cRGD-directed, NIR-responsive and robust AuNR/PEG–PCL hybrid nanoparticles for targeted chemotherapy of glioblastoma in vivo

Abstract

cRGD-directed, NIR-responsive and robust AuNR/PEG–PCL hybrid nanoparticles (cRGD-HNs) were designed and developed for targeted chemotherapy of human glioma xenografts in mice. As expected, cRGD-HNs had excellent colloidal stability. The in vitro release studies showed that drug release from DOX-loaded cRGD-HNs (cRGD-HN-DOX) was minimal under physiological conditions but markedly accelerated upon NIR irradiation at a low power density of 0.2 W/cm², due to photothermally induced phase transition of PCL regime. MTT assays showed that the antitumor activity of cRGD-HN-DOX in α_vβ₃ integrin over-expressed human glioblastoma U87MG cells was greatly boosted by mild NIR irradiation, which was significantly more potent than non-targeting HN-DOX counterpart under otherwise the same conditions and was comparable or superior to free DOX, supporting receptor-mediated endocytosis mechanism. The in vivo pharmacokinetics studies showed that cRGD-HN-DOX had much longer circulation time than free DOX. The in vivo imaging and biodistribution studies revealed that cRGD-HN-DOX could actively target human U87MG glioma xenograft in nude mice. The therapeutic studies in human U87MG glioma xenografts exhibited that cRGD-HN-DOX in combination with NIR irradiation completely inhibited tumor growth and possessed much lower side effects than free DOX. The Kaplan–Meier survival curves showed that all mice treated with cRGD-HN-DOX plus NIR irradiation survived over an experimental period of 48 days while control groups treated with PBS, cRGD-HN-DOX, cRGD-HNs with NIR irradiation, free DOX, or HN-DOX with NIR irradiation (non-targeting control) had short life spans of 15–40 days. Ligand-directed AuNR/PEG–PCL hybrid nanoparticles with evident tumor-targetability as well as superior spatiotemporal and rate control over drug release have emerged as an appealing platform for cancer chemotherapy in vivo.

Introduction

Biodegradable micelles based on amphiphilic block copolymers of poly(ethylene glycol) (PEG) and aliphatic polyesters such as poly(ε-caprolactone) (PCL), polylactide (PLA), and poly(lactide-co-glycolide) (PLGA) are among the most studied nanocarriers for targeted and controlled delivery of poorly water soluble chemotherapeutic agents including paclitaxel (PTX) and doxorubicin (DOX) [1], [2], [3]. It is interesting to note that a couple of micellar drugs such as Genexol-PM (PTX formulation based on PEG-b-PLA micelles) and BIND-014 (micellar docetaxel formulation based on PEG-PDLLA or PEG-PLGA targeting to prostate cancer) have been translated to the clinical trials [4], [5]. The current micellar drugs are, however, often associated with problems of low in vivo stability, premature drug release, inefficient tumor cell uptake, as well as slow drug release in the tumor site and inside the tumor cells, which would not only greatly reduce their therapeutic efficacy but also induce systemic side effects [6], [7], [8].

In the past years, different strategies have been explored to improve the targetability and in vivo drug release behaviors of biodegradable micelles. For example, crosslinked micelles have been developed to achieve better in vivo stability and prolonged circulation time, thereby enhancing drug accumulation in the tumor site via the enhanced permeability and retention (EPR) effect [9], [10]. In particular, stimuli-sensitive degradable crosslinked micelles that are de-crosslinked or hydrolyzed in response to the reducing environment in the cytosol or acidic pH in the endo/lysosomal compartments are of interest, as they not only possess enhanced stability but also trigger drug release inside the tumor cells [11], [12], [13], [14], [15]. Several groups reported that stimuli-sensitive degradable crosslinked micelles exhibit improved antitumor efficacy and reduced systemic toxicity in vivo as compared to the stimuli-insensitive or non-crosslinked controls [16], [17], [18], [19], [20]. It should be noted, however, that these sophisticated micelles are typically based on new functional block copolymers, which usually involve multi-step synthesis. Moreover, drug release from stimuli-sensitive crosslinked micelles inside the tumor cells is often slow and lacks control. Very recently, we reported that micelles prepared by coating gold nanorods (AuNRs) with PEG-b-PCL-lipoic acid ester (PEG–PCL–LA) block copolymer while possessing high stability exhibited excellent temporal, spatial and rate control over drug release via mild NIR irradiation, resulting in effective inhibition of both drug-sensitive and drug-resistant (acquired drug resistance, ADR) cancer cells [21]. Interestingly, Jerome et al. reported similar results almost at the same time [22]. Lipoic acid is a natural anti-oxidant that is produced by the human body [23], [24]. Lipoic acid with a high affinity to gold surface has widely been used to stabilize gold nanoparticles in aqueous environment [25], [26], [27], [28]. In our lab, based on ring-opening polymerizability of lipoic ring to form linear polydisulfide in the presence of catalytic amount of dithiothreitol, we have prepared reduction-sensitive reversibly crosslinked micelles and nanoparticles from various polymeric conjugates of lipoic acid [29], [30], [31], [32], [33].

In this paper, we report on cRGD-directed AuNR/PEG–PCL hybrid nanoparticles (cRGD-HNs) for targeted chemotherapy of glioblastoma in mice (Scheme 1). cRGD has a high affinity with the α_vβ₃ integrin receptors over-expressed on angiogenic endothelial cells as well as tumor cells such as malignant glioma cells, breast cancer cells, bladder cancer cells and prostate cancer cells, which render cRGD a unique molecular ligand for targeted cancer chemotherapy [34], [35]. Several groups reported that cRGD-functionalized nanoparticles exhibited enhanced antitumor effects toward glioblastoma in vitro and in vivo as compared to the non-targeting counterpart [36], [37], [38], [39]. Herein, cRGD-HNs were designed with the following features: (i) they have defined shape and size and are particularly robust in circulation minimizing premature drug release and improving in vivo pharmacokinetics, as AuNRs are covalently coated with a layer of PEG–PCL–LA copolymer; (ii) they can be recognized and taken up by glioblastoma cells via the receptor-mediated endocytosis mechanism; and (iii) drug release can be remotely controlled by NIR laser, which triggers drug release into tumor extracellular environment as well as inside the target tumor cells, resulting in enhanced tumor penetration and potent antitumor effect. Here, preparation of DOX-loaded cRGD-HNs (cRGD-HN-DOX), specificity and antitumor activity toward human glioblastoma U87MG cells, in vivo pharmacokinetics and biodistribution, as well as therapeutic effects in human glioblastoma xenografts in mice were investigated.