Fluorescent molecules, their calcium phosphate nanoparticles and methods of making and using the same

Abstract

The application discloses fluorescent molecules, calcium phosphate nanoparticles thereof, and a preparation method and application thereof. Specifically, the application discloses a compound as shown in formula I, or a salt thereof. The calcium phosphate nanoparticles formed by the fluorescent molecules provided by the application have both improved fluorescence quantum yield and metabolic rate.

Description

Technical Field

[0001] This invention relates to the field of organic compound probes, and mainly to fluorescent molecules, their calcium phosphate nanoparticles, and their preparation methods and uses. Background Technology

[0002] Fluorescence imaging technology, due to its significant advantages such as high sensitivity, high spatiotemporal resolution, and real-time performance, is considered one of the most effective methods for visualizing biological processes, diagnosing tumors, and image-guided surgery, and has received widespread clinical application and attention. Currently, near-infrared I (NIR-I, 650-900nm) fluorescent dyes (such as FDA-approved drugs like indocyanine green (ICG)) are widely used in basic and clinical guidance. However, due to wavelength limitations, existing clinical probes have poor tissue penetration and cannot play a role in deep tissue imaging during actual surgical applications. In contrast, near-infrared II (NIR-II, 900-1700nm) fluorescent probes, due to their longer wavelengths, exhibit reduced light scattering and autofluorescence in tissues, resulting in deeper tissue penetration (~10mm), higher spatial resolution (~3mm), and a higher signal-to-noise ratio (SBR) (>5), giving them a significant technological advantage in the field of deep tissue imaging in clinical applications.

[0003] Despite the superior optical properties that give NIR-II fluorescent probes great development potential, two technical challenges still limit their clinical translation. First, the low fluorescence quantum yield (QY): low fluorescence quantum yield often leads to low fluorescence intensity and difficulty in resolving small structures, thus affecting imaging accuracy and posing significant difficulties for clinical translation. Second, existing NIR-II fluorescent probes are difficult to metabolize in vivo, resulting in serious long-term liver accumulation, raising concerns about biosafety in clinical applications and becoming another bottleneck for clinical translation.

[0004] To address these issues, some strategies have been reported, such as using small-molecule coupling with PEG side chains to increase water solubility and improve metabolic efficiency (Antaris, AL, H. Chen, K. Cheng, et al., A small-molecule dye for NIR-II imaging. [J]. Nature Materials, 2016. 15(2): p. 235-242.). However, this approach results in energy dissipation due to molecular rotation in polar solvents such as water, leading to very low probe brightness and significantly reducing imaging performance. There are also some strategies to increase QY: for example, Dai et al. (Antaris, AL, H. Chen, S. Diao, et al., A high quantum yield molecule-protein complex fluorophore for near-infrared II imaging. [J]. Nat Commun, 2017.8: p. 15269) found that binding a fluorophore to fetal calf serum (FBS) can form a stable protein-fluorophore complex. Because the intramolecular rotation of the fluorophore is restricted and the intermolecular interaction between the core structure of the fluorophore and the surrounding water is reduced, the QY of the complex is usually higher than that of the fluorophore alone. Some representative complexes, such as CH-4T and FD1080 (Antaris, AL, H. Chen, S. Diao, et al., A high-quantum yield molecule-protein complex fluorophore for near-infrared II imaging. [J]. Nat Commun, 2017. 8: p. 15269; Li, B., L. Lu, M. Zhao, et al., An Efficient 1064nm NIR-II Excitation Fluorescent Molecular Dye for Deep-Tissue High-Resolution Dynamic Bioimaging. [J]. Angew Chem Int Ed Engl, 2018. 57(25): p. 7483-7487.), have achieved orders-of-magnitude improvements over single molecules. The DAD fluorophore structure modified with aggregation-induced emission (AIE) groups is also an effective method to improve QY in polar solvents.Although these strategies have achieved good results in preclinical studies, various problems still exist in clinical applications, such as the complex composition of FBS, severe accumulation and long-term retention of AIE molecules in the liver. In summary, there are currently no reported NIR-II fluorophores with both high fluorescence brightness and rapid metabolism. Therefore, it is urgent to simultaneously address the two major problems of low fluorescence intensity and slow metabolism in the field of NIR-II imaging.

[0005] In recent years, lipid calcium phosphate (LCP) nanoparticles have been used to encapsulate various therapeutic drugs. Phosphate-containing prodrugs, nucleotides, siRNAs, and DNA are ideal drugs for encapsulation in LCP NPs because the phosphate groups on these molecules co-precipitate with calcium ions to form a pH-sensitive calcium phosphate (CaP) core. However, existing calcium phosphate nanoparticles are mainly used to encapsulate and deliver small molecule drugs and nucleic acid drugs, and have not been applied to the encapsulation of near-infrared II dyes. Current methods for encapsulating ICG with calcium phosphate nanoparticles have not significantly improved the fluorescence quantum yield. Summary of the Invention

[0006] The technical problem this invention aims to solve is to overcome the limitation of existing fluorescent probes that cannot simultaneously possess both high quantum yield and fast metabolic rate. This invention provides a novel fluorescent molecule, its calcium phosphate nanoparticles, and their preparation methods and applications. The calcium phosphate nanoparticles formed from the fluorescent molecule provided by this invention exhibit significantly improved fluorescence quantum yield and metabolic rate.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0008] This invention provides a compound as described in Formula I, or a salt thereof.

[0009]

[0010] in,

[0011] L 1 for

[0012] R 1 for

[0013] R 3 and R 4 Each L is independently substituted with one or more carboxyl groups, wherein the L is C2-C. 11 alkyl;

[0014] R 5 and R 6 Each independently

[0015] In one implementation scheme, R 3and R 4 In the context of C2-C 11 Alkyl groups can be independently C2-C. 11 Straight-chain alkyl, such as C2-C6 straight-chain alkyl, for example ethyl, n-propyl, n-butyl, n-pentyl or n-hexyl, preferably n-propyl.

[0016] In one implementation scheme, R 3 and R 4 In this context, the carboxyl group may be located at C2-C. 11 The alkyl terminus.

[0017] In one implementation scheme, R 3 and R 4 In this context, the number of carboxyl groups can be one.

[0018] In one implementation scheme, R 3 and R 4 Each can be independently -(CH2)3-COOH.

[0019] In one implementation scheme, R 1 for

[0020] In one embodiment, the compound represented by Formula I is a compound represented by Formula I-1 or I-2:

[0021]

[0022] In one embodiment, the compound represented by Formula I is any one of the following compounds:

[0023]

[0024]

[0025]

[0026] In one embodiment, the salt of the compound of formula I is its calcium salt.

[0027] The present invention also provides a method for preparing the compound as described in Formula I, which is any one of the following methods:

[0028] Method 1, when the compound shown in Formula I is the compound shown in Formula I-1, comprises the following steps: preparing I-1 by acid hydrolysis of the compound shown in Formula I-1-1 in a solvent;

[0029]

[0030] In compound I-1-1, R1-1 Independently -(CH2) n1 -R, n1 is 1, 2, 3 or 4, R is TMS, TES, TBDMS, TBDPS, DIPS, DPS or TIPDS; in compounds as shown in Formula I-1, R 3 and R 4 Each is independently -L-COOH;

[0031] Method 2, when the compound shown in Formula I is the compound shown in Formula I-2, comprises the following steps: reacting compound I-2-1 with POCl3 in a solvent and mixing with water;

[0032]

[0033] Where L 1 The definition is as described above.

[0034] In method 1, the acid can be a commonly used acid in this type of acidolysis reaction in the art, such as trifluoroacetic acid.

[0035] In method 1, the solvent for the acidolysis reaction can be a commonly used solvent for such reactions in the art, such as a halocarbon solvent, preferably dichloromethane.

[0036] Preferably, method 1 further includes post-processing, which includes the following steps: after the acidolysis reaction is completed, quenching and recrystallization are performed.

[0037] In method 1, the quenching in the post-processing can be quenching with quenching reagents commonly used in this type of operation in the art, preferably quenching with ether solvents, such as diethyl ether.

[0038] In method 1, the solvent for recrystallization in the post-processing is preferably an ether solvent, such as diethyl ether.

[0039] Preferably, method 2 includes the following steps: in a solvent, under the action of an alkali, compound I-2-1 reacts with POCl3, and is then mixed with water.

[0040] In method 2, the solvent can be a commonly used solvent for such reactions in the art, such as an ether solvent, preferably tetrahydrofuran; the base can be a commonly used base for such reactions in the art, such as an organic amine, preferably triethylamine.

[0041] Preferably, method 2 further includes a post-processing step, which includes the following steps: precipitation with acid and recrystallization.

[0042] In method 2, during the post-treatment, the acid is preferably an inorganic acid, such as hydrochloric acid; the molar concentration of the acid is preferably 1-3 mol / L, for example 2 mol / L.

[0043] In method 2, the solvent for recrystallization in the post-processing is preferably an ether solvent, such as tetrahydrofuran.

[0044] The present invention also provides compounds as shown in Formula I-1-1 and Formula I-2-1:

[0045]

[0046] The definitions of each substituent are as described above.

[0047] In one embodiment, the compound shown in Formula I-1-1 is The compound shown in formula I-2-1 is

[0048] The present invention also provides a compound as shown in Formula I-1-2,

[0049]

[0050] The definitions of each substituent are as described above.

[0051] In one embodiment, the compound shown in Formula I-1-2 is

[0052] The present invention also provides an organocalcium phosphate nanoparticle with a drug loading of 2%-4.5%, comprising the calcium salt of the compound shown in Formula I, wherein the drug loading is the mass ratio of the compound shown in Formula I to the organocalcium phosphate nanoparticle (the compound shown in Formula I is the free acid corresponding to the calcium salt of the compound shown in Formula I).

[0053] In one embodiment, the calcium salt of the compound represented by Formula I in the organophosphate nanoparticles has a mass percentage of 2.1% to 4.6%.

[0054] In one embodiment, the particle size of the organocalcium phosphate nanoparticles is 8-90 nm, where the particle size is the actual size of the nanoparticles under a transmission electron microscope (TEM), and the testing method is conventional in the art.

[0055] In one embodiment, the hydration diameter of the organocalcium phosphate nanoparticles is 10–100 nm. This hydration diameter is determined using dynamic light scattering (DLS) analysis, measuring the particle size of the composite particles in solution, i.e., the hydrated particle size. The hydrated particle size includes the nanoparticles and a solvation layer. The testing method is conventional in the art.

[0056] In one embodiment, the percentage of the organic calcium phosphate nanoparticles with a hydration diameter in the range of 35 to 65 nm is 50% to 80%, wherein 35 to 65 nm means ≥35 nm and <65 nm.

[0057] In one embodiment, the percentage of the organic calcium phosphate nanoparticles with a hydration diameter in the range of 10 to 35 nm is 5% to 15%, wherein 10 to 35 nm means ≥10 nm and <35 nm.

[0058] In one embodiment, the percentage of the organic calcium phosphate nanoparticles with a hydration diameter in the range of 65 to 100 nm is 15%-35%, wherein 65 to 100 nm means ≥65 nm and <100 nm.

[0059] In one embodiment, the percentage of the organic calcium phosphate nanoparticles with a hydration diameter in the range of 35-65 nm is 50%-80%, the percentage of the hydration diameter in the range of 10-35 nm is 55%-15%, and the percentage of the hydration diameter in the range of 65-100 nm is 15%-35%.

[0060] In one embodiment, the absorption wavelength of the organocalcium phosphate nanoparticles is 600–1000 nm.

[0061] In one embodiment, the emission wavelength of the organocalcium phosphate nanoparticles is 900–1600 nm.

[0062] In one embodiment, the maximum absorption wavelength of the organocalcium phosphate nanoparticles is 720–820 nm, for example, 773 nm.

[0063] In one embodiment, the maximum emission wavelength of the organocalcium phosphate nanoparticles is 1000-1300 nm, for example, 1150 nm.

[0064] In one embodiment, the drug loading of the organophosphate nanoparticles is 2.15%.

[0065] In one embodiment, the organophosphate nanoparticles may further comprise calcium phosphate.

[0066] In one embodiment, the mass percentage of calcium phosphate in the organophosphate nanoparticles is 6% to 10%.

[0067] In one embodiment, the organophosphate nanoparticles may further comprise substance A and substance B, wherein substance A is a salt formed by an anion in an anionic surfactant and calcium ions, and substance B is an amphiphilic compound.

[0068] In one embodiment, the mass percentage of substance A is 15% to 18%.

[0069] In one embodiment, the mass percentage of substance B is 68% to 72%.

[0070] In one embodiment, the organocalcium phosphate nanoparticles comprise a calcium salt of a compound as shown in Formula I, calcium phosphate, substance A, and substance B.

[0071] In one embodiment, the organocalcium phosphate nanoparticles are composed of a calcium salt of a compound as shown in Formula I, calcium phosphate, substance A, and substance B.

[0072] In one embodiment, the organocalcium phosphate nanoparticles are composed of a calcium salt of a compound as shown in Formula I, calcium phosphate, substance A, and substance B, wherein the mass percentage of the calcium salt of the compound as shown in Formula I is 2.1%-4.6%, the mass percentage of calcium phosphate is 6%-10%, the mass percentage of substance A is 15%-18%, and the mass percentage of substance B is 68%-72%.

[0073] In one embodiment, the anionic surfactant may be an anionic surfactant commonly used in this type of structure in the art, preferably a phosphate ester anionic surfactant, such as dioleoyl phosphatidyl acid (DOPA), dilauroyl phosphatidyl acid (DLPA), 1,2-dimyristoyl phosphatidyl acid (DMPA), 1-palmitoyl-2-oleoyl phosphatidyl acid (POPA), 1,2-distearyl phosphatidyl acid (DSPA), 2-dispalmitoyl phosphatidyl acid (DPPA), phosphatidylglycerol (16:0Lyso PG), 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), 1-stearoyl-2-oleoyl phosphatidyl acid (SOPA), 1,2-disorcinyl-sn-glycero-3-phosphate (DEPA), and more specifically, dioleoyl phosphatidyl acid (DOPA, CAS: 108392-02-5).

[0074] In one embodiment, the amphiphilic compound may be an amphiphilic compound commonly used in the art for this type of structure, such as phospholipids, amino lipids, and sphingolipids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, distearylphosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearylphosphatidylcholine, dioleoylphosphatidylcholine, or dithioylphosphatidylcholine.

[0075] In one embodiment, the amphiphilic compound is a phospholipid, such as a PEGylated derivative of a phospholipid, or, for example... Among them, m5 and m6 are each independently 15, 16, 17, 18, 19 or 20, preferably 16.

[0076] In one embodiment, the amphiphilic compound is DSPE-mPEG, preferably DSPE-PEG-2000.

[0077] In one embodiment, in A, the anionic surfactant is RPO3M2;

[0078] Where R is

[0079] m1, m2, m3 and m4 are each independently 6, 7, 8, 9 or 10, preferably 7;

[0080] M is an alkali metal, preferably Na.

[0081] In one embodiment, the organocalcium phosphate nanoparticles comprise a calcium salt of the compound shown in Formula I, the phosphate, substance A, and substance B, wherein the calcium salt of the compound shown in Formula I and calcium phosphate are mixed to form a coprecipitate, substance A coats the outer surface of the coprecipitate to form a nanoparticle core, and substance B coats the outer surface of substance A.

[0082] In one embodiment, the hydration diameter of the nanoparticle core is 3–45 nm.

[0083] In one embodiment, the percentage of calcium phosphate nanoparticle cores with a hydration diameter in the range of 8 to 12 nm is 50% to 80%, where 8 to 12 nm means ≥8 nm and <12 nm.

[0084] In one embodiment, the percentage of calcium phosphate nanoparticle cores with a hydration diameter in the range of 3 to 8 nm is 10% to 25%, where 3 to 8 nm means ≥3 nm and <8 nm.

[0085] In one embodiment, the percentage of calcium phosphate nanoparticle cores with a hydration diameter in the range of 12 to 45 nm is 15% to 35%, where 12 to 45 nm means ≥12 nm and <45 nm.

[0086] In one embodiment, the calcium phosphate nanoparticle core has a hydration diameter in the range of 8-12 nm, a hydration diameter in the range of 10-25%, and a hydration diameter in the range of 12-45 nm, comprising 50%-80% of the total core, 10%-25% of the total core, and 15%-35% of the total core, comprising a hydration diameter in the range of 12-45 nm.

[0087] In one embodiment, the organocalcium phosphate nanoparticles are synthesized using a reverse microemulsion method. This invention also provides a method for preparing calcium phosphate nanoparticles, comprising the following steps:

[0088] ①: A solution containing calcium ions is mixed with a solution containing the compound shown in Formula I above to obtain mixture a;

[0089] ②: Mix the mixture a described in ① with anionic surfactant and demulsifier to obtain a precipitate;

[0090] ③: Mix the precipitate described in ② with the amphiphilic compound and water;

[0091] In ①, the solution containing calcium ions is a mixture of "aqueous solution of soluble calcium salt" and "organic solvent containing nonionic surfactant", referred to as the calcium phase;

[0092] The solution containing the compound shown in Formula I is a mixture of "aqueous phosphate solution of the compound shown in Formula I" and "an organic solvent containing a nonionic surfactant", referred to as the phosphorus phase.

[0093] The operation and reaction conditions of the preparation method can be the conventional operation and reaction conditions in the preparation methods of this type of nanoparticle in the art, and preferably the conventional operation and reaction conditions in the reverse microemulsion method. In this application, the following operation and reaction conditions are preferred:

[0094] In ①, the molar ratio of the compound as shown in Formula I to the calcium ion substance can be 1:(1500-4000), preferably 1:(3000-3500), for example 1:3348.

[0095] Preferably, in ①, the mixing involves adding the "solution containing the compound shown in Formula I" to the "solution containing calcium ions".

[0096] Preferably, in step ①, after mixing, the mixture is stirred for, for example, 8 to 15 minutes, preferably 10 minutes.

[0097] The soluble calcium salt may be selected from one or more of calcium chloride, calcium nitrate, calcium bicarbonate, calcium bisulfate, calcium bisulfite, calcium hypochlorite, calcium bromide, calcium iodide, calcium chlorate, calcium perchlorate, and calcium permanganate, preferably calcium chloride.

[0098] The concentration of the aqueous solution of the soluble calcium salt can be 2 to 3 mol / L, for example, 2.5 mol / L.

[0099] The volume ratio of the "aqueous solution of soluble calcium salt" to the "organic solvent containing nonionic surfactant" can be 0.3:(8-12), for example 0.3:10.

[0100] Preferably, the calcium phase is obtained by mixing the "aqueous solution of soluble calcium salt" with the "organic solvent containing nonionic surfactant" and stirring for 3 to 8 minutes, for example, 5 minutes.

[0101] The pH of the calcium phase can be 5.6-8.0, for example, 5.6.

[0102] The phosphate aqueous solution of the compound shown in Formula I is prepared by dissolving the compound shown in Formula I in a phosphate aqueous solution as a solute and a phosphate aqueous solution as a solvent. The phosphate can be a commonly used soluble phosphate in the art, such as sodium dihydrogen phosphate and / or disodium hydrogen phosphate, preferably disodium hydrogen phosphate. In the phosphate aqueous solution of the compound shown in Formula I, the concentration of the solvent can be 0.005–0.025 mol / L, for example, 0.02 mol / L.

[0103] In one embodiment, the phosphate aqueous solution of the compound as shown in Formula I is used as the solute and the phosphate aqueous solution as the solvent. The molar ratio of the compound as shown in Formula I to the phosphate ions in the solvent can be 1:(10-40), preferably 1:(10-30), for example 1:27.

[0104] The volume ratio of the "phosphate aqueous solution of the compound as shown in Formula I" to the "organic solvent containing a nonionic surfactant" is 0.3:(8-12), preferably 0.3:10.

[0105] Preferably, the phosphorus phase is obtained by mixing "an aqueous solution of a phosphate of a compound as shown in Formula I" with "an organic solvent containing a nonionic surfactant" and then stirring, wherein the stirring time is preferably 3 to 8 minutes, more preferably 5 minutes; preferably, the mixing is performed by adding "a phosphate solution of a compound as shown in Formula I" dropwise to "an organic solvent containing a nonionic surfactant".

[0106] The pH of the phosphorus phase can be 2.0-8.0, preferably 2.0.

[0107] In the aforementioned "organic solvent containing a nonionic surfactant," the organic solvent may be a hydrocarbon solvent and / or an alcohol solvent. The hydrocarbon solvent may be cyclohexane. The alcohol solvent may be n-hexanol.

[0108] In one embodiment, the organic solvent in the "organic solvent containing a nonionic surfactant" may be cyclohexane and / or n-hexanol, preferably cyclohexane.

[0109] In the "organic solvent containing nonionic surfactant", when the organic solvent is a mixture of hydrocarbon solvent and alcohol solvent, the volume ratio of the hydrocarbon solvent to the alcohol solvent is preferably (7-8):1, for example 7.5:1.

[0110] In the “organic solvent containing a nonionic surfactant”, the nonionic surfactant may be an alkylphenol polyoxyethylene ether, more such as polyethylene glycol octylphenyl ether (triton x100) and / or polyoxyethylene (5) nonylphenyl ether (Igepal CO-520), and more preferably Igepal CO-520.

[0111] In one embodiment, the Igepal CO-520 is sourced from SIGMA Corporation in the United States, with a number-average molecular weight of 441 and a linear molecular formula of (C2H4O)n·C 15 H 24 O,n~5.

[0112] In the "organic solvent containing a nonionic surfactant", the volume ratio of the organic solvent to the nonionic surfactant is preferably (2-7):1, for example 2.3:1 or 5.7:1, and more preferably 2.3:1.

[0113] The volume ratio of the "organic solvent containing nonionic surfactant" in the phosphorus phase to the "organic solvent containing nonionic surfactant" in the calcium phase is preferably (0.8-1.2):1, for example, 1:1.

[0114] Preferably, step ② involves mixing the mixture a described in step ① with an anionic surfactant to obtain mixture b, and then mixing it with a demulsifier to obtain a precipitate. Preferably, mixing the mixture a described in step ① with the anionic surfactant involves adding the anionic surfactant dropwise to the mixture a described in step ①. Preferably, step ② involves mixing the mixture a described in step ① with the anionic surfactant, stirring to obtain mixture b, and then mixing it with a demulsifier. The stirring time is preferably 25-35 minutes, more preferably 30 minutes. Preferably, the volume ratio of the demulsifier to the mixture b is 1:(0.8-1.2), more preferably 1:1.

[0115] In step ②, the anionic surfactant may be as described in any of the preceding embodiments.

[0116] In step ②, the anionic surfactant is preferably a solution thereof, and the solvent in the solution is preferably a halocarbon solvent, such as chloroform. The concentration of the solution is preferably 0.02–0.03 mol / L, more preferably 0.026 mol / L. The volume ratio of the solution to the mixture a in step ① is preferably 1:(120–150), more preferably 1:137.

[0117] In one embodiment, the molar ratio of the anionic surfactant to the compound shown in Formula I can be (8-30):1, for example (8-20):1, preferably 18:1.

[0118] In step ②, the demulsifier is preferably an alcohol solvent, such as ethanol.

[0119] Preferably, in step ②, after obtaining the precipitate, the precipitate is collected and washed with a demulsifier, and the number of washings is preferably 2 to 4 times, more preferably 3 times.

[0120] In ③, the amphiphilic compound is as described above.

[0121] Preferably, step ③ involves dissolving the precipitate described in step ②, mixing it with the amphiphilic compound to obtain mixture c, and then mixing it with water. Preferably, mixing with water involves pouring mixture c into water. The volume ratio of mixture c to water is preferably 1:(2.5-4), more preferably 1:3. The solvent used to dissolve the precipitate is preferably an organic solvent, such as an ether solvent, preferably tetrahydrofuran. The volume-to-mass ratio of the solvent used to dissolve the precipitate to the precipitate is 1-2 ml / mg, preferably 1 ml / mg.

[0122] The mass ratio of the amphiphilic compound to the precipitate described in ② can be (2-3):1, for example, 2.5:1.

[0123] Preferably, the amphiphilic compound participates in the reaction in the form of an amphiphilic compound solution. In the amphiphilic compound solution, the solvent is preferably an organic solvent, such as an ether solvent, and more preferably tetrahydrofuran. In the amphiphilic compound solution, the concentration of the amphiphilic compound is preferably 0.005–0.015 mol / L, for example, 0.012 mol / L.

[0124] After step ③ is completed, post-processing can be performed through the following steps: concentration.

[0125] Preferably, the concentration involves filtering the mixture obtained in step ③ and concentrating the filtrate. Preferably, the organic solvent present in the mixture obtained in step ③ is removed before filtration. The filtration is preferably performed using a micromembrane, for example, a 0.22 μm micromembrane; the concentration is preferably performed using an ultrafiltration device.

[0126] After concentration, the resulting calcium phosphate nanoparticles can be dispersed in water for later use.

[0127] In one embodiment, the particle size, hydration diameter, absorption wavelength, maximum absorption wavelength, emission wavelength, maximum emission wavelength, drug loading, and composition of the calcium phosphate nanoparticles are the same as those described for the organophosphate nanoparticles.

[0128] In one embodiment, the method for preparing the calcium phosphate nanoparticles includes the following steps:

[0129] A calcium phase was obtained by mixing and stirring a calcium chloride solution with a cyclohexane / Igepal CO-520 (7:3, V:V) solution. A Na₂HPO₄ solution of the compound shown in Formula I was added dropwise to the cyclohexane / Igepal CO-520 (7:3, V:V) solution, and the mixture was stirred to form a phosphorus phase. The phosphorus phase was added to the calcium phase and stirred, followed by the addition of DOPA solution and stirring. Ethanol was added to precipitate the precipitate, which was collected and washed with ethanol. The precipitate was redissolved in tetrahydrofuran and mixed with a tetrahydrofuran solution of DSPE-PEG-2000. The mixture was then quickly poured into water.

[0130] The present invention also provides calcium phosphate nanoparticles prepared by the above-described method for preparing calcium phosphate nanoparticles.

[0131] The present invention also provides the use of the above-described compound as shown in Formula I, or a salt thereof, in the preparation of calcium phosphate nanoparticles.

[0132] The present invention also provides the use of the above-described compound as shown in Formula I, its salt, or the above-described calcium phosphate nanoparticles in the preparation of fluorescent probes.

[0133] In one embodiment, the probe is a near-infrared II fluorescent probe used for tumor and vascular imaging.

[0134] In one implementation, the tumor and vascular imaging is tumor and vascular imaging of experimental animals.

[0135] The present invention also provides a near-infrared II fluorescent probe comprising a compound as shown in Formula I, its salt, or the above-described calcium phosphate nanoparticles.

[0136] In this invention, the near-infrared II fluorescent probe refers to a conventional near-infrared II fluorescent probe in the art, such as a fluorescent probe with an emission wavelength in the range of 900 to 1700 nm.

[0137] The above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0138] The reagents and raw materials used in this invention are all commercially available.

[0139] The positive and progressive effects of this invention are as follows: By introducing phosphate groups into compounds as shown in Formula I-3 or I-4, this invention obtains compounds as shown in Formula I, which are co-precipitated with calcium ions to form calcium phosphate nanoparticles. The calcium ions encapsulate the molecular dye and restrict its rotation, cleverly preventing the TICT effect of the compound, thereby improving the fluorescence quantum yield and also enhancing the metabolic rate. Attached Figure Description

[0140] Figure 1 The absorption and emission spectra of LJ-2P and LJ-2P NPs are shown.

[0141] Figure 2 The slopes of fluorescence quantum yield for LJ-2P, LJ-2P NPs, and reference IR26 are given.

[0142] Figure 3 The slopes of fluorescence quantum yield for LJ-4P, LJ-4P NPs, and reference IR26 are given.

[0143] Figure 4 DLS and TEM characterization of LJ-2P NPs kernels in tetrahydrofuran.

[0144] Figure 5 DLS and TEM characterization of LJ-2P NPs in water.

[0145] Figure 6 To achieve a power density of 90 mW / cm² 2 The photostability curves of LJ-2P, LJ-2P NPs and ICG for 60 min under 808nm laser irradiation.

[0146] Figure 7Cell viability after 24 h of incubation of 4T1 and 293T cells with different concentrations of LJ-2P or LJ-2P NPs.

[0147] Figure 8 NIR-II fluorescence imaging of tumor vessels in BALB / c nude mice using LJ-2P NPs (power: 70mW / cm²) 2 Exposure time: 50ms, 1000nm LP filter. Dosage: 25mg / kg, scale bar: 5mm.

[0148] Figure 9 The Gaussian function of the cross section is fitted along the yellow line in Figure (8) to fit the data profile.

[0149] Figure 10 NIR-II fluorescence imaging of tumor vessels in BALB / c nude mice using LJ-2P NPs (power: 70mW / cm²) 2 Exposure time: 200ms, 1150nm LP filter. Dosage: 25mg / kg, scale bar: 5mm.

[0150] Figure 11 The Gaussian function of the cross section is fitted along the yellow line in Figure (10) to fit the data profile.

[0151] Figure 12 For 808nm laser (power: 70mW / cm) 2 Signal-to-noise ratio of vascular imaging in mice after tail vein injection of LJ-2P NPs under excitation conditions (exposure time: 50ms, 1000nm LP filter, 1150nm LP filter). Dosage: 25mg / kg, scale bar: 5mm.

[0152] Figure 13 For 808nm laser (power: 70mW / cm) 2 NIR-II fluorescence imaging of LJ-2P NPs in mice with subcutaneous 4T1 breast tumors was performed at different time points under the excitation of an exposure time of 50ms and a 1000nm LP filter. The dose was 25mg / kg.

[0153] Figure 14 For based on Figure 13 The ratio of mean NIR-II fluorescence signal in tumors to normal tissues at each time point. Data are plotted as mean ± SD; n = 6.

[0154] Figure 15 Thirty-six hours after tail vein injection of LJ-2P NPs, primary organs (heart, liver, spleen, lung, kidney) and tumors were harvested for H&E staining. Dosage: 25 mg / kg, scale bar: 100 μm.

[0155] Figure 16 Release characteristics of LJ-2P NPs in release media with different pH values.

[0156] Figure 17 In vivo fluorescence imaging of BALB / c nude mice injected with LJ-2P on days 0, 1, 2, 4, 6, 8, 11, and 14 (808 nm laser excitation, 90 mW / cm²). 2 Exposure time 100ms, 1000nm LP filter), dose: 25mg / kg.

[0157] Figure 18 for Figure 17 Statistical calculation of fluorescence signals (all intensities normalized to day 0).

[0158] Figure 19 In vivo fluorescence imaging of BALB / c nude mice injected with LJ-2P NPs on days 0, 1, 2, 4, 6, 8, 11, and 14 (808 nm laser excitation, 90 mW / cm²). 2 Exposure time 70ms, 1000nm LP filter), dose: 25mg / kg.

[0159] Figure 20 for Figure 19 Statistical calculation of fluorescence signals (all intensities normalized to day 0). Detailed Implementation

[0160] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0161] The various instruments used in the experiment and their models are shown in the table below:

[0162]

[0163] Example 1: Synthesis of LJ-2P

[0164]

[0165] Compound 1: Acyl chloride (0.72 g, 9.2 mmol) was added to a 100 mL round-bottom flask containing AlCl3 (1.53 g, 11.4 mmol) and anhydrous dichloromethane (40 mL) with stirring. When all solids in the flask disappeared, 2-bromofluorene (1.879 g, 7.7 mmol) was added dropwise, and the mixture was stirred at room temperature until the initial substrate was completely converted (monitored by TLC). The mixture was poured into ice / water and extracted with EA (40 mL × 3). The combined organic layers were washed with brine and dried over anhydrous Na2SO4. The solvent was removed under vacuum, and the crude product was purified by silica gel column chromatography, eluting with PE and EA, to give compound 1 (2 g, yield: 91%) as white crystals.

[0166] Compound 2: Compound 1 (3.432 g, 12 mmol) and potassium iodide (199 mg, 1.2 mmol) were dissolved in dimethyl sulfoxide (40 mL) under nitrogen protection. Methyl 4-bromobutyrate (4.752 g, 26.4 mmol) was added to the reaction mixture, followed by potassium hydroxide (3.366 g, 60 mmol). The green reaction mixture was stirred at room temperature for 12 hours and quenched with water. The mixture was acidified to pH 5 with 2 M HCl solution and extracted with EA (3 × 150 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated by rotary evaporator. Compound 2 was obtained without further purification as a yellow solid (4.84 g, yield: 88%).

[0167] Compound 3: Compound 2 (3 g, 6.55 mmol) and 2-trimethylsilylethanol (1.936 g, 16.4 mmol) were dissolved in DCM (40 mL) and DMF (10 mL) under nitrogen protection and cooled to 0 °C in an ice bath. DCC (3.378 g, 16.4 mmol) and DMAP (0.16 g, 1.31 mmol) were added to the reaction mixture. The reaction mixture was then heated to ambient temperature and stirred for 12 hours, and finally quenched with water. After dilution with dichloromethane (100 mL) to remove DCC, the mixture was filtered, and the filtrate was washed with saturated brine (3 × 100 mL) and dried over anhydrous Na₂SO₄. After removing the solvent, the crude mixture was purified by silica gel column chromatography using a gradient elution of PE and EA to give compound 3 as a yellow solid (2.41 g, yield: 56%).

[0168] Compound 4: To a chloroform (40 mL) solution containing compound 3 (3.29 g, 5 mmol) and trifluoroacetic acid (421 μl, 647 mg, 3.75 mmol), m-chloroperoxybenzoic acid (mCPBA) (2.588 g, 15 mmol) was added, and the mixture was stirred overnight at room temperature. After the reaction was complete (monitored by thin-layer chromatography (TLC), the reaction mixture was washed successively with saturated Na₂SO₃ and saturated NaHCO₃, and then extracted with DCM. The combined organic layers were washed with saturated brine and dried over anhydrous Na₂SO₄. The crude product was purified by silica gel column chromatography, eluting with PE and EA, to give target compound 4 as a yellow solid (2.798 g, yield: 83%).

[0169] Compound 5: 10 mL of 10% NaOH solution was added to a 50 mL round-bottom flask containing compound 4 (1.348 g, 2 mmol) and EtOH (10 mL). The mixture was stirred at room temperature for 3 minutes and quenched with water. The pH of the solution was adjusted to 5–6 using 6 M HCl, and then extracted with EA. The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The crude product was purified by silica gel column chromatography using a gradient elution of PE and EA to give the target compound 5 as a yellow solid (1.035 g, yield: 84%).

[0170] Compound 6: Bis((triphenylphosphine)palladium(II)) dichloride (141 mg, 0.2 mmol) was added to a solution of compound 5 (1.232 g, 2 mmol), bis(pinacol)diboron (0.609 g, 2.4 mmol), and KOAc (0.471 g, 4.8 mmol) in dioxane (60 mL). The reaction mixture was then heated in an oil bath at 110 °C for 2 hours under nitrogen protection. After cooling the reaction mixture to room temperature, 60 mL of ethyl acetate was added, and the solids were removed by filtration. The combined organic layers were washed with water (3 × 60 mL) and saturated brine (150 mL) and dried over anhydrous Na₂SO₄. The crude product was purified by silica gel column chromatography using a gradient elution of PE and EA to give the target compound 6 as a yellow transparent liquid (0.626 g, yield: 46%).

[0171] Compound 8: A solution of compound 6 (170 mg, 0.25 mmol) and compound 7 (66.4 mg, 0.2 mmol) in THF (5 mL) was bubbled under nitrogen for 5 minutes. Cesium carbonate (160 mg, 0.5 mmol) in 0.5 mL of distilled water and 1,1'-bis(diphenylphosphino)ferrocene palladium(II) dichloromethane complex (9 mg, 0.01 mmol) were added to the above reaction mixture under nitrogen. The mixture was heated in an oil bath at 70 °C in the absence of oxygen for 12 hours. After cooling to room temperature, 50 mL of ethyl acetate was added, and the solid was removed by filtration. The combined organic layers were washed with saturated brine (3 × 50 mL) and dried over anhydrous Na₂SO₄, and the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography using a gradient elution of PE and THF to give target compound 6 as a purple solid (0.117 g, yield: 73%).

[0172] Compound 9: Zinc powder (392 mg, 6 mmol) and NH4Cl (96.3 mg, 1.8 mmol) were added to a solution of Compound 8 (81 mg, 0.05 mmol) in dichloromethane (7.2 mL) and 90% methanol (11.4 mL). After stirring for 2 hours under nitrogen protection at room temperature, the solution was filtered through a diatomaceous earth mat, diluted with dichloromethane, and washed with water, saturated NaHCO3 aqueous solution, and saturated brine. The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give a yellow solid, which could be used in the next step without further purification. Under nitrogen protection, N-thionylaniline (0.2 mL, 1.8 mmol, 247 mg) and trimethylchlorosilane (0.3 mL, 3.5 mmol, 377 mg) were added to a deep yellow solution in anhydrous pyridine (2 mL). The mixture was heated in an oil bath at 80 °C for 16 hours. The reaction mixture was cooled to room temperature and poured into ice water. Extraction was performed with 50 mL of EA, followed by washing with 0.1 M HCl (2 × 50 mL) and saturated brine (50 mL). The organic phase was dried over anhydrous Na₂SO₄. The solvent was removed under vacuum. The crude product was purified by silica gel column chromatography using a gradient elution with PE and EA to give the target compound 10 as a dark green solid (25 mg, two-step yield: 32%).

[0173] Compound 10: A solution of compound 9 (15.8 mg, 0.01 mmol) in anhydrous THF (2 mL) was cooled to 0 °C in an ice bath, and then phosphorus oxychloride (93 μl, 153 mg, 1 mmol) was added under nitrogen protection. The reaction mixture was allowed to return to room temperature and reacted for 30 min, quenched with ice water, extracted with 10 mL of EA, and washed with saturated brine (2 × 20 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to give a dark green solid, which could be used in the next step without further purification. The dark green solution in anhydrous DCM (2 mL) was cooled to 0 °C, and then trifluoroacetic acid (2 mL) was added under nitrogen protection. The reaction mixture was then heated to ambient temperature and stirred for 1 hour, quenched with anhydrous diethyl ether at 0 °C, and recrystallized from anhydrous diethyl ether to obtain a green solid (10 mg, two-step yield: 78%).

[0174] 1 H NMR (500MHz, DMSO-d6) δ7.82(s,4H),7.74(s,4H),7.22(s,4H),4.50(s,4H),4.36(s,4H),1.99(s,18H),1.23(s,6H).

[0175] 31 P NMR (162MHz, DMSO-d6) δ-6.50.

[0176] MALDI-TOF-MS calculation: C 60 H 53 N4O 20 P2S4 + ([M+H)) + ):1339.16, Characterization:1339.2.

[0177] Example 2: Synthesis of LJ-4P

[0178]

[0179] Compound 11: Weigh 2.042 g (14.4 mmol) of 3,4-ethylenedioxythiophene and add it to 20 mL of dry THF. Place the solution in a 100 mL two-necked flask and immerse it in a dry ice and acetone bath. Purge the gas three times with N2. Add 5.4 mL of 2.5 M nBuLi to the solution using a syringe and react for 1 h. Add 3.8 mL (15.6 mmol) of SnBu3Cl to the solution slowly using a syringe and react for 2.5 h at room temperature. Dilute with 30 mL of EA, wash three times with saturated ammonium chloride water, remove water with anhydrous Na2SO4, and then rotary evaporate to obtain compound 1 (5.711 g, yield: 95%).

[0180] Compound 12: Compound 1 (5.711 g, 13.35 mmol) and 4,7-dibromo-5,6-dinitrobenzodithiodiazole (2.03 g, 5.3 mmol) were weighed and added to 30 mL of dry THF. The mixture was purged three times. Then, Pd(PPh3)4 (372 mg, 0.52 mmol) was added, and the mixture was purged three more times. The mixture was heated to reflux at 70 °C under nitrogen and stirred for 12 h. When TLC showed that the starting material was completely consumed and a new main site was formed, the mixture was cooled to room temperature. 100 mL of saturated potassium fluoride was added and stirred at room temperature for 1 h. The mixture was diluted with EA, washed three times with saturated potassium fluoride, dried over anhydrous Na2SO4, and filtered to remove Na2SO4. The filtrate was concentrated on a rotary evaporator to obtain a mixture. The mixture was purified by chromatography (PE / EA = 4 / 1, v / v) to obtain orange-red compound 2 (798 mg, yield: 29.7%).

[0181] Compound 13: Compound 2 (250 mg, 0.494 mmol) and NBS (295 mg, 1.66 mmol) were weighed into a 250 mL light-protected two-necked flask. 6 mL of DMF, 3 mL of MeCN, and 3 mL of 48% HBr were added. The mixture was heated to 70 °C and reacted for 3 h. Then, NBS (147 mg, 0.82 mmol) was added and reacted for 40 min. This reaction was repeated three times. After cooling to room temperature, 80 mL of 2 M / L HCl was added, and the mixture was reacted at room temperature for 2 h. The mixture was filtered, washed with water, and washed with methanol to obtain a deep red compound 3 (267 mg, 79.2%).

[0182] Compound 14: 4-Iodoanisole (17.55 g, 78 mmol), aniline (2.45 g, 26 mmol), 1,10-phenanthroline (0.9 g, 5.2 mmol), potassium tert-butoxide (20.64 g, 195 mmol), and CuI (4.75 g, 26 mmol) were added to 200 mL of toluene. The mixture was purged with nitrogen three times, heated to 100 °C, and reacted under nitrogen for 10 h. When TLC showed complete consumption of the starting material and the formation of a new main site, the reaction solution was cooled to room temperature, extracted with EA, washed three times with saturated brine, dried over anhydrous Na₂SO₄, and filtered to remove Na₂SO₄. The filtrate was concentrated on a rotary evaporator to obtain a mixture. The mixture was purified by chromatography (PE / DCM = 4 / 1, v / v) to obtain an orange-yellow compound 4 (7.5 g, yield: 94.6%).

[0183] Compound 15: Compound 4 (1 g, 3.28 mmol) was dissolved in 20 ml of dichloromethane and cooled to 0 °C in an ice bath. NBS (580 mg, 3.28 mmol) was slowly added in three batches in an ice bath and reacted for 4 h. When TLC showed that the starting material was completely consumed and a new main site was formed, 10 ml of water was added to quench the reaction. The mixture was extracted with dichloromethane, dried over anhydrous Na2SO4, filtered to remove Na2SO4, and the filtrate was concentrated on a rotary evaporator to produce compound 5 (1 g, yield: 94.7%).

[0184] Compound 16: Compound 5 (2.36 g, 6.16 mmol) was dissolved in 20 mL of chloroform and cooled to 0 °C in an ice bath. Boron tribromide (24 mL, 24.64 mmol) was extracted and dissolved in 40 mL of dichloromethane and slowly added dropwise over 1 hour. The reaction was allowed to proceed at room temperature for 16 hours. When TLC showed complete consumption of the starting material and the formation of a new principal site, 10 mL of methanol was added to remove unreacted boron tribromide. The reaction solution was cooled to room temperature, extracted with EA, washed three times with saturated sodium bicarbonate water, dried over anhydrous Na₂SO₄, and filtered to remove Na₂SO₄. The filtrate was concentrated on a rotary evaporator to obtain a mixture. The mixture was purified by chromatography (PE / EA = 4 / 1, v / v) to give yellow compound 6 (1.4 g, yield: 76%).

[0185] Compound 17: Compound 6 (1.92 g, 5 mmol) was dissolved in 30 mL of dry dioxane along with pinacol diboronate (1.524 g, 6 mmol), Pd(Ph3P)2Cl2 (351 mg, 0.5 mmol), and potassium acetate (1.175 g, 12 mmol). The reaction mixture was stirred overnight at 110 °C under nitrogen protection. The reaction was quenched with water, and the cooled solution was extracted with EA. The solution was dried over anhydrous Na2SO4, filtered to remove Na2SO4, and the filtrate was concentrated on a rotary evaporator to obtain a mixture. The mixture was purified by chromatography (PE / EA = 3 / 1, v / v) to give yellow compound 7 (1.5 g, yield: 69.6%).

[0186] Compound 18: Compound 3 (400 mg, 0.6 mmol) and Compound 7 (650 mg, 1.5 mmol) were weighed and placed in a 100 mL three-necked flask. 30 mL of dry THF was added, followed by the dissolution of cesium carbonate (393 mg, 1.2 mmol) in 1 mL of water. The solution was added to the flask, and the gas was purged three times. PdCl2(dppf)CH2Cl2 (98 mg, 0.12 mmol) was added and the gas was purged three times. The mixture was refluxed overnight at 70 °C under nitrogen protection. The reaction solution changed from red to blue. The red spot disappeared and the blue spot appeared on the TLC plate. After cooling, water was added, and the cooled solution was extracted with EA. The solution was dried over anhydrous Na2SO4, filtered to remove Na2SO4, and the filtrate was concentrated on a rotary evaporator to form a mixture. The mixture was purified by chromatography (DCM / MeOH = 20 / 1, v / v) to give blue compound 4 (470 mg, yield: 69.1%).

[0187] Compound 19: Zinc powder (4.91 g, 74.9 mmol) and NH4Cl (650 mg, 12.153 mmol) were added to a solution of compound 8 dissolved in DCM (380 mg, 0.34 mmol) and 90% methanol at 0 °C. The mixture was purged with nitrogen three times and stirred at room temperature for 4 h. When TLC showed complete consumption of the starting material and the formation of a new orange fluorescent host site, the reaction solution was filtered through diatomaceous earth, washed with methanol and DCM, and the solvent was removed by rotary evaporation. The DCM was dissolved, dried over anhydrous Na2SO4, filtered to remove Na2SO4, and the solvent was removed by rotary evaporation. PNSO (2.839 g, 21.1 mmol) and TMSCl (3.12 g, 29.575 mmol) were added to achieve the desired yield. 25 mL of pyridine was added, and the mixture was reacted overnight at 80 °C for 16 h under nitrogen protection. The solution turned green. Once TLC showed complete consumption of the starting material and the formation of a new principal site, the reaction solution was poured into a large amount of dilute hydrochloric acid, extracted with EA, and washed 4-5 times with dilute hydrochloric acid to remove excess pyridine. 10 ml of 2M / L hydrochloric acid and 10 ml of methanol were added, and the mixture was stirred at room temperature for 2 hours to remove TMS. The solution was washed 4-5 times with saturated sodium bicarbonate to remove excess hydrochloric acid, dried over anhydrous Na2SO4, and filtered to remove Na2SO4. The filtrate was concentrated on a rotary evaporator to obtain a mixture. The mixture was purified by chromatography (DCM / MeOH = 20 / 1, v / v) to obtain green compound 5 (98 mg, yield: 26.7%).

[0188] Compound 20: Weigh 100 mg of LTC-4OH (0.09765 mmol) and dissolve it in 10 mL of dry THF. Place the solution in an ice bath and add 1 mL of triethylamine (11.72 mmol). Purge the gas three times with nitrogen. Slowly add 728 μl of POCl3 (9.765 mmol). The solution changes from green to bright green immediately upon addition. Purge the gas three more times and react at room temperature for 1 h. Quench the reaction solution with ice water and precipitate THF with 2 M / L hydrochloric acid. Recrystallize to give compound 10 (50 mg, yield: 50%).

[0189] 1 H NMR (400MHz, DMSO-d6) δ7.64(d,J=8.4Hz,4H),7.14(d,J=8.6Hz,8H),7.06(d,J=8.6Hz,8H),6.94(d,J=8.5Hz,4H),4.40(s,4H),4.30(s,4H).

[0190] 31 P NMR (162MHz, DMSO-d6) δ -5.98.

[0191] MALDI-TOF-MS calculation: C 54 H 40 N6O 20 P4S4 + ([M] + ):1344.01, Characterization:1344.0.

[0192] Example 3: Preparation of LJ-2P NPs, where LJ-2P NPs are calcium phosphate nanoparticles of LJ-2P.

[0193] LJ-2P NPs were synthesized via a reverse microemulsion method. 300 μL of 2.5 M CaCl2 was mixed with 10 mL of cyclohexane / Igepal CO-520 (70:30, V:V) solution (oil phase) and stirred for 5 min to form the Ca phase (pH = 5.6). 0.3 mg of LJ-2P was weighed into 300 μL of 20 mM Na2HPO4 and added dropwise to 10 mL of cyclohexane / Igepal CO-520 (70:30, V:V) solution (oil phase), and stirred for 5 min to form the P phase (pH adjusted to 2 with 2 M HCl). The P phase was added to the Ca phase and stirred for 10 min, followed by the addition of 150 μL of a 20 mg / mL DOPA chloroform solution and stirring for 30 min. 20 mL of ethanol was added to precipitate the LCP core. The precipitate was collected by centrifugation and washed three times with ethanol; this precipitate constitutes the LJ-2P NP core. TEM analysis of the LJ-2P NPs core showed a particle size of approximately 5 nm, while DLS measurements indicated a hydration diameter of 9 nm. Figure 4As shown.

[0194] Finally, the LJ-2P NPs core was redissolved in 1 ml of THF to form a solution of the LJ-2P NPs core. Then, 2.5 times the mass of the LCP core was added to DSPE-PEG-2000 (DSPE-PEG-2000 dissolved in THF, with a DSPE-PEG-2000 to THF mass-to-volume ratio of 10 mg / ml). The solution was then quickly poured into 3 times the volume of water to form a solution of LJ-2P NPs. After removing THF under reduced pressure, the suspension was filtered through a 0.22 μm micromembrane, concentrated by ultrafiltration, and finally dispersed in 600 μL of water for later use.

[0195] TEM showed that the LJ-2P NPs had a particle size of approximately 25 nm, while DLS measurements indicated a hydration diameter of 43 nm. Figure 5 As shown.

[0196] Determination of encapsulation efficiency of LJ-2P NPs: 0.1 mg of LJ-2P was dissolved in 1 ml of lysis buffer (0.2 M AA, 1% Triton X100, 10 mM EDTA), and serially diluted 7 times with the lysis buffer. The absorbance was measured at 808 nm UV absorbance, and a standard curve was plotted. LJ-2P NPs prepared from 0.3 mg of LJ-2P were dissolved in 1 ml of lysis buffer, then diluted 6 times with the lysis buffer, and incubated for 5 min to allow nanoparticle lysis. The absorbance was measured and incorporated into the standard curve to calculate the actual mass of LJ-2P and the encapsulation efficiency. The final encapsulation efficiency of LJ-2P NPs was 92%, and the drug loading was 2.15%.

[0197] Example 4: Determination of absorption and fluorescence emission spectra of LJ-2P and LJ-2P NPs

[0198] (1) Determination of absorption spectra of LJ-2P and LJ-2P NPs: Accurately weigh 1.388 mg of LJ-2P and 1.388 mg of LJ-2P nanoparticles and dissolve them in 1 ml of PBS as stock solution; dilute 100 times with PBS buffer. Take 2 ml of the above liquid and add it to a 1 cm quartz cuvette. Use a DU-700 spectrophotometer to scan the absorption spectrum, save the scan data and perform normalization plotting.

[0199] Test results are as follows Figure 1 As shown: LJ-2P has an absorption wavelength of 650-1000nm, with a maximum absorption wavelength of 736nm; LJ-2P NPs have an absorption wavelength of 650-1000nm, with a maximum absorption wavelength of 773nm.

[0200] (2) Determination of fluorescence emission spectra of LJ-2P and LJ-2P NPs: Accurately weigh 1.388 mg of LJ-2P and 1.388 mg of LJ-2P nanoparticles and dissolve them in 1 ml of PBS as a stock solution; dilute 100 times with PBS buffer. Add 2 ml of the above liquid to a 1 cm quartz cuvette, and use an excitation light of 804 nm and an excitation power of 90 mW / cm. 2 The exposure time was 100ms, and the fluorescence spectrum was measured using a Fuxiang FX2000 spectrometer. The scan data was saved and then normalized for plotting.

[0201] Test results are as follows Figure 1 As shown: The emission wavelength of LJ-2P is between 900-1500nm, with a maximum emission wavelength of 1059nm; the emission wavelength of LJ-2P NPs is between 900-1500nm, with a maximum emission wavelength of 1029nm.

[0202] Example 5: Determination of fluorescence quantum yield (QY)

[0203] The QY of molecules and LCP NPs was determined based on a previously reported method (Ye, F., W. Huang, C. Li, et al., Near-Infrared Fluorescence / Photoacoustic Agent with an Intensifying Optical Performance for Imaging-Guided Effective Photothermal Therapy. [J]. Advanced Therapeutics, 2020. 3(12): p. 2000-170.). First, five solutions of different concentrations with absorbance ranging from 0.05 to 0.1 were prepared, and the corresponding fluorescence spectra were measured. A linear relationship between the integrated fluorescence intensity and absorbance values ​​was fitted using a first-order function. The QY of the fluorophore was obtained based on the following formula:

[0204]

[0205] QY samp QY represents the dye to be tested. refe QY (0.5%) represents IR-26, S represents the slope, and n represents the refractive index of the solvent.

[0206] The results showed that the fluorescence quantum yield of LJ-2P was 0.14%, while that of LJ-2P NPs was 5.12%, representing a 36.57-fold increase compared to LJ-2P. Figure 2As shown, the fluorescence quantum yield of LJ-4P is 0.02%, while that of LJ-4PNPs is 0.25%, representing a 12.5-fold increase compared to LJ-4P. Figure 3 As shown.

[0207] Example 6: Light Stability Measurement

[0208] LJ-2P and LJ-2P NPs with the same absorbance were dissolved in water and irradiated with an 808 nm laser (90 mW / cm2) for 1 hour, with ICG used as a reference. Emission spectra were measured every 5 minutes, and the data were expressed as the ratio of the total fluorescence intensity at a set time point (I) to its initial value (I0) as a function of irradiation time.

[0209] The results showed that LJ-2P and LJ-2P NPs were treated with 90mW / cm². 2 After one hour of laser irradiation, the fluorescence intensity remained unchanged, indicating that both exhibit strong photostability. In contrast, the reference molecule ICG decayed by 100% within 60 minutes. Figure 6 As shown.

[0210] Example 7: Measurement of Cell Viability by MTT Assay

[0211] (1) Cell culture:

[0212] 4T1 and 293T cells were obtained from the China Type Culture Collection (ATCC). All cells were cultured in a humidified atmosphere at 37°C and 5% CO2. 4T1 tumor cells were cultured in RPMI 1640 medium containing 10% FBS, 100 IU mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin. 293T cells were cultured in complete medium (MEM, Gibco) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin.

[0213] (2) MTT assay for cell viability:

[0214] Cell viability was assessed for 293T and 4T1 cells. PBS buffer (100 μL) was added to the outermost wells of a 96-well plate. Cell culture solution (100 μL) was added to the remaining 80 wells, and cells were cultured for 24 hours. Different concentrations of LJ-2P and LJ-2P NPs solutions were prepared using culture medium. Different concentrations of LJ-2P and LJ-2P NPs culture medium solutions (100 μL) were added to the cells. After 24 hours of incubation, the 96-well plate was removed, the medium containing LJ-2P and LJ-2P NPs was discarded, and 120 μL of MTT solution was added. After 4 hours of incubation, the MTT solution was removed, and 150 μL of DMSO was added. Absorbance was measured at 490 nm using a SpectraMax i3 microplate reader. The following formula was used to calculate cell viability: Cell viability = (Average absorbance value of the treated group / Average absorbance value of the control group).

[0215] The results are as follows Figure 7 As shown, LJ-2P and LJ-2P NPs exhibited low cytotoxicity in different cell types (4T1, 293T), with cell viability approaching 100% even at concentrations as high as 30 μM, demonstrating the good biocompatibility and low cytotoxicity of LJ-2P and LJ-2P NPs.

[0216] Example 8: In vitro release of LJ-2P NPs

[0217] 1 ml of LJ-2P NPs was placed in a dialysis bag, and 30 ml of release medium at different pH values ​​was added. The bag was then incubated at 37°C and 140 rpm on a shaker to simulate an in vitro release environment. 1 ml of release medium was collected at 5 min, 15, 30, 45 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 36 h, and replenished to maintain a constant 30 ml release medium. Calcium ion concentration was measured using ICP-OES, and a release curve was plotted.

[0218] Release curve (e.g.) Figure 16 (As shown) This indicates the Ca2+ of LJ-2P NPs at release times of 30 min and pH values ​​of 7.4 and 5.0. 2+ The cumulative dissolution rates were 8% and 80%, respectively. Subsequently, in the release medium at pH 7.4, the solubility of LJ-2P NPs slowly increased to 14% within 36 hours, while in the release medium at pH 5.0, the solubility of LJ-2P NPs reached 89%. These results demonstrate that LJ-2P NPs exhibit acid-sensitive properties and can cleave and release LJ-2P in acidic environments, laying the foundation for in vivo experiments.

[0219] Example 9: NIR-II Imaging Method

[0220] (1) Establishment of a 4T1-luciferase in situ breast cancer mouse model:

[0221] BALB / c nude mice were anesthetized, and a 1 cm incision was made in the midline of the thorax under aseptic conditions. Bilateral mammary fat pads were bluntly dissected for cell seeding. 4T1-luciferase-treated breast cancer cells were cultured in complete culture medium, and after trypsin digestion, the concentration was adjusted to 2 × 10⁻⁶. 7 / mL, suspended in PBS. Then, 50μL of the mixed cell suspension was seeded into the third bilateral mammary fat pads of BALB / c nude mice (1×10⁹ / mL per side). 6 In a single cell, a noticeable skin protrusion appeared at the injection site. The incision was then sutured, and the tumor burden in mice was assessed by monitoring the whole-animal bioluminescence imaging.

[0222] (2) NIR-II imaging method:

[0223] 200 μL of LJ-2P NPs or LJ-2P (0.25 mg / mL) were intravenously injected into BALB / c nude mice. Mice were anesthetized with a 2 L / min oxygen flow of 2% isoflurane during injection and imaging. NIR-II fluorescence images were collected using a two-dimensional InGaAs array (Suzhou Optics) for photon collection in NIR-II, with excitation light provided by an 808 nm diode laser. The light emitted by the animals was coupled to an InGaAs camera through a 1000 nm or 1150 nm long-pass filter for NIR-II imaging.

[0224] LJ-2P NPs imaging tumor imaging results show (e.g.) Figure 8 , Figure 10 , Figure 12 and Figure 13 (As shown) It exhibits good tumor angiography and tumor accumulation capabilities. Tumor angiography results show a full width at half maximum (FWHM) of 0.352 mm under a 1000 nm long-pass filter. Figure 9 (As shown); with a long-pass filter at 1150nm, the full width at half maximum (FWHM) is 0.314mm (as shown). Figure 11 (As shown). The time variation of LJ-2P NPs accumulation at the tumor site was then measured. Due to the high permeability and retention (EPR) effect of solid tumors, LJ-2P NPs peaked at 3 hours, with a signal-to-noise ratio of 4.84 between tumor and normal tissue. Subsequently, as the tumor was cleared from the body, the brightness of the tumor site gradually decreased (as shown). Figure 14 (As shown).

[0225] Both LJ-2P NPs and LJ-2P showed good metabolic efficiency. Following tail vein injection of LJ-2P NPs, the liver metabolized 80% within 24 hours and completely metabolized it within 8 days (e.g., ...). Figure 19 and Figure 20(As shown). After tail vein injection of LJ-2P, the liver metabolizes 80% in 4 days and completely metabolizes it in 14 days (as shown). Figure 17 and Figure 18 (As shown). These benefits are due to the acid-sensitive properties of calcium phosphate nanoparticles and the good water solubility of LJ-2P.

[0226] Example 10 Histological Analysis

[0227] Tumors and major organs were obtained from BALB / c nude mice and fixed in EDTA / formalin solution. After sectioning, various tissue samples were stained with H&E.

[0228] Staining revealed no specific changes in major organs, demonstrating the good biocompatibility of LJ-2P NPs, such as... Figure 15 As shown.

[0229] Comparative Example 1: Unlike LJ-2P and LJ-4P, the following molecules also contain phosphate groups, but cannot form calcium phosphate nanoparticles.

[0230]

[0231] Comparative Example 2: The following molecules containing phosphate groups are synthesized slowly, require an alkaline environment during synthesis, and their structures are easily destroyed.

[0232]

Claims

1. A compound, or a salt thereof, wherein the compound is or .

2. A method for preparing the compound as described in claim 1, characterized in that, It is any of the following methods: Method 1, when the compound is a compound as shown in Formula I-1, comprises the following steps: preparing I-1 by acid hydrolysis of the compound as shown in Formula I-1-1 in a solvent; ； In compound I-1-1, R 1-1 Independently -(CH2) n1 -R, n1 is 1, 2, 3 or 4, R is TMS, TES, TBDMS, TBDPS, DIPS, DPS or TIPDS; L 1 for L is -(CH2)3-; Method 2, when the compound is a compound as shown in Formula I-2, comprises the following steps: reacting compound I-2-1 with POCl3 in a solvent, and then mixing with water; L 1 for .

3. The method for preparing the compound according to claim 2, characterized in that, It meets one or more of the following conditions: (1) In method 1, the acid is trifluoroacetic acid; (2) In method 1, the solvent is a halogenated hydrocarbon solvent.

4. The method for preparing the compound according to claim 2, characterized in that, In Method 1, the solvent is dichloromethane.

5. The method for preparing the compound according to claim 2, characterized in that, The method 1 further includes post-processing, which includes the following steps: after the acidolysis reaction is completed, quenching and recrystallization are performed.

6. The method for preparing the compound according to claim 5, characterized in that, It meets one or two of the following conditions: (1) In the method 1, the quenching in the post-processing is quenching with an ether solvent; (2) In the method 1, the solvent for recrystallization in the post-processing is an ether solvent.

7. The method for preparing the compound according to claim 5, characterized in that, It meets one or two of the following conditions: (1) In the method 1, the quenching in the post-processing is quenching with ether; (2) In the method 1, the solvent for recrystallization in the post-processing is diethyl ether.

8. The method for preparing the compound according to claim 2, characterized in that, The method 2 includes the following steps: in a solvent, under the action of an alkali, compound I-2-1 reacts with POCl3, and is then mixed with water.

9. The method for preparing the compound according to claim 2, characterized in that, In method 2, the solvent is an ether solvent.

10. The method for preparing the compound according to claim 2, characterized in that, In method 2, the solvent is tetrahydrofuran.

11. The method for preparing the compound according to claim 8, characterized in that, In method 2, the base is an organic amine.

12. The method for preparing the compound according to claim 8, characterized in that, In method 2, the base is triethylamine.

13. The method for preparing the compound according to claim 2, characterized in that, The method 2 further includes a post-processing step, which includes the following steps: precipitation with acid and recrystallization.

14. The method for preparing the compound according to claim 13, characterized in that, It meets one or more of the following conditions: (1) In the method 2, the acid in the post-processing is an inorganic acid; (2) In method 2, the molar concentration of the acid in the post-treatment is 1~3 mol / L; (3) In the method 2, the solvent for recrystallization in the post-processing is an ether solvent.

15. The method for preparing the compound according to claim 13, characterized in that, It meets one or more of the following conditions: (1) In the method 2, the acid in the post-processing is hydrochloric acid; (2) In method 2, the molar concentration of the acid in the post-treatment is 2 mol / L; (3) In method 2, the solvent for recrystallization in the post-processing is tetrahydrofuran.

16. A compound as shown in Formula I-1-1, I-1-2 or Formula I-2-1: The compound shown in Formula I-1-1 is ; The compound shown in Formula I-1-2 is ; The compound shown in Formula I-2-1 is .

17. An organocalcium phosphate nanoparticle, characterized in that, It contains a calcium salt of the compound as described in claim 1, and has a drug loading of 2%-4.5%, wherein the drug loading is the mass ratio of the compound to the organophosphate calcium nanoparticles.

18. The organocalcium phosphate nanoparticles as described in claim 17, characterized in that, It meets one or more of the following conditions: (1) The calcium salt of the compound has a mass percentage of 2.1% to 4.6%; (2) The particle size of the organic calcium phosphate nanoparticles is 8~90nm; (3) The hydration diameter of the organic calcium phosphate nanoparticles is 10~100 nm; (4) The absorption wavelength of the organic calcium phosphate nanoparticles is 600~1000nm; (5) The emission wavelength of the organic calcium phosphate nanoparticles is 900~1600nm; (6) The organic calcium phosphate nanoparticles further comprise calcium phosphate; (7) The organic calcium phosphate nanoparticles further comprise substance A and substance B; substance A is a salt formed by an anion in an anionic surfactant and calcium ions, and substance B is an amphiphilic compound.

19. The organocalcium phosphate nanoparticles as described in claim 17, characterized in that, It meets one or more of the following conditions: (1) The percentage of the organic calcium phosphate nanoparticles with a hydration diameter in the range of 35~65 nm is 50%~80%; (2) The maximum absorption wavelength of the organic calcium phosphate nanoparticles is 773 nm; (3) The maximum emission wavelength of the organic calcium phosphate nanoparticles is 1150 nm; (4) The organophosphate nanoparticles further comprise calcium phosphate; the calcium phosphate has a mass percentage of 6% to 10%; (5) The organophosphate nanoparticles further comprise substance A and substance B; substance A is a salt formed by anion and calcium ions in an anionic surfactant, and substance B is an amphiphilic compound; the mass percentage of substance A is 15%~18%; (6) The organophosphate nanoparticles further comprise substance A and substance B; substance A is a salt formed by an anion in an anionic surfactant and calcium ions, and substance B is an amphiphilic compound; the mass percentage of substance B is 68%~72%.

20. The organocalcium phosphate nanoparticles as described in claim 17, characterized in that, It meets one or more of the following conditions: (1) The percentage of the organic calcium phosphate nanoparticles with a hydration diameter in the range of 10~35 nm is 5%~15%; (2) The maximum absorption wavelength of the organic calcium phosphate nanoparticles is 720~820nm; (3) The maximum emission wavelength of the organic calcium phosphate nanoparticles is 1000~1300 nm; (4) The organic calcium phosphate nanoparticles comprise the calcium salt of the compound, calcium phosphate, substance A and substance B, wherein the calcium salt of the compound and calcium phosphate are mixed to form a coprecipitate, substance A is a salt formed by an anion and calcium ions in an anionic surfactant, substance B is an amphiphilic compound, substance A coats the outer surface of the coprecipitate to form the nanoparticle core, and substance B coats the outer surface of substance A.

21. The organocalcium phosphate nanoparticles as described in claim 17, characterized in that, The percentage of the organic calcium phosphate nanoparticles with a hydration diameter in the range of 65-100 nm is 15%-35%.

22. The organocalcium phosphate nanoparticles as described in claim 17, characterized in that, The organocalcium phosphate nanoparticles comprise the calcium salt of the compound, calcium phosphate, substance A, and substance B. The calcium salt and calcium phosphate of the compound are mixed to form a co-precipitate. Substance A is a salt formed by an anion in an anionic surfactant and calcium ions. Substance B is an amphiphilic compound. Substance A coats the outer surface of the co-precipitate to form a nanoparticle core, and substance B coats the outer surface of substance A. The hydration diameter of the nanoparticle core is 3-45 nm.

23. The organocalcium phosphate nanoparticles as described in claim 17, characterized in that, The organocalcium phosphate nanoparticles comprise the calcium salt of the compound, calcium phosphate, substance A, and substance B. The calcium salt and calcium phosphate of the compound are mixed to form a co-precipitate. Substance A is a salt formed by an anion in an anionic surfactant and calcium ions. Substance B is an amphiphilic compound. Substance A coats the outer surface of the co-precipitate, forming a nanoparticle core. Substance B coats the outer surface of substance A. The percentage of nanoparticle cores with a hydration diameter in the range of 8-12 nm is 50%-80%.

24. The organocalcium phosphate nanoparticles as described in claim 23, characterized in that, The percentage of nanoparticle cores with hydration diameters in the range of 3-8 nm is 10%-25%.

25. The organocalcium phosphate nanoparticles as described in claim 23, characterized in that, The percentage of nanoparticle cores with hydration diameters in the range of 12-45 nm is 15%-35%.

26. The organocalcium phosphate nanoparticles as described in claim 23, characterized in that, It meets one or more of the following conditions: (1) The drug loading of the organophosphate nanoparticles is 2.15%; (2) The anionic surfactant is a phosphate ester anionic surfactant; (3) The amphiphilic compounds are phospholipids, amino lipids and sphingolipids.

27. The organocalcium phosphate nanoparticles as described in claim 18, characterized in that, It meets one or two of the following conditions: (1) When the organophosphate nanoparticles contain substance A, the anionic surfactant is DLPA, DMPA, POPA, DSPA, DPPA, 16:0 Lyso PG, POPG, SOPA, DEPA or DOPA. (2) When the organophosphate nanoparticles contain substance B, the amphiphilic compound is a phospholipid, an amino lipid, and a sphingolipid, and the phospholipid is phosphatidylcholine, phosphatidylethanolamine, distearylphosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearylphosphatidylcholine, dioleoylphosphatidylcholine, or dithioylphosphatidylcholine.

28. The organocalcium phosphate nanoparticles as described in claim 18, characterized in that, It meets one or more of the following conditions: (1) The organocalcium phosphate nanoparticles are composed of the calcium salt of the compound, calcium phosphate, substance A and substance B; (2) When the organophosphate nanoparticles contain substance B, the amphiphilic compound is a phospholipid; (3) When the organophosphate nanoparticles contain substance A, the anionic surfactant is RPO3M2; wherein R is m1, m2, m3 and m4 are each independently 6, 7, 8, 9 or 10; M is an alkali metal; (4) The organic calcium phosphate nanoparticles were synthesized by reverse microemulsion method.

29. The organocalcium phosphate nanoparticles as described in claim 18, characterized in that, It meets one or two of the following conditions: (1) When the organophosphate nanoparticles contain substance B, the amphiphilic compound is a PEGylated derivative of phospholipid; (2) When the organophosphate nanoparticles contain substance A, the anionic surfactant is RPO3M2; wherein R is m1, m2, m3 and m4 are each independently 7; M is Na.

30. The organocalcium phosphate nanoparticles as described in claim 18, characterized in that, When the organophosphate nanoparticles contain substance B, the amphiphilic compound is DSPE-mPEG.

31. The organocalcium phosphate nanoparticles as described in claim 18, characterized in that, When the organophosphate nanoparticles contain substance B, the amphiphilic compound is DSPE-PEG-2000.

32. A method for preparing calcium phosphate nanoparticles, characterized in that, It includes the following steps: ①: A solution containing calcium ions is mixed with a solution containing the compound as described in claim 1 to obtain mixture a; ②: Mix the mixture a described in ① with anionic surfactant and demulsifier to obtain a precipitate; ③: Mix the precipitate described in ② with the amphiphilic compound and water; In ①, the solution containing calcium ions is a mixture of "aqueous solution of soluble calcium salt" and "organic solvent containing nonionic surfactant", referred to as the calcium phase; The solution containing the compound as described in claim 1 is " The mixture of the phosphate aqueous solution of the compound as described in claim 1 and the organic solvent containing a nonionic surfactant is referred to as the phosphorus phase.

33. The method for preparing calcium phosphate nanoparticles as described in claim 32, characterized in that, It meets one or more of the following conditions: (1) In ①, the ratio of the amount of the compound to the amount of calcium ions is 1:(1500~4000). (2) In ①, the mixing is to add "a solution containing the compound as described in claim 1" to "a solution containing calcium ions"; (3) In ①, after mixing, the mixture is stirred to obtain mixture a; (4) The soluble calcium salt is selected from one or more of calcium chloride, calcium nitrate, calcium bicarbonate, calcium bisulfate, calcium bisulfite, calcium hypochlorite, calcium bromide, calcium iodide, calcium chlorate, calcium perchlorate and calcium permanganate. (5) The concentration of the aqueous solution of the soluble calcium salt is 2~3 mol / L; (6) The volume ratio of the "aqueous solution of soluble calcium salt" to the "organic solvent containing nonionic surfactant" is 0.3:(8~12); (7) The calcium phase is obtained by mixing the "aqueous solution of soluble calcium salt" with the "organic solvent containing nonionic surfactant" and stirring. (8) The pH of the calcium phase is 5.6-8.0; (9) The phosphate is sodium dihydrogen phosphate and / or disodium hydrogen phosphate; (10) The phosphate aqueous solution of the compound, i.e., the compound is used as the solute and the phosphate aqueous solution is used as the solvent, wherein the concentration of the solvent is 0.005~0.025mol / L; (11) The phosphate aqueous solution of the compound is used as the solute and the phosphate aqueous solution is used as the solvent, wherein the molar ratio of the compound to the phosphate ions in the solvent is 1:(10~40). (12) The volume ratio of the "phosphate aqueous solution of the compound" to the "organic solvent containing a nonionic surfactant" is 0.3:(8~12); (13) The phosphorus phase is obtained by mixing "an aqueous solution of the phosphate of the compound" with "an organic solvent containing a nonionic surfactant" and stirring. (14) The pH of the phosphorus phase is 2.0-8.0; (15) In the "organic solvent containing nonionic surfactant", the organic solvent is a hydrocarbon solvent and / or an alcohol solvent; (16) In the "organic solvent containing nonionic surfactant", when the organic solvent is a mixture of hydrocarbon solvent and alcohol solvent, the volume ratio of the hydrocarbon solvent and alcohol solvent is (7~8):1; (17) In the "organic solvent containing a nonionic surfactant", the nonionic surfactant is an alkylphenol polyoxyethylene ether; (18) In the "organic solvent containing nonionic surfactant", the volume ratio of the organic solvent to the nonionic surfactant is (2~7):1; (19) The volume ratio of the "organic solvent containing nonionic surfactant" in the phosphorus phase to the "organic solvent containing nonionic surfactant" in the calcium phase is (0.8~1.2):1; (20) ② refers to mixing the mixture a mentioned in ① with an anionic surfactant to obtain mixture b, and then mixing it with a demulsifier to obtain a precipitate; (21) The anionic surfactant as described in any one of claims 26-29; (22) The molar ratio of the anionic surfactant to the compound is (8~30):1; (23) In the second part, the anionic surfactant is its solution; (24) In the second part, the demulsifier is an alcohol solvent; (25) In step ②, after obtaining the precipitate, the precipitate is collected and washed with a demulsifier; (26) In the third step, the amphiphilic compound is as described in any one of claims 28-31; (27) ③ refers to dissolving the precipitate described in ②, mixing it with an amphiphilic compound to obtain a mixture c, and then mixing it with water; (28) The mass ratio of the amphiphilic compound to the precipitate described in ② is (2~3):1; (29) The amphiphilic compound participates in the reaction in the form of an amphiphilic compound solution; (30) After step ③ is completed, post-processing is performed through the following steps: concentration; (31) The particle size, hydration diameter, absorption wavelength, maximum absorption wavelength, emission wavelength, maximum emission wavelength, drug loading and composition of the calcium phosphate nanoparticles are as described in any one of claims 17 to 31.

34. The method for preparing calcium phosphate nanoparticles as described in claim 32, characterized in that, It meets one or more of the following conditions: (1) In ①, the ratio of the amount of the compound to the amount of calcium ions is 1:(3000~3500). (2) In ①, after mixing, the mixture is stirred to obtain mixture a, and the stirring time is 8 to 15 minutes; (3) The soluble calcium salt is calcium chloride; (4) The concentration of the aqueous solution of the soluble calcium salt is 2.5 mol / L; (5) The volume ratio of the "aqueous solution of soluble calcium salt" to the "organic solvent containing nonionic surfactant" is 0.3:10; (6) The calcium phase is obtained by mixing the "aqueous solution of soluble calcium salt" with the "organic solvent containing nonionic surfactant" and stirring for 3 to 8 minutes. (7) The pH of the calcium phase is 5.6; (8) The phosphate mentioned is disodium hydrogen phosphate; (9) The phosphate aqueous solution of the compound, i.e., the compound is used as the solute and the phosphate aqueous solution is used as the solvent, wherein the concentration of the solvent is 0.02 mol / L; (10) A phosphate aqueous solution of the compound, wherein the compound is used as a solute and the phosphate aqueous solution is used as a solvent, and the molar ratio of the compound to the phosphate ions in the solvent is 1:(10~30). (11) The volume ratio of the "phosphate aqueous solution of the compound" to the "organic solvent containing a nonionic surfactant" is 0.3:10; (12) The phosphorus phase is obtained by mixing "an aqueous solution of the phosphate of the compound" with "an organic solvent containing a nonionic surfactant" and stirring for 3 to 8 minutes. (13) The pH of the phosphorus phase is 2.0; (14) In the "organic solvent containing nonionic surfactant", the organic solvent is a hydrocarbon solvent and / or an alcohol solvent; the hydrocarbon solvent is cyclohexane; (15) In the "organic solvent containing nonionic surfactant", the organic solvent is a hydrocarbon solvent and / or an alcohol solvent; the alcohol solvent is n-hexanol; (16) In the "organic solvent containing nonionic surfactant", when the organic solvent is a mixture of hydrocarbon solvent and alcohol solvent, the volume ratio of the hydrocarbon solvent to the alcohol solvent is 7.5:1; (17) In the “organic solvent containing nonionic surfactant”, the nonionic surfactant is polyethylene glycol octylphenyl ether (triton x100) and / or polyoxyethylene (5) nonylphenyl ether. (18) In the “organic solvent containing nonionic surfactant”, the volume ratio of the organic solvent to the nonionic surfactant is 2.3:1 or 5.7:1; (19) The volume ratio of the "organic solvent containing nonionic surfactant" in the phosphorus phase to the "organic solvent containing nonionic surfactant" in the calcium phase is 1:1; (20) ② refers to mixing the mixture a described in ① with an anionic surfactant to obtain mixture b, and then mixing it with a demulsifier to obtain a precipitate; the mixing of the mixture a described in ① with an anionic surfactant refers to adding the anionic surfactant dropwise to the mixture a described in ①. (21) The molar ratio of the anionic surfactant to the compound is (8~20):1; (22) In the ② above, the anionic surfactant is its solution, and the solvent in the solution is a halocarbon solvent; (23) In the second part, the demulsifier is ethanol; (24) In step ②, after obtaining the precipitate, the precipitate is collected and washed with a demulsifier, and the washing is performed 2 to 4 times. (25) ③ refers to dissolving the precipitate described in ②, mixing it with an amphiphilic compound to obtain a mixture c, and then mixing it with water; the latter means pouring the mixture c into water. (26) The mass ratio of the amphiphilic compound to the precipitate described in ② is 2.5:1; (27) The amphiphilic compound participates in the reaction in the form of an amphiphilic compound solution; the solvent in the amphiphilic compound solution is an organic solvent; (28) After the completion of step ③, post-processing is carried out through the following steps: concentration; the concentration is to filter the mixture obtained in step ③ and concentrate the filtrate.

35. The method for preparing calcium phosphate nanoparticles as described in claim 32, characterized in that, It meets one or more of the following conditions: (1) In ①, the molar ratio of the compound to the calcium ion is 1:3348; (2) In ①, after mixing, the mixture is stirred to obtain mixture a, and the stirring time is 10 minutes; (3) The calcium phase is obtained by mixing the "aqueous solution of soluble calcium salt" with the "organic solvent containing nonionic surfactant" and stirring for 5 minutes. (4) The phosphate aqueous solution of the compound is used as the solute and the phosphate aqueous solution is used as the solvent, and the molar ratio of the compound to the phosphate ions in the solvent is 1:

27. (5) The phosphorus phase is obtained by mixing "an aqueous solution of the phosphate of the compound" with "an organic solvent containing a nonionic surfactant" and stirring for 5 minutes. (6) In the "organic solvent containing a nonionic surfactant", the nonionic surfactant is Igepal CO-520; (7) In the "organic solvent containing nonionic surfactant", the volume ratio of the organic solvent to the nonionic surfactant is 2.3:1; (8) Mix the mixture a described in ① with the anionic surfactant, stir to obtain mixture b, and then mix with the demulsifier; (9) The molar ratio of the anionic surfactant to the compound is 18:1; (10) In the second part, the anionic surfactant is a solution thereof, and the solvent in the solution is chloroform; (11) In step ②, after obtaining the precipitate, the precipitate is collected and washed with a demulsifier, and the washing is performed 3 times; (12) ③ refers to dissolving the precipitate described in ②, mixing it with an amphiphilic compound to obtain a mixture c, and then mixing it with water; the further mixing with water refers to pouring the mixture c into water; the volume ratio of the mixture c to the water is 1:(2.5~4). (13) The amphiphilic compound participates in the reaction in the form of an amphiphilic compound solution; the solvent in the amphiphilic compound solution is an ether solvent; (14) After the completion of step ③, post-processing is carried out through the following steps: concentration; the concentration is to filter the mixture obtained in step ③ and concentrate the filtrate; before the filtration, the organic solvent present in the mixture obtained in step ③ is removed.

36. The method for preparing calcium phosphate nanoparticles as described in claim 32, characterized in that, It meets one or more of the following conditions: (1) The phosphorus phase is obtained by mixing "aqueous solution of phosphate of the compound" with "organic solvent containing nonionic surfactant" and stirring; the mixing is by adding "phosphate solution of the compound" dropwise to "organic solvent containing nonionic surfactant"; (2) In the "organic solvent containing a nonionic surfactant", the nonionic surfactant is Igepal CO-520; the number average molecular weight is 441, and the linear molecular formula is (C2H4O)n • C 15 H 24 O, n~5; (3) Mix the mixture a described in ① with the anionic surfactant, stir to obtain mixture b, and then mix with the demulsifier. The stirring time is 25-35 minutes. (4) In the second part, the anionic surfactant is a solution thereof, and the concentration of the solution is 0.02~0.03 mol / L. (5) ③ refers to dissolving the precipitate described in ②, mixing it with an amphiphilic compound to obtain a mixture c, and then mixing it with water; the volume ratio of the mixture c to the water is 1:

3. (6) The amphiphilic compound participates in the reaction in the form of an amphiphilic compound solution; the solvent in the amphiphilic compound solution is tetrahydrofuran; (7) After the completion of step ③, post-processing is carried out through the following steps: concentration; the concentration is to filter the mixture obtained in step ③ and concentrate the filtrate; the filtration is to use micromembrane filtration.

37. The method for preparing calcium phosphate nanoparticles as described in claim 32, characterized in that, It meets one or more of the following conditions: (1) Mix the mixture a described in ① with an anionic surfactant and stir to obtain mixture b, then mix with a demulsifier for 30 minutes; (2) In the second part, the anionic surfactant is a solution of itself, and the solvent in the solution is chloroform; the concentration of the solution is 0.026 mol / L. (3) ③ refers to dissolving the precipitate described in ②, mixing it with an amphiphilic compound to obtain a mixture c, and then mixing it with water; the solvent used to dissolve the precipitate is an organic solvent. (4) The amphiphilic compound participates in the reaction in the form of an amphiphilic compound solution; the concentration of the amphiphilic compound in the amphiphilic compound solution is 0.005-0.015 mol / L; (5) After the completion of step ③, post-processing is carried out through the following steps: concentration; the concentration is to filter the mixture obtained in step ③ and concentrate the filtrate; the filtration is to filter with a micromembrane, wherein the micromembrane is a 0.22 μm micromembrane.

38. The method for preparing calcium phosphate nanoparticles as described in claim 32, characterized in that, It meets one or more of the following conditions: (1) Mix the mixture a described in ① with an anionic surfactant and stir to obtain mixture b, and then mix with a demulsifier; the volume ratio of the demulsifier to the mixture b is 1: (0.8~1.2). (2) In ②, the anionic surfactant is its solution; the volume ratio of the solution to the mixture a in ① is 1:(120~150). (3) ③ refers to dissolving the precipitate described in ②, mixing it with an amphiphilic compound to obtain a mixture c, and then mixing it with water; the solvent used to dissolve the precipitate is an ether solvent; (4) The amphiphilic compound participates in the reaction in the form of an amphiphilic compound solution; the concentration of the amphiphilic compound in the amphiphilic compound solution is 0.012 mol / L; (5) After the completion of step ③, post-processing is carried out through the following steps: concentration; the concentration is concentration using an ultrafiltration device.

39. The method for preparing calcium phosphate nanoparticles as described in claim 32, characterized in that, It meets one or more of the following conditions: (1) Mix the mixture a described in ① with an anionic surfactant and stir to obtain mixture b, and then mix with a demulsifier; the volume ratio of the demulsifier to the mixture b is 1:

1. (2) In ②, the anionic surfactant is its solution; the volume ratio of the solution to the mixture a in ① is 1:137; (3) ③ refers to dissolving the precipitate described in ②, mixing it with an amphiphilic compound to obtain a mixture c, and then mixing it with water; the solvent used to dissolve the precipitate is tetrahydrofuran.

40. The method for preparing calcium phosphate nanoparticles as described in claim 32, characterized in that, The ③ step involves dissolving the precipitate described in ②, mixing it with an amphiphilic compound to obtain a mixture c, and then mixing it with water; the volume-to-mass ratio of the solvent used to dissolve the precipitate to the precipitate is 1~2 ml / mg.

41. The method for preparing calcium phosphate nanoparticles as described in claim 32, characterized in that, The third step involves dissolving the precipitate described in step ②, mixing it with an amphiphilic compound to obtain a mixture c, and then mixing it with water; the volume-to-mass ratio of the solvent used to dissolve the precipitate to the precipitate is 1 ml / mg.

42. The method for preparing calcium phosphate nanoparticles according to any one of claims 32-41, characterized in that, The process includes the following steps: mixing a calcium chloride solution with a cyclohexane:Igepal CO-520 solution at a volume ratio of 7:3 to obtain a calcium phase; adding a Na2HPO4 solution of the compound dropwise to the cyclohexane:Igepal CO-520 solution at a volume ratio of 7:3, mixing and stirring to form a phosphorus phase; adding the phosphorus phase to the calcium phase and stirring, then adding DOPA solution dropwise and stirring; adding ethanol to precipitate, collecting the precipitate, and washing the precipitate with ethanol; redissolving the precipitate in tetrahydrofuran, mixing it with a tetrahydrofuran solution of DSPE-PEG-2000, and quickly pouring the mixture into water.

43. Calcium phosphate nanoparticles prepared by the method for preparing calcium phosphate nanoparticles according to any one of claims 32 to 42.

44. The use of a compound as described in claim 1, or a salt thereof, in the preparation of calcium phosphate nanoparticles.

45. The use of a compound as claimed in claim 1, its salt, calcium phosphate nanoparticles as claimed in any one of claims 17 to 31, or calcium phosphate nanoparticles as claimed in claim 43 in the preparation of a near-infrared fluorescent probe.

46. ​​The application as described in claim 45, characterized in that, The probe described is a near-infrared II fluorescent probe used for tumor and vascular imaging.

47. The application as described in claim 46, characterized in that, The tumor and vascular imaging described refers to tumor and vascular imaging in experimental animals.

48. A near-infrared II fluorescent probe comprising the compound of claim 1, its salt, calcium phosphate nanoparticles of any one of claims 17-31, or calcium phosphate nanoparticles of claim 43.

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