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Effect of using amino acids in the freeze-drying of siRNA lipoplexes on gene knockdown in cells after reverse transfection.

ABSTRACT: Recently, small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) have become a crucial research tool for studying gene function. Easy and reliable siRNA transfection with a large set of siRNAs is required for the successful screening of gene function. Reverse (Rev)-transfection with freeze-dried siRNA lipoplexes is validated for siRNA transfection with a large set of siRNAs in a multi-well plate. In our previous study, it was shown that Rev-transfection with siRNA lipoplexes freeze-dried in disaccharides or trisaccharides resulted in long-term stability with a high level of gene-knockdown activity. In the present study, the effects of amino acids used as cryoprotectants in the freeze-drying of siRNA lipoplexes on gene knockdown via Rev-transfection were assessed. A total of 15 types of amino acids were used to prepare freeze-dried siRNA lipoplexes, and it was found that the freeze-drying of siRNA lipoplexes with amino acid concentrations >100 mM strongly suppressed targeted gene expression regardless of the amino acid type; however, some amino acids strongly upregulated or downregulated gene expression in the cells transfected with negative control siRNA. Amongst the amino acids tested, the presence of asparagine showed specific gene-knockdown activity, forming large cakes after freeze-drying and retaining a favorable siRNA lipoplex size after rehydration. These findings provide valuable information regarding amino acids as cryoprotectants for Rev-transfection using freeze-dried siRNA lipoplexes for the efficient delivery of siRNA into cells.

SUBMITTER: Tang M

PROVIDER: S-EPMC8329998 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Vacuum freeze-drying of biological materials is one of the best methods of water removal, with final products of highest quality. The solid state of water during freeze-drying protects the primary structure and the shape of the products with minimal volume reduction. In addition, the lower temperatures in the process allow maximal nutrient and bioactive compound retention. This technique has been successfully applied to diverse biological materials, such as meats, coffee, juices, dairy products, cells, and bacteria, and is standard practice for penicillin, hormones, blood plasma, vitamin preparations, etc. Despite its many advantages, having four to ten times more energy requirements than regular hot air drying, freeze-drying has always been recognized as the most expensive process for manufacturing a dehydrated product. The application of the freeze-drying process to plant-based foods has been traditionally dedicated to the production of space shuttle goods, military or extreme-sport foodstuffs, and specialty foods such as coffee or spices. Recently, the market for 'natural' and 'organic' products is, however, strongly growing as well as the consumer's demand for foods with minimal processing and high quality. From this perspective, the market for freeze-dried plant-based foods is not only increasing but also diversifying. Freeze-dried fruits and vegetables chunks, pieces, or slices are nowadays majorly used in a wide range of food products such as confectionaries, morning cereals, soups, bakeries, meal boxes, etc. Instant drinks are prepared out of freeze-dried tea, coffee, or even from maple syrup enriched with polyphenol concentrated extracts from trees. The possibilities are endless. In this review, the application of freeze-drying to transform plant-based foods was analyzed, based on the recent research publications on the subject and personal unpublished data. The review is structured around the following related topics: latest applications of freeze-drying to plant-based foods, specific technological problems that could be found when freeze-drying such products (i.e., presence of cuticle; high sugar or lipid concentration), pretreatments and intensification technologies employed in freeze-drying of plant-based foods, and quality issues of these freeze-dried products.

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Effect of using amino acids in the freeze-drying of siRNA lipoplexes on gene knockdown in cells after reverse transfection.

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