Controlled drug delivery of antileishmanial chalcones from Layer-by-Layer (LbL) self assembled PSS/PDADMAC thin films

Abstract

Layer-by-Layer (LbL) approach was applied for the encapsulation of antileishmanial drugs viz. chalcones (3-mB-4′-HC and 3-DC-4′-HC) to study their release properties at pH 7.4 from a polyelectrolyte self assembled multilayer thin film. The LbL self assembly was achieved by alternate adsorption of oppositely charged polyelectrolytes, poly(styrene-4-sulfonic acid) sodium salt (PSS) and poly(diallyldimethylammonium) chloride (PDADMAC) on planar quartz substrate. The growth of the multilayer self assembly as well as loading and release of the drugs were studied by UV-Visible spectroscopy. Both the chalcones, 3-mB-4′-HC and 3-DC-4′-HC have shown controlled and sustained release up to 224 and 824 minutes respectively. Kinetic fitting of the data confirmed that the process of drug release from the self assembly followed pseudo second order kinetics (R² ≥ 0.99).

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1. Introduction

There has been a great deal of research in recent years towards improving drug delivery and selectivity through the macromolecular-based drug carriers.¹ Controlled drug delivery has been proven to bear certain advantages over the conventional drug administration. More so in the cases where the drug has to target localized tissues thereby reducing damage to healthy tissues of the subject and its concentration is to be maintained with respect to time for prolonged effect. In all these cases the release of the drug and its localization is achieved through a drug carrier. Therefore the carrier and its structural features are also important for the best outcome of the method of controlled delivery. With the recent advances in the synthesis and deposition methods of biocompatible polymers, several drug delivery techniques have been emerged. Amongst these, controlled drug delivery through encapsulation of drugs has successfully been attained for a number of therapeutics viz. anti inflammatory agents,² anticancer drugs,³ steroids,⁴ proteins,⁵ antibiotics,^6,7 hormones,⁸ anesthetics⁹ and vaccines.^10,11 However self assemblies developed by Layer-by-Layer (LbL) method have mostly been applied to the delivery of anticancer drugs. This is due to the fact that stimuli responsive release of the drug can be achieved for cancerous tissues owing to the difference in the pH between cancer cells and their healthy counterparts.^12,13 There are few reports available for the delivery of anti-inflammatory drugs,¹⁴ enzymes,^15–19 hormones²⁰ and proteins.^21,22 However, delivery of antileishmanial drugs through LbL self assembled drug delivery vehicles is still lacking.

The effective delivery of the drugs by the LbL technique is dependent upon the properties of the multilayer self assembly such as its mechanical strength and permeability which in turn lies in the very nature of the deposition technique, the nature of the materials used, temperature and pH of the solution.¹⁵ There are many parameters which control the film thickness, surface area, drug concentration and possibility of films for release of multiple drugs.²³ The LbL deposited carriers proved to be most useful in the treatment of dermatological diseases, burns and in military applications for handling the situations arising from acute microbial attack through skin in case of biological warfare.

Leishmaniasis has been identified as a major and increasing public health problem particularly in Africa, Asia and Latin America. Leishmaniasis is a group of diseases caused by trypanosomatids from the genus Leishmania. It is one of the most neglected tropical diseases with a major impact on the poorest. Leishmania parasites live a dual-form life cycle as either a flagellar promastigote in the sand fly vector or an amastigote form as an obligate intracellular parasite of macrophagedendritic cells in human. Depending on the tropism, the disease can be characterized by at least four syndromes, cutaneous leishmaniasis (CL), muco-cutaneous leishmaniasis (MCL), visceral leishmaniasis (VL), also known as kala-azar in the Indian subcontinent or black fever. This is the most severe form of the disease being fatal if untreated and post kala-azar dermal leishmaniasis (PKDL). Transmission occurs in 88 tropical and subtropical countries due to the presence of sand fly vector. At present, approximately 350 million people are at risk from various forms of leishmaniasis all over the world and each year 500 000 new cases of VL arise.^24,25 Many therapeutic agents have been used to cure leishmaniasis to date. These include antimonial agents i.e. liposomal Sb(V) vesicles^26–28 and various drug formulations of amphotericin B (AmB). However, these drugs are in general toxic, expensive and require long term treatment. Again, a large scale clinical resistance to these drugs has also been reported.^29–32 Therefore the search for a new, more potent and safer drug for the treatment of leishmaniasis had resulted with the identification of licochalcone A³³ [Fig. 1(A)]. This is the first oxygenated chalcone which have shown strong antileishmanial activity. Structure–activity relationship (SAR) studies with these types of compounds revealed that antileishmanial activity is favored by chalcones having more hydrophilic character³⁴ and most active members are found among 4′-hydroxychalcones [Fig. 1(B)].

Fig. 1 (A) Licochalcone A, (B) general chemical structure of chalcones having antileishmanial activity.

In the present study, polyelectrolyte coatings of poly(styrene-4-sulfonic acid) sodium salt (PSS) and poly(diallyldimethylammonium) chloride (PDADMAC) has been used for the encapsulation of the chalcones such as 3-mB-4′-HC and 3-DC-4′-HC (Fig. 2) which were fabricated by the LbL method.^35–37 The controlled release properties of the chalcones were studied in the release medium Phosphate Buffer Saline (PBS) at physiological condition (pH 7.4).

Fig. 2 Structure of chalcones (A) 3-mB-4′-HC and (B) 3-DC-4′-HC.

2. Experimental

2.1. Materials

Poly(styrene-4-sulfonic acid) sodium salt (PSS M_w ∼ 70 000), poly(diallyl dimethyl ammonium chloride) (PDADMAC M_w ∼ 200 000–350 000, 20% aq. solution), 3-bromobenzaldehyde, 2, 4-dichlorobenzaldehyde and 4′-hydroxy acetophenone were purchased from Sigma Aldrich, USA. Sodium chloride, sodium hydroxide and methanol were from S. D. Fine Chem. Ltd., India. Spectroscopic grade ethanol was obtained from Brampton, Ontario, Canada. All other chemicals except methanol were used as obtained without further purification. Methanol was distilled before use. Deionized Milli-Q water with resistivity ∼18.2 MΩ cm or greater was used throughout the experiments.

2.2. Instruments

¹H NMR spectra were obtained from Bruker DPX 400 FT NMR spectrometer at 400 MHz. ¹³C NMR spectra were recorded on the same instrument at 100 MHz. All the NMR spectra were acquired in DMSO-d⁶ at 27 °C. Mass spectra were recorded on a Micromass Q-ToF Micro high resolution mass spectrometer equipped with electro spray ionization (ESI) on Masslynx 4.0 data acquisition system. ESI was used in +ve ionization mode. IR spectra were acquired from a Bruker Tensor 27 FTIR spectrometer. UV-Visible analysis was carried out on UV-Visible spectrophotometer, Unicam UV 300 model using quartz plates (1′ × 1′ × 1 mm) for coated samples. SEM images were obtained from ESEM-EDAX instrument, Quanta 400 model. The pH of the buffer solution was measured by Mettler-Toledo Seven Easy pH meter equipped with Inlab@ ExpertPro glass electrode with an accuracy of ±0.01 units. The pH meter was calibrated at 25 °C using the two point calibration method using commercially available Mettler-Toledo standard buffer solutions pH 7.00 and 9.21.

2.3. Method

2.3.1. Synthesis of antileishmanial chalcones. Chalcones were synthesized by Claisen–Schmidt condensation according to the reported procedure³⁸ with little modification (Scheme 1). In brief, a methanolic solution of 4′-hydroxy acetophenone (1.36 g, 0.01 moles) was made alkaline by addition of 20 mL 10% aq. NaOH followed by addition of substituted benzaldehyde (1.85 g, 0.01 moles). The reaction mixture was stirred at room temperature for 24 h. The resulting solution was then cooled and neutralized with dilute HCl. The precipitate appeared was filtered off, washed repeatedly with cold water, dried and recrystallized from methanol. 3-mB-4′HC; Yield: 1.99 g, 65%, mp: 174–176 °C and 3-DC-4′HC; Yield: 1.80 g, 61%, mp: 228–230 °C.

Scheme 1 Synthesis of chalcones 3-mB-4′HC and 3-DC-4′HC.

2.3.2. Preparation of solutions for the LbL self assembly. A solution of PSS with a concentration of 1 mg mL⁻¹ was prepared by dissolving 50 mg of PSS in deionized Milli-Q water (50 mL) with salt concentration 0.15 M NaCl. Similarly 50 mL aq. solution of PDADMAC was prepared by dissolving 250 μL (1 mg mL⁻¹) of the polymer solution in deionized Milli-Q water with same salt concentration (0.15 M NaCl).

2.3.3. Fabrication of the LbL self assembly on quartz substrate. The process of self assembly formation is illustrated in Fig. 3 (A).

Fig. 3 (A) Schematic of PSS/PDADMAC LbL self-assembly formation on a planar quartz surface, (B) electrostatic interaction between substrate–polyelectrolyte interface and in oppositely charged polyelectrolyte molecules.

The quartz plates were cleaned by overnight treatment with Piranha solution (H₂SO₄ : H₂O₂: 7 : 3) followed by frequent washing of the plates with deionized Milli-Q water. Subsequently these plates were dried under nitrogen flow. Piranha treatment generated a negative surface potential on the quartz substrate. For adsorption of first layer on to the plate, the plate was immersed into PDADMAC solution for 2 h. It was then dipped into water for 0.5 minutes to remove unadsorbed PDADMAC followed by gentle drying under nitrogen flow. Further the plate adsorbed with PDADMAC was dipped into PSS solution for 30 minutes to allow adsorption of next layer having opposite charge to the first layer. Again the plate was allowed for washing and drying steps similar to the first layer. This process resulted in the formation of one bilayer, Fig. 3 (B). This cycle was repeated until adsorption of 10 bilayers on the plate obtained. The growth of the film was monitored by UV-Visible spectroscopy.

2.3.4. Loading of drugs. Solution of the drug (50 mL) was prepared by dissolving appropriate chalcone (0.25 mg mL⁻¹) in 50% aq. ethanol. The plate containing PSS-PDADMAC thin film was immersed into the drug solution for different time intervals followed by washing and drying under nitrogen flow. Excess drug content from the surface of the film was removed by washing with deionized water. The amount of drug loaded was monitored by UV-Visible spectroscopy after each loading–washing–drying step.

2.3.5. In vitro drug release from multilayer self-assembly. The in vitro drug release of chalcones was studied in phosphate buffer saline (PBS pH 7.4) at 27 °C. The drug loaded film on the quartz substrate was incubated in 50 mL PBS (pH 7.4) for different time intervals. Each step was followed by washing with deionized water and drying under nitrogen flow. After each drying step the release of drug was measured spectrophotometrically.

2.4. Characterization

2.4.1. Structural identification of chalcones. The synthesized chalcones were characterized by their ¹H-NMR, ¹³C-NMR, FT-IR and Mass spectral data (see ESI).

2.4.2. Monitoring of the LbL self assembly. The growth of the thin film formed by LbL adsorption of polyelectrolytes was monitored by UV-Visible spectroscopy. The surface morphology of the thin film and thickness of a single layer was measured by Scanning Electron Microscopy (SEM). FT-IR spectroscopy was performed to analyze the nature of the interaction between oppositely charged polyelectrolytes within the self assembly.

2.4.3. Loading and release. Drug loading onto the film and release through it was studied by UV-Visible spectrophotometric analysis by placing the quartz plate into the plate holder inside the instrument after each loading and release step. Each measurement was performed in triplicate and the data were presented as averaged value with respective standard error. The characteristic peak of individual chalcone was identified by plotting a calibration curve for each compound. This was carried out by measuring the absorbance for the drug at its analytical wavelength (λ_max) up to a certain amount of drug concentrations in aqueous ethanolic solution (see ESI† for calibration curves).

3. Results and discussion

3.1. Fabrication of the LbL self assembly

The phenomenon of self assembly formation is the complexation (ion pairing) between oppositely charged polyelectrolyte fragments in association with release of counter ions and water. The driving force for this process is an electrostatic interaction which is the result of two types of interactions termed as intrinsic³⁷ and extrinsic³⁹ compensation. The complexation of polyelectrolytes is known to be virtually a thermal process.^40,41 The overall free energy change of complexation is obtained mainly from the increase in entropy of the system resulting from the release of counter ions.⁴⁰

Formation of self assembly can be tailored by modifying the charge density on the polyelectrolyte. This is generally achieved by altering the pH of the dipping solution (in case of weak polyelectrolytes) or by using copolymers in different ratios of ionic groups. Moreover salt concentration of the dipping solution is the key factor in determining the electrostatic interactions. At higher salt concentration or at low polymer charge density, the electrostatic repulsion between different polyelectrolyte fragments is reduced thereby favoring adsorption.

3.1.1. Scanning Electron Microscopy.
Surface morphology. The LbL self assembly of PSS-PDADMAC is amorphous in nature and followed well patterned structure [Fig. 4(A)]. The self-assembly results from successive strong electrostatic interactions between the sulfonate ion of PSS and quaternary ammonium ion of PDADMAC, and the positive charge of PDADMAC layer with the negative charge of quartz surface. Consequently, the balance between these two, governs the layer growth and thickness. Apart from electrostatic interactions, non-covalent interactions such as hydrophobic interactions between aromatic ring of PSS and aliphatic group and allyl residues of PDADMAC also play an important role in determining the morphology of the resulted film.

Fig. 4 (A) Surface morphology of PSS/PDADMAC multilayer film; 56 layers. Inset illustrating the random adsorption pattern of the polyelectrolyte layers. (B) Cross sectional SEM micrograph of the film. Thickness of one layer is calculated to be ≈ 11 nm.

The random pattern of the film was due to the absence of a straight chain conformation of the PDADMAC molecules. The polymers are able to change their conformation during adsorption with an associated rearrangement of the polymer chains at the surface. The adsorbed chain can be separated into three parts depending upon their contact with the surface. Those parts of the polymer chain adsorbed with all the fragments at the surface, are called trains. A loop connects two trains and this part is not adsorbed at the interface. The tails are the free ends of a polymer chain directed towards the solution. They arrange themselves in a state of higher stability with increased entropy and form a highly interpenetrated structure. In this study, PDADMAC being a weak polyelectrolyte formed multiple loops during its adsorption on to the substrate and on the PSS layer. This was responsible for the less organized structure of the film and higher order of the layer thickness.

Thickness of the film. For a multilayer of PSS/PDADMAC (at polyelectrolyte concentration 10 mM and salt concentration 1.0 M NaCl) the thickness t can be measured by following equation,

t = ∑t_n = t₁ + ∑t⁺_n + ∑t⁻_n (1)

where, t₁ is the thickness of first layer. The thickness increments, t_n⁺ and t_n ^– are for odd PDADMAC and even PSS layers respectively. The overall thickness of a film is the sum of t⁺_n and t⁻_n. The thickness increments can be represented as,where Φ is the factor by which the charge of the last-added polymer compensates to that present on the previous one. If Φ = 1, the opposite polymer charges are in exact stoichiometric ratio. Φ − 1 is the surface overcompensation level. l_cp is characteristic length from the surface of the multilayer to the bulk. It is used in determining the spreading of polymer charge on a particular polyelectrolyte. The thickness increment from each layer is limited at the beginning of buildup due to reduction of the excess charge by the substrate represented by the right most term in eqn (2) and (3). At sufficient thicknesses (smaller l_cp), the growth is a linear function of number of layers.⁴²

In this study a linear growth of the film up to 20 layers (10 bilayers) was observed which was monitored by UV-Visible spectroscopy (Fig. 5). However, the thickness of the film was difficult to calculate for this 20 layered film from SEM micrograph and hence the number of polyelectrolyte layers was increased up to 56. In this case an exponential growth of the film was observed resulting in a comparatively thick film [Fig. 4(B)] with thickness of 625 nm. From this observation an approximate thickness for one layer was estimated to be 11 nm. These observations were in close agreement with the earlier reports.⁴³

Fig. 5 UV-Visible spectral analysis for monitoring the growth of LbL self assembled multilayer thin film of PSS and PDADMAC up to 10 bilayers. Inset showing linear growth pattern.

3.1.2. FT-IR analysis. FT-IR spectrum of the self-assembly (Fig. 6) illustrated the presence of characteristic functional group frequencies only from the component polymers. In the spectrum of PSS (red), two closely spaced characteristic peaks appeared at 1184 cm⁻¹ and 1042 cm⁻¹ which were due to asymmetric and symmetric stretching vibrations of SO₃⁻ group respectively.⁴⁴ Similarly in the spectrum of PDADMAC (blue), a medium intensity peak appeared at 1474 cm⁻¹ which was characteristic fundamental frequency for –CH₃ bending vibrations of PDADMAC. These peaks were evidently visualized in the spectrum of LbL self assembly (green). However, absence of any new peak in the LbL self assembly clearly ruled out the involvement of covalent interactions between the two materials.

Fig. 6 FT-IR spectrum of PSS-PDADMAC self assembly and individual polyelectrolytes.

3.1.3. UV-Visible spectroscopy. The LbL self-assembly was fabricated up to 10 bilayers and the growth was monitored by UV-Visible spectroscopy by taking PSS λ_max at 224 nm as reference peak. The UV-Visible analysis showed that there was a linear increase in the absorption with the increase in the number of layers in the film (Fig. 5).

3.2. Loading of the drugs

Loading profile of 3-mB-4′-HC followed an increasing pattern up to 125 minutes after which further entrapment of the drug was not observed (Fig. 7). This might be due to the saturation of the multilayer by the drug molecules. Since 3-mB-4′-HC did not have any polar groups that could interact electrostatically with the self assembly, only hydrophobic interactions could be possible between the alkyl and phenyl groups present in the polyelectrolyte and the aromatic group of the chalcone. This hydrophobic interaction was responsible for the entrapment of drug molecules within the self assembly. Because there was zero initial concentration of the drug in the self-assembly, the diffusion process allowed the drug molecules to move towards the self assembly from the bulk solution of the drug. This process continued until saturation point of the self assembly was attained. This led to the attainment of equilibrium point between the LbL self assembly and the bulk solution of the drug.

Fig. 7 Loading profile of 3-mB-4′-HC.

Since these interactions are weak in nature therefore drug loading process is almost governed by the diffusion phenomenon. It may be proved on comparing the loading profile at different time intervals (30 and 45 minutes) that the amount of the drug loaded was lower than its previous value (15 minutes). It was due to the fact that washing solution did not contain any drug and during washing some of the drug molecules got released into it. In addition, the drug loading was also not up to a considerable extent and hence the drug released into the washing solution ultimately resulted in an overall decrease in the loading profile. But it was not observed on increasing the drug loading time (incubation period) from 15 to 40 minutes. Here some of the drug molecules were lost into the washing solution but this was not significant as compared to the amount of drug loaded at that particular time. This led to an overall increase in the loading profile.

The loading profile of the other drug molecule i.e., 3-DC-4′-HC was also performed in a similar manner but it was found irregular due to the fact that the drug was released into the washing solution during the washing step.

3.3. Release of the drugs

To study the release profile, the drug was loaded by overnight incubation of the multilayer self assembly into the drug solution for both the drugs (3-mB-4′-HC and 3-DC-4′-HC) and their sustained release was monitored in the PBS.

The release was measured as % release within a certain period of time. It was calculated indirectly from their respective absorbance values (A) which is directly proportional to the concentration (C) of the drug in the solution (Beer–Lambert's law), i.e.

A = ∈ C l (4)

and,where, C₀ ≈ A₀ = Absorbance of the drug into the self-assembly at t = 0, C_t ≈ A_t = absorbance of the drug into the self-assembly after certain time interval t.

The release profile of the drugs was monitored at different time intervals in order to study their release kinetics. The release profiles were found to follow pseudo second order kinetics in the case of both the drugs.

3.3.1. Release of 3-mB-4′-HC. The release of this drug was studied up to 584 minutes starting from 2 minutes. The measurements were carried out at 2, 5, 8, 11, 14, 19, 24, 29, 34, 44, 59, 74, 104, 164, 224, 284, 344 and 584 minutes (Fig. 8).

Fig. 8 Release profile of 3-mB-4′-HC. Inset showing expanded profile up to 74 minutes.

The drug was slowly released in to the medium with an initial release of 15.32%. Total 90.21% of the drug was released within 224 minutes following which it became constant. Further, no burst release was observed which generally is a common phenomenon result from desorption of the drug molecules that were adsorbed onto the outer layer of the film. It was found that the rate of diffusion of the drug was dependent on its concentration i.e., with higher drug concentration the rate of diffusion was greater. The inset of Fig. 8 showed that almost 80% of drug got released within 1 hour followed by a gradual decrease of the rate of diffusion. This resulted in the slow release of the drug from the film in the following time period.

3.3.2. Release of 3-DC-4′-HC. The release of 3-DC-4′-HC was also performed in PBS. The total release was found 90.28% within a period of 824 minutes after which the release profile became steady (Fig. 9). The time intervals were similar to that of 3-mB-4′-HC. In addition, three more measurements were taken for this drug at 704, 824 and 944 minutes respectively.

Fig. 9 Release profile of 3-DC-4′-HC. Inset showing expanded profile up to 74 minutes.

From this study, it was hypothesized that these drug molecules did not interact via strong interactions such as electrostatic or covalent bonding with the polyelectrolyte molecules of the thin film. For such interactions to occur there must be either an ionic moiety (electrostatic interactions) or a suitable functional group for covalent bond formation present in the drug molecule. Though the chalcones have a 4′-hydroxy group on ring B and halogen atoms i.e. –Br in case of 3-mB-4′-HC and two –Cl atoms in 3-DC-4′-HC on the periphery of ring A however, covalent interactions were quite difficult due to stabilization of the drug molecules in the presence of two aromatic rings and α, β-unsaturated keto groups. Thus the possibility of covalent interaction was also ruled out as the medium was aqueous and the experimental conditions were mild. Thus only non-covalent type of interactions viz. hydrogen bonding, hydrophobic interactions and van-der-Walls interactions were possible for the encapsulation of the drug molecules within the self assembly. The hydrogen bonding was supposed to take place by interaction of –OH group (H-bond donor), at the 4′ position of the chalcones and the SO₃⁻ of PSS (H-bond acceptor). Further, the aromatic rings of the chalcone could interact with the aromatic system of PSS by π–π stacking interactions which could be further assisted by hydrophobic interactions between the phenyl groups of the drug molecule and the alkyl or allyl groups of PDADMAC.

3.4. Kinetic fitting for the release data of 3-mB-4′-HC and 3-DC-4′-HC

The data obtained from these studies were fitted into six different kinetic models such as zero order, first order, pseudo second order, Higuchi, Korsmeyer–Peppas and Hixson–Crowell model. The best fitting results were found for pseudo second order model. The accuracy of the fitting was determined on the basis of the value of correlation coefficient (R²) for individual model. Only in the case of pseudo second order model (Fig. 10) the correlation coefficient was found to be 0.999 (R² = 0.999) and for all other models it was <0.90.

Fig. 10 Pseudo second order kinetic fitting, (A) 3-mB-4′HC and (B) 3-DC-4′HC.

The kinetic rate equation for pseudo second order reaction can be written as follows,

where k is the pseudo second order rate constant, q_e is the % drug release at equilibrium i.e. maximum amount of the drug released and q_t is % drug release at a particular time t. On separating the variables in eqn (6), it results in eqn (7) as,

Integrating this for the boundary conditions, t = 0 to t = t and q_t = 0 to q_t = q_t, gives eqn (8),

Eqn (8) is the integrated rate law for a second-order reaction which can be rearranged to obtain eqn (9) which has a linear form as represented below,^45,46

with intercept 1/kq_e² and slope 1/q_e.

The pseudo second order rate constant, k was calculated from the intercept of the kinetic fitting profile. The values of k were found to be 1.753 × 10⁻³ g mg⁻¹ min⁻¹ and 5.78 × 10⁻⁴ g mg⁻¹ min⁻¹ respectively for 3-mB-4′-HC and 3-DC-4′-HC.

4. Conclusion

In conclusion, we have studied controlled and sustained release for antileishmanial chalcones 3-mB-4′-HC and 3-DC-4′-HC up to 224 minutes and 824 minutes respectively. From this study, 3-DC-4′-HC was found to be more suitable for prolonged therapy of Leishmaniasis. The drugs were not capable of interacting with the self assembly either through electrostatic interactions or covalent interactions. Therefore the loading and release was mainly diffusion based. However hydrogen bonding and hydrophobic interactions might contribute to the sustained release of the drugs.

Acknowledgements

Authors acknowledge the Director, Defence R&D Establishment, Gwalior for his keen interest throughout the research work. UMB is thankful to Defence Research and Development Organization (DRDO) New Delhi, India, and Indian Institute of Science, Bangalore, for financial support.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3ra44611e

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