Functionalized dendrimer-based delivery of angiotensin type 1 receptor siRNA for preserving cardiac function following infarction

Abstract

Cardiovascular disease (CVD) is the leading cause of death throughout the world and much pathology is associated with upregulation of inflammatory genes. Gene silencing using RNA interference is a powerful tool in regulating gene expression, but its application in CVDs has been prevented by the lack of efficient delivery systems. We report here the development of tadpole dendrimeric materials for siRNA delivery in a rat ischemia-reperfusion (IR) model. Angiotensin II (Ang II) type 1 receptor (AT1R), the major receptor that mediates most adverse effects of Ang II, was chosen to be the silencing targeting. Among the three tadpole dendrimers synthesized, the oligo-arginine conjugated dendrimer loaded with siRNA demonstrated effective down-regulation in AT1R expression in cardiomyocytes in vitro. When the dendrimeric material was applied in vivo, the siRNA delivery prevented the increase in AT1R levels and significantly improved cardiac function recovery compared to saline injection or empty dendrimer treated groups after IR injury. These experiments demonstrate a potential treatment for dysfunction caused by IR injury and may represent an alternative to AT1R blockade.

Introduction

Cardiovascular disease (CVD) is the leading cause of death worldwide and with aging populations, incidence is on the rise. Myocardial infarction (MI) is class of CVD that accounts for 1 in every 6 deaths in the US alone, with roughly 1.5 million events annually [1]. Those that survive the initial insult, characterized by regional loss of tissue and function have a high probability of developing heart failure in subsequent years. With the global numbers on the rise, new treatments are greatly needed.

CVDs are controlled and influenced by numerous factors in the “cardiovascular continuum” [2], [3]. One important hormone system involved is the Renin-Angiotensin-Aldosterone System (RAAS). The activation of RAAS following cardiovascular ischemic injury is supposed to promote blood pressure recovery, but its continuous stimulation can cause vasoconstriction, vascular and cardiac hypotrophy, and fibrosis [4]. Angiotensin II (Ang II), an octa-peptide hormone, which is the end-product of the RAAS, regulates most effects of RAAS [5]. Its overexpression following MI leads to cardiomyocyte death and hypertrophy, vascular smooth muscle growth, and fibrosis, all of which cause adverse cardiac remodeling, progressive ventricular dysfunction, and finally heart failure [6]. Therefore, the inhibition of Ang II activation has become a common target for CVD therapy [7].

The Ang II type 1 receptor (AT1R) is the essential mediator of the well-known adverse effects of Ang II, and its activation results in worsened cardiac functions after injury [6], [8]. In contrast, the type 2 receptor (AT2R), another important receptor, is generally considered as a protective receptor, leading to improved function recovery [9], [10], [11]. By suppressing AT1R activity, the negative effects from Ang II could be reduced, while AT2R activity could be exacerbated, bringing more beneficial effects [12]. Currently, angiotensin receptor blockers (ARBs) are used following MI to restore blood pressure and possibly prevent fibrosis, and improve cardiac function in laboratories and in clinic [13], [14], [15], [16], [17]. Despite these interesting data, previous studies have shown that the suppression of AT1R at the gene expression level is superior to AT1R pharmacological blockade [18], [19], likely because the simple blockade interrupts the feedback loop in RAAS, causing renin and Ang II levels to increase in plasma, which may further activate the whole system. Such studies show a need for RNA interference (RNAi) therapeutics to selectively block receptor expressions following MI, specifically small interfering RNA (siRNA), one of the most efficient initiators of RNAi.

The development of siRNA delivery systems for cardiac tissue is one challenge that needs to be overcome for RNAi therapeutics to be successful in treating CVDs, as the non-phagocytic nature of cardiomyocytes set up a high barrier for the delivery of small RNA molecules. Bull et al. reported the usage of peptide-conjugated polymers for siRNA delivery in a cardiac myoblast cell line in vitro, but no in vivo efficiency was verified [20], [21], [22]. SiRNA–albumin conjugates have also been used in cardiac gene silencing in vivo by Lau et al., however a high dose (1–5 mg/kg) was required to induce a 40% silencing effect [23]. Due to the lack of efficiency, especially low efficiency in vivo, new delivery systems for cardiac tissue are highly in demand.

Among current non-viral delivery systems, dendrimeric materials have been regarded promising in siRNA delivery [24]. They are mono-disperse molecules and have tunable structures and properties. The rich cationic peripheries on dendrimers allow strong charge interaction with anionic siRNA molecules, forming stable particles to fight against nuclease digestion. Meanwhile, the buffering amines inside dendrimers can induce a unique “proton sponge” effect, which can trigger endosomal escape and release siRNA cargoes into cytoplasm. The inherent advantages of dendrimeric materials make them suitable for constructing siRNA delivery systems; however their application in cardiac tissue has not been developed.

In this study, we aim to develop a non-cytotoxic and efficient “tadpole” siRNA delivery system for cardiac tissue based on dendrimeric materials, combining the strengths of a cationic dendrimer and a cell penetrating peptide (CPP) to enhance uptake by cardiomyocytes. Two types of CPPs, oligo arginine (R9) and TAT, were linked to the reduced thiol end to cystamine core G4.0 poly(amido amine) (PAMAM) through a polyethylene glycol (PEG) crosslinker, separately. This design allows the PAMAM moiety to regulate siRNA complexation and endosomal escape, CPP improves cell internalization, and the PEG segments and the disulfide linkage between the three parts should enhance biocompatibility. The properties and performance of the dendrimeric materials were first evaluated in isolated cardiomyocytes in vitro, and we chose AT1R as the target to exploit its potential cardiovascular application in a rat ischemia-reperfusion (IR) model.