Assessment of in vivo systemic toxicity and biodistribution of iron-doped silica nanoshells.

Silica nanoparticles are an emerging class of biomaterials which may be used as diagnostic and therapeutic tools for biomedical applications. In particular, hollow silica nanoshells are attractive due to their hollow core. Approximately 70% of a 500 nm nanoshell is hollow, therefore more particles can be administered on a mg/kg basis compared to solid nanoparticles. Additionally, their nanoporous shell permits influx/efflux of gases and small molecules. Since the size, shape, and composition of a nanoparticle can dramatically alter its toxicity and biodistribution, the toxicology of these nanomaterials was assessed. A single dose toxicity study was performed in vivo to assess the toxicity of 500 nm iron-doped silica nanoshells at clinically relevant doses of 10-20 mg/kg. This study showed that only a trace amount of silica was detected in the body 10 weeks post-administration. The hematology, biochemistry and pathological results show that the nanoshells exhibit no acute or chronic toxicity in mice.

Assessment of cortical bone with clinical and ultrashort echo time sequences.

We describe the use of ultrashort echo time (UTE) sequences and fast spin echo sequences to assess cortical bone using a clinical 3T scanner. Regular two- and three-dimensional UTE sequences were used to image both bound and free water in cortical bone. Adiabatic inversion recovery prepared UTE sequences were used to image water bound to the organic matrix. Two-dimensional fast spin echo sequences were used to image free water. Regular UTE sequences were used together with bicomponent analysis to measure T*2s and relative fractions of bound and free water components in cortical bone. Inversion recovery prepared UTE sequences were used to measure the T*2 of bound water. Saturation recovery UTE sequences were used to measure the T1 of bone water. Eight cadaveric human cortical bone samples and a lower leg specimen were studied. Preliminary results show two distinct components in UTE detected signal decay, a single component in inversion recovery prepared UTE detected signal decay, and a single component in saturation recovery UTE detected signal recovery. Regular UTE sequences appear to depict both bound and free water in cortical bone. Inversion recovery prepared UTE sequences appear to depict water bound to the organic matrix. Two-dimensional fast spin echo sequences appear to depict bone structure corresponding to free water in large pores.

mTOR and HIF-1alpha-mediated tumor metabolism in an LKB1 mouse model of Peutz-Jeghers syndrome.

Peutz-Jeghers syndrome (PJS) is a familial cancer disorder due to inherited loss of function mutations in the LKB1/ STK11 serine/threonine kinase. PJS patients develop gastrointestinal hamartomas with 100% penetrance often in the second decade of life, and demonstrate an increased predisposition toward the development of a number of additional malignancies. Among mitogenic signaling pathways, the mammalian-target of rapamycin complex 1 (mTORC1) pathway is hyperactivated in tissues and tumors derived from LKB1-deficient mice. Consistent with a central role for mTORC1 in these tumors, rapamycin as a single agent results in a dramatic suppression of preexisting GI polyps in LKB1+/- mice. However, the key targets of mTORC1 in LKB1-deficient tumors remain unknown. We demonstrate here that these polyps, and LKB1- and AMPK-deficient mouse embryonic fibroblasts, show dramatic up-regulation of the HIF-1alpha transcription factor and its downstream transcriptional targets in an rapamycin-suppressible manner. The HIF-1alpha targets hexokinase II and Glut1 are up-regulated in these polyps, and using FDG-PET, we demonstrate that LKB1+/- mice show increased glucose utilization in focal regions of their GI tract corresponding to these gastrointestinal hamartomas. Importantly, we demonstrate that polyps from human Peutz-Jeghers patients similarly exhibit up-regulated mTORC1 signaling, HIF-1alpha, and GLUT1 levels. Furthermore, like HIF-1alpha and its target genes, the FDG-PET signal in the GI tract of these mice is abolished by rapamycin treatment. These findings suggest a number of therapeutic modalities for the treatment and detection of hamartomas in PJS patients, and potential for the screening and treatment of the 30% of sporadic human lung cancers bearing LKB1 mutations.

Direct imaging and quantification of carotid plaque calcification.

Carotid plaque calcification normally appears as a signal void with clinical MR sequences. Here, we describe the use of an adiabatic inversion recovery prepared two-dimensional ultrashort echo time sequence to image and characterize carotid plaque calcification using a clinical 3-T scanner. T(1), T 2*, and free water content were measured for seven carotid samples, and the results were compared with micro-CT imaging. Conventional gradient echo and fast spin echo images were also acquired for comparison. Correlations between T(1), T 2*, free water concentration, and mineral density were performed. There was a close correspondence between inversion recovery prepared two-dimensional ultrashort echo time morphologic and micro-CT appearances. Carotid plaque calcification varied significantly from sample to sample, with T(1) s ranging from 94 ± 19 to 328 ± 21 msec, T 2*s ranging from 0.31 ± 0.12 to 2.15 ± 0.25 msec, and free water concentration ranging from 5.7 ± 2.3% to 16.8 ± 3.4%. There was a significant positive correlation between T(1)(R = 0.709; P < 0.074), T 2* (R = 0.816; P < 0.025), and free water concentration, a negative correlation between T(1) (R = 0.773; P < 0.042), T 2* (R = 0.948; P < 0.001) and CT measured mineral density, and a negative correlation between free water concentration (R = 0.936; P < 0.002) and mineral density.

Validation of ultrasound contrast destruction imaging for flow quantification.

Our purpose was to validate in vitro a kinetic flow model based on microbubble signal decay curve. Using a 3.5 MHz transducer and phase-inversion (1.8 MHz central transmit frequency), a renal dialysis cartridge oriented vertically was imaged in the transverse plane as 1:1000 dilution of AF0150 was infused at 50, 100, 200, 300 and 400 mL/min. Ten gray-scale images were acquired at each infusion rate using 2.5, 5 and 10 frames/s at 100%, 40%, 15% or 1% of maximum transmit power. Video-intensity measured on each 10 images was fit to a kinetic model using Sigma Plot that yielded microbubble concentration, velocity and destruction per frame. These were correlated with the experimental conditions. At 100% power, video-intensity on the first frame (microbubble concentration at equilibrium) was similar for all flow and frame rates. The model fit the experimental data for all flows at 10 frames/s and for flows lower than 400 and 100 mL/min at 5 frames/s and 2.5 frames/s, respectively. The calculated flow was similar to the experimental flow rates, regardless of technique (r(2) = 0.98). Microbubble fraction destroyed per frame was similar for all flow and frame rates and increased linearly with transmit power (r(2) > 0.98). These results suggest that using appropriate power and frame rate for a given flow rate, estimates of fractional blood volume, flow and destruction fraction can be calculated from the decay curve using 10 frames that can be acquired in 1 to 4 s.

Angiogenesis model for ultrasound contrast research: exploratory study.

RATIONALE AND OBJECTIVES: To optimize an angiogenesis model for imaging research that is stable and can be imaged several times over the angiogenic time course. MATERIALS AND METHODS: Mice and rats received two injections of 0.4 mL of extract of basement membrane matrix (Matrigel; Becton Dickinson Labware, Bedford, MA) in the subcutaneous spaces on either side of the spine. One of the two Matrigel plugs in each animal had either 0.1 microg/mL of basic fibroblast growth factor (bFGF) (11 mice), 1.0 microg/mL of bFGF (12 mice, 5 rats), or 1.0 microg/mL of bFGF and 60 U/mL of heparin (11 mice). Three to 12 days after implantation, animals were imaged before and after the administration of up to four injections of 0.1 mL AF0150. Phase inversion imaging was used on a Siemens Elegra (Siemens ultrasound, Issaquah, WA) equipped with a 13 MHz VFX transducer. Three observers subjectively assessed the pattern of enhancement using a four-point scale. The Matrigel plugs were then removed and two observers graded the angiogenic response on a four-point scale. Ten Matrigel plugs, five with 1.0 microg/mL bFGF and five without, were evaluated histologically following immunohistochemical staining with anti-CD31. RESULTS: The angiogenic response was greater in Matrigel plugs with 1.0 than with 0.1 microg/mL of bFGF. Heparin did not increase the angiogenic response. Vessels were predominantly at the periphery of the plugs with variable central penetration. Plugs appeared anechoic and homogeneous on ultrasound. Contrast enhancement within the plug occurred in 44% of mice with an angiogenic response at or after day 6 and the enhancement increased with the angiogenic response. In the others, peripheral enhancement could not be distinguished from the enhancement of surrounding tissues that were also hyperemic. The thicker rat skin interfered with plug assessment. CONCLUSION: A stable angiogenesis model without the complexity of tumors is described. This model offers the opportunity to image the development and/or inhibition of angiogenesis. Neovasculature in Matrigel was detectable using ultrasound contrast. Quantitative studies correlating the degree of enhancement to microvascular density will be determined in subsequent studies.

Gadolinium-DTPA-dextran: a macromolecular MR blood pool contrast agent.

RATIONALE AND OBJECTIVES: Magnetic resonance (MR) imaging blood pool agents offer numerous advantages for vascular and tumor imaging. The purpose of this study was to test gadolinium-diethylenetriaminepentaacetate-dextran ([Gd]DTPA-dextran) as a new water soluble macromolecular blood pool agent for MR imaging. MATERIALS AND METHODS: [Gd]DTPA-dextran (187 gadolinium atoms per dextran, molecular weight 165 kD, diameter 17.6 nm) was synthesized. Fifteen anesthetized New Zealand White rabbits with thigh VX2 tumors were scanned in a knee coil at 1.5T. Coronal 3D MR angiographic sequences were obtained before and at several time points up to 72 hours after the intravenous bolus injection of [Gd]DTPA-dextran providing gadolinium at either 0.05 (n = 4) or 0.1 mmol/kg (n = 8) or [Gd]DTPA-bismethylamide (BMA) providing gadolinium at 0.1 mmol/kg (n = 3). Time enhancement curves for aorta, cava, and tumor rim were compared by univariate General Linear Model. RESULTS: Contrast enhancement of cava and aorta relative to a water phantom were significantly greater at all time points after either dose of [Gd]DTPA-dextran than after [Gd]DTPA-BMA (P < 0.01). Tumor rim enhancement was less intense for either dose of [Gd]DTPA-dextran at peak than for [Gd]DTPA-BMA (P < 0.05). Tumor rim enhancement with both doses of [Gd]DTPA-dextran became equivalent to that of [Gd]DTPA-BMA at one hour and was greater at 24 hours (P < 0.05). CONCLUSION: [Gd]DTPA-dextran is a new macromolecular MR contrast agent that can be synthesized to carry a high density of gadolinium atoms without intra-molecular cross-linking. It provides significantly greater vascular residence time than a conventional gadolinium chelate and shows promise for MR blood pool imaging.

Angiogenesis: noninvasive quantitative assessment with contrast-enhanced functional US in murine model.

PURPOSE: To evaluate quantitative functional ultrasonography (US) in a murine gel model by using microbubble destruction kinetics to determine whether parametric indices provided with US could help assess angiogenesis. MATERIALS AND METHODS: Institutional Animal Subjects Committee approved experiments and procedures. In 36 normal mice, two 0.4-mL gel implants were placed subcutaneously on either side of spine. One implant contained 0.5, 1.0, or 1.5 microg human basic fibroblast growth factor (bFGF) per milliliter of gel. Functional US quantitative analysis of angiogenesis with microbubble contrast agent was performed on days 3, 6, 9, and 12; histologic data were collected. Time-intensity curve of implant was fitted to mathematic decay model to calculate fractional blood volume and fraction of blood replaced per unit of time. Microvascular density (MVD) and percentage of microvascular area (MVA) were measured after anti-CD31 staining. Spearman rank order correlation was used in analyses. RESULTS: bFGF-containing implants induced MVD of eight, 35, 42, and 42 vessels per square millimeter on days 3, 6, 9, and 12, respectively; in controls, MVD was four vessels/mm2 (P<.05 on days 6, 9, and 12). bFGF-containing implants induced percentage MVA of 2%, 5%, 20%, and 27%, respectively; in controls, it was 0.5% (P<.05). Maximum enhancement was significantly increased in bFGF implants (23.3 gray level+/-14.1 [standard deviation]) compared with controls (11.0+/-5.5, P<.001). Implants containing bFGF showed poor correlations between fractional blood volume and MVD (r2=0.42) or percentage MVA (r2=0.51) at US. There was no correlation between microbubble velocity and MVD (r2<0.05) or percentage MVA (r2<0.13). CONCLUSION: Functional US perfusion parameters do not correlate with current histologic indices for quantifying angiogenesis. MVD, as a histologic quantitative measurement of angiogenesis, may not be an appropriate standard for contrast-enhanced imaging that relies on perfused neovessels.

Time course and role of morphine dose and concentration in intrathecal granuloma formation in dogs: a combined magnetic resonance imaging and histopathology investigation.

BACKGROUND: Intrathecal morphine infusion leads to intrathecal granulomas. In dogs, the authors examined time course of granuloma formation and the role of concentration in granuloma development. METHODS: Dogs were prepared with lumbar intrathecal catheters and vest-mounted pumps. To define the time course of granuloma formation, serial magnetic resonance imaging was performed in animals receiving 10 or 31 days of morphine infusion (12.5 mg/ml at 40 microl/h). At these times, morphine was removed from the infusate, and further magnetic resonance images were acquired over 14-35 additional days. To assess dose versus concentration, dogs received 28-day infusions of vehicle, 12 mg morphine/day as 12.5 mg/ml at 40 microl/h, or 1.5 mg/ml at 334 microl/h (12 mg/day) for 28 days. Additional dogs received 3 mg/day as 12.5 mg/ml at 10 mul/h. RESULTS: Serial magnetic resonance images in dogs receiving morphine (12.5 mg/ml at 40 microl/h) revealed pericatheter-enhancing tissues as early as 3 days with a prominent signal by 10 days. Removal of morphine reduced the mass volume within 7 days. At a fixed infusion rate, the incidence of granuloma formation with the continuous intrathecal infusion of morphine ranged from 0 in vehicle-treated dogs to 100% in dogs treated with 12.5 mg/ml at 40 microl/h (12 mg/day). Infusion of 12 mg/day at 1.5 mg/ml (334 microl/h) resulted in granuloma in one of four animals. The authors found that infusion of morphine in different concentrations at a fixed rate resulted in a dose-dependent increase in concentration, with the granuloma-producing, dose-yielding lumbar cerebrospinal fluid morphine concentrations around 40 microg/ml. CONCLUSIONS: Serial magnetic resonance imaging showed a rapid formation and regression of the masses initiated by intrathecal morphine infusion. These masses are dependent on local concentration.

Model to quantify lymph node enhancement on indirect sonographic lymphography.

OBJECTIVE: Our goal was to develop a reliable technique that has minimal operator dependence for quantifying lymph node enhancement to test and optimize new sonography contrast formulations. MATERIALS AND METHODS: Twenty healthy rabbits were studied using five agents, labeled A-G. Agents D and E were the same agent and agents F and G were Imagent, studied blindly to test reproducibility. One milliliter of contrast agent was injected into each hind footpad. A 13-MHz transducer was fixed over the popliteal node, which was imaged at a 4.8-MHz central transmit frequency using phase-inversion technology at 100% power and one frame per second. Immediately after each injection, the footpad was massaged 12 times for 30 sec each time and then imaged after each massage to assess the number of times the node could be refilled from each injection. Lymph node video intensity was measured, and the degree of enhancement was evaluated using analysis of variance with the massage number and the agent used as independent variables. RESULTS: Lymph node enhancement was observed after the first massage with all agents. Degree of enhancement was least with agents A and B, intermediate with agents D and F, and greatest with agent C. Agent A was effective after the first two massages, agent B after the first four, agent C after all 12, agent D after the first eight, and agent F after the first nine. Performance of agents D and F was similar to that of their duplicates, E and G. CONCLUSION: We established a reproducible technique to quantify lymph node enhancement that can distinguish between different agents. The differences in performance suggest that it is possible to optimize agent formulation for indirect sonographic lymphography.