Cationic nanocarrier for penetrating fibrocartilage and its preparation method and application

Abstract

The present invention discloses a cationic nanocarrier for penetrating fibrocartilage and its preparation method and application, which belongs to the field of biomedicine technology. The preparation method comprises the following steps: mixing an ethylenediamine solution and a Boc-Lys(Fmoc)-OH solution, adding HATU and HOAt for activation, adding DIPEA for further activation, stirring the reaction, adding glacial ether and standing to obtain a precursor; mixing the precursor and a piperidine solution, adding glacial ether and n-hexane and standing to obtain DGL; reacting DGL in a mixed solution of TFA, DCM and TIS to obtain de-Boc DGL, and reacting the de-Boc DGL with phenylboronic acid in a solvent. The cationic nanocarrier prepared by the present invention can reversibly bind to glycosaminoglycans in cartilage and penetrate the full layer of condylar cartilage of the temporomandibular joint while carrying siRNA to target deep chondrocytes, giving lysosomal escape ability, promoting chondrocyte uptake, and improving siRNA transfection efficiency.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a cationic nanocarrier for penetrating fibrocartilage, and a preparation method and application thereof. Background Art

[0002] Articular cartilage, particularly the surface fibrocartilage of the temporomandibular joint condyle, is dense. Therefore, existing delivery systems for intra-articular injections for osteoarthritis are unable to penetrate the cartilage, resulting in limited drug penetration and inability to fully exert therapeutic effects. Especially in early-stage temporomandibular joint osteoarthritis, condylar cartilage lesions begin in the fibrous layer and exhibit fibrosis and thickening of type I collagen in the fibrocartilage layer, further hindering drug penetration.

[0003] Furthermore, the density of glycosaminoglycans distributed within the staggered collagen fiber network increases with cartilage depth, significantly reducing the pore size available for drug penetration, affecting drug transport and targeted binding to deep chondrocytes, and reducing the efficiency of chondrocyte transfection. On the other hand, glycosaminoglycans carry a large amount of fixed negative charge, providing an opportunity to utilize electrostatic interactions to enhance drug transport in cartilage.

[0004] Cationic nanocarriers can penetrate the fibrocartilage barrier and enhance tissue binding and penetration through reversible electrostatic interactions with anionic cartilage tissue. However, existing nanocarriers face significant challenges in cellular uptake, gene delivery, and lysosomal escape, resulting in insufficient transfection efficiency in cartilage cells. Therefore, developing cationic nanocarriers that can penetrate fibrocartilage and enhance chondrocyte transfection efficiency is a key challenge currently underway in the treatment of osteoarthritis (OA). Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a cationic nanocarrier for penetrating fibrocartilage and its preparation method and application, so as to solve the problems of existing nanocarriers in terms of cell uptake, gene delivery and lysosomal escape, and insufficient transfection efficiency of cells in cartilage.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] The first aspect of the present invention provides a method for preparing a cationic nanocarrier for penetrating fibrocartilage, comprising the following steps:

[0008] (1) First, mix the ethylenediamine solution and the Boc-Lys(Fmoc)-OH solution evenly, then add HATU and HOAt for activation, then add DIPEA for further activation, finally stir the reaction, add icy ether and let it stand, collect the precipitate and dry it to obtain the polylysine precursor;

[0009] (2) first dissolving the polylysine precursor obtained in step (1) in a solvent, adding a piperidine solution and stirring, then adding glacial ether and n-hexane and allowing to stand, collecting the precipitate and drying it to obtain polylysine;

[0010] (3) The polylysine obtained in step (2) is stirred in a mixed solution of trifluoroacetic acid, dichloromethane and triisopropylsilane, then added to icy ether and allowed to stand, and the precipitate is collected and dried to obtain de-tert-butyloxycarbonyl polylysine;

[0011] (4) The de-tert-butyloxycarbonyl polylysine obtained in step (3) and 4-(bromomethyl)phenylboronic acid are mixed in a solvent for reaction to obtain the product.

[0012] The beneficial effects of the present invention are as follows: the preparation method of the present invention is simple, and a cationic nanocarrier is prepared by modifying polylysine with 4-(bromomethyl)phenylboronic acid through grafting modification. The carrier can retain the cationic properties of polylysine, can reversibly bind to the anionic glycosaminoglycans contained in the cartilage, and can penetrate the entire layer of the condylar cartilage of the temporomandibular joint while carrying siRNA to target deep-layer chondrocytes, and endow them with lysosomal escape ability, thereby promoting the uptake of grafted modified DGL by chondrocytes, improving the transfection efficiency of the siRNA, and can accurately regulate the expression of target genes and proteins according to the targets screened by sequencing, and is suitable for promotion and application.

[0013] Furthermore, the ethylenediamine solution is prepared by the following method:

[0014] Prepared by stirring ethylenediamine and DMF at -5~5℃ under argon atmosphere for 5-20 min.

[0015] Furthermore, in step (1), the mixing temperature is -5~5°C; the activation temperature is -5~5°C, and the time is 5-20 min; the activation temperature is -5~5°C, and the time is 3-7 min; the stirring reaction temperature is room temperature, and the time is 15-20 h; the standing temperature is 1-10°C, and the time is 1-3 h.

[0016] Furthermore, in step (1), the volume ratio of glacial ether to the solution after stirring the reaction is (2-4): (1-2).

[0017] Furthermore, in step (2), the concentration of the polylysine precursor solution is 50-80 g / L, and the solvent is DMF; the concentration of the piperidine solution is 10%-20%, and the solvent is DMF; the volume ratio of the polylysine precursor solution to the piperidine solution is (5-10):(5-10); and the volume ratio of glacial ether to n-hexane is (0.8-1.2):(0.8-1.2).

[0018] Furthermore, in step (2), the stirring time is 20-40 min; the standing temperature is 1-10° C., and the standing time is 2-4 h.

[0019] Furthermore, in step (2), the volume ratio of the total volume of glacial ether and n-hexane to the reaction solution obtained after stirring is (4-6): (1-2).

[0020] Furthermore, in step (3), the mass volume ratio of the mixed solution consisting of polylysine and trifluoroacetic acid, dichloromethane and triisopropylsilane is 30-40 g:500-1000 mL; the volume ratio of trifluoroacetic acid, dichloromethane and triisopropylsilane is (40-60):(35-55):(3-7); the stirring reaction temperature is -5~5°C, and the time is 3-5 h.

[0021] Furthermore, in step (4), the molar ratio of polylysine to 4-(bromomethyl)phenylboronic acid is (100-200):1, and the solvent is methanol.

[0022] Preferably, the molar ratio of polylysine to 4-(bromomethyl)phenylboronic acid in step (4) is 150:1.

[0023] Furthermore, in step (4), the reaction temperature is 40-60° C. and the reaction time is 40-60 h.

[0024] The second aspect of the present invention provides a cationic nanocarrier for penetrating fibrocartilage, which is prepared by the above preparation method.

[0025] The third aspect of the present invention provides the use of the above-mentioned cationic nanocarrier for penetrating fibrocartilage in the preparation of osteoarthritis siRNA drugs.

[0026] A fourth aspect of the present invention provides an osteoarthritis siRNA drug, which is prepared by mixing the above-mentioned cationic nanocarrier for penetrating fibrocartilage with siRNA for siRNA loading.

[0027] Furthermore, the mass ratio of the cationic nanocarrier to the siRNA is (5-20):1.

[0028] Preferably, the mass ratio of cationic nanocarrier to siRNA is 10:1.

[0029] The present invention has the following beneficial effects:

[0030] The present invention prepares a cationic nanocarrier capable of penetrating cartilage fibers by modifying polylysine with 4-(bromomethyl)phenylboronic acid. The carrier degrades monomers into amino acids required by the body, is non-toxic, highly controllable, and has good biosafety. The diameter before and after grafting modification is smaller than the pores of condylar cartilage, allowing it to penetrate dense type I collagen fibers. The PBA-DGL obtained by grafting modification contains cations and can achieve targeted binding with anion-rich glycosaminoglycan components in the deep layer of cartilage through electrostatic interactions, facilitating penetration into the deep layer of cartilage to exert therapeutic effects. The cationic nanocarrier prepared by the present invention can carry negatively charged gene therapy drugs such as siRNA or microRNA, efficiently penetrate the fibrous cartilage layer, and has clinical translation potential to improve the transfection efficiency and targeting effect of condylar chondrocytes in the treatment of temporomandibular joint OA. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of poly-lysine modification and loading of siRNA to penetrate condylar fibrocartilage and transfect chondrocytes;

[0032] Figure 2 IR spectra of the cationic nanocarriers prepared in Example 1 and Comparative Example 1;

[0033] Figure 3 The cationic nanocarriers prepared in Example 1 and Comparative Example 1 are 1 H NMR spectrum;

[0034] Figure 4 Lens characterization images and particle size distribution images of cationic nanocarriers prepared in Example 1 and Comparative Example 1, wherein (a) is the lens characterization image of DGL and PBA-DGL, (b) and (c) are the particle size distribution images of DGL and PBA-DGL, respectively;

[0035] Figure 5 The agarose gel electrophoresis characterization results of the cationic nanocarriers loaded with siRNA prepared in Example 1 and Comparative Example 1, wherein (a) is DGL@siRNA and (b) is PBA-DGL@siRNA;

[0036] Figure 6 Lens characterization images and particle size distribution images of cationic nanocarriers loaded with siRNA prepared in Example 1 and Comparative Example 1, wherein (a) is the lens characterization image of DGL@siRNA and PBA-DGL@siRNA, (b) and (c) are the particle size distribution images of DGL@siRNA and PBA-DGL@siRNA, respectively;

[0037] Figure 7 The quantitative results of the particle size of the cationic nanocarriers prepared in Example 1 and Comparative Example 1 before and after loading siRNA;

[0038] Figure 8 The zeta potential test results of the cationic nanocarriers prepared in Example 1 and Comparative Example 1 before and after loading siRNA are shown, where (a)-(d) are DGL, PBA-DGL, DGL@siRNA, and PBA-DGL@siRNA, respectively;

[0039] Figure 9 Schematic diagram of fluorescence penetration detection in cartilage explants;

[0040] Figure 10 Figure 2 shows the penetration effect of PBA-DGL@siRNA and pure siRNA on condylar cartilage explants, where (a) is a fluorescence image and (b) is a topographic quantitative image.

[0041] Figure 11 AFM morphology of condylar fibrocartilage in TMJOA model and normal rat model;

[0042] Figure 12 Figure 2 shows the penetration effect of PBA-DGL@siRNA and pure siRNA on condylar fibrocartilage in vivo, where (a) is a fluorescence image and (b) is a topographic quantitative image.

[0043] Figure 13 Fluorescence images of the cationic nanocarriers prepared in Example 1 and Comparative Example 1, loaded with FAM-siRNA, transfected into condylar chondrocytes in the TMJOA model 24 hours after transfection;

[0044] Figure 14 Flow cytometric analysis of the cationic nanocarriers prepared in Example 1 and Comparative Example 1 carrying FAM-siRNA for transfection into condylar chondrocytes in the TMJOA model 24 hours after transfection;

[0045] Figure 15 This is an analysis diagram of the mechanism of cationic nanocarriers prepared in Example 1 and Comparative Example 1 promoting lysosomal escape by transfecting condylar chondrocytes with lysotracker probes;

[0046] Figure 16 This is a diagram showing the results of biosafety analysis of the cationic nanocarriers prepared in Example 1 when applied to rats. DETAILED DESCRIPTION

[0047] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not intended to limit the scope of the invention. In the embodiments, if specific conditions are not specified, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0048] Example 1:

[0049] A method for preparing a cationic nanocarrier for penetrating fibrocartilage comprises the following steps:

[0050] (1) First, ethylenediamine (EDA, 45 mmol, 3 mL) and dimethylformamide (DMF, 500 mL) were stirred at 0 °C under argon for 10 min to obtain an ethylenediamine solution; Boc-Lys(Fmoc)-OH (150 mmol, 62 g) and DMF (500 mL) were mixed evenly to obtain a Boc-Lys(Fmoc)-OH solution; then, the ethylenediamine solution and the Boc-Lys(Fmoc)-OH solution were mixed evenly at 0 °C, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 150 mmol, 57 g) and 1-hydroxy-7-azabenzotriazole (HOAt, 150 mmol, 20.4 g) were added and stirred for 10 min for activation; then, N,N-diisopropylethylamine (DIPEA, 600 mmol, 105 mL) was added and activation was continued for 5 min. min; finally, the temperature was raised to room temperature and stirred for 18 h, 3 L of icy ether was added to the reaction solution, and the mixture was allowed to stand at 4°C for 2 h. The precipitate was collected by centrifugation and dried in vacuo to obtain 50 g of DGL precursor.

[0051] (2) 50 g of the DGL precursor obtained in step (1) was dissolved in 800 mL of DMF at room temperature, followed by addition of 800 mL of a 20% piperidine solution in DMF and stirring for 30 min. The mixture was poured into 5 L of glacial ether and n-hexane in a volume ratio of 1:1, and allowed to stand at 4°C. The precipitate was collected after centrifugation and vacuum dried to obtain 35 g of DGL.

[0052] (3) 35 g of DGL obtained in step (2) was stirred in a mixed solution of trifluoroacetic acid (TFA), dichloromethane (DCM) and triisopropylsilane (TIS) (the volume ratio of TFA, DCM and TIS was 50:45:5) under ice bath conditions for 4 h, 3 L of ice ether was added and the mixture was allowed to stand at -20°C. The precipitate was collected after centrifugation and vacuum dried to obtain 25 g of Boc-free DGL.

[0053] (4) The de-Boc DGL obtained in step (3) and 4-(bromomethyl)phenylboronic acid (PBA) were reacted in methanol at a molar concentration ratio of 150:1 at 50°C for 48 h. The obtained product was fully freeze-dried to obtain PBA-modified DGL (PBA-DGL), which is a cationic nanocarrier for penetrating fibrocartilage.

[0054] Schematic diagram of polylysine modification and siRNA loading for chondrocyte transfection Figure 1 shown.

[0055] Example 2:

[0056] A method for preparing a cationic nanocarrier for penetrating fibrocartilage comprises the following steps:

[0057] (1) First, ethylenediamine (EDA, 45 mmol, 3 mL) and dimethylformamide (DMF, 900 mL) were stirred at 0 °C under argon for 15 min to obtain ethylenediamine solution; Boc-Lys(Fmoc)-OH (150 mmol, 62 g) and DMF (600 mL) were mixed evenly to obtain Boc-Lys(Fmoc)-OH solution; then, the ethylenediamine solution and Boc-Lys(Fmoc)-OH solution were mixed evenly at 0 °C, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 150 mmol, 57 g) and 1-hydroxy-7-azabenzotriazole (HOAt, 150 mmol, 20.4 g) were added and stirred for 20 min for activation; then, N,N-diisopropylethylamine (DIPEA, 600 mmol, 105 mL) was added to continue activation for 7 min. min; finally, the temperature was raised to room temperature and stirred for 20 h, 3 L of icy ether was added to the reaction solution, and the mixture was allowed to stand at 4°C for 2 h. The precipitate was collected by centrifugation and dried in vacuo to obtain the DGL precursor.

[0058] (2) The DGL precursor obtained in step (1) was dissolved in 500 mL of DMF at room temperature, and then 500 mL of a DMF solution with a concentration of 20% piperidine was added and stirred for 30 min. The mixture was poured into 5 L of glacial ether and n-hexane in a volume ratio of 1:1, and allowed to stand at 4°C. The precipitate was collected after centrifugation and vacuum dried to obtain DGL.

[0059] (3) 30 g of DGL obtained in step (2) was added to 700 mL of a mixed solution of trifluoroacetic acid (TFA), dichloromethane (DCM) and triisopropylsilane (TIS) (the volume ratio of TFA, DCM and TIS was 50:45:5) and stirred in an ice bath for 4 h. 3 L of ice ether was added and the mixture was allowed to stand at -20°C. The precipitate was collected after centrifugation and dried in vacuo to obtain Boc-free DGL.

[0060] (4) The Boc-modified DGL obtained in step (3) and 4-(bromomethyl)phenylboronic acid (PBA) were reacted in methanol at a molar concentration ratio of 100:1 at 50°C for 48 h. The obtained product was fully freeze-dried to obtain PBA-modified DGL (PBA-DGL), which is a cationic nanocarrier for penetrating fibrocartilage.

[0061] Example 3:

[0062] A method for preparing a cationic nanocarrier for penetrating fibrocartilage comprises the following steps:

[0063] (1) First, ethylenediamine (EDA, 45 mmol, 3 mL) and dimethylformamide (DMF, 300 mL) were stirred at 0 °C under argon for 10 min to obtain an ethylenediamine solution; Boc-Lys(Fmoc)-OH (150 mmol, 62 g) and DMF (1000 mL) were mixed evenly to obtain a Boc-Lys(Fmoc)-OH solution; then, the ethylenediamine solution and the Boc-Lys(Fmoc)-OH solution were mixed evenly at 0 °C, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 150 mmol, 57 g) and 1-hydroxy-7-azabenzotriazole (HOAt, 150 mmol, 20.4 g) were added and stirred for 15 min for activation; then, N,N-diisopropylethylamine (DIPEA, 600 mmol, 105 mL) was added and activation was continued for 5 min. min; finally, the temperature was raised to room temperature and stirred for 15 h, 3 L of icy ether was added to the reaction solution, and the mixture was allowed to stand at 4°C for 2 h. The precipitate was collected by centrifugation and dried in vacuo to obtain the DGL precursor.

[0064] (2) The DGL precursor obtained in step (1) was dissolved in 900 mL of DMF at room temperature, and then 900 mL of a DMF solution with a concentration of 20% piperidine was added and stirred for 30 min. The mixture was poured into 5 L of glacial ether and n-hexane in a volume ratio of 1:1, and allowed to stand at 4°C. The precipitate was collected after centrifugation and vacuum dried to obtain DGL.

[0065] (3) 32 g of DGL obtained in step (2) was mixed with 600 mL of a mixed solution of trifluoroacetic acid (TFA), dichloromethane (DCM) and triisopropylsilane (TIS) (the volume ratio of TFA, DCM and TIS was 50:45:5) and stirred in an ice bath for 4 h. 3 L of ice ether was added and the mixture was allowed to stand at -20°C. The precipitate was collected after centrifugation and dried in vacuo to obtain Boc-free DGL.

[0066] (4) The de-Boc DGL obtained in step (3) and 4-(bromomethyl)phenylboronic acid (PBA) were reacted in methanol at a molar concentration ratio of 200:1 at 50°C for 48 h. The obtained product was fully freeze-dried to obtain PBA-modified DGL (PBA-DGL), which is a cationic nanocarrier for penetrating fibrocartilage.

[0067] Comparative Example 1:

[0068] A method for preparing a cationic nanocarrier comprises the following steps:

[0069] (1) First, ethylenediamine (EDA, 45 mmol, 3 mL) and dimethylformamide (DMF, 500 mL) were stirred at 0 °C under argon for 10 min to obtain an ethylenediamine solution; Boc-Lys(Fmoc)-OH (150 mmol, 62 g) and DMF (500 mL) were mixed evenly to obtain a Boc-Lys(Fmoc)-OH solution; then, the ethylenediamine solution and the Boc-Lys(Fmoc)-OH solution were mixed evenly at 0 °C, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 150 mmol, 57 g) and 1-hydroxy-7-azabenzotriazole (HOAt, 150 mmol, 20.4 g) were added and stirred for 10 min for activation; then, N,N-diisopropylethylamine (DIPEA, 600 mmol, 105 mL) was added and activation was continued for 5 min. min; finally, the temperature was raised to room temperature and stirred for 18 h, 3 L of icy ether was added to the reaction solution, and the mixture was allowed to stand at 4°C for 2 h. The precipitate was collected by centrifugation and dried in vacuo to obtain 50 g of DGL precursor.

[0070] (2) 50 g of the DGL precursor obtained in step (1) was dissolved in 800 mL of DMF at room temperature, and then 800 mL of a DMF solution with a concentration of 20% piperidine was added and stirred for 30 min. The mixture was poured into 5 L of icy ether and n-hexane in a volume ratio of 1:1, and allowed to stand at 4°C. The precipitate was collected after centrifugation and vacuum dried to obtain 35 g of DGL, i.e., a cationic nanocarrier.

[0071] Test Example 1:

[0072] (1) FTIR infrared spectroscopy was performed on the cationic nanocarriers prepared in Example 1 and Comparative Example 1. 1 H NMR and transmission electron microscopy characterization analysis, the experimental results are as follows Figure 2-Figure 4 shown.

[0073] like Figure 2 As shown in the FTIR infrared spectrum of the modified PBA-DGL, we can see that 1348 cm -1 The characteristic peaks of B−O stretching vibration mode and the 1612 cm -1 v The (C=C) characteristic peak proved that 4-(bromomethyl)phenylboronic acid PBA was successfully coupled to the cationic nanocarrier DGL.

[0074] like Figure 3 As shown, 1 The H NMR results show that after PBA modification, the only new peak (δ = 7-9 ppm range) comes from the aromatic hydrogen unique to PBA.

[0075] like Figure 4 As shown in the results, DGL and PBA-DGL are both relatively uniform spherical, the particle size distribution of DGL is concentrated around 20nm, and the particle size of PBA-DLG prepared after PBA modification increases to a certain extent, and the distribution changes are as follows Figure 4 As shown in Figures (b) and (c).

[0076] (2) The ability of the cationic nanocarriers prepared in Example 1 and Comparative Example 1 to carry siRNA was tested by agarose gel electrophoresis. The cationic nanocarriers prepared in Comparative Example 1 and Example 1 and siRNA were mixed in 100 μL DMEM medium (Gibco) at a mass ratio of 1:1, 2:1, 5:1, 10:1 and 20:1 at room temperature for 30 minutes to load siRNA. The mixture was then characterized by agarose gel electrophoresis. The experimental results are shown in FIG. Figure 5 shown.

[0077] like Figure 5 As shown, the cationic nanocarriers prepared in Comparative Example 1 and Example 1 both have the ability to carry siRNA, and cationic nanocarriers and siRNA in different ratios show different electrophoretic imaging effects. When the ratio of DGL and siRNA prepared in Comparative Example 1 is 5:1 or higher, all siRNA can be carried. Although the PBA-DGL obtained after modification in Example 1 has a certain degree of weakened siRNA complexing ability, it can still carry siRNA after increasing the ratio, which is manifested as PBA-DGL can achieve full siRNA carrying when the ratio of PBA-DGL and siRNA is 10:1 or higher.

[0078] (3) The morphology and particle size of PBA-DGL@siRNA prepared by mixing DGL and siRNA at a mass ratio of 5:1 and PBA-DGL@siRNA prepared by mixing PBA-DGL and siRNA at a mass ratio of 10:1 were observed by transmission electron microscopy and quantitatively analyzed, and zeta potential was tested. The experimental results are as follows: Figure 6-Figure 8 shown.

[0079] Figure 6 The results showed that DGL@siRNA and PBA-DGL@siRNA were still spherical. From the particle size distribution and quantitative statistical results ( Figure 7 ) It can be seen that the particle size of DGL@siRNA and PBA-DGL@siRNA formed after binding with siRNA has increased compared with that before binding. The zeta potential detection results show that ( Figure 8 ), the zeta potential of the modified PBA-DGL decreased, and the zeta potential of DGL@siRNA and PBA-DGL@siNRA formed after binding with siRNA also decreased, but still remained positive.

[0080] (4) Based on the positive charge characteristics of the PBA-DGL prepared in Example 1, its ability to bind to the negatively charged glycosaminoglycan chains in the cartilage and penetrate the condylar cartilage was tested (the schematic diagram of the fluorescence penetration test of the cartilage explant is shown in FIG. Figure 9 As shown in the figure): Normal porcine mandibular condylar cartilage with the closest morphology, structure and tissue composition to humans was obtained. The cartilage explant was modified to fit into a single-channel cylindrical culture dish. Cy5-siRNA containing a fluorescent group was combined with PBA-DGL (mass ratio of 1:10) and then incubated with the condylar cartilage explant. The effect of pure cy5-siRNA on penetrating fibrocartilage was compared. The results are shown in the figure. Figure 10 shown.

[0081] like Figure 10 As shown, at 12 hours, the red fluorescent group in the pure Cy5-siRNA group remained largely confined to the cartilage surface, while the red fluorescence in the PBA-DGL@siRNA group gradually penetrated the cartilage. Over time, at 24 hours, the PBA-DGL@siRNA group completely penetrated the cartilage layer. Quantitative topographic analysis results demonstrated that the PBA-DGL@siRNA group possessed superior penetrating properties into the condylar fibrocartilage.

[0082] (5) To evaluate the condylar cartilage penetration effect of PBA-DGL@siRNA in rats, the right temporomandibular joint area was first prepared and disinfected. After that, 50 μL of saline solution containing 2 mg of sodium iodoacetate was injected into the right joint cavity of the mouse to establish a temporomandibular osteoarthritis (TMJOA) model.

[0083] One week after modeling, the condylar fibrocartilage of rat models in normal and OA states was observed using atomic force microscopy (AFM). Figure 11 ) morphology. The results showed that the surface of the condylar fibrocartilage in the OA group was significantly rougher than that in the normal group, and was uneven. The pores in the condyle of the normal group were no longer obvious in the OA group, indicating that the changes caused by the early OA state increase the difficulty of drug penetration, reflecting the importance of using the cationic nanocarrier PBA-DGL to achieve deep targeting and penetrate dense barriers.

[0084] After the cy5-siRNA containing fluorescent groups was combined with PBA-DGL, it was injected into the right joint cavity of the rat TMJOA model. Compared with pure cy5-siRNA, the penetration effect of the condylar fibrocartilage in vivo was Figure 12 As shown, over time, the depth of fluorescence penetration into the condylar fibrocartilage increased in the PBA-DGL@siRNA group compared to the pure siRNA group. Quantitative topographical analysis demonstrated that the PBA-DGL@siRNA group possessed superior condylar fibrocartilage penetration properties when injected intra-articularly in vivo.

[0085] (6) Detection of transfection ability of PBA-DGL loaded with siRNA and analysis of the mechanism of improving transfection efficiency

[0086] Condylar chondrocytes were extracted from the TMJOA model and cultured in the primary culture medium. The PBA-DGL prepared in Example 1 of the present invention and the DGL prepared in Comparative Example 1 were loaded with FAM-siRNA carrying a green fluorescent group and transfected. The cells were observed after 24 h. The experimental results are shown in FIG. Figure 13 and Figure 14 shown.

[0087] like Figure 13 As shown, compared with the blank control group and the DGL group, the PBA-DGL group produced a good chondrocyte transfection effect, and the green FAM fluorescent group in the cells was more obvious, proving that PBA-DGL is more conducive to being taken up by chondrocytes.

[0088] like Figure 14 The flow cytometry results showed that the number of fluorescently labeled transfected chondrocytes in the PBA-DGL group was significantly more than that in the blank control group and the DGL group, verifying the promoting effect of PBA-modified DGL on siRNA transfection of chondrocytes under OA conditions.

[0089] Based on the co-staining and co-localization analysis of lysotracker probe (red fluorescence), the results are as follows Figure 15 As shown in the figure, compared with the DGL group, the co-localization area of ​​the green FAM fluorescent group carried by the nanocarrier in the PBA-DGL group and the lysosome was significantly reduced (0.31 vs. 0.53), indicating that PBA modification has the effect of promoting the lysosomal escape of siRNA, preventing it from being captured by intracellular lysosomes, thereby improving the transfection efficiency.

[0090] (7) Biosafety

[0091] The effects of the vector system on the main organs (heart, liver, spleen, lung and kidney) of rats were detected by HE staining. The experimental results are as follows Figure 16 shown.

[0092] The results showed that compared with the untreated blank control group, the PBA-DGL prepared by the present invention had no abnormalities in major organs after in vivo application. The cell and tissue morphology in the heart, liver, spleen, lung and kidney was normal, and no abnormal manifestations such as cell degeneration and necrosis, matrix destruction, etc. were observed.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a cationic nanocarrier for penetrating fibrocartilage, characterized in that: The following steps are involved: (1) First, mix the ethylenediamine solution and the Boc-Lys(Fmoc)-OH solution evenly, then add HATU and HOAt for activation, then add DIPEA for further activation, finally stir the reaction, add icy ether and let it stand, collect the precipitate and dry it to obtain the polylysine precursor; (2) first dissolving the polylysine precursor obtained in step (1) in a solvent, adding a piperidine solution and stirring, then adding glacial ether and n-hexane and allowing to stand, collecting the precipitate and drying it to obtain polylysine; (3) The polylysine obtained in step (2) is stirred in a mixed solution of trifluoroacetic acid, dichloromethane and triisopropylsilane, then added to icy ether and allowed to stand, and the precipitate is collected and dried to obtain de-tert-butyloxycarbonyl polylysine; (4) mixing the de-tert-butyloxycarbonyl polylysine obtained in step (3) and 4-(bromomethyl)phenylboronic acid in a solvent to react to obtain; Wherein, the concentration of the ethylenediamine solution in step (1) is 50-150 mmol / L; the concentration of the Boc-Lys(Fmoc)-OH solution is 150-250 mmol / L; and the volume ratio of the ethylenediamine solution to the Boc-Lys(Fmoc)-OH solution is (3-7):(5-10).

2. The method for preparing a cationic nanocarrier for penetrating fibrocartilage according to claim 1, characterized in that: The solvent of the ethylenediamine solution in step (1) is DMF; the solvent of the Boc-Lys(Fmoc)-OH solution is DMF.

3. The method for preparing a cationic nanocarrier for penetrating fibrocartilage according to claim 1, characterized in that: The mixing temperature in step (1) is -5~5°C; the activation temperature is -5~5°C, and the time is 5-20 min; the activation temperature is -5~5°C, and the time is 3-7 min; the stirring reaction temperature is room temperature, and the time is 15-20 h; the standing temperature is 1-10°C, and the time is 1-3 h.

4. The method for preparing a cationic nanocarrier for penetrating fibrocartilage according to claim 1, characterized in that: In the step (2), the concentration of the polylysine precursor solution is 50-80 g / L, and the solvent is DMF; the concentration of the piperidine solution is 10%-20%, and the solvent is DMF; the volume ratio of the polylysine precursor solution to the piperidine solution is (5-10):(5-10); and the volume ratio of glacial ether to n-hexane is (0.8-1.2):(0.8-1.2).

5. The method for preparing a cationic nanocarrier for penetrating fibrocartilage according to claim 1, characterized in that: The stirring time in step (2) is 20-40 min; the standing temperature is 1-10°C and the standing time is 2-4 h.

6. The method for preparing a cationic nanocarrier for penetrating fibrocartilage according to claim 1, characterized in that: In the step (3), the mass volume ratio of the mixed solution consisting of polylysine and trifluoroacetic acid, dichloromethane and triisopropylsilane is 30-40 g:500-1000 mL; the volume ratio of trifluoroacetic acid, dichloromethane and triisopropylsilane is (40-60):(35-55):(3-7); the stirring reaction temperature is -5~5°C and the time is 3-5 h.

7. The method for preparing a cationic nanocarrier for penetrating fibrocartilage according to claim 1, characterized in that: In step (4), the molar ratio of polylysine to 4-(bromomethyl)phenylboronic acid is (100-200):1, the solvent is methanol; the reaction temperature is 40-60°C, and the reaction time is 40-60 h.

8. A cationic nanocarrier for penetrating fibrocartilage, characterized in that: The method is prepared according to any one of claims 1 to 7.

9. Use of the cationic nanocarrier for penetrating fibrocartilage according to claim 8 in the preparation of osteoarthritis siRNA drugs.

10. An osteoarthritis siRNA drug, characterized in that The cationic nanocarrier for penetrating fibrocartilage according to claim 8 is mixed with siRNA for siRNA loading.

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