CN114306747A - A kind of small-caliber cardiovascular implant and preparation method thereof - Google Patents

Abstract

The invention discloses a small-caliber cardiovascular implant and a preparation method thereof, belonging to the technical field of biological medicine, and the key points of the technical scheme are as follows: comprises a cardiovascular implant body and a double-layer responsive gel incubated on the outer surface of the cardiovascular implant body; the inner layer of the double-layer responsive gel is ATP responsive hydrogel; the outer layer of the double-layer responsive gel is ROS responsive hydrogel; the preparation method of the small-caliber cardiovascular implant comprises the steps of constructing a small-caliber cardiovascular implant body, preparing ATP response molecules and ROS response molecules, preparing ATP response hydrogel mixed liquor, ATP response hydrogel mixed liquor and ROS response hydrogel mixed liquor, and sequentially gelatinizing the surface of the cardiovascular implant body to obtain the small-caliber cardiovascular implant. The invention is mainly used for preparing the small-caliber cardiovascular implant, and the vascular implant can maintain good intimal form and thickness for a long time under the high-sugar environment, thereby improving the success rate and long-term patency rate of the small-caliber artificial blood vessel transplantation under diabetes.

Description

A kind of small-caliber cardiovascular implant and preparation method thereof

technical field

The invention relates to the technical field of biomedicine, in particular to a small-caliber cardiovascular implant and a preparation method thereof.

Background technique

Cardiovascular disease is a serious threat to human health, and about 170,000 people die from cardiovascular disease every year. Due to peripheral vascular occlusion, coronary artery bypass grafting, hemodialysis, arteriovenous fistula, etc., the demand for small-bore cardiovascular implants (<6mm) is increasing worldwide. However, thrombosis and intimal hyperplasia are serious problems after small-bore cardiovascular implants. Thrombosis is mainly controlled by drugs, but intimal hyperplasia leads to restenosis of vascular grafts, severely shortening their patency time and limiting further clinical application of small-bore cardiovascular implants.

Small-bore cardiovascular implant transplantation is an important therapeutic approach for the treatment of diabetes complicated by small and medium vascular disease. However, dyslipidemia and accumulation of advanced glycation end products can lead to severe intimal hyperplasia in vascular implants, which in turn leads to rapid graft stenosis. Therefore, preventing intimal hyperplasia and enabling their long-term patency in diabetes has become a key issue to be addressed.

Intimal hyperplasia is characterized by abnormal migration and proliferation of vascular smooth muscle cells. Although many studies have focused on inhibiting vascular smooth muscle cell proliferation-related signaling pathways, such as TGF-β, by blocking vascular smooth muscle cell proliferation, the control of intimal hyperplasia is still limited. Vascular smooth muscle cells can be divided into two subtypes: contractile and synthetic. The transition from contractile to synthetic phenotype is a common pathological feature of postoperative restenosis, suggesting that abnormal phenotypic transition of vascular smooth muscle cells is the key to the study of intimal hyperplasia. From a structural or functional point of view, nerves and blood vessels are intertwined. With the exception of capillaries, almost all vascular networks are innervated by sympathetic nerve fibers. Nerves regulate vascular activity and nutrient exchange. It has been reported that nerve fibers branch multiple times and innervate vascular smooth muscle cells in the form of varicose veins. In addition, finger-like axon terminals can grab vascular smooth muscle cells to form synaptosomes, and nerve cells release exosomes containing active substances through synaptosomes, and these exosomes are simultaneously captured and absorbed by smooth muscle cells. However, growth inhibition induced by changes in gene expression associated with hyperglycemia leads to disorganized nerve fiber architecture, distal degenerative neuropathy, and axonal damage that inhibit nerve fiber growth and ingrowth into vascular implants. Also, hyperglycemia reduced the activity of Rab35, a protein that controls exosome transport in neurons.

In order to solve the above problems, a small-diameter cardiovascular implant and a preparation method thereof are provided on the basis of the prior art.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a small-diameter cardiovascular implant and a preparation method thereof, which can maintain a good intimal shape and thickness for a long time in a high-glucose environment, and prevent the occurrence of abnormal intimal hyperplasia caused by abnormal intimal hyperplasia. It can improve the success rate and long-term patency rate of small-diameter artificial blood vessel transplantation under diabetes.

The above-mentioned technical purpose of the present invention is achieved through the following technical solutions:

A small-caliber cardiovascular implant, comprising a cardiovascular implant body and a double-layer responsive gel incubated on the outer surface of the cardiovascular implant body; the inner layer of the double-layer responsive gel is ATP-responsive water gel, the ATP-responsive hydrogel contains DENND1A protein; the outer layer of the bilayer-responsive gel is a ROS-responsive hydrogel, and the ROS-responsive hydrogel contains axon guidance molecules- 1.

Further: the preparation method of the ATP-responsive hydrogel is: mixing the eight-arm PEG azide and the ATP-responsive molecule according to a molar mass ratio of 1:3-1:5, and then mixing 1%-10% DENND1A protein, The gel can be formed by reacting at room temperature for 5-20 minutes;

The preparation method of the ROS-responsive hydrogel is as follows: the eight-arm PEG azide and the ROS-responsive molecule are mixed in a molar mass ratio of 1:3 to 1:5, and then mixed with 1% to 10% of axon guidance molecule-1 , the gel can be formed by reacting at room temperature for 5-20 min.

Further, the small-diameter cardiovascular implant is tubular, with a diameter of 0.5-4 mm and a length of 0.5-20 cm.

The present invention also provides a preparation method of a small-caliber cardiovascular implant, the steps are as follows:

(1) Construction of small-caliber cardiovascular implant body;

(2) Preparation of ATP-responsive molecules and ROS-responsive molecules;

(3) Preparation of ATP-responsive hydrogel mixture using eight-arm PEG azide, ATP-responsive molecule and DENND1A protein; preparation of ROS-responsive water using eight-arm PEG azide, ROS-responsive molecule and axon guidance molecule-1 Gel mix.

(4) The ATP-responsive hydrogel mixture and the ROS-responsive hydrogel mixture are sequentially gelled on the surface of the cardiovascular implant body to obtain a small-diameter cardiovascular implant.

Further: the preparation method of the cardiovascular implant body described in step (1) is as follows:

First, use decellularization solution to remove cells in the isolated blood vessel; then use nuclease to remove the nucleic acid in the isolated blood vessel to obtain a vascular matrix material; then incubate collagen and vascular endothelial growth factor (VEGF) on the surface of the vascular matrix material ( VEGF) to obtain the cardiovascular implant body.

Further: the specific preparation steps of the cardiovascular implant body are:

A. The decellularization solution is 0.025%～0.25% trypsin, and the decellularization solution and the isolated blood vessel are shaken and reacted at 0℃～37℃ for 30min～90min to complete decellularization; the nuclease is DNase and RNase, the nuclease reacts with isolated blood vessels at 0°C to 37°C for 10 minutes to 45 minutes, to complete denucleation, and to obtain blood vessel matrix materials;

B. After washing the vascular matrix material with PBS solution, incubate 10%-50% collagen on the surface of the vascular matrix material; )) cross-linking for 12-36 hours to obtain the cardiovascular implant body.

Further: the ATP response molecule described in step (2) is prepared by reacting ATP nucleic acid aptamer, ssDNA1 and ssDNA2 according to the ratio of 1:1:1～1:2:2 at 0℃～50℃ for 6h～24h. to make.

Further: the sequence of the ATP nucleic acid aptamer is CACCTGGGGGAGTATTGCGGAGGAAGGTT; the sequence of the ssDNA1 is 5'-ACTCCCCCAGGTGTTTTTT- _NH2 -DBCO-3'; the sequence of the ssDNA2 is 5'-DBCO- _NH2 -TTTTTTTAACCTTCCTCCGC- 3'.

Further: the ROS response molecule described in step (3) is aminothio ketal and dibenzocyclooctyne (DBCO) according to the molar mass ratio of 1:1 to 1:3, and the reaction is stirred for 30min at 0°C to 75°C ~ 2h from.

Further: the specific steps of forming the ATP-responsive hydrogel mixture and the ROS-responsive hydrogel mixture on the surface of the cardiovascular implant body in step (4) in turn are as follows:

a. First tie both ends of the cardiovascular implant body with a 1-0 surgical thread, then immerse it in the ATP-responsive hydrogel mixture, and then take it out to cover the surface of the cardiovascular implant body A thin layer of mixed solution I, waiting for the thin layer of mixed solution I to react to form a gel;

b. The cardiovascular implant body is then immersed in the ROS-responsive hydrogel mixture, and then taken out, so that the surface of the cardiovascular implant body is covered with a thin layer of mixture II, waiting for the After the thin-layer mixed solution II reacted to form a gel, the surgical threads at both ends of the vascular implant were untied.

Technical principle: In the pathological environment of diabetes, the regenerated smooth muscle cells on the cardiovascular graft are damaged by high glucose, the level of intracellular ROS increases, protein carbonylation, and the cells change from a differentiated phenotype to a dedifferentiated phenotype, and abnormality occurs. Proliferation and migration, and due to the influence of high glucose environment, the repair of peripheral nerves is slowed down and stagnant, and it is difficult for nerves to participate in the process of angiogenesis and homeostasis maintenance.

In this technical solution, DENND1A and Axon Guidance Factor-1 are respectively modified on the outer surface of the cardiovascular implant by responsive hydrogel wrapping, and the changes of the surrounding biochemical environment are used to trigger the release of the two, reducing the smooth muscle in situ. Cells are damaged by superoxide under high glucose, inhibit the abnormal proliferation of the intima, and promote long-term patency in the environment of diabetes. Nerve axon guidance molecule-1 is triggered by the release of a large number of ROS generated by the inflammatory response after vascular implant transplantation, which promotes and induces the rapid repair of nerves and grows into the vascular graft in the early stage of transplantation; on the other hand, it is also related to the vascular graft. Pre-incubated vascular endothelial growth factor together promotes early endothelialization of cardiovascular grafts, increasing the success rate of vascular graft engraftment.

DENND1A is triggered by the neurotransmitter-ATP released after nerve ingrowth. After entering the neuron, it converts Rab35 to the activated state to enhance the neuronal exosome secretion activity that was originally inhibited by the high-glucose environment. Neurons transport peroxidase-1 protein to smooth muscle cells through exosomes, eliminate ROS in smooth muscle cells, reduce high-glucose injury, maintain smooth muscle cells in a dedifferentiated phenotype, inhibit intimal hyperplasia, and improve small-caliber cardiovascular implantation Long-term patency rates in diabetes.

To sum up, the present invention has the following beneficial effects:

1. The double-layer responsive hydrogel is completely and uniformly covered on the surface of the decellularized vascular implant, and the small-diameter cardiovascular implant has good biocompatibility and obvious biological function, and the outer layer of ROS In response to the ROS generated by the hydrogel first in response to the inflammatory response, the disintegration released the axon guidance factor-1 in it, inducing nerve growth into the cardiovascular implant, and the inner layer of the ATP-responsive hydrogel protected the DENND1A activity in it .

2. After nerve growth, the neurotransmitter ATP is released, and the inner layer of glue triggers the release of DENND1A, which acts on neurons and increases the secretion level of neuronal exosomes that are inhibited under high glucose, and neuronal exosomes are implanted by blood vessels. Uptake by regenerated smooth muscle cells, the peroxidase-1 contained in it reduces the level of ROS in smooth muscle cells, reduces high-glucose injury, inhibits the excessive proliferation, migration and intimal hyperplasia of smooth muscle cells under high glucose, and maintains smooth muscle cells Cell differentiation phenotype and normal intimal morphology improved the transplantation success rate and long-term patency rate of small-bore cardiovascular implants in diabetes.

3. It has a good application prospect in the field of clinical treatment of diabetic cardiovascular transplantation.

Description of drawings

1 is a schematic structural diagram of gel formation and response triggering of the bilayer responsive hydrogel in Example 1 of the present invention;

Fig. 2 is the fluorescence microscopy result of the bilayer responsive hydrogel covering the outer surface of the decellularized cardiovascular implant in Example 1 of the present invention;

Fig. 3 is the verification result of the infrared spectrum of the responsive hydrogel gelation of the present invention;

Figure 4 shows the gel formation and hydrolysis of the ROS-responsive hydrogel in the present invention;

Fig. 5 is the experimental result of maintaining the activity of the hydrogel-wrapped fluorescent dye in the present invention;

Fig. 6 is the small animal in vivo imaging experiment result after the cardiovascular implant in the present invention is transplanted;

Fig. 7 is the internal morphology (scanning electron microscope observation) of two kinds of responsive hydrogels in the present invention;

Fig. 8 is the cytotoxicity test result of the hydrogel in the present invention;

Fig. 9 is the cytotoxicity test result of Axon Guidance Molecule-1 and DENND1A in the present invention;

Fig. 10 is the experimental result of hydrogel sustained-release protein of the present invention;

Figure 11 is a graph showing the results of changes in ROS levels in the vascular implants of the present invention;

Figure 12 is the appearance of the cardiovascular graft in the present invention (30d after implantation);

Figure 13 is a graph showing the results of immunofluorescence staining of nerves (cross-sections) in the vascular implant of the present invention (PGP9.5 is a nerve marker);

Figure 14 is a graph showing the results of immunofluorescence staining of nerves (longitudinal sections) in the vascular implant of the present invention (PGP9.5 and beta-tublin3 are nerve markers);

Figure 15 is a graph showing the results of blood flow rate of the cardiovascular graft in the present invention (30d after implantation);

Fig. 16 is the result picture of the H&E staining section of the cardiovascular graft in the present invention (30d after implantation, the dotted line in the enlarged image is the boundary line of each layer of membrane);

Figure 17 is a graph showing the results of immunofluorescence staining of cardiovascular grafts in the present invention (CD31 is an endothelial cell marker);

Figure 18 is a graph of the detection results of the ATP content of the cardiovascular graft in the present invention;

Figure 19 is a graph showing the results of Prdx-1 immunofluorescence staining of cardiovascular grafts in the present invention;

Figure 20 is the appearance of the cardiovascular graft in the present invention (60d after implantation);

Figure 21 is a graph showing the results of blood flow velocity of the cardiovascular graft in the present invention (60d after implantation);

Fig. 22 is a graph showing the results of Masson staining of the cardiovascular graft in the present invention. (60d after implantation, the dotted line in the enlarged image is the boundary line of each layer of film);

Figure 23 is an immunohistochemical staining image of the cardiovascular graft of the present invention (60d after implantation, the position indicated by the arrow shows the smooth muscle cells of intimal hyperplasia);

Figure 24 is the appearance of the cardiovascular graft in the present invention (90d after implantation);

Figure 25 is a result diagram of blood flow velocity in the present invention (90d after implantation);

Figure 26 is a CTA image result of the cardiovascular graft in the present invention (the red arrow indicates the transplanted cardiovascular graft);

Figure 27 is a graph showing the results of H&E staining of the cardiovascular graft in the present invention (90d after implantation, the dotted line in the enlarged image is the boundary line of each layer of membrane);

Fig. 28 is a graph showing the results of Masson's staining section of the cardiovascular graft in the present invention; (90d after implantation, the dotted line in the enlarged image is the boundary line of each layer of membrane);

Figure 29 is a graph showing the results of immunofluorescence staining of cardiovascular grafts in the present invention (Calponin is a differentiated smooth muscle marker, and Thrombospondin is a dedifferentiated smooth muscle marker);

Figure 30 shows the results of immunohistochemical staining of type 1 collagen of the cardiovascular graft of the present invention.

Detailed ways

Below in conjunction with accompanying drawing and embodiment, the present invention is described in further detail:

Embodiment 1: a preparation method of a small-caliber cardiovascular implant, as shown in Figure 1 and Figure 2, the steps are as follows:

(1) Construction of small-caliber cardiovascular implant body;

1) Take the small-diameter blood vessels from the same or different sources. The small-diameter blood vessels in this example are obtained from the carotid artery of rats and processed. First, the blood vessels obtained from the materials are cut into small sections of 0.5-1 cm, and the blood vessels are carried out in normal saline. Rinse, wash away the blood involved in it.

2) Carry out decellularization and removal of residual nucleic acid: use 0.025%~0.05% trypsin for 40min at 0℃~37℃; remove nucleic acid with RNase and DNase, and shake the reaction at 0℃~37℃ for 10min~45min , complete the nucleic acid removal, and obtain the vascular matrix material.

3) The vascular matrix material is modified with 10%-50% collagen, and then cross-linked with vascular growth factor (100-500ng/ml, in vitro) at 0℃～37℃ for 12～36h.

4) Then ligate both ends of the small-diameter vascular implant, and use sterile filter paper to blot the surface dry to obtain the cardiovascular implant body.

(2) Preparation of ATP-responsive molecules and ROS-responsive molecules, the steps are as follows:

1) ATP-responsive molecules are formed by reacting ATP aptamer, ssDNA1 and ssDNA2 in a ratio of 1:1:1 to 1:2:2 at 0°C to 50°C for 6h to 24h; The sequence (SEQ ID NO: 1) is CACCTGGGGGAGTATTGCGGAGGAAGGTT; the sequence (SEQ ID NO: 2) of ssDNA1 is 5'-ACTCCCCCAGGTGTTTTTT-NH2-DBCO-3'; the sequence (SEQ ID NO: 3) of ssDNA2 is 5'-DBCO- NH ₂ -TTTTTAACCTTCCTCCGC-3';

2) The ROS response molecule is aminothio ketal and dibenzocyclooctyne (DBCO) according to the molar mass ratio of 1:1～1:3, and the reaction is stirred at 0℃～75℃ for 30min～2h.

(3) Preparation of ATP-responsive hydrogel mixture using eight-arm PEG azide, ATP-responsive molecule and DENND1A protein; preparation of ROS-responsive water using eight-arm PEG azide, ROS-responsive molecule and axon guidance molecule-1 gel mixture;

1) The preparation steps of the ATP-responsive hydrogel mixture are as follows: Mix the eight-arm PEG azide and the ATP-responsive molecule according to a molar mass ratio of 1:3 to 1:5, and then add 1% to 10% of DENND1A protein at room temperature. The gel can be formed by reacting for 5-20 minutes;

2) The preparation method of the ROS-responsive hydrogel is as follows: the eight-arm PEG azide and the ROS-responsive molecule are mixed in a molar mass ratio of 1:3 to 1:5, and then mixed with 1% to 10% of the axon guidance molecule-1. , the gel can be formed by reacting at room temperature for 5-20 min.

(4) The ATP-responsive hydrogel mixture and the ROS-responsive hydrogel mixture were sequentially gelled on the surface of the cardiovascular implant body to obtain a small-diameter cardiovascular implant; the ATP-responsive hydrogel mixture and the ROS-responsive hydrogel mixture The specific steps of forming the gelatinous hydrogel mixture on the surface of the cardiovascular implant body in turn are as follows:

a. First ligate and seal both ends of the cardiovascular implant body with 1-0 surgical thread, absorb the surface water, and then immerse it into the ATP-responsive molecule, BSA-Alexa568 (red fluorescence, 10 μg ~ 100 μg/ml) Then take it out, cover the surface of the cardiovascular implant body with a thin layer of mixed solution, and cover the outer surface of the cardiovascular implant body with a thin layer of mixed solution I, and wait for the thin layer to mix Liquid Ⅰ is reacted at 0℃～37℃ for 15～45min to form gel;

b. The cardiovascular implant body is then immersed in a mixture containing ROS responsive molecules, FITC (green fluorescence, 10-100 micrograms/ml) and axon guidance molecule-1, and then taken out to make the cardiovascular implantation The surface of the object body is covered with a thin layer of mixed solution II, and after the thin layer of mixed solution II is reacted at 0°C to 37°C for 15 to 45 minutes to form a gel, the surgical wires at both ends of the vascular implant are untied, that is, in the vascular implant. A bilayer responsive hydrogel was formed on the surface of the body to obtain a small-bore cardiovascular implant. Small-bore cardiovascular implants are tubular, with a diameter of 0.5 to 4 mm and a length of 0.5 to 20 cm.

Analysis of results: After decellularization of the rat common carotid artery (1 cm in length, 1 mm in diameter), the cells (blue) were removed, and the collagen (red) was completely retained. Figure 2 shows the binding of the inner and outer hydrogels encapsulating fluorescent molecules to the outer surface of the outer membrane. The double-layered gel structure of the blood vessel cross-section was observed under a fluorescence microscope, demonstrating that the inner layer of ATP-responsive hydrogel containing BSA-FITC and the outer layer of ROS-responsive hydrogel containing BSA-Alexa568 adhered to the decellularized vascular matrix. on the outer membrane.

The present invention detects the properties of the prepared small-caliber cardiovascular implant, and the detection results are as follows:

(1) After freeze-drying the eight-arm PEG azide, ROS-responsive hydrogel and ATP-responsive hydrogel, the characteristic absorption bands of their functional groups were measured by FT-IR spectroscopy. Figure 3 shows that the characteristic absorption at 2105 of the azide group in the eight-arm PEG disappears after the addition of the two crosslinkers separately, indicating that the alkyne-azide reaction was successful in both hydrogels of.

(2) Determination of the response ability and sustained release ability of the hydrogel in vitro. Prepare 500 μl of ROS-responsive hydrogel at the bottom of a 5 ml EP tube and take pictures. After adding 200 μl of 10 mM H ₂ O ₂ and reacting at 37° C. for 2 h, the EP tube was inclined at about 60° and then photographed. ROS-responsive hydrogels were prepared in 48-well plates (200 μl of hydrogel per well, containing 10 mg/ml BSA). The control group was treated with deionized water (100 μl per well), while the experimental group was treated with hydrogen peroxide (100 μl per well, 3 mm). Then, the BSA concentration in the supernatant was measured for 0-30 days using a BSA-ELISA detection kit.

The results shown in Figure 4 and Figure 10 show that the monomer formed a hydrogel at 25°C, and the hydrogel became liquid again after the addition of H ₂ O ₂ . Therefore, ROS-responsive hydrogels containing BSA _were treated with ₃ mM H2O2. _The daily release rate of BSA in the H2O2 group was approximately 3.6% ± 0.7% within 30 days, which indicated a sustained response and stable release of _hydrogen peroxide from the hydrogel.

(3) Determination of protein stability in hydrogels. During the preparation of small-bore vessels modified with responsive hydrogels, BSA-FITC and BSA-Alexa 568 were incorporated into ATP- and ROS-responsive hydrogels, respectively. Vascular implants were soaked in PBS. We then cut 8 μm frozen tissue sections on days 0, 9, 14, 21 and 30. Images were taken by fluorescence microscopy. The small-diameter blood vessels modified with hydrogel Dir (Dir, 300 μg/ml, Retai Biotech) were implanted into rats. On days 0, 9, 14, 21 and 30, rats were anesthetized and neck skin preparations were performed and imaged with IVIS spectroscopy (Perkinlemer). Filter parameters were set to 760 nm emission and 710 nm excitation.

The results are shown in Figure 5 and Figure 6. After a period of observation, the hydrogel-modified small-bore cardiovascular implant had strong green fluorescence on the 9th and 14th days, and on the 30th day. This fluorescence remained unchanged in vitro and in vivo, indicating that the hydrogel could maintain sustained release in vivo for more than 1 month, thus providing sufficient time for nerve fibers to grow into small-bore vascular implants.

(4) Pore structure and cytotoxicity of hydrogels. The two responsive hydrogels were frozen and fractured by immersion in liquid nitrogen. After gold spraying, the internal structure of the hydrogel was observed by scanning electron microscope (SEM, Zeiss). Human umbilical vein endothelium (1×104 per well) was inoculated in a 96-well culture plate. Neuronal cells cultured for 1 and 3 days were assessed for 24h gel hydrogels (30, 50 and 100 mg/ml, 10 μl per well), Axon Guidance Molecules-1 and DENND1A using the MTT assay kit (Trevigen). Cytotoxicity. Absorbance at 570 nm was measured using a microplate reader.

The results are shown in Fig. 7, Fig. 8, Fig. 9, the hydrogel synthesized by the present invention has a uniform loose structure for increasing its response contact area and promoting the ingrowth of nerve fibers. PEG hydrogels showed little cytotoxicity to sensitive neurons, and Axon Guidance Molecule-1 and DENND1A did not show significant cytotoxicity even when their concentrations reached 500 ng/ml and 2000 ng/ml, respectively.

The present invention also detects and observes that the small-caliber cardiovascular implant maintains normal intimal shape and blood flow for a long time under the diabetic environment, and the results are as follows:

(1) Small-caliber vascular implants were implanted in Sprague-Dawley rats. All animal manipulations and experiments were reviewed and approved by the Army Medical University Animal Ethics Committee. Female Sprague-Dawley (SD) rats (equivalent, 230-250 g) were anesthetized with 1% sodium pentobarbital (ip, 40 mg/kg). The room temperature was kept at 20°C. The common carotid artery of the rat was clipped on both sides and incised in the center, then the incised ends on both sides were passed through the homemade cuff and turned over. Then, both ends of the prepared small-caliber vascular implant (length: 1 cm, diameter: 1 mm, immersed in 50 U/ml heparin sodium) were anastomosed with the end of the carotid artery. Ligate the vascular seam at the cuff and release the vascular clip. After recovery, the rats were intraperitoneally injected with heparin sodium (1000U/kg, Aladdin) for 4 days. On the 15th day, an experimental diabetic rat model was established by injecting streptozotocin. The rats were divided into the following 5 groups according to the response system and functional factors included in the hydrogel of small-bore vascular implants: Group 1: control group (blank gel + blank gel); Group: gel Netrin1+gel-DEND1A; Group 3: ROS gel-Netrin1+empty gel; Group 4: ROS-gel-Netrin1+gel-DENND1A; Group 5: ROS-gel-Netrin1+ATP gel- d1A.

(2) On days 1, 3, 7, and 14, the small-bore vascular implants were removed in the control group, and then decellularized using 0.1% SDS for 2 h at room temperature. After 5 washes with PBS, it was homogenized in a glass homogenizer (150 μl/cm). ROS content in vascular implants was detected using a ROS detection kit. The results in Figure 14 show that reactive oxygen species are generated due to inevitable local inflammation, and these ROS can trigger the release of axon-guiding molecule-1 from the outer hydrogel of small-caliber vascular implants implanted in diabetic rats.

(3) Small-bore vascular implants were labeled with anti-PGP9.5 and anti-β-tubulin 3 antibodies to detect nerve fibers growing in the adventitia 30 days after implantation. Statistical analysis was performed on the fluorescence signals of each group. As shown in Figure 12 and Figure 13, on the 30th day after implantation, a large number of nerve-specific markers PGP9.5 and β3-tubulin were seen in the adventitia of the small-caliber vascular implants in group 3, confirming that the nerve fibers had been repaired and reconstructed. The number of nerve fibers in group 3 was significantly increased. Compared with group 2, group 1 of small-bore vascular implants not modified with Axon Guidance Molecules-1 had no nerve fibers. The nerve fiber density was highest in group 3.

(4) On days 30, 60 and 90, the rats were anesthetized and neck skin preparations were performed. Coat the rat neck evenly with ultrasonic gel. For color Doppler ultrasound (Esaote), the frequency was set to 13.0 MHz, the depth was set to 2.5 cm, the mechanical index was set to M10.4, and the gray scale was set to 46. After several consecutive cardiac cycles were observed, the Doppler mode was selected to acquire the ultrasound spectrum of the common carotid artery, and the images were saved. Measurement of peak carotid velocity and blood flow with small-bore cardiovascular implants. Figure 15 and Figure 16 show that, excluding the control group, on the 30th day, the blood flow velocity of the other four groups was higher than 80.31±6.24cm/s.

(5) H&E and Masson's trichrome staining of paraffin sections. Following the above experiments, small bore cardiovascular implants were fixed with 4% paraformaldehyde for 3 hours at 4°C and then placed in 30% sucrose overnight. Subsequently, small-bore cardiovascular implants were embedded in paraffin, sectioned (5 μm thick), deparaffinized in xylene and rehydrated in an ethanol gradient, followed by Masson’s trichrome staining (60 days, 90 days) , H&E staining (30 days, 60 days, 90 days), and immunohistochemistry and immunofluorescence staining. Figures 17 and 18 show that the small-bore cardiovascular implants completed endothelialization in all groups and also formed feeder vessels on the vessels. The ROS-responsive axon guidance molecule-1 release system plays an important role in inducing the integration of nerve fibers into small-bore cardiovascular implants. Ensuring early open endothelialization of small-bore cardiovascular implants is the basis for further research on the relationship between nerve remodeling and VSMC function.

(6) On day 30, the small bore cardiovascular implants of each group were removed and processed into tissue homogenates as above. Prepare the working solution and mix with the vascular tissue homogenate at a ratio of 1:1 according to the instructions of the ATP assay kit. Luminescence intensity (ATP content) was measured by a microplate reader with a photometer function. The results in Figure 19 show that after implantation on day 30, the small-bore cardiovascular implants in groups 3, 4, and 5 had higher ATP content and correlated with nerve fiber density. This result suggests that innervating small-bore cardiovascular implants contain sufficient ATP to initiate degradation of the internal hydrogel and release of DEND1A.

(7) For immunofluorescence staining, small bore cardiovascular implants were fixed with 4% paraformaldehyde for 1 hour at 4°C. After washing with PBS, permeabilization and blocking were performed with 0.125% Triton X-100 (Solarbio, 20 min, room temperature) and 10% goat serum (Solarbio, 1 h, room temperature), respectively. The samples were then incubated overnight at 4°C with primary antibodies, including mouse anti-active Rab35 (Abeam, 1:500), rabbit anti-β3-tubulin (Abeam, 1:500), mouse anti-α-SMA ( Boster, 1:300), rabbit anti-CD-31 (Abcam, 1:500), mouse anti-α-SMA (Boster, 1:300), rabbit anti-prdx-1 (KleanAB, 1:500) and mouse anti- PGP9.5 (Bioss, 1:200). Alexa-Fluor 488- and Alexa-Fluor 568 conjugated antibodies (Invitrogen) were used as secondary antibodies. After incubation with primary antibody, cells and frozen sections were incubated with secondary antibody for 30 min at 37°C. Nuclei were stained with 4',6-diamino-2-phenylindole (DAPI, Invitrogen) for 15 minutes at room temperature. Confocal microscopy images were acquired using a Leica SP8 confocal microscope and analyzed using ImageJ.

Immunohistochemical staining was performed on 5 μm paraffin sections of small-bore cardiovascular implants (days 60 and 90). After routine deparaffinization and hydration, TEVG sections _were incubated with ₃ % H2O2 for 10 min at room temperature. After washing with PBS, infiltrate and block as described above, then incubate sections with primary mouse anti-α-SMA (Boster, 1:300) or anti-COL1 (Bioss, 1:200) antibodies in a humidified chamber at 4°C placed for 48h. HRP IgG goat anti-rabbit antibody (Invitrogen, 1:800) was used as secondary antibody (1 h at 37°C). Diaminobenzidine (DAB, Boster) was used for color development, followed by hematoxylin (Boster) immunohistochemical images were obtained with light microscopy (Olympus) and analyzed with ImageJ.

The results in Figure 20 show that there was more Prdx-1 on the small-bore cardiovascular implants in group 5 than the small-bore cardiovascular implants in groups 4 and 3, which demonstrated the inner layer of hydrocoagulation. Gel degradation leads to DENND1A promoting neuroexosome secretion under hyperglycemic environment in vivo. Figures 22, 23, and 24 show that, 60 days after implantation, small-bore cardiovascular implants in groups 1 and 2 had more severe intimal hyperplasia and necrosis, decreased blood flow velocity, and increased intima-media ratio to 2.33±0.21 and 0.92±0.085. In contrast, groups 3, 4, and 5 had small-bore cardiovascular implants with a blood flow velocity greater than 76.25 ± 6.47 cm/s, mild intimal hyperplasia, IMTR below 0.51 ± 0.047, and smooth muscle cell migration to small-bore cardiovascular implants material and keep it unobstructed.

Masson staining test showed that the proportion of intimal collagen fibers in groups 1 and 2 exceeded 25.7 ± 2.1%, while the proportion of collagen fibers in groups 3, 4 and 5 decreased to a maximum of 18.6 ± 1.9%, It is suggested that nerve reconstruction inhibits the proliferation of vascular smooth muscle cells and the secretion of collagen fibers. In particular, the proportion of intimal collagen in group 5 was only 15.8 ± 1.3%, indicating that DENND1A-promoted neural exosomes could inhibit the secretion of collagen from vascular smooth muscle cells in the neointima. At day 60, the small-bore cardiovascular implants in groups 3, 4, and 5 nearly completed vascular smooth muscle remodeling, which was positively correlated with nerve fiber density within the small-bore cardiovascular implants. However, groups 3 and 4 had some degree of intimal hyperplasia. At the same time, a large number of chaotically arranged vascular smooth muscle cells were also observed in the thickened neointima in the small-bore cardiovascular implants of groups 1 and 2.

(8) Figures 26 to 30 show the properties and structures of each group of small-diameter implants when transplanted for 90 d. Aneurysm production was observed in group 1 small-bore cardiovascular implants 90 days after surgery, likely due to the inability of small-bore cardiovascular implants to resist blood pressure, which lacked vascular smooth muscle remodeling. H&E staining and carotid ultrasound scans revealed severe occlusion of Group 1 and Group 2 small-bore cardiovascular implants in rats. CT angiography (CTA) results showed the blood flow status of the carotid arteries and showed that the stenosis of the small-bore cardiovascular implants in groups 1 and 2 was more severe than in the other groups. From the 30th day, the intimal hyperplasia and lumen obstruction of the small-caliber cardiovascular implants in group 3 gradually increased, the blood flow velocity decreased to 41.90±5.22cm/s on the 90th day, and the IMTR was 0.74±0.087. Changes in blood flow velocity and IMTR were smaller than those in the control group. Few significant intimal hyperplasia was observed in group 5. At the 90th day, the proportion of collagen fibers in the intima of group 5 was still 15.8±1.3%, which was significantly lower than that of the other four groups. These results suggest that DENND1A-activated neuroexosome secretion enhances the ability of small-bore cardiovascular implants to resist intimal hyperplasia and maintain long-term patency. In addition, the reconstituted vascular smooth muscle cells in the small-bore cardiovascular implants of group 3 had a distinct synthetic phenotype, and a large amount of type I collagen was also present in the vascular intima. In contrast, group 4 had significantly higher contractile phenotypic markers and significantly lower collagen type I content in smooth muscle cells within the vascular implants. In group 5, vascular smooth muscle cells showed a distinct contractile phenotype, and the secretion of type I collagen was normal. All these results suggest that small-bore cardiovascular implants and on-demand programmable bilayer response release systems can improve neuromodulation of smooth muscle cells within vascular implants under hyperglycemic conditions, reduce hyperglycemia injury, and prevent vascular smooth muscle cells Abnormal phenotypic transformation and inhibition of vascular intimal hyperplasia to promote long-term patency of small-diameter implants under diabetes.

This specific embodiment is only an explanation of the present invention, and it does not limit the present invention. Those skilled in the art can make modifications without creative contribution to the present embodiment as needed after reading this specification, but as long as the rights of the present invention are used All claims are protected by patent law.

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<120> A small-caliber cardiovascular implant and its preparation method

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Claims (10)

1. a small-caliber cardiovascular implant, characterized in that: comprising a cardiovascular implant body and a double-layer responsive gel incubated on the outer surface of the cardiovascular implant body; the double-layer responsive gel is The inner layer is an ATP-responsive hydrogel, and the ATP-responsive hydrogel contains DENND1A protein; the outer layer of the bilayer-responsive gel is a ROS-responsive hydrogel, and the ROS-responsive hydrogel contains Axon Guidance Molecule-1. 2. a kind of small-caliber cardiovascular implant according to claim 1, is characterized in that, the preparation method of described ATP response hydrogel is: by eight-arm PEG azide and ATP response molecule according to molar mass ratio Mix at 1:3～1:5, then mix in 1%～10% DENND1A protein, and react at room temperature for 5～20min to form gel; The preparation method of the ROS-responsive hydrogel is as follows: the eight-arm PEG azide and the ROS-responsive molecule are mixed in a molar mass ratio of 1:3 to 1:5, and then mixed with 1% to 10% of axon guidance molecule-1 , the gel can be formed by reacting at room temperature for 5-20 min. 3 . The small-diameter cardiovascular implant according to claim 1 , wherein the small-diameter cardiovascular implant is tubular, with a diameter of 0.5-4 mm and a length of 0.5-20 cm. 4 . 4. a preparation method of small-caliber cardiovascular implant, is characterized in that, step is as follows: (1) Construction of small-caliber cardiovascular implant body; (2) Preparation of ATP-responsive molecules and ROS-responsive molecules; (3) Preparation of ATP-responsive hydrogel mixture using eight-arm PEG azide, ATP-responsive molecule and DENND1A protein; preparation of ROS-responsive water using eight-arm PEG azide, ROS-responsive molecule and axon guidance molecule-1 gel mixture; (4) The ATP-responsive hydrogel mixture and the ROS-responsive hydrogel mixture are sequentially gelled on the surface of the cardiovascular implant body to obtain a small-diameter cardiovascular implant. 5. the preparation method of a kind of small-caliber cardiovascular implant according to claim 4, is characterized in that, the preparation method of the cardiovascular implant body described in step (1) is as follows: First, use decellularization solution to remove cells in the isolated blood vessels; then use nuclease to remove nucleic acids from the isolated blood vessels to obtain a vascular matrix material; then incubate collagen and vascular endothelial growth factor (VEGF) on the surface of the vascular matrix material ( VEGF) to obtain the cardiovascular implant body. 6. the preparation method of a kind of small-caliber cardiovascular implant according to claim 5, is characterized in that, the concrete preparation step of described cardiovascular implant body is: A. The decellularization solution is 0.025%～0.25% trypsin, and the decellularization solution and the isolated blood vessel are shaken and reacted at 0℃～37℃ for 30min～90min to complete decellularization; the nuclease is DNase and RNase, the nuclease reacts with the isolated blood vessel at 0°C to 37°C for 10 min to 45 min to complete the denuclease and obtain the blood vessel matrix material; B. After washing the vascular matrix material with PBS solution, incubate 10%-50% collagen on the surface of the vascular matrix material; )) cross-linking for 12-36 h to obtain the cardiovascular implant body. 7. the preparation method of a kind of small-caliber cardiovascular implant according to claim 4, is characterized in that: the described ATP response molecule of step (2) is by ATP nucleic acid aptamer, ssDNA1 and ssDNA2 according to 1:1 The ratio of :1～1:2:2 is formed by reacting at 0℃～50℃ for 6h～24h. 8. the preparation method of a kind of small-caliber cardiovascular implant according to claim 7, is characterized in that: the sequence of described ATP nucleic acid aptamer is CACCTGGGGGAGTATTGCGGAGGAAGGTT; The sequence of described ssDNA1 is 5'-ACTCCCCCAGGTGTTTTTT-NH ₂ -DBCO-3'; the sequence of the ssDNA2 is 5'-DBCO- _NH2 -TTTTTTTAACCTTCCTCCGC-3'. 9. the preparation method of a kind of small-caliber cardiovascular implant according to claim 4, is characterized in that: the ROS response molecule described in step (3) is aminothioketal and dibenzocyclooctyne (DBCO ) according to the molar mass ratio of 1:1 to 1:3, at 0°C to 75°C, and stirred for 30 minutes to 2 hours. 10. The preparation method of a small-diameter cardiovascular implant according to claim 4, wherein the ATP-responsive hydrogel mixed solution and the ROS-responsive hydrogel mixed solution described in step (4) are sequentially The specific steps of forming glue on the surface of the cardiovascular implant body are as follows: a. First tie both ends of the cardiovascular implant body with 1-0 surgical thread, then immerse it in the ATP-responsive hydrogel mixture, and then take it out to cover the surface of the cardiovascular implant body A thin layer of mixed solution I, waiting for the thin layer of mixed solution I to react to form a gel; b. The cardiovascular implant body is then immersed in the ROS-responsive hydrogel mixture, and then taken out, so that the surface of the cardiovascular implant body is covered with a thin layer of mixture II, waiting for the After the thin-layer mixed solution II reacted to form a gel, the surgical threads at both ends of the vascular implant were untied.

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