Urinary system implant loaded with nanoparticle coating and application of urinary system implant

Abstract

The invention discloses a urinary system implant loaded with a nanoparticle coating and application of the urinary system implant, and belongs to the technical field of biological medicine. The urinary system implant comprises a support carrier and a nano-particle coating loaded on the support carrier, the nano-particle coating contains siRNA (small interfering Ribonucleic Acid) of a targeted TGF (Transforming Growth Factor)-beta 1 gene, and the siRNA is wrapped in nano-particles. The siRNA can be transfected to ureteral tissues and inhibit the expression of TGF-beta 1, so that the occurrence of ureteral stenosis is inhibited. According to the invention, the urinary system implant is combined with an RNA interference technology and a nanoparticle delivery system, and in-vivo and in-vitro experiments prove that the urinary system implant loaded with the targeted TGF-beta1-siRNA nanoparticle compound has the effect of inhibiting the ureterostenosis, and can effectively inhibit the formation of the ureterostenosis around the urinary system implant; wide clinical application prospects are realized.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a urinary system implant loaded with a nanoparticle coating and application thereof. Background Art

[0002] Ureteral stenosis refers to the narrowing of the ureteral lumen caused by scar repair after ureteral injury, most of which are caused by iatrogenic injury. It is a difficult disease that can lead to postoperative complications. If not intervened in time, severe ureteral stenosis will prevent urine discharge, leading to upper urinary tract obstruction and hydronephrosis. At present, the treatment options for ureteral stenosis include endoscopic treatment, laser endarterectomy and balloon dilatation, which often require the placement of ureteral stents in the body. Ureteral stents can stabilize the ureter, prevent ureteral obstruction and promote urine flow, but they also bring side effects, such as crystal deposition. Therefore, the success rate and complete resolution rate of the above treatment options are limited, and better treatment options are still being explored. Some innovative treatment methods explored in recent years have not yet been promoted and applied in clinical practice due to the lack of large-sample follow-up studies. Therefore, ureteral stenosis has not been fundamentally solved in clinical practice. At present, based on the existing treatment status of ureteral stenosis, the main research direction is to prevent the formation of ureteral stenosis caused by iatrogenic injury, including preventing the formation of ureteral stenosis around ureteral stents. Summary of the invention

[0003] The purpose of the present invention is to provide a urinary system implant loaded with nanoparticle coating and its application to solve the problems existing in the above-mentioned prior art. The present invention combines ureteral stent with RNA interference technology and nanoparticle delivery system, and verifies the effect of ureteral stent loaded with targeted TGF-β1-siRNA nanoparticle complex on inhibiting ureteral stenosis through in vivo and in vitro experiments. It can effectively inhibit the formation of ureteral stenosis around ureteral stent, and has broad clinical application prospects in inhibiting ureteral stenosis caused by iatrogenic ureteral injury.

[0004] To achieve the above object, the present invention provides the following solutions:

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

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

[0007] The present invention encapsulates siRNA targeting the TGF-β1 gene in nanoparticles and loads the nanoparticles on the ureteral stent. The siRNA can be effectively transfected into the ureteral tissue and inhibit the expression of TGF-β1, thereby inhibiting the occurrence of ureteral stenosis.

[0008] Optionally, the sequences of the sense strand and antisense strand of the siRNA are shown in SEQ ID NOs. 1 to 2, respectively.

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

[0010] The siRNA targeting the TGF-β1 gene is encapsulated in the nanoparticles to prepare a nanoparticle / siRNA complex;

[0011] placing the urinary system implant in a PBS buffer containing dopamine hydrochloride for modification to obtain a modified urinary system implant;

[0012] The modified urinary system implant is placed in the 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 by a double emulsification method.

[0014] Optionally, in the nanoparticle / siRNA complex, the molar ratio of the amino group on the nanoparticle to the phosphate group in the siRNA, calculated 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, and the modification time is 3 hours.

[0017] The present invention also provides the use of the siRNA or the delivery system in preparing a product for inhibiting ureteral stenosis, wherein the ureteral stenosis is ureteral stenosis caused by iatrogenic injury.

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

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

[0020] The present invention combines a ureteral stent with RNA interference technology and a nanoparticle delivery system, and verifies the effect of a ureteral stent loaded with a targeted TGF-β1-siRNA nanoparticle complex on inhibiting ureteral stenosis through in vivo and in vitro experiments. 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0023] Figure 2 The figures show the inhibitory effects of different siRNAs targeting TGF-β1 on the expression of TGF-β1; A: fluorescent staining images of ureteral epithelial cells (scale bar = 200 μm); B and C: Western blot experimental detection and statistical results of the expression level of TGF-β1 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; A: Dynamic particle size distribution of NP / TGF-β1-siRNA complex; B: Gel retardation experiment of 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 24, 48 and 72 hours of co-culture with ureteral epithelial cells;

[0025] Figure 4Figure 2 is a graph of in vivo and in vitro transfection efficiency; A: Fluorescence expression of EGFP in ureteral epithelial cells of the unmodified ureteral stent group; B is the NP / TGF-β1-siRNA complex-coated ureteral stent of the NP / TGF-β1-siRNA complex-coated ureteral stent group; C: Flow cytometry analysis to detect the transfection efficiency of ureteral epithelial cells in the unmodified ureteral stent group and the NP / TGF-β1-siRNA complex-coated ureteral stent group; D: Statistical graph of transfection efficiency analysis results by flow cytometry;

[0026] Figure 5 Scanning electron microscopy 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 images of ureteral tissue one week after nanoparticle / TGF-β1-siRNA ureteral stent implantation;

[0028] Figure 7 The diagram shows the placement of rabbit ureteral stents and the formation of a ureteral injury model; A: placement of different groups of ureteral stents in the rabbit ureter; B: use of vascular clamps to create ureteral injury; C: formation of a rabbit ureteral injury model;

[0029] Figure 8 These are anatomical images of specimens from different treatment groups four weeks after injury formation;

[0030] Fig. 9 HE staining of ureteral tissue sections in different treatment groups four weeks after injury formation;

[0031] Fig.10 Masson staining of ureteral tissue sections in different treatment groups four weeks after injury formation;

[0032] Fig.11 These are immunohistochemical staining images of ureteral tissue sections in different treatment groups four weeks after injury formation. DETAILED DESCRIPTION

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

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

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

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

[0039] In the present invention, nanoparticles act as a medium between siRNA and ureteral stents, encapsulating therapeutic drug TGF-β1-siRNA, so that siRNA can be loaded on the ureteral stent. The ureteral stent is soaked in DA solution (5mM phosphate buffer containing 0.5mg / mL dopamine hydrochloride, pH=8.5) to improve adhesion, thereby adsorbing nanoparticle / TGF-β1-siRNA complex. The NP / TGF-β1-siRNA complex prepared in this way is encapsulated in the ureteral stent and placed in the body, which can prevent the formation of ureteral stenosis around the ureteral stent.

[0040] The present invention is described in detail below with reference to specific embodiments.

[0041] Example

[0042] 1. Materials and Methods

[0043] 1. Construction of siRNA targeting TGF-β1

[0044] Based on the gene sequence of rabbit TGF-β1 precursor in Gene Bank, three siRNAs targeting TGF-β1 and one negative control siRNA were constructed. The sense and antisense sequences of siRNA1 were 5'-GCGUCUAUAUGCUGUUUAA-3' (SEQ ID NO.1) and 5'-UUAAACAGCAUAGACGC-3' (SEQ ID NO.2), respectively. The sense and antisense sequences of siRNA2 were 5'-GGCUCAACAUCUACACAGU-3' (SEQ ID NO.3) and 5'-ACUGU GUAGAUGUUGAGCC-3' (SEQ ID NO.4), respectively. The sense and antisense sequences of siRNA3 were 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, whose sense strand was 5'-UUCUCCGAACGUGUCA CGU-3' (SEQ ID NO.7) and antisense strand was 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO.8). The silencing effect of TGF-β1 was detected and the target siRNA was screened by Western blot experiment.

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

[0046] The rabbit ureteral tissue was quickly cut into pieces, washed with physiological saline, and placed in a mixture of collagenase and trypsin for oscillation digestion. The tissue blocks were regularly ground with a pipette to collect the separated cells, wash the cells and remove the digestive enzymes. The cells were suspended in a medium supplemented with serum and inoculated into a plastic culture dish. One week later, the cultured ureteral epithelial cells were transferred to a 24-well culture plate with a coverslip. When the cells grew to cover about 80% of the culture area, the coverslip was removed and washed with PBS. Fix with methanol at -20°C for 5 minutes, rinse with PBS, add 10% goat serum, block at 37°C for 30 minutes, and rinse with PBS again. Add a broad spectrum keratin (PCK) antibody and incubate at 4°C overnight. Add goat anti-rabbit FITC (secondary antibody, 1:100 dilution), react at 37°C for 1 hour, and rinse with PBS. After sealing with 90% glycerol, the staining results were observed under a fluorescence microscope.

[0047] 3. Preparation of Nanoparticle / TGF-β1-siRNA Complex

[0048] The present invention adopts double emulsification method to construct NP (nanoparticle) / TGF-β1-siRNA complex. First, 400mg polylactic acid-co-glycolic acid (PLGA, Mw=40,000-75,000, St.Louis, USA) is dissolved in 4mL dichloromethane (DCM), then 12mL of 7% (w / v) polyvinyl alcohol (PVA, Mw=14,160) aqueous solution is added, ultrasonic emulsification is performed for 1 minute by ultrasonic crusher, and then the above mixture is added to 200mL 1% (w / v) PVA aqueous solution, and emulsification is continued for 3 minutes to complete the preparation of double emulsion. The emulsion is stirred at room temperature overnight until the remaining dichloromethane is completely evaporated. At 4°C, the nanoparticles are separated by centrifugation at 12000rpm for 5 minutes. Finally, the nanoparticles are washed twice with deionized water and dispersed in a deionized water solution to obtain a nanoparticle suspension. The nanoparticles were modified with polyethyleneimine to give them a positive charge, TGF-β1-siRNA solution (20 μM) was added, and the optimal N / P value of the nanoparticles and siRNA was screened through a gel retardation experiment to obtain the TGF-β1siRNA-loaded nanoparticle (NP / TGF-β1siRNA) complex with the best binding effect.

[0049] 4. Characterization of Nanoparticles and Cell Viability Detection

[0050] The particle size of the NP / TGF-β1 siRNA complex was measured by the dynamic light scattering (DLS) method. In addition, the cytotoxicity of the NP / TGF-β1 siRNA complex was detected by the CCK8 method. The NP / TGF-β1 siRNA complex and the cell suspension (SV-HUC-1, PC101) homogenized after centrifugation were added to a 96-well plate (0.5 × 10 cells / well).4 cells) and cultured at 37°C and 5% CO 2 When the cells were cultured in an incubator for 24 h, 48 h, and 72 h, 10 μL of CCK8 solution (10 mg / mL, Sigma) was added to each well of the culture medium. After the second addition of CCK8 solution, the 96-well plate was cultured in an incubator for another 4 hours. Subsequently, the absorbance value of each well was measured at 450 nm using an ELISA reader. The blank control was a cell group without the addition of the complex. The experiment was repeated three times.

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

[0052] 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 prepare the modified ureteral stent. Subsequently, the stent was washed twice with deionized water and then placed in the pre-prepared NP / TGF-β1-siRNA suspension. Finally, the surface morphology of the composite-coated ureteral stent was characterized using a scanning electron microscope (SEM, Hitachi, S-3400N, Tokyo, Japan).

[0053] 6. In vitro transfection efficiency

[0054] FAM fluorescent group modified siRNA nanoparticle complex (synthesized by Jima Gene) was used for in vitro transfection experiments. In order to detect the in vitro transfection efficiency, the FAM fluorescent group modified siRNA nanoparticle complex was placed in a cell culture medium and incubated with human ureteral epithelial cells. After 6 hours, the culture medium was replaced with DMEM culture medium containing 10% FBS and continued to be cultured for 48 hours. Subsequently, the cells were washed 3 times with 1mL PBS and observed in the FITC channel (excitation wavelength 488nm, emission wavelength 518nm) by fluorescence microscopy (Leica DMR 3000; Leica Microsystem, Bensheim, Germany). Finally, the cells were collected and blown off, suspended in PBS, and then the in vitro transfection efficiency was detected using a FACSCalibur flow cytometer (BD FACSCalibur, BD Bioscience, San Jose, CA).

[0055] 7. In vivo transfection experiments

[0056] Rhodamine B was used for in vivo transfection experiments. Rhodamine B was mixed into PBS for the preparation of Rhodamine B-labeled NP / TGF-β1-siRNA complexes. 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 added to 4.5 mL of 1.5% PVA and ultrasonically emulsified in an ice bath for 1 minute to obtain a primary emulsion. Then, ultrasonically emulsified in an ice bath for 3 minutes again to complete the preparation of a double emulsion. Subsequently, the emulsion was stirred at room temperature overnight until the remaining dichloromethane was completely evaporated. The 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 a phosphate buffered saline (PBS, 5mM, pH=8.5) containing dopamine hydrochloride (0.5mg / mL) and stirred at room temperature for 3 hours to obtain a modified ureteral stent. After the stent was washed twice with deionized water, it was placed in a pre-prepared suspension of rhodamine B-labeled NP / TGF-β1-siRNA. Unmodified ureteral stents and ureteral stents coated with rhodamine B-labeled NP / TGF-β1-siRNA complexes were placed in rabbits for one week. Finally, the distribution of nanoparticles in ureteral tissue was observed.

[0057] 8. Animal model preparation

[0058] Twenty-five healthy male New Zealand rabbits aged 20 weeks were selected as the experimental model, each weighing about 3.0 kg. The 25 rabbits were divided into five groups: (1) sham operation 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 generally anesthetized by injection of Suminxin II (2 mg / kg) + Shutai 50 (15 mg / kg) and then fixed on the operating table. In the supine position, the abdominal cavity of the rabbit was exposed layer by layer, and the adipose tissue was bluntly separated. The ureter was exposed behind the colon and mesentery, and a section of the ureter about 1 cm long was freed and longitudinally cut on it. 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 vascular clamp. The abdominal cavity was then closed and opened again 48 hours later to remove the vascular clamp. At this time, ureteral strictures had formed in the rabbits, and ceftizoxime was injected daily for 3 consecutive days to prevent infection. After 4 weeks, all rabbits were killed, and their ureters and kidneys were collected and preserved for further research.

[0059] 9. Histopathological evaluation

[0060] The bilateral ureters were fixed with 10% formalin solution. The tissues were then dehydrated and embedded in paraffin, sliced ​​at a thickness of 5 μm, and stained with hematoxylin-eosin (HE). The tissue samples were evaluated pathologically.

[0061] 10. Masson staining

[0062] After dewaxing with xylene, the sections were washed with ethanol and hydrated. According to the instructions, the sections of the five groups of samples were stained with Weigert iron hematoxylin and Ponceau S dye solution in turn, and washed with phosphomolybdic acid solution. Subsequently, the sections were stained with aniline blue solution and washed with weak acid solution. Finally, before microscopic observation, the sections were dehydrated, routinely transparentized, and sealed with neutral gum.

[0063] 11. Immunohistochemical staining

[0064] First, the stored tissue samples were dewaxed with xylene and dehydrated with alcohol. To inhibit the activity of endogenous peroxidase, the tissue samples were incubated with hydrogen peroxide, and nonspecific binding sites were blocked with serum. Subsequently, the tissue samples were incubated with the primary antibody in a 4°C refrigerator overnight and washed three times with PBS. With the help of a microscope (Leica DMR 000, Leica Microsystems, Bensheim, Germany), we clearly observed and evaluated the tissue images. Finally, the staining intensity of TGF-β1, type I collagen, and type III collagen was detected.

[0065] 12. Western blotting experiment

[0066] Proteins were extracted from the ureter. Protein samples were separated on a precast 15% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane after electrophoresis. The PVDF membrane was blocked for 2 hours at room temperature in a mixture of 5% skim milk powder and TBST (50mM Tris-HCl, 150mM NaCl and 0.1% Tween-20, pH 7.6). Subsequently, the membrane was incubated 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)) in a 4°C refrigerator shaker overnight. Afterwards, the membrane was washed three times with TBST and incubated with secondary antibodies in a 4°C refrigerator shaker overnight. After washing with TBST for 3 times, the target bands and internal reference were imaged by Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA). The expression levels of the bands were detected using ImageJ software, and the results were normalized to GADPH.

[0067] 13. Statistical analysis

[0068] Descriptive data are presented as mean ± SD. The differences between different groups were analyzed by independent sample Student's t test. The differences between the three groups were analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism9 software. The statistical significance level was set at p < 0.05.

[0069] 2. Results

[0070] 1. siRNA targeting TGF-β1

[0071] Figure 2 The inhibitory effects of three different siRNAs targeting TGF-β1 on TGF-β1 expression. Figure 2 A shows that ureteral epithelial cells were successfully extracted and identified from rabbit ureters by immunofluorescence. In order to detect three siRNAs targeting TGF-β1 and negative control siRNA and screen out the one with the best interference effect, the expression level of TGF-β1 was detected by Western blotting experiment two days after transfection. Figure 2 B and Figure 2 As shown in C, TGF-β1-siRNA1 has the best inhibitory effect, so TGF-β1-siRNA1 was selected as the therapeutic drug. 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 Experiment and Cell Viability Detection

[0073] The particle size of NP / TGF-β1-siRNA complex was detected by dynamic light scattering (DLS), and the average particle size was found to be 187.70±52.20nm. Figure 3 As shown in A. The complexation of siRNA and PLGA nanoparticles was detected by gel retardation experiment, and different NP / TGF-β1-siRNA ratios were considered, such as Figure 3 As shown in B. The complex showed better blocking effect when the N / P (molar ratio of amino groups on nanoparticles to phosphate groups of siRNA) ratio was 6:1 NP / TGF-β1-siRNA, so this ratio was used in subsequent experiments, indicating that the NP / TGF-β1-siRNA complex has gene binding ability. The degradability of polylactic acid-glycolic acid copolymer (PLGA) ensures the sustained release of TGF-β1-siRNA. Figure 3 As shown in Figure C, the release amount on the 5th day was about 23%, and the release amount on the 28th day was about 79%, proving that TGF-β1-siRNA has sustained release characteristics. The cytotoxicity of NP / TGF-β1-siRNA complex to human ureteral epithelial cells was detected by CCK8 experiment. CCK8 solution was added to each well at 24h, 48h and 72h. No significant cytotoxicity difference was observed compared with the blank control group without adding the complex (such as Figure 3 This provides a basis for further experiments.

[0074] 3. In vitro transfection efficiency

[0075] The in vitro transfection efficiency was detected by FAM-modified siRNA. The NP / TGF-β1-siRNA complex-coated ureteral stent significantly increased the expression level of EGFP, while no EGFP expression was observed in the unmodified ureteral stent (e.g. Figure 4 In order to eliminate the interference of irrelevant factors, the experiment was carried out under the same transfection conditions, and the percentage of EGFP-positive cells was measured by flow cytometry analysis 48 hours later 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 Nanoparticle / TGF-β1-siRNA Complex Coated Ureteral Stents

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

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

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

[0080] 6. Rabbit ureteral injury model and gross specimen changes

[0081] Figure 7 The in vivo ureteral injury model induced by vascular clamp was demonstrated. Twenty-five rabbits were divided into five groups and all were sacrificed after four weeks of feeding. Figure 8 The complete specimens of kidneys and ureters and renal coronal section specimens of five groups (normal group, model group, unmodified ureteral stent group, TGF-β1-siRNA ureteral stent group and negative control group) were displayed four weeks after the injury was formed. Compared with the healthy kidney normal group, the ureteral stenosis group showed obvious swelling, indicating severe ureteral obstruction. The edema in the negative control group was slightly improved, and the inflammatory edema in the ureteral stent group and siRNA nanoparticle stent group was significantly reduced.

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

[0083] Fig. 9 The results showed that compared with the sham group (Sham), the model group (US) induced severe ureteral stenosis due to the injury caused by the vascular clamp. In addition, the stenosis in the unmodified ureteral stent group was alleviated. More importantly, the ureteral stenosis of rabbits using the nanoparticle / TGF-β1-siRNA complex-coated ureteral stent was significantly improved.

[0084] Masson staining showed that collagen fibers were clearly stained blue. The ureteral structures of the sham group were clear, and the proportion of blue fibrous tissue was small; while in the US group, collagen fibers proliferated in large quantities, occupying most of the lumen area. In addition, the fibrosis of the unmodified ureteral stent group and the negative control group was reduced, while the fibrosis of the nanoparticle / TGF-β1-siRNA complex-coated ureteral stent group was significantly improved (e.g. Fig.10 shown).

[0085] The present invention also performed immunohistochemical analysis. Since a large number of TGF-β1-positive cells were observed, the number and staining intensity of TGF-β1-positive cells were significantly increased in the US group, US+unmodified ureteral stent group and negative control group, while the number and staining intensity of TGF-β1-positive cells were significantly reduced in the TGF-β1-siRNA ureteral stent group. Fig.11 .

[0086] 8. Western Blot Analysis

[0087] In the model group, unmodified ureteral stent group, nanoparticle / TGF-β1-siRNA complex coated ureteral stent group and negative control group, the expression of TGF-β1 protein, type I collagen and type III collagen were all higher than those in the normal group. The increase in TGF-β1 and protein expression in the TGF-β1-siRNA ureteral stent group was the smallest, that is, after the nanoparticle / TGF-β1-siRNA complex coated ureteral stent, the expression of TGF-β1 protein was significantly reduced, thus affecting the expression of fibrosis-related proteins.

[0088] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A urinary system implant loaded with a nanoparticle coating, characterized in that: The urinary system implant comprises a support carrier and a nanoparticle coating loaded on the support carrier, wherein the nanoparticle coating contains siRNA targeting TGF-β1 gene, and the siRNA is wrapped in nanoparticles.

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

3. The urinary system implant according to claim 1, characterized in that: The sequences of the sense strand and antisense strand of the siRNA are shown in SEQ ID NOs. 1 to 2, respectively.

4. A method for preparing a urinary system implant according to any one of claims 1 to 3, characterized in that: The following steps are involved: The siRNA targeting the TGF-β1 gene is encapsulated in the nanoparticles to prepare a nanoparticle / siRNA complex; placing the urinary system implant in a PBS buffer containing dopamine hydrochloride for modification to obtain a modified urinary system implant; The modified urinary system implant is placed in the suspension of the nanoparticle / siRNA complex to obtain the ureteral stent loaded with the nanoparticle coating.

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

6. The preparation method according to claim 4, characterized in that: In the nanoparticle / siRNA complex, the molar ratio of the amino group on the nanoparticle to the phosphate group in the siRNA, calculated as N and P, is 6:

1.

7. The preparation method according to claim 4, characterized in that: 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.

8. The preparation method according to claim 4, characterized in that: The modification was achieved by stirring, and the modification time was 3 h.

9. Use of the urinary system implant according to any one of claims 1 to 3 in the preparation of a product for inhibiting ureteral stenosis, characterized in that: The ureteral stenosis is ureteral stenosis caused by iatrogenic injury.

10. The use according to claim 9, characterized in that: The ureteral stenosis caused by iatrogenic injury includes ureteral stenosis formed around a ureteral stent.

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