| Atherosclerosis?AS?is a typical chronic inflammatory vascular disease.It is the predominant pathological onset of ischemic cardio-cerebrovascular diseases,including cerebrovascular disease,coronary heart disease,and peripheral arterial disease.AS seriously endangers human health.Recently,oral lipid-lowering or anti-inflammatory drugs are clinically used to inhibit the progression of AS.However,these traditional oral drug therapies still face certain issues,such as poor utilization rates,slow therapeutic effects,and severe side effects.Nanodrug-based drug delivery system can increase drug solubility,prolong drug half-life,improve its in vivo biodistribution,and reduce the side effects.Nanodrug has great potential in the clinical diagnosis and therapy for AS.However,due to the complexity of nanocarrier surface,it is extremely hard for nanodrug to be recognized and approved by organisms and subsequently be phagocytized by the immune system as a foreign matter,which fails to deliver drugs to the targeted site.Consequently,the complex physiological systems can greatly reduce the efficacy of targeted drug delivery by nanoparticles.Therefore,the construction of biomimetic nanodrug with targeted delivery and sustained drug release function based on pathological characteristics of AS is the key to achieve high efficiency and low toxicity of AS treatment.In this study,we focused on the basic pathological character of EPR effects and high expression of adhesion molecules in the inflammatory endothelium at the atherosclerotic lesion area.The macrophage membrane?MM?with the function of immune escape and specific binding ability to inflammatory endothelium was coated on the surface of rapamycin-loaded poly?lactic-co-glycolic acid?copolymer?PLGA?nanoparticles?RAPNPs?to prepare MM-coated nanodrug?MM/RAPNPs?.The results showed that MM/RAPNPs have sustained drug release,long-term blood circulation and efficient AS targeting.The biomimetic nanodrug?MM/RAPNPs?could effectively inhibit the progression of AS and demonstrated high safety in long term treatment.In detail,the main contents and conclusions of this study are as follows:?1?RAPNPs were prepared by the nano-precipitation method,in which PLGA,a material approved by US food and drug administration?FDA?,was selected as an excellent carrier and rapamycin?RAP?was chosen as an effective anti-atherosclerotic agent.The hydrodynamic diameter,surface zeta potential,morphology,drug loading and encapsulation efficiency of RAPNPs were determined.Through the nano-precipitation method,we could successfully prepare RAPNPs nanodrug,which possessed uniform nano-scale size and good dispersibility.Moreover,RAPNPs could significantly increase the solubilization of RAP and had high drug loading or encapsulation efficiency.Subsequently,MM/RAPNPs were prepared by the co-extrusion method and the property of MM/RAPNPs were characterized in vitro by size and zeta potential analysis,TEM detection,stability test,phagocytosis colocalization,membrane orientation identification,protein composition analysis,protein characterization,and drug release behavior.The results indicated that the MM/RAPNPs can be successfully fabricated by co-extrusion method.MM/RAPNPs had a typical“core-shell”structure,right-side-out membrane coating,uniform nano-scale size,good dispersibility and good stability in aqueous solution.In addition,MM/RAPNPs retained the protein composition and key function proteins?Integrin?4?1and CD47?on the surface of the nanodrug.The in vitro drug release study showed that MM/RAPNPs could slowly release RAP.Compared with the RAPNPs,MM/RAPNPs showed a slightly slower RAP release profile,thus achieving sustained drug release.?2?The hemocompatibility and biological function of MM/RAPNPs were determined in vitro.The hemolysis tests showed that both RAPNPs and MM/RAPNPs did not cause hemolysis.Measurement of platelets?-granule membrane protein in serum indicated that MM/RAPNPs did not cause the activation of platelets,so MM/RAPNPs demonstrated good hemocompatibility.The protein adsorption experiment showed that MM/RAPNPs could effectively inhibit the formation of“protein corona”in serum,benefiting from the MM coating,which promoted the stability of nanodrug in vivo.The macrophage phagocytosis tests showed that phagocytosis of nanoparticles by macrophage was time-dependence.The uptake of MM/DiDNPs by macrophages was about 2.3 times lower than that of DiDNPs after 4 h incubation,which indicated that MM/RAPNPs had the potential to inhibit phagocytosis by the immune system.The phagocytosis experiment of MM/DiDNPs by inflammatory endothelial cells?ECs?demonstrated that MM coating could significantly enhance the phagocytosis of MM/DiDNPs,which indicated that MM/RAPNPs had the potential to target inflammatory ECs at the atherosclerotic lesion area.Furthermore,in vitro anti-proliferation test of MM/RAPNPs against macrophages and smooth muscle cells?SMCs?showed that MM/RAPNPs could effectively inhibit proliferation of macrophages and SMCs.In general,MM-coated biomimetic nanodrug had good hemocompatibility,could inhibit the phagocytosis of macrophages,enhanced the phagocytosis of inflammatory ECs,and effectively inhibited the proliferation of macrophages and SMCs.?3?The therapeutic efficacy and safety of MM/RAPNPs against AS were studied by in vivo animal experiments.The results showed that the blood circulation time of MM-camouflaged nanoparticles was significantly higher than that of naked nanoparticles.It indicated that the MM coating on the surface of nanoparticles could avoid the removal of nanoparticles by the immune system,which laid a foundation for the accumulation of nanoparticles in the atherosclerotic lesion area.The MM coating could enhance the ability of nanoparticles to target atherosclerotic lesions area.In ApoE-/-mice fed with high fat diet,MM/DiDNPs had better ability to specifically target atherosclerotic lesion area than DiDNPs.Ex vivo fluorescence imaging showed that the accumulation of MM/DiDNPs in the atherosclerotic lesions increased by about 2.2-fold compared to the bare nanoparticles.In addition,compared to naked nanoparticles,MM-coated nanoparticles could reduce the accumulation in the liver,kidneys,and lungs.Next,the anti-atherosclerosis potential of MM/RAPNPs was evaluated in ApoE-/-mice.MM-coated nanodrug could effectively inhibit the progression of AS.After treatment with MM/RAPNPs for one month,the atherosclerotic plaques significantly reduced in the aorta,and only small atherosclerotic plaques were observed in the area of aortic root.The atherosclerotic lesion area decreased to only about 6.59%in the aorta,and significantly attenuated the extent of plaques in the atherosclerotic prone areas such as the aortic arch and abdominal aorta.In addition,the composition of plaques in aortic root sections was detected by ORO and immunohistochemistry staining.The results showed that MM/RAPNPs could significantly inhibited the lipid deposition and expression level of collagen in the atherosclerotic plaque to alleviate plaque deterioration.The MM/RAPNPs also significantly reduced the formation of necrotic areas,the expression level of MMP-9 and kept the integrality of endothelium in AS lesions,which maintained the stability of plaque,and reduced the risk of plaque vulnerability.Mover,MM/RAPNPs can significantly decreased the number of macrophages and smooth muscle cells in AS lesions,which inhibited the progression of AS.Finally,the long-term safety evaluation showed that MM/RAPNPs did not cause severe side effects and demonstrated excellent biocompatibility,which provided a promising strategy for long-term therapy of chronic vascular diseases.In summary,the MM/RAPNPs based on biomimetic nanodrug had excellent characteristics of nanodrug that improved the solubilization of RAP,achieved high drug loading efficiency,and facilitated sustained drug release.In addition,the biomimetic nanodrug further synergized the function of MM that can escape the immune system and actively target the atherosclerotic lesion area.Thus,MM/RAPNPs based on biomimetic nanodrug achieved long-acting blood circulation and targeted drug delivery of AS,which could efficiently and safely inhibit the progression of AS.This biomimetic nanodrug may be considered as a feasible candidate for a new class of safe and effective targeted drug delivery system for chronic inflammatory disease management. |
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