Efficacy of RNA interference knockdown using aerosolized short interfering RNAs bound to nanoparticles in three diverse aphid species.

RNA interference (RNAi) has emerged as a promising method for validating gene function; however, its utility in nonmodel insects has proven problematic, with delivery methods being one of the main obstacles. This study investigates a novel method of RNAi delivery in aphids, the aerosolization of short interfering RNA (siRNA)-nanoparticle complexes. By using nanoparticles as a siRNA carrier, the likelihood of cellular uptake is increased, when compared to methods previously used in insects. To determine the efficacy of this RNAi delivery system, siRNAs were aerosolized with and without nanoparticles in three aphid species: Acyrthosiphon pisum, Aphis glycines and Schizaphis graminum. The genes targeted for knockdown were carotene dehydrogenase (tor), which is important for pigmentation in Ac. pisum, and branched chain-amino acid transaminase (bcat), which is essential in the metabolism of branched-chain amino acids in all three aphid species. Overall, we observed modest gene knockdown of tor in Ac. pisum and moderate gene knockdown of bcat in Ap. glycines along with its associated phenotype. We also determined that the nanoparticle emulsion significantly increased the efficacy of gene knockdown. Overall, these results suggest that the aerosolized siRNA-nanoparticle delivery method is a promising new high-throughput and non-invasive RNAi delivery method in some aphid species.

Additional results for "joint entropy of continuously differentiable ultrasonic waveforms" [J. Acoust. Soc. Am. 133(1), 283-300 (2013)].

Previous results on the use of joint entropy for detection of targeted nanoparticles accumulating in the neovasculature of MDA435 tumors [Fig. 7 of M. S. Hughes et al., J. Acoust. Soc. Am. 133, 283-300 (2013)] are extended, with sensitivity improving by nearly another factor of 2. This result is obtained using a "quasi-optimal" reference waveform in the computation of the joint entropy imaging technique used to image the accumulating nanoparticles.

Joint entropy of continuously differentiable ultrasonic waveforms.

This study is based on an extension of the concept of joint entropy of two random variables to continuous functions, such as backscattered ultrasound. For two continuous random variables, X and Y, the joint probability density p(x,y) is ordinarily a continuous function of x and y that takes on values in a two dimensional region of the real plane. However, in the case where X=f(t) and Y=g(t) are both continuously differentiable functions, X and Y are concentrated exclusively on a curve, γ(t)=(f(t),g(t)), in the x,y plane. This concentration can only be represented using a mathematically "singular" object such as a (Schwartz) distribution. Its use for imaging requires a coarse-graining operation, which is described in this study. Subsequently, removal of the coarse-graining parameter is accomplished using the ergodic theorem. The resulting expression for joint entropy is applied to several data sets, showing the utility of the concept for both materials characterization and detection of targeted liquid nanoparticle ultrasonic contrast agents. In all cases, the sensitivity of these techniques matches or exceeds, sometimes by a factor of two, that demonstrated in previous studies that employed signal energy or alternate entropic quantities.

Thrombin-inhibiting perfluorocarbon nanoparticles provide a novel strategy for the treatment and magnetic resonance imaging of acute thrombosis.

BACKGROUND: As a regulator of the penultimate step in the coagulation cascade, thrombin represents a principal target of direct and specific anticoagulants. OBJECTIVE: A potent thrombin inhibitor complexed with a colloidal nanoparticle was devised as a first-in-class anticoagulant with prolonged and highly localized therapeutic impact conferred by its multivalent thrombin-absorbing particle surface. METHODS: PPACK (Phe[D]-Pro-Arg-Chloromethylketone) was secured covalently to the surface of perfluorocarbon-core nanoparticle structures. PPACK and PPACK nanoparticle inhibition of thrombin were assessed in vitro via thrombin activity against a chromogenic substrate. In vivo antithrombotic activity of PPACK, heparin, non-functionalized nanoparticles and PPACK nanoparticles was assessed through intravenous (i.v.) administration prior to acute photochemical injury of the common carotid artery. Perfluorocarbon particle retention in extracted carotid arteries from injured mice was assessed via (19) F magnetic resonance spectroscopy (MRS) and imaging (MRI) at 11.7 T. Activated partial thromboplastin time (APTT) measurements determined the systemic effects of the PPACK nanoparticles at various times after injection. RESULTS: An optical assay verified that PPACK nanoparticles exceeded PPACK's intrinsic activity against thrombin. Application of an in vivo acute arterial thrombosis model demonstrated that PPACK nanoparticles outperformed both heparin (P=0.001) and uncomplexed PPACK (P = 0.0006) in inhibiting thrombosis. (19) F MRS confirmed that PPACK nanoparticles specifically bound to sites of acute thrombotic injury. APTT normalized within 20 min of PPACK nanoparticles injection. CONCLUSIONS: PPACK nanoparticles present thrombin-inhibiting surfaces at sites of acutely forming thrombi that continue to manifest local clot inhibition even as systemic effects rapidly diminish and thus represent a new platform for localized control of acute thrombosis.

A nanoscale Ti∕GaAs metal-semiconductor hybrid sensor for room temperature light detection.

We report an individually addressable Ti∕GaAs metal-semiconductor hybrid optical nanosensor with positive photoresistance and a sensitivity that increases as the device dimensions shrink. The underlying physics relates to the crossover from ballistic to diffusive transport of the photoinduced carriers and the geometric enhancement of the effect associated with a Schottky-barrier-coupled parallel metal shunt layer. For a 250 nm device under 633 nm illumination we observe a specific detectivity of D(*)=5.06×10(11) cm √Hz∕W with a dynamic response of 40 dB.

Theragnostics for tumor and plaque angiogenesis with perfluorocarbon nanoemulsions.

Molecular imaging agents are extending the potential of noninvasive medical diagnosis from basic gross anatomical descriptions to complicated phenotypic characterizations based upon the recognition of unique cell-surface biochemical signatures. Although originally the purview of nuclear medicine, "molecular imaging" is now studied in conjunction with all clinically relevant imaging modalities. Of the myriad of particles that have emerged as prospective candidates for clinical translation, perfluorocarbon nanoparticles offer great potential for combining targeted imaging with drug delivery, much like the "magic bullet" envisioned by Paul Ehrlich 100 years ago. Perfluorocarbon nanoparticles, once studied in Phase III clinical trials as blood substitutes, have found new life for molecular imaging and drug delivery. The particles have been adapted for use with all clinically relevant modalities and for targeted drug delivery. In particular, their intravascular constraint due to particle size provides a distinct advantage for angiogenesis imaging and antiangiogenesis therapy. As perfluorocarbon nanoparticles have recently entered Phase I clinical study, this review provides a timely focus on the development of this platform technology and its application for angiogenesis-related pathologies.

Real-time calculation of a limiting form of the Renyi entropy applied to detection of subtle changes in scattering architecture.

Previously a new method for ultrasound signal characterization using entropy H(f) was reported, and it was demonstrated that in certain settings, further improvements in signal characterization could be obtained by generalizing to Renyi entropy-based signal characterization I(f)(r) with values of r near 2 (specifically r=1.99) [M. S. Hughes et al., J. Acoust. Soc. Am. 125, 3141-3145 (2009)]. It was speculated that further improvements in sensitivity might be realized at the limit r-->2. At that time, such investigation was not feasible due to excessive computational time required to calculate I(f)(r) near this limit. In this paper, an asymptotic expression for the limiting behavior of I(f)(r) as r-->2 is derived and used to present results analogous to those obtained with I(f)(1.99). Moreover, the limiting form I(f,infinity) is computable directly from the experimentally measured waveform f(t) by an algorithm that is suitable for real-time calculation and implementation.

Application of Renyi entropy for ultrasonic molecular imaging.

Previous work has demonstrated that a signal receiver based on a limiting form of the Shannon entropy is, in certain settings, more sensitive to subtle changes in scattering architecture than conventional energy-based signal receivers [M. S. Hughes et al., J. Acoust. Soc. Am. 121, 3542-3557 (2007)]. In this paper new results are presented demonstrating further improvements in sensitivity using a signal receiver based on the Renyi entropy.

Fibrin-targeted perfluorocarbon nanoparticles for targeted thrombolysis.

BACKGROUND: Reperfusion of the ischemic brain is the most effective therapy for acute stroke, restoring blood flow to threatened tissues. Thrombolytics, such as recombinant tissue plasminogen activator, administered within 3 h of symptom onset can improve neurologic outcome, although the potential for adverse hemorrhagic events limits its use to less than 3% of acute ischemic stroke patients. Targeting of clot-dissolving therapeutics has the potential to decrease the frequency of complications while simultaneously increasing treatment effectiveness, by concentrating the available drug at the desired site and permitting a lower systemic dose. OBJECTIVES: We aimed to develop a fibrin-specific, liquid perfluorocarbon nanoparticle that is surface modified to deliver the plasminogen activator streptokinase. We also aimed to evaluate its effectiveness for targeted thrombolysis in vitro using quantitative acoustic microscopy. METHODS: Human plasma clots were formed in vitro and targeted with streptokinase-loaded nanoparticles, control nanoparticles or a mixture of both. Depending on the treatment group, clots were then exposed to either phosphate-buffered saline (PBS), PBS with plasminogen or PBS with plasminogen and free streptokinase. Spatially registered ultrasound scans were performed at 15-min intervals for 1 h to quantify changes in clot morphology and backscatter. RESULTS: Nanoparticles bound to the clot significantly increased the acoustic contrast of the targeted clot surface, permitting volumetric estimates. Profile plots of detected clot surfaces demonstrated that streptokinase-loaded, fibrin-targeted perfluoro-octylbromide nanoparticles in the presence of plasminogen induced rapid fibrinolysis (<60 min) without concurrent microbubble production and cavitation. Streptokinase-loaded or fibrin-targeted control nanoparticles insonified in PBS did not induce clot lysis. Morphologic changes in the treated group were accompanied by temporal and spatial changes in backscatter. Ultrasound exposure had no effect on the digestion process. Effective concentrations of targeted streptokinase were orders of magnitude lower than equivalently efficacious levels of free drug. Moreover, increasing competitive inhibition of fibrin-bound streptokinase nanoparticles reduced clot lysis in a monotonic fashion. As little as 1% surface targeting of streptokinase nanoparticles produced significant decreases in clot volumes (approximately 30%) in 1 h. CONCLUSION: This new nanoparticle-based thrombolytic agent provides specific and rapid fibrinolysis in vitro and may have a clinical role in early reperfusion during acute ischemic stroke.

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.

Occult cardiac contractile dysfunction in dystrophin-deficient children revealed by cardiac magnetic resonance strain imaging.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an inherited disease characterized by early onset of skeletal muscle degeneration and progressive weakness. Although dilated cardiomyopathy may occur during adolescence, it is often undetected early in its course because of physical inactivity and generalized debilitation. The purpose of this study was to apply the technique of cardiac magnetic resonance (CMR) tagging to detect occult cardiac dysfunction in young subjects with DMD by measuring myocardial strain and torsion. METHODS AND RESULTS: Thirteen DMD pediatric subjects without clinically apparent heart disease and 9 age-matched healthy males were recruited. Each was scanned on a 1.5-T clinical scanner to acquire contiguous short-axis planes from the apex to the mitral valve plane and then 3 tagged images at base, midventricle, and apex. Global and segmental myocardial net twist and circumferential strain were computed with the use of 2D homogeneous strain analysis. Ventricular torsion was computed by normalizing net twist by the distance from apex to mitral valve plane. DMD patients exhibited normal left ventricular volumes and ejection fractions but manifested reduced midventricular and basal cross-sectional global circumferential strain compared with the reference group (P<0.005). These alterations also appeared in segmental analyses in the septal, anterior, lateral, and inferior walls (P<0.05). CONCLUSIONS: In patients predisposed to cardiomyopathies because of dystrophinopathy, occult regional cardiac dysfunction can be diagnosed with CMR tagging. This method of strain imaging analysis may offer a sensitive approach for delineating the presence and progression of cardiovascular disease and for assessing therapies designed to modulate the onset and course of heart failure.

Echocardiographic characterization of fundamental mechanisms of abnormal diastolic filling in diabetic rats with a parameterized diastolic filling formalism.

Abnormalities of diastolic function (DF) precede systolic dysfunction in diabetic cardiomyopathy. Transmitral Doppler flow analysis is the primary method for noninvasively assessing DF. We used model-based Doppler E-wave analysis to evaluate diastolic function differences between normal and diabetic rat hearts. Control rats and those with diabetes underwent echocardiography with analysis by traditional Doppler indexes and by the parameterized diastolic filling (PDF) formalism, generating 3 parameters, x0, c, and k, that uniquely characterize each E-wave. Significant intergroup differences in the E/A ratios (P <.01), isovolumic relaxation times (P <.01), and the modeling parameter c (P <.05) were found. There were no significant differences in shortening fraction, deceleration time, myocardial collagen content, or the parameters x0 and k between diabetic and control rats. These results indicate that differences in diastolic function may be noninvasively quantified and that diabetic hearts may exhibit defects in uncoupling of the contractile apparatus without concomitant increases in chamber stiffness.

Temperature dependence of ultrasonic enhancement with a site-targeted contrast agent.

Molecular imaging contrast agents specifically detect the biochemical "signatures" of disease before anatomical manifestations are apparent. Sensitive and specific localization of fibrin both in vivo and in vitro has been demonstrated with the use of a ligand-directed liquid perfluorocarbon nanoparticle. Since the acoustic properties of perfluorocarbons are known to vary with temperature, it was hypothesized that temperature could be used to augment the magnitude of enhancement imparted by targeted nanoparticles. Accordingly, the acoustic backscatter of two different substrates, nitrocellulose membrane and human plasma clot, targeted by the nanoparticles was measured at temperatures ranging from 27 degrees to 47 degrees C in 5 degrees C increments. Classic avidin-biotin interactions were utilized to couple biotinylated nanoparticles to avidin-conjugated nitrocellulose membranes. Ultrasonic contrast enhancement of the nitrocellulose membrane at 25 MHz, measured by acoustic microscopy, increased from 2.0+/-0.3 dB at 27 degrees C to 3.7+/-0.4 at 47 degrees C. In a similar experiment, antifibrin nanoparticles bound to human plasma clots also exhibited temperature-dependent ultrasonic signal enhancement ranging from 13.9+/-1.5 dB at 27 degrees C to 18.1+/-1.5 dB at 47 degrees C. The increase in ultrasonic contrast enhancement measured was well described by a simple, acoustic transmission line model with temperature-dependent impedance. These results suggest that temperature-dependent changes in acoustic backscatter may be used to further differentiate tissues targeted with site-specific nanoparticles from surrounding normal soft tissues.

Targeted ultrasonic contrast agents for molecular imaging and therapy.

Targeted contrast agents are expanding the detectability and diagnosis of pathology from a strict anatomic to biochemical basis. Moreover, these new agents, in their various forms, offer the potential for site-specific drug and gene delivery, ie, the "magic bullet" first postulated by Paul Erhlich 100 years ago. The ability to direct drugs to the molecular signatures of disease, to confirm noninvasively their presence at the site-of-interest, and to quantify the adequacy of local drug concentration at the time of treatment, ie, rational targeted drug delivery, offers exciting new clinical paradigms in the near future.

Novel MRI contrast agent for molecular imaging of fibrin: implications for detecting vulnerable plaques.

BACKGROUND: Molecular imaging of thrombus within fissures of vulnerable atherosclerotic plaques requires sensitive detection of a robust thrombus-specific contrast agent. In this study, we report the development and characterization of a novel ligand-targeted paramagnetic molecular imaging agent with high avidity for fibrin and the potential to sensitively detect active vulnerable plaques. METHODS AND RESULTS: The nanoparticles were formulated with 2.5 to 50 mol% Gd-DTPA-BOA, which corresponds to >50 000 Gd(3+) atoms/particle. Paramagnetic nanoparticles were characterized in vitro and evaluated in vivo. In contradistinction to traditional blood-pool agents, T1 relaxation rate as a function of paramagnetic nanoparticle number was increased monotonically with Gd-DTPA concentration from 0.18 mL. s(-1). pmol(-1) (10% Gd-DTPA nanoparticles) to 0.54 mL. s(-1). pmol(-1) for the 40 mol% Gd-DTPA formulations. Fibrin clots targeted in vitro with paramagnetic nanoparticles presented a highly detectable, homogeneous T1-weighted contrast enhancement that improved with increasing gadolinium level (0, 2.5, and 20 mol% Gd). Higher-resolution scans and scanning electron microscopy revealed that the nanoparticles were present as a thin layer over the clot surface. In vivo contrast enhancement under open-circulation conditions was assessed in dogs. The contrast-to-noise ratio between the targeted clot (20 mol% Gd-DTPA nanoparticles) and blood was approximately 118+/-21, and that between the targeted clot and the control clot was 131+/-37. CONCLUSIONS: These results suggest that molecular imaging of fibrin-targeted paramagnetic nanoparticles can provide sensitive detection and localization of fibrin and may allow early, direct identification of vulnerable plaques, leading to early therapeutic decisions.

Age-related alterations of cardiac tissue microstructure and material properties in Fischer 344 rats.

The cardiac aging process is accompanied by global mechanical dysfunction that reflects increased myocardial stiffness. Accordingly, age-related changes in microscopic material properties of myocardium were delineated with high-frequency ultrasound (US) (30 to 44 MHz) tissue characterization methods for aging Fischer 344 rats at 6 (adult), 18 (aged), and 24 (senescent) months of age. The excised lateral wall of the left ventricle of rats (n = 10 per group) was insonified with a 50-MHz acoustic microscope for determination of integrated backscatter, backscatter coefficient and attenuation coefficient. Histological and biochemical analyses for collagen content and cardiac myocyte diameter were performed. Collagen concentration increased progressively with age, with the greatest increments occurring from 6 to 18 months (38.0 +/- 6.3 to 53.0 +/- 7.1 mg/g dry wt), and leveling off at 24 months (60.0 +/- 7.4 mg/g dry wt). Tissue microscopic material properties also changed progressively from 6 to 24 months of age, as determined by US methods: integrated backscatter increased (-44.7 +/- 1.8 vs. -40.8 +/- 1.9 dB, p < 0.05), attenuation increased (47.1 +/- 5.9 to 65.3 +/- 7.8 dB/cm, p < 0.05), and the backscatter coefficient increased (0.73 +/- 0.16 x 10(-5) to 3.76 +/- 1.6 x 10(-5) cm(-1), p < 0.05), from 6 to 24 months of age in each case. Age-related alterations in indices of cardiac microscopic material properties were closely correlated with the changes in cardiac microstructure. Ultrasonic tissue characterization may prove to be a sensitive tool to monitor changes in the cardiac microstructure, such as increased collagen deposition, that occur within age-related diastolic dysfunction.

Can infections be imaged in implanted devices?

Approximately 30,000 to 60,000 patients per year in the United States are candidates for heart transplants, mechanical assist devices, or both. These procedures, devices, and the associated short- and long-term care required are expensive and command significant utilization of health care resources. Because device related infection and thrombosis are potentially devastating complications of implanted device utilization, early diagnosis of infection, thrombosis, or both, would be helpful for initiation of early therapy to prevent untoward clinical events. Therefore, the development of a robust imaging technology for identification of infection could be cost effective if early assessment, diagnosis, and therapy of infected devices led to improvements in morbidity and mortality.

High-resolution MRI characterization of human thrombus using a novel fibrin-targeted paramagnetic nanoparticle contrast agent.

In this study, the sensitivity of a novel fibrin-targeted contrast agent for fibrin detection was defined in vitro on human thrombus. The contrast agent was a lipid-encapsulated perfluorocarbon nanoparticle with numerous Gd-DTPA complexes incorporated into the outer surface. After binding to fibrin clots, scanning electron microscopy of treated clots revealed dense accumulation of nanoparticles on the clot surfaces. Fibrin clots with sizes ranging from 0.5-7.0 mm were imaged at 4.7 T with or without treatment with the targeted contrast agent. Regardless of sizes, untreated clots were not detectable by T(1)-weighted MRI, while targeted contrast agent dramatically improved the detectability of all clots. Decreases in T(1) and T(2) relaxation times (20-40%) were measured relative to the surrounding media and the control clots. These results suggest the potential for sensitive and specific detection of microthrombi that form on the intimal surfaces of unstable atherosclerotic plaque.