CN110665018B - Cu2MoS4@ BSA nano material and preparation method and application thereof - Google Patents

Cu2MoS4@ BSA nano material and preparation method and application thereof Download PDF

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CN110665018B
CN110665018B CN201910912882.1A CN201910912882A CN110665018B CN 110665018 B CN110665018 B CN 110665018B CN 201910912882 A CN201910912882 A CN 201910912882A CN 110665018 B CN110665018 B CN 110665018B
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易昌凤
张宏量
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    • A—HUMAN NECESSITIES
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    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A61K49/143—Peptides, e.g. proteins the protein being an albumin, e.g. HSA, BSA, ovalbumin
    • A—HUMAN NECESSITIES
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Abstract

The invention provides Cu2MoS4The @ BSA nano material and the preparation method and application thereof are characterized in that bovine serum albumin is used as a template, ammonium tetrathiomolybdate and cuprous chloride are used as raw materials, and the nano material is prepared by a microwave-assisted biomimetic mineralization method. Preparation of the obtained Cu2MoS4@ BSA nanomaterial, nanocrystal dispersed and uniform particle size, average particle size of about 2.1 nm; has excellent biocompatibility, and can effectively avoid the elimination of phagocytes in organisms, thereby achieving a longer body fluid circulation period. The Cu2MoS4The @ BSA nano material has a T1 magnetic resonance imaging function, and the contrast effect is more obvious along with the increase of the concentration of the nano particles; the Cu2MoS4The @ BSA nanomaterial can respond to photothermal agents of near-infrared II-region laser after being coated by a photosensitizer.

Description

Cu2MoS4@ BSA nano material and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological targeting nano-medicines, in particular to Cu2MoS4@ BSA nano material and its preparation method and application.
Background
The photothermal therapy (PTT) is widely concerned about due to the characteristics of small toxic and side effects, high targeting, good treatment effect and the like, and becomes a research hotspot in recent years. In recent years, many materials having near-infrared absorption have attracted attention in the PTT field, such as precious metal nanomaterials including gold nanorods, gold nanoclusters, and gold nanostars, carbon-based materials including graphene and carbon nanotubes, small organic molecules having near-infrared absorption, such as porphyrin and indocyanine green (ICG), conjugated polymers, such as Polyaniline (PANI), Polydopamine (PDA), and polypyrrole (PPy), and copper sulfide and molybdenum disulfide (MoS)2) And transition metal sulfide-based materials. At present, most photo-thermal agents are heated by laser radiation of 808nm, but according to the American national standard Z136.1-2007, the laser with the wavelength of 1000-1100 nm in the near infrared II region irradiates deeper than the laser with other wavelengthsThe depth is deeper. In addition, the skin can bear an energy density of 1W/cm at most2Compared with the common laser with the wavelength of 808nm at present, the laser with the wavelength of 1000-1100 nm can only bear 0.33W/cm at most on the skin2. Therefore, it is necessary to construct photothermal agents that respond to near-infrared region II laser light to achieve treatment of deeper tumors.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides Cu2MoS4The invention relates to a @ BSA nano material and a preparation method and application thereof, and the invention synthesizes Cu protected by bovine serum albumin by using a microwave-assisted biomimetic mineralization method and taking bovine serum albumin BSA as a template and ammonium tetrathiomolybdate ATTM and cuprous chloride CuCl as raw materials2MoS4Nanocrystal (Cu)2MoS4@ BSA), the nanomaterial has T1 magnetic resonance imaging function, and the contrast effect is more obvious with the increase of the concentration of the nanoparticles; cu2MoS4The @ BSA nanomaterial can respond to photothermal agents of near-infrared II-region laser after being coated by a photosensitizer.
The invention is realized by the following steps:
an object of the present invention is to provide a Cu2MoS4The preparation method of the @ BSA nano material takes bovine serum albumin as a template, takes ammonium tetrathiomolybdate and cuprous chloride as raw materials, and adopts a microwave-assisted biomimetic mineralization method to prepare the material.
The method specifically comprises the following steps:
step 1, respectively dissolving BSA and ammonium tetrathiomolybdate in deionized water to prepare a BSA aqueous solution and an ammonium tetrathiomolybdate aqueous solution;
step 2, adding the ammonium tetrathiomolybdate aqueous solution into the BSA aqueous solution, and uniformly stirring to obtain a BSA-ATTM mixed system;
step 3, adding CuCl into the injector rapidly, and dissolving the injected ammonia water solution to prepare Cu (NH)3)2A Cl solution;
step 4, the Cu (NH) prepared in the step 33)2Injecting a Cl solution into the BSA-ATTM mixed system obtained in the step 2 to obtain a mixed solution, and mixing the mixed solutionHeating the mixed solution in a microwave reactor to 36.5-37.5 ℃, reacting at a constant temperature for a period of time, heating to 80-100 ℃, reacting for a period of time, and dialyzing to obtain Cu2MoS4@ BSA nanomaterial.
The second purpose of the invention is to provide Cu prepared by the method2MoS4@ BSA nanomaterial.
The Cu2MoS4The @ BSA nanomaterial is in a P crystal form, and the X-ray powder diffraction pattern of the crystal has absorption peaks at 17 +/-0.3, 29 +/-0.3, 32 +/-0.3, 38 +/-0.3, 48 +/-0.3 and 57 +/-0.3 when the reflection angle is 2 theta.
It is a further object of the present invention to provide the Cu2MoS4The application of the @ BSA nano material in the nuclear magnetic resonance T1 weighted image-guided PTT/PDT diagnostic agent.
The invention has the beneficial effects that:
1. the invention provides a Cu2MoS4The preparation method of the @ BSA nano material creatively adopts a microwave-assisted biomimetic mineralization method, takes bovine serum albumin as a template, and takes ammonium tetrathiomolybdate and cuprous chloride as raw materials for preparation; preparation of the obtained Cu2MoS4@ BSA nanomaterial, nanocrystal dispersed and uniform particle size, average particle size of about 2.1 nm; has excellent biocompatibility, and can effectively avoid the elimination of phagocytes in organisms, thereby achieving a longer body fluid circulation period.
2. The invention provides a Cu2MoS4The @ BSA nanomaterial has a T1 magnetic resonance imaging function, and the contrast effect is more obvious with the increase of the concentration of nanoparticles, so that the @ BSA nanomaterial can be applied to a nuclear magnetic resonance T1 weighted image-guided PTT/PDT diagnostic agent. Cu2MoS4The @ BSA nanomaterial can respond to a photothermal agent of near-infrared II-region laser after being coated by a photosensitizer, and the photosensitizer endows the near-infrared II-region laser with a photodynamic function to combine photodynamic therapy, so that a good tumor treatment effect can be achieved.
Drawings
FIG. 1 shows Cu provided in Experimental example 1 of the present invention2MoS4A transmission electron micrograph of the @ BSA nanomaterial;
FIG. 2 shows Cu provided in Experimental example 1 of the present invention2MoS4A hydrated particle size diagram of @ BSA nanomaterial;
FIG. 3 shows Cu provided in Experimental example 2 of the present invention2MoS4XPS analysis of @ BSA nanomaterials; wherein, the a, b and c are respectively the analysis graphs of elements Cu, Mo and S;
FIG. 4 shows Cu provided in Experimental example 2 of the present invention2MoS4The ultraviolet absorption pattern of the @ BSA nano material;
FIG. 5 shows Cu provided in Experimental example 3 of the present invention2MoS4The @ BSA nano material has a crystal structure characterized by XRD;
fig. 6 shows the MRI imaging results provided by the application example of the present invention.
Detailed Description
Example 1
Cu of the present example2MoS4The preparation method of the @ BSA nano material specifically comprises the following steps:
step 1, dissolving 250mgBSA in 5mL deionized water to prepare a BSA aqueous solution with the concentration of 50 mg/mL; dissolving 0.2mmol of ammonium tetrathiomolybdate in 10mL of deionized water to prepare an ammonium tetrathiomolybdate aqueous solution with the concentration of 0.04 mmol/mL;
step 2, dropwise adding the ammonium tetrathiomolybdate aqueous solution into the BSA aqueous solution, stirring (magnetically stirring for 1h), and uniformly mixing to obtain a BSA-ATTM mixed system which is brownish red; the volume ratio of the BSA aqueous solution to the ammonium tetrathiomolybdate aqueous solution is 1: 1;
step 3, quickly adding 0.4mmol of CuCl into an injector, and injecting 10mL of ammonia water solution for dissolving to obtain Cu (NH)3)2A Cl solution; NH in the ammonia solution3And H2The volume ratio of O is 1: 1;
step 4, the Cu (NH) prepared in the step 33)2Injecting a Cl solution into the BSA-ATTM mixed system obtained in the step 2 to obtain a mixed solution (the volume ratio is 1:1), wherein the solution gradually turns into dark red; placing the mixed solution in a microwave reactor (power is 800W), heating to 37 ℃, reacting for 2 hours at constant temperature, then heating to 90 ℃, reacting for 30 minutes, and turning the solution into dark brown; with deionized water (pH 4) for 2 days (molecular weight cut-off 14000 Da), and then for 1 day with PBS buffer (pH 7.4); to obtain Cu2MoS4@ BSA nanomaterial. The average particle size was about 2.1 nm.
Example 2
Cu of the present example2MoS4The preparation method of the @ BSA nano material specifically comprises the following steps:
step 1, dissolving BSA in deionized water to prepare a BSA aqueous solution with the concentration of 30 mg/mL; dissolving ammonium tetrathiomolybdate in deionized water to prepare an ammonium tetrathiomolybdate aqueous solution with the concentration of 0.02 mg/mL;
step 2, dropwise adding the ammonium tetrathiomolybdate aqueous solution into the BSA aqueous solution, stirring (magnetically stirring for 0.5h), and uniformly mixing to obtain a BSA-ATTM mixed system which is brownish red; the volume ratio of the BSA aqueous solution to the ammonium tetrathiomolybdate aqueous solution is 1: 3;
step 3, adding CuCl into the injector rapidly, and dissolving the injected ammonia water solution to prepare Cu (NH)3)2A Cl solution; NH in the ammonia solution3And H2The volume ratio of O is 0.5: 1.5; the molar volume ratio of the CuCl to the ammonia water solution is 0.2 mmol: 12 mL;
step 4, the Cu (NH) prepared in the step 33)2Injecting the Cl solution into the BSA-ATTM mixed system obtained in the step 2 to obtain a mixed solution, wherein the solution gradually becomes dark red; placing the mixed solution in a microwave reactor (power is 600W), heating to 36.5 ℃, reacting for 1h at constant temperature, then heating to 80 ℃, reacting for 50min, and changing the solution into dark brown; dialyzing against deionized water (pH 4) for 2 days (molecular weight cut-off 14000 Da) and then against PBS buffer (pH 7.4) for 1 day; to obtain Cu2MoS4@ BSA nanomaterial. The average particle size was about 2.1 nm.
Example 3
Cu of the present example2MoS4The preparation method of the @ BSA nano material specifically comprises the following steps:
step 1, dissolving BSA in deionized water to prepare a BSA aqueous solution with the concentration of 70 mg/mL; dissolving ammonium tetrathiomolybdate in deionized water to prepare an ammonium tetrathiomolybdate aqueous solution with the concentration of 0.06 mg/mL;
step 2, dropwise adding the ammonium tetrathiomolybdate aqueous solution into the BSA aqueous solution, stirring (magnetically stirring for 4 hours), and uniformly mixing to obtain a BSA-ATTM mixed system which is brownish red; the volume ratio of the BSA aqueous solution to the ammonium tetrathiomolybdate aqueous solution is 3: 1;
step 3, adding CuCl into the injector rapidly, and dissolving the injected ammonia water solution to prepare Cu (NH)3)2A Cl solution; NH in the ammonia solution3And H2The volume ratio of O is 1.5: 0.5; the molar volume ratio of the CuCl to the ammonia water solution is 0.6 mmol: 8 mL;
step 4, the Cu (NH) prepared in the step 33)2Injecting the Cl solution into the BSA-ATTM mixed system obtained in the step 2 to obtain a mixed solution, wherein the solution gradually becomes dark red; placing the mixed solution in a microwave reactor (power is 1000W), heating to 37.5 ℃, reacting for 3 hours at constant temperature, heating to 100 ℃, reacting for 10 minutes, and turning the solution into dark brown; dialyzing against deionized water (pH 4) for 2 days (molecular weight cut-off 14000 Da) and then against PBS buffer (pH 7.4) for 1 day; to obtain Cu2MoS4@ BSA nanomaterial. The average particle size was about 2.1 nm.
Experimental example 1
The Cu obtained in example 1 was subjected to a Transmission Electron Microscope (TEM)2MoS4The morphology of the @ BSA nanomaterial is characterized. Firstly, placing a sample in absolute ethyl alcohol, dispersing the sample by ultrasonic oscillation, dripping the dispersed liquid on a small copper net coated with a carbon film, and airing. The morphology of the tested sample was characterized at different resolutions, and the results are shown in fig. 1. As is clear from FIG. 1, the average particle size was about 2.1nm, the size was uniform, and the dispersibility was good.
Measurement of the Cu obtained in example 12MoS4The hydrated particle size of the @ BSA nanomaterial, as shown in FIG. 2, was about 2.1nm in average particle size, consistent with the results of transmission electron microscopy. The surface of the nano particles is successfully synthesized, and the nano crystals are dispersed and have uniform particle size.
Experimental example 2
Example 1 Cu obtained2MoS4The ultraviolet absorption diagram of the @ BSA nanomaterial is shown in FIG. 4; analysis of Cu by XPS2MoS4The major elements in @ BSA and their compound states, the results are shown in FIG. 3: as shown in a, Cu 2p1/2 and Cu 2p3/2 are located at 952.7eV and 932.8eV, indicating that Cu is present in the nanoparticle; as shown in panel b, Mo 3d3/2 and Mo 3d5/2 correspond to 232.8eV and 230.0eV, indicating that Mo is present in the nanoparticle; as shown in the c diagram, S2 p1/2 and S2 p3/2 are at 162.8eV and 161.9eV, and all peaks are assigned to Cu respectively+，Mo6+And S2-Indicating that S is present in the nanoparticle. In conclusion, the invention successfully prepares Cu2MoS4@ BSA nanomaterial.
Experimental example 3
Characterization of Cu prepared in example 1 by XRD2MoS4The structure of the crystal form of @ BSA, as shown in FIG. 5, the Cu2MoS4The @ BSA nanomaterial is in a P crystal form, and the X-ray powder diffraction pattern of the crystal has absorption peaks at 17 +/-0.3, 29 +/-0.3, 32 +/-0.3, 38 +/-0.3, 48 +/-0.3 and 57 +/-0.3 when the reflection angle is 2 theta. Diffraction angles 2 θ of 17 °, 29 °, 32 °, 38 °, 48 ° and 57 ° correspond to Cu2MoS4The (002), (112), (103), (202), (204) and (312) crystal planes of (A) show that the Cu of p phase is synthesized by the biomimetic mineralization method2MoS4。
Application example
Both in vitro and in vivo T1 weighted MRI were performed on a clinical 3.0T MR scanner. Example 1 Cu obtained2MoS4The longitudinal relaxation rate (r1) of @ BSA was calculated from a linear fit of the reciprocal of the longitudinal relaxation time to the molar concentration of Gd. Taking Cu of different concentrations2MoS4@ BSA was placed in centrifuge tubes and scanned to obtain in vitro T1 weighted MR images. For in vitro MRI experiments, T1-weighted MRI employs a spin echo pulse sequence with an echo Time (TE) of 45ms, a repetition Time (TR) of 1000 ms, a field of view (FOV) of 150mm, and a layer thickness of 1.5 mm.
The MRI results are shown in FIG. 6, the Cu2MoS4The @ BSA nano-particle has the function of T1 magnetic resonance imaging and is accompanied with the nano-particleThe contrast effect is more obvious due to the increase of the concentration.
The invention is not to be considered as limited to the particular embodiments shown, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. Cu2MoS4The preparation method of the @ BSA nano material is characterized in that bovine serum albumin is used as a template, ammonium tetrathiomolybdate and cuprous chloride are used as raw materials, and a microwave-assisted biomimetic mineralization method is adopted to prepare the nano material.
2. The method according to claim 1, wherein the method comprises the steps of:
step 1, respectively dissolving BSA and ammonium tetrathiomolybdate in deionized water to prepare a BSA aqueous solution and an ammonium tetrathiomolybdate aqueous solution;
step 2, adding the ammonium tetrathiomolybdate aqueous solution into the BSA aqueous solution, and uniformly stirring to obtain a BSA-ATTM mixed system;
step 3, adding CuCl into an injector rapidly, and injecting ammonia water solution for dissolving to prepare Cu (NH)3)2A Cl solution;
step 4, the Cu (NH) prepared in the step 33)2Injecting a Cl solution into the BSA-ATTM mixed system obtained in the step 2 to obtain a mixed solution, placing the mixed solution into a microwave reactor, heating the mixed solution to 36.5-37.5 ℃, reacting at a constant temperature for a period of time, heating the mixed solution to 80-100 ℃, reacting for a period of time, and dialyzing to obtain Cu2MoS4@ BSA nanomaterial.
3. The method according to claim 2, wherein in the step 1, the concentration of the aqueous BSA solution is 30-70 mg/mL; the concentration of the ammonium tetrathiomolybdate aqueous solution is 0.02-0.06 mol/mL.
4. The method according to claim 2, wherein in the step 2, the volume ratio of the BSA aqueous solution to the ammonium tetrathiomolybdate aqueous solution is 1-3: 1 to 3.
5. The method of claim 2, wherein step 2 is a dropwise addition of the aqueous ammonium tetrathiomolybdate solution; magnetic stirring is adopted for stirring, and the stirring time is 0.5-4 h.
6. The method according to claim 2, wherein in step 3, NH is contained in the aqueous ammonia solution3And H2The volume ratio of O is 0.5-1.5: 0.5 to 1.5; the molar volume ratio of the CuCl to the ammonia water solution is 0.2-0.6 mmol: 8-12 mL.
7. The preparation method according to claim 2, wherein in the step 4, the power of the microwave reactor is 600-1000W; the constant-temperature reaction time at 36.5-37.5 ℃ is 1-3 h; the reaction time is 10-50 min at 80-100 ℃.
8. The method according to claim 2, wherein the dialysis in step 4 comprises the following steps: dialyzing for 1-5 days by using deionized water with the pH value of 4, and then dialyzing for 1-3 days by using PBS buffer solution with the pH value of 7.4.
9. Cu obtainable by a process according to any one of claims 1 to 82MoS4@ BSA nanomaterial.
10. Cu according to claim 92MoS4The application of the @ BSA nano material in non-disease diagnosis and treatment of nuclear magnetic resonance T1 weighted image-guided PTT/PDT diagnostic agents.
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CN105999309A (en) * 2016-05-24 2016-10-12 天津大学 Protein biological template-based gadolinium-doped copper sulfide nano-particles and preparation method thereof

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