EGCG (epigallocatechin gallate) and amino hyaluronic acid-based nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses nanoparticles based on EGCG (epigallocatechin gallate) and amino hyaluronic acid as well as a preparation method and application of the nanoparticles, and belongs to the technical field of medical materials. The post-preparation method comprises the following steps: 1) dissolving EGCG and aminated hyaluronic acid in deionized water to obtain a mixed solution; (2) adding a formaldehyde solution into the mixed solution, uniformly mixing, and carrying out stirring reaction to obtain a pre-product; and 3) centrifuging the pre-product, washing and drying to obtain the nanoparticles based on EGCG and amino hyaluronic acid. According to the invention, EGCG and aminated hyaluronic acid are chemically coupled through amino groups and phenolic hydroxyl groups to form particles, and the targeting capability of hyaluronic acid on CD44 receptors highly expressed by activated macrophages in sepsis can be utilized; meanwhile, EGCG is assembled into nanoparticles, so that the in-vivo circulation time can be prolonged, and the bioavailability can be improved. And combined targeted therapy of pyroptosis inhibition, inflammation resistance and oxidation resistance can be realized for sepsis, so that the treatment effect of sepsis is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical materials, and particularly relates to a nanoparticle based on EGCG and amino hyaluronic acid, a preparation method thereof, and an application thereof. Background Art

[0002] Pyroptosis is a new form of programmed cell death (PCD) mediated by the gasdermin protein family that targets the membrane and induces cytoplasmic membrane pore formation. Pyroptosis is usually activated through the NLRP3 inflammasome or the non-canonical inflammasome, and the inflammasome will further activate caspase-1. The activated caspase-1 will directly cause the cleavage of gasdermin D protein (GSDMD) at the junction of the N-terminal domain (GSDMD-NT) and the auto-inhibitory C-terminal domain (GSDMD-CT), initiating pyroptosis. The cleaved GSDMD-NT binds to the cytoplasmic membrane and oligomerizes to form pores of 10-14 nm, disrupting the cell osmotic pressure, resulting in cell lysis and death, and further releasing cell contents and pro-inflammatory cytokines activated by caspase-1 during pyroptosis.

[0003] Pyroptosis plays a crucial role in the occurrence and development of sepsis. Pyroptosis induces the rupture of the cytoplasmic membrane to release a large number of cytokines, aggravating multi-organ damage, and ultimately leading to sepsis death. During the occurrence and development of sepsis, pyroptosis mainly occurs in macrophages, further aggravating the inflammatory response and inducing multi-organ dysfunction. Among them, in sepsis-induced liver injury, the incidence of pyroptotic cells in the liver is about 18.19%, while the incidence of pyroptosis in liver macrophages is 16.29%, indicating that pyroptosis in liver macrophages dominates among the cells undergoing pyroptosis. Pyroptosis is also an important way of macrophage death in sepsis. Studies have shown that 39% of the death of peritoneal macrophages in a cecal ligation and puncture model (CLP) of septic mice is caused by pyroptosis, and inhibiting pyroptosis of lung macrophages in sepsis can reduce sepsis-induced lung injury.

[0004] Based on the above mechanism, it can be seen that in sepsis, the inflammation-induced pyroptosis process not only directly causes macrophage death but also releases pro-inflammatory cytokines extracellularly, aggravating the inflammatory response. Therefore, in the treatment of sepsis, inhibiting macrophage pyroptosis is beneficial to controlling sepsis. However, the problems existing in the treatment of sepsis by inhibiting pyroptosis currently are: (1) Most of the drugs used to inhibit pyroptosis are small molecule drugs and polypeptide drugs, which have a short circulation time in the body circulation and low bioavailability. (2) Using a single drug to inhibit pyroptosis only plays the role of inhibiting pyroptosis and does not have the function of targeting macrophages undergoing pyroptosis.

[0005] Epigallocatechin gallate (abbreviated as EGCG) is the most effective active ingredient in tea polyphenols of natural origin. It can inhibit pyroptosis by inhibiting the oligomerization of GSDMD protein, which mediates pore formation and membrane rupture, and has potential in the treatment of sepsis. However, as a small molecule drug, EGCG also has problems of low bioavailability and lack of targeting. Therefore, designing EGCG into targeted particles for the treatment of sepsis is a way to solve the above problems. Summary of the Invention

[0006] For this reason, the main object of the present invention is to provide a nanoparticle based on EGCG and amino hyaluronic acid and its preparation method; The present invention also provides the application of the nanoparticle based on EGCG and amino hyaluronic acid in the treatment of sepsis by inhibiting pyroptosis.

[0007] The object of the present invention is achieved by the following technical solutions: A preparation method of a nanoparticle based on EGCG and amino hyaluronic acid, comprising the following steps: 1) Dissolve EGCG and amino-functionalized hyaluronic acid in deionized water to obtain a mixed solution; 2) Add a formaldehyde solution to the mixed solution, mix evenly and stir to react to obtain a pre-product; 3) After centrifuging, washing and drying the pre-product, the nanoparticle based on EGCG and amino hyaluronic acid is obtained.

[0008] The synthesis process of the nanoparticle based on EGCG and amino hyaluronic acid is as follows: In some specific embodiments, in step 1), the mass concentration of EGCG is 0.5 - 10 mg / mL, and the mass concentration of amino-functionalized hyaluronic acid is 0.5 - 10 mg / mL; and the mass ratio of EGCG to amino-functionalized hyaluronic acid is 2 - 6:1.

[0009] In some specific embodiments, the dosage of the formaldehyde solution in step 2) is 0.1 - 0.5 μL / mL.

[0010] In some specific embodiments, the process conditions of the stirring reaction in step 2) are: stirring and reacting at 25 - 37 °C at a rotation speed of 500 - 1000 rpm for 0.5 - 12 h.

[0011] In some specific embodiments, the process conditions of the centrifugation in step 3) are: centrifuging at a rotation speed of 8000 - 12000 rpm for 5 - 15 min.

[0012] As the same inventive concept, the present invention also discloses nanoparticles based on EGCG and amino hyaluronic acid prepared by the described preparation method, the particle size of the nanoparticles is 250-300 nm, and the surface of the nanoparticles is negatively charged.

[0013] Furthermore, the particle size of the nanoparticles is 279.4 nm.

[0014] As the same inventive concept, the present invention also discloses the application of the nanoparticles based on EGCG and amino hyaluronic acid in the preparation of drugs for treating sepsis.

[0015] In some specific embodiments, the nanoparticles based on EGCG and amino hyaluronic acid can inhibit pyroptosis.

[0016] Compared with the prior art, the present invention has at least the following advantages: 1) In the present invention, EGCG and amino-functionalized hyaluronic acid are formed into particles through chemical coupling of amino groups and phenolic hydroxyl groups. The nanoparticles can utilize the targeting ability of hyaluronic acid to the CD44 receptor highly expressed on activated macrophages in sepsis; at the same time, EGCG is assembled into nanoparticles. Compared with small molecule drugs, due to the increase in its particle size, glomerular filtration can be effectively avoided, and rapid renal clearance can be avoided; at the same time, the negative charge on the surface of the nanoparticles can reduce the interaction with blood proteins, and ultimately the in vivo circulation time can be increased and the bioavailability can be improved. In addition, hyaluronic acid has anti-inflammatory and antioxidant activities. Combining with EGCG with pyroptosis inhibitory activity to form nanoparticles can achieve combined targeted therapy of inhibiting pyroptosis, anti-inflammation and antioxidant for sepsis, and improve the treatment effect of sepsis.

[0017] 2) Both EGCG and hyaluronic acid that make up the particles of the present invention are natural products, which can be obtained from natural sources and have excellent biocompatibility.

[0018] 3) The EGCG / HA-NH2 particles of the present invention are only composed of two components, EGCG and HA-NH2. The composition is simple and the preparation method is simple. And both components play a role in the treatment of the diseases to which they are applied, and no other components that do not play a therapeutic effect are introduced to prepare the particles, avoiding the in vivo metabolic burden and potential toxicity problems caused by the presence of redundant components after the particles are delivered to the target site in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0020] Figure 1Appearance and morphology diagram of the nanoparticles in the present invention; among them, (a) photograph of EGCG / HA-NH2 nanoparticles; (b) particle size distribution of EGCG / HA-NH2 nanoparticles. Figure 2 Zeta potential test diagram of EGCG / HA-NH2 nanoparticles in the present invention. Figure 3 After co-incubating different concentrations of EGCG / HA-NH2 nanoparticles with THP-1 cells and RAW 264.7 cells for 48 h in the present invention, CCK-8 was used to detect cell viability. Figure 4 Observation of the effect of EGCG / HA-NH2 nanoparticles in inhibiting pyroptosis of THP-1 cells induced by LPS and Nigericin for 5 h (scale bar = 200 μm) in the present invention. Figure 5 PI staining of THP-1 (a) and Raw264.7 (b) cells after treatment with EGCG / HA-NH2 nanoparticles in the present invention for 5 h to observe the effect of pyroptosis induced by LPS and Nigericin. Detailed description of specific embodiments

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are only descriptive and not restrictive, and the protection scope of the present invention cannot be limited thereby.

[0022] When expressing a certain quantity, concentration or other value or parameter in the form of a range, a preferred range, or a preferred upper limit and lower limit of a value, it should be understood that any range formed by combining any upper limit of the range or a preferred value with any lower limit of the range or a preferred value is specifically disclosed, regardless of whether the range is specifically disclosed. Unless otherwise indicated, the numerical range values listed herein include the endpoints of the range and all integers and fractions within the range.

[0023] Unless otherwise specified, all percentages, parts, ratios, etc. in this article are by weight.

[0024] The materials, methods and embodiments in this article are all exemplary and should not be construed as restrictive unless otherwise specified.

[0025] In the following embodiments, the main raw material EGCG was purchased from Aladdin, product number E107404; hyaluronic acid was purchased from Kerrhui, product number BR102812; and the rest of the reagents were all purchased from Aladdin.

[0026] The test methods adopted in the following embodiments include: By separately testing the main properties of each test sample, the successful preparation, biosafety, and in vitro therapeutic effect performance of EGCG / HA-NH2 nanoparticles are demonstrated; the main properties tested in this application include particle size and potential, cytotoxicity, intracellular inhibition of pyroptosis effect, etc.

[0027] 1) Particle size and potential test; Use a Malvern Zetasizer Nano device to detect the particle size and Zeta potential of the sample.

[0028] 2) Cytotoxicity test Perform a cytotoxicity test on the sample according to the CCK-8 method. The principle is that CCK-8 evaluates cell viability by detecting the dehydrogenase activity in the mitochondria of living cells: the dehydrogenase in living cells can reduce the water-soluble tetrazolium salt WST-8 to orange-yellow formazan, and the amount of its generation is positively correlated with the number of living cells. The cell viability can be quantified by measuring the absorbance at a wavelength of 450 nm. This method does not require cell lysis, is easy to operate, and has high sensitivity.

[0029] 3) Intracellular inhibition of pyroptosis effect test Use a NIKON TS2R inverted microscope and a NIKON Ti2A inverted fluorescence microscope to test the intracellular inhibition of pyroptosis effect of the sample by photographing the morphology of the cells after sample treatment after inducing pyroptosis and performing fluorescence staining.

[0030] Example 1 This example provides a preparation method of nanoparticles (EGCG / HA-NH2) based on EGCG and amino hyaluronic acid, which includes the following steps: 1) Preparation of amino hyaluronic acid (HA-NH2): Dissolve 200 mg of sodium hyaluronate (molecular weight 100000) in 60 mL of deionized water by magnetic stirring at 500 rpm, and add 30-fold molar equivalent of ethylenediamine hydrochloride (2.504 g); then add 540 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 540 mg of N-hydroxysuccinimide (NHS), and adjust the pH to 7 with 0.1 mol / L hydrochloric acid and sodium hydroxide; then stir magnetically at 500 rpm for 12 h, and dialyze the product with a 3000 Da dialysis bag in deionized water for 3 days, changing the water every 12 h; after freeze-drying the obtained HA-NH2 product, store it in a refrigerator at 4°C to obtain amino hyaluronic acid (HA-NH2); 2) Preparation of EGCG / HA-NH2: Dissolve 2 mg of EGCG and 1 mg of the prepared HA-NH2 in 1 mL of deionized water, add 0.4 μL of formaldehyde solution, and stir magnetically at 500 rpm for 2 h at room temperature; then centrifuge at 10000 rpm for 10 min. After removing the supernatant, wash by centrifugation with deionized water 3 times. The obtained EGCG / HA-NH2 product is freeze-dried to obtain nanoparticles based on EGCG and amino hyaluronic acid (EGCG / HA-NH2), which are stored in a refrigerator at 4 °C for later use; In this example, the photograph of the prepared nanoparticles based on EGCG and amino hyaluronic acid (EGCG / HA-NH2) after washing is shown in the appendix Figure 1 as shown in a. Compared with the raw material HA-NH2 and EGCG solutions, the reaction solution shows a white turbid state, indicating the formation of nanoparticles; Dissolve the freeze-dried EGCG / HA-NH2 nanoparticles in deionized water to a 1 mg / mL solution, and use Malvern, Zetasizer Nano ZS90 to detect the particle size and Zeta potential of the nanoparticles. The particle size test diagram of the nanoparticles is shown in Figure 1 b. It can be seen from the figure that the obtained nanoparticles have a particle size of 279.4 nm detected by a Malvern laser particle size analyzer; The test diagram of the Zeta potential of the nanoparticles is shown in Figure 2 as shown. It can be seen from the figure that the Zeta potential of the nanoparticles in this application is -27 mV, indicating that the surface of the prepared nanoparticles is negatively charged.

[0031] Example 2 This example verified the safety of EGCG / HA-NH2 nanoparticles, specifically: Materials and test methods: Using human monocytic leukemia cells (THP-1) and mouse mononuclear macrophage leukemia cells (RAW 264.7) as cell models, the cytotoxicity of EGCG / HA-NH2 nanoparticles was detected, specifically including the following steps: Inoculate THP-1 cells in a 96-well plate using RPMI-1640 complete medium. The medium contains 10 ng / mL phorbol myristate acetate (PMA) to induce cell adhesion for 24 h, with 10000 cells per well; Inoculate RAW264.7 cells in a 96-well plate using DMEM complete medium, with 10000 cells per well; The EGCG / HA-NH2 nanoparticles were dissolved in PBS at concentrations of 20, 50, 100, and 500 μg / mL. Different concentrations of EGCG / HA-NH2 nanoparticles were co-incubated with THP-1 and RAW 264.7 cells in an incubator at 37 °C for 48 h, and then CCK-8 dye was added for staining for 1.5 h. The cytotoxicity of EGCG / HA-NH2 nanoparticles in vitro cells at 48 h was detected by measuring cell viability using an enzyme-linked immunosorbent assay (ELISA) with CCK-8.

[0032] Results and Discussion: The results were as Figure 3 shown. As can be seen from the figure, after treatment with EGCG / HA-NH2 nanoparticles at 100 μg / mL and below, the activities of THP-1 and RAW264.7 cells did not decrease to 80%. According to the standard of ISO 10993-5-2009, a cell survival rate ≥ 70% (relative to the negative control group) is generally considered to have no cytotoxic reaction. Therefore, the EGCG / HA-NH2 nanoparticles in this application have no cytotoxicity and have good biocompatibility.

[0033] Example 3 This example tested that the EGCG / HA-NH2 nanoparticles can effectively inhibit pyroptosis, specifically as follows: Materials and Test Methods: Using THP-1 as a cell model, a pyroptotic cell model was induced and constructed using lipopolysaccharide (LPS) and nigericin to detect the inhibitory effect of EGCG / HA-NH2 nanoparticles on pyroptosis. The specific steps are as follows: THP-1 cells were seeded in a 24-well plate using RPMI-1640 complete medium containing 10 ng / mL phorbol 12-myristate 13-acetate (PMA) to induce cell adhesion for 24 h, with 200000 cells per well. First, the cells were stimulated with 1 μg / mL LPS for 3 h, and then a pyroptotic cell model was co-induced using 1 μg / mL LPS and 0.02 mM nigericin. At the same time, 100 μg / mL of EGCG / HA-NH2 nanoparticles dissolved in PBS was added, and the occurrence of cell pyroptosis was observed under a microscope at 0, 1, 3, and 5 h after incubation.

[0034] Results and Discussion: The results were as Figure 4As shown, it can be seen from the figure that within 0 - 5 h after the co - induction of pyroptosis by LPS and Nigericin, the cell morphology of the 100 μg / mL EGCG / HA - NH2 nanoparticle treatment group showed little difference from that of the uninduced cell group. However, in the group without treatment after induction, cell swelling and many bubble - like protrusions characteristic of pyroptosis could be observed. The results indicate that EGCG / HA - NH2 nanoparticles can effectively inhibit the pyroptosis induced by the combination of LPS and Nigericin.

[0035] Example 4 In this example, the inhibitory effect of the EGCG / HA - NH2 nanoparticles on pyroptosis was tested, specifically as follows: Using THP - 1 and RAW264.7 as cell models, a pyroptotic cell model was induced by lipopolysaccharide (LPS) and nigericin (Nigericin). After staining with propidium iodide (PI), the inhibitory effect of EGCG / HA - NH2 nanoparticles on pyroptosis was detected. The specific steps are as follows: THP - 1 cells were inoculated into 24 - well plates using RPMI - 1640 complete medium containing 10 ng / mL phorbol 12 - myristate 13 - acetate (PMA) to induce cell adhesion for 24 h, with 200,000 cells per well; RAW264.7 cells were inoculated into 96 - well plates using DMEM complete medium, with 200,000 cells per well.

[0036] First, cells were stimulated with 1 μg / mL LPS for 3 h, then a pyroptotic cell model was co - induced using 1 μg / mL LPS and 0.02 mM Nigericin, and 100 μg / mL EGCG / HA - NH2 nanoparticles dissolved in PBS were added simultaneously. At the 3rd h after incubation, 1.5 μL of 1.5 mM PI dye was added to each well, and after staining in a 37°C incubator for 20 min, pictures were taken using an inverted fluorescence microscope.

[0037] Results and Discussion: The results are as Figure 5As shown, it can be seen from the figure that since PI dye cannot penetrate intact cell membranes, and the cell membranes undergoing pyroptosis are ruptured, resulting in the penetration of PI dye through the cell membranes for staining. The occurrence of cell pyroptosis can be detected by measuring PI uptake. At 3 h after co-inducing cell pyroptosis with LPS and Nigericin, no excessive PI dye uptake was observed in the cells of the 100 μg / mL EGCG / HA-NH2 particle treatment group in both THP-1 and RAW264.7 cells, which was close to the level of the non-induced group. However, a large amount of PI dye uptake and staining were observed in the untreated group after induction. The results further indicated that EGCG / HA-NH2 nanoparticles could effectively inhibit LPS- and Nigericin-induced cell pyroptosis.

[0038] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered by the scope of the claims and the specification of the present invention.

Claims

1. A preparation method of nanoparticles based on EGCG and amino hyaluronic acid, characterized in that, It includes the following steps: 1) Dissolve EGCG and amino-functionalized hyaluronic acid in deionized water to obtain a mixed solution; 2) Add a formaldehyde solution to the mixed solution, mix evenly, and then carry out a stirring reaction to obtain a pre-product; 3) After centrifuging, washing, and drying the pre-product, nanoparticles based on EGCG and amino hyaluronic acid are obtained.

2. The preparation method of the nanoparticles based on EGCG and amino hyaluronic acid according to claim 1, characterized in that, In step 1), the mass concentration of EGCG is 0.5 - 10 mg / mL, the mass concentration of amino-functionalized hyaluronic acid is 0.5 - 10 mg / mL, and the mass ratio of EGCG to amino-functionalized hyaluronic acid is 2 - 6:

1.

3. The preparation method of the nanoparticles based on EGCG and amino hyaluronic acid according to claim 1, characterized in that, In step 2), the dosage of the formaldehyde solution is 0.1 - 0.5 μL / mL.

4. The preparation method of the nanoparticles based on EGCG and amino hyaluronic acid according to claim 1, wherein The process conditions of the stirring reaction in step 2) are: stirring reaction at a speed of 500 - 1000 rpm for 0.5 - 12 h at 25 - 37 °C.

5. The preparation method of the nanoparticles based on EGCG and amino hyaluronic acid according to claim 4, characterized in that, The process conditions of the centrifuging in step 3) are: centrifuging at a speed of 8000 - 12000 rpm for 5 - 15 min.

6. A nanoparticle based on EGCG and amino hyaluronic acid prepared by the preparation method according to any one of claims 1-5, characterized in that, The particle size of the nanoparticles is 279.4 nm, and the surface of the nanoparticles is negatively charged.

7. Application of nanoparticles based on EGCG and amino hyaluronic acid in the preparation of drugs for treating sepsis.

8. The application according to claim 7, wherein The nanoparticles based on EGCG and amino hyaluronic acid can inhibit pyroptosis.

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