Nitric Oxide-Delivering High-Density Lipoprotein-like Nanoparticles as a Biomimetic Nanotherapy for Vascular Diseases

Abstract

Disorders of blood vessels cause a range of severe health problems. As a powerful vasodilator and cellular second messenger, nitric oxide (NO) is known to have beneficial vascular functions. However, NO typically has a short half-life and is not specifically targeted. On the other hand, high-density lipoproteins (HDLs) are targeted natural nanoparticles (NPs) that transport cholesterol in the systemic circulation and whose protective effects in vascular homeostasis overlap with those of NO. Evolving the AuNP-templated HDL-like nanoparticles (HDL NPs), a platform of bioinspired HDL, we set up a targeted biomimetic nanotherapy for vascular disease that combines the functions of NO and HDL. A synthetic S-nitrosylated (SNO) phospholipid (1,2-dipalmitoyl-sn-glycero-3-phosphonitrosothioethanol) was synthesized and assembled with S-containing phospholipids and the principal protein of HDL, apolipoprotein A-I, to construct NO-delivering HDL-like particles (SNO HDL NPs). SNO HDL NPs self-assemble under mild conditions similar to natural processes, avoiding the complex postassembly modification needed for most synthetic NO-release nanoparticles. In vitro data demonstrate that the SNO HDL NPs merge the functional properties of NO and HDL into a targeted nanocarrier. Also, SNO HDL NPs were demonstrated to reduce ischemia/reperfusion injury in vivo in a mouse kidney transplant model and atherosclerotic plaque burden in a mouse model of atherosclerosis. Thus, the synthesis of SNO HDL NPs provides not only a bioinspired nanotherapy for vascular disease but also a foundation to construct diversified multifunctional platforms based on HDL NPs in the future.

Keywords: S-nitrosylation; biomimetic; high-density lipoprotein-like nanoparticles; nanotherapy; nitric oxide-delivering; vascular disease.

Figure 1.

Preparation, properties and functions of SNO HDL NPs.

Figure 2.

Characterization of DPPNOTE. A) FTIR spectra of DPPTE (blue solid-line) and DPPNOTE (red solid-line). B) Raman spectra of DPPTE (blue solid-line) and DPPNOTE (red solid-line). C) Normalized UV-Vis spectra of DPPTE (blue solid-line with an inlet of a picture of DPPTE powder), GSNO [brown solid-line with inlets of a magnified spectrum (by 125 folds) ranged from 500 ~ 600 nm and a picture of GSNO powder] and DPPNOTE [red solid-line with inlets of a magnified spectrum (by 83 folds) ranged from 500 ~ 600 nm and a picture of DPPNOTE powder]. D) Mass spectra of DPPTE (narrow blue bars, the dominant peak ascribe to the molecular ion, [DPPTE + NH₃]⁺) and DPPNOTE (broad red bars, the dominant peak ascribe to the molecular ion, [DPPNOTE + NH₃]⁺).

Figure 3.

Characterization of SNO HDL NPs. A) Zeta potentials of HDL NPs (blue solid-line) and SNO HDL NPs (red solid-line). B) Normalized UV-Vis spectra of bare 5 nm AuNPs (brown solid-line), HDL NPs (blue solid-line) and SNO HDL NPs DPPTE (red solid-line). C) AFM images of HDL NPs and SNO HDL NPs.

Figure 4.

In vitro characterization of NO Release from SNO HDL NPs. A) NO release from SNO HDL NPs (red solid-line) and DPPNOTE (brown solid-line) at 37^oC. B) GSH -induced NO release from SNO HDL NPs at 4 °C and 37 °C.

Figure 5.

In vitro characterization of SNO HDL NP delivery of NO and function. A) Competitive binding of SNO HDL NPs and HDL NPs to macrophage cells. Left- representative histogram of DiI HDL NP fluorescence of THP-1 cells treated with DiI HDL NPs alone, or in combination with unlabeled HDL NPs or SNO HDL NPs. Right- median fluorescent intensities for each treatment group. B, C) Release of NO to J774 macrophages (B) and differentiated THP-1 macrophages (C) treated with DAF FM diacetate. From left to right: PBS, DPPNOTE (2 μM), HDL NP (25 nM), and SNO HDL NP (25 nM). Green- DAF FM diacetate. Blue- nuclei (DAPI). Images taken at 40X magnification. D) Quantiblue assay of THP-1 Dual cells treated with 5ng/ mL LPS and increasing concentrations of DPPNOTE, HDL NP or SNO HDL NPs. *p<0.05 vs. all other treatment groups. E, F) Cholesterol efflux from ³H-cholesterol loaded macrophages to SNO HDL NPs. J774 macrophages (E) and differentiated THP-1 macrophages (F) were loaded with ³H-cholesterol prior to addition of cholesterol acceptors for 24 hrs. G) SNO HDL NPs reduced transwell migration of AoSMCs compared with controls, PBS, DPPNOTE alone and HDL NPs (*p<0.0001 v. all other treatment groups).

Figure 6.

SNO HDL NPs as therapy for IRI and atherosclerosis. A) Plasma creatinine levels of mouse kidney transplant recipients on day 2 post transplantation (*p=0.0113 v. PBS control). B) Quantification of apoptosis (TUNEL) staining in kidney transplant recipients (*p<0.0001 vs. PBS). C) Quantification of immunocytochemistry staining for Gr-1, a neutrophil marker, in kidney transplant recipients (*p=0.0175. **p=0.0041. ***p=0.008). D) ApoE knockout mice were fed a high fat diet for 12 weeks, then administered PBS, HDL NPs or SNO HDL NPs 3×/ week for an additional 6 weeks. Atherosclerotic lesions were visualized using Sudan Red staining. The atherosclerotic area was calculated by measuring the area of Sudan Red staining, divided by the total aortic area (*p=0.0149. **p<0.0001. ***p=0.0118). E) Representative images of aortas from mice treated with PBS, HDL NP or SNO HDL NPs. Red fluorescent staining indicates atherosclerotic plaques. Images taken at 6X magnification.