High Throughput Screening Method for Identification of New Lipofection Reagents

Abstract

Lipofection, the transfer of genetic material into cells by means of cationic lipids, is of growing interest for in vitro and in vivo approaches. In order to identify ideal lipofection reagents in a HTS, we have developed an automated lipofection method for the transfer of reporter genes into cells and for determination of the lipofection results. The method has specifically been designed and optimized for 96-well microtiter plates and can successfully be carried out by a pipetting robot with accessory equipment. It consists of two separate parts: (1) pretransfection (preparation of liposomes, formation of lipoplexes, and lipoplex transfer to the cells) and (2) posttransfection (determination of the reporter enzyme activity and the protein content of the transfected cells). Individual steps of the lipofection method were specifically optimized - for example, lipoplex formation and incubation time as well as cell lysis, cell cultivating, and the reporter gene assay. The HTS method facilitates characterization of the transfection properties (efficiency and cytotoxicity) of large numbers of (cationic) lipids in various adherent cell types.

The Phospholipid Vesicle-Based Drug Permeability Assay: 5. Development toward an Automated Procedure for High-Throughput Permeability Screening

“…By design, the robot is equipped with four pipetting needles and is able to do all pipetting steps, as well as to move the trays and the filter inserts. 8 For moving of single inserts, a custom-made extension of the gripper was used. A UV-absorbance reader (Tecan Infinite M200), which is able to measure in the wavelength range from 230 to 1000 nm, was connected to the robot.…”

Combinatorial Synthesis of New Cationic Lipids and High-Throughput Screening of Their Transfection Properties

“…Determination of the transfection properties: Determination of the transfection behavior of the new lipids was performed by using an automated assay system as described in detail by Regelin et al [17] In brief, the method consists of two parts: 1) the preparation of liposomes and formation of lipoplexes from the different lipids, and 2) cell transfection and determination of transfection properties.…”

“…The approach described here comprises synthesis of systematically modified cationic lipids by solid-phase combinatorial chemistry, and subsequent testing of the novel lipids for their transfection behavior towards various target cells in a high-throughput screening (HTS). [17] The goal was to identify promising new transfection lipids and, in further steps, to develop optimized lipofection protocols for different cell types.…”

High-Throughput Automation of Endosomolytic Polymers for mRNA Delivery

“…Consequently high-throughput (HT) screening can be useful for the development of novel, multivariant delivery systems. Automated HT screening of polymeric and lipid transfection agents has been pioneered by both Langer (synthesis and transfection efficiency) and Massing (toxicity and transfection efficiency), respectively. , Schubert and co-workers further established automated screening of DNA containing polyplexes by screening multiple biological properties such as polyplex stability, cytotoxicity, and transfection in multi wellplates …”

“…Automated HT screening of polymeric and lipid transfection agents has been pioneered by both Langer (synthesis and transfection efficiency) and Massing (toxicity and transfection efficiency), respectively. 22,23 Schubert and co-workers further established automated screening of DNA containing polyplexes by screening multiple biological properties such as polyplex stability, cytotoxicity, and transfection in multi wellplates. 24 There has been extensive work in the development of various delivery materials for siRNA, mRNA, and DNA.…”