An implant device interface system, method and use thereof for achieving rapid cell adhesion and controlled release

Abstract

The application discloses an implant device interface system capable of realizing rapid cell adhesion and controllable release, a method and application, and relates to the technical field of biomedical materials and tissue engineering. The interface system comprises a first reaction group fixed on the surface of an implant device and a second reaction group inserted into the membrane of target cells through a membrane anchoring molecule; the membrane anchoring molecule comprises a cleavable linker; the first and second reaction groups constitute a complementary system such as click chemistry or biological orthogonality. The system realizes rapid adhesion within 0.5-6 hours, the cell migration rate increases after 6 hours of triggering, and a monolayer is formed within 12-48 hours, the cell tight junction is enhanced, and the blood compatibility is good (hemolysis rate < 5%). The system is applicable to different materials and cell types, and is suitable for surface cell construction and biological integration improvement of small-diameter artificial blood vessels, vascular stents, catheters, valves and other devices.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and tissue engineering technology, and more specifically, to an implantable device interface system, method, and application that enables rapid cell adhesion and controlled release. Background Technology

[0002] Cardiovascular disease is one of the leading causes of death worldwide. Small-diameter artificial blood vessels (≤6 mm) have important applications in coronary artery bypass grafting and peripheral artery reconstruction. However, their clinical performance has long been limited by problems such as early thrombosis, restenosis, and intimal hyperplasia. The core reason is that it is difficult for a stable and continuous endothelial cell layer to form in the lumen of artificial blood vessels in a timely manner. The lumen of natural blood vessels is covered by a complete endothelial layer, which has important functions such as anticoagulation, anti-inflammation, and maintaining blood flow homeostasis. However, commonly used artificial blood vessel materials such as ePTFE, PET, PCL, and their copolymers are highly hydrophobic, chemically inert, and lack cell adhesion sites, making it difficult for endothelial cells to attach and spread, and they are prone to detachment under the impact of blood flow.

[0003] Existing strategies for promoting endothelialization mainly rely on immobilizing bioactive molecules (such as RGD peptides, fibronectin, and laminin), but these strategies suffer from problems such as insufficient adhesion strength, uncontrollable directionality and density, easy degradation, or being washed away by blood flow. They cannot provide sufficient selectivity and strength in the early stages to resist the challenges of the in vivo environment. Furthermore, cell behavior on material surfaces is dynamic: rapid adhesion is needed in the early stages, migration and rearrangement are required in the middle stages, and tight junctions are needed in the later stages to establish a functional endothelial layer. Direct, irreversible immobilization methods can only satisfy initial adhesion but inhibit later migration, while soft hydrogel materials, although biocompatible, have insufficient adhesion. This makes "strong adhesion" and "migration" a contradictory requirement that is difficult to satisfy simultaneously.

[0004] Click chemistry and bioorthogonal reactions have attracted attention in the functionalization of biomaterials due to their high selectivity and mild reaction conditions, especially the copper-free SPAAC reaction (azide / tornethylene) and the tetrazine-tornene reaction, which can occur rapidly under near-physiological conditions. However, the coupling bonds formed by these reactions are generally stable, and if used directly as a long-term cell fixation method, they may inhibit the necessary migration and monolayer formation in the later stages. Cell membrane insertion labeling technology can insert PEG molecules with functional groups into the cell membrane through lipid anchors (such as DSPE and cholesterol), endowing cells with new interfacial reactivity. Combined with chemical functionalization of the material surface, it can significantly enhance selective cell adhesion, but current research has failed to combine "cell-side cleavable linkers" with "material-side controllable click groups" to form a reversible system. The rational endothelialization process of artificial blood vessels requires dynamic regulation: early strong adhesion, timely release, migration rearrangement, and tight junction formation.

[0005] Therefore, a cell-material interface system that can achieve "anchoring before release" through chemical bond switching is an important direction for overcoming the early failure of small-diameter artificial blood vessels. Summary of the Invention

[0006] The purpose of this invention is to provide an implantable device interface system, method, and application that enables rapid cell adhesion and controlled release. It aims to address the common problems of insufficient cell adhesion, difficulty in rapid endothelial layer formation, and limited cell migration in the early stages of use of small-diameter artificial blood vessels and other implantable devices. This is achieved by constructing a dynamically controllable cell adhesion system based on a "dissociable intercalation marker + clickable chemical pairing interface," enabling full-process regulation of cell adhesion, controlled release, and monolayer rearrangement.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides an implantable device interface system capable of achieving rapid cell adhesion and controlled release. The interface system includes a first reactive group immobilized on the surface of the implantable device, and a second reactive group inserted into the target cell membrane via a membrane anchoring molecule. The membrane anchoring molecule includes a cleavable linker. The first reactive group and the second reactive group constitute a click chemistry or biological positive complement system. The click chemistry or biological positive complement system is selected from at least one of the following: azide-strainyne reaction systems (e.g., DBCO, BCN, BARAC, DIFO paired with an azide group), tetrazine-strainyne reaction systems (e.g., TCO, norbornene paired with a tetrazine group), mercapto-ene reaction systems, mercapto-maleimide reaction systems, aldehyde-amino condensation reaction systems, aldehyde-hydrazine hydrazone formation reaction systems, or aldehyde-aminooxy oxime formation reaction systems. The cleavable linker is selected from at least one of disulfide bonds, acid-sensitive linkers, photosensitizing linkers, enzyme-sensitive linkers, or host-guest competitive linkers.

[0008] In an optional embodiment, the membrane anchoring molecule contains a hydrophobic anchoring segment selected from at least one of phospholipids, sterols, or C16–C22 aliphatic chains; And / or, the phospholipids include 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine, or 1,2-dioleoyl-sn-glycerol-3-phosphate choline; the sterols include cholesterol.

[0009] In an optional embodiment, the first reactive group and / or the second reactive group are respectively connected to the implantation device surface or membrane anchoring molecules via a hydrophilic flexible spacer arm. This hydrophilic flexible spacer arm helps to reduce steric hindrance, improve reaction accessibility, and improve cell compatibility. The hydrophilic flexible spacer arm is polyethylene glycol, a polyethylene glycol derivative, or a copolymer thereof. And / or, the molecular weight of the polyethylene glycol is 0.35-20 kDa.

[0010] In an optional embodiment, the apparent density of the first reactive group on the surface of the implantable device is 0.05-50 pmol / cm³. 2 .

[0011] In an optional embodiment, the substrate of the implantable device is selected from at least one of polytetrafluoroethylene, expanded polytetrafluoroethylene, polyethylene terephthalate, polyurethane, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid copolymer, polycaprolactone, poly-L-lactic-co-caprolactone copolymer, polyvinylidene fluoride, or metal; wherein the metal includes at least one of stainless steel, nickel-titanium alloy, and cobalt-chromium alloy, and the surface of the metal has a coating; And / or, the implantable device is a small-diameter artificial blood vessel, vascular stent, covering membrane, catheter, valve, or occluder.

[0012] In an optional embodiment, the target cell is at least one of endothelial cells, smooth muscle cells, fibroblasts, epithelial cells, or mesenchymal stem cells.

[0013] Secondly, the present invention provides a method for forming a continuous cell monolayer on the inner surface of an implantable device, using the aforementioned implantable device interface system, comprising the following steps: constructing a functional layer immobilized with the first reactive group on the inner surface of the implantable device; inserting the second reactive group into the target cell membrane through a membrane anchoring molecule containing the cleavable linker; under physiological conditions, bringing the functional layer immobilized with the first reactive group into contact with the cell containing the inserted second reactive group, achieving cell adhesion through a rapid bioorthogonal reaction of the click chemistry complementary system; culturing the adhered cells until a continuous cell monolayer is formed; applying a triggering condition corresponding to the cleavable linker to cleave the cleavable linker, thereby releasing the cells and completing their migration and rearrangement, to obtain a continuous, stable cell monolayer with tight connections.

[0014] In an optional embodiment, the concentration of the membrane-anchoring molecule is 1-500 μM, the action time is 1-60 min, and the cell seeding density is 1×10⁻⁶. 4 -1×10 6 cells / cm 2 .

[0015] In an optional embodiment, the first reactive group is immobilized on the surface of the implantable device by at least one of the following methods: plasma treatment, ultraviolet / ozone treatment, silanization modification, catechol or dopamine self-polymerization modification, initiation graft polymerization, dip coating or spray coating, electrospinning coating combined with photocrosslinking or layer-by-layer self-assembly. And / or, the triggering condition is selected from at least one of the following: an acidic buffer solution with pH 5.5-7.0, a 0.05-50 mM reducing agent, 320-420 nm light, a proteolytic enzyme, or a host-guest competitor.

[0016] Thirdly, the present invention provides a small-diameter artificial blood vessel, the inner surface of which includes the first reactive functional layer in the above-mentioned implantable device interface system that enables rapid cell adhesion and controlled release. The small-diameter artificial blood vessel is used in conjunction with a membrane anchoring marker, which includes a cleavable linker and carries a second reactive group complementary to the first reactive group, for achieving rapid cell adhesion and controlled release in vitro or implanted in vivo. The inner diameter of the small-diameter artificial blood vessel is 2-6 mm.

[0017] Fourthly, the present invention provides a kit for rapid cell adhesion and controlled release, comprising: A membrane anchoring marker comprising a cleavable linker and carrying a second reactive group, the second reactive group being configured to undergo a click chemical reaction with a first reactive group immobilized on the surface of an implantable device; The detection reagent comprises a fluorescent probe or a quantitative detection reagent paired with the first reactive group; At least one triggering solution; and instructions for use; The triggering solution is selected from at least one of acidic triggering solutions, reducing triggering solutions, enzyme triggering solutions, or competing guest triggering solutions.

[0018] The present invention has the following beneficial effects: This invention achieves compatibility between strong cell adhesion and reversible release. It provides rapid, high-intensity cell anchoring through click chemical pairs and utilizes the controlled cleavage of cleavable linkers in membrane anchoring molecules to achieve timely cell release. The system preferably employs azide-torsynylene or tetraazine-torsynylene click chemical pairs, exhibiting high selectivity and rapid reaction kinetics, and eliminating the need for metal catalysis, thus avoiding potential toxicity to cell viability. This invention is not limited to copper-free click chemical systems and can also be adapted to mercapto-ene, mercapto-maleimide, aldehyde-amino / oxime / hydrazone, and other systems, thereby improving applicability to different device substrates and cell types. The triggering conditions for the cleavable linkers are mild; cell release can be achieved under reducing environments, weakly acidic pH, light exposure, enzymatic stimulation, or the action of competing guests without affecting cell viability. Released cells can migrate along the material surface, rearrange, and restore the continuous distribution of tight junction proteins, which is beneficial for the formation of a functional endothelial cell monolayer, thereby reducing the risk of thrombosis, restenosis, and intimal hyperplasia. This system is highly versatile and can be adapted to various implantable device substrates and cell types. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of cell labeling in the present invention; Figure 2 This is the HPLC detection result of the disulfide bond breaking triggered by the TCEP reducing agent, which is a cleavable membrane anchoring molecule, in this invention; Figure 3 This is a schematic diagram of fluorescence microscopy verification of endothelial cell labeling and controllable triggering of bond-breaking release membrane anchoring tags in this invention; Figure 4 This is a schematic diagram illustrating the construction of the vascular stent substrate in this invention; Figure 5 This is a schematic diagram of fluorescence micrograph verification of vascular stent substrate containing azide label in this invention; Figure 6 These are fluorescence images and quantitative results of endothelial cell adhesion during the rapid adhesion experiment (0.5-4 h) in this invention; Figure 7 To compare the monolayer coverage effect after cell release is triggered at 6 h and cultured for another 24 h, CFDA-SE staining was performed on the cells. Figure 8 Fluorescent photographs comparing the adsorption effects of the endothelialized material (P / G-Endo) in this invention on fluorescently labeled albumin and fibrinogen. Figure 9 The hemolysis test of the endothelialized material (P / G-Endo) in this invention showed good blood compatibility. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] The following is a detailed description of the implantable device interface system, method, and application provided by the present invention, which enables rapid cell adhesion and controlled release.

[0023] Based on existing technologies, there has been no attempt to explore a strategy of introducing azide groups on the material surface, introducing DBCO intercalation labeling agents with cleavable bonds (such as disulfide bonds) on the cell side, and utilizing SPAAC for rapid adhesion and disulfide bond-triggered cleavage to achieve reversible adhesion and rearrangement. Therefore, this invention proposes a system that provides a novel technical solution for achieving cell monolayering on the surface of biomedical implantable devices such as "small-diameter artificial blood vessels with rapid, controllable, and functional endothelialization," thereby improving the biocompatibility of implantable materials.

[0024] Specifically, a first reactive group is introduced on the material surface, and a second reactive group is introduced on the cell membrane surface through a hydrophobic lipid anchoring method. This allows cells to rapidly and selectively adhere to the material surface under physiological conditions through a tension-promoted azido-alkyne cycloaddition reaction (SPAAC). At a predetermined time, the cleavable nature of disulfide bonds is utilized to gently trigger (such as TCEP, GSH, etc.) the cells to release their chemical anchoring, restore their migration ability, and form a continuous monolayer, thereby meeting the dynamic requirements of endothelialization.

[0025] In a first aspect, the present invention provides an implantable device interface system that enables rapid cell adhesion and controlled release. The interface system includes a first reactive group immobilized on the surface of the implantable device and a second reactive group inserted into the target cell membrane via a membrane anchoring molecule, wherein the membrane anchoring molecule includes a cleavable linker. Furthermore, the first reactive group and the second reactive group constitute a click chemistry complementary system.

[0026] In some preferred embodiments, the click chemical complementary system is selected from at least one of the following: azide-strainyne click chemical pairs (DBCO, BCN, BARAC, DIFO, etc.), tetrazine-strainyne click chemical pairs (TCO, norbornene), mercapto-alkene reaction system, mercapto-maleimide reaction system, aldehyde-amino condensation reaction system, aldehyde-hydrazine forming hydrazone reaction system, or aldehyde-aminooxy forming oxime reaction system.

[0027] In some preferred embodiments, the cleavable linker is selected from at least one of disulfide bonds (-SS-), acid-sensitive linkers, photosensitive linkers, enzyme-sensitive linkers, or host-guest competitive linkers. The "reversible / controlled release" of this invention is achieved through cleavable linkers or competitive interactions; the click-generate bond itself is typically irreversible.

[0028] The cleavable linker is a disulfide bond (-SS-), which is cleavable in 0.1-20 mM reducing agents (TCEP, GSH, DTT, L...). It can be cleaved under the action of cysteine.

[0029] In some preferred embodiments, the membrane anchoring molecule contains a hydrophobic anchoring fragment selected from at least one of phospholipids, sterols, or C16–C22 aliphatic chains; Phospholipids include 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), or 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC); cholesterol can be used as a sterol.

[0030] It should be noted that the first reactive group and / or the second reactive group are respectively connected to the implantation device surface or membrane anchoring molecules via a hydrophilic flexible spacer arm. This hydrophilic flexible spacer arm helps reduce steric hindrance, improve reaction accessibility, and enhance cell compatibility; wherein the hydrophilic flexible spacer arm is polyethylene glycol (PEG), a polyethylene glycol derivative, or a copolymer thereof, and the molecular weight of PEG is 0.35-20 kDa.

[0031] The reactive ends of the membrane anchoring molecule carry click groups that are complementary to the device surface, preferably DSPE-PEG(2 / 5 / 10 kDa)-SS-DBCO.

[0032] In an optional embodiment, the substrate of the implantable device is selected from at least one of polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene (ePTFE), polyethylene terephthalate (PET), polyurethane (PU), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid copolymer (PLGA), polycaprolactone (PCL), poly-L-lactic-co-caprolactone copolymer (PLCL), polyvinylidene fluoride (PVDF), or a metal; wherein the metal includes at least one of stainless steel, nickel-titanium alloy, and cobalt-chromium alloy, and the surface of the metal has a coating; And / or, the implantable device is a small-diameter artificial blood vessel, vascular stent, covering membrane, catheter, valve, or occluder.

[0033] In some preferred embodiments, the target cells are at least one of endothelial cells, smooth muscle cells, fibroblasts, epithelial cells, or mesenchymal stem cells.

[0034] Secondly, the present invention provides a method for forming a continuous cell monolayer on the inner surface of an implantable device, using the above-mentioned implantable device interface system, comprising the following steps: S1. Construct a functional layer on the inner surface of the implantable device that is immobilized with the first reactive group; S2. Insert the second reactive group into the target cell membrane through a membrane anchoring molecule containing the cleavable linker; S3. Under physiological conditions, the functional layer immobilized with the first reactive group is brought into contact with the cell with the second reactive group inserted, and cell adhesion is achieved through the rapid bioorthogonal reaction of the click chemical complementary system. S4. Culture the adhesion cells until a continuous cell monolayer is formed; S5. Apply a triggering condition corresponding to the cleavable linker to cause the cleavable linker to cleave, thereby releasing the cells and completing their migration and rearrangement to obtain a continuous, stable cell monolayer with tight connections.

[0035] In some preferred embodiments, the concentration of the membrane anchoring molecule is 1-500 μM, which can be 1 μM, 10 μM, 50 μM, 100 μM, 200 μM, 300 μM, 400 μM or 500 μM, etc., preferably 10-100 μM; the action time is 1-60 min, which can be 1 min, 10 min, 20 min, 30 min, 40 min, 50 min or 60 min, etc., preferably 10-30 min.

[0036] In some preferred embodiments, the cell seeding density is 1×10⁻⁶. 4 -1×10 6 cells / cm 2 Furthermore, the shear stress in cell culture is 1-40 dyn / cm. 2 Preferably 5-30 dyn / cm 2 .

[0037] Tight junction-related proteins are continuously distributed in the cell monolayer, including ZO-1, VE-cadherin, and CD31.

[0038] In some preferred embodiments, the first reactive group is immobilized on the surface of the implantable device by means of at least one of the following: plasma treatment (O2, Ar, etc.), ultraviolet (UV) / ozone treatment, silanization modification, catechol or dopamine self-polymerization modification, initiation graft polymerization, dip coating or spray coating, electrospinning coating combined with photocrosslinking or layer-by-layer self-assembly.

[0039] In some preferred embodiments, the triggering condition is at least one of the following: an acidic buffer solution with pH 5.5-7.0, a 0.05-50 mM reducing agent, 320-420 nm light, a proteolytic enzyme, or a host-guest competitor.

[0040] The reducing agent is preferably tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or glutathione (GSH), the dosage of the reducing agent is preferably 0.5-10 mM, and the treatment time is 10-60 min.

[0041] The preferred intensity of 320-420 nm light is 1-50 mW / cm². 2 The processing time is 1-30 minutes.

[0042] Thirdly, the present invention provides a small-diameter artificial blood vessel, the inner surface of which includes the first reactive functional layer in the above-mentioned implantable device interface system that enables rapid cell adhesion and controlled release. The small-diameter artificial blood vessel is used in conjunction with a membrane anchoring marker, which includes a cleavable linker and carries a second reactive group complementary to the first reactive group, for achieving rapid cell adhesion and controlled release in vitro or implanted in vivo. The small-diameter artificial blood vessel has an inner diameter of 2-6 mm and meets the compliance and burst pressure requirements of ISO 7198.

[0043] It should be noted that the preferred embodiment of the present invention includes the following steps in its technical solution: (1) Material segment functionalization: A polycaprolactone / methacrylamide gelatin composite (PCL / GelMA) functional layer containing azide side groups is constructed on the surface of artificial blood vessels or devices by electrospinning or coating. (2) Cell terminology, such as Figure 1 As shown: Dibenzocyclooctylene (DBCO) is presented on the outer side of the cell membrane using membrane anchoring molecules with cleavable linkers, such as distearate-polyethylene glycol-disulfide-dibenzocyclooctylene (DSPE-PEG-SS-DBCO) or cholesterol-polyethylene glycol-disulfide-dibenzocyclooctylene (Chol-PEG-SS-DBCO). (3) Click coupling: The cells and the material surface undergo a rapid SPAAC reaction, achieving efficient and highly selective initial adhesion; (4) Triggered release: By adding a biocompatible reducing agent (such as 0.5-5 mM TCEP or GSH) to cleave disulfide bonds, the cells are gradually defixed; (5) Cell rearrangement and monolayer formation: After the cells are released from chemical constraints, they migrate and rearrange, and form a continuous and functional endothelial cell layer within 24 hours.

[0044] Fourthly, the present invention provides a kit for rapid cell adhesion and controlled release, comprising: A membrane anchoring marker comprising a cleavable linker and carrying a second reactive group, the second reactive group being configured to undergo a click chemical reaction with a first reactive group immobilized on the surface of an implantable device; The detection reagent comprises a fluorescent probe or a quantitative detection reagent paired with the first reactive group; At least one triggering solution; and instructions for use; The triggering solution is selected from at least one of acidic triggering solutions, reducing triggering solutions, enzyme triggering solutions, or competing guest triggering solutions.

[0045] Furthermore, the implantable device interface system of the present invention, which enables rapid cell adhesion and controlled release, can also be applied to the cellular construction of vascular implantable devices, especially for the preparation process of in vitro pre-endothelialization.

[0046] And the application of the implantable device interface system in the preparation of vascular implantable devices, wherein the vascular implantable devices are used to reduce the risk of early implantation thrombosis, promote endothelialization, and reduce at least one of intimal hyperplasia.

[0047] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0048] Example 1 A method for forming a continuous cell monolayer on the inner surface of an implantable device includes the following steps: The substrates and reagents used are as follows: medical-grade polycaprolactone (PCL), gelatin (approximately 240 g Bloom), methacrylic anhydride (MAA), azide-containing side-chain monomer AzMA, photoinitiator LAP, hexafluoroisopropanol (HFIP), tris(2-carboxyethyl)phosphine (TCEP), and glutathione (GSH); the cells are human umbilical vein endothelial cells (HUVEC) or equivalent sources, and the culture medium is EGM-2 or equivalent medium; other reagents include phosphate-buffered saline (PBS), paraformaldehyde (PFA), fluorescent probes (Az-Cy5 or DBCO-Cy5), DAPI, and CFDA-SE.

[0049] S1. Preparation of methacrylamide gelatin (GelMA): Dissolve 10 g of gelatin in PBS at 50℃ to prepare a 10% solution. Slowly add 0.5-1.0 mL / g of methacrylic anhydride to the solution. React at 50℃ for 1-3 hours. After terminating the reaction, dialyze the solution using a dialysis bag with a molecular weight cutoff of 12-14 kDa for 5-7 days, changing the solution 3-4 times daily. Freeze-dry to obtain GelMA. Simultaneously, prepare AzMA containing an azide monomer: React 3-azidopropyl-1-amine with methacrylamide chloride in dichloromethane at 0℃. Continue the reaction at room temperature for 12-14 hours. Wash successively with dilute acid, water, and dilute alkali, dry with anhydrous magnesium sulfate, and rotary evaporate to obtain AzMA. Confirm the structure by 1H NMR or HPLC-MS.

[0050] S2. Preparation of the cleavable membrane anchoring agent Chol-PEG-SS-DBCO: Cholesterol-polyethylene glycol-carboxyl (Chol-PEG-COOH, molecular weight approximately 3400) was dissolved in N,N-dimethylformamide, activated with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide for 0.5 hours, and DBCO-SS-NH2 was slowly added dropwise. The reaction was carried out at room temperature for 12-24 hours under nitrogen protection. After precipitation with diethyl ether at -20 °C, dialyzing with a dialysis bag with a molecular weight cutoff of 1-3 kDa for 24-48 hours, and lyophilization, the product was confirmed by 1H NMR spectroscopy and HPLC. After treatment with different concentrations of TCEP, the cleavable bond breaking of the membrane anchoring agent was verified by HPLC, as shown in the results. Figure 2 As shown.

[0051] S3. A PCL / GelMA / AzMA composite fiber scaffold is constructed using coaxial electrospinning technology, such as... Figure 4 As shown: PCL was dissolved in hexafluoroisopropanol at a concentration of 15 wt.% as the core spinning solution; GelMA and AzMA were dissolved in hexafluoroisopropanol at a total concentration of 10-15 wt.%, with the molar percentage of AzMA relative to the methacrylyl group in GelMA being 1-10 mol%, and 0.05-0.5 wt.% photoinitiator LAP was added as the shell spinning solution. Spinning parameters were set as follows: voltage 12-20 kV, receiving distance 10-20 cm, core and shell flow rates 0.2-1.0 mL / h each, ambient humidity 20-60%RH, and receiving drum speed 200-800 rpm. After collecting the fiber membrane, it was irradiated with light at a wavelength of 365-405 nm and an intensity of 5-30 mW / cm. 2 Crosslinking was performed for 0.5-10 minutes to obtain a crosslinked shell structure with azide side groups on the surface. The surface azide groups were titrated with a DBCO-Cy5 fluorescent probe to establish a fluorescence intensity-surface density standard curve, and the apparent density of azide was measured to be 0.2-10 pmol / cm³. 2 This was further corroborated by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy.

[0052] S4. Cell membrane labeling of human umbilical vein endothelial cells: Human umbilical vein endothelial cells were co-incubated with 50 μM hol-PEG-SS-DBCO at 37 ℃ for 30 min, and washed three times with PBS to remove unbound labeling agent. To verify the membrane labeling efficiency, the labeled cells were reacted with 50 μM Az-Cy5 for 30 min. Confocal laser scanning microscopy and flow cytometry confirmed successful DBCO anchoring on the cell membrane surface. After 10 minutes of treatment with 0.5 mM TCEP reducing agent, DBCO labeling was significantly weakened, as shown in the results. Figure 3 As shown.

[0053] The azide-modified fiber membrane was cut into pieces measuring 1.4 cm × 0.7 cm, and then sized into 7.5 × 10⁻⁶ pieces. 5 cells / cm 2 DBCO-labeled human umbilical vein endothelial cells were seeded at a density of [missing information]. During the rapid adhesion phase (0.5-6 hours), samples were taken at 0.5, 1, 2, 4, and 6 hours. Cell coverage was quantified using CFDA-SE live cell staining and DAPI nuclear staining for fluorescence imaging. Figure 5 As shown in the figure. The results showed that cells achieved rapid and uniform adhesion within 6 hours. Triggered release and monolayer formation experiments were then performed: 6 hours after adhesion, cells were treated with 0.5-5 mM TCEP for 30 minutes, washed with PBS, and then replaced with complete culture medium. Live-cell imaging was used to continuously record for 15 hours (e.g., 6-21 hours) to analyze cell migration speed, migration trajectory, and population coverage growth curves. The results are shown in the figure. Figure 6 As shown in the figure. The results indicate that after TCEP triggers the cleavage of disulfide bonds, cells gain the ability to migrate, gradually rearrange along the fiber surface, and form a continuous monolayer.

[0054] Immunofluorescence staining was performed on the formed cell monolayer. Cells were fixed with 4% paraformaldehyde for 15 minutes, incubated overnight at 4°C with ZO-1 primary antibody (1:400), and then incubated at room temperature for 2 hours with fluorescently labeled secondary antibody (1:500). Cell nuclei were counterstained with DAPI. Results are shown below. Figure 7 As shown in the image, ZO-1 exhibits a continuous linear distribution, indicating tight junctions between cells. Further validation was performed using qPCR: Total RNA was extracted using the Trizol method, and 1 μg of RNA was reverse transcribed into cDNA. This cDNA was then amplified using a two-step SYBR Green method (95℃ for 30 seconds; 40 cycles: 95℃ for 5 seconds, 60℃ for 30 seconds). -ΔΔCt The relative expression levels of genes such as ZO-1, OCLN, and MMP2 were calculated using the method, and the results are as follows: Figure 8 As shown in the figure, the results indicate that the expression of tight junction-related genes was significantly upregulated in the cell monolayer after triggered release and rearrangement.

[0055] The blood compatibility of the constructed artificial blood vessel material was evaluated according to ISO 10993-4 standard, including tests for hemolysis rate, platelet adhesion and activation (CD62P), complement activation (C3a, SC5b-9), and fibrinogen adsorption. The results are as follows: Figure 9 As shown in the figure. The results show that the interface system has good blood compatibility.

[0056] The above results demonstrate that the interface system constructed in this invention can achieve rapid adhesion, mild and controllable release, and functional monolayering of endothelial cells, making it suitable for in vitro pre-endothelialization of small-diameter artificial blood vessels. It has the potential to reduce the risk of early thrombosis, promote endothelialization, and reduce intimal hyperplasia.

[0057] Comparative Example 1 This comparative example provides a method for forming a continuous cell monolayer on the inner surface of an implantable device. The steps are the same as in Example 1, except that: no first reactive group surface: a PCL / GelMA surface without azide or other first reactive groups is used to contact DBCO-SS labeled cells.

[0058] The results showed that the cells mainly relied on non-specific adhesion, and the early coverage and erosion resistance were significantly lower than those in Example 1, indicating that the complementary reactive groups on the material side play an important role in rapid and highly selective adhesion.

[0059] Comparative Example 2 This comparative example provides a method for forming a continuous cell monolayer on the inner surface of an implantable device. The steps are the same as in Example 1, except that: no cleavable linker membrane anchoring agent is used: the surface is modified with azide and contacted with Chol-PEG-DBCO labeled cells, wherein the membrane anchoring agent does not contain disulfide bonds or other cleavable linkers.

[0060] The results showed that cells could adhere rapidly in the early stage, but the cell migration ability was not significantly improved after the triggering treatment, and the monolayer rearrangement was limited, indicating that the cleavable linker plays a key role in the subsequent controlled release and migration rearrangement.

[0061] Comparative Example 3 This comparative example provides a method for forming a continuous cell monolayer on the inner surface of an implantable device. The steps are the same as in Example 1, except that: no triggered release is performed: the same system as in Example 1 is used for cell adhesion, but no TCEP or other triggering conditions are applied after adhesion.

[0062] The results showed that cells could adhere stably in the early stages, but the length and coverage of the migration trajectory increased less in the later stages, indicating that timely triggering of release is beneficial to cell rearrangement and the formation of continuous monolayers.

[0063] In summary, the three elements of this invention—complementary reactive groups on the material side, cleavable membrane anchoring molecules on the cell side, and timely triggered release—are indispensable and possess synergistic technical effects. Without material-side reactive groups, only non-specific adhesion can be relied upon, resulting in a significant decrease in early coverage and erosion resistance; without cleavable linkers, cells cannot effectively respond to trigger signals, limiting migration and monolayer rearrangement; even with the first two elements, without applying triggering conditions, cells struggle to achieve subsequent controllable migration and continuous monolayer formation. Therefore, these three elements respectively ensure rapid selective adhesion, release potential, and precise trigger control, synergistically achieving rapid adhesion and controllable release effects that traditional single-adhesion or non-cleavable fixation systems cannot simultaneously obtain.

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An implantable device interface system capable of achieving rapid cell adhesion and controlled release, characterized in that, The interface system includes a first reactive group immobilized on the surface of the implantable device and a second reactive group inserted into the target cell membrane via a membrane anchoring molecule; the membrane anchoring molecule includes a cleavable linker; the first reactive group and the second reactive group constitute a click chemistry or biological positive complement system, wherein the click chemistry or biological positive complement system is selected from at least one of the following: azide-strainyne reaction system, tetrazine-strainyne reaction system, mercapto-ene reaction system, mercapto-maleimide reaction system, aldehyde-amino condensation reaction system, aldehyde-hydrazine forming hydrazone reaction system, or aldehyde-aminooxy forming oxime reaction system; the cleavable linker is selected from at least one of the following: disulfide bond, acid-sensitive linker, photosensitive linker, enzyme-sensitive linker, or host-guest competitive linker.

2. The implantable device interface system according to claim 1, characterized in that, The membrane anchoring molecule contains a hydrophobic anchoring fragment, which is selected from at least one of phospholipids, sterols, or C16–C22 aliphatic chains; And / or, the phospholipids include 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine, or 1,2-dioleoyl-sn-glycerol-3-phosphate choline; the sterols include cholesterol.

3. The implantable device interface system according to claim 1, characterized in that, The first reactive group and / or the second reactive group are respectively connected to the surface of the implanted device or the membrane anchoring molecules via a hydrophilic flexible spacer arm; the hydrophilic flexible spacer arm is polyethylene glycol, a polyethylene glycol derivative or a copolymer thereof. And / or, the molecular weight of the polyethylene glycol is 0.35-20 kDa.

4. The implantable device interface system according to claim 1, characterized in that, The apparent density of the first reactive group on the surface of the implantable device is 0.05-50 pmol / cm³. 2 .

5. The implantable device interface system according to claim 1, characterized in that, The substrate of the implantable device is selected from at least one of polytetrafluoroethylene, expanded polytetrafluoroethylene, polyethylene terephthalate, polyurethane, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid copolymer, polycaprolactone, poly-L-lactic-co-caprolactone copolymer, polyvinylidene fluoride, or metal; wherein the metal includes at least one of stainless steel, nickel-titanium alloy, and cobalt-chromium alloy, and the surface of the metal has a coating. And / or, the implantable device is a small-diameter artificial blood vessel, vascular stent, covering membrane, catheter, valve, or occluder.

6. The implantable device interface system according to claim 1, characterized in that, The target cells are at least one of endothelial cells, smooth muscle cells, fibroblasts, epithelial cells, or mesenchymal stem cells.

7. A method for forming a continuous cell monolayer on the inner surface of an implantable device, characterized in that, The implantable device interface system described in any one of claims 1-6 is adopted. The procedure includes the following steps: constructing a functional layer immobilized with the first reactive group on the inner surface of an implantable device; inserting the second reactive group into the target cell membrane via a membrane anchoring molecule containing the cleavable linker; under physiological conditions, bringing the functional layer immobilized with the first reactive group into contact with the cell containing the second reactive group, achieving cell adhesion through a rapid bioorthogonal reaction of the click chemistry complementary system; culturing the adhered cells until a continuous cell monolayer is formed; applying a triggering condition corresponding to the cleavable linker to cleave the cleavable linker, thereby releasing the cells and completing their migration and rearrangement, resulting in a continuous, stable, and tightly connected cell monolayer. The concentration of the membrane-anchoring molecule is 1-500 μM, and the action time is 1-60 min; the cell seeding density is 1×10⁻⁶. 4 -1×10 6 cells / cm 2 .

8. A method for forming a continuous cell monolayer on the inner surface of an implantable device according to claim 7, characterized in that, The first reactive group is immobilized on the surface of the implantable device by at least one of the following methods: plasma treatment, ultraviolet / ozone treatment, silanization modification, catechol or dopamine self-polymerization modification, initiation graft polymerization, dip coating or spray coating, electrospinning coating combined with photocrosslinking or layer-by-layer self-assembly. And / or, the triggering condition is selected from at least one of the following: an acidic buffer solution of pH 5.5-7.0, a 0.05-50 mM reducing agent, 320-420 nm light, a proteolytic enzyme, or a host-guest competitor.

9. A small-diameter artificial blood vessel, characterized in that, Its inner surface includes a first reactive functional layer of the implantable device interface system according to any one of claims 1-6; the small-diameter artificial blood vessel is used in conjunction with a membrane anchoring marker, the membrane anchoring marker includes a cleavable linker and carries a second reactive group complementary to the first reactive group, for achieving rapid cell adhesion and controlled release in vitro or in vivo; the inner diameter of the small-diameter artificial blood vessel is 2-6 mm.

10. A kit for rapid cell adhesion and controlled release, characterized in that, include: A membrane anchoring marker comprising a cleavable linker and carrying a second reactive group, the second reactive group being configured to undergo a click chemical or bioorthogonal reaction with a first reactive group immobilized on the surface of an implantable device; The detection reagent comprises a fluorescent probe or a quantitative detection reagent paired with the first reactive group or the second reactive group; At least one triggering solution; And the instruction manual; The triggering solution is selected from at least one of acidic triggering solutions, reducing triggering solutions, enzyme triggering solutions, light triggering condition descriptions, or competing object triggering solutions.

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