Swelling, Rupture and Endosomal Escape of Biological Nanoparticles Per Se and Those Fused with Liposomes in Acidic Environment.

Biological nanoparticles (NPs), such as extracellular vesicles (EVs), exosome-mimetic nanovesicles (EMNVs) and nanoghosts (NGs), are perspective non-viral delivery vehicles for all types of therapeutic cargo. Biological NPs are renowned for their exceptional biocompatibility and safety, alongside their ease of functionalization, but a significant challenge arises when attempting to load therapeutic payloads, such as nucleic acids (NAs). One effective strategy involves fusing biological NPs with liposomes loaded with NAs, resulting in hybrid carriers that offer the benefits of both biological NPs and the capacity for high cargo loads. Despite their unique parameters, one of the major issues of virtually any nanoformulation is the ability to escape degradation in the compartment of endosomes and lysosomes which determines the overall efficiency of nanotherapeutics. In this study, we fabricated all major types of biological and hybrid NPs and studied their response to the acidic environment observed in the endolysosomal compartment. In this study, we show that EMNVs display increased protonation and swelling relative to EVs and NGs in an acidic environment. Furthermore, the hybrid NPs exhibit an even greater response compared to EMNVs. Short-term incubation of EMNVs in acidic pH corresponding to late endosomes and lysosomes again induces protonation and swelling, whereas hybrid NPs are ruptured, resulting in the decline in their quantities. Our findings demonstrate that in an acidic environment, there is enhanced rupture and release of vesicular cargo observed in hybrid EMNVs that are fused with liposomes compared to EMNVs alone. This was confirmed through PAGE electrophoresis analysis of mCherry protein loaded into nanoparticles. In vitro analysis of NPs colocalization with lysosomes in HepG2 cells demonstrated that EMNVs mostly avoid the endolysosomal compartment, whereas hybrid NPs escape it over time. To conclude, (1) hybrid biological NPs fused with liposomes appear more efficient in the endolysosomal escape via the mechanism of proton sponge-associated scavenging of protons by NPs, influx of counterions and water, and rupture of endo/lysosomes, but (2) EMNVs are much more efficient than hybrid NPs in actually avoiding the endolysosomal compartment in human cells. These results reveal biochemical differences across four major types of biological and hybrid NPs and indicate that EMNVs are more efficient in escaping or avoiding the endolysosomal compartment.

An Overview of Adalimumab Therapy for Ankylosing Spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a chronic inflammatory disease known for causing pain, stiffness, and reduced mobility in the axial skeleton. Adalimumab, a tumor necrosis factor (TNF) inhibitor, has emerged as a promising therapeutic option for AS. METHODS: This systematic review involved a comprehensive search of randomized controlled trials related to AS treatment, conducted in major databases such as MEDLINE, Google Scholar, and PubMed. The search terms encompassed ankylosing spondylitis, adalimumab, methotrexate, other non-biologic DMARDs, glucocorticoids, NSAIDs, and analgesics. A total of 14 randomized controlled trials with 4,500 participants were included in the review. RESULTS: The review's results revealed that adalimumab demonstrated notable superiority when compared to a placebo. It effectively reduced disease activity, improved physical function, and lowered inflammatory markers such as C-reactive protein and erythrocyte sedimentation rate. Adalimumab demonstrated a favorable safety profile, with adverse events comparable to those observed with placebo. CONCLUSION: Based on the results, adalimumab is deemed an effective treatment for AS, showcasing its potential as a first-line therapeutic option. Notably, no significant increase in adverse events was observed compared to placebo. However, the conclusion emphasizes the need for further studies with extended follow-up durations to ascertain the long-term efficacy and safety of adalimumab in AS management. This systematic review provides valuable insights supporting the use of adalimumab in the treatment of AS and underscores the importance of ongoing investigations into its long-term effects to optimize its clinical utilization in AS patients.

HPTLC and FTIR Fingerprinting of Olive Leaves Extracts and ATR-FTIR Characterisation of Major Flavonoids and Polyphenolics.

The aim of this study was to analyse the effect of spontaneous microbial maceration on the release and extraction of the flavonoids and phenolics from olive leaves. Bioprofiling based on thin-layer chromatography effect-directed detection followed by ATR-FTIR spectroscopy proved to be a reliable and convenient method for simultaneous comparison of the extracts. Results show that fermentation significantly enhances the extraction of phenolic compounds and flavonoids. The polyphenolic content was increased from 6.7 µg GAE (gallic acid equivalents) to 25.5 µg GAE, antioxidants from 10.3 µg GAE to 25.3 µg GAE, and flavonoid content from 42 µg RE (rutin equivalents) to 238 µg RE per 20 µL of extract. Increased antioxidant activity of fermented ethyl acetate extracts was attributed to the higher concentration of extracted flavonoids and phenolic terpenoids, while increased antioxidant activity in fermented ethanol extract was due to increased extraction of flavonoids as extraction of phenolic compounds was not improved. Lactic acid that is released during fermentation and glycine present in the olive leaves form a natural deep eutectic solvent (NADES) with significantly increased solubility for flavonoids.

HPTLC and ATR/FTIR Characterization of Antioxidants in Different Rosemary Extracts.

The effect of spontaneous fermentation by lactic acid bacteria on the extraction yield of bioactive compounds and antioxidant activity from rosemary leaf extracts was investigated using high-performance thin-layer chromatography (HPTLC). Brining and spontaneous fermentation with lactic acid bacteria more than doubled extraction of polyphenolics and antioxidants from the rosemary leaves. The results show that lactic acid fermentation enhances antioxidant activity in extracts by increasing the total phenolic content but does not increase extraction of phytosterols. Increased extraction of phenolic oxidants during fermentation assisted extraction, results from the in situ generated natural eutectic solvent from the plant sample. ATR-FTIR spectra from the bioactive bands suggests that this increased antioxidant activity is associated with increased extraction of rosmarinic acid, depolymerised lignin, abietane diterpenoids and 15-hydroxy-7-oxodehydroabietic acid.

The effect of extractive lacto-fermentation on the bioactivity and natural products content of Pittosporum angustifolium (gumbi gumbi) extracts.

Pittosporum angustifolium, known as gumbi gumbi, is a native Australian plant, which has traditionally been used as an Aboriginal medicine. This study investigates the effect of different solvents and extractive fermentation on the content and natural products composition of Pittosporum angustifolium extracts, and compares their antioxidant activity, in vitro α-amylase inhibition, and anti-inflammatory properties. Anti-inflammatory activity of the extracts was determined by measuring the inhibition of nitric oxide (NO) production. Extracts were characterised with FTIR-ATR spectroscopy, and screened for antioxidant activities and α-amylase inhibitory activity via High-performance thin-layer chromatography (HPTLC)-Effect-directed analysis (EDA) with direct bioautography. HPTLC combined with chemical derivatization and bioassays was used for EDA screening. The results show that lactic acid fermentation of gumbi gumbi leaves boosts the antioxidant activity in extracts by increasing the total phenolic content, but does not affect (increase or decrease) α-amylase inhibitory activity or nitrogen scavenging/anti-inflammatory activity. Analysis of the ATR-FTIR spectra from the band at RF = 0.85 that inhibits α-amylase, suggests that fatty acid esters are responsible for the enzyme inhibition; both saturated fatty acid esters in unfermented extracts and unsaturated fatty acid esters in fermented extracts. The ATR-FTIR spectra of the polyphenolics in fermented extracts (RF = 0.15-0.20) suggests the presence of soluble lignin fragments (i.e. lignins depolymerized into monomers and oligomers during the fermentation process).

Analytical Strategies in Lipidomics for Discovery of Functional Biomarkers from Human Saliva.

Human saliva is increasingly being used and validated as a biofluid for diagnosing, monitoring systemic disease status, and predicting disease progression. The discovery of biomarkers in saliva biofluid offers unique opportunities to bypass the invasive procedure of blood sampling by using oral fluids to evaluate the health condition of a patient. Saliva biofluid is clinically relevant since its components can be found in plasma. As salivary lipids are among the most essential cellular components of human saliva, there is great potential for their use as biomarkers. Lipid composition in cells and tissues change in response to physiological changes and normal tissues have a different lipid composition than tissues affected by diseases. Lipid imbalance is closely associated with a number of human lifestyle-related diseases, such as atherosclerosis, diabetes, metabolic syndromes, systemic cancers, neurodegenerative diseases, and infectious diseases. Thus, identification of lipidomic biomarkers or key lipids in different diseases can be used to diagnose diseases and disease state and evaluate response to treatments. However, further research is needed to determine if saliva can be used as a surrogate to serum lipid profiles, given that highly sensitive methods with low limits of detection are needed to discover salivary biomarkers in order to develop reliable diagnostic and disease monitoring salivary tests. Lipidomic methods have greatly advanced in recent years with a constant advance in mass spectrometry (MS) and development of MS detectors with high accuracy and high resolution that are able to determine the elemental composition of many lipids.

An improved extraction protocol for therapeutic dabigatran monitoring using HPLC-MS/MS.

A new sample extraction protocol was developed for pharmacokinetic studies of dabigatran with high-performance liquid chromatography separation - electrospray ionization time-of-flight mass spectrometry analysis. After protein precipitation with acetonitrile, free dabigatran and its metabolites are separated into water phase by water-dichloromethane liquid-liquid extraction to purify the sample from proteins and endogenous lipophilic compounds. Chromatographic separation was achieved on an Agilent Zorbax SB-CN column (150 × 4.6 mm, 5 µm)) using 0.1% aqueous solution of formic acid and acetonitrile (80:20) as the mobile phase. Agilent Zorbax SB-CN column was selected to improve sample resolution and to avoided early elution of dabigatran previously seen when using a C18 column. The extended calibration curve was constructed from 5 to 1000 ng/L while precision and accuracy were assessed at four levels across the linear dynamic ranges. Within-run precision was <5.6% and the between-run precision was <3.9%. The method accuracy ranged from 89.8% to 104.4%. The developed method was successfully applied to 30 patient samples to evaluate antithrombotic efficacy and anticoagulant activity of dabigatran following knee endoprosthesis surgery.