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A comparative study on long-term MTX controlled release from intercalated nanocomposites for nanomedicine applications
Colloids and Surfaces B: Biointerfaces 106 (2013) 135–139
Contents lists available at SciVerse ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A comparative study on long-term MTX controlled release from intercalated nanocomposites for nanomedicine applications Iuliana Florentina Alexa a , Cristina Giorgiana Pastravanu a , Maria Ignat a,b,∗ , Evelini Popovici a a b
“Alexandru Ioan Cuza” University, Faculty of Chemistry, Iasi, Romania Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania
a r t i c l e
i n f o
Article history: Received 26 September 2012 Received in revised form 17 December 2012 Accepted 15 January 2013 Available online 23 January 2013 Keywords: Mesoporous materials LDH Drug delivery Methotrexate Sustained release
a b s t r a c t The feasibility of some mesoporous materials such as SBA-15 and MCM-41 silica, LDH (layered double hydroxide) (Mg3 Al-NO3 ) and MC (mesoporous carbon) have been comparatively evaluated for oral drug delivery applications, in order to broaden the range of matrices and implicitly to develop the class of drug delivery systems based on diffusion mechanism. As well known, methotrexate (MTX) is used widely to treat various neoplastic diseases such as acute lymphoblast leukemia, lymphoma and solid cancers and autoimmune diseases such as psoriasis and rheumatoid arthritis. The commercially available formulations of this drug have disadvantages due to the traditional release process that occurs in the body. Thus, this work is focused on the long-term controlled MTX delivery because this one could eliminate over or underdosing, could maintain drug levels in desired range, could increase patient compliance and prevent the side effects. Therefore, the mesoporous materials are used and efficient MTX-delivery systems, based on above-mentioned mesoporous materials, are successfully prepared by intercalation. The obtained drug carriers were tested in the controlled MTX-drug release process and the influence of the pore morphology and geometry on MTX release profiles was extensively studied comparatively. The prepared MTX delivery systems were characterized by FTIR and UV–vis spectroscopy, N2 sorption measurements. Then, the data obtained from the in vitro release studies have been analyzed, and in order to evaluate the MTX-release mechanism and kinetics, the Korsmeyer–Peppas equation has been applied. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Due to their unique features, organic–inorganic nanocomposites have been recognized as one of the most promising research fields in materials chemistry. Intercalation reactions are particularly important as they can be utilized to dramatically change the chemical, electronic, optical, and magnetic properties of a host matrix [1]. Considering the increasing demands for effective drugs, some studies to develop drug delivery systems have been attempted. Recently, several reports proposed a wide range of bioinorganic hybrid systems, in which bio- or bio-functionalized molecules have been combined with inorganic mesoporous materials, as potential drug delivery systems [2]. The first proposed drug delivery system with a mesoporous structure was MCM-41 in 2001. Since then, the
∗ Corresponding author at: Petru Poni Institute of Macromolecular Chemistry, Aleea Grigore Ghica Voda 41a , 700487 Iasi, Romania. Tel.: +40 746505227; fax: +40 232 211299. E-mail address: [email protected] (M. Ignat). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.01.022
relationship between their textural and structural properties, as well as the drug adsorption/release properties have been described [3–5]. Generally, mesoporous materials have received much attention because of their applications, including the biomedical field. Therefore, mesoporous matrices are potential drug carriers due to their specific features [3] as: the ordered pore network that is important, due to its homogeneous size, allowing a high control of the drug loading and the release kinetics; the high total pore volume that allows to host pharmaceuticals; the high surface area, implying high potential for drug adsorption, and surface functional groups that allow a better control over drug loading and release. There are several reports that propose some bio-inorganic hybrid systems, in which bio or bio-functionalized molecules are combined with inorganic materials, as potential drug delivery systems. Such systems that meet al above described criteria are silica materials, as MCM-41 and SBA-15, and mesoporous carbon materials. Methotrexate is and anticancer drug that could be incorporated in an organic–inorganic nanocomposite, such as methotrexate – mesoporous matrix and its possible applications in a controlled release process, are listed below. For example, Renu et al.
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reported the effects of the chemical structure alterations of polyester-co-polyether (PEPE) dendrimers on the methotrexate (MTX) encapsulation and Singh and Hildgen investigated the release process of MTX [6]. Other authors reported the remarkable intercalation of MTX into layered double hydroxides (LDH) by a successfully ion-exchange technique [2,7]. Thematic argument approached in the present paper is given by the fact that the above-mentioned mesoporous materials are ideal matrices for drug delivery systems. To the best of our knowledge, these materials have not been tested yet as drug delivery systems for MTX [8]. Thus, these types of systems could provide highly efficient drug delivery and reduce the side effects, especially when dealing with drugs that are presumed to kill cancer cells but can also kill healthy cells. But, Thassu demonstrated that the adverse effects in such controlled release systems could be reduced or prevented [9]. The aim of this work is focused on the diversification of the nature of matrix-type mesoporous materials used for MTX drug delivery system preparation and their uses as single dose oral formulations with extended release.
mesoporous carbon was obtained after silica’s framework dissolution in HF. 2.4. Synthesis of MCM-41 matrices The synthesis of MCM-41 (using cetyl trimethylammonium bromide – CTAB) is based on the delayed neutralization process reported previously [15,16]. In a typical procedure, 15.5 g CTAB was added to distilled water. The suspension was heated and stirred vigorously until the clear solution is formed. Then fumed silica and 24 mL TEAOH (tetraethylammonium hydroxide) were added into the above solution and the obtained mixture was stirred continuously for 2 h at 70 ◦ C. After that, the solution was aged for 24 h at room temperature. The obtained gel was transferred in a Teflon autoclave and kept at 140 ◦ C for 48 h. The as-synthesized sample was air-dried at room temperature overnight and then calcined in air to remove the soft template at 550 ◦ C for 6 h (heating rate of 1 ◦ C/min). 2.5. Anticancer drug models
2. Materials and methods In this study, we used as host mesoporous matrices MgAl-LDH (layered double hydroxide), SBA-15 and MCM-41 (mesoporous silicas), and MC (mesoporous carbon). For the first time, mesoporous carbon material is involved in such system and studied in comparison with the rest of mesoporous materials. All mesoporous matrices were synthesized in our laboratory as follows.
MTX (methotrexate), purchased from Sigma–Aldrich (MFCD00150847) (CAS number 133073-73-1) is an allosteric inhibitor of dihydrofolate reductase (DHFR), the enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate. Since tetrahydrofolate is required for purine and pyrimidine synthesis, methotrexate treatment results in the inhibition of DNA and RNA synthesis.
2.1. Synthesis of MgAl-LDH matrices
2.6. MTX intercalation in mesoporous materials
0.3 mol of Mg(NO3 )2 ·6H2 O and 0.1 mol Al(NO3 )3 ·9H2 O dissolved in 200 mL of ultra-pure water, have been co-precipitated under N2 atmosphere to avoid, or at least minimize, the contamination with atmospheric CO2 . A solution of NH4 OH (35%) was simultaneously added to adjust the pH to 10 (±0.1). The resulting gel was aged under nitrogen atmosphere at 80 ◦ C for 24 h [10]. Then, the obtained mixture was filtered and the resulting white solid was extensively washed with ultra-pure water and left to dry overnight at 80 ◦ C.
In order to obtain the drug delivery systems LDH-MTX, SBA15-MTX, MCM-41-MTX, and MC-MTX, in a MTX solution have been introduced the inorganic matrices and the intercalation process into LDH structure occurred by anion exchange route and into SBA-15, MCM-41 and MC structures by impregnation process, respectively. By a gram of each type of dried mesoporous material was mixed with 100 mL of MTX aqueous solution (50 mg/L) and stirred vigorously at room temperature for 3 days under N2 atmosphere. The obtained mixture was filtered and the resulting solid was dried and used for subsequent investigations.
2.2. Synthesis of SBA-15 matrices The triblock copolymer Pluronic P123 (BTC-Benelux, La Hulpe, Belgium) was dissolved in HCl, 2 M and ultra-pure water. After 2 h of magnetic stirring, the TEOS (tetraethyl orthosilicate) was added. The mole ratio of components is SiO2 :P123:HCl:H2 O1 = 1.0:0.016:6.8:179. The obtained mixture was aged at 40 ◦ C under magnetic stirring [11–13], and after 24 h the temperature was raised up to 100 ◦ C for the next 48 h. Finally, the powder was filtered, washed with ultra-pure water, dried and calcined at 550 ◦ C for 6 h (heating rate of 1 K/min) in order to remove the organic from the pores. 2.3. Synthesis of MC matrices In a typical synthesis of mesoporous carbon, a solution obtained by mixing 1 mL of glycerol with 4 mL of water is added to 1 g of hot SBA-15 silica. A drop of H2 SO4 was added to the solution in order to ensure a better glycerol polymerization. After 1 h, the mixture was placed in the oven and kept at 100 ◦ C for 6 h, then at 160 ◦ C for another 6 h. The polymer–silica composite were pyrolyzed in a N2 flow at 800 ◦ C (heating rate of 1 ◦ C/min) and kept under these conditions for 5 h to carbonize the polymer [14]. The
2.7. Characterization The infrared spectra were obtained on a SPECORD Carl Zeiss Jena FT-IR spectrometer (KBr pellets). The morphology and particle sizes of the synthesized mesoporous matrices have been investigated and the obtained results are presented in Supplementary information. SEM images were obtained with a QuantaTM Scanning Electron Microscope (operating at an accelerating voltage of 30 kV) and Vega II LSH microscope (with accelerating voltage of 30 kV-Tescan Company). Size distribution and mean diameter measurements of particles were recorded using a SALD-7001 type Laser Diffraction Particle Size Analyzer (Shimadzu, Japan). Porosity and surface areas were performed on a NOVA 2200e system using nitrogen as the absorbate at liquid nitrogen temperature (−196 ◦ C). All samples were out-gassed under vacuum, for 6 h at room temperature before adsorption measurements. The surface areas were calculated using the BET (Brunauer–Emmet–Teller) equation in the range of relative pressure of 0.05–0.35. Pore volume was taken at the relative pressure value of 0.95. Pore size distributions were calculated from the adsorption branches of
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Fig. 1. The FT-IR spectra.
the N2 sorption isotherms using the BJH (Barett–Joyner–Halenda) model. UV–vis spectroscopy measurements were carried out using a Shimadzu, Japan spectrophotometer equipped with an integrating sphere attachment. The analysis range was from 200 to 800 nm. 2.8. In vitro drug release In all experiments, the release profile of MTX was obtained by soaking drug-loaded powders in a solution of simulated intestinal fluid (pH = 7.4, aqueous solution of PBS – phosphate buffer saline). Updated literature studies show that at pH = 5, a rapid methotrexate release is registered, while at pH = 7, slower drug releases result [17]. The MTX release profiles of LDH-MTX, SBA-15-MTX, MCM-41MTX and MC-MTX systems in simulated media was performed by adding 0.1 g of solid in 100 mL of PBS. The mixture was continuously stirred (75 rpm) and kept at 37 ± 1 ◦ C for a period of 30 h. A sample of 10 mL was withdrawn at predetermined intervals and centrifuged [18–20] and the concentration of the released MTX was measured using UV-Vis spectrophotometer at 257.5 nm.
Fig. 2. UV–vis absorption spectra for MTX solutions after removal of solid phase: (A) MTX-commercial drug, (B) MCM-41-MTX, (C) LDH-MTX, (D) MC-MTX and (E) SBA-15-MTX.
MTX molecule. As result, the FT-IR spectra demonstrate that MTX has been incorporated into mesoporous matrices. 3.2. Nitrogen adsorption/desorption isotherms The obtained results from the nitrogen adsorption/desorption isotherms and the corresponding pore size distributions for LDH, MCM-41, SBA-15 and MC host matrices, before and after MTX loadings, were systematized in Table 1. As could be observed, for all systems, the specific surface areas, the total pore volumes and the pore sizes of the host matrices decrease when MTX is loaded. This conclusion is available for all systems, meaning that the pores of the host matrices were occupied by MTX molecules. This confirms that MTX was intercalated between brucite layers of MgAl-LDH and loaded inside of the mesoporous channels of MCM-41, SBA-15 and MC networks.
3. Results and discussion 3.3. UV–vis absorption spectra 3.1. FT-IR measurements The FT-IR study is based on the fact that MTX molecules have specific functional groups which are characterized by specific rotational and/or vibrational frequencies, corresponding to discrete energy levels (vibration modes) [21]. FT-IR spectra of the MTXloaded matrices, further gave a direct demonstration of that the MTX is present in all four systems (LDH-MTX, SBA-15-MTX, MCM41-MTX and MC-MTX). Therefore, all IR spectra exhibit peaks that are characteristic for MTX drug molecules (Fig. 1). The FT-IR spectra of all samples show a broad band around 3580 cm−1 , due to OH stretching of water. After loading, the MTX characteristic absorption bands appear in the IR spectra. The presence of the peak at 1274 cm−1 is due to the asymmetric and symmetric CH3 bending vibrations, and in the low-frequency region, the presence of the band at 1201 cm−1 is attributed to C O vibration. A weak shoulder appears also at 1616 cm−1 that could be assigned to the C O and NH stretching, all characteristic to the
Fig. 2 shows the UV–vis absorption spectra of MTX solutions after loadings on mesoporous materials used as host matrices. The spectrum of the initial MTX solution was compared with the spectra obtained on the solutions after being in contact with solid mesoporous matrices. The decreasing concentration of MTX drug in the solutions support that MTX molecules have been retained on mesoporous materials. As could be observed, SBA-15 silica retained the largest amount of MTX, while MCM-41 silica retained the smallest amount. This is in good concordance with the nitrogen sorption measurements and the retained amount of MTX drug increase with the increasing pore diameter [22] in the series MCM41 < LDH < CM < SBA-15. 3.4. Toxicity of the synthesized materials Using the Kärber method, in a pharmacodynamic laboratory, the toxicities of the unloaded and MTX-loaded synthesized
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Table 1 Textural properties of simple supports and drug delivery systems and the toxicity values. Sample
Surface area (m2 /g)
Pore volume (cm3 /g)
Pore diameter (nm)
DL50 (mg/kg body)
LDH LDH-MTX SBA-15 SBA-15-MTX MCM-41 MCM-41-MTX MC MC-MTX
60.33 42.52 794.5 466.3 1310.1 583.6 1022.6 964.3
0.06 0.01 1.07 0.68 0.31 0.27 1.5 1.14
2.66 2.41 3.79 3.32 1.87 1.18 3.28 2.38
7410 7465 7369 7345 7260 7165 7410 7365
materials were determined and their values are listed in Table 1. The obtained values are in the range of 5000–15,000 mg/kg (Hodge and Sterner toxicity scale) meaning that the prepared host matrices and nanocomposites are practically non-toxic [23]. 3.5. Drug release results The main experimental part of this work is based on the compared drug release processes, in a synthetic stomach media as PBS (phosphate buffer solution) [24], from the synthesized nanocomposite systems. Also, with respect to the pharmaceutical formulations of MTX, a compared study has been done. The obtained results for the MTX-release process demonstrate that MTX-encapsulated LDH, SBA-15, MCM-41 and MC nanomaterials could be used as efficient controlled drug delivery systems. The prepared nanocomposites have been used as drug delivery systems attempting to control drug concentrations in the target media with the aim of maintaining the constant level of MTX-drug in stomach over an extended period. For example, in Fig. 3, is shown the methotrexate release rate from nanostructured systems in PBS. From Fig. 3, it is easily observed that even after 20 h of release process, the MTX-drug continues to be released. The released MTX quantities, after 4 h of release, were found to be: 18.6 wt.% for SBA-15-MTX, 22.6 wt.% for LDH-MTX, 30.5 wt.% MCM-41-MTX, 21.8 wt.% for MC-MTX, and 54.2 wt.% for the commercial MTX. But, after 6 h, the quantity of released MTX from the mesoporous matrices is lower than that from the commercially available drug system. So, we conclude that the mesoporous
Fig. 3. Release synthetic in stomach media (PBS).
materials synthesized during this study are suitable as drug carriers, as well as controllable drug delivery systems. The best MTX delivery systems, according to our study, are the SBA-15 and LDH matrices. This could be explained by the formation of stronger bonds between MTX and SBA-15/LDH framework than in the case of MCM-41 or MC. But, the last two host matrices may provide a longer constant release, allowing a better control of therapeutic effect. In order to evaluate the kinetics and mechanism of MTX release, we applied the Korsmeyer–Peppas [25] and Higuchi [26] equations to the obtained results. Fig. 4 shows the graphical representation of the released MTX vs time, from nanocomposite systems prepared by us comparative to
Fig. 4. Graphic representation of “n” using the Korsmeyer–Peppas model F = ktn .
I.F. Alexa et al. / Colloids and Surfaces B: Biointerfaces 106 (2013) 135–139
Fig. 5. Higuchi release model of MTX sustained release formulation. Table 2 Release parameters of MTX sustained release tablets. Systems
Higuchi
Korsmeyer–Peppas
Parameters in PBS
R2
KH (h−1/2 )
R2
Value “n”
K (h−n )
MCM-41-MTX LDH-MTX MC-MTX SBA-15-MTX
0.9262 0.9058 0.9091 0.9298
16.71 13.70 14.99 8.493
0.9860 0.9169 0.9677 0.9482
0.74 0.84 0.88 0.92
0.187 0.178 0.135 0.194
commercial MTX formulation. In order to find out the mechanism of MTX release, the first 60% of MTX release data (at pH = 7.4) are fitted with the Korsmeyer–Peppas model (F = ktn ) (Fig. 4) and the calculated “n” value revealed that the MTX release mechanism of the commercial formulation (n = 0.54) indicates anomalous diffusion or non-fickian diffusion mechanism [27]. The same conclusion is available for the MTX-delivery systems as MCM-41-MTX (n = 0.74), LDH-MTX (n = 0.84), and MC-MTX (n = 0.88). In the case of SBA-15MTX system, the value of n is about 0.92, meaning that the MTX release mechanism occurs through the super case II transport. It is observed that, as the “n” exponent increases, the released quantity of MTX decreases. Applying the Higuchi kinetic model, Fig. 5 shows the plots giving the linearization curves. The calculated correlation coefficients (R2 ) are summarized in Table 2 and compared with the values of the same parameter obtained by the Korsmeyer–Peppas model. It can be concluded that, the release profiles of the MTX nanocomposite systems prepared in this work, could be best explained by Korsmeyer–Peppas model, where the correlation coefficient R2 values are a little bit higher: 0.9482 (SBA-15-MTX), 0.9677 (MC-MTX), 0.9169 (LDH-MTX) and 0.9860 (MCM-41-MTX), respectively, than that in the case of Higuchi model. 4. Conclusions The aim of the present study was to determine the feasibility of the applicable MTX-mesoporous materials nanocomposites as intercalated storage and transport materials, and mainly as controlled drug delivery systems. This aim has been achieved first by determining the adsorption capacities of the purposed mesoporous materials for MTX-drug. It was observed that, SBA-15 and MC mesoporous materials have significantly higher adsorption capacities, than the ones achieved with LDH and MCM-41 matrices. The enhanced anticancer efficacy of MTX with SBA-15, MCM-41, LDH and MC has been studied with respect to the drug concentration and incubation time. It was found that the obtained nanocomposite delivery systems have a very low toxicity, and based on that we are able to confirm that the used matrices, as SBA15, MCM-41, LDH and MC have low toxicity, and they do not harm the healthily or cancer cells. Therefore, the biocompatible SBA-15,
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MCM-41, LDH and MC are excellent host materials for encapsulating various anticancer drugs (as MTX in this work) and can play a role as a non-viral delivery carrier for target delivery and/or controlled release. MTX-drug release kinetics of the prepared formulations fit best to Korsmeyer–Peppas mathematical model and drug release mechanism, due to the “n” value, appears to be a complex mechanism of swelling, diffusion and erosion. In a system of long-term controlled release, individuals could reduce the amount of ingested drugs, reducing the stress factor and improving their quality of life. In the near future, in order to demonstrate that the synthesized nanocomposites are perfect controlled MTX-delivery systems, we plan to test them in vivo, then to perform a large number of pharmacological tests in rats and analysis of macrophages from mousses. Acknowledgments This work was supported by the European Social Fund in Romania, under the responsibility of the Managing Authority for the POSDRU 2007–2013 [grant POSDRU/88/1.5/S/47646]. Part of the research leading to the conclusions of this work has received funding from the European Union’s Seventh Framework Programmer (FP7/2007–2013) under grant agreement no. 264115 – STREAM. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.01.022. References [1] J.R. Bertino, Ode to methotrexate, J. Clin. Oncol. 11 (1993) 5. [2] J.H. Choy, S.Y. Kwak, Y.J. Jeong, J.S. Park, Angew. Chem. Int. Ed. 39 (2000) 4041–4045. [3] M. Vallet-Regí, F. Balas, D. Arcos, Angew. Chem. Int. Ed. 46 (2007) 7548–7558. [4] M. Vallet-Regi, J. Intern. Med. 267 (2009) 22–43. ˜ J. Román, M.V. Cabanas, ˜ R.M. Vallet, Acta Biomater. 6 (2010) 1288–1296. [5] J. Pena, [6] R.D. Singh, P. Hildgen, Biomaterials 28 (2007) 3140–3152. [7] J.M. Oh, M. Park, T.K. Sang, J.J. Young, G.K. Yong, J.C. Ho, J. Phys. Chem. Solids 67 (2006) 1024–1027. [8] S. Wang, Microporous Mesoporous Mater. 117 (2009) 1–9. [9] D. Thassu, M. Deleers, Y. Pathak, Dugs and Pharmaceutical Sciences, Informa Healthcare USA, Inc., New York, 2007, p 6. [10] I.F. Alexa, R.F. Popovici, M. Ignat, E. Popovici, V.A. Voicu, Digest J. Nanomater. Biostruct. 6 (2011) 1091–1101. [11] R.F. Popovici, E.M. Seftel, G.D. Mihai, E. Popovici, A.V. Voicu, J. Pharm. Sci. 100 (2011) 704–714. [12] R.F. Popovici, I.F. Alexa, O. Novac, N. Vrinceanu, E. Popovici, C.E. Lupusoru, A.V. Voicu, Digest J. Nanomater. Biostruct. 6 (2011) 1619–1630. [13] I.F. Alexa, M. Ignat, R.F. Popovici, D. Timpu, E. Popovici, Int. J. Pharm. 436 (2012) 111–119. [14] M. Ignat, C.J. VanOers, J. Vernimmen, M. Mertensc, S. Potgieter-Vermaak, V. Meynen, E. Popovici, P. Cool, Carbon 48 (2010) 1609–1618. [15] K. Ulbrich, V. Subr, Adv. Drug Deliv. Rev. 56 (2004) 1023–1050. [16] H.P. Lin, C.Y. Mou, Science 273 (1996) 765–768. [17] Z. Ying, J. Tuo, X.Z. Ren, Colloids Surf. B: Biointerfaces 44 (2005) 104–109. [18] L. Tao, K.E. Uhrich, J. Colloid Interface Sci. 298 (2006) 102–110. [19] C. Salerno, A.M. Carlucci, C. Bregni, AAPS PharmSciTech 11 (2010) 986–993. [20] T. Wang, M. Li, H. Gao, W. Yan, J. Colloid Interface Sci. 353 (2011) 107–115. [21] H.S. Dong, I.J. Young, G.K. Don, J.J. Min, J.M. Kyeong, N.J. Woon, Colloids Surf. B: Biointerfaces 69 (2009) 157–163. [22] R. Mellaerts, C.A. Aerts, J.V. Humbeeck, P. Augustijns, G.V. Mooter, J.A.R. Martens, R. Mols, J.A.G. Jammaer, C.A. Aerts, P. Annaert, V.J. Humbeeck, Eur. J. Pharm. Biopharm. 69 (2008) 223. [23] http://www.ccohs.ca/oshanswers/chemicals/ld50.html [24] W. Zhou, J. Feijen, J. Control. Release 132 (2008) e35–e36. [25] S. Dash, P.N. Murthy, P. Chowdhury, L. Nath, Acta Poloniae Pharm. – Drug Res. 67 (2010) 217–223. [26] A. Hahn, G. Brandes, P. Wagener, S. Barcikowski, J. Control. Release 154 (2011) 164–170. [27] C. Ferrero, D. Massuelle, E. Doelker, J. Control. Release 141 (2010) 223–233.
Contents lists available at SciVerse ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A comparative study on long-term MTX controlled release from intercalated nanocomposites for nanomedicine applications Iuliana Florentina Alexa a , Cristina Giorgiana Pastravanu a , Maria Ignat a,b,∗ , Evelini Popovici a a b
“Alexandru Ioan Cuza” University, Faculty of Chemistry, Iasi, Romania Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania
a r t i c l e
i n f o
Article history: Received 26 September 2012 Received in revised form 17 December 2012 Accepted 15 January 2013 Available online 23 January 2013 Keywords: Mesoporous materials LDH Drug delivery Methotrexate Sustained release
a b s t r a c t The feasibility of some mesoporous materials such as SBA-15 and MCM-41 silica, LDH (layered double hydroxide) (Mg3 Al-NO3 ) and MC (mesoporous carbon) have been comparatively evaluated for oral drug delivery applications, in order to broaden the range of matrices and implicitly to develop the class of drug delivery systems based on diffusion mechanism. As well known, methotrexate (MTX) is used widely to treat various neoplastic diseases such as acute lymphoblast leukemia, lymphoma and solid cancers and autoimmune diseases such as psoriasis and rheumatoid arthritis. The commercially available formulations of this drug have disadvantages due to the traditional release process that occurs in the body. Thus, this work is focused on the long-term controlled MTX delivery because this one could eliminate over or underdosing, could maintain drug levels in desired range, could increase patient compliance and prevent the side effects. Therefore, the mesoporous materials are used and efficient MTX-delivery systems, based on above-mentioned mesoporous materials, are successfully prepared by intercalation. The obtained drug carriers were tested in the controlled MTX-drug release process and the influence of the pore morphology and geometry on MTX release profiles was extensively studied comparatively. The prepared MTX delivery systems were characterized by FTIR and UV–vis spectroscopy, N2 sorption measurements. Then, the data obtained from the in vitro release studies have been analyzed, and in order to evaluate the MTX-release mechanism and kinetics, the Korsmeyer–Peppas equation has been applied. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Due to their unique features, organic–inorganic nanocomposites have been recognized as one of the most promising research fields in materials chemistry. Intercalation reactions are particularly important as they can be utilized to dramatically change the chemical, electronic, optical, and magnetic properties of a host matrix [1]. Considering the increasing demands for effective drugs, some studies to develop drug delivery systems have been attempted. Recently, several reports proposed a wide range of bioinorganic hybrid systems, in which bio- or bio-functionalized molecules have been combined with inorganic mesoporous materials, as potential drug delivery systems [2]. The first proposed drug delivery system with a mesoporous structure was MCM-41 in 2001. Since then, the
∗ Corresponding author at: Petru Poni Institute of Macromolecular Chemistry, Aleea Grigore Ghica Voda 41a , 700487 Iasi, Romania. Tel.: +40 746505227; fax: +40 232 211299. E-mail address: [email protected] (M. Ignat). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.01.022
relationship between their textural and structural properties, as well as the drug adsorption/release properties have been described [3–5]. Generally, mesoporous materials have received much attention because of their applications, including the biomedical field. Therefore, mesoporous matrices are potential drug carriers due to their specific features [3] as: the ordered pore network that is important, due to its homogeneous size, allowing a high control of the drug loading and the release kinetics; the high total pore volume that allows to host pharmaceuticals; the high surface area, implying high potential for drug adsorption, and surface functional groups that allow a better control over drug loading and release. There are several reports that propose some bio-inorganic hybrid systems, in which bio or bio-functionalized molecules are combined with inorganic materials, as potential drug delivery systems. Such systems that meet al above described criteria are silica materials, as MCM-41 and SBA-15, and mesoporous carbon materials. Methotrexate is and anticancer drug that could be incorporated in an organic–inorganic nanocomposite, such as methotrexate – mesoporous matrix and its possible applications in a controlled release process, are listed below. For example, Renu et al.
136
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reported the effects of the chemical structure alterations of polyester-co-polyether (PEPE) dendrimers on the methotrexate (MTX) encapsulation and Singh and Hildgen investigated the release process of MTX [6]. Other authors reported the remarkable intercalation of MTX into layered double hydroxides (LDH) by a successfully ion-exchange technique [2,7]. Thematic argument approached in the present paper is given by the fact that the above-mentioned mesoporous materials are ideal matrices for drug delivery systems. To the best of our knowledge, these materials have not been tested yet as drug delivery systems for MTX [8]. Thus, these types of systems could provide highly efficient drug delivery and reduce the side effects, especially when dealing with drugs that are presumed to kill cancer cells but can also kill healthy cells. But, Thassu demonstrated that the adverse effects in such controlled release systems could be reduced or prevented [9]. The aim of this work is focused on the diversification of the nature of matrix-type mesoporous materials used for MTX drug delivery system preparation and their uses as single dose oral formulations with extended release.
mesoporous carbon was obtained after silica’s framework dissolution in HF. 2.4. Synthesis of MCM-41 matrices The synthesis of MCM-41 (using cetyl trimethylammonium bromide – CTAB) is based on the delayed neutralization process reported previously [15,16]. In a typical procedure, 15.5 g CTAB was added to distilled water. The suspension was heated and stirred vigorously until the clear solution is formed. Then fumed silica and 24 mL TEAOH (tetraethylammonium hydroxide) were added into the above solution and the obtained mixture was stirred continuously for 2 h at 70 ◦ C. After that, the solution was aged for 24 h at room temperature. The obtained gel was transferred in a Teflon autoclave and kept at 140 ◦ C for 48 h. The as-synthesized sample was air-dried at room temperature overnight and then calcined in air to remove the soft template at 550 ◦ C for 6 h (heating rate of 1 ◦ C/min). 2.5. Anticancer drug models
2. Materials and methods In this study, we used as host mesoporous matrices MgAl-LDH (layered double hydroxide), SBA-15 and MCM-41 (mesoporous silicas), and MC (mesoporous carbon). For the first time, mesoporous carbon material is involved in such system and studied in comparison with the rest of mesoporous materials. All mesoporous matrices were synthesized in our laboratory as follows.
MTX (methotrexate), purchased from Sigma–Aldrich (MFCD00150847) (CAS number 133073-73-1) is an allosteric inhibitor of dihydrofolate reductase (DHFR), the enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate. Since tetrahydrofolate is required for purine and pyrimidine synthesis, methotrexate treatment results in the inhibition of DNA and RNA synthesis.
2.1. Synthesis of MgAl-LDH matrices
2.6. MTX intercalation in mesoporous materials
0.3 mol of Mg(NO3 )2 ·6H2 O and 0.1 mol Al(NO3 )3 ·9H2 O dissolved in 200 mL of ultra-pure water, have been co-precipitated under N2 atmosphere to avoid, or at least minimize, the contamination with atmospheric CO2 . A solution of NH4 OH (35%) was simultaneously added to adjust the pH to 10 (±0.1). The resulting gel was aged under nitrogen atmosphere at 80 ◦ C for 24 h [10]. Then, the obtained mixture was filtered and the resulting white solid was extensively washed with ultra-pure water and left to dry overnight at 80 ◦ C.
In order to obtain the drug delivery systems LDH-MTX, SBA15-MTX, MCM-41-MTX, and MC-MTX, in a MTX solution have been introduced the inorganic matrices and the intercalation process into LDH structure occurred by anion exchange route and into SBA-15, MCM-41 and MC structures by impregnation process, respectively. By a gram of each type of dried mesoporous material was mixed with 100 mL of MTX aqueous solution (50 mg/L) and stirred vigorously at room temperature for 3 days under N2 atmosphere. The obtained mixture was filtered and the resulting solid was dried and used for subsequent investigations.
2.2. Synthesis of SBA-15 matrices The triblock copolymer Pluronic P123 (BTC-Benelux, La Hulpe, Belgium) was dissolved in HCl, 2 M and ultra-pure water. After 2 h of magnetic stirring, the TEOS (tetraethyl orthosilicate) was added. The mole ratio of components is SiO2 :P123:HCl:H2 O1 = 1.0:0.016:6.8:179. The obtained mixture was aged at 40 ◦ C under magnetic stirring [11–13], and after 24 h the temperature was raised up to 100 ◦ C for the next 48 h. Finally, the powder was filtered, washed with ultra-pure water, dried and calcined at 550 ◦ C for 6 h (heating rate of 1 K/min) in order to remove the organic from the pores. 2.3. Synthesis of MC matrices In a typical synthesis of mesoporous carbon, a solution obtained by mixing 1 mL of glycerol with 4 mL of water is added to 1 g of hot SBA-15 silica. A drop of H2 SO4 was added to the solution in order to ensure a better glycerol polymerization. After 1 h, the mixture was placed in the oven and kept at 100 ◦ C for 6 h, then at 160 ◦ C for another 6 h. The polymer–silica composite were pyrolyzed in a N2 flow at 800 ◦ C (heating rate of 1 ◦ C/min) and kept under these conditions for 5 h to carbonize the polymer [14]. The
2.7. Characterization The infrared spectra were obtained on a SPECORD Carl Zeiss Jena FT-IR spectrometer (KBr pellets). The morphology and particle sizes of the synthesized mesoporous matrices have been investigated and the obtained results are presented in Supplementary information. SEM images were obtained with a QuantaTM Scanning Electron Microscope (operating at an accelerating voltage of 30 kV) and Vega II LSH microscope (with accelerating voltage of 30 kV-Tescan Company). Size distribution and mean diameter measurements of particles were recorded using a SALD-7001 type Laser Diffraction Particle Size Analyzer (Shimadzu, Japan). Porosity and surface areas were performed on a NOVA 2200e system using nitrogen as the absorbate at liquid nitrogen temperature (−196 ◦ C). All samples were out-gassed under vacuum, for 6 h at room temperature before adsorption measurements. The surface areas were calculated using the BET (Brunauer–Emmet–Teller) equation in the range of relative pressure of 0.05–0.35. Pore volume was taken at the relative pressure value of 0.95. Pore size distributions were calculated from the adsorption branches of
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Fig. 1. The FT-IR spectra.
the N2 sorption isotherms using the BJH (Barett–Joyner–Halenda) model. UV–vis spectroscopy measurements were carried out using a Shimadzu, Japan spectrophotometer equipped with an integrating sphere attachment. The analysis range was from 200 to 800 nm. 2.8. In vitro drug release In all experiments, the release profile of MTX was obtained by soaking drug-loaded powders in a solution of simulated intestinal fluid (pH = 7.4, aqueous solution of PBS – phosphate buffer saline). Updated literature studies show that at pH = 5, a rapid methotrexate release is registered, while at pH = 7, slower drug releases result [17]. The MTX release profiles of LDH-MTX, SBA-15-MTX, MCM-41MTX and MC-MTX systems in simulated media was performed by adding 0.1 g of solid in 100 mL of PBS. The mixture was continuously stirred (75 rpm) and kept at 37 ± 1 ◦ C for a period of 30 h. A sample of 10 mL was withdrawn at predetermined intervals and centrifuged [18–20] and the concentration of the released MTX was measured using UV-Vis spectrophotometer at 257.5 nm.
Fig. 2. UV–vis absorption spectra for MTX solutions after removal of solid phase: (A) MTX-commercial drug, (B) MCM-41-MTX, (C) LDH-MTX, (D) MC-MTX and (E) SBA-15-MTX.
MTX molecule. As result, the FT-IR spectra demonstrate that MTX has been incorporated into mesoporous matrices. 3.2. Nitrogen adsorption/desorption isotherms The obtained results from the nitrogen adsorption/desorption isotherms and the corresponding pore size distributions for LDH, MCM-41, SBA-15 and MC host matrices, before and after MTX loadings, were systematized in Table 1. As could be observed, for all systems, the specific surface areas, the total pore volumes and the pore sizes of the host matrices decrease when MTX is loaded. This conclusion is available for all systems, meaning that the pores of the host matrices were occupied by MTX molecules. This confirms that MTX was intercalated between brucite layers of MgAl-LDH and loaded inside of the mesoporous channels of MCM-41, SBA-15 and MC networks.
3. Results and discussion 3.3. UV–vis absorption spectra 3.1. FT-IR measurements The FT-IR study is based on the fact that MTX molecules have specific functional groups which are characterized by specific rotational and/or vibrational frequencies, corresponding to discrete energy levels (vibration modes) [21]. FT-IR spectra of the MTXloaded matrices, further gave a direct demonstration of that the MTX is present in all four systems (LDH-MTX, SBA-15-MTX, MCM41-MTX and MC-MTX). Therefore, all IR spectra exhibit peaks that are characteristic for MTX drug molecules (Fig. 1). The FT-IR spectra of all samples show a broad band around 3580 cm−1 , due to OH stretching of water. After loading, the MTX characteristic absorption bands appear in the IR spectra. The presence of the peak at 1274 cm−1 is due to the asymmetric and symmetric CH3 bending vibrations, and in the low-frequency region, the presence of the band at 1201 cm−1 is attributed to C O vibration. A weak shoulder appears also at 1616 cm−1 that could be assigned to the C O and NH stretching, all characteristic to the
Fig. 2 shows the UV–vis absorption spectra of MTX solutions after loadings on mesoporous materials used as host matrices. The spectrum of the initial MTX solution was compared with the spectra obtained on the solutions after being in contact with solid mesoporous matrices. The decreasing concentration of MTX drug in the solutions support that MTX molecules have been retained on mesoporous materials. As could be observed, SBA-15 silica retained the largest amount of MTX, while MCM-41 silica retained the smallest amount. This is in good concordance with the nitrogen sorption measurements and the retained amount of MTX drug increase with the increasing pore diameter [22] in the series MCM41 < LDH < CM < SBA-15. 3.4. Toxicity of the synthesized materials Using the Kärber method, in a pharmacodynamic laboratory, the toxicities of the unloaded and MTX-loaded synthesized
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Table 1 Textural properties of simple supports and drug delivery systems and the toxicity values. Sample
Surface area (m2 /g)
Pore volume (cm3 /g)
Pore diameter (nm)
DL50 (mg/kg body)
LDH LDH-MTX SBA-15 SBA-15-MTX MCM-41 MCM-41-MTX MC MC-MTX
60.33 42.52 794.5 466.3 1310.1 583.6 1022.6 964.3
0.06 0.01 1.07 0.68 0.31 0.27 1.5 1.14
2.66 2.41 3.79 3.32 1.87 1.18 3.28 2.38
7410 7465 7369 7345 7260 7165 7410 7365
materials were determined and their values are listed in Table 1. The obtained values are in the range of 5000–15,000 mg/kg (Hodge and Sterner toxicity scale) meaning that the prepared host matrices and nanocomposites are practically non-toxic [23]. 3.5. Drug release results The main experimental part of this work is based on the compared drug release processes, in a synthetic stomach media as PBS (phosphate buffer solution) [24], from the synthesized nanocomposite systems. Also, with respect to the pharmaceutical formulations of MTX, a compared study has been done. The obtained results for the MTX-release process demonstrate that MTX-encapsulated LDH, SBA-15, MCM-41 and MC nanomaterials could be used as efficient controlled drug delivery systems. The prepared nanocomposites have been used as drug delivery systems attempting to control drug concentrations in the target media with the aim of maintaining the constant level of MTX-drug in stomach over an extended period. For example, in Fig. 3, is shown the methotrexate release rate from nanostructured systems in PBS. From Fig. 3, it is easily observed that even after 20 h of release process, the MTX-drug continues to be released. The released MTX quantities, after 4 h of release, were found to be: 18.6 wt.% for SBA-15-MTX, 22.6 wt.% for LDH-MTX, 30.5 wt.% MCM-41-MTX, 21.8 wt.% for MC-MTX, and 54.2 wt.% for the commercial MTX. But, after 6 h, the quantity of released MTX from the mesoporous matrices is lower than that from the commercially available drug system. So, we conclude that the mesoporous
Fig. 3. Release synthetic in stomach media (PBS).
materials synthesized during this study are suitable as drug carriers, as well as controllable drug delivery systems. The best MTX delivery systems, according to our study, are the SBA-15 and LDH matrices. This could be explained by the formation of stronger bonds between MTX and SBA-15/LDH framework than in the case of MCM-41 or MC. But, the last two host matrices may provide a longer constant release, allowing a better control of therapeutic effect. In order to evaluate the kinetics and mechanism of MTX release, we applied the Korsmeyer–Peppas [25] and Higuchi [26] equations to the obtained results. Fig. 4 shows the graphical representation of the released MTX vs time, from nanocomposite systems prepared by us comparative to
Fig. 4. Graphic representation of “n” using the Korsmeyer–Peppas model F = ktn .
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Fig. 5. Higuchi release model of MTX sustained release formulation. Table 2 Release parameters of MTX sustained release tablets. Systems
Higuchi
Korsmeyer–Peppas
Parameters in PBS
R2
KH (h−1/2 )
R2
Value “n”
K (h−n )
MCM-41-MTX LDH-MTX MC-MTX SBA-15-MTX
0.9262 0.9058 0.9091 0.9298
16.71 13.70 14.99 8.493
0.9860 0.9169 0.9677 0.9482
0.74 0.84 0.88 0.92
0.187 0.178 0.135 0.194
commercial MTX formulation. In order to find out the mechanism of MTX release, the first 60% of MTX release data (at pH = 7.4) are fitted with the Korsmeyer–Peppas model (F = ktn ) (Fig. 4) and the calculated “n” value revealed that the MTX release mechanism of the commercial formulation (n = 0.54) indicates anomalous diffusion or non-fickian diffusion mechanism [27]. The same conclusion is available for the MTX-delivery systems as MCM-41-MTX (n = 0.74), LDH-MTX (n = 0.84), and MC-MTX (n = 0.88). In the case of SBA-15MTX system, the value of n is about 0.92, meaning that the MTX release mechanism occurs through the super case II transport. It is observed that, as the “n” exponent increases, the released quantity of MTX decreases. Applying the Higuchi kinetic model, Fig. 5 shows the plots giving the linearization curves. The calculated correlation coefficients (R2 ) are summarized in Table 2 and compared with the values of the same parameter obtained by the Korsmeyer–Peppas model. It can be concluded that, the release profiles of the MTX nanocomposite systems prepared in this work, could be best explained by Korsmeyer–Peppas model, where the correlation coefficient R2 values are a little bit higher: 0.9482 (SBA-15-MTX), 0.9677 (MC-MTX), 0.9169 (LDH-MTX) and 0.9860 (MCM-41-MTX), respectively, than that in the case of Higuchi model. 4. Conclusions The aim of the present study was to determine the feasibility of the applicable MTX-mesoporous materials nanocomposites as intercalated storage and transport materials, and mainly as controlled drug delivery systems. This aim has been achieved first by determining the adsorption capacities of the purposed mesoporous materials for MTX-drug. It was observed that, SBA-15 and MC mesoporous materials have significantly higher adsorption capacities, than the ones achieved with LDH and MCM-41 matrices. The enhanced anticancer efficacy of MTX with SBA-15, MCM-41, LDH and MC has been studied with respect to the drug concentration and incubation time. It was found that the obtained nanocomposite delivery systems have a very low toxicity, and based on that we are able to confirm that the used matrices, as SBA15, MCM-41, LDH and MC have low toxicity, and they do not harm the healthily or cancer cells. Therefore, the biocompatible SBA-15,
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MCM-41, LDH and MC are excellent host materials for encapsulating various anticancer drugs (as MTX in this work) and can play a role as a non-viral delivery carrier for target delivery and/or controlled release. MTX-drug release kinetics of the prepared formulations fit best to Korsmeyer–Peppas mathematical model and drug release mechanism, due to the “n” value, appears to be a complex mechanism of swelling, diffusion and erosion. In a system of long-term controlled release, individuals could reduce the amount of ingested drugs, reducing the stress factor and improving their quality of life. In the near future, in order to demonstrate that the synthesized nanocomposites are perfect controlled MTX-delivery systems, we plan to test them in vivo, then to perform a large number of pharmacological tests in rats and analysis of macrophages from mousses. Acknowledgments This work was supported by the European Social Fund in Romania, under the responsibility of the Managing Authority for the POSDRU 2007–2013 [grant POSDRU/88/1.5/S/47646]. Part of the research leading to the conclusions of this work has received funding from the European Union’s Seventh Framework Programmer (FP7/2007–2013) under grant agreement no. 264115 – STREAM. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.01.022. References [1] J.R. Bertino, Ode to methotrexate, J. Clin. Oncol. 11 (1993) 5. [2] J.H. Choy, S.Y. Kwak, Y.J. Jeong, J.S. Park, Angew. Chem. Int. Ed. 39 (2000) 4041–4045. [3] M. Vallet-Regí, F. Balas, D. Arcos, Angew. Chem. Int. Ed. 46 (2007) 7548–7558. [4] M. Vallet-Regi, J. Intern. Med. 267 (2009) 22–43. ˜ J. Román, M.V. Cabanas, ˜ R.M. Vallet, Acta Biomater. 6 (2010) 1288–1296. [5] J. Pena, [6] R.D. Singh, P. Hildgen, Biomaterials 28 (2007) 3140–3152. [7] J.M. Oh, M. Park, T.K. Sang, J.J. Young, G.K. Yong, J.C. Ho, J. Phys. Chem. Solids 67 (2006) 1024–1027. [8] S. Wang, Microporous Mesoporous Mater. 117 (2009) 1–9. [9] D. Thassu, M. Deleers, Y. Pathak, Dugs and Pharmaceutical Sciences, Informa Healthcare USA, Inc., New York, 2007, p 6. [10] I.F. Alexa, R.F. Popovici, M. Ignat, E. Popovici, V.A. Voicu, Digest J. Nanomater. Biostruct. 6 (2011) 1091–1101. [11] R.F. Popovici, E.M. Seftel, G.D. Mihai, E. Popovici, A.V. Voicu, J. Pharm. Sci. 100 (2011) 704–714. [12] R.F. Popovici, I.F. Alexa, O. Novac, N. Vrinceanu, E. Popovici, C.E. Lupusoru, A.V. Voicu, Digest J. Nanomater. Biostruct. 6 (2011) 1619–1630. [13] I.F. Alexa, M. Ignat, R.F. Popovici, D. Timpu, E. Popovici, Int. J. Pharm. 436 (2012) 111–119. [14] M. Ignat, C.J. VanOers, J. Vernimmen, M. Mertensc, S. Potgieter-Vermaak, V. Meynen, E. Popovici, P. Cool, Carbon 48 (2010) 1609–1618. [15] K. Ulbrich, V. Subr, Adv. Drug Deliv. Rev. 56 (2004) 1023–1050. [16] H.P. Lin, C.Y. Mou, Science 273 (1996) 765–768. [17] Z. Ying, J. Tuo, X.Z. Ren, Colloids Surf. B: Biointerfaces 44 (2005) 104–109. [18] L. Tao, K.E. Uhrich, J. Colloid Interface Sci. 298 (2006) 102–110. [19] C. Salerno, A.M. Carlucci, C. Bregni, AAPS PharmSciTech 11 (2010) 986–993. [20] T. Wang, M. Li, H. Gao, W. Yan, J. Colloid Interface Sci. 353 (2011) 107–115. [21] H.S. Dong, I.J. Young, G.K. Don, J.J. Min, J.M. Kyeong, N.J. Woon, Colloids Surf. B: Biointerfaces 69 (2009) 157–163. [22] R. Mellaerts, C.A. Aerts, J.V. Humbeeck, P. Augustijns, G.V. Mooter, J.A.R. Martens, R. Mols, J.A.G. Jammaer, C.A. Aerts, P. Annaert, V.J. Humbeeck, Eur. J. Pharm. Biopharm. 69 (2008) 223. [23] http://www.ccohs.ca/oshanswers/chemicals/ld50.html [24] W. Zhou, J. Feijen, J. Control. Release 132 (2008) e35–e36. [25] S. Dash, P.N. Murthy, P. Chowdhury, L. Nath, Acta Poloniae Pharm. – Drug Res. 67 (2010) 217–223. [26] A. Hahn, G. Brandes, P. Wagener, S. Barcikowski, J. Control. Release 154 (2011) 164–170. [27] C. Ferrero, D. Massuelle, E. Doelker, J. Control. Release 141 (2010) 223–233.