Tetrandrine and baicalin self-assembled nanoparticles as well as preparation method and application thereof
By preparing self-assembled nanoparticles of tetrandrine and baicalin, the problems of low solubility and hepatotoxicity were solved, enhancing the therapeutic effect of silicosis and achieving more efficient improvement in lung function and safety.
Patent Information
- Application Number
- CN202511239652.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-01
- Publication Date
- 2025-12-23
AI Technical Summary
Tetrandrine and baicalin have problems with low solubility and limited bioavailability when treating silicosis. Traditional combined administration methods cannot fully exert their synergistic effect, and tetrandrine has the risk of liver and kidney toxicity.
Tetrandrine and baicalin were used to prepare self-assembled nanoparticles. They were dissolved in deionized water by a specific molar ratio and concentration combination, and purified by constant temperature stirring, cooling and dialysis to form self-assembled nanoparticles, which improved solubility and reduced hepatotoxicity and nephrotoxicity.
It significantly improves the solubility of tetrandrine and baicalin, enhances the therapeutic effect of silicosis, reduces respiratory rate, improves lung function and fibrosis, reduces the risk of liver and kidney toxicity, and achieves better therapeutic results.
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Figure CN121177227A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to the self-assembled nanoparticles of tetrandrine and baicalin, their preparation methods, and applications. Background Technology
[0002] Silicosis, also known as pneumoconiosis, is an occupational disease caused by long-term inhalation of dust containing free silica, characterized by widespread nodular fibrosis in the lungs. It is the most common, fastest-progressing, and most serious type of pneumoconiosis. Silicosis is mainly caused by occupational exposure, commonly seen in the following industries: mining and quarrying: workers are exposed to rock dust containing silica; tunnel excavation and digging: large amounts of free silica dust are generated during operations; quartz powder factories, glass factories, and refractory material factories: high concentrations of dust are released during production processes. Pathogenesis: After inhalation, some dust is deposited in the alveoli and phagocytosed by alveolar macrophages, releasing inflammatory factors such as TNF-α and IL-1. The toxicity of silica leads to macrophage degeneration and necrosis, releasing fibrotic factors that stimulate fibroblast proliferation and collagen fiber production. Repeated stimulation leads to pulmonary fibrosis, forming nodular fibrotic lesions. As the nodules increase and merge, lung function gradually declines, ultimately affecting gas exchange. There is currently no cure for silicosis and no specific drugs. Treatment focuses on slowing disease progression, relieving symptoms, and improving quality of life. Commonly used medications include silicone and tetrandrine to slow the progression of fibrosis.
[0003] Tetrandrine (TET) is one of the commonly used drugs in clinical practice for the treatment of silicosis. Its therapeutic effect has been verified in long-term practice, mainly in delaying disease progression and inhibiting nodule enlargement. Tetrandrine is an alkaloid extracted from the traditional Chinese medicine Stephania tetrandra. Its core anti-silicosis mechanism includes: inhibiting dust damage to macrophages: reducing inflammatory factors such as TNF-α and IL-1 released after macrophages phagocytose silica dust, thus alleviating chronic inflammatory response in lung tissue; and inhibiting collagen synthesis: reducing fibroblast activity, decreasing the excessive synthesis of collagen and other fibrosis-related substances, and delaying the progression of silicosis nodules and interstitial fibrosis.
[0004] Tetrandrine has been used in my country for over 50 years, and in 2018 it became a recommended drug in the consensus of Chinese pneumoconiosis experts. However, the efficacy of tetrandrine in treating pneumoconiosis has not yet met expectations, thus necessitating the search for new treatment options.
[0005] Baicalin BA, based on its anti-inflammatory, antioxidant, and hepatoprotective properties, may be used as an adjunct therapy in the comprehensive treatment of silicosis, in combination with tetrandrine and other drugs to enhance anti-inflammatory and anti-fibrotic effects while reducing the hepatotoxicity and nephrotoxicity of tetrandrine. However, both drugs suffer from low solubility and limited bioavailability, making it difficult to fully realize their synergistic effects using traditional simple combination therapy. Summary of the Invention
[0006] To address the aforementioned technical problems and enhance the therapeutic effect of tetrandrine on silicosis, this invention provides a self-assembled nanoparticle of tetrandrine and baicalin, its preparation method, and its application.
[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A self-assembled nanoparticle of tetrandrine and baicalin, comprising the following materials: tetrandrine and baicalin.
[0008] Preferably, the molar ratio of tetrandrine to baicalin is 1:0.5 - 1:3.
[0009] The most preferred molar ratio of tetrandrine to baicalin is 1:2.
[0010] Preferably, the concentrations of tetrandrine and baicalin are 0.56-2.24 mM and 1.12-4.48 mM, respectively.
[0011] The most preferred concentrations of tetrandrine and baicalin are 1.12 mM and 2.24 mM, respectively.
[0012] A method for preparing self-assembled nanoparticles of tetrandrine and baicalin includes the following steps: weighing tetrandrine and baicalin according to the required molar ratio, dissolving them together in deionized water, stirring at 100°C for 1 hour, naturally cooling to room temperature to complete the self-assembly process, centrifuging to remove large aggregates, and dialysis purification to obtain self-assembled nanoparticles.
[0013] Application of self-assembled nanoparticles of tetrandrine and baicalin: They can be used for the treatment of silicosis.
[0014] The beneficial effects of this invention are as follows: Compared with the prior art, the self-assembled nanoparticles of tetrandrine and baicalin significantly improve the solubility of both tetrandrine and baicalin; the self-assembled nanoparticles of tetrandrine and baicalin can significantly reduce the respiratory rate of silicosis rats and enhance the improvement effects on lung function, silicosis nodules, alveolar structure, lung index, hydroxyproline content, and silicosis fibrosis; more importantly, the self-assembled nanoparticles of this invention show significantly better therapeutic effects than the tetrandrine-only group even when the dosage of tetrandrine is only half that of the tetrandrine-only group; at the same time, even when the drug loading of tetrandrine in the self-assembled nanoparticles is increased to twice the level of the tetrandrine-only group, no liver or kidney toxicity is observed, and its serum biochemical indicators, including creatinine, urea nitrogen, aspartate aminotransferase, and alanine aminotransferase, are maintained at the levels of the normal control group; in stark contrast, significant liver and kidney toxicity occurs when tetrandrine is used alone. Therefore, the self-assembled nanoparticles of tetrandrine and baicalin of the present invention not only effectively eliminate the risk of hepatotoxicity and nephrotoxicity of tetrandrine, but also achieve the goal of obtaining better therapeutic effect while reducing the dosage of tetrandrine, and have significant advantages in clinical application. Attached Figure Description
[0015] Figure 1 The turbidity and Tyndall effect of the self-assembled systems of tetrandrine and baicalin at different molar ratios in this invention are investigated.
[0016] Figure 2 The particle size and morphology of the self-assembled systems of tetrandrine and baicalin at different concentrations are shown.
[0017] Figure 3 The appearance of TET, BA, and the TET / BA SANPs of the present invention under the same conditions.
[0018] Figure 4 Characterization of the self-assembled nanoparticle structure of tetrandrine and baicalin in this invention.
[0019] Figure 5 This invention demonstrates the effect of self-assembled nanoparticles of tetrandrine and baicalin on improving lung function and histopathology in patients with silicosis.
[0020] Figure 6 This invention demonstrates the effect of self-assembled nanoparticles of tetrandrine and baicalin on improving silicosis fibrosis.
[0021] Figure 7 The results of liver and kidney toxicity testing of the self-assembled nanoparticles of tetrandrine and baicalin of this invention are shown. Detailed Implementation
[0022] To make the technical objectives, technical solutions, and technical effects of the present invention easier to understand, the technical solutions of the present invention will now be further described in conjunction with the accompanying drawings and embodiments.
[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0024] The abbreviations in the following examples represent the following substances: Tetrandrine: TET, Baicalin: BA, Tetrandrine and Baicalin self-assembled nanoparticles: TET / BA SANPs, TET / BA SANPs low dose group: TB-L, TET / BASANPs medium dose group: TB-M, TET / BA SANPs high dose group: TB-H.
[0025] A self-assembled nanoparticle of tetrandrine and baicalin A self-assembled nanoparticle of tetrandrine and baicalin, comprising the following materials: tetrandrine and baicalin, preferably with a molar ratio of tetrandrine to baicalin of 1:0.5-1:3, and most preferably with a molar ratio of tetrandrine to baicalin of 1:2. Preferably, the concentrations of tetrandrine and baicalin are 0.56-2.24 mM and 1.12-4.48 mM, respectively. Most preferably, the concentrations of tetrandrine and baicalin are 1.12 mM and 2.24 mM, respectively.
[0026] A method for preparing self-assembled nanoparticles of tetrandrine and baicalin A method for preparing self-assembled nanoparticles of tetrandrine and baicalin includes the following steps: (1) Weigh out tetrandrine and baicalin according to the required molar ratio; (2) Dissolve tetrandrine and baicalin together in deionized water; (3) Stir at a constant temperature of 100℃ for 1 hour; (4) Allow the solution to cool naturally to room temperature to complete the self-assembly process; (5) Centrifuge the self-assembled nanoparticle suspension at 5000 rpm for 10 minutes to remove large aggregates; (6) Collect the supernatant and dialyze in deionized water for 9 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, changing the dialysate every 3 hours; (7) A suspension of self-assembled nanoparticles of tetrandrine and baicalin was obtained.
[0027] Screening of the preparation ratio of tetrandrine-baicalin self-assembled nanoparticles To systematically investigate the self-assembly ratio of TET and BA, this invention prepared and screened ten self-assembly systems with different molar ratios: 1:0.5, 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, 1:2.25, 1:2.5, 1:2.75, and 1:3. Figure 1 As shown in Figure 1A, the experimental results show that solutions with a molar ratio in the range of 1:0.5-1:2 form a transparent pale yellow solution after heating and cooling, and the precipitation gradually decreases with increasing BA ratio. When the molar ratio is in the range of 1:2.25-1:3, the solution becomes turbid and separates into layers after heating and cooling, with 1:2 being the critical ratio for both transparency and turbidity. As shown in Figure 1B, laser irradiation detection revealed that in systems with a molar ratio of 1:0.5-1:2, the Tyndall effect increases with increasing BA ratio, but when the BA ratio exceeds 2, the laser penetration is significantly hindered. Based on the above experimental results, the optimal molar ratio of TET to BA is determined to be 1:2.
[0028] Preparation and concentration screening of self-assembled nanoparticles of tetrandrine and baicalin Based on the determination of the optimal molar ratio, to further clarify the optimal concentration for self-assembly, this invention tested the particle size and dispersibility of the system with a molar ratio of 1:2 at four different concentrations. Figure 2 As shown in Figure 2A, the dynamic light scattering (DLS) results indicate that when the TET concentration is 0.56 mM and the BA concentration is 1.12 mM, the nanoparticle size is 354.64 nm and the polydispersity index (PDI) is 0.64. When the TET concentration is increased to 1.12 mM and the BA concentration is correspondingly increased to 2.24 mM, the nanoparticle size decreases to 328 nm and the PDI decreases to 0.283. However, when the TET concentration is further increased to 1.68 mM (corresponding to a BA concentration of 3.36 mM) and further increased to 2.24 mM (corresponding to a BA concentration of 4.48 mM), multiple peaks appear in the nanoparticle size distribution curves, as shown in Figure 2CD. Therefore, the nanoparticles formed when the TET concentration is 1.12 mM, the BA concentration is 2.24 mM, and the molar ratio is 1:2 are the most uniform. At this point, the Zeta potential of the self-assembled nanoparticles is -5.38 mV, as shown in Figure 2E. The scanning electron microscope (SEM) observation results also show that TET / BA SANPs are spherical with an average particle size of 208 nm, as shown in Figure 2F.
[0029] Solubility of self-assembled nanoparticles of tetrandrine and baicalin Studies have reported low solubilities for both TET and BA, at 0.015 mg / mL and 0.043 mg / mL, respectively. However, in this invention, the 1:2 molar ratio self-assembly system significantly increased the solubility of TET to 0.697 mg / mL, a 46.5-fold increase, and the solubility of BA to 0.998 mg / mL, an approximately 23.2-fold increase. As shown in Figure 3, solutions of free monomeric TET and BA at the same concentration remained turbid, while the self-assembled nanoparticle solution was transparent, showing a significant difference from the individual TET and BA solutions.
[0030] Characterization of the self-assembled nanoparticle structure of tetrandrine and baicalin Elucidating the intermolecular interactions is crucial for understanding the self-assembly process of TET / BA SANPs. For example... Figure 4 As shown in Figure A, UV-Vis spectroscopy confirmed the formation of TET / BA SANPs. The experimental results showed that the characteristic absorption peak of TET was at 280 nm, while the characteristic absorption peaks of BA were at 276 nm and 317 nm. The characteristic absorption peaks of TET / BA SANPs were at 277 nm and 317 nm, indicating the presence of both BA and TET within the TET / BA SANPs.
[0031] Fourier transform infrared spectroscopy (FTIR) provided direct evidence for specific intermolecular interactions. In the hydroxyl region, BA showed multiple peaks at 3727, 3550, 3489, and 3398 cm⁻¹, originating from the phenolic and glucuronic acid hydroxyl groups, respectively, while TET showed a single hydroxyl peak at 3435 cm⁻¹. After self-assembly, these independent hydroxyl peaks converged to form a single broad absorption peak at 3435 cm⁻¹ in TET / BASANPs, exhibiting a maximum redshift of 292 cm⁻¹ relative to BA's strongest peak at 3727 cm⁻¹, confirming extensive hydrogen bonding between BA and TET. Furthermore, the COC stretching vibration peak of the BA glucuronic acid ring split from a single peak at 1066 cm⁻¹ into two peaks at 1120 and 1080 cm⁻¹. This peak split indicates a significant conformational change in the glycosyl moiety of BA during self-assembly, suggesting that the BA glucuronic acid ring actively participates in intermolecular interactions with the TET molecule, as shown in Figure 4B.
[0032] Powder X-ray diffraction (XRD) analysis showed that BA and TET alone have high crystallinity, and BA and TET exhibit obvious diffraction peaks. However, in the XRD pattern of TET / BA SANPs formed by self-assembly, the number of diffraction peaks decreased significantly, and the peak shapes broadened. This indicates that the crystallinity of TET / BA SANPs decreased, forming a more amorphous self-assembled structure, as shown in Figure 4C.
[0033] ¹H-NMR spectroscopy provided crucial evidence for understanding the precise interaction sites between BA and TET. In the spectra of the self-assembled nanoparticles, the H-5" (δ 4.07–4.09 ppm) signal of the BA glycoside moiety shifted to δ 3.98–4.00 ppm. Since H-5" is adjacent to the 6"-COOH carboxyl group, this high-field shift suggests that the carboxyl group may be involved in hydrogen bonding interactions with the nitrogen atom of TET. Furthermore, the disappearance of the 2", 3", and 4"-OH signals (δ 5.30–5.51 ppm) further confirms that multiple hydroxyl groups are involved in hydrogen bonding with TET. Of particular note, the 6-OH signal at δ 8.66 ppm in BA completely disappeared after self-assembly; simultaneously, the 2-NCH3 in TET shifted from δ 2.51 ppm to δ 2.68 ppm. This low-field shift indicates a possible interaction between the nitrogen atom adjacent to the N-methyl group and the 6-OH group in BA, as shown in Figure 4D. Combining the results of the above characterization methods, it is clear that when the feed ratio of TET to BA is 1:2, the two can form self-assembled nanoparticles through non-covalent interactions.
[0034] The effects of tetrandrine and baicalin self-assembled nanoparticles on improving lung function and histopathology in silicosis. To investigate the ameliorative effect of TET / BA SANPs on silicosis, we established a rat model of silicosis by intratracheal instillation of micron-sized SiO2 particles. Pulmonary function tests showed that, compared with the control group (C), the tidal volume (TV) of the model group (M) was significantly reduced. Compared with the model group (M), the TV of the TET and BA groups increased, but this was not statistically significant. However, the TV of all three doses of TET / BA SANPs groups increased significantly. Figure 5 A. Furthermore, compared to the control group C, the relative respiratory time (Repf) in the model group M was significantly increased, while it decreased in the TET and BA groups, but this was not statistically significant. All three doses of TET / BA SANPs significantly reduced Repf. Figure 5 B. Furthermore, the respiratory rate F in the model group M was significantly higher than that in the control group C, and was significantly reduced in all intervention groups, with the low-dose TET / BA SANPs group showing a significantly lower rate than the TET and BA groups, such as... Figure 5 C. Lung index test results showed that the lung index of the model group M was significantly higher than that of the control group C, while the lung index of each intervention group was significantly lower. Among them, the lung index of the low-dose TET / BA SANPs group was significantly lower than that of the TET and BA groups. Figure 5 D.
[0035] Microscopic observation revealed numerous silicosis nodules and alveolar fusion in the lung tissue of the model group M. In the intervention group, nodule formation was reduced, and alveolar damage was repaired. However, compared with the three different doses of TET / BA SANPs groups, the TET group had limited effect on improving alveolar structure, such as... Figure 5 E. Mean linear intercept (MLI) analysis showed that the MLI value was significantly higher in the model group compared to the control group (C), and significantly lower in all intervention groups. Notably, the MLI values of the three doses of TET / BA SANPs were significantly lower than those of the TET and BA groups, as shown in the figure. Figure 5 F. Mean alveolar number (MAN) analysis showed a significant decrease in M in the model group, no significant change in the TET group, while the BA group and the low-dose and medium-dose TET / BA SANPs groups showed a significant increase in M compared to the model group and the TET group. Figure 5 G. Overall, although both the TET and BA groups showed improvements in lung function, lung index, nodule reduction, and alveolar structure, the TET / BA SANPs group demonstrated better results.
[0036] The effect of tetrandrine and baicalin self-assembled nanoparticles on improving silicosis fibrosis To comprehensively evaluate the anti-fibrotic effect of TET / BA SANPs, this invention analyzed changes in hydroxyproline (HYP) and fibrosis-related biomarkers in lung tissue. HYP analysis showed that HYP levels in the lung tissue of the model group (M) were significantly increased compared to the control group (C), while HYP levels were significantly decreased in all intervention groups. HYP levels in the medium- and high-dose TET / BA SANPs groups were significantly lower than those in the TET and BA groups, as shown in the following figures. Figure 6 A. Sirius red staining showed that, compared with the control group C, the collagen level in the model group M was significantly increased, while the collagen level in all intervention groups was significantly decreased. Collagen deposition in the three different doses of TET / BA SANPs groups was significantly lower than that in the TET group, such as... Figure 6 Immunohistochemical analysis showed a significant increase in type I collagen COL-1 expression in model group M. Compared with model M, all intervention groups showed a significant decrease in COL-1 expression, with the medium-dose TET / BA SANPs group showing a significantly lower expression than the TET group. α-SMA expression was also significantly increased in model group M, and compared with model group M, α-SMA expression was significantly decreased in all intervention groups. The low-dose and medium-dose TET / BA SANPs groups showed significantly lower expression than the TET group. Figure 6 B, 6D-E. These analyses indicate that TET / BA SANPs have a superior anti-fibrotic effect compared to TET and BA alone, and can effectively reduce collagen deposition and inhibit the expression of fibrosis-related proteins.
[0037] Safety assessment of self-assembled nanoparticles containing tetrandrine and baicalin To assess the safety of self-assembled nanoparticles, this invention measured serum creatinine (Cr), blood urea nitrogen (BUN), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels in rats of each group. The results showed that compared to the control group (C), the model group (M) did not exhibit significant changes in Cr, BUN, AST, and ALT levels, indicating that the silicosis model did not cause liver or kidney toxicity. However, the TET group showed significantly elevated BUN, AST, and ALT levels, while these parameters did not show significant changes in the BA group. Compared to the TET group, the medium-dose TET / BA SANPs group contained the same dose of TET, and the high-dose group contained twice the dose of TET. The BUN levels in the three different doses of TET / BA SANPs groups were comparable to those in the control group (C), with no significant increase. Figure 7 AB. Similarly, serum transaminase analysis showed that AST and ALT levels in the three different doses of TET / BA SANPs groups were comparable to those in control group C, such as... Figure 7 CD. These results indicate that TET / BASANPs did not show significant hepatotoxicity or nephrotoxicity and were safer than TET alone in the treatment of silicosis.
[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A self-assembled nanoparticle of tetrandrine and baicalin, characterized in that, It includes the following materials: tetrandrine and baicalin.
2. The self-assembled nanoparticles of tetrandrine and baicalin according to claim 1, characterized in that, The molar ratio of tetrandrine to baicalin is 1:0.5–1:
3.
3. The self-assembled nanoparticles of tetrandrine and baicalin according to claim 2, characterized in that, The molar ratio of tetrandrine to baicalin is 1:
2.
4. The self-assembled nanoparticles of tetrandrine and baicalin according to claim 3, characterized in that, The concentrations of tetrandrine and baicalin were 0.56-2.24 mM and 1.12-4.48 mM, respectively.
5. The self-assembled nanoparticles of tetrandrine and baicalin according to claim 4, characterized in that, The concentrations of tetrandrine and baicalin were 1.12 mM and 2.24 mM, respectively.
6. The method for preparing self-assembled nanoparticles of tetrandrine and baicalin as described in any one of claims 1-5, characterized in that, The preparation method includes the following steps: S1: Weigh out tetrandrine and baicalin according to the required molar ratio; S2: Dissolve tetrandrine and baicalin together in deionized water; S3: Stir at 100°C for 1 hour; S4: Allow the solution to cool naturally to room temperature to complete the self-assembly process; S5: Centrifugation to remove large aggregates; S6: Collect the supernatant and dialyze it to obtain self-assembled nanoparticles.
7. The application of the self-assembled nanoparticles of tetrandrine and baicalin prepared by the method of preparing the self-assembled nanoparticles of tetrandrine and baicalin as described in any one of claims 1-5 or claim 6.