The promoted delivery of RRM2 siRNA to vascular smooth muscle cells through liposome-polycation-DNA complex conjugated with cell penetrating peptides

Highlights

• RRM2-CLPD could significantly inhibited RRM2 expression by ∼80% in vascular smooth muscle cells (VSMCs).

• RRM2-CLPD was able to effectively bind to VSMCs.

• RRM2-CLPD was able to inhibit the proliferation and migration of VSMCs.

Abstract

Peripheral vascular disease (PVD) is a prevalent vascular disease that affect a large number of patients. The establishment of optimal treatments to mitigate the intimal hyperplasia (IH)-induced restenosis would help relieve the health burden of the PVD. Ribonucleotide reductase M2 (RRM2) is critical to cellular migration and proliferation. We have previously demonstrated that suppression of RRM2 expression could substantially inhibit hepatocellular carcinoma cell proliferation and migration. We hereby developed RRM2 small interfering RNA (siRNA)-loaded cell penetrating peptides-conjugated liposome-polycation-DNA complex (LPD) (RRM2-CLPD), aiming to inhibit the migration and proliferation of vascular smooth muscle cells (VSMCs) crucial for IH. RRM2-CLPD is of a small size (∼150 nm) and high siRNA encapsulation efficiency (∼90%). Further, we demonstrated that RRM2-CLPD could significantly inhibited RRM2 gene and protein expression by ∼80%. Notably, RRM2-CLPD was able to effectively bind to VSMCs, resulting in significant cellular proliferation and migration inhibition. Taken together, RRM2-CLPD represent a very promising treatment for IH.

Introduction

The prevalence of peripheral vascular disease (PVD), is continuing to rise. The worldwide prevalence of PVD is estimated to be almost 10%, and PVD affects about 15–20% in people over 70 years of age [1]. Endovascular interventions, which offer a lower risk alternative to open surgery, are minimally invasive procedures of low risk, and have increasingly pervasive treatment for PVD [2]. Although endovascular interventions have high initial success rates of >90%, they are linked with higher re-intervention rates due to chronic restenosis induced by intimal hyperplasia (IH) [2]. It is reported that, IH-induced restenosis often occurs in more than 60% of endovascular cases [3,4]. Thus, it is crucial to develop an optimal treatment to impede IH-induced restenosis.

The pathology of IH is characterized by the dysfunctional proliferation and migration of vascular smooth muscle cells (VSMCs) in the vessel wall [5]. After vascular endothelial injury occurs, inflammatory cells infiltrate and VSMCs migrate, proliferate, and secret extracellular matrix, resulting in IH and chronic restenosis. The migration, proliferation, and secretion of extracellular matrix of VSMCs constitute the basic pathological features of IH [6]. It is crucial to inhibit the migration and proliferation of VSMCs to treat IH [7,8].

Ribonucleotide reductase M2 (RRM2) plays an important role in DNA synthesis and repair [9]. There is accumulating evidence that the altered expression level of RRM2 could have a substantial impact on tumor initiation, progression and metastasis, suggesting its role as a possible cancer therapeutic target [10]. Two RRM2 inhibitors (GTI-2040 and gemcitabine) have entered clinical trial or application [11]. We previously have demonstrated that the expression of RRM2 was higher in hepatocellular carcinoma (HCC) compared with non-HCC tissue, and suppression of RRM2 expression could substantially inhibit HCC cell proliferation and migration, indicating that RRM2 is a promising target in HCC therapy [12]. Since RRM2 is critical for cellular migration and proliferation, we hypothesize that the suppression of RRM2 expression in VSMCs could substantially inhibit the migration and proliferation of VSMCs. However, there have been few data reporting the therapeutic efficacy of RRM2 suppression in IH so far.

Gene therapy represents a promising treatment for IH [13,14]. Hall S et al. developed viral vectors for gene therapy in IH [13]. Fisher RK et al. developed small interfering RNA (siRNA)-loaded neutral PEGylated liposomes for targeting vascular smooth muscle cells [14]. It is noteworthy that gene therapy using siRNA represents a potential treatment for various diseases, and an elegant alternative to toxic chemotherapy with few effects [15]. Since siRNA are unable to overcome cellular barriers to find their targets, nanoparticles-based formulations have emerged as effective carriers to facilitate siRNA delivery to cells and clinical development of siRNA-based therapy [12,[16], [17], [18]]. In our previous studies, we developed targeted LPD (liposome-polycation-DNA complex) with the EGFR (epidermal growth factor receptor) antibody to effectively deliver siRNA to breast cancer and liver cancer overexpressing EGFR [12,16,17].

Cell penetrating peptides (CPPs), are short peptides that could increase the cellular uptake of a series of cargoes such as nanoparticles [19,20]. Jha D et al. have demonstrated that CyLoP-1, a cationic cysteine-rich CPP, could efficiently deliver agents to cytosolic targets [21]. Herein, to deliver RRM2 siRNA to VSMCs efficiently, we have developed RRM2 siRNA loaded CCPs-conjugated LPD (CLPD) as molecular nanocarriers in VSMCs. We hypothesized that CLPD could efficiently deliver RRM2 siRNA to VSMCs, resulting in much enhanced therapeutic effect compared with nontargeted controls. The targeting efficacy, gene silencing ability, inhibitory effects of cellular proliferation and migration in VSMCs of CLPD were investigated thoroughly.