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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.

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Selective and efficient silencing effect of VHPK-CCL-anti-miR-712 in inflamed endothelium in d-flow regions of mice. At 2 days following partial carotid ligation, ApoE–/– mice were tail-vein injected with VHPK-CCL-anti-miR-712, VHPK-CCL-mismatched control (VHPK-CCL-anti-m.m) or CCL-anti-miR-712 (1 mg/kg). Mice were sacrificed 2 days later and endothelial-enriched RNA was extracted from the RCA and LCA. Expression of miR-712 (A) and its target genes TIMP3 and RECK (C, E) was determined by qPCR. RNA from the left over (LO) samples (containing media and adventitia) were also tested for expression of miR-712 (B) and its target genes TIMP3 and RECK (D, F) for comparison. Expression of miRNA and mRNAs was normalized to RNU6b and 18S, respectively as internal controls. n = 5, data shown as mean ± s.e.m; *p < 0.05 as determined by Student’s t-test. In some studies, frozen sections of the RCA and LCA (n = 5 each) were prepared and immunostained with TIMP3 (red) (G); and in situ gelatinase activity assay using DQ-gelatin (green) (H) was performed as shown by the representative images (n = 5). Inset shows zoomed sections of endothelial regions showing expression of TIMP3 under different treatment conditions. L = lumen. Internal elastic lamina (green autofluorescence), nuclei (blue).
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fig4: Selective and efficient silencing effect of VHPK-CCL-anti-miR-712 in inflamed endothelium in d-flow regions of mice. At 2 days following partial carotid ligation, ApoE–/– mice were tail-vein injected with VHPK-CCL-anti-miR-712, VHPK-CCL-mismatched control (VHPK-CCL-anti-m.m) or CCL-anti-miR-712 (1 mg/kg). Mice were sacrificed 2 days later and endothelial-enriched RNA was extracted from the RCA and LCA. Expression of miR-712 (A) and its target genes TIMP3 and RECK (C, E) was determined by qPCR. RNA from the left over (LO) samples (containing media and adventitia) were also tested for expression of miR-712 (B) and its target genes TIMP3 and RECK (D, F) for comparison. Expression of miRNA and mRNAs was normalized to RNU6b and 18S, respectively as internal controls. n = 5, data shown as mean ± s.e.m; *p < 0.05 as determined by Student’s t-test. In some studies, frozen sections of the RCA and LCA (n = 5 each) were prepared and immunostained with TIMP3 (red) (G); and in situ gelatinase activity assay using DQ-gelatin (green) (H) was performed as shown by the representative images (n = 5). Inset shows zoomed sections of endothelial regions showing expression of TIMP3 under different treatment conditions. L = lumen. Internal elastic lamina (green autofluorescence), nuclei (blue).

Mentions: Next, we determined if anti-miR-712 delivery using VHPK-CCLs can effectively silence increased miR-712 expression in inflamed LCA endothelium of partially ligated mice. For this study, VHPK-CCL-anti-miR-712, VHPK-CCL-mismatched control, or nontargeted-CCL-anti-miR-712 (1 mg/kg each) were injected into C57BL/6 mice via the tail vein 4 and 5 days post partial carotid ligation. At 7 days post partial ligation, endothelial-enriched RNA was isolated from the RCA and LCA, respectively. The expression of miR-712 was increased in the LCA endothelium (EC-enriched) compared to the RCA (Figure 4A) which was significantly silenced in endothelium only in the mice treated with VHPK-CCL-anti-miR-712 but not in VHPK-CCL-mismatched or nontargeting CCL-anti-miR-712 controls. Interestingly, however, this silencing effect of VHPK-CCL-anti-miR-712 was observed only in endothelial layer but not in the media and adventitia (Figure 4A and B). The mechanism for the lack of anti-miR-712 effect in the media and adventitia is currently unclear, but correlates with the lack of anti-miR-712-Alexa555 in the media (SI Figure S6).


Multifunctional Nanoparticles Facilitate Molecular Targeting and miRNA Delivery to Inhibit Atherosclerosis in ApoE – / – Mice
Selective and efficient silencing effect of VHPK-CCL-anti-miR-712 in inflamed endothelium in d-flow regions of mice. At 2 days following partial carotid ligation, ApoE–/– mice were tail-vein injected with VHPK-CCL-anti-miR-712, VHPK-CCL-mismatched control (VHPK-CCL-anti-m.m) or CCL-anti-miR-712 (1 mg/kg). Mice were sacrificed 2 days later and endothelial-enriched RNA was extracted from the RCA and LCA. Expression of miR-712 (A) and its target genes TIMP3 and RECK (C, E) was determined by qPCR. RNA from the left over (LO) samples (containing media and adventitia) were also tested for expression of miR-712 (B) and its target genes TIMP3 and RECK (D, F) for comparison. Expression of miRNA and mRNAs was normalized to RNU6b and 18S, respectively as internal controls. n = 5, data shown as mean ± s.e.m; *p < 0.05 as determined by Student’s t-test. In some studies, frozen sections of the RCA and LCA (n = 5 each) were prepared and immunostained with TIMP3 (red) (G); and in situ gelatinase activity assay using DQ-gelatin (green) (H) was performed as shown by the representative images (n = 5). Inset shows zoomed sections of endothelial regions showing expression of TIMP3 under different treatment conditions. L = lumen. Internal elastic lamina (green autofluorescence), nuclei (blue).
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fig4: Selective and efficient silencing effect of VHPK-CCL-anti-miR-712 in inflamed endothelium in d-flow regions of mice. At 2 days following partial carotid ligation, ApoE–/– mice were tail-vein injected with VHPK-CCL-anti-miR-712, VHPK-CCL-mismatched control (VHPK-CCL-anti-m.m) or CCL-anti-miR-712 (1 mg/kg). Mice were sacrificed 2 days later and endothelial-enriched RNA was extracted from the RCA and LCA. Expression of miR-712 (A) and its target genes TIMP3 and RECK (C, E) was determined by qPCR. RNA from the left over (LO) samples (containing media and adventitia) were also tested for expression of miR-712 (B) and its target genes TIMP3 and RECK (D, F) for comparison. Expression of miRNA and mRNAs was normalized to RNU6b and 18S, respectively as internal controls. n = 5, data shown as mean ± s.e.m; *p < 0.05 as determined by Student’s t-test. In some studies, frozen sections of the RCA and LCA (n = 5 each) were prepared and immunostained with TIMP3 (red) (G); and in situ gelatinase activity assay using DQ-gelatin (green) (H) was performed as shown by the representative images (n = 5). Inset shows zoomed sections of endothelial regions showing expression of TIMP3 under different treatment conditions. L = lumen. Internal elastic lamina (green autofluorescence), nuclei (blue).
Mentions: Next, we determined if anti-miR-712 delivery using VHPK-CCLs can effectively silence increased miR-712 expression in inflamed LCA endothelium of partially ligated mice. For this study, VHPK-CCL-anti-miR-712, VHPK-CCL-mismatched control, or nontargeted-CCL-anti-miR-712 (1 mg/kg each) were injected into C57BL/6 mice via the tail vein 4 and 5 days post partial carotid ligation. At 7 days post partial ligation, endothelial-enriched RNA was isolated from the RCA and LCA, respectively. The expression of miR-712 was increased in the LCA endothelium (EC-enriched) compared to the RCA (Figure 4A) which was significantly silenced in endothelium only in the mice treated with VHPK-CCL-anti-miR-712 but not in VHPK-CCL-mismatched or nontargeting CCL-anti-miR-712 controls. Interestingly, however, this silencing effect of VHPK-CCL-anti-miR-712 was observed only in endothelial layer but not in the media and adventitia (Figure 4A and B). The mechanism for the lack of anti-miR-712 effect in the media and adventitia is currently unclear, but correlates with the lack of anti-miR-712-Alexa555 in the media (SI Figure S6).

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.


Related in: MedlinePlus