Enhanced drug delivery via hyperthermal membrane disruption using targeted gold nanoparticles with PEGylated Protein-G as a cofactor.

Gold nanoparticles (GNPs) with near infrared (NIR) plasmon resonance have been promisingly used in photothermal cancer therapy as a less invasive treatment. Recombinant Protein-G (ProG) was PEGylated to act as a cofactor to immobilize immunoglobulins (IgGs) on GNPs by the Fc region, resulting in optimal orientation of IgGs for efficient cancer targeting. In-vitro studies showed that HER-2 overexpressing breast cancer cells, SK-BR-3, were efficiently targeted and ablated at a laser power of 900 J/cm(2) (5 W/cm(2) for 3 min). However, as a means of enhancing treatment efficacy by increasing cellular sensitivity to chemotherapeutic agents, we showed that GNP exposure to lower power laser resulted in small disruptions of cell membrane due to localized hyperthermia. This did not lead to cell death but provided a mechanism for killing cancer cells by providing enhanced uptake of drug molecules thus leading to a new avenue for hyperthermia-anticancer drug combined cancer therapeutics. FROM THE CLINICAL EDITOR: PEGylated recombinant Protein-G was used as a cofactor to optimize the orientation of IgGs providing "target seeking" properties to gold nanoparticles used in photothermal cancer therapy. The system demonstrated excellent properties in cancer therapy, with the hope and expectation of future clinical translation.

Near-infrared-resonant gold/gold sulfide nanoparticles as a photothermal cancer therapeutic agent.

The development and optimization of near-infrared (NIR)-absorbing nanoparticles for use as photothermal cancer therapeutic agents has been ongoing. This work exploits the properties of gold/gold sulfide NIR-absorbing nanoparticles (approximately 35-55 nm) that provide higher absorption (98% absorption and 2% scattering for gold/gold sulfide versus 70% absorption and 30% scattering for gold/silica nanoshells) as well as potentially better tumor penetration. The ability to ablate tumor cells in vitro and efficacy for photothermal cancer therapy is demonstrated, and an in vivo model shows significantly increased long-term, tumor-free survival. Furthermore, enhanced circulation and biodistribution is observed in vivo. This class of NIR-absorbing nanoparticles has the potential to improve upon photothermal tumor ablation for cancer therapy.

Nano-C60 cytotoxicity is due to lipid peroxidation.

This study examines the biological effects of water-soluble fullerene aggregates in an effort to evaluate the fundamental mechanisms that contribute to the cytotoxicity of a classic engineered nanomaterial. For this work we used a water-soluble fullerene species, nano-C60, a fullerene aggregate that readily forms when pristine C60 is added to water. Nano-C60 was cytotoxic to human dermal fibroblasts, human liver carcinoma cells (HepG2), and neuronal human astrocytes at doses>or= 50 ppb (LC50=2-50 ppb, depending on cell type) after 48 h exposure. This water-soluble nano-C60 colloidal suspension disrupts normal cellular function through lipid peroxidation; reactive oxygen species are responsible for the membrane damage. Cellular viability was determined through live/dead staining and LDH release. DNA concentration and mitochondrial activity were not affected by the nano-C60 inoculations to cells in culture. The integrity of cellular membrane was examined by monitoring the peroxy-radicals on the lipid bilayer. Subsequently, glutathione production was measured to assess the cell's reaction to membrane oxidation. The damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes. With the addition of an antioxidant, L-ascorbic acid, the oxidative damage and resultant toxicity of nano-C60 was completely prevented.

Evaluation of uptake and distribution of gold nanoparticles in solid tumors.

Although nanotherapeutics offer a targeted and potentially less toxic alternative to systemic chemotherapy in cancer treatment, nanotherapeutic transport is typically hindered by abnormal characteristics of tumor tissue. Once nanoparticles targeted to tumor cells arrive in the circulation of tumor vasculature, they must extravasate from irregular vessels and diffuse through the tissue to ideally reach all malignant cells in cytotoxic concentrations. The enhanced permeability and retention effect can be leveraged to promote extravasation of appropriately sized particles from tumor vasculature; however, therapeutic success remains elusive partly due to inadequate intra-tumoral transport promoting heterogeneous nanoparticle uptake and distribution. Irregular tumor vasculature not only hinders particle transport but also sustains hypoxic tissue kregions with quiescent cells, which may be unaffected by cycle-dependent chemotherapeutics released from nanoparticles and thus regrow tumor tissue following nanotherapy. Furthermore, a large proportion of systemically injected nanoparticles may become sequestered by the reticuloendothelial system, resulting in overall diminished efficacy. We review recent work evaluating the uptake and distribution of gold nanoparticles in pre-clinical tumor models, with the goal to help improve nanotherapy outcomes. We also examine the potential role of novel layered gold nanoparticles designed to address some of these critical issues, assessing their uptake and transport in cancerous tissue.

A high yield, one-pot dialysis-based process for self-assembly of near infrared absorbing gold nanoparticles.

HYPOTHESIS: A facile, dialysis-based synthesis of stable near infrared (nIR) absorbing plasmonic gold nanoparticles (λmax=650-1000 nm) will increase the yield of nIR particles and reduce the amount of gold colloid contaminant in the product mixture. EXPERIMENTS: Chloroauric acid and sodium thiosulfate were reacted using a dialysis membrane as a reaction vessel. Product yield and composition was determined and compared to traditional synthesis methods. The product particle distribution, yield, and partitioning of gold between dispersed product and membrane-adsorbed gold were determined. FINDINGS: The synthesis results in polydisperse particle suspensions comprised of 70% spheroid-like particles, 27% triangular plates, and 3% rod-like structures with a 3% batch-to-batch variation and a prominent nIR absorption band with λmax=650-1000 nm. The amount of small gold colloid (λmax=530 nm; d<10 nm) in the isolated product was reduced by 96% compared to traditional methods. Additionally, 91.1% of the gold starting material is retained in the solution-based nanoparticle mixture while 8.2% is found on the dialysis membrane. The synthesis results in a quality ratio (QR=Abs(nIR)/Abs(530)) of 1.7-2.4 (twice that of previous techniques) and 14.3 times greater OD∗ml yield of the nIR-absorbing nanoparticle fraction.

Detection of Phosphatidylcholine-Coated Gold Nanoparticles in Orthotopic Pancreatic Adenocarcinoma using Hyperspectral Imaging.

Nanoparticle uptake and distribution to solid tumors are limited by reticuloendothelial system systemic filtering and transport limitations induced by irregular intra-tumoral vascularization. Although vascular enhanced permeability and retention can aid targeting, high interstitial fluid pressure and dense extracellular matrix may hinder local penetration. Extravascular diffusivity depends upon nanoparticle size, surface modifications, and tissue vascularization. Gold nanoparticles functionalized with biologically-compatible layers may achieve improved uptake and distribution while enabling cytotoxicity through synergistic combination of chemotherapy and thermal ablation. Evaluation of nanoparticle uptake in vivo remains difficult, as detection methods are limited. We employ hyperspectral imaging of histology sections to analyze uptake and distribution of phosphatidylcholine-coated citrate gold nanoparticles (CGN) and silica-gold nanoshells (SGN) after tail-vein injection in mice bearing orthotopic pancreatic adenocarcinoma. For CGN, the liver and tumor showed 26.5 ± 8.2 and 23.3 ± 4.1 particles/100 µm2 within 10 µm from the nearest source and few nanoparticles beyond 50 µm, respectively. The spleen had 35.5 ± 9.3 particles/100 µm2 within 10 µm with penetration also limited to 50 µm. For SGN, the liver showed 31.1 ± 4.1 particles/100 µm2 within 10 µm of the nearest source with penetration hindered beyond 30 µm. The spleen and tumor showed uptake of 22.1 ± 6.2 and 15.8 ± 6.1 particles/100 µm2 within 10 µm, respectively, with penetration similarly hindered. CGH average concentration (nanoparticles/µm2) was 1.09 ± 0.14 in the liver, 0.74 ± 0.12 in the spleen, and 0.43 ± 0.07 in the tumor. SGN average concentration (nanoparticles/µm2) was 0.43 ± 0.07 in the liver, 0.30 ± 0.06 in the spleen, and 0.20 ± 0.04 in the tumor. Hyperspectral imaging of histology sections enables analysis of phosphatidylcholine-coated gold-based nanoparticles in pancreatic tumors with the goal to improve nanotherapeutic efficacy.

Infrared light-absorbing gold/gold sulfide nanoparticles induce cell death in esophageal adenocarcinoma.

Gold nanoparticles and near infrared-absorbing light are each innocuous to tissue but when combined can destroy malignant tissue while leaving healthy tissue unharmed. This study investigated the feasibility of photothermal ablation therapy for esophageal adenocarcinoma using chitosan-coated gold/gold sulfide (CS-GGS) nanoparticles. A rat esophagoduodenal anastomosis model was used for the in vivo ablation study, and three human esophageal cell lines were used to study the response of cancer cells and benign cells to near infrared light after treatment with CS-GGS. The results indicate that both cancerous tissue and cancer cells took up more gold nanoparticles and were completely ablated after exposure to near infrared light. The benign tissue and noncancerous cells showed less uptake of these nanoparticles, and remained viable after exposure to near infrared light. CS-GGS nanoparticles could provide an optimal endoluminal therapeutic option for near infrared light ablation of esophageal cancer.

Enhanced penetration into 3D cell culture using two and three layered gold nanoparticles.

Nano-scale particles sized 10-400 nm administered systemically preferentially extravasate from tumor vasculature due to the enhanced permeability and retention effect. Therapeutic success remains elusive, however, because of inhomogeneous particle distribution within tumor tissue. Insufficient tumor vascularization limits particle transport and also results in avascular hypoxic regions with non-proliferating cells, which can regenerate tissue after nanoparticle-delivered cytotoxicity or thermal ablation. Nanoparticle surface modifications provide for increasing tumor targeting and uptake while decreasing immunogenicity and toxicity. Herein, we created novel two layer gold-nanoshell particles coated with alkanethiol and phosphatidylcholine, and three layer nanoshells additionally coated with high-density-lipoprotein. We hypothesize that these particles have enhanced penetration into 3-dimensional cell cultures modeling avascular tissue when compared to standard poly(ethylene glycol) (PEG)-coated nanoshells. Particle uptake and distribution in liver, lung, and pancreatic tumor cell cultures were evaluated using silver-enhancement staining and hyperspectral imaging with dark field microscopy. Two layer nanoshells exhibited significantly higher uptake compared to PEGylated nanoshells. This multilayer formulation may help overcome transport barriers presented by tumor vasculature, and could be further investigated in vivo as a platform for targeted cancer therapies.

Targeted cancer therapy by immunoconjugated gold-gold sulfide nanoparticles using Protein G as a cofactor.

Gold-gold sulfide nanoparticles (GGS-NPs) fabricated from chloroauric acid and sodium thiosulfate show unique near infrared (NIR) absorption that renders them as a promising candidate for photothermal cancer therapy. To improve targeting efficiency, we developed a versatile method to allow ordered immunoconjugation of antibodies on the surfaces of these nanoparticles via a PEGylated recombinant Protein G (ProG). The PEGylated ProG was prepared with orthopyridyldisulfide-polyethylene glycol-succinimidyl valerate, average MW 2000 (OPSS-PEG-SVA), to first allow the self-assembly of ProG on the nanoparticles, subsequently antibodies were added to this construct to enable active targeting. The bioconjugated GGS-NPs were characterized by TEM, NIR-spectra, dynamic light scattering and modified immunoassay. In in vitro studies, the ProG-conjugated GGS-NPs with bound mouse anti c-erbB-2 (HER-2) immunoglobulin G (IgG) successfully targeted the HER-2 overexpressing breast cancer cell, SK-BR-3. Extensive cell death was observed for the targeted SK-BR-3 line at a low laser power of 540 J (3 W cm(-2) for 3 min) while the control breast cancer cell (low expressing HER-2), HTB-22 survived. Using PEGylated ProG as a cofactor for immobilization of antibodies offers a promising strategy to functionalize various IgGs on nanoparticles for engineering their biomedical applications in cancer therapeutics.

Tunability and stability of gold nanoparticles obtained from chloroauric Acid and sodium thiosulfate reaction.

In the quest for producing an effective clinically relevant therapeutic agent, scalability, repeatability, and stability are paramount. In this paper, gold nanoparticles (GNPs) with precisely controlled near infrared (NIR) absorption are synthesized by a single step reaction of HAuCl4 and Na2S2O3, without assistance of additional templates, capping reagents or seeds. The anisotropy in the shape of gold nanoparticles offers high NIR absorption making it therapeutically relevant. The synthesized products consist of GNPs with different shape and size, including small spherical colloid gold particles and non-spherical gold crystals. The NIR absorption wavelengths and particle size increase with increasing molar ratio of HAuCl4/Na2S2O3. Non-spherical gold particles can be further purified and separated by centrifugation to improve the NIR absorbing fraction of particles. In-depth studies reveal that GNPs with good structural and optical stability only form in a certain range of the HAuCl4/Na2S2O3 molar ratio, whereas higher molar ratios result in unstable GNPs, which lose their NIR absorption peak due to decomposition and reassembly via Ostwald ripening. Tuning the optical absorption of the gold nanoparticles in the NIR regime via a robust and repeatable method will improve many applications requiring large quantities of desired NIR absorbing nanoparticles.

EphrinA I-targeted nanoshells for photothermal ablation of prostate cancer cells.

Gold-coated silica nanoshells are a class of nanoparticles that can be designed to possess strong absorption of light in the near infrared (NIR) wavelength region. When injected intravenously, these nanoshells have been shown to accumulate in tumors and subsequently mediate photothermal treatment, leading to tumor regression. In this work, we sought to improve their specificity by targeting them to prostate tumor cells. We report selective targeting of PC-3 cells with nanoshells conjugated to ephrinA I, a ligand for EphA2 receptor that is overexpressed on PC-3 cells. We demonstrate selective photo-thermal destruction of these cells upon application of the NIR laser.

Temperature-sensitive hydrogels with SiO2-Au nanoshells for controlled drug delivery.

Silica-gold (SiO(2)-Au) nanoshells are a new class of nanoparticles that consist of a silica dielectric core that is surrounded by a gold shell. These nanoshells are unique because their peak extinctions are very easily tunable over a wide range of wavelengths particularly in the near infrared (IR) region of the spectrum. Light in this region is transmitted through tissue with relatively little attenuation due to absorption. In addition, irradiation of SiO(2)-Au nanoshells at their peak extinction coefficient results in the conversion of light to heat energy that produces a local rise in temperature. Thus, to develop a photothermal modulated drug delivery system, we have fabricated nanoshell-composite hydrogels in which SiO(2)-Au nanoshells of varying concentrations have been embedded within temperature-sensitive hydrogels, for the purpose of initiating a temperature change with light. N-isopropylacrylamide-co-acrylamide (NIPAAm-co-AAm) hydrogels are temperature-sensitive hydrogels that were fabricated to exhibit a lower critical solution temperature (LCST) slightly above body temperature. The resulting composite hydrogels had the extinction spectrum of the SiO(2)-Au nanoshells in which the hydrogels collapsed reversibly in response to temperature (50 degrees C) and laser irradiation. The degree of collapse of the hydrogels was controlled by the laser fluence as well as the concentration of SiO(2)-Au nanoshells. Modulated drug delivery profiles for methylene blue, insulin, and lysozyme were achieved by irradiation of the drug-loaded nanoshell-composite hydrogels, which showed that drug release was dependent upon the molecular weight of the therapeutic molecule.

Near-infrared resonant nanoshells for combined optical imaging and photothermal cancer therapy.

Metal nanoshells are core/shell nanoparticles that can be designed to either strongly absorb or scatter within the near-infrared (NIR) wavelength region ( approximately 650-950 nm). Nanoshells were designed that possess both absorption and scattering properties in the NIR to provide optical contrast for improved diagnostic imaging and, at higher light intensity, rapid heating for photothermal therapy. Using these in a mouse model, we have demonstrated dramatic contrast enhancement for optical coherence tomography (OCT) and effective photothermal ablation of tumors.

Overexpression of lysyl oxidase to increase matrix crosslinking and improve tissue strength in dermal wound healing.

In this study, we aimed to increase crosslinking in collagen and elastin in the extracellular matrix through overexpression of lysyl oxidase (LO) in order to improve mechanical strength in dermal wounds during healing. We had used a gene activated matrix (GAM) approach to locally deliver plasmid DNA (pDNA) complexed with polyethylenimine (PEI) in collagen gels at the wound site for localized and sustained transfection of cells involved in the healing process. We first demonstrated in vitro that PEI-pDNA complexes in collagen gels could be taken up and expressed by cultured fibroblasts for at least 20 days. In vitro studies showed that fibroblast-seeded GAMs with the LO transgene exhibited over a 3-fold increase in mechanical strength as compared with a green fluorescent protein (GFP)-transgene control. Addition of an inhibitor of LO abolished this increase. We applied this system in a rat dermal wound healing model and showed that treatment with LO-producing GAMs led to significantly enhanced mechanical strength of the wound site.

Immunonanoshells for targeted photothermal ablation of tumor cells.

Consisting of a silica core surrounded by a thin gold shell, nanoshells possess an optical tunability that spans the visible to the near infrared (NIR) region, a region where light penetrates tissues deeply. Conjugated with tumor-specific antibodies, NIR-absorbing immunonanoshells can preferentially bind to tumor cells. NIR light then heats the bound nanoshells, thus destroying the targeted cells. Antibodies can be consistently bound to the nanoshells via a bifunctional polyethylene glycol (PEG) linker at a density of approximately 150 antibodies per nanoshell. In vitro studies have confirmed the ability to selectively induce cell death with the photothermal interaction of immunonanoshells and NIR light. Prior to incubation with anti-human epidermal growth factor receptor (HER2) immunonanoshells, HER2-expressing SK-BR-3 breast carcinoma cells were seeded alone or adjacent to human dermal fibroblasts (HDFs). Anti-HER2 immunonanoshells bound to HER2-expressing cells resulted in the death of SK-BR-3 cells after NIR exposure only within the irradiated area, while HDFs remained viable after similar treatment since the immunonanoshells did not bind to these cells at high levels. Control nanoshells, conjugated with nonspecific anti-IgG or PEG, did not bind to either cell type, and cells continued to be viable after treatment with these control nanoshells and NIR irradiation.

Metal nanoshells.

Metal nanoshells are a new class of nanoparticles with highly tunable optical properties. Metal nanoshells consist of a dielectric core nanoparticle such as silica surrounded by an ultrathin metal shell, often composed of gold for biomedical applications. Depending on the size and composition of each layer of the nanoshell, particles can be designed to either absorb or scatter light over much of the visible and infrared regions of the electromagnetic spectrum, including the near infrared region where penetration of light through tissue is maximal. These particles are also effective substrates for surface-enhanced Raman scattering (SERS) and are easily conjugated to antibodies and other biomolecules. One can envision a myriad of potential applications of such tunable particles. Several potential biomedical applications are under development, including immunoassays, modulated drug delivery, photothermal cancer therapy, and imaging contrast agents.

Near infrared laser-tissue welding using nanoshells as an exogenous absorber.

BACKGROUND AND OBJECTIVE: Gold nanoshells are a new class of nanoparticles that can be designed to strongly absorb light in the near infrared (NIR). These particles provide much larger absorption cross-sections and efficiency than can be achieved with currently used chemical chromophores without photobleaching. In these studies, we have investigated the use of gold nanoshells as exogenous NIR absorbers to facilitate NIR laser-tissue welding. STUDY DESIGN/MATERIALS AND METHODS: Gold nanoshells with peak extinction matching the NIR wavelength of the laser being used were manufactured and suspended in an albumin solder. Optimization work was performed on ex vivo muscle samples and then translated into testing in an in vivo rat skin wound-healing model. Mechanical testing of the muscle samples was immediately performed and compared to intact tissue mechanical properties. In the in vivo study, full thickness incisions in the dorsal skin of rats were welded, and samples of skin were excised at 0, 5, 10, 21, and 32 days for analysis of strength and wound healing response. RESULTS: Mechanical testing of nanoshell-solder welds in muscle revealed successful fusion of tissues with tensile strengths of the weld site equal to the uncut tissue. No welding was accomplished with this light source when using solder formulations without nanoshells. Mechanical testing of the skin wounds showed sufficient strength for closure and strength increased over time. Histological examination showed good wound-healing response in the soldered skin. CONCLUSIONS: The use of nanoshells as an exogenous absorber allows the usage of light sources that are minimally absorbed by tissue components, thereby, minimizing damage to surrounding tissue and allowing welding of thicker tissues.