DRUG DELIVERY NANOTECHNOLOGY APPLIED TO METHOTREXATE
CONTROLLED RELEASE IN HUMAN OSTEOSARCOMA: IN VITRO TEST
Pretti, T. S.2,4, Souza, M. A.2, Santos, H. T.2, Nascimento, A. C. G. G.2, Santos, F. J.2, Fraga,
A. F.2, Jafelicci, M. J.3, Marques, R. F. C.1
1
Institute of Science and Technology, Federal University of Alfenas, Poços de Caldas (MG), Brazil
2
Procell Biologics, São Carlos (SP), Brazil
3
Institute of Chemistry, State University of São Paulo, Araraquara (SP), Brazil
4
Interunits Postgraduate Program in Bioengineering, University of São Paulo, São Carlos (SP), Brazil
E-mail: rodrigo@unifal-mg.edu.br
Abstract. Nowadays there is an increasing search for new efficiently treatments and with fewer side effects for
cancer therapy. For this, drug delivery system has the potential to overcome the limitations associated with the
systemic distribution of conventional chemotherapeutic agents, reducing the dosage and the adverse side effects
associated with the use of non-specific cytotoxic drugs to healthy tissues (SUN; LEE; ZHANG; 2008). The use of
biodegradable polymer as drug delivery system has lot of advantages: prolonging the blood circulation time of
drug, solubilization of hydrophobic drugs, reduction of adverse effects of anticancer drugs to healthy cells
(LEGRAND et al., 2007; LAVASANIFAR; SAMMUEL; KWON; 2002). So, the aim of this study was to test the
controlled release and cytotoxicity of the drug methotrexate entrapped into a biocompatible and biodegradable
amphiphilic copolymer, in human osteosarcoma cell. The amount of methotrexate released was measure
according to UV absorbance intensity at 303 nm at 20, 35, 37, 40 and 42 ºC. It was observed with the increase
of temperature the drug release also increased, being released about 44% at 42 ºC. The in vitro cytotoxicity test
was carried out using MTT assay on MG63 human osteosarcoma cells. According to the test, the drug delivery
system was efficient, as confirmed by the decreasing in cell viability of about 7.42% in 1 µg/mL and 91.07% in
10 µg/mL of the complex mPEG-PCL-NP-MTX. Thus, it could be concluded that the complex copolymeric,
magnetic nanoparticle and methotrexate was efficient as a device to drug entrapment and its release, being
enough to induce a large decreasing in cell viability of human osteosarcoma cell.
Keywords: Drug delivery, Methotrexate, Osteosarcoma.
1. INTRODUCTION
Magnetic nanoparticles (NPs), more particularly magnetite (Fe3O4), have been
extensively used for biomedical applications due to its high stability in biologic systems and
low toxicity, acting as cell targeting, cell separation, drug delivery, hyperthermia and
magnetic resonance (YOUNG et al, 2009; MARQUES et al, 2008). In drug controlled release,
magnetic nanoparticles are especially required due to allow an effective release mechanism of
the agent within the cell by its heating potential when in presence of alternated magnetic
fields. In order to improve its efficiency in drug delivery, NPs should be linked to chemistry
or biologics molecules which have strong affinity for specific cells and high efficiency to
internalize the nanoparticles (KOHLER et al, 2005). In cancer therapy, nanoparticles have the
potential to overcome the limitations associated with the systemic distribution of conventional
chemotherapeutic agents, decreasing dosage and side effects related with the use of nonspecific cytotoxic drugs by healthy tissues (SUN; LEE; ZHANG; 2008). However, to be used
in drug delivery devices and also to have an optimal potential application of nanoparticles in
biologics systems it is necessary stabilize them to avoid agglomeration.
For this porpose, amphiphilic block copolymers consisting of hydrophilic and
hydrophobic segment have attracted much attention because of their unique phase behavior in
aqueous media which increase their potential applications as drug delivery systems (ZHANG;
ZHUO, 2005). Polymeric thermo-sensitives nanoparticle micelles can be assembled from
amphiphilic copolymers segments, magnetic nanoparticles and drug. The hydrophobic
segment creates the inner core of the micelle, while the hydrophilic segment creates the outer
shell in an aqueous media (KWON; KATAOKA; 1995). Polymeric micelles can be used as a
drug delivery device by either physically entrapping the drug in the core (hydrophobic drugs
can be trapped inside the micelle by hydrophobic interactions), or by chemically conjugating
the drug to the hydrophobic block prior to micelle formation (LAVASANIFAR; SAMMUEL;
KWON; 2002). The advantages of polymeric micelles as drug delivery systems are: prolong
the blood circulation time of drug, passive tumor targeting, small particle size, solubilization
of hydrophobic drugs, reduction of the adverse effects of anticancer drugs and preservation of
bioactive agents within the micellar core for long durations, etc (LEGRANDet al., 2007; SEO
et al.; 2007; ZHANG; ZUO; 2005).
As a model drug for delivery in cancer therapy, Methotrexate (MTX) was chosen for
this study. MTX is a folate antimetabolite, which is widely used in the treatment of
malignancies including osteosarcoma, childhood acute lymphocytic leukemia, non-Hodgkin’s
lymphoma, Hodgkin’s disease, head and neck cancer, lung cancer, breast cancer, psoriasis,
choriocarcinoma and related trophoblastic tumors (SEO et al., 2009). Folates act as onecarbon carriers in a set of interconversions of metabolic cycles of purine, thymidine,
methionine, histidine and serine biosynthesis. Such activity makes folates essential for normal
growth and maturation. Thus, the use of fotates analogs, e.g. antifolates, can inhibit cell
growth by disruption of folate metabolism and can be established as targeted chemotherapy
(BRZEZINSKA; WINSKA; BALINSKA, 2000). This disruption of folic acid metabolism by
MTX can result in exhaustion of folate coenzymes and in a cessation of the biosynthesis of
deoxythymidylate monophosphate and purines. In addition, MTX can interfere with various
other anabolic and catabolic processes (WIDEMANN; ADAMSON, 2006).
MTX incorporated into core–shell type copolymeric nanoparticles of mPEG-PCL
copolymer on NPs surfaces had the potential to provide a novel type of MTX-delivery carrier.
Such novel types of polymeric drug delivery systems would diminish the adverse side-effects
of MTX and increase delivery efficiency to tumors (SEO et al., 2009). Therefore, as there are
high quantities of folate receptors into tumor cells comparing to healthy cells, the aim of this
study was to analyze the cytotoxicity potential presented by this system, through in vitro tests
with osteosarcoma human cells.
2. MATERIALS AND MHETODS
2.1. MATERIALS
Poly(ethyleneglycol) mono methylether-ε-Caprolactone (mPEG-PCL) copolymer
synthetized in laboratory, Methotrexate, Magnetite, Osteosarcoma MG63, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Minimum Essential Medium Eagle.
2.2. METHODS
SYNTHESIS OF THE SAMPLE mPEG-PCL-MTX.
All procedure was carried in soft illumination and all the recipients were covered with
aluminum sheet due to MTX degradation in light. A solution of 0.2 mg/ml of mPEG-PCL
copolymer was heated until 45 ° C. To this heated solution were added 30 mg/ml of an initial
methotrexate solution 90 mg/mL in 0.1 N NaOH, and was left under magnetic stirring for 2
hours at 45 °C. After this period, the resulting solution was cooled to 0 ° C and then was
dialyzed in water for 24 h under stirring, with water being exchanged every 3 h. During the
initial 12 h of dialysis, at each change of water an aliquot was taken to evaluate how much of
drug remained in copolymer micelles, through analysis by UV absorbance in a
spectrophotometer (Cary 5000, Varian, Australia) at 303 nm. Values were calculated
according to the standard curve of methotrexate.
IN VITRO DRUG RELEASE.
After dialysis, the dispersion containing mPEG-PCL-MTX was gradually heated at 20,
35, 37, 40 and 42 ºC. Aliquots of 3 ml were withdrawn from the supernatant at each
temperature described above. The amount of MTX released from micelles was determined
according to the UV absorbance intensity at 303 nm. The values were calculated according to
standard curve of the Methotrexate.
CITOTOXICITY TEST.
The in vitro complex copolymer mPEG-PCL-MTX-NP cytotoxicity was tested using
MTT assay on MG63 human osteosarcoma cells. Cells were seeded in 96 well plates in
Minimum Essential Medium Eagle, Alpha Modification (α-MEM, Invitrogen) without serum,
supplemented by 100 U/mL penicillin/ streptomycin (Invitrogen), at 37 °C in humidified
atmosphere containing 5% CO2. After overnight attachment, the cells were exposed to the test
substances (1 µg/mL and 10 µg/mL) for additional 24 h and methyl tetrazolium (MTT) assay
was performed as previously described (MOSMANN, 1983). Briefly, the extracts were
replaced by 90 µL of α-MEM and 10 µL of MTT solution (5 mg/mL), remaining in the
incubator for 4 h. Thereafter, the media was replaced by acidified isopropanol (0.04 N HCl) to
dissolve the formazan crystals. The absorbance was measured by microplate reader (Tp
Reader; Thermoplate, Nanshan District, Shenzhen, China) at 570 nm.
3. RESULTS AND DISCUSSION
3.1. METHOTREXATE ENTRAPMENT AND RELEASE
The potential application of the controlled drug release was evaluated by the
investigation of the drug release profile. Water analysis of dialyze by UV absorbance in a
spectrophotometer at 303 nm, indicated that the system allowed 29.57 µg/mL of MTX
entrapment, which correspond to 98.6% of the initial drug concentration.
The MTX release profiles from the copolymeric micelles were performed in five
different temperatures (20, 35, 37, 40 and 42 °C). As illustrated in the release curve (Fig. 1), it
was observed that with increasing temperature the amount of drug released also increased.
When the temperature increases up to 42 °C, it was observed a major rise of the drug released
reaching 44% of 98. 6% entrapped into the copolymers.
% recovered (µg/mL)
44,0
43,5
43,0
42,5
42,0
41,5
20
25
30
35
40
45
o
Temperature ( C)
Figure 1: Release curve with percentage of MTX released of the mPEG-PCL during heating
at 20, 35, 37, 40 and 42 ºC at 303 nm.
The heating and cooling procedures of mPEG-PCL solution allowed methotrexate
fixation into the copolymer, inducing the interactions between the copolymer and the drug.
Also, during dialysis, self-assembly of copolymers took place and entrapped the drug into the
micelle, at same time, the membrane of dialysis allowed the removal of unloaded drug and/or
copolymer. Hydrophobic interactions may be responsible for entrapment of the drug in the
copolymer as hydrophobic drug like methotrexate can be intermolecularly bonded to
hydrophobic PCL copolymer chains (CHEN et al., 2008). Zhang et al. (2005) indicated that
hydrophobic core composed of PCL segment has good drug permeability. This result is in
accord to results reported by Wei et al. (2009), which shows high percentage entrapment of
drug in the NP-Copolymers during the dialysis procedure.
From release curve it can be observed that with the rising of temperature the
concentration of the drug increased, indicating that the liberation occurred. Chen et al. (2008)
also showed the same performance with the increase of temperature. And they explain that the
drug release behavior could be due some factor of structural change of the micelles which
resulted in micelle deformation (CHEN et al., 2008).
3.2. CYTOTOXICITY ON HUMAN OSTEOSARCOMA
Viable cells have the ability to reduce MTT from a yellow water-soluble dye to a dark
blue insoluble formazan product (RASTOGI et al.; 2011). Formazan crystals were dissolved
in DMSO and quantified by measuring the absorbance of the solution at 570 nm, and the
resultant value was related to the number of living cells. Figure 2 shows a dose-dependent
reduction in MTT absorbance in cells treated with 1 and 10 µg/ml of complex for 24 h. Cells
treated with 1 and 10 µg/mL of mPEG-PCL-MTX-NP showed 92.58 % and 8.93 % of cells
viability, respectively. The results of test realized just with magnetic nanoparticle (NP) in
both concentrations (1 and 10 µg/ml), showed that there was no cytotoxicity to osteosarcoma
human, proving the biocompatibility of the magnetite to biologics systems. The MTT results
also indicated the efficiency of the drug delivery of mPEG-PCL-MTX-NP devices.
(a)
100
(b)
(c)
(d)
% Cells Viability
80
60
40
20
(e)
0
X
Products Concentrations
Figure2: MTT assay showing viability of MG63 human osteosarcoma after 24 h upon various
treatments: a) Control group, b) magnetic nanoparticle 1 µg/mL, c) magnetic nanoparticle 10
µg/mL, d) mPEG-PCL-MTX-NP 1 µg/mL and e) mPEG-PCL-MTX-NP 10 µg/mL.
The use of drug delivery system is a great opportunity to increase the action of
chemotherapy agent and at the same time, reducing its side effects on healthy cells. However,
identification of specific targeting agents or drugs that can be effectively released from the
system of drug delivery inside target cells remains a challenge (KOHLER et al.; 2005). In this
work, the choice of Methotrexate as chemotherapy agent was based in its similar properties to
folic acid, and because the osteosarcoma and other tumor cells over expressed folate receptors
on the cell membranes.
There were some studies that related the cell viability to the rate of methotrexate within
the cell. In this case, the authors developed MTX conjugates consisting of poly(ethylene
glycol) which retains a higher concentration of MTX within the cell, and these conjugates
have been shown to increase cellular cytotoxicity and increase cellular mortality
(RIEBESSEL et al.; 2002).
The results indicated that release of drug from mPEG-PCL-MTX-NP was efficient as
drug delivery device. It can be also observed a loss viability dose-dependent of MTX. When
10µg/mL of mPEG-PCL-MTX-NP was used there was a decrease in cell number. Dosedependent behaviour of MTX have been also shown by Seo et al. (2009), whose study have
evaluated the efficiency of MTX incorporated in polymeric nanoparticles of methoxy
poly(ethylene glycol)-grafted chitosan for treatment of melanoma cells. They observed with
increasing concentration of 0.0001 to 10 µg/mL there was a significant reduction in the
viability of these cells, being 10 µg/mL the most efficient concentration.
4. CONCLUSIONS
The drug delivery system of an amphiphilic block copolymer consisting of poly
(ethylenglycol) methyl ether – poly (ε-caprolactone), a hydrophobic drug methotrexate and
magnetic nanoparticles (magnetite) present a potential for treatment of human osteosarcoma.
The copolymer efficiently entrapped the drug onto the inner core by hydrophobics
interactions, corresponding to 98.6% of initial concentration of drug, and about 44% were
released when temperature increased up to 42 ºC. This amount released was enough to cause
cellular death of about 91% of human osteosarcoma cells when concentration of the drug was
10 µg/mL, proving the efficiency of drug release system.
ACKNOWLEDGMENTS
This work was financially supported by the Conselho Nacional de Desenvolvimento
Científico e Tecnológico and Procell Biologics.
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