Comparison of anti-tumor efficacy of paclitaxel delivered in nano- and microparticles

Abstract

This research compares the anti-tumor efficacy of paclitaxel delivered intratumorally in PLGA nanoparticles, microparticles, or the commercial Paclitaxel Injection^®. The hypothesis of the research is that larger PLGA microparticles adhere to mucus on the cell surface, release paclitaxel locally, and enhance cellular association of paclitaxel. PLGA-paclitaxel particles of mean diameters 315 nm, 1 μm, and 10 μm were prepared and their drug content, in vitro release, and cellular association of paclitaxel into 4T1 cells quantified. These particles were injected intratumorally into tumor xenografts, and the tumor volumes monitored over 13 days. Mean tumor volumes of the groups that received placebo and the 315 nm nanoparticles increased 2 and 1.5 times, respectively. Tumor growth was arrested in groups that received 1 μm and 10 μm microparticles. Additional cell culture studies were performed to test the hypothesis. The size-dependent increase in cellular concentration of paclitaxel was independent of duration of incubation of PLGA particles with 4T1 cells, and was enhanced 1.5 times by coating the particles or 4T1 cells with mucin. These particles were not internalized by clathrin-mediated endocytosis or macropinocytosis. In conclusion, PLGA microparticles sustained drug release, increased cellular concentration, and enhanced anti-tumor efficacy of paclitaxel compared to nanoparticles and Paclitaxel Injection^®.

Introduction

In 2009, it is expected that breast cancer will account for 27% of all the newly diagnosed cancer cases in women (Jemal et al., 2009). Paclitaxel is approved to treat breast and ovarian cancer and has also demonstrated anti-cancer activity against non-small cell lung cancer as well as Kaposi's sarcoma (Tulpule et al., 2002). Paclitaxel stabilizes microtubules against depolymerization, resulting in altered cellular functions during mitosis leading to apoptosis (Spencer and Faulds, 1994).

It is well documented that the low aqueous solubility of paclitaxel creates formulation challenges. The first commercially available intravenous formulation of paclitaxel is prepared by dissolving the drug in a 50:50 mixture of Cremophor^® EL (polyethoxylated castor oil) and dehydrated alcohol (Panchagnula, 1998). However, Cremophor^® EL was reported to cause anaphylactic hypersensitivity reactions, hyperlipidaemia, neurotoxicity, and alteration of paclitaxel's pharmacokinetics (Gelderblom et al., 2001). In 2005, the FDA approved Abraxane^®, a solvent-free, albumin-bound paclitaxel, for the treatment of metastatic breast cancer. Although Abraxane^® addressed the disadvantages of Cremophor, during phase III clinical trials, patients developed neutropenia and sensory neuropathy (Gradishar et al., 2005). Alternate delivery systems for paclitaxel have been, and continue to be investigated including emulsions, micelles, liposomes, cyclodextrins, implants, pastes and prodrugs (Singla et al., 2002).

Intratumoral chemotherapy provides sustained, localized, and elevated drug concentrations with low systemic toxicity and may prevent post-operative metastasis (Goldberg et al., 2002). An ideal formulation for intratumoral administration must have the following characteristics: (1) be injectable into the tumor mass, (2) provide sustained, predictable drug release, (3) be stable in the tumor milieu, (4) and be non-toxic. Therefore, injecting particulate delivery systems directly into tumors, or the resected tumor space could enhance the anti-tumor efficacy compared to systemic intravenous injection. Poly D, L-(lactide-co-glycolide) [PLGA] has been widely used as a polymeric vehicle for controlled release of hydrophilic and hydrophobic drugs. The formulation parameters affecting the particle size, drug loading, and in vitro release of hydrophobic drugs from the PLGA particles have been reviewed (Wischke and Schwendeman, 2008). While the effect of size of the PLGA particles on the in vitro drug release of encapsulated drugs has been well investigated (Klose et al., 2008, Budhian et al., 2008), the impact of PLGA particle size on the anti-tumor efficacy of the encapsulated drug following intratumoral administration has not been compared.

It has been previously shown that when delivered in nanoparticles, paclitaxel was more cytotoxic than the commercially available formulation (Chen et al., 2001, Fonseca et al., 2002). In an intratumoral chemotherapy study using emulsified wax/Brij^® 78 nanoparticles containing paclitaxel, a 50% reduction in tumor volume after 19 days was reported compared to the same dose of paclitaxel in solution (Koziara et al., 2006). Lapidus et al. (2004) reported a 50% reduction in tumor volume after intratumoral administration of paclitaxel in polilactofate (Paclimer^®) microparticles. More recently, Xie et al. (2007) reported that intratumoral injection of paclitaxel-loaded PLGA microparticles (42.72 μm) reduced tumor weight by 59.9% over a 21-day treatment period, compared to paclitaxel injection. These authors also reported that Vascular Endothelial Growth Factor (VEGF) expression was significantly lower in PLGA microparticle-treated tumors suggesting that this delivery system may enhance the anti-angiogenic activity of paclitaxel. Another study reported that when delivered in microparticles, paclitaxel exhibited greater anti-tumor efficacy than the commercial product (Harper et al., 1999, Lapidus et al., 2004). When co-injected with Lewis Lung Carcinoma Cells into the subcutaneous tissue, paclitaxel-containing PLGA microparticles of size range 1–5 μm significantly inhibited tumor growth and cell proliferation compared to placebo-PLGA microparticles (Azouz et al., 2008). In vitro cell culture studies of paclitaxel-loaded PLGA microparticles of the size range 0.5–5 μm demonstrated a dose-dependent cytotoxicity on human uterine cancer cells, with IC₅₀ values similar to that of Taxol^® (Hamoudeh et al., 2008).

Based on this literature, it can be concluded that greater cellular accumulation of paclitaxel is achieved when delivered in either nanoparticles or microparticles compared to solution. However, a comparison of the in vitro cellular association, cytotoxicity, and anti-tumor efficacy of paclitaxel-containing PLGA nanoparticles and microparticles has not been previously reported. We reported that a greater cellular association of paclitaxel into 4T1, Caco-2, and Cor-L23/R cells was achieved when delivered in microparticles compared to nanoparticles (De et al., 2005). It was hypothesized the larger microparticles adhere to mucus on the cell surface, enhancing the cellular association of paclitaxel. This hypothesis was supported by additional experiments using confocal microscopy in which it was demonstrated that microparticles adhere to the surface of 4T1 cells (De et al., 2005). Therefore, the specific aims of this study were to (1) compare the efficacy of paclitaxel delivered locally in nanoparticles, microparticles and the commercially available formulation administered intravenously and intratumorally in a mouse model using the 4T1 murine mammary adenocarcinoma cell line; and, (2) to investigate the mechanism responsible for the increased cellular association of paclitaxel delivered in larger microparticles compared to nanoparticles. Placebo and paclitaxel-containing PLGA nanoparticles and microparticles were administered intratumorally into xenografts developed on balb/c mice and the tumor growth was monitored over 13 days. To investigate the mechanism underlying the hypothesis that the observed size-dependent increase in cellular association of paclitaxel delivered in larger microparticles is due to the adhesion of microparticles to mucus, in vitro cellular association studies were performed after (a) different durations of incubation of the PLGA particles with the 4T1 cells, (b) use of metabolic inhibitors.