- Email: [email protected]
Synthesis and characterization of micelles as carriers of non-steroidal anti-inflammatory drugs (NSAID) for application in breast cancer therapy
Accepted Manuscript Title: Synthesis and Characterization of Micelles as carriers of Non-steroidal anti-inflammatory drugs (NSAID) for application in breast cancer therapy Author: Jo˜ao G. Marques V´ıtor M. Gaspar Elisabete Costa Catarina M. Paquete Il´ıdio J. Correia PII: DOI: Reference:
S0927-7765(13)00605-X http://dx.doi.org/doi:10.1016/j.colsurfb.2013.09.037 COLSUB 6038
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
31-5-2013 10-9-2013 18-9-2013
Please cite this article as: J.G. Marques, V.M. Gaspar, E. Costa, C.M. Paquete, I.J. Correia, Synthesis and Characterization of Micelles as carriers of Non-steroidal antiinflammatory drugs (NSAID) for application in breast cancer therapy, Colloids and Surfaces B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.09.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Ac
Page 1 of 26
Highlights
Chitosan amphiphilic derivatives self-assembly into nanosized micellar carriers
•
Micelles are capable of encapsulating non-steroidal anti-inflammatory drugs (NSAIDs)
•
Ibuprofen delivery into breast cancer cells shows anti-proliferative potential
•
Use of cost-effective NSAID-micelles can be a promising approach for cancer therapy
Ac ce pt e
d
M
an
us
cr
ip t
•
1
Page 2 of 26
Synthesis and Characterization of Micelles as carriers of Non-steroidal anti-inflammatory drugs (NSAID) for application in breast cancer therapy
ip t
João G. Marques1, Vítor M. Gaspar1, Elisabete Costa1, Catarina M. Paquete2 and Ilídio J. Correia1* 1
cr
CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6200-506, Covilhã, Portugal
2
us
ITQB-UNL - Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa
an
Av. da Republica, Estação Agronómica Nacional, 2780-157 Oeiras, Portugal
Ac ce pt e
d
M
*Corresponding author: Professor Dr. Ilídio Correia Avenida Infante D. Henrique, 6200-506, Covilhã, Portugal E-mail: [email protected]
2
Page 3 of 26
Abstract Non-steroidal anti-inflammatory drugs (NSAIDS) are emerging as a particularly valuable class of drugs due to their recently reported anti-tumoral activity in colorectal cancer. However, despite this tremendous potential, their bioavailability at the tumor
ip t
microenvironment remains rather limited. To overcome this issue, in this work we synthesized biocompatible micellar nanocarriers composed of amphiphilic chitosan to deliver ibuprofen into breast cancer cells and evaluate its anti-tumor activity, while avoiding
cr
side-effects. Our results reveal that the formulations produced herein self-assembly into spherical micelles with suitable sizes for tumor accumulation (108 – 252 nm). Furthermore,
us
by using a vortex-sonication method, ibuprofen was successfully encapsulated with high efficiency. Cell uptake studies show that ibuprofen-loaded micelles are readily internalized
an
by tumor cells and deliver their cargo in the intracellular compartment as demonstrated by confocal microscopy images. This fact led to a remarkable reduction in cancer cell viability (< 13%), at a relatively low drug dosage, illustrating the anti-tumoral activity of ibuprofen
M
when delivered to breast cancer cells. These findings demonstrate the promising potential of chitosan micelles as carriers of cost-effective NSAIDS for application in breast cancer
Ac ce pt e
d
therapy.
Keywords
Chitosan; Micelles; NSAIDs; ibuprofen; Anti-proliferative activity; Breast Cancer.
3
Page 4 of 26
1. Introduction Nowadays the continuous worldwide increase of cancer-related fatalities compels both the scientific and medical community towards the development of new anti-cancer
ip t
therapeutics that may improve patient survival rates even at late stages of cancer development [1]. Interestingly, the present advances in antineoplastic treatments are ever more focused not only on eliminating the primary tumor and niches of malignant cells, but
cr
also on reducing systemic cytotoxicity [2, 3].
Recently, nanotechnology has revolutionized the development of proficient delivery
us
systems that improve the pharmacodynamics and pharmacokinetics of chemotherapeutic drugs. However, the existing cutting-edge therapeutics still rely on highly toxic and
an
expensive active pharmaceutical ingredients (API) restricting their widespread application [4]. In this context, NSAIDS commonly associated with cancer prevention, have also been recently reported to be involved in cancer ablation [5-7]. Particularly, in the past couple of
M
years, NSAIDS such as aspirin [7], celecoxib [8], and ibuprofen have shown to have relevant anti-cancer activity [6]. Indeed, as reported by Baek and coworkers, 2002, ibuprofen has demonstrated the capacity to inhibit malignant cell proliferation through the
d
pro-apoptotic NSAID activated gene (NAG-1) [9]. Nevertheless, alike other drugs, it is
Ac ce pt e
crucial to reduce NSAIDS gut toxicity [10], first pass metabolism, and also increase their bioavailability on the target tumor tissues [11]. To overcome these issues, different classes of delivery vehicles that include polymeric micelles [12, 13], liposomes [14], silica nanoparticles [15], or cell penetrating peptides [16] are currently under development to transport numerous anti-tumoral drugs. Particularly, polymeric micelles comprised by hydrophobic and hydrophilic moieties are of vast interest due to their self-assembly characteristics into nanosized micellar structures, and exceptional drug loading capacity [17, 18]. Such property depends on the size and rearrangement of the hydrophobic moieties that eventually form an inner particle core which acts as a highly efficient reservoir for hydrophobic drugs like NSAIDS [19]. Thermodynamic stability is an additional advantage of hydrophobically modified polymers that comprise micellar nanodevices [20, 21]. These characteristics are crucial for drug delivery since micelles must assure the protection of their payload until being localized inside the target cells [22]. Nanocarriers comprised of hydrophobically functionalized chitosan are one of the most promising drug delivery systems for application in cancer therapy as recently demonstrated by Pan et al.,
4
Page 5 of 26
2013, with the delivery of the anti-tumoral drug paclitaxel [23]. Chitosan exclusive biocompatibility, biodegradability and cationic charge render it a unique biomaterial for nanoparticle synthesis [24]. In addition to these intrinsic features, the versatile structure of chitosan unlocks the possibility to manipulate its configuration by chemical grafting. To promote the synthesis of micelle nanocarriers different hydrophobic moieties including
ip t
deoxycholic acid (DOCA) [25, 26], oleoyl fatty acids [27] or 5β-cholanic acid [28], have been grafted onto chitosan backbone. DOCA, an amphiphilic moiety a constituent of bile
cr
acid, is particularly advantageous due to its high biocompatibility and micelle forming capacity in aqueous environments [29, 30]. Additional biomolecules, including amino
us
acids, have also been inserted in chitosan as our group recently described [31]. Grafting biocompatible amino acids improves chitosan solubility and facilitates nanoparticle cellular uptake [32]. Amino acid-mediated self-assembly can also be achieved when hydrophobic
an
moieties are included in chitosan, leading to the formation of an amphiphilic polymer capable of encapsulating hydrophobic drugs [32]. Leucine (Leu) addition is particularly valuable since chitosan-Leu nanoparticles present small sizes and superior cellular uptake
M
in comparison to other hydrophobic amino acids such as Valine [33]. From this standpoint, in this work we optimized the synthesis of nanosized micelles formulated with different amphiphilic polymers, explored NSAIDS drug loading capacity
d
and also the anti-tumoral effectiveness of different ibuprofen-loaded carriers in breast
Ac ce pt e
cancer cells. The inclusion of DOCA or Leu in chitosan backbone led to the formation of self-assembled micelles with considerable loading capacity and high anti-tumoral activity, particularly evident for the CS-DOCA formulations.
5
Page 6 of 26
2. Materials and Methods 2.1. Materials Chitosan hydrochloride (CS) (Protasan UP CL 113, MW ≈ 110 kDa) was purchased from (Novamatrix, Sandvika, Norway). L-Leucine was purchased from Affimetrix Inc. (Santa
ip t
Clara, USA). Acetic acid (CH3COOH) was obtained from Panreac (Barcelona, Spain). 3α, 12α-dihydroxy-5β-cholanate (Deoxycholic acid (DOCA)), Dulbecco’s Modified Eagle’s (DMEM),
N-Hydroxysuccinimide
(NHS),
N-(3-Dimethylaminopropyl)-N´-
cr
Medium
ethylcarbodiimide hydrochloride (EDC), Resazurin, Rhodamine B Isothiocyanate (RITC),
us
and cellulose dialysis membrane were obtained from Sigma–Aldrich (Sintra, Portugal). MCF-7 (ATCC® HTB-22) mammary gland adenocarcinoma cell line was obtained from ATCC (Middlesex, UK). Hoechst 33342® and Cell Light Bacman 2.0® (Actin-GFP) where
an
obtained from Invitrogen (Carlsbad, USA). The Pyrene fluorescent probe was acquired from TCI (Tokyo Chemical Industry, Co., LTD., Japan). Additional reagents were used
2.2. Synthesis of Amphiphilic Chitosan
M
without further purification.
The inclusion of DOCA or Leu hydrophobic moities in chitosan backbone was
d
accomplished by using an amino coupling chemistry as previously described in the
Ac ce pt e
literature [34] and as represented in Figure 2A. Initially, the carboxylic end groups of DOCA (1.48 mmol, 0.59 g), or Leu (1.04 mmol, 0.14 g), were selectively activated in a 1 % v/v CH3COOH for 5 min, by using EDC (1.63 mmol, 0.313 g) and NHS (0.93 mmol, 0.107 g). Immediately afterwards, the amine reactive DOCA or Leu were added to a chitosan solution (250 mg, V = 25 mL) in CH3COOH (1 % v/v). The solution was then vigorously stirred for 24 h at room temperature. The raw product was then dialyzed (12 000 – 14 000 MWCO) in methanol for three days to remove unreacted hydrophobic species. Following this primary purification, the amphiphilic polymer was dialyzed against double distilled water for 2 days. During this process the dialysate was renewed several times. The modified polymer was then freeze-dried (Scanvac CoolSafe™, ScanLaf A/S, Denmark) for 24 h and stored at 4 ºC. From this point onwards the synthesized chitosan is termed CSDOCA and CS-Leu accordingly to its modification.
6
Page 7 of 26
2.3. Characterization of hydrophobic chitosan derivatives The characterization of the synthesis process of the amphiphilic polymers was performed through proton (1H) NMR spectroscopy by using a Brüker Advance III 400 MHz spectrometer (Brüker Scientific Inc, USA). For NMR analysis freeze dried polymer samples
ip t
were dissolved in 1 mL of D2O/Acetic Acid (95% / 5%) and sonicated for 15 min. The 1H homonuclear spectra were acquired at a constant temperature of 298 K with a pulse program that records the water signal (zg, Brüker Scientific Inc.). The data was recorded
cr
with a spectral width of 8.00 kHz. All the spectra were processed in the TOPSPIN 3.1 software (Brüker Scientific Inc), with a line broadening of 3 Hz. Exponential window
us
function was also used to increase signal-to-noise ratio and eliminate possible acquisition artifacts. The inclusion of both DOCA and Leu was additionally confirmed by Fourier Transform Infrared Spectroscopy (FTIR). Prior to analysis all the freeze-dried samples
an
were dehydrated in an oven for 48 h. The FTIR spectra were then acquired in a Nicolet iS10 spectrometer (Thermo Scientific Inc., USA) by recording 512 scans with a spectral width ranging from 4000 cm-1 to 600 cm-1, and a spectral resolution of 4 cm-1. Baseline
M
subtraction and data analysis was performed in the OMNIC Spectra software (Thermo Scientific). The chemical composition of the CS-DOCA and CS-Leu polymers was also
d
determined by Energy-dispersive X-ray spectroscopy (EDX). For this analysis 10 mg of polymer samples were pressed into homogenous tablets. The samples were then
Ac ce pt e
analyzed with a EDX Rontec equipment by scanning random areas during 100 seconds. These experiments were performed in triplicate. The degree of substitution of the hydrophobically modified polymers was determined by NMR [35], FTIR [36], and EDX [37], in order to provide a more accurate quantification.
2.4. Determination of critical micelle concentration The critical micelle concentration (CMC) of the different amphiphilic polymers was determined by fluorescence spectroscopy using the pyrene encapsulation method as previously described by Laek and co-workers, 2013 [38]. To determine the CMC different polymer concentrations ranging from 1 to 1000 µg/mL were used to encapsulate pyrene (0.6 µM). The fluorescence was then monitored in a Spectramax Gemini XS spectrofluorometer (Molecular Devices LLC, USA). To record the differences in pyrene fluorescence an excitation wavelength of λex 333 nm and λex 335 nm and an emission
7
Page 8 of 26
wavelength of λem 390 nm were used. The results obtained are presented as ratio of the fluorescence intensity of the pyrene excitation peaks (Iλex 335 / Iλex 333). 2.5. Self-assembly of micelle nanocarriers
ip t
The self-assembly of the chitosan micelles was promoted by using a stirring-sonication methodology as previously described elsewhere [24, 39]. Briefly, a stock solution of modified chitosan was prepared by dissolving the polymer in 1% (v/v) CH3COOH to a final
cr
concentration of 10 mg/mL. Subsequently, different dilutions where prepared from this stock solution to promote the formation of micelles. Ibuprofen was dissolved in MetOH (5
us
mg/mL) and then added to the chitosan solution in a ratio of 1:10. The mixture was then vortexed and sonicated (Bransonic® 5510 bath sonicator). The micelles where recovered
an
afterwards by centrifugation at 27000 rpm for 1 h. 2.6. Ibuprofen encapsulation efficiency
M
The micelle encapsulation capacity was determined by Uv-vis spectrophotometry by using an UV-vis Spectrophotometer Shimadzu - 1700 (Shimadzu Inc., Japan). To study different encapsulation efficiencies a range of 0.1 to 1 mg/mL micelle concentrations were used.
d
The micelles were then recovered as above mentioned and the remaining ibuprofen in the
Ac ce pt e
supernatant quantified at λ = 263 nm. The encapsulation efficiency was determined by using the following equation:
Encapsulati on Efficiency (%) =
[ibuprofen] initial − [ibuprofen] supernatant × 100 [ibuprofen] initial
(1)
2.7. Micelle physicochemical characterization After self-assembly and drug encapsulation micelle size and zeta potential were determined by dynamic light scattering (DLS). Prior to analysis micelles were resuspended in 500 µL of double distilled water and analyzed immediately. Sample analysis was performed at 25 °C by using a disposable folded capillary cell. All sample measurements were performed in a Nano ZS instrument (Malvern Instruments, Worcestershire, UK) equipped
with
a
He-Ne
633
nm
laser.
Micelle
size
was
determined
by
8
Page 9 of 26
Cumulants/Correlogram analysis. Zeta (ζ) potential was computed by using the Smoluchowski model (F[Ka] =1.50) included in the Zetasizer software (v 6.32).
2.8. Analysis of micelle morphological characteristics The micelle morphological features were visualized by Scanning electron microscopy
ip t
(SEM). Prior to image acquisition, micelle suspensions were stained with the electron dense phosphotungstic acid (PTA) anionic stain for 5 min, and afterwards dispersed in a cover glass (ø = 15 mm). All samples were dried overnight in an oven to remove residual
cr
water. The cover glasses were then coated with gold using a plasma sputter coater (Emitech K550, Emitech Ltd, UK) and observed on a Hitachi S-2700 (Tokyo, Japan)
an
2.9. Evaluation of polymer and micelle biocompatibility
us
electron microscope configured with optimal detection settings and magnifications.
To evaluate polymer and micelle cytotoxicity, MCF-7 cells were initially seeded at a density of 8 x 103 cells/well in a 96-well flat bottom culture plates, containing DMEM-F12
M
supplemented with 10% FBS. Adherent cells were grown at 37 °C, in an incubator with an humidified atmosphere, containing 5 % CO2. The following day, the culture medium was removed and cells were incubated with different concentrations of amphiphilic polymers
d
(CS-DOCA and CS-Leu), to a final concentration ranging between 5 and 200 µg/mL. In
Ac ce pt e
addition, to determine the influence of the micelle production process, MCF-7 cells were also incubated with blank micelles. Cell cytotoxicity was monitored by using the Resazurin assay. This method uses a highly sensitive and non-toxic reagent (Resazurin) that is reduced to a fluorescent substrate (Resorufin) by intracellular enzymes. Briefly, to perform this evaluation the culture medium was replaced at pre-determined periods (24 and 48h) and the cells were incubated with 10 % (v/v) of Resazurin during 4 h, at 37 ºC and 5 % CO2 in the dark. Fluorescence measurements were then performed in a plate reader spectrofluorometer (Spectramax Gemini XS, Molecular Devices LLC, USA) at an excitation/emission wavelength of 560/590 nm respectively. Ethanol treated cells were used as positive controls and cells without samples were used as negative controls.
2.10. Characterization of micelle uptake in tumor cells Micelle uptake capacity in MCF-7 malignant cells was studied by confocal laser scanning microscopy (CLSM). Prior to CLSM experiments CS-DOCA and CS-Leu were
9
Page 10 of 26
fluorescently labeled with RITC. Polymer labeling was promoted by the addition of RITC (100 mM) to 1 % (w/v) CS solution. The reaction proceeded for 24 h under stirring at room temperature (RT). The labelled polymers were then precipitated with 1M NaOH and recovered by centrifugation at 3000 rpm, 15 min, RT. Excess RITC was then removed by repeated precipitation, centrifugation, and washing until no fluorescence was detected in
ip t
the supernatant. Afterwards the CS-DOCA-RITC and CS-Leu-RITC polymers were freezedried and used to produce ibuprofen loaded micelles as previously described. For the
cr
visualization of micelle uptake 20 x 103 MCF-7 cells were seeded in µ-Slide 8 well Ibidi imaging plates (Ibidi GmbH, Germany), and transfected with the Backman Cell Light 2.0®
us
Actin-GFP probe one day before the experiments. The following day the cells were incubated with the different micelle formulations for 4 h, fixed in 4 % paraformaldehyde (15 min, RT) and washed with PBS. The cell nucleus was labeled with Hoechst 33342® (2
an
µM), for 10 min at RT. Imaging experiments were performed in a Zeiss LSM 710 confocal microscope (Carl Zeiss SMT Inc., USA), where consecutive z-stacks were acquired. 3D version) software’s.
M
reconstruction and image analysis was performed in Zeiss Zen 2010 and Huygens (trial
d
2.11. Anti-proliferative activity of ibuprofen-loaded micelles The anti-proliferative activity of the different micelles was determined by the Resazurin
Ac ce pt e
assay to evaluate cell viability. Initially, 8 x 103 MCF-7 cells were seeded in 96 well plates, 24 h before the experiment in DMEM-F12 – 10 % FBS. In the following day, the culture medium was replaced and the cells were incubated with ibuprofen loaded micelles, at a final drug concentration of 1.67 mM. Cell viability was determined at various time periods (24, 48 and 72 h) by incubating MCF-7 cells with the Resazurin reagent and measuring fluorescence intensity as described before.
2.12. Statistical analysis
One-way analysis of variance (ANOVA) with the Student-Newman-Keuls test was used to compare different groups. A value of p<0.05 is considered statistically significant. Data analysis was executed in GraphPad Prism v.5.0 software (Trial version, GraphPad Software, CA, USA).
10
Page 11 of 26
3. Results and discussion 3.1. Synthesis and characterization of amphiphilic chitosan
ip t
The synthesis of amphiphilic chitosan through the inclusion of DOCA or Leu moieties by using amine coupling chemistry was initially verified by FTIR analysis. As demonstrated in Figure 1 the slight increase in the amide I band at approximately 1650
cr
cm-1 results from the amidation and simultaneous grafting of DOCA into the polymer backbone. A similar peak increase as obtained for Leu amino acid, indicating its
Ac ce pt e
d
M
an
the synthesis process schematized in Figure 2 A.
us
successful inclusion in the chitosan structure (Figure 1), and evidencing the feasibility of
Figure 1. FTIR analysis of amphiphilic chitosan derivatives. Representative spectra of CS, CS-DOCA and CS-Leu.
Further characterization of the chemical composition of CS-DOCA was also performed by 1H NMR analysis. Figure 2 B shows the acetyl group (-NH-O-CH3) protons from the acetylated residues in chitosan (δ ≈ 2.0 ppm, a). The protons of the C2 carbon of chitosan appear at δ ≈ 3.0 ppm (-CH2-, a). The proton signals of C3 to C6 appear in the
11
Page 12 of 26
region of δ ≈ 3.5- 3.9 ppm (H3 to H6 and H6’, b and c). The presence of only the methyl (CH3) protons in the spectra of DOCA (δ ≈ 2.0 ppm) shown in Figure 2 B is correlated with the self-aggregation of the hydrophobic DOCA moieties in D2O, a fact that leads to proton signal shielding as reported by Gao and co-workers 2008 [40]. Figure 2 B also shows the existence of characteristic proton peaks that are assigned to the DOCA hydrophobic
ip t
moieties, namely to its methylene-methine envelope (δ = 1 – 2.5 ppm, d) [40, 41]. The 1H NMR spectra of DOCA in CDCl3 is provided as supplementary information (Figure S1).
cr
This spectra shows additional characteristic peaks of DOCA (Supplementary information). In addition the CS-Leu and CS-DOCA spectra are also provided as supplementary
us
information (Figure S1). Figure 2 C shows the methyl (δ ≈ 0.8 ppm, d) protons that correspond to the anomeric carbon of the hydrophobic amino acid [41]. The degree of substitution (DS) of chitosan to its amphiphilic derivatives was determined by using FTIR,
Ac ce pt e
d
M
an
EDX and NMR (Table 1).
12
Page 13 of 26
13
Page 14 of 26
d
Ac ce pt e us
an
M
cr
ip t
Figure 2. 1H NMR spectra (D2O/Acetic acid 95% / 5%) of the different formulations of amphiphilic chitosan. (A) Schematics of CS amphiphilic derivatives synthesis, (B) CSDOCA, DOCA and CS (top-to-bottom) (C) CS-Leu, L-Leu, CS (top-to-bottom). The results shown in table 1 demonstrate that the DS determined is similar
ip t
regardless of the used method, with no statistically significant difference observed. The CS-DOCA derivatives present a higher DS in comparison to those of CS-Leu.
presented as mean ± s.d., n=3. DS (FTIR)*
DS ( H NMR)*
DS (EDX)*
CS-Leu
0.202 ± 0.143
0.240 ± 0.068
0.259 ± 0.01
CS-DOCA
0.869 ± 0.162
0.933 ± 0.043
0.873 ± 0.009
us
Amphiphilic Derivatives
an
*
1
cr
Table 1. Determination of the amphiphilic derivatives DS by various techniques. Data is
Not statistically different (One-way ANOVA, with Neuman-Keuls post-hoc test, p<0.001.
M
3.2. Micelle self-assembly and ibuprofen encapsulation
The determination of the CMCs for the different formulations is presented in Figure 3. The results demonstrate that CS-DOCA and CS-Leu self-assemble into micelles at
Ac ce pt e
d
relatively low polymer concentrations, 0.101 mg/mL and 0.065 mg/mL, respectively.
Figure 3. CMC determination of CS amphiphilic derivatives. (A) CS-DOCA and (B) CSLeu. Our findings demonstrate that the CS-Leu derivatives self-assembly into micelle carriers with lower polymer concentrations when compared with the CS-Leu derivatives reported in the literature (0.090 mg/mL) [38]. The CS-DOCA derivatives also possess very
14
Page 15 of 26
low CMC, comparable to that previously reported in the literature [42], a fact that is attributed to the high degree of DOCA substitution. Overall, these CMC results indicate that the self-assembled carriers are highly stable in aqueous solutions since low CMC values indicate that these micellar systems have a compact hydrophobic core, a feature that is responsible for micelles stabilization as described by Owen et al, 2012 [43].
ip t
Following micelle synthesis the ibuprofen encapsulation capacity of the selfassembled amphiphilic polymers was also initially evaluated. The FTIR spectra of drug-
cr
loaded micelle powder formulations shows the ibuprofen characteristic high intensity bands at 1730 cm-1 (C=O stretching) and 2921 cm-1 (CH3 asymmetric stretching),
us
evidencing the successful encapsulation of NSAIDS inside the micellar carriers (Figure 4 A). The determination of ibuprofen encapsulation in micellar carriers was performed by Uvvis spectrophotometry, since ibuprofen shows a concentration dependent absorbance in
an
PBS with a maximum absorbance peak at λ= 263 nm (Figure 4 B). The quantitative results of ibuprofen encapsulation obtained by Uv-vis analysis demonstrate that the CS-DOCA carriers formulated at various micelle concentrations have high encapsulation efficiency
M
(Figure 4 C). Particularly, the CS-DOCA0.3 formulation has the highest encapsulation (>66 %) in comparison with the other CS-DOCA micelles. The CS-Leu delivery systems also have high NSAID encapsulation efficiencies (>69 %) in the various concentrations studied
d
(Figure 4 D). Moreover, no significant differences in encapsulation were observed for the
Ac ce pt e
different types of micellar nanocarriers.
15
Page 16 of 26
ip t cr us an M d Ac ce pt e
Figure 4. NSAID encapsulation analysis. (A) FTIR spectra of blank micelles and ibuprofen-loaded micelles; (B) Uv-vis analysis of ibuprofen NSAIDS at various concentrations in PBS; (C and D) Encapsulation efficiencies of CS-DOCA and CS-Leu respectively. n=3; *p<0.05; n.s. not significant.
3.3. Physicochemical characterization of ibuprofen-loaded micelles The results of characterization performed by DLS and the most stable micelle
formulations are shown in Figure 5. The CS-DOCA0.3 and CS-DOCA0.4 micelles possess similar particle size distributions as demonstrated by size histograms. DOCA-based micelles have narrow size range between 171-173 nm, and both formulations show similar positive surface charge (Figure 5). Regarding morphological features, CS-DOCA micelles reveal uniform spherical shapes (Figure 5 C).
16
Page 17 of 26
ip t cr us an M
Figure 5. Physicochemical characterization of ibuprofen-loaded micelles. (A and B)
d
Micelle size and zeta potential of CS-DOCA0.3 and CS-DOCA0.4 formulations, respectively.
Ac ce pt e
(C) Representative SEM image of CS-DOCA0.4 micelles. (D and E) Micelle size and zeta potential of CS-Leu0.6 and CS-Leu0.7. (F) Representative SEM image of CS-Leu0.6 micelles. Concerning amino acid substituted CS, the CS-Leu0.6 formulation yields the
smallest sized particles in comparison with all other formulations tested (Figure 5 D). These particular micelles possess a highly negative surface charge indicating its colloidal stability, whilst the CS-Leu0.7 micelles present a slightly negative zeta potential (Figure 5 E). Leu amphiphilies also present a characteristic spherical morphology as demonstrated by SEM images (Figure 5 F), a valuable characteristic since it increases nanocarriers cell uptake rate as recently demonstrated by Herd et al., 2013 [44]. Moreover, it is relevant to highlight that both CS-DOCA and CS-Leu NSAIDS formulations self-assembly into micelles with sizes suitable for passive accumulation in tumor tissues [45].
3.4. Evaluation of amphiphilic chitosan biocompatibility
17
Page 18 of 26
The biocompatibility of the amphiphilic chitosan derivatives was screened by incubating tumor cells with growing polymer concentrations. All cells presented high viability, even at the maximum polymer concentration of 200 µg/mL (Figure 6). Particularly, for the CSDOCA amphiphilies, cell proliferation occurs at 48 h in comparison with non-incubated cells (Figure 6 A). For CS-Leu amphiphilies also slight MCF-7 cell proliferation occurs at
ip t
both 24 h and 48 h (Figure 6 B). To fully characterize polymer biocompatibility, the effect of micelle formulation conditions was also investigated in order to address whether this could
cr
negatively influence MCF-7 cell viability and mask NSAIDS anti-proliferative activity. As shown in Figure 6 C and D, the administration of blank micelles comprised of CS-DOCA or
us
CS-Leu formulations does not affect MCF-7 cell viability neither at 24 h, nor at 48 h of incubation. These significant findings evidence the suitability of using the self-assembly
Ac ce pt e
d
M
an
methodology to engineer micellar carriers for therapeutic applications.
Figure 6. Evaluation of the cytotoxic profile of CS amphiphilic polymer derivatives. (A and B) Cytotoxicity evaluation of CS-DOCA and CS-Leu polymers at various concentrations,
18
Page 19 of 26
respectively. (C and D) Cytotoxicity evaluation of CS-DOCA and CS-Leu blank micelle formulations. n=5; *p<0.05; n.s. not significant.
ip t
3.5. Cellular uptake of ibuprofen-loaded micelles
Cell uptake capacity of CS-DOCA and CS-Leu formulations was visualized through
cr
CLSM by using RITC as a model of poorly water-soluble fluorescent probe. As shown in Figure 7, CS-DOCA-RITC micelles are readily observed in MCF-7 cell cytoplasm after 4 h
us
of micelle administration. This intracellular localization is evident for both CS-DOCA and CS-Leu micelles. Fluorescence images also shown that CS-DOCA0.4 micelles are
an
internalized to a slightly higher extent than the CS-DOCA0.3 formulation, or the CS-Leu
Ac ce pt e
d
M
micelles (Figure 7B).
Figure 7. 3D CLSM images of micelle uptake in cancer cells. (A and B) CS-DOCA formulations; (C and D) CS-Leu formulations. Green channel: Actin-GFP; Blue channel: Hoechst 33342® stained nucleus; Red Channel: RITC-loaded micelles. White arrows indicate RITC-loaded micelles. The intracellular localization of both CS-DOCA and CS-Leu micelles is achieved after the micellar carriers transpose the extracellular barriers, namely the extracellular
19
Page 20 of 26
membrane. The amphiphilic character of these delivery systems promotes the establishment of both electrostatic and hydrophobic interactions at the micelle-membrane interface [46]. The cell uptake of micellar carriers is a complex process in which the various uptake routes could be involved, however it is widely proposed that endocytosis is the major route of internalization for micelles [46]. Regarding the possible internalization
ip t
mechanism of the hydrophobically modified chitosan micelles produced herein, previous reports in the literature, namely the work of Nam and co-workers describes that
cr
internalization of these carriers might occur through clathrin and caveolae-mediated endocytosis and also macropinocytosis [47]. In this particular report, chitosan was also
us
hydrophiobic modified with 5β-cholanic acid moieties [47]. This report thus indicates that these routes of internalization might also be responsible for the cellular uptake of CSDOCA and CS-Leu micelles.
an
It is also important to emphasize that the localization of CS-DOCA and CS-Leu micelles inside MCF-7 cells is of critical importance since ibuprofen biological targets, namely cyclooxygenases 1 and 2 (COX-1 and COX-2) exert their activity in the cytoplasm
M
[48]. Thus, the micelle mediated delivery to the intracellular compartment increases NSAID bioavailability at the target location and may contribute for a markedly decrease of the
d
short-term ulcerogenic potential of this drug [49].
Ac ce pt e
3.6. In vitro anti-tumoral activity of ibuprofen-loaded micelles The anti-tumoral activity of ibuprofen (NSAIDS) in breast cancer cells was initially
studied by incubating MCF-7 with free drug to determine IC 50 and evaluate a possible decrease in cell viability. Our results demonstrate that free Ibuprofen is capable of reducing MCF-7 cell viability to 50 % when administered at a concentration of 0.212 ± 0.0876 mM (Figure 8 A).
20
Page 21 of 26
Figure 8. Evaluation of ibuprofen anti-tumoral activity in MCF-7 breast cancer cells. (A) IC 50 determination; (B) ibuprofen-loaded CS-DOCA micelles; (C) Ibuprofen-loaded CS-Leu micelles. n=5; *p<0.05; n.s. not significant. The determined IC50 is higher than that reported in the literature for MCF-7 cells (2.78 µM)
ip t
[50], such suggests that the tested breast cancer cells have probably acquired a more resistant phenotype during cell culture passages. Even so, these findings illustrate the actual anti-proliferative activity of cost-effective NSAIDS. Following IC 50 estimation, the
cr
cells were incubated with different micelle derivatives. The administration of ibuprofenloaded micelles during 24 h resulted in a decreased tumor cell viability in all amphiphilic
us
formulations, in comparison with non-treated cells (Figure 8B and 8C). Though, at this time point the anti-proliferative capacity of free ibuprofen was higher than that of the micellar carriers (Figure 8 B and C). Yet, the free drug formulation suffers from low bioavailability
an
and non-specific toxicity as described beforehand [10]. Furthermore, during the course of the assay, clear differences are observed between the different amphiphilies, with the CS-
M
DOCA-ibuprofen micelles presenting a strikingly higher anti-proliferative activity, particularly at 72 h in both CS-DOCA0.3 and CS-DOCA0.4 formulations (Figure 8 B and C). Furthermore, as demonstrated by Figure 8B these particular formulations decrease tumor
d
cell viability to levels comparable with those of the free drug. Actually, our findings are in agreement with those reported by Baek and co-workers, 2002, that described the anti-
Ac ce pt e
proliferative potential of ibuprofen in various malignant cell lines [51]. Interestingly, it should also be emphasized that the anti-tumoral potential of this straightforward approach is comparable with that obtained with standard chemotherapeutic drugs such as Doxorubicin (Dox) [52]. In fact, as recently reported by Ke et al., 2013, the delivery of free Dox reduces MCF-7 viability to approximately 20 %, after 48 h [52]. Although obtained at a later time frame, the DOCA-ibuprofen micellar carriers achieve similar results. This crucial finding indicates the therapeutic potential of these micelles as carriers for this widely used NSAID.
21
Page 22 of 26
Conclusion In this study we have synthesized biofunctionalized chitosan derivatives comprised of natural hydrophobic moieties in order to imprint an amphiphilic character on the native
ip t
polymer backbone. Chitosan functionalization with DOCA and Leu moieties unlocked the possibility to engineer self-assembly delivery systems that encapsulate poor water soluble NSAIDS with high efficiency. The amphiphilic polymers self-assembled into nanosized
cr
micelles under mild conditions and maintained the characteristic high biocompatibility of unmodified chitosan. More importantly, both micellar formulations were capable of
us
delivering ibuprofen inside tumor cells and elicit an anti-tumoral effect comparable with that of generally used chemotherapeutics.
an
Overall we have shown that is possible to produce a relatively inexpensive and highly biocompatible delivery system that effectively reduces breast cancer cell viability by administering a widely used NSAID. Furthermore, since this system is highly versatile it
M
could be included in a near future in other delivery platforms such as microcarriers or
Ac ce pt e
Acknowledgments
d
nanofibers for possible combinatorial drug delivery.
The authors would like to thank to Eng. Ana Paula for the acquisition of SEM images. This work was supported by the Portuguese Foundation for Science and Technology (FCT), (PTDC/EME-TME/103375/2008, PTDC/EBB-BIO/114320/2009, PEstC/SAU/UI0709/2011). Vítor M. Gaspar, also acknowledges a PhD fellowship from FCT (SFRH/BD/80402/2011).
22
Page 23 of 26
References
Ac ce pt e
d
M
an
us
cr
ip t
[1] R. Wang, P.S. Billone and W.M. Mullett, Nanomedicine in Action: An Overview of Cancer Nanomedicine on the Market and in Clinical Trials, Journal of Nanomaterials, 2013 (2013). [2] C. Varma, Molecular Targeted Therapy: Cancer Therapy Of The Future, International Journal of Medicine and Molecular Medicine, (2012). [3] D.J. Burgess, Therapy: Enhancing efficacy by reducing side effects, Nature Reviews Cancer, 12 (2012) 377-377. [4] L.E. Schnipper, N.J. Meropol and D.W. Brock, Value and cancer care: toward an equitable future, Clinical cancer Research, 16 (2010) 6004-6008. [5] J. Cuzick, F. Otto, J.A. Baron, P.H. Brown, J. Burn, P. Greenwald, J. Jankowski, C. La Vecchia, F. Meyskens and H.J. Senn, Aspirin and non-steroidal anti-inflammatory drugs for cancer prevention: an international consensus statement, The lancet oncology, 10 (2009) 501-507. [6] D.P. Cronin-Fenton, L. Pedersen, T.L. Lash, S. Friis, J.A. Baron and H.T. Sorensen, Prescriptions for selective cyclooxygenase-2 inhibitors, non-selective non-steroidal anti-inflammatory drugs, and risk of breast cancer in a population-based case-control study, Breast cancer research : BCR, 12 (2010) R15. [7] M.A. Hossain, D.H. Kim, J.Y. Jang, Y.J. Kang, J.H. Yoon, J.O. Moon, H.Y. Chung, G.Y. Kim, Y.H. Choi, B.L. Copple and N.D. Kim, Aspirin induces apoptosis in vitro and inhibits tumor growth of human hepatocellular carcinoma cells in a nude mouse xenograft model, International journal of oncology, 40 (2012) 1298-1304. [8] A.T. Koki and J.L. Masferrer, Celecoxib: a specific COX-2 inhibitor with anticancer properties, Cancer Control, 9 (2002) 28-35. [9] S.J. Baek, L.C. Wilson, C.H. Lee and T.E. Eling, Dual function of nonsteroidal anti-inflammatory drugs (NSAIDs): inhibition of cyclooxygenase and induction of NSAID-activated gene, Journal of Pharmacology and Experimental Therapeutics, 301 (2002) 1126-1131. [10] P. Prakash, A. Sayyed-Ahmad, Y. Zhou, D.E. Volk, D.G. Gorenstein, E. Dial, L.M. Lichtenberger and A.A. Gorfe, Aggregation Behavior of Ibuprofen, cholic acid and dodecylphosphocholine micelles, Biochimica et Biophysica Acta (BBA)-Biomembranes, 1818 (2012) 3040-3047. [11] U.A. Boelsterli, H.J. Zimmerman and A. Kretz-Rommel, Idiosyncratic Liver Toxicity of Nonsteroidal Antiinflammatory Drugs: Molecular Mechanisms and Pathology, Critical Reviews in Toxicology, 25 (1995) 207-235. [12] H. Otsuka, Y. Nagasaki and K. Kataoka, PEGylated nanoparticles for biological and pharmaceutical applications, Advanced drug delivery reviews, 55 (2003) 403-419. [13] A. Rösler, G.W.M. Vandermeulen and H.-A. Klok, Advanced drug delivery devices via selfassembly of amphiphilic block copolymers, Advanced Drug Delivery Reviews, 53 (2012) 95–108. [14] M.L. Tan, P.F.M. Choong and C.R. Dass, Recent developments in liposomes, microparticles and nanoparticles for protein and peptide drug delivery, Peptides, 31 (2010) 184-193. [15] X. Li, Y. Chen, M. Wang, Y. Ma, W. Xia and H. Gu, A mesoporous silica nanoparticle – PEI – Fusogenic peptide system for siRNA delivery in cancer therapy, Biomaterials, 34 (2013) 1391-1401. [16] I. Nakase, Y. Konishi, M. Ueda, H. Saji and S. Futaki, Accumulation of arginine-rich cellpenetrating peptides in tumors and the potential for anticancer drug delivery in vivo, Journal of Controlled Release, 159 (2012) 181-188. [17] H. Li, M. Huo, J. Zhou, Y. Dai, Y. Deng, X. Shi and J. Masoud, Enhanced oral absorption of paclitaxel in N-deoxycholic acid-N, O-hydroxyethyl chitosan micellar system, Journal of pharmaceutical sciences, 99 (2010) 4543-4553.
23
Page 24 of 26
Ac ce pt e
d
M
an
us
cr
ip t
[18] F. Wang, D. Zhang, C. Duan, L. Jia, F. Feng, Y. Liu, Y. Wang, L. Hao and Q. Zhang, Preparation and characterizations of a novel deoxycholic acid–O-carboxymethylated chitosan–folic acid conjugates and self-aggregates, Carbohydrate Polymers, 84 (2011) 1192-1200. [19] A.A. Haroun, N. El-Halawany, C. Loira-Pastoriza and P. Maincent, Synthesis and in vitro release study of ibuprofen-loaded gelatin graft copolymer nanoparticles, Drug development and industrial pharmacy, (2012) 1-5. [20] K. Kim, S. Kwon, J.H. Park, H. Chung, S.Y. Jeong, I.C. Kwon and I.-S. Kim, Physicochemical Characterizations of Self-Assembled Nanoparticles of Glycol Chitosan−Deoxycholic Acid Conjugates, Biomacromolecules, 6 (2005) 1154-1158. [21] A. Albanese, P.S. Tang and W.C. Chan, The effect of nanoparticle size, shape, and surface chemistry on biological systems, Annual review of biomedical engineering, 14 (2012) 1-16. [22] J.S. Gebauer, M. Malissek, S. Simon, S.K. Knauer, M. Maskos, R.H. Stauber, W. Peukert and L. Treuel, Impact of the Nanoparticle–Protein Corona on Colloidal Stability and Protein Structure, Langmuir, 28 (2012) 9673-9679. [23] Z. Pan, Y. Gao, L. Heng, Y. Liu, G. Yao, Y. Wang and Y. Liu, Amphiphilic N-(2, 3dihydroxypropyl)-chitosan-cholic acid micelles for paclitaxel delivery, Carbohydrate Polymers, 94 (2013) 394-399. [24] S.Y. Chae, S. Son, M. Lee, M.K. Jang and J.W. Nah, Deoxycholic acid-conjugated chitosan oligosaccharide nanoparticles for efficient gene carrier, Journal of Controled Release, 109 (2005) 330-344. [25] H. Zhou, W. Yu, X. Guo, X. Liu, N. Li, Y. Zhang and X. Ma, Synthesis and Characterization of Amphiphilic Glycidol− Chitosan− Deoxycholic Acid Nanopar cles as a Drug Carrier for Doxorubicin, Biomacromolecules, 11 (2010) 3480-3486. [26] Y.H. Jin, H.Y. Hu, M.X. Qiao, J. Zhu, J.W. Qi, C.J. Hu, Q. Zhang and D.W. Chen, pH-sensitive chitosan-derived nanoparticles as doxorubicin carriers for effective anti-tumor activity: preparation and in vitro evaluation, Colloids and surfaces. B, Biointerfaces, 94 (2012) 184-191. [27] J. Zhang, X.G. Chen, Y.Y. Li and C.S. Liu, Self-assembled nanoparticles based on hydrophobically modified chitosan as carriers for doxorubicin, Nanomedicine: Nanotechnology, Biology and Medicine, 3 (2007) 258-265. [28] J.-H. Kim, Y.-S. Kim, S. Kim, J.H. Park, K. Kim, K. Choi, H. Chung, S.Y. Jeong, R.-W. Park and I.-S. Kim, Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel, Journal of controlled release, 111 (2006) 228-234. [29] J.Y. Lee, S.H. Lee, M.H. Oh, J.S. Kim, T.G. Park and Y.S. Nam, Prolonged Gene Silencing by siRNA/chitosan-g-deoxycholic acid Polyplexes Loaded within Biodegradable Polymer Nanoparticles, Journal of Controlled Release, 162 (2012) 407–413. [30] K. Lee, J.H. Kim, I. Kwon and S. Jeong, Self-aggregates of deoxycholic acid-modified chitosan as a novel carrier of adriamycin, Colloid & Polymer Science, 278 (2000) 1216-1219. [31] VM Gaspar , JG Marques , F Sousa, RO Louro, JA Queiroz and C. IJ, Biofunctionalized nanoparticles with pH-responsive and cell penetrating blocks for gene delivery, Nanotechnology (2013) in press. [32] L. Casettari, D. Vllasaliu, J. KW Lam, M. Soliman and L. Illum, Biomedical applications of amino acid-modified chitosans: A review, Biomaterials, 33 (2012) 7565–7583. [33] B. Layek and J. Singh, Amino acid grafted chitosan for high performance gene delivery: comparison of amino acid hydrophobicity on vector and polyplex characteristics, Biomacromolecules, 14 (2013) 485-494. [34] Y. Kim, H. Jiang, Y. Choi, I. Park, M. Cho and C. Cho, Polymeric Nanoparticles of Chitosan Derivatives as DNA and siRNA Carriers, Chitosan for Biomaterials I, 243 (2011) 1-21.
24
Page 25 of 26
Ac ce pt e
d
M
an
us
cr
ip t
[35] C. Pau-Roblot, E. Petit, C. Sarazin, J. Courtois, B. Courtois, J.N. Barbotin and J.P. Séguin, Studies of low molecular weight samples of glucuronans with various acetylation degree, Biopolymers, 64 (2002) 34-43. [36] <2005 Yang - Synthesis of Diblock Copolymers Consisting.pdf>. [37] A. Pestov, N. Zhuravlev and Y.G. Yatluk, Synthesis in a gel as a new procedure for preparing carboxyethyl chitosan, Russian Journal of Applied Chemistry, 80 (2007) 1154-1159. [38] B. Layek and J. Singh, Amino Acid Grafted Chitosan for High Performance Gene Delivery: Comparison of Amino Acid Hydrophobicity on Vector and Polyplex Characteristics, Biomacromolecules, (2013). [39] K.Y. Lee, I.C. Kwon, W.H. Jo and S.Y. Jeong, Complex formation between plasmid DNA and self-aggregates of deoxycholic acid-modified chitosan, Polymer, 46 (2005) 8107-8112. [40] F.-P. Gao, H.-Z. Zhang, L.-R. Liu, Y.-S. Wang, Q. Jiang, X.-D. Yang and Q.-Q. Zhang, Preparation and physicochemical characteristics of self-assembled nanoparticles of deoxycholic acid modifiedcarboxymethyl curdlan conjugates, Carbohydrate Polymers, 71 (2008) 606-613. [41] H. Zhou, W. Yu, X. Guo, X. Liu, N. Li, Y. Zhang and X. Ma, Synthesis and Characterization of Amphiphilic Glycidol−Chitosan−Deoxycholic Acid Nanopar cles as a Drug Carrier for Doxorubicin, Biomacromolecules, 11 (2010) 3480-3486. [42] H. Li, M. Huo, J. Zhou, Y. Dai, Y. Deng, X. Shi and J. Masoud, Enhanced oral absorption of paclitaxel in N-deoxycholic acid-N, O-hydroxyethyl chitosan micellar system, Journal of Pharmaceutical Sciences, 99 (2010) 4543-4553. [43] S.C. Owen, D.P. Chan and M.S. Shoichet, Polymeric micelle stability, Nano Today, 7 (2012) 5365. [44] H. Herd, N. Daum, A.T. Jones, H. Huwer, H. Ghandehari and C.-M. Lehr, Nanoparticle Geometry and Surface Orientation Influences Mode of Cellular Uptake, ACS nano, 7 (2013) 1961– 1973. [45] T. Stylianopoulos, EPR-effect: utilizing size-dependent nanoparticle delivery to solid tumors, Therapeutic delivery, 4 (2013) 421-423. [46] R. Savic, A. Eisenberg and D. Maysinger, Block copolymer micelles as delivery vehicles of hydrophobic drugs: micelle-cell interactions, Journal of drug targeting, 14 (2006) 343-355. [47] H.Y. Nam, S.M. Kwon, H. Chung, S.-Y. Lee, S.-H. Kwon, H. Jeon, Y. Kim, J.H. Park, J. Kim, S. Her, Y.-K. Oh, I.C. Kwon, K. Kim and S.Y. Jeong, Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles, Journal of Controlled Release, 135 (2009) 259-267. [48] D. Wang and R.N. DuBois, Prostaglandins and Cancer, Gut, 55 (2006) 115-122. [49] J.L. Wallace, B. Reuter, C. Cicala, W. McKnight, M. Grisham and G. Cirino, Novel nonsteroidal anti-inflammatory drug derivatives with markedly reduced ulcerogenic properties in the rat, Gastroenterology, 107 (1994) 173. [50] M. Barkume, P. Tayade, P. Vavia and M. Indap, Antiproliferative effect of aspirin and its inclusion complex on human MCF-7 and K-562 cancer cells in vitro, Indian Journal of Pharmaceutical Sciences, 65 (2003) 486-491. [51] S.J. Baek, L.C. Wilson, C.-H. Lee and T.E. Eling, Dual function of nonsteroidal antiinflammatory drugs (NSAIDs): inhibition of cyclooxygenase and induction of NSAID-activated gene, Journal of Pharmacology and Experimental Therapeutics, 301 (2002) 1126-1131. [52] C.-J. Ke, W.-L. Chiang, Z.-X. Liao, H.-L. Chen, P.-S. Lai, J.-S. Sun and H.-W. Sung, Real-time visualization of pH-responsive PLGA hollow particles containing a gas-generating agent targeted for acidic organelles for overcoming multi-drug resistance, Biomaterials, 34 (2013) 1-10.
25
Page 26 of 26
S0927-7765(13)00605-X http://dx.doi.org/doi:10.1016/j.colsurfb.2013.09.037 COLSUB 6038
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
31-5-2013 10-9-2013 18-9-2013
Please cite this article as: J.G. Marques, V.M. Gaspar, E. Costa, C.M. Paquete, I.J. Correia, Synthesis and Characterization of Micelles as carriers of Non-steroidal antiinflammatory drugs (NSAID) for application in breast cancer therapy, Colloids and Surfaces B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.09.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Ac
Page 1 of 26
Highlights
Chitosan amphiphilic derivatives self-assembly into nanosized micellar carriers
•
Micelles are capable of encapsulating non-steroidal anti-inflammatory drugs (NSAIDs)
•
Ibuprofen delivery into breast cancer cells shows anti-proliferative potential
•
Use of cost-effective NSAID-micelles can be a promising approach for cancer therapy
Ac ce pt e
d
M
an
us
cr
ip t
•
1
Page 2 of 26
Synthesis and Characterization of Micelles as carriers of Non-steroidal anti-inflammatory drugs (NSAID) for application in breast cancer therapy
ip t
João G. Marques1, Vítor M. Gaspar1, Elisabete Costa1, Catarina M. Paquete2 and Ilídio J. Correia1* 1
cr
CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6200-506, Covilhã, Portugal
2
us
ITQB-UNL - Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa
an
Av. da Republica, Estação Agronómica Nacional, 2780-157 Oeiras, Portugal
Ac ce pt e
d
M
*Corresponding author: Professor Dr. Ilídio Correia Avenida Infante D. Henrique, 6200-506, Covilhã, Portugal E-mail: [email protected]
2
Page 3 of 26
Abstract Non-steroidal anti-inflammatory drugs (NSAIDS) are emerging as a particularly valuable class of drugs due to their recently reported anti-tumoral activity in colorectal cancer. However, despite this tremendous potential, their bioavailability at the tumor
ip t
microenvironment remains rather limited. To overcome this issue, in this work we synthesized biocompatible micellar nanocarriers composed of amphiphilic chitosan to deliver ibuprofen into breast cancer cells and evaluate its anti-tumor activity, while avoiding
cr
side-effects. Our results reveal that the formulations produced herein self-assembly into spherical micelles with suitable sizes for tumor accumulation (108 – 252 nm). Furthermore,
us
by using a vortex-sonication method, ibuprofen was successfully encapsulated with high efficiency. Cell uptake studies show that ibuprofen-loaded micelles are readily internalized
an
by tumor cells and deliver their cargo in the intracellular compartment as demonstrated by confocal microscopy images. This fact led to a remarkable reduction in cancer cell viability (< 13%), at a relatively low drug dosage, illustrating the anti-tumoral activity of ibuprofen
M
when delivered to breast cancer cells. These findings demonstrate the promising potential of chitosan micelles as carriers of cost-effective NSAIDS for application in breast cancer
Ac ce pt e
d
therapy.
Keywords
Chitosan; Micelles; NSAIDs; ibuprofen; Anti-proliferative activity; Breast Cancer.
3
Page 4 of 26
1. Introduction Nowadays the continuous worldwide increase of cancer-related fatalities compels both the scientific and medical community towards the development of new anti-cancer
ip t
therapeutics that may improve patient survival rates even at late stages of cancer development [1]. Interestingly, the present advances in antineoplastic treatments are ever more focused not only on eliminating the primary tumor and niches of malignant cells, but
cr
also on reducing systemic cytotoxicity [2, 3].
Recently, nanotechnology has revolutionized the development of proficient delivery
us
systems that improve the pharmacodynamics and pharmacokinetics of chemotherapeutic drugs. However, the existing cutting-edge therapeutics still rely on highly toxic and
an
expensive active pharmaceutical ingredients (API) restricting their widespread application [4]. In this context, NSAIDS commonly associated with cancer prevention, have also been recently reported to be involved in cancer ablation [5-7]. Particularly, in the past couple of
M
years, NSAIDS such as aspirin [7], celecoxib [8], and ibuprofen have shown to have relevant anti-cancer activity [6]. Indeed, as reported by Baek and coworkers, 2002, ibuprofen has demonstrated the capacity to inhibit malignant cell proliferation through the
d
pro-apoptotic NSAID activated gene (NAG-1) [9]. Nevertheless, alike other drugs, it is
Ac ce pt e
crucial to reduce NSAIDS gut toxicity [10], first pass metabolism, and also increase their bioavailability on the target tumor tissues [11]. To overcome these issues, different classes of delivery vehicles that include polymeric micelles [12, 13], liposomes [14], silica nanoparticles [15], or cell penetrating peptides [16] are currently under development to transport numerous anti-tumoral drugs. Particularly, polymeric micelles comprised by hydrophobic and hydrophilic moieties are of vast interest due to their self-assembly characteristics into nanosized micellar structures, and exceptional drug loading capacity [17, 18]. Such property depends on the size and rearrangement of the hydrophobic moieties that eventually form an inner particle core which acts as a highly efficient reservoir for hydrophobic drugs like NSAIDS [19]. Thermodynamic stability is an additional advantage of hydrophobically modified polymers that comprise micellar nanodevices [20, 21]. These characteristics are crucial for drug delivery since micelles must assure the protection of their payload until being localized inside the target cells [22]. Nanocarriers comprised of hydrophobically functionalized chitosan are one of the most promising drug delivery systems for application in cancer therapy as recently demonstrated by Pan et al.,
4
Page 5 of 26
2013, with the delivery of the anti-tumoral drug paclitaxel [23]. Chitosan exclusive biocompatibility, biodegradability and cationic charge render it a unique biomaterial for nanoparticle synthesis [24]. In addition to these intrinsic features, the versatile structure of chitosan unlocks the possibility to manipulate its configuration by chemical grafting. To promote the synthesis of micelle nanocarriers different hydrophobic moieties including
ip t
deoxycholic acid (DOCA) [25, 26], oleoyl fatty acids [27] or 5β-cholanic acid [28], have been grafted onto chitosan backbone. DOCA, an amphiphilic moiety a constituent of bile
cr
acid, is particularly advantageous due to its high biocompatibility and micelle forming capacity in aqueous environments [29, 30]. Additional biomolecules, including amino
us
acids, have also been inserted in chitosan as our group recently described [31]. Grafting biocompatible amino acids improves chitosan solubility and facilitates nanoparticle cellular uptake [32]. Amino acid-mediated self-assembly can also be achieved when hydrophobic
an
moieties are included in chitosan, leading to the formation of an amphiphilic polymer capable of encapsulating hydrophobic drugs [32]. Leucine (Leu) addition is particularly valuable since chitosan-Leu nanoparticles present small sizes and superior cellular uptake
M
in comparison to other hydrophobic amino acids such as Valine [33]. From this standpoint, in this work we optimized the synthesis of nanosized micelles formulated with different amphiphilic polymers, explored NSAIDS drug loading capacity
d
and also the anti-tumoral effectiveness of different ibuprofen-loaded carriers in breast
Ac ce pt e
cancer cells. The inclusion of DOCA or Leu in chitosan backbone led to the formation of self-assembled micelles with considerable loading capacity and high anti-tumoral activity, particularly evident for the CS-DOCA formulations.
5
Page 6 of 26
2. Materials and Methods 2.1. Materials Chitosan hydrochloride (CS) (Protasan UP CL 113, MW ≈ 110 kDa) was purchased from (Novamatrix, Sandvika, Norway). L-Leucine was purchased from Affimetrix Inc. (Santa
ip t
Clara, USA). Acetic acid (CH3COOH) was obtained from Panreac (Barcelona, Spain). 3α, 12α-dihydroxy-5β-cholanate (Deoxycholic acid (DOCA)), Dulbecco’s Modified Eagle’s (DMEM),
N-Hydroxysuccinimide
(NHS),
N-(3-Dimethylaminopropyl)-N´-
cr
Medium
ethylcarbodiimide hydrochloride (EDC), Resazurin, Rhodamine B Isothiocyanate (RITC),
us
and cellulose dialysis membrane were obtained from Sigma–Aldrich (Sintra, Portugal). MCF-7 (ATCC® HTB-22) mammary gland adenocarcinoma cell line was obtained from ATCC (Middlesex, UK). Hoechst 33342® and Cell Light Bacman 2.0® (Actin-GFP) where
an
obtained from Invitrogen (Carlsbad, USA). The Pyrene fluorescent probe was acquired from TCI (Tokyo Chemical Industry, Co., LTD., Japan). Additional reagents were used
2.2. Synthesis of Amphiphilic Chitosan
M
without further purification.
The inclusion of DOCA or Leu hydrophobic moities in chitosan backbone was
d
accomplished by using an amino coupling chemistry as previously described in the
Ac ce pt e
literature [34] and as represented in Figure 2A. Initially, the carboxylic end groups of DOCA (1.48 mmol, 0.59 g), or Leu (1.04 mmol, 0.14 g), were selectively activated in a 1 % v/v CH3COOH for 5 min, by using EDC (1.63 mmol, 0.313 g) and NHS (0.93 mmol, 0.107 g). Immediately afterwards, the amine reactive DOCA or Leu were added to a chitosan solution (250 mg, V = 25 mL) in CH3COOH (1 % v/v). The solution was then vigorously stirred for 24 h at room temperature. The raw product was then dialyzed (12 000 – 14 000 MWCO) in methanol for three days to remove unreacted hydrophobic species. Following this primary purification, the amphiphilic polymer was dialyzed against double distilled water for 2 days. During this process the dialysate was renewed several times. The modified polymer was then freeze-dried (Scanvac CoolSafe™, ScanLaf A/S, Denmark) for 24 h and stored at 4 ºC. From this point onwards the synthesized chitosan is termed CSDOCA and CS-Leu accordingly to its modification.
6
Page 7 of 26
2.3. Characterization of hydrophobic chitosan derivatives The characterization of the synthesis process of the amphiphilic polymers was performed through proton (1H) NMR spectroscopy by using a Brüker Advance III 400 MHz spectrometer (Brüker Scientific Inc, USA). For NMR analysis freeze dried polymer samples
ip t
were dissolved in 1 mL of D2O/Acetic Acid (95% / 5%) and sonicated for 15 min. The 1H homonuclear spectra were acquired at a constant temperature of 298 K with a pulse program that records the water signal (zg, Brüker Scientific Inc.). The data was recorded
cr
with a spectral width of 8.00 kHz. All the spectra were processed in the TOPSPIN 3.1 software (Brüker Scientific Inc), with a line broadening of 3 Hz. Exponential window
us
function was also used to increase signal-to-noise ratio and eliminate possible acquisition artifacts. The inclusion of both DOCA and Leu was additionally confirmed by Fourier Transform Infrared Spectroscopy (FTIR). Prior to analysis all the freeze-dried samples
an
were dehydrated in an oven for 48 h. The FTIR spectra were then acquired in a Nicolet iS10 spectrometer (Thermo Scientific Inc., USA) by recording 512 scans with a spectral width ranging from 4000 cm-1 to 600 cm-1, and a spectral resolution of 4 cm-1. Baseline
M
subtraction and data analysis was performed in the OMNIC Spectra software (Thermo Scientific). The chemical composition of the CS-DOCA and CS-Leu polymers was also
d
determined by Energy-dispersive X-ray spectroscopy (EDX). For this analysis 10 mg of polymer samples were pressed into homogenous tablets. The samples were then
Ac ce pt e
analyzed with a EDX Rontec equipment by scanning random areas during 100 seconds. These experiments were performed in triplicate. The degree of substitution of the hydrophobically modified polymers was determined by NMR [35], FTIR [36], and EDX [37], in order to provide a more accurate quantification.
2.4. Determination of critical micelle concentration The critical micelle concentration (CMC) of the different amphiphilic polymers was determined by fluorescence spectroscopy using the pyrene encapsulation method as previously described by Laek and co-workers, 2013 [38]. To determine the CMC different polymer concentrations ranging from 1 to 1000 µg/mL were used to encapsulate pyrene (0.6 µM). The fluorescence was then monitored in a Spectramax Gemini XS spectrofluorometer (Molecular Devices LLC, USA). To record the differences in pyrene fluorescence an excitation wavelength of λex 333 nm and λex 335 nm and an emission
7
Page 8 of 26
wavelength of λem 390 nm were used. The results obtained are presented as ratio of the fluorescence intensity of the pyrene excitation peaks (Iλex 335 / Iλex 333). 2.5. Self-assembly of micelle nanocarriers
ip t
The self-assembly of the chitosan micelles was promoted by using a stirring-sonication methodology as previously described elsewhere [24, 39]. Briefly, a stock solution of modified chitosan was prepared by dissolving the polymer in 1% (v/v) CH3COOH to a final
cr
concentration of 10 mg/mL. Subsequently, different dilutions where prepared from this stock solution to promote the formation of micelles. Ibuprofen was dissolved in MetOH (5
us
mg/mL) and then added to the chitosan solution in a ratio of 1:10. The mixture was then vortexed and sonicated (Bransonic® 5510 bath sonicator). The micelles where recovered
an
afterwards by centrifugation at 27000 rpm for 1 h. 2.6. Ibuprofen encapsulation efficiency
M
The micelle encapsulation capacity was determined by Uv-vis spectrophotometry by using an UV-vis Spectrophotometer Shimadzu - 1700 (Shimadzu Inc., Japan). To study different encapsulation efficiencies a range of 0.1 to 1 mg/mL micelle concentrations were used.
d
The micelles were then recovered as above mentioned and the remaining ibuprofen in the
Ac ce pt e
supernatant quantified at λ = 263 nm. The encapsulation efficiency was determined by using the following equation:
Encapsulati on Efficiency (%) =
[ibuprofen] initial − [ibuprofen] supernatant × 100 [ibuprofen] initial
(1)
2.7. Micelle physicochemical characterization After self-assembly and drug encapsulation micelle size and zeta potential were determined by dynamic light scattering (DLS). Prior to analysis micelles were resuspended in 500 µL of double distilled water and analyzed immediately. Sample analysis was performed at 25 °C by using a disposable folded capillary cell. All sample measurements were performed in a Nano ZS instrument (Malvern Instruments, Worcestershire, UK) equipped
with
a
He-Ne
633
nm
laser.
Micelle
size
was
determined
by
8
Page 9 of 26
Cumulants/Correlogram analysis. Zeta (ζ) potential was computed by using the Smoluchowski model (F[Ka] =1.50) included in the Zetasizer software (v 6.32).
2.8. Analysis of micelle morphological characteristics The micelle morphological features were visualized by Scanning electron microscopy
ip t
(SEM). Prior to image acquisition, micelle suspensions were stained with the electron dense phosphotungstic acid (PTA) anionic stain for 5 min, and afterwards dispersed in a cover glass (ø = 15 mm). All samples were dried overnight in an oven to remove residual
cr
water. The cover glasses were then coated with gold using a plasma sputter coater (Emitech K550, Emitech Ltd, UK) and observed on a Hitachi S-2700 (Tokyo, Japan)
an
2.9. Evaluation of polymer and micelle biocompatibility
us
electron microscope configured with optimal detection settings and magnifications.
To evaluate polymer and micelle cytotoxicity, MCF-7 cells were initially seeded at a density of 8 x 103 cells/well in a 96-well flat bottom culture plates, containing DMEM-F12
M
supplemented with 10% FBS. Adherent cells were grown at 37 °C, in an incubator with an humidified atmosphere, containing 5 % CO2. The following day, the culture medium was removed and cells were incubated with different concentrations of amphiphilic polymers
d
(CS-DOCA and CS-Leu), to a final concentration ranging between 5 and 200 µg/mL. In
Ac ce pt e
addition, to determine the influence of the micelle production process, MCF-7 cells were also incubated with blank micelles. Cell cytotoxicity was monitored by using the Resazurin assay. This method uses a highly sensitive and non-toxic reagent (Resazurin) that is reduced to a fluorescent substrate (Resorufin) by intracellular enzymes. Briefly, to perform this evaluation the culture medium was replaced at pre-determined periods (24 and 48h) and the cells were incubated with 10 % (v/v) of Resazurin during 4 h, at 37 ºC and 5 % CO2 in the dark. Fluorescence measurements were then performed in a plate reader spectrofluorometer (Spectramax Gemini XS, Molecular Devices LLC, USA) at an excitation/emission wavelength of 560/590 nm respectively. Ethanol treated cells were used as positive controls and cells without samples were used as negative controls.
2.10. Characterization of micelle uptake in tumor cells Micelle uptake capacity in MCF-7 malignant cells was studied by confocal laser scanning microscopy (CLSM). Prior to CLSM experiments CS-DOCA and CS-Leu were
9
Page 10 of 26
fluorescently labeled with RITC. Polymer labeling was promoted by the addition of RITC (100 mM) to 1 % (w/v) CS solution. The reaction proceeded for 24 h under stirring at room temperature (RT). The labelled polymers were then precipitated with 1M NaOH and recovered by centrifugation at 3000 rpm, 15 min, RT. Excess RITC was then removed by repeated precipitation, centrifugation, and washing until no fluorescence was detected in
ip t
the supernatant. Afterwards the CS-DOCA-RITC and CS-Leu-RITC polymers were freezedried and used to produce ibuprofen loaded micelles as previously described. For the
cr
visualization of micelle uptake 20 x 103 MCF-7 cells were seeded in µ-Slide 8 well Ibidi imaging plates (Ibidi GmbH, Germany), and transfected with the Backman Cell Light 2.0®
us
Actin-GFP probe one day before the experiments. The following day the cells were incubated with the different micelle formulations for 4 h, fixed in 4 % paraformaldehyde (15 min, RT) and washed with PBS. The cell nucleus was labeled with Hoechst 33342® (2
an
µM), for 10 min at RT. Imaging experiments were performed in a Zeiss LSM 710 confocal microscope (Carl Zeiss SMT Inc., USA), where consecutive z-stacks were acquired. 3D version) software’s.
M
reconstruction and image analysis was performed in Zeiss Zen 2010 and Huygens (trial
d
2.11. Anti-proliferative activity of ibuprofen-loaded micelles The anti-proliferative activity of the different micelles was determined by the Resazurin
Ac ce pt e
assay to evaluate cell viability. Initially, 8 x 103 MCF-7 cells were seeded in 96 well plates, 24 h before the experiment in DMEM-F12 – 10 % FBS. In the following day, the culture medium was replaced and the cells were incubated with ibuprofen loaded micelles, at a final drug concentration of 1.67 mM. Cell viability was determined at various time periods (24, 48 and 72 h) by incubating MCF-7 cells with the Resazurin reagent and measuring fluorescence intensity as described before.
2.12. Statistical analysis
One-way analysis of variance (ANOVA) with the Student-Newman-Keuls test was used to compare different groups. A value of p<0.05 is considered statistically significant. Data analysis was executed in GraphPad Prism v.5.0 software (Trial version, GraphPad Software, CA, USA).
10
Page 11 of 26
3. Results and discussion 3.1. Synthesis and characterization of amphiphilic chitosan
ip t
The synthesis of amphiphilic chitosan through the inclusion of DOCA or Leu moieties by using amine coupling chemistry was initially verified by FTIR analysis. As demonstrated in Figure 1 the slight increase in the amide I band at approximately 1650
cr
cm-1 results from the amidation and simultaneous grafting of DOCA into the polymer backbone. A similar peak increase as obtained for Leu amino acid, indicating its
Ac ce pt e
d
M
an
the synthesis process schematized in Figure 2 A.
us
successful inclusion in the chitosan structure (Figure 1), and evidencing the feasibility of
Figure 1. FTIR analysis of amphiphilic chitosan derivatives. Representative spectra of CS, CS-DOCA and CS-Leu.
Further characterization of the chemical composition of CS-DOCA was also performed by 1H NMR analysis. Figure 2 B shows the acetyl group (-NH-O-CH3) protons from the acetylated residues in chitosan (δ ≈ 2.0 ppm, a). The protons of the C2 carbon of chitosan appear at δ ≈ 3.0 ppm (-CH2-, a). The proton signals of C3 to C6 appear in the
11
Page 12 of 26
region of δ ≈ 3.5- 3.9 ppm (H3 to H6 and H6’, b and c). The presence of only the methyl (CH3) protons in the spectra of DOCA (δ ≈ 2.0 ppm) shown in Figure 2 B is correlated with the self-aggregation of the hydrophobic DOCA moieties in D2O, a fact that leads to proton signal shielding as reported by Gao and co-workers 2008 [40]. Figure 2 B also shows the existence of characteristic proton peaks that are assigned to the DOCA hydrophobic
ip t
moieties, namely to its methylene-methine envelope (δ = 1 – 2.5 ppm, d) [40, 41]. The 1H NMR spectra of DOCA in CDCl3 is provided as supplementary information (Figure S1).
cr
This spectra shows additional characteristic peaks of DOCA (Supplementary information). In addition the CS-Leu and CS-DOCA spectra are also provided as supplementary
us
information (Figure S1). Figure 2 C shows the methyl (δ ≈ 0.8 ppm, d) protons that correspond to the anomeric carbon of the hydrophobic amino acid [41]. The degree of substitution (DS) of chitosan to its amphiphilic derivatives was determined by using FTIR,
Ac ce pt e
d
M
an
EDX and NMR (Table 1).
12
Page 13 of 26
13
Page 14 of 26
d
Ac ce pt e us
an
M
cr
ip t
Figure 2. 1H NMR spectra (D2O/Acetic acid 95% / 5%) of the different formulations of amphiphilic chitosan. (A) Schematics of CS amphiphilic derivatives synthesis, (B) CSDOCA, DOCA and CS (top-to-bottom) (C) CS-Leu, L-Leu, CS (top-to-bottom). The results shown in table 1 demonstrate that the DS determined is similar
ip t
regardless of the used method, with no statistically significant difference observed. The CS-DOCA derivatives present a higher DS in comparison to those of CS-Leu.
presented as mean ± s.d., n=3. DS (FTIR)*
DS ( H NMR)*
DS (EDX)*
CS-Leu
0.202 ± 0.143
0.240 ± 0.068
0.259 ± 0.01
CS-DOCA
0.869 ± 0.162
0.933 ± 0.043
0.873 ± 0.009
us
Amphiphilic Derivatives
an
*
1
cr
Table 1. Determination of the amphiphilic derivatives DS by various techniques. Data is
Not statistically different (One-way ANOVA, with Neuman-Keuls post-hoc test, p<0.001.
M
3.2. Micelle self-assembly and ibuprofen encapsulation
The determination of the CMCs for the different formulations is presented in Figure 3. The results demonstrate that CS-DOCA and CS-Leu self-assemble into micelles at
Ac ce pt e
d
relatively low polymer concentrations, 0.101 mg/mL and 0.065 mg/mL, respectively.
Figure 3. CMC determination of CS amphiphilic derivatives. (A) CS-DOCA and (B) CSLeu. Our findings demonstrate that the CS-Leu derivatives self-assembly into micelle carriers with lower polymer concentrations when compared with the CS-Leu derivatives reported in the literature (0.090 mg/mL) [38]. The CS-DOCA derivatives also possess very
14
Page 15 of 26
low CMC, comparable to that previously reported in the literature [42], a fact that is attributed to the high degree of DOCA substitution. Overall, these CMC results indicate that the self-assembled carriers are highly stable in aqueous solutions since low CMC values indicate that these micellar systems have a compact hydrophobic core, a feature that is responsible for micelles stabilization as described by Owen et al, 2012 [43].
ip t
Following micelle synthesis the ibuprofen encapsulation capacity of the selfassembled amphiphilic polymers was also initially evaluated. The FTIR spectra of drug-
cr
loaded micelle powder formulations shows the ibuprofen characteristic high intensity bands at 1730 cm-1 (C=O stretching) and 2921 cm-1 (CH3 asymmetric stretching),
us
evidencing the successful encapsulation of NSAIDS inside the micellar carriers (Figure 4 A). The determination of ibuprofen encapsulation in micellar carriers was performed by Uvvis spectrophotometry, since ibuprofen shows a concentration dependent absorbance in
an
PBS with a maximum absorbance peak at λ= 263 nm (Figure 4 B). The quantitative results of ibuprofen encapsulation obtained by Uv-vis analysis demonstrate that the CS-DOCA carriers formulated at various micelle concentrations have high encapsulation efficiency
M
(Figure 4 C). Particularly, the CS-DOCA0.3 formulation has the highest encapsulation (>66 %) in comparison with the other CS-DOCA micelles. The CS-Leu delivery systems also have high NSAID encapsulation efficiencies (>69 %) in the various concentrations studied
d
(Figure 4 D). Moreover, no significant differences in encapsulation were observed for the
Ac ce pt e
different types of micellar nanocarriers.
15
Page 16 of 26
ip t cr us an M d Ac ce pt e
Figure 4. NSAID encapsulation analysis. (A) FTIR spectra of blank micelles and ibuprofen-loaded micelles; (B) Uv-vis analysis of ibuprofen NSAIDS at various concentrations in PBS; (C and D) Encapsulation efficiencies of CS-DOCA and CS-Leu respectively. n=3; *p<0.05; n.s. not significant.
3.3. Physicochemical characterization of ibuprofen-loaded micelles The results of characterization performed by DLS and the most stable micelle
formulations are shown in Figure 5. The CS-DOCA0.3 and CS-DOCA0.4 micelles possess similar particle size distributions as demonstrated by size histograms. DOCA-based micelles have narrow size range between 171-173 nm, and both formulations show similar positive surface charge (Figure 5). Regarding morphological features, CS-DOCA micelles reveal uniform spherical shapes (Figure 5 C).
16
Page 17 of 26
ip t cr us an M
Figure 5. Physicochemical characterization of ibuprofen-loaded micelles. (A and B)
d
Micelle size and zeta potential of CS-DOCA0.3 and CS-DOCA0.4 formulations, respectively.
Ac ce pt e
(C) Representative SEM image of CS-DOCA0.4 micelles. (D and E) Micelle size and zeta potential of CS-Leu0.6 and CS-Leu0.7. (F) Representative SEM image of CS-Leu0.6 micelles. Concerning amino acid substituted CS, the CS-Leu0.6 formulation yields the
smallest sized particles in comparison with all other formulations tested (Figure 5 D). These particular micelles possess a highly negative surface charge indicating its colloidal stability, whilst the CS-Leu0.7 micelles present a slightly negative zeta potential (Figure 5 E). Leu amphiphilies also present a characteristic spherical morphology as demonstrated by SEM images (Figure 5 F), a valuable characteristic since it increases nanocarriers cell uptake rate as recently demonstrated by Herd et al., 2013 [44]. Moreover, it is relevant to highlight that both CS-DOCA and CS-Leu NSAIDS formulations self-assembly into micelles with sizes suitable for passive accumulation in tumor tissues [45].
3.4. Evaluation of amphiphilic chitosan biocompatibility
17
Page 18 of 26
The biocompatibility of the amphiphilic chitosan derivatives was screened by incubating tumor cells with growing polymer concentrations. All cells presented high viability, even at the maximum polymer concentration of 200 µg/mL (Figure 6). Particularly, for the CSDOCA amphiphilies, cell proliferation occurs at 48 h in comparison with non-incubated cells (Figure 6 A). For CS-Leu amphiphilies also slight MCF-7 cell proliferation occurs at
ip t
both 24 h and 48 h (Figure 6 B). To fully characterize polymer biocompatibility, the effect of micelle formulation conditions was also investigated in order to address whether this could
cr
negatively influence MCF-7 cell viability and mask NSAIDS anti-proliferative activity. As shown in Figure 6 C and D, the administration of blank micelles comprised of CS-DOCA or
us
CS-Leu formulations does not affect MCF-7 cell viability neither at 24 h, nor at 48 h of incubation. These significant findings evidence the suitability of using the self-assembly
Ac ce pt e
d
M
an
methodology to engineer micellar carriers for therapeutic applications.
Figure 6. Evaluation of the cytotoxic profile of CS amphiphilic polymer derivatives. (A and B) Cytotoxicity evaluation of CS-DOCA and CS-Leu polymers at various concentrations,
18
Page 19 of 26
respectively. (C and D) Cytotoxicity evaluation of CS-DOCA and CS-Leu blank micelle formulations. n=5; *p<0.05; n.s. not significant.
ip t
3.5. Cellular uptake of ibuprofen-loaded micelles
Cell uptake capacity of CS-DOCA and CS-Leu formulations was visualized through
cr
CLSM by using RITC as a model of poorly water-soluble fluorescent probe. As shown in Figure 7, CS-DOCA-RITC micelles are readily observed in MCF-7 cell cytoplasm after 4 h
us
of micelle administration. This intracellular localization is evident for both CS-DOCA and CS-Leu micelles. Fluorescence images also shown that CS-DOCA0.4 micelles are
an
internalized to a slightly higher extent than the CS-DOCA0.3 formulation, or the CS-Leu
Ac ce pt e
d
M
micelles (Figure 7B).
Figure 7. 3D CLSM images of micelle uptake in cancer cells. (A and B) CS-DOCA formulations; (C and D) CS-Leu formulations. Green channel: Actin-GFP; Blue channel: Hoechst 33342® stained nucleus; Red Channel: RITC-loaded micelles. White arrows indicate RITC-loaded micelles. The intracellular localization of both CS-DOCA and CS-Leu micelles is achieved after the micellar carriers transpose the extracellular barriers, namely the extracellular
19
Page 20 of 26
membrane. The amphiphilic character of these delivery systems promotes the establishment of both electrostatic and hydrophobic interactions at the micelle-membrane interface [46]. The cell uptake of micellar carriers is a complex process in which the various uptake routes could be involved, however it is widely proposed that endocytosis is the major route of internalization for micelles [46]. Regarding the possible internalization
ip t
mechanism of the hydrophobically modified chitosan micelles produced herein, previous reports in the literature, namely the work of Nam and co-workers describes that
cr
internalization of these carriers might occur through clathrin and caveolae-mediated endocytosis and also macropinocytosis [47]. In this particular report, chitosan was also
us
hydrophiobic modified with 5β-cholanic acid moieties [47]. This report thus indicates that these routes of internalization might also be responsible for the cellular uptake of CSDOCA and CS-Leu micelles.
an
It is also important to emphasize that the localization of CS-DOCA and CS-Leu micelles inside MCF-7 cells is of critical importance since ibuprofen biological targets, namely cyclooxygenases 1 and 2 (COX-1 and COX-2) exert their activity in the cytoplasm
M
[48]. Thus, the micelle mediated delivery to the intracellular compartment increases NSAID bioavailability at the target location and may contribute for a markedly decrease of the
d
short-term ulcerogenic potential of this drug [49].
Ac ce pt e
3.6. In vitro anti-tumoral activity of ibuprofen-loaded micelles The anti-tumoral activity of ibuprofen (NSAIDS) in breast cancer cells was initially
studied by incubating MCF-7 with free drug to determine IC 50 and evaluate a possible decrease in cell viability. Our results demonstrate that free Ibuprofen is capable of reducing MCF-7 cell viability to 50 % when administered at a concentration of 0.212 ± 0.0876 mM (Figure 8 A).
20
Page 21 of 26
Figure 8. Evaluation of ibuprofen anti-tumoral activity in MCF-7 breast cancer cells. (A) IC 50 determination; (B) ibuprofen-loaded CS-DOCA micelles; (C) Ibuprofen-loaded CS-Leu micelles. n=5; *p<0.05; n.s. not significant. The determined IC50 is higher than that reported in the literature for MCF-7 cells (2.78 µM)
ip t
[50], such suggests that the tested breast cancer cells have probably acquired a more resistant phenotype during cell culture passages. Even so, these findings illustrate the actual anti-proliferative activity of cost-effective NSAIDS. Following IC 50 estimation, the
cr
cells were incubated with different micelle derivatives. The administration of ibuprofenloaded micelles during 24 h resulted in a decreased tumor cell viability in all amphiphilic
us
formulations, in comparison with non-treated cells (Figure 8B and 8C). Though, at this time point the anti-proliferative capacity of free ibuprofen was higher than that of the micellar carriers (Figure 8 B and C). Yet, the free drug formulation suffers from low bioavailability
an
and non-specific toxicity as described beforehand [10]. Furthermore, during the course of the assay, clear differences are observed between the different amphiphilies, with the CS-
M
DOCA-ibuprofen micelles presenting a strikingly higher anti-proliferative activity, particularly at 72 h in both CS-DOCA0.3 and CS-DOCA0.4 formulations (Figure 8 B and C). Furthermore, as demonstrated by Figure 8B these particular formulations decrease tumor
d
cell viability to levels comparable with those of the free drug. Actually, our findings are in agreement with those reported by Baek and co-workers, 2002, that described the anti-
Ac ce pt e
proliferative potential of ibuprofen in various malignant cell lines [51]. Interestingly, it should also be emphasized that the anti-tumoral potential of this straightforward approach is comparable with that obtained with standard chemotherapeutic drugs such as Doxorubicin (Dox) [52]. In fact, as recently reported by Ke et al., 2013, the delivery of free Dox reduces MCF-7 viability to approximately 20 %, after 48 h [52]. Although obtained at a later time frame, the DOCA-ibuprofen micellar carriers achieve similar results. This crucial finding indicates the therapeutic potential of these micelles as carriers for this widely used NSAID.
21
Page 22 of 26
Conclusion In this study we have synthesized biofunctionalized chitosan derivatives comprised of natural hydrophobic moieties in order to imprint an amphiphilic character on the native
ip t
polymer backbone. Chitosan functionalization with DOCA and Leu moieties unlocked the possibility to engineer self-assembly delivery systems that encapsulate poor water soluble NSAIDS with high efficiency. The amphiphilic polymers self-assembled into nanosized
cr
micelles under mild conditions and maintained the characteristic high biocompatibility of unmodified chitosan. More importantly, both micellar formulations were capable of
us
delivering ibuprofen inside tumor cells and elicit an anti-tumoral effect comparable with that of generally used chemotherapeutics.
an
Overall we have shown that is possible to produce a relatively inexpensive and highly biocompatible delivery system that effectively reduces breast cancer cell viability by administering a widely used NSAID. Furthermore, since this system is highly versatile it
M
could be included in a near future in other delivery platforms such as microcarriers or
Ac ce pt e
Acknowledgments
d
nanofibers for possible combinatorial drug delivery.
The authors would like to thank to Eng. Ana Paula for the acquisition of SEM images. This work was supported by the Portuguese Foundation for Science and Technology (FCT), (PTDC/EME-TME/103375/2008, PTDC/EBB-BIO/114320/2009, PEstC/SAU/UI0709/2011). Vítor M. Gaspar, also acknowledges a PhD fellowship from FCT (SFRH/BD/80402/2011).
22
Page 23 of 26
References
Ac ce pt e
d
M
an
us
cr
ip t
[1] R. Wang, P.S. Billone and W.M. Mullett, Nanomedicine in Action: An Overview of Cancer Nanomedicine on the Market and in Clinical Trials, Journal of Nanomaterials, 2013 (2013). [2] C. Varma, Molecular Targeted Therapy: Cancer Therapy Of The Future, International Journal of Medicine and Molecular Medicine, (2012). [3] D.J. Burgess, Therapy: Enhancing efficacy by reducing side effects, Nature Reviews Cancer, 12 (2012) 377-377. [4] L.E. Schnipper, N.J. Meropol and D.W. Brock, Value and cancer care: toward an equitable future, Clinical cancer Research, 16 (2010) 6004-6008. [5] J. Cuzick, F. Otto, J.A. Baron, P.H. Brown, J. Burn, P. Greenwald, J. Jankowski, C. La Vecchia, F. Meyskens and H.J. Senn, Aspirin and non-steroidal anti-inflammatory drugs for cancer prevention: an international consensus statement, The lancet oncology, 10 (2009) 501-507. [6] D.P. Cronin-Fenton, L. Pedersen, T.L. Lash, S. Friis, J.A. Baron and H.T. Sorensen, Prescriptions for selective cyclooxygenase-2 inhibitors, non-selective non-steroidal anti-inflammatory drugs, and risk of breast cancer in a population-based case-control study, Breast cancer research : BCR, 12 (2010) R15. [7] M.A. Hossain, D.H. Kim, J.Y. Jang, Y.J. Kang, J.H. Yoon, J.O. Moon, H.Y. Chung, G.Y. Kim, Y.H. Choi, B.L. Copple and N.D. Kim, Aspirin induces apoptosis in vitro and inhibits tumor growth of human hepatocellular carcinoma cells in a nude mouse xenograft model, International journal of oncology, 40 (2012) 1298-1304. [8] A.T. Koki and J.L. Masferrer, Celecoxib: a specific COX-2 inhibitor with anticancer properties, Cancer Control, 9 (2002) 28-35. [9] S.J. Baek, L.C. Wilson, C.H. Lee and T.E. Eling, Dual function of nonsteroidal anti-inflammatory drugs (NSAIDs): inhibition of cyclooxygenase and induction of NSAID-activated gene, Journal of Pharmacology and Experimental Therapeutics, 301 (2002) 1126-1131. [10] P. Prakash, A. Sayyed-Ahmad, Y. Zhou, D.E. Volk, D.G. Gorenstein, E. Dial, L.M. Lichtenberger and A.A. Gorfe, Aggregation Behavior of Ibuprofen, cholic acid and dodecylphosphocholine micelles, Biochimica et Biophysica Acta (BBA)-Biomembranes, 1818 (2012) 3040-3047. [11] U.A. Boelsterli, H.J. Zimmerman and A. Kretz-Rommel, Idiosyncratic Liver Toxicity of Nonsteroidal Antiinflammatory Drugs: Molecular Mechanisms and Pathology, Critical Reviews in Toxicology, 25 (1995) 207-235. [12] H. Otsuka, Y. Nagasaki and K. Kataoka, PEGylated nanoparticles for biological and pharmaceutical applications, Advanced drug delivery reviews, 55 (2003) 403-419. [13] A. Rösler, G.W.M. Vandermeulen and H.-A. Klok, Advanced drug delivery devices via selfassembly of amphiphilic block copolymers, Advanced Drug Delivery Reviews, 53 (2012) 95–108. [14] M.L. Tan, P.F.M. Choong and C.R. Dass, Recent developments in liposomes, microparticles and nanoparticles for protein and peptide drug delivery, Peptides, 31 (2010) 184-193. [15] X. Li, Y. Chen, M. Wang, Y. Ma, W. Xia and H. Gu, A mesoporous silica nanoparticle – PEI – Fusogenic peptide system for siRNA delivery in cancer therapy, Biomaterials, 34 (2013) 1391-1401. [16] I. Nakase, Y. Konishi, M. Ueda, H. Saji and S. Futaki, Accumulation of arginine-rich cellpenetrating peptides in tumors and the potential for anticancer drug delivery in vivo, Journal of Controlled Release, 159 (2012) 181-188. [17] H. Li, M. Huo, J. Zhou, Y. Dai, Y. Deng, X. Shi and J. Masoud, Enhanced oral absorption of paclitaxel in N-deoxycholic acid-N, O-hydroxyethyl chitosan micellar system, Journal of pharmaceutical sciences, 99 (2010) 4543-4553.
23
Page 24 of 26
Ac ce pt e
d
M
an
us
cr
ip t
[18] F. Wang, D. Zhang, C. Duan, L. Jia, F. Feng, Y. Liu, Y. Wang, L. Hao and Q. Zhang, Preparation and characterizations of a novel deoxycholic acid–O-carboxymethylated chitosan–folic acid conjugates and self-aggregates, Carbohydrate Polymers, 84 (2011) 1192-1200. [19] A.A. Haroun, N. El-Halawany, C. Loira-Pastoriza and P. Maincent, Synthesis and in vitro release study of ibuprofen-loaded gelatin graft copolymer nanoparticles, Drug development and industrial pharmacy, (2012) 1-5. [20] K. Kim, S. Kwon, J.H. Park, H. Chung, S.Y. Jeong, I.C. Kwon and I.-S. Kim, Physicochemical Characterizations of Self-Assembled Nanoparticles of Glycol Chitosan−Deoxycholic Acid Conjugates, Biomacromolecules, 6 (2005) 1154-1158. [21] A. Albanese, P.S. Tang and W.C. Chan, The effect of nanoparticle size, shape, and surface chemistry on biological systems, Annual review of biomedical engineering, 14 (2012) 1-16. [22] J.S. Gebauer, M. Malissek, S. Simon, S.K. Knauer, M. Maskos, R.H. Stauber, W. Peukert and L. Treuel, Impact of the Nanoparticle–Protein Corona on Colloidal Stability and Protein Structure, Langmuir, 28 (2012) 9673-9679. [23] Z. Pan, Y. Gao, L. Heng, Y. Liu, G. Yao, Y. Wang and Y. Liu, Amphiphilic N-(2, 3dihydroxypropyl)-chitosan-cholic acid micelles for paclitaxel delivery, Carbohydrate Polymers, 94 (2013) 394-399. [24] S.Y. Chae, S. Son, M. Lee, M.K. Jang and J.W. Nah, Deoxycholic acid-conjugated chitosan oligosaccharide nanoparticles for efficient gene carrier, Journal of Controled Release, 109 (2005) 330-344. [25] H. Zhou, W. Yu, X. Guo, X. Liu, N. Li, Y. Zhang and X. Ma, Synthesis and Characterization of Amphiphilic Glycidol− Chitosan− Deoxycholic Acid Nanopar cles as a Drug Carrier for Doxorubicin, Biomacromolecules, 11 (2010) 3480-3486. [26] Y.H. Jin, H.Y. Hu, M.X. Qiao, J. Zhu, J.W. Qi, C.J. Hu, Q. Zhang and D.W. Chen, pH-sensitive chitosan-derived nanoparticles as doxorubicin carriers for effective anti-tumor activity: preparation and in vitro evaluation, Colloids and surfaces. B, Biointerfaces, 94 (2012) 184-191. [27] J. Zhang, X.G. Chen, Y.Y. Li and C.S. Liu, Self-assembled nanoparticles based on hydrophobically modified chitosan as carriers for doxorubicin, Nanomedicine: Nanotechnology, Biology and Medicine, 3 (2007) 258-265. [28] J.-H. Kim, Y.-S. Kim, S. Kim, J.H. Park, K. Kim, K. Choi, H. Chung, S.Y. Jeong, R.-W. Park and I.-S. Kim, Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel, Journal of controlled release, 111 (2006) 228-234. [29] J.Y. Lee, S.H. Lee, M.H. Oh, J.S. Kim, T.G. Park and Y.S. Nam, Prolonged Gene Silencing by siRNA/chitosan-g-deoxycholic acid Polyplexes Loaded within Biodegradable Polymer Nanoparticles, Journal of Controlled Release, 162 (2012) 407–413. [30] K. Lee, J.H. Kim, I. Kwon and S. Jeong, Self-aggregates of deoxycholic acid-modified chitosan as a novel carrier of adriamycin, Colloid & Polymer Science, 278 (2000) 1216-1219. [31] VM Gaspar , JG Marques , F Sousa, RO Louro, JA Queiroz and C. IJ, Biofunctionalized nanoparticles with pH-responsive and cell penetrating blocks for gene delivery, Nanotechnology (2013) in press. [32] L. Casettari, D. Vllasaliu, J. KW Lam, M. Soliman and L. Illum, Biomedical applications of amino acid-modified chitosans: A review, Biomaterials, 33 (2012) 7565–7583. [33] B. Layek and J. Singh, Amino acid grafted chitosan for high performance gene delivery: comparison of amino acid hydrophobicity on vector and polyplex characteristics, Biomacromolecules, 14 (2013) 485-494. [34] Y. Kim, H. Jiang, Y. Choi, I. Park, M. Cho and C. Cho, Polymeric Nanoparticles of Chitosan Derivatives as DNA and siRNA Carriers, Chitosan for Biomaterials I, 243 (2011) 1-21.
24
Page 25 of 26
Ac ce pt e
d
M
an
us
cr
ip t
[35] C. Pau-Roblot, E. Petit, C. Sarazin, J. Courtois, B. Courtois, J.N. Barbotin and J.P. Séguin, Studies of low molecular weight samples of glucuronans with various acetylation degree, Biopolymers, 64 (2002) 34-43. [36] <2005 Yang - Synthesis of Diblock Copolymers Consisting.pdf>. [37] A. Pestov, N. Zhuravlev and Y.G. Yatluk, Synthesis in a gel as a new procedure for preparing carboxyethyl chitosan, Russian Journal of Applied Chemistry, 80 (2007) 1154-1159. [38] B. Layek and J. Singh, Amino Acid Grafted Chitosan for High Performance Gene Delivery: Comparison of Amino Acid Hydrophobicity on Vector and Polyplex Characteristics, Biomacromolecules, (2013). [39] K.Y. Lee, I.C. Kwon, W.H. Jo and S.Y. Jeong, Complex formation between plasmid DNA and self-aggregates of deoxycholic acid-modified chitosan, Polymer, 46 (2005) 8107-8112. [40] F.-P. Gao, H.-Z. Zhang, L.-R. Liu, Y.-S. Wang, Q. Jiang, X.-D. Yang and Q.-Q. Zhang, Preparation and physicochemical characteristics of self-assembled nanoparticles of deoxycholic acid modifiedcarboxymethyl curdlan conjugates, Carbohydrate Polymers, 71 (2008) 606-613. [41] H. Zhou, W. Yu, X. Guo, X. Liu, N. Li, Y. Zhang and X. Ma, Synthesis and Characterization of Amphiphilic Glycidol−Chitosan−Deoxycholic Acid Nanopar cles as a Drug Carrier for Doxorubicin, Biomacromolecules, 11 (2010) 3480-3486. [42] H. Li, M. Huo, J. Zhou, Y. Dai, Y. Deng, X. Shi and J. Masoud, Enhanced oral absorption of paclitaxel in N-deoxycholic acid-N, O-hydroxyethyl chitosan micellar system, Journal of Pharmaceutical Sciences, 99 (2010) 4543-4553. [43] S.C. Owen, D.P. Chan and M.S. Shoichet, Polymeric micelle stability, Nano Today, 7 (2012) 5365. [44] H. Herd, N. Daum, A.T. Jones, H. Huwer, H. Ghandehari and C.-M. Lehr, Nanoparticle Geometry and Surface Orientation Influences Mode of Cellular Uptake, ACS nano, 7 (2013) 1961– 1973. [45] T. Stylianopoulos, EPR-effect: utilizing size-dependent nanoparticle delivery to solid tumors, Therapeutic delivery, 4 (2013) 421-423. [46] R. Savic, A. Eisenberg and D. Maysinger, Block copolymer micelles as delivery vehicles of hydrophobic drugs: micelle-cell interactions, Journal of drug targeting, 14 (2006) 343-355. [47] H.Y. Nam, S.M. Kwon, H. Chung, S.-Y. Lee, S.-H. Kwon, H. Jeon, Y. Kim, J.H. Park, J. Kim, S. Her, Y.-K. Oh, I.C. Kwon, K. Kim and S.Y. Jeong, Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles, Journal of Controlled Release, 135 (2009) 259-267. [48] D. Wang and R.N. DuBois, Prostaglandins and Cancer, Gut, 55 (2006) 115-122. [49] J.L. Wallace, B. Reuter, C. Cicala, W. McKnight, M. Grisham and G. Cirino, Novel nonsteroidal anti-inflammatory drug derivatives with markedly reduced ulcerogenic properties in the rat, Gastroenterology, 107 (1994) 173. [50] M. Barkume, P. Tayade, P. Vavia and M. Indap, Antiproliferative effect of aspirin and its inclusion complex on human MCF-7 and K-562 cancer cells in vitro, Indian Journal of Pharmaceutical Sciences, 65 (2003) 486-491. [51] S.J. Baek, L.C. Wilson, C.-H. Lee and T.E. Eling, Dual function of nonsteroidal antiinflammatory drugs (NSAIDs): inhibition of cyclooxygenase and induction of NSAID-activated gene, Journal of Pharmacology and Experimental Therapeutics, 301 (2002) 1126-1131. [52] C.-J. Ke, W.-L. Chiang, Z.-X. Liao, H.-L. Chen, P.-S. Lai, J.-S. Sun and H.-W. Sung, Real-time visualization of pH-responsive PLGA hollow particles containing a gas-generating agent targeted for acidic organelles for overcoming multi-drug resistance, Biomaterials, 34 (2013) 1-10.
25
Page 26 of 26