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Aggregation of monoclonal antibodies (mAbs) is the driving force for their undesirable immunogenic effects. There are multiple factors responsible for aggregation in therapeutic proteins. One significant cause is the process-related shear and interfacial stress generated due to impellers and stirrers. This investigation focuses on understanding the possible aggregation arising upon stirring mAb formulations using stirrers made of different materials. We used quantitative laser diffraction (qLD) to monitor and quantify the stirring induced formation of submicron and subvisible aggregates in the size range from 100 nm to 10 µm. We analysed various aspects of aggregate generation, such as onset of aggregation, particle size distribution, and concentration of aggregates generated using stirrers of different materials. We observed that mixing with stainless steel stirrers resulted in a quicker onset of aggregation and led to significantly higher concentrations of aggregates. Analysis of the stirred samples using dynamic light scattering (DLS) and background imaging technique (BMI) were conducted to complement the qLD analysis. All the three techniques resulted in a similar trend, showing presence of larger and higher quantities of aggregates in steel stirred samples, as compared to those stirred using PEEK and glass. Additionally, we performed SEC-HPLC to quantify the soluble fraction of monomer and recorded that the least amount was present in the steel stirred samples. This work highlights the need for optimizing the materials used for fabricating the stirrers/impellers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Nanoemulsions are very interesting systems as they offer capacity to encapsulate both hydrophilic and lipophilic molecules in a single particle, as well as the controlled release of chemical moieties initially entrapped in the internal droplets. In this study, we propose a new two-step modified ultrasound-assisted phase inversion approaches-phase inversion temperature (PIT) and self-emulsification, to prepare stable o/w nanoemulsions from a fully water-dilutable microemulsion template for the transdermal delivery of loratadine (a hydrophobe and as Ostwald ripening inhibitor). Firstly, the primary water-in-oil microemulsion concentrate (w/o) was formed using loratadine in the oil phase (oleic acid or coconut oil) and Tween 80 in the aqueous phase and by adjusting the PIT around 85 °C followed by stepwise dilution with water at 25 °C to initiate the formation the nanoemulsions (o/w). To assure the long-term stability, a brief application of low frequency ultrasound was employed. Combining the two low energy methods resulted in nanoemulsions prepared by mixing constant surfactant/oil ratios above the PIT with varying water volume fraction (self-emulsification) during the PIT by stepwise dilution. The kinetic stability was evaluated by measuring the droplet size with time by dynamic light scattering (DLS). The droplet size ranged 15-43 nm and did not exceed 100 nm over the period of 6 months indicating the system had high kinetic stability. Cryo-TEM showed that the nanoemulsions droplets were monodispersed and approaching micellar structure and scale. All nanoemulsions had loratadine crystals formed within 20 days after preparation, which tended to sediment during storage. Nanoemulsions improved the in vitro permeation of loratadine through porcine skin up to 20 times compared to the saturated solution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Econazole nitrate (ECN) is a weakly basic drug with very low aqueous solubility that hampers its permeation through biological membranes and results in low ECN bioavailability. Formation of drug/cyclodextrin (drug/CD) inclusion complexes is a formulation technology that can be applied to enhance drug solubility in aqueous media. The aim of this study was to determine the effect of CD complexation and pH adjustments on the ECN solubility. The ECN pH-solubility and ECN/CD phase-solubility profiles were determined. The solubility of ECN in aqueous acidic solutions containing α-cyclodextrin (αCD) was relatively high and much higher than in aqueous γ-cyclodextrin (γCD) solutions under same conditions. The complexation efficiency of the ECN/CD complex was relatively low for the unionized drug. Formation of ECN/CD inclusion complex was verified by proton nuclear magnetic resonance spectroscopy. Formation of ECN/CD complexes enhanced the drug stability during autoclaving. γCD complexes self-assembled to form nano- and microparticles whereas αCD complexes had negligible tendency to self-assemble. Formation of CD complex nano- and microparticles was investigated by dynamic light scattering and by drug permeation through semipermeable membranes of different molecular weight cut-off. The largest aggregate fraction was observed for the unionized ECN in aqueous pH 7.5 solution containing high CD concentration, that is 10% (w/v) CD. It was shown that in acidic solutions ECN/αCD can enhance the antifungal activity to filamentous fungi. This was associated with the increased ECN solubility and increase of readily available ECN molecules in aqueous αCD solutions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Doxorubicin liposomal formulations were characterized for the particle size distribution and size-based distribution of lipids and doxorubicin (DOX). An asymmetric flow-field flow fractionation (AF4) method was developed and employed for size-based fractionation of the doxorubicin liposomal formulations. Liposome size-fractions collected from AF4 were analyzed for particle size using nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). Three doxorubicin liposomal formulations (DLFs) were compared for liposome size distributions. We did not observe any statistically significant variation in D10, D50 and D90 value of size distributions of three fomulations. Lipids and DOX compositions in liposomal fractions from AF4 were determined using liquid chromatography-mass spectrometry (LC-MS) method. The lipid compositions are close to be constant as a function of liposomal size. The drug to lipid ratio was close to be constant for all the size fractions suggesting drug loading may be proportional to membrane surface area of liposomes as membrane surface area is proportional to the amount of lipid in a given liposome. This analytical method could be used in liposomal based drug development as a tool for comparison of generic versions against the reference standard drug., (Published by Elsevier B.V.)

Background: Perfluorocarbon-nanoemulsions (PFC-NE) made of PFC and phospholipids (PL) by homogenization are optimal for in vivo- 19 F labelling of monocytes and subsequently of inflamed tissues in magnetic resonance imaging (MRI). Necessary requirements for in vivo use of PFC-NE are sterility, suitable droplet sizes and the absence of immune activating liposomes, which are a typical byproduct of the homogenization process., Methods and Results: To meet these requirements, we developed an aseptic in-vial preparation technique for PFC-NE based on dual centrifugation (DC) by testing different PFC/phospholipid ratios as well as the application of additives. Two different PFC, perfluorooctylbromide (PFOB) and perfluoro-15-crown-5-ether (PFCE), were investigated. Particle sizes were assessed by dynamic light scattering and NE morphology by cryoTEM. DC homogenization was optimal when using an excess of PL (8.7 % (m/m) of utilized PFC, z-ave: 180 nm, pdi: 0.2). A purification approach by centrifugation was implemented to remove liposomes formed from the excess of PL during homogenization. The purification success was proven by phospholipid assay and PFC quantification via density and sound velocity measurements., Conclusion: DC in combination with a short centrifugation is a fast and reliable way of small-scale aseptic PFC-NE production for 19 F MRI passive-targeting experiments of monocytes and inflamed tissues., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Antigenic drift of the influenza A virus requires that vaccine production is targeted to the strains circulating each year. Live-attenuated influenza A vaccine manufacturing is used to produce intact virions with the surface antigens of the circulating strains. Influenza A typically contains a large percentage (>90%) of non-infective virions. The ribonucleoprotein (RNP) content, virion structure, and aggregation are factors that are thought to have an impact on infectivity. However, these factors are difficult to study because of the intrinsic variability in virion size, shape and overall structural integrity. Negative stain TEM for total particle counts and cryoTEM for detailed size/structural analysis are established benchmark techniques for virus characterization. Other methods may be valuable for certain sample types or circumstances. The aim of this work is to establish a benchmark comparison between orthogonal biophysical techniques for particle counts, population size distribution, structural integrity, and aggregate levels. NTA and FFF-MALS rapidly provided total counts, size distribution, and aggregate/elongated virion content. CryoTEM with size analysis and fraction counting yielded detailed information about the pleomorphism of the sample. The structural integrity of virions was inferred from multi-signal AUC-SV and CryoTEM. The current work provides a comparative assessment and a baseline for the selection of biophysical tools for the determination of particle counts, aggregation and pleomorphic characteristics of influenza A virus., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In vivo fate of nanomaterials is influenced by the particle size among other parameters. Thus, Health Agencies have identified the size of nanomaterial as an essential physicochemical property to characterize. This parameter can be explored by dynamic light scattering (DLS) that is described in the ISO standard 22412:2008(E) and is one of the methods recognized by Health Agencies. However, no protocol of DLS size measurement has been validated over a large range of size so far. In this work, we propose an extension of validation of a protocol of size measurement by DLS previously validated with certified reference materials (CRM) at 60 and 203nm. The present work reports robustness, precision and trueness of this protocol that were investigated using CRM at 100 and 400nm. The protocol was robust, accurate and consistent with the ISO standard over the whole range of size that were considered. Expanded uncertainties were 4.4 and 3.6% for CRM at 100 and 400nm respectively indicating the reliability of the protocol. The range of application of the protocol previously applied to the size measurement of liposomes and polymer nanoparticles was extended to inorganic nanomaterial including silica nanoparticles., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Solid particulate matter introduced into the bloodstream as a result of parenteral drug administration can produce serious pathological conditions. Particulate matter that cannot be eliminated by pre-infusion filtration is often the result of drug precipitation that occurs when certain parenteral formulations are mixed with blood. A new device is designed to model the mixing of drug formulations with flowing blood utilizing a uniquely designed flow cell and a CCD camera to view the formulation as it is mixed with a blood surrogate in real time. The performance of the proposed device is measured using 3 commercially available parenteral formulations previously tested using a validated in vitro model., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study aimed to evaluate the potential of a novel glycoside non-ionic surfactant synthesized and characterized in our laboratory for increased oral bioavailability of Cefixime. The surfactant was synthesized by simple etherification of bergenin with bromoundecane and characterized by (1)H NMR and mass spectroscopy (MS). Biocompatibility of the surfactant (BRM-BG) was assessed by in-vitro cytotoxicity against NIH/3T3 cells and human blood hemolysis. In-vivo acute toxicity was evaluated in mices. Cefixime loaded BRM-BG niosomes were investigated for drug entrapment efficiency using HPLC and surface morphology and vesicle size by atomic force microscopy (AFM) and dynamic light scattering (DLS). The in-vivo oral bioavailability and pharmacokinetics studies were carried out using rabbits. Cefixime loaded BRM-BG vesicles were spherical in the size range of 178.66±8.17nm with a polydipersity index (PDI) of 0.20±0.01, offering an entrapment efficiency as high as 78.4±0.83%. When the surfactant was applied on NIH 3T3 tissue culture, as high as 90.77±3.15% and 86.86±3.02%, cell viability at 1000μg/mL concentration after 24 and 48h respectively were observed. The surfactant also caused 5.49±1.62% haemolysis and was found to be safe at a dose up to 2000mg/kg. In-vivo drug plasma concentration (Cmax) was found to be 9.69±1.22μg/mL, much higher than that resulting from the intake of commercial suspension and capsules. BRM-BG demonstrated to be safe and effective as carrier of Cefixime following oral dosing in rabbits. The BRM-BG surfactant delivery nano-system is relatively safe and in animal models it is an appropriate carrier for Cefixime, offering enhanced bioavailability compared to commercially available formulations of the drug., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Insulin (INS) was incorporated into complexes with the block polyelectrolyte quaternized poly[3,5-bis(dimethylaminomethylene)hydroxystyrene]-b-poly(ethylene oxide) (QNPHOSEO), which is a cationic-neutral block polyelectrolyte. Light scattering techniques are used in order to examine the size, the size distribution and the ζ-potential of the nanocarriers in aqueous and biological media, which are found to depend on the ratio of the components and the physicochemical parameters during and after complex preparation. Circular dichroism and infrared spectroscopy, employed to investigate the structure of the complexed INS, show no alteration of protein structure after complexation. In vitro release profiles of the entrapped protein are found to depend on the ratio of the components and the solution conditions used during preparation of the complexes., (Copyright © 2015 Elsevier B.V. All rights reserved.)