Multifunctional nano-particle targeting abdominal aortic aneurysm and preparation method and application thereof

Abstract

The application provides a multifunctional nano particle for targeting abdominal aortic aneurysm and a preparation method and application thereof, and belongs to the field of nanobiomedicine, wherein polyphenol oxidation self-polymer nanoparticles are used as carriers, doxycycline is loaded, and a targeting ligand cRGD is modified on the surface of the nano carrier; the neovasculature in the media and adventitia of AAA sites helps accumulation of the nanoparticles in the abdominal aortic aneurysm, and the integrin αν3 beta receptor highly expressed on the surface of the diseased cell membrane can recognize the cRGD with high affinity, and then mediate the targeted endocytosis of the nanoparticles; after intravenous injection, the nanoparticles can be effectively enriched in the lesion site and achieve long-term retention, and can release DC in response to the high ROS level in the tumor microenvironment; the drug is rapidly released to take effect, and the non-specific toxic side effects are reduced; the free radical scavenging capacity of the nanoparticles can be synergized with the DC activity to treat AAA through multiple mechanisms such as anti-inflammatory, antioxidant, macrophage repolarization promotion, anti-apoptosis and calcification inhibition, and MMPs inhibition.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanobiomedicine, and particularly relates to a multifunctional nano particle for targeting abdominal aortic aneurysm, and a preparation method and application thereof. BACKGROUND

[0002] Abdominal aortic aneurysm (AAA) is a life-threatening progressive vascular disease, but there is no effective drug control method at present. The risk of aneurysm rupture of larger or irregular aneurysm is extremely high, and the mortality rate is more than 80% once it ruptures. With the increase of population aging and the incidence of hypertension, the incidence and mortality of AAA are increasing year by year. Surgical intervention is currently the only effective method for the clinical treatment of AAA. For AAA that does not meet the surgical indications (usually refers to aneurysm greater than 3 cm and less than 5.5 cm), surgery is not possible, and patients are usually advised to monitor the changes of aneurysm regularly. However, small AAA also has the risk of rupture, and regular monitoring requires contrast examination, which leads to the risk of liver and kidney damage. Therefore, it is imperative to develop effective drugs that can reduce the expansion of AAA and prevent the rupture of AAA.

[0003] The occurrence and development of AAA are related to a series of pathological changes, including inflammatory cell infiltration, increased matrix metalloproteinase (MMP) levels, excessive production of reactive oxygen species, intimal and medial calcification, neovascularization, vascular smooth muscle cell (VSMC) damage and aortic elastic lamina degradation. Therefore, in view of the pathological changes in the AAA site, the development of corresponding regulatory drugs is a hot spot of current research.

[0004] Although doxycycline (DC) has a good effect on inhibiting MMPs and has achieved promising results in preclinical studies, it has not provided beneficial effects in clinical trials. In addition, due to the non-specific distribution of DC, it usually produces adverse reactions after taking. In addition, the poor water solubility limits the clinical application of DC. More importantly, the single mechanism of action of doxycycline makes it difficult to target the complex pathological changes of AAA and achieve multidimensional disease treatment, thus the therapeutic effect is poor. Therefore, there is an urgent need for innovative drug delivery strategies to reduce the side effects of DC while enhancing its therapeutic effect to promote clinical translation. SUMMARY

[0005] To solve the above technical problems, the application provides a multifunctional nano particle for targeting abdominal aortic aneurysm, a preparation method and application thereof, a carrier is a polyphenol oxidation self-polymerization nano particle, and doxycycline is adsorbed on the surface, the enrichment of the nano particle at the AAA lesion site is increased by 5 times through the modification of PEG-cRGD, and the targeting delivery is realized by recognizing the highly expressed integrin αν3β receptor on the lesion cell membrane. The nano drug can release doxycycline under the trigger of high-level ROS in AAA, and realize the synergy with the strong antioxidant effect of the nano carrier itself, so as to play multiple activities such as anti-inflammatory, antioxidant and inhibition of MMPs, realize the treatment of AAA, and at the same time, through the delivery of the nano particle carrier, the liver and kidney toxicity induced by doxycycline can be significantly reduced, and good biological safety is displayed.

[0006] To achieve the above purpose, the application first provides a functional nano particle for targeting abdominal aortic aneurysm, the nano particle comprises a polyphenol oxidation self-polymerization nano particle, doxycycline and a targeting ligand cRGD, the polyphenol oxidation self-polymerization nano particle is used as a carrier, the targeting ligand cRGD is first modified on the surface, and then doxycycline is adsorbed on the surface.

[0007] Based on a general inventive concept, the application further provides a preparation method of the multifunctional nano particle for targeting abdominal aortic aneurysm, comprising the following steps:

[0008] S1, preparing a carrier polyphenol oxidation self-polymerization nano particle: epigallocatechin gallate is dissolved in a buffer solution with a pH of 7.4-8.5, water bath stirring is carried out at room temperature, an epigallocatechin gallate mixed solution is obtained, divalent manganese ion solution is added dropwise, constant temperature water bath stirring is carried out, and then the precipitate is collected by centrifugation, the precipitate is washed with a buffer solution, and the precipitate is ultrasonically redissolved, so that the polyphenol oxidation self-polymerization nano particle is obtained;

[0009] S2, modifying a targeting ligand cRGD: the system prepared in S1 is added with mercapto-polyethylene glycol-cyclic arginine-glycine-aspartic acid, constant temperature water bath stirring is carried out, and a targeting ligand cRGD-polyphenol oxidation self-polymerization nano particle is constructed;

[0010] S3, adsorbing doxycycline: the system prepared in S2 is added with doxycycline, constant temperature water bath stirring is carried out, and the multifunctional nano particle for targeting abdominal aortic aneurysm is obtained.

[0011] Preferably, the buffer solution in the step S1 is HEPES buffer solution, and the final concentration of the HEPES buffer solution is 10-15 mM; the water bath stirring time is 1 min, the centrifugation speed is 16,000 rpm, and the centrifugation time is 20 min.

[0012] Preferably, the molar ratio of epigallocatechin gallate to divalent manganese ion in the step S1 is 1:5-5:1.

[0013] As preferred, the metal manganese ion solution in step S1 is MnCl2 solution, the final concentration of the metal manganese ion after dropping is 0.5-12.5 mM, the temperature of the constant temperature water bath is 25-35℃, and the time of the vigorous stirring is 0.5-12 h.

[0014] As preferred, the molar ratio of the mercapto-polyethylene glycol-cyclic arginine-glycine-aspartic acid to doxycycline is 1:4.5.

[0015] As preferred, the temperature of the constant temperature water bath stirring in the S2 step is 37℃, the time of the constant temperature water bath stirring is 2 h, the temperature of the constant temperature water bath stirring in the S3 step is 37℃, and the time of the constant temperature water bath stirring is 2 h.

[0016] Based on one general inventive concept, the scheme also provides an application of the multifunctional nano-particle targeting abdominal aortic aneurysm in inhibiting the progression of abdominal aortic aneurysm drugs.

[0017] The mechanism of the nano-particle prepared by the scheme in inhibiting abdominal aortic aneurysm is as follows:

[0018] The mechanism is as shown in Figure 1 The new blood vessels in the intima and adventitia of AAA sites help the accumulation of nano-particles in the aneurysm, and the integrin αν3β receptor highly expressed on the membrane surface of the lesion cells (including VSMC and macrophages) can recognize cRGD with high affinity, and then mediate the targeted endocytosis of the nano-particles. After intravenous injection, the nano-particles can effectively enrich in the AAA site and achieve long-term retention, and release DC in response to the high ROS level in the AAA microenvironment, so as to make the drug release quickly and reduce its non-specific toxic side effects. The free radical scavenging ability of the nano-particle itself can cooperate with the DC activity, and the nano-delivery system is proved to treat AAA through multiple mechanisms such as anti-inflammatory, antioxidant and inhibition of MMPs at the cell and animal levels.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] (1) The nano-particle carrier prepared by the scheme is a polyphenol oxidation self-polymerization nano-particle, and doxycycline is adsorbed on the surface. Through the modification of PEG-cRGD, the enrichment of the nano-particle in the AAA lesion site can be increased by 5 times, and the integrin αν3β receptor highly expressed on the membrane of the lesion cells is recognized to achieve targeted delivery.

[0021] (2) The nano-particle prepared by the scheme can release doxycycline under the trigger of high level of ROS in AAA, and realize cooperation with the strong antioxidant effect of the nano-carrier itself, so as to play multiple activities such as anti-inflammatory, antioxidant, promotion of macrophage repolarization, anti-apoptosis and calcification, and inhibition of MMPs, and realize the treatment of AAA.

[0022] (3) By the delivery of nanoparticle carriers, doxycycline-induced hepatorenal toxicity can be significantly reduced, showing good biosafety. This study provides a potential targeted nanoparticle drug for the treatment of abdominal aortic aneurysm, and also provides a design reference for the development of targeted drugs for other vascular diseases. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0024] Figure 1 Working mechanism diagram of multifunctional targeted abdominal aortic aneurysm nanoparticles prepared in Example 1;

[0025] Figure 2 Characteristics of nanoparticles in Experimental Example 1, A is a synthesis schematic diagram of cRGD-TPNs, B is a particle size diagram of TPNs, cRGD-TPNs and cRGD-TPNs / DC NPs, C is a potential diagram of TPNs, cRGD-TPNs and cRGD-TPNs / DC NPs; D is a UV-vis ultraviolet-visible absorption spectrum detection of thiol concentration; E is a TEM diagram and element scanning diagram of cRGD-TPNs; F is a synthesis schematic diagram of cRGD-TPNs / DC; G is the encapsulation efficiency of cRGD-TPNs / DC under different DC feeding concentrations; H is a TEM diagram and element scanning diagram of cRGD-TPNs / DC; I is the colloidal stability of cRGD-TPNs / DC in different media; J is the drug release of cRGD-TPNs / DC under different conditions;

[0026] Figure 3 The broad-spectrum free radical scavenging ability of multifunctional targeted abdominal aortic aneurysm nanoparticles cRGD-TPNs / DC in Experimental Example 2, A is the ABST free radical scavenging rate of different concentrations of cRGD-TPNs / DC, B is the change of the color of each free radical solution with the increase of the concentration of cRGD-TPNs / DC; C is the change of the ultraviolet absorption of ·O2 - free radicals, D is the free radical scavenging rate of ·O2 - free radicals, E is the change of the ultraviolet absorption of ·NO free radicals, F is the free radical scavenging rate of ·NO free radicals, G is the change of the ultraviolet absorption of ·OH free radicals, H is the free radical scavenging rate of ·OH free radicals;

[0027] Figure 4For the cell targeting uptake of multifunctional targeting abdominal aortic aneurysm nanometer particles cRGD-TPNs / DC in experimental example 3, A is fluorescence microscope and B is flow cytometry to observe the uptake of VSMC to different nanoparticles, D is flow cytometry to observe the uptake of RAW264.7 to different nanoparticles, C is fluorescence quantification results of VSMC to different nanoparticles, E is fluorescence quantification results of RAW264.7 to different nanoparticles, F is intracellular fluorescence map of VSMC incubated with cRGD-TPNs / DC NPs for different time, G is intracellular fluorescence map of RAW264.7 incubated with cRGD-TPNs / DC NPs for different time, H and I are Western-blot analysis of the expression of MMP9 in different treatment groups and its gray quantification, K, L are Western-blot analysis of the expression of MMP2 in different treatment groups and its gray quantification, J is qPCR detection of the expression of MMP9, M is qPCR detection of the expression of MMP2;

[0028] Figure 5 For the intracellular antioxidant, anti-inflammatory properties and induction of macrophage repolarization activity of cRGD-TPNs / DC NPs in experimental example 4, A-D are fluorescence microscope to observe the fluorescence images of General ROS, ·O2 - , ·OH / ONOO-, ·NO free radicals in RAW264.7 cells after different treatments, E-H are fluorescence quantification results, I is fluorescence microscope picture, J is flow cytometry to detect the intracellular free radical level of VSMC after different treatments, K is quantitative result, L is qPCR to detect the mRNA level of TNF-a in macrophages after different treatments, M is qPCR to detect the mRNA level of IL-6 in macrophages after different treatments, N is immunofluorescence analysis of the expression of CD206 and iNOS in RAW264.7 cells after different treatments; O is qPCR to detect the expression of CD206 in macrophages after different treatments, P is qPCR to detect the expression of iNOS in macrophages after different treatments;

[0029] Figure 6 For the anti-VSMC apoptosis and calcification activity research of cRGD-TPNs / DC NPs in experimental example 5, A is intracellular calcification staining of VSMC after different treatments under microscope and digital camera, B is flow cytometry to detect the apoptosis of VSMC cells after different treatments, C is apoptosis intensity numerical quantification, D-E are intracellular calcification staining of VSMC cells after different treatments under microscope and digital camera, F is calcification intensity numerical quantification, G is CCK8 to detect the cell viability of VSMC cells after different treatments;

[0030] Figure 7For the in vivo targeting performance of cRGD-TPNs / DC NPs in Experimental Example 6, A is the establishment of an AAA mouse model and a schematic diagram of tissue distribution and treatment administration, B is the in vitro imaging of the aorta of normal mice and AAA mice after being administered different treatments for 24 h, C is the quantitative fluorescence intensity, D-E are in vitro imaging diagrams of the aorta and main internal organs of AAA mice at different time points after being administered cRGD-TPNs-Cy5.5 NPs, and F is a fluorescence quantification diagram;

[0031] Figure 8 For the in vivo therapeutic effect of cRGD-TPNs / DC NPs in Experimental Example 7, A is the observation of the abdominal aorta diameter by digital camera and color ultrasound, B is the quantitative value measured by color ultrasound, C is the HE staining pathological changes of the abdominal aorta tissue of mice in different treatment groups, the EVG detection of elastin layer damage of the aorta wall, and the alizarin red detection of vascular wall calcification deposition, and D is the TUNEL detection of the apoptosis of the aorta vascular wall;

[0032] Figure 9 For the in vivo mechanism of cRGD-TPNs / DC NPs in Experimental Example 8, A is the Western blot detection of the expression of MMP9 and MMP2 in different treatment groups, B-C are the gray value quantification of MMP9 and MMP2, D is the immunohistochemical detection of the expression of MMP2 and MMP9 in the abdominal aorta tissue of different treatment groups, E-F are the levels of H2O2 and MDA in the serum of different treatment groups, G-H are the qPCR detection of the mRNA expression levels of TNF-ɑ and IL-1β in the abdominal aorta tissue of different treatment groups, and I is the immunofluorescence analysis of the expression of CD206 and iNOS;

[0033] Figure 10 For the in vivo safety of cRGD-TPNs / DC NPs in Experimental Example 9, A is the hemolysis determination of cRGD-TPNs / DC NPs at different concentrations, B is the body weight change of mice in different treatment groups within 28 days, C-F are the levels of BUN, CR, ALT, and AST in the serum of mice after different treatments, and G is the HE section staining of the main internal organs of mice after different treatments;

[0034] Figure 11 For the cell viability after TPNs, DC, and cRGD-TPNs / DC NPs are taken up by cells in Experimental Example 3, A is the cell viability after TPNs are taken up by cells, B is the cell viability after DC is taken up by cells, and C is the cell viability after cRGD-TPNs / DC NPs are taken up by cells;

[0035] Figure 12For the in vivo mechanism of cRGD-TPNs / DC NPs in Experimental Example 8, the expression of MMP2 / MMP9 in the abdominal aorta vascular wall tissue after Ang II treatment was analyzed by mRNA qPCR, A is the expression of MMP9 in the tissue, B is the expression of MMP2 in the tissue. DETAILED DESCRIPTION

[0036] To make the technical problems, technical solutions and advantages of the present application clearer, the following will be described in detail with reference to the drawings and specific embodiments.

[0037] The following examples are used to illustrate the present application, but not to limit the scope of the present application. Modifications or replacements of the methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application.

[0038] If not specifically indicated, the technical means used in the examples is the conventional means familiar to those skilled in the art; if not specifically indicated, the reagents used in the examples are commercially available.

[0039] Example 1

[0040] Preparation of multifunctional targeted abdominal aortic aneurysm nanoparticles

[0041] S1, preparation of carrier polyphenol oxidation self-polymerization nanoparticles TPNs: take 3 mL of 100 mM HEPES buffer solution with pH 7.4 into a 100 mL beaker, add 25.65 mL of ultrapure water, add 750 μL of 100 mM EGCG solution, water bath at room temperature for 1 min to get a mixed solution, then drop 600 μL of 100 mM MnC12 solution, 25℃ constant temperature water bath and vigorous stirring for 1 h, take the reaction liquid to centrifuge at 16,000 rpm for 20 min to collect the nanoparticles, wash twice with HEPES buffer (10 mM, pH 7.4), ultrasonic redissolution of the precipitate, and the polyphenol self-polymerization nanoparticles TPNs are obtained;

[0042] S2, modification of targeting ligand cRGD: add 20 μL of 5 mM thiol-polyethylene glycol-cyclic arginine-glycine-aspartic acid (SH-PEG-cRGD) to the system prepared in S1, 37℃ constant temperature water bath stirring for 2 h, to construct cRGD-TPNs;

[0043] S3, adsorption of doxycycline DC: add 20 μL of 22.5 mM doxycycline (DC) to the system prepared in S2, 37℃ constant temperature water bath stirring for 2 h, to obtain multifunctional targeted abdominal aortic aneurysm nanoparticles multifunctional targeted abdominal aortic aneurysm nanoparticles cRGD-TPNs / DC;

[0044] Figure 2 A is the synthesis schematic diagram of cRGD-TPNs,Figure 2 F is a schematic diagram of cRGD-TPNs / DC synthesis.

[0045] Experimental Example 1

[0046] The properties of nanoparticles TPNS, cRGD-TPNs, and cRGD-TPNs / DC were investigated.

[0047] The results are as follows Figure 2 As shown in Figure A, after step S1, the solution color changed from colorless to dark, indicating successful synthesis of TPNs. Compared to TPNs, the particle size of cRGD-TPNs was slightly increased. Figure 2 B), while the zeta potential was significantly reduced ( Figure 2 C) indicates that SH-PEG-cRGD has successfully bonded to the surface of nanoparticles.

[0048] The bonding rate of SH-PEG-cRGD was quantitatively characterized using a thiol reagent kit. After treatment, cRGD-TPNs exhibited a typical thiol absorption peak at 380 nm. Figure 2 D), based on this, the bonding amount of SH-PEG-cRGD was calculated to be 0.296 μmol / ml.

[0049] Transmission electron microscopy revealed that cRGD-TPNs are uniformly sized, near-spherical nanoparticles with an average particle size of approximately 200 nm. Figure 2 E). Further elemental analysis revealed that the structure is mainly composed of C and O elements, consistent with the chemical structure of its EGCG monomer. Additionally, a small amount of residual Mn was observed, possibly due to Mn... 2+ Surface adsorption is caused by strong coordination with -OH in the structure.

[0050] After adsorption of DC, the DLS particle size of the nanoparticles increased slightly. Figure 2 B), and a significant reduction in surface negative charge was observed. Figure 2 C), this is due to the positive charge of DC. Quantitative characterization by UV-Vis spectrophotometry revealed that the nanoparticles have a strong adsorption effect on DC; even at a dosage of 500 μg / ml, the encapsulation efficiency still reached 81%. Figure 2 H). Transmission electron microscopy revealed no significant structural changes or aggregation of the nanoparticles after DC adsorption. Figure 2 G). Elemental analysis revealed significant nitrogen enrichment in cRGD-TPNs / DC, further confirming the successful adsorption of DC. Figure 2 G).

[0051] The nanoparticles were incubated in medium, PBS, normal saline, FBS, H2O, HEPES at 37℃, and the DLS particle size was dynamically measured. It was found that cRGD-TPNs / DC remained stable within 48h, and the particle size did not change significantly Figure 2 I), indicating that the nanoparticles had good colloidal stability in various physiological matrices.

[0052] Subsequently, the in vitro release behavior of the drug was determined. Under the buffer conditions of pH 7.4, cRGD-TPNs / DC exhibited typical sustained-release characteristics, and the cumulative release amount of the drug was 16% within 48h Figure 2 J). It was shown that the drug could be stably adsorbed on the nanoparticles during the in vivo circulation, reducing the premature leakage of the drug.

[0053] In the AAA microenvironment, there is a high concentration of H2O2 locally due to oxidative stress. Therefore, by adding different concentrations of H2O2, the AAA microenvironment was simulated. The results showed that after the addition of H2O2, the release rate of the drug was significantly improved, confirming the stimulus-responsive drug release characteristics of the nanoparticles Figure 3 J). Therefore, cRGD-TPNs / DC can stably adsorb drugs in the blood circulation, and quickly release drugs when reaching the AAA site, thereby prolonging the half-life of the drug, reducing the toxic side effects of the drug, and better exerting the therapeutic effect.

[0054] Experimental Example 2

[0055] Investigation of the broad-spectrum free radical scavenging ability of multifunctional targeted abdominal aortic aneurysm nanoparticles cRGD-TPNs / DC

[0056] ABST detection kit was used to evaluate its ability to scavenge total RONS. ABST can generate stable free radicals under the oxidative conditions of (NH4)2S2O8, and exhibit a UV absorption peak at 732 nm.

[0057] The results showed that with the increase of the concentration of cRGD-TPNs / DC NPs, the UV absorption gradually decreased, from which the free radical scavenging rate Figure 3 A), Overall, the free radical scavenging ability of cRGD-TPNs / DC NPs was linearly related to the concentration. When the concentration reached 125 μg / ml, the scavenging ability could be as high as 81.89%.

[0058] Further investigation of the broad-spectrum scavenging ability of the nanoparticles on other physiological related free radicals, including ·OH, ·O2 - and ·NO. Under the treatment of the kit, different types of RONS solution were purple. With the increase of the concentration of cRGD-TPNs / DC NPs, the color of the solution gradually faded Figure 3B). When the concentration of nanoparticles reached 125 μg / ml, the solution color became significantly lighter. To quantify the free radical scavenging ability, the UV-Vis absorption spectra of different free radicals were measured. The results showed that with the increase of cRGD-TPNs / DC NPs concentration, the UV absorption gradually decreased. Figure 3 C, 3E, 3G). When the concentration of cRGD-TPNs / DC NPs reaches 125 μg / ml, O2 - The scavenging rates of NO and ·OH were 54%, 67%, and 83%, respectively. Figure 4 D, 3F, 3H).

[0059] In summary, cRGD-TPNs / DC NPs TPNs loaded with DC still possess broad-spectrum free radical scavenging capabilities.

[0060] Experimental Example 3

[0061] Investigating the cellular uptake of multifunctional nanoparticles cRGD-TPNs / DCs targeting abdominal aortic aneurysms and their intracellular inhibition of MMPs activity.

[0062] (1) Cellular targeted uptake characteristics of cRGD-TPNs / DC

[0063] VSMC and RAW264.7 cells were used as experimental cells. The nanosystems were fluorescently labeled with FITC, and the cell nuclei were stained with DAPI. Confocal microscopy revealed weak intracellular signals in the TPNs / DC NPs. Figure 4 A) The negatively charged PEG-modified nanoparticles may hinder cellular endocytosis. However, the fluorescence intensity of cRGD-TPNs / DC NPs modified with cRGD significantly increased. This is likely due to the high expression of the cRGD receptor (integrin αν3β receptor) on the surface of VSMC cells, which mediates cellular uptake of NPs. To confirm this, VSMCs were pretreated with free SH-PEG-cRGD, resulting in a significant decrease in fluorescence signal, clarifying the important role of cRGD and its receptor recognition in nanoparticle endocytosis.

[0064] The same inverse method was used to evaluate the uptake of nanoparticles by RAW264.7. Unactivated RAW264.7 showed weak uptake of both cRGD-TPNs / DC and TPNs / DC NPs. Figure 4A). But after polarization by LPS treatment, RAW264.7 selectively enhanced the uptake of cRGD-TPNs / DC NPs, and this difference was due to the increased expression of integrin αν3β receptor on the surface of macrophage membrane after LPS induction, which promoted the uptake of TPNs-PEG-cRGD / DC NPs by the integrin αν3β receptor binding with the ligand on the surface of TPNs-PEG-cRGD / DC NPs. The fluorescence signal was weakened again by pretreatment of the cells with free SH-PEG-cRGD.

[0065] The fluorescence intensity of cell uptake was quantitatively analyzed by flow cytometry, and it was found that VSMC could significantly enhance the uptake of cRGD-TPNs / DC NPs, but this enhanced fluorescence signal could be blocked by free SH-PEG-cRGD Figure 4 B, C). The uptake ability of polarized macrophages to cRGD-TPNs / DC NPs was 4.9 times that of macrophages cultured under normal conditions Figure 4 D, E). Similarly, this enhanced uptake signal could also be blocked by SH-PEG-cRGD. Overall, the results of flow cytometry were highly consistent with those of confocal microscopy, confirming that cRGD modification could enhance the targeting of nanoparticles to VSMC.

[0066] The uptake kinetics of cells to nanoparticles was evaluated by observing the intracellular fluorescence signal at different time points, and the results showed that the intracellular green fluorescence of VSMC Figure 4 F) and RAW264.7 Figure 11 G) gradually increased with time, indicating that the uptake ability had a certain time dependence. The cytotoxicity of nanoparticles after being taken up was determined by MTT, and the results showed that the cRGD-modified TPN carrier had low toxicity, and the cell viability was still higher than 80% after incubation at a concentration of 150 μg / ml for 24 h Figure 4 Free DC could cause certain cytotoxicity at a concentration higher than 20 μg / ml. However, when DC was loaded into the nanoparticle structure, its toxicity was reduced, which was due to the slow release of drugs in the nanoparticles.

[0067] (2) Inhibition of MMPs activity in cells by cRGD-TPNs / DC

[0068] The expression levels of MMP9 and MMP2 protein in cells were detected by Western blot, and after LPS treatment, the intracellular MMP9 and MMP2 of VSMC were significantly up-regulated Figure 4H, K), suggesting the activation of macrophages. The expression and secretion of MMP9 and MMP2 can lead to the degradation of matrix, promoting the progression of AAA. cRGD-TPNs / DC NPs treatment can significantly inhibit the expression of MMP9 and MMP2, demonstrating the therapeutic potential of nanoparticles for AAA.

[0069] Notably, cRGD-TPNs NPs without DC encapsulation also have certain regulatory ability of MMPs, which may be due to the strong anti-inflammatory and antioxidant effects of TPNs. Therefore, DCs can achieve functional synergy with TPNs to jointly regulate MMPs. Further gray quantification of protein expression Figure 4 I, L), and the mRNA level was determined by qPCR Figure 5 J, M). The above quantitative results all confirm the effective regulatory ability of cRGD-TPNs / DC NPs on MMPs.

[0070] Experimental Example 4

[0071] Investigation of intracellular antioxidant, anti-inflammatory properties and induction of macrophage repolarization activity of cRGD-TPNs / DC NPs

[0072] RAW264.7 cells were activated by LPS, and the intensity of intracellular general ROS was detected by free radical fluorescent probe. The results showed that after LPS induction treatment, there was obvious fluorescence signal in the cells Figure 5 A). However, after adding cRGD-TPNs / DC NPs and cRGD-TPNs NPs, the fluorescence signal was significantly weakened. This indicates the free radical scavenging activity of nanoparticles. In order to further explore the activity of scavenging a wide range of free radicals in cells, we used 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA), dihydroethidium (DHE) and hydroxyphenyl fluorescein (HPF) to detect hydrogen peroxide, O2 - and NO, respectively. Fluorescence microscopy showed that cRGD-TPNs / DC NPs and cRGD-TPNs NPs could reduce fluorescence to background level Figure 5 A-D). Flow cytometry was used to quantify fluorescence intensity, and the results all showed obvious ROS elimination Figure 5 E-H).

[0073] Using the same method, we measured the free radical scavenging activity of nanoparticles in VSMC cells. Intracellular free radical level was induced by H2O2, and dihydroethidium (DHE) fluorescent probe was used for fluorescence tracing. Observation by fluorescence microscope Figure 5I) and flow cytometry detection (5J-K) confirmed that nanoparticles can significantly reduce intracellular fluorescence signals. Overall, cRGD-TPNs / DC NPs can widely scavenge various types of oxidative free radicals in different cells, demonstrating the ability of nanoparticles to effectively protect cells from oxidative damage.

[0074] The expression levels of representative inflammatory factors, including TNF-ɑ and IL-1β, which play an important role in inducing macrophage infiltration into the aortic wall, were determined. By qRCR determination, it was found that the secretion of inflammatory factors in LPS-activated macrophages was significantly up-regulated Figure 5 L, M), but TNF-ɑ and IL-1β can be reduced to normal levels after treatment with nanoparticles. In comparison, cRGD-TPNs / DC NPs have stronger anti-inflammatory ability than cRGD-TPNs NPs, especially for IL-1β, which is mainly due to the anti-inflammatory effect of DCs, so TPNs loaded with DCs have stronger inhibitory effect on inflammation.

[0075] Given the strong antioxidant and anti-inflammatory effects of cRGD-TPNs / DC NPs, we further investigated their regulatory effects on macrophage phenotypes. By immunofluorescence determination, it was found that after LPS stimulation, the expression of M1 marker iNOS was significantly up-regulated, while the expression of M2 marker CD206 was reduced Figure 5 N), indicating that LPS successfully stimulated M1 polarization. After treatment with nanoparticles, the fluorescence signal of iNOS was significantly weakened, while the fluorescence signal of CD206 was increased. In order to more accurately determine the polarization state of macrophages, we used qPCR experiments to detect the mRNA expression of iNOS and CD206 in different treatment groups, and the results were consistent with the trend of the fluorescence microscope image Figure 6 O, P). Compared with cRGD-TPNs NPs, cRGD-TPNs / DC NPs have stronger regulatory effects on iNOS and CD206. Therefore, TPNs nanocarriers can synergize with DCs to promote the M1-TO-M2 repolarization of macrophages.

[0076] Experimental Example 5

[0077] Investigation of the anti-VSMC apoptosis and calcification activity of cRGD-TPNs / DC NPs

[0078] VSMC apoptosis and calcification are important factors for abdominal aortic vascular wall damage. By induction with H2O2, the microenvironment of AAA disease can be simulated to induce cell apoptosis. Quantitative determination by flow cytometry can observe that the number of apoptotic cells significantly increases after H2O2 treatment Figure 6A). However, the apoptosis level of cells treated with NPs was significantly decreased. Similarly, TPNs NPs could synergize with DCs, and cRGD-TPNs / DC NPs had the strongest anti-apoptosis ability, reducing the number of apoptotic cells by 5-fold Figure 6 B). CCK8 assay further confirmed the protective effect of NPs on H2O2-induced apoptosis of VSMCs Figure 6 C).

[0079] To investigate the anti-calcification effect of NPs, we induced intracellular calcification of VSMCs by Ca / Pi, and labeled the calcification precipitates by alizarin red staining Figure 6 D, E, F). After treatment with NPs, the color precipitate was significantly weakened, confirming the anti-calcification effect of NPs. This was mainly due to the free radical scavenging activity of NPs, which effectively improved oxidative stress and inhibited intracellular calcification levels. Similarly, due to the encapsulation of DCs, cRGD-TPNs / DC NPs had stronger anti-calcification activity than cRGD-TPNs NPs. CCK8 assay proved that cRGD-TPNs / DC NPs had the best cell protection effect in this model Figure 7 G).

[0080] In summary, the combination of TPNs and DCs can synergistically exert anti-apoptosis and anti-calcification effects, thereby protecting the abdominal aortic vascular wall from injury.

[0081] Experimental Example 6

[0082] Investigation of the in vivo targeting performance of cRGD-TPNs / DC NPs

[0083] An AAA model was established by continuously pumping Ang II (1 μg / kg / min) into ApoE- / - mice for 28 days Figure 7 A), to verify the in vivo targeting ability of NPs in AAA mice, Cy5.5-labeled NPs were injected intravenously on the 28th day of modeling, and normal mice were used as controls. The aorta was stripped and imaged after 24 hours of administration. cRGD-TPNs NPs had no obvious fluorescence in the abdominal aortic vascular wall of normal mice, but had obvious fluorescence signals in the lesion site of AAA mice Figure 7 B). In contrast, no obvious fluorescence signal was observed at the AAA site for TPNs NPs, suggesting that the upregulation of integrin αν3β receptors in the AAA site mediated the retention of cRGD-modified NPs at the lesion site.

[0084] Pretreatment with free SH-PEG-cRGD via the tail vein of AAA mice blocked the accumulation of cRGD-TPNs in the AAA region. Quantitative analysis of the fluorescence intensity of the AAA aortic vessel wall revealed that the fluorescence signal of the cRGD-TPNs group was 5 times that of TPNs. Figure 7 C), and this enhancement effect can be completely blocked by SH-PEG-cRGD pretreatment.

[0085] To investigate the persistence of the enrichment, we measured the fluorescence intensity of the aortic wall and viscera of AAA mice at different time points. Even 24 hours after drug administration, high fluorescence intensity was still observed in the lesion sites of the abdominal aorta. Figure 7 D), indicating that the nanoparticles remained in this location for a relatively long time. Simultaneously, strong fluorescence signals were also observed in liver and kidney tissues after 24 hours, suggesting long circulation of the nanoparticles. Figure 7 E). Within 48 hours of administration, fluorescence in the aortic lesion site and major organs was significantly reduced (E). Figure 7 The results (DF) indicate that the nanoparticles can be slowly metabolized and cleared. These results demonstrate that SH-PEG-cRGD modification can endow nanoparticles with long-term cycling characteristics and enable them to actively target and accumulate in AAA lesion tissue.

[0086] Experimental Example 7

[0087] Investigating the in vivo efficacy of cRGD-TPNs / DC NPs

[0088] The treatment process is as follows Figure 8 As shown in Figure A, the drug was administered every two days during the modeling process. The aortic diameter was measured using ultrasound. Mice were then euthanized, and the AAA was isolated for further investigation. Compared to the normal group and the control group receiving daily Saline infusion, the Ang II group showed a significantly larger abdominal aortic diameter. Figure 8 A) indicates the successful construction of the disease model. Treatment with free DCs and cRGD-TPNs reduced the diameter of AAA in mice. Figure 8 B). Among the various treatment groups, cRGD-TPNs / DC NPs showed the most significant effect. For further visual observation, measurements were taken after aortic dissection, and the quantitative treatment efficacy was ranked as cRGD-TPNs / DC NPs > cRGD-TPNs > DC.

[0089] To further evaluate the protective effect of nanoparticles against pathological damage to the AAA vessel wall, systematic pathological characterization was performed, including H&E staining, EVG staining, and alizarin red staining. Figure 8C). Compared with the control and saline groups, the aneurysmal aortic wall of the model group still had some degree of damage after DC treatment, with elastic lamina rupture and damage, and obvious calcification. After treatment with nanoparticles, the damage to the aortic wall was significantly alleviated, with elastin being preserved and calcification being significantly reduced. Similarly, cRGD-TPNs / DC NPs showed the best therapeutic effect. Using TUNEL staining, the apoptosis of the abdominal aortic vessel wall was verified, further confirming the in vivo anti-apoptotic activity of the nanoparticles Figure 9 D).

[0090] The above results all confirm the good in vivo anti-AAA efficacy of cRGD-TPNs / DC NPs.

[0091] Experimental Example 8

[0092] Investigation of the in vivo mechanism of action of cRGD-TPNs / DC NPs

[0093] After confirming the efficacy of cRGD-TPNs / DC NPs, we further explored its mechanism of action. After treatment with Ang II, the expression levels of MMP2 and MMP9 proteins in the abdominal aortic vessel wall tissue were significantly up-regulated Figure 9 A), which is consistent with the pathological changes of AAA. Free DCs have a slight alleviating effect on the up-regulation of MMP2 and MMP9. In contrast, nanoparticles have more obvious activity, with cRGD-TPNs / DC NPs being the most effective. Through gray protein quantification Figure 9 B, C), the trend was further confirmed, indicating the synergistic regulation of nanoparticles and DCs on MMPs in vivo. The expression levels of MMP2 / MMP9 were analyzed by protein immunohistochemistry Figure 12 D) and mRNA qPCR Figure 9 ), and the results were consistent with the above.

[0094] Given the ROS scavenging and inflammation inhibiting activity of nanoparticles, we measured the expression levels of representative oxidative stress markers H2O2 and MDA in the plasma of mice Figure 9 E, F), and the expression levels of inflammatory factors TNF-ɑ and IL-1β in tissues Figure 9 G, H). After modeling by treatment with Ang II, the levels of oxidative stress and inflammatory factors in mice were significantly up-regulated. However, under the treatment of cRGD-TPNs / DC NPs, these indicators all returned to normal levels.

[0095] Subsequently, the phenotype of macrophages in AAA was further investigated by staining the macrophage markers CD206 and iNOS. The fluorescence of CD206 in the abdominal aortic wall of the model group was significantly stronger than that of the control group, while iNOS showed the opposite trend, which was due to the M1 polarization of macrophages in pathological conditions. However, through the treatment of cRGD-TPNs / DC NPs, both CD206 and iNOS returned to normal levels Figure 10 I). The above results confirmed that cRGD-TPNs / DC NPs still possessed the activities of regulating MMPs levels, anti-inflammatory, antioxidant, and inducing macrophage repolarization in vivo, achieving AAA treatment through multiple mechanisms.

[0096] Experimental Example 9

[0097] Investigation of the in vivo safety of cRGD-TPNs / DC NPs

[0098] The in vivo safety of nanomedicines is an important indicator for evaluating their clinical transformation potential. Therefore, we systematically evaluated the biological safety of cRGD-TPNs / DC NPs. The hemolysis rate of cRGD-TPNs / DC NPs at different concentrations was less than 5% ( Figure 10 A), which proved its good blood compatibility. During the treatment period, all mice had normal diet and water intake and normal activity, and there was no significant difference in weight gain between groups ( Figure 10 B). By detecting the serum biochemical indicators of mice, it was found that BUN, CR, AST, and ALT all abnormally increased after free DC treatment ( Figure 10 C-F), suggesting that DC can cause acute liver and kidney toxicity. This is also a bottleneck for the clinical transformation of DC. However, cRGD-TPNs / DC NPs did not show related toxicity, which may be due to the slow release of drugs mediated by nanocarriers, reducing the toxicity of drugs. In order to further confirm, hematoxylin and eosin staining of the main organs of mice was also observed, and liver and kidney damage caused by DC treatment was also observed ( ​ G). However, no cRGD-TPNs / DC NPs-related toxicity was observed at the same dose and treatment method, which again proved that the encapsulation of nanoparticles can effectively reduce the systemic toxicity of DC, and the biological safety of nanoparticles.

[0099] The above only describes the preferred embodiments of the present application patent, and the protection scope of the present application patent is not limited to the above-mentioned embodiments. For those skilled in the art, the improvements and changes obtained without departing from the technical concept of the present application patent should also be considered as the protection scope of the present application patent.

Claims

1. A multifunctional nanoparticle targeting abdominal aortic aneurysm, characterized in that, The nanoparticles include polyphenol oxidized self-polymerized nanoparticles, doxycycline, and the targeting ligand cRGD. The polyphenol oxidized self-polymerized nanoparticles serve as a carrier, and the surface is first modified with the targeting ligand cRGD, and then the surface is adsorbed with doxycycline. The preparation method of the multifunctional nanoparticles targeting abdominal aortic aneurysms includes the following steps: S1. Preparation of carrier polyphenol oxidized self-polymerized nanoparticles: Epigallocatechin gallate was dissolved in a buffer solution with pH 7.4-8.5 and stirred in a water bath at room temperature to obtain a mixture of epigallocatechin gallate and divalent manganese ion solution. The mixture was then stirred in a constant temperature water bath and centrifuged to collect the precipitate. The precipitate was washed with buffer solution and then sonicated to redissolve the precipitate to obtain polyphenol oxidized self-polymerized nanoparticles. S2, Modified Targeting Ligand cRGD: Thiol-polyethylene glycol-cyclic arginine-glycine-aspartic acid was added to the system prepared in S1 and stirred in a constant temperature water bath to construct the targeting ligand cRGD-polyphenol oxidative self-polymerized nanoparticles. S3, Adsorption of Doxycycline: Doxycycline was added to the system prepared in S2 and stirred in a constant temperature water bath to obtain multifunctional nanoparticles targeting abdominal aortic aneurysms.

2. A method for preparing multifunctional targeted abdominal aortic aneurysm nanoparticles as described in claim 1, characterized in that, Includes the following steps: S1. Preparation of carrier polyphenol oxidized self-polymerized nanoparticles: Epigallocatechin gallate was dissolved in a buffer solution with pH 7.4-8.5 and stirred in a water bath at room temperature to obtain a mixture of epigallocatechin gallate and divalent manganese ion solution. The mixture was then stirred in a constant temperature water bath and centrifuged to collect the precipitate. The precipitate was washed with buffer solution and then sonicated to redissolve the precipitate to obtain polyphenol oxidized self-polymerized nanoparticles. S2, Modified Targeting Ligand cRGD: Thiol-polyethylene glycol-cyclic arginine-glycine-aspartic acid was added to the system prepared in S1 and stirred in a constant temperature water bath to construct the targeting ligand cRGD-polyphenol oxidative self-polymerized nanoparticles. S3, Adsorption of Doxycycline: Doxycycline was added to the system prepared in S2 and stirred in a constant temperature water bath to obtain multifunctional nanoparticles targeting abdominal aortic aneurysms.

3. The preparation method according to claim 2, characterized in that, In step S1, the buffer solution is HEPES buffer with a final concentration of 10-15 mM; the stirring time in the water bath at room temperature is 1 min, the centrifugation speed is 16,000 rpm, and the centrifugation time is 20 min.

4. The preparation method according to claim 2, characterized in that, In step S1, the molar ratio of epigallocatechin gallate to divalent manganese ions is 1:5 to 5:

1.

5. The preparation method according to claim 2, characterized in that, In step S1, the manganese ion solution is a MnCl2 solution, the final concentration of the manganese ions after dropwise addition is 0.5~12.5mM, the temperature of the constant temperature water bath is 25℃~35℃, and the time of vigorous stirring is 0.5~12h.

6. The preparation method according to claim 2, characterized in that, The molar ratio of the mercapto-polyethylene glycol-cyclic arginine-glycine-aspartic acid to doxycycline is 1:4.

5.

7. The preparation method according to claim 2, characterized in that, In step S2, the temperature of the constant temperature water bath for stirring is 37°C, and the stirring time is 2 hours. In step S3, the temperature of the constant temperature water bath for stirring is 37°C, and the stirring time is 2 hours.

8. The use of a multifunctional nanoparticle targeting abdominal aortic aneurysm as described in claim 1, or a multifunctional nanoparticle targeting abdominal aortic aneurysm prepared by any one of the preparation methods described in claims 2-7, in the preparation of a drug to inhibit the progression of abdominal aortic aneurysm.

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