Huan Xie

In vivo PET imaging and biodistribution of radiolabeled gold nanoshells in rats with tumor xenografts.

Here we report the radiolabeling of gold nanoshells (NSs) for PET imaging in rat tumor model. A conjugation method was developed to attach NSs with the radionuclide, (64)Cu. The resulting conjugates showed good labeling efficiency and stability in PBS and serum. The pharmacokinetics of (64)Cu-NS and the controls ((64)Cu-DOTA and (64)Cu-DOTA-PEG2K) were determined in nude rats with a head and neck squamous cell carcinoma xenograft by radioactive counting. Using PET/CT imaging, we monitored the in vivo distribution of (64)Cu-NS and the controls in the tumor-bearing rats at various time points after their intravenous injection. PET images of the rats showed accumulation of (64)Cu-NSs in the tumors and other organs with significant difference from the controls. The organ biodistribution of rats at 46h post-injection was analyzed by radioactive counting and compared between the (64)Cu-NS and the controls. Different clearance kinetics was indicated. Neutron activation analysis (NAA) of gold concentration was performed to quantify the amount of NSs in major tissues of the dosed rats and the results showed similar distribution. Overall, PET images with (64)Cu had good resolution and therefore can be further applied to guide photothermal treatment of cancer.

Integrin αvβ3-targeted gold nanoshells augment tumor vasculature-specific imaging and therapy

Purpose Gold nanoshells (NSs) have already shown great promise as photothermal actuators for cancer therapy. Integrin αvβ3 is a marker that is specifically and preferentially overexpressed on multiple tumor types and on angiogenic tumor neovasculature. Active targeting of NSs to integrin αvβ3 offers the potential to increase accumulation preferentially in tumors and thereby enhance therapy efficacy. Methods Enzyme-linked immunosorbent assay (ELISA) and cell binding assay were used to study the in vitro binding affinities of the targeted nanoconjugate NS–RGDfK. In vivo biodistribution and tumor specificity were analyzed using 64Cu-radiolabeled untargeted and targeted NSs in live nude rats bearing head and neck squamous cell carcinoma (HNSCC) xenografts. The potential thermal therapy applications of NS–RGDfK were evaluated by subablative thermal therapy of tumor xenografts using untargeted and targeted NSs. Results ELISA and cell binding assay confirmed the binding affinity of NS–RGDfK to integrin αvβ3. Positron emission tomography/computed tomography imaging suggested that tumor targeting is improved by conjugation of NSs to cyclo(RGDfK) and peaks at ~20 hours postinjection. In the subablative thermal therapy study, greater biological effectiveness of targeted NSs was implied by the greater degree of tumor necrosis. Conclusion The results presented in this paper set the stage for the advancement of integrin αvβ3-targeted NSs as therapeutic nanoconstructs for effective cancer therapy.

Effect of intratumoral administration on biodistribution of 64Cu-labeled nanoshells

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Development of PLGA-based itraconazole injectable nanospheres for sustained release

Purpose Itraconazole (ITZ) is a synthetic triazole antifungal agent, which is widely used for treatment and prevention of fungal infections. The purpose of this study is to develop ITZ-loaded poly(lactic-co-glycolic acid) (PLGA) nanospheres (PLGA-ITZ-NS) as a new sustained-release formulation for intravenous ITZ administration. Materials and methods PLGA-ITZ-NS were prepared by a nanoprecipitation method and optimized by modifying the surfactant poloxamer 188 concentration and PLGA:ITZ ratio. Their physicochemical properties, including size, zeta potential, external morphology and encapsulation efficiency, were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM) and high performance liquid chromatography (HPLC). The effect of the different selected lyoprotectants with various concentrations on NS particles size and surface charge were also assessed. Rapid and sensitive HPLC and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed to determine ITZ concentrations in formulation and in rat plasma, respectively. Pharmacokinetics of the optimum PLGA-ITZ-NS formulation was compared with the former commercial Sporanox® injection formulation using rats as the animal model. Noncompartmental pharmacokinetic parameters were obtained by WinNonlin® software. Results Optimal PLGA-ITZ-NS had a mean particle size of about 200 nm with a high homogeneity (polydispersity index ≈0.2), favorable zeta potential (approximately −20 to −30 mV) and encapsulation efficiency (72%). In addition, 2% w/v sucrose was selected as a lyoprotectant for NS freeze-drying. The newly developed LC-MS/MS assay was validated and found to be accurate and precise. The in vivo study showed that the NS formulation has a similar systemic bioavailability to Sporanox® while providing a sustained plasma level (> 100 ng/mL) for up to 24 hours after intravenous administration. Conclusion Our newly developed PLGA-ITZ-NS has shown great sustained release and comparable bioavailability with Sporanox®, therefore having the potential to be an alternative injectable formulation of ITZ.

Development and Validation of a Sensitive LC/MS/MS Method for the Determination of γ-Tocotrienol in Rat Plasma: Application to Pharmacokinetic Studies

γ-Tocotrienol has attracted much attention owing to its multiple health benefits. This study developed and validated a simple, specific, sensitive and reliable LC/MS/MS method to analyze γ-tocotrienol in rat plasma. Plasma samples (50 μL) were extracted with internal standard solution (25 ng/mL of itraconazole) in acetonitrile (200 μL) with an average recovery of 44.7% and an average matrix effect of -2.9%. The separation of γ-tocotrienol and internal standard from the plasma components was achieved with a Waters XTerra® MS C(18) column with acetonitrile-water as mobile phase. Analysis was performed under positive ionization electrospray mass spectrometer via the multiple reaction monitoring. The standard curve was linear over a concentration range of 10-1000 ng/mL with correlation coefficient values >0.997. The method was validated with intra- and inter-day accuracy (relative error) ranging from 1.79 to 9.17% and from 2.16 to 9.66%, respectively, and precision (coefficient of variation) ranged from 1.94 to 9.25% and from 2.37 to 10.08%, respectively. Short-term stability, freeze-thaw stability and the processed sample stability tests were performed. This method was further applied to analyze γ-tocotrienol plasma concentrations in rats at various time points after administration of a 2 mg/kg single intravenous dose, and a pharmacokinetic profile was successfully obtained.

Solution formulation development and efficacy of MJC13 in a preclinical model of castration-resistant prostate cancer

Abstract MJC13, a novel FKBP52 targeting agent, has potential use for the treatment of castration-resistant prostate cancer. The purpose of this work was to develop a solution formulation of MJC13, and obtain its efficacy profile in a human prostate cancer xenograft mouse model. Preformulation studies were conducted to evaluate the physicochemical properties. Co-solvent systems were evaluated for aqueous solubility and tolerance. A human prostate cancer xenograft mouse model was established by growing 22Rv1 prostate cancer cells in C.B-17 SCID mice. The optimal formulation was used to study the efficacy of MJC13 in this preclinical model of castrate-resistant prostate cancer. We found that MJC13 was stable (at least for 1 month), highly lipophilic (logP = 6.49), poorly soluble in water (0.28 µg/mL), and highly plasma protein bound (>98%). The optimal formulation consisting of PEG 400 and Tween 80 (1:1, v/v) allowed us to achieve a MJC13 concentration of 7.5 mg/mL, and tolerated an aqueous environment. After twice weekly intratumoral injection with 10 mg/kg MJC13 in this formulation for four consecutive weeks, tumor volumes were significantly reduced compared to vehicle-treated controls.

Determination of proline in human serum by a robust LC-MS/MS method: Application to identification of human metabolites as candidate biomarkers for esophageal cancer early detection and risk stratification

Altered serum proline levels are related to cancer metabolism. This study developed and validated a LC-MS/MS method to analyze proline in human serum. Surrogate blank serum, coupled with stable isotope l-proline-(13) C5 ,(15)  N as internal standard, was used for generating standard curves ranging from 2.5 to 100 μg/mL. Proline was extracted from serum samples using methanol. A Phenomenex Lux 5u Cellulose-1 column (250 × 4.6 mm) was used for chromatographic separation with 40% methanol in 0.05% formic acid aqueous solution as a mobile phase. Mass detection was performed under positive ionization electrospray. Intra- and inter-day accuracy and precision were <10%. The extraction recovery and matrix factor were 99.17 and 1.47%, respectively. Our study showed that the chiral column had high specificity and selectivity for separating proline from serum components. The assay was successfully applied for the quantification of human serum proline levels from 30 esophageal cancer patients and 30 healthy volunteers. Statistical analyses showed significantly lower levels of serum proline in the patients as compared with the healthy volunteers (p-value = 0.011). We report here a simple, specific and reproducible LC-MS/MS method for the quantification of proline in human serum as a potential screening biomarker for esophageal cancer.

Radiolabeled gold nanoshells for in vivo imaging: Example of methodology for initial evaluation of biodistribution of a novel nanoparticle

Nanotechnology is an interdisciplinary research effort bridging many scientific fields from physics and chemistry to engineering, biology, and medicine. The result of such interconnections is holding great potential for the early detection, diagnosis, and personalized treatment of disease. The nanoscale range at which nanosystems operate, i.e., one-thousandth smaller than a human cell, can offer facile transport across the human body and intracellular interactions with many cell components that would otherwise be inaccessible. Imaging for early detection and diagnosis of diseases using newly emerged nanoparticles such as quantum dots (QDs) [21], carbon nanotubes (CNTs) [19], nanoshells [10], paramagnetic nanoparticles [34], and others [6, 9] have been an area of interest over the last few years. Carbon nanotubes are a type of nanomaterial that offers unique intrinsic properties that make them very interesting candidates as imaging contrast agents. Their identification in the early 1990s [12] and their further biomedical development [18] opened a new era in the development of novel delivery systems for therapeutics and diagnostics. Carbon nanotubes are mainly classified as single-walled (SWNTs)

Abstract 2889: Development of a novel nanoconstruct for tumor hypoxia photothermal therapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL PURPOSE: Hypoxia is a common feature of solid tumors that occurs across a wide variety of malignancies and significantly reduces tumor sensitivity to radiation and chemotherapy. Carbonic anhydrase IX (CAIX), an extracellular protein that is overexpressed in hypoxic zones within many human tumors, has emerged as a promising biomarker. To pursue focal targeted therapy of tumor hypoxia, we prepared and characterized a novel nanoconstruct composed of gold nanorods (GNR), which show superior capability to assist tumor photothermal ablation, and anti-CAIX antibody (CAIX-Ab), which binds specifically to CAIX. EXPERIMENTS: GNR with aspect ratio (length 24 nm to width 8nm) of 3 were conjugated to CAIX-Ab through bifunctional polyethylene glycol (PEG). The conjugates were characterized by monitoring the absorption peak shift as well as size and zeta potential changes for each step of the preparation. The stabilities of the GNR, GNR-PEG and GNR/CAIX-Ab were challenged by 1X PBS. The binding affinity and specificity of the GNR/CAIX-Ab were evaluated with ELISA using CAIX protein and cell adhesion assay using HT29 human colorectal cells (overexpresing CAIX) and NIH-3T3 cells (no expressing CAIX, control). The photothermal cell ablation was performed on HT29 cells treated with medium (control), GNR-PEG (control) and GNR/CAIX-Ab. RESULTS: Obvious shifts of the absorption peaks of GNR at the NIR region and particle size and surface charge changes suggested the success of the conjugation. After challenging by 1XPBS, bare GNR formed aggregations and lost the peak at 745 nm while GNR-PEG and GNR/CAIX-Ab remained stable as their absorption peaks around 750 nm were preserved. This is an important finding suggesting that our conjugated GNR will be stable for in vivo work. From the sandwich ELISA study, we obtained a standard linear curve of absorbance vs. CAIX-Ab concentration with R2 of 0.997 and linear curve of absorbance vs. GNR/CAIX-Ab concentration with R2 of 0.999. And we found that the negative control GNR-PEG had no binding to the CAIX protein. We calculated the number of CAIX-Ab per GNR to be 1.47 ± 0.2. In cell adhesion assay, neither GNR-PEG nor GNR/CAIX-Ab had detectable binding to 3T3 cells; but with HT29 cells, GNR-PEG still showed no binding while GNR/CAIX-Ab bound specifically and avidly to the cells. Photothermal ablation of HT29 cells revealed complete cell death after treatment of GNR/CAIX-Ab while the cells remained alive when treated with medium or GNR-PEG. CONCLUSION: These findings suggested that GNR/CAIX-Ab is a stable and effective nanoconstruct appropriate for focal targeting and photothermal ablation of tumor hypoxia. This new photothermal therapy has the great potential as an alternative or complementary approach for standard cancer treatment to eliminate residual or recurrent tumor cells, especially within the hypoxic areas that are resistant to traditional therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2889. doi:1538-7445.AM2012-2889

Hypoxia-targeted gold nanorods for cancer photothermal therapy

Tumor hypoxia is a well-recognized driver of resistance to traditional cancer therapies such as chemotherapy and radiation therapy. We describe development of a new nanoconstruct composed of gold nanorods (GNRs) conjugated to carbonic anhydrase IX (CAIX) antibody that specifically binds to CAIX, a biomarker of hypoxia, to facilitate targeting tumor hypoxic areas for focused photothermal ablation. Physicochemical characterization studies confirmed the size, shape, monodispersity, surface charge, and serum stability of the GNRs. Enzyme-linked immunosorbent assays and cellular binding and uptake studies confirmed successful conjugation of antibody to the GNRs and specificity for CAIX. Near-infrared irradiation of CAIX-overexpressing cells treated with GNR/anti-CAIX resulted in significantly higher cell death than cells treated with control GNRs. In vivo biodistribution studies using hyperspectral imaging and inductively coupled plasma mass spectrometry confirmed intravenous administration results not only in greater accumulation of GNR/anti-CAIX in tumors than control GNRs but also greater penetration into hypoxic areas of tumors. Near-infrared ablation of these tumors showed no tumor regression in the sham-treated group, regression but recurrence in the non-targeted-GNR group, and complete tumor regression in the targeted-GNR group. GNR/anti-CAIX nanoconstructs show promise as hypoxia targeting and photothermal ablation agents for cancer treatment.