Preventing diet-induced obesity in mice by adipose tissue transformation and angiogenesis using targeted nanoparticles

Abstract

The incidence of obesity, which is recognized by the American Medical Association as a disease, has nearly doubled since 1980, and obesity-related comorbidities have become a major threat to human health. Given that adipose tissue expansion and transformation require active growth of new blood vasculature, angiogenesis offers a potential target for the treatment of obesity-associated disorders. Here we construct two peptide-functionalized nanoparticle (NP) platforms to deliver either Peroxisome Proliferator-Activated Receptor gamma (PPARgamma) activator rosiglitazone (Rosi) or prostaglandin E2 analog (16,16-dimethyl PGE2) to adipose tissue vasculature. These NPs were engineered through self-assembly of a biodegradable triblock polymer composed of end-to-end linkages between poly(lactic-coglycolic acid)-b-poly(ethylene glycol) (PLGA-b-PEG) and an endothelial-targeted peptide. In this system, released Rosi promotes both transformation of white adipose tissue (WAT) into brown-like adipose tissue and angiogenesis, which facilitates the homing of targeted NPs to adipose angiogenic vessels, thereby amplifying their delivery. We show that i.v. administration of these NPs can target WAT vasculature, stimulate the angiogenesis that is required for the transformation of adipose tissue, and transform WAT into brown-like adipose tissue, by the up-regulation of angiogenesis and brown adipose tissue markers. In a diet-induced obese mouse model, these angiogenesis-targeted NPs have inhibited body weight gain and modulated several serological markers including cholesterol, triglyceride, and insulin, compared with the control group. These findings suggest that angiogenesis-targeting moieties with angiogenic stimulator-loaded NPs could be incorporated into effective therapeutic regimens for clinical treatment of obesity and other metabolic diseases.

Keywords: adipose tissue; angiogenesis; nanoparticle; targeting; transformation.

Fig. 1.

NP design and characterization. (A) A schematic representation of the WAT browning process through a positive feedback drug delivery system. Released Rosi and PGE2 promote transformation of WAT into brown-like adipose tissue and stimulate angiogenesis. This facilitates the homing of targeted NPs to adipose angiogenic vessels, thereby amplifying their delivery and hence expediting the WAT browning process. (B) Chemical structure of PLGA-b-PEG-Peptide/Rosiglitazone NPs (NPs). The particle consists of two components: (i) an outer PEG surface with a targeting peptide iRGD or P3, which can bind to angiogenic vessels through Integrinavb3/b5 receptors and WAT vasculature through the membrane protein prohibitin, respectively,and (ii) a PLGA hydrophobic core that serves two functions: first, acting as a polymer matrix loaded with Rosiglitazone, and second, promoting Rosiglitazone molecule retention inside the NP core and controlling drug release. Nontargeted and targeted NPs encapsulating Rosiglitazone were formulated via a single-step emulsion method. (C) Representative TEM image of the iRGD-NP-Rosi. (D) In vitro release profile of Rosiglitazone from NP-Rosi and iRGD-NP-Rosi. (E) Size distribution of the iRGD-NP-Rosi measured by dynamic light scattering.

Fig. S1.

The reaction scheme to synthesize peptide-NP constructs.

Fig. 2.

NPs stimulate angiogenesis in vitro and target to adipose tissue in vivo. Stromal vascular fragments (SVF) were isolated from inguinal WAT and were treated with free Rosi and NP-encapsulated Rosi (NP-Rosi). (A) Relative expression levels of Integrinαv in SVF from inguinal WAT (ingWAT) were measured by qRT-PCR (n = 3 per group). (B) Relative expression levels of Integrinß3 in SVF from ingWAT were measured by qRT-PCR PCR (n = 3 per group). (C) Proliferation of SVF from ingWAT induced by Rosi and NP-Rosi treatments PCR (n = 6 per group). (D) CD31⁺ SVF was sorted and stained with Integrinαv antibody (red), Isolectin B4 (green), and DAPI (blue). C57BL/6 mice were treated with Rosi, NP-Rosi, iRGD-NP-Rosi, or P3-NP-Rosi for 2 wk (n = 3). (Scale bar, 50 μm.) (E) IngWAT, epiWAT, and livers were dissected, and representative samples were imaged under the IVIS imaging system. (Scale bars, 1 cm.) (F) Inguinal WAT and epididymal WAT were dissected, and representative samples were photographed.

Fig. 3.

IRGD- and P3-NP-Rosi induced adipose tissue transformation and angiogenesis in vivo. C57BL/6 mice received treatments with Rosi, NP-Rosi, iRGD-NP-Rosi, or P3-NP-Rosi for 15 d. (A) Sections of ingWAT were stained with hematoxylin and eosin to demonstrate the general histology of adipose tissues. (Scale bar, 50 μm.) (C) Portions of ingWAT were immune-stained with anti-CD31 antibody (red), and 3D images were captured under a confocal microscope. (Scale bar, 50 μm.) (D) Sections of ingWAT were stained with Isolectin B4 (brown) and hematoxylin (blue). (Scale bar, 50 μm.) (B) Sections of ingWAT were stained with anti-UCP1 antibody (brown) and hematoxylin (blue). (Scale bar, 50 μm.) (E) Density of CD31⁺ blood vessel area per optical field was quantified from confocal images. Data represent means ± SEM from nine samples from four mice in each group. (F) Quantification of average size of adipocytes from different treatment groups. Data represent means ± SEM from nine samples from four mice in each group. (G) Quantification of numbers of isolectin-positive vessels per adipocyte of ingWAT. Data represent means ± SEM from nine samples from four mice in each group.

Fig. S2.

IRGD- and P3-NP-Rosi induced adipose tissue transformation and angiogenesis and up-regulated both BAT and angiogenesis markers in epididymal WAT. (A) Sections of epididymal WAT from various treatment groups were stained with hematoxylin and eosin (Upper) and anti-CD31 antibody (red; Lower). Total RNAs were isolated from epididymal WAT from various treatment groups. Expression levels of UCP1 (B), CIDEA (C), DIO2 (D), and VEGFR2 (E) were quantified by qRT-PCR. Data are means ± SEM from three to four mice in each group.

Fig. 4.

IRGD- and P3-NP-Rosi up-regulated BAT and angiogenesis markers in inguinal WAT. Total RNAs were isolated from inguinal WAT from various treatment groups. Expression levels of UCP1 (A), CIDEA (B), DIO2 (C), VEGFR2 (D), early-stage VEGF (E), and late-stage VEGF (F) were quantified by qRT-PCR. Data are means ± SEM from three to four mice in each group.

Fig. S3.

IRGD-NP-PGE2 and P3-NP-PGE2 induced adipose tissue transformation and angiogenesis in vivo. (A) WATs from C57BL/6 mice treated with PGE2, NP-PGE2, iRGD-NP-PGE2, and P3-NP-PGE2 were stained with anti-CD31 antibody (red). (B) Quantification of CD31-positive blood vessel area in inguinal WAT per optical field. Data are means ± SEM from six samples in each group. (C) Quantification of CD31-positive blood vessel area in epididymal WAT per optical field. Data are means ± SEM from six samples in each group. (D) Relative expression level of Ucp1in inguinal WAT from mice treated with PGE2, NP-PGE2, iRGD-NP-PGE2, and P3-NP-PGE2. Data are means ± SEM from three to four mice in each group. (E) Relative expression level of Ucp1 in epididymal WAT from mice treated with PGE2, NP-PGE2, iRGD-NP-PGE2, and P3-NP-PGE2. Data are means ± SEM from three to four mice in each group.

Fig. 5.

Antiobesity effect of iRGD-NP-Rosi and P3-NP-Rosi in diet-induced obese (DIO) mice. DIO mice received treatments (intravenously injection at the drug dose of 80 mg/kg, every second day) with Rosi, NP-Rosi, iRGD-Rosi, and P3-NP-Rosi for 25 d. (A) Representative mice were photographed at the experiment end point. (B) Relative body weight increases of nontreated mice or mice receiving Rosi or NP-Rosi. Data are relative means ± SEM from three to four mice in each group. (C) Relative body weight increases of nontreated mice or mice receiving iRGD-NP-Rosi or P3-NP-Rosi. Data are relative means ± SEM from three to four mice in each group. (D) Average food intake per mouse per day. Data are means ± SEM from three to four mice in each group. (E) Inguinal WATs were stained with hematoxylin and eosin to illustrate histological structure (Upper) and immune-stained with anti-CD31 antibody (red; Lower) to illustrate vasculature. (Scale bars, 50 μm.) (F) Quantification of CD31-positive blood vessels area per optical field. Data are means ± SEM from nine samples in each group. (G) Relative expression levels of Ucp1 of inguinal WATs from DIO mice treated with Rosi, NP-Rosi, iRGD-NP-Rosi, and P3-NP-Rosi. Data are means ± SEM from three to four mice in each group. (H) Relative expression levels of Vegfr2 of inguinal WATs from DIO mice treated with Rosi, NP-Rosi, iRGD-NP-Rosi, and P3-NP-Rosi. Data are means ± SEM from three to four mice in each group.

Fig. 6.

BAT, vascular markers, and serological parameters from DIO mice were altered by of iRGD-NP-Rosi and P3-NP-Rosi treatments. Quantification of serum levels of cholesterol (A), triglyceride (B), free fatty acid (FFA) (C), glucose (D), and insulin (E) from fasting DIO mice treated with Rosi, NP-Rosi, iRGD-NP-Rosi, and P3-NP-Rosi. Data are means ± SEM from three to four mice in each group. (F) Insulin resistance was calculated as insulin level × FFA level. Data are means ± SEM from three to four mice in each group.

Fig. S4.

NP constructs do not affect body weight and food intake for wild-type C57BL/6 mice. C57BL/6 mice received treatments with NP and iRGD-NP for 21 d. (A) Relative body weight increases of nontreated mice or mice receiving NP or iRGD-NP. Data are relative means ± SEM from three to four mice in each group. (B) Average food intake per mouse per day. Data are means ± SEM from three to four mice in each group.

Fig. S5.

Structure of PLGA-PEG-iRGD-FAM and associated ¹H NMR spectrum (Dimethyl Sulfoxide-d₆).