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.

Sensitive ultrasonic delineation of steroid treatment in living dystrophic mice with energy-based and entropy-based radio frequency signal processing.

Duchenne muscular dystrophy is a severe wasting disease, involving replacement of necrotic muscle tissue by fibrous material and fatty infiltrates. One primary animal model of this human disease is the X chromosome-linked mdx strain of mice. The goals of the present work were to validate and quantify the capability of both energy and entropy metrics of radio-frequency ultrasonic backscatter to differentiate among normal, dystrophic, and steroid-treated skeletal muscle in the mdx model. Thirteen 12-month-old mice were blocked into three groups: 4 treated mdx-dystrophic that received daily subcutaneous steroid (prednisolone) treatment for 14 days, 4 positive-control mdx-dystrophic that received saline injections for 14 days, and 5 negative-control animals. Biceps muscle of each animal was imaged in vivo using a 40-MHz center frequency transducer in conjunction with a Vevo-660 ultrasound system. Radio-frequency data were acquired (1 GHz, 8 bits) corresponding to a sequence of transverse images, advancing the transducer from "shoulder" to "elbow" in 100-micron steps. Data were processed to generate both "integrated backscatter" (log energy), and "entropy" (information theoretic receiver, H(f)) representations. Analyses of the integrated-backscatter values delineated both treated-and untreated-mdx biceps from normal controls (p < 0.01). Complementary analyses of the entropy images differentiated the steroid-treated and positive-control mdx groups (p < 0.01). To our knowledge, this study represents the first reported use of quantitative ultrasonic characterization of skeletal muscle in mdx mice. Successful differentiation among dystrophic, steroid-treated, and normal tissues suggests the potential for local noninvasive monitoring of disease severity and therapeutic effects.

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 +/- 0.07 dB/(cm MHz) (mean +/- one standard deviation) for freshly excised to 0.85 +/- 0.08 dB/(cm MHz) for formalin-fixed myocardium. In contrast, results for parallel insonification exhibit considerable overlap (1.88 +/- 0.17 for freshly excised and 1.75 +/- 0.19 dB/(cm MHz) 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.

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.

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.

The frequency dependence of ultrasonic velocity and the anisotropy of dispersion in both freshly excised and formalin-fixed myocardium.

The objectives of this study were to measure the frequency dependence of the ultrasonic velocity in myocardium and to quantify the frequency dependence of phase velocity as a function of the insonification angle relative to the predominant direction of the myofibers. Broadband phase spectroscopy data were acquired, spanning a frequency range of 3 to 8 MHz. Measurements were made on 36 tissue specimens cored from 12 freshly excised lamb hearts and were repeated after fixation with formalin. Measured phase velocities were found to be well characterized by a logarithmic fit. For freshly excised myocardium, the dispersion over the 3 to 8 MHz bandwidth was dependent on the direction of insonification, ranging from 1.2 m/s change for perpendicular insonification (across the myofibers) to 3.7 m/s for parallel insonification (along the myofibers). The effects of formalin-fixation resulted in a significant increase in dispersion for perpendicular insonification, but did not appreciably alter the dispersion for parallel insonification.

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.

Estimating myocardial attenuation from M-mode ultrasonic backscatter.

The objective of this investigation was to determine whether measurements of myocardial attenuation can be obtained from analyses of M-mode images. We exploited the inherent anisotropy of myocardial properties as a means of systematically varying the attenuation to evaluate this M-mode image-based method for myocardial tissue characterization. A commercially available ultrasonic imaging system was used to acquire M-mode images of 24 excised cylindrical specimens from six formalin-fixed sheep hearts that were analyzed using video signal analysis. Data were compensated for the presence of bright intramural myocardial echoes, a potentially significant contributor to uncertainty in measurements of attenuation from backscattered ultrasound. The estimated attenuation coefficient in dB/cm at an effective center frequency of 2.75 MHz as a function of angle of insonification for measurements obtained from analyses of M-mode images is presented. Given a linear frequency-dependence of attenuation in myocardial tissue over frequencies ranging from 1.5 MHz to 8 MHz, as has been previously reported, M-mode image-based analyses were used to estimate the slope of attenuation. Results showed slopes of attenuation (over a -10 dB transmit bandwidth of 1.875 MHz to 3.75 MHz) ranging from 1.00 +/- 0.07 to 1.81 +/- 0.08 dB/(cm.MHz) for perpendicular and parallel insonification, respectively. These values were in good agreement with contemporaneously measured values (0.99 +/- 0.02 to 1.77 +/- 0.04 dB/(cm.MHz)) obtained over a frequency bandwidth of 4 MHz to 7 MHz using a through-transmission radio-frequency-based approach. These data suggest that robust measurements of myocardial attenuation can be obtained from analyses of M-mode images and that this method may be diagnostically feasible in the clinical setting.

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]

Influence of bright intramural echoes on estimates of ultrasonic attenuation from backscattered ultrasound in excised myocardium.

The purpose of this study was to quantitate the influence of bright intramural echoes on estimates of myocardial attenuation from analyses of backscattered ultrasound. To achieve this, M-mode image-based measurements of the inherent anisotropic properties of myocardial attenuation were performed on rotating myocardial specimens. The approach was to use a commercially-available ultrasonic imaging system to acquire M-mode images of 24 excised cylindrical specimens from six formalin-fixed lamb hearts for data analysis using a video signal analysis technique. As a control, through-transmission rf-based measurements were performed concurrently using a pair of focused, single-element ultrasonic transducers. We devised an objective approach to compensate M-mode results for the presence of bright intramural echoes that makes use of the rotational symmetry of the measurements. A comparison of the uncompensated and compensated estimates of attenuation shows that the effect of bright intramural echoes under the conditions of this study increases the average error in M-mode results by approximately 240% compared with that observed when such effects are minimized by compensation. For both uncompensated and compensated M-mode results, increased temporal averaging shows only a modest reduction in average error. These data suggest that for measurements of attenuation from backscattered ultrasound using M-mode images, the effects of bright intramural echoes can be a significant source of error despite increased temporal averaging and therefore may require compensation.