A two-photon triptolide nanomedicine for treating gouty arthritis and a preparation method and application thereof

Abstract

The application discloses a nano drug formed by triptolide and a TPA-(BT-TPE)3 fluorescent probe with an AIE effect, and a preparation method and application thereof. The two-photon triptolide nano drug is a nanoparticle, which is formed by triptolide and the TPA-(BT-TPE)3 fluorescent probe through a drug solubilization and controlled release agent assembly. The preparation method of the two-photon triptolide nano drug comprises the following steps: a) raw material dissolution: triptolide, the TPA-(BT-TPE)3 fluorescent probe and a drug solubilization and controlled release agent are dissolved in an organic solvent to form a premix; b) nano precipitation: under stirring, the premix is added into a poor solvent of triptolide, and stirring and evaporation are performed until the organic solvent is completely removed, so that a reaction mixture is obtained; and c) post-treatment: the reaction mixture is purified and concentrated to obtain a finished product. The application can not only improve the treatment effect of triptolide, but also provide intuitive basis for disease diagnosis and treatment effect tracking by using two-photon imaging technology.

Description

Technical Field

[0001] This invention relates to the technical field of drug preparation, and in particular to the technical field of two-photon triptolide nanomedicine. Background Technology

[0002] Gouty arthritis (GA) is an acute joint disease caused by metabolic abnormalities, characterized by sudden, severe joint pain, redness, and swelling. The pathological basis of this disease is long-term purine metabolism disorder, leading to elevated blood uric acid levels. When the concentration of uric acid in the blood becomes excessively saturated, monosodium urate (MSU) crystals are formed and deposited in the joints and surrounding tissues, thereby inducing an acute inflammatory response. In clinical treatment, colchicine, nonsteroidal anti-inflammatory drugs (NSAIDs), and glucocorticoids are commonly used. Although these drugs can effectively inhibit inflammation and relieve pain, they still have the problems of easy relapse after discontinuation and a high incidence of adverse reactions.

[0003] Triptolide (TPL) is a key active ingredient extracted from the traditional Chinese medicine Tripterygium wilfordii Hook.f. Existing research indicates that TPL can treat gouty arthritis through a dual pathway: the PI3K / Akt signaling pathway regulates macrophage polarization, inhibiting M1 polarization and reducing inflammatory factor secretion while promoting M2 polarization; additionally, it inhibits neutrophil activation and recruitment, and regulates neutrophil death by modulating extracellular trap formation, autophagy, and apoptosis, thereby alleviating tissue damage and inflammatory responses caused by neutrophil activation (Du Y, Zhang Y, Jiang Z, Xu L, Ru J, Wei S, Chen W, DongR, Zhang S, Jia T. Triptolide alleviates acute gouty arthritis caused by monosodium urate crystals by modulating macrophage polarization and neutrophil activity. Immunol Lett. 2024 Oct;269:106907. doi: 10.1016 / j.imlet.2024.106907. Epub 2024 Aug 8. PMID: 39122094); However, while the epoxy groups in the TPL structure endow it with activity, they also lead to defects such as poor water solubility, low bioavailability, short half-life, and toxicity to organs such as the liver and kidneys (which limits the further clinical application of TPL).

[0004] Drug carriers can improve the solubility and targeting of triptolide (TPL) and reduce its toxicity. Currently, the development of triptolide drug carriers mainly focuses on liposomes and hydrogels, such as a functionalized modified triptolide nanoliposome, preparation method and use (CN118831177A), a triptolide-gold nanoparticle / hyaluronic acid composite hydrogel and its preparation and application (CN113577016B), and a reactive oxygen species-responsive triptolide prodrug immunomodulatory hydrogel formulation, preparation method and application (CN120168394A). However, most of these triptolide drug carriers suffer from complex carrier synthesis processes, making large-scale production difficult, and their in vivo metabolic mechanisms are not yet fully understood, requiring further exploration and optimization.

[0005] Furthermore, existing drugs for gouty arthritis often fail to achieve non-invasive in vivo bioimaging of the site of inflammation. Summary of the Invention

[0006] This invention discloses a nanomedicine formed by assembling triptolide with a TPA-(BT-TPE)3 fluorescent probe with AIE effect, its preparation method and application. It can not only improve the therapeutic effect of triptolide, but also provide intuitive evidence for disease diagnosis and efficacy tracking by using two-photon imaging technology.

[0007] To achieve the above objectives, the present invention provides the following technical solution: Two-photon triptolide nanomedicine consists of nanoparticles formed by assembling triptolide and TPA-(BT-TPE)3 fluorescent probe (a fluorescent probe with AIE effect) through a drug solubilizing and controlled-release phase.

[0008] Preferably, the average particle size is 43–615 nm, the PDI is 0.40–0.45, and the Zeta potential is -18.9 mV–-33.6 mV.

[0009] Furthermore, the average particle size is 43–550 nm.

[0010] Furthermore, the average particle size is 78–220 nm.

[0011] Preferably, the TPA-(BT-TPE)3 fluorescent probe has the following molecular structure: .

[0012] The TPA-(BT-TPE)3 fluorescent probe is a high-performance fluorescent labeling tool designed based on the two-photon absorption (TPA) principle. It can be applied to fields such as bioimaging, medical diagnosis, and monitoring of dynamic cell processes. Compared with traditional single-photon fluorescent probes, this two-photon probe can achieve fluorescence emission through near-infrared (NIR) or long-wavelength excitation light, and has advantages such as deeper tissue penetration, lower phototoxicity, and higher spatial resolution. It is particularly suitable for in vivo tissue and three-dimensional imaging research. This application combines triptolide with the TPA-(BT-TPE)3 fluorescent probe with AIE effect, thereby utilizing the minimal autofluorescence interference and deeper imaging penetration of the TPA-(BT-TPE)3 fluorescent probe to complete non-invasive in vivo bioimaging of the inflammation site. In other words, this design can not only significantly improve the drug efficacy of triptolide, but also use fluorescence to achieve non-invasive in vivo bioimaging of the inflammation site.

[0013] The preparation method of the two-photon triptolide nanomedicine described above includes the following steps: a) Raw material dissolution: Tripterygium wilfordii, TPA-(BT-TPE)3 fluorescent probe and drug solubilizer and controlled release agent are dissolved together in an organic solvent to form a premix; b) Nanoprecipitate: Under stirring, a premixed solution was added to the poor solvent of triptolide, and the mixture was stirred and evaporated until the organic solvent was completely removed to obtain the reaction mixture; c) Post-processing: The reaction mixture is purified and concentrated to obtain the final product.

[0014] This invention utilizes a nanoprecipitation method to prepare nanoparticles, and simultaneously introduces a drug solubilizer and controlled-release agent to load triptolide, enabling triptolide to be released slowly at the affected area. This improves the solubility and bioavailability of triptolide on the one hand, and reduces its toxicity to normal cells on the other, ultimately effectively enhancing the therapeutic effect of the drug on gouty arthritis.

[0015] Preferably, in step a), the source of triptolide is not limited, and commercial triptolide extract (with a purity of more than 90%) can be used.

[0016] Preferably, in step a), the mass ratio of TPA-(BT-TPE)3 fluorescent probe to drug solubilizer and controlled-release agent is controlled at 1:6 to 20, and the added mass of triptolide does not exceed 10% of the added mass of drug solubilizer and controlled-release agent; wherein, the average particle size of the two-photon triptolide nanoparticles will vary with different ratios of TPA-(BT-TPE)3 fluorescent probe to drug solubilizer and controlled-release agent.

[0017] Preferably, in step a), the drug solubilizing and controlled-release agent is poloxamer and / or a synthetic block copolymer, and the organic solvent is one or a combination of several of tetrahydrofuran, methanol, dimethyl sulfoxide, anhydrous ethanol, ethyl acetate, and chloroform; in step b), the poor solvent for triptolide is water and / or PBS buffer; wherein, poloxamer, through its amphiphilic nature, can self-assemble into water-dispersible NPs with a hydrophilic surface and internally loaded triptolide and TPA-(BT-TPE)3 fluorescent probes, thereby effectively solving the problem that hydrophobic functional substances are difficult to stably disperse and transport in aqueous solutions, and bringing better stability, biocompatibility, and longer circulation time to the treatment of gout.

[0018] Furthermore, the synthetic block copolymer is PLGA-PEG or PCL-PEG.

[0019] Furthermore, double-distilled water can be used as a poor solvent for triptolide.

[0020] Preferably, in step a), the synthesis of the TPA-(BT-TPE)3 fluorescent probe involves first mixing tris[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborane-2-yl)phenyl]amine, Br-BT-TPE, Pd(PPh3)4, and an alkaline co-reactant in a liquid reaction medium. After several cycles of freezing-vacuuming-thawing to remove gas, the medium is refilled with inert gas and heated under reflux for 6–18 h. Then, the gas is quenched and purified and concentrated to obtain the finished product (i.e., the orange-red solid TPA-(BT-TPE)3 fluorescent probe).

[0021] Furthermore, the tris[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborane-2-yl)phenyl]amine, Br-BT-TPE, Pd(PPh3)4, and alkaline co-reactant are mixed together in a ratio of 0.05–0.15: 0.25–0.35: 0.0045–0.0065: 1.5–3.5, and the concentration of the tris[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborane-2-yl)phenyl]amine is controlled at 3.33 mmol / L.

[0022] Furthermore, the alkaline co-reactant is K2CO3, the liquid reaction medium is a mixed solution of THF and water in a ratio of 3 to 5:1, and the inert gas is nitrogen.

[0023] Furthermore, the purification and concentration operations involve first extracting the organic layer several times, and then sequentially drying, filtering, concentrating, and separating the collected organic layer by chromatography.

[0024] Preferably, in step b), the organic solvent is removed by stirring evaporation, and the stirring evaporation time is 2 days to 1 month.

[0025] Preferably, in step c), the purification and concentration operation involves centrifuging the remaining reagent after evaporating the organic solvent and then concentrating the supernatant.

[0026] The above-described two-photon triptolide nanomedicine is used in the preparation of gout inhibitory drugs and / or non-invasive in vivo bioimaging of inflammatory sites (i.e., in vivo drug visualization via non-invasive in vivo fluorescence imaging).

[0027] Preferably, the gout suppressant is an injectable formulation; furthermore, there are no particular restrictions on pharmaceutically acceptable excipients for the gout suppressant, as long as they do not damage the nanoparticle structure of the two-photon triptolide nanoparticle drug.

[0028] The beneficial effects of this invention are: 1) Dual-function integration: This invention integrates treatment and imaging by assembling triptolide and TPA-(BT-TPE)3 fluorescent probe with AIE effect into nanoparticles. It can not only treat gout with triptolide, but also perform high-resolution imaging monitoring of inflammation sites under non-invasive in vivo conditions through two-photon imaging technology. At the same time, it can evaluate the distribution and accumulation of drugs in the body in real time during drug administration, providing strong support for early diagnosis, disease assessment and personalized treatment. It can significantly improve the accuracy and controllability of diagnosis and treatment, and has broad clinical translation potential and application prospects. 2) Significantly improved drug performance: This invention effectively improves the water solubility and bioavailability of triptolide through the synergistic effect of nano-processing and drug solubilizing and controlled-release agents, solving the problems of poor absorption and limited efficacy caused by low solubility in clinical applications; at the same time, triptolide is released at the lesion site in a sustained-release form, reducing fluctuations in blood drug concentration and reducing toxicity to normal organs such as the liver and kidneys, thus significantly improving drug safety while ensuring efficacy. 3) Advanced preparation process: This invention uses a nanoprecipitation method to dissolve triptolide, TPA-(BT-TPE)3 fluorescent probe and drug solubilizer and controlled-release agent in an organic solvent, then mix them with a poor solvent, and finally remove the organic solvent by stirring and evaporation to obtain the target nanoparticles. The overall process is simple, the operating conditions are mild, the equipment requirements are low and the repeatability is good, which is very convenient for large-scale production. At the same time, this invention can also effectively control the formation process and final properties of nanoparticles by adjusting the raw material ratio and process parameters, so as to ensure the stability and consistency between product batches. 4) Excellent formulation properties: The nanoparticles prepared by this invention have a controllable particle size in the range of 43 to 615 nm, and a PDI of 0.40 to 0.45 (indicating uniform particle size and good dispersibility); the Zeta potential is between -18.9 mV and -33.6 mV, which endows the formulation with good colloidal stability and makes it less prone to aggregation; in addition, the nanoparticles have good dispersibility and storage stability, and can maintain structural integrity in physiological environment, which is conducive to efficient drug delivery and efficacy.

[0029] The features and advantages of the present invention will be described in detail through embodiments and in conjunction with the accompanying drawings. Attached Figure Description

[0030] Figure 1 This is a chemical structure diagram of triptolide, TPA-(BT-TPE)3 fluorescent probe, and poloxamer; Figure 2 This is a diagram illustrating the chemical reaction process for the synthesis of the TPA-(BT-TPE)3 fluorescent probe; Figure 3 This is a diagram illustrating the molecular orbitals, molecular configuration, and surface electrostatic potential of the TPA-(BT-TPE)3 fluorescent probe. Figure 4 This is a diagram illustrating the AIE properties of the TPA-(BT-TPE)3 fluorescent probe; Figure 5 The TEM and DLS particle size distribution and zeta potential distribution of the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL synthesized in Example 1 are shown. Figure 6 These are TEM and DLS particle size distribution and Zeta potential distribution of the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL synthesized in Example 2; Figure 7 These are TEM and DLS particle size distribution and Zeta potential distribution of the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL synthesized in Example 3; Figure 8 This is the ultraviolet spectrum of the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL synthesized in Example 2; Figure 9 This is the fluorescence spectrum of the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL synthesized in Example 2; Figure 10 This is the PL chromaticity diagram of the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL synthesized in Example 2; Figure 11These are images of RAW264.7 macrophages taking up the nanomedicine TPA-(BT-TPE)3@TPL at different time points in Experiment Example 2; Figure 12 This is the result of the RAW264.7 macrophage cck8 validation of the nanomedicine TPA-(BT-TPE)3@TPL in Experiment Example 3; Figure 13 These are in vivo fluorescence images of mice in Experiment 1 at different times after injection of the nanomedicine TPA-(BT-TPE)3@TPL. Detailed Implementation

[0031] Example 1: The preparation method of the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL includes the following steps: a) Raw material dissolution: Dissolve 1 mg triptolide, 2.2 mg TPA-(BT-TPE)3 fluorescent probe and 14.6 mg drug solubilizer and controlled release agent (poloxam) in 2 ml of organic solvent (tetrahydrofuran) to form a premix; b) Nanoprecipitation: Place 10 ml of the poor solvent of triptolide (double-distilled water) on a magnetic stirrer, and add the premixed solution dropwise while stirring. Evaporate for 3 days to allow the organic solution to evaporate completely, and obtain the reaction mixture. c) Post-processing: The reaction mixture is purified and concentrated to obtain the finished product (i.e., the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL).

[0032] The synthesis steps of the TPA-(BT-TPE)3 fluorescent probe: First, in a 50 ml three-necked flask, tris[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborane-2-yl)phenyl]amine (62.7 mg, 0.10 mmol), Br-BT-TPE (174.0 mg, 0.32 mmol), Pd(PPh3)4 (6.3 mg, 0.0055 mmol) and K2CO3 (343.4 mg, 2.49 mmol) were dissolved in THF (24 mL) and water (6 mL). The mixture was placed in a solution of (mL), and after three cycles of freezing-vacuuming-thawing to degas the mixture, nitrogen was introduced. The reaction mixture was then heated under reflux for 12 h. Subsequently, cold water was injected to quench the reaction, and the mixture was extracted multiple times with chloroform / water. All organic layers were then collected and dried with anhydrous magnesium sulfate (MgSO4). After filtration, the mixture was concentrated by rotary evaporation under reduced pressure. The residue was separated by silica gel column chromatography (using PE / DCM (2 / 1, V / V) as eluent to give an orange-red solid TPA-(BT-TPE)3). The final yield was 60.0 mg (36.4%).

[0033] The chemical structures of triptolide, TPA-(BT-TPE)3 fluorescent probe, and poloxamer are as follows: Figure 1 Parts A, B, and C are shown; this TPA-(BT-TPE)3 fluorescent probe is a novel small-sized organic semiconductor material with AIE properties. The chemical reaction formula for its synthesis can be found in [reference needed]. Figure 2 Molecular orbitals, molecular geometry, and surface electrostatic potential (ESP) can be found in [reference needed]. Figure 3 Part A, Part B, and Part C.

[0034] Furthermore, to investigate the AIE properties of the aforementioned TPA-(BT-TPE)3 fluorescent probe, the TPA-(BT-TPE)3 fluorescent probe was first dissolved in tetrahydrofuran to prepare a 2 μM stock solution. Different proportions of water were gradually added to form mixed solutions with different water contents fw (vol%) (fw = 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95%). Then, a fluorescence spectrophotometer was used to fix the excitation wavelength and measure the emission spectra at different fw (vol%), observing the change in fluorescence intensity with increasing water content. Figure 4 The results shown in Part A indicate that the PL spectrum changed significantly with increasing water volume fraction; this suggests that the luminescence properties of the TPA-(BT-TPE)3 fluorescent probe are highly sensitive to the water ratio; according to Figure 4 The results shown in Part B indicate that the THF / water mixture undergoes a transition from the TICT state to the AIE state as the water volume fraction increases. Specifically, at low water volume fractions, the PL intensity is low (due to fluorescence quenching caused by intramolecular charge transfer); when the water volume fraction reaches a certain value (around 40%), the PL intensity increases significantly (indicating aggregation-induced emission).

[0035] Next, the two-photon triptolide nanoparticles TPA-(BT-TPE)3@TPL prepared in this embodiment were subjected to transmission electron microscopy (TEM), DLS particle size analysis, and zeta potential distribution analysis, and the results can be found in the following figures. Figure 5 The A, B, and C parts of the two-photon triptolide nanoparticle TPA-(BT-TPE)3@TPL nanoparticles were analyzed. Dynamic light scattering tests revealed that the nanoparticles exhibited a size range of 122–615 nm, with uniform size and dispersion. Furthermore, based on… Figure 5 As can be seen from part C, the zeta potential of the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL is -33.8mV.

[0036] Example 2: The amounts of TPA-(BT-TPE)3 fluorescent probe and poloxamer were adjusted to 1.1 mg and 20 mg, respectively, with other parameters the same as in Example 1.

[0037] Next, the two-photon triptolide nanoparticles TPA-(BT-TPE)3@TPL prepared in this embodiment were subjected to transmission electron microscopy (TEM), DLS particle size analysis, and zeta potential distribution analysis, and the results can be found in the following figures. Figure 6 The two-photon triptolide nanoparticles TPA-(BT-TPE)3@TPL are divided into parts A, B, and C. Dynamic light scattering tests show that the nanoparticles have a size of 43–550 nm, are uniform in size, and are evenly dispersed. Furthermore, according to… Figure 6 As can be seen from part C, the zeta potential of the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL is -18.9mV.

[0038] In this embodiment, the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL was also subjected to ultraviolet spectroscopy, fluorescence spectroscopy, and PL colorimetric analysis, and the results are as follows: Figure 8 , Figure 9 and Figure 10 As shown; the ultraviolet spectrum of the triptolide nanomedicine TPA-(BT-TPE)3@TPL indicates that this material has a characteristic absorption peak at 460 nm, which can be used for bioimaging; according to Figure 9 It can be seen that the emission spectrum reaches its peak at 572 nm; according to Figure 10 As can be seen, the color coordinates corresponding to the CIE 1931 chromaticity diagram are located in the transition region from yellow to orange.

[0039] Example 3: The amounts of triptolide, TPA-(BT-TPE)3 fluorescent probe, and poloxamer were adjusted to 0.2 mg, 1.5 mg, and 20 mg, respectively, with other parameters the same as in Example 1.

[0040] Next, the two-photon triptolide nanoparticles TPA-(BT-TPE)3@TPL prepared in this embodiment were subjected to transmission electron microscopy (TEM), DLS particle size analysis, and zeta potential distribution analysis, and the results can be found in the following figures. Figure 7 The two-photon triptolide nanoparticles TPA-(BT-TPE)3@TPL are divided into parts A, B, and C. Dynamic light scattering tests show that the nanoparticles have a size of 78–220 nm, are uniform in size, and are evenly dispersed. Furthermore, according to… Figure 7As can be seen from part C, the zeta potential of the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL is -22.7mV.

[0041] Experimental Example 1: In vivo fluorescence imaging experiments were conducted on mice using the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL prepared in Example 2. Mice were injected via tail vein with the two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL prepared in Example 2, and in vivo fluorescence imaging was performed at 30 min, 5 h, 7 h, 24 h, 48 h, 72 h, and 96 h.

[0042] The results are as follows Figure 13 As shown, the distribution and metabolism of the triptolide nanomedicine TPA-(BT-TPE)3@TPL in mice were dynamically demonstrated. During the period from 30 min to 96 h, the fluorescence intensity increased at the joint site, indicating that the triptolide nanomedicine TPA-(BT-TPE)3@TPL accumulated at the affected area.

[0043] Experimental Example 2: Cell uptake assays of RAW264.7 macrophages were performed using the two-photon triptolide nanoparticle TPA-(BT-TPE)3@TPL prepared in Example 3. Specifically, RAW264.7 macrophages were first seeded onto 35mm laser confocal microplates, and then 1ml of the two-photon triptolide nanoparticle TPA-(BT-TPE)3@TPL prepared in Example 3 and 1ml of complete culture medium were incubated with the cells for 0h, 30min, 2h, and 4h, respectively. Subsequently, the culture medium was removed and the cells were subjected to PBS washing, paraformaldehyde fixation, glycine quenching, permeabilization, PBS washing, DAPI staining in the dark, and PBS washing again, followed by laser confocal imaging of the cells.

[0044] The results are as follows Figure 11 As shown, the triptolide nanomedicine TPA-(BT-TPE)3@TPL can be internalized into RAW264.7 macrophages within 30 min, and the signal of the triptolide nanomedicine TPA-(BT-TPE)3@TPL is enhanced with the extension of time.

[0045] Experimental Example 3: The two-photon triptolide nanomedicine TPA-(BT-TPE)3@TPL prepared in Example 3 was used to conduct a nanomedicine cytotoxicity test; specifically, RAW264.7 macrophages were first subjected to a cytotoxicity test at a concentration of 2 × 10⁻⁶ cells. 5Cells were seeded per well in a 96-well plate, with each well containing 1 μg / mL LPS for induction. After 24 h of culture, the old culture medium was discarded, and DMEM medium containing the two-photon triptolide nanodrug TPA-(BT-TPE)3@TPL prepared in Example 3 was added sequentially at concentrations of 0 ng / ml, 2 ng / ml, 4 ng / ml, 8 ng / ml, 12 ng / ml, 14 ng / ml, 16 ng / ml, 32 ng / ml, and 64 ng / ml. At the same time, n=6 and control wells were set up, which contained only DMEM without the drug. After 24 h of culture, CCK-8 was added, and after 4 h of incubation, the absorbance was measured at 490 nm. The cell viability and IC50 were calculated based on the absorbance ratio of the drug-containing wells to the control wells.

[0046] The specific results are as follows: Figure 12 As shown, the results indicated that when the concentration of the two-photon triptolide nanoparticle TPA-(BT-TPE)3@TPL was 4 ng / ml, the cell viability was 95.47%; while when the concentration of the two-photon triptolide nanoparticle TPA-(BT-TPE)3@TPL was 8 ng / ml, the cell viability was 58.86%. This indicates that a concentration of TPA-(BT-TPE)3@TPL of 8.448 ng / ml produced cytotoxicity. Therefore, when the concentration of TPA-(BT-TPE)3@TPL was less than 4 ng / ml, the cytotoxicity was negative. In other words, the safe dose of the two-photon triptolide nanoparticle TPA-(BT-TPE)3@TPL is 4 ng / ml.

[0047] The above embodiments are illustrative of the present invention and are not intended to limit the present invention. Any simple modifications to the present invention are within the scope of protection of the present invention.

Claims

1. A two-photon triptolide nanomedicine, characterized in that: The nanoparticles are formed by assembling triptolide and TPA-(BT-TPE)3 fluorescent probe with AIE effect through a drug solubilizing and controlled-release phase.

2. The two-photon triptolide nanomedicine as described in claim 1, characterized in that: The average particle size is 43–615 nm with a PDI of 0.40–0.45 and a Zeta potential of -18.9 mV–-33.6 mV.

3. The method for preparing two-photon triptolide nanomedicine as described in claim 1 or 2, characterized in that, Includes the following steps: a) Raw material dissolution: Tripterygium wilfordii, TPA-(BT-TPE)3 fluorescent probe and drug solubilizer and controlled release agent are dissolved together in an organic solvent to form a premix; b) Nanoprecipitate: Under stirring, a premixed solution was added to the poor solvent of triptolide, and the mixture was stirred and evaporated until the organic solvent was completely removed to obtain the reaction mixture; c) Post-processing: The reaction mixture is purified and concentrated to obtain the final product.

4. The preparation method of the two-photon triptolide nanomedicine as described in claim 3, characterized in that: In step a), the mass ratio of TPA-(BT-TPE)3 fluorescent probe to drug solubilizer and controlled-release agent is controlled at 1:6 to 20, and the added mass of triptolide does not exceed 10% of the added mass of drug solubilizer and controlled-release agent.

5. The preparation method of the two-photon triptolide nanomedicine as described in claim 3, characterized in that: In step a), the drug solubilizing and controlled-release agent is poloxamer and / or a synthetic block copolymer, and the organic solvent is one or a combination of several of tetrahydrofuran, methanol, dimethyl sulfoxide, anhydrous ethanol, ethyl acetate and chloroform; in step b), the poor solvent for triptolide is water and / or PBS buffer.

6. The method for preparing the two-photon triptolide nanomedicine as described in claim 3, characterized in that: In step a), the synthesis of the TPA-(BT-TPE)3 fluorescent probe involves first mixing tris[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborane-2-yl)phenyl]amine, Br-BT-TPE, Pd(PPh3)4, and an alkaline co-reactant in a liquid reaction medium. After several cycles of freezing-vacuuming-thawing to degas the medium, it is refilled with inert gas and heated under reflux for 6–18 h. Then, it is quenched and purified and concentrated to obtain the final product.

7. The preparation method of the two-photon triptolide nanomedicine as described in claim 6, characterized in that: The tris[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborane-2-yl)phenyl]amine, Br-BT-TPE, Pd(PPh3)4, and basic co-reactant were mixed together in a ratio of 0.05–0.15: 0.25–0.35: 0.0045–0.0065: 1.5–3.5, and the concentration of the tris[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborane-2-yl)phenyl]amine was controlled at 3.33 mmol / L.

8. The method for preparing the two-photon triptolide nanomedicine as described in claim 6, characterized in that: The alkaline co-reactant is K2CO3, the liquid reaction medium is a mixed solution of THF and water in a ratio of 3 to 5:1, and the inert gas is nitrogen.

9. The application of the two-photon triptolide nanomedicine as described in claim 1 or 2 in the preparation of gout inhibitory drugs and / or non-invasive in vivo bioimaging of inflammatory sites.

Images