Antonio Greco

Effectiveness of Functionalized Nanosystems for Multimodal Molecular Sensing and Imaging in Medicine

Successful employment of multimodal molecular imaging for cancer targeting entails the development of safe nanoparticle contrast agents (NPCAs), detects at least by two nonionizing imaging techniques. This paper presents a quantitative assessment of the effectiveness of both pure silica nanospheres (SiNSs) and composite silica/superparamagnetic NPCAs as scatterers for low-frequency diagnostic ultrasound (US) (3 MHz) in very low range of concentrations (1.5–5 mg/mL). Iron oxide (IO) and FePt-IO nanocrystals are employed for SiNS magnetic coating. Different samples of NPCA-containing agarose gel are US imaged through a commercially available system and acquired data are processed through a dedicated prototypal platform to extract image backscatter information and perform evaluation of the image gray level. The pure silica NPCAs confirms recent reports for higher concentrations at higher frequencies. The FePt-IO-coated NPCAs show similar behavior, although with lower values of image backscatter, with a marked effectiveness peak for 330-nm SiNSs, particularly useful for tumor targeting purposes. Finally, the IO-coated SiNSs presented a marked lowering of US enhancement potential and a peak efficiency for a particle diameter of 660 nm. The extent of US backscatter reduction is found to be a function of the number of magnetic nanoparticles per mL of NPCA-containing gel and decreased with increasing NPCA concentrations. These results broadened our knowledge of dual-mode molecular imaging of deep tumors, employing US, and magnetic resonance techniques for the accurate, safe and early detection of cancer cells located in internal organs.

Multiparametric Evaluation of the Acoustic Behavior of Halloysite Nanotubes for Medical Echographic Image Enhancement

Halloysite nanotubes (HNTs) are nanomaterials composed of double layered aluminosilicate minerals characterized by a wide range of medical applications. Nonetheless, systematic investigations of their imaging potential are still poorly documented. This paper shows a parametric assessment of the effectiveness of HNTs as scatterers for safe ultrasound (US)-based molecular imaging. Quantitative evaluation of average signal enhancement produced by HNTs with varying set up configuration was performed. The influence of different levels of power (20%, 50%, and 80%) of the signal emitted by clinical equipment was determined, to assess the efficacy of different HNT concentrations (1.5, 3, and 5 mg/mL) at conventional ultrasonic frequencies (5.7-7 MHz), even in case of specific limitation regarding US mechanical interaction with target tissues. Different samples of HNT containing agarose gel were imaged through a commercially available echographic system and acquired data were processed through a dedicated prototypal platform to extract the average ultrasonic signal amplitude. The rate of signal enhancement achieved by different concentration values was quantified and the contribution of frequency increment was separately evaluated. Despite influencing the level of mechanical excitation on HNTs and tissues, our results demonstrated how increasing the power of the emitted signal negatively affected the measured backscatter. Conversely, noticeable improvements in signal backscatter could be achieved incrementing HNT concentration and the echographic frequency employed; specifically the signal enhancement over the used concentration range could be improved by averagely 20%, corresponding to 4.86 ± 0.80 (a.u.), when employing the higher value of echographic frequency.

An Innovative Ultrasound Signal Processing Technique to Selectively Detect Nanosized Contrast Agents in Echographic Images

The aim of this paper was to optimize the employment of a novel algorithm for acquisition and processing of medical ultrasound (US) signals to facilitate its clinical translation. The implemented procedure is dedicated to selective enhancement of nanoparticle (NP) contrast agents in echographic images and is based on the differences in US signal backscatter between NP-containing targets and more homogeneous objects. Previous preliminary studies verified the feasibility of this approach on silica nanospheres (SiNSs) dispersed at a constant volume concentration (0.7%) in agarose gel samples. The present extended these evaluations, addressing two issues of direct clinical interest: 1) safety: SiNSs were coated with a biocompatible layer made of polyethylene glycol (PEG) and the adopted NP volume concentration was reduced to 0.2%, which is in the nontoxic range and 2) reproducibility: a different phantom configuration was used, to verify the independence of algorithm performance from a specific target region shape. The obtained results demonstrated that the proposed method can be effectively applied to enhance the presence of PEG-coated SiNSs in the diameter range 160-660 nm at a low and biocompatible volume concentration: the combined employment of a phantom with a different geometry and a lower concentration of PEG-coated NPs, in fact, caused only slight variations in the suppression patterns of noncontrast echoes, without affecting the final diagnostic effectiveness of the investigated contrast detection scheme. This approach also provides specific advantages with respect to the available measurement techniques dedicated to the enhancement of targeted US contrast agents for molecular imaging purposes.

Nanocomposites for multimodal molecular imaging

Early and accurate diagnosis of tumors requires the combined adoption of different imaging modalities with molecular sensitivity. A successful employment of multimodal molecular imaging is related to the development of smart fully-biodegradable nanoparticle contrast agents (NPCAs), detectable by at least two non-ionizing imaging techniques and suitably sized for tumor targeting. After a short overview of recent findings obtained by our research group in the development and characterization of novel NPCAs, this paper shows for the first time a quantitative assessment of the effectiveness of both a pure silica NPCA and a composite silica/superparamagnetic NPCA as scatterers of low-frequency diagnostic ultrasound (3 MHz) in very low volume concentrations (0.1–0.2%). The pure silica NPCA confirmed the behavior recently reported for higher concentrations at higher frequencies. The composite NPCA followed the same behavior, showing a marked effectiveness peak for a particle diameter of 330 nm, which represents a particularly useful size for tumor targeting purposes. These results open new exciting perspectives for dual-mode molecular imaging of deep tumors, combining ultrasound and magnetic resonance techniques for the accurate, safe and early detection of cancer cells located in internal organs.

A novel dual-frequency method for selective ultrasound imaging of targeted nanoparticles

Current methods for ultrasound (US) molecular imaging suffer the lack of image processing techniques specifically designed to identify the newer nanosized contrast agents (CAs). The available pulse sequences and signal analysis methods for US contrast detection, in fact, were developed for the older microbubble CAs, whose acoustic properties differ significantly from those of nanoparticles. This work illustrates the implementation and experimental testing of a new contrast detection scheme, tailored to enhance the contribution of solid nanosized CAs in echographic images. The proposed protocol, including a novel pulse sequence and a two-step image processing algorithm, was evaluated on a phantom consisting of silica nanospheres dispersed into an agarose gel matrix that was imaged through a conventional echographic transducer. Obtained results demonstrated the capability of selectively suppressing non-contrast echoes, without any loss in spatial resolution and maintaining the characteristics of real-time imaging, therefore showing very promising perspectives for clinical applications.

OP0003 Fracture Risk Prediction: Comparative Evaluation of Ultrasound-Based Fragility Score and DXA-Measured BMD Against Frax®

Background A high percentage of fragility fractures occur in subjects showing low bone strength in presence of a normal bone mineral density (BMD). Consequently, the approach to bone health assessment and fracture prevention is gradually changing, moving the focus from the identification of osteoporotic patients (based on BMD thresholds) towards the detection of individuals at high risk of fracture. Currently, the only validated tool that provides a precise quantification of the osteoporotic fracture risk is FRAX®, which is based on age, sex, body mass index (BMI) and a series of clinical risk factors, taking into account epidemiological data that are specific for patient country. FRAX® calculation can also include femoral neck BMD measured by dual X-ray absorptiometry (DXA), since it has been shown that integration of BMD and clinical risk factors provides improved fracture predictions. Nevertheless, DXA availability is limited by the typical issues related to ionizing radiation employment (high costs, need of dedicated structures with certified operators, long-term safety risks). Objectives Aim of this work was to compare the performance of DXA-measured BMD and a novel ultrasound (US) parameter measured on lumbar spine in the estimation of osteoporotic fracture risk as calculated by FRAX®. Methods 80 female patients [40-80 years; BMI (body mass index) ≤30 kg/m2] were enrolled for the study. Each of them answered the FRAX® questionnaire and underwent the following diagnostic examinations: a conventional DXA investigation of lumbar spine and proximal femur (Hologic Discovery) and an abdominal US scan of lumbar spine. US data were analyzed by an innovative algorithm that processed both echographic images and “raw” radiofrequency (RF) signals, providing as final output a new parameter named Fragility Score (F.S.), which quantifies bone strength. For each patient, FRAX®10-year probability of a major osteoporotic fracture was calculated both with and without femoral neck BMD inclusion. Pearson coefficient (r) was used to compare the performance of F.S. and BMD values in the estimation of fracture risk provided by FRAX®. Results Fracture risk provided by FRAX® with femoral BMD included in the calculation showed a good correlation with F.S. (r =0.70, p<0.001) and, as expected, with femoral neck BMD (r = -0.72, p<0.001), while the correlation was weaker for lumbar BMD (r = -0.44, p<0.001). Furthermore, F.S. was the only considered diagnostic parameter that kept an appreciable correlation with FRAX® predictions even when their calculation did not include femoral BMD (r =0.52 for F.S., r = -0.21 for femoral BMD, r = -0.07 for lumbar BMD; p<0.001 for all). Conclusions The proposed US-based F.S. alone showed a good correlation with osteoporotic fracture risk calculated by FRAX® integrated with femoral neck BMD, which represents the most reliable value. The performance of F.S. in fracture risk estimation was significantly better than lumbar BMD and comparable with femoral BMD. Therefore, F.S. is a suitable candidate to be employed for fracture risk prediction in primary healthcare settings. Acknowledgements This work was partially funded by FESR P.O. Apulia Region 2007-2013 – Action 1.2.4, grant n. 3Q5AX31 (ECHOLIGHT Project) Disclosure of Interest None declared

Ex-vivo measurements of quantitative ultrasound and micro-CT parameters on intact human femoral heads

The purpose of this work was to study the relationships between quantitative ultrasound (QUS) parameters and the microstructure properties of human proximal femur samples. QUS data acquisition was achieved by means of a custom-developed experimental set-up, which allowed the insonification of excised femoral heads along 30 different directions, each time including both the trabecular region and the cortical layer in their physiologic morphological configuration. Two QUS parameters, Integrated Reflection Coefficient (IRC) and Apparent Integrated Backscatter (AIB), were measured by means of both single-element transducers at two different frequencies (2.25 MHz and 3.5 MHz) and a clinically-available 128-element convex probe. The obtained data were compared with local structural properties of the bone samples as quantified by high-resolution micro-computed tomography (micro-CT). The corresponding results showed a strong correlation between trabecular bone volume fraction and AIB (|r| up to 0.81) and an appreciable linear correlation between cortical bone density and IRC (|r| up to 0.59). QUS parameter values measured by single-element transducers were optimally reproduced when the clinically-available probe was employed. This provides the proposed approach with an interesting potential for a prompt clinical translation as a possible new tool for osteoporosis diagnosis, especially considering that the insonification of the whole femoral head was performed in its physiological shape with all its components (cartilage, cortical layer, trabecular region).

Experimental assessment of gold nanorods for optoacoustic imaging in a tissue-mimicking phantom

Plasmon-resonant gold nanorods (AuNRs) are of great interest for optoacoustic imaging, due to their capacity of generating ultrasound (US) signals when irradiated by Near Infrared light. In fact, detection of emitted acoustic waves could lead to micron-scale resolution imaging and early diagnosis of tumor masses. Nonetheless, until now, no relevant studies have assessed the optoacoustic behavior of AuNRs under repeatable experimental conditions. A new experimental set-up including a novel custom-designed tissue-mimicking phantom was developed for this study, in order to quantify the contribution of independent parameters to the optoacoustic signal (OAS) produced by AuNRs. Analysis of the OAS recorded by 2 US probes demonstrated a direct proportionality between signal amplitude and both AuNR concentration and laser intensity. Moreover, the optimal and maximum duration of laser exposure were determined through a quantitative analysis of progressive degradation of AuNRs under irradiation.

Automatic ultrasound technique to measure angle of progression during labor

To evaluate the accuracy and reliability of an automatic ultrasound technique for assessment of the angle of progression (AoP) during labor.

A new ultrasound parameter for osteoporosis diagnosis: Clinical validation on normal- and under-weight women

Aim of this work was to evaluate the effectiveness of a recently introduced ultrasound (US) method for osteoporosis diagnosis, when extensively used in a clinical context to investigate adult women of variable age. A total of 384 female patients (46-65 years; body mass index <; 25 kg/m2) underwent a spinal dual X-ray absorptiometry (DXA) and an abdominal US scan of lumbar spine, acquiring both echographic images and unprocessed radiofrequency signals. US data were analyzed through a new fully automatic algorithm, which performed a series of spectral and statistical analyses to calculate the parameter called Osteoporosis Score (O.S.). Diagnostic effectiveness of O.S. was assessed through a direct comparison with DXA measurements (assumed as the gold standard reference), quantifying the agreement between the two methods through accuracy calculation, Cohens kappa (k) and Pearson correlation coefficient (r). The overall accuracy of O.S.-based diagnoses resulted 84.6%, ranging from a minimum of 81.7% for the oldest patients (aged in 61-65 y) to a maximum of 87.2% for the youngest patients (aged in 46-50 y). Cohens kappa showed an analogous trend, confirming a significant agreement between DXA and US-based diagnoses along the whole considered age interval (k=0.758, p<;0.0001). A good correlation was also found between O.S.-derived BMD values and corresponding DXA measurements (r=0.72, p<;0.001). These results demonstrated that US-measured O.S. is significantly correlated with spinal BMD in normal- and under-weight adult women belonging to a wide age interval. Therefore, the routine clinical application of this innovative approach to osteoporosis diagnosis can be envisioned.