Preparation method of upconversion fluorescent probe with strong cell penetration and strong solution dispersibility

Abstract

The application discloses a preparation method of a cell-penetrating strong-solution and strong-dispersion upconversion fluorescent probe and application thereof. Compared with the prior art, the cell-penetrating peptide modified upconversion nanoprobe can significantly improve the efficiency of nanoparticles entering living cells on one hand, and compared with polypeptide modified rare earth nanoparticles coupled with traditional modification and functionalization methods and not desalted ions, the cell-penetrating peptide modified upconversion nanoprobe can not only show strong particle dispersion and chemical stability in extracellular, but also show stronger single-particle dispersion in intracellular. The method provided by the application can enter different subcellular regions in cells by coupling different cell-penetrating peptide molecules, and has multiple potential application values.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of nanomaterials, and particularly relates to a preparation method of an upconversion fluorescent probe with strong cell penetration and strong solution dispersibility and application thereof. BACKGROUND

[0002] Rare earth doped upconversion nanoparticles (UCNPs) can absorb several low-energy long-wavelength photons and then emit a high-energy short-wavelength photon to realize multi-photon luminescence. UCNPs have the advantages of near-infrared excitation, large anti-Stokes shift, stable luminescence, and narrow emission peak, and are widely used in the field of biomedical research. However, the prepared UCNPs have hydrophobic oleic acid ligands on the surface, and the polypeptide modified rare earth nanoparticles prepared by traditional modification and functionalization methods often have poor dispersibility, which is difficult to meet the application requirements. In order to improve the performance of nanomaterials, new surface modification and functionalization methods need to be developed to improve the dispersibility and functionality.

[0003] The cell membrane can effectively prevent exogenous substances from entering the cell, ensuring the intracellular homeostasis and the orderly progress of life activities, but this also makes it difficult for exogenous molecules to pass through the cell membrane, limiting the observation and regulation of intracellular life activities. In order to deliver more exogenous substances into cells, a series of carriers or ligands such as cell penetrating peptides (CPPs), liposomes, fatty acids, polymers or surfactants have been developed for cell uptake of exogenous substances. Among them, cell penetrating peptides can promote the entry of drugs, nucleic acids and other molecules into cells, and have high application value. By utilizing the optical properties of UCNPs and then combining with cell penetrating peptide modification, the efficiency of UCNPs probes entering cells can be significantly improved, and the intracellular microenvironment can be observed and characterized. SUMMARY

[0004] The purpose of the present application is to overcome the problems of poor sample dispersibility, easy agglomeration and poor cell penetration caused by the existing functionalization treatment method of upconversion nanoparticles, and to provide a preparation method of cell penetrating peptide modified upconversion nanofluorescent probes and application thereof. The cell penetrating peptide modified nanoparticles obtained by the method of the present application have strong monodispersity, and the efficiency of UCNPs probes entering cells is improved, which has high application value and provides an efficient way for cell penetrating peptides to promote the entry of UCNPs into cells.

[0005] To achieve the above-mentioned purpose, the present application provides a preparation method of cell penetrating peptide modified upconversion nanofluorescent probes, which comprises the following steps:

[0006] S1. preparing NaYF4 matrix upconversion nanoparticles with uniform dispersion and adjustable size by co-precipitation method;

[0007] S2 reacting the rare earth nanoparticles with NOBF4 to remove the oleic acid ligand on the surface of the nanoparticles and coating with PAA to obtain water-soluble PAA-UCNPs with carboxyl groups on the surface;

[0008] S3 covalently coupling a desalination cell-penetrating peptide molecule to the PAA molecule by mediation of EDC and Sulfo-NHS to prepare cell-penetrating peptide-modified upconversion nanoparticles.

[0009] It should be noted that in the step S1, 1 mmol of rare earth acetate aqueous solution, 7.5 mL of oleic acid and 17.5 mL of octadecene are mixed, and after stirring at 150°C for 40 min, the temperature is lowered to room temperature, then 0.148 g of ammonium fluoride solid and 0.761 g of sodium oleate solid are mixed and added to the reaction system, and after stirring at 40°C for 2 h, the temperature is raised to 270°C, and the reaction is stirred for 1.5 h under argon protection to obtain oleic acid-coated UCNPs. After the reaction is completed, the solution is lowered to room temperature, the sample is washed with anhydrous ethanol and cyclohexane, and finally the upconversion nanoparticles are dispersed in cyclohexane.

[0010] It should be noted that in the step S2, the oleic acid ligand on the surface of the nanoparticles is removed with NOBF4, and then coated with PAA, which includes dispersing 20 mg of rare earth nanoparticle sample in 5 mL of cyclohexane, then dissolving 20 mg of NOBF4 in 5 mL of DMF, mixing the NOBF4 solution with the sample solution, stirring for 10 minutes to remove the oleic acid molecules on the surface of the particles to obtain ligand-free rare earth nanoparticle samples; then 5 mL of chloroform is added and centrifuged, the resulting precipitate is washed with a mixed solution of DMF and chloroform, and the resulting transparent precipitate sample is dispersed in pure water; 100 mg of PAA is dispersed in 6 mL of deionized water, then 10 mg of the above ligand-free rare earth particle aqueous solution is added dropwise to the PAA solution, and then stirring is continued for 2 hours to allow the PAA molecules to fully coat the surface of the particles to obtain PAA-UCNPs samples; then centrifuged, and the product is redispersed in water or buffer.

[0011] It should be noted that in the step S3, the cell-penetrating peptide molecule is coupled to the carboxyl group on the surface of the particles by mediation of EDC and Sulfo-NHS; including, first taking 1 mg of PAA-UCNPs in MES solution, adding 10 mg of EDC in MES solution and 15 mg of Sulfo-NHS in MES solution in turn, stirring vigorously for 2 h to obtain Sulfo-NHS-coupled PAA-UCNPs; then centrifuged, and the precipitate is dispersed in 1 mL of HEPES buffer; 50 μg of cell-penetrating peptide is added to the above solution, and the reaction is oscillated at 4°C for 3 h to couple the cell-penetrating peptide to the carboxyl group of PAA by displacement reaction, and finally centrifuged, and the precipitate is redispersed in 1 mL of HEPES buffer or cell culture medium.

[0012] It should be noted that the size of the upconversion nanoprobe can be precisely controlled by the reaction temperature, and the nanoparticles with a particle size of 7nm, 9nm and 11nm can be obtained by reacting at 250℃, 260℃ and 270℃, respectively.

[0013] Further, the application also provides the upconversion nanoprobe obtained by the preparation method of the upconversion nanoprobe modified by the cell penetrating peptide.

[0014] It should be noted that the cell penetrating peptide coupled on the surface of the nanoprobe is a salt-free product, and the type of the cell penetrating peptide is determined according to different use requirements.

[0015] Further, the application further provides the application of the upconversion nanoprobe obtained by the preparation method of the upconversion nanoprobe modified by the cell penetrating peptide, that is, the upconversion nanoprobe is used for fluorescence detection of a microenvironment of living cells.

[0016] The modification method of the application can be used to select different functional cell penetrating peptides for modification to obtain different functions, or multiple cell penetrating peptides can be used to prepare multifunctional rare earth nanoparticles. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a schematic diagram of the principle of the application;

[0018] Figure 2 is an electron microscope image of the prepared oleic acid upconversion nanoparticles;

[0019] Figure 3 is a fluorescence imaging diagram of the nanoparticles after coupling with TAT penetrating peptide in a multiphoton microscope, and the sparse single light emitting center proves that the upconversion nanoparticles modified by the cell penetrating peptide have good dispersibility;

[0020] Figure 4 is an aggregated state of the TAT penetrating peptide coupled with non-dephosphorylated ions, which is difficult to enter the inside of the cell. The TAT penetrating peptide coupled with dephosphorylated ions has good dispersibility in the cell, and is easier to enter the cell than the PAA-UCNPs, and the number of the nanoparticles entering the cell is obviously increased. DETAILED DESCRIPTION

[0021] The technical solutions of the present application will be described in detail below with reference to the drawings. It should be noted that the embodiments described are based on the technical solutions of the present application, and a detailed specific implementation is given, but the protection scope of the present application is not limited to the listed embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0022] The present application is a preparation method of a cell-penetrating peptide modified up-conversion nanofluorescent probe, which comprises the following steps:

[0023] S1. A NaYF4 matrix up-conversion nanoparticle with uniform dispersion and adjustable size is prepared by co-precipitation method;

[0024] S2. The rare earth nanoparticle is reacted with NOBF4 to remove the oleic acid ligand on the surface of the particle, and PAA is used for coating to obtain a water-soluble PAA-UCNPs with carboxylated surface;

[0025] S3. The cell-penetrating peptide molecules are covalently coupled to the PAA molecules by EDC and Sulfo-NHS mediation to prepare the cell-penetrating peptide modified up-conversion nanoparticle.

[0026] Further, in the step S1 of the present application, 1 mmol of rare earth acetate aqueous solution, 7.5 mL of oleic acid and 17.5 mL of octadecene are mixed, and after stirring at 150℃ for 40 min, the temperature is lowered to room temperature. Then, 0.148 g of ammonium fluoride solid and 0.761 g of sodium oleate solid are mixed and added to the reaction system. After stirring at 40℃ for 2 h, the temperature is raised to 270℃, and the reaction is stirred for 1.5 h under argon protection to obtain oleic acid coated UCNPs. After the reaction is completed, the solution is lowered to room temperature, the sample is washed with anhydrous ethanol and cyclohexane, and finally the up-conversion nanoparticles are dispersed in cyclohexane.

[0027] Further, in the step S2 of the present application, the oleic acid ligand on the surface of the nanoparticle is removed with NOBF4, and then PAA is used for coating. Specifically, 20 mg of rare earth nanoparticle sample is dispersed in 5 mL of cyclohexane, then 20 mg of NOBF4 is dissolved in 5 mL of DMF, the NOBF4 solution is mixed with the sample solution, and the reaction is stirred for 10 min to remove the oleic acid molecules on the surface of the particle to obtain a ligand-free rare earth nanoparticle sample. Then 5 mL of chloroform is added and centrifuged, the obtained precipitated product is washed with a mixed solution of DMF and chloroform, and the obtained transparent precipitated sample is dispersed in pure water. 100 mg of PAA is dispersed in 6 mL of deionized water, then 10 mg of the above ligand-free rare earth particle aqueous solution is added dropwise to the PAA solution, and then the stirring is continued for 2 h to allow the PAA molecules to fully coat the surface of the particle to obtain a PAA-UCNPs sample. Then, centrifugation is performed, and the product is redispersed in water or buffer.

[0028] Further, in the step S3 of the present application, the cell penetrating peptide molecule is coupled to the carboxyl on the surface of the particle by EDC and Sulfo-NHS mediation; including, first taking 1 mg of PAA-UCNPs in MES solution, adding 10 mg of EDC in MES solution and 15 mg of Sulfo-NHS in MES solution in turn, stirring vigorously for 2 h, obtaining Sulfo-NHS coupled PAA-UCNPs; then centrifuging, dispersing the precipitate in 1 mL of HEPES buffer; adding 50 μg of cell penetrating peptide to the above solution, oscillating the reaction at 4℃ for 3 h, coupling the cell penetrating peptide to the carboxyl of PAA through displacement reaction, and finally centrifuging, redispersing the precipitate in 1 mL of HEPES buffer or cell culture medium.

[0029] Further, the size of the upconversion nanoprobe of the present application can be precisely controlled by the reaction temperature, and well-dispersed nanoparticles with particle sizes of 7 nm, 9 nm and 11 nm can be obtained by reactions at 250℃, 260℃ and 270℃, respectively.

[0030] Further, the present application also provides an upconversion nanoprobe obtained by the preparation method of the cell penetrating peptide modified upconversion nanoprobe, specifically, the cell penetrating peptide molecule is covalently coupled to the carboxyl on the surface of the upconversion nanoparticle, which can significantly improve the efficiency of the upconversion nanoprobe into living cells, and has stronger dispersibility and stability, and can enter different subcellular regions by coupling different cell penetrating peptide molecules.

[0031] Further, the cell penetrating peptide coupled to the surface of the nanoprobe of the present application is preferably a salt-free product, and the type of cell penetrating peptide is determined according to different use requirements.

[0032] Further, the present application further provides an application of the upconversion nanoprobe obtained by the preparation method of the cell penetrating peptide modified upconversion nanoprobe, i.e., the upconversion nanoprobe is used for fluorescence detection of the microenvironment of living cells.

[0033] Example 1

[0034] The present embodiment provides a preparation method of an upconversion fluorescence probe with strong cell penetrating ability and strong solution dispersibility, including the specific steps of preparation of rare earth nanoparticles, surface modification and coupling of cell penetrating peptides.

[0035] Step 1: First, NaYF4:20Yb2Er nanoparticles were prepared: Using co-precipitation method, 3.9 mL Y(CH3COO)3(0.2 M), 1 mL Yb(CH3COO)3(0.2 M), 0.1 mL Er(CH3COO)3(0.2 M) aqueous solution, 7.5 mL oleic acid and 17.5 mL octadecene were added into a round bottom flask, after stirring at 150 °C for 40 min, the solution was cooled to room temperature, then 0.148 g of ammonium fluoride solid and 0.761 g of sodium oleate solid were added into the reaction system, after stirring at 40 °C for 2 h, the temperature was increased to 300 °C, and the reaction was stirred for 1.5 h under argon protection to obtain oleic acid-coated UCNPs. After the reaction was completed, the solution was cooled to room temperature, and the sample was washed with anhydrous ethanol and cyclohexane, and finally 8-10 nm rare earth nanoparticles were dispersed in cyclohexane.

[0036] Step 2: 20 mg of rare earth nanoparticle sample was dispersed in 5 mL of cyclohexane, then 20 mg of NOBF4 was dissolved in 5 mL of DMF, the NOBF4 solution was mixed with the sample solution, and the reaction was stirred for 10 min to remove the oleic acid molecules on the surface of the particles to obtain a ligand-free rare earth nanoparticle sample. Then 5 mL of chloroform was added and centrifuged, and the resulting precipitated product was washed with a mixed solution of DMF and chloroform, and the resulting transparent precipitated sample was dispersed in pure water. Then 100 mg of PAA was dispersed in 6 mL of deionized water, and then 10 mg of the above ligand-free rare earth particle aqueous solution was added dropwise into the PAA solution, and then the stirring was continued for 2 h to allow the PAA molecules to fully coat the surface of the particles to obtain a PAA-UCNPs sample; then centrifuged, and the product was redispersed in water or buffer.

[0037] Step 3: Take 1 mg of PAA-UCNPs in MES solution, add 10 mg of EDC in MES solution and 15 mg of Sulfo-NHS in MES solution in turn, and stir vigorously for 2 h to obtain Sulfo-NHS coupled PAA-UCNPs; then centrifuged, and the precipitate was dispersed in 1 mL of HEPES buffer. Then 50 ug of TAT penetrating peptide was added to the above Sulfo-NHS coupled PAA-UCNPs solution, and the reaction was carried out by oscillation at 4 °C for 3 h to couple the cell penetrating peptide to the carboxyl group of PAA through displacement reaction. Finally, centrifuged, and the precipitate was redispersed in 1 mL of HEPES buffer or cell culture medium.

[0038] Example 2

[0039] Step 1: Preparation of NaYF4:20Yb2Ho as upconversion particles: 3.9 mL Y(CH3COO)3(0.2 M), 1 mL Yb(CH3COO)3(0.2 M), 0.1 mL Ho(CH3COO)3(0.2 M) aqueous solution, 7.5 mL oleic acid and 17.5 mL octadecene were added into a round bottom flask, after stirring at 150 °C for 40 min, the solution was cooled to room temperature, then 0.148 g of ammonium fluoride solid and 0.761 g of sodium oleate solid were added into the reaction system, after stirring at 40 °C for 2 h, the temperature was increased to 300 °C, and the reaction was stirred for 1.5 h under argon protection to obtain oleic acid-coated UCNPs. After the reaction was completed, the solution was cooled to room temperature, and the sample was washed with anhydrous ethanol and cyclohexane, and finally 8-10 nm rare earth nanoparticles were dispersed in cyclohexane.

[0040] Step 2 is the same as step in example 1.

[0041] Step 3: 1 mg of PAA-UCNPs in MES solution was taken, 10 mg of EDC in MES solution and 15 mg of Sulfo-NHS in MES solution were added in turn, and stirred vigorously for 2 h to obtain Sulfo-NHS coupled PAA-UCNPs; then centrifuged, and the precipitate was dispersed in 1 mL of HEPES buffer. Then 50 μg of Pep-1 was added to the above Sulfo-NHS coupled PAA-UCNPs solution, and the reaction was carried out at 4 °C for 3 h, and the cell penetrating peptide was coupled to the carboxyl group of PAA by displacement reaction. Finally, centrifugation was performed, and the precipitate was re-dispersed in 1 mL of HEPES buffer or cell culture medium.

[0042] For those skilled in the art, various corresponding changes can be made according to the above technical solutions and concepts, and all these changes should be included in the protection scope of the claims of the present application.

Claims

1. A method for preparing an upconversion fluorescent probe with strong cell penetration and strong solution dispersibility, characterized in that, The method includes the following steps: S1 prepared uniformly dispersed and size-tunable NaYF4 matrix upconversion nanoparticles using a coprecipitation method. Specifically, the method included: mixing 1 mmol of rare earth acetate aqueous solution, 7.5 mL of oleic acid, and 17.5 mL of octadecene, stirring at 150 °C for 40 min, and then cooling to room temperature. Subsequently, 0.148 g of ammonium fluoride solid and 0.761 g of sodium oleate solid were mixed and added to the reaction system. After stirring at 40 °C for 2 h, the temperature was raised to 270 °C, and the reaction was carried out under argon protection for 1.5 h to obtain oleic acid-coated UCNPs. S2 reacted upconversion nanoparticles with nitrosotetrafluoroborate (NOBF4) to remove oleic acid ligands from the particle surface, and then coated them with PAA to obtain surface-carboxylated water-soluble PAA-UCNPs. The process involved removing oleic acid ligands from the nanoparticle surface with NOBF4, followed by PAA coating. This included dispersing 20 mg of the upconversion nanoparticle sample in 5 mL of cyclohexane, dissolving 20 mg of NOBF4 in 5 mL of N,N-dimethylformamide (DMF), mixing the NOBF4 solution with the sample solution, and stirring for 10 minutes to remove oleic acid molecules from the particle surface, obtaining ligand-free upconversion nanoparticles. Subsequently, 5 mL of chloroform was added and centrifuged. The resulting precipitate was washed with a mixture of DMF and chloroform, and the resulting transparent precipitate was dispersed in pure water. 100 mg of PAA was dispersed in 6 mL of deionized water, and then 10... The aqueous solution of the above ligand-free upconversion nanoparticles was added dropwise to the PAA solution, and then the mixture was stirred for 2 hours to allow the PAA molecules to be fully coated on the particle surface, thus obtaining the PAA-UCNPs sample; then the product was redispersed in water or buffer solution by centrifugation. S3 prepared upconversion nanoparticles modified with desalination cell-penetrating peptides by covalently coupling desalination cell-penetrating peptide molecules to polyacrylic acid (PAA) molecules through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxythiosuccinimide (Sulfo-NHS). Specifically, the desalination cell-penetrating peptide molecules were coupled to the carboxyl groups on the particle surface through EDC and Sulfo-NHS. The process included: first, taking 1 mg of PAA-UCNPs in MES solution, adding 10 mg of EDC in MES solution and 15 mg of Sulfo-NHS in MES solution sequentially, stirring vigorously for 2 h to obtain Sulfo-NHS-coupled PAA-UCNPs; then centrifuging and dispersing the precipitate in 1 mL of HEPES buffer; adding 50 μg of desalination cell-penetrating peptide to the above solution, and reacting with shaking at 4 °C for 3 hours. h, the desalted cell-penetrating peptide is coupled to the carboxyl group of PAA through a displacement reaction, and finally centrifuged to redisperse the precipitate in 1 mL HEPES buffer or cell culture medium.

2. The method for preparing the upconversion fluorescent probe with strong cell penetration and strong solution dispersibility according to claim 1, characterized in that, The size of the upconversion nanoprobe can be precisely controlled by the reaction temperature. Reacting at 250℃, 260℃, and 270℃ can yield well-dispersed nanoparticles with particle sizes of 7 nm, 9 nm, and 11 nm, respectively.

3. An upconversion nanoprobe obtained using the preparation method of the upconversion fluorescent probe with strong cell penetration and strong solution dispersibility as described in claim 1, characterized in that, Desalinated cell-penetrating peptide molecules can be covalently coupled to the carboxyl groups on the surface of upconversion nanoparticles, which can significantly improve the efficiency of upconversion fluorescent nanoprobes entering living cells. They also have stronger dispersibility and stability, and can enter subcellular regions within cells by coupling with different cell-penetrating peptide molecules.

4. The application of the upconversion nanoprobe according to claim 3 in fluorescence detection of the living cell microenvironment.

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