A dye-sensitized rare-earth luminescent nanoprobe resistant to phosphate quenching, its preparation method and application

Abstract

The application discloses a dye-sensitized rare earth luminescent nano probe against phosphate quenching and a preparation method and application thereof, and belongs to the technical field of nanometer material biology; different from the structure of the previous probe, the application utilizes the reaction of the NHS group in the near-infrared dye structure and the NH2 on the bisphosphonic acid group ligand to form an amide bond for coupling, and with the help of the strong coordination group phosphonic acid group of the bisphosphonic acid group ligand, the inorganic nanocrystal doped with rare earth ions is stably anchored, meanwhile, a protective layer is constructed on the probe surface through the hydrophobic interaction of the monophosphonic acid group ligand and the amphiphilic molecule, and the dye-sensitized rare earth luminescent nano probe with up-conversion and down-conversion emission characteristics is formed; the dye-sensitized rare earth luminescent nano probe has high luminescence stability in aqueous solution, PBS solution and ATP or ADP solution, and through the introduction of the monophosphonic acid ligand, the antenna sensitization effect of the dye is protected, the dye has high near-infrared two-region emission characteristics, and can be applied to in-vivo biological imaging.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials biotechnology, specifically relating to a dye-sensitized rare-earth luminescent nanoprobe resistant to phosphate quenching, its preparation method, and its application. Background Technology

[0002] At the end of the 20th century, scientists began exploring rare-earth-doped nanomaterials. Most rare-earth ion-doped nanoprobes possess narrow absorption and emission spectra, and initially, attention was focused on their upconversion luminescence properties from longer infrared wavelengths to shorter wavelengths. However, short-wavelength visible light (400–700 nm) is easily absorbed and scattered when passing through biological tissues, thus only suitable for in vitro diagnostics and cellular-level bioimaging. Compared to visible or ultraviolet light, near-infrared light (NIR, 700–1700 nm) can penetrate biological tissues more deeply, making it ideal for deep-level in vivo imaging. Therefore, the near-infrared window of rare-earth downconversion luminescence, as a relatively "transparent" optical window to biological tissues, possesses deeper tissue penetration and a higher imaging signal-to-noise ratio due to the weaker absorption and scattering of biological tissues and lower autofluorescence, especially in the 1000 nm–1700 nm NIR II region. Among these, 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 luminescent center in rare earth-doped nanoparticle-transferred luminescent 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 fields such as bioimaging, optical sensing, and disease treatment.

[0003] However, due to the small absorption cross-section of rare-earth ions and the limitation of parity-forbidden transitions in the 4f-4f energy level, the luminescence efficiency of rare-earth-doped nanoparticles is relatively low. Developing high-brightness luminescence systems has always been an important aspect of rare-earth-doped nanoparticle research and is also key to applying rare-earth-doped nanoprobes clinically. With the development of nanotechnology, the synthesis process and performance of rare-earth near-infrared probes have been further improved. Commonly used rare-earth luminescence modulation strategies include dye sensitization, elemental doping, core-shell structure design, and surface modification.

[0004] Dye-sensitized rare-earth luminescent nanoprobes play a crucial role in biomedical research, particularly in the early diagnosis of diseases. Due to the inherent material properties of rare-earth nanocrystals, such as low toxicity, large anti-Stokes shift, and near-infrared excitation, they offer significant advantages in bioimaging, including high imaging depth, low autofluorescence, and a high signal-to-background ratio. Furthermore, thanks to the dye-sensitization amplification effect, the upconversion and downconversion luminescence of rare-earth ions are significantly enhanced, broadening the application of probes in bioimaging, such as vascular imaging and tumor imaging. With this unique advantage, dye-sensitized rare-earth luminescent nanoprobes occupy an important place in the field of bioimaging.

[0005] However, research has revealed that dye-sensitized rare-earth luminescent nanoprobes synthesized using conventional methods are susceptible to quenching in more complex biological environments, failing to achieve the expected results. Firstly, when dye nanocrystal complexes formed by direct coordination of dyes with rare-earth ions via carboxyl or sulfonic acid groups are dispersed in PBS, the binding force between phosphates and lanthanide ions in PBS is much stronger than that of the dye, causing the dye to detach from the rare-earth nanocrystals and significantly weakening the dye antenna sensitization effect. Secondly, the surface of conventionally dye-sensitized rare-earth nanocrystals, constructed with a hydrophobic protective layer formed by the hydrophobic interactions of oleic acid ligands and amphiphilic molecules, is unstable and subject to water quenching in biological environments containing phosphate, ATP, or ADP.

[0006] Chinese patent CN113845914A, filed on September 29, 2021, discloses a method for enhancing dye-sensitized rare-earth upconversion luminescence. Cyanide dyes interact with upconversion nanoparticles via sulfonate and carboxylic acid groups, achieving dye-sensitized upconversion luminescence in ethanol or DMF. Through electrostatic shielding of positive and negative charges, the aggregation of cyanide dyes on the surface of the upconversion nanoparticles is suppressed, increasing the composite ratio of cyanide dyes to nanoparticles and enhancing the intensity of dye-sensitized rare-earth upconversion luminescence. However, this probe cannot solve the problem of quenching in phosphate solutions, leading to unstable luminescence. Chinese patent CN117229779A, filed on August 9, 2023, discloses a near-infrared cyanine dye-sensitized rare-earth upconversion luminescent nanoprobe, its preparation method, and its application. This probe modifies the conjugated portion of the cyanine dye with sulfonate or carboxylate coordination groups, rather than being linked to the conjugated portion of the dye via a long carbon chain. This shortens the distance between the dye and rare-earth ions, improving energy transfer efficiency and achieving highly efficient upconversion luminescence. However, this probe can only be used in non-aqueous solvents such as cyclohexane, dichloromethane, and dimethyl sulfoxide.

[0007] Therefore, due to the limitations of complex aqueous environments containing phosphates, there is an urgent need to develop a dye-sensitized rare earth probe that exhibits excellent luminescence performance, high luminescence stability, and can protect the dye antenna sensitization effect in aqueous solutions, PBS solutions, and ATP or ADP solutions, so as to promote the application of rare earth probes in bioimaging. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a dye-sensitized rare-earth luminescent nanoprobe resistant to phosphate quenching, its preparation method, and its application. By utilizing the strong binding effect between rare-earth ions and phosphonic acid ligands, it effectively solves the problem of unstable luminescence caused by the quenching of previous dye-sensitized rare-earth luminescent probes in phosphate solutions, and realizes a stable and efficient dye-sensitized antenna effect of dye-sensitized rare-earth probes in aqueous and phosphate solutions.

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

[0010] One objective of this 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 bisphosphonate ligand, thereby anchoring the strong coordinating phosphonate group of the bisphosphonate ligand onto an inorganic nanocrystal doped with rare-earth ions. Simultaneously, a hydrophobic protective layer is constructed on the probe surface through the interaction between the monophosphonate ligand and amphiphilic molecules.

[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 alendronate (ADA).

[0013] Furthermore, the average particle size of the rare earth nanocrystals is 1–200 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 upconversion and downconversion 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] Further, the amphiphilic molecule is any one of EO-PO type polyether (F127), distearate phosphatidylethanolamine-polyethylene glycol (DSPE-mPEG), distearate phosphatidylethanolamine-polyethylene glycol amino (DSPE-PEG-NH2), distearate phosphatidylethanolamine-polyethylene glycol carboxyl (DSPE-PEG-COOH), and polyoxyethylene sorbitan monooleate (Tween-80).

[0017] Furthermore, the dye-sensitized rare-earth luminescent nanoprobe exhibits highly efficient near-infrared II emission characteristics.

[0018] The second objective of this 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, rare earth nanocrystals dissolved in chloroform and V 水 :V 乙醇 A 3:2 alendronate solution was mixed at a volume ratio of 1:1, hydrochloric acid was added dropwise to adjust the pH to 1-2, stirred at room temperature for 30 min, and the precipitate was collected after centrifugation to obtain intermediate product A.

[0020] S2. Disperse intermediate product A and dye in N,N-dimethylformamide DMF solution, add 1 mol / L 4-dimethylaminopyridine DMAP, stir for 6 h to obtain intermediate product B;

[0021] S3. Add the monophosphonic acid ligand dropwise into intermediate product B and mix and stir for 1 hour to obtain intermediate product C.

[0022] S4. Dissolve the amphiphilic molecule in dichloromethane solution, mix it with intermediate product C and stir for 6 hours to obtain intermediate product D;

[0023] S5. Rotary evaporation of intermediate product D removes the solvent dichloromethane. After the dichloromethane has completely evaporated, it is redispersed in an aqueous solution.

[0024] S6. The intermediate product D, which was redispersed in aqueous solution after centrifugation in S5, was washed with deionized water to obtain the dye-sensitized rare earth luminescent nanoprobe.

[0025] Further, the mass ratio of the rare earth nanocrystals and alendronate solution in step S1 is (1-30):1000.

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

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

[0028] The third objective of this invention is to provide an application of a dye-sensitized rare-earth luminescent nanoprobe resistant to phosphate quenching in in vivo bioimaging.

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

[0030] 1. This invention is the first to innovate a dye-sensitized rare-earth luminescent nanoprobe resistant to phosphate quenching. It utilizes the reaction between the NHS group in the near-infrared dye structure and the NH2 on the bisphosphonate ligand to form a stable sulfonamide bond for coupling. Thus, the strong coordinating phosphonate group of the bisphosphonate ligand is used to firmly anchor the inorganic nanocrystals doped with rare-earth ions. At the same time, a monophosphonate ligand with a hydrophobic long chain is introduced on the probe surface, which interacts with the amphiphilic molecule to form a stable hydrophobic protective layer, enhancing the protection of the dye. This results in a novel dye-sensitized rare-earth luminescent nanoprobe with both upconversion and downconversion emission characteristics.

[0031] 2. The dye-sensitized rare-earth luminescent nanoprobe described in this invention exhibits excellent luminescence performance and high luminescence stability in aqueous solutions, PBS solutions, and ATP or ADP solutions. Furthermore, the strong binding interaction between rare-earth ions and phosphonic acid ligands protects the antenna sensitization effect of the dye, demonstrating an anti-quenching effect against phosphate. This effectively solves the problem of unstable luminescence caused by easy quenching of previous dye-sensitized rare-earth luminescent probes in phosphate solutions, achieving a stable and efficient dye-sensitized antenna effect in aqueous solutions and phosphate solutions.

[0032] 3. The dye-sensitized rare-earth luminescent nanoprobes described in this invention exhibit the same enhancement effects of dye-sensitized upconversion and downconversion luminescence in various phosphate solutions as in aqueous solutions, possessing unique advantages for application in complex biological environments. They also possess highly efficient near-infrared II emission characteristics, effectively improving the brightness and clarity of in vivo fluorescence imaging. These dye-sensitized rare-earth luminescent nanoprobes can serve as important tools for in vivo bioimaging, suitable for widespread application in the field of bioimaging technology, and provide significant reference potential for in vivo multi-target visualization and detection. Attached Figure Description

[0033] Figure 1 This is a 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 images show the sensitized upconversion emission spectra of the rare-earth luminescent nanoprobes prepared in Example 1 and Comparative Examples 1-2 of this invention.

[0035] Figure 3The images show the sensitization upconversion and downconversion emission spectra of the rare earth luminescent nanoprobes prepared in Example 1 and Comparative Example 1 of this invention dispersed in aqueous solution and PBS solution.

[0036] Figure 4 The images show the sensitization upconversion and downconversion emission spectra of the rare earth luminescent nanoprobes prepared in Comparative Examples 1 and 3 of this invention dispersed in aqueous solution and PBS solution.

[0037] Figure 5 The bar chart shows the retention rates of rare earth luminescent nanoprobes prepared in Example 1 and Comparative Example 1 of this invention in 0.1 mM and 1.0 mM ATP or ADP solutions, representing the enhanced upconversion and downconversion luminescence under dye sensitization.

[0038] Figure 6 The bar chart shows the retention rates of rare earth luminescent nanoprobes prepared in Comparative Examples 1 and 3 of this invention in 0.1 mM and 1.0 mM ATP or ADP solutions, representing the enhanced upconversion and downconversion luminescence under dye sensitization.

[0039] Figure 7 For the performance testing of this invention, LNP-ODPA-ADA-Cy7.5@F127(1) and LNP-OA@F127(2) probes were used to perform in vivo vascular imaging on normal mice 30 seconds after intravenous injection. Inset: Enlarged view of abdominal vascular image in near-infrared region II, scale bar = 10 mm. Detailed Implementation

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

[0041] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] Example 1

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

[0045] S1. 30 mg of oil-soluble NaGdF4 nanocrystals doped with Nd, Yb, and Er ions dissolved in chloroform and 0.375 mg of alendronic acid solution (V) 水 :V 乙醇 =3:2) Mix in a volume ratio of 1:1, add hydrochloric acid to adjust the pH to 2, stir at room temperature for 30 min, centrifuge and take the precipitate to obtain intermediate product A;

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

[0047] S3. Add 1.0 mg ODPA dropwise to intermediate product B, mix and stir for 1 h to obtain intermediate product C;

[0048] S4. Dissolve 120 mg of F127 in dichloromethane solution, mix with intermediate product C and stir for 6 h to obtain intermediate product D;

[0049] S5. Rotary evaporate the intermediate product D to remove the solvent dichloromethane. After the dichloromethane has completely evaporated, redisperse it in the aqueous solution.

[0050] S6. The intermediate product D, which was redispersed in the aqueous solution after centrifugation in S5, was 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, which is prepared by the following steps:

[0053] S1. 30 mg of oil-soluble NaGdF4 nanocrystals doped with Nd, Yb, and Er ions dissolved in chloroform and 0.375 mg of alendronic acid solution (V) 水 :V 乙醇 =3:2) Mix in a volume ratio of 1:1, add hydrochloric acid to adjust the pH to 1, stir at room temperature for 30 min, centrifuge and take the precipitate to obtain intermediate product A;

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

[0055] S3. Add 1.0 mg HPA dropwise to intermediate product B, mix and stir for 1 h to obtain intermediate product C;

[0056] S4. Dissolve 120 mg of F127 in dichloromethane solution, mix with intermediate product C and stir for 6 h to obtain intermediate product D;

[0057] S5. Rotary evaporate the intermediate product D to remove the solvent dichloromethane. After the dichloromethane has completely evaporated, redisperse it in the aqueous solution.

[0058] S6. The intermediate product D, which was redispersed in aqueous solution after centrifugation in S5, was 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, which is prepared by the following steps:

[0061] S1. 30 mg of oil-soluble NaGdF4 nanocrystals doped with Nd, Yb, and Er ions dissolved in chloroform and 0.375 mg of alendronic acid solution (V) 水 :V 乙醇 =3:2) Mix in a volume ratio of 1:1, add hydrochloric acid to adjust the pH to 2, stir at room temperature for 30 min, centrifuge and take the precipitate to obtain intermediate product A;

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

[0063] S3. Add 1.0 mg DOPA dropwise to intermediate product B, mix and stir for 1 h to obtain intermediate product C;

[0064] S4. Dissolve 15 mg of DSPE-mPEG in dichloromethane solution, mix with intermediate product C and stir for 6 h to obtain intermediate product D;

[0065] S5. Rotary evaporate the intermediate product D to remove the solvent dichloromethane. After the dichloromethane has completely evaporated, redisperse it in the aqueous solution.

[0066] S6. The intermediate product D, which was redispersed in the aqueous solution after centrifugation in S5, was 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, the preparation method of which includes the following steps:

[0069] S1. 30 mg of oil-soluble NaGdF4 nanocrystals doped with Nd, Yb, and Er ions dissolved in chloroform and 0.375 mg of alendronic acid solution (V) 水 :V 乙醇 =3:2) Mix in a volume ratio of 1:1, add hydrochloric acid to adjust the pH to 1, stir at room temperature for 30 min, centrifuge and take the precipitate to obtain intermediate product A;

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

[0071] S3. Add 1.0 mg TDPA dropwise to intermediate product B, mix and stir for 1 h to obtain intermediate product C;

[0072] S4. Dissolve 15 mg DSPE-NHS in dichloromethane solution, mix with intermediate product C and stir for 6 h to obtain intermediate product D;

[0073] S5. Rotary evaporate the intermediate product D to remove the solvent dichloromethane. After the dichloromethane has completely evaporated, redisperse it in the aqueous solution.

[0074] S6. The intermediate product D, which was redispersed in the aqueous solution after centrifugation in S5, was 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, which is prepared by the following steps:

[0077] S1. 30 mg of oil-soluble NaGdF4 nanocrystals doped with Nd, Yb, and Er ions dissolved in chloroform and 0.375 mg of alendronic acid solution (V) 水 :V 乙醇 =3:2) Mix in a volume ratio of 1:1, add hydrochloric acid to adjust the pH to 2, stir at room temperature for 30 min, centrifuge and take the precipitate to obtain intermediate product A;

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

[0079] S3. Add 1.0 mg HDPA dropwise to intermediate product B, mix and stir for 1 hour to obtain intermediate product C;

[0080] S4. Dissolve 15 mg of DSPE-PEG-COOH in dichloromethane solution, mix with intermediate product C and stir for 6 h to obtain intermediate product D;

[0081] S5. Rotary evaporate the intermediate product D to remove the solvent dichloromethane. After the dichloromethane has completely evaporated, redisperse it in the aqueous solution.

[0082] S6. The intermediate product D, which was redispersed in aqueous solution after centrifugation in S5, was 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, the preparation method of which includes the following steps:

[0085] S1. 30 mg of oil-soluble NaGdF4 nanocrystals doped with Nd, Yb, and Er ions dissolved in chloroform and 0.375 mg of alendronic acid solution (V) 水 :V 乙醇 =3:2) Mix in a volume ratio of 1:1, add hydrochloric acid to adjust the pH to 1, stir at room temperature for 30 min, centrifuge and take the precipitate to obtain intermediate product A;

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

[0087] S3. Add 1.0 mg HDPA dropwise to intermediate product B, mix and stir for 1 hour to obtain intermediate product C;

[0088] S4. Dissolve 15 mg of DSPE-PEG-NH2 in dichloromethane solution, mix with intermediate product C and stir for 6 h to obtain intermediate product D;

[0089] S5. Rotary evaporate the intermediate product D to remove the solvent dichloromethane. After the dichloromethane has completely evaporated, redisperse it in the aqueous solution.

[0090] S6. The intermediate product D, which was redispersed in the aqueous solution after centrifugation in S5, was 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 phosphate-quench-resistant dye-sensitized rare-earth luminescent nanoprobe LNP-DOPA-ADA-IRDye 800CW@Tween-80, comprising the following steps:

[0093] S1. 30 mg of oil-soluble NaGdF4 nanocrystals doped with Nd, Yb, and Er ions dissolved in chloroform and 0.375 mg of alendronic acid solution (V) 水 :V 乙醇 =3:2) Mix in a volume ratio of 1:1, add hydrochloric acid to adjust the pH to 2, stir at room temperature for 30 min, centrifuge and take the precipitate to obtain intermediate product A;

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

[0095] S3. Add 1.0 mg DOPA dropwise to intermediate product B, mix and stir for 1 h to obtain intermediate product C;

[0096] S4. Dissolve 50 mg of Tween-80 in dichloromethane solution, mix with intermediate product C and stir for 6 h to obtain intermediate product D;

[0097] S5. Rotary evaporate the intermediate product D to remove the solvent dichloromethane. After the dichloromethane has completely evaporated, redisperse it in the aqueous solution.

[0098] S6. The intermediate product D, redispersed in aqueous solution after centrifugation in S5, is washed 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 rare-earth luminescent nanoprobe LNP-OA@F127, which is prepared by 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. Stir for 6 h to obtain product one.

[0103] S2. The product is removed from the solvent dichloromethane by rotary evaporation. After the dichloromethane has 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 surface-mounted monophosphonic acid ligand and an amphiphilic molecule forming a hydrophobic protective layer. The experimental reagents and conditions used in this comparative example are exactly the same as in Example 1, except that step S3 is omitted, resulting in 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 with a hydrophobic protective layer constructed from oleic acid ligands and amphiphilic molecules on its surface. The preparation method includes the following steps:

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

[0110] S2. Mix intermediate product A with a dichloromethane solution containing 120 mg F127 and sonicate for 5 min to obtain intermediate product B.

[0111] S3. Rotary evaporation of intermediate product B removes the solvent dichloromethane. After the dichloromethane has completely evaporated, it is redispersed in an aqueous solution.

[0112] S4. The intermediate product B, which was redispersed in the aqueous solution after centrifugation in S3, was 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 upconversion emission spectrum of the samples under 808nm near-infrared laser excitation was monitored using an FLS980 spectrometer.

[0116] Test results are as follows Figure 2As shown, compared to the probe LNP-OA@F127 in Comparative Example 1, the probe LNP-ODPA-ADA-Cy7.5@F127 in Example 1 exhibits significantly enhanced dye-sensitized upconversion in aqueous solution, while the enhancement in sensitization of the probe LNP-ADA-Cy7.5@F127 in Comparative Example 2 is negligible. The results indicate that the dye-sensitized rare-earth luminescent nanoprobe described in this invention exhibits 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. The visible upconversion and near-infrared II downconversion emission spectra of the samples under 808nm near-infrared laser excitation were monitored using an FLS 980 spectrometer.

[0119] Test results are as follows Figure 3 As shown in Figure 4, compared to the probe LNP-OA@F127 in Comparative Example 1, the sensitization upconversion and downconversion enhancement of the probe LNP-ODPA-ADA-Cy7.5@F127 in Example 1 were almost identical, while the sensitization upconversion and downconversion enhancement of the probe LNP-OA-IR-808@F127 in Comparative Example 3 were weak.

[0120] At the same time, such as Figure 5 As shown in Figure 6, the probe LNP-ODPA-ADA-Cy7.5@F127 of Example 1 maintained over 86% sensitization upconversion and downconversion luminescence enhancement in aqueous solutions of ATP and ADP. In contrast, the probe LNP-OA-IR-808@F127 of Example 3 exhibited a significantly greater quenching effect in ATP or ADP solutions, with a minimum quenching rate of 13.2%. These results demonstrate that the dye-sensitized rare-earth luminescent nanoprobes of this invention possess excellent luminescence performance and high luminescence stability in aqueous solutions, PBS solutions, and ATP or ADP solutions, exhibiting unique advantages for application in complex biological environments.

[0121] 3. Bioimaging Application Testing

[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 images of the samples in mice were taken using a small animal imaging system under 808nm laser excitation.

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

[0124] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A dye-sensitized rare earth luminescent nanoprobes against quenching by phosphate, characterized in that, The dye-sensitized rare earth luminescent nanoprobes are coupled with near-infrared dyes modified by N-hydroxysuccinimide and bisphosphonic ligands, so that the strong coordination group phosphonic acid group of the bisphosphonic ligand is anchored on the inorganic nanocrystals doped with rare earth ions, and a hydrophobic protective layer is constructed on the surface of the probe through the interaction of the monophosphonic ligand and the amphiphilic molecule; 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; The bisphosphonic ligand is alendronate ADA; The rare earth nanocrystal is an inorganic nanocrystal doped with Nd, Yb and Er ions, which can be sensitized by a near-infrared dye to realize up-conversion and down-conversion luminescence, and has an average particle size of 1-200 nm; The monophosphonic ligand is any one of n-hexyl phosphonic acid, di-n-octyl phosphonic acid, n-tetradecyl phosphonic acid, n-hexadecyl phosphonic acid, and n-octadecyl phosphonic acid; The amphiphilic molecule is any one of EO-PO type polyether, distearoyl phosphatidyl ethanolamine-polyethylene glycol, distearoyl phosphatidyl ethanolamine polyethylene glycol amino, distearoyl phosphatidyl ethanolamine polyethylene glycol carboxyl, and polyoxyethylene sorbitan monooleate.

2. A method for preparing the dye-sensitized rare earth luminescent nanoprobes against quenching by phosphate according to claim 1, characterized in that, The method comprises the following steps: S1, rare earth nanocrystals dissolved in chloroform and V 水 : V 乙醇 = 3:2 alendronate solution was mixed 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 min, and the precipitate was obtained after centrifugation, which was the 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 h to obtain an intermediate product B; S3, dropping the monophosphonic ligand into the intermediate product B, mixing and stirring for 1 h to obtain an intermediate product C; S4, dissolving the amphiphilic molecule in a dichloromethane solution, mixing and stirring with the intermediate product C for 6 h to obtain an intermediate product D; S5, rotary evaporation of the intermediate product D to remove the solvent dichloromethane, and then redispersed in an aqueous solution after the dichloromethane is volatilized; S6, centrifuging the intermediate product D redispersed in the aqueous solution in S5, washing with deionized water to obtain the dye-sensitized rare earth luminescent nanoprobes.

3. The method for preparing a phosphate-quench-resistant dye-sensitized rare-earth luminescent nanoprobe according to claim 2, characterized in that, The mass ratio of the rare earth nanocrystal and the alendronate solution in step S1 is (1-30):1000; the mass ratio of the alendronate solution and the monophosphonic ligand in steps S1 and S3 is (1-40):

100.

4. The method according to claim 2, wherein the method is characterized by, The mass ratio of the amphiphilic molecule and the rare earth nanocrystal in step S4 is (1-10):

100.

5. The dye-sensitized rare earth luminescent nanoprobes of claim 1 are applied to in vivo biological imaging.

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