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Effect of pre?freezing and saccharide types in freeze?drying of siRNA lipoplexes on gene?silencing effects in the cells by reverse transfection.

ABSTRACT: Our previous study reported that reverse (Rev)?transfection with small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) freeze?dried in trehalose or sucrose solution resulted in high gene?silencing activity in cells. The current study investigated whether pre?freezing or saccharide types present during the freeze?drying of siRNA lipoplexes affected gene?silencing in cells after Rev?transfection. Three types of cationic cholesterol derivatives and three types of dialkyl or trialkyl cationic lipids were used for the preparation of cationic liposomes. Additionally, six types of siRNA lipoplexes were vacuum?dried in trehalose or sucrose solution without a pre-freezing process in multi?well plates. A strong gene?silencing activity after Rev?transfection was observed regardless of the cationic lipid types in the cationic liposomes. It was also investigated whether saccharide types in the freeze?drying of siRNA lipoplexes affected gene?silencing after Rev?transfection. siRNA lipoplexes freeze?dried in monosaccharides (glucose, fructose, galactose or mannose), disaccharides (maltose, lactose, lactulose or cellobiose) and trisaccharide solution (raffinose or melezitose) demonstrated high gene?silencing activity. However, following Rev?transfection with siRNA lipoplexes freeze?dried in monosaccharides or trisaccharides, certain saccharides induced cytotoxicity and/or off?target effects. The results of the current study indicated that disaccharides may be suitable for the preparation of vacuum?dried or freeze?dried siRNA lipoplexes for Rev?transfection.

SUBMITTER: Tang M

PROVIDER: S-EPMC7453497 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Effect of pre‑freezing and saccharide types in freeze‑drying of siRNA lipoplexes on gene‑silencing effects in the cells by reverse transfection.

Tang Min M Hu Subin S Hattori Yoshiyuki Y

Molecular medicine reports 20200805 4

Our previous study reported that reverse (Rev)‑transfection with small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) freeze‑dried in trehalose or sucrose solution resulted in high gene‑silencing activity in cells. The current study investigated whether pre‑freezing or saccharide types present during the freeze‑drying of siRNA lipoplexes affected gene‑silencing in cells after Rev‑transfection. Three types of cationic cholesterol derivatives and three types of dialkyl or t ...[more]

PMID: 32945442

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Project description:Nonviral siRNA vectors prepared by the direct mixing of siRNA and mixtures of an asymmetric N(4),N(9)-diacyl spermine conjugate, N(4)-linoleoyl-N(9)-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), with either cholesterol or DOPE, at various molar ratios of the neutral lipids, are reported. The effects of varying the lipid formulation and changing the N/P charge ratio on the intracellular delivery of siRNA to HeLa cells and on the siRNA-mediated gene silencing of a stably expressed reporter gene (EGFP) were evaluated. The presence of either cholesterol or DOPE in the mixture resulted in a marked increase in the delivery of the siRNA as well as enhanced EGFP silencing as evaluated by FACS. A LinOS/Chol 1:2 mixture resulted in the highest siRNA delivery and the most efficient EGFP silencing (reduced to 20%) at N/P = 3.0. Lowering the amount of siRNA from 15 pmol to 3.75 pmol, thus increasing the N/P charge ratio to 11.9, resulted in decreasing the amount of delivered siRNA, while the efficiency of gene silencing was comparable to that obtained with 15 pmol (N/P = 3.0) of siRNA. Mixtures of symmetrical N(4),N(9)-dioleoyl spermine (DOS) with cholesterol at 1:2 molar ratio showed less siRNA delivery than with LinOS/Chol at N/P = 3.0 (15 pmol of siRNA), and comparable delivery at N/P = 11.9 (3.75 pmol of siRNA). The EGFP silencing was comparable with LinOS and with DOS when mixed with cholesterol 1:2 (lipoplexes prepared with 15 pmol of siRNA), but LinOS mixtures showed better EGFP silencing when the siRNA was reduced to 3.75 pmol. Lipoplex particle size determination by DLS of cholesterol mixtures was 106-118 nm, compared to 194-356 nm for lipoplexes prepared with the spermine conjugates only, and to 685 nm for the LinOS/DOPE 1:1 mixture. Confocal microscopy showed successful siRNA delivery of red tagged siRNA and quantitative EGFP knockdown in HeLa EGFP cells; Z-stack photomicrographs showed that the delivered siRNA is distributed intracellularly. Cryo-TEM of siRNA LinOS/Chol 1:2 lipoplexes shows the formation of multilamellar spheres with a size of ∼100 nm, in good agreement with the particle size measured by DLS. The constant distance between lamellar repeats is ∼6 nm, with the electron-dense layers fitting a monolayer of siRNA. AlamarBlue cell viability assay showed that the lipoplexes resulted in cell viability ≥81%, with LinOS/Chol 1:2 mixtures resulting in cell viabilities of 89% and 94% at siRNA 15 nM and 3.75 nM respectively. These results show that lipoplexes of siRNA and LinOS/Chol mixtures prepared by the direct mixing of the lipid mixture and siRNA, without any preceding preformulation steps, result in enhanced siRNA delivery and EGFP knockdown, with excellent cell viability. Thus, LinOS/Chol 1:2 mixture is a promising candidate as a nontoxic nonviral siRNA vector.

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.

Dataset Information

Effect of pre?freezing and saccharide types in freeze?drying of siRNA lipoplexes on gene?silencing effects in the cells by reverse transfection.

Publications

Effect of pre‑freezing and saccharide types in freeze‑drying of siRNA lipoplexes on gene‑silencing effects in the cells by reverse transfection.

Similar Datasets

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