Functionalization of liposomes with ApoE-derived peptides affects cellular uptake and drug transport to atherosclerotic plaque

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However, there remains an apparent gap between laboratory discovery and clinical translation of nanomaterials. Therefore, we constructed a new nanomaterial that selected liposomes as drug-delivery carriers. and modified with Apolipoprotein E (ApoE) peptides which may alleviate atherosclerotic plaque to seek a novel idea about treatment of atherosclerosis . Methods We successfully constructed nanoparticles loaded with Atorvastatin and modified them with aE15A (ApoE peptide) (Lip@A@15A). In vitro, the study of cellular uptake of nanomaterials by macrophages was detected by Confocal laser scanning microscopy (CLSM), and concentrations of inflammatory factors were detected using Enzyme-linked Immunosorbent Assay. In vivo, ApoE-/- mice were used to construct atherosclerotic models that were treated with many reagents, including Lip@A@15A, in which the stability of atherosclerotic plaque in aortas and the macrophage-derived foam cells were observed by oil “O” and Masson staining. Results Lip@A@15A showed low toxicity in an in vitro cytotoxicity test and had the highest phagocytic efficiency by macrophages compared to liposomes and nanoparticles loaded with atorvastatin (Lip@A). Therefore, the function of lipid phagocytosis and the levels of IL-1βand TNF-αin macrophages treated with Lip@A@15A were the lowest compared with that of macrophages treated with the other three reagents (P<0.05). More importantly, Lip@A@15A may be localized in the aortic intima. Lip@A@15A markedly improved plaque stability and inhibited plaque rupture compared with mice treated with other reagents. Conclusion Relative to Lip@A, Lip@A@15A more significantly enhanced endocytosis by macrophages and impeded inflammatory factors from macrophages. In addition, it actively targeted atherosclerotic plaques and improved plaque stability. ApoE peptides Atorvastatin Liposome Atherosclerosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction For decades, atherosclerotic cardiovascular disease (ASCVD) has been a severe burden on the healthcare system worldwide[ 1 ]. It is the leading cause of death in China, accounting for more than 40% of the deaths[ 2 , 3 ]. Atherosclerosis (AS) is a complex disease process and is a pathological disease characterized by vascular injury, chronic inflammation, lipid accumulation, and immune disorder[ 4 , 5 ]. Vascular endothelial cell dysfunction, induced by various conditions, facilitates low-density lipoprotein (LDL) accumulation in the intima, where LDL can undergo oxidative. Afterward, macrophages proliferate and uptake oxidized-low-density lipoprotein (ox-LDL) to form foam cells and release inflammatory factors[ 6 , 7 ]. As the disease progresses, the deposition of lipids will lead to the thickening of arterial walls and, over time, may cause lumen narrowing of the arteries at different levels[ 8 ]. Eventually, in a condition of a sizeable necrotic core covered by thin fibrous caps, the plaques are prone to rupture, which may cause acute cardiovascular events[ 9 – 11 ]. At present, drug therapy remains the crucial clinical treatment for atherosclerosis[ 9 , 12 ]. As an essential drug for the treatment of atherosclerosis, long-term use of statins may elicit serious side effects and low compliance in patients [ 13 , 14 ]. With the rapid development of nanotechnology, nanomedicine has become a strategy to solve the problem. Compared to conventional drugs, nanomaterials could enhance the therapeutic effect[ 15 , 16 ], bioavailability[ 17 ], and target specificity[ 18 , 19 ]. Furthermore, nanomaterials have impressive potential as drug carriers[ 20 – 22 ]. Advances in nanomaterials have opened new avenues for the treatment of cardiovascular diseases. The application of nanocarriers has been extensively considered in cardiology[ 23 ]. Although nanomaterials provide robust drug delivery systems for the treatment of AS, nanomedicine faces many obstacles in clinical translation[ 24 , 25 ]. Due to interacting with the bio-environment in the body complicatedly[ 20 ], traditional nanomaterials are characterized by easy clearance, off-targeting, and uncontrolled release[ 26 – 28 ]. Biomimetic nanomaterials have characteristics, such as self-marking, natural tropism, and cell entry mechanisms, to enhance the therapeutic effect of drugs. So, the nature-inspired nanocarriers are expected to overcome drug delivery barriers for AS treatment with optimized natural properties [ 24 ]. Apolipoprotein E (ApoE) is a member of the soluble apolipoprotein family and a significant component of lipoproteins. It is a vital ligand of lipoproteins and clears triglycerides and cholesterol[ 29 ]. Studies show that ApoE has a significant anti-atherosclerosis role[ 30 – 32 ]. It acts synergistically with apolipoprotein A-I in facilitating cholesterol efflux from foam cells[ 33 ], reversely transports cholesterol to the liver by specifically binding to LDL receptors (LDLR)[ 34 ], and regulates inflammatory and immune responses[ 32 ]. It has also been reported that ApoE binds to LDLR at the N-terminal domain of amino acid residues[ 29 , 35 ]. LDLR is expressed in various tissues including vascular endothelial cells, smooth muscle cells, macrophages, and hepatocytes broadly, and participates in lipid metabolism and atherosclerosis development[ 36 – 38 ]. The LDLR family has identical sequences, including A-type repeats, epidermal growth factor (EGF)-like domains, transmembrane domains, and cytoplasmic domains. The A-type repeats are located at the amino termini, consisting of seven homologous sequences (LR1-LR7), which act as a binding region for ApoE[ 39 , 40 ]. So considering these challenges for clinical application, we selected liposomes which are one of the most common organic vehicles with stable physicochemical properties and high drug-loading capacity.. It has been extensively applied in the biomedical field because of its advantages of non-immunogenicity, sustained-release of medicines, and non-toxicity in vivo[ 41 , 42 ].Liposomal doxorubicin was the first nanomaterial approved by the Food and Drug Administration (FDA) for clinical treatment[ 24 , 43 ]. Given the specific binding between ApoE and LDLR, we designed liposomes modified with a peptide targeting ApoE loaded with atorvastatin (Lip@A@15A) to improve macrophage endocytosis and inhibit the release of inflammatory factors. In addition, it further targets atherosclerotic plaques and increases plaque stability. Due to the practical clinical transformation ability of liposomes and the anti-atherosclerotic effect of ApoE, Lip@A@15A may provide novel and effective ideas for the clinical treatment of atherosclerosis. Results Constructing nanomaterial of liposome modified with peptide targeting ApoE(Lip@A@15A) Liposomes, liposomes with atorvastatin (Lip@A), and liposomes with fluorescent Cy5 (Lip@Cy5) were prepared by thin-film dispersion, and the drug content (DC) and encapsulation ratio (EE) were 10.63% and 92.43%.the outcome was determined using the following equation. DC% = loaded atorvastatin/total solid content of liposomes × 100% = 27.73/260.8 * 100% = 10.63%. EE% = atorvastatin loaded/atorvastatin dose × 100% = 27.73/30 × 100% = 92.43%. The synthesized ApoE peptide containing an amino group was combined with carboxyl drug-carrying liposomes to form Lip@A@15A. The polypeptide-coupling rate of Lip@A@15A was 93% (Table 1 ). Table 1 The coupling rate of Lip@A modified with polypeptides was 93.2%. Group Number of unconjugated peptides(mg) Total peptides(mg) Number of conjugated peptides(mg) Conjugated rate of peptide (%) Lip@A 0.51 7.5 6.99 93.2 Lipsome 0.68 7.5 6.82 90.9 The spherical morphology and dispersity of the nanomaterials were observed using transmission electron microscopy (TEM). The liposomes Lip@A and Lip@A@15A had spherical particles of uniform size and even dispersity (Fig. 1 a). The dynamic light scattering further detected that the particle size of different nanomaterials was about 89.96 nm to 114.5 nm, with PDI values from 0.209 to 0.354 and negative zeta potential between − 13.52 to -36.27 mv (Fig. 1 b, c). The nanomaterials of Lip@A@15A were effectively devoured by RAW264.7 cells Macrophages play a major role in the development of atherosclerosis, drug uptake, and targeted therapy. The endocytic nanomaterials in RAW264.7 cells were observed using CLSM. After incubation with liposomes, Lip@A, and Lip@A@15A (containing Cy5) for a certain time, the fluorescence intensity of Lip@A@15A in macrophages was more obvious than that in the liposome and Lip@A groups (Fig. 2 a). Quantification by flow cytometry further confirmed that the ratio of macrophages carrying nanomaterials in the Lip@A@15A group was significantly higher than that in the liposomes and Lip@A groups, which was time-dependent (P < 0.001) (Fig. 2 b, c). Lip@A@15A inhibited the formation of foam cells and the levels of inflammatory factors secreted by macrophages The formation and necrosis of foam cells induced by ox-LDL is a crucial element in the AS process and is the fundamental cause of the formation of atherosclerotic plaques and core necrosis. Therefore, we investigated the effect of the nanomaterials on the cellular uptake of ox-LDL by RAW264.7 cells. As shown in( Fig. 3 a, b), atorvastatin and Lip@Aand Lip@A@15A could inhibit ox-LDL-stimulated accumulation of lipid droplets in macrophages, and Lip@A@15A could better reduce lipid accumulation (P < 0.001). Besides, the level of inflammatory factors including TNF-α, IL-1β, and MCP-1 was lower in Lip@A@15A treated macrophages than in atorvastatin-treated cells (P < 0.001) (Fig. 3 c). These results indicate that atorvastatin-loaded nanomaterials are more potent at suppressing foam cell formation and inflammatory factor production than atorvastatin alone. Lip@A@15A was especially targeting and localizing on Aortic intima To determine the targetability of the nanomaterials, C57BL/6 and ApoE-/- mice were injected with the nanomaterials via the tail vein. More Lip@A@15A appeared in aortic vessels of C57BL/6 mice at 3h after injection compared to Lip@A (P < 0.05). However, no time-dependent effects were observed (Fig. 4 a, b). In ApoE-/- mice, the intensity of Lip@A@15A in the aortic vessels was higher than that of Lip@A at every time point (3h, 6h, 12h), and Lip@A@15A in the aorta increased as the treatment time was prolonged(P < 0.001) (Fig. 4 c, d). These results illustrate that the nanomaterials could accurately target atherosclerotic plaques and had increasing effects as the treatment time increased. Lip@A@15A inhibited atherosclerotic plaque in AS models To detect the effects of Lip@A@15A on AS treatment, ApoE-/- mice were used to construct an AS model and treated with various nanomaterials, including Lip@A@15A. The entire aorta and aortic roots were harvested and stained with ORO. The ORO-positive areas in the vessels of AS models treated with Lip@A@15A were the smallest compared to those of the other two AS models treated with PBS or Lip@A. Lip@A also alleviated lipid enrichment in the aorta compared with the control group (P < 0.001) (Fig. 5 a). Consistent with these results, ORO staining of the aortic root also showed the most significant anti-atherosclerotic function of Lip@A@15A (P < 0.001). EVG staining represents the integrity of elastic fibers. The EVG-stained areas were low and discontinuous in the PBS-or Lip@A-treated AS mice. Interestingly, the EVG-stained areas were significantly larger and more continuous in the Lip@A@15A-treated AS mice than in the other two groups (P < 0.05). Mac-3-stained areas in mice treated with Lip@A or Lip@A@15A were darker than those in PBS-treated mice, indicating that collagen fibers were augmented by Lip@A or Lip@A@15A. In addition, the aortic root was stained for MCP-1, a monocyte chemokine, and a pro-inflammatory factor. The expression of MCP-1 was reduced in Lip @A-treated mouse plaques compared with that in the PBS-treated group. Mice treated with Lip@A@15A showed the lowest MCP-1 expression in the plaques compared to the other two groups (Fig. 5 b). These results indicated that Lip@A@15A had a more prominent anti-atherosclerotic effect than Lip@A. Safety evaluation of nanomaterials We measured the toxicity of these nanomaterials in RAW264.7 macrophages. The effects of Lip@A@15A on cell viability were very low and showed no significant dose-dependence, even at 100 µg/mL, the cell viability was still above 70%, indicating that Lip@A@15A had low cytotoxicity (Fig. 6 a). After injecting the nanomaterials into the tail vein, immunohistochemical staining of the important organs of the mice showed no obvious cellular tissue damage, indicating that the effect was safe in vivo (Fig. 6 b) Discussion As drug carrier systems, nanomaterials can be effectively applied in the treatment of cardiovascular diseases due to its superior physicochemical properties[ 23 ]. However, given the dilemma of clinical translation, the design of nanomaterials is particularly critical.[ 44 ]. The modification of peptides is one of the common nanodesign options [ 45 , 46 ]. Since liposomes have been successfully used in clinical tumor therapy, we choose ApoE peptides that binds specifically to LDLR to modify liposomes. in order to increase targeting and possibility of clinical translation of nanomaterials. Therefore, we synthesized Lip@A@15A, which was efficiently and rapidly taken up by macrophages. Ingested Lip@A@15A dramatically decreased ox-LDL uptake, lipid infiltration, macrophage-derived foam cell formation, and cellular inflammatory responses. In an in vivo study, Lip@A@15A accumulated in atherosclerotic plaques further alleviated atherosclerotic plaques in the aortic vessels and postponed the development of atherosclerosis in ApoE-/- mice. These results suggested that relative to Lip@A, Lip@A@15A had more significant uptake by macrophages to penetrate atherosclerotic plaques and suppress formation of foam cells and release of inflammatory factors from macrophages. In addition, Lip@A@15A enhanced atherosclerotic plaque stability, lowered cholesterol crystals, diminished the inflammatory response, and increased the amount of collagen and elastic fibers, suggesting a stronger anti-atherogenic effect than in the control group. At the same time, the safety was verified in vivo and in vitro. In conclusion, we demonstrated that Lip@A@15A can improve microevironment of macrophages, and it is a prospective therapeutic drug for the treatment of atherosclerosis that enhances the bioavailability of statins, aortic targeting, and therapeutic efficacy. Furthermore, the adverse effects and clinical dose of atorvastatin were reduced by Lip@A@15A, which improved patient compliance and promoted statin use in atherosclerotic therapy. Methods(EXPERIMENTAL SECTION) Materials Lecithin, Cholesterol (Aladdin), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), Atorvastatin (McLean), peptides (flame trite), N-hydroxysuccinimide (NHS), 1- Ethyl − 3- (3-dimethylaminopropyl) carbondiimide hydrochloride (EDC, Sigma). anhydrous dimethylsulfoxide (DMSO), fatty acid methyl ester sulfonate (MES), anhydrous dimethylformamide (DMF), chloroform (Tris-Hcl) Human high-oxidized low-density lipoprotein (ox-LDL), Cyanine5 NHS ester (Cy5), Recombinant murine interferon-γ (TNF-γ). Synthesis of Atorvastatin-loaded liposome modified peptide (Lip@A@15A). 1. Synthesis of Atorvastatin loaded liposome (Lip@A). Lip@A was prepared using a thin-film dispersion method. Atorvastatin, lecithin, DSPE-mPEG-COOH, cholesterol, and chloroform were dissolved adequately by ultrasonication, followed by evaporation at 70 ℃ at 30 rpm. Then the solution was hydrated at 60 ℃ at 17 rpm for 40 min and then homogenized by ultrasonic probes for 9 min under ice bath, followed by washing. After passing through a 0.22µm microporous membrane, the Lip@A was prepared. 2. Synthesis of peptide ( 15A). The ApoE domain, which spans residues 136–150 in the N-terminal domain, is critical for the specific binding to LDLR. The amino acid sequence LGQSTEEIRARLSTH was queried using UniProtKB and synthesized by a company (Jiangsu Qiangyao). 3. Drug content(DC)and encapsulation efficiency(EE) . The amount of Atorvastatin was determined by high-performance liquid chromatography (HPLC) using an Apollo C18 column at a UV absorption wavelength of 240 nm. The drug loading and encapsulation efficiencies of Atorvastatin in the liposomes were calculated. 4. Preparation of peptide-modified Lip@A. Lip@A was conjugated to MES and diluted with deionized water. The peptide and EDC were then added. The reaction was performed overnight at 37°C on a shaker at 150 rpm. After centrifugation and washing, the supernatant was used to measure the peptide coupling rate using bicinchoninic acid (BCA), and the remaining Lip@A@15A was collected. Fluorescence spectra The fluorescence spectra of Cy5 and Cy5 modified liposome (Lip@Cy5) were obtained using a fluorescence spectrophotometer. Characterization of Nanoparticles Size, size distribution profiles, polydispersity index (PDI), and zeta-potential of nanoparticles were quantified using a Malvern Zetasizer NanoZS instrument at 25°C. Transmission electron microscopy (TEM) was performed using a JEM-1400 PLUS microscope (JEOL, Tokyo, Japan). Cytotoxicity Evaluation Mouse macrophages (RAW264.7) were cultured in 96-well plates in Dulbecco ’ s modified Eagle’s medium (DMEM) with fetal bovine serum (FBS), penicillin, and streptomycin. After the cells were treated with various concentrations of the nanomaterials, cell viability was assayed using the Cell Counting Kit-8 assay (CCK-8). The study of cellular uptake of nanomaterials RAW264.7 cells were cultured in 12-well plates and incubated for 12h in medium different nanomaterials containing Cy5. After centrifugation and washing, the cells were harvested for flow cytometry. RAW264.7 cells were cultured in confocal dishes and incubated for 12h in different medium nanomaterials containing Cy5. Then RAW264.7 cells were stained with 4, 6-Diamidino-2-phenylindole (DAPI). Confocal laser scanning microscopy (CLSM) was performed to acquire fluorescent images. Inhibition of generation of foam cells and Anti-Inflammatory functions of nanomaterials RAW264.7 cells were cultured in 24-well plates and treated with medium, 100 ng/mL LPS and 10ng/mL IFN-γ and nanomaterials, separately. Supernatants were harvested and concentrations of Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), and Monocyte chemotactic protein-1 (MCP-1) were detected using Enzyme-linked Immunosorbent Assay (ELISA). Finally, all groups were incubated with ox-LDL and stained with Oil Red O (ORO) and hematoxylin. The cells were observed under a light microscope. Animals All the procedures and protocols were approved by the Institutional Animal Ethics Committee of the National Center for Nanoscience and Technology. Male C57BL/6 and Apolipoprotein E-deficient (ApoE-/-) mice (approximately 6 weeks old) were fed a high-fat and high-cholesterol diet. Mice were purchased from Beijing Viton Lever. Distribution and anti-atherosclerosis effect of nanomaterials in Vivo C57BL/6 and ApoE-/-mice were fed a high-fat, high-cholesterol diet for 3 months. The nanomaterials were then administered via intravenous (i.v.) injection to mice. The mice were euthanized and the aortas and vital organs, including the heart, liver, spleen, lung, and kidney, were separated and imaged using an In Vivo Imaging System (IVIS). ORO Staining and immunohistochemistry of plaque of entire aortas and aortic root ApoE-/- mice were randomly assigned and different treatments were conducted for additional 2 months. The mice were intravenously injected with saline or various nanomaterials. The Mice were euthanized, and the entire aorta and aortic root were collected and stained with ORO. The aortic roots were immunohistochemically stained with Verhoeff’s Van Gieson (EVG), macrophage differentiation antigen-3 (Mas-3), and monocyte chemotactic protein-1 (MCP-1). Statistical analysis All data are presented as mean ± standard error (SE), The ANOVA test for experiments with multiple groups, while a two-tailed, unpaired t-test was used for data with two groups. * means P < 0.05, ** means P < 0.01, *** means P < 0.001. Declarations Acknowledgments We would like to thank the teachers of the CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics for their guidance in the design of the experiment, and all members of the laboratory for their help with the experiment. Authors’ contributions CL and ZTJ jointly designed and completed the tests and analyzed the results. CL, XZM, and TXX collated, consulted, and wrote the paper. HHX reviewed paper. Availability of data and materials Data will be made available on request. Funding This study was supported by the Hospital-level Scientific Research Project in the Affiliated Hospital of North Sichuan Medical College (2020ZD002) , Scientific Research Project of Nanchong Municipal Science and Technology Bureau, Sichuan Province (19SXHZ0192) and project for the First-class Pharmaceutical Sciences of North Sichuan Medical College (CBY21-YLXK03）. 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Li W, Gonzalez KM, Chung J, Kim M, Lu J. Surface-modified nanotherapeutics targeting atherosclerosis. Biomater Sci. 2022;10:5459–71. Additional Declarations No competing interests reported. Supplementary Files floatimage1.jpeg Graphical Abstract: Lip@A@15A targets and alleviates atherosclerotic plaque. Synthesis of Lip@A@15A: Liposome-loaded atorvastatin (Lip@A) modified with ApoE peptide (LGQSTEEIRARLSTH). (b) By binding with LDLR, Lip@A@15A is more easily swallowed by macrophages, and delivers atorvastatin to foam cells, reduces the release of inflammatory factors (TNF-α、IL-1β、MCP-1) and stabilizes atherosclerotic plaque. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4237089","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":291207321,"identity":"18fb1e08-28ac-4590-b70e-0cc1a9013f1d","order_by":0,"name":"ling chen","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"ling","middleName":"","lastName":"chen","suffix":""},{"id":291207323,"identity":"c4c2d458-e027-49cf-8ee8-e09378010f37","order_by":1,"name":"tingjun zhang","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"tingjun","middleName":"","lastName":"zhang","suffix":""},{"id":291207324,"identity":"450c62fa-1978-4b76-8684-14e5467e1da5","order_by":2,"name":"Cai Rong","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Cai","middleName":"","lastName":"Rong","suffix":""},{"id":291207325,"identity":"1f9cba76-92d5-4fd2-9f36-9b4f91f32e1b","order_by":3,"name":"zhengming xu","email":"","orcid":"","institution":"North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"zhengming","middleName":"","lastName":"xu","suffix":""},{"id":291207327,"identity":"ac172582-f043-4290-9471-079ef0c67ce0","order_by":4,"name":"xiaoxue tang","email":"","orcid":"","institution":"North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"xiaoxue","middleName":"","lastName":"tang","suffix":""},{"id":291207328,"identity":"0b413590-39b9-427f-8b4e-833331fc5040","order_by":5,"name":"Houxiang Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACAwkGhgNAWg7KZyZeizFpWkAgsYFoLebSPYYHfu6oTd/OfzrxA0OFdWID+9kDeLVYzjmWcLD3zPHcnTNyN0swnElPbODJS8DvsBvJBw7wth3L3XCDdxsDY9vhxAYJHgMCWhIbDv5tO5ZucP4sUMs/orQkHzjM21aTYHAgF6ilgSgtaQmHZdsOGG64AfRLwrF04zaeHEJacow/vm2rkwc6bOOHDzXWsv3sZ/BrgYLDECoBiNmIUQ8EdUSqGwWjYBSMghEJAHjaTN6KfSKRAAAAAElFTkSuQmCC","orcid":"","institution":"Academician Workstation, Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":true,"prefix":"","firstName":"Houxiang","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2024-04-08 14:15:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4237089/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4237089/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54745566,"identity":"e2f8ca65-0b34-4d8f-8071-6cdade000465","added_by":"auto","created_at":"2024-04-16 07:12:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":500364,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-4237089/v1/631685bf92e3fc1b48d30de3.png"},{"id":54745569,"identity":"3d641e13-0bfc-4a7c-b78c-7a3acc66e0ab","added_by":"auto","created_at":"2024-04-16 07:12:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":256014,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-4237089/v1/40ed2c1b53a536241636b001.png"},{"id":54745113,"identity":"26dd7c3f-145d-46bc-810b-d9c3e9964871","added_by":"auto","created_at":"2024-04-16 07:04:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":638826,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-4237089/v1/74bbc0409cb510fa97573f04.png"},{"id":54745115,"identity":"7843a77f-6245-46ba-9968-14ba0fed95e6","added_by":"auto","created_at":"2024-04-16 07:04:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":556202,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-4237089/v1/f4439b59cf3e5bae09ed14d9.png"},{"id":54746168,"identity":"57893bb9-f6fc-495c-9db1-c61c19d4a6b6","added_by":"auto","created_at":"2024-04-16 07:20:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1483889,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-4237089/v1/f23406444caf33b604f63e6f.png"},{"id":54745567,"identity":"351a1962-3ea4-4ff7-b977-6c2f27d1ea90","added_by":"auto","created_at":"2024-04-16 07:12:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2480383,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-4237089/v1/0c015926429fac86e8a738bf.png"},{"id":56345120,"identity":"c3d73baf-4227-4a3c-a849-da9572151130","added_by":"auto","created_at":"2024-05-13 02:02:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2773361,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4237089/v1/444595b5-67f7-42de-b11f-4cc820b08c32.pdf"},{"id":54745112,"identity":"da9acbce-77e6-47d4-99fe-bbb9e830fa1e","added_by":"auto","created_at":"2024-04-16 07:04:26","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":117963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract: \u0026nbsp;\u003c/strong\u003eLip@A@15A targets and alleviates atherosclerotic plaque.\u003c/p\u003e\n\u003cp\u003eSynthesis of Lip@A@15A: Liposome-loaded atorvastatin (Lip@A) modified with ApoE peptide (LGQSTEEIRARLSTH). (b) By binding with LDLR, Lip@A@15A is more easily swallowed by macrophages, and delivers atorvastatin to foam cells, reduces the release of inflammatory factors (TNF-α、IL-1β、MCP-1) and stabilizes atherosclerotic plaque.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4237089/v1/12798bf497f7b4a9a087131c.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functionalization of liposomes with ApoE-derived peptides affects cellular uptake and drug transport to atherosclerotic plaque","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFor decades, atherosclerotic cardiovascular disease (ASCVD) has been a severe burden on the healthcare system worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is the leading cause of death in China, accounting for more than 40% of the deaths[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Atherosclerosis (AS) is a complex disease process and is a pathological disease characterized by vascular injury, chronic inflammation, lipid accumulation, and immune disorder[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Vascular endothelial cell dysfunction, induced by various conditions, facilitates low-density lipoprotein (LDL) accumulation in the intima, where LDL can undergo oxidative. Afterward, macrophages proliferate and uptake oxidized-low-density lipoprotein (ox-LDL) to form foam cells and release inflammatory factors[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As the disease progresses, the deposition of lipids will lead to the thickening of arterial walls and, over time, may cause lumen narrowing of the arteries at different levels[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Eventually, in a condition of a sizeable necrotic core covered by thin fibrous caps, the plaques are prone to rupture, which may cause acute cardiovascular events[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. At present, drug therapy remains the crucial clinical treatment for atherosclerosis[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. As an essential drug for the treatment of atherosclerosis, long-term use of statins may elicit serious side effects and low compliance in patients [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. With the rapid development of nanotechnology, nanomedicine has become a strategy to solve the problem. Compared to conventional drugs, nanomaterials could enhance the therapeutic effect[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], bioavailability[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and target specificity[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Furthermore, nanomaterials have impressive potential as drug carriers[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Advances in nanomaterials have opened new avenues for the treatment of cardiovascular diseases. The application of nanocarriers has been extensively considered in cardiology[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Although nanomaterials provide robust drug delivery systems for the treatment of AS, nanomedicine faces many obstacles in clinical translation[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Due to interacting with the bio-environment in the body complicatedly[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], traditional nanomaterials are characterized by easy clearance, off-targeting, and uncontrolled release[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Biomimetic nanomaterials have characteristics, such as self-marking, natural tropism, and cell entry mechanisms, to enhance the therapeutic effect of drugs. So, the nature-inspired nanocarriers are expected to overcome drug delivery barriers for AS treatment with optimized natural properties [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eApolipoprotein E (ApoE) is a member of the soluble apolipoprotein family and a significant component of lipoproteins. It is a vital ligand of lipoproteins and clears triglycerides and cholesterol[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Studies show that ApoE has a significant anti-atherosclerosis role[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. It acts synergistically with apolipoprotein A-I in facilitating cholesterol efflux from foam cells[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], reversely transports cholesterol to the liver by specifically binding to LDL receptors (LDLR)[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and regulates inflammatory and immune responses[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt has also been reported that ApoE binds to LDLR at the N-terminal domain of amino acid residues[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. LDLR is expressed in various tissues including vascular endothelial cells, smooth muscle cells, macrophages, and hepatocytes broadly, and participates in lipid metabolism and atherosclerosis development[\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The LDLR family has identical sequences, including A-type repeats, epidermal growth factor (EGF)-like domains, transmembrane domains, and cytoplasmic domains. The A-type repeats are located at the amino termini, consisting of seven homologous sequences (LR1-LR7), which act as a binding region for ApoE[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. So considering these challenges for clinical application, we selected liposomes which are one of the most common organic vehicles with stable physicochemical properties and high drug-loading capacity.. It has been extensively applied in the biomedical field because of its advantages of non-immunogenicity, sustained-release of medicines, and non-toxicity in vivo[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].Liposomal doxorubicin was the first nanomaterial approved by the Food and Drug Administration (FDA) for clinical treatment[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Given the specific binding between ApoE and LDLR, we designed liposomes modified with a peptide targeting ApoE loaded with atorvastatin (Lip@A@15A) to improve macrophage endocytosis and inhibit the release of inflammatory factors. In addition, it further targets atherosclerotic plaques and increases plaque stability. Due to the practical clinical transformation ability of liposomes and the anti-atherosclerotic effect of ApoE, Lip@A@15A may provide novel and effective ideas for the clinical treatment of atherosclerosis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstructing nanomaterial of liposome modified with peptide targeting ApoE(Lip@A@15A)\u003c/h2\u003e \u003cp\u003eLiposomes, liposomes with atorvastatin (Lip@A), and liposomes with fluorescent Cy5 (Lip@Cy5) were prepared by thin-film dispersion, and the drug content (DC) and encapsulation ratio (EE) were 10.63% and 92.43%.the outcome was determined using the following equation.\u003c/p\u003e \u003cp\u003eDC% = loaded atorvastatin/total solid content of liposomes \u0026times; 100% = 27.73/260.8 * 100% = 10.63%.\u003c/p\u003e \u003cp\u003eEE% = atorvastatin loaded/atorvastatin dose \u0026times; 100% = 27.73/30 \u0026times; 100% = 92.43%.\u003c/p\u003e \u003cp\u003eThe synthesized ApoE peptide containing an amino group was combined with carboxyl drug-carrying liposomes to form Lip@A@15A. The polypeptide-coupling rate of Lip@A@15A was 93% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe coupling rate of Lip@A modified with polypeptides was 93.2%.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of unconjugated peptides(mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal peptides(mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNumber of conjugated peptides(mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eConjugated rate of peptide (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLip@A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e93.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLipsome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e90.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe spherical morphology and dispersity of the nanomaterials were observed using transmission electron microscopy (TEM). The liposomes Lip@A and Lip@A@15A had spherical particles of uniform size and even dispersity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The dynamic light scattering further detected that the particle size of different nanomaterials was about 89.96 nm to 114.5 nm, with PDI values from 0.209 to 0.354 and negative zeta potential between \u0026minus;\u0026thinsp;13.52 to -36.27 mv (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe nanomaterials of\u003c/b\u003e Lip@A@15A \u003cb\u003ewere effectively devoured by RAW264.7 cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMacrophages play a major role in the development of atherosclerosis, drug uptake, and targeted therapy. The endocytic nanomaterials in RAW264.7 cells were observed using CLSM. After incubation with liposomes, Lip@A, and Lip@A@15A (containing Cy5) for a certain time, the fluorescence intensity of Lip@A@15A in macrophages was more obvious than that in the liposome and Lip@A groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Quantification by flow cytometry further confirmed that the ratio of macrophages carrying nanomaterials in the Lip@A@15A group was significantly higher than that in the liposomes and Lip@A groups, which was time-dependent (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLip@A@15A inhibited the formation of foam cells and the levels of inflammatory factors secreted by macrophages\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe formation and necrosis of foam cells induced by ox-LDL is a crucial element in the AS process and is the fundamental cause of the formation of atherosclerotic plaques and core necrosis. Therefore, we investigated the effect of the nanomaterials on the cellular uptake of ox-LDL by RAW264.7 cells. As shown in( Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b), atorvastatin and Lip@Aand Lip@A@15A could inhibit ox-LDL-stimulated accumulation of lipid droplets in macrophages, and Lip@A@15A could better reduce lipid accumulation (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Besides, the level of inflammatory factors including TNF-α, IL-1β, and MCP-1 was lower in Lip@A@15A treated macrophages than in atorvastatin-treated cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). These results indicate that atorvastatin-loaded nanomaterials are more potent at suppressing foam cell formation and inflammatory factor production than atorvastatin alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLip@A@15A was especially targeting and localizing on Aortic intima\u003c/h2\u003e \u003cp\u003eTo determine the targetability of the nanomaterials, C57BL/6 and ApoE-/- mice were injected with the nanomaterials via the tail vein. More Lip@A@15A appeared in aortic vessels of C57BL/6 mice at 3h after injection compared to Lip@A (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no time-dependent effects were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). In ApoE-/- mice, the intensity of Lip@A@15A in the aortic vessels was higher than that of Lip@A at every time point (3h, 6h, 12h), and Lip@A@15A in the aorta increased as the treatment time was prolonged(P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). These results illustrate that the nanomaterials could accurately target atherosclerotic plaques and had increasing effects as the treatment time increased.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLip@A@15A inhibited atherosclerotic plaque in AS models\u003c/h2\u003e \u003cp\u003eTo detect the effects of Lip@A@15A on AS treatment, ApoE-/- mice were used to construct an AS model and treated with various nanomaterials, including Lip@A@15A. The entire aorta and aortic roots were harvested and stained with ORO. The ORO-positive areas in the vessels of AS models treated with Lip@A@15A were the smallest compared to those of the other two AS models treated with PBS or Lip@A. Lip@A also alleviated lipid enrichment in the aorta compared with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Consistent with these results, ORO staining of the aortic root also showed the most significant anti-atherosclerotic function of Lip@A@15A (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). EVG staining represents the integrity of elastic fibers. The EVG-stained areas were low and discontinuous in the PBS-or Lip@A-treated AS mice. Interestingly, the EVG-stained areas were significantly larger and more continuous in the Lip@A@15A-treated AS mice than in the other two groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Mac-3-stained areas in mice treated with Lip@A or Lip@A@15A were darker than those in PBS-treated mice, indicating that collagen fibers were augmented by Lip@A or Lip@A@15A. In addition, the aortic root was stained for MCP-1, a monocyte chemokine, and a pro-inflammatory factor. The expression of MCP-1 was reduced in Lip @A-treated mouse plaques compared with that in the PBS-treated group. Mice treated with Lip@A@15A showed the lowest MCP-1 expression in the plaques compared to the other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). These results indicated that Lip@A@15A had a more prominent anti-atherosclerotic effect than Lip@A.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSafety evaluation of nanomaterials\u003c/h2\u003e \u003cp\u003eWe measured the toxicity of these nanomaterials in RAW264.7 macrophages. The effects of Lip@A@15A on cell viability were very low and showed no significant dose-dependence, even at 100 \u0026micro;g/mL, the cell viability was still above 70%, indicating that Lip@A@15A had low cytotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). After injecting the nanomaterials into the tail vein, immunohistochemical staining of the important organs of the mice showed no obvious cellular tissue damage, indicating that the effect was safe in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs drug carrier systems, nanomaterials can be effectively applied in the treatment of cardiovascular diseases due to its superior physicochemical properties[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, given the dilemma of clinical translation, the design of nanomaterials is particularly critical.[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The modification of peptides is one of the common nanodesign options [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Since liposomes have been successfully used in clinical tumor therapy, we choose ApoE peptides that binds specifically to LDLR to modify liposomes. in order to increase targeting and possibility of clinical translation of nanomaterials.\u003c/p\u003e \u003cp\u003eTherefore, we synthesized Lip@A@15A, which was efficiently and rapidly taken up by macrophages. Ingested Lip@A@15A dramatically decreased ox-LDL uptake, lipid infiltration, macrophage-derived foam cell formation, and cellular inflammatory responses. In an in vivo study, Lip@A@15A accumulated in atherosclerotic plaques further alleviated atherosclerotic plaques in the aortic vessels and postponed the development of atherosclerosis in ApoE-/- mice. These results suggested that relative to Lip@A, Lip@A@15A had more significant uptake by macrophages to penetrate atherosclerotic plaques and suppress formation of foam cells and release of inflammatory factors from macrophages. In addition, Lip@A@15A enhanced atherosclerotic plaque stability, lowered cholesterol crystals, diminished the inflammatory response, and increased the amount of collagen and elastic fibers, suggesting a stronger anti-atherogenic effect than in the control group. At the same time, the safety was verified in vivo and in vitro. In conclusion, we demonstrated that Lip@A@15A can improve microevironment of macrophages, and it is a prospective therapeutic drug for the treatment of atherosclerosis that enhances the bioavailability of statins, aortic targeting, and therapeutic efficacy. Furthermore, the adverse effects and clinical dose of atorvastatin were reduced by Lip@A@15A, which improved patient compliance and promoted statin use in atherosclerotic therapy.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Methods(EXPERIMENTAL SECTION)","content":"\u003ch2\u003eMaterials\u003c/h2\u003e\n\u003cp\u003eLecithin, Cholesterol (Aladdin), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), Atorvastatin (McLean), peptides (flame trite), N-hydroxysuccinimide (NHS), 1- Ethyl \u0026minus;\u0026thinsp;3- (3-dimethylaminopropyl) carbondiimide hydrochloride (EDC, Sigma). anhydrous dimethylsulfoxide (DMSO), fatty acid methyl ester sulfonate (MES), anhydrous dimethylformamide (DMF), chloroform (Tris-Hcl) Human high-oxidized low-density lipoprotein (ox-LDL), Cyanine5 NHS ester (Cy5), Recombinant murine interferon-\u0026gamma; (TNF-\u0026gamma;).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of Atorvastatin-loaded liposome modified peptide (Lip@A@15A).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003e1. Synthesis of Atorvastatin loaded liposome (Lip@A).\u003c/strong\u003e Lip@A was prepared using a thin-film dispersion method. Atorvastatin, lecithin, DSPE-mPEG-COOH, cholesterol, and chloroform were dissolved adequately by ultrasonication, followed by evaporation at 70 ℃ at 30 rpm. Then the solution was hydrated at 60 ℃ at 17 rpm for 40 min and then homogenized by ultrasonic probes for 9 min under ice bath, followed by washing. After passing through a 0.22\u0026micro;m microporous membrane, the Lip@A was prepared.\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Synthesis of peptide (\u003c/strong\u003e15A). The ApoE domain, which spans residues 136\u0026ndash;150 in the N-terminal domain, is critical for the specific binding to LDLR. The amino acid sequence LGQSTEEIRARLSTH was queried using UniProtKB and synthesized by a company (Jiangsu Qiangyao).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Drug content(DC)and encapsulation efficiency(EE)\u003c/strong\u003e. The amount of Atorvastatin was determined by high-performance liquid chromatography (HPLC) using an Apollo C18 column at a UV absorption wavelength of 240 nm. The drug loading and encapsulation efficiencies of Atorvastatin in the liposomes were calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Preparation of peptide-modified Lip@A.\u003c/strong\u003e Lip@A was conjugated to MES and diluted with deionized water. The peptide and EDC were then added. The reaction was performed overnight at 37\u0026deg;C on a shaker at 150 rpm. After centrifugation and washing, the supernatant was used to measure the peptide coupling rate using bicinchoninic acid (BCA), and the remaining Lip@A@15A was collected.\u003c/p\u003e\n\u003ch2\u003eFluorescence spectra\u003c/h2\u003e\n\u003cp\u003eThe fluorescence spectra of Cy5 and Cy5 modified liposome (Lip@Cy5) were obtained using a fluorescence spectrophotometer.\u003c/p\u003e\n\u003ch2\u003eCharacterization of Nanoparticles\u003c/h2\u003e\n\u003cp\u003eSize, size distribution profiles, polydispersity index (PDI), and zeta-potential of nanoparticles were quantified using a Malvern Zetasizer NanoZS instrument at 25\u0026deg;C. Transmission electron microscopy (TEM) was performed using a JEM-1400 PLUS microscope (JEOL, Tokyo, Japan).\u003c/p\u003e\n\u003ch2\u003eCytotoxicity Evaluation\u003c/h2\u003e\n\u003cp\u003eMouse macrophages (RAW264.7) were cultured in 96-well plates in Dulbecco\u003csup\u003e\u0026rsquo;\u003c/sup\u003es modified Eagle\u0026rsquo;s medium (DMEM) with fetal bovine serum (FBS), penicillin, and streptomycin. After the cells were treated with various concentrations of the nanomaterials, cell viability was assayed using the Cell Counting Kit-8 assay (CCK-8).\u003c/p\u003e\n\u003ch2\u003eThe study of cellular uptake of nanomaterials\u003c/h2\u003e\n\u003cp\u003eRAW264.7 cells were cultured in 12-well plates and incubated for 12h in medium different nanomaterials containing Cy5. After centrifugation and washing, the cells were harvested for flow cytometry. RAW264.7 cells were cultured in confocal dishes and incubated for 12h in different medium nanomaterials containing Cy5. Then RAW264.7 cells were stained with 4, 6-Diamidino-2-phenylindole (DAPI). Confocal laser scanning microscopy (CLSM) was performed to acquire fluorescent images.\u003c/p\u003e\n\u003ch2\u003eInhibition of generation of foam cells and Anti-Inflammatory functions of nanomaterials\u003c/h2\u003e\n\u003cp\u003eRAW264.7 cells were cultured in 24-well plates and treated with medium, 100 ng/mL LPS and 10ng/mL IFN-\u0026gamma; and nanomaterials, separately. Supernatants were harvested and concentrations of Tumor Necrosis Factor-\u0026alpha; (TNF-\u0026alpha;), Interleukin-1\u0026beta; (IL-1\u0026beta;), and Monocyte chemotactic protein-1 (MCP-1) were detected using Enzyme-linked Immunosorbent Assay (ELISA). Finally, all groups were incubated with ox-LDL and stained with Oil Red O (ORO) and hematoxylin. The cells were observed under a light microscope.\u003c/p\u003e\n\u003ch2\u003eAnimals\u003c/h2\u003e\n\u003cp\u003eAll the procedures and protocols were approved by the Institutional Animal Ethics Committee of the National Center for Nanoscience and Technology. Male C57BL/6 and Apolipoprotein E-deficient (ApoE-/-) mice (approximately 6 weeks old) were fed a high-fat and high-cholesterol diet. Mice were purchased from Beijing Viton Lever.\u003c/p\u003e\n\u003ch2\u003eDistribution and anti-atherosclerosis effect of nanomaterials in Vivo\u003c/h2\u003e\n\u003cp\u003eC57BL/6 and ApoE-/-mice were fed a high-fat, high-cholesterol diet for 3 months. The nanomaterials were then administered via intravenous (i.v.) injection to mice. The mice were euthanized and the aortas and vital organs, including the heart, liver, spleen, lung, and kidney, were separated and imaged using an In Vivo Imaging System (IVIS).\u003c/p\u003e\n\u003ch2\u003eORO Staining and immunohistochemistry of plaque of entire aortas and aortic root\u003c/h2\u003e\n\u003cp\u003eApoE-/- mice were randomly assigned and different treatments were conducted for additional 2 months. The mice were intravenously injected with saline or various nanomaterials. The Mice were euthanized, and the entire aorta and aortic root were collected and stained with ORO. The aortic roots were immunohistochemically stained with Verhoeff\u0026rsquo;s Van Gieson (EVG), macrophage differentiation antigen-3 (Mas-3), and monocyte chemotactic protein-1 (MCP-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE), The ANOVA test for experiments with multiple groups, while a two-tailed, unpaired t-test was used for data with two groups. * means P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** means P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** means P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the teachers of\u0026nbsp;the CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics\u0026nbsp;for their guidance in the design of the experiment, and all\u0026nbsp;members of the laboratory for their help with the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCL and ZTJ jointly designed and completed the tests and analyzed the results.\u0026nbsp;CL, XZM,\u0026nbsp;and TXX collated, consulted, and wrote\u0026nbsp;the paper. HHX reviewed paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by\u0026nbsp;the Hospital-level Scientific Research Project in the Affiliated\u003c/p\u003e\n\u003cp\u003eHospital of North Sichuan Medical College\u0026nbsp;(2020ZD002) ,\u0026nbsp;Scientific Research Project of Nanchong Municipal Science and Technology Bureau, Sichuan Province (19SXHZ0192)\u0026nbsp;and project for the First-class Pharmaceutical Sciences of North Sichuan Medical College (CBY21-YLXK03）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by the\u0026nbsp;CAS Center for Excellence at the Nanoscience National Center for Nanoscience and Technology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSong P, Fang Z, Wang H, Cai Y, Rahimi K, Zhu Y, et al. 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Apolipoprotein E in Cardiometabolic and Neurological Health and Diseases. IJMS. 2022;23:9892. \u003c/li\u003e\n\u003cli\u003eGetz G, Reardon C. Apoprotein E and Reverse Cholesterol Transport. IJMS. 2018;19:3479. \u003c/li\u003e\n\u003cli\u003eMahley RW, Weisgraber KH, Huang Y. Apolipoprotein E: structure determines function, from atherosclerosis to Alzheimer\u0026rsquo;s disease to AIDS. Journal of Lipid Research. 2009;50:S183\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eFerence BA, Kastelein JJP, Ray KK, Ginsberg HN, Chapman MJ, Packard CJ, et al. Association of Triglyceride-Lowering \u003cem\u003eLPL\u003c/em\u003e Variants and LDL-C\u0026ndash;Lowering \u003cem\u003eLDLR\u003c/em\u003e Variants With Risk of Coronary Heart Disease. JAMA. 2019;321:364. \u003c/li\u003e\n\u003cli\u003eAlves AC, Azevedo S, Benito-Vicente A, Gra\u0026ccedil;a R, Galicia-Garcia U, Barros P, et al. LDLR variants functional characterization: Contribution to variant classification. 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Liposomes as biocompatible and smart delivery systems \u0026ndash; the current state. Advances in Colloid and Interface Science. 2022;309:102757. \u003c/li\u003e\n\u003cli\u003eTenchov R, Bird R, Curtze AE, Zhou Q. Lipid Nanoparticles─From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement. ACS Nano. 2021;15:16982\u0026ndash;7015. \u003c/li\u003e\n\u003cli\u003eZou D, Yang P, Liu J, Dai F, Xiao Y, Zhao A, et al. Exosome-Loaded Pro-efferocytic Vascular Stent with Lp-PLA \u003csub\u003e2\u003c/sub\u003e -Triggered Release for Preventing In-Stent Restenosis. ACS Nano. 2022;16:14925\u0026ndash;41. \u003c/li\u003e\n\u003cli\u003eGong F, Wang Z, Mo R, Wang Y, Su J, Li X, et al. Nano-sponge-like liposomes remove cholesterol crystals for antiatherosclerosis. J Control Release. 2022;349:940\u0026ndash;53. \u003c/li\u003e\n\u003cli\u003eLi W, Gonzalez KM, Chung J, Kim M, Lu J. Surface-modified nanotherapeutics targeting atherosclerosis. Biomater Sci. 2022;10:5459\u0026ndash;71. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ApoE peptides, Atorvastatin, Liposome, Atherosclerosis","lastPublishedDoi":"10.21203/rs.3.rs-4237089/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4237089/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNanomedicine has gathered significant attention in atherosclerosis. However, there remains an apparent gap between laboratory discovery and clinical translation of nanomaterials. Therefore, we constructed a new nanomaterial that selected liposomes as drug-delivery carriers. and modified with Apolipoprotein E (ApoE) peptides which may alleviate atherosclerotic plaque to seek a novel idea about treatment of atherosclerosis .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe successfully constructed nanoparticles loaded with Atorvastatin and modified them with aE15A (ApoE peptide) (Lip@A@15A). In vitro, the study of cellular uptake of nanomaterials by macrophages was detected by Confocal laser scanning microscopy (CLSM), and concentrations of inflammatory factors were detected using Enzyme-linked Immunosorbent Assay. In vivo, ApoE-/- mice were used to construct atherosclerotic models that were treated with many reagents, including Lip@A@15A, in which the stability of atherosclerotic plaque in aortas and the macrophage-derived foam cells were observed by oil “O” and Masson staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLip@A@15A showed low toxicity in an in vitro cytotoxicity test and had the highest phagocytic efficiency by macrophages compared to liposomes and nanoparticles loaded with atorvastatin (Lip@A). Therefore, the function of lipid phagocytosis and the levels of IL-1βand TNF-αin macrophages treated with Lip@A@15A were the lowest compared with that of macrophages treated with the other three reagents (P\u0026lt;0.05). More importantly, Lip@A@15A may be localized in the aortic intima. Lip@A@15A markedly improved plaque stability and inhibited plaque rupture compared with mice treated with other reagents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelative to Lip@A, Lip@A@15A more significantly enhanced endocytosis by macrophages and impeded inflammatory factors from macrophages. In addition, it actively targeted atherosclerotic plaques and improved plaque stability.\u003c/p\u003e","manuscriptTitle":"Functionalization of liposomes with ApoE-derived peptides affects cellular uptake and drug transport to atherosclerotic plaque","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-16 07:04:21","doi":"10.21203/rs.3.rs-4237089/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f9be4a80-196b-4967-b759-01029a8543f7","owner":[],"postedDate":"April 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-13T01:54:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-16 07:04:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4237089","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4237089","identity":"rs-4237089","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}