morphology evolution, intercalation and release ... - Springer Link

Front. Mater. Sci. 2017, 11(4): 395–409 https://doi.org/10.1007/s11706-017-0400-1

RESEARCH ARTICLE

Layered double hydroxide using hydrothermal treatment: morphology evolution, intercalation and release kinetics of diclofenac sodium Mathew JOY (✉)1,2, Srividhya J. IYENGAR1,2, Jui CHAKRABORTY2, and Swapankumar GHOSH (✉)1 1 Project Management Division, CSIR-Central Glass & Ceramic Research Institute, Kolkata-700032, India 2 Bioceramics & Coating Division, CSIR-Central Glass & Ceramic Research Institute, Kolkata-700032, India

© Higher Education Press and Springer-Verlag GmbH Germany 2017

ABSTRACT: The present work demonstrates the possibilities of hydrothermal transformation of Zn-Al layered double hydroxide (LDH) nanostructure by varying the synthetic conditions. The manipulation in washing step before hydrothermal treatment allows control over crystal morphologies, size and stability of their aqueous solutions. We examined the crystal growth process in the presence and the absence of extra ions during hydrothermal treatment and its dependence on the drug (diclofenac sodium (DicNa)) loading and release processes. Hexagonal plate-like crystals show sustained release with ~90% of the drug from the matrix in a week, suggesting the applicability of LDH nanohybrids in sustained drug delivery systems. The fits to the release kinetics data indicated the drug release as a diffusion-controlled release process. LDH with rod-like morphology shows excellent colloidal stability in aqueous suspension, as studied by photon correlation spectroscopy. KEYWORDS: drug loading

layered double hydroxide; crystal morphology; hydrothermal treatment;

Contents 1 Introduction 2 Experimental 2.1 Materials 2.2 Synthesis of LCp 2.3 Synthesis of LHa 2.4 Synthesis of LHb 2.5 Preparation of drug-loaded samples 2.6 Instrumental characterization 2.7 Ion chromatography Received July 21, 2017; accepted September 8, 2017 E-mails: [email protected] (S.G.), [email protected] (M.J.)

2.8 Electron microscopy 2.9 Dynamic light scattering (DLS) 2.10 Thermal analyses 2.11 In vitro drug release study 3 Results and discussion 3.1 X-ray studies 3.2 IR spectroscopy 3.3 Electron microscopy 3.4 Thermal studies 3.5 Zeta potential measurements 3.6 Hydrodynamic size data 3.7 Analysis of release data 4 Conclusions Acknowledgements References Supplementary information

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Front. Mater. Sci. 2017, 11(4): 395–409

Introduction

Growing crystals in different crystal morphologies through various techniques has acquired great attention in recent years [1]. Many approaches like sacrificial template driven growth [2], solvothermal synthesis [3], high-pressure solution growth [4] and molecular capping [5] are often employed to realize various synthetic conditions for the growing demand in crystals synthesis. Among them, environment friendly low temperature aqueous hydrothermal process has great potential in altering the nano-level morphology through a slow and steady crystal growth in presence of the autogenous steam pressure [6]. Under hydrothermal conditions, crystal size, crystallization kinetics and even morphology of products are highly sensitive to reaction parameters like pH, ionic concentration and temperature. Layered double hydroxide (LDH) as a controlled drug release carrier has attracted great attention recently due to their advantages like biocompatibility [7], low cytotoxicity [8], good drug encapsulation potential [9], and full protection to the loaded drugs [10]. The LDH structure can be described by the general formula [M12+– x n– 2+ M3+ and M3+ are divalent x (OH)2]Ax/n$mH2O where M and trivalent metal cations, and An– is counter anion [11]. Structurally, LDH consists of brucite-like layers of edge sharing M(OH)6 octahedra, and the isomorphous replacement of a fraction of M2+ with M3+ leads to positively charged layers which are neutralized by the counter anions located in the hydrated interlayer galleries [12]. The excellent anion exchange property of LDH affords the reversible insertion and desertion of a number of organic molecules, biomolecular nucleic acids into the LDH structure [13]. In addition, there is ample scope for fine tuning the intercalation and de-intercalation processes by employing LDHs with different nano-morphologies and metal-ion combinations [10,14]. The ability of these materials as drug-delivery vehicle for the pharmaceutical agents like NSAID (non-steroidal anti-inflammatory) [9], anticancerous [15–16] and anticardiovascular drugs [17] has been clearly demonstrated. The height/length of the anionic drug molecules is normally 2–3 folds greater than the natural gallery heights of the nitrate/chloride interlayered LDHs. Therefore, during the intercalation process, the galleries will prop-up to a certain height in order to accommodate the drug in the restricted interlayer space. The precise positioning of drugs

in the interlayer gallery may be inclined at an angle to the hydroxide surface of the layers. After administration of the LDH-drug hybrid, the drug is released via de-intercalation process through ion exchange reaction with chloride or phosphate ions present in the biological fluids. Normally, the initial rate of the release process is very high, as the LDH host has great affinity towards phosphate and chloride ions. The poor kinetics control of the drug release in LDH material is a serious drawback for a drug delivery vehicle since pharmaceutical formulations has to maintain pharmacologically active drug levels for sufficiently long periods [18]. In order to overcome this issue, some modifications in the LDH synthesis protocol or postsynthesis surface modification with polymers or surfactants have been reported. The LDH particles are either initially surface modified by employing surfactants or polymers followed by intercalation with the drug molecules or by intercalating the LDH with drug molecules first with subsequent encapsulation of the LDH-drug hybrid particles [19–20]. Gunawan et al. synthesized unusual coral-like LDH systems through methanol/dodecyl sulfate (DS) system and then exchanged DS anions with drugs [21]. In another report, fenbufen-intercalated LDHs as the core was coated with enteric polymers such as Eudragit as a shell exhibited controlled release of drugs in vitro conditions which models the passage of the material through the gastrointestinal tract [22]. We reported another method of blocking the burst release of drug from LDH nanocarrier by imparting a poly(lactic-co-glycolic acid) (PLGA) coating over the drug-LDH hybrid [19]. Surface modified LDH nanocomposites are reported to improve drug immobilization [20], which sometimes questions the role of LDH as a controlled release system. In addition, the presence of such materials decreases the surface to volume ratio of the nanoparticle and thus reduces the drug:carrier ratio as well as the biocompatibility of the delivery system. The precipitation of LDH from the metal salt solution using an alkali, generally, do not have any control over the nucleation rate, growth and homogeneity which makes the size, morphology and aggregation control difficult [23]. Control over structural and textural properties of the final products has been recently attempted with a post hydrothermal treatment [24]. In fact, an earlier work reported the synthesis of stable homogeneous suspension of Mg–Al LDH using hydrothermal post treatment [25]. There are only few reports of drug incorporation in the hydrothermally processed LDH which studied the effect of synthetic

Mathew JOY et al. LDH using hydrothermal treatment: morphology evolution, intercalation and ...

parameters e.g., role of ions, pH etc. in the formation of LDH crystals in aqueous medium and its consequent effect in the drug intercalation capacity [26–29]. Aqueous coprecipitation produces highly aggregated sheet-like LDH crystals which has no colloidal stability in aqueous suspension. Nanoparticulate drug delivery systems based on penetration of biological barriers and controlled release of the drug involves fairly stable suspension of nanoparticulate drug carriers [30]. In this article, we demonstrate the fabrication of LDH structures by a combined co-precipitation process followed by hydrothermal treatment to improve the crystallinity and tailor the size of the LDH particles. The suspension after co-precipitation has been used for the subsequent hydrothermal treatment. Interestingly, the hydrothermal treatment before or after the washing process produced different morphologies with widely different properties. The colloid properties of the suspensions have been thoroughly investigated using photon correlation spectroscopy (PCS). Drug release kinetics from the LDH carrier was studied after intercalating LDH with diclofenac sodium (Dic-Na) in representative in vitro conditions. Particles obtained after autoclaving the washed precipitate showed rod-like morphologies with improved stability in aqueous solution and poor drug loading capacity, whereas the particles with unwashed procedure showed the usual plate-like hexagonal morphology and exhibited extended release up to ~9 d. LDH crystal shapes have been confirmed by electron microscopy as well as X-ray diffraction (XRD). The structural features have been ascertained from Fourier transform infrared spectroscopy (FTIR) and the analysis of X-ray data. The fabrication methods have been correlated with the surface characteristics of nanoparticles, stability of the suspension, and drug loading which indicates prospective sustained release of drug.

2

chemicals utilized were used as received without further purification. The water was decarbonated by purging with ultrapure nitrogen gas. 2.2

Synthesis of LCp

Zn2Al-NO3-LDH with the molar ratio of n(Zn):n(Al) = 2:1 was synthesized by a simple co-precipitation method. Solution A was prepared by dissolving Zn(NO3)2$6H2O and Al(NO3)3$9H2O into 20 mL water with the ratio of n(Zn):n(Al) = 2:1 (4 mmol Zn(NO3)2$6H2O and 2 mmol Al(NO3)3$9H2O). Solution B (0.15 mol/L NaOH) was prepared by dissolving appropriate amount of NaOH in 100 mL water. Co-precipitation was done by introducing solution A into solution B rapidly under vigorous stirring over a magnetic stirrer. The pH of the resulting solution was 10.2. The slurry was stirred for another 30 min at room temperature to ensure complete precipitation. The reaction was conducted in a 3-neck round-bottom flask under a flow of nitrogen to avoid intrusion of atmospheric CO2 into the LDH layer structure. The slurry was divided into three parts. The white gelatinous precipitate from one of them (part A) was separated by centrifugation and washed several times to remove impurities, dried and aged at 80°C overnight and is denoted as LCp. 2.3

Synthesis of LHa

The white gelatinous precipitate in the slurry (part B) was separated by centrifugation and washed several times to free from impurities/extra electrolytes. The precipitate was subsequently dispersed in 100 mL water and transferred into a Teflon lined stainless steel high pressure bomb and autoclaved at 100°C for 16 h at autogeneous steam pressure by placing the bomb in a hot-air oven. After natural cooling, LDH particles were separated via centrifugation at 7000 g rcf, dried and aged at 80°C overnight. The product is denoted as LHa.

Experimental 2.4

2.1

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Synthesis of LHb

Materials

Zinc nitrate hexahydrate (Zn(NO3)2$6H2O), aluminium nitrate nonahydrate (Al(NO3)3$9H2O), and sodium hydroxide (NaOH) were purchased from Merck, India. Dic-Na has been procured from Sigma Aldrich. Ultrapure deionized water (Millipore, specific resistivity 18.2 MW) was used in all the syntheses stated above and the

In this method, co-precipitated LDH slurry (part C) was directly used for hydrothermal treatment under conditions same as for the synthesis of LHa. The ionic strength due to salt formation was ~0.11 mol/L in this slurry. After cooling, the particles were separated via centrifugation at 7000 g rcf, dried and aged at 80°C overnight. The product is denoted as LHb.

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Preparation of drug-loaded samples

Dic-Na drug was intercalated into LDH layers through the anion exchange method. The intercalation was done by adding the drug solution (1.69 mmol in 20 mL) dropwise into a slurry containing 500 mg LDH dispersed in 20 mL water in nitrogen atmosphere. The resulting solution was kept at 60°C for 72 h with slow stirring. The drug-loaded particles were separated by centrifugation and washed with 1:1 ethanol–water mixture. Finally, the solids were freezedried (EYEL4, FDU2200, Japan) at 82°C and 20 Pa pressure. The drug-loaded LCp, LHa and LHb samples are denoted as LCpDic, LHaDic and LHbDic, respectively. 2.6

Instrumental characterization

The XRD patterns of the sample were obtained using a Philips X’PERT PRO diffractometer with Ni-filtered Cu Kα radiation (l = 1.5406 Å) using 30 mA current at 40 kV. The step size of 0.04 was used in the scan range 5°–70° (2θ). The preferred orientation of the crystallographic planes was estimated as texture coefficient [31]. FTIR spectra of the sample were recorded using the KBr pellet method (sample:KBr = 1:100) on a Perkin–Elmer Spectrum 100 spectrophotometer in the 400–4000 cm–1 range. 2.7

Ion chromatography

The concentration of carbonate, nitrate and chloride ions for the anion exchange study was carried out using an ion chromatograph (792 Basic IC, Metrohm AG, Switzerland). Metrosep Organic Acids-250 (6.1005.200), Metrohm, Switzerland, separation column was connected for the detection of carbonate ions. The ion chromatograph was calibrated using standard sodium carbonate solutions. Nitrate and chloride ions were detected using a Metrosep A Supp 5-250 (6.1006.530) along with Metrosep A Supp 4/ 5 Guard Column (6.1006.500), Metrohm, Switzerland. Thermostated electrical conductivity was used as the mode of detection in all the cases. For the anion exchange study, 200 mg of bare LDH sample were dispersed in 100 mL, 100 ppm (1 ppm = 10–6) of sodium chloride solution. The solution was placed overnight in a tightly closed condition to avoid the absorption of atmospheric carbon dioxide. The supernatant liquid was separated and filtered using luer lock syringe with 0.2 μm filter paper. About 3.2 mmol/L Na2CO3 and 1 mmol/L NaHCO3 solutions were used as the mobile phase with 50 mmol/L H2SO4 suppressor solution for suppressed conductivity detection [32]. The instrument

was calibrated using standard solutions of sodium chloride. Zn and Al molar ratio in the products were determined by inductively coupled plasma atomic emission spectrometer (ICP-AES) (Spectro Analytical Instrument). 2.8

Electron microscopy

The nanoparticle morphology and the size were investigated by high resolution transmission electron microscopy (HR-TEM) using a FEI Tecnai 30 G2 S-Twin microscope operated at 300 kV and equipped with a Gatan CCD camera and also a field emission scanning electron microscope (FESEM) (Carl Zeiss SMT AG SUPRA 35VP, Germany). The elemental composition of the samples (data not shown here) was studied using energy dispersive X-ray spectroscopy (EDX) attached to the FESEM. The samples for microscopy were prepared by dispersing the specimens in deionized water and diluted 1:5 (v/v) with the same at room temperature. 2.9

Dynamic light scattering (DLS)

The zeta potential, hydrodynamic size and the colloidal stability of the dispersed LDH particles were measured by photon correlation spectroscopy using a Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK). The surface charge was estimated by measuring the zeta potential of their dispersion in water by laser-Doppler velocimetry technique. The change in zeta potential of LDH nanoparticles and their drug-loaded counterparts were measured in a wide range of pH. The instrument uses a 4 mW He–Ne laser of wavelength 632.6 nm and a scattering angle of 173°. 2.10

Thermal analyses

Thermogravimetric and differential thermal analyses (TG and DTA) of sample powders were carried out using a NETZSCH STA 409 CD thermal analyzer. The thermograms were collected using a heating ramp of 10°C/min in the temperature range of 30°C–1100°C. Powder contact angles were measured with a KRUSS GmbH instrument (easy drop DSA20E model) using 6 µL water droplets under ambient condition. 2.11

In vitro drug release study

In vitro release of diclofenac drug from the intercalated LDHs was carried out using dissolution test apparatus (Electrolab TDT-146 08L Mumbai, India). 0.2 g of drug


loaded samples was dispersed in 1000 mL phosfate buffer saline (pH 7.4) maintained at 37.2°C with constant stirring at the speed of 100 r/min. At specific time intervals, 10 mL of aliquot was withdrawn for estimating drug and replenished immediately [19]. The suspensions have been filtered through a 0.2 μm Nylon 66 filter paper and 10 µL filtrate was injected into the chromatograph (HPLC 820 Metrohm AG, Herisau, Switzerland). The retention time of the diclofenac drug in the column was recorded against the absorbance of the same using the UV-Vis detector (Bischoff Lambda 1010, Switzerland, with l in the 190– 800 nm range). The mobile phase was 20 mmol/L phosfate buffer (pH 7.0)/acetonitrile (65:35), at the flow rate of 1 mL/min. The HPLC was calibrated using standard solutions of diclofenac in phosfate buffer saline (pH 7.4) in the concentration range of 10–100 ppm.

3

Results and discussion

3.1

X-ray studies

Figure 1 shows XRD patterns of the pristine LCp and hydrothermally treated LHb specimens as well as their drug-loaded counterparts. Sharp and intense basal reflections are characteristic of this group of materials. According to the standard card of Zn-Al LDH (JCPDS card No. 38-0486), all the peaks belong to Zn-Al LDH with the R3m rhombohedral symmetry. Above 30° (2θ), the relative intensities of diffraction peaks, namely (101) and (104) planes with d-values of 2.65 and 2.5 Å respectively, are much stronger in LHa (Fig. S1), suggesting that LHa tends to grow preferentially along (101) and (104) direction as already been reported [33]. Therefore, the post synthesis autoclaving has changed the LDH structure. However, the XRD pattern of LHb remains unchanged and is quite similar to that of LCp. The position of pronounced diffraction peaks (003) and (006) translates the interlayer spacing and therefore on the anions present in the interlayer (Fig. 1). The lattice parameters and interlayer space of the samples, calculated from the positions of the (00l) and (110) peaks, are provided in Table 1. XRD patterns of LHbDic and LCpDic show a shift of d00l reflections towards lower 2θ angles suggesting an increase in interlayer spacing. The enhanced d-spacing is an indication of the successful intercalation of Dic-Na into the interlayer galleries of LDH. It is also interesting to note that LHbDic produces relatively higher crystalline peaks

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Fig. 1 XRD patterns of LCp, LHb and their drug-loaded counterparts, LCpDic and LHbDic, showing high intense basal reflections.

than that in LCpDic owing to better ordering in stacking of layers induced by the hydrothermal treatment during crystal growth [9]. After subtracting the layer thickness of the brucite sheet (4.8 Å) from the interlayer spacing [34], the space (17.7 Å) available in the interlayer for diclofenac is ~1.2 times greater than the size of the guest molecule (14.2 Å). Therefore, we propose that the guest ions are oriented in a bilayer fashion, with carboxylate ion close to the positive brucite-like layers as illustrated in Scheme 1. LHa did not produce any gallery expansion (Table 1). This is correlated with the TG data in a later section. Drug molecules in the LDH structures attain a stable conformation due to the drug to metal hydroxide interaction and drug to drug interaction which result in extended release of drug [17]. However, the cell parameter of a is almost same in all set of samples, since there is no cation impurities into the hexagonal layer structure and the molar ratio of n(Zn):n(Al) remain almost unchanged [9]. 3.2

IR spectroscopy

Further confirmation of the structure of LDH and the intercalated structure of the drug-LDH hybrid was obtained from FTIR analysis and is provided in Fig. 2. The spectra for LCp, LHa and LHb exhibit typical features of layered double hydroxide. The broad absorption band between 3000 and 3600 cm–1 is assigned as the stretching of Hbonded – OH groups in the LDH layers. The strong absorption band at 1386 cm–1 is assigned to the NO3– stretching vibration, whereas the weak bands at ~600–800 cm–1 correspond to the bending vibrations of the nitrate groups. The bending vibration of water molecule (H – O – H) was observed at 1636 cm–1. The carbonate

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Table 1 Calculated lattice parameters and interlayer spacing of various LDH samples Sample

Cell parameters

d(003)/Å

d(006)/Å

l/Å

22.8

7.60

3.8

7.59

3.07

65.88

21.9

11.22

22.2

3.06

22.59

7.53

3.75

7.5

LHaDic

3.06

23.04

7.68

3.82

7.6

LHb

3.07

26.8

8.93

4.42

8.8

LHbDic

3.07

67.05

22.35

11.38

22.5

a/Å

c/Å

LCp

3.07

LCpDic LHa

Note: The interlayer spacing, l, can be calculated as: l = 0.5 (d(003) + 2d(006)).

stretch at ~3300 cm–1 of diclofenac sodium remains invisible in the spectrum as it is overlaid by the broad absorption band of OH vibrations from hydroxide layer of LDH. It is noteworthy that the intensity of diclofenac peaks (in marked rectangle) in LHaDic is not as intense as the peaks observed with LCpDic and LHbDic which indicates indirectly the low drug intercalation efficiency of LHa. 3.3

Scheme 1 (a) The sketch showing probable orientation of DicNa drug in the interlayer gallery of LHbDic. (b) The chemical structure of Dic-Na.

vibration is observed at 1352 cm–1 as shoulder peak in the intense nitrate peak. The intense peaks at ~1572 and 1391 cm–1 in pure Dic Na are due to symmetric and asymmetric stretching of COO– group. After the ion exchange reaction, the intensity of IR bands for nitrate decreased and the peaks related to drug becomes discernible in the 700–1600 cm–1 range. The diclofenacloaded LDH spectra (Fig. 2) show – COOH stretch at 1578 cm–1, – COOH bend at ~1293 cm–1, aromatic C – Cl stretching at 775 and 746 cm–1, and characteristic aromatic peaks at 1499 and 1446 cm–1 [35]. The distinctive NH

Fig. 2 FTIR spectra of pristine and diclofenac-loaded LDHs.

Electron microscopy

Few selected electron micrographs ca. FESEM of LCp and TEM of LHa and LHb are shown in Fig. 3. The FESEM image of LCp shows clusters of aggregated LDH nanoparticles. The TEM image of LHa presents spindlelike particles resulted from the preferential growth of the (101) and (104) planes, in agreement with the XRD result (Fig. S1 in ESI). In addition, selected area electron diffraction (SAED) pattern (inset to Fig. 3(b)) demonstrates clear Debye–Scherrer rings corresponding to (101) and (104) planes ascertaining the above observation. The HR-TEM image of LHb contains large sheets of LDH crystals growing orthogonal to the (00l) direction and its SAED pattern (inset to Fig. 3(c)) shows Debye–Scherrer rings of (003) and (006) facets which exemplify the usual hexagonal crystal structure of LDH as was already shown by the XRD results (Fig. 1). The mechanism of the crystal growth in LHb can be explained by considering the electrostatics that played when the crystal nuclei are in solution. The high charge density of LDH leads to the electrostatic interaction between the layers which initiates the aggregation process and assists the crystal growth mechanism. Electrostatic interactions among the crystals during hydrothermal treatment have been manipulated by the washing step following precipitation. The washing process prior to autoclaving controls the local environment of the crystals during hydrothermal treatment which modulates the crystal-growth process. The illustration of the possible morphological evolution of the large hexagonal LHb nanostructures and LHb-drug hybrid is shown in


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Fig. 3 (a) FESEM image of LCp. (b)(c) Bright-field TEM images of hydrothermally prepared LHa and LHb respectively when viewed in the [001] zone axis. Insets of (b) and (c) show the SAED patterns.

examination of the rod type crystal structure using XRD and electron microscopy suggests dissolution and recrystallization as the best mechanism to explain the growth in to the elongated crystal [33]. 3.4

Scheme 2 Schematic representation of the formation of LHb crystals and the loading of drug into the sample.

Scheme 2. The unwashed slurry on hydrothermal treatment with large excess of impurity ions localized on to the positively charged LDH crystal surfaces. At high temperature and pressure, the total interaction between the particles will be governed by Brownian motion and electrostatics. However, if the concentration of salt ion is high like in the case of LHb crystals, shrinkage of electric layer occurs and short range van der Waals forces keep them together. This results in the more perfect stacking of the LHb layers. Moreover, this also helps in the further growth of crystals in the a-b direction, more uniform distribution of metal ions in the brucite layers and a more regular crystallite shape. In the case of thoroughly washed crystals factors other than electrostatics may play the key role, particularly at neutral pH, and elevated temperature/ pressure for relatively longer reaction times. Here, positively charged LDH crystals can escape from the aggregates through Brownian motion and adopt a different pathway for crystal nucleation and growth [25]. Close

Thermal studies

TG and DTA profiles of pristine and drug-loaded LDH specimens, carried out under air purge (thermooxidative) is presented in Fig. 4 (partly supplied in Fig. S2 in ESI). The detailed analyses of the TG and chloride ion saturation data are provided in Table 2. The loading of drug in LDH samples was computed from the thermogravimetric data as already reported [36]. Pristine LHb powder has a total loss of ~26% comprised of adsorbed and structural water. The thermal decomposition of LHbDic has three distinct steps involving a total loss of ~49%. The TG patterns of LHb and LHbDic are almost similar till 200°C. The first peak in LHbDic at ~100°C is due to the removal of adsorbed water molecules from the LDH layers (panel (a)) and its corresponding endothermic effect can be seen in the DTA curve (panel (b)). As the temperature was further increased, there were significant changes as loss at that stage depends on the amount of structural water component along with the intercalated diclofenac in the structure [37]. The second loss occurs between 120°C and 220°C due to the dehydration of interlayer region and removal of adsorbed gaseous molecules that has been trapped from the ambient during synthesis and intercalation process (CO2). Another small loss in weight between 230°C and 320°C is observed in all samples of LDH. Costa et al. identified this as the partial dehydroxylation of hydrotalcite layers [38]. The remaining mass loss in pristine LDH above 330°C is due to the complete dehydroxylation of hexagonal layers [39]. On detailed analysis, the loading of drug (Dic) into LHb

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Fig. 4 (a) TG and (b) DTA plots of LHb (i), LHbDic (ii) and pure Dic-Na (iii). The derivative of TG data of LHbDic is shown as dotted line.

crystals. In such structures, water molecules coordinate to the layer edges rather than in the basal planes and leave more easily as they face less diffusional resistance [40]. The small loss of 3.3% at ~330°C in LHa is due to the removal of NO3– in the layers which has been replaced by drug anions in LHaDic. LCp powders have 1% surface adsorbed moisture which on drug loading increased to ~4.8% (the highest among all the drug-loaded LDH powders) in LCpDic (Fig. S2) and varied according to the degree of hydration of the sample. The DTA curves of all the LDH specimens exhibit this change as an endothermic peak at ~100°C (DTA of LHa and LCp samples are not provided here). The drug loading capacity was also affirmed by the HPLC analysis is provided in Table S2 in ESI. The drug loading in the above specimens can be correlated with the chloride saturation ion chromatographic (anion exchange) studies conducted with LHa, LHb and LCp which suggests that anion exchange capacity is following the order LHa < LCp < LHb and is supported by the drug loading data. LHb crystals have grown under the 0.11 mol/L ionic strength during autoclaving, with resultant small particle sizes with high ion exchange capacity. 3.5

is calculated as 30.1% along with 16.5% structural moieties and ~2% adsorbed moisture. XRD data (Fig. 1) have shown an enhancement of d-spacing of 8.8 Å in LHb to 22.5 Å in LHbDic due to the intercalation of Dic-Na into the layered structure. Drug loading in the precipitated LCpDic is ~19.6% which is of the same order as already reported [11]. TG patterns of LHa and LHaDic (Fig. S2) are almost similar except that the total weight loss of 16.8% in pristine LHa increased to ~23.4% in LHaDic, indicating a poor loading of ~7.7% drug, coexisting with 13.7% structural moeties (Fig. S2 in ESI). During initial thermal events, the surface as well as structural water content of LDH-Ha and its drug intercalates decaying rapidly than other samples. This may be related to the elongated morphology of the

Zeta potential measurements

The change in zeta potential of the pristine as well as drugloaded LDH suspensions in a wide range of pH are shown in Fig. 5. Zeta potential measures the electric potential at the boundary of the double layer. Due to the positive charge of brucite layers, LDH attracts a thin layer of ions of opposite charge on to its surface in aqueous suspension and these ions in the proximity of particle surface forms an electric double layer structure (http://cdn.shopify.com/s/ files/1/0257/8237/files/nanoComposix_Guidelines_for_Zeta_Potential_Analysis_of_Nanoparticles.pdf). Natural pH of LDH suspension was measured as ~6.5. LDH particles are structurally unstable below its natural pH and get dissolved when it is subjected to any pH falling condition. For the zeta potential data against pH of the

Table 2 Analyses of thermogravimetry data and chloride ion saturation into the LDH structure Sample

Weight remaining/%

Adsorbed moisture/%

Weight content of structural ions/%

LHb

74.0

2.0

24.0

LHbDic

51.0

2.4

16.5

LCp

71.0

1.0

28.0

LCpDic

54.2

4.8

21.4

LHa

83.2

1.9

14.9

LHaDic

76.6

2.0

13.7

Drug loading/%

Chloride loading/(meq$g–1) 0.59

30.1 0.56 19.6 0.073 7.7


Fig. 5 Zeta potentials of various LDH and drug-loaded samples with different pH values varying from neutral to alkaline.

slurry, the particles were dispersed in water and subsequently its pH was increased by the dropwise addition of 0.01 mol/L NaOH solution. All bare LDH samples exhibit ζ values in the range from + 38 to – 30 mV in the pH range of 6–13. The colloidal dispersion (LHa) exhibited high degree stability at ζ values greater than + 25 mV. There was no apparent aggregation above this ζ value; however at pH 10.8 where ζ value for LHa drops to zero, a rapid precipitation of colloid was observed. So it can be concluded that LHa is stable below its isoelectric point. Both the hydrothermally treated samples have point of zero charge (pHpzc) at pH 10.8 whereas LCp neutralize its surface charge at pH 9.8. The ζ-value of LHbDic and LCpDic at its natural pH is around zero, suggesting anionic drug molecules displaced the labile nitrate ions, thereby, neutralizing the charge imbalance in the LDH structure. But the ζ value in LHaDic at natural pH is 26.5 mV, suggesting only few dichlofenac has entered into the LDH structure as confirmed by XRD, FTIR and TG. All LDH samples were found to be stable only at their natural pH, except LHa which shows some stability up to its isoelectric point. The sedimentation at higher pH values can be explained by considering the factors like ionic concentration and its relation to the electric double layer thickness. The electric potential drops approximately following E / expð – κÞ

(1)

and κ is given by " κ¼

F2

X i

ðCi Zi2 Þ

εr ε0 R g T

#1=2 (2)

where 1/κ is known as the Debye–Hückel screening strength and to describe the thickness of the double layer, F is the Faraday’s constant, ε0 is the permittivity of

403

vacuum, εr is the dielectric constant of solvent, and Ci and Zi are respectively concentration and valence of counter ions of type i. This equation shows that the electric potential decreases with the increase in concentration of the ions (http://depts.washington.edu/solgel/pages/courses/ MSE_502/Electrostatic_Stabilization.html). At higher pH, ionic content in the solution are quite high which is described by the higher conductivity values (Fig. S3 in ESI). As the ionic concentration increases, the thickness of the double layer (repulsive barrier) decreases. Due to the combination of Brownian motion and van der Waals attraction forces, particles come close to each other and may stick to form an aggregate. 3.6

Hydrodynamic size data

Autocorrelation function against time as well as the temperature trend of hydrodynamic size and polydispersity index (PDI) is shown in Fig. 6. Single-exponential decay of the correlation function of the scattered intensity G2(t) is observed for the LHa suspension (PDI 0.278) indicating the monodisperse nature of the distribution. During the initial time (t = 0) and even after ~10 μs time, the correlation between the scattered photons remain high (G2(t) ~ 1.0) as the particles do not have chance to move to a great extent from the initial state indicating they are sluggish due to large size (Zav 655 nm) and keeps similar correlation up to ~100 μs in LHb, followed by exponential decay in correlation to about zero [41]. The decay process for LHb is rather longer that in LHa. In order to understand the heat induced clustering, few measurements were conducted by varying the slurry temperature from 20°C to 90°C. When temperature for LHa suspension was increased from 20°C to 40°C, the hydrodynamic size 216 nm grew to 222 nm and decreased down to 212 nm on increasing the temperature further from 40°C to 60°C (Fig. 6). Similar trend (sinusoidal) was repeated on further increase of temperature. We did not observe any stability related issues in the suspension while increasing the temperature. This may be due to the poor fit to the autocorrelation function as translational diffusion and rotational diffusion involved in the correlation decay process of LHa rods. The stability of the suspensions as observed by change in hydrodynamic size and photon count rate as a function of time is depicted in Fig. S6. We observed that the LHa suspension is stable on ageing at room temperature up to 3 d. After that larger particles start settling along with drop in photon count rate as well as size.

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release of intercalated drugs is crucial in the toxicity control of certain anticancerous drugs [42]. LHaDic shows a burst release of ~96% in short span of ~7 h time and its entire drug load (97%) was released in 16 h. The release profile of LCpDic shows that ~50% of the incorporated drug was released in 1 h and 90% drug release took ~40 h, whereas LHbDic hybrid particle released 50% of the drug in 6 h, ~78% in 60 h and 92% in a week giving clear indication of the ability of hydrothermally produced LHb in sustained drug release. Powder specimens of LHb and LHbDic demonstrate its hydrophilic surface property with contact angle of 37° and 33°, respectively (Fig. 7). The increased hydrophilicity of LHbDic is due to the strong hydrophilic nature of Dic-Na. Therefore, it is expected that Dic-Na will be released from ZnAl-LDH structure into PBS of pH 7.4 relatively faster than the same for any hydrophobic counterparts. Fluvastatin drug release from MgAl-fluva was reported to be slightly slower in the beginning (50% and 80% drug releases in 15 and 60 h, respectively).

Fig. 6 (a) The decay of auto correlation function with time obtained from DLS for LHa (i) and LHb (ii). The inset shows the hydrodynamic size of LHa (dotted line) and LHb. (b) Zav size and PDI as a function of temperature for LHa.

The stability of drug-loaded LHaDic was only few hours. The hydrodynamic size of LHbDic was measured as 637 nm, (Zav of LHb was 655 nm), showing almost no growth of LDH crystal on drug loading in LHb (Fig. S6 in ESI). 3.7

Analysis of release data

The cumulative release of Dic-Na from the LDH matrix of LHaDic, LHbDic and LCpDic was monitored by HPLC as a function of the reaction time at a constant temperature of 37°C, and microscopic images of water droplets while the contact angle measurement are presented in Fig. 7. The drug anions could be recovered intact from intercalates after the release process as we did not observed any additional peaks (for any chemically modified products) in the HPLC data along with diclofenac peak (not shown here). The BET (Brunauer–Emmett–Teller) surface area (Fig. S7 in SI) of LHaDic is 57.9 m2/g which is higher than those of LCpDic (11.9 m2/g) and LHbDic (12.0 m2/g). The creation of surface area in LHaDic is due to the presence of pores where amount of drug entered into the structure was relatively less and this can be correlated with the ζ results, as at the neutral pH LHaDic shows positive ζ values due to the charge imbalance. Sustained, controlled and slow

Fig. 7 (a) Release profiles of diclofenac drug from LHaDic (i), LCpDic (ii) and LHbDic (iii). Contact angle microscopic images of water droplets in (b) LHb and (c) LHbDic surface.

It may be noted that fluvastatin drug intercalated LDH hybrid is slightly hydrophobic as the drug contains large hydrocarbon tails [17]. Burst release from drug carrier was reported to be arrested by coating the MgAl-LDH–MTX nanoparticles with PLGA and only 13% release of MTX drug was achieved in 8 h and ~60% in 2 d [19].


To understand the release mechanism of guest molecule, we tried to fit the observed release data into appropriate kinetic models. Here it should be remembered that the significant drug loading was achieved in LHb and LCp (LHa has only 7.7% loading) and hence release kinetics of LHbDic and LCpDic hybrids will be discussed in this section. The mathematical forms and correlation coefficient values obtained from the fits are given in Table 3. A good drug carrier is assessed on the basis of its drug loading capacity and the ability to sustain its release over about appreciable time. The fits to the release data of LHbDic and LCpDic following different kinetic models are provided in Figs. 8 and 9, respectively. These models describe different physical processes. The first-order model describes a system where the release is dependent on the dissolution of the host material. The parabolic model details a diffusion-controlled process, whereas the Elovich model represent a complex process which covers a number of processes, including bulk and surface diffusion, and the activation/deactivation of catalytic sites. The R-P equation

405

relates exponentially the drug release to the release time in which n is the release exponent indicative of the drug release mechanism. The value of n < 0.45 corresponds to the drug diffusion control; n > 0.89 is attributed to the dissolution of LDH matrix; 0.45 < n < 0.89 is due to the cooperation of drug diffusion and LDH matrix dissolution. All the models fit the release data reasonably well except the first-order model for LHbDic (Fig. 8). This is an indication that drug release from LHbDic is a diffusioncontrolled process. The value of n obtained from the R-P equation is 0.214 (n < 0.45), which is confirmative of the drug diffusion control of LHbDic. For LCpDic fits are poorer for the first-order model as well as the parabolic diffusion model (Fig. 9). The value for exponent n obtained from the R-P equation is 0.118 (n < 0.45), so the release process follows a diffusioncontrolled process. We believe that the slower and sustained release of drug from LHbDic compared to that from LCpDic is strongly related to better ordering in crystal stacking in LHb as evident from the XRD pattern and the lateral dimensions of

Table 3 Linear correlation coefficient (R2) values of different kinetic models for LHbDic and LCpDic Kinetic model First-order model Parabolic diffusion model Elovich model Ritger–Peppas equation

Mathematical form

Parameter, R2 LHbDic

LCpDic

ln(Ct/C0) = – kdt

0.872

0.823

(1 – Ct/C0)/t = kdt–0.5 + a

0.973

0.800

1 – Ct/C0 = alnt + b

0.985

0.979

Ct/C0 = kd(t – a)n

0.991

0.986

Notes: C0 represents the amount of sample at time t = 0, Ct is the amount of drug at time t, kd is the rate of release, and a, b and n are constants.

Fig. 8 Fits to drug release data of LHbDic for different kinetic models: (a) first-order model; (b) parabolic diffusion model; (c) Elovich model; (d) R-P equation.

Fig. 9 Fits to drug release data of LCpDic for different kinetic models: (a) first-order model; (b) parabolic diffusion model; (c) Elovich model; (d) R-P equation.

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LDH. In the case of LHaDic, the drug molecules were attached on the periphery of the LDH structure leading to the fast and complete release in a short span of time.

[4] Bockowski M, Grzegory I, Krukowski S, et al. Directional crystallization of GaN on high-pressure solution grown substrates by growth from solution and HVPE. Journal of Crystal Growth, 2002, 246(3–4): 194–206

4

Conclusions

ZnAl-LDH nanoparticles have been fabricated under hydrothermal processing condition with different processing parameters. The role of extra ions during autoclaving on the growth pattern of the LDH crystals is systematically studied. XRD patterns showed that the expansion in the interlayer spacing of LHbDic is ~22.5 Å, slightly higher to that of LCpDic. On the basis of TGA and HPLC data, the loading of diclofenac in LHbDic was found to be ~34 wt.% which is higher than that in LCpDic and comparable with recent reported data. More importantly, sustained drug release could be achieved without any significant burst release. Such slow and sustained release has been demonstrated in the literature only with the usage of polymer or enteric coating on LDH systems. So here, this bottleneck has been broken by the inclusion of hydrothermal treatment and explored the possibility to use pristine LDH directly as drug delivery systems. The hydrodynamic data of LHa obtained from the DLS measurement clearly shows its good stability in water, but its drug loading efficiency is very poor and showing undesirable burst release kinetics at physiological condition. Acknowledgements The authors are grateful to the Director, Central Glass & Ceramic Research Institute, Kolkata for permission and extending facilities to carry out the above work. MJ and SJI acknowledge UGC and CSIR for their fellowships. We thank 12 FYP CSIR Network project ESC0103 for funding the DLS facility. Staff members of electron microscopy, XRD, FTIR and Central Instrumentation Facility are also acknowledged for their assistance in obtaining data.

[5] Cara C, Musinu A, Mameli V, et al. Dialkylamide as both capping agent and surfactant in a direct solvothermal synthesis of magnetite and titania nanoparticles. Crystal Growth & Design, 2015, 15(5): 2364–2372 [6] Ortiz-Landeros J, Gómez-Yáñez C, López-Juárez R, et al. Synthesis of advanced ceramics by hydrothermal crystallization and modified related methods. Journal of Advanced Ceramics, 2012, 1(3): 204–220 [7] Xu Z P, Lu G Q. Layered double hydroxide nanomaterials as potential cellular drug delivery agents. Pure and Applied Chemistry, 2006, 78(9): 1771–1779 [8] Choi G, Kim S Y, Oh J M, et al. Drug-ceramic 2-dimensional nanoassemblies for drug delivery system in physiological condition. Journal of the American Ceramic Society, 2012, 95 (9): 2758–2765 [9] del Arco M, Gutiérrez S, Martín C V, et al. Synthesis and characterization of layered double hydroxides (LDH) intercalated with non-steroidal anti-inflammatory drugs (NSAID). Journal of Solid State Chemistry, 2004, 177(11): 3954–3962 [10] Khan A I, Lei L, Norquist A J, et al. Intercalation and controlled release of pharmaceutically active compounds from a layered double hydroxide. Chemical Communications, 2001, (22): 2342– 2343 [11] Chakraborty M, Dasgupta S, Soundrapandian C, et al. Methotrexate intercalated ZnAl-layered double hydroxide. Journal of Solid State Chemistry, 2011, 184(9): 2439–2445 [12] Zhang F, Zhao L, Chen H, et al. Corrosion resistance of superhydrophobic layered double hydroxide films on aluminum. Angewandte Chemie International Edition, 2008, 47(13): 2466– 2469 [13] Zhang K, Xu Z P, Lu J, et al. Potential for layered double hydroxides-based, innovative drug delivery systems. International

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Table S1 Sample

Journal, 2014, 8(1): 47 (8 pages)

Supplementary information

Composition by weight /% Zn

Al

Nitrate

LCp

43.63

8.62

18.6

0.56

LHa

43.53

5.95

13.34

0.26

LHb

43.45

8.34

17.89

0.64

hydroxide nanocomposite for drug delivery systems; bio-distribution, toxicity and drug activity enhancement. Chemistry Central

Summary of chemical analysis data of pristine LDH Carbonate

Determination of diclofenac loading by HPLC The percentage loading of diclofenac was estimated by HPLC analysis. 10 mg of LDH-Dic was dispersed in 20 mL PBS solution and sonicated for 10 min to release the dicolfenac from the LDH. The extract was filtered and injected into the HPLC instrument to quantify the loading. Table S2 Sample

Drug content value calculated from HPLC data Drug content /%

LHaDic

5.7

LHbDic

32.45

LCpDic

22.5

Fig. S1 The XRD pattern of LHa. The vertical drop-lines correspond to JCPDS No. 38-0486 shown for comparison of reflection positions and intensity.

Fig. S3 The conductivity changes of LHb suspension while varying the pH of surrounding medium from 6 to 13.

Fig. S2 TG curves: (a) LHa (i), LHaDic (ii); (b) LCp (iii), LCpDic (iv). The derivatives of TG data of LHaDic and LCpDic are shown as dotted lines.

Fig. S4 The high-quality phase data associated with LHb samples from positive to negative with zeta potential while varying the pH value of surrounding medium from 6 to13.