Three-dimensional intra-tumor turbulent flow device with repairing function and preparation method of three-dimensional intra-tumor turbulent flow device

Abstract

The invention belongs to the technical field of intra-tumor turbulent flow scaffolds, and particularly relates to a three-dimensional intra-tumor turbulent flow device with a repairing function and a preparation method thereof, the preparation method comprises the following steps: S1, preparing a metal turbulent flow scaffold and an extracellular matrix raw material; s2, carrying out surface modification on the metal turbulent flow bracket through oxygen plasma treatment or dilute acid etching; s3, preparing an extracellular matrix solution; s4, coating the surface of the pretreated metal stent with an extracellular matrix solution in a spraying, dipping or spin-coating manner; and S5, performing low-temperature drying or freeze drying on the coated stent, performing cross-linking treatment by using a cross-linking agent, and repeatedly eluting by using a phosphate buffer solution after cross-linking to obtain the three-dimensional intra-tumor turbulent flow device with the repairing function. According to the metal stent, the core turbulence function is guaranteed to assist tumor treatment, the ECM coating makes up the defect that metal has no biological activity, and the dual purposes of improving the tumor treatment effect and reducing the treatment side effect are truly achieved.

Description

Technical Field

[0001] This invention belongs to the field of intratumoral turbulence stent technology, and particularly relates to a three-dimensional intratumoral turbulence device with repair function and its preparation method. Background Technology

[0002] Intratumoral disruptive devices are important adjunctive instruments for the local treatment of solid tumors (such as liver cancer and lung cancer). The core of these devices utilizes a metal scaffold made of nickel-titanium alloy or medical-grade stainless steel to disrupt the imbalance between blood flow and drug distribution within the tumor, promoting drug penetration and improving the energy uniformity of local treatments (such as ablation and radiotherapy). Clinical needs have evolved from "simple disruptive effects" to "integrated disruptive and repair mechanisms"—requiring a metal scaffold to ensure mechanical stability to maintain the disruptive function while possessing biorepair capabilities to reduce the risk of tissue damage, inflammation, and thrombosis after implantation. However, existing metal substrates lack bioactivity, making the "integrated fabrication of metal scaffolds and biorepair coatings" a core research direction, requiring consideration of coating adhesion, bioactivity, toxicity control, and process repeatability.

[0003] Currently, related technologies fall into two categories. The first is pure metal flow disruptor devices, which only optimize the scaffold structure (such as porosity and shape memory) to achieve flow disruption. They lack bio-coatings, and the surface treatment is limited to basic cleaning without activation or modification, thus lacking repair capabilities. The second is devices with non-ECM coatings, which use ordinary polymers or non-specific protein coatings. These only improve compatibility but cannot promote tissue regeneration. The coating process lacks parameter optimization (such as uncontrolled spraying pressure and number of dipping layers) and effective surface activation. Crosslinking (high concentration of glutaraldehyde) and sterilization (high dose of gamma rays) can easily lead to toxic residues or coating inactivation. In addition, ECM coating technology in the field of tissue engineering is not adapted to the mechanical and implantation requirements of metal flow disruptor scaffolds and cannot be transferred for application.

[0004] Existing technologies have several shortcomings: First, they lack biorepair capabilities; pure metal devices are inactive, and non-ECM coatings cannot promote tissue regeneration through the active sites of ECM, easily leading to delayed healing and inflammatory complications. Second, the coatings do not adhere firmly; there is no effective surface activation (such as oxygen plasma treatment) or a low-residue, high-binding-strength cross-linking system (such as EDC / NHS), making the coatings prone to detachment and posing safety hazards. Third, there are high levels of toxic residues; high concentrations of glutaraldehyde are not fully eluted and there is no residue detection, harming normal cells. Fourth, the coating process has poor adaptability; a single method cannot meet the requirements of different coating thicknesses, limiting clinical applicability. Fifth, ECM activity is easily lost; uncontrolled solution parameters (pH, temperature) and improper sterilization destroy activity. Sixth, turbulence and repair cannot be simultaneously addressed; the process is not integrated and optimized, easily resulting in one aspect being neglected while the other is addressed. Summary of the Invention

[0005] The purpose of this invention is to provide a three-dimensional intratumoral turbulence disturbance device with repair function and its preparation method, so as to solve the above-mentioned problems.

[0006] To achieve the above objectives, the present invention provides the following solution: A method for preparing a three-dimensional intratumoral turbulence disturbance device with repair function includes: S1. Prepare the metal turbulence scaffold and extracellular matrix materials; S2. Surface modification of the metal flow-disrupting support is performed by oxygen plasma treatment or dilute acid etching. S3. Prepare the extracellular matrix solution; S4. Apply the extracellular matrix solution to the pretreated metal scaffold surface by spraying, dipping, or spin coating. S5. The coated scaffold is dried at low temperature or freeze-dried, and then cross-linked with a cross-linking agent. After cross-linking, it is repeatedly eluted with phosphate buffer to obtain a three-dimensional intratumoral disturbance device with repair function.

[0007] Preferably, ultrasonic cleaning is performed before using the metal baffle in step S2 to achieve sterility and contamination-free operation.

[0008] Preferably, the parameters for the oxygen plasma treatment in step S2 are: power 30-100W, treatment time 30-180s, and oxygen flow rate 10-20sccm.

[0009] Preferably, the dilute acid etching in step S2 uses 5-10% dilute sulfuric acid or 3-5% dilute hydrochloric acid, with an etching time of 30-60 seconds, and is neutralized to pH 7.0-7.4 with 0.1M NaOH solution after etching.

[0010] Preferably, in step S3, sterile deionized water, phosphate buffer, or 0.05-0.1M dilute acetic acid is selected as the solvent according to the type of extracellular matrix raw material to dissolve the extracellular matrix and adjust the concentration to 0.5-20 mg / mL, adjust the pH of the solution to 7.2-7.6, and control the operating temperature to 4-25℃.

[0011] Preferably, the spraying parameters in step S4 are: pressure 10-30psi, spraying distance 10-20cm, multiple short-distance uniform sprayings, and curing at 4°C for 5-10min after each spraying. The parameters for the impregnation are: impregnation time 0.5-5 min, withdrawal speed 1-3 mm / s, and repeated impregnation 2-3 times; The parameters for spin coating are: rotation speed 1000-4000 rpm, rotation time 30-120 s.

[0012] Preferably, the crosslinking agent in step S5 is EDC / NHS, Genipin, or glutaraldehyde. When glutaraldehyde is selected, the residual aldehyde groups are blocked with a 1% w / v glycine solution after crosslinking.

[0013] Preferably, in step S5, the phosphate buffer solution is eluted at least 3 times, with each elution lasting 10-30 minutes.

[0014] A three-dimensional intratumoral turbulence disturbance device with repair function is prepared by the preparation method of the three-dimensional intratumoral turbulence disturbance device with repair function.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: The preparation method provided in this invention revolves around the integrated design of a metal intratumoral disruptive scaffold and an ECM repair coating. First, the metal scaffold undergoes ultrasonic cleaning and plasma or acid etching pretreatment, followed by an EDC / NHS low-residue crosslinking process. This ensures the ECM coating firmly adheres to the scaffold surface, effectively preventing coating detachment caused by blood flow within the body. Crucially, the method uses highly active ECM raw materials and precisely controls the pH, temperature, freeze-drying, and sterilization conditions of the ECM solution to maximize the preservation of ECM's bioactivity, thereby achieving the repair function for peritumoral tissue damage. Simultaneously, the reagents are of analytical purity and sterilely filtered, and residues are rigorously tested after crosslinking, fully complying with the safety standards for medical implantable devices. Furthermore, the three coating processes—spraying, dipping, and spin coating—are flexibly adaptable to different coating thicknesses and scaffold structural requirements, avoiding limitations imposed by a single process. Ultimately, the metal scaffold ensures the core disruptive function to aid tumor treatment, while the ECM coating compensates for the lack of bioactivity in metals, truly achieving the dual goals of improving tumor treatment efficacy and reducing treatment side effects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 A schematic diagram of the metal aerodynamic support structure; Figure 2 A schematic diagram of the structure during the installation of the metal spoiler bracket. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] This invention provides a robustly attached ECM liquid film / thin-layer coated metal turbulence device with bioremediation function, specifically comprising: S1. Prepare raw materials and equipment Material: The metal flow-disrupting stent is fabricated using medical-grade nickel-titanium alloy (phase transformation temperature 32-37℃, conforming to GB / T24627-2021 standard) or medical-grade 316L stainless steel (conforming to GB / T13810-2017 standard). The specific structure is as follows: Figure 1-2 As shown, after being woven / stamped, it is first rinsed with clean water; ECM raw materials are medical-grade decellularized small intestinal mucosa ECM powder (decellularization rate ≥99%, endotoxin content ≤0.5EU / mg) and recombinant collagen (Type I, purity ≥95%). The solvents / buffers were sterile deionized water (conductivity ≤10μS / cm), PBS (pH 7.2-7.4, containing 0.01% (w / v) sodium azide for preservation), MES buffer (pH 5.0-6.0), and 0.05-0.1M dilute acetic acid (analytical grade, filtered through a 0.22μm sterile filter membrane). The crosslinking agent selected is medical-grade EDC (purity ≥98%) / NHS (purity ≥98%), with Genipin (medical grade, purity ≥95%) and 0.01%-0.05% (v / v) very low concentration glutaraldehyde (medical grade, an additional 1% (w / v) glycine solution is required to block residual aldehyde groups) as alternatives. Equipment selection: Low-pressure spraying device (nozzle diameter 0.2-0.5mm), immersion device (with uniform speed lifting function), spin coater (speed accuracy ±10rpm), freeze dryer (controllable temperature range -80℃ to 50℃, vacuum degree ≤1Pa), Class 10,000 clean bench (compliant with ISO14644-1 standard), medical-grade ultrasonic cleaner (power 50-300W, controllable temperature 25-40℃), ethylene oxide sterilizer (compliant with GB18279.1-2015 standard).

[0020] S2, Metal Surface Pretreatment The molded metal bracket was placed in an ultrasonic cleaning tank and then ultrasonically cleaned with medical-grade neutral detergent (0.5% (w / v) sodium dodecyl sulfate solution, sterilized), followed by ultrasonic cleaning with sterile deionized water and ultrasonic degreasing with 75% (v / v) medical ethanol for 5 minutes. Afterward, it was transferred to a clean workbench and dried with sterile air.

[0021] For the roughening and activation components, one of the two options can be selected: Plasma treatment (physical activation): Place the dried support into the oxygen plasma treatment chamber and set the parameters: power 30-100W, oxygen flow rate 10-20sccm, treatment time 30-180s. After the treatment is completed, immediately fill the chamber with sterile nitrogen for protection and remove the support. Acid etching (chemical roughening): Immerse the stent in 5%-10% (v / v) dilute sulfuric acid or 3%-5% (v / v) dilute hydrochloric acid (both analytical grade, aseptically treated) and etch at room temperature for 30-60 seconds. Immediately afterward, transfer it to 0.1M sterile NaOH solution for 5 minutes to neutralize. Then rinse with sterile deionized water until the eluent pH is 7.0-7.4 (tested with precision pH test paper (accuracy ±0.1), and take at least 3 eluent samples from the stent surface). Finally, repeat the drying operation in step 1.

[0022] After treatment by both methods, the product must be applied to the subsequent coating process within 10 minutes to prevent the surface active groups from becoming inactive.

[0023] Preparation of S3 and ECM solutions Solvent selection and dissolution: If it is natural collagen, slowly add the ECM raw material to 0.05-0.1M dilute acetic acid and stir magnetically at 4°C (100-200 rpm) for 2-4 hours until completely dissolved, avoiding the generation of bubbles during the process; if it is recombinant collagen, dissolve it directly in PBS (pH 7.2-7.4) at 4°C with stirring for 1-2 hours.

[0024] Concentration and purification: Adjust the concentration according to the coating requirements: 0.5-5.0 mg / mL (0.05%-0.5% w / v) for thin, uniform coatings, and 5-20 mg / mL for thick, sponge-like coatings; After preparation, the solution must be vacuum filtered through a 0.22 μm sterile filter membrane (hydrophilic, uniform pore size) to remove impurities and undissolved particles (to prevent clogging of the coating nozzle or causing coating bulges).

[0025] pH and temperature control: Before coating, the acid-soluble collagen solution is slowly neutralized with 0.1M sterile NaOH solution (neutralization rate controlled at 1 drop / 10s, with stirring while adding), and the pH is monitored in real time with a precision pH meter until it reaches 7.2-7.6; the ECM solution dissolved in PBS is kept at pH 7.2-7.4; the operating temperature is controlled at 4-25℃ throughout the process.

[0026] If the solution viscosity is insufficient (<50 cP, measured with a rotational viscometer), add 0.1%-0.5% (w / v) medical-grade low molecular weight PVA (molecular weight 10000-20000 Da) or PEG (molecular weight 4000 Da), and stir at 4℃ for 30 min until homogeneous. After addition, it needs to be verified by a cytotoxicity test (L929 cell survival rate ≥90%, in compliance with GB / T16886.5-2017 standard).

[0027] S4, ECM coating process This process can be implemented in three ways: (1) Spraying method (preparing thin film coating, suitable for support grid structure) The pretreated metal scaffold is fixed onto a medical-grade silicone clamp (non-cytotoxic and non-adhesive to ECM), placed in a Class 100 clean bench, and the parameters of the low-pressure spraying device are adjusted as follows: pressure 10-30 psi (≈0.7-2.0 bar), spray distance 10-20 cm, and nozzle moving speed 5-10 mm / s (moving at a uniform speed along the scaffold axis). The "multiple short spray" method is adopted, with each spray amount controlled at 0.1-0.2 mL / cm² of the support surface area. After spraying, the support is transferred to a 4℃ refrigerator for curing for 5-10 min. A total of 3-5 layers are sprayed, with the dry film thickness of each layer controlled at 5-10 μm (measured with a film thickness gauge), and the total dry film thickness is 15-50 μm (it is necessary to ensure that the turbulent pores of the support are not blocked and the porosity is maintained at ≥70%). After each layer is sprayed, the uniformity of the coating is observed with an optical microscope. If local accumulation occurs, the spray distance needs to be adjusted and re-sprayed.

[0028] (2) Impregnation method (preparing a thicker coating or full coverage, suitable for the entire stent) Pour the ECM solution into a sterile impregnation tank, ensuring the liquid level completely covers the stent. Slowly immerse the stent in the solution for 30 seconds to 5 minutes (30 seconds to 2 minutes for 0.5-5.0 mg / mL solution, and 2-5 minutes for 5-20 mg / mL solution). Activate the uniform lifting function of the impregnation device and remove the support at a speed of 1-3 mm / s (to avoid uneven coating thickness caused by gravity flow); after removal, hang the support vertically in the clean workbench and allow it to air dry naturally for 10-20 minutes (laminar air velocity 0.3-0.5 m / s); repeat the impregnation coating 2-3 times, and control the total dry film thickness to 50-100 μm (using a film thickness gauge). After each drying, check the integrity of the coating with an optical microscope. Only if there is no cracking or peeling can the next impregnation coating be carried out.

[0029] (3) Spin coating method (for preparing standardized thin films, suitable for support components with a diameter ≤10mm and a surface flatness ≤0.1mm) Secure the support components with medical-grade silicone clamps (ensure they are firmly fixed and do not shift during rotation), and adjust the spin coater parameters as follows: rotation speed 1000-4000 rpm (1000-2000 rpm corresponds to a dry film thickness of 8-15 μm, 3000-4000 rpm corresponds to a dry film thickness of 3-8 μm), rotation time 30-120 s (the higher the speed, the shorter the rotation time can be). Add ECM solution evenly to the surface of the support (0.1-0.2 mL per cm² surface area, avoid excessive addition which may cause accumulation at the edges), and start the spin coater immediately; after spin coating is completed, place the part in a 4℃ drying oven to dry for 30-60 min (relative humidity ≤30%).

[0030] S5, Drying and Crosslinking Curing (1) Drying process Preserving ECM bioactivity and porous structure (preferred): Place the coated scaffold in a freeze dryer and set the freeze-drying curve: ① Pre-freeze at -40℃ for 2-4 hours (ensure the ECM solution is completely frozen and free of free water); ② Reduce the vacuum to 1-5 Pa and raise the temperature to -20℃ and maintain for 8-12 hours; ③ Raise the temperature to 25℃ and maintain for 4-6 hours; After drying, use a halogen moisture analyzer to ensure that the coating moisture content is ≤5%; For non-porous structures: Place the support under the sterile laminar flow of a Class 100 clean bench (wind speed 0.3-0.5m / s), air dry at room temperature for 4-6 hours, or slowly dry in a 4℃ drying oven for 8-12 hours, with the moisture content controlled at 5%-10% (avoid over-drying to prevent coating cracking).

[0031] (2) Crosslinking curing Preferred EDC / NHS crosslinking (low residue, high biocompatibility): Immerse the dried scaffold in MES buffer (pH 5.0-6.0, aseptic) containing 10-50 mMMEDC and 5-20 mMMNHS, and react in a sterile, sealed container at 20-25°C in the dark for 2-24 hours (0.5-5.0 mg / mL coating reaction for 2-8 hours, 5-20 mg / mL coating reaction for 12-24 hours, gently shaking the container once every 4 hours during the reaction). After the reaction is complete, the stent is transferred to sterile PBS (pH 7.2-7.4) for elution. The volume of the elution buffer should be more than 10 times the volume of the stent each time (to ensure thorough elution). Elute 3 times, 10-30 min each time, and finally rinse once with sterile deionized water. Alternatively, Genipin crosslinking can be used (mild and suitable for ECMs sensitive to EDC): Immerse in 0.01%-0.5% (w / v) Genipin solution (prepared with PBS, aseptically treated), react at 20-25°C in the dark for 24-48 hours (the reaction time is extended to 48-72 hours when the temperature is below 20°C), and then elute as in the EDC / NHS protocol. Alternatively, crosslinking with glutaraldehyde (use with caution, only when other crosslinking agents are not suitable): Immerse in 0.01%-0.05% (v / v) glutaraldehyde solution (aseptic treatment), react at room temperature in the dark for 1-2 hours; after elution, block with 1% (w / v) sterile glycine solution for 30 minutes (to neutralize residual aldehyde groups), then elute twice with PBS for 15 minutes each time; S6. Sterilization and Packaging The dried and cross-linked cured device is aseptically packaged to obtain the desired product.

[0032] This invention focuses on the integrated design of a metal intratumoral flow-disrupting scaffold and an ECM repair coating. First, the metal scaffold undergoes ultrasonic cleaning and plasma or acid etching pretreatment, followed by an EDC / NHS low-residue crosslinking process. This ensures the ECM coating adheres firmly to the scaffold surface, effectively preventing coating detachment caused by blood flow. Crucially, the method uses highly active ECM raw materials and precisely controls the pH, temperature, freeze-drying, and sterilization conditions of the ECM solution to maximize the preservation of ECM's bioactivity, thereby achieving repair of peritumoral tissue damage. Simultaneously, the reagents are of analytical grade and sterilely filtered, and residues are rigorously tested after crosslinking, fully complying with the safety standards for medical implantable devices. Furthermore, the three coating processes—spraying, dipping, and spin coating—are flexibly adaptable to different coating thicknesses and scaffold structural requirements, avoiding limitations imposed by a single process. Ultimately, the metal scaffold ensures the core flow-disrupting function to aid tumor treatment, while the ECM coating compensates for the lack of bioactivity in metals, truly achieving the dual goals of improving tumor treatment efficacy and reducing side effects.

[0033] In the description of this invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this invention, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0034] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a three-dimensional intratumoral turbulence disturbance device with repair function, characterized in that, include: S1. Prepare the metal turbulence scaffold and extracellular matrix materials; S2. Surface modification of the metal flow-disrupting support is performed by oxygen plasma treatment or dilute acid etching. S3. Prepare the extracellular matrix solution; S4. Apply the extracellular matrix solution to the pretreated metal scaffold surface by spraying, dipping, or spin coating. S5. The coated scaffold is dried at low temperature or freeze-dried, and then cross-linked with a cross-linking agent. After cross-linking, it is repeatedly eluted with phosphate buffer to obtain a three-dimensional intratumoral disturbance device with repair function.

2. The method for preparing a three-dimensional intratumoral turbulence disturbance device with repair function according to claim 1, characterized in that, Before using the metal baffle in step S2, ultrasonic cleaning is performed to achieve sterility and prevent contamination.

3. The method for preparing a three-dimensional intratumoral turbulence disturbance device with repair function according to claim 1, characterized in that, The parameters for the oxygen plasma treatment in step S2 are: power 30-100W, treatment time 30-180s, and oxygen flow rate 10-20sccm.

4. The method for preparing a three-dimensional intratumoral turbulence disturbance device with repair function according to claim 1, characterized in that, The dilute acid etching in step S2 uses 5-10% dilute sulfuric acid or 3-5% dilute hydrochloric acid, with an etching time of 30-60 seconds. After etching, the solution is neutralized to pH 7.0-7.4 with 0.1M NaOH solution.

5. The method for preparing a three-dimensional intratumoral turbulence-disrupting device with repair function according to claim 1, characterized in that, In step S3, depending on the type of extracellular matrix raw material, sterile deionized water, phosphate buffer, or 0.05-0.1M dilute acetic acid is selected as the solvent to dissolve the extracellular matrix and adjust the concentration to 0.5-20 mg / mL. The pH of the solution is adjusted to 7.2-7.6, and the operating temperature is controlled at 4-25℃.

6. The method for preparing a three-dimensional intratumoral turbulence-disrupting device with repair function according to claim 1, characterized in that, The spraying parameters in step S4 are: pressure 10-30psi, spraying distance 10-20cm, multiple short-distance uniform sprayings, and curing at 4℃ for 5-10min after each spraying. The parameters for the impregnation are: impregnation time 0.5-5 min, withdrawal speed 1-3 mm / s, and repeated impregnation 2-3 times; The parameters for spin coating are: rotation speed 1000-4000 rpm, rotation time 30-120 s.

7. The method for preparing a three-dimensional intratumoral turbulence disturbance device with repair function according to claim 1, characterized in that, The crosslinking agent in step S5 is EDC / NHS, Genipin, or glutaraldehyde. When glutaraldehyde is selected, the residual aldehyde groups are blocked with a 1% w / v glycine solution after crosslinking.

8. The method for preparing a three-dimensional intratumoral turbulence disturbance device with repair function according to claim 1, characterized in that, In step S5, the phosphate buffer solution is used to elute at least 3 times, with each elution lasting 10-30 minutes.

9. A three-dimensional intratumoral turbulence disturbance device with repair function, characterized in that, It is prepared by the method of preparing the three-dimensional intratumoral turbulence device with repair function as described in any one of claims 1-8.

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