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. 2013;8(1):e54795.
doi: 10.1371/journal.pone.0054795. Epub 2013 Jan 22.

Local Delivery of Cannabinoid-Loaded Microparticles Inhibits Tumor Growth in a Murine Xenograft Model of Glioblastoma Multiforme

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Local Delivery of Cannabinoid-Loaded Microparticles Inhibits Tumor Growth in a Murine Xenograft Model of Glioblastoma Multiforme

Dolores Hernán Pérez de la Ossa et al. PLoS One. .
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Abstract

Cannabinoids, the active components of marijuana and their derivatives, are currently investigated due to their potential therapeutic application for the management of many different diseases, including cancer. Specifically, Δ(9)-Tetrahydrocannabinol (THC) and Cannabidiol (CBD) - the two major ingredients of marijuana - have been shown to inhibit tumor growth in a number of animal models of cancer, including glioma. Although there are several pharmaceutical preparations that permit the oral administration of THC or its analogue nabilone or the oromucosal delivery of a THC- and CBD-enriched cannabis extract, the systemic administration of cannabinoids has several limitations in part derived from the high lipophilicity exhibited by these compounds. In this work we analyzed CBD- and THC-loaded poly-ε-caprolactone microparticles as an alternative delivery system for long-term cannabinoid administration in a murine xenograft model of glioma. In vitro characterization of THC- and CBD-loaded microparticles showed that this method of microencapsulation facilitates a sustained release of the two cannabinoids for several days. Local administration of THC-, CBD- or a mixture (1:1 w:w) of THC- and CBD-loaded microparticles every 5 days to mice bearing glioma xenografts reduced tumour growth with the same efficacy than a daily local administration of the equivalent amount of those cannabinoids in solution. Moreover, treatment with cannabinoid-loaded microparticles enhanced apoptosis and decreased cell proliferation and angiogenesis in these tumours. Our findings support that THC- and CBD-loaded microparticles could be used as an alternative method of cannabinoid delivery in anticancer therapies.

Conflict of interest statement

Competing Interests: GW Pharmaceuticals funded part of the research that was performed in GV's laboratory. GV is a PLOS ONE Editorial Board member. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Characterization of cannabinoide-loaded microparticles.
(A) Scanning electron microscopy (500X) of blank, CBD- and THC-loaded PCL MPs. Representative microphotographs of the three types of MPs are shown. (B) Particle size distribution of blank, CBD- and THC-loaded microspheres. Results correspond to microsphere diameter determined by percentage volume distribution. (C) Cannabinoid release profiles of THC and CBD-loaded PCL microspheres. For the in vitro release studies, microspheres were incubated in PBS pH 7.4-Tween®80 0.1% (v/v) and maintained in a shaking incubator at 37°C. At predetermined time intervals supernatants were withdrawn and media was replaced. The concentration of CBD or THC in the release medium was quantified by HPLC. Data correspond to the cumulative amount of each cannabinoid released at the indicated time points, and are expressed as mean percentage of released cannabinoid relative the total amount of cannabinoid loaded into the microspheres ± s.d (n = 3).
Figure 2
Figure 2. Cannabinoid-loaded microparticles reduce the growth of U87MG cell-derived tumour xenografts.
(A) Effect of the local administration of placebo MPs, THC-loaded MP (75 mg of MP containing approximately 6.15 mg of THC per administration, one administration every 5 days), CBD-loaded MP (75 mg of MP containing approximately 6.7 mg of CBD per administration, one administration every 5 days), a mixture (1∶1 w:w) of THC- and CBD-loaded MP (37.5 mg of THC-loaded MP and 37.5 mg of CBD-loaded MP per administration, one administration every 5 days), THC (15 mg/kg/day corresponding to 0.5 mg THC per day), CBD (15 mg/kg/day corresponding to 0.5 mg THC per day) or THC + CBD (7.5 mg/kg/day of THC and 7.5 mg/kg/day CBD corresponding to 0.25 mg of THC and 0.25 mg of CBD per day) on the growth of U87MG cell-derived tumor xenografts. No significant differences were found between tumours treated with vehicle in solution or placebo MPs and these data were represented together. For the sake of clarity, comparisons between the effect of THC-loaded MPs and THC in solution (B), CBD-loaded MPs and CBD in solution (C), and THC-loaded MPs + CBD-loaded MPs and THC + CBD in solution (D) on the growth of U87MG cell-derived tumour xenografts are shown. Results are expressed as the mean fold increase ± SEM relative to vehicle treated tumors on the day one of the treatment. (n = 7). Tumours treated with THC-loaded MPs, CBD loaded MPs, a mixture of THC-loaded MPs and CBD-loaded MPs were significantly different (** p<0.01) from vehicle/placebo MPs-treated tumours. Tumours treated with THC in solution, CBD in solution or a mixture of THC and CBD in solution were also significantly different (p<0.01) from vehicle/placebo-treated tumours from day 14 until the end of the treatment (signs of significance are omitted for clarity). No significant differences were found among any of the treatments with cannabinoid-loaded microparticles and any of the treatments with cannabinoids in solution.
Figure 3
Figure 3. Cannabinoid-loaded microparticles reduce the weight of U87MG cell-derived tumour xenografts.
(A) Effect of the local administration of placebo MPs, THC-loaded MP (75 mg of MP containing approximately 6.15 mg of THC per administration, one administration every 5 days), CBD-loaded MP (75 mg of MP containing approximately 6.7 mg of CBD per administration, one administration every 5 days), a mixture (1∶1 w:w) of THC- and CBD-loaded MP (37.5 mg of THC-loaded MP and 37.5 mg of CBD-loaded MP per administration, one administration every 5 days), THC (15 mg/kg/day corresponding to 0.5 mg THC per day), CBD (15 mg/kg/day corresponding to 0.5 mg THC per day) or THC + CBD (7.5 mg/kg/day of THC and 7.5 mg/kg/day CBD corresponding to 0.25 mg of THC and 0.25 mg of CBD per day) on tumour weight on the last day of the treatment. (B) Photographs of representative tumors of each experimental condition. (n = 7; ** p<0.01 from vehicle/placebo MPs-treated tumours).
Figure 4
Figure 4. Cannabinoid loaded microparticles activate apoptosis and inhibit proliferation and angiogenesis of U87 MG cell-derived tumour xenografts.
Effect of THC-loaded MP, CBD-loaded MP and a mixture of THC- and CBD-loaded MP on cell proliferation (as determined by KI67 immunostaining; A), apoptosis (as determined by TUNEL; B) and angiogeneis (as determined by CD31 immnunostaining; C) of U87MG cell-derived tumor xenografts. Values on the lower right corner of each panel correspond to the percentage of KI67-positive cells relative to the total number of nuclei in each section ± s.d. (A), the percentage of TUNEL-positive cells relative to the total number of nuclei in each section ± s.d. (B) or the CD31-stained area normalized to the total number of nuclei in each section (mean fold change ± s.d.; C) (10 sections of 3 different tumors from each condition were analyzed; ** p<0.01 from vehicle-treated tumors; # p<0.05 from CBD-loaded MP-treated tumors.

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This work was supported by grants from the Spanish Ministry of Science and Innovation (MICINN) (PS09/01401 to GV), Comunidad Autónoma de Madrid (PR1/06-14474-B to AITS) and Complutense University (CCG07-UCM/BIO-2824 to AITS). DH was recipient of a FPU fellowship from MICINN. ML was sequentially recipient of a “Juan de la Cierva” contract, a postdoctoral contract from Spanish Ministry of Education and Science and a postdoctoral contract from Comunidad de Madrid. ST was recipient of a research training contract from Comunidad de Madrid. GW Pharmaceuticals funded part of the research that was performed in GV's laboratory. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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