Extracellular vesicles (EVs) have emerged as a promising drug delivery system. Connectosomes are a specialized type of EVs that contain connexins in their membranes. Connexin is a surface transmembrane protein that forms connexin hemichannels. When a connexin hemichannel on a connectosome docks with another connexin hemichannel of a target cell, they form a gap junction that allows direct intracellular delivery of therapeutic cargos from within the connectosome to the cytoplasm of the recipient cell. In the present study, we tested the feasibility of converting connectosomes into dry powders by (thin-film) freeze-drying to enable their potential storage in temperatures higher than the recommended -80 °C, while maintaining their activity. Connectosomes were isolated from a genetically engineered HeLa cell line that overexpressing connexin-43 subunit protein tagged with red fluorescence protein. To facilitate the testing of the function of the connectosomes, they were loaded with calcein green dye. Calcein green-loaded connectosomes were thin-film freeze-dried with trehalose alone or trehalose and a polyvinylpyrrolidone polymer as lyoprotectant(s) to produce amorphous powders with high glass transition temperatures (>100 °C). Thin-film freeze-drying did not significantly change the morphology and structure of the connectosomes, nor their particle size distribution. Based on data from confocal microscopy, flow cytometry, and fluorescence spectrometry, the connexin hemichannels in the connectosomes reconstituted from the thin-film freeze-dried powder remained functional, allowing the passage of calcein green through the hemichannels and the release of the calcein green from the connectosomes when the channels were opened by chelating calcium in the reconstituted medium. The function of connectosomes was assessed after one month storage at different temperatures. The connexin hemichannels in connectosomes in liquid lost their function when stored at -19.5 ± 2.2 °C or 6 ± 0.5 °C for a month, while those in dry powder form remained functional under the same storage conditions. Finally, using doxorubicin-loaded connectosomes, we showed that the connectosomes reconstituted from thin-film freeze-dried powder remained pharmacologically active. These findings demonstrate that thin-film freeze-drying represents a viable method to prepare stable and functional powders of EVs that contain connexins in their membranes.

OBJECTIVE: This paper investigates the critical role of material thickness in freeze-dried pellets for enhancing the storage stability of encapsulated bacteria. Freeze dried material of varying thicknesses obtained from different annealing durations is quantified using Scanning Electron Microscopy (SEM) and X-ray microtomography (μCT), the material thickness is then correlated to the storage stability of the encapsulated cells.
METHODS: A formulation comprising of sucrose, maltodextrin, and probiotic cells is quenched in liquid nitrogen to form pellets. The pellets undergo different durations of annealing before undergoing freeze-drying. The material thickness is quantified using SEM and μCT. Storage stability in both oxygen-rich and oxygen-poor environments is evaluated by measuring CFU counts and correlated with the pellet structure.
RESULTS: The varying annealing protocols produce a range of material thicknesses, with more extensive annealing resulting in thicker materials. Storage stability exhibits a positive correlation with material thickness, indicating improved stability with thicker materials. Non-annealed pellets exhibit structural irregularities and inconsistent storage stability, highlighting the impracticality of avoiding annealing in the freeze-drying process.
CONCLUSIONS: Extensive annealing not only enhances the storage stability of probiotic products but also provides greater control over the freeze-drying process, ensuring homogeneous and reproducible products. This study underscores the importance of material thickness in freeze-dried pellets for optimizing storage stability for probiotic formulations, and emphasize the necessity of annealing as a critical step in freeze-drying quenched pellets to achieve desired structural and stability outcomes.

Monoclonal antibodies (mAbs) represent a promising modality for the prevention and treatment of viral infections. For infections that initiate from the respiratory tract, direct administration of specific neutralizing mAbs into lungs has advantages over systemic injection of the same mAbs. Herein, using AUG-3387, a human-derived mAb with high affinity to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its various variants, as a model mAb, we formulated the mAb into dry powders by thin-film freeze-drying, confirmed that the AUG-3387 mAb reconstituted from the dry powders retained their integrity, high affinity to the SARS-CoV-2 spike protein receptor binding domain (RBD), as well as ability to neutralize RBD-expressing pseudoviruses. Finally, we showed that one of the AUG-3387 mAb dry powders had desirable aerosol properties for pulmonary delivery into the lung. We concluded that thin-film freeze-drying represents a viable method to prepare inhalable powders of virus-neutralizing mAbs for pulmonary delivery into the lung.

The effects of MRS, whey protein and blueberry alone, and mixed fermentation on the survival rate of lactic acid bacteria under various freeze-drying conditions were investigated. The surface structure of the freeze-dried powders was also investigated to explore the anti-freezing protection mechanism of mixed whey protein and blueberry fermentation on the bacteria. It was found that the mixed fermentation medium of blueberry and whey protein has a protective effect on the freeze-drying bacteria and is better than the traditional MRS and whey protein medium. The optimal concentration of blueberry juice addition was 9%. The survival rate of the pre-freezing temperature at -80 °C was higher than at -20 °C after the pre-freezing and freeze-drying processes. The freeze-drying thickness of 0.3 cm could improve the survival rate of the bacteria. The Fourier transform infrared spectroscopy results indicated the interaction between the whey protein, anthocyanins, and the surface composition of the lactic acid bacteria.

Bio-aerogels have emerged as promising materials for energy storage, providing a sustainable alternative to conventional aerogels. This review addresses their syntheses, properties, and characterization challenges for use in energy storage devices such as rechargeable batteries, supercapacitors, and fuel cells. Derived from renewable sources (such as cellulose, lignin, and chitosan), bio-based aerogels exhibit mesoporosity, high specific surface area, biocompatibility, and biodegradability, making them advantageous for environmental sustainability. Bio-based aerogels serve as electrodes and separators in energy storage systems, offering desirable properties such as high specific surface area, porosity, and good electrical conductivity, enhancing the energy density, power density, and cycle life of devices. Recent advancements highlight their potential as anode materials for lithium-ion batteries, replacing non-renewable carbon materials. Studies have shown excellent cycling stability and rate performance for bio-aerogels in supercapacitors and fuel cells. The yield properties of these materials, primarily porosity and transport phenomena, demand advanced characterization methods, and their synthesis and processing methods significantly influence their production, e.g., sol-gel and advanced drying. Bio-aerogels represent a sustainable solution for advancing energy storage technologies, despite challenges such as scalability, standardization, and cost-effectiveness. Future research aims to improve synthesis methods and explore novel applications. Bio-aerogels, in general, provide a healthier path to technological progress.

With the increase of oily wastewater discharge and the growing demand for clean water supply, high throughput green materials for oil-water separation with anti-pollution and self-cleaning ability are urgently needed. Herein, the polysaccharide-based composite aerogels of CMC/SA@TiO2-MWCNTs (CSTM) with fast photo-driven self-cleaning ability have been prepared by a simple freeze-drying and ionic cross-linking strategy. The introduction of TiO2 /MWCNTs nanocomposites effectively improves the underwater oleophobic and mechanical properties of polysaccharide aerogels and enables their photo-driven self-cleaning ability for efficient oil-water separation and purification of complex oily wastewater. For immiscible oil-water mixtures, a high separation flux of about 7650 L m-2 h-1 and a separation efficiency of up to 99.9 % was obtained. For surfactant-stabilized oil-in-water emulsion, a flux of 3952 L m-2 h-1 was achieved with a separation efficiency of up to 99.3 %. More importantly, the excellent photoluminescent self-cleaning ability and low oil adhesion contribute to the high contamination resistance, excellent reusability, and robust durability of CSTM aerogel. With the advantages of simple preparation, remarkable performance, and recyclability, this aerogel is expected to provide a green, economical, and scalable solution for the purification of oily wastewater.

Freeze-drying is a preservation method known for its effectiveness in dehydrating food products while minimizing their deterioration. However, protein denaturation and oxidation during freezing and drying can degrade the quality of meat and aquatic products. Therefore, finding the strategies to ensure the dried products\' sensory, functional, and nutritional attributes is crucial. This study aimed to summarize protein denaturation mechanisms and overall quality changes in meat and aquatic products during freezing and drying, while also exploring methods for quality control. Different freeze-drying conditions result in varying levels of oxidation and functionality in meat and aquatic products, leading to changes in quality, such as altered fatty and amino acid compositions, protein digestibility, and sensory attributes. To obtain high-quality dried products by freeze-drying, several parameters should be considered, including sample type, freezing and drying temperatures, moisture content, pulverization effects, and storage conditions.

Recombinant human acidic fibroblast growth factor (rh-aFGF) is a widely used biological product, but it is unstable and its biological activity is easy to decrease. In order to maintain the long-term stability and biological activity of rh-aFGF, based on the response surface method, the freeze-drying characterization and cell proliferation rate of rh-aFGF freeze-dried powder were evaluated by scoring and Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay in this study. The optimal concentrations of trehalose, glycine and BSA were optimized, and the optimal formulation was verified by regression experiment. The results showed that trehalose, glycine and BSA had significant effects on the characterization of lyophilized rh-aFGF and cell proliferation. The optimal formulation of 5.7% trehalose, 2.04% glycine and 1.98%BSA combined with rh-aFGF could achieve the optimal freeze-dried characterization and biological activity. Using the best formulation to verify, the freeze-dried formability index of the freeze-dried powder was 23.35, and the rate of cell proliferation was 43.59%, which was close to the expected 23 and 41.69%. This study determined a freeze-dried formulation of rh-aFGF that meets the requirements of freeze-dried formalization integrity and maintains biological activity, providing reliable support for the subsequent development of related drugs.

Biopharmaceuticals are labile biomolecules that must be safeguarded to ensure the safety, quality, and efficacy of the product. Batch freeze-drying is an established means of manufacturing solid biopharmaceuticals but alternative technologies such as spray-drying may be more suitable for continuous manufacturing of inhalable biopharmaceuticals. Here we assessed the feasibility of spray-drying Olipudase alfa, a novel parenteral therapeutic enzyme, by evaluating some of its critical quality attributes (CQAs) in a range of excipients, namely, trehalose, arginine (Arg), and arginine hydrochloride (Arg-HCl) in the sucrose/methionine base formulation. The Arg-HCl excipient produced the best gain in CQAs of spray-dried Olipudase with a 63% reduction in reconstitution time and 83% reduction in the optical density of the solution. Molecular dynamics simulations revealed the atomic-scale mechanism of the protein-excipient interactions, substantiating the experimental results. The Arg-HCl effect was explained by the calculated thermal stability and structural order of the protein wherein Arg-HCl acted as a crowding agent to suppress protein aggregation and promote stabilization of Olipudase post-spray-drying. Therefore, by rational selection of appropriate excipients, our experimental and modelling dataset confirms spray-drying is a promising technology for the manufacture of Olipudase and demonstrates the potential to accelerate development of continuous manufacturing of parenteral biopharmaceuticals.

This study addressed the critical issue of food waste, particularly focusing on carrot pomace, a by-product of carrot juice production, and its potential reutilization. Carrot pomace, characterized by high dietary fiber content, presents a sustainable opportunity to enhance the functional properties of food products. The effects of physical pretreatments-high shearing (HS), hydraulic pressing (HP), and their combination (HSHP)-alongside two drying methods (freeze-drying and dehydration) on the functional, chemical, and physical properties of carrot pomace were explored. The results indicated significant enhancements in water-holding capacity, fat-binding capacity, and swelling capacity, particularly with freeze-drying. Freeze-dried pomace retained up to 33% more carotenoids and demonstrated an increase of up to 22% in water-holding capacity compared to dehydrated samples. Freeze-dried pomace demonstrated an increase of up to 194% in fat-binding capacity compared to dehydrated samples. Furthermore, HSHP pretreatment notably increased the swelling capacity of both freeze-dried (54%) and dehydrated pomace (35%) compared to pomace without pretreatments. Freeze-drying can enhance the functional properties of dried carrot pomace and preserve more carotenoids. This presents an innovative way for vegetable juice processors to repurpose their processing by-products as functional food ingredients, which can help reduce food waste and improve the dietary fiber content and sustainability of food products.