Layered double hydroxides: A review

Abstract

Combination of two-dimensional layered materials and intercalation technique offers a new area for developing nanohybrids with desired functionality. Layered double hydroxides (LDHs) are mineral and synthetic materials with positively charged brucite type layers of mixed metal hydroxides. Exchangeable anions located in interlayer spaces compensate for positive charge of brucite type layer. Since most biomolecules are negatively charged, can be incorporated between LDHs. A number of cardiovascular, anti-inflammatory agents are either carboxylic acids or carboxylic derivatives and could be ion exchanged with LDHs to have controlled release. LDHs have technological importance in catalysis, separation technology, medical science and nanocomposite material engineering. Introduction Since living matter is composed of biological nanomachines and nanostructures, biology and medicine could be prime field for application of nanotechnology 1 . In particular, combination of two-dimensional layered material and intercalation technique offers new area for developing nanohybrids with desired functionality. Nanohybrids have composites function and most biomolecules (nucleoside monophosphates and ATP) that are negatively charged can be incorporated between hydroxide layers as charge compensating anions through ion exchange. Layered double hydroxides [LDHs] are also called anionic clays; mineral of this family is Hydrotalcite (Mg-Al-CO 3). LDHs have technological importance in catalysis, separation technology, optics, medical science and nanocomposite material engineering 2 .

Full text

NALAWADE et al: LAYERED DOUBLE HYDROXIDES: A REVIEW 267
Journal of Scientific & Industrial Research
Vol. 68, April 2009, pp.267-272
*Author for correspondence
Tel: 9769244623
E-mail: rshirlekar@bvcop.com
Layered double hydroxides: A review
P Nalawade, B Aware, V J Kadam and R S Hirlekar*
Bharati Vidyapeeth’s College of Pharmacy, Sec-8, C B D Belapur, Navi Mumbai 400 614, India
Received 19 February 2008; revised 31 December 2008; accepted 15 January 2009
Combination of two-dimensional layered materials and intercalation technique offers a new area for developing nanohybrids
with desired functionality. Layered double hydroxides (LDHs) are mineral and synthetic materials with positively charged
brucite type layers of mixed metal hydroxides. Exchangeable anions located in interlayer spaces compensate for positive
charge of brucite type layer. Since most biomolecules are negatively charged, can be incorporated between LDHs. A number of
cardiovascular, anti-inflammatory agents are either carboxylic acids or carboxylic derivatives and could be ion exchanged
with LDHs to have controlled release. LDHs have technological importance in catalysis, separation technology, medical
science and nanocomposite material engineering.
Keywords: Anticancer drugs, Intercalation, Layered double hydroxides (LDHs), Nanobiohybrides, Nanotechnology
Introduction
Since living matter is composed of biological
nanomachines and nanostructures, biology and
medicine could be prime field for application of
nanotechnology
1
. In particular, combination of two-
dimensional layered material and intercalation
technique offers new area for developing nanohybrids
with desired functionality. Nanohybrids have
composites function and most biomolecules (nucleoside
monophosphates and ATP) that are negatively charged
can be incorporated between hydroxide layers as charge
compensating anions through ion exchange. Layered
double hydroxides [LDHs] are also called anionic clays;
mineral of this family is Hydrotalcite (Mg-Al-CO
3
).
LDHs have technological importance in catalysis,
separation technology, optics, medical science and
nanocomposite material engineering
2
.
Layered Double Hydroxides (LDHs)
Chemical composition of LDH (Fig. 1) is generally
expressed as
M (II)
1-x
M (III)
x
(OH)
2
(A
n-
)
x/n
× yH
2
O,
where, M (II) =divalent cation, M (III) =trivalent
cation, A =interlayer anion, n- =charge on inerlayer
ion, and x and y are fraction constants.
Inorganic or organic anions can be introduced
between hydroxide layer by ion exchange or
precipitation
3
. LDHs containing magnesium and
aluminum have already been used as an antacid and
antipepsin agent; therefore, LDH is quite
biocompatible. Novel biohybrids of LDH and
biomolecules [ATP or nucleoside monophosphate] are
designed and organized artificially on nanometer scale
to provide opportunities for reservoir and delivery
carriers of functional biomolecules in gene therapy and
drug delivery.
LDHs can act as soluble inorganic vectors for
different genes and DNA biomolecules. Negatively
charged biomolecules intercalated in gallery spaces
would gain extra stabilization energy due to
electrostatic interaction between cationic brucite like
layers and anionic biomolecules. Such biomolecules
incorporated between hydroxide layers can be
intentionally dissolved in an acidic media, which offers
a way of recovering encapsulated or intercalated
biomolecules
5
. Hosting of biologically active
molecules inside LDHs can act as a ‘chemical flask-
jacket’, protecting host from degradation. Additionally,

268 J SCI IND RES VOL 68 MARCH 2009
hosting of a negatively charged species could provide
improved ways of drugs and genetic material to be
introduced into cells. If ingested, biomolecules-LDH
nano-hybrid can move across mucous membrane of
intestine into bloodstream. Neutral hybrid can then
enter cells by moving across negatively charged cell
membrane without repulsive electrostatic interactions
that would be experienced by guest anion alone. Once
inside the cell, LDH is broken down by lysosomes
resulting in intercalate release.
LDH materials, being unstable in acidic conditions,
do not survive for long in stomach. However, given a
suitable enteric coating, slow-release of drugs into
small intestine could be realized leading to effective
delivery of fragile genetic materials into cells.
Antisense Therapy
Antisense DNA, a potential gene specific
therapeutic agent, can be intercalated in LDH to form
Bio –LDH nanohybrid (Fig. 2), which protects
intercalated antisense molecule from degradation and
also improves cell penetration. Bio-LDH nanohybrid
also avoids specific aptameric effects (leading to non-
specific binding of antisense oligonucleotides). Once
LDH-antisense hybrids entered cell, hydroxide layers are
removed by dissolution in lysosomes, where pH is slightly
acidic and encapsulated biomolecules are released into
cell
6
.
Preparation of Layered Double Hydroxides (LDHs)
Reconstruction Method
Metal salts are calcinated at 500°C for 4 h in
nitrogen at a heating rate of 5°C/min. This solid is
then added to solution containing decarbonated water
with guest molecule. pH (7-8)is adjusted by NaOH.
Then, precipitate aged at room temperature, filtered,
washed with decarbonated water thoroughly and
finally dried under vacuum
7
.
Co-precipitation Method
Typically, a mixed solution of two different metal
salts in decarbonated water is added dropwise over
hours to an aqueous solution containing organic guest
species under nitrogen atmosphere with vigorous
stirring. During titration, solution pH (7-8) is adjusted
with 0.1 N NaOH to induce co-precipitation. Then
precipitate, aged at room temperature for 24 h, is
filtered, washed with decarbonated water thoroughly
and finally dried under vacuum
7
. Biomolecules LDH
hybrids can be prepared by ion-exchanging interlayer
anion of LDH with biomolecules. Co precipitation
method is more useful (yield, 3 times) than
reconstruction method.
Characterization of Layered Double Hydroxides (LDHs)
Stoichiometry of each biomolecule–LDH hybrid
can be determined by elemental analysis (CHN),
thermogravimetry (TG), and inductively coupled
plasma spectrometry (ICP). Synthesis of each hybrid
can be confirmed by XRD measurement using Ni-
filtered Cu-Ka radiation with a graphite diffracted
beam monochromator. Infrared spectra (IR) can be
obtained with FT-IR spectrometer by standard KBr
disk method. Crystal structure of biomolecule–LDH
hybrids can be studied by X-ray diffraction carried
on biomolecules (CMP, AMP, GMP and ATP). Taking
into account brucite-like LDH sheets (4.8 A°), gallery
heights of biomolecule–LDH hybrids were estimated
Fig. 1 - A schematic illustration of LDHs structure (Metal hydroxides layer located on top and bottom layers
while anion layer located in middle
4
)

NALAWADE et al: LAYERED DOUBLE HYDROXIDES: A REVIEW 269
to be: CMP, 9.7; AMP, 12.1; GMP, 13.6; and ATP,
14.6 A°. It means that nucleotides tend to have a
monolayer arrangement. Anionic substituents
(phosphate groups) are reported
8
to orient towards
LDH layers to maximize electrostatic attraction.
Considering charge density of layers, about 22
intercalates are perpendicularly arranged to hydroxide
layer. Schematic molecular arrangement in interlayer
of LDH is based on basal spacing and molecular size
of corresponding intercalates.
Transfer Efficiency and Cellular Uptake of LDH-Biomolecule
Hybrid
Biomolecules are well stabilized in LDH lattice,
and can be, if necessary, deintercalated by ion-
exchange reaction with other anions or atmospheric
CO
2
. These features will allow LDHs to be applied as
new drug or gene carriers if transfer efficiency of
biohybrids to target organs or cells is proved. To
elucidate transfer efficiency, isotope-labeled [
32
P]
ATP–LDH hybrid was prepared by ion exchange and
uptake of such hybrids by eukaryotic cells was
monitored with respect to incubation time.
Exogenously introduced ATP–LDH hybrid can enter
into HL-60 cells effectively within a relatively short
time. Transfer efficiency was found to be higher (up
to 25-fold) after 2 h of incubation, than that of ATP
only, where after 4 h of incubation, uptake amount of
hybrids becomes lower (below 12-fold). Triphosphate
group of [
32
P] ATP has a negative charge, which
inhibits [
32
P] ATP from being internalized in cell
through negatively charged cell walls. In contrast,
hybridization between ATP and LDH neutralizes
surface charge of anionic phosphate groups in ATP
due to cationic charge of LDH, which leads to
favorable endocytosis of cells, and results in enhanced
transfer efficiency
9
.
Longer the incubation time in a CO
2
atmosphere,
more ATP will be released from interlayer space of
hydroxide lattice. In spite of this, transfer efficiency
Fig. 2 - Schematic illustration of hybridization and expected transfer mechanism of bio
LDH nanohybrid into a cell
6

270 J SCI IND RES VOL 68 MARCH 2009
of hybrid remains higher than that of ATP only (up to
4-fold) after 24 h of incubation. Thus hybridization
between cationic layers and anionic biomolecules
greatly enhances transfer efficiency of biomolecules
to mammalian cells or organs.
Controlled Drug Delivery using LDHs
Addition of one of LDHs to a solution of chosen
pharmaceutical in water at room temperature results
in intercalation of these molecules between sheets of
host. LDHs are able to swell by up to 20 A°
to
accommodate size of new guest molecules. LDHs
possess antacid and antipepsin properties. Proprietary
antacids products (TALCID
TM
and ALTACITE
TM
)
contain LDH [Mg
6
Al
2
(OH)
16
]CO
3
3
.
Drugs [Diclofenac
(DIC), Ibuprofen, Naproxen, Gemfibrozil,
2-Propylpentonoic acids, 4-Biphenylacetic acid, and
Tolfenamic acid] are reversibly intercalated into LDHs.
A number of cardiovascular, anti-inflammatory agents
are either carboxylic acids or carboxylic derivatives,
could be ion exchange intercalated in a LDH to have
controlled release.
Apart from the potential of using these materials to
deliver drug in-vivo, it will be possible to control the
point of release and pharmacokinetic profile by selection
of metal ions in host layers. Antacid performance and
pH stability is also controllable by choice of metal layers,
which restricts molecular interactions and dynamics and
should improve long-term stability. In addition, improved
taste qualities of formulation are predicted.
Intercalating into hydrotalcite (HTIc) modifies DIC
release. Interlayer region of this matrix can be considered
a micro vessel, in which drug may be stored and released
by a deintercalation process due to the ions present in
small intestine
10-12
.
Release of DIC depends on diffusion
through particle and not on drug concentration. In vitro
studies show that drug is released by a deintercalation
process due to exchange of drug with ions present in
dissolution medium. At pH 7.5, drug release from HTIc-
DIC is slower than that from physical mixture and is
complete after 9 h. Kinetic analysis shows importance
of diffusion through particle in controlling drug release
rate. Hence, reversible intercalation of number of active
cardiovascular and anti-inflammatory agents into LDHs
can lead to novel tune able drug delivery system.
Anticancer Drug Therapy using LDHs
Folic acid derivatives [Folinic acid and Methotrexate
(MTX)] have been hybridized with LDHs by ion-
exchange reaction. MTX is used in therapy for different
forms of cancers. But, very short plasma half-life of
MTX necessitates administering a high dose that could
lead to drug resistance and nonspecific toxicities in
normal proliferating cells. Intercalation of MTX into
LDH protects MTX from deterioration during
transportation, whereas anion exchange along with acid
dissolution may result in controlled release. LDH also
probably affect permeability of MTX through cell
barrier, leading to significant enhancement. X-ray
diffraction patterns and spectroscopic analysis indicate
that these molecules intercalate into hydroxide interlayer.
Cellular uptake test of MTX-LDH hybrid is carried out
in fibroblast (human tendon) and SaOS-2 cell
(Osteosarcoma, human) by in vitro MTT [3-(4, 5-
dimethyl thiazol-2-yl) 2-diphenyl tetrazolium bromide]
assay. Initial proliferation of SaOS-2 cell is more strongly
suppressed by MTX-LDH hybrid than with MTX alone.
Thus LDH acts as biocompatible delivery matrix for
drugs and facilitates a significant increase in delivery
efficiency
13
.
Campothecin (CPT), an inhibitor of topoisomerase I
(enzyme involved in replication of DNA), has been
studied as a treatment for several forms of cancer. CPT
is pentacyclic indole alkaloid, with terminal ring
converting readily between lactone in acidic
environments (pH < 5) to carboxylate (pH < 8) form.
For CPT to be active, lactone form must dominate. Active
form, however, is only slightly soluble in water, leading
to poor dispersions in physiological solutions as well as
difficulties in efficient dose delivery. Study
14
demonstrated new delivery for non-ionic, insoluble drugs
such as Campothecin. Drug is loaded into a micelle,
which is then intercalated into nanometer galleries of
LDHs. This complex provides similar cytotoxic
characteristics to naked drug, but nanohybrids can be
administered in a dose-controlled fashion due to good
dispersion of complexes in water. Also, threefold
increase in solubility is observed as compared to naked
drug. Ability to attach targeting biomolecules to outside
surface of hybrids as well as potential controlled release
properties of complexes indicate that these hybrids may
be used for specific delivery of poorly water-soluble,
non-ionic drugs.
Improved stability of Vitamins
Vitamins [retinoic acid (Vit A), ascorbic acid (Vit C)
and tocopherol (Vit E)] that are very sensitive to light,

NALAWADE et al: LAYERED DOUBLE HYDROXIDES: A REVIEW 271
temperature, oxygen etc. can be stabilized by
intercalating into LDHs
15
.
Avoids Side Effects of Drugs
Organic UV ray absorbents, used as sun care products,
may pose a safety problem in high concentration use,
when they tend to be absorbed in body through skin.
This problem may be solved by intercalation of organic
UV ray absorbents in nanospaces of LDHs
16
.
Nonsteroidal anti-inflammatory drugs, used in rheumatic
treatment, produce side effects such as gastric-duodenal
ulcer formation. Intercalation of Indomethacin with
LDHs reduces gastric damage
17
.
Intercalation of Amino Acids and Peptides in LDHs
DNA is anionic macromolecule and is expected to
be intercalated by ion-exchange
method
5,6
. Amino
acids exist as zwitterions and are neutral (pH 7).
Therefore, intercalation of amino acids and protein
are expected to be difficult. By using co precipitation
method and reconstruction method, it is sometimes
possible to intercalate neutral molecules, which could
not be intercalated by ion-exchange method. Recently,
intercalation of some amino acids into Zn–Al and Mg–
Al LDHs by co precipitation method has been
reported
18
.
Because amino acid exits as zwitterions in
interlayer space of LDH, another anion (OH
-
or CO
3
2-
) must be intercalated at the same time for electrical
neutrality of LDH-amino acid that is co intercalation.
Therefore, amino acid would be interacted with
positive LDH layer not by Coulomb force but by
hydrogen bonding. MASS-NMR spectrum of LDH-
glycine and LDH-leucine suggested no deformation
of LDH layer by reconstruction reaction, and distance
between layers just changed. In amino acids with
larger hydrophobic group, molecule arranges as
bilayer structure in LDH as evidenced by XRD. In
other amino acids, long axis of amino acid molecule
is parallel to LDH. XRD pattern and MASS-NMR
spectrum of LDH-aspartame showed interlayer
distance of 2.2 nm, and a sharp signal at 9 ppm,
suggesting bilayer structure of aspartame in interlayer
space. This bilayer structure is reasonable because
phenylalanine also shows bilayer structure because
of large hydrophobic phenyl group. Amino acid was
easily deintercalated in H
2
O, because interaction
between positive LDH layer and zwitterions is not so
strong hydrogen bonding. This co-intercalation
mechanism was also true for peptides. Although easy
release of amino acid in H
2
O suggests the problem
for controlled release formulation, it shows that LDH/
amino acid could be used as amino acid reservoir and
adsorbent
19
.
Conclusions
Hybridization of drug or a biomolecule with LDH
results in remarkable transfer efficiency and stability.
So, LDH hybrids can be useful as reservoirs and
carriers for genes, drugs, and other functional
molecules. LDH will allow many diseases to be
monitored, diagnosed and treated in minimally
invasive way and it thus holds great promise of
improving health and prolonging life. LDH might very
well be next breakthrough in drug delivery system.
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