Targeted polyelectrolyte complex micelles treat vascular complications in vivo

Abstract

Vascular disease is a leading cause of morbidity and mortality in the United States and globally. Pathological vascular remodeling, such as atherosclerosis and stenosis, largely develop at arterial sites of curvature, branching, and bifurcation, where disturbed blood flow activates vascular endothelium. Current pharmacological treatments of vascular complications principally target systemic risk factors. Improvements are needed. We previously devised a targeted polyelectrolyte complex micelle to deliver therapeutic nucleotides to inflamed endothelium in vitro by displaying the peptide VHPKQHR targeting vascular cell adhesion molecule 1 (VCAM-1) on the periphery of the micelle. This paper explores whether this targeted nanomedicine strategy effectively treats vascular complications in vivo. Disturbed flow-induced microRNA-92a (miR-92a) has been linked to endothelial dysfunction. We have engineered a transgenic line (miR-92a^EC-TG /Apoe^-/- ) establishing that selective miR-92a overexpression in adult vascular endothelium causally promotes atherosclerosis in Apoe^-/- mice. We tested the therapeutic effectiveness of the VCAM-1-targeting polyelectrolyte complex micelles to deliver miR-92a inhibitors and treat pathological vascular remodeling in vivo. VCAM-1-targeting micelles preferentially delivered miRNA inhibitors to inflamed endothelial cells in vitro and in vivo. The therapeutic effectiveness of anti-miR-92a therapy in treating atherosclerosis and stenosis in Apoe^-/- mice is markedly enhanced by the VCAM-1-targeting polyelectrolyte complex micelles. These results demonstrate a proof of concept to devise polyelectrolyte complex micelle-based targeted nanomedicine approaches treating vascular complications in vivo.

Keywords: atherosclerosis; nanomedicine; nanoparticle; stenosis; vascular remodeling.

Fig. 1.

Increased atherosclerosis in Apoe^−/− mice by endothelial-specific overexpression of miR-92a. (A) Construction of miR-92a^EC-TG /Apoe^−/− mice in which miR-92a expression is induced in adult vascular endothelium by tamoxifen injections. The triple transgenic mice (miR-92a^EC-TG /Apoe^−/− ) were engineered by crossing Apoe^−/− mice with miR-92a^EC-TG mice. The miR-92a^EC-TG line was generated by breeding the inducible miR-92a transgenic line (miR-92a^TG ) with the Cdh5(PAC)-CreERT2 mice with an inducible Cre recombinase under the control of the vascular endothelial cadherin (Cdh5) promoter. The miR-92a^TG line was generated by inserting the sequence of mmu-miR-92a precursor in the ROSA26 locus in the C57BL/6 mice. (B) Induction of miR-92a in adult vascular endothelial cells by tamoxifen injections. (C) Increased miR-92a expression in lung endothelial cells isolated from tamoxifen-injected miR-92a^EC-TG /Apoe^−/− mice compared to lung endothelial cells isolated from tamoxifen-injected Cdh5(PAC)-CreERT2/Apoe^−/− (CDH5-Cre/Apoe^−/− ) mice (n = 4 to 5 biological samples). (D) Increased miR-92a expression in the endothelium-enriched intima of carotid artery in tamoxifen-injected miR-92a^EC-TG /Apoe^−/− mice compared to tamoxifen-injected CDH5-Cre/Apoe^−/− mice (n = 7 biological samples). (E and F) Increased atherosclerosis in the aortic root in tamoxifen-injected miR-92a^EC-TG /Apoe^−/− mice compared to tamoxifen-injected CDH5-Cre/Apoe^−/− mice (n = 7 biological samples). (G) The plasma cholesterol was not significantly affected by endothelial miR-92a overexpression in tamoxifen-injected miR-92a^EC-TG /Apoe^−/− mice compared to tamoxifen-injected CDH5-Cre/Apoe^−/− mice (n = 8 biological samples). Statistical significance was determined by multiple unpaired one-tailed t tests. All error bars are means ± SD. n.s., not significant. *P ≤ 0.05; ***P ≤ 0.001.

Fig. 2.

The formulation and characterizations of VCAM-1–targeting polyelectrolyte complex micelles which encapsulate miRNA inhibitor. (A) The illustration of formation of VCAM-1–targeting polyelectrolyte complexes encapsulating miRNA inhibitors in the core. (B) Condensation of miRNA inhibitor by the VHPKQHR-PEG-K30 at the mass ratio (polymer/miRNA inhibitor) of 2 or above demonstrated by an agarose gel retardation assay. (C) Encapsulation of miRNA inhibitor by the VHPKQHR-PEG-K30 demonstrated by a EthBr competitive binding assay. (D) ζ-Potentials of the VHPKQHR-PEG-K30 and VCAM-1–targeting micelles encapsulating miRNA inhibitor (n = 3). (E) ITC therogram of the assembling of VCAM-1–targeting polyelectrolyte complex micelles encapsulating miRNA inhibitor. (F) A negatively stained TEM image of miRNA inhibitor-encapsulated, VCAM-1–targeting polyelectrolyte complex micelles. (G) The hydrodynamic diameter of the miRNA inhibitor-encapsulated, VCAM-1–targeting polyelectrolyte complex micelles. Statistical significance was determined by multiple unpaired one-tailed t tests. All error bars are means ± SD. ***P ≤ 0.001.

Fig. 3.

Delivery of miRNA inhibitor to inflamed cultured vascular endothelial cells by VCAM-1–targeting polyelectrolyte complex micelles in vitro. (A) Increased inflammation, demonstrated by elevated expression of VCAM-1, E-Selectin, CCL2, and IL-6 in HAEC treated with LPS (n = 3 biological samples). (B) Representative confocal images demonstrating the cellular uptake of Dye 547–labeled miRNA inhibitor Ctrl delivered by VCAM1-targeting polyelectrolyte complex micelles in LPS-treated HAEC but not in quiescent control HAEC. Limited cellular uptake of Dye 547–labeled miRNA inhibitor Ctrl, delivered by nontargeting polyelectrolyte complex micelles, in LPS-treated or control HAEC. (C) Quantitative analyses of confocal images showing significantly increased cellular uptake of Dye 547–labeled miRNA inhibitor Ctrl delivered by VCAM-1–targeting micelles when compared to nontargeting micelles in LPS-treated HAEC but not in quiescent control HAEC. (D) Increased cellular uptake of Dye 547–labeled miRNA inhibitor Ctrl, detected by flow cytometry, by VCAM-1–targeting polyelectrolyte complex micelles compared to nontargeting micelles in LPS-treated HAEC. (E) Reduced cellular uptake of Dye 547–labeled miRNA inhibitor Ctrl delivered by VCAM-1–targeting micelles in LPS-stimulated HAEC resulting from the pretreatment with excess free VCAM-1–targeting peptides. Statistical significance was determined by multiple unpaired one-tailed t tests. All error bars are means ± SD. n.s., not significant. ***P ≤ 0.001.

Fig. 4.

Delivery of miRNA inhibitors to activated vascular endothelium by VCAM-1–targeting polyelectrolyte complex micelles in vivo. (A) A diagram depicting the partial carotid ligation in the LCA in Apoe^−/− mice and tail-vein injections of Dye 547–labeled miRNA inhibitor Ctrl in the naked form or encapsulated in the polyelectrolyte complex micelles. (B) The experimental design. (C) En face images of ligated left carotid arteries in Apoe^−/− mice subjected to an injection of Dye 547–labeled miRNA inhibitors Ctrl in the naked form or encapsulated in micelles. Green: CD31; blue: nuclei; red: Dye 547. (D) Quantitative analyses of en face images showing significantly increased endothelial uptake of Dye 547–labeled miRNA inhibitor Ctrl delivered by VCAM-1–targeting micelles when compared to nontargeting micelles or the naked form of miRNA inhibitor in the ligated mouse carotid artery. (E) En face images of nonligated right carotid arteries (RCA) in Apoe^−/− mice subjected to an injection of Dye 547–labeled miRNA inhibitor Ctrl in the naked form or encapsulated in micelles. Green: CD31; blue: nuclei; red: Dye 547. All error bars are means ± SD. *P ≤ 0.05; **P ≤ 0.01.

Fig. 5.

Enhanced therapeutic effectiveness of the anti–miR-92a therapy treating atherosclerosis in Apoe^−/− mice by VCAM-1–targeting polyelectrolyte complex micelles. (A) The experimental design. (B) Representative images of aortic root lesions in Apoe^−/− mice subjected to an injection of miR-92a inhibitor (8 mg/kg body weight) in the naked form or encapsulated in VCAM-1–targeting micelle or miRNA inhibitor control delivered by VCAM-1–targeting micelles. (C) Aortic root lesion quantifications in Apoe^−/− mice subjected to an injection of miR-92a inhibitor (8 mg/kg body weight) in the naked form or encapsulated in VCAM-1–targeting micelles or miRNA inhibitor control delivered by VCAM-1–targeting micelles (n = 5 to 7 biological samples). (D) Body weights and (E) cholesterol levels of Apoe^−/− mice subjected to an injection of miR-92a inhibitor (8 mg/kg body weight) in the naked form or encapsulated in VCAM-1–targeting micelles or miRNA inhibitor control delivered by VCAM-1–targeting micelles (n = 5 to 7 biological samples). (F) Representative images of aortic root lesions in Apoe^−/− mice subjected to an injection of miR-92a inhibitor (4 mg/kg body weight) in the naked form or encapsulated in VCAM-1–targeting micelles or miRNA inhibitor control delivered by VCAM-1–targeting micelles. (G) Aortic root lesion quantifications in Apoe^−/− mice subjected to an injection of miR-92a inhibitor (4 mg/kg body weight) in the naked form or encapsulated in VCAM-1–targeting micelles or miRNA inhibitor control delivered by VCAM-1–targeting micelles) (n = 7 to 8 biological samples). (H) Body weights and (I) cholesterol levels of Apoe^−/− mice subjected to an injection of miR-92a inhibitor (4 mg/kg body weight) in the naked form or encapsulated in VCAM-1–targeting micelles or miRNA inhibitor control delivered by VCAM1-targeting micelles (n = 7 to 8 biological samples). Statistical significance was determined by multiple unpaired one-tailed t tests. All error bars are means ± SD. n.s., not significant. **P ≤ 0.01; ***P ≤ 0.001.

Fig. 6.

Enhanced therapeutic effectiveness of the anti–miR-92 therapy treating disturbed flow-induced vascular remodeling in Apoe^−/− mice by VCAM-1–targeting polyelectrolyte complex micelles. (A) The experimental design. (B) Representative images of disturbed flow-induced vascular remodeling in the ligated LCA in Apoe^−/− mice subjected to injections of miR-92a inhibitor (three injections of 2 mg/kg body weight) in the naked form or encapsulated in VCAM-1–targeting micelles or miRNA inhibitor control delivered by VCAM-1–targeting micelles. (C) Representative cryosection images of the ligated LCA and nonligated RCA in Apoe^−/− mice subjected to injections of miR-92a inhibitor (three injections of 2 mg/kg body weight) in the naked form or encapsulated in VCAM-1–targeting micelles or miRNA inhibitor control delivered by VCAM-1–targeting micelles. (D) Lesion quantifications of the ligated LCA in Apoe^−/− mice subjected to injections of miR-92a inhibitor (three injections of 2 mg/kg body weight) in the naked form or encapsulated in VCAM1-targeting micelles or miRNA inhibitor control delivered by VCAM-1–targeting micelles (n = 6 biological samples). (E) Serum cholesterol levels and (F) body weights of Apoe^−/− mice subjected to injections of miR-92a inhibitor (three injections of 2 mg/kg body weight) in the naked form or encapsulated in VCAM-1–targeting micelles or miRNA inhibitor control delivered by VCAM-1–targeting micelles (n = 6 biological samples). Statistical significance was determined by multiple unpaired one-tailed t tests. All error bars are means ± SD. n.s., not significant. ***P ≤ 0.001.

Fig. 7.

Schematic diagram depicting that VCAM-1–targeting polyelectrolyte complex micelles deliver miR-92a inhibitor to inflamed endothelial cells treating pathological vascular remodeling induced by local disturbed blood flow.