Monodisperse Sub-100 nm Au Nanoshells for Low-Fluence Deep-Tissue Photoacoustic Imaging.

Nanoparticles with high absorption cross sections will advance therapeutic and bioimaging nanomedicine technologies. While Au nanoshells have shown great promise in nanomedicine, state-of-the-art synthesis methods result in scattering-dominant particles, mitigating their efficacy in absorption-based techniques that leverage the photothermal effect, such as photoacoustic (PA) imaging. We introduce a highly reproducible synthesis route to monodisperse sub-100 nm Au nanoshells with an absorption-dominant optical response. Au nanoshells with 48 nm SiO2 cores and 7 nm Au shells show a 14-fold increase in their volumetric absorption coefficient compared to commercial Au nanoshells with dimensions commonly used in nanomedicine. PA imaging with Au nanoshell contrast agents showed a 50% improvement in imaging depth for sub-100 nm Au nanoshells compared with the smallest commercially available nanoshells in a turbid phantom. Furthermore, the high PA signal at low fluences, enabled by sub-100 nm nanoshells, will aid the deployment of low-cost, low-fluence light-emitting diodes for PA imaging.

Ablation of Venous Malformations by Photothermal Therapy with Intravenous Gold Nanoshells.

Venous malformations (VMs) consist of hugely enlarged and dysmorphic veins. These lesions cause significant disfigurement, pain, and complications such as bleeding and coagulopathy. Pharmacotherapy for the treatment of VMs has limited efficacy and potentially limiting toxicity. Current treatment for patients with VMs entails life-long pharmacotherapy or surgical procedures. Here we explored whether intravenously administered agents can be used to destroy VMs by photothermal therapy (PTT), using gold nanoshells (AuNSs) that generated heat following irradiation with near-infrared (NIR) light. In a murine model of VMs, intravenous AuNSs accumulated within the VMs. Irradiation of the VMs induced marked regression and even elimination. Nanoparticle-based photothermal therapy can provide effective therapy for VMs, which are otherwise relatively refractory to treatment.

Synthetic Immunogenic Cell Death Mediated by Intracellular Delivery of STING Agonist Nanoshells Enhances Anticancer Chemo-immunotherapy.

Many favorable anticancer treatments owe their success to the induction immunogenic cell death (ICD) in cancer cells, which results in the release of endogenous danger signals along with tumor antigens for effective priming of anticancer immunity. We describe a strategy to artificially induce ICD by delivering the agonist of stimulator of interferon genes (STING) into tumor cells using hollow polymeric nanoshells. Following intracellular delivery of exogenous adjuvant, subsequent cytotoxic treatment creates immunogenic cellular debris that spatiotemporally coordinate tumor antigens and STING agonist in a process herein termed synthetic immunogenic cell death (sICD). sICD is indiscriminate to the type of chemotherapeutics and enables colocalization of exogenously administered immunologic adjuvants and tumor antigens for enhanced antigen presentation and anticancer adaptive response. In three mouse tumor models, sICD enhances therapeutic efficacy and restrains tumor progression. The study highlights the benefit of delivering STING agonists to cancer cells, paving ways to new chemo-immunotherapeutic designs.

Inorganic Nanoshell-Stabilized Liquid Metal for Targeted Photonanomedicine in NIR-II Biowindow.

Gallium and gallium-based alloys, typical types of liquid metals with unique physiochemical properties, are emerging as a next generation of functional materials in versatile biomedical applications. However, the exploration of their biomedical performance is currently insufficient, and their intrinsic low oxidative resistance is a key factor blocking their further clinical translation. Herein, we report on the surface engineering of liquid metal-based nanoplatforms by an inorganic silica nanoshell based on a novel but facile sonochemical synthesis for highly efficient, targeted, and near-infrared (NIR)-triggered photothermal tumor hyperthermia in the NIR-II biowindow. The inorganic silica-shell engineering of liquid metal significantly enhances the photothermal performance of the liquid metal core as reflected by enhanced NIR absorption, improved photothermal stability by oxidation protection, and abundant surface chemistry for surface-targeted engineering to achieve enhanced tumor accumulation. Systematic in vitro cell-level evaluation and in vivo tumor xenograft assessment demonstrate that (Arg-Gly-Asp) RGD-targeted and silica-coated nanoscale liquid metal substantially induces phototriggered cancer-cell death and photothermal tumor eradication, accompanied by high in vivo biocompatibility and easy excretion out of the body. This work provides the first paradigm for surface-inorganic engineering of liquid metal-based nanoplatforms for achieving multiple desirable therapeutic performances, especially for combating cancer.

Smart Plasmonic Nanozyme Enhances Combined Chemo-photothermal Cancer Therapy and Reveals Tryptophan Metabolic Apoptotic Pathway.

Regulation of the tumor microenvironment is considered to be an intelligent strategy for cancer therapeutics, but the related metabolic pathways of cell apoptosis still remain a great challenge. Herein, by applying multifunctional carbon dot-decorated Ag/Au bimetallic nanoshells (CDs-Ag/Au NSs, CAANSs) nanoprobes as smart plasmonic nanozymes for combined chemo-photothermal cancer therapy, we achieved a high efficiency in cancer cell therapy and revealed a tryptophan (Trp) metabolic apoptotic pathway. In addition to high photothermal conversion efficiency, the CAANSs can act as a smart nanozyme to catalyze intracellular H2O2 to the cytotoxic reactive oxygen species (ROS) of superoxide anion (·O2-), in response to mild acidic cancerous cell microenvironment to damage cellular DNA. More importantly, the Trp metabolic pathway during the combined chemo-photothermal therapy has revealed that the Trp participates in an oxidative stress process, which can be decomposed to produce H2O2 and further formed into superoxide anions to kill cells under the catalytic nanomedicine process. The current work provides an effective platform for cancer therapeutics and is promising for cancer-related molecular biology studies.

Detection and Inhibition of Bacteria on a Dual-Functional Silver Platform.

Detection and inhibition of bacteria are universally required in clinics and daily life for health care. Developing a dual-functional material is challenging and in demand, engaging advanced applications for both defined bioanalysis and targeted biotoxicity. Herein, magnetic silver nanoshells are designed as a multifunctional platform for the detection and inhibition of bacteria. The optimized magnetic silver nanoshells enable direct laser desorption/ionization mass spectrometry based metabolic analysis of bacteria (≈10 µL-1 ), in complex biofluids. The serum infection process (0-10 h) is monitored by statistics toward clinical classification. Moreover, magnetic silver nanoshells facilitate surface adhesion on bacteria due to nanoscale surface roughness and thus display long-term antibacterial effects. Bacteria metabolism is studied with metabolic biomarkers (e.g., malate and lysine) identified during inhibition, showing cell membrane destruction and dysfunctional protein synthesis mechanisms. This work not only guides the design of material-based approaches for bioanalysis and biotoxicity, but contributes to bacteria-related diagnosis by using specific metabolic biomarkers for sensitive detection and new insights by monitoring metabolomic change of bacteria for antibacterial applications.

Comprehensive Evaluation of Degradable and Cost-Effective Plasmonic Nanoshells for Localized Photothermolysis of Cancer Cells.

Integrating the concept of biodegradation and light-triggered localized therapy in a functional nanoformulation is the current approach in onco-nanomedicine. Morphology control with an enhanced photothermal response, minimal toxicity, and X-ray attenuation of polymer-based nanoparticles is a critical concern for image-guided photothermal therapy. Herein, we describe the simple design of cost-effective and degradable polycaprolactone-based plasmonic nanoshells for the integrated photothermolysis as well as localized imaging of cancer cells. The gold-deposited polycaprolactone-based plasmonic nanoshells (AuPCL NS) are synthesized in a scalable and facile way under ambient conditions. The synthesized nanoshells are monodisperse, fairly stable, and highly inert even at five times (250 µg/mL) the therapeutic concentration in a week-long test. AuPCL NS are capable of delivering standalone photothermal therapy for the complete ablation of cancer cells without using any anticancerous drugs and causing toxicity. It delivers the same therapeutic efficacy to different cancer cell lines, irrespective of their chemorefractory status and also works as a potential computed tomography contrast agent for the integrated imaging-directed photothermal cancer therapy. High biocompatibility, degradability, and promising photothermal efficacy of AuPCL NS are attractive aspects of this report that could open new horizons of localized plasmonic photothermal therapy for healthcare applications.

Evaluation of theranostic perspective of gold-silica nanoshell for cancer nano-medicine: a numerical parametric study.

Using gold-silica nanoshell as a reference nano-agent, this work has performed preliminary numerical parametric study to investigate the feasibility and if feasible the efficiency of using a single nano-agent to achieve theranostic goals. In total, seven generics of gold-silica nanoshells have been tested including the R[50,10] (radius of the silica core is 50 nm and thickness of the gold shell is 10 nm), R[40,15], R[55,25], R[40,40], R[75,40], R[104,23], and R[154,24] nanoshells. A planar tissue model has been constructed as the platform for parametric study. For mathematical modeling, radiant transport equation (RTE) has been applied to describe the interactions among laser lights, the hosting tissue, and the hosted nanoshells and Penne's bio-heat equation has been applied to describe the hyperthermia induced by such interactions. Effects of different nanoshell generics on the diffuse reflectance signal and hyperthermia temperature transition have been simulated, basing on which the potential of a certain nanoshell generic as theranostic nano-agent has been evaluated. It has been found that it is highly feasible for gold-silica nanoshells to be engineered for theranostic purpose and nanoshell generics that are preferentially scattering should be explored for good theranostic candidates. On the condition that nanoshell generic with the right optical properties has been located, a moderate nanoshell retention in the target tissue site is already sufficient to induce effective theranostic effects, which indicates that theranostic nano-medicine might not have a stringent requirement for the delivery technique. Among nanoshells that have been tested, the R[55,25] nanoshell seems to be a promising candidate as theranostic nano-agent. Further testing on it is highly recommended. Nanoshells that are preferentially absorbing such as the R[50,10] and R[40,15] nanoshells are efficient photothermal agent and could be used for therapeutic purpose only. However, it is not recommended that preferentially absorbing nanoshells being used for theranostic purpose due to possible negative effects such nanoshells might bring to the diffuse reflectance signal.

Multifunctional Porous Iron Oxide Nanoagents for MRI and Photothermal/Chemo Synergistic Therapy.

Nanoagents of integrating multiple imaging and therapeutic modalities have attracted tremendous attention for biomedical applications. Herein, we synthesize porous hollow Fe3O4 as a theranostic agent for MRI and combined photothermal/chemo cancer therapy. The as-prepared porous iron oxide nanoagents allow for T2-weighted MR imaging. Interestingly, we demonstrate that the porous structure endows the nanoagents an outstanding photothermal property for cancer cell killing, in comparison with other types of iron oxide nanomaterials. Under the exposure of an NIR laser, the heat produced by porous Fe3O4 can accelerate the release of the loaded drug (e.g., DOX) to enhance chemotherapeutic efficacy, promoting the ablation of cancer cells with synergistic photothermal/chemotherapy.

Gold nanoshell-based betulinic acid liposomes for synergistic chemo-photothermal therapy.

A novel synthesis approach is first developed to fabricate a multifunctional smart nanodrug delivery system: gold nanoshell-coated betulinic acid liposomes (AuNS-BA-Lips) mediated by a glutathione. The AuNS-BA-Lips exhibited good size distribution (149.4±2.4nm), preferable photothermal conversion ability and synergistic chemo-photothermal therapy. Additionally, the absorption wavelength of AuNS-BA-Lips showed a significantly red-shifted to near infrared (NIR) region, which can strongly absorbed NIR laser and efficiently convert it into localized heat, thus providing controlled drug release and antitumor thermotherapy. Moreover, the nanocarriers excited by NIR light significantly promoted cell uptake compared to those without irradiation, resulting in an enhanced intracellular drug accumulation. Upon NIR irradiation, the AuNS-BA-Lips showed highly efficient antitumor effects on tumor-bearing mice with an inhibition rate of 83.02%, thus demonstrating a remarkable synergistic therapeutic effect of chemotherapy and thermotherapy. Therefore, this work provides new insight into developing a multifunctional antitumor drug.

Aspect ratios of gold nanoshell capsules mediated melanoma ablation by synergistic photothermal therapy and chemotherapy.

Nanomaterial-mediated photothermal therapy has shown great potential to fulfill the unmet medical needs for treatment of tumors. In this study, a rod-like gold nanoshell capsule, which can offer both photothermal therapy and chemotherapy, is synthesized and applied for the treatment of melanoma. This nano-platform is made by developing a gold nanoshell on rod-like mesoporous silica nanoparticles with different aspect ratios, and it was found that the aspect ratio significantly influenced the cellular uptake and tumor distribution of the nanoparticles. The gold nanoshell capsules with a moderate aspect ratio are found to be efficiently taken up by melanoma cells and are able to penetrate tumor tissues, resulting in the effective ablation of highly malignant melanomas when used along with mild laser irradiation and a single treatment. This study demonstrates that the optimization of the aspect ratio is indispensable to further development of this nanoplatform for antitumor therapy. FROM THE CLINICAL EDITOR: The combination of hyperthermia and chemotherapeutic agents has been investigated as a new approach for the treatment of malignant melanoma. It appears that the aspect ratio may play an important role in the treatment efficacy. In this article, the authors studied how the AR influenced the cellular uptake and the optimal AR for antitumor effects.

Special issue of ChinaNanomedicine 2015.

The special issue of ChinaNanomedicine 2015 is the invited contributions from the attendees of the 1st International Conference on Nanomedicine (ChinaNanomedicine 2015, Hangzhou, China), the inaugural conference of the Chinese Society for Nanomedicine. The content includes using natural virus capsids and capsid-mimicking nanostructures for efficient drug delivery, gold nanoshell capsules for photothermal therapy and chemotherapy, codelivery of drugs and genes for cancer therapy as well as nanomedicine applications in antibacterial and anti-Alzheimer treatments, and rapid detection of early osteogenic differentiation.

Numerical investigation of thermal response of laser-irradiated biological tissue phantoms embedded with gold nanoshells.

The work presented in this paper focuses on numerically investigating the thermal response of gold nanoshells-embedded biological tissue phantoms with potential applications into photo-thermal therapy wherein the interest is in destroying the cancerous cells with minimum damage to the surrounding healthy cells. The tissue phantom has been irradiated with a pico-second laser. Radiative transfer equation (RTE) has been employed to model the light-tissue interaction using discrete ordinate method (DOM). For determining the temperature distribution inside the tissue phantom, the RTE has been solved in combination with a generalized non-Fourier heat conduction model namely the dual phase lag bio-heat transfer model. The numerical code comprising the coupled RTE-bio-heat transfer equation, developed as a part of the current work, has been benchmarked against the experimental as well as the numerical results available in the literature. It has been demonstrated that the temperature of the optical inhomogeneity inside the biological tissue phantom embedded with gold nanoshells is relatively higher than that of the baseline case (no nanoshells) for the same laser power and operation time. The study clearly underlines the impact of nanoshell concentration and its size on the thermal response of the biological tissue sample. The comparative study concerned with the size and concentration of nanoshells showed that 60nm nanoshells with concentration of 5×1015mm-3 result into the temperature levels that are optimum for the irreversible destruction of cancer infected cells in the context of photo-thermal therapy. To the best of the knowledge of the authors, the present study is one of the first attempts to quantify the influence of gold nanoshells on the temperature distributions inside the biological tissue phantoms upon laser irradiation using the dual phase lag heat conduction model.

Investigation of thermal distribution for pulsed laser radiation in cancer treatment with nanoparticle-mediated hyperthermia.

In this paper, we have simulated the efficacy of gold/gold sulfide (GGS) nanoshells in NIR laser hyperthermia to achieve effective targeting for tumor photothermal therapy. The problem statement takes into account the heat transfer with the blood perfusion through capillaries, and pulsed laser irradiation during the hyperthermia. Although previous researchers have used short laser pulses (nanosecond and less), in order to prevent heat leakage to the neighbor tissues, we have examined the effect of millisecond pulses, as the extent of the target volume to which hyperthermia is induced is usually larger and also the lasers with this specification are more available. A tumor with surrounding tissue was simulated in COMSOL software (a finite element analysis, solver and simulation software) and also in a phantom made of agarose and intralipid. The tumor was irradiated by 10, 20 and 30 laser pulses with durations of 15, 50 and 200ms and fluences of 20, 40 and 60J/cm(2). Experimental tests performed on a phantom prove the ability of the applied numerical model to capture the temperature distribution in the target tissue. We have shown that our simulation permits prediction of treatment outcome from computation of thermal distribution within the tumor during laser hyperthermia using GGS nanoshells and millisecond pulsed laser irradiation. The advantage of this simulation is its simplicity as well as its accuracy. Although, to develop the model completely for a given organ and application, all the parameters should be estimated based on a real vasculature of the organ, physiological conditions, and expected variation in those physiological conditions for that application in the organ.

Hollow iron-silica nanoshells for enhanced high intensity focused ultrasound.

BACKGROUND: High intensity-focused ultrasound (HIFU) is an alterative ablative technique currently being investigated for local treatment of breast cancer and fibroadenomas. Current HIFU therapies require concurrent magnetic resonance imaging monitoring. Biodegradable 500 nm perfluoropentane-filled iron-silica nanoshells have been synthesized as a sensitizing agent for HIFU therapies, which aid both mechanical and thermal ablation of tissues. In low duty cycle high-intensity applications, rapid tissue damage occurs from mechanical rather than thermal effects, which can be monitored closely by ultrasound obviating the need for concurrent magnetic resonance imaging. MATERIALS AND METHODS: Iron-silica nanoshells were synthesized by a sol-gel method on polystyrene templates and calcined to yield hollow nanoshells. The nanoshells were filled with perfluoropentane and injected directly into excised human breast tumor, and intravenously (IV) into healthy rabbits and Py8119 tumor-bearing athymic nude mice. HIFU was applied at 1.1 MHz and 3.5 MPa at a 2% duty cycle to achieve mechanical ablation. RESULTS: Ex vivo in excised rabbit livers, the time to visually observable damage with HIFU was 20 s without nanoshells and only 2 s with nanoshells administered IV before sacrifice. Nanoshells administered IV into nude mice with xenograft tumors were activated in vivo by HIFU 24 h after administration. In this xenograft model, applied HIFU resulted in a 13.6 ± 6.1 mm(3) bubble cloud with the IV injected particles and no bubble cloud without particles. CONCLUSIONS: Iron-silica nanoshells can reduce the power and time to perform HIFU ablative therapy and can be monitored by ultrasound during low duty cycle operation.

Combined concurrent photodynamic and gold nanoshell loaded macrophage-mediated photothermal therapies: an in vitro study on squamous cell head and neck carcinoma.

BACKGROUND AND OBJECTIVE: Treatment modalities, such as hyperthermia and photodynamic therapy (PDT) have been used in the treatment of a variety of head and neck squamous cell carcinoma (HNSCC), either alone or as an adjuvant therapy. Macrophages loaded with gold nanoshells, which convert near-infrared light to heat, can be used as transport vectors for photothermal hyperthermia of tumors. The purpose of this study was to investigate the effects of combined macrophage mediated photothermal therapy (PTT) and PDT on HNSCC cells. STUDY DESIGN/MATERIALS AND METHODS: Gold nanoshell loaded rat macrophages either alone or combined with human FaDu squamous cells in hybrid monolayers were subjected to PTT, PDT, or a simultaneous combination of the two light treatments. Therapies were given concurrently employing two laser light sources of λ = 670 nm (PDT) and λ = 810 nm (PTT), respectively. RESULTS: Significant uptake of gold nanospheres (AuNS) by rat alveolar macrophages was observed thus providing the rationale for their use as delivery vectors. Viability of the AuNS-loaded Ma was reduced to 35 and 12% of control values at an irradiance of 14 or 28 W/cm(2) administered over a 5 minute period respectively. No significant cytotoxicity was observed for empty Ma for similar PTT exposure. AlPcS2a mediated PDT at a fluence level of 0.25 J/cm(2) and PTT at 14 W/cm(2) irradiance had little effect on cell viability for the FaDu/Ma (ratio 2:1) hybrid monolayers. In contrast, combined treatment reduced the cell viability to less than 40% at these same laser power settings. CONCLUSIONS: The results of this study provide proof of concept for the use of macrophages as a delivery vector of AuNS for photothermal enhancement of the effects of PDT on squamous cell carcinoma. A significant synergy was demonstrated with combined PDT and PTT compared to each modality applied separately.

Feasibility of selective nanoparticle-assisted photothermal treatment for an embedded liver tumor.

Using the finite volume method, the present work numerically explored the feasibility of extending nanoparticle-assisted photothermal therapy (PPTT) from treating subcutaneous tumors to treating organ tumors, particularly tumors growing in the clearance organ liver. To serve this purpose, a superficially embedded liver tumor and its immediate surrounding medium were selected as the study object. A 633-nm laser beam of 1- W/cm(2) intensity externally irradiated the tumor. The matching gold-silica nanoshell with a 16-nm silica core and a 5-nm-thick gold shell was used as the photothermal agent. The nanoshell retention ratio was varied to simulate different levels of nanoshell tumor discriminations. Laser light distributions, conversions from photon energy to heat, and tissues' thermal responses to the generated heat within the study object were analyzed. It was found that although nanoshells have enhanced the thermal transportation, they also restricted the optical transportation of PPTT. This indicates that laser delivery is more demanding for PPTT than for the conventional laser therapy. For the investigated case, when the nanoshell retention ratio was in the range of 2/1-4/1, the therapeutic effects were optimal: a confined medium temperature hyperthermia (47-55 °C) was achieved in the liver tumor while impacts on the surrounding health liver tissues were only marginal. When then nanoshell retention ratio was 8/1 or higher, about half of the liver tumor was ablated. However, some of the surrounding healthy liver tissues were sacrificed as well. The therapeutic effects of PPTT depend nonlinearly on the nanoshell tumor discriminations. Better tumor discriminations do not necessarily result in better PPTT therapeutic effects.

Theranostic nanoshells: from probe design to imaging and treatment of cancer.

Recent advances in nanoscience and biomedicine have expanded our ability to design and construct multifunctional nanoparticles that combine targeting, therapeutic, and diagnostic functions within a single nanoscale complex. The theranostic capabilities of gold nanoshells, spherical nanoparticles with silica cores and gold shells, have attracted tremendous attention over the past decade as nanoshells have emerged as a promising tool for cancer therapy and bioimaging enhancement. This Account examines the design and synthesis of nanoshell-based theranostic agents, their plasmon-derived optical properties, and their corresponding applications. We discuss the design and preparation of nanoshell complexes and their ability to enhance the photoluminescence of fluorophores while maintaining their properties as MR contrast agents. In this Account, we discuss the underlying physical principles that contribute to the photothermal response of nanoshells. We then elucidate the photophysical processes that induce nanoshells to enhance the fluorescence of weak near-infrared fluorophores. Nanoshells illuminated with resonant light are either strong optical absorbers or scatterers, properties that give rise to their unique capabilities. These physical processes have been harnessed to visualize and eliminate cancer cells. We describe the application of nanoshells as a contrast agent for optical coherence tomography of breast carcinoma cells in vivo. Our recent studies examine nanoshells as a multimodal theranostic probe, using these nanoparticles for near-infrared fluorescence and magnetic resonance imaging (MRI) and for the photothermal ablation of cancer cells. Multimodal nanoshells show theranostic potential for imaging subcutaneous breast cancer tumors in animal models and the distribution of tumors in various tissues. Nanoshells also show promise as light-triggered gene therapy vectors, adding temporal control to the spatial control characteristic of nanoparticle-based gene therapy approaches. We describe the fabrication of DNA-conjugated nanoshell complexes and compare the efficiency of light-induced and thermally-induced release of DNA. Double-stranded DNA nanoshells also provide a way to deliver small molecules into cells: we describe the delivery and light-triggered release of DAPI (4',6-diamidino-2-phenylindole), a dye molecule used to stain DNA in the nuclei of cells.

Evaluation of the toxicity of intravenous delivery of auroshell particles (gold-silica nanoshells).

Gold nanoshells (155 nm in diameter with a coating of polyethylene glycol 5000) were evaluated for preclinical biocompatibility, toxicity, and biodistribution as part of a program to develop an injectable device for use in the photothermal ablation of tumors. The evaluation started with a complete good laboratory practice (GLP) compliant International Organization for Standardization (ISO)-10993 biocompatibility program, including cytotoxicity, pyrogenicity (US Pharmacopeia [USP] method in the rabbit), genotoxicity (bacterial mutagenicity, chromosomal aberration assay in Chinese hamster ovary cells, and in vivo mouse micronucleus), in vitro hemolysis, intracutaneous reactivity in the rabbit, sensitization (in the guinea pig maximization assay), and USP/ISO acute systemic toxicity in the mouse. There was no indication of toxicity in any of the studies. Subsequently, nanoshells were evaluated in vivo by intravenous (iv) infusion using a trehalose/water solution in a series of studies in mice, Sprague-Dawley rats, and Beagle dogs to assess toxicity for time durations of up to 404 days. Over the course of 14 GLP studies, the gold nanoshells were well tolerated and, when injected iv, no toxicities or bioincompatibilities were identified.