Hydroxyapatite nanocrystals as a smart, pH sensitive, delivery system for kiteplatin

Article abstract of DOI:10.1039/c6dt01976e

Hydroxyapatite (HA) nanocrystals are important inorganic constituents of biological hard tissues in vertebrates and have been proposed as a bone substitute or a coating material for prostheses in biomedicine. Hydroxyapatite is also amenable for its capacity to bind to a great variety of biomolecules and therapeutic agents. As drug carriers, apatite nanoparticles also have the advantage of pH dependent solubility and low toxicity. Thus HA nanoparticles are negligibly soluble at physiological pH but their dissolution is accelerated at lower pH such as that typically found in the vicinity of tumors. In the present study we have investigated the adsorption on and the release from biomimetic HA nanoparticles of two platinum derivatives of cis-1,4-diaminocyclohexane ([PtX₂(cis-1,4-DACH)], X₂ = Cl₂ (1) and 1,1-cyclobutanedicarboxylate (CBDCA, 2)). The first of the two compounds proved to be active against colon cancer cells also resistant to oxaliplatin. The release has been investigated as a function of pH to mimic the different physiological environments of healthy tissues and tumors, and the in vitro cytotoxicity of the releasates from the HA matrices has been assessed against various human cancer cell lines. The results fully confirmed the potential of 1-loaded HA nanoparticles as bone-specific drug delivery devices.

Full text of DOI:10.1039/c6dt01976e

Dalton
Transactions
View Article Online
View Journal
PAPER
Hydroxyapatite nanocrystals as a smart,
pH sensitive, delivery system for kiteplatin
Cite this: DOI: 10.1039/c6dt01976e
a
a
b
c
Marco Lelli, Norberto Roveri, Cristina Marzano, James D. Hoeschele,
d
d
b
d
Alessandra Curci, Nicola Margiotta,* Valentina Gandin* and Giovanni Natile
Hydroxyapatite (HA) nanocrystals are important inorganic constituents of biological hard tissues in ver-
tebrates and have been proposed as a bone substitute or a coating material for prostheses in biomedicine.
Hydroxyapatite is also amenable for its capacity to bind to a great variety of biomolecules and therapeutic
agents. As drug carriers, apatite nanoparticles also have the advantage of pH dependent solubility and low
toxicity. Thus HA nanoparticles are negligibly soluble at physiological pH but their dissolution is acceler-
ated at lower pH such as that typically found in the vicinity of tumors. In the present study we have investi-
gated the adsorption on and the release from biomimetic HA nanoparticles of two platinum derivatives
2 2 2
of cis-1,4-diaminocyclohexane ([PtX (cis-1,4-DACH)], X = Cl (1) and 1,1-cyclobutanedicarboxylate
(CBDCA, 2)). The first of the two compounds proved to be active against colon cancer cells also resistant
Received 17th May 2016,
Accepted 28th June 2016
to oxaliplatin. The release has been investigated as a function of pH to mimic the different physiological
environments of healthy tissues and tumors, and the in vitro cytotoxicity of the releasates from the HA
matrices has been assessed against various human cancer cell lines. The results fully confirmed the
potential of 1-loaded HA nanoparticles as bone-specific drug delivery devices.
DOI: 10.1039/c6dt01976e
www.rsc.org/dalton
Calcium phosphate is the most important inorganic con-
stituent of biological hard tissues in vertebrates. In the form
of carbonate-containing hydroxyapatite (HA), calcium phos-
Introduction
5,6
1
,2
Nanoparticles are promising tools to target tumor cells.
Nanoparticulate delivery systems offer the opportunity to phate is present in bone, teeth and cartilaginous tissues.
prolong circulation in the bloodstream and hence increase the Bones are the most typical calcified tissues in organisms and
possibility of accumulation at the tumor sites by taking advan- their shape and size are optimized to achieve best protection
tage of the Enhanced Permeability and Retention (EPR) effect, and mechanical support for the body. The size of bone crys-
to specifically target cells or tissues of interest via conjugation tals, despite variations due to different methods of preparation
with ligands specific for cell surface biomolecules, to respond and analysis of the sample, is generally in the range of nano-
to local stimuli in vivo (such as changes in pH and temperature), meters (30–50 nm length, 15–30 nm width, and 2–10 nm thick-
7
,8
and to overcome the cell membrane barrier and avoid degra- ness). It has been recently demonstrated that the nanometer
3
dation by lysosomes. Ideally, a delivery system consists of: (i) size of mineral particles is used in nature to ensure optimum
9
,10
non-toxic starting materials and degradation products, (ii) strength and maximum tolerance of flaws.
improved uptake achieved by a size in the range of 10–100 nm Based on the similarities between synthetic HA and in-
and a large surface area, (iii) colloidal stability of the delivery organic crystals present in natural bones, synthetic HA has
system (to avoid agglomeration during circulation in vivo), (iv) been proposed as a bone substitute and/or replacement in bio-
long clearance times (to permit adequate time for the delivery medicine either in the form of an implant material or as an
system to reach target cells), (v) controlled and sustained release agent for surface modification of prostheses to enhance the
6
of the active agent, and (vi) targetability of the delivery system calcification process of teeth and bones. Research effort has
4
(
to enable delivery of particles to selected cells).
also been devoted to learn more about the chemical process of
1
1
formation of this mineral in vivo. Several methodologies have
been proposed for the synthesis of nanocrystalline hydroxy-
apatite, the most used are sol–gel synthesis, co-precipitation,
a
b
Bioecoactive S.r.l, via San Donato n.5, 40050 Granarolo, Italy
Dipartimento di Scienze del Farmaco, Università di Padova, Via Marzolo 5,
mechanochemical synthesis, hydrothermal reaction, and
3
c
5131 Padova, Italy. E-mail: valentina.gandina@unipd.it
12–15
vibro-milling from natural bones.
Dimensions, mor-
Department of Chemistry, Eastern Michigan University, 48197 Ypsilanti, MI, USA
d
phology, crystallinity and surface properties are the major
physico-chemical properties which need to be tailored for opti-
Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Via E. Orabona
4
, 70125 Bari, Italy. E-mail: nicola.margiotta@uniba.it
This journal is © The Royal Society of Chemistry 2016
Dalton Trans.

View Article Online
Paper
Dalton Transactions
mizing the specific biomedical application of synthetic HA named kiteplatin (1 in Chart 1), that contains an isomeric
1
6
nanocrystals.
form of the oxaliplatin diamine ligand 1R,2R-DACH. In par-
Hydroxyapatite is also amenable for its capability to bind a ticular, interest in this compound stems from its activity
1
7–20
wide variety of biological molecules
and therapeutic against cisplatin (ovarian cancer cells 2008/C13*) and oxalipla-
2
1,22
agents, thus matching a major challenge for advanced tin sensitive and resistant cell lines (colon cancer cells LoVo/
bone substitute biomaterials, that is their functionalization LoVo-OXP). Tested in vivo in a syngeneic murine solid tumor,
3
5
2
3–31
with bioactive molecules.
Moreover, the adsorption and the Lewis Lung Carcinoma (LLC), kiteplatin showed an anti-
release properties of the loaded bioactive molecules can be tumor effect similar to that of cisplatin and, most importantly,
optimized by acting on chemical and structural characteristics was less toxic than the reference metallo-drug.
2
8
of the HA substrate.
In the present study our aim was to assess if the above
As drug carriers, apatite nanoparticles also have the advan- described characteristics of HA nanoparticles could be
tage of lower toxicity than silica nanoparticles, quantum dots, exploited to achieve a specific delivery and accumulation of
carbon nanotubes, or magnetic particles and a greater kiteplatin (or its CBDCA derivative) at the acidic tumor sites
stability/robustness than organic liposomes. For instance, con- (where HA nanoparticles dissolve).
trary to liposomes and other micelle-based carriers, which are
subjected to dissipation below specific critical concentrations tion on and the release from biomimetic HA nanocrystals of
a clear obstacle to their intravenous administration), HA nano- kiteplatin and of its 1,1-cyclobutanedicarboxylate derivative
particles are negligibly soluble in blood and can be used as [Pt(CBDCA)(cis-1,4-DACH)] (2 in Chart 1). The release has been
Therefore, we have preliminarily investigated the adsorp-
(
3
2
reliable drug delivery systems.
investigated as a function of pH to mimic the different physio-
Interestingly, the dissolution of calcium phosphates is logical environments of healthy tissues (including blood) and
accelerated by low pH, such as that typically found in the vicin- tumors. The in vitro cytotoxicity of the releasates from the in-
ity of tumors, providing an advantage in the delivery of drugs organic matrices has also been investigated.
into malignant zones. They are also able to permeate the cell
membrane and dissolve affording their ionic constituents
2
+
3−
(
Ca and PO
4
), which are already present in a relatively high
Materials and methods
concentration (1–5 mM) in cells and in the bloodstream. Their
dissolution prevents the undesirable accumulation of nano-
particles in cells and tissues, a flaw often encountered with
other inorganic and metallic nanoparticle systems. Finally, HA
nanoparticles have been recently shown to inhibit colon
cancer HCT116 cells by inducing large production of inflam-
matory cytokines leading to decreased cell proliferation. This
effect is correlated with the low crystallinity of the HA powders
that results in higher dissolution in the physiological medium
Commercial reagent grade chemicals and solvents were used
as received without further purification. cis-1,4-Diaminocyclo-
hexane (cis-1,4-DACH) was purchased from TCI-Europe.
1,1-Cyclobutanedicarboxylic acid (CBDCA) was purchased from
Sigma (Milan). cis-[PtCl (DMSO) ] (DMSO = dimethylsulfoxide)
2
2
2 4
was prepared from an aqueous solution of K PtCl and
3
6
DMSO. Electrospray ionisation mass spectrometry (ESI-MS)
was performed with an electrospray interface and an ion trap
mass spectrometer (1100 Series LC/MSD Trap system Agilent,
Palo Alto, CA).
3
3
and increased calcium uptake in the tumor cells.
Platinum(II) based drugs, such as cisplatin, carboplatin and
oxaliplatin (Chart 1) are the first line of treatment for many
types of malignancies, including testicular, ovarian and lung
Elemental analyses were carried out with a Eurovector EA
1
3
000 CHN instrument. H-NMR spectra were recorded on a
1
3
4
Bruker Avance DPX 300 MHz. H chemical shifts were refer-
cancers. A valuable new candidate platinum anticancer drug
is [PtCl (cis-1,4-DACH)] (DACH = diaminocyclohexane), also
enced using the internal residual peak of the solvent (DMSO-
2
1
d
6
: 2.50 ppm, D
2
O = 4.80 ppm for H).
Synthesis of [Pt(CBDCA)(cis-1,4-DACH)] (compound 2)
Pt(CBDCA)(cis-1,4-DACH)] was prepared following the pro-
cedure already reported in the literature with slight modifi-
[
3
7
cations. CBDCA (944.13 mg, 6.55 mmol) was dissolved in
water (270 mL) and neutralized with Ag O (1.52 g, 6.55 mmol).
2
After stirring in the dark at room temperature for 20 min, the
dicarboxylate solution was treated with cis-[PtCl
2 2
(DMSO) ]
(
2.77 g, 6.55 mmol) and the resulting suspension was kept
under stirring for 10 min at 70 °C and then for 24 h at room
temperature in the dark. The suspension was filtered (to separ-
ate the AgCl precipitate) and the resulting solution was con-
centrated to 20 mL under reduced pressure. The solution was
then kept at 70 °C for 10 min and then allowed to cool down
to room temperature and, successively, kept at 4 °C overnight,
Chart 1 Structures of platinum drugs currently approved by FDA (cis-
platin, carboplatin, and oxaliplatin), kiteplatin (1), and [Pt(CBDCA)(cis-
1
,4-DACH)] (2).
Dalton Trans.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Dalton Transactions
Paper
meanwhile a white crystalline solid of cis-[Pt(CBDCA)(DMSO)
2
]
water and then a few droplets of the slurry were deposited on
precipitated from the solution. The solid was isolated by fil- holey-carbon foils supported on conventional copper microgrids.
tration of the mother liquor, washed with cold water, and Scanning electron microscopy (SEM) investigations were
dried in vacuo (2.91 g, 5.89 mmol, yield = 90%). Anal. Calcu- carried out using a ZEISS EVO MA 10 instrument with an EDX
lated for [Pt(CBDCA)(DMSO) ] (C H O S Pt): C, 24.34; H, Probe for microanalysis.
2
10 18 6 2
1
3
(
.68%. Found: C, 24.40, H, 3.88%. H-NMR (ppm, D
s, 3H), 2.80 (t, JH−H = 7.90 Hz, 4H), 1.94 (quintet, JH−H = 7.90 on an aluminium stub and successfully coated with gold to
Hz, 2H). ESI-MS, m/z 516 [M + Na] .
cis-[Pt(CBDCA)(DMSO) ] (2.91 g, 5.89 mmol) was dissolved
in water (140 mL) and treated with a solution of cis-1,4-DACH
694 μL, 5.89 mmol) in H O (22 mL). The resulting aqueous
2
O): 3.58
Before the analysis, the sample was fixed with apposite glue
3
3
+
increase the conductivity of the sample.
2
FT-IR analysis
(
2
solution was kept under stirring at 90 °C for 1.5 h. The solu- Infrared spectra were recorded on a Nicolet 380 FT-IR spectro-
tion was filtered while hot and then allowed to cool to room meter (Thermo Scientific, Baltimore, MD, USA) equipped with
temperature. After concentration to a small volume (5 mL) a commercial attenuated total reflectance accessory. The FT-IR
under reduced pressure, the solution was kept at 4 °C over- spectra were recorded from 4000 to 400 cm− at 2 cm resolu-
night. The off-white precipitate was recovered by filtration of tion using an IFS 66v/S spectrometer (Bruker Corporation,
the mother liquor, washed with 3 mL ice-cold water and dried Karlsruhe, Germany) and KBr pellets. Spectra were collected by
under vacuum. It proved to be [Pt(CBDCA)(cis-1,4-DACH)] averaging 256 scans at 4 cm− resolution.
1
−1
1
(1.06 g, 2.35 mmol; yield = 40%). Anal. Calc. for [Pt(CBDCA)
(
5
cis-1,4-DACH)]·H O (C H O N Pt): C, 30.70; H, 4.72; N,
.97%. Found: C, 30.33; H, 4.78; N, 5.86%. H-NMR (ppm,
2
12 22 5 2
1
Structural analysis
3
D
2
O): 3.16 (pseudotriplet,
J
Pt−H = 100.83 Hz, 2H), 2.89 (t, X-ray powder patterns were collected using an Analytical X’Pert
3
3
JH−H = 7.90 Hz, 4H), 1.90 (q, J
= 7.90 Hz, 2H), 1.76 (s, 8H). Pro equipped with an X’Celerator detector powder diffracto-
H−H
−
ESI-MS, m/z 450 [M − H] .
meter using Cu Kα radiation generated at 40 kV and 40 mA.
The instrument was configured with a 1° divergence and
Synthesis of [PtCl
2
(cis-1,4-DACH)] (kiteplatin, compound 1)
38
0
.2 mm receiving slits.
The samples were prepared using the front loading of stan-
dard aluminium sample holders which are 1 mm deep,
Pt(CBDCA)(cis-1,4-DACH)] (1.06 g, 2.35 mmol) was suspended 20 mm long and 15 mm wide. The degree of crystallinity was
Kiteplatin was prepared following the procedure already
reported in the literature with slight modifications.
[
3
7
in 95 mL of 1.2 M HCl and stirred for two days at room tem- evaluated according to the formula:
perature in the dark. The obtained yellow suspension was
Crystallinity ¼ ðX=YÞ ꢀ 100
dried by evaporation of the solvent under reduced pressure
and the yellow residue was washed with 6 mL of ice-cold water where X = net area of diffracted peaks, and Y = net area of
and dried under vacuum. The obtained yellow crystalline diffracted peaks + background area.
product proved to be [PtCl (cis-1,4-DACH)] (0.733 g,
The crystal domain size along the c direction was calculated
(cis-1,4-DACH)] applying Scherrer’s formula:
6 2 2
(C H14Cl N Pt): C, 18.96; H, 3.71; N, 7.37%. Found: C, 18.85,
2
1
.93 mmol; yield = 82%). Anal. Calc. for [PtCl
2
0
:94λ
2
1
2
ꢀ
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ꢁ
L
¼
H, 3.72, N, 7.29%. H-NMR (ppm, DMSO-d ): 4.90 (s, J
7
=
ð002Þ
6
Pt−H
2
cos θð Δr ꢁ Δ0 Þ
3
0.00 Hz, 4H), 2.95 (pseudotriplet, JPt−H = 90.10 Hz, 2H),
1
.73–1.66 (br, 4H), 1.48–1.43 (br, 4H). ESI-MS, m/z 402 [M + where θ is the diffraction angle for the plane (002), Δr and Δ
0
+
Na] .
are the widths in radiants of reflection (002) at half height for
the synthesized and the reference HA materials, respectively,
and λ = 1.5405 Å.
Synthesis of HA nanocrystals
39
HA nanocrystals were prepared from an aqueous suspension
of Ca(OH)
addition of aqueous H
2
(1.35 M, 1 L) and CaCO
3
(0.1 M, 50 mL) by slow
Determination of specific surface area
3
PO (1.26 M, 600 mL) at room tempera-
4
ture under stirring. The reaction mixture was kept under stir- Measurements were performed using a Sorpty 1750 instrument
ring for 24 hours at room temperature and the resulting (Carlo Erba) using N absorption at 77 K and the well-known
2
4
0
homogeneous suspension of HA nanocrystals was used directly Brunauer, Emmett, and Teller procedure.
to load the compounds under investigation without any
further pre-treatment.
Platinum determination
Morphological characterization
The content of platinum loaded onto HA biomimetic nanocrys-
Transmission electron microscopy (TEM) investigations were tals was determined by ICP measurements with an HORIBA
carried out using a Philips CM 100 instrument (80 kV). The JOBIN YVON ULTIMA 2 ICP-AES instrument. Aliquots of the
powdered samples were ultrasonically dispersed in ultrapure sample were dissolved in 10 mL concentrated nitric acid.
This journal is © The Royal Society of Chemistry 2016
Dalton Trans.

View Article Online
Paper
Dalton Transactions
Adsorption and release kinetics of platinum complexes from
HA nanoparticles
sphere using the following culture media containing 10% fetal
calf serum (Euroclone, Milan, Italy), antibiotics (50 units per
mL penicillin and 50 μg mL− streptomycin) and 2 mM L-gluta-
mine: (i) McCoy’s (Euroclone, Milan, Italy) medium, sup-
plemented with 0.1% gentamicin for U2OS and U2OS/Pt cells;
1
An aliquot (1.5 mL) of a solution of the platinum complex
−1
under investigation (1 mg mL ) was added to 10 mL of a
homogeneous HA suspension.
(
ii) Nutrient Mixture F-12 Ham (Sigma Chemical Co.) for A549
The HA suspension was maintained in a bascule bath at
and LoVo cells.
3
7 °C under stirring for 24 hours. At scheduled times, aliquots
(
1 mL) of the supernatant (that was well separated from the
Cytotoxicity assays
solid phase by 4 min of centrifugation at 8000 rpm) were taken
off and the platinum content was determined by ICP-AES
measurements.
In the release studies, an aliquot of Pt-loaded HA nano-
particles was suspended in physiological-like solution having
different pH values (5.0 and 5.5) in order to mimic the environ-
ment surrounding a tumor mass. At scheduled times, aliquots
The growth inhibitory effect toward tumor cells was evaluated
by means of the MTT assay. Briefly, (5–8) × 10 cells per well,
depending upon the growth characteristics of the cell line,
were seeded in 96-well microplates in a growth medium
3
(
100 μL). After 24 h, the medium was removed and replaced
with fresh media containing the compound to be investigated
at the appropriate concentration. Triplicate cultures were
established for each treatment. After 72 h, each well was
treated with 10 μL of a 5 mg mL− MTT saline solution and,
following 5 h of incubation, 100 μL of a sodium dodecylsulfate
(1 mL) of the supernatant (that was well separated from the
solid phase by 4 min of centrifugation at 8000 rpm) were taken
off and the amount of platinum in the solution was deter-
mined by ICP-AES measurements.
1
(
SDS) solution in 0.01 M HCl were added. After overnight incu-
bation, the cell growth inhibition was determined by measur-
ing the absorbance of each well at 570 nm using a Bio-Rad
Experiments with cultured human cells
680 microplate reader. The mean absorbance for each drug
The Pt(II) complexes kiteplatin (1) and [Pt(CBDCA)(cis-1,4- dose was expressed as a percentage of the absorbance of the
DACH)] (2) used in cell experiments were dissolved in 0.9% control untreated well and plotted vs. the drug concentration.
NaCl solution just before use.
Data were fitted to a dose–response curve and IC50 values, the
drug concentrations that decrease the mean absorbance at
Preparation of releasates from HA nanocrystals loaded with
570 nm to 50% of those in the untreated, control wells, were
1
and 2
calculated with the four-parameter logistic model (4PL). The
For the release experiments, two cell culture media (Nutrient final value is the mean ± S.D. of at least three independent
Mixture F-12 Ham and McCoy’s) at physiological pH (7.4) and experiments performed in triplicate.
at acidic pH 5.0 or 5.5 (the latter two obtained by addition of
citrate buffer 0.1 M) were used. Subsequently, 10 mg of
unloaded HA, 1-loaded HA or 2-loaded HA were suspended in
the culture media and, after 15 s of treatment in a vortex
apparatus, the suspensions were maintained in a shaking bath
Results and discussion
Synthesis of the Pt complexes
at 37 °C. At different time points (0, 5, 30, 60, 120, 240 and Compound 2 is a kiteplatin analog containing the 1,1-cyclobu-
3
60 min), aliquots of the supernatant were collected. Before tanedicarboxylate leaving ligand and may be considered
proceeding with the cell culture treatments, the pH of the related to kiteplatin as carboplatin is related to cisplatin. The
releasates was adjusted to the value of 7.4 to avoid cytotoxicity synthesis of kiteplatin was modified with respect to that pre-
stemming from acidic pH. For each sample, the amount of viously reported in the literature by using a dilute solution of
platinum released from the HA nanocrystals was quantified by HCl to perform substitution of CBDCA by two chlorido
Graphite Furnace Atomic Absorption Spectrometry (GF-AAS) ligands. The use of dilute HCl allowed increasing the yield of
using a Varian AA Duo graphite furnace atomic absorption the reaction (+16%) by reducing the amount of the tetrachlor-
spectrometer (Varian, Palo Alto, CA, USA) at 324.7 nm. The ido Pt(IV)-derivative typically formed as a side product in the
37
calibration curve was obtained using known concentrations of published procedure where concentrated HCl was used. The
standard solutions purchased from Sigma Chemical Co.
spectroscopic and spectrometric characterization of the Pt
complexes were consistent with the literature data.
Cell cultures
Biomimetic HA nanocrystals
Human colon (LoVo) and lung (A549) carcinoma cell lines
were obtained from American Type Culture Collection (ATCC, The synthesized biomimetic hydroxyapatite nanocrystals had
Rockville, MD). The human osteosarcoma cell line U2OS and composition, structure, morphology, dimension, surface area,
its cisplatin resistant subline, U2OS/Pt cells, were kindly pro- and bio-reactivity very close to those of bone inorganic
vided by Prof. F. Zunino (Istituto Nazionale per lo Studio e la nanocrystals.
Cura dei Tumori, Milan, Italy). Cell lines were maintained in
The powder X-ray diffraction pattern of the synthesized
the logarithmic phase at 37 °C in a 5% carbon dioxide atmo- nanocrystals (Fig. 1A) exhibits not-well defined diffraction
Dalton Trans.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Dalton Transactions
Paper
Fig. 1 (A) XRD spectra of biomimetic HA nanocrystals prepared in this
work; (B) XRD spectra of reference Bone-HA.
maxima that indicate a relatively low degree of crystallinity,
Fig. 3 Elementary analysis performed with EDX probe on biomimetic
which can be estimated to be about 30–32% according to the HA nanocrystals. In evidence the presence of calcium (Ca) and phos-
phorus (P) in the typical ratio Ca/P of biomimetic HA.
calculation method described by Scherrer’s formula (see the
Materials and methods section). This value is close to that of
about 28% determined by the X-ray diffraction pattern of
natural, deproteinated bone nanocrystals (Fig. 1B).
The diffraction maxima of the powder X-ray diffraction pat-
terns of the biomimetic HA nanocrystals are characteristic of
the hydroxyapatite single phase (JCPDS 01-089-6439).
The lamellar morphology of these nanocrystals mimics
bone HA. In Fig. 2A is reported the transmission electron
microscopy (TEM) image of the synthesized HA nanocrystals.
The scanning electron microscopy (SEM) image reported in
Fig. 2B shows how the biomimetic HA nanocrystals spon-
taneously aggregate in micrometric clusters. The EDX analysis
(Fig. 3) indicates a Ca/P ratio for the synthesized biomimetic
hydroxyapatite nanocrystals very close to that of natural bone
hydroxyapatite crystals (the ratio close to 1.5).
The FTIR pattern of the synthesized HA nanocrystals is
reported in Fig. 4 where it is possible to observe the adsorption
−
1
bands of the phosphate group at 1033 and 1097 cm and the
Fig. 4 FT-IR spectra of biomimetic HA nanocrystals. In evidence the
at 880 cm . The characteristic adsorption bands of hydroxyapatite.
2
−
−1
characteristic adsorption band of CO
3
Fig. 2 (A) Transmission Electron Microscopy (TEM) image of biomimetic HA nanocrystals with acicular morphology, (B) Scanning Electron
Microscopy (SEM) image of spontaneously formed micrometric aggregates of biomimetic HA nanocrystals.
This journal is © The Royal Society of Chemistry 2016
Dalton Trans.

View Article Online
Paper
Dalton Transactions
sion of biomimetic-HA nanocrystals immediately after their
synthesis. In this way, the formation of micrometric clusters
that partially reduce the reactivity of biomimetic HA nanocrys-
tals is avoided and, as the plots show, an important adsorption
of the Pt-complex on the surface of the HA nanocrystals takes
place in a relatively short time.
Fig. 5 also shows that the adsorption kinetics have a similar
trend for the two complexes and after 24 h of incubation (in
the figure we report the kinetics only up to 360 min) the per-
centage of the complex adsorbed on the HA nanoparticles,
with respect to that initially present in solution, is 89% and
95% for 1 and 2, respectively. A comparison between the
adsorption profiles of the two platinum complexes clearly indi-
cates that the loading of the complexes on the HA nanocrystals
most likely occurs by chemical-adsorption of the compounds
on the surface of the nanocrystals.
Fig. 5 Kinetics of drug adsorption (complexes 1 and 2) on biomimetic
HA nanocrystals.
Release of 1 and 2 from Pt-loaded HA nanocrystals
For the drug release studies, buffer solutions of different
is about 5% of the total, resembling the acidity (pH 5.0 or 5.5) were used. The low pH values mimic
2
−
amount of CO
3
amount of carbonate (4–8%) present in bone nanocrystals.
those typically present in cancer tissues. The release experi-
Synthesized HA nanocrystals exhibit a high bioactivity, due
2
−1
to the large surface area (about 120 m
g ), and a surface
calcium deficiency as in bone HA nanocrystals. At pH 7.4 syn-
thesized HA nanocrystals exhibit a negative electrostatic
surface potential (zeta potential of −20.5 ± 1.5 mV) that would
inhibit the occurrence of protein–protein interactions.
Adsorption of 1 and 2 on HA nanocrystals
The adsorption experiments were performed at 37 °C in an
aqueous medium and monitored for a period of 24 hours. The
adsorbed amount of platinum was determined by difference
between the initial Pt content in the supernatant solution and
that found at different time intervals.
Fig. 5 shows the adsorption profiles of the two different
platinum complexes on HA nanocrystals in water. This experi-
ment was performed using an innovative procedure, that is
treatment with the Pt complexes of the homogeneous suspen-
Fig. 7 Platinum released from 1-loaded HA or 2-loaded HA suspended
in cell culture media (F-12 Ham, panel A, and McCoy’s panel B) at pH 7.4
or acidic pH 5.0 and 5.5. At different time points (0, 5, 30, 60, 120, 240
and 360 min) aliquots of the supernatant were collected and the plati-
num content in each sample was quantified by means of GF-AAS analy-
sis. Error bars indicate SD.
Fig. 6 Released Pt percentages from HA nanocrystals loaded with
complexes 1 and 2. Two pH values (5.0 and 5.5) were considered.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Dalton Transactions
Paper
ments were performed at 37 °C and monitored for a period of 5.0 and 5.5. Biomimetic HA nanocrystals are more soluble at
4 hours (in Fig. 6 we report the kinetics only up to 360 min). lower pH, pH 5 probably favors the dissolution of the nano-
2
The platinum released from HA (M) was determined by ICP crystals and, as a consequence, also the release of the Pt com-
analysis of the supernatant solution as a function of time. The plexes chemisorbed on the HA surface. Remarkably, the
data plotted in Fig. 6 are expressed in terms of M/Mtot × 100 amounts of complex released from HA are 2.5 and 1.5 times
where Mtot corresponds to the amount of complex initially greater at pH 5.0 compared to pH 5.5 for complexes 1 and 2,
bound to the HA nanocrystals.
respectively. Although in both cases we observed an initial
Complex 1 exhibits a markedly faster release rate and a burst in the release of the Pt compound followed by a constant
greater released amount with respect to complex 2 both at pH and sustained release, the trend observed is opposite to the
Fig. 8 Cytotoxicity tests. Cells (5–8 × 104 mL) were treated for 72 h with cell culture media used to investigate the Pt-release from 1-loaded HA
nanocrystals (A) or 2-loaded HA nanocrystals (B) at different time points. Cytotoxicity was assessed by MTT test. Data are the means of at least three
independent experiments. Error bars indicate SD.
This journal is © The Royal Society of Chemistry 2016
Dalton Trans.

View Article Online
Paper
Dalton Transactions
adsorption kinetics, with kiteplatin (1) being less adsorbed should be tested in vivo for which the time scale is much
and more released than 2.
longer than for in vitro tests on cell cultures.
In order to test if the releasates from the Pt-loaded HA
nanocrystals are endowed with cytotoxic activity, the Pt-loaded
HA nanocrystals were allowed to release their platinum
content into cell culture media (McCoy’s and Nutrient Mixture
Conclusion
F-12 Ham) conditioned at physiological pH (7.4) and at acidic HA nanoparticles are good delivery devices that we have
pH (5.0 and 5.5). The platinum content in cell culture media recently developed for the local treatment of bone tumors by
incubated with unloaded HA, 1-loaded HA or 2-loaded HA was loading Pt-bisphosphonate complexes. HA nanoparticles of
assessed by means of GF-AAS analysis (Fig. 7).
suitable dimensions could also be administered by intravenous
Both 1-loaded and 2-loaded HA nanocrystals release plati- injection and could accumulate at the tumor site via the EPR
num in the cell culture media in a time- and pH-dependent effect. The acidic environment of tumor tissues may favor the
manner. At physiological pH both 1-loaded and 2-loaded HA dissolution of HA and the release of the Pt complexes. In this
release a very scarce amount of platinum, however, at acidic work we have exploited the adsorption of Pt complexes 1 and 2
pH, the release of platinum is far greater for 1-loaded HA (with on HA nanoparticles. Complex 1, in particular, has a broad
a maximum release achieved at pH 5.0) than for 2-loaded HA spectrum of activity against tumors. The two Pt(cis-1,4-DACH)
for which the release of platinum remains rather low, even at complexes have been loaded onto the HA nanoparticles soon
pH 5.0. Interestingly, the trend was similar for McCoy’s and after their preparation as water suspension. In this way, the lyo-
for Nutrient Mixture F-12 Ham cell culture media. It was con- philization step has been avoided and with it the possibility of
firmed that a lower pH favors the dissolution of the nanocrys- formation of micrometric aggregates of nanoparticles that
tals and, as a consequence, also the release of the Pt results in a decrease of drug adsorption on the nanoparticle
complexes chemisorbed on the HA surface.
surface. The adsorption of the complexes was found to be
rather fast and the amount of complex loaded very high in both
cases with that of 2 slightly greater than that of 1.
Cell growth inhibitory effect of releasates
The releasates at pH 5.5 and 5.0 from the HA nanocrystals
An opposite trend (1 > 2) was observed for the release kine-
loaded with the Pt complexes were subsequently tested for tics performed at physiological pH (7.4) and at acidic pH (5.0
their cytotoxic potential towards human colon (LoVo) and lung and 5.5) to simulate the conditions that are usually observed
(A549) cancer cells as well as against human osteosarcoma in the tissues surrounding a tumor mass. The drug release
cells sensitive (U2OS) and resistant (U2OS/Pt) to cisplatin. The from HA nanoparticles was pH-sensitive. For 1 the release was
latter pair of cell lines was selected since synthetic biomimetic far greater at acidic pH than at pH 7.4 and, remarkably, quite a
HA nanocrystals are specifically designed to act as carrier of big difference was observed between pH 5.5 and pH 5.0. In the
Pt-drugs in the treatment of bone tumors. In U2OS/Pt cells case of compound 2 the release remained low even at pH 5.0.
cisplatin resistance is associated with reduced drug uptake,
The drug releasates were tested against some selected
reduced formation of DNA lesions, defect in the DNA mismatch human cells derived from solid tumors, also including a cis-
4
1
repair, and upregulation of β-DNA polymerase. The cell viabi- platin resistant phenotype. Releasates from 1-loaded HA nano-
lity results (MTT test after 72 h of exposure time) are depicted crystals resulted to be the most cytotoxic against all tested cell
in Fig. 8. The releasates from unloaded HA nanocrystals lines, being also able to overcome cisplatin resistance.
proved to be non-effective against all tested cell lines (data not
As the pH in tumor tissues is far more acidic (pH = 5–6)
shown). On the contrary, the releasate from 1-loaded HA than in the physiological environment (pH = 7.4), our results
shows a good in vitro antiproliferative effect against all tested suggest that the unique characteristics of HA nanoparticles
cell lines. The cytotoxic potential increases in a time-depen- can be exploited for the development of tumor-targeting pH-
dent manner, with releasates at 360 min being the most sensitive nanoparticles.
effective in decreasing cancer cell viability. Taken together,
These preliminary results are of foremost importance since
cytotoxicity results and platinum quantification data strongly they suggest the potential of kiteplatin-loaded HA nanocrystals
confirm that the cytotoxicity of the releasates strictly depends as bone-specific drug delivery devices to be used for the treat-
upon the platinum content which, in turn, is modulated by ment of bone tumors upon local implantation. Moreover, HA
the pH of the cell culture media.
nanoparticles having suitable dimensions could be adminis-
Our results clearly show that releasates of complex 1 are tered by injection and unlike the blood stream, where the high
extremely effective in decreasing cell viability, pointing to kite- pH value (7.4) will guarantee the stability of the nanocarrier, at
platin as the most effective platinum(II) DACH derivative. Inter- the tumor site the acidic pH will promote activation by dis-
estingly, loaded HA nanoparticles are similarly effective solution of the nanocarrier and release of the Pt drug.
against osteosarcoma sensitive and resistant cells, thus attest-
The in vivo investigation that we have planned to perform
ing the ability of the compounds to overcome cisplatin resist- will hopefully show a potentiated effect due to the dissolution
ance. It was rather disappointing to observe the scarce of the HA nanoparticles at the tumor site and consequent
effectiveness of compound 2, probably due to its very low release of the platinum drug in addition to high concen-
2
+
3−
release from the HA nanovectors. However, this compound trations of Ca and PO4 that, as mentioned in the Introduc-
Dalton Trans.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Dalton Transactions
Paper
tion section, can induce the production of inflammatory cyto- 18 X. M. Liu, H. T. Lee, R. A. Reinhardt, L. A. Marky and
kines that can contribute to the inhibition of tumor cell
proliferation.
D. Wang, J. Controlled Release, 2007, 122, 54.
19 X. Cheng and L. Kuhn, Int. J. Nanomed., 2007, 2, 667–674.
2
2
2
2
0 A. Barroug and M. Glimcher, J. Orthop. Res., 2002, 20, 274–
280.
Acknowledgements
1 T. Xu, N. Zhang, H. L. Nichols, D. Shi and X. Wen, Mater.
Sci. Eng., 2007, 27, 579–594.
2 M. Stigtera, J. Bezemera, K. de Grootb and P. Layrollec,
J. Controlled Release, 2004, 99, 127–137.
3 N. Nocerino, A. Fulgione, M. Iannaccone, L. Tomasetta,
F. Ianniello, F. Martora, M. Lelli, N. Roveri, F. Capuano and
R. Capparelli, Int J Nanomed., 2014, 9, 1175–1184.
4 N. Roveri and M. Iafisco, Nanotechnol., Sci. Appl., 2010, 3,
The authors wish to thank the RBAP114AMK, RINAME
Project (funds for selected research topics), the C.I.R.C.M.S.B.
(Interuniversity Consortium for Research on Chemistry of
Metals in Biological Systems), Qualifu project, Chemical
Center Srl and BioEcoActive s.r.l. The Authors thank Dr Salvatore
Savino (University of Bari) for assistance in the preparation of
the platinum compounds.
2
2
107–125.
5 M. Benedetti, F. De Castro, D. Migoni, A. Romano, T. Verri,
M. Lelli, N. Roveri and F. P. Fanizzi, J. Inorg. Biochem.,
References
2016, 157, 73–79.
2
6 M. Benedetti, D. Antonucci, F. De Castro, C. R. Girelli,
M. Lelli, N. Roveri and F. P. Fanizzi, J. Inorg. Biochem.,
1
2
3
4
5
6
7
8
9
S. Pietronave, M. Iafisco, D. Locarno, L. Rimondini and
M. Maria Prat, J. Appl. Biomater. Biomech., 2009, 7, 77–89.
N. Roveri, P. Barbara and M. Iafisco, Expert Opin. Drug
Delivery, 2008, 5, 861–877.
2015, 153, 279–283.
2
7 A. Rimola, Y. Sakhno, L. Bertinetti, M. Lelli, G. Martra and
P. Ugliengo, J. Phys. Chem. Lett., 2011, 2, 1390–1394.
8 B. Palazzo, M. Iafisco, M. Laforgia, N. Margiotta, G. Natile,
C. L. Bianchi, D. Walsh, S. Mann and N. Roveri, Adv. Funct
Mater., 2007, 17, 2180–2188.
9 M. Iafisco, B. Palazzo, M. Marchetti, N. Margiotta,
R. Ostuni, G. Natile, M. Morpurgo, V. Gandin, C. Marzano
and N. Roveri, J. Mater. Chem., 2009, 19, 8385–8392.
0 M. Iafisco, B. Palazzo, G. Martra, N. Margiotta,
S. Piccinonna, G. Natile, V. Gandin, C. Marzano and
N. Roveri, Nanoscale, 2012, 4, 206–217.
H. Hillaireau and P. Couvreur, Cell. Mol. Life Sci., 2009, 66,
2
2
873–2896.
J. H. Adair, M. P. Parette, E. I. Altınoğlu and M. Kester, ACS
Nano, 2010, 4, 4967–4970.
F. C. M. Driessens, J. W. E. van Dijk and J. M. P. M.
Borggreven, Calcif. Tissue Int., 1978, 26, 127–136.
M. Vallet-Regí and J. M. Gonzalez-Calbet, Prog. Solid State
Chem., 2004, 32, 1–31.
L. Wang, G. H. Nancollas, Z. J. Henneman, E. Klein and
S. Weiner, Biointerphases, 2006, 1, 106–111.
H. A. Lowenstam and S. Weiner, On Biomineralization,
Oxford University Press, USA, 1989.
2
3
3
3
3
1 M. Iafisco and N. Margiotta, J. Inorg. Biochem., 2012, 117,
2
37–242.
2 V. Uskokovic and D. P. Uskokovic, J. Biomed. Mater. Res.,
011, 96, 152–191.
3 S. Dey, M. Das and V. K. Balla, Mater. Sci. Eng. C, 2014, 39,
36–339.
H. Gao, B. Ji, I. L. Jager, E. Arz and P. Fratzl, Proc. Natl.
Acad. Sci. U. S. A., 2003, 100, 5597–5660.
2
1
0 H. S. Gupta, J. Seto, W. Wagermaier, P. Zaslansky,
3
P. Boesecke and P. Fratzl, Proc. Natl. Acad. Sci. U. S. A.,
3
3
4 L. Kelland, Nat. Rev. Cancer, 2007, 7, 573–584.
5 N. Margiotta, C. Marzano, V. Gandin, D. Osella, M. Ravera,
E. Gabano, J. A. Platts, E. Petruzzella, J. D. Hoeschele and
G. Natile, J. Med. Chem., 2012, 55, 7182–7192.
2
006, 103, 17743–17746.
1
1
1 S. Weiner and L. Addadi, J. Mater. Chem., 1997, 7, 689–702.
2 T. A. Kuriakose, S. N. Kalkura, M. Palanichamy, D. Arivuoli,
K. Dierks, G. Bocellif and C. Betzel, J. Cryst. Growth, 2004,
3
3
3
3
4
4
6 Yu. N. Kukushkin, Yu. E. Vyaz’menskii, L. I. Zorina and Yu.
L. Pazukhina, Zh. Neorg. Khim., 1968, 13, 1595–1599.
7 R. Ranaldo, N. Margiotta, F. P. Intini, C. Pacifico and
G. Natile, Inorg. Chem., 2008, 47, 2820–2830.
8 B. V. Almelo, X’PERT HIGH SCORE (version 2.0a), PANalyti-
cal, The Netherlands, 2004.
9 H. P. Klug and L. E. Alexander, X-Ray Diffraction Procedures,
John Wiley and Sons, New York, 1954.
0 S. Brunauer, P. H. Emmett and E. Teller, J. Am. Chem. Soc.,
2
63, 517–523.
1
1
3 A. C. Tas, Biomaterials, 2000, 21, 1429–1438.
4 L. W. Suchanek, P. Shuk, K. Byrappa, R. E. Riman,
K. S. TenHuisen and V. F. Janas, Biomaterials, 2002, 23,
6
99–710.
1
1
5 A. Ruksudjarit, K. Pengpat, G. Rujijanagul and
T. Tunkasiri, Curr. Appl. Phys., 2008, 8, 270–272.
6 N. Roveri and B. Palazzo, in Tissue, Cell and Organ Engineering
(Nanotechnologies for the Life Sciences), ed. C. S. S. R. Kumar,
1938, 60, 309–319.
Wiley-VCH, Weinheim, 2006, pp. 283–307.
7 M. Iafisco, B. Palazzo, G. Falini, M. Di Foggia, S. Bonora,
S. Nicolis, L. Casella and N. Roveri, Langmuir, 2008, 24,
1 S. Mazzega Sbovata, F. Bettio, M. Mozzon, R. Bertani,
A. Venzo, F. Benetollo, R. A. Michelin, V. Gandin and
C. Marzano, J. Med. Chem., 2007, 50, 4775–4784.
1
4
924–4930.
This journal is © The Royal Society of Chemistry 2016
Dalton Trans.