Pharmaceutical nanotechnology
In vitro and in vivo evaluation of Δ9-tetrahidrocannabinol/PLGA nanoparticles for cancer chemotherapy

https://doi.org/10.1016/j.ijpharm.2015.04.054Get rights and content

Abstract

Nanoplatforms can optimize the efficacy and safety of chemotherapy, and thus cancer therapy. However, new approaches are encouraged in developing new nanomedicines against malignant cells. In this work, a reproducible methodology is described to prepare Δ9-tetrahidrocannabinol (Δ9-THC)-loaded poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles against lung cancer. The nanoformulation is further improved by surface functionalization with the biodegradable polymers chitosan and poly(ethylene glycol) (PEG) in order to optimize the biological fate and antitumor effect. Mean nanoparticle size (≈290 nm) increased upon coating with PEG, CS, and PEG–CS up to ≈590 nm, ≈745 nm, and ≈790 nm, respectively. Surface electrical charge was controlled by the type of polymeric coating onto the PLGA particles. Drug entrapment efficiencies (≈95%) were not affected by any of the polymeric coatings. On the opposite, the characteristic sustained (biphasic) Δ9-THC release from the particles can be accelerated or slowed down when using PEG or chitosan, respectively. Blood compatibility studies demonstrated the adequate in vivo safety margin of all of the PLGA-based nanoformulations, while protein adsorption investigations postulated the protective role of PEGylation against opsonization and plasma clearance. Cell viability studies comparing the activity of the nanoformulations against human A-549 and murine LL2 lung adenocarcinoma cells, and human embryo lung fibroblastic MRC-5 cells revealed a statistically significant selective cytotoxic effect toward the lung cancer cell lines. In addition, cytotoxicity assays in A-549 cells demonstrated the more intense anticancer activity of Δ9-THC-loaded PEGylated PLGA nanoparticles. These promising results were confirmed by in vivo studies in LL2 lung tumor-bearing immunocompetent C57BL/6 mice.

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.

References (49)

  • A. Kumari et al.

    Biodegradable polymeric nanoparticles based drug delivery systems

    Colloids Surf. B Biointerfaces

    (2010)
  • L. Martín-Banderas et al.

    Engineering of Δ9-tetrahydrocannabinol delivery systems based on surface modified-PLGA nanoplatforms

    Colloids Surf. B Biointerfaces

    (2014)
  • R.J. McKallip et al.

    Targeting CB2 cannabinoid receptors as a novel therapy to treat malignant lymphoblastic disease

    Blood

    (2002)
  • E. Moreno et al.

    Targeting CB2-GPR55 receptor heteromers modulates cancer cell signaling

    J. Biol. Chem.

    (2014)
  • N. Nafee et al.

    Relevance of the colloidal stability of chitosan/PLGA nanoparticles on their cytotoxicity profile

    Int. J. Pharm.

    (2009)
  • B. Patel et al.

    PEG–PLGA based large porous particles for pulmonary delivery of a highly soluble drug, low molecular weight heparin

    J. Controlled Release

    (2012)
  • S. Parveen et al.

    Long circulating chitosan/PEG blended PLGA nanoparticles for tumor drug delivery

    Eur. J. Pharm.

    (2011)
  • K. Sempf et al.

    Adsorption of plasma proteins on uncoated PLGA nanoparticles

    Eur. J. Pharm. Biopharm.

    (2013)
  • S. Takeda et al.

    Δ9-Tetrahydrocannabinol enhances MCF-7 cell proliferation via cannabinoid receptor-independent signaling

    Toxicology

    (2008)
  • L. Thiele et al.

    Competitive adsorption of serum proteins at microparticles affects phagocytosis by dendritic cells

    Biomaterials

    (2003)
  • M.G. Traber et al.

    Vitamin E: antioxidant and nothing more

    Free Radical Biol. Med.

    (2007)
  • V.-T. Tran et al.

    Protein-loaded PLGA–PEG–PLGA microspheres: A tool for cell therapy

    Eur. J. Pharm. Sci.

    (2012)
  • H. Vihola et al.

    Cytotoxicity of thermosensitive polymers poly(N-isopropylacrylamide), poly(N-vinylcaprolactam) and amphiphilically modified poly(N-vinylcaprolactam)

    Biomaterials

    (2005)
  • C.E. Astete et al.

    Antioxidant poly(lactic-co-glycolic) acid nanoparticles made with α-tocopherol-ascorbic acid surfactant

    ACS Nano

    (2011)
  • Cited by (43)

    • Cannabis extract-loaded lipid and chitosan-coated lipid nanoparticles with antifungal activity

      2024, Colloids and Surfaces A: Physicochemical and Engineering Aspects
    • Chitosan-based nano drug delivery system for lung cancer

      2023, Journal of Drug Delivery Science and Technology
    • Preparation and characterization of full-spectrum cannabis extract loaded poly(thioether-ester) nanoparticles: In vitro evaluation of their antitumoral efficacy

      2023, Colloids and Surfaces A: Physicochemical and Engineering Aspects
      Citation Excerpt :

      The revelations of the endocannabinoid system have provided the opportunity to understand Cannabis potentialities for the treatment of many diseases [2] such as chronic pain, nausea, eating disorders, glaucoma, neurodegeneration, multiple sclerosis, cancer, Alzheimer's disease, epilepsy, stress, and anxiety [3,6,8–13]. In fact, delta-9-tetrahydrocannabinol (THC) is supposed to have the capacity to inhibit angiogenesis and consequently cell growth of tumor cells leading to their death [14]. Interestingly, THC has been shown to exert antitumoral activity through tribbles homolog 3-dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis and the subsequent stimulation of autophagy-mediated cancer cell death [15,16].

    • Colon cancer therapy with calcium phosphate nanoparticles loading bioactive compounds from Euphorbia lathyris: In vitro and in vivo assay

      2022, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      Thus, the efficacy and therapeutic effect of a natural extracts can be modulated by controlling the relative concentrations of the bioactive compounds present in it [26]. The high incidence of cancer worldwide has aroused enormous interest in the search of new natural extracts with potential to prevent and suppress cancer proliferation [27,28]. Euphorbia, a therapeutic resource of traditional Chinese medicine, has attracted great interest for its recognized anticancer properties.

    • Selective Targeting of the Novel CK-10 Nanoparticles to the MDA-MB-231 Breast Cancer Cells

      2022, Journal of Pharmaceutical Sciences
      Citation Excerpt :

      Kidney clearance happens characteristically for molecules of a size less than 50 kDa (e.g. siRNA). Consequently, loading such drug load into nanosystems with a hydrodynamic size ≥ 10.00 nm can support escaping glomerular filtration and excretion in urine, thus enhancing the drug load circulation period.14-20 Definite biological surroundings can be reached through passive targeting, which is typically a size-dependent phenomenon.

    View all citing articles on Scopus
    View full text