SiRNA for improving sliding capacity after tendon injury, medicine as well as preparation method and application of siRNA

Abstract

The invention relates to the technical field of biological medicine, in particular to siRNA for improving sliding capacity after tendon injury, a medicine and a preparation method and application of the siRNA and the medicine. Double-stranded siRNA is designed according to a gene sequence of TGF-beta1, the effect of the siRNA on adhesion after tissue repair is verified, and it is found that the siRNA can reduce adhesion after tissue healing. SiRNA is loaded on a nanoparticle hydrogel composite system, and after damaged tissues are treated, the effect of obviously improving sliding is achieved. The invention also provides an siRNA-loaded nanoparticle hydrogel composite system (siRNA + hydrogel carrier), which utilizes the slow release characteristic of nanoparticles and the controllable degradation of hydrogel to realize the controllable release of siRNA, increase the local administration efficiency and reduce the adhesion of tissues after healing.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an siRNA for improving the sliding ability of tendon after injury, a drug, and a preparation method and application thereof. Background Art

[0002] Tendons are defined as connective tissue that connects muscle to bone. They are an essential component of the musculoskeletal system, responsible for storing and transferring energy during movement. Tendons are also key elements of the human locomotor system. Tendon injuries, primarily caused by trauma or overuse, are a common occupational hazard and also frequently occur during sports activities. Although clinical management and prognosis prediction for tendon surgery have achieved some success, complications such as tendon adhesions and restricted sliding remain. Methods are needed to reduce adhesions and improve sliding after tendon repair.

[0003] TGF-β1 is a multifunctional cell activity regulator with the highest proportion and strongest activity in the TGF-β family. It can promote fibroblast proliferation and the expression of extracellular matrix proteins. Previous studies have shown that TGF-β1 is expressed throughout the entire process of tendon repair. It can promote tendon repair in the early stages, but its overexpression in the middle and late stages of healing can lead to fibrotic adhesions around the tendon. Specific inhibition of TGF-β1 expression in the early stages of tendon healing using gene-loaded nanoparticles can effectively reduce postoperative tendon adhesions, but tendon healing strength is also affected. Therefore, there is an urgent need to design a carrier that can release gene-loaded nanoparticles on demand, withholding them in the early stages and releasing them in the middle and late stages when adhesions form to inhibit TGF-β1 expression, thereby inhibiting adhesions without affecting tendon healing strength. Summary of the Invention

[0004] The present invention aims to provide an siRNA, a drug, and its preparation method and application for improving tendon gliding ability after tendon injury, thereby addressing the aforementioned problems of the prior art. The siRNA provided by the present invention can reduce adhesion after damaged tissue repair. Simultaneously, the nanoparticle-hydrogel composite system loaded with the siRNA utilizes the sustained-release properties of the nanoparticles and the controlled-release properties of the hydrogel to achieve controlled release of the siRNA, increasing the efficiency of local drug delivery and reducing adhesion after damaged tissue repair.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an siRNA (si-387) for improving the sliding ability of tendon after injury. The siRNA comprises a positive chain with a nucleotide sequence as shown in SEQ ID NO.1 and an antisense chain with a nucleotide sequence as shown in SEQ ID NO.2.

[0007] The present invention provides the use of the above-mentioned siRNA in preparing a medicine for improving tendon injury.

[0008] Preferably, the drug achieves the effect of improving tendon injury by inhibiting tendon adhesion formation and improving sliding function.

[0009] The present invention provides a medicine for improving tendon injury, which comprises the above-mentioned siRNA.

[0010] Further preferably, the drug further comprises a hydrogel carrier.

[0011] Further preferably, the drug achieves the effect of improving tendon injury by inhibiting tendon adhesion formation and improving sliding function.

[0012] Further preferably, the preparation method of the hydrogel carrier comprises the following steps:

[0013] The polylactic acid-co-glycolic acid solution and the polyvinyl alcohol solution are uniformly mixed and emulsified to obtain a primary emulsion;

[0014] The primary emulsion and the polyvinyl alcohol solution are uniformly mixed, and sequentially subjected to emulsification treatment, stirring treatment and centrifugation treatment to obtain PLGA nanoparticles;

[0015] The PLGA nanoparticles and the polyethyleneimine solution are mixed evenly, and then incubated to obtain PEI-modified PLGA nanoparticles;

[0016] Sodium diatomite, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were mixed evenly, and then mixed with tK to obtain tK-SA solution;

[0017] After the tK-SA solution and titanium dioxide are evenly mixed, calcium chloride solution is added to obtain the hydrogel carrier.

[0018] Further preferably, the volume ratio of the polylactic acid-co-glycolic acid solution to the polyvinyl alcohol solution is 1:(1-10);

[0019] and / or, the volume ratio of the PLGA nanoparticles to the polyethyleneimine solution is 20:1;

[0020] And / or, the molar ratio of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide is 2:2:1; based on the molar mass of the sodium diatomite in the mixed solution of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide, the molar ratio of the mixed solution of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide to the tK is 500:1;

[0021] And / or, the volume mass ratio of the tK-SA solution to the titanium dioxide is 1 mL:1 mg; the volume ratio of the mixed solution obtained by uniformly mixing the tK-SA solution and titanium dioxide to the calcium chloride solution is 1:1.

[0022] The present invention provides a siRNA-carrying nanoparticle-hydrogel composite system, which comprises the above-mentioned siRNA and a hydrogel carrier.

[0023] Preferably, the preparation method of the hydrogel carrier comprises the following steps:

[0024] The polylactic acid-co-glycolic acid solution and the polyvinyl alcohol solution are uniformly mixed and emulsified to obtain a primary emulsion;

[0025] The primary emulsion and the polyvinyl alcohol solution are uniformly mixed, and sequentially subjected to emulsification treatment, stirring treatment and centrifugation treatment to obtain PLGA nanoparticles;

[0026] The PLGA nanoparticles and the polyethyleneimine solution are mixed evenly, and then incubated to obtain PEI-modified PLGA nanoparticles;

[0027] Sodium diatomite, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were mixed evenly, and then mixed with tK to obtain tK-SA solution;

[0028] After the tK-SA solution and titanium dioxide are evenly mixed, calcium chloride solution is added to obtain the hydrogel carrier.

[0029] Preferably, the volume ratio of the polylactic acid-co-glycolic acid solution to the polyvinyl alcohol solution is 1:3;

[0030] and / or, the volume ratio of the PLGA nanoparticles to the polyethyleneimine solution is 20:1;

[0031] And / or, the molar ratio of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide is 2:2:1; based on the molar mass of the sodium diatomite in the mixed solution of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide, the molar ratio of the mixed solution of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide to the tK is 500:1;

[0032] And / or, the volume mass ratio of the tK-SA solution to the titanium dioxide is 1 mL:1 mg; the volume ratio of the mixed solution obtained by uniformly mixing the tK-SA solution and titanium dioxide to the calcium chloride solution is 1:1.

[0033] The present invention provides a method for preparing the above-mentioned nanoparticle hydrogel composite system, comprising the following steps:

[0034] The polylactic acid-co-glycolic acid solution and the polyvinyl alcohol solution are uniformly mixed and emulsified to obtain a primary emulsion;

[0035] The primary emulsion and the polyvinyl alcohol solution are uniformly mixed, and sequentially subjected to emulsification treatment, stirring treatment and centrifugation treatment to obtain PLGA nanoparticles;

[0036] The PLGA nanoparticles and the polyethyleneimine solution are mixed evenly, and then incubated to obtain PEI-modified PLGA nanoparticles;

[0037] Sodium diatomite, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were mixed evenly, and then mixed with tK to obtain tK-SA solution;

[0038] After the tK-SA solution and titanium dioxide are evenly mixed, calcium chloride solution is added to obtain the hydrogel carrier;

[0039] The phosphate buffer containing the siRNA is mixed with the hydrogel carrier to obtain the nanoparticle hydrogel composite system.

[0040] Preferably, the volume ratio of the polylactic acid-co-glycolic acid solution to the polyvinyl alcohol solution is 1:(1-10);

[0041] and / or, the volume ratio of the PLGA nanoparticles to the polyethyleneimine solution is 20:1;

[0042] And / or, the molar ratio of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide is 2:2:1; based on the molar mass of the sodium diatomite in the mixed solution of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide, the molar ratio of the mixed solution of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide to the tK is 500:1;

[0043] And / or, the volume mass ratio of the tK-SA solution to the titanium dioxide is 1 mL:1 mg; the volume ratio of the mixed solution obtained by uniformly mixing the tK-SA solution and titanium dioxide to the calcium chloride solution is 1:1.

[0044] The present invention provides application of the nanoparticle hydrogel composite system in preparing a medicine for improving tendon injury.

[0045] Further preferably, the nanoparticle-hydrogel composite system achieves the effect of improving tendon injury by inhibiting tendon adhesion formation and improving sliding function.

[0046] As an additional solution, the present invention also provides the use of the siRNA or the nanoparticle hydrogel composite system in the preparation of a drug for reducing adhesion after tissue healing.

[0047] Further preferably, the tissue includes Achilles tendon tissue.

[0048] As an additional solution, the present invention also provides a method for reducing adhesion after tissue healing, comprising the steps of using the nanoparticle hydrogel composite system to treat damaged tissue and reduce adhesion after tissue healing.

[0049] Further preferably, the tissue includes Achilles tendon tissue.

[0050] Further preferably, the damage includes a fracture.

[0051] As an additional solution, the present invention provides a sustained-release nanoparticle loaded with the above-mentioned siRNA, wherein the carrier is a polylactic acid-glycolic acid (PLGA) copolymer.

[0052] Further preferably, the sustained-release nanoparticles can reduce the degree of adhesion after tendon injury.

[0053] As an additional solution, the present invention provides a sustained-release nanoparticle hydrogel system loaded with the above-mentioned siRNA, comprising nanoparticles loaded with siRNA and a hydrogel that can be cleaved in response to ultrasound.

[0054] Further preferably, the hydrogel has good stability and biocompatibility in vivo and in vitro, and can be cleaved in response to ultrasound to release its internal substances and nanoparticles loaded with siRNA.

[0055] More preferably, the particle size of the nanoparticles is 100-200 nm.

[0056] As an additional solution, the present invention provides a method for preparing the above-mentioned siRNA-loaded sustained-release nanoparticle hydrogel system, comprising the following steps:

[0057] a) Nanoparticles were synthesized using a double emulsion method. First, 100 mg of PLGA was dissolved in 1 mL of DCM. Subsequently, 3 mL of a 7% (w / v) PVA solution was slowly added to the PLGA solution, and the mixture was emulsified using an ultrasonic device to produce a primary emulsion. Afterwards, 50 mL of a 1% (w / v) PVA solution was added to the primary emulsion, and then re-emulsified using an ultrasonic device to form a double emulsion. The resulting emulsion was stirred at room temperature for 24 hours and centrifuged at 13,000 rpm for 5 minutes to separate the PLGA nanoparticles.

[0058] b) A cationic polymer tK, which is cleavable by ROS, was synthesized by polymerizing oligoamines with an acrylamide-thioketal crosslinker. Subsequently, SA, EDC, and NHS were added to distilled water at a molar ratio of 2:2:1 and stirred for more than 10 minutes to activate the carboxyl groups on the SA molecules. tK molecules were then introduced into the solution at a molar ratio of SA to tK of 500:1 and stirred continuously for at least 6 hours to obtain a tK-SA solution.

[0059] c) After synthesizing the tK-SA solution, TiO2 was added at a ratio of 1 mg:1 mL and stirred for more than 10 minutes to obtain a SA-tK / TiO2 solution, and the SA-tK / TiO2 solution was mixed with a 1 mg / mL CaCl2 solution in an equal volume ratio to quickly form a hydrogel;

[0060] d) After the hydrogel is synthesized, phosphate buffered saline (PBS) containing the siRNA-loaded sustained-release nanoparticles is mixed into the hydrogel to obtain the siRNA-loaded sustained-release nanoparticle hydrogel system.

[0061] The present invention discloses the following technical effects:

[0062] The present invention designed a double-stranded siRNA (si-387) based on the TGF-β1 gene sequence and verified its effect on adhesion after tissue repair, finding that the siRNA can reduce adhesion after tissue healing. When the siRNA is loaded onto a nanoparticle hydrogel composite system and applied to damaged tissue, it significantly improves the slippage effect.

[0063] The present invention also provides a nanoparticle hydrogel composite system (siRNA+hydrogel carrier) loaded with siRNA, which utilizes the sustained-release properties of nanoparticles and the controlled degradation of hydrogel to achieve controlled release of siRNA, increase the efficiency of local administration, and reduce adhesion after tissue healing. The nanoparticle hydrogel composite system loaded with siRNA of the present invention has a significant improvement in the sliding ability after treating injured tendons. The nanoparticle hydrogel composite system loaded with siRNA provided by the present invention can be stably present in vivo, and the release of nanoparticles in vivo can be controlled by artificial intervention, thereby achieving controlled release of drugs, increasing local administration, and improving therapeutic effects.

[0064] At the same time, the present invention designs and prepares a new type of ultrasonic responsive hydrogel carrier (tK-SA / TiO2-Ca 2+ , hereinafter referred to as hydrogel), this hydrogel can control the release of its encapsulated drug at a specific time and place in response to medical ultrasound (M-US), thereby improving the accuracy of drug release. In the present invention, the hydrogel is composed of sodium alginate (SA) modified with tK (which can be cleaved by ROS), TiO2, CaCl2, etc. TiO2 produces ROS after ultrasonic treatment. This hydrogel exhibits good stability and biocompatibility in vitro and in vivo, and can be degraded by M-US to release the encapsulated substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0066] Figure 1 The morphology (A) and particle size distribution (B) of three different PLGA nanoparticles;

[0067] Figure 2 The H NMR spectrum (A) and FTIR spectrum (B) of tK-SA; D2O is heavy water;

[0068] Figure 3 is the change of hydrogel modulus before and after ultrasonic treatment for 120s; where A is the hydrogel modulus before ultrasonic treatment, and B is the hydrogel modulus after ultrasonic treatment for 120s;

[0069] Figure 4 tK-SA-Ca 2+ , hydrogel and SEM images of siRNA@NPs-hydrogel; A is tK-SA-Ca 2+A is the SEM image of NPs-hydrogel, B is the SEM image of hydrogel, and C is the SEM image of siRNA@NPs-hydrogel;

[0070] Figure 5 is the stability of the hydrogel; where A is the stability after standing for 0-60 days, B is the absorbance after standing for 0-60 days, and C is the stability after immersion in a water bath at 25, 37, and 45°C for 30 min;

[0071] Figure 6 The cleavage of hydrogel in vitro and in vivo after M-US treatment; A is the cleavage of hydrogel at an acoustic intensity of 2 W / cm 2 , B is the dissolution of the hydrogel after treatment with M-US at a frequency of 4 Hz, C is the image of the hydrogel changes after subcutaneous ultrasound treatment for 0, 30, 60, 90, and 120 seconds, and D is the cleavage image of the hydrogel obtained by diagnostic ultrasound at 0, 30, 60, 90, and 120 seconds after M-US treatment. The hydrogel is represented by a white circle in the figure;

[0072] Figure 7 Figure 4 shows the release of hydrogels in vitro and in vivo after M-US treatment; A shows the release curves of hydrogels after M-US treatment for 0, 30, 60, 90, and 120 seconds in vitro; B shows the release of Rhodamine B nanoparticles around the tendon in hydrogels after M-US treatment for 0, 30, 60, 90, and 120 seconds. Images were analyzed using cross-sectional and longitudinal views.

[0073] Figure 8 is the biocompatibility of the nanoparticle-hydrogel; wherein, A is the H&E staining image of the heart, liver, spleen, lung and kidney of SD rats after the nanoparticle-hydrogel was placed in the body for 2 weeks, B is the H&E staining image of the heart, liver, spleen, lung and kidney of SD rats after the nanoparticle-hydrogel was placed in the body for 3 weeks, C is the hemolysis experiment image of NPs and siRNA@NPs, D is the statistical graph of the hemolysis experiment of NPs and siRNA@NPs, EG is the statistical graph of the tenocyte survival rate measured by CCK-8 after the hydrogel and NPs-hydrogel were placed in a culture dish containing tenocytes for 24, 48 and 72 hours;

[0074] Figure 9 Zeta potential and statistical graph of nanoparticles; A is the Zeta potential of unmodified PLGA nanoparticles, B is the Zeta potential of PEI-modified PLGA nanoparticles, C is the Zeta potential of siRNA@NPs, and D is the Zeta potential statistical graph;

[0075] Figure 10 For agarose gel electrophoresis analysis;

[0076] Figure 11is the transfection efficiency of siRNA-loaded nanoparticles; where A is the tenocytes not transfected with FAM-siRNA, B is the tenocytes transfected with FAM-siRNA treated with M-US, and C is the tenocytes transfected with FAM-siRNA;

[0077] Figure 12 Anatomical images of the tendon at 2 weeks (A) and 3 weeks (B) after surgery.

[0078] Figure 13 Figure 3: Changes in tendon sliding function in SD rats after surgery. Figure A shows the flexion angle of the middle toe at different loads of the repaired tendon after 2 weeks: 0, 5, 10, 15, and 20 g. Figure B shows the flexion angle of the middle toe at different loads of the repaired tendon after 3 weeks: 0, 5, 10, 15, and 20 g. Figure C shows the flexion angle of the middle toe at different loads after 2 weeks: 0-5 g, 0-10 g, 0-15 g, and 0-20 g. Figure D shows the flexion angle of the middle toe at different loads after 3 weeks: 0-5 g, 0-10 g, 0-15 g, and 0-20 g.

[0079] Figure 14 The ultimate strength of the tendon at 2 weeks (A) and 3 weeks (B) after surgery;

[0080] Figure 15 This is the H&E staining of the tissue at week 2 and week 3 after tendon injury.

[0081] Figure 16 This is the Masson staining of the tissue at week 2 and week 3 after tendon injury.

[0082] Figure 17 The expression of TGF-β1 around the tendon during tendon repair was regulated by slowly cleaving the hydrogel by M-US to release si-387@NPs 1 week after tendon injury. The expression of TGF-β1 around the tendon was regulated on the protein expression of the damaged tendon and surrounding tissues during tendon repair. Among them, AC are protein blotting and statistical graphs of TGF-β1 in different tissues, D is the immunohistochemical image of TGF-β1, Col1a1 and Col3a1 protein expression in the tissue at week 2, E, H and J are the immunohistochemical statistics of Col1a1 protein, Col3a1 protein and TGF-β1 protein expression in the tissue at week 2, G is the immunohistochemical image of TGF-β1, Col1a1 and Col3a1 protein expression in the tissue at week 3, F, I and K are the immunohistochemical statistics of Col1a1 protein, Col3a1 protein and TGF-β1 protein expression in the tissue at week 3. DETAILED DESCRIPTION

[0083] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0084] Unless otherwise specified, the materials used in the present invention are all purchased conventionally by those skilled in the art, and the methods used in the present invention are all well known to those skilled in the art.

[0085] Example 1

[0086] 1. Experimental methods

[0087] 1.1 Preparation of PLGA Nanoparticles

[0088] Preparation of unmodified PLGA nanoparticles (UnmodifiedNPs, UNP): Nanoparticles were synthesized using the double emulsion method. First, 100 mg of PLGA (poly(lactic-co-glycolic acid)) was dissolved in 1 mL of DCM (dichloromethane). Subsequently, 3 mL of a 7% (w / v) PVA (polyvinyl alcohol) solution was slowly added to the PLGA solution to obtain an emulsified mixture. The ultrasonic condition parameters were set as follows: frequency: 50 kHz, time: 2 min, wavelength: 60 W. Under these condition parameters, the emulsified mixture was emulsified using an ultrasonic device to produce a primary emulsion. Next, 50 mL of a 1% (w / v) PVA solution was added to the primary emulsion, and then re-emulsified using an ultrasonic device to form a double emulsion (ultrasonic condition parameters were the same as above) to obtain an emulsion. The resulting emulsion was stirred at room temperature for 24 hours and centrifuged at 13,000 rpm for 5 minutes to separate the unmodified PLGA nanoparticles.

[0089] Preparation of PEI-modified PLGA nanoparticles (PEI-modifiedNPs, NPs): Nanoparticles were synthesized using a double emulsion method. First, 100 mg of PLGA (polylactic acid-co-glycolic acid) was dissolved in 1 mL of DCM (dichloromethane). Subsequently, 3 mL of 7% (w / v) PVA (polyvinyl alcohol) solution was slowly added to the PLGA solution to obtain an emulsified mixture. The ultrasonic condition parameters were set as follows: frequency: 50 kHz, time: 2 min, wavelength: 60 W. Under these condition parameters, the emulsified mixture was emulsified using an ultrasonic device to produce a primary emulsion. Next, 50 mL of 1% (w / v) PVA solution was added to the primary emulsion, and then re-emulsified using an ultrasonic device to form a double emulsion (the ultrasonic condition parameters were the same as above) to obtain an emulsion. The resulting emulsion was stirred at room temperature for 24 hours and centrifuged at 13,000 rpm for 5 minutes to separate the unmodified PLGA nanoparticles. Afterwards, 1 mL of an unmodified aqueous solution of nanoparticles (10 mg / mL) was mixed with 50 μL of a 10 mg / mL PEI (polyethyleneimine) solution and incubated at room temperature for 15 minutes to obtain PEI-modified PLGA nanoparticles. To better load the negatively charged siRNA onto the PLGA nanoparticles, the present invention added a positively charged PEI solution to the PLGA nanoparticle solution to form PEI-modified PLGA nanoparticles.

[0090] 1.2 Synthesis of Thioketal-Alginate (tK-SA)

[0091] The ROS-cleavable cationic polymer tK was synthesized and provided by Xi'an Ruixi Biotechnology Co., Ltd. This is the new ROS-responsive thioketal linker (TK) described on the company's website. Subsequently, SA (sodium alginate), EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), and NHS (N-hydroxysuccinimide) were added to distilled water at a molar ratio of 2:2:1 and stirred for at least 10 minutes to activate the carboxyl groups on the SA molecules, yielding a SA solution. The cationic polymer tK was then introduced into the SA solution at a molar ratio of 500:1, with continuous stirring for at least 6 hours to yield the tK-SA solution.

[0092] 1.3 Synthesis of hydrogel

[0093] After synthesizing the tK-SA solution, TiO2 (titanium dioxide) was added at a ratio of 1 mL:1 mg and stirred for more than 10 minutes to obtain the SA-tK / TiO2 solution. Finally, the SA-tK / TiO2 solution was mixed with a CaCl2 (calcium chloride) solution (1 mg / mL) at an equal volume ratio to quickly form a hydrogel.

[0094] 1.4 Synthesis of siRNA-loaded nanoparticle-hydrogel (siRNA@NPs-hydrogel, abbreviated as siRNA@NPs)

[0095] After the hydrogel is synthesized, phosphate buffer solution (PBS) containing nanoparticles (the density of nanoparticles in the phosphate buffer solution is 20nM / uL) is mixed into the hydrogel to obtain nanoparticle-loaded siRNA@NPs-hydrogel. That is, the phosphate buffer solution containing nanoparticles and the hydrogel are mixed in a volume ratio of 1:100 to obtain the nanoparticle-loaded siRNA@NPs-hydrogel.

[0096] 1.5. Detection of Nanoparticles and Hydrogel Morphology

[0097] First, a small amount of nanoparticles (unmodified PLGA nanoparticles and PEI-modified PLGA nanoparticles), hydrogel (hydrogel obtained in step "1.3"), and si-387@NPs-hydrogel solution were adhered to the sample holder using conductive glue. The sample was then placed in a freeze dryer and freeze-dried overnight at -40°C. After the sample was sprayed with gold, it could be placed in a scanning electron microscope for observation.

[0098] 1.6. Determination of Nanoparticle Size

[0099] The nanoparticles were diluted to a concentration of 0.1 mg / mL in deionized water. The average hydrodynamic diameter of the nanoparticles was then analyzed by dynamic light scattering (DLS) to determine the size range of the nanoparticles.

[0100] 1.7 Determination of H NMR Spectrum

[0101] The nuclear magnetic resonance spectra were measured using a Bruker nuclear magnetic resonance instrument at 25°C with deuterated water as the solvent, and the unit is ppm.

[0102] 1.8. Fourier transform infrared spectroscopy

[0103] SA, tK, and tK-SA samples were freeze-dried (using the same method as step 1.5) and then mixed with KBr (potassium bromide) and pressed into pellets, or placed directly on an ATR (attenuated total reflectance) accessory. Fourier transform infrared spectra of SA, tK, and tK-SA were measured on an Infinity AR60 spectrometer. The spectra were taken between 450 and 4000 cm -1 64 scans were recorded with a resolution of 2 cm -1 .

[0104] 1.9. Determination of rheological properties of hydrogels

[0105] The rheological properties of the hydrogels were measured before and after ultrasound treatment using a rheometer to analyze their rheological behavior in response to ultrasound. The evolution of gelation was plotted logarithmically, with the intersection of G' and G" indicating the transition from a liquid state (G' < G") to a solid state (G' > G"). The changes in the rheological properties of the hydrogels were measured before and after 2 minutes of ultrasound treatment.

[0106] 1.10. Testing of hydrogel stability

[0107] Crystal violet dye was mixed into the hydrogel. The hydrogel was placed in an Eppendorf tube at 24°C in the dark. The tube was inverted on a table and distilled water was added to the bottom of the inverted tube. The color of the hydrogel and the liquid at the bottom of the tube were observed after 30 and 60 days, respectively, and the absorbance of the liquid was measured at 595 nm using a microplate reader. Furthermore, the prepared hydrogel was placed in an Eppendorf tube and the tube was placed in a constant temperature water bath for 30 minutes at 25°C, 37°C, and 45°C. After the bath, the hydrogel morphology was observed to determine any changes.

[0108] 1.11. Detection of Ultrasonic Responsiveness of Hydrogels

[0109] In vitro, the prepared hydrogels were placed in the ep tube and under the skin respectively, and the sound intensity was 2W / cm 2 The morphological changes of the hydrogel were observed after treatment at a frequency of 4 Hz for 30s, 60s, 90s and 120s. The supernatant was centrifuged at 1000 rpm for 5 min and the ROS content in the supernatant was measured using a ROS kit. In vivo, the hydrogel was implanted subcutaneously in SD rats and treated with M-US at an acoustic intensity of 2 W / cm 2 The morphological changes of the hydrogel in vivo were observed by diagnostic ultrasound after treatment at a frequency of 4 Hz for 30s, 60s, 90s and 120s.

[0110] 1.12. Detection of hydrogel release efficiency in vitro and in vivo

[0111] In vitro, after mixing si-387@NPs into the hydrogel obtained in step “1.3”, the prepared si-387@NPs-hydrogel was placed in an ep tube and the M-US was used at an acoustic intensity of 2 W / cm 2, processed at a frequency of 4 Hz for 15s, 30s, 45s, 60s, 75s, 90s, 105s and 120s, and centrifuged at 1000rpm for 5min. The RNA content in the supernatant in the ep tube was detected using a spectrophotometer, and compared with the RNA content added to the hydrogel to detect the release efficiency of the hydrogel in vitro. In vivo, rhodamine-B nanoparticles were mixed into the hydrogel to prepare an SD rat tendon injury model, and the prepared rhodamine-B nanoparticle-hydrogel mixed system was wrapped in the SD rat tendon. One week after the operation, some SD rats were killed to obtain the tendons and surrounding tissues. Thereafter, M-US was used at the injured tendon of the rat every two days at an acoustic intensity of 2W / cm 2 The rats were treated with ultrasound at a frequency of 4 Hz for 30 seconds until sacrifice. The remaining SD rats were sacrificed three days after ultrasound. Tendons and surrounding tissues were harvested from the SD rats. These tissues were sectioned, fluorescently stained, and observed under a microscope.

[0112] 1.13. Testing of the biocompatibility of si-387@NPs-hydrogel in vivo and in vitro

[0113] The biocompatibility testing of si-387@NPs-hydrogel in vitro and in vivo is divided into nanoparticle hemolysis test and si-387@NPs-hydrogel in vitro and in vivo biocompatibility test. The details are as follows:

[0114] Nanoparticle Hemolysis Experiment: Blood was drawn from the cardiac apex of SD rats and placed in a test tube. The blood was anticoagulated with sodium citrate and diluted at a ratio of 10 mL of 0.9% sodium chloride solution per 8 mL of blood to obtain diluted rat blood. Three groups were established: a negative control group (10 mL of 0.9% sodium chloride solution) (Saline group), a positive control group (10 mL of distilled water, H2O), and an experimental group (5 g of test sample + 10 mL of sodium chloride solution). The experimental group was further divided into two subgroups: the NPs group (NPs-hydrogel, si-387@NPs-hydrogel) and the siRNA@NPs group. The test tubes were incubated in a thermostatted shaker at 37 ± 1°C for 30 minutes without shaking. Diluted rat blood was then added to the test tube at a ratio of 0.2:10 and mixed thoroughly. The mixture was incubated at a constant temperature for 60 minutes. Subsequently, the contents of the centrifuge tube were centrifuged at 800 × g for 5 minutes, and the supernatant was aspirated. Absorbance was measured at 545 nm using a spectrophotometer. Absorbance values were designated as follows: A for the experimental group, B for the negative control group, and C for the positive control group. Hemolysis rate was calculated using the formula: (AB) / (CB) × 100%. This experiment was repeated three times.

[0115] In vitro biocompatibility of the material: The in vitro biocompatibility of the material was mainly detected by Cell Counting Kit-8 (CCK-8). In short, the experimental groups were divided into three different categories: control group (Control), hydrogel group (hydrogel obtained in step "1.3") and si-387@NPs-hydrogel (NPs-hydrogel). Tendon cells were seeded in a six-well plate, and hydrogels or si-387@NPs-hydrogels with a diameter of approximately 1 cm were added to the corresponding groups. After 24, 48 and 72 hours of treatment, the CCK8 solution was diluted to 100 μM in DMEM (containing 10% fetal bovine serum) culture medium. Subsequently, 2 mL of the diluted CCK8 solution was added to each well and incubated at 37°C for 2 hours. The effect of the material on cell viability was judged by measuring the absorbance of the CCK8 solution at 450 nm using a microplate reader.

[0116] In vivo biocompatibility of materials: The in vivo compatibility of materials is mainly determined by testing the changes in blood indicators and major organs of SD rats after implantation of the materials, as follows:

[0117] (1) Routine blood examination and blood biochemical test: A tendon injury model was established in SD rats. The experimental groups consisted of a control group (Control), an empty nanoparticle hydrogel group (NC, hydrogel obtained in step “1.3”), a low-dose si-387@NPs-hydrogel group (Low, dosage of 6 μL / tendon), a medium-dose si-387@NPs-hydrogel group (Mid, dosage of 2 μL / tendon), and a high-dose si-387@NPs-hydrogel group (High, dosage of 24 μL / tendon). Six SD rats were used in each experimental group. Routine and biochemical blood analysis was performed on the blood samples of the rats at 2 and 3 weeks after surgery. The following parameters were analyzed: aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (Crea), red blood cell (RBC) count, white blood cell (WBC) count, and platelet (PLT) count.

[0118] (2) Changes in the main organs of SD rats: The main organs such as the heart, liver, spleen, lung, and kidney of the above rats were fixed in 4% formaldehyde solution for 24 hours, and then dehydrated to prepare for paraffin embedding. After embedding, the tissue was cut into 5 μm thick sections. The paraffin sections were deparaffinized with xylene twice, each for 10 minutes, anhydrous ethanol twice, each for 5 minutes, 90% ethanol and 75% ethanol once, each for 5 minutes, and then washed with tap water. Dewaxed sections of the tissue were obtained. Next, the dewaxed sections were stained with hematoxylin and eosin (H&E), that is, the dewaxed sections were stained in hematoxylin staining solution for 3 minutes and then rinsed with tap water. The differentiation process should be carried out using hematoxylin differentiation solution for 5 seconds. The sections were then rinsed with tap water. Anti-blue staining was performed using hematoxylin anti-blue solution (manufacturer: Seville, product number: G1005) for 5 seconds, and then rinsed with tap water. The sections were then dehydrated in a gradient of ethanol, starting with 85% ethanol for 5 minutes, followed by 95% ethanol for an additional 5 minutes. They were then stained with an ethanol-soluble eosin solution for 5 minutes, followed by a second round of dehydration in absolute ethanol for 5 minutes. The sections were then treated with xylene twice, each for 5 minutes, and sealed with neutral glue. Finally, the sections were placed under a microscope for observation.

[0119] 1.14. Detection of Zeta Potential of Nanoparticles

[0120] The Zeta potential of nanoparticles is determined using electrophoresis. The nanoparticle solution to be tested is injected into an electrophoresis cell with voltage applied at both ends. The migration velocity of the colloidal particles is then measured using laser Doppler velocimetry. The Zeta potential of the solution to be tested is then calculated based on the relationship between the Zeta potential and the migration velocity.

[0121] 1.15 Gel retardation test

[0122] 0.4 mL of unmodified PLGA nanoparticle solution (10 mg / mL) was mixed with PEI (100 mg) in deionized water, and the resulting solution was then added to a siRNA (SEQ ID NO. 1 and SEQ ID NO. 2 mixed in equal proportions) solution at different ratios of "molar number of amine groups of PEI / molar number of phosphate groups of DNA" (N / P) and gently vortexed. The siRNA nanoparticle complexes with different N / P ratios were incubated at room temperature for 20 minutes. The complexes were then electrophoresed in a 1% tris-ethyl acetate EDTA (TAE) agarose gel at 90 V for 30 minutes. Images were acquired using a gel imaging system. GelRed was used to examine the interaction between siRNA and nanoparticles to determine the optimal N / P ratio.

[0123] 1.16. Construction of TGF-β1 siRNA-loaded nanoparticle-hydrogel system

[0124] 1.16.1. Construction of siRNA

[0125] Double-stranded siRNA was designed based on the gene sequence of SD rat TGF-β1 and synthesized by Heyuan Biotechnology Co., Ltd. The specific sequence is shown in Table 1.

[0126] Table 1 Three double-stranded siRNA gene sequences of SD rat TGF-β1

[0127]

[0128] 1.16.2. Construction of siRNA-loaded Nanoparticles (siRNA@NPs)

[0129] In order to load negatively charged siRNA onto nanoparticles, the present invention adds a positively charged polyethyleneimine (PEI) aqueous solution to an unmodified PLGA nanoparticle solution to form PEI-modified PLGA nanoparticles, and then constructs siRNA-loaded nanoparticles (siRNA@NPs). The specific steps are as follows:

[0130] 1 mL of unmodified PLGA nanoparticle aqueous solution (10 mg / mL) was mixed with 50 μL of PEI solution (10 mg / mL) and incubated at room temperature for 15 min. The resulting mixture was then added to 500 μL of siRNA-387 solution (500 ng / μL, the mass ratio of the sense chain to the antisense chain was 1:1) and incubated at room temperature for 15 min to form siRNA-loaded@NPs.

[0131] 1.17. Culture of Primary SD Rat Tenocytes

[0132] Tendon cells were extracted using a digestion method. A collagenase I solution at a concentration of 5 mg / mL was prepared using phosphate-buffered saline (PBS). After euthanizing the rat, the tendon was removed and placed in a PBS solution containing penicillin and streptomycin. The trimmed tendon tissue was immersed in the collagenase I solution and digested at 37°C for 2 hours, gently shaking every 10-20 minutes to ensure sufficient contact between the tendon tissue and the collagenase I solution. After 2 hours, the solution was filtered using a cell sieve. The sample was then centrifuged at 1000×g for 5 minutes. The supernatant was removed and the cells were inoculated into a culture dish containing DMEM (containing 10% FBS).

[0133] 1.18. Transfection of Tenocytes and Detection of Transfection Efficiency.

[0134] The FAM-siRNA nanoparticles were transfected into SD rat tendon cells as follows: tenocytes were seeded in a 24-well plate, and when the cell confluence was above 80%, a solution of FAM-siRNA nanoparticles (FAM-si-387@NPs, which differ from si-387@NPs only in that the sense and antisense strands of the siRNA in FAM-si-387@NPs were modified with FAM) was added to the wells. The solution was replaced after 8 hours, and green fluorescence was observed 24 hours after transfection and the transfection efficiency was determined by flow cytometry. In addition, in order to confirm whether the FAM-siRNA nanoparticles can retain the ability to transfect SD rat tendon cells after M-US ultrasound, the present invention subjected the FAM-siRNA nanoparticle solution to M-US ultrasound treatment for 120 seconds and then transfected it into the tendon cells. The group without transfection of FAM-siRNA nanoparticle solution was set as the Control group, the group with FAM-siRNA nanoparticle solution after M-US ultrasound was set as the NC group, and the group with FAM-siRNA nanoparticle solution without M-US ultrasound was set as the siRNA group.

[0135] 1.19. Western Blot Experiment

[0136] 1.19.1. Protein extraction

[0137] 1) Extraction of cell proteins

[0138] Culture cells in a 6-well plate. Add 500 μL of protein lysis buffer and 5 μL of PMSF (phenylmethylsulfonyl fluoride) solution to the plate. Place on ice and shake on a shaker for 30 minutes. Gently pipette the lysis buffer to ensure it comes into contact with the cells. After 30 minutes, scrape the cells and transfer them to a 1.5 mL EP tube. Centrifuge at 12,000 rpm at 4°C for 5 minutes and collect the supernatant.

[0139] 2) Tissue protein extraction

[0140] Place the tissue sample in a grinder and add 1 mL of protein lysate (containing 10 μL PMSF (phenylmethylsulfonyl fluoride)) to the grinder for grinding at the rate of 1 mL of protein lysate (containing 10 μL PMSF (phenylmethylsulfonyl fluoride)) per 100 mg of tissue. After grinding, centrifuge at 4°C, 12,000 rpm for 15 minutes, and then aspirate the supernatant and transfer it to a 1.5 mL EP tube.

[0141] 3) Protein denaturation

[0142] Add Loading Buffer to the cell or tissue lysate and boil in boiling water for 5 minutes to denature the protein.

[0143] 1.19.2, Glue preparation:

[0144] 1) Clean the glass plate before use, make sure the bottom is flush, clamp the glass plate, clamp it on the rack, add double distilled water and observe for leaks.

[0145] 2) Prepare the separation gel first, and slowly add the separation gel from one side with a pipette until it reaches the bottom line of the upper frame.

[0146] 3) Seal with isopropyl alcohol. Wait for about 30 minutes for the gel to solidify. Pour out the isopropyl alcohol, rinse with ddH2O, and then dry with a paper towel.

[0147] 4) Add concentrated glue and insert the comb.

[0148] 5) After gelling, remove the gel and the plate and store them in a 4°C refrigerator.

[0149] Table 2 Reagents and dosages required for separation gel and stacking gel

[0150]

[0151]

[0152] Note: The amounts here are rounded off.

[0153] 1.19.3. SDS-PAGE electrophoresis:

[0154] (1) Install the gel plate tightly in the electrophoresis tank and remove the comb.

[0155] (2) Start from the left marker and add samples in sequence.

[0156] (3) Add an appropriate amount of electrophoresis buffer to ensure the formation of a current loop.

[0157] (4) Select a voltage of 90 V for the stacking gel and 120 V for the separation gel.

[0158] 1.19.4. Transfer (wet transfer of protein gel to PVDF membrane):

[0159] (1) Prepare an ice box and three large glass dishes, add methanol, ddH2O, and transfer solution respectively.

[0160] (2) Place the following in order from positive to negative: cotton pad, filter paper, adhesive strip, membrane, filter paper, cotton pad. The membrane is first placed in methanol solution for depolarization, then placed in ddH2O, and finally placed in the transfer solution.

[0161] (3) Place the entire membrane transfer apparatus in ice water and adjust the membrane transfer current to 200 mA and time to 120 min.

[0162] 1.19.5. Closure:

[0163] The membrane was removed from the transfer tank, placed face up in a box containing 5% skim milk powder, and blocked on a shaker at room temperature for 2 h. The membrane was then washed three times with TBST, each time for 3 min.

[0164] 1.19.6 Antibody incubation:

[0165] (1) Dilute the primary antibody in TBST: incubate at 4°C overnight, then wash the membrane three times with TBST.

[0166] (2) Dilute the secondary antibody in TBST: incubate at 4°C overnight, then wash the membrane three times with TBST.

[0167] 1.19.7. Development: The bands were detected and their grayscale values were analyzed using the Odyssey Infrared Imaging System (Li-COR Biosciences).

[0168] 1.20 Construction and Repair Evaluation of the Palmar Tendon Injury Model in SD Rats

[0169] 1.20.1. Construction of the palmar tendon injury model in SD rats

[0170] After anesthesia and disinfection, the palmar tendons of SD rats were sharply cut, and the tendon rupture site was repaired with a modified two-strand Kessler suture method plus one circle of peripheral suture using 5-0 nylon suture with a needle. The surface skin was sutured with 3-0 nylon thread, and no fixation was performed after surgery.

[0171] 1.20.2 Experimental Grouping

[0172] All animal experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals developed by Nantong University Hospital and approved by the Animal Ethics Committee of Nantong University.

[0173] The present invention used 6-week-old male SD rats from the Experimental Animal Center of Nantong University, Nantong, China. A total of 50 SD rats were randomly divided into 5 groups: surgical repair group (control group, Control), NPs-hydrogel group (NC, hydrogel obtained in step "1.3"), low-dose si-387@NPs-hydrogel group (6 μL / tendon) (Low), medium-dose si-387@NPs-hydrogel (12 μL / tendon) (Mid) and high-dose si-387@NPs-hydrogel (24 μL / tendon) (High).

[0174] Before surgical intervention, rats were anesthetized by inhalation of aerosolized isoflurane and injected intraperitoneally with 3% sodium pentobarbital at a dose of 1.6 mL / kg. Surgery was initiated 3–5 minutes after surgery to allow the anesthetic to take effect. To minimize the risk of intraoperative bleeding, a solution of 50% lidocaine hydrochloride and 1% epinephrine (0.1 mL / palm) was injected into the palm to establish a palmar tendon injury model in SD rats.

[0175] The Control group did not receive any additional treatment. The NC group was treated with the hydrogel obtained in step "1.3" at the repair site, while the Low, Mid, and High groups were treated with low, medium, and high doses of si-387@NPs-hydrogel, respectively. Starting from one week after surgery, the palms of the hands were ultrasonically treated every day, 15s / palm×day, until the rats were killed. Starting from the second week (2W), 5 rats were randomly selected from each group for euthanasia, and 10 tendon specimens were collected from each group. At the end of the third week (3W), the remaining rats in each group were euthanized, and an additional 10 tendon specimens were collected from each group. At the second and third weeks, specimens from each group were randomly selected for histological analysis (four specimens per group) and biomechanical testing (six specimens per group).

[0176] 1.20.3 Mechanical Testing

[0177] Two and three weeks after surgery, the surface skin was first incised with a scalpel. Centering on the tendon bundle, the tendon was carefully separated from the surrounding skin and muscle tissue. The tibia and fibula were removed, leaving the distal end of the tendon attached to the ankle bone. The proximal muscle tissue was removed, offset 1.5 cm from the incision area. The healing strength was then tested using an Instron biomechanical tester. The two sides of the injured tendon were secured in upper and lower fixtures. The upper fixture was pulled upward at a rate of 2.5 cm / min until the tendon ruptured. The maximum load the tendon could withstand was recorded as a force-displacement curve, representing the healing strength.

[0178] 1.20.4. Sliding degree detection

[0179] The rat's hind legs were disarticulated at the knee joint, exposing the proximal tendon of the flexor digitorum longus (FDL). The palm of the hand was secured, and weights were placed on the other end of the tendon, increasing in 5g increments from 5 to 20g. The toe flexion angle was assessed using a goniometer and statistically analyzed. The flexion angles of each group were photographed.

[0180] 1.21. Tissue H&E and Masson staining

[0181] 1.21.1. Preparation of tissue sections and dewaxing of sections

[0182] Fresh tissue was excised and fixed in 4% formaldehyde for 24 hours, then dehydrated and embedded in paraffin. After embedding, the tissue was cut into 5 μm thick sections. Paraffin sections were deparaffinized using xylene twice for 10 minutes each, anhydrous ethanol twice for 5 minutes each, and 90% ethanol and 75% ethanol once for 5 minutes each. The sections were then washed with tap water to obtain dewaxed tissue sections.

[0183] 1.21.2 Hematoxylin and eosin (H&E) staining

[0184] The dewaxed sections were incubated in hematoxylin staining solution for 3 minutes and then rinsed with tap water. Differentiation was performed using hematoxylin differentiation solution for 5 seconds. The solution was then rinsed off with tap water. Anti-blue staining was performed using hematoxylin anti-blue solution for 5 seconds and then rinsed with tap water. The sections were then dehydrated in an ethanol gradient, starting with 85% ethanol for 5 minutes, and then the ethanol gradient was increased to 95% ethanol for another 5 minutes. The sections were then stained with ethanol-soluble eosin solution for 5 minutes and dehydrated for a second time in anhydrous ethanol for 5 minutes. The sections were treated with xylene twice for 5 minutes each and then sealed with neutral glue.

[0185] 1.21.3 Masson staining

[0186] Dewaxed sections were stained using the Masson kit. Dewaxed sections were stained with Weigert's iron hematoxylin solution for 10 minutes, then differentiated with acidic ethanol solution for 10 seconds and washed with water. The sections were then deblued with Masson's bluing solution, washed with water, and then rinsed with distilled water for 1 minute. Next, sections were stained with Ponceau fuchsin solution for 10 minutes and rinsed with weak acid working solution for 1 minute. The sections were stained with aniline blue solution for 2 minutes and rinsed with weak acid working solution for 1 minute. After washing, the sections were dehydrated in 95% ethanol and anhydrous ethanol three times for 10 seconds each. Finally, the sections were cleared in xylene three times for 2 minutes each and mounted with neutral gum.

[0187] 1.22 Tissue Immunohistochemistry (IHC) Staining

[0188] Soak the dewaxed sections in 3% hydrogen peroxide for 10 minutes. Place the antigen retrieval solution in a water bath and heat to boiling point. Incubate the sections in this solution for 2-3 minutes, then remove and cool naturally. The present invention washes the sections three times with PBS buffer, then adds blocking solution and incubates at 4°C for 12 hours. After the blocking step, add the primary antibody solution (anti-TGF-β1 antibody, 1:1000; anti-Col1a1 antibody, 1:1000; anti-Col3a1 antibody, 1:1000) and incubate at 4°C for 16-18 hours. Wash the sections three times with PBS buffer. Subsequently, incubate the cells with IRDye800-labeled anti-rabbit / mouse IgG secondary antibody solution (1:10000) at 4°C for 16-18 hours. Then wash the sections three times with PBS. Add DAB staining solution and incubate for 2 minutes, then wash three times with PBS. Then incubate the sections in hematoxylin and eosin staining solution for 1 minute and rinse with tap water for 1 hour. Finally, the sections were dehydrated and sealed with neutral glue.

[0189] 2. Experimental results

[0190] 2.1 Morphology and size distribution of three different PLGA nanoparticles

[0191] First, the morphology of three different PLGA nanoparticles was observed by SEM. Figure 1 Figure A shows the microscopic morphology of three different PLGA nanoparticles. It can be observed that after freeze-drying, the three types of nanoparticles, namely unmodified PLGA nanoparticles (UNP), PEI-modified PLGA nanoparticles, and siRNA@NPs, all showed uniform spherical shapes with basically the same shape. According to the ruler, the diameter of the nanoparticles was greater than 100 nm. The particle size of the three different PLGA nanoparticles was measured by DLS analysis, as shown in Figure 1. Figure 1 As shown in Figure 2B, the average particle sizes of three different PLGA nanoparticles were 128.6 ± 36.9 nm, 138.7 ± 34.4 nm, and 145.6 ± 39.8 nm.

[0192] 2.2. H NMR spectrum and FTIR spectrum of tK-SA

[0193] SA, tK and tK-SA were characterized by H NMR spectroscopy. Figure 2 The nuclear magnetic resonance 1H spectrum of tK-modified alginate shown in Figure A shows the characteristic peaks of tK: methyl (CH3) peak at 0.926ppm, methylene (S-CH2) peak at 1.74ppm, and methylene (NH2-CH2) peak, indicating that tK was successfully modified on alginate. SA, tK and tK-SA were characterized by Fourier transform infrared spectroscopy. Figure 2 The B in the figure is located at 3480 and 3450 cm -1 The peaks at 1620 cm-1 are the hydrogen bonds of the hydroxyl groups in SA and the amino groups in tK. However, due to the conjugation between tK and SA, these two peaks disappear in tK-SA. -1 The peak at can be attributed to the stretching vibration of the carbonyl group.

[0194] 2.3. Rheological properties of the hydrogel obtained in step “1.3” before and after ultrasound

[0195] In order to confirm whether the hydrogel will be completely cleaved in response to ultrasonic treatment, the present invention measured the modulus of the hydrogel before and after ultrasonic treatment. Figure 3 As shown, the hydrogel was subjected to an acoustic intensity of 2 W / cm 2 The modulus of the hydrogel was measured after 120s of M-US treatment at a frequency of 4 Hz and compared with the modulus of the hydrogel before ultrasound. The results showed that the hydrogel changed from solid to liquid before and after ultrasound.

[0196] 2.4 tK-SA-Ca 2+ Morphology of hydrogel and siRNA@NPs-hydrogel

[0197] tK-SA-Ca was observed by SEM. 2+ , hydrogel (gel obtained in step “1.3”) and siRNA@NPs-hydrogel morphology, Figure 4 A in the equation is the addition of Ca into the tK-SA solution. 2+ Prepared tK-SA-Ca 2+ The morphology (after synthesizing the tK-SA solution in step "1.3", the obtained tK-SA solution and CaCl2 solution (1 mg / mL) were mixed in an equal volume ratio) was obtained. 2+ It is a three-dimensional porous structure. Figure 4 B in the formula is the addition of Ca 2+ The morphology of the hydrogel prepared by / TiO2 is a three-dimensional porous structure. Figure 4 C in the figure is the morphology of siRNA@NPs-hydrogel. Its structure is similar to that of hydrogel under low-power microscope, but a large number of nanoparticles can be observed on the surface after magnification.

[0198] 2.5 Stability of siRNA@NPs-hydrogel

[0199] To determine whether the siRNA@NPs-hydrogel would be degraded due to other factors (such as heat generated during ultrasound, time, etc.), the present invention conducted a series of experiments to evaluate its stability. The experiments showed that the siRNA@NPs-hydrogel did not degrade or release its contents after being stored at room temperature and protected from light for 30 and 60 days ( Figure 5 To investigate whether the degradation of the siRNA@NPs-hydrogel is affected by changes in body temperature or heat generated during ultrasound, the present invention examined its thermal stability. In vitro experimental results showed that after immersion in a water bath at 25, 37, and 45°C for 30 minutes, the siRNA@NPs-hydrogel remained intact ( Figure 5 C).

[0200] 2.6 Ultrasound responsiveness of siRNA@NPs-hydrogel in vivo and in vitro

[0201] In order to determine whether siRNA@NPs-hydrogel will be degraded after responding to M-US and its ability to release the contents after cleavage, the present invention tested the ultrasound response ability and in vitro and in vivo release efficiency of siRNA@NPs-hydrogel. Figure 6 As shown in Figure A, siRNA@NPs-hydrogel was exposed to an acoustic intensity of 2 W / cm 2, and gradually degraded after treatment with M-US at a frequency of 4 Hz. Finally, the hydrogel was completely degraded after 120s. The present invention detected the ROS content in the degraded siRNA@NPs-hydrogel. The results showed that after ultrasound, the ROS content in the siRNA@NPs-hydrogel gradually increased ( Figure 6 Subsequently, the present invention removed a piece of SD rat skin and placed the siRNA@NPs-hydrogel on the skin for transcutaneous ultrasound. The results showed that the siRNA@NPs-hydrogel could still respond to M-US and be degraded within 120 seconds ( Figure 6 Finally, the present invention implanted siRNA@NPs-hydrogel subcutaneously in SD rats, performed transcutaneous ultrasound at the implanted siRNA@NPs-hydrogel site, and used a small animal imaging ultrasound instrument to detect the degradation of the hydrogel in vivo. The results showed that siRNA@NPs-hydrogel can also respond to M-US and degrade in vivo ( Figure 6 D) in.

[0202] 2.7. Release of siRNA@NPs-hydrogel in vitro and in vivo

[0203] To confirm whether siRNA@NPs-hydrogel releases its loaded substances after cleavage and the release conditions of the loaded substances, the present invention tested the ability of siRNA@NPs-hydrogel to release its loaded substances in vitro and in vivo. The results showed that in vitro, after 120s of M-US treatment of siRNA@NPs-hydrogel, the release efficiency of siRNA@NPs-hydrogel was as high as about 93% ( Figure 7 To more fully understand the in vivo release of siRNA@NPs-hydrogels after M-US treatment, we examined cross-sectional and longitudinal sections of tendons under a fluorescence microscope. The results showed that siRNA@NPs-hydrogels loaded with rhodamine B nanoparticles continuously degraded in vivo after M-US treatment, releasing the rhodamine B nanoparticles into the surrounding area ( Figure 7 B) in.

[0204] 2.8 Biocompatibility of Nanoparticle-Hydrogels in Vivo and In Vitro

[0205] In order to verify the biocompatibility, the present invention conducted a series of verifications. In vitro, the present invention tested whether the nanoparticles used in the experiment would cause hemolysis and the biocompatibility of siRNA@NPs-hydrogel by nanoparticle hemolysis test and CCK-8 test respectively. Among them, the results of the nanoparticle hemolysis test showed that the nanoparticles used in the experiment did not cause hemolysis ( Figure 8C and D in the figure), so the nanoparticles used in the present invention are safe. Similarly, in order to further evaluate the effect of nanoparticle-hydrogel on the survival rate of tendon cells, the present invention placed the hydrogel and siRNA@NPs-hydrogel in a six-well plate with tendon cells and continued to culture them. The survival of tendon cells was detected by CCK-8 at 24, 48 and 72 hours. The CCK-8 test showed that the survival rate of tendon cells was not significantly affected 24, 48 and 72 hours after hydrogel implantation ( Figure 8 EG in). However, biological systems are complex, and relying solely on in vitro experimental data may result in erroneous results. Therefore, the empty nanoparticle hydrogel group and the hydrogel loaded with siRNA@NPs (si-387@NPs-hydrogel) (Low, Mid, High groups) were implanted into SD rats to explore their effects on SD rats. At 2 weeks and 3 weeks after implantation, the rats were euthanized, and blood and visceral samples were collected for analysis. Among them, the blood sample analysis involved a variety of biomarkers, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (TP), albumin (ALB), albumin-globulin ratio (A / G), blood urea nitrogen (BUN) and creatinine (Crea). In addition, the present invention also measured the number of red blood cells (RBC), white blood cells (WBC) and platelets (PLT) in the blood (Tables 3 and 4). In view of the differences between species, it is unreasonable to use human blood biochemistry and standard blood indicators to evaluate the unique blood parameters of rats. Therefore, the present invention refers to the relevant blood indicators unique to SD rats for evaluation. The results showed that, like the SD rats without the addition of bioactive materials (NH group), the blood indicators of the SD rats in both the hydrogel and the siRNA@NPs-loaded hydrogel were within the standard range, indicating that the nanoparticle-hydrogel had good biocompatibility both in vivo and in vitro.

[0206] Table 3 Blood biochemistry and blood routine indicators in the second week

[0207]

[0208] Table 4 Blood biochemistry and blood routine indicators at week 3

[0209]

[0210] In addition, the present invention examined the structure and inflammatory changes of major organs by sectioning tissue and using hematoxylin and eosin (H&E) staining. At 2 and 3 weeks, H&E staining of myocardial tissue in all groups revealed the presence of numerous capillaries between myocardial cells, surrounded by single endothelial cells. Endothelial cell nuclei appeared as blue spindle-shaped cells, while intercalated discs between adjacent myocardial fibers exhibited a darker staining pattern. H&E staining of the liver revealed dark staining of hepatocyte nuclei, light staining of hepatocyte cytoplasm, and uniform staining of sinusoidal endothelial cells and Kupffer cells. The spleen showed a distinct boundary between the red and white pulp, with the latter exhibiting slightly increased staining intensity. This region contained numerous red blood cells, lymphocytes, macrophages, plasma cells, and other cellular components. Furthermore, the architecture of the splenic cords and sinusoids appeared normal. H&E staining of the lungs revealed that the alveoli maintained a round or oval shape. The alveolar walls were regular and tightly packed, while the pulmonary vessels and bronchioles exhibited tubular structures. H&E staining of the kidneys revealed clear renal tissue architecture. The glomeruli are located within the renal capsule and are round or oval in shape. They are composed of clusters of capillaries enclosed in the glomerular capsule. The renal tubules are well-preserved, with uniform diameters and neatly arranged cells within the tubular walls. In summary, no pathological changes were observed in the H&E-stained sections of the above organs, including the absence of inflammatory cell infiltration, vasodilation, congestion, and tissue edema ( Figure 8 These results indicate that the siRNA@NPs-loaded hydrogel has good biocompatibility and does not cause organ damage.

[0211] 2.9 Zeta Potential of Nanoparticles

[0212] The present invention measured the unmodified PLGA nanoparticles (UNPs) ( Figure 9 A), PEI-modified PLGA nanoparticles (NPs) ( Figure 9 B) and nanoparticles bound to siRNA (siRNA@NPs) ( Figure 9 The Zeta potentials of the three nanoparticles were -21.13±7.55mV, 41.67±11.56mV and 33.27±10.92mV, respectively. Figure 9 D). The experimental results show that PEI-modified nanoparticles can bind to the negatively charged cell membrane and then enter the cell through the mutual attraction between positive and negative charges.

[0213] 2.10 Optimal Combination Ratio of Nanoparticles and siRNA

[0214] In order to evaluate the optimal binding ratio of nanoparticles to siRNA, the present invention uses gel electrophoresis retardation experiments to detect changes in gene bands after nanoparticles bind to siRNA at different N / P ratios. Figure 10As shown, when the N / P ratio is 1:1, 2:1, and 4:1, the gene band is not completely blocked. When the N / P ratio is 6:1, the gene band is significantly blocked, indicating that the N / P ratio of 6:1 is the optimal ratio for the combination of nanoparticles and siRNA.

[0215] 2.11 Transfection efficiency of siRNA-loaded nanoparticles

[0216] After 24 h of transfection, the present invention observed and recorded the untransfected ( Figure 11 A) and after transfection ( Figure 11 B) Fluorescence and corresponding bright field images of tendon cells to evaluate transfection efficiency. Subsequently, the present invention detected the transfection efficiency of siRNA-loaded nanoparticles by flow cytometry. The results showed that the nanoparticles can successfully transfect siRNA into tendon cells with a transfection efficiency of approximately 99.2%. At the same time, M-US ultrasonic treatment did not affect the ability of the nanoparticles to transfect siRNA into tendon cells (the transfection efficiency was approximately 98.1%) ( Figure 11 C).

[0217] 2.12. Inhibiting TGF-β1 expression in the middle and late stages of tendon repair reduces adhesions around the tendon after repair

[0218] 2 weeks after surgery ( Figure 12 A) and 3 weeks ( Figure 12 B) Anatomical images of the tendon show enhanced fiber alignment and a wider gap between the tendon and surrounding tissue in all three treatment groups, with the most pronounced effect in the Mid group.

[0219] 2.13. Inhibiting TGF-β1 expression in the middle and late stages of tendon repair improves tendon sliding function after repair

[0220] In order to evaluate the efficacy of the treatment, the present invention tested the sliding ability of the tendon after surgery. At the 2nd and 3rd week after treatment, the flexion angle of the middle finger of each group of rats was measured under loads of 0, 5, 10, 15 and 20 g. Two weeks after surgery, the change in the flexion angle of the middle finger of the Mid group was significantly greater than that of the control group in all weight changes (0-5, 0-10, 0-15 and 0-20 g). Figure 13 A in Figure 3), with the 0-20g group showing the most significant change (P<0.001). In addition, both the low-dose (P=0.166) and high-dose (P=0.403) groups showed improvement in the flexion angle of the middle toe. Three weeks after surgery, the changes in the flexion angle of the middle toe in all three groups exceeded those in the control group ( Figure 13 B), of which the 0-20g group had the most significant effect. Among the 3 groups, the medium dose group showed the most significant improvement in sliding (P = 0.0003), followed by the High group (P = 0.001) and the Low group (P = 0.0079) ( Figure 13 C and D in the above example).

[0221] 2.14. Inhibiting TGF-β1 expression during the middle and late stages of tendon repair does not reduce tendon strength after repair

[0222] The present invention conducted statistics on the maximum tensile force of the healed tendon, and the results were as follows: after 2 weeks of treatment, the maximum tensile force of the Control group was 20.6±4.5N; the maximum tensile force of the NC group was 17.9±3.2N; the maximum tensile force of the Low group was 20.7±4.2N; the maximum tensile force of the Mid group was 19.9±3.3N; and the maximum tensile force of the High group was 19.7±3.62N. Figure 14 Similarly, after 3 weeks of treatment, tendon strength was as follows: Control group: 31.2±2.93N; NC group: 31.5±3.7N; Low group: 30.0±2.6N; Mid group: 31.3±3.5N; High group: 31.4±3.4N ( Figure 14 B), no significant differences were observed among the groups.

[0223] 2.15. Inhibiting TGF-β1 expression in the middle and late stages of tendon repair promotes the reduction of inflammatory cell infiltration around the tendon

[0224] After dewaxing each group of tendon slices, the present invention performed H&E staining on them. Figure 15 As shown, inflammatory cell infiltration and adhesion were reduced in the three treatment groups compared with the Control group at 2 and 3 weeks after surgery, among which the Mid group showed the most significant effect.

[0225] 2.16. Inhibiting TGF-β1 expression in the middle and late stages of tendon repair reduces the expression of peritendon fibroblasts

[0226] After dewaxing each group of tendon slices, the present invention performed Masson staining on them. Figure 16 As shown, 2 and 3 weeks after surgery, the fibroblasts in the three treatment groups decreased and the collagen expression was reduced compared with the Control group, among which the Mid group showed the most obvious performance.

[0227] 2.17. Inhibiting TGF-β1 expression in the middle and late stages of tendon repair reduces the expression and distribution of TGF-β1 and related proteins in tendons

[0228] The present invention detected the expression of TGF-β1 in the tendons of each group by western blot. The results showed that there was no significant difference in protein expression between the Control group and the NC group. Compared with the Control group, the TGF-β1 protein level in the Mid group was significantly reduced at 2 weeks and 3 weeks ( Figure 17 AC in).

[0229] Next, the present invention dewaxed each group of tendon slices and performed immunohistochemical staining ( Figure 17 The protein expression in the tendons of each group was evaluated. There was no significant difference in protein expression between the Control and NC groups. Compared with the Control group, the levels of Col1a1, Col3a1, and TGF-β1 proteins around the tendons of the three treatment groups (Low, Mid, and High) were significantly reduced at 2 and 3 weeks after surgery, with the Mid group showing the greatest reduction ( Figure 17 EK in ).

[0230] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An siRNA for improving tendon sliding ability after tendon injury, characterized in that The siRNA includes a sense strand having a nucleotide sequence as shown in SEQ ID NO.1 and an antisense strand having a nucleotide sequence as shown in SEQ ID NO.

2.

2. Use of the siRNA according to claim 1 in the preparation of a medicament for improving tendon injury.

3. The use according to claim 2, characterized in that The drug achieves the effect of improving tendon injury by inhibiting tendon adhesion formation and improving sliding function.

4. A drug for improving tendon injury, characterized in that: The drug comprises the siRNA according to claim 1.

5. A nanoparticle hydrogel composite system loaded with siRNA, characterized in that: The nanoparticle-hydrogel composite system comprises the siRNA according to claim 1 and a hydrogel carrier.

6. The nanoparticle hydrogel composite system according to claim 5, characterized in that: The preparation method of the hydrogel carrier comprises the following steps: The polylactic acid-co-glycolic acid solution and the polyvinyl alcohol solution are uniformly mixed and emulsified to obtain a primary emulsion; The primary emulsion and the polyvinyl alcohol solution are uniformly mixed, and sequentially subjected to emulsification treatment, stirring treatment and centrifugation treatment to obtain PLGA nanoparticles; The PLGA nanoparticles and the polyethyleneimine solution are mixed evenly, and then incubated to obtain PEI-modified PLGA nanoparticles; Sodium diatomite, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were mixed evenly, and then mixed with tK to obtain tK-SA solution; After the tK-SA solution and titanium dioxide are evenly mixed, calcium chloride solution is added to obtain the hydrogel carrier.

7. The nanoparticle hydrogel composite system according to claim 6, characterized in that: The volume ratio of the polylactic acid-co-glycolic acid solution to the polyvinyl alcohol solution is 1:(1-10); and / or, the volume ratio of the PLGA nanoparticles to the polyethyleneimine solution is 20:1; And / or, the molar ratio of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide is 2:2:1; based on the molar mass of the sodium diatomite in the mixed solution of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide, the molar ratio of the mixed solution of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide to the tK is 500:1; And / or, the volume mass ratio of the tK-SA solution to the titanium dioxide is 1 mL:1 mg; the volume ratio of the mixed solution obtained by uniformly mixing the tK-SA solution and titanium dioxide to the calcium chloride solution is 1:

1.

8. The method for preparing the nanoparticle hydrogel composite system according to claim 6, characterized in that: The following steps are involved: The polylactic acid-co-glycolic acid solution and the polyvinyl alcohol solution are uniformly mixed and emulsified to obtain a primary emulsion; The primary emulsion and the polyvinyl alcohol solution are uniformly mixed, and sequentially subjected to emulsification treatment, stirring treatment and centrifugation treatment to obtain PLGA nanoparticles; The PLGA nanoparticles and the polyethyleneimine solution are mixed evenly, and then incubated to obtain PEI-modified PLGA nanoparticles; Sodium diatomite, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were mixed evenly, and then mixed with tK to obtain tK-SA solution; After the tK-SA solution and titanium dioxide are evenly mixed, calcium chloride solution is added to obtain the hydrogel carrier; The phosphate buffer containing the siRNA according to claim 1 is mixed with the hydrogel carrier to obtain the nanoparticle hydrogel composite system.

9. The preparation method according to claim 8, characterized in that The volume ratio of the polylactic acid-co-glycolic acid solution to the polyvinyl alcohol solution is 1:(1-10); and / or, the volume ratio of the PLGA nanoparticles to the polyethyleneimine solution is 20:1; And / or, the molar ratio of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide is 2:2:1; based on the molar mass of the sodium diatomite in the mixed solution of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide, the molar ratio of the mixed solution of the sodium diatomite, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the N-hydroxysuccinimide to the tK is 500:1; And / or, the volume mass ratio of the tK-SA solution to the titanium dioxide is 1 mL:1 mg; the volume ratio of the mixed solution obtained by uniformly mixing the tK-SA solution and titanium dioxide to the calcium chloride solution is 1:

1.

10. Use of the nanoparticle hydrogel composite system according to any one of claims 5 to 7 in the preparation of a drug for improving tendon injury.

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