Steven L. Baldwin

Molecularly targeted nanocarriers deliver the cytolytic peptide melittin specifically to tumor cells in mice, reducing tumor growth

The in vivo application of cytolytic peptides for cancer therapeutics is hampered by toxicity, nonspecificity, and degradation. We previously developed a specific strategy to synthesize a nanoscale delivery vehicle for cytolytic peptides by incorporating the nonspecific amphipathic cytolytic peptide melittin into the outer lipid monolayer of a perfluorocarbon nanoparticle. Here, we have demonstrated that the favorable pharmacokinetics of this nanocarrier allows accumulation of melittin in murine tumors in vivo and a dramatic reduction in tumor growth without any apparent signs of toxicity. Furthermore, direct assays demonstrated that molecularly targeted nanocarriers selectively delivered melittin to multiple tumor targets, including endothelial and cancer cells, through a hemifusion mechanism. In cells, this hemifusion and transfer process did not disrupt the surface membrane but did trigger apoptosis and in animals caused regression of precancerous dysplastic lesions. Collectively, these data suggest that the ability to restrain the wide-spectrum lytic potential of a potent cytolytic peptide in a nanovehicle, combined with the flexibility of passive or active molecular targeting, represents an innovative molecular design for chemotherapy with broad-spectrum cytolytic peptides for the treatment of cancer at multiple stages.

Properties of an entropy-based signal receiver with an application to ultrasonic molecular imaging

Qualitative and quantitative properties of the finite part, H(f), of the Shannon entropy of a continuous waveform f(t) in the continuum limit are derived in order to illuminate its use for waveform characterization. Simple upper and lower bounds on H(f), based on features of f(t), are defined. Quantitative criteria for a priori estimation of the average-case variation of H(f) and log E(f), where E(f) is the signal energy of f(t) are also derived. These provide relative sensitivity estimates that could be used to prospectively choose optimal imaging strategies in real-time ultrasonic imaging machines, where system bandwidth is often pushed to its limits. To demonstrate the utility of these sensitivity relations for this application, a study designed to assess the feasibility of identification of angiogenic neovasculature targeted with perfluorocarbon nanoparticles that specifically bind to alpha(v)beta3-integrin expression in tumors was performed. The outcome of this study agrees with the prospective sensitivity estimates that were used for the two receivers. Moreover, these data demonstrate the ability of entropy-based signal receivers when used in conjunction with targeted nanoparticles to elucidate the presence of alpha(v)beta3 integrins in primordial neovasculature, particularly in acoustically unfavorable environments.

Measurements of the anisotropy of ultrasonic attenuation in freshly excised myocardium.

Echocardiography requires imaging of the heart with sound propagating at varying angles relative to the predominant direction of the myofibers. The degree of anisotropy of attenuation can significantly influence ultrasonic imaging and tissue characterization measurements in vivo. This study quantifies the anisotropy of attenuation of freshly excised myocardium at frequencies typical of echocardiographic imaging. Results show a significantly larger anisotropy than previously reported in specimens of locally unidirectional myofibers. Through-transmission radio frequency-based measurements were performed on specimens from 12 ovine and 12 bovine hearts. Although ovine hearts are closer in size to human, the larger size of bovine hearts offers the potential for specimens in which myofibers are more nearly unidirectionally aligned. The attenuation coefficient increased approximately linearly with frequency. The mean slope of attenuation with frequency was 3-4 times larger for propagation parallel than for perpendicular to the myofibers. At perpendicular insonification, slopes between ovine and bovine myocardium were approximately equal. However, attenuation in bovine specimens was larger for angles approaching parallel. The difference in results for parallel appears consistent with what might be expected from increased myofiber curvature associated with smaller lamb hearts. Quantitative knowledge of anisotropy of attenuation may be useful in understanding mechanisms underlying the interaction of ultrasound with myocardium.

Measurements of the anisotropy of ultrasonic velocity in freshly excised and formalin-fixed myocardial tissue.

The objective of this study was to quantify the anisotropy of ultrasonic velocity in freshly excised myocardial tissue and to examine the effects of formalin-fixation. Through-transmission radio-frequency-based measurements were performed on ovine and bovine myocardial specimens from 24 different hearts. A total of 81 specimens were obtained from specific locations within each heart to investigate the possibility of regional differences in anisotropy of velocity in the left ventricular wall and septum. No regional differences were observed for either lamb or cow myocardial specimens. In addition, no specific species-dependent differences were observed between ovine and bovine myocardium. Average values of velocity at room temperature for perpendicular and parallel insonification were 1556.9 +/- 0.6 and 1565.2 +/- 0.7 m/s (mean +/- standard error), respectively, for bovine myocardium (N=45) and 1556.3 +/- 0.6 and 1564.7 +/- 0.7 m/s for ovine myocardium (N=36). Immediately after measurements of freshly excised myocardium, ovine specimens were fixed in formalin for at least one month and then measurements were repeated. Formalin-fixation appears to increase the overall velocity at all angles of insonification and to increase the magnitude of anisotropy of velocity.

Transmural variation of myocardial attenuation measured with a clinical imager

The objective of this study was to quantify the transmural variation in attenuation for the septal and lateral walls of the heart. Our approach was to utilize a commercially available ultrasonic imaging system to acquire images of excised sections of eight sheep hearts with an orientation similar to that encountered in the apical four-chamber view. The measured values (mean +/-SE) of the slope of attenuation for the transmural regions of the septum are: 1.40 +/-0.11, 0.99 +/-0.09, and 1.85 +/-0.16 (dB/cm/MHz) for the left subendocardial, midmyocardial, and right subendocardial zones, respectively. The analogous data from the lateral wall are: 1.42 +/-0.11, 0.83 +/-0.07, and 1.20 +/- 0.16 (dB/cm/MHz) for the subendocardial, midmyocardial, and subepicardial zones, respectively. These data demonstrate that ultrasonic attenuation associated with the septum and the lateral wall, when imaged in a manner similar to that of the apical four-chamber view, is anisotropic.

18FDG PET-CT imaging detects arterial inflammation and early atherosclerosis in HIV-infected adults with cardiovascular disease risk factors

BackgroundPersistent vascular inflammation has been implicated as an important cause for a higher prevalence of cardiovascular disease (CVD) in HIV-infected adults. In several populations at high risk for CVD, vascular 18Fluorodeoxyglucose (18FDG) uptake quantified using 3D-positron emission-computed tomography (PET-CT) has been used as a molecular level biomarker for the presence of metabolically active proinflammatory macrophages in rupture-prone early atherosclerotic plaques. We hypothesized that 18FDG PET-CT imaging would detect arterial inflammation and early atherosclerosis in HIV-infected adults with modest CVD risk.MethodsWe studied 9 HIV-infected participants with fully suppressed HIV viremia on antiretroviral therapy (8 men, median age 52 yrs, median BMI 29 kg/m2, median CD4 count 655 cells/μL, 33% current smokers) and 5 HIV-negative participants (4 men, median age 44 yrs, median BMI 25 kg/m2, no current smokers). Mean Framingham Risk Scores were higher for HIV-infected persons (9% vs. 2%, p < 0.01). 18FDG (370 MBq) was administered intravenously. 3D-PET-CT images were obtained 3.5 hrs later. 18FDG uptake into both carotid arteries and the aorta was compared between the two groups.ResultsRight and left carotid 18FDG uptake was greater (P < 0.03) in the HIV group (1.77 ±0.26, 1.33 ±0.09 target to background ratio (TBR)) than the control group (1.05 ± 0.10, 1.03 ± 0.05 TBR). 18FDG uptake in the aorta was greater in HIV (1.50 ±0.16 TBR) vs control group (1.24 ± 0.05 TBR), but did not reach statistical significance (P = 0.18).ConclusionsCarotid artery 18FDG PET-CT imaging detected differences in vascular inflammation and early atherosclerosis between HIV-infected adults with CVD risk factors and healthy HIV-seronegative controls. These findings confirm the utility of this molecular level imaging approach for detecting and quantifying glucose uptake into inflammatory macrophages present in metabolically active, rupture-prone atherosclerotic plaques in HIV infected adults; a population with increased CVD risk.

Backscatter imaging and myocardial tissue characterization

The goal of myocardial ultrasonic tissue characterization is to complement two-dimensional and Doppler echocardiography by providing information (such as assessment of regional viability based on localized values of backscatter) beyond that derived from an assessment of myocardial dimensions and motion. Quantitative backscatter imaging can be subdivided into three broad areas: (1) direct applications, in which specific pathologies are identified and monitored, (2) indirect applications, in which quantitative techniques designed for use in tissue characterization serve to expand the role of echocardiography, and (3) contributions to the understanding of cardiac structure and function.

Elastic stiffness coefficients (c11, c33, and c13) for freshly excised and formalin-fixed myocardium from ultrasonic velocity measurements

The goal of this study was to measure elastic stiffness coefficients of freshly excised and subsequently formalin-fixed myocardial tissue. Our approach was to measure the angle-dependent phase velocities associated with the propagation of a longitudinal ultrasonic wave (3-8 MHz) in ovine myocardium using phase spectroscopy techniques and to interpret the results in the context of orthotropic and transversely isotropic models describing the elastic properties of myocardium. The phase velocity results together with density measurements were used to obtain the elastic stiffness coefficients c11, c33, and c13 for both symmetries. The results for the elastic stiffness coefficients c11, c33, and c13 are the same for both symmetries. Measurements for freshly excised myocardium and the same tissue after a period of formalin fixation were compared to examine the impact of fixation on the elastic stiffness coefficients.

Effects of region-of-interest length on estimates of myocardial ultrasonic attenuation and backscatter

Measurements of tissue properties using an image-based technique that makes use of an external reference may have the potential for practical clinical implementation in echocardiography. The objective of this study was to quantify the ability of this technique to distinguish myocardial attenuation and backscatter properties for specific lengths of the region-of-interest (ROI). We chose to exploit the anisotropic properties of the myocardium as a model for distinguishing tissue with different acoustic properties. Excised lateral wall segments from seven healthy adult sheep hearts were imaged using a commercially available (Philips/ATL) clinical scanner operating in the fundamental imaging mode with a linear array (L 7-4). Statistical and receiver operating characteristic (ROC) analyses were used to evaluate the ability of the video signal analysis method to differentiate midmyocardial from subendocardial regions based on measurements of the acoustic properties for specific lengths of the ROI. Results demonstrate that the ability to distinguish tissue properties increases with ROI length for both slope of attenuation and backscatter coefficient measurements. Statistically significant differences were observed for measurements utilizing the ROI lengths as short as 0.4 cm with corresponding progressively increasing areas under the ROC curves for increasing ROI lengths. [NIH R37 HL40302]

Ultrasonic Detection of the Anisotropy of Protein Cross Linking in Myocardium at Diagnostic Frequencies

Increased myocardial stiffness in aging and diabetes that may result in pathologies such as diastolic dysfunction has been attributed, in part, to an increase in cross linking of extracellular matrix proteins such as collagen. With the development of new approaches to cardiovascular therapy, it becomes increasingly important to develop noninvasive approaches for monitoring changes in myocardial cross linking. The objective of this study was to use ultrasound at frequencies used in clinical echocardiography to measure changes in myocardial attenuation resulting from increased cross linking as a function of angle of insonification over a complete rotation. Through- transmission radiofrequency-based measurements were performed on 36 specimens from 12 freshly excised ovine hearts at room temperature, which were then fixed in formalin to induce protein cross linking prior to repeated measurements. For angles near perpendicular to the myofiber direction, the measured slope of attenuation increased from 0.52 plusmn 0.07 dB/(cmldrMHz) (mean plusmn one standard deviation) for freshly excised to 0.85 plusmn 0.08 dB/(cmldrMHz) for formalin-fixed myocardium. In contrast, results for parallel insonification exhibit considerable overlap (1.88 plusmn 0.17 for freshly excised and 1.75 plusmn 0.19 dB/(cmldrMHz) for formalin- fixed myocardium). Results of this study suggest that the response of the extracellular collagenous matrix to changes in cross linking is directionally dependent. The anisotropy of ultrasonic attenuation thus may provide an approach for noninvasive monitoring of the extent and progression of myocardial disease associated with changes in protein cross linking. Accounting for effects due to anisotropy may be essential for the future detection of such changes using ultrasonic attenuation in vivo.