Dendronized heparin−doxorubicin conjugate based nanoparticle as pH-responsive drug delivery system for cancer therapy

Abstract

Heparin drug conjugates are currently investigated as excellent candidates for drug delivery vehicles. In this study, we report the preparation and characterization of dendronized heparin–doxorubicin (heparin–DOX) conjugate as pH-sensitive drug delivery vehicle by combination of the features of dendrimer and heparin. Dynamic light scattering (DLS) and transmission electron microscope (TEM) studies demonstrated the dendronized heparin–DOX conjugate self–assembled into compact nanoparticles with negatively charged surface. The nanoparticles with 9.0 wt% (weight percent) of doxorubicin (DOX) showed pH-sensitive property due to the faster drug release rate at pH 5.0 and slow release rate at pH 7.4 aqueous. The nanoparticles were shown to effectively kill cancer cells in vitro. Notablely, the nanoparticles resulted in strong antitumor activity, high antiangiogenesis effects and induced apoptosis on the 4T1 breast tumor model due to the evidences from mice weight shifts, tumor weights, tumor growth curves, immunohistochemical assessment and histological analysis. It's also noteworthy that dendronized heparin and its nanoparticle with drug demonstrated no significant toxicity to healthy organs of both tumor–bearing and healthy mice, which was confirmed by histological analysis compared with free drug DOX. The dendronized heparin–DOX conjugate based nanopatilce with high antitumor activity and low side effects may be therefore a potential nanoscale drug delivery vehicle for breast cancer therapy.

Introduction

Nanoparticles, as drug delivery vehicles for cancer therapy, are rapidly progressing and are being implemented to overcome the limitations of conventional chemotherapeutic agents such as nonspecific biodistribution and targeting in the body, poor water solubility and low therapeutic indices [1], [2]. Nanoscale drug delivery vehicles provide enhanced antitumor efficacy and reduced side effects, owing to their properties such as higher accumulation in tumors via the enhanced permeability and retention (EPR) effect and active cellular uptake [1], [3]. Among the emergent nanoparticles, lipids (liposomes) and polymers (polymeric nanoparticles, micelles and dendrimers) likely have the great potential clinical impact for the foreseeable future [4], [5], [6], [7]. However, although those lipids and polymers based nanoparticles demonstrate many advantages as drug delivery systems, there are still many limitations to address such as instability in circulation, inadequate tissue distribution and toxicity.

Dendrimers with nanometer dimensions have provided potential drug delivery vehicles due to their features such as their precise and monodisperse size, low polydispersity, modifiable surface functionality, water solubility and multivalency, resulting in a number of possible advantages, such as pharmacokinetic advantages of typical colloidal or macromolecular delivery systems [8], [9]. The fate of injected nanoparticles, including dendrimer based drug delivery, suitable for cancer therapy depends on their sizes and surface characteristics [1], [10], [11], [12], [13], [14]. However, this kind of drug delivery with ideal antitumor properties in vivo has not been sufficiently achieved, since the reported dendrimers with size less than 10 nm are rapidly cleared from the circulation through extravasation or renal clearance [10]. Increasing generation and surface modification can increase the sizes of the dendrimers, which leads 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 [15]. Simultaneously, the high generation dendrimer leads to side effects due to their slow degradation [15], [16].

In order to prepare dendrimer or dendritic polymer based nanoparticles with larger sizes and well–defined molecular objects, dendronization strategy has been utilized to prepare nanoscopic objects via connecting dendron to polymer [17], [18], in which polymer was used as a polyfunctional and polydisperse core, and the new polymers with this architecture were referred to as ‘dendronized polymers’ [19], [20]. The dendronized polymers can self–assemble into nanoparticles taking advantage of dendrimer and linear polymer [21], [22]. The dendronization and self–assembly can overcome the synthetic challenges associated with preparation of dendrimer based nanoparticles with satisfied sizes.

Heparin, a non-cytotoxic, biodegradable, rich in animal tissues and water–soluble natural polysaccharide belonging to the family of glycosaminoglycans [23], has been widely used as an anticoagulant drug owing to its ability to accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade [24], [25], as well as antitumor drug delivery carriers due to its ability to inhibiting tumor growth and metastasis by interacting with tumor related factors such as selectins, heparanases, and growth factors [26], [27], [28], [29], [30], [31], [32]. The antitumor drug was conjugated to heparin or encapsulated into the heparin nanoparticles [33], [34], [35], [36], [37], [38], [39]. However, the major problems are the unsuitable particle sizes for cancer therapy and poor stability. Based on above observations, our question here was if the dendronized heparin–DOX conjugate can self–assemble into nanoparticle and be suitable as nanoscale drug delivery with significant antitumor efficacy as well as good biosafety by combination of the features of dendrimer and heparin. However, currently, few studies on dendronized heparin based nanoparticles as antitumor drug delivery has been reported.

In this study, we described the preparation and characterization of dendronized heparin–DOX conjugate as pH-stimuli and nanoscale drug delivery system for breast tumor therapy. Its antitumor efficacy and biosafety were assessed well. The heparin was dendronized with low generation dendron via click reaction; DOX was conjugated to the surface of dendron through pH-sensitive hydrazone bond, resulting in compact nanoparitcle via the self–assembly, as shown in Fig. 1. The in vitro and in vivo characteristics of nanoparticle as pH-stimuli drug delivery system, such as size, zeta potential, drug release, antitumor efficacy, antiangiogenic effect and toxicity, were evaluated, which showed the dendronized heparin–DOX conjugate based nanoparticle provide a potential drug delivery vehicle for breast cancer therapy.