Elsevier

Pharmacological Research

Volume 141, March 2019, Pages 451-465
Pharmacological Research

A novel nanoformulation of PLGA with high non-ionic surfactant content improves in vitro and in vivo PTX activity against lung cancer

https://doi.org/10.1016/j.phrs.2019.01.013Get rights and content

Abstract

Paclitaxel (PTX), a chemotherapy agent widely used to treat lung cancer, is characterised by high toxicity, low bioavailability and the need to use of excipients with serious side effects that limit its use. Paclitaxel encapsulation into nanoparticles (NPs) generates drug pharmacokinetic and pharmacodynamic advantages compared to free PTX. In this context, a NP carrier formed from a copolymer of lactic acid and glycolic acid (PLGA) has demonstrated high biocompatibility and low toxicity and therefore being approved by FDA to be used in humans. We synthesised a new PLGA NP and loaded it with PTX to improve drug efficacy and reduce side effects. This nanoformulation showed biocompatibility and no toxicity to human immune system. These NPs favor the intracellular uptake of PTX and enhance its antitumor effect in human and murine lung cancer cells, with up to 3.6-fold reductions in the PTX’s IC50. Although PLGA NPs did not show any inhibitory capacity against P-glycoprotein, they increased the antitumor activity of PTX in cancer stem cells. Treatment with PLGA-PTX NPs increased apoptosis and significantly reduced the volume of the tumorspheres derived from A549 and LL2 cells by up to 36% and 46.5%, respectively. Biodistribution studies with PLGA-PTX NPs revealed an increase in drug circulation time, as well as a greater accumulation in lung and brain tissues compared to free PTX. Low levels of PTX were detected in the dorsal root ganglion with PLGA-PTX NPs, which could exert a protective effect against peripheral neuropathy. In vivo treatment with PLGA-PTX NPs showed a greater decrease in tumor volume (44.6%) in immunocompetent mice compared to free PTX (24.4%) and without increasing the toxicity of the drug. These promising results suggest that developed nanosystem provide a potential strategy for improving the chemotherapeutic effect and reducing the side effects of PTX.

Introduction

Lung cancer is the leading cause of cancer death worldwide [1]. Paclitaxel (PTX), a drug of choice in lung cancer chemotherapy [2,3], is a diterpene alkaloid that promotes polymerisation and microtubule assembly by specifically binding to the β-tubulin subunit of tubulin causing the kidnapping in the G2 phase of the cell cycle [4,5]. However, the administration of PTX has several disadvantages which limit its antitumor activity and result in treatment failure. In fact, the two most common chronic toxicities associated with PTX are painful peripheral neuropathy [6,7] and haematological toxicity due to bone marrow depression, which affect more than 60% of treated patients and lead to treatment suspension (temporarily or permanently) or dose reduction and therefore decreased antitumor efficacy [7,8]. In addition, PTX shows low solubility in water (˜ 0.4 μg/ml), so the drug formulation requires the use of Cremophor® EL and ethanol which induce hypersensitivity reactions, neutropenia or peripheral neuropathy [8,9]. Finally, PTX also undergoes rapid elimination and has a low bioavailability [10,11] and drug resistance may develop mediated by several mechanisms including P-glycoprotein (P-gp) which considerably reduces its antitumor effect [[12], [13], [14]]. Thus, new pharmaceutical technology strategies must be developed to enhance the effect of PTX, eliminate the need for solvents and increase its specificity.

A potential means of overcoming PTX’s limitations is by encapsulating it in nanoparticles (NPs), which protect drugs from degradation and multidrug resistance (MDR) mechanisms such as P-gp [14,15]. Nanoparticles promote the incorporation of the drug into cells and its accumulation in tumor as a result of increased permeability and retention (EPR) that usually characterize these tissues. In addition, the use of nanoparticles may improve drug solubility, stability and bioavailability [[16], [17], [18]]. Furthermore, NPs can be modified to target specific cell types, so they accumulate in tumors rather than healthy tissues [17,19]. Numerous types of NPs have been developed to improve PTX efficacy including the albumin-bound PTX NP Abraxane® (Nab-PTX), the first formulation approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) to treat patients with non-small cell lung cancer (NSCLC) [3,4,20]. However, although Nab-PTX increases the maximum tolerated dose of paclitaxel compared to commercial Taxol®, thus circumventing the need for Cremophor and improving tumor drug accumulation, this formulation still has several limitations associated with its clinical use [21]. In fact, In fact, besides its high production costs, Nab-PTX replicates some of the most undesirable side effects of Taxol®, such as hypersensitivity reactions, neutropaenia, neuropathy, myalgia, anaemia and thrombocytopaenia [[22], [23], [24]]. In addition, Nab-PTX may induce a more severe degree of peripheral neuropathy than Taxol® [25]. PTX has been formulated as Lipusu®, a liposomal preparation of cholesterol and lecithin for the treatment of ovarian, breast, NSCLC, gastric and head and neck cancers with a lower associated toxicity than free PTX. Two liposomes associated with PTX, LEP-ETU and EndoTag-1, are currently being assayed in clinical trials for the treatment of breast cancer [5,26,27]. Furthermore, two polymeric micelles associated with PTX have been approved for clinical use in some Asian countries for the treatment of lung cancer (Nanoxel®) and metastatic breast cancer and locally advanced or metastatic NSCLC (Genexol-PM®). The latter enlarges PTX’s therapeutic window without increasing its toxicity [28]. Biodegradable polymers conjugated with PTX, such as the polyglutamic acid Opaxio™, are also being studied in a phase III clinical trial for ovarian and lung cancer. This formulation increases the half-life of PTX and releases the drug from the polymer network through the action of lysosomal proteases [5]. However, at present, of all the formulations mentioned above only Abraxane is used clinically in the USA and Europe [21]; therefore, new PTX formulations need to be developed to improve its antitumor efficiency and reduce its side effects. In this context, poly-lactic and glycolic acid (PLGA), approved by the FDA as a biocompatible polymer, represents an ideal carrier for developing new cytotoxic nanoformulations due to its low toxicity and biodegradation by generating lactic acid and glycolic acid monomers which are easily metabolized [18,29].

In this work we prepared a new and promising PLGA NP formulation (PLGA-PTX NPs) to improve the antitumor activity of PTX against lung cancer while also reducing its side effects. We used human and murine lung cancer cell lines and immunocompetent C57BL/6 mice to perform the in vitro and in vivo studies, respectively. Our results showed that PLGA-PTX NPs provide a significant in vitro and in vivo therapeutic advantage over free PTX, including excellent biocompatibility, a significant increase in PTX antiproliferative effect in lung cancer cells and multicellular tumor spheroids (MTS), and intense activity against lung cancer stem cells (CSCs). In addition, in vivo PLGA-PTX NPs treatment achieved a greater reduction in tumor volume than free PTX and without increasing drug toxicity. Interestingly, PLGA-PTX NPs increased the presence of PTX in lung tissue and decreased the drug concentration in dorsal root ganglion (DRG) related to peripheral neuropathy. Thus, PLGA-PTX NPs represent a new possibility for lung cancer treatment as they improve the antitumor efficacy of PTX and reduce its side effects, which are the main limitations to its clinical use.

Section snippets

Materials

Resomer® RG 504H (PLGA 50:50, molecular weight, MW: 48,000 Da; inherent viscosity: 0.53 dL/g) was obtained from Boehringer-Ingelheim (Germany). All other chemicals used were of analytical quality from Sigma-Aldrich (USA), except for PTX (Tocris Cookson Ltd., UK), and threalose (VWR International Eurolab S.L., Spain). Acetone, acetonitrile, methanol, dimethyl sulfoxide (DMSO), and acetic acid were of high-performance liquid chromatography (HPLC) grade from Panreac Química (Spain). Water used in

Particle size, surface electrical charge and storage conditions

Synthesis of all the PLGA-based nanoparticle formulations was based on a nanoprecipitation methodology [30], and we studied the influence of initial amount of PTX (5, 10 or 15% w/w) on particle size and surface electrical charge. PLGA-PTX NPs were characterised by a spherical shape and an average diameter under 250 nm with a coefficient of variation (CV%) of less than 15% and polydispersity index (PdI) below 0.3, which suggest a homogeneous particle size distribution (Table 1).

The surface

Conclusions

We used nanoprecipitation to synthesise a new PLGA NP loaded with PTX as an alternative means to improve the treatment of lung cancer. These biodegradable nanocarriers had a negative charge, a size below 250 nm and good biocompatibility even at the highest concentration used. In vitro assays demonstrated a significant increase in PTX activity against lung cancer cells in culture and an apoptosis increase in MTS derived from A549 and LL2 cells. What is more, PLGA-PTX NPs increased drug

Conflict of interest

The authors declare no conflict of interest

Acknowledgements

This work was funded by Consejería de Salud de la Junta de Andalucía (projects P11-CTS-7649, PI-0476-2016 and PI-0102-2017) and by Granada University (project PP2015-13 and financial Groups 09/112016). This work was also partially supported by grant of Junta de Andalucía (PI-0038-2014). L.M.-B. is especially grateful for the financial support from V Plan Propio (University of Seville). The research grant (FPU) from Ministerio de Educación Cultura y Deporte (Government of Spain) and

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