Cancer theranostics with gold nanoshells - Future Medicine

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Cancer theranostics with gold nanoshells

Gold nanoshells (AuNSs) present a vivid example of integrating nanoscience in order to solve a biomedical problem. AuNSs exhibit tunable surface plasmon resonance, which can be tuned to the near-infrared region in order to realize optimal tissue penetration. The highly efficient light-to-heat transformation by AuNSs during laser irradiation causes thermal damage to the tumor without damaging healthy organs. Transient nanobubbles can form around AuNSs during laser treatment and induce mechanical stress specifically in tumor cells. AuNSs also serve as a versatile platform for the delivery of various diagnostic and therapeutic agents. In this article, we describe the physicochemical properties of AuNSs in the context of their design, preparation and application in cancer theranostics. Ultimately, we look beyond the current research on AuNSs and discussed future challenges to their successful translation into clinical use. Keywords: cancer imaging • drug delivery • gold nanoshells • hyperthermia • photoacoustic • siRNA • surface plasmon resonance • thermal ablation • tumor penetration • tumor targeting

Cancer was the second-leading cause of death in the USA in 2010 [1] . Although recent advances in the understanding of cancer’s mechanisms, diagnosis and therapy have decreased the risk of death from the disease, it remains a major health problem in the USA. More than 1.6 million new cases are predicted to be diagnosed in 2014, with an anticipated overall mortality rate of 35%. Notably, several types of cancer are highly lethal, with 5-year overall survival rates below 20%; these include pancreatic, liver, lung and esophageal cancers [1] . Timely diagnosis and effective treatment are imperative for improving patient survival [2] . Recent progress in nanomedicine has led to the design and application of multifunctional nanoplatforms that are integrated with diagnostic and therapeutic modalities. Compared with conventional small-molecular theranostic agents, these nanometric agents exhibit promising properties, including reduced toxicity, tumor targeting and simultaneous monitoring of treatment response [3] . Among the

10.2217/NNM.14.136 © 2014 Future Medicine Ltd

Jun Zhao1, Michael Wallace2 & Marites P Melancon*,2,3 Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA 2 Department of Interventional Radiology – Unit 1471, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA 3 Graduate School for Biomedical Science, The University of Texas at Houston, 6767 Bertner Avenue, Houston, TX 77030, USA *Author for correspondence: Tel.: +1 713 794 5387 mmelancon@ mdanderson.org 1

numerous nanoformulations that have been developed to date, gold nanoshells (AuNSs) have emerged as highly effective theranostic agents by themselves, as well as versatile carriers for other theranostic agents. The unique surface plasmon resonance (SPR) of AuNSs offers an optical resonance that can be tuned to near-infrared (NIR) regions [4] . Such plasmonic effects also endow AuNSs with lightinduced thermal and mechanical effects [3,5] . Similar to other Au nanoparticles (AuNPs), AuNSs have the additional merit of biocompatibility and benefit from a well-established chemistry for surface modification [6] . In this article, we provide an up-to-date overview of AuNSs as used in cancer diagnosis, therapy and drug and gene delivery. We discuss the theranostic effects of AuNSs, both alone and as delivery carriers for other theranostic agents. Preparation of AuNS Currently, there are two common methods for preparing core–shell AuNSs [2,6] . The

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Review Zhao, Wallace & Melancon first is the formation of the Au shell on the surface of a nonsacrificial template, such as silica nanoparticles (Figure 1A) . In this method, the silica surfaces are first attached with Au nanoseeds (2 nm in size), which function as growth nuclei to form a continuous Au shell around the silica core (Si@AuNS). The second method is the formation of hollow AuNSs (HAuNS) via the galvanic replacement of a metallic core (e.g., Co and Ag) [7,8] , the reduction potentials of which are more negative than AuCl4- /Au. The addition of HAuCl4 results in a HAuCl4-to-Au reduction at the core surface. The Au shell continues to grow at the expense of the metallic core and eventually forms a HAuNS (Figure 1B) . Synthesized AuNSs are often coated with hydrophilic polymers (e.g., PEG) in order to increase their colloidal stability. Further modifications include tagging tumortargeting ligands, fluorophores and radioisotope imaging tracers [9] . Using the two methods described above, a myriad of AuNSs with miscellaneous components have been prepared, including superparamagnetic iron oxide (SPIO) [10] , quantum dots [11] , perfluorocarbons [12] , biodegradable poly(lactic-co-glycolic acid) [13] , liposomes [14] and chemotherapy drugs [15] . In general, AuNSs are a versatile nanometric platform that can accommodate multiple theranostic modalities for treating cancer.

including tunable SPR wavelengths, laser-induced heating and laser-induced mechanical effects. Tunable SPR wavelengths

The SPR wavelength of AuNSs can be tuned from visible to NIR and far-infrared regions by changing their core-to-shell ratio. Such tunability was first established in theory by Neeves and Birnboim using a nanoshell model with a metallic shell and dielectric core [16] . In accordance with theoretical studies, Oldenburg et al. prepared a series of AuNSs that had 60-nm silica cores and 5–20-nm Au shells (Figure 2A) [17] . The SPR peak red-shifted for over 300 nm as the Au shell thinned. Further calculations predicted the SPR to be up to 10 μm as the core-to-shell ratio increased to almost 1000 (Figure 2B) . However, the practical range of SPR is limited by synthetic techniques and challenges during biomedical applications. For example, the SPR of AuNS clusters is of special interest because AuNSs easily aggregate in the presence of proteins and cells once they are injected in vivo. The optical properties of AuNS clusters differ from those of isolated AuNSs because of the strong optical and electromagnetic interactions between adjacent nanoshells. Liu et al. found that the SPR of 3D clusters was blue-shifted compared with that of isolated AuNSs [18] ; Hirsch et al. reported that AuNS dimers that were 10–40 nm apart had a red-shift compared with that of isolated AuNSs [19] .

SPR of AuNS AuNSs has sparked wide interest because of their unique optical performance. During laser irradiation, a coherent oscillation of the conduction band electrons is induced at the Au shell, a phenomenon known as SPR. SPR confers AuNSs with interesting properties, A Hydrolysis SiO2

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As a laser travels through dispersed AuNSs, its energy is attenuated via both absorption and scattering. The energy absorbed by AuNSs can be transformed into heat. The temperature increase (ΔT) on the surface

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Figure 1. Schemes of gold nanoshell preparation. (A) AuNSs deposited on a nonsacrificial SiO2 template. (B) Hollow AuNS synthesis by galvanic replacement of the gold core. APTES: 3-aminopropyltriethoxysilane; AuNS: Gold nanoshell; EG: Ethylene glycol; PVP: Polyvinylpyrrolidone. Reproduced with permission from [2,6] . For color figures please see online at: http://www.futuremedicine.com/doi/full/10.2217/NNM.14.136

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of an individual AuNS in aqueous solution can be calculated using the following equation [20] : 3T =

vabs I 4rReq blwater

where σabs is the absorption cross-section, I is the intensity of the incident laser, Req is the radius of a sphere with the same volume as the AuNS, β is the thermal capacitance coefficient and κwater is the thermal conductivity of water. The calculation shows that ΔT = 2.3 × 10-4 K for AuNSs. Therefore, a laser with a power density of almost 104 W/cm2, which is usually a pulsed laser, is required in order to achieve a 1-K temperature increase on the surface of an individual AuNS. Laser-induced heating of a AuNS dispersion, on the other hand, does

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not require a high-powered laser. Indeed, a continuous laser with a low power density can readily heat a AuNS dispersion to above 50°C (Figure 2C) [21] . Richardson et al. reported that, under these circumstances, the collective heating of AuNSs was the main mechanism by which the temperature increased [22] . Compared with organic dyes, AuNSs are more efficient in light-to-heat conversion because their absorption cross-sections (σabs) is several orders of magnitude higher than those of organic dyes, such as rhodamine 6G [23] . AuNSs are also less susceptible to photobleaching than organic dyes. Larger AuNSs tend to have a higher extinction cross-section (σext ) [24] , but not necessarily a higher σabs, since larger AuNSs also have more scattering. It should be noted that the surB 1000

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Figure 2. Unique properties of gold nanoshells conferred by surface plasmon resonance. (A & B) Tunable surface plasmon resonance of AuNSs, (C) light-to-heat conversion and laser-induced PNBs around AuNSs. Scattering images show (D) AuNSs and (E) produced PNBs. (F) There is an energy threshold to produce PNBs with a pulsed laser. AuNS: Gold nanoshell; PNB: Plasmonic nanobubble. Reproduced with permission from [17,21,27] .


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Review Zhao, Wallace & Melancon rounding tissue may be damaged during photothermal ablation if a large portion of laser energy is scattered by AuNSs [25] . Sikdar et al. calculated that optical thermal ablation could be achieved using Si@AuNS with a silica core of 50–100 nm in size and an Au shell of 3–10 nm in thickness [25] . Meanwhile, another theoretical study found that HAuNSs were more efficient than Si@AuNS at thermal ablation, probably because of their higher absorption efficiency [26] . Pulsed laser-induced thermomechanical effect

Irradiation of AuNSs in aqueous solution with a pulsed laser can generate plasmonic nanobubbles (PNBs), which produce a much stronger scattering signal than AuNSs alone (Figure 2D vs E) [27] . The energy threshold of the incident laser to generate minimal PNBs (EPNB ; Figure 2F) is determined by two factors: the energy to heat water beyond the explosive boiling point and the energy to overcome the surface tension pressure of the vapor–water interface at the AuNS surface. Since the surface tension pressure is inversely proportional to the radius of the interface curvature, which is determined by the geometry of AuNSs, larger AuNSs or AuNS clusters tend to have lower surface tension pressure and consequently lower EPNB than smaller or isolated AuNSs. This difference in EPNB was used to generate PNBs in specific cells or subcellular organelles [28] . The size of PNBs can be tuned by changing the fluence of the pulsed laser. It should be noted that a pulsed laser delivers extremely high-powered irradiation during each pulse (lasting from 10 –9 to 10 –12 s), while the overall energy output remains low. As a result, the overall temperature of AuNS dispersion usually remains ambient. On the other hand, continuous lasers are not as efficient in generating PNBs. Lukianova-Hleb et al. found that a continuous laser at an intensity as high as 3.0 × 104 W/cm2 did not generate PNBs around AuNSs [27] . AuNS for tumor targeting & penetration AuNSs provide a versatile platform for delivering imaging tracers, anticancer drugs and genetic agents to tumors. As drug delivery systems, it is imperative for nanoparticles to have specific accumulation in tumors and minimal uptake in other healthy organs. Prolonged blood circulation is observed with nanoparticles that are approximately 20–200 nm in size and coated with stealth polymers (e.g., PEG). Nanoparticles smaller than 20 nm, especially those smaller than 5 nm, are quickly excreted via the kidneys, while those larger than 200 nm or with an insufficient surface coating can be trapped in reticuloendothelial systems, such as the liver and spleen [29] . Tumor-specific accumulation can occur via passive or active tumor targeting. Passive tumor targeting,

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also known as the enhanced permeation and retention effect, makes use of the leaky tumor vasculature and lack of intratumoral lymphatic drainage [30] . Nanoparticles with an adequate size and prolonged blood circulation can extravasate from blood vessels and accumulate in the tumor over time [31,32] . On the other hand, active tumor targeting can be achieved by conjugating nanoparticles with tumor-specific ligands, including small molecules, peptides, aptamers and monoclonal antibodies [33] . These actively targeting nanoparticles (T-NPs) can bind to tumor cells with a high affinity and be endocytosed. Many in vitro studies have confirmed that T-NPs, including actively targeting AuNSs, have higher uptake in tumor cells than their nontargeting counterparts [8] . Many in vivo studies have also found that T-NPs have higher uptake in tumors [34,35] . However, for in vivo experiments, this uptake in the tumor is not the same as the uptake in tumor cells. Indeed, detailed studies of their intratumoral distributions indicated that T-NPs have difficulty accessing tumor cells that are far from the vasculature; their high affinity for tumor cells causes them to become trapped by the first tumor cells they encounter, preventing them from penetrating into the depth of the tumor [36,37] . The tumor penetration of T-NPs depends on their size, conjugation with the targeting ligands and the tumor environment. Perrault et al. studied the intratumoral distribution of PEGcoated AuNPs with sizes of 20–100 nm [38] . While the 20-nm AuNPs diffused into the tumor depth, most 100-nm AuNPs were confined to the vicinity of the blood vessels. Lee et al. studied the effects of size and targeting ligands on the intratumoral distribution of polymeric micelles (Figure 3) [39] . Smaller micelles demonstrated greater tumor penetration (46 μm for 25-nm micelles vs 20 μm for 60-nm micelles), whereas micelles conjugated with targeting ligands demonstrated reduced tumor penetration (34 μm for targeted micelles vs 46 μm for nontargeted micelles). Since the size of AuNSs ranges from 50 to 200 nm in most cases, it is improbable that they would reach the tumor cells that are far from the blood vessels. Therefore, additional effort is required for AuNSs to deliver diagnostic or therapeutic agents into such remote regions. AuNSs for cancer imaging AuNSs as standalone imaging tracers

Direct imaging of AuNSs in a biological system has been based on its scattering and absorption properties. In vitro dark-field microscopy has been commonly used to study the intracellular trafficking of AuNSs [40] . 3D images of AuNS-injected tumors were obtained using optical coherent tomography based on the strong scattering of AuNSs [41,42] . The strong absorption of



AuNSs could be used to image breast cancer via diffuse optical tomography [43] . Park et al. used Si@AuNS as the contrast agent for two photon-induced photoluminescence imaging [44] . A heterogeneous distribution of nanoshells in the xenograft tumor was visualized through luminescence imaging. Another imaging application of AuNSs based on their ability as radiopaque agents for x-ray imaging. Hainfeld et al. investigated the signal enhancement of tumors in mice injected with 1.9-nm diameter AuNPs [45] . The results showed that blood vessels that are less than 100 μm were visualized clearly within the tumor and has longer accumulation times in the tumor compared with the iodine contrast agents. Once irradiated by a pulsed laser, AuNSs undergo thermoelastic expansion and generate ultrasonic waves that can be recorded for photoacoustic tomography A

(PAT) [46] . AuNSs are an excellent PAT contrast agent because they absorbs NIR light that has optimal tissue penetration. AuNSs are also resistant to laserinduced photobleaching. A comparison of Si@AuNSs and HAuNSs showed that HAuNSs have better signal enhancement for PAT since HAuNSs have a higher absorption coefficient [47] . HAuNS-mediated PAT has been used to image the vasculature and tumors [9,47] . Figure 4A & B shows PAT images of the mouse cerebral cortex before and 2 h after the administration of HAuNSs, respectively [47] . Only large vessels were visible without HAuNSs, while high-clarity images showing small vessels were obtained with HAuNSs (Figure 4A–C) . Lu et al. explored HAuNS-mediated PAT by conjugating HAuNSs with a targeting peptide, arginine-glycine-aspartic acid. The resultant HAuNSs had higher signal intensity in tumors (Figure 4E) than B

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Figure 4. Hollow gold nanoshell-mediated photoacoustic tomography for imaging the brain vasculature and glioblastoma.(A) Brain vasculature image without HAuNSs; (B) image with HAuNSs; and (C) anatomy of a mouse cerebral cortex. (D) Glioblastoma image using nontargeting HAuNSs; (E) image using targeting HAuNSs; and (F) anatomy of a mouse brain with U87 glioblastoma. HAuNSs: Hollow gold nanoshells; L: Left; PA: Photoacoustic. Reproduced with permission from [9,47] .

nontargeting HAuNSs (Figure 4D) [9] . The PAT image was well correlated with the anatomy of the brain (Figure 4F) . AuNSs as carriers for imaging tracers

AuNSs have been tagged with numerous tracers for tumor imaging, including NIR fluorescence imaging, PET, single-photon emission computed tomography (SPECT) and MRI [2] . AuNSs labeled with radioisotopes can be used for nuclear imaging, including PET and SPECT. Xie et al. used 64Cu-labeled Si@AuNSs for the PET imaging of a human head and neck squamous cell carcinoma (SCC-4) xenograft in rats. The tumor became visible on PET imaging 1 h after injection of the nanoshells [48] . The signal intensity at the tumor sites reached a plateau 24 h postinjection and lasted until 48 h postinjection. AuNSs were also labeled with 111 In for SPECT imaging [49] . MRI is a powerful and noninvasive modality for cancer diagnosis, providing high-resolution images. SPIO nanoparticles are a popular MRI contrast agent based on their high relaxivity, excellent contrast and low toxicity. AuNSs loaded with SPIO have been tested

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in a number of preclinical studies [50–52] . Dong et al. prepared 150-nm SPIO@polymer@AuNSs containing a SPIO-embedded polystyrene-b-poly(acrylic acid) core [50] . The r2 relaxivity of the nanoshell was 270 mM/Fe/s. In vivo MRI imaging was tested in mice bearing MCF-7 breast cancer subcutaneous xenografts. The T2-weighted image of the tumor sites showed a 38% decrease in T2 signal intensity after injection of the nanoshells. Melancon et al. prepared SPIO@Si@AuNSs for the MRI of mice bearing A431 human epidermoid carcinoma subcutaneous xenografts [51] . The SPIO was embedded in a silica core, which was then wrapped with a Au shell. The T2* values of the tumor sites decreased to approximately 70% at 24 h after injection of the nanoshells. Chen et al. conjugated neutrophil gelatinase-associated lipocalin to SPIO-loaded AuNSs, and used them for the imaging of pancreatic cancer in AsPC-1 xenografts in mice [52] . The in vivo imaging showed higher tumor contrast by using the neutrophil gelatinase-associated lipocalin-conjugated nanoshells compared with nontargeting pegylated nanoshells. Very recently, Dong et al. synthesized a SPIO@hybrid@AuNS, which can



be used for magnetic resonance-guided photothermal therapy [50] . In their study, using mice bearing MCF-7 xenografts, the authors were able to show in vivo and in vitro decreases in T2 signals on MRI after the injection of the nanoparticle, as well as increases in temperature in the tumors injected with the agent with laser treatment [50] . NIR fluorescence has proven to be a promising modality for noninvasive imaging. However, fluorescence quenching is often observed when a fluorophore is directly attached to the AuNS surface. Bardhan et al. found that such quenching could be reversed by adding spacers between AuNSs and fluorophores [53] . Indeed, a 40-fold enhancement of fluorescence intensity and a quantum yield as high as 86% were observed when human serum albumin was used as the spacer. Such enhancements were attributed to the enhanced light absorption by the fluorophore, modification of the radiative decay and the enhanced coupling efficiency of the emission to the far field. Similar results were obtained using silica as the spacer [54] . The enhancement was 50-fold for 7-nm spacing and sevenfold for a 42-nm spacer. Such AuNS-mediated enhancement is optimal when the SPR of AuNSs is tuned to the emission wavelength of the fluorophore [55] . AuNSs for cancer therapy Thermal ablation & hyperthermia

AuNSs generate heat as a result of irradiation by a continuous laser. The temperature increase (ΔT) depends on the laser power and AuNS concentration. Both high-temperature thermal ablation and low-temperature hyperthermia have been used for cancer treatment. Conventional thermal ablation techniques, such as high-intensity focused ultrasound, radiofrequency and microwave- and laser-induced ablation, are hindered by the potential damage to surrounding healthy tissues and by heat dissipation by adjacent blood vessels [56] . AuNS-mediated thermal ablation, on the other hand, is advantageous in several aspects. AuNSs absorb NIR lasers that can penetrate tissue, enabling a noninvasive and extracorporeal delivery of energy. The thermal damage occurs only in tissues that contain AuNSs; those that do not are spared. Therefore, by specifically targeting AuNSs to tumor cells by conjugating tumorspecific ligands, tumor-specific thermal ablation can be achieved. The ΔT during AuNS-mediated thermal ablation can reach 30–50°C. Nguyen et al. reported that >95% of cells underwent necrotic cell death during AuNS-mediated thermal ablation [57] . Melancon et al. prepared SPIO-loaded AuNSs and conjugated them with C225, an anti-EGF receptor (anti-EGFR)


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antibody [21] . The resultant SPIO@AuNS–C225 was specifically taken up by EGFR-overexpressing head and neck cancer cells (Figure 5A vs B & C) . The thermal damage was confined to the cells treated with C225–conjugated SPIO@AuNSs (Figure 5D), but not those treated with nontargeting SPIO@AuNSs or blocked with free C225 (Figure 5E &F) . Antitumor efficacy was also observed in animal studies. Hirsch et al. evaluated AuNS-mediated thermal ablation in a mouse xenograft under MRI guidance [56] . The average intratumoral temperature increase was 30–60°C, with a distribution of temperature along the laser trajectory. A histological analysis revealed that tissue damage was colocalized with regions containing AuNSs. Unlike direct cell killing by thermal ablation, hyperthermia (40–46°C) induces cellular biochemical alterations, such as changes in protein conformations, protein unfolding, exposure of protein hydrophobic domains and aggregation with other proteins that are not directly affected by hyperthermia [58] . Diagaradjane et al. reported that AuNS-mediated hyperthermia enhanced tumor response to radiation therapy by increasing tumor perfusion [59] . Consequently, the tumors had fewer FaDu A

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Figure 5. Gold nanoshell-induced thermal ablation in vitro. (A–C) Selective binding of AuNSs to EGF receptorpositive FaDu cells. Cells were incubated with (A) C225– SPIO@AuNSs, (B) PEG–SPIO@AuNSs or (C) C225 plus C225–SPIO@AuNSs for 30 min at 37°C. Cells were stained with DAPI (nuclei, blue). Light-scattering images of AuNSs were pseudocolored green. (D–F) Cell viability after various treatments. Cells within yellow lines were treated with a NIR laser. Live cells were stained green with calcein AM. (D) C225–SPIO@AuNSs with NIR laser, (E) blocking group (C225 + C225–SPIO@AuNSs) with NIR laser and (F) PEG–SPIO@ AuNSs with NIR laser. AuN: Gold nanoshell; DAPI: 4’,6-Diamidino-2-phenylindole; NIR: Near-infrared; SPIO: Superparamagnetic iron oxide. Reproduced with permission from [18] .


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Review Zhao, Wallace & Melancon hypoxic regions that were resistant to radiation therapy. The tumor doubling time was significantly slowed by combination therapy: 29 days for combination therapy versus 17 days for radiation therapy alone. Recently, hyperthermia has been used to treat cancer stem cells (CSCs). CSCs are critical for cancer treatment because of their resistance to most therapies and ability to cause tumor relapse and metastasis [60] . Hyperthermia can deplete CSC populations, decrease the expression of stemness-related proteins and reduce tumorigenicity [61–63] . Atkinson et al. found that AuNS-induced hyperthermia sensitized breast CSCs to radiation therapy [64] . The CSCs were first identified and confirmed by limiting dilution transplantation. As few as ten CSCs generated tumors in mice [64] . Radiation alone enriched CSCs by 30%, while combination therapy with hyperthermia reduced the CSC population by more than 70% (Figure 6A). These results were confirmed using the mammosphere assay (Figure 6B), indicating that CSCs were depleted by combination therapy. The radiosensitization was attributed to the disruption of DNA damage repair, since more DNA double-strand break foci were present after combination therapy (Figure 6C & D). A histological analysis revealed that radiation therapy alone resulted in tumors with more aggressive undifferentiated phenotypes (Figure 6F) as compared to control (Figure 6E), while combination therapy generated tumors with a more differentiated phenotype (Figure 6G). Bubble-induced mechanical treatment of cancer

AuNSs can generate PNBs upon irradiation by pulsed lasers. These PNBs can be used to kill cancer cells and enhance intracellular drug delivery [65] . PNBs expand quickly and then collapse, producing a mechanical impact that can disrupt the membranes of cells or intracellular organelles. If it is strong enough, this disruption kills cells in a short time. Since this mechanical effect is confined to the cells that contain AuNSs, highly selective tumor cell killing is possible [28,66] . Wagner et al. prepared AuNSs that were conjugated with C225, an anti-EGFR antibody [66] . The resultant C225-conjugated AuNSs were specifically bound (Figure 7A) and internalized (Figure 7B) by EGFRexpressing C4–2B prostate cancer cells. A pulsed laser then created PNBs, the lethality of which was tuned by the incident laser influence. Sublethal PNBs did not disrupt the cell morphological characteristics or the integrity of the cell membranes (Figure 7C), while lethal PNBs induced blebs at the cell membrane and subsequent cell death (Figure 7D) . The effect of PNBs on cancer cells was visualized in real time using brightlight and fluoresence microscopy. C4–2B cells were preincubated with DiI fluorescent dye, which stained the viable cells. The sublethal PNBs did not induce

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changes in cell morphological characteristics or loss of fluorescent signals (Figure 7E & F) . By contrast, lethal PNBs created blebs at the cell membrane (Figure 7G), and the DiI signals were attenuated within a few hours as a result of cell death (Figure 7H) . Lukianova-Hleb et al. evaluated specific cell killing in a mixed cell culture model with HN31 squamous cell carcinoma cells and NOM9 normal cells [67] . AuNSs were conjugated with anti-EGFR antibodies and were therefore selectively taken up by HN31 cells. Upon laser irradiation, AuNSs produced PNBs that were captured by scattering (Figure 7J) . Subsequent cell staining revealed that cell death was restricted only to those cells with PNBs, while adjacent cells experieneced no damage (Figure 7I–K) . Such selective cell killing was attributed to the difference in EPNB (see ‘Pulsed laser-induced thermomechanical effect’ section). HN31 cells had higher concentrations of intracellular AuNSs, which likely formed clusters in endosomes, while NOM9 cells only had minimal amounts of isolated AuNSs. Since the EPNB for AuNS clusters was lower than that for isolated AuNSs, a selective production of PNBs was achieved by tuning the laser influence. This resulted in tumor-specific killing. The disruption of cell membranes by AuNS-induced PNBs has been explored as an approach to the intracellular delivery of chemotherapy drugs and genetic agents. Drug delivery into multidrug-resistant cancer cells has been challenging for decades. Chemotherapy drugs can be actively transported out of multidrug resistant cells that have P-glycoprotein expression [68] . This drug efflux pathway can be circumvented using drug-loaded nanoparticles, but the intracellular drug release from nanoparticles is often slow and relies on the physiological environment (e.g., pH or redox potential). Lukianova-Hleb et al. used AuNS-induced sublethal PNBs for drug delivery [67] . These PNBs did not kill cells, but rather transiently opened pores at the cell membrane in order to facilitate the influx of chemotherapy drugs, enhancing drug delivery. Similar levels of cell death were reached using tenfold lower drug concentrations in the presence of such sublethal PNBs. This PNB-enhanced drug delivery was limited to cells that had taken up AuNSs. Lukianova-Hleb et al. prepared CD117-targeting AuNSs and incubated them with a mixture of CD117+ and CD117- human stem cells [69] . After incubation, the cells were exposed to extra cellular DNA plasmids (pHIV-7-green fluorescent protein [GFP]). CD117+ cells were selectively transfected with GFP signals, while CD117- cells were not affected. PNBs also facilitated the drug release of nanoparticles and their endosomal escape. Anderson et al. prepared liposomes that were loaded with AuNSs



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Figure 7. Gold nanoshells produced plasmonic nanobubbles with laser irradiation and specifically killed C4–2B cells that had taken up gold nanoshells. (A–D) Scanning electron microscopy images: (A) cells with AuNSs attached to the cell membrane; (B) cells with internalized AuNSs; (C) cells treated with sublethal PNBs; and (D) cells treated with lethal PNBs. (E & F) Cells treated with nonlethal PNBs: (E) optical and (F) fluorescence images. Cells were preincubated with DiI (green) dye that stained only the viable cells. (G & H) Cells treated with lethal PNBs: (G) optical and (H) fluorescence images. C4–2B cells were seeded in a plate, (I) preincubated with AuNSs and treated with a near-infrared laser in order to induce lethal PNBs. (J) PNBs were visualized by scattering imaging (green). (K) After treatment, dead cells were distinguished using trypan blue. AuNS: Gold nanoshell; NP: Nanoparticle; PNB: Plasmonic nanobubble. Reproduced with permission from [66,67] .

and dye-labeled proteins, the latter as a mimic drug [70] . Irradiation with a pulsed laser generated PNBs around AuNSs, which disrupted the lipid bilayer of the liposome, thereby releasing the dye-labeled proteins (Figure 8A) . Lukianova-Hleb et al. incubated HN31 cancer cells with AuNSs and Doxil® (ALZA Corporation, OH, USA) simultaneously [71] . Both AuNSs and Doxil were conjugated with panitumumab, an antiEGFR antibody, and were thus actively endocytosed into the endosome (Figure 8B & C) . HN31 cells were then subjected to a pulsed laser in order to generate sublethal PNBs. The generated PNBs disrupted the liposome and endosome and subsequently released doxorubicin into the cytoplasm. The results showed

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that larger PNBs, created with a stronger laser, were more effective at releasing doxorubicin and led to a higher cytotoxicity (Figure 8D) . AuNSs as carriers for anticancer drugs & genetic agents

HAuNSs exhibit interesting properties as drug carriers because of their hollow structure [3,72,73] . You et al. prepared doxorubicin-loaded HAuNS (Figure 9A) [74] . Because of the hollow structure, as much as 63% (by weight) of doxorubicin was loaded in HAuNSs, as confirmed by ultraviolet-visible spectroscopy (Figure 9B) . A continuous NIR laser, used at 4.0 W/cm2 for 5 min, led to the release of 25% of the loaded amount.



A

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Figure 8. Plasmonic nanobubbles-induced drug release and endosome escape.(A) AuNS-induced PNBs released the drug from the liposome (AuNSs: orange sphere; drug: blue open circle). (B) Drugs and AuNSs colocalized in the endosome after being actively endocytosed by cells. (C) Fluorescence microscopy images of doxorubicin (red) and AuNS (blue) colocalization. (D) Cell viability measured 72 h after PNB treatment at different sizes (lifetimes) of PNBs: black (no PNBs), green (21 ± 7 ns PNBs), red (32 ± 8 ns PNBs) and blue (51 ± 7 ns PNBs). AuNS: Gold nanoshell; NP: Nanoparticle; PNB: Plasmonic nanobubble. Reproduced with permission from [70,71] .

Repeated laser treatment induced further releases, albeit of smaller amounts of doxorubicin, probably because less doxorubicin remained in the HAuNSs. In comparison, only 11% of doxorubicin was released after 2 days of incubation in phosphate-buffered saline at ambient temperature [74] . Lee et al. studied the in vivo release of doxorubicin from HAuNS–doxorubicin and intratumoral AuNS-mediated heating [75] . Laser irradiation released doxorubicin from AuNSs inside tumors, showing stronger fluorescence signals than the nonlaser control (Figure 9C) . The intratumoral distribution of doxorubicin-loaded HAuNSs was monitored by PAT (Figure 9D) . Since the PAT signal intensity was related to temperature, a real-time map of intratumoral temperature was obtained. The peak temperature reached approximately 54°C after 3 min of laser irradiation, which was sufficient to induce thermal ablation (Figure 9E) . You et al. went on to prepare HAuNS–doxorubicin that was conjugated with an EphB4 binding peptide (T-HAuNS–doxorubicin;


EphB4 is overexpressed in several tumor types, such as prostate, breast, bladder, lung, colon, gastric and ovarian cancers) [76] . The resultant T-HAuNS–doxorubicin showed selective uptake in ovarian cancer xenografts in mice; its tumor uptake was 50–100% higher than in its nontargeting counterpart. The in vivo antitumor efficacy showed a disappearance of the tumor after chemothermal therapy using T-HAuNS–doxorubicin (Figure 9F) . A histological analysis showed the presence of scar tissue and a lack of residual tumor cells in mice treated with T-HAuNS–doxorubicin plus laser (Figure 9G) . RNAi (i.e., the use of siRNA or antisense ssDNA oligonucleotides to disrupt the function of specific genes and knock down the expression of their encoded proteins) has been proven to be an effective treatment for cancer [77] . However, siRNA and antisense DNA cannot voluntarily enter cells because of their negative charges [78] . Currently, there are two methods for loading siRNA/ssDNA onto AuNSs. In the first method,


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B 0.10 O

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Figure 9. Hollow gold nanoshell–doxorubicin for photochemotherapy. (A) Schematic illustration of HAuNS–doxorubicin. (B) Ultravioletvisible spectra of doxorubicin (red), HAuNSs (green) and HAuNS–doxorubicin (burgundy). (C) In vivo monitoring of doxorubicin release after laser treatment. (D) Photoacoustic signals (red) of HAuNSs in tumor xenografts. (E) Intratumoral temperature increase after treatment with HAuNSs and a near infrared laser. (F) Tumor growth curve after various treatments. (G) Histological analysis of tumor sections after various treatments. DOX: Doxorubicin; HAuNS: Hollow gold nanoshell; T-DOX@ HAuNS: DOX-encapsulated HAuNS with EphB4 as targeting ligand (T). Reproduced with permission from [73–76] .

AuNSs are coated with a layer of positively charged polymers, followed by condensing the negatively charged siRNA/ssDNA via ionic interactions. Huschka et al. prepared AuNSs coated with poly-l-lysine and used them to load siRNA/ssDNA labeled with Alexa Fluor® (Life Technologies, CA, USA) 488 onto AuNSs (Figure 10A) [79] . The intracellular release of siRNA/

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ssDNA was monitored by fluorescence microscopy, since the fluorescence signals were no longer quenched after release. Irradiation with a continuous laser resulted in the cytoplasmic distribution of fluorescence signals, indicating that siRNA/ssDNA was not only released from AuNSs, but they also escaped from endosomes (Figure 10B) . The low-power laser’s insignificant



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Figure 10. Gold nanoshells for gene delivery. (A) Schematic illustration of gene loading by precoating gold nanoshells with a silica core with cationic poly(L-lysine). (B) Treatment with a near-infrared laser induced gene release in gold nanoshells with a silica core (green). (C) Near-infrared laser treatment improved the downregulation of green fluorescent protein. NS-PLL: Nanoshell coated with poly(L-lysine) Reproduced with permission from [79] .

heating was still able to disrupt endosomes and allow AuNS–siRNA/ssDNA to escape, probably because of the production of reactive oxygen species [80] . The prepared system was tested in H1299 cells that expressed destabilized GFP and RFP. Compared with the nonlaser-treated group, laser-treated samples exhibited 23 and 31% more GFP downregulation for antisense ssDNA and siRNA, respectively (Figure 10C) . The second method for loading siRNA/ssDNA onto AuNSs is to covalently conjugate thiol-tagged siRNA to AuNSs via the well-characterized Au–S bond. Lu et al. prepared HAuNSs that were covalently conjugated with thiol-tagged anti-NF-κB siRNA and folic acid (F–HAuNS–siRNA) [81] . The release of siRNA upon laser irradiation was confirmed by an elemental analysis that revealed that the nitrogen and phosphorus content disappeared after the laser treatment. Gel electrophoresis revealed that the released siRNA had a similar molecular weight to the free siRNA, indicating selective cleavage of the Au–S bond without degradation in the siRNA backbones. The release mechanism was attributed to the thermal effect and the ‘hot electron’ nonthermal effect [82] . Tumor targeting was realized by the folate ligand. Immunohistochemical staining confirmed that laser-treated F–HAuNS–siRNA significantly downregulated NF-κB expression. Quantified data were confirmed using an immunoblotting assay. AuNSs for clinical applications AuNSs have great potential for the imaging and therapy of patients with cancer. AuNSs have been shown to


detect cancers using minimally invasive in vivo imaging, such as two photon-induced photoluminescence, diffuse optical tomography, x-ray imaging and photoacoustic imaging. From the therapeutic standpoint, AuNSs can mediate heating through hyperthermia or thermal ablation, can be made to release drugs and therapeutic DNA/RNA and can selectively target areas of interest using specific ligands. However, at present, Si@AuNSs are currently involved in two pilot clinical investigations under the trade name Aurolase® (Nanospectra Biosciences Inc., TX, USA): first, in the treatment of primary and/or metastatic lung cancer, and second, in the treatment of refractory and/or recurrent head and neck cancer. According to clinicaltrials.gov, both studies would have been completed by February 2014. However, no results have been reported to date. The AuNS profiles in terms of biocompatibility, toxicity and in vivo stability have been investigated for potential clinical applications. Gad et al. performed a thorough evaluation of Si@AuNSs based on International Organization for Standardization (ISO) standards for biomedical devices [83] . Si@AuNSs had negative results in evaluations of genotoxicity, cytotoxicity, hemocompatibility, tissue tolerance and pyrogenicity. When injected intravenously at massive doses (∼10× the predicted effective therapeutic dose), no subacute systemic toxicity was found in rats, nor was significant acute systemic toxicity found in albino Swiss mice or beagle dogs. Biodistribution studies at up to 28 days postinjection revealed that approxi-


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Review Zhao, Wallace & Melancon mately 85% of injected Si@AuNSs were entrapped in reticuloendothelial system organs, including the liver and spleen, while no significant amounts of nanoparticles were present in other tissues. Long-term clearance studies found that the nanoparticles were not excreted from the body at 404 days postinjection. A chronic toxicity study in beagle dogs exhibited no noted clinical observations over 10 months postinjection. However, differences in food consumption ere observed, in the order of nontreatment control > low dose (2 × 7.5 ml/kg) > high dose (2 × 15 ml/kg). Histopathology found black pigmentation in Kupffer cells (liver), macrophages (spleen) and some lymph nodes. The pigmentation was dose dependent and was mild for the low-dose group and moderate for the high-dose group. Although Gad et al. found that Si@AuNSs remained intact in animal bodies after an extended time [83] , the stabilities of other AuNSs in vivo are yet to be examined. Recently, Goodman et al. found a fragmentation of Ag@AuNSs in vivo, which was affected by the size of nanoshells and the pH of the environment [84] . Smaller nanoshells broke more readily, probably due to their thinner shells, while the pH sensitivity was attributed to the dissolution of Ag remnants at an acidic pH. In addition, the presence of human serum also led to the degradation of nanoshells. On the other hand, the structural changes of AuNSs during laser irradiation have been well documented. Akchurin et al. demonstrated the complete destruction of AuNS structure after a single laser pulse at 10 mJ by transmission electron microscopy [85] . Determining the fate of these structure-compromised AuNSs in vivo requires further safety evaluation in order to minimize the risk to patient health. Conclusion & future perspective In this article, we have summarized the use of AuNSs as standalone theranostic agents against cancer and as drug delivery platforms in combination with other

theranostic modalities. The unique SPR of AuNSs has important properties, such as tunable SPR absorption, light-to-heat conversion and light-induced mechanical effects. The well-characterized Au chemistry of AuNSs, in combination with the versatile nanometric structures, confers them with promising performance as drug delivery systems. AuNSs in cancer theranostics are perfect examples of the evolution of a multidisciplinary nanoscience that encompasses chemistry, material science, physics, biology and oncology. However, more evaluations are needed before AuNS-mediated cancer therapy can be further advanced. For example, the remnants of the sacrificial cores of HAuNSs remains a topic that has not been well addressed. Such residual core components (e.g., Ag or Co) could cause additional toxicity that may hinder the clinical translation of AuNSs. The tumor-specific delivery of AuNSs, especially the penetration of tumors with poor vasculature or dense stroma, has yet to be elucidated. In addition, the clearance of AuNSs has not been determined. Since most AuNS formulations are nondegradable, the kinetics of their clearance may pose a risk for patients. Considering the existing health condition of cancer patients, the excessive accumulation of AuNSs is not desirable. The design of a novel degradable AuNS formulation may alleviate concerns regarding chronic toxicity. Further understanding of these important issues will facilitate the bench-to-bedside translation of AuNS-mediated theranostics. Financial & competing interests disclosure This work was supported in part by grants from NIH NCI (Award # 2 P50 CA127001-06) SPORE in Brain Cancer Career Development Award (to MP Melancon) and the John S Dunn Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. The authors would like to thank A Sutton for editing the manuscript.

Executive summary • Gold nanoshells (AuNSs) confer antitumor efficacy through laser-induced thermal and thermomechanical effects. • AuNSs enable tumor imaging via photoacoustic effects, as well as their high absorption and scattering properties. • AuNSs serve as nanoscale carriers for both imaging tracers and therapeutic agents, with potential for active tumor targeting. • As a drug delivery platform, the physicochemical properties of the AuNSs have to be optimized in order to achieve satisfactory tumor penetration. • Simultaneous imaging and therapy is possible with the use of AuNSs. • AuNSs are biocompatible and well tolerated in experimental animals, although the difficulty of clearance may present chronic hazards.

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