Characterization of a Biochemical Mouse Model of Primary Aldosteronism for Thermal Therapies.

Introduction: Aldosterone-producing adenoma (APA) is the most common cause of endocrine-related hypertension but surgery is not always feasible. Current medical interventions are associated with significant side effects and poor patient compliance. New APA animal models that replicate basic characteristics of APA and give physical and biochemical feedback are needed to test new non-surgical treatment methods, such as image-guided thermal ablation. Methods: A model of APA was developed in nude mice using HAC15 cells, a human adrenal carcinoma cell line. Tumor growth, aldosterone production, and sensitivity to angiotensin II were characterized in the model. The utility of the model was validated via treatment with microwave ablation and characterization of the resulting physical and biochemical changes in the tumor. Results: The APA model showed rapid and relatively homogeneous growth. The tumors produced aldosterone and steroid precursors in response to angiotensin II challenge, and plasma aldosterone levels were significantly higher in tumor bearing mice two hours after challenge verses non-tumor bearing mice. The model was useful for testing microwave ablation therapy, reducing aldosterone production by 80% in treated mice. Conclusion: The HAC15 model is a useful tumor model to study and develop localized treatment methods for APA.

T1-mapping characterization of two tumor types.

T1 mapping is a quantitative method to characterize tissues with magnetic resonance imaging in a quick and efficient manner. It utilizes the relaxation rate of protons to depict the underlying structures within the imaging frame. While T1-mapping techniques are used with some frequency in areas such as cardiac imaging, their application for understanding malignancies and identifying tumor structures has yet to be thoroughly investigated. Utilizing a saturation recovery method to acquire T1 maps for two different tumor models has revealed that longitudinal relaxation mapping is sensitive enough to distinguish between normal and malignant tissue. This is seen even with decreased signal/noise ratios using small voxel sizes to obtain high-resolution images. In both tumor models, it was revealed that relaxation mapping recorded significantly different relaxation values between regions encapsulating the tumor, muscle, kidney, or spleen, as well as between the cell lines themselves. This indicates a potential future application of relaxation mapping as a method to fingerprint various stages of tumor development and may prove a useful measure to identify micro-metastases.

In Vitro Measurement and Mathematical Modeling of Thermally-Induced Injury in Pancreatic Cancer Cells.

Thermal therapies are under investigation as part of multi-modality strategies for the treatment of pancreatic cancer. In the present study, we determined the kinetics of thermal injury to pancreatic cancer cells in vitro and evaluated predictive models for thermal injury. Cell viability was measured in two murine pancreatic cancer cell lines (KPC, Pan02) and a normal fibroblast (STO) cell line following in vitro heating in the range 42.5-50 °C for 3-60 min. Based on measured viability data, the kinetic parameters of thermal injury were used to predict the extent of heat-induced damage. Of the three thermal injury models considered in this study, the Arrhenius model with time delay provided the most accurate prediction (root mean square error = 8.48%) for all cell lines. Pan02 and STO cells were the most resistant and susceptible to hyperthermia treatments, respectively. The presented data may contribute to studies investigating the use of thermal therapies as part of pancreatic cancer treatment strategies and inform the design of treatment planning strategies.

Extracellular vesicles production and proteomic cargo varies with incubation time and temperature.

Extracellular vesicles (EVs) are heterogenous populations of proteolipid bi-layered vesicles secreted by cells as an important biological process. EVs cargo can reflect the cellular environmental conditions in which cells grow. The use of serum-free conditioned media to harvest EVs leads to stress-mediated cellular changes with longer incubation time and impacts EV production and functionality. This study aims to explore the role of incubation time and temperature on EV production and proteomic cargo. For this purpose, an optimized ultrafiltration-size exclusion chromatography-based technique is developed, which isolates small EVs ranging from 130 to 220 nm. The result shows higher EVs production in cancerous cells (K7M2) compared to noncancerous cells (NIH/3T3), which increases with longer incubation time and elevated temperature. Mass spectrometry-based proteomic characterization of EVs showed incubation time and temperature-dependent proteomic profile. A set of enriched EV proteins were identified in EVs isolated at nutrient-stress (72 h incubation time) and heat-stress (40 °C incubation temperature) environment. Enrichment of Serpinb1a in EVs isolated in heat stress was further validated via immunoblot. Gene enrichment analysis revealed that enriched EV proteins following nutrient stress were involved in negative regulation of transcription, response to oxidative stress, and protein folding. Likewise, enriched EV proteins following heat stress were involved in oxaloacetate and aspartate metabolism, and glutamate catabolic process. EVs isolated under nutrient stress showed pro-proliferative activity whereas EVs isolated under heat stress showed anti-proliferative activity. Our results show that incubation time and temperature can alter EV production, its proteomic cargo, and functionality, which can be used to design need-based standard isolation parameters for reproducible EV research.

Enzyme Nanoscale Interactions with Manganese Zinc Sulfide Give Insight into Potential Antiviral Mechanisms and SARS-CoV-2 Inhibition.

Recent interest in nanomedicine has skyrocketed because of mRNA vaccine lipid nanoparticles (LNPs) against COVID-19. Ironically, despite this success, the innovative nexus between nanotechnology and biochemistry, and the impact of nanoparticles on enzyme biochemical activity is poorly understood. The studies of this group on zinc nanoparticle (ZNP) compositions suggest that nanorod morphologies are preferred and that ZNP doped with manganese or iron can increase activity against model enzymes such as luciferase, DNA polymerase, and ß-galactosidase (ß-Gal), with the latter previously being associated with antimicrobial activity. SARS-CoV-2 encodes several of these types of oxido-reductase, polymerase, or hydrolase types of enzymes, and while metamaterials or nanoparticle composites have become important in many fields, their application against SARS-CoV-2 has only recently been considered. Recently, this group discovered the antiviral activity of manganese-doped zinc sulfide (MnZnS), and here the interactions of this nanoparticle composite with ß-Gal, angiotensin converting enzyme (ACE), and human ACE2 (hACE2), the SARS-CoV-2 receptor, are demonstrated. Low UV, circular dichroism, and zeta potential results confirm their enzyme interaction and inhibition by fluorometric area under the curve (AUC) measurements. The IC50 of enzyme activity varied depending on the manganese percentage and surface ranging from 20 to 50 µg/mL. MnZnS NPs give a 1-2 log order inhibition of SARS-CoV-2; however, surface-capping with cysteine does not improve activity. These data suggest that Mn substituted ZNP interactions to hACE2 and potentially other enzymes may underlie its antiviral activity, opening up a new area of pharmacology ready for preclinical translation.

System for delivering microwave ablation to subcutaneous tumors in small-animals under high-field MRI thermometry guidance.

PURPOSE: Bio-effects following thermal treatments are a function of the achieved temperature profile in tissue, which can be estimated across tumor volumes with real-time MRI thermometry (MRIT). Here, we report on expansion of a previously developed small-animal microwave hyperthermia system integrated with MRIT for delivering thermal ablation to subcutaneously implanted tumors in mice. METHODS: Computational models were employed to assess suitability of the 2.45 GHz microwave applicators for delivering ablation to subcutaneous tumor targets in mice. Phantoms and ex-vivo tissues were heated to temperatures in the range 47-67 °C with custom-made microwave applicators for validating MRIT with the proton resonance frequency shift method against fiberoptic thermometry. HAC15 tumors implanted in nude mice (n = 6) were ablated in vivo and monitored with MRIT in multiple planes. One day post ablation, animals were euthanized, and excised tumors were processed for viability assessment. RESULTS: Average absolute error between temperatures from fiberoptic sensors and MRIT was 0.6 °C across all ex-vivo ablations. During in-vivo experiments, tumors with volumes ranging between 5.4-35.9 mm3 (mean 14.2 mm3) were ablated (duration: 103-150 s) to achieve 55 °C at the tumor boundary. Thermal doses ≥240 CEM43 were achieved across 90.7-98.0% of tumor volumes for four cases. Ablations were incomplete for remaining cases, attributed to motion-affected thermometry. Thermal dose-based ablative tumor coverage agreed with viability assessment of excised tumors. CONCLUSIONS: We have developed a system for delivering microwave ablation to subcutaneous tumors in small animals under MRIT guidance and demonstrated its performance in-vivo.

Increased volumes of lobule VI in a valproic acid model of autism are associated with worse set-shifting performance in male Long-Evan rats.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a skewed sex-based diagnostic ratio. While males are at a higher risk for ASD, it is critical to understand the neurobiology of the disorder to develop better treatments for both males and females. Our prior work has demonstrated that VPA (valproic acid) treated offspring had impaired performance on an attentional set-shifting task. The current study used MRI and regions of interest analyses to measure the volumes of cerebellar subregions in VPA and controls rats that had participated in the attentional set-shifting task. VPA males had significantly more volume in lobule VI compared to male controls. VPA female rats had significantly less volume in lobules I, IV and X compared to female controls. In addition, it was revealed that decreases in volume for VPA females was associated with worse performance. Males with increases in lobule VI were also impaired on the set-shifting task. Similar volumetric differences within the cerebellum have been observed in humans with ASD, which suggests that the VPA model is capturing some of the same brain changes observed in humans with ASD, and that these changes in volume may be impacting cognition.

Enhanced charge separation in TiO2/nanocarbon hybrid photocatalysts through coupling with short carbon nanotubes.

The interfacial contact between TiO2 and graphitic carbon in a hybrid composite plays a critical role in electron transfer behavior, and in turn, its photocatalytic efficiency. Herein, we report a new approach for improving the interfacial contact and delaying charge carrier recombination in the hybrid by wrapping short single-wall carbon nanotubes (SWCNTs) on TiO2 particles (100 nm) via a hydration-condensation technique. Short SWCNTs with an average length of 125 ± 90 nm were obtained from an ultrasonication-assisted cutting process of pristine SWCNTs (1-3 µm in length). In comparison to conventional TiO2-SWCNT composites synthesized from long SWCNTs (1.2 ± 0.7 µm), TiO2 wrapped with short SWCNTs showed longer lifetimes of photogenerated electrons and holes, as well as a superior photocatalytic activity in the gas-phase degradation of acetaldehyde. In addition, upon comparison with a TiO2-nanographene "quasi-core-shell" structure, TiO2-short SWCNT structures offer better electron-capturing efficiency and slightly higher photocatalytic performance, revealing the impact of the dimensions of graphitic structures on the interfacial transfer of electrons and light penetration to TiO2. The engineering of the TiO2-SWCNT structure is expected to benefit photocatalytic degradation of other volatile organic compounds, and provide alternative pathways to further improve the efficiency of other carbon-based photocatalysts.

Temperature estimation for MR-guided microwave hyperthermia using block-based compressed sensing.

Mild hyperthermia has been clinically employed as an adjuvant for radiation/chemotherapy and is under investigation for precise thermally-mediated delivery of cancer therapeutic agents. Magnetic Resonance Imaging (MRI) facilitates non-invasive, real-time spatial thermometry for monitoring and guiding hyperthermia procedures. Long image acquisition time during MR-guided hyperthermia may fail to capture rapid changes in temperature. This may lead to unwanted heating of healthy tissue and/or temperature rise above hyperthermic range. We have developed a block-based compressed sensing approach to reconstruct volumetric MR-derived microwave hyperthermia temperature profiles using a subset of measured data. This algorithm exploits the sparsity of MR images due to the presence of inter- and intra-slice correlation of hyperthermic MR-derived temperature profiles. We have evaluated the performance of our developed algorithm on a phantom and in vivo in mice using previously implemented microwave applicators. This algorithm reconstructs 3D temperature profiles with PSNR of 33 dB - 49 dB in comparison to the original profiles. In summary, this study suggests that microwave hyperthermia induced temperature profiles can be reconstructed using subsamples to reduce MR image acquisition time.

Using Naturally Occurring Bioluminescent Enzymes to Track Specific Cell Populations.

Luminol-based bioluminescence imaging allows noninvasive tracking of oxidatively active cells such as neutrophils. Luminol is given intravenously or intraperitoneally, followed by bioluminescence imaging at 425 nm. Here we describe a method for tracking neutrophil extravasation into an inflammatory site, especially focusing on mammary carcinoma.

Development of a Gene Delivery System Composed of a Cell-Penetrating Peptide and a Nontoxic Polymer.

Major concerns have arisen with respect to using viral vectors for gene therapies. Collateral effects include cancer resistance, development of new cancers, and even systemic deaths. For this reason, researchers have focused on the alternative of using nonviral nanocarriers for gene therapy. In this study, a gene delivery nanocarrier was developed, comprising a cell-penetrating peptide called WTAS as a primary nanocarrier and a poly(ß-amino ester) (PBAE) polymer as a secondary nanocarrier. Here, the PBAE polymer is used to protect the WTAS peptide from early degradation while further facilitating the transportation into cells. WTAS is a peptide that penetrates cell nuclei within a few minutes after exposure, which makes it an ideal candidate to transport genetic materials. The PBAE-WTAS nanocarrier was assembled and tested against three cell lines (NSC, B16F10, and GL26). Cytotoxic studies demonstrated the relatively low toxicity of the PBAE-WTAS nanocarrier and PBAE-WTAS loaded with green fluorescent protein (GFP) plasmid DNA (pDNA@PBAE-WTAS) against all three cell lines. Cell transfection experiments were carried out using GL26 cells. These studies demonstrated a very high transfection rate of PBAE-WTAS loaded with GFP plasmid DNA, leading to virtually complete transfection (> 90%). In conclusion, we report a very promising gene delivery nanocarrier, which can be further modified to transport a variety of genetic materials for targeted therapy of multiple diseases.

Peptide Nanosponges Designed for the Delivery of Perillyl Alcohol to Glioma Cells.

Peptide nanosponges of low polydispersity are spontaneously formed from trigonal supramolecular building blocks in aqueous buffers, which feature cationic and/or anionic oligopeptides (n = 5-20) and a hydrophobic unit. In contrast to classical liposomes/vesicles, nanosponges feature interwoven hydrophilic and hydrophobic nanodomains and are readily taken up by mammalian cells. Perillyl alcohol is known to be a simple, but effective small molecule drug against glioma multiforme. However, its efficacy is limited by a poor bioavailability. In order to make perillyl alcohol bioavailable, two nanosponges consisting of 10 aspartates, to which perillyl alcohol is attached by means of an ester bond, and 20 lysines or arginines (type (D-POH)10K20 and (D-POH)10R20) were synthesized, purified, and characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). These nanosponges were then tested in cell cultures of murine glioma cells (GL26) and murine neural progenitor cells (NPC) because the latter was previously utilized in cell-based cancer therapy. The two nanosponges exhibited significantly different biophysical properties (size distribution and ζ potentials). Consequently, different efficacies in killing GL26 and NPC were observed in serum-containing culture media. The results from these experiments confirmed that the type (D-POH)10K20 nanosponge is a promising candidate for the (cell-mediated) cytotherapy of glioblastoma.

Early detection of pancreatic cancers in liquid biopsies by ultrasensitive fluorescence nanobiosensors.

Numerous proteases, such as matrix metalloproteinases (MMPs), cathepsins (CTS), and urokinase plasminogen activator (UpA), are dysfunctional (that is, over- or under-expressed) in solid tumors, when compared to healthy human subjects. This offers the opportunity to detect early tumors by liquid biopsies. This approach is of particular advantage for the early detection of pancreatic cancer, which is a "silent killer". We have developed fluorescence nanobiosensors for ultrasensitive (sub-femtomolar) arginase and protease detection, consisting of water-dispersible Fe/Fe3O4 core/shell nanoparticles and two tethered fluorescent dyes: TCPP (Tetrakis(4-carboxyphenyl)porphyrin) and cyanine 5.5. Upon posttranslational modification or enzymatic cleavage, the fluorescence of TCPP increases, which enables the detection of proteases at sub-femtomolar activities utilizing conventional plate readers. We have identified an enzymatic signature for the detection of pancreatic adenocarcinomas in serum, consisting of arginase, matrix metalloproteinase-1, -3, and - 9, cathepsin-B and -E, urokinase plasminogen activator, and neutrophil elastase, which is a potential game-changer.

An integrated platform for small-animal hyperthermia investigations under ultra-high-field MRI guidance.

PURPOSE: Integrating small-animal experimental hyperthermia instrumentation with magnetic resonance imaging (MRI) affords real-time monitoring of spatial temperature profiles. This study reports on the development and preliminary in vivo characterisation of a 2.45 GHz microwave hyperthermia system for pre-clinical small animal investigations, integrated within a 14 T ultra-high-field MRI scanner. MATERIALS AND METHODS: The presented system incorporates a 3.5 mm (OD) directional microwave hyperthermia antenna, positioned adjacent to the small-animal target, radiating microwave energy for localised heating of subcutaneous tumours. The applicator is integrated within the 30 mm bore of the MRI system. 3D electromagnetic and biothermal simulations were implemented to characterise hyperthermia profiles from the directional microwave antenna. Experiments in tissue mimicking phantoms were performed to assess hyperthermia profiles and validate MR thermometry against fibre-optic temperature measurements. The feasibility of delivering in vivo hyperthermia exposures to subcutaneous 4T1 tumours in experimental mice under simultaneous MR thermometry guidance was assessed. RESULTS: Simulations and experiments in tissue mimicking phantoms demonstrated the feasibility of heating 21-982 mm3 targets with 8-12 W input power. Minimal susceptibility and electrical artefacts introduced by the hyperthermia applicator were observed on MR imaging. MR thermometry was in excellent agreement with fibre-optic temperatures measurements (max. discrepancy ≤0.6 °C). Heating experiments with the reported system demonstrated the feasibility of heating subcutaneous tumours in vivo with simultaneous MR thermometry. CONCLUSIONS: A platform for small-animal hyperthermia investigations under ultra-high-field MR thermometry was developed and applied to heating subcutaneous tumours in vivo.

Peptide nanosponges designed for rapid uptake by leukocytes and neural stem cells.

The structure of novel binary nanosponges consisting of (cholesterol-(K/D) n DEVDGC)3-trimaleimide units possessing a trigonal maleimide linker, to which either lysine (K)20 or aspartic acid (D)20 are tethered, has been elucidated by means of TEM. A high degree of agreement between these findings and structure predictions through explicit solvent and then coarse-grained molecular dynamics (MD) simulations has been found. Based on the nanosponges' structure and dynamics, caspase-6 mediated release of the model drug 5(6)-carboxyfluorescein has been demonstrated. Furthermore, the binary (DK20) nanosponges have been found to be virtually non-toxic in cultures of neural progenitor cells. It is of a special importance for the future development of cell-based therapies that DK20 nanosponges were taken up efficiently by leucocytes (WBC) in peripheral blood within 3 h of exposure. The percentage of live cells among the WBC was not significantly decreased by the DK20 nanosponges. In contrast to stem cell or leucocyte cell cultures, which have to be matched to the patient, autologous cells are optimal for cell-mediated therapy. Therefore, the nanosponges hold great promise for effective cell-based tumor targeting.

Rationally designed peptide nanosponges for cell-based cancer therapy.

A novel type of supramolecular aggregate, named a "nanosponge" was synthesized through the interaction of novel supramolecular building blocks with trigonal geometry. The cholesterol-(K/D)nDEVDGC)3-trimaleimide unit consists of a trigonal maleimide linker to which homopeptides (either K or D) of variable lengths (n=5, 10, 15, 20) and a consensus sequence for executioner caspases (DEVDGC) are added via Michael addition. Upon mixing in aqueous buffer cholesterol-(K)nDEVDGC)3-trimaleimides and a 1:1 mixture of cholesterol-(K/D)nDEVDGC)3-trimaleimides form stable nanosponges, whereas cholesterol-(D)nDEVDGC)3-trimaleimide is unable to form supramolecular aggregates with itself. The structure of the novel nanosponges was investigated through explicit solvent and then coarse-grained molecular dynamics (MD) simulations. The nanosponges are between 80 nm and several micrometers in diameters and virtually non-toxic to monocyte/macrophage-like cells.

Synergy of Iron Chelators and Therapeutic Peptide Sequences Delivered via a Magnetic Nanocarrier.

Here, we report the synthesis, characterization, and efficacy study of Fe/Fe3O4-nanoparticles that were co-labeled with a tumor-homing and membrane-disrupting oligopeptide and the iron-chelator Dp44mT, which belongs to the group of the thiosemicarbazones. Dp44mT and the peptide sequence PLFAERL(D[KLAKLAKKLAKLAK])CGKRK were tethered to the surface of Fe/Fe3O4 core/shell nanoparticles by utilizing dopamine-anchors. The 26-mer contains two important sequences, which are the tumor targeting peptide CGKRK, and D[KLAKLAK]2, known to disrupt the mitochondrial cell walls and to initiate programmed cell death (apoptosis). It is noteworthy that Fe/Fe3O4 nanoparticles can also be used for MRI imaging purposes in live mammals. In a first step of this endeavor, the efficacy of this nanoplatform has been tested on the highly metastatic 4T1 breast cancer cell line. At the optimal ratio of PLFAERD[KLAKLAK]2CGKRK to Dp44mT of 1 to 3.2 at the surface of the dopamine-coated Fe/Fe3O4-nanocarrier, the IC50 value after 24 h of incubation was found to be 2.2 times lower for murine breast cancer cells (4T1) than for a murine fibroblast cell line used as control. Based on these encouraging results, the reported approach has the potential of leading to a new generation of nanoplatforms for cancer treatment with considerably enhanced selectivity towards tumor cells.

Membrane Fusion-Mediated Gold Nanoplating of Red Blood Cell: A Bioengineered CT-Contrast Agent.

Red blood cells (RBCs) are the natural resident of the vascular lumen, therefore delivery of any agents within the vascular lumen could benefit by unique natural transporting features of RBCs. RBCs continuously circulate for ∼100 days before being sequestered in the spleen, they only extravasate at sites of vascular hemorrhage. Taking advantages of these features, we engineered RBC as a carrier in order to design a unique delivery system capable of delivering X-ray computed tomography (CT) contrast agents, gold nanoparticles (AuNPs), thereby acting as CT-contrast agent. A strategic membrane fusion technique was used to engineer the surface of RBC with gold nanoparticles in this in vitro study without altering its shape, size, and surface properties.

A nanobiosensor for the detection of arginase activity.

A nanobiosensor for arginase detection was designed and synthesized. It features a central dopamine-coated iron/iron oxide nanoparticle to which sulfonated cyanine 7.0 is tethered via a stable amide bond. Cyanine 5.5 is linked to the N-terminal of the peptide sequence GRRRRRRRG. Arginine (R) reacts to ornithine (O) in the presence of arginase. Based on calibration with commercially obtained arginase II, the limit of detection (LOD) is picomolar. It is noteworthy that the nanobiosensor for arginase detection does not show a fluorescence increase when incubated with the enzyme NO-reductase, which also uses arginase as substrate, but is indicative of an inflammatory response by the host to cancer and infections. Arginase activity was determined in a syngeneic mouse model for aggressive breast cancer (4T1 tumors in BALB/c mice). It was found that the arginase activity is systemically enhanced, but especially pronounced in the active tumor regions.

Insights from Theory and Experiment on the Photochromic spiro-Dihydropyrrolo-Pyridazine/Betaine System.

We elucidated the photochromic spiro-4a,5-dihydropyrrolo[1,2-b]pyridazine/betaine (DPP/betaine) system by comparing state-of-the-art density functional theory calculations with nanosecond/millisecond UV-vis absorption spectroscopy, as well as steady-state absorption and cyclization kinetics. Time-dependent density functional theory calculations are employed to examine the transformations occurring after photoexcitation. This study shows that the photochromic spiro-4a,5-dihydropyrrolo[1,2-b]pyridazine and spiro-1,8a-dihydroindolizine (DHI) systems react according to similar pathways. However, notable differences exist. Although photoexcitation of the spiro-DPP system also leads to cis-betaines, which then isomerize to trans-betaines, we found two distinct classes of cis isomers (cis-betaine rotamer-1 and cis-betaine rotamer-2), which do not exist in spiro-1,8a-dihydroindolizine. Similar to our previous study on the spiro-DHI/betaine system, a complicated potential-energy landscape between cis and trans isomers exists in the spiro-DPP system, consisting of a network of transition states and intermediates. Because the spiro-DPP/betaine is even more complicated than the spiro-DHI/betaine system, (substituted) photochromic systems featuring a 4a,5-dihydropyrrolo[1,2-b]pyridazine functional unit will require thorough in silico design to function properly as logical gates or in devices for information storage.