Near-infrared luminous drug-loaded nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses near-infrared luminous drug-loaded nanoparticles as well as a preparation method and application thereof. The preparation method of the drug-loaded nanoparticles comprises the following steps: S1, dissolving aggregation-induced emission molecules NDA-NH2 and a drug in a solvent to obtain a drug-containing NDA-NH2 solution; s2, dissolving oxidized hyaluronic acid in ultrapure water to obtain an oxidized hyaluronic acid aqueous solution; and S3, mixing the drug-containing NDA-NH2 solution with the oxidized hyaluronic acid aqueous solution under stirring, carrying out ultrasonic treatment, and after the reaction is finished, carrying out dialysis and freeze-drying to prepare the near-infrared luminous drug-loaded nanoparticles. Oxidized hyaluronic acid is used as a base material, NDA-NH2 is modified on an oxidized hyaluronic acid chain through a Schiff base reaction, and drug-loaded nanoparticles are formed through self-assembly by providing a hydrophobic core. The drug-loaded nano particle has excellent near-infrared imaging capability and photo-thermal effect, can be used as an imaging unit, a treatment unit and a controlled release unit at the same time, and realizes functional combination of near-infrared two-region imaging, photo-thermal and controllable drug release.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a near-infrared luminescent drug-loaded nanoparticle, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a latent autoimmune inflammatory disease primarily characterized by joint involvement, gradually destroying joint function and ultimately leading to permanent disability. Conventional treatments for RA include medication and surgery. Early disease control and maintenance therapy can effectively slow disease progression and improve patients' quality of life. Disease-modifying antirheumatic drugs (DMARDs) are key medications in RA treatment. These drugs work by modulating the immune system, inhibiting inflammatory responses and the development of joint damage. However, RA patients often have severe synovial hyperplasia, inflammatory cell infiltration, and abnormal angiogenesis in their joints, creating a dense pathological microenvironment. This highly dense tissue structure not only severely hinders the effective penetration and accumulation of DMARDs at the lesion site but also leads to drug accumulation in non-target tissues during systemic administration, easily triggering serious adverse reactions.

[0003] In recent years, near-infrared II (NIR-II) optics has demonstrated unique advantages in disease diagnosis and treatment. Its deep tissue penetration and high signal-to-noise ratio imaging offer new possibilities for the precise diagnosis and treatment of rheumatoid arthritis. Phototherapy-integrated nanomedicine, as an emerging research direction, attempts to integrate diagnostic and therapeutic functions into a single nanoplatform to achieve real-time visualization and precise intervention of lesions. However, there is a physical contradiction between photothermal effects and NIR-II fluorescence emission: photothermal effects mainly rely on converting absorbed light energy into heat energy through non-radiative relaxation (such as molecular vibrations), while NIR-II fluorescence emission requires light energy to be released through radiative relaxation (photon emission). These two processes are competitive; therefore, the stronger the non-radiative relaxation process, the lower the efficiency and intensity of fluorescence emission. This leaves the development of an integrated nanosystem possessing both high photothermal conversion efficiency and good NIR-II imaging performance as a gap in current research. Therefore, developing a highly efficient integrated nanomedicine platform that can simultaneously achieve precise targeting of joint inflammation, real-time NIR-II imaging monitoring, and photothermal / drug synergistic therapy is expected to improve the precision and efficacy of rheumatoid arthritis treatment. Summary of the Invention

[0004] To overcome the shortcomings of the existing technology, the present invention aims to provide a near-infrared luminescent drug-loaded nanoparticle with excellent NIR-II imaging capability and near-infrared photothermal effect. It can simultaneously serve as an imaging unit, a treatment unit, and a controlled release unit, realizing the functional combination of near-infrared II imaging, photothermal effect, and controlled drug release. When applied to the treatment of rheumatoid arthritis, it can target inflamed joint sites, diagnose and report inflammatory sites under near-infrared II imaging, and destroy proliferating synovial cells in inflamed joints through photothermal action and controllably release anti-arthritis drugs, thus achieving photothermal-drug combined therapy and realizing the efficient integration of real-time imaging and treatment of arthritis.

[0005] Another object of the present invention is to provide a method for preparing the near-infrared luminescent drug-loaded nanoparticles.

[0006] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for preparing near-infrared luminescent drug-loaded nanoparticles, comprising the following steps: S1. The aggregation-induced emission molecule NDA-NH2 and the drug are dissolved in a solvent to obtain a drug-containing NDA-NH2 solution; the structural formula of the NDA-NH2 is as follows: ; S2. Dissolve oxidized hyaluronic acid in ultrapure water to obtain an aqueous solution of oxidized hyaluronic acid; S3. The drug-containing NDA-NH2 solution was mixed with an aqueous solution of oxidized hyaluronic acid under stirring and ultrasonic treatment. After the reaction was completed, the near-infrared luminescent drug-loaded nanoparticles were prepared by dialysis and freeze-drying.

[0007] Further, in step S1, the concentration of NDA-NH2 in the drug-containing NDA-NH2 solution is 0.2-1 mg / mL, and the concentration of the drug is 0.2-1 mg / mL.

[0008] The NDA-NH2 of the present invention can be obtained commercially or by self-production. The NDA-NH2 can be prepared according to the method disclosed in Chinese patent application CN202510199792.8.

[0009] Further, in step S1, the drug is an anti-arthritis drug; the anti-arthritis drug is selected from at least one of resveratrol, methotrexate, sulfasalazine, leflunomide, and ellamod.

[0010] Further, in step S1, the solvent is selected from at least one of tetrahydrofuran, toluene, dimethyl sulfoxide, and chloroform.

[0011] Further, in step S2, the concentration of the oxidized hyaluronic acid aqueous solution is 0.2-10 mg / mL.

[0012] Further, in step S2, the oxidation degree of the oxidized hyaluronic acid is 5-60%. The oxidation degree is determined by NMR: the oxidized hyaluronic acid is dissolved in pure water at a concentration of 10 mg / mL, and 0.5 M t-BC solution (the amount used is 10 times the amount of NaIO4 used in the aldehyde modification process) is added; the mixture is stirred at 700 rpm for 1 hour at room temperature; then, an equimolar amount of 0.5 M NaBH3CN aqueous solution equal to t-BC is added, and the reaction is carried out for 24 hours; the sample is purified by dialysis through a 1000 molecular weight cutoff membrane and then freeze-dried; the freeze-dried product is then subjected to… 1 The degree of aldehyde modification, or oxidation degree, was calculated by comparing the tert-butyl substituent signal (1.4 ppm, 9H) with the hyaluronic acid acetylamino signal (1.9 ppm, 3H) using ¹H NMR spectroscopy.

[0013] Further, in step S2, the preparation method of the oxidized hyaluronic acid includes the following steps: adding an aqueous solution of hyaluronic acid to an aqueous solution of sodium periodate under stirring, controlling the molar ratio of repeating units of sodium periodate to hyaluronic acid to be 1:10~10:1, then placing the reaction system under light-protected conditions for reaction, after the reaction is completed, adding ethylene glycol to terminate the oxidation, purifying by dialysis, and lyophilizing to obtain oxidized hyaluronic acid.

[0014] Preferably, in the method for preparing oxidized hyaluronic acid, the concentration of the hyaluronic acid aqueous solution is 5-50 mg / mL.

[0015] Preferably, in the method for preparing oxidized hyaluronic acid, the concentration of the sodium periodate aqueous solution is 5-50 mg / mL.

[0016] Preferably, in the method for preparing oxidized hyaluronic acid, the reaction time is 12-48 hours.

[0017] Preferably, in the method for preparing oxidized hyaluronic acid, the amount of ethylene glycol used is not less than the amount of sodium periodate. The preferred mass ratio of ethylene glycol to sodium periodate is (15-25):1.

[0018] Preferably, in the method for preparing oxidized hyaluronic acid, the dialysis purification involves using double-distilled water as the dialysis solution and detecting the residual periodate in the dialysis fluid using a 1% silver nitrate solution until no precipitate is formed, at which point the purification is complete.

[0019] Further, in step S3, the mass ratio of NDA-NH2 to oxidized hyaluronic acid is 1:(1-20).

[0020] Furthermore, in step S3, the stirring speed is 250-800 rpm; the reaction time is 4-24 h. Furthermore, in step S3, the dialysis is performed using deionized water as the dialysis solution.

[0021] Secondly, the present invention provides near-infrared luminescent drug-loaded nanoparticles, which are prepared by the preparation method described in the present invention.

[0022] Thirdly, the present invention provides the application of the near-infrared luminescent drug-loaded nanoparticles described above in the preparation of pharmaceutical formulations for the diagnosis or treatment of rheumatoid arthritis.

[0023] The present invention has the following beneficial effects: This invention uses oxidized hyaluronic acid as the base material, modifying the oxidized hyaluronic acid chain with aggregation-induced emission molecule NDA-NH2 via a Schiff base reaction, and forming drug-loaded nanoparticles by providing a hydrophobic core for self-assembly. These drug-loaded nanoparticles possess excellent near-infrared imaging capabilities and near-infrared photothermal effects, and can simultaneously serve as imaging, treatment, and controlled release units, achieving a functional combination of near-infrared II imaging, photothermal therapy, and controlled drug release. When applied to the treatment of rheumatoid arthritis, they can target inflamed joint sites, diagnose and report inflammatory sites under near-infrared II imaging guidance, and destroy proliferating synovial cells in inflamed joints through photothermal action, releasing anti-arthritis drugs in a controlled manner, thus achieving a photothermal-drug combined therapy. This achieves efficient integration of real-time imaging and treatment of arthritis, and is expected to improve the precision and efficacy of rheumatoid arthritis treatment. Attached Figure Description

[0024] Figure 1 This is a transmission electron microscope image of the drug-loaded nanoparticles RES-NDA@HA from Example 2.

[0025] Figure 2 The Fourier transform infrared spectra of the NDA@HA nanoparticles in Example 1 are shown.

[0026] Figure 3 For Example 1, the nanoparticles NDA@HA 1 H NMR spectrum Figure 4 The absorption and emission spectra of the drug-loaded nanoparticles RES-NDA@HA in Example 2 are shown.

[0027] Figure 5 The emission spectra of the drug-loaded nanoparticles RES-NDA@HA in Example 2 are shown at different pH values.

[0028] Figure 6 The drug release curves of the drug-loaded nanoparticles RES-NDA@HA in Example 2 are shown at different temperatures and pH values.

[0029] Figure 7 Example 2: Inflammatory joint-targeting imaging of drug-loaded nanoparticles RES-NDA@HA.

[0030] Figure 8 Example 2: Inflammation inhibition test of drug-loaded nanoparticles RES-NDA@HA. Detailed Implementation

[0031] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0032] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0033] Unless otherwise specified, the temperature in the embodiments of the present invention is room temperature or ambient temperature; room temperature or ambient temperature refers to 25±1℃.

[0034] The aggregation-induced emission molecule NDA-NH2 in this embodiment of the invention was prepared according to the method disclosed in Chinese patent application CN202510199792.8.

[0035] The preparation of oxidized hyaluronic acid (OHA) in this embodiment of the invention is as follows: Oxidized hyaluronic acid: Hyaluronic acid (HA) (Sangon Biotech, A614332) was dissolved in double-distilled water at a concentration of 10 mg / mL at room temperature. Then, 15 mL of a double-distilled aqueous solution containing 26.7 mg / mL sodium periodate (NaIO4) was slowly added while continuously stirring, maintaining a 1:1 molar ratio of NaIO4 to HA repeating units. The reaction system was then placed in the dark and reacted at room temperature for 24 hours. After the reaction was complete, 0.5 mL of ethylene glycol was added to terminate the oxidation process. Double-distilled water was used as the dialysate for purification. The residual periodate in the dialysate was detected using 1% silver nitrate solution until no precipitate formed, indicating successful purification. Finally, the purified solution was freeze-dried to obtain a solid oxidized hyaluronic acid product with an oxidation degree of 62%.

[0036] The functional groups of the product were characterized using Fourier transform infrared spectroscopy (FTIR). The freeze-dried sample was ground into powder, placed in the sample well, gently pressed flat, and then scanned and analyzed within the wavenumber range of 700-2200 cm⁻¹. Figure 2 As shown. Example

[0037] This embodiment provides near-infrared luminescent nanoparticles NDA@HA, which are prepared by the following method: S1. Dissolve 1 mg of aggregation-induced luminescence molecule NDA-NH2 in 2 mL of tetrahydrofuran (THF) to obtain an NDA-NH2 solution; S2. Dissolve 20 mg of oxidized hyaluronic acid in 10 mL of ultrapure water to obtain an aqueous solution of oxidized hyaluronic acid; S3. The two solutions were rapidly mixed under stirring at 600 rpm and subjected to ultrasonic treatment at 200 W for 3 minutes. The mixture was then magnetically stirred at 600 rpm for 12 hours. After the reaction was completed, the mixture was dialyzed in deionized water for 72 hours and then freeze-dried to obtain nanoparticles NDA@HA.

[0038] The nanoparticles NDA@HA from Example 1 were characterized using Fourier transform infrared spectroscopy (FTIR), and the FTIR results are as follows: Figure 2 As shown, 1730cm -1 1640cm -1 New peaks appeared at the respective locations, corresponding to aldehyde groups and Schiff base carbon-nitrogen double bonds, indicating that sodium periodate oxidized hyaluronic acid to produce aldehyde groups and that a Schiff base reaction occurred.

[0039] The nanoparticles NDA@HA from Example 1 were characterized using proton nuclear magnetic resonance (HMR) spectroscopy. The HMR spectroscopy results are as follows: Figure 3 As shown, the NDA@HA sample exhibits two peaks at 6.8 ppm and 7.1 ppm, corresponding to the hydrogen atoms on the benzene ring of the NDA-NH2 molecule, indicating that the Schiff base reaction occurred. Example

[0040] This embodiment provides a near-infrared luminescent drug-loaded nanoparticle RES-NDI@HA, which differs from Example 1 only in that in step S1, 1 mg of aggregation-induced emission molecule NDA-NH2 and 1 mg of resveratrol are simultaneously dissolved in 2 mL of tetrahydrofuran (THF) to obtain a drug-containing NDA-NH2 solution; the remaining preparation conditions are the same as in Example 1; and the drug-loaded nanoparticle RES-NDI@HA is prepared.

[0041] Figure 4 The absorption and emission spectra of the drug-loaded nanoparticles RES-NDA@HA in Example 2 are shown. These nanoparticles exhibit a maximum absorption peak at 780 nm and bright near-infrared II emission.

[0042] Relevant performance tests: Example 2: pH response property test of drug-loaded nanoparticles RES-NDA@HA.

[0043] The emission spectra of RES-NDA@HA were tested in buffer solutions with different pH values. The results are as follows: Figure 5 As shown, the emission spectrum of RES-NDA@HA nanoparticles redshifts and weakens when pH decreases.

[0044] II. Example 2: Drug release test of drug-loaded nanoparticles RES-NDA@HA.

[0045] The in vitro drug release behavior of RES-NDA@HA was studied using dynamic dialysis: a nanogel solution loaded with resveratrol (5 mg / mL) was placed in a dialysis bag (molecular weight cutoff = 100 kDa), and then immersed in PBS buffer (pH 7.4 or pH 5.0). Release experiments were conducted at 25, 37, and 42°C with gentle shaking at 200 rpm. At predetermined time points, 2 mL of the external release medium was removed, and an equal volume of fresh PBS buffer was added. The concentration of released resveratrol was determined using a UV-Vis spectrophotometer.

[0046] The results are as follows Figure 6 The figure shows drug release curves at different temperatures and pH values. The drug release rate increases significantly with increasing temperature or decreasing pH.

[0047] III. Example 2: Inflammatory joint-targeting imaging of drug-loaded nanoparticles RES-NDA@HA.

[0048] The drug-loaded nanoparticles RES-NDA@HA from Example 2 were diluted to 2.0 mg / mL with PBS, and 100 µL was injected intravenously into the tail vein of rheumatoid arthritis rats. NIR-II fluorescence images of the inflamed joints were captured at different time points. The results are as follows: Figure 7 As shown, the fluorescence signal of the nanoparticles at the inflamed joint gradually increased over time, reaching a peak at 12 hours, indicating effective accumulation and targeting of the inflamed site.

[0049] IV. Example 2: Inflammation inhibition test of drug-loaded nanoparticles RES-NDA@HA.

[0050] The drug-loaded nanoparticles from Example 1 were diluted with PBS to 2.0 mg / mL, and 100 µL was injected intravenously into the tail vein of rheumatoid arthritis rats. Twenty-four hours later, the inflamed joints were irradiated with laser for 10 minutes. After treatment, the inflamed joints were removed, embedded in paraffin, sectioned, and subjected to relevant staining procedures. Results are as follows... Figure 8 As shown, under laser irradiation, the nanoparticles can inhibit the expression of inflammatory factors and alleviate damage to inflamed joints and cartilage through the combined effects of photothermal and drug action.

[0051] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing near-infrared luminescent drug-loaded nanoparticles, characterized in that, Includes the following steps: S1. The aggregation-induced emission molecule NDA-NH2 and the drug are dissolved in a solvent to obtain a drug-containing NDA-NH2 solution; the structural formula of the NDA-NH2 is as follows: ， S2. Dissolve oxidized hyaluronic acid in ultrapure water to obtain an aqueous solution of oxidized hyaluronic acid; S3. The drug-containing NDA-NH2 solution was mixed with an aqueous solution of oxidized hyaluronic acid under stirring and ultrasonic treatment. After the reaction was completed, the near-infrared luminescent drug-loaded nanoparticles were prepared by dialysis and freeze-drying.

2. The preparation method according to claim 1, characterized in that, In step S1, the concentration of NDA-NH2 in the drug-containing NDA-NH2 solution is 0.2-1 mg / mL, and the concentration of the drug is 0.2-1 mg / mL.

3. The preparation method according to claim 1, characterized in that, In step S1, the drug is an anti-arthritis drug; the anti-arthritis drug is selected from at least one of resveratrol, methotrexate, sulfasalazine, leflunomide, and ellamod; the solvent is selected from at least one of tetrahydrofuran, toluene, dimethyl sulfoxide, and chloroform.

4. The preparation method according to claim 1, characterized in that, In step S2, the concentration of the oxidized hyaluronic acid aqueous solution is 0.2-10 mg / mL.

5. The preparation method according to claim 1, characterized in that, In step S2, the degree of oxidation of the oxidized hyaluronic acid is 5-60%.

6. The preparation method according to claim 1, characterized in that, In step S2, the preparation method of oxidized hyaluronic acid includes the following steps: adding an aqueous solution of hyaluronic acid to an aqueous solution of sodium periodate under stirring, controlling the molar ratio of repeating units of sodium periodate to hyaluronic acid to be 1:10~10:1, then placing the reaction system under light-protected conditions for reaction, adding ethylene glycol to terminate oxidation after the reaction is completed, purifying by dialysis, and lyophilizing to obtain oxidized hyaluronic acid; The concentration of the hyaluronic acid aqueous solution is 5-50 mg / mL; The concentration of the sodium periodate aqueous solution is 5-50 mg / mL.

7. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of NDA-NH2 to oxidized hyaluronic acid is 1:(1-20).

8. The preparation method according to claim 1, characterized in that, In step S3, the stirring speed is 250-800 rpm; the reaction time is 4-24 h. The dialysis was performed using deionized water for 24-72 hours.

9. A near-infrared luminescent drug-loaded nanoparticle, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The use of the near-infrared luminescent drug-loaded nanoparticles according to claim 9 in the preparation of pharmaceutical formulations for the diagnosis or treatment of rheumatoid arthritis.

Images