Orthogonal pipelines for lipid nanoparticle evaluation

Ribonucleic acid (RNA) drugs pose promising candidates for gene therapy in treatment resistant conditions and rare diseases. The FDA approval of siRNA Onpattro® in 2018 mRNA-LNP Spikevax® and Comirnaty® COVID-19 vaccinations in 2021[1] ignited research interests as these were the first siRNA and mRNA candidates to utilize lipid nanoparticles (LNPs) as a drug delivery platform. As the RNA-LNP research field is rapidly growing, robust, high-resolution separation techniques coupled to in-line detectors are required to analyze particle key quality attributes and accelerate the successful clinical translation of RNA-LNP therapies. Asymmetric-Flow Field Flow Fraction (AF4) and Size Exclusion Chromatography (SEC) are robust approaches for the characterization of oligo-LNPs [2, 3]. AF4 utilizes perpendicular field induction and particle diffusion-based separation, whereas SEC uses LNP-stationary phase interactions for separation. The goal of this study was to develop separation pipelines for the high-resolution analysis of LNPs. Briefly, we prepared (6Z,9Z,28Z,31Z)-heptatriaconta6,9,28,31-tetraen-19-yl-4-(dimethylamino)- butanoate:cholesterol: 1,2-distearoyl-sn-glycero-3-phosphocholine: 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (MC3:CHOL:DSPC:DMG-PEG2000) LNPs and 8-[(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester:cholesterol: 1,2-distearoyl-sn-glycero-3-phosphocholine: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (SM-102:CHOL:DSPC:DMG-PEG2000) using microfluidics at 50:38.5:10:1.5 mol% ratio. Here, we performed AF4, combined with in-line dynamic light scattering, multi-angle light scattering, and UV detection. Using these detectors, we measured key particle quality attributes including particle size, polydispersity index (PDI), and shape factor. The properties were evaluated alongside oligo-LNP samples that had not been subjected to separation. Manufacture of LNPs using microfluidics-based analysis led to PolyA MC3-LNPs in the 56.5 nm ± 1.2 nm size range with a corresponding PDI of 0.12 ± 0.02, and PolyA SM-102-LNPs of 48.5 nm ± 1.1 nm with a PDI of 0.10 ± 0.01. Our findings show the presence of sub-populations within LNP samples, which cannot be detected using routine particle metrology techniques such as nanoparticle tracking analysis and dynamic light scattering. Our results highlight the need for developing more high-resolution approaches for the analysis of LNPs and linking these to input materials and process parameter design

Go with the flow- High resolution lipid nanoparticle metrology

Asymmetric-Flow Field Flow Fraction (AF4) is a rapidly growing analytical technique for the separation and in-line analysis of RNA-loaded lipid nanoparticles (RNA-LNPs). This high resolution, robust technique will facilitate the clinical translation of new RNA-LNP formulations by evaluating nanoparticle critical quality attributes (CQAs) and enhance the knowledge between RNA-LNP design, manufacture, and associated physical chemical properties [1]. The use of high-resolution analytical techniques can bridge existing knowledge gaps and accelerate the successful clinical translation of RNA-LNP therapies. The aim of this work was to develop a method for the characterization of RNA-LNPs using Frit Inlet (FI) AF4-Multi angle light scattering (MALS)-UV to determine LNP particle size distribution

Higher or lower? : the resolution of analytical pipelines for the evaluation of lipid nanoparticle critical quality attributes

Dynamic light scattering (DLS) is a routinely used analytical technique to measure the size distribution of a colloidal sample. Nanoparticle tracking analysis (NTA) has more recently emerged as an orthogonal technique to determine particle size and estimated concentration, although widespread use of NTA for lipid nanoparticle (LNP) analysis is not reported. Here, we use DLS and NTA to screen the stability of oligonucleotide drug loaded lipid nanoparticles (oligo-LNPs) at ultra-low storage temperatures (-80 °C) in the presence and absence of a cryoprotectant (20 % sucrose, w/v) as a case study to highlight differences in technique resolution. NTA was able to detect additional LNP subpopulations samples for all measured samples, whereas DLS was unable to detect these subpopulations. Our study conveys that the use of orthogonal sizing techniques can support early-stage product development, where a more in-depth analysis of formulation and process parameters on LNP stability would support comprehensive understanding of variable defining product stability

Evaluation of the critical quality attributes of lipid nanoparticles stored under different conditions

Lipid nanoparticles (LNPs) are emerging new modalities for mRNA therapeutics which have been in the spotlight for the past decade. Since these are relatively new drug delivery systems compared to conventional medicines, new analytical techniques for the robust characterization of their critical quality attributes (CQAs) are needed [1]. It has been reported that several stimuli can affect the stability of the LNPs such as leakage of the nucleic acid cargo from the nanoparticle and LNP aggregation, resulting in low translation efficiency. Hence, understanding the duration of stability is key during formulation development. The aim of the present study is to evaluate the stability of PolyA-LNPs: 1) Stored at different temperatures (4oC and 25oC); 2) Dialysed in the absence and presence of cryoprotectant sucrose. We measured the impact of the above storage conditions on LNP physicochemical parameters

Physicochemical characterisation of poly(A) lipid nanoparticles : effect of cryoprotectant and temperature storage conditions

Lipid nanoparticles (LNPs) are emerging new modalities for mRNA therapeutics which have been in the spotlight for the past decade. Since these are relatively new drug delivery systems compared to conventional medicines, new analytical techniques for the robust characterization of their critical quality attributes (CQAs) are needed [1]. It has been reported that several stimuli can affect the stability of the LNPs such as leakage of the nucleic acid cargo from the nanoparticle and LNP aggregation, resulting in low translation efficiency [2]. Hence, understanding the duration of stability is key during formulation development. The aim of the present study is to evaluate the stability of PolyA-LNPs: 1) Stored at different temperatures (4oC and 25oC); 2) Dialysed in the absence and presence of cryoprotectant sucrose. We measured the impact of the above storage conditions on LNP physicochemical parameters

GSuite HyperBrowser: integrative analysis of dataset collections across the genome and epigenome

Background: Recent large-scale undertakings such as ENCODE and Roadmap Epigenomics have generated experimental data mapped to the human reference genome (as genomic tracks) representing a variety of functional elements across a large number of cell types. Despite the high potential value of these publicly available data for a broad variety of investigations, little attention has been given to the analytical methodology necessary for their widespread utilisation. Findings: We here present a first principled treatment of the analysis of collections of genomic tracks. We have developed novel computational and statistical methodology to permit comparative and confirmatory analyses across multiple and disparate data sources. We delineate a set of generic questions that are useful across a broad range of investigations and discuss the implications of choosing different statistical measures and null models. Examples include contrasting analyses across different tissues or diseases. The methodology has been implemented in a comprehensive open-source software system, the GSuite HyperBrowser. To make the functionality accessible to biologists, and to facilitate reproducible analysis, we have also developed a web-based interface providing an expertly guided and customizable way of utilizing the methodology. With this system, many novel biological questions can flexibly be posed and rapidly answered. Conclusions: Through a combination of streamlined data acquisition, interoperable representation of dataset collections, and customizable statistical analysis with guided setup and interpretation, the GSuite HyperBrowser represents a first comprehensive solution for integrative analysis of track collections across the genome and epigenome. The software is available at: https://hyperbrowser.uio.no.This work was supported by the Research Council of Norway (under grant agreements 221580, 218241, and 231217/F20), by the Norwegian Cancer Society (under grant agreements 71220’PR-2006-0433 and 3485238-2013), and by the South-Eastern Norway Regional Health Authority (under grant agreement 2014041).Peer Reviewe

Ballota saxatilis from Jordan: Evaluation of Essential Oil Composition and Phytochemical Profiling of Crude Extracts and Their In-Vitro Antioxidant Activity

The chemical composition of essential oil extracted from the aerial parts of Ballota saxatilis Sieber ex C.Presl from Jordan has been elucidated by gas chromatography–mass spectrometry (GC-MS). Additionally, aqueous methanol (BsA), Butanol (BsB) and water (BsW) extracts were screened for their total phenol content (TPC), total flavonoid content (TFC), and antioxidant activities using the 2,2 Diphenyl-1-picrylhydrazyl (DPPH) and 2,2-Azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) methods. The most potent extracts were screened for their phenolic acids and flavonoid content using liquid the chromatography–mass spectrometry (LC-MS) technique. The results indicated that the essential oil predominantly contained cis-pinane (14.76%), β-caryophyllene (8.91%) and allo-aromadendrene epoxide (6.39%). Among the different extracts investigated, the BsB fraction had the most TPC and TFC (455.79 ± 1.03 µg gallic acid/g dry extract; 272.62 ± 8.28 µg quercetin/g dry extract, respectively) and had the best radical and radical cation scavenging activities, as determined using the DPPH and ABTS methods. Quantitative and qualitative LC-MS analyses of BsA and BsB using LC-MS revealed each of the kaempferol-3-O-rutinoside (30.29%), chrysoeriol-7-glucoside (7.93%) and luteolin 7-o-glucoside (7.76%) as the main constituents of the BsA fraction. The BsB fraction was rich in 7,4′-dimethoxy-3-hydroxyflavone (34.68%), kaempferol-3,7,4′-trimethyl ether (29.17%) and corymbosin (9.66%) and lower concentration levels of kaempferol-3-O-rutinoside (1.63%) and chrysoeriol-7-glucoside (0.51%)

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Single‐dose ibuprofen induced Stevens–Johnson Syndrome

Key Clinical Message Ibuprofen single dose may rarely induce Stevens–Johnson Syndrome, emphasizing the vital need for heightened vigilance in healthcare and public awareness for safer medication practices. Abstract Stevens–Johnson Syndrome (SJS) is a severe and potentially life‐threatening skin disorder associated with certain medications, including ibuprofen. We present a case of a 45‐year‐old woman who developed SJS following a single dose of ibuprofen. Despite its rarity, this case underscores the importance of heightened vigilance in healthcare and public awareness regarding the potential risks of commonly used medications. Prompt recognition of SJS symptoms and immediate medical intervention are crucial for patient outcomes. Healthcare providers should exercise caution when prescribing ibuprofen, particularly in patients with a history of adverse drug reactions. This case emphasizes the need for ongoing monitoring, patient education, and informed decision‐making to promote medication safety and optimal patient care

Case-control association between CCT-associated variants and keratoconus in a Saudi Arabian population

10.1186/s12952-015-0029-5Journal of Negative Results in BioMedicine1412