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

D. Mathe (7162364)

D.S. Veres (7162367)

Diwei Zhou (1251321)

K. Djanashvili (7162361)

K. Szigeti (7162370)

M.A. Green (7162355)

P.J. Blower (7162358)

W. Zhang (820655)

X. Cui (7162352)

Y. Yan (6513260)

English

Loughborough University Institutional Repository

9332 | Chem. Commun., 2015, 51, 9332--9335 This journal is©The Royal Society of Chemistry 2015Cite this:Chem. Commun., 2015,51, 9332Al(OH)3 facilitated synthesis of water-soluble,magnetic, radiolabelled and fluorescenthydroxyapatite nanoparticles†X. Cui,‡a M. A. Green,*ab P. J. Blower,*a D. Zhou,c Y. Yan,d W. Zhang,eK. Djanashvili,e D. Mathe,f D. S. Veresg and K. SzigetigMagnetic and fluorescent hydroxyapatite nanoparticles were synthe-sised using Al(OH)3-stabilised MnFe2O4 or Fe3O4 nanoparticles asprecursors. They were readily and efficiently radiolabelled with 18F.Bisphosphonate polyethylene glycol polymers were utilised toendow the nanoparticles with excellent colloidal stability in waterand to incorporate cyclam for high affinity labelling with 64Cu.Molecular imaging techniques, including magnetic resonanceimaging (MRI), positron emission tomography (PET), single photonemission computed tomography (SPECT) and fluorescence opticalimaging, play an increasingly important role in clinical diagnosisand management of disease, as well as medical and biologicalresearch. Multimodal imaging recently has gained attentionbecause of its potential to overcome the limitations of individualimaging modalities and to provide more accurate and completephysiological information at sites of disease.1–3 Numerous nano-particles (NPs) have been studied as multimodal imaging contrastagents due to their multi-functionality and potential for surfacemodification.4–7 An adequate multimodal nanoparticulate contrastagent must be multifunctional, biocompatible and colloidallystable. The NPs should be uniform in morphology and size,so that they share similar in vivo behaviour, and chemicallystable to ensure that the signal of each modality reflects thesame anatomic position.Hydroxyapatite (HA) has attracted much interest as the basis ofmultifunctional probes very recently,8–11 because of its biocompat-ibility and high affinity for fluoride which allows facile labelling withthe positron emitter 18F. Fluorescent HA can be obtained by eitherdoping with rare earth cations8,11 or by conjugation with organicdyes.10 HA is not an ideal fluorescent host matrix, so luminescentrare earth dopedHA nanocrystal requires up to 20% replacement ofOH by F (maximum theoretical value for fluoride substitution),to minimise the quenching of the excited sate of rare earthcations.8,12,13 As a result, such HA is no longer suitable for 18Fradiolabelling. It has been reported that magnetic iron oxide NPscan be deposited on the surface of HA aggregates or NPs viathermolysis11 or a wet chemistry approach.9 One problem thatremained unsolved for both synthetic approaches is how toeffectively isolate the desired Fe3O4–HA composites from theunwanted iron oxides and HA nanoparticles. Moreover, all thesemultifunctional HA NPs suffer from the problem of aggregation orlarge size to some extent, which is an obstacle for their biologicalor medical applications. In this work, we present a novel synthesisof magnetic and fluorescent HA nanocomposites with uniformsize and morphology, and excellent colloidal stability in water byusing Fe3O4 nanoparticles stabilised with Al(OH)3 as a template.The radiolabelling, magnetic and optical properties were investi-gated, to demonstrate potential for application as tri-modalprobes for MR, PET and optical imaging.Our strategy is to synthesise HA using water-soluble magneticFe3O4@Al(OH)3 or MnFe2O4@Al(OH)3 NPs as templates. The advan-tages of this approach is the small hydrodynamic size of thetemplate particles and their excellent colloidal stability, providedby the Al(OH)3 layer as reported previously.14 More importantly, thelayer of Al(OH)3 can be readily removed as it is soluble under basicpH conditions. The design incorporated bisphosphonate poly-ethylene glycol (BP-PEG) polymers (Scheme 1) to stabilise NPsafter the formation of HA on the surface, to take advantage of theoutstanding binding affinity of bisphosphonates to HA.a King’s College London, Division of Imaging Sciences and Biomedical Engineering,4th Floor of Lambeth wing, St Thomas Hospital, London SE1 7EH, UK.E-mail: philip.blower@kcl.ac.uk, mark.a.green@kcl.ac.ukb King’s College London, Department of Physics, Strand Campus, London,WC2R 2LS, UKc Department of Mathematical Science, Loughborough University, Loughborough,LE11 3TU, UKd School of Chemistry, Nottingham University, Nottingham, NG7 2RD, UKe Department of Biotechnology, Delft University of Technology, Julianalaan, 136,2628 BL, Delft, The Netherlandsf CROmed Ltd, Baross u. 91-95, H-1047, Budapest, Hungaryg Department of Biophysics and Radiation Biology, Semmelweis University, IX,Tu¨zolto´ u. 37-47, H1094, Budapest, Hungary† Electronic supplementary information (ESI) available: Conjugation of NPs withdyes, radiolabelling for NPs, NMR spectra, XRD, IR, zeta potential, DLS sizedistribution, TEM images and TGA data of NPs, fluorescent images of NPs. SeeDOI: 10.1039/c5cc02259b‡ Present address: Department of Materials, South Kensington campus, ImperialCollege London, SW7 2AZ, UK.Received 17th March 2015,Accepted 28th April 2015DOI: 10.1039/c5cc02259bwww.rsc.org/chemcommChemCommCOMMUNICATIONOpen Access Article. Published on 28 April 2015. Downloaded on 22/06/2015 14:01:38.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View IssueThis journal is©The Royal Society of Chemistry 2015 Chem. Commun., 2015, 51, 9332--9335 | 9333Bisphosphonate amine 1 was obtained via a slightly modifiedversion of the previously reported protocol.15 PEG carboxylic acidswere obtained by oxidation of corresponding PEG polymers withCrO3/H2SO4 via the reported protocol.16 The bisphosphonate (BP)or 1,4,8,11-tetraazacyclotetradecane (cyclam) were grafted to PEGpolymeric chain via amide formation mediated by N,N0-dicyclo-hexylcarbodiimide (DCC), followed by deprotection with bromo-trimethylsilane (TMBS).17 BP-PEG-Me 2 and BP-PEG-cyclam 3were purified by dialysis for over 24 hours using a membranewith a cut-off size of 3500 Da to remove unconjugated smallmolecules such as bisphosphonate amine 1 and 1,4,8,11-tetra-azacyclotetradecane. The conjugation of bisphosphonate and PEGwas confirmed by the change in chemical shift in the 31P NMRspectrum (from 20 ppm for free bisphosphonate to 12.8 ppm forBP-PEG, see ESI†).Nanoparticulate precursors MnFe2O4@Al(OH)3 and Fe3O4@Al(OH)3 were obtained via a method reported by our grouppreviously.14 Typically, 4 ml Fe3O4@Al(OH)3 colloids (concentrationof Fe3O4, ca. 8 mg ml1) and 200 mg BP-PEG-Me polymers 2 wereplaced in a 500 ml flask containing 300 ml water. Under stirring,4 ml 0.1 mol l1 NaH2PO4 and 4 ml 0.2 mol l1 CaCl2 aqueoussolutions were added sequentially. This light brown solution wasrefluxed overnight after the addition of 30 ml 28% ammonia water.The NPs were collected by centrifugation at 2000 g for 30 minutes,re-dissolved in 10 ml water and freeze dried.The X-ray powder diffraction (XRD) pattern of NPs indicatesco-existence of HA and Fe3O4 (or MnFe2O4). TEM images showan olive-like morphology for Fe3O4@HA and MnFe2O4@HA NPs,which is significantly different both in size and in morphologyfrom pure HA NPs synthesised under the same conditions (Fig. 1).Particle analysis by TEM gave amean size of 60.3 nm (major axis)29.7 nm (minor axis) for Fe3O4@HA NPs (Fig. 1c and d).MnFe2O4@HA NPs displayed a similar aspect ratio to Fe3O4@HANPs, but the size was almost doubled. These results indicate animportant role of BP-PEG-Me in reducing particle size.The hydrodynamic size of NPs during the synthesis was mon-itored by dynamic light scattering (DLS) experiments. No obviouschange was observed after adding the solutions of NaH2PO4 andCaCl2 into the solution of Fe3O4@Al(OH)3 NPs, and it remainedaround 80 nm. This leads to a hypothesis that phosphate anionsadsorb on the highly positive charged surface of Fe3O4@Al(OH)3 NPand then react with the subsequently added Ca2+ to form calciumphosphate around the NPs. At the elevated temperature and basicsolution environment, the outer layer of calcium phosphate wasconverted to crystallised HA, meanwhile the Al(OH)3 was dissolved,resulting in the formation of Fe3O4@HA NPs. The diminished peakdue to –OH around 3300–3500 cm1 in the IR spectrum, togetherwith XRD patterns, confirm the replacement of Al(OH)3 layerby HA. The changes in surface potential of NPs could not bemonitored by measuring the zeta potential during the process,since the polymeric PEG imposes a thick hydration layer on NPsurface and zeta potential no longer correlates to the surfacepotential. Therefore the surface potential was monitored in theabsence of BP-PEG-Me, and a significant decrease in zeta potentialwas observed after the addition of NaH2PO4 solution, from 42.5 mVto 24.9 mV. This is presumed to be due to the adsorption ofphosphate anions on the surface, since the changes in pH shouldbe negligible in this case. The zeta potential slightly increased backto 27.1 mV after adding the CaCl2 solution, indicating a reactionof calcium cations and phosphate anions. Similar results werealso observed for MnFe2O4@HA NPs. In this synthesis approach,the positively charged Al(OH)3 layer is essential for the formationof MnFe2O4@HA or Fe3O4@HA. Using the MnFe2O4 NPs colloidsinstead of MnFe2O4@Al(OH)3 as precursors, a simple mixture ofmagnetic MnFe2O4 and non-magnetic HA NPs was obtained,identifiable as two kinds of NPs with apparently differentmorphology and size on TEM images.Both MnFe2O4@HA and Fe3O4@HA NPs were coated byBP-PEG-Me polymers 2 during the synthesis, as confirmed byca. 18% mass loss on thermogravimetric analysis (TGA). Due tothe strong interactions between the bisphosphonate group of 2Scheme 1 Synthesis of bisphosphonate PEG, (i) dropwise addition ofPOCl3 at 7.5 1C under N2, extract with CH2Cl2/H2O/CH2Cl2 under basic/acidic/basic conditions; (ii) CrO3/H2SO4, 24 hours; (iii) react with 1 andN,N0-dicyclohexylcarbodiimide (DCC), CH2Cl2, 18 h then with TMBS,remove solvent, stir in methanol; (iv) CrO3/H2SO4, 24 hours; (v) react with 1and DCC, CH2Cl2, 18 hours; (vi) react with 1,4,8,11-tetraazacyclotetradecaneand DCC, CH2Cl2, 18 hours, then with TMBS, remove solvent, stir in methanolfor 2 h.Fig. 1 TEM images (a) and (b) and size distribution (c) and (d) (major andminor axis, respectively) of Fe3O4@HA NPs synthesised from Fe3O4@Al(OH)3in the presence of BP-PEG-Me; (e) TEM image of MnFe2O4@HA NPssynthesised in absence of BP-PEG-Me 2; (f) TEM image of pure HA NPs.Communication ChemCommOpen Access Article. Published on 28 April 2015. Downloaded on 22/06/2015 14:01:38.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online9334 | Chem. Commun., 2015, 51, 9332--9335 This journal is©The Royal Society of Chemistry 2015and MnFe2O4@HA and Fe3O4@HA NPs,17,18 both NPs exhibitlong-term colloidal stability in aqueous solution, even in highionic strength environment such as PBS. The hydrodynamicsize of Fe3O4@HA and MnFe2O4@HA NPs remained at 50.7 nmand 60.3 nm, respectively, for over two months (Fig. 2a). Theexcellent colloidal stability and small hydrodynamic size ofMnFe2O4@HA and Fe3O4@HA make them potentially suitablefor biological or medical applications.Unsurprisingly, because of the high affinity of fluoridefor HA,19 both NPs exhibit a high radiolabelling efficiency with18F-fluoride, up to 88.3  0.5% for 0.3 mg MnFe2O4@HA NPsand 92.1  0.1% for 0.3 mg Fe3O4@NaYF4 NPs (Fig. 2b).Labelling and purification was readily achieved in less than23 min. To provide a means of incorporating the positron emitter64Cu, the NPs were sonicated in 1 mgml1 BP-PEG-cyclam solutionfor 30 minutes to allow replacement of a fraction of BP-PEG-Meby BP-PEG-cyclam, and free BP-PEG-polymers were removed bycentrifugation before mixing with radioactivity. The resulting par-ticles showed a high 64Cu radiolabelling efficiency in a short time(o5 minutes) (Fig. 2b). Both NPs display essentially the magneticproperties of Fe3O4 or MnFe2O4 NPs and are active on MR images(Fig. 2c and d). The transverse (r2) and longitudinal (r1) relaxivitiesof Fe3O4@HA NPs were measured to be 150.2  3.2 mM1 s1and 1.9  0.1 mM1 s1, respectively, at 3 T magnetic field. Asexpected, the relaxivities of NPs could be improved by altering theratio of magnetic component and non-magnetic HA, since r2 isproportional to the volume fraction of magnetic component.20 Forexample, the r2 of MnFe2O4@HA NPs could be dramaticallyimproved from 105.7  3.5 mM1 s1 to 246.5  15.9 mM1 s1by doubling the amount of MnFe2O4@Al(OH)3 while keeping theamount of NaH2PO4 and CaCl2 solutions the same during thesynthesis. High transverse relaxivity of these magnetic hydroxy-apatite NPs as well as a high ratio of r2/r1 demonstrate theirpotential application as T2 contrast agents on MR imaging.Fluorescent HA is normally produced either by doping withrare earth cations (Eu or Tb),8,11,21 or by conjugation with organicdyes.10,22 Here we conjugated the fluorescent dyes Maria blueand Alexa Fluors covalently to the NPs surface using sodiumpamidronate as an aminobisphosphonate linker (see ESI†). Theamine group of pamidronate is reactive for NHS ester dyes toform stable amide bonds while its bisphosphonate group inter-acts strongly with the Ca or Fe at the surface of NPs; dyes arethus linked to NPs without the risk of leakage. Fluorescentspectra in Fig. 3 show an emission at 455 nm for the conjugatesof Fe3O4@HA and Maria blue under excitation at 365 nm, andan emission at 517 nm for the Alexa Fluors 488 conjugates underexcitation at 488 nm. More importantly, the fluorescence of theseconjugated NPs is stable and strong even after being stored atroom temperature for over one month, implying the potentialapplications as optical contrast.In summary, we have presented a facile approach to synthe-sise magnetic and fluorescent hydroxyapatite nanoparticles witha well-defined morphology and uniform size, using Al(OH)3-stabilised Fe3O4 or MnFe2O4 NPs as templates. The change fromthe highly positively charged surface and base-solubility of theAl(OH)3 layer to the neutral or slightly negative zeta potentialand acid solubility of the Fe3O4@HA particles is likely tooffer alternative biological properties. These NPs are promisingcandidates for development as tri-modal probes for MR, PET andoptical imaging, since they display excellent colloidal stabilityand high radiolabelling efficiency both for 64Cu and for 18F, aswell as fluorescent and magnetic properties. Radiolabelling withother metallic radioisotopes will also be achievable by replacingcyclam with corresponding chelators. This synthesis approachallows us to tune the magnetic properties of particles by alteringthe ratio of precursors, without decreasing the radiolabellingefficiency or fluorescent property. The flexible conjugation methodensures that dyes with different wavelengths could be selectedfor different applications. The synthesis strategy for conjuga-tion of BP-PEG-cyclam can also be applied to the conjugation ofBP-PEG with peptides or antibodies, leading to the applicationin targeted imaging.We acknowledge Mr Dirk Kru¨ger from Division of ImagingScience and Biomedical engineering, King’s College London,for his works on the magnetic relaxivitity measurement. Thisresearch was supported by the Centre of Excellence in MedicalEngineering Centre funded by the Wellcome Trust and EPSRCunder grant number WT088641/Z/09/Z, and the Kings CollegeFig. 2 (a) DLS size distribution of 1 mg ml1 Fe3O4@HA NPs in aqueoussolution; (b) radiolabelling of MnFe2O4@HA and Fe3O4@HA NPs with18F-fluoride and 64CuCl2 (the latter after sonicating the particles with BP-PEG-cyclam); (c) T1 and T2 weighted MR images of the solution containingFe3O4@HA NPs, and (d) relaxivities of Fe3O4@HA NPs. Concentration ofiron in the solution was measured by ICP-MS.Fig. 3 Emission and excitation spectra of aqueous solutions of Fe3O4@HAconjugates with (a) Maria blue and (b) Alexa Fluors 488.ChemComm CommunicationOpen Access Article. Published on 28 April 2015. Downloaded on 22/06/2015 14:01:38.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineThis journal is©The Royal Society of Chemistry 2015 Chem. Commun., 2015, 51, 9332--9335 | 9335London and UCL Comprehensive Cancer Imaging Centre fundedby the CRUK and EPSRC in association with the MRC and DoH(England), and by the National Institute for Health Research(NIHR) Biomedical Research Centre at Guy’s and St Thomas’NHS Foundation Trust and King’s College London. The viewsexpressed are those of the author(s) and not necessarily those ofthe NHS, the NIHR or the Department of Health.Notes and references1 N. E. Bolus, R. George, J. Washington and B. R. Newcomer, J. Nucl.Med. Technol., 2009, 37, 63–71; quiz 72-63.2 M. Baker, Nature, 2010, 463, 977–980.3 Z. Ali, A. Z. Abbasi, F. Zhang, P. Arosio, A. Lascialfari, M. F. Casula,A. Wenk, W. Kreyling, R. Plapper, M. Seidel, R. Niessner, J. Knoell,A. Seubert and W. J. Parak, Anal. Chem., 2011, 83, 2877–2882.4 M. Baker, Nat. Methods, 2010, 7, 957–962.5 R. T. M. de Rosales, R. Tavare, R. L. Paul, M. Jauregui-Osoro,A. Protti, A. Glaria, G. Varma, I. Szanda and P. J. Blower, Angew.Chem., Int. Ed., 2011, 50, 5509–5513.6 L. Sandiford, A. Phinikaridou, A. Protti, L. K. Meszaros, X. Cui, Y. Yan,G. Frodsham, P. A. Williamson, N. Gaddum, R. M. Botnar, P. J. Blower,M. A. Green and R. T. M. de Rosales, ACS Nano, 2012, 7, 500–512.7 M. Lewin, N. Carlesso, C. H. Tung, X. W. Tang, D. Cory,D. T. Scadden and R. Weissleder, Nat. Biotechnol., 2000, 18, 410–414.8 J. Hui and X. Wang, Chem. – Eur. J., 2011, 17, 6926–6930.9 H.-C. Wu, T.-W. Wang, M. C. Bohn, F.-H. Lin and M. Spector,Adv. Funct. Mater., 2010, 20, 67–77.10 H. Liu, F. Chen, P. Xi, B. Chen, L. Huang, J. Cheng, C. Shao, J. Wang,D. Bai and Z. Zeng, J. Phys. Chem. C, 2011, 115, 18538–18544.11 J. Pan, J. Zhang, L. Wang and D. Wan, Chem. Commun., 2014, 50,14010–14012.12 R. X. Yan and Y. D. Li, Adv. Funct. Mater., 2005, 15, 763–770.13 J. W. Stouwdam and F. van Veggel, Nano Lett., 2002, 2, 733–737.14 X. Cui, S. Belo, D. Kru¨ger, Y. Yan, R. T. M. de Rosales, M. Jauregui-Osoro, H. Ye, S. Su, D. Mathe, N. Kova´cs, I. Horva´th, M. Semjeni,K. Sunassee, K. Szigeti, M. A. Green and P. J. Blower, Biomaterials,2014, 35, 5840–5846.15 G. Olive and A. Jacques, Phosphorus, Sulfur Silicon Relat. Elem., 2003,178, 33–46.16 B. S. Lele and M. G. Kulkarni, J. Appl. Polym. Sci., 1998, 70, 883–890.17 V. Kubicek, J. Rudovsky, J. Kotek, P. Hermann, L. V. Elst, R. N. Muller,Z. I. Kolar, H. T. Wolterbeek, J. A. Peters and I. Lukes, J. Am.Chem. Soc., 2005, 127, 16477–16485.18 R. Torres Martin de Rosales, C. Finucane, S. J. Mather andP. J. Blower, Chem. Commun., 2009, 4847–4849.19 M. Jauregui-Osoro, P. A. Williamson, A. Glaria, K. Sunassee,P. Charoenphun, M. A. Green and P. J. Blower, Dalton Trans.,2011, 40, 6226–6237.20 Q. L. Vuong, J.-F. Berret, J. Fresnais, Y. Gossuin and O. Sandre, Adv.Healthcare Mater., 2012, 1, 502–512.21 O. A. Graeve, R. Kanakala, A. Madadi, B. C. Williams and K. C. Glass,Biomaterials, 2010, 31, 4259–4267.22 M. Neumeier, L. A. Hails, S. A. Davis, S. Mann andM. Epple, J. Mater.Chem., 2011, 21, 1250–1254.Communication ChemCommOpen Access Article. Published on 28 April 2015. Downloaded on 22/06/2015 14:01:38.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

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

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

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