Enhanced antitumor efficacy by cyclic RGDyK-conjugated and paclitaxel-loaded pH-responsive polymeric micelles

Abstract

Cyclic RGDyK (cRGDyK)-conjugated pH-sensitive polymeric micelles were fabricated for targeted delivery of paclitaxel to prostate cancer cells based on pH-sensitive copolymer poly(2-ethyl-2-oxazoline)-poly(d,l-lactide) (PEOz-PLA) and cRGDyK-PEOz-PLA to enhance antitumor efficacy. The prepared micelles with an average diameter of about 28 nm exhibited rapid release behavior at endo/lysosome pH, effectively enhanced the cytotoxicity of paclitaxel to PC-3 cells by increasing the cellular uptake, which was correlated with integrin α_vβ₃ expression in tumor cells. The active targeting activity of the micelles was further confirmed by in vivo real time near-infrared fluorescence imaging in PC-3 tumor-bearing nude mice. Moreover, the active targeting and pH-sensitivity endowed cRGDyK-conjugated micelles with a higher antitumor effect in PC-3 xenograft-bearing nude mice compared with unmodified micelles and Taxol with negligible systemic toxicity. Therefore, these results suggested that cRGDyK-conjugated pH-sensitive polymeric micelles may be a promising delivery system for efficient delivery of anticancer drugs to treat integrin α_vβ₃-rich prostate cancers.

Introduction

Currently, androgen ablation therapy remains the first choice of prostate cancer treatment, however, the cells gradually acquire hormone-resistance as the disease progresses [1]. After endocrine therapy, chemotherapy with drugs such as paclitaxel and docetaxel is adopted as the primary clinical treatment regimen [2]. However, the benefit of chemotherapy is limited due to its serious systemic toxicity. To overcome this inconvenience, many new and innovative strategies to entrap antitumor drugs in different types of nanocarriers have been developed including liposomes, microemulsion, nanoparticles, and polymer-drug conjugates. Among them, polymeric micelles present great potential to improve water solubility of anticancer drugs, prolong blood circulation time, and enhance their accumulation at tumor sites by the enhanced permeability and retention (EPR) effect and therefore have attracted considerable attention [3], [4], [5]. However, their efficiency of passive targeting to tumor by EPR effect is limited. Consequently, recognition and uptake of micelle delivery system by tumor cells remain a considerable challenge [2], [6], [7], [8], which highlights the urgent need for more effective design strategies.

The use of various targeting ligands on the surface of nanocarriers, being recognized by their specific receptors/antigens on tumor cell surface, has been demonstrated to promote cellular uptake [9]. Cyclic Arg-Gly-Asp-Tyr-Lys (cRGDyK) has high affinity to integrin α_vβ₃, a tumor angiogenesis biomarker overexpressed in tumor neovasculature and most tumor cells [10], [11]. It was reported that cRGDyK conjugated onto the nanocarriers facilitated their uptake by tumor cells via integrin-mediated endocytosis, thereby enhancing the cytotoxicity of antitumor drug-loaded nanocarriers against tumor cells [11], [12], [13], [14]. Herein, we focused on cRGDyK as a candidate decoration.

Another major concern is that slow release of anticancer drug from nanocarriers in tumor cells may result in a low level of intracellular free drug concentration and thereby induce limited antitumor effect [3], [15]. Even worse, maintaining a low level of free drug concentration in tumor cells for a long time may cause occurrence of drug resistance. Consequently, to ensure the delivery of anticancer drug to tumor site with sufficient drug concentration, nanocarriers, such as polymeric micelles, can be used for increased drug stability in circulation and rapid drug release in the tumor. The phenomenon of rapid release might be achieved by targeted polymeric micelles with a triggered release mechanism that responds to the pH or enzymes inside the cells [16], [17]. Further, ligand-modified nanocarriers are generally internalized into tumor cells via an endocytic pathway with an experience of a pH gradient from 5.5–6.5 in endosomes to 4.5–5.0 in lysosomes in their intracellular trafficking pathway, thereby leading to an inferior antitumor efficacy due to the degradation of the cargos by the lysosome enzymes [18]. Therefore, it is very important to facilitate drug escape from endo/lysosome vesicles [19], [20], and pH-responsive polymeric micelles may be suitable for intracellular drug delivery. As known, pH-responsive hydrophobic polyacids or polybases usually aggregate to form an inner core of polymeric micelles that convey pH-sensitivity to drug release. For example, the protonation of poly(l-histidine) frequently constituting the hydrophobic core of mixed PEG-poly(l-histidine)/PEG-poly(d,l-lactic acid) micelles results in destabilization of micelle cores and expedient drug release [20]. However, polymeric micelles with pH-responsive outer shell have rarely been reported.

Here, we aimed to overcome the current limitations mentioned above. cRGDyK-conjugated pH-sensitive polymeric micelles were designed based on pH-sensitive diblock copolymer poly(2-ethyl-2-oxazoline)-poly(d,l-lactide) (PEOz-PLA) and cRGDyK-PEOz-PLA for integrating the merits of ligand-modified polymeric micelles for enhanced accumulation at tumor site and increased uptake by tumor cells, and pH-sensitive polymeric micelles for rapid intracellular drug release and endo/lysosome escape. We hypothesized that the designed micelles would be endowed with tumor cell-targeting ability and pH-response to intracellular compartments and thereby an effective delivery system for anticancer drugs to treat prostate cancers.