Morphological reconstruction during cell regeneration in the ciliate Spirostomum ambiguum.

When the ciliate Spirostomum ambiguum is transected into two pieces, both fragments regenerate and proliferate. In the anterior fragments, which have lost their contractile vacuoles due to transection, new contractile vacuoles were formed at their posterior ends in a few minutes. When the cells were cut into three pieces, new contractile vacuoles were formed in the anterior and middle fragments, both at their posterior ends. Thus, the anterior-posterior axis of S. ambiguum was maintained after transection. Morphological repair, including the formation of the contractile vacuole, was also observed when only the anteriormost portion was transected to cut out a small fragment that did not contain part of the macronucleus. Scanning electron microscopy was performed to observe changes in the shape of the cleavage surface of S. ambiguum during the wound healing process. Within minutes after cutting, the cut surface was covered with a cilia-free membrane, preventing leakage of cytoplasmic contents. The surface of the cut area then rounded with time and was covered with cilia, completing the repair of the cut area in about one day.

Quantification of Residual Water in Pharmaceutical Frozen Solutions via 1H Solid-state NMR.

Freezing is essential for the stability of biological drug substances and products, particularly in frozen solution formulations and during the primary drying of lyophilized preparations. However, the unfrozen segment within the frozen matrix can alter solute concentration, ionic strength, and stabilizer crystallization, posing risks of increased biophysical instability and faster chemical degradation. While quantifying the unfrozen water content is important for designing stable biopharmaceuticals, there is a lack of analytical techniques for in situ quantitative measurements. In this study, we introduce a 1H magic angle spinning NMR technique to identify the freezing point (Tice) and quantify mobile water content in frozen biologics, applying this method to analyze the freezing of a commercial high-concentration drug product, Dupixent®. Our results demonstrate that water freezing is influenced by buffer salt properties and formulation composition, including the presence of sugar cryoprotectants and protein concentration. Additionally, the 1H chemical shift can probe pH in the unfrozen phase, potentially predicting the microenvironmental acidity in the frozen state. Our proposed methodology provides fresh insights into the analysis of freeze-concentrated solutions, enhancing our understanding of the stability of frozen and lyophilized biopharmaceuticals.

Effects of ultrasonication and freeze-thaw pretreatments on the vacuum freeze-drying process and quality characteristics of apricot (Prunus armeniaca L. cv. Diaoganxing).

The combination of pretreatment and vacuum freeze-drying (VFD) technology is an effective technique for extending the shelf life of apricots, reducing costs and energy consumption. However, the impact of pretreatment on the freeze-drying and quality characteristics of apricots is still unclear. The effects of ultrasound (US), freeze-thaw (FT), and their combination (FT-US) on water migration and quality characteristics of apricot slices on VFD were studied. LR-NMR and SEM showed that pretreatment significantly reduced the time (19.05%-33.33%) and energy consumption (17.67%-35.66%) of the VFD process. Compared with the control group, the US, FT, and FT-US improved the color, texture, rehydration ability, and flavor of apricot slices. Among them, FT-US retained the most biologically active substances and antioxidant capacity, with the highest sensory score. Overall, FT-US pretreatment induced changes in the microstructure and chemistry of apricots, which contributed to the production of high-quality VFD apricot slices.

Porous Functionally Graded Scaffold prepared by a single-step freeze-drying process. A bioinspired approach for wound care.

Nowadays, chronic wounds are the major cause of morbidity worldwide and the healthcare costs related to wound care are a billion-dollar issue; chronic wounds involve a non-healing process that makes necessary the application of advanced wound dressings to promote skin integrity recovery. Functionally Graded Scaffolds (FGSs) are currently driving interest as promising candidates in mimicking the skin tissue environment and, thus, in enhancing a faster and more effective wound healing process. Aim of the present work was to design and develop a porous FGS based on κ-carrageenan (κCG) for the management of chronic skin wounds; a freeze-drying process was optimized to obtain in a single-step a three-layered FGS characterized by a pore size gradient functional to mimic the structure of native skin tissue. In addition to κCG, arginine and whey protein isolate were used as multifunctional agents for FGS preparation; these substances can not only intervene in some stages of wound healing but are able to establish non-covalent interactions with κCG, which were responsible for the production of layers with different pore size, water content capability and mechanical properties. Cell migration, adhesion and proliferation within the FGS structure were evaluated in vitro on fibroblasts and FGS wound healing potential was also studied in vivo on a murine model.

Novel fast dissolving freeze dried sublingual baicalin tablets for enhanced hepatoprotective effect: in-vitro characterization, cell viability, and in-vivo evaluation.

Baicalin (BG), a natural product, has been used in the prevention and treatment of drug-induced liver injury (DILI); however, its poor solubility and extensive liver metabolism limit its pharmacological use. The aim of the present study was the formulation of fast-dissolving freeze-dried sublingual tablets (FFSTs) to increase BG dissolution, avoid first-pass metabolism, and overcome swallowing difficulties. FFSTs were prepared following a 23 factorial design. The effect of three independent variables namely matrix former, Maltodextrin, concentration (4%, and 6%), binder concentration (2%, and 3%), and binder type (Methocel E5, and Methocel E15) on the FFSTs' in-vitro disintegration time and percentage dissolution was studied along with other tablet characteristics. Differential scanning calorimetry, scanning electron microscopy, in-vitro HepG2 cell viability assay, and in-vivo characterization were also performed. F8 (6% Maltodextrin, 2% Mannitol, 2% Methocel E5), with desirability of 0.852, has been furtherly enhanced using 1%PEG (F10). F10 has achieved an in-vitro disintegration time of 41 secs, and 60.83% in-vitro dissolution after 2 min. Cell viability assay, in-vivo study in rats, and histopathological studies confirmed that pretreatment with F10 has achieved a significant hepatoprotective effect against acetaminophen-induced hepatotoxicity. The outcome of this study demonstrated that FFSTs may present a patient-friendly dosage form against DILI.

Mouse Brain Tissue Preparation for Scanning Electron Microscopy.

Scanning electron microscopy (SEM) is used to observe the surface structure of an object by irradiating an electron beam onto the sample and detecting the reflected and emitted electrons. Because of its large depth of focus, SEM can provide the three-dimensional structure of small surfaces that cannot be observed using an optical microscope. Furthermore, the cross-sectional structure of the tissue can be observed by freeze-cracking. Observing the ultrastructure of organisms that contain large amounts of water in their bodies while maintaining high resolution is challenging; however, this has recently become possible. Here, we explain the fixation and freeze-cracking method for mouse brain samples.

Effect of guar gum, gelatin, and pectin on moisture changes in freeze-dried restructured strawberry blocks.

This study aimed to investigate the effects of edible gum addition on moisture changes in freeze-dried restructured strawberry blocks (FRSB), which involved five groups: the control, 1.2% guar gum, 1.2% gelatin, 1.2% pectin, and the composite group with 0.5% guar gum, 0.5% gelatin, and 0.45% pectin. The results indicated that the drying rates of the five groups of FRSB presented similar early acceleration and later deceleration trends. Moisture content in FRSB was linearly predicted by peak area of low field nuclear magnetic resonance with R2 higher than 0.90 for all the five groups. The FRSB samples in the gelatin and composition groups formed a denser porous structure and had a lower hygroscopicity after four days of storage. This study provides a theoretical basis for controlling the processing of FRSB.

Dual Functionality of Poloxamer 188 in Freeze-Dried Protein Formulations: A Stabilizer in Frozen Solutions and a Bulking Agent in Lyophiles.

Poloxamer 188 (P188) was hypothesized to be a dual functional excipient, (i) a stabilizer in frozen solution to prevent ice-surface-induced protein destabilization and (ii) a bulking agent to provide elegant lyophiles. Based on X-ray diffractometry and differential scanning calorimetry, sucrose, in a concentration-dependent manner, inhibited P188 crystallization during freeze-drying, while trehalose had no such effect. The recovery of lactate dehydrogenase (LDH), the model protein, was evaluated after reconstitution. While low LDH recovery (∼60%) was observed in the lyophiles prepared with P188, the addition of sugar improved the activity recovery to >85%. The secondary structure of LDH in the freeze-dried samples was assessed using infrared spectroscopy, and only moderate structural changes were observed in the lyophiles formulated with P188 and sugar. Thus, P188 can be a promising dual functional excipient in freeze-dried protein formulations. However, P188 alone does not function as a lyoprotectant and needs to be used in combination with a sugar.

Design of an Oral Tablet Containing Furosemide Nanoparticles with Elevated Bioavailability.

The solubility and permeability of the Biopharmaceutics Classification System (BCS) class IV drugs, such as furosemide (FUR), are low. Thus, the oral bioavailability of these drugs needs to be augmented. Here, we aimed to design orally disintegrating tablets containing FUR nanoparticles to improve bioavailability after oral administration. The FUR nanoparticles were generated by bead-milling in water containing 0.5% methylcellulose and 0.5% 2-hydroxypropyl-ß-cyclodextrin (w/w%). Particle size was approximately 47-350 nm (mean particle size, 188 nm). An orally disintegrating tablet (FUR-NP tablet) comprising FUR nanoparticles (1%) was successfully produced by employing suspensions outlined above that incorporated additives (4% D-mannitol, 0.4% polyvinylpyrrolidone, and 16% gum Arabic, w/w%), followed by freeze-drying. The FUR-NP tablet disaggregated after only 5 s in water, liberating nano-sized FUR particles (172 nm). Experiments using rats showed the absorption of the FUR-NP tablet was significantly improved by comparison with a FUR tablet containing microparticles. In summary, the orally disintegrating tablet containing FUR nanoparticles markedly enhanced the bioavailability of FUR. We anticipate this formulation will also improve the bioavailability of other BCS class IV drugs.

Effect of Annealing on Visible-Bubble Formation and Stability Profiles of Freeze-Dried High Concentration Omalizumab Formulations.

In the clinical application of freeze-dried highly concentrated omalizumab formulations, extensive visible bubbles (VBs) can be generated and remain for a long period of time in the reconstitution process, which greatly reduces the clinical use efficiency. It is necessary to understand the forming and breaking mechanism of VBs in the reconstitution process, which is a key factor for efficient and safe administration of biopharmaceutical injection. The effects of different thermal treatments on the volume of VBs and stability of omalizumab, mAb-1, and mAb-2 were investigated. The internal microvoids of the cake were characterized by scanning electron microscopy and mercury intrusion porosimetry. Electron paramagnetic resonance was applied to obtain the molecular mobility of the protein during annealing. A large number of VBs were generated in the reconstitution process of unannealed omalizumab and remained for a long period of time. When annealing steps were added, the volume of VBs was dramatically reduced. When annealed at an aggressive temperature (i.e., -6 °C), although the volume of VBs decreased, the aggregation and acidic species increased significantly. Thus, our observations highlight the importance of setting an additional annealing step with a suitable temperature, which contributes to reducing the VBs while maintaining the stability of the high concentration freeze-dried protein formulation.

Innovative freeze-drying technique in the fabrication of dissolving microneedle patch: Enhancing transdermal drug delivery efficiency.

Microneedle patch (MNP) has become a hot research topic in the field of transdermal drug delivery due to its ability to overcome the stratum corneum barrier. Among the various types of microneedles, dissolving microneedles represent one of the most promising transdermal delivery methods. However, the most used method for preparing dissolving microneedles, namely microfabrication, suffers from issues such as long drying time, susceptibility to humidity, and large batch-to-batch variability, which limit the development of dissolving microneedles. In this study, we report for the first time a method for preparing dissolving microneedles using freeze-drying technology. We screened substrates suitable for freeze-dried microneedle patch (FD-MNP) and used coating technology to enhance the mechanical strength of FD-MNP, allowing them to meet the requirements for skin penetration. We successfully prepared FD-MNP using hyaluronic acid as the substrate and insulin as the model drug. Scanning electron microscopy revealed that the microneedles had a porous structure. After coating, the mechanical strength of the microneedles was 0.61 N/Needle, and skin penetration rate was 97%, with a penetration depth of 215 µm. The tips of the FD-MNP dissolved completely within approximately 60 s after skin penetration, which is much faster than conventional MNP (180 s). In vitro transdermal experiments showed that the FD-MNP shortened the lag time for transdermal delivery of rhodamine 123 and insulin compared to conventional MNP, indicating a faster transdermal delivery rate. Pharmacological experiments showed that the FD-MNP lowered mouse blood glucose levels more effectively than conventional MNP, with a relative pharmacological availability of 96.59 ± 2.84%, higher than that of conventional MNP (84.34 ± 3.87%), P = 0.0095. After storage under 40℃ for two months, the insulin content within the FD-MNP remained high at 95.27 ± 4.46%, which was much higher than that of conventional MNP (58.73 ± 3.71%), P < 0.0001. In conclusion, freeze-drying technology is a highly valuable method for preparing dissolving microneedles with potential applications in transdermal drug delivery.

Freezing-mediated formation of supraproteins using depletion forces.

Hypothesis Long-acting formulations such as microparticles, injectable depots and implantable devices can realize spatiotemporally controlled delivery of protein drugs to extend their therapeutic in vivo half-lives. To efficiently encapsulate the protein drugs into such drug delivery systems, (sub)micron-sized protein particles are needed. The formation of micronized supraproteins can be induced through the synergistic combination of attractive depletion forces and freezing. The size of the supraproteins can be fine-tuned from submicron to several microns by adjusting the ice crystallization rate through the freeze-quench depth, which is set by the target temperature. Methods Supraprotein micron structures were prepared from protein solutions under various conditions in the presence and absence of nonadsorbing polyethylene glycol. Scanning electron microscopy and dynamic light scattering were employed to determine the sizes of the supraproteins and real-time total internal reflection fluorescent microscopy was used to follow the supraprotein formation during freezing. The protein secondary structure was measured before and after micronization by circular dichroism. A phase diagram of a protein-polyethylene glycol mixture was theoretically predicted to investigate whether the depletion interaction can elucidate the phase behavior. Findings Micronized protein supraparticles could be prepared in a controlled manner by rapid freeze-drying of aqueous mixtures of bovine serum albumin, horseradish peroxidase and lysozyme mixed with polyethylene glycol. Upon freezing, the temperature quench initiates a phase separation process which is reminiscent of spinodal decomposition. This demixing is subsequently arrested during droplet phase separation to form protein-rich microstructures. The final size of the generated protein microparticles is determined by a competition between phase separation and cooling rate, which can be controlled by target temperature. The experimental phase diagram of the aqueous protein-polyethylene glycol dispersion aligns with predictions from depletion theory for charged colloids and nonadsorbing polymers.

Tableting behavior of freeze and spray-dried excipients in pharmaceutical formulations.

Most of biopharmaceuticals, in their liquid form, are prone to instabilities during storage. In order to improve their stability, lyophilization is the most commonly used drying technique in the pharmaceutical industry. In addition, certain applications of biopharmaceutical products can be considered by oral administration and tablets are the most frequent solid pharmaceutical dosage form used for oral route. Thus, the tableting properties of freeze-dried products used as cryo and lyoprotectant could be a key element for future pharmaceutical developments and applications. In this study, we investigated the properties that might play a particular role in the specific compaction behavior of freeze-dried excipients. The tableting properties of freeze-dried trehalose, lactose and mannitol were investigated and compared to other forms of these excipients (spray-dried, commercial crystalline and commercial crystalline milled powders). The obtained results showed a specific behavior in terms of compressibility, tabletability and brittleness for the amorphous powders obtained after freeze-drying. The comparison with the other powders showed that this specific tableting behavior is linked to both the specific texture and the physical state (amorphization) of these freeze-dried powders.

Sustainable cellulose foams for all-weather high-performance radiative cooling and building insulation.

Passive daytime radiative cooling (PDRC) as a zero-energy-consumption cooling technique offers rich opportunities in reducing global energy consumption and mitigating CO2 emissions. Developing high-performance PDRC coolers with practical applicability based on sustainable materials is of great significance, but remains a big challenge. Herein, polyvinyl alcohol (PVA) and esterified cellulose (EC) extracted from sawdust were used as raw materials to construct foams by using a dual-crosslinking assisted-unidirectional freeze-drying strategy followed by hydrophobic surface modification. The resultant PVA/EC (PEC) foams with ideal hierarchical macropore structure displayed various excellent features, such as low thermal conductivity (26.2 mW·m-1·K-1), high solar reflectance (95 %) and infrared emissivity (0.97), superhydrophobicity as well as high mechanical properties. The features allowed the PEC foams to be used as radiative coolers with excellent PDRC performance and thermal insulating materials. A maximum sub-ambient temperature drops of 10.2 °C could be achieved for optimal PEC foams. Building simulations indicated that PEC foams could save 55.8 % of the energy consumption for Xi'an. Our work would give inspiration for designing various types of PDRC coolers, including but certainly not limited to foams-based radiative coolers.

Lyophilization for bacteria preservation: a promising approach for Yersinia pestis strains from an unique collection in Brazil (Fiocruz-CYP).

Yersinia pestis, the causative agent of plague, is a highly virulent bacterium that poses a significant threat to human health. Preserving this bacterium in a viable state is crucial for research and diagnostic purposes. This paper presents and evaluates a simple lyophilization protocol for the long-term storage of Y. pestis strains from Fiocruz-CYP, aiming to explore its impact on viability and long-term stability, while replacing the currently used methodologies. The lyophilization tests were conducted using the non-virulent Y. pestis strain EV76, subjected to the lyophilization process under vacuum conditions. Viability assessment was performed to evaluate the effects of lyophilization and storage conditions on Y. pestis under multiple temperature conditions (- 80 °C, - 20 °C, 4-8 °C and room temperature). The lyophilization protocol employed in this study consistently demonstrated its efficacy in maintaining high viability rates for Y. pestis samples in a up to one year follow-up. The storage temperature that consistently exhibited the highest recovery rates was - 80 °C, followed by - 20 °C and 4-8 °C. Microscopic analysis of the post-lyophilized cultures revealed preserved morphological features, consistent with viable bacteria. The high viability rates observed in the preserved samples indicate the successful preservation of Y. pestis using this protocol. Overall, the presented lyophilization protocol provides a valuable tool for the long-term storage of Y. pestis, offering stability, viability, and functionality. By refining the currently used methods of lyophilization, this protocol can improve long-term preservation for Y. pestis strains collections, facilitating research efforts, diagnostic procedures, and the development of preventive and therapeutic strategies against plague.

Soft and flexible polyvinyl alcohol/pullulan aerogels with fast and high water absorption capacity for facial mask substrates.

Facial mask substrates commonly used in skincare are often considered unhealthy and environmentally unfriendly due to their composition of premoistened nonwovens containing various preservatives. This study aims to address this issue by developing a preservative-free degradable aerogel made from polyvinyl alcohol (PVA)/pullulan (PUL) using a unidirectional freeze-drying method. The aerogels had ordered three-dimensional porous structures and exhibited desirable mechanical properties. They were soft and flexible in both dry and wet states, and their Young's moduli were comparable to that of human skin. The aerogels had high porosity, ranging from 93.0 % to 95.1 %, and exhibited a high water absorption rate and water absorption capacity (ranging from 7.5 g/g to 10.1 g/g). After 30 min of water evaporation, the aerogels showed excellent moisture retention, ranging from 88 % to 93 %. Additionally, the PVA/PUL aerogel efficiently loaded and released active ingredients, such as rapidly releasing ascorbic acid (> 90 % within 30 min). These findings suggest that the PVA/PUL aerogel has potential as a material for facial mask substrates.

Examining the Effect of Freezing Temperatures on the Survival Rate of Micro-Encapsulated Probiotic Lactobacillus acidophilus LA5 Using the Flash Freeze-Drying (FFD) Strategy.

This work proposes a novel drying method suitable for probiotic bacteria, called flash freeze-drying (FFD), which consists of a cyclic variation in pressure (up-down) in a very short time and is applied during primary drying. The effects of three FFD temperatures (-25 °C, -15 °C, and -3 °C) on the bacterial survival and water activity of Lactobacillus acidophilus LA5 (LA), previously microencapsulated with calcium alginate and chitosan, were evaluated. The total process time was 900 min, which is 68.75% less than the usual freeze-drying (FD) time of 2880 min. After FFD, LA treated at -25 °C reached a cell viability of 89.94%, which is 2.74% higher than that obtained by FD, as well as a water activity of 0.0522, which is 55% significantly lower than that observed using FD. Likewise, this freezing temperature showed 64.72% cell viability at the end of storage (28 days/20 °C/34% relative humidity). With the experimental data, a useful mathematical model was developed to obtain the optimal FFD operating parameters to achieve the target water content in the final drying.

Nasal delivery of encapsulated recombinant ACE2 as a prophylactic drug for SARS-CoV-2.

Angiotensin-converting enzyme 2 (ACE2) is responsible for cell fusion with SARS-CoV viruses. ACE2 is contained in different areas of the human body, including the nasal cavity, which is considered the main entrance for different types of airborne viruses. We took advantage of the roles of ACE2 and the nasal cavity in SARS-CoV-2 replication and transmission to develop a nasal dry powder. Recombinant ACE2 (rhACE2), after a proper encapsulation achieved via spray freeze drying, shows a binding efficiency with spike proteins of SARS-CoV-2 higher than 77 % at quantities lower than 5 µg/ml. Once delivered to the nose, encapsulated rhACE2 led to viability and permeability of RPMI 2650 cells of at least 90.20 ± 0.67 % and 47.96 ± 4.46 %, respectively, for concentrations lower than 1 mg/ml. These results were validated using nasal dry powder containing rhACE2 to prevent or treat infections derived from SARS-CoV-2.

Dynamic intervention to enhance the stability of PEGylated Ibrutinib loaded lipidic nano-vesicular systems: transitioning from colloidal dispersion to lyophilized product.

Liposomes being a promising colloidal system facilitates delivery of drugs with limited pharmacokinetic properties to achieve desirable clinical applications. However, development of a stable liposomal system is always challenging due to multiple complexities involved. Aqueous instability of liposomes and impact of various process and formulation parameters can lead to serious alteration of its therapeutic performance. In the proposed work, the authors aim to develop stable Ibrutinib-loaded liposomes using lyophilization and Quality-by-Design and assess their long-term stability. Ibrutinib-loaded liposomes were developed and optimized using Quality-by-Design technique and were further PEGylated and characterized for the same. Effect of cryoprotectants during lyophilization and other parameters are evaluated to obtain a robust formulation. The stability studies were conducted upto 6 months at various storage conditions to evaluate the effect of lyophilization. The impact of formulation, processing and lyophilization parameters on physicochemical properties of developed liposomal systems were evaluated and are critically discussed. Liquid dispersion exhibited a %degradation of 16-36% at 25 °C/60% RH which was reduced for less than 1% in lyophilized formulation for 6 months. Critical analysis and assessment of various parameters lead to identification of optimum conditions to manufacture this drug product and also opens way forward for further evaluation and translational possibilities.

Shelf Life and Functional Quality of Almond Bagasse Powders as Influenced by Dehydration and Storing Conditions.

Almond bagasse resulting after the production of almond-based drinks represents a promising by-product with potential for use as a functional ingredient. To facilitate its utilization, the stability of this material can be achieved through dehydration processes such as hot air drying or freeze-drying. Nevertheless, owing to its high fat content, almond bagasse is prone to lipid oxidation, which could result in undesirable quality. Therefore, the objective of this work was to assess the impact of dehydration (by hot air drying at 60 and 70 °C and by freeze-drying) and storage (at room temperature and in accelerated conditions) on the functional quality and stability of almond bagasse powder. Throughout the dehydration process, it was observed that antioxidant compounds were preserved without significant differences among dehydration treatments. These compounds increased over the storage period, especially in the samples treated with hot air. Regarding antiradical capacity, the hot-air-dried samples showed higher values than the freeze-dried ones, although in all cases, it increased during storage. For total phenols in samples air-dried at 70 °C, increases of more than 50% were observed. The acidity and peroxide index were increased in the extended storage period, although they did not reach critical values. Samples stored for 180 days showed peroxide values ranging from 10 to 12.8 meq O2/kg dry matter for samples stored at room temperature and from 14.7 to 23 meq O2/kg dry matter for samples subjected to accelerated storage.