[interface science and technology] clay surfaces - fundamentals and applications volume 1 ||...

CATIONIC AND ANIONIC CLAYS FOR BIOLOGICAL APPLICATIONS

JIN-HO CHOY* and MAN PARK

National Nanohybrid Materials Laboratory (NNML)

School of Chemistry and Molecular Engineering

Seoul National University, Seoul, 151-747 - KOREA

* E-mail: [email protected]

Clay Surfaces: Fundamentals and Applications F. Wypych and K. G. Satyanarayana (editors) �9 2004 Elsevier Ltd. All rights reserved.

404 J. Choy and M. Park

1 - I n t r o d u c t i o n

Clay minerals have attracted a great deal of attention from a very wide range of scientific and industrial fields because both of natural abundance and of exhaustless and unlimited potentials [1-7]. Incessant researches have been conducted on the fundamental properties like basic structures, formation and weathering of framework structure, swelling (hydration), ion exchange capacity, etc. all of which leads to elucidation of their important roles in many academic fields like agriculture, biology, geology, and inorganic and environmental sciences [8-16]. Of course, they are extensively utilized in innumerable industrial fields like ceramics, paper, paint, plastics, drilling fluids, foundry bondants, chemical carders, liquid barriers, decolorization, catalysis, adsorption, and so on. In particular, their biological applications for curative and protective purposes are as old as mankind itself, which clearly assures biocompatibility. To our surprise, there is still the increasing interest in further development of their applications for biological purposes, especially for pharmaceutical, cosmetic, and even medical purposes.

A variety of clay minerals are found in nature. In general, clay minerals could be roughly divided into three classes by ion exchange property [17]. These are non- ionic, cationic, and anionic clays. Non-ionic clays with no true ion exchange capacity include kaolinite, serpentine, chlorite, illite, pyrophyllite, and talc. Cationic clays with cation exchange capacity comprise many alumino-silicate clays such as vermiculites, smectites, and swelling micas. And the last class with anionic exchange capacity is represented by layered double hydroxides. The former two classes of clay minerals are widespread in nature, but fairly difficult to be synthesized. Whereas, the last class is rare in nature, but easily and inexpensively synthesized. Although crystal structure is a major criterion of the detailed classification into a number of groups and subgroups, all their structures are based on two-dimensional framework in common. And for the most part, they are usually hydrated or to be easily hydrated, which leads to one of the important features along with ion exchange properties. In fact, these two properties are most intensively studied, and also play a crucial role in biological applications, the key topic in this chapter. Therefore, this chapter mainly deals with biological applications of two classes of clays, cationic and anionic ones, describing their structure, physical and chemical properties, and preparation.

2 - C a t i o n i c c l ays Biological applications of cationic clays have been found in many fields such

as pharmaceutics, medicine, cosmetics, spas, food, fodder, and pesticide into which cationic clays are directly involved as active principles or excipients [16, 18-20]. In these applications, their important roles are originated from the physical and chemical properties such as high adsorption capacity, plastic (soft) framework, chemical inertness and low or null toxicity. Although a few synthetic clays like laponite are employed in some cases, natural clays are generally preferred partially due to favorable concept of 'natural' and partially due to difficulty and high cost of their syntheses.

2.1 - S t r u c t u r e a n d c l a s s i f i c a t i o n

Negative charge of cationic clay minerals could be generated from two different mechanisms, namely, isomorphic substitution and breakage of oxygen bridge [17. 21-22]. The former develops permanent negative charge in framework that is not

Cationic and Anionic Clays for Biological Applications 405

affected by bulk environments like pH and particle size, whereas the latter leads to temporary charge (or pH-dependent charge) derived from deprotonation of hydroxyl groups on cleaved edge or defect site of layer framework. The clay minerals possessing only temporary charge could belong to non-ionic clays rather than cationic clays because of their fundamental structure being non-ionic. On the other hand, the clay minerals like illite and muscovite could be also described as non-ionic clays due to lack of cation exchange property although they have isomorphic substitution in their framework. Thus, cationic clays considered here are arbitrarily confined to those, which allow their interlayer cations to be easily exchanged by other cationic species in solution, which make it possible for us to extract their common features such as mica- type 2:1 structure, exothermic hydration, high internal surface area, high cation exchange capacity, some degree of cation selectivity, and typically interlayer displacement.

Fundamental crystal structure of cationic clays is very similar to framework of mica. Unit layer consists of one octahedral sheet sandwiched between two tetrahedral sheets, as shown in Figure 1 [2-6].

Figure 1 - Crystal structure of2:1 aluminosilicate clay

The cations in the tetrahedral sheet are typically Si 4§ and AI 3+, while those in the octahedral sheet are A13+, Fe 3§ Mg 2+ Fe 2+ and etc. Because cations of both tetrahedral (Si 4+) and/or octahedral (AI 3+, Fe 3§ Mg 2+) sheets could be isomorphically substituted by lower valence cations, layer framework develops permanent negative charge that is usually delocalized over layer surface. Net negative charge is electrostatically neutralized by the cations in interlayer space between layers. And these interlayer cations prefer to be hydrated for thermodynamic reasons. In general, degree of net negative charge is evaluated by cation exchange capacity expressed in centiequivalent of exchangeable cation per kilogram of sample [ 17].

The cationic clays could be divided into several groups by charge density, as demonstrated in Table 1. Each group could be further separated by various criteria like octahedral occupancy, cation species of octahedral site and charge site, as described in Table 2 [ 17, 21-22].


Table 1 - Class i f i ca t ion o f the 2:1 clays

Layer charge per Group unit cell name

i

0.0 Pyrophillite

Talc

0.5-1.2 Smectite

1.2-1.8 Vermiculite

-_~_2.0 Mica

=-z-4.0 Brittle mica

Variable Chlorite

Octahedral ~ Species

Dioctahedral Pyrophillite

Trioctahedral Talc

Dioctahedral Montmorillonite, beidellite

Trioctahedral Hectorite, saponite

Dioctahedral Dioctahedral vermiculite

Trioctahedral Trioctahedral vermiculite

Dioctahedral Muscovite, paragonite

Trioctahedral Phlogopite, biotite

Dioctahedral Margarite

Trioctahedral Clintonite

Dioctahedral Donbassite

Trioctahedral Clinochlore

Table 2 - S u b g r o u p s of Smect i te

Subgroup Charge site

Montmorillonite Octahedral

Ideal composition Mint(Moet)(Mtetra)Ol(On)m.nH20

. i i

Mx(A12.xMgx)(Si4)Olo(OH)2.nH20

Beidellite Tetrahedral Mx(A12)(Si4_xAlx)O lo(OH)2.nH20

Nontronite Tetrahedral Mx(Fe23+)(Si4.xAlx)Olo(OH)2.nH20

Saponite Tetrahedral Mx(Mg3)(Si4.xAlx)O lo(OH)2.nH20

Hectorite Octahedral Mx(Mg3.xLix)(Si4)Olo(OH)2.nH20

2.2 - P r e p a r a t i o n Cationic clays for biological purposes are prepared mostly from natural sources

with a few exceptions. Although there are the great variations in quality requirements for each application, biological applications typically require more strict and higher purity compared with that of other applications. Especially, the clays to be applied for pharmaceutics, medicine, and cosmetics should meet the strict requirements about phase purity, chemical composition, particle size, and kinds of impurity [16, 23]. However, overall quality of natural clays mainly depends on origin of the deposit. General purification methods for natural clays mainly depend on physical and magnetic


separations including sieving, filtration, and sedimentation. For some specific applications, acidification is also frequently employed.

Synthetic cationic clays is available even though their practical applications are currently very limited. General methods for synthesis of clays are based on hydrothermal treatment of precursors at various temperatures under autogenous pressure. Several smectites are successfully synthesized in a high purity, including beidellite, hectorite (the trade name Laponite), saponite, and stevensite. However, most synthetic methods have some disadvantages like long reaction time (often several weeks to even months), high temperature (up to 300-600~ and relatively low crystallinity [24-25].

2.3 - P h a r m a c e u t i c a l A p p l i c a t i o n s

Cationic clays possess high sorption ability, high internal surface area, high cation exchange capacity and typically interlayer displacement from which most pharmaceutical applications have been benefited [16,26-27]. Traditionally, smectites have been most widely employed as both active principle and excipient for various pharmaceutical purposes [28-29]. As the active principle, major therapeutic effects of smectites are to eliminate the excess water in the feces as antidiarrhoeaics and to protect skin mechanically against the physical or chemical substances generated from both external and internal sources as dermatological protectors. Bentonite-water mixtures are known to be the best materials for spas to alleviate joint pain occurring between flares of joint disease. Local temperature elevation along with ion exchanges gives rise to pain-relieving effects. Their major function derives from high swelling properties and ability to retain large water percentages [28-29]. Slow delivery of heat to the underlying tissues ensures that temperatures up to 50~ can be applied without causing tissue injury [28]. On the other hand, as the excipient, they play an important role in dispersing active principles due to their ability to increase in volume in the presence of water, in buffeting abrupt change of acidity, and in stabilizing emulsion, polar gel and suspension because of their colloidal characteristics to avoid the segregation of the pharmaceutical formulation's components. Although there are still innumerous efforts to enhance and expand the roles of clays in these traditional applications, recent attention rapidly shifts to exploration of their new potentials such as drug carrier, protecting matrix, release controlling agent and chemical modifier. These new attempts are dealt in detail here because they are indeed of hot issues in current times.

New potentials of cationic clays for pharmaceutical applications arise mostly from recent hybridization of drugs with the clays either in ordered or disordered manner. Traditional applications mainly rely on the inherent physical, rheological, and chemical properties of the clay [16,30]. On the other hand, new potentials from hybridization take advantage of complexation of inorganic and organic compounds that leads to interesting characteristics of organic-inorganic hybrid distinguished from that of each component in simple physical mixtures. Hybridization is reported to offer the fascinating features such as protected delivery, controlled release, enhanced water solubility, and increased dispersion ability. Furthermore, it has a feasibility of target delivery [19,31-33]. In spite of these promising prospects suggested thus far, not so many works appear in literatures to date mainly because hybridization approaches have quite recently attracted intense attention.

The recent studies on complexation of drug and clay are rapidly increased. A


great number of studies have focused on preparation and characterization of surfactant- clay hybrids to facilitate the advanced formulations of various drugs [17,19,34]. Polyethylene oxide and N-isopropylacrylamide are the representative organic polymers for the hybrids. On the other hand, only a few studies have been carried out on direct complexation of drug with clays. Ito et al recently found that complexation of indomethacin, an anti-inflammatory and as an analgesic agent, with smectite enhanced its penetration rate through skin [19]. Enhanced permeability was rationalized by the increase in both stability of amorphous indomethacin and water solubility by their complexation with smectite. Lin et al intercalated 5-fluorouracil, effective chemotherapeutic agent for colorectal cancer, into montmorillonite to diminish its severe side effect through its in situ release [17]. Lee and Fu reported that release properties of drugs could be controlled by loading them into nanocomposites of N- isopropylacrylamide and montmorillonite. Release property of loaded drug could be controlled by electrostatic interaction between the drug and nanocomposite.

Electrostatic attraction decreases the release ratio, while electrostatic repulsion increases the release ratio. Our laboratory also recently found that hybridization of poorly water-soluble intraconozole, antifungal agent, with smectites led to remarkable improvement of its water solubility and bioavailability (Figure 2). It was suggested that molecular arrangement of intraconozole within nanosized interlayer space of smectites was greatly contributed to these enhancements.

Figure 2 - Effect of clays on pharmacokinetic parameters of itraconazole after oral administration. After magnabrite (Mag) or Montmorillonite (Mmt) was reacted with itraconazole (Itra) at a weight ratio of clay 0.7/Itra 0.3, the resulted solid was coated with either hydroxypropylmethyl cellulose (hpmc) or hydroxypropyl cellulose (hpc) at a weight ratio of solid 0.7/polymer 0.3. The parameters were compared with those of commercial Sporanox (maximum blood concentration (Cmax): 223 ng/ml; time to reach Cmax (Tmax) : 1.8 hr).


Figure 3 - Stabilization of emulsions by solid particles. (a) stabilization by envelopes of particles around the oil droplets, (b) stabilization encapsulation oil in a three- dimensional network of particles

2 .4 - C o s m e t i c a p p l i c a t i o n

Cationic clays have been widely employed as thickener and emulsion stabilizer in cosmetics [35-36]. They are also used as active principles for adsorption of substances such as greases, toxins, etc. and for antiperspiration to give the skin opacity, remove shine and cover blemishes. These applications are mainly based on high cation exchange capacity, excellent swelling property along with remarkable hydration ability, and structural plasticity. Particular attention has been given to the organic polymer-clay nanocomposites in which organic polymers are complexated with the layers of clay by chemical interactions like electrostatic attraction, hydrogen bonding, and Van der Waals interaction. The complexation enables one not only to enhance the above inherent properties but also to impart new functionalities, especially organophilicity of clay, high stability of organic component, and new rheological properties [37]. A recent review by Ray and Okamoto describes the detailed properties of various polymer/layered silicate nanocomposites [38-39]. Depending on the strength of interfacial interactions between the polymer matrix and layered silicate, they classified three different types of nanocomposites, intercalated, intercalated and flocculated, and exfoliated ones (Figure 4).

Figure 4 - Possible polymer~inorganic nanocomposites.


These unique combinations of hydrophilic inorganic layers and hydrophobic organic polymers at sublattice and molecular level facilitate loading or incorporating chemically diverse active compounds into a broad range of cosmetic formulation. Furthermore, long lasting and high effectiveness could be also achieved by controlling their release property.

2.5 - Agricultural applications to animal feed and pesticides Clay minerals have been incorporated in animal feeds for multiple purposes

mainly due to high binding (processing) ability, water sorption capacity, and cation retention capacity [40]. In animal feed, they act as an anti-caking and pelletizing aid in nonmedicated feeds, as an adsorbent of mycotoxins and gastrointestinal gases, and as a consolidating additive of feces [41]. Especially, incessant attention has been given to mycotoxin detoxication by the various clays. Phillips et al analyzed the in vitro binding capacities of different adsorbents, which were representative of the major chemical classes of aluminosilicates [42-43]. They claimed that smectites are the suitable candidate clay for in vivo trials concerning the prevention of aflatoxicosis in chicken because smectites were shown to have a high affinity for aflatoxin B 1. The complex of smectite-aflatoxin B 1 was stable at temperatures of 25 and 37~ in a pH range of 2-10, and in an eluotropic series of solvents. A chemically modified montmorillonite also reported to exhibit a high binding capacity for zearalenone of 108 mg/g [44]. The clay modified with cetylpyridinium or hexadecyltrimethylammonium resulted in an increased hydrophobicity of the clay surface that led to a high affinity to the hydrophobic zearalenone. There is also an attempt of virus adsorption to clays, and the viruses most studied include poliovirus, encephalomyocarditis virus and reovirus. Although adsorbed viruses were not deactivated completely, these results suggested a potential of clay additive to give a distinct advantage in prevention of virus infection [20,45].

Application of clays to pesticide formulation is another important subject nowadays because both active ingredient and adjuvants of pesticide formulations cause serious environmental problems [46-48]. It is desperately pursued that a minimum amount of a pesticide exerts the maximum effect at the right moment and place. In order to increase the pesticide efficiency and to reduce their leaching into related environments like air and water, it has been suggested that reversible complexation of active pesticide ingredient with clay minerals would be one of the feasible solutions. However, most of researches have focused on adsorption of pesticides by clay minerals for their removal from water and immobilization in soils. To date, there are only a few attempts to explore the potential of clay minerals as carriers in pesticide formulations. E1-Nahhal et al reported that adsorption of alachlor and metolachlor on a bentonite was very efficient when the clay mineral was modified by cation exchange with benzyltrimethyl ammonium (BTMA) ions [49,50]. The raw bentonite only adsorbed 3% of the amount of metolachlor added, whereas bentonite preadsorbed with 0.5 mmol BTMA and 0.8 mmol BTMA adsorbed, 25% and 20% of the amount added, respectively, to suggest a high feasibility for slow release formulation. Carrizosa also suggested that primary alkylammonium saturated clays were suitable as potential sorbents for slow release formulation [51]. These bentazone-modified clay complexes released 20 to 80% of their bentazone content. In addition to slow release formulations, clays also turned out to be very effective in protecting unstable pesticides against


volatilization and photodegradation that consequently forced farmers to increase frequency and dose of herbicide application. Recently, Lagaly evaluated feasibility of pesticide-clay formulations based on the binding mechanisms between pesticides and clays, and suggested a wide range of pesticides to be stably complexated with clays by direct electrostatic interaction as well as by induced interactions through surface modification of clays [52].

3 -Anionic clays: layered double hydroxides Anionic clays are typically included into layered double hydroxides (LDHs)

because no other layered clays have been found to possess exchangeable anions in their interlayer space. Similar to cationic clays, their interlayer could accommodate a variety of anionic guest components, which leads to a broad spectrum of potential biological applications such as pharmaceutics, medicine, cosmetics, and pesticides [53-57]. However, their practical applications to biological fields are currently very limited compared with those of cationic clays mainly due to shortage of accumulated understanding in their characteristics [58]. The representative commercial product based on LDH is an antacid made of hydrotalcite. As a matter of fact, their potentials for biological applications [59-63] are recently explored in spite of extensive studies on their catalytic activity [64-67] and anion exchange behavior [68]. It is worthy to note the trend that more and more attention has been given to their potentials for a wide range of biological fields.

3.1 -Structure and classification Fundamental structure of LDH is based on hydrotalcite. The structure of

hydrotalcites was first elucidated by Allmann for the CO3-Mg,Fe system (pyroaurite and sjOgrenite) in 1968 [69] (Figure 5). Depending on the arrangement of an octahedral layer and an interlayer, two polytypes of layer structure are well recognized. The one (hydrotalcite) has a rhombohedral unit cell containing three stacked repeat units, whereas the other (manasseite) has a hexagonal unit cell containing two stacked repeat units [70-71]. These two polytypes exhibit the same local topology of the layer- interlayer bonding, but different in the long-distance layer-layer interactions.

Figure 5 - Layer stacking of LDHs.


On the other hand, two dimensional lattice structure could be constructed in two distinct types: hydrotalcite-type and hydrocalumite-type (Figure 6). The general

[M 1.xM x(OH)2] (A)~:,,,nH20, where the M n+ formula of hydrotalcite-type LDHs is 2+ 3+ - m- are metal cations (M 2+ = Mg 2+, Zn 2+, Ni 2+, Cu 2+, etc., M 3+ = A13+, Fe 3+, etc.) and A m- are interlayer anions (A m-= CO32-, NO3, 5042, and other anionic species) [69-71 ].

The layer structure of LDHs is constructed with a stacking of brucite structure of Mg(OH)2 in which Mg(OH)6 octahedra are connected through edge sharing into 2- dimensional sheets with layer thickness of 4.8 A. Some of divalent cations in the brucite layer are substituted by trivalent cations such as A13§ which develop permanent cationic layer charge (Figure 7).

Figure 6- Layer structures o f hydrotalcite (a) and hydrocalumite (b)

Figure 7 - The structure of LDHs (a) brucite layer (b) LDH layer.

They have a wide range of chemical compositions. In addition, their layered structures exhibit a variety of stacking faults to generate many different polytypes of crystals. On the other hand, Ca-containing LDHs exhibit hydrocalumite structure with corrugated brucite-like main layers [72]. Ca atoms are hepta-coordinated with six hydroxides and one interlayered water, which are edge-shared with octahedral trivalent metal cations. The framework layer is formed in an almost fixed molar ratio of 2 Ca2§ 3§ which results in the general formula of [Ca2M(OH)6] § A mH20. Also, the kinds of M 3§ of hydroxide layer is very limited, typically Fe 3§ and A13§ This unique


layer framework results in the distinct XRD pattern distinguished from the other usual LDHs, usually leading to better crystallinity. The interlayer space of LDHs is occupied by charge-balancing anions that are typically bound to the layer through hydrogen bonding with water molecules. Exchangeability of interlayered anions depends on their electrostatic interaction with positively charged layer. Except for carbonate ion, most organic and inorganic anions are known to be exchangeable. Thus LDHs are widely applicable to various supramolecular structure or heterogeneous hybrid systems.

Another unique group of LDHs are layered hydroxide salts with anion exchange capacity [73-77]. The layered hydroxide salts can be classified into two structural types, based on the structure of either zinc or copper hydroxide nitrate with the typical compositions Zns(OH)8(NO3)2)'2H20 and Cu2(OH)3NO3 (Figure 8) [77]. Interlayered nitrate is exchangeable. And a fraction of Zn or Cu cations could be isomorphically substituted for other divalent cations. These hydroxide salts are fundamentally built of brucite-like layers in which one forth of octahedral sites is vacant. Particularly, vacant zinc sites are occupied with tetrahedral zinc cations. Their theoretical anion-exchange capacity is similar to those of other LDHs (2-5 meq/g).

Figure 8- The structure of Zn5(OH)8(NO3)2"2H20 and Cue(OH)3N03.

3.2- Preparation LDHs are easily and economically synthesized in high purity and yield, which

greatly increases the potential of LDH for various applications. In spite of great heterogeneity in framework composition, LDHs are exclusively synthesized by precipitation of metal hydroxides, typically coprecipitation of mixed metal cations by base titration either with or without hydrothermal treatment that usually enhances crystalline property. Because the contamination with air-born carbonate ions frequently occurs during synthesis procedures, a special caution is needed to prepare carbonate- free LDHs. On the other hand, two dimensional heterostructured LDH hybrids could be prepared by three different techniques, coprecipitation, anion exchange and reconstruction ones (Figure 9). Intensive attention has been given to coprecipitation technique because it allows lattice engineering through fine tuning of framework charge density, fi'amework composition, anion properties, treatment condition, and etc. [70-71 ].


Figure 9 - Strategic illustration for preparation of LDH-derived complexes and their application; (a) C60-LDH hybrid (b) metal complex-LDH hybrid (c) isomeric organic molecue-LDH hybrid and (d) bulky organic molecule-LDH hybrid and their delaminated product

3 .3 - P h a r m a c e u t i c a l A p p l i c a t i o n s

Pharmaceutical applications of LDHs mainly depend on acid buffering effect and anion exchange property. Hydrotalcite-derived antacid and antipeptic are representative of their applications in pharmaceutics [55,78-81]. Hydrotalcite was also explored as potential adsorbents of intestinal phosphate [82-84]. In addition to simple acid buffering and anion adsorption ability, hydrotalcite was also suggested to have barrier properties similar to those of gastric mucous, and to afford mucosal protection by its ability to maintain or mimic the barrier properties of gastric mucous gel. Unfortunately, LDHs could be found in a narrow range of pharmaceutics currently, unlike natural cationic clays. However, recent advance in hybridization technique has brought about a dramatic increase in the attention to their pharmaceutical potentials.

Pioneering works have been carried out by Choy et al. They demonstrated excellent potentials of LDHs as a reservoir and delivery carrier for genes and drugs by hybridizing with DNA and As-myc, and etc. [59,62]. X-ray diffraction analyses showed that the interlayer distance of LDH increased fi'om 0.87 nm (for NO3) to 2.39 nm (DNA), 1.94 nm (ATP), 1.88 nm (FITC), and 1.71 nm (As-myc), respectively, upon intercalating corresponding biomolecules into hydroxide layers (Figure 10).

They clearly showed the excellent efficiency of LDH as gene and drug delivery carder as well as protective role. The intercalated DNA was safely protected against harsh conditions such as strong alkaline to weak acidic environments and against DNase attack. [59] HL-60 cells treated with As-myc/LDH hybrids exhibited time dependent


inhibition on cell proliferation, indicating nearly 65 % of inhibition on the growth compared to the untreated cells, alter 4 days (Figure 11). On the other hand, LDH itself was noncytotoxic towards HL-60, indicating its biocompatibility, and thus the suppression effect of cancer cell growth is solely from As-myc/LDH. [62].

Figure 10 - Powder X-ray diffraction patterns for (a) the pristine LDH, (b) DNA-LDH, (c) A TP-LDH, (d) FITC-LDH, and (e) As-myc-LDH

Figure 11 - Effect of As-myc/LDH hybrids and As-myc only on the growth of HL-60 cells.


Controlled cells are incubated without any treatment. The final concentration of each material was 20 ~tM. These remarkable results have evolved into the advanced concepts of LDHs for pharmaceutical applications, that is, degradable inorganic drug delivery system and targeted delivery. The drugs intercalated into LDHs are easily released by ubiquitous carbonate ion due to extremely high affinity. Furthermore, distinguished from other inorganic matrixes like cationic clays, not only could LDH be prepared from biocompatible compositions with arbitrarily tailored physical and chemical properties but also it is completely decomposed by acidic body fluids. These characteristics could be exploited for novel target delivery system for a wide spectrum of drugs. The schematic illustration was also proposed, based on the above experimental results along with inherent characteristics of LDH (Figure 12).

Figure 12 - The schematic diagram of bio-LDH hybrid (a) pristine LDH host (b) biomolecules (c) bio-LDH hybrid for biomolecule reservoir or carrier (d) cellular uptake mechanism of bio-LDH hybrid for biomolecule carrier cell-line by apoptosis. Lanel; control, Lane 2; LDH, Lane 3; MTX, Lane 4; MTX-LDH.


Figure 13- Electrophoresis analysis for detection of DNA ladder formation of SaOS-2

Figure 14 - The effect o f MTX-LDH on normal cell growth at the concentration of 5. 0

t~g/ml.


Quite recently, Choy et al also reported a successful application of drug-LDH hybrid to in vitro cancer treatment in which LDH played an essential role in protective delivery of methotrexate (MTX) [85] (Figure 13,14). The initial proliferation of SaOS-2 cell was more strongly suppressed by treatment with MTX-LDH hybrid than with MTX alone. A series of genetic and efficacy analyses indicated that LDH did not exert any appreciable harmful effects on both normal and cancer cells, and that the treatment mechanism of MTX was not affected by hybridization. These results strongly suggested that LDH not only plays a role as a biocompatible delivery matrix for drugs but also facilitates a protective delivery to result in a significant increase in the delivery efficiency. Furthermore, Kriven together with Choy et al [86] evaluated the in vivo sai~y of LDH for adult male Sprague Dawley rats, and found that LDHs could be vein- injected without any considerable effects on tissues and organs below a dose rate of 100 mg/Kg. LDHs, when extravascular, are locally irritating and elicit an inflammatory response around the precipitated particles at a dose rate of more than 200 mg/Kg.

The pioneering works of Choy et al have led to a rapid increase in the research on pharmaceutical applications of LDHs [59-63]. Khan et al showed that a series of pharmaceutically active compounds including diclofenac, gemfibrozil, ibuprofen, naproxen, 2-propylpentanoic acid, 4-biphenylacetic acid and tolfenamic acid could be reversibly intercalated into LDH for their storage and controlled release [55]. Ambrogi et al have reported the hybridization of LDH with ibuprofen exhibiting antiinflammatory activity through which its controlled release as well as water- solubility could be significantly enhanced [83]. Recently, Ambrogi et al also found that hybridization with LDHs resulted in a significant increase in solubility of the nonsteroidal anti-inflammatory drugs such as indomethacin, tiaprofenic acid, and ketoprofen [86]. In gastric juice of pH 1.2, their LDH hybrids exhibited much higher (up to twice in case of tiaprofenic acid) solubility than their free forms did. They suggested that the enhancement of solubility resulted from the lack in crystallinity of intercalated drugs, which is directly released in ionic form by the fast dissolution of LDHs in acidic medium. Increase in water-solubility of poorly soluble drugs also plays a crucial role in drug bioavailability and hence in drug formulation. Many different approaches have been developed to improve drug solubility, which include introduction of polar or ionizable group, preparation of soluble prodrugs, use of polymorphs and of amorphous form of the drug, complexation, formation of inclusion compound, solid dispersions, etc. Compared with these approaches, hybridization with LDHs offers outstanding advantages like established safety, simplicity, cost effectiveness, and high dispersion property.

Current researches for the pharmaceutical applications are still concentrated mainly on simple intercalation and disintercalation of various drugs in LDHs. The drugs to be intercalated into LDHs are widely expanding from simple anionic forms to neutral and zwitterionic forms. However, there are the very limited reports on in vitro activities of the intercalated drugs. It is not so far away for LDHs to be practically utilized as a next generation drug carrier for a broad spectrum of drugs.

3 .4 - C o s m e t i c a p p l i c a t i o n s

Practical application of LDHs to cosmetics have not been found so far although LDHs are reported to offer many fascinating aspects like high adsorption capacity to remove skin exudates and to encapsulate skin-sensitive coloring and UV-screening


agents, excellent anion exchange ability to protectively deliver active substances for anti-wrinkling and skin-regenerating, and also stabilizing potentials to improve rheological properties of various formulations, especially emulsion. Even though very limited, there are dozens of references to explore the potentials of LDHs for cosmetic purposes.

Choy et.al, reported the intercalation of vitamins A, E, and C into zinc-based double salt (ZBS) for their safe storage and delivery [53-54]. In particular, they carried out further study on ascobate-LDH hybrid for controlled release and safe delivery. Ascorbate was intercalated into ZBS by coprecipitation route, and then the hybrid was coated with silica to enhance ascorbate stability and dispersion property of the hybrid. Futhermore, they showed better skin permeation efficacy of intercalated ascorbate. Figure 15 shows the X-ray diffraction patterns of the vitamin C-zinc hydroxide hybrid obtained during the first encapsulation process (a) and the silica modified one (vitamin C-inorganic hybrid) and SEM of (b) [53]. The primary L-ascorbic acid-inorganic hybrid exhibited a layer character with the basal spacing of 14.5 A to indicate an intercalation with 1:1 layer sequence along the c-axis, where L-ascorbate molecules were encapsulated by inorganic layers as depicted in the inset.

Figure 15- (A) Powder X-ray diffraction patterns of a) vitamin C-zinc hydroxide hybrid and (b) Vitabrid-C and (B) scanning electron micrograph of Vitabrid-C powder

The silica deposition on the primary L-ascorbic acid-inorganic hybrid gave rise to a drastic suppression of long range ordering, as indicated by X-ray amorphous property of the modified hybrid. It was also noticed that the vitamin C molecules encapsulated in the interlayer space of inorganic layers were released in a time- controlled manner gradually by foreign chloride anions via ion-exchange process in an aqueous solution of 0.08 % NaC1. The released vitamin C was confirmed to be the pure one by its UV-Vis spectrum. In skin permeation test, the overall features of permeation patterns are quite similar one another, suggesting the similar penetration mechanism irrespective of the sample forms. However, the absolute amounts of permeated vitamin C after 24 hrs are more or less different with the following order; the modified hybrid powder (12.0 mg/cm 2) > the modified hybrid powder in w/o emulsion (10.4 mg/cm 2) > pure vitamin C in o/w emulsion (7.9 mg/cm2). This clearly indicated that the inorganically encapsulated vitamin C shows higher penetration rate than the pure


vitamin C.

The proposed releasing and delivering mechanism of vitamin C molecules in vitamin C-inorganic hybrid is schematically represented in Figure 16. In vitamin C- inorganic hybrid, the vitamin C molecules are adsorbed and immobilized between inorganic layers with positive surface charge, and further coated with nano-sized silica particles, forming a nanoporous shell structure.

Figure 16 - The proposed releasing and delivering mechanism o f vitamin C in Vitabrid- C

Due to its well developed nanoporous structure, the vitamin C-inorganic hybrid absorbs effectively the skin wastes, serums, and sweats discharged from the human skin. Actually, the hybrid shows a large oil absorption capacity more than 150 %. The absorption of chemical species such as NaC1 and fatty acids in sweat and skin wastes into the nanopores of the hybrid gives rise to a release of vitamin C in the pore by the exchange reaction between them, in such a way that the vitamin C molecules could be slowly diffused out from the inorganic shell and delivered into the epidermis in skin.

There are couples of other reports dealing with LDHs for cosmetic applications [56,88]. Hussen et al intercalated naphthol blue black into Mg-A1 LDH for ~ its encapsulation, which may make its formulation more easy and broad. They attempted to encapsulate the human skin several organic UV absorbents such as 4-hydroxy-3- methoxybenzoic acid, 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid, 4-hydroxy- 3-methoxycinnamic acid, 4,40-diaminostilbene-2,20-disulfonic acid, p-aminobenzoic acid and urocanic acid by the intercalation into Zn2A1 layered double hydroxides (Zn/A1-LDHs).

They found that the oxidation catalytic activity of the intercalated UV adsorbents for the air oxidation of castor oil greatly decreased along with enhancement of the UV absorption ability. Further elucidation on potential of LDH and consequent applications would be actively exploited in near future.


3 . 5 - Agricul tural appl icat ions Layered double hydroxides are one of the idealized inorganic matrices for a

wide range of agricultural fields because not only could their framework be decomposed into plant nutrients, but also their structures offer charming features such as accommodation and controlled release of various active anionic agro-substances, high buffering capacity, high water retention ability, and acid-neutralizing potential. However, their agricultural applications within biological scope are rarely found up to now although cationic clays have been widely utilized. Only a few references are available. One of the main reasons seems to be due to the fact that cationic clays are naturally abundant and cheap. For another reason, demand on anionic clays in agriculture was not so high and urgent enough to search for them. However, the present situation has changed since so many anionic compounds derived from agriculture are contaminating soil and water environments, intensively cultivated soils develop acidic property extensively, and advances in various techniques lead both to increased demand on anionic clays and to their cost-effective availability. In fact, the attempts to remove anionic pesticides by adsorption to LDHs have steadily increased recently, which is not a main topic here.

LDHs possess the excellent potential as green carrier for plant nutrients, pesticides, and growth regulators and as active principle in animal feeds, although currently not so many researches are undergoing. Komarneni et al [89] suggested nitrate-LDH as a potential slow-release fertilizer by synthesizing nitrate-LDH in ambient condition without any considerable contamination of carbonate-LDH. Recently, a plant growth regulator a-naphthaleneacetate (NAA) was intercalated through coprecipitation route by Hussein et.al [90] to explore the protected storage and controlled release in natural environments. More attention has been given to pesticide formulation that consists mainly of various organic solvents. Lakraimi et al [57] prepared pesticide-LDH hybrid with 2,4-dichlorophenoxyacetate, a broad leaf herbicide, by ion exchange reaction with chloride form of ZnA1-LDH for slow-release formulation. It is expected, once their nontoxicity is confirmed, that LDHs could be found in animal feeds soon or later because their acid neutralizing potential and high anion adsorption capacity are highly required to animal feeds as a complement to cationic clays.

4 - Conc lus ive remarks

Clays have served human life in various ways, and their contribution will be further expanded in future. To date, many natural clays have been incorporated into various commercial products as simple additives or adjuvants; whereas, their uses as active principles are very limited. However, recent attention has been rapidly shifted to their advanced applications. In particular, a new interdisciplinary field is to emerge from protective and controlled delivery of various functional components with both natural and synthetic clays.

An increasing number of bio-clay hybrids are continuously design-made and ready to be applied as the new types of delivery systems. It is easily expected that more and more bio-clay hybrids will be applied to in vivo study sooner or later. This trend also renews the interest in synthetic clays because their physical and chemical identities could be precisely controlled. Therefore, human life will further benefit from intensive exploration on biological potentials of clays.


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