Materials Science and Engineering C 45 (2014) 297–305

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Facile synthesis of methotrexate intercalated layered double hydroxides: Particle control, structure and bioassay explore De-Ying Tian a, Zhen-Lei Liu a, Shu-Ping Li a,⁎, Xiao-Dong Li a,b,⁎⁎ a b

Jiangsu Key Laboratory of Biofunctional Material, College of Chemistry and Material Science, Nanjing Normal University, Nanjing 210023, PR China Shenzhen Research Institute of Xiamen University, Shenzhen 518057, PR China

a r t i c l e

i n f o

Article history: Received 20 June 2014 Received in revised form 2 September 2014 Accepted 13 September 2014 Available online 16 September 2014 Keywords: Methotrexate Layered double hydroxides Nanohybrids Release properties Bioassay test

a b s t r a c t To study the influence of particle size on drug efficacy and other properties, a series of methotrexate intercalated layered double hydroxides (MTX/LDHs) were synthesized through the traditional coprecipitation method, using a mixture of water and polyethylene glycol (PEG-400) as the solvent. To adjust the particle size of MTX/LDHs, the dropping way, the volume ratio of water to PEG-400 and different hydrothermal treatment time changed accordingly, and the results indicate that the particle size can be controlled between 90 and 140 nm. Elemental C/H/N and inductive coupled plasma (ICP) analysis indicated that different synthesis conditions almost have no effect on the compositions of the nanohybrids. X-ray diffraction (XRD) patterns manifested the successful intercalation of MTX anions into the LDH interlayers, and it's also found out that different volume ratios of water to PEG-400 and variable dropping way can affect the crystallinity of the final samples, i.e., the volume ratio of 3:1 and pH decreasing are proved to be optimum conditions. Furthermore, both antiparallel monolayer and bilayers adopting different orientations are suggested for four samples from XRD results. Fourier transform infrared spectroscopy and MTX anions in the interlayer of the nanohybrids. (FTIR) investigations proved the coexistence of CO2− 3 MTX/LDH particles exhibited hexagonal platelet morphology with round corner and different dropping ways can affect the morphology greatly. Moreover, a DSC study indicated that longer time treatment can weaken the bond between the MTX anions and LDH layers. The kinetic release profiles told us that larger MTX/LDH particles have enhanced the ability of LDH layers to protect interlayer molecules. At last, the bioassay study indicated that the nanohybrids with larger diameters have higher tumor suppression efficiency. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Layered double hydroxides (LDHs) are a class of anionic clays which can be represented by the general formula of [M2 +1 − xM3 +x (OH)2]x+An−x / n·mH2O, where M3+ and M2+ represent the tri- and divalent metal ions, respectively, and An− represents the exchangeable anions located in the interlayers [1,2]. LDHs are the only known inorganic materials with positive layer charge, their structural units are made from stacks of positively charged octahedral sheets [M2 +1 − xM3 +x(OH)2]x +, exchangeable interlayer anions (An−) as well as water molecules. The net positive charge, which is due to the substitution of M2 + with M3 + in the brucite-like metal hydroxide M(OH)2, is balanced by the negative charge from the interlayer anions (An−). Various amounts of water (mH2O) are hydrogen bonded to the hydroxide layers or to the interlayer anions, thus forming the 3-D layered structure [3].

⁎ Corresponding author. ⁎⁎ Correspondence to: X.D. Li, Jiangsu Key Laboratory of Biofunctional Material, College of Chemistry and Material Science, Nanjing Normal University, Nanjing 210023, PR China. E-mail address: [email protected] (S.-P. Li).

http://dx.doi.org/10.1016/j.msec.2014.09.024 0928-4931/© 2014 Elsevier B.V. All rights reserved.

Indeed, thanks to the intrinsic properties of LDHs such as adsorption behavior, anionic exchange capacities and good biocompatibility, many processes starting from pristine LDH precursors have already been developed to generate enzyme-LDH biohybrids [4,5]. Also, LDHs have been widely used as drug and biomaterial delivery systems [6,7]. For instance, many anti-inflammatory drugs such as fenbufen, diclofenac, ibuprofen and camptothecin have been intercalated into the interlayers of LDHs to form drug/LDH nanohybrids [8,9]. In fact, drug/LDH nanohybrids can undergo the following process very slowly under physiological conditions (pH = 7.4 or lower) to release the drug, i.e.: drug-Mg–Al-LDHs + H+ → Mg2+ + Al3+ + drug + H2O. In this process, the drug can be released slowly, which prolongs its effect. Additionally, the pH is buffered, such as under the weakly acidic conditions within the endosomes and lysosomes after the drug/LDHs are taken up by the cells [10]. For this project, we chose the drug methotrexate for incorporation into the LDH interlayers. Methotrexate (MTX for short, the structure is shown in Fig. 1) is one of the antifolate drugs that can effectively deactivate the metabolism of diseased cells through programmed cell death or apoptosis, and it has been applied to certain human cancers such as

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Fig. 1. Chemical structure of MTX molecular (C20H22N8O5).

osteosarcoma (bone cancer) and leukemia [11]. Unfortunately, the very short plasma half-life and high efflux rate of MTX compared to the influx rate have required a high administration dose, which places many restrictions on its clinical applications. Recently, many improvements have been attempted on this drug, and synthesis of MTX intercalated LDH nanohybrids has proved to be one of the most well-established methods. Oh et al. [11,12] reported that MTX/LDHs showed better drug efficacy than MTX itself in terms of drug concentration, and the nanohybrids have higher efficiency in tumor suppression than MTX alone. The conventional method to prepare drug/LDH nanohybrids is by using the coprecipitation method at a varied or constant pH, followed by aging at a certain temperature [13–15]. However, the as-prepared nanohybrids are usually loose powders of irregular aggregates that impede the further application. On the other hand, particle size control is another important issue when LDH materials are used in application [16]. Recently, we have reported a facile way to regulate and control the particle size of MTX/LDH nanohybrids between 80 and 300 nm [17]. However, large particles are not suitable in clinical application since most of the drugs enter the body through intravenous injection. Therefore, in this paper the particle size was accurately controlled between 90 and 140 nm. At last, the influence of particle size on the suppression effect of the tumor cells was emphatically studied, to help us develop a new dosage form in the future.

treated at 80 °C for 24 h, and the final samples were called as (a) VH2O/PEG-400 = 5:1; (b) VH2O/PEG-400 = 4:1; (c) VH2O/PEG-400 = 3:1; and (d) VH2O/PEG-400 = 2:1, respectively. The process by changing the dropping way was as follows, the mixed salt solution, containing 0.032 mol/L Mg2+ and 0.016 mol/L Al3+ dissolved in the solvent of water/PEG-400 with a volume ratio of 3:1, was first prepared and signed as solution A′. MTX was dissolved into 15 mL 10% NH3·H2O to obtain a 0.05 mol/L solution and signed as solution B. Then, solutions A′ and B were mixed with different dropping ways, and the final solution was adjusted to pH 9.5 by adding a certain amount of 10% NH3·H2O. Followed by vigorous stirring for 1 h at 60 °C, the products were washed several times with deionized water and ethanol, while N2 gas was bubbled into the solutions throughout the coprecipitation procedure. Finally, the product was transferred into a Telfon-lined stainless steel autoclave and hydrothermally treated at 80 °C for 48 h, and the final samples were called as (e) the solution B was dropped to solution A′ at a constant rate of 3 mL/min; (f) the solutions A′ and B were dropped simultaneously at a constant rate of 3 mL/min; and (g) the solution A′ was dropped to solution B at a constant rate of 3 mL/min. 2.3. Drug-loading capacity To determine the amount of MTX loaded into the LDHs, 0.01 g MTX/ LDHs were dissolved completely into a HCl solution (pH = 1.2) and diluted with H2O to 500 mL in a volumetric flask. Under these conditions, it can be assumed that 100% of the MTX is released from the MTX/LDH nanohybrids. The concentration of the MTX was determined by monitoring the absorbance at λmax = 306 nm with UV–vis spectroscopy. It must be mentioned that the concentration was calculated by regression analysis according to the standard curve obtained from a series of standard solutions of MTX in HCl solution. Finally, the intercalated amount of MTX was calculated and designated as AIn. These data were collected in triplicate and presented in Tables 1 and 2. 2.4. In vitro drug release

2. Materials and methods 2.1. Experimental Materials Polyethylene glycol-400 (PEG-400) was purchased from Shanghai Lingfeng Chemical Reagent Co., and MTX was from Zhejiang Province, Huzhou Prospect Pharmaceutical Co. Human lung adenocarcinoma cells (A549) purchased from the Chinese Academy of Sciences (Shanghai, CN) were used in this study. All chemicals used were of analytical grade or of the highest purity available. 2.2. Synthesis of pristine LDHs and MTX/LDH nanohybrids with different sizes Pristine Mg–Al-NO3-LDHs were prepared by the traditional coprecipitation method and were used as reference materials [18]. The MTX/LDH nanohybrids with different particle sizes were prepared in a mixture of water and PEG-400 by changing the dropping way and the volume ratio of water to PEG-400. The process by changing the volume ratio of water to PEG-400 was as follows: the mixed salt solution, containing 0.032 mol/L Mg2+ and 0.016 mol/L Al3 + dissolved in the mixed solvent of water/PEG-400 with different volume ratios, was first prepared and signed as solution A. MTX was dissolved into 15 mL 10% NH3·H2O to obtain a 0.05 mol/L solution and signed as solution B. Then, solution A was added to solution B at a constant rate of 3 mL/min, and the final solution was adjusted to pH 9.5 by adding a certain amount of 10% NH3·H2O. Followed by vigorous stirring for 1 h at 60 °C, the products were washed several times with deionized water and ethanol, while N2 gas was bubbled into the solutions throughout the coprecipitation procedure. Finally, the product was transferred into a Teflon-lined stainless steel autoclave and hydrothermally

To evaluate the release property of MTX from the MTX/LDH nanohybrids, the in vitro drug release test was performed as follows: 0.02 g of MTX/LDHs was added into 500 mL of phosphate buffered solution (pH = 7.4) in a closed glass bottle at a constant temperature of 37 °C. At a selected time after addition of the nanohybrids, 4 mL of the solution was withdrawn and centrifuged. A portion of the supernatant was used for the measurement, and the concentration of MTX was measured by UV–vis spectroscopy at λmax = 306 nm. Finally, the release profiles were plotted as the relative release percentages of MTX against time. The dissolution medium was maintained at constant volume by replacing the samples with a fresh dissolution medium [19]. These data were collected in triplicate and presented in Fig. 6A. 2.5. In vitro bioassay Cells were routinely cultured at 37 °C in a humidified atmosphere with 5% CO2 in 75 cm2 flasks containing 10 mL of Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 100 U/mL penicillin and 100 mg/mL streptomycin. When the cells were grown to 80–90% cellular confluence, the fault culture cells were differentiated with trypsin-EDTA and then washed twice with PBS (pH = 7.4), which was previously prepared. Then, the cells were diluted with a volume of DMEM containing 10% FBS. For cell proliferation and viability studies, the cells were seeded onto 96-well plates and were incubated overnight at 37 °C under a 5% CO2 atmosphere. After that, the medium in the wells was replaced with fresh medium containing MTX/LDH nanohybrids and further incubated for 24 h. The effect of the MTX/LDH nanohybrids on cell proliferation was determined using an MTT (a yellow tetrazole) assay [20]. Briefly, after the supernatant was removed, 10 μL of MTT (5 mg·mL−1 in PBS, pH 7.4) stock

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Table 1 Chemical composition of key elements of the MTX/LDH nanohybrids. Sample

Mg%

Al%

C%

N%

H%

Chemical formulab

AIn/%a

a c e g

12.21 11.40 12.50 12.36

6.15 5.87 6.09 6.15

26.83 26.11 26.17 25.85

12.05 12.39 12.05 12.33

3.87 3.98 3.89 2.89

[Mg0.66Al0.34(OH)2](C20H20N8O5)0.14·(CO3)0.03·0.09H2O [Mg0.66Al0.34(OH)2](C20H20N8O5)0.14·(CO3)0.03·0.12H2O [Mg0.67Al0.33(OH)2](C20H20N8O5)0.13·(CO3)0.04·0.05H2O [Mg0.67Al0.33(OH)2](C20H20N8O5)0.14·(CO3)0.03·0.03H2O

49.70 50.80 45.64 48.78

± ± ± ±

1.76 (51.10 1.14 (48.90 1.46 (48.35 1.58 (49.37

± ± ± ±

2.58) 2.37) 3.48) 2.67)

Data points represent mean ± SD (n = 3). a AIn represents the intercalated amounts of MTX in various MTX/LDH nanohybrids. b The values in brackets are the results obtained by C/H/N elemental analysis and the values outside the brackets are from UV–vis measurements.

solution and 90 μL DMEM with no FBS were added into each well and further incubated for 4 h at 37 °C. During the incubation, MTT was reduced to insoluble purple formazan by mitochondrial reductase in the living cells. Afterwards, the product was dissolved with 100 μL of dimethylsulfoxide (DMSO). Absorbance was recorded at 570 nm on a microplate reader (Thermo MK3, USA). The MTT assays were also performed with the cells being cultured with different incubation time and various MTX/LDH concentrations, and then the metabolic activities of A549 cells cultured with different MTX/LDH samples were also obtained. The data were collected in triplicate and presented in Fig. 7. 2.6. Characterization XRD patterns were obtained with a D/max-2500PC rotating anode Xray powder diffractometer (Rigaku Co., Japan), using Cu Kα radiation (λ = 1.5406 Å). The samples were scanned from 2° to 70° at a scanning rate of 1°/min. FTIR spectra were recorded on a Bruker Tensor 27 spectrometer (Germany) in the frequency range of 400–4000 cm−1 using KBr pellets (with a weight ratio of sample to KBr being 1:100). Transmission electron microscopy (TEM) images were obtained using an H-7650HITACHI (Hitachi Medical Co., Japan) instrument operating at 200 kV to observe the morphologies. The particle size distribution was characterized using a Sirion 200 (FEI Co., Holland) Scanning Electron Microscopy (SEM) instrument. The thermogravimetric/differential scanning calorimetry (TG–DSC) was measured on a STA-449C (Netzsch Co., Germany) with a heating rate of 10 °C/min in N2 atmosphere. The compositions of the nanohybrids were determined by elemental C/ H/N analysis performed by a Vario EL III elemental analyzer (Elementar Co., Germany) and inductively coupled plasma (ICP) analysis using a JA1100 (Jarrell-Ash Co., USA). The release properties of the MTX/LDHs were collected at specific time intervals and performed at 306 nm with a scanning rate of 24,000 nm/min by a Cary 50 UV–vis (Varian Co., USA) spectrophotometer. Zeta potentials were obtained by the Zetasizer 3000 system (Malvern Instruments Ltd., England) with a pH value of LDH suspension being 9.5 ± 0.1 and a weight percentage of 0.1%. 3. Results and discussion 3.1. Elemental chemical analyses From the elemental analyses, it was found out that different synthesis conditions had almost no effect on the compositions of the

nanohybrids. The chemical compositions and intercalated MTX amounts (AIn) of the MTX/LDH nanohybrids were shown in Table 1. In addition, AIn values were obtained from both UV–vis measurements and chemical elemental analyses, and AIn results obtained by UV–vis measurements are close to those obtained by C/H/N analysis. Shown in Table 1, AIn values for various MTX/LDH nanohybrids remain almost unchanged in the range of allowable error. The appearance of trace CO2− was often observed, even when attempts were made to exclude 3 atmospheric carbon dioxide, due to the high affinity of the LDH layers for carbonate anions [21]. The mean particle diameters are summarized in Table 2 and the value was calculated from the corresponding size distributions from SEM statistical results, and the data were collected in triplicate. Shown from Table 2, the diameters increase with the prolonging of the hydrothermal treatment.

3.2. XRD patterns of MTX/LDH nanohybrids Fig. 2 shows the XRD patterns of pristine NO3-LDHs, together with MTX/LDH nanohybrids with different particle sizes. For the pristine NO3-LDHs, the three intense lines in the low 2θ region correspond to diffractions by planes (003), (006) and (012) [22]. The interlayer distance d(003) value of the pristine LDHs is 0.86 nm, showing the same value as reported for the NO3-LDHs [22]. Intercalation of guest anions results in the shift of (003) reflection to lower 2θ angle values (a—3.15°, b—3.56°, c—3.76°, d—3.46°, e—3.21°, f—3.30°, and g—3.59°), corresponding to an increase of the basal spacing. After intercalation, the crystallinity was not reduced, indicating that the nanohybrids still showed a well-organized stacking arrangement. No change was observed in the value of the unit cell parameter a (Table 2), calculated from the position of (110) reflection near 2θ = 60° and varies with the M2+/M3+ ratio, suggesting that the Mg/Al ratio in the layers is not affected by different synthesis conditions [17]. This is consistent with the previous results from chemical analysis. It is known that peak area can be used to compare the crystallinity of the samples [23], and the areas for well-defined peaks are proportional to their heights. Accordingly, the heights of the XRD peaks are proportional to their crystallization. As seen in Fig. 2(A), larger particles (i.e., samples e, f and g) show higher peak intensities and sharper peak width, indicating good crystallinity [24,25]. Also from Fig. 2(A),different volume ratios of water to PEG-400 can affect the crystallinity of the final samples, and sample c has the sharp diffraction peak, indicating good crystallinity as well, this conclusion is consistent with other reports

Table 2 Characteristic data of MTX/LDH nanohybrids in different synthesis conditions. Samples

Hydrothermal treatment time (h)

Particle size (nm)

Basal spacing (nm)

Interlayer spacing (nm)

d(110) (nm)

Zeta potential (mV)

AIn (%)

a b c d e f g

24 24 24 24 48 48 48

100 96 93 94 135 128 137

2.28 1.98 1.89 2.07 2.72 2.60 2.42

1.80 1.50 1.41 1.59 2.24 2.12 1.94

0.15 0.15 0.15 0.15 0.15 0.15 0.15

+25.20 +23.00 +28.40 +20.00 +25.41 +35.65 +32.83

49.70 47.60 50.80 43.90 45.64 46.53 48.78

Data points represent mean ± SD (n = 3). The values of AIn are from UV–vis measurements.

± ± ± ± ± ± ±

10 8 7 11 9 11 10

± ± ± ± ± ± ±

1.76 2.03 1.14 1.36 1.46 1.89 1.58

300

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Fig. 2. (A) XRD patterns of NO3-LDH and MTX/LDH nanohybrids synthesized at different volume ratios of PEG-400 to water; (B) XRD patterns of MTX/LDH nanohybrids synthesized at different dropping ways.

[17]. As a result, the higher amount of PEG-400, not the better crystallinity, a reasonable volume ratio of water to PEG should be controlled during the synthesis process (i.e., 3:1). As for the influence of different dropping ways, it can affect crystallinity as well (see Fig. 2B). During the formation of sample e, the pH value increased gradually, and then the excess metal cations were easy to be adsorbed on the surface of the precipitation, as a result the crystal growth was hampered, leading to the loose precipitations of irregular aggregates, so the XRD peak is low. Sample f was obtained under the condition of constant pH value, and then it can avoid the heterogeneous phenomenon and the precipitation process became more stable, leading to the formation of final samples with high crystallinity. While the formation of sample g was through the pH decreasing process, on one hand, the MTX anions had enough opportunity to enter the LDH interlayer, on the other hand, the formation of MTX/LDH nanohybrids was always under alkaline condition, and therefore it is advantageous for final samples to increase the crystallinity. To sum up, the dropping way of pH decreasing is beneficial for the formation of final samples with high crystallinity. The basal spacing obtained from the XRD results is also displayed in Table 2, and the larger value of the nanohybrids compared with pristine NO3-LDHs indicates the successful intercalation of guest MTX into the interlayers of the LDHs. From X-ray diffraction studies, the orientation of the intercalated species can be roughly estimated. The molecular size of MTX was calculated to be 2.12 nm [9], given that the thickness of the LDH layers is 0.48 nm, the gallery height of MTX/LDHs measured from X-ray diffraction peaks is 1.80, 1.50, 1.41, 1.59, 2.24, 2.12 and 1.94 nm for samples a–g, respectively. When the arrangement of MTX anions in the LDH interlayer was mentioned, there are many explanations. Latterini et al. [26] proposed that these organic molecules can be located in the interlayers with their aromatic rings perpendicular or declined to the brucite-like layers and with their carboxylic groups alternatively pointing toward the upper and lower layers, forming a monolayer of molecules partially superposed, while other authors [27–29] suggest that the anions are located forming bilayers adopting different orientations but with their carboxylic groups pointing toward the brucite-like layers, favoring in this way the electrostatic interactions. As for our system studied, both antiparallel monolayer and bilayers adopting different orientations are suggested, and shown in Scheme 1. For sample e, MTX anions are arranged as declining bilayers since the gallery height is larger than the molecular size of MTX (2.12 nm). For sample f, COO− group of MTX/LDHs is thought to attach onto the positively charged LDH layer, an antiparallel pattern is anticipated, and MTX anions are arranged as a monolayer that allows their longest dimension to be oriented perpendicularly from the interlayer mineral surfaces. Such arrangement permits an enhanced electrostatic interaction between the anionic terminal carboxylates of MTX and cationic

hydroxide layers [30]. For other samples (i.e., samples c–h), MTX molecules are intercalated as a declining antiparallel monolayer, shown in Scheme 1. Moreover, the presence of a diffraction peak at approximately 25° clearly indicates the occurrence of π–π stacking of the benzene ring in MTX during the intercalation [17,22]. 3.3. FTIR analysis of MTX/LDH nanohybrids Vibrational spectroscopy is a useful tool to study guest intercalated LDH hybrids, because it can be used as a probe for the interactions between the guest anions, the host layers and the interlayer water [31]. The FTIR spectra for the MTX/LDH nanohybrids in comparison with pristine NO3-LDHs and MTX are illustrated in Fig. 3. The intense absorption band centered at 3460 cm− 1 which appears in the nanohybrids and NO3-LDHs is due to the stretching vibration of the OH− groups for the hydroxide layer or interlayer water molecules. The bending vibrations at 1637 and 1386 cm−1 found in the spectra of NO3-LDHs correspond to the deformation mode of the interlayer water (δH2O) and NO− 3 , respectively [9]. As for MTX, the intense absorption band centered at 3400 cm−1 is due to the stretching vibration of the \NH groups, and the most characteristic absorption band for the carboxylate group is due to the antisymmetric COO− vibration in the region of 1570– 1660 cm−1 [32]. Furthermore, the peaks at 1208 and 1100 cm−1 belong to the C\N stretching frequencies of primaryamine and tertiaryamine in MTX. An intense peak at 1624 cm−1 for the MTX/LDH nanohybrids indicates that the COO− groups have been intercalated into the LDH interlayers, and the bond centered at 1450 cm−1 corresponds to the symmetric stretching mode of the COO− group. Comparisons between the spectra of MTX and MTX/LDHs show that MTX in the LDH interlayers does not cause any significant changes in positions of the major IR bands, except for the disappearance or weakening of the peak at 1208 cm−1 for MTX/LDHs. This change results from the fact that the interlayer gallery of LDHs restricts the stretching movement of electrons around C atoms. The weak vibration at 1380 cm−1 in the spectra for the MTX/LDHs proved the coexistence of CO2− 3 and MTX anions in the interlayer of the nanohybrids. 3.4. Morphology and particle size control The morphology images and particle size distribution histograms of MTX/LDH nanohybrids are shown in Fig. 4, and size distributions are obtained from SEM statistical results. Nanohybrids obtained from the mixture of water and PEG all exhibit sphere-like particles, and spherical structure is beneficial to the application of drug carriers [33]. Shown from Fig. 4, the monodispersity of the nanohybrids is significantly improved compared with those obtained in pure H2O solvent [30]. LDH

301

g f e d

1380 1208

MTX

1624 1450

Transmittance/%

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c b a NO3-LDHs

Sample e

4000 3500 3000 2500 2000 1500 1000

500

Wavenumber/cm-1 Fig. 3. FTIR spectra of NO3-LDH, MTX and MTX/LDH nanohybrids at different synthesis conditions.

Sample f

Other samples Scheme 1. Different arrangement of MTX anions in LDH layers.

particles, prepared by the coprecipitation method, typically show preferential growth of ab faces (i.e., perpendicular to the stacking direction) and exhibit hexagonal platelet morphology on the microscale. The intercalation of MTX also strongly influences the textural properties of LDHs, and the assemblies of MTX anions and LDH hosts give rise to hexagonal platelet morphology with round corner. Shown from Fig. 4, compared with the effect of the volume ratio of water to PEG-400, different dropping ways can affect the morphology greatly. Particles obtained from the dropping way of pH decreasing (sample g) have regular morphology and good monodispersity, whereas particles from the pH increasing method (sample e) are irregular and a little aggregated. During the formation of sample e, pH value increased

gradually, and most of time the formation of MTX/LDH nanohybrids was under an acidic condition. It's well known that the acidic medium will hinder the full growth of LDH particles, as a result to form irregular aggregates. Sample f was obtained under the condition of constant pH value, and then it can avoid the heterogeneous phenomenon and the morphology was improved. While the formation of sample g was through the pH decreasing process, and the formation of MTX/LDH nanohybrids was always under an alkaline condition, and therefore it is advantageous for final samples to grow and develop fully, and then the morphologies and monodispersity will be improved greatly. In a word, the dropping way of pH decreasing is beneficial for the formation of regular and monodispersed MTX/LDH nanohybrids. As far as the change of the particle size has been concerned, it was found out that both volume ratio of water to PEG-400 and dropping way can influence it. The mechanism of how PEG molecules affect the formation of MTX/LDH nanohybrids is described as follows: nonionized PEG molecules will form chain-like structures due to the assembly in water, and these chain-like structures will adhere to the surface of particles, preventing the collisions and coagulations [34,17]. However, low concentration of PEG-400 cannot provide effective control on the particle growth and then irregular particles occurred (see samples a and b in Fig. 4). Generally speaking, the addition of PEG will give rise to the decrease of the particle size and it's attributed to the fact that the emergence of PEG will lead to decreasing the dielectric constant of the solvent, resulting in the decrease of solubility of the particles [35]. Of course, our result proved this idea, i.e., the particle size decreased with the increase of PEG addition (see samples a, b and c of Fig. 4). While too high concentration of PEG-400 will restrict the particle growth on the contrary, as the growth space is compressed by extra PEG molecular and the decrease of the particle size with the addition of PEG disappeared (see sample d of Fig. 4). In conclusion, the most regular and smaller particles were obtained at the optimized procedure, i.e. volume ratio of water to PEG-400 being 3:1 (sample c of Fig. 4). The mechanism of different dropping ways influencing the particle size can be explained briefly, particles under the pH decreasing process can grow up fully and develop themselves sufficiently, while particles at constant pH and the pH increasing process always suffer from the interference of acid attack, as a result, the biggest particles occurred in the pH decreasing process. It's well known that particle size control is the most important issue when LDH particles are used in application. Reverse microemulsions have been used to control the growth of MTX/LDH hybrids, and the particles between 80 and 110 nm can be obtained by adjusting the synthesis conditions [36]. Recently, a facile way to regulate the particle size of

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20

a-100nm

25

Fraction/%

Fraction/%

f-128nm

20

15

15

10

10

5

5 0 50

100

150

200

Particle diameter/nm

0 0

75

b-96nm

25

150

225

300

Particle diameter/nm

e-135nm

15

20

10

5

0 0

50

100

150

200

Particle diameter/nm

c-93nm

25

Fraction/%

Fraction/%

20

15

10

5

Fraction/%

20

0 0

15

50

100

150

200

250

300

Particle diameter/nm

10

5

0

0

20

40

60

80

100

120

140

160

g-137nm

180

Particle diameter/nm

d-94nm Fraction/%

25

Fraction/%

30

20

20

15

10 10

5

0 0

0

0

30

60

90

120

150

50

100

150

200

250

300

Particle diameter/nm

180

Particle diameter/nm

Fig. 4. TEM images and particle size distribution of MTX/LDH nanohybrids at different synthesis conditions.

MTX/LDH nanohybrids between 80 and 300 nm by controlling hydrothermal treatment conditions has been reported [17]. However, it was reported that LDH particles with a size about 100 nm and individually separated in a stable suspension are desirable for gene and biomaterial carriers [16], then as a result, the proper way to accurately control the particle size is in urgent need. Compared with the method of reverse microemulsions, our controlling approach is easily accessible and environmentally friendly. 3.5. Thermal analysis of MTX/LDH nanohybrids The DSC and TG curves for the MTX/LDH nanohybrids are shown in Fig. 5(A) and (B), which also show the data of pristine NO3-LDHs for comparisons. From Fig. 5(A), two clear endothermic peaks are observed at 207 °C and 385 °C for the pristine LDHs. The first peak results from the removal of water physisorbed on the external surface as well as water intercalated into the interlayer galleries while the second arises from dehydroxylation of the layers and decomposition of the interlayer anions [37]. The DSC curves of the nanohybrids are almost identical to that of the pristine LDHs, the first peak is attributed to the removal of water and the second one results from dehydroxylation of the layers and decomposition of the MTX anions. The temperatures of the second endothermic are 366 °C, 375 °C, 376 °C, 381 °C, 357 °C, 361 °C and 363 °C for samples a–g, respectively. Careful observation on the DSC curves of the nanohybrids reveals that their thermal properties can be changed by different synthesis conditions. When prolonging the

hydrothermal treatment at the same temperature (i.e., samples c and g), the value of the second endothermic peak becomes lowered as well, and this change suggests that the MTX anions become more weakly bonded to the LDH layers, which indicating that longer time treatment will weaken the bond between the MTX anions and LDH layers. The TG curve of the pristine NO3-LDHs clearly shows a two-step reaction, which is accordant with the results of DSC. In the first reaction, from room temperature up to ca. 230 °C, a 12.9% weight loss was observed from the total weight, corresponding to the removal of the adsorbed and intercalated water. The second weight loss for LDHs involved was approximately 32.1%, corresponding to the dehydroxylation of the layers and the decomposition of nitrate anions. This two-step behavior became merged for the MTX/LDHs and a continuous weight loss from 200 to 700 °C was observed. The weight loss of MTX anions was still observed above 450 °C, implying that the chemical stability of MTX is enhanced significantly in the confined region of the LDH galleries compared with that of its pristine form (the decomposition temperature of pure MTX is 185–204 °C). 3.6. Zeta potentials and AIn of MTX/LDH nanohybrids Zeta potential is an important parameter for characterizing the stability of colloidal dispersion, the zeta potentials of the nanohybrids changed from +23 mV to +35 mV, shown in Table 2, indicating these systems are relatively stable. All results point to the conclusion that long hydrothermal treatment favors the stability of MTX/LDHs. A

D.-Y. Tian et al. / Materials Science and Engineering C 45 (2014) 297–305

A

The release mechanism of MTX from the nanohybrids is very complicated and not completely understood until now. There are two types of mechanisms of drug release, an ion-exchange process and the dissolution of LDH layers in buffered solution [38]. According to the literature, four equations are often used for the release of MTX/LDH nanohybrids:

0.5 a c e g

0.0

Heat flow/(mW/mg)

-0.5

303

b d f NO3-LDH

-1.0

(1) The first-order model expresses the release from systems where dissolution rate depends on the MTX amount in the LDH hybrids and can be generally written as

-1.5 -2.0 -2.5

ln ð1−Mt =M∞ Þ ¼ −k1 t:

-3.0

(2) The Higuchi model describes whether the Fickian diffusion is the rate limiting step, and can be generally expressed as

207°C

-3.5

385°C

-4.0

100

200

300

400

500

600

700

Temperature/°C

:

(3) The Ritger–Peppas (R–P) equation is often used to explain drug diffusion and dissolution of the LDH layers. A value of n b 0.45 corresponds to the drug diffusion control, which is based on the ion exchange process; n N 0.89 is attributed to the dissolution of LDHs; and 0.45 b n b 0.89 is due to the cooperation of drug diffusion and LDH dissolution.

B

Weight Loss/%

1=2

Mt =M∞ ¼ kH t

a b c d e f g

Mt =M∞ ¼ kt NO3-LDHs

n

(4) The parabolic diffusion model elucidates that the release process is controlled by a diffusion process such as intraparticle diffusion or surface diffusion and the equation is as follows

20%

−0:5

ðMt =M∞ Þ=t ¼ kP t

100

200

300

400

500

600

700

þ b:

800

Temperature/°C Fig. 5. (A) The DSC curves for NO3-LDH and MTX/LDH nanohybrids at different synthesis conditions. (B) The TG curves for NO3-LDH and MTX/LDH nanohybrids at different synthesis conditions.

unified change tendency of AIn with the variation of particle size was not obtained, it can be concluded from our results that the particle size has no influence on the drug capacity, at least in our study range.

3.7. In vitro drug release and release kinetics Here, the in vitro release profiles of MTX from MTX/LDHs are shown in Fig. 6A. A rapid release of MTX from the nanohybrids occurs at the initial stage, after which a slower release of MTX is observed. The t0.5 s (the time for release fraction of 50%) was 20 min, 40 min, 90 min, 70 min, 80 min, 53 min and 38 min for samples a to g, respectively. The burst release fractions (defined as the release fraction in the initial 100 min) were 76%, 72%, 54%, 57%, 52%, 59% and 61%, respectively. From the above information, it is obvious that MTX was released quickly in the initial period for samples a, b and g, when the MTX/LDH nanohybrids were placed in phosphate buffered solution. While when sustained release to 500 min, the released fraction changed to 100%, 93% and 74% for samples a, b and g, respectively. Consequently, the drug release of sample g belongs to quick first and afterwards slow process, and this character is quite suitable for the clinical application: the first quick release can kill the cancer cells heavily and then the slow and controlled release can maintain the daily dosage, to avoid frequent medication taken. Careful observation found out that bigger particles (samples e, f and g) present much better controlled-release property than those of small ones (samples a, b, c and d), and this result is accordant with our previous conclusion [17].

In these equations, Mt / M∞, t, and k are the fractional drug release, release time, and the corresponding release rate constant, respectively, and a and b are constants whose chemical significance is not clearly resolved [38–40]. On the basis of the four kinetic models above, the fitting results of drug release profiles are given in Fig. 6B and the corresponding linear correlation coefficients (R) are shown in Table 3. In general the firstorder model and Higuchi model are not suitable to explain the release process reflected by the fact that modeling data points do not form a straight line and small R values. While the R–P model and the parabolic diffusion model give more reasonable fitting coefficients of R = 0.96– 0.99, indicating that these two models can better describe the release mechanism. Careful examination on the results of data fitting for R–P model (Table 3), the release of samples c and d, with values of n between 0.45 and 0.89, belongs to cooperation of drug diffusion and LDH dissolution, while the release of other samples belongs to the drug diffusion via ion exchange. Together with the previous synthesis conditions, the LDH layers of the larger nanohybrids have the stronger protective effect on the interlayer molecules although they were synthesized at the same dropping way and the identical volume ratio of water to PEG-400 (see samples c and g). From a clinical perspective, samples c and d are not suitable as the easy dissolution of LDH layers. 3.8. In vitro bioassay of MTX/LDH nanohybrids The anticancer efficiency of various MTX/LDH nanohybrids compared with MTX itself was evaluated by bioassay test. Fig. 7A shows the concentration dependent of cell viability when the nanohybrids and MTX act on A549. As expected, an increase in MTX/LDH concentration leads to the lower cell viability and more cell death. We observed a decrease to 81.93% cell viability at a MTX concentration of 120 μg·mL−1, while the cell viability decreases to 77.84%, 70.69%, 72.41%, 73.41%, 62.41%, 59.46% and 57.47%, respectively for samples a–g at a

304

D.-Y. Tian et al. / Materials Science and Engineering C 45 (2014) 297–305

A

100 90 80

Release/%

70 60

a c e

50 40

b d f g

30 20 10 0

0

100

200

300 Time/min

400

B

500

1.0 0.9

0.0

0.7

Acoording to first-order eq.

-1.2

0.6

a b c d a'' b'' c''

-1.8 -2.4

Mt/M

-ln(Mt/M )

0.8 -0.6

0.4 0.2 0.1 0.0

100

200

300

400

500

Acoording to Higuchi eq.

0.3

-3.0 0

a b c d a'' b'' c''

0.5

600

0

5

10

15

t/min

20

25

t1/2

0.06

According to Parabolic diffusion eq.

(Mt/M )/t

0.0

ln(Mt/M )

-0.4 -0.8 a b c d a'' b'' c''

-1.2 -1.6

Acoording to R-P eq.

-2.0

0.03

a b c d a'' b'' c''

0.00

-2.4 1

2

3

4

5

6

7

lnt

0.0

0.1

0.2

-0.5

0.3

0.4

0.5

t

Fig. 6. (A) Release profiles of MTX/LDH nanohybrids at different synthesis conditions. Error bars represent standard error (n = 3). (B) Plots of different kinetic models of first-order eq., Higuchi eq., R–P eq. and parabolic diffusion eq. for the release of MTX from MTX/LDH nanohybrids at different synthesis conditions.

concentration of 120 μg·mL−1. It makes it clear that MTX/LDH nanohybrids have higher tumor suppression efficiency compared to MTX itself and larger MTX/LDH particles have higher tumor suppression efficiency. Table 3 The release data of MTX/LDH nanohybrids fitting to different kinetic equations. Samples

a b c d e f g

First-order eq.

Higuchi eq.

Ritger–Peppas eq.

Parabolic diffusion eq.

R

R

R

n

R

0.8212 0.8023 0.7687 0.7680 0.8114 0.7757 0.7232

0.9544 0.9478 0.9625 0.9667 0.9626 0.9449 0.9086

0.9932 0.9824 0.9812 0.9782 0.9942 0.9857 0.9666

0.2184 0.2924 0.4429 0.5315 0.2378 0.2348 0.2183

0.9776 0.9815 0.9815 0.9800 0.9883 0.9881 0.9883

Fig. 7B shows the time-dependent anticancer effects of MTX/LDH nanohybrids at the concentration of 120 μg·mL−1 in comparison with MTX. The overall features reveal that both MTX and MTX/LDH nanohybrids gradually suppress the tumor cell growth in both time and concentration dependent manner. As shown in Fig. 7B, the time dependent anticancer effects of MTX and various MTX/LDH nanohybrids at 120 μg·mL−1 are quite different, since MTX/LDH nanohybrids are much faster than MTX in terms of efficacy. One important thing to be mentioned here is that larger MTX/LDH nanohybrids show higher efficacy than those small ones even at a very early stage and all nanohybrids show higher efficacy than MTX itself, whereas it takes about 3 days for MTX to achieve the maximum value. This time difference between MTX and MTX/LDH nanohybrids is attributed to the different permeation ways through cell membrane for both drugs [41]. Our study also indicates that the nanohybrids with larger diameters have the stronger ability to engulf more amount of MTX in the early stage, then having higher tumor suppression efficiency.

D.-Y. Tian et al. / Materials Science and Engineering C 45 (2014) 297–305

A

At last, the bioassay study indicates that the nanohybrids with larger diameters have higher tumor suppression efficiency.

100

Cell viability(%)

95 90

Acknowledgments

85

The authors are grateful for the financial support of this project by the National Natural Science Foundation of China (21073093 and 21273116), the special fund for the Development of Strategic Emerging Industries in Shenzhen City (JCYJ20120618155457353) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

80

MTX a b c d e f g

75 70 65 60

References

55 0

20

40

60

80

100

120

Drug concentration/ug.mL-1

B

90 MTX b d f

80 70

Cell viability(%)

305

a c e g

60 50 40 30 20 10 0

1

2

3

Time/day Fig. 7. (A) The viability of the cells versus various concentrations of MTX/LDH nanohybrids from 0 to 120 μg·mL−1 under 1 day incubation time. The cell viability date was from three separated experiments. (B) The viability of the cells versus various incubation time from 1 to 3 days under the concentration of 120 μg·mL−1. The cell viability date was from three separated experiments.

4. Conclusions In conclusions, a series of MTX/LDHs with different sizes were prepared by changing the synthesis conditions such as the dropping way, volume ratio of water to PEG-400 and hydrothermal treatment time, and the particle sizes in the range of 90–140 nm can be achieved. TEM images indicated that all particles exhibited hexagonal platelet morphology with round corner and a DSC study showed that longer time treatment will weaken the bond between the MTX anions and LDH layers. The kinetic release profiles told us that larger MTX/LDH particles have enhanced the ability of LDH layers to protect interlayer molecular.

[1] P.S. Braterman, Z.P. Xu, F. Yarberry, Handbook of Layered Materials, Marcel Dekker, New York, 2004, p. 373. [2] N.N. Das, J. Konar, M.K. Mohanta, S.C. Srivastara, J. Colloid Interface Sci. 270 (2004) 1–8. [3] J.M. Oh, S.H. Hwang, J.H. Choy, Solid State Ionics 151 (2002) 285. [4] N. Touisni, F. Charmantray, V. Helaine, C. Forano, L. Hecquet, C. Mousty, Colloids Surf. B 112 (2013) 452–459. [5] M.S. Gasser, Colloids Surf. B 73 (2009) 103–109. [6] A.I. Khan, L. Lei, A.J. Norquist, D. O'Hare, Chem. Commun. 22 (2001) 2342. [7] J.H. Choy, S.Y. Kwak, Y.J. Jeong, J.S. Park, Angew. Chem. Int. Ed. 39 (2000) 4141. [8] Z. Gu, A.C. Thomas, Z.P. Xu, J.H. Campbell, G.Q. Lu, Chem. Mater. 20 (2008) 3715. [9] J.H. Choy, J.S. Jung, J.M. Oh, M. Park, J. Jeong, Y.K. Kang, O.J. Han, Biomaterials 25 (2004) 3059. [10] Z.P. Xu, G.Q. Lu, Pure Appl. Chem. 78 (2006) 1771. [11] J.M. Oh, S.J. Choi, S.T. Kim, J.H. Choy, Bioconjugate Chem. 17 (2006) 1411. [12] J.M. Oh, M. Park, S.T. Kim, J.Y. Jung, Y.G. Kang, J.H. Choy, J. Phys. Chem. Solids 67 (2006) 1024. [13] P.X. Zhi, S. Gregory, Q.L. Chao, Q.L. Gao, J. Phys. Chem. B 110 (2006) 16923. [14] D.G. Evans, X. Duan, Chem. Commun. 6 (2006) 485. [15] M.P. Kapoor, Y. Matsumura, Catal. Today 93 (2004) 287. [16] S.J. Choi, J.M. Oh, J.H. Choy, J. Mater. Chem. 18 (2008) 615–620. [17] X.Q. Zhang, M.G. Zeng, S.P. Li, X.D. Li, Colloids Surf. B 117 (2014) 98–106. [18] K. Wosikowski, E. Biedermann, B. Rattel, N. Breiter, P. Jank, R. Löser, G.J. Peters, Clin. Cancer Res. 9 (2003) 1917. [19] A.A. AL-Kahtani, B.S. Sherigara, Colloids Surf. B 115 (2014) 132–138. [20] P.R. Twentyman, M. Luscombe, Br. J. Cancer 56 (1987) 279. [21] C. Chakraborty, K. Dana, S. Malik, J. Phys. Chem. C 115 (2010) 1996. [22] C.X. Liu, W.G. Hou, L.F. Li, Y. Li, S.J. Liu, J. Solid State Chem. 181 (2008) 1792. [23] S. Kannan, A. Narayanan, C.S. Swamy, J. Mater. Sci. 31 (1996) 2353. [24] S.P. Li, Colloids Surf. A 290 (2006) 56. [25] Y.F. Wang, H.Z. Gao, J. Colloid Interface Sci. 301 (2006) 19. [26] L. Latterini, M. Nocchetti, G.G. Aloisi, U. Costantino, F. Elisei, Inorg. Chem. Acta 360 (2007) 728–740. [27] M. del Arco, S. Gutiérrez, C. Martín, V. Rives, J. Rocha, J. Solid State Chem. 177 (2004) 3954–3962. [28] A.I. Khan, L. Lei, A.J. Norquist, D. O'Hare, Chem. Commun. (2001) 2342–2343. [29] H. Zhang, K. Zou, S. Guo, X. Duan, J. Solid State Chem. 179 (2006) 1792–1799. [30] F.F. Cen, S.P. Li, J. Dispersion Sci. Technol. 31 (2010) 1055. [31] Z.P. Xu, H.C. Zeng, J. Phys. Chem. B 105 (2001) 1743. [32] C. Li, L. Wang, D.G. Evans, X. Duan, Ind. Eng. Chem. Res. 48 (2009) 2162. [33] Y. Kuang, L. Zhao, S. Zhang, F.Z. Zhang, M.D. Dong, S.L. Xu, Materials 3 (2010) 5220–5235. [34] J. Wang, Z.M. Peng, Y.J. Huang, Q.W. Chen, J. Crystal Growth 263 (2004) 616. [35] H.I. Chen, H.Y. Chang, Colloids Surf. A-Physicochem. Eng. Asp. 242 (2004) 61–69. [36] Z.L. Liu, D.Y. Tian, S.P. Li, X.D. Li, T.H. Lu, Int. J. Pharm. 473 (2014) 414–425. [37] F. Cavani, F. Trifiro, A. Vaccari, Catal. Today 11 (1991) 173. [38] L. Serra, J. Doménech, N.A. Peppas, Biomaterials 27 (2006) 5440. [39] F.S. Li, L. Jin, J.B. Han, M. Wei, C.J. Li, Ind. Eng. Chem. Res. 48 (2009) 5590. [40] R. Bhaskar, R.S.R. Murthy, B.D. Miglani, K. Viswanathan, J. Pharm. 28 (1986) 581. [41] S.J. Choi, J.M. Oh, J.H. Choy, J. Inorg. Biochem. 103 (2009) 463.