The potential of self-assembled, pH-responsive nanoparticles of mPEGylated peptide dendron–doxorubicin conjugates for cancer therapy

Abstract

Nanoparticles, such as dendritic polymers, are currently investigated as excellent candidates for drug delivery vehicles. In this study, we report the preparation and characterization of mPEGylated peptide dendron–doxorubicin (dendron–DOX) conjugate based vehicle carrying 14.0 wt% (weight percent) of doxorubicin (DOX). Dynamic light scattering (DLS), scanning electron microscope (SEM) and transmission electron microscope (TEM) studies demonstrated the dendron–DOX conjugate self-assembled into nanoscale particles with neutral charged surface. The globular morphology and compact nanoparticle with diameter around 80 nm were observed by SEM and TEM. The release rates of DOX from the nanoparticle at pH 5.0 were much faster than those at pH 7.4 due to the pH-sensitive cleavage of the hydrazone bonds. The nanoparticle was shown to effectively kill cancer cells in vitro. Importantly, the nanoparticle resulted in strong antitumor activity and induced apoptosis on the 4T1 breast tumor model. In vivo toxicity evaluation demonstrated that drug-free dendron and drug-loading nanoparticle provided good biosafety in healthy or tumor-bearing mice, because no significant systematic toxicity was revealed via histological analysis. The functionalized peptide dendron–DOX conjugate based nanoparticle may be therefore a potential candidate for drug delivery vehicle for cancer therapy.

Introduction

Chemotherapy has been heavily investigated for the treatment of numerous cancers. However, one of the biggest challenges in this field is the lack of highly efficient and safe drug delivery vehicles. Nanoparticles, as nanoscale drug delivery vehicles [1], [2], are emerging as a class of therapeutics for cancer, suggesting enhanced efficacy, while simultaneously reducing side effects, owing to properties such as higher accumulation in tumors via the enhanced permeability and retention (EPR) effect and active cellular uptake [1], [3]. Among the emergent nanotechnology platforms, liposomes and polymer technologies likely have the great potential clinical impact for the foreseeable future [4]. Currently, natural polymers and synthetic polymers [5], [6], [7], such as dendritic polymers [8], have been widely prepared as nanoscale drug delivery vehicles for cancer therapy.

Dendritic polymers [9], [10], including dendrimers and dendrons [11], [12], [13], [14], have provided a potential alternative route to the development of drug delivery vehicles due to their versatility, precise nanostructure, low polydispersity, controllable molecular size, highly adaptable surface chemistry and chemically stable molecular entities with great flexibility [13], [15], [16], which have demonstrated a number of possible advantages, such as drug targeting and pharmacokinetic advantages of typical colloidal or macromolecular delivery systems [16], [17]. Recently, peptide dendritic polymers with attractive characteristics are currently under investigation as drug delivery systems due to their properties similar to proteins, such as good biocompatibility, water solubility and resistance to proteolytic digestion. However, the development of dendrimer–drug conjugates with ideal antitumor properties in vivo has not been sufficiently addressed, since the reported dendrimer based conjugates with size less than 10 nm are rapidly cleared from the circulation through extravasation or renal clearance [18].

The nanoparticles, including dendrimer/dendron-based drug delivery systems, suitable for cancer therapy depend on the size and surface properties [1], [9], [18], [19], [20], [21]. For dendrimers or dendrons, two methods, including increasing generation and surface modification via covalently connection, can be used to increase molecular weights and sizes of the dendrimers, which lead to longer blood circulation and higher antitumor efficacy. However, it's not easy to prepare higher generation dendrimers due to the steric hindrance to chemical reactions [22]. Simultaneously, the high generation dendrimer (over 5 generation) can cause side effects due to their slow degradation [22], [23]. In order to enhance the blood circulation of dendrimers, as well as biocompatibility, polyethylene glycol (PEG), which can extend retention time in blood by decreasing non-specific interactions with endogenous components and macrophages, has been used to modified the surface of dendrimers to form a new kind of dendritic structures. The PEGylated dendrimers [24], [25], as drug delivery [26], [27], [28], [29] or imaging probes [30], have demonstrated increased drug loading and solubility, longer blood circulation and high accumulation in tumor tissue via the enhanced permeability and retention (EPR) effect [24]. In addition, PEGylation is one preferred method for avoiding premature clearance of nanoparticles by the reticuloendothelial system (RES) [5].

Another important issue regarding the development of dendritic polymers for specific applications is their ability to self-assemble into nanostructures [31], [32], [33], [34], [35], [36], [37], [38]. After encapsulation of drug, the nanostructures provided potential as drug delivery vehicles. The self-assembly can overcome the synthetic challenges associated with preparation of dendrimer [36]. In our group [39] and others [40], the degradable dendrimers/dendrons were aggregated to nanocarriers for gene delivery, demonstrated the dendrimers/dendrons self-assembly significantly enhanced DNA binding and efficiency at transporting DNA into cells compared to dendrimer or dendron alone. Based on above observations, our question here was if the mPEGylated peptide dendron–DOX conjugate can aggregate to nanoparticle and be suitable as nanoscale drug delivery with good biosafety as well as significant antitumor efficacy. However, currently, few studies on peptide dendron based nanoparticles as antitumor drug delivery have been reported.

In this study, we described the preparation and characterization of mPEGylated peptide dendron–DOX conjugate as pH-stimuli drug delivery system for breast tumor therapy. Its biosafety and antitumor efficacy were assessed well. The tail of protected dendron synthesized by divergent strategy in liquid phase with high yield was modified with mPEG via click reaction; DOX, as a widely used antitumor chemotherapeutic drug, was conjugated to another tail of the dendron through pH-sensitive hydrazone bond, resulting in compact nanoparitcle via the self-assembly governed by dendron–DOX itself, as shown in Fig. 1. The in vitro and in vivo characteristics of nanoparticle as pH-stimuli drug delivery system, such as size and morphology, zeta potential, drug release, antitumor efficacy and toxicity, were evaluated, which showed the mPEGylated peptide dendron–DOX conjugate based nanoparticle may be as a potential drug delivery vehicle for breast cancer therapy.