Chlorogenic acid-asiasaponin vi self-assembled nanoparticles and preparation method and application thereof

Abstract

This invention discloses a chlorogenic acid-dipsacin VI self-assembled nanoparticle, its preparation method, and its applications, belonging to the fields of nanobiomaterials and biomedicine. The nanoparticles are formed by the self-assembly of chlorogenic acid (CGA) and dipsacin VI (AVI) in a solvent through intermolecular hydrogen bonding networks, electrostatic interactions, aromatic ring interactions, and hydrophobic effects. The method of this invention is simple and mild, and the resulting CA nanoparticles exhibit good stability under physiological conditions and can be conditionally released in a pathological microenvironment. These nanoparticles solve the problem of instability of CGA and AVI under conventional conditions, and can synergistically exert multiple functions such as antioxidation, anti-inflammation, osteogenic differentiation promotion, and angiogenesis promotion, with excellent biocompatibility. These nanoparticles can be widely used in the preparation of antioxidants, anti-inflammatory drugs, biomaterials that promote osteogenic differentiation and angiogenesis, health products, cosmetics, and pharmaceuticals, possessing significant biomedical application value.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomaterials and biomedicine, specifically relating to a chlorogenic acid-dipsacin VI self-assembled nanoparticle, its preparation method, and its application. Background Technology

[0002] Chlorogenic acid is a polyphenolic compound widely found in natural plants such as Eucommia ulmoides, coffee, and honeysuckle. Its molecular structure contains caffeoyl and quinic acid groups, which endow it with significant antioxidant, anti-inflammatory, and self-assembly chelating abilities. Due to its good biocompatibility, chlorogenic acid has broad application prospects in the biomedical field, and can be used for functional material construction, inflammatory microenvironment regulation, and bioactivity enhancement. However, chlorogenic acid has poor stability in normal environments and is prone to oxidative degradation, making it difficult to maintain its bioactivity for a long time, which greatly limits its practical application in the biopharmaceutical field. Dipsacus saponin VI is an important triterpenoid saponin derived from Dipsacus genus medicinal materials. It has been reported to promote osteoblast maturation and differentiation, enhance bone formation, and is associated with BMP-2 upregulation and activation of signaling pathways such as p38 / ERK. Studies have also shown that it can promote osteogenic differentiation of bone marrow mesenchymal stem cells in osteoporosis models and is associated with pathways such as PI3K / AKT.

[0003] To improve the stability, solubility, dispersibility, and in vivo availability of natural active ingredients, carrier delivery and nanostructuring are common strategies. Meanwhile, in recent years, carrier-free small-molecule self-assembled nanosystems have attracted attention because they can improve solubility, stability, and bioavailability while reducing the introduction of exogenous carriers. Furthermore, saponin molecules possess amphiphilic structural characteristics and can serve as nanocarriers to construct or participate in self-assembly systems. However, to date, there are no research reports, either domestically or internationally, on constructing carrier-free co-assembled nanosystems using chlorogenic acid and Dipsacus saponin VI as dual-active co-building units; research on the synergistic application of these two co-assembled nanosystems for bone repair and skin wound repair is also completely lacking. Moreover, there is a lack of standardized methods for constructing co-assembled nanosystems that can be prepared under mild processing conditions, possess long-term storage stability, exhibit lesion microenvironment-responsive release characteristics, and are reproducible and scalable. Therefore, developing carrier-free co-assembled nanoparticles based on chlorogenic acid and Dipsacus saponin VI, and establishing a stable, controllable, and industrializable preparation method, has important scientific value and application prospects for overcoming the drug-likeness bottleneck of these two substances, exploring their synergistic therapeutic potential, and promoting the clinical translation of active ingredients in traditional Chinese medicine. Summary of the Invention

[0004] The first technical problem to be solved by this invention is to provide a chlorogenic acid-dipsacin VI self-assembled nanoparticle, which constructs a stable nanostructure through intermolecular non-covalent interactions, thereby improving the dispersibility and storage stability of the two active ingredients in an aqueous system, and providing a formulation basis for accelerated release in an acidic environment, thus providing a new solution for the research and development of biomaterials and drugs related to bone tissue regeneration and repair.

[0005] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned chlorogenic acid-dipsacin VI self-assembled nanoparticles.

[0006] The third technical problem to be solved by the present invention is to provide the application of the above-mentioned chlorogenic acid-dipsacin VI self-assembled nanoparticles.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A method for preparing chlorogenic acid-dipsacin VI self-assembled nanoparticles includes the following steps: (1) The oil phase containing chlorogenic acid and bisacodyl VI was added dropwise to the aqueous phase to carry out solvent replacement and reaction, and the reaction solution was obtained. (2) The reaction solution obtained in step (1) was centrifuged, washed and resuspended to obtain a dispersion of self-assembled nanoparticles; then a freeze-drying protectant was added and freeze-dried to obtain chlorogenic acid-dipsacin VI self-assembled nanoparticles. Steps (1) to (2) are all carried out under light-protected conditions.

[0009] In some embodiments, in step (1), the oil phase containing chlorogenic acid and dipsacin VI is prepared by the following method: chlorogenic acid is dissolved in an organic solvent to obtain a chlorogenic acid mother liquor; dipsacin VI is dissolved in an organic solvent to obtain a dipsacin VI mother liquor; the chlorogenic acid mother liquor and the dipsacin VI mother liquor are mixed to obtain an oil phase; preferably, the oil phase containing chlorogenic acid and dipsacin VI is prepared under light-protected conditions.

[0010] In some embodiments, the organic solvent is ethanol or methanol, preferably ethanol; and / or, the concentration of the chlorogenic acid mother liquor is 1~50 mg / mL, preferably 10 mg / mL; and / or, the concentration of the dipsacin VI mother liquor is 1~50 mg / mL, preferably 10 mg / mL; the molar ratio of the chlorogenic acid to the dipsacin VI in the oil phase is 0.5:1~3:1, preferably 3:1.

[0011] In some embodiments, in step (1), the volume ratio of the oil phase to the aqueous phase is 1:10~20, preferably 1:10; and / or, the aqueous phase is water or a buffer solution with a pH value of 6.5~7.4; and / or, the dropwise addition is carried out at a rate of 0.05~0.5 mL / min under stirring conditions of 500~1000 rpm.

[0012] Preferably, the dropping is performed at a rate of 0.1 mL / min under stirring at 800 rpm.

[0013] In some embodiments, in step (1), the reaction is carried out at a speed of 300-800 rpm (preferably 500 rpm) for a time of 1-6 h, preferably 2 h.

[0014] In some embodiments, in step (2), the centrifugation is performed at a speed of 8000~15000 rpm (preferably 12000 rpm) for 10~30 min (preferably 20 min); and / or, the washing is performed by centrifugation with water for 1~5 times, preferably 3 times; and / or, the resuspension is performed by resuspension with water; and / or, the concentration of self-assembled nanoparticles in the self-assembled nanoparticle dispersion is 5~10 mg / mL, preferably 10 mg / mL.

[0015] In some embodiments, in step (2), the freeze-drying protectant is any one or more of trehalose, sucrose, or mannitol; and / or, the mass-to-volume ratio of the freeze-drying protectant to the self-assembled nanoparticle dispersion is 1 g:100 mL ~ 10 g:100 mL, preferably 5 g:100 mL; and / or, the freeze-drying is performed at a temperature of -60 to -90°C for 12 to 48 hours; and / or, after adding the freeze-drying protectant, a pre-freezing is performed before freeze-drying, wherein the pre-freezing is performed at a temperature of -60 to -90°C for 2 to 3 hours.

[0016] This invention also protects the chlorogenic acid-dipsacin VI self-assembled nanoparticles prepared by the above preparation method.

[0017] In some embodiments, the chlorogenic acid-dipsacin VI self-assembled nanoparticles have an average particle size of 139-175 nm and a zeta potential of -11.0-9.0 mV.

[0018] This invention also protects the use of the above-mentioned chlorogenic acid-dipsacin VI self-assembled nanoparticles in the preparation of antioxidant products, and / or anti-inflammatory products, and / or osteogenic differentiation promoting products, and / or angiogenesis promoting products; the products are any one of biomaterials, health products, cosmetics or pharmaceuticals.

[0019] Beneficial effects:

[0020] (1) The chlorogenic acid-dipsacin VI self-assembled nanoparticles provided by the present invention have good adaptability to the inflammatory microenvironment. They can achieve comprehensive intervention on the pathological microenvironment by regulating the local immune inflammatory response and oxidative stress level in inflammatory lesions, which is beneficial to the repair of lesion tissue.

[0021] (2) Achieving synergistic effects of multiple biological activities: the antioxidant and anti-inflammatory properties of chlorogenic acid and the osteogenic and angiogenic activities of Dipsacus saponin VI have a synergistic effect. Compared with chlorogenic acid alone, its antioxidant capacity, anti-inflammatory effect, osteogenic capacity and angiogenic capacity are significantly improved.

[0022] (3) Excellent biosafety: Cell experiments have confirmed that the nanoparticles have no significant cytotoxicity to bone marrow mesenchymal stem cells in the concentration range of 0~200 μg / mL.

[0023] (4) The preparation method is simple and mild: the reaction is carried out at room temperature, without the need for high temperature, high pressure and toxic and harmful reagents. The operation is simple, the cost is low, and it is easy to scale up production. The whole process is carried out in the dark to avoid the degradation of the chlorogenic acid oxidation characteristics and ensure product quality.

[0024] (5) It can be widely used in the development of biomaterials and drugs related to tissue repair, inflammation regulation, bone formation and angiogenesis. Attached Figure Description

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0026] Figure 1 Typical images of the self-assembled nanoparticles prepared in Example 1 under SEM and TEM.

[0027] Figure 2 The intensity distribution diagram is shown in the dynamic light scattering experiment of the self-assembled nanoparticles prepared in Example 1.

[0028] Figure 3 Characteristic Fourier transform infrared absorption spectra of the self-assembled nanoparticles prepared by chlorogenic acid, dipsaponin VI and Example 1;

[0029] Figure 4 XRD patterns of the self-assembled nanoparticles prepared from chlorogenic acid, dipsaponin VI and Example 1;

[0030] Figure 5 Zeta potential distribution of self-assembled nanoparticles prepared with chlorogenic acid, dipsacus saponin VI and Example 1;

[0031] Figure 6 The following is a free radical scavenging test of the self-assembled nanoparticles prepared in Example 1. In the figure, (A) shows the ABTS scavenging capacity data; (B) shows the DPPH scavenging capacity data; (C) shows the PITO scavenging capacity data; and (D) shows the oxygen free radical scavenging efficiency and quantification graph.

[0032] Figure 7 PDI test results for the self-assembled nanoparticles prepared with chlorogenic acid, bisacodyl VI and Example 1 under storage conditions at 4°C;

[0033] Figure 8 The cumulative release-time curves of chlorogenic acid in release media at different pH values ​​for the nanoparticles prepared in Example 1;

[0034] Figure 9 Image showing the biocompatibility assessment of self-assembled nanoparticles prepared in Example 1 in CCK-8 cell viability.

[0035] Figure 10 The graph shows the results of assessing cell viability in a biocompatibility test using live / dead cell fluorescence staining of the self-assembled nanoparticles prepared in Example 1.

[0036] Figure 11 The image shows the results of the hemolysis experiment to evaluate blood compatibility in the biocompatibility test of the self-assembled nanoparticles prepared in Example 1.

[0037] Figure 12 The reactive oxygen species (ROS) assay was performed on the self-assembled nanoparticles prepared in Example 1 in bone marrow mesenchymal stem cell culture. (A) is a typical image from the ROS assay; (B) is a typical image from the JC-1 staining assay.

[0038] Figure 13 Anti-inflammatory experiments were conducted on the self-assembled nanoparticles prepared in Example 1 in macrophage culture. (AB) show typical immunofluorescence images and quantitative results of CD86 and CD206; (C) shows PCR data for IL-6, iNOS, TNF-α, and IL-10.

[0039] Figure 14 Typical staining images of the self-assembled nanoparticles prepared in Example 1 during ALP and ARS staining experiments in bone marrow mesenchymal stem cell culture;

[0040] Figure 15 The image shows the scratch assay results of the self-assembled nanoparticles prepared in Example 1 interfering with the migration of human umbilical vein endothelial cells.

[0041] Figure 16 Figure 1 shows the Transwell experiment results of the self-assembled nanoparticles prepared in Example 1 interfering with the migration of human umbilical vein endothelial cells.

[0042] Figure 17 The figure shows the results of the tube formation experiment on the self-assembled nanoparticles prepared in Example 1 interfering with the migration of human umbilical vein endothelial cells. Detailed Implementation

[0043] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings of the embodiments of the present invention. It should be understood that the described embodiments are only some implementations of the present invention, and not all embodiments. All other embodiments derived by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the claims of the present invention.

[0044] Unless otherwise specified, the present invention does not impose any special restrictions on the source of the raw materials, and commercially available products well known to those skilled in the art can be used.

[0045] Example 1: Preparation and characterization of chlorogenic acid-dipsacin VI self-assembled nanoparticles

[0046] In this invention, CGA and AVI are mixed with a solvent and subjected to a coordination reaction to prepare chlorogenic acid-dipsacin VI self-assembled nanoparticles (CA NPs).

[0047] The specific steps are as follows:

[0048] (1) Under light-protected conditions, weigh 10 mg of CGA and dissolve it in anhydrous ethanol (1 mL concentration 10 mg / mL), stir until homogeneous to prepare CGA stock solution; under light-protected conditions, weigh 10 mg of AVI and dissolve it in anhydrous ethanol (1 mL concentration 10 mg / mL), stir until completely dissolved to prepare AVI stock solution; mix the two stock solutions according to the molar ratio of chlorogenic acid to Dipsacus saponin VI of 3:1 and vortex for 30 s to obtain a mixed solution;

[0049] (2) Under light-protected and stirring conditions (800 rpm), 2 mL of the mixed solution was slowly added dropwise to 20 mL of deionized water (pH 6.5-7.4) at a rate of 0.1 mL / min. After the addition was completed, stirring was continued for 2 hours (500 rpm) to obtain the reaction solution.

[0050] (3) Purification and post-treatment: Under light-protected conditions, the reaction solution was centrifuged (12000 rpm, 20 minutes) and the supernatant was discarded. The precipitate was resuspended in deionized water and centrifuged and washed three times to remove unassembled monomers. After the last centrifugation, the supernatant was discarded and the obtained solid precipitate was resuspended in deionized water to obtain a CA NPs dispersion (10 mg / mL). Trehalose, a freeze-drying protectant, was added to the CA NPs dispersion. The amount of trehalose added was based on the final volume of the CA NPs dispersion, and the mass-volume ratio (w / v) was 5% (i.e., 5 g of trehalose was added to every 100 mL of CA NPs dispersion). After mixing, the mixture was pre-frozen at -80℃ for 2 hours and then freeze-dried for 24 hours to obtain self-assembled nanoparticles.

[0051] The chlorogenic acid-dipsacin VI self-assembled nanoparticles prepared in this embodiment were characterized and analyzed, and the results are as follows:

[0052] 1. Morphological observation experiment

[0053] Morphological characterization was performed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), such as... Figure 1 Microscopic images show that CA NPs exhibit a spherical nanoscale composite structure.

[0054] 2. Dynamic light scattering experiment

[0055] Intensity detection using dynamic light scattering (DLS) technology Figure 2 Test data shows that the average particle size of CA NPs is 159.96±3.23nm, and the particle distribution is relatively uniform.

[0056] 3. Fourier transform infrared spectroscopy

[0057] Two mg of the CA NPs solid powder prepared in Example 1 was dispersed in 200 μL of anhydrous ethanol. 20 μL of this dispersion was drop-coated onto a clean wafer and allowed to air dry at room temperature to form a uniform film. CGA and AVI films were prepared at the same concentration. The nanoparticles prepared in Example 1 were coated onto the wafer and dried at 4000–400 cm⁻¹. -1 The sample was scanned with infrared light within the wavenumber range to obtain the characteristic infrared absorption spectrum of the sample. Figure 3 Test data show that CGA, AVI, and their self-assembled products (CANPs) all exhibit their respective typical functional group absorption characteristics. CGA and AVI show absorption characteristics at 3200-3500 cm⁻¹. -1 All regions exhibit broad -OH stretching vibration absorption bands, originating from the polyphenolic hydroxyl, carboxyl hydroxyl, and saponin sugar chain polyhydroxyl structures, respectively. Compared to the raw material, CA NPs are at approximately 3400 cm⁻¹. -1The -OH absorption band at this point broadens significantly, accompanied by changes in intensity and peak shape, suggesting enhanced intermolecular hydrogen bonding after self-assembly, resulting in a more stable hydrogen bond network. (Approximately 1700 cm⁻¹) -1 C=O stretching vibration-related absorptions (mainly attributed to the carboxyl / ester structure of CGA) can be observed at this region. The peak shape of CA NPs in this region shows passivation and subtle changes, indicating a change in the microenvironment of the C=O group, which may be involved in hydrogen bonding or embedded in a self-assembled structure. Furthermore, at approximately 1500 cm⁻¹... -1 Distinguished characteristic changes were observed in CA NPs in the vicinity (absorption region related to aromatic ring skeletal vibration and conjugated structure), suggesting altered molecular packing and possibly enhanced π-π interactions or hydrophobic association; these changes were observed in the 1000-1100 cm⁻¹ region. -1 Within the range, AVI glycan-related CO / COC stretching vibration absorptions are still retained in CA NPs, and at ≈1030 cm⁻¹ -1 The presence of a distinct characteristic peak indicates that the saponin sugar chain structure remains intact in the self-assembled product. In summary, CA NPs simultaneously retain the characteristic absorption bands of CGA and AVI, and exhibit peak shape / intensity changes in key regions such as -OH and C=O, proving that there is a significant non-covalent interaction between CGA and AVI (mainly hydrogen bonding, possibly accompanied by hydrophobic association and π-π stacking), thereby driving their co-assembly to form a stable nanoparticle structure.

[0058] 4. XRD Experiment

[0059] Take 50 mg of the CA NPs lyophilized powder prepared in Example 1, flatten it in the groove of the sample holder of the X-ray diffractometer to form a flat surface, load it into the diffractometer, and scan it at a Cu Kα ray source, voltage 40 kV, current 30-40 mA, scanning range 5°~90° (2θ), and scanning speed 2-5° / min. Record the XRD intensity of the sample at different diffraction angles. The obtained spectra ( Figure 4 The results show that CGA exhibits multiple sharp and intense diffraction peaks, indicating its high crystallinity and crystalline state; while AVI is dominated by broad and blunt diffuse peaks, suggesting its lower crystallinity and amorphous or low-crystallinity characteristics. Compared with the two raw materials, the diffraction curves of CA NPs show smoother diffuse peaks overall, and the characteristic crystal peaks of CGA are significantly weakened to the point of almost disappearing. This indicates that after self-assembly to form nanoparticles, the long-range ordered lattice structure of CGA is significantly destroyed and tends to become amorphous or molecularly dispersed, suggesting that there is a strong intermolecular interaction between CGA and AVI, driving the formation of new low-order assembly structures.

[0060] 5. Zeta potential detection experiment

[0061] Dispersions (2 mg / mL) of CGA, AVI, and CA NPs obtained in Example 1 were prepared, and the zeta potentials were measured at 25°C using a potentiometer. Experimental data showed that ( Figure 5 The average Zeta potential of CGA is approximately -12.02 mV, the average Zeta potential of AVI is approximately +3.30 mV, and the average Zeta potential of CA NPs is approximately -10.58 mV. This indicates that coordination occurs between the two, resulting in a reduction of negative charge after synthesis, indicating successful synthesis.

[0062] Example 2

[0063] The method for preparing chlorogenic acid-dipsacin VI self-assembled nanoparticles in this embodiment is the same as that in Example 1, except that the molar ratio of chlorogenic acid to dipsacin VI in step (1) is 0.5:1.

[0064] Example 3

[0065] The method for preparing chlorogenic acid-dipsacin VI self-assembled nanoparticles in this embodiment is the same as that in Example 1, except that the molar ratio of chlorogenic acid to dipsacin VI in step (1) is 1:1.

[0066] Example 4

[0067] The method for preparing chlorogenic acid-dipsacin VI self-assembled nanoparticles in this embodiment is the same as that in Example 1, except that the molar ratio of chlorogenic acid to dipsacin VI in step (1) is 2:1.

[0068] Example 5 Free Radical Scavenging Test

[0069] A certain amount of ABTS solution was mixed with potassium persulfate solution and stored in the dark for 12 hours to allow ABTS to be completely oxidized to ABTS. + This was used as the working stock solution for ABTS. ABTS was prepared using phosphate buffer (pH=7.4, 0.01 mM). + The solution is diluted to an appropriate concentration so that its absorbance at a specified wavelength is approximately 734 nm, which is ABTS. + Working solution. Then, CA NPs sample solutions prepared at different molar ratios (CGA:AVI = 0.5:1, 1:1, 2:1, 3:1) with 0.2 mL of 50 μg / mL were mixed with 2 mL of ABTS. + The working solution was mixed thoroughly and incubated at room temperature in the dark for 30 min. The absorbance at 734 nm was then measured, and the results are as follows. Figure 6 As shown in Figure A, CANPs, with a feed molar ratio of CGA:AVI = 3:1, ABTS + It has the lowest content and the highest clearance rate.

[0070] CA NPs sample solutions prepared at different molar ratios (CGA:AVI = 0.5:1, 1:1, 2:1, 3:1) with a concentration of 50 μg / mL were mixed with 2 mL of 0.04 mg / mL DPPH radical ethanol solution. A blank control was prepared by adding 2 mL of ethanol to 2 mL of DPPH solution. The mixtures were incubated at room temperature in the dark for 30 min, and the absorbance was measured at 519 nm. The results are as follows: Figure 6 As shown in Figure B, CANPs at a CGA:AVI molar ratio of 3:1, DPPH + It has the lowest content and the highest clearance rate.

[0071] CA NPs sample solutions prepared at different molar ratios (CGA:AVI = 0.5:1, 1:1, 2:1, 3:1) with a concentration of 50 μg / mL were mixed with 2 mL of 0.04 mg / mL PTIO radical solution (prepared with 50 mM phosphate-buffered saline PBS, pH 7.4). A blank control was prepared by adding 2 mL of ethanol to 2 mL of PTIO solution. After incubation at 37 °C for 2 h in the dark, the absorbance was measured at 557 nm. The results are as follows: Figure 6 As shown in C, CA NPs under the condition of CGA:AVI = 3:1 molar ratio, PTIO + It has the lowest content and the highest clearance rate.

[0072] The above results indicate that CA NPs have reactive oxygen species scavenging capabilities.

[0073] To evaluate the antioxidant enzyme activity of the nanoparticles (CA NPs) prepared in Example 1, hydroxyl radicals (•OH) and superoxide anion radicals (•O2) were selected. - ) and singlet oxygen ( 1 The in vitro scavenging capacity of three typical reactive oxygen species (ROS) models (O2) was tested, and all reactions were carried out at room temperature (25°C).

[0074] Hydroxyl radicals (•OH): generated using a Fenton reaction system. A 200 μL solution of CA NPs nanoparticles (1 mg / mL) was mixed with an equal volume of a mixed solution (containing 0.2 mM FeSO4 and 2 mM H2O2 to a final concentration) in phosphate-buffered saline (PBS, 50 mM, pH 7.4). The reaction system was immediately and thoroughly mixed with an equal volume of 100 mM spin trapping agent 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO).

[0075] Superoxide anion radical (•O2) -The nanoparticles were generated using a xanthine / xanthine oxidase (X / XO) system. A solution of nanoparticles (200 μL, 1 mg / mL) was mixed with a PBS solution (50 mM, pH 7.4) containing a final concentration of 0.2 mM xanthine and 0.1 U / mL xanthine oxidase, and an equal volume of 100 mM DMPO spin trapping agent was immediately added.

[0076] Singlet oxygen ( 1 O2: Generated using the photosensitizer Rose Bengal. A nanoparticle solution (200 μL, 1 mg / mL) was mixed with a PBS solution (50 mM, pH 7.4) containing a final concentration of 20 μM Rose Bengal in a transparent EPR quartz capillary tube. The mixture was irradiated for 5 minutes under a 500 W halogen tungsten lamp to generate ¹O2, followed immediately by the addition of a spin trapping agent, 2,2,6,6-tetramethylpiperidine (TEMP), at a final concentration of 50 mM.

[0077] Signal acquisition was then performed using a Bruker A300 electron paramagnetic resonance spectrometer. The general instrument parameters were set as follows: center field strength 3510 G, scan width 100 G, microwave power 20 mW, modulation frequency 100 kHz, modulation amplitude 1.0 G, and scan time 30 s. The EPR signal intensity (based on peak height or peak area in a single scan) of the corresponding characteristic spin adducts (DMPO-OH adduct, DMPO-OOH adduct, or TEMP-¹O2 adduct) of each system after nanoparticle interaction was measured and compared with the signal intensity of the positive control group (containing only the free radical generating system and scavenger). The free radical scavenging rate was calculated using the following formula:

[0078] Clearance rate (%) = [1 - (I_sample / I_control)] × 100%

[0079] Where I_sample represents the EPR signal intensity of the sample group, and I_control represents the EPR signal intensity of the positive control group.

[0080] Test data show that the nanoparticles obtained in Example 1 (molar ratio of CGA:AVI = 3:1) exhibit significant oxygen free radical scavenging ability and excellent antioxidant enzyme activity. Figure 6 D).

[0081] Example 6: Stability Testing Experiments of CGA, AVI, and CA NPs

[0082] Weigh out 10 mg of CGA, 10 mg of AVI, and 10 mg of CA NPs respectively, and add 10 mL of phosphate buffer (pH 7.4, simulating the physiological microenvironment) to each. Vortex to fully disperse or dissolve them, obtaining CGA monomer solution, AVI monomer solution, and CA NPs dispersion (all concentration 1 mg / mL). Store the above three groups of samples at 4℃ in the dark. Samples were taken at regular intervals on days 1, 3, 5, 7, 9, 11, and 14. The polydispersity index (PDI) of each sample was determined using dynamic light scattering (DLS) technology. Before the measurement, the sample was appropriately diluted to a suitable concentration, gently shaken to mix, and then poured into a quartz cuvette. After equilibration at 254℃ for 2 minutes, the measurement was performed. Each sample was measured three times, and the average value was taken. Test data showed that the PDI values ​​of CGA and AVI monomer solutions increased with incubation time under simulated physiological conditions, and on day 14, the PDI values ​​significantly increased to above 0.3. This indicates that the two monomers exist only as free molecules or random aggregates in aqueous solution, failing to form a homogeneous and stable colloidal dispersion system, resulting in poor physical dispersion stability. In contrast, the PDI value of the CA NPs dispersion fluctuated by less than 0.1 within the same incubation period, consistently remaining below 0.2, demonstrating excellent monodispersity. This confirms that the self-assembled nanoparticles constructed in this invention possess excellent colloidal stability, effectively solving the problem of poor dispersion stability of CGA and AVI monomers. Figure 7 ).

[0083] Example 7 pH-responsive CGA release experiment

[0084] 10 mg of CA NPs prepared in Example 1 were placed in 10 mL of release media with different pH values ​​(phosphate buffer solutions with pH values ​​of 5.5 and 7.4, simulating pathological and physiological microenvironments). The solutions were incubated at a constant temperature of 37°C with shaking. At predetermined time points (0–24 h), the supernatant was collected to determine the concentration of CGA in the solution (UV spectrophotometry), and a cumulative release concentration-time curve was plotted. Test data showed that the CGA release rate in the pathological microenvironment at pH 5.5 was significantly higher than that in the physiological microenvironment at pH 7.4, exhibiting pH-responsive release characteristics. Figure 8 Compared to traditional non-responsive bone repair drugs, the pH-responsive release properties of CA NPs enable targeted release to lesions, providing an ideal drug choice for constructing a multifunctional bone repair delivery system.

[0085] Example 8 Biosafety Experiment

[0086] 1. CCK-8 cell viability assay

[0087] Bone marrow mesenchymal stem cells (BMSCs) were routinely cultured and passaged using a dedicated mesenchymal stem cell culture medium (Cyagen, HUXMA-90011). BMSCs were then cultured and passaged at a rate of 2 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL / well in 96-well plates. The cells were incubated at 37°C with 5% CO2 for 24 h to allow complete cell adhesion. The old culture medium was discarded, and 100 μL of fresh culture medium containing different concentrations (2.5, 5, 10, 20, 50, 100, 200 μg / mL) of CA NPs (prepared in Example 1) was added to each well. A blank control (culture medium only) was also included. After co-incubating the cells with the nanoparticles for 24 h, cell viability was assessed using a CCK-8 assay, and the absorbance of each well was measured at 450 nm using a microplate reader. The results showed that CA NPs nanoparticles had no significant inhibitory effect on BMSC cell viability within the concentration range of 0-200 μg / mL. Figure 9 ).

[0088] 2. Live / Dead Cell Fluorescence Staining Assay

[0089] Bone marrow mesenchymal stem cells (BMSCs) were used at a rate of 2 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL / well in 96-well plates, with 100 μL of mesenchymal stem cell culture medium per well. Cells were cultured at 37°C and 5% CO2 for 24 h to allow complete cell adhesion. The old culture medium was discarded, and 100 μL of fresh culture medium containing CGA (20 μg / mL), AVI (20 μg / mL), and CA NPs (20 μg / mL, prepared in Example 1) were added. A blank control (culture medium only) was also included. After co-incubating the cells with the nanoparticles for 3 days, cell viability was assessed using live / dead cell fluorescence staining. The results showed that the self-assembled nanoparticles obtained in Example 1 had good cell compatibility, and the cell viability was not significantly different from the control group. Figure 10 ).

[0090] 3. Hemolysis test

[0091] Fresh anticoagulated whole blood was collected and centrifuged at 1500-3000 rpm for 5-10 min to separate red blood cells. The supernatant was discarded, and the cells were resuspended in PBS or 0.9% NaCl and washed three times until the supernatant was clear. A 5% red blood cell suspension was then prepared. CANPs prepared in Example 1 (and control samples CGA and AVI) were mixed with the red blood cell suspension in equal volumes and incubated at 37 ℃ in the dark for 1-3 h. After incubation, the cells were centrifuged (1500-3000 rpm, 5-10 min), and the supernatant was measured at 540 nm to determine the absorbance corresponding to bilirubin release. Deionized water or 0.1% Triton X-100 was used as a positive control (complete hemolysis), and PBS or 0.9% NaCl was used as a negative control (virtually no hemolysis). The hemolysis rate was calculated using the formula:

[0092]

[0093] in A s The absorbance of the sample group. A n This is the absorbance for the negative control. A p The absorbance is for the positive control.

[0094] The results show (e.g.) Figure 11 The hemolysis rate in the positive control group was close to 100%, indicating that the system could achieve complete hemolysis; the hemolysis rate in the negative control group was approximately 4%–5%. The hemolysis rates in the CGA, AVI, and CA NPs groups remained at a low level of approximately 4%–5%, comparable to the negative control and significantly lower than the positive control. Furthermore, the supernatant was basically clear and the erythrocyte sediment was intact upon visual inspection, with no obvious red hemolytic supernatant observed. This indicates that CA NPs did not cause significant erythrocyte hemolysis under these experimental conditions, demonstrating good blood compatibility and biosafety.

[0095] Example 9 Intracellular Antioxidant Stress Experiment

[0096] ROS test (reactive oxygen species level): BMSCs were administered at 2 × 10⁻⁶ 4Cultured at a density of 1000 samples per well in 12-well plates, with 1 mL of culture medium (DMEM low-glucose + 10% FBS + 1% penicillin / streptomycin) added to each well. After adherent culture, the culture medium was discarded, and the following groups were treated: blank control group (routine culture), model group (1 μg / mL LPS), CGA intervention group (1 μg / mL LPS + 20 μg / mL CGA), AVI intervention group (1 μg / mL LPS + 20 μg / mL AVI), and CA NPs intervention group prepared in Example 1 (1 μg / mL LPS + 20 μg / mL CA NPs). All treatment reagents were prepared with culture medium. After 24 hours of intervention culture in each group, 1 mL of DCFH-DA probe working solution with a final concentration of 10 μM was added to each well, and the plates were incubated in the dark for 30 minutes. After washing with PBS, the plates were observed under a fluorescence microscope.

[0097] Test data showed that CA NPs treatment significantly reduced LPS-induced intracellular ROS levels, and its green fluorescence intensity was weaker than that of the CGA group, indicating that CA NPs have excellent antioxidant activity and can protect cells from oxidative damage by scavenging excess ROS. Figure 12 A).

[0098] JC-1 staining (mitochondrial membrane potential): BMSCs were stained with 2×10⁻⁶... 4 Cultured at a density of cells / well in 12-well plates, the culture medium was discarded after adherent culture, and the following groups were treated: blank control group (routine culture), model group (1 μg / mL LPS), CGA intervention group (1 μg / mL LPS + 20 μg / mL CGA), AVI intervention group (1 μg / mL LPS + 20 μg / mL AVI), and CA NPs intervention group prepared in Example 1 (1 μg / mL LPS + 20 μg / mL CA NPs). All treatment reagents were prepared with culture medium. After 24 hours of intervention culture, each group was washed with PBS, and incubated with JC-1 staining working solution (1.0 mL per well, final concentration 2 μM) for 30 minutes. After washing, the samples were observed under a fluorescence microscope.

[0099] Test data showed that the red / green fluorescence ratio of cells decreased significantly after LPS induction, indicating a decline in mitochondrial membrane potential. Compared with CGA or AVI single-drug treatment, the red / green fluorescence ratio of the CA NPs treatment group recovered more significantly, indicating that CA NPs can synergistically maintain mitochondrial membrane potential stability, alleviate LPS-induced mitochondrial dysfunction, and thus exert a stronger antioxidant stress effect. Figure 12 B).

[0100] Example 10 Cell Anti-inflammatory Experiment

[0101] 1. Immunofluorescence staining experiments of M1 (CD86) and M2 (CD206)

[0102] Macrophages (RAW264.7) were sized at a rate of 5 × 10⁻⁶. 5 Cells were seeded at a density of cells / well in 24-well plates containing cell spreaders. After adherent culture, the old culture medium was discarded, and the following groups were treated: blank control group (routine culture), model group (1 μg / mL LPS), CGA intervention group (1 μg / mL LPS + 20 μg / mL CGA), AVI intervention group (1 μg / mL LPS + 20 μg / mL AVI), and CA NPs intervention group prepared in Example 1 (1 μg / mL LPS + 20 μg / mL CA NPs). All treatment reagents were prepared with culture medium (0.5 mL). After 24 hours of culture following intervention, cells in each group were washed with PBS, fixed with 4% paraformaldehyde at room temperature for 30 min, washed three times with PBS, permeabilized with 0.5% Triton X-100 at room temperature for 10 min, and blocked with 3% BSA at room temperature for 1 h. The blocking solution was removed, and primary antibodies diluted with 1% TBST (rabbit anti-CD86, 1:500; rabbit anti-CD206, 1:400; rabbit anti-TNF-α, 1:1000; rabbit anti-IL-10, 1:800) were added and incubated overnight at 4°C. Cells were washed three times with PBS, and species-matched secondary antibodies labeled with Fluor 488 or Fluor 594 (1:1000 dilution) were added and incubated at room temperature in the dark for 1 h. Cells were washed three times with PBS, and nuclei were stained with 1 μg / mL DAPI solution for 5 min. After washing with PBS, the cells were mounted with anti-fluorescence quenching mounting medium, and images were acquired using a laser confocal microscope. The fluorescence intensity was used to reflect the changing trends of different inflammatory factors.

[0103] The results are as follows Figure 13 As shown in A and B, the expression of CD86, the M1 polarization-specific marker of macrophages in the CA NPs group, was significantly weaker, while the expression of CD206, the M2 polarization-specific marker of macrophages, was significantly stronger, indicating that the self-assembled nanoparticles obtained in Example 1 have the ability to regulate macrophage polarization and have good anti-inflammatory capabilities.

[0104] 2. Quantitative assays of IL-6, iNOS, TNF-α, and IL-10 using polymerase chain reaction (PCR)

[0105] Macrophages (RAW264.7) were sized at a rate of 5 × 10⁻⁶. 5Cells were seeded at a density of 1 cell / well in 24-well plates. After adherent culture, the old culture medium was discarded, and the cells were divided into the following groups: blank control group (routine culture), model group (1 μg / mL LPS), CGA intervention group (1 μg / mL LPS + 20 μg / mL CGA), AVI intervention group (1 μg / mL LPS + 20 μg / mL AVI), and CA NPs intervention group prepared in Example 1 (1 μg / mL LPS + 20 μg / mL CA NPs). All treatment reagents were prepared with culture medium (0.5 mL). After 24 hours of culture, the cells were washed with PBS, and total RNA was extracted using TRIzol. The RNA concentration and purity were measured. An equal amount of RNA was reverse transcribed to synthesize cDNA, and real-time quantitative PCR was performed using the SYBR Green system to detect the mRNA expression levels of inflammation-related genes IL-6, iNOS, TNF-α, and IL-10.

[0106] The results are as follows Figure 13 As shown in Figure C, the expression of inflammatory markers (IL-6, iNOS, TNF-α) in the CA NPs group decreased significantly, while the expression of the anti-inflammatory marker IL-10 increased significantly, indicating that the nanoparticles obtained in Example 1 have good anti-inflammatory capabilities.

[0107] Example 11: ALP and ARS staining experiments to evaluate the effect of CA NPs on osteogenic differentiation capacity of BMSCs.

[0108] BMSCs were 2 × 10 4 Cells were seeded per well in 24-well plates. After cell adhesion and growth to approximately 70%–80% confluence, the medium was replaced with osteogenic induction medium for induction culture. The following groups were established: blank control group (routine culture), model group (1 μg / mL LPS), CGA intervention group (1 μg / mL LPS + 20 μg / mL CGA), AVI intervention group (1 μg / mL LPS + 20 μg / mL AVI), CGA and AVI physical mixed intervention group (1 μg / mL LPS + 20 μg / mL CGA + 20 μg / mL AVI), and CA NPs intervention group prepared in Example 1 (1 μg / mL LPS + 20 μg / mL CA NPs). All treatment reagents were prepared in 0.5 mL culture medium. Alkaline phosphatase (ALP) expression is a key marker of osteoblast activity and the early stages of bone formation. To assess osteogenic activity, ALP staining was performed 14 days after induction culture.

[0109] Test data showed that, compared with the blank control group, LPS treatment significantly reduced ALP staining intensity and ALP activity in BMSCs, suggesting that inflammatory stimulation inhibits early osteogenic differentiation. Compared with the LPS group, the CGA group, AVI group, and physical mixture of CGA and AVI could restore ALP expression to some extent, while the CA NPs group showed the deepest ALP staining and the highest ALP activity, indicating that CA NPs have a more significant protective and promoting effect on early osteogenic differentiation of BMSCs under the LPS-induced inflammatory environment. Figure 14 )

[0110] Cells in each group were cultured under osteogenic induction conditions until the mineralized nodule formation stage, and then stained with Alizarin Red S (ARS).

[0111] Test data showed that the CA NPs group exhibited the strongest alizarin red staining and the highest calcium deposition, indicating that it had the best reversal effect on LPS-induced osteogenic inhibition and could most effectively promote BMSC mineralization and mature osteogenic differentiation. (e.g.) Figure 14 )

[0112] Example 12: Scratch test to evaluate the effect of CA NPs on the migration ability of HUVECs

[0113] HUVECs were used at 2 × 10⁻⁶ per well. 4 Cells were seeded at a density of 1000 μL in each well of a 12-well plate, and cultured for 24 h until complete confluence. Using a 200 μL sterile pipette tip perpendicular to the bottom of the well, a straight line was drawn evenly on the monolayer of cells. The cells were then gently washed three times with PBS to remove any detached cells. Immediately after the intervention, 1 mL of drug-containing medium was added to each well according to the assigned groups. After group intervention, the cells were placed in an incubator for further culture. The same location was photographed under a microscope at 0 h and 24 h after the drawing. The experimental groups were as follows: blank control group (routine culture), model group (1 μg / mL LPS), CGA intervention group (1 μg / mL LPS + 20 μg / mL CGA), AVI intervention group (1 μg / mL LPS + 20 μg / mL AVI), physical mixed intervention group of CGA and AVI (1 μg / mL LPS + 20 μg / mL CGA + 20 μg / mL AVI), and CA NPs intervention group prepared in Example 1 (1 μg / mL LPS + 20 μg / mL CA NPs). All treatment reagents were prepared with culture medium.

[0114] Test data showed that the CA NPs group exhibited significantly improved scratch healing speed, indicating that the self-assembled nanoparticles obtained in Example 1 possessed good cell migration promotion capabilities (e.g., Figure 15 ).

[0115] Example 13: Transwell experiment to evaluate the effect of CA NPs on the migration ability of HUVECs

[0116] HUVECs were 2×10 4 Seeded at a density of cells / well in Transwell chambers (upper chamber), and cultured adherently, the following groups were treated: blank control group (routine culture), model group (1 μg / mL LPS), CGA intervention group (1 μg / mL LPS + 20 μg / mL CGA), AVI intervention group (1 μg / mL LPS + 20 μg / mL AVI), physical mixed intervention group of CGA and AVI (1 μg / mL LPS + 20 μg / mL CGA + 20 μg / mL AVI), and CA NPs intervention group prepared in Example 1 (1 μg / mL LPS + 20 μg / mL CA NPs). All treatment reagents were prepared with culture medium (0.5 mL). During the treatment, culture medium containing 10% FBS was added to the lower chamber as a source of chemokines. After incubation for 24 h, unmigrated cells in the upper chamber were gently wiped away with a cotton swab, washed with PBS, fixed with 4% paraformaldehyde, and stained with crystal violet. Multiple fields of view were randomly selected and the number of cells that migrated through the membrane was counted under a microscope as an indicator of cell migration ability.

[0117] Test data showed that the number of vascular endothelial cells migrating in the CA NPs group was significantly higher than that in other groups, indicating that the nanoparticles obtained in Example 1 have a significant ability to promote vascular endothelial cell migration, thereby exerting a pro-angiogenic effect (e.g., Figure 16 ).

[0118] Example 14: In vitro tube formation experiment to evaluate the effect of CA NPs on the angiogenic capacity of HUVECs

[0119] The molten Matrigel was evenly spread at low temperature onto the bottom of a 96-well plate (50 μL per well), and incubated at 37°C for 30–60 min to allow polymerization to form a gel layer. HUVECs were then added at a rate of 2 × 10⁻⁶. 4Cells were resuspended in low-concentration serum medium (ECM endothelial cell culture medium) (50 μL per well) and seeded onto a gel layer. After seeding, cells were divided into the following groups: blank control group (routine culture), model group (1 μg / mL LPS), CGA intervention group (1 μg / mL LPS + 20 μg / mL CGA), AVI intervention group (1 μg / mL LPS + 20 μg / mL AVI), CGA and AVI physical mixed intervention group (1 μg / mL LPS + 20 μg / mL CGA + 20 μg / mL AVI), and CA NPs intervention group prepared in Example 1 (1 μg / mL LPS + 20 μg / mL CA NPs). All treatment reagents were prepared in 50 μL of culture medium. The culture plates were then incubated at 37°C in a 5% CO2 incubator for 8 hours. After incubation, the culture medium was discarded, and the cells were washed once with PBS. Add 100 μL of 4% paraformaldehyde to each well and fix at room temperature for 15 min. After washing with PBS, add 100 μL of 2 μM Calcein-AM staining solution and incubate at 37°C in the dark for 30 min. Wash twice with PBS. After staining the cytoskeleton, photograph different fields of view using a fluorescence microscope.

[0120] Test data showed that the CA NPs group formed significantly more vascular ring structures than other groups, indicating that the nanoparticles obtained in Example 1 have a good ability to promote angiogenesis. Figure 17 ).

[0121] This invention provides a concept and method for the preparation and application of chlorogenic acid-dipsacin VI self-assembled nanoparticles. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing chlorogenic acid-dipsacin VI self-assembled nanoparticles, characterized in that, Includes the following steps: (1) The oil phase containing chlorogenic acid and bisacodyl VI was added dropwise to the aqueous phase to carry out solvent replacement and reaction, and the reaction solution was obtained. (2) The reaction solution obtained in step (1) was centrifuged, washed and resuspended to obtain a dispersion of self-assembled nanoparticles; then a freeze-drying protectant was added and freeze-dried to obtain chlorogenic acid-dipsacin VI self-assembled nanoparticles. Steps (1) to (2) are all carried out under light-protected conditions.

2. The preparation method according to claim 1, characterized in that, In step (1), the oil phase containing chlorogenic acid and dipsacin VI is prepared by the following method: chlorogenic acid is dissolved in an organic solvent to obtain chlorogenic acid mother liquor; dipsacin VI is dissolved in an organic solvent to obtain dipsacin VI mother liquor; the chlorogenic acid mother liquor and the dipsacin VI mother liquor are mixed to obtain the oil phase; preferably, the oil phase containing chlorogenic acid and dipsacin VI is prepared under light-protected conditions.

3. The preparation method according to claim 2, characterized in that, The organic solvent is ethanol or methanol; and / or, the concentration of the chlorogenic acid mother liquor is 1~50 mg / mL; and / or, the concentration of the bisacodyl VI mother liquor is 1~50 mg / mL; the molar ratio of the chlorogenic acid to the bisacodyl VI in the oil phase is 0.5:1~3:

1.

4. The preparation method according to claim 1, characterized in that, In step (1), the volume ratio of the oil phase to the aqueous phase is 1:10~20; and / or, the aqueous phase is water or a buffer solution with a pH value of 6.5~7.4; and / or, the dropwise addition is carried out at a rate of 0.05~0.5 mL / min under stirring conditions of 500~1000 rpm.

5. The preparation method according to claim 1, characterized in that, In step (1), the reaction is carried out at a speed of 300-800 rpm for 1-6 hours.

6. The preparation method according to claim 1, characterized in that, In step (2), the centrifugation is performed at a speed of 8000~15000 rpm for 10~30 min; and / or the washing is performed by centrifugation with water for 1~5 times; and / or the resuspension is performed by resuspension with water; and / or the concentration of self-assembled nanoparticles in the self-assembled nanoparticle dispersion is 5~10 mg / mL, preferably 10 mg / mL.

7. The preparation method according to claim 1, characterized in that, In step (2), the freeze-drying protectant is any one or more of trehalose, sucrose, or mannitol; and / or, the mass-to-volume ratio of the freeze-drying protectant to the self-assembled nanoparticle dispersion is 1 g:100 mL ~ 10 g:100 mL, preferably 5 g:100 mL; and / or, the freeze-drying is performed at a temperature of -60 to -90°C for 12 to 48 hours; and / or, after adding the freeze-drying protectant, a pre-freezing is performed before freeze-drying, wherein the pre-freezing is performed at a temperature of -60 to -90°C for 2 to 3 hours.

8. Chlorogenic acid-dipsacin VI self-assembled nanoparticles prepared by the preparation method according to any one of claims 1 to 7.

9. The chlorogenic acid-dipsacin VI self-assembled nanoparticles according to claim 8, characterized in that, The chlorogenic acid-dipsacin VI self-assembled nanoparticles have an average particle size of 139~175 nm and a zeta potential of -11.0 ~ -9.0 mV.

10. The use of the chlorogenic acid-dipsacin VI self-assembled nanoparticles according to claim 8 in the preparation of antioxidant products, and / or anti-inflammatory products, and / or osteogenic differentiation promoting products, and / or angiogenesis promoting products; wherein the products are any one of biomaterials, health products, cosmetics or pharmaceuticals.

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