Engineering Crystallinity Gradients for Tailored CaO2 Nanostructures: Enabling Alkalinity-Reinforced Anticancer Activity with Minimized Ca2+/H2O2 Production.

CaO2 nanoparticles (CNPs) can produce toxic Ca2+ and H2O2 under acidic pH, which accounts for their intrinsic anticancer activity but at the same time raises safety concerns upon systemic exposure. Simultaneously realizing minimized Ca2+/H2O2 production and enhanced anticancer activity poses a dilemma. Herein, we introduce a "crystallinity gradient-based selective etching" (CGSE) strategy, which is realized by creating a crystallinity gradient in a CNP formed by self-assembled nanocrystals. The nanocrystals distributed in the outer layer have a higher crystallinity and thus are chemically more robust than those distributed in the inner layer, which can be selectively etched. CGSE not only leads to CNPs with tailored single- and double-shell hollow structures and metal-doped compositions but more surprisingly enables significantly enhanced anticancer activity as well as tumor growth inhibition under limited Ca2+/H2O2 production, which is attributed to an alkalinity-reinforced lysosome-dependent cell death pathway.

Respiratory mucosal vaccination of peptide-poloxamine-DNA nanoparticles provides complete protection against lethal SARS-CoV-2 challenge.

The ongoing SARS-CoV-2 pandemic represents a brutal reminder of the continual threat of mucosal infectious diseases. Mucosal immunity may provide robust protection at the predominant sites of SARS-CoV-2 infection. However, it remains unclear whether respiratory mucosal administration of DNA vaccines could confer protective immune responses against SARS-CoV-2 challenge due to insurmountable barriers posed by the airway. Here, we applied self-assembled peptide-poloxamine nanoparticles with mucus-penetrating properties for pulmonary inoculation of a COVID-19 DNA vaccine (pSpike/PP-sNp). The pSpike/PP-sNp not only displays superior gene transfection and favorable biocompatibility in the mouse airway, but also promotes a tripartite immunity consisting of systemic, cellular, and mucosal immune responses that are characterized by mucosal IgA secretion, high levels of neutralizing antibodies, and resident memory phenotype T-cell responses in the lungs of mice. Most importantly, immunization with pSpike/PP-sNp completely eliminates SARS-CoV-2 infection in both upper and lower respiratory tracts and enables 100% survival rate of mice following lethal SARS-CoV-2 challenge. Our findings indicate PP-sNp is a promising platform in mediating DNA vaccines to elicit all-around mucosal immunity against SARS-CoV-2.

Surface chemistry of spiky silica nanoparticles tailors polyethyleneimine binding and intracellular DNA delivery.

Cellular delivery of DNA using silica nanoparticles has attracted great attention. Typically, polyethyleneimine (PEI) is used to form a silica/PEI composite vector. Understanding the interactions at the silica and PEI interface is important for successful DNA delivery and transfection, especially for silica with different surface functionality. Herein, we report that a higher content of hydrogen boning formed between PEI molecules and phosphonate modified silica nanoparticles could slow down the PEI dissolution from the freeze-dried solid composites into aqueous solution than the bare silica counterpart. The pronounced PEI retention ability through phosphonation of silica nanoparticles effectively improves the transfection efficiency due to the high DNA binding affinity extracellularly, effective lysosome escape and high nuclear entry of both PEI and DNA intracellularly. Our study provides a fundamental understanding on designing effective silica-PEI-based nano-vectors for DNA delivery applications.

Mesoporous sodium four-coordinate aluminosilicate nanoparticles modulate dendritic cell pyroptosis and activate innate and adaptive immunity.

Pyroptosis is a programmed cell death widely studied in cancer cells for tumour inhibition, but rarely in dendritic cell (DC) activation for vaccine development. Here, we report the synthesis of sodium stabilized mesoporous aluminosilicate nanoparticles as DC pyroptosis modulators and antigen carriers. By surface modification of sodium-stabilized four-coordinate aluminium species on dendritic mesoporous silica nanoparticles, the resultant Na-IVAl-DMSN significantly activated DC through caspase-1 dependent pyroptosis via pH responsive intracellular ion exchange. The released proinflammatory cellular contents further mediated DC hyperactivation with prolonged cytokine release. In vivo studies showed that Na-IVAl-DMSN induced enhanced cellular immunity mediated by natural killer (NK) cells, cytotoxic T cells, and memory T cells as well as humoral immune response. Our results provide a new principle for the design of next-generation nanoadjuvants for vaccine applications.

Nanotechnologies in Delivery of DNA and mRNA Vaccines to the Nasal and Pulmonary Mucosa.

Recent advancements in the field of in vitro transcribed mRNA (IVT-mRNA) vaccination have attracted considerable attention to such vaccination as a cutting-edge technique against infectious diseases including COVID-19 caused by SARS-CoV-2. While numerous pathogens infect the host through the respiratory mucosa, conventional parenterally administered vaccines are unable to induce protective immunity at mucosal surfaces. Mucosal immunization enables the induction of both mucosal and systemic immunity, efficiently removing pathogens from the mucosa before an infection occurs. Although respiratory mucosal vaccination is highly appealing, successful nasal or pulmonary delivery of nucleic acid-based vaccines is challenging because of several physical and biological barriers at the airway mucosal site, such as a variety of protective enzymes and mucociliary clearance, which remove exogenously inhaled substances. Hence, advanced nanotechnologies enabling delivery of DNA and IVT-mRNA to the nasal and pulmonary mucosa are urgently needed. Ideal nanocarriers for nucleic acid vaccines should be able to efficiently load and protect genetic payloads, overcome physical and biological barriers at the airway mucosal site, facilitate transfection in targeted epithelial or antigen-presenting cells, and incorporate adjuvants. In this review, we discuss recent developments in nucleic acid delivery systems that target airway mucosa for vaccination purposes.

A Method for Reducing Variability Across Dual-Energy CT Manufacturers in Quantification of Low Iodine Content Levels.

BACKGROUND. Clinical use of the dual-energy CT (DECT) iodine quantification technique is hindered by between-platform (i.e., across different manufacturers) variability in iodine concentration (IC) values, particularly at low iodine levels. OBJECTIVE. The purpose of this study was to develop in an anthropomorphic phantom a method for reducing between-platform variability in quantification of low iodine content levels using DECT and to evaluate the method's performance in patients undergoing serial clinical DECT examinations on different platforms. METHODS. An anthropomorphic phantom in three body sizes, incorporating varied lesion types and scanning conditions, was imaged with three distinct DECT implementations from different manufacturers at varying radiation exposures. A cross-platform iodine quantification model for correcting between-platform variability at low iodine content was developed using the phantom data. The model was tested in a retrospective series of 30 patients (20 men, 10 women; median age, 62 years) who each underwent three serial contrast-enhanced DECT examinations of the abdomen and pelvis (90 scans total) for routine oncology surveillance using the same three DECT platforms as in the phantom. Estimated accuracy of phantom IC values was summarized using root-mean-square error (RMSE) relative to known IC. Between-platform variability in patients was summarized using root-mean-square deviation (RMSD). RMSE and RMSD were compared between platform-based IC (ICPB) and cross-platform IC (ICCP). ICPB was normalized to aorta and portal vein. RESULTS. In the phantom study, mean RMSE of ICPB across platforms and other experimental conditions was 0.65 ± 0.18 mg I/mL compared with 0.40 ± 0.08 mg I/mL for ICCP (38% decrease in mean RMSE; p < .05). Intrapatient between-platform variability across serial DECT examinations was higher for ICPB than ICCP (RMSD, 97% vs 88%; p < .001). Between-platform variability was not reduced by normalization of ICPB to aorta (RMSD, 97% vs 101%; p = .12) or portal vein (RMSD, 97% vs 97%; p = .81). CONCLUSION. The developed cross-platform method significantly decreased between-platform variability occurring at low iodine content with platform-based DECT iodine quantification. CLINICAL IMPACT. With further validation, the cross-platform method, which has been implemented as a web-based app, may expand clinical use of DECT iodine quantification, yielding meaningful IC values that reflect tissue biologic viability or treatment response in patients who undergo serial examinations on different platforms.

Virtual Unenhanced Dual-Energy CT Images Obtained with a Multimaterial Decomposition Algorithm: Diagnostic Value for Renal Mass and Urinary Stone Evaluation.

Background Virtual unenhanced (VUE) images obtained by using a dual-energy CT (DECT) multimaterial decomposition algorithm hold promise for diagnostic use in the abdomen in lieu of true unenhanced (TUE) images. Purpose To assess VUE images obtained from a DECT multimaterial decomposition algorithm in patients undergoing renal mass and urinary stone evaluation. Materials and Methods In this retrospective Health Insurance Portability and Accountability Act-compliant study, DECT was performed in patients undergoing evaluation for renal mass or urinary stone. VUE images were compared quantitatively to TUE images and qualitatively assessed by four independent radiologists. Differences in attenuation between VUE and TUE images were summarized by using 95% limits of agreement. Diagnostic performance in urinary stone detection was summarized by using area under the receiver operating characteristic curve, sensitivity, and specificity. Results A total of 221 patients (mean age ± standard deviation, 61 years ± 14; 129 men) with 273 renal masses were evaluated. Differences in renal mass attenuation between VUE and TUE images were within 3 HU for both enhancing masses (95% limits of agreement, -3.1 HU to 2.7 HU) and nonenhancing cysts (95% limits of agreement, -2.9 HU to 2.5 HU). Renal mass classification as enhancing mass versus nonenhancing cyst did not change (reclassification rate of enhancing masses, 0% [0 of 78]; 95% CI: 0, 5; reclassification rate of nonenhancing cysts, 0% [0 of 193]; 95% CI: 0, 2) with use of VUE in lieu of TUE images. Among 166 urinary stones evaluated, diagnostic performance of VUE images for stone detection was lower compared with that of TUE images (area under the receiver operating characteristic curve, 0.79 [95% CI: 0.73, 0.84] vs 0.93 [95% CI: 0.91, 0.95]; P < .001) due to reduced sensitivity of VUE for detection of stones 3 mm in diameter or less compared with those greater than 3 mm (sensitivity, 23% [25 of 108; 95% CI: 12, 40] vs 88% [126 of 144; 95% CI: 77, 94]; P < .001). Conclusion Compared with true unenhanced images, virtual unenhanced (VUE) images were unlikely to change renal mass classification as enhancing mass versus nonenhancing cyst. Diagnostic performance of VUE images remained suboptimal for urinary stone detection due to subtraction of stones 3 mm or less in diameter. © RSNA, 2021 Online supplemental material is available for this article. See also the editorial by Sosna in this issue.

Use of dual-energy CT for renal mass assessment.

Although dual-energy CT (DECT) may prove useful in a variety of abdominal imaging tasks, renal mass evaluation represents the area where this technology can be most impactful in abdominal imaging compared to routinely performed contrast-enhanced-only single-energy CT exams. DECT post-processing techniques, such as creation of virtual unenhanced and iodine density images, can help in the characterization of incidentally discovered renal masses that would otherwise remain indeterminate based on post-contrast imaging only. The purpose of this article is to review the use of DECT for renal mass assessment, including its benefits and existing limitations. KEY POINTS: • If DECT is selected as the scanning mode for most common abdominal protocols, many incidentally found renal masses can be fully triaged within the same exam. • Virtual unenhanced and iodine density DECT images can provide additional information when renal masses are discovered in the post-contrast-only setting. • For renal mass evaluation, virtual unenhanced and iodine density DECT images should be interpreted side-by-side to troubleshoot pitfalls that can potentially lead to erroneous interpretation.

Building a dual-energy CT service line in abdominal radiology.

As the access of radiology practices to dual-energy CT (DECT) has increased worldwide, seamless integration into clinical workflows and optimized use of this technology are desirable. In this article, we provide basic concepts of commercially available DECT hardware implementations, discuss financial and logistical aspects, provide tips for protocol building and image routing strategies, and review radiation dose considerations to establish a DECT service line in abdominal imaging. KEY POINTS: • Tube-based and detector-based DECT implementations with varying features and strengths are available on the imaging market. • Thorough assessment of financial and logistical aspects is key to successful implementation of a DECT service line. • Optimized protocol building and image routing strategies are of critical importance for effective use and seamless inception of DECT in routine clinical workflows.

Calcium Stimulation Test for Insulinoma Localization in an End-stage Renal Disease Patient on Diazoxide.

Insulinomas are rare, and even rarer in patients with end-stage renal disease (ESRD). Clear criteria for the biochemical diagnosis of insulinomas in patients with renal failure have not been established, and hypoglycemia is often attributed to the renal disease itself, frequently leading to a delay in diagnosis. We describe a case of a patient who presented with asymptomatic recurrent hypoglycemia during hemodialysis. Disease progression and biochemical testing strongly suggested an insulinoma. Computed tomography (CT) of the abdomen and pelvis, 111In-pentetreotide scintigraphy and endoscopic ultrasound did not localize a pancreatic tumor. A calcium stimulation test was performed while the patient was taking diazoxide due to severe hypoglycemia with fasting for a couple of hours without treatment. The test showed a marked increase in insulin after calcium infusion in the dorsal pancreatic artery, localizing the tumor to the body and tail of the gland. Exploratory surgery easily identified a tumor at the body of the pancreas and pathology confirmed an insulin-secreting pancreatic neuroendocrine tumor. On follow-up, there was resolution of the hypoglycemia. We review the challenges of diagnosing an insulinoma in ESRD and describe a successful intra-arterial calcium stimulation test done in an ESRD patient while continuing diazoxide.

A General Strategy to Design Highly Fluorogenic Far-Red and Near-Infrared Tetrazine Bioorthogonal Probes.

Highly fluorogenic tetrazine bioorthogonal probes emitting at near-infrared wavelengths are in strong demand for biomedical imaging applications. Herein, we have developed a strategy for forming a palette of novel Huaxi-Fluor probes in situ, whose fluorescence increases hundreds of times upon forming the bioorthogonal reaction product, pyridazine. The resulting probes show large Stokes shifts and high quantum yields. Manipulating the conjugate length and pull-push strength in the fluorophore skeleton allows the emission wavelength to be fine-tuned from 556 to 728 nm. The highly photo-stable and biocompatible probes are suitable for visualizing organelles in live cells without a washing step and for imaging of tumors in live small animals to depths of 500 µm by two-photon excitation.

3D T2-weighted and Gd-EOB-DTPA-enhanced 3D T1-weighted MR cholangiography for evaluation of biliary anatomy in living liver donors.

PURPOSE: To investigate whether the addition of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced 3D T1-weighted MR cholangiography (T1w-MRC) to 3D T2-weighted MRC (T2w-MRC) improves the confidence and diagnostic accuracy of biliary anatomy in living liver donors. METHODS: Two abdominal radiologists retrospectively and independently reviewed pre-operative MR studies in 58 consecutive living liver donors. The second-order bile duct visualization on T1w- and T2w-MRC images was rated on a 4-point scale. The readers also independently recorded the biliary anatomy and their diagnostic confidence using (1) combined T1w- and T2w-MRC, and (2) T2w-MRC. In the 23 right lobe donors, the biliary anatomy at imaging and the imaging-predicted number of duct orifices at surgery were compared to intra-operative findings. RESULTS: T1w-MRC had a higher proportion of excellent visualization than T2w-MRC, 66% vs. 45% for reader 1 and 60% vs. 31% for reader 2. The median confidence score for biliary anatomy diagnosis was significantly higher with combined T1w- and T2w-MRC than T2w-MRC alone for both readers (Reader 1: 3 vs. 2, p < 0.001; Reader 2: 3 vs. 1, p < 0.001). Compared to intra-operative findings, the accuracy of imaging-predicted number of duct orifices using combined T1w-and T2w-MRC was significantly higher than that using T2w-MRC alone (p = 0.034 for reader 1, p = 0.0082 for reader 2). CONCLUSION: The addition of Gd-EOB-DTPA-enhanced 3D T1w-MRC to 3D T2w-MRC improves second-order bile duct visualization and increases the confidence in biliary anatomy diagnosis and the accuracy in the imaging-predicted number of duct orifices acquired during right lobe harvesting.

Adult living donor liver imaging.

Adult living donor liver transplantation (LDLT) is increasingly used for the treatment of end-stage liver disease. The three most commonly harvested grafts for LDLT are left lateral segment, left lobe, and right lobe grafts. The left lateral segment graft, which includes Couinaud's segments II and III, is usually used for pediatric recipients or small size recipients. Most of the adult recipients need either a left or a right lobe graft. Whether a left or right lobe graft should be harvested from the donors depends on estimated graft and donor remnant liver volume, as well as biliary and vascular anatomy. Detailed preoperative assessment of the potential donor liver volumetrics, biliary and vascular anatomy, and liver parenchyma is vital to minimize risks to the donors and maximize benefits to the recipients. Computed tomography (CT) and magnetic resonance imaging (MRI) are currently the imaging modalities of choice in the preoperative evaluation of potential donors. This review provides an overview of key surgical considerations in LDLT that the radiologists must be aware of, and imaging findings on CT and MRI that the radiologists must convey to the surgeons when evaluating potential donors for LDLT.

IDH1 Mutation Induces Reprogramming of Pyruvate Metabolism.

Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of IDH1 mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized (13)C-magnetic resonance spectroscopy ((13)C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-(13)C-pyruvate to 5-(13)C-glutamate, relative to cells expressing wild-type IDH1. (13)C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-(13)C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by IDH1 mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the IDH1 mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications.

Glioma cells with the IDH1 mutation modulate metabolic fractional flux through pyruvate carboxylase.

BACKGROUND: Over 70% of low-grade gliomas carry a heterozygous R132H mutation in the gene coding for isocitrate dehydrogenase 1 (IDH1). This confers the enzyme with the novel ability to convert α-ketoglutarate to 2-hydroxyglutarate, ultimately leading to tumorigenesis. The major source of 2-hydroxyglutarate production is glutamine, which, in cancer, is also a source for tricarboxylic acid cycle (TCA) anaplerosis. An alternate source of anaplerosis is pyruvate flux via pyruvate carboxylase (PC), which is a common pathway in normal astrocytes. The goal of this study was to determine whether PC serves as a source of TCA anaplerosis in IDH1 mutant cells wherein glutamine is used for 2-hydroxyglutarate production. METHODS: Immortalized normal human astrocytes engineered to express heterozygous mutant IDH1 or wild-type IDH1 were investigated. Flux of pyruvate via PC and via pyruvate dehydrogenase (PDH) was determined by using magnetic resonance spectroscopy to probe the labeling of [2-¹³C]glucose-derived ¹³C-labeled glutamate and glutamine. Activity assays, RT-PCR and western blotting were used to probe the expression and activity of relevant enzymes. The Cancer Genome Atlas (TCGA) data was analyzed to assess the expression of enzymes in human glioma samples. RESULTS: Compared to wild-type cells, mutant IDH1 cells significantly increased fractional flux through PC. This was associated with a significant increase in PC activity and expression. Concurrently, PDH activity significantly decreased, likely mediated by significantly increased inhibitory PDH phosphorylation by PDH kinase 3. Consistent with the observation in cells, analysis of TCGA data indicated a significant increase in PC expression in mutant IDH-expressing human glioma samples compared to wild-type IDH. CONCLUSIONS: Our findings suggest that changes in PC and PDH may be an important part of cellular adaptation to the IDH1 mutation and may serve as potential therapeutic targets.

Hyperpolarized [1-13C] glutamate: a metabolic imaging biomarker of IDH1 mutational status in glioma.

Mutations of the isocitrate dehydrogenase 1 (IDH1) gene are among the most prevalent in low-grade glioma and secondary glioblastoma, represent an early pathogenic event, and are associated with epigenetically driven modulations of metabolism. Of particular interest is the recently uncovered relationship between the IDH1 mutation and decreased activity of the branched-chain amino acid transaminase 1 (BCAT1) enzyme. Noninvasive imaging methods that can assess BCAT1 activity could therefore improve detection of mutant IDH1 tumors and aid in developing and monitoring new targeted therapies. BCAT1 catalyzes the transamination of branched-chain amino acids while converting α-ketoglutarate (α-KG) to glutamate. Our goal was to use (13)C magnetic resonance spectroscopy to probe the conversion of hyperpolarized [1-(13)C] α-KG to hyperpolarized [1-(13)C] glutamate as a readout of BCAT1 activity. We investigated two isogenic glioblastoma lines that differed only in their IDH1 status and performed experiments in live cells and in vivo in rat orthotopic tumors. Following injection of hyperpolarized [1-(13)C] α-KG, hyperpolarized [1-(13)C] glutamate production was detected both in cells and in vivo, and the level of hyperpolarized [1-(13)C] glutamate was significantly lower in mutant IDH1 cells and tumors compared with their IDH1-wild-type counterparts. Importantly however, in our cells the observed drop in hyperpolarized [1-(13)C] glutamate was likely mediated not only by a drop in BCAT1 activity, but also by reductions in aspartate transaminase and glutamate dehydrogenase activities, suggesting additional metabolic reprogramming at least in our model. Hyperpolarized [1-(13)C] glutamate could thus inform on multiple mutant IDH1-associated metabolic events that mediate reduced glutamate production.