High-fidelity KKH variant of Staphylococcus aureus Cas9 nucleases with improved base mismatch discrimination.

The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9's REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9's DNA- and guide RNA- interacting residues to optimize its editing fidelity.

SPG7 Is an Essential and Conserved Component of the Mitochondrial Permeability Transition Pore.

Mitochondrial permeability transition is a phenomenon in which the mitochondrial permeability transition pore (PTP) abruptly opens, resulting in mitochondrial membrane potential (ΔΨm) dissipation, loss of ATP production, and cell death. Several genetic candidates have been proposed to form the PTP complex, however, the core component is unknown. We identified a necessary and conserved role for spastic paraplegia 7 (SPG7) in Ca(2+)- and ROS-induced PTP opening using RNAi-based screening. Loss of SPG7 resulted in higher mitochondrial Ca(2+) retention, similar to cyclophilin D (CypD, PPIF) knockdown with sustained ΔΨm during both Ca(2+) and ROS stress. Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7-CypD binding prevented Ca(2+)- and ROS-induced ΔΨm depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site.

Quality of Life Associated with Nausea and Vomiting from Anthracycline-Based Chemotherapy: A Pooled Data Analysis from Three Prospective Trials.

BACKGROUND: There is limited work on the impact of chemotherapy-induced nausea and vomiting (CINV) on quality of life (QoL) in adriamycin-cyclophosphamide (AC)-treated patients with breast cancer. The objectives of the study were the following: (a) to confirm if symptoms of CINV led to lower QoL during AC; (b) to evaluate the pattern of changes in patients' QoL during multiple cycles of AC; and (c) to assess if the QoL in an earlier cycle affected the QoL in subsequent cycles of AC. MATERIALS AND METHODS: This is a secondary pooled data analysis that included 303 Chinese patients with breast cancer who received 1,177 cycles of adjuvant AC in three prospective antiemetic studies. QoL data were based on Functional Living Index-emesis (FLIE) scored over three to four AC cycles. CINV symptoms assessed included "no significant nausea" (NSN), "significant nausea" (SN), "no vomiting" (NoV), "vomiting" (V), and complete response (CR). RESULTS: Across all AC cycles, the mean scores for the FLIE nausea domain for patients who experienced NSN versus SN were 10.92 versus 53.92, respectively (p < .0001), with lower scores indicating better QoL; the mean scores for the FLIE vomiting domain for patients who experienced NoV versus V were 1.44 versus 19.11, respectively (p < .0001), with similar results across subsequent cycles. Analysis of the effect of the QoL in cycle 1 on the QoL of subsequent cycles revealed the following: for the nausea domain, among patients who had cycle 1 FLIE scores ≥ versus < the mean, the corresponding scores in cycle 2 were 6.87 versus 36.71 (p < .0001); whereas those for cycle 3 were 7.07 versus 36.87 (p < .0001); and those for cycle 4 were 5.92 versus 21.48 (p < .0001). Similar findings were observed for the vomiting domain. Netupitant + palonosetron- or aprepitant/olanzapine-based antiemetics had significantly better QoL outcomes. CONCLUSION: CINV had a significant impact on the QoL of patients with breast cancer treated with AC over multiple cycles. IMPLICATIONS FOR PRACTICE: In this post-hoc analysis of three prospective studies on chemotherapy-induced nausea and vomiting (CINV), quality of life (QoL) using contemporary antiemetic regimens in Chinese breast cancer patients receiving doxorubicin-cyclophosphamide (AC) was evaluated. During the first and subsequent AC cycles, QoL was significantly better for patients who did not experience vomiting or significant nausea. QoL in an earlier cycle affected the QoL in subsequent AC cycles. Furthermore, recent regimens involving olanzapine/aprepitant or netupitant-palonosetron were associated with a positive impact in QoL. Antiemetic guideline-consistent practice and higher clinician awareness of the impact of CINV on QoL can further mitigate the negative effects of CINV on QoL.

Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.

OBJECTIVE: Identification of patients with high-risk asymptomatic carotid plaques remains an elusive but essential step in stroke prevention. Inflammation is a key process in plaque destabilization and a prelude to clinical sequelae. There are currently no clinical imaging tools to assess the inflammatory activity within plaques. This study characterized inflammation in atherosclerosis using dual-targeted microparticles of iron oxide (DT-MPIO) as a magnetic resonance imaging (MRI) probe. METHODS: DT-MPIO were used to detect and characterize inflammatory markers, vascular cell adhesion molecule 1 (VCAM-1). and P-selectin on (1) tumor necrosis factor-α-treated cells by immunocytochemistry and (2) aortic root plaques of apolipoprotein-E deficient mice by in vivo MRI. Furthermore, apolipoprotein E-deficient mice with focal carotid plaques of different phenotypes were developed by means of periarterial cuff placement to allow in vivo molecular MRI using these probes. The association between biomarkers and the magnetic resonance signal in different contrast groups was assessed longitudinally in these models. RESULTS: Immunocytochemistry confirmed specificity and efficacy of DT-MPIO to VCAM-1 and P-selectin. Using this in vivo molecular MRI strategy, we demonstrated (1) the DT-MPIO-induced magnetic resonance signal tracked with VCAM-1 (r = 0.69; P = .014), P-selectin (r = 0.65; P = .022), and macrophage content (r = 0.59; P = .045) within aortic root plaques and (2) high-risk inflamed plaques were distinguished from noninflamed plaques in the murine carotid artery within a practical clinical imaging time frame. CONCLUSIONS: These molecular MRI probes constitute a novel imaging tool for in vivo characterization of plaque vulnerability and inflammatory activity in atherosclerosis. Further development and translation into the clinical arena will facilitate more accurate risk stratification in carotid atherosclerotic disease in the future.

Changes in physiological signal entropy in patients with obstructive sleep apnoea: a systematic review.

Background and Objective.Obstructive sleep apnoea (OSA) affects an estimated 936 million people worldwide, yet only 15% receive a definitive diagnosis. Diagnosis of OSA poses challenges due to the dynamic nature of physiological signals such as oxygen saturation (SpO2) and heart rate variability (HRV). Linear analysis methods may not fully capture the irregularities present in these signals. The application of entropy of routine physiological signals offers a promising method to better measure variabilities in dynamic biological data. This review aims to explore entropy changes in physiological signals among individuals with OSA.Approach.Keyword and title searches were performed on Medline, Embase, Scopus, and CINAHL databases. Studies had to analyse physiological signals in OSA using entropy. Quality assessment used the Newcastle-Ottawa Scale. Evidence was qualitatively synthesised, considering entropy signals, entropy type, and time-series length.Main results.Twenty-two studies were included. Multiple physiological signals related to OSA, including SpO2, HRV, and the oxygen desaturation index (ODI), have been investigated using entropy. Results revealed a significant decrease in HRV entropy in those with OSA compared to control groups. Conversely, SpO2and ODI entropy values were increased in OSA. Despite variations in entropy types, time scales, and data extraction devices, studies using receiver operating characteristic curves demonstrated a high discriminative accuracy (>80% AUC) in distinguishing OSA patients from control groups.Significance. This review highlights the potential of SpO2entropy analysis in developing new diagnostic indices for patients with OSA. Further investigation is needed before applying this technique clinically.

Association between aspartate aminotransferase-to-alanine aminotransferase ratio and insulin resistance among US adults.

OBJECTIVE: To determine whether a low aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio (AST/ALT ratio) is associated with insulin resistance among those without liver dysfunction. METHODS: In this cross-sectional study of the National Health and Nutrition Examination Survey (NHANES) 2011-2016, we included 2747 (1434 male and 1313 nonpregnant female) adults ≥20 years without evidence of liver dysfunction (ALT<30 in male and <19 in female, negative viral serologies, no excess alcohol consumption, no elevated transferrin saturation, AST/ALT <2). Serum AST/ALT ratio was categorized into sex-specific quartiles (female: <1.12, 1.12-1.29, 1.29-1.47, ≥1.47 and male: <0.93, 0.93-1.09, 1.09-1.26, ≥1.26). The primary outcome was insulin resistance, as determined by Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index score ≥3. Covariate-adjusted odds ratios (ORs) were estimated. Study analysis completed from 13 March 2020 to 21 April 2021. RESULTS: Among the 2747 individuals, 33% had insulin resistance. Those in the lowest quartile (Q1) of AST/ALT had 75% higher adjusted odds of insulin resistance compared to the highest quartile (Q4) [aOR (95% confidence interval (CI), 1.75 (1.20-2.57)]. This association was more pronounced in those with elevated BMI [Q1 vs. Q4; BMI ≥ 25: 2.29 (1.58-3.33), BMI < 25: 0.66 (0.26-1.69); NAFLD per Fatty Liver Index ≥ 60: 2.04 (1.21-3.44), No NAFLD: 1.68 (0.94-3.01)]. CONCLUSION: Lower AST/ALT ratio is associated with increased insulin resistance among those with healthy-range ALT, especially in those with BMI greater than or equal to 25 kg/m2.

Identification of optimal contemporary antiemetic prophylaxis for doxorubicin-cyclophosphamide chemotherapy in Chinese cancer patients: post-hoc analysis of 3 prospective studies.

OBJECTIVE: Chemotherapy-induced nausea and vomiting (CINV) are common with doxorubicin-cyclophosphamide (AC) chemotherapy. Recommended antiemetic regimens incorporate neurokinin-1 receptor antagonist (NK1RA), 5-hydroxytryptamine type-3 receptor antagonist (5HT3RA), corticosteroid, and dopamine antagonists. This post-hoc analysis compared results of 3 prospective antiemetic studies conducted among Chinese breast cancer patients who received (neo)adjuvant AC, in order to identify optimal antiemetic prophylaxis. METHODS: A total of 304 patients were included: Group 1, ondansetron/dexamethasone (D1); Group 2, aprepitant/ondansetron/dexamethasone (D1); Group 3, aprepitant/ondansetron/dexamethasone (D1-3); Group 4, aprepitant/ondansetron/dexamethasone (D1-3)/olanzapine; and Group 5, netupitant/palonosetron/dexamethasone (D1-3). Antiemetic efficacies of Groups 3, 4, and 5 during cycle 1 of AC were individually compared with Group 1. In addition, emesis outcomes of patients in Groups 3 and 5, and those of Groups 2 and 3, were compared. RESULTS: When comparing efficacies of a historical doublet (5HT3RA/dexamethasone) with triplet antiemetic regimens (NK1RA/5HT3RA/dexamethasone) with/without olanzapine, complete response (CR) percentages and quality of life (QOL) in overall phase of cycle 1 AC were compared between Group 1 and the other groups: Group 1 vs. 3, 41.9% vs. 38.3% (P = 0.6849); Group 1 vs. 4, 41.9% vs. 65.0% (P = 0.0107); and Group 1 vs. 5, 41.9% vs. 60.0% (P = 0.0460). Groups 4 and 5 achieved a better QOL. When comparing netupitant-based (Group 3) with aprepitant-based (Group 5) triplet antiemetics, CR percentages were 38.3% vs. 60.0%, respectively (P = 0.0176); Group 5 achieved a better QOL. When comparing 1 day (Group 2) vs. 3 day (Group 3) dexamethasone, CR percentages were 46.8% and 38.3%, respectively (P = 0.3459); Group 3 had a worse QOL. CONCLUSIONS: Aprepitant-containing triplets were non-superior to doublet antiemetics. Netupitant-containing triplets and adding olanzapine to aprepitant-containing triplets were superior to doublets. Netupitant/palonosetron/dexamethasone was superior to aprepitant/ondansetron/dexamethasone. Protracted administration of dexamethasone provided limited additional benefit.

A Combinatorial CRISPR-Cas9 Screen Identifies Ifenprodil as an Adjunct to Sorafenib for Liver Cancer Treatment.

Systematic testing of existing drugs and their combinations is an attractive strategy to exploit approved drugs for repurposing and identifying the best actionable treatment options. To expedite the search among many possible drug combinations, we designed a combinatorial CRISPR-Cas9 screen to inhibit druggable targets. Coblockade of the N-methyl-d-aspartate receptor (NMDAR) with targets of first-line kinase inhibitors reduced hepatocellular carcinoma (HCC) cell growth. Clinically, HCC patients with low NMDAR1 expression showed better survival. The clinically approved NMDAR antagonist ifenprodil synergized with sorafenib to induce the unfolded protein response, trigger cell-cycle arrest, downregulate genes associated with WNT signaling and stemness, and reduce self-renewal ability of HCC cells. In multiple HCC patient-derived organoids and human tumor xenograft models, the drug combination, but neither single drug alone, markedly reduced tumor-initiating cancer cell frequency. Because ifenprodil has an established safety history for its use as a vasodilator in humans, our findings support the repurposing of this drug as an adjunct for HCC treatment to improve clinical outcome and reduce tumor recurrence. These results also validate an approach for readily discovering actionable combinations for cancer therapy. SIGNIFICANCE: Combinatorial CRISPR-Cas9 screening identifies actionable targets for HCC therapy, uncovering the potential of combining the clinically approved drugs ifenprodil and sorafenib as a new effective treatment regimen.

Structure, photophysical and electrochemical properties, biomolecular interactions, and intracellular uptake of luminescent cyclometalated iridium(III) dipyridoquinoxaline complexes.

A series of luminescent cyclometalated iridium(III) dipyridoquinoxaline complexes [Ir(N--C)(2)(N--N)](PF(6)) (HN--C = 1-phenylpyrazole, Hppz, N--N = dipyrido[3,2-f:2',3'-h]quinoxaline, dpq (1a), 2-(n-butylamido)dipyrido[3,2-f:2',3'-h]quinoxaline, dpqa (1b); HN--C = 7,8-benzoquinoline, Hbzq, N--N = dpq (2a), dpqa (2b); HN--C = 2-phenylquinoline, Hpq, N--N = dpq (3a), dpqa (3b)) has been synthesized and characterized. Cyclic voltammetric studies revealed a reversible or quasi-reversible iridium(IV/III) oxidation couple at about +1.13 to +1.32 V and a reversible diimine reduction couple at about -1.10 to -1.29 V versus SCE. Upon photoexcitation, all the complexes displayed intense and long-lived green to orange triplet metal-to-ligand charge-transfer ((3)MLCT) (dpi(Ir) --> pi*(dpq or dpqa)) emission in aprotic organic solvents at room temperature and in low-temperature glass. In aqueous solution, these complexes were only weakly emissive or even non-emissive. The lipophilicity of all the complexes has been determined by reversed-phase HPLC. The cytotoxicity of these iridium(III) complexes toward the human cervix epithelioid carcinoma (HeLa) and Madin-Darby canine kidney (MDCK) cell lines has been evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cellular uptake of the complexes by MDCK cells has been examined by laser-scanning confocal microscopy. Most importantly, apparent nucleolar staining was observed after the cells were treated by the complexes. The interactions of these complexes with proteins, DNA, and RNA have also been studied by emission titrations and SDS-PAGE gel staining. The results revealed that the complexes bound to the hydrophobic pockets of proteins, intercalated into the base-pairs of double-stranded DNA, but did not appear to interact with RNA.

Clinical Manifestations of Hepatic Encephalopathy.

Hepatic encephalopathy (HE) occurs in patients with acute-on-chronic liver disease. It has a wide progression of symptoms, with its initial presentation being subtle. The symptoms of HE mainly affect mental status, the musculoskeletal system, and mood/behavior. Its severity ranges from minor disturbances in sleep-wake cycle to the patient being comatose. HE is categorized based on 4 main features: the underlying disease, the severity of manifestations, the time course, and whether precipitating factors are present. The severity of the manifestations is classically identified using the West Haven Criteria. There are several other clinical tests, but they require further validation.

Laboratory Abnormalities of Hepatic Encephalopathy.

Currently, there is no gold standard serologic or imaging modality to detect hepatic encephalopathy (HE). It is a clinical diagnosis gathered from the history and physical. Imaging is nonspecific; however, PET and MRI have shown areas of utility, but are not widely available, cost-efficient, or necessary for diagnosis. Electroencephalogram has shown promise as it can be used in conjunction with the Portal Systemic Hepatic Encephalopathy Score test to diagnose minimal HE. Further research on these techniques would need to be performed to identify strict criteria and cutoffs for diagnosing HE as well as associated sensitivities and specificities.

Learning curve for EUS-guided biliary drainage: What have we learned?

BACKGROUND AND OBJECTIVE: EUS-guided-biliary drainage (EUS-BD) is an efficacious and safe option for patients who fail ERCP. EUS-BD is a technically challenging procedure. The aim of this study was to define the learning curve for EUS-BD. METHODS: Consecutive patients undergoing EUS-BD by a single operator were included for a prospective registry over 6 years. Demographics, procedural information, adverse events, and follow-up data were collected. Nonlinear regression and CUSUM analyses were conducted for the learning curve. Technical success was defined as successful stent placement. Clinical success was defined as resolution of jaundice and/or at least a 30% reduction in the pretreatment bilirubin level within a week after placement or normalization of bilirubin within 30 days. RESULTS: Seventy-two patients were included in the study (53% male, mean age 67 years). Technical success was achieved in 69 patients (96%). Clinical success was achieved in 59/69 patients (86%). Seven patients (10%) had adverse events including bleeding (n = 6) and liver abscess (n = 1). The median procedural time was 59 min (range 36-138 min). This was achieved at the 32nd procedure. Procedural durations were further reduced to 50 min and below after the 50th procedure in a nonlinear pattern. This suggests that procedural durations approach a potential plateau after 100 cases. CONCLUSION: Endoscopists experienced in EUS-BD are expected to achieve a reduction in procedural time over successive cases, with efficiency reached at 59 min and a learning rate of 32 cases. Continued improvement is demonstrated with additional experience, with mastery suggested after approximately 100 cases.

Dataset on chemotherapy-induced nausea and vomiting (CINV) and quality of life (QOL) during multiple chemotherapy cycles among a Chinese breast cancer patient population who were randomized to antiemetic regimens with or without olanzapine.

Chemotherapy-induced nausea and vomiting (CINV) are highly distressing symptoms for cancer patients undergoing cytotoxic chemotherapy. This dataset was obtained from a homogenous group of Chinese breast cancer patients who were uniformly planned to receive a highly emetogenic (neo)adjuvant chemotherapy regimen, consisting of doxorubicin and cyclophosphamide (commonly known as AC). Patients were being randomized to one of the two antiemetic regimens: aprepitant, ondansetron and dexamethasone with (the Olanzapine arm) or without olanzapine (the Standard arm). Patients underwent self-reported diaries and questionnaires to record their nausea and vomiting symptoms, use of rescue medication as well as their quality of life (QOL). The primary and secondary endpoints have focused on efficacy analysis during the first cycle of AC chemotherapy; the results have been reported in The Breast [1]. In this Data in Brief article, we provide outcome of the analysis of data collected during multiple cycles of chemotherapy. The data reported here include the proportion of patients with "Complete Response", "Complete Protection" and "Total Control" of emesis in the acute (0-24 h), delayed (24-120 h) and overall periods (0-120 h), as well as QOL data during all the 4 cycles of AC.

Patient-derived tumor organoid predicts drugs response in glioblastoma: A step forward in personalized cancer therapy?

Despite significant medical advances, glioblastoma multiforme (GBM) remains a formidable therapeutic challenge. Advent of targeted capture sequencing and patients-derived organoid cultures may hold the key to scientifically sound individualized treatment approaches. We report on our initial experience of using the combination of these two technologies to create a tailored approach of systemic therapies for a patient with GBM, which challenges the conventional treatment paradigm, as well as specifically highlighting the complexities of such an approach and the potential for a more favorable treatment outcome.

AI-doscopist: a real-time deep-learning-based algorithm for localising polyps in colonoscopy videos with edge computing devices.

We have designed a deep-learning model, an "Artificial Intelligent Endoscopist (a.k.a. AI-doscopist)", to localise colonic neoplasia during colonoscopy. This study aims to evaluate the agreement between endoscopists and AI-doscopist for colorectal neoplasm localisation. AI-doscopist was pre-trained by 1.2 million non-medical images and fine-tuned by 291,090 colonoscopy and non-medical images. The colonoscopy images were obtained from six databases, where the colonoscopy images were classified into 13 categories and the polyps' locations were marked image-by-image by the smallest bounding boxes. Seven categories of non-medical images, which were believed to share some common features with colorectal polyps, were downloaded from an online search engine. Written informed consent were obtained from 144 patients who underwent colonoscopy and their full colonoscopy videos were prospectively recorded for evaluation. A total of 128 suspicious lesions were resected or biopsied for histological confirmation. When evaluated image-by-image on the 144 full colonoscopies, the specificity of AI-doscopist was 93.3%. AI-doscopist were able to localise 124 out of 128 polyps (polyp-based sensitivity = 96.9%). Furthermore, after reviewing the suspected regions highlighted by AI-doscopist in a 102-patient cohort, an endoscopist has high confidence in recognizing four missed polyps in three patients who were not diagnosed with any lesion during their original colonoscopies. In summary, AI-doscopist can localise 96.9% of the polyps resected by the endoscopists. If AI-doscopist were to be used in real-time, it can potentially assist endoscopists in detecting one more patient with polyp in every 20-33 colonoscopies.

A randomized study of olanzapine-containing versus standard antiemetic regimens for the prevention of chemotherapy-induced nausea and vomiting in Chinese breast cancer patients.

OBJECTIVES: Chemotherapy-induced nausea and vomiting (CINV) are distressing symptoms. This randomized study evaluated the antiemetic efficacies of standard antiemetic regimen with/without olanzapine. PATIENTS AND METHODS: Eligible patients were chemotherapy-naive Chinese breast cancer patients who were planned for (neo)adjuvant doxorubicin/cyclophosphamide. Antiemetic regimen for all studied population included aprepitant, ondansetron and dexamethasone; patients were randomized to Olanzapine (with olanzapine) or Standard arms (without olanzapine). Patients filled in self-reported diaries and completed visual analogue scales for nausea, as well as Functional Living Index-Emesis questionnaires. Blood profiles including fasting glucose and lipids were monitored. RESULTS: 120 patients were randomized. In Cycle 1 doxorubicin/cyclophosphamide, the Olanzapine arm had significantly higher rates of "Complete Response" than the Standard arm: 65.0% vs 38.3% in the overall period (p = 0.0035), 70.0% vs 51.7% in the acute period (p = 0.0397) and 92.9% vs 74.2% in the delayed period (p = 0.0254). Olanzapine arm also had significantly higher rates of "No significant nausea" and "No nausea" during all 3 time-frames and better QOL. Similar findings were also revealed throughout multiple cycles. Pre-study abnormalities in glucose and lipids occurred in 39.7% and 34.2% of the studied population respectively; there were no differences in these parameters between the two arms at end-of-study assessment. CONCLUSION: The addition of olanzapine to standard aprepitant-based antiemetic regimen provides clinically meaningful improvement in controlling CINV. This was associated with a positive impact on QOL and tolerable toxicity profiles among Chinese breast cancer patients receiving doxorubicin/cyclophosphamide chemotherapy. Further studies on metabolic profiles of breast cancer patients are warranted.

Promoting the Delivery of Nanoparticles to Atherosclerotic Plaques by DNA Coating.

Many nanoparticle-based carriers to atherosclerotic plaques contain peptides, lipoproteins, and sugars, yet the application of DNA-based nanostructures for targeting plaques remains infrequent. In this work, we demonstrate that DNA-coated superparamagnetic iron oxide nanoparticles (DNA-SPIONs), prepared by attaching DNA oligonucleotides to poly(ethylene glycol)-coated SPIONs (PEG-SPIONs), effectively accumulate in the macrophages of atherosclerotic plaques following an intravenous injection into apolipoprotein E knockout (ApoE-/-) mice. DNA-SPIONs enter RAW 264.7 macrophages faster and more abundantly than PEG-SPIONs. DNA-SPIONs mostly enter RAW 264.7 cells by engaging Class A scavenger receptors (SR-A) and lipid rafts and traffic inside the cell along the endolysosomal pathway. ABS-SPIONs, nanoparticles with a similarly polyanionic surface charge as DNA-SPIONs but bearing abasic oligonucleotides also effectively bind to SR-A and enter RAW 264.7 cells. Near-infrared fluorescence imaging reveals evident localization of DNA-SPIONs in the heart and aorta 30 min post-injection. Aortic iron content for DNA-SPIONs climbs to the peak (∼60% ID/g) 2 h post-injection (accompanied by profuse accumulation in the aortic root), but it takes 8 h for PEG-SPIONs to reach the peak aortic amount (∼44% ID/g). ABS-SPIONs do not appreciably accumulate in the aorta or aortic root, suggesting that the DNA coating (not the surface charge) dictates in vivo plaque accumulation. Flow cytometry analysis reveals more pronounced uptake of DNA-SPIONs by hepatic endothelial cells, splenic macrophages and dendritic cells, and aortic M2 macrophages (the cell type with the highest uptake in the aorta) than PEG-SPIONs. In summary, coating nanoparticles with DNA is an effective strategy of promoting their systemic delivery to atherosclerotic plaques.

MRI detection of endothelial cell inflammation using targeted superparamagnetic particles of iron oxide (SPIO).

BACKGROUND: There is currently no clinical imaging technique available to assess the degree of inflammation associated with atherosclerotic plaques. This study aims to develop targeted superparamagnetic particles of iron oxide (SPIO) as a magnetic resonance imaging (MRI) probe for detecting inflamed endothelial cells. METHODS: The in vitro study consists of the characterisation and detection of inflammatory markers on activated endothelial cells by immunocytochemistry and MRI using biotinylated anti-P-selectin and anti-VCAM-1 (vascular cell adhesion molecule 1) antibody and streptavidin conjugated SPIO. RESULTS: Established an in vitro cellular model of endothelial inflammation induced with TNF-α (tumor necrosis factor alpha). Inflammation of endothelial cells was confirmed with both immunocytochemistry and MRI. These results revealed both a temporal and dose dependent expression of the inflammatory markers, P-selectin and VCAM-1, on exposure to TNF-α. CONCLUSION: This study has demonstrated the development of an in vitro model to characterise and detect inflamed endothelial cells by immunocytochemistry and MRI. This will allow the future development of contrast agents and protocols for imaging vascular inflammation in atherosclerosis. This work may form the basis for a translational study to provide clinicians with a novel tool for the in vivo assessment of atherosclerosis.