Synthesis, Radiolabelling and In Vitro Imaging of Multifunctional Nanoceramics

Abstract Molecular imaging has become a powerful technique in preclinical and clinical research aiming towards the diagnosis of many diseases. In this work, we address the synthetic challenges in achieving lab‐scale, batch‐to‐batch reproducible copper‐64‐ and gallium‐68‐radiolabelled metal nanoparticles (MNPs) for cellular imaging purposes. Composite NPs incorporating magnetic iron oxide cores with luminescent quantum dots were simultaneously encapsulated within a thin silica shell, yielding water‐dispersible, biocompatible and luminescent NPs. Scalable surface modification protocols to attach the radioisotopes 64Cu (t1/2=12.7 h) and 68Ga (t1/2=68 min) in high yields are reported, and are compatible with the time frame of radiolabelling. Confocal and fluorescence lifetime imaging studies confirm the uptake of the encapsulated imaging agents and their cytoplasmic localisation in prostate cancer (PC‐3) cells. Cellular viability assays show that the biocompatibility of the system is improved when the fluorophores are encapsulated within a silica shell. The functional and biocompatible SiO2 matrix represents an ideal platform for the incorporation of 64Cu and 68Ga radioisotopes with high radiolabelling incorporation.


General Information
Characterisation of small molecule compounds was carried out using 1 H NMR and Mass Spectrometry. The 1 H NMR spectra were obtained on a 300 MHz Bruker Ultra Shield Spectrometer. All compounds for NMR analysis were dissolved in DMSO-d6-and referenced according to the residual solvent peak. Mass spectrometry was carried out using the microTOF (ESI-TOF) at the University of Bath.

Powder X-ray diffraction
The analyses of the crystalline structure and the phase identification were performed by X-ray diffraction (XRD Bruker D8 ADVANCE, Madison, WI) with a monochromatized source of Cu Kα1 radiation (λ = 1.5406 nm) at 1.6 kW (40 kV, 40 mA); samples were prepared by placing a drop of a concentrated ethanol dispersion of particles onto a single crystal silicon plate.

TEM/EDX
All images were taken on a JEOL JEM1200, a transmission electron microscope with an operating voltage ranging from 40 to 120 keV and all elemental analysis were carried out on an Oxford Energy Dispersive X-ray Spectrometer (EDS). All spectra were calibrated against a copper standard. In EDX, copper and nickel peaks were consistently observed between 7 and 10 keV as a result of the grid used to place the samples on.

In vitro fluorescence imaging
The cells were cultured at 37 ºC in a humidified atmosphere in air and diluted once confluence had been reached. Culture occurred in Eagle's Minimum Essential Medium (EMEM) for FeK4, HeLa, CHO cells. The media contained foetal calf serum (FCS) (15% for FeK4), 0.5% penicillin/streptomycin and 1% L-Glutamine. Surplus supernatant containing dead cell matter and the excess protein was aspirated. The live adherent cells were then washed with 2 x 10 mL aliquots of phosphate buffer saline (PBS) solution to remove any remaining media containing FCS, which inactivates trypsin. Cells were resuspended in solution by incubation in 3 mL of trypsin-PBS solution (0.25% trypsin) for 5 min at 37 ºC.
After trypsinisation, 5 mL of medium containing serum was added to inactivate the trypsin and the solution was centrifuged for 5 min (1000 rpm, 25 ºC) to remove any remaining dead cell matter. The supernatant liquid was aspirated and 5 mL of medium was added to the cell matter left behind. Cells were counted using a haemocytometer and then seeded as appropriate.
Cells were seeded as 10,000 cells per well and incubated for at least 48 h prior to microscopy. The wells were twice washed either PBS or SFM warmed to 37 ºC.
The working distance of a 60x objective lens is 0.17 mm (40x = 0.2 mm); therefore cells were seeded onto wells of which the coverslip was the bottom of the well. Wells were pre-coated with poly-d-lysine to equally maximise cell attachment and minimise detachment during washing steps.
PC-3 cells were cultured at 37 OC in 5 % CO2 atmosphere and diluted once a suitable confluency had been obtained. PC-3 cells were cultured in RPMI 1640 containing 10 % heat activated foetal calf serum (FCS), 0.5 % penicillin/streptomycin (10000 mg/ml) and 200 mM L-glutamine. The medium contained no fluorescent indicator dyes such as phenol red and was therefore suitable for use in fluorescence imaging studies. The excess supernatant containing dead cell constituents and excess proteins and metabolites was aspirated. The viable live adherent cells were washed with 2x10 ml aliquots of PBS to remove residual media containing FCS. Cells were then re-suspended in 10 ml PBS with additional 2-5 ml Trypsin and incubated for a further 5 minutes at 37 OC. After trypsinisation, 5 ml of medium containing 10 % serum was added to inactivate the trypsin and the suspension centrifuged for 5 minutes (1000 rpm) to remove residual dead cell constituents. The resulting supernatant was aspirated and 5 ml of medium was added. Cells were counted using a haemocytometer and seeded as appropriate.
Fresh DMEM (10% FCS) was added to the suspended cells to give a sufficient concentration of cells (ca. 300000 cells/mL). The cells were plated in a Petri dish with a glass coverslip (MaTek) and left for 24 h to adhere before fluorescence imaging measurements were made.

Confocal Microscopy was performed using a Nikon A1Rsi Laser Scanning Confocal
Microscope System fitted with 60X oil objective lens, equipped with three lasers (405.0, 488.0, and 561.0 nm). The microscope was also fitted with a motorized piezo z-stage, halogen lamp and mercury lamp for epifluorescence microscopy. All images were processed using functions within the NIS elements software package.
Two-photon excitation experiments were performed at the Rutherford Appleton Laboratory A mode locked Mira titanium sapphire laser (Coherent Lasers Ltd, USA), generating 180 fs pulses at 75 MHz and emitting light at a wavelength of 710-970 nm was used for the 2photon excitation. The laser was pumped by a solid state continuous wave 532 nm laser (Verdi V18, Coherent Laser Ltd), with the oscillator fundamental output of 915 ± 2 nm or 810 ± 2 nm. The laser beam was focused to a diffraction limited spot through a water immersion ultraviolet corrected objective (Nikon VC x60, NA1.2) and specimens illuminated at the microscope stage of a modified Nikon TE2000-U with UV transmitting optics. The focused laser spot was raster scanned using an XY galvanometer (GSI Lumonics). Fluorescence emission was collected and passed through a coloured glass (BG39) filter and detected by fast microchannel plate photomultiplier tube used as the detector (R3809-U, Hamamatsu, Japan).
These were linked via a TCSPC PC module SPC830. Lifetime calculations were obtained using SPCImage analysis software (Becker and Hickl, Germany) or Edinburgh Instruments F900 TCSPC analysis software.

Relative fluorescence quantum yields (QY) determination
UV/visible and fluorescence spectra were recorded on Perkin Elmer Lambda S50 UV/Vis and a Perkin Elmer LS55 Luminescence spectrometer, respectively. Room temperature fluorescence QY was calculated according to the following equation: In this equation subscripts r refers to the reference (anthracene), while s is referred to Cd0.1Zn0.9Se and Fe3O4/Cd0.1Zn0.9Se@SiO2. Φr and Φs are the fluorescence QY of anthracene (0.36), and unknown (Cd0.1Zn0.9Se and Fe3O4/Cd0.1Zn0.9Se@SiO2, respectively). A is the absorbance of the solution, E is the corrected emission intensity, I is the relative intensity of the exciting light and n is the average refractive index of the solutions.

Synthesis of iron oxide nanoparticles for a microemulsion method of coating
10 ml of 1 M FeCl3 were mixed with 2.5 ml of 2 M FeCl2 dissolved in 2 M HCl. Both solutions were freshly prepared with deoxygenated water before use. Immediately after being mixed under nitrogen, the solution containing the iron chlorides was added to 125 ml of potassium hydroxide solution (0.7 M) under vigorous mechanical stirring, and under nitrogen atmosphere. After 30 min, the black precipitate formed was separated magnetically using a standard permanent magnet and washed with water (3×250 ml). Finally, oleic acid (5 mmol) was dissolved in 5 ml of acetone and was dropwise added. The crystal structure of the black powder was investigated by X-ray diffraction. Figure S1 shows a crystalline single-phase pattern corresponding to Fe3O4 (ICDD file no. 86-2368) with no perceivable traces of other phases. Figure S1. X-ray diffraction pattern for a) the synthesised Fe3O4 nanoparticles and b) Fe3O4@SiO2 MNPs.

Coating of MNPs with a silica shell using microemulsion method
44.60 g of polyoxyethylene(5)isooctylphenyl ether (IGEPAL CA-520) was dispersed in 700 ml of cyclohexane. Then, 200 mg of Fe3O4 nanoparticles dispersed in cyclohexane (20 mg·ml -1 ) was added. The mixture was stirred until it became transparent. After this step, 9.44 ml of ammonium hydroxide (29% aqueous solution) was added to form a reverse microemulsion. Finally, 7.70 ml of tetraethylorthosilicate (TEOS) was added. The solution was gently stirred for 16 h. The nanocomposite was precipitated with methanol and separated by magnetic decantation. The X-ray diffractogram of the obtained powder confirms that the Fe3O4 phase remains unaffected by the microemulsion method ( Figure S1b).

Synthesis of dye-doped silica coated MNPs
0.223 g of polyoxyethylene(5)isooctylphenyl ether was dispersed in 3.5 ml of cyclohexane.
Then, 1.0 mg of Fe3O4 dispersed in cyclohexane (20 mg·ml -1 ) was added. The mixture was stirred until it became transparent. After this step, 45 µl of ammonium hydroxide (29 % aqueous solution), containing different amounts of dye (methylene blue 10 mg·ml -1 , fluorescein 20 mg·ml -1 , rhodamine B 10 mg·ml -1 , Rubpy 40 mg·ml -1 ) was added to form a reverse microemulsion. Finally, 39 µl of TEOS was added. The solution was gently stirred for 16 h. The nanocomposite was precipitated with methanol and separated by magnetic decantation.
Then, 1.0 mg of Fe3O4 dispersed in cyclohexane (20 mg·ml -1 ) was added, followed by 50 µl of QD (Lumidot CdSe 5 mg·ml -1 ). The mixture was stirred until it became transparent. After this step, 45 µl of ammonium hydroxide (29% aqueous solution) was added to form a reverse microemulsion. Finally, 39 µl of TEOS was added. The solution was gently stirred for 16 h.
The nanocomposite was precipitated with methanol and separated by magnetic decantation.

Synthesis of Cd0.1Zn0.9Se QDs
Stock solutions for Se and ZnEt2 were prepared in a glovebox under atmosphere of argon (Ar). Cadmium stearate (0.2044 g, 0.3 mmol), stearic acid (0.1707 g, 0.6 mmol), TOPO (5.0 g), and ODA (5.0 g) were added to a flask, and the mixture was heated, under stirring, to 330 °C under Ar flow until a clear solution formed. At this temperature, a solution containing 0.1184 g of Se (1.5 mmol) dissolved in TOP was injected into the reaction flask and the temperature was set at 290 °C. After 5-10 min.
under stirring, the heating was removed to stop the reaction and allow the flask to cool to room temperature. After 1h, the mixture of CdSe and organic ligands was heated up to 300 °C again. An aliquot (3 mL) of the as-prepared crude CdSe reaction mixture, containing 0.1 mmol of CdSe, were transferred in a three-neck Schlenk flask and heated at 300 °C. At this temperature, 0.450 mL of ZnEt2 (TOP solution, 0.2 M) and 0.450 mL of Se (TOP solution, 0.2 M) were injected. After the addition, the reaction mixture was heated for 6 min, and then heat was removed to stop the reaction. Once the mixture reached room temperature, 9 mL of chloroform was added under stirring.
Quantum dots were precipitated in a mixture 1:1 of methanol/acetone and isolated by centrifugation and decantation. The same mixture of methanol/acetone (5×25 mL) was used to wash the QDs from the excess of organic ligands. Finally, Cd0.1Zn0.9Se nanocrystals were dispersed in 9 ml of n-hexane and characterized by optical spectroscopy.

Synthesis of hydrophilic Cd0.1Zn0.9Se QDs modified silica coated MNPs
In a typical experiment, 0.223 g of polyoxyethylene(5)isooctylphenyl ether was dispersed in 3.5 ml of cyclohexane. Then, 1.0 mg of Fe3O4 dispersed in cyclohexane (20 mg·ml -1 ) was added, followed by 50 µl of QD (water dispersible, 1.50 mg/ml). The mixture was stirred until it became transparent. After this step, 45 µl of ammonium hydroxide (29% aqueous solution) was added to form a reverse microemulsion. Finally, 39 µl of TEOS was added. The solution was gently stirred for 16 h. The nanocomposite was precipitated with methanol and separated by magnetic decantation.

Synthesis of 64 Cu modified and silica-coated MNPs
In this experiment, the positron-emitting radiotracer [ 64 Cu]Cu(OAc)2 (prepared by Drs Paul Burke and Frank Aigbirhio from Wolfson Brain Imaging Centre in Cambridge using a standard method) was used in order to establish a general method to incorporate radioactive materials in silica shells. Different amounts of [ 64 Cu]Cu(OAc)2 (from 100 µl of 100 MBq stock solutions at pH 8.4, using 10 mM NaOAc solutions as the support for the carrier-added radioactivity) was added (in different addition sequences) at several different stages during the coating process in order to optimize the encapsulation conditions, followed by aqueous washing and magnetic separation and evaluation of the radioactivity associated with the MNPs and residual phases. Taking into account the half-life (12.701 h) of the 64 Cu radionuclide, the radiolabeling yield, denoted here 'encapsulation factor', for all samples were estimated. The reaction conditions used and corresponding results are summarized in Tables S1 and S2 below. There is a difference between samples a-c (method 1) and d-f (method 2): smaller encapsulation factor can be simply explained by shorter time of reaction of [ 64 Cu]Cu(OAc)2 in the reverse microemulsion system. Table S1. Summary of the procedures for rapid preparation of 64 Cu-modified MNPs and reaction conditions for the 6 different approaches to encapsulation, resulting in Samples a-f after magnetic separation.      as the mobile phase.
The reaction was carried out for 40 min at 90 °C (using the vortex every 10 min). In a typical encapsulation experiment (method 2: c,d), 0.223 g of polyoxyethylene (5) isooctylphenyl ether (IGEPAL CA-520) was dispersed in 3.5 ml of cyclohexane. Then, in a vortex. Next, 45 μl of ammonium hydroxide (29 % aqueous solution) was added to form a reverse microemulsion, followed by 15 μl of tetraethylorthosilicate (TEOS). Lastly

General synthesis
Compounds 1-3 were synthesised using a method described by Dilworth et al. [Inorg. Chem. 2007, 46, 465−485]. For Compounds 4 and 5, the synthesis was simply adapted to suit the change in metal centres (e.g. Ga and In), the reactions are depicted in Scheme S1. Compound 2 was reacted with the zinc, gallium and indium reagents in the appropriate ratios as described in the experimental section, in methanol, refluxed for four hours and then collected by filtration. For compounds 4 and 5 the reagents GaCl3 and InCl3 were weighed out in a glove box to prevent hydrolysis or H2O complexation of the anhydrous reagents used.
Upon the addition of Zn(OAc)·2H2O to the Compound 2 there was an immediate colour change to yellow, which was retained in the solid product for Compound 3. For Compound 4 a bright orange solid was collected and a further fraction isolated as a brown/orange oil. For Compound 5 again a small amount of solid was collected and a second fraction was obtained, an orange/brown oil from the filtrate.

Synthesis of ligands and metal complexes
Adapted synthesis of Compound 2 Step (1): 4-methyl thiosemicarbazone (1.25 g, 11.9 mmol) was added to 100 ml of deionised water and vigorously stirred at 0 ºC. 5 drops of concentrated HCl was then added followed by the rapid Step (2): Thiocarbohydrazide (0.3664 g, 3.5 mmol) was added to 20 ml of ethanol and the suspension stirred at 50 ºC. 1 equivalence of compound emerging from Step (1)

Synthesis of Compound 6
Compound 3 acted as a precursor to the analogous copper complex in a transmetallation reaction. One equivalent of Compound 3 (0.034 g, 0.12 mmol) and two equivalents of copper acetate (0.044 mg, 0.24 mmol) were stirred in a methanol under reflux for 24 hours and the product was isolated by filtration.

Synthesis of Copper(II) (thiosemicarbazone) complex
Compound 6 was synthesised using a transmetallation reaction from the analogous Compound 3. Two equivalents of Cu(OAc)2 were added to Compound 3 in methanol and then stirred under reflux for 24 hours. As Compound 3 was the precursor to this reaction and Compound 3 had not been produced in any considerable quantity very little solid was collected from this reaction.    In comparison to the unchanged reverse microemulsion method, the addition of aqueous solution of 64 Cu(OAc)2 affects the size and shape and agglomeration state of resulting nanocomposites, likely due to the loss of water/oil equilibrium in reverse microemulsion.

TEM imaging and EDX analysis of organic and inorganic compoundsdopped silica NPs and MNPs
These MNPs were imaged post-decay after a 3 weeks period in an aqueous environment. The samples a-c appeared to be rather aggregated. Better results were obtained when 64 Cu(OAc)2 solution was added after the pre-coating period (samples d-f). There is a clear difference between nanoparticles d-f.
The best quality nanoparticles (sample e) were obtained when the smallest amount of the aqueous phase containing 64 Cu(OAc)2 stock solution was added (25 µl containing 25 MBq activity, 84% radiochemical incorporation in the 'chelate-free' method).

Dynamic Light Scattering (DLS)
DLS measurements were performed by using a Zetasizer Nano S dynamic light scattering instrument (Malvern instruments). DLA spectra were recorded at room temperature (25 o C), using a standard laser with the incident length of 633 nm and a power of 4 mW. Fe3O4 and Fe3O4/Cd0.1Zn0.9Se@SiO2 samples were prepared in methanol and analysed in disposal semimicro PS cuvettes (Fiscerbrand ® ). Cd0.1Zn0.9Se dispersions were prepared in chloroform and analysed in a Hellma ® quartz cuvette. All the particles sizes here reported are an average of diameters and relative percentage of three-time measurements (Table S8). Figure S23. DLS of particles with size and perceptual distributions of Fe3O4 (black line), Cd0.1Zn0.9Se (red line) and Fe3O4/Cd0.1Zn0.9Se @SiO2 (blue line).

Fluorescence Spectroscopy
The fluorescence studies were carried out using solutions at concentrations of 100 μM in HPLC-grade DMSO.
2D contour plots Compounds 2-5 were taken of in order to determine the excitation wavelength that produces the most intense fluorescence emission are shown in Figures S26-S29, where excitation wavelength was plotted against emission wavelength. Compound 2 was included as a way of comparing the variation in fluorescence between the free bis(thiosemicarbazone) ligand and the chelated metal complexes.
The intensity of the fluorescence for Compound 4 and 5 is greater with respect to Compound 2, whereas the fluorescence intensity for Compound 2 has decreased. Therefore these results show chelation of zinc to the bis(thiosemicarbazone) causes a decrease in fluorescence, whereas the chelation of gallium and indium causes an increase in chelation. Overall, however, the fluorescence intensity for all the Compounds 2-5 is rather weak. The plots show that each of the Compounds 2-5 has fluorescent properties that vary slightly in terms of intensity and emission wavelength, although all the compounds seem to produce the most intense fluorescence when excited with light of wavelength 390 nm.
When designing fluorescent probes it is important to ensure that the fluorescence emission of the probe is different to the excitation wavelength of any biological autofluorescence within the cell. It has generally been found that cells contain molecules that become fluorescent when excited with light in the UV/Vis region. Therefore fluorescent emission up to 500 nm is preferable. Table S9 shows that the most intense fluorescent emissions for Compounds 2-5 all fall in the region of 500 nm, making the compounds suitable for use as fluorescent probes. Table S9. Excitation wavelength required for the most intense fluorescence emission at the wavelength specified. It is clear from Figure S30 that Compound 4 has the most intense fluorescence when excited with light of wavelength 400 nm. There is little difference between the fluorescence intensities of Compounds 2 and 4, and it is again clearly shown that Compound 3 produces the weakest fluorescence.