Al(OH)3 facilitated synthesis of water-soluble, magnetic, radiolabelled and fluorescent hydroxyapatite nanoparticles

Article abstract of DOI:10.1039/c5cc02259b

Magnetic and fluorescent hydroxyapatite nanoparticles were synthesised using Al(OH)₃-stabilised MnFe₂O₄ or Fe₃O₄ nanoparticles as precursors. They were readily and efficiently radiolabelled with ¹⁸F. Bisphosphonate polyethylene glycol polymers were utilised to endow the nanoparticles with excellent colloidal stability in water and to incorporate cyclam for high affinity labelling with ⁶⁴Cu.

Full text of DOI:10.1039/c5cc02259b

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Commun., 2015, DOI: 10.1039/C5CC02259B.
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Al(OH) Facilitated Synthesis of Water-soluble,
3
Magnetic, Radiolabelled and Fluorescent
Hydroxyapatite Nanoparticles
Cite this: DOI: 10.1039/x0xx00000x
ξ a
a, b
a
c
d
e
Received 00th January 2012,
Accepted 00th January 2012
X. Cui, M. A. Green,
∗
f
P. J. Blower,
∗
D. Zhou, Y. Yan, W. Zhang, K.
e
g
g
Djanashvili, D. Mathe, D. S. Veres, and K. Szigeti
DOI: 10.1039/x0xx00000x
www.rsc.org/
ꢀ
ꢀ
Magnetic and fluorescent hydroxyapatite nanoparticles were to 20% replacement of OH by F (maximum theoretical value for
fluoride substitution), to minimise the quenching of the excited sate
synthesised using Al(OH) -stabilised MnFe O or Fe O4
3
2
4
3
8, 12, 13
of rare earth cations.
for F radiolabelling . It has been reported that magnetic iron oxide
As a result, such HA is no longer suitable
nanoparticles as precursors. They were readily and efficiently
18
ꢀ
1
8
radiolabelled with F. Bisphosphonate polyethylene glycol
polymers were utilised to endow the nanoparticles with
NPs can be deposited on the surface of HA aggregates or NPs via
11
9
thermolysis or a wet chemistry approach. One problem that
excellent colloidal stability in water and to incorporate remained unsolved for both synthetic approaches is how to
6
4
cyclam for high affinity labelling with Cu.
effectively isolate the desired Fe O /HA composites from the
3 4
unwanted iron oxides and HA nanoparticles. Moreover, all these
multifunctional HA NPs suffer from the problem of aggregation or
Molecular imaging techniques, including magnetic resonance large size to some extent, which is an obstacle for their biological or
imaging (MRI), positron emission tomography (PET), single photon medical applications. In this work, we present a novel synthesis of
emission computed tomography (SPECT) and fluorescence optical magnetic and fluorescent HA nanocomposites with uniform size and
imaging, play an increasingly important role in clinical diagnosis and morphology, and excellent colloidal stability in water by using
management of disease, as well as medical and biological research. Fe O nanoparticles stabilised with Al(OH) as a template. The
3
4
3
Multimodal imaging recently has gained attention because of its radiolabelling, magnetic and optical properties were investigated, to
potential to overcome the limitations of individual imaging demonstrate potential for application as triꢀmodal probes for MR,
modalities and to provide more accurate and complete physiological PET and optical imaging.
1
ꢀ3
information at sites of disease. Numerous nanoparticles (NPs)
have been studied as multimodal imaging contrast agents due to their
4
ꢀ7
multiꢀfunctionality and potential for surface modification.
An
adequate multimodal nanoparticulate contrast agent must be
multifunctional, biocompatible and colloidally stable. The NPs
should be uniform in morphology and size, so that they share similar
in vivo behaviour, and chemically stable to ensure that the signal of
each modality reflects the same anatomic position.
Hydroxyapatite (HA) has attracted much interest as the basis of
8
ꢀ11
multifunctional probes very recently,
because of its
biocompatibility and high affinity for fluoride which allows facile
1
8
labelling with the positron emitter F. Fluorescent HA can be
8
,11
obtained by either doping with rare earth cations
or by
1
0
conjugation with organic dyes . HA is not an ideal fluorescent host
matrix, so luminescent rare earth doped HA nanocrystal requires up
This journal is © The Royal Society of Chemistry 2012
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DOI: 10.1039/C C0225
Jou5Crnal Na9Bme
polymeric chain via amide formation mediated by N,N’ꢀ
dicyclohexylcarbodiimide (DCC), followed by deprotection with
17
bromotrimethylsilane (TMBS) . BPꢀPEGꢀMe 2 and BPꢀPEGꢀ
cyclam 3 were purified by dialysis for over 24 hours using a
membrane with a cutꢀoff size of 3500 Da to remove unconjugated
small molecules such as bisphosphonate amine 1 and 1,4,8,11ꢀ
tetraazacyclotetradecane. The conjugation of bisphosphonate and
3
1
PEG was confirmed by the change in chemical shift in the P NMR
spectrum (from 20 ppm for free bisphosphonate to 12.8 ppm for BPꢀ
PEG, see Supporting Information).
Nanoparticulate
precursors
MnFe O @Al(OH)
and
2
4
3
Fe O @Al(OH) were obtained via a method reported by our group
3
4
3
14
previously.
Typically,
4
ml Fe O @Al(OH)
colloids
3
4
3
(
concentration of Fe O , ca. 8 mg/ml) and 200 mg BPꢀPEGꢀMe
3
4
polymers 2 were placed in a 500 ml flask containing 300 ml water.
Under stirring, 4 ml 0.1 mol/l NaH PO and 4 ml 0.2 mol/l CaCl
2
4
2
aqueous solutions were added sequentially. This light brown solution
was refluxed overnight after the addition of 30 ml 28% ammonia
water. The NPs were collected by centrifugation at 2000 g for 30
minutes, reꢀdissolved in 10 ml water and freeze dried.
Scheme 1: synthesis of bisphosphonate PEG, i) dropwise addition of
o
POCl at ꢀ7.5 C under N , extract with CH Cl /H O/CH Cl under
3
2
2
2
2
2
2
basic/acidic/basic conditions; ii) CrO /H SO , 24 hours; iii) react
3
2
4
with 1 and N, N’ꢀdicyclohexylcarbodiimide (DCC), CH Cl , 18 h
2
2
The Xꢀray powder diffraction (XRD) pattern of NPs indicates coꢀ
existence of HA and Fe O (or MnFe O ). TEM images show an
then with TMBS, remove solvent, stir in methanol; iv) CrO /H SO ,
3
2
4
3
4
2
4
2
1
4 hours; v) react with 1 and DCC, CH Cl ,18 hours; vi) react with
2 2
oliveꢀlike morphology for Fe O @HA and MnFe O @HA NPs,
3 4 2 4
,4,8,11ꢀtetraazacyclotetradecane and DCC, CH Cl , 18 hours, then
2
2
which is significantly different both in size and in morphology from
pure HA NPs synthesised under the same conditions (Fig. 1).
Particle analysis by TEM gave a mean size of 60.3 nm (major axis) x
with TMBS, remove solvent, stir in methanol for 2 h.
2
9.7 nm (minor axis) for Fe O @HA NPs (Fig. 1c&d).
3 4
MnFe O @HA NPs displayed a similar aspect ratio to Fe O @HA
2
4
3
4
NPs, but the size was almost doubled. These results indicate an
important role of BPꢀPEGꢀMe in reducing particle size.
The hydrodynamic size of NPs during the synthesis was
monitored by dynamic light scattering (DLS) experiments. No
obvious change was observed after adding the solutions of NaH PO
2
4
and CaCl into the solution of Fe O @Al(OH) NPs, and it remained
2
3
4
3
around 80 nm. This leads to a hypothesis that phosphate anions
adsorb on the highly positive charged surface of Fe O @Al(OH)
3
4
3
2+
NP and then react with the subsequently added Ca to form calcium
phosphate around the NPs. At the elevated temperature and basic
solution environment, the outer layer of calcium phosphate was
converted to crystallised HA, meanwhile the Al(OH) was dissolved,
3
Fig. 1 TEM images a) and b) and size distribution c) and d) (major resulting in the formation of Fe
O @HA NPs. The diminished peak
3
4
ꢀ
1
and minor axis, respectively) of Fe O @HA NPs synthesised from due to –OH around 3300ꢀ3500 cm in the IR spectrum, together
3
4
Fe O @Al(OH) in the presence of BPꢀPEGꢀMe; e) TEM image of with XRD patterns, confirm the replacement of Al(OH)
layer by
3
4
3
3
MnFe O @HA NPs synthesised in absence of BPꢀPEGꢀMe 2; f) HA. The changes in surface potential of NPs could not be monitored
2
4
TEM image of pure HA NPs.
by measuring the zeta potential during the process, since the
polymeric PEG imposes a thick hydration layer on NP surface and
Our strategy is to synthesise HA using waterꢀsoluble magnetic zeta potential no longer correlates to the surface potential. Therefore
Fe O @Al(OH) or MnFe O @Al(OH) NPs as templates. The the surface potential was monitored in the absence of BPꢀPEGꢀMe,
3
4
3
2
4
3
advantages of this approach is the small hydrodynamic size of the and a significant decrease in zeta potential was observed after the
template particles and their excellent colloidal stability, provided by addition of NaH PO solution, from 42.5 mV to 24.9 mV. This is
2
4
1
4
the Al(OH) layer as reported previously. More importantly, the presumed to be due to the adsorption of phosphate anions on the
3
layer of Al(OH) can be readily removed as it is soluble under basic surface, since the changes in pH should be negligible in this case.
3
pH conditions. The design incorporated bisphosphonate The zeta potential slightly increased back to 27.1 mV after adding
polyethylene glycol (BPꢀPEG) polymers (Scheme 1) to stabilise NPs the CaCl2 solution, indicating a reaction of calcium cations and
after the formation of HA on the surface, to take advantage of the phosphate anions. Similar results were also observed for
outstanding binding affinity of bisphosphonates to HA.
MnFe O @HA NPs. In this synthesis approach, the positively
2 4
charged Al(OH)3 layer is essential for the formation of
Bisphosphonate amine 1 was obtained via a slightly modified MnFe O @HA or Fe O @HA. Using the MnFe O NPs colloids
2
4
3
4
2
4
1
5
version of the previously reported protocol. PEG carboxylic acids instead of MnFe O @Al(OH) as precursors, a simple mixture of
2
4
3
were obtained by oxidation of corresponding PEG polymers with magnetic MnFe O4 and nonꢀmagnetic HA NPs was obtained,
2
1
6
CrO /H SO via the reported protocol. The bisphosphonate (BP) or identifiable as two kinds of NPs with apparently different
3
2
4
1
,4,8,11ꢀtetraazacyclotetradecane (cyclam) were grafted to PEG morphology and size on TEM images.
2
| J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5CC02259B
_
hydroxyapatite NPs as well as a high ratio of r /r
2 1
potential application as T contrast agents on MR imaging.
2
Fig. 3 Emission and excitation spectra of aqueous solutions of
®
Fe O @HA conjugates with a) Maria blue and b) Alexa Fluor 488.
3
4
Fluorescent HA is normally produced either by doping with rare
8, 11, 21
earth cations (Eu or Tb),
or by conjugation with organic
10, 22
dyes.
Here we conjugated the fluorescent dyes Maria blue and
®
Alexa Fluor covalently to the NPs surface using sodium
pamidronate as an aminobisphosphonate linker (see Supplementary
Information). The amine group of pamidronate is reactive for NHS
ester dyes to form stable amide bonds while its bisphosphonate
group interacts strongly with the Ca or Fe at the surface of NPs; dyes
are thus linked to NPs without the risk of leakage. Fluorescent
spectra in Fig. 3 show an emission at 455 nm for the conjugates of
Fig. 2 a) DLS size distribution of 1 mg/ml Fe O @HA NPs in
3
4
aqueous solution; b) 1 Radiolabelling of MnFe O @HA and
2
4
8
64
Fe O @HA NPs with Fꢀfluoride and CuCl2 (the latter after
3
4
sonicating the particles with BPꢀPEGꢀcyclam); c) T1 and T2
weighted MR images of the solution containing Fe O @HA NPs,
3
4
and d) relaxivities of Fe O @HA NPs. Concentration of iron in the
3
4
Fe O @HA and Maria blue under excitation at 365 nm, and an
solution was measured by ICPꢀMS.
3
4
®
emission at 517 nm for the Alexa Fluor 488 conjugates under
Both MnFe O @HA and Fe O @HA NPs were coated by BPꢀ excitation at 488 nm. More importantly, the fluorescence of these
2
4
3
4
PEGꢀMe polymers 2 during the synthesis, as confirmed by ca. 18% conjugated NPs is stable and strong even after being stored at room
mass loss on thermogravimetric analysis (TGA). Due to the strong temperature for over one month, implying the potential applications
interactions between the bisphosphonate group of
2
and as optical contrast.
both NPs exhibit longꢀ
1
7, 18
MnFe O @HA and Fe O @HA NPs,
2
4
3
4
In summary, we have presented a facile approach to synthesise
magnetic and fluorescent hydroxyapatite nanoparticles with a wellꢀ
term colloidal stability in aqueous solution, even in high ionic
strength environment such as PBS. The hydrodynamic size of
Fe O @HA and MnFe O @HA NPs remained at 50.7 nm and 60.3
defined morphology and uniform size, using Al(OH) ꢀstabilised
3
3
4
2
4
Fe O or MnFe O NPs as templates. The change from the highly
nm, respectively, for over two months (Fig. 2a). The excellent
colloidal stability and small hydrodynamic size of MnFe O @HA
3
4
2
4
positively charged surface and baseꢀsolubility of the Al(OH) layer
3
2
4
to the neutral or slightly negative zeta potential and acid solubility of
and Fe O @HA make them potentially suitable for biological or
3
4
the Fe O @HA particles is likely to offer alternative biological
medical applications.
3
4
properties. These NPs are promising candidates for development as
triꢀmodal probes for MR, PET and optical imaging, since they
19
Unsurprisingly, because of the high affinity of fluoride for HA,
18
both NPs exhibit a high radiolabelling efficiency with Fꢀfluoride, display excellent colloidal stability and high radiolabelling
64
18
up to 88.3±0.5% for 0.3 mg MnFe O @HA NPs and 92.1±0.1% for efficiency both for Cu and for F, as well as fluorescent and
2
4
0
.3 mg Fe O @NaYF NPs (Fig. 2b). Labelling and purification was magnetic properties. Radiolabelling with other metallic radioisotopes
3 4 4
readily achieved in less than 23 min. To provide a means of will also be achievable by replacing cyclam with corresponding
6
4
incorporating the positron emitter Cu, the NPs were sonicated in 1 chelators. This synthesis approach allows us to tune the magnetic
mg/ml BPꢀPEGꢀcyclam solution for 30 minutes to allow properties of particles by altering the ratio of precursors, without
replacement of a fraction of BPꢀPEGꢀMe by BPꢀPEGꢀcyclam, and decreasing the radiolabelling efficiency or fluorescent property. The
free BPꢀPEGꢀpolymers were removed by centrifugation before flexible conjugation method ensures that dyes with different
mixing with radioactivity. The resulting particles showed a high wavelengths could be selected for different applications. The
64
Cu radiolabelling efficiency in a short time (< 5 minutes) (Fig. 2b). synthesis strategy for conjugation of BPꢀPEGꢀcyclam can also be
Both NPs display essentially the magnetic properties of Fe O or applied to the conjugation of BPꢀPEG with peptides or antibodies,
MnFe O NPs and are active on MR images (Fig. 2c&d). The leading to the application in targeted imaging.
3
4
2
4
transverse (r ) and longitudinal (r ) relaxivities of Fe O @HA NPs
2
1
3
4
ꢀ
1
ꢀ1
ꢀ1 ꢀ1
This research was supported by the Centre of Excellence in
Medical Engineering Centre funded by the Wellcome Trust and
EPSRC under grant number WT088641/Z/09/Z, and the Kings
College London and UCL Comprehensive Cancer Imaging Centre
funded by the CRUK and EPSRC in association with the MRC and
DoH (England), and by the National Institute for Health Research
were measured to be 150.2±3.2 mM s and 1.9±0.1 mM s ,
respectively, at 3T magnetic field. As expected, the relaxivities of
NPs could be improved by altering the ratio of magnetic component
and nonꢀmagnetic HA, since r is proportional to the volume fraction
2
2
0
of magnetic component . For example, the r of MnFe O @HA NPs
2
2
4
ꢀ
1 ꢀ1
could be dramatically improved from 105.7±3.5 mM s to
46.5±15.9 mM s by doubling the amount of MnFe O @Al(OH)
ꢀ
1 ꢀ1
(NIHR) Biomedical Research Centre at Guy's and St Thomas' NHS
2
2 4 3
Foundation Trust and King's College London.
while keeping the amount of NaH PO and CaCl solutions the same
2
4
2
during the synthesis. High transverse relaxivity of these magnetic
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DOI: 10.1039/C C0225
Jou5Crnal Na9Bme
Notes and references
a
King’s College London, Division of Imaging Sciences and Biomedical
th
Engineering, 4 Floor of Lambeth wing, St Thomas Hospital, London
SE1 7EH, UK.
b
King’s College London, Department of Physics, Strand Campus,
London, WC2R 2LS, UK
c
Department of Mathematical Science, Loughborough University,
Loughborough, LE11 3TU, UK
d
School of Chemistry, Nottingham University, Nottingham, NG7 2RD,
UK
e
Department of Biotechnology, Delft University of Technology,
Julianalaan, 136, 2628 BL, Delft, the Netherlands
f
CROmed Ltd, Baross u. 91ꢀ95, Hꢀ1047, Budapest, Hungary
Department of Biophysics and Radiation Biology, Semmelweis
g
University, IX, Tüzoltó u. 37ꢀ47, H0194, Budapest, Hungary
ξ
present address: Department of Materials, South Kensington campus,
Imperial College London, SW7 2AZ, UK
∗
To whom correspondence should be addressed: philip.blower@kcl.ac.uk
and mark.a.green@kcl.ac.uk
Electronic Supplementary Information (ESI) available: conjugation of
NPs with dyes, radiolabelling for NPs, NMR spectra, XRD, IR, zeta
potential, DLS size distribution, TEM images and TGA data of NPs,
fluorescent images of NPs. See DOI: 10.1039/c000000x/
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