WO2024125091A1 - Method for preparing ureteral stent tube coated with pirfenidone-carrying nanoparticle composite - Google Patents
Method for preparing ureteral stent tube coated with pirfenidone-carrying nanoparticle composite Download PDFInfo
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- WO2024125091A1 WO2024125091A1 PCT/CN2023/126065 CN2023126065W WO2024125091A1 WO 2024125091 A1 WO2024125091 A1 WO 2024125091A1 CN 2023126065 W CN2023126065 W CN 2023126065W WO 2024125091 A1 WO2024125091 A1 WO 2024125091A1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
- A61L29/04—Macromolecular materials
- A61L29/06—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
- A61L31/10—Macromolecular materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention relates to the technical field of ureteral stent tube preparation, and in particular to a method for preparing a ureteral stent tube with a composite coating of pirfenidone-loaded nanoparticles.
- Ureteral injury repair is usually fibrous scar repair, and the pathological characteristics are massive proliferation of fibroblasts, excessive deposition of collagen and proteoglycans in the extracellular matrix, resulting in disordered collagen fiber arrangement.
- the main animal models of ureteral injury at home and abroad are unilateral ureteral complete obstruction models and electrocoagulation injury models. Therefore, we attempted to use a rabbit ureteral injury model that is closer to ureteral stenosis caused by electrocoagulation under clinical ureteroscopy to further explore the pathogenesis of ureteral stenosis.
- ureteral stents There are many methods for treating ureteral stenosis, including endoscopy, balloon dilatation, laparoscopy or open surgery for ureteroplasty. After surgery, a ureteral stent is usually placed in the ureter. Although ureteral stents have a certain effect in dilating the ureter and preventing stenosis, some patients still experience mild stenosis again after removing the ureteral stent. Therefore, there is an urgent need to develop more effective stents to prevent ureteral stenosis in clinical practice. In view of the above defects, it is necessary to design a preparation method for ureteral stents with a composite coating of pirfenidone nanoparticles.
- the object of the present invention is to provide a method for preparing a ureteral stent tube with a composite coating of pirfenidone-loaded nanoparticles, so as to solve the problems raised in the above-mentioned background technology.
- the present invention provides the following technical solution: a method for preparing a ureteral stent tube with a composite coating of pirfenidone nanoparticles, comprising the following steps:
- the ureteral stent was placed in phosphate buffered saline containing dopamine hydrochloride and incubated;
- step S4 washing the polydopamine-modified ureteral stent obtained in step S3 twice with deionized water, and then immersing the stent in a suspension of pirfenidone nanoparticles to form a ureteral stent loaded with a pirfenidone nanoparticle coating;
- nanoparticle complex was labeled with rhodamine B to detect the distribution of pirfenidone nanoparticles on the ureteral stent.
- step S1 the preparation of the suspension containing pirfenidone nanoparticles comprises the following steps:
- S11 first, phosphate buffered saline, pirfenidone and polylactic-co-glycolic acid are emulsified in dichloromethane, and ultrasonicated for 1 min using an ultrasonic homogenizer to obtain a mixture 1;
- step S12 then, adding the polyvinyl alcohol solution to the mixture 1 obtained in step S11 and ultrasonically forming an emulsion;
- step S13 stirring the emulsion obtained in step S12 at room temperature for 24 hours to completely evaporate the dichloromethane to obtain nanoparticles;
- step S14 centrifuging the nanoparticles obtained in step S13 at a speed of 12,000 rpm and below 4° C. for 5 min;
- step S6 the preparation of the nanoparticle complex labeled with rhodamine B comprises the following steps:
- step S22 incubating the ureteral stent in step S4 in a phosphate buffer containing 0.5 mg/ml dopamine hydrochloride, and then stirring at room temperature for 3 hours to form a polydopamine-modified ureteral stent;
- the preparation of the suspension of rhodamine B labeled nanoparticles comprises the following steps:
- the nanoparticles are washed three times with deionized water and finally suspended in deionized water to obtain a Rhodamine B labeled nanoparticle suspension.
- the concentration of dopamine hydrochloride is 0.5 mg/ml, and the pH value of the phosphate buffer is 8.5.
- step S3 the stirring time is 3 hours.
- the preparation method of the ureteral stent tube loaded with pirfenidone nanoparticle composite coating of the present invention is that we can soak the commonly used ureteral stent tube in the clinic in an alkaline solution containing dopamine to form a layer of adhesive coating on the surface of the stent, and then incubate the dopamine-modified stent tube with the nanoparticle/pirfenidone complex to form a ureteral stent tube loaded with pirfenidone nanoparticle coating.
- the use of nanoparticles as a drug delivery platform.
- the pirfenidone nanoparticle composite coating was applied to the ureteral stent, which could inhibit ureteral stenosis by reducing the expression of transforming growth factor- ⁇ 1 and the deposition of collagen. Therefore, we believe that the use of nanoparticle/PFD composite-coated ureteral stents is an effective method to prevent ureteral stenosis caused by iatrogenic operations.
- the present invention provides a technical solution: a method for preparing a ureteral stent tube with a composite coating of pirfenidone-loaded nanoparticles, comprising the following steps:
- the ureteral stent was placed in phosphate buffered saline containing dopamine hydrochloride and incubated;
- step S4 washing the polydopamine-modified ureteral stent obtained in step S3 twice with deionized water, and then immersing the stent in a suspension of pirfenidone nanoparticles to form a ureteral stent loaded with a pirfenidone nanoparticle coating;
- nanoparticle complex was labeled with rhodamine B to detect the distribution of pirfenidone nanoparticles on the ureteral stent.
- step S1 the preparation of the suspension containing pirfenidone nanoparticles comprises the following steps:
- step S12 then, adding polyvinyl alcohol to the mixture 1 obtained in step S11 to form an emulsion;
- step S13 the emulsion obtained in step S12 is re-emulsified by ultrasonic wave in an ice bath for 3 minutes, and then stirred at room temperature for 24 hours to completely evaporate the dichloromethane to obtain nanoparticles;
- step S14 centrifuging the nanoparticles obtained in step S13 at a speed of 12,000 rpm and below 4° C. for 5 min;
- step S6 the preparation of the nanoparticle complex labeled with rhodamine B comprises the following steps:
- step S22 incubating the ureteral stent in step S4 in a phosphate buffer containing 0.5 mg/ml dopamine hydrochloride, and then stirring at room temperature for 3 hours to form a polydopamine-modified ureteral stent;
- step S21 the preparation of the suspension of rhodamine B labeled nanoparticles comprises the following steps:
- the nanoparticles are washed three times with deionized water and finally suspended in deionized water to obtain a Rhodamine B labeled nanoparticle suspension.
- step S2 the concentration of dopamine hydrochloride is 0.5 mg/ml, and the pH value of the phosphate buffer is 8.5.
- step S3 the stirring time is 3 hours.
- the cytotoxicity of the nanoparticle/pirfenidone complex-coated ureteral stent was then determined by the CCK8 method.
- the determination method is as follows: a 20 mm long sterile ureteral stent was cut into 2 mm lengths and placed in a 96-well plate. Next, the cell suspension was added to the 96 wells. After 24 h and 48 h of culture, 10 mg/ml of CCK8 dye solution was added to each well and cultured for another 4 h at 37 °C and 5% carbon dioxide. The absorbance of each well was measured at 450 nm using an enzyme-linked immunosorbent assay. Using untreated cells as the control, the cell survival rate was 100%. The experiment was performed in triplicate.
- the experimental animals were 20-week-old male New Zealand white rabbits. The animal experiment was approved by the Experimental Animal Ethics Committee of Nantong University (approval number: S20210301-991).
- the experimental rabbits were randomly divided into a scald modeling group, an unmodified ureteral stent treatment group, and an NP/pirfenidone ureteral stent treatment group, with 3 rabbits in each group. All rabbits were injected intramuscularly with Su Mian Xin II (1 ml/kg) + Shu Tai 50 (0.4 ml/kg). After general anesthesia, the rabbit was fixed on the operating table in a supine position.
- ureteral tissue was freed, a longitudinal incision was made in the middle of the ureter, and an unmodified ureteral stent and an NP/pirfenidone ureteral stent were placed, respectively, and then thermal injury was performed with an electrocoagulation guide wire.
- the animals were killed 2 weeks after surgery, and the ureteral stricture segment and the treatment segment (about 1 cm) were obtained to compare the changes in gross specimens and the expression of related cytokines.
- each rabbit was killed by air embolism.
- the ureters and kidneys of both rabbits were then completely removed.
- the tissues were preserved in a 10% formalin solution.
- the specimens were dehydrated, embedded in paraffin, and cut into 5-micron-thick sections.
- Hematoxylin-eosin (H&E) staining was used to observe changes in ureteral endothelial cells and lumen area.
- Immunohistochemistry was used to detect the expression level of TGF- ⁇ 1 in ureteral tissue. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide at room temperature for 5–10 min. The water bath was set at 100°C; the sections were placed in citrate buffer (Dako, Glostrup, Denmark) for 5 min for antigen retrieval. The slides were washed with PBS three times for 5 min each and blocked with 5% BSA for 2 h. The sections were then incubated with rabbit anti-TGF- ⁇ 1 antibody (1:500, 21898-1-AP, proteintech) at 4°C overnight. Next, the slides were washed with PBS and incubated with secondary antibodies for 1 h.
- the sections were stained with DAB for color development and then counterstained with hematoxylin.
- the sections were mounted with neutral gum, and the tissue images were observed and evaluated using a microscope (Leica DMR3000, 234 Leica Microsystems, Bensheim, Germany).
- Total protein of ureteral tissues in the US group, US+ureteral stent group, and US+NP/pirfenidone ureteral stent group was extracted for western blot analysis. Protein samples were separated on SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. Then, they were washed with TBST buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1% Tween-20, pH 7.6) and blocked with 5% skim milk powder in TBST for 2 h.
- PVDF polyvinylidene difluoride
- the preparation method of the ureteral stent tube loaded with pirfenidone nanoparticle composite coating of the present invention is to soak the commonly used ureteral stent tube in the clinic in an alkaline solution containing dopamine, form a layer of adhesive coating on the surface of the stent, and then incubate the dopamine-modified stent tube with the nanoparticle/pirfenidone complex to form a ureteral stent tube loaded with pirfenidone nanoparticle coating.
- the use of nanoparticles as a drug delivery platform.
- the terms “set”, “install”, “connect”, “connect”, and “fix” should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements.
- the specific meanings of the above terms in the present invention can be understood according to specific circumstances.
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- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Surgery (AREA)
- Heart & Thoracic Surgery (AREA)
- Vascular Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Disclosed is a method for preparing a ureteral stent tube coated with a pirfenidone-carrying nanoparticle composite. The method comprises the following steps: S1, firstly, preparing a pirfenidone-carrying nanoparticle suspension in advance; S2, incubating the ureteral stent tube in a phosphate buffer containing dopamine hydrochloride; S3, stirring at room temperature for a specified duration to give a polydopamine-modified ureteral stent tube; and S4, washing the polydopamine modified ureteral stent tube obtained in step S3 twice with deionized water and then soaking the stent in the pirfenidone-carrying nanoparticle suspension. By coating the ureteral stent tube with a pirfenidone-carrying nanoparticle composite coating, the present invention inhibits ureteral stenosis by reducing the expression of transforming growth factor TGF-β1 and the deposition of collagen. Therefore, we believe that the use of the nanoparticle/PFD composite-coated ureteral stent tube is an effective method to prevent ureteral stenosis caused by iatrogenic operations.
Description
本发明涉及输尿管支架管制备技术领域，具体涉及载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法。The invention relates to the technical field of ureteral stent tube preparation, and in particular to a method for preparing a ureteral stent tube with a composite coating of pirfenidone-loaded nanoparticles.
近年来，随着输尿管镜技术的不断应用，伴随而来的输尿管损伤也越来越多。据报道，国内外发生输尿管药物损伤的概率为60％-94％。特别是，由于对上尿路结石使用输尿管镜检查，医源性操作引起输尿管狭窄的发生率不断增加。因此，如何预防医源性操作引起的输尿管狭窄，尤其是输尿管镜手术后的输尿管狭窄，是当今泌尿外科医生迫切需要解决的问题。In recent years, with the continuous application of ureteroscopy, the accompanying ureteral injuries have also increased. It is reported that the probability of ureteral drug injury at home and abroad is 60%-94%. In particular, due to the use of ureteroscopy for upper urinary tract stones, the incidence of iatrogenic ureteral stenosis has continued to increase. Therefore, how to prevent iatrogenic ureteral stenosis, especially ureteral stenosis after ureteroscopy, is an urgent problem that urologists need to solve today.
输尿管损伤修复通常是纤维瘢痕修复，病理特征是成纤维细胞大量增殖，胶原和细胞外基质中蛋白多糖过度沉积，导致胶原纤维排列紊乱。研究表明，TGF-β1在成纤维细胞中高表达，是与瘢痕形成关系最密切的细胞因子。在这项研究中，我们试图通过使用动物输尿管损伤模型来模拟输尿管镜操作造成的输尿管损伤，以进一步探讨输尿管狭窄的机制。目前，国内外主要的输尿管损伤动物模型有单侧输尿管完全梗阻模型和电凝损伤模型。因此，我们试图利用一种更接近于临床输尿管镜下电凝操作所致的输尿管狭窄的兔输尿管损伤模型，进一步探讨输尿管狭窄的发病机制。Ureteral injury repair is usually fibrous scar repair, and the pathological characteristics are massive proliferation of fibroblasts, excessive deposition of collagen and proteoglycans in the extracellular matrix, resulting in disordered collagen fiber arrangement. Studies have shown that TGF-β1 is highly expressed in fibroblasts and is the cytokine most closely related to scar formation. In this study, we attempted to simulate ureteral injury caused by ureteroscopy using an animal ureteral injury model to further explore the mechanism of ureteral stenosis. At present, the main animal models of ureteral injury at home and abroad are unilateral ureteral complete obstruction models and electrocoagulation injury models. Therefore, we attempted to use a rabbit ureteral injury model that is closer to ureteral stenosis caused by electrocoagulation under clinical ureteroscopy to further explore the pathogenesis of ureteral stenosis.
治疗输尿管狭窄的方法很多，包括内窥镜、球囊扩张术、腹腔镜或开放手术进行输尿管成形术等。手术后，输尿管支架管通常放置在输尿管内。尽管输尿管支架管在扩张输尿管和预防狭窄方面有一定的作用，但仍有部分患者在取下输尿管支架管后再次出现轻度狭窄。因此，临床上迫切需要开发更有效的支架管来预防输尿管狭窄。鉴于以上缺陷，实有必要设计载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法。There are many methods for treating ureteral stenosis, including endoscopy, balloon dilatation, laparoscopy or open surgery for ureteroplasty. After surgery, a ureteral stent is usually placed in the ureter. Although ureteral stents have a certain effect in dilating the ureter and preventing stenosis, some patients still experience mild stenosis again after removing the ureteral stent. Therefore, there is an urgent need to develop more effective stents to prevent ureteral stenosis in clinical practice. In view of the above defects, it is necessary to design a preparation method for ureteral stents with a composite coating of pirfenidone nanoparticles.
发明内容Summary of the invention
本发明的目的在于提供载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法，以解决上述背景技术中提出的问题。The object of the present invention is to provide a method for preparing a ureteral stent tube with a composite coating of pirfenidone-loaded nanoparticles, so as to solve the problems raised in the above-mentioned background technology.
为实现上述目的，本发明提供如下技术方案：载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法，包括以下步骤：To achieve the above object, the present invention provides the following technical solution: a method for preparing a ureteral stent tube with a composite coating of pirfenidone nanoparticles, comprising the following steps:
S1，首先，预先制备载有吡非尼酮纳米颗粒的悬浮液；S1, first, a suspension of pirfenidone-loaded nanoparticles is prepared in advance;
S2，然后，将输尿管支架管置于含盐酸多巴胺的磷酸盐缓冲液中孵育；S2, then, the ureteral stent was placed in phosphate buffered saline containing dopamine hydrochloride and incubated;
S3，接下来，在室温下搅拌一定时间，形成聚多巴胺修饰的输尿管支架管；
S3, next, stirring at room temperature for a certain period of time to form a polydopamine-modified ureteral stent;
S4，将步骤S3中得到聚多巴胺修饰的输尿管支架管，用去离子水清洗两次，然后将支架浸泡在吡非尼酮纳米颗粒的悬浮液中，形成负载吡非尼酮纳米颗粒涂层的输尿管支架管；S4, washing the polydopamine-modified ureteral stent obtained in step S3 twice with deionized water, and then immersing the stent in a suspension of pirfenidone nanoparticles to form a ureteral stent loaded with a pirfenidone nanoparticle coating;
S5，用扫描电子显微镜观察包裹纳米颗粒的输尿管支架管的表面形态；S5, the surface morphology of the ureteral stent coated with nanoparticles was observed using scanning electron microscopy;
S6，最后，通过罗丹明B来标记纳米粒子复合物，对吡非尼酮纳米粒子在输尿管支架管上的分布进行检测。S6. Finally, the nanoparticle complex was labeled with rhodamine B to detect the distribution of pirfenidone nanoparticles on the ureteral stent.
优选的，在步骤S1中，制备载有吡非尼酮纳米颗粒的悬浮液的制备，包括以下步骤：Preferably, in step S1, the preparation of the suspension containing pirfenidone nanoparticles comprises the following steps:
S11，首先，将磷酸盐缓冲盐水、吡非尼酮和聚乳酸-羟基乙酸，乳化在二氯甲烷中，使用超声波均质机，超声1min得到混合物一；S11, first, phosphate buffered saline, pirfenidone and polylactic-co-glycolic acid are emulsified in dichloromethane, and ultrasonicated for 1 min using an ultrasonic homogenizer to obtain a mixture 1;
S12，然后，在步骤S11得到的混合物一中加入聚乙烯醇溶液超声以形成乳状液；S12, then, adding the polyvinyl alcohol solution to the mixture 1 obtained in step S11 and ultrasonically forming an emulsion;
S13，然后，将步骤S12得到的乳状液在室温下搅拌24h，使二氯甲烷完全蒸发，得到纳米颗粒；S13, then, stirring the emulsion obtained in step S12 at room temperature for 24 hours to completely evaporate the dichloromethane to obtain nanoparticles;
S14，将步骤S13得到的纳米颗粒以低于4℃的12,000转/min的速度离心5min；S14, centrifuging the nanoparticles obtained in step S13 at a speed of 12,000 rpm and below 4° C. for 5 min;
S15，最后，用去离子水洗涤纳米颗粒多次，并将其悬浮在去离子水中得到载有吡非尼酮纳米颗粒的悬浮液。S15, finally, washing the nanoparticles with deionized water for multiple times, and suspending them in deionized water to obtain a suspension of pirfenidone-loaded nanoparticles.
优选的，在步骤S6中，罗丹明B来标记纳米粒子复合物的制备，包括以下步骤：Preferably, in step S6, the preparation of the nanoparticle complex labeled with rhodamine B comprises the following steps:
S21，首先，对含有罗丹明B标记纳米颗粒悬浮液进行预先制备；S21, first, a suspension containing rhodamine B labeled nanoparticles is prepared in advance;
S22，然后，将步骤S4中输尿管支架管孵育在含有0.5mg/ml盐酸多巴胺的磷酸盐缓冲液中，然后在室温度下搅拌3h，形成聚多巴胺修饰的输尿管支架管；S22, then, incubating the ureteral stent in step S4 in a phosphate buffer containing 0.5 mg/ml dopamine hydrochloride, and then stirring at room temperature for 3 hours to form a polydopamine-modified ureteral stent;
S23，将聚多巴胺修饰的输尿管支架管用去离子水清洗两次，然后浸泡在步骤S21预先制备的罗丹明B标记纳米颗粒悬浮液中，形成罗丹明B纳米颗粒/吡非尼酮涂层输尿管支架管。S23, washing the polydopamine-modified ureteral stent twice with deionized water, and then soaking it in the rhodamine B-labeled nanoparticle suspension prepared in advance in step S21 to form a rhodamine B nanoparticle/pirfenidone-coated ureteral stent.
优选的，在步骤S21中，罗丹明B标记纳米颗粒悬浮液的制备，包括以下步骤：Preferably, in step S21, the preparation of the suspension of rhodamine B labeled nanoparticles comprises the following steps:
S211，首先，将100ul的磷酸盐缓冲盐水和罗丹明B，在2ml的含20mg PLGA的二氯甲烷中乳化，加入，在冰浴中超声0.5min，得混合物二；S211, first, emulsify 100 ul of phosphate buffered saline and rhodamine B in 2 ml of dichloromethane containing 20 mg PLGA, add, and ultrasonicate in an ice bath for 0.5 min to obtain mixture 2;
S212，然后，向混合物二中加入4.5ml的1.5％PVA并超声以形成复乳；S212, then, adding 4.5 ml of 1.5% PVA to the mixture 2 and sonicating to form a double emulsion;
S213，接下来，将乳状液在室温下搅拌24h，以完全蒸发二氯甲烷，纳米颗粒以低于4℃的12,000转/min的速度离心5min；S213, next, the emulsion was stirred at room temperature for 24 h to completely evaporate the dichloromethane, and the nanoparticles were centrifuged at 12,000 rpm for 5 min at below 4 °C;
S214，最后，纳米颗粒用去离子水洗三次，最后悬浮在去离子水中，得罗丹明B标记纳米颗粒悬浮液。
S214, finally, the nanoparticles are washed three times with deionized water and finally suspended in deionized water to obtain a Rhodamine B labeled nanoparticle suspension.
优选的，在步骤S2中，盐酸多巴胺的浓度为0.5mg/ml，磷酸盐缓冲液PH值为8.5。Preferably, in step S2, the concentration of dopamine hydrochloride is 0.5 mg/ml, and the pH value of the phosphate buffer is 8.5.
优选的，在步骤S3中，搅拌的搅拌时长为3h。Preferably, in step S3, the stirring time is 3 hours.
与现有技术相比，本发明载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法，我们可以将临床常用的输尿管支架管浸泡在含有多巴胺的碱性溶液中，在支架表面形成一层粘附性的涂层，然后将多巴胺修饰的支架管与纳米颗粒/吡非尼酮复合物孵育，形成负载吡非尼酮的纳米颗粒涂层的输尿管支架管。在输尿管狭窄的治疗中，使用纳米颗粒作为药物输送平台的报道很少。在本研究中，我们创新性地将纳米颗粒/吡非尼酮复合物负载到输尿管支架管上，并通过纳米颗粒的生物降解和控制释放，检测了纳米颗粒/吡非尼酮复合物包裹的输尿管支架管的生物相容性、释放特性和组织分布。最后，我们通过病理切片染色和Western Blot检测大体解剖改变、胶原沉积和各种纤维蛋白的表达，评价纳米颗粒/吡非尼酮复合物涂层输尿管支架管抑制输尿管纤维化的疗效，以达到预防输尿管狭窄的效果，通过吡非尼酮纳米粒子复合涂层涂覆在输尿管支架管上，可以通过减少转化生长因子β1的表达和胶原蛋白的沉积来抑制输尿管狭窄，因此，我们认为使用纳米粒子/PFD复合涂层输尿管支架是预防医源性操作引起的输尿管狭窄的有效方法。Compared with the prior art, the preparation method of the ureteral stent tube loaded with pirfenidone nanoparticle composite coating of the present invention is that we can soak the commonly used ureteral stent tube in the clinic in an alkaline solution containing dopamine to form a layer of adhesive coating on the surface of the stent, and then incubate the dopamine-modified stent tube with the nanoparticle/pirfenidone complex to form a ureteral stent tube loaded with pirfenidone nanoparticle coating. In the treatment of ureteral stenosis, there are few reports on the use of nanoparticles as a drug delivery platform. In this study, we innovatively loaded the nanoparticle/pirfenidone complex onto the ureteral stent tube, and through the biodegradation and controlled release of the nanoparticles, the biocompatibility, release characteristics and tissue distribution of the ureteral stent tube wrapped with the nanoparticle/pirfenidone complex were detected. Finally, we evaluated the efficacy of nanoparticle/pirfenidone complex-coated ureteral stents in inhibiting ureteral fibrosis by detecting gross anatomical changes, collagen deposition, and the expression of various fibrins through pathological section staining and Western Blot, so as to achieve the effect of preventing ureteral stenosis. The pirfenidone nanoparticle composite coating was applied to the ureteral stent, which could inhibit ureteral stenosis by reducing the expression of transforming growth factor-β1 and the deposition of collagen. Therefore, we believe that the use of nanoparticle/PFD composite-coated ureteral stents is an effective method to prevent ureteral stenosis caused by iatrogenic operations.
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
本发明提供一种技术方案：载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法，包括以下步骤：The present invention provides a technical solution: a method for preparing a ureteral stent tube with a composite coating of pirfenidone-loaded nanoparticles, comprising the following steps:
S1，首先，预先制备载有吡非尼酮纳米颗粒的悬浮液；S1, first, a suspension of pirfenidone-loaded nanoparticles is prepared in advance;
S2，然后，将输尿管支架管置于含盐酸多巴胺的磷酸盐缓冲液中孵育；S2, then, the ureteral stent was placed in phosphate buffered saline containing dopamine hydrochloride and incubated;
S3，接下来，在室温下搅拌一定时间，形成聚多巴胺修饰的输尿管支架管；S3, next, stirring at room temperature for a certain period of time to form a polydopamine-modified ureteral stent;
S4，将步骤S3中得到聚多巴胺修饰的输尿管支架管，用去离子水清洗两次，然后将支架浸泡在吡非尼酮纳米颗粒的悬浮液中，形成负载吡非尼酮纳米颗粒涂层的输尿管支架管；S4, washing the polydopamine-modified ureteral stent obtained in step S3 twice with deionized water, and then immersing the stent in a suspension of pirfenidone nanoparticles to form a ureteral stent loaded with a pirfenidone nanoparticle coating;
S5，用扫描电子显微镜观察包裹纳米颗粒的输尿管支架管的表面形态；S5, the surface morphology of the ureteral stent coated with nanoparticles was observed using scanning electron microscopy;
S6，最后，通过罗丹明B来标记纳米粒子复合物，对吡非尼酮纳米粒子在输尿管支架管上的分布进行检测。
S6. Finally, the nanoparticle complex was labeled with rhodamine B to detect the distribution of pirfenidone nanoparticles on the ureteral stent.
其中，在步骤S1中，制备载有吡非尼酮纳米颗粒的悬浮液的制备，包括以下步骤：Wherein, in step S1, the preparation of the suspension containing pirfenidone nanoparticles comprises the following steps:
S11，首先，将磷酸盐缓冲盐水、吡非尼酮和聚乳酸-羟基乙酸，乳化在二氯甲烷中，使用超声波均质机，冰浴1min得到混合物一；S11, first, phosphate buffered saline, pirfenidone and poly(lactic-co-glycolic acid) were emulsified in dichloromethane, and an ultrasonic homogenizer was used to ice-bathe for 1 min to obtain a mixture 1;
S12，然后，在步骤S11得到的混合物一中加入的聚乙烯醇以形成乳状液；S12, then, adding polyvinyl alcohol to the mixture 1 obtained in step S11 to form an emulsion;
S13，然后，将步骤S12得到的乳状液在冰浴中用超声波再次乳化3min，然后在室温下搅拌24h，使二氯甲烷完全蒸发，得到纳米颗粒；S13, then, the emulsion obtained in step S12 is re-emulsified by ultrasonic wave in an ice bath for 3 minutes, and then stirred at room temperature for 24 hours to completely evaporate the dichloromethane to obtain nanoparticles;
S14，将步骤S13得到的纳米颗粒以低于4℃的12,000转/min的速度离心5min；S14, centrifuging the nanoparticles obtained in step S13 at a speed of 12,000 rpm and below 4° C. for 5 min;
S15，最后，用去离子水洗涤纳米颗粒多次，并将其悬浮在去离子水中得到载有吡非尼酮纳米颗粒的悬浮液。S15, finally, washing the nanoparticles with deionized water for multiple times, and suspending them in deionized water to obtain a suspension of pirfenidone-loaded nanoparticles.
其中，在步骤S6中，罗丹明B来标记纳米粒子复合物的制备，包括以下步骤：Wherein, in step S6, the preparation of the nanoparticle complex labeled with rhodamine B comprises the following steps:
S21，首先，对含有罗丹明B标记纳米颗粒悬浮液进行预先制备；S21, first, a suspension containing rhodamine B labeled nanoparticles is prepared in advance;
S22，然后，将步骤S4中输尿管支架管孵育在含有0.5mg/ml盐酸多巴胺的磷酸盐缓冲液中，然后在室温度下搅拌3h，形成聚多巴胺修饰的输尿管支架管；S22, then, incubating the ureteral stent in step S4 in a phosphate buffer containing 0.5 mg/ml dopamine hydrochloride, and then stirring at room temperature for 3 hours to form a polydopamine-modified ureteral stent;
S23，将聚多巴胺修饰的输尿管支架管用去离子水清洗两次，然后浸泡在步骤S21预先制备的罗丹明B标记纳米颗粒悬浮液中，形成罗丹明B纳米颗粒/吡非尼酮涂层输尿管支架管。S23, washing the polydopamine-modified ureteral stent twice with deionized water, and then soaking it in the rhodamine B-labeled nanoparticle suspension prepared in advance in step S21 to form a rhodamine B nanoparticle/pirfenidone-coated ureteral stent.
其中，在步骤S21中，罗丹明B标记纳米颗粒悬浮液的制备，包括以下步骤：Wherein, in step S21, the preparation of the suspension of rhodamine B labeled nanoparticles comprises the following steps:
S211，首先，将100ul的磷酸盐缓冲盐水和罗丹明B，在2ml含20mg PLGA的二氯甲烷中乳化，加入，在冰浴中超声0.5min，得混合物二；S211, first, emulsify 100 ul of phosphate buffered saline and rhodamine B in 2 ml of dichloromethane containing 20 mg PLGA, add, and ultrasonicate in an ice bath for 0.5 min to obtain mixture 2;
S212，然后，向混合物二中加入4.5ml的1.5％PVA并超声以形成复乳；S212, then, adding 4.5 ml of 1.5% PVA to the mixture 2 and sonicating to form a double emulsion;
S213，接下来，将乳状液在室温下搅拌24h，以完全蒸发二氯甲烷，纳米颗粒以低于4℃的12,000转/min的速度离心5min；S213, next, the emulsion was stirred at room temperature for 24 h to completely evaporate the dichloromethane, and the nanoparticles were centrifuged at 12,000 rpm for 5 min at below 4 °C;
S214，最后，纳米颗粒用去离子水洗三次，最后悬浮在去离子水中，得罗丹明B标记纳米颗粒悬浮液。S214, finally, the nanoparticles are washed three times with deionized water and finally suspended in deionized water to obtain a Rhodamine B labeled nanoparticle suspension.
其中，在步骤S2中，盐酸多巴胺的浓度为0.5mg/ml，磷酸盐缓冲液PH值为8.5。Wherein, in step S2, the concentration of dopamine hydrochloride is 0.5 mg/ml, and the pH value of the phosphate buffer is 8.5.
其中，在步骤S3中，搅拌的搅拌时长为3h。Wherein, in step S3, the stirring time is 3 hours.
然后通过CCK8法测定纳米颗粒/吡非尼酮复合物涂层输尿管支架管的细胞毒性。其测定方法为：将20mm长的无菌输尿管支架管切成2mm长，放入96孔板中。接下来，将细胞悬浮液加入96孔。培养24h和48h后，每孔加入10毫克/毫升的CCK8染料溶液，继续在37℃、5％二氧化碳下培养4h。使用酶联免疫吸附实验在450nm处测量每一孔的吸光度。以未经处理的细胞为对照，细胞存活率为100％。实验一式三份进行。The cytotoxicity of the nanoparticle/pirfenidone complex-coated ureteral stent was then determined by the CCK8 method. The determination method is as follows: a 20 mm long sterile ureteral stent was cut into 2 mm lengths and placed in a 96-well plate. Next, the cell suspension was added to the 96 wells. After 24 h and 48 h of culture, 10 mg/ml of CCK8 dye solution was added to each well and cultured for another 4 h at 37 °C and 5% carbon dioxide. The absorbance of each well was measured at 450 nm using an enzyme-linked immunosorbent assay. Using untreated cells as the control, the cell survival rate was 100%. The experiment was performed in triplicate.
输尿管损伤的动物实验模型
Animal experimental model of ureteral injury
实验动物为20周龄雄性新西兰大白兔。动物实验获得了南通大学实验动物伦理委员会(批准号：S20210301-991)的批准。实验兔随机分为烫伤造模组、未改良输尿管支架管治疗组、NP/吡非尼酮输尿管支架管治疗组，每组3只。所有兔均肌肉注射速眠新Ⅱ(1ml/kg)+舒泰50(0.4ml/kg)。全身麻醉后，兔仰卧固定在手术台上。取腹部中间切口，逐层切开皮肤、皮下组织、腹直肌层，打开腹膜。进入腹腔后，推开降结肠和小肠系膜，钝性分离脂肪组织，一段长约1厘米的输尿管被游离出来。将LK-3型电凝导丝插入输尿管，用10W电凝电灼输尿管3秒钟，拔除导丝。放回小肠结肠系膜，关闭腹腔。术后三天，每日肌肉注射头孢菌素预防感染。The experimental animals were 20-week-old male New Zealand white rabbits. The animal experiment was approved by the Experimental Animal Ethics Committee of Nantong University (approval number: S20210301-991). The experimental rabbits were randomly divided into a scald modeling group, an unmodified ureteral stent treatment group, and an NP/pirfenidone ureteral stent treatment group, with 3 rabbits in each group. All rabbits were injected intramuscularly with Su Mian Xin II (1 ml/kg) + Shu Tai 50 (0.4 ml/kg). After general anesthesia, the rabbit was fixed on the operating table in a supine position. Make a middle incision in the abdomen, cut the skin, subcutaneous tissue, and rectus abdominis layer by layer, and open the peritoneum. After entering the abdominal cavity, push away the descending colon and small intestinal mesentery, bluntly separate the adipose tissue, and a section of ureter about 1 cm long is freed. Insert the LK-3 electrocoagulation guide wire into the ureter, electrocauterize the ureter with 10W electrocoagulation for 3 seconds, and remove the guide wire. Put the small intestinal colon mesentery back and close the abdominal cavity. For three days after surgery, cephalosporin was injected intramuscularly daily to prevent infection.
在治疗组中，与上述方法相同，游离出一段长1cm的输尿管组织，在输尿管中部做一个纵向切口，分别放置个未改良的输尿管支架管和NP/吡非尼酮输尿管支架管，然后用电凝导丝进行热损伤。术后2周处死动物，取输尿管狭窄段和治疗段(约1cm)，比较大体标本的变化和相关细胞因子的表达。In the treatment group, a 1-cm-long ureteral tissue was freed, a longitudinal incision was made in the middle of the ureter, and an unmodified ureteral stent and an NP/pirfenidone ureteral stent were placed, respectively, and then thermal injury was performed with an electrocoagulation guide wire. The animals were killed 2 weeks after surgery, and the ureteral stricture segment and the treatment segment (about 1 cm) were obtained to compare the changes in gross specimens and the expression of related cytokines.
HE染色和免疫组织化学染色分析HE staining and immunohistochemical staining analysis
在输尿管损伤及支架置入2周后，每只兔子用空气栓塞法处死。然后将兔的双侧输尿管和肾脏全部摘除。组织保存在10％的福尔马林溶液中。标本脱水，石蜡包埋，切成5微米厚的切片。用苏木精-伊红(H&E)进行染色，观察输尿管内皮细胞和管腔面积的变化。Two weeks after ureteral injury and stent placement, each rabbit was killed by air embolism. The ureters and kidneys of both rabbits were then completely removed. The tissues were preserved in a 10% formalin solution. The specimens were dehydrated, embedded in paraffin, and cut into 5-micron-thick sections. Hematoxylin-eosin (H&E) staining was used to observe changes in ureteral endothelial cells and lumen area.
应用免疫组织化学方法检测输尿管组织中TGF-β1的表达水平。用3％的过氧化氢在室温下孵育5-10min以阻断内源性过氧化物酶的活性。水浴设定为100℃；将切片置于柠檬酸缓冲液(达科，格洛斯特鲁普，丹麦)中5min，进行抗原修复。用PBS清洗载玻片3次，每次5min，用5％的BSA封堵玻片2h。然后将切片与兔抗转化生长因子β1抗体(1:500,21898-1-AP,proteintech)在4℃孵育过夜。接下来，用PBS清洗玻片，并用二抗孵育1h。将切片用DAB染色显色，然后用苏木精反染。用中性胶封住切片，用显微镜(莱卡DMR3000,234徕卡微系统，德国本斯海姆)观察和评价组织图像。Immunohistochemistry was used to detect the expression level of TGF-β1 in ureteral tissue. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide at room temperature for 5–10 min. The water bath was set at 100°C; the sections were placed in citrate buffer (Dako, Glostrup, Denmark) for 5 min for antigen retrieval. The slides were washed with PBS three times for 5 min each and blocked with 5% BSA for 2 h. The sections were then incubated with rabbit anti-TGF-β1 antibody (1:500, 21898-1-AP, proteintech) at 4°C overnight. Next, the slides were washed with PBS and incubated with secondary antibodies for 1 h. The sections were stained with DAB for color development and then counterstained with hematoxylin. The sections were mounted with neutral gum, and the tissue images were observed and evaluated using a microscope (Leica DMR3000, 234 Leica Microsystems, Bensheim, Germany).
Western Blot分析Western Blot Analysis
提取US组、US+输尿管支架管组和US+NP/吡非尼酮输尿管支架管组的输尿管组织总蛋白，进行蛋白质印迹分析。蛋白质样品在SDS-PAGE凝胶上分离，转移到聚偏二氟乙烯(PVDF)膜上。然后用TBST缓冲液(50mM Tris-HCl，100mM NaC l，0.1％吐温-20，pH 7.6)清洗，并用5％脱脂奶粉在TBST中封闭2h。然后，用兔抗转化生长因子β1抗体(1：1000,21898-1-AP，Protetech)、兔抗I型胶原抗体(1：2000，GB114197，ServicBio)和兔抗胶原III型抗体(1：1000，GB111323，ServicBio)在4℃下孵育过夜。所有实验均重复三次。第二天，取一抗，将膜在室温下用TBST洗涤3次，10min，将
膜在4℃下与二抗孵育过夜。膜的洗涤方法同上，然后用奥德赛红外成像系统((LICOR,Lincoln,NE,USA)对蛋白质条带进行可视化。用ImageJ软件测定蛋白条带的表达，并将蛋白条带的表达归一化为GAPDH。Total protein of ureteral tissues in the US group, US+ureteral stent group, and US+NP/pirfenidone ureteral stent group was extracted for western blot analysis. Protein samples were separated on SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. Then, they were washed with TBST buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1% Tween-20, pH 7.6) and blocked with 5% skim milk powder in TBST for 2 h. Then, they were incubated overnight at 4°C with rabbit anti-transforming growth factor β1 antibody (1:1000, 21898-1-AP, Protetech), rabbit anti-type I collagen antibody (1:2000, GB114197, ServicBio), and rabbit anti-type III collagen antibody (1:1000, GB111323, ServicBio). All experiments were repeated three times. The next day, the primary antibody was taken and the membrane was washed three times with TBST at room temperature for 10 min. The membrane was incubated with secondary antibodies overnight at 4°C. The membrane was washed as above, and then the protein bands were visualized using the Odyssey infrared imaging system (LICOR, Lincoln, NE, USA). The expression of protein bands was determined using ImageJ software and normalized to GAPDH.
统计分析Statistical Analysis
所有数值均以均数±标准差(SD)表示。组间差异采用非配对t检验。用GraphPadPrism9软件进行单因素方差分析(ANOVA)。P值<0.05有统计学意义。All values are expressed as mean ± standard deviation (SD). Unpaired t-test was used to analyze the differences among groups. One-way analysis of variance (ANOVA) was performed using GraphPad Prism 9 software. P value < 0.05 was considered statistically significant.
综上所述，本发明载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法，将临床常用的输尿管支架管浸泡在含有多巴胺的碱性溶液中，在支架表面形成一层粘附性的涂层，然后将多巴胺修饰的支架管与纳米颗粒/吡非尼酮复合物孵育，形成负载吡非尼酮的纳米颗粒涂层的输尿管支架管。在输尿管狭窄的治疗中，使用纳米颗粒作为药物输送平台的报道很少。在本研究中，我们创新性地将纳米颗粒/吡非尼酮复合物负载到输尿管支架管上，并通过纳米颗粒的生物降解和控制释放，检测了纳米颗粒/吡非尼酮复合物包裹的输尿管支架管的生物相容性、释放特性和组织分布。最后，我们通过病理切片染色和WesternBlot检测大体解剖改变、胶原沉积和各种纤维蛋白的表达，评价纳米颗粒/吡非尼酮复合物涂层输尿管支架管抑制输尿管纤维化的疗效，以达到预防输尿管狭窄的效果。In summary, the preparation method of the ureteral stent tube loaded with pirfenidone nanoparticle composite coating of the present invention is to soak the commonly used ureteral stent tube in the clinic in an alkaline solution containing dopamine, form a layer of adhesive coating on the surface of the stent, and then incubate the dopamine-modified stent tube with the nanoparticle/pirfenidone complex to form a ureteral stent tube loaded with pirfenidone nanoparticle coating. In the treatment of ureteral stenosis, there are few reports on the use of nanoparticles as a drug delivery platform. In this study, we innovatively loaded the nanoparticle/pirfenidone complex onto the ureteral stent tube, and detected the biocompatibility, release characteristics and tissue distribution of the ureteral stent tube wrapped with the nanoparticle/pirfenidone complex through biodegradation and controlled release of nanoparticles. Finally, we evaluated the efficacy of the nanoparticle/pirfenidone complex-coated ureteral stent tube in inhibiting ureteral fibrosis by pathological section staining and Western Blot detection of gross anatomical changes, collagen deposition and the expression of various fibrins, so as to achieve the effect of preventing ureteral stenosis.
在本发明的描述中，除非另有明确的规定和限定，术语“设置”、“安装”、“相连”、“连接”、“固定”应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系。对于本领域的普通技术人员而言，可以具体情况理解上述术语在本发明中的具体含义。In the description of the present invention, unless otherwise clearly specified and limited, the terms "set", "install", "connect", "connect", and "fix" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims (6)
- 载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法，其特征在于：包括以下步骤：The method for preparing a ureteral stent tube with a composite coating of pirfenidone-loaded nanoparticles is characterized by comprising the following steps:S1，首先，预先制备载有吡非尼酮纳米颗粒的悬浮液；S1, first, a suspension of pirfenidone-loaded nanoparticles is prepared in advance;S2，然后，将输尿管支架管置于含盐酸多巴胺的磷酸盐缓冲液中孵育；S2, then, the ureteral stent was placed in phosphate buffered saline containing dopamine hydrochloride and incubated;S3，接下来，在室温下搅拌一定时间，形成聚多巴胺修饰的输尿管支架管；S3, next, stirring at room temperature for a certain period of time to form a polydopamine-modified ureteral stent;S4，将步骤S3中得到聚多巴胺修饰的输尿管支架管，用去离子水清洗两次，然后将支架浸泡在吡非尼酮纳米颗粒的悬浮液中，形成负载吡非尼酮纳米颗粒涂层的输尿管支架管；S4, washing the polydopamine-modified ureteral stent obtained in step S3 twice with deionized water, and then immersing the stent in a suspension of pirfenidone nanoparticles to form a ureteral stent loaded with a pirfenidone nanoparticle coating;S5，用扫描电子显微镜观察包裹纳米颗粒的输尿管支架管的表面形态；S5, the surface morphology of the ureteral stent coated with nanoparticles was observed using scanning electron microscopy;S6，最后，通过罗丹明B来标记纳米粒子复合物，对吡非尼酮纳米粒子在输尿管支架管应用体内后在组织中分布情况进行检测。S6. Finally, the nanoparticle complex was labeled with rhodamine B to detect the distribution of pirfenidone nanoparticles in tissues after ureteral stent application in vivo.
- 根据权利要求1所述载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法，其特征在于：在步骤S1中，制备载有吡非尼酮纳米颗粒的悬浮液的制备，包括以下步骤：The method for preparing a ureteral stent tube with a composite coating of pirfenidone nanoparticles according to claim 1, characterized in that: in step S1, preparing a suspension containing pirfenidone nanoparticles comprises the following steps:S11，首先，将磷酸盐缓冲盐水、吡非尼酮和聚乳酸-羟基乙酸，乳化在二氯甲烷中，使用超声波均质机，超声1min得到混合物一；S11, first, phosphate buffered saline, pirfenidone and polylactic-co-glycolic acid are emulsified in dichloromethane, and ultrasonicated for 1 min using an ultrasonic homogenizer to obtain a mixture 1;S12，然后，在步骤S11得到的混合物一中加入聚乙烯醇溶液并超声乳化3min；S12, then, adding polyvinyl alcohol solution to the mixture 1 obtained in step S11 and ultrasonically emulsifying for 3 minutes;S13，然后，将步骤S12得到的乳状液在室温下搅拌24h，使二氯甲烷完全蒸发，得到纳米颗粒；S13, then, stirring the emulsion obtained in step S12 at room temperature for 24 hours to completely evaporate the dichloromethane to obtain nanoparticles;S14，将步骤S13得到的纳米颗粒以低于4℃的12,000转/min的速度离心5min；S14, centrifuging the nanoparticles obtained in step S13 at a speed of 12,000 rpm and below 4° C. for 5 min;S15，最后，用去离子水洗涤纳米颗粒多次，并将其悬浮在去离子水中得到载有吡非尼酮纳米颗粒的悬浮液。S15, finally, washing the nanoparticles with deionized water for multiple times, and suspending them in deionized water to obtain a suspension of pirfenidone-loaded nanoparticles.
- 根据权利要求2所述载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法，其特征在于：在步骤S6中，罗丹明B来标记纳米粒子复合物的制备，包括以下步骤：The method for preparing a ureteral stent tube with a composite coating of pirfenidone-loaded nanoparticles according to claim 2, characterized in that: in step S6, the preparation of the nanoparticle complex labeled with rhodamine B comprises the following steps:S21，首先，对含有罗丹明B标记纳米颗粒悬浮液进行预先制备；S21, first, a suspension containing rhodamine B labeled nanoparticles is prepared in advance;S22，然后，将步骤S4中输尿管支架管孵育在含有0.5mg/ml盐酸多巴胺的磷酸盐缓冲液中，然后在室温度下搅拌3h，形成聚多巴胺修饰的输尿管支架管；S22, then, incubating the ureteral stent in step S4 in a phosphate buffer containing 0.5 mg/ml dopamine hydrochloride, and then stirring at room temperature for 3 hours to form a polydopamine-modified ureteral stent;S23，将聚多巴胺修饰的输尿管支架管用去离子水清洗两次，然后浸泡在步骤S21预先制备的罗丹明B标记纳米颗粒悬浮液中，形成罗丹明B纳米颗粒/吡非尼酮涂层输尿管支架管。 S23, washing the polydopamine-modified ureteral stent twice with deionized water, and then soaking it in the rhodamine B-labeled nanoparticle suspension prepared in advance in step S21 to form a rhodamine B nanoparticle/pirfenidone-coated ureteral stent.
- 根据权利要求3所述载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法：在步骤S21中，罗丹明B标记纳米颗粒悬浮液的制备，包括以下步骤：According to the method for preparing the ureteral stent tube with a composite coating of pirfenidone-loaded nanoparticles as claimed in claim 3: in step S21, the preparation of a suspension of rhodamine B-labeled nanoparticles comprises the following steps:S211，首先，将100ul的磷酸盐缓冲盐水和罗丹明B，在2ml的含20mg PLGA的二氯甲烷中乳化，在冰浴中超声0.5min，得混合物二；S211, first, emulsify 100 ul of phosphate buffered saline and rhodamine B in 2 ml of dichloromethane containing 20 mg PLGA, and ultrasonicate for 0.5 min in an ice bath to obtain mixture 2;S212，然后，向混合物二中加入4.5ml的1.5％PVA并超声，以形成复乳；S212, then, adding 4.5 ml of 1.5% PVA to the mixture 2 and sonicating to form a double emulsion;S213，接下来，将乳状液在室温下搅拌24h，以完全蒸发二氯甲烷，纳米颗粒以低于4℃的12,000转/min的速度离心5min；S213, next, the emulsion was stirred at room temperature for 24 h to completely evaporate the dichloromethane, and the nanoparticles were centrifuged at 12,000 rpm for 5 min at below 4 °C;S214，最后，纳米颗粒用去离子水洗三次，最后悬浮在去离子水中，得罗丹明B标记纳米颗粒悬浮液。S214, finally, the nanoparticles are washed three times with deionized water and finally suspended in deionized water to obtain a Rhodamine B labeled nanoparticle suspension.
- 根据权利要求1所述载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法，其特征在于：在步骤S2中，盐酸多巴胺的浓度为0.5mg/ml，磷酸盐缓冲液PH值为8.5。The method for preparing a ureteral stent tube with a composite coating of pirfenidone-loaded nanoparticles according to claim 1, characterized in that: in step S2, the concentration of dopamine hydrochloride is 0.5 mg/ml, and the pH value of the phosphate buffer is 8.5.
- 根据权利要求1所述载吡非尼酮纳米粒子复合涂层输尿管支架管的制备方法，其特征在于：在步骤S3中，搅拌的搅拌时长为3h。 The method for preparing a ureteral stent tube with a composite coating of pirfenidone-loaded nanoparticles according to claim 1, characterized in that: in step S3, the stirring time is 3 hours.
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| CN115970069A (en) * | 2022-12-13 | 2023-04-18 | 南通大学附属医院 | Preparation method of ureteral stent tube carrying pirfenidone nanoparticle composite coating |
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| EP3638221B1 (en) * | 2017-06-16 | 2021-09-29 | Avexxin AS | Compositions and methods for treatment of a fibrotic disease |
| CN211911903U (en) * | 2019-11-26 | 2020-11-13 | 中国科学院金属研究所 | Ureteral stent for inhibiting recurrent stricture stimulation symptoms and calculus |
| JP2023509760A (en) * | 2020-01-08 | 2023-03-09 | シンシス セラピューティクス，インコーポレイテッド | ALK5 inhibitor complexes and uses thereof |
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| CA2880011A1 (en) * | 2012-07-24 | 2014-01-30 | Mark William Surber | Aerosol pirfenidone and pyridone analog compounds and uses thereof |
| CN109893682A (en) * | 2019-04-16 | 2019-06-18 | 东北大学 | A kind of degradable metal ureter bracket and preparation method with composite construction |
| CN110464882A (en) * | 2019-07-29 | 2019-11-19 | 东华大学 | A kind of ureter rack tube and preparation method thereof that hydrophilic antimicrobial is degradable |
| WO2021054526A1 (en) * | 2019-09-20 | 2021-03-25 | 의료법인 성광의료재단 | Nanoparticles with special coating and use thereof |
| CN113663142A (en) * | 2021-09-03 | 2021-11-19 | 中国科学院长春应用化学研究所 | Multifunctional coating suitable for urinary medical instruments, and preparation method and application thereof |
| CN115970069A (en) * | 2022-12-13 | 2023-04-18 | 南通大学附属医院 | Preparation method of ureteral stent tube carrying pirfenidone nanoparticle composite coating |
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| CN115970069A (en) | 2023-04-18 |
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