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The finite difference time domain (FDTD) method is widely used as a computational tool for development, validation, and optimization of emerging microwave breast cancer detection and treatment techniques. When expressed in terms of Debye parameters, dispersive breast tissue dielectric properties can be efficiently incorporated into FDTD codes. Previously, we experimentally characterized the dielectric properties of a large number of excised normal and malignant breast tissue samples from 0.5 to 20 GHz. We subdivided the large database of normal tissue data into three groups based on the percent adipose tissue present in a particular sample. In addition, we formed a group of all cancer samples that contained at least 30% malignant tissue. We summarized the data using one-pole Cole-Cole models that were rigorously fit to the median dielectric properties of the three normal tissue groups and one malignant tissue group. In this letter, we present computationally simpler one- and two-pole Debye models that retain the high accuracy of the Cole-Cole models. Model parameters are derived for two sets of frequency ranges: the entire measurement frequency range from 0.5 to 20 GHz, and the 3.1-10.6 GHz FCC band allocated for ultrawideband medical applications. The proposed Debye models provide a means for creating computationally efficient FDTD breast models with realistic wideband dielectric properties derived from the largest and most comprehensive experimental study conducted to date on human breast tissue. [ABSTRACT FROM PUBLISHER]

Introduction: Lipid nanoparticles (LNPs) are one of the most clinically advanced candidates for delivering nucleic acids to target cell populations, such as hepatocytes. Once LNPs are endocytosed, they must release their nucleic acid cargo into the cell cytoplasm. For delivering messenger RNA (mRNA), delivery into the cytosol is sufficient; however, for delivering DNA, there is an added diffusional barrier needed to facilitate nuclear uptake for transcription and therapeutic effect., Method: Here, we use fluorescence microscopy to investigate the intracellular fate of different LNP formulations to determine the kinetics of localization to endosomes and lysosomes. LNPs used in the studies were prepared via self-assembly using a NanoAssemblr for microfluidic mixing. As the content of polyethylene glycol (PEG) within the LNP formulation influences cellular uptake by hepatocyte cells, the content and hydrocarbon chain length within the formulation were assessed for their impact on intracellular trafficking. Standard LNPs were then formed using three commercially available ionizable lipids, Dlin-MC3-DMA (MC3), Dlin-KC2-DMA (KC2), and SS-OP. Plasmid DNA (pDNA) and mRNA were used, more specifically with a mixture of Cyanine 3 (Cy3)-labeled and green fluorescence protein (GFP) producing plasmid DNA (pDNA) as well as Cy5-labeled GFP producing mRNA. After formulation, LNPs were characterized for the encapsulation efficiency of the nucleic acid, hydrodynamic diameter, polydispersity, and zeta potential. All standard LNPs were ~100 nm in diameter and had neutral surface charge. All LNPs resulted in encapsulation efficiency greater than 70%. Confocal fluorescence microscopy was used for the intracellular trafficking studies, where LNPs were incubated with HuH-7 hepatocyte cells at times ranging from 0-48 h. The cells were antibody-stained for subcellular components, including nuclei, endosomes, and lysosomes., Result: Analysis was performed to quantify localization of pDNA to the endosomes and lysosomes. LNPs with 1.5 mol% PEG and a hydrocarbon chain C14 resulted in optimal endosomal escape and GFP production. Results from this study demonstrate that a higher percentage of C14 PEG leads to smaller LNPs with limited available phospholipid binding area for ApoE, resulting in decreased cellular uptake. We observed differences in the localization kinetics depending on the LNP formulation type for SS-OP, KC2, and MC3 ionizable lipids. The results also demonstrate the technique across different nucleic acid types, where mRNA resulted in more rapid and uniform GFP production compared to pDNA delivery., Conclusion: Here, we demonstrated the ability to track uptake and the sub-cellular fate of LNPs containing pDNA and mRNA, enabling improved screening prior to in vivo studies which would aid in formulation optimization., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Design of safe and effective synthetic nucleic acid delivery vectors such as polycation/DNA or polycation/siRNA complexes (polyplexes) will be facilitated by quantitative understanding of the mechanisms by which such materials escort cargo from the cell surface to the nucleus. In particular, the mechanisms of cellular internalization by various endocytosis pathways and subsequent endocytic vesicle trafficking have been shown to strongly affect nucleic acid delivery efficiency. Fluorescence microscopy and subcellular fractionation methods are commonly employed to follow intracellular trafficking of biomolecules and nanoparticulate delivery systems such as polyplexes. However, it is difficult to obtain quantitative data from microscopy and subcellular fractionation is experimentally difficult and low throughput. We have developed a method for quantifying the transport of polyplexes through important endocytic vesicles. The method is based on polymerization of 3,3'-diaminobenzidine by endocytosed horseradish peroxidase, causing an increase in the vesicle density, resistance to being solubilized by detergent and quenching of fluorophores within the vesicles, which makes them easy to separate and quantify. Using this method in HeLa cells, we have observed polyethylenimine/siRNA polyplexes initially appearing in early endosomes and rapidly moving to other compartments within 30min post-transfection. At the same time, we observed the kinetics of accumulation of the polyplexes in lysosomes at a similar rate. The results from the new method are consistent with similar measurements by confocal fluorescence microscopy and subcellular fractionation of endocytic vesicles on a Percoll gradient. The relative ease of this new method will aid investigation of gene delivery mechanisms by providing the means to rapidly quantify endocytic trafficking of polyplexes and other vectors., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose: siRNA may be delivered as electrostatic complexes with cationic lipids (lipoplexes) or polycations (polyplexes). The purpose of this project was to determine the effect of cellular internalization mechanism(s) on siRNA-mediated gene silencing efficiency., Methods: Lipoplexes were formed comprising siRNA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol and dioleoyl phosphatidylethanolamine (DOPE), and polyplexes comprised siRNA with polyethylenimine (PEI). During transfections, specific uptake mechanisms were inhibited by pharmacological agents and RNAi-mediated knockdown of proteins involved in various endocytosis pathways. Confocal fluorescence microscopy further elucidated the predominant endocytic pathways of siRNA delivery via colocalization of vectors with endocytic vesicle markers., Results: Inhibition of macropinocytosis (MP), caveolin-mediated endocytosis (CvME), flotillin-mediated endocytosis (FME) and knockdown of ARF6 significantly decreased PEI/siRNA-mediated gene silencing. Inhibition of endocytosis pathways, however, had negligible effect on lipoplex uptake and gene silencing mediated by lipoplexes. Rather, internalization of lipoplexes and subsequent siRNA-mediated gene silencing occurred via an energy-independent process., Conclusions: MP, CvME and FME, but not the acidified clathrin-mediated pathway, lead to effective gene silencing by PEI/siRNA polyplexes. Lipoplexes, in contrast, deliver siRNA primarily by direct fusion of the liposomal and cellular membranes. These results provide a new understanding of the mechanisms of siRNA delivery materials in HeLa cells and may aid in design of more effective RNAi strategies.

Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Although there are several hazards for patients with implanted pacemakers and defibrillators in the magnetic resonance imaging (MRI) environment, evaluation of lead electrode heating is the most complex because of the many influencing variables: patient size, anatomy, body composition, patient position in the bore, scan sequence (radiofrequency power level), lead routing, and lead design. Although clinical studies are an important step in demonstrating efficacy, demonstrating safety through clinical trials alone is not practical because of this complexity., Objective: The purpose of this study was to develop a comprehensive modeling framework to predict the probability of pacing capture threshold (PCT) change due to lead electrode heating in the MRI environment and thus provide a robust safety evaluation., Methods: The lead heating risk was assessed via PCT change because this parameter is the most clinically relevant measure of lead heating. The probability for PCT change was obtained by combining the prediction for power at the electrode-tissue interface obtained via simulations with a prediction for PCT change as a function of radiofrequency power obtained via an in vivo canine study., Results: The human modeling framework predicted that the probability of a 0.5-V PCT change due to an MRI scan for the Medtronic CapSureFix MRI SureScan model 5086 MRI leads is <1/70,000 for chest scans and <1/10,000,000 for either head scans or lower torso scans., Conclusion: The framework efficiently models millions of combinations, delivering a robust evaluation of the lead electrode heating hazard. This modeling approach provides a comprehensive safety evaluation that is impossible to achieve using phantom testing, animal studies, or clinical trials alone., (© 2013 Heart Rhythm Society Published by Heart Rhythm Society All rights reserved.)

Tissue engineering strategies for the intervertebral disc (IVD) have traditionally focused either on the annulus fibrosus (AF) or the nucleus pulposus (NP) in isolation, or have simply compared AF cells and NP cells in identical culture conditions. Recently, others in the field have become aware of the advantage of combining the AF and NP into a more comprehensive strategy to address IVD tissue engineering, and have introduced biomimetic approaches to either AF or NP tissue engineering. Here, we introduced a new method for developing a biomimetic, cell-seeded IVD by electrospinning circumferentially-orientated polycaprolactone fibres (AF analogue), seeding them with cells (porcine chondrocytes) and then gelling a cell-agarose solution in the centre (NP analogue). Scanning electron microscopy images demonstrated a high degree of fibre alignment and, along with fluorescent actin staining, confirmed a preferred orientation of cells in the direction of the fibres. Viability assays and histology collectively demonstrated that cells were viable and well-distributed around the interface between the NP and AF regions. In addition, mechanical testing confirmed that the composite IVD scaffolds had higher moduli than the agarose hydrogels alone. As we enter the new decade and the fields of AF and NP tissue engineering begin to merge into a new interfacial and functional IVD tissue-engineering field, approaches such as the method presented here will serve as the foundation for continuously advancing technology that we ultimately endeavour to bring to the clinic for the treatment of patients severely afflicted by degenerative disc disease., (Copyright © 2011 John Wiley & Sons, Ltd.)

Techniques utilizing electromagnetic energy at microwave and optical frequencies have been shown to be promising for breast cancer detection and diagnosis. Since different biophysical mechanisms are exploited at these frequencies to discriminate between healthy and diseased tissue, combining these two modalities may result in a more powerful approach for breast cancer detection and diagnosis. Toward this end, we performed microwave dielectric spectroscopy and optical diffuse reflectance spectroscopy measurements at the same sites on freshly excised normal breast tissues obtained from reduction surgeries at the University of Wisconsin Hospital, using microwave and optical probes with very similar sensing volumes. We found that the microwave dielectric constant and effective conductivity are correlated with tissue composition across the entire measurement frequency range (|r| approximately 0.5-0.6, p<0.01) and that the optical absorption coefficient at 460 nm and optical scattering coefficient are correlated with tissue composition (|r| approximately 0.4-0.6, p<0.02). Finally, we found that the optical absorption coefficient at 460 nm is correlated with the microwave dielectric constant and effective conductivity (r=-0.55, p<0.01). Our results suggest that combining optical and microwave modalities for analyzing breast tissue samples may serve as a crosscheck and provide complementary information about tissue composition.

The development of microwave breast cancer detection and treatment techniques has been driven by reports of substantial contrast in the dielectric properties of malignant and normal breast tissues. However, definitive knowledge of the dielectric properties of normal and diseased breast tissues at microwave frequencies has been limited by gaps and discrepancies across previously published studies. To address these issues, we conducted a large-scale study to experimentally determine the ultrawideband microwave dielectric properties of a variety of normal, malignant and benign breast tissues, measured from 0.5 to 20 GHz using a precision open-ended coaxial probe. Previously, we reported the dielectric properties of normal breast tissue samples obtained from reduction surgeries. Here, we report the dielectric properties of normal (adipose, glandular and fibroconnective), malignant (invasive and non-invasive ductal and lobular carcinomas) and benign (fibroadenomas and cysts) breast tissue samples obtained from cancer surgeries. We fit a one-pole Cole-Cole model to the complex permittivity data set of each characterized sample. Our analyses show that the contrast in the microwave-frequency dielectric properties between malignant and normal adipose-dominated tissues in the breast is considerable, as large as 10:1, while the contrast in the microwave-frequency dielectric properties between malignant and normal glandular/fibroconnective tissues in the breast is no more than about 10%.

Hepatic malignancies have historically been treated with surgical resection. Due to the shortcomings of this technique, there is interest in other, less invasive, treatment modalities, such as microwave hepatic ablation. Crucial to the development of this technique is the accurate knowledge of the dielectric properties of human liver tissue at microwave frequencies. To this end, we characterized the dielectric properties of in vivo and ex vivo normal, malignant and cirrhotic human liver tissues from 0.5 to 20 GHz. Analysis of our data at 915 MHz and 2.45 GHz indicates that the dielectric properties of ex vivo malignant liver tissue are 19 to 30% higher than normal tissue. The differences in the dielectric properties of in vivo malignant and normal liver tissue are not statistically significant (with the exception of effective conductivity at 915 MHz, where malignant tissue properties are 16% higher than normal). Also, the dielectric properties of in vivo normal liver tissue at 915 MHz and 2.45 GHz are 16 to 43% higher than ex vivo. No statistically significant differences were found between the dielectric properties of in vivo and ex vivo malignant tissue (with the exception of effective conductivity at 915 MHz, where malignant tissue properties are 28% higher than normal). We report the one-pole Cole-Cole parameters for ex vivo normal, malignant and cirrhotic liver tissue in this frequency range. We observe that wideband dielectric properties of in vivo liver tissue are different from the wideband dielectric properties of ex vivo liver tissue, and that the in vivo data cannot be represented in terms of a Cole-Cole model. Further work is needed to uncover the mechanisms responsible for the observed wideband trends in the in vivo liver data.

The efficacy of emerging microwave breast cancer detection and treatment techniques will depend, in part, on the dielectric properties of normal breast tissue. However, knowledge of these properties at microwave frequencies has been limited due to gaps and discrepancies in previously reported small-scale studies. To address these issues, we experimentally characterized the wideband microwave-frequency dielectric properties of a large number of normal breast tissue samples obtained from breast reduction surgeries at the University of Wisconsin and University of Calgary hospitals. The dielectric spectroscopy measurements were conducted from 0.5 to 20 GHz using a precision open-ended coaxial probe. The tissue composition within the probe's sensing region was quantified in terms of percentages of adipose, fibroconnective and glandular tissues. We fit a one-pole Cole-Cole model to the complex permittivity data set obtained for each sample and determined median Cole-Cole parameters for three groups of normal breast tissues, categorized by adipose tissue content (0-30%, 31-84% and 85-100%). Our analysis of the dielectric properties data for 354 tissue samples reveals that there is a large variation in the dielectric properties of normal breast tissue due to substantial tissue heterogeneity. We observed no statistically significant difference between the within-patient and between-patient variability in the dielectric properties.

The development of ultrawideband (UWB) microwave diagnostic and therapeutic technologies, such as UWB microwave breast cancer detection and hyperthermia treatment, is facilitated by accurate knowledge of the temperature- and frequency-dependent dielectric properties of biological tissues. To this end, we characterize the temperature-dependent dielectric properties of a representative tissue type-animal liver-from 0.5 to 20 GHz. Since discrete-frequency linear temperature coefficients are impractical and inappropriate for applications spanning wide frequency and temperature ranges, we propose a novel and compact data representation technique. A single-pole Cole-Cole model is used to fit the dielectric properties data as a function of frequency, and a second-order polynomial is used to fit the Cole-Cole parameters as a function of temperature. This approach permits rapid estimation of tissue dielectric properties at any temperature and frequency.

We propose and characterize oil-in-gelatin dispersions that approximate the dispersive dielectric properties of a variety of human soft tissues over the microwave frequency range from 500 MHz to 20 GHz. Different tissues are mimicked by selection of an appropriate concentration of oil. The materials possess long-term stability and can be employed in heterogeneous configurations without change in geometry or dielectric properties due to osmotic effects. Thus, these materials can be used to construct heterogeneous phantoms, including anthropomorphic types, for narrowband and ultrawideband microwave technologies, such as breast cancer detection and imaging systems.

We have demonstrated functional optical coherence tomography (fOCT) for neural imaging by detecting scattering changes during the propagation of action potentials through neural tissue. OCT images of nerve fibers from the abdominal ganglion of the sea slug Aplysia californica were taken before, during, and after electrical stimulation. Images acquired during stimulation showed localized reversible increases in scattering compared with those acquired before stimulation. Motion-mode OCT images of nerve fibers showed transient scattering changes from spontaneous action potentials. These results demonstrate that OCT is sensitive to the optical changes in electrically active nerve fibers.