Albumin-based up-conversion nanoparticles and preparation method thereof

Abstract

The invention provides an up-conversion nanoparticle based on albumin and a preparation method thereof, the up-conversion nanoparticle takes albumin as an assembly element, in the presence of ultrasonic stimulation and an organic solvent, the albumin exposes an internal hydrophobic cavity in a reversible manner, and through multi-stage self-assembly of albumin and TTA-UC molecular pairs, the up-conversion nanoparticle is prepared. According to the present invention, the albumin-based up-conversion nano-particles are prepared, the up-conversion nano-particles can instantaneously produce TTA-UC in air and an aqueous solution, the preparation method does not depend on an exogenous material, the biocompatibility is good, the particle size of the prepared up-conversion nano-particles is 7-55 nm, the absorption peak of the up-conversion nano-particles is 350-750 nm, and the up-conversion nano-particles do not produce serious light scattering; and the reliability of an analysis result is greatly improved.

Description

Technical Field

[0001] The present application relates to the technical field of nanomaterial preparation, and in particular to an albumin-based upconversion nanoparticle and a preparation method thereof. Background Art

[0002] The triplet-triplet annihilation luminescence (TTA-UC) system is a bimolecular system consisting of a triplet photosensitizer (donor) and a triplet annihilation agent (acceptor). 2 ), has potential applications in biophotonics, photocatalysis, chemical metrology and nanomaterial design. The bimolecular system of TTA-UC can only be realized in deoxygenated organic solvents and cannot be directly applied in aqueous systems. Therefore, the development of triplet-triplet annihilation upconversion nanoparticles (TTA-UC NPs) that are stable in aqueous phase is an important idea to expand the performance application of TTA-UC.

[0003] As a new type of photon upconversion material, TTA-UC NPs have a high upconversion quantum efficiency (ηUC) and a low excitation power density (mW / cm 2 ) and tunable excitation / emission wavelengths, which enable it to be used in important fields such as biosensing, imaging, photoredox catalysis and 3D printing. This also puts higher requirements on the synthesis of TTA-UCNPs. The current method for preparing TTA-UC NPs relies on exogenous materials, has poor biocompatibility, and has a particle size of more than 100nm, with severe light scattering, which greatly reduces the reliability of the analysis results. Developing a synthetic method for TTA-UC NPs with small particle size, good biocompatibility and simple operation steps is still a huge challenge.

[0004] TTA-UC NPs without exogenous substances have great potential in biomedical applications. The currently developed TTA-UC NPs preparation method is mainly to encapsulate the photosensitizer / annihilator by SiO2 shell or use amphiphilic polymer self-assembly (Iyisan, B, Thiramanas, R, Nazarova, N, Avlasevich, Y, Mailander, V, Baluschev, S, Landfester, K. Temperature Sensing in Cells Using Polymeric Upconversion Nanocapsules. Biomacromolecules 2020, 21 (11): 4469-4478.). The raw materials used in these nanoparticles are all exogenous substances and have certain biological toxicity. The analysis and test results show that the nanoparticles prepared by these methods are large in size, have severe light scattering, poor photostability and the particle morphology is susceptible to the critical micelle concentration (CMC), and it is impossible to achieve accurate chemometric measurement when applied to complex biological systems. In addition, the absorption of the TTA-UC NPs prepared so far is in the visible light region and is easily interfered by background signals. Summary of the invention

[0005] In view of this, the present application provides an albumin-based upconversion nanoparticle and a preparation method thereof, which solves the technical problems that the method for preparing TTA-UC NPs in the prior art completely relies on exogenous materials, has poor biocompatibility, and has a particle size exceeding 100nm, has severe light scattering, and greatly reduces the reliability of the analysis results.

[0006] According to the first aspect of the present application, the present application provides an albumin-based upconversion nanoparticle, which can instantly generate TTA-UC in air and aqueous solution under laser irradiation, and the particle size of the upconversion nanoparticle is 7nm to 55nm.

[0007] In one embodiment, the absorption peak of the upconversion nanoparticles is 350-750 nm.

[0008] According to the second aspect of the present application, the present application provides a method for preparing albumin-based upconversion nanoparticles, which comprises the following preparation steps: step S1, dissolving a certain amount of solid albumin in ultrapure water to obtain a first mixed solution; step S2, dissolving a certain amount of photosensitizer and annihilator in a volatile solvent to obtain a second mixed solution; step S3: after mixing the first mixed solution and the second mixed solution, ultrasonically subjecting the mixed first mixed solution and the second mixed solution to ultrasound for 10 to 20 minutes to obtain an emulsion system; step S4: continuing to ultrasonically subject the emulsion system to ultrasound for 20 to 60 minutes to volatilize the organic solvent in the emulsion system to obtain an albumin upconversion nanoparticle solution.

[0009] In one embodiment, in step S1, the solid albumin includes one of bovine serum and human serum, the mixing temperature of the first mixed solution is 0-60° C., and the concentration of the albumin in the first mixed solution is 0.07 mg / mL-100 mg / mL.

[0010] In one embodiment, in step S2, the absorption peak of the photosensitizer is adjusted to a range of 350 to 750 nm; the triplet energy level of the photosensitizer ( 3 Sen*) 3 Sen*> 3 An*, and 2× 3 An*> 1 An*; in the step S2, the triplet energy level of the annihilator molecule ( 3 An*) 3 Sen*> 3 An*, and 2× 3 An*> 1 An*; the molar concentration of the annihilation agent is less than 1000 μM; in the step S2, the volatile solvent includes one of chloroform, dichloromethane, toluene, n-hexane and an alkane having less than 6 carbon atoms and being immiscible with water; in the step S2, the mixing temperature of the second mixed solution is less than the boiling point of the volatile solvent.

[0011] In one embodiment, the photosensitizer includes one of PtTNP, PdTNP, PdTPBP, PtTPBP, PtOEP, and PdOEP; the annihilator includes one of diketopyrrolopyrrole (DPP) and its derivatives, fluoroborane dipyrrole (Bodipy) and its derivatives, perylene (Py) and its derivatives, anthracene and its derivatives, and cou and its derivatives.

[0012] In one embodiment, in step S3, the temperature of the emulsion system is 0-60° C., the concentration of the photosensitizer in the second mixed solution is 0.01-100 μM; and the concentration of the annihilator in the second mixed solution is 10-1000 μM.

[0013] In one embodiment, in step S4, the concentration of albumin in the prepared upconversion nanoparticle solution is 0.07 mg / mL to 100 mg / mL.

[0014] In one embodiment, the annihilation agent DPP is formed with benzonitrile and diisopropyl succinate as substrates under strong base and FeCl3 as catalyst to form compound 1, and then alkylated with compound 2 and bromobutyl as substrates to obtain compound 3, and finally compound 3 and 2,6-dimethylphenylboron are reacted under palladium catalysis to obtain An-4.

[0015] In one embodiment, benzonitrile and diisopropyl succinate are used as substrates, and under the action of strong base Na and 2-Me-2-BuOH, and with FeCl3 as a catalyst, the reaction is carried out at 120°C for 8 to 14 hours to form compound 2; compound 2 and butyl bromide are used as substrates, and 18-Crown-6, K2CO3 and KI are mixed in deoxygenated DMF, and reacted at 120°C for 8 to 14 hours to obtain compound 3; then compound 3, 2,6-dimethylphenylboric acid, tetrakis(triphenylphosphine)palladium and K2CO3 are dissolved in deoxygenated toluene / ethanol / water, the volume ratio of toluene / ethanol / water is 2:2:1, and stirred at 100°C for 8 to 14 hours to obtain a first solution, the first solution is cooled to room temperature, filtered with saturated brine and purified by silica gel column chromatography to obtain product An4.

[0016] In one embodiment, the structure of the annihilator An can be a derivative of various annihilator monomers (such as An-4, a derivative of DPP).

[0017] In one embodiment, the preparation reaction formula of the annihilation agent An4 is as follows:

[0018]

[0019] The present application provides an albumin-based upconversion nanoparticle and a preparation method thereof, comprising the following preparation steps: step S1, dissolving a certain amount of solid albumin in ultrapure water to obtain a first mixed solution; step S2, dissolving a certain amount of photosensitizer and annihilator in a volatile solvent to obtain a second mixed solution; step S3: after mixing the first mixed solution and the second mixed solution, ultrasonically subjecting the mixed first mixed solution and the second mixed solution to 10 to 20 minutes under ultrasonic conditions to obtain an emulsion system; step S4: continuing to ultrasonically subject the emulsion system to 20 to 60 minutes under ultrasonic conditions to volatilize the organic solvent in the emulsion system to obtain an albumin upconversion nanoparticle solution. The present application uses albumin as an assembly unit. Under ultrasonic stimulation and the presence of an organic solvent, albumin exposes the internal hydrophobic cavity in a reversible manner. The albumin-based upconversion nanoparticle is prepared through multi-level self-assembly of albumin and TTA-UC molecular pairs. The upconversion nanoparticles can instantly generate TTA-UC in the air and aqueous solution. The absorption peak of the upconversion nanoparticles is 350-750nm. The preparation method does not rely on exogenous materials and has good biocompatibility. The particle size of the prepared upconversion nanoparticles is 7nm-55nm. The absorption peak of the upconversion nanoparticles is 350-750nm. It will not produce serious light scattering, which greatly improves the reliability of the analysis results. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Shown is a schematic diagram of the preparation process of upconversion nanoparticles provided in Examples 1-7 of the present application;

[0021] Figure 2 Shown is a particle size distribution diagram of nanoparticles prepared from different concentrations of An-4 annihilation agents provided in Examples 1-7 of the present application;

[0022] Figure 3 Shown are transmission electron microscopy images of nanoparticles of different concentrations provided in Example 4 of the present application;

[0023] Figure 4 Shown is the up-conversion luminescence spectrum of nanoparticles prepared from the annihilation agent An-4 and perylene (Py-26) provided in the present application;

[0024] Figure 5 Shown is the power-dependent upconversion luminescence spectrum and threshold power of the PtTNP / An-4 protein nanoparticles provided in Example 4 of the present application;

[0025] Figure 6 Shown is the up-conversion luminescence spectrum of PtTNP / An-4 protein nanoparticles before and after freeze-drying provided in Example 4 of the present application;

[0026] Figure 7Shown is a comparison chart of the biological toxicity of PtTNP / An-4 protein nanoparticles to MCF-7 cells provided in Example 4 of the present application. DETAILED DESCRIPTION

[0027] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0028] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0029] Unless otherwise specified, the reagents used in this embodiment are all commercially available products.

[0030] Example 1

[0031] The preparation method of the albumin upconversion nanoparticles based on Example 1 comprises the following steps:

[0032] S1. Weigh 80 mg of bovine serum albumin (Solabol) and dissolve it in 2 mL of ultrapure water to obtain a first mixed solution;

[0033] S2. Weigh 20 μg of photosensitizer (PtTNP) and 0.22 μmol of annihilator (phenyl-substituted diketopyrrolopyrrole (An-4)) and dissolve them in 1 mL of chloroform to obtain a second mixed solution. The concentration of annihilator An-4 in the second mixed solution is 110 μM.

[0034] S3, after mixing the first mixed solution and the second mixed solution, ultrasonically subjecting the mixed first mixed solution and the second mixed solution to the ultrasonic cleaning machine for 10 minutes to obtain an emulsion system;

[0035] S4. Continue to ultrasonicate the emulsion system in an ultrasonic cleaning machine for 20 to 60 minutes to evaporate the chloroform in the emulsion system until the solution becomes clear, thereby obtaining albumin upconversion nanoparticles.

[0036] The preparation steps of the annihilator (An-4) are as follows: using benzonitrile and diisopropyl succinate as substrates, under the action of strong base Na and 2-Me-2-BuOH, and using FeCl3 as a catalyst, react at 120°C for 8 to 14 hours to form compound 2; using compound 2 and butyl bromide as substrates, and mixing 18-Crown-6, K2CO3 and KI in deoxygenated DMF, and reacting at 120°C for 12 hours to obtain compound 3; then dissolving compound 3, 2,6-dimethylphenylboric acid, tetrakis(triphenylphosphine)palladium and K2CO3 in deoxygenated toluene / ethanol / water, the volume ratio of toluene / ethanol / water is 2:2:1, and stirring at 100°C for 12 hours to obtain a first solution, cooling the first solution to room temperature, filtering with saturated brine and purifying by silica gel column chromatography to obtain the product An4.

[0037] Example 2

[0038] The preparation method of the albumin upconversion nanoparticles according to Example 2 comprises the following steps:

[0039] S1. Weigh 80 mg of bovine serum albumin (Solabol) and dissolve it in 2 mL of ultrapure water to obtain a first mixture;

[0040] S2. Weigh 20 μg of photosensitizer (PtTNP) and 0.35 μmol of annihilator (phenyl-substituted diketopyrrolopyrrole (An-4), An-4 is a derivative of DPP) and dissolve them in 1 mL of chloroform to obtain a second mixed solution. The concentration of the annihilator An-4 in the second mixed solution is 175 μM.

[0041] S3, after mixing the first mixed solution and the second mixed solution, ultrasonically subjecting the mixed first mixed solution and the second mixed solution to the ultrasonic cleaning machine for 10 minutes to obtain an emulsion system;

[0042] S4. Continue to ultrasonicate the emulsion system in an ultrasonic cleaning machine for 20 to 60 minutes to evaporate the chloroform in the emulsion system until the solution becomes clear, thereby obtaining albumin upconversion nanoparticles.

[0043] The preparation steps of the annihilator (An-4) are as follows: using benzonitrile and diisopropyl succinate as substrates, under the action of strong base Na and 2-Me-2-BuOH, and using FeCl3 as a catalyst, react at 120°C for 8 to 14 hours to form compound 2; using compound 2 and butyl bromide as substrates, and mixing 18-Crown-6, K2CO3 and KI in deoxygenated DMF, and reacting at 120°C for 12 hours to obtain compound 3; then dissolving compound 3, 2,6-dimethylphenylboric acid, tetrakis(triphenylphosphine)palladium and K2CO3 in deoxygenated toluene / ethanol / water, the volume ratio of toluene / ethanol / water is 2:2:1, and stirring at 100°C for 12 hours to obtain a first solution, cooling the first solution to room temperature, filtering with saturated brine and purifying by silica gel column chromatography to obtain the product An4.

[0044] Example 3

[0045] The preparation method of the albumin upconversion nanoparticles according to Example 3 comprises the following steps:

[0046] S1. Weigh 80 mg of bovine serum albumin (Solabol) and dissolve it in 2 mL of ultrapure water to obtain a first mixture;

[0047] S2. Weigh 20 μg of photosensitizer (PtTNP) and 0.548 μmol of annihilator (phenyl-substituted diketopyrrolopyrrole (An-4)) and dissolve them in 1 mL of chloroform to obtain a second mixed solution. The concentration of the annihilator An-4 in the second mixed solution is 274 μM.

[0048] S3, after mixing the first mixed solution and the second mixed solution, ultrasonically subjecting the mixed first mixed solution and the second mixed solution to the ultrasonic mixer for 10 minutes to obtain an emulsion system;

[0049] S4. Continue to ultrasonicate the emulsion system for 20 to 60 minutes under ultrasonic conditions to evaporate the chloroform in the emulsion system until the solution becomes clear, thereby obtaining albumin upconversion nanoparticles.

[0050] The preparation steps of the annihilator (An-4) are as follows: using benzonitrile and diisopropyl succinate as substrates, under the action of strong base Na and 2-Me-2-BuOH, and using FeCl3 as a catalyst, react at 120°C for 8 to 14 hours to form compound 2; using compound 2 and butyl bromide as substrates, and mixing 18-Crown-6, K2CO3 and KI in deoxygenated DMF, and reacting at 120°C for 12 hours to obtain compound 3; then dissolving compound 3, 2,6-dimethylphenylboric acid, tetrakis(triphenylphosphine)palladium and K2CO3 in deoxygenated toluene / ethanol / water, the volume ratio of toluene / ethanol / water is 2:2:1, and stirring at 100°C for 12 hours to obtain a first solution, cooling the first solution to room temperature, filtering with saturated brine and purifying by silica gel column chromatography to obtain the product An4.

[0051] Example 4

[0052] The preparation method of the albumin upconversion nanoparticles according to Example 4 comprises the following steps:

[0053] S1. Weigh 80 mg of bovine serum albumin (Solabol) and dissolve it in 2 mL of ultrapure water to obtain a first mixture;

[0054] S2. Weigh 20 μg of photosensitizer (PtTNP) and 0.768 μmol of annihilator (phenyl-substituted diketopyrrolopyrrole (An-4)) and dissolve them in 1 mL of chloroform to obtain a second mixed solution. The concentration of annihilator An-4 in the second mixed solution is 384 μM.

[0055] S3, after mixing the first mixed solution and the second mixed solution, ultrasonically subjecting the mixed first mixed solution and the second mixed solution to the ultrasonic cleaning machine for 10 minutes to obtain an emulsion system;

[0056] S4. Continue to ultrasonicate the emulsion system in an ultrasonic cleaning machine for 20 to 60 minutes to evaporate the chloroform in the emulsion system until the solution becomes clear, thereby obtaining albumin upconversion nanoparticles.

[0057] The preparation steps of the annihilator (An-4) are as follows: using benzonitrile and diisopropyl succinate as substrates, under the action of strong base Na and 2-Me-2-BuOH, and using FeCl3 as a catalyst, react at 120°C for 8 to 14 hours to form compound 2; using compound 2 and butyl bromide as substrates, and mixing 18-Crown-6, K2CO3 and KI in deoxygenated DMF, and reacting at 120°C for 12 hours to obtain compound 3; then dissolving compound 3, 2,6-dimethylphenylboric acid, tetrakis(triphenylphosphine)palladium and K2CO3 in deoxygenated toluene / ethanol / water, the volume ratio of toluene / ethanol / water is 2:2:1, and stirring at 100°C for 12 hours to obtain a first solution, cooling the first solution to room temperature, filtering with saturated brine and purifying by silica gel column chromatography to obtain the product An4.

[0058] Example 5

[0059] The preparation method of the albumin upconversion nanoparticles according to Example 5 comprises the following steps:

[0060] S1. Weigh 80 mg of bovine serum albumin (Solabol) and dissolve it in 2 mL of ultrapure water to obtain a first mixture;

[0061] S2. Weigh 20 μg of photosensitizer (PtTNP) and 0.876 μmol of annihilator (phenyl-substituted diketopyrrolopyrrole (An-4)) and dissolve them in 1 mL of chloroform to obtain a second mixed solution. The concentration of the annihilator An-4 in the second mixed solution is 438 μM.

[0062] S3, after mixing the first mixed solution and the second mixed solution, ultrasonically subjecting the mixed first mixed solution and the second mixed solution to the ultrasonic cleaning machine for 10 minutes to obtain an emulsion system;

[0063] S4. Continue to ultrasonicate the emulsion system in an ultrasonic cleaning machine for 20 to 60 minutes to evaporate the chloroform in the emulsion system until the solution becomes clear, thereby obtaining albumin upconversion nanoparticles.

[0064] The preparation steps of the annihilator (An-4) are as follows: using benzonitrile and diisopropyl succinate as substrates, under the action of strong base Na and 2-Me-2-BuOH, and using FeCl3 as a catalyst, react at 120°C for 8 to 14 hours to form compound 2; using compound 2 and butyl bromide as substrates, and mixing 18-Crown-6, K2CO3 and KI in deoxygenated DMF, and reacting at 120°C for 12 hours to obtain compound 3; then dissolving compound 3, 2,6-dimethylphenylboric acid, tetrakis(triphenylphosphine)palladium and K2CO3 in deoxygenated toluene / ethanol / water, the volume ratio of toluene / ethanol / water is 2:2:1, and stirring at 100°C for 12 hours to obtain a first solution, cooling the first solution to room temperature, filtering with saturated brine and purifying by silica gel column chromatography to obtain the product An4.

[0065] Example 6

[0066] The preparation method of the albumin upconversion nanoparticles according to Example 6 comprises the following steps:

[0067] S1. Weigh 80 mg of bovine serum albumin (Solabol) and dissolve it in 2 mL of ultrapure water to obtain a first mixture;

[0068] S2. Weigh 20 μg of photosensitizer (PtTNP) and 0.986 μmol of annihilator (phenyl-substituted diketopyrrolopyrrole (An-4)) and dissolve them in 1 mL of chloroform to obtain a second mixed solution. The concentration of the annihilator An-4 in the second mixed solution is 493 μM.

[0069] S3, after mixing the first mixed solution and the second mixed solution, ultrasonically subjecting the mixed first mixed solution and the second mixed solution to the ultrasonic cleaning machine for 10 minutes to obtain an emulsion system;

[0070] S4. Continue to ultrasonicate the emulsion system in an ultrasonic cleaning machine for 20 to 60 minutes to evaporate the chloroform in the emulsion system until the solution becomes clear, thereby obtaining albumin upconversion nanoparticles.

[0071] The preparation steps of the annihilator (An-4) are as follows: using benzonitrile and diisopropyl succinate as substrates, under the action of strong base Na and 2-Me-2-BuOH, and using FeCl3 as a catalyst, react at 120°C for 8 to 14 hours to form compound 2; using compound 2 and butyl bromide as substrates, and mixing 18-Crown-6, K2CO3 and KI in deoxygenated DMF, and reacting at 120°C for 12 hours to obtain compound 3; then dissolving compound 3, 2,6-dimethylphenylboric acid, tetrakis(triphenylphosphine)palladium and K2CO3 in deoxygenated toluene / ethanol / water, the volume ratio of toluene / ethanol / water is 2:2:1, and stirring at 100°C for 12 hours to obtain a first solution, cooling the first solution to room temperature, filtering with saturated brine and purifying by silica gel column chromatography to obtain the product An4.

[0072] Example 7

[0073] The preparation method of the albumin upconversion nanoparticles according to Example 7 comprises the following steps:

[0074] S1. Weigh 80 mg of bovine serum albumin (Solabol) and dissolve it in 2 mL of ultrapure water to obtain a first mixture;

[0075] S2. Weigh 25 μg of photosensitizer (PdTPBP) and 0.35 μmol of annihilator (perylene (Py-26)) and dissolve them in 1 mL of chloroform to obtain a second mixed solution. The concentration of photosensitizer PdTPBP in the second mixed solution is 6.4 μM.

[0076] S3, after mixing the first mixed solution and the second mixed solution, ultrasonically subjecting the mixed first mixed solution and the second mixed solution to the ultrasonic cleaning machine for 10 minutes to obtain an emulsion system;

[0077] S4. Continue to ultrasonicate the emulsion system in an ultrasonic cleaning machine for 20 to 60 minutes to evaporate the chloroform in the emulsion system until the solution becomes clear, thereby obtaining albumin upconversion nanoparticles.

[0078] Experimental Example 1

[0079] Figure 1 Shown is Figure 1 Shown is a schematic diagram of the preparation process of upconversion nanoparticles provided in Examples 1-7 of the present application. Figure 2 The graph shows the particle size distribution of nanoparticles prepared by using different concentrations of An-4 annihilation agent provided in Examples 1-6 of the present application; the concentrations of the annihilation agent (An-4) are 110 μM, 175 μM, 274 μM, 384 μM, 438 μM and 493 μM, respectively. Figure 1 as well as Figure 2As shown, the prepared upconversion nanoparticles can form stable protein nanoparticles at different concentrations of photosensitizer / annihilator, and when the concentration of annihilator is lower than 400 μM, the self-assembly of bovine serum albumin and photosensitizer / annihilator produces protein nanoparticles of less than 10 nm. When the concentration of annihilator exceeds 450 μM, protein nanoparticles of greater than 50 nm are produced.

[0080] Figure 3 The following are transmission electron micrographs of nanoparticles of different concentrations provided in Example 4 of the present application. The protein nanoparticles prepared in Example 4 were added to water to prepare suspensions of different concentrations. The negative staining method was used to prepare transmission electron microscopy samples, and the samples were placed under a transmission electron microscope (HITACHI HT7800) for observation. The concentrations of the diluted nanoparticles were A, 40 mg / mL; B, 20 mg / mL; C, 1.25 mg / mL; D, 0.4 mg / mL; E, 0.1 mg / mL; and F, 0.07 mg / mL. Figure 3 It can be seen that the prepared nanoparticles are spherical, and the protein nanoparticles can form large-scale assemblies as the concentration increases. The diameter of the nanoparticles is 7 to 55 nm.

[0081] Experimental Example 2

[0082] Figure 4 The absorption spectrum and up-conversion luminescence spectrum of the protein nanoparticles prepared by using the annihilator An-4 and perylene (Py-26) in Examples 1-7; Figure 5 Shown is the power-dependent upconversion luminescence spectrum and threshold power of the PtTNP / An-4 protein nanoparticles provided in Example 4 of the present application; Figure 4 and Figure 5 It can be seen that the prepared protein nanoparticles can directly produce upconversion luminescence in aqueous solution and air without removing oxygen from the system. Since the absorption of the upconversion nanoparticles is consistent with the absorption spectrum range of the photosensitizer encapsulated therein, and its emission is consistent with the emission spectrum range of the annihilator, the protein nanoparticles also broaden the adjustable range of absorption / emission.

[0083] Experimental Example 3

[0084] The protein nanoparticles prepared in Example 4 were evenly distributed in 10 mL centrifuge tubes (0.5 mL / tube), and rapidly cooled to a solid state with liquid nitrogen. The frozen protein upconversion nanoparticles were placed in a freeze dryer and freeze-dried in a vacuum environment of -50°C for 4 hours to obtain protein upconversion nanoparticle solids. Subsequently, ultrapure water was added again to dissolve the nanoparticles, and the concentration was kept constant. The upconversion luminescence spectra of the protein upconversion nanoparticles before and after freeze-drying were measured. Figure 6The figure shows the up-conversion luminescence spectra of PtTNP / An-4 protein nanoparticles before and after freeze-drying provided in Example 4 of the present application; Figure 6 It can be seen that the upconversion luminescence intensity of the prepared protein nanoparticles has no obvious change before and after freeze-drying, indicating that the upconversion performance of the nanoparticles is not lost.

[0085] Experimental Example 4

[0086] The protein nanoparticles prepared in Example 4 were added to water to prepare suspensions of different concentrations, which were added to MCF-7 cells in a 96-well plate and co-cultured for 12 h. The biological toxicity of protein upconversion nanoparticles of different concentrations to MCF-7 was detected by CCK-8 cytotoxicity experiment. The concentrations were 0 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL and 10 mg / mL, respectively. Figure 7 The figure shows the comparison of the biological toxicity of PtTNP / An-4 protein nanoparticles to MCF-7 cells provided in Example 4 of the present application. Figure 7 It can be seen that PtTNP / An-4 protein upconversion nanoparticles have good biocompatibility with MCF-7 cells.

[0087] The present application uses protein as the assembly unit. Under ultrasonic stimulation and the presence of organic solvents, BSA exposes internal hydrophobic cavities in a reversible manner, and these cavities provide a structural basis for the formation of protein upconversion nanoparticles. Protein upconversion nanoparticles are prepared by multi-level self-assembly of BSA and TTA-UC molecular pairs. The prepared nanoparticles are small in size, stable in structure, uniformly dispersed, and have good biocompatibility. In addition, this upconversion nanoparticle can instantly produce TTA-UC in air and aqueous solution under laser irradiation, and the absorption peak is adjustable at 350 to 750 nm, which also broadens the adjustable range of absorption / emission. The protein upconversion nanoparticles synthesized by this method can also be stored after freeze-drying, which provides a new idea for solving the problem of long-term stable storage of nanoparticles.

[0088] Many proteins in organisms form a natural spatial system due to their complex helical and folded structures and specific assembly methods. The polypeptide chain of the protein forms the secondary structure of the protein through α-helix or β-folding, and the tertiary structure of each polypeptide chain has a specific three-dimensional distribution and is connected to form the quaternary structure of the protein. Studies have shown that the natural amphiphilic cavity structure inside the protein can loosen the higher-level structure under certain external forces or chemical reactions without denaturing the protein. Therefore, it is a new design idea for TTA-UC NPs to directly construct nanoparticles based on endogenous substances by drawing on the natural multi-level assembly method of proteins.

[0089] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An albumin-based upconversion nanoparticle, characterized in that: Under laser irradiation, the up-conversion nanoparticles can instantly generate TTA-UC in the air and aqueous solution, and the particle size of the up-conversion nanoparticles is 7nm-55nm.

2. The albumin-based upconversion nanoparticle according to claim 1, characterized in that: The absorption peak of the up-conversion nanoparticles is 350-750nm.

3. A method for preparing albumin-based upconversion nanoparticles, characterized in that: The method comprises the following preparation steps: Step S1, dissolving a certain amount of solid albumin in ultrapure water to obtain a first mixed solution; Step S2, dissolving a certain amount of photosensitizer and annihilator in a volatile solvent to obtain a second mixed solution; Step S3: After mixing the first mixed solution and the second mixed solution, ultrasonically subjecting the mixed first mixed solution and the second mixed solution to the ultrasonic condition for 10 to 20 minutes to obtain an emulsion system; Step S4: continue to ultrasonicate the emulsion system for 20 to 60 minutes under ultrasonic conditions to volatilize the organic solvent in the emulsion system and obtain an albumin upconversion nanoparticle solution.

4. The method for preparing albumin-based upconversion nanoparticles according to claim 3, characterized in that: In the step S1, the solid albumin includes one of bovine serum and human serum, the mixing temperature of the first mixed solution is 0 to 60°C, and the concentration of the albumin in the first mixed solution is 0.07 mg / mL to 100 mg / mL.

5. The method for preparing albumin-based upconversion nanoparticles according to claim 3, characterized in that: In step S2, the absorption peak of the photosensitizer is adjusted to a range of 350 to 750 nm; the triplet energy level of the photosensitizer ( 3 Sen*) 3 Sen*> 3 An*, and 2× 3 An*> 1 An*; In step S2, the triplet energy level of the annihilator molecule ( 3 An*) 3 Sen*> 3 An*, and 2× 3 An*> 1 An*; the molar concentration of the annihilator is less than 1000 μM; In step S2, the volatile solvent includes one of chloroform, dichloromethane, toluene, n-hexane, and an alkane having less than 6 carbon atoms and being immiscible with water; In the step S2, the mixing temperature of the second mixed solution is lower than the boiling point of the volatile solvent.

6. The method for preparing albumin-based upconversion nanoparticles according to claim 5, characterized in that: The photosensitizer includes one of PtTNP, PdTNP, PdTPBP, PtTPBP, PtOEP, and PdOEP; The annihilator includes one of diketopyrrolopyrrole (DPP) and its derivatives, boron dipyrrole (Bodipy) and its derivatives, perylene (Py) and its derivatives, anthracene and its derivatives, and cou and its derivatives.

7. The method for preparing albumin-based upconversion nanoparticles according to claim 6, characterized in that: In the step S3, the temperature of the emulsion system is 0-60° C., the concentration of the photosensitizer in the second mixed solution is 0.01-100 μM; and the concentration of the annihilator in the second mixed solution is 10-1000 μM.

8. The method for preparing albumin-based upconversion nanoparticles according to claim 3, characterized in that: In the step S4, the concentration of albumin in the prepared upconversion nanoparticle solution is 0.07 mg / mL to 100 mg / mL.

9. The method for preparing albumin-based upconversion nanoparticles according to claim 6, characterized in that: The annihilation agent DPP is prepared by using benzonitrile and diisopropyl succinate as substrates and FeCl3 as a catalyst in the presence of a strong base to form compound 1, and then using compound 2 and bromobutyl as substrates for alkylation to obtain compound 3, and finally compound 3 and 2,6-dimethylphenylborane are reacted under the catalysis of palladium to obtain An-4.

10. The method for preparing albumin-based upconversion nanoparticles according to claim 9, characterized in that: Using benzonitrile and diisopropyl succinate as substrates, under the action of strong bases Na and 2-Me-2-BuOH, and with FeCl3 as a catalyst, the reaction was carried out at 120°C for 8 to 14 hours to form compound 2; Compound 2 and bromobutyl are used as substrates, and 18-Crown-6, K2CO3 and KI are mixed in deoxygenated DMF, and reacted at 120°C for 8 to 14 hours to obtain compound 3; Then, compound 3, 2,6-dimethylphenylboric acid, tetrakis(triphenylphosphine)palladium and K2CO3 are dissolved in deoxygenated toluene / ethanol / water, the volume ratio of toluene / ethanol / water is 2:2:1, and stirred at 100°C for 8 to 14 hours to obtain a first solution. The first solution is cooled to room temperature, filtered with saturated brine and purified by silica gel column chromatography to obtain product An4.

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