Layered double hydroxide: Inorganic organic conjugate nanocarrier for methotrexate

Layered double hydroxide: Inorganic organic conjugate nanocarrier for methotrexate

Journal of Physics and Chemistry of Solids 72 (2011) 779–783 Contents lists available at ScienceDirect Journal of Physics and Chemistry of Solids jo...

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Journal of Physics and Chemistry of Solids 72 (2011) 779–783

Contents lists available at ScienceDirect

Journal of Physics and Chemistry of Solids journal homepage:

Layered double hydroxide: Inorganic organic conjugate nanocarrier for methotrexate Manjusha Chakraborty a, Sudip Dasgupta a, Poulomi Bose a, Akhilesh Misra b, Tapan Kumar Mandal b, Manoj Mitra c, Jui Chakraborty a,n, Debabrata Basu a a

Bioceramics & Coating Division, Central Glass & Ceramic Research Institute, Kolkata 700032, India Dept. of Veterinary, Pharmacology and Toxicology, West Bengal University of Animal and Fisheries Sciences, 68 K.B. Sarani, Belgachia Road, Kolkata 37, India c Metallurgical and Materials Engineering, Jadavpur University, Kolkata 700032, India b

a r t i c l e i n f o


Article history: Received 26 February 2010 Received in revised form 23 December 2010 Accepted 17 March 2011 Available online 7 April 2011

Layered double hydroxide (LDH)–methotrexate (MTX) nanohybrids were successfully synthesized using ex situ and in situ processes. X-ray diffraction patterns of the synthesized nanopowders revealed that intercalated MTX molecules were stabilized in tilted longitudinal conformation into the hydroxide interlayer space. Two separate hydroxyl peaks were found in the FTIR spectra of LDH–MTX nanopowders suggesting successful intercalation of the MTX molecule into LDH matrix. The synthesized powders were further characterized using transmission electron microscopy (TEM) and selected area electron diffraction (SAED) pattern. HRTEM images showed an increase in interlayer spacing in hydrothermally crystallized LDH–MTX nanohybrids as compared to pristine LDH. The study showed that depending on the synthesis route used to synthesize LDH–MTX nanohybrid, its particle size as well as morphology can be varied at nano scale. & 2011 Elsevier Ltd. All rights reserved.

Keywords: A. Ceramics A. Inorganic compounds A. Nanostructures B. Chemical synthesis C. Electron microscopy

1. Introduction Inorganic–inorganic or organic–inorganic hybrid systems have tremendous potentials for controlled and targeted delivery of drugs and biomolecules. These kinds of hybrid systems require biocompatible inorganic matrixes for safe retention as well as controlled delivery of drugs. Layered double hydroxides are considered to be one of the most promising inorganic matrices for gene or drug delivery because of their low toxicity, high reserving capacity and enhanced cellular uptake behavior [1–4]. Many biologically important molecules including genes or drugs can easily be incorporated into the cationic framework of layered double hydroxide [5]. Layered double hydroxides (LDHs), commonly known as hydrotalcite (HT)-like materials, consist of brucite-like layers containing hydroxides of metal cations (M2 þ and M3 þ ). There are exchangeable anions (An-) and a variable amount of water molecules attached through H- bonding in the gallery space [6,7]. The chemical composition of LDH can be represented by the þ general formula [M21–x M3x þ (OH)2]x þ (An–)x/n  mH2O, where the value of x is equal to the molar ratio of M2 þ /(M2 þ þM3 þ ), generally lies in the range of 0.2–0.33. Thus, M2 þ /M3 þ molar ratio would vary from 2.0 to 4.0 and m is equal to 1  3x/2 [8,9]. n

Corresponding author. E-mail address: [email protected] (J. Chakraborty).

0022-3697/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpcs.2011.03.012

As a result, a large class of isostructural materials, with widely varied physicochemical properties can be obtained by changing the nature of the metal cations, the molar ratios of M2 þ /M3 þ as well as the type of interlayer anions. In this case, we report the synthesis of Mg aluminate LDH with Mg/Al atomic ratio nearly equal to 2 which has the highest layer positive charge density having the potential to incorporate maximum amount of anions. Due to the swelling and de-swelling properties, LDH has widely been studied as host materials in intercalation and de-intercalation reactions [10]. Because of its anion exchange capacity, many negatively charged pharmaceutically active compounds have been intercalated into the interlayers of LDH to ensure controlled and sustained release of incorporated biomolecules. Different kind of drugs, in their anionic form, has been incorporated into LDHs. These include nonsteroidal anti-inflammatory drugs such as ibuprofen, fenbufen, naproxen [11–14], anti-metabolic drugs such as 5-fluorouracil [15], anti-cancer drugs such as folate derivatives, methotrexate [3,16,17], cardiovascular drugs for example 4-biphenylacetic acid [14], etc. The incorporated drugs inside LDH nanocarriers would possess higher resistance to enzymatic degradation. Again, different cell lines are found to take up LDH nanoparticles very quickly inside the cellular compartments [17,18]. MTX has been found to be quite effective for the treatment of certain human cancer such as osteosarcoma, leukemia, etc. [19].


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The activity of dihydrofolate reductase (DHFR) is inhibited by MTX. DHFR catalyzes the reduction of the dihydrofolate to the tetrahydrofolate in a folate cycle coupled with DNA synthesis and cell proliferation. Thus, the influx of MTX into the cytosol eventually results in the cell apoptosis. Again, in course of cell permeation and intracellular trafficking, MTX remains intact inside the LDH nanocarriers and can be released in the cytoplasm of cancer cells in a stable and constant manner because of its anion exchange capacity in acidic pH. MTX–LDH nanohybrids can be used as a reservoir of MTX molecules [3] and synthesized using different wet chemical routes. Depending on the synthesis route, particle size, morphology and crystallinity of LDH–MTX nanohybrid can vary. Here we investigate the synthesis of LDH-MTX nanopowders using coprecipitation, anion exchange and hydrothermal ex situ methods. The synthesized nanopowders were characterized using XRD, FTIR, TEM and DLS technique.

2. Materials and methods 2.1. Materials Magnesium nitrate hexahydrate (Mg(NO3)2  6H2O), aluminum nitrate nonahydrate (Al(NO3)3  9H2O) and ammonium hydroxide (NH4OH) were purchased from Sigma Aldrich and Sun laboratory (Bangalore, India) supplied methotrexate (MTX powder form, calibration standard drug). 2.2. Synthesis of MgAl LDH intercalated with MTX 2.2.1. Ex situ ion-exchange method LDH was chemically synthesized following an ion-exchange method. Briefly, an aqueous solution (40 ml) containing NH4OH was added dropwise to a solution containing Mg(NO3)2  6H2O (0.8461 g, 0.66 mol) and Al(NO3)3  9H2O (0.6189 g, 0.33 mol) such that Mg:Al molar ratio was maintained at 2:1. The reaction was carried out in nitrogen atmosphere at a pH of 10.0. The mixed solution was stirred for 1 h to get white gelatinous precipitate of LDH which was subsequently washed with ammoniacal water thrice after consecutive centrifugation and resuspension. Methotrexate solution of 0.3987 gm/10 ml was added to LDH suspension at pH 10 with continuous stirring for 12 h to get LDH–MTX composite NPS, which was then washed with water twice and dried in vacuum oven at 50 1C.

2.2.4. Preparation of sample for HPLC analysis Ten milligram of the ZnAlLDH–MTX sample was taken in 50 ml volumetric flask and suspended in distilled water. It was acidified with concentrated hydrochloric acid and shaken properly. Finally volume was made with distilled water. This is the stock solution. At each time point, 1 ml of the sample was pipetted out (replenishing the same volume each time to keep the total volume of the stock constant), filtered through 0.2 mm membrane filter and an aliquot of 20 ml of the filtered sample was analyzed by HPLC using tris buffer (0.1 M KH2PO4 and 0.01 M Tris HCl) acetonitrile and methanol (in 80:10:10 (v/v)) as eluent. This buffer was membrane filtered (as above) and degassed prior to use. 2.3. Characterization techniques and procedure Powder X-ray diffraction patterns were obtained from Siemens D 501 X-ray diffractometer using CuKa radiation fitted with graphite scattered beam monochromator. The samples were scanned from 21 to 151 in steps of 0.031 with a count time of 2 s at each point. Fourier transform infrared spectra were obtained with a Perkin-Elmer spectrum 100 spectrophotometer at a resolution of 2 cm  1 and averaging 10 scans in the 400–4000 cm  1 region on pressed KBr pellets. Particle size distributions were performed on a Malvern zetasizer 2000 using the accessory for powder. Particle size and morphology of LDH nanoparticles were studied using transmission electron microscopy (FEI, Netherland). The elemental analyses (EA) for the percentage of carbon, hydrogen and nitrogen (CHN) content of all the three samples were carried out using 2400 series II CHN analyzer, Perkin Elmer, USA. High Performance Liquid Chromatography (HPLC) (Shimadzu LC-20AT, Japan) was used to determine the release of methotrexate from LDH nano vehicle in PBS (phosphate buffer saline) at pH 7.4.

3. Results and discussion The powder XRD patterns of MTX–LDH samples were compared with pristine LDH in Fig. 1. The (0 0 3) spacing for pristine ˚ As MTX was intercalated into LDH, LDH was found to be 8.02 A. (0 0 3) peak was shifted towards lower angle because of increase in d003-spacing. For LDH–MTX nanohybrids the d003 basal spacing ˚ the values are similar to that was calculated to be 21.26 A, reported by Gordijo et al. [20]. Assuming the sheet thickness of

2.2.2. In situ co-precipitation method First, a methotrexate solution at pH 10 was prepared. As mentioned above mixture of Mg(NO3)2  6H2O and Al(NO3)3  9H2O in 2:1 molar ratio was prepared. Now, the mixed metal nitrate solution was added dropwise to methotrexate solution at pH 10 and stirred continuously for 12 h. The pH of the solution was adjusted to 10 by dropwise addition of dilute ammonia. The solution was centrifuged and washed with ammoniacal water thrice. The yellowish residue was dried in a vacuum oven at 50 1C. 2.2.3. Ex situ hydrothermal process Layer double hydroxide nanoparticles were prepared at Mg:Al molar ratio of 2:1 as mentioned above in Section 2.2.1. The washed and dried LDH powder was hydrothermally treated at 150 1C for 24 h. To the hydrothermally treated LDH powder suspension (aqueous medium) methotrexate solution was added and stirred for 12 h. Methotrexate containing LDH suspension was then centrifuged and washed thrice and finally dried in a vacuum oven at 50 1C.

Fig. 1. X-ray diffraction patterns of (a) LDH, (b) in situ synthesized LDH-MTX, (c) ex situ synthesized LDH–MTX and (d) hydrothermally synthesized ex situ LDH-MTX nanopowders.

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˚ the estimated gallery height in brucite-like LDH equals to 4.8 A, ˚ the LDH–MTX complex was found to be 16.46 A˚ (21.26 4.8) A. Taking into account of the longitudinal molecular length of MTX to be 21.2 A˚ [3], a model of a bilayer anti-parallel arrangement of tilted MTX anions anchored to the brucite-like layers is suitably proposed [21,22]. The calculation of molecular length of MTX is based on a similar calculation as has been elaborated in the method for determination of the length and cross sectional area of a molecule of stearic acid (reference link: stone/2090/stearic.htm). The interaction of MTX with LDH was primarily through H-bonding between NH2 or COOH in MTX and OH groups in brucite layer. From XRD data it is also evident that LDH–MTX nanopowder synthesized using in situ synthesis route resulted in powders with the lowest crystallinity whereas nanopowders synthesized using hydrothermal ex situ process showed the highest crystallinity. Crystallinity of LDH–MTX composite plays a major role in particle dissolution and subsequent release of intercalated drug molecule from the matrix. The FTIR spectra of MTX–LDH hybrids are compared with that of pristine LDH in Fig. 2. The stretching band of both hydroxide and interlayer water molecule was found at approximately 3400 cm  1. For ex situ and in situ synthesized LDH–MTX samples, two separate peaks, one very broad peak at 3410 cm  1 and another very sharp intense peak at 3695 cm  1, for stretching vibration of hydroxyl groups were found. The peak at higher wave number of 3695 cm  1 appeared due to hydroxyl groups that were hydrogen bonded to MTX molecule, whereas the broad peak at lower wave number was due to free hydroxyl group in brucite layer. The peak at 2950 cm  1 indicated typical (CH)n stretching vibrations of MTX [3]. The strong absorption band at 1381 cm  1 signifies the presence of NO3 in LDH intensity of which was decreased in LDH–MTX samples. This suggests that MTX intercalation resulted in partial removal of NO3 in LDH. In the MTX– LDH, the absorption bands at 1614 and 1361 cm  1 corresponded to the stretching vibrations of C¼C in the backbone of the aromatic ring [23–26]. The peaks at 1550 and 1399 cm  1 were unambiguously assigned to anti-symmetric (nas) and symmetric (ns) stretching vibrations of the –COO  group of MTX anion [3,23]. The peaks and bands in the lower wave number region from 400 to 800 cm  1, were due to M–O vibration and M–OH bending in the brucite-like layers [27]. The peak due to Mg–O

Fig. 2. FTIR spectroscopy of (a) MTX, (b) LDH, (c) ex situ synthesized LDH–MTX, (d) hydrothermally synthesized ex situ LDH–MTX and (e) in situ synthesized LDH–MTX nanopowders.


Fig. 3. Particle size distribution in LDH and LDH–MTX nanopowders synthesized through different routes.

vibration mode was found at 451 cm  1, whereas the peak at 551 cm  1 indicated the presence of Al–O vibration mode. The particle size of LDH and LDH–MTX nanohybrids synthesized using different wet chemical routes was evaluated using dynamic light scattering (DLS) technique as depicted in Fig. 3. The pristine LDH showed a particle size in the range of 200–215 nm whereas the LDH–MTX nanohybrids synthesized using in situ process showed the lowest average particle size in the range of 40–45 nm. The hydrothermally synthesized pristine LDH exhibited the highest average particle size of 458 nm, although the particle size reduced to 295 and 342 nm for the LDH–MTX nanohybrids synthesized ex situ and by ex situ hydrothermal methods as above. The MTX molecule added during in situ synthesis process arrested the growth of LDH nanocrystals and resulted in the synthesis of LDH–MTX nanohybrids with the lowest particle size. Again hydrothermally synthesized pristine LDH nanoparticles showed an average particle size of 458 nm which was higher than the average particle size of 295 nm found in LDH–MTX nanohybrids synthesized using ex situ process. Hydrothermal crystallization promoted growth in LDH nanocrystal and resulted in higher particle sized pristine LDH nanopowders. The particle size measured using DLS technique was found to be higher than that is evident from TEM images in Fig. 4. The aspect ratio of all the synthesized nanopowders found to be quite high. DLS measures the hydrodynamic radius of nanoparticles taking into account of both translational and rotational motion of nanoparticles in suspended medium. As the hydrodynamic radius of gyration of LDH and LDH–MTX nanohybrids was much higher compared to the distance traversed during translational motion, a higher particle size value measured using DLS technique is quite expected. The morphology of individual nanohybrid particles was further studied using TEM as in Fig. 4. LDH–MTX nanopowders synthesized using in situ process showed lower aspect ratio as compared to LDH–MTX nanohybrids synthesized using the ex situ process. The MTX molecule added to the reaction mixture during the in situ synthesis process restricted the growth of nanocrystals in the z-direction and resulted in nanoparticles with lower aspect ratio. At higher magnification the layered structure of both pristine LDH and hydrothermally synthesized ex situ LDH–MTX were observed. The dark and bright stripes represented the mixed hydroxide layer and the interlayers with exchangeable organic MTX and inorganic NO3 and CO3 . The average d003 spacing was calculated by measuring 5–7 repeated layer-interlayer units as


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Fig. 4. Transmission electron micrographs of (a) hydrothermally treated LDH, (b) ex situ synthesized hydrothermally treated LDH–MTX, (c) LDH–MTX synthesized in situ and (d) LDH–MTX synthesized ex situ.

Fig. 5. HRTEM images of (a) hydrothermally treated LDH (b) ex situ synthesized hydrothermally treated LDH–MTX.

shown in Fig. 5. The pristine LDH (Fig. 5(a)) showed an average d003 ˚ which is close to the d003 value determined spacing of 7.95 A, ˚ in Fig. 1. The layer structure of LDH was using XRD (8.02 A), preserved after the incorporation of MTX anions to form MTX– LDH complex as evident from Fig. 5(b). The average value of d003 of MTX–LDH was found to be 21.11 A˚ in Fig. 5(b). This in turn is close to our XRD data of d003 of 21.26 A˚ in Fig. 1. Thus it was confirmed that the increase in interlayer distance was caused by the exchange of larger MTX anions with NO3 . Fig. 6(a) exhibits the chemical structure of methotrexate. It is a folate antimetabolite and an analog of aminopterin, which is also derived from folic acid. The molecular structure of methotrexate differs from folic acid in that it has a hydroxyl group in place of the 4-amino group on the pteridine ring and there is no methyl group at the N10 position. Methotrexate is a weak bicarboxylic acid with a pKa of 4.8–5.5. Fig. 6(b) exhibits the schematic representation of MTX intercalation in between the layers of LDH. The gallery height (basal spacing- thickness of brucite ˚ is less than the molecular length layers¼ 21.26–4.8 A˚ ¼16.46 A) ˚ Hence, to accommodate MTX within the layer of MTX, 21.1 A. space of LDH, it is placed at a tilted conformation here. From the EA data (Table 1), based on the carbon % present in the samples, the amount of MTX loading in sample A (ex situ hydrothermal) was found to be 422.504 mg/g, approximately

Fig. 6. (a) Chemical structure and IUPAC nomenclature of methotrexate drug (C20H22N8O5). (b) Schematic representation of the slanting conformation of methotrexate within the layers of LDH.

42.25 wt%, for sample B (in situ coprecipitation), it was 103.76 mg/g, approximately 10.376% and for sample C (ex situ ion exchange), it was 99.4 mg/g, approximately 9.94 wt% (from the elemental analyses (CHN) of all the three samples, as shown in Table 1) [28]. The ex situ hydrothermal sample exhibited nearly four (04) times drug loading in comparison to the other two samples. Fig. 7 shows the release profile of all the three samples at pH 7.4. The release rate of the samples varies at the first 10 h, commensurate with our drug loading data: for sample A and B450% release of drug takes place at the first 10 h whereas in case of C, it is around 40%. Although sample B and C have comparable drug content, the former exhibits a higher % release of drug on account of presence of a greater amount of loosely bound surface adsorbed drug due to incorporation of MTX in the precursor solution during its synthesis as mentioned in Section 2.2.2. The entire drug is released within a period of 170 h (47 days) maximum for all the three samples. The time span in between 50% and 90% release of drug (t50 and t90: 1090 h approximately) of samples A, B and C indicate controlled release profile for the same. Work is in progress to understand the exact mechanism of the release and the variation in release profiles of the individual samples and will be presented in details in our next communication.

4. Conclusions Mg Al–LDH nanohybrids intercalated with MTX were synthesized using in situ coprecipitation, ex situ and hydrothermal ex situ methods. The nanopowders were characterized using XRD, FTIR, DLS, TEM, EA and HPLC. XRD study showed an increase in d003 -spacing from 8.02 A˚ for pristine LDH to 21.26 A˚ for LDH–MTX due to MTX intercalation, which was consistent with TEM observations. FTIR spectra showed the splitting of hydroxyl bands in LDH into two peaks due to intercalation of MTX in LDH–MTX nanohybrids. Depending on the synthesis route the particle size of LDH-MTX nanohybrid varied from 40 to 458 nm. LDH–MTX nanohybrid synthesized using in situ process exhibited lower aspect ratio as compared to ex situ synthesized LDH–MTX nanopowder. The ex situ hydrothermal sample exhibited nearly four (04) times drug loading in comparison to the other two samples. The release

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Table 1 CHN Analysis of the samples. Sample name

Carbon (%)

Hydrogen (%)

Nitrogen (%)

Mg–Al–LDH–MTX (hydrothermally synthesized ex situ LDH–MTX) Mg–Al–LDH–MTX (in situ synthesized LDH–MTX) Mg–Al–LDH–MTX (ex situ synthesized LDH–MTX) Mg–Al–LDH

22.32 5.48 5.25 0.41

3.87 3.47 3.62 2.38

10.24 2.07 3.60 4.20

Sample weight: 1.706 g in all the samples given below.

120 % Release of Drug

A 100



C A. Hydrothermally synthesized ex situ LDH-MTX


B. In situ synthesized LDH-MTX


C. Ex situ synthesized LDH-MTX

20 0 0







Time (hrs) Fig. 7. Release of MTX at pH 7.4 of the Mg–Al LDH MTX samples: A Hydrothermally synthesized ex situ LDH–MTX, B in situ synthesized LDH–MTX and C ex situ synthesized LDH–MTX.

behavior also corroborates the above observation and show a controlled release profile of the drug delivery formulations.

Acknowledgement The authors are grateful to the Director, Central Glass and Ceramic Research Institute, Kolkata, India for providing his permission to carry on the above work. Thanks are due to all other support staffs Mr. Soumitra and Miss Kajari of CGCRI, Kolkata who made this work possible. We are indeed indebted to the 11th 5 year plan CSIR Network Program NWP 0035 for all the kind support and the financial assistance for undertaking this work. References [1] Z.P. Xu, Q.Z. Zeng, G.Q. Lu, A.B. Yu, Chem. Eng. Sci. 61 (2006) 1027–1040. [2] J.H. Choy, S.Y. Kwak, Y.J. Jeong, J.S. Park, Angew. Chem. Int. Ed. 39 (2000) 4042–4045.

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