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, Δ9-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.

Citation: Hernán Pérez de la Ossa D, Lorente M, Gil-Alegre ME, Torres S, García-Taboada E, Aberturas MdR, et al. (2013) Local Delivery of Cannabinoid-Loaded Microparticles Inhibits Tumor Growth in a Murine Xenograft Model of Glioblastoma Multiforme. PLoS ONE 8(1): e54795. https://doi.org/10.1371/journal.pone.0054795 Editor: Natarajan Aravindan, University of Oklahoma Health Sciences Center, United States of America Received: October 29, 2012; Accepted: December 14, 2012; Published: January 22, 2013 Copyright: © 2013 Hernán Pérez de la Ossa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: 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. 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.

Introduction Δ9-Tetrahydrocannabinol (THC), the main active component of the hemp plant Cannabis sativa [1], exerts a wide variety of biological effects by mimicking endogenous substances – the endocannabinoids – that bind to and activate specific cannabinoid receptors [2]. So far, two G protein–coupled cannabinoid-specific receptors have been cloned and characterized from mammalian tissues: CB 1 , abundantly expressed in the brain and at many peripheral sites, and CB 2, expressed in the immune system and also present in some neuron subpopulations and glioma cells [2], [3]. One of the most active areas of research in the cannabinoid field is the study of the potential application of cannabinoids in the treatment of different pathologies [4], [5]. Among these therapeutic applications, cannabinoids are being investigated as anti-tumoral agents [6], [7]. Thus, cannabinoid administration curbs the growth of several types of tumor xenografts in rats and mice [6], [7] including gliomas [8]–[10]. Based on this preclinical evidence, a pilot clinical trial has been recently run to investigate the anti-tumor action of THC on recurrent gliomas [11]. The mechanism of THC anti-tumoral action relies on the ability of this compound to: (i) promote the apoptotic death of cancer cells (ii) to inhibit tumour angiogenesis and (iii) to reduce the migration of cancer cells [6]. Aside from THC, C. sativa produces approximately 70 other cannabinoids although, unlike THC, many of them exhibit little affinity for CB receptors [5], [12]. Of interest, at least one of these components, namely cannabinol (CBD), has been shown to reduce the growth of different types of tumor xenografts including gliomas [13]–[17]. Although the mechanism of CBD anti-tumoral action has not been completely clarified yet, it has been proposed that CBD-induced apoptosis relies on an increased production of reactive oxygen species (ROS) [13], a mechanism that seems to operate also in glioma cells [14], [15]. To note, co-administration of THC and CBD – an option that is being therapeutically explored also for other applcations [5], [12]; has been shown to promote cancer cell death and reduce the growth of glioma xenografts [18], [19]. One of the factors limiting the efficacy of anticancer treatments is the difficulty to reach effective concentration of antineoplasic agents at the tumour site. For example, the poor water solubility of certain anticancer agents such as paclitaxel or camptothecin hinders their application and complicates direct parenteral administration. In the case of cannabinoids, several pharmaceutical preparations have been developed and approved for cannabinoid administration including oral capsules of THC (Marinol®, Unimed Pharmaceuticals Inc.) and of its synthetic analogue nabilone (Cesamet®, Meda Pharmaceuticasl) and an oro-mucosal spray of standardized cannabis extract (Sativex®, GW Pharmaceuticals). These formulations have been approved for several clinical applications [5], [20]. Specifically, cannabinoids are well-known to exert palliative effects in cancer patients [5], [20]. The best-established use is the inhibition of chemotherapy-induced nausea and vomiting [5], [6] (Marinol® and Cesamet®). Cannabinoids also inhibit pain, and Sativex® has been already approved in Canada and is currently subject of large-scale Phase III clinical trials for managing cancer-associated pain. However, from the perspective of the utilization of cannabinoid-based medicines as antineoplastic agents, one of the issues that needs to be clarified is whether systemic administration of cannabinoids allows reaching effective concentrations of these highly lipid soluble agents [21] at the tumor site without enhancing undesired side affects [5], [6]. Local administration of polymeric implants for interstitial sustained release of anti-neoplasic agents allows enhancing the concentration of anticancer active substances in the proximity of the tumour [22]–[26] and could be an alternative strategy to systemic delivery at least for certain types of cancer. The aim of the present study was therefore to evaluate the antitumor efficacy of biodegradable polymeric microparticles allowing the controlled release of the phytocannabinoids THC and CBD. Our findings show that administration of cannabinoid-loaded microparticles reduces the growth of glioma xenografts supporting that this method of administration could be exploited for the design of cannabinoid-based anticancer treatments.

Materials and Methods Ethics statement animal work This study was carried out in strict accordance with the Spanish regulation for the care and use of laboratory animals. The protocol was approved by the committee on animal experimentation of Complutense University (Permits Number: CEA-1334; CEA-67/2012; CEA-75/2012). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Materials Δ9-tetrahidrocannabinol (THC) and cannabidiol (CBD) were from THC Pharm GmbH (Frankfurt, Germany), poly-ε-caprolactone (PCL) (Mw: 42,500), polyvinyl alcohol (PVA, MW = 30,000–70,000) and Sigmacote® were from Sigma-Aldrich (St. Louis, MO, USA). Methylene chloride (DCM) (HPLC grade) and dimethylsulfoxide (DMSO) were from Panreac (Barcelona, Spain). All chemicals and reagents were used as received. In order to avoid cannabinoid binding to labware, materials were pre-treated with Sigmacote®. Cannabinoid solution For in vivo administration to mice, cannabinoid solutions were prepared at 1% (v/v) DMSO in 100 µL of PBS supplemented with 5 mg/mL of bovine serum albumin. No significant influence of the vehicle was observed on any of the variables determined in this study. Microparticles preparation Biodegradable polymeric microparticles (MPs) were prepared by the oil-in-water emulsion solvent evaporation technique. Briefly, 50 mg of drug and 500 mg of polymer were dissolved in 5 mL of methylene chloride. Subsequently, the organic solution was poured onto 250 mL of a 0.5% PVA aqueous solution under stirring at 3000 rpm for 6 min. The resulting O/W emulsion was then stirred for 3 h to evaporate the organic solvent. Finally, the resulting MPs were washed with distilled water, filtrated (0.45 µm membrane filters) and freeze-dried. Vitamin E acetate (5%) was added to the organic solution when preparing THC-loaded MPs in order to avoid THC oxidation. Blank MPs were prepared using the same procedure but without adding cannabinoids. Microparticles morphology and size distribution Scanning electron microscopy (JSM 6400, Tokyo, Japan) was used to evaluate the shape and the surface morphology of the blank, CBD- or THC-loaded PCL MPs. Particle size distribution was analyzed using a Microtrac® SRA 150 Particle Size Analyzer (Leeds & Northrup Instruments, Ireland). Samples were prepared by resuspending 5 mg of MPs in distilled deionized water. The results correspond to microsphere diameter determined by percentage volume distribution. Analytical method High performance liquid chromatography was used to quantify the cannabinoid loaded in the microspheres and the amount of cannabinoid released at different time-points. HP1050 series instrument (Hewlett Packard) using a Mediterranea®Sea C18 column (150*4.6 mm, 5 μm) (Teknokroma, Barcelona, Spain) equipped with a UV detector set at 228 nm was used. The isocratic elution was prepared with methanol:acetonitrile: water (52∶30∶18) adjusted to pH 4.5 with acetic acid as mobile phase at a flow rate of 1.8 mL/min. Drug content and encapsulation efficiency Briefly, 10 mg of MPs were dissolved with 1 mL of methylene chloride. Subsequently, mobile phase was added to the solution in order to precipitate the polymer and extract the cannabinoid. Samples were filtered prior to analysis by HPLC. The encapsulation efficiency was obtained by calculating the percent of total cannabinoid loaded in the microspheres, divided by the initial cannabinoid added during the preparation of the microspheres. In vitro release of CBD and THC from 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 (n = 3). 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. The percentage of drug released was presented as a cumulative curve. Cell culture U87MG human glioma cells were obtained from ATCC. Cells were cultured in DMEM containing 10% FBS and maintained at 37°C in a humidified atmosphere with 5% CO 2 . Nude Mouse Xenograft Model of Human Glioma Tumors were generated in athymic nude mice (Harlan Laboratories). The animals were injected subcutaneously on the right flank with 5*106 U87 human glioma cells in 0.1 ml of PBS supplemented with 0.1% glucose. Tumors were measured using an external caliper, every day of treatment, and volume was calculated by the formula: 4π/3 *(length/2) *(width/2)2. When tumors reached a volume of 200 mm3, mice were randomly distributed into 8 experimental groups and treated daily with vehicle of the corresponding cannabinoid in solution or with blank or cannabinoid-loaded MPs at a dose of 75 mg MPs every 5 days. Mice were monitored daily for health status and for tumor volumes. After 22 days of treatment mice were sacrified and tumors were removed, measured and weighted. The remaining microspheres were removed, freeze-dried and analyzed for drug content. Immunofluorescence from tumor samples Samples from tumors xenografts were dissected and frozen. Sections (10 µm) were permeabilized, blocked to avoid nonspecific binding with 10% goat antiserum and 0.25% TritonX-100 in PBS for 90 min, and subsequently incubated with rabbit polyclonal anti-KI67 (1∶300; Neomarkers; 4°C, o/n), or mouse monoclonal anti-CD31 (1∶200; Cymbus Biotechnology LTD; 4°C, o/n) antibodies. Next, sections were washed and further incubated with the corresponding Alexa-594-conjugated secondary antibodies (Invitrogen; 90 min, room temperature). Nuclei were stained with Hoechst 33342 (Invitrogen; 10 min, room temperature) and mounted with Mowiol (Merck, Darmstadt, Germany). Fluorescence images were acquired using an Axiovert 135 microscope(Carl Zeiss, Thornwood, NY, USA). Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) was done using the in situ cell death detection kit (Roche). Statistics Statistical analysis for tumor volume data were performed by ANOVA with a post hoc analysis by the Student-Neuman-Keuls test.

Discussion One of the strategies that are currently under investigation to improve the efficacy of anticancer treatments is the utilization of drug carrier systems facilitating the local delivery of antineoplasic agents. Among these drug carrier systems, polymeric MPs have drawn much attention owing to their ability to control drug release, improve the therapeutic effect, prolong the biological activity, and decrease the administration frequency of several anti-neoplasic agents [27]–[29]. THC and CBD – two phytocannabinoids with potent anti-cancer activity – can be efficiently encapsulated into biodegradable PCL microspheres [30]. Our data show that PCL microspheres permit continuous release of these drugs and that its administration every 5 days to tumour-bearing mice reduces the growth of glioma xenografts with similar efficacy than a daily local administration of these cannabinoids in solution. Furthermore, results show that using this frequency of administration a significant fraction of the two cannabinoids is still present in the MPs at the end of the treatment. These observations suggest that effective concentrations of THC and CBD could be reached at the tumour site using a higher dosing interval. Of note, different observations suggest that the doses of THC required to produce its cell death-promoting effect in cancer cells (IC 50 of around 1.5 to 6 μM in vitro depending on the type of cancer cell and the conditions of cell culture) are higher than the ones required for other actions of this agent or other CB 1 receptor agonists in non-transformed cells [6]. Thus, reaching effective concentrations of THC at the tumour site using a systemic route of administration may require increasing the doses of THC administered to humans, which would enhance the risk of undergoing the undesired side effects of THC derived from its binding to CB1 receptors present in different brain regions. Local administration of cannabinoid-loaded MPs can help to circumvent this problem as their administration in the proximity of the tumour would ensure that effective concentrations of THC are reached at the therapeutically relevant site without enhancing acutely the levels of this agent in the brain regions responsible for its pyschoactivity. In addition, in this study we also found that the anticancer efficacy of the individual treatments with THC-loaded MP (containing approximately 6.15 mg of THC per administration) or CBD-loaded MP (containing approximately 6.7 mg of CBD per administration) is similar to that produced by co-administration of a mixture (1∶1 w:w) of THC- and CBD-loaded MPs (containing approximately 3.075 mg of THC and 3.75 mg of CBD per administration). These results are in line with previous observations by our laboratory [18], and suggest that rather than producing a synergistic effect, the combined administration of sub-maximal doses of THC and CBD could help to reduce the doses of these compounds required to produce their inhibitory effects on tumour growth. Cannabinoids have been shown to produce a potent anticancer action in different types of tumour xenografts including some of the ones that exhibit a higher resistance to standard chemotherapies such as gliomas [8]–[10], pancreatic adenocarcinomas [31] and hepatocellular carcinomas [32], three tumour types that are susceptible of being treated with drug-loaded MPs [33]–[41]. This anticancer action of cannabinois is based on the ability of these compounds to enhance apoptosis, inhibit proliferation of cancer cells and inhibit tumour angiogenesis. Data presented here confirm that these mechanisms of action are activated in glioma xenografts upon administration of MPs loaded with THC, CBD or the combination of the two types of MPs. Although additional research should clarify whether a similar effect can be produced in other types of tumour xenografts, and whether MPs loaded with THC, CBD or its combination are equally efficacious in different tumour types and sub-types, these observations strongly support that microencapsulation could be a promising strategy to optimize the utilization of cannabinoids as anticancer agents. Of interest, we have recently found that the combined administration of THC or THC + CBD [18] (but not CBD, S Torres, M Lorente and G Velasco unpublished observations) with temozolomide synergistically reduces the growth of glioma xenografts. The findings presented here now provide a rational for the design of novel anticancer strategies based on the use of cannabinoid-loaded MPs in combinational therapies.

Conclusions Data presented in this manuscript show for the first time that in vivo administration of microencapsulated cannabinoids efficiently reduces tumor growth thus providing a proof of concept for the utilization of this formulation in cannabinoid-based anti-cancer therapies.

Acknowledgments We thank the “Luis Bru” UCM Microscopy Research Support Centre for valuable technical and professional assistance.

Author Contributions Conceived and designed the experiments: GV AITS ML DH. Performed the experiments: DH ML MEG-A ST EG-T MRA JM. Analyzed the data: DH ML MEG-A GV. Contributed reagents/materials/analysis tools: MEG-A MRA JM AITS. Wrote the paper: GV DH ML.