Investigating the stabilisation of IFN-α2a by replica exchange molecular dynamics simulation.

Current biopharmaceutical drugs are mainly a class of peptides or proteins that play an essential role in the treatment of many diseases. Such peptides/proteins are usually thermally unstable and may lose their bioactivity when exposed to ambient conditions. Therefore, they are not suitable for long-term storage. Lyophilisation is the most common method to prolong shelf life of solid peptide/protein drugs; however, the freeze-drying process can lead to irreversible damage. In the present study, human interferon-alpha 2a (IFN-α2a) was selected as a model protein drug; four disaccharides (ß-lactose, ß-maltose, sucrose, and trehalose) were selected as bioactive protectants. We investigated the effects of different protectants on IFN-α2a under various ambient conditions (vacuum, dry state, and aqueous solution) using replica exchange molecular dynamics simulation. The protective effect of ß-maltose on IFN-α2a was the highest in aqueous solution and dry state, ß-lactose showed a poor protective effect in all three conditions, the performance of sucrose was good in all conditions, and trehalose showed a better protective effect under vacuum conditions and in aqueous solution. Disaccharides form H-bonds with water, thereby preventing water from the tertiary structure of proteins. Trehalose forms strong H-bonds with water which explains its extraordinary stability.

Effect of oleic acid on the viability of different freeze-dried Lactiplantibacillus plantarum strains.

Freeze drying is one of the most convenient ways to preserve microorganisms, but in the freeze-drying process, strains will inevitably suffer varying degrees of damage under different conditions. The deterioration of cell membrane integrity is one of the main forms of damage. The type and ratio of fatty acids in the cell membrane affect its characteristics. Therefore, it is worth investigating whether certain fatty acids can increase freeze-drying resistance. In this study, we found that adding a low concentration of oleic acid to a cryoprotectant could increase survival rate of strains of Lactiplantibacillus plantarum following freeze drying, and the optimal concentration of oleic acid was determined to be 0.001%. When 0.001% oleic acid was added to phosphate-buffered saline, the freeze-drying survival rate of L. plantarum increased by up to 6.63 times. Adding 0.001% oleic acid to sorbitol, the survival rate of L. plantarum increased by as much as 3.65 times. The 0.001% oleic acid-sucrose cryoprotectant resulted in a freeze-drying survival rate of L. plantarum of about 90%, a 2.26-fold improvement compared with sucrose alone. Although the effect of oleic acid depends on the cryoprotectants used and the strain treated, addition of oleic acid showed significant improvement overall. Further experiments showed that adding a low concentration of oleic acid to the cryoprotectants improved the freeze-drying survival rate of L. plantarum by maintaining cell membrane integrity and lactate dehydrogenase activity. Our findings provide a new strategy for safeguarding bacterial viability in commonly used cryoprotectants by the addition of a common food ingredient, which may be extensively applied in the food industry.

Polysaccharides can improve the survival of Lactiplantibacillus plantarum subjected to freeze-drying.

Freeze-drying is one of the most commonly used methods of bacteria preservation. During this process, cryoprotectants can greatly reduce cellular damage. Micromolecular cryoprotectants have been widely adopted but have limited selectivity and protective effects. Therefore, explorations of other types of cryoprotectants are needed. This study aimed to explore the possibility of the macromolecular cryoprotectants and combinations of cryoprotectants to maintain bacterial activity. We found that the survival rate of Lactiplantibacillus plantarum AR113 after freeze-drying was 19% higher in the presence of soy polysaccharides than with trehalose, the best-performing micromolecular cryoprotectant. Moreover, a 90.52% survival rate of L. plantarum WCFS1 was achieved using the composite cryoprotectant containing soy polysaccharide and trehalose, which increased by 31.48 and 36.47% compared with adding solely trehalose or soy polysaccharide, respectively. These results demonstrate that macromolecular and micromolecular cryoprotectants have similar effects, and that combinations of macromolecular and micromolecular cryoprotectants have better protective effects. We further observed that the composite cryoprotectant can increase Lactobacilli survival by improving cell membrane integrity and lactate dehydrogenase activity. Our finding provides a new type of cryoprotectant that is safer and more effective, which can be extensively applied in the relevant food industry.

[Investigation on bioactive protective function of amino acids to insulin by molecular simulation].

Heat sensitive protein medicines are increasingly exhibiting their critical importance on treatment of various diseases at present. But their popularization and application meet a great challenge because of their heat instability. In the present study, insulin was taken as a heat sensitive protein medicine and amino acid as bio-protective agent in order to investigate if these amino acids can protect the insulin from losing its bioactivity due to desiccation. The experiment was performed by using replica exchange molecular simulation (REMD) method and Gromacs software with Gromos96 (53a6) force field. The REMD results indicated that these amino acids could protect the bioactive structure of insulin during desiccation. The configurations of the protected insulin were preserved very well. Those results proved that amino acid is a kind of good bioactive protective agent for the heat sensitive protein medicines.

A molecular simulation study of the protection of insulin bioactive structure by trehalose.

Biopharmaceuticals are proteins with a crucial role in the treatment of many diseases. However, these protein medicines are often thermally labile and therefore unsuitable for long-term application and storage, as they tend to lose their activity under ambient conditions. Desiccation is one approach to improving protein stability, but the drying process itself can cause irreversible damage. In the current study, insulin was chosen as an example of a thermally sensitive biopharmaceutical to investigate whether the disaccharide, trehalose, can prevent loss of structural integrity due to drying. The experiment was performed using replica exchange molecular simulation and Gromacs software with a Gromos96 (53a6) force field. The results indicate that trehalose preserves the bioactive structure of insulin during drying, consistent with the use of trehalose as a protectant for thermally sensitive biopharmaceuticals. For instance, at the water content of 1.77%, insulin without any protectants yields the highest RMSD value as 0.451 nm, then the RMSD of insulin in presence of trehalose only ca. 0.100 nm.

[Investigation on bioactive protection of LEA protein for insulin by molecular simulation in the low-temperature drying process].

Nowadays various protein medicines are increasingly playing significant roles in the treatment of many diseases, but the bioactive structures of such kinds of protein medicines are unstable because they are heat sensitive. Therefore, it is very important to explore a protective method and to explain the protective mechanism of protein medicines. In the present research, insulin was chosen as a heat-sensitive protein medicine, and a Group 3 late embryogenesis abundant (LEA) protein was chosen as its bioactive protectant during desiccation. The results of replica exchange molecular dynamics simulation suggest that comparing with insulin without any protection, the bioactive 3D structure and secondary structure of the insulin protected by LEA protein were preserved very well. All analyzing results proved that the LEA protein was a good bioactive protectant for heat sensitive protein medicines.

[Molecular simulation research on aggregation of insulin].

In the present research, molecular simulation and quantum chemistry calculations were combined to investigate the thermal stability of three kinds of insulin aggregations and the effect of Zn (II) ion coordination on these aggregations. The results of molecular simulation indicated that the three insulin dimers in the same sphere closed hexamer had synergistic stability. It is the synergistic stability that enhances the structural and thermal stability of insulin, preserves its bioactivity during production, storage, and delivery of insulin formulations, and prolongs its halflife in human bodies. According to the results of quantum chemistry calculations, each Zn (II)-N (Im-insulin) bond energy can reach 73.610 kJ/mol for insulin hexamer and 79.907 kJ/mol for insulin tetramer. However, the results of Gibbs free energy changes still indicats that the coordination of zinc (II) ions is unfavorable for the formation of insulin hexamer, because the standard Gibbs free energy change of the coordinate reaction of zinc (II) ions associated with the formatting insulin hexamer is positive and increased.

Investigation on formulation and preparation of adenovirus encoding human endostatin lyophilized powders.

A recombinant adenovirus encoding human endostatin gene, E10A, has finished phase II trials for head and neck cancer. However, the rigid storage temperature (-80°C) and the toxicity of glycerol in the E10A liquid preparation limited its clinical application. In this study, lyophilization was applied to develop a stable E10A lyophilized powder without glycerol that is able to maintain biological activity at 4°C and suitable for intravenous administration. The E10A lyophilized formulations composed of nontoxic and already clinically used excipients were characterized in terms of the pH change during freezing, the eutectic melting temperature (T(eu)) and the collapse temperature (T(c)). Freeze thawing tests were carried out to examine the protective effect of various excipients during freezing. Mannitol and its combinations with sucrose or inulin showed effective protection of E10A. The E10A lyophilized powders were analyzed by particle size measurement, residual humidity quantification, infectivity assay and gene expression level. An optimized formulation (formulation I1) yielded a good recovery of 76% of the starting infectivity after lyophilization and 89% of the original infectivity after storage at 4°C for 180 days. Also the gene expression capability of E10A in formulation I1 was maintained after lyophilization. In addition, it was found that the matrix of amorphous excipients, mannitol combinations with sucrose or inulin, was indispensible in protecting E10A against the stress of freezing and dehydration. Hereby, the E10A lyophilized powder with eliminated glycerol toxicity and improved stability could enhance the applicability of E10A for cancer gene therapy through intravenous administration.