Analysis of montmorillonite clay as a vehicle in platinum anticancer drug delivery

Article abstract of DOI:10.1016/j.ica.2014.07.009

As a proof-of-concept study, the platinum anticancer complex [(1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)]chloride, PHENSS, was loaded into montmorillonite (MMT) clay to evaluate its utility as a drug delivery vehicle. Loading is complete within one hour and the total amount of PHENSS that can be loaded into the clay is based on the PHENSS solution concentration in which the MMT is suspended. From a PHENSS solution concentration of 30 mM, a maximum loading of 0.257 mmol per gram of MMT can be achieved. The pH of the solution also has an effect with a solution pH of 6 giving maximum loading of PHENSS. Metal complex release from the MMT was examined using the dialysis bag and dispersion methods. PHENSS is incompletely released from MMT; after 4 h just 47% has been released from the clay using the dialysis method and 30% using the dispersion method. The release is also very fast with a half-life of just 10-16 min. The MMT was shown to have a negative effect on the in vitro cytotoxicity of PHENSS in the human breast cancer cell lines MCF-7 and MDA-MB-231, presumably due to the incomplete release of the metal complex from the clay. Overall the results demonstrate that MMT is not a suitable slow release vehicle for PHENSS, although it may still be of use to other platinum complexes and drugs.

Full text of DOI:10.1016/j.ica.2014.07.009

Inorganica Chimica Acta 421 (2014) 513–518
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Analysis of montmorillonite clay as a vehicle in platinum anticancer
drug delivery
⇑
Michael G. Apps, Alaina J. Ammit, Alice Gu, Nial J. Wheate
Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 April 2014
Received in revised form 23 June 2014
Accepted 1 July 2014
Available online 17 July 2014
As a proof-of-concept study, the platinum anticancer complex [(1,10-phenanthroline)(1S,2S-diaminocy-
clohexane)platinum(II)]chloride, PHENSS, was loaded into montmorillonite (MMT) clay to evaluate its
utility as a drug delivery vehicle. Loading is complete within one hour and the total amount of PHENSS
that can be loaded into the clay is based on the PHENSS solution concentration in which the MMT is sus-
pended. From a PHENSS solution concentration of 30 mM, a maximum loading of 0.257 mmol per gram of
MMT can be achieved. The pH of the solution also has an effect with a solution pH of 6 giving maximum
loading of PHENSS. Metal complex release from the MMT was examined using the dialysis bag and dis-
persion methods. PHENSS is incompletely released from MMT; after 4 h just 47% has been released from
the clay using the dialysis method and 30% using the dispersion method. The release is also very fast with
a half-life of just 10–16 min. The MMT was shown to have a negative effect on the in vitro cytotoxicity of
PHENSS in the human breast cancer cell lines MCF-7 and MDA-MB-231, presumably due to the incom-
plete release of the metal complex from the clay. Overall the results demonstrate that MMT is not a suit-
able slow release vehicle for PHENSS, although it may still be of use to other platinum complexes and
drugs.
Keyword:
Clay
Montmorillonite
Phenanthroline
Platinum
Cancer
Cytotoxicity
Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction
bind to DNA via intercalation [8], their mechanism of action is not
known and is thought to potentially be derived from binding to
The many limitations of platinum-based chemotherapy drugs,
like their severe side effects [1] and the ability of cancers to
develop drug resistance [2], may mean that they are eventually
replaced in the clinic by more actively targeted and selective drugs
[3]. To provide a future for platinum drugs, it is therefore impor-
tant to develop ways to improve their effectiveness, safety and
patient tolerability.
Recently a family of new phenanthroline-based anticancer com-
plexes has been developed by Aldrich-Wright and group at the
University of Western Sydney [4,5], Totaling over 70 in number,
all contain a derivative of 1,10-phenanthroline and a chiral amine
ancillary ligand. The complexes containing the 1S,2S-diaminocy-
clohexane ligand are the most cytotoxic with IC50 values up to
100-fold lower than cisplatin [6]. The complex [(1,10-phenathro-
mitochondria and/or cell cycle proteins [9–12].
One of the major drawbacks to all platinum-based anticancer
drugs is their very short blood plasma residence time. The highest
concentration of cisplatin is achieved in a very short period of time
after administration (potentially as short as just 5 min) after which
the plasma concentration drops rapidly as it is cleared by the body
[13,14]. As anticancer drugs are most effective when they are able
to circulate in the blood stream for significantly longer periods
(hours to days) then slow release delivery methods are of particu-
lar interest. Examples of slow release platinum formulations
include: hydrogels [15], micelles and polymers [16], nanoparticles
[17], carbon nanotubes [18] and macrocycles [13]. Whilst effective,
these new types of delivery vehicles are not without their own
pharmaceutical and regulatory drawbacks and the use of natural
materials which are already generally regarded as safe, instead of
new synthetic vehicles, provides an advantage in developing new
delivery systems [19].
line)(1S,2S-diaminocyclohexane)platinum(II)]chloride,
abbrevi-
ated as PHENSS (Fig. 1), is one of the most potent complexes in
this class and has displayed in vivo cytotoxicity using a PC3 human
prostate xenograft [7]. Whilst this class of metal complex is able to
Clay has been used in medicine for thousands of years either to
treat poisoning or infection and was administered orally or topi-
cally [20]. Currently, clays and other minerals are used in pharma-
ceutical formulations as lubricants, desiccants, disintegrants,
diluents and binders, pigments and opacifiers, emulsifying, thick-
⇑
Corresponding author. Fax: +61 2 9351 4391.
E-mail address: nial.wheate@sydney.edu.au (N.J. Wheate).
http://dx.doi.org/10.1016/j.ica.2014.07.009
0020-1693/Ó 2014 Elsevier B.V. All rights reserved.

514
M.G. Apps et al. / Inorganica Chimica Acta 421 (2014) 513–518
O
OH
Cl
Cl
O-
-O
Cl
Cl
Cl
Cl
Pt
OH
O
Pt
K2
NH2
H2N
+
+H3N
NH3
N
Cl
Cl
Pt
N
N
N
N
NH2
H2N
2Cl
Pt
NH2
H2N
50-80 o
C
Fig. 1. The modified synthetic method for PHENSS using the D-tartrate salt of 1S,2S-diaminocyclohexane as the starting reagent. The ligand is converted to the free base by
triethylamine and then reacted with K2[PtCl4] to produce [PtCl2(S,S-dach)]. PHENSS is synthesized by the reaction of 1,10-phenanthroline with [PtCl2(S,S-dach)] in hot water.
ening and anticaking agents, flavor correctors and isotonic agents
[21]. There has also been growing interest in the use of clays in
drug delivery as slow release vehicles [22–28]. Montmorillonite
(MMT) is a common clay named for the area in France where this
smectite-type mineral is found. The clay is composed of Al2O3-
Á4SiO4ÁH2OÁxH2O which forms layers with negatively charged sur-
face groups [29] and drugs can be intercalated between these
anionic clay layers [23,30]. As a flat, dicationic drug, PHENSS is a
perfect candidate for drug delivery using MMT. PHENSS can poten-
tially intercalate between the layers of the clay where binding is
stabilized by electrostatic interactions.
In this paper we report the first investigation of a naturally
occurring clay as a slow release delivery vehicle for a platinum-
based anticancer complex. Factors affecting the loading capacity
of PHENSS into MMT including time, solution pH and metal com-
plex concentration were investigated using UV–Vis spectropho-
tometry. The slow release properties of the PHENSS-MMT
complex were examined using two methods: the dialysis bag and
dispersion methods. Finally, the effect of the clay on the in vitro
cytotoxicity of PHENSS was examined using the human breast can-
cer cell lines MCF-7 and MDA-MB-231.
purchased from ATCC. All water was obtained from an SG Ultra
clear water system.
2.2. Synthesis of PHENSS
The metal complex was made using a modification to a method
previously reported [6]. Equimolar amounts of K2[PtCl4] and the D-
tartrate salt of 1S,2S-diaminocyclohexane (S,S-dach) were com-
bined in water (10 mL per 100 mg of K2[PtCl4]). Four mole equiva-
lents of triethylamine were added and the solution stirred at room
temperature for 24 h to give [PtCl2(S,S-dach)] as a yellow coloured
precipitate. The precipitate was collected by vacuum filtration,
then washed with water, methanol and allowed to air dry. The
[PtCl2(S,S-dach)] (400 mg) was stirred with one mole equivalent
of 1,10-phenanthroline in 800 mL of water at 80 °C until all solid
had dissolved. The solution was then allowed to cool to ꢀ50 °C
and stirred overnight at this temperature. For purification, the
solution was rotary evaporated to near dryness and filtered to
remove platinum metal. The remaining solution was eluted on a
C-18 reverse phase Sep-Pak, the eluant collected and rotary evap-
orated to dryness to produce a yellow coloured powder. 1H NMR
(400 MHz, D2O, ppm): 8.64, d, 2H; 8.49, d, 2H; 7.78, d, 2H; 7.76,
d, 2H; 7.59, s, 2H; 2.66, dd, 2H; 2.13, d, 2H; 1.56, d, 2H; 1.43, q,
2H; 1.16, m, 2H. 13C NMR (400 MHz, D2O): 150.9, 145.8, 140.5,
130.0, 127.5, 126.4, 61.7, 32.0, 23.8 ppm. Electrospray ionization
mass spectrometry, Expected [M] = 489.15 m/z; Found, [MÀH+]+
2. Experiment
2.1. Materials
488.08 m/z. UV–Vis (H2O, 100
0.773;
= 30920 MÀ1 cmÀ1
lM): 227 nm, 0.884; 277 nm,
K2[PtCl4] was purchased from Precious Metals Online. Cisplatin,
MTT, montmorillonite, 1S,2S-diaminocyclohexane D-tartrate, 1,10-
phenanthroline and triethylamine were purchased from Sigma
Aldrich. C-18 reverse phase Sep-Paks (500 mg; 3,000 MWCO) were
purchased from Waters. Simulated gastric fluid (SGF) was made
according to the British Pharmacopeia 2013: 2.002 g NaCl,
3.228 g pepsin, 7 mL of 37% w/v HCl and water to a total volume
of 1 L. The breast cancer cell lines MCF-7 and MDA-MB-231 were
e
.
2.3. Clay purification
MMT (10 g) was prepared by suspending the clay in 100 mL of
water and stirring overnight. The clay was then collected by vac-
uum filtration and washed with water before it was resuspended

M.G. Apps et al. / Inorganica Chimica Acta 421 (2014) 513–518
515
in a 200 mL 0.5 M NaCl solution and stirred overnight. Finally, the
3. Results and discussion
clay was filtered, washed with water to remove excess salt, air
dried for 3 h and oven dried (80 °C) for a further 2 h.
3.1. Metal complex synthesis
The synthetic method for PHENSS, and its derivatives, requires
the use of enantiometrically pure 1S,2S-diaminocyclohexane (S,S-
dach). In this form the ligand costs AUD$190 per gram (based on
November 2013 prices). We have now modified this method to
use the D-tartrate salt of the 1S,2S-diaminocyclohexane ligand
which costs just AUD$20 per gram. As a tartrate salt the ligand is
a dication, but can be converted to the free base by the addition
of four equivalents of triethylamine (Fig. 1). At the same time, K2-
[PtCl4] is also added to the S,S-dach solution; as the free base of
S,S-dach is generated it reacts with the platinum to form [PtCl2(-
S,S-dach)]. As a single step, one pot reaction the intermediate
[PtCl2(S,S-dach)] is produced in almost stoichiometric yield and
at significantly lower cost compared with the old method. The
[PtCl2(S,S-dach)] crystallizes from solution and is collected by vac-
uum filtration at which point the triethylammonium chloride/tar-
trate and KCl are washed from the product. The [PtCl2(S,S-dach)] is
then reacted with 1,10-phenanthroline to yield PHENSS which is
purified by elution through a C-18 reverse phase column and
rotary evaporated to dryness. The characterisation data for the
metal complex (1H and 13C NMR, ESI-MS and UV–Vis) are consis-
tent with the proposed compound and identical to the values pre-
viously reported [6].
2.4. UV–Visible spectrophotometry
UV–Vis was performed on a Shimadzu UV-1800 with paired
1 cm quartz cells. PHENSS calibration graphs were generated using
5 to 40 lM samples with maximum absorption measured at
277 nm. Sample analysis for intercalation kinetics, concentration
and pH loading and drug release experiments were performed by
centrifuging the PHENSS loaded MMT suspensions at 13000 rpm
for 3 min. The supernatant solutions were removed and the con-
centration of free PHENSS used to determine the amount of
PHENSS loaded/remaining in the clay.
2.5. Intercalation kinetics
Loading of PHENSS into MMT was studied over time periods
from 1 to 50 h. PHENSS (1 mL, 8.5 mM) was stirred with MMT
(25 mg) in eppendorf tubes in water. At time intervals, the solu-
tions from individual eppendorf tubes were centrifuged and the
PHENSS loading was determined by UV–Vis spectrophotometry.
2.6. Effect of pH of PHENSS loading
Eight samples of MMT (25 mg) were suspended in 1 mL of
PHENSS solution (8.5 mM) with pH values between 3 and 10. The
solutions were stirred for several hours before PHENSS loading
was examined by UV–Vis spectrophotometry.
3.2. PHENSS loading as a function of time, concentration and solution
pH
Prior to the loading of PHENSS, the MMT clay was washed with
water and then washed with 0.5 M NaCl to ensure all cations
within the structure were Na+. Excess NaCl was removed by wash-
ing with more water before the clay was air and oven dried. A sin-
gle batch of MMT was produced in this way and used for all
subsequent experiments with PHENSS.
The loading of PHENSS into MMT was measured as a function of
time. The clay was suspended in a solution of water and then
PHENSS predissolved in water was added with continuous stirring.
At intervals, an aliquot of the PHENSS loaded MMT suspension was
removed and centrifuged. The amount of PHENSS loaded into the
clay was determined by measuring the amount of unbound
PHENSS. The results indicate that absorption of the PHENSS onto
the clay is rapid and complete within 1 h, which is consistent with
the results obtained for the drug timolol and MMT [30].
2.7. Effect of PHENSS concentration on loading
PHENSS was dissolved in 1 mL of water at various concentra-
tions (3, 5, 10, 19, 30 mM) with 25 mg of MMT in eppendorf tubes
in triplicate and stirred overnight before PHENSS loading was
examined by UV–Vis spectrophotometry.
2.8. PHENSS release study – dispersion method
The PHENSS loaded MMT (25 mg) was stirred in 60 mL of SGF
solution at 37.5 °C. Aliquots (1 mL) were taken at time intervals
of between 20 min and 5 h and PHENSS release was examined by
UV–Vis spectrophotometry.
As PHENSS loading is a dynamic and reversible process the
amount of metal complex loaded into the clay will, in part, be
determined by the equilibrium between the free and bound spe-
cies. The use of higher PHENSS solution concentrations should
yield higher loading into the clay. Total loading in the clay is either
limited by the maximum concentration of PHENSS in solution or
the number of potential binding sites in the clay.
The loading of PHENSS was therefore examined by suspending
MMT in solutions of PHENSS at metal complex concentrations
between 3 and 30 mM. The results indicate a direct relationship
between PHENSS concentration and loading of the metal complex
into MMT. The loading of PHENSS increases with increasing metal
complex concentration to the limit of PHENSS solubility: 30 mM
(Fig. 2). At this highest concentration the loading of PHENSS is
0.257 mmol per gram of MMT. This result is significantly lower
than the loading capacity for other drugs, like timolol maleate,
which has almost double the loading capacity at 0.499 mmol per
gram of MMT [30].
2.9. PHENSS release – dialysis bag method
Dialysis sacks were allowed to equilibrate in SGF before PHENSS
loaded MMT (25 mg) suspended in 1 mL of SGF was added inside
the dialysis sacks. Each dialysis sack was then placed in a reservoir
of SGF (100 mL) which was maintained at 37 °C with stirring. At
time intervals, 3 mL aliquots were taken from the SGF reservoir
and
PHENSS
concentration
determined
by
UV–Vis
spectrophotometry.
2.10. Cytotoxicity
Cell respiration, an indicator of cell viability, was determined by
the mitochondrial-dependent reduction of 3-(3,4-dimethylthiazol-
2yl)-,5-diphenyl tetrazolium bromide (MTT) to formazan. MTT
assays were performed according to Guh et al. [31]. The cytotoxic-
ities of MMT, PHENSS and PHENSS loaded MMT were compared
with cisplatin in the MCF-7 and MDA-MB-231 human breast can-
cer cell lines using 72 h drug exposure times.
Finally, the loading of PHENSS into MMT was examined as a
function of solution pH. Potentially, the pH of the solution has an

516
M.G. Apps et al. / Inorganica Chimica Acta 421 (2014) 513–518
Fig. 2. The loading capacity of PHENSS in MMT (moles metal complex per gram of clay) as a function of PHENSS concentration. The results demonstrate increasing loading as
concentration increases up to the limit of PHENSS solubility (30 mM).
ability to affect the metal complex loading due to competitive cat-
ion binding to the clay’s anionic groups at low pH by H+ and at high
pH by Na+ or K+ depending on the base used to prepare the solu-
tion. As such, the loading of PHENSS was examined between pH
3 and 10. The results indicate that within the error of the experi-
ment there is no significant difference in loading between pHs 4
to 10. Only at a very low pH of 3 has the loading capacity of the clay
dropped; at this pH the loading is only 65% compared with the
loading at pH 6.
Overall the results indicate that PHENSS can be loaded into
MMT, but the extent of loading is dependent on both the PHENSS
concentration and the pH of the solution in which the clay is
dispersed.
via burst release. As such, we hypothesize that despite the appar-
ent slow release shape of the graphs, the PHENSS release profile
is rather a measurement of the diffusion of PHENSS out of the dial-
ysis bag and not diffusion out of the clay.
The release of PHENSS out of the clay, as examined using the
dialysis bag method, is also incomplete. The maximum percentage
release of PHENSSS is 47% which is achieved after four hours. This
result is consistent with the pH loading experiments completed in
Section 3.2. At a pH of 3, the PHENSS loading was just 65% of the
maximum loading observed at pH 6. Given that the SGF solution
used in the release studies was pH 1.2, then the incomplete release
is very similar to the results observed for the loading experiment.
Release is not based on kinetics but on competitive binding with
other solution cations; thus, burst release is observed.
In measuring PHENSS release using the dispersion method, the
PHENSS-MMT complex was suspended in SGF with stirring. At reg-
ular time intervals, an aliquot of the solution was withdraw and
centrifuged to determine the concentration of released metal com-
plex. The results indicate PHENSS release from MMT using the dis-
persion method has similar release kinetics to the dialysis bag
method. The PHENSS is burst released with a half-life of less than
10 min. The release of the metal complex is also incomplete by this
method with only 30% of the PHENSS in solution after more than
four hours.
Overall, the results indicate that PHENSS is rapidly released
from the clay, most likely under burst kinetics. It is unclear why
this is the case when other drugs demonstrate slow release. The
results may indicate that the PHENSS is not intercalated between
the clay layers but instead may only be absorbed onto the surface
of the clay. If this is the case, then there is no barrier to metal com-
plex release and the PHENSS dissolution into the SGF is dictated
simply by its solubility, diffusion kinetics and competitive binding
by solution cations.
3.3. Metal complex release rate
There are various ways in which drug release from a delivery
vehicle can be measured and there is considerable debate as to
which measures are the most reliable and representative of biolog-
ical systems [32,33]. In this work we examined the rate of release
of PHENSS from MMT using two different methods: the dialysis
bag and the dispersion methods. Both experiments were com-
pleted using simulated gastric fluid (SGF) as the dissolution media
as the particulate nature of clay makes it unsuitable as a delivery
vehicle in intravenous injections. In traditional medical use, clay
is orally administered, and as an excipient has been found suitable
for the production of oral tablets [29].
In examining metal complex release using the dialysis bag
method samples of PHENSS loaded MMT were placed into dialysis
sacks as a suspension in SGF. The sacks were then further placed in
reservoirs of SGF and the PHENSS concentration in the reservoir
fluid measured at intervals using UV–Vis spectrophotometry
(Fig. 3). From the data there is a very small, but statistically signif-
icant (P < 0.05), difference in the release half-lifes of free PHENSS
(no clay; t1/2 = 11.9 0.78 min) compared with PHENSS loaded
MMT (t1/2 = 16.3 0.80 min). However, these half-lifes are very
much shorter compared with literature release rates for other
drugs using the same clay; timolol release has a half-life of approx-
imately 1.5 h and irinotecan has a half-life of approximately
1.25 h) [23,30]. Given that PHENSS and PHENSS loaded MMT have
near identical half-lifes it demonstrates that the PHENSS is most
likely not slowly released from the clay but instead enters solution
3.4. In vitro cytotoxicity
Whilst platinum drugs are not used in the clinic for the treat-
ment of breast cancers, PHENSS has shown high cytotoxicity in
breast cancer cells lines. As such, the drug can potentially be devel-
oped as an alternative to current breast cancer chemotherapy
drugs. The effect of MMT on the in vitro cytotoxicity of PHENSS
was examined using the MCF-7 and MDA-MB-231 human breast

M.G. Apps et al. / Inorganica Chimica Acta 421 (2014) 513–518
517
Fig. 3. The release profiles of free PHENSS (N, no clay) and PHENSS loaded MMT (ꢀ), showing the similar release half-life profiles for both formulations and the incomplete
release of metal complex from the clay, even after four hours. The apparent slow release profiles are a function of the slow diffusion of PHENSS out of the dialysis sacks and
not slow release of the metal complex from the clay.
Even though PHENSS could be loaded into the clay in a solution
concentration and pH dependent manner, the metal complex does
not appear to intercalate between the clay layers and is burst
released upon suspension in simulated gastric fluid. The results
indicate that MMT is unsuitable as a slow release vehicle for
PHENSS and most likely for other 1,10-phenathroline based drugs;
however, MMT clay may still have application for other platinum
based drugs. For instance, MMT may be useful for the delivery of
the mono-aquated form of cisplatin, which is cationic. It may also
be useful for highly cationic drugs, like BBR3464 [36], where bind-
ing into MMT may be facilitated by the multiple electrostatic
bonds that can be formed between the drug and the clay. Alterna-
tively, other clays may provide better drug absorption between
their layers. Examples of other clays which can be examined
include: laponite, beidellite, nontronite, saponite, kaolinite, halloy-
site and hisingerite [25].
Table 1
The effect of MMT on the in vitro cytotoxicity of PHENSS in the human breast cancer
cell lines MCF-7 and MDA-MB-231. The values given are for inhibition concentration
50 (IC50) which is defined as the concentration of drug or complex required to inhibit
cell growth by 50% compared with untreated cells.
Drug/complex
IC50
(lM)
MCF-7
MDA-MB-231
Cisplatina
MMT
PHENSS
13.3 1.32
>50
0.31 0.12
1.73 0.22
55 4.8
11.3 0.65
0.64 0.23
0.90 0.002
PHENSS-MMT
a
Data taken from Refs. [34,35].
cancer cell lines (Table 1). Free PHENSS is 4- to 85-fold more cyto-
toxic to these cell lines than cisplatin, which is consistent with the
high PHENSS activity observed in other animal and human cancer
cell lines, such as murine leukaemia L1210 (2.5-fold more cyto-
toxic) and human prostate PC3 (10-fold more cytotoxic) [7].
The results indicate that MMT has a measurable negative effect
on the cytotoxicity of PHENSS. In the MCF-7 cell line the PHENSS
cytotoxicity drops 5.5-fold and in the MCA-MB-231 cell line the
cytotoxicity drops 1.4-fold. Given that PHENSS is burst released
from the clay, the drop in cytotoxicity is not likely due to slower
release and uptake of the metal complex, but more likely it can
be explained in part by the incomplete release of PHENSS from
the clay. With just 30–47% PHENSS being released from the clay
this would be expected to affect significantly the metal complex’s
measured cytotoxicity.
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In this work a proof-of-concept study was undertaken to deter-
mine the suitability of montmorillonite as a slow release delivery
vehicle for the phenanthroline-based platinum complex PHENSS.

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