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

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DOI: 10.1371/journal.pone.0054795 · Source: PubMed
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.
Local Delivery of Cannabinoid-Loaded Microparticles
Inhibits Tumor Growth in a Murine Xenograft Model of
Glioblastoma Multiforme
Dolores Herna
´
nPe
´
rez de la Ossa
1.
, Mar Lorente
2,3.
, Maria Esther Gil-Alegre
1,5
, Sofı
´
a Torres
2
,
Elena Garcı
´
a-Taboada
2
,Marı
´
a del Rosario Aberturas
4
, Jesu
´
s Molpeceres
4
, Guillermo Velasco
2,3
*
.
,
Ana Isabel Torres-Sua
´
rez
1,5.
1 Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, Complutense University, Madrid, Spain, 2 Department of Biochemistry and Molecular
Biology I, School of Biology, Complutense University, Madrid, Spain, 3 Instituto de Investigacio
´
n Sanitaria del Hospital Clı
´
nico San Carlos, Madrid, Spain, 4 Department of
Pharmacy and Pharmaceutical Technology, School of Pharmacy, Alcala
´
University, Madrid, Spain, 5 Instituto de Farmacia Industrial, Complutense University, Madrid, Spain
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, D
9
-Tetrahydrocan-
nabinol (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-e-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: Herna
´
nPe
´
rez de la Ossa D, Lorente M, Gil-Alegre ME, Torres S, Garcı
´
a-Taboada E, 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. doi: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: ß 2013 Herna
´
nPe
´
rez 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 Auto
´
noma 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.
* E-mail: gvd@bbm1.ucm.es
. These authors contributed equally to this work.
Introduction
D
9
-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
PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e54795
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 pharmaceu-
tical preparations have been developed and approved for
cannabinoid administration including oral capsules of THC
(MarinolH, Unimed Pharmaceuticals Inc.) and of its synthetic
analogue nabilone (CesametH, Meda Pharmaceuticasl) and an oro-
mucosal spray of standardized cannabis extract (SativexH,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] (MarinolH and CesametH). Cannabi-
noids also inhibit pain, and SativexH 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
D
9
-tetrahidrocannabinol (THC) and cannabidiol (CBD) were
from THC Pharm GmbH (Frankfurt, Germany), poly-e-capro-
lactone (PCL) (Mw: 42,500), polyvinyl alcohol (PVA,
MW = 30,000–70,000) and SigmacoteH 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 SigmacoteH.
Cannabinoid solution
For in vivo administration to mice, cannabinoid solutions were
prepared at 1% (v/v) DMSO in 100
mL 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
mm
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 MicrotracH 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 MediterraneaHSea C18
column (150*4.6 mm, 5 mm) (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-TweenH80 0.1% (v/v) and maintained in a shaking
incubator at 37uC (n = 3). At predetermined time intervals
supernatants were withdrawn and media was replaced. The
concentration of CBD or THC in the release medium was
Cannabinoid Microparticles Inhibit Tumor Growth
PLOS ONE | www.plosone.org 2 January 2013 | Volume 8 | Issue 1 | e54795
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-TweenH80 0.1% (v/v) and maintained
in a shaking incubator at 37uC. 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 6 s.d
(n = 3).
doi:10.1371/journal.pone.0054795.g001
Cannabinoid Microparticles Inhibit Tumor Growth
PLOS ONE | www.plosone.org 3 January 2013 | Volume 8 | Issue 1 | e54795
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
37uC 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*10
6
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: 4p/3 *(length/2) *(width/2)
2
. When
tumors reached a volume of 200 mm
3
, 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
mm) 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; 4uC, o/n), or mouse monoclonal
anti-CD31 (1:200; Cymbus Biotechnology LTD; 4uC, o/n)
antibodies. Next, sections were washed and further incubated
with the corresponding Alexa-594-conjugated secondary antibod-
ies (Invitrogen; 90 min, room temperature). Nuclei were stained
with Hoechst 33342 (Invitrogen; 10 min, room temperature) and
mounted with Mowiol (Merck, Darmstadt, Germany). Fluores-
cence 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.
Results
Preparation and characterization of cannabinoid-loaded
microparticles
In order to evaluate the potential anticancer efficacy of
microencapsulated cannabinoids, we prepared biodegradable
polymeric poly-e-caprolactone (PCL) microparticles (MPs) con-
taining THC or CBD by using the oil-in-water emulsion solvent
evaporation technique. Microparticles prepared by this procedure
were spherical, showed a smooth surface (Figure 1A) and had an
average size of 50 mm (Figure 1B). The encapsulation efficiencies
of CBD and THC into PCL MPs were 99.0965.14% and
84.55613.6%, respectively. The release profile for the two types of
MPs was characterized by a continuous release of CBD or THC
for 13 days including a five-day initial burst release-phase during
which 64% and 79% respectively of the total CBD or THC
present in the MPs was released (Figure 1C and Table 1).
Evaluation of the anticancer activity of cannabinoid-
loaded microparticles
To investigate the potential anticancer activity of the above-
described cannabinoid-loaded MPs, we generated tumor xeno-
grafts by injecting subcutaneously U87MG cells (a well-established
cellular model of glioma, that has been widely used to investigate
the anticancer action of cannabinoids in this type of tumors [8,10])
into the right flank of immnunodeficient mice. Once the tumours
reached a 200–250 mm
3
volume, animals were treated every 5
days with blank MPs (prepared in the absence of cannabinoids) or
with microparticles loaded with THC or CBD. In addition, as the
combined administration of submaximal doses of THC and CBD
(1:1) has been shown to reduce the growth of glioma xenografts
[18], animal were also treated with a mixture of THC and CBD
MPs.(1:1 w:w) In the same experiment, another set of tumours was
treated daily with a single peritumoral injection of a solution
containing vehicle, THC, CBD or a mixture of THC and CBD
(1:1). As shown in Figure 2 administration every five days of
cannabinoid-loaded microparticles (THC, CBD or THC + CBD)
reduced tumor growth at the same extent than daily treatment
with THC, CBD or THC + CBD in solution (Figure 2A–D). A
similar effect was observed when the weight of the tumors on the
last day of the treatment was analyzed (Figure 3A and 3B).
To note, animals treated with cannabinoids in solution and with
cannabinoid-loaded MPs received approximately the same
amount of cannabinoids along the treatment (Table 2). Thus, we
found that 59 % of the initial amount of CBD present in CBD-
loaded MPs (5.360.22 mg CBD/100 mg of MPs of the initial
8.9360.13 mg CBD/100 mg MPs) and 58% of THC present in
THC-loaded MPs (4.7360.13 mg THC/100 mg MPs of the
initial 8.2160.07 mg THC/100 mg MPs) were still present in the
MPs remaining at the site of injection at the end of the experiment
(Table 2). Taken together, these observations support that
administration of cannabinoid-loaded MPs every five days reduces
tumor growth with the same efficiency than a daily injection of
Table 1. In vitro analysis of the amount of CBD or THC
released from cannabinoid-loaded microparticles.
Time (days) mg CBD mg THC
1 1.55 2.99
2 2.27 3.39
3 2.94 4.24
5 4.28 4.87
7 5.51 5.28
10 6.34 5.78
13 6.66 6.00
16 6.68 6.11
20 6.70 6.16
Microspheres were incubated in PBS pH 7.4-TweenH80 0.1% (v/v) and
maintained in a shaking incubator at 37uC. 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. Results correspond
to the cumulative amounts of cannabinoid released in vitro from 75 mg MP.
doi:10.1371/journal.pone.0054795.t001
Cannabinoid Microparticles Inhibit Tumor Growth
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cannabinoids in solution and suggest that effective concentrations
of cannabinoids could be reached at the tumour site using a lower
frequency of MPs administration.
Treatment with cannabinoid-loaded microparticles
activates apoptosis and inhibits tumor angiogensis
The mechanism of cannabinoid anticancer action relies on the
ability of these compounds to promote cancer cell death via
stimulation of apoptosis and inhibit cancer cell proliferation
and tumour angiogenesis [6]. Therefore, we analyzed whether
these mechanisms were activated in the tumour xenografts that
had been treated with cannabinoid-loaded MPs. Unlike tumors
that have been treated with blank MPs, treatment of U87-
derived xenografts with THC- or CBD-loaded MPs or with a
mixture of THC and CBD MPs reduced cancer cell proliferation
(as determined by Ki67 immunostaing, Figure 4A), enhanced
apoptosis (as determined by TUNEL; Figure 4B) and decreased
tumour vascularization (as determined by immunostaining with
the endothelial cell marker CD31, Figure 4C). These observa-
tions confirm that cannabinoid microencapsulation does not
interfere with the mechanism by which these agents inhibit
tumor growth.
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 administra-
tion 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
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 6 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.
doi:10.1371/journal.pone.0054795.g002
Cannabinoid Microparticles Inhibit Tumor Growth
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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 mM 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 administra-
tion) 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 chemother-
apies such as gliomas [8–10], pancreatic adenocarcinomas [31]
and hepatocellular carcinomas [32], three tumour types that are
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).
doi:10.1371/journal.pone.0054795.g003
Table 2. Amount of THC or CBD administered to mice and released at the end of the treatment from cannabinoid-loaded
microparticles.
Cannabinoid-loaded MPs* Cannabinoid-loaded MPs* Cannabinoid-loaded MPs* Cannabinoids in solution
Total amount of cannabinoid
administered (mg per animal)
Total amount of cannabinoid
remaining on day 22 (mg per
animal)
Estimated amount of
cannabinoid released (mg per
animal)
Total amount of cannabinoid
administered (mg per animal)
THC 24.64 mg THC 14.19 mg THC 10.44 mg THC 10.5 mg THC
CBD 26.79 mg CBD 15.9 mg CBD 10.89 mg CBD 10.5 mg THC
*Animals received 75 mg of cannabinoid-loaded MPs every 5 days (corresponding to a total amount of 300 mg of microparticles per animal).
doi:10.1371/journal.pone.0054795.t002
Cannabinoid Microparticles Inhibit Tumor Growth
PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e54795
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
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 6 s.d. (A), the percentage of TUNEL-positive cells relative to the total number of nuclei in each section 6 s.d. (B) or
the CD31-stained area normalized to the total number of nuclei in each section (mean fold change 6 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.
doi:10.1371/journal.pone.0054795.g004
Cannabinoid Microparticles Inhibit Tumor Growth
PLOS ONE | www.plosone.org 7 January 2013 | Volume 8 | Issue 1 | e54795
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.
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    • "Microcapsules (1–250 µm diameter) may be prepared through a variety of techniques [46], carrying larger drug loads than nanoparticles, as well as allowing a better control over sustained release profiles [47]. Based on this fact, several anticancer agents have been encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microcapsules (Figure 1) [48][49][50]. PLGA is a suitable option for developing controlled-release delivery devices due to its biocompatibility, biodegradability and the gradual release of drugs over a long period of time [39,46]. Here we present a PLGA-microencapsulated nor-β-lapachone formulation prepared by the emulsification/solvent evaporation method, which reduces the drug liposolubility and enables subsequent in vivo studies. "
    [Show abstract] [Hide abstract] ABSTRACT: Prostate cancer is one of the most common malignant tumors in males and it has become a major worldwide public health problem. This study characterizes the encapsulation of Nor-β-lapachone (NβL) in poly(d,l-lactide-co-glycolide) (PLGA) microcapsules and evaluates the cytotoxicity of the resulting drug-loaded system against metastatic prostate cancer cells. The microcapsules presented appropriate morphological features and the presence of drug molecules in the microcapsules was confirmed by different methods. Spherical microcapsules with a size range of 1.03 ± 0.46 μm were produced with an encapsulation efficiency of approximately 19%. Classical molecular dynamics calculations provided an estimate of the typical adsorption energies of NβL on PLGA. Finally, the cytotoxic activity of NβL against PC3M human prostate cancer cells was demonstrated to be significantly enhanced when delivered by PLGA microcapsules in comparison with the free drug.
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    • "The exogenous cannabinoids THC and cannabidiol (CBD) reduced tumor growth in animal models [27,31,828384. THC acts as an agonist of GPR18, CB1 and CB2 whereas cannabidiol is an antagonist of GPR55, and an agonist of GPR18 and GPR119. "
    [Show abstract] [Hide abstract] ABSTRACT: Endocannabinoids including anandamide and 2-arachidonoylglycerol are involved in cancer pathophysiology in several ways, including tumor growth and progression, peritumoral inflammation, nausea and cancer pain. Recently we showed that the endocannabinoid profiles are deranged during cancer to an extent that this manifests in alterations of plasma endocannabinoids in cancer patients, which was mimicked by similar changes in rodent models of local and metastatic cancer. The present topical review summarizes the complexity of endocannabinoid signaling in the context of tumor growth and metastasis.
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    • "Cannabinol (CBD) and Δ 9 -Tetrahydrocannabinol (THC) are two phytocannabinoids that have been shown to reduce the growth of glioma xenographs in rats and mice [62]. When encapsulated in PCL microspheres, a 64 and 79% release of CBD and THC was achieved, respectively, after five days followed by a sustained release through day 13 [62]. 3.5. "
    [Show abstract] [Hide abstract] ABSTRACT: The grim prognosis for patients diagnosed with malignant gliomas necessitates the development of new therapeutic strategies for localized and sustained drug delivery to combat tumor drug resistance and regrowth. Here we introduce drug encapsulated aerosolized microspheres as a biodegradable, intelligent glioma therapy (DREAM BIG therapy). DREAM BIG therapy is envisioned to deliver three chemotherapeutics, temporally staged over one year, via a bioadhesive, biodegradable spray directly to the brain surgical site after tumor excision. In this proof-of-principle article exploring key components of the DREAM BIG therapy prototype, rhodamine B (RB) encapsulated poly(lactic-co-glycolic acid) and immunoglobulin G (IgG) encapsulated poly(lactic acid) microspheres were formulated and characterized. The encapsulation efficiency of RB and IgG and the release kinetics of the model drugs from the microspheres were elucidated in addition to the release kinetics of RB from poly(lactic-co-glycolic acid) microspheres formulated in a degradable poly(N-isopropylacrylamide) solution. The successful aerosolized application onto brain tissue ex-vivo demonstrated the conformal adhesion of the RB encapsulated poly(lactic-co-glycolic acid) microspheres to the convoluted brain surface mediated by the thermoresponsive carrier, poly(N-isopropylacrylamide). These preliminary results suggest the potential of the DREAM BIG therapy for future use with multiple chemotherapeutics and microsphere types to combat gliomas at a localized site. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
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