Article Synthesis and Characterization of Citrate-Stabilized Gold-Coated Superparamagnetic Iron Oxide Nanoparticles for Biomedical Applications
René Stein 1,*, Bernhard Friedrich 1 , Marina Mühlberger 1, Nadine Cebulla 1, Eveline Schreiber 1, Rainer Tietze 1, Iwona Cicha 1, Christoph Alexiou 1, Silvio Dutz 2 , Aldo R. Boccaccini 3 and Harald Unterweger 1,* 1 Department of Otorhinolaryngology-Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kroener-Fresenius-Stiftung-Professorship, Universitätsklinikum, 91054 Erlangen, Germany; [email protected] (B.F.); [email protected] (M.M.); [email protected] (N.C.); [email protected] (E.S.); [email protected] (R.T.); [email protected] (I.C.); [email protected] (C.A.) 2 Institute of Biomedical Engineering and Informatics, Technische Universität Ilmenau, 98693 Ilmenau, Germany; [email protected] 3 Institute of Biomaterials, University of Erlangen-Nuremberg, 91058 Erlangen, Germany; [email protected] * Correspondence: [email protected] (R.S.); [email protected] (H.U.)
Academic Editor: Ashok Kakkar Received: 31 August 2020; Accepted: 24 September 2020; Published: 26 September 2020
Abstract: Surface-functionalized gold-coated superparamagnetic iron oxide nanoparticles (Au-SPIONs) may be a useful tool in various biomedical applications. To obtain Au-SPIONs, gold salt was precipitated onto citrate-stabilized SPIONs (Cit-SPIONs) using a simple, aqueous one-pot technique inspired by the Turkevich method of gold nanoparticle synthesis. By the further stabilization of the Au-SPION surface with additional citrate (Cit-Au-SPIONs), controllable and reproducible Z-averages enhanced long-term dispersion stability and moderate dispersion pH values were achieved. The citrate concentration of the reaction solution and the gold/iron ratio was found to have a major influence on the particle characteristics. While the gold-coating reduced the saturation magnetization to 40.7% in comparison to pure Cit-SPIONs, the superparamagnetic behavior of Cit-Au-SPIONs was maintained. The formation of nanosized gold on the SPION surface was confirmed by X-ray diffraction measurements. Cit-Au-SPION concentrations of up to 100 µg Fe/mL for 48 h had no cytotoxic effect on Jurkat cells. At a particle concentration of 100 µg Fe/mL, Jurkat cells were found to take up Cit-Au-SPIONs after 24 h of incubation. A significantly higher attachment of thiol-containing L-cysteine to the particle surface was observed for Cit-Au-SPIONs (53%) in comparison to pure Cit-SPIONs (7%).
Keywords: nanoparticles; superparamagnetic iron oxide nanoparticles (SPIONs); gold coating; thiol-binding; surface functionalization; characterization; cytotoxicity
1. Introduction Gold nanoparticles (Au-NPs) or gold-coated nanoparticles have extraordinary material properties that make them particularly interesting for biomedical applications. In particular, the surface-binding property of gold gives rise to a wide variety of possible utilizations. Simple and strong binding motifs on Au-NPs are found, e.g., for thiols or disulfide compounds [1–3]. Selenides, isothiocyanates and phosphines also show a stable surface attachment. In addition, amines and carboxylates exhibit an
Molecules 2020, 25, 4425; doi:10.3390/molecules25194425 www.mdpi.com/journal/molecules Molecules 2020, 25, 4425 2 of 23 affinity with the gold surface as well [4]. The functionalization of gold surfaces is far from being fully understood and novel functionalization strategies are still being discovered that lead to very stable and selective linkers, as recently described by De Jesus et al. in the form of new N-heterocyclic carbenes [5]. In applications as bioprobes, gold surfaces can be functionalized with antibodies or DNA aptamers, thus binding the corresponding complementary molecules. For the subsequent detection of these interactions, the enzyme-mediated growth or aggregation of Au-NPs is employed [6]. In the same way, components of viruses can be bound as antigens to gold nanoparticles to develop improved vaccination strategies [7]. Furthermore, surface modifications can be used, e.g., to create drug delivery systems when functionalized with therapeutics [8–11], as well as for photothermal therapy for cancer [12–15]. The biodistribution of pure Au-NPs is, however, hardly controllable, which hampers the accumulation of the particles in the target tissue and therefore their efficacy during application [16–18]. Due to their magnetic properties, superparamagnetic iron oxide nanoparticles (SPIONs) are likewise promising for biomedical applications. Modification of their surface enables SPIONs to be used, e.g., as contrast agents in magnetic resonance imaging [19,20] or as drug delivery systems for the magnetic drug targeting of tumors and vascular injuries [21–23]. By combining the highly magnetic properties of SPIONs with an easy-to-functionalize gold surface, a fundamental hybrid carrier system (Au-SPIONs) can be generated [24–27], on which molecules designed for various medical applications can be attached. Hence, applications such as photothermal or drug delivery therapy may not be limited by the unspecific accumulation of the particles in the tissue as Au-SPIONs can be magnetically guided for a control of their biodistribution and localization of their efficacy in the targeted tissue. Furthermore, Au-SPIONs may exhibit enhanced biocompatibility in comparison to mere SPIONs due to their bioinert gold surface. The functionalization and corresponding biomedical application can only be successful with a simple, reliable and reproducible aqueous synthesis that delivers nanoparticles with the appropriate qualities. Many of the procedures found in the literature, however, are based on non-aqueous SPION synthesis; e.g., thermal decomposition or micro-emulsion methods [24,28–32], which require potentially harmful substances or solvents in steps of their synthesis. For aqueous and non-harmful procedures [27,33–35], no studies were published, which shows the systematic optimization of the synthesis parameters and the evaluation of their effects on Au-SPION properties such as particle size, dispersion stability and reproducibility. By modifying and adapting the method of Elbialy et al. [34], we present in this study a simple, aqueous synthesis procedure for a highly reproducible and size controllable production of citrate-stabilized gold-coated SPIONs (Cit-Au-SPIONs). This research highly focuses on providing an understanding for the effects of various synthesis parameters on the produced nanoparticles as they are systematically investigated. The synthesized particles are physicochemically characterized and further tested for cytotoxicity to ensure biocompatibility. As a proof of concept, the thiol group containing amino acid L-cysteine is attached to the citrate-stabilized gold surface to show the facile thiol–gold binding motif and surface functionalization of Cit-Au-SPIONs, which may be used in various medical purposes.
2. Results
2.1. Characterization of Cit-SPIONs, Au-SPIONs and Cit-Au-SPIONs During the reduction of gold onto Cit-SPIONs to generate SPION cores with a surrounding gold shell, the color of the particle dispersion changed from black to a deep wine red, as seen in Figure1a. There were no major differences in color between non-stabilized Au-SPIONs and Cit-Au-SPIONs, which have additional citrate on their gold shell. This was further confirmed by ultraviolet–visible spectroscopy measurements, which are shown in Figure A1. On scanning electron microscopy (SEM) images, Cit-Au-SPION clusters (Figure1d) resemble the spherical clusters seen in pure Cit-SPIONs (Figure1b), whereas non-stabilized Au-SPIONs form large irregularly shaped structures (Figure1c). Molecules 2020, 25, 4425 3 of 23
MoleculesMolecules 20202020,, 2255,, xx FORFOR PEERPEER REVIEWREVIEW 33 ofof 2323 Accordingly, dynamic light scattering (DLS) data (Figure2a) display a large increase in the Z-average Accordingly,Accordingly, dynamicdynamic lightlight scatteringscattering ((DLSDLS)) datadata ((FigureFigure 2a2a)) displaydisplay aa largelarge increaseincrease inin thethe ZZ--averageaverage from 107 3 nm with a corresponding polydispersity index (PDI) of 0.15 0.03 in pure Cit-SPIONs fromfrom 107107± ±± 33 nmnm withwith aa correspondingcorresponding polydispersitypolydispersity indexindex ((PDIPDI)) ofof 0.150.15 ±± 0.030.03 inin purepure CitCit--SPIONsSPIONs to 405 83 nm with a corresponding PDI of 0.25 0.06 in non-stabilized Au-SPIONs. As shown in toto 405405± ±± 8383 nmnm withwith aa correspondingcorresponding PDIPDI ofof 0.250.25 ±±± 0.060.06 inin nonnon--stabistabillizedized AuAu--SPIONsSPIONs.. AsAs shownshown inin Figure2b, a huge deviation in the Z-average was observed between di fferent Au-SPION batches as well FigureFigure 22b,b, aa hhugeuge deviationdeviation inin thethe ZZ--averageaverage waswas observedobserved betweenbetween differentdifferent AuAu--SPIONSPION batchesbatches asas as during the storage for 21 days. The presence of additional citrate ions on Cit-Au-SPIONs reduced wellwell asas duringduring thethe storagestorage forfor 2121 daysdays.. TheThe presencepresence ofof additionaladditional citratecitrate ionsions onon CitCit--AuAu--SPIONsSPIONs the Z-average to 152 5 nm with a corresponding PDI of 0.19 0.01, improved the reproducibility in reducedreduced the the Z Z--averageaverage± to to 152 152 ±± 55 nmnm withwith a a corresponding corresponding± PDI PDI of of 0.190.19 ±± 0.010.01,, improvedimproved thethe sizereproducibilityreproducibility of different particle inin size size batchesofof different different and enhanced particle particle batches batches the long-term and and enhancedenhanced stability theduring the longlong storage.--termterm stability stability Cit-Au-SPIONs during during exhibitedstorage.storage. Cit Cit similar--AuAu--SPIONsSPIONs storage stabilityexhibited exhibited as similar similar pure Cit-SPIONs, storage storage stability stability with onlyasas pure pure small Cit Cit changes--SPIONsSPIONs in,, withwith their only only Z-averages small small overchangeschanges 21 days. inin theirtheir In contrast, ZZ--averagesaverages non-stabilized overover 2121 daysdays Au-SPIONs.. InIn contrast,contrast, sedimented nonnon--stabilizedstabilized out AuAu of dispersion--SPIONsSPIONs sedimensedimen after onetteded week outout ofof of storagedispersiondispersion at 4 after◦afterC, as oneone shown wweekeek in ofof Figure storstorageage A2 atat. 44 °C°C,, asas shownshown inin FigureFigure A2A2..
FigureFigureFigure 1. 1. 1. ((aa)) ParticleParticle dispersions dispersions of pure pure citrate-stabilizedcitratecitrate--stabilizedstabilized ( ((Cit)-superparamagneticCitCit))--superparamagneticsuperparamagnetic iron iron iron oxide oxide oxide nanoparticlesnanoparticlesnanoparticles (SPIONs) (SPIONs)(SPIONs) (left),(left),(left), Au-SPIONsAuAu--SPIONsSPIONs (middle) (middle) andand CitCit Cit-Au-SPIONs--AuAu--SPIONsSPIONs (right)(right) (right) asas as wellwell well asas as scanningscanning scanning electronelectronelectron microscopy micro microscopyscopy (SEM) (SEM) (SEM) images images images of ofof ( b ( ()bb) pure) pure pure Cit-SPION Cit Cit--SPIONSPION clusters, clusters, clusters, (c )((cc Au-SPION)) AuAu--SPIONSPION clusters clusters clusters and and and (d ) Cit-Au-SPION((dd)) CitCit--AuAu--SPIONSPION clusters. clusters.clusters.
FigureFigureFigure 2. 22.. ( (aa)) Mean Mean ZZ-averages Z--averagesaverages over over 21 21 days for CitCit-SPIONs, Cit--SPIONSPIONs,s, Au Au Au-SPIONs--SPIONsSPIONs and and and Cit Cit Cit-Au-SPIONs;--AuAu--SPIONs;SPIONs; (b((bb))) Z-Averages ZZ--AveragesAverages of of of Cit-SPIONs, Cit Cit--SPIONs,SPIONs, Au-SPIONs Au Au--SPIONsSPIONs and and and Cit-Au-SPIONs Cit Cit--AuAu--SPIONsSPIONs during duri duri storagengng storage storage for di for forfferent different different time points. time time Statisticallypoints.points. StatisticallyStatistically significant significantsignificant changes changeschanges are marked areare markedmarked with * for withwithp < ** 0.05.forfor pp << 0.05.0.05.
ToToTo investigate investigateinvestigate whether whetherwhether thethethe SPIONsSPIONsSPIONs werewere coatedcoated with with gold gold oror whetherwhether purepure pure goldgold gold nanoparticlesnanoparticles nanoparticles formedformedformed without without without any any any attachment attachment attachment to to the the SPIONs, SPIONs, a a magnet magnet was was pla pla placedcedced next next next to to to the the the Au Au Au-SPION--SPIONSPION and and and Cit-Au-SPIONCitCit--AuAu--SPIONSPION dispersions. dispersions. dispersions. The TheThe supernatants supernatant supernatant ofss both of of both both samples samples samples were werecolorless were colorlesscolorless after 8 ah, afterfter which 88 hh indicated,, which which thatindicatedindicated any nanosized thatthat anyany goldnanosizednanosized is attached goldgold isis to attachedattached the SPIONs toto thethe and SPIONsSPIONs can be andand manipulated cancan bebe manipulatedmanipulated using a magnet usingusing (Figure aa magnetmagnet A3 ). At((FigureFigure an adjusted A3 A3).). AtAt pH a ann of adjusted adjusted 7.0, Cit-SPIONs, pHpH of of 7.0, 7.0,Au-SPIONs CitCit--SPIONs,SPIONs, and Au Au-- Cit-Au-SPIONsSPIONsSPIONs and and Cit Cit exhibit--AuAu--SPIONsSPIONs a ζ-potential exhibit exhibit a ofa ζζ48.0--potentialpotential6.3, ofof43.5 −−48.048.0 ±±0.6 6.3,6.3, and −−43.543.548.6 ±± 0.60.6 andand0.3 mV,−−48.648.6 respectively, ±± 0.30.3 mV,mV, respectivelyrespectively as listed in,, asas Table listedlisted1. inin The TableTable pH 11 values.. TheThe pHpH of − ± − ± − ± thevaluevalue particless ooff the the dispersions particle particle dispersions dispersions after synthesis after after were synthesis synthesis 8.27 were were0.07, 8.278.27 2.89 ±± 0.07,0.07,0.14 2.89 and2.89 6.21±± 0.140.14 0.14 and and for6.216.21 Cit ±± 0.140.14 SPIONs, for for ± ± ± Au-SPIONsCCitit SPIONs,SPIONs, and AuAu-- Cit-Au-SPIONs,SPIONsSPIONs andand CitCit-- respectively.AuAu--SPIONsSPIONs,, respectivelyrespectively..
Molecules 2020, 25, x FOR PEER REVIEW 4 of 23 Molecules 2020, 25, 4425 4 of 23 Table 1. Comparison of the basic parameters of Cit-SPIONs, Au-SPIONs and Cit-Au-SPIONs.
Table 1. Comparison ofZ- theAvg. basic parametersPDI ofζ Cit-SPIONs,-Potential @ Au-SPIONspH 7 pH V andalue Cit-Au-SPIONs.Relative Susceptibility
in nm in a.u. in mV in a.u. in a.u. Z-Avg. PDI ζ-Potential @ pH 7 pH Value Relative Susceptibility Cit-SPIONs in nm 107 ±in 3 a.u. 0.15 ± 0.03in mV–48.0 ± 6.3 in a.u.8.27 ± 0.07 in100% a.u. Cit-SPIONs 107 3 0.15 0.03 –48.0 6.3 8.27 0.07 100% Au-SPIONs ± 405 ± 83± 0.25 ± 0.06 ± −43.5 ± 0.6 ±2.89 ± 0.14 94% ± 2% Au-SPIONs 405 83 0.25 0.06 43.5 0.6 2.89 0.14 94% 2% ± ± − ± ± ± Cit-Au-SPIONs 152 5 0.19 0.01 48.6 0.3 6.21 0.14 89% 2% Cit-Au-SPIONs ± 152 ± 5 ± 0.19 ± 0.01− ± −48.6 ± 0.3 ±6.21 ± 0.14 89%± ± 2% Z-avg.: Z-average; PDI: polydispersity index; Cit: citrate; Au: gold; SPIONs: superparamagnetic iron oxideZ-avg.: nanoparticles. Z-average; PDI: polydispersity index; Cit: citrate; Au: gold; SPIONs: superparamagnetic iron oxide nanoparticles Fourier-transform infrared spectroscopy (FTIR) measurements, shown in Figure3, were used to Fourier-transform infrared spectroscopy (FTIR) measurements, shown in Figure 3, were used to investigate the citrate stabilization on the particles. Cit-SPIONs, Au-SPIONs and Cit-Au-SPIONs all investigate the citrate stabilization on the particles. Cit-SPIONs, Au-SPIONs and Cit-Au-SPIONs all showed a characteristic iron-oxygen (Fe-O) band between 550 and 650 cm 1 [36,37]. Characteristic showed a characteristic iron-oxygen (Fe-O) band between 550 and 650 cm−−1 [36, 37]. Characteristic peaks of deprotonated carboxylic acid groups (COO ) at ~844, ~904 and 1397 cm 1, as well as a broad peaks of deprotonated carboxylic acid groups (COO−−) at ~844, ~904 and 1397 cm−−1, as well as a broad band between 1550 and 1750 cm 1, were found in all three particle samples [38–40]. Peaks that could be band between 1550 and 1750 cm− −1, were found in all three particle samples [38-40]. Peaks that could related to hydroxyl groups (C-OH) oscillations at ~1070 and ~1275 cm 1 [38–40] were absent, or clearly be related to hydroxyl groups (C-OH) oscillations at ~1070 and ~1275− cm−1 [38-40] were absent, or less pronounced, in the spectrum of non-stabilized Au-SPIONs when compared to the spectra of clearly less pronounced, in the spectrum of non-stabilized Au-SPIONs when compared to the spectra Cit-SPIONs and Cit-Au-SPIONs. Furthermore, the relative intensities between the Fe-O band and the of Cit-SPIONs and Cit-Au-SPIONs. Furthermore, the relative intensities between the Fe-O band and COO features in the Au-SPION spectrum were lower in comparison to the relative intensities of the the COO− − features in the Au-SPION spectrum were lower in comparison to the relative intensities of features in the Cit-SPION and in the Cit-Au-SPION spectrum. As an example, the ICOO/IFe-O ratio for the features in the Cit-SPION and in the Cit-Au-SPION spectrum. As an example, the ICOO/IFe-O ratio the 904 cm 1 COO peak was calculated to be 0.039, 0.025 and 0.166 for Cit-SPION, Au-SPIONs and for the 904− cm−1 COO− − peak was calculated to be 0.039, 0.025 and 0.166 for Cit-SPION, Au-SPIONs Cit-Au-SPIONs, respectively. and Cit-Au-SPIONs, respectively.
Figure 3. 3. FTIR spectra of of Cit-SPIONs, Cit-SPIONs, Au-SPIONs Au-SPIONs and and Cit-Au-SPIONs. Cit-Au-SPIONs. Peak identificationidentification was performedperformed usingusing [[3636–-40]40]..
Due to the enhancedenhanced reproducibility and controllability,controllability, thethe citratecitrate stabilizationstabilization ofof Au-SPIONsAu-SPIONs waswas setset as as a a standard standard procedureprocedure andand only Cit-Au-SPIONsCit-Au-SPIONs were analyzedanalyzed in further experiments. Both, Cit-SPIONs Cit-SPIONs and and Cit-Au-SPIONs, Cit-Au-SPIONs, exhibited exhibited superparamagnetic superparamagne behavior,tic behavior, as illustrated as illustrated in Figure 4 ina. TheFigure HC 4avalues. The areHC values 0.221 kA ar/em 0.221 for Cit-SPIONskA/m for Cit and-SPIONs 0.225 kA and/m 0.225 forCit-Au-SPIONs. kA/m for Cit-Au However,-SPIONs. aHowever, large diff aerence large indifference their saturation in their magnetizationsaturation magnetization was detected. was After detected gold-coating,. After gold the-coating, saturation the magnetizationsaturation magnetization decreased todecreased 40.7% from to 40.7 54 to% 22from Am 542/ kg.to 22 Am2/kg.
Molecules 2020, 25, 4425 5 of 23 Molecules 2020, 25, x FOR PEER REVIEW 5 of 23
FigureFigure 4. 4.(a )(a Magnetization) Magnetization curvescurves andand ((bb)) XRDXRD measurements of of Cit Cit-SPIONs-SPIONs and and Cit Cit-Au-SPIONs.-Au-SPIONs. PeakPeak identification identification was was performed performed using using [[41,41,42 42]]. .
SimilarSimilar results results were were observed observed whenwhen examining examining the the volumetric volumetric susceptibility susceptibility of of the the samples. samples. RelativeRelative values, values, referring referring to to pure pure Cit-SPIONs Cit-SPIONs as as 100%,100%, showedshowed a reduct reductionion to to 94% 94% ± 2%2% and and 89 89%% ± 2%2% ± ± inin Au-SPIONs Au-SPIONs and and Cit-Au-SPIONs, Cit-Au-SPIONs, respectively. respectively. TheThe X-ray X-ray di diffractionffraction (XRD) (XRD) pattern pattern ofof Cit-SPIONsCit-SPIONs ( (FigureFigure 4b4b)) revealed revealed peaks peaks at at 30.2, 30.2, 35.6, 35.6, 43.1, 43.1, 57.0 and 62.7°. The detected peaks showed accordance with characteristic peaks of magnetite and 57.0 and 62.7◦. The detected peaks showed accordance with characteristic peaks of magnetite and maghemitemaghemite found found in in the the literature literature [[41,41,42 42]].. TheThe hklhkl values for for the the respective respective peaks peaks were were (220), (220), (311), (311), (400), (511) and (440). The lattice parameter and crystallite size of Cit-SPIONs was calculated with the (400), (511) and (440). The lattice parameter and crystallite size of Cit-SPIONs was calculated with the most intense peak at 35.6°. The lattice parameter of the sample was 8.37 Å . The crystallite size was most intense peak at 35.6 . The lattice parameter of the sample was 8.37 Å. The crystallite size was found to be about 16 nm◦. found to be about 16 nm. The analysis of Cit-Au-SPIONs revealed the appearance of an additional gold peak at 38.1° and The analysis of Cit-Au-SPIONs revealed the appearance of an additional gold peak at 38.1 and the broadening of the peak at 43.1°, while the characteristic peaks of Cit-SPIONs were still present.◦ theThe broadening gold lattice of parameter the peak was at 43.1 calculated◦, while to the be characteristic4.09 Å by using peaks the peak of Cit-SPIONs at 38.1°. The were gold stillcrystallite present. Thesize gold was lattice determined parameter to be was 29 nm. calculated to be 4.09 Å by using the peak at 38.1◦. The gold crystallite size wasFor determined an additional to beconfirmation 29 nm. of the gold formation on the SPION surface, energy dispersive X-rayFor an spectroscopy additional (EDX)confirmation was performed of the gold on formation Cit-SPIONs on the and SPION Cit-Au surface,-SPIONs, energy as shown dispersive in X-rayFigure spectroscopy A4. A peak (EDX) related was to performedgold was found on Cit-SPIONs in the spectrum and Cit-Au-SPIONs, of Cit-Au-SPIONs. as shown The Au/Fe in Figure ratio A4 . A peak(wt/wt related) on Cit- toAu gold-SPIONs was was found calculated in the spectrumto be 0.23 ± of 0.03. Cit-Au-SPIONs. The Au/Fe ratio (wt/wt) on Cit-Au-SPIONsTransmission was calculated electron microscopy to be 0.23 (TEM)0.03. images of Cit-SPIONs and Cit-Au-SPIONs show ± aggregatesTransmission of particles electron independent microscopy of the (TEM) gold-coating, images as of depicted Cit-SPIONs in Figure and A5 Cit-Au-SPIONs. show aggregates of particles independent of the gold-coating, as depicted in Figure A5. 2.2. Investigation of Parameters Affecting Cit-Au-SPION Synthesis 2.2. Investigation of Parameters Affecting Cit-Au-SPION Synthesis 2.2.1. Citrate Concentration 2.2.1. Citrate Concentration By increasing the citrate concentration in the Cit-Au-SPION dispersion, the Z-average decreased fromBy 405 increasing ± 83 to 318 the ± citrate 60, 167 concentration ± 5 and 152 ± in5 nm the for Cit-Au-SPION 0.0, 0.1, 0.5 and dispersion, 1.0 mg citrate the Z-average/mL, respectively decreased, fromas shown 405 83 in toFigure 318 5a60,. The 167 corresponding5 and 152 PDI5 nm values for 0.0, were 0.1, 0.25 0.5 and± 0.06, 1.0 0.25 mg ± citrate 0.03, /0.21mL, ± respectively, 0.01 and ± ± ± ± as0. shown19 ± 0.01. in FigureWith the5a. increase The corresponding in the citrate concentration, PDI values were particle 0.25 batches0.06, became 0.25 0.03,more 0.21reproducible0.01 and ± ± ± 0.19with0.01. less heterogeneous With the increase particle in the sizes citrate and concentration,smaller deviation particles in the batches Z-average became between more each reproducible other as ± withwell less as heterogeneousduring the storage particle time over sizes 21 and days smaller (Figure deviations 5b). Z-averages in the changed Z-average only between by 14 and each 9 nm other asduring well as 21 during days forthe 0.5 storage and 1.0 time mg over citrate 21/mL, days respectively. (Figure5b). T Z-averageshe ζ-potential changed at pH only 7 showed by 14 no and 9 nmsignificant during differences 21 days for even 0.5 andwhen 1.0 compared mg citrate to/ mL,non-stabiliz respectively.ed Au-SPIONs. The ζ-potential The unadjusted at pH 7 pH showed value no significantof the particle differences dispersion, even however, when compared increased to as non-stabilized more citrate was Au-SPIONs. added. The ThepH rose unadjusted from 2.89 pH ± 0.14 value to 3.19 ± 0.25, 5.52 ± 0.17 and 6.21 ± 0.14 for 0.0, 0.1, 0.5 and 1.0 mg citrate/mL (Table 2). Volumetric of the particle dispersion, however, increased as more citrate was added. The pH rose from 2.89 0.14 susceptibilities did not reveal an influence of the citrate concentration. The Au/Fe ratio (wt/wt) of ±the to 3.19 0.25, 5.52 0.17 and 6.21 0.14 for 0.0, 0.1, 0.5 and 1.0 mg citrate/mL (Table2). Volumetric particles± was kept± at 0.30 ± 0.04 for± all samples, as shown in Figure A6.
Molecules 2020, 25, 4425 6 of 23
Molecules 2020, 25, x FOR PEER REVIEW 6 of 23 susceptibilities did not reveal an influence of the citrate concentration. The Au/Fe ratio (wt/wt) of the particles was kept at 0.30 0.04 for all samples, as shown in Figure A6. Molecules 2020, 25, x FOR PEER± REVIEW 6 of 23
Figure 5. Influence of the citrate concentration in Cit-Au-SPION dispersions on (a) the Z-average and (b) the particle stability during 21 days of storage at 4 °C. Statistically significant changes are marked with * for p < 0.05. FigureFigure 5. 5.Influence Influence of of the the citrate citrate concentration concentration in in Cit-Au-SPION Cit-Au-SPION dispersions dispersions on on ( a()a) the the Z-average Z-average and and (b(Tableb) the) the particle 2.particle Comparison stability stability of during during the basic 21 21 days parametersdays of of storage storage of Cit at at- 4Au 4◦ C.°C.-SPIONs Statistically Statistically with significantvaried significant citrate changes changes concentrations. are are marked marked withwith * * for forp p< <0.05. 0.05. Citrate Conc. Z-Avg. PDI ζ-Potential @ pH 7 pH Value Relative Susceptibility Table 2. Comparison of the basic parameters of Cit-Au-SPIONs with varied citrate concentrations. in mgTable/mL 2. Comparisonin nm of the inbasic a.u. parameters ofin Cit mV-Au -SPIONs within a.u. varied citrate concentrations.in a.u.
Citrate0.0 Conc. 405Z-Avg. ± 83 0.25PDI ± 0.06 ζ-Potential−43.5 ± 0.6 @ pH 7 2.89pH ± Value 0.14 Relative94% Susceptibility ± 2% Citratein mg/ mLConc. Zin-A nmvg. inPDI a.u. ζ-Potentialin mV @ pH 7 pHin a.u.Value Relativein S a.u.usceptibility in mg0.1 /mL 318in ±nm 60 0.25in ± a.u.0.03 −44.9in ±mV 2.5 3.19in ± 0.25a.u. 89%in ± 4%a.u. 0.0 405 83 0.25 0.06 43.5 0.6 2.89 0.14 94% 2% ± ± − ± ± ± 0.10.0 318405 ± 6083 0.250.25 ± 0.030.06 −444.93.5 ± 2.50.6 3.192.89 ±0.25 0.14 89%94% ±4% 2% 0.5 167 ±± 5 0.21 ± 0.01 −4− 7.6 ±± 2.0 5.52 ±± 0.17 90% ±± 2% 0.5 167 5 0.21 0.01 47.6 2.0 5.52 0.17 90% 2% ± ± − ± ± ± 1.01.00.1 152318 ±± 5605 0.19 0.25 ± ± 0.01 0.010.03 −4−448.68.64.9 ± ±0.3 0.32.5 6.21 6.213.19 ± 0.14±0.14 0.25 89% 89%89% ± 2% ±2% 4% ± ± − ± ± ± 0.5 167Conc.: Conc.:± 5 concentration; concentration;0.21 ± 0.01 Z Z-avg.:-avg.: Z Z-average;-−4ave7.6rage; ± 2.0 PDI:PDI: polydispersity polydispersity5.52 ± 0.17 index. index . 90% ± 2%
2.2.2.2.2.2. ReactionReaction1.0 TimeTime152 ± 5 0.19 ± 0.01 −48.6 ± 0.3 6.21 ± 0.14 89% ± 2% Conc.: concentration; Z-avg.: Z-average; PDI: polydispersity index. ExtendingExtending thethe reactionreaction timetime from 15 to 60 and 300 min resulted resulted in in an an increased increased Z Z-average-average from from 152152 ± 55 toto 165165 ± 6 and 170 ± 66 nm, nm, respectively respectively (Figure (Figure 66a),a), but but it it did did not not affect a ffect the the hydrodynamic hydrodynamic size size 2.2.2.± Reaction± Time ± duringduring storagestorage (Figure(Figure6 6b).b).No Nostatistically statistically significantsignificant changeschanges in the PDI, ζζ--potential,potential, pH pH value value and and volumetricvolumetricExtending susceptibilitysusceptibility the reaction werewere time foundfound from asas 15thethe to reactionreaction 60 and times300 min were resulted variedvaried in ( (TableTable an increased3 3).). Z-average from 152 ± 5 to 165 ± 6 and 170 ± 6 nm, respectively (Figure 6a), but it did not affect the hydrodynamic size during storage (Figure 6b). No statistically significant changes in the PDI, ζ-potential, pH value and volumetric susceptibility were found as the reaction times were varied (Table 3).
FigureFigure 6.6.Influence Influence of of the the reaction reaction time time during during Cit-Au-SPION Cit-Au-SPION synthesis synthesis on on (a ()a the) the Z-average Z-average and and (b ) the(b) particlethe particle stability stability during during 21 days 21 ofdays storage of storage in a fridge. in a fridge. Statistically Statistically significant significant changes changes are marked are withmarked * for withp < 0.05.* for p < 0.05. Figure 6. Influence of the reaction time during Cit-Au-SPION synthesis on (a) the Z-average and (b) the particle stability during 21 days of storage in a fridge. Statistically significant changes are marked with * for p < 0.05.
Molecules 2020, 25, x FOR PEER REVIEW 7 of 23
Table 3. Comparison of the basic parameters of Cit-Au-SPIONs with varied reaction times. Molecules 2020, 25, 4425 7 of 23 Reaction Time Z-Avg. PDI ζ-Potential @ pH 7 pH Value Relative Susceptibility in min in nm in a.u. in mV in a.u. in a.u. Table 3. Comparison of the basic parameters of Cit-Au-SPIONs with varied reaction times. 15 152 ± 5 0.19 ± 0.01 −48.6 ± 0.3 6.21 ± 0.14 89% ± 2% Reaction Time Z-Avg. PDI ζ-Potential @ pH 7 pH Value Relative Susceptibility in min60 in165 nm ± 6 0.18in a.u.± 0.01 −5in0.0 mV ± 0.9 6.25in ± a.u. 0.13 84%in a.u.± 3% 15 152 5 0.19 0.01 48.6 0.3 6.21 0.14 89% 2% 300 170± ± 6 0.19± ± 0.01 −−48.2 ± 1.8 6.34 ± 0.19 85% ± 2% 60 165 6 0.18 0.01 50.0 0.9 6.25 0.13 84% 3% ± ± − ± ± ± 300 170 6 0.19Z-avg.:0.01 Z-average;48.2 PDI: 1.8polydispersity 6.34 index0.19. 85% 2% ± ± − ± ± ± Z-avg.: Z-average; PDI: polydispersity index. 2.2.3. Reaction Temperature and Precursor Concentration 2.2.3.By Reaction changing Temperature the reaction and temperature Precursor Concentration measured in an oil bath surrounding the reaction flask fromBy 90 changing to 110 and the 130 reaction °C , no temperature statistically measured significant in an changes oil bath were surrounding found for the the reaction Z-average, flask fromPDI, 90ζ-potential, to 110 and pH130 ◦ valueC, no statistically and volumetric significant susceptibility changes were (data found shown for thein Figure Z-average, A7 PDI,and ζTable-potential, A1). pHSimilarly, value and changing volumetric the susceptibilityiron and gold (data precursor shown inconcentration Figure A7 and together Table A1 resulted). Similarly, in only changing minor thechanges. iron and Upon gold doubling precursor the concentration precursor conce togetherntration resulteds, no in statistical only minorly significant changes. Upon changes doubling in the theZ-average, precursor PDI, concentrations, ζ-potential and no statistically volumetric significant susceptibility changes were in found the Z-average, (Table A2 PDI,). Hζalving-potential the andprecursor volumetric concentration susceptibility led to were a decrease found in (Table particle A2 ).size Halving to 120 ± the 10 precursornm (Figure concentration A8) and in PDI led value to a decreaseto 0.16 ±in 0.01. particle The sizeζ-potential to 120 and10 nmvolumetric (Figure A8 susceptibility) and in PDI were value unaffected to 0.16 0.01. by the The 50%ζ-potential decrease and in ± ± volumetricthe precursor susceptibility concentration. were However, unaffected with by the increasing 50% decrease precursor in the concentrations, precursor concentration. the pH value However, of the withparticle increasing dispersion precursor significantly concentrations, decreased the pH from value 6.59 of ± the 0.13 particle for 50% dispersion to 6.21 significantly ± 0.14 for decreased 100% and from5.50 ± 6.59 0.17 for0.13 200% for 50% iron to and 6.21 gold0.14 concentration for 100% and. 5.50 0.17 for 200% iron and gold concentration. ± ± ± 2.2.4.2.2.4. GoldGold ConcentrationConcentration ParticlesParticles synthesizedsynthesized withwith 50%,50%, 100%100% andand 200%200% goldgold concentrationconcentration showedshowed anan AuAu/Fe/Fe ratioratio ofof 0.150.15 ± 0.05, 0.30 0.30 ± 0.020.02 and and 0.56 0.56 ± 0.01,0.01, respectivel respectively.y. An increase An increase in the in Z the-average Z-average from from103 ± 1031 to 1521 ± to 5 ± ± ± ± 152and 2395 and ± 49 239 nm49 for nm 50%, for 50%, 100% 100% and and 200% 200% gold gold concentration concentration were were observed observed,, as as illustrated illustrated in ± ± inFigure Figure 7a7.a. By By doubling doubling the the gold gold concentration, concentration, the reproducibility ofof thethe synthesissynthesis decreaseddecreased and and largelarge deviationsdeviations inin thethe Z-averageZ-average appearedappeared betweenbetween thethe particleparticle batchesbatches andand duringduring theirtheir 2121 daysdays storagestorage time,time,as asdepicted depictedin inFigure Figure7 b.7b. Halving Halving the the gold gold concentration concentration resulted resulted in in a a more more narrow narrow PDI PDI value,value, whilewhile doublingdoubling thethe goldgold concentrationconcentration revealedrevealed largelarge deviationsdeviations inin thethe PDI.PDI. TheTheζ ζ-potential-potential andand volumetricvolumetric susceptibilitysusceptibility werewere notnot influencedinfluenced byby thethe goldgold saltsalt concentration,concentration, whilewhile raisingraising thethe concentrationconcentration of of gold gold decreased decreased the the pH pH value value of of the the particle particle dispersion dispersion from from 6.66 6.66 ±0.06 0.06 to to 6.21 6.21 ±0.14 0.14 ± ± andand 5.055.05 ± 0.100.10 forfor 50%,50%, 100%100% andand 200%200% ofof gold,gold, respectivelyrespectively (Table(Table4 ).4). ±
FigureFigure 7. 7.Influence Influence of of the the gold gold concentration concentration of Cit-Au-SPIONs of Cit-Au-SPIONs on (a ) on the ( Z-averagea) the Z-average and (b) theand particle (b) the stabilityparticle duringstability 21 during days of 21 storage days of in storage a fridge. in Statisticallya fridge. Statistically significant significant changes are changes marked are with marked * for pwith< 0.05. * for p < 0.05.
Molecules 2020, 25, x FOR PEER REVIEW 8 of 23
Molecules 2020, 25, 4425 8 of 23 Table 4. Comparison of the basic parameters of Cit-Au-SPIONs with varied gold concentrations.
Gold Z-Avg. PDI ζ-Potential @ pH 7 pH Value Relative Susceptibility Table 4. Comparison of the basic parameters of Cit-Au-SPIONs with varied gold concentrations. Concentration in nm in a.u. in mV in a.u. in a.u. Gold Z-Avg. PDI ζ-Potential @ pH 7 pH Value Relative Susceptibility 50% 103 ± 1 0.15 ± 0.01 −50.3 ± 0.9 6.66 ± 0.06 87% ± 9% Concentration in nm in a.u. in mV in a.u. in a.u. 50%100% 103 1521 ± 5 0.150.19 0.01± 0.01 −450.38.6 ± 0.30.9 6.21 6.66 ± 0.140.06 89% 87% ± 2%9% ± ± − ± ± ± 100% 152 5 0.19 0.01 48.6 0.3 6.21 0.14 89% 2% ± ± − ± ± ± 200%200% 239 23949 ± 490.14 0.14 0.06± 0.06 −446.76.7 ± 1.51.5 5.05 5.05 ± 0.100.10 86% 86% ± 3%3% ± ± − ± ± ± ZZ-avg.:-avg.: Z Z-average;-average; PDI:PDI: polydispersity polydispersity index. index
2.3.2.3. Cell ToxicityToxicity andand UptakeUptake ofof Cit-Au-SPIONsCit-Au-SPIONs SterileSterile Cit-Au-SPIONsCit-Au-SPIONs werewere usedused forfor cellcell testing.testing. The important physicochemicalphysicochemical characteristicscharacteristics suchsuch as as the the Z-average Z-averag ande and susceptibility susceptibility of Cit-Au-SPIONsof Cit-Au-SPIONs did not did change not change after autoclaving, after autoclaving as shown, as inshown Table in5. Table 5.
TableTable 5.5. ComparisonComparison ofof thethe Z-averageZ-average andand volumetricvolumetric susceptibilitysusceptibility ofof Cit-Au-SPIONsCit-Au-SPIONs beforebefore andand afterafter autoclaving.autoclaving.
Z-avg.Z-avg. PDIPDI VolumetricVolumetric Susceptibility Susceptibility 10×1 03−3 × − in nmin nm inin a.u. a.u. inin a.u. a.u. Cit-Au-SPIONsCit-Au-SPIONs 1451452 ± 2 0.170.17 0.01± 0.01 5.05.0 ±0.3 0.3 ± ± ± Sterile Cit-Au-SPIONs 141 1 0.17 0.01 5.1 0.1 ± ± ± SterileZ-avg.: Cit Z-average;-Au-SPIONs Cit: citrate; Au:141 gold; ± 1 SPIONs: 0.17 superparamagnetic ± 0.01 iron oxide5.1 nanoparticles.± 0.1
Z-avg.: Z-average; Cit: citrate; Au: gold; SPIONs: superparamagnetic iron oxide nanoparticles Jurkat cell viability results by Annexin A5 fluorescein isothiocyanate (FITC) conjugate Jurkat cell viability results by Annexin A5 fluorescein isothiocyanate (FITC) conjugate (AxV)/propidium iodide (PI) staining are presented in Figure8a. Viable cells (AxV −PI−) are illustrated (AxV)/propidium iodide (PI) staining+ are presented in Figure 8a. Viable cells (AxV−PI−) are illustrated+ in green, early apoptotic cells (AxV PI−) in blue and late apoptotic or necrotic cells (PI ) in red. in green, early apoptotic cells (AxV+PI−) in blue and late apoptotic or necrotic cells (PI+) in red. When When comparing cells treated with H2O as a control to cells treated with different concentrations of Cit-Au-SPIONs,comparing cells no treated statistically with H significant2O as a control changes to in cells cell viability treated with were different observed concentrations up to the highest of Cit-Au-SPIONCit-Au-SPIONs, concentration no statistical evenly significant after 48 h. change At 100sµ ing Fecell/mL, viability cell viability were observed of 90% 1%up wasto the observed. highest ± MoreCit-Au diluted-SPION Cit-Au-SPION concentration dispersions even after exhibit 48 h. At viabilities 100 µg ofFe 92%/mL, cell0.3%, viability 89% 3%, of 90% 91% ± 1% andwas ± ± ± 91%observed1%. for More 10, 25, diluted 50 and Cit 75-Auµg-SPION Fe/mL, respectively.dispersions exhibit viabilities of 92% ± 0.3%, 89% ± 3%, 91% ±± 1% and 91% ± 1% for 10, 25, 50 and 75 μg Fe/mL, respectively.
FigureFigure 8.8. (a) Jurkat cell viability evaluated by Annexin A5 fluorescein fluorescein isothiocyanate (FITC) conjugate (AxV)(AxV)/propidium/propidium iodide iodide (PI) (PI) staining staining after 48 after h of incubation 48 h of incubation with different with Cit-Au-SPION different Citconcentrations-Au-SPION andconcentrations (b) Cit-Au-SPION and (b) uptake Cit-Au into-SPION Jurkat uptake cells evaluated into Jurkat by cells Lucifer evaluated Yellow stainingby Lucifer after Yellow 24 and staining 48 h of incubationafter 24 and with 48 h di offf incubationerent concentrations with different of Cit-Au-SPIONs. concentrations Statistically of Cit-Au-SPIONs. significant Statistically changes are significant marked withchanges * for arep < marked0.05. with * for p < 0.05.
Molecules 2020, 25, 4425 9 of 23 Molecules 2020, 25, x FOR PEER REVIEW 9 of 23
ToTo investigate investigate the the uptake uptake of Cit-Au-SPIONs of Cit-Au-SPIONs into the into cells, theLucifer cells, Yellow Lucifer staining Yellow was staining performed. was performed.The resulting The mean resulting fluorescence mean fluorescence index is displayed index inis Figuredisplayed8b for in iron Figure concentrations 8b for iron ofconcentrations 10, 25, 50, 75 ofand 10, 100 25, µ50,g/ mL75 and at incubation 100 μg/mL times at incubation of 24 and times 48 h. of In 24 comparison and 48 h. In to comparison the H2O control, to the H a2 significantO control, aincrease significant in the increase mean in fluorescence the mean fluorescence index was detected index was only detected at iron only concentrations at iron concentrations of 100 µg/mL. of 100After μg 24/mL. h, the After mean 24 fluorescence h, the mean index fluorescence reached values index of reached 1.13 0.04 values for H ofO 1.13 and ± 1.34 0.04 0.03 for forH2O 100 andµg ± 2 ± 1.iron34 ±/mL. 0.03 Comparable for 100 μg iron values/mL. wereComparable measured values after were 48 h measured with 1.12 after0.05 48 for h with H O 1.12 and ± 1.34 0.05 for0.02 H for2O ± 2 ± and100 µ1.34g iron ± 0.02/mL. for 100 μg iron/mL.
2.4.2.4. L L-Cysteine-Cysteine B Bindinginding to to Cit Cit-Au-SPIONs-Au-SPIONs CitCit-SPIONs-SPIONs and and Cit-Au-SPIONs Cit-Au-SPIONs were were incubated incubated with with L-cysteine L-cysteine solution solution in order in to order test whether to test whetherthe specific the bindingspecific of bind thiol-containinging of thiol-containing molecules molecules to the gold to surface the gold occurs surface in higher occurs amounts in higher as amountscompared as to compared iron oxide to surfaces. iron oxide By analyzing surfaces. theBy supernatantsanalyzing the of supernatants Cit-SPIONs and of Cit Cit-Au-SPIONs-SPIONs and Citusing-Au Ellman-SPIONs0s reagentusing Ellman after the′s incubation reagent after with the 1 mM incubation L-cysteine, with the 1 relative mM L binding-cysteine, of L-cysteine the relative to bindingthe particles of L-cysteine was calculated, to the particles as depicted was in calculated Figure9. , as depicted in Figure 9.
FigureFigure 9. 9. RelativeRelative L L-cysteine-cysteine binding binding to to Cit Cit-SPIONs-SPIONs and and Cit Cit-Au-SPIONs-Au-SPIONs after after incubation incubation in in a a 1 1 mM mM LL-cysteine-cysteine solution solution for for 2 2 h. h. Stati Statisticallystically significant significant changes changes are are marked marked with with * * for for pp << 0.05.0.05.
ForFor Cit Cit-SPIONs,-SPIONs, thethe relativerelative L-cysteine L-cysteine binding binding increased increased with with higher higher particle particle concentrations concentrations from 2.3% 0.1% to 5.4% 0.4% and 6.6% 0.2% for concentrations of 93, 186 and 272 µg Fe/mL, respectively. from ±2.3% ± 0.1% to± 5.4% ± 0.4% and± 6.6% ± 0.2% for concentrations of 93, 186 and 272 µg Fe/mL, Cit-Au-SPIONs exhibited significantly higher relative cysteine bindings of 46.4% 0.7%, 50.8% 0.2% respectively. Cit-Au-SPIONs exhibited significantly higher relative cysteine bindings± of 46.4% ±± 0.7%, and 52.6% 0.3% for concentrations of 93, 186 and 272 µg Fe/mL and 25, 50 and 75 µg Au/mL. 50.8% ± 0.2%± and 52.6% ± 0.3% for concentrations of 93, 186 and 272 µg Fe/mL and 25, 50 and 75 µg Au/mL. 3. Discussion 3. DiscussionThis study aimed to create size controllable, colloidal stable, reproducible and non-cytotoxic gold-coatedThis study SPIONs aimed that to create show sizepromising control propertieslable, colloidal for attaching stable, reproducible thiol-containing and non molecules-cytotoxic on goldtheir-coated surface. SPIONs that show promising properties for attaching thiol-containing molecules on their surface.3.1. Cit-SPION Characterization
3.1. CitMere-SPION Cit-SPIONs Characterization were synthesized following the protocol of Mühlberger et al. modified by a dialysis step instead of magnetic separation to remove excess citrate that might interfere during Mere Cit-SPIONs were synthesized following the protocol of Mühlberger et al. modified by a the gold reduction step. In comparison to SPIONs reported in that study (hydrodynamic size of dialysis step instead of magnetic separation to remove excess citrate that might interfere during the 58 nm, PDI of 0.149) [43], Cit-SPIONs exhibited a larger hydrodynamic size (107 nm) and a slightly gold reduction step. In comparison to SPIONs reported in that study (hydrodynamic size of 58 nm, narrower size distribution (0.129). Through the dialysis, excess citrate was removed from the dispersion, PDI of 0.149) [43], Cit-SPIONs exhibited a larger hydrodynamic size (107 nm) and a slightly narrower which could have caused an instability of dispersed Cit-SPION cores. The small cores could have size distribution (0.129). Through the dialysis, excess citrate was removed from the dispersion, which therefore aggregated onto larger clusters causing an increase in average hydrodynamic size and a could have caused an instability of dispersed Cit-SPION cores. The small cores could have therefore decrease in PDI. The large aggregation of SPION clusters found on SEM images can be explained by aggregated onto larger clusters causing an increase in average hydrodynamic size and a decrease in some inhomogeneity during the freeze-drying sample preparation as the solvent sublimates. PDI. The large aggregation of SPION clusters found on SEM images can be explained by some inhomogeneity during the freeze-drying sample preparation as the solvent sublimates.
Molecules 2020, 25, 4425 10 of 23
3.2. Gold-Coating of Cit-SPIONs
During the reduction of chloroauric acid (HAuCl4) onto Cit-SPIONs to create Au-SPIONs, the SPION dispersions0 color turned from black to a deep red, which could indicate the formation of nanosized gold [44,45]. Non-stabilized Au-SPIONs exhibited insufficient size controllability, reproducibility and long-term stability. Thus, Au-SPIONs were additionally stabilized with citrate after the gold reduction. The resulting Cit-Au-SPIONs showed improved reproducibility, long-term stability in dispersion and moderate dispersion pH values. During the reduction of HAuCl4 to solid gold, the acidic HAuCl4 and the stabilizing citrate on the particle surfaces are consumed and H+ is generated as a byproduct of the reaction [46]. The consequential decrease in pH value is especially pronounced for non-stabilized Au-SPIONs, while the addition of citrate to Cit-Au-SPIONs may buffer the generated H+, thus causing the observed significant increase in the dispersions0 pH value. Furthermore, the strong decrease in the pH value of Au-SPIONs could indicate that citrate ions that stabilize the Cit-SPION cores react almost completely with the added HAuCl4 solution. Therefore, the citrate is consumed which leaves the synthesized Au-SPIONs without surface stabilization. The resulting agglomeration of Au-SPIONs could lead to the increase in the Z-average and PDI. By adding more citrate after the gold-coating, the formed gold-coated SPIONs can be re-stabilized, which is demonstrated by more a controllable and stable Z-average, smaller PDI and higher pH value of Cit-Au-SPIONs. FTIR measurements further provided evidence that citrate was mostly consumed on non-stabilized Au-SPIONs. The disappearance of peaks related to C-OH oscillations and the decrease in relative intensities of COO− related peaks can be explained by the consumption of citrate, which has three carboxyl groups and one hydroxyl group. During gold reduction, the citrate is converted to dicarboxy acetone [44,46]. Dicarboxy acetone has two carboxyl groups, but no hydroxyl group. Thus, features related to C-OH disappear and COO− peaks are less pronounced for non-stabilized Au-SPIONs. After the addition of citrate in Cit-Au-SPIONs, the intensities of these features reappear, which can indicate the stabilization with citrate. The comparison of the magnetic behavior of pure Cit-SPIONs and Cit-Au-SPIONs revealed a decrease in the saturation magnetization to 40.7% after the gold-coating. Similarly, the volumetric susceptibility dropped by ~10% after the coating process. Both findings could be caused by the shielding of the superparamagnetic iron oxide cores with a paramagnetic gold layer, which might reduce the total magnetic behavior of Cit-Au-SPIONs. Elbialy et al. also reported a change of the magnetic properties after the gold-coating, whereby their particles decreased to 32.8% of the starting saturation magnetization after the coating process, dropping from 58 to 19 Am2/kg [34]. In a direct comparison with this study, citrate stabilization may have contributed to slightly enhanced saturation magnetization, which was 22 Am2/kg in our Cit-Au-SPIONs. The XRD peaks detected for Cit-SPIONs and Cit-Au-SPIONs were in accordance with the characteristic peaks of magnetite and maghemite found in the literature [41]. For Cit-Au-SPIONs, two additional peaks at 38.1◦ and 44.5◦ were detected, which are correlated to characteristic gold peaks [42]. Thus, XRD data indicate the formation of crystalline gold with a lattice parameter of 4.09 Å calculated using the peak at 38.1◦. This value is in line with the lattice parameter found in the literature (4.08 Å) [47]. The iron oxide lattice parameter of Cit-SPIONs was calculated using the peak at 35.6◦ and had a value of 8.37 Å. This value is comparable to the lattice parameters of magnetite (8.40 Å) and maghemite (8.35 Å) reported in the literature [41,48]. The crystallite size was found to be about 16 nm, which is below the critical size of 20 nm for superparamagnetic behavior in magnetite and maghemite [49,50]. Elbialy et al. reported a crystallite size of 10 nm for their similarly synthesized SPIONs [34]. The iron precursor ratio of these particles was different in comparison to Cit-SPIONs, however, which could have caused the differences in crystallite size. The gold crystallite size was observed to be 29 nm. This value is comparable with the size of pure Au-NPs synthesized with the Turkevich method [51,52]. Hence, it could be assumed that individual Au-NPs had formed during the reduction and that these Au-NPs interacted with the SPION Molecules 2020, 25, 4425 11 of 23 cores. Elbialy et al. estimated their gold shell around the SPIONs to be 10–15 nm in thickness [34]. The calculation of the crystallite size of the iron oxide core by using the (311) iron oxide peak in the Cit-Au-SPION XRD pattern, however, resulted in a value of 35 nm instead of 16 nm when calculated from the Cit-SPION pattern. The difference in the iron oxide cores0 crystallite sizes could be due to the overlap of the (311) iron oxide peak and the (111) gold peak. Further analysis, such as high-resolution transmission electron microscopy, will be necessary in the future to examine the exact structure of the gold coating.
3.3. Factors Influencing the Gold-Coating Process When investigating the factors influencing the particle synthesis and coating, the citrate concentration as well as the gold concentration in the reaction liquid seem to have a major effect on the controllability of the Z-average, reproducibility and long-term stability of Cit-Au-SPIONs. While an increase in citrate concentration led to smaller hydrodynamic sizes, enhanced long-term stability, and a more neutral pH value with better reproducibility, increasing the gold concentration on SPIONs showed opposite effects. Longer reaction times resulted in an increase in the Z-average, which might be explained by the Ostwald ripening of the particles at elevated temperatures. Changing the reaction temperature did not have any effect on Cit-Au-SPIONs. Lower temperatures might have an influence, but further experiments would be necessary to test the potential effects. At 130 ◦C, the reaction solution started to boil. Therefore, there is no use in a further increase in the reaction temperature. Doubling the precursor concentration for iron and gold did not influence the particles except for a decrease in the pH value of the dispersion due to the increased formation of H+ during the gold reduction. In contrast, halving the precursor concentrations resulted in smaller hydrodynamic sizes of the particles and an increase in the pH. A strong influence was found when the Fe/Au ratio was varied. With an increase in the Au/Fe ratio, the hydrodynamic size of the particles increased while size controllability and reproducibility could not be maintained. Higher gold concentrations decreased the pH value of the particle dispersion presumably due to the higher H+ concentration which was generated during gold reduction.
3.4. Cytotoxicty and Uptake into Cells of Cit-Au-SPIONs No statistically significant cell toxicity was found when Jurkat cells were incubated for 48 h with Cit-Au-SPION concentrations up to 100 µg Fe/mL. Cell viability was comparable between the H2O control and the highest concentration of Cit-Au-SPIONs (100 µg Fe/mL) after 48 h of incubation. Jurkat cells were chosen for primary experiments as they are non-adherent T cells present in blood that will have contact with the particles during a potential application. Mühlberger et al. investigated the viability of EL4 T lymphocytes from mouse lymphoma treated with non-dialyzed citrate-stabilized SPIONs. At iron concentrations of 100 µg/mL, they reported cell viabilities of 87%, 80% and >70% for time points at 0, 24 and 48 h, respectively. [43]. Although EL4 and Jurkat cells are similar cell types, a direct comparison is not possible. Nevertheless, coating SPIONs with gold might decrease the slightly toxic effects of pure SPIONs on cells. After the gold-coating, the particle size of the SPIONs increases, which reduces the probability of particle uptake into the cells and inducing harmful effects inside the cell. Furthermore, gold can be considered as a bioinert material which intrinsically shows weak interactions with biological tissue and cells [53]. Therefore, a gold shell could prevent harmful interactions of the SPION core with the cell even after internalization. Results of Lucifer Yellow staining indicate that Cit-Au-SPIONs are incorporated into Jurkat cells only if the particle concentration is sufficiently high. Lower concentrations seemed to show no cellular uptake of the particles. A similar effect was described by Mühlberger et al. regarding their non-dialyzed citrate-stabilized SPIONs [43]. The cellular uptake of their particles in EL4 cells was observed at iron concentrations of 50 µg/mL and higher. Further experiments with higher particle concentrations and primary human cells are necessary to reach solid conclusions, however, on the toxicity and uptake of Cit-Au-SPIONs in different cell types. Molecules 2020, 25, 4425 12 of 23
3.5. L-Cysteine Binding Increasing the concentration of the particles from a relative concentration of 25 to 50 and 75 µg gold/mL led to an increase in L-cysteine attachment in Cit-SPION and Cit-Au-SPION samples. As the concentration of the particles increase, there is more available surface for the L-cysteine to attach. Thus, the relative surface binding of L-cysteine increases in both samples as the particle concentration is raised. Low amounts of L-cysteine might non-specifically bind to the iron oxide surface [54] or show some interaction with the stabilizing citrate on Cit-SPIONs [55]. The amount of relative surface binding on Cit-Au-SPIONs, however, was significantly higher than the binding on pure Cit-SPIONs. The specific attraction of the thiol group of the L-cysteine to the gold surface seems to be stronger than the unspecific attraction to the iron oxide surface via the carboxylic acid or amino group. Therefore, L-cysteine could be bound to Cit-Au-SPIONs in larger amounts than to pure Cit-SPIONs. These data indicate that Cit-Au-SPIONs constitute promising candidate particles for binding thiol-containing molecules or drugs.
4. Materials and Methods
4.1. Materials Iron (III) chloride hexa-hydrate (99%) and iron (II) chloride tetra-hydrate (99%) were purchased from Merck (Darmstadt, Germany). Ammonia (NH3, 25%), sodium citrate (99%), potassium bromide (KBr) (spectroscopy grade), dimethyl sulfoxide (DMSO, 99.5%), L-cysteine (98%), disodium hydrogen phosphate (99%) and Ellman0s reagent (98%) were purchased from Carl Roth (Karlsruhe, Germany). Chloroauric acid (HAuCl4, 99.9%), propidium iodide (PI, 94%) and Lucifer Yellow CH dipotassium salt (LY) were purchased from Sigma-Aldrich (Taufkirchen, Germany). RPMI 1640 medium, GlutaMAX supplement, Hoechst 33342 (Hoe), Annexin A5 fluorescein isothiocyanate (FITC) conjugate (AxV) and DiIC1(5) (1,10-dimethyl-3,3,30,30-tetramethylindodicarbocyanine iodide, DiI) were purchased from Thermo Fisher (Waltham, MA, USA). Fetal calf serum (FCS) was purchased from Biochrom (Berlin, Germany). T cell leukemia cells Jurkat (ACC 282) were purchased from DSMZ (German collection of Microorganisms and Cell Cultures, Braunschweig, Germany). Ringer0s solution was purchased from Fresenius Kabi (Bad Homburg, Germany). Deionized water was produced using a Merck Milli-Q purification system. All reagents were used without further purification.
4.2. Synthesis of Citrate-Stabilized SPIONs (Cit-SPIONs) Citrate-stabilized SPIONs (Cit-SPIONs) were synthesized according to the protocol described by Mühlberger et al. [43], modified by dialysis instead of magnetic separation to remove excess citrate. In a typical synthesis procedure, iron (II) and iron (III) salts were dissolved in H2O to achieve a solution containing 13.2 mg iron/mL. After deoxygenizing with argon, 25% NH3 (aq) was rapidly injected into the vigorously stirred iron salt solution. The now blackened SPION dispersion was further stirred for 10 min at room temperature. Next, 15 mL of a 293.3 mg/mL sodium citrate solution was added to the dispersion while mixing at 400 rpm. The temperature was raised to 90 ◦C and the dispersion was stirred for 30 min under reflux. After cooling to room temperature, the dispersion was transferred to dialysis tubes. The dispersion was dialyzed in H2O for 6 h. The dialyzed particles were filtered through a 0.2 µm filter membrane and stored at 4 ◦C until further use. These particles are referred to as Cit-SPIONs. All particle batches were synthesized in triplicate (n = 3).
4.3. Synthesis of Gold-Coated SPIONs Gold-coating of Cit-SPIONs was achieved by following the procedure of Elbialy et al. with some adaptations [34]. In a regular synthesis, 35 mL of a diluted Cit-SPION dispersion was added to a three-necked flask. The iron concentration of the Cit-SPION dispersion was previously adjusted using H2O. The dispersion was deoxygenized in argon for 5 min. The temperature was raised to 110 ◦C while stirring. Different amounts of a HAuCl4 solution were injected in one shot to the vigorously stirred Molecules 2020, 25, 4425 13 of 23
SPION dispersion. The dispersion turned deeply red during stirring for 15 min at 110 ◦C. After cooling to room temperature, the dispersion was transferred into glass flasks. The particles are further referred to as Au-SPIONs. All particle batches were synthesized in triplicate (n = 3).
4.4. Synthesis of Citrate-Stabilized Gold-Coated SPIONs To achieve an enhanced long-term stability and size control over Au-SPIONs, the above-mentioned procedure was extended by further stabilizing the particles using sodium citrate to create Cit-Au-SPIONs. After the addition of the HAuCl4 solution to the Cit-SPION dispersion and stirring for 15 min at 110 ◦C as described above, different amounts of a citrate solution were added. The dispersion was further stirred for 30 s and then cooled to room temperature. All particle batches were synthesized in triplicate (n = 3).
4.5. Investigation of Synthesis Parameters for Cit-Au-SPIONs To investigate the effects of different parameters on Cit-Au-SPIONs during synthesis, the process parameters were varied. The citrate concentration in the final Cit-Au-SPION dispersion was adjusted to 0.1, 0.5 and 1.0 mg/mL and compared to non-stabilized Au-SPIONs to examine the additional stabilization with citrate. Secondly, while the citrate concentration was kept at 1.0 mg/mL, the reaction time varied from 15 to 60 and 300 min. Furthermore, the reaction temperature, measured in an oil bath surrounding the reaction flask, decreased from 110 to 90 ◦C and increased to 130 ◦C. The overall dilution of the reaction solution was adjusted to 50%, 100% and 200% iron and gold precursor concentrations in relation to the Cit-Au-SPION synthesis procedure. Lastly, only the gold concentration was modified to 50%, 100% and 200%, while the iron concentration was kept at 100% in relation to the Cit-Au-SPION synthesis procedure. All particles were synthesized in individual triplicates (n = 3).
4.6. Physicochemical Characterization
4.6.1. Atomic Emission Spectroscopy (AES) The iron content in Cit-SPIONs and the iron and gold content in Au-SPIONs and Cit-Au-SPIONs was determined by AES measurements (Agilent 4200 MP-AES, Agilent Technologies, Santa Clara, CA, USA). AES samples were prepared by dissolving Cit-SPIONs in nitric acid while Au-SPIONs and Cit-Au-SPIONs were dissolved in a 1:1 (v/v) mixture of nitric acid and hydrochloric acid and the dissolved particles were diluted with H2O.
4.6.2. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDX) The size and shape of Cit-SPIONs, Au-SPIONs and Cit-Au-SPIONs were determined using a scanning electron microscope (Auriga, Zeiss, Oberkochen, Germany). The samples were prepared by freeze-drying (Alpha 1–2 LD plus, Martin Christ, Osterode am Harz, Germany) previously diluted SPION dispersions for 24 h on silicon sample holders. The EDX detector (Silicon Drift Detector (SDD)-X-MaxN, Oxford Instruments, Tubney Woods, Abingdon, United Kingdom) is part of the SEM. Therefore, the samples used in the SEM could also be used for the EDX analysis.
4.6.3. Transmission Electron Microscopy (TEM) Transmission electron microscopy (TEM) images were taken using a CM30 TEM/STEM (Philips, The Netherlands) operating at 300 kV. Specimens were prepared by drop casting diluted nanoparticle dispersions onto carbon-coated copper grids (Plano, Germany). For the CCD camera, a Tietz Fast Scan-F114 (Tietz Video and Image Processing Systems GmbH, Gauting, Germany) was used. Molecules 2020, 25, 4425 14 of 23
4.6.4. Dynamic Light Scattering (DLS) The hydrodynamic size of the SPIONs was determined by DLS using a Zetasizer Nano (Malvern instruments, Worcestershire, United Kingdom). Samples were prepared by diluting the dispersions with H2O. The iron concentration of Cit-SPIONs was adjusted to 50 µg/mL. The iron concentration of Au-SPIONs and Cit-Au-SPIONs was adjusted to 25 µg/mL. The measurement was conducted at 25 ◦C. The long-term stability of the SPION dispersions was determined by measuring the hydrodynamic size over three weeks. Time points were set to 0, 1, 2, 4, 7, 14 and 21 days, with day 0 being measured directly after the synthesis procedure was finished.
4.6.5. ζ-Potential Measurement The ζ-potential of the SPIONs was investigated using the Zetasizer Nano (Malvern instruments, Worcestershire, United Kingdom). SPION dispersions were diluted with H2O to an iron concentration of 50 (Cit-SPIONs) or 25 µg/mL (Au-SPIONs and Cit-Au-SPIONs). The pH value was adjusted to 7.0 by the dropwise addition 1% hydrochloric acid or 1 M sodium hydroxide.
4.6.6. Susceptibility Measurement To verify the magnetic properties of the synthesized SPIONs, the magnetic susceptibility was investigated by using a magnetic susceptibility meter (MS2G, Bartington Instruments, Witney, UK). The SPION dispersions were diluted with H2O to an iron concentration of 1 mg/mL for the measurement.
4.6.7. Vibrating Sample Magnetometry (VSM) VSM measurements (Micromag 3900, Lake Shore Cryotronics, Montgomery, Westervill, OH, USA) were used to examine the saturation magnetization of Cit-SPIONs and Cit-Au-SPIONs at a field of 955 kA/m. Freeze-dried powder samples were used.
4.6.8. Fourier Transform Infrared Spectroscopy (FTIR) For the investigation of citrate stabilization and gold formation, the samples were characterized with Fourier transform infrared spectroscopy. Potassium bromide pellets containing 1% (wt/wt) of sample were analyzed using a FTIR spectrometer (Alpha-P, Bruker, Billerica, MA, USA). OPUS software (Bruker, Billerica, USA) was used for background subtraction and baseline correction.
4.6.9. Ultraviolet–Visible Spectroscopy (UV–Vis) The absorption spectra of aqueous Cit-SPION, Au-SPION and Cit-Au-SPION dispersions with a concentration of 25 µg Fe/mL were measured using a UV–Vis Spectrophotometer (Libra S22, Biochrom, Cambridge, United Kingdom). The measurement range was set between 250 and 700 nm with a step size of 2 nm.
4.6.10. X-ray Diffraction (XRD) To further analyze the gold formation on the SPIONs, an X-ray diffraction (MiniFlex 600, Rigaku, Tokyo, Japan) was performed. Adequate amounts of Cit-SPIONs and Cit-Au-SPIONs were freeze-dried to receive about 30 mg of particle powder. The powder was dispersed in ethanol and dripped onto a sample holder for XRD analysis. A Cu Kα1 X-ray source with a wavelength of 1.5406 Å and a step size of 0.03◦/s was used. The distance of the atomic planes dhkl calculated by Bragg0s law (1) together with Equation (2) [56] was used to receive the lattice parameter a of the samples.
λ d = (1) hkl 2 sin(θ) × a dhkl = p , (2) h2 + k2 + l2 Molecules 2020, 25, 4425 15 of 23 where λ is the wavelength, θ is the diffraction angle and h, k, l are the Miller indices of the diffraction plane. With the usage of the Debye-Scherrer formula (3), the crystallite size dcry of the samples was calculated. 0.9 λ dcry = × , (3) FWHM cos(θ) × where FWHM represents the full-width at half maximum value of the XRD peaks [56].
4.7. In Vitro Toxicity For the investigation of the potential toxic effects of produced Cit-Au-SPIONs on the cell viability of the T cell leukemia cell line Jurkat (ACC 282), flow cytometry was used according to the protocols described by Mühlberger et al. [43]. Cit-Au-SPIONs were sterilized by autoclaving and analyzed afterwards to ensure comparable particle properties to non-sterile particles. Jurkat cells were cultured in RPMI medium supplemented with 10% fetal calf serum and 1% L-glutamine at 37 ◦C in a humidified 5% CO atmosphere. In total, 900 µL of a Jurkat cell suspension (1.5 105 cells) was transferred 2 × into each well of a 24-well plate, and 100 µL of prediluted sterile Cit-Au-SPION dispersions was added to the wells. The final iron concentration in the wells was set to 10, 25, 50, 75 and 100 µg/mL. The nanoparticle-treated samples were incubated at 37 ◦C in a humidified 5% CO2 atmosphere and were analyzed at 0, 24 and 48 h after the addition of the SPIONs. Sterile H2O was used as a negative control, while 2% and 5% of sterile DMSO were used as positive controls for cell death.
4.7.1. Cell Viability A Hoe/AxV/PI/DiI staining solution containing 1 µL/mL Hoe of a 10 mg/mL solution, 2 µL/mL AxV, 66.7 ng/mL PI and 0.4 µL/mL DiI of a 10 µM solution in Ringer0s solution was freshly prepared at each time point. In total, 250 µL of the as prepared staining solution was added to 50 µL of a well-mixed cell sample. The stained cell sample was incubated under light protection at 4 ◦C for 20 min before analyzing its fluorescence with a flow cytometer (Gallios, Beckman Coulter, Krefeld, Germany).
4.7.2. Particle Uptake into Cells To investigate whether particles enter the cell or whether they are adsorbed onto the cell membrane, Lucifer Yellow staining was performed as described by Mühlberger et al. [43]. Lucifer Yellow staining was performed under light protection. In the procedure, 20 µL of a 2 mg/mL Lucifer Yellow stock solution was added to 18 mL cell suspension containing 3 106 cells. In total, 900 µL of the stained cell × suspension (1.5 105 cells) was transferred into each of 18 wells of a 24-well plate. A total of 100 µL of × prior diluted sterile Cit-Au-SPION dispersions were added to each well. The final iron concentration in the wells was set to 10, 25, 50, 75 and 100 µg/mL. The final concentration of Lucifer Yellow in each well was 2 µg/mL. The samples were incubated under light protection at 37 ◦C in a humidified 5% CO2 atmosphere. To differentiate between aggregated particles and cells of various viability, the Lucifer Yellow stained cell samples were further stained with Hoe, PI and DiI. The Hoe/PI/DiI staining solution containing 1 µL/mL Hoe of a 10 mg/mL solution, 66.7 ng/mL PI and 0.4 µL/mL DiI of a 10 µM solution in Ringer0s solution was freshly prepared at each time point. A total of 250 µL of the as prepared staining solution was added to 50 µL of a well-mixed Lucifer Yellow-stained cell sample. The stained cell samples were incubated under light protection at 4 ◦C for 20 min before analyzing their fluorescence with flow cytometry. Flow cytometry data were analyzed using the Kaluza software (Version 2.1, Beckman Coulter, Krefeld, Germany).
4.8. L-Cysteine Binding on Cit-Au-SPIONs L-cysteine was dissolved in a 100 mM sodium phosphate buffer (pH 8) to generate a 10 mM L-cysteine solution. Adequate amounts of the Cit-Au-SPION dispersion were added to the sodium Molecules 2020, 25, 4425 16 of 23 phosphate buffer to yield 0.9 mL of buffered Cit-Au-SPION dispersions. A total of 0.1 mL of the 10 mM L-cysteine solution was added to the 0.9 mL buffered Cit-Au-SPION dispersions to achieve samples containing 1 mM L-cysteine with Au concentrations of 25, 50 and 75 µg/mL in the total sample volume. The samples were incubated at room temperature for 2 h while being mildly mixed at 250 rpm. As a negative control, pure Cit-SPIONs were used. The concentrations of the Cit-SPION samples were adjusted to fit the exact iron concentrations of the three Cit-Au-SPION samples. After 2 h of incubation, all samples were transferred in Vivaspin® tubes (100,000 MWCO) and centrifuged for 30 min at 10,000 rcf. The clear supernatants were used in the Ellman assay, which was conducted according to the manufacturers0 instructions Thermo Scientific Ellman0s reagent (Product No. 22582). A total of 250 µL of the prior made phosphate buffer was added into individual wells of a 96-well plate. Subsequently, 5 µL of a 4 mg/mL Ellman0s reagent phosphate buffer solution was added to each well, followed by the addition of 25 µL of the test samples and controls. The whole well plate was incubated for 15 min under gentle shaking. The absorption of the individual wells was analyzed at a wavelength of 412 nm using a plate reader (FilterMax F5, Molecular Devices, Biberach an der Riß, Germany).
4.9. Statistical Analysis
Statistical significance was examined by Student0s t-test with a p-value of 0.05. Statistical significances are marked with an asterisk.
5. Conclusions This study presented a novel SPION system with thiol-binding ability as a fundamental carrier system for various medical applications, such as intravascular drug delivery or photothermal therapy. Citrate-stabilized SPION cores were coated with a gold shell by the adaptation of a simple, aqueous and moderate procedure reported in the literature. Reaction parameters such as citrate-stabilization, reaction time, reaction temperature, precursor concentration and gold concentration were varied to investigate their influences on the particle properties and to generate a reproducible and controllable nanosystem. Low cytotoxicity in Jurkat cells and an enhanced attachment of thiol-containing L-cysteine confirmed the potential of Cit-Au-SPIONs for medical applications requiring specifically designed surface functionalization. The surface attachment of large thiol-containing molecules and the binding capacity of, e.g., drugs onto Cit-Au-SPIONs must be confirmed in future studies. Furthermore, hemocompatibility tests will be necessary to ensure that the particles have no negative impact on the whole blood and blood components. In further studies, the magnetic attraction of the SPIONs towards the targeted tissue must be likewise evaluated.
Author Contributions: Conceptualization, R.S. and H.U.; methodology, R.S., B.F. and H.U.; validation, R.S. and H.U.; formal analysis, R.S. and H.U.; investigation, R.S., N.C., E.S. and S.D.; resources, C.A.; data curation, R.S.; writing—original draft preparation, R.S.; writing—review and editing, H.U., B.F., M.M., S.D., R.T., A.R.B., I.C. and C.A.; visualization, R.S. and M.M.; supervision, B.F., A.R.B., M.M. and H.U.; project administration, H.U. and R.T.; funding acquisition, I.C. and C.A. All authors have read and agreed to the published version of the manuscript. Funding: The authors thank the Margarete Ammon Foundation, Munich, the Manfred-Roth-Stiftung, Fürth, Germany and the Forschungsstiftung Medizin am Universitätsklinikum Erlangen, Erlangen, Germany for the support. This work was funded in part by the Deutsche Forschungsgemeinschaft (German Research Foundation; CI 162/2-3). Conflicts of Interest: The authors declare no conflict of interest. Molecules 2020, 25, x FOR PEER REVIEW 17 of 23 Molecules 2020, 25, x FOR PEER REVIEW 17 of 23
Funding: The authors thank the Margarete Ammon Foundation, Munich, the Manfred-Roth-Stiftung, Fürth, Funding: The authors thank the Margarete Ammon Foundation, Munich, the Manfred-Roth-Stiftung, Fürth, Germany and the Forschungsstiftung Medizin am Universitätsklinikum Erlangen, Erlangen, Germany for the Germany and the Forschungsstiftung Medizin am Universitätsklinikum Erlangen, Erlangen, Germany for the support. This work was funded in part by the Deutsche Forschungsgemeinschaft (German Research Foundation; support. This work was funded in part by the Deutsche Forschungsgemeinschaft (German Research Foundation; MoleculesCI 162/2-20203). , 25, 4425 17 of 23 CI 162/2-3). Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Appendix Appendix
Figure A1. UV–Vis measurements of Cit-SPIONs, Au-SPIONs and Cit-Au-SPIONs showing an FigureFigure A1. A1. UVUV–Vis–Vis measurements measurements of of Cit Cit-SPIONs,-SPIONs, Au Au-SPIONs-SPIONs and and Cit- Cit-Au-SPIONsAu-SPIONs showing showing an intensity peak at a wavelength of 520 nm for both gold-coated SPION independent of additional intensityan intensity peak peak at a wavelength at a wavelength of 520 ofnm 520 for bothnm for gold both-coated gold-coated SPION independent SPION independent of additional of citrate-stabilization. citrateadditional-stabilization. citrate-stabilization.
Molecules 2020, 25, x FOR PEER REVIEW 18 of 23 Figure A2. Dispersion stability of Au Au-SPIONs-SPIONs and Cit-Au-SPIONsCit-Au-SPIONs after one week of storage at 4 ◦°CC.. Figure A2. Dispersion stability of Au-SPIONs and Cit-Au-SPIONs after one week of storage at 4 °C .
Figure A3. Magnetic separation of Au-SPIONs and Cit-Au-SPIONs after 8 h showed clear supernatants, Figure A3. Magnetic separation of Au-SPIONs and Cit-Au-SPIONs after 8 h showed clear which indicated that the gold was reduced onto the SPION cores instead of forming unattached supernatants, which indicated that the gold was reduced onto the SPION cores instead of forming nanosized gold that would appear red in the supernatant. unattached nanosized gold that would appear red in the supernatant.
Figure A4. Energy dispersive X-ray spectra of (a) Cit-SPIONs and (b) Cit-Au-SPIONs. A gold related peak appeared after gold coating process.
Figure A5. Transmission electron microscopy (TEM) images of (a) Cit-SPIONs and (b) Cit-Au-SPIONs.
Molecules 2020, 25, x FOR PEER REVIEW 18 of 23
Molecules 2020, 25, x FOR PEER REVIEW 18 of 23
Figure A3. Magnetic separation of Au-SPIONs and Cit-Au-SPIONs after 8 h showed clear Figure A3. Magnetic separation of Au-SPIONs and Cit-Au-SPIONs after 8 h showed clear Moleculessupernatants,2020, 25, 4425 which indicated that the gold was reduced onto the SPION cores instead of forming18 of 23 supernatants, which indicated that the gold was reduced onto the SPION cores instead of forming unattached nanosized gold that would appear red in the supernatant. unattached nanosized gold that would appear red in the supernatant.
Figure A4. Energy dispersive X-ray spectra of (a) Cit-SPIONs and (b) Cit-Au-SPIONs. A gold related Figure A4. Energy dispersive X-rayX-ray spectra of (a)) Cit-SPIONsCit-SPIONs andand ((bb)) Cit-Au-SPIONs.Cit-Au-SPIONs. A gold related peak appeared after gold coating process. peakpeak appearedappeared afterafter goldgold coatingcoating process.process.
Figure A5. Transmission electron microscopy (TEM) images of (a) Cit-SPIONs and (b) Cit-Au-SPIONs. Figure A5. Transmission electron microscopy (TEM) images of (a) Cit-SPIONs and (b) Cit-Au-SPIONs. FigureTable A1.A5. ComparisonTransmission of the electron parameters microscopy of Cit-Au-SPIONs (TEM) with images varied of reaction (a) Cit temperatures.-SPIONs and Reaction (b) Cit Temp.-Au-SPIONs.Z-Avg. PDI ζ-Potential @ pH 7 pH Value Relative Susceptibility in ◦C in nm in a.u. in mV in a.u. in a.u.
90 145 4 0.18 0.01 50.8 1.3 6.14 0.16 87% 4% ± ± − ± ± ± 110 152 5 0.19 0.01 48.6 0.3 6.21 0.14 89% 2% ± ± − ± ± ± 130 146 11 0.19 0.01 46.9 2.6 6.09 0.20 96% 3% ± ± − ± ± ± Temp.: temperature; Z-avg.: Z-average; PDI: polydispersity index.
Table A2. Comparison of the parameters of Cit-Au-SPIONs with varied precursor concentrations.
Precursor Z-Avg. PDI ζ-Potential @ pH 7 pH Value Relative Susceptibility Concentration in nm in a.u. in mV in a.u. in a.u. 50% 120 10 0.16 0.01 45.9 2.6 6.59 0.13 93% 0% ± ± − ± ± ± 100% 152 5 0.19 0.01 48.6 0.3 6.21 0.14 89% 2% ± ± − ± ± ± 200% 146 6 0.17 0.01 47.8 1.3 5.50 0.17 93% 15% ± ± − ± ± ± Z-avg.: Z-average; PDI: polydispersity index. Molecules 2020, 25, 4425 19 of 23 Molecules 2020, 25, x FOR PEER REVIEW 19 of 23
FigureFigure A6. A6.Gold Gold to to iron iron ratios ratios of of synthesized synthesized Cit-Au-SPIONs Cit-Au-SPIONs with with varied varied ( a()a) citrate citrate concentration concentration in in thethe particle particle dispersion, dispersion (, b(b)) reaction reaction times, times, ( c()c) reaction reaction temperatures, temperatures, ( d(d)) precursor precursor concentrations concentrations and and (e()e) gold gold concentrations. concentrations. TheThe targetedtargeted AuAu/Fe/Fe ratioratio forfor ((aa–)d–()d was) was 0.30. 0.30. For For (e ()e the) the targeted targeted ratios ratios were were 0.15,0.15, 0.30 0.30 and and 0.60 0.60 for for 50%, 50%, 100% 100% and and 200%, 200%, respectively. respectively. Statistically Statistically significant significant changes changes are are marked marked withwith * * for forp p< <0.05. 0.05.
Molecules 2020, 25, x FOR PEER REVIEW 20 of 23
Molecules 2020, 25, 4425 20 of 23 Molecules 2020, 25, x FOR PEER REVIEW 20 of 23
Figure A7. Influence of the reaction temperature measured in an oil bath surrounding the reaction flask on (a) the Z-average and (b) the particle stability during 21 days of storage in a fridge. Statistically significant changes are marked with * for p < 0.05.
Table A1. Comparison of the parameters of Cit-Au-SPIONs with varied reaction temperatures.
Reaction Temp. Z-Avg. PDI ζ-Potential @ pH 7 pH Value Relative Susceptibility in °C in nm in a.u. in mV in a.u. in a.u.
90 145 ± 4 0.18 ± 0.01 −50.8 ± 1.3 6.14 ± 0.16 87% ± 4%
110 152 ± 5 0.19 ± 0.01 −48.6 ± 0.3 6.21 ± 0.14 89% ± 2%
FigureFigure130 A7.A7. InfluenceInfluence146 ± ofof 11 thethe 0.19 reactionreaction ± 0.01 temperaturetemperature −46.9 measuredmeasured ± 2.6 inin anan6.09 oiloil ± bathbath0.20 surroundingsurrounding96% thethe ± reaction3%reaction flaskflask on on ( a ()a the) the Z-average Z-average and and (b) the (b) particle the particle stability stability during 21 during days of 21 storage days of in astorage fridge. in Statistically a fridge. Temp.: temperature; Z-avg.: Z-average; PDI: polydispersity index. significantStatistically changes significant are markedchangeswith are marked * for p < with0.05. * for p < 0.05.
Table A1. Comparison of the parameters of Cit-Au-SPIONs with varied reaction temperatures.
Reaction Temp. Z-Avg. PDI ζ-Potential @ pH 7 pH Value Relative Susceptibility in °C in nm in a.u. in mV in a.u. in a.u.
90 145 ± 4 0.18 ± 0.01 −50.8 ± 1.3 6.14 ± 0.16 87% ± 4%
110 152 ± 5 0.19 ± 0.01 −48.6 ± 0.3 6.21 ± 0.14 89% ± 2%
130 146 ± 11 0.19 ± 0.01 −46.9 ± 2.6 6.09 ± 0.20 96% ± 3% Temp.: temperature; Z-avg.: Z-average; PDI: polydispersity index.
FigureFigure A8. A8. InfluenceInfluence of the precursor precursor concentration concentration (iron and gold) gold) of of Cit-Au-SPIONs Cit-Au-SPIONs on on ( (aa)) the the Z-averageZ-average andand ((bb)) thethe particleparticle stabilitystability duringduring 2121 daysdays ofof storagestorage inin aa fridge.fridge. StatisticallyStatistically significantsignificant changeschanges areare markedmarked withwith ** forfor pp< < 0.05.
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Sample Availability: Samples of the compounds are not available from the authors.
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