Phosphate-quenching-resistant dye-sensitized rare earth luminescent nanoprobe as well as preparation method and application thereof

Abstract

The invention discloses a phosphate quenching resistant dye-sensitized rare earth luminescent nanoprobe as well as a preparation method and application thereof, and belongs to the technical field of nanomaterial biology. Different from the structure of a conventional probe, an NHS group in a near-infrared dye structure reacts with NH2 on a diphosphonic acid group ligand to form an amido bond for coupling, and a strong coordination group phosphonic acid group of the diphosphonic acid group ligand is firmly anchored on an inorganic nanocrystal doped with rare earth ions; meanwhile, a protective layer is constructed on the surface of the probe through the hydrophobic interaction of a monophosphonic acid ligand and amphiphilic molecules, and the dye-sensitized rare earth luminescent nanoprobe with up-conversion and down-transfer emission characteristics is formed. The dye-sensitized rare earth luminescent nanoprobe has high luminescent stability in an aqueous solution, a PBS solution and an ATP or ADP solution, has efficient near-infrared two-region emission characteristic by introducing a monophosphate ligand to protect the antenna sensitization effect of the dye, and can be applied to in-vivo biological imaging.

Description

Technical Field

[0001] The invention belongs to the field of nanomaterial biotechnology, and specifically relates to a dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching, and a preparation method and application thereof. Background Art

[0002] At the end of the 20th century, scientists began to explore rare earth-doped nanomaterials. Most rare earth ion-doped nanoprobes have narrow absorption and emission spectra. Initially, people focused on their upconversion luminescence properties from longer infrared to shorter wavelengths. However, short-wavelength visible light (400-700nm) is easily absorbed and scattered when passing through biological tissues, so it can only meet the needs of in vitro diagnosis and cellular-level biological imaging. Compared with visible light or ultraviolet light, near-infrared light (NIR, 700-1700nm) can penetrate biological tissues more deeply, making it very suitable for deep in vivo imaging. Therefore, the near-infrared window of down-transferred luminescence of rare earths, as a relatively "transparent" optical window for biological tissues, has deeper tissue penetration and higher imaging signal-to-noise ratio due to the weaker absorption and scattering of biological tissues and lower autofluorescence, especially the near-infrared second-zone fluorescence of 1000nm-1700nm. Among them, Pr 3+ 、Nd 3+ 、Ho 3+ , Er 3+ 、Tm 3+ Because its emission wavelength is in the spectral range of 1000nm-1700nm, it is a commonly used luminescence center in rare earth doped nanoparticles down-transfer luminescence probes. With the advantages of low toxicity, high chemical stability, narrow-band emission and long fluorescence lifetime, rare earth doped nanoparticles have been increasingly used in bioimaging, optical sensing, disease treatment and other fields.

[0003] However, due to the small absorption cross section of rare earth ions and the restriction of parity forbidden transition of 4f-4f energy level, the luminescence efficiency of rare earth doped nanoparticles is low. The development of high brightness luminescence system has always been an important part of the research of rare earth doped nanoparticles. It is also the key to the application of rare earth doped nanoprobes in clinical practice. With the development of nanotechnology, the synthesis process and performance of rare earth near-infrared probes have been further improved. Common rare earth luminescence modulation strategies include dye sensitization, element doping, core-shell structure design, surface modification, etc.

[0004] Among them, dye-sensitized rare earth luminescent nanoprobes play an important role in biomedical research, especially for early diagnosis of diseases. Since rare earth nanocrystals have low toxicity, large anti-Stokes shift, near-infrared light excitation and other material properties, they have outstanding advantages in biological imaging, such as high imaging depth, low autofluorescence characteristics and high signal-to-background ratio. And thanks to the dye-sensitized amplification effect, the up-conversion and down-transfer luminescence of rare earth ions are significantly enhanced, which broadens the application of probes in biological imaging, such as vascular imaging, tumor imaging, etc. With this unique advantage, dye-sensitized rare earth luminescent nanoprobes occupy an important place in the field of biological imaging.

[0005] However, as research has found, in the face of more complex biological environments, dye-sensitized rare earth luminescent nanoprobes synthesized by conventional methods are easily affected and obviously cannot achieve the expected effect. First, when the dye nanocrystal complex formed by direct coordination of the dye with the rare earth ion through the carboxyl or sulfonic acid group is dispersed in PBS, the binding force between the phosphate in PBS and the lanthanide ion is much greater than that of the dye, causing the dye to detach from the rare earth nanocrystal and greatly weakening the dye antenna sensitization effect. In addition, the surface of conventional dye-sensitized rare earth nanocrystals is a hydrophobic protective layer constructed by the hydrophobic interaction of oleic acid ligands and amphiphilic molecules, which is also unstable in biological environments containing phosphate, ATP or ADP and is quenched by water.

[0006] A Chinese patent with publication number CN113845914A and application date September 29, 2021 discloses a method for enhancing dye-sensitized rare earth upconversion luminescence. Cyanine dyes interact with upconversion nanoparticles through sulfonate and carboxylic acid to achieve dye-sensitized upconversion luminescence in ethanol or DMF. The electrostatic shielding effect of positive and negative charges inhibits the aggregation of cyanine dyes on the surface of upconversion nanoparticles, increases the composite ratio of cyanine dyes to nanoparticles, and enhances the intensity of dye-sensitized rare earth upconversion luminescence. However, the probe cannot solve the problem of unstable luminescence caused by easy quenching in phosphate solution. The Chinese patent with publication number CN117229779A and application date of August 9, 2023 discloses a near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe, preparation method and application thereof. The probe modifies the conjugated part of the cyanine dye with a sulfonate or carboxylate coordination group, rather than connecting to the conjugated part of the dye through a long carbon chain structure, thereby shortening the distance between the dye and the rare earth ion, improving the energy transfer efficiency, and achieving efficient upconversion luminescence. However, the probe can only be used in non-aqueous solvents such as cyclohexane, dichloromethane, and dimethyl sulfoxide.

[0007] Therefore, limited by the complex aqueous environment containing phosphate, it is urgent to develop a dye-sensitized rare earth probe with excellent luminescence performance, high luminescence stability and the ability to protect the dye antenna sensitization effect in aqueous solution, PBS solution, and ATP or ADP solution, so as to promote the application of rare earth probes in biological imaging. Summary of the invention

[0008] In order to solve the problems existing in the prior art, the present invention provides a dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching, and a preparation method and application thereof. The strong binding effect between rare earth ions and phosphonic acid ligands is utilized to effectively solve the problem that the previous dye-sensitized rare earth luminescent probe is quenched in phosphate solution, resulting in unstable probe luminescence, and the dye-sensitized rare earth probe is realized. The stable and efficient dye-sensitized antenna effect of the dye-sensitized rare earth probe in aqueous solution and phosphate solution is achieved.

[0009] The technical solution of the present invention is as follows:

[0010] One of the purposes of the present invention is to provide a dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching. The dye-sensitized rare earth luminescent nanoprobe utilizes a near-infrared dye modified with N-hydroxysuccinimide to couple with a bisphosphonic acid ligand, so that the strong coordinating group phosphonic acid group of the bisphosphonic acid ligand is anchored on an inorganic nanocrystal doped with a rare earth ion, and a hydrophobic protective layer is constructed on the probe surface through the interaction between the monophosphonic acid ligand and the amphiphilic molecule.

[0011] Furthermore, the near-infrared dye is any one of IR783-NHS, IR806-NHS, IR820-NHS, ICG-NHS, Sulfo-ICG-NHS, Cy7.5-NHS, Sulfo-Cy7.5-NHS, and IRDye 800CW-NHS.

[0012] Furthermore, the bisphosphonate ligand is alendronic acid ADA.

[0013] Furthermore, the average particle size of the rare earth nanocrystals is 1 to 200 nm.

[0014] Furthermore, the rare earth nanocrystals are inorganic nanocrystals doped with Nd, Yb and Er ions, which can be sensitized by near-infrared dyes to achieve up-conversion and down-transfer luminescence.

[0015] Furthermore, the monophosphonic acid ligand is any one of n-hexylphosphonic acid (HPA), di-n-octylphosphonic acid (DOPA), n-tetradecylphosphonic acid (TDPA), n-hexadecylphosphonic acid (HDPA), and n-octadecylphosphonic acid (ODPA).

[0016] Furthermore, the amphiphilic molecule is any one of EO-PO type polyether (F127), distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-mPEG), distearoyl phosphatidylethanolamine polyethylene glycol amino (DSPE-PEG-NH2), distearoyl phosphatidylethanolamine polyethylene glycol carboxyl (DSPE-PEG-COOH), and polyoxyethylene sorbitan monooleate (Tween-80).

[0017] Furthermore, the dye-sensitized rare earth luminescent nanoprobe has efficient near-infrared second-zone emission characteristics.

[0018] The second object of the present invention is to provide a method for preparing a dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching, comprising the following steps:

[0019] S1. Dissolve rare earth nanocrystals and V in chloroform 水 :V 乙醇 =3:2 alendronic acid solution in a volume ratio of 1:1, hydrochloric acid was added dropwise to adjust the pH to 1-2, stirred at room temperature for 30 minutes, and the precipitate was collected after centrifugation to obtain intermediate product A;

[0020] S2, dispersing the intermediate product A and the dye in a solvent N,N-dimethylformamide DMF solution, adding 1 mol / L 4-dimethylaminopyridine DMAP, and stirring for 6 hours to obtain the intermediate product B;

[0021] S3, dropping the monophosphonic acid ligand into the intermediate product B, mixing and stirring for 1 hour to obtain the intermediate product C;

[0022] S4, dissolving the amphiphilic molecule in a dichloromethane solution, mixing with the intermediate product C and stirring for 6 hours to obtain an intermediate product D;

[0023] S5, rotary evaporating the intermediate product D to remove the solvent dichloromethane, and redispersing it in an aqueous solution after the dichloromethane has evaporated;

[0024] S6. The intermediate product D in the aqueous solution after redispersion in S5 is centrifuged and washed with deionized water to obtain the dye-sensitized rare earth luminescent nanoprobe.

[0025] Furthermore, the mass ratio of the rare earth nanocrystals to the alendronic acid solution in step S1 is (1-30):1000.

[0026] Furthermore, in steps S1 and S3, the mass ratio of the alendronic acid solution to the monophosphonic acid ligand is (1-40):100.

[0027] Furthermore, in step S4, the mass ratio of the amphiphilic molecules to the rare earth nanocrystals is (1-10):100.

[0028] The third object of the present invention is to provide a dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching for use in in vivo biological imaging.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] 1. The present invention is the first to innovate a dye-sensitized rare earth luminescent nanoprobe that is resistant to phosphate quenching. The NHS group in the near-infrared dye structure reacts with the NH2 on the bisphosphonic acid ligand to form a stable sulfonamide bond for coupling, thereby firmly anchoring the probe to the inorganic nanocrystal doped with rare earth ions with the help of the strong coordinating group phosphonic acid group of the bisphosphonic acid ligand. At the same time, a monophosphonic acid ligand with a hydrophobic long chain is introduced on the probe surface to form a stable hydrophobic protective layer through hydrophobic-hydrophobic interaction with the amphiphilic molecules, thereby enhancing the protection of the dye and forming a new dye-sensitized rare earth luminescent nanoprobe with upconversion and down-transfer emission characteristics.

[0031] 2. The dye-sensitized rare earth luminescent nanoprobe described in the present invention has excellent luminescent performance and high luminescent stability in aqueous solution, PBS solution, and ATP or ADP solution. The antenna sensitization effect of the dye is protected by the strong binding effect between rare earth ions and phosphonic acid ligands, and it exhibits anti-quenching effect on phosphate, which effectively solves the problem that the previous dye-sensitized rare earth luminescent probes are easily quenched in phosphate solutions, resulting in unstable luminescence, and realizes the stable and efficient dye-sensitized antenna effect of the dye-sensitized rare earth probes in aqueous solution and phosphate solution.

[0032] 3. The dye-sensitized rare earth luminescent nanoprobe of the present invention has the same dye-sensitized up-conversion and down-transfer luminescence enhancement effect in various phosphate solutions as in aqueous solution, and has the unique advantage of being applied in complex biological environments. It also has efficient near-infrared second-zone emission characteristics, which can effectively improve the brightness and clarity of in vivo fluorescence imaging. The dye-sensitized rare earth luminescent nanoprobe can be used as an important tool for in vivo biological imaging, suitable for wide application in the field of biological imaging technology, and provides a huge reference potential for in vivo multi-target visualization and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the synthetic structure of the dye-sensitized rare earth luminescent nanoprobe prepared in Example 1 of the present invention;

[0034] Figure 2 The sensitized up-conversion emission spectra of the rare earth luminescent nanoprobes prepared in Example 1 of the present invention and Comparative Examples 1-2;

[0035] Figure 3The sensitized up-conversion and down-transfer emission spectra of the rare earth luminescent nanoprobes prepared in Example 1 of the present invention and Comparative Example 1 dispersed in an aqueous solution and a PBS solution;

[0036] Figure 4 The sensitized up-conversion and down-transfer emission spectra of the rare earth luminescent nanoprobes prepared in Comparative Examples 1 and 3 of the present invention dispersed in aqueous solution and PBS solution;

[0037] Figure 5 The bar graph is a retention rate bar graph of the dye-sensitized enhanced up-conversion and down-transfer luminescence of the rare earth luminescent nanoprobes prepared in Example 1 of the present invention and Comparative Example 1 in 0.1 mM and 1.0 mM ATP or ADP solutions;

[0038] Figure 6 The bar graph is a retention rate bar graph of the dye-sensitized enhanced up-conversion and down-transfer luminescence of the rare earth luminescent nanoprobes prepared in Comparative Example 1 and Comparative Example 3 of the present invention in 0.1 mM and 1.0 mM ATP or ADP solutions;

[0039] Figure 7 In vivo vascular imaging of normal mice 30 seconds after intravenous injection using LNP-ODPA-ADA-Cy7.5@F127(1) and LNP-OA@F127(2) probes in the performance test of the present invention, inset: magnified view of the near-infrared second zone abdominal vascular image, scale bar = 10 mm. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with preferred embodiments. The endpoints of the ranges disclosed in the present invention and any values ​​are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be deemed to be specifically disclosed herein.

[0041] The experimental methods in the following examples, unless otherwise specified, are conventional methods and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] Example 1

[0044] This embodiment provides a dye-sensitized rare earth luminescent nanoprobe LNP-ODPA-ADA-Cy7.5@F127 resistant to phosphate quenching, and the preparation method thereof comprises the following steps:

[0045] S1. Dissolve oil-soluble Nd, Yb and Er ion-doped NaGdF4 nanocrystals (30 mg) and 0.375 mg alendronic acid solution (V 水 :V 乙醇 =3:2) in a volume ratio of 1:1, hydrochloric acid was added dropwise to adjust the pH to 2, stirred at room temperature for 30 min, and the precipitate was collected after centrifugation to obtain intermediate product A;

[0046] S2, disperse the intermediate product A and Cy7.5-NHS in a solvent DMF solution, add 1 mol / L DMAP, and stir for 6 h to obtain the intermediate product B;

[0047] S3, dropping 1.0 mg of ODPA into the intermediate product B, mixing and stirring for 1 hour, to obtain an intermediate product C;

[0048] S4, dissolving 120 mg of F127 in a dichloromethane solution, mixing with the intermediate product C and stirring for 6 h to obtain the intermediate product D;

[0049] S5, rotary evaporating the intermediate product D to remove the solvent dichloromethane, and redispersing it in an aqueous solution after the dichloromethane has evaporated;

[0050] S6. After redispersion in S5, the intermediate product D in the aqueous solution is centrifuged and washed with deionized water to obtain the dye-sensitized rare earth luminescent nanoprobe LNP-ODPA-ADA-Cy7.5@F127.

[0051] Example 2

[0052] This embodiment provides a dye-sensitized rare earth luminescent nanoprobe LNP-HPA-ADA-IR820@F127 resistant to phosphate quenching, and the preparation method thereof comprises the following steps:

[0053] S1. Dissolve oil-soluble Nd, Yb and Er ion-doped NaGdF4 nanocrystals (30 mg) and 0.375 mg alendronic acid solution (V 水 :V 乙醇 =3:2) in a volume ratio of 1:1, hydrochloric acid was added dropwise to adjust the pH to 1, stirred at room temperature for 30 min, and the precipitate was collected after centrifugation to obtain intermediate product A;

[0054] S2, disperse the intermediate product A and IR820-NHS in a solvent DMF solution, add 1 mol / L DMAP, and stir for 6 h to obtain the intermediate product B;

[0055] S3, dropping 1.0 mg HPA into the intermediate product B, mixing and stirring for 1 hour, to obtain an intermediate product C;

[0056] S4, dissolving 120 mg of F127 in a dichloromethane solution, mixing with the intermediate product C and stirring for 6 h to obtain the intermediate product D;

[0057] S5, rotary evaporating the intermediate product D to remove the solvent dichloromethane, and redispersing it in an aqueous solution after the dichloromethane has evaporated;

[0058] S6. After redispersion in S5, the intermediate product D in the aqueous solution is centrifuged and washed with deionized water to obtain the dye-sensitized rare earth luminescent nanoprobe LNP-HPA-ADA-IR820@F127.

[0059] Example 3

[0060] This embodiment provides a dye-sensitized rare earth luminescent nanoprobe LNP-DOPA-ADA-IR783@DSPE-mPEG resistant to phosphate quenching, and the preparation method thereof comprises the following steps:

[0061] S1. Dissolve oil-soluble Nd, Yb and Er ion-doped NaGdF4 nanocrystals (30 mg) and 0.375 mg alendronic acid solution (V 水 :V 乙醇 =3:2) in a volume ratio of 1:1, hydrochloric acid was added dropwise to adjust the pH to 2, stirred at room temperature for 30 min, and the precipitate was collected after centrifugation to obtain intermediate product A;

[0062] S2, disperse the intermediate product A and IR783-NHS in a solvent DMF solution, add 1 mol / L DMAP, and stir for 6 h to obtain the intermediate product B;

[0063] S3, dropping 1.0 mg DOPA into the intermediate product B, mixing and stirring for 1 hour, to obtain an intermediate product C;

[0064] S4, dissolving 15 mg of DSPE-mPEG in a dichloromethane solution, mixing with the intermediate product C and stirring for 6 h to obtain an intermediate product D;

[0065] S5, rotary evaporating the intermediate product D to remove the solvent dichloromethane, and redispersing it in an aqueous solution after the dichloromethane has evaporated;

[0066] S6. After redispersion in S5, the intermediate product D in the aqueous solution is centrifuged and washed with deionized water to obtain the dye-sensitized rare earth luminescent nanoprobe LNP-DOPA-ADA-IR783@DSPE-mPEG.

[0067] Example 4

[0068] This embodiment provides a dye-sensitized rare earth luminescent nanoprobe LNP-TDPA-ADA-ICG@DSPE-NHS resistant to phosphate quenching, and the preparation method thereof comprises the following steps:

[0069] S1. Dissolve oil-soluble Nd, Yb and Er ion-doped NaGdF4 nanocrystals (30 mg) and 0.375 mg alendronic acid solution (V 水 :V 乙醇 =3:2) in a volume ratio of 1:1, hydrochloric acid was added dropwise to adjust the pH to 1, stirred at room temperature for 30 min, and the precipitate was collected after centrifugation to obtain intermediate product A;

[0070] S2, disperse the intermediate product A and ICG-NHS in a solvent DMF solution, add 1 mol / L DMAP, and stir for 6 h to obtain the intermediate product B;

[0071] S3, dropping 1.0 mg TDPA into the intermediate product B, mixing and stirring for 1 hour, to obtain an intermediate product C;

[0072] S4, dissolving 15 mg of DSPE-NHS in a dichloromethane solution, mixing with the intermediate product C and stirring for 6 h to obtain an intermediate product D;

[0073] S5, rotary evaporating the intermediate product D to remove the solvent dichloromethane, and redispersing it in an aqueous solution after the dichloromethane has evaporated;

[0074] S6. After redispersion in S5, the intermediate product D in the aqueous solution is centrifuged and washed with deionized water to obtain the dye-sensitized rare earth luminescent nanoprobe LNP-TDPA-ADA-ICG@DSPE-NHS.

[0075] Example 5

[0076] This embodiment provides a dye-sensitized rare earth luminescent nanoprobe LNP-HDPA-ADA-IR806@DSPE-PEG-COOH resistant to phosphate quenching, and the preparation method thereof comprises the following steps:

[0077] S1. Dissolve oil-soluble Nd, Yb and Er ion-doped NaGdF4 nanocrystals (30 mg) and 0.375 mg alendronic acid solution (V 水 :V 乙醇 =3:2) in a volume ratio of 1:1, hydrochloric acid was added dropwise to adjust the pH to 2, stirred at room temperature for 30 min, and the precipitate was collected after centrifugation to obtain intermediate product A;

[0078] S2, disperse the intermediate product A and IR806-NHS in a solvent DMF solution, add 1 mol / L DMAP, and stir for 6 h to obtain the intermediate product B;

[0079] S3, dropping 1.0 mg of HDPA into the intermediate product B, mixing and stirring for 1 hour, to obtain an intermediate product C;

[0080] S4, dissolving 15 mg of DSPE-PEG-COOH in a dichloromethane solution, mixing with the intermediate product C and stirring for 6 h to obtain an intermediate product D;

[0081] S5, rotary evaporating the intermediate product D to remove the solvent dichloromethane, and redispersing it in an aqueous solution after the dichloromethane has evaporated;

[0082] S6. After redispersion in S5, the intermediate product D in the aqueous solution is centrifuged and washed with deionized water to obtain the dye-sensitized rare earth luminescent nanoprobe LNP-HDPA-ADA-IR806@DSPE-PEG-COOH.

[0083] Example 6

[0084] This embodiment provides a dye-sensitized rare earth luminescent nanoprobe LNP-HDPA-ADA-Sulfo-Cy7.5@DSPE-PEG-NH2 resistant to phosphate quenching, and the preparation method thereof comprises the following steps:

[0085] S1. Dissolve oil-soluble Nd, Yb and Er ion-doped NaGdF4 nanocrystals (30 mg) and 0.375 mg alendronic acid solution (V 水 :V 乙醇 =3:2) in a volume ratio of 1:1, hydrochloric acid was added dropwise to adjust the pH to 1, stirred at room temperature for 30 min, and the precipitate was collected after centrifugation to obtain intermediate product A;

[0086] S2, disperse the intermediate product A and Sulfo-Cy7.5-NHS in a solvent DMF solution, add 1 mol / L DMAP, and stir for 6 h to obtain the intermediate product B;

[0087] S3, dropping 1.0 mg of HDPA into the intermediate product B, mixing and stirring for 1 hour, to obtain an intermediate product C;

[0088] S4, dissolving 15 mg of DSPE-PEG-NH2 in a dichloromethane solution, mixing with the intermediate product C and stirring for 6 h to obtain an intermediate product D;

[0089] S5, rotary evaporating the intermediate product D to remove the solvent dichloromethane, and redispersing it in an aqueous solution after the dichloromethane has evaporated;

[0090] S6. After redispersion in S5, the intermediate product D in the aqueous solution is centrifuged and washed with deionized water to obtain the dye-sensitized rare earth luminescent nanoprobe LNP-HDPA-ADA-Sulfo-Cy7.5@DSPE-PEG-NH2.

[0091] Example 7

[0092] This embodiment provides a method for preparing a dye-sensitized rare earth luminescent nanoprobe LNP-DOPA-ADA-IRDye 800CW@Tween-80 that is resistant to phosphate quenching, comprising the following steps:

[0093] S1. Dissolve oil-soluble Nd, Yb and Er ion-doped NaGdF4 nanocrystals (30 mg) and 0.375 mg alendronic acid solution (V 水 :V 乙醇 =3:2) in a volume ratio of 1:1, hydrochloric acid was added dropwise to adjust the pH to 2, stirred at room temperature for 30 min, and the precipitate was collected after centrifugation to obtain intermediate product A;

[0094] S2, disperse the intermediate product A and IRDye 800CW-NHS in a solvent DMF solution, add 1 mol / L DMAP, and stir for 6 h to obtain the intermediate product B;

[0095] S3, dropping 1.0 mg DOPA into the intermediate product B, mixing and stirring for 1 hour, to obtain an intermediate product C;

[0096] S4, dissolving 50 mg of Tween-80 in a dichloromethane solution, mixing with the intermediate product C and stirring for 6 h to obtain an intermediate product D;

[0097] S5, rotary evaporating the intermediate product D to remove the solvent dichloromethane, and redispersing it in an aqueous solution after the dichloromethane has evaporated;

[0098] S6, centrifuge the intermediate product D in the aqueous solution after redispersion in S5, wash it with deionized water to obtain the dye-sensitized rare earth luminescent nanoprobe LNP-DOPA-ADA-IRDye

[0099] 800CW@Tween-80.

[0100] Comparative Example 1

[0101] This comparative example provides a dye-free sensitized rare earth luminescent nanoprobe LNP-OA@F127, the preparation method of which comprises the following steps:

[0102] S1. Dissolve 30 mg of oil-soluble rare earth nanocrystals in dichloromethane and mix with a chloroform solution containing 120 mg of F127, and stir for 6 h to obtain product 1.

[0103] S2, the product is subjected to rotary evaporation to remove the solvent dichloromethane, and after the dichloromethane is completely evaporated, it is redispersed in an aqueous solution;

[0104] S3. After step S2 is completed, centrifuge and wash with deionized water to obtain the probe LNP-OA@F127.

[0105] Comparative Example 2

[0106] This comparative example provides a rare earth luminescent nanoprobe lacking a monophosphonic acid ligand and an amphiphilic molecule to construct a hydrophobic protective layer on the surface. The experimental reagents and conditions used in this comparative example are exactly the same as those in Example 1, except that step S3 is not performed to obtain the probe LNP-ADA-Cy7.5@F127.

[0107] Comparative Example 3

[0108] This comparative example provides a rare earth luminescent nanoprobe LNP-OA-IR808@F127 having an oleic acid ligand and an amphiphilic molecule on the surface to construct a hydrophobic protective layer, and the preparation method thereof comprises the following steps:

[0109] S1, dispersing 30 mg of oil-soluble rare earth nanocrystals and IR808 in solvent dichloromethane, dispersing and stirring for 1 h to obtain intermediate product A;

[0110] S2, mixing the intermediate product A with a dichloromethane solution containing 120 mg of F127, and ultrasonicating for 5 min to obtain the intermediate product B;

[0111] S3, rotary evaporating the intermediate product B to remove the solvent dichloromethane, and after the dichloromethane is completely evaporated, redispersing it in an aqueous solution;

[0112] S4. After redispersion in S3, the intermediate product B in the aqueous solution was centrifuged and washed twice with deionized water to obtain the probe LNP-OA-IR808@F127.

[0113] Performance Testing

[0114] 1. Luminescence performance test in aqueous solution

[0115] The probe LNP-ODPA-ADA-Cy7.5@F127 prepared in Example 1, the probe LNP-OA@F127 prepared in Comparative Example 1, and the probe LNP-ADA-Cy7.5@F127 prepared in Comparative Example 2 were dispersed in an aqueous solution, and the visible up-conversion emission spectra of the samples under 808 nm near-infrared laser excitation were monitored using an FLS980 spectrometer.

[0116] The test results are as follows Figure 2As shown, compared with the probe LNP-OA@F127 of comparative example 1, the dye-sensitized upconversion of the probe LNP-ODPA-ADA-Cy7.5@F127 of example 1 in aqueous solution is significantly enhanced, and the sensitization enhancement of the probe LNP-ADA-Cy7.5@F127 of comparative example 2 is negligible. The results show that the dye-sensitized rare earth luminescent nanoprobe of the present invention has excellent luminescence performance in aqueous solution, and the antenna sensitization effect of the dye is protected by introducing the monophosphate ligand ODPA.

[0117] 2. Phosphate quenching resistance test

[0118] The probe LNP-ODPA-ADA-Cy7.5@F127 prepared in Example 1, the probe LNP-OA@F127 prepared in Comparative Example 1, and the probe LNP-OA-IR-808@F127 prepared in Comparative Example 3 were dispersed in aqueous solution and PBS solution, respectively, and the visible up-conversion and near-infrared second-region down-shift emission spectra of the samples under 808nm near-infrared laser excitation were monitored by FLS 980 spectrometer.

[0119] The test results are as follows Figure 3 As shown in Figure 4, compared with the probe LNP-OA@F127 of Comparative Example 1, the sensitized upconversion and downtransfer enhancements of the probe LNP-ODPA-ADA-Cy7.5@F127 of Example 1 in water and PBS solutions are almost the same, while the sensitized upconversion and downtransfer enhancements of the probe LNP-OA-IR-808@F127 of Comparative Example 3 in PBS solution are weak.

[0120] At the same time, if Figure 5 As shown in Figure 6, the probe LNP-ODPA-ADA-Cy7.5@F127 of Example 1 can maintain more than 86% of the sensitized up-conversion and down-transfer luminescence enhancement in the aqueous solution in the solution environment of ATP and ADP, and the quenching effect of the probe LNP-OA-IR-808@F127 of Example 3 in the ATP or ADP solution is significant, and the minimum is reduced to 13.2%. The results show that the dye-sensitized rare earth luminescent nanoprobe of the present invention has excellent luminescence performance, high luminescence stability in aqueous solution, PBS solution and ATP or ADP solution, and has unique advantages in application in complex biological environments.

[0121] 3. Biological imaging application test

[0122] The probe LNP-ODPA-ADA-Cy7.5@F127 prepared in Example 1 and the probe LNP-OA@F127 prepared in Comparative Example 1 were dispersed in an aqueous solution, and vascular imaging photos of the samples in mice were taken under 808 nm laser excitation using a small animal imager.

[0123] The test results are as follows Figure 7 As shown, the mouse vascular imaging of the probe LNP-ODPA-ADA-Cy7.5@F127 in Example 1 is more obvious and brighter. The results show that the near-infrared second region emission of the probe LNP-ODPA-ADA-Cy7.5@F127 is stronger, and the sensitization of Cy7.5 in vivo can significantly enhance the near-infrared second region emission characteristics of LNP-OA@F127.

[0124] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching, characterized in that: The dye-sensitized rare earth luminescent nanoprobe utilizes a near-infrared dye modified with N-hydroxysuccinimide to couple with a bisphosphonic acid ligand, so that the strong coordination group phosphonic acid group of the bisphosphonic acid ligand is anchored on the inorganic nanocrystal doped with rare earth ions, and at the same time, a hydrophobic protective layer is constructed on the probe surface through the interaction between the monophosphonic acid ligand and the amphiphilic molecule.

2. The dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching according to claim 1, characterized in that: The near-infrared dye is any one of IR783-NHS, IR806-NHS, IR820-NHS, ICG-NHS, Sulfo-ICG-NHS, Cy7.5-NHS, Sulfo-Cy7.5-NHS, and IRDye 800CW-NHS.

3. The dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching according to claim 1, characterized in that: The bisphosphonic acid ligand is alendronic acid ADA.

4. The dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching according to claim 1, characterized in that: The rare earth nanocrystal is an inorganic nanocrystal doped with Nd, Yb and Er ions, can be sensitized by near-infrared dyes to realize up-conversion and down-transfer luminescence, and has an average particle size of 1 to 200 nm.

5. The dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching according to claim 1, characterized in that: The monophosphonic acid ligand is any one of n-hexylphosphonic acid, di-n-octylphosphonic acid, n-tetradecylphosphonic acid, n-hexadecylphosphonic acid and n-octadecylphosphonic acid.

6. The dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching according to claim 1, characterized in that: The amphiphilic molecule is any one of EO-PO type polyether, distearoyl phosphatidylethanolamine-polyethylene glycol, distearoyl phosphatidylethanolamine polyethylene glycol amino group, distearoyl phosphatidylethanolamine polyethylene glycol carboxyl group, and polyoxyethylene sorbitan monooleate.

7. A method for preparing a dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1. Dissolve rare earth nanocrystals and V in chloroform 水 :V 乙醇 =3:2 alendronic acid solution in a volume ratio of 1:1, hydrochloric acid was added dropwise to adjust the pH to 1-2, stirred at room temperature for 30 minutes, and the precipitate was collected after centrifugation to obtain intermediate product A; S2, dispersing the intermediate product A and the dye in a solvent N,N-dimethylformamide DMF solution, adding 1 mol / L 4-dimethylaminopyridine DMAP, and stirring for 6 hours to obtain the intermediate product B; S3, dropping the monophosphonic acid ligand into the intermediate product B, mixing and stirring for 1 hour to obtain the intermediate product C; S4, dissolving the amphiphilic molecule in a dichloromethane solution, mixing with the intermediate product C and stirring for 6 hours to obtain an intermediate product D; S5, rotary evaporating the intermediate product D to remove the solvent dichloromethane, and redispersing it in an aqueous solution after the dichloromethane has evaporated; S6. The intermediate product D in the aqueous solution after redispersion in S5 is centrifuged and washed with deionized water to obtain the dye-sensitized rare earth luminescent nanoprobe.

8. The method for preparing a dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching according to claim 7, characterized in that: The mass ratio of the rare earth nanocrystals to the alendronic acid solution in step S1 is (1-30):1000; the mass ratio of the alendronic acid solution to the monophosphonic acid ligand in steps S1 and S3 is (1-40):

100.

9. The method for preparing a dye-sensitized rare earth luminescent nanoprobe resistant to phosphate quenching according to claim 7, characterized in that: The mass ratio of the amphiphilic molecules to the rare earth nanocrystals in step S4 is (1-10):

100.

10. Use of the phosphate-quenching-resistant dye-sensitized rare earth luminescent nanoprobe according to any one of claims 1 to 6 in in vivo bioimaging.

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