Gold nanoshell-localized photothermal ablation of prostate tumors in a clinical pilot device study.

Biocompatible gold nanoparticles designed to absorb light at wavelengths of high tissue transparency have been of particular interest for biomedical applications. The ability of such nanoparticles to convert absorbed near-infrared light to heat and induce highly localized hyperthermia has been shown to be highly effective for photothermal cancer therapy, resulting in cell death and tumor remission in a multitude of preclinical animal models. Here we report the initial results of a clinical trial in which laser-excited gold-silica nanoshells (GSNs) were used in combination with magnetic resonance-ultrasound fusion imaging to focally ablate low-intermediate-grade tumors within the prostate. The overall goal is to provide highly localized regional control of prostate cancer that also results in greatly reduced patient morbidity and improved functional outcomes. This pilot device study reports feasibility and safety data from 16 cases of patients diagnosed with low- or intermediate-risk localized prostate cancer. After GSN infusion and high-precision laser ablation, patients underwent multiparametric MRI of the prostate at 48 to 72 h, followed by postprocedure mpMRI/ultrasound targeted fusion biopsies at 3 and 12 mo, as well as a standard 12-core systematic biopsy at 12 mo. GSN-mediated focal laser ablation was successfully achieved in 94% (15/16) of patients, with no significant difference in International Prostate Symptom Score or Sexual Health Inventory for Men observed after treatment. This treatment protocol appears to be feasible and safe in men with low- or intermediate-risk localized prostate cancer without serious complications or deleterious changes in genitourinary function.

Initial Evaluation of the Safety of Nanoshell-Directed Photothermal Therapy in the Treatment of Prostate Disease.

To evaluate the clinical safety profile for the use of gold nanoshells in patients with human prostate cancer. This follows on the nonclinical safety assessment of the AuroShell particles reported previously. Twenty-two patients, with biopsy diagnosed prostate cancer, underwent nanoshell infusion and subsequent radical prostatectomy (RRP). Fifteen of these patients had prostates that were additionally irradiated by a single-fiber laser ablation in each prostate hemisphere prior to RRP. Patients in the study were assessed at 9 time points through 6 months postinfusion. Adverse events were recorded as reported by the patients and from clinical observation. Blood and urine samples were collected at each patient visit and subjected to chemical (16 tests), hematological (23 tests), immunological (3 tests, including total PSA), and urinalysis (8 tests) evaluation. Temperature of the anterior rectal wall at the level of the prostate was measured. The study, recorded 2 adverse events that were judged attributable to the nanoparticle infusion: (1) an allergic reaction resulting in itching, which resolved with intravenous antihistamines, and (2) in a separate patient, a transient burning sensation in the epigastrium. blood/hematology/urinalysis assays indicated no device-related changes. No change in temperature of the anterior rectal wall was recorded in any of the patients. The clinical safety profile of AuroShell particles is excellent, matching nonclinical findings. A recent consensus statement suggested that the published literature does not support a preference for any ablation technique over another.(1) Now that clinical safety has been confirmed, treatment efficacy of the combined infusion plus laser ablation in prostate will be evaluated in future studies using imaging modalities directing the laser against identified prostate tumors.

Suppression of the reticuloendothelial system using λ-carrageenan to prolong the circulation of gold nanoparticles.

AIM: Gold nanoparticles are employed for imaging and treatment of surgically inaccessible tumors owing to their inherent optical absorption and ability to extravasate through intravenous distribution. These nanoparticles are cleared from the blood by the reticuloendothelial system (RES) as expected given their size. MATERIALS & METHODS: This study demonstrates the effects of RES blockade through the intravenous administration of λ-carrageenan, resulting in a decrease in the median clearance rate from 18.9 (95% CrI: 15.9-22.6) to 11.2 (95% CrI: 8.8-13.9) µl/min and an increase in nanoparticle circulation half-life t(½)( = 264 ± 73 vs 160 ± 22 min; p < 0.01). RESULTS: This 59.3% decrease in clearance is greater than the 15% previously reported for liposomes [ 1 ]. CONCLUSION: The primary benefit of nontoxic RES blockade is to increase the circulation time, where traditional particle modification is ineffective or impractical.

MR temperature imaging of nanoshell mediated laser ablation.

Minimally invasive thermal therapy using high-power diode lasers is an active area of clinical research. Gold nanoshells (AuNS) can be tuned to absorb light in the range used for laser ablation and may facilitate more conformal tumor heating and sparing of normal tissue via enhanced tumor specific heating. This concept was investigated in a xenograft model of prostate cancer (PC-3) using MR temperature imaging (MRTI) in a 1.5T scanner to characterize the spatiotemporal temperature distribution resulting from nanoparticle mediated heating. Tumors with and without intravenously injected AuNS were exposed to an external laser tuned to 808 nm for 180 sec at 4 W/cm(2) under real-time monitoring with proton resonance frequency shift based MRTI. Microscopy indicated that these nanoparticles (140-150 nm) accumulated passively in the tumor and remained close to the tumor microvasculature. MRTI measured a statistically significant (p < 0.001) increase in maximum temperature in the tumor cortex (mean = 21 ± 7°C) in +AuNS tumors versus control tumors. Analysis of the temperature maps helped demonstrate that the overall distribution of temperature within +AuNS tumors was demonstrably higher versus control, and resulted in damage visible on histopathology. This research demonstrates that passive uptake of intravenously injected AuNS in PC-3 xenografts converts the tumor vasculature into a potent heating source for nanoparticle mediated ablation at power levels which do not generate significant damage in normal tissue. When used in conjunction with MRTI, this has implications for development and validation of more conformal delivery of therapy for interstitial laser ablations.

Selective nanoparticle-directed ablation of the canine prostate.

BACKGROUND AND OBJECTIVES: Prostate cancer is the most frequent cancer type and the second most common cause of cancer death among US men. This study, adapted a previously reported nanoparticle-directed photothermal treatment of brain tumors to the treatment of prostate disease by using normal canine prostate in vivo, directly injected with a suspension of nanoparticles as a proxy for prostate tumor, and by developing laser dosimetry for prostate which is marginally ablative in native tissue, yet producing photothermal coagulation in prostate tissue containing nanoparticles. METHODS: Canine prostates were exposed by surgical laparotomy and directly injected with suspensions of nanoparticles (nanoshells) and irradiated by a NIR laser source delivered percutaneously by an optical fiber catheter and isotropic diffuser. The photothermal lesions were permitted to resolve for up to 8 days, at which time each animal was euthanized, necropsied, and the prostate taken for histopathological and elemental analysis. RESULTS: Nanoparticles were retained for up to 4 hours in prostate and served as a proxy for prostate tumor. A marginally ablative laser dose of 3.0 W for 3 minutes was developed which would yield 4 mm-radius coagulo-necrotic lesions if nanoparticles were present. CONCLUSION: We have shown that the addition of nanoshells to native tissue, combined with a marginally ablative laser dose can generate ablative thermal lesions, and that the radial extent of the thermal lesions is strictly confined to within ∼4 mm of the optical fiber with sub-millimeter uncertainty. This, in turn, suggests a means of precise tumor ablation with an ability to obviate damage to critical structures limited primarily by the precision with which the optical fiber applicator can be placed. In so doing, it should be possible to realize a precise, nerve bundle and urethra sparing prostate cancer treatment using a minimally invasive, percutaneous approach.

Three-wavelength murine photoplethysmography for estimation of vascular gold nanorod concentration.

Nanoparticle-assisted photo-thermal (NAPT) ablation has become a new and attractive modality for the treatment of cancerous tumors. This therapy exploits the passive accumulation of intravenously delivered optically resonant metal nanoparticles into tumors, however, the circulating bioavailability of these particles is often unknown. We present a non-invasive optical device capable of monitoring the circulation of optically resonant gold nanorods. The device, referred to as a pulse photometer, uses the technique of multi-wavelength photoplethysmography. We simultaneously report the circulation of gold nanorods and oximetry for six hours post-injection in mice with no anesthesia and remove the probe when not collecting data. The instrument shows good agreement (R(2) = 0.903, n = 30) with ex vivo spectrophotometric analysis of blood samples. The real-time feedback provided has a strong potential for reducing variability and thus improving the efficacy of similar clinical therapies.

Murine photoplethysmography for in vivo estimation of vascular gold nanoshell concentration.

There is an urgent clinical need to monitor the intravenous delivery and bioavailability of circulating nanoparticles used in cancer therapy. This work presents the use of photoplethysmography for the noninvasive real-time estimation of vascular gold nanoshell concentration in a murine subject. We develop a pulse photometer capable of accurately measuring the photoplethysmogram in mice and determining the ratio of pulsatile changes in optical extinction between 805 and 940 nm, commonly referred to as R. These wavelengths are selected to correspond to the extinction properties of gold nanoshells. Six 30-s measurements (5 min, 2, 4, 6, 8, 10 h) are taken under light anesthesia to observe the change in R as the nanoparticles clear from the circulation. Our model describes the linear fit (R(2)=0.85) between R and the concentration of nanoparticles measured via ex vivo spectrophotometric and instrumental neutron activation analysis. This demonstrates the utility of this technique in support of clinical nanoparticle therapies.

Intra-organ Biodistribution of Gold Nanoparticles Using Intrinsic Two-photon Induced Photoluminescence.

BACKGROUND AND OBJECTIVES: Gold nanoparticles (GNPs) such as gold nanoshells (GNSs) and gold nanorods (GNRs) have been explored in a number of in vitro and in vivo studies as imaging contrast and cancer therapy agents due to their highly desirable spectral and molecular properties. While the organ-level biodistribution of these particles has been reported previously, little is known about the cellular level or intra-organ biodistribution. The objective of this study was to demonstrate the use of intrinsic two-photon induced photoluminescence (TPIP) to study the cellular level biodistribution of GNPs. STUDY DESIGN/MATERIALS AND METHODS: Tumor xenografts were created in twenty-seven male nude mice (Swiss nu/nu) using HCT 116 cells (CCL-247, ATCC, human colorectal cancer cell line). GNSs and GNRs were systemically injected 24 hr. prior to tumor harvesting. A skin flap with the tumor was excised and sectioned as 8 µm thick tissues for imaging GNPs under a custom-built multiphoton microscope. For multiplexed imaging, nuclei, cytoplasm, and blood vessels were demonstrated by hematoxylin and eosin (H&E) staining, YOYO-1 iodide staining and CD31-immunofluorescence staining. RESULTS: Distribution features of GNPs at the tumor site were determined from TPIP images. GNSs and GNRs had a heterogeneous distribution with higher accumulation at the tumor cortex than tumor core. GNPs were also observed in unique patterns surrounding the perivascular region. While most GNSs were confined at the distance of approximately 400 µm inside the tumor edge, GNRs were shown up to 1.5 mm penetration inside the edge. CONCLUSIONS: We have demonstrated the use of TPIP imaging in a multiplexed fashion to image both GNPs and nuclei, cytoplasm, or vasculature simultaneously. We also confirmed that TPIP imaging enabled visualization of GNP distribution patterns within the tumor and other critical organs. These results suggest that direct luminescence-based imaging of metal nanoparticles holds a valuable and promising position in understanding the accumulation kinetics of GNPs. In addition, these techniques will be increasingly important as the use of these particles progress to human clinical trials where standard histopathology techniques are used to analyze their effects.

Near-infrared narrow-band imaging of gold/silica nanoshells in tumors.

Gold nanoshells (GNS) are a new class of nanoparticles that can be optically tuned to scatter or absorb light from the near-ultraviolet to near-infrared (NIR) region by varying the core (dielectric silica)/shell (gold) ratio. In addition to spectral tunability, GNS are inert and bioconjugatable, making them potential labels for in vivo imaging and therapy of tumors. We report the use of GNS as exogenous contrast agents for enhanced visualization of tumors using narrow-band imaging (NBI). NBI takes advantage of the strong NIR absorption of GNS to distinguish between blood and nanoshells in the tumor by imaging in narrow wavelength bands in the visible and NIR, respectively. Using tissue-simulating phantoms, we determined the optimum wavelengths to enhance contrast between blood and GNS. We then used the optimum wavelengths for ex vivo imaging of tumors extracted from human colon cancer xenograft bearing mice injected with GNS. Systemically delivered GNS accumulated passively in tumor xenografts by the enhanced permeability and retention (EPR) effect. Ex vivo NBI of tumor xenografts demonstrated heterogeneous distribution of GNS with a clear distinction from the tumor vasculature. The results of this study demonstrate the feasibility of using GNS as contrast agents to visualize tumors using NBI.

In-vivo photoacoustic microscopy of nanoshell extravasation from solid tumor vasculature.

In this study, high resolution backward-mode photoacoustic microscopy (PAM) is used to noninvasively image progressive extravasation and accumulation of nanoshells within a solid tumor in vivo. PAM takes advantage of the strong near-infrared absorption of nanoshells and their extravasation tendency from leaky tumor vasculatures for imaging. Subcutaneous tumors are grown on immunocompetent BALB/c mice. Polyethylene glycol (PEGylated) nanoshells with a peak optical absorption at approximately 800 nm are intravenously administered. With an 800-nm laser source, a prescan prior to nanoshell injection is taken to determine the background that is free of nanoshell accumulation. After injection, the 3-D nanoshell distribution at the tumor foci is monitored by PAM for 6 h. Experimental results show that accumulated nanoshells delineate the tumor position. Nanoshell accumulation is heterogeneous in tumors: more concentrated within the tumor cortex and largely absent from the tumor core. Because nanoshells have been recently demonstrated to enhance thermal therapy of subcutaneous tumors, we anticipate that PAM will be an important aid before, during, and after nanoshell thermal therapy.

Feasibility study of particle-assisted laser ablation of brain tumors in orthotopic canine model.

We report on a pilot study showing a proof of concept for the passive delivery of nanoshells to an orthotopic tumor where they induce a local, confined therapeutic response distinct from that of normal brain resulting in the photothermal ablation of canine transmissible venereal tumor (cTVT) in a canine brain model. cTVT fragments grown in severe combined immunodeficient mice were successfully inoculated in the parietal lobe of immunosuppressed, mixed-breed hound dogs. A single dose of near-IR (NIR)-absorbing, 150-nm nanoshells was infused i.v. and allowed time to passively accumulate in the intracranial tumors, which served as a proxy for an orthotopic brain metastasis. The nanoshells accumulated within the intracranial cTVT, suggesting that its neovasculature represented an interruption of the normal blood-brain barrier. Tumors were thermally ablated by percutaneous, optical fiber-delivered, NIR radiation using a 3.5-W average, 3-minute laser dose at 808 nm that selectively elevated the temperature of tumor tissue to 65.8 +/- 4.1 degrees C. Identical laser doses applied to normal white and gray matter on the contralateral side of the brain yielded sublethal temperatures of 48.6 +/- 1.1 degrees C. The laser dose was designed to minimize thermal damage to normal brain tissue in the absence of nanoshells and compensate for variability in the accumulation of nanoshells in tumor. Postmortem histopathology of treated brain sections showed the effectiveness and selectivity of the nanoshell-assisted thermal ablation.

Two-photon-induced photoluminescence imaging of tumors using near-infrared excited gold nanoshells.

Gold nanoshells (dielectric silica core/gold shell) are a novel class of hybrid metal nanoparticles whose unique optical properties have spawned new applications including more sensitive molecular assays and cancer therapy. We report a new photo-physical property of nanoshells (NS) whereby these particles glow brightly when excited by near-infrared light. We characterized the luminescence brightness of NS, comparing to that of gold nanorods (NR) and fluorescent beads (FB). We find that NS are as bright as NR and 140 times brighter than FB. To demonstrate the potential application of this bright two-photon-induced photoluminescence (TPIP) signal for biological imaging, we imaged the 3D distribution of gold nanoshells targeted to murine tumors.

A High-Voltage Integrated Circuit Engine for a Dielectrophoresis-based Programmable Micro-Fluidic Processor.

A high-voltage (HV) integrated circuit has been demonstrated to transport droplets on programmable paths across its coated surface. This chip is the engine for a dielectrophoresis (DEP)-based micro-fluidic lab-on-a-chip system. This chip creates DEP forces that move and help inject droplets. Electrode excitation voltage and frequency are variable. With the electrodes driven with a 100V peak-to-peak periodic waveform, the maximum high-voltage electrode waveform frequency is about 200Hz. Data communication rate is variable up to 250kHz. This demonstration chip has a 32×32 array of nominally 100V electrode drivers. It is fabricated in a 130V SOI CMOS fabrication technology, dissipates a maximum of 1.87W, and is about 10.4 mm × 8.2 mm.

Dielectrophoresis-based programmable fluidic processors.

Droplet-based programmable processors promise to offer solutions to a wide range of applications in which chemical and biological analysis and/or small-scale synthesis are required, suggesting they will become the microfluidic equivalents of microprocessors by offering off-the-shelf solutions for almost any fluid based analysis or small scale synthesis problem. A general purpose droplet processor should be able to manipulate droplets of different compositions (including those that are electrically conductive or insulating and those of polar or non-polar nature), to control reagent titrations accurately, and to remain free of contamination and carry over on its reaction surfaces. In this article we discuss the application of dielectrophoresis to droplet based processors and demonstrate that it can provide the means for accurately titrating, moving and mixing polar or non-polar droplets whether they are electrically conductive or not. DEP does not require contact with control surfaces and several strategies for minimizing surface contact are presented. As an example of a DEP actuated general purpose droplet processor, we show an embodiment based on a scaleable CMOS architecture that uses DEP manipulation on a 32 x 32 electrode array having built-in control and switching circuitry. Lastly, we demonstrate the concept of a general-purpose programming environment that facilitates droplet software development for any type of droplet processor.

Droplet-based chemistry on a programmable micro-chip.

We describe the manipulation of aqueous droplets in an immiscible, low-permittivity suspending medium. Such droplets may serve as carriers for not only air- and water-borne samples, contaminants, chemical reagents, viral and gene products, and cells, but also the reagents to process and characterise these samples. We present proofs-of-concept for droplet manipulation through dielectrophoresis by: (1). moving droplets on a two-dimensional array of electrodes, (2). achieving dielectrically-activated droplet injection, (3). fusing and reacting droplets, and (4). conducting a basic biological assay through a combination of these steps. A long-term goal of this research is to provide a platform fluidic processor technology that can form the core of versatile, automated, micro-scale devices to perform chemical and biological assays at or near the point of care, which will increase the availability of modern medicine to people who do not have ready access to modern medical institutions, and decrease the cost and delays associated with that lack of access.

DIELECTROPHORESIS BASED MICRO FLOW CYTOMETRY.

We report a simplified flow cytometer design that makes use of negative dielectrophoresis (DEP) for particle focusing and integrated optical and AC impedance detectors to enable an inexpensive, compact and robust system for cell and particle characterization. This straightforward, modular design could be applied as a standalone instrument or as a particle detector in an integrated micro total analysis system.