Tetrahedral nucleic acid nano delivery system of chicoric acid and application thereof in gout treatment

Abstract

The invention belongs to the field of biological medicine, and discloses a chicoric acid (CA)-loaded tetrahedral framework nucleic acid (tFNA) nano preparation for treating acute gouty arthritis. According to the preparation, tFNA formed by self-assembly is taken as a carrier, and a natural active ingredient CA is loaded to form a CA-tFNA compound. The compound overcomes the defects that CA is poor in water solubility and low in bioavailability, and existing drugs are weak in targeting and large in side effect. Through the targeted delivery and synergistic effect of tFNA, the preparation can be effectively enriched in inflammatory joints, significantly inhibit NF-kappa B signal pathways, and down-regulate the expression of key proinflammatory factors such as TNF-alpha and IL-1beta, so that joint swelling and synovial tissue damage are relieved, and repair is promoted. Experiments show that the traditional Chinese medicine composition is remarkable in curative effect and good in safety, and a novel efficient treatment strategy is provided for acute gouty arthritis.

Description

TECHNICAL FIELD

[0001] The present application relates to a tetrahedral framework nucleic acid (tFNA) loaded with chicoric acid for use in a therapeutic pharmaceutical composition for acute gouty arthritis and its application, belonging to the technical field of medicine. BACKGROUND

[0002] Acute gouty arthritis (AGA) is an autoimmune inflammatory disease triggered by the deposition of monosodium urate crystals (MSU). Its incidence is increasing globally, especially in middle-aged men and postmenopausal women. With the changes in people's lifestyle and the popularity of high-purine diet, the incidence of the disease is becoming younger and younger, and it has become one of the metabolic diseases that seriously affect public health and quality of life. The typical clinical manifestations of AGA are sudden onset of severe pain, redness, heat and dysfunction in the joints, often involving the first metatarsophalangeal joint, ankle joint, knee joint and other parts. Repeated attacks can lead to joint deformity, bone destruction and even disability. It is also closely related to cardiovascular disease, diabetes, chronic kidney disease and other complications, causing heavy burden to the patient's family and the social medical system.

[0003] At present, the pathogenesis of AGA has not been fully elucidated, but it is generally believed that MSU crystals are the key trigger of the disease. When the body's uric acid level exceeds the solubility threshold, MSU crystals will deposit in the joint cavity and surrounding tissues, triggering an inflammatory cascade reaction by activating innate immune cells such as macrophages and neutrophils. Specifically, MSU crystals can bind to pattern recognition receptors such as Toll-like receptors (TLRs), NOD-like receptor protein 3 (NLRP3) inflammasomes on the surface of macrophages, prompting the release of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and other pro-inflammatory cytokines. These cytokines further recruit inflammatory cells such as neutrophils to infiltrate the joint tissue, release lysosomal enzymes, reactive oxygen species (ROS) and other inflammatory mediators, exacerbate joint synovial inflammation and tissue damage, form an "inflammatory amplification cycle", and lead to acute onset of the disease. Therefore, inhibiting NLRP3 inflammasome activation, reducing the release of pro-inflammatory cytokines and clearing MSU crystals are key targets for the treatment of AGA.

[0004] The current clinical drugs for treating AGA mainly include non-steroidal anti-inflammatory drugs (NSAIDs), colchicine and glucocorticoids, and allopurinol and febuxostat. Although NSAIDs and colchicine can quickly relieve pain symptoms, they have obvious gastrointestinal side effects (such as nausea, vomiting, gastric ulcer) and the risk of liver and kidney function damage, and colchicine has a narrow therapeutic window and is easy to cause poisoning. Glucocorticoids have a significant short-term effect, but long-term use can cause immunosuppression, osteoporosis, and elevated blood sugar, and are not suitable for patients with infections or diabetes. Uric acid-lowering drugs can reduce the recurrence of AGA by inhibiting uric acid production or promoting uric acid excretion, but they cannot effectively control the inflammatory response during acute episodes, and some patients have drug allergies or poor efficacy. In addition, existing drugs are difficult to achieve the dual effects of "rapid anti-inflammatory analgesia" and "repairing joint damage", and lack of targeting, which can easily cause toxic side effects to normal tissues. Therefore, developing new AGA treatment strategies with high targeting, multiple action targets, and low toxic side effects has become a research hotspot and urgent need in the field of biological medicine.

[0005] Tetrahedral Framework Nucleic Acids (tFNAs) are a kind of three-dimensional nanostructure formed by self-assembly of four single-stranded DNA with specific sequences through base complementary pairing, which has good biocompatibility, stability and modifiability, and shows great application potential in drug delivery, gene therapy and immune regulation. Compared with traditional linear nucleic acids or liposomes, tFNAs have a unique tetrahedral spatial structure, which can passively target inflammatory tissues by enhancing the EPR effect, and can actively target through surface modification of targeting ligands to improve drug delivery efficiency. In addition, studies have found that tFNAs themselves have certain anti-inflammatory activity, which can reduce inflammation by regulating macrophage polarization and inhibiting inflammasome activation, providing a new way for the treatment of inflammatory diseases.

[0006] Cichoric acid (CA) is a natural phenolic acid compound extracted from plants such as chicory and echinacea, which has rich pharmacological activities, including anti-inflammatory, antioxidant, immune regulation and tissue repair effects. Modern pharmacological studies have shown that CA can exert anti-inflammatory effects by inhibiting the NF-κB signaling pathway, reducing the expression of pro-inflammatory cytokines (IL-1β, TNF-α), and removing ROS; at the same time, CA can also promote fibroblast proliferation and collagen synthesis, and accelerate the repair of damaged tissues. However, CA has the disadvantages of poor water solubility, low bioavailability, and fast in vivo metabolism, which limit its clinical application. By combining CA with tFNAs and using tFNAs as a drug carrier, not only can the solubility and stability of CA be improved, and its targeted delivery capacity be enhanced, but also the anti-inflammatory activity of tFNAs and the multi-effect pharmacological action of CA can be synergistically exerted, realizing the synergistic treatment effect of "carrier-drug" and providing a new, efficient and low-toxic drug composition for the treatment of acute gouty arthritis. Based on the above background, it has important scientific significance and clinical value to develop a tetrahedral framework nucleic acid loaded with cichoric acid for the treatment of acute gouty arthritis and its application. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings of existing drugs for treating acute gouty arthritis and the defects of CA as a drug, and to provide a tetrahedral framework nucleic acid (tFNA) loaded with cichoric acid for treating acute gouty arthritis, which has the characteristics of good efficacy and high safety. At the same time, the present application also provides a preparation method of the drug composition and its application in preparing a drug for treating acute gouty arthritis.

[0008] The specific technical solutions of the present application are as follows: Step 1: Composition of the drug composition: the tetrahedral framework nucleic acid drug loaded with cichoric acid is composed of cichoric acid (CA) and tetrahedral framework nucleic acid (tFNA), which is abbreviated as CA-tFNAs.

[0009] In the CA-tFNAs complex, the molar ratio of tetrahedral framework nucleic acid to cichoric acid is 1:200. Among them, the tetrahedral framework nucleic acid is formed by self-assembly of four single-stranded DNAs through base complementary pairing, and the sequences of the four single-stranded DNAs are as follows: Single-stranded DNA 1: 5'-ATCCATAGATACGTCCATAGATACGT-3'; Single-stranded DNA 2: 5'-ACGTATCTATGGACGTATCTATGGAT-3'; Single-stranded DNA 3: 5'-ATCCATAGATACGTCCATAGATACGT-3'; Single-stranded DNA 4: 5'-ACGTATCTATGGACGTATCTATGGAT-3') self-assemble to form.

[0010] Step 2: Preparation method of pharmaceutical composition: (1) Preparation of tFNAs: four oligonucleotide chains were dissolved in enzyme-free water at the same concentration. Then the four single-stranded DNAs were dissolved in TM buffer (50 mM MgCl2 and 10 mM Tris-HCl, pH 8.0) at an equimolar ratio. After mixing, the DNA tetrahedral framework was synthesized by setting the annealing program in the PCR instrument, rapidly heating to 95℃ for 10 min, and then rapidly cooling to 4℃.

[0011] (2) Preparation of CA-tFNAs complex: chicoric acid was prepared into a 10 mg / mL stock solution with DMSO, and added to the tFNAs solution at a ratio of 1:200, and shaken at room temperature for 6H; (3) Step 3: Formulation and mechanism of action: The preferred formulation is an intra-articular injection, which targets the inflammatory site, inhibits the NF-κB pathway, removes ROS to reduce the release of pro-inflammatory factors, and promotes joint tissue repair.

[0012] The present application aims to solve the limitations of existing treatment methods for acute gouty arthritis (AGA), and provides a new type of therapeutic drug composition with high efficiency, low toxicity and multi-target synergy. The current AGA treatment drugs have obvious deficiencies: the side effects of symptomatic drugs such as non-steroidal anti-inflammatory drugs and colchicine are significant, long-term use of glucocorticoids can easily cause immunosuppression, uric acid-lowering drugs cannot quickly control acute inflammation, and existing drugs generally lack targeting, making it difficult to achieve the dual effects of “rapid anti-inflammatory analgesia” and “joint tissue repair”. Based on this, the present application uses tetrahedral framework nucleic acid (tFNAs) as a carrier to load chicoric acid (CA) to construct a drug composition. tFNAs have excellent biocompatibility, targeting and anti-inflammatory potential, and can be passively targeted to inflammatory joints through the EPR effect, chicoric acid has anti-inflammatory, antioxidant and tissue repair activity, can remove ROS and inhibit the NF-κB pathway, but has the defects of poor water solubility and low bioavailability.

[0013] The present application improves the pharmacokinetic properties of CA through the advantages of tFNAs carrier, and simultaneously exerts the synergistic effect of the two: tFNAs target the delivery of CA to the inflammatory site, reducing the toxic side effects on normal tissues; both of them block the inflammatory cascade reaction, reduce the release of pro-inflammatory factors, relieve joint swelling and synovial membrane damage, and promote the repair of damaged tissues. Ultimately, it achieves efficient treatment of AGA, provides a new drug with targeting, synergistic anti-inflammatory and tissue repair functions for clinical use, solves the existing treatment pain points, and improves the treatment effect and quality of life of patients. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Figure 3 is a schematic diagram showing the degree of swelling of the mouse ankle joint after modeling, and the results show that the model mice have the highest degree of ankle joint swelling.

[0015] Figure 2 Figure 4 is a line graph showing the effect of the drug on the joint swelling of the model mice, wherein the horizontal axis represents the administration time (h), and the vertical axis represents the swelling degree (mm), and the curve shows that CA-tFNAs has the best effect on swelling.

[0016] Figure 3 Figure 5 is a HE staining graph of the synovial membrane (x200), wherein A is the blank group, B is the model group, C is the free CA group, D is the tFNAs group, and E is the CA-tFNAs group, and the results show that CA-tFNAs significantly improves the pathological damage of the synovial membrane.

[0017] Figure 4 Figure 6 is an immunofluorescence micrograph of LPS-treated RAW264.7 cells (cytoskeleton: green; nucleus: blue; NF-κB P65, TNF-α, IL-1β and iNOS: red).

[0018] Figure 5 Figure 7 is an HE staining picture of the heart, liver, spleen, lung and kidney tissues of the mice after 48 hours of administration, and the results show that there is no obvious tissue damage, indicating that the drug has good safety. DETAILED DESCRIPTION

[0019] Example 1: Preparation and characterization of tFNAs Step 1 Reagent preparation: Single-stranded DNA 1-4 was synthesized by Shanghai Shengong Bioengineering Co., Ltd., and purified by HPLC; TM buffer (50 mM MgCl2and 10 mM Tris-HCl, pH 8.0); sterile water for injection was sterilized by 0.22 μm filter membrane.

[0020] Step 2 Preparation process: four single-stranded DNAs were dissolved in enzyme-free water at the same concentration. Then, ssDNA and TM buffer (50 mM MgCl2and 10 mM Tris-HCl, pH 8.0) were mixed. TFNAs were successfully synthesized by heating (95°C, 10 min) and then cooling (4°C, 20 min).

[0021] Step 3 Characterization detection: The three-dimensional tetrahedral morphology of CA-TFNAs was observed by taking a transmission electron microscope (TEM), and the average side length was measured.

[0022] Example 2: Preparation of CA-tFNAs complex with different molar ratios and determination of loading rate Step 1 Chicoric acid mother liquor preparation: A certain amount of chicoric acid is weighed and dissolved in dimethyl sulfoxide to prepare a chicoric acid solution of a certain concentration.

[0023] Step 2 Complex preparation: The pretreated chicoric acid solution is added to the tetrahedral framework nucleic acid solution, and the chicoric acid is loaded onto the tetrahedral framework nucleic acid by stirring at room temperature for 6 hours; after the reaction is completed, the loaded product is purified by centrifugation or dialysis to obtain chicoric acid-loaded tetrahedral framework nucleic acid CA-TFNAs.

[0024] Step 3 Loading rate determination: Chicoric acid encapsulation rate and release behavior verification: 30 kDa ultrafiltration tube is used for centrifugation to remove residual Cur. The absorbance of CA is detected at a wavelength of 330 nm. The encapsulation and binding of CA in CA-TFNAs are further studied by spectral analysis.

[0025] Example 3: Preparation and stability investigation of intra-articular injection In PBS (pH 7.4) at 37°C, take samples at regular intervals and detect the concentration of released chicoric acid, draw the release curve, and evaluate the release kinetics.

[0026] Example 4: Comparison experiment of in vitro anti-inflammatory activity Step 1: Cell culture: RAW264.7 macrophages are purchased from the Chinese Academy of Sciences Cell Bank and cultured in DMEM medium containing 10% fetal bovine serum (FBS, Gibco), 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37°C, 5% CO2 incubator. The logarithmic growth phase cells are used for experiments.

[0027] Step 2 Cell crawling and grouping: The cells are inoculated on sterile coverslips in 24-well plates at a density of 2×10 4 6 cells / well, and cultured for 24 h before being divided into 5 groups, each with 3 replicate wells: blank group (normal cells), model group (LPS induction), free CA group (75 μM), tFNAs group (250 nM), CA-tFNAs group (CA 75 μM + tFNAs 250 nM).

[0028] Step 3: Model establishment and drug administration: LPS (final concentration 1 μg / mL) is added to all groups except the blank group to establish an inflammatory model for 24 h; then the corresponding drugs are added to each drug administration group, and the blank and model groups are added with an equal amount of medium, and the culture is continued for 12 h.

[0029] Step 4 Immunofluorescence staining: ①Fixation: remove the coverslips, wash with PBS for 3 times, 5 min each time; 4% paraformaldehyde room temperature fixation for 20 min, PBS wash for 3 times; ②Permeabilization: 0.1% Triton X-100 room temperature incubation for 15 min, PBS wash for 3 times; ③Blocking: 5% BSA room temperature blocking for 1 h; ④Primary antibody incubation: add rabbit anti-mouse NF-κB P65 antibody (1:200, Sevile), TNF-α antibody (1:200, Wanleibios), IL-1β antibody (1:400, Wanleibios), iNOS antibody (1:200, Wanleibios) respectively, 4°C incubation overnight; ⑤Secondary antibody incubation: after PBS wash for 3 times, add rhodamine-labeled goat anti-rabbit IgG (1:100, Zhongshanjingqiao), room temperature incubation for 1 h in the dark; ⑥Nuclei staining: DAPI staining solution (1:1000) room temperature incubation for 5 min in the dark, PBS wash for 3 times; ⑦Mounting: anti-fluorescence quenching mounting medium mounting.

[0030] Step 5 Image acquisition and analysis: laser confocal microscope (Zeiss LSM 880) was used to observe and collect images, 5 random fields were selected, and the fluorescence intensity (IOD / Area) was analyzed by ImageJ software. The results showed that compared with the model group, the fluorescence intensity of TNF-α, IL-1β, iNOS, NF-κB P65 in the CA-tFNAs group was reduced by 92.0%, 80.6%, 47.7% and 84.6% respectively (all P<0.01), the fluorescence intensity of TNF-α, IL-1β, iNOS, NF-κB P65 in the free CA group was reduced by 18.5%, 49.1%, 27.9% and 12.1% respectively (P<0.05), and the fluorescence intensity of TNF-α, IL-1β, iNOS, NF-κB P65 in the tFNAs group was reduced by 87.9%, 59.3%, 26.6% and 57.4% respectively (P<0.05). It is indicated that the CA-tFNAs complex can significantly inhibit the nuclear translocation of NF-κB P65 and the expression of inflammatory factors and inflammatory mediators, and the anti-inflammatory activity is better than that of free CA or tFNAs alone.

[0031] Example 5: in vivo therapeutic effect and safety evaluation Step 1: experimental animals: 60 male Kunming mice, 6-8 weeks old, weighing 20-22 g, the feeding environment temperature is 22-25°C, humidity is 50%-60%, 12h light and dark cycle, free feeding and drinking.

[0032] Step 2: model establishment: 20μL MSU suspension (25mg / mL, ultrasonic treatment for 30min to make the particles uniform) was injected into the right hind ankle joint cavity of the mice, and the same amount of normal saline was injected into the left hind ankle joint. 2h after injection, the joint diameter was measured. If the mouse ankle joint swelled, it was considered that the model was successfully established.

[0033] Step 3: Experimental grouping and drug administration: 60 Kunming mice were randomly divided into 6 groups, namely blank group, model group, CA group, tFNAs group, CA-tFNAs group, and colchicine (positive drug) group Step 4: In vivo efficacy evaluation: ①Joint swelling degree: Before administration (0h), 0h, 2h, 4h, 8h, 24h and 48h after administration, the inner and outer diameters of the right ankle joint of the hind limb were measured by vernier caliper (accuracy 0.01mm), and the swelling degree was calculated as diameter after administration-diameter before administration. The results showed that the swelling rapidly increased after 2h of administration, reached a peak (2.5mm) around 4-6h, and then gradually subsided, but still maintained a certain degree of swelling (1.2mm) at 48h, indicating that MSU successfully induced ankle joint swelling in mice. The control group maintained a swelling increase of about 0mm within 0-48h, with no obvious swelling reaction. CA group, tFNA group, CA-tFNAs group, and positive drug group: these intervention groups showed varying degrees of inhibition of ankle joint swelling, and the anti-inflammatory and detumescence effect of the MSU+CA-tFNAs group was particularly prominent: the swelling peak was significantly lower than that of other intervention groups, and the swelling increase was only about 0.5mm at 48h. This result indicates that tFNA as a carrier can effectively improve the bioavailability of CA, enhance its delivery efficiency, targeting and duration in the inflammatory local, and thus more efficiently inhibit MSU-mediated ankle joint inflammatory swelling.

[0034] ②Pathological histological examination: the mice were sacrificed 48h after administration, the right hind ankle joint tissue was taken, fixed with 4% paraformaldehyde for 24h, decalcified, paraffin-embedded, 5μm sectioning, HE staining, and observed under an optical microscope. The results showed that the synovial tissue of the model group was significantly hyperplastic, hyperemic and edematous, with a large number of neutrophil infiltration; the synovial structure of the CA-tFNAs group was basically normal, with only a small amount of inflammatory cells; the synovial hyperplasia of the CA group and tFNA group was slight, with fewer inflammatory cells.

[0035] Step 5: Safety evaluation: during the administration period, the general state (diet, activity, body weight) of the mice was observed, and no abnormalities were found; 48h after administration, the heart, liver, spleen, lung and kidney tissues of the mice were taken, and pathological changes were observed by HE staining, and no obvious tissue damage was found, indicating that the drug safety was good.

[0036] The present application belongs to the technical field of biological medicine, and specifically relates to a tetrahedral framework nucleic acid drug composition loaded with chicoric acid, and application of the drug composition in preparation of an acute gouty arthritis treatment drug.

Claims

1. A pharmaceutical composition for treating acute gouty arthritis, characterized in that, The pharmaceutical composition comprises a tetrahedral backbone nucleic acid complex loaded with chicoric acid, wherein the tetrahedral backbone nucleic acid is formed by the self-assembly of four single-stranded DNA molecules through complementary base pairing, and the chicoric acid is loaded non-covalently on the surface or in the internal cavity of the tetrahedral backbone nucleic acid.

2. A method for preparing the pharmaceutical composition as described in claim 1, characterized in that, Includes the following steps: (1) Four single-stranded DNA strands were self-assembled into a tetrahedral backbone nucleic acid by heating and annealing in a magnesium-containing buffer solution; (2) The chicoric acid solution was mixed with the tetrahedral backbone nucleic acid solution and the mixture was shaken at room temperature to load the chicoric acid onto the tetrahedral backbone nucleic acid to obtain the CA-tFNA complex.

3. The use of the pharmaceutical composition according to claim 1 in the preparation of a medicament for treating acute gouty arthritis, characterized in that, The drug is administered via intra-articular injection to suppress inflammatory responses, reduce joint swelling, and promote tissue repair.

4. The pharmaceutical composition according to claim 1, characterized in that, The sequences of the four single-stranded DNA strands are as follows: Single-stranded DNA 1: 5′-ATCCATAGATACGTCCATAGATACGT-3′; Single-stranded DNA 2: 5′-ACGTATCTATGGACGTATCTATGGAT-3′; Single-stranded DNA 3: 5′-ATCCATAGATACGTCCATAGATACGT-3′; Single-stranded DNA 4: 5′-ACGTATCTATGGACGTATCTATGGAT-3′.

5. The pharmaceutical composition according to claim 1, characterized in that, The molar ratio of the tetrahedral backbone nucleic acid to chicoric acid is 1:50-1:500, preferably 1:

200.

6. The preparation method as described in claim 2, characterized in that, The buffer solution mentioned in step (1) is a TM buffer solution containing 50 mM MgCl2 and 10 mM Tris-HCl with a pH of 8.0; the heating-annealing procedure is: incubation at 95°C for 10 minutes, followed by rapid cooling to 4°C.

7. The preparation method according to claim 2, characterized in that, The chicoric acid solution described in step (2) was prepared using DMSO, and the reaction time was 6 hours with shaking.

8. The application as described in claim 3, characterized in that, The pharmaceutical composition exerts its therapeutic effect through one or more mechanisms, including inhibiting the NF-κB signaling pathway, reducing the expression of TNF-α, IL-1β, and iNOS, and scavenging reactive oxygen species.

9. The application as described in claim 3, characterized in that, The pharmaceutical composition is used to simultaneously achieve rapid relief of acute joint swelling and long-term repair of joint tissues.

10. A preparation for treating acute gouty arthritis, characterized in that, The formulation comprises the CA-tFNA complex as described in claim 1 and a pharmaceutically acceptable carrier, wherein the formulation is an injectable, preferably an intra-articular injection.

Images