Paclitaxel-clusters coated with hyaluronan as selective tumor-targeted nanovectors

Abstract

Paclitaxel (PTX) is a widely used anti-tumor agent in the treatment of solid tumors. Lack of selective strategies to target PTX into tumor cells together with poor solubility necessitating detergent, are severe clinical limitations. To address these hurdles, we devised a strategy that utilized PTX insolubility, mixing it with lipids that self-assemble into nanoparticle-like clusters. These clusters were then coated with hyaluronan, a glycosaminoglycan (GAG), and termed PTX-GAGs. These particles, delivered PTX selectively into tumor cells in a CD44-dependent manner. Injected systemically to mice bearing solid tumors, the PTX-GAGs showed high safety profile and tumor accumulation. Tumor progression was exponential upon treatment with free PTX or PTX in albumin nanoparticles (the FDA-approved Taxol^® and Abraxane^®, respectively). Under the same conditions, PTX-GAGs induced tumor arrest and were as potent as a 4-fold higher Taxol^® dose. Our findings suggest GAGs merit further investigation as vehicles for taxanes, and may be applicable as carriers in other therapeutic settings.

Introduction

Paclitaxel (PTX), a microtubules stabilizer that causes mitotic arrest, has shown broad–spectrum activity in many solid tumors including ovarian, breast, AIDS-related Kaposi’s sarcoma, lung, head and neck and bladder [1], [2]. The FDA-approved formulation of PTX-Cre (Taxol^®), requires dissolving the paclitaxel in Cremophor^® EL (polyoxyethylated castor oil) and ethanol (CrEL) [3]. However, the use of Taxol^® requires premedication with corticosteroids and antihistamines to reduce risks of hypersensitivity reaction [4], [5], [6], [7]. It can also cause neutropenia [8], and prolonged, peripheral neuropathy, which may be associated with axonal degradation [6], [7]. In addition to serious toxicities, CrEL may negatively impact efficacy by limiting tumor penetration through the formation of large polar micelles, which can lead to nonlinear pharmacokinetics and decreased unbound drug fraction [6], [8].

Among carriers for paclitaxel made from biological materials, liposomes have been well-studied as carriers for PTX, although drug loading into liposomes is relatively low (≤10% (w/w)) [9], [10], [11], [12], [13], [14]. Recently, a new formulation was approved by the FDA, albumin-bound PTX (nab™-PTX; Abraxane^®) forming nanoparticles (∼135 nm in diameter). This formulation, consisting of unmodified PTX and human albumin, is CrEL-free. By eliminating CrEL from its formulation, nab-PTX reduces risks of hypersensitivity reactions, does not require premedication, and can be given over a shorter period without special intravenous tubing [15]. However, it is reported to have low PTX loading yield, similar to liposomes and minimal improvement in efficacy [16]. Both liposomes and nab™-PTX are non-targeted drug carriers that utilize the enhanced permeability and retention effect (EPR) [17], [18] to accumulate at close proximity to the tumors.

The frequent overexpression of the hyaluronan (HA) receptors CD44 and CD168 (RHAMM) on many types of tumors opens new avenues for targeting by the naturally-occurring high-molecular weight HA [19], [20], [21], [22]. HA, a naturally-occurring glycosaminoglycan, is one of the major components of the extracellular matrix (ECM). It is found in many tissues such as skin, joint tissue (in synovial fluid) and eyes [23], [24]. HA is known as a bioadhesive compound capable of binding with high affinity to both cell surface and intracellular receptors, to ECM components and to itself [25], [26]. In cancer cells, binding of HA to its receptors is involved in tumor growth and spreading. CD44 regulates cancer cells proliferation and metastatic processes [27], [28]. In addition, disruption of HA–CD44 binding was shown to reduce tumor progression [29], [30], [31]. Administration of exogenous HA resulted in arrest of tumor spreading [31]. HA is a non-toxic and non-immunogenic compound, already approved for use in eye surgery, joint therapy and wound healing [32]. Coating small unilamellar liposomes with HA stabilizes these particles in a cycle of lyophilization and rehydration [33], provides selective targeting to tumors expressing the HA receptors [19], [20], [34], and presents a scaffold for conjugation of other ligands to the surface for further improving the selectivity to cell surface receptors [35].

Here, we report on tumor-targeted nanoparticles, constructed of PTX-phosphatidylethanolamine (PE) clusters covalently coated with HA. The terms used for the particles themselves (i.e., drug-free) and when loaded with PTX are GAGs and PTX-GAGs, respectively [36]. In contrast to other phospholipids such as phosphatidylcholine (PC) or phosphatidylserine (PS) that form lipid bilayers closed into circular particle like liposomes, PE by itself does not form a liposome, but other lamellar shapes. The variety of shapes depends on several factors such as the lipid side chains, concentration, temperature, pressure, hydration level, pH, and salt concentration [37], [38], [39], [40], [41], [42]. For the present task we selected dilauroyl-PE (DLPE). The saturated chains are more stable than unsaturated chains that tend to oxidize into toxic peroxi-lipids. Being relatively-short, the DL chains impart additional advantages: (i) relatively low gel-liquid crystalline transition temperature (Tm = 43 °C) [43], indicating homogenous particle suspensions may be achieved without the need for overheating that may harm incorporated drugs, and (ii) higher affinity, compared to longer chains, to PTX [14], [44].

Herein, we report studies at the molecular, cellular and whole animal levels of organization. The molecular studies included preparation, physicochemical, thermal analyses and ultrastructure characterization of the PTX-GAG particles. The in vitro studies in cancer cells included GAGs-cell binding and internalization, as well the cytotoxicity of PTX-GAGs. Biological effects of the PTX-GAG particles, studied in BALB/c mice, included evaluations of liver enzyme release and inductions of cytokines and of interferon response. PTX-GAGs retention in circulation, biodistribution, weight changes and efficacy were studied in BALB/c mice bearing a solid s.c. tumor (CT-26). Control systems were usually saline, drug-free GAGs and Taxol, and the addition of Abraxane^® for the in vivo efficacy studies.