then 0.09 g of Mn(NO 3 ) 3 6H 2 O was added, and the , stirred for 0.5h. Then, 0.09g of urotropine was added, and the stirring reaction was continued for 2h. After the reaction was completed, the product was collected by centrifugation and washed three times with water and ethanol. Then, the virus-like silicon mesoporous nanoparticle template was etched in NaOH aqueous solution.
  • the specific operation was as follows: the reaction product was dispersed in 2mol/L NaOH aqueous solution, placed in an oven at 60°C for 24h, and then washed three times with water and ethanol to obtain the final
  • the product is virus-like hollow mesoporous manganese oxide particles, which are dried and used for future use.
  • a and B in FIG. 2 are transmission electron micrographs of the virus-like silicon mesoporous nanoparticles prepared in this example and the virus-like hollow mesoporous manganese oxide synthesized on the basis of this, respectively.
  • this example successfully prepared a virus-like silicon mesoporous nanoparticle structure, the interior of which is a mesoporous structure, and clusters of tubules grow on the surface.
  • the overall structure is a virus-like structure, and its mesoporous and tubular bodies can be used.
  • the virus-like hollow mesoporous manganese oxide prepared by the subsequent hard template method successfully replicated the structure of the virus-like silicon mesoporous nanoparticles, which is similar to the virus shell, so it has rapid cell invasion and drug loading Function.
  • a and B in Fig. 3 are the rare earth nanocrystals (MnO 2 -IR1064@ ⁇ -NaErF 4 : 2%Ho@NaYF 4 ) TEM images and near-infrared two-b region excited rare earth nanocrystals ( ⁇ -NaErF 4 :2%Ho@NaYF 4 nanocrystalline core-shell structure) and composite probe (MnO 2 -IR1064@ ⁇ -NaErF 4 : Fluorescence spectrum of 2%Ho@NaYF 4 core-shell structure).
  • Figure 4 shows the rare earth nanocrystals (MnO 2 -IR1064@ ⁇ -NaErF 4 :2%Ho@NaYF 4 ) excited by the near-infrared 2b region of the virus-like hollow manganese oxide loaded with IR1064 prepared in this example.
  • the transmission electron micrographs of the precipitate were obtained by centrifugation after incubation for 0 minutes, 30 minutes and 120 minutes under the conditions of 5.5 and 7.0.
  • the results in the figure show that the rare earth nanocrystals of this example can be used as composite probes, and the virus-like hollow manganese oxide will be degraded in the slightly acidic environment of the tumor, releasing ⁇ -NaErF 4 :2%Ho@NaYF 4 nanocrystals And IR1064, the fluorescence of ⁇ -NaErF 4 :2%Ho@NaYF 4 nanocrystals will not be absorbed by IR1064, the fluorescence recovery can be used for imaging, and the released manganese ions can be used for chemokinetic therapy of tumor metastases.
  • Tissue penetration test dilute 20% fat emulsion to 1% with water for use. Take a hexagonal small dish, and add the rare earth nanocrystal (MnO 2 -IR1064@ ⁇ -NaErF 4 :2%Ho@NaYF 4 ) and ICG solution prepared in Example 3 with the same fluorescence intensity into it. Take a petri dish with a diameter of 3.5 cm, place the above-mentioned hexagonal small dishes with samples on the bottom of the petri dish, and fix them with adhesive tape. Put the petri dish containing the rare earth nanocrystal prepared in Example 3 under the InGaAs imager, 1532nm excitation, 880nm filter, 20mm exposure and take pictures. Use a pipette gun to drop fat emulsions with a concentration of 1% in different thicknesses (0mm, 2mm, 4mm, 6mm, 8mm, 10mm), and then photograph until no hexagonal dish is observed.
  • the rare earth nanocrystal MnO 2 -IR1064@
  • Figure 5 is a comparison chart of the rare earth nanocrystals (MnO 2 -IR1064@ ⁇ -NaErF 4 :2%Ho@NaYF 4 ) prepared in Example 3 prepared in this example and the clinically approved ICG penetration depth (the steps are as follows) .
  • the penetration depth of the rare earth nanocrystals (MnO 2 -IR1064@ ⁇ -NaErF 4 :2%Ho@NaYF 4 ) prepared in this example can reach 10 mm;
  • the penetration of ICG 6mm, the rare earth nanocrystals (MnO 2 -IR1064@ ⁇ -NaErF 4 :2%Ho@NaYF 4 ) prepared in this example have significant advantages in tissue penetration,
  • As a composite probe or contrast agent it can realize fluorescence imaging of tumor microenvironment response, navigate tumor resection and photodynamic therapy of metastases, so as to completely remove tumors, thereby improving the postoperative survival rate of patients, and providing support for the treatment of malignant tumors. Provide reference for clinical treatment.

Abstract

A virus-like hollow oxide loaded near-infrared two-b region excited rare earth nanocrystal, a preparation method therefor and an application thereof. An Er and Ho doped NaErF4:2％Ho@NaYF 4 rare earth nanomaterial is first synthesized by means of a hydrothermal method, and then, by means of an amidation reaction, the nanomaterial is modified on the surface of a virus-like hollow manganese oxide in IR1064, so as to obtain a fluorescent composite probe.

Description

一种类病毒空心氧化物负载近红外二b区激发的稀土纳米晶及其制备方法和应用A rare earth nanocrystal excited by a near-infrared second b region supported by a virus-like hollow oxide and its preparation method and application
本申请要求享有2021年9月16日向中国国家知识产权局提交的，专利申请号为202111096606.6，发明名称为“一种类病毒空心氧化锰负载近红外二b区激发的稀土纳米晶及其制备方法和应用”的在先申请的优先权权益。所述在先申请的全文通过引用的方式结合于本申请中。This application claims to be entitled to the patent application number submitted to the State Intellectual Property Office of China on September 16, 2021. The patent application number is 202111096606.6, and the title of the invention is "a rare earth nanocrystal excited by a near-infrared second b region loaded with a virus-like hollow manganese oxide and its preparation method and Priority benefit of an earlier application. The entirety of said prior application is incorporated by reference into this application.
技术领域technical field
本发明属于生物医学纳米材料领域，具体涉及一种类病毒空心氧化物负载近红外二b区激发的稀土纳米晶及其制备方法和用于手术导航和术后化动力治疗的应用。The invention belongs to the field of biomedical nanomaterials, and in particular relates to a rare earth nanocrystal excited by a near-infrared 2b region supported by a virus-like hollow oxide, a preparation method thereof, and an application for surgical navigation and postoperative chemodynamic therapy.
背景技术Background technique
目前针对癌症的治疗方法中，完整彻底的手术切除是首选的最普遍、最理想的方法。但是目前对于肿瘤的术前常规检查手段中由于自身分辨率的限制，因而难以有效的判断和发现肿瘤的位置、边缘和微转移，且手术切除后尚残余的肿瘤细胞会致使患者术后肿瘤的复发。据此，需要一种高效的成像手段以用于在手术切除过程中实时、准确、客观精准的定位肿瘤的位置和边界、并发现微转移灶，从而辅助临床医师彻底的切除肿瘤，以减少患者术后的复发率，这对于提高肿瘤患者的术后生存率具有重要意义。Among the current treatment methods for cancer, complete and complete surgical resection is the most common and ideal method of choice. However, due to the limitation of its own resolution in the current routine preoperative examination methods for tumors, it is difficult to effectively judge and find the location, margin and micrometastasis of the tumor, and the remaining tumor cells after surgical resection will lead to postoperative tumor progression in patients. relapse. Accordingly, there is a need for an efficient imaging method for real-time, accurate, objective and precise positioning of the tumor location and boundaries during surgical resection, and for the detection of micrometastases, thereby assisting clinicians in completely resecting the tumor and reducing the number of patients. Postoperative recurrence rate, which is of great significance for improving the postoperative survival rate of cancer patients.
荧光探针由于其响应速度快、检测灵敏度高，因此荧光成像已成为早期肿瘤诊断、辅助手术导航、检测复发或残留病灶、疗效监测等最有效的途径之一。当前荧光手术导航的探针主要集中在第一窗口近红外光的范围内(the first window near-infrared region，NIR I，650～900nm)。由于NIR I的荧光组织穿透性、信噪比的限制，因此不适用于位于脏器深部的肿瘤，例如脑瘤、卵巢肿瘤、肝肿瘤和淋巴转移灶等的荧光成像。研究表明：相比于NIR I的荧光，第二窗口近红外光(the second window near-infrared region，NIR II，1000～1700nm)具有更深的组织穿透深度、更高的信噪比、更低的光损伤。因此，NIR II荧光成像可以用于精准定位肿瘤位置、清晰的观察肿瘤边缘并进行高效切除。Due to the fast response speed and high detection sensitivity of fluorescent probes, fluorescence imaging has become one of the most effective ways for early tumor diagnosis, assisted surgical navigation, detection of recurrence or residual lesions, and efficacy monitoring. The current probes for fluorescent surgical navigation are mainly concentrated in the range of the first window near-infrared region (NIR I, 650-900nm). Due to the limitations of fluorescence tissue penetration and signal-to-noise ratio of NIR I, it is not suitable for fluorescence imaging of tumors located in deep organs, such as brain tumors, ovarian tumors, liver tumors, and lymphatic metastases. Studies have shown that compared with the fluorescence of NIR I, the second window near-infrared region (NIR II, 1000-1700nm) has a deeper tissue penetration depth, a higher signal-to-noise ratio, and a lower of light damage. Therefore, NIR II fluorescence imaging can be used to precisely locate the tumor location, clearly observe the tumor margin and perform efficient resection.
采用智能纳米载体将药物输送到肿瘤部位，并利用肿瘤组织与正常组织之间的差异，在肿瘤特有的微环境或标志物的刺激下将药物特异的、可控释放到癌变位置，能够提高肿瘤治 疗的疗效、并降低毒副作用。因此，可以通过肿瘤细胞的特异性，实现药物分子的精准、可控释放。氧化锰被广泛应用于肿瘤治疗，其发生降解释放出的锰离子可以在肿瘤酸性环境中产生单线态氧，进行化动力治疗，也可以用于核磁共振成像。此外，具有粗糙表面的类病毒二氧化硅纳米颗粒，由于细胞的粘附作用，可以大大增加细胞的摄取率。因此，亟需发展一种方法简单的可用于构筑类病毒金属介孔氧化物的新方法，以提高肿瘤细胞摄取和生物安全性，对于生物应用和临床转化具有非常重要的意义。Use smart nanocarriers to deliver drugs to tumor sites, and use the difference between tumor tissues and normal tissues to release drugs specifically and controllably to cancerous sites under the stimulation of tumor-specific microenvironment or markers, which can improve tumor treatment efficacy and reduce side effects. Therefore, the precise and controllable release of drug molecules can be achieved through the specificity of tumor cells. Manganese oxide is widely used in the treatment of tumors. The manganese ions released by its degradation can generate singlet oxygen in the acidic environment of tumors, perform chemodynamic therapy, and can also be used for nuclear magnetic resonance imaging. In addition, virus-like silica nanoparticles with rough surfaces can greatly increase the uptake rate by cells due to cell adhesion. Therefore, it is urgent to develop a new method that can be used to construct virus-like metal mesoporous oxides with simple methods to improve tumor cell uptake and biosafety, which is of great significance for biological applications and clinical transformation.
发明内容Contents of the invention
为了改善上述技术问题，本发明提供一种稀土纳米晶，所述稀土纳米晶包括载体及负载于载体表面的稀土核壳纳米颗粒。In order to improve the above technical problems, the present invention provides a rare earth nanocrystal, which includes a carrier and rare earth core-shell nanoparticles loaded on the surface of the carrier.
根据本发明的实施方案，所述稀土纳米晶中，载体与稀土核壳纳米颗粒的质量比为1:(1-2)，优选质量比为1:(1-1.5)，示例性为1:1、1:1.2、1:1.5、1:2。According to an embodiment of the present invention, in the rare earth nanocrystals, the mass ratio of the carrier to the rare earth core-shell nanoparticles is 1:(1-2), preferably the mass ratio is 1:(1-1.5), exemplarily 1: 1, 1:1.2, 1:1.5, 1:2.
根据本发明的实施方案，所述载体与稀土核壳纳米颗粒之间通过价键连接，例如通过酰胺键连接。According to an embodiment of the present invention, the carrier is connected to the rare earth core-shell nanoparticles through a valence bond, for example, through an amide bond.
根据本发明的实施方案，所述载体具有氨基，其与所述稀土核壳纳米颗粒中的羧基形成酰胺键。According to an embodiment of the present invention, the carrier has amino groups that form amide bonds with carboxyl groups in the rare earth core-shell nanoparticles.
根据本发明的实施方案，所述稀土纳米晶在肿瘤弱酸性微环境中能选择性降解，优选为在肿瘤弱酸性微环境中能选择性降解释放出锰离子(Mn 2+)、铁离子(Fe 2+)。 According to an embodiment of the present invention, the rare earth nanocrystals can be selectively degraded in the weakly acidic microenvironment of the tumor, and preferably can be selectively degraded to release manganese ions (Mn 2+ ), iron ions ( Fe 2+ ).
根据本发明的实施方案，所述载体为表面修饰有氨基官能团化合物的空心球。例如，所述载体为氨基化修饰的空心氧化锰和/或空心氧化铁。According to an embodiment of the present invention, the carrier is a hollow sphere whose surface is modified with an amino functional group compound. For example, the carrier is aminated hollow manganese oxide and/or hollow iron oxide.
优选地，所述带有氨基官能团化合物可以为氨基硅烷APTES((3-Aminopropyl)-triethoxysilane)。Preferably, the compound with amino functional group may be aminosilane APTES ((3-Aminopropyl)-triethoxysilane).
优选地，所述载体的空腔中负载有荧光淬灭分子。例如，所述荧光淬灭分子可以为近红外吸收剂，如IR1064。又如，所述荧光淬灭分子的负载量为载体总重量的10～15％，示例性为10％、12％、15％。Preferably, fluorescence quenching molecules are loaded in the cavity of the carrier. For example, the fluorescence quencher molecule can be a near-infrared absorber, such as IR1064. As another example, the loading amount of the fluorescence quencher molecule is 10-15% of the total weight of the carrier, exemplarily 10%, 12%, and 15%.
优选地，所述空心氧化锰为纳米颗粒，其具有类病毒形貌，可以快速被肿瘤细胞吞噬，实现肿瘤的精准成像和治疗。Preferably, the hollow manganese oxide is nanoparticles, which have a virus-like shape and can be quickly phagocytosed by tumor cells to achieve precise imaging and treatment of tumors.
本发明的类病毒空心氧化锰在肿瘤酸性微环境中会降解并坍塌，释放出的锰离子可用于术后肿瘤转移灶的化动力治疗或用于肿瘤的核磁共振成像；其释放出荧光淬灭分子IR1064后，稀土纳米颗粒的荧光恢复，因而可以用于肿瘤的荧光成像手术导航切除。The virus-like hollow manganese oxide of the present invention will degrade and collapse in the acidic microenvironment of the tumor, and the released manganese ions can be used for chemodynamic therapy of postoperative tumor metastases or for nuclear magnetic resonance imaging of tumors; it releases fluorescence quenching After the molecule IR1064, the fluorescence of rare earth nanoparticles is restored, so it can be used for surgically guided resection of tumors with fluorescence imaging.
根据本发明的实施方案，所述稀土核壳纳米颗粒包括核纳米颗粒、至少一层包覆于所述核纳米颗粒外部的壳层、以及修饰于所述壳层外部的含羧基官能团的物质，所述核纳米颗粒和壳层分别独立地选自AREF 4，其中：A为Na或K，RE为Er、Ho、Gd和Y中的至少一种。 According to an embodiment of the present invention, the rare earth core-shell nanoparticles include core nanoparticles, at least one shell covering the outside of the core nanoparticles, and a carboxyl functional group-containing substance modified on the outside of the shell, The core nanoparticle and the shell layer are independently selected from AREF 4 , wherein: A is Na or K, RE is at least one of Er, Ho, Gd and Y.
优选地，所述含羧基官能团的物质可以为含羧基官能团的聚合物。Preferably, the carboxyl functional group-containing substance may be a carboxyl functional group-containing polymer.
优选地，A为Na。Preferably, A is Na.
优选地，所述核纳米颗粒和壳层中的RE元素不同。优选地，所述核纳米颗粒中，RE为Er、Ho中的两种；所述壳层纳米颗粒中，RE为Y。Preferably, the RE elements in the core nanoparticles and the shell are different. Preferably, in the core nanoparticles, RE is two of Er and Ho; in the shell nanoparticles, RE is Y.
优选地，所述核纳米颗粒中，Ho的掺杂量为1～5％，示例性为1％、2％、5％。Preferably, in the core nanoparticles, the doping amount of Ho is 1-5%, exemplarily 1%, 2%, 5%.
优选地，所述稀土核壳纳米颗粒的粒径为24～25.5nm，示例性为24nm、24.13nm、25nm、25.25nm、25.5nm。Preferably, the particle size of the rare earth core-shell nanoparticles is 24-25.5nm, exemplarily 24nm, 24.13nm, 25nm, 25.25nm, 25.5nm.
优选地，所述核纳米颗粒的粒径和壳层的厚度之比为1:(1～1.5)，示例性为1:1、1:1.18、1:1.5。Preferably, the ratio of the particle size of the core nanoparticles to the thickness of the shell layer is 1:(1˜1.5), exemplarily 1:1, 1:1.18, 1:1.5.
优选地，所述核纳米颗粒的粒径为17～18nm，示例性为17.09nm、17.5nm、17.95nm。Preferably, the particle diameter of the core nanoparticles is 17-18nm, exemplarily 17.09nm, 17.5nm, 17.95nm.
优选地，所述的含羧基官能团的物质可以选自PEG聚合物、小分子酸类或者含羧基的高分子聚合物，例如可以为磷脂聚乙二醇(DSEP-PEG 2000-COOH)、马来酰胺酸和聚丙烯酸等中的至少一种。 Preferably, the carboxyl functional group-containing substance can be selected from PEG polymers, small molecular acids or carboxyl-containing polymers, such as phospholipid polyethylene glycol (DSEP-PEG 2000 -COOH), Malay At least one of amic acid and polyacrylic acid, etc.
本发明的含羧基官能团的聚合物可以增强材料的生物相容性，减少细胞毒性。The polymer containing carboxyl functional group of the present invention can enhance the biocompatibility of materials and reduce cytotoxicity.
优选地，所述稀土核壳纳米颗粒具有近红外一区(650-900nm)及近红外二区(1000-1700nm)光学造影功能。Preferably, the rare earth core-shell nanoparticles have optical imaging functions in the first near-infrared region (650-900nm) and the second near-infrared region (1000-1700nm).
根据本发明的实施方案，所述稀土纳米晶呈单分散且尺寸均一。优选地，所述稀土纳米晶的平均粒径约为24～26nm，示例性为24.13nm、24.69nm、25nm、25.25nm。According to an embodiment of the present invention, the rare earth nanocrystals are monodisperse and uniform in size. Preferably, the average particle size of the rare earth nanocrystals is about 24-26 nm, exemplarily 24.13 nm, 24.69 nm, 25 nm, 25.25 nm.
根据本发明的实施方案，所述稀土纳米晶可以被近红外二b区激发，产生近红外一区、近红外二区荧光发射。According to an embodiment of the present invention, the rare earth nanocrystals can be excited by the near-infrared region 2b to generate fluorescence emission in the near-infrared region 1 and near-infrared region 2 .
例如，所述稀土纳米晶在1532nm激发下可以发射出660nm的近红外一区的红光。以用于近红外一区荧光成像，且可作为近红外二区成像的参比。For example, the rare earth nanocrystals can emit red light in the near-infrared region of 660 nm when excited at 1532 nm. It can be used for fluorescence imaging in the first near-infrared region, and can be used as a reference for imaging in the second near-infrared region.
例如，所述稀土纳米晶在1532nm激发下可以发射出1180nm的荧光。以用于近红外二区荧光成像导航肿瘤的手术切除，解决了当前手术导航中分辨率低、穿透浅的问题，可用于深层组织无背景荧光成像。For example, the rare earth nanocrystals can emit fluorescence at 1180 nm when excited at 1532 nm. It can be used for surgical resection of tumors guided by near-infrared two-zone fluorescence imaging, which solves the problems of low resolution and shallow penetration in current surgical navigation, and can be used for background-free fluorescence imaging of deep tissues.
根据本发明的实施方案，所述稀土核壳纳米颗粒可以为β-NaErF 4:Ho@NaYF 4。 According to an embodiment of the present invention, the rare earth core-shell nanoparticles may be β-NaErF 4 :Ho@NaYF 4 .
本发明的稀土纳米晶颗粒可以实现荧光成像导航肿瘤切除、术后转移灶的荧光成像指导、 肿瘤光动力增强、肿瘤的核磁共振成像，大大提高了肿瘤的清除效率，为临床恶性肿瘤的治疗提供指导。The rare earth nanocrystalline particles of the present invention can realize fluorescence imaging navigation tumor resection, fluorescence imaging guidance of postoperative metastatic lesions, tumor photodynamic enhancement, and tumor nuclear magnetic resonance imaging, greatly improving the removal efficiency of tumors, and providing clinical treatment for malignant tumors. guide.
本发明还提供上述稀土纳米晶的制备方法，包括将载体与稀土核壳纳米颗粒进行反应，制备得到所述稀土纳米晶。The present invention also provides a method for preparing the above-mentioned rare earth nanocrystals, which includes reacting a carrier with rare earth core-shell nanoparticles to prepare the rare earth nanocrystals.
根据本发明的实施方案，所述稀土核壳纳米颗粒与载体的质量比为2～5:1，示例性为2:1、3:1、4:1、5:1。According to an embodiment of the present invention, the mass ratio of the rare earth core-shell nanoparticles to the carrier is 2-5:1, exemplarily 2:1, 3:1, 4:1, 5:1.
根据本发明的实施方案，所述稀土核壳纳米颗粒的制备方法包括以下步骤：According to an embodiment of the present invention, the preparation method of the rare earth core-shell nanoparticles comprises the following steps:
(1)将稀土盐加入油酸和十八烯的混合溶液中，然后加入碱金属氟化物溶液、碱性溶液进行反应，得到所述核纳米颗粒；(1) adding the rare earth salt into the mixed solution of oleic acid and octadecene, then adding an alkali metal fluoride solution and an alkaline solution to react to obtain the nuclear nanoparticles;
(2)向步骤(1)的产物中加入RE-OA和A-TFA-OA前驱体溶液，进行反应，制备得到所述稀土核壳纳米颗粒。(2) Adding RE-OA and A-TFA-OA precursor solution to the product of step (1) for reaction to prepare the rare earth core-shell nanoparticles.
优选地，步骤(2)至少进行一次，得到至少包覆一层壳层的纳米颗粒。示例性地，步骤(2)可以进行1次、2次、3次或更多次；Preferably, step (2) is performed at least once to obtain nanoparticles coated with at least one shell layer. Exemplarily, step (2) can be carried out 1 time, 2 times, 3 times or more;
其中，A为Na或K；RE为Er、Ho、Gd和Y中的至少一种，优选为Y。Wherein, A is Na or K; RE is at least one of Er, Ho, Gd and Y, preferably Y.
优选地，所述碱金属氟化物指碱金属离子或者与碱金属离子性质相似的阳离子和F离子形成的化合物。例如，所述碱金属离子为Na +、K +，所述与碱金属离子性质相似的阳离子为NH 4 +。 Preferably, the alkali metal fluoride refers to an alkali metal ion or a compound formed of a cation similar to an alkali metal ion and a F ion. For example, the alkali metal ions are Na + , K + , and the cations with properties similar to alkali metal ions are NH 4 + .
优选地，所述碱金属氟化物溶液为NaF、NH 4F和KF的溶液中的一种，优选为NH 4F的溶液。 Preferably, the alkali metal fluoride solution is one of NaF, NH 4 F and KF solutions, preferably NH 4 F solution.
优选地，步骤(1)中，所述稀土盐中的稀土离子和碱金属氟化物的摩尔比为1:(2-5)，示例性为1:4。Preferably, in step (1), the molar ratio of the rare earth ion and the alkali metal fluoride in the rare earth salt is 1:(2-5), exemplarily 1:4.
优选地，所述稀土盐中的稀土离子为Er离子、Ho离子和Y离子中的至少一种，优选为Er离子、Ho离子中的两种。Preferably, the rare earth ion in the rare earth salt is at least one of Er ion, Ho ion and Y ion, preferably two of Er ion and Ho ion.
优选地，所述Er离子、Ho离子的用量的摩尔比为(48～49.5):(0.5～2)，示例性为49.5:0.5、49:1、48:2。Preferably, the molar ratio of the Er ions and Ho ions used is (48-49.5):(0.5-2), exemplarily 49.5:0.5, 49:1, 48:2.
优选地，步骤(1)中，还包括将所述核纳米颗粒分散于溶剂中，得到核纳米颗粒分散液；例如，所述溶剂为环己烷。例如，所述核纳米颗粒分散液的浓度可以为0.05～0.2mol/L，示例性为0.1mol/L。Preferably, in step (1), it also includes dispersing the nuclear nanoparticles in a solvent to obtain a nuclear nanoparticle dispersion; for example, the solvent is cyclohexane. For example, the concentration of the nuclear nanoparticle dispersion liquid may be 0.05˜0.2 mol/L, exemplarily 0.1 mol/L.
优选地，在步骤(1)中，所述稀土盐为稀土氯化物、稀土硝酸盐或稀土醋酸盐，优选为稀土醋酸盐。Preferably, in step (1), the rare earth salt is rare earth chloride, rare earth nitrate or rare earth acetate, preferably rare earth acetate.
例如，所述稀土氯化物为HoCl 3、ErCl 3和YCl 3中的至少一种。 For example, the rare earth chloride is at least one of HoCl 3 , ErCl 3 and YCl 3 .
例如，所述稀土硝酸盐为Ho(NO 3) 3、Er(NO 3) 3和Y(NO 3) 3中的至少一种。 For example, the rare earth nitrate is at least one of Ho(NO 3 ) 3 , Er(NO 3 ) 3 and Y(NO 3 ) 3 .
例如，所述稀土醋酸盐为Ho(CH 3COO) 3、Er(CH 3COO) 3和Y(CH 3COO) 3中的至少一种。 For example, the rare earth acetate is at least one of Ho(CH 3 COO) 3 , Er(CH 3 COO) 3 and Y(CH 3 COO) 3 .
优选地，在步骤(1)中，所述碱性溶液为氢氧化钠溶液或氢氧化钾溶液。Preferably, in step (1), the alkaline solution is sodium hydroxide solution or potassium hydroxide solution.
优选地，所述碱金属氟化物溶液、碱性溶液中所采用的溶剂为甲醇。Preferably, the solvent used in the alkali metal fluoride solution and the alkaline solution is methanol.
优选地，在步骤(1)中，所述稀土盐的总量为0.5～2mmol，示例性为0.5mmol、1mmol、2mmol。Preferably, in step (1), the total amount of the rare earth salt is 0.5-2 mmol, exemplarily 0.5 mmol, 1 mmol, 2 mmol.
优选地，在步骤(1)中，所述油酸和十八烯的体积比为1:(2～3)，示例性为1:2、1:2.5、1:3。Preferably, in step (1), the volume ratio of oleic acid to octadecene is 1:(2-3), exemplarily 1:2, 1:2.5, 1:3.
优选地，在步骤(1)中，还包括对加入稀土盐后的油酸和十八烯的混合溶液进行加热搅拌，以去除体系中的水和氧气。例如，所述加热搅拌的温度为130～150℃，示例性为130℃、140℃、150℃。例如，所述加热搅拌的时间为20～40min，示例性为20min、30min、40min。进一步地，所述加热搅拌在真空条件下进行。Preferably, in step (1), heating and stirring the mixed solution of oleic acid and octadecene after adding the rare earth salt is also included, so as to remove water and oxygen in the system. For example, the temperature of the heating and stirring is 130-150°C, exemplarily 130°C, 140°C, 150°C. For example, the heating and stirring time is 20-40 minutes, exemplarily 20 minutes, 30 minutes, 40 minutes. Further, the heating and stirring are carried out under vacuum conditions.
优选地，在步骤(1)中，还包括对加入碱金属氟化物溶液、碱性溶液后的混合溶液进行搅拌成核。例如，所述搅拌成核采用二级加热方式。Preferably, in step (1), it also includes stirring the mixed solution after adding the alkali metal fluoride solution and the alkaline solution to nucleate. For example, the stirring nucleation adopts a two-stage heating method.
优选地，所述二级加热方式包括：Preferably, the secondary heating method includes:
第一级加热的温度为40～60℃，示例性为40℃、50℃、60℃；第一级加热的时间为0.5～2h，示例性为0.5h、1h、2h；The temperature of the first stage heating is 40-60°C, exemplarily 40°C, 50°C, 60°C; the time of the first stage heating is 0.5-2h, exemplarily 0.5h, 1h, 2h;
第二级加热的温度为60～80℃，示例性为60℃、70℃、80℃；第二级加热的时间为0.5～2h，示例性为0.5h、1h、2h。The temperature of the second stage heating is 60-80°C, exemplarily 60°C, 70°C, 80°C; the time of the second stage heating is 0.5-2h, exemplarily 0.5h, 1h, 2h.
优选地，所述制备方法还包括对步骤(1)制得的核纳米颗粒混合液进行固液分离得到反应产物的过程。例如，所述固液分离的方法为向核纳米颗粒混合液中加入沉淀剂进行沉淀，并离心得到固体产物。又如，所述沉淀剂可以为乙醇。Preferably, the preparation method further includes a process of solid-liquid separation of the nuclear nanoparticle mixture prepared in step (1) to obtain a reaction product. For example, the solid-liquid separation method is to add a precipitating agent to the mixture of nuclear nanoparticles for precipitation, and centrifuge to obtain a solid product. As another example, the precipitating agent may be ethanol.
优选地，所述制备方法还包括对步骤(1)固液分离得到的反应产物进行洗涤。例如，洗涤的溶剂可以为无水乙醇。Preferably, the preparation method further includes washing the reaction product obtained from the solid-liquid separation in step (1). For example, the washing solvent can be absolute ethanol.
优选地，步骤(2)中，在向步骤(1)的产物中加入RE-OA和A-TFA-OA前驱体溶液前，还包括将所述核纳米颗粒分散液加入油酸(OA)和十八烯的混合溶液。例如，所述核纳米颗粒分散液与油酸和十八烯的混合溶液的体积比为1:(1-3)，示例性为1:2。又如，油酸和十八烯的混合溶液中，油酸和十八烯的体积比为1:(1-2)，示例性为1:1.5。Preferably, in step (2), before adding RE-OA and A-TFA-OA precursor solution to the product of step (1), it also includes adding oleic acid (OA) and mixed solution of octadecene. For example, the volume ratio of the nuclear nanoparticle dispersion to the mixed solution of oleic acid and octadecene is 1:(1-3), exemplarily 1:2. As another example, in the mixed solution of oleic acid and octadecene, the volume ratio of oleic acid and octadecene is 1:(1-2), exemplarily 1:1.5.
优选地，在步骤(2)中，所述RE-OA前驱体溶液的浓度为0.05～0.2mol/L，示例性为0.05mol/L、0.1mol/L、0.2mol/L。Preferably, in step (2), the concentration of the RE-OA precursor solution is 0.05-0.2 mol/L, exemplarily 0.05 mol/L, 0.1 mol/L, 0.2 mol/L.
根据本发明示例性的实施方案，所述RE-OA前驱体溶液由包括将含有RE元素的盐溶于油 酸、十八烯的混合溶剂中制备得到。优选地，所述混合溶剂中，油酸、十八烯的体积比为1:(1～2)，示例性为1:1、1:1.5、1:2。进一步地，还包括对混合溶液进行加热搅拌。例如，所述加热的温度可以为130～150℃，示例性为130℃、140℃、150℃；所述加热的时间为0.5～2h，示例性为0.5h、1h、2h。又如，所述含有RE元素的盐为稀土氯化物、稀土硝酸盐或稀土醋酸盐，优选为氯化物。According to an exemplary embodiment of the present invention, the RE-OA precursor solution is prepared by dissolving a salt containing RE elements in a mixed solvent of oleic acid and octadecene. Preferably, in the mixed solvent, the volume ratio of oleic acid to octadecene is 1:(1-2), exemplarily 1:1, 1:1.5, 1:2. Further, heating and stirring the mixed solution is also included. For example, the heating temperature may be 130-150°C, exemplarily 130°C, 140°C, 150°C; the heating time is 0.5-2h, exemplarily 0.5h, 1h, 2h. As another example, the salt containing RE element is rare earth chloride, rare earth nitrate or rare earth acetate, preferably chloride.
优选地，在步骤(2)中，所述A-TFA-OA前驱体溶液的浓度为0.3～0.5mol/L，示例性为0.3mol/L、0.4mol/L、0.5mol/L。Preferably, in step (2), the concentration of the A-TFA-OA precursor solution is 0.3-0.5 mol/L, exemplarily 0.3 mol/L, 0.4 mol/L, 0.5 mol/L.
根据本发明示例性的实施方案，所述A-TFA-OA前驱体溶液由包括将含有A元素的三氟乙酸盐(即A-TFA)溶于油酸中制备得到。优选地，所述A-TFA-OA前驱体溶液的制备，还包括对混合溶液进行搅拌。According to an exemplary embodiment of the present invention, the A-TFA-OA precursor solution is prepared by dissolving trifluoroacetate salt containing A element (ie, A-TFA) in oleic acid. Preferably, the preparation of the A-TFA-OA precursor solution also includes stirring the mixed solution.
优选地，在步骤(2)中，所述RE-OA前驱体溶液和A-TFA-OA前驱体溶液以交替间隔的方式加入步骤(1)制得的核纳米颗粒分散液中。例如，所述RE-OA前驱体溶液和A-TFA-OA前驱体溶液交替加入的次数至少各为一次。示例性地，交替各加入1次、2次或更多次；优选交替各加入三次。又如，间隔的时间可以为10～20min，示例性为10min、15min、20min。Preferably, in step (2), the RE-OA precursor solution and the A-TFA-OA precursor solution are added to the nuclear nanoparticle dispersion prepared in step (1) at alternating intervals. For example, the number of alternate additions of the RE-OA precursor solution and the A-TFA-OA precursor solution is at least one. Exemplarily, each is added alternately 1 time, 2 times or more; preferably alternately added three times. As another example, the time interval may be 10-20 minutes, exemplarily 10 minutes, 15 minutes, 20 minutes.
优选地，所述稀土盐中的稀土离子与步骤(2)中的RE-OA前驱体溶液中的稀土离子(RE)的摩尔比为1:(1～10)，优选为1:(6～8)，示例性为1:1、1:2、1:5、1:6、1:7.5、1:8、1:10。Preferably, the molar ratio of the rare earth ion in the rare earth salt to the rare earth ion (RE) in the RE-OA precursor solution in step (2) is 1:(1～10), preferably 1:(6～ 8), exemplarily 1:1, 1:2, 1:5, 1:6, 1:7.5, 1:8, 1:10.
优选地，步骤(1)、步骤(2)的反应在惰性气氛下进行。例如，所述惰性气氛为氮气、氩气。Preferably, the reactions of step (1) and step (2) are carried out under an inert atmosphere. For example, the inert atmosphere is nitrogen, argon.
优选地，在步骤(1)、(2)中，所述反应的温度相同或不同，彼此独立地为200-400℃，示例性为200℃、280℃、300℃、400℃。优选地，在步骤(1)中，所述反应的时间为40-80min，示例性为40min、60min、80min。Preferably, in steps (1) and (2), the reaction temperatures are the same or different, and are independently 200-400°C, exemplarily 200°C, 280°C, 300°C, 400°C. Preferably, in step (1), the reaction time is 40-80 min, exemplarily 40 min, 60 min, 80 min.
优选地，在步骤(1)、(2)中，所述反应的升温速率为5-20℃/min，示例性为5℃/min、10℃/min、20℃/min。Preferably, in steps (1) and (2), the heating rate of the reaction is 5-20°C/min, exemplarily 5°C/min, 10°C/min, 20°C/min.
优选地，所述制备方法还包括步骤(3)：将步骤(2)得到的至少包覆一层壳层的纳米颗粒与含羧基官能团的物质(例如聚合物)在有机溶剂中混匀，静置至有机溶剂挥发，然后加水分散，离心分离出所述羧基修饰的稀土核壳纳米颗粒。Preferably, the preparation method further includes step (3): mixing the nanoparticles coated with at least one shell layer obtained in step (2) with a substance (such as a polymer) containing a carboxyl functional group in an organic solvent, and statically Place until the organic solvent volatilizes, then add water to disperse, and centrifuge to separate the carboxyl-modified rare earth core-shell nanoparticles.
优选地，在步骤(3)中，有机溶剂为氯仿、环己烷、正己烷、四氢呋喃，优选为氯仿。Preferably, in step (3), the organic solvent is chloroform, cyclohexane, n-hexane, tetrahydrofuran, preferably chloroform.
优选地，在步骤(3)中，离心分离转速为8000～20000rpm，示例性为8000rpm、10000rpm、15000rpm、17500rpm、20000rpm。进一步地，所述离心的时间为20～40min，示例性为20min、30min、40min。Preferably, in step (3), the rotational speed of centrifugation is 8000-20000rpm, exemplarily 8000rpm, 10000rpm, 15000rpm, 17500rpm, 20000rpm. Further, the centrifugation time is 20-40 minutes, for example, 20 minutes, 30 minutes, 40 minutes.
进一步地，在步骤(3)中，所述至少包覆一层壳层的纳米颗粒与含羧基官能团的物质(例 如聚合物)的用量比为0.1mmol:(20-30)mg，示例性为0.1mmol:20mg、0.1mmol:25mg、0.1mmol:30mg。Further, in step (3), the dosage ratio of the nanoparticles coated with at least one shell layer and the substance (such as a polymer) containing carboxyl functional groups is 0.1 mmol: (20-30) mg, exemplarily as 0.1mmol: 20mg, 0.1mmol: 25mg, 0.1mmol: 30mg.
根据本发明示例性的实施方案，所述稀土纳米晶的制备方法，包括如下步骤：According to an exemplary embodiment of the present invention, the preparation method of the rare earth nanocrystals includes the following steps:
(A1)β-NaErF 4:2％Ho核的制备 (A1) Preparation of β-NaErF 4 :2%Ho core
将Er(CH 3CO 2) 3·4H 2O、Ho(CH 3CO 2) 3·4H 2O加入油酸和十八烯的混合溶液中混合均匀，在真空条件下加热搅拌(去除体系中的水以及氧气)，而后冷却至室温，得到澄清透明溶液；然后向所述澄清透明溶液中加入氟化铵以及氢氧化钠的甲醇溶液，在40～60℃条件下保持0.5～2h充分搅拌成核，升温至60～80℃，并于真空条件下保持0.5～2h(除去多余的甲醇、氧气以及水分子)；随后，在氩气保护下于300℃下反应50～70min；反应完成后，固液分离得到β-NaErF 4:2％Ho核，洗涤后将其分散在环己烷中，得到β-NaErF 4:2％Ho核纳米颗粒分散液； Add Er(CH 3 CO 2 ) 3 4H 2 O and Ho(CH 3 CO 2 ) 3 4H 2 O into the mixed solution of oleic acid and octadecene, mix well, heat and stir under vacuum (remove the water and oxygen), and then cooled to room temperature to obtain a clear and transparent solution; then, ammonium fluoride and methanol solution of sodium hydroxide were added to the clear and transparent solution, kept at 40-60°C for 0.5-2h and fully stirred to form core, heated to 60-80°C, and kept under vacuum conditions for 0.5-2h (to remove excess methanol, oxygen and water molecules); then, under the protection of argon, reacted at 300°C for 50-70min; after the reaction was completed, Solid-liquid separation to obtain β-NaErF 4 : 2% Ho core, after washing, disperse it in cyclohexane to obtain β-NaErF 4 : 2% Ho core nanoparticle dispersion;
(A2)前驱体的制备(A2) Preparation of precursors
①Y-OA前驱体的合成：将YCl 3溶于油酸和十八烯的混合溶剂中，在保持真空的条件下加热搅拌制得澄清透明的Y-OA前驱体溶液； ① Synthesis of Y-OA precursor: Dissolve YCl 3 in a mixed solvent of oleic acid and octadecene, heat and stir under vacuum conditions to obtain a clear and transparent Y-OA precursor solution;
②Na-TFA-OA前驱体的合成：将三氟乙酸钠溶于油酸，然后抽真空搅拌均匀直至完全溶解，得到淡黄色透明前驱体油酸溶液；②Synthesis of Na-TFA-OA precursor: Dissolve sodium trifluoroacetate in oleic acid, then vacuumize and stir evenly until completely dissolved to obtain light yellow transparent precursor oleic acid solution;
(A3)NaYF 4的包裹采用连续层层生长法制备 (A3) The wrapping of NaYF 4 is prepared by continuous layer growth method
首先将β-NaErF 4:2％Ho核纳米颗粒分散液加入油酸和十八烯的混合溶液中，在真空下，将环己烷从体系中除去，再通入氩气，升温，然后交替加入Y-OA前驱体溶液和Na-TFA-OA前驱体溶液，制备得到所述稀土纳米晶。 First, β-NaErF 4 : 2% Ho core nanoparticle dispersion is added to the mixed solution of oleic acid and octadecene, under vacuum, cyclohexane is removed from the system, and then argon is introduced, the temperature is raised, and then alternately Adding Y-OA precursor solution and Na-TFA-OA precursor solution to prepare the rare earth nanocrystal.
根据本发明的实施方案，所述载体的制备方法，包括以类病毒硅介孔纳米颗粒为模板，与锰盐、乌洛托品反应后，经碱处理除去类病毒硅介孔纳米颗粒模板，即制得类病毒空心氧化锰(即其为介孔氧化锰)。According to an embodiment of the present invention, the preparation method of the carrier includes using the virus-like silicon mesoporous nanoparticle as a template, reacting with manganese salt and urotropine, and removing the virus-like silicon mesoporous nanoparticle template through alkali treatment, That is, the virus-like hollow manganese oxide (ie, it is mesoporous manganese oxide) is obtained.
根据本发明示例性的实施方案，所述类病毒硅介孔纳米颗粒由包括正硅酸四乙酯与十六烷基三甲基溴化铵(CTAB)、三乙胺的原料反应制备得到。According to an exemplary embodiment of the present invention, the viroid silicon mesoporous nanoparticles are prepared by reacting raw materials including tetraethylorthosilicate, cetyltrimethylammonium bromide (CTAB) and triethylamine.
优选地，所述正硅酸四乙酯与十六烷基三甲基溴化铵(CTAB)均以溶液形式加入反应体系中。例如，先分别配制含有正硅酸四乙酯的环己烷溶液和十六烷基三甲基溴化铵(CTAB)的水溶液。优选地，先将十六烷基三甲基溴化铵(CTAB)的水溶液与三乙胺混合，再与正硅酸四乙酯的环己烷溶液混合，反应得到所述类病毒硅介孔纳米颗粒。Preferably, both the tetraethyl orthosilicate and cetyltrimethylammonium bromide (CTAB) are added to the reaction system in the form of a solution. For example, a cyclohexane solution containing tetraethylorthosilicate and an aqueous solution of cetyltrimethylammonium bromide (CTAB) are first prepared respectively. Preferably, an aqueous solution of cetyltrimethylammonium bromide (CTAB) is mixed with triethylamine, and then mixed with a cyclohexane solution of tetraethyl orthosilicate to react to obtain the viroid silicon mesoporous nanoparticles.
根据本发明一个示例性的实施方案，所述正硅酸四乙酯与十六烷基三甲基溴化铵(CTAB)、三乙胺的用量比为5.3mL:2g:1mL。According to an exemplary embodiment of the present invention, the dosage ratio of tetraethyl orthosilicate to cetyltrimethylammonium bromide (CTAB) and triethylamine is 5.3mL:2g:1mL.
根据本发明一个示例性的实施方案，所述三乙胺的浓度可以为10～30％，示例性为10％、25％、30％。According to an exemplary embodiment of the present invention, the concentration of the triethylamine may be 10-30%, exemplarily 10%, 25%, 30%.
优选地，所述类病毒硅介孔纳米颗粒的制备方法还包括对制得的类病毒硅介孔纳米颗粒进行固液分离的步骤。例如，所述固液分离可以采用本领域已知手段，比如过滤、离心。Preferably, the preparation method of the virus-like silicon mesoporous nanoparticles further includes a step of solid-liquid separation of the prepared virus-like silicon mesoporous nanoparticles. For example, the solid-liquid separation can be performed by means known in the art, such as filtration and centrifugation.
优选地，所述制备方法还包括对固液分离得到的反应产物进行洗涤。例如，洗涤所用的溶剂可以为水、乙醇。又如，所述洗涤的次数可以为一次、两次、三次。Preferably, the preparation method further includes washing the reaction product obtained by solid-liquid separation. For example, the solvent used for washing can be water, ethanol. As another example, the number of times of washing may be one, two, or three times.
优选地，所述类病毒硅介孔纳米颗粒与锰盐、乌洛托品的反应在溶剂体系下进行。例如，先将所述类病毒硅介孔纳米颗粒分散于水中，再向其中加入锰盐、乌洛托品。又如，类病毒硅介孔纳米颗粒分散液的浓度为1～3mg/mL，示例性为1mg/mL、2mg/mL、3mg/mL。Preferably, the reaction of the viroid silicon mesoporous nanoparticles with manganese salt and hexatropine is carried out in a solvent system. For example, the virus-like silicon mesoporous nanoparticles are first dispersed in water, and then manganese salt and urotropine are added thereto. As another example, the concentration of the viroid silicon mesoporous nanoparticle dispersion is 1-3 mg/mL, exemplarily 1 mg/mL, 2 mg/mL, 3 mg/mL.
根据本发明一个示例性的实施方案，所述类病毒硅介孔纳米颗粒与锰盐、乌洛托品的质量比为10:9:9。示例性地，所述锰盐可以为Mn(NO 3) 2·6H 2O。 According to an exemplary embodiment of the present invention, the mass ratio of the viroid silicon mesoporous nanoparticles to the manganese salt and hexatropine is 10:9:9. Exemplarily, the manganese salt may be Mn(NO 3 ) 2 ·6H 2 O.
根据本发明一个示例性的实施方案，所述类病毒硅介孔纳米颗粒与锰盐、乌洛托品反应的温度为80～100℃，示例性为80℃、90℃、100℃。进一步地，反应的时间为3～5h，示例性为3h、4h、5h。According to an exemplary embodiment of the present invention, the reaction temperature of the virus-like silicon mesoporous nanoparticles with the manganese salt and urotropine is 80-100°C, exemplarily 80°C, 90°C, 100°C. Further, the reaction time is 3-5 hours, exemplarily 3 hours, 4 hours, 5 hours.
根据本发明一个示例性的实施方案，所述碱处理采用的碱液为NaOH水溶液。优选地，所述碱处理的温度为60～80℃，示例性为60℃、75℃、80℃。进一步地，所述碱液的浓度为1～3mol/L，示例性为1mol/L、2mol/L、3mol/L。According to an exemplary embodiment of the present invention, the alkali solution used in the alkali treatment is NaOH aqueous solution. Preferably, the temperature of the alkali treatment is 60-80°C, exemplarily 60°C, 75°C, 80°C. Further, the concentration of the lye is 1-3 mol/L, exemplarily 1 mol/L, 2 mol/L, 3 mol/L.
优选地，所述载体的制备方法还包括对制得的类病毒介孔氧化锰反应液进行固液分离的步骤。例如，所述固液分离可以采用本领域已知手段，比如过滤、离心。Preferably, the preparation method of the carrier further includes a step of solid-liquid separation of the prepared viroid mesoporous manganese oxide reaction liquid. For example, the solid-liquid separation can be performed by means known in the art, such as filtration and centrifugation.
优选地，所述载体的制备方法还包括对固液分离得到的反应产物进行洗涤。例如，洗涤所用的溶剂可以为水、乙醇。又如，所述洗涤的次数可以为一次、两次、三次。Preferably, the preparation method of the carrier further includes washing the reaction product obtained by solid-liquid separation. For example, the solvent used for washing can be water, ethanol. As another example, the number of times of washing may be one, two, or three times.
优选地，所述载体的制备方法还包括将洗涤后的反应产物与含有氨基官能团的化合物进行反应，以制得表面修饰有氨基官能团的类病毒空心氧化锰。Preferably, the preparation method of the carrier further includes reacting the washed reaction product with a compound containing an amino functional group, so as to prepare a virus-like hollow manganese oxide whose surface is modified with an amino functional group.
优选地，所述载体的制备方法还包括将上述表面修饰有氨基官能团的类病毒空心氧化锰与荧光淬灭分子进行反应，以制备得到负载有荧光淬灭分子表面修饰有氨基官能团的类病毒空心氧化锰。例如，所述表面修饰有氨基官能团的类病毒空心氧化锰与荧光淬灭分子的质量比为(3～5):1，示例性为3:1、4:1、5:1。又如，所述反应的时间为不少于12h，示例性为24h。Preferably, the preparation method of the carrier also includes reacting the above-mentioned virus-like hollow manganese oxide with amino functional groups on its surface and fluorescent quencher molecules to prepare virus-like hollow manganese oxides loaded with fluorescent quencher molecules and modified with amino functional groups on the surface. manganese oxide. For example, the mass ratio of the viroid hollow manganese oxide surface-modified with amino functional groups to the fluorescence quencher molecule is (3-5):1, exemplarily 3:1, 4:1, 5:1. As another example, the reaction time is not less than 12 hours, exemplarily 24 hours.
优选地，所述载体的制备方法还包括对上述负载有荧光淬灭分子表面修饰有氨基官能团的类病毒空心氧化锰进行固液分离的步骤。例如，所述固液分离可以采用本领域已知手段，比如离心。Preferably, the preparation method of the carrier further includes the step of solid-liquid separation of the above-mentioned virus-like hollow manganese oxide loaded with fluorescence quenching molecules and modified with amino functional groups on the surface. For example, the solid-liquid separation can be performed by means known in the art, such as centrifugation.
根据本发明的实施方案，所述载体的制备方法包括如下步骤：According to an embodiment of the present invention, the preparation method of the carrier includes the following steps:
(S1)两相法合成类病毒硅介孔纳米颗粒；(S1) Synthesis of virus-like silicon mesoporous nanoparticles by two-phase method;
将十六烷基三甲基溴化铵(CTAB)溶于水中，然后加入三乙胺、环己烷和正硅酸四乙酯的混合溶液，经反应制得所述类病毒硅介孔纳米颗粒；Cetyltrimethylammonium bromide (CTAB) is dissolved in water, then a mixed solution of triethylamine, cyclohexane and tetraethylorthosilicate is added, and the virus-like silicon mesoporous nanoparticles are prepared by reaction ;
(S2)类病毒空心氧化锰的制备(S2) Preparation of viroid hollow manganese oxide
加热搅拌条件下，向类病毒硅介孔纳米颗粒分散液中加入Mn(NO 3) 2·6H 2O、乌洛托品，反应后离心洗涤，最后在NaOH水溶液中刻蚀掉类病毒介孔硅模板，以制得类病毒空心氧化锰；再将类病毒空心氧化锰与含有氨基官能团的化合物进行反应，以制得表面修饰有氨基官能团的类病毒空心氧化锰； Under the condition of heating and stirring, add Mn(NO 3 ) 2 6H 2 O and urotropine to the dispersion liquid of viroid silicon mesoporous nanoparticles, centrifuge and wash after reaction, and finally etch viroid mesoporous pores in NaOH aqueous solution Silicon template to prepare viroid hollow manganese oxide; then react viroid hollow manganese oxide with a compound containing amino functional group to prepare viroid hollow manganese oxide with amino functional group on the surface;
(S3)负载IR1064的类病毒空心氧化锰的制备(S3) Preparation of virus-like hollow manganese oxide loaded with IR1064
将步骤(S2)制得的类病毒空心氧化锰颗粒溶于水中，然后再加入IR1064，室温下搅拌反应，然后离心去除多余的IR1064，从而制得负载IR1064的类病毒空心氧化锰颗粒。Dissolving the viroid hollow manganese oxide particles prepared in step (S2) in water, then adding IR1064, stirring the reaction at room temperature, and then centrifuging to remove excess IR1064, so as to prepare the viroid hollow manganese oxide particles loaded with IR1064.
根据本发明的实施方案，所述稀土纳米晶的制备方法中，所述载体与稀土核壳纳米颗粒的反应还包括加入活化剂。例如，所述活化剂可以为碳二亚胺(EDC)、N-羟基琥珀酰亚胺(NHS)和二甲基乙酰胺(DMAC)中的至少一种；示例性地，所述活化剂为EDC和NHS。According to an embodiment of the present invention, in the preparation method of the rare earth nanocrystal, the reaction between the carrier and the rare earth core-shell nanoparticle further includes adding an activator. For example, the activator can be at least one of carbodiimide (EDC), N-hydroxysuccinimide (NHS) and dimethylacetamide (DMAC); Exemplarily, the activator is EDC and NHS.
优选地，所述活化剂活化的过程在pH＝8.0～9.0(示例性pH＝8.5)的溶液中进行。进一步地，所述活化的时间为不低于10h，示例性为12h。Preferably, the activation process of the activator is carried out in a solution with pH=8.0-9.0 (exemplary pH=8.5). Further, the activation time is not less than 10 h, exemplarily 12 h.
优选地，所述稀土核壳纳米颗粒和活化剂的质量比为1:3～5。Preferably, the mass ratio of the rare earth core-shell nanoparticles to the activator is 1:3-5.
优选地，稀土核壳纳米颗粒与载体的质量比为2～5:1。Preferably, the mass ratio of the rare earth core-shell nanoparticles to the carrier is 2-5:1.
本发明还提供一种复合探针，其含有上述稀土纳米晶。The present invention also provides a composite probe, which contains the above-mentioned rare earth nanocrystals.
本发明还提供上述稀土纳米晶或复合探针在手术导航(如荧光成像导航肿瘤切除)、术后化动力治疗、术后转移灶的荧光指导、肿瘤的核磁共振成像等领域中制备和/或作为造影剂的应用。The present invention also provides the preparation and/or preparation of the above-mentioned rare earth nanocrystals or composite probes in the fields of surgical navigation (such as fluorescence imaging navigation tumor resection), postoperative chemodynamic therapy, fluorescence guidance of postoperative metastases, and nuclear magnetic resonance imaging of tumors. Application as a contrast agent.
例如，所述稀土纳米晶在制备和/或作为近红外二区光学造影剂/核磁共振造影剂的应用。For example, the preparation and/or application of the rare earth nanocrystal as an optical contrast agent in the second near-infrared region/nuclear magnetic resonance contrast agent.
优选地，所述近红外二区光学造影剂应用于血管显像和***显影中。Preferably, the optical contrast agent in the second near-infrared region is used in blood vessel imaging and lymph node imaging.
优选地，当作为近红外二区光学造影剂时，其发射波长为1000-1700nm，激发波长为700-1100nm。Preferably, when used as an optical contrast agent in the second near-infrared region, its emission wavelength is 1000-1700 nm, and its excitation wavelength is 700-1100 nm.
本发明还提供一种造影剂，含有上述稀土纳米晶。The present invention also provides a contrast agent, which contains the above-mentioned rare earth nanocrystals.
本发明的有益效果:Beneficial effects of the present invention:
(1)本发明利用水热溶剂热法合成了近红外二b区激发、近红外二区发射的稀土纳米晶体；并以类病毒介孔二氧化硅为硬模板，优选合成了类病毒空心氧化锰；并进一步在类病毒空心氧化锰的空腔中负载荧光淬灭分子IR1064；随后通过羧基化反应修饰稀土纳米晶体，氨基化反应修饰类病毒空心氧化锰，最后通过氨基-羧基之间的缩合反应，将稀土纳米晶体修饰到类病毒空心氧化锰表面，以构筑成可用于手术导航和术后光动力治疗的复合探针。由于氧化锰具有酸性环境响应性，因而可以实现转移灶的化动力治疗，同时锰离子可用于核磁共振成像，并恢复释放出的稀土纳米晶的荧光。本发明的材料具有高组织穿透性、无背景自荧光干扰、高分辨率等优点，因而可用于无背景干扰、分辨率高及组织穿透力强的临床肿瘤的早期诊断、肿瘤近红外二区的荧光成像、导航肿瘤切除以及指导转移灶的化动力治疗。(1) The present invention utilizes the hydrothermal solvothermal method to synthesize rare earth nanocrystals excited in the near-infrared 2b region and emitted in the near-infrared 2 region; manganese; and further load the fluorescence quenching molecule IR1064 in the cavity of the viroid hollow manganese oxide; then modify the rare earth nanocrystals by carboxylation reaction, modify the viroid hollow manganese oxide by amination reaction, and finally pass the condensation between the amino-carboxyl groups Reaction, modifying rare earth nanocrystals to the surface of virus-like hollow manganese oxide to construct a composite probe that can be used for surgical navigation and postoperative photodynamic therapy. Because manganese oxide is responsive to acidic environments, chemokinetic therapy of metastases can be achieved, and manganese ions can be used for nuclear magnetic resonance imaging and restore the fluorescence of the released rare earth nanocrystals. The material of the present invention has the advantages of high tissue penetration, no background autofluorescence interference, and high resolution, so it can be used for early diagnosis of clinical tumors without background interference, high resolution, and strong tissue penetration. Fluorescent imaging of areas, navigating tumor resection, and guiding chemodynamic therapy of metastases.
(2)本发明利用水热溶剂热法，通过掺杂调节合成了近红外二b区激发，近红外二区、红光发射的、单分散且尺寸均一的稀土纳米晶，从而实现了肿瘤组织的高穿透、高分辨率成像和肿瘤转移灶的光动力治疗。(2) The present invention utilizes the hydrothermal solvothermal method to synthesize rare earth nanocrystals excited by the near-infrared 2b region, near-infrared 2 region, red light emitting, monodisperse and uniform in size through doping adjustment, thereby realizing tumor tissue High-penetration, high-resolution imaging and photodynamic therapy of tumor metastases.
(3)本发明合成的类病毒空心氧化锰纳米颗粒，可以被肿瘤细胞大量吞噬，并在肿瘤细胞微酸环境中发生降解，以释放出单线态氧和锰离子，因而可用于肿瘤的化动力治疗和核磁共振成像，以实现肿瘤的精准治疗。(3) The virus-like hollow manganese oxide nanoparticles synthesized by the present invention can be phagocytized by tumor cells in large quantities, and degrade in the slightly acidic environment of tumor cells to release singlet oxygen and manganese ions, so they can be used for the chemical kinetics of tumors. Therapy and MRI for precise tumor treatment.
(4)本发明合成的类病毒空心氧化锰负载近红外二b区激发的稀土纳米晶复合探针，能够实现肿瘤微环境响应的荧光成像、导航肿瘤切除和转移灶的光动力治疗，以将肿瘤彻底清除，从而提高患者的术后生存率，并为恶性肿瘤的临床治疗提供借鉴。(4) The virus-like hollow manganese oxide-loaded rare earth nanocrystal composite probe excited by the near-infrared 2b region can realize fluorescence imaging of tumor microenvironment response, navigation tumor resection and photodynamic therapy of metastases, so as to The tumor is completely removed, thereby improving the survival rate of patients after surgery, and providing reference for the clinical treatment of malignant tumors.
附图说明Description of drawings
图1中A、B分别为实施例1制得的近红外二b区激发的稀土纳米晶的核(β-NaErF 4:2％Ho)和β-NaErF 4:2％Ho@NaYF 4纳米晶核壳结构的透射电镜图。 A and B in Fig. 1 are the nuclei (β-NaErF 4 : 2% Ho) and β-NaErF 4 : 2% Ho@NaYF 4 nanocrystals of the near-infrared two-b region excited rare earth nanocrystals prepared in Example 1, respectively TEM image of the core-shell structure.
图2中A、B分别为实施例2制得的类病毒硅介孔纳米颗粒和类病毒空心氧化锰的透射电镜图。A and B in FIG. 2 are the transmission electron micrographs of the virus-like silicon mesoporous nanoparticles and the virus-like hollow manganese oxide prepared in Example 2, respectively.
图3中A、B分别为实施例3制得的负载IR1064的类病毒空心氧化锰表面修饰近红外二b区激发的稀土纳米晶(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4)的透射电镜图、及近红外二b区激发的稀土纳米晶(β-NaErF 4:2％Ho@NaYF 4纳米晶核壳结构和复合探针(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4)的荧光光谱图。 A and B in Fig. 3 are the rare earth nanocrystals (MnO 2 -IR1064@β-NaErF 4 : 2%Ho@NaYF 4 ), the transmission electron microscope image, and the rare earth nanocrystal (β-NaErF 4 :2%Ho@NaYF 4 nanocrystal core-shell structure and composite probe (MnO 2 -IR1064@β-NaErF 4 : Fluorescence spectrum of 2%Ho@NaYF 4 ).
图4为实施例3制得的负载IR1064的类病毒空心氧化锰表面修饰近红外二b区激发的稀土纳米晶(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4)分别在pH＝5.5和7.0条件下，孵育不同时间 后的透射电镜图。 Figure 4 shows the rare earth nanocrystals (MnO 2 -IR1064@β-NaErF 4 :2%Ho@NaYF 4 ) excited by the near-infrared 2b region on the surface of the virus-like hollow manganese oxide loaded with IR1064 prepared in Example 3 at pH = 5.5 and 7.0 conditions, transmission electron micrographs after incubation for different times.
图5为实施例3制得的负载IR1064的类病毒空心氧化锰表面修饰近红外二b区激发的稀土纳米晶(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4)和临床批准的ICG穿透深度在不同激光照射下的荧光成像对比图。 Figure 5 shows the rare earth nanocrystals (MnO 2 -IR1064@β-NaErF 4 : 2%Ho@NaYF 4 ) and clinical approval of the surface-modified IR1064-loaded hollow manganese oxide prepared in Example 3 excited by the near-infrared 2b region Comparison of fluorescence imaging of ICG penetration depth under different laser irradiation.
具体实施方式Detailed ways
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies realized based on the above contents of the present invention are covered within the scope of protection intended by the present invention.
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.
实施例1Example 1
β-NaErF 4:2％Ho@NaYF 4核壳结构纳米晶的制备： Preparation of β-NaErF 4 :2%Ho@NaYF 4 core-shell structure nanocrystals:
首先将摩尔比为49:1的Er(CH 3CO 2) 3·4H 2O(408.123mg)，Ho(CH 3CO 2) 3·4H 2O(9.000mg)，一共1mmol稀土盐原料，6mL油酸，15mL十八烯依次加入100mL三口瓶中并混合均匀，在真空条件下加热至140℃，搅拌保持30min，去除体系中的水以及氧气，而后冷却至室温，得到澄清透明溶液；将4mmol氟化铵、2.5mmol氢氧化钠分别溶于5mL甲醇中，快速混合后加入上述澄清透明的溶液中，在50℃条件下保持1h充分搅拌成核，然后升温至70℃，并于真空条件下保持60min(除去多余的甲醇、氧气以及水分子)；随后，向溶液体系中通入高纯氩气，同时加热到300℃(升温速率10℃/min)，并保持60min。(整个制备过程中一直保持磁力搅拌)。最后将反应后的体系冷却至室温，再加入15mL无水乙醇进行沉淀，高速离心得到产物。将得到的NaErF 4:2％Ho纳米晶用无水乙醇洗涤、3000rpm,10min离心分离，并将分离得到的固体产物分散于环己烷中，得到β-NaErF 4:2％Ho纳米颗粒分散液，冷冻保存待用，分散液浓度为0.1mol/L。 First, Er(CH 3 CO 2 ) 3 4H 2 O (408.123mg), Ho(CH 3 CO 2 ) 3 4H 2 O (9.000mg) with a molar ratio of 49:1, a total of 1mmol rare earth salt raw material, 6mL Add oleic acid and 15mL octadecene into a 100mL three-necked flask in turn and mix well, heat to 140°C under vacuum, keep stirring for 30min, remove water and oxygen in the system, and then cool to room temperature to obtain a clear and transparent solution; 4mmol Dissolve ammonium fluoride and 2.5mmol sodium hydroxide in 5mL methanol respectively, mix quickly and add to the above clear and transparent solution, keep stirring at 50°C for 1 hour to form a nucleate, then raise the temperature to 70°C, and Keep it for 60 minutes (to remove excess methanol, oxygen and water molecules); then, pass high-purity argon into the solution system while heating to 300°C (heating rate 10°C/min) and keep it for 60 minutes. (Magnetic stirring was maintained throughout the preparation). Finally, the reacted system was cooled to room temperature, then 15 mL of absolute ethanol was added for precipitation, and the product was obtained by high-speed centrifugation. The obtained NaErF 4 : 2% Ho nanocrystals were washed with absolute ethanol, centrifuged at 3000 rpm for 10 min, and the separated solid product was dispersed in cyclohexane to obtain a β-NaErF 4 : 2% Ho nanoparticle dispersion , cryopreserved for later use, and the concentration of the dispersion was 0.1mol/L.
采用连续层层生长法(one-pot successive layer-by-layer protocol)制备β-NaErF 4:2％Ho@NaYF 4核壳结构纳米晶： β-NaErF 4 : 2%Ho@NaYF 4 core-shell structure nanocrystals were prepared by one-pot successive layer-by-layer protocol:
钇-油酸(Y-OA)前驱体的合成：将5.0mmol的YCl 3，油酸(20.0mL)，十八烯(30.0mL)加入100mL三口烧瓶中混合，在保持真空的条件下升温至140℃，搅拌保持60min，保持体系处于高度无水氧环境。随后将合成的配合物溶液冷却降温，最后获得澄清透明Y-OA前驱体溶液(0.1mol/L)； The synthesis of yttrium-oleic acid (Y-OA) precursor: the YCl 3 of 5.0mmol, oleic acid (20.0mL), octadecene (30.0mL) are added in the 100mL three-necked flask and mixed, and the temperature is raised to 140°C, keep stirring for 60 minutes, and keep the system in a highly anhydrous and oxygen-free environment. Subsequently, the synthesized complex solution was cooled down to obtain a clear and transparent Y-OA precursor solution (0.1mol/L);
三氟乙酸钠-油酸(Na-TFA-OA)前驱体的合成：将16.0mmol三氟乙酸钠、40mL油酸加入100mL三口瓶中混合，然后置于室温条件下，抽真空搅拌均匀直至完全溶解，得到淡黄色透明Na-TFA-OA前驱体溶液(0.4mol/L)；Synthesis of sodium trifluoroacetate-oleic acid (Na-TFA-OA) precursor: add 16.0mmol sodium trifluoroacetate and 40mL oleic acid into a 100mL three-neck flask and mix, then place at room temperature, vacuumize and stir until completely Dissolved to obtain light yellow transparent Na-TFA-OA precursor solution (0.4mol/L);
核壳结构β-NaErF 4:2％Ho@NaYF 4的制备：首先将4.0mL油酸、6mL十八烯依次加入50mL三口瓶中并混合均匀，然后向混合溶液中加入2.5mL上述合成的β-NaErF 4:2％Ho纳米颗粒分散液(0.25mmol)；在真空下，将环己烷从体系中除去，再向三口瓶中通入氩气，将温度升温至280℃(升温速率为20℃/min)，然后交替的加入Y-OA前驱体溶液(0.10mol/L，1.0mL)和Na-TFA-OA(0.40mol/L，0.50mL)前驱体溶液，两种前驱体溶液之间的滴加时间间隔为15min，上述两种前驱体溶液滴加完成后即制得包覆一层壳层的纳米颗粒；总共包覆三层(即重复交替的加入Y-OA前驱体溶液(0.10mol/L，1.0mL)和Na-TFA-OA(0.40M，0.50mL)前驱体溶液三次)，每层之间的包覆时间间隔为15min，通过组数来控制壳层厚度。反应结束后，将反应混合溶液冷却至室温，将得到的β-NaErF 4:2％Ho@NaYF 4稀土纳米晶用无水乙醇3000rpm，10min离心洗涤分离，并将分离得到的β-NaErF 4:2％Ho@NaYF 4稀土纳米晶固体产物分散于环己烷中冷冻存储待用。 Preparation of core-shell structure β-NaErF 4 : 2%Ho@NaYF 4 : First, add 4.0mL oleic acid and 6mL octadecene into a 50mL three-necked flask and mix well, and then add 2.5mL of the above-synthesized β -NaErF 4 : 2%Ho nanoparticle dispersion (0.25mmol); Under vacuum, cyclohexane is removed from the system, and then argon is fed into the there-necked flask, and the temperature is raised to 280°C (the heating rate is 20 ℃/min), then alternately add Y-OA precursor solution (0.10mol/L, 1.0mL) and Na-TFA-OA (0.40mol/L, 0.50mL) precursor solution, between the two precursor solutions The dripping time interval is 15min. After the above two kinds of precursor solutions are added dropwise, the nanoparticles coated with a shell layer are obtained; a total of three layers are coated (that is, repeated and alternately added Y-OA precursor solution (0.10 mol/L, 1.0mL) and Na-TFA-OA (0.40M, 0.50mL) precursor solution three times), the coating time interval between each layer is 15min, and the thickness of the shell layer is controlled by the number of groups. After the reaction, the reaction mixed solution was cooled to room temperature, and the obtained β-NaErF 4 : 2% Ho@NaYF 4 rare earth nanocrystals were washed and separated by centrifugation at 3000 rpm with absolute ethanol for 10 min, and the separated β-NaErF 4 : The 2% Ho@NaYF 4 rare earth nanocrystalline solid product was dispersed in cyclohexane and stored frozen until use.
图1中A、B分别为本实施例制得的近红外二b区激发的稀土纳米晶的核(即A图中的核层纳米晶β-NaErF 4:2％Ho)和核壳结构纳米晶(即B图中的核壳结构纳米晶β-NaErF 4:2％Ho@NaYF 4)的透射电镜图。从图中可以看出，核层和核壳层的纳米晶体均具有结构均一的β形结构；且粒径大小均一，其中核层的粒径约为17.52±0.43nm，核-壳结构的稀土纳米晶的粒径大小约为24.69±0.52nm。 In Fig. 1, A and B are respectively the nucleus of the rare earth nanocrystal excited by the near-infrared 2b region prepared in this embodiment (that is, the core layer nanocrystal β-NaErF 4 :2%Ho in the figure A) and the core-shell structure nanocrystal TEM image of the crystal (namely the core-shell structure nanocrystal β-NaErF 4 :2%Ho@NaYF 4 in Figure B). It can be seen from the figure that both the nanocrystals of the core layer and the core-shell layer have a uniform β-shaped structure; and the particle size is uniform. The particle size of the nanocrystal is about 24.69±0.52nm.
实施例2Example 2
负载IR1064的类病毒空心介孔氧化锰的制备：Preparation of virus-like hollow mesoporous manganese oxide loaded with IR1064:
(1)两相法合成类病毒硅介孔纳米颗粒；(1) Synthesis of virus-like silicon mesoporous nanoparticles by two-phase method;
在100mL烧瓶中加入60mL超纯水，1.5g十六烷基三甲基溴化铵(CTAB)，溶解之后在60℃条件下搅拌0.5h后，加入25％三乙胺0.75mL。0.5h之后加入16mL环己烷和4mL正硅酸四乙酯混合溶液，反应48h后，通过10000rpm，10min离心获得沉淀，再用水和乙醇各洗涤三次获得类病毒硅介孔纳米颗粒。Add 60mL of ultrapure water and 1.5g of cetyltrimethylammonium bromide (CTAB) into a 100mL flask, dissolve and stir at 60°C for 0.5h, then add 0.75mL of 25% triethylamine. After 0.5h, a mixed solution of 16mL cyclohexane and 4mL tetraethylorthosilicate was added, and after 48h of reaction, the precipitate was obtained by centrifugation at 10000rpm for 10min, and then washed three times with water and ethanol to obtain viroid silicon mesoporous nanoparticles.
(2)类病毒空心介孔氧化锰的制备(2) Preparation of virus-like hollow mesoporous manganese oxide
将步骤(1)制备好的100mg类病毒硅介孔纳米颗粒超声分散于50mL去离子水中，然后加入0.09g Mn(NO 3) 3·6H 2O，并于90℃油浴中，600rpm转速下，搅拌0.5h。然后，加入0.09g 乌洛托品，继续搅拌反应2h。待反应结束后，离心分离收集产物，并用水、乙醇各洗涤三次。然后在NaOH水溶液中刻蚀掉类病毒硅介孔纳米颗粒模板，具体操作如下：将反应产物分散到2mol/L NaOH水溶液中，60℃烘箱中放置24h，然后用水、乙醇各洗涤三次，得到最终产物类病毒空心介孔氧化锰粒子，干燥后备用。 100 mg of virus-like silicon mesoporous nanoparticles prepared in step (1) were ultrasonically dispersed in 50 mL of deionized water, then 0.09 g of Mn(NO 3 ) 3 6H 2 O was added, and the , stirred for 0.5h. Then, 0.09g of urotropine was added, and the stirring reaction was continued for 2h. After the reaction was completed, the product was collected by centrifugation and washed three times with water and ethanol. Then, the virus-like silicon mesoporous nanoparticle template was etched in NaOH aqueous solution. The specific operation was as follows: the reaction product was dispersed in 2mol/L NaOH aqueous solution, placed in an oven at 60°C for 24h, and then washed three times with water and ethanol to obtain the final The product is virus-like hollow mesoporous manganese oxide particles, which are dried and used for future use.
(3)负载IR1064的类病毒空心氧化锰的制备(3) Preparation of viroid hollow manganese oxide loaded with IR1064
将步骤(2)制得的20mg类病毒空心介孔氧化锰颗粒分散于20mL水中，然后再加入5mg IR1064，室温下搅拌24h，通过3000rpm离心5min去除多余的IR1064，从而成功获得负载IR1064的类病毒空心氧化锰颗粒。Disperse 20 mg of viroid hollow mesoporous manganese oxide particles prepared in step (2) in 20 mL of water, then add 5 mg of IR1064, stir at room temperature for 24 hours, and centrifuge at 3000 rpm for 5 minutes to remove excess IR1064, thus successfully obtaining viroids loaded with IR1064 Hollow manganese oxide particles.
图2中A、B分别为本实施例制备得到的类病毒硅介孔纳米颗粒和以此为基础合成的类病毒空心介孔氧化锰的透射电镜图。从图中可以看出，本实施例成功制备了类病毒硅介孔纳米颗粒结构，其内部为介孔结构，表面生长簇小管，其整体为类病毒结构，同时其介孔和管状体可以用于负载药物分子；通过后续的硬模板法制备的类病毒空心介孔氧化锰，成功地复制了类病毒硅介孔纳米颗粒的结构，类似于病毒外壳，因而具有快速的细胞侵染和药物负载功能。A and B in FIG. 2 are transmission electron micrographs of the virus-like silicon mesoporous nanoparticles prepared in this example and the virus-like hollow mesoporous manganese oxide synthesized on the basis of this, respectively. As can be seen from the figure, this example successfully prepared a virus-like silicon mesoporous nanoparticle structure, the interior of which is a mesoporous structure, and clusters of tubules grow on the surface. The overall structure is a virus-like structure, and its mesoporous and tubular bodies can be used The virus-like hollow mesoporous manganese oxide prepared by the subsequent hard template method successfully replicated the structure of the virus-like silicon mesoporous nanoparticles, which is similar to the virus shell, so it has rapid cell invasion and drug loading Function.
实施例3Example 3
负载IR1064的空心病毒氧化锰表面修饰近红外二b区激发的稀土纳米晶的复合探针的制备：Preparation of compound probes of rare earth nanocrystals excited by near-infrared 2b region excitation of IR1064-loaded hollow virus manganese oxide surface:
(1)将实施例1制得的油酸包裹的β-NaErF 4:2％Ho@NaYF 4核壳结构纳米晶(0.1mmol)分散于5mL氯仿中，然后加入1mL含有25mg DSEP-PEG 2000-COOH的氯仿溶液。此混合溶液在玻璃瓶中搅拌24h后，氯仿就会自发的在空气氛围下蒸发掉。待氯仿蒸发完毕，将玻璃瓶置于50℃烘箱0.5h以促进氯仿的进一步蒸发。最后向上述亲水性的羧基磷脂修饰的β-NaErF 4:2％Ho@NaYF 4核壳结构纳米晶颗粒中加入5mL去离子水，并通过超声，多余的羧基磷脂通过超速离心机(17500rpm，30min)清洗至少三次。离心洗涤后存在的一些大的团聚体可以通过0.22μm滤网去除。最终可以获得分散在水中的羧基功能化的β-NaErF 4:2％Ho@NaYF 4核壳结构纳米晶； (1) Disperse the oleic acid-wrapped β-NaErF 4 : 2% Ho@NaYF 4 core-shell structure nanocrystals (0.1 mmol) prepared in Example 1 in 5 mL of chloroform, and then add 1 mL containing 25 mg DSEP-PEG 2000 - COOH in chloroform. After the mixed solution was stirred in a glass bottle for 24 hours, the chloroform evaporated spontaneously in the air atmosphere. After the chloroform evaporated, put the glass bottle in an oven at 50°C for 0.5h to promote the further evaporation of chloroform. Finally, add 5 mL of deionized water to the β-NaErF modified by the above-mentioned hydrophilic carboxyl phospholipids: 2% Ho@NaYF 4 core-shell structure nanocrystal particles, and by ultrasonication, excess carboxyl phospholipids are passed through an ultracentrifuge (17500rpm, 30min) to wash at least three times. Some large aggregates present after centrifugal washing can be removed through a 0.22 μm filter. Finally, the carboxyl-functionalized β-NaErF 4 : 2%Ho@NaYF 4 core-shell nanocrystals dispersed in water can be obtained;
(2)将100mg实施例2制得的负载IR1064的类病毒空心氧化锰颗粒分散于50mL乙醇当中，并于80℃油浴中，搅拌0.5h后加入100μL氨基硅烷APTES((3-Aminopropyl)-triethoxysilane)，反应12h后收样，并用乙醇和水分别3000rpm，10min洗涤三次，获得氨基修饰的负载IR1064的类病毒空心氧化锰颗粒；(2) Disperse 100 mg of the virus-like hollow manganese oxide particles loaded with IR1064 prepared in Example 2 in 50 mL of ethanol, stir in an oil bath at 80°C for 0.5 h, and then add 100 μL of aminosilane APTES ((3-Aminopropyl)- triethoxysilane), after reacting for 12h, collect the sample, and wash three times with ethanol and water respectively at 3000rpm and 10min, to obtain amino-modified IR1064-loaded virus-like hollow manganese oxide particles;
(3)将步骤(1)制得的羧基功能化的β-NaErF 4:2％Ho@NaYF 4核壳结构纳米晶与步骤(2)制得的氨基修饰的负载IR1064的类病毒空心氧化锰颗粒(质量比为3:1)在含有8mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和10mg N-羟基琥珀酰亚胺(NHS)，pH＝8.5的溶液中反应缩合12h，以制得负载IR1064的类病毒空心氧化锰表面修饰近红外二b区激发的稀土纳米晶(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4)。 (3) The carboxyl-functionalized β-NaErF 4 : 2%Ho@NaYF 4 core-shell structure nanocrystals prepared in step (1) and the amino-modified IR1064-loaded viroid hollow manganese oxide prepared in step (2) Granules (mass ratio 3:1) in the presence of 8 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mg N-hydroxysuccinimide (NHS) , reacted and condensed in a solution of pH=8.5 for 12 hours to prepare rare earth nanocrystals excited by the near-infrared 2b region of the virus-like hollow manganese oxide loaded with IR1064 (MnO 2 -IR1064@β-NaErF 4 :2%Ho@NaYF 4 ).
图3中A、B分别为本实施例制得的负载IR1064的类病毒空心氧化锰表面修饰近红外二b区激发的稀土纳米晶(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4)的透射电镜图及近红外二b区激发的稀土纳米晶(β-NaErF 4:2％Ho@NaYF 4纳米晶核壳结构)和复合探针(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4核壳结构)的荧光光谱图。从A图可以观察到，β-NaErF 4:2％Ho@NaYF 4纳米晶核壳结构可以成功附着于类病毒空心氧化锰表面，由此证明了本实施例氨基羧基缩合反应的可靠性。从B图可以看出：本实施例制备的MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4稀土纳米晶在1532nm的激发下，在可见光660nm和近红外二区均有很强的发射；由于类病毒空心氧化锰内部负载了IR1064，其具有较强的吸光作用，因此稀土纳米晶的荧光被淬灭。 A and B in Fig. 3 are the rare earth nanocrystals (MnO 2 -IR1064@β-NaErF 4 : 2%Ho@NaYF 4 ) TEM images and near-infrared two-b region excited rare earth nanocrystals (β-NaErF 4 :2%Ho@NaYF 4 nanocrystalline core-shell structure) and composite probe (MnO 2 -IR1064@β-NaErF 4 : Fluorescence spectrum of 2%Ho@NaYF 4 core-shell structure). It can be observed from Figure A that the β-NaErF 4 :2%Ho@NaYF 4 nanocrystalline core-shell structure can be successfully attached to the surface of the virus-like hollow manganese oxide, thus proving the reliability of the aminocarboxyl condensation reaction in this example. It can be seen from Figure B that the MnO 2 -IR1064@β-NaErF 4 :2%Ho@NaYF 4 rare earth nanocrystals prepared in this example have a strong emission in the visible light 660nm and near-infrared regions under the excitation of 1532nm. Emission: Since the virus-like hollow manganese oxide is loaded with IR1064, which has a strong light absorption effect, the fluorescence of the rare earth nanocrystals is quenched.
图4为本实施例制得的负载IR1064的类病毒空心氧化锰表面修饰近红外二b区激发的稀土纳米晶(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4)在pH＝5.5和7.0条件下孵育0分钟，30分钟和120分钟后离心获得沉淀物的透射电镜图。图中结果表明：本实施例的稀土纳米晶能够作为复合探针，在肿瘤的微酸环境中，类病毒空心氧化锰会发生降解，释放出β-NaErF 4:2％Ho@NaYF 4纳米晶和IR1064，β-NaErF 4:2％Ho@NaYF 4纳米晶的荧光不会被IR1064吸收，荧光恢复可以用于成像，而释放的锰离子可以用于肿瘤转移灶的化动力治疗。 Figure 4 shows the rare earth nanocrystals (MnO 2 -IR1064@β-NaErF 4 :2%Ho@NaYF 4 ) excited by the near-infrared 2b region of the virus-like hollow manganese oxide loaded with IR1064 prepared in this example. The transmission electron micrographs of the precipitate were obtained by centrifugation after incubation for 0 minutes, 30 minutes and 120 minutes under the conditions of 5.5 and 7.0. The results in the figure show that the rare earth nanocrystals of this example can be used as composite probes, and the virus-like hollow manganese oxide will be degraded in the slightly acidic environment of the tumor, releasing β-NaErF 4 :2%Ho@NaYF 4 nanocrystals And IR1064, the fluorescence of β-NaErF 4 :2%Ho@NaYF 4 nanocrystals will not be absorbed by IR1064, the fluorescence recovery can be used for imaging, and the released manganese ions can be used for chemokinetic therapy of tumor metastases.
组织穿透实验：将20％的脂肪乳液用水稀释至1％以备用。取六方形小皿，内部分别加入相同荧光强度的实施例3制备的稀土纳米晶(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4)和ICG溶液。取3.5cm直径的培养皿，将上述带有样品的六方小皿分别至于培养皿底，用胶带固定住。将含有实施例3制备的稀土纳米晶的培养皿放在InGaAs成像仪下，1532nm激发，880nm滤光片，20mm曝光拍照。用移液枪滴加不同厚度(0mm、2mm、4mm、6mm、8mm、10mm)的浓度为1％的脂肪乳液，然后拍摄，直至观察不到六方皿。 Tissue penetration test: dilute 20% fat emulsion to 1% with water for use. Take a hexagonal small dish, and add the rare earth nanocrystal (MnO 2 -IR1064@β-NaErF 4 :2%Ho@NaYF 4 ) and ICG solution prepared in Example 3 with the same fluorescence intensity into it. Take a petri dish with a diameter of 3.5 cm, place the above-mentioned hexagonal small dishes with samples on the bottom of the petri dish, and fix them with adhesive tape. Put the petri dish containing the rare earth nanocrystal prepared in Example 3 under the InGaAs imager, 1532nm excitation, 880nm filter, 20mm exposure and take pictures. Use a pipette gun to drop fat emulsions with a concentration of 1% in different thicknesses (0mm, 2mm, 4mm, 6mm, 8mm, 10mm), and then photograph until no hexagonal dish is observed.
设置ICG为对照组，808nm激发，880nm滤光片，20mm曝光拍照；重复上述实验。通过image J软件分析信噪比，得出本实施例制备的二b区激发的稀土纳米晶(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4)相比于临床批准的ICG在穿透性和信噪比方面的优势性。 Set ICG as the control group, 808nm excitation, 880nm filter, 20mm exposure to take pictures; repeat the above experiment. The signal-to-noise ratio was analyzed by image J software, and it was concluded that the rare earth nanocrystals (MnO 2 -IR1064@β-NaErF 4 :2%Ho@NaYF 4 ) prepared in this example prepared in this example were compared with the clinically approved ICG in Superiority in terms of penetration and signal-to-noise ratio.
图5为本实施例制得的实施例3制备的稀土纳米晶(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4)和临床批准的ICG穿透深度(步骤如下)的对比图。从图中得出，本实施例制得的稀土纳米晶(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4)的穿透深度可达10mm；相比于传统的临床比准的ICG 6mm的穿透性，本实施例制得的二b区激发的稀土纳米晶(MnO 2-IR1064@β-NaErF 4:2％Ho@NaYF 4)在组织穿透上有显著的优势性，其作为复合探针或造影剂，能够实现肿瘤微环境响应的荧光成像、导航肿瘤切除和转移灶的光动力治疗，以将肿瘤彻底清除，从而提高患者的术后生存率，并为恶性肿瘤的临床治疗提供借鉴。 Figure 5 is a comparison chart of the rare earth nanocrystals (MnO 2 -IR1064@β-NaErF 4 :2%Ho@NaYF 4 ) prepared in Example 3 prepared in this example and the clinically approved ICG penetration depth (the steps are as follows) . It can be seen from the figure that the penetration depth of the rare earth nanocrystals (MnO 2 -IR1064@β-NaErF 4 :2%Ho@NaYF 4 ) prepared in this example can reach 10 mm; The penetration of ICG 6mm, the rare earth nanocrystals (MnO 2 -IR1064@β-NaErF 4 :2%Ho@NaYF 4 ) prepared in this example have significant advantages in tissue penetration, As a composite probe or contrast agent, it can realize fluorescence imaging of tumor microenvironment response, navigate tumor resection and photodynamic therapy of metastases, so as to completely remove tumors, thereby improving the postoperative survival rate of patients, and providing support for the treatment of malignant tumors. Provide reference for clinical treatment.
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The embodiments of the present invention have been described above. However, the present invention is not limited to the above-mentioned embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

  1. 一种稀土纳米晶，其特征在于，所述稀土纳米晶包括载体及负载于载体表面的稀土核壳纳米颗粒。A rare earth nanocrystal, characterized in that the rare earth nanocrystal includes a carrier and rare earth core-shell nanoparticles loaded on the surface of the carrier.
  2. 如权利要求1所述的稀土纳米晶，其特征在于，所述稀土纳米晶中，载体与稀土核壳纳米颗粒的质量比为1:(1-2)。The rare earth nanocrystal according to claim 1, characterized in that, in the rare earth nanocrystal, the mass ratio of the carrier to the rare earth core-shell nanoparticle is 1:(1-2).
    优选地，所述载体与稀土核壳纳米颗粒之间通过价键连接，例如通过酰胺键连接。Preferably, the carrier is connected to the rare earth core-shell nanoparticles through a valence bond, such as an amide bond.
    优选地，所述载体具有氨基，其与稀土核壳纳米颗粒中的羧基形成酰胺键。Preferably, the carrier has amino groups, which form amide bonds with carboxyl groups in the rare earth core-shell nanoparticles.
    优选地，所述稀土纳米晶在肿瘤弱酸性微环境中能选择性降解，优选为在肿瘤弱酸性微环境中能选择性降解释放出锰离子(Mn 2+)、铁离子(Fe 2+)。 Preferably, the rare earth nanocrystals can be selectively degraded in the weakly acidic microenvironment of the tumor, preferably can be selectively degraded in the weakly acidic microenvironment of the tumor to release manganese ions (Mn 2+ ), iron ions (Fe 2+ ) .
    优选地，所述载体为表面修饰有氨基官能团化合物的空心球。Preferably, the carrier is a hollow sphere whose surface is modified with an amino functional group compound.
    优选地，所述载体为氨基化修饰的空心氧化锰和/或空心氧化铁。Preferably, the carrier is aminated hollow manganese oxide and/or hollow iron oxide.
    优选地，所述载体的空腔中负载有荧光淬灭分子。Preferably, fluorescence quenching molecules are loaded in the cavity of the carrier.
    所述稀土核壳纳米颗粒包括核纳米颗粒、至少一层包覆于所述核纳米颗粒外部的壳层以及修饰于所述壳层外部的含羧基官能团的物质，所述核纳米颗粒和壳层分别独立地选自AREF 4，其中：A为Na或K，RE为Er、Ho、Gd和Y中的至少一种。 The rare earth core-shell nanoparticles include core nanoparticles, at least one shell coated on the outside of the core nanoparticles, and a carboxyl functional group-containing substance modified on the outside of the shell, the core nanoparticles and the shell are independently selected from AREF 4 , wherein: A is Na or K, and RE is at least one of Er, Ho, Gd and Y.
    优选地，核纳米颗粒和壳层中的RE元素不同。Preferably, the RE elements in the core nanoparticles and the shell are different.
    优选地，所述核纳米颗粒中，RE为Er、Ho中的两种；所述壳层纳米颗粒中，RE为Y。Preferably, in the core nanoparticles, RE is two of Er and Ho; in the shell nanoparticles, RE is Y.
    优选地，所述核纳米颗粒中，Ho的掺杂量为1～5％。Preferably, the doping amount of Ho in the core nanoparticles is 1-5%.
    优选地，所述稀土核壳纳米颗粒的粒径为24～25.5nm。Preferably, the particle diameter of the rare earth core-shell nanoparticles is 24-25.5 nm.
    优选地，所述核纳米颗粒的粒径和壳层的厚度之比为1:(1～1.5)。Preferably, the ratio of the particle size of the core nanoparticles to the thickness of the shell layer is 1:(1˜1.5).
    优选地，所述核纳米颗粒的粒径为17～18nm。Preferably, the particle diameter of the core nanoparticles is 17-18 nm.
    优选地，所述的含羧基官能团的物质选自PEG聚合物、小分子酸类或者含羧基的高分子聚合物。Preferably, the carboxyl functional group-containing substance is selected from PEG polymers, small molecular acids or carboxyl-containing polymers.
  3. 权利要求1或2所述稀土纳米晶的制备方法，其特征在于，所述制备方法包括将载体与稀土核壳纳米颗粒进行反应，制备得到所述稀土纳米晶。The method for preparing rare earth nanocrystals according to claim 1 or 2, characterized in that the preparation method comprises reacting the carrier with rare earth core-shell nanoparticles to prepare the rare earth nanocrystals.
    优选地，所述稀土核壳纳米颗粒与载体的质量比为2～5:1。Preferably, the mass ratio of the rare earth core-shell nanoparticles to the carrier is 2-5:1.
  4. 如权利要求3所述的制备方法，其特征在于，所述稀土核壳纳米颗粒的制备方法，包括以下步骤：The preparation method according to claim 3, wherein the preparation method of the rare earth core-shell nanoparticles comprises the following steps:
    (1)将稀土盐加入油酸和十八烯的混合溶液中，然后加入碱金属氟化物溶液、碱性溶液进 行反应，得到所述核纳米颗粒；(1) Rare earth salt is added in the mixed solution of oleic acid and octadecene, then add alkali metal fluoride solution, alkaline solution to react, obtain described core nanoparticle;
    (2)向步骤(1)的产物中加入RE-OA和A-TFA-OA前驱体溶液，进行反应，制备得到所述稀土核壳纳米颗粒。(2) Adding RE-OA and A-TFA-OA precursor solution to the product of step (1) for reaction to prepare the rare earth core-shell nanoparticles.
    优选地，步骤(2)至少进行一次，得到至少包覆一层壳层的纳米颗粒。Preferably, step (2) is performed at least once to obtain nanoparticles coated with at least one shell layer.
    其中，A为Na或K，RE为Er、Ho、Gd和Y中的至少一种。Wherein, A is Na or K, RE is at least one of Er, Ho, Gd and Y.
  5. 如权利要求4所述的制备方法，其特征在于，所述碱金属氟化物溶液为NaF、NH 4F和KF的溶液中的一种。 The preparation method according to claim 4, wherein the alkali metal fluoride solution is one of NaF, NH 4 F and KF solutions.
    优选地，步骤(1)中，所述稀土盐中的稀土离子和碱金属氟化物的摩尔比为1:(2-5)。Preferably, in step (1), the molar ratio of the rare earth ion and the alkali metal fluoride in the rare earth salt is 1:(2-5).
    优选地，所述稀土盐中的稀土离子为Er离子、Ho离子和Y离子中的至少一种，优选为Er离子、Ho离子中的两种。Preferably, the rare earth ion in the rare earth salt is at least one of Er ion, Ho ion and Y ion, preferably two of Er ion and Ho ion.
    优选地，所述Er离子、Ho离子的用量的摩尔比为(48～49.5):(0.5～2)。Preferably, the molar ratio of the Er ions and Ho ions used is (48-49.5):(0.5-2).
  6. 如权利要求4或5所述的制备方法，其特征在于，步骤(2)中，在向步骤(1)的产物中加入RE-OA和A-TFA-OA前驱体溶液前，还包括将所述核纳米颗粒分散液加入油酸和十八烯的混合溶液。The preparation method according to claim 4 or 5, characterized in that, in step (2), before adding RE-OA and A-TFA-OA precursor solution to the product of step (1), it also includes adding the A mixed solution of oleic acid and octadecene was added to the nuclear nanoparticle dispersion.
    优选地，所述核纳米颗粒分散液与油酸和十八烯的混合溶液的体积比为1:(1-3)。Preferably, the volume ratio of the nuclear nanoparticle dispersion to the mixed solution of oleic acid and octadecene is 1:(1-3).
    优选地，油酸和十八烯的混合溶液中，油酸和十八烯的体积比为1:(1-2)。Preferably, in the mixed solution of oleic acid and octadecene, the volume ratio of oleic acid and octadecene is 1:(1-2).
    优选地，在步骤(2)中，所述RE-OA前驱体溶液和A-TFA-OA前驱体溶液以交替间隔的方式加入步骤(1)制得的核纳米颗粒分散液中。Preferably, in step (2), the RE-OA precursor solution and the A-TFA-OA precursor solution are added to the nuclear nanoparticle dispersion prepared in step (1) at alternating intervals.
    优选地，所述RE-OA前驱体溶液和A-TFA-OA前驱体溶液交替加入的次数至少各为一次。Preferably, the number of alternate additions of the RE-OA precursor solution and the A-TFA-OA precursor solution is at least one.
    优选地，所述稀土盐与步骤(2)中的RE-OA前驱体溶液中的稀土离子和油酸的摩尔比为1:(6～8)。Preferably, the molar ratio of the rare earth salt to the rare earth ion and oleic acid in the RE-OA precursor solution in step (2) is 1:(6-8).
    优选地，在步骤(1)、(2)中，所述反应的温度相同或不同，彼此独立地为200-400℃，所述反应的时间为40-80min。Preferably, in steps (1) and (2), the reaction temperature is the same or different, independently of each other is 200-400°C, and the reaction time is 40-80min.
    优选地，所述制备方法还包括步骤(3)：将步骤(2)得到的至少包覆一层壳层的纳米颗粒与含羧基官能团的物质在有机溶剂中混匀，静置至有机溶剂挥发，然后加水分散，离心分离出所述羧基修饰的稀土核壳纳米颗粒。Preferably, the preparation method further includes step (3): mixing the nanoparticles coated with at least one shell obtained in step (2) with the carboxyl functional group-containing substance in an organic solvent, and standing until the organic solvent volatilizes , and then add water to disperse, and centrifuge to separate the carboxy-modified rare earth core-shell nanoparticles.
    优选地，在步骤(3)中，所述至少包覆一层壳层的纳米颗粒与含羧基官能团的物质的用量比为0.1mmol:(20-30)mg。Preferably, in step (3), the dosage ratio of the nanoparticles coated with at least one shell layer to the substance containing carboxyl functional group is 0.1 mmol: (20-30) mg.
  7. 如权利要求3-6任一项所述的制备方法，其特征在于，所述载体的制备方法，包括以类病毒硅介孔纳米颗粒为模板，与锰盐、乌洛托品反应后，经碱处理除去类病毒硅介孔纳米颗 粒模板，即制得类病毒空心氧化锰。The preparation method according to any one of claims 3-6, characterized in that, the preparation method of the carrier comprises using the virus-like silicon mesoporous nanoparticles as a template, reacting with manganese salts and urotropine, Alkali treatment removes the virus-like silicon mesoporous nanoparticle template to prepare the virus-like hollow manganese oxide.
    优选地，所述类病毒硅介孔纳米颗粒由包括正硅酸四乙酯与十六烷基三甲基溴化铵(CTAB)、三乙胺反应制备得到。Preferably, the virus-like silicon mesoporous nanoparticles are prepared by reacting tetraethylorthosilicate, cetyltrimethylammonium bromide (CTAB) and triethylamine.
    优选地，所述类病毒硅介孔纳米颗粒与锰盐、乌洛托品反应的温度为80～100℃，反应的时间为3～5h。Preferably, the reaction temperature of the virus-like silicon mesoporous nanoparticles with manganese salt and urotropine is 80-100° C., and the reaction time is 3-5 hours.
    优选地，所述载体的制备方法还包括将洗涤后的反应产物与含有氨基官能团的化合物进行反应，以制得表面修饰有氨基官能团的类病毒空心氧化锰。Preferably, the preparation method of the carrier further includes reacting the washed reaction product with a compound containing an amino functional group, so as to prepare a virus-like hollow manganese oxide whose surface is modified with an amino functional group.
    优选地，所述载体的制备方法还包括将上述表面修饰有氨基官能团的类病毒空心氧化锰与荧光淬灭分子进行反应，以制备得到负载有荧光淬灭分子表面修饰有氨基官能团的类病毒空心氧化锰。Preferably, the preparation method of the carrier also includes reacting the above-mentioned virus-like hollow manganese oxide with amino functional groups on its surface and fluorescent quencher molecules to prepare virus-like hollow manganese oxides loaded with fluorescent quencher molecules and modified with amino functional groups on the surface. manganese oxide.
    优选地，所述表面修饰有氨基官能团的类病毒空心氧化锰与荧光淬灭分子的质量比为(3～5):1。Preferably, the mass ratio of the viroid hollow manganese oxide surface-modified with amino functional groups to the fluorescence quencher molecule is (3-5):1.
    优选地，所述载体的制备方法，包括如下步骤：Preferably, the preparation method of the carrier comprises the following steps:
    (S1)两相法合成类病毒硅介孔纳米颗粒；(S1) Synthesis of virus-like silicon mesoporous nanoparticles by two-phase method;
    将十六烷基三甲基溴化铵(CTAB)溶于水中，然后加入三乙胺、环己烷和正硅酸四乙酯的混合溶液，经反应制得所述类病毒硅介孔纳米颗粒；Cetyltrimethylammonium bromide (CTAB) is dissolved in water, then a mixed solution of triethylamine, cyclohexane and tetraethylorthosilicate is added, and the virus-like silicon mesoporous nanoparticles are prepared by reaction ;
    (S2)类病毒空心氧化锰的制备(S2) Preparation of viroid hollow manganese oxide
    加热搅拌条件下，向类病毒硅介孔纳米颗粒分散液中加入Mn(NO 3) 2·6H 2O、乌洛托品，反应后离心洗涤，最后在NaOH水溶液中刻蚀掉类病毒介孔硅模板，以制得类病毒空心氧化锰；再将类病毒空心氧化锰与含有氨基官能团的化合物进行反应，以制得表面修饰有氨基官能团的类病毒空心氧化锰； Under the condition of heating and stirring, add Mn(NO 3 ) 2 6H 2 O and urotropine to the dispersion liquid of viroid silicon mesoporous nanoparticles, centrifuge and wash after reaction, and finally etch viroid mesoporous pores in NaOH aqueous solution Silicon template to prepare viroid hollow manganese oxide; then react viroid hollow manganese oxide with a compound containing amino functional group to prepare viroid hollow manganese oxide with amino functional group on the surface;
    (S3)负载IR1064的类病毒空心氧化锰的制备(S3) Preparation of virus-like hollow manganese oxide loaded with IR1064
    将步骤(S2)制得的类病毒空心氧化锰颗粒溶于水中，然后再加入IR1064，室温下搅拌反应，然后离心去除多余的IR1064，从而制得负载IR1064的类病毒空心氧化锰颗粒。Dissolving the viroid hollow manganese oxide particles prepared in step (S2) in water, then adding IR1064, stirring the reaction at room temperature, and then centrifuging to remove excess IR1064, so as to prepare the viroid hollow manganese oxide particles loaded with IR1064.
  8. 如权利要求3-7任一项所述的制备方法，其特征在于，所述稀土纳米晶的制备方法中，所述载体与稀土核壳纳米颗粒的反应还包括加入活化剂。The preparation method according to any one of claims 3-7, characterized in that, in the preparation method of the rare earth nanocrystals, the reaction between the carrier and the rare earth core-shell nanoparticles further includes adding an activator.
    优选地，所述活化剂可以为碳二亚胺(EDC)、N-羟基琥珀酰亚胺(NHS)和二甲基乙酰胺(DMAC)中的至少一种。Preferably, the activator may be at least one of carbodiimide (EDC), N-hydroxysuccinimide (NHS) and dimethylacetamide (DMAC).
    优选地，所述稀土核壳纳米颗粒和活化剂的质量比为1:3～5。Preferably, the mass ratio of the rare earth core-shell nanoparticles to the activator is 1:3-5.
  9. 一种复合探针，其特征在于，其含有权利要求1或2所述的稀土纳米晶和/或权利要求3-8 任一项所述的制备方法制得的稀土纳米晶。A composite probe, characterized in that it contains the rare earth nanocrystal according to claim 1 or 2 and/or the rare earth nanocrystal prepared by the preparation method according to any one of claims 3-8.
  10. 权利要求1或2所述的稀土纳米晶、权利要求3-8任一项所述的制备方法制得的稀土纳米晶、和/或权利要求9所述的复合探针在手术导航、术后化动力治疗、术后转移灶的荧光指导、肿瘤的核磁共振成像等领域中制备和/或作为造影剂的应用。The rare earth nanocrystals described in claim 1 or 2, the rare earth nanocrystals prepared by the preparation method described in any one of claims 3-8, and/or the composite probe described in claim 9 are used in surgical navigation, postoperative Preparation and/or application as a contrast agent in the fields of chemodynamic therapy, fluorescence guidance of postoperative metastases, and nuclear magnetic resonance imaging of tumors.
PCT/CN2022/119211 2021-09-16 2022-09-16 Virus-like hollow oxide loaded near-infrared two-b region excited rare earth nanocrystal, preparation method therefor and application thereof WO2023041005A1 (en)

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