**7.1 ApoA-I mimetic peptides**

*Apolipoproteins, Triglycerides and Cholesterol*

**6.2 HDL-P measurement**

measure of HDL than HDL-C.

application [151].

**6.3 HDL: nature's nanoparticles**

(PLTP), hepatic lipase (HL), and CETP [141, 142].

HDL2a- and TG-rich lipoproteins, resulting in the formation of HDL2b subspecies. These latter particles are then transformed back to HDL3c by hydrolysis of TG and phospholipids as a result of the combined action of phospholipid transfer protein

Experimental studies pointed out that the widely used measurements of HDL-C

Compared to the artificial nanocarriers (e.g., liposomes, micelles, inorganic, and polymeric nanoparticles), HDL-based drug delivery strategies have unique features that deliver drugs, peptides/proteins, nucleic acids, and imaging agents targeted to various organs more efficiently [147]. These attributes of HDL include ultrasmall size (8–12 nm), high tolerability in humans (up to 8 g of protein per infusion), long circulating half-life (12–24 h), and intrinsic targeting properties to specific recipient cells [148, 149]. A statin-loaded rHDL nanoparticle remarkably inhibits the inflammatory responses in atherosclerotic plaque [150]. Moreover, nanoparticle-labeled HDL might be used to evaluate the stability of atherosclerotic plaque through magnetic resonance imaging (MRI) after intraperitoneal

Furthermore, HDL is the natural anticancer drug delivery system for tumor imaging and treatment, which provides tumor-selective delivery of anticancer agents while reducing harmful off-target effects [152, 153]. Therefore, utilizing HDL nanoparticles would revolutionize the future strategy for the management of a broad range of cancers. Synthetic HDL nanoparticles could act synergistically and lessen the amount of mitotane/etoposide/cisplatin needed for anticancer efficacy in adrenocortical carcinoma [154]. The binding of anticancer drug valrubicin with rHDL increases the water solubility of valrubicin, which appears ideally suited for extended applications, including systemic cancer chemotherapy [155]. Artificial HDL nanoparticles using a gold nanoparticle induce B lymphoma cell apoptosis through SR-BI-mediated cholesterol starvation and selectively inhibit B-cell

lymphoma growth in mice [156]. In addition, after delivering anti-angiogenic RNAi to endothelial cells, HDL strongly attenuates neovascularization *in vivo* and reduces

tumor growth, which might be a potential treatment for cancer [157].

levels may have obvious limitations, and the quantitative evaluation of HDL-P might be a more robust biomarker for assessing HDL functions and predicting CVD risk [11, 140]. A number of epidemiological and clinical trials, including the Heart Protection Study (HPS) [143], the Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin (JUPITER) [144], and the Multi-Ethnic Study of Atherosclerosis (MESA) [145], demonstrated that HDL-P is a stronger and more independent predictor of CVD risk than HDL-C. In JUPITER study, investigators evaluated the relationship of HDL-C and HDL-P in more than 10,000 subjects with CVD risk. JUPITER showed a significant inverse association between HDL-P and CVD risk. In contrast, HDL-C is not associated with CVD risk in statin-treated patients after adjustment for additional lipoprotein parameters [144]. MESA also found that HDL-P is a significant predictor of incident CVD events and carotid intima-media thickening (cIMT), even adjusting for HDL-C levels and other CVD confounders [145]. In addition, HDL-P is an independent predictor of major adverse cardiovascular events (MACE) among patients undergoing angiography [146]. Therefore, HDL-P might provide a more accurate and reliable

**22**

Studies found that the interaction between apoA-I and ABCA1 is not sequencespecific and instead the amphipathic helices of apoA-I are identified as the key structural motifs [158]. To further understand these helices, a model of 18-amino acid peptide (18A) was developed, which is not identical in sequence to any of the individual helices of apoA-I [159]. 18A is referred to as 2F because it contains two phenylalanine (F) residues, which could solubilize phospholipids and activate LCAT. Many peptides were further designed on the basis of 2F to enhance the biological activities of the peptides [160, 161]. Among them, 4F, containing four F residues, is the most well-studied peptide, which significantly reduces atherosclerotic lesion in apoE knockout and LDL-R null mice [162, 163]. The ability of 4F to promote cholesterol efflux was also noted, although it is not as effective as lipid-free apoA-I [164]. L-4F, synthesized with natural L-amino acids, is effective but not stable when administered orally, presumably due to its susceptibility to proteolysis in the intestine [162]. This problem was circumvented by fabrication of D-4F with D-amino acids, which displays the similar biological properties to L-4F and exerts significant antiatherogenic effects upon oral administration [162]. D-4F protects endothelial cells against ox-LDL-induced injury by antagonizing the downregulation of pigment epithelium-derived factor (PEDF) [105]. We also found that D-4F alleviates ox-LDL-induced oxidative stress and promotes endothelial repair through the eNOS/HO-1 pathway [165]. Besides, D-4F accelerates vasodilatation and restrains atherosclerosis by regulating phospholipid metabolites and decreasing plasma LysoPC in LDL-R null mice [166]. Furthermore, D-4F decreases the myocardial infarction area in hyperglycemia mice through promoting NO release and decreasing ROS generation in endothelial cells [167]. Metabolomic analysis showed that D-4F alleviates ox-LDL-induced oxidative stress and abnormal glycolysis in endothelial cells [168].

In addition, 6F is also bioactive even made from L-amino acids and presented orally [169]. End-blocked 6F is more hydrophobic than 4F, more effectively activates LCAT, and is at least as effective in binding oxidized lipids [170, 171]. The 5A peptide possesses many functional attributes of native apoA-I including cholesterol efflux, inhibition of LDL oxidation, and suppression of inflammation [172]. Additionally, 5A reduces atherosclerosis and prevents the induction of asthma in mouse models [173, 174].

#### **7.2 ApoE-mimetic peptides**

ApoE is a multifunctional apolipoprotein that associates with VLDL, LDL, and subsets of HDL. It participates in the clearance of these lipoproteins from plasma, by serving as ligand for LDL-R and its family of related receptors. Like apoA-I, it is also active in RCT and has anti-inflammatory and anti-oxidative activities [175]. These properties are believed to contribute to the antiatherogenic functions of apoE. Mimetic peptides derived from apoE have been developed. AT1–5261 is an apoE-mimetic peptide containing 25 amino acids [176]. In the lipid-free state, ATI-5261 efficiently promotes ABCA1-mediated cholesterol efflux. When the peptide is complexed with phospholipids, it is still capable of promoting cholesterol efflux in a partially ABCA1-dependent fashion [176].

#### **7.3 Dual-domain peptides**

The rationale for constructing the dual-domain peptide (Ac-hE18A-NH2) is that 18A promotes the association of lipid-free apoE ligand-binding sequence with lipoproteins. The peptide Ac-hE18A-NH2 lowers plasma cholesterol levels in hyperlipidemic mice [177, 178]. *In vitro* studies, Ac-hE18A-NH2 also decreases monocyte adhesion to endothelial cells, attenuates LPS-induced inflammatory responses in HUVECs, and reduces lipid hydroperoxides in LDL [178]. Compared to 4F, Ac-hE18A-NH2 peptide was also shown to promote cholesterol efflux, improve endothelial dysfunctions, and lower plasma lipid hydroperoxides [179]. 4F binds oxidized lipid with high affinity, and Ac-hE18A-NH2 rapidly reduces plasma cholesterol levels, including lowering VLDL and LDL levels [177].
