Construction method and application of triptolide nano delivery system

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a construction method and application of a triptolide nano delivery system. The invention constructs a triptolide nano delivery system TN-CP NPs with temperature-sensitive response and free radical scavenging functions, and the effectiveness and safety of the triptolide nano delivery system TN-CP NPs in treatment of rheumatoid arthritis are verified through in-vivo and in-vitro experiments. In a CIA mouse model, the system significantly relieves joint swelling, inhibits inflammatory factor expression, improves joint tissue pathological damage, effectively reduces hepatotoxicity and testicular toxicity caused by TN, and does not show obvious hematological or systemic toxicity. Therefore, the TN-CP NPs provides a safe and effective novel nano strategy for realizing high-efficiency and low-toxicity delivery of TN and precise treatment of RA through the synergistic effect of temperature-sensitive controlled release and oxidation resistance, and also provides a reference thought for clinical conversion of other natural medicines with strong activity but high toxicity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the construction method and application of a triptolide nanodelivery system. Background Technology

[0002] rheumatoid arthritis ( Rheumatoid Arthritis Joint rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovial membrane. Its pathological process is accompanied by synovial hyperplasia, progressive destruction of articular cartilage and bone tissue, which can eventually lead to joint deformity and loss of function, seriously affecting the patient's quality of life.

[0003] Currently, clinical treatment mainly relies on nonsteroidal anti-inflammatory drugs (NSAIDs) to relieve symptoms and disease-modifying antirheumatic drugs (DMARDs) to control disease progression. However, traditional drugs generally have poor targeting, low bioavailability, and long-term use can easily cause side effects such as gastrointestinal damage, abnormal liver and kidney function, and immunosuppression, making it difficult to achieve precise and safe treatment for RA.

[0004] With the interdisciplinary integration of nanomedicine and materials science, stimulus-responsive nanodrug delivery systems, possessing the characteristic of "on-demand drug release," offer a new strategy for overcoming the bottlenecks in RA treatment. The local microenvironment of RA-affected joints exhibits significant pathological differences. For example, the temperature at inflamed sites is elevated due to increased metabolism, typically 2-3°C higher than normal tissue. Enhanced anaerobic glycolysis in synovial cells leads to a slightly acidic local pH (pH 6.0-6.5). These unique physiological and pathological signals provide ideal targeted triggering conditions for constructing environmentally responsive nanocarriers.

[0005] Among them, thermosensitive polymers based on N-isopropylacrylamide (NIPAM) have a low critical solution temperature (LCST) close to physiological temperature, allowing for phase transition at the local temperature of the RA joint and achieving effective drug release. Furthermore, the pathogenesis of RA is closely related to oxidative stress imbalance; during inflammation, macrophages and neutrophils are activated and release large amounts of reactive oxygen species (such as hydroxyl radicals •OH and superoxide anions O2). - (etc.), further exacerbating synovial inflammation and tissue damage. Therefore, nanocarriers with ROS scavenging function can enhance the therapeutic effect of RA through a synergistic effect of "anti-inflammatory + antioxidant".

[0006] Tripterygium wilfordii lactone ( Triptonide TN, an active monomer extracted from the traditional Chinese medicine Tripterygium wilfordii, possesses potent anti-inflammatory and immunosuppressive activities, showing promising potential in the treatment of rheumatoid arthritis (RA). However, TN has extremely poor water solubility, low bioavailability, and strong toxicity to the liver and reproductive system, which greatly limits its clinical application.

[0007] To solve the above problems, there is an urgent need to construct a nano-carrier with temperature responsiveness and free radical scavenging ability to realize effective delivery of TN and reduce toxicity. SUMMARY

[0008] The present application aims to provide a triptolide ketone nano delivery system with temperature sensitivity and free radical scavenging function, and the nano delivery system provided by the present application can significantly relieve joint swelling, inhibit inflammatory factor expression, improve joint tissue pathological damage, and effectively reduce liver toxicity and testicular toxicity caused by TN, without showing obvious hematological or systemic toxicity.

[0009] To achieve the above-mentioned purpose, the present application provides the following technical solutions: The present application aims to provide a triptolide ketone nano delivery system with temperature sensitivity and free radical scavenging function, and the nano delivery system provided by the present application can significantly relieve joint swelling, inhibit inflammatory factor expression, improve joint tissue pathological damage, and effectively reduce liver toxicity and testicular toxicity caused by TN, without showing obvious hematological or systemic toxicity.

[0010] The present application also provides a method for constructing the triptolide ketone nano delivery system as described above, which comprises synthesizing a temperature-sensitive copolymer P(VIM x -co-NIPAM y -co-TEMPO z ) by using a RAFT polymerization method, and adding PHEMA to load TN to prepare nanoparticles TN-CP NPs.

[0011] Preferably, the RAFT polymerization method is to mix a macromolecular RAFT reagent, VIM, NIPAM, TEMPO and AIBN as an initiator in a ratio of 1:(30-100):(5-50):(10-80):(0.01-1), and then dissolve in 1,4-dioxane, stir for 40 min under nitrogen; then react at 70℃ for 24 h, separate the obtained precipitate by precipitating the polymer solution with cold petroleum ether; and vacuum dry at 40℃ to obtain P(VIM x -co-NIPAM y -co-TEMPO z ).

[0012] Preferably, the mass ratio of the temperature-sensitive copolymer, PHEMA and TN is 1:(0.05-0.02):(0.5-2.5).

[0013] The present application also provides a pharmaceutical composition comprising the triptolide ketone nano delivery system prepared by the above method, and a pharmaceutically acceptable carrier or excipient.

[0014] The application also provides application of the triptolide nano delivery system or the nano delivery system prepared by the method to preparation of a medicine for treating rheumatoid arthritis.

[0015] Preferably, the medicine can relieve joint swelling, inhibit expression of inflammatory factors and / or improve pathological damage of joint tissues.

[0016] The application also provides application of the triptolide nano delivery system or the nano delivery system prepared by the method to preparation of a medicine for reducing liver toxicity and / or testicular toxicity of triptolide.

[0017] Compared with the prior art, the application has the following beneficial effects: The application provides a triptolide (TN) nano delivery system (TN-CP NPs) with temperature-sensitive response and free radical scavenging functions, and a temperature-sensitive copolymer P(VIMx-co-NIPAMy-co-TEMPOz) (PC) is synthesized by RAFT polymerization, and TN is loaded in PHEMA to prepare nanoparticles.

[0018] The TN-CP NPs prepared by the application have good particle size distribution, high encapsulation efficiency, temperature-sensitive drug release characteristics and significant hydroxyl radical scavenging capacity. In a CIA mouse model, the system significantly relieves joint swelling, inhibits expression of inflammatory factors, improves pathological damage of joint tissues, and effectively reduces liver toxicity and testicular toxicity caused by TN, and does not show obvious hematological or systemic toxicity.

[0019] The TN-CP NPs prepared by the application realize efficient and low-toxicity delivery of TN through synergistic effect of temperature-sensitive controlled release and antioxidation, provide a safe and effective new nano strategy for precise treatment of RA, and also provide a reference idea for clinical transformation of other natural medicines with strong activity but high toxicity. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0021] Figure 1 The figure shows the synthesis process of CP.

[0022] Figure 2(A) 1H-NMR spectra, (B) FTIR spectra, (C) XRD diffractograms, and (D) DSC data of VIM, TEMPO, NIPAM, MIX, and CP.

[0023] Figure 3 Particle size, PDI and zeta potential stability of nanoparticles prepared at different ratios.

[0024] Figure 4 (A) is a TEM image of TN-CP nanoparticles; (B) is a particle size distribution schematic; (C) is zeta potential; (D) is a standard curve in HPLC; (E) is a drug release curve of TN-CP nanoparticles at different temperatures; (F) is a drug release curve of TN-CP nanoparticles under different pH conditions; (G-H) are TN-CP nanoparticles in different media.

[0025] Figure 5 (A) is the absorption-time curve of ·OH-mediated oxidative degradation of methylene blue over time; (B) is the absorption-time curve of methylene blue after the addition of CP; (C) is the absorption-time curve of methylene blue after the addition of -CP nanoparticles.

[0026] Figure 6 (A) is the change in foot volume and clinical arthritis score during administration; (B) is the change in foot volume and clinical arthritis score on the last day; (C) is the effect of TN-CP nanoparticles on histopathological changes; (D) is the effect of TN-CP nanoparticles on inflammatory factors.

[0027] Figure 7 (A) is AST, ALT, BUN, CRE of CP at different concentrations; (B) is hemolytic activity evaluation.

[0028] Figure 8 H&E staining of heart tissue, liver tissue, spleen tissue, lung tissue, kidney tissue, and testis tissue.

[0029] Figure 9 (A) is the change in organ index of experimental mice during administration; (B) is the change in body weight of experimental mice during administration; (C) is the hematology of experimental mice. DETAILED DESCRIPTION

[0030] The purpose of the present application is to provide a triptolide ketone nano delivery system with temperature-sensitive responsiveness and free radical scavenging function, which is used for delivering triptolide ketone, and the nano delivery system is TN-CP NPs loaded with triptolide ketone.

[0031] The application also provides a construction method of the triptolide ketone nano delivery system, and the method comprises the following steps: synthesizing a temperature-sensitive copolymer P(VIM x -co-NIPAM y -co-TEMPO z ) by using a RAFT polymerization method, and adding PHEMA to prepare TN-CP NPs by loading TN.

[0032] Preferably, the RAFT polymerization method is to mix the macromolecular RAFT agent, VIM, NIPAM, TEMPO and AIBN as an initiator in a ratio of 1: (30-100): (5-50): (10-80): (0.01-1), and then dissolve in 1,4-dioxane, stir for 40 min under nitrogen; then react at 70 DEG C for 24 h, separate the obtained precipitate by precipitating the polymer solution with cold petroleum ether; and dry at 40 DEG C under vacuum to obtain P(VIM x -co-NIPAM y -co-TEMPO z ).

[0033] Preferably, the RAFT polymerization method is to mix the macromolecular RAFT agent, VIM, NIPAM, TEMPO and AIBN as an initiator in a ratio of 1: (30-100): (5-50): (10-80): (0.01-1), preferably 1:70:10:20:0.1.

[0034] Preferably, the mass ratio of the temperature-sensitive copolymer, PHEMA and TN is 1: (0.05-0.02): (0.5-2.5), preferably 1:0.1:1.

[0035] The application also provides a pharmaceutical composition comprising the triptolide ketone nano delivery system prepared by the above method and a pharmaceutically acceptable carrier or excipient.

[0036] The application also provides an application of the triptolide ketone nano delivery system or the nano delivery system prepared by any of the above methods in the preparation of a drug for treating rheumatoid arthritis.

[0037] Preferably, the drug can relieve joint swelling, inhibit expression of inflammatory factors and / or improve pathological damage of joint tissues.

[0038] The application also provides an application of the triptolide ketone nano delivery system or the nano delivery system prepared by any of the above methods in the preparation of a drug for reducing liver toxicity and / or testis toxicity of triptolide ketone.

[0039] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in conjunction with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0040] The production process, experimental method or detection method involved in the embodiments of the present application are all conventional methods in the prior art without special instructions, and their names and / or abbreviations are all conventional names in the field, which are very clear and explicit in the relevant application field, and the skilled in the art can understand the conventional process steps and apply the corresponding equipment according to the conventional conditions or the conditions recommended by the manufacturer.

[0041] The various instruments, equipment, raw materials or reagents used in the embodiments of the present application have no special restrictions on the source, and are all conventional products that can be purchased through normal commercial channels, or can be prepared according to the conventional methods well known to those skilled in the art.

[0042] Triptolide ketone was purchased from Chen Guang Biological Technology Co., Ltd.; 4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid (DTTCP) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; 5-2,2,6,6-tetramethylpiperidinyloxy-4-methacrylate (TEMPO), N-isopropyl acrylamide (NIPAM), 1-vinylimidazole (VIM), 2,2-azobisisobutyronitrile (AIBN), poly (2-hydroxyethyl methacrylate) (PHEMA), ferrous sulfate heptahydrate, methylene blue were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; 6-hydrogen peroxide was purchased from Shanghai Mokang Biological Technology Co., Ltd.; 7-anhydrous methanol, anhydrous ethanol were purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; 8-acetonitrile (HPLC grade), methanol (HPLC grade) were purchased from Honeywell Trading (Shanghai) Co., Ltd.; 9-ready-to-use dialysis bag was purchased from Beijing Ruida Henghui Technology Development Co., Ltd.; 10-DMEM high-sugar medium, Triton X-100 were purchased from Beijing Solabio Technology Co., Ltd.; 11-fetal bovine serum (FBS) was purchased from American Invitrogen Corporation; 12-PBS buffer, paraformaldehyde were purchased from Wuhan Sevier Biological Technology Co., Ltd.; 13-sodium heparin was purchased from Shanghai Macklin Biochemical Technology Co., Ltd.; 14-physiological saline was purchased from Henan Kelun Pharmaceutical Co., Ltd.; 15-tween-80 was purchased from Tianjin Kemio Chemical Reagent Co., Ltd.

[0043] Preparation of TN-CP NPs 1.1 Synthesis of P(VIM x -co-NIPAM y -co-TEMPO z ) and characterization The block copolymer P(VIM 70 -co-NIPAM 10 -co-TEMPO 20 ) was synthesized by RAFT polymerization method. The high molecular RAFT agent, 1-vinylimidazole (VIM), N-isopropyl acrylamide (NIPAM), 2,2,6,6-tetramethylpiperidinooxy-4-methacrylate (TEMPO) and AIBN as initiator were mixed in the ratio of 1:70:10:20:0.1 to obtain VIM, NIPAM, TEMPO block with different molecular weights.

[0044] The above ingredients were dissolved in 1,4-dioxane, the solution was stirred under nitrogen for 40 min, then reacted at 70°C for 24 h, the polymer solution was precipitated with cold petroleum ether, the obtained precipitate was separated and dried under vacuum at 40°C to obtain P(VIM 70 -co-NIPAM 10 -co-TEMPO 20 )(CP).

[0045] The structure of the prepared P(VIM 70 -co-NIPAM 10 -co-TEMPO 20 ) was characterized by 1 H-NMR, infrared spectrum (IR), X-ray powder diffraction (XRD) and thermogravimetric analysis (TGA), and the reaction schematic diagram is shown in Figure 1 .

[0046] The results of characterization are shown in Figure 2 , the VIM infrared spectrum appears at about 1647 cm -1 nearby, which is the stretching vibration absorption peak of C=N double bond on the imidazole ring; about 3113 cm -1 , the stretching vibration absorption peak of unsaturated C-H bond, which is the characteristic absorption of C-H bond on the vinyl group, indicating that there is an unsaturated vinyl structure in the molecule.

[0047] The TEMPO spectrum appears at about 2960 cm -1 , the stretching vibration absorption peak of saturated C-H bond, because there are a large number of saturated alkyl structures in the TEMPO molecule.

[0048] The NIPAM spectrum appears at about 1630 cm-1 There is a strong absorption peak corresponding to the stretching vibration of C=O bond in amide group, which is an important feature of NIPAM amide function.

[0049] Compared with other infrared spectra, the infrared of CP has both traces of absorption peaks from relevant functional groups of each monomer and new characteristic absorption. In the range of 1600-1700 cm -1 , it integrates the absorption characteristics of imidazole ring C=N and amide C=O, and in the range of 3292 cm -1 , there is a wide and blunt absorption peak, which is the O-H stretching vibration absorption peak of free carboxyl group, indicating that CP has the expected molecular structure characteristics.

[0050] TEMPO has a very strong characteristic diffraction peak at low angles (10°-20°), and the rest of the angle diffraction peaks are relatively weak and few, indicating that TEMPO has a certain crystal structure, and the strong peak is the diffraction signal of its characteristic crystal face.

[0051] NIPAM has multiple diffraction peaks in the low-angle region, and the peak shape is relatively complex, indicating that the crystal structure or crystal form of NIPAM is different from TEMPO, and there are multiple crystal face diffractions.

[0052] The diffraction peaks of MIX are the superposition of the diffraction peaks of TEMPO and NIPAM, indicating that the crystal structures of the two substances in the mixture are still retained, and there is no obvious crystal form transformation or formation of a new single crystal.

[0053] The diffraction intensity of CP is very low, almost no obvious sharp diffraction peak, showing a "steamed bun peak" like diffuse scattering characteristics, which shows that the crystallinity of CP is very low, and it tends to be amorphous or non-crystalline structure.

[0054] TEMPO has a stable mass in the whole temperature range (from room temperature to about 800℃), with almost no obvious mass loss, indicating that TEMPO has good thermal stability.

[0055] NIPAM has a rapid mass loss at a lower temperature (about 100-200℃), and then the mass remains stable, indicating that NIPAM has undergone thermal decomposition at this temperature range, and the remaining substance has good thermal stability.

[0056] The mass loss trend of MIX is similar to that of NIPAM, but the initial temperature and rate of mass loss are different from those of NIPAM, which may be due to the interaction between the two substances in the mixture affecting the thermal decomposition behavior.

[0057] The CP mass loss is divided into multiple stages, and there is a certain mass loss in the lower temperature range. As the temperature rises, subsequent mass loss processes occur, indicating that the thermal decomposition of CP is a multi-step process, and its thermal stability is different from TEMPO, NIPAM, etc.

[0058] 1.2 To determine the effect of the carrier-drug ratio on the nanoparticles, the particle size and Zeta potential of the nanoparticles prepared at different ratios were measured in aqueous solution at room temperature for seven days to explore their stability. It was found that the particle size and PDI stability of each ratio group were good within seven days. Figure 3

[0059] Among them, the particle size distribution of the 1:0.1:1 group is around 100 nm, the particle size distribution is more uniform, and the Zeta potential is relatively high negative compared with the other three groups. Therefore, under all conditions, the carrier-drug ratio of 1:0.1:1 (PHEMA:TN:CP) is selected to prepare nanoparticles for subsequent experiments.

[0060] 1.3 Preparation of TN-CP NPs CP and poly(2-hydroxyethyl methacrylate) (PHEMA) were weighed and dissolved in methanol, and triptolide TN was weighed and dissolved in ethanol. The mass ratio of PHEMA:TN:CP was 1:0.1:1, and the three were slowly added to 3 mL of pure water, and stirred at 800 r / min under room temperature for 24 h. After stirring, the sample solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed for 24 h, and the water was changed every 6 h. After dialysis, the sample solution was filtered through a 0.45 μm water-based microporous filter to obtain TN-CP NPs.

[0061] Example 2 Characterization of TN-CP NPs 2.1 Particle size, zeta potential and morphology An appropriate amount of TN-CP NPs solution prepared in Example 1 was taken, and the particle size, PDI and Zeta potential were measured at 25°C using Nano brook 90PlusPALS particle size potential analyzer; and the morphology of the nanoparticles was observed by transmission electron microscope (TEM).

[0062] The results are shown in Figure 4 A, the prepared nanoparticles are spherical and uniformly distributed; as shown in Figure 4 B, the particle size of TN-CP NPs is 106.37±3.44 nm, and the PDI is 0.120±0.022. It can be seen that the narrow particle size distribution further verifies the uniformity of the nanoparticles; as shown in Figure 4 ​As shown in FIG. C, the zeta potential was measured to be -30.77 ± 4.23 mV, indicating that the nanoparticles had good stability.

[0063] 2.2 Encapsulation efficiency (EE) and drug loading (DL) The standard curve of TN was established by HPLC method: TN standard solutions with mass concentrations of 1, 5, 10, 20, 40, 60, 80, 100, 120, 150, and 200 μg / mL were prepared, respectively, filtered through a 0.22 μm microporous filter, and then subjected to HPLC chromatographic analysis. The chromatographic conditions were set as follows: the mobile phase was acetonitrile / water (45:55, V / V); the column temperature was 30°C; the flow rate was 1.0 mL / min; and the detection wavelength was 217 nm.

[0064] The linear regression equation of the peak area of TN and the concentration measured by high performance liquid chromatography is as follows: Figure 4 As shown in FIG. D, y = 12257x + 9698.3, R 2 = 0.9995.

[0065] 200 μL of the TN-CP NPs solution was taken, mixed with 800 μL of ethanol, and subjected to ultrasonic treatment for 20 min to obtain a demulsified nanoparticle solution. The obtained solution was filtered through a 0.22 μm filter, and the content of TN in the nanoparticles was measured by HPLC analysis. Another 1 mL of the TN-CP NPs solution was subjected to freeze-drying, and the mass of the dried solid was weighed to determine the total mass of the nanoparticles.

[0066] The EE and DL of TN were calculated according to the following formula: Encapsulation efficiency (EE) = content of TN in nanoparticles / dosage × 100%; Drug loading (DL) = content of TN in nanoparticles / mass of freeze-dried nanoparticles × 100%; According to the above calculation formula, the encapsulation efficiency of TN in the TN-CP NPs was 89.43 ± 0.21%, and the drug loading was 2.97 ± 0.007%.

[0067] 2.3 In vitro release study The dialysis method was used to study the in vitro release kinetics of the TN-CP NPs: an appropriate amount of TN-CP NPs was sealed in a dialysis bag with a molecular weight cutoff of 1000 Da, and was placed in a PBS buffer system with pH 7.4 and pH 6.4, respectively, and the buffer contained 0.5% (V / V) Tween-80. The system was incubated in a 37°C constant temperature incubator, and 1 mL of dialysate was taken at 0.5, 1, 2, 3, 4, 6, 12, 24, and 48 h, respectively. The content of TN in the dialysate was determined by HPLC to explore the influence of different pH on the release performance of TN-CP NPs at the same temperature.

[0068] To investigate the effect of different temperatures on the release performance of TN-CP NPs, another set of experiments was designed: the dialysis bag (molecular weight cut-off 1000 Da) containing TN-CP NPs was placed in the PBS buffer system containing 0.5% (V / V) Tween-80 at pH 7.4, and incubated in a constant temperature incubator at 25°C. The content was determined according to the above method.

[0069] The results are shown in Figure 4 E, TN-CP NPs showed different drug release behaviors at 25°C and 37°C at pH 7.4, and the cumulative drug release rate at 37°C was significantly higher than that at 25°C; Further comparison of the release curves at 37°C in different pH environments (pH 5.5 and pH 7.4) showed that Figure 4 F, the time to reach the peak of drug release at pH 7.4 was shorter than that at pH 6.4; this result showed that the drug release behavior of TN-CP NPs was mainly regulated by the temperature-responsive component NIPAM group, which dominated the drug release process at higher temperature, and the effect of pH condition on the release rate was relatively limited.

[0070] 2.4 In vitro stability The in vitro stability of TN-CP NPs was evaluated by particle size and Zeta potential. TN-CP NPs were added to FBS and DMEM medium containing 10% FBS in a constant temperature incubator at 37°C. The particle size, PDI and Zeta potential were measured at 100 r / min for 2, 4, 6, 8, 10, 12, 24, 36 and 48 h.

[0071] To evaluate its storage stability, the stability of TN-CP NPs in water was measured at room temperature, i.e. the particle size, potential and PDI were measured and recorded every day within 7 days of incubation at 25°C. The results are shown in Figure 4 G-H; As shown in Figure 4 G, in aqueous solution, the particle size of the nanoparticles was in the range of 104.75±6.99 nm, the PDI value was stable at 0.112±0.018, and the Zeta potential was -22.31±3.19 mV, with little fluctuation, indicating that the particles were uniformly dispersed and the system had good stability.

[0072] As shown in Figure 5H, in FBS, the size of TN-CP NPs did not change significantly within 24 h, but increased continuously after 24 h, and the size increased from 114.05 ± 3.92 nm to 268.67 ± 11.38 nm at 48 h, indicating that FBS had a significant effect on the dispersion and stability of TN-CP NPs.

[0073] In DMEM medium containing 10% FBS, the size of TN-CP NPs showed a slow upward trend and then tended to be stable, and finally stabilized at 217.52 ± 7.70 nm, which indicated that the nanoparticles still had good stability in this physiological environment and did not appear to be significantly aggregated or degraded.

[0074] The above results show that different environmental media have a significant effect on the stability of TN-CP NPs.

[0075] 2.5 Free radical scavenging study of TN-CP NPs According to the principle that Fenton reagent (Fe 2＋ / H2O2) generates hydroxyl radicals (•OH) under acidic conditions, methylene blue (MB) was used as a marker to study whether TN-CP NPs had scavenging effect on •OH.

[0076] The FeSO4•7H2O and H2O2 reaction system was prepared under acidic conditions, and MB aqueous solution was added to the system, and the color change was observed, and the absorbance was measured every half hour. In addition, a group of experiments was designed, that is, TN-CP NPs were added to the above system, and the change of MB absorbance in the presence of TN-CP NPs was observed to study whether TN-CP NPs had scavenging effect on •OH.

[0077] The results are shown in Figure 5 A, compared with MB alone, when MB coexists with •OH, its absorbance gradually decreases with time within 1.5 h, accompanied by obvious discoloration, indicating that •OH has strong oxidizing ability and can cause MB to degrade.

[0078] As shown in Figure 5 B, when CP is present, MB is added to the Fenton reaction system, and it is found that the absorption peak of MB has almost no change within 1 h: the peak height, peak strength, and peak position are consistent; this indicates that •OH does not combine with MB in the presence of CP, which indirectly indicates that CP has the function of capturing hydroxyl radicals, so that the free radicals can no longer degrade MB.

[0079] The reaction can also be used to evaluate the ability of TN-CP NPs to capture free radicals, as shown in Figure 6 C.

[0080] In the presence of TN-CP NPs, MB was added to the Fenton reaction system, and its absorption spectrum did not change significantly within 1 h: the peak intensity, peak position and peak shape remained stable, which indicated that MB did not undergo obvious degradation. This phenomenon indirectly indicated that TN-CP NPs could effectively capture hydroxyl radicals, thereby inhibiting the oxidation reaction between ·OH and MB, proving that it had significant free radical scavenging ability.

[0081] Example 3 In vivo therapeutic effect on CIA mice 3.1 Establishment of model Establishment of collagen-induced arthritis (CIA) mouse model: 2 mg / mL bovine type II collagen was emulsified with an equal amount of complete Freund's adjuvant. At the first immunization, 0.05 mg of bovine type II collagen emulsion was injected subcutaneously at the base of the mouse tail, and 0.1 mg of emulsion was injected intradermally at the left and right ankle joints. Three weeks after the initial immunization, all mice were injected with bovine type II collagen and incomplete Freund's adjuvant in the same way to maintain the inflammatory effect. When the mouse foot joints showed significant swelling, limited movement, and other typical inflammatory reactions, the model was determined to be successfully constructed.

[0082] 3.2 Grouping and management The CIA mice were randomly divided into 5 groups (n=10), namely Control group, Model group, TN group (100 µg / kg), CP group, and TN-CP NPs group (equivalent to 100 µg / kg of TN group).

[0083] The drug was injected through the tail vein every two days. During the treatment period, body weight, foot volume, and limb arthritis index were measured every two days. After treatment, the orbital venous blood was collected for subsequent experiments, and then the mice were sacrificed according to the institutional guidelines. The ankle joints, heart, liver, spleen, lungs, kidneys, and testes were collected for histological analysis.

[0084] The results are shown in Figure 6 As shown in A-B, the foot volumes of the arthritis mice in the treatment groups were similar, except for the model group and the control group. At the end of the experiment, the foot volumes of the rats in each treatment group were smaller than those in the model group, and the foot volume of the rats in the TN-CP NPs group was smaller than that in the TN group, indicating that TN-CP NPs played a better therapeutic effect through the synergistic effect of TN and CP.

[0085] 3.3 Effect of TN-CP NPs on histopathological changes of joint tissue The ankle joints were fixed with 4% paraformaldehyde overnight and decalcified with 10% EDTA solution for 40 days. After dehydration, the samples were embedded in paraffin and cut into 4 μm-thick sections. The tissue sections were prepared, stained with hematoxylin and eosin (H&E), and observed under an optical microscope. The symptoms of arthritis were described by foot volume and clinical arthritis score.

[0086] Results are shown in Figure 6 As shown in FIG. C, the bone and cartilage tissue of the model group was severely damaged, the boundary was blurred, and the inflammatory cell infiltration was very obvious. The bone and synovium of the treated mice were relatively complete, and no inflammatory cell infiltration was observed in the mice treated with TN-CP NPs and CP, indicating that TN-CP NPs and CP can effectively reduce the pathological changes in the inflamed joints.

[0087] 3.4 Effect of TN-CP NPs on inflammatory cytokines The blood samples were placed in centrifuge tubes, and after standing at room temperature for 30 min, centrifuged at 4°C, 3000 rpm for 15 min to obtain serum. According to the instructions, the levels of TNF-α, IL-6 and IL-1β in the serum of mice were detected by ELISA kit.

[0088] Inflammatory cytokines in inflamed joints are usually released into the blood, so the inhibitory effect of TN-CP NPs on the production of inflammatory cytokines was verified by measuring the levels of TNF-α, IL-6 and IL-1β in the serum.

[0089] Results are shown in Figure 7 Results show that TN, CP and TN-CP NPs can all reduce the levels of IL-1β, IL-6 and TNF-α in CIA mice compared with the Model group; among them, TN-CP NPs have the strongest inhibitory effect on inflammatory cytokines, indicating that TN-CP NPs may play an anti-inflammatory role through the synergistic effect of TN and CP.

[0090] Example 4 In vivo safety evaluation 4.1 CP hemolysis experiment 1 mL of fresh mouse whole blood was collected, mixed and centrifuged at 3000 rpm for 10 min, and the supernatant was discarded. The lower layer was transferred to a 10 mL centrifuge tube, mixed with normal saline, and centrifuged under the same conditions to discard the supernatant. Repeat washing 3-5 times until the supernatant is colorless and transparent to completely remove the plasma. Finally, 1 mL of red blood cells was taken and diluted to 2% suspension with PBS, and stored at 4°C.

[0091] The CP sample was accurately weighed and diluted to prepare a concentration gradient of 100-1000 μg / mL (100, 200, 300, 500, 800, 1000 μg / mL). 100 μL of each concentration solution was mixed with 900 μL of 2% red blood cell suspension, incubated at 37°C for 1 h, centrifuged at 3000 rpm for 10 min, and 100 μL of supernatant was taken after centrifugation and placed in a 96-well plate. The absorbance was measured at 540 nm, and the hemolysis rate was calculated according to the following formula: A sample is the absorbance of the sample; A negative is the absorbance of the supernatant incubated with PBS, which is the negative control; A positive is the absorbance of the supernatant incubated with Triton X-100, which is the positive control.

[0092] The results are shown in Figure 9 B, which is a photograph of red blood cell hemolysis, with 1% Triton X-100 as the positive control group. The hemolysis rate is 100%, and PBS is the negative control group. The absorbance measured at each concentration was substituted into the hemolysis rate formula, and the results showed that the hemolysis rate of CP at each concentration was less than 5%, within the safe hemolysis rate range, indicating that CP had no hemolytic toxicity.

[0093] 4.2 Blood routine test To evaluate the potential toxicity of each administration group on the blood system, blood routine test was performed on the blood of mice. The content of red blood cells (RBC), hemoglobin (HGB), and platelets (PLT) in the blood of each group of mice was monitored using a fully automatic blood cell analyzer.

[0094] The results are shown in Figure 7 C. Compared with the Control group, TN-CP NPs had no significant effect on red blood cells (RBC), hemoglobin (HGB), and platelets (PLT) in the body, indicating that TN-CP NPs would not cause adverse reactions in blood and had no significant toxicity.

[0095] 4.3 Liver and kidney function test According to the manufacturer's instructions, the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE), and blood urea nitrogen (BUN) in serum were determined using standard kits.

[0096] The results are shown in Figure 8As shown in FIG. 4A, the ALT and AST levels of the TN group were significantly higher than those of the Control group, indicating that the TN group caused liver injury in mice. Compared with the TN group, the ALT level of the TN-CP NPs group was significantly reduced, indicating that this intervention could improve the liver cell damage caused by TN. The AST level of the TN-CP NPs group was not significantly lower than that of the Model and TN groups, which needs to be further verified by histopathology. Compared with the Control group, the CRE and BUN levels of all treatment groups did not change significantly, indicating that TN-CP NPs did not cause kidney damage.

[0097] 4.4 Histopathological examination The heart, liver, spleen, lung, kidney, and testis tissues were fixed with 4% paraformaldehyde overnight, dehydrated, and then embedded in paraffin to cut 4-μm-thick sections. The tissue sections were prepared, stained with hematoxylin and eosin (H&E, Sigma-Aldrich), and observed under an optical microscope.

[0098] The results are shown in FIGS. 4B to 4G. Figure 9 As shown in FIG. 4B, compared with the Control group, the myocardial fibers of the Model and each administration group were arranged in order, and the size was uniform without obvious rupture or disorder. The cell nucleus was normal in shape, and the interstitial proportion was normal without obvious fibrosis.

[0099] The liver pathological analysis showed that, compared with the Control group, the liver cells of the TN group were arranged in disorder, and the liver tissue had various-sized vacuoles, which was caused by the liver cell steatosis induced by TN. The liver lesions of the TN-CP NPs group were significantly reduced, indicating that TN-CP NPs could reduce the liver damage induced by TN.

[0100] In the spleen tissue sections, compared with the Control group, the cell density of the TN group was significantly reduced, the red pulp structure was disordered, and the white pulp appeared atrophy, indicating that TN caused spleen damage. The cell density of the CP and TN-CP NPs groups was close to that of the Control group, and the structure was relatively complete, indicating that CP could improve the damage induced by TN.

[0101] In the lung tissue sections, compared with the Control group, the cells of each experimental group were clear, the alveolar wall was thin, the vascular structure was complete, there was no effusion or exudate in the alveolar cavity, and the alveolar wall cells were arranged in order, indicating that TN-CP NPs were non-toxic to lung tissue.

[0102] The kidney pathological analysis showed that, compared with the Control group, the kidney tissue morphology of each experimental group was relatively regular, and the cells were arranged in order, indicating that TN-CP NPs were non-toxic or low-toxic to kidney tissue.

[0103] The testis histopathological sections showed that the testis morphology of TN group was irregular, and the number of spermatogenic cells and sperm cells in the seminiferous tubules was less than that of the Control group, indicating that TN had certain testicular toxicity. Compared with the TN group, the pathological changes of the testis of the CP group and the TN-CP NPs group were significantly reduced.

[0104] The organ coefficients of the mice in each experimental group and the body weight changes during the administration period were as shown in Table 2. ​ As can be seen from Table 2, compared with the Control group, the organ coefficients of other organs of each group had no significant difference, and the body weight changes of the mice in each group were also relatively stable, indicating that TN-CP NPs did not produce obvious systemic toxicity.

[0105] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A trichothecene nanodelivery system with temperature-sensitive responsiveness and free radical scavenging function, characterized in that, The nano delivery system is used for delivering triptolide ketone, and the nano delivery system is a nano particle TN-CP NPs loaded with triptolide ketone.

2. A method for constructing the nano-delivery system of triptolide as claimed in claim 1, characterized in that, The method comprises synthesizing a temperature-sensitive copolymer P(VIM x -co-NIPAM y -co-TEMPO z ), and preparing nanoparticles TN-CP NPs by adding PHEMA to load TN.

3. The method of claim 2, wherein, The RAFT polymerization method is to mix the high molecular RAFT agent, VIM, NIPAM, TEMPO and AIBN as an initiator in a ratio of 1:(30-100):(5-50):(10-80):(0.01-1), then dissolve in 1,4-dioxane, stir for 40 min under nitrogen; then react at 70°C for 24 h, separate the obtained precipitate by precipitating the polymer solution with cold petroleum ether; dry under vacuum at 40°C to obtain P(VIM x -co-NIPAM y -co-TEMPO z ).

4. The method of claim 2, wherein, The mass ratio of the temperature-sensitive copolymer, PHEMA and TN is 1:(0.05-0.02):(0.5-2.5).

5. A pharmaceutical composition, characterized by, The triptolide ketone nano delivery system prepared by the method of any one of claims 2-4, and a pharmaceutically acceptable carrier or excipient.

6. The use of the triptolide ketone nano delivery system of claim 1 or the nano delivery system prepared by the method of any one of claims 2-4 in the preparation of a drug for treating rheumatoid arthritis.

7. Use according to claim 6, characterized in that, The drug can relieve joint swelling, inhibit expression of inflammatory factors and / or improve pathological damage of joint tissue.

8. The use of the triptolide ketone nano delivery system of claim 1 or the nano delivery system prepared by the method of any one of claims 2-4 in the preparation of a drug for reducing liver toxicity and / or testicular toxicity of triptolide.

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