1.Introduction Atherosclerosis, characterized by the build-up of fatty plaques within the artery walls, is a major cause of death worldwide.1,2 Monocyte-derived macrophages play a pivotal role in the atherogenic process as the modulators of lipid metabolism and inflammatory responses.3,4 Aberrant cholesterol accumulation in macrophages due to the uptake of oxidized low density lipoprotein (oxLDL) results in foam cells formation, activates atherogenic inflammatory cascade, and eventually causes arterial damage and plaque rupture. Therefore, the intracellular lipid deposition of macrophages is pursued as therapeutic target to prevent atherosclerosis.Scavenger receptors (SRs)-mediated uptake of oxLDL into macrophages is being recognized as critical to lipid accumulation within the intima. Several SRs for oxLDL have been identified in macrophages, such as cluster of differentiation 36 (CD36), scavenge receptor type-A (SR-A) and lectin-like oxLDL receptor-1 (LOX-1).5,6 Although CD36 and SR-A are generally recognized as the principal contributors to cholesterol uptake in macrophages, conflicting results were reported regarding to the antiatherosclerotic efficiency of SR-A and CD36 knockout.7-9 LOX-1, an up-regulated receptor in activated macrophages is correlated with tissue factor expression and apoptosis, indicating the involvement of LOX-1 in initial stages of atherosclerosis and plaque instability.10-13 Overexpression of LOX-1 was shown to cause lipid deposition in the coronary arteries of apolipoprotein E (apoE)-deficient mice, and less pronounced development of atherosclerotic plaques was observed in LDL receptor-deficient mice with genetic deletion of LOX-1.14,15 Considering the important roles of LOX-1 in the pathogenesis of atherosclerosis, down-regulating the LOX-1 level in macrophages is appealing in atherosclerosis therapy. Small interfering RNAs (siRNA) with the capability to silence the expression of targeted protein is tremendously attractive for disease treatment. Blocking the expression of LOX-1 on macrophages via LOX-1 specific siRNA might be a promising strategy for atherosclerosis intervention. Nevertheless, naked siRNA is unfavorable for systemic delivery because of its inherent limitations such as negative charge, large molecular weight, instability, rapid elimination, poor cellular internalization and less tissue selectivity.16 To overcome the limitations, engineered vehicles have been designed as delivery carriers for stable encapsulation, protection, and intracellular trafficking.17 Among them, cell-penetrating peptides (CPPs) are one of the most popular and efficient vectors for intracellular transport.18,19 CPPs are a class of diverse peptides, typically with 5-30 amino acids, and can be taken up by cells via multiple pathways, such as direct translocation across the membrane bilayer and endocytosis-mediated uptake.20,21 However, compelling evidences manifested that CPPs had strong non-specific binding because other molecules can attach onto them and lead to the binding to non-targeted cells. As a result, this issue is probably one of the major drawbacks for the use of CPPs as delivery vehicles in vivo.22 Therefore, alternative strategies are needed to promote cell-specific delivery, such as further modification with an appropriate target ligand to elicit cell surface binding.23Cell adhesion molecule CD44 is overexpressed on the surface of impaired endothelium in atherosclerotic lesions. The primary endogenous ligand of CD44 is hyaluronic acid (HA), a naturally existing mucopolysaccharide composed of tandem disaccharide repeats of beta;-1,4-D-glucuronic acid-beta;-1,3-D-N-acetylglucosamine. As reported, HA-decorated nanocarriers could efficiently evade recognition by reticuloendothelial system (RES), owing to the similar molecular structure to polyethylene glycol (PEG). Moreover, HA is biodegradable, non-toxic, nonimmunogenic and non-inflammatory, allowing it to be widely applied in drug delivery.24-28 The up-regulation of CD44 at inflammatory sites, the high affinity of HA to CD44 and the intrinsic attributes of HA make it appealing to utilize HA for plaque targeting.30-322.Overview of the Research In view of the increased permeability of impaired endothelium and wide availability of hyaluronidase (HAase) in extracellular matrix of plaque, we developed HA-coated CPPs/siRNA nanoparticles (NPs) and hypothesized that the delivery mechanism of NPs from blood to macrophages is an ingenious multistage-targeting process. At the initial stage, in virtue of the HA coating, NPs preferentially target atherosclerotic lesions by adhering to the CD44 on the impaired endothelial cells. Subsequently, NPs translocated across the permeable endothelium via the gap between injured endothelial cells, a process that was similar to enhanced permeation and retention effect (EPR). The capping HA layer would be degraded by HAase within plaque at the subsequent stage, allowing the exposure of naked siRNA-loaded CPPs nanocomplexes (NCs) to foam cells. Then, siRNA could be efficiently delivered into foam cells via the uptake pathways of CPPs (Scheme 1).29 Noteworthy, part of the nanoparticles might be internalized by endothelial cells when interaction with CD44. We mainly focused on the translocation of nanoparticles via intercellular gap of endothelial cells and the final uptake by macrophages for gene delivery to macrophages and anti-atherosclerotic therapy.3.Future DirectionsWe will construct siRNA-condensed CPPs nanocomplexes with HA coating via electrostatic adsorption for effective siRNA delivery into macrophages within the atherosclerotic plaques. The physicochemical properties, such as particle size, zeta potential, stability test, and particle morphology wil be elaborately characterized. Furthermore, in vitro cell viability, cellular uptake behavior, internalization mechanisms, and in vitro gene silencing efficacy will be assessed. The anti-atherosclerotic efficacies involving intracellular lipid dispositions and DiI-oxLDL uptake will also be investigated. In vivo studies, including atherosclerotic lesions targeting property and atheroprotective efficacy will be comprehensively examined in apoE-deficient mice.4.The Implications of the ResearchThe results of this study will provide valuable advice for research and development of gene delivery systems targeting cardiovascular diseases and provide a new idea for the treatment of atherosclerosis.REFERENCES(1) Hansson, G. K.; Libby, P. The Immune Response in Atherosclerosis: a Double-Edged Sword. Nat. Rev. Immunol. 2006, 6, 508-519. (2) Weber, C.; Noels, H. Atherosclerosis: Current Pathogenesis and Therapeutic Options. Nat. Med. 2011, 17, 1410-1422.(3) Tabas, I. Macrophage Death and Defective Infammation Resolution in Atherosclerosis. Nat. Rev. Immunol. 2010, 10, 36-46. (4) Moore, K. J.; Sheedy, F. J.; Fisher, E. A. Macrophages in Atherosclerosis: a Dynamic Balance. Nat. Rev. Immunol. 2013, 13, 709-721.(5) Chistiakov, D. A.; Bobryshev, Y. V.; Orekhov, A. N. Macrophage-Mediated Cholesterol Handling in Atherosclerosis. J. Cell. Mol. Med. 2016, 20, 17-28. (6) Kzhyshkowska, J.; Neyen, C.; Gordon, S. Role of Macrophage Scavenger Receptors in Atherosclerosis. Immunobiology 2012, 217, 492-502. (7) Makinen, P. I.; Lappalainen, J. P.; Heinonen, S. E.; Leppanen, P.; Lahteenvuo, M. T.; Aarnio, J. V.; Heikkila, J.; Turunen, M. P.; Yla-Herttuala, S. Silencing of Either SR-A or CD36 Reduces Atherosclerosis in Hyperlipidaemic Mice and Reveals Reciprocal Upregulation of these Receptors. Cardiovasc. Res. 2010, 88, 530-538. (8) Moore, K. 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