Layered double hydroxides as effective carrier for anticancer drugs and
tailoring of release rate through interlayer anions

Sudipta Senapati, Ravi Thakur, Shiv Prakash Verma, Shivali Duggal,

Durga Prasad Mishra, Parimal Das, T. Shripathi, Mohan Kumar, Dipak Rana,
Pralay Maiti

PII: S0168-3659(16)30013-X
DOI: doi: 10.1016/j.jconrel.2016.01.016
Reference: COREL 8072

To appear in: Journal of Controlled Release

Received date: 16 September 2015

Revised date: 28 December 2015
Accepted date: 11 January 2016

Please cite this article as: Sudipta Senapati, Ravi Thakur, Shiv Prakash Verma, Shivali
Duggal, Durga Prasad Mishra, Parimal Das, T. Shripathi, Mohan Kumar, Dipak Rana,
Pralay Maiti, Layered double hydroxides as effective carrier for anticancer drugs and
tailoring of release rate through interlayer anions, Journal of Controlled Release (2016),
doi: 10.1016/j.jconrel.2016.01.016

This is a PDF file of an unedited manuscript that has been accepted for publication.
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 ACCEPTED MANUSCRIPT

Layered Double Hydroxides as Effective Carrier for Anticancer Drugs and

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Tailoring of Release Rate through Interlayer Anions

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Sudipta Senapati,1 Ravi Thakur,2 Shiv Prakash Verma,3 Shivali Duggal,2 Durga Prasad

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*1
Mishra,2 Parimal Das,3 T. Shripathi,4 Mohan Kumar,5 Dipak Rana,6 and Pralay Maiti
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1
School of Materials Science and Technology, Indian Institute of Technology (Banaras Hindu
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University), Varanasi 221 005, India

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2
Cell Death Research Laboratory, Division of Endocrinology, CSIR-Central Drug Research
Institute, Lucknow 226031, India
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3
Centre for Genetic Disorders, Faculty of Science, Banaras Hindu University, Varanasi
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221005, India
4
UGC-DAE CSR, University Campus, Khandwa Road, Indore, 452 001, India
5
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Department of Pathology, Institute of Medical Science, Banaras Hindu University, Varanasi

221005, India
6
Industrial Membrane Research Institute, Department of Chemical and Biological
Engineering, University of Ottawa, 161 Louis Pasteur St., Ottawa, ON, Canada KIN 6N5

*
Correspondence should be made to Pralay Maiti (pmaiti.mst@itbhu.ac.in)

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Abstract

Hydrophobic anticancer drug, raloxifene hydrochloride (RH) is intercalated into a series of

magnesium aluminum layered double hydroxides (LDHs) with various charge density anions

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through ion exchange technique for controlled drug delivery. The particle nature of the LDH

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in presence of drug is determined through electron microscope and surface morphology. The

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release of drug from the RH intercalated LDHs was made very fast or sustained by altering

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the exchangeable anions followed by the modified Freundlich and parabolic diffusion

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models. The drug release rate is explained from the interactions between the drug and LDHs

along with order-disorder structure of drug intercalated LDHs. Nitrate bound LDH exhibits
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greater interaction with drug and sustained drug delivery against the loosely interacted

phosphate bound LDH-drug, which shows fast release. Cell viability through MTT assay
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suggests drug intercalated LDHs as better drug delivery vehicle for cancer cell line against
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poor bioavailability of the pure drug. In vivo study with mice indicates the differential tumor

healing which becomes fast for greater drug release system but the body weight index clearly
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hints at damaged organ in case of fast release system. Histopathological experiment confirms

the damaged liver of the mice treated either with pure drug or phosphate bound LDH-drug,
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fast release system, vis-à-vis normal liver cell morphology for sluggish drug release system

with steady healing rate of tumor. These observations clearly demonstrate that nitrate bound

LDHs nanoparticle is a potential drug delivery vehicle for anticancer drugs without any side

effect.

Key words: Layered double hydroxide; controlled release; anti tumor activity;

histopathology.

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Introduction

Chemotherapy is considered to be an effective practice for the treatments of cancer

worldwide. The drugs or chemotherapeutic agents which kill predominantly the rapidly

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growing cancerous cells in addition to the normal cells are considered as cytotoxic in nature.

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Hence, the most common side effects of chemotherapy are myelosuppression (decreased

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production of blood cell), mucositis (inflammation to the digestive system) and alopecia (hair

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loss). Chemotherapy related toxicities occur within hours or days of drug administration

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resulting nausea, vomiting and anemia caused by the sudden exposure of drugs molecules to

the body parts. Drugs associated with chemotherapy have often severe side effects that limit
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their efficacy. Another serious obstacle for many effective cancer drugs is their poor water

solubility and very low bioavailability of most hydrophobic anticancer drugs which require a
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vehicle for their practical uses. Therefore, an efficient drug delivery system is much needed
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which can overcome these drawbacks and improve the clinical therapy.

In last few decades, many drug delivery systems have been developed to enhance the
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efficiency of chemotherapeutics. Among these, polymers (polymer nanoparticles and

micelles) and lipids (liposomes) are the prominent ones [1‒3]. Inorganic nanocarriers are
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becoming strong competitors in recent years due to their great advantages, such as large

surface area, better loading capacity of drug, better bioavailability, lower toxic side effects,

controlled release of drug and unlike polymer-based nanoparticles they can tolerate most

organic solvents [4‒6]. Layered double hydroxides (LDHs) are one of the promising

inorganic nanocarriers that have several attractive features for their uses in the delivery of

negatively charged drugs. LDHs are a family of anionic clay materials, generally represented

by the general formula [MII 1−xMIII x (OH) 2] x+

(An−) x/n.yH2O, where MII is a divalent metal

ion, such as Mg2+, Ca2+, Zn2+, etc., MIII is a trivalent metal ion, such as Al3+, Cr3+, Fe3+, Co3+,

etc. and An− is an anion, such as Cl−, CO32−, NO3 −, etc [7]. Nowadays LDHs are well known

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materials due to its other interesting properties such as excellent anion exchange capacity [8],

good biocompatibility, low toxicity [9], ability to make stable suspension leading to inject

able drug delivery vehicles [10]. It has widespread applications in the fields of catalysis [11],

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flame-retardant nanofiller for polymers [12], photo/UV stability of polymeric materials [13],

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ion scavenger [14], bone joint implant materials [15], and improvement of mechanical

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properties of polymers [16]. The layers of LDH possess positive charges which are counter

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balanced by the anions present in the interlayer spacing and those anions are exchangeable

with other suitable negatively charged species. The interlayer anion exchange capability of

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LDHs can be used to intercalate negatively charged functional biomolecules such as vitamins
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[17], amino acids [18], DNA [19], siRNA [20] and drugs [21, 22] within the LDH layers.

Thus, LDHs can be used as drug vehicle and may be used to regulate the release of drug from
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the drug intercalated LDH. Sustained drug release systems have the advantages that they
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reduce the frequency of dose and thereby improve patient compliances, develop drug

utilization, minimize fluctuations in plasma and serum level, and more importantly reduce the
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adverse side effects. Similarly, in fast release systems, therapeutic blood concentration can
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quickly be achieved especially for those drugs which have very short lives. Raloxifene
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hydrochloride (RH) is a potent antitumor drug but its poor bioavailability, arising from low

solubility in water, restricts its uses [23].

In the present work, a series of LDHs have been synthesized through coprecipitation

techniques using various anions. A representative hydrophobic anticancer drug, raloxifene

hydrochloride (RH) has been intercalated into layered double hydroxides using ion-exchange

method. The mechanisms involved in the drug release process have been studied for the

series of LDH having different anions as the counter ions. This investigation addresses the

process of controlled release (both fast and/or sustained release) system as required by

altering the intercalated anions. The in vitro and in vivo drug release characteristics from drug

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loaded LDHs in comparison to pristine drug has been evaluated taking antitumor efficacy and

toxicity as the major parameters. A new drug delivery vehicle with controlled release (both

fast and slow) has been developed to overcome the insolubility problem of particular

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anticancer drugs (RH) and to reduce its adverse side effects.

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Experimental

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Materials

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The anti-tumor drug, Raloxifene hydrochloride (RH), was purchased from Sigma-Aldrich.

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Mg(NO3)2·6H2O, Al(NO3)3·9H2O, NaNO3, Na2CO3, and Na3PO4 were obtained from Merck

India Ltd. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, HiMedia,

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India), Dulbecco’s Modified Eagle Medium (DMEM), penicillin and streptomycin were also

obtained from Sigma, USA. Fetal bovine serum was purchased from Gibco. Dimethyl
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sulphoxide (DMSO) was purchased from Merck India Ltd. All the materials were used as
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received.

Synthesis of layered double hydroxide (LDHs)

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The Mg-Al-LDHs were synthesized by using coprecipitation method [24]. Mixture solution

of 0.5 M Mg(NO3)2·6H2O and 0.25 M Al(NO3)3·9H2O with [Mg2+]/[Al3+] molar ratio of 2:1
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in 100 ml deionized water was prepared first. 100 ml of 1.5 M NaNO3, Na2CO3, and Na3PO4

were prepared separately and were kept in five-neck flat bottom flask. Five necks of the flask

were fitted with two burettes, one condenser, one gas inlet-outlet and one pH meter probe.

The solutions were degassed by purging purified nitrogen. Previously prepared mixture

solution of Mg(NO3)2·6H2O and Al(NO3)3·9H2O was added drop wise into NaNO3, Na2CO3,

and Na3PO4 separately and the solutions were stirred vigorously during mixing. NaOH

solution (0.5 M), fitted in one burette, was added drop wise to the three different above

mentioned solutions until the pH reached the value of 10. The whole solution was stirred

continuously at 60 C for 14 h. The appearance of white gelatinous precipitate indicated the

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formation of MgAl-LDHs having three different intercalated anions (NO3LDH, CO3LDH,

PO4LDH and are abbreviated as LN, LC and LP, respectively). The precipitates were

recovered by centrifugation (5000 rpm, RT) and were washed thoroughly with deionized

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water followed by drying in air oven at 50 oC for 36 h.

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Preparation of drug intercalated LDHs

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Drug (Raloxifene hydrochloride; RH) intercalated LDHs with desired drug loading (5, 15,

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and 30% of the intercalated anions in the pristine LDHs were replaced with RH anions) were

prepared through the anion exchange method [25]. For example, to prepare 15% RH

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intercalated in LN (NO3-LDH), 1 g LN was dispersed in 75 ml of deionized water and was
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placed in a three-neck round bottom flask followed by degassing through nitrogen gas

purging for 20 min. NaOH (1 M) solution was added to the above solutions until the pH

reached 9. Then, 0.15 g of drug was added into it and was stirred vigorously at 60 C for 12
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h. The resulting precipitates were centrifuged, washed thoroughly with water, dried at 60 C

and stored. The RH intercalated LDHs are abbreviated as LN-R, LC-R and LP-R using NO3-
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LDH, CO3-LDH and PO4-LDH, respectively.

Determination of intercalated drug

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Spectrophotometric technique was carried out to determine the total amount of intercalated

drug [24]. A known amount of drug intercalated LDH was dissolved in 6 M HCl followed by

dilution with phosphate buffer of pH ~7.4 and the solution was stirred at a moderate rate for 8

h at 50 C. The solution was then filtered and the concentration of drug was determined by

UV absorption at 295 nm. The concentration of RH was determined using a standard curve of

known concentrations of RH.

Characterization

X-ray Diffraction (XRD) Powder XRD patterns were examined by using an advance wide-

angle X-ray diffractometer with Cu-K radiation and a graphite monochromator

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(wavelength, nm, Rigaku, MiniFlex-600, Japan). Patterns were recorded at

diffraction angle 2 from 3 to 70 at the scanning rate of 3 min-1.

FTIR FTIR was performed in the transmittance mode at room temperature from 400-4000

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cm-1 on a Thermo Scientific FTIR (NICOLET-6700) with a resolution of 2 cm-1 using the

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KBr pellet technique.

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UV- Vis Spectroscopy UV-Visible measurements were performed by using Jasco-V-650

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spectrophotometer, Japan, operating in the spectral range of 190 - 1100 nm.

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TGA The thermogravimetric analysis (TGA) was carried out using a Mettler-Toledo

thermogravimetric/differential thermal analyzer (TGA/DTA). The samples ( 8 mg) were

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mounted in an alumina crucible and were heated from 30 to 650 C at the heating rate of 20

min-1 in nitrogen atmosphere.

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Differential Scanning Calorimetry (DSC) The thermal behavior of the pristine drug, pure

LDHs and drug intercalated LDHs was determined through DSC using Mettler 832 at a scan
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rate of 10 min-1. The DSC was calibrated with indium before use.
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X-ray photoelectron spectroscopy (XPS) XPS measurements were performed using a VSW
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made photoelectron spectrometer at UGC-DAE-CSR, Indore, India. The spectra were

recorded using Al-K radiations with incident energy of 1486.6 eV. Binding energies in all

XPS spectra were calibrated using C1s peak (284.6 eV). Measurements were conducted

under ultrahigh vacuum ( 4.410−10 Torr).

Particle size and zeta potential: The average particle size (z-average size), polydispersity

index (PDI) and zeta potential of the pristine LDHs and drug intercalated LDHs were

measured by using Horiba nanoparticle analyzer SZ-100 instrument (Horiba Scientific,

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Japan) at 25 C under a fixed angle of 90. The samples were diluted to a suitable

concentration (0.3 mg/ml) with double distilled high purity water before measurement.

Morphological investigations: Images of pristine LDHs particles and drug loaded LDH

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particles were taken using a JEOL JSM-2010 transmission electron microscope (TEM) at the

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accelerating voltage of 200 kV. The morphology of the pristine LDHs and its drug

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intercalated LDHs were investigated by using filed emission scanning electron microscopy

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(SEM) and atomic force microscopy (AFM). The surface morphology of the thin film was

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examined through FESEM ZEISS SUPRATM 40 instrument operated at 5 kV. All the samples

were gold coated by means of a sputtering apparatus before observation in FESEM. Solver
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scanning probe controlled NT-MDT multimode atomic force microscope, Russia, was used in

tapping mode with the tip mounted on a 100 µm long single beam cantilever with resonant
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frequency of 240–255 kHz and the corresponding spring constant of 11.5 N m–1.
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Drug release study The dissolution test was performed at 37 C by suspending drug
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intercalated LDH in 100 ml phosphate buffer saline of pH 7.4. Aliquots (1 ml) of supernatant
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were taken at regular intervals, and the drug content was determined through UV absorption
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at λmax = 295 nm. Further, to understand the kinetics for the release behavior, several kinetic

models were applied (details given in the supplementary document along with the respective

equations) and fitted with the in vitro drug release profiles.

Biocompatibility

Cell Culture HeLa (Human cervical cancer cell line) cells and murine breast cancer cells

4T1 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium containing 10%

heat inactivated fetal bovine serum, 100 U/ml penicillin and 100 µg/ ml streptomycin. The

temperature of the culture was maintained at 37 C in a CO2 incubator with 5% CO2 supply.

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Cell viability The in vitro anti-tumor effect of LDHs, pristine drug and drug intercalated

LDHs against HeLa cell was investigated through cell viability test. The percentage of viable

cells was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

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assay [26]. Briefly, cells grown to 70-90% confluence onto the 96-well culture plates in 0.1

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ml of DMEM were used for all the treatment in triplicate for MTT assay. Drug intercalated

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LDH, pristine drug and pure LDHs were added to the wells in increasing concentrations

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corresponding to 10 and 100 g/ml and were incubated for 24, 48, and 72 h time intervals at

37 C. After incubation, media containing drug loaded nanomaterial was replaced with fresh

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medium containing 0.5 mg/ml MTT to each well and was incubated at 37 C for 4 hrs to
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produce formazan. This purple coloured dye was then dissolved in DMSO and the

absorbance was taken using spectrophotometer (Amersham) at 570 nm wavelength. The %

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cell viability was calculated using the formula:

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
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where, C is the optical density of ‘control’ representing HeLa cells incubated in medium

alone and T is the optical density of test specimen representing HeLa cells treated with the
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corresponding samples.

Fluorescence imaging HeLa cells were seeded in 12 well plate on cover slips as described

earlier and grown upto 60-80% confluence. Cells were treated with 20 g/ml concentration of

pristine LDH, drug intercalated LDHs and equivalent amount of pure drug (RH) and were

incubated at 37 C in CO2 incubator. Cells on coverslips were stained with 100 µg/ml

Acridine Orange and 100 µg/ml Ethidium Bromide, The images were captured through

confocal microscope (LSM780, Carl Zeiss. Germany).

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Animal studies

Animals Female Balb/c mice were reared at CSIR-Central Drug Research Institute, Lucknow

for the in vivo studies and were conducted in accordance with the principles and standard

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procedures approved by the Institutional Animal Ethics Committee (IEAC) of the CSIR-

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Central Drug Research Institute.

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In vivo antitumor efficacy The anti-cancer potential of the drug loaded LDHs and pure drug

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was determined with healthy female Balb/c mice (20 ± 2 g). In brief, subconfluent 4T1 cells

were trypsinized, washed twice, and harvested by centrifugation at 1000×g for 5 minutes. A

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single-cell suspension was prepared in phosphate-buffered saline (PBS). Cells (1 × 106) were
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injected subcutaneously into the mammary fat pad of 4- to 5-week-old female Balb/c mice.

When the average tumor size reached 50 mm3, mice were divided into six treatment groups
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consisting of five mice per group: Vehicle (PBS), LN (pristine LDH), RH (pure drug), LNR,
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LCR, and LPR dissolved in PBS. Pristine RH and drug intercalated LDHs were

administered intra-intraperitoneally (i.p) at 30 mg drug / Kg body weight and equivalent

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amount in drug intercalated LDH using 26 gauge needles for 22 days with a two days interval
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between subsequent doses. The tumor volume was calculated using the equation:
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tumor volume (mm3) = 1/2 (L  W  W),

where, L is length and W is width. The body weight and tumor volume were recorded every

two days after the first injection.

Histological assessment Mice were sacrificed and main organs (liver, kidney and spleen)

were dissected for further immunohistochemical staining at day 21 after first injection. The

excised organs were fixed in 10 % (v/v) buffered formalin solution, dehydrated with a graded

ethanol series and embedded in paraffin. The tissues were then sliced into sections 4-6 m in

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thickness and were stained with hematoxylin and eosin (H & E). These (H & E) stained

assays were then monitored using light microscopy at 200 magnification.

Pharmacokinetic study 6‒7 weeks-old male Sprague–Dawley rats, weighing 220±20 g,

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were procured from the National Laboratory Animal Center, CDRI (Lucknow, India). The

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study protocol was approved by the Local Animal Ethics Committee of CSIR-Central Drug

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Research Institute and all the animal studies were carried out in accordance with the approved

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guidelines and regulations. Pure RH and RH intercalated LDH (LN-R) formulations were

injected intraperitoneally at the dose of 30 mg drug / Kg body weight and equivalent amount

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in drug intercalated LN-R, to four mice of each group. Blood samples (~ 0.35 ml) were
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collected from the retro-orbital plexus into heparinized microfuge tubes at a regular time

interval up to 72 h of post-dosing. Non-compartmental pharmacokinetics analysis was done

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using Phoenix WinNonlin (version 6.3, Pharsight Corporation, USA) software to calculate
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the pharmacokinetic parameters (area under the curve (AUC); mean elimination half-life

( 1/2) etc.). The observed maximum plasma concentration (Cmax) and the time to reach the
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maximum plasma concentration (tmax) were obtained from the observed concentration versus
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time profiles and the area under the curve (AUCo-t) was calculated by using standard
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procedure [27].

Biochemical assays Heparinized blood samples (50μl /mouse) were obtained on day 0 and at

the end of treatment by sub mandibular bleeding. The clinical chemistry of the blood

including parameters for: aspartate aminotransferase (AST), alanine aminotransferase (ALT),

blood urea nitrogen (BUN), White blood cells (WBC), hemoglobin concentration (Hgb) and

Platelets (PLT) were obtained using the Hemagen AnalystVR Benchtop Chemistry System

(Hemagen Diagnostics, Inc. Columbia, USA).

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Statistical analysis

Results of cytotoxicity and in vivo studies were expressed as the mean value standard

deviation. Statistical comparisons were performed by single factorial analysis of variance

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(one-way ANOVA with t-test). P values * < 0.05 were considered significant.

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Results and discussion

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Morphological changes due to drug intercalation

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The shape and sizes of LDH particles have been determined through transmission electron

microscopy. Discrete particles have been observed in pure LN as hexagonal platelet like
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shape with the lateral dimension of 84±3 nm while predominantly stacking of platelets and

some agglomerated morphology have been noticed in the corresponding drug intercalated
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LDH (LN-R) of lateral dimension 94±4 nm (Figure 1a). The intercalation of the drug
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molecules enhances the stacking force (cohesive force) between the layers and the extensive

edge-on hydrogen bonding of the ordered structure makes the system agglomerated [28]. This
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is to mention that the shape and size of the as prepared LDHs are similar to earlier published

works obtained through bright field TEM images [29]. Morphologies of the other LDHs
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(LP/LPR) and their drug intercalation are similar to LN system (Supplementary Figure S1).

The surface morphology through FESEM of LN and LNR is shown in Figure 1b. LN

exhibits a plate-like morphology of dimension 95±2 nm while agglomerates of compact and

non-porous granular structure of size 106±3 nm are clearly visible after the intercalation of

drug in LNR. Moreover, the presence of Mg and Al metal in LDHs has been confirmed

through EDAX measurements (Supplementary Figure S2). Figure 1c shows the AFM

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a. b.
LN LN-R
LN LN-R

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200 nm 200 nm

100 nm 100 nm

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c. 220 nm d.

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LN
LN LN-R
LN-R
1260
1597

Transmittance / a.u
450

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200 200
Height profile / nm
Height profile / nm

1260
150 150
1597
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100 100 551
LN-R
50 50
R 1630 676 447
1384
0
0 200 400 600
0
0 200 400 600 LN
Length / nm Length / nm 2000 1500 1000 500
-1
wavenumber / cm
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Figure 1: (a) Bright field transmission electron micrographs of LN and LNR; (b) FESEM

micrographs of LN and LNR; (c) AFM topographs of LN and LNR with height profile,
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and, (d) FTIR spectra of pristine LN, free drug and LNR.
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images of the dried suspension indicating larger particle size in LN-R (100 nm) as compared
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to pure LN (90 nm). Height profiles also indicate the relatively smoother surface in LN-R vis-

à-vis pure LN. However, platelet nature of LDH is obtained which agglomerate extensively

after drug intercalation. The particle sizes of LN and LNR are found to be 145±6 and 174±8

nm, with polydispersity index (PDI) of 0.18 and 0.20, respectively. The particle size and their

distribution of other LDHs and their drug intercalation species (LP/LPR) are similar to LN

system and are reported in Table 1.

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~ 80 nm


Cationic brucite-like
O + 1 nm [M2+ M 3+ (OH) ] x+ layers
1−x x 2

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Drug molecules LDH

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NO3 H 2O

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~ 80 nm

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2 nm
O
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Drug intercalated LDH
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Figure 2: Schematic model representing the structure of LDH and drug intercalation within

LDH layers.
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Table 1: Particle size, distribution (PDI) and Zeta potential for pristine LDHs and drug
intercalated LDHs. Values are presented as mean  SE (n = 3).

Sample Particle dimension (nm) PDI Zeta potential (mV)

LN 145 ± 6 0.18±0.07 41.3

LN‒R 174± 8 0.20± 0.08 21.4

LP 151 ±7 0.19±0.07 42.1

LP‒R 188±10 0.23±0.09 22.6

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Intercalation of raloxifene into LDH interlayers is also confirmed by comparing the

FTIR spectra for LNR, in comparison with pristine LN and raloxifene hydrochloride salt

(Figure 1d). The spectrum of pristine LN shows the intense broadband around 3445 cm−1

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associated with the stretching vibration of the hydroxyl groups of LDH layers and interlayer

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water molecules. The band at 1630 cm−1 is due to bending vibration of interlayer water

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molecules. The sharp band at 1384 cm−1 corresponds to stretching vibration of NO3−

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groups. In the lower wavenumber range, the peak at 551 cm−1 is assigned to MO and

MOH stretching vibrations in the brucite-like layers of the LDH. The band at 447 cm−1 is a

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characteristic peak of Mg2AlLDH [28]. In the spectrum of pure raloxifene hydrochloride,
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the characteristic peaks at 1643 cm−1 (C=O stretching), 1598 cm−1 (–C–O–C– stretching),

1466 cm−1 (–S– benzothiophene), 1260 cm−1 (alkyl–O–phenyl stretching) and 905 cm−1
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(benzene ring) are observed, which agree well with the previous literature [30]. LN–R
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exhibits the diluted (low intense) characteristic peak of nitrate group at 1384 cm −1 confirming

the replacement of nitrates ions with the raloxifene ions and the other prominent drug peaks.
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LC shows characteristic peaks at 1640 cm−1 (bending vibration of interlayer water), 1366

cm−1 (antisymmetric 3 vibration of CO32), 678 cm−1 (overlapping of the antisymmetric 3

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vibration of CO32- and M–O stretching vibration) and 551 cm−1 (translational mode of M–

OH) [15] (Supplementary Figure S3). Similarly, LP shows the characteristic peaks at 1098

cm−1 (asymmetric P–O str.), 1035 cm−1 (symmetric P–O str.), 965 cm−1 (asymmetric P–OH

str.), and 535 cm−1 (asymmetric O–P–O str.) [31]. The drug intercalated LC–R and LP–R

show the characteristic peaks of low intense (diluted) carbonate and phosphate groups along

with the drug peaks, confirming that the intercalation of raloxifene ions inside layers.

However, the drug is intercalated within the layered LDH and the schematic representation of

LDH layers along with drug intercalation has been shown in Fig. 2 mentioning the position of

anion, intercalated water molecules and relative dimension of LDHs.

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Structure and level of intercalation

The XRD patterns of LN, RH and LNR are shown in Figure 3a. The reflections from (003)

basal planes represent the thickness of the brucite layer plus the interlayer spacing and are a

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function of the size and orientation of the intercalated anions [32]. The diffraction peaks

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obtained from (003), (006) and (009) crystal planes as well as two well separated (110) and

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(113) peaks indicate both the pristine LN and LC are well-crystallized [33]. However, the

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degree of crystallinity in pristine LP is somewhat lower than that of LN and LC as the (003),

(006) and (009) basal planes are not very sharp along with merging of the (110) and (113)

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peaks. The (003) basal spacing in LN, LC and LP are 0.85, 0.75, and 0.73 nm, respectively,
MA
indicating gradual decrease of basal spacing with increasing charge density of anions, from

(Figure 3a). The (003) and (006) peaks become weaker and slightly
D

broader after the intercalation of drug inside the layers suggesting lower crystallinity. Further,
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a new set of basal reflections (indicated with the ‘’ mark in Figure 3a) appeared at 2 

6.67o, which indicates the intercalation of RH into the LDH layers having the (003) spacing
P
CE

of 1.32 nm for LNR, and 1.34 nm both for LCR and LPR. Thus, the expansions of basal

spacing are 0.47, 0.58 and 0.61 nm for LNR, LCR and LPR, respectively, for similar
AC

extent of drug intercalation (~15 %) in all the LDHs. Now it is important to understand the

LDH basal spacing depending on the charge density of anions. Considering the similar

positive charge in the layers, the number of anions goes down with its increasing charge state

and it is believed that the number of carbonate and phosphate ions is halved and one-third in

LC and LP, respectively, as compared to LN where nitrate ions have the single charge state.

Therefore, reduced numbers of interlayer anions decrease the basal spacing for LC and LP

gradually with respect to LN. On the other hand, similar basal spacing is obtained in all drug

intercalated LDHs (1.32 – 1.34 nm) when the extent of drug was kept constant i.e the number

of intercalate remains same for 15% drug intercalation. This is to mention that raloxifene has

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a. LN-R b. S O
LN O
O S
O

1. 32 nm
O O
N
O

0. 85 nm
O O
N
O
N
Raloxifene
(003)

( 009 )
O
S O

( 012 )
(006)

(113)
(110)
(015)

T
O S
O
Controlled release N O
O

IP
O

LC-R LN = H2O O N

 O
S O

LC = NO3

R
Intensity / a.u

LNR

SC
( 009 )
(006)
(003)

(113)
(110)
( 012 )
(015)

NU
LP-R O
S O

1. 34 nm
O S
LP O O

0. 73 nm
O
O N O

O
N
Raloxifene O N
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O
O S

S O
Controlled release O

= H2O3 O N
LP
= PO4
D

10 20 30 40 50 60 70
2 / degree
LPR
P TE

Figure 3: (a) XRD patterns of LN, LNR; LC, LCR; and LP, LPR. ‘’ marks indicates the
CE

new set of basal reflections originates from RH intercalated LDH phases; (b) a schematic

representation of LDH structure before and after drug intercalation in LN and LP.
AC

two negative sites for intercalation. However, LDH with phosphate ion as exchangeable

anion has been synthesized for the first time while the basal spacing of LN and LC has

similar spacing as reported in the literature [34, 28]. LNR and LCR have ordered structure

while in LPR, the drug molecules are intercalated into the LP layers with a disordered

arrangement and based on the XRD patterns, the nature of intercalation has been shown in

Figure 3b leading to two different types of nanostructure. Now, it appears that LP-R has

more disordered structure or open to the environment as compared to the LN-R (intercalated

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 ACCEPTED MANUSCRIPT

and ordered) raising different drug releasing behavior. XRD pattern of pure RH shows its

crystalline nature with the characteristics peak of 13.46, 14.52, 20.98, 22.62 and 24.1o, which

agree well with the literature [30] while the absence of its peak in intercalated LDH suggest

T
that RH is intercalated into the interlayer galleries rather than simple binding to the surface or

IP
adsorbed (Supplementary Figure S4). This is to mention that the dispersion of LDH or drug

R
intercalated LDH is very stable.

SC
In vitro controlled release: Variations using different anions

Anticancer drug delivery has been studied with raloxifene intercalated LDHs of varying

NU
nanostructure to investigate the kinetics of drug release through UV-Vis spectroscopy. Drug
MA
release profiles for LNR, LCR and LPR with 15 wt.% drug intercalation are presented in

Figure 4a. LP-R exhibits very fast release of drug; 80% release occurs in just 1 h and total
D

release takes place at ~7 h. On the other hand, LNR follows a biphasic elution profile, with
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relatively slow release rate ( 65% release occurs in the first 6 h) followed by a sustained

release (100 % in 42 h from the initial stage). A similar biphasic release pattern is also
P
CE

observed for LCR, in which more than 80% of drug is released at the first 5 h and full

release takes around 26 h. A gradual sustained release is clearly observed for

AC

containing LDH. The fast release from LP-R (phosphate ion based LDH) is

anticipated from its disordered pattern where the drug intercalated layers are exposed more

towards the releasing media while ordered intercalated structure restricts the release of drug

from LN-R (Figure 4b). The interaction issues will be uncovered in next section. However,

the extent of drug intercalation does not affect much the nature of release profile and has been

shown in Supplementary Figure S5 with varying drug content (5% and 30% w/w) in LDHs.

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a. b.

100 O O O
Slow release
Cummulative release %

80
O O LNR system O O

Cummulative release %
60 100

T
75
40 50

IP
LP-R 25
20 LC-R
0 O Fast release
LN-R 0 2 4 6 8

R
t/h
0
0 10 20 30 40 50
t/h LPR system

SC
c. d.
0.0

NU
0.45
log (1- Mt / Mo )

(1- Mt / Mo ) / t
-0.4
0.30
MA
-0.8 LP-R 0.15
LP-R
LC-R
LC-R
LN-R
LN-R
D

0.00
-1.2 -0.6 0.0 0.6 0.2 0.4 0.6 0.8
- 0.5
log (t) t
TE

Figure 4: (a) In vitro drug release profiles for LNR, LCR and LPR; Inset figure also
P

describes the release pattern of the above mentioned systems in the time frame of 0-8 h. the
CE

results presented are mean ± standard deviation (SD) values obtained from three independent
AC

experiments (b) Schematic representation of drug release behaviour from LNR and LPR

systems; Plots of to kinetic models (c) modified Freundlich model and (d) parabolic diffusion

model for the release of raloxifene from LDH in the two stages, release pattern for stage I (0

to 70% release) is better fitted by modified Freundlich model, whereas release pattern of

stage II (70 to 100% release) is most suited by parabolic diffusion model.

This is to mention that although the percentage of drug release remains same but the quantity

of actual drug released will be different.

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To understand the release mechanism of the drug (RH) from the intercalated LDHs,

five different kinetic models (zero-order, first-order, parabolic diffusion, modified Freundlich

and Elovich model) have been used. The rate constants and the linear correlation coefficient

T
(r2) values obtained from the linear fittings are given in Table 2. Among the present models,

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zero- and the first-order models give poor r2 values ranging from 0.565 to 0.879 and are not

R
found suitable to explain release mechanism. On the contrary, the parabolic diffusion model,

SC
the modified Freundlich and the Elovich models explain the release phenomena more

reasonably. To gain more insights of the release behaviour, the whole release process can be

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subdivided into two stages: 1) the rapid release stage I (up to 70% cumulative release)
MA
followed by, 2) the slow release stage II (70-100% release). Stages I and II are best fitted

with the modified Freundlich and parabolic diffusion models, respectively, with a high linear

correlation coefficients (r2 ~ 0.980.99) (Figure 4c,d). The modified Freundlich model
D
TE

describes heterogeneous diffusion from the flat surfaces via both diffusion controlled and ion-

exchange phenomena, better suitable for initial release while the parabolic diffusion model
P

describes the release of the drug RH from the drug intercalated LDHs as a diffusion
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controlled process through intra-particle diffusion or surface diffusion. The Elovich model
AC

explains a number of process including bulk and surface diffusion, as well as activation and

deactivation of catalytic surfaces [35, 36]. These simulation results suggest strongly that the

release of the intercalated RH from the LDHs layers is a mix-effect including dissolution of

nanoparticle and ion-exchange between the intercalated anions in the lamellar host and the

phosphate anions in the buffer solution. In stage I, most of the R ions from intercalated drug

(RH) dissociate from the surface of LDHs and diffuse into the medium solution via anion

exchange, responsible for the rapid drug release, whereas diffusion of the drug anions from

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Table 2: Rate Constants and Linear Correlation Coefficients (r2 ) Obtained by Fitting the RH
Release Data from LNR, LCR and LPR.

T
LNR LCR LPR
kinetic models

IP
I II III I II III I II III
zero-order model

R
k0 11.20 2.13 45.24 2.68 60.2 9.76
r2 0.847 0.707 0.914 0.527 0.942 0.676

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first-order model
k1 0.227 0.086 0.740 0.170 1.220 0.492
0.887 0.923 0.901 0.931 0.911 0.936

NU
r2
parabolic diffusion model
kd 0.39 0.349 0.345 0.685 0.627 0.667 1.26 0.966 1.139
MA
r2 0.925 0.996 0.946 0.955 0.993 0.969 0.961 0.992 0.976
modified Freundlich model
model
km 0.333 0.525 0.288 0.617 0.669 0.380 0.727 0.779 0.645
1.26 0.966
0.9821.139
D

r2 0.991 0.932 0.987 0.976 0.914 0.981 0.969 0.950

Elovich model
TE

ke 0.178 0.155 0.172 0.326 0.108 0.164 0.250 0.110 0.175

r2 0.991 0.982 0.932 0.987 0.976 0.914 0.981 0.969 0.950

P

I For 070 % release. II For 70100 % release. III For the whole release process (0100 %).
CE

0.987 0.976 0.914 0.981 0.969 0.950

AC

the interlayer space of LDH is the rate-controlling step in stage II, that takes a longer time

[32, 33]. This biphasic model prediction is consistent with the release process of drug from

the LDH layers. The release rate constants (kd) of the LNR is found to be less than that of

LCR, which in turn less as compared to LPR using parabolic diffusion model (Table 2).

Similar trends of release rate constants (kd) is also found for the calculation using modified

Freundlich and the Elovich models. All these observations indicate that the drugs intercalated

in LNR are more difficult to get release as compared to LCR, whereas the release kinetics

are more sluggish than LPR. Interactions between drug and LDHs might play a role for the

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fast or sluggish release. Moreover, pH of the media also plays an important role but we have

studied only in PBS system (pH~7.4). Similarly, drug release mechanisms are influenced by

the addition of anions in the release medium. Giacomelli et al. investigated the release

T
kinetics of ibuprofen from ibuprofen-LDH complex as a function of the anion type (chloride,

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acetate, and phosphate) and concentration of the anions [37]. The release of ibuprofen in the

R
medium containing phosphate anion was faster in comparison to chloride and acetate anions

SC
present in the medium. However, one can tune the drug release rate by altering the various

charge densities of anions for the preparation of LDHs.

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Interactions between drug and LDH: Spectroscopic evidence
MA
The nature of interactions between LDH and drug molecules can be understood from the X-

ray photoelectron spectroscopy (XPS). A significant shift in the XPS Al 2p peak has been
D

observed to higher binding energy side in LN-R as compared to pure LN (73.65 to 74.2 eV,
TE

i.e., ∆ BE = 0.55 eV) against the meager increment noticed in LP-R vis-à-vis LP (74.68 to

74.77 eV, i.e., ∆ BE = 0.09 eV) strongly suggest the greater interaction between drug and
P

LDH in LN-R as compared to LP-R (Figure 5a). The change in binding energy observed in
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LC-R with respect to pure LC is moderate (73.86 to 74.08 eV i.e., ∆ BE = 0.22 eV). Similar
AC

results are also observed for Mg 2p peak and the differences in binding energies are 0.01,

0.15 and 0.39 eV for LP-R, LC-R and LN-R systems, respectively, as compared to their

respective pure LDHs (Figure 5b). XPS results clearly indicate that drug molecules strongly

interact with LN while the extent of interactions goes down in LC and LP gradually [38‒41].

Figure 5c exhibits the comparative solid state UV-Vis spectra of pristine LDH, pure

drug and drug intercalated all three LDHs. Pure RH has the characteristic absorption band at

225, 266 and 364 nm while pure LN shows strong absorption peaks at 221 and 293 nm. On

the other hand, LN–R exhibits absorption peaks at 226, 295 and 392 nm indicating strong red

shift of the peaks especially for the 266 and 364 bands where Δλ is 28 nm against the

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relatively lower shift (Δλ ~ 19 nm) observed for LP-R. Further, the absorption band of RH

appears at 286 nm in LC-R with a red shifting of ~22 nm (Supplementary Figure S6). The

red shift of the absorption band is an indicator of interaction while the relatively greater

T
shifting in LN-R indicates stronger interactions as compared to LC-R and LP-R. Both the

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a. Al 2p c.

R
LP-R
74.77 LP
295 LN-R
226
392 RH

SC
266 LN
74.68 225
364
Intensity / a.u

74.08 LC-R
221
LC

73.86

NU
Absorbance / a.u
293
74.2 LN-R
73.65 LN
MA
68 72 76 80 84 LP-R
289 383
B.E / e.V. 226
266 RH
225 LP
364
b. Mg 2p
207
50.58 LP-R
D

LP
50.57
TE
Intensity / a.u

49.89 LC-R 284

49.74 LC
P

200 300 400 500

50.09 LN-R wavelength / nm
CE

49.70 LN

40 45 50 55 60
B.E / e.V.
AC

Figure 5: (a) Al 2p and (b) Mg 2p XPS spectra for pristine LDHs and drug intercalated

LDHs; and (c) Comparison of UV-Vis absoption spectra for pure drug, pristine NO3LDH

and PO4LDH system and drug intercalated LDHs.

XPS and UV-vis studies clearly indicate that the order of interactions between drug and

LDHs is LN-R > LC-R > LP-R and based on this order of interactions, one can explain well

the fast cumulative drug release using LP-R and sustained release associated with LN-R.

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Now, it is pertinent to understand the reason behind the variation of interactions in all three

cases where drug and basic constituents of LDHs are same with the changes in exchangeable

anions. The charge densities of the anions in this study alter from nitrate to carbonate to

T
phosphate and the electrostatic interaction between triply charged phosphate ion and rest of

IP
layers is maximum which causes the interaction between drug and layer in LP-R lowest. In

R
contrary, singly charged nitrate is attracted by the layer in a relatively meagre way and then

SC
drug molecules interact with the rest of the layer in greater way leading to the above order

(LN-R > LC-R > LP-R ) of interactions between drug and LDHs.

NU
MA
Thermal behavior: Interactions and stability
D

Figure 6a compares the DSC thermograms of pure drug and all three drug intercalated LDHs
TE

(LNR, LCR and LPR). Crystalline nature of the pure drug is evident from its well defined

endothermic peak (melting temperature, Tm) at 264 C. The melting temperature of the drug
P
CE

intercalated in different LDHs reduces as compared to pure drug and the Tm gradually

decreases in the order of LPR (232.3 C), LCR (211.7 C) and LNR (194.2 C) showing
AC

significant reduction of Tm in intercalated systems vis-à-vis pure drug (264 oC). Further, the

heat of fusion, ΔH associated with the melting of drug systematically reduces to 75 and 64

J.g-1 for LP-R and LN-R, respectively, from its original value of 85 J.g-1 for pure drug.

Reduction in melting temperature and decreased heat of fusion are considered to be strongly

interactive in nature in diluted systems like this case. However, drug intercalated LDHs are

strongly interactive systems and the extent of interaction decreases in the order of LN-R >

LC- R > LP-R as revealed from the relative reduction in Tm and ΔH. This is to mention that

ΔH value of LCR is relatively high (132 J.g-1) as compared to LN-R or LP-R and is

explained from the simultaneous degradation of carbonate species of LC in addition to the

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regular melting of drug as evident from the DSC thermogram of pure LC (Supplementary

Figure S7). On the other hand, considerable degradation is not observed either in LN or LP.

a. b.
LN
1.0

T
LN-R LN-R
LC-R RH
o LP-R 0.8

IP
194.2 C
64 Jg
-1 RH
Heat flow / a.u

0.6

R
o
211.7 -1C

Weight fraction
132 Jg 0.4

SC
232.3oC 0.2
-1
75 Jg
1.0 LP
LP-R
0.8 RH
264.3o C

NU
-1
85 Jg
0.6
50 100 150 200 250 300
T / oC 0.4
MA
0.2

100 200 300 400 500 600

T / oC
D
TE

Figure 6: (a) DSC thermograms for free drugs and drug intercalated LDHs. and (b) TGA
P

thermograms NO3LDH and for PO4LDH system.

CE

Hence, in addition to XPS and UV studies, DSC measurements also indicate the relative
AC

interactions between drug molecule and LDHs is in the order of LN-R > LC-R > LP-R.

Figure 6b shows the comparative weight loss behavior of LDH, drug intercalated

LDH and pure drug under heat treatment in nitrogen atmosphere. Three stages of weight loss

are clearly observed for pure LN and LP. The first step of weight loss occurs in the

temperature range of 100 C with  4% weight loss and is associated to the loss of adsorbed

water molecules along with hydrogen bonded water within the LDH layers [42]. The second

stage of weight loss occurs between 170 - 380 C and is attributed mainly to dehydroxylation

of LDH host layers with 16% weight loss. The third stage of weight loss takes place in the

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temperature region of 380 to 540 C which accounts for 12% weight loss resulting from the

elimination of interlayer NO3 ions. Thus, the final decomposition products are MgO and

Al2O3 at a temperature of 650 C with total weight loss  32% [43]. On the other hand, pure

T
drug follows single stage degradation in the temperature range of 280–450 C while drug

IP
intercalated LN-R exhibits 5wt.% loss at a temperature of 177 oC. On contrary, 5 wt.% loss

R
occurs in LP-R at ~114 oC indicating considerable lower thermal stability of LP-R as

SC
compared to LN-R. The greater thermal stability of LN-R is presumably due to the ordered

NU
stacking patterns where organic drug molecules are shielded by the inorganic layers while the

drug molecules are exposed in the disordered structure of LP-R causing lower degradation
MA
temperature. The details of degradation behaviour of all the systems have been reported in the

supplementary Figure S8.

D

Biocompatibility: Cell viability study

TE

So far, new biomaterials have been reported which deliver drug in a controlled way (both fast
P

and sustained release) by changing the exchangeable anion during the preparation of LDHs. It
CE

is necessary that the material (pure LDHs) should be biocompatible but drug intercalated
AC

LDHs should be able to kill the affected cells depending on the release kinetics of the drug.

The in vitro anti-cancer efficiencies of pure drug (RH) and RH intercalated LDHs (20 µg/ml)

have been evaluated using HeLa cell lines. The IC50 (half maximal inhibitory concentration)

is a measure of effectiveness of a particular drug. The IC50 of RH against HeLa cells was

measured to be 0.79 nM. The biocompatibility of pure LDHs has been tested with growing

number of cells with time implying good biocompatibility of the LDH nanoparticles

(supplementary Figure S9). Viability of the cells seeded on the tissue culture surface was

taken as a control and Figure 7a shows the time-dependent cell viability determined through

MTT assay. The cell viability is found to be decreased with time for pure drug and drug

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intercalated LDHs. Interestingly, the drug intercalated LDHs have higher cancer suppression

efficiency as compared to pure drug as observed from the lower cell viability of the drug

intercalated LDHs vis-à-vis pure drug which raises the lower bioavailability of pure drug and

T
instead indicate the efficacy of the LDH as better drug vehicle.

IP
a. b. Day 1 Day 2 Day 3

R
Control

RH

SC
100
  LN-R


LC-R
LP-R

% Cell viability

80

 

RH
NU
60  

40

LN-R
MA
20

0
day1 day2 day3
LC-R
D
TE

LP-R
P
CE
AC

Figure 7: (a) In vitro cytotoxicity of free drug and drug intercalated LDHs against HeLa cells

with different time intervals; the results presented are mean ± standard deviation (SD) values

obtained from three independent experiments, where P  0.05, P  0.01, P  0.001

(b) fluorescent images of AO/EB staining of control, free drug, and drug intercalated LDHs.

Cell membranes are negatively charged in nature and it controls the movements of substances

in and out of the cells. The approaches of negatively charged drug molecule towards cell are

repelled by the negatively charged cell membrane resulting poor bioavailibilty of pure drug

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as only a few drug molecules can be taken up by the cell [44, 45]. Therefore, pure RH

showed a low cytotoxicity against HeLa cells. On the other hand, drug intercalated LDH

molecules exhibit positive charge as evident from the zeta potential measurements (2123

T
mV) (Table 1). Thus, the drug intercalated LDH particles can approach easily and adhere to

IP
the negatively charged cell membrane via electrostatic interactions and, thereby, enhances the

R
bioavailability of drug towards the cell in a more facile way [46]. Individual LDH having

SC
crystallite dimension of 50–300 nm may be taken up by the cell via endocytosis while

slightly agglomerated LDH of sizes ≥500 nm is consumed via phagocytosis [47]. Thus, a

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larger number of drug molecules/drug intercalated LDH molecules can pass through the cell
MA
membrane and, thereby, showing a higher cytotoxicity against HeLa cells as compared to

pure drug arising from its poor bioavailability. Further, the cell viability decreases in the
D

order of LN-R > LC-R > LP-R following the relative rate of drug release from the drug
TE

intercalated LDHs. This is to mention that the amount of pure drug and the total amount of

drug intercalated inside LDHs were kept constant for comparison purpose and LP-R exhibits
P

the lowest cell viability due to its greater release for a particular time period (e.g. the cell
CE

viabilities after 24 hours of exposure are 58 (*P<0.05), 60 (*P<0.05), 80 (*P<0.05) and 95%
AC

(**P<0.05) for LPR, LCR, LNR and pure drug, respectively, for a drug concentration of

20 g/ml in all the cases). Further incubation for 48 and 72 hours showed a decreasing

tendency in cell viability keeping the order remains same.

The relative cell viability using different drug intercalated LDHs, pristine LDH and

pure drug is also confirmed through fluorescence imaging of the HeLa cell after staining with

acridine orange and ethidium bromide. The health of the growing cells was monitored as a

function of time (after 1, 2, and 3 days of incubation) for similar concentration of 20 g/ml.

Acridine orange and ehidium bromide distinguish the normal cells from apoptotic cells based

on the permeability of cell membrane. After binding with DNA, acridine orange exhibits

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green fluorescence while ethidium bromide exhibits red fluorescence. Usually, dead cells are

permeable to both the acridine orange and ethidium bromide and fluoresce red while the

living cells permeate only acridine orange and fluoresce in green [48‒50]. Figure 7b

T
indicates a large number of cell deaths in LP-R at different time (yellow to red colored cells

IP
depending on early or late apoptosis) while number density of healthy cells is more LN-R

R
where less amount of drug is being released for a particular time period which kills less

SC
number of cells. In the control system, in absence of any drug, the cell health is perfect as

indicated by the green cells only. The cell number density is highest in case pure drug, though

NU
some cell apoptosis is noticed indicating poor killing performance of pure drug as compared
MA
to drug intercalated LDHs. Thus, cell viability and morphology studies strongly suggest the

better efficacy of killing cancer cell by the drug intercalated LDH vis-à-vis pure drug. So, it is
D

expected to have the similar behavior in anti-tumor activity in vivo in animal studies.
P TE

In vivo antitumor activity and histological analysis: Effect of controlled release

CE

To evaluate the antitumor activity of drug intercalated LDHs in vivo, LPR, pure RH, pristine
AC

LDH and PBS have been injected intra-intraperitoneally (i.p) in Balb/c mice after creating

similar size (~50 mm3) of syngenic 4T1 tumor in them. Tumor growth and systemic toxicity

have been monitored routinely throughout the experiment. Figure 8a shows the original

tumor size (0 day) and the changes in their shape and size after 21 days of treatment with

various indicated agents. Excised tumor volume has been shown in Figure 8b indicating

diminished size in the mice treated with pure drug and drug intercalated LDHs against the

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a.
0 day 21 days b.

1cm

T
R IP
c. d.
2.5 30

SC
Relative tumor volume

LN-R LDH
Relative tumor volume

Control 1.00

Relative Body weight

RH 20

2.0 LP-R 10

0
0 5 10
t / day
15 20 0.96
1.5

NU

1.0 0.92 LN-R 
LP-R

MA
0.5 LN
0.88

Control
 RH 
0.0
0 5 10 15 20 0 5 10 15 20
t / day t / day
D
P TE

Figure 8: In vivo antitumor effect and systemic toxicity of pure RH and drug intercalated
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LDHs in comparison to control. (a) Photographs of the mice of different treatment groups at 0

day and at 21 days, (b) excised 4T1 solid tumors at the 22nd day, (c) relative changes in tumor
AC

volume of pure drug and drug intercalated LDHs with time, inset figure represents relative

changes in tumor volume of pristine LN and control (PBS) treated groups, and (d) changes in

body weight of the members of the different treatment groups with time, P  0.05, P 

0.01, P  0.001.

aggravated size when treated with pure LDH and control (treated with PBS). Pure RH and

RH intercalated LDHs result in a lowering of tumor volume as compared to control vehicles

(PBS or LDH NPs; inset figure of Fig. 8c) clearly indicates the efficacy of the drug released

in vivo for reducing the tumor size (Figure 8c). The downward tendency of the relative

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tumor volume of LN-R has started late as compared to LP-R presumably due to slow drug

release kinetics in LN-R system. However, LPR exhibits the best antitumor effect due to the

smallest tumor volume during the therapy which is attributed to the faster release of RH from

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the LPR nanohybrid.

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It is well known that the changes in body weight are the general indicators for the

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activity of the potentially toxic chemicals [51]. In order to investigate the adverse effects of

different therapy treatments, changes in body weights of the tumor-bearing mice have been

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evaluated after the administrations (Figure 8d). It was observed that, body weight of the mice
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administered with pure RH drug decreases steeply as compared to the group treated with drug

intercalated LDHs presumably due to the serious side effect of pure drug. Further, amongst
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the drug intercalated LDHs, body weight of mice treated with LNR is higher as compared to
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the LPR treated group, although the tumor sizes of LPR treated group are smaller than

those of LNR treated group. In other words, the body organs appear to be healthy in LN-R
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treated mice even though the healing rate is bit slow as against the faster healing rate in LP-R

treated mice which cause damage to other organs, as indicated from greater loss of body
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weights. This is worthy to mention that body weight loss in the mice treated with LP-R are

comparable to the control but there is significant size reduction by the use LP-R against the

greater dimension of tumor size in control over the experiment time. Histopathology is a

measure of cell health of other body organs when the animal is administered with drug for a

period of time. All mice were sacrificed on day 21 and the tumors and the main organs were

excised for volume measurement and histological analysis wherever applicable. Furthermore,

hematoxylin and eosin (H&E) stained sections of the main organs including liver, kidney and

spleen have thoroughly been investigated to confirm any potential toxicity of pure drug and

drug intercalated LDHs (Figure 9). Micrograph of liver tissues of mice administered with

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Control RH LP-R LN-R

a.
Liver

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b.
Kidney

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c.

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Spleen

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Figure 9: Histological analysis of (a) liver (b) kidney and (c) spleen of tumor bearing mice
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treated with control (saline), pure RH, LNR and LPR (all tissues: 200). The analysis
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revealed that the free drug resulting in bile ductular proliferation and congested portal vein in
portal triad (shown by red arrows). Mice that administered LPR shows dilated venous
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radical with mild congestion (red arrows). Mice treated with pure drug shows cloudy
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degeneration of the tubular epithelial cells in the kidney (shown by blue arrows). A slight
damage of tubular cells also observed for mice treated with LPR (blue arrows). However,
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other organs of mice that administered saline and drug intercalated LDH nanoparticles
exhibits no significant toxicity.

saline (control) shows normal architectural hepatocytes, which are large in size, hexagonal in

shape with more or less centrally located one nuclei and homogenous cytoplasm. In contrast,

the liver of mice treated with pure drug shows portal triad having bile ductular proliferation,

congested portal vein (indicated by arrows) and a partial effacement of normal architecture of

the liver. Mice treated with LPR shows dilated venous radical with mild congestion.

However, no obvious liver toxicity of the mice treated with LNR is observed. Further, a

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cloudy degeneration of the tubular epithelial cells in the kidney is observed for those mice

treated with pure drug. A slight damage of tubular cells is also observed for mice treated with

LPR. However, there is no obvious kidney toxicity of the mice treated with LNR. This is

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to mention that the mice administered with saline (control) also show normal morphology of

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kidney. Further, no significant histopathological abnormalities or lesions are observed in

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spleen. This is worth mentioning that pristine LDHs have no systemic effects as demonstrated

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by clinical chemistry and histopathology [10] and the side effect in liver and kidney is caused

by the released drug either from pure drug or drug intercalated LP-R. Now, it is clear that

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pure drug exposed to blood directly or drug intercalated LP (LP-R), which release the drug
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very fast, engenders strong side effect specially on liver and kidney although the rate of

tumor healing are moderately fast in both the cases. In contrast, LN-R has no side effect but
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heal the tumor in relatively slow pace proving a much better drug release vehicle for cancer
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treatment.

The pharmacokinetic behaviors of pure RH and one representative RH intercalated

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LDH (LN‒R) were assessed in male Sprague–Dawley rats. The mean elimination half-lives
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(t1/2) were found to be considerably higher for LN‒R treated rats (25 hrs) as compared to pure
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drug treated group (18 hrs). The mean plasma AUCo–72 in animals treated with pure RH and

LN‒R was 644±32 and 724±36 h ng ml‒1, respectively. The highest plasma concentrations

( max) for pure RH and LN‒R were found to be 45± 6 and 71 ± 11 ng/ml, respectively. The

time to achieve maximum plasma concentration (tmax) for pure RH and LN‒R were 3.0 and

3.7 h, respectively. The pharmacokinetic parameters are summarized in supplementary Table

S1. Thus, in vivo pharmacokinetic studies clearly indicate the better efficiency of drug

embedded LDH as compared to pure drug.

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Biochemical parameters of blood: Effect of released drug

Biochemical profile forms the data base for most investigations and may have specificity for

an organ or pathological process. Individual biochemical evaluation can be used for

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therapeutic drug testing. Biochemical parameters of the treated mice blood are presented in

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the supplementary Table S2. The lowest concentration of hemoglobin (11.4 mg/dL) has been

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detected in pure drug treated group. On the other hand, the highest hemoglobin concentration

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(12.6 mg/dL) and the best blood parameters have been detected in pristine LN and LNR

treated mice groups. The hemoglobin concentration in the LPR and control groups has

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nearly the similar values (12.3 mg/dL). In the present study, liver function was evaluated by
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measuring of liver enzymes AST and ALT levels. The ratio ALT/ AST is a more sensitive

indicator for hepatic injury [52]. The physiological values of AST and ALT activity in serum
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for mice are ~128 and ~32 U/L, respectively [53]. An increase in the activities of AST and
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ALT in plasma is attributed mainly to the leakage of these enzymes from the liver cytosol

into the blood stream which gives an indication of the hepatotoxic effect [54]. The decrease
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in the liver AST and ALT activities may be due to liver dysfunction and disturbance in the
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synthesis of these enzymes [54]. This ratio of ALT/ AST has increased (0.336) in the mice
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treated with pure drugs while this ratio has decreased for LPR, LNR and LN (~0.305) in

comparison to the control (322). These results indicate that pure RH induces hepatotoxicity

by repeated administration. Therefore, it is concluded that the hepatotoxic effect of the pure

drug can be reduced significantly by intercalating the drug into LDH layers. The renal

function has also been monitored by measuring the BUN levels. An elevation in this

biomarker level has been observed in the mice treated with pure drug (63±4.8 mg/dL)

indicating dysfunction of the kidney. However, BUN levels are nearly equal for LN, LNR,

LPR and control (56 – 60 mg/dL). There are no significant differences in plasma WBC and

PLT counts of all the investigated groups of mice. Hence, the biochemical parameters clearly

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suggest the toxicity appear in the blood of mice when treated with pure drug while the

toxicity greatly reduced after using drug intercalated inside LDHs confirming LDH as the

better drug delivery vehicle which releases drug in a sustained manner for the healing of

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tumor in animal system. This is worth mentioning that the damaged liver/kidney actually

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increase the level of ALT/BUN in blood sample of the mice treated with pure drug or LP-R

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while slow release of drug from LN-R does not exhibit any organ damage and thereby the

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normal biochemical parameters has clearly been observed.

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In summary, layered double hydroxides have been used as a novel drug delivery

vehicle which delivers drug in very wide range of its rate (fast or sustained) by altering the
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exchangeable interlayer anions. Slow or fast releasing systems using LDH as carrier have

applied to cancerous cell line for their demonstration in vitro to exhibit the relative release
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rate of drug against the poor bioavailability of pure drug. Animal studies indicate the relative
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healing rate depending on the drug release rate while the body weight index of the mice

clearly shows the damaged organs in case of fast releasing system including pure drug which
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have further been confirmed through histopathological and biochemical experiments. In gist,

an effective drug delivery vehicle has been discovered with steady rate of cancer / tumor
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healing without any side effect.

Conclusion

Three different Mg-Al based layered double hydroxides (LDHs) have been synthesized with

varying interlayer exchangeable anions ( and raloxifene drug was

intercalated within the LDH layers through ion exchange reactions. The LDH particle

dimension and surface morphology were determined through electron microscopy and atomic

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force microscopy suggesting stacking and agglomeration in presence of drug. Controlled drug

delivery has been achieved with very fast rate with phosphate bound LDH-drug (LP-R) while

sustained delivery is obtained using nitrate based LDH (LN-R). The differential drug delivery

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rate has been explained through interactions between drug molecule and LDHs through XPS

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and UV-vis studies and ordered-disordered structure of drug-LDH conjugates, obtained using

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XRD. Stronger interactions lead to sluggish delivery in LN-R against relatively weak

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interaction in LP-R, responsible for fast release. In vitro anticancer studies demonstrate that

drug intercalated LDHs efficiently inhibit the growth of HeLa cells as compared to pure drug.

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Amongst the drug intercalated LDHs, LPR exhibits better tumor suppression efficiency
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while body weight loss index suggests the damage of organs. In contrast, LN-R shows slight

slow healing of tumor while it exhibits minimum body weight loss indicating a better drug
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delivery vehicle. Histograms of different organs strongly suggest the damaged liver cell of
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mice treated with fast release vehicle (pure drug and LP-R) while no damage occurs in mice

liver cell treated with LN-R or slow release vehicle. Further, analyses of biochemical
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parameters also demonstrate that drug intercalated LDHs have less toxic effects than that of
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pure drug. Interestingly, side effects as measured from body weight measurements and
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histological assessment indicate that drug intercalated LDHs, especially slow releasing

system, are safer materials as compared to pure drug.

Acknowledgement

The authors acknowledge the receipt of funding for his fellowship from the University Grant

Commission, India. Authors acknowledge the Interdisciplinary School of Life Sciences

(ISLS) Banaras Hindu University, Varanasi for Confocal microscopy facility.

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Graphical Abstract

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Layered Double Hydroxides as Effective Carrier for Anticancer Drugs and

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Tailoring of Release Rate through Interlayer Anions

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Sudipta Senapati,1 Ravi Thakur,2 Shiv Prakash Verma,3 Durga Prasad Mishra,2 Parimal Das,3
T. Shripathi,4 Mohan Kumar,5 Dipak Rana,6 and Pralay Maiti*1
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Drug intercalated layered double hydroxide as the novel carrier for anti-cancer drug without
any side effect against poor bioavailability and remarkable side effect of pure drug.
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Weight loss
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Damaged liver
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Pure drug

O O
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No weight loss
O O

Normal liver
Intercalated drug

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