CN113331990B

CN113331990B - Drug-loaded elastic degradable artificial blood vessel and construction method thereof

Info

Publication number: CN113331990B
Application number: CN202110436446.9A
Authority: CN
Inventors: 吴炜; 陈婉丽; 张思倩
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2021-04-22
Filing date: 2021-04-22
Publication date: 2023-04-07
Anticipated expiration: 2041-04-22
Also published as: CN113331990A

Abstract

The invention relates to a drug-loaded elastic degradable artificial blood vessel and a construction method thereof, and the steps are as follows: preparing a PGS tube core by using a sodium citrate tubular salt mold to form a PGS polymer-sodium citrate template; mixing the collagen spinning solution loaded with the drug and the PCL spinning solution by an electrostatic spinning technology and winding the mixture on a PGS polymer-citric acid sodium salt template to form a drug-loaded mixed sheath layer; the tubular stent is slightly atomized by the atomizer to obtain the elastic degradable vascular stent convenient for suturing, and the anticoagulation of the transplant in the PGS porous stent, which is rich in sodium citrate, is ensured. In the invention, sodium citrate is used as a salt mold pore-forming agent to replace NaCl, the use of the sodium citrate avoids the drug-loading loss caused by a water washing step in the preparation process before the implant is implanted, and meanwhile, because the sodium citrate has anticoagulation property, the sodium citrate is beneficial to preventing the formation of blood clots at the side of the lumen after the implant is implanted, and is beneficial to maintaining the patency of the implant to a certain extent.

Description

Drug-loaded elastic degradable artificial blood vessel and construction method thereof

Technical Field

The invention relates to the technical field of biomedical equipment, in particular to a drug-loaded elastic degradable artificial blood vessel and a construction method thereof.

Background

Vascular disease is the leading disease leading to death, both in developed and developing countries. The progression of the disease is often associated with narrowing or obstruction of the blood vessel, ultimately leading to inadequate blood supply and tissue damage. Common vascular diseases are coronary heart disease, cerebrovascular disease, peripheral arterial disease and deep vein thrombosis. Common vascular procedures for treating vascular disease may include endovascular procedures such as angioplasty, stenting, or atherectomy to enlarge a stenosed vessel or to clear an obstruction. In addition, a vascular graft may be used to replace or establish a collateral bypass around the site of injury or occlusion. Despite the great advances and increasing popularity of endovascular surgery in recent decades, vascular grafting remains common and is considered the best choice for patients who need to rebuild long-term blood transport solutions. At present, the preferred graft for vascular grafting is an autologous artery or vein. However, autologous veins are limited in clinical use due to their limited source, possibly poor quality, and their availability which may also contribute to certain donor site morbidity.

In addition to autologous vessels, clinically synthetic vascular grafts have also been shown to be satisfactory replacements for damaged vessels, and these grafts have shown satisfactory long-term efficacy at large diameters (> 8 mm), e.g. aortic iliac substitutes can achieve patency levels of around 90%. However, in small diameter vessels (< 6 mm), the use of synthetic vascular grafts is very limited due to the low patency rate. In small-caliber blood vessels such as coronary arteries, the inguinal artery (below the inguinal ligament), and the tendon sheath artery (below the knee), the effect of grafting autologous blood vessels has been demonstrated to be superior to synthetic blood vessels. In coronary artery bypass grafting, the patency rate of 1 year using a Polytetrafluoroethylene (PTFE) catheter is 60%, while the patency rate of over 95% using autologous vein grafting. After 2 years, the patency rate of the polytetrafluoroethylene implant is reduced to only 32%, and the patency rate of the autologous vein implant is still kept above 90%. In the collateral bypass operation established above the knee joint, the result shows that the patency rate of 5-year PTFE transplantation is only 59%, while 78% can be reached using autologous veins. Synthetic vascular grafts will only be selected if no other suitable autologous vessels are available.

Failure of vascular grafts is often associated with thrombosis, intimal hyperplasia, atherosclerosis, or infection. In view of the limitations of the existing blood vessel grafts, the construction of an ideal blood vessel graft suitable for small-caliber artery transplantation is of great significance for vascular surgery. The ideal blood vessel stent can reflect the biomechanical characteristics of blood vessels, can be used as a platform for cell attachment and proliferation, and can regenerate a functional structure similar to a natural blood vessel in vivo. The composite artificial blood vessel transplant which is formed by taking a fast degradable elastomer PGS (glycerol sebacate) as an inner layer stent and preparing a PCL (polycaprolactone) spinning sheath layer from an outer layer through an electrostatic spinning technology can realize short-term fast endothelialization and full-layer muscular remodeling in a rat body to form a new artery structure similar to a natural artery. However, the transplant is extremely dependent on the regeneration potential of the recipient, and when the transplant is transplanted into large animals such as sheep and dogs, the transplant has low patency rate, and the reconstruction effect is not ideal, so that a great gap exists from clinical application. In order to further improve the patency and reconstruction success rate of the elastically degradable vascular graft in a large animal body, the graft is subjected to loading of bioactive factors and related drugs in order to obtain more enhanced biological functions. Since PGS is rapidly degraded in vivo within two weeks, loading the drug to the PCL sheath becomes the only option in order to obtain a long-term drug sustained release effect. However, in the preparation process of the porous PGS inner layer scaffold, naCl is generally used as a pore-forming agent in the salt mold method, and due to the procoagulant property of NaCl itself, complete water washing and desalting treatment are required after the preparation of the PGS-PCL spinning sheath, thereby preventing NaCl from remaining in the scaffold. Meanwhile, before the implant is implanted, the heparin anticoagulation treatment of the material is required. The two steps are easy to cause the loss of the drug load of the PCL, and are not beneficial to the exertion of the drug effect. Drug loading of elastic degradable vascular stents remains an unsolved technical problem.

Disclosure of Invention

The invention aims to provide a drug-loaded elastic degradable artificial blood vessel and a construction method thereof, overcomes the defects of the prior art, and solves the problem of low drug loading efficiency on a rapid degradable elastomer.

The technical scheme adopted by the invention is as follows:

the construction method of the drug-loaded elastic degradable artificial blood vessel is characterized by comprising the following steps:

the method comprises the following steps:

the method comprises the following steps: preparing a PGS tube core by using a sodium citrate tubular salt mold to form a PGS polymer-sodium citrate template;

step two: mixing the collagen spinning solution loaded with the drug and the PCL spinning solution by an electrostatic spinning technology and winding the mixture on a PGS polymer-citric acid sodium salt template to form a drug-loaded mixed sheath layer;

step three: the tubular stent is slightly atomized by the atomizer to obtain the elastic degradable vascular stent convenient for suturing, and the anticoagulation of the transplant in the PGS porous stent, which is rich in sodium citrate, is ensured.

The first step is specifically as follows:

guiding the sodium citrate salt particles into a mould by utilizing a circular membrane with a uniform columnar tube core, uniformly vibrating to enable the salt particles to be tightly filled into gaps, carrying out atomization treatment and then guiding out a salt tube to manufacture a tubular salt mould consisting of the sodium citrate salt particles;

preparing a 10% and 15% mixed solution of a polytrimethylene sebacate prepolymer by using a tetrahydrofuran solution, dripping the 15%, 10% and 15% mixed solution on a sodium citrate tubular salt mold in sequence, and after the sodium citrate tubular salt mold is completely saturated, putting a PGS prepolymer-sodium citrate tubular salt mold mixture into a vacuum drying oven to form a PGS polymer-sodium citrate tubular stent.

The second step is specifically as follows:

dissolving PCL granules in 2,2,2-trifluoroethanol to obtain 14% PCL-trioxymethylene solution;

dissolving collagen in hexafluoroisopropanol to obtain 8% collagen-hexafluoroisopropanol solution, and mixing with polypeptide solution with concentration of 2.0mg/mL at volume ratio of 4:1;

the two injectors respectively extract a PCL-trivolvulum ethanol solution and a collagen-hexafluoroisopropanol solution containing polypeptide, and then respectively fix the PCL-trivolvulum ethanol solution and the collagen-hexafluoroisopropanol solution on the two injection pump motors;

putting the two motors and the salt template into an electrostatic spinning system, aligning the salt template to the center of the receiving sheet, enabling the salt template to be vertical to a spinning needle head and enabling the salt template to be parallel to the ground;

turning on the power supply of the rotating motor and the switch of the injection pump, and mixing and electrospinning the PCL solution and the collagen polypeptide mixed solution on a PGS sodium citrate inner core rotating at the rotating speed of 120RPM for 3 minutes at the speed of 2.5ml/h and the speed of 1 ml/h.

The third step is specifically as follows:

atomizing the composite tubular object containing the PCL-drug-loaded collagen sheath and the PGS-citric acid sodium salt model scaffold for a short time to obtain a state which is relatively easy to suture, carrying out ultraviolet disinfection and sterilization, and storing at-80 ℃ for later use.

The drug-loaded elastic degradable artificial blood vessel constructed by the method.

The drug-loaded elastic degradable artificial blood vessel is of a double-layer elastic vascular composite structure and sequentially comprises a PGS citric acid inner tube core and a drug-loaded mixed spinning sheath layer from inside to outside.

The PGS sodium citrate tube core is a tubular structure made of polysebacate glycerol ester material on the basis of taking sodium citrate as a salt molding pore agent.

The mixed spinning sheath is formed by PCL spinning and drug-loaded collagen spinning through an electrostatic spinning and blending technology.

The inner diameter of the drug-loaded elastic degradable artificial blood vessel is 1mm, and the outer diameter of the drug-loaded elastic degradable artificial blood vessel is 1.5mm.

The thickness of the drug-loaded spinning sheath is 20-30 μm.

The invention has the following advantages:

1. the sodium citrate is used as the anticoagulant in the preparation process of the vascular graft for the first time, the sodium citrate is used as the hole making agent for replacing NaCl in a salt mold method, the use of the sodium citrate avoids the drug loading loss caused by a water washing step in the preparation process before the graft is implanted, and meanwhile, due to the anticoagulation property of the sodium citrate, the sodium citrate is favorable for preventing the formation of blood clots on the side of a lumen after the graft is implanted into the body, and is favorable for keeping the smoothness of the graft to a certain extent.

2. The invention obtains the in vivo anticoagulation, instant degradation and biological activity blood vessel transplant with high-efficiency polypeptide slow-release function by constructing the double-layer elastic blood vessel composite home material of PGS sodium citrate tube core-PCL collagen drug-carrying sheath layer, and obtains the improvement effect superior to the prior artificial blood vessel material in the animal body. The intravascular stent drug-loaded platform can be loaded with various bioactive factors and drugs, and lays a foundation for subsequent application in the research of a vascular regeneration mechanism and clinical research.

3. The invention greatly reduces the loss of the loaded drug in the preparation process, and the activity of the drug is well reserved.

Drawings

FIG. 1 is a schematic diagram of preparation of PGS sodium citrate tube core-PCL collagen drug-carrying sheath stent material.

FIG. 2 is a schematic diagram of the step of blending the PCL collagen drug-loaded sheath layer by electrostatic spinning.

FIG. 3 is a scanning electron micrograph.

Wherein, (a) is the cross section of the bracket, (b) is the junction of the spinning layer and the internal PGS sodium citrate, and (c) is the microscopic representation of the scanning electron microscope of the spinning sheath layer under the high power lens; (d) is a general diagram of the stent material; (e) is an atomic force microscope image of the spinning sheath; (f) The mechanical comparison of the PGS sodium citrate scaffold with the general PGS salt-model scaffold was performed.

Fig. 4 is a graph of the in vitro release profile of the drug-loaded stent and the fourier spectrometer spectral analysis of the drug-loaded stent.

Wherein, (a) is a drug-loaded stent in vitro sustained-release curve; (b) Is a Fourier spectrometer spectrum analysis chart of the drug-loaded stent.

FIG. 5 is a comparative illustration of Sca-1+ vascular precursor cells after implantation in two groups of blood vessels.

Wherein, (a) the immunofluorescence staining of Sca-1+ vascular precursor cells after two groups of vascular grafts are implanted in animals for 2 weeks; (b) Comparison of numbers of Sca-1+ pro-vascular cells recruited for two groups of vascular grafts (n = 3): p is less than 0.01.

Fig. 6 is a schematic diagram of the longitudinal section of the neovascular tissue and the coverage rate of the CD31+ endothelial cells on the inner side of the lumen after the two groups of blood vessels are implanted.

Wherein, (a) after two groups of vascular grafts are implanted in an animal body for 2 weeks, the CD31+ endothelial cells of the longitudinal section of the neovascular tissue are subjected to immunofluorescence staining; (b) Comparison of the coverage of CD31+ endothelial cells on the inside of the lumen 2 weeks after implantation of the vascular grafts in the animals of both groups.

FIG. 7 is a scanning electron microscope image of the surface topography of the anastomotic site, the quarter and the middle of the inner side of the lumen of the neovascular tissue after two groups of vascular grafts are implanted in the animal body for 2 weeks.

Detailed Description

The present invention will be described in detail with reference to specific embodiments.

The invention relates to a construction method of a drug-loaded elastic degradable artificial blood vessel, which comprises the following steps:

The first step is specifically as follows:

guiding the sodium citrate salt particles into a mold by utilizing a circular film with a uniform columnar tube core, uniformly vibrating to enable the salt particles to be tightly filled into gaps, carrying out atomization treatment and then guiding out a salt tube to manufacture a tubular salt mold consisting of the sodium citrate salt particles;

The second step is specifically as follows:

dissolving PCL granules in 2,2,2-trifluoroethanol to obtain a 14% PCL-trivoltol solution;

The third step is specifically as follows:

The drug-loaded elastic degradable artificial blood vessel constructed by the method is of a double-layer elastic blood vessel assembly composite structure, and sequentially comprises a PGS citric acid inner tube core and a drug-loaded mixed spinning sheath layer from inside to outside. The PGS sodium citrate tube core is a tubular structure made of polysebacate glycerol ester material on the basis of taking sodium citrate as a salt molding pore agent. The mixed spinning sheath is formed by PCL spinning and drug-loaded collagen spinning through an electrostatic spinning and blending technology. The inner diameter of the drug-loaded elastic degradable artificial blood vessel is 1mm, and the outer diameter is 1.5mm. The thickness of the drug-loaded spinning sheath is 20-30 μm.

In order to verify the clinical effect of the drug-loaded elastic degradable artificial blood vessel constructed by the invention, the invention constructs a rat abdominal aorta defect transplantation model, and the process is as follows: rats were anesthetized (5% isoflurane induced, 2% isoflurane maintained) and fixed in supine position on a sterile drape on the operating table, the anaesthetic mask and limbs were fixed with adhesive tape, the abdominal hair was shaved off, the operating area was disinfected with iodophor gauze twice and then draped over a hole towel to fully expose the field of view of the operating area. The vascular forceps slightly draw the skin on the two sides of the leucorrhea line, the scalpel vertically feeds along the leucorrhea line to cut the skin, and the tissue forceps separate the skin from the lower muscle layer. The muscular layers on both sides of the abdominal lineae are gently pulled by the vascular forceps again, the abdominal muscles are lifted to avoid damaging the internal organs of the abdomen, and a longitudinal incision with the length of about 3cm is made by extending the abdominal lineae to the middle with scissors. After exposure of the abdominal cavity, the small intestine was opened with a sterile cotton swab soaked with normal saline and covered with gauze soaked with normal saline to prevent damage. The surgical field was then maintained with a small dilator to completely expose the abdominal aorta and vena cava. Adjusting a microscope to a surgical area, wherein the visual field is clear, the magnification is moderate, carefully separating the abdominal aorta and the vena cava by using fiber forceps under the microscope, avoiding branches as much as possible and dissociating a section of abdominal aorta (about 10 mm), clamping two ends of the abdominal aorta by using vascular clamps respectively, cutting a section of abdominal aorta with the length of about 5mm, extracting a proper amount of heparin sodium saline by using a 1ml syringe, carefully flushing residual blood in a broken end lumen, taking out a sterilized vascular graft, anastomosing the graft with the end of the artery at two ends by using a 9-0 microscopic suture, and suturing about 10 needles at one end. Sequentially loosening the artery clamps at the far end and the near end, observing the blood leakage at the anastomotic stoma and the pulsation state of the transplant, flushing the abdominal cavity with normal saline after the state is stable, returning the small intestine to the original position and sequentially suturing the muscle and the skin, and carrying out anticoagulant treatment before and after the operation.

And (4) analyzing results:

as shown in FIG. 3, the mechanical results show that the vascular stent prepared by the method of the present invention has better mechanical properties and good suture property compared with the traditional PGS-PCL stent.

As shown in figure 4, the intravascular stent polypeptide prepared by the method of the invention is successfully loaded on a sheath layer, and has long-term in vitro slow release efficiency, and the slow release amount can reach 60 percent of the total loading amount.

As shown in FIG. 5, the vascular stent prepared by the method of the present invention showed a significantly enhanced recruitment efficiency of vascular precursor cells (Sca-1 + cells) compared to the blank control group after 2 weeks of in vivo transplantation.

As shown in 6,7, the intravascular stent prepared by the method of the present invention has significantly enhanced efficiency of rapid endothelialization of the inner surface of the graft lumen, compared to the blank control group, after 2 weeks of in vivo transplantation.

The invention is not limited to the examples, and any equivalent changes to the technical solution of the invention by a person skilled in the art after reading the description of the invention are covered by the claims of the invention.

Claims

1. The construction method of the drug-loaded elastic degradable artificial blood vessel is characterized by comprising the following steps:

the method comprises the following steps:

the method comprises the following steps: preparing a PGS tube core by using a sodium citrate tubular salt mold to form a PGS polymer-sodium citrate tubular stent inner core;

step two: mixing and winding a collagen spinning solution loaded with a medicament and a PCL spinning solution on an inner core of the PGS polymer-citric acid sodium salt tubular stent by using an electrostatic spinning technology to form a medicament-loaded mixed sheath layer;

2. The construction method of the drug-loaded elastic degradable artificial blood vessel according to claim 1, which is characterized in that:

the first step is specifically as follows:

guiding sodium citrate salt particles into a circular mold with a uniform columnar tube core, vibrating uniformly to enable the salt particles to be tightly filled into gaps, carrying out atomization treatment, and guiding out a salt tube to manufacture a sodium citrate tubular salt mold formed by the sodium citrate salt particles;

preparing a 10% and 15% mixed solution of a polytrimethylene sebacate prepolymer by using a tetrahydrofuran solution, sequentially dripping the 15%, 10% and 15% mixed solution on a sodium citrate tubular salt mold, and after the sodium citrate tubular salt mold is completely saturated, putting a PGS prepolymer-sodium citrate tubular stent mixture into a vacuum drying oven to form a PGS polymer-sodium citrate tubular stent core.

3. The construction method of the drug-loaded elastic degradable artificial blood vessel according to claim 2 is characterized in that:

the second step is specifically as follows:

dissolving PCL granules in 2,2,2-trifluoroethanol to obtain 14% PCL-trioxymethylene solution; dissolving collagen in hexafluoroisopropanol to obtain 8% collagen-hexafluoroisopropanol solution, and mixing the solution with polypeptide solution with concentration of 2.0mg/mL at volume ratio of 4:1;

placing the two motors and the tubular support inner core into an electrostatic spinning system, aligning the tubular support inner core to the center of the receiving sheet, enabling the tubular support inner core to be vertical to a spinning needle head and enabling the tubular support inner core to be parallel to the ground;

and turning on a power supply of a rotating motor and a switch of a syringe pump, and carrying out mixed electrospinning on the inner core of the PGS polymer-sodium citrate tubular scaffold rotating at the rotating speed of 120RPM for 3 minutes by using the PCL solution and the collagen polypeptide mixed solution at the speed of 2.5mL/h and the collagen polypeptide mixed solution at the speed of 1 mL/h.

4. The construction method of the drug-loaded elastic degradable artificial blood vessel according to claim 3, wherein the construction method comprises the following steps:

the third step is specifically as follows:

atomizing the composite tubular object containing the PCL-drug-loaded collagen sheath layer and the PGS polymer-sodium citrate salt tubular stent inner core for a short time to obtain a state which is relatively easy to suture, sterilizing by ultraviolet rays, and storing at-80 ℃ for later use.

5. The drug-loaded elastic degradable artificial blood vessel constructed by the method of claim 4.

6. The drug-loaded elastic degradable artificial blood vessel of claim 5, wherein:

the drug-loaded elastic degradable artificial blood vessel is of a double-layer elastic blood vessel-mounted composite structure and sequentially comprises a PGS sodium citrate tube core and a drug-loaded spinning sheath layer from inside to outside.

7. The drug-loaded elastic degradable artificial blood vessel of claim 6, wherein: the PGS sodium citrate tube core is a tubular structure made of polysebacate glycerol ester material on the basis of taking sodium citrate as a salt molding pore agent.

8. The drug-loaded elastic degradable artificial blood vessel of claim 7, wherein:

the drug-loaded spinning sheath layer is formed by PCL spinning and drug-loaded collagen spinning through an electrostatic spinning blending technology.

9. The drug-loaded elastic degradable artificial blood vessel of claim 8, wherein:

the inner diameter of the drug-loaded elastic degradable artificial blood vessel is 1mm, and the outer diameter is 1.5mm.

10. The drug-loaded elastic degradable artificial blood vessel of claim 9, wherein: the thickness of the drug-loaded spinning sheath is 20-30 μm.