A nanoparticle-coated urinary implant and uses thereof

Abstract

The application discloses a kind of nanoparticle coating loaded urinary system implants and its application, belong to the field of biomedicine.The urinary system implant includes support carrier and nanoparticle coating loaded on the support carrier, the siRNA of targeting TGF-β1 gene is contained in the nanoparticle coating, and the siRNA is wrapped in nanoparticle.The siRNA can be transfected to ureter tissue and inhibit the expression of TGF-β1, to inhibit the occurrence of ureteral stricture.The application combines RNA interference technology and nanoparticle delivery system by urinary system implant, and the effect of the urinary system implant loaded with TGF-β1-siRNA nanoparticle complex for inhibiting ureteral stricture is verified by in-vivo and in-vitro experiments, can effectively inhibit the formation of ureteral stricture around urinary system implant, and has wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a urinary system implant with a nanoparticle coating and its application. Background Technology

[0002] Ureteral stricture refers to narrowing of the ureteral lumen caused by scar repair following ureteral injury, with the majority of cases resulting from iatrogenic injury. This is a challenging condition that can lead to postoperative complications. Without timely intervention, severe ureteral stricture can prevent urine flow, leading to upper urinary tract obstruction and hydronephrosis. Current treatment options for ureteral stricture include endoscopic procedures, laser endarterectomy, and balloon dilation, which often require the placement of a ureteral stent. While ureteral stents stabilize the ureter, prevent obstruction, and promote urine flow, they can also cause side effects such as crystal deposition. Therefore, the success rate and complete resolution rate of these treatments are limited, and better treatment options are still under investigation. Some innovative treatment methods explored in recent years have not yet been widely adopted clinically due to a lack of large-scale follow-up studies. Therefore, ureteral stricture remains a fundamental problem in clinical practice. Currently, based on the existing treatment of ureteral stricture, the main research direction is to prevent the formation of ureteral stricture caused by iatrogenic injury, including preventing the formation of ureteral stricture around ureteral stents. Summary of the Invention

[0003] The purpose of this invention is to provide a urinary system implant loaded with a nanoparticle coating and its application, thereby addressing the problems existing in the prior art. This invention integrates a ureteral stent with RNA interference technology and a nanoparticle delivery system. In vitro and in vivo experiments have verified the effect of the ureteral stent loaded with a TGF-β1-siRNA-targeting nanoparticle complex in inhibiting ureteral stenosis. It can effectively inhibit the formation of ureteral stenosis around the ureteral stent and has broad clinical application prospects in inhibiting ureteral stenosis caused by iatrogenic ureteral injury.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] The present invention provides a urinary system implant loaded with a nanoparticle coating. The urinary system implant includes a support carrier and a nanoparticle coating loaded on the support carrier. The nanoparticle coating contains siRNA targeting the TGF-β1 gene, and the siRNA is encapsulated in nanoparticles.

[0006] Optionally, the support carrier includes a ureteral stent.

[0007] This invention encapsulates siRNA targeting the TGF-β1 gene in nanoparticles and loads it onto a ureteral stent. The siRNA can effectively transfect into ureteral tissue and inhibit the expression of TGF-β1, thereby inhibiting the occurrence of ureteral stricture.

[0008] Optionally, the sense and antisense strand sequences of the siRNA are shown in SEQ ID NO.1 to 2, respectively.

[0009] The present invention also provides a method for preparing the aforementioned urinary system implant, comprising the following steps:

[0010] A nanoparticle / siRNA complex was prepared by encapsulating siRNA targeting the TGF-β1 gene in nanoparticles.

[0011] The urinary system implant was modified by placing it in PBS buffer containing dopamine hydrochloride to obtain the modified urinary system implant;

[0012] The modified urinary system implant was placed in a suspension of the nanoparticle / siRNA complex to obtain the ureteral stent loaded with the nanoparticle coating.

[0013] Optionally, the siRNA is encapsulated in the nanoparticles using a double emulsification method.

[0014] Optionally, in the nanoparticle / siRNA complex, the molar ratio of amino groups on the nanoparticles to phosphate groups in the siRNA, expressed as N and P, is 6:1.

[0015] Optionally, in the PBS buffer containing dopamine hydrochloride, the concentration of dopamine hydrochloride is 0.5 mg / mL and the concentration of PBS is 5 mM.

[0016] Optionally, the modification is achieved by stirring for 3 hours.

[0017] The present invention also provides the use of the siRNA or the delivery system described herein in the preparation of products for inhibiting ureteral stricture, wherein the ureteral stricture is caused by iatrogenic injury.

[0018] Optionally, the ureteral stricture caused by iatrogenic injury includes ureteral stricture formed around a ureteral stent.

[0019] The present invention discloses the following technical effects:

[0020] This invention combines ureteral stent with RNA interference technology and a nanoparticle delivery system. In vitro and in vivo experiments have verified the effect of ureteral stent loaded with TGF-β1-siRNA nanoparticle complex on inhibiting ureteral stenosis. It can effectively inhibit the formation of ureteral stenosis around the ureteral stent and has broad clinical application prospects in inhibiting ureteral stenosis caused by iatrogenic ureteral injury. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Schematic diagram of the fabrication and research mechanism of NP / TGF-β1-siRNA complex coated ureteral stent;

[0023] Figure 2 The inhibitory effects of different siRNAs targeting TGF-β1 on TGF-β1 expression are shown. A: Fluorescent staining image of ureteral epithelial cells (scale bar = 200 μm); B and C: Western blot plots and statistical results of TGF-β1 expression levels after using negative control siRNA, TGF-β1-siRNA1, TGF-β1-siRNA2, and TGF-β1-siRNA3.

[0024] Figure 3 Characterization of TGF-β1-siRNA-coated ureteral stents and siRNA release characteristics; where A: dynamic particle size distribution of the NP / TGF-β1-siRNA complex; B: gel retardation experiment of the NP / TGF-β1-siRNA complex at different N / P ratios; C: in vitro release curve of TGF-β1-siRNA from nanoparticle-coated ureteral stents in PBS at pH 7.4; D: in vitro cytotoxicity analysis of different experimental groups after co-culturing with ureteral epithelial cells for 24, 48, and 72 hours.

[0025] Figure 4The graph shows the in vitro and in vivo transfection efficiency. A: Fluorescent expression of EGFP in ureteral epithelial cells of the unmodified ureteral stent group; B: NP / TGF-β1-siRNA complex-coated ureteral stent group; C: Transfection efficiency of ureteral epithelial cells in the unmodified ureteral stent group and the NP / TGF-β1-siRNA complex-coated ureteral stent group as analyzed by flow cytometry; D: Statistical graph of transfection efficiency results analyzed by flow cytometry.

[0026] Figure 5 Scanning electron microscope images of unmodified ureteral stents, nanoparticle / TGF-β1-siRNA ureteral stents, and nanoparticle / TGF-β1-siRNA ureteral stents after friction treatment;

[0027] Figure 6 Immunofluorescence staining image of ureteral tissue one week after implantation of nanoparticle / TGF-β1-siRNA ureteral stent;

[0028] Figure 7 The diagram shows the placement of ureteral stents in rabbits and the formation of a ureteral injury model. A: Placement of different groups of ureteral stents in the rabbit ureter; B: Induction of ureteral injury using vascular clamps; C: Formation of the rabbit ureteral injury model.

[0029] Figure 8 Anatomical images of specimens from different treatment groups four weeks after lesion formation;

[0030] Figure 9 HE staining images of ureteral tissue sections from different treatment groups four weeks after the injury occurred;

[0031] Figure 10 Masson staining images of ureteral tissue sections from different treatment groups four weeks after the injury occurred;

[0032] Figure 11 Immunohistochemical staining images of ureteral tissue sections from different treatment groups four weeks after the formation of the lesion. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] This invention established a rabbit ureteral stenosis model, implanted a ureteral stent loaded with TGF-β1-siRNA for treatment, and compared the histopathological changes and related gene and protein expression of ureteral stenosis in different groups to evaluate the therapeutic effect. The results confirmed that the combination of ureteral stent and RNA interference technology with a nanoparticle delivery system has broad clinical application prospects in inhibiting ureteral stenosis. The research idea and experimental process of this invention are as follows: Figure 1 As shown.

[0039] In this invention, nanoparticles act as a medium between siRNA and a ureteral stent, encapsulating the therapeutic drug TGF-β1-siRNA, thereby enabling the siRNA to be loaded onto the ureteral stent. The ureteral stent is immersed in DA solution (5 mM phosphate buffer containing 0.5 mg / mL dopamine hydrochloride, pH 8.5) to improve adhesion, thereby adsorbing the nanoparticle / TGF-β1-siRNA complex. The NP / TGF-β1-siRNA complex thus prepared and encapsulated in the ureteral stent, when placed in vivo, can prevent the formation of ureteral stricture around the ureteral stent.

[0040] The present invention will now be described in detail with reference to specific embodiments.

[0041] Example

[0042] I. Materials and Methods

[0043] 1. Constructing siRNA targeting TGF-β1

[0044] Based on the gene sequence of rabbit TGF-β1 precursor from Gene Bank, three siRNAs targeting TGF-β1 and one negative control siRNA were constructed. The sense and antisense strand sequences of siRNA1 are 5'-GCGUCUAUAUGCUGUUUAA-3' (SEQ ID NO.1) and 5'-UUAAACAGCAUAGACGC-3' (SEQ ID NO.2), respectively. The sense and antisense strand sequences of siRNA2 are 5'-GGCUCAACAUCUACACAGU-3' (SEQ ID NO.3) and 5'-ACUGU GUAGAUGUUGAGCC-3' (SEQ ID NO.4), respectively. The sense and antisense strand sequences of siRNA3 are 5'-CCAC CAUUCACAGCAUGAA-3' (SEQ ID NO.5) and 5'-UUCAUGCUGUGAAUGGUGG-3' (SEQ ID NO.6), respectively. In addition, a negative control siRNA was constructed, with the sense strand being 5'-UUCUCCGAACGUGUCA CGU-3' (SEQ ID NO.7) and the antisense strand being 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO.8). Western blot experiments were used to detect the silencing effect of TGF-β1 and to screen for target siRNAs.

[0045] 2. Primary culture and identification of rabbit ureteral epithelial cells

[0046] Rabbit ureteral tissue was rapidly minced, washed with physiological saline, and then placed in a mixture of collagenase and trypsin for digestion by agitation. The tissue blocks were periodically ground with a pipette to collect isolated cells, which were then washed to remove the digestive enzymes. The cells were suspended in serum-added culture medium and seeded into plastic culture dishes. After one week, the cultured ureteral epithelial cells were transferred to 24-well culture plates with coverslips. When the cells covered approximately 80% of the culture area, the coverslips were removed and the cells were washed with PBS. The cells were fixed with methanol at -20°C for 5 minutes, washed with PBS, and then blocked with 10% goat serum at 37°C for 30 minutes, followed by another wash with PBS. Broad-spectrum keratin (PCK) antibody was added and incubated overnight at 4°C. Goat anti-rabbit FITC (secondary antibody, 1:100 dilution) was added, and the mixture was reacted at 37°C for 1 hour, followed by a wash with PBS. The cells were mounted with 90% glycerol and the staining results were observed under a fluorescence microscope.

[0047] 3. Preparation of nanoparticle / TGF-β1-siRNA complex

[0048] This invention employs a dual emulsification method to construct an NP (nanoparticle) / TGF-β1-siRNA complex. First, 400 mg of polylactic acid-glycolic acid copolymer (PLGA, Mw = 40,000-75,000, St. Louis, USA) was dissolved in 4 mL of dichloromethane (DCM). Then, 12 mL of a 7% (w / v) aqueous solution of polyvinyl alcohol (PVA, Mw = 14,160) was added, and the mixture was ultrasonically emulsified for 1 minute. Next, the mixture was added to 200 mL of a 1% (w / v) aqueous solution of PVA, and emulsification was continued for 3 minutes to complete the preparation of the dual emulsion. The emulsion was stirred overnight at room temperature until the remaining dichloromethane was completely evaporated. The nanoparticles were separated by centrifugation at 12,000 rpm for 5 minutes at 4°C. Finally, the nanoparticles were washed twice with deionized water and dispersed in a deionized aqueous solution to obtain a nanoparticle suspension. By modifying nanoparticles with polyethyleneimine to give them a positive charge, adding a TGF-β1-siRNA solution (20 μM), and then screening the optimal N / P value of nanoparticles and siRNA through gel retardation experiments, the TGF-β1-siRNA-loaded nanoparticle (NP / TGF-β1-siRNA) complex with the best binding effect was obtained.

[0049] 4. Characterization of nanoparticles and detection of cell viability

[0050] The particle size of the NP / TGF-β1siRNA complex was measured using dynamic light scattering (DLS). Furthermore, the cytotoxicity of the NP / TGF-β1siRNA complex was assessed using the CCK8 assay. The NP / TGF-β1siRNA complex and centrifuged and homogenized cell suspension (SV-HUC-1, PC101) were added to 96-well plates (0.5 × 10⁶ cells per well).4 Cells were cultured in 96-well plates at 37°C and 5% CO2 for 24, 48, and 72 hours. At these times, 10 μL of CCK8 solution (10 mg / mL, Sigma) was added to each well. After the second addition of CCK8 solution, the plates were incubated for another 4 hours. The absorbance of each well was then measured at 450 nm using a microplate reader. The blank control group consisted of cells without the added complex. The experiment was repeated three times.

[0051] 5. Preparation and characterization of NP / TGF-β1-siRNA complex-coated ureteral stents

[0052] A modified ureteral stent was prepared by placing it in phosphate-buffered saline (PBS, 5 mM, pH 8.5) containing dopamine hydrochloride (0.5 mg / mL) and stirring at room temperature for 3 hours. The stent was then washed twice with deionized water and placed in a pre-prepared NP / TGF-β1-siRNA suspension. Finally, the surface morphology of the composite-coated ureteral stent was characterized using scanning electron microscopy (SEM, Hitachi, S-3400N, Tokyo, Japan).

[0053] 6. In vitro transfection efficiency

[0054] FAM-modified siRNA nanoparticles (synthesized by Gemma Biosciences) were used for in vitro transfection experiments. To assess in vitro transfection efficiency, the FAM-modified siRNA nanoparticles were incubated with human ureteral epithelial cells in cell culture medium. After 6 hours, the medium was replaced with DMEM containing 10% FBS, and the cells were cultured for another 48 hours. Subsequently, the cells were washed three times with 1 mL PBS and observed under a fluorescence microscope (Leica DMR 3000; Leica Microsystems, Bensheim, Germany) in the FITC channel (excitation wavelength 488 nm, emission wavelength 518 nm). Finally, the cells were collected and aspirated to suspend them in PBS, and the in vitro transfection efficiency was assessed using a FACSCalibur flow cytometer (BD FACSCalibur, BD Bioscience, San Jose, CA).

[0055] 7. In vivo transfection experiment

[0056] Rhodamine B was used for in vivo transfection experiments. Rhodamine B was mixed into PBS to prepare a rhodamine B-labeled NP / TGF-β1-siRNA complex. 20 mg of PLGA was dissolved in 2 mL of dichloromethane (DCM). Next, 100 μL of PBS containing NP / TGF-β1-siRNA was added, and the mixture was then added to 4.5 mL of 1.5% PVA and sonicated on ice for 1 minute to obtain a primary emulsion. This was then sonicated again on ice for 3 minutes to complete the preparation of a secondary emulsion. The emulsion was then stirred overnight at room temperature until the remaining dichloromethane was completely evaporated. Nanoparticles were collected by centrifugation at 12,000 RPM for 5 minutes at 4°C, washed twice with deionized water, and dispersed in deionized water to obtain a rhodamine B-labeled NP / TGF-β1-siRNA suspension. Subsequently, the ureteral stent was placed in phosphate-buffered saline (PBS, 5 mM, pH 8.5) containing dopamine hydrochloride (0.5 mg / mL) and stirred at room temperature for 3 hours to obtain the modified ureteral stent. After washing the stent twice with deionized water, it was placed in a pre-prepared suspension of rhodamine B-labeled NP / TGF-β1-siRNA. Both the unmodified ureteral stent and the rhodamine B-labeled NP / TGF-β1-siRNA complex-coated ureteral stent were implanted in rabbits for one week. Finally, the distribution of nanoparticles in the ureteral tissue was observed.

[0057] 8. Animal model preparation

[0058] Twenty-five healthy 20-week-old New Zealand male rabbits, each weighing approximately 3.0 kg, were selected as experimental models. The 25 rabbits were divided into five groups: (1) sham surgery group, (2) ureteral stricture model group (US), (3) US + unmodified ureteral stent group, (4) US + TGF-β1-siRNA ureteral stent group, and (5) negative control group. Before the experiment, the rabbits were anesthetized by injection of Supra-Symplocyl II (2 mg / kg) + Supra-Symplocyl 50 (15 mg / kg) and then fixed on the operating table. In the supine position, the abdominal cavity of the rabbits was exposed layer by layer, and the adipose tissue was bluntly dissected. The ureter was exposed behind the colon and mesentery, a section of the ureter about 1 cm long was freed and longitudinally cut on it, and different ureteral stents were inserted according to the experimental group. In order to form ureteral stricture, the ureteral site where the ureteral stent was placed was clamped with a hemostat. The abdominal cavity was then closed, and the hemostat was removed 48 hours later when the abdominal cavity was reopened. At this point, ureteral stricture had formed in the rabbits, and they were given daily injections of cefazolin for three consecutive days to prevent infection. Four weeks later, all rabbits were euthanized, and their ureters and kidneys were collected and preserved for further research.

[0059] 9. Histopathological evaluation

[0060] Both ureters were fixed in 10% formalin solution. The tissues were then dehydrated and paraffin-embedded, sectioned to a thickness of 5 micrometers, and stained with hematoxylin-eosin (HE). Pathological evaluation of the tissue samples was performed.

[0061] 10. Masson staining

[0062] After dewaxing with xylene, the sections were washed with ethanol and hydrated. Following the instructions, sections from the five sample groups were stained sequentially with Weigert iron hematoxylin and Ponceau S staining solutions, and then washed with phosphomolybdic acid solution. Subsequently, the sections were stained with aniline blue solution and washed with a weak acid solution. Finally, before microscopic observation, the sections were dehydrated, routinely cleared, and mounted with neutral resin.

[0063] 11. Immunohistochemical staining

[0064] First, the stored tissue samples were dewaxed with xylene and dehydrated with alcohol. To inhibit endogenous peroxidase activity, the tissue samples were co-incubated with hydrogen peroxide while non-specific binding sites were blocked with serum. Subsequently, the tissue samples were incubated overnight with primary antibody at 4°C and washed three times with PBS. With the aid of a microscope (Leica DMR 000, Leica Microsystems, Bensheim, Germany), we were able to clearly observe and evaluate the tissue images. Finally, the staining intensity of TGF-β1, type I collagen, and type III collagen was measured.

[0065] 12. Western blotting experiment

[0066] Proteins were extracted from the ureter. Protein samples were separated on a pre-prepared 15% SDS-polyacrylamide gel, electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane, and then blocked at room temperature for 2 hours in a mixture of 5% skim milk powder and TBST (50 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20, pH 7.6). Subsequently, the membrane was incubated overnight at 4°C with anti-rabbit primary antibodies (anti-TGF-β1 antibody (1:1000, 21898-1-AP, ProteinTech), anti-type I collagen antibody (1:2000, GB114197, ServiceBio), and anti-type III collagen antibody (1:1000, GB111323, ServiceBio)). Afterward, the membrane was washed three times with TBST and then incubated overnight at 4°C with secondary antibody. After washing three more times with TBST, the target bands and internal controls were imaged using an Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA). The band expression levels were detected using ImageJ software, and the results were normalized to GADPH.

[0067] 13. Statistical Analysis

[0068] Descriptive data are expressed as mean ± standard deviation (mean ± SD). Differences between groups were analyzed using the independent samples Student's test. Differences among the three groups were analyzed using one-way ANOVA with GraphPad Prism 9 software. The statistical significance level was set at p < 0.05.

[0069] II. Results

[0070] 1. siRNA targeting TGF-β1

[0071] Figure 2 The inhibitory effects of three different siRNAs targeting TGF-β1 on TGF-β1 expression were studied. Figure 2 As shown in section A, ureteral epithelial cells were successfully extracted and identified from rabbit ureters using immunofluorescence. To detect three siRNAs targeting TGF-β1 and a negative control siRNA, and to screen for the one with the best interference effect, the expression level of TGF-β1 was detected by Western blotting two days after transfection. Finally, as... Figure 2 B and Figure 2 As shown in Figure C, TGF-β1-siRNA1 exhibited the best inhibitory effect; therefore, TGF-β1-siRNA1 was selected as the therapeutic agent. Furthermore, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Figure 2It was also found that the negative control siRNA was unable to inhibit the expression of TGF-β1 compared with the siRNA targeting TGF-β1.

[0072] 2. Characterization of nanoparticles, gel retardation assay, and cell viability detection

[0073] The particle size of the NP / TGF-β1-siRNA complex was detected by dynamic light scattering (DLS), and its average particle size was found to be 187.70 ± 52.20 nm. Figure 3 As shown in A. The complexation of siRNA and PLGA nanoparticles was detected using gel retardation assays, considering different NP / TGF-β1-siRNA ratios, such as... Figure 3 As shown in Figure B. The complex exhibited better blocking effect at an N / P (molar ratio of amino groups on the nanoparticles to phosphate groups of siRNA) ratio of 6:1 NP / TGF-β1-siRNA, and this ratio was used in subsequent experiments, demonstrating that the NP / TGF-β1-siRNA complex possesses gene-binding capacity. The degradability of polylactic-co-glycolic acid copolymer (PLGA) ensured the sustained release of TGF-β1-siRNA. Figure 3 As shown in Figure C, the release rate was approximately 23% on day 5 and approximately 79% on day 28, demonstrating the sustained release characteristic of TGF-β1-siRNA. The cytotoxicity of the NP / TGF-β1-siRNA complex against human ureteral epithelial cells was assessed using a CCK8 assay. CCK8 solution was added to each well at 24h, 48h, and 72h. No significant difference in cytotoxicity was observed compared to the blank control group without the complex (e.g., ...). Figure 3 As shown in D), this provides a basis for further experiments.

[0074] 3. In vitro transfection efficiency

[0075] In vitro transfection efficiency was assessed using FAM-modified siRNA. The NP / TGF-β1-siRNA complex-coated ureteral stent significantly increased EGFP expression levels, while no EGFP expression was observed on the unmodified ureteral stent (e.g., ...). Figure 4 (As shown in A and B). To eliminate interference from irrelevant factors, experiments were performed under the same transfection conditions, and the percentage of EGFP-positive cells was measured by flow cytometry after 48 hours to obtain quantitative data. Figure 4 As shown in C and D, the transfection efficiency of the unmodified ureteral stent was 0.03%, while the transfection efficiency of the NP / TGF-β1-siRNA complex-coated ureteral stent was 78.43%, demonstrating the high transfection efficiency of nanoparticles as siRNA carriers.

[0076] 4. Characterization of ureteral stent coated with nanoparticle / TGF-β1-siRNA complex

[0077] The distribution and adsorption of the NP / TGF-β1-siRNA complex on the surface of the ureteral stent were observed using scanning electron microscopy. Figure 5 The three sets of results showed that after the ureteral stent was placed in the body, most of the nanoparticle complex remained adsorbed on the stent surface. These three sets were: unmodified ureteral stent, ureteral stent loaded with nanoparticle / TGF-β1-siRNA complex, and ureteral stent loaded with nanoparticle / TGF-β1-siRNA complex and subjected to friction treatment.

[0078] 5. Distribution of nanoparticles / TGF-β1-siRNA complex in the ureter

[0079] like Figure 6 As shown, after one week of treatment with the ureteral stent coated with the NP / TGF-β1-siRNA complex, the diffusion of the red fluorescent marker in the ureteral tissue was clearly observed. This indicates that the nanoparticles carrying NP / TGF-β1-siRNA have penetrated into the ureteral tissue.

[0080] 6. Ureteral injury model in rabbits and changes in gross specimens

[0081] Figure 7 An in vivo ureteral injury model induced by hemostats was demonstrated. Twenty-five rabbits were divided into five groups and all were euthanized after four weeks of rearing. Figure 8 This study presents complete kidney and ureter specimens, as well as coronal section specimens of the kidneys, four weeks after lesion formation in five groups (normal group, model group, unmodified ureteral stent group, TGF-β1-siRNA ureteral stent group, and negative control group). The ureteral stenosis group showed significant swelling compared to the healthy kidney group, indicating severe ureteral obstruction. The negative control group showed mild improvement in edema, while the ureteral stent group and the siRNA nanoparticle stent group showed significant reduction in inflammatory edema.

[0082] 7. HE staining, Masson staining analysis, and immunohistochemical results of TGF-β1, type I collagen, and type III collagen.

[0083] Figure 9 The results showed that, compared to the sham group, the model group (US) experienced severe ureteral stenosis due to damage caused by the vascular clamp. Furthermore, the stenosis in the unmodified ureteral stent group was alleviated. More importantly, the rabbit ureteral stenosis was significantly improved using a ureteral stent coated with a nanoparticle / TGF-β1-siRNA complex.

[0084] Masson staining revealed that collagen fibers were clearly stained blue. In the sham-operated group, the ureteral layers were clearly defined, with a smaller proportion of blue fibrous tissue; however, in the US group, collagen fibers proliferated extensively, occupying most of the luminal area. Furthermore, fibrosis was reduced in the unmodified ureteral stent group and the negative control group, while fibrosis was significantly improved in the nanoparticle / TGF-β1-siRNA complex-coated ureteral stent group (e.g., ...). Figure 10 (As shown).

[0085] Immunohistochemical analysis was also performed in this invention. Due to the observation of a large number of TGF-β1 positive cells, the number and staining intensity of TGF-β1 positive cells were significantly enhanced in the US group, the US+ unmodified ureteral stent group, and the negative control group. However, in the TGF-β1-siRNA ureteral stent group, both the number and staining intensity of TGF-β1 positive cells were significantly reduced. Figure 11 .

[0086] 8. Western Blot Analysis

[0087] In the model group, the unmodified ureteral stent group, the nanoparticle / TGF-β1-siRNA complex-coated ureteral stent group, and the negative control group, the expression of TGF-β1 protein, type I collagen, and type III collagen were all increased compared with the normal group. However, the increase in TGF-β1 and protein expression was the smallest in the TGF-β1-siRNA ureteral stent group, indicating that TGF-β1 protein expression was significantly reduced after the nanoparticle / TGF-β1-siRNA complex was coated on the ureteral stent, thereby affecting the expression of fibrosis-related proteins.

[0088] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A urinary system implant with a nanoparticle coating, characterized in that, The urinary system implant includes a support carrier and a nanoparticle coating loaded on the support carrier, wherein the nanoparticle coating contains siRNA targeting the TGF-β1 gene, and the siRNA is encapsulated in the nanoparticles; The sense and antisense strand sequences of the siRNA are shown in SEQ ID NO.1-2, respectively; The method for preparing the urinary system implant includes the following steps: A nanoparticle / siRNA complex was prepared by encapsulating siRNA targeting the TGF-β1 gene in nanoparticles. The support vector was placed in a PBS buffer containing dopamine hydrochloride for modification to obtain a modified support vector; The modified support carrier is placed in a suspension of the nanoparticle / siRNA complex to obtain the urinary system implant with the nanoparticle coating.

2. The urinary system implant according to claim 1, characterized in that, The support carrier includes a ureteral stent.

3. A method for preparing a urinary system implant according to claim 1 or 2, characterized in that, Includes the following steps: A nanoparticle / siRNA complex was prepared by encapsulating siRNA targeting the TGF-β1 gene in nanoparticles. The support vector was placed in a PBS buffer containing dopamine hydrochloride for modification to obtain a modified support vector; The modified support carrier is placed in a suspension of the nanoparticle / siRNA complex to obtain the urinary system implant with the nanoparticle coating.

4. The preparation method according to claim 3, characterized in that, The siRNA is encapsulated in the nanoparticles using a double emulsification method.

5. The preparation method according to claim 3, characterized in that, In the nanoparticle / siRNA complex, the molar ratio of amino groups on the nanoparticles to phosphate groups in the siRNA, expressed as N and P, is 6:

1.

6. The preparation method according to claim 3, characterized in that, The dopamine hydrochloride-containing PBS buffer contains dopamine hydrochloride at a concentration of 0.5 mg / mL and PBS at a concentration of 5 mM.

7. The preparation method according to claim 3, characterized in that, The modification is achieved by stirring, and the modification time is 3 hours.

8. The use of a urinary system implant according to claim 1 or 2 in the preparation of a product for inhibiting ureteral stricture, characterized in that, The ureteral stricture mentioned is a ureteral stricture caused by iatrogenic injury.

9. The application according to claim 8, characterized in that, The ureteral stricture caused by iatrogenic injury includes ureteral stricture formed around a ureteral stent.

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