Preparation Of Platelet Membrane Biomimetic Nanoparticles And Combination With UTMD To Treat Atherosclerosis

The management of atherosclerosis（AS）and its associated adverse cardiovascular events still remains a significant clinical challenge.This study proposed a new therapeutic strategy for AS by combining the pharmacological properties of rapamycin（RAP）,the compatibilization effects of nanoparticles（NPs）the biological properties of platelet membrane（PLT）,and the physical effects of ultrasound-targeted microbubble destruction（UTMD）.Platelet membranes are used to coat RAP-loaded PLGA（poly lactic-co-glycolic acid）NPs to prepare a novel platelet membrane-coated biomimetic nanodrug PLT-RAP@NPs.The therapeutic effect of PLT-RAP@NPs combined with UTMD technology on AS was evaluated through a series of in vitro and in vivo experiments.This study consists of the following four parts:Part 1: Preparation and characterization of PLT-RAP@NPsObjective: To optimize the prescription and synthesis of NPs to form a new platelet membrane-coated biomimetic nanodrug,and to conduct characterization experiments.Methods: RAP was loaded onto polymer PLGA by single emulsification-solvent evaporation technique,and a nanodrug RAP@NPs was synthesized.Platelet membrane vesicles extracted by density-gradient centrifugation and repeated freeze-thaw cycles were used to prepare platelet membrane-coated biomimetic nanodrug PLT-RAP@NPs by ultrasonic oscillation method.The particle size,potential and stability of nanodrug were detected by dynamic light scattering technique.The microscopic morphology of the nanodrug was observed by transmission electron microscopy.High-performance liquid chromatography was used to detect the encapsulation efficiency and drug loading efficiency of RAP in various proportions of nanodrug to determine the best drug-loading protocol for RAP.Protein expression was determined using gel electrophoresis and western blot.Results: The average particle size of nanodrug increased from 256 nm to 287 nm and the Zeta potential decreased from-18.5 m V to-15.6 m V,with the encapsulation of platelet membrane vesicles.When RAP@NPs and PLT-RAP@NPs were kept in fetal bovine serum and phosphate-buffered saline（PBS）solution at room temperature for 7 days,the particle size and surface potential remained relatively constant,both in the range of plus/minus 10 %.The drug encapsulation efficiency reached the highest when 100 mg PLGA and 3 mg RAP entered the organic solvent;the encapsulation efficiency and drug loading efficiency were 60.35% and 2.18%,respectively.PLT-RAP@NPs were transparent and spherical,and showed a uniform size and clear core-shell structure.The surface of the core was coated with a film.Gel electrophoresis showed that PLT-RAP@NPs had similar presentation of protein bands to fresh platelets and platelet membrane vesicles,and their glycoprotein content was 90.3% of that of platelet membrane vesicles.Western blot assay showed that the expression levels of PLT-RAP@NPs’ s characteristic proteins（CD47,Integrin β1 and GP Ib）were comparable with those of fresh platelets and platelet membrane vesicles.Conclusions: The nano-sized biomimetic nanodrug PLT-RAP@NPs with uniform size of particles,good dispersion,stable structure,negative surface potential,and high encapsulation efficiency was successfully prepared.PLT-RAP@NPs was successfully coated with platelet membrane,with platelet membrane proteins and characteristic functional proteins remained largely intact,providing a good structural basis for the realization of biological functions.Part 2: In vitro evaluation of PLT-RAP@NPs combined with UTMDObjective: To assess the immune escape ability,targeting and adhesion properties,cytotoxicity,and blood compatibility of PLT-RAP@NPs in vitro,and the RAP release of PLT-RAP@NPs in the presence of UTMD.Methods: Di I dye was encapsulated into PLGA to form NPs Di I@PLGA and PLT-Di I@PLGA instead of the nanodrug RAP@NPs and PLT-RAP@NPs for fluorescence detection,and then the new NPs were co-incubated with macrophages（RAW264.7）,foam cells,and endothelial cells（HUVEC）in vitro.The phagocytosis and adhesion of these cells to the NPs were observed and analyzed by fluorescence microscope.For evaluating cell proliferation and cytotoxicity,RAW264.7macrophages and vascular smooth muscle cells（VSMC）were treated with fresh DMEM containing free RAP,RAP@NPs and PLT-RAP@NPs at various RAP concentrations separately.CCK-8 method was then used to evaluate the cell viability.Hemolysis test was used to evaluate the blood compatibility of RAP@NPs and PLT-RAP@NPs with various RAP concentrations.In vitro dialysis andhigh-performance liquid chromatography were used to evaluate RAP release of PLT-RAP@NPs in the presence of UTMD.Results: After RAW264.7 cells were co-incubated with Di I@PLGA and PLT-Di I@PLGA separately for 2 h,the orange-red fluorescence intensity of the Di I@PLGA group was significantly stronger than that of the PLT-Di I@PLGA group,while the opposite result was obtained when the foam cells were co-incubated with the NPs for 2 h（both P < 0.05）.After TNF-a stimulated HUVEC up-regulated the expression of von Willebrand factor（v WF）and then co-incubated with Di I@PLGA and PLT-Di I@PLGA for 2 h,a significant overlap between orange-red fluorescence and green fluorescence（of v WF）in the PLT-Di I@PLGA group was seen,while no significant such overlap was seen in the Di I@PLGA group（P < 0.05）.With the increase of RAP concentration,the cell viability of the free RAP group gradually decreased,while the cell proliferation of RAP@NPs and PLT-RAP@NPs groups was less affected.Slow release of RAP in RAP@NPs group and PLT-RAP@NPs group was seen,and their release amount after 72 h was 42.12 % and 33.74 %,respectively.After combination with UTMD,their RAP release was raised to 75.57 % and 67.54 %,respectively.Conclusion: PLT-RAP@NPs can inhibit the phagocytosis of macrophages,enhance the uptake of foam cells,and improve the adhesion to endothelial cells and collagen through the biological functions of platelet membrane,and therefore achieve the capacity to immune escape and good passive and active targeting abilities.Due to the encapsulation of NPs and platelet membrane coating,PLT-RAP@NPs present slow RAP release and mild cytotoxicity,which can prevent the toxic side effects of high systemic drug concentration on normal tissue cells and reduce adverse reactions.Their combination with UTMD can promote RAP release,elevate the local drug concentration,and inhibit the proliferation of macrophages and VSMC,thereby achieving better therapeutic efficacy.Besides,the hemolysis rate of various concentrations of nanodrug is less than 5%,indicating PLT-RAP@NPs can be used for intravenous administration.Part 3: In vivo targeting experiment of PLT-RAP@NPs combined with UTMDObjective: To investigate the in vivo pharmacokinetics,circulation,and biodistribution of PLT-RAP@NPs,and to validate their targeting and adhesion at atherosclerotic plaques in vivo when combined with UTMD.Methods: The successful AS modeling was confirmed by ultrasonic examination and post-anatomical microscope observation.（i）IR780 labeled RAP@NPs and PLT-RAP@NPs solutions were injected into the C57BL/6 mice via the tail vein,followed by measurement of the fluorescence intensity at various time points.In addition,the in vivo pharmacokinetics and circulation were assessed by ultraviolet visible spectrophotometry.（ii）IR780 labeled RAP@NPs and PLT-RAP@NPs were injected into the C57BL/6 mice and Apo E^-/-model mice via the tail vein.These mice were sacrificed 2 h after injection and their vital organs were removed for quantitation of fluorescence intensity by small animal optical imaging system.（iii）The isolated aortae of Apo E^-/-model mice were randomized into three groups and separately immersed in PBS,RAP@NPs and PLT-RAP@NPs solutions,followed by fluorescence imaging of these aortae after 5-min immersion.（iv）Apo E^-/-model mice were randomized into five groups,PBS group,RAP@NPs group,PLT-RAP@NPs group,RAP@NPs+Sono Vue+US group,and PLT-RAP@NPs+Sono Vue+US group.The mice of these groups were injected with the corresponding solution via the tail vein.In the latter two groups,ultrasound cavitation instrument（1 MHz,2 W/cm²,40%,30 s）was used immediately after injection.The whole aorta of each mouse was removed 2 h after injection,followed by quantitation of fluorescence intensity using small animal optical imaging system.Results:（i）The fluorescence intensity in the blood of C57BL/6 mice decreased with time after injection of the NPs solutions.At 48 h after injection,the fluorescence signal in the blood of RAP@NPs-treated mice could hardly be detected,while the fluorescence intensity in the PLT-RAP@NPs-treated mice was about 28%.PLT-RAP@NPs had significantly longer half-life and lower clearance rate in vivo as compared with RAP@NPs（both P < 0.05）.（ii）IR780 labeled NPs were mainly accumulated in livers and lungs of C57BL/6 mice and Apo E^-/-model mice,followed by spleens,kidneys,and hearts,and no fluorescence signal was detected in brain tissues.The fluorescence intensity in liver and lung of PLT-RAP@NPs group was significantly weaker than RAP@NPs group（both P < 0.05）.（iii）Among Apo E^-/- model mice,fluorescence imaging showed no fluorescence signal in the PBS group,weak scattered signal in the RAP@NPs group and moderate fluorescence intensity in the PLT-RAP@NPs group.The fluorescence intensity of the whole aorta in the PLT-RAP@NPs group was significantly stronger than that in the RAP@NPs group or PBS group（both P < 0.05）.（iv）Among the five Apo E^-/-model mice groups,PLT-RAP@NPs+Sono Vue+US group had the strongest aggregation of fluorescent NPs in multiple places throughout the whole aorta,especially in the aortic arch.The fluorescence intensity of the whole aorta was significantly stronger than that of the other groups（all P < 0.05）.Conclusions: The platelet membrane-coated nanodrug PLT-RAP@NPs displays more optimized pharmacokinetic parameters,with significantly longer half-life and lower clearance rate,as well as longer presentation in the blood circulation.In C57BL/6 mice and Apo E^-/-model mice,PLT-RAP@NPs mainly accumulate in the liver and lung,and platelet membrane coating can reduce the aggregation of the NPs in the reticuloendothelial system in vivo.PLT-RAP@NPs combined with UTMD have better performance in targeting atherosclerotic lesions,and can achieve efficient targeted delivery of RAP to atherosclerotic plaques.Part 4: In vivo evaluation of PLT-RAP@NPs combined with UTMD in AS treatmentObjective: To verify that PLT-RAP@NPs combined with UTMD can effectively delay the progression and enhance the stability of atherosclerotic plaques in vivo.Methods: The successfully modeled AS Apo E^-/-mice were randomized into six groups（5 mice per group）,PBS group,free RAP group,RAP@NPs group,PLT-RAP@NPs group,RAP@NPs+Sono Vue+US group,and PLT-RAP@NPs+Sono Vue+US group.The treatment cycle was 30 days,and the injection of RAP at a dose of 0.7 mg/kg via the tail vein was conducted every 3 days.Ultrasonic cavitation instrument（1 MHz,2 W/cm²,40 %,30 s）was used immediately after the injection of NPs solution and Sono Vue solution in the latter two groups.Tissue samples of Apo E^-/-model mice were collected after the whole treatment,followed by histopathological examination（H&E staining,oil red O staining,Masson’s trichrome staining and toluidine blue staining for mouse aortae;immunohistochemical analysisof CD68,α-SMA,MMP-9 and CD31）and biological safety examination（body weight,blood lipid,blood biochemistry,and H&E staining of vital organs）.Results: The total plaque area of PBS group,free RAP group,RAP@NPs group,PLT-RAP@NPs group,RAP@NPs+Sono Vue+US group and PLT-RAP@NPs+Sono Vue+US group was about 26.30%,22.50 %,18.57 %,12.53 %,10.86 % and7.05 %,respectively.PLT-RAP@NPs+Sono Vue+US group was significantly better than other groups in terms of total plaque area（all P < 0.05）.The lipid content,collagen content and the relative numbers of macrophages and VSMC in the plaques of aortae of the six groups gradually decreased,while PLT-RAP@NPs+Sono Vue+US group presented significantly more decrease as compared with other groups（all P <0.05）.The decrease extent in platelet membrane-coated groups was higher than that in the uncoated groups,and the combination treatment with UTMD achieved significantly better results（both P < 0.05）.In terms of plaque stability,necrotic core size of the plaques at aortic sinus in the six groups was gradually reduced,along with a gradually decreased expression in MMP-9（responsible for degradation of the extracellular matrix of plaques）level and a gradually increased expression in CD31（a regulator of endothelial junctional integrity）level.Significantly better results were achieved in PLT-RAP@NPs+Sono Vue+US group as compared with other groups（all P < 0.05）.All mice survived at the end of treatment,and no significant decrease was seen in their body weight.No significant difference was seen between groups in the changes in serum lipid profile（LDL,TG,TC and HDL）,in the levels of serum ALT,ASL,BUN and CRE,and in pathological changes in the H&E-stained sections of vital organs.Conclusion: PLT-RAP@NPs combined with UTMD can significantly reduce plaque size,lipid content,and necrotic core formation,improve plaque composition,inhibit inflammatory reaction,and maintain endothelial integrity,which can effectively delay the progression and enhance the stability of atherosclerotic plaques,and lower the risk of plaque rupture.Their combination can serve as a safe and effective new modality for AS treatment.Good therapeutic efficacy of the combination of platelet membrane-coated biomimetic nanodrug PLT-RAP@NPs with UTMD for AS is achieved by combining the pharmacological properties of RAP,the compatibilization effect of NPs,the biological properties of platelet membrane,and the physical effects of UTMD.