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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.


VCAM1-dependent delivery of VHPK-CCL-anti-miR-712 and its silencing efficiency in iMAECs. (A) Anti-miR-712 labeled with Alexa555 (200 nM) was delivered to iMAECs in 2 different ways, VHPK-CCL-anti-miR-712 or CCL-anti-miR-712 for 30 min. iMAECs were pretreated with or without TNFα (3 ng/mL) to induce VCAM1. Fluorescence microscopy imaging of iMAECs shows delivery of anti-miR-712 (red) in the TNFα-treated VHPK-CCL group (iv). (B–F) To test the silencing efficiency of 3 different nanoparticles, iMAECs pretreated without or with TNFα were incubated with VHPK-CCL-anti-miR-712, VHPK-CCL-mismatched, and CCL-anti-miR-712 for 24 h. (B) VCAM1 induction in TNFα-treated cells by qPCR. (C) Basal miR-712 expression that was reduced in VHPK-CCL-anti-miR-712 treated cells. (D–F) iMAECs transfected with pre-miR-712 (20 nM) plus TNFα were further incubated with the lipid nanoparticles for 24 h and expression of miR-712 (D), TIMP3 (E) and RECK (F) was determined by qPCR and normalized to 18S. n = 5, data shown as mean ± s.e.m; *p < 0.05 as determined by Student’s t-test.
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fig2: VCAM1-dependent delivery of VHPK-CCL-anti-miR-712 and its silencing efficiency in iMAECs. (A) Anti-miR-712 labeled with Alexa555 (200 nM) was delivered to iMAECs in 2 different ways, VHPK-CCL-anti-miR-712 or CCL-anti-miR-712 for 30 min. iMAECs were pretreated with or without TNFα (3 ng/mL) to induce VCAM1. Fluorescence microscopy imaging of iMAECs shows delivery of anti-miR-712 (red) in the TNFα-treated VHPK-CCL group (iv). (B–F) To test the silencing efficiency of 3 different nanoparticles, iMAECs pretreated without or with TNFα were incubated with VHPK-CCL-anti-miR-712, VHPK-CCL-mismatched, and CCL-anti-miR-712 for 24 h. (B) VCAM1 induction in TNFα-treated cells by qPCR. (C) Basal miR-712 expression that was reduced in VHPK-CCL-anti-miR-712 treated cells. (D–F) iMAECs transfected with pre-miR-712 (20 nM) plus TNFα were further incubated with the lipid nanoparticles for 24 h and expression of miR-712 (D), TIMP3 (E) and RECK (F) was determined by qPCR and normalized to 18S. n = 5, data shown as mean ± s.e.m; *p < 0.05 as determined by Student’s t-test.

Mentions: Since the VHPK-conjugated nanoparticles are known to be internalized via endocytosis,25,39,40 we first determined whether VHPK-CCL-anti-miR-712 can be internalized into endothelial cells. For this study, anti-miR-712 labeled with Alexa555 (easier to detect by fluorescence microscopy than FAM against autofluorescence background in arteries) was encapsulated into either VHPK-CCLs (VHPK-CCL-anti-miR-712-Alexa555) or CCLs (CCL-anti-miR-712-Alexa555) as a nontargeting control. Mouse endothelial cells (iMAECs) were pretreated with TNFα (3 ng/mL), a well-known inducer of VCAM1 expression on the endothelial surface, and incubated with VHPK-CCL-anti-miR-712-Alexa555 or CCL-anti-miR-712-Alexa555 for 30 min and imaged using fluorescence microscopy. When compared to CCL-anti-miR-712, however, VHPK-CCL-anti-miR-712 was abundantly observed in the cytosol of TNFα-treated endothelial cells but not in untreated cells, suggesting its VCAM1-dependent intracellular delivery (Figure 2A).


Multifunctional Nanoparticles Facilitate Molecular Targeting and miRNA Delivery to Inhibit Atherosclerosis in ApoE – / – Mice
VCAM1-dependent delivery of VHPK-CCL-anti-miR-712 and its silencing efficiency in iMAECs. (A) Anti-miR-712 labeled with Alexa555 (200 nM) was delivered to iMAECs in 2 different ways, VHPK-CCL-anti-miR-712 or CCL-anti-miR-712 for 30 min. iMAECs were pretreated with or without TNFα (3 ng/mL) to induce VCAM1. Fluorescence microscopy imaging of iMAECs shows delivery of anti-miR-712 (red) in the TNFα-treated VHPK-CCL group (iv). (B–F) To test the silencing efficiency of 3 different nanoparticles, iMAECs pretreated without or with TNFα were incubated with VHPK-CCL-anti-miR-712, VHPK-CCL-mismatched, and CCL-anti-miR-712 for 24 h. (B) VCAM1 induction in TNFα-treated cells by qPCR. (C) Basal miR-712 expression that was reduced in VHPK-CCL-anti-miR-712 treated cells. (D–F) iMAECs transfected with pre-miR-712 (20 nM) plus TNFα were further incubated with the lipid nanoparticles for 24 h and expression of miR-712 (D), TIMP3 (E) and RECK (F) was determined by qPCR and normalized to 18S. n = 5, data shown as mean ± s.e.m; *p < 0.05 as determined by Student’s t-test.
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fig2: VCAM1-dependent delivery of VHPK-CCL-anti-miR-712 and its silencing efficiency in iMAECs. (A) Anti-miR-712 labeled with Alexa555 (200 nM) was delivered to iMAECs in 2 different ways, VHPK-CCL-anti-miR-712 or CCL-anti-miR-712 for 30 min. iMAECs were pretreated with or without TNFα (3 ng/mL) to induce VCAM1. Fluorescence microscopy imaging of iMAECs shows delivery of anti-miR-712 (red) in the TNFα-treated VHPK-CCL group (iv). (B–F) To test the silencing efficiency of 3 different nanoparticles, iMAECs pretreated without or with TNFα were incubated with VHPK-CCL-anti-miR-712, VHPK-CCL-mismatched, and CCL-anti-miR-712 for 24 h. (B) VCAM1 induction in TNFα-treated cells by qPCR. (C) Basal miR-712 expression that was reduced in VHPK-CCL-anti-miR-712 treated cells. (D–F) iMAECs transfected with pre-miR-712 (20 nM) plus TNFα were further incubated with the lipid nanoparticles for 24 h and expression of miR-712 (D), TIMP3 (E) and RECK (F) was determined by qPCR and normalized to 18S. n = 5, data shown as mean ± s.e.m; *p < 0.05 as determined by Student’s t-test.
Mentions: Since the VHPK-conjugated nanoparticles are known to be internalized via endocytosis,25,39,40 we first determined whether VHPK-CCL-anti-miR-712 can be internalized into endothelial cells. For this study, anti-miR-712 labeled with Alexa555 (easier to detect by fluorescence microscopy than FAM against autofluorescence background in arteries) was encapsulated into either VHPK-CCLs (VHPK-CCL-anti-miR-712-Alexa555) or CCLs (CCL-anti-miR-712-Alexa555) as a nontargeting control. Mouse endothelial cells (iMAECs) were pretreated with TNFα (3 ng/mL), a well-known inducer of VCAM1 expression on the endothelial surface, and incubated with VHPK-CCL-anti-miR-712-Alexa555 or CCL-anti-miR-712-Alexa555 for 30 min and imaged using fluorescence microscopy. When compared to CCL-anti-miR-712, however, VHPK-CCL-anti-miR-712 was abundantly observed in the cytosol of TNFα-treated endothelial cells but not in untreated cells, suggesting its VCAM1-dependent intracellular delivery (Figure 2A).

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 (&sim;1400 molecules, &gt;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.