WO2015009544A1 - Rna interference of fabp4 for the treatment of atherosclerosis - Google Patents

Abstract

Methods of making anionic liposomal FABP4 siRNA, and an anionic liposomal atherosclerosis related siRNA delivery system, for targeting atherosclerosis. A method of making an anionic liposomal siRNA comprising: mixing a lipid-A and a lipid-B to form a lipid mixture comprising lipid-A and lipid-B; and mixing: (i) the lipid mixture, (ii) an siRNA, and (iii) a solvent to form a liposome. In another embodiment the siRNA further comprises a fluorescent tag. In some embodiments of the method of making an anionic liposomal siRNA, lipid-A comprises DMPG, in other embodiment, lipid-S comprises DMPC.

Description

RNA INTERFERENCE OF FABP4 FOR

THE TREATMENT OF ATHEROSCLEROSIS

BACKGROUND

Field of the Disclosure

[0001] The disclosure herein generally relates to methods of making anionic liposomal FABP4siRNA. More particularly, the disclosure relates to an anionic liposomal atherosclerosis related siRNA delivery system.

Background of the Technology

[0002] Atherosclerosis, is a chronic inflammatory disease, is the principal cause of heart attack, and stroke, thus being responsible for 30% of all deaths in the United States [1-3].

[0003] Atherosclerosis is characterized as a systemic, progressive disease process in which the arterial wall thickens through a process of inflammation [4-7], oxidative stress, and dyslipidemia [8-10].

[0004] This process leads to plaque formation and flow limitation in the vessel lumen. Early and intermediate-stage atherosclerotic plaque is characterized by thin fibrous caps, lipid-laden cores, inflammation, and endothelial dysfunction [1 1 ,12]. As a result, local vascular permeability increases due to alterations in endothelial cell endocytosis [13], damage to the glycocalyx layer [14], and the loosening of gap junctions between cells [13]. The increase in vascular permeability leads to increases in paracellular transport of macromolecules and endothelial cell endocytosis. Similar alterations of vascular permeability occur during tumor growth in cancers [15].

[0005] Liposomes have been used in the diagnosis and treatment of cancer [16,17] and, to a lesser extent, cardiovascular disease [18-20]. The aqueous cores of liposomes have the ability to concentrate hydrophilic compounds for imaging [21-23]. Moreover, liposomes have been used successfully in molecular imaging such as single photon emission computed 58 tomography [21-23] and as nuclear isotope imaging agents to enhance local tissue imaging [24]. Liposomal uptake by tumors has also been shown to occur, and is speculated to be the result of altered vascular permeability; however, no mechanism has been confirmed, although it has also been demonstrated that anionic liposomes have been taken up into the atheromas of Watanabe heritable hyperiipidemic rabbits within lipid pools [20], however the description of treatment using Watanabe rabbits demonstrate the effectiveness of liposomal penetration into atherosclerotic plaque but did not characterize the plaque or cellular distribution such as the clear co-localization with macrophages and FABP4. It would therefore, be desirable to utilize these increases in vascular and endothelial cell permeability to develop a method of localized delivery of therapeutic agents to treat cancer and atherosclerosis, utilizing liposomes that can be manipulated via modification of their physicochemical properties.

BRIEF SUMMARY OF THE DISCLOSED EMBODIMENTS

[0006] These and other needs in the art are addressed by an embodiment of the method a method of making an anionic liposomal siRNA comprising: mixing a lipid-A and a lipid-B to form a lipid mixture comprising lipid-A and lipid-B; and mixing: (i) the lipid mixture, (ii) an siRNA, and (iii) a solvent to form a liposome.

[0007] In some embodiments of the method of making an anionic liposomal siRNA , the siRNA comprises FABP4siRNA, in further embodiments the siRNA comprises 4 kinds of FABP4siRNA reagent, and in another embodiment the siRNA further comprises a fluorescent tag.

[0008] In some embodiments of the method of making an anionic liposomal siRNA, lipid-A comprises DMPG, in other embodiment, lipid-B comprises DMPC. In a further embodiment, lipid-A and lipid-B are in a molar ratio of between about 9.99: 0.01 to about 0.01 : 9.99, and in a still further embodiment lipid-A and lipid-B are in a molar ratio of between about 3 to about 7.

[0009] In some embodiments of the method of making an anionic liposomal siRNA, the lipid mixture; siRNA; and said solvent are mixed in a ratio of about 10:1 :40. In another embodiment, the solvent comprises tertiary butanol. In another embodiment of the method of making an anionic liposomal siRNA, the liposome comprises an anionic charge, in a further embodiment, said liposome is between .01 microns to about 1000 microns. In a further embodiment, said liposomes further comprising a nanogold label, and in a still further embodiment the liposomes are about 2pm to about 7 m. In one embodiment of the method of making an anionic liposomal siRNA the liposomes are further lyophilized and stored at -20 °C. In one embodiment a method of delivering a siRNA to an atherosclerotic plaque is described, the method comprising:1 ) administering an anionic liposomal delivery system to a patient comprising the plaque, wherein the liposomal delivery system comprises:(a) an anionic liposome, wherein the liposome comprises a siRNA, and a lipid mixture; and 2) targeting macrophage cells comprising the plaque with the liposome, wherein the targeting silences FABP4 and stables the plaque. In another embodiment, administration is intravenous or intra-arterial.

[0010] In one embodiment a method of delivering a siRNA to an atherosclerotic plaque, the liposomes comprise a mixture of a lipid-A, and a lipid-B, in another embodiment, the liposome further comprise a solvent, in a further embodiment, lipid- A comprises DMPG, and in a still further embodiment lipid-B comprises DMPC. In one embodiment a method of delivering a siRNA to an atherosclerotic plaque, lipid-A and lipid-B are in a molar ratio of between about 9.99: 0.01 to about 0.01 : 9.99, in a further embodiment lipid-A and lipid-B are in a molar ratio of between about 3 to about 7.

[0011] In one embodiment a method of delivering a siRNA to an atherosclerotic plaque, the siRNA comprises FABP4siRNA, in another embodiment, the siRNA further comprises a fluorescent tag, in a further embodiment, the lipid mixture; siRNA; and said solvent are mixed in a ratio of about 10:1 :40,and in a still further embodiment, targeting further requires clatherin mediated endocytosis.

[0012] In one embodiment a method of visualizing liposomal uptake by atherosclerotic plaques is described, comprising:(1 ) administering to a patient comprising the plaque a fluorescently labeled anionic liposome, wherein the liposome comprises a fluorescent tagged siRNA; and (2) visualizing said labeled anionic liposome, and identifying the location of the liposomes.

BRIEF DESCRIPTION OF DRAWINGS

[0013] For a detailed description of the disclosed embodiments of the invention, reference will now be made to the accompanying drawings, wherein:

[0014] Figure 1 (A) is a Light microscopy analysis of Oil Red O-stained atherosclerotic plaque comprising highly complex lesions in ApoE-/- mouse aortic tissue (20X magnification), in accordance with an embodiment of this invention;

[0015] Figure 1 (B-C) is a TEM (Transmission electric microscopy) analysis of an atherosclerotic plaque showing the presence of foam cells and cholesterol monohydrate crystals (showing foam cell lesions in atherosclerotic plaque (4000X and 10.000X magnification, respectively); scale bars = 10 μm in (B) and 2 μm in (C). MFC, macrophage derived foam cells; CC, cholesterol monohydrate crystal; P, plaque; L, lumen, in accordance with an embodiment of this invention;

[0016] Figure 2 (A-C) provides a study of the uptake and distribution of anionic liposomes in atherosclerotic plaque in the aortic tissue of ApoE-/- mice, A non- silencing siRNA sequence (Sequence 1 : UACAAUAGUCAGUCGGAUUCUUCAAACUGGGCGUGGAACACUAAUAAGCAAG CAAUUGGAAAGUCGACCACAAUAA) which was purchased from Thermo Scientific was tagged with Alexa Fluor 488 dye (excitation, 495 nm; emission, 519 nm) 409 was incorporated into liposomes and used to determine liposome uptake and distribution in 410 atherosclerotic plaque of aortic tissue. Confocal microscopy images (63X magnification) showing (A) the absence of fluorescence in atherosclerotic plaque of a PBS-injected control mouse and (B, C) the uptake of fluorescently labeled liposomes into atherosclerotic plaque 30 minutes (B) and 60 minutes (C) after the injection of liposomes, seen as green punctuate structures; tissue sections were counterstained with 4', 6'-diamidino-2-phenylindole (DAPI) nucleic acid stain; scale bars = 50 μm. L, lumen; P, plaque, in accordance with an embodiment of this invention;

[0017] Figure 3 (A-D): illustrate the distribution of anionic liposomes in the atherosclerotic plaque of ApoE-/- mouse aortic tissue. Confocal microscopy images (63X magnification) showing fluorescently labeled liposomes (A, green) and immunofluorescence staining for CD68+ macrophages (B, red) in atherosclerotic plaque, liposomes accumulated in macrophage-rich areas (C) shows an overlay (yellow) of images depicted in (A) and (B) showing the colocalization of liposomes with macrophages; (D) transmission electron microscopy analysis (20.000X magnification) showing the accumulation of macrophages in atherosclerotic plaque; scale bar = 2 μm; Li, liposome; N, nucleus, all in accordance with embodiments of this invention;

[0018] Figure 4 (A-B) illustrate the effect of amantadine on the uptake of fluorescently labeled liposomes into human coronary artery endothelial cells (HCAECs); (A) is a confocal microscopy analysis (63X magnification) showing the uptake of fluorescently labeled liposomes into HCAECs after 30 minutes of incubation; as indicated, some cells were pretreated with amantadine for 20 minutes and/or TNF-a for 1 hour; (B) shows results of flow cytometry analysis showing the quantification of fluorescently labeled liposome uptake in HCAECs, some cells were pretreated with amantadine for 20 minutes or TNF-a for 1 hour; bars represent the mean ± standard error (n=3; *p<0.05) in accordance with embodiments of this invention; and

[0019] Figure 5 illustrates that Amantadine inhibits TNF-a induced clathrin overexpression; confocal microscopy analysis (63X magnification) shows clathrin expression (green punctuate structures) in (A) untreated HCAECs; (B) HCAECs pretreated with TNF-a for 3hrs; and (C) HCAECs pretreated with 1 mM amantadine for 20 minutes and TNF-a and for 3hrs; (D) immunoblot analysis showing results similar to those above; β-actin was used as loading control (HC, heavy chain; Amt, amantadine), in accordance with an embodiment of this invention.

DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS

[0020] The following discussion is directed to various embodiments of the invention. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be exemplary of that embodiment, and not intended to intimate that the scope of the disclosure, including the claims, is limited to that embodiment.

[0021] Certain terms are used throughout the following description and claims to refer to particular features or components. As one skilled in the art will appreciate, different persons may refer to the same feature or component by different names. This document does not intend to distinguish between components or features that differ in name but not in function. In the following discussion and in the claims, the terms "including" and "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "including, but not limited to... ." The term "substantially" generally means mostly, near completely, or approximately entirely. As used herein, the term "about," when used in conjunction with a percentage or other numerical amount, means plus or minus 10% of that percentage or other numerical amount. For example, the term "about 80%," would encompass 80% plus or minus 8%. Further, all references cited herein are incorporated in their entirety. [0022] Atherosclerosis is a chronic inflammatory disease, characterized as having a systemic, progressive disease process, in which the arterial wall thickens through a process of inflammation, oxidative stress, and dyslipidemia. This process leads to plaque formation, and flow limitation in the vessel lumen.

[0023] As described above, early and intermediate-stage atherosclerotic plaque is characterized by thin fibrous caps, lipid-laden cores, inflammation, and endothelial dysfunction. As a result, local vascular permeability increases due to alterations in endothelial cell endocytosis, damage to the glycocalyx layer, and the loosening of gap junctions between cells. The increase in vascular permeability leads to increases in paracellular transport of macromolecules and endothelial cell endocytosis. These increases in vascular and endothelial cell permeability in some embodiments, may provide favorable conditions for the localized delivery of therapeutic agents to treat cancer and atherosclerosis.

[0024] In some embodiments described herein Liposomes are manipulated, wherein their size and external charge may be specified. The aqueous cores of liposomes have the ability to concentrate hydrophilic compounds, thus in some embodiments herein described, liposomes target select tissues and deliver sufficient quantities of compounds for imaging studies, further their malleable nature may allow for the local delivery of novel therapeutic compounds directly to the atherosclerotic plaque.

[0025] Embodiments of the methods herein described have been used to show distribution of the anionic liposomes of this invention, in atherosclerotic plaque in the aortic tissues of apolipoprotein E-deficient (ApoE- -) mice. In some embodiments, to facilitate the tracking of the liposomes, liposomes contain fluorescently labeled nonsilencing siRNA which can be tracked using confocal microscopy. In some embodiments, as further described in the examples below, confocal microscopy analysis showed the uptake of anionic liposomes into atherosclerotic plaque in ApoE-/- mouse aortic tissue and the co-localization of these liposomes with macrophages which are the primary metabolically active component of atherosclerotic plaque and play a pivotal role in plaque instability. In another embodiment described herein, transmission electron microscopy analysis further revealed the accumulation of the anionic liposomes in macrophages. [0026] In further embodiments herein described, the mechanism of anionic liposome absorption into local endothelial cells, was investigated. The role of clathrin-mediated endocytosis in human coronary artery endothelial cells (HCAECs) treated with or without the inflammatory cytokine tumor necrosis factor (TNF)-a, was used in this investigation. Pretreatment with amantadine, an inhibitor of clathrin- mediated endocytosis, significantly decreased the uptake of liposomes in HCAECs treated with or without TNF-a by 77% and 46%, respectively.

[0027] Furthermore, in other embodiments of the methods herein described, immunoblot analysis showed that endogenous clathrin expression was significantly increased in HCAECs stimulated with TNF-a but was inhibited by amantadine, thus indicated that clathrin-mediated endocytosis is partly responsible for the uptake of liposomes by endothelial cells. Some embodiments herein described indicate that anionic liposomes target the macrophage-rich areas of plaque in ApoE-/- mice and may be used lead in the development of novel diagnostic and therapeutic strategies for treating vulnerable plaque in humans.

Experimental Procedures

[0028] ApoE-/-mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The animal 90 experimental protocol was approved by the Animal Welfare Committee (Permit number AWC 1 1-069) at The University of Texas Health Science Center at Houston.

Liposome Preparation

[0029] Liposomes were prepared with DMPC (1 ,2-dimyristoyl-sn-glycero-3- phosphocholine) and DMPG (1 ,2-dimyristoyl-sn-glycero-3-phosphoglycerol) (Avanti Polar Lipids, Inc., Alabaster, AL, USA) 96 in a 7:3 molar ratio. Lipids and a nonsilencing siRNA sequence tagged with Alexa Fluor dye (Qiagen, 97 Valencia, CA, USA) were mixed in excess tertiary butanol, and Tween 20 was added. This mixture was then lyophilized and stored at -20 °C until use. Before in vivo administration, this preparation was hydrated with 1X phosphate-buffered saline (PBS) at a concentration of 50 pg/ml to achieve the desired dose in a 100 μΙ injection. A nonsilencing siRNA sequence tagged with Alexa Fluor (green), which does not share any sequence homology with any known human mRNA sequences, was incorporated into the liposomes and used as a label for the identification and quantification of uptake and distribution in tissues in vivo. [0030] Liposomes were prepared as described above, with DMPC (1 ,2-dimyristoyl- sn-glycero-3-phosphocholine) and DMPG (1 ,2-dimyristoyl-sn-glycero-3- phosphoglycerol) in a 7:3 molar ratio. DMPC is neutral and DMPG has a negative charge.

[0031] As showed in Table 1 , the Zeta potential analysis data, shows the liposomes described herein have strong negative charges. Liposome particle sizes are in a range of about 200 nm to about 300 nm with the average size of about 235 nm.

[0032] Table 1 : Zeta potential of liposomes made by embodiments herein described

[0033] Table 2: shows the multimodal size distribution of liposomes formed by an embodiment of the method herein described.

Animal Procedure and Delivery of Liposomes

[0034] Eleven ApoE-/- mice (9-11 weeks old, C57BL/6 background) were randomly divided into the control group (n=5) and test group (n=6). The mice were fed a normal chow diet. The mice were anesthetized with 2% isoflurane and placed in dorsal recumbency. A topical hair removal cream was applied to the femoral area and rinsed off once the hair was removed. Topical lidocaine was applied to the skin over the femoral vein as a vasodilator. A small incision was made to allow visualization of the femoral vein. A slow injection of 100 μί PBS (control group) or liposomes (5 pg in 100 μί PBS; test group) was administered. A small amount of GLUture (Abbott Laboratories, Abbott Park, IL, USA) was applied to the incision for closure. While still anesthetized, the mice were euthanized by exsanguination via cardiac puncture 30 or 60 minutes (n=3 per group) later for the test group and 60 minutes (n=5) later for the control group. The aorta of each mouse was collected for immunofluorescence staining and transmission electron microscopy (TEM) analyses. Tissue Processing and Atherosclerotic Lesion Detection

[0035] Aortas were divided into two sections. One section was fixed for immunofluorescence staining in 4% paraformaldehyde in PBS (pH 7.4) overnight at 4°C. The tissue was then rinsed in 15% sucrose for 24 hours at 4°C. The plaque tissues were identified, cut into 5-mm segments, embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Sakura Finetek, Torrance, CA, USA), and stored at -80°C before slides were made. The second section of tissue was fixed for TEM analysis in 3% glutaraldehyde in PBS (pH 7.4). Slides containing 5 to 8 μητι of thin sections of OCT-embedded ApoE-/- mouse aortic tissues were cut with a Leica CM1800 Cryostat (Leica, Bensheim, Germany), placed on Fisherbrand #15 Superfrost Plus positively charged slides, and stored at -80°C overnight. For assessment of the atherosclerotic lesions, the slides were stained with Oil Red O (Sigma-Aldrich, St. Louis, MO, USA), and images were observed with an Olympus 1X71 brightfield microscope.

Confocal Fluorescence Microscopy

[0036] To determine the uptake and distribution of anionic liposomes in atherosclerotic plaque in ApoE-/- mouse aortic tissue, the slides were washed with PBS and counterstained with 4', 6'-diamidino-2-phenylindole (DAPI) nucleic acid stain. Images were observed on a Leica SP5 confocal system with a 134 Leica TCS SP5 II microscope (Leica Microsystems, Mannheim, Germany), DAPI and FITC filters, and LAS AF image acquisition software. For colocalization analysis, the slides were incubated in 1 % 136 BSA/10% normal goat serum in 0.1 % PBS-Tween 20 for 1 h to permeabilize the tissue and block nonspecific protein-protein interactions. The slides were then incubated with rabbit polyclonal anti-CD68 (sc-9139; Santa Cruz Biotechnology, Santa Cruz, CA; 1 :100 dilution) at 4°C overnight, washed three times with 1X PBS, and incubated with anti-rabbit Texas red conjugated secondary antibody (#6719, Abcam, Cambridge, UK; 1 :1000 dilution) at room temperature for 60 minutes. Images were analyzed on a Leica SP5 confocal system with a Leica TCS SP5 II microscope as described above.

Transmission Electron Microscopy

[0037] To further characterize the distribution of liposomes in atherosclerotic plaque, tissue sections were initially fixed with 3% glutaraldehyde in PBS (pH 7.4) and post-fixed in 1 % osmium tetroxide. Dehydration was carried out in a series of graded alcohol washes (70%, 80%, 90%, and 100% ethanol), followed by two acetone washes. The samples were infiltrated with Epon plastic resin, embedded, and cut into 1 μιτι-thick sections with an RMC MTXL Ultra Microtome (Boeckeler Instruments, Tucson, AZ, USA) for low-resolution images. Furthermore, the plaque sections were cut into 60 to 80 nm-thick sections for ultra-structure images. The thin sections were stained for Uranyl Acetate and Lead Citrate. Images were acquired with a JEOL JeM-1230 transmission electron microscope (JEOL, Tokyo, Japan). Cell Culture and Treatment

[0038] HCAECs were purchased from Lonza Walkersville, Inc. (Walkersville, MD, USA). Cells were cultured in EBM-2 (Lonza) medium. Liposomes were added to serum-free incubation medium at a final concentration of 150 pg/ml total lipids. To measure liposome uptake, medium was replaced by liposome containing medium or medium without liposomes (control) and incubated for 30 minutes. In the TNF-a treatment experiment, cells were incubated with TNF-a (R&D System, 100 ng/ml) for 1 or 3 hours to analyze clathrin expression. Then, the cells were washed, and incubated with liposomes. To evaluate the uptake of liposomes by the clathrin- mediated pathway, cells were pretreated with 1 rti amantadine (Sigma-Aldrich) for 20 minutes before incubation with TNF-a, as described above. To evaluate the effects of amantadine on clathrin expression, cells treated with TNF-a or pretreated with amantadine followed by TNF-a were incubated with rabbit anti-clathrin primary antibody and anti-rabbit FITC conjugated secondary antibody (#6717, Abeam, 1 :1000 dilution). The uptake of fluorescent liposomes and clathrin expression were visualized by using a Leica SP5 confocal system with a Leica TCS SP5 II microscope as described above. For the quantitative analysis of fluorescence, cells were trypsinized, resuspended in ice-cold PBS, and analyzed by using a BD LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with a blue laser (488 nm). Automated compensation was performed by using FACSDiva Version 6.1.3 software.

Immunoblotting Assay

[0039] To prepare total cellular extracts, cells were isolated after treatment and sonicated in RIPA solution (Thermo Scientific, Rockford, IL, USA). Protein concentrations were measured by using the BCA protein assay reagent (Pierce, Rockford IL, USA). Proteins were separated by using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (25 μg protein/lane) and transferred to Immun- Blot PVDF membrane/filter paper (Bio-Rad, Hercules, CA, USA), which was then incubated with polyclonal anti-clathrin heavy-chain antibody (#2410, Cell Signaling Technology Inc., Danvers, MA, USA). Subsequently, the blot was incubated with goat anti-rabbit horse radish peroxidase-labeled secondary antibody (Bio-Rad). Proteins were detected by using Pierce enhanced chemiluminescence Western blotting substrate, β-actin (A1978, Sigma-Aldrich) was used as loading control.

Statistical Analysis

[0040] Data are representative of 3 experiments or replicates and are expressed as the mean ± standard error. Experimental groups were compared by using a Student f test. A p-value of less than 0.05 was considered statistically significant.

[0041] The following examples of processing conditions and parameters are given for the purpose of illustrating certain exemplary embodiments of the present invention.

Examples

[0042] EXAMPLE 1 : Liposomal uptake in Atherosclerotic Plaque. Light microscopy analysis of Oil Red O-stained atherosclerotic plaque (Figure 1A) revealed highly complex lesions within the plaque (p). Furthermore, TEM analysis of the atherosclerotic plaque showed the presence of foam cells (derived from macrophage cells) and cholesterol monohydrate crystals (cc) (Figure 1 B and C). To examine the uptake and distribution of the anionic liposomes (described herein) in atherosclerotic plaque in the aortic tissue of ApoE-/- mice immunofluorescence staining was performed after the injection of PBS-control (Figure 2A) or fluorescently labeled liposomes (Figure 2B and 2C). At 30 minutes (Figure 2B) and 60 (Figure 2C) minutes after injection, a significant amount of fluorescently labeled liposome was seen in the atherosclerotic plaque (p) of all ApoE-/- mice, whereas no fluorescence was detected in atherosclerotic plaque after the injection of PBS (Figure 2A). Liposomes were most abundant in acellular regions of atherosclerotic plaque, as demonstrated by the absence of DAPI staining in these regions.

[0043] EXAMPLE 2: Colocalization of Macrophages and Fluorescently Labeled Liposomes in Atherosclerotic Plaque. In some embodiments of the method herein described, metabolically active components of atherosclerotic plaque such as macrophages preferentially uptake negatively charged particles. To determine whether atherosclerosis associated macrophages expressing CD68 co-localized with fluorescently labeled liposomes in atherosclerotic plaque, confocal fluorescence imaging of atherosclerotic plaque in the aortic tissue of ApoE-/- mice was performed. CD68 staining indicated the presence of macrophages in the plaque area (3B), and fluorescently labeled liposomes were visible in the macrophage-rich area of plaque (3C). Fluorescently labeled liposomes and CD68+ macrophages colocalized primarily in the adventitia lipid rich areas of plaque (Figure 3A-C). To further confirm the distribution of anionic liposomes. In a further embodiment of the methods herein described, TEM analysis was conducted. As shown in Figure 3D, liposomes accumulated within macrophages in atherosclerotic plaque, indicating an association between liposomal uptake and areas of metabolically active plaque.

[0044] EXAMPLE 3: The Role of Clathrin-mediated Endocytosis in Liposome Uptake by HCAECs. In one embodiment, the clathrin-mediated endocytosis as a pathway for the uptake of liposomes by HCAECs, and the effects of amantadine, (an independent inhibitor of clathrin mediated endocytosis [35,36] on liposome uptake in HCAECs) were studied. Further, to examine the effects of amantadine under inflammatory conditions, HCAECs were treated with TNF-a, a key cytokine in the recruitment and activation of inflammatory cells.

[0045] The cytotoxicity of amantadine and found that a concentration of 1 mM amantadine was not toxic to HCAECs (<5% of cell death); thus, this concentration was used in subsequent experiments. Confocal microscopy analysis showed that the uptake of liposomes was greater in HCAECs treated with TNF-a for 1 hour than in untreated HCAECs (liposomes only) (Figure 4A). The pretreatment of HCAECs with amantadine reduced the uptake of liposomes in both TNF-a-treated or untreated HCAECs (Figure 4A). The quantification of fluorescently labeled liposomes by flow cytometry (expressed as the % of viable cells) showed that there was a 3.4-fold increase of the uptake of liposomes by HCAECs incubated with TNF-a for one hour as compared to no treatment (liposomes only) (Figure 4B). Furthermore, in untreated HCAECs or HCAECs stimulated with TNF-a pretreatment with amantadine significantly reduced liposome uptake by 46% and 77%, respectively (Figure 4B). These results suggest that clathrin-mediated endocytosis may be responsible for the uptake of liposomes by HCAECs.

[0046] EXAMPLE 4: Analysis of Clathrin Expression in HCAECs. In a further embodiment, confocal microscopy analysis was used to show that TNF-a-induced an increase in the uptake of liposomes which was mediated by an increase in clathrin expression. Basal expression of endogenous clathrin in HCAECs was low (Figure 5A). However, in cells stimulated with TNF-a for 3 hours, clathrin expression was significantly increased (Figure 5B). Amantadine greatly inhibited clathrin expression in TNF-a-stimulated cells (Figure 5C). The results of immunoblot analysis were fully consistent with the results of confocal microscopy (Figure 5D), these data indicate that the TNF-a-induced increase in liposome uptake which was mediated by clathrin-dependent endocytosis.

[0047] In one embodiment of the method herein described, the uptake of anionic liposomes into atherosclerotic plaque in the aortic tissue of ApoE-/- mice and that the liposomes co-localize with, and accumulate in macrophages was evidenced in for example Figure 2.

[0048] In a further embodiment of the methods herein described, the uptake of anionic liposomes into HCAECs was shown to occur through clathrin-mediated endocytosis (Figures 4 and 5). Macrophages play a pivotal role in plaque instability, which is directly involved in triggering acute coronary syndromes including but not limited to: unstable angina, acute myocardial infarction, and sudden coronary death [37-41]. Thus, some embodiments the an anionic liposome herein described, may be used as liposomal drug delivery system for therapeutically targeting the inhibition of macrophage infiltration in atherosclerotic plaque, and may thus be used to promote plaque stabilization.

[0049] In ApoE-/- mice, foam cell lesions develop as early as 8 weeks [28]. In another embodiment of the method herein described, it is evident that the atherosclerotic plaques in the aortas of ApoE-/- mice consist of highly complex lesions and showed evidence of foam cell accumulation. Furthermore, in some embodiments of the method herein described, it was observed that the accumulation of liposomes in macrophages within atherosclerotic plaque, indicate that the negative charge of the liposomes may lead to preferential phagocytosis of the liposomes by macrophages via their scavenger receptors, which is in turn followed by conversion into macrophage-derived foam cells and a lipid-rich area.

[0050] In some embodiments, anionic liposomes have several advantages that positively charged liposomes do not. Namely, positively charged liposomes have been shown to be toxic as a carrier and tend to attach to arterial walls [42,43]. In contrast, the anionic liposomes described herein are passively absorbed through atherosclerotic areas of the arterial wall as a result of altered permeability caused by glycocalyx loss. For liposomes to be taken up into atherosclerotic plaque, they must first cross the local endothelial barrier. Clathrin and caveolar-mediated endocytosis are the most commonly reported processes of cellular uptake [42-46]. Clathrin- coated vesicles are the best characterized of the vesicular carriers and provide a specific delivery system for the uptake of extracellular material [47]. Thus in some embodiments described herein, the uptake of liposomes into HCAECs was low under normal conditions but was significantly increased in HCAECs stimulated with TNF-a. Furthermore, amantadine, an independent inhibitor of clathrin-mediated endocytosis, significantly. ¹²

[0051] While embodiments have been shown and described, modifications thereof can be made by one skilled in the art without departing from the scope or teachings herein. The embodiments describe herein are exemplary only and are not limiting. Many variations and modifications of the systems, apparatus, and processes described herein are possible and are within the scope of the invention. For example, the relative dimensions of various parts, the materials from which the various parts are made, and other parameters can be varied. Accordingly, the scope of protection is not limited to the embodiments described herein, but is only limited by the claims that follow, the scope of which shall include all equivalents of the subject matter of the claims. The references listed below are herein incorporated in their entirety: References:

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Claims

CLAIMS What is claimed is:

1. A method of making an anionic liposomal siRNA comprising:

1 ) mixing a lipid-A and a lipid-B to form a lipid mixture comprising lipid-A and lipid-B; and

2) mixing: (i) the lipid mixture, (ii) an siRNA, and (iii) a solvent to form a liposome.

2. The method of claim 1 , wherein said siRNA comprises FABP4siRNA.

3. The method of claim 1 , wherein said siRNA further comprises a fluorescent tag.

4. The method of claim 1 , wherein said lipid-A comprises DMPG; and said lipid- B comprises DMPC.

5. The method of claim 1 , wherein lipid-A and lipid-B are in a molar ratio of between about 3 to about 7.

6. The method of claim 1 wherein said lipid mixture; siRNA; and said solvent are mixed in a ratio of about 10:1 :40.

7. The method of claim 1 , wherein said liposome comprises an anionic charge.

8. A method of delivering a siRNA to an atherosclerotic plaque, the method comprising:

1 ) administering an anionic liposomal delivery system to a patient comprising said plaque, wherein said liposomal delivery system comprises an anionic liposome, wherein said liposome comprises an siRNA, and a lipid mixture; and 2) targeting macrophage cells comprising said plaque with said liposome, wherein said targeting silences FABP4 and stables said plaque. The method of claim 8, wherein said liposomes comprise a mixture of a lipid- A; and a lipid-B. The method of claim 8, wherein said liposome further comprise a solvent. The method of claim 9, wherein said lipid-A comprises DMPG; and said lipid- B comprises DMPC. The method of claim 8, wherein said siRNA comprises FABP4siRNA. The method of claim 8, wherein said siRNA further comprises a fluorescent tag. The method of claim 8 wherein said lipid mixture; siRNA; and said solvent are mixed in a ratio of about 10:1 :40. A method of visualizing liposomal uptake by atherosclerotic plaques, comprising:

(1 ) administering to a patient comprising said plaque a fluorescently labeled anionic liposome, wherein said liposome comprises a fluorescent tagged siRNA; and

(2) visualizing said labeled anionic liposome, and identifying the location of said liposomes.