Quercetin-loaded cascaded targeting nanoparticles as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: (1) synthesizing FA-PEG-PAMAM, weighing a polyamide-amine dendritic polymer, dissolving the polyamide-amine dendritic polymer in a PBS solution, weighing folic acid-polyethylene glycol-activated ester, dissolving the folic acid-polyethylene glycol-activated ester in SMSO, mixing the two solutions, carrying out a stirring reaction under a dark condition, and carrying out filtration, washing and drying so as to obtain the quercetin-loaded cascaded targeted nano-particles; and dialyzing at normal temperature in a dark place. (3) synthesizing FA-PTP (at) Que by using FA-PTP loaded drug quercetin, dissolving FA-PTP in a DMSO solution, adding quercetin, carrying out a stirring reaction in a dark place, then carrying out dialysis in a dark place at a normal temperature, and replacing the dialysate once every 6 hours; and centrifuging the dialysis product, retaining the supernatant, and freeze-drying to obtain a yellowish-brown solid product FA-PTP-Que. According to the invention, the active chemotaxis of the nano-carrier to inflammatory tissues and the active targeting of the nano-carrier to effector cells are combined, and the nano-carrier can accurately play a role in each barrier level in different microenvironments, so that the systematicness and continuity of multi-layer barriers in the inflammatory microenvironment are overcome.

Description

Technical Field

[0001] The invention relates to the technical field of biopolymer materials, and in particular to quercetin-loaded cascade targeted nanoparticles and a preparation method and application thereof. Background Art

[0002] Inflammation is considered to be a natural protective mechanism of the human body against irritating trauma. When tissues are damaged by toxins or infected by bacteria, they enter a normal self-protection and damage repair stage. However, during the repair process, if damage repair is not carried out in a timely manner, the inflammatory response will cause functional disorders such as cytokine storms, further leading to continuous tissue damage. Currently, small molecule drugs are the most commonly used treatments for inflammatory diseases. However, the physicochemical properties of traditional small molecule drugs are poor, and there are multiple levels of biological barriers in the body, which prevents the drugs from achieving the expected therapeutic effect at the site of action at a sufficient concentration, which in turn may have side effects on normal tissues and organs. Among the new treatments for inflammatory diseases, nano drug delivery systems (NDDS) have become the first choice to replace small molecule drugs, because they can improve and develop new routes of administration, reduce toxicity, reduce side effects, improve biodistribution, and prolong the drug action cycle by improving drug solubility, drug release efficiency, diffusion rate, bioavailability, and immunogenicity.

[0003] However, increasing evidence shows that the lack of systematic and continuous penetration of multiple barriers in the microenvironment during NDDS administration greatly reduces the target enrichment and bioavailability of drugs, which limits the current treatment of inflammatory diseases. Therefore, improving the systematic and continuous perspectives of NDDS to overcome the multi-level barriers of the inflammatory microenvironment is a potential strategy to solve the problem of drug delivery. Summary of the invention

[0004] In order to solve the above technical problems, the first purpose of the present invention is to provide a method for preparing quercetin-loaded cascade targeted nanoparticles, the second purpose is to provide quercetin-loaded cascade targeted nanoparticles, and the third purpose is to provide applications thereof. The active chemotaxis of nanocarriers to inflammatory tissues and active targeting to effector cells are combined, and they can accurately play a role at each barrier level in different microenvironments, overcoming the systematicity and continuity of multi-level barriers in inflammatory microenvironments.

[0005] To achieve the above first object, the present invention is implemented by the following technical scheme: a method for preparing quercetin-loaded cascade targeted nanoparticles, characterized in that the preparation is carried out according to the following steps:

[0006] (1) Synthesis of FA-PEG-PAMAM: weigh polyamide-amine dendrimer (PAMAM) and dissolve it in PBS solution; weigh folic acid-polyethylene glycol-activated ester (FA-PEG1000-NHS) and dissolve it in SMSO; mix the two solutions and stir them in a dark environment; after the reaction, dialyze at room temperature in a dark environment, replace the dialyzate every 12 hours, and freeze-dry the sample to obtain FA-PEG-PAMAM;

[0007] (2) Synthesis of FA-PTP

[0008] FA-PEG-PAMAM was dissolved in PBS, and an excess of methoxypolyethylene glycol ketone thioacetal (mPEG5000-TK) was added for reaction, followed by 3-hydroxy-2-butanone (EDCL) and N-hydroxysuccinimide (NHS). After stirring in the dark, the mixture was dialyzed at room temperature in the dark, the dialyzate was replaced every 6 hours, and the product FA-PTP was obtained by freeze drying.

[0009] (3) Synthesis of FA-PTP@Que by loading FA-PTP with drug quercetin

[0010] FA-PTP was dissolved in DMSO solution, quercetin (Que) was added, the reaction was stirred in the dark, and then dialyzed at room temperature in the dark, with the dialysate replaced every 6 hours; the dialyzed product was centrifuged, the supernatant was retained, and freeze-dried to obtain a yellow-brown solid product FA-PTP@Que.

[0011] In step (1), the molar ratio of folic acid-polyethylene glycol-activated ester to polyamide-amine dendrimer is 5:1 to 10:1.

[0012] In step (1), a dialysis bag with a molecular weight cutoff of 3500Da is selected and the dialysis bag is soaked in DD water; the reaction solution is placed in the dialysis bag and both ends are clamped with dialysis bag clamps; the dialysis bag is placed in deionized water and dialyzed in the dark at room temperature for 3 days, and the dialysate is replaced every 12 hours.

[0013] The molar ratio of methoxy polyethylene glycol ketalthioate to FA-PEG-PAMAM was 10:1, and the mass ratio of the added amount of EDCL and NHS to methoxy polyethylene glycol ketalthioate was 1:10.

[0014] In step (2), a dialysis bag with a molecular weight cutoff of 12000Da was selected and the dialysis was performed for 3 days.

[0015] In step (3), the mass ratio of FA-PTP to quercetin is 20:1-20:10.

[0016] In step (3), a dialysis bag with a molecular weight cutoff of 3500 Da was selected and the dialysis was performed for 3 days.

[0017] A method for preparing quercetin-loaded cascade targeted nanoparticles is provided to prepare quercetin-loaded cascade targeted nanoparticles.

[0018] Application of quercetin-loaded cascade-targeted nanoparticles in the preparation of anti-inflammatory drugs.

[0019] Reactive oxygen species (ROS) are the most common inflammatory mediators. It has been reported that ROS are maintained at low levels in healthy tissues, but significantly increased in inflamed tissues. This significant upregulation suggests that ROS can act as an inducer of chemotaxis targets. Therefore, nanocarriers constructed based on this concentration difference can actively chemotaxis in inflamed tissues and are expected to break the endothelial barrier.

[0020] Folate receptor (FRS) is abundantly expressed in pro-inflammatory macrophages. As the natural ligand of FRS, folic acid (FA) is a target molecule for macrophage internalization, which endows nanocarriers with the ability to target and actively break the internalization barrier of effector cells. We constructed a cascade-structured nanocarrier that combines the active chemotaxis of nanocarriers to inflammatory tissues with the active targeting of effector cells, and can precisely act at each barrier level in different microenvironments, overcoming the systematic and continuous nature of the multi-level barriers in the inflammatory microenvironment. Quercetin is loaded into FA-PTP (FA-PTP@Que). Quercetin is a natural flavonoid widely found in fruits, herbs and vegetables. It has been shown to have a wide range of beneficial effects and bioactivities, including anti-inflammatory, antioxidant and neuroprotective. This scheme is expected to achieve effective delivery of quercetin and play a role in reducing ROS levels and anti-inflammatory effects.

[0021] The nanoparticles (FA-PTP@Que) constructed in this scheme are constructed based on PAMAM dendrimers. The first layer structure is a hydration shielding layer (mPEG-TK) for reactive oxygen species (ROS) chemotaxis, and the second layer structure is a macrophage regulatory system (FA-PEG-NHS) modified by a targeting molecule, and loaded with the anti-inflammatory drug quercetin. The hydrophilic structure of the shielding layer effectively prevents protein adsorption, prolongs blood circulation, and reduces immune clearance. During in vivo delivery, it first escapes the phagocytosis of mononuclear phagocytes and overcomes the phagocytic barrier of the mononuclear phagocyte system (MPS). Subsequently, the difference in ROS concentration between tissues is used to enrich in the inflamed tissue to overcome the endothelial barrier. After enrichment in the inflamed tissue, the nanocarrier triggers a cascade reaction to the local excess of ROS. The primary structure collapses, exposing the secondary functional structure, and finally overcoming the barrier of effector cell internalization. The prepared shielded cascade targeted nanocarrier achieves good targeting and therapeutic properties by matching the systemic and continuous multi-level barriers of the inflammatory microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Schematic diagram of the synthesis of PAMAM-PEG-FA and FA-PTP.

[0023] Figure 2 FTIR graphs of PAMAM, FA-PEG-PAMAM and FA-PTP.

[0024] Figure 3 1H-NMR analysis of PAMAM, FA-PEG-NHS, FA-PEG-PAMAM, mPEG-TK and FA-PTP.

[0025] Figure 4 Zeta potential diagram of PAMAM, FA-PEG-PAMAM and FA-PTP.

[0026] Figure 5 The drug loading efficiency (LE%) and encapsulation efficiency (EE%) of FA-PTP and quercetin at the mass ratios of 20:1, 20:5, and 20:10, respectively.

[0027] Figure 6 This is a fluorescence image of the expression of folate receptor β in Raw 264.7 cells under the stimulation of lipopolysaccharide (LPS).

[0028] Figure 7 A. Immunofluorescence of PTP and FA-PTP targeting macrophages under different conditions. Scale bar: 20 μm. B. Flow cytometry detection of PTP and FA-PTP targeting macrophages under different conditions. C. Quantitative analysis of flow cytometry detection of PTP and FA-PTP targeting macrophages under different conditions.

[0029] Figure 8 A. DCFH-A staining is used to evaluate the ROS scavenging ability of FA-PTP@Que. Scale bar: 20 μm. B. Flow cytometry was used to detect the ROS scavenging ability of FA-PTP@Que.

[0030] Fig. 9 Real-time quantitative PCR analysis of the expression of related inflammatory genes after FA-PTP@Que treated Raw 264.7.

[0031] Fig.10A. Immunofluorescence changes of CD86, a marker of M1 macrophages, after Raw264.7 was treated with different treatments. Scale bar: 50 μm. B. Immunofluorescence changes of CD206, a marker of M2 macrophages, after Raw264.7 was treated with different treatments. Scale bar: 50 μm. C. Flow cytometry results of CD86, a marker of M1 macrophages, after Raw264.7 was treated with different treatments. D. Flow cytometry results of CD206, a marker of M2 macrophages, after Raw264.7 was treated with different treatments.

[0032] Fig.11 A. Fluorescence intensity of Free-FITC, FA-PAMAM-FITC, and FA-PTP-FITC in joint tissues of arthritic mice. B. Quantitative analysis of A. Reactive oxygen species scavenging by FA-PTP@Que.

[0033] Fig.12 A. The establishment of the arthritis mouse model and the treatment process; B. The weight change of mice during the entire treatment process; C. The view of the joints of mice in different groups after the treatment. D. The statistical graph and statistical analysis of the joint thickness of mice in different groups during the treatment; E. The joint scores and statistical analysis of mice in different groups during the treatment.

[0034] Fig.13 The expression of TNF-α and IL-6 inflammatory factors in joint tissues in different treatment groups.

[0035] Fig.14 A. Flow chart of modeling and treatment of colitis mice. B. Weight change during treatment. C. Final DAI scores of different treatment groups. D. Statistical graph of colon length of different treatment groups. E. Colon view of different treatment groups.

[0036] Fig.15 The expression of TNF-α and IL-6 inflammatory factors in colon tissue in different treatment groups. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0038] Example 1

[0039] The preparation method of quercetin-loaded cascade targeted nanoparticles is prepared according to the following steps:

[0040] 1. Synthesis of FA-PEG-PAMAM

[0041] Weigh 14.2 mg (1 mM) of PAMAM (polyamidoamine dendrimer) solid using a precision balance and dissolve it in 5 mL of PBS solution. Dissolve PAMAM completely in PBS by ultrasonic vibration. Weigh 20 mg (10 mM) of brown FA-PEG1000-NHS (folic acid-polyethylene glycol-activated ester) powder and dissolve it in 5 mL of DMSO. Place 5 mL of PAMAM solution and 5 mL of FA-PEG1000-NHS solution in a 25 mL round-bottom flask and stir magnetically for 24 h in a dark environment. Select a dialysis bag with a molecular weight cutoff of 3500Da, cut the dialysis bag of appropriate length and soak it in DD (RNase-free water); put the solution in the round-bottom flask into the dialysis bag, and clamp both ends with dialysis bag clamps; place the dialysis bag in a 2L deionized water beaker, dialyze for 3 days in the dark at room temperature, and replace the dialysate every 12 hours; finally, freeze-dry the sample for 48 hours to obtain a brown solid product FA-PEG-PAMAM, which is stored in a -20° refrigerator.

[0042] 2. Synthesis of FA-PTP

[0043] The product FA-PEG-PAMAM from the previous step was dissolved in 10 mL PBS and placed in a round-bottom flask. 50 mg mPEG5000-TK (10 mM methoxypolyethylene glycol ketalthioate) white powder was added at an excess molar ratio of 10:1. After reacting for 5 minutes, 5 mg EDCL (3-hydroxy-2-butanone) and 5 mg NHS (N-hydroxysuccinimide) were added successively. The mixture was stirred magnetically for 30 minutes under light-proof conditions. A dialysis bag with a molecular weight cutoff of 12000 Da was selected (the dialysis bag was cut to a suitable length and then soaked in DD (RNase-free water); the solution in the round-bottom flask was placed in the dialysis bag, and both ends were clamped with dialysis bag clamps; the dialysis bag was placed in a 2 L deionized water beaker), dialyzed at room temperature and light-proof for 3 days, and the dialysate was replaced every 6 hours; finally, the sample was freeze-dried for 48 hours to obtain a brown solid product FA-PTP, which was stored in a -20 ° refrigerator.

[0044] 3. Synthesis of FA-PTP@Que by loading FA-PTP with drug quercetin

[0045] DMSO and PBS were mixed in a volume ratio of 1:9, and quercetin Que was dissolved in the above solution to prepare concentration solutions of 0, 25, 50, 100, 400 and 800 μg / mL, respectively. The absorbance of the solution at a wavelength of 374 nm was tested using an ELISA reader and the standard curve of quercetin was calculated.

[0046] A certain amount of FA-PTP was dissolved in DMSO solution, and the corresponding mass of quercetin was added according to the mass ratio of FA-PTP (mg): Que (mg) = 20:1, 20:5, and 20:10, respectively, and magnetic stirring was carried out in the dark for 24 hours. A dialysis bag with a molecular weight cutoff of 3500Da was selected (cut the dialysis bag of appropriate length and then soaked in DD (RNase-free water); the solution in the round-bottom flask was filled into the dialysis bag, and the two ends were clamped with dialysis bag clamps; the dialysis bag was placed in a 2L deionized water beaker), and dialyzed at room temperature in the dark for 3 days, and the dialysate was replaced every 6 hours. The dialyzed product was centrifuged at 3000rpm for 5 minutes, and the supernatant was retained and freeze-dried for 48 hours to obtain a yellow-brown solid product FA-PTP@Que.

[0047] The obtained product was redissolved in PBS containing 10% DMSO. The absorbance of the solution at a wavelength of 374 nm was detected using an enzyme marker to calculate the encapsulation efficiency (EE%) and drug loading efficiency (LE%).

[0048] EE (%) = (loaded quercetin Que mass / added quercetin Que mass) * 100%

[0049] LE (%) = (mass of loaded quercetin Que / total mass of loaded quercetin Que and FA-PTP) * 100%.

[0050] Study Results

[0051] 1. Characterization of Nanoparticles:

[0052] The synthesis of FA-PTP was confirmed by FT-IR and 1H-NMR characterization. Figure 2 As shown, compared with the spectrum of PAMAM, a peak at 1098 cm appeared in the FA-PEG-PAMAM conjugate. -1 The absorption peak caused by C=O stretching vibration. In FA-PTP conjugate, the vibration peak of sulfur and methylene (-S-CH2-) appears at 1343 cm -1 These results indicate that FA-PEG-NHS and mPEG-TK were successfully modified onto PAMAM.

[0053] In the 1H-NMR results ( Figure 3 ), the peak at 3.6ppm belongs to the proton in the PEG group, and the peak at 6.5-9.0ppm belongs to the phenyl ring on the folic acid, indicating that the synthesis of the FA-PEG-PAMAM conjugate is successful. In addition, the peak at 1.5ppm belongs to the -SH group in mPEG-TK, further indicating that mPEG-TK is conjugated with FA-PTP, indicating the successful preparation of FA-PTP.

[0054] 2. Surface charge of FA-PEG-PAMAM and FA-PTP

[0055] The surface charge of nanocarriers is crucial to their in vivo stability, and relatively high charges lead to better dispersibility. Figure 4 As shown in Figure 2, we used DLS to evaluate the Zeta potential of PAMAM, FA-PEG-PAMAM, and FA-PTP. The Zeta potential of PAMAM was +4.27±0.48mV. After modification with FA-PEG-NHS, the charge of FA-PEG-PAMAM increased to +23.13±0.17mV. After modification with mPEG-TK, the charge of FA-PTP decreased slightly, and the Zeta potential was +19.02±0.59mV. FA-PTP exhibited a relatively high positive charge and had relatively good dispersibility.

[0056] 3. Drug loading rate and encapsulation efficiency of FA-PTP@Que

[0057] The broad-spectrum anti-inflammatory drug quercetin (Que) was used as an experimental drug. The hydrophobic drug quercetin can be effectively encapsulated in the internal cavity of FA-PTP through hydrophobic interaction. Figure 5 As shown in the figure, we explored the encapsulation efficiency and drug loading rate of FA-PTP:Que (20:1, 20:5, 20:10) at three different mass ratios, and determined the most suitable drug loading ratio of FA-PTP@Que. When the mass ratio increased from 20:1 to 20:10, the drug loading rate of Que increased from 7.09% (±0.16%) to 19.95% (±0.45%), but the encapsulation efficiency decreased from 85.11% (±1.96%) to 47.88% (±1.09%). Therefore, we adopted a more balanced ratio, using a mass ratio of 20:5, at which time the encapsulation efficiency of FA-PTP@Que was 69.88% (±4.54%) and the drug loading rate was 16.88% (±1.09%).

[0058] 4. Targeting effect of nanocarriers on macrophages

[0059] 4.1 Expression of folate receptors on the surface of macrophages

[0060] Raw 264.7 cells polarize to pro-inflammatory macrophages and release inflammatory cytokines under the stimulation of lipopolysaccharide (LPS). The cell membrane of pro-inflammatory macrophages highly expresses folate receptor β (FR-β). We evaluated FR-β on the cell surface by immunofluorescence staining, such as Figure 6 As shown, compared with the control group, the green fluorescence of FR-β on the surface of Raw 264.7 increased significantly after LPS treatment, confirming that FR-β on the surface of macrophages was upregulated under LPS stimulation.

[0061] 4.2 Targeting verification of FA-PEG-PAMAM and FA-PTP on macrophages

[0062] We set up different experimental conditions to explore the targeting of FA-PTP. The particles were labeled with FITC and the cell nuclei were stained with DAPI to observe the cell location. Figure 7 As shown in A, in the experimental group without LPS stimulation, no obvious intracellular green fluorescence was observed for both the nanoparticles with the secondary structure targeting molecule FA (FA-PTP) and the nanoparticles without the secondary structure targeting molecule FA (PTP). In the experimental group activated by LPS, obvious green fluorescence was observed for FA-PTP compared with PTP. It is worth noting that cells pretreated with free FA can competitively bind to FR-β on the cell membrane surface. In this case, almost no intracellular green fluorescence was observed for both. To further confirm the targeting effect of FA-PTP, we performed flow cytometry and quantified the fluorescence intensity. The results of flow cytometry were highly consistent with the results of immunofluorescence ( Figure 7 B, Figure 7 C). These results indicate that the design of the primary structure can effectively protect the activity of the targeting molecule. After the primary structure collapses and the secondary structure is exposed, the targeting molecule in the secondary structure of FA-PTP can enhance the uptake of nanocarriers by macrophages. Nanocarriers lacking a secondary functional structure are difficult to achieve effective cell targeting.

[0063] 5. Biological effects of nanoparticles

[0064] 5.1. Reactive oxygen species removal

[0065] like Figure 8 As shown in A, macrophages showed strong ROS signals in response to LPS stimulation, and strong green fluorescence was detected in the cells. When the cells were pretreated with FA-PTP@Que, the observed ROS signals were significantly weakened, while there was no significant change in the FA-PTP without quercetin group. Quantitative analysis by flow cytometry showed that the ROS expression level in the FA-PTP@Que group was much lower than that in the LPS treatment group, and it was statistically significant ( Figure 8 B). The experimental results in this section confirmed that FA-PTP@Que has a scavenging effect on ROS.

[0066] 5.2. Inhibition of inflammatory gene expression

[0067] LPS-stimulated macrophages secrete related inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and CD86 protein. Fig. 9As shown in the figure, the expression level of inflammation-related genes in the LPS-treated group was significantly higher than that in the control group and was statistically significant. After being treated with FA-PTP@Que, the expression level of inflammation-related genes was significantly decreased and was statistically significant. The experimental results show that FA-PTP@Que has a significant inhibitory effect on LPS-induced inflammatory factors and has anti-inflammatory activity in vitro.

[0068] 5.3. Regulation of macrophage repolarization toward an anti-inflammatory phenotype

[0069] In general, macrophages are divided into two functional subsets: classically activated pro-inflammatory M1 macrophages and alternatively activated anti-inflammatory M2 macrophages. Immunofluorescence staining and flow cytometry revealed that FA-PTP@Que increased the expression of M2 macrophage markers (CD206) and reduced the expression of M1 macrophage markers (CD86). Fig.10 A and Fig. 9 As shown, CD86 (red fluorescence) is marked as M1, CD206 (green fluorescence) is marked as M2. After LPS treatment of macrophages, the red fluorescence of CD86 increased significantly. After FA-PTP@Que treatment of macrophages, the red fluorescence intensity of CD86 decreased, and the green fluorescence of CD206 increased, indicating that FA-PTP@Que can effectively repolarize macrophages from M1 to M2. The quantitative results of flow cytometry showed that after FA-PTP@Que treatment, the fluorescence intensity of CD86 decreased, and the fluorescence intensity of CD206 increased and was statistically significant ( Fig.10 C and Fig.10 D). Macrophage polarization experiments verified that FA-PTP@Que could regulate the repolarization of macrophages to develop an anti-inflammatory phenotype.

[0070] 5.4. Results of Nanoparticle Treatment on Arthritis Mice

[0071] To evaluate the targeting ability of shielded cascade nanocarriers at the site of inflammation, we injected FITC-labeled FA-PTP-FITC, FA-PEG-PAMAM-FITC (without mPEG-TK modification), and free FITC into mice and performed optical imaging of the fluorescence signal using IVIS. Fig.11 As shown in A, 4 hours after intravenous injection, the FA-PTP group showed a strong fluorescence signal in the inflamed joints compared with the Free-FITC and FA-PEG-PAMAM groups. The quantitative results showed that the fluorescence intensity of the FA-PTP group was significantly higher than that of the other two control groups, and it was statistically significant ( Fig.11 B). The experimental results show that the cascade structure design of FA-PTP can effectively improve the targeting efficiency of nanocarriers in arthritic mice.

[0072] The induction and treatment of arthritis can be seen in Fig.12 A. We divided arthritic mice into four groups and treated them with saline, FA-PTP, free quercetin, and FA-PTP@Que. The therapeutic effect was determined by evaluating various parameters including body weight, paw thickness, arthritis score, histological evidence of inflammation, and changes in the expression levels of pro-inflammatory cytokines. No significant differences in body weight occurred throughout the treatment ( Fig.12 B). At the end of the treatment period, the paws of the Saline group, FA-PTP group, and Free-Que group showed obvious redness and swelling, and the FA-PTP@Que group showed significant improvement in paw redness and swelling ( Fig.12 C). The degree of swelling in the mouse paws was quantified by examining the joint thickness, as Fig.12 As shown in D, during the entire treatment process, the paw thickness of the FA-PTP@Que group gradually decreased compared with the other control groups, and this was statistically significant. In addition, FA-PTP@Que significantly reduced the clinical score of arthritis, and the results of each group were consistent with the joint thickness and statistically significant ( Fig.12 E).

[0073] Extracting RNA from joint tissues of different treatment groups to detect levels of related inflammatory factors is another indicator for evaluating the therapeutic effect. Fig.13 As shown in the figure, TNF-α and IL-6 were still highly expressed in the Saline group and FA-PTP group. After treatment with Free-Que and FA-PTP@Que, the expression levels of TNF-α and IL-6 decreased. Among them, FA-PTP@Que had a significant therapeutic effect, and the gene expression level decreased significantly and statistically significant. The results of the in vivo inflammatory factor detection experiment showed that FA-PTP@Que had good anti-inflammatory activity in vivo.

[0074] 5.5. Results of Nanoparticles in Treating Colitis in Mice

[0075] Given that FA-PTP has a good targeting effect on colitis, we evaluated the therapeutic effect of FA-PTP@Que in a colitis mouse model. Colitis mice were randomly assigned to four groups: saline, FA-PTP, Free Que, and FA-PTP@Que groups, and a negative control group was set up. Fig.14 As shown in A, a 7-day treatment was carried out while the model was being constructed. Body weight change is an indicator for evaluating the severity of colitis. Fig.14 As shown in Figure B, compared with the yin-yang control group, the body weight of the experimental group induced by DSS colitis decreased significantly, among which FA-PTP@Que had the lowest body weight loss on day 7. The disease activity index score DAI is the gold standard for colitis disease assessment, such as Fig.14 As shown in Figure C, the DAI score of FA-PTP@Que was the lowest at the end of treatment, and it was statistically significant compared with other experimental groups. After the mice were killed at the end of treatment, the colon lengths of different groups were measured, as shown in Figure 4. Fig.14 D and Fig.14 As shown in Figure E, the colon length after treatment with Saline, FA-PTP and Free Que was significantly shortened compared with the control group, and the colon length after treatment with FA-PTP@Que was slightly shortened. The in vivo experimental results showed that FA-PTP@Que can have a good therapeutic effect on DSS-induced colitis and greatly improve the treatment evaluation of colitis.

[0076] Extracting RNA from joint tissues of different treatment groups to detect levels of related inflammatory factors is another indicator for evaluating the therapeutic effect. Fig.15 As shown in the figure, TNF-α and IL-6 were still highly expressed in the Saline combined with FA-PTP group. After treatment with Free-Que and FA-PTP@Que, the expression levels of TNF-α and IL-6 decreased. Among them, FA-PTP@Que had a significant therapeutic effect, and the gene expression level decreased significantly and statistically significant. The results of the in vivo inflammatory factor detection experiment showed that FA-PTP@Que had good anti-inflammatory activity in vivo.

[0077] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing quercetin-loaded cascade targeted nanoparticles, characterized in that: Prepare according to the following steps: (1) Synthesis of FA-PEG-PAMAM: weigh polyamide-amine dendrimer (PAMAM) and dissolve it in PBS solution; weigh folic acid-polyethylene glycol-activated ester (FA-PEG1000-NHS) and dissolve it in SMSO; mix the two solutions and stir them in a dark environment; after the reaction, dialyze at room temperature in a dark environment, replace the dialyzate every 12 hours, and freeze-dry the sample to obtain FA-PEG-PAMAM; (2) Synthesis of FA-PTP FA-PEG-PAMAM was dissolved in PBS, and an excess of methoxypolyethylene glycol ketone thioacetal (mPEG5000-TK) was added for reaction, followed by 3-hydroxy-2-butanone (EDCL) and N-hydroxysuccinimide (NHS). After stirring in the dark, the mixture was dialyzed at room temperature in the dark, the dialyzate was replaced every 6 hours, and the product FA-PTP was obtained by freeze drying. (3) Synthesis of FA-PTP@Que by loading FA-PTP with drug quercetin FA-PTP was dissolved in DMSO solution, quercetin was added, the reaction was stirred in the dark, and then dialyzed at room temperature in the dark, with the dialysate replaced every 6 hours; the dialyzed product was centrifuged, the supernatant was retained, and freeze-dried to obtain a yellow-brown solid product FA-PTP@Que.

2. The method for preparing quercetin-loaded cascade targeted nanoparticles according to claim 1, characterized in that: In step (1), the molar ratio of folic acid-polyethylene glycol-activated ester to polyamide-amine dendrimer is 5:1 to 10:

1.

3. The method for preparing quercetin-loaded cascade targeted nanoparticles according to claim 2, characterized in that: In step (1), a dialysis bag with a molecular weight cutoff of 3500Da is selected and the dialysis bag is soaked in DD water; the reaction solution is placed in the dialysis bag and both ends are clamped with dialysis bag clamps; the dialysis bag is placed in deionized water and dialyzed in the dark at room temperature for 3 days, and the dialysate is replaced every 12 hours.

4. The method for preparing quercetin-loaded cascade targeted nanoparticles according to any one of claims 1 to 3, characterized in that: The molar ratio of methoxy polyethylene glycol ketalthioate to FA-PEG-PAMAM was 10:1, and the mass ratio of the added amount of EDCL and NHS to methoxy polyethylene glycol ketalthioate was 1:

10.

5. The method for preparing quercetin-loaded cascade targeted nanoparticles according to claim 4, characterized in that: In step (2), a dialysis bag with a molecular weight cutoff of 12000Da was selected and the dialysis was performed for 3 days.

6. The method for preparing quercetin-loaded cascade targeted nanoparticles according to claim 5, characterized in that: In step (3), the mass ratio of FA-PTP to quercetin is 20:1-20:

10.

7. The method for preparing quercetin-loaded cascade targeted nanoparticles according to claim 6, characterized in that: Select a dialysis bag with a molecular weight cutoff of 3500Da and dialyze for 3 days. 8 . Quercetin-loaded cascade targeted nanoparticles prepared by the method for preparing quercetin-loaded cascade targeted nanoparticles according to any one of claims 1 to 7 .

9. Use of the quercetin-loaded cascade targeted nanoparticles according to claim 8 in the preparation of anti-inflammatory drugs.

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