Curcumin-cyclodextrin-polylactic acid-glycolic acid copolymer composite nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of pharmaceutical preparations, and particularly discloses curcumin-cyclodextrin-polylactic acid-glycolic acid copolymer composite nanoparticles as well as a preparation method and application thereof. According to the nanoparticles, curcumin is used as an active component, and cyclodextrin and a polylactic acid-glycolic acid copolymer are used as a carrier of a nano skeleton. The core of the preparation method is mainly as follows: firstly, curcumin and cyclodextrin form an inclusion compound in an organic solvent, then the inclusion compound is entrapped in a nanoparticle skeleton formed by a polylactic acid-glycolic acid copolymer, and finally, a nanoparticle colloidal solution is formed. The prepared composite nanoparticles are small in particle size, uniform in distribution, high in encapsulation efficiency and good in stability, the solubility, bioavailability and anti-tumor activity of curcumin can be remarkably improved, and the composite nanoparticles have wide application prospects in breast cancer treatment drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a preparation method of curcumin-cyclodextrin-polylactic-glycolic acid copolymer composite nanoparticles and its application in anti-tumor drugs. BACKGROUND

[0002] Curcumin (CUR) is a natural polyphenolic compound extracted from turmeric, which has anti-inflammatory, antioxidant and anti-tumor activities, and shows good application prospects in the field of breast cancer treatment. However, curcumin has low water solubility, poor chemical stability in physiological environment and rapid metabolism, which leads to extremely low oral bioavailability and seriously limits its wide clinical application. In order to improve the physicochemical properties and pharmacokinetic behavior of curcumin, various strategies have been tried in the prior art. Cyclodextrin (CD) has a special cyclic cavity structure of "outer hydrophilic and inner hydrophobic", which can be used as a molecular inclusion carrier to form an inclusion compound with hydrophobic drugs, thereby effectively improving the solubility and stability of the drugs. On the other hand, nano medicine delivery system (NDDS) has significant advantages in improving drug targeting, prolonging in vivo circulation time and reducing toxic side effects as a frontier technology in pharmacy. Therefore, nano medicine delivery system based on cyclodextrin has gradually attracted widespread attention.

[0003] Polylactic-glycolic acid copolymer (PLGA) is a biodegradable polymer material, which is widely used to construct nanoparticles and has good biocompatibility and controlled release characteristics. Cyclodextrin improves the solubility and stability of drugs through inclusion, and then is encapsulated by PLGA polymer to form a composite system with a double-layer protection structure, which is expected to further improve the delivery efficiency and therapeutic effect of drugs. However, through reasonable prescription design and process optimization, it is still a technical problem to be solved in the field to construct a composite nano delivery system with high encapsulation efficiency, small particle size, high stability and excellent in vitro and in vivo anti-tumor effect, which can overcome the bottlenecks of curcumin in solubility, targeting and therapeutic effect. SUMMARY

[0004] The present application aims to overcome the deficiencies of existing curcumin delivery systems in solubility, stability, targeting and therapeutic effect, and provides a novel curcumin composite nanoparticle and its preparation method and use. The nanoparticle combines curcumin-cyclodextrin inclusion compound with polylactic-glycolic acid copolymer nanoparticle carrier system, aiming to improve the encapsulation efficiency, stability and anti-tumor effect of curcumin, and reduce its toxic side effects, thereby providing an effective formulation option for breast cancer treatment.

[0005] To achieve the above-mentioned purpose of the application, the following technical solutions are adopted in the application.

[0006] In a first aspect, the application provides a curcumin-cyclodextrin-poly(lactic-co-glycolic acid) composite nanoparticle.

[0007] The active ingredient of the inclusion complex is curcumin, and the carrier system is composed of cyclodextrin and poly(lactic-co-glycolic acid). The steps are as follows:

[0008] Step one, dissolve cyclodextrin in an organic solvent, then slowly add an appropriate amount of curcumin powder and ultrasonic to completely dissolve to form an inclusion complex.

[0009] Step two, add poly(lactic-co-glycolic acid) to the inclusion solution and vortex ultrasonic to form an organic phase.

[0010] Step three, slowly drop the organic phase into a polyvinyl alcohol aqueous solution.

[0011] Step four, use a probe ultrasonic instrument to ultrasonic.

[0012] Step five, magnetic stirring to volatilize the organic solvent to form a curcumin-cyclodextrin-poly(lactic-co-glycolic acid) composite nanoparticle.

[0013] Among them, the type of cyclodextrin is β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, 2,6-di-o-methyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin.

[0014] Among them, the type of organic solvent is dichloromethane, acetone and chloroform.

[0015] Among them, the concentration of poly(lactic-co-glycolic acid) polymer is 10-30 mg / mL.

[0016] Among them, the concentration of emulsifier polyvinyl alcohol is 0.5-2% (w / v).

[0017] Preferably, the cyclodextrin in step one is methyl-β-cyclodextrin.

[0018] Preferably, the organic solvent in steps one and two is dichloromethane.

[0019] Preferably, the concentration of poly(lactic-co-glycolic acid) in step two is 20 mg / mL.

[0020] Preferably, the concentration of polyvinyl alcohol in step three is 1% (w / v).

[0021] Preferably, the power of the probe ultrasonic in step four is 105 W amplitude for 5 min of emulsification ultrasonic.

[0022] Preferably, the power of the magnetic stirring in step five is 400 rpm, and the stirring time is 4 h.

[0023] In a second aspect, the application provides a use of curcumin-cyclodextrin-poly (lactic-co-glycolic acid) composite nanoparticles in treating breast cancer.

[0024] Compared with the prior art, the application has the following advantages:

[0025] The prepared curcumin nanoparticles have a smooth surface, a uniform dispersed regular spherical structure, and the characteristics of high encapsulation efficiency and high stability, and the in-vitro drug release behavior exhibits significant pH responsiveness, that is, the drug release amount is significantly improved in a slightly acidic tumor environment, and less released under physiological conditions, which is helpful to realize targeted therapy and enhance the anti-tumor effect. The application effectively overcomes the problems of poor solubility and low bioavailability of curcumin, and the curcumin nanoparticles are more easily taken up by cells and have a stronger inhibitory effect on tumor cell growth than pure curcumin. In addition, the preparation method of the application is simple, convenient and easy to operate. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The solubility graph of curcumin and different cyclodextrins in Example 2.

[0027] Figure 2 The particle size distribution graph of the nanoparticles under the optimal prescription of Examples 3-5.

[0028] Figure 3 The electron microscope graph of the nanoparticles in Example 6.

[0029] Figure 4 The storage stability graph of the nanoparticles in Example 7.

[0030] Figure 5 The cytotoxicity result graph of the nanoparticles in Example 8.

[0031] Figure 6 The flow cytometry result graph of the cell uptake of the nanoparticles in Example 8.

[0032] Figure 7 The confocal microscope observation graph of the cell uptake and distribution of the nanoparticles in Example 8.

[0033] Figure 8 The cell migration imaging graph of the nanoparticles in Example 8.

[0034] Figure 9 The cell migration rate result graph of the nanoparticles in Example 8.

[0035] Figure 10 The anti-tumor effect graph of the nanoparticles in Example 9.

[0036] Figure 11 Tumor inhibition effect diagram of nanoparticles in Example 9.

[0037] Figure 12 Tumor tissue H&E staining diagram of mice after administration of nanoparticles in Example 9. DETAILED DESCRIPTION

[0038] The application will be further described in detail below by specific examples in conjunction with the accompanying drawings. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application.

[0039] Example 1: Solubility test of curcumin raw material in water.

[0040] An excess of curcumin raw material (about 10 mg) was added to 10 mL of ultrapure water, and placed in a constant temperature shaking incubator, shaken at 37°C and 150 rpm for 72 h. The supernatant of the above curcumin supersaturated aqueous solution was filtered through a 0.22 μm water-based microporous filter, and the filtrate was used to determine the concentration of curcumin by high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition Chapter 0512). The results showed that the saturation solubility of curcumin in ultrapure water was 7.847 ± 1.004 ng / mL, which belongs to insoluble drugs.

[0041] Example 2: Phase solubility test of curcumin and cyclodextrin.

[0042] An appropriate amount of β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, methyl-β- cyclodextrin and 2,6-di-o-methyl-β-cyclodextrin powder was precisely weighed, and ultrapure water was used to prepare cyclodextrin aqueous solutions with concentrations of 2, 4, 6, 8, 10 and 12 mM. 8 mL of different concentrations of cyclodextrin aqueous solution was taken, and an excess of curcumin raw material was added, and shaken at 37°C and 150 rpm for 72 h. The supernatant was diluted and determined by high performance liquid chromatography. The phase solubility diagram was drawn with the molar concentration of cyclodextrin as the abscissa and the molar concentration of curcumin as the ordinate. The apparent binding constant (Kc) and Gibbs free energy (△G) were calculated according to the phase solubility diagram.

[0043] As shown in Table 1, Figure 1As shown, the solubility of curcumin in β-CD and its derivatives increased with the increase of their concentrations, and the phase solubility curves were AL type with a slope less than 1, indicating the formation of 1:1 stoichiometric inclusion complex. The binding constants (Kc) of all systems were greater than 5000, indicating good stability of the inclusion complex, among which the inclusion complex with methyl-β-CD as the carrier had the strongest binding capacity (Kc = 98799 M-1). In addition, the Gibbs free energy (ΔG) of all inclusion processes was negative, proving that the inclusion process can proceed spontaneously. In particular, compared with β-CD, the ΔG value of the derivative system decreased (difference ≥ 3.32 kJ / mol), which indicated that the chemical modification group effectively promoted the spontaneous formation and thermodynamic stability of the inclusion complex.

[0044] Table 1. Binding constants and free energies calculated according to the phase solubility curves.

[0045]

[0046] Example 3: Screening of the concentration of polylactic acid-glycolic acid copolymer.

[0047] The concentration of polyvinyl alcohol was fixed at 1% (w / v), the organic solvent was dichloromethane, and the concentration of the polymer polylactic acid-glycolic acid copolymer was changed to 10 mg / mL, 20 mg / mL, and 30 mg / mL. 0.3 mL of polylactic acid-glycolic acid copolymer organic phase was slowly dropped into 3 mL of polyvinyl alcohol aqueous phase, and curcumin-nanoparticles and flavin-cyclodextrin-nanoparticles were prepared after ultrasonic emulsification and magnetic stirring. The optimal concentration of polylactic acid-glycolic acid copolymer was screened by particle size and encapsulation efficiency.

[0048] As shown in Table 2, with the increase of the concentration of polylactic acid-glycolic acid copolymer, the particle size of the prepared nanoparticles decreased, and the drug encapsulation efficiency steadily increased. This phenomenon can be attributed to the fact that a higher polymer concentration effectively inhibits the Oswald ripening effect, thereby facilitating the formation of smaller nanoparticles. Taking into account the overall situation, 20 mg / mL was chosen as the optimal concentration of polylactic acid-glycolic acid copolymer.

[0049] Table 2. Effect of PLGA concentration on nanoparticle particle size, PDI, zeta potential, and encapsulation efficiency (EE%).

[0050]

[0051] Example 4: Screening of the concentration of polyvinyl alcohol.

[0052] The optimal concentration of polylactic-co-glycolic acid, the organic solvent of dichloromethane, and the concentration of emulsifier polyvinyl alcohol of 0.5%, 1%, and 2% (w / v) were determined according to the results of Example 3. The curcumin-nanoparticles and curcumin-cyclodextrin-nanoparticles were prepared by the emulsification solvent evaporation method, and the particle size and encapsulation efficiency were determined.

[0053] As shown in Table 3, the particle size of the nanoparticles continuously decreased with the increase of the concentration of polyvinyl alcohol, which was attributed to the decrease of the interfacial tension. The encapsulation efficiency first increased and then decreased. The optimal value appeared near the critical micelle concentration. When the concentration exceeded the critical micelle concentration, the increase of the viscosity of the aqueous phase was not conducive to the dispersion and formation of the nanoparticles, resulting in the decrease of the yield and encapsulation efficiency. Therefore, 1% (w / v) was selected as the optimal concentration of polyvinyl alcohol.

[0054] Table 3. Effect of the concentration of PVA on the particle size, PDI, zeta potential, and encapsulation efficiency (EE%) of the nanoparticles.

[0055]

[0056] Example 5: Screening of the optimal type of organic solvent.

[0057] The optimal concentration of polylactic-co-glycolic acid and polyvinyl alcohol and the type of organic solvent of dichloromethane, acetone, and trichloromethane were determined according to the results of Example 3 and Example 4. The curcumin-nanoparticles and curcumin-cyclodextrin-nanoparticles were prepared by the emulsification solvent evaporation method, and the particle size and encapsulation efficiency were determined.

[0058] As shown in Table 4, both dichloromethane and trichloromethane could be used to prepare nanoparticles with small particle size and high encapsulation efficiency. Considering the volatility and toxicity of the solvent, dichloromethane was determined as the suitable substitute for trichloromethane due to its better safety and operational convenience. When acetone was used as the organic solvent, it would quickly diffuse into the aqueous phase during the emulsification process due to its water-miscible property, resulting in the loss of the drug and the heterogeneous aggregation of the polymer, and finally leading to the significant increase of the particle size and the significant decrease of the encapsulation efficiency. Therefore, dichloromethane was selected as the optimal organic solvent.

[0059] Table 4. Effect of the organic solvent on the particle size, PDI, zeta potential, and encapsulation efficiency (EE%) of the nanoparticles.

[0060]

[0061] Based on the results of the prescription screening of Examples 3-5, the present application determines the optimal process conditions for preparing curcumin nanoparticles: the concentration of polylactic acid-glycolic acid copolymer is 20 mg / mL, the concentration of polyvinyl alcohol is 1% (w / v), and the organic solvent is dichloromethane. Under the optimized conditions, both the curcumin nanoparticles and the curcumin-cyclodextrin inclusion complex nanoparticles exhibit excellent performance. The particle size of the curcumin-nanoparticles is 155.6 ± 9.9 nm, the polydispersity index (PDI) is 0.147 ± 0.059, the potential is -6.55 ± 1.41 mV, and the encapsulation efficiency is 62.39% ± 4.60%. The particle size of the curcumin-cyclodextrin-nanoparticles is 153.3 ± 12.9 nm, the PDI is 0.142 ± 0.056, the potential is -10.22 ± 1.38 mV, and the encapsulation efficiency is increased to 75.51% ± 0.97%. As shown in FIG. 1, the particle size distribution of both types of nanoparticles is relatively narrow, and the average particle size of the curcumin-cyclodextrin-nanoparticles is smaller, indicating that the introduction of cyclodextrin makes the nanoparticle structure more compact, thereby significantly improving the encapsulation efficiency and physical stability of the preparation. Figure 2

[0062] Example 6: Scanning electron microscopy of curcumin-cyclodextrin nanoparticles.

[0063] The morphology of the nanoparticles was observed by scanning electron microscopy. Before testing, the nanoparticle solution was uniformly blown and fixed, and gold was sprayed to make it conductive, and then the respective scanning electron microscopy images were obtained.

[0064] The curcumin-nanoparticles and curcumin-cyclodextrin-nanoparticles solutions were characterized by laser irradiation method, and the results are shown in FIG. 2. Figure 3 As shown in the upper left of FIG. 2, both solutions exhibit a clear Tyndall effect, confirming the formation and stable existence of the nanoscale colloidal particles. Further observation of the microstructure by scanning electron microscopy (SEM) showed that both types of nanoparticles were uniformly dispersed, with clear outlines, smooth surfaces, and regular spherical structures. It was also observed that the drug was wrapped by a compact and translucent polylactic acid-glycolic acid copolymer outer membrane. It is worth noting that the nanoparticle size observed by SEM is slightly smaller than the measurement results by the dynamic laser particle size distribution instrument. This difference is due to the difference in measurement principles, as SEM is a static and dry state observation of a small number of particles, while the particle size instrument is a dynamic hydrodynamic diameter measurement of a large number of particles in a hydrated state in solution. These results together prove that the present application successfully prepared nanoparticles with excellent morphology.

[0065] Example 7: Storage stability study of curcumin-cyclodextrin nanoparticles.

[0066] The curcumin-nanoparticles and curcumin-cyclodextrin-nanoparticles solutions were sealed and stored at room temperature, and samples were taken every day for 14 days to determine the particle size, potential, and PDI values. ​

[0067] The results are as follows Figure 4 The results show that both types of nanoparticles maintained a stable colloidal state during the observation period, with no significant sedimentation. Their key quality properties remained stable: particle size fluctuated between 140-160 nm, PDI ranged from 0.1 to 0.2, and Zeta potential ranged from -4 to -12 mV. Notably, in the later stages of storage (after day 8), the curcumin-cyclodextrin-nanoparticles exhibited superior stability. Their higher absolute Zeta potential provided stronger electrostatic repulsion, effectively inhibiting particle aggregation. Therefore, their particle size only increased slightly, and the overall PDI value was lower, resulting in better dispersion uniformity. This demonstrates that the introduction of cyclodextrin further improved the long-term storage stability of the nanoparticles by enhancing the steric or electrostatic stability of the system.

[0068] Example 8: In vitro antitumor study of curcumin-cyclodextrin nanosystem.

[0069] Procedure 1: Cytotoxicity assay.

[0070] Using a murine breast cancer cell (4T1) model, the cell density was 5000 cells per well. The calculated cell suspension and culture medium were mixed, and 100 μL was injected into each well of a 96-well plate. The plates were incubated for 24 h. After adhesion, the culture medium was removed, and 100 μL of curcumin solutions at concentrations of 80, 60, 40, 20, 10, and 5 μg / mL, curcumin / cyclodextrin inclusion complex solutions, curcumin-nanoparticle solutions, and curcumin-cyclodextrin-nanoparticle solutions were added to each well (6 replicates per concentration). The plates were incubated for 24 h and 48 h, respectively. After removing the culture medium, 110 μL of a CCK-8 / culture medium mixture (containing 10 μL CCK-8) was added to each well. After 1 h of incubation, the absorbance at 450 nm was measured for each well. The highest and lowest values ​​were removed, and the cell viability (%) was calculated. The half-maximal inhibitory concentration (IC50) was calculated using GraphPadPrism statistical software. 50 ).

[0071] Results Table 5 Figure 5 The results show that the curcumin-nanoparticles and curcumin-cyclodextrin-nanoparticles prepared in this invention exhibit significantly better anti-proliferative effects than free curcumin and its cyclodextrin inclusion complex. Within a treatment time of 48 h, the IC50 values ​​of curcumin-nanoparticles and curcumin-cyclodextrin-nanoparticles were... 50 The concentrations decreased to 5.471 μg / mL and 9.631 μg / mL, respectively, significantly lower than other groups, which fully demonstrates the significant advantage of the sustained-release properties of polylactic acid-glycolic acid copolymer nanoparticles in long-term treatment. Particularly noteworthy is that although the IC50 of cyclodextrin-nanoparticles... 50The cyclodextrin-inclusion-nanoparticle was slightly smaller, but under high concentration administration conditions (40-80 μg / mL), the curcumin-cyclodextrin-nanoparticle exhibited the strongest cell killing effect, and could suppress cell viability to an extremely low level of 3.82%-5.39%, which was significantly better than the 11.50%-12.95% of the curcumin-nanoparticle group. This phenomenon showed that the curcumin-cyclodextrin-nanoparticle, while achieving effective sustained release, its higher drug loading or better cell uptake efficiency enabled it to exert more thorough therapeutic effect at high doses, and exhibited excellent clinical application potential.

[0072] Table 5. Half inhibitory concentration (IC50) of different administration groups at 24 h and 48 h 50 ).

[0073]

[0074] Operation 2: Quantitative analysis of cell uptake by flow cytometry.

[0075] 4T1 cells were inoculated in a 6-well plate at a density of 1×10 6 cells / well (2 mL per well), incubated for 24 h, and then the medium was carefully aspirated after adhesion, washed with PBS for 3 times, and then 2 mL of serum-free medium (blank group) and 20 μg / mL free curcumin, curcumin / cyclodextrin inclusion complex, curcumin-nanoparticle and curcumin-cyclodextrin-nanoparticle drug solutions (administration groups) diluted with medium were added to each well, respectively, and placed in a CO2 incubator for 4 h. After 4 h, the 6-well plate was removed to remove the drug solution, washed with PBS for 3 times, 0.5 mL of 0.25% trypsin was added to each well, and the digestion was terminated after the cytoplasm began to retract and round, and then the cells were gently blown off and transferred to a centrifuge tube, centrifuged at 800 rpm / min for 5 min to collect the cells, resuspended with PBS and centrifuged for 2 times, 0.5 mL of PBS was added to resuspend the cells, and then placed in a 1.5 mL centrifuge tube for testing (stored in an ice box), and the fluorescence intensity in the living cells was detected by flow cytometry.

[0076] The drug uptake behavior of 4T1 cells was evaluated by flow cytometry, and the results are shown in Table 6. Figure 6The cell fluorescence intensity of curcumin-cyclodextrin inclusion complex group was the highest in the short term of 4 hours, indicating that its uptake rate was the fastest; curcumin-cyclodextrin-nanoparticle was the second, and the free curcumin group was the weakest, which was consistent with the characteristics of poor water solubility and difficult to be effectively taken up by cells. Curcumin-nanoparticle and curcumin-cyclodextrin-nanoparticle were negatively charged polylactic acid-glycolic acid copolymer nanoparticles, and there was electrostatic repulsion between them and the same negatively charged cell membrane, resulting in low uptake rate. However, the uptake intensity of curcumin-cyclodextrin-nanoparticle was significantly higher than that of curcumin-nanoparticle, which indicated that the introduction of cyclodextrin effectively improved the cell uptake behavior of nanoparticles, which may be related to the fact that cyclodextrin modification enhanced the rigidity of nanoparticles. The uptake results were mutually confirmed by the cytotoxicity experiment, and curcumin / cyclodextrin inclusion complex had fast initial uptake but strong short-term toxicity. Curcumin-cyclodextrin-nanoparticle combined with "improved cell uptake" and "polylactic acid-glycolic acid copolymer-mediated slow release effect", which was expected to achieve better and more durable anti-proliferation effect in long-term treatment.

[0077] Operation 3: Confocal microscope qualitative observation of cell uptake.

[0078] Curcumin has a spontaneous fluorescence property (Ex = 488 nm, Em = 520 nm), so curcumin can be directly used as a fluorescent marker to detect the uptake of drugs by breast cancer cells. 4T1 cells were seeded in confocal dishes at a density of 1.2 x 10 5 After 24 hours of cell adhesion, serum-free medium (control group) and 20 μg / mL of free curcumin, curcumin / cyclodextrin inclusion complex, curcumin-nanoparticle and curcumin-cyclodextrin-nanoparticle drug solutions (1 mL) were added. After 8, 12 and 24 hours of incubation in the dark in the incubator, the drug solutions were removed, washed twice with PBS, fixed with 4% tissue cell fixative (1 mL) for 15 minutes, and then washed twice with PBS. Then, DAPI (200 μL) staining solution was added under dark conditions for 5 minutes, and then DAPI (Ex = 364 nm, Em = 454 nm) was discarded. After washing twice with PBS, 0.5 mL of PBS was added, and the fluorescence in the cells was observed under a laser confocal microscope (CLSM).

[0079] The distribution and pro-apoptotic effect of each drug administration group in 4T1 cells were observed by confocal laser scanning microscopy Figure 7). The results showed that all curcumin-containing groups were taken up by the cells, and the intracellular green fluorescence intensity increased with time (except for the free curcumin group). More importantly, the morphological changes of the cells clearly revealed the differences in drug release kinetics of the different formulations. At 8 h, the free curcumin group showed a significant decrease in cell number due to the rapid action of the drug. The curcumin-cyclodextrin inclusion complex and curcumin-nanoparticle groups showed moderate and fast signs of apoptosis (cell rounding and aggregation), respectively. The curcumin-cyclodextrin-nanoparticle group showed the least morphological changes in the cells, indicating the slowest drug release. By 12 h, the curcumin-cyclodextrin-nanoparticle group still showed a relatively stable cell number and morphology, further verifying its slow-release characteristics. At the 24 h endpoint, the curcumin-cyclodextrin-nanoparticle group showed the most significant decrease in cell number and morphological changes in apoptosis. This dynamic process confirmed that the curcumin-cyclodextrin-nanoparticle successfully combined fast cellular uptake with persistent slow-release behavior, ultimately exhibiting the strongest pro-apoptotic effect under long-term action, and its long-acting antitumor effect was superior to that of the other groups.

[0080] Operation 4: Cell scratch test to observe cell migration.

[0081] Uniformly marked lines were drawn on the back of a 6-well plate with a ruler, about every 0.5-1 cm, and at least 3 lines per well. Cells in the logarithmic growth phase were centrifuged, counted, and inoculated in the 6-well plate at a density of 5 x 10 5 cells / well (2 mL per well) and shaken. When the cells grew and fused to 100%, the scratch treatment was performed, and 200 μL of a sterilized gun head was used to scratch the back of the marked line vertically, so that the scratch intersected the marked line. The adherent cells were washed twice with PBS to remove the scratched cells, and serum-free medium (control group), 20 μg / mL of free curcumin, curcumin / cyclodextrin inclusion complex, curcumin-nanoparticle, and curcumin-cyclodextrin-nanoparticle solutions were added for treatment. Incubation was performed at 37°C for 0, 24, and 48 h, and the cell migration was observed under a microscope. ImageJ software was used to calculate the scratch area at 0, 24, and 48 h as A0and A t , and the relative migration rate was calculated.

[0082] The inhibitory effect of each sample on the migration of 4T1 cells (a triple-negative breast cancer model) was evaluated by a cell scratch test. The results Figure 8 , 9The results showed that all curcumin-containing groups inhibited cell migration, but their duration of action differed significantly. At 24 hours, free curcumin and the curcumin-cyclodextrin inclusion complex exhibited the strongest early migration inhibition rate due to rapid drug release. Curcumin-nanoparticles and curcumin-cyclodextrin-nanoparticles, due to their sustained-release properties, showed relatively lower early inhibition rates. However, at the 48-hour endpoint, curcumin-cyclodextrin-nanoparticles demonstrated superior long-term inhibitory effects, with no significant increase in cell migration rate, indicating the most stable inhibitory effect. In contrast, the free curcumin group showed a relative increase in migration rate of approximately 20%, and the curcumin-cyclodextrin and curcumin-nanoparticle groups also showed an increase of approximately 10%. This demonstrates that curcumin-cyclodextrin-nanoparticles, through sustained and slow drug release, can provide a more durable and stable anti-migration effect, possessing significant potential therapeutic value for inhibiting the key pathological process of cancer metastasis.

[0083] Example 9: Evaluation of the in vivo antitumor activity and safety of the nanoparticle system.

[0084] Operation 1: Establishment and administration of an in situ breast cancer model.

[0085] Thirty-six female BALB / c mice, aged 6-8 weeks and weighing approximately 18-22g, were selected. Hair was removed from the area around the fourth pair of nipples of the mice the day before the experiment. On the day of the experiment, 4T1 cells were digested, centrifuged, resuspended in PBS, washed, counted, and finally diluted to 100μL PBS containing 5×10⁻⁶ cells. 6 Four T1 cells were temporarily stored at 0℃. The dosage of anesthetic for each mouse was calculated at 16 mL / kg. Mice were anesthetized by intraperitoneal injection of 2% Avertin working solution. After disinfection with 75% alcohol, the skin near the fourth pair of nipples was grasped with forceps. After expelling air from the syringe, the needle was inserted 5-10 mm into the mammary fat pad with the tip pointing downwards towards the nipple. Tumor cells were slowly injected into the fat pad. After injection, the needle tip was carefully rotated and slowly withdrawn. The puncture site was gently pressed with a cotton swab for 10-20 seconds. Observation was maintained for one week until the tumor volume reached 40-60 mm². 3 Administer the medication.

[0086] Operation 2: In vivo anti-tumor efficacy study.

[0087] Thirty-six mice were divided into six groups: a control group, a model group, a free curcumin group, a curcumin-cyclodextrin inclusion complex group, a curcumin-nanoparticle group, and a curcumin-cyclodextrin-nanoparticle group. The drugs were administered via tail vein injection at a dose of 5 mg / kg every other day. The model group received only an equal volume of 0.9% saline. Tumor length (a) and width (b) were measured using calipers every other day, and tumor volume (V) was calculated.

[0088] The results are as follows Figure 10As shown, after 14 days of treatment, the curcumin-cyclodextrin-nanoparticle of the present application showed extremely significant tumor inhibition effect compared with the model group (normal saline) and the free curcumin group. At the end of treatment, the tumor volume of the curcumin-cyclodextrin-nanoparticle group was only 6.51 times the initial volume, and its inhibition effect was significantly better than that of the curcumin-cyclodextrin inclusion group (9.28 times) and the curcumin-nanoparticle group (11.37 times). Notably, the free curcumin group did not show obvious therapeutic effect, confirming that it is easily rapidly cleared in vivo. While the curcumin-cyclodextrin-nanoparticle successfully overcomes this defect through the slow release effect of the nanoparticle and the optimization of the drug behavior by cyclodextrin, and exhibits the strongest tumor growth inhibition capacity. This in vivo efficacy result is highly consistent with the in vitro cell uptake and pharmacodynamics experiment results, and together proves the outstanding advantages of the curcumin-cyclodextrin-nanoparticle in achieving efficient anti-tumor therapy.

[0089] The analysis results of tumor mass after treatment Figure 11 further quantified the efficacy advantage of the present application. The data showed that the tumor inhibition rates of each administration group were in the order of: curcumin-cyclodextrin-nanoparticle (39.35% ± 5.98%) > curcumin-cyclodextrin inclusion (26.15% ± 17.51%) > curcumin-nanoparticle (12.86% ± 9.20%) > free curcumin (10.14% ± 3.37%). Most importantly, there was a statistically significant difference in the inhibition rate between the curcumin-cyclodextrin-nanoparticle group and the free curcumin group and the curcumin-nanoparticle group. This result confirmed that the curcumin-cyclodextrin-nanoparticle has the best in vivo anti-tumor efficacy. Its significant therapeutic effect can be attributed to the synergy of multiple advantages: first, the appropriate nanoparticle size enables it to be efficiently enriched in tumor tissues through the enhanced permeability and retention (EPR) effect. Second, the introduction of cyclodextrin helps to improve the uptake efficiency of the nanoparticle by tumor cells. Third, the polylactic acid-glycolic acid copolymer carrier realizes the slow and sustained release of the drug in the tumor site, thereby exerting a more persistent therapeutic effect.

[0090] Operation 3: H&E staining of pathological tissues.

[0091] The excised tumor was rapidly placed in an EP tube containing 4% tissue cell fixative within 20 min, the volume of tissue fixative should be 7 times of the tumor volume, and the tumor was fixed at 4°C for 1 day. After fixation, the tumor tissue sample was embedded with paraffin, and the frozen section was dyed with H&E (hematoxylin and eosin) dye, and the tumor section was observed under a microscope. The specific steps are as follows: the fixed tumor sample was cut into small pieces and placed in a tissue embedding box, the fixative was washed away with running water, the tissue was slowly dehydrated by alcohol with a gradient from low concentration to high concentration (75%-100%), and then the tissue block was transparentized in xylene for two times. The transparentized tissue block was immersed in a 65°C paraffin solution for 1-2 h, after the immersion was completed, the tumor tissue was moved into an embedding frame containing paraffin solution for embedding and waxing, and the tissue was cut into a 2-4 μm thin section with a tissue section machine, and then the tissue section was quickly moved into pure water at 42°C to expand the tissue section, and after the tissue section was fully expanded, the tissue section was fished out with a glass slide, and the section sample was placed in a 37°C oven for drying, and after the water was completely volatilized, H&E staining was performed.

[0092] The results are shown in Table 1. Figure 12 As shown in Table 1, the morphological characteristics of tumor cell proliferation and lymphocyte infiltration were observed in the model group, the free curcumin group, the curcumin-cyclodextrin inclusion complex group, the curcumin-nanoparticle group, and the curcumin-cyclodextrin-nanoparticle group. The tumor cells in each group were irregular in shape and different in size, the nucleus was enlarged and showed obvious nuclear division, and the nuclear division was most extensive in the model group, the free curcumin group, and the curcumin-cyclodextrin inclusion complex group. Compared with the above groups, the curcumin-nanoparticle and curcumin-cyclodextrin-nanoparticle treated groups showed more significant cell shrinkage morphology, indicating that the tumor cells underwent necrosis or apoptosis. In addition, the tumor cells in the model group were closely arranged, while the tumor tissue cell gap was significantly increased in the curcumin-nanoparticle and curcumin-cyclodextrin-nanoparticle treated groups. In particular, the curcumin-cyclodextrin-nanoparticle group showed more apoptosis areas in the tumor tissue. The above results show that the nanoparticle preparation, especially the curcumin-cyclodextrin-nanoparticle, shows better anti-tumor activity. It should be noted that since the tumor necrosis area is usually accompanied by inflammatory cell infiltration, and the inhibition ability of curcumin monotherapy on tumor growth and metastasis is limited, more macrophages and neutrophil infiltration are observed in the model group and each treatment group, showing significant inflammatory response.

[0093] The above examples are only examples of the specific embodiments of the present application, and are not limitations of the present application. Any modification, equivalent replacement or improvement made by those skilled in the art within the scope of the core principles of the present application and the protection scope defined in the claims should be included in the protection scope of the present application.

Claims

1. A curcumin-cyclodextrin-polylactic acid-glycolic acid copolymer composite nanoparticle, characterized in that, Its active ingredient is curcumin, and the carrier components include cyclodextrin and biodegradable polylactic acid-glycolic acid copolymer. The cyclodextrin forms an inclusion complex with curcumin, which is loaded into the nanoparticle framework formed by the polylactic acid-glycolic acid copolymer.

2. The curcumin-cyclodextrin-polylactic acid-glycolic acid copolymer composite nanoparticles as described in claim 1, characterized in that, The cyclodextrin is selected from any one of β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, 2,6-di-o-methyl-β-cyclodextrin, and sulfobutyl-β-cyclodextrin.

3. The curcumin-cyclodextrin-polylactic acid-glycolic acid copolymer composite nanoparticles as described in claim 1, characterized in that, The molar ratio of curcumin to cyclodextrin is 1:

1.

4. The method for preparing curcumin-cyclodextrin-polylactic acid-glycolic acid copolymer composite nanoparticles as described in claim 1, characterized in that, Includes the following steps: Step 1: Dissolve cyclodextrin in an organic solvent by sonication, then slowly add an appropriate amount of curcumin powder and sonicate until completely dissolved to form an inclusion complex. Then add polylactic acid-glycolic acid copolymer to the inclusion complex solution and vortex sonicate to form an organic phase. Step 2: Dissolve the emulsifier polyvinyl alcohol in water to form a polyvinyl alcohol aqueous solution. Step 3: Under stirring conditions, the organic phase obtained in Step 1 is added dropwise to the aqueous phase obtained in Step 2. The mixture is then ultrasonically emulsified to form a primary emulsion. Subsequently, the organic solvent is evaporated and removed by continuous stirring, and the mixture is solidified into nanoparticles to obtain a colloidal solution of the composite nanoparticles.

5. The method for preparing nanoparticles as described in claim 4, characterized in that: The cyclodextrin is β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, 2,6-di-o-methyl-β-cyclodextrin, and sulfobutyl-β-cyclodextrin. Preferably, the cyclodextrin is methyl-β-cyclodextrin.

6. The method for preparing nanoparticles as described in claim 4, characterized in that: The concentration of the polylactic acid-glycolic acid copolymer is 10–30 mg / mL. Preferably, the concentration of the polylactic acid-glycolic acid copolymer is 20 mg / mL.

7. The method for preparing nanoparticles as described in claim 4, characterized in that: The concentration of the emulsifier polyvinyl alcohol is 0.5-2% (w / v). Preferably, the concentration of polyvinyl alcohol is 1% (w / v).

8. The method for preparing nanoparticles as described in claim 4, characterized in that: The organic solvents are dichloromethane, acetone, and chloroform. Dichloromethane is preferred.

9. The curcumin-cyclodextrin-polylactic acid-glycolic acid copolymer composite nanoparticles as described in claim 1, characterized in that: The curcumin-cyclodextrin-polylactic acid-glycolic acid copolymer composite nanoparticles are preferably suitable for use in the field of breast cancer.

Images