Burko D.V.¹, Kucharskaya T.A.¹, Kvach S.V.¹, Zinchenko À.I.^1,2

¹Institute of Microbiology, National Academy of Sciences of Belarus, Minsk

²International Sakharov Environmental University, Minsk

intercalation and release of pharmaceutically useful diadenosine tetraphosphate from a layered double hydroxide

Layered double hydroxides (LDHs), also called “anionic clays” are a family of materials that have attracted increasing attention in recent years due to their technological importance in catalysis, separation technology, optics, medical science, and nanocomposite materials engineering. LDHs consist of positively charged metal hydroxide layers, in which the anions (along with water) are stabilized in order to compensate the positive layer charges. The general chemical formula of LDH clays is written as [M^II_1−xM^III_x(OH)₂]^x+(Aⁿ⁻)_x/n·yH₂O, where M^II is a divalent metal ion, such as Mg²⁺, Ca²⁺, Zn²⁺, etc., M^III is a trivalent metal ion, such as Al³⁺, Cr³⁺, Fe³⁺, Co³⁺, etc. and Aⁿ⁻ is an anion of any type with charge n, such as Cl⁻, CO₃²⁻, NO₃⁻, etc. [1]. The electrostatic interactions and hydrogen bonds between layers and contents of the gallery hold the layers together, forming a three-dimensional structure. There are a number of combinations of divalent and trivalent cations that can form LDHs. For these ions, the only requirement is that their radii are not too different from those of Mg²⁺ and Al³⁺. The anions occupy the interlayer region of these layered crystalline materials. The remarkable behavior of LDHs is their high reactivity toward various organic anions, which can exchange as much as 80–100% of the interlayer anions.

LDHs have historically been of interest as catalysts, ceramic precursors, traps for anionic pollutans, catalyst supports, ion exchangers, and additives for polymers. More recently, the successful synthesis of LDH materials on the nanometer scale has paved the way for many novel applications particularly in the field of nanomedicine. For example, LDH materials are increasingly explored as controlled release systems [2–4]. Up to now many kinds of anions were intercalated in the LDH interlayer gallery, such as common inorganic anions, organic anions, polymeric anions, complex anions, macrocyclic ligands and their metal complexes, polyoxalates, and biochemical anions (amino acids, CMP, AMP, ATP and nucleic acid fragments).

In the present study, we prepared artificially a novel bio-inorganic nanohybrid of LDH and biomolecule (pharmaceutically useful diadenosine tetraphosphate [5, 6], Ap₄A) and investigated the possibility of using intercalated LDH as a drug delivery system.

Materials and methods. Materials. Ap₄A sample was synthesized from ATP enzymatically as previously described in paper [7]. Twice-distilled water from which carbon dioxide was removed by boiling was used in all experiments.

Preparation of LDH/Ap₄A nanohybrid particles. The LDH/Ap₄A nanohybrid with Mg:Al ratio of 2:1 was synthesized by coprecipitation and crystallization at 60°C in the presence of ammonium hydroxide. Briefly, a requisite amount of Mg(NO₃)₂·6H₂O and Al(NO₃)₃·9H₂O were dissolved in 10 ml of water. The resultant solution was added dropwise to 10 ml of 10 mM NH₄OH solution containing 10 mM Ap₄A at 25°C while stirring vigorously. The pH of mixture was maintained about 10. The resultant reaction slurry was aged at 55°C for 12 h. The resulting white precipitate was collected by centrifugation and washed five times with water, and finally, with acetone. All the samples were air-dried at 60°C for 5–6 h. To obtain LDH nanoparticles without Ap₄A (LDH/NO₃) the above procedure was repeated without addition of Ap₄A to solution of NH₄OH.

Characterization of LDH/Ap₄A nanohybrids. As-obtained LDH nanoparticles were imaged using a JEOL JSM-2010 transmission electron microscope at the acceleration voltage of 200 kV.

The loading capacity of Mg,Al–LDH/Ap₄A nanoparticles and the efficiency of this process under different experimental conditions were determined by UV spectroscopy. The intercalated amount of Ap₄A in the LDH/Ap₄A nanohybrids was determined by 1202 Shimadzu Corporation model UV-vis spectroscopy using the following method. A known weight of the nanohybrids was placed in a 10 ml volumetric flask, then 0.5 ml 6 M HCl solution was added, and the balance filled with phosphate buffer solution (0.02 M). Then the concentration of Ap₄A in solution was determined by monitoring the absorbance at λ=260 nm (ε=30800 M^-1·cm^-1) with UV-vis spectroscopy to calculate the intercalated amount of Ap₄A into the nanohybrids.

Release of Ap₄A from LDH/Ap₄A nanohybrids. To measure the amount of Ap₄A released from LDH/Ap₄A nanohybrids, the in vitro drug release test was performed at 25ºC by stirring powdered LDH/Ap₄A nanohybrids (0.032 g) in 20 ml either a pH 4.4 or 7.5 0.05 M phosphate-citrate buffer solution [8]. Aliquots (1 ml) of the suspension were taken at desired time intervals, centrifuged and the Ap₄A content of supernatant was determined by UV absorbtion at λ=260 nm to calculate the release amount of Ap₄A from the nanohybrids. The percentage released at each time point was expressed as a fraction of the total amount of Ap₄A.

Results and discussion. The LDH, containing Ap₄A (LDH/Ap₄A) was prepared by coprecipitation from a mixed aqueous solution containing Mg²⁺, Al³⁺ and Ap₄A. In order to survey the optimal conditions for intercalation, various reaction conditions, that is, temperature, reaction time, concentration and pH, were examined. Based on these experiments, it was concluded that the optimal intercalation conditions of Ap₄A are as follows: Mg:Al molar ratio of 2:1, room temperature, pH 10, and 12 h aging. The maximum amount of Ap₄A in the intercalation compound was around 0.83 mmol per 1 g of LDH.

The morphology and size of LDH/Ap₄A nanoparticles have been estimated by electron microscopy. Transmission electron microscopy image of LDH/Ap₄A nanoparticles is shown in Fig. 1.

Fig. 1. Transmission electron microscopy image of LDH/Ap₄A nanoparticles

It should be noted the presence of small hexagonal platelets having a diameter of about 200 nm together with some larger platelets (about 350 nm), likely due to the formation of aggregates.

The intercalated Ap₄A was quantitatively recovered from the host lattice by treatment of the LDH/Ap₄A nanoparticles with 0.2 M HCl. Therefore it was concluded that Ap₄A is intercalated into LDH without decomposition.

In order to investigate the possibility of using intercalated LDH as a drug delivery system, deintercalation of Ap₄A was examined. Typical release kinetic curves of Ap₄A from the LDH/Ap₄A nanoparticles at different pH are shown in Fig. 2. As can be seen from Fig. 2, the physical mixture LDH/NO₃ and Ap₄A exposed to buffer solution (pH 7.5) release Ap₄A quickly, the release being complete within 5 min. The release rate of Ap₄A from the nanohybrid is obviously lower than that from the physical mixture. In addition, the release rate of Ap₄A from the nanohybrid is obviously dependent on pH, and the release rate at pH 7.5 is remarkable lower than that at pH 4.4.

Fig. 2. Release of Ap₄A from LDH/Ap₄A nanoparticles

in 0.05 M phosphate-citrate buffer solution at pH 4.4 and pH 7.5

The lower release rate of Ap₄A from LDH/Ap₄A nanohybrids at pH 7.5 indicates that the LDH/Ap₄A nanohybrids are indeed a potential drug delivery system. Such a discrepancy of the release rate at pH 4.4 and pH 7.5 may be due to a possible difference in mechanism for the release of Ap₄A from the nanohybrid [9]. At acidic pH, LDHs begin to dissolve. This would indicate that release of an interlayer molecule should occur mainly through the removal of inorganic host. At above pH 7, the LDH should be more stable, and as a result, release may be attributed to the restricted motion of Ap₄A molecules arising from steric effect of LDHs and the electrostatic interaction between Ap₄A anions and positively charged LDHs layers. That is to say, the mechanism of release in the pH 4.4 environment should be through both the dissolution of LDH layers and the ion exchange; while for the pH 7.5 release, the mechanism should be primary through ion exchange with the ions in the buffer solution [9].

In summary, in this work we for the first time successfully show that Ap₄A can be reversible intercalated into LDH. Therefore, LDH would be attractive candidate of the studied dinucleotide carrier.

Acknowledgements. The work was supported by a grant from the Belarus State Research Program «Fundamental Basics of Biotechnologies».

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