Pharmaceutical nanotechnologyIn vitro and in vivo evaluation of Δ9-tetrahidrocannabinol/PLGA nanoparticles for cancer chemotherapy
Graphical abstract
Introduction
Δ9-Tetrahidrocannabinol (Δ9-THC) has attracted special interest in oncology given their well-known palliative effects and antitumor activity (Ramer and Hinz, 2008, Aviello et al., 2012, Velasco et al., 2012, Solinas et al., 2013). In fact, Δ9-THC has been described to inhibit tumor angiogenesis and cell growth in malignant tissues, thus causing cell death (McKallip et al., 2002, Casanova et al., 2003, Blázquez et al., 2004, Bifulco et al., 2006, Ramer et al., 2012, Hernán Pérez de la Ossa et al., 2013a, Machado-Rocha et al., 2014).
Unfortunately, and despite oral aerosols, transdermal patches, and suppositories have been proposed (Hernán Pérez de la Ossa et al., 2013a), up to now the development of an effective and safety (Δ9-THC)-based formulation remains to be accomplished. Probably, reasons beneath the challenge are the high instability, oily-resin nature, low water solubility (≈2.8 mg/L), and low bioavailability of the compound (Brownjohn and Ashton, 2012). To beat the challenge, (Δ9-THC)-loaded microparticulate systems based on the polymer poly(ϵ-caprolactone) have been engineered by oil-in-water emulsion-solvent evaporation with promising results against cancer (Hernán Pérez de la Ossa et al., 2012, Hernán Pérez de la Ossa et al., 2013a, Hernán Pérez de la Ossa et al., 2013b). However, the micrometer size of the Δ9-THC particulate formulation (≈20–50 μm) is expected to limit the clinical outcome (Decuzzi et al., 2009).
As an alternative, poly(d,l-lactide-co-glycolide) nanoparticles (PLGA NPs) have been investigated (Martín-Banderas et al., 2014). This PLGA-based formulation was also used as nanocarrier for synthetic cannabinoid receptor agonist 13 (CB13) molecules (Durán-Lobato et al., 2013, Martín-Banderas et al., 2012). However, a definitive in vitro and in vivo proof of concept of the possibilities of this (Δ9-THC)-loaded nanoparticulate formulation is needed.
Therefore, this work is devoted to the development of PLGA-based nanocarriers as delivery systems for Δ9-THC. Concretely, PLGA NPs, PLGA NPs surface coated with poly(ethylene glycol) (PEGylated PLGA NPs), PLGA NPs embedded within a chitosan (CS) shell (chitosan-coated PLGA NPs), and PEGylated chitosan-coated PLGA NPs were investigated. Vitamin E molecules were incorporated to the formulations to enhance the stability of Δ9-THC against oxidation. Geometry and surface electrical charge measurements, blood compatibility and protein adsorption characterizations, and the in vitro evaluation of the Δ9-THC loading and release capabilities revealed that the PEGylated PLGA nanosystem was the more adequate formulation for the parenteral administration of Δ9-THC (see below). Finally, the in vitro anticancer activities of (Δ9-THC)-loaded PEGylated PLGA NPs were evaluated in murine LL2 and human A-549 lung cancer cell lines. The human embryo lung fibroblastic MRC-5 cell line was used as control. Regarding the in vivo investigation of the antitumor potential of (Δ9-THC)-loaded PEGylated PLGA NPs, LL2 lung tumor-bearing immunocompetent C57BL/6 mice was used to that aim.
Section snippets
Materials
Δ9-THC was provided by THC Pharm (Frankfurt am Main, Germany). PLGA (Resomer® RG 502, PLGA 50:50, molecular weight: 12,000 Da, inherent viscosity: 0.24 dL/g) was obtained from Boehringer-Ingelheim (Ingelheim, Germany). Low molecular weight chitosan, Span® 60, Tween® 80, Pluronic® F-68, ethylenediaminetetraacetic acid (EDTA), trypsin, sodium citrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton® X-100, ammonium oxalate, sodium dodecyl sulphate (SDS), monoclonal
Particle geometry and surface electrical charge
Mean particle size of PLGA NPs is increased from ≈300 nm to ≈600 nm or ≈750 nm when a polymeric coating is incorporated onto their surface, i.e., PEG or chitosan, respectively (Table 1). It has been attributed such kind of increase in size to polymer deposition onto the NP surface in the form of multi-layers (Nafee et al., 2009, Parveen and Sahoo, 2011). In all cases the coefficient of variation is in the range of ≈15%, this indicating that the particle distributions were almost homogeneous.
Conclusions
A reproducible nanoprecipitation technique has been proposed to prepare PLGA-based nanoformulations loaded with Δ9-THC. The nanosystems are characterized by an adequate in vivo safety margin, high drug loading efficiencies, and sustained drug release properties. Surface modification with PEG chains can improve the Δ9-THC vehiculization capabilities and reduce protein adsorption (and thus probably the in vivo opsonization processes). Vitamin E has been advantageously included in the
Acknowledgements
The research leading to these results has received funding from projects P09-CTS-5029 (Consejería de Innovación, Junta de Andalucía, Spain), PE-2012-FQM-694 (Consejería de Innovación, Junta de Andalucía, Spain), and FIS 11/02571 (Instituto de Salud Carlos III, Spain). L.M.-B. is especially grateful for the financial support from Junta de Andalucía (España). We also thank Microscopy and Biology Services of CITIUS (University of Seville) for technical assistance.
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