Suspensions of antibiotics in self-emulsifying oils as a novel approach to formulate eye drops with substances which undergo hydrolysis in aqueous environment.

The aim of this study was to develop eye-drops with cefuroxime (CEF) sodium or vancomycin (VAN) hydrochloride, antibiotics that are instable in water. Anhydrous self-emulsifying oils (SEO) are proposed as a carrier and antibiotics are suspended. In the contact with tear fluid, the formulation should transform into emulsion, with fast dissolution of an antibiotic. CEF or VAN (5% w/w) was suspended in SEO carriers prepared by dissolving surfactants (Tween 20 or Span 80 5% w/w) in Miglyol, castor oil, or olive oil. Formulations with or without sodium citrate (2% w/w) were compared. Six-months or 1-year stability tests were carried out at 40 °C. The content of CEF and VAN was evaluated using HPLC and the potency of the antibiotic was assessed with agar diffusion method. In contact with water, drug particles suspended in SEO dissolved rapidly and o/w emulsion was formed. After 1-year at 40 °C, the content of degradation products was at most 0.5% in CEF and 4.0% in VAN formulations. The agar diffusion assay has shown that CEF and VAN loaded into SEO retained its potency against the sensitive microorganisms comparable to an aqueous solution. Therefore, SEO can be used as a novel carrier for the active substances which may not require improved solubility or absorption but need to be protected from moisture. This is a formulation that can be produced on industrial scale, with no limitation of stability or drug concentration.

System-agnostic prediction of pharmaceutical excipient miscibility via computing-as-a-service and experimental validation.

We applied computing-as-a-service to the unattended system-agnostic miscibility prediction of the pharmaceutical surfactants, Vitamin E TPGS and Tween 80, with Copovidone VA64 polymer at temperature relevant for the pharmaceutical hot melt extrusion process. The computations were performed in lieu of running exhaustive hot melt extrusion experiments to identify surfactant-polymer miscibility limits. The computing scheme involved a massively parallelized architecture for molecular dynamics and free energy perturbation from which binodal, spinodal, and mechanical mixture critical points were detected on molar Gibbs free energy profiles at 180 °C. We established tight agreement between the computed stability (miscibility) limits of 9.0 and 10.0 wt% vs. the experimental 7 and 9 wt% for the Vitamin E TPGS and Tween 80 systems, respectively, and identified different destabilizing mechanisms applicable to each system. This paradigm supports that computational stability prediction may serve as a physically meaningful, resource-efficient, and operationally sensible digital twin to experimental screening tests of pharmaceutical systems. This approach is also relevant to amorphous solid dispersion drug delivery systems, as it can identify critical stability points of active pharmaceutical ingredient/excipient mixtures.

Illuminating a biologics development challenge: systematic characterization of CHO cell-derived hydrolases identified in monoclonal antibody formulations.

Monoclonal antibodies (mAb) and other biological drugs are affected by enzymatic polysorbate (PS) degradation that reduces product stability and jeopardizes the supply of innovative medicines. PS represents a critical surfactant stabilizing the active pharmaceutical ingredients, which are produced by recombinant Chinese hamster ovary (CHO) cell lines. While the list of potential PS-degrading CHO host cell proteins (HCPs) has grown over the years, tangible data on industrially relevant HCPs are still scarce. By means of a highly sensitive liquid chromatography-tandem mass spectrometry method, we investigated seven different mAb products, resulting in the identification of 12 potentially PS-degrading hydrolases, including the strongly PS-degrading lipoprotein lipase (LPL). Using an LPL knockout CHO host cell line, we were able to stably overexpress and purify the remaining candidate hydrolases through orthogonal affinity chromatography methods, enabling their detailed functional characterization. Applying a PS degradation assay, we found nine mostly secreted, PS-active hydrolases with varying hydrolytic activity. All active hydrolases showed a serine-histidine-aspartate/glutamate catalytical triad. Further, we subjected the active hydrolases to pH-screenings and revealed a diverse range of activity optima, which can facilitate the identification of residual hydrolases during bioprocess development. Ultimately, we compiled our dataset in a risk matrix identifying PAF-AH, LIPA, PPT1, and LPLA2 as highly critical hydrolases based on their cellular expression, detection in purified antibodies, active secretion, and PS degradation activity. With this work, we pave the way toward a comprehensive functional characterization of PS-degrading hydrolases and provide a basis for a future reduction of PS degradation in biopharmaceutical drug products.

Surfactant-Polymer Complexation and Competition on Drug Nanocrystal Surfaces Control Crystallinity.

Nanosizing drug crystals has emerged as a successful approach to enabling oral bioavailability, as increasing drug crystal surface area improves dissolution kinetics and effective solubility. Recently, bottom-up methods have been developed to directly assemble nanosized crystals by leveraging polymer and surfactant excipients during crystallization to control crystal size, morphology, and structure. However, while significant research has investigated how polymers and other single additives inhibit or promote crystallization in pharmaceutical systems, there is little work studying the mechanistic interactions of multiple excipients on drug crystal structure and the extent of crystallinity, which can influence formulation performance. This study explores how the structure and crystallinity of a model hydrophobic drug crystal, fenofibrate, change as a result of competitive interfacial chemisorption between common nonionic surfactants (polysorbate 80 and sorbitan monooleate) and a surface-active polymer excipient (methylcellulose). Classical molecular dynamics simulations highlight how key intermolecular interactions, including surfactant-polymer complexation and surfactant screening of the crystal surface, modify the resulting crystal structure. In parallel, experiments generating drug nanocrystals in hydrogel thin films validate that drug crystallinity increases with an increasing weight fraction of surfactant. Simulation results reveal a connection between accelerated dynamics in the bulk crystal and the experimentally measured extent of crystallinity. To our knowledge, these are the first simulations that directly characterize structural changes in a drug crystal as a result of excipient surface composition and relate the experimental extent of crystallinity to structural changes in the molecular crystal. Our approach provides a mechanistic understanding of crystallinity in nanocrystallization, which can expand the range of orally deliverable small molecule therapies.

Stabilization of zein nanoparticles with tween-80 and fucoidan for encapsulation of eugenol via a nozzle simulation chip.

Eugenol (EU), a natural bioactive compound found in various plants, offers numerous health benefits, but its application in the food and pharmaceutical industry is limited by its high volatility, instability, and low water solubility. Therefore, this study aimed to utilize the surface coating technique to develop zein-tween-80-fucoidan (Z-T-FD) composite nanoparticles for encapsulating eugenol using a nozzle simulation chip. The physicochemical characteristics of the composite nanoparticles were examined by varying the weight ratios of Z, T, and FD. Results showed that the Z-T-FD weight ratio of 5:1:15 exhibited excellent colloidal stability under a range of conditions, including pH (2-8), salt concentrations (10-500 mmol/L), heating (80 °C), and storage (30 days). Encapsulation of EU into Z-T-FD nanoparticles (0.5:5:1:15) resulted in an encapsulation efficiency of 49.29 ± 1.00%, loading capacity of 0.46 ± 0.05%, particle size of 205.01 ± 3.25 nm, PDI of 0.179 ± 0.006, and zeta-potential of 37.12 ± 1.87 mV. Spherical structures were formed through hydrophobic interaction and hydrogen bonding, as confirmed by Fourier transform infrared spectroscopy and molecular docking. Furthermore, the EU-Z-T-FD (0.5:5:1:15) nanoparticles displayed higher in vitro antioxidant properties (with DPPH and ABTS radical scavenging properties at 75.28 ± 0.16% and 39.13 ± 1.22%, respectively), in vitro bioaccessibility (64.78 ± 1.37%), and retention rates under thermal and storage conditions for EU compared to other formulations. These findings demonstrate that the Z-T-FD nanoparticle system can effectively encapsulate, protect, and deliver eugenol, making it a promising option for applications in the food and pharmaceutical industries.

Safety and Immunogenicity of a DNA Vaccine With Subtype C gp120 Protein Adjuvanted With MF59 or AS01B: A Phase 1/2a HIV-1 Vaccine Trial.

BACKGROUND: An effective vaccine is required to end the HIV pandemic. We evaluated the safety and immunogenicity of a DNA (DNA-HIV-PT123) vaccine with low- or high-dose bivalent (TV1.C and 1086.C glycoprotein 120) subtype C envelope protein combinations, adjuvanted with MF59 or AS01B. METHODS: HIV Vaccine Trials Network (HVTN)108 was a randomized, placebo-controlled, double-blind, phase 1/2a trial conducted in the United States and South Africa. HIV-negative adults were randomly assigned to 1 of 7 intervention arms or placebo to assess DNA prime with DNA/protein/adjuvant boosts, DNA/protein/adjuvant co-administration, and low-dose protein/adjuvant regimens. HVTN111 trial participants who received an identical regimen were also included. Outcomes included safety and immunogenicity 2 weeks and 6 months after final vaccination. RESULTS: From June 2016 to July 2018, 400 participants were enrolled (N = 334 HVTN108, N = 66 HVTN111); 370 received vaccine and 30 received placebo. There were 48 grade 3 and 3 grade 4 reactogenicity events among 39/400 (9.8%) participants, and 32 mild/moderate-related adverse events in 23/400 (5.8%) participants. All intervention groups demonstrated high IgG response rates (>89%) and high magnitudes to HIV-1 Env gp120 and gp140 proteins; response rates for AS01B-adjuvanted groups approached 100%. V1V2 IgG magnitude, Fc-mediated functions, IgG3 Env response rates, and CD4+ T-cell response magnitudes and rates were higher in the AS01B-adjuvanted groups. The AS01B-adjuvanted low-dose protein elicited greater IgG responses than the higher protein dose. CONCLUSIONS: The vaccine regimens were generally well tolerated. Co-administration of DNA with AS01B-adjuvanted bivalent Env gp120 elicited the strongest humoral responses; AS01B-adjuvanted regimens elicited stronger CD4+ T-cell responses, justifying further evaluation.ClinicalTrials.gov registration: NCT02915016, registered 26 September 2016.

Poloxamer-188 as a wetting agent for microfluidic resistive pulse sensing measurements of extracellular vesicles.

INTRODUCTION: Microfluidic resistive pulse sensing (MRPS) can determine the concentration and size distribution of extracellular vesicles (EVs) by measuring the electrical resistance of single EVs passing through a pore. To ensure that the sample flows through the pore, the sample needs to contain a wetting agent, such as bovine serum albumin (BSA). BSA leaves EVs intact but occasionally results in unstable MRPS measurements. Here, we aim to find a new wetting agent by evaluating Poloxamer-188 and Tween-20. METHODS: An EV test sample was prepared using an outdated erythrocyte blood bank concentrate. The EV test sample was diluted in Dulbecco's phosphate-buffered saline (DPBS) or DPBS containing 0.10% BSA (w/v), 0.050% Poloxamer-188 (v/v) or 1.00% Tween-20 (v/v). The effect of the wetting agents on the concentration and size distribution of EVs was determined by flow cytometry. To evaluate the precision of sample volume determination with MRPS, the interquartile range (IQR) of the particles transit time through the pore was examined. To validate that DPBS containing Poloxamer-188 yields reliable MRPS measurements, the repeatability of MRPS in measuring blood plasma samples was examined. RESULTS: Flow cytometry results show that the size distribution of EVs in Tween 20, in contrast to Poloxamer-188, differs from the control measurements (DPBS and DPBS containing BSA). MRPS results show that Poloxamer-188 improves the precision of sample volume determination compared to BSA and Tween-20, because the IQR of the transit time of EVs in the test sample is 11 µs, which is lower than 56 µs for BSA and 16 µs for Tween-20. Furthermore, the IQR of the transit time of particles in blood samples with Poloxamer-188 are 14, 16, and 14 µs, which confirms the reliability of MRPS measurements. CONCLUSION: The solution of 0.050% Poloxamer-188 in DPBS does not lyse EVs and results in repeatable and unimpeded MRPS measurements.

Performance of ß-carotene-loaded nanostructured lipid carriers under dynamic in vitro digestion system: Influence of the emulsifier type.

A better understanding of how emulsifier type could differently influence the behavior of nanostructured lipid carriers (NLC) under the gastrointestinal digestion process, as well as at the cellular level, is of utmost importance for the NLC-based formulations' optimization and risk assessment in the food field. In this study, NLC composed by fully hydrogenated soybean and high-oleic sunflower oils were prepared using soy lecithin (NLC Lß) or Tween 80 (NLC Tß) as an emulsifier. ß-Carotene was entrapped within NLC developed as a promising strategy to overcome ß-carotene's low bioavailability and stability. The effect of emulsifier type on the digestibility of ß-carotene-loaded NLC was evaluated using an in vitro dynamic digestion model mimicking peristalsis motion. The influence of ß-carotene-loaded NLC on cell viability was assessed using Caco-2 cells in vitro. NLC Tß remained stable in the gastric compartment, presenting particle size (PS) similar to the initial NLC (PS: 245.68 and 218.18 nm, respectively), while NLC Lß showed lower stability (PS > 1000 nm) in stomach and duodenum phases. NLC Tß also provided high ß-carotene protection and delivery capacity (i.e., ß-carotene bioaccessibility increased 10-fold). Based on the results of digestion studies, NLC Tß has shown better physical stability during the passage through the in vitro dynamic gastrointestinal system than NLC Lß. Moreover, the developed NLC did not compromise cell viability up to 25 µg/mL of ß-carotene. Thus, the NLC developed proved to be a biocompatible structure and able to incorporate and protect ß-carotene for further food applications. PRACTICAL APPLICATION: The findings of this study hold significant implications for industrial applications in terms of developing nanostructured lipid carriers from natural raw materials widely available and used to produce other lipid-based products in the food industry, as an alternative to synthetic ones. In this respect, the ß-carotene-loaded NLC developed in this study would find a great industrial application in the food industry, which is in constant search to develop functional foods capable of increasing the bioavailability of bioactive compounds.

Dessecting the toxicological profile of polysorbate 80 (PS80): comparative analysis of constituent variability and biological impact using a zebrafish model.

Polysorbate 80, commonly abbreviated as PS80, is a widely used pharmaceutical excipient renowned for its role as a solubilizer and stabilizer in drug formulations. Although PS80 is essential for various pharmaceutical applications, particularly in the formulation of injectable drugs, it has been implicated in a range of adverse reactions. However, due to the complexity of the composition of PS80, the differences in the types and contents of the constituents of PS80 from different manufacturers increase the probability or likelihood of their uneven quality. Addressing the complete spectrum of PS80's components is challenging; thus, most studies to date have examined PS80 as a singular entity. This approach, however, carries a degree of uncertainty, as it overlooks the unique composition and concentration of components within the PS80 used in experiments, which may not reflect the actual diversity in commercially available PS80 products. Recognizing the critical need to understand how PS80's composition influences biological effects and toxicity, our study aims to bridge this knowledge gap. By doing so, we can clarify how different PS80 compositions from various manufacturers might affect the quality of pharmaceutical formulations, and also guide excipient manufacturers toward producing higher-quality PS80. Such insights could further facilitate a more targeted application of PS80 in drug development. Building on our previous work, we isolated and prepared two key components of PS80-polyoxyethylene sorbitan monooleate (PSM) and polyoxyethylene isosorbide monooleate (PIM)-and conducted a systematic comparison. We evaluated the acute, hemolytic, and target organ toxicity of two different PS80 samples, as well as PSM and PIM, using a zebrafish model. Our research also delved into the potential mechanisms behind the observed toxicological effects, providing an in-depth understanding of PS80's impact on biological systems.The results show that PS80, PSM, and PIM resulted in developmental anomalies in larval zebrafish. The primary organs of acute toxicity in zebrafish exposed to PS80 and its typical components PSM and PIM include the cardiovascular system, kidneys, intestines, skin, and liver. Notably, PIM further induced severe pericardial edema and erythrocyte hemolysis, thereby affecting blood flow. The samples also instigated oxidative damage by disrupting the redox equilibrium in the larvae. Compared to PS80, both PSM and PIM induced greater oxidative damage, with PIM notably causing significantly higher lipid oxidation, suggesting that oxidative stress may play a crucial role in polysorbate80-induced toxicity. Furthermore, our study found that PS80 could induce alterations in DNA conformation. The findings underscore the necessity for excipient regulators to establish comprehensive quality standards for Polysorbate 80 (PS80). By implementing such standards, it is possible to minimize the clinical risks associated with the variability in PS80 composition, ensuring safer pharmaceutical products for patients.

Stress-mediated polysorbate 20 degradation and its potential impact on therapeutic proteins.

PURPOSE: Polysorbates are the most commonly used surfactants in formulations to stabilize therapeutic proteins against interfacial stresses. Polysorbates can undergo oxidative or enzyme-mediated hydrolytic degradation to produce free fatty acids (FFAs) and subvisible particles in formulations. To determine which product related variables contribute to PS20 degradation, we investigated the effects of storage temperature, formulation, pH, presence of hydrolytic enzymes, and specific fatty acid composition on different grades of PS20 in relation to their PS20 degradation profile and consequently the quality of protein drug products. METHODS: Bevacizumab and T-DM1 were reformulated in the freshly prepared therapeutic protein formulations containing either compendial PS20 or non-compendial PS20 with high % lauric acid and spiked with exogenous esterase or lipase. The release of FFAs and formation of particles were monitored at 4°C and 37°C. Protein quality was assessed for secondary structures, purity, and biological activity. RESULTS: Hydrolytic release of FFAs and formation of subvisible particles were found to be dependent on grades of PS20, types of enzymes used, incubation temperature, and pH. Esterase- or lipase-mediated degradation of PS20 and formation of subvisible particles in drug formulation showed no significant impact on the biological activity and stability of therapeutic proteins against degradation or aggregation. CONCLUSIONS: Our study suggests that degradation of PS20 and formation of FFA particles depend on the fatty acid composition of PS20, types of hydrolytic enzymes, pH, and temperature. The presence of FFA subvisible particles showed no significant impact on the purity and biological activity of the therapeutic proteins under the tested conditions.

Ultraviolet spectrophotometry as method to determine the concentration of ß-myrcene released from chitosan in aqueous medium.

Myrcene (ß-myrcene), found in essential oils from plant species such as hops and cannabis, has many advantageous properties, but its use is limited due to volatility and low solubility in water. One way to circumvent these limitations is to encapsulate the essential oils in a polymer matrix. However, these hydrophobic molecules are difficult to quantify when dispersed in water. Seeking to study the release of this terpene in drug release tests from polymeric matrices, this work aimed to develop an easy and cheap UV spectrophotometric method for the quantification of ß-myrcene in aqueous medium. To achieves this goal, samples were prepared in 0.05% (w/v) polysorbate 80 solution, with concentrations of ß-myrcene ranging from 0.01% to 0.1% (v/v), and were analyzed at 226 nm. Each sample was analyzed in triplicate and repeated on three different days, to evaluate the repeatability of the results. The results were subjected to Q, F and Student's t-tests. The regression parameters obtained for ß-myrcene were above 0.99 and through statistical analysis, it was possible to confirm the repeatability for the results. The values of the limits of detection and quantification indicated that the method is not affected by intrinsic factors of the equipment. The results of accuracy, robustness and selectivity showed recovery rates within acceptable limits. This demonstrates that the quantification of ß-myrcene in aqueous medium by UV spectrophotometry is feasible.

Acceleration a yeast-based biodegradation process of polyethylene terephthalate microplastics by Tween 20: Efficiency, by-product analysis, and metabolic pathway Prediction.

Polyethylene terephthalate is a widely produced plastic polymer that exhibits considerable biodegradation resistance, making its derived microplastics ubiquitous environmental pollutants. In this study, a new yeast strain (Vanrija sp. SlgEBL5) was isolated and found to have lipase and esterase-positive capabilities for degrading polyethylene terephthalate microplastics. This isolate changed the microplastic surface charge from -19.3 to +31.0 mV and reduced more than 150 µm of its size in addition to reducing the intensity of the terephthalate, methylene, and ester bond functional groups of the polymer in 30 days. Tween 20 as a chemical auxiliary treatment combined with biodegradation increased the microplastic degradation rate from 10 to 16.6% and the thermal degradation rate from 85 to 89%. Releasing less potentially hazardous by-products like 1,2 diethyl-benzene despite the higher abundance of long-chain n-alkanes, including octadecane and tetracosane was also the result of the bio + chemical treatment. Altogether, the findings showed that Vanrija sp. SlgEBL5 has the potential as a biological treating agent for polyethylene terephthalate microplastics, and the simultaneous bio + chemical treatment enhanced the biodegradation rate and efficiency.

A mechanistic kinetic model for lipid oxidation in Tween 20-stabilized O/W emulsions.

Models predicting lipid oxidation in oil-in-water (O/W) emulsions are a requirement for developing effective antioxidant solutions. Existing models do, however, not include explicit equations that account for composition and structural features of O/W emulsions. To bridge this gap, a mechanistic kinetic model for lipid oxidation in emulsions is presented, describing the emulsion as a one-dimensional three phase (headspace, water, and oil) system. Variation in oil droplet sizes, overall surface area of oil/water interface, oxidation of emulsifiers, and the presence of catalytic transition metals were accounted for. For adequate predictions, the overall surface area of oil/water interface needs to be determined from the droplet size distribution obtained by dynamic and static light scattering (DLS, SLS). The kinetic model predicted well the formation of oxidation products in both mono- and polydisperse emulsions, with and without presence of catalytic transition metals.

Integrating population and single-cell variations in vaccine responses identifies a naturally adjuvanted human immune setpoint.

Multimodal single-cell profiling methods can capture immune cell variations unfolding over time at the molecular, cellular, and population levels. Transforming these data into biological insights remains challenging. Here, we introduce a framework to integrate variations at the human population and single-cell levels in vaccination responses. Comparing responses following AS03-adjuvanted versus unadjuvanted influenza vaccines with CITE-seq revealed AS03-specific early (day 1) response phenotypes, including a B cell signature of elevated germinal center competition. A correlated network of cell-type-specific transcriptional states defined the baseline immune status associated with high antibody responders to the unadjuvanted vaccine. Certain innate subsets in the network appeared "naturally adjuvanted," with transcriptional states resembling those induced uniquely by AS03-adjuvanted vaccination. Consistently, CD14+ monocytes from high responders at baseline had elevated phospho-signaling responses to lipopolysaccharide stimulation. Our findings link baseline immune setpoints to early vaccine responses, with positive implications for adjuvant development and immune response engineering.

Characterization of Freezing Processes in Drug Substance Bottles by Ice Core Sampling.

Freezing of biological drug substance (DS) is a critical unit operation that may impact product quality, potentially leading to protein aggregation and sub-visible particle formation. Cryo-concentration has been identified as a critical parameter to impact protein stability during freezing and should therefore be minimized. The macroscopic cryo-concentration, in the following only referred to as cryo-concentration, is majorly influenced by the freezing rate, which is in turn impacted by product independent process parameters such as the DS container, its size and fill level, and the freezing equipment. (At-scale) process characterization studies are crucial to understand and optimize freezing processes. However, evaluating cryo-concentration requires sampling of the frozen bulk, which is typically performed by cutting the ice block into pieces for subsequent analysis. Also, the large amount of product requirement for these studies is a major limitation. In this study, we report the development of a simple methodology for experimental characterization of frozen DS in bottles at relevant scale using a surrogate solution. The novel ice core sampling technique identifies the axial ice core in the center to be indicative for cryo-concentration, which was measured by osmolality, and concentrations of histidine and polysorbate 80 (PS80), whereas osmolality revealed to be a sensitive read-out. Finally, we exemplify the suitability of the method to study cryo-concentration in DS bottles by comparing cryo-concentrations from different freezing protocols (-80°C vs -40°C). Prolonged stress times during freezing correlated to a higher extent of cryo-concentration quantified by osmolality in the axial center of a 2 L DS bottle.

Optimized quantification of fatty acids in complex media using advanced analytical techniques.

RATIONALE: Various medium formulations contain essential fatty acids at concentrations ranging from 10 to 100 mg/L. Accurate and precise lipid measurement in media is crucial for monitoring media quality and conducting studies on lipids in the context of cell culture. This study employed two-dimensional gas chromatography (GC × GC) analyses to offer enhanced resolution, sensitivity, and separation performance compared to GC. METHODS: Quantification of fatty acid methyl esters (FAMEs) in a medium was conducted using GC × GC combined with a high-resolution mass spectrometer and flame ionization detector, considering potential interference from nonionic surfactant Tween 80, which was precipitated and removed by optimizing the concentration of cobalt thiocyanate (CTA) solution during pretreatment. This advanced analytical approach enabled identification of cis and trans isomers of identical molecular weights and determination of the location and number of double bonds in the same carbon number structure. RESULTS: Our analysis identified 36 FAMEs within the C6-C24 region, and a 5% CTA solution was optimal for efficient removal of Tween 80 during lipid extraction. Additionally, this advanced method minimized FAME contamination and loss during pretreatment, thereby significantly reducing the sample volume required to detect trace levels of FAMEs. This improvement led to a fatty acid recovery rate of 106% while maintaining the average relative standard deviation for the target FAMEs of about 3%. CONCLUSIONS: Our research paves the way for future investigation into medium quality control and the role of fatty acids in cell culture. This offers the possibility for economical and effective trace quantification of fatty acids in complex media.

Influence of Surfactant on the Skin Permeation of Methylisothiazolinone and Methylchloroisothiazolinone.

Patch tests are often used in safety evaluations to identify the substance causing skin irritation, but the same substance can sometimes give positive or negative results depending on the test conditions. Here, we investigated differences in the skin penetration of two test compounds under different application conditions. We studied the effects of the anionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant polysorbate 80 (PS) on skin penetration of the preservatives methylisothiazolinone (MT) and methylchloroisothiazolinone (MCT), which are used in cosmetics such as shampoos. The skin permeation of MT was enhanced by SDS but was unchanged by PS. Skin impedance decreased in the presence of SDS whereas PS had the same effect as the control aqueous solution, suggesting that SDS reduction of the barrier function of skin affects the permeation of MT, a hydrophilic drug. Application of a mixture of MCT and MT in the presence of SDS did not affect the skin permeation of MCT whereas the permeation of MT was enhanced by SDS, indicating that the skin permeation of MCT is less affected by SDS than is MT. Thus, attention should be paid to the possible effect of co-solutes, especially hydrophilic drugs.

Quantification of Biopharmaceutically Relevant Nonionic Surfactant Excipients Using Benchtop qNMR.

Nonionic surfactant excipients (NISEs) are commonly added to biologics formulations to mitigate the effects of stress incurred by the active biotherapeutic during manufacturing, transport, and storage. During manufacturing, NISEs are added by dilution of a stock solution directly into a protein formulation, and their accurate addition is critical in maintaining the quality and integrity of the drug product and thus ensuring patient safety. This is especially true for the common NISEs, polysorbates 20 and 80 (PS20 and PS80, respectively) and poloxamer 188 (P188). With the increasing diversity of biologic modalities within modern pharmaceutical pipelines, there is thus a critical need to develop and deploy convenient and user-accessible analytical techniques that can rapidly and reliably quantify these NISEs under biopharmaceutically relevant conditions. We thus pursued 60 MHz benchtop quantitative NMR (qNMR) as a nondestructive and user-friendly analytical technique for the quantification of PS20, PS80, and P188 under such conditions. We demonstrated the ability of benchtop qNMR (1) to quantify simulated PS20, PS80, and P188 stock solutions representative of those used during the drug substance (DS) formulation step in biomanufacturing and (2) to quantify these NISEs at and below their target concentrations (≤0.025% w/v) directly in biologics formulations containing histidine, sucrose, and one of three biotherapeutic modalities (monoclonal antibody, antibody-drug conjugate, and Fc-fusion protein). Our results demonstrate that benchtop qNMR offers a fit-for-purpose, reliable, user-friendly, and green analytical route by which NISE of interest to the biopharmaceutical industry may be readily and reliably quantified. We conclude that benchtop qNMR has the potential to be applied to other excipient formulation components in the presence of various biological modalities as well as the potential for routine integration within analytical and QC laboratories across pharmaceutical development and manufacturing sites.

O1/W/O2 double emulsion gels based on nanoemulsions and Pickering particles for co-encapsulating quercetin and cyanidin: A functional fat substitute.

An O1/W/O2 double emulsion gel, as a functional fat substitute and based on nanoemulsions and hydrophobic Pickering particles, is prepared by two-step emulsification to co-encapsulate hydrophilic cyanidin and hydrophobic quercetin. Nanoemulsions loading quercetin are fabricated by Tween-80 and combining high-speed and high-pressure emulsification. Phytosterol nanoparticles stabilize the W-O2 interface of the secondary emulsion to load cyanidin in the W phase. The concentration of Tween-80 is optimized as 0.3% by the droplet size and viscosity of nanoemulsions. The structural stability of double emulsion gels will be weakened along with the increase of nanoemulsions, showing lower modulus and encapsulation efficiency (EE) and bigger droplets. In double emulsion gels, the EE of quercetin and cyanidin reaches 93% and 85.6%, respectively. Analysis of molecular interaction indicates that Tween-80 would decrease the in-situ hydrophobicity of phytosterol nanoparticles by hydrogen bonding adsorption, thereby weakening the emulsification. The pH-chromic 3D printing of double emulsion gels is designed according to the pH sensitivity of cyanidin. Texture profile analysis is performed to test the textural properties of 3D-printed objects. The simulated digestion is conducted on double emulsion gels. The double emulsion gel with fewer nanoemulsions is beneficial for protecting quercetin and improving the delivery due to the higher structural stability, while that with more nanoemulsions is conducive to the digestion of cyanidin and camellia oil due to weakened semi-solid properties. This double emulsion gel further simulates fat tissues by co-encapsulating hydrophilic and hydrophobic substances, promoting the application of fat substitutes in the food industry.

Long-term exposure to dietary emulsifier Tween 80 promotes liver lipid accumulation and induces different-grade inflammation in young and aged mice.

With the advent of industrialization, there has been a substantial increase in the production and consumption of ultra-processed foods (UPFs). These processed foods often contain artificially synthesized additives, such as emulsifiers. Emulsifiers constitute approximately half of the total amount of food additives, with Tween 80 being a commonly used emulsifier in the food industry. Concurrently, China is undergoing significant demographic changes, transitioning into an aging society. Despite this demographic shift, there is insufficient research on the health implications of food emulsifiers, particularly on the elderly population. In this study, we present novel findings indicating that even at low concentrations, Tween 80 suppressed the viability of multiple cell types. Prolonged in vivo exposure to 1 % Tween 80 in drinking water induced liver lipid accumulation and insulin resistance in young adult mice under a regular chow diet. Intriguingly, in mice with high-fat diet (HFD) induced metabolic dysfunction-associated steatotic liver disease (MASLD), this inductive effect was masked. In aged mice, liver lipid accumulation was replicated under prolonged Tween 80 exposure. We further revealed that Tween 80 induced inflammation in both adult and aged mice, with a more pronounced inflammation in aged mice. In conclusion, our study provides compelling evidence that Tween 80 could contribute to a low-grade inflammation and liver lipid accumulation. These findings underscore the need for increasing attention regarding the consumption of UPFs with Tween 80 as the emulsifier, particularly in the elderly consumers.