A novel drug-loaded matrix, its preparation method and application

Abstract

The application provides a drug-loaded substrate, which is modified with a coating layer containing chitosan and triamcinolone acetonide. The combination of triamcinolone acetonide and chitosan in the drug-loaded substrate of the application can significantly promote the healing of the epithelial mucosa of the urinary tract and digestive tract and inhibit scar formation while inhibiting bacteria, and is a good material for preparing medical devices such as catheters.

Description

Technical Field

[0001] This application belongs to the field of medical materials technology. Specifically, this application provides a novel drug-loaded matrix, its preparation method, and its application. Background Technology

[0002] The urothelial and digestive tract epithelium form the outermost barrier of the urinary and digestive systems, but various environmental factors such as accidental trauma, surgery, and infection can damage these mucosa. Damaged mucosa may form scars during the repair process. There are many types of scars, including immature scars, mature scars, hypertrophic scars, and keloids. These scars can cause partial or complete narrowing of the urethra or digestive tract, leading to urinary or digestive tract obstruction and serious diseases.

[0003] From a cellular and tissue perspective, normal urethral and digestive tract mucosa are composed of different types of epithelial cells. However, in cases of epithelial mucosal damage from any cause, during scar formation, fibroblasts become abnormally active, causing collagen synthesis to exceed its breakdown rate. This not only leads to disordered collagen fiber arrangement but also alters the secretion of certain cytokines, exacerbating scar formation and persistence.

[0004] To treat scars while preventing infection, surgical methods are currently the primary clinical approach. While methods such as scar excision and autologous patch transplantation are effective, they are invasive, require long treatment periods, and carry the risk of scar recurrence. There is a need in this field to develop novel materials in this area. Summary of the Invention

[0005] On the one hand, this application provides a drug-loaded matrix, which is a PVC material modified with a coating containing chitosan and triamcinolone.

[0006] Furthermore, the method for preparing the drug-loaded matrix includes:

[0007] (1) Clean and dry the PVC material;

[0008] (2) Prepare dopamine hydrochloride solution using a buffer solution with a pH greater than 8;

[0009] (3) Immerse the PVC material treated in step (1) into the hydrochloric acid dopamine solution obtained in step (2), react in an open manner, wash and dry to obtain PVC material with polydopamine layer.

[0010] (4) Prepare a solution containing 0.5-2% w / v chitosan, 0.2-1% w / v triamcinolone, 5-20% w / v PEGMA and 0.5-2% w / v ammonium persulfate using acetic acid solution;

[0011] (5) Immerse the PVC material with polydopamine layer obtained in step (3) into the solution prepared in step (4), seal it and react, then clean and dry to obtain the drug-loaded matrix.

[0012] Furthermore, the method for preparing the drug-loaded matrix includes:

[0013] (1) Clean and dry the PVC material;

[0014] (2) Prepare a 1-4 mg / mL dopamine hydrochloride solution using Tris-HCl buffer with a pH greater than 8;

[0015] (3) Immerse the PVC material treated in step (1) into the hydrochloric acid dopamine solution obtained in step (2), and react in an open environment for 6-12 hours. Then clean and dry to obtain PVC material with a polydopamine layer.

[0016] (4) Prepare a solution containing 0.5-2% w / v chitosan, 0.2-1% w / v triamcinolone, 5-20% w / v PEGMA, 0.5-2% w / v ammonium persulfate and 0.05-0.5% w / v or v / v surfactant using a 0.5-2% v / v acetic acid solution;

[0017] (5) Immerse the PVC material with polydopamine layer obtained in step (3) into the solution prepared in step (4), seal it and react for 6-12 hours, then clean and dry to obtain the drug-loaded matrix.

[0018] Furthermore, the surfactant is Span 20, Tween 20, rhamnolipin, or sophorolipid.

[0019] Furthermore, the method for preparing the drug-loaded matrix includes:

[0020] (1) Clean and dry the PVC material;

[0021] (2) Prepare a 2 mg / mL dopamine hydrochloride solution using a pH 8.5, 50 mM Tris-HCl buffer.

[0022] (3) Immerse the PVC material treated in step (1) into the hydrochloric acid dopamine solution obtained in step (2), react in the open for 8 hours, clean and dry to obtain PVC material with polydopamine layer.

[0023] (4) Prepare a solution containing 1% w / v chitosan, 0.3% w / v triamcinolone, 10% w / v PEGMA, 1% w / v ammonium persulfate and 0.1% v / v rhamnolipid using a 1% v / v acetic acid solution;

[0024] (5) Immerse the PVC material with polydopamine layer obtained in step (3) into the solution prepared in step (4), seal it and react for 8 hours, then clean and dry to obtain the drug-loaded matrix.

[0025] Furthermore, the cleaning in steps (1) and (5) is performed using distilled water and anhydrous ethanol.

[0026] Furthermore, the drying in steps (1), (3) and (5) is carried out at 40 degrees Celsius.

[0027] On the other hand, this application provides a method for preparing the above-mentioned drug-loaded matrix.

[0028] On the other hand, this application provides the application of the above-mentioned drug-loaded matrix in the preparation of biliary stents, urinary catheters, covered esophageal stents or ureteral stents.

[0029] On the other hand, this application provides a urinary catheter made using the above-described drug-loaded matrix.

[0030] The order of addition and dissolution method in the solution preparation process of this application can be adjusted by those skilled in the art according to general practices in the art. Stirring, shaking, sonication and other methods can be used to help the solution form or to make the components disperse evenly.

[0031] In this application, PVC is an abbreviation for poly(vinyl chloride), a material commonly used in various medical bags, tubes, and devices. PEGMA is an abbreviation for polyethylene glycol methacrylate. Attached Figure Description

[0032] Figure 1 To show the activity of normal fibroblasts and scar fibroblasts after incubation with the drug-loaded matrix; the four daily bar data, from top to bottom, are normal fibroblasts of drug-loaded matrix 2, scar fibroblasts of drug-loaded matrix 2, normal fibroblasts of drug-loaded matrix 1, and scar fibroblasts of drug-loaded matrix 1.

[0033] Figure 2 The effect of the drug-loaded matrix on window healing in Examples 1 and 2 (dotted line for Example 2, square line for Example 1).

[0034] Figure 3 These are endoscopic images of the urethra of a male rabbit after using a conventional urinary catheter, as provided by this invention.

[0035] Figure 4 This is an X-ray image of the urethra of a male rabbit after a standard urinary catheter was used.

[0036] Figure 5 This is a microscopic image of HE staining of urethral tissue from a male rabbit after using a conventional urinary catheter, provided by the present invention.

[0037] Figure 6 This is an endoscopic image of the urethra after using the drug-loaded substrate in Example 1 of the present invention in the urethra of a male rabbit.

[0038] Figure 7 This is an X-ray fluoroscopic image of the urethra of a male rabbit using the drug-loaded substrate in Example 1 of the present invention.

[0039] Figure 8 This is a microscopic image of HE staining of the urethral tissue of a male rabbit using the drug-loaded matrix provided in Example 1 of this invention.

[0040] Figure 9 This is an endoscopic image of the urethra after using the drug-loaded matrix in Example 4 of the present invention in the urethra of a male rabbit.

[0041] Figure 10 This is an X-ray fluoroscopic image of the urethra after using the drug-loaded matrix in Example 4 of the present invention on the male rabbit urethra.

[0042] Figure 11 This is a microscopic HE-stained image of the urethral tissue behind the urethra in Example 4 of the application of the drug-loaded matrix in the male rabbit urethra provided by the present invention. Detailed Implementation

[0043] The following embodiments are for illustrative purposes only and do not constitute a limitation on the scope of protection of this application. The scope of protection of this application is determined solely by the claims.

[0044] Example 1: Basic Preparation Method of Drug-Loaded Matrix

[0045] (1) Clean the PVC substrate with distilled water and anhydrous ethanol in sequence, and then dry it at 60 degrees Celsius for later use.

[0046] (2) Weigh 1g of chitosan powder (molecular weight 1000) and add it to 100mL of 1% v / v acetic acid solution. Stir continuously until a uniform and transparent 1% w / v chitosan solution is obtained.

[0047] (3) Weigh 0.5g of triamcinolone acetonide and add it to the chitosan solution obtained in step (2). Stir under ice bath stirring conditions and then use ultrasonic treatment to disperse the triamcinolone acetonide evenly to obtain a solution containing 1% w / v chitosan and 0.5% w / v triamcinolone acetonide.

[0048] (4) The PVC matrix treated in step (1) is immersed in the solution prepared in step (3) and vacuum impregnated for 60 minutes. The PVC matrix sample is placed at room temperature for 1 hour and then dried at 40 degrees Celsius to finally obtain drug-loaded matrix 1.

[0049] Example 2: Improved method for preparing drug-loaded matrix

[0050] The drug release and retention effects in drug-loaded matrices prepared by simple soaking and drying are insufficient. To address this issue, the applicant attempted to use a polydopamine-mediated PEGMA coating as a carrier to accommodate chitosan and triamcinolone acetonide.

[0051] (1) Clean the PVC substrate with distilled water and anhydrous ethanol in sequence, and then dry it at 60 degrees Celsius for later use.

[0052] (2) Prepare a 2 mg / mL dopamine hydrochloride solution using 50 mM Tris-HCl buffer at pH 8.5;

[0053] (3) Add the PVC matrix treated in step (1) to the hydrochloric acid dopamine solution obtained in step (2), and react with stirring at room temperature for 8 hours. After washing with distilled water and anhydrous ethanol, dry at 40 degrees Celsius.

[0054] (4) Weigh 1g of chitosan powder (molecular weight 1000) and add it to 100mL of 1% v / v acetic acid solution. Stir continuously until a uniform and transparent 1% w / v chitosan solution is obtained.

[0055] (5) Weigh 0.5g of triamcinolone acetonide and add it to the chitosan solution obtained in step (4). Stir under ice bath stirring conditions and then use ultrasonic treatment to disperse the triamcinolone acetonide evenly to obtain a solution containing 1% w / v chitosan and 0.5% w / v triamcinolone acetonide.

[0056] (6) Weigh 10g of PEGMA and 1g of ammonium persulfate, add them to the solution obtained in step (5), and stir to dissolve;

[0057] (7) The PVC matrix sample obtained in step (3) is immersed in the solution prepared in step (6), sealed and reacted at 60 degrees Celsius for 8 hours. After washing with distilled water and anhydrous ethanol, it is dried at 40 degrees Celsius to finally obtain the drug-loaded matrix 2.

[0058] Example 3: Effects of drug-loaded matrix on cell activity and wound healing

[0059] Normal fibroblasts and scar fibroblasts were seeded into 96-well plates and incubated in a cell culture incubator for 24 hours. After 24 hours, the cells were washed twice with DPBS to remove loose cells and other impurities. The cells were then incubated with two groups of cells: one group was incubated with the novel drug delivery system described in Example 1, and the other group was incubated with the drug group from Example 2. This incubation was to evaluate the effect of the novel drug delivery system on cell viability.

[0060] After the incubation process was completed, CCK-8 solution was added to each well, and incubation continued for 4 hours. This was to further assess the cell viability. After 4 hours of incubation, the absorbance (OD) values ​​of each well were read using a microplate reader at a wavelength of 450 nm. These data allowed for the calculation and assessment of cell viability. Experimental results showed (e.g.) Figure 1 As shown in Example 2): Compared with Example 1, the novel drug delivery system in Example 2 can better maintain the activity of normal fibroblasts, while having a significant inhibitory effect on scar fibroblasts. The polydopamine-mediated PEGMA-coated triamcinolone and chitosan can produce a better synergistic effect in vivo, effectively inhibiting the activity of scar fibroblasts.

[0061] from Figure 2 The data clearly show that, compared to Example 1, mice treated with the drug-loaded matrix of Example 2 experienced significantly faster wound healing. Particularly noteworthy is that by day 15 of treatment, the wounds in Example 2 had completely healed. More significantly, after one month of treatment, the wounds in the Example 2 group showed no scarring whatsoever, whereas, in contrast, the wounds in the mice of Example 1 exhibited significant scarring.

[0062] Example 4: Further Improvements to the Drug-Loaded Matrix Coating

[0063] Further research showed that the antibacterial effects of drug-loaded matrix 1 and drug-loaded matrix 2 decreased significantly with immersion in liquids, especially urine, indicating insufficient long-term performance when used as materials in prolonged contact with liquids, such as urinary catheters. The applicant further investigated the addition of appropriate surfactants during coating formation to improve the interwoven structure and hydrophilicity of the coating.

[0064] Using the drug-loaded matrix preparation method in Example 2, after adding various surfactants in step (6), the antibacterial effect of the obtained material and its antibacterial effect after soaking in artificial urine for 7 days were verified.

[0065] The strains tested for antibacterial efficacy included Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231. The test method referred to GB / T 20944.3-2008 shaking method: three 1cm×1cm drug-loaded substrates were placed in a 250mL flask, inoculated with bacterial solution, shaken, and the liquid in the flask was aspirated and incubated on a plate for 48 hours (Staphylococcus aureus, Escherichia coli) or 72 hours (Candida albicans). The colony count (cfu / ml) was recorded and the inhibition rate was calculated. A result below 85% was considered as having no antibacterial effect.

[0066] Changes in antibacterial properties after adding different surfactants

[0067]

[0068] The results showed that both the original and improved drug-loaded matrix 2 had significant inhibitory effects on the three bacteria. However, the antibacterial effect of the original drug-loaded matrix 2 completely disappeared after soaking in artificial urine for 7 days. The addition of Tween and Span surfactants maintained the antibacterial effect against Escherichia coli, but it was significantly less effective against Staphylococcus aureus and Candida albicans than the addition of rhamnolipin. Sophorolipid, which is also a biosurfactant, did not play any beneficial role at all.

[0069] Example 5: Actual animal experiments on drug-loaded matrix materials

[0070] New Zealand male rabbits of equal volume and weight were selected as experimental subjects and divided into three groups. All groups underwent the same degree of plasma electrocoagulation injury in the bulbous urethra. Immediately after injury, a corresponding urinary catheter was inserted into each group and securely fixed. The three groups of rabbits were housed in the same environment for two weeks. Afterwards, endoscopy and X-ray fluoroscopy were performed, and the rabbits were euthanized. Urethral tissue from the injured area was then collected for HE staining.

[0071] Group 1: After electrocoagulation injury, a regular urinary catheter was left in place for two weeks. Endoscopic examination and imaging were performed on the male rabbit urethra. X-ray fluoroscopy was performed on the male rabbit urethra and imaging was also performed. Urethral tissue was taken from the male rabbits, stained with hematoxylin and eosin (HE), and observed under a microscope.

[0072] Group 2: After electrocoagulation injury, a urinary catheter containing the drug-loaded matrix of Example 1 was left in place for two weeks. The male rabbit urethra was examined endoscopically and images were taken. The male rabbit urethra was fluoroscopically examined and images were taken. Male rabbit urethral tissue was taken, stained with hematoxylin and eosin (HE), and observed under a microscope.

[0073] Group 3: After electrocoagulation injury, the drug-loaded matrix of Example 4 (drug-loaded matrix 2 + 0.1% v / v rhamnolipin) was left in place for two weeks. The male rabbit urethra was examined by endoscopy and the images were taken. The male rabbit urethra was fluoroscopically examined and the images were taken. The male rabbit urethra tissue was taken and stained with HE. The male rabbit urethra tissue was observed under a microscope.

[0074] like Figures 3 to 11As shown, it can be clearly observed that the use of triamcinolone acetonide is effective in inhibiting scar formation and reducing inflammatory response. Through comparative experiments of three groups, the drug-loaded matrix prepared in Example 4 of the present invention showed a more significant effect in inhibiting scar formation and reducing inflammatory response. In particular, in X-ray fluoroscopic images, the patency of the urethra in the third group of male rabbits was significantly better than that in the second group, and both groups were better than the first group, indicating that the novel drug-loaded matrix has a better effect on preventing urethral scar formation. In addition, in microscopic observation after HE staining, the fibrosis and inflammatory cell infiltration of the urethral tissue of the third group of male rabbits were significantly less than those in the second group, and both groups were better than the first group, further proving that the novel drug-loaded matrix prepared in Example 1 of the present invention has superiority in inhibiting scar formation and inflammatory response. These experimental results all show that compared with the use of triamcinolone acetonide alone and triamcinolone acetonide plus chitosan, the application of the novel drug-loaded matrix can better prevent and treat urethral scar formation in male rabbits.

Claims

1. The application of a drug-loaded matrix in the preparation of a urinary catheter with anti-scarring and anti-inflammatory effects, characterized in that, The method for preparing the drug-loaded matrix includes: (1) Clean and dry the PVC material; (2) Prepare a 2 mg / mL dopamine hydrochloride solution using a pH 8.5, 50 mM Tris-HCl buffer; (3) Immerse the PVC material treated in step (1) into the hydrochloric acid dopamine solution obtained in step (2), react at room temperature for 8 hours, clean and dry to obtain PVC material with polydopamine layer. (4) Prepare a solution containing 1% w / v chitosan, 0.3% w / v triamcinolone, 10% w / v PEGMA, 1% w / v ammonium persulfate and 0.1% v / v rhamnolipid using a 1% v / v acetic acid solution; wherein the chitosan has a molecular weight of 1000; (5) Immerse the PVC material with polydopamine layer obtained in step (3) into the solution prepared in step (4), seal it and react at 60°C for 8 hours, then clean and dry to obtain the drug-loaded matrix.

2. The application according to claim 1, wherein the cleaning in steps (1) and (5) is performed using distilled water and anhydrous ethanol.

3. The application according to claim 1, wherein the drying in steps (1), (3) and (5) is carried out at 40°C.

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