Carrier-free nano-particles with pH / ROS dual response as well as preparation method and application of carrier-free nano-particles

Abstract

The invention discloses a carrier-free nano-particle with pH / ROS dual response as well as a preparation method and application of the carrier-free nano-particle. The preparation method comprises the following steps: enabling quinolizidine alkaloid and flavonol to be subjected to self-assembly, so as to prepare quinolizidine alkaloid-flavonol carrier-free nano particles; modifying the quinolizidine alkaloid-flavonol carrier-free nano particles by adopting a phenylboronic acid-hyaluronic acid conjugate, so as to prepare nano particles modified by the phenylboronic acid-hyaluronic acid conjugate; and coating the surfaces of the nano-particles modified by the phenylboronic acid-hyaluronic acid conjugate with a pH sensitive polymer to prepare the pH / ROS dual-response carrier-free nano-particles. The sophocarpidine-kaempferol carrier-free nanoparticles with pH / ROS dual response, prepared by the invention, have the advantages of good targeting property, high biocompatibility, small toxicity and adverse reaction and the like, meanwhile, the nanoparticles have a good anti-inflammatory effect, and a new choice is provided for the treatment of ulcerative colitis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to carrier-free nanoparticles with pH / ROS dual response, and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory intestinal disease that primarily affects the rectum and colon, with lesions presenting a continuous, diffuse distribution. Its pathogenesis is complex, involving multiple factors, including immune imbalance, intestinal microbiome dysbiosis, environmental factors, and genetic susceptibility. Currently, the main drugs used clinically to treat UC include aminosalicylic acids, glucocorticoids, immunosuppressants, and biologics. However, these drugs have limited efficacy, significant side effects, and the tendency to develop drug resistance with long-term use.

[0003] Matrine is derived from the leguminous plant Sophora flavescens and is an alkaloid. Kaempferol is a natural flavonoid compound that is widely found in fruits, vegetables, cereals and other plants. Matrine and kaempferol are two natural compounds with significant pharmacological activities, with anti-inflammatory, anti-tumor and antioxidant effects respectively. However, their low bioavailability, poor stability and easy degradation in the gastrointestinal tract limit their clinical application. Traditional nanocarriers such as liposomes, microcapsules, nanoemulsions, polymer nanoparticles, etc. have problems such as complex preparation process, high cost and toxic side effects caused by lack of target specificity.

[0004] In summary, the development of a simple, efficient, safe and responsive matrine-kaempferol nanodelivery system is of great significance for improving the therapeutic effect of ulcerative colitis. Summary of the Invention

[0005] The main purpose of the present invention is to provide a carrier-free nanoparticle with pH / ROS dual response and its preparation method and application, so as to overcome the shortcomings of the existing technology.

[0006] To achieve the aforementioned object of the invention, the technical solutions adopted by the present invention include:

[0007] The present invention provides a method for preparing carrier-free nanoparticles with pH / ROS dual response, which comprises:

[0008] Quinolizidine alkaloids and flavonols are self-assembled to prepare quinolizidine alkaloid-flavonol carrier-free nanoparticles;

[0009] The quinolizidine alkaloid-flavonol carrier-free nanoparticles are modified with a phenylboronic acid-hyaluronic acid conjugate to prepare phenylboronic acid-hyaluronic acid conjugate-modified nanoparticles;

[0010] Furthermore, a pH-sensitive polymer is coated on the surface of the nanoparticles modified with the phenylboronic acid-hyaluronic acid conjugate to prepare carrier-free nanoparticles with pH / ROS dual response.

[0011] The present invention also provides carrier-free nanoparticles with pH / ROS dual response obtained by the aforementioned preparation method, which include: quinolizidine alkaloid-flavonol carrier-free nanoparticles as the core, phenylboronic acid-hyaluronic acid conjugates modified on the surface of quinolizidine alkaloid-flavonol carrier-free nanoparticles, and a pH-sensitive polymer coated on the outer layer.

[0012] The embodiments of the present invention also provide the use of the aforementioned carrier-free nanoparticles with pH / ROS dual response in the preparation of drugs for preventing and / or treating intestinal diseases.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: the matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response prepared by the present invention have the advantages of good targeting, high biocompatibility, low toxicity and adverse reactions, etc. At the same time, the matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response exhibit good anti-inflammatory effects, providing a new option for the treatment of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 This is a transmission electron micrograph of matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response prepared in Example 5 of the present invention;

[0016] Figure 2 The cytotoxicity graphs of the samples prepared in Example 5 and Comparative Example 1 of the present invention are shown;

[0017] Figure 3a-Figure 3b Graph showing pH / ROS response test results of the nanoparticles of Example 5 and Comparative Examples 1-3 of the present invention;

[0018] Figure 4 This is a comparison chart of the effects of the samples prepared in Example 5 of the present invention and Comparative Example 1 on the colon length of mice;

[0019] Figure 5a-5fThis is a graph showing the effects of the samples prepared in Example 5 of the present invention and Comparative Example 1 on the levels of Lcn-2, MPO, TNF-α, IL-6, IL-10 and MCP-1 inflammatory factors in mice induced by DSS. DETAILED DESCRIPTION

[0020] In view of the shortcomings of the prior art, the inventors of this case, after long-term research and extensive practice, have proposed the technical solution of the present invention. The technical solution of the present invention will be clearly and completely described below. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making any creative effort shall fall within the scope of protection of the present invention.

[0021] Specifically, as one aspect of the technical solution of the present invention, a method for preparing carrier-free nanoparticles with pH / ROS dual response involves:

[0022] Quinolizidine alkaloids and flavonols are self-assembled to prepare quinolizidine alkaloid-flavonol carrier-free nanoparticles;

[0023] The quinolizidine alkaloid-flavonol carrier-free nanoparticles are modified with a phenylboronic acid-hyaluronic acid conjugate to prepare phenylboronic acid-hyaluronic acid conjugate-modified nanoparticles;

[0024] Furthermore, a pH-sensitive polymer is coated on the surface of the nanoparticles modified with the phenylboronic acid-hyaluronic acid conjugate to prepare carrier-free nanoparticles with pH / ROS dual response.

[0025] In some preferred embodiments, the preparation method specifically comprises:

[0026] dissolving quinolizidine alkaloids and flavonols in methanol respectively to form a quinolizidine alkaloid methanol solution and a flavonol methanol solution, and adjusting the pH value of the flavonol methanol solution to 6.8-7.0 with a pH regulator;

[0027] Furthermore, the quinolizidine alkaloid methanol solution and the flavonol methanol solution are mixed, and phosphate buffer is added and stirred at 50-70° C. for 2-4 hours, followed by dialysis, centrifugation, washing, and vacuum drying to obtain quinolizidine alkaloid-flavonol carrier-free nanoparticles.

[0028] Furthermore, the quinolizidine alkaloids include matrine and / or sophoridine, but are not limited thereto.

[0029] Furthermore, the flavonols include any one or more combinations of kaempferol, quercetin, and myricetin, but are not limited thereto.

[0030] Furthermore, the molar ratio of the quinolizidine alkaloids to the flavonols is 1:3 to 3:1.

[0031] Furthermore, the pH adjuster includes an organic base and / or an inorganic base, but is not limited thereto.

[0032] Furthermore, the pH adjuster includes any one or more combinations of sodium hydroxide, sodium carbonate, sodium bicarbonate, and potassium dihydrogen phosphate, but is not limited thereto.

[0033] Furthermore, the pH regulator includes a sodium hydroxide solution with a concentration of 4-8 mg / mL.

[0034] Furthermore, the molecular weight cut-off of the dialysis bag used for the dialysis is 1.0-3.5 kDa, and the dialysis time is 24-72 h.

[0035] Furthermore, the centrifugal speed used in the centrifugation is 8000-12000 rpm, and the centrifugal time is 10-20 min.

[0036] In some preferred embodiments, the preparation method specifically includes: mixing quinolizidine alkaloid-flavonol carrier-free nanoparticles with a phenylboronic acid-hyaluronic acid conjugate solution, adding a CaCl2 solution and reacting at 25-37°C for 20-60 minutes, and then centrifuging to obtain phenylboronic acid-hyaluronic acid conjugate-modified nanoparticles.

[0037] Furthermore, the preparation method of the phenylboronic acid-hyaluronic acid conjugate comprises:

[0038] Dissolve hyaluronic acid in water, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and stir at room temperature for 1-3 hours to obtain a hyaluronic acid solution;

[0039] 3-Aminophenylboronic acid was dissolved in DMF, and then added dropwise to the hyaluronic acid solution and the pH value was adjusted to 5.0-6.8. The mixture was then reacted at 25-37° C. for 24-48 hours. Finally, the mixture was purified by dialysis and freeze-dried to obtain a phenylboronic acid-hyaluronic acid conjugate.

[0040] Furthermore, the mass volume concentration of the phenylboronic acid-hyaluronic acid conjugate solution is 0.5-1.5 (w / v, gdmL)%.

[0041] Furthermore, the concentration of the CaCl2 solution is 0.05-0.15 mmol / L.

[0042] Furthermore, the centrifugal speed used in the centrifugation is 8000-12000 rpm, and the centrifugal time is 10-30 min.

[0043] In some preferred embodiments, the preparation method specifically includes: dispersing the phenylboronic acid-hyaluronic acid conjugate modified nanoparticles in an ethanol solution of a pH-sensitive polymer, stirring at 25-37°C for 1-3 hours, and then spray drying to obtain carrier-free nanoparticles with pH / ROS dual response.

[0044] Furthermore, the pH sensitive polymer includes any one or more combinations of Eudragit S100, pectin, sodium alginate, and carboxymethyl chitosan, but is not limited thereto.

[0045] Furthermore, the mass volume concentration of the pH sensitive polymer ethanol solution is 1-3 (w / v, g / mL)%.

[0046] Furthermore, the inlet temperature of the spray drying is 80-120°C, and the outlet temperature is 40-60°C.

[0047] In some more specific embodiments, the method for producing matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response comprises the following steps:

[0048] (1) Weighing appropriate amounts of matrine and kaempferol, respectively, and dissolving them in appropriate amounts of methanol solution, adjusting the pH value of the methanol solution of kaempferol to 6.8-7.0, and stirring the two solutions, slowly adding them to phosphate buffer, and reacting them under constant temperature and stirring for 2-4 hours to facilitate self-assembly of matrine and kaempferol to obtain a mixed solution;

[0049] (2) placing the mixed solution of step (1) in a dialysis bag and dialyzing with ultrapure water to remove unassembled matrine, kaempferol and organic solvent, changing the ultrapure water every 4 hours to obtain a self-assembled solution of matrine and kaempferol;

[0050] (3) centrifuging the matrine and kaempferol self-assembly solution prepared in step (2), collecting the precipitate, washing it three times with ultrapure water, and freeze-drying it in a vacuum to obtain matrine-kaempferol carrier-free nanoparticles. The matrine-kaempferol carrier-free nanoparticles are mainly maintained by electrostatic interaction, π-π stacking, and hydrogen bonding.

[0051] (4) Synthesis of phenylboronic acid-hyaluronic acid (PBA-HA) conjugate: Hyaluronic acid (HA, 1 g) was dissolved in 50 mL of deionized water, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.5 mmol) and N-hydroxysuccinimide (NHS, 0.5 mmol) were added. The mixture was stirred at room temperature for 2 h to activate the carboxylic acid group. 3-Aminophenylboronic acid (PBA-NH2, 0.8 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF) and slowly added dropwise to the HA solution. The pH was adjusted to 5.5. After 24 h of reaction, the mixture was purified by dialysis (MWCO 3.5 kDa, deionized water, 48 h) and freeze-dried to obtain the PBA-HA conjugate.

[0052] (5) The matrine-kaempferol carrier-free nanoparticles prepared in step (3) were mixed with the PBA-HA conjugate solution, CaCl2 was added, magnetic stirring was performed for 30 minutes, and the unadsorbed PBA-HA was removed by centrifugation (10000 rpm, 20 minutes) to obtain PBA-HA modified nanoparticles; the PBA-HA modified nanoparticles were dispersed in Eudragit S100 ethanol solution, magnetic stirring was performed for 1 hour, and spray drying was performed to form an outer layer coating structure to obtain matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response.

[0053] Preferably, the molar ratio of matrine to kaempferol in step (1) is 1:3, 1:2, 1:1, 2:1 or 3:1, and the pH of the phosphate buffer solution is 6.8 and the temperature is 60°C.

[0054] Preferably, the pH regulator in step (1) is an organic or inorganic base, such as sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium dihydrogen phosphate, etc., and a further preferred pH regulator is a 5 mg / mL sodium hydroxide solution.

[0055] Preferably, the molecular weight cut-off of the dialysis bag in step (2) is 3.5 kDa, and the dialysis time is 24 h.

[0056] Preferably, the centrifugal speed in step (3) is 10000 rpm, and the centrifugal time is 10-20 min, for example, 10 min, 15 min, 20 min.

[0057] Preferably, the matrine-kaempferol carrier-free nanoparticles in step (3) are mainly maintained by electrostatic interaction, π-π stacking and hydrogen bonding.

[0058] Preferably, the concentration of the PBA-HA solution in step (5) is 0.5%-1.5% (w / v, g / mL), and the concentration of the calcium chloride solution is 0.05-0.15 mM.

[0059] Preferably, the concentration of the Eudragit S100 ethanol solution in step (5) is 1-3% (w / v, gdmL), and the spray drying conditions are an inlet temperature of 100°C and an outlet temperature of 50°C.

[0060] This method utilizes matrine and kaempferol to self-assemble into carrier-free nanoparticles, a method that is environmentally friendly, simple, economical, and highly safe. Furthermore, the resulting nanoparticles are surface-modified with PBA-HA and coated with Eudragit S100, making them dual-responsive to pH and ROS, enhancing their therapeutic efficacy for inflammatory bowel disease.

[0061] Another aspect of the embodiments of the present invention further provides carrier-free nanoparticles with pH / ROS dual response obtained by the aforementioned preparation method, which include: quinolizidine alkaloid-flavonol carrier-free nanoparticles as the core, phenylboronic acid-hyaluronic acid conjugates modified on the surface of quinolizidine alkaloid-flavonol carrier-free nanoparticles, and a pH-sensitive polymer coated on the outer layer.

[0062] The carrier-free nanoparticles with pH / ROS dual response in the present invention are self-assembled by matrine and kaempferol through intermolecular forces to form a carrier-free core, and the surface is modified by phenylboronic acid-hyaluronic acid (PBA-HA) and coated with Eudragit S100, and are designed to have pH / ROS dual response and targeted delivery characteristics. Among them, the pH response is achieved by the dissolution of the outer layer Eudragit S100 in the neutral environment of the colon (pH ≥ 7.0) to achieve localized release, and the ROS response depends on the breakage of the PBA ester bond in a highly reactive oxygen (H2O2) environment to trigger the precise release of the drug; hyaluronic acid (HA) targets the CD44 receptor at the inflammatory site, and phenylboronic acid (PBA) binds to intestinal mucosal mucin, and the dual mechanism significantly improves the lesion enrichment efficiency. In addition, the nanoparticles can significantly improve the therapeutic effect of ulcerative colitis by regulating the immune microenvironment, reducing proinflammatory factors and repairing the integrity of the intestinal barrier. Its efficacy is better than that of the commonly used clinical drug mesalazine (5-ASA). The nanoparticles prepared by the present invention have the characteristics of uniform particle size, good dispersibility, high stability, excellent biocompatibility and high safety, providing new options and important technical support for the development of carrier-free nanoparticle delivery systems and the efficient treatment of ulcerative colitis.

[0063] Another aspect of the embodiments of the present invention further provides the use of the aforementioned carrier-free nanoparticles with pH / ROS dual response in the preparation of drugs for preventing and / or treating intestinal diseases.

[0064] Furthermore, the intestinal disease is inflammatory bowel disease, which includes ulcerative colitis.

[0065] The present invention discloses a carrier-free nanoparticle with pH / ROS dual response, matrine-kaempferol, which has a therapeutic effect on ulcerative colitis. The carrier-free nanoparticle can show a good therapeutic effect on ulcerative colitis by balancing the intestinal barrier and the immune microenvironment, and has good safety and biocompatibility, and has great potential in clinical application.

[0066] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and the accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0067] The experimental materials used in the following examples, unless otherwise specified, can be purchased from conventional biochemical reagent companies.

[0068] Example 1 Screening of the Ratio of Matrine-Kaempferol Carrier-Free Nanoparticles

[0069] Matrine-kaempferol carrier-free nanoparticles were prepared according to the following method using matrine-kaempferol molar ratios of 1:3, 1:2, 1:1, 2:1, and 3:1 as treatment groups. The particle size, ζ-potential, and polydispersity index (PDI) of the matrine-kaempferol carrier-free nanoparticles at different ratios were measured to evaluate the stability and uniformity of the nanoparticles (see Table 1).

[0070] Preparation method: (1) Weigh matrine and kaempferol in different molar ratios (1:3, 1:2, 1:1, 2:1, 3:1) and dissolve them in an appropriate amount of methanol solution, adjust the pH value of the kaempferol solution to 6.8-7.0 using 5 mg / mL sodium hydroxide solution, then mix the matrine solution and the kaempferol solution and stir them evenly, slowly add them to 10 mL of 60°C phosphate buffer (pH 6.8), and react under constant temperature stirring for 2-4 hours to facilitate self-assembly of matrine and kaempferol to obtain a matrine-kaempferol mixed solution;

[0071] (2) placing the mixed solution of step (1) into a dialysis bag (MWCO = 3.5 kDa) and dialyzing it with ultrapure water for 24 h to remove unassembled matrine, kaempferol, and organic solvent, replacing the ultrapure water every 4 h to obtain a solution of matrine and kaempferol;

[0072] (3) centrifuging the matrine and kaempferol self-assembly solution prepared in step (2), collecting the precipitate, washing it three times with ultrapure water, and freeze-drying it in a vacuum to obtain matrine-kaempferol carrier-free nanoparticles.

[0073] Table 1 Particle size, ζ-potential and PDI of nanoparticles formed by different molar ratios of matrine and kaempferol

[0074]

[0075] As shown in Table 1, the molar ratio of matrine to kaempferol affects the particle size, potential, and polydispersity index of the unsupported nanoparticles. When the molar ratio of matrine to kaempferol was 1:1, the resulting nanoparticles had the smallest particle size of 267.7 ± 23.6 nm, demonstrating enhanced permeability and retention at the inflamed site of ulcerative colitis. Nanoparticles in the 100-500 nm range were better able to utilize the retention effect and target inflamed tissue. Nanoparticles formed at other molar ratios all had a particle size range of 680-930 nm, which is generally larger, making them more susceptible to capture by the immune system or less able to penetrate inflamed tissue. Furthermore, the zeta potential and polydispersity index (PDI) also reflect the stability and uniformity of the nanoparticle system. A zeta potential greater than or equal to 30 mV and a polydispersity index less than or equal to 0.3 indicate a stable and uniform system, conditions met when the molar ratio of matrine to kaempferol was 1:1. Therefore, a molar ratio of matrine to kaempferol of 1:1 was selected as the optimal ratio for preparing matrine-kaempferol carrier-free nanoparticles.

[0076] Example 2 Preparation of matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response

[0077] 2.1 Preparation of matrine-kaempferol carrier-free nanoparticles

[0078] (1) Matrine and kaempferol in a molar ratio of 1:1 were weighed and dissolved in an appropriate amount of methanol solution, and the pH value of the kaempferol solution was adjusted to 6.8-7.0 using a 5 mg / mL sodium hydroxide solution. The matrine solution and the kaempferol solution were then mixed and stirred evenly, and then slowly added to 10 mL of 60°C phosphate buffer (pH 6.8). The mixture was reacted under constant temperature stirring for 2-4 hours to facilitate self-assembly of matrine and kaempferol, thereby obtaining a matrine-kaempferol mixed solution;

[0079] (2) placing the mixed solution of step (1) into a dialysis bag (MWCO = 3.5 kDa) and dialyzing it with ultrapure water for 24 h to remove unassembled matrine, kaempferol, and organic solvent, replacing the ultrapure water every 4 h to obtain a solution of matrine and kaempferol;

[0080] (3) centrifuging the matrine and kaempferol self-assembly solution prepared in step (2), collecting the precipitate, washing it three times with ultrapure water, and freeze-drying it in a vacuum to obtain matrine-kaempferol carrier-free nanoparticles.

[0081] 2.2 Preparation of carrier-free matrine-kaempferol nanoparticles with pH / ROS dual response

[0082] Synthesis of Phenylboronic Acid-Hyaluronic Acid (PBA-HA) Conjugate: Dissolve hyaluronic acid (HA, 1 g) in 50 mL of deionized water. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.5 mmol) and N-hydroxysuccinimide (NHS, 0.5 mmol). Stir at room temperature for 2 h to activate the carboxylic acid groups. Dissolve 3-aminophenylboronic acid (PBA-NH2, 0.8 mmol) in 10 mL of N,N-dimethylformamide (DMF) and slowly add dropwise to the HA solution. Adjust the pH to 5.5. After reacting for 24 h, dialysis purification (MWCO 3.5 kDa, deionized water, 48 h) and freeze-drying to obtain the PBA-HA conjugate.

[0083] The carrier-free nanoparticles prepared in step (3) were mixed with a 1% (w / v) PBA-HA solution, 0.15 mM CaCl2 was added, and magnetic stirring was performed for 30 minutes. The unadsorbed PBA-HA was removed by centrifugation (10,000 rpm, 20 minutes) to obtain PBA-HA-modified nanoparticles. The modified particles were dispersed in a 2% (w / v) Eudragit S100 ethanol solution and magnetic stirring was performed for 1 hour. Spray drying (inlet temperature 100°C, outlet temperature 50°C) was performed to form an outer layer coating structure, and matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response were obtained.

[0084] Example 3 Preparation of matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response

[0085] 3.1 Preparation of matrine-kaempferol carrier-free nanoparticles

[0086] (1) Matrine and kaempferol in a molar ratio of 1:1 were weighed and dissolved in an appropriate amount of methanol solution, and the pH value of the kaempferol solution was adjusted to 6.8-7.0 using a 5 mg / mL sodium hydroxide solution. The matrine solution and the kaempferol solution were then mixed and stirred evenly, and then slowly added to 10 mL of 60°C phosphate buffer (pH 6.8). The mixture was reacted under constant temperature stirring for 2-4 hours to facilitate self-assembly of matrine and kaempferol, thereby obtaining a matrine-kaempferol mixed solution;

[0087] (2) placing the mixed solution of step (1) into a dialysis bag (MWCO = 3.5 kDa) and dialyzing it with ultrapure water for 24 h to remove unassembled matrine, kaempferol, and organic solvent, replacing the ultrapure water every 4 h to obtain a solution of matrine and kaempferol;

[0088] (3) Centrifuging the matrine and kaempferol white assembly solution prepared in step (2), collecting the precipitate, washing it three times with ultrapure water, and freeze-drying it in a vacuum to obtain matrine-kaempferol carrier-free nanoparticles.

[0089] 3.2 Preparation of carrier-free matrine-kaempferol nanoparticles with pH / ROS dual response

[0090] Synthesis of Phenylboronic Acid-Hyaluronic Acid (PBA-HA) Conjugate: Dissolve hyaluronic acid (HA, 1 g) in 50 mL of deionized water. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.5 mmol) and N-hydroxysuccinimide (NHS, 0.5 mmol). Stir at room temperature for 2 h to activate the carboxylic acid group. Dissolve 3-aminophenylboronic acid (PBA-NH2, 0.8 mmol) in 10 mL of N,N-dimethylformamide (DMF) and slowly add dropwise to the HA solution. Adjust the pH to 5.5. After reacting for 24 h, dialysis purification (MWCO 3.5 kDa, deionized water, 48 h) and freeze-drying to obtain the PBA-HA conjugate.

[0091] The carrier-free nanoparticles prepared in step (3) were mixed with a 1.5% (w / v) PBA-HA solution, 0.1 mM CaCl2 was added, and magnetic stirring was performed for 30 minutes. The unadsorbed PBA-HA was removed by centrifugation (10,000 rpm, 20 minutes) to obtain PBA-HA-modified nanoparticles. The modified particles were dispersed in a 1% (w / v) Eudragit S100 ethanol solution and magnetic stirring was performed for 1 hour. Spray drying (inlet temperature 100°C, outlet temperature 50°C) was performed to form an outer layer coating structure, and matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response were obtained.

[0092] Example 4 Preparation of matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response

[0093] 4.1 Preparation of matrine-kaempferol carrier-free nanoparticles

[0094] (1) Matrine and kaempferol in a molar ratio of 1:1 were weighed and dissolved in an appropriate amount of methanol solution, and the pH value of the kaempferol solution was adjusted to 6.8-7.0 using a 5 mg / mL sodium hydroxide solution. The matrine solution and the kaempferol solution were then mixed and stirred evenly, and then slowly added to 10 mL of 60°C phosphate buffer (pH 6.8). The mixture was reacted under constant temperature stirring for 2-4 hours to facilitate self-assembly of matrine and kaempferol, thereby obtaining a matrine-kaempferol mixed solution;

[0095] (2) placing the mixed solution of step (1) into a dialysis bag (MWCO = 3.5 kDa) and dialyzing it with ultrapure water for 24 h to remove unassembled matrine, kaempferol, and organic solvent, replacing the ultrapure water every 4 h to obtain a solution of matrine and kaempferol;

[0096] (3) centrifuging the matrine and kaempferol self-assembly solution prepared in step (2), collecting the precipitate, washing it three times with ultrapure water, and freeze-drying it in a vacuum to obtain matrine-kaempferol carrier-free nanoparticles.

[0097] 4.2 Preparation of matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response

[0098] Synthesis of Phenylboronic Acid-Hyaluronic Acid (PBA-HA) Conjugate: Dissolve hyaluronic acid (HA, 1 g) in 50 mL of deionized water. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.5 mmol) and N-hydroxysuccinimide (NHS, 0.5 mmol). Stir at room temperature for 2 h to activate the carboxylic acid groups. Dissolve 3-aminophenylboronic acid (PBA-NH2, 0.8 mmol) in 10 mL of N,N-dimethylformamide (DMF) and slowly add dropwise to the HA solution. Adjust the pH to 5.5. After reacting for 24 h, dialysis purification (MWCO 3.5 kDa, deionized water, 48 h) and freeze-drying to obtain the PBA-HA conjugate.

[0099] The carrier-free nanoparticles prepared in step (3) were mixed with a 1% (w / v) PBA-HA solution, 0.05 mM CaCl2 was added, and magnetic stirring was performed for 30 min. The unadsorbed PBA-HA was removed by centrifugation (10,000 rpm, 20 min) to obtain PBA-HA-modified nanoparticles. The modified particles were dispersed in a 3% (w / v) Eudragit S100 ethanol solution and magnetic stirring was performed for 1 h. Spray drying (inlet temperature 100°C, outlet temperature 50°C) was performed to form an outer layer coating structure, and matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response were obtained.

[0100] Example 5 Preparation of matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response

[0101] 5.1 Preparation of matrine-kaempferol carrier-free nanoparticles

[0102] (1) Matrine and kaempferol at a molar ratio of 1:1 were weighed and dissolved in an appropriate amount of methanol solution, and the pH value of the kaempferol solution was adjusted to 6.8-7.0 using a 5 mg / mL sodium hydroxide solution. The matrine solution and the kaempferol solution were then mixed and stirred evenly, and then slowly added to 10 mL of 60°C phosphate buffer (pH 6.8). The mixture was reacted under constant temperature stirring for 2-4 hours to facilitate self-assembly of matrine and kaempferol, thereby obtaining a matrine-kaempferol mixed solution;

[0103] (2) placing the mixed solution of step (1) into a dialysis bag (MWCO = 3.5 kDa) and dialyzing it with ultrapure water for 24 h to remove unassembled matrine, kaempferol, and organic solvent, replacing the ultrapure water every 4 h to obtain a solution of matrine and kaempferol;

[0104] (3) centrifuging the matrine and kaempferol self-assembly solution prepared in step (2), collecting the precipitate, washing it three times with ultrapure water, and freeze-drying it in a vacuum to obtain matrine-kaempferol carrier-free nanoparticles.

[0105] 5.2 Preparation of matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response

[0106] Synthesis of Phenylboronic Acid-Hyaluronic Acid (PBA-HA) Conjugate: Dissolve hyaluronic acid (HA, 1 g) in 50 mL of deionized water. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.5 mmol) and N-hydroxysuccinimide (NHS, 0.5 mmol). Stir at room temperature for 2 h to activate the carboxylic acid group. Dissolve 3-aminophenylboronic acid (PBA-NH2, 0.8 mmol) in 10 mL of N,N-dimethylformamide (DMF) and slowly add dropwise to the HA solution. Adjust the pH to 5.5. After reacting for 24 h, dialysis purification (MWCO 3.5 kDa, deionized water, 48 h) and freeze-drying to obtain the PBA-HA conjugate.

[0107] The carrier-free nanoparticles prepared in step (3) were mixed with 1% (w / v) PBA-HA solution, 0.1 mM CaCl2 was added, and magnetic stirring was performed for 30 min. The non-adsorbed PBA-HA was removed by centrifugation (10,000 rpm, 20 min) to obtain PBA-HA modified nanoparticles. The modified particles were dispersed in 2% (w / v) Eudragit S100 ethanol solution and magnetic stirring was performed for 1 h. Spray drying (inlet temperature 100 ° C, outlet temperature 50 ° C) was performed to form an outer layer coating structure to obtain matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response. The morphology under transmission electron microscopy is shown as follows: Figure 1 .

[0108] Example 6 Preparation of matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response

[0109] 6.1 Preparation of matrine-kaempferol carrier-free nanoparticles

[0110] (1) Matrine and kaempferol in a molar ratio of 1:1 were weighed and dissolved in an appropriate amount of methanol solution, and the pH value of the kaempferol solution was adjusted to 6.8-7.0 using a 5 mg / mL sodium hydroxide solution. The matrine solution and the kaempferol solution were then mixed and stirred evenly, and then slowly added to 10 mL of 60°C phosphate buffer (pH 6.8). The mixture was reacted under constant temperature stirring for 2-4 hours to facilitate self-assembly of matrine and kaempferol, thereby obtaining a matrine-kaempferol mixed solution;

[0111] (2) placing the mixed solution of step (1) into a dialysis bag (MWCO = 3.5 kDa) and dialyzing it with ultrapure water for 24 h to remove unassembled matrine, kaempferol, and organic solvent, replacing the ultrapure water every 4 h to obtain a solution of matrine and kaempferol;

[0112] (3) centrifuging the matrine and kaempferol self-assembly solution prepared in step (2), collecting the precipitate, washing it three times with ultrapure water, and freeze-drying it in a vacuum to obtain matrine-kaempferol carrier-free nanoparticles.

[0113] 6.2 Preparation of carrier-free matrine-kaempferol nanoparticles with pH / ROS dual response

[0114] Synthesis of Phenylboronic Acid-Hyaluronic Acid (PBA-HA) Conjugate: Dissolve hyaluronic acid (HA, 1 g) in 50 mL of deionized water. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.5 mmol) and N-hydroxysuccinimide (NHS, 0.5 mmol). Stir at room temperature for 2 h to activate the carboxylic acid group. Dissolve 3-aminophenylboronic acid (PBA-NH2, 0.8 mmol) in 10 mL of N,N-dimethylformamide (DMF) and slowly add dropwise to the HA solution. Adjust the pH to 5.5. After reacting for 24 h, dialysis purification (MWCO 3.5 kDa, deionized water, 48 h) and freeze-drying to obtain the PBA-HA conjugate.

[0115] The carrier-free nanoparticles prepared in step (3) were mixed with a 0.5% (w / v) PBA-HA solution, 0.1 mM CaCl2 was added, and magnetic stirring was performed for 30 min. The unadsorbed PBA-HA was removed by centrifugation (10,000 rpm, 20 min) to obtain PBA-HA-modified nanoparticles. The modified particles were dispersed in a 3% (w / v) Eudragit S100 ethanol solution and magnetic stirring was performed for 1 h. Spray drying (inlet temperature 100°C, outlet temperature 50°C) was performed to form an outer layer coating structure, and matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response were obtained.

[0116] Example 7 Preparation of matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response

[0117] 7.1 Preparation of matrine-kaempferol carrier-free nanoparticles

[0118] (1) Matrine and kaempferol in a molar ratio of 1:1 were weighed and dissolved in an appropriate amount of methanol solution, and the pH value of the kaempferol solution was adjusted to 6.8-7.0 using a 5 mg / mL sodium hydroxide solution. The matrine solution and the kaempferol solution were then mixed and stirred evenly, and then slowly added to 10 mL of 60°C phosphate buffer (pH 6.8). The mixture was reacted under constant temperature stirring for 2-4 hours to facilitate self-assembly of matrine and kaempferol, thereby obtaining a matrine-kaempferol mixed solution;

[0119] (2) placing the mixed solution of step (1) into a dialysis bag (MWCO = 3.5 kDa) and dialyzing it with ultrapure water for 24 h to remove unassembled matrine, kaempferol, and organic solvent, replacing the ultrapure water every 4 h to obtain a solution of matrine and kaempferol;

[0120] (3) centrifuging the matrine and kaempferol self-assembly solution prepared in step (2), collecting the precipitate, washing it three times with ultrapure water, and freeze-drying it in a vacuum to obtain matrine-kaempferol carrier-free nanoparticles.

[0121] 7.2 Preparation of carrier-free matrine-kaempferol nanoparticles with pH / ROS dual response

[0122] Synthesis of Phenylboronic Acid-Hyaluronic Acid (PBA-HA) Conjugate: Dissolve hyaluronic acid (HA, 1 g) in 50 mL of deionized water. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.5 mmol) and N-hydroxysuccinimide (NHS, 0.5 mmol). Stir at room temperature for 2 h to activate the carboxylic acid group. Dissolve 3-aminophenylboronic acid (PBA-NH2, 0.8 mmol) in 10 mL of N,N-dimethylformamide (DMF) and slowly add dropwise to the HA solution. Adjust the pH to 5.5. After reacting for 24 h, dialysis purification (MWCO 3.5 kDa, deionized water, 48 h) and freeze-drying to obtain the PBA-HA conjugate.

[0123] The carrier-free nanoparticles prepared in step (3) were mixed with a 1% (w / v) PBA-HA solution, 0.1 mM CaCl2 was added, and magnetic stirring was performed for 30 minutes. The unadsorbed PBA-HA was removed by centrifugation (10,000 rpm, 20 minutes) to obtain PBA-HA-modified nanoparticles. The modified particles were dispersed in a 1% (w / v) Eudragit S100 ethanol solution and magnetic stirring was performed for 1 hour. Spray drying (inlet temperature 100°C, outlet temperature 50°C) was performed to form an outer layer coating structure, and matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response were obtained.

[0124] Comparative Example 1

[0125] Preparation of matrine-kaempferol carrier-free nanoparticles

[0126] (1) Matrine and kaempferol in a molar ratio of 1:1 were weighed and dissolved in an appropriate amount of methanol solution, and the pH value of the kaempferol solution was adjusted to 6.8-7.0 using a 5 mg / mL sodium hydroxide solution. The matrine solution and the kaempferol solution were then mixed and stirred evenly, and then slowly added to 10 mL of 60°C phosphate buffer (pH 6.8). The mixture was reacted under constant temperature stirring for 2-4 hours to facilitate self-assembly of matrine and kaempferol, thereby obtaining a matrine-kaempferol mixed solution;

[0127] (2) placing the mixed solution of step (1) into a dialysis bag (MWCO = 3.5 kDa) and dialyzing it with ultrapure water for 24 h to remove unassembled matrine, kaempferol, and organic solvent, replacing the ultrapure water every 4 h to obtain a solution of matrine and kaempferol;

[0128] (3) centrifuging the matrine and kaempferol self-assembly solution prepared in step (2), collecting the precipitate, washing it three times with ultrapure water, and freeze-drying it in a vacuum to obtain matrine-kaempferol carrier-free nanoparticles.

[0129] Comparative Example 2

[0130] 2.1 Preparation of matrine-kaempferol carrier-free nanoparticles

[0131] (1) Matrine and kaempferol in a molar ratio of 1:1 were weighed and dissolved in an appropriate amount of methanol solution, and the pH value of the kaempferol solution was adjusted to 6.8-7.0 using a 5 mg / mL sodium hydroxide solution. The matrine solution and the kaempferol solution were then mixed and stirred evenly, and then slowly added to 10 mL of 60°C phosphate buffer (pH 6.8). The mixture was reacted under constant temperature stirring for 2-4 hours to facilitate self-assembly of matrine and kaempferol, thereby obtaining a matrine-kaempferol mixed solution;

[0132] (2) placing the mixed solution of step (1) into a dialysis bag (MWCO = 3.5 kDa) and dialyzing it with ultrapure water for 24 h to remove unassembled matrine, kaempferol, and organic solvent, replacing the ultrapure water every 4 h to obtain a solution of matrine and kaempferol;

[0133] (3) centrifuging the matrine and kaempferol self-assembly solution prepared in step (2), collecting the precipitate, washing it three times with ultrapure water, and freeze-drying it in a vacuum to obtain matrine-kaempferol carrier-free nanoparticles.

[0134] 2.2 Preparation of pH-responsive matrine-kaempferol carrier-free nanoparticles

[0135] Matrine-kaempferol carrier-free nanoparticles were dispersed in a 2% (w / v) Eudragit S100 ethanol solution and magnetically stirred for 1 hour. The solution was then spray-dried (inlet temperature 100°C, outlet temperature 50°C) to form an outer coating structure, yielding pH-responsive matrine-kaempferol carrier-free nanoparticles.

[0136] Comparative Example 3

[0137] 3.1 Preparation of matrine-kaempferol carrier-free nanoparticles

[0138] (1) Matrine and kaempferol at a molar ratio of 1:1 were weighed and dissolved in an appropriate amount of methanol solution, and the pH value of the kaempferol solution was adjusted to 6.8-7.0 using a 5 mg / mL sodium hydroxide solution. The matrine solution and the kaempferol solution were then mixed and stirred evenly, and then slowly added to 10 mL of 60°C phosphate buffer (pH 6.8). The mixture was reacted under constant temperature stirring for 2-4 hours to facilitate self-assembly of matrine and kaempferol, thereby obtaining a matrine-kaempferol mixed solution;

[0139] (2) placing the mixed solution of step (1) into a dialysis bag (MWCO = 3.5 kDa) and dialyzing it with ultrapure water for 24 h to remove unassembled matrine, kaempferol, and organic solvent, replacing the ultrapure water every 4 h to obtain a solution of matrine and kaempferol;

[0140] (3) centrifuging the matrine and kaempferol self-assembly solution prepared in step (2), collecting the precipitate, washing it three times with ultrapure water, and freeze-drying it in a vacuum to obtain matrine-kaempferol carrier-free nanoparticles.

[0141] 3.2 Preparation of ROS-responsive matrine-kaempferol carrier-free nanoparticles

[0142] Synthesis of Phenylboronic Acid-Hyaluronic Acid (PBA-HA) Conjugate: Dissolve hyaluronic acid (HA, 1 g) in 50 mL of deionized water. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.5 mmol) and N-hydroxysuccinimide (NHS, 0.5 mmol). Stir at room temperature for 2 h to activate the carboxylic acid group. Dissolve 3-aminophenylboronic acid (PBA-NH2, 0.8 mmol) in 10 mL of N,N-dimethylformamide (DMF) and slowly add dropwise to the HA solution. Adjust the pH to 5.5. After reacting for 24 h, dialysis purification (MWCO 3.5 kDa, deionized water, 48 h) and freeze-drying to obtain the PBA-HA conjugate.

[0143] The carrier-free nanoparticles prepared in step (3) were mixed with a 1% (w / v) PBA-HA solution, 0.1 mM CaCl2 was added, and magnetic stirring was performed for 30 minutes. The unadsorbed PBA-HA was removed by centrifugation (10,000 rpm, 20 minutes) to obtain ROS-responsive matrine-kaempferol carrier-free nanoparticles.

[0144] Cytotoxicity assay

[0145] The cytotoxicity of matrine, kaempferol, comparative examples 1-3, and Example 5 was evaluated by CCK-8 test as follows:

[0146] RAW264.7 cells were evenly transferred to a 96-well plate at a density of 5000 cells per well, and cytotoxicity tests were performed when the cell morphology was good and the number was cultured to about 80%. Different samples were prepared into working solutions with final concentrations of 20μM, 40μM, 60μM, 80μM and 100μM for testing. 100μL of the working solution of each sample with different concentrations was added to a 96-well plate, cultured for 24 hours, and the culture solution was discarded and washed 3 times with sterile PBS. Then, 10μL of CCK-8 reaction solution was added to each well and incubated for another 1 hour. Finally, the absorbance value at a wavelength of 450nm was measured using a microplate reader to determine cell viability. The control group consisted of culture medium without the addition of hydrogel, and each experimental group was repeated 3 times.

[0147] The results of cytotoxicity evaluation are shown in Figure 2 .like Figure 2 As shown, after treating RAW264.7 cells with matrine, kaempferol, matrine-kaempferol carrier-free nanoparticles, and matrine-kaempferol carrier-free nanoparticles with pH / ROS dual responsiveness at concentrations ranging from 10-24 μM, the cell viability remained greater than 90%. Therefore, the nanoparticles of Example 5 have good biocompatibility and do not produce toxic side effects on organisms.

[0148] pH / ROS response test

[0149] The pH / ROS response test was performed using the nanoparticles of Example 5 and Comparative Examples 1-3:

[0150] The nanoparticles were dispersed in simulated gastric fluid (pH 1.2, 37°C) (1 mg / mL) and shaken for 2 hours. 1 mL of samples were taken at 0.5 h, 1 h, and 2 h, centrifuged at 12,000 rpm for 10 min, and the supernatant was passed through a membrane (0.22 μm). The release of matrine and kaempferol was detected by HPLC. The particles were then transferred to simulated colonic fluid (pH 7.4) and divided into an experimental group (containing 1 mM H2O2) and a control group (without H2O2). The shaking was continued for 24 hours, and samples were taken at 4 h, 8 h, and 24 h, respectively. The release of matrine and kaempferol was determined after centrifugation through a membrane. The test results can be found in Figure 3a-Figure 3b .

[0151] like Figure 3a-Figure 3b As shown, in Comparative Example 1 (uncoated) and Comparative Example 3 (PBA-HA modified only), the release of kaempferol and matrine in the nanoparticles was as high as 30% or more at pH 1.2 due to the lack of Eudragit S100 coating. However, the release rates of kaempferol and matrine in Comparative Example 2 (Eudragit S100 coated) and Example 5 (dual response) were both less than 5%, indicating that the pH-sensitive layer effectively blocked gastric acid erosion and protected the nanoparticles from entering the intestine smoothly. In addition, at pH 7.4, the release rates of kaempferol and matrine from the nanoparticles in Comparative Example 2 after 24 hours were 32.65% and 28.33%, respectively; compared with the conditions of pH 7.4 + H2O2, the release amount was not significantly improved, indicating that the nanoparticles lacked a ROS triggering mechanism. In Example 5, at pH 7.4, the release rates of kaempferol and matrine were only 25.48% and 24.65%, respectively. However, at pH 7.4 + H2O2, the release rates were both over 85%, demonstrating the ROS responsiveness of the PBA ester bond. Therefore, these results demonstrate that the combination of a pH-responsive layer (Eudragit S100) and a ROS-responsive layer (PBA-HA) can achieve dual "targeting + triggering" control, enhancing the therapeutic efficacy of colitis.

[0152] Animal experiments

[0153] Animal experiments were conducted using the matrine-kaempferol carrier-free nanoparticles with pH / ROS dual response of Example 5 and the matrine-kaempferol carrier-free nanoparticles of Comparative Example 1:

[0154] Treatment of colitis mice: mice injected with only saline served as the blank group (saline), mice induced with dextran sulfate sodium (DSS) only (DSS), DSS mice treated with mesalazine only (5-ASA), DSS mice treated with matrine only (MAT), and DSS mice treated with kaempferol only (QUE) served as the control group. After a seven-day treatment period, the colon length and inflammatory factor changes of the mice were compared to evaluate the therapeutic effect on ulcerative colitis. The test results are shown in the table. Figure 4 and Figure 5a-5f .

[0155] like Figure 4As shown, the colon length of mice treated with the nanoparticles of Example 5 was significantly longer than that of mice treated with the nanoparticles of Comparative Example 1, matrine or kaempferol alone. In addition, the colon length of mice treated with the nanoparticles of Comparative Example 1 was longer than that of mice treated with matrine or kaempferol alone. This shows that after matrine and kaempferol are co-assembled to form nanoparticles, they have a synergistic effect. However, the Eudragit S100 coating layer on the surface of the nanoparticles of Example 5 can protect matrine and kaempferol from degradation by gastric acid and digestive enzymes, while the PBA-HA modification of the inner layer can trigger precise drug release through ROS response. Among them, HA targets the CD44 receptor, and PBA binds to intestinal mucosal mucin, thereby increasing the enrichment rate at the inflammatory site, thereby having a better therapeutic effect on ulcerative colitis.

[0156] like Figure 5a-5f As shown, the levels of inflammatory cytokines Lcn-2, MPO, TNF-α, IL-6, IL-10 and MCP-1 in the mice treated with Example 5 were significantly lower than those in the DSS group, MAT group, QUE group and Comparative Example 1, indicating that the nanoparticles of Example 5 can better treat ulcerative colitis due to their colon targeting.

[0157] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments using other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0158] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical variations made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing carrier-free nanoparticles with pH / ROS dual response, characterized in that: include: Quinolizidine alkaloids and flavonols are self-assembled to prepare quinolizidine alkaloid-flavonol carrier-free nanoparticles; The quinolizidine alkaloid-flavonol carrier-free nanoparticles are modified with a phenylboronic acid-hyaluronic acid conjugate to prepare phenylboronic acid-hyaluronic acid conjugate-modified nanoparticles; Furthermore, a pH-sensitive polymer is coated on the surface of the nanoparticles modified with the phenylboronic acid-hyaluronic acid conjugate to prepare carrier-free nanoparticles with pH / ROS dual response.

2. The preparation method according to claim 1, characterized in that Specifically include: dissolving quinolizidine alkaloids and flavonols in methanol respectively to form a quinolizidine alkaloid methanol solution and a flavonol methanol solution, and adjusting the pH value of the flavonol methanol solution to 6.8-7.0 with a pH regulator; Furthermore, the quinolizidine alkaloid methanol solution and the flavonol methanol solution are mixed, and phosphate buffer is added and stirred at 50-70° C. for 2-4 hours, followed by dialysis, centrifugation, washing, and vacuum drying to obtain quinolizidine alkaloid-flavonol carrier-free nanoparticles.

3. The preparation method according to claim 2, wherein: The quinolizidine alkaloids include matrine and / or sophoridine; and / or the flavonols include any one or more of kaempferol, quercetin, and myricetin; and / or, the molar ratio of the quinolizidine alkaloid to the flavonol is 1:3 to 3:1; And / or, the pH adjuster includes an organic base and / or an inorganic base; preferably, the pH adjuster includes any one or more combinations of sodium hydroxide, sodium carbonate, sodium bicarbonate, and potassium dihydrogen phosphate; preferably, the pH adjuster includes a sodium hydroxide solution with a concentration of 4-8 mg / mL; And / or, the dialysis bag used for the dialysis has a molecular weight cutoff of 1-3.5 kDa, and the dialysis time is 24-72 h; And / or, the centrifugal speed used in the centrifugation is 8000-12000 rpm, and the centrifugation time is 10-20 min.

4. The preparation method according to claim 1, characterized in that Specifically include: The quinolizidine alkaloid-flavonol carrier-free nanoparticles were mixed with a phenylboronic acid-hyaluronic acid conjugate solution, and then a CaCl2 solution was added and reacted at 25-37°C for 20-60 minutes, and then centrifuged to obtain phenylboronic acid-hyaluronic acid conjugate-modified nanoparticles.

5. The preparation method according to claim 4, characterized in that The preparation method of the phenylboronic acid-hyaluronic acid conjugate comprises: Dissolve hyaluronic acid in water, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and stir at room temperature for 1-3 hours to obtain a hyaluronic acid solution; 3-Aminophenylboronic acid is dissolved in DMF, then added dropwise to the hyaluronic acid solution and the pH value is adjusted to 5.0-6.8, followed by reaction at 25-37° C. for 24-48 hours, and finally purified by dialysis and freeze-dried to obtain a phenylboronic acid-hyaluronic acid conjugate.

6. The preparation method according to claim 4, characterized in that: The mass volume concentration of the phenylboronic acid-hyaluronic acid conjugate solution is 0.5-1.5 (w / v)%; and / or, the concentration of the CaCl2 solution is 0.05-0.15 mmol / L; And / or, the centrifugal speed used in the centrifugation is 8000-12000 rpm, and the centrifugation time is 10-30 min.

7. The preparation method according to claim 1, characterized in that Specifically include: The phenylboronic acid-hyaluronic acid conjugate modified nanoparticles are dispersed in an ethanol solution of a pH-sensitive polymer, stirred at 25-37° C. for 1-3 hours, and then spray-dried to prepare carrier-free nanoparticles with pH / ROS dual response.

8. The preparation method according to claim 7, characterized in that: The pH sensitive polymer includes any one or more combinations of Eudragit S100, pectin, sodium alginate, and carboxymethyl chitosan; And / or, the mass volume concentration of the ethanol solution of the pH sensitive polymer is 1-3 (w / v)%; And / or, the inlet temperature of the spray drying is 80-120°C, and the outlet temperature is 40-60°C.

9. The carrier-free nanoparticles with pH / ROS dual response prepared by the preparation method according to any one of claims 1 to 8, characterized in that: include: Quinolizidine alkaloid-flavonol carrier-free nanoparticles as cores, phenylboronic acid-hyaluronic acid conjugates modified on the surface of quinolizidine alkaloid-flavonol carrier-free nanoparticles, and pH-sensitive polymers coated on the outer layer.

10. Use of the carrier-free nanoparticles with pH / ROS dual response according to claim 9 in the preparation of a drug for preventing and / or treating intestinal diseases; preferably, the intestinal disease is inflammatory bowel disease, including ulcerative colitis.

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