Biocompatible gold nanoclusters: synthetic strategies and biomedical prospects

The Nanogold (Au₆₇NCs, 1.4 nm) is detectable by TEM only if is deposited alone onto an ultra-thin carbon film. When buried into a slice of cellular specimen, the Nanogold is unfortunately not discernable. Nevertheless, it can be revealed using a silver enhancement strategy. Takizawa et al carried out immunochemistry experiments on ultra-thin cryo-sections and compared the quality of the EM immunolabeling of lactoferrin into human neutrophils using either 1.4 nm Nanogold-antibody conjugates or 5–10 nm AuNPs-antibody conjugates [30]. It was observed that the 1.4 nm nanoprobes penetrate much deeper into cryo-sectioned neutrophils than the 5–10 nm nanoprobes, enhancing de facto the epitope detection accuracy. Moreover, the quality of the images was in large favor to Au₆₇NCs, justifying the use of a procedure that incorporates an extra silver enhancement step. The enhancement takes place due to silver deposition onto gold surface, which increases the size of the gold core, and hence its opacity to electrons [31].

In recent years, it has been made possible to analyze the same specimen by fluorescent microscopy and EM, correlating the position and field of observation. This new technique named correlative microscopy [32] relies on antibody conjugates probes that can be detected by fluorescence and EM [33]. In this context, the team of Hainfeld prepared a dual system affording both fluorescence and opacity to EM. The group discovered that the AuNCs quenches the fluorescence of fluorescein when the fluorophore was directly attached to the AuNCs-bound ligands phosphine [28]. However, the quenching of fluorescence decreased by increasing the distance between the gold and fluorophore. A fluorescent Nanogold-Fab' was therefore obtained by conjugating the fluorophore (e.g. fluorescein) to the Fab'. This bimodal nanoprobe designed for correlative fluorescence to EM microscopy was named Fluoronanogold (FNG) [34, 35].

Takizawa et al used the FNG in immunochemistry studies and performed correlative microscopy experiments in cells and tissues [36, 37]. The FNG was used as a secondary antibody to investigate the lactoferrin distribution in human neutrophils [36]. A correlation between the fluorescent and electron-opaque images was done before and after silver enhancement of the Nanogold. The results showed a clear improvement of the image resolution and offered better insights of the analyzed samples. Other applications of FNG were found for labeling the microtubules in phagocytic leukocytes [38] or myeloperoxidase in ultra-thin cryo-section of human neutrophils [37]. Overall FNG proved to be a powerful labeling nanoprobe for correlative microscopy.

Hainfeld et al investigated the properties of the phosphine-coated AuNCs and their ability to sensitize tumor cells to radiation in vivo [39]. The phosphine-coated AuNCs of 1.9 nm were intravenously administrated into tumor-bearing mice. The results demonstrated that these phosphine-coated AuNCs mostly accumulated into tumors and not in the liver probably due to their chemical structure. Moreover, the treated mice showed to benefit from the radiation treatment, indicating that the phosphine-coated AuNCs can sensitize the tumor cells to radiation. It has been suggested that the phosphine-coated AuNCs can enhance the therapeutic efficiency by providing a local augmented Au concentration in tumor and thus absorbing x-ray more efficiently than the organic surrounding molecules. In this context, the phosphine-coated AuNCs radiotherapy enhancement takes advantages of the direct interactions Au/radiation waves during treatment while the nanoclusters is hit with high energy. Subsequently the phosphine-coated AuNCs becomes locally a new source of radiation emitting high energy electrons that will generate free radicals and ionizations damaging the cancer cells [40]. The results of this study were reproduced in several other investigations (squamous cell [41], brain [42], subcutaneous [40]) and did not reveal any toxicity of the phosphine-coated AuNCs.

The thiolate-protected AuNCs (Au₁₀₂ and Au₁₄₄) were used as nanoprobes for imaging supramolecular structures in solution. They offered a better contrast in comparison to the Nanogold because the gold core is more substantial. Hakkinen and his coworkers reported the site-specific covalent conjugation of functionalized atomically mono-dispersed AuNCs with 1.5 nm metal core to viral surfaces of enteroviruses echovirus 1 (EV1) and coxsackievirus B3 (CVB3) [55]. Some benzoic acids of Au₁₀₂(pMBA)₄₄ were reacted with a heterobifunctional spacer in order to equip the nanocluster with a maleimide group for further coupling reaction to free cysteines present on the surface of viral particles. Several AuNCs were covalently conjugated to EV1 and CVB3 and the resulting labeled viral particles were observed by TEM (figure 2). Covalent attachments of the AuNCs on the viral particle did not appear to diminish the viral infectivity. This property permitted to investigate the structure-function relationships of enteroviruses (in cellulo) and follows the onset of viral genome release (e.g. entry mechanism into cells and virus uncoating). It was latter demonstrated that a compound binding to the viral particle can also be linked to AuNCs and label its binding pocket onto the viral particle [56]. The approach offered new images on the sites of the enteroviruses EV1 and CVA9 where the compounds bound. Thiolate-protected AuNCs were also used to facilitate the characterization of a sophisticated protein delivery system as described by Postupalenko et al [57]. An analogous AuNCs was also observed to be easily equipped with nuclear localization signal (NLS) and shuttled into the nuclei of living cells in a peptide-dependent manner without cell toxicity [58]. The results demonstrate that these AuNCs can be used inside the cell and open the way to other intracellular delivery opportunities.

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Bowman et al demonstrated that the 2 nm AuNCs having the exact formula Au₁₄₄(SC₆H₄COOH)₅₂ could transform a weakly binding and biologically inactive molecule into a multivalent conjugate that can inhibit HIV-1 fusion to human cells [59]. The Au₁₄₄(SC₆H₄COOH)₅₂ was conjugated to SDC-1721 known as a CCR5 antagonist which functions as principal entry co-receptor for most commonly transmitted strains of HIV-1. The average number of SDC-1721 molecules per AuNCs was 12. The antiviral activity of AuNCs-SDC-1721 was determined on phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells that were previously infected with the CCR5-targeting HIV-1 clone JR-CSF or JR-FL. The results showed that JR-CSF was less sensitive compared to JR-FL and proved as well that a therapeutically inactive monovalent small organic molecule coated with AuNCs can become an effective inhibitor of HIV fusion.

Rotello et al reported a direct cytosolic delivery of CRISPR/Cas9 using nanoassemblies containing 2 nm AuNCs, Cas9 protein and sgRNA [60]. The prepared delivery nanosystem showed high delivery efficiency and acceded to selective gene editing. The same system was further used to knock out SIRP-α in macrophages to turn of the 'don't eat me' signal triggering thus the phagocytosis of cancer cells [61]. These experimental observations open the way to new immunotherapeutic strategies for cancer therapy.

Zhang et al reported that the gluthathione-coated AuNCs namely Au_10–12(GSH)_10–12 was uptaken in larger amount by tumor cells than healthy tissues and evaluated their ability to sensitize tumor cells to gamma radiation [62]. The Au_10–12(GSH)_10–12 were administered by intraperitoneal injection into U14 tumor-bearing mice and subsequently irradiated by gamma radiation. After 23 days (d) the Au_10–12(GSH)_10–12 and radiation co-treated group displayed tumors with 57% decreased volume in comparison to the control groups (figure 3(a)). To get further insights on the in vivo behavior of Au_10–12(GSH)_10–12, the mice were analyzed for gold content at 24 h and 23 d post injection (figure 3(b)). The Au_10–12(GSH)_10–12 clearly accumulated into the tumor at 24 h and were observed in most blood-accessible organs. At 23 d post-injection, the Au_10–12(GSH)_10–12 were still present into the tumors but were cleared out from all other organs, indicating that they might be administrated on long term without risk and undesired accumulation in some tissues.

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The group of Feldheim performed in vivo toxicity, biodistribution and clearance studies of non-fluorescent AuNCs-GSH of 1.2 ± 0.9 nm in mice following a subcutaneous administration at different concentrations [63]. No morbidity was observed even at the highest concentration of 60 μM used in a 4 weeks-experiment. At lower concentrations (10 and 20 μM), AuNCs-GSH were rapidly cleared out from the body by kidney filtration but at increased concentration (60 μM) the AuNCs-GSH were uptaken in part by the reticulo endothelial system (RES). All together these data suggest that AuNCs-GSH can be indeed effectively cleared out from the body by glomerular filtration but that the pharmacokinetic profile becomes less straightforward when the blood starts to be saturated with these AuNCs.

The group of Ackerson studied the ADME and PK properties of five well defined Au₂₅(SR)₁₈ and Au₁₀₂(SR)₄₄ nanoclusters with different SR ligand shells in a murine model [15]. Each AuNCs was prepared and protected with three different ligands: the as-synthesized ligand shell Au₂₅(GSH)₁₈, Au₁₀₂(p-MBA)₄₄ and three partially ligand exchanged shells with tetraethylene glycol grafted in various amounts: Au₂₅(GSH)₉[S(CH₂)₆(EG)₄OH]₉, Au₂₅(GSH)₆[S(CH₂)₆(EG)₄OH]₁₂ and Au₁₀₂(pMBA)₂₅[S(CH₂)₁₁(EG)₄OH]₁₉. After intravenous administration, the distribution and excretion properties were surveyed. The nature of the surface coating dramatically altered the biodistribution and excretion profile. For instance, the Au₂₅(GSH)₁₈ accumulated more in the kidneys while those substituted with tetraethylene glycol accumulated in the liver and kidneys. The Au₁₀₂(p-MBA)₄₄ and Au₁₀₂(pMBA)₂₅[S(CH₂)₁₁(EG)₄OH]₁₉ were primarily located in the liver and spleen following then a renal and hepatic excretion.

Rotello and coworkers analyzed the impact of the surface coating on the cellular uptake of 2 nm AuNCs [64]. The 2 nm AuNCs as well as 4 and 6 nm AuNPs-dodecanthiol were synthesized and then functionalized with anionic, zwitterionic and cationic head groups ligands by exchange place reaction. The cellular uptake performed in HeLa cells showed that the cationic head groups caused the most cellular internalization and the uptake increased as the size increased. For the anionic and zwitterionic AuNPs, the uptakes were moderate and decreased as the size increased. Thereafter, the same group has studied the in vivo distribution of AuNCs (2 nm) with cationic, neutral or anionic terminated head groups after i.v. administration into mice [65]. The neutral AuNCs were partially uptaken by the RES into the spleen and liver but were also excreted by the glomeruli. The anionic AuNCs were efficiently excreted by the glomeruli, whereas the cationic AuNCs were accumulating into the kidneys.

The impact of size on tiopronin-coated AuNCs during the cellular internalization pathway was also investigated by Huang et al [66]. The 2 and 6 nm AuNCs-tiopronin were prepared according to the Murray's procedure [67, 68]. A 15 nm AuNPs-tiopronin were prepared in parallel by reacting AuNPs-citrate with tiopronin. TEM analysis showed that 2 nm AuNCs-tiopronin generate higher cellular uptake in cancer cells than the other AuNPs-tiopronin. The in vivo pharmacokinetic and biodistribution studies were then examined after a single intravenous injection into tumor-bearing mice. The 2 and 6 nm AuNCs-tiopronin were observed to strongly accumulate into the tumor with little accumulation in any other organs. In contrast, the 15 nm AuNPs-tiopronin were detected in spleen and liver [67]. An additional work performed by Simpson et al revealed that the 2.5 nm AuNCs-tiopronin unfortunately engendered harmful side effects in mice that were treated with concentrations above 20 μM [69]. These harmful side effects were latter reduced by grafting PEG on the surface of AuNCs at a 10% ratio [70].

The ability of AuNCs to deliver an antitumoral drug (Doxorubicin, DOX) was studied [79]. The AuNCs capped with HS-C₁₁-EG₆-OCH₂COOH and HS-C₁₁-EG₆-OH at a 1:2 ratio were covalently linked to DOX using an EDC/sulfo-NHS coupling reaction. Before conjugation, the AuNCs did not display any absorbance at wavelengths >400 nm, while after coupling, the new AuNCs-DOX displayed a maximum excitation peak at 475 nm. Confocal imaging of experiments performed on A549 cell line with 1 μg ml⁻¹ DOX or AuNCs-DOX showed that red dots were mainly found inside the cell after 1 h of incubation. After 3 h of incubation, the red fluorescent dots were increased in number in the cytoplasm and could also be found into the cell nuclei. Chatterjee et al prepared fluorescent AuNCs using deoxy guanosine 5'-triphosphate (dGTP) as protecting ligand [80]. The generated orange fluorescent AuNCs-DGTP of 2 nm (λ_ex = 325 nm, λ_em = 590 nm, QY = 2.16%) were able to encapsulate an anticancerous drug (cis-platin, Pt) and the resulting nanodelivery system was further coated with PEG. When delivered to HeLa cells, the AuNCs-DGTP-Pt-PEG could be tracked within the cells using the fluorescence of AuNCs and were observed to induce cell apoptosis due to cis-platin delivery.

Fernandez et al have studied the effects of fluorescent AuNCs-(Zw, ZwMUA and mPEG) on the intracellular accumulation and immune response of human derived-monocyte dendritic cells (DCs) [81]. The study demonstrated that the cellular uptake of AuNCs bearing zwitterionic ligands was more effective than PEGylated ones. The group also compared these AuNCs to AuNPs (12 nm) bearing the same type of ligands. The results showed that AuNCs-Zw induced low cytotoxicity and strong immunosupressive response (Th1/Treg pattern) associated with DC maturation state whereas AuNPs-Zw provoked a cytotoxic response, involving Natural Killer cells (CD56).

The tripeptide GSH is a popular ligand for synthesizing AuNCs with well-defined morphologies. Additionally, GSH plays an important role in the cell metabolism by keeping the cellular potential in a reduced state. Schaaff et al have developed a synthetic approach that led to the preparation of Au₂₅(GSH)₁₈ nanoclusters [82]. In this method GSH was solubilized in water, mixed with a methanolic solution of HAuCl₄ then 15 min later with an aqueous solution of NaBH₄. Negishi et al produced several series of AuGSH_n (n = 10 − 39) of various sizes and masses [83]. The group demonstrated by PAGE analysis and ESI mass spectroscopy that the most stable AuNCs was Au₂₅(GSH)₁₈ (10 437 Da). The strong stability of Au₂₅(GSH)₁₈ was explained by the mechanism of thiolate-induced stabilization that implies a preferential formation of a specific type of AuNCs. Moreover, a selective stabilization of certain AuGSH_n involves an electron donation from Au core to GSH ligands shell but with a smaller impact compared to Au(I)-thiolate complexes.

Luo et al developed strong fluorescent orange-emitting AuNCs using GSH as ligand [13]. The synthesis was straightforward and consisted in mixing aqueous solutions of GSH and HAuCl₄ heated at 70 °C for 24 h. The GSH is the 'reducing-cum-protecting' reactant meaning that GSH is used both as a reducing and protecting ligand of the generated AuNCs. It was observed that the physicochemical properties of the resulting AuNCs heavily rely on the reaction temperature and the GSH/Au input ratio. Indeed, the preparation of fluorescent Au(GSH)_n is a multi-step process that involves 3 main phases. The first phase is the reduction of Au(III) to Au(I) by the sulfur of GSH (or GSSG) and a concomittant formation of Au(I)-thiolate or Au(I)-X complexes (where X = non-thiolate ligand). The second phase consists in the selective reduction of Au(I)-X complexes to Au(0) atoms and caging of the Au(0) core by Au(I)-thiolate complexes. The reduction of Au(I)-X complexes to Au(0) can also be performed using GSSG at 70 °C. In the third phase, an aggregation of Au(0)-on-Au(I)-thiolate occurs. Further characterization indicated that the photoluminescence originated from the Au(I)-GSH complexes [13].

Wu et al studied the photoluminescent properties of [Au₂₅GSH₁₈]^q where q is the charge of the cluster and provided evidences that the photoluminescent properties originated from Au(I)-thiolate complexes [84]. They were also able to set up general rules for tailoring the photoluminescent properties of AuNCs by increasing the electron donating capacity of the ligand via modification of the ligand's structure and by increasing the electro-positivity of the metal core for AuNCs to sustain multiple charge states.

3.2.1.2.1. In vitro fluorescence imaging

3.2.1.3.1. Photodynamic therapy

3.2.1.3.2. In vivo delivery and fluorescence imaging

The capacity of AuNCs to deliver into cells hybrid mixtures of protein and nucleic acids has been reported by Wang et al [91]. A multifunctional nanosystem containing AuNCs has been employed for CRISP/Cas9 protein delivery and targeted gene editing (single guide RNA, sgRNA). Fluorescent AuNCs were firstly prepared by reducing Au(III) with GSH. Some surface-bound GSH were then substituted with a thiolated-TAT peptide to decorate the nanosystem with transduction domains [92]. The positively charged TAT-AuNCs were next associated to negatively charged Cas9 protein and sgRNA-coding DNA plasmid to form a ternary [TAT-AuNCs/Cas9 protein/DNA] complex named GCP. Since the GCP was made with excess DNA plasmid, its overall charge was negative. Thus, for enhanced uptake into cells, the ternary complex was further protected with a polyethylene glycol–phospholipid layer forming a LGCP nanosystem. Further on, the experiments using fluorescently labeled DNA plasmid showed indeed that this nanosystem was uptaken by the A375 cells. To demonstrate the cytosolic release and trafficking of the Cas9/DNA plasmid into the nucleus, in vivo experiments were performed on mice grafted with tumors. The LGCP was delivered to the subcutaneous tumor by intratumoral injection aiming to destruct the polo-like kinase I (PLK-1) gene. At 16 d postinjection it was observed that the LGCP diminished the production of PLK-1 (>70% down-regulation) resulting in inhibition of the tumor growth by 75% in mice.

Another system that combines intracellular delivery with fluorescent detection property was developed by crosslinking AuNCs-GSH with cationic polymers such as poly(allyl amine hydrochloride) (PAH) or polyethylenimine (PEI). The crosslinking yielded nanogels with pH-dependent swelling properties and a 3 to 4-fold fluorescent enhancement due to aggregation-induced emission (figure 5(a)) [93]. The nanogels were loaded with peptides (2 kDa) or antibodies (IgG; 150 kDa) giving rise to protein delivery nanosystems that could be tracked using various imaging techniques (electron and fluorescence intensity; life time microscopy) as well as FCM (figure 5(b)).

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Detection of the nuclei of living cells is highly important in imaging analysis. To selectively label the nucleus, a peptide H₂N-CCYRGRKKRRQRRR was designed to contain two different sequences in its structure. The first sequence (CCY) had the role in facilitating the AuNCs nucleation and the other one (RGRKKRRQRRR) was responsible for the transduction and nuclear accumulating properties as constituted from a NLS derived from protein HIV-1 (TAT) (figure 6) [92, 94]. After synthesis, the AuNCs-CCYTAT were incubated with three different cell lines: HeLa, GES-1 (human gastric mucosa cell) and MRC-5 (human embryonic lung fibroblast cells). The results showed that the AuNCs-CCYTAT were accumulating into the nuclei of cells but with efficiency that varies function of the cell type. Following a similar synthetic procedure, Liu et al fused the CCY sequence to the SV40 NLS sequence to obtain a tridecapeptide (CCYGGPKKKRKVG) that could lead to a fluorescent AuNCs, which was assigned Au₂₅NCs formula according to its photoluminescent properties and MS spectrum [95]. When incubated with cells, the fluorescent Au₂₅NCs-tridecapeptides (λ = 645 nm) was observed in perinuclear compartments and caused cell death. The cell death was explained from the propention of Au₂₅NCs-tridecapeptide to associate to TrxR1, suppressing its activity in the cytoplasm. This leads to an increased level of reactive oxygen species that induces the up-regulation of the activated poly(ADP-ribose) polymerase (PARP) which allows the tumor cell apoptosis to take place in an Au dose dependent manner (figure 7).

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De la Rica et al designed AuNCs-peptides with emission in the NIR region [96]. The employed peptide was synthesized as C(GRP)₃ and used as a 'template-ligand' yielding AuNCs-C(GRP)₃. The resulting AuNCs-C(GRP)₃ revealed an emission peak at 680 nm (λ_ex = 440 nm). In agreement with MALDI-TOF MS and PAGE analysis, it was concluded that the AuNCs had the Au₁₆-C(GRP)₃ formula. When added to human primary fibroblast cells, these Au₁₆-C(GRP)₃ were detected using their NIR emission properties by confocal microscopy. Additionally, Au₁₆-C(GRP)₃ did not induce acute toxicity up to 1 mg ml⁻¹ but started to accumulate inside subcellular compartments.

Chen et al developed AuNCs with high bluish-green fluorescence (480 nm) using histidine as reducing and coordinating ligand [103]. The resulting AuNCs-His of 1.4 nm size were obtained after 5 d of stirring at 4 °C. Since the 480 nm wavelength does not penetrate deeply into tissue, the AuNCs-His were further equipped with a NIR organic dye (MPA) [104]. Using this novel nanoprobe, it was possible to track the biodistribution of AuNCs-His-MPA by NIR fluorescence imaging in tumor-bearing nude mice model (MCF-7). The authors showed that the in vivo retention time of AuNCs-His-MPA was enhanced in comparison to NIR dye alone, observation that gives the opportunity to perform real time monitoring of tumors in living animals.

A core-hollow and shell-porous poly(acrylic acid) nanogel (PAA) was used as template for in situ reduction of gold salts in aqueous environment followed by nucleation and growth of AuNCs of 1.7 nm [105]. The generated AuNCs-PAA-Ngs displayed intense fluorescence at 825 nm and good properties for in vivo imaging with deep penetration. The nanogel was subcutaneously injected into healthy mouse to track the fluorescence (λ_ex = 704 nm, λ_em = 745 nm) in the subcutaneous tissue under NIR excitation. Since bright fluorescence has been observed, further experiments were performed on subcutaneous hepatic H22 tumor-bearing mice. A first fluorescence signal was registered 10 min postinjection that was correlated with good fluorescence contrast distinguishing thus the tumor from surrounding tissues. An accumulation of AuNCs-PAA-Ngs took place in different organs mainly liver and spleen but the organs were considerably cleared out within 96 h postinjection.

Kong et al synthesized bovine pancreatic ribonuclease A (RNAse A)-AuNCs for cancer imaging applications [131]. The resulting RNAse A-AuNCs displayed a fluorescence emission in the NIR range (λ_em = 682 nm) with a good QY ≈ 12%, a long fluorescence time (≈1.5 μs) and a large Stokes shift (≈210 nm). When observed by TEM, the solution contained 1.6 nm dense spherical entities (likely the AuNCs). Some carboxylates present on the protein surface of AuNC-RNase-A were further condensed to vitamin B12 (vitB₁₂) using an EDC/NHS coupling strategy. The VitB₁₂-RNase-A-AuNCs was then used for simultaneous targeting and imaging of cancerous cells (Caco-2) that internalized and transcitosed this nanoprobe by the intrinsic VitB₁₂ uptake system. The specificity of VitB₁₂-RNase-A-AuNCs consists in the potential of performing targeted oral delivery due to the presence of VitB₁₂ that follows uptake pathways via digestive system. Thus, a different type of nanoprobe delivery (oral delivery) that is more confortable could be coupled with in vivo imaging applications.

Fluorescent cytochrome c-AuNCs (cyt c-AuNCs) were prepared and used for delivery of cyt c to cytoplasm and mitochondria as well as for imaging of live lung and breast cells [132]. Cyt c is a membrane impermeable protein that is difficult to deliver to cytoplasm where plays a crucial role in cell apoptosis. The experiments showed that cyt c-AuNCs once entered the cell start to exchange with GSH present in cytoplasm displacing and releasing cyt c. For an accurate monitoring of cyt c delivery inside cells, an organic fluorophore (Alexa Fluor 594) was covalently linked to the already prepared fluorescent cyt c-AuNCs. The experiments showed that cyt c-AuNCs were able to deliver cyt c within approx. 60 min in four different cells lines and image mitochondria by FRET.

Blue-fluorescence emitting Lyz-AuNCs were loaded with DOX and employed for optical imaging of live cancer and non-cancer cells as well as for drug delivery [133]. Uptake of DOX-Lys-AuNCs was 15–20 times higher in A549 and MCF7 cancer cell lines than the uptake in non-cancer lung fibroblast WI38 and breast epithelial MCF10A cell lines. This enhanced uptake was accompanied by an increased cell death, indicating that lysozyme can be used as a vehicle for targeting cancer cell lines.

Li et al synthesized FA-BSA-AuNCs for selective targeting FA receptors overexpressed on the surface of some tumor cells [134]. Condensation of FA onto BSA-AuNCs produced a non-fluorescent FA-BSA-AuNCs likely because the FA was close enough to BSA-AuNCs to promote fluorescence quenching. However, the fluorescence was recovered upon binding of FA to its receptors displayed on the cell surface, indicating that FA-BSA-AuNCs can be used as a turn-on probe for monitoring the presence of FA receptors expressing cells. Two complementary techniques (dark field and fluorescence imaging) were used to determinate the targeting ability and intracellular uptake of FA-BSA-AuNCs into the MGC803 gastric cancer cell line [135]. The dark field scattering displayed images with cells having an intense homogeneous cytoplasmic golden color due to the presence of BSA-AuNCs. When analyzed by fluorescence (λ_em = 588 nm), an intense cytoplasmic red color around the nucleus was observed, indicating the accumulation of FA-BSA-AuNCs in the cytoplasm. Thus, the two imaging techniques showed complementary and confirmed the results. In a different study, BSA-AuNCs were used for the encapsulation and delivery of a hydrophilic anticancerous drug (DOX) [136]. Quantitative analysis using two-photon fluorescent microscopy showed that the fluorescent species were mostly found into the cell nuclei revealing weaker (in the cytoplasm) and brighter red luminescence (mainly in nucleus) for the HeLa cells incubated with DOX-BSA-AuNCs.

Wu et al used BSA-AuNCs (λ_em = 640 nm) for fluorescence detection of tumors in vivo [137]. The BSA-AuNCs were intravenously injected (tail vein) into nude Balb/c mice to vizualize the fluorescence in the superficial vasculature of the whole body. This fluorescence persisted for several hours (up to 5 h) and decreased after 24 h except in the liver and bladder. No sign of toxicity was recorded over the 4 weeks period of treatment in mice. In the same study, mice bearing two types of tumors (MDA-MB-45 or HeLa) were intravenously injected with BSA-AuNCs (figure 9). The fluorescence accumulated over time in the tumor (ratio fluorescence tumor/muscle = 15), principally due to the small size of the BSA-AuNCs that can avoid RES and target the tumor through enhanced permeability and retention effect. Zhang et al performed in vitro experiments with the BSA-AuNCs on two types of cancer cells (MCF-7, HeLa) and one normal cell line (L102) [126]. After 2 h of incubation with BSA-AuNCs, MCF-7 and HeLa displayed a large number of red emitting BSA-AuNCs specifically attached to the cell membrane and the intensity of fluorescence as the time of exposure increased. In contrast, L102 did not display any staining of the cell membrane or cellular uptake for the same incubation time and concentrations of BSA-AuNCs. Further in vivo experiments were performed in mice bearing MCF-7 or Hela tumors. The intratumoral delivery showed fluorescence 60 min after injection and was almost cleared out 24 h later.

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Hu et al developed FA-BSA-AuNCs (λ_em = 655 nm) for active-targeting abilities and bioimaging applications [138]. In cancer tissues, this type of nanoprobe can strongly stain cancer cells due to FA that will target its receptors overexpressed on the surface of certain type of cancer cells. The FA-BSA-AuNCs confirmed its feasibility for faster (1 h versus 4 h) tumor detection/diagnosis by simultaneous microscopic imaging with bright field and fluorescent images in the same section since the two images are reciprocally complementary. In a different study FA-BSA-AuNCs has been covalently conjugated to an anticancerous drug cis-platin (Pt) yielding the Pt-FA-BSA-AuNCs [139]. The nanoprobe was tested on a highly aggressive 4T1 breast cancer cell line and its orthotropic tumor model. It was observed that the Pt-FA-BSA-AuNCs could selectively accumulate inside the tumor and cancer cells. The in vitro results showed a significant cellular apoptosis upon intracellular activation of the cis-platin prodrug. The in vivo studies displayed high efficacy to simultaneously inhibit the growth and lung metastasis of 4T1 breast cancer [139].

BSA-AuNCs were also utilized by Su et al to design a nanogel for targeted drug delivery at tumor site [140]. A thermo and pH-responsive poly(N-isopropyl acrylamide-co-acrylic acid) nanogel was synthesized and then DOX was encapsulated into the negatively charged swollen networks by electrostatic adsorption at pH 7.4. The as-prepared BSA-AuNCs (2 nm) were conjugated onto the surface of the nanogel, then a tumor targeting peptide (iRGD) was covalently attached on the surface of BSA. The in vitro experiments confirmed that the surface modification with iRGD enhanced the intracellular uptake of DOX-iRGD-BSA-AuNCs nanogel in the vein endothelial cells (HUVEC) and the extravascular tumor (B16). The DOX-iRGD-BSA-AuNCs nanogel could be detected and tracked in vivo using the fluorescence of BSA-AuNCs and the release of DOX in tumor could be controlled by the pH-responsive properties of the nanogel. Following a similar strategy, a core–shell AuNCs nanocarrier was employed to deliver a hydrophobic anticancerous drug (camptothecin) (table 1) [141].

The FA-Lys-AuNCs with strong NIR fluorescence (QY of 19.61%) could avoid biological interference and proved to be an efficient contrasting agent for NIRF/CT dual-modal bioimaging [142]. The in vitro studies were performed on HeLa cells (FR positive cell line) and revealed NIR fluorescence of FA-Lys-AuNCs in the cytoplasm after 4 h of incubation. The whole-body time depended imaging was studied by performing in vivo fluorescence of Lys-AuNCs on nude mice. The results confirmed that this nanoprobe is suitable for in vivo optical imaging. The FA-Lys-AuNCs was tested on tumor-bearing mouse demonstrating the ability of FA to target its receptor. The in vivo CT of a Kumming mouse before and after being administered with Lys-AuNCs showed positive signal enhancement in the liver and kidney due to the accumulation of the nanoprobe in these organs.

BSA-AuNCs with NIR fluorescence (λ_em = 645 nm) and robust x-ray attenuation have been reported by Wang et al [143]. To study the in vivo localization of BSA-AuNCs, the saline (control) and BSA-AuNCs (experimental) injected mice at 2 h post injection were sacrificed to generate ex vivo NIR images of the main organs. Only the organs containing BSA-AuNCs showed strong fluorescence (liver and kidneys) and weaker fluorescence (lungs and spleen). Further CT imaging has been accomplished by a comparison experiment between BSA-AuNCs and a commercial iodine based contrast agent (iopromide). As shown in figure 10(a) the HU values increase with the concentration of the contrast materials, with lower concentrations of BSA-AuNCs matching the HU values of much higher concentrations of iopromide. The CT imaging showed visualization of the major organs (figure 10(c)). Moreover, the BSA-AuNCs are excreted in the urine, resulting in clear visualization of the calyces, pelvis, ureters and bladder (figure 10(d)). Thus label free BSA-AuNCs may be suitable for diagnosis of renal abnormalities on CT.

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Insulin-AuNCs with red fluorescence (λ_em = 670 nm) were reported by Liu et al [122]. The confocal microscopy using fluorescence staining showed that insulin-AuNCs were internalized by C2C12 (mouse myoblast) without specific distribution in the cytoplasm. Interesting was to discover that insulin-AuNCs posses a strong x-ray CT signal displayed at a concentration of 30 mg ml⁻¹ as well as when entrapped within C2C12 cells. These observations show that insulin-AuNCs could be used as a bimodal nanoprobe that can function as fluorescent and/or a contrasting agent for x-ray tomography. Additional insights on insulin-AuNCs fluorescence properties were reported by Garcia et al [144].

Recently, Ghahremani et al reported encouraging results with AS1411-BSA-AuNCs used as radiosensitizer for megavoltage radiation therapy of 4T1 breast cancer cells [145]. AS1411 is an aptamer that binds to nucleolin, a protein highly overexpressed in breast cancer cells. The AS1411 targets the nucleolin via AS1411 aptamer and the internalization follows shuttling mechanism from cell membrane to the nucleus. The experimental results showed indeed a high uptake and accumulation of AS1411-BSA-AuNCs in cancer cells. Further experiments performed with AS1411-BSA-AuNCs demonstrated its radiosensing efficacy even when low doses of radiations were used.

A significant study was performed to compare in vivo renal clearance, and biodistribution of the GSH-Au₂₅NCs and BSA-Au₂₅NCs adduct [146]. The blood plasma experiments showed that GSH-AuNCs were more stable than BSA-AuNCs. The biodistribution showed that BSA-Au₂₅NCs mainly accumulated in the liver and spleen and the GSH-Au₂₅NCs had low concentration in all organs. It was observed that 36% of GSH-Au₂₅NCs were cleared out of the body after 24 h but only 1% of BSA-Au₂₅NCs were cleared in the same interval of time. Additionally, 94% and 5% of GSH-Au₂₅NCs and BSA-Au₂₅NCs were metabolized after 28 d, respectively. The study demonstrated that the BSA-Au₂₅NCs adduct accumulate in liver and spleen whereas the GSH-Au₂₅NCs were almost cleared by kidney and have induced no toxicity responses.