Self-Assembled Gold Nanoclusters for Bright Fluorescence Imaging and Enhanced Drug Delivery Akram Yahia-Ammar,† Daniel Sierra,‡ Fabienne Mérola,§ Niko Hildebrandt,† and Xavier Le Guével*,‡ †
NanoBioPhotonics, Institut d’Electronique Fondamentale, Université Paris-Saclay, Université Paris-Sud, CNRS, 91400 Orsay, France Therapeutic Nanosystems, The Andalusian Centre for Nanomedicine and Biotechnology, BIONAND, 29590 Málaga, Spain § Laboratoire de Chimie Physique, Université Paris-Saclay and Université Paris-Sud, CNRS, 91400 Orsay, France ‡
S Supporting Information *
ABSTRACT: Nanoparticles combining enhanced cellular drug delivery with efficient fluorescence detection are important tools for the development of theranostic agents. Here, we demonstrate this concept by a simple, fast, and robust protocol of cationic polymermediated gold nanocluster (Au NCs) self-assembly into nanoparticles (NPs) of ca. 120 nm diameter. An extensive characterization of the monodisperse and positively charged NPs revealed pH-dependent swelling properties, strong fluorescence enhancement, and excellent colloidal and photostability in water, buffer, and culture medium. The versatility of the preparation is demonstrated by using different Au NC surface ligands and cationic polymers. Steady-state and time-resolved fluorescence measurements give insight into the aggregation-induced emission phenomenon (AIE) by tuning the Au NC interactions in the self-assembled nanoparticles using the pH-dependent swelling. In vitro studies in human monocytic cells indicate strongly enhanced uptake of the NPs compared to free Au NCs in endocytic compartments. The NPs keep their assembly structure with quite low cytotoxicity up to 500 μg Au/mL. Enhanced drug delivery is demonstrated by loading peptides or antibodies in the NPs using a one-pot synthesis. Fluorescence microscopy and flow cytometry confirmed intracellular colocalization of the biomolecules and the NP carriers with a respective 1.7-fold and 6.5-fold enhanced cellular uptake of peptides and antibodies compared to the free biomolecules. KEYWORDS: gold nanoclusters, self-assembly, nanogel, aggregation-induced-emission, delivery system, fluorescence enhancement
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Metal quantum clusters, so-called nanoclusters (NCs), have gained high interest in the field of nanomedicine for biosensing,8,9 bioimaging,9−11 and recently for therapy.12−14 NCs are generally synthesized by rather simple methods using small organic molecules or polymeric or biological templates to stabilize them in solution.15,16 Their ultrasmall size (10 μM) or of a highly concentrated Au-GSH sol (gold concentration > 500 μg/mL) lead to some cross-linked and hyperbranched particles (Figure S2). The individual Au NCs with an average diameter lower than 2 nm could be nicely visualized (Figure 1c) with similar size to free Au-GSH (Figure 1d) and EDS confirmed their gold nature (Figures 1, S1). Dynamic light scattering (DLS) measurements of Au-GSHPAH in deionized water confirmed a relatively narrow size distribution around 120 nm (Figure 2a). Adjusting the pH of Au-GSH and PAH solutions to pH 7, hence controlling the electrostatic interactions between the polyelectrolyte and the Au NCs, was found to be optimal to form relatively monodisperse particles without aggregation. These selfassembled NPs are positively charged in solutions for pH< 8 (Figure 2b) as expected due to the presence of the high content of amino groups in PAH (pKa = 8.3). Au-GSH-PAH show good stability in PBS buffer and in complete medium (CM) (RPMI1640 + 10%FCS) with a surface charge decreasing from 35.6 mV in water to 7.3 mV in PBS (10 mM, pH 7.4) and
Figure 2. (a) DLS measurement of Au-GSH and Au-GSH-PAH dispersed in water. (b) Zeta potential of Au-GSH-PAH in PBS buffer (10 mM) at different pH. (c) PAGE analysis of concentrated Au-GSH and Au-GSH-PAH sols. (d) DLS measurement of AuGSH-PAH in PBS buffer (10 mM) at different pH.
to −5.89 mV in CM (Figure S3). This decrease could be associated with the neutralization effect of salt in buffer and the formation of a protein corona around the self-assembled particles in CM. DLS measurements of Au-GSH-PAH at different dilution did not show any change in particle size underlining the strong physical cross-linking between the Au NCs. The presence of a very bright Au NC fluorescence band at higher molecular weight compared to Au-GSH in PAGE electrophoresis (Figure 2c) provided further evidence for efficient cross-linking between Au NCs by the polyelectrolyte. B
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Figure 3. (a) Excitation and emission spectra of Au-GSH (dashed lines) and Au-GSH-PAH (solid lines) sols at the similar Au NC concentrations. (b) Fluorescence emission of Au-GSH and Au-GSH-PAH solutions under UV illumination (λexc = 366 nm). (c) Bathochromic effect (∼10 nm) on fluorescence emission spectra of Au-GSH in the presence of PAH. (d) Fluorescence enhancement (λexc/λem= (450 nm)/ (600 nm)) of the self-assembled NPs as a function of the PAH concentration at different particle concentrations. (e) Fluorescence emission (λexc = 450 nm) and (f) absorbance spectra of Au-GSH-PAH (50 μg of gold/mL) prepared at different PAH concentrations.
27.8% C; 5.1% H; 9.2% N; 3.1% S; 20.3% Au. The molar ratio of Au:S = 1:0.95 for Au-GSH-PAH, calculated from ICP analysis, remained similar to Au-GSH (Au:S = 1:1), which indicates that PAH does not modify the covalent binding between the thiol groups of GSH and the Au NCs. Optical Properties. The fluorescence excitation and emission spectra of Au-GSH and Au-GSH-PAH are depicted in Figure 3a. For both samples, we observed a broad emission band centered around 600 nm and the presence of multiple excitation peaks at 370 nm (3.35 eV), 405 nm (3.06 eV), 455 nm (2.72 eV), 495 nm (2.50 eV), and 515 nm (2.41 eV), which have been assigned to intra- and interelectronic transitions in the metal core and between the ligand and the metal surface of Au NCs.18,32 The addition of PAH had a strong effect on the fluorescence properties with an enhancement of the signal intensity accompanied by a blue shift (∼10 nm) of the emission peak (Figures 3a−e). Fluorescence measurements were performed at different PAH and Au-GSH concentrations (Figures 3d,e) and the results show an almost 4-fold enhancement of the fluorescence intensity for the selfassembled Au-GSH-PAH prepared with 50 μg of gold/mL Au-GSH and 2.90 μM PAH. This is transduced by an increase of the quantum yield from 7% for Au-GSH to almost 25% for Au-GSH-PAH (using fluorescein as reference). The fluorescence intensity is decreasing for high PAH and high Au-GSH concentrations, probably due to the sedimentation of aggregated species. This was confirmed by the decrease of the absorption band in the UV region for sols containing high PAH concentrations (Figure 3f) and by DLS measurements. Fluorescence enhancement of Au-GSH-PAH is almost
The pH-sensitivity of the morphology of the self-assembled NPs is most probably due to Coulombic interactions between Au-GSH and PAH. To investigate pH-dependent swelling properties, Au-GSH-PAH were dispersed in buffer solutions at different pH, and their size was determined by DLS. Multiple peaks at the lowest pH (pH 4, Figure 2) suggest the presence of several species with sizes below 100 nm and a peak around 10 μm. At this pH, the isoelectric point of the carboxyl groups of Au-GSH is reached, which leads to the self-aggregation of AuGSH and to the instability of the system Au-GSH-PAH inducing strong particle aggregation. An increase of the pH led to a continuous shift of the mean particle size from 80 nm (at pH 6) to 350 nm (at pH 11), which is reversible by adjusting the pH in the range 6 to 10. The low polydispersity index obtained for each measurement (PDI < 0.2) suggests that the size increase is related to swelling of the self-assembled NPs rather than their aggregation. This observation of size enhancement over pH makes sense considering the reduction of the electrostatic interaction when amino groups of PAH reach the pKa ∼ 8.3, which relaxes the scaffold of the selfassembled NPs. These results of particle size expansion under basic conditions are in agreement with previously reported hydrogel nanoparticles composed of cationic polymers.5,6 To determine the composition of Au-GSH-PAH, we used thermogravimetry (Figure S4), elemental analysis, and inductively coupled plasma mass spectrometry (ICP). The percentage of organic content in Au-GSH-PAH was estimated at 65.2% corresponding to a ∼14% increase related to the PAH contribution when compared to Au-GSH. Elemental analysis and ICP of Au-GSH-PAH revealed the following composition: C
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ACS Nano unchanged selecting different excitation wavelengths and remains at 3-fold increase even for the most diluted samples (Figure S5). This suggests that scattering of the self-assembled particles is not the major contribution to the fluorescence enhancement. The amplitude-averaged fluorescence lifetime of Au-GSHPAH was 2.2 μs (calculated from three individual lifetimes of 2.7 μs (78%), 0.4 μs (18%), and 0.03 μs (4%)) and remained unchanged when selecting different emission wavelengths (λ = 600 and 670 nm) and 405 nm laser excitation. The high contribution of the long component for Au NCs emitting in the range of 600 to 850 nm has been previously observed and attributed to multiple metal−ligand energy transfer between degenerate triple states.24,27,33−35 Fluorescence measurements obtained in PBS and in CM over a period of 24 h demonstrated the high photophysical stability of Au-GSH-PAH (Figure S6). Only high NaCl level led to a small decrease of the fluorescence intensity (Figure S7). Effect of Cross-Linking on the Optical Properties. The phenomenon of AIE for Au NCs has been recently reported by Xie’s team31 and could be originated from intra-/interaurophilic interaction or electrostatic interaction depending on the nanosystem studied. The relatively high monodispersity and good stability of the self-assembled Au NC NPs in solution offer a good model to investigate AIE thanks to the fine-tuning capability by pH-dependent particle swelling (vide supra). As shown in Figure 3d, addition of PAH induced a strong fluorescence enhancement, which reached saturation at around 3 μM PAH and slightly decreases for higher concentrations. We commented earlier that fluorescent enhancement was not strongly influenced by the dilution factor (Figure S5) and Au NCs were still protected by glutathione (ICP data). Those observations suggest then that the main contribution to the AIE is not related to particle scattering or intra-auriphilic interactions but rather to electrostatic interactions between Au NCs. The cross-linking between Au NCs led to an increase of the amplitude averaged fluorescence lifetime (Figure 4a) from 1.7 μs for Au-GSH to 2.2 μs for Au-GSH-PAH. This increase was mainly caused by the long lifetime component (from 2.2 μs (70%) to 2.7 μs (78%)) and has been attributed by us and others to multiple intra- and interelectronic transitions involving ligand-to-metal charge transfer.26 This lifetime increase reflects only a part of the fluorescence quantum yield increase (from 7 to 25%), which suggests that the AIE also suppresses static quenching effects of the Au NCs. The effect of pH on the fluorescence intensity is distinct between Au-GSH and Au-GSH-PAH. For instance, fluorescence intensity increased continuously for Au-GSH under basic condition, whereas it reached a maximum at pH 6−8 for AuGSH-PAH (Figures 4b and S8). High pH conditions favor electron transfer between gold and sulfur atoms for Au-GSH but the contribution of AIE for Au-GSH-PAH is directly dependent on the electrostatic interaction between the carboxyl groups of GSH and the amino groups of PAH. At pH > pKa (8.3) fluorescence intensity decreased due to the weak crosslinking interaction that is associated with the particle swelling. Lowering the pH to 6.5 enabled recovery of the initial fluorescence intensity confirming the reversibility of particle swelling (Figure S9). Amplitude average fluorescence lifetime of Au-GSH-PAH decrease from 2.0 μs at pH 6 to 1.6 μs at pH 9, whereas there was only neglectable change (1.7 to 1.6 μs) for Au-GSH (Figure S10). For strongly acidic pH, the selfassembled NPs were more instable in solution and led to a
Figure 4. (a) Fluorescence lifetime decays of Au-GSH (blue line) and Au-GSH-PAH (red line) sols. λexc/λem = 405 nm/607 ± 8 nm. (b) Fluorescence emission of AuGSH and Au-GSH-PAH as a function of the pH. (c) Excitation spectra selecting λem = 680 nm and (d) emission spectra selecting λexc = 455 nm of AuGSH (dashed line) and Au-GSH-PAH (solid line) sols at the same gold concentration.
fluorescence decrease. These results show multiple evidence how the strength of the electrostatic interaction between the self-assembled Au NCs enhances the fluorescence emission and the fluorescence lifetime. In an elegant study, Wu et al. suggested that the increase of fluorescent emission, in their case from self-assembled Cu NCs in films, could be partially related to the reduction of the intramolecular vibration and rotation of the capping ligands that could reduce the nonradiative relaxation of excited states.29 By normalizing the excitation and emission spectra of AuGSH and Au-GSH-PAH (Figure 4c and d), we were able to identify a significant increase of the narrow excitation/emission bands λexc/λem = (455 nm)/(680 nm) with a full width at halfmaximum (fwhm) around 20 nm in the presence of PAH. It should be mentioned that whereas sharp excitation peaks were detected for Au NCs and self-assembled Au-NCs, no absorbance band at λ ∼ 520 nm (Figure 3f), typical of the surface plasmon resonance (SPR) for Au NPs, was observed for both samples. The cross-linking between Au NCs by the polyelectrolyte will be then not sufficient to generate the plasmon resonance condition36 keeping the molecular-like properties of the metal NCs. By increasing pH and therefore reducing the cross-linking between Au NCs, this effect was suppressed. Jin et al. reported the presence of a band at 2.63 eV (∼470 nm) for Au25(SH)−18 NCs,37 which is very close to our excitation peak, being partly associated with interband (sp → d) transitions between the orbitals of Au and S. They also demonstrated the influence of the nature of the ligand to the fluorescence emission of Au NCs in the red-NIR region.26 Thus, our observations suggest that Au NC cross-linking has a strong effect on the ligand-to-metal charge transfer and both radiative and nonradiative recombination rates of charge carriers could be responsible of the QY enhancement. Theoretical calculations and spectroscopic investigations may be a useful tool to further understand how the AIE phenomenon affects the electronic structure of Au NCs. D
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ACS Nano Versatility of the Method. To demonstrate the versatility of this method to prepare self-assembled particles from Au NCs in aqueous solution and at room temperature, we choose two additional Au NCs previously described in the literature: (i) Au-Zw, which are Au NCs stabilized by a zwitterionic ligand,23,27 and (ii) Au-GSH-NIR,13 which are Au NCs stabilized by glutathione but with a bigger metal core size than Au-GSH and exhibiting fluorescence emission at λem ≈ 800 nm. By optimizing the PAH concentration we could achieve the preparation of spherical self-assembled NPs Au-ZwPAH and Au-GSH-NIR-PAH (see TEM and DLS measurements in Figures S11, S12). Fluorescence intensity of Au-ZwPAH is similar to Au-Zw and for Au-GSH-NIR-PAH an intensity enhancement of only up to around 1.3-fold compared with Au-GSH-NIR could be found. This behavior can be explained by the low affinity between zwitterionic ligands and PAH and the lower photostability of Au-GSH-NIR (Figure S13). To also show the applicability of our method for other cationic polymers, we selected polyethylenimine (PEI), a polymer widely used in pharmacology to design nanogels.38,39 Using PEI instead of PAH with a similar molecular weight led to comparable self-assembled NPs to Au-GSH-PAH with a mean particle size around 100−150 nm and with a relatively high monodispersity and stability (Figures S11, S12). In addition, around a 3-fold fluorescence enhancement could be realized for Au-GSH-PEI in comparison to Au-GSH (Figure S13). To verify if the molecular weight of the polymer has an effect on the self-assembled morphology and on the AIE, PEI with three different molecular weights (Mn ∼ 20 000; 4000; and 1200) were used as cross-linking agent. We observed a weaker fluorescence enhancement at ∼1.8-fold for the lower molecular weights compared to Au-GSH-PEI at 3-fold prepared with PEI (Mn ∼ 20 000) (Figure S14). Surprisingly, there was no evidence of particle size change observed by TEM and DLS between Au-GSH-PEI samples independently of PEI molecular weight. We hypothesize that a stronger AIE effect selecting the bigger cationic polymer could be related to the higher binding of Au NCs per chains, which increase the close packing of NCs in the self-assembled particles. In Vitro Studies with Loaded and Unloaded SelfAssembled Au NC NPs. An important application of such pH-mediated size-controllable NPs may be theranostics. Therefore, we investigated the drug loading and delivery capability of our self-assembled NPs. THP1 cells were incubated with Au-GSH-PAH and Au-GSH at different concentration (25−250 μg of gold/mL in CM) for 24 h. Confocal laser scanning microscopy (CLSM) and flow cytometry experiments show a clear uptake enhancement for Au-GSH-PAH compared to Au-GSH with an accumulation in the cytoplasm (Figures S15, S16). This behavior is most probably related to the positive surface charge of Au-GSHPAH, a parameter that is often reported to promote strong cellular uptake.40−42 TEM images (Figures 5 and S17) confirmed the accumulation of Au-GSH-PAH in the cytoplasm and inside different multilaminar vesicles. The morphology of the self-assembled NPs is not degraded and does not present any aggregation inside the cells. We could also clearly distinguish the single Au NCs in all self-assembled NPs. Thus, the cross-linking between Au NCs is strong enough to keep the integrity of those NPs inside the cells. Au-GSH also show accumulation in vesicles but with random aggregation (Figure S18). Although fluorescence lifetime imaging micros-
Figure 5. (a−d) TEM images of TPH1 cells at different magnifications incubated with Au-GSH-PAH (50 μg of gold/mL in CM) for 24 h.
copy (FLIM) of microsecond lifetime fluorophores is difficult because it requires long detection times, we were able to show microsecond fluorescence decays for both Au-GSH and AuGSH-PAH and could find a significantly longer fluorescence decay of Au-GSH-PAH compared to Au-GSH, which is in agreement with the lifetime measurement in solution and confirmed the stability of the cross-linking between the Au NCs inside the cells (Figure S19). In order to demonstrate drug delivery capability, we select two types of biomolecules, a peptide (∼2 kDa) and an IgG antibody (∼150 kDa). Alexa Fluor 647 dye peptide or antibody (Ab) conjugates (see experimental section for conjugation procedure) were incorporated in the NPs again by a very simple method. Fluorescent peptide and Ab were added in solution with Au-GSH before adding the polyelectrolyte PAH to form biomolecule loaded self-assembled NPs. The full characterization of the loaded particles Au-GSH-PAH-peptide and Au-GSH-PAH-Ab concerning their size, stability, and optical properties are reported in the Supporting Information (Figures S20−S22). Cytotoxicity assays were performed using propridium iodide on THP1 cells incubated with loaded and unloaded Au-GSH-PAH at particle concentration between 25 and 500 μg of gold/mL for 24 h (Figure S23). Only the highest particle concentration for all type of self-assembled particles seems to induce a toxicity (below 80% of viability), which could be attributed to the cytotoxic effect of the cationic polymer PAH.43,44 The main aim of our theranostic study was to show that loading of the biomolecules into the self-assembled NPs can enhance their transport in/on cells and to determine the colocalization of the nanocarrier and the biomolecules after 24 h incubation. We therefore performed experiments with loaded and unloaded Au-GSH-PAH particles at 25 μg of gold/mL in CM with THP1 cell lines for 24 h. Moreover, various control samples with only peptide and only Ab in equivalent concentrations to the loaded Au-GSH-PAH and without any E
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Figure 6. Flow cytometry measurements of THP1 cells incubated with unloaded Au-GSH-PAH and loaded Au-GSH-PAH-peptide and AuGSH-PAH-Ab (25 μg of gold/mL in CM) for 24 h. Controlled samples with free peptide and antibody (Ab) in equivalent biomolecule concentration than loaded particles and without particles (negative control) are presented as well. Detection of the fluorescence signal in cells from (a,b) the labeled biomolecules (peptide, Ab − Alexa Fluor fluorescence signal) and from (c,d) the self-assembled Au NCs (Au NC fluorescence signal).
for the cell surface, which might have caused the more efficient accumulation in the cytoplasm. Regarding the fluorescence signal observed from the free peptide in cells, it might be originated either from (i) peptides located close/onto the surface of the self-assembled particles and release due to the change of electrostatic interaction in intracellular environment or from (ii) free peptides already released before to penetrate in cells.
particles or biomolecules were performed. The results obtained by flow cytometry illustrated in Figure 6a−b show statistically consistent data with a 1.7-fold enhanced fluorescence intensity for the peptides and 6.5-fold enhancement for the Abs in THP1 cells when loaded in the self-assembled Au NCs compared to free peptides and Abs, respectively. Thus, Au-GSH-PAH successfully promoted biomolecule uptake into THP1 cells. Detection of the Au NC fluorescence using flow cytometry and the same loaded and unloaded samples (Figure 6c,d) showed similar intensities of Au-GSH-PAH, Au-GSH-PAH-peptide, and Au-GSH-PAH-Ab with slightly enhanced fluorescence intensities for the biomolecule-loaded NPs. The combined cytometry results of biomolecule and Au NC fluorescence detection show that the biomolecules were most probably carried by the particles into the cells. The localization of the uptaken NPs and biomolecules was further investigated by looking at single cells with CLSM (Figures 7 and S24). Similar to the cytometry results the microscopy images show more intense fluorescence signals of both biomolecules when loaded to the NPs. The results also indicate that Au-GSH-PAH-peptide NPs are uptaken in the cytoplasm with a clear colocalization of the peptide and the NPs. In the case of Au-GSH-PAH-Ab, we also observed a good correlation between the fluorescence signal emitted by the Ab and by the NPs but with a stronger accumulation onto the cell membrane. This last result is not surprising considering the much larger size of the Ab and the absence of Ab specificity to enter into the cell. The ease for Au-GSH-PAH-Ab to accumulate/aggregate onto the cell surface could also explain the strong signal detected by flow cytometry (Figure 6a). On the contrary, the peptide with a smaller size could be incorporated more easily inside the particles, being “invisible”
CONCLUSION Monodisperse and stable self-assembled Au NC NPs with sizes around 100−150 nm could be prepared in a very simple and fast procedure. The NPs possess pH-dependent (pH range 6 to 10) swelling and shrinking properties and positive surface charge for pH < 8. The cross-linking between Au NCs led to around a 4-fold fluorescence enhancement compared to free Au NCs accompanied by an increase of the fluorescence lifetime. The demonstration of the AIE effect involving electrostatic interaction inside the self-assembled particles could be confirmed by controlling the particle swelling and followed by steady-state and time-resolved fluorescence detection. Multiple imaging techniques (TEM, CLSM, FLIM) and flow cytometry demonstrated efficient cellular uptake without compromising the integrity of the self-assembly inside the cytoplasm. Peptides and antibodies could be loaded in the NPs using a simple one-pot synthesis approach and led to a strongly enhanced biomolecule uptake (1.7-fold for peptides and 6.5fold for Abs) with a clear colocalization of the NP carrier and the biomolecules. The versatility to design self-assembled Au NCs was demonstrated by selecting Au NCs stabilized by different molecules (glutathione, zwitterions) and two different cationic polyelectrolytes (PAH, PEI). The successful preparaF
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hydrochloride) (PAH: Mw ∼ 17 000; 2 mg/mL ∼ 119 μM; pH 7) dropwise to an aqueous solution of Au-GSH NCs diluted at different concentration (25 to 250 μg of gold/mL; pH 7) and stirred for 90 min. In a typical experiment, 50 μL PAH (∼119 μM) was added to 2 mL Au-GSH (50 μg of gold/mL). Solutions were then dialyses with a 15 kDa cutoff for 48 h to remove free Au-GSH and kept refrigerated before use. Similar protocols were used to prepare: (i) Au-Zw-PAH, and Au-GSHNIR-PAH using Au-Zw and Au-GSH-NIR (25−250 μg of gold/mL; pH 7) respectively instead of Au-GSH and (ii) the self-assembled particles Au-GSH-PEI using polethylenimine (PEI: Mn ∼ 20 000; 119 μM; pH 7) instead of PAH. For the experiment of Au-GSH-PEI selecting PEI of different molecular weight: Mn ∼ 20 000; Mn ∼ 4000; PEI Mn ∼ 1200, the same conditions were used as previously described above. See the Supporting Information for the synthesis of Au-Zw and AuGSH-NIR. Peptide and antibody loading in Au-GSH-PAH. The 21 amino acids peptide TRALNNVNRIGNCCAPPVAGG (∼2 kDa) and the Donkey IgG antibody (Ab) (∼150 kDa) were labeled with an excess of Alexa fluor 647-succinimidyl ester (Alexa647) in sodium bicarbonate buffer (0.1 M; pH 8.3) following standard protocol and washed several times with PBS buffer (10 mM; pH 7.4) to remove free Alexa647. In optimized experiments, 25 μL of Alexa647-peptide (500 μM) or Alexa647-Ab (500 μM) was added to 2 mL Au-GSH (50 μg of gold/mL) and stirred for 15 min before adding PAH (50 μL; 119 μM) and let stirred for another 90 min in the dark. Solutions were then dialyzed with a 15 kDa cutoff membrane for 48 h to remove free Au-GSH and biomolecules, then concentrated to 250 μg of gold/mL and kept refrigerated before use. Cell Culture. A monocyte-like cell line (THP1), was cultured in complete medium (CM) consisting of Roswell Park Memorial Institute 1640 medium (Life Technologies, Invitrogen, U.S.A.) supplemented with 10% fetal calf serum (FCS; Life Technologies, U.S.A.), penicillin−streptomycin (100 U/ mL), and gentamicin (1.25 U/mL) and was kept at 37 °C and 5% CO2. THP1 cells were maintained at densities below 1 × 106 cells/mL and diluted in CM to 1 × 105 cell/mL prior to experimental treatment.
Figure 7. CLSM images of THP1 cells incubated with Au-GSHPAH, and with the loaded particles Au-GSH-PAH-peptide and AuGSH-PAH-Ab (25 μg of gold/mL in CM) for 24 h. Controlled experiments were performed with free fluorescent peptide (peptide) and antibody (Ab) at equivalent biomolecule concentration than the loaded particles. Particles are visible with the NC channel (green, 550−620 nm), peptide and antibody with the Alexa647 channel (red, 650−800 nm), and merge both channels with and without bright field (BF). Bars: 5 μm.
tion of self-assembled Au NC NPs with all the different material combinations confirmed the robustness of this method. Our results present a very promising approach to easily produce selfassembly NPs from metal NCs with different types of polymer as cross-linking agent, exhibiting for example stimuli responsive properties such as temperature, enzymatic or redox reaction. The multimodal imaging properties of the NPs, the strong interaction of Au NCs with light or X-ray irradiation, and the ability of the NP drug carriers to dissemble into nontoxic ultrasmall Au particles (
