Controlled rate slow freezing with lyoprotective agent to retain the integrity of lipid nanovesicles during lyophilization

We designed a novel lyophilization method using controlled rate slow freezing (CSF) with lyoprotective agent (LPA) to achieve intact lipid nanovesicles after lyophilization. During the freezing step, LPA prevented water supercooling, and the freezing rate was controlled by CSF. Regulating the freezing rate by various liquid media was a crucial determinant of membrane disruption, and isopropanol (freezing rate of 0.933 °C/min) was the optimal medium for the CSF system. Lyophilized lipid nanovesicle using both CSF and LPA retained 92.9% of the core material and had uniform size distributions (Z-average diameter = 133.4 nm, polydispersity index = 0.144), similar to intact vesicles (120.7 nm and 0.159, respectively), after rehydration. Only lyophilized lipid nanovesicle using both CSF and LPA showed no changes in membrane fluidity and polarity. This lyophilization method can be applied to improve storage stability of lipid nanocarriers encapsulating drugs while retaining their original activity.

An Advanced Lyophilization Toward Intact Lipid Nanovesicles: Liquid-mediated Freezing With Cryoprotectant to Retain the Integrity of Lipid Nanovesicles

A novel lyophilization method using liquid-mediated freezing (LMF) with cryoprotectant (CPA) was designed to achieve intact lipid nanovesicles after lyophilization. During the freezing step, CPA prevented water supercooling, and the freezing rate was controlled by LMF. Regulating the freezing rate by various liquid media was a crucial determinant of membrane disruption, and isopropanol (freezing rate of 0.933°C/min) was the optimal medium for the LMF system. Lyophilized lipid nanovesicle using both LMF and CPA retained 92.9% of the core material and had uniform size distributions (Z-average diameter = 135.5 nm, polydispersity index = 0.074), similar to intact vesicles (112.3 nm and 0.184, respectively), after rehydration. Only lyophilized lipid nanovesicle using both LMF and CPA showed no changes in membrane fluidity and polarity. This lyophilization method can be applied to improve storage stability of lipid nanocarriers encapsulating drugs such as a mRNA vaccine, while retaining its original activity.

Influence of Microwave Radiation on the Freezing Rate of Water in a Heat Pump Installation

The article presents the data obtained as a result of an experiment to determine the effect of microwave radiation on the freezing rate of water in a heat pump installation are presented. (Research purpose) The research purpose is in experimentally evaluating the effect of microwave radiation on the speed of the water-ice phase transition to increase the efficiency of the heat pump unit by increasing the rate of water crystallization. (Materials and methods) The main criterion for conducting the experiment was the speed of the water-ice phase transition of ordinary water and water that passed through microwave radiation. The article presents an experimental installation for conducting experiments, consisting of a 90-liter freezer, a Danfoss TLES4F compressor with a cooling capacity of 91 Watts, a programmable Arduino controller with four connected sealed DS18B20 temperature sensors, a water tank made of food- grade plastic. The article presents the scheme of the experimental installation. The water was treated with microwave radiation for 12 seconds, the thickness of the water layer was 4-5 millimeters, and the power of the magnetron used was 750 Watts. (Results and discussion) There was conducted 20 experiments on obtaining thermal energy using the water-ice phase transition. Ten experiments with ordinary filtered water and ten experiments with water subjected to microwave radiation. (Conclusions) Water subjected to uniform microwave radiation cools to 0 degrees Celsius 23 minutes earlier than water that has passed only filtration, and performs a phase transition to a solid state 74 minutes faster. Microwave radiation can be used to increase the efficiency of a heat pump using the energy of the water-ice phase transition by accelerating the production of thermal energy from the heat carrier to the heat supply system.

Effects of freezing rate on structural changes in l-lactate dehydrogenase during the freezing process

Freezing is a common method for improving enzyme storage stability. During the freezing process, the freezing rate is an important parameter that can affect protein stability. However, there is limited information on the denaturation mechanisms and protein conformational changes associated with the freezing rate. In this study, the effects of freezing rate on activity loss and conformational changes in a model enzyme, l-lactate dehydrogenase, were evaluated. Enzyme solutions were frozen at various rates, from 0.2 to 70.6 °C/min, and ice seeding was conducted to reduce supercooling. The results demonstrated that fast freezing results in activity loss, structural changes, and aggregation. The residual activities at freezing rates of 0.2, 12.8, and 70.6 °C/min were 77.6 ± 0.9%, 64.1 ± 0.4%, and 44.8 ± 2.0%, respectively. As the freezing rate increased, the degree of dissociation and unfolding increased significantly, as determined using blue native-polyacrylamide gel electrophoresis and fluorescence spectroscopy. Moreover, a large number of amyloid aggregates were detected in samples frozen at a fast freezing rate (70.6 °C/min). The enzyme inactivation mechanism induced by fast freezing was proposed in terms of increased dehydration at the enzyme surface and an ice/unfroze solution interface, which could be helpful to establish a common understanding of enzyme inactivation during the freezing process.