Construction Of Nano-calcium Phosphate-rapamycin Coated Balloon And Research Of Its Therapeutic Effect On Atherosclerosis

Cardiovascular disease（CVD）is currently one of the main causes of death among urban and rural residents in my country.As the atherosclerosis（AS）plaque is unstable and the plaque ruptures,the blood vessels are completely blocked,which can cause acute ischemic infarction in heart,and induce arrhythmia and heart failure.At present,percutaneous coronary stent implantation and balloon expansion are important treatments for acute myocardial infarction.However,the treatment of stent implantation is likely to cause the intima tear and trigger the process of damage repair.Besides,permanent stent implantation is likely to cause excessive vascular smooth muscle cells（SMCs）over-proliferation and intravascular restenosis,which severely limits its long-term therapeutic effect.As an emerging interventional therapy technology,drug-coated balloon（DCB）has shown great advantages in inhibiting vascular smooth muscle proliferation and acute thrombosis.Compared with traditional stent intervention technology,the advantages of drug-coated balloons include 1)avoiding of the permanent stent implantation,maintaining of original anatomical structure and blood hemodynamics;2)no chronic stimulation by polymer carrier and decrease of inflammatory response;3)uniform and high-concentrated drug-distribution in a short period of time,and no long-term inflammation and systemic adverse reactions.Therefore,the drug coated balloon treatment is becoming the most important interventional therapy in the future.Due to the super lipophilicity and crystallinity of paclitaxel,the drug-coated balloons are all paclitaxel-coated currently.For example,advance 18PTX drug-coated balloon from Cook company is coated with a single layer of paclitaxel.During the operation,the drug crystals are pressed into the blood vessel wall by balloon expansion,so that the drug enters the blood vessel tissue and has a certain slow-release effect.However,due to the systemic toxicity of paclitaxel,which is not conducive to advanced endothelialization of blood vessels.Therefore,it is critical to search a new generation of coating drug.Rapamycin（RAP）is a macrolide antibiotic immunosuppressant,which is often used clinically to suppress immune response.In recent years,it has been found that it has excellent efficacy of anti-neointimal hyperplasia,safer implication and wider treatment range,which making rapamycin one of the promising new generation drugs for drug-coated balloon.By interacting with m TOR,rapamycin blocks the signal pathway mediated by it,reduces the phosphorylation level of the downstream molecule rp S6,thereby inhibiting the proliferation and migration of smooth muscle cells.However,compared with paclitaxel,rapamycin is less lipophilic,which leading to low vascular transfer efficiency and slower tissue absorption.Additionally,rapamycin has poor crystallinity,which make it hard to form a stable coating on the surface of the balloon.Therefore,it is critical to improve the vascular absorption efficiency of rapamycin from drug coated balloons.The application of nanotechnology to transform or modify drugs,and the construction of nano-drug composite coatings are expected to regulate the physical properties of the drugs and improve the tissue penetration of the drugs,the redistribution ability and the retention time of the drugs,thereby improving the defects of rapamycin-coated balls application.First of all,nanoparticle carrier promotes the release of rapamycin from the balloon surface and transfer to the vascular wall tissue during the balloon expansion process.Nanoparticle carrier also improves the rapamycin retension on the balloon surface and decreases the drug loss in the blood stream.We also improve the tissue penetration ability of the drug by regulating the size effect of nanomaterials,which helps the drug enter the cell as soon as possible,and reduces the impact of high shear force in the blood vessel on drug absorption.Secondly,the formation of a nanomaterial-rapamycin complex can realize the slow release of the drug,solve the problem of"bursting release"of the balloon-coated drug,eliminate the"peak-valley"phenomenon of the blood drug concentration,and effectively prevent the adverse reactions caused by high blood concentration of drug.Recently,some studies using nanomaterials such as polymers or metal oxides as rapamycin carriers to produce drug-eluted stents,which showed that these stents continuously inhibit the proliferation of smooth muscle cells.However,polymer nanomaterials have the problem of poor blood compatibility,which may cause inflammatory response and systemic toxicity.Metal oxide nanomaterials cannot be degraded in the body,and heavy metal ions may cause endothelial damage and increase activity of platelets and neutrophils.Therefore,nanomaterials with both degradability and safety is important for preparing nanomaterial-rapamycin composite coating balloons.Organisms use their own powerful regulatory capabilities to induce and control their construction of inorganic materials in the body.This process is called biomineralization.Some reports showed that biomimetic mineralization can effectively protect protein drugs from protease degradation and greatly improve the physical and chemical stability of proteins;calcium phosphate formed by mineralization has p H-responsive properties,and the calcium phosphate-protein structure achieves property of slow protein release.Besides,the mineralized calcium phosphate also mediates protein drugs into the cell.In addition,calcium phosphate has excellent p H responsiveness,which can effectively dissolve and release rapamycin in the acidic environment of lusosomes;rapamycin can be embedded in the process of biomimetic mineralization.This structure improves the rapamycin stability and facilitates the sustained release in the body.Therefore,biomimetic calcium phosphate（Ca P）is considered to prepare nanoparticle calcium phosphate-rapamycin（nCaP-RAP）composite coating,thereby prolonging the the retention time of rapamycin in the blood vessel and the penetration ability in the tissue.The calcium phosphate-rapamycin was prepared by the method of biomimetic mineralization.By adjusting the size,morphology,surface charge,hydrophilicity/hydrophobicity and other properties of the composite nanomaterials,the effects of different physical and chemical properties on calcium phosphate rapamycin,the drug loading,stability and release efficiency,we established a simple and stable method for nanomaterial preparation;Then we evaluated the blood compatibility of nanoparticle calcium phosphate rapamycin and its effect on vascular smooth muscle cell to clarify its mechanism;In addition,we constructed a new type of balloon coated with nanoparticle calcium phosphate rapamycin to evaluate its release efficiency,blood compatibility and the therapeutic effects in atherosclerosis model.This research provides practical technologies and scientific solutions for understanding and solving the bottleneck problem of drug coated balloon in clinical application by the biomimetic nanotechnology.PartⅠ Construction and characteristics test of nCaP-RAPObjectivenCaP-RAP was prepared by biomimetic mineralization method,and its physical-chemical properties,release efficiency and biocompatibility were evaluated in vitro;MethodsnCaP-RAP was prepared by adding different concentrations of rapamycin and Ca Cl₂to the DMEM medium for mineralization reaction.By adjusting the size,morphology,surface charge,hydrophilicity/hydrophobicity and other properties of the composite nanomaterials,we evaluated the effects of different physical and chemical properties on the drug loading,stability and drug release of calcium phosphate-rapamycin composites for establishing a simple and stable method of nanomaterial preparation.ResultsThrough continuous exploration,the mineralization reaction in DMEM solution was carried out for 4 hours to obtain nanoparticles with good dispersibility and uniform size under the 20m M concentration of Ca Cl₂.The result of electron microscope observation showed uniformity of nCaP-RAP,and the size of the nanoparticles is about200nm.ConclusionnCaP-RAP shows good biocompatibility and was prepared for subsequent experiments.Part Ⅱ nCaP-RAP inhibits smooth muscle cell proliferation and migration in vitroObjectivenCa P-RAP was prepared in DMEM medium.We observed the phagocytic effect of this nanoparticles in smooth muscle cells in vitro,compared and evaluated the both effects of rapamycin and nCaP-RAP on smooth muscle cell growth,proliferation and migration inhibition,and the expression of vascular calcification-related genes of smooth muscle cells.MethodsnCaP-RAP were prepared by adding rapamycin and Ca Cl2 to the DMEM medium for mineralization reaction according to the application conditions obtained from the previous exploration.The CCK-8 experiment was used to compare the inhibitory effects of simple rapamycin and nCaP-RAP on smooth muscle cell proliferation.In addition,western-blot assay and confocal microscopy were used to verify the inhibitory effects of rapamycin and nCaP-RAP on m TOR and downstream target phosphorylation.In addition,we used real time-PCR（RT-PCR）to further verify the regulatory effect of nCaP-RAP on smooth muscle calcification-related proteins.ResultsThe CCK-8 experiment results showed that compared with rapamycin,nCaP-RAP showed a stronger inhibitory effect on smooth muscle cell proliferation.Under the condition of low drug concentration（200n M）,rapamycin cannot inhibit the proliferation of smooth muscle cells.However,nCaP-RAP reduced 30% of smooth muscle cell proliferation under the 200 n M concentration of rapamycin.Western-blot and immunofluorescence results showed that calcium phosphate-rapamycin has significant increased effect in inhibiting the phosphorylation of m TOR and rp S6.In addition,calcium phosphate does not affect the expression of calcification-related proteins in smooth muscle cells.ConclusionsnCa P-RAP increases the phagocytic efficiency of smooth muscle cells and delays the degradation rate of rapamycin in the cytoplasm,thereby enhancing the inhibitory effect of rapamycin on the phosphorylation of m TOR and its downstream targets,which consequently reduce the proliferation of smooth muscle cells.Part Ⅲ Application of nCaP-RAP coated balloon in New Zealand rabbit iliac artery balloon injury modelObjectiveThrough high-fat diet combined with balloon injury surgery,the New Zealand rabbit illiac artery atherosclerosis model was successfully established.In addition,the solution of nCaP-RAP was sprayed onto the surface of the blank balloon by ultrasonic spraying,and the therapeutic effect of the nanoparticles was verified on the atherosclerosis model.MethodsBy balloon injury of the illiac artery endothelium combined with a high-fat diet to induce lipid precipitation,the model of illiac artery atherosclerosis was successfully established.The animals were randomly seperated into naked balloon group,calcium phosphate coated balloon group,rapamycin coated balloon group and nCaP-RAP balloon group.Two weeks after balloon injury,4 different drug-coated balloons were implanted and expanded in vivo.Angiography was performed to evaluate the vascular stenosis before and 2 weeks after administration.Additionally,oil red staining,HE staining,Masson staining,immunofluorescence and RT-PCR and other technical means were aloso performed to evaluate the therapeutic effects of these drug-coated balloons.ResultsAlthough low-concentration rapamycin balloon therapy reduces the inflammatory response in the plaque,it cannot inhibit the growth of lipid deposition and neointimal hyperplasia in the plaque,which indicated no prevention of the lumen stenosis caused by balloon injury.However,under the same drug concentration,nCaP-RAP showed a stronger inhibitory effect on inflammation and neointimal hyperplasia,and the deposition of collagen fibers,which effectively reducing stenosis by balloon injury and high-fat diet treatment.ConclusionsnCaP-RAP enhances the absorption efficiency of rapamycin in the vascular wall tissue and the residence time in the cytoplasm,thereby enhancing the inhibitory effect of rapamycin on phosphorylated m TOR.Therefore,nCaP-RAP shows a better effect on inhibiting the atherosclerotic plaque growth.