A fibrotic microenvironment-responsive polymer, drug-loaded nanoparticles made therefrom, and methods of making and using the same

Abstract

This invention belongs to the pharmaceutical field, specifically relating to a fibrosis-responsive microenvironment polymer, drug-loaded nanoparticles made from the polymer, its preparation method, and its applications. The fibrosis-responsive microenvironment polymer of this invention can serve as a carrier for drug encapsulation and delivery, exhibiting intelligent drug release characteristics, targeting performance, and excellent safety. Drug-loaded nanoparticles made from the aforementioned polymer and drugs can effectively alleviate organ fibrosis, demonstrating significantly better therapeutic effects than free drugs and showing very promising clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a polymer that responds to a fibrotic microenvironment, drug-encapsulated nanoparticles made therefrom, and their preparation methods and uses. Background Technology

[0002] Organ fibrosis is the terminal pathological stage of many chronic diseases, characterized by excessive deposition of the extracellular matrix (ECM) and progressive destruction of normal tissue structure, ultimately leading to organ failure. This pathological process can affect almost all solid organs, constituting a significant global health burden, with liver fibrosis and kidney fibrosis being the most common and damaging. Drugs for treating organ fibrosis, such as tripterygium wilfordii (Celastrol, Cel), are natural compounds with significant anti-inflammatory and anti-fibrotic activities, showing great potential in treating organ fibrosis. However, due to its poor water solubility, low bioavailability, and systemic toxicity, traditional administration methods are insufficient to maximize its efficacy.

[0003] Currently, effective treatments for organ fibrosis remain scarce in clinical practice, often making organ transplantation the only option for end-stage patients. This limitation stems primarily from two key challenges: first, existing therapies cannot precisely target pathogenic cells; and second, they cannot deliver drugs on demand within the complex and dynamic microenvironment of fibrotic lesions. Traditionally, fibrosis is considered an organ-specific damage response, but recent studies have shown that the fibrosis process in different organs shares highly common molecular and cellular mechanisms.

[0004] Numerous studies have demonstrated that myofibroblast activation is a central event in the progression of multi-organ fibrosis. Although their cellular origin varies by organ—primarily hepatic stellate cells in the liver and renal interstitial fibroblasts in the kidneys—these cells exhibit high phenotypic and functional consistency, including upregulated expression of α-smooth agonistin, enhanced ECM secretion, and increased contractility. Recent studies have found that receptors such as fibronectin and integrin αvβ3 are overexpressed in myofibroblasts of both liver and kidney fibrosis, and drug delivery systems targeting these common receptors have shown initial potential for simultaneous targeting of fibrotic liver and kidneys.

[0005] Currently, drug delivery systems for treating organ fibrosis mainly include nanomicelles and liposomes. A pH-sensitive lutein-chitosan nanomicelle loaded with drugs (Cel@LU-CA-CS) utilizes the slightly acidic environment of tumor or inflamed tissues to release the drug, significantly improving drug stability in vivo and demonstrating good anti-fibrotic effects in mouse models. Nanostructured lipid carriers (such as nanostructured lipid carriers and plant-derived lipid-reconstructed multi-chambered vesicle nanocarriers) can effectively reduce collagen deposition in the lungs, and these nanoliposome carriers exhibit excellent biocompatibility.

[0006] However, while current nanodelivery systems for treating organ fibrosis have made significant progress in solubility, targeting, and toxicity control, they still have the following shortcomings before clinical translation: First, there are defects in the synthesis of nanocarriers. Multi-step self-assembly processes using polymers and liposomes are sensitive to temperature, solvent residue, and stirring rate. Batch drift often occurs between laboratory-scale trials and GMP-compliant large-scale trials in terms of particle size, encapsulation efficiency, and drug loading, leading to a lack of unified quality evaluation standards and introducing variables into subsequent toxicology and clinical studies. Second, drug loading is relatively low. Conventional carriers such as polymer micelles and liposomes have limited encapsulation efficiency for hydrophobic small molecules, often resulting in low drug loading. The current "high carrier-low drug" phenomenon leads to large single-dose volumes and high excipient exposure, increasing the risk of excipient-related adverse reactions. Thirdly, release controllability is poor. Protein carriers (albumin, gelatin, etc.) rely on enzyme degradation to release the drug, and significant differences in protease activity between individuals easily lead to polarization between "rapid release-overexposure" and "slow release-undertreatment." Some pH / reduction response systems also become inaccurate due to differences in the lesion microenvironment, making true on-demand release difficult to achieve. Fourthly, there are potential toxicity risks. Some polymers (such as PEG) may trigger immune responses or antibody production, while materials such as chitosan may also exhibit cytotoxicity at high doses. Therefore, there is an urgent need to discover a ligand that combines excellent safety and high targeting ability to identify receptors that are commonly highly expressed by activated myofibroblasts in different organs, thereby establishing a targeted therapy strategy applicable to multi-organ fibrosis. Summary of the Invention

[0007] To address the problems of existing technologies, this invention provides a polymer responsive to fibrous microenvironment, drug-encapsulated nanoparticles made therefrom, a preparation method thereof, and applications.

[0008] A fibrotic microenvironment-responsive polymer, the polymer being a copolymer of chondroitin sulfate as the backbone and modified with aminophenylboronic acid and triglyceride monostearate.

[0009] Preferably, the polymer comprises chondroitin sulfate structural units modified with the following branches:

[0010] , where n = 10~50.

[0011] The above structural formulas are used to illustrate the molecular structure of the branched chondroitin sulfate structural units, and do not imply that all structural units in the polymer of the present invention are modified with branches. In fact, the polymer of the present invention has a certain degree of grafting. In the embodiments of the present invention, the grafting degree of aminophenylboronic acid can be 5%-10%. In specific embodiments, the grafting degree of aminophenylboronic acid is preferably 6.7%-8.6%, and more preferably 6.7%, 8.4%, or 8.6%. The grafting degree of aminophenylboronic acid can be determined by ultraviolet spectrophotometry.

[0012] The aforementioned polymer of the present invention can be prepared by the following method: chondroitin sulfate and aminophenylboronic acid are mixed and reacted to obtain chondroitin sulfate-aminophenylboronic acid copolymer; then the chondroitin sulfate-aminophenylboronic acid copolymer is blended and reacted with triglyceride monostearate to obtain chondroitin sulfate-aminophenylboronic acid-triglyceride monostearate copolymer.

[0013] Specifically, the preparation method of the above-mentioned polymer includes the following steps:

[0014] Step 1: Chondroitin sulfate and aminophenylboronic acid are reacted under the action of a condensing agent to obtain chondroitin sulfate-aminophenylboronic acid copolymer.

[0015] Step 2: Mix chondroitin sulfate-aminophenylboronic acid copolymer with triglyceride monostearate and react to obtain chondroitin sulfate-aminophenylboronic acid-triglyceride monostearate copolymer.

[0016] Preferred,

[0017] In step 1, the chondroitin sulfate-aminophenylboronic acid copolymer is obtained by polymerizing the following components in parts by weight:

[0018] 50-100 parts of chondroitin sulfate

[0019] 10-40 parts of aminophenylboronic acid;

[0020] In step 2, the chondroitin sulfate-aminophenylboronic acid-polyglycerol monostearate copolymer is obtained by polymerization of the following components in parts by weight:

[0021] 10-20 parts of chondroitin sulfate-aminophenylboronic acid copolymer

[0022] 1-10 parts of triglyceride monostearate.

[0023] Preferred,

[0024] In step 1, the chondroitin sulfate-aminophenylboronic acid copolymer is obtained by polymerizing the following components in parts by weight: 50 parts chondroitin sulfate,

[0025] 17.3 parts of aminophenylboronic acid;

[0026] In step 2, the chondroitin sulfate-aminophenylboronic acid-polyglycerol monostearate copolymer is obtained by polymerization of the following components in parts by weight:

[0027] 10 parts of chondroitin sulfate-aminophenylboronic acid copolymer

[0028] 7.5 parts of triglyceride monostearate.

[0029] The above-mentioned polymers are used in the preparation of drug carriers.

[0030] A drug-loaded nanoparticle, the nanoparticle comprising the aforementioned polymer and active ingredient.

[0031] Preferably, the active ingredient is a drug for treating organ fibrosis.

[0032] The above-mentioned method for preparing drug-loaded nanoparticles includes the following steps: mixing the polymer with the active ingredient, stirring, sonicating, dialysis, and centrifuging to obtain the final product.

[0033] The above-mentioned drug-encapsulated nanoparticles are used in the preparation of drugs for treating organ fibrosis.

[0034] This invention provides a novel drug carrier—a chondroitin sulfate-aminophenylboronic acid-triglyceride monostearate (CS-PBA-TGMS, CPT) polymer—which can spontaneously assemble into nanoparticles and achieve highly efficient drug loading. The drug-loaded nanoparticles can intelligently release drugs within the pathological microenvironment of organ fibrosis. Furthermore, the drug-loaded nanoparticles of this invention exhibit good targeting properties for both fibrotic liver and fibrotic kidneys, with particularly excellent targeting performance in fibrotic kidneys. Regarding efficacy and systemic toxicity, the drug-loaded nanoparticles of this invention significantly alleviate liver fibrosis and kidney damage, demonstrating significantly better efficacy than free drugs. Therefore, the polymer CPT of this invention, as a nanoparticle drug delivery carrier for organ fibrosis drugs, achieves intelligent controlled-release and targeted drug delivery with good safety, and has excellent application prospects.

[0035] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0036] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0037] Figure 1 The synthetic route (A) and the 1H NMR spectrum (B) of the CPT polymer of the present invention are shown.

[0038] Figure 2 Transmission electron microscopy (TEM) image of Cel@CPT nanoparticles (A), statistical results of DLS detection (B), and statistical graph of particle size and polydispersity index (PDI) of CPT and Cel@CPT nanoparticles at different time points after incubating the nanoparticles in PBS buffer containing 10% fetal bovine serum for 24 h (C).

[0039] Figure 3 Transmission electron microscopy images (A) of drug-loaded nanoparticles incubated for 24 hours in a pathological microenvironment, showing blank Cel@CPT nanoparticles and Cel@CPT nanoparticles incubated with 10 mM AAPH (oxidant) and / or 2 μg / mL MMP-9 at 37°C for 24 hours, respectively; cumulative release curves of triptolide from Cel@CPT nanoparticles under different environments in in vitro responsive release experiments (B); and statistical graph of drug release from fibrotic tissue homogenates after co-incubation of Cel@CPT nanoparticles with homogenates of fibrotic liver or kidney tissue (C).

[0040] Figure 4 The average fluorescence intensity of CPT nanoparticles (CPT-Cy5.5) labeled with Cy5.5 fluorescent dye in the human hepatic stellate cell line LX-2 under resting and pathological activation states is shown in Figure (A). The CD44 gene was knocked down in LX-2 cells after transfection with CD44-specific siRNA (CD44-siRNA) and negative control siRNA (control-siRNA), respectively (B). The average fluorescence intensity of nanoparticle uptake in cells after CD44-siRNA knockdown is shown in Figure (C).

[0041] Figure 5 The average fluorescence intensity of CPT nanoparticles labeled with Cy5.5 fluorescent dye (CPT-Cy5.5) in the rat fibroblast cell line NRK-49F under resting and pathological activation states is shown in Figure (A). The CD44 gene was knocked down in NRK-49F cells after transfection with CD44-specific siRNA (CD44-siRNA) and negative control siRNA (control-siRNA), respectively (B). The average fluorescence intensity of nanoparticle uptake in cells after CD44-siRNA knockdown is shown in Figure (C).

[0042] Figure 6The images show the in vitro fluorescence of Cel@CPT nanoparticles in organs of fibrotic mice (A); a semi-quantitative statistical graph of the fluorescence intensity of Cel@CPT nanoparticles in the liver of fibrotic mice (B); and a statistical graph of the average fluorescence intensity of the cellular distribution of Cel@CPT nanoparticles in the fibrotic liver (C).

[0043] Figure 7 The image shows the in vitro fluorescence of Cel@CPT nanoparticles in the kidneys of fibrotic mice (A); the semi-quantitative statistical graph of the fluorescence intensity of Cel@CPT nanoparticles in the kidneys of fibrotic mice (B).

[0044] Figure 8 Statistical diagram of hydroxyproline content in the liver of mice with liver fibrosis after treatment (A); immunohistochemical staining of the liver of mice with liver fibrosis after treatment with Sirius red and α-SMA (B); qPCR analysis (C); Western blot analysis (D); serum aspartate aminotransferase level in model mice (E); serum alanine aminotransferase level in model mice (F).

[0045] Figure 9 Statistical graph of liver hydroxyproline content in treated mice with renal fibrosis (A); Sirius red and Masson staining of kidneys in treated mice with renal fibrosis (B); qPCR analysis (C); Western blot analysis (D); Serum creatinine and serum urea levels in the blood of model mice (E).

[0046] Figure 10 Hematological analysis of a liver fibrosis model (A); Statistical graph of the levels of lactate dehydrogenase and creatine kinase isoenzymes, markers of myocardial injury (B).

[0047] Figure 11 Hematological analysis of a renal fibrosis model (A); Statistical graph of the levels of lactate dehydrogenase and creatine kinase isoenzymes, markers of myocardial injury (B). Detailed Implementation

[0048] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0049] Example 1 Chondroitin sulfate-aminophenylboronic acid-polyglycerol monostearate (CPT) copolymer

[0050] Prepared according to the polymer synthesis route diagram of the present invention ( Figure 1A). Chondroitin sulfate (Solarbio, C9160) (CS, 50 mg, 0.005 mmol) was dissolved in 2 mL of double-distilled water, followed by the addition of condensing agents EDC (28.7 mg, 0.15 mmol) and NHS (17.3 mg, 0.15 mmol). After activation for 30 minutes, the activated CS solution was added dropwise to an aqueous solution of 4-aminophenylboronic acid (PBA) (17.3 mg, 0.1 mmol, dissolved in 1 mL of double-distilled water), and the reaction was stirred at room temperature for 24 hours. The reaction solution was dialyzed (molecular weight cutoff 3500 kDa), centrifuged, and freeze-dried to obtain the CS-PBA copolymer. Dissolve 10 mg of CS-PBA in 1 mL of double-distilled water and slowly add it to 4 mL of DMSO solution containing triglyceride monostearate (TGMS, 7.5 mg, 0.015 mmol). Adjust the pH to 8.0, stir the reaction, and then dialyze and freeze dry according to the above steps to finally obtain the CS-PBA-TGMS (CPT) copolymer.

[0051] The structure of CPT was confirmed by hydrogen nuclear magnetic resonance (NMR) spectrum. Figure 1 B): 1B is the 1H NMR spectrum of CPT. The characteristic peak of -NHCOCH3- on the CS skeleton is at δ = 1.91 ppm. The characteristic peak of the benzene ring on PBA appears at δ = 7-8 ppm, indicating that PBA has been successfully modified on CS. The peak at 1.18 ppm is attributed to the methylene group of TGMS, indicating that TGMS has been conjugated with CS−PBA.

[0052] Example 2 Chondroitin sulfate-aminophenylboronic acid-polyglycerol monostearate (CPT) copolymer

[0053] Chondroitin sulfate (CS, 50 mg, 0.005 mmol) was dissolved in 2 mL of double-distilled water, followed by the addition of condensing agent EDC (20 mg, 0.104 mmol) and NHS (15 mg, 0.13 mmol). The mixture was stirred and activated at room temperature for 30 minutes. The activated CS solution was then added dropwise to an aqueous solution of 4-aminophenylboronic acid (PBA) (10 mg, 0.057 mmol, dissolved in 1 mL of double-distilled water), and the reaction was stirred at room temperature for 24 hours. The reaction solution was dialyzed (molecular weight cutoff 3500 kDa), centrifuged, and freeze-dried to obtain the CS-PBA copolymer. The CS-PBA copolymer (10 mg) prepared above was dissolved in 1 mL of double-distilled water. Separately, triglyceride monostearate (TGMS, 1 mg, 0.002 mmol) was dissolved in 4 mL of DMSO. Under stirring, the CS-PBA aqueous solution was slowly added dropwise to the TGMS DMSO solution. After adjusting the pH to 8.0 and stirring the reaction, the reaction was carried out by dialysis and freeze-drying as described above, and finally the CS-PBA-TGMS (CPT) copolymer was obtained.

[0054] Example 3 Chondroitin sulfate-aminophenylboronic acid-polyglycerol monostearate (CPT) copolymer

[0055] Chondroitin sulfate (CS, 75 mg, 0.0075 mmol) was dissolved in 2 mL of double-distilled water, followed by the addition of condensing agent EDC (25 mg, 0.13 mmol) and NHS (17.3 mg, 0.15 mmol). The mixture was stirred and activated at room temperature for 30 minutes. The activated CS solution was then added dropwise to an aqueous solution of 4-aminophenylboronic acid (PBA) (25 mg, 0.144 mmol, dissolved in 1 mL of double-distilled water), and the reaction was stirred at room temperature for 24 hours. The reaction solution was dialyzed (molecular weight cutoff 3500 kDa), centrifuged, and freeze-dried to obtain the CS-PBA copolymer. The prepared CS-PBA copolymer (15 mg) was dissolved in 1 mL of double-distilled water. Triglyceride monostearate (TGMS, 5 mg, 0.01 mmol) was dissolved in 4 mL of DMSO. The CS-PBA aqueous solution was slowly added dropwise to the TGMS DMSO solution while stirring. After adjusting the pH to 8.0 and stirring the reaction, the reaction was carried out by dialysis and freeze-drying as described above, and finally the CS-PBA-TGMS (CPT) copolymer was obtained.

[0056] Example 4 Chondroitin sulfate-aminophenylboronic acid-polyglycerol monostearate (CPT) copolymer

[0057] Chondroitin sulfate (CS, 100 mg, 0.01 mmol) was dissolved in 2 mL of double-distilled water, followed by the addition of condensing agent EDC (30 mg, 0.156 mmol) and NHS (20 mg, 0.17 mmol), and the mixture was stirred at room temperature for 30 minutes to activate. The activated CS solution was then added dropwise to an aqueous solution of 4-aminophenylboronic acid (PBA) (40 mg, 0.23 mmol, dissolved in 1 mL of double-distilled water), and the reaction was stirred at room temperature for 24 hours. The reaction solution was dialyzed (molecular weight cutoff 3500 kDa), centrifuged, and freeze-dried to obtain the CS-PBA copolymer. The prepared CS-PBA copolymer (20 mg) was dissolved in 1 mL of double-distilled water. Triglyceride monostearate (TGMS, 10 mg, 0.02 mmol) was dissolved in 4 mL of DMSO. The CS-PBA aqueous solution was slowly added dropwise to the TGMS DMSO solution while stirring. After adjusting the pH to 8.0 and stirring the reaction, the reaction was carried out by dialysis and freeze-drying as described above, and finally the CS-PBA-TGMS (CPT) copolymer was obtained.

[0058] Example 5 Preparation of drug-loaded nanoparticles

[0059] The CPT material used in this embodiment was prepared according to the method in Example 1.

[0060] 1. Preparation of triptolide nanoparticles (Cel@CPT):

[0061] 1 mg of triptolide was dissolved in 200 μL of dimethyl sulfoxide (DMSO) to form a drug solution, which was then added dropwise to 4 mL of a DMSO / water mixture (volume ratio 1:3) containing 10 mg of CPT polymer material under stirring. The mixture was then magnetically stirred and sonicated (120 W power, 5 minutes), followed by dialysis for 24 hours to completely remove free drug and organic solvent (MW = 3500 kDa). Finally, the dialysate was centrifuged at 4500 rpm for 10 minutes, and the supernatant was collected to obtain Cel@CPT drug-loaded nanoparticles.

[0062] Particle size and stability characterization of Cel@CPT nanoparticles: Blank CPT nanoparticles and drug-loaded Cel@CPT nanoparticles were prepared. The particle size, polydispersity index (PDI), and zeta potential of the nanoparticles in water were measured using a laser particle size analyzer (DLS). The results are shown in Table 1. 2 mL of each nanoparticle solution (1 mg / mL) was stored at room temperature, and DLS analysis was performed on days 1, 3, and 5 to observe the stability of the nanoparticles. Simultaneously, to assess the stability of the nanoparticles in serum, all nanoparticles were incubated in PBS buffer containing 10% fetal bovine serum for 24 h, and the changes in particle size and PDI of CPT and Cel@CPT nanoparticles were monitored at different time points.

[0063] The results showed that the average particle size of both blank CPT and Cel@CPT nanoparticles was less than 150 nm, and the PDI was less than 0.25. Transmission electron microscopy (TEM) revealed that the nanoparticles were uniformly spherical. Figure 2 A). The particle size of all nanoparticles did not change significantly within 5 days at room temperature. Figure 2 B). The particle size increased slightly over 24 hours, but the average diameter remained below 200 nm. Figure 2 C) indicates that the nanoparticles have good stability.

[0064] Table 1

[0065]

[0066] 2. Preparation of nanoparticles loaded with triptolide (TP@CPT):

[0067] 1 mg of triptolide was dissolved in 200 µL of dimethyl sulfoxide (DMSO) to form a drug solution, which was then added dropwise to 4 mL of DMSO / water mixed solvent (volume ratio 1:3) containing 10 mg of CPT polymer material under stirring. The mixture was then magnetically stirred and sonicated with a probe (power 120 W, time 5 minutes). After 24 hours of dialysis (MW = 3500 kDa), the dialysate was centrifuged at 4500 rpm for 10 minutes, and the supernatant was collected to obtain TP@CSS drug-loaded nanoparticles.

[0068] 3. Preparation of pirfenidone-loaded nanoparticles (PFD@CPT):

[0069] 1 mg pirfenidone (PFD) was dissolved in 100 µL of dimethyl sulfoxide (DMSO), and 10 mg CPT was dissolved in 1 mL of DMSO. The two solutions were mixed, and 3 mL of deionized water was slowly added under sonication. After dialysis for 24 hours (MW = 3500 kDa), the mixture was centrifuged (4500 rpm, 10 min), and the supernatant was collected to obtain PFD@CPT.

[0070] 4. Preparation of curcumin-loaded nanoparticles (Cur@CPT):

[0071] 1 mg curcumin (Cur) was dissolved in 100 µL dimethyl sulfoxide (DMSO), and 10 mg CPT was dissolved in 1 mL DMSO. The two solutions were mixed, and 3 mL of deionized water was slowly added under sonication. After dialysis for 24 hours (MW = 3500 kDa), the mixture was centrifuged (4500 rpm, 10 min), and the supernatant was collected to obtain Cur@CPT.

[0072] Comparative Example 1: Preparation of chondroitin sulfate-stearic acid (CSS) copolymer and its non-responsive nanoparticles loaded with triptolide (Cel@CSS)

[0073] To verify the key role of specific structural units in CPT materials in their "dual responsiveness", this invention simultaneously prepared a non-responsive control material—chondroitin sulfate-stearic acid (CSS) copolymer and its non-responsive nanoparticles (Cel@CSS) loaded with triptolide—for comparative studies.

[0074] Preparation of CSS: Chondroitin sulfate (Solarbio, C9160) (CS, 50 mg, 0.005 mmol), EDC (28.7 mg, 0.15 mmol), DMAP (14.6 mg, 0.12 mmol), and ethylenediamine (EDA; 3.91 mg, 0.05 mmol) were dissolved in 4 mL of DMF and stirred at room temperature for 24 hours. The reaction solution was dialyzed (molecular weight cutoff 3500 kDa), centrifuged, and freeze-dried to obtain CS-EDA. Subsequently, stearic acid (SA, 21.37 mg, 0.075 mmol) was dissolved in DMSO and activated with EDC (28.7 mg, 0.15 mmol) and DMAP (14.6 mg, 0.12 mmol) for 30 minutes. Then, it was stirred and reacted with 20 mg CS-EDA dissolved in 4 mL DMSO / water (3:1, v / v) at room temperature for 48 hours. The crude product was dialyzed and lyophilized to obtain CSS.

[0075] Preparation of Cel@CSS: 1 mg triptolide was dissolved in 200 µL of dimethyl sulfoxide (DMSO) to form a drug solution, which was then added dropwise to 4 mL of DMSO / water mixed solvent (volume ratio 1:3) containing 10 mg of CSS polymer material under stirring. The mixture was then magnetically stirred and ultrasonically treated with a probe (power 120 W, time 5 minutes), followed by dialysis for 24 hours to completely remove free drug and organic solvent (MW = 3500 kDa). Finally, the dialysate was centrifuged at 4500 rpm for 10 minutes, and the supernatant was collected to obtain Cel@CSS drug-loaded nanoparticles.

[0076] The beneficial effects of the present invention are illustrated below through experimental examples.

[0077] Experiment Example 1: Screening Experiment for Raw Material Feed Ratio

[0078] To optimize the feed ratio in the CPT preparation process, this experimental example compares the effects of different feed ratios on the product in two reaction steps. This experimental example modifies the amount of raw materials used in Example 1, while keeping other reaction conditions the same as in Example 1.

[0079] 1. Screening based on the feeding ratio of CS and PBA

[0080] As a key copolymer in subsequent reactions, the PBA grafting degree of CS-PBA directly affects the boric acid ligand density and final properties of the product. Therefore, this invention first optimized the feed ratio of CS to PBA: fixing the amounts of chondroitin sulfate CS (50 mg, approximately 0.005 mmol) and condensing agents EDC / NHS (0.15 mmol each), while varying the feed mass of 4-aminophenylboronic acid (PBA). The specific steps are as follows:

[0081] CS (50 mg) was dissolved in 2 mL of double-distilled water, and EDC (28.7 mg) and NHS (17.3 mg) were added. The mixture was activated at room temperature for 30 minutes. The activated CS solution was then added dropwise to different masses of PBA aqueous solutions (dissolved in 1 mL of water). The reaction mixture was stirred at room temperature for 24 hours. The reaction solution was dialyzed (MWCO 3500 Da), centrifuged, and freeze-dried to obtain CS-PBA products with different PBA dosages. The PBA grafting degree of the products was determined by ultraviolet spectrophotometry, and the results are as follows:

[0082] Table 2

[0083]

[0084] When the molar ratio of CS to PBA is 1:20, the resulting CS-PBA copolymer exhibits the highest degree of PBA grafting, indicating that the condensation reaction between the carboxyl and amino groups is most complete and efficient under these conditions. Therefore, this ratio was selected as the optimal condition for the first step of the reaction, and the CS-PBA synthesized under these conditions will be used for subsequent bonding with TGMS.

[0085] 2. Screening of CS-PBA and TGMS feed ratio

[0086] To obtain CPT copolymers capable of self-assembling into ideal nanoparticles (small size and uniform distribution), this experimental example investigated the effect of the feed ratio of the hydrophilic segment CS-PBA to the strongly hydrophobic segment TGMS in the second step of the reaction on the self-assembly properties of the final product. With a fixed feed amount of 10 mg for CS-PBA, the effect of different TGMS feed masses on the hydrated particle size (Dh) and dispersibility (PDI) of the ultimately self-assembled nanoparticles was examined. The specific procedures are as follows:

[0087] Five 10 mg portions of CS-PBA were accurately weighed and placed in five separate reaction flasks. Each portion was dissolved in 1 mL of double-distilled water to prepare solution A. TGMS was accurately weighed at mass values ​​of 1.0 mg, 2.5 mg, 5.0 mg, 7.5 mg, and 10.0 mg, and each TGMS mass was dissolved in 4 mL of DMSO to prepare solution B. While stirring, each portion of solution A was added dropwise to its corresponding portion of solution B. The pH of the mixture was adjusted to 8.0, and the reaction was carried out at room temperature for 24 hours. The reaction solution was dialyzed (MWCO 3500 Da), centrifuged, and freeze-dried to obtain CPT copolymers with different TGMS feed amounts, labeled CPT-1.0, CPT-2.5, CPT-5.0, CPT-7.5, and CPT-10.0 according to the TGMS mass.

[0088] Each of the above CPT copolymers was dispersed in double-distilled water at the same concentration (1 mg / mL), and after ultrasonic treatment with a probe, its hydrated particle size distribution and polydispersity index were determined by dynamic light scattering method.

[0089] Table 3

[0090]

[0091] In summary, when the CS-PBA feed amount is fixed at 10 mg, the optimal TGMS feed amount is 7.5 mg. Under these conditions, the mass ratio of CS-PBA to TGMS is 10:7.5. The CPT copolymer synthesized at this ratio can self-assemble into nanoparticles with small particle size and uniform distribution.

[0092] Experimental Example 2: Responsive Release of Cel@CPT Nanoparticles

[0093] To verify the intelligent drug release characteristics of the Cel@CPT nanoparticles prepared in Example 5 above in a pathological microenvironment, the following in vitro responsive release experiment was conducted. First, the Cel@CPT nanoparticles were co-incubated with 10 mM AAPH (oxidant) and / or 2 μg / mL MMP-9 at 37°C for 24 hours, and their morphological changes were observed by transmission electron microscopy (TEM). Subsequently, the drug release kinetics were quantitatively assessed by dialysis. One mL of free Cel or Cel@CPT nanoparticle solution was placed in a dialysis bag with a molecular weight cutoff of 3500 kDa and immersed in 30 mL of release medium simulating fibrosis conditions: (1) PBS containing 10 mM AAPH; (2) TNCB buffer containing 2 μg / mL MMP-9 (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.05% Brij-35, 1 mM APMA and 1 mg / mL α-chymotrypsin); (3) TNCB buffer containing both 10 mM AAPH and 2 μg / mL MMP-9. The system was incubated at 37°C under light-protected conditions with shaking at 100 rpm. 100 μL samples were taken at specified time points (with an equal amount of fresh medium added simultaneously), and the cumulative release of triptolide was determined by HPLC. To further simulate a real pathological environment, Cel@CPT nanoparticles (1 mL, 100 μg / mL Cel) were co-incubated with homogenates of fibrotic liver or kidney tissue and placed in a dialysis bag. The samples were then subjected to shaking dialysis at 37°C with PBS containing 0.2% Tween 80 (pH 7.4) as the receiving solution. After 24 hours, the samples were centrifuged at 12,000 rpm for 10 minutes, and the content of triptolide in the supernatant was determined by HPLC.

[0094] The results showed that treatment with AAPH or MMP-9 alone caused significant structural damage to the nanoparticles; however, when exposed to both stimuli simultaneously, the nanoparticles underwent near-complete disintegration. Figure 3 A). Free drug release was rapid, exceeding 70% within 6 hours; while Cel@CPT release was slow in standard PBS, with a cumulative release of approximately 30% over 48 hours. Under single stimulation with AAPH or MMP-9, the cumulative release over 48 hours increased to approximately 70%; when subjected to dual stimulation with both AAPH and MMP-9, both the rate and extent of drug release were significantly enhanced, with a cumulative release of nearly 90% over 48 hours. Figure 3 B). Cel@CPT showed significantly higher drug release in fibrotic tissue homogenate compared to normal conditions, demonstrating its effective response to the overexpression of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) in fibrotic lesions, achieving on-demand drug release. Figure 3 C).

[0095] Experimental Example 3: Uptake of Cel@CPT nanoparticles on myofibroblasts

[0096] To evaluate the targeting ability of the Cel@CPT nanoparticles prepared in Example 5 above against myofibroblasts, cellular uptake studies were conducted in the human hepatic stellate cell line LX-2 and the rat fibroblast cell line NRK-49F. First, experiments were performed on the human hepatic stellate cell line LX-2. CPT nanoparticles labeled with Cy5.5 fluorescent dye (CPT-Cy5.5) were used for treatment at a working concentration of 100 ng / mL. Experimental groups included LX-2 cells in a resting state (-TGF-β1) and a pathologically activated state (+TGF-β1, pretreated with 2 ng / mL for 48 hours), with free Cy5.5 as a control. Mean fluorescence intensity (MFI) of the cells was measured by flow cytometry. To investigate the molecular mechanism of this selective uptake, a competitive inhibition experiment was further conducted: Before adding CPT-Cy5.5 nanoparticles, activated LX-2 cells were pre-incubated with an excess of free chondroitin sulfate (CS) for 30 minutes to competitively saturate the CD44 receptors on the cell surface. To provide more conclusive evidence, gene knockout verification was performed using RNA interference technology: TGF-β1-stimulated LX-2 cells were transfected with CD44-specific siRNA (CD44-siRNA) and negative control siRNA (control-siRNA), respectively. After confirming that the CD44 gene was effectively knocked down ( Figure 4 (B) Cells were treated with CPT-Cy5.5 nanoparticles and then analyzed by flow cytometry. Following this, experiments were performed on the NRK-49F rat fibroblast cell line, using the same procedures as the human hepatic stellate cell line LX-2.

[0097] The results showed that in resting LX-2 cells, the uptake of CPT-Cy5.5 nanoparticles increased only slightly compared to free Cy5.5. However, in TGF-β1-activated LX-2 cells, the uptake of CPT-Cy5.5 nanoparticles was approximately twice that of free Cy5.5. Figure 4 A) indicates that the nanoparticles have a significantly enhanced targeted uptake capacity on activated hepatic stellate cells. Flow cytometry analysis showed that, compared with the control siRNA group, the nanoparticle uptake in the CD44-siRNA knockout group was significantly reduced (A). Figure 4 (C), thus confirming that the CD44 receptor is a key target mediating the targeted uptake of Cel@CPT nanoparticles. In NRK-49F cells, TGF-β1-activated NRK-49F cells also showed significantly enhanced uptake of CPT-Cy5.5 nanoparticles, and this enhancement effect could also be competitively inhibited by free CS (Cy5.5). Figure 5 A) and the knockout of the CD44 gene ( Figure 5Effective blocking of B and C). The above results indicate that Cel@CPT nanoparticles have enhanced targeting ability against myofibroblasts.

[0098] Experimental Example 4: Distribution of Cel@CPT nanoparticles in a mouse model of liver fibrosis

[0099] Based on the in vitro confirmation of the good targeting effect of nanoparticles on myofibroblasts, the in vivo targeting performance of Cel@CPT nanoparticles prepared in Example 5 was further evaluated using a CCl4-induced liver fibrosis mouse model. Male C57BL / 6 mice aged 8-10 weeks and weighing 20-25 g were used to establish a liver fibrosis model by intraperitoneal injection of a corn oil solution containing 20% ​​(v / v) CCl4 (0.75 mL / kg) twice a week for 8 weeks. Free Cy5.5 or CPT-Cy5.5 (1 mg / kg) was intravenously injected into fibrotic mice and normal control mice. Four hours later, the mice were sacrificed, and the heart, liver, spleen, lungs, and kidneys were harvested. In vitro fluorescence images were acquired using an IVIS Spectrum imaging system, and the fluorescence intensity of Cel@CPT nanoparticles in the liver of fibrotic mice was semi-quantitatively analyzed. To clarify the cellular distribution characteristics of the nanoparticles in liver tissue, flow cytometry was used to analyze different cells isolated from the fibrotic liver.

[0100] The results showed that in vivo imaging revealed the highest fluorescence accumulation of nanoparticles in fibrotic livers. Figure 6 A). Semi-quantitative analysis showed that the fluorescence intensity of the nanoparticle group in fibrotic liver was approximately 5 times that of the free dye group ( Figure 6 B). Flow cytometry analysis showed that CPT nanoparticles were mainly enriched in α-SMA-positive hepatic stellate cells, and their fluorescence intensity was 20 times higher than that of free Cy5.5. Figure 6 C). Notably, the accumulation of nanoparticles in hepatocytes was significantly lower than that of free Cy5.5, indicating that they possess selective delivery properties that target parenchymal cells and tend towards pathogenic myofibroblasts. Figure 6 C). The above results indicate that Cel@CPT nanoparticles have enhanced targeting ability against fibrotic liver in in vivo experiments.

[0101] Experimental Example 5: Distribution of Cel@CPT nanoparticles in a mouse model of renal fibrosis

[0102] A renal fibrosis model was established using 8-10 week old male C57BL / 6 mice weighing 20-25 g via a single intraperitoneal injection of folic acid (250 mg / kg) dissolved in sodium bicarbonate buffer (0.3 mol / L, pH 7.4). Similarly, free Cy5.5 or CPT-Cy5.5 (1 mg / kg) was intravenously injected into fibrotic mice and normal control mice. Four hours later, key tissues (heart, liver, spleen, lung, and kidney) were collected for in vitro fluorescence imaging analysis.

[0103] The results showed that the fibrotic kidneys treated with Cel@CPT nanoparticles prepared in Example 5 exhibited the strongest fluorescence signal. Figure 7 A). Semi-quantitative analysis showed that the fluorescence intensity of the CPT group in fibrotic kidneys was 14 times that of free Cy5.5 (A). Figure 7 (B) indicates that CPT nanoparticles exhibit significant targeting performance in the fibrotic kidney model. These results demonstrate that Cel@CPT nanoparticles possess enhanced targeting ability against fibrotic kidneys in in vivo experiments.

[0104] Experimental Example 6: Therapeutic Effect of Cel@CPT Nanoparticles on Liver Fibrosis in Mice

[0105] Male C57BL / 6 mice aged 8-10 weeks and weighing 20-25 g were used to establish a liver fibrosis model by intraperitoneal injection of CCl4 solution diluted with corn oil at a volume ratio of 1:4, twice a week for 8 weeks. From week 5, drugs were administered via tail vein injection three times a week for 4 weeks. Mice were randomly assigned to receive the following treatments: saline, blank CPT nanoparticles, free triptolide Cel (0.5 mg / kg), non-responsive drug-loaded nanoparticles Cel@CSS (0.5 mg / kg), responsive drug-loaded nanoparticles Cel@CPT (prepared according to Example 5, 0.5 mg / kg), and low-dose responsive drug-loaded nanoparticles Cel@CPT (prepared according to Example 5, 0.25 mg / kg), three times a week via tail vein injection. The CCl4 model treatment lasted for 4 weeks. After treatment, blood and tissue samples were collected for biochemical and molecular analysis.

[0106] Results showed that assessment of liver hydroxyproline content (a quantitative indicator of collagen deposition) revealed the lowest hydroxyproline content in the Cel@CPT nanoparticle treatment group, with no significant difference between the two doses. Figure 8 A). Further immunohistochemical staining with Sirius red and α-SMA revealed a significant reduction in collagen deposition in the livers of model mice in the Cel@CPT-treated group. Figure 8 B). qPCR analysis confirmed that the expression of fibrosis and inflammation-related genes was significantly downregulated after Cel@CPT treatment. Figure 8C). Western blot analysis further confirmed that Cel@CPT nanoparticles significantly reduced α-SMA and Col1a1 protein levels (C). Figure 8 D). Regarding liver injury markers, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were significantly elevated in the CCl4 model group (D). Figure 8 (E, F) Cel@CPT nanoparticle treatment significantly reduced serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. These results indicate that the Cel@CPT nanoparticles provided by this invention have an enhanced therapeutic effect on liver fibrosis.

[0107] Experimental Example 7: Therapeutic Effect of Cel@CPT Nanoparticles on Mice with Renal Fibrosis

[0108] The therapeutic effect of the Cel@CPT nanoparticles prepared in Example 5 was evaluated in a folic acid-induced renal fibrosis model. A renal fibrosis model was established using 8-10 week old male C57BL / 6 mice weighing 20-25 g via a single intraperitoneal injection of folic acid (250 mg / kg). The treatment groups were consistent with those in the liver fibrosis mouse treatment experiment, with all groups receiving folic acid via tail vein injection three times a week for 3 weeks. Kidney tissue and blood samples were collected after treatment for relevant tests.

[0109] The results showed that the Cel@CPT nanoparticle treatment group had the lowest renal hydroxyproline content, which effectively reduced collagen deposition levels. Figure 9 A). Sirius red and Masson staining results showed that the Cel@CPT treatment group alleviated collagen deposition to the greatest extent. Figure 9 B). qPCR analysis showed that Cel@CPT treatment significantly inhibited the mRNA expression of inflammatory cytokines and fibrosis markers (B). Figure 9 C). Western blot analysis confirmed that Cel@CPT treatment reduced α-SMA and Col1a1 protein levels to the lowest level. Figure 9 D). Blood sample test results showed that Cel@CPT treatment restored elevated serum creatinine (CRE) and blood urea nitrogen (BUN) levels to normal. Figure 9 (E), effectively reversing renal dysfunction. In summary, Cel@CPT significantly alleviated renal fibrosis and renal injury in model mice.

[0110] Experimental Example 8: Cel@CPT Nanoparticles Reduce Systemic Toxicity of Tripterygium Witch Hazel

[0111] Hematological analysis was performed on the liver fibrosis model mice and kidney fibrosis model mice that underwent group treatment. The blood samples of the liver fibrosis model mice were from Experiment 6 above, and the blood samples of the kidney fibrosis model mice were from Experiment 7 above.

[0112] The results showed that in the liver fibrosis treatment model, free triptolide significantly increased lymphocyte, leukocyte, and neutrophil counts, while all nanoparticle formulations showed a decreasing trend in toxicity. Among them, the Cel@CPT half-dose group (0.25 mg / kg) had the lowest hematological toxicity. Figure 10 A); Free triptolide also caused elevated levels of myocardial injury markers lactate dehydrogenase and creatine kinase isoenzymes, while no such toxic reactions were observed in the Cel@CPT treatment group ( Figure 10 B). In a renal fibrosis treatment model, Cel@CPT treatment also significantly reduced the hematologic toxicity of triptolide (B). Figure 11 A) and cardiotoxicity ( Figure 11 B). This demonstrates that using the CPT of the present invention as a carrier to encapsulate triptolide for drug delivery can effectively reduce the systemic toxicity of triptolide.

[0113] In summary, the CPT polymer of this invention, when used as a carrier to encapsulate drugs for drug delivery, can effectively reduce the systemic toxicity of drugs and has excellent safety. In terms of controlled release, the drug-encapsulated nanoparticles (Cel@CPT) made from the CPT polymer of this invention have intelligent drug release characteristics, and Cel@CPT exhibits significant targeting performance in the fibrosis model. In terms of efficacy, Cel@CPT of this invention promotes the restoration of liver damage indicators to normal levels, effectively reverses renal dysfunction, and significantly alleviates liver fibrosis and renal fibrosis.

Claims

1. Use of nanoparticles loaded with tripterine in the preparation of a medicament for treating organ fibrosis, characterized in that, The nanoparticles contain polymers that respond to fibrous microenvironment and triptolide; The polymer is a copolymer with chondroitin sulfate as the backbone and modified with aminophenylboronic acid and triglyceride monostearate; The polymer is obtained by reacting chondroitin sulfate and aminophenylboronic acid to obtain a chondroitin sulfate-aminophenylboronic acid copolymer, and then by blending the chondroitin sulfate-aminophenylboronic acid copolymer with triglyceride monostearate. The chondroitin sulfate-aminophenylboronic acid copolymer is obtained by polymerizing the following components in parts by weight: 50 parts chondroitin sulfate, 17.3 parts of aminophenylboronic acid; The polymer is obtained by polymerizing the following components in parts by weight: 10 parts of chondroitin sulfate-aminophenylboronic acid copolymer 7.5 parts of polyglycerol monostearate; The polymer comprises the following branched chondroitin sulfate structural units: wherein n = 10-50; The grafting degree of the aminophenylboronic acid is 8.6%.

2. According to the use of claim 1, the organ fibrosis is liver fibrosis and kidney fibrosis.

3. The use according to claim 2, wherein the organ fibrosis is renal fibrosis.

4. Use according to claim 1, characterized in that, The preparation method of the nanoparticles loaded with triptolide includes the following steps: mixing the polymer with triptolide, stirring, sonicating, dialysis, and centrifuging to obtain the nanoparticles.

Images