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Preparation and characterization of Au nanoparticles capped with mercaptocarboranyl clusters† Ana M. Cioran,a Francesc Teixidor,a Željka Krpetić,b Mathias Brustb and Clara Viñas*a The preparation of 3–4 nm and 10 nm gold nanoparticles capped with neutral carborane-based mercaptocarboranes, via two different preparative routes, is reported. The resulting boron-enriched nanomaterials exhibit complete dispersibility in water, opening the way for the use of these monolayer protected clusters (MPCs) in medical applications, such as boron neutron capture therapy (BNCT). These
Received 11th October 2013, Accepted 15th November 2013
newly prepared MPCs have been characterized by FTIR, 1H and
11
B NMR spectroscopy, UV-visible, cen-
DOI: 10.1039/c3dt52872c
trifugal particle sizing (CPS), and, in some cases, inductively coupled plasma atomic emission spectrometry (ICP-AES). Water dispersibility exhibited by these MPCs allowed the study of the cellular uptake
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by HeLa cells.
Introduction The extensive use of gold nanoparticles (Au NPs) in biological applications is due to their ease of preparation and surface functionalization and their unique physicochemical properties such as excellent absorbance and scattering of light.1 As a result of these properties as well as their usual stability in biological fluids,2 Au NPs have become potential candidates to be used as tools for the controlled release of active agents, cell labeling, targeted drug delivery,3 medical imaging,4 cancer diagnostic and therapy,5 and biological sensors,6 amongst others. Likewise, reports claim7 that such structures afford up to a 10-fold increase in the concentration of the drug in the brain, a lessened burst effect, slow clearance and improved half-life. Moreover, research reports have also shown that surface size plays a large role in the therapeutic effect of Au NPs.8 The modification of the surfaces of gold nanoparticles and macroscopic gold surfaces represents a chemical tool frequently used in the preparation of materials with properties that reflect a transitional phase between the molecular and bulk levels.9
a Institut de Ciència de Materials de Barcelona, Campus U.A.B., 08193 Bellaterra, Spain. E-mail: [email protected] b Department of Chemistry, University of Liverpool, Crown Street, Liverpool L69 7ZD, UK † Electronic supplementary information (ESI) available: 11B{1H} and 11B NMR spectra of Au nanoparticles capped with ligands 1, 2, 3 and 5. FTIR spectra of 3 nm MPCs capped with mercaptocarborane and ligands 1–5. FTIR spectra of 10 nm MPCs capped with ligands 2 and 3. See DOI: 10.1039/c3dt52872c
5054 | Dalton Trans., 2014, 43, 5054–5061
Thiolate-stabilized gold nanoparticles, commonly referred to as monolayer protected clusters (MPCs), have opened the way to novel interdisciplinary research due to their ease of preparation and high ambient stability, enabling experimental studies that had previously not been accessible to experimental research, such as reversible metal–insulator transitions10 or NMR spectroscopy of thin films of self-assembled monolayers.11 The practical importance of MPCs is evidenced, for example, by their current role in the development of artificial nose-type gas sensors with potential applications in lung cancer diagnostics based on breath analysis.12 A facile synthesis of nanoparticles composed of gold clusters coated with thiolate monolayers13,14 has attracted extensive use. Understanding reactivities of MPC monolayers and developing efficient strategies to functionalize them is key for their application in areas such as catalysis and chemical sensing.15 We recently reported the preparation of Au NPs capped with mercaptocarborane, 1-SH-1,2-closo-C2B10H11, performed in a single-phase reaction (methanol) by reduction of tetrachloroauric acid with sodium borohydride in the presence of mercaptocarborane as a stabilizing agent.16 These newly obtained nanoparticles exhibited unique properties regarding adaptive dispersibility in polar and non-polar solvents ( phasetransfer), storage of ionic and electronic charge, ion exchange and cellular uptake by direct membrane penetration.16 In this paper we investigate whether the presence of a second substituent on the carborane cluster carbon (Cc) adjacent to the one bonded to the thiol group or the direct bonding between the Cc and the thiol group are decisive factors when considering the phase transfer and other unusual properties mentioned
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above.16 Finally, the preparation methods giving rise to different nanoparticle sizes were also considered to be of importance when determining the factors inducing these properties. The resulting nanomaterials could be interesting as highly boron-enriched agents and therefore could be used in boron neutron capture therapy (BNCT).
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0.9 L min−1, a coolant flow of 13.0 L min−1 and an auxiliary flow of 0.8 L min−1. Line selection for each element was: Au 242.795 nm/267.595 nm; S 180.731 nm/182.034 nm and B 249.773 nm/249.677 nm. Typical RSD values were between 1 and 1.5% indicating a stable plasma with consistent sample injection and aspiration.
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Synthesis of 1-HSCH2CH2CH2-2-CH3-1,2-closo-C2B10H10 (5)
Experimental section Materials and methods Commercial o-carborane, 1-methyl-o-carborane and 1-phenylo-carborane were sublimed under high vacuum at 0.01 mmHg prior to use. Solvents were degassed under vacuum to remove the dissolved oxygen by repeating three times the freeze– pump–thaw procedure. A 1.6 M solution of n-butyllithium in n-hexane was used as purchased from Sigma Aldrich. 1-SH2-CH3-1,2-closo-C2B10H10 (1),17 1-SH-2-C6H5-1,2-closo-C2B10H10 (2),18 1,2-(SH)2-1,2-closo-C2B10H10 (3),19 1-HSCH2CH2-2-CH31,2-closo-C2B10H10 (4)20 and 1-ClCH2CH2CH2-2-CH3-1,2-closoC2B10H10 21 were synthesised as previously reported. All reactions were carried out under a nitrogen atmosphere employing Schlenk techniques. Microanalyses were performed using a Perkin-Elmer 240 B microanalyser. FTIR spectra were obtained using a Perkin-Elmer Spectrum One spectrophotometer, which covers a wavelength range from 4000 to 400 cm−1. 1H-NMR (300.13 MHz) and 11B-NMR (96.29 MHz) spectra were recorded on a Bruker ARX 300WB spectrometer. Chemical shift values for 1H-NMR spectra were referenced to an internal standard of Si(CH3)4 in deuterated solvents. Chemical shift values for 11 B-NMR spectra were referenced relative to external BF3·OEt2. UV-vis spectroscopy was carried out using a Shimadzu UV-vis 1700 spectrophotometer at 23 °C using 1 cm quartz cuvettes. Particle sizing by analytical centrifugation Particle size and distribution were estimated using a CPS disc centrifuge DC24000 (CPS Instruments Inc.). For measurements, the speed was set to 24 000 rpm, and the centrifuge disc was successively filled with a density gradient liquid (8–24% w/w sucrose dissolved in milliQ water) leaving it to stabilize for 1 hour prior to analysis. The disc was filled successively in nine steps, starting with the dilution of the highest density. Prior to the analysis of the nanoparticles, calibration was performed using as a calibration standard 0.377 μm PVC particles (Analytik Ltd). All nanoparticle samples were sonicated for 3 minutes before injection into the disc centrifuge. The size of the particles was calculated using CPS Software (9.5c) for the density of 6.5 g cm−3.
To a solution of 1-ClCH2CH2CH2-2-CH3-1,2-closo-C2B10H10 (183 mg, 0.78 mmol) in dry diethyl ether, maintained at 0 °C for 30 min, thiourea (65 mg, 0.86 mmol) was added. The resulting solution was allowed to reach r.t. and stirred at 25 °C for 1 hour. After this time, 10 cm3 of water were added. The mixture was thoroughly shaken and the two layers separated. The organic layer was dried over MgSO4. The filtrate was evaporated to give a yellow solid. Yield: 157 mg, 0.68 mmol, 88%. FTIR (cm−1): ν (C–H)alkyl 2988, 2902, ν (B–H) 2593, 2560, ν (δ(CH2) 1442, 1390). 1H-NMR (CD3COCD3) δ ( ppm): 3.61 (s, 1H, S–H), 2.95 (d, 1J (H,H) = 8.4 Hz, 2H, CH2), 2.92 (d, 1J (H,H) = 8.7 Hz, 2H, CH2), 2.53 (s, 3H, CH3), 2.33 (dd, 1J (H,H) = 8.4 Hz, 1 J (H,H) = 8.7 Hz, 2H, CH2). 1H-{11B}-NMR (CD3COCD3) δ ( ppm): 3.61 (s, 1H, S–H), 2.95 (d, 1J (H,H) = 8.4 Hz, 2H, CH2), 2.92 (d, 1J (H,H) = 8.7 Hz, 2H, CH2), 2.53 (s, 3H, CH3), 2.33 (dd, 1 J (H,H) = 8.4 Hz, 1J (H,H) = 8.7 Hz, 2H, CH2), 2.70–2.53 (br s, 10H, B–H). 11B-NMR (CD3COCD3) δ ( ppm): −3.3 (d, 1J (B,H) = 130 Hz, 1B), −4.7 (d, 1J (B,H) = 149 Hz, 1B), −7.8 (d, 1J (B,H) = 132 Hz), −8.5 (d, 1J (B,H) = 132 Hz), −9.5 (d, 1J (B,H) = 150 Hz). The 11B-NMR spectrum pattern corresponds to 1 : 1 : 8. Preparation of 3 nm thiocarborane-capped MPCs. General procedure 0.283 mmol of the respective mercaptocarborane (Chart 1) and 111 mg of chloroauric acid (0.283 mmol) were dissolved in 60 mL of methanol. 64 mg of sodium borohydride (1.700 mmol), previously dissolved in 30 mL of methanol, were immediately added under vigorous stirring. The mixture was stirred at r.t. for 10 min before the solvent was removed by rotary evaporation. The resulting dark-brown residue was first thoroughly washed with diethyl ether to remove excess mercaptocarborane and then dissolved in isopropanol and filtered to remove the remaining sodium borohydride and other
ICP-AES Quantitative analysis of Au, S and B by atomic emission spectroscopy was carried out using a Spectro Ciros ICP-AES radial view spectrometer. The machine set-up is comprised of a cross flow nebuliser, a double pass (Scott) spray chamber and a peristaltic pump. Instrument parameters from optimisation were: plasma power 1360 W with a nebulising flow of
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Chart 1
Schematic drawings of closo mercaptocarboranes 1–5.
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insoluble contaminants. After rotary evaporation of the isopropanol, the final product was obtained as a dark-brown solid. Characterization of MPCs capped with 1: FTIR (cm−1): ν = 2564.52 (vs, νB–H). 1H{11B}-NMR (300 MHz, CD3COCD3) δ ( ppm): 2.44 (br s, B–H), 2.29 (br s, B–H), 1.3 (br s, B–H), 1.89 (br s, B–H); the overlapping of the signals does not allow us to do spectrum integration (see ESI†). 11B-NMR (96 MHz, CD3COCD3) δ ( ppm): −5.7 (d, 1J (B–H) = 163 Hz), −7.3 (d, 1 J (B–H) = 144 Hz), −8.9 (d, 1J (B–H) = 150 Hz), −10.2 (d, 1J (B– H) = 125 Hz); the overlapping of the signals do not allow us to do spectrum integration (see ESI†). CPS: 3–4 nm. Characterization of MPCs capped with 2: FTIR (cm−1): ν = 2576.86 (vs, νB–H). 11B-NMR (96 MHz, CD3COCD3) δ ( ppm): −2.9, −4.4, −7.0, −8.9, −10.9, −12.8; peaks integration was not possible (see ESI†). CPS: 4–5 nm. Characterization of MPCs capped with 3: FTIR (cm−1): ν = 2564.52 (vs, νB–H). 11B-NMR (96 MHz, CD3COCD3) δ ( ppm): −7.5 (br s, 8B), −12.4 (1J (B–H) = 130 Hz, 2B). CPS: 4–6 nm. Characterization of MPCs capped with 4: FTIR (cm−1): ν = 2589.20 (vs, νB–H). 11B-NMR (96 MHz, CD3COCD3) δ ( ppm): −5.7, −7.2, −10.3; integration was not possible. CPS: 4–6 nm. Characterization of MPCs capped with 5: 11B-NMR (96 MHz, CD3COCD3) δ ( ppm): −5.6 (d, 1J (B–H) = 164 Hz, 2B), −10.3 (d, 1J (B–H) = 124 Hz, 8B). Preparation of 10 nm mercaptocarborane-capped MPCs. General procedure 0.032 mmol of mercaptocarboranes 1–5 were dissolved in 6 mL acetone, followed by the dropwise addition of this solution onto 40 mL of previously prepared aqueous dispersion of 10 nm Au particles prepared following the Turkevich citrate reduction method.22 The resulting mixture was shaken for 1 hour at room temperature, and then centrifuged four times for 20 minutes at 13 500 rpm and 10 °C using equal volumes of water and ether to remove the unreacted mercaptocarboranes and the resulting citrate. Once dried, the NPs were no longer dispersible in water or any of the common solvents. MPC phase transfer experiments Phase transfer of MPCs from water to diethyl ether has been carried out by acidification of the aqueous phase with dilute hydrochloric acid as was reported by us previously.16 However, unlike the case of the formerly reported mercaptocarborane, no gas evolution was noticed, nor did the gold nanoparticles transfer from the aqueous phase to the organic layer.
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nanoparticles, Na+ ions are retained close to the gold surface compensating for the negative charge of the particles. 1-SH-1,2-closo-C2B10H11 has the thiol group directly bonded to the carbon of the cluster (Cc) and an H atom bonded to the adjacent Cc. At this point, we wanted to examine, first, whether a second substituent on the adjacent Cc is capable of altering electronically or sterically the first thiol, and second, whether the SH group must be directly bonded to the Cc as a necessary requirement for the MPCs to exhibit the phase transfer property. To investigate whether the presence of a second substituent on the adjacent carbon of the carborane cluster (Cc) is a decisive factor for the phase transfer property, the preparation of the new Au NPs capped with mercaptocarboranes, 1–3, was performed in a single-phase reaction (methanol) by reduction of tetrachloroauric acid with sodium borohydride in the presence of the corresponding mercaptocarborane as a stabilizing agent. To investigate whether direct bonding between the Cc and the thiol group is necessary, synthesis of new compounds (4, 5) that would separate the o-carborane cluster from the thiol group through an alkyl spacer was required.
Synthesis and spectroscopic studies of mercaptocarboranyl derivative, 5 The synthetic way employed for the synthesis of thiocarborane 5 is based on the nucleophilic attack of the carboranyl group on the C–I bond, with the formation of lithium iodide salt, which shifts the equilibrium to the preparation of the chlorinated derivative as an intermediate.21 This compound is then reacted with thiourea and subsequently treated with a solution of sodium hydroxide to yield the desired species, as illustrated in Scheme 1. Regarding the NMR spectroscopic characterization of 5 (Fig. 1), the signal which appears at the lowest field value, 3.58 ppm, belongs to the H bonded to sulfur, whereas the peak at 2.93 ppm is characteristic to the CH2– groups bonded to the two electron withdrawing moieties within the molecule, namely the o-carborane cluster and the sulfur atom. The proximity to these fragments determines the deshielding of the CH2– groups and, as a direct consequence, the downfield shift as depicted in Fig. 1a. The 11B–{1H}-NMR spectrum corroborates the fact that the closo structure is kept during the synthesis, as indicated by the FTIR analysis, which shows a broad absorption band at approximately 2560 cm−1 (see ESI†).
Results and discussion We previously reported that nanoparticles capped with 1-SH-1,2-closo-C2B10H1116 displayed a unique phase transfer property, namely the reversible transfer of the particles from water to diethyl ether, achieved by acidification of the aqueous phase with dilute hydrochloric acid or from ether to water by addition of sodium borohydride. To explain such an unusual property we inferred that the particles act both as electron and ion storage devices. We demonstrated that, in as prepared
5056 | Dalton Trans., 2014, 43, 5054–5061
Scheme 1
Synthesis of 1-HSCH2CH2CH2-2-CH3-1,2-closo-C2B10H10, 5.
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Fig. 1 (a) Comparison between 1H-NMR (black) and 1H{11B}-NMR (red) for 5; (b) 11B{1H}-NMR spectrum of 5 (black) and its corresponding Au MPCs (blue).
Preparation and characterization of Au NPs capped with boron clusters containing mercaptocarborane ligands by reducing with sodium borohydride Ligands 1 and 2 have the Cc directly bonded to the S–H group while the H bonded to the adjacent Cc was replaced by a Me or Ph group, respectively. Ligand 3 has two thiol groups directly bonded to the two cluster Cc. Ligands 4 and 5 have a spacer between the cluster unit and the thiol moiety, in this case an alkyl chain. Having defined the preparative targets, the ratios of reagents were established for mercaptocarborane, 1-SH-1,2closo-C2B10H11, and the reaction conditions were applied as described for the preparation of 3 nm MPCs capped with closo o-carborane-based mercaptocarboranes, 1–5. Thus the preparations of Au NPs capped with 1–5 were performed in a single-phase reaction (methanol) by reduction of tetrachloroauric acid with sodium borohydride in the presence of the proper mono-thiol o-carborane derivative (Scheme 2). The Au NPs obtained, although soluble in water, acetone, and isopropanol, did not display the same phase transfer properties as the ones functionalized with 1-SH-1,2-closo-C2B10H11. The MPCs were characterized by FTIR, 1H and 11B-NMR, UV-vis, and, in some cases, CPS and ICP-AES.
Scheme 2
Formation of 3 nm Au nanoparticles capped with 2.
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The comparison between FTIR spectra of the starting material 2 and its corresponding MPCs is depicted in Fig. 2. FTIR spectroscopy confirms the existence of the boron-based ligands in the structure of MPCs due to strong broad absorptions at 2577 cm−1, a typical indicator of the presence of B–H bonds of a closo cluster attesting that the mercaptocarboranes retain the initial closo structure throughout the MPCs preparation. The NMR analysis completes the characterization of the MPCs, by showing that the S–H is no longer present in the 1 H-NMR spectra after the functionalization of the gold nanoparticles, proving that the bonding of sulfur to the gold core
Fig. 2 Comparison between the FTIR spectra of neutral thioligand 2 (green) and its corresponding MPCs (blue).
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Fig. 3 (a) 1H{11B}-NMR and (b) (blue).
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11
B{1H}-NMR (spectra recorded in d6-acetone); comparison between the starting thioligand 1 (green) and its MPCs
did indeed take place. Regarding the 11B-NMR comparison, the broadening of the boron signals as well as the shift of about 2 ppm towards higher field values indicates the interaction between the ligand and the gold core. Moreover, the NMR spectrum confirms once again the preservation of the closo structure (Fig. 1b and 3). Another characterization method that brings additional information concerning the synthesis of the sought MPCs is UV-vis spectroscopy. As depicted in Fig. 4, the UV-vis spectra of the MPCs stabilized with ligands 1–5 show, in agreement with the theoretical calculations performed by Mulvaney,23 that the MPCs produced are smaller than 3 nm (no surface
plasmon band) for all neutral ligands except for 5 that leads to the synthesis of somewhat larger MPCs and ligand 3 that leads to a broad band centered at approx. 550 nm that is indicative of aggregation and may be due to the presence of the two sulfur atoms that may allow some degree of cross linking (Fig. 4). The particle size distribution was determined as indicated at the experimental section and shown in Fig. 5 for MPCs functionalized with compounds 1–3. Quantitative elemental analysis by ICP-AES was also employed to determine the Au : carborane ratio, since it provides rapid and precise means of simultaneously monitoring most of the elements present in the sample. The results are depicted in Table 1, and show that there is an average of 3 Au
Fig. 5 Size distribution of 3 nm MPCs functionalized with compounds 1 (a), 2 (b) and 3 (c).
Table 1
ICP-AES analysis of 3 nm MPCs of mercaptocarboranes 1–3
Experimental values Fig. 4 UV-vis spectra of 3 nm MPCs of 1–5. Note the clear indication of particle aggregation in the case of ligand 3. Ligand 5 appears to lead to slightly larger particles as evidenced by the shoulder at 520 nm, i.e. the onset of plasmon absorption.
5058 | Dalton Trans., 2014, 43, 5054–5061
Sample
Au
S
B
Theoretical S : B ratio
MPCs of 1 MPCs of 2 MPCs of 3
3 2.8 4
0.91 0.93 1.61
9.4 10.8 9.25
1 : 10 1 : 10 2 : 10
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Scheme 3
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Formation of 10 nm Au nanoparticles capped with 2 via citrate ligands exchange.
atoms for every neutral mercaptocarborane suggesting that a typical nanoparticle would contain in the order of 300 Au atoms and 100 carborane ligands. Hence, the first conclusion to be drawn based on these results is that in order for the MPCs to exhibit phase transfer properties it is required to have the thiol group directly bonded to the carborane cage as well as absence of a bulky substituent at the adjacent carbon of the cluster. The mercaptocarborane that displays the phase transfer has a Cc–H unit at the adjacent carbon. A further question to be answered concerning the requirements for the phase transfer property had to do with the preparative procedure. Is the Au NPs preparation method significant for such an unusual property? To address this, we looked at the well-known 10 nm Au NPs capped with citrate, reported by Turkevich,22 with the objective of exchanging the citrate ligands for mercaptocarboranes. The procedure for preparing water-soluble gold nanoparticles of approximately 10 nm in diameter by exchanging initial citrate ligands for boron-rich clusters is depicted in Scheme 3. After purification, the wine-red water-dispersible product was characterized by FTIR, UV-vis, CPS and, in some cases, ICP-AES. The coordination of thioligand to the gold core was observed using FTIR spectroscopy. Strong broad absorptions at 2562–2570 cm−1 are present in both the pure neutral ligands (1–5) and their corresponding final MPCs, due to B–H stretches, also supporting a closo cluster structure (Fig. 6).24 Additionally, the typical plasmon band present at about 520 nm in the UV-visible analysis (Fig. 7) indicates the absence of aggregation before and after modification with ligands (1–5), also supported by the red color of the dispersions. In relation to the characterization of the MPCs by analytical centrifugation, Fig. 8 shows a small decrease in the apparent particle size upon the formation of the new ligand shell. This is an artifact due to the decreased density of the ligand-capped nanoparticle with respect to that of pure metallic gold used by the analysis program to compute the particle size. The effect of the decrease in density overcompensates that of the increased particle size resulting in an actual decrease in the effective particle size measured by the instrument. In the presence of an
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Fig. 6 FTIR spectra of 1 before (black) and after (red) functionalization of 10 nm gold nanoparticles by ligand interchange.
Fig. 7 UV-vis spectra of MPCs functionalized via ligand exchange with neutral carborane-based mercaptocarboranes.
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Fig. 9 TEM images of the uptake of 10 nm mercaptocarborane-capped MPCs by HeLa cells. It is evident from the distribution of the particles that they are confined to vesicles although the vesicle membranes are not discernible in these images.
Fig. 8 Size distribution of 10 nm Au NPs capped with carborane-based mercaptocarboranes 1–5 by a Centrifugal Particle Size analyzer, CPS. Note the apparent decrease in particle size upon the attachment of the ligand. This is due to the decrease in particle density that overcompensates the effect of increased size.
adequate calibration sample (not available here) this can be corrected, and the method can then reveal quantitatively minute differences in particle size resulting from differences in ligand length that can be as small as a single carbon atom.25 The ICP-AES analysis, summarized in Table 2, indicates that for every carborane cluster (10 boron atoms), there are approximately 10 ± 1 Au atoms suggesting that a typical nanoparticle contained in the order of 30 000 gold atoms26 and 3000 carborane ligands. Given the size of the particle this experimentally determined number of ligands is very high, which suggests that either it was not possible by repeated centrifugation to remove all excess ligands, or, in addition to a compact monolayer, there are further carborane units associated with each nanoparticle. This may be beneficial from the point of view of creating boron rich particles for medical applications and deserves further studies. Although these newly obtained 10 nm MPCs capped with closo mercaptocarboranes 1–5 were dispersible in organic solvents but most importantly in water, there was, once again, no reversible phase transfer phenomenon as in the case of the 3 nm Au NP 1-SH-2-C2B10H11 capped MPCs.
Table 2 ICP-AES of mercaptocarboranes
10 nm
MPCs
functionalized
with
neutral
Experimental values Sample
Au
B
MPCs of mercaptocarborane MPCs of 1 MPCs of 2 MPCs of 3 MPCs of 5
9.5 9.4 9.1 10.9 11.9
10.18 10.58 10.85 9.9 9.4
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Finally, cellular uptake studies of 10 nm MPCs capped with the mercaptocarborane ligands were performed on HeLa cells, a common human fibroblast cell line, with an incubation time of four hours. The experimental procedures were identical to those reported previously.16 The corresponding TEM images are shown in Fig. 9. The MPCs were found to be taken up by the cells and, as most nanoparticles, strictly remained confined to endocytic vesicles, in contrast to the previously reported behavior of the 3 nm particles that also exhibited phase transfer behaviour. Those were capable of crossing membrane boundaries and were found freely in the cytoplasm as well as in the nucleus and even inside the mitochondria. It is likely that the ability to phase transfer by adjusting the ionic and electronic charge of the particle is linked to the ability to cross biological membranes. Hence, none of the particles reported here had this remarkable property. It can thus be inferred that the preparation method for the MPCs as well as the size of the resulting MPCs are also important for the conservation of phase transfer properties, alongside with the presence of a Cc–H bond and the direct bonding of the thiol group to the carborane cluster.
Conclusions In conclusion, we reported the synthesis and structural characterisation of a new mercapto compound as well as the preparation and characterization of 3–5 nm and 10 nm gold nanoparticles functionalized with neutral boron-based mercaptocarboranes. The properties shown by these MPCs were not similar to the ones observed when using 1-SH-1,2-closo-C2B10H11 as a capping agent. Based on these and earlier results, the requirements for the reversible phase transfer of the particles from aqueous to organic layer, and the related remarkable ability to cross biological membranes, are as follows: the S–H group directly bonded to the carborane cage combined with the presence of a Cc–H bond within the cluster and a MPC size of about 3 nm in diameter.
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Acknowledgements This work was supported by Generalitat de Catalunya (2009/ SGR/00279), Ministerio de Ciencia e Innovación (CTQ201016237). A. M. Cioran thanks MEC for a FPU predoctoral grant (AP2007-01723). M. B. and Z. K. acknowledge support from the European Community Seventh Framework Programme (FP7NMP-2010-EU-MEXICO) and CONACYT under grant agreement no. 263878 and 125141, respectively. Access to a CPS disc centrifuge was generously provided by the Knowledge Centre for Materials Chemistry (KCMC) at the University of Liverpool. The authors wish to thank George Miller for conducting the ICP-AES measurements and Prof. Ian A. Prior for access to the TEM and cell culture facilities at the University of Liverpool.
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10 (a) C. P. Collier, R. J. Saykally, J. J. Shiang, S. E. Henrichs and J. R. Heath, Science, 1998, 277, 1978; (b) B. M. Quinn, I. Prieto, S. K. Haram and A. J. Bard, J. Phys. Chem. B, 2001, 105, 7474. 11 (a) A. Badia, W. Gao, S. Singh, L. Demers, L. Cuccia and L. Reven, Langmuir, 1996, 12, 1262; (b) A. Badia, L. Demers, L. Dickinson, F. G. Morin, R. B. Lennox and L. Reven, J. Am. Chem. Soc., 1997, 119, 11104; (c) A. C. Templeton, D. E. Cliffel and R. W. Murray, J. Am. Chem. Soc., 1999, 121, 7081; (d) A. Badia, R. B. Lennox and L. Reven, Acc. Chem. Res., 2000, 33, 475; (e) R. L. Donkers, Y. Songa and R. W. Murray, Langmuir, 2004, 20, 4703. 12 (a) H. Wohltjen and A. W. Snow, Anal. Chem., 1998, 70, 2856; (b) Y. Joseph, A. Peic, X. D. Chen, J. Michl, T. Vossmeyer and A. Yasuda, J. Phys. Chem. C, 2007, 111, 12855; (c) O. Barash, N. Peled, F. R. Hirsch and H. Haick, Small, 2009, 5, 2618; (d) G. Peng, U. Tisch, O. Adams, M. Hakim, N. Shehada, Y. Y. Broza, S. Billan, R. AbdahBortnyak, A. Kuten and H. Haick, Nat. Nanotechnol., 2009, 4, 669; (e) E. Chow, T. R. Gengenbach, L. Wieczorek and B. Raguse, Sens. Actuators, B, 2010, 143, 704; (f) E. Covincton, F. I. Bohrer, C. Xu, E. T. Zellers and C. Kurdak, Lab Chip, 2010, 22, 3058. 13 M. Brust, J. Fink, D. Bethell, D. J. Schiffrin and C. J. Kiely, J. Chem. Soc., Chem. Commun., 1995, 1655. 14 M. Brust, M. Walker, D. Bethell, D. J. Schiffrin and R. Whyman, J. Chem. Soc., Chem. Commun., 1994, 801. 15 A. C. Templeton, W. P. Wuelfing and R. W. Murray, Acc. Chem. Res., 2000, 33, 27. 16 A. M. Cioran, A. D. Musteti, F. Teixidor, Ž. Krpetić, I. A. Prior, Q. He, C. J. Kiely, M. Brust and C. Viñas, J. Am. Chem. Soc., 2012, 134(1), 212. 17 C. Viñas, R. Benakki, F. Teixidor and J. Casabó, Inorg. Chem., 1995, 34, 3844. 18 F. Teixidor, J. Rius, A. M. Romerosa, C. Miravitlles, L. Escriche, E. Sanchez, C. Viñas and J. Casabó, Inorg. Chim. Acta, 1990, 176, 287. 19 H. D. Smith Jr., C. O. Obenland and S. Papetti, Inorg. Chem., 1966, 5(6), 1013. 20 R. Kivekäs, J. Pedrajas, F. Teixidor and R. Sillanpää, Acta. Crystallogr., Sect. C: Cryst. Struct. Commun., 1998, 54, 1716. 21 F. Teixidor, S. Gómez, M. Lamrani, C. Viñas, R. Sillanpää and R. Kivekäs, Organometallics, 1997, 16, 1278. 22 (a) J. Turkevich, P. C. Stevenson and J. Hillier, Discuss. Faraday Soc., 1951, 11, 55; (b) G. Frens, Nat. Phys. Sci., 1973, 241, 20. 23 P. Mulvaney, Langmuir, 1996, 12, 788. 24 L. A. Leites, Chem. Rev., 1992, 92, 279. 25 Ž. Krpetić, A. M. Davidson, M. Volk, R. Lévy, M. Brust and D. L. Cooper, ACS Nano, 2013, 7, 8881. 26 A typical 10 nm gold nanoparticle contains approximately 30 000 Au atoms.
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Preparation and characterization of Au nanoparticles capped with mercaptocarboranyl clusters† Ana M. Cioran,a Francesc Teixidor,a Željka Krpetić,b Mathias Brustb and Clara Viñas*a The preparation of 3–4 nm and 10 nm gold nanoparticles capped with neutral carborane-based mercaptocarboranes, via two different preparative routes, is reported. The resulting boron-enriched nanomaterials exhibit complete dispersibility in water, opening the way for the use of these monolayer protected clusters (MPCs) in medical applications, such as boron neutron capture therapy (BNCT). These
Received 11th October 2013, Accepted 15th November 2013
newly prepared MPCs have been characterized by FTIR, 1H and
11
B NMR spectroscopy, UV-visible, cen-
DOI: 10.1039/c3dt52872c
trifugal particle sizing (CPS), and, in some cases, inductively coupled plasma atomic emission spectrometry (ICP-AES). Water dispersibility exhibited by these MPCs allowed the study of the cellular uptake
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by HeLa cells.
Introduction The extensive use of gold nanoparticles (Au NPs) in biological applications is due to their ease of preparation and surface functionalization and their unique physicochemical properties such as excellent absorbance and scattering of light.1 As a result of these properties as well as their usual stability in biological fluids,2 Au NPs have become potential candidates to be used as tools for the controlled release of active agents, cell labeling, targeted drug delivery,3 medical imaging,4 cancer diagnostic and therapy,5 and biological sensors,6 amongst others. Likewise, reports claim7 that such structures afford up to a 10-fold increase in the concentration of the drug in the brain, a lessened burst effect, slow clearance and improved half-life. Moreover, research reports have also shown that surface size plays a large role in the therapeutic effect of Au NPs.8 The modification of the surfaces of gold nanoparticles and macroscopic gold surfaces represents a chemical tool frequently used in the preparation of materials with properties that reflect a transitional phase between the molecular and bulk levels.9
a Institut de Ciència de Materials de Barcelona, Campus U.A.B., 08193 Bellaterra, Spain. E-mail: [email protected] b Department of Chemistry, University of Liverpool, Crown Street, Liverpool L69 7ZD, UK † Electronic supplementary information (ESI) available: 11B{1H} and 11B NMR spectra of Au nanoparticles capped with ligands 1, 2, 3 and 5. FTIR spectra of 3 nm MPCs capped with mercaptocarborane and ligands 1–5. FTIR spectra of 10 nm MPCs capped with ligands 2 and 3. See DOI: 10.1039/c3dt52872c
5054 | Dalton Trans., 2014, 43, 5054–5061
Thiolate-stabilized gold nanoparticles, commonly referred to as monolayer protected clusters (MPCs), have opened the way to novel interdisciplinary research due to their ease of preparation and high ambient stability, enabling experimental studies that had previously not been accessible to experimental research, such as reversible metal–insulator transitions10 or NMR spectroscopy of thin films of self-assembled monolayers.11 The practical importance of MPCs is evidenced, for example, by their current role in the development of artificial nose-type gas sensors with potential applications in lung cancer diagnostics based on breath analysis.12 A facile synthesis of nanoparticles composed of gold clusters coated with thiolate monolayers13,14 has attracted extensive use. Understanding reactivities of MPC monolayers and developing efficient strategies to functionalize them is key for their application in areas such as catalysis and chemical sensing.15 We recently reported the preparation of Au NPs capped with mercaptocarborane, 1-SH-1,2-closo-C2B10H11, performed in a single-phase reaction (methanol) by reduction of tetrachloroauric acid with sodium borohydride in the presence of mercaptocarborane as a stabilizing agent.16 These newly obtained nanoparticles exhibited unique properties regarding adaptive dispersibility in polar and non-polar solvents ( phasetransfer), storage of ionic and electronic charge, ion exchange and cellular uptake by direct membrane penetration.16 In this paper we investigate whether the presence of a second substituent on the carborane cluster carbon (Cc) adjacent to the one bonded to the thiol group or the direct bonding between the Cc and the thiol group are decisive factors when considering the phase transfer and other unusual properties mentioned
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above.16 Finally, the preparation methods giving rise to different nanoparticle sizes were also considered to be of importance when determining the factors inducing these properties. The resulting nanomaterials could be interesting as highly boron-enriched agents and therefore could be used in boron neutron capture therapy (BNCT).
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0.9 L min−1, a coolant flow of 13.0 L min−1 and an auxiliary flow of 0.8 L min−1. Line selection for each element was: Au 242.795 nm/267.595 nm; S 180.731 nm/182.034 nm and B 249.773 nm/249.677 nm. Typical RSD values were between 1 and 1.5% indicating a stable plasma with consistent sample injection and aspiration.
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Synthesis of 1-HSCH2CH2CH2-2-CH3-1,2-closo-C2B10H10 (5)
Experimental section Materials and methods Commercial o-carborane, 1-methyl-o-carborane and 1-phenylo-carborane were sublimed under high vacuum at 0.01 mmHg prior to use. Solvents were degassed under vacuum to remove the dissolved oxygen by repeating three times the freeze– pump–thaw procedure. A 1.6 M solution of n-butyllithium in n-hexane was used as purchased from Sigma Aldrich. 1-SH2-CH3-1,2-closo-C2B10H10 (1),17 1-SH-2-C6H5-1,2-closo-C2B10H10 (2),18 1,2-(SH)2-1,2-closo-C2B10H10 (3),19 1-HSCH2CH2-2-CH31,2-closo-C2B10H10 (4)20 and 1-ClCH2CH2CH2-2-CH3-1,2-closoC2B10H10 21 were synthesised as previously reported. All reactions were carried out under a nitrogen atmosphere employing Schlenk techniques. Microanalyses were performed using a Perkin-Elmer 240 B microanalyser. FTIR spectra were obtained using a Perkin-Elmer Spectrum One spectrophotometer, which covers a wavelength range from 4000 to 400 cm−1. 1H-NMR (300.13 MHz) and 11B-NMR (96.29 MHz) spectra were recorded on a Bruker ARX 300WB spectrometer. Chemical shift values for 1H-NMR spectra were referenced to an internal standard of Si(CH3)4 in deuterated solvents. Chemical shift values for 11 B-NMR spectra were referenced relative to external BF3·OEt2. UV-vis spectroscopy was carried out using a Shimadzu UV-vis 1700 spectrophotometer at 23 °C using 1 cm quartz cuvettes. Particle sizing by analytical centrifugation Particle size and distribution were estimated using a CPS disc centrifuge DC24000 (CPS Instruments Inc.). For measurements, the speed was set to 24 000 rpm, and the centrifuge disc was successively filled with a density gradient liquid (8–24% w/w sucrose dissolved in milliQ water) leaving it to stabilize for 1 hour prior to analysis. The disc was filled successively in nine steps, starting with the dilution of the highest density. Prior to the analysis of the nanoparticles, calibration was performed using as a calibration standard 0.377 μm PVC particles (Analytik Ltd). All nanoparticle samples were sonicated for 3 minutes before injection into the disc centrifuge. The size of the particles was calculated using CPS Software (9.5c) for the density of 6.5 g cm−3.
To a solution of 1-ClCH2CH2CH2-2-CH3-1,2-closo-C2B10H10 (183 mg, 0.78 mmol) in dry diethyl ether, maintained at 0 °C for 30 min, thiourea (65 mg, 0.86 mmol) was added. The resulting solution was allowed to reach r.t. and stirred at 25 °C for 1 hour. After this time, 10 cm3 of water were added. The mixture was thoroughly shaken and the two layers separated. The organic layer was dried over MgSO4. The filtrate was evaporated to give a yellow solid. Yield: 157 mg, 0.68 mmol, 88%. FTIR (cm−1): ν (C–H)alkyl 2988, 2902, ν (B–H) 2593, 2560, ν (δ(CH2) 1442, 1390). 1H-NMR (CD3COCD3) δ ( ppm): 3.61 (s, 1H, S–H), 2.95 (d, 1J (H,H) = 8.4 Hz, 2H, CH2), 2.92 (d, 1J (H,H) = 8.7 Hz, 2H, CH2), 2.53 (s, 3H, CH3), 2.33 (dd, 1J (H,H) = 8.4 Hz, 1 J (H,H) = 8.7 Hz, 2H, CH2). 1H-{11B}-NMR (CD3COCD3) δ ( ppm): 3.61 (s, 1H, S–H), 2.95 (d, 1J (H,H) = 8.4 Hz, 2H, CH2), 2.92 (d, 1J (H,H) = 8.7 Hz, 2H, CH2), 2.53 (s, 3H, CH3), 2.33 (dd, 1 J (H,H) = 8.4 Hz, 1J (H,H) = 8.7 Hz, 2H, CH2), 2.70–2.53 (br s, 10H, B–H). 11B-NMR (CD3COCD3) δ ( ppm): −3.3 (d, 1J (B,H) = 130 Hz, 1B), −4.7 (d, 1J (B,H) = 149 Hz, 1B), −7.8 (d, 1J (B,H) = 132 Hz), −8.5 (d, 1J (B,H) = 132 Hz), −9.5 (d, 1J (B,H) = 150 Hz). The 11B-NMR spectrum pattern corresponds to 1 : 1 : 8. Preparation of 3 nm thiocarborane-capped MPCs. General procedure 0.283 mmol of the respective mercaptocarborane (Chart 1) and 111 mg of chloroauric acid (0.283 mmol) were dissolved in 60 mL of methanol. 64 mg of sodium borohydride (1.700 mmol), previously dissolved in 30 mL of methanol, were immediately added under vigorous stirring. The mixture was stirred at r.t. for 10 min before the solvent was removed by rotary evaporation. The resulting dark-brown residue was first thoroughly washed with diethyl ether to remove excess mercaptocarborane and then dissolved in isopropanol and filtered to remove the remaining sodium borohydride and other
ICP-AES Quantitative analysis of Au, S and B by atomic emission spectroscopy was carried out using a Spectro Ciros ICP-AES radial view spectrometer. The machine set-up is comprised of a cross flow nebuliser, a double pass (Scott) spray chamber and a peristaltic pump. Instrument parameters from optimisation were: plasma power 1360 W with a nebulising flow of
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Chart 1
Schematic drawings of closo mercaptocarboranes 1–5.
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insoluble contaminants. After rotary evaporation of the isopropanol, the final product was obtained as a dark-brown solid. Characterization of MPCs capped with 1: FTIR (cm−1): ν = 2564.52 (vs, νB–H). 1H{11B}-NMR (300 MHz, CD3COCD3) δ ( ppm): 2.44 (br s, B–H), 2.29 (br s, B–H), 1.3 (br s, B–H), 1.89 (br s, B–H); the overlapping of the signals does not allow us to do spectrum integration (see ESI†). 11B-NMR (96 MHz, CD3COCD3) δ ( ppm): −5.7 (d, 1J (B–H) = 163 Hz), −7.3 (d, 1 J (B–H) = 144 Hz), −8.9 (d, 1J (B–H) = 150 Hz), −10.2 (d, 1J (B– H) = 125 Hz); the overlapping of the signals do not allow us to do spectrum integration (see ESI†). CPS: 3–4 nm. Characterization of MPCs capped with 2: FTIR (cm−1): ν = 2576.86 (vs, νB–H). 11B-NMR (96 MHz, CD3COCD3) δ ( ppm): −2.9, −4.4, −7.0, −8.9, −10.9, −12.8; peaks integration was not possible (see ESI†). CPS: 4–5 nm. Characterization of MPCs capped with 3: FTIR (cm−1): ν = 2564.52 (vs, νB–H). 11B-NMR (96 MHz, CD3COCD3) δ ( ppm): −7.5 (br s, 8B), −12.4 (1J (B–H) = 130 Hz, 2B). CPS: 4–6 nm. Characterization of MPCs capped with 4: FTIR (cm−1): ν = 2589.20 (vs, νB–H). 11B-NMR (96 MHz, CD3COCD3) δ ( ppm): −5.7, −7.2, −10.3; integration was not possible. CPS: 4–6 nm. Characterization of MPCs capped with 5: 11B-NMR (96 MHz, CD3COCD3) δ ( ppm): −5.6 (d, 1J (B–H) = 164 Hz, 2B), −10.3 (d, 1J (B–H) = 124 Hz, 8B). Preparation of 10 nm mercaptocarborane-capped MPCs. General procedure 0.032 mmol of mercaptocarboranes 1–5 were dissolved in 6 mL acetone, followed by the dropwise addition of this solution onto 40 mL of previously prepared aqueous dispersion of 10 nm Au particles prepared following the Turkevich citrate reduction method.22 The resulting mixture was shaken for 1 hour at room temperature, and then centrifuged four times for 20 minutes at 13 500 rpm and 10 °C using equal volumes of water and ether to remove the unreacted mercaptocarboranes and the resulting citrate. Once dried, the NPs were no longer dispersible in water or any of the common solvents. MPC phase transfer experiments Phase transfer of MPCs from water to diethyl ether has been carried out by acidification of the aqueous phase with dilute hydrochloric acid as was reported by us previously.16 However, unlike the case of the formerly reported mercaptocarborane, no gas evolution was noticed, nor did the gold nanoparticles transfer from the aqueous phase to the organic layer.
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nanoparticles, Na+ ions are retained close to the gold surface compensating for the negative charge of the particles. 1-SH-1,2-closo-C2B10H11 has the thiol group directly bonded to the carbon of the cluster (Cc) and an H atom bonded to the adjacent Cc. At this point, we wanted to examine, first, whether a second substituent on the adjacent Cc is capable of altering electronically or sterically the first thiol, and second, whether the SH group must be directly bonded to the Cc as a necessary requirement for the MPCs to exhibit the phase transfer property. To investigate whether the presence of a second substituent on the adjacent carbon of the carborane cluster (Cc) is a decisive factor for the phase transfer property, the preparation of the new Au NPs capped with mercaptocarboranes, 1–3, was performed in a single-phase reaction (methanol) by reduction of tetrachloroauric acid with sodium borohydride in the presence of the corresponding mercaptocarborane as a stabilizing agent. To investigate whether direct bonding between the Cc and the thiol group is necessary, synthesis of new compounds (4, 5) that would separate the o-carborane cluster from the thiol group through an alkyl spacer was required.
Synthesis and spectroscopic studies of mercaptocarboranyl derivative, 5 The synthetic way employed for the synthesis of thiocarborane 5 is based on the nucleophilic attack of the carboranyl group on the C–I bond, with the formation of lithium iodide salt, which shifts the equilibrium to the preparation of the chlorinated derivative as an intermediate.21 This compound is then reacted with thiourea and subsequently treated with a solution of sodium hydroxide to yield the desired species, as illustrated in Scheme 1. Regarding the NMR spectroscopic characterization of 5 (Fig. 1), the signal which appears at the lowest field value, 3.58 ppm, belongs to the H bonded to sulfur, whereas the peak at 2.93 ppm is characteristic to the CH2– groups bonded to the two electron withdrawing moieties within the molecule, namely the o-carborane cluster and the sulfur atom. The proximity to these fragments determines the deshielding of the CH2– groups and, as a direct consequence, the downfield shift as depicted in Fig. 1a. The 11B–{1H}-NMR spectrum corroborates the fact that the closo structure is kept during the synthesis, as indicated by the FTIR analysis, which shows a broad absorption band at approximately 2560 cm−1 (see ESI†).
Results and discussion We previously reported that nanoparticles capped with 1-SH-1,2-closo-C2B10H1116 displayed a unique phase transfer property, namely the reversible transfer of the particles from water to diethyl ether, achieved by acidification of the aqueous phase with dilute hydrochloric acid or from ether to water by addition of sodium borohydride. To explain such an unusual property we inferred that the particles act both as electron and ion storage devices. We demonstrated that, in as prepared
5056 | Dalton Trans., 2014, 43, 5054–5061
Scheme 1
Synthesis of 1-HSCH2CH2CH2-2-CH3-1,2-closo-C2B10H10, 5.
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Fig. 1 (a) Comparison between 1H-NMR (black) and 1H{11B}-NMR (red) for 5; (b) 11B{1H}-NMR spectrum of 5 (black) and its corresponding Au MPCs (blue).
Preparation and characterization of Au NPs capped with boron clusters containing mercaptocarborane ligands by reducing with sodium borohydride Ligands 1 and 2 have the Cc directly bonded to the S–H group while the H bonded to the adjacent Cc was replaced by a Me or Ph group, respectively. Ligand 3 has two thiol groups directly bonded to the two cluster Cc. Ligands 4 and 5 have a spacer between the cluster unit and the thiol moiety, in this case an alkyl chain. Having defined the preparative targets, the ratios of reagents were established for mercaptocarborane, 1-SH-1,2closo-C2B10H11, and the reaction conditions were applied as described for the preparation of 3 nm MPCs capped with closo o-carborane-based mercaptocarboranes, 1–5. Thus the preparations of Au NPs capped with 1–5 were performed in a single-phase reaction (methanol) by reduction of tetrachloroauric acid with sodium borohydride in the presence of the proper mono-thiol o-carborane derivative (Scheme 2). The Au NPs obtained, although soluble in water, acetone, and isopropanol, did not display the same phase transfer properties as the ones functionalized with 1-SH-1,2-closo-C2B10H11. The MPCs were characterized by FTIR, 1H and 11B-NMR, UV-vis, and, in some cases, CPS and ICP-AES.
Scheme 2
Formation of 3 nm Au nanoparticles capped with 2.
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The comparison between FTIR spectra of the starting material 2 and its corresponding MPCs is depicted in Fig. 2. FTIR spectroscopy confirms the existence of the boron-based ligands in the structure of MPCs due to strong broad absorptions at 2577 cm−1, a typical indicator of the presence of B–H bonds of a closo cluster attesting that the mercaptocarboranes retain the initial closo structure throughout the MPCs preparation. The NMR analysis completes the characterization of the MPCs, by showing that the S–H is no longer present in the 1 H-NMR spectra after the functionalization of the gold nanoparticles, proving that the bonding of sulfur to the gold core
Fig. 2 Comparison between the FTIR spectra of neutral thioligand 2 (green) and its corresponding MPCs (blue).
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Fig. 3 (a) 1H{11B}-NMR and (b) (blue).
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11
B{1H}-NMR (spectra recorded in d6-acetone); comparison between the starting thioligand 1 (green) and its MPCs
did indeed take place. Regarding the 11B-NMR comparison, the broadening of the boron signals as well as the shift of about 2 ppm towards higher field values indicates the interaction between the ligand and the gold core. Moreover, the NMR spectrum confirms once again the preservation of the closo structure (Fig. 1b and 3). Another characterization method that brings additional information concerning the synthesis of the sought MPCs is UV-vis spectroscopy. As depicted in Fig. 4, the UV-vis spectra of the MPCs stabilized with ligands 1–5 show, in agreement with the theoretical calculations performed by Mulvaney,23 that the MPCs produced are smaller than 3 nm (no surface
plasmon band) for all neutral ligands except for 5 that leads to the synthesis of somewhat larger MPCs and ligand 3 that leads to a broad band centered at approx. 550 nm that is indicative of aggregation and may be due to the presence of the two sulfur atoms that may allow some degree of cross linking (Fig. 4). The particle size distribution was determined as indicated at the experimental section and shown in Fig. 5 for MPCs functionalized with compounds 1–3. Quantitative elemental analysis by ICP-AES was also employed to determine the Au : carborane ratio, since it provides rapid and precise means of simultaneously monitoring most of the elements present in the sample. The results are depicted in Table 1, and show that there is an average of 3 Au
Fig. 5 Size distribution of 3 nm MPCs functionalized with compounds 1 (a), 2 (b) and 3 (c).
Table 1
ICP-AES analysis of 3 nm MPCs of mercaptocarboranes 1–3
Experimental values Fig. 4 UV-vis spectra of 3 nm MPCs of 1–5. Note the clear indication of particle aggregation in the case of ligand 3. Ligand 5 appears to lead to slightly larger particles as evidenced by the shoulder at 520 nm, i.e. the onset of plasmon absorption.
5058 | Dalton Trans., 2014, 43, 5054–5061
Sample
Au
S
B
Theoretical S : B ratio
MPCs of 1 MPCs of 2 MPCs of 3
3 2.8 4
0.91 0.93 1.61
9.4 10.8 9.25
1 : 10 1 : 10 2 : 10
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Scheme 3
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Formation of 10 nm Au nanoparticles capped with 2 via citrate ligands exchange.
atoms for every neutral mercaptocarborane suggesting that a typical nanoparticle would contain in the order of 300 Au atoms and 100 carborane ligands. Hence, the first conclusion to be drawn based on these results is that in order for the MPCs to exhibit phase transfer properties it is required to have the thiol group directly bonded to the carborane cage as well as absence of a bulky substituent at the adjacent carbon of the cluster. The mercaptocarborane that displays the phase transfer has a Cc–H unit at the adjacent carbon. A further question to be answered concerning the requirements for the phase transfer property had to do with the preparative procedure. Is the Au NPs preparation method significant for such an unusual property? To address this, we looked at the well-known 10 nm Au NPs capped with citrate, reported by Turkevich,22 with the objective of exchanging the citrate ligands for mercaptocarboranes. The procedure for preparing water-soluble gold nanoparticles of approximately 10 nm in diameter by exchanging initial citrate ligands for boron-rich clusters is depicted in Scheme 3. After purification, the wine-red water-dispersible product was characterized by FTIR, UV-vis, CPS and, in some cases, ICP-AES. The coordination of thioligand to the gold core was observed using FTIR spectroscopy. Strong broad absorptions at 2562–2570 cm−1 are present in both the pure neutral ligands (1–5) and their corresponding final MPCs, due to B–H stretches, also supporting a closo cluster structure (Fig. 6).24 Additionally, the typical plasmon band present at about 520 nm in the UV-visible analysis (Fig. 7) indicates the absence of aggregation before and after modification with ligands (1–5), also supported by the red color of the dispersions. In relation to the characterization of the MPCs by analytical centrifugation, Fig. 8 shows a small decrease in the apparent particle size upon the formation of the new ligand shell. This is an artifact due to the decreased density of the ligand-capped nanoparticle with respect to that of pure metallic gold used by the analysis program to compute the particle size. The effect of the decrease in density overcompensates that of the increased particle size resulting in an actual decrease in the effective particle size measured by the instrument. In the presence of an
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Fig. 6 FTIR spectra of 1 before (black) and after (red) functionalization of 10 nm gold nanoparticles by ligand interchange.
Fig. 7 UV-vis spectra of MPCs functionalized via ligand exchange with neutral carborane-based mercaptocarboranes.
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Fig. 9 TEM images of the uptake of 10 nm mercaptocarborane-capped MPCs by HeLa cells. It is evident from the distribution of the particles that they are confined to vesicles although the vesicle membranes are not discernible in these images.
Fig. 8 Size distribution of 10 nm Au NPs capped with carborane-based mercaptocarboranes 1–5 by a Centrifugal Particle Size analyzer, CPS. Note the apparent decrease in particle size upon the attachment of the ligand. This is due to the decrease in particle density that overcompensates the effect of increased size.
adequate calibration sample (not available here) this can be corrected, and the method can then reveal quantitatively minute differences in particle size resulting from differences in ligand length that can be as small as a single carbon atom.25 The ICP-AES analysis, summarized in Table 2, indicates that for every carborane cluster (10 boron atoms), there are approximately 10 ± 1 Au atoms suggesting that a typical nanoparticle contained in the order of 30 000 gold atoms26 and 3000 carborane ligands. Given the size of the particle this experimentally determined number of ligands is very high, which suggests that either it was not possible by repeated centrifugation to remove all excess ligands, or, in addition to a compact monolayer, there are further carborane units associated with each nanoparticle. This may be beneficial from the point of view of creating boron rich particles for medical applications and deserves further studies. Although these newly obtained 10 nm MPCs capped with closo mercaptocarboranes 1–5 were dispersible in organic solvents but most importantly in water, there was, once again, no reversible phase transfer phenomenon as in the case of the 3 nm Au NP 1-SH-2-C2B10H11 capped MPCs.
Table 2 ICP-AES of mercaptocarboranes
10 nm
MPCs
functionalized
with
neutral
Experimental values Sample
Au
B
MPCs of mercaptocarborane MPCs of 1 MPCs of 2 MPCs of 3 MPCs of 5
9.5 9.4 9.1 10.9 11.9
10.18 10.58 10.85 9.9 9.4
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Finally, cellular uptake studies of 10 nm MPCs capped with the mercaptocarborane ligands were performed on HeLa cells, a common human fibroblast cell line, with an incubation time of four hours. The experimental procedures were identical to those reported previously.16 The corresponding TEM images are shown in Fig. 9. The MPCs were found to be taken up by the cells and, as most nanoparticles, strictly remained confined to endocytic vesicles, in contrast to the previously reported behavior of the 3 nm particles that also exhibited phase transfer behaviour. Those were capable of crossing membrane boundaries and were found freely in the cytoplasm as well as in the nucleus and even inside the mitochondria. It is likely that the ability to phase transfer by adjusting the ionic and electronic charge of the particle is linked to the ability to cross biological membranes. Hence, none of the particles reported here had this remarkable property. It can thus be inferred that the preparation method for the MPCs as well as the size of the resulting MPCs are also important for the conservation of phase transfer properties, alongside with the presence of a Cc–H bond and the direct bonding of the thiol group to the carborane cluster.
Conclusions In conclusion, we reported the synthesis and structural characterisation of a new mercapto compound as well as the preparation and characterization of 3–5 nm and 10 nm gold nanoparticles functionalized with neutral boron-based mercaptocarboranes. The properties shown by these MPCs were not similar to the ones observed when using 1-SH-1,2-closo-C2B10H11 as a capping agent. Based on these and earlier results, the requirements for the reversible phase transfer of the particles from aqueous to organic layer, and the related remarkable ability to cross biological membranes, are as follows: the S–H group directly bonded to the carborane cage combined with the presence of a Cc–H bond within the cluster and a MPC size of about 3 nm in diameter.
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Acknowledgements This work was supported by Generalitat de Catalunya (2009/ SGR/00279), Ministerio de Ciencia e Innovación (CTQ201016237). A. M. Cioran thanks MEC for a FPU predoctoral grant (AP2007-01723). M. B. and Z. K. acknowledge support from the European Community Seventh Framework Programme (FP7NMP-2010-EU-MEXICO) and CONACYT under grant agreement no. 263878 and 125141, respectively. Access to a CPS disc centrifuge was generously provided by the Knowledge Centre for Materials Chemistry (KCMC) at the University of Liverpool. The authors wish to thank George Miller for conducting the ICP-AES measurements and Prof. Ian A. Prior for access to the TEM and cell culture facilities at the University of Liverpool.
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