CN115414536A

CN115414536A - Gene elution balloon and preparation method and application thereof

Info

Publication number: CN115414536A
Application number: CN202211053750.6A
Authority: CN
Inventors: 崔取金; 郭力友; 夏洁; 赵晟
Original assignee: Suzhou Zhongtian Medical Device Technology Co ltd
Current assignee: Suzhou Zhongtian Medical Device Technology Co ltd
Priority date: 2022-08-29
Filing date: 2022-08-29
Publication date: 2022-12-02

Abstract

The invention relates to a gene elution balloon and a preparation method and application thereof, wherein the gene elution balloon comprises a balloon body, a hydrophilic coating and a nano coating for coating therapeutic genes, wherein the hydrophilic coating and the nano coating are sequentially arranged on the surface of the balloon body; the nanocoating includes cationic liposomes and a therapeutic gene. The gene elution balloon has the characteristics of high efficiency and safety, has small and uniform particle size, can quickly penetrate through a cell membrane to be absorbed after the balloon is expanded, greatly improves the bioavailability of a medicament, and ensures that a therapeutic gene can be loaded as much as possible. The raw materials related to the gene elution balloon can be biodegraded, the whole body toxicity can not be generated to a human body, the gene elution balloon has good biocompatibility, small side effect to the human body, strong targeting property and controllable treatment process, and the time of effective concentration can be prolonged due to the slow release effect of the cationic liposome carrier.

Description

Gene elution balloon and preparation method and application thereof

Technical Field

The invention relates to the technical field of gene eluting balloons, in particular to a gene eluting balloon as well as a preparation method and application thereof.

Background

Cardiovascular disease is still the most common cause of death worldwide, accounting for approximately 30% of all deaths. Atherosclerosis leads to the development of restricted blood flow lesions, resulting in impaired oxygen supply to the tissues, and clinical symptoms such as angina pectoris or intermittent claudication. In addition, unstable lesions of plaque rupture and thrombosis lead to Myocardial Infarction (MI), resulting in significant morbidity and mortality. Although the widespread use of Drug Eluting Stents (DES) may contribute to some degree to cardiovascular disease, in-stent restenosis (ISR), delayed arterial healing and thrombosis remain important clinical complications.

Neointimal growth is a multifactorial response to mechanical vascular injury in Percutaneous Coronary Intervention (PCI). The radial forces required to expand the balloon catheter and place the stent can result in rupture or tearing of atherosclerotic plaque, endothelial cells, and intimal and medial layers of the arterial wall. This results in a cascade of release of cytokines and growth factors, expression of adhesion molecules, recruitment and infiltration of macrophages and other inflammatory cells, proliferation and migration of VSMCs, and deposition of ECM. Neointimal development leads to restenosis, reduced coronary blood flow leading to recurrent symptoms requiring revascularization, and in some cases thrombotic occlusion leading to myocardial infarction.

CN109966564A discloses a medicine carrying balloon, its medicine carrying balloon that discloses include the sacculus and in proper order range upon range of in the medicine carrying layer and the protective layer of the surface of sacculus, contain the gene in the medicine carrying layer, the protective layer covers the medicine carrying layer, just the material of protective layer is selected from at least one in polyvinyl alcohol, polyvinylpyrrolidone, chitosan, hydroxypropyl-beta-cyclodextrin, hydroxyethyl cellulose, sodium carboxymethylcellulose, dextran, arabic gum, sodium alginate, collagen, soybean protein and polyethylene glycol stearate. The protective layer can protect the medicine carrying layer well, can avoid the excessive loss of gene in the transportation process, and the material of protective layer is selected from at least one of the above-mentioned substances, makes this medicine carrying sacculus reach pathological change position after, during the expansion of medicine carrying sacculus, the protective layer can dissolve or drop fast, in order to release the gene fast.

Gene Eluting Balloons (GEBs) are an effective strategy to prevent ISR by delivering therapeutic genes from the balloon surface to the vessel wall through a vector. The large biomolecule delivery of GEB can address the shortcomings of the current stent platform or catheter based delivery systems: for example, the active drugs have insufficient concentration at the pathological part, the compounds diffuse to other organs at the far end, and the problems of insufficient cell specificity targeting participating in thrombosis, inflammation and intimal hyperplasia after vascular injury and the like can be classified according to the action mode of gene products and cell targets, and the targeted treatment can be performed.

In view of the above, it is important to develop a gene eluting balloon that is highly efficient and safe.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a gene elution balloon and a preparation method and application thereof, the gene elution balloon has small and uniform particle size, can quickly penetrate through a cell membrane to be absorbed after the balloon is expanded, the bioavailability of a medicament is greatly improved, and related raw materials of the gene elution balloon can be biodegraded without generating toxicity on the whole body of a human body.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a gene elution balloon, which comprises a balloon body, and a hydrophilic coating and a nano coating for coating a therapeutic gene, which are sequentially arranged on the surface of the balloon body;

the nanocoating includes cationic liposomes and a therapeutic gene.

In the invention, the used carrier is the cationic liposome, the composition, the structure and the cell membrane of the cationic liposome are highly similar, and the cationic liposome has good biocompatibility, can help therapeutic genes to smoothly pass through the cell membrane, and improves the bioavailability of the therapeutic genes; the cationic liposome is biodegradable in vivo, and the product can be absorbed by human body or discharged by metabolism, without generating toxicity; moreover, the cationic liposome can play a certain slow release role, and the effective concentration time is prolonged; compared with active medicines, the therapeutic gene has better biocompatibility and smaller side effect on human body; compared with medicines, the targeting of the gene is stronger, the treatment on the target part is more targeted, and the treatment process is more controllable; therefore, the gene elution balloon has the characteristics of high efficiency and safety.

In the invention, through GEB delivery, the cationic liposome is used as a vector of the therapeutic gene to mediate into a target spot, and the therapeutic gene can be prevented from being digested too quickly by nuclease in vivo. In addition, the cationic liposome is used as a carrier, so that better penetration at a target point can be promoted, and meanwhile, the carrier is used as a temporary storage of a therapeutic gene, so that long-acting and uniform release of the gene is realized, the release kinetics are improved, and adverse systemic side effects are better controlled. Moreover, the raw materials involved in the gene elution balloon can be biodegraded, and the whole body toxicity to a human body can not be generated.

Preferably, the hydrophilic coating has a thickness of 10-500 μm, such as 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, and the like.

Preferably, the nanocoating has a thickness of 200-300 μm, such as 220 μm, 240 μm, 260 μm, 280 μm, and the like.

The raw material of the cationic liposome comprises cationic lipid.

The cationic lipid molecule consists of four parts: head, linker, linkage, and hydrophobic tail, the polar head acts to bind the liposome to DNA, the liposome-DNA complex to the cell membrane or other components inside the cell, and the cationic liposome with a multivalent polar head group or with multiple positively charged polar heads is more efficient in transfection, probably because it binds more strongly to DNA. The length of the connecting chain can influence the interaction of the cationic nanoliposome and the surface of the mucosa, thereby influencing the transfection activity. Generally, the cationic nano-liposome with the long connecting chain can obviously enhance the interaction with the mucosal surface, and the transfection efficiency is high. The connecting bond is an important component of lipoid molecules and determines the chemical stability and biodegradability of the cationic nanoliposome.

The cationic liposome is used as a carrier, so that nano particles can be quickly released and enter target cells after the saccule is expanded, the therapeutic gene is prevented from being digested too quickly by nuclease in vivo, the transfection rate of the therapeutic gene in the vascular intima is improved, and the cationic liposome is a biodegradable material which is nontoxic to a human body and has important significance for preventing cardiovascular and cerebrovascular restenosis, delayed artery healing and thrombosis.

Preferably, the first and second liquid crystal display panels are, the cationic lipid includes trimethyl-2, 3-dioleyloxypropylammonium chloride (DOTMA), trimethyl-2, 3-dioleyloxypropylammonium bromide (DOTAP), dimethyl-2, 3-dioleyloxypropyl-2- (2-spermimido) ethylammonium bromide (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyloctadecylammonium bromide (DDAB), dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropylammonium bromide (DORI), dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropylammonium bromide (DORIE), dimethyl-3-hydroxypropyl-2, 3-dioleyloxypropylammonium bromide (DORIE-HP), dimethyl-4-hydroxybutyl-2, 3-dioleyloxypropylammonium bromide (DOHB-RIE), dimethyl-5-hydroxypentyl-2, 3-dioleyloxypropylammonium bromide (DOHPC), dimethyl-2-alkoxypropylammonium bromide (DMDPRI-3-DPRI), dimethyldodecylpropylammonium bromide (DPRIE-3, DMHPIEE-2, 3-bis-octadecyloxypropylammonium bromide (DMDPIE), any one of or a combination of at least two of N- (2-sperminylamido) -N ', N' -dioctadecyl glycinamide (DOGS), 1, 2-dioleoyl-3-succinyl-sn-glycerocholine ester (DOSC), 3 β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol), lipid poly-L-lysine (LPLL), stearylamine (SA), wherein typical but non-limiting combinations include: a combination of DOTAP and DOTMA, a combination of DOTMA and DC-Chol, a combination of DOTAP, DOTMA and DC-Chol, and the like.

In the present invention, the cationic liposome is preferably of the above kind because: firstly, their size, shape, charge and tunability of the target molecule implies reliable transport capacity; secondly, all the cationic liposomes can form a compound with DNA, and the molecular volume of the DNA is greatly compressed, so that the cationic liposomes can effectively enter cells for transfection, and simultaneously, possible immune toxicity caused by viral vectors can be avoided.

Preferably, the therapeutic gene comprises any one of Vascular Endothelial Growth Factor (VEGF), nitric oxide synthase (eNOS), antisense oligonucleotides, prostacyclin (PGIS), 7ND, or Tissue Factor (TF), or a combination of at least two thereof, wherein typical but non-limiting combinations include: a combination of VEGF and eNOS, a combination of antisense oligonucleotide, PGIS and 7ND, a combination of antisense oligonucleotide, PGIS, 7ND and TF, and the like.

Preferably, the mass percentage of the therapeutic gene is 0.1% -10%, such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, etc., based on 100% of the total mass of the cationic liposome.

According to the gene elution balloon, the particle size is small and uniform, the enough specific surface area ensures enough amount of loaded therapeutic genes, the conventional gene elution balloon has the maximum therapeutic gene loading amount of 0.5%, and the therapeutic genes in the gene elution balloon can reach a level far higher than that in the prior art.

In a second aspect, the present invention provides a method for preparing the gene eluting balloon of the first aspect, the method comprising the steps of:

(1) Dissolving cationic lipid in an organic solvent to form a solution, removing the solvent to form a cationic lipid film, adding water to mix with the cationic lipid film, and then forming a cationic liposome suspension under the action of ultrasound;

(2) Mixing and incubating the cationic liposome suspension with a therapeutic gene;

(3) Filtering the suspension by adopting a microporous membrane, and adding an additive to form a coating suspension;

(4) And arranging coating suspension on the balloon body containing the hydrophilic coating, and curing to form a nano coating for coating the therapeutic gene to obtain the gene eluting balloon.

Preferably, in step (1), the organic solvent comprises chloroform.

Preferably, the solution has a mass concentration of 1-100mg/mL, such as 5mg/mL, 10mg/mL, 20mg/mL, 40mg/mL, 60mg/mL, 80mg/mL, and the like.

Preferably, the means for removing the organic solvent comprises rotary evaporation.

Illustratively, the rotary evaporation is performed in a rotary evaporator.

Preferably, the pressure of the rotary evaporation is 10 to 70kPa, such as 15kPa, 20kPa, 30kPa, 40kPa, 50kPa, 60kPa, and the like.

Preferably, the temperature of the rotary evaporation is 45-70 ℃, such as 50 ℃, 55 ℃, 60 ℃, 65 ℃ and the like.

Preferably, the rotary steaming time is 10-100min, such as 20min, 40min, 60min, 80min and the like.

Preferably, the time of the ultrasound is 10-60min, such as 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, and the like.

Illustratively, the ultrasound is performed in a probe sonicator.

Preferably, the water is present in an amount of 50 to 500 parts by mass, for example 100 parts, 150 parts, 200 parts, 250 parts, 300 parts, 350 parts, 400 parts, 450 parts, etc., based on 100 parts by mass of the cationic lipid film as a whole.

Preferably, in step (2), after the mixing, further incubation is included.

In the invention, cationic lipid is prepared into nano particles by an ultrasonic-double emulsion method, the nano particles and therapeutic genes are incubated to form composite nano particles, and the nano particles are filtered by a microporous filter membrane to have small and uniform particle size. The nano carrier can effectively improve the loading capacity of the loaded medicine due to the high specific surface area and the small particle size of the nano carrier, greatly promotes the penetrating power of the medicine, and simultaneously, the nano particles can be effectively eliminated through blood and tissues, so that the specific damage degree of the medicine to cells can be reduced.

Preferably, the incubation time is 20-80min, such as 30min, 40min, 50min, 60min, 70min, etc.

Preferably, in step (3), the pore size of the microporous membrane is 0.4 to 1 μm, e.g., 0.45 μm, 0.8 μm, etc.

Preferably, the microporous membrane further comprises mixing the filtered suspension with an additive after filtration.

Preferably, the additive comprises any one or a combination of at least two of fructose, glucose, sucrose, lactose, maltose, erythritol, threitol, arabitol, ribitol, mannitol, galactitol, fucitol, iditol, inositol, heptatol, isomalt, maltitol, lactitol, maltotriose, voglibose, xylitol, sorbitol, or polyethylene glycol, wherein typical but non-limiting combinations include: fructose, glucose, sucrose and lactose, arabitol, ribitol, mannitol, galactitol, fucoitol and iditol, lactitol, maltotriose, voglibose, xylitol, sorbitol and polyethylene glycol, and the like.

Preferably, in step (4), the hydrophilic coating is disposed in a manner including: and (3) arranging the raw material of the hydrophilic coating on the surface of the balloon to finish the arrangement of the hydrophilic coating.

Preferably, the raw material of the hydrophilic coating includes polyacrylamide.

Preferably, the setting comprises spraying and drying.

Illustratively, the hydrophilic coating is disposed in a manner including: uniformly spraying polyacrylamide with the mass concentration of 0.5-2% (such as 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8% and the like) on the surface of the balloon by a spraying device, wherein the spraying amount is 15-25 mu g/mm ² (e.g., 16. Mu.g/mm) ² 、18μg/mm ² 、20μg/mm ² 、22μg/mm ² 、24μg/mm ² Etc.), after the spraying is finished, the mixture is placed in a vacuum drying oven to be dried for more than 20 hours (such as 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, etc.) under the conditions of 0.1-1 atm (such as 0.2atm, 0.4atm, 0.6atm, 0.8atm, etc.) and 40-70 ℃ (such as 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, etc.).

Preferably, the curing temperature is 40-70 ℃, such as 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ and the like.

As a preferable technical scheme, the preparation method comprises the following steps:

(1) Mixing cationic lipid with an organic solvent to form a solution with the mass concentration of 1-100mg/mL;

then rotationally steaming the solution for 10-100min under the conditions that the pressure is 10-70kPa and the temperature is 45-70 ℃, and removing the organic solvent to form a cationic lipid film;

then carrying out ultrasonic treatment on the cationic lipid film and water for 10-60min, and mixing and dispersing to form the suspension;

(2) Mixing the suspension with the therapeutic gene, and incubating for 20-80min to form a suspension containing the therapeutic gene;

(3) Filtering the suspension containing the therapeutic gene by a microporous membrane with the aperture of 0.4-1 mu m to form a coating suspension;

(4) And arranging a coating suspension on the balloon body containing the hydrophilic coating, and curing at 40-70 ℃ to form a nano coating for coating the therapeutic gene to obtain the gene eluting balloon.

In a third aspect, the present invention provides a medical device comprising a gene eluting balloon according to the first aspect.

Compared with the prior art, the invention has the following beneficial effects:

(1) The gene elution balloon disclosed by the invention is small and uniform in particle size, can quickly penetrate through a cell membrane to be absorbed after the balloon is expanded, greatly improves the bioavailability of a medicament, and ensures that therapeutic genes can be loaded as much as possible.

(2) The raw materials of the gene elution balloon can be biodegraded, the whole body toxicity can not be generated to a human body, the gene elution balloon has good biocompatibility, small side effect to the human body, strong targeting property and controllable treatment process, and the time of effective concentration can be prolonged due to the slow release effect of the cationic carrier.

(3) The gene elution balloon has the characteristics of high efficiency and safety, and has important significance for preventing cardiovascular and cerebrovascular restenosis, delayed artery healing and thrombosis.

(4) The preparation process is simple, efficient, stable and convenient to operate.

(5) The average particle size of the gene elution balloon is within 667nm, the particle size distribution is between 40 nm and 800nm, the bioavailability is more than 11% in 24h, and the coating integrity is more than 67% after the test.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitation of the present invention.

In the present invention, purchase information of a part of raw materials according to each embodiment is as follows:

a balloon body: the material is Pebax when being produced in the medical treatment of the middle-aged and the elderly;

DOTAP: purchased from Sigma-Aldrich under the trademark of ≥ 98% (TLC), powder;

DOTMA: purchased from Sigma-Aldrich;

DC-Chol: purchased from Sigma-Aldrich;

VEGF: purchased from Sigma-Aldrich;

eNOS: purchased from Sigma-Aldrich;

PGIS: purchased from Sigma-Aldrich.

Example 1

The embodiment provides a gene eluting balloon, which comprises a balloon body, a hydrophilic coating and a nano coating, wherein the hydrophilic coating and the nano coating coat the therapeutic gene are sequentially arranged on the surface of the balloon body;

the nanocoating includes cationic liposomes and a therapeutic gene.

The thickness of the hydrophilic coating is 100 μm.

The thickness of the nano coating is 300 mu m.

The gene elution balloon is prepared by a method comprising the following steps:

(1) Dissolving DOTAP in chloroform to prepare a solution with the concentration of 30 mg/mL;

transferring the solution in the first step to a rotary evaporator, performing rotary evaporation for 90min under the conditions of 50kPa and 50 ℃, and removing chloroform to form a lipid membrane;

adding 40mL of deionized water to disperse the lipid membrane in the second step, and uniformly dispersing the liposome by using a probe ultrasonic instrument to form a suspension;

(2) Adding 10 μ g VEGF into the suspension, and incubating at 37 deg.C for 60min to form therapeutic gene-containing suspension;

(3) Filtering the suspension with a 0.8-micron microporous filter membrane to remove particles with overlarge particle size, and then adding 1.6g of sorbitol to form a spraying suspension;

(4) Uniformly spraying the spraying suspension on the outer surface of the balloon by ultrasonic spraying equipment, wherein the outer surface of the balloon is subjected to hydrophilic treatment in advance, and the hydrophilic treatment process comprises the following steps: uniformly spraying polyacrylamide with the mass concentration of 1% on the surface of the balloon by spraying equipment, wherein the spraying amount is 20 mu g/mm ² After the spraying is finished, the mixture is placed in a vacuum drying oven to be dried for 24 hours under the conditions of 0.5atm and 60 ℃;

and then curing the sprayed balloon at 55 ℃ to obtain the gene eluting balloon.

Example 2

The embodiment provides a gene elution balloon, which comprises a balloon body, a hydrophilic coating and a nano coating, wherein the hydrophilic coating and the nano coating coat the therapeutic gene are sequentially arranged on the surface of the balloon body;

the nanocoating includes cationic liposomes and a therapeutic gene.

The thickness of the hydrophilic coating is 100 μm.

The thickness of the nano coating is 300 mu m.

The gene eluting balloon is prepared by the following method, and the method comprises the following steps:

(1) Dissolving DOTMA in dichloromethane to prepare a solution with the concentration of 40 mg/mL;

transferring the solution to a rotary evaporator, carrying out rotary evaporation for 100min under the conditions of 60kPa and 50 ℃, and removing dichloromethane to form a lipid membrane;

(2) Adding 10 μ g of eNOS to said emulsion and incubating at 37 ℃ for 60min to form a suspension containing the therapeutic gene;

(3) Filtering the suspension by a 0.8-micron microporous filter membrane to remove particles with overlarge particle size, and then adding 1.6g of polyethylene glycol to form a spraying suspension;

(4) Uniformly spraying the spraying suspension on the outer surface of the balloon by ultrasonic spraying equipment, wherein the outer surface of the balloon is subjected to hydrophilic treatment in advance, and the hydrophilic treatment process comprises the following steps: uniformly spraying polyacrylamide with the mass concentration of 0.5 percent on the surface of the balloon by spraying equipment, wherein the spraying amount is 25 mu g/mm ² After the spraying is finished, the mixture is placed in a vacuum drying oven to be dried for 30 hours under the conditions of 0.1atm and 4 ℃;

Example 3

the nanocoating includes cationic liposomes and a therapeutic gene.

The thickness of the hydrophilic coating is 100 μm.

The thickness of the nano coating is 300 mu m.

(1) Dissolving DC-Chol in chloroform to prepare a solution with the concentration of 30 mg/mL;

then transferring the solution to a rotary evaporator, carrying out rotary evaporation for 90min under the conditions of 50kPa pressure and 50 ℃, and removing chloroform to form a lipid membrane;

then adding 40mL of deionized water to disperse the lipid membrane, and uniformly dispersing the liposome by using a probe ultrasonic instrument to form suspension;

(2) Adding 10 μ g of PGIS to the emulsion, and incubating at 37 ℃ for 60min to form a therapeutic gene-containing suspension;

(3) Filtering the suspension with a 0.8-micron microporous filter membrane to remove particles with overlarge particle size, and adding 0.8g of sucrose and 0.8g of polyethylene glycol to form a spraying suspension;

(4) Uniformly spraying the spraying suspension on the balloon by ultrasonic spraying equipmentThe outer surface of the balloon is subjected to hydrophilic treatment in advance, and the hydrophilic treatment process comprises the following steps: uniformly spraying 2% polyacrylamide on the surface of the balloon by a spraying device, wherein the spraying amount is 15 mu g/mm ² After the spraying is finished, the mixture is placed in a vacuum drying oven to be dried for 22 hours under the conditions of 1atm and 70 ℃;

Example 4

This example is different from example 1 in that DOTAP was replaced with LPLL of equal mass, and the rest is the same as example 1.

Example 5

This example is different from example 1 in that DOTAP was directly dispersed in deionized water in step (1), and the rest was the same as example 1.

Comparative example 1

This comparative example differs from example 1 in that DOTMA was replaced with laureth-23 of equal mass, the remainder being the same as in example 1.

Comparative example 2

This comparative example differs from example 1 in that: DOTAP, VEGF, chloroform and water were directly mixed and dispersed, and then incubated at 37 ℃ for 60min to form a spray suspension, and the rest was the same as in example 1.

Performance testing

The gene-eluting balloons described in examples 1 to 5 and comparative examples 1 to 2 were subjected to the following tests:

(1) Particle size distribution and average particle size measurement: the particle size distribution and average particle size of the nanoparticles were determined using Masteriser 2000E + zs90 laser particle sizer.

(2) Bioavailability of therapeutic genes: animal experiments, the gene-coated balloon and a control blank balloon simulate the operation process of the balloon in the human body in the abdominal aorta of a rabbit, and the capacities of the gene-coated stent and a control group sample are both 10. 2 patients are killed at 12h, 24h, 2d, 4d and 7d after operation respectively, blood vessels of a lesion part are taken out, the blood vessels are washed by physiological saline, 1mg of blood vessel tissues are taken out from a sacculus expansion part, RNA is extracted for RT-PCR amplification, and the result is observed by 1.5 percent agarose gel electrophoresis.

(3) Layer firmness: the gene coating saccule is sent into an external vascular flushing simulation device through a guide sheath, a far-end passage catheter and a guide wire, and the conveying process that the medicine saccule enters the body and is conveyed to a target position is simulated. Adopting PBS to simulate blood, controlling the flow rate to be 35mL/min, carrying out water bath at 37 ℃, controlling the simulation flushing time to be 3min, immediately taking out the drug coating saccule from the target position after the simulation flushing time is 3min, and observing the coating integrity on the surface of the saccule by using an electron microscope.

The test results are summarized in table 1.

TABLE 1

The data in the table 1 are analyzed, so that the average particle size of the gene elution balloon is within 667nm, the particle size distribution is between 40 nm and 800nm, the bioavailability is over 11 percent in 24 hours, and the integrity of the coating is over 67 percent after the test; the gene elution balloon has small and uniform particle size, can quickly penetrate through a cell membrane to be absorbed after the balloon is expanded, greatly improves the bioavailability of the medicine, and simultaneously, all materials in the method can be biodegraded without generating toxicity on the whole body of a human body. In addition, the preparation process is simple, efficient, stable and convenient to operate.

As can be seen from the analysis of comparative example 1 and example 1, the performance of comparative example 1 is inferior to that of example 1, and the performance of the gene eluting balloon formed by the coordination of the cationic liposome and the therapeutic gene is proved to be better.

As can be seen from the analysis of comparative example 2 and example 1, comparative example 2 is inferior to example 1 in performance, and it is confirmed that the cationic liposome and the therapeutic gene are formed into emulsion and suspension in sequence in the preparation, i.e., the gene eluting balloon formed by the ultrasonic-multiple emulsion method has better performance.

As can be seen from the analysis of example 4 and example 1, example 4 is inferior in performance to example 1, and it was confirmed that the performance of the gene-eluting balloon formed by the cationic liposome of the present invention is preferable.

Analysis of example 5 and example 1 revealed that example 5 is inferior to example 1 in performance, and that formation of a solution and then formation of an emulsion in the cationic liposome yielded a gene-eluting balloon with better performance.

The present invention is illustrated in detail by the examples given above, but the present invention is not limited to the details given above, which means that the present invention is not limited to the details given above. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. A gene elution balloon is characterized by comprising a balloon body, a hydrophilic coating and a nano coating, wherein the hydrophilic coating and the nano coating coat the therapeutic gene are sequentially arranged on the surface of the balloon body;

the nanocoating includes cationic liposomes and a therapeutic gene.

2. A gene elution balloon as claimed in claim 1 wherein the hydrophilic coating has a thickness of 10-500 μ ι η;

preferably, the thickness of the nanocoating is 200-300 μm.

3. A gene elution balloon as claimed in claim 1 or 2 wherein the material of the cationic liposome comprises a cationic lipid;

preferably, the first and second liquid crystal display panels are, the cationic lipid comprises trimethyl-2, 3-dioleyloxypropyl ammonium chloride, trimethyl-2, 3-dioleyloxypropyl ammonium bromide, dimethyl-2, 3-dioleyloxypropyl-2- (2-spermicarbonamido) ethyl ammonium trifluoroacetate, trimethyldodecyl ammonium bromide, trimethyltetradecyl ammonium bromide, trimethylhexadecyl ammonium bromide, dimethyloctadecyl ammonium bromide, dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropyl ammonium bromide, dimethyl-3-hydroxypropyl-2, 3-dioleyloxypropyl ammonium bromide, dimethyl-4-hydroxybutyl-2, 3-dioleyloxypropyl ammonium bromide, dimethyl-5-hydroxypentyl-2, 3-dioleyloxypropyl ammonium bromide, dimethyl-2-hydroxyethyl-2, 3-dicetyl-alkoxypropyl ammonium bromide, dimethyl-2-hydroxyethyl-2, 3-dicetyl-oxypropylammonium bromide, dimethyl-2-hydroxyethyl-2, 3-dioctadecyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2, 3-ditetradecyloxypropyl ammonium bromide, N-dicarbamoylamide, N ' -dioctadecyl glycinamide, 1, 2-dioleoyl-3-succinyl-sn-glycerocholine ester, 3 beta- [ N- (N ', N ' -dimethylaminoethyl) carbamoyl ] cholesterol, lipid poly-L-lysine, any one or a combination of at least two of stearylamine;

preferably, the therapeutic gene comprises any one of vascular endothelial growth factor, nitric oxide synthase, antisense oligonucleotide, prostacyclin, 7ND, or tissue factor, or a combination of at least two thereof;

preferably, the mass percentage of the therapeutic gene is 0.1-10% based on the total mass of the cationic liposome as 100%.

4. A method of manufacturing a gene elution balloon according to any one of claims 1 to 3, wherein the method of manufacturing comprises the steps of:

(1) Dissolving cationic lipid in an organic solvent to form a solution, removing the organic solvent to form a cationic lipid film, adding water to mix with the cationic lipid film, and then forming a cationic liposome suspension under the action of ultrasound;

5. The method according to claim 4, wherein in the step (1), the organic solvent comprises chloroform;

preferably, the mass concentration of the solution is 1-100mg/mL;

preferably, the means for removing the organic solvent comprises rotary evaporation;

preferably, the pressure of the rotary evaporation is 10-70kPa;

preferably, the temperature of the rotary evaporation is 45-70 ℃;

preferably, the rotary steaming time is 10-100min;

preferably, the time of the ultrasonic treatment is 10-60min;

preferably, the mass of the water is 50 to 500 parts based on 100 parts of the total mass of the cationic liposome membrane.

6. The method according to claim 4 or 5, wherein in the step (2), the mixing further comprises incubation;

preferably, the incubation time is 20-80min.

7. The production method according to any one of claims 4 to 6, wherein in the step (3), the pore diameter of the microporous membrane is 0.4 to 1 μm;

preferably, after the microporous membrane is filtered, mixing the filtered suspension with an additive;

preferably, the additive comprises any one or a combination of at least two of fructose, glucose, sucrose, lactose, maltose, erythritol, threitol, arabitol, ribitol, mannitol, galactitol, fucitol, iditol, inositol, heptatol, isomalt, maltitol, lactitol, maltotriose, voglibose, xylitol, sorbitol, or polyethylene glycol.

8. The production method according to any one of claims 4 to 7, wherein in the step (4), the hydrophilic coating is provided in such a manner as to include: arranging the raw material of the hydrophilic coating on the surface of the balloon to complete the arrangement of the hydrophilic coating;

preferably, the raw material of the hydrophilic coating comprises polyacrylamide;

preferably, the setting mode comprises spraying and drying;

preferably, the temperature of the curing is 40-70 ℃.

9. The production method according to any one of claims 4 to 8, characterized by comprising the steps of:

(1) Mixing the cationic liposome with an organic solvent to form a solution with the mass concentration of 1-100mg/mL;

then, carrying out ultrasonic treatment on the cationic lipid film and water for 10-60min, and mixing and dispersing to form the cationic liposome suspension;

(2) Mixing the cationic liposome suspension with the therapeutic gene, and incubating for 20-80min to form a suspension containing the therapeutic gene;

10. A medical device comprising the gene eluting balloon of any one of claims 1-3.