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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

Targeted delivery of VHPK-CCLs to inflamed endothelium expressing VCAM1 in d-flow regions. (A) The partial carotid ligation surgery in the left common carotid artery (LCA) and contralateral RCA along with the naturally occurring d-flow (lesser curvature, LC) and s-flow (greater curvature, GC) regions in the aortic arch are indicated. (B) VCAM1 antibody staining (red) in arterial endothelium was carried out using the LCA and RCA as well as the aortic arches (GC and LC) obtained from C57BL/6 mice at 4 days post partial carotid ligation surgery. Shown are representative (n = 3) en face confocal images of VCAM1 (red), DAPI (blue) and internal elastic lamina (IEL, green autofluorescence). (C) At 4 days postpartial ligation, anti-miR-712 was conjugated with Alexa555 and mice were tail-vein injected with VHPK-CCL-anti-miR-712 or CCL-anti-miR-712 (1 mg/kg). One hour later, the carotids and aortic arch were dissected out and en face confocal imaged. Representative images (n = 3) showed that Alexa555 (red) was visible in mouse arteries injected with VHPK-CCL-Alexa only in d-flow regions (LCA and LC). DAPI (blue) and IEL (green autofluorescence).
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fig3: Targeted delivery of VHPK-CCLs to inflamed endothelium expressing VCAM1 in d-flow regions. (A) The partial carotid ligation surgery in the left common carotid artery (LCA) and contralateral RCA along with the naturally occurring d-flow (lesser curvature, LC) and s-flow (greater curvature, GC) regions in the aortic arch are indicated. (B) VCAM1 antibody staining (red) in arterial endothelium was carried out using the LCA and RCA as well as the aortic arches (GC and LC) obtained from C57BL/6 mice at 4 days post partial carotid ligation surgery. Shown are representative (n = 3) en face confocal images of VCAM1 (red), DAPI (blue) and internal elastic lamina (IEL, green autofluorescence). (C) At 4 days postpartial ligation, anti-miR-712 was conjugated with Alexa555 and mice were tail-vein injected with VHPK-CCL-anti-miR-712 or CCL-anti-miR-712 (1 mg/kg). One hour later, the carotids and aortic arch were dissected out and en face confocal imaged. Representative images (n = 3) showed that Alexa555 (red) was visible in mouse arteries injected with VHPK-CCL-Alexa only in d-flow regions (LCA and LC). DAPI (blue) and IEL (green autofluorescence).

Mentions: We first confirmed expression of VCAM1 as a marker of inflammation in various regions of mouse arterial endothelium exposed to d-flow or s-flow by en face confocal imaging. As we showed previously,2 the mouse partial carotid ligation surgery induces d-flow in the left carotid artery (LCA) while the contralateral right carotid artery (RCA) continues to experience s-flow (Figure 3A). VCAM1 protein expression (shown in red) significantly increased in the LCA endothelium compared to the RCA at 4-days post partial carotid ligation surgery (Figure 3B). Further, expression of VCAM1 was significantly increased in the lesser curvature (LC) region of the aortic arch (naturally occurring pro-atherogenic d-flow region) compared to the greater curvature (GC) region (naturally occurring athero-resistant s-flow region) in control C57BL/6 mice as well (Figure 3A and B). These results confirmed that VCAM1 expression was significantly higher in endothelial cells exposed to d-flow conditions induced either by surgery or naturally in curved arteries.


Multifunctional Nanoparticles Facilitate Molecular Targeting and miRNA Delivery to Inhibit Atherosclerosis in ApoE – / – Mice
Targeted delivery of VHPK-CCLs to inflamed endothelium expressing VCAM1 in d-flow regions. (A) The partial carotid ligation surgery in the left common carotid artery (LCA) and contralateral RCA along with the naturally occurring d-flow (lesser curvature, LC) and s-flow (greater curvature, GC) regions in the aortic arch are indicated. (B) VCAM1 antibody staining (red) in arterial endothelium was carried out using the LCA and RCA as well as the aortic arches (GC and LC) obtained from C57BL/6 mice at 4 days post partial carotid ligation surgery. Shown are representative (n = 3) en face confocal images of VCAM1 (red), DAPI (blue) and internal elastic lamina (IEL, green autofluorescence). (C) At 4 days postpartial ligation, anti-miR-712 was conjugated with Alexa555 and mice were tail-vein injected with VHPK-CCL-anti-miR-712 or CCL-anti-miR-712 (1 mg/kg). One hour later, the carotids and aortic arch were dissected out and en face confocal imaged. Representative images (n = 3) showed that Alexa555 (red) was visible in mouse arteries injected with VHPK-CCL-Alexa only in d-flow regions (LCA and LC). DAPI (blue) and IEL (green autofluorescence).
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fig3: Targeted delivery of VHPK-CCLs to inflamed endothelium expressing VCAM1 in d-flow regions. (A) The partial carotid ligation surgery in the left common carotid artery (LCA) and contralateral RCA along with the naturally occurring d-flow (lesser curvature, LC) and s-flow (greater curvature, GC) regions in the aortic arch are indicated. (B) VCAM1 antibody staining (red) in arterial endothelium was carried out using the LCA and RCA as well as the aortic arches (GC and LC) obtained from C57BL/6 mice at 4 days post partial carotid ligation surgery. Shown are representative (n = 3) en face confocal images of VCAM1 (red), DAPI (blue) and internal elastic lamina (IEL, green autofluorescence). (C) At 4 days postpartial ligation, anti-miR-712 was conjugated with Alexa555 and mice were tail-vein injected with VHPK-CCL-anti-miR-712 or CCL-anti-miR-712 (1 mg/kg). One hour later, the carotids and aortic arch were dissected out and en face confocal imaged. Representative images (n = 3) showed that Alexa555 (red) was visible in mouse arteries injected with VHPK-CCL-Alexa only in d-flow regions (LCA and LC). DAPI (blue) and IEL (green autofluorescence).
Mentions: We first confirmed expression of VCAM1 as a marker of inflammation in various regions of mouse arterial endothelium exposed to d-flow or s-flow by en face confocal imaging. As we showed previously,2 the mouse partial carotid ligation surgery induces d-flow in the left carotid artery (LCA) while the contralateral right carotid artery (RCA) continues to experience s-flow (Figure 3A). VCAM1 protein expression (shown in red) significantly increased in the LCA endothelium compared to the RCA at 4-days post partial carotid ligation surgery (Figure 3B). Further, expression of VCAM1 was significantly increased in the lesser curvature (LC) region of the aortic arch (naturally occurring pro-atherogenic d-flow region) compared to the greater curvature (GC) region (naturally occurring athero-resistant s-flow region) in control C57BL/6 mice as well (Figure 3A and B). These results confirmed that VCAM1 expression was significantly higher in endothelial cells exposed to d-flow conditions induced either by surgery or naturally in curved arteries.

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