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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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Related in: MedlinePlus

Treatment with VHPK-CCL-anti-miR-712 inhibits atherosclerosis in ApoE–/– mice. ApoE–/– mice received the partial carotid ligation surgery plus high-fat diet for 2 weeks to rapidly develop atherosclerosis in the LCA. At 4, 5, 8, and 11 days post partial ligation surgery, mice were injected with VHPK-CCL-anti-miR-712, VHPK-CCL-anti-miR-mismatched control, or CCL-anti-miR-712 (1 mg/kg anti-miR-712 dose each injected in 150 μL via tail-vein). Mice were sacrificed at 2 weeks postsurgery, arterial trees dissected out for bright field imaging and histological evaluation (A) and plaque size quantification by image analysis (B). n = 7 mice each, data shown as mean ± s.e.m; *p < 0.05 as determined by 1-way ANOVA. Following bright field imaging, LCA and RCA were frozen sectioned (from the mid regions indicated by the arrows) and stained with Oil-Red-O for further immunohistochemical examination as shown by representative images (A). (C) VHPK-CCL-anti-miR-712 did not affect miR-712 expression in other tissues and cells. Following extraction of arteries, various tissues and whole blood were also obtained from these mice, total RNA extracted, and expression of miR-712 in liver, kidneys, lungs, spleen, thymus, bone marrow and PBMCs was determined by qPCR. n = 7 mice each; data shown as mean ± s.e.m.
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fig5: Treatment with VHPK-CCL-anti-miR-712 inhibits atherosclerosis in ApoE–/– mice. ApoE–/– mice received the partial carotid ligation surgery plus high-fat diet for 2 weeks to rapidly develop atherosclerosis in the LCA. At 4, 5, 8, and 11 days post partial ligation surgery, mice were injected with VHPK-CCL-anti-miR-712, VHPK-CCL-anti-miR-mismatched control, or CCL-anti-miR-712 (1 mg/kg anti-miR-712 dose each injected in 150 μL via tail-vein). Mice were sacrificed at 2 weeks postsurgery, arterial trees dissected out for bright field imaging and histological evaluation (A) and plaque size quantification by image analysis (B). n = 7 mice each, data shown as mean ± s.e.m; *p < 0.05 as determined by 1-way ANOVA. Following bright field imaging, LCA and RCA were frozen sectioned (from the mid regions indicated by the arrows) and stained with Oil-Red-O for further immunohistochemical examination as shown by representative images (A). (C) VHPK-CCL-anti-miR-712 did not affect miR-712 expression in other tissues and cells. Following extraction of arteries, various tissues and whole blood were also obtained from these mice, total RNA extracted, and expression of miR-712 in liver, kidneys, lungs, spleen, thymus, bone marrow and PBMCs was determined by qPCR. n = 7 mice each; data shown as mean ± s.e.m.

Mentions: We tested whether VHPK-CCL-anti-miR-712 treatment can inhibit atherosclerosis development using the partial carotid ligation model using ApoE–/– mice. As shown previously,2,9 partial carotid ligation surgery plus a high-fat diet rapidly induced robust atherosclerosis (indicated with white arrow) in the LCA within 2 weeks, as shown by bright field imaging (top) and cross-sectional Oil-Red-O stained images of LCA and RCA (mid and bottom panels) (Figure 5A). Intravenous injection of VHPK-CCL-anti-miR-712 (1 mg/kg, twice a week for 2 weeks) significantly reduced atherosclerotic lesion development (Figure 5A and B), compared to the mismatched or nontargeting controls. Further, lipid profiling showed no significant difference between the three CCL groups, suggesting that the antiatherogenic effect of VHPK-CCL-anti-miR-712 was not due to changes in the lipid profile (SI Table S2).


Multifunctional Nanoparticles Facilitate Molecular Targeting and miRNA Delivery to Inhibit Atherosclerosis in ApoE – / – Mice
Treatment with VHPK-CCL-anti-miR-712 inhibits atherosclerosis in ApoE–/– mice. ApoE–/– mice received the partial carotid ligation surgery plus high-fat diet for 2 weeks to rapidly develop atherosclerosis in the LCA. At 4, 5, 8, and 11 days post partial ligation surgery, mice were injected with VHPK-CCL-anti-miR-712, VHPK-CCL-anti-miR-mismatched control, or CCL-anti-miR-712 (1 mg/kg anti-miR-712 dose each injected in 150 μL via tail-vein). Mice were sacrificed at 2 weeks postsurgery, arterial trees dissected out for bright field imaging and histological evaluation (A) and plaque size quantification by image analysis (B). n = 7 mice each, data shown as mean ± s.e.m; *p < 0.05 as determined by 1-way ANOVA. Following bright field imaging, LCA and RCA were frozen sectioned (from the mid regions indicated by the arrows) and stained with Oil-Red-O for further immunohistochemical examination as shown by representative images (A). (C) VHPK-CCL-anti-miR-712 did not affect miR-712 expression in other tissues and cells. Following extraction of arteries, various tissues and whole blood were also obtained from these mice, total RNA extracted, and expression of miR-712 in liver, kidneys, lungs, spleen, thymus, bone marrow and PBMCs was determined by qPCR. n = 7 mice each; data shown as mean ± s.e.m.
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fig5: Treatment with VHPK-CCL-anti-miR-712 inhibits atherosclerosis in ApoE–/– mice. ApoE–/– mice received the partial carotid ligation surgery plus high-fat diet for 2 weeks to rapidly develop atherosclerosis in the LCA. At 4, 5, 8, and 11 days post partial ligation surgery, mice were injected with VHPK-CCL-anti-miR-712, VHPK-CCL-anti-miR-mismatched control, or CCL-anti-miR-712 (1 mg/kg anti-miR-712 dose each injected in 150 μL via tail-vein). Mice were sacrificed at 2 weeks postsurgery, arterial trees dissected out for bright field imaging and histological evaluation (A) and plaque size quantification by image analysis (B). n = 7 mice each, data shown as mean ± s.e.m; *p < 0.05 as determined by 1-way ANOVA. Following bright field imaging, LCA and RCA were frozen sectioned (from the mid regions indicated by the arrows) and stained with Oil-Red-O for further immunohistochemical examination as shown by representative images (A). (C) VHPK-CCL-anti-miR-712 did not affect miR-712 expression in other tissues and cells. Following extraction of arteries, various tissues and whole blood were also obtained from these mice, total RNA extracted, and expression of miR-712 in liver, kidneys, lungs, spleen, thymus, bone marrow and PBMCs was determined by qPCR. n = 7 mice each; data shown as mean ± s.e.m.
Mentions: We tested whether VHPK-CCL-anti-miR-712 treatment can inhibit atherosclerosis development using the partial carotid ligation model using ApoE–/– mice. As shown previously,2,9 partial carotid ligation surgery plus a high-fat diet rapidly induced robust atherosclerosis (indicated with white arrow) in the LCA within 2 weeks, as shown by bright field imaging (top) and cross-sectional Oil-Red-O stained images of LCA and RCA (mid and bottom panels) (Figure 5A). Intravenous injection of VHPK-CCL-anti-miR-712 (1 mg/kg, twice a week for 2 weeks) significantly reduced atherosclerotic lesion development (Figure 5A and B), compared to the mismatched or nontargeting controls. Further, lipid profiling showed no significant difference between the three CCL groups, suggesting that the antiatherogenic effect of VHPK-CCL-anti-miR-712 was not due to changes in the lipid profile (SI Table S2).

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