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Multifunctional Nanoparticles Facilitate Molecular Targeting and miRNA Delivery to Inhibit Atherosclerosis in ApoE – / – Mice

View Article: PubMed Central - PubMed

ABSTRACT

The current study presents an effective and selective multifunctional nanoparticle used to deliver antiatherogenic therapeutics to inflamed pro-atherogenic regions without off-target changes in gene expression or particle-induced toxicities. MicroRNAs (miRNAs) regulate gene expression, playing a critical role in biology and disease including atherosclerosis. While anti-miRNA are emerging as therapeutics, numerous challenges remain due to their potential off-target effects, and therefore the development of carriers for selective delivery to diseased sites is important. Yet, co-optimization of multifunctional nanoparticles with high loading efficiency, a hidden cationic domain to facilitate lysosomal escape and a dense, stable incorporation of targeting moieties is challenging. Here, we create coated, cationic lipoparticles (CCLs), containing anti-miR-712 (∼1400 molecules, >95% loading efficiency) within the core and with a neutral coating, decorated with 5 mol % of peptide (VHPK) to target vascular cell adhesion molecule 1 (VCAM1). Optical imaging validated disease-specific accumulation as anti-miR-712 was efficiently delivered to inflamed mouse aortic endothelial cells in vitro and in vivo. As with the naked anti-miR-712, the delivery of VHPK-CCL-anti-miR-712 effectively downregulated the d-flow induced expression of miR-712 and also rescued the expression of its target genes tissue inhibitor of metalloproteinase 3 (TIMP3) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) in the endothelium, resulting in inhibition of metalloproteinase activity. Moreover, an 80% lower dose of VHPK-CCL-anti-miR-712 (1 mg/kg dose given twice a week), as compared with naked anti-miR-712, prevented atheroma formation in a mouse model of atherosclerosis. While delivery of naked anti-miR-712 alters expression in multiple organs, miR-712 expression in nontargeted organs was unchanged following VHPK-CCL-anti-miR-712 delivery.

No MeSH data available.


Related in: MedlinePlus

Preparation and characterization of coated-cationic lipoparticles (CCLs) and VHPK-CCLs encapsulating anti-miR-712. (A) The hydrophilic anti-miR-712 in water and hydrophobic DOTAP in chloroform are brought to interaction by addition of methanol to induce the Bligh–Dyer monophase and create the hydrophobic DOTAP-anti-miR-712 complex. Following the addition of water and chloroform to produce a biphasic solution, the hydrophobic complex is extracted in the organic phase and added to a mixture of neutral and PEG lipids. A stable water-in-oil microemulsion was induced upon addition of water. Gradual removal of chloroform resulted in formation of CCLs with asymmetric lipid bilayers entrapping anti-miR-712 within the core. The VCAM1 targeting was achieved by postinsertion of VHPK-LPP into the outer leaflet of CCL-anti-miR-712. (B) Diameter and zeta potential of nontargeted-CCL-anti-miR-712 and VHPK-CCL-anti-miR-712. (C) Electron microscopy image of CCL-anti-miR-712.
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fig1: Preparation and characterization of coated-cationic lipoparticles (CCLs) and VHPK-CCLs encapsulating anti-miR-712. (A) The hydrophilic anti-miR-712 in water and hydrophobic DOTAP in chloroform are brought to interaction by addition of methanol to induce the Bligh–Dyer monophase and create the hydrophobic DOTAP-anti-miR-712 complex. Following the addition of water and chloroform to produce a biphasic solution, the hydrophobic complex is extracted in the organic phase and added to a mixture of neutral and PEG lipids. A stable water-in-oil microemulsion was induced upon addition of water. Gradual removal of chloroform resulted in formation of CCLs with asymmetric lipid bilayers entrapping anti-miR-712 within the core. The VCAM1 targeting was achieved by postinsertion of VHPK-LPP into the outer leaflet of CCL-anti-miR-712. (B) Diameter and zeta potential of nontargeted-CCL-anti-miR-712 and VHPK-CCL-anti-miR-712. (C) Electron microscopy image of CCL-anti-miR-712.

Mentions: The CCL synthesis and characterization are described in detail in the Material and Methods and Supporting Information. Briefly, anti-miR-712 was encapsulated into a formulation of DOTAP:DSPE-PEG2k:HSPC:chol (9.3:3.1:52.6:35, molar ratio) in a multistep process (Figure 1A). First, an electrostatic interaction was formed between the negatively charged oligonucleic acid and the positively charged lipid, DOTAP. By employing the concept of Bligh and Dyer monophase,29 the hydrophilic oligonucleic acids were extracted into the organic phase and formed hydrophobic anti-miR-712-DOTAP particles. Anti-miR-712 is a 15-mer oligonucleic acid and stoichiometrically can bind to up to 15 mol of DOTAP. To optimize the various steps involved in the loading procedure and maximize the loading of anti-miR-712, anti-miR-712 was first labeled with carboxyfluorescein (anti-miR-712-FAM). In a series of pilot studies, the amount of DOTAP was kept constant at 0.72 mg (1.03 μmol) and the amount of FAM-labeled anti-miR-712 (anti-miR-712-FAM) was gradually increased to fully react with DOTAP. We found that with 480 μg of anti-miR-712-FAM, 95% of the total was extracted into the organic phase and thus loaded into the particles.


Multifunctional Nanoparticles Facilitate Molecular Targeting and miRNA Delivery to Inhibit Atherosclerosis in ApoE – / – Mice
Preparation and characterization of coated-cationic lipoparticles (CCLs) and VHPK-CCLs encapsulating anti-miR-712. (A) The hydrophilic anti-miR-712 in water and hydrophobic DOTAP in chloroform are brought to interaction by addition of methanol to induce the Bligh–Dyer monophase and create the hydrophobic DOTAP-anti-miR-712 complex. Following the addition of water and chloroform to produce a biphasic solution, the hydrophobic complex is extracted in the organic phase and added to a mixture of neutral and PEG lipids. A stable water-in-oil microemulsion was induced upon addition of water. Gradual removal of chloroform resulted in formation of CCLs with asymmetric lipid bilayers entrapping anti-miR-712 within the core. The VCAM1 targeting was achieved by postinsertion of VHPK-LPP into the outer leaflet of CCL-anti-miR-712. (B) Diameter and zeta potential of nontargeted-CCL-anti-miR-712 and VHPK-CCL-anti-miR-712. (C) Electron microscopy image of CCL-anti-miR-712.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581466&req=5

fig1: Preparation and characterization of coated-cationic lipoparticles (CCLs) and VHPK-CCLs encapsulating anti-miR-712. (A) The hydrophilic anti-miR-712 in water and hydrophobic DOTAP in chloroform are brought to interaction by addition of methanol to induce the Bligh–Dyer monophase and create the hydrophobic DOTAP-anti-miR-712 complex. Following the addition of water and chloroform to produce a biphasic solution, the hydrophobic complex is extracted in the organic phase and added to a mixture of neutral and PEG lipids. A stable water-in-oil microemulsion was induced upon addition of water. Gradual removal of chloroform resulted in formation of CCLs with asymmetric lipid bilayers entrapping anti-miR-712 within the core. The VCAM1 targeting was achieved by postinsertion of VHPK-LPP into the outer leaflet of CCL-anti-miR-712. (B) Diameter and zeta potential of nontargeted-CCL-anti-miR-712 and VHPK-CCL-anti-miR-712. (C) Electron microscopy image of CCL-anti-miR-712.
Mentions: The CCL synthesis and characterization are described in detail in the Material and Methods and Supporting Information. Briefly, anti-miR-712 was encapsulated into a formulation of DOTAP:DSPE-PEG2k:HSPC:chol (9.3:3.1:52.6:35, molar ratio) in a multistep process (Figure 1A). First, an electrostatic interaction was formed between the negatively charged oligonucleic acid and the positively charged lipid, DOTAP. By employing the concept of Bligh and Dyer monophase,29 the hydrophilic oligonucleic acids were extracted into the organic phase and formed hydrophobic anti-miR-712-DOTAP particles. Anti-miR-712 is a 15-mer oligonucleic acid and stoichiometrically can bind to up to 15 mol of DOTAP. To optimize the various steps involved in the loading procedure and maximize the loading of anti-miR-712, anti-miR-712 was first labeled with carboxyfluorescein (anti-miR-712-FAM). In a series of pilot studies, the amount of DOTAP was kept constant at 0.72 mg (1.03 μmol) and the amount of FAM-labeled anti-miR-712 (anti-miR-712-FAM) was gradually increased to fully react with DOTAP. We found that with 480 μg of anti-miR-712-FAM, 95% of the total was extracted into the organic phase and thus loaded into the particles.

View Article: PubMed Central - PubMed

ABSTRACT

The current study presents an effective and selective multifunctional nanoparticle used to deliver antiatherogenic therapeutics to inflamed pro-atherogenic regions without off-target changes in gene expression or particle-induced toxicities. MicroRNAs (miRNAs) regulate gene expression, playing a critical role in biology and disease including atherosclerosis. While anti-miRNA are emerging as therapeutics, numerous challenges remain due to their potential off-target effects, and therefore the development of carriers for selective delivery to diseased sites is important. Yet, co-optimization of multifunctional nanoparticles with high loading efficiency, a hidden cationic domain to facilitate lysosomal escape and a dense, stable incorporation of targeting moieties is challenging. Here, we create coated, cationic lipoparticles (CCLs), containing anti-miR-712 (∼1400 molecules, >95% loading efficiency) within the core and with a neutral coating, decorated with 5 mol % of peptide (VHPK) to target vascular cell adhesion molecule 1 (VCAM1). Optical imaging validated disease-specific accumulation as anti-miR-712 was efficiently delivered to inflamed mouse aortic endothelial cells in vitro and in vivo. As with the naked anti-miR-712, the delivery of VHPK-CCL-anti-miR-712 effectively downregulated the d-flow induced expression of miR-712 and also rescued the expression of its target genes tissue inhibitor of metalloproteinase 3 (TIMP3) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) in the endothelium, resulting in inhibition of metalloproteinase activity. Moreover, an 80% lower dose of VHPK-CCL-anti-miR-712 (1 mg/kg dose given twice a week), as compared with naked anti-miR-712, prevented atheroma formation in a mouse model of atherosclerosis. While delivery of naked anti-miR-712 alters expression in multiple organs, miR-712 expression in nontargeted organs was unchanged following VHPK-CCL-anti-miR-712 delivery.

No MeSH data available.


Related in: MedlinePlus