The Emerging Role of Exosome Nanoparticles in Regenerative Medicine
Harishkumar Madhyastha, Durgesh Nandini Chauhan in Nanopharmaceuticals in Regenerative Medicine, 2022
Methods for accommodation of small molecules, protein, or nucleic acids into the exosomes are: (1) physical methods such as electroporation (in the range of 150–700 V), sonication, extrusion procedures, freeze–thaw cycles, incubation at RT (with or without the use of saponin permeabilisation), or other temperature. The small-sized molecules can cross the lipid bilayer of exosome by simple incubation. (2) Chemical methods such as transfection by Lipofectamine 2000. This method is frequently used for siRNA (small interference RNA) packaging into the exosomes. (3) Biological methods called transfection of exosome-producing cells. In this method, the parent cells are genetically modified to overexpress a certain gene. The overexpressed gene would ultimately be collected into the parent cell-derived exosomes. For example, various sources of Mesenchymal Stem Cells (MSCs) were transfected with miR-146b and the exosomes containing the miR-146a cargo were collected from culture media (Johnsen et al. 2014).
Methods in Molecular Biology
Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman in Molecular Imaging in Oncology, 2008
Like transformation process, transfection typically involves opening transient pores or holes in the cell plasma membrane, to allow the uptake of material. There are various methods of introducing foreign DNA into a eukaryote cell. They fall into three categories, namely, (i) biochemical methods using calcium phosphate, diethylaminoethyl (DEAE)-dextran, and several cationic liposome-based transfection agents; (ii) physical methods including electroporation and direct injection; and (iii) transduction mediated by viruses. In the liposome-based transfection technique, lipid-based agents (Lipofectamine 2000, Superfect, FuGEN6, Poly-l-lysine) form complexes with DNA. The resultant lipid-coated DNA is taken into the cell using nonreceptor-mediated endocytosis.
Cationic Lipid-Based Gene Delivery: An Update
Kenneth L. Brigham in Gene Therapy for Diseases of the Lung, 2020
On the contrary, it is known that some cationic polymers can condense DNA into small particles of 15-100 nm in size (34,35). These DNA/polymer complexes, however, are relatively inefficient at transfection, unless they are accompanied by agents, such as chloroquine, that perturb endosome maturation, or treatments like osmotic shock or viral fusion proteins which destroy endosomal membranes (35-38). Since it is generally believed that DOPE-containing cationic liposomes have intrinsic cell membrane fusion activity (1), an interesting question was asked: Would a combination of polycations, which have superior DNA condensation capability, and cationic liposomes, which can rupture endosomes, result in a new generation of DNA delivery systems? To explore such a possibility, Gao and Huang recently tested several high-molecular-weight cationic polymers for their ability to modulate DNA/lipid complex formulation and to improve gene transfection activity (39). Several polymers such as polylysine or protamine significantly potentiated transfection activity by twofold to 28-fold in a number of cell types with several cationic liposomes in vitro, including DC-chol/DOPE and Lipofectin, particularly at lower than the usual optimal ratios of lipid to DNA used for transfection. Polylysine potentiated transfection was less for LipofectAMINE, a liposome formulation that condenses DNA well. Mechanistic studies revealed that polycations could drastically reduce the size of DNA-lipid complexes and that DNA, in the form of a polycation/liposome complex, became more resistant to nuclease activity present in fetal bovine serum. Interestingly, ultracentrifugation analysis of the DNA/polylysine/DC-chol liposome complexes on a sucrose density gradient revealed that purified liposome/polylysine/DNA (LPD) complexes contain small, condensed structures of 30-70 nm in size. These purified particles are three- to ninefold more potent in transfection compared to unpurified complexes based on the same amount of DNA. Electron microscopic images of these purified particles clearly showed that lipidic structures were associated with a proportion of the particles.
Ultrasound-targeted microbubble destruction mediated miR-492 inhibitor suppresses the tumorigenesis in non-small cell lung cancer
Published in Annals of Medicine, 2021
Wendi Zou, Yan Wang, Qingqing Song, Qianqian Li, Jie Ren, Xiaoyu Liu, Wei Cui
Lipofectamine, which has been widely and commonly used for gene transfection into cultured cells [38]. The study by Shi et al. showed that a high level of transfection and transduction efficiency using Lipofectamine 3000 transfection reagent compared with Lipofectamine 2000 or FuGENE 6 reagents could be achieved [39]. In addition, studies have confirmed that Lipofectamine 3000™ had the least impact on cell morphology and viability [40]. Thus, UTMD and Lipofectamine 3000 (Invitrogen, California, USA) were combined in this study. Based on the cell experimental results, we speculated that both approaches may yield a synergistic utility such that cell transfection efficiencies WERE superimposed to significantly improve transfection efficiency. However, the efficiency of the UTMD technique was affected by the ultrasound irradiation conditions. Thus, the application of UTMD needs to be explored under conditions with different loading products and different cell strains.
Delivering CRISPR: a review of the challenges and approaches
Published in Drug Delivery, 2018
Christopher A. Lino, Jason C. Harper, James P. Carney, Jerilyn A. Timlin
Perhaps the most commonly used lipid nanoparticle system is the commercially available Lipofectamine. Lipofectamine is a cationic liposome formulation that complexes to negatively charged nucleic acids, allowing fusion of the complex with negatively-charged cell membranes and endocytosis. Lipofectamine has been used to deliver Cas9- and sgRNA-encoding plasmid DNA to human pluripotent stem cells to generate a model for Immunodeficiency, Centromeric region instability, Facial anomalies syndrome (ICF) syndrome with 63% transfection efficiency (Horii et al., 2013), transfect human cells with an all-in-one expression cassette with up to seven sgRNAs and a Cas9 nuclease/nickase (Sakuma et al., 2014), correct the cystic fibrosis transmembrane conductor receptor locus in cultured intestinal stem cells of cystic fibrosis patients (Schwank et al., 2013), introduce modular ‘AND’ gate circuits based on CRISPR/Cas9 that detects bladder cancer cells, inhibits bladder cancer cell growth, induces apoptosis, and decreases cell motility (Liu et al., 2014), and deliver Cas9:sgRNA RNP in vivo to modify the hair cells within mouse inner ear (Zuris et al., 2015).
Advances in siRNA delivery in cancer therapy
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Aishwarya Singh, Piyush Trivedi, Narendra Kumar Jain
Lipoplexes are one of the most attractive nonviral vectors for plasmid and siRNA delivery [31]. The transfection mechanism of liposomes involves static interactions between negatively charged nucleic acids and cationic lipids as shown in Figure 4(b). Once mixed along, they spontaneously form lipoplexes [32,33]. Cationic lipids (100–300 nm in size) can protect siRNA from enzymatic degradation and increase the circulating half-life and uptake by cells. Cationic lipids such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and N-{1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTMA), along with helper lipids such as DOPE, are often used to form cationic liposomes and complex with negatively charged deoxyribonucleic acid and siRNA, resulting in high in vitro transfection efficiency [34]. Commercially available formulations like lipofectamine 2000 are used for in vitro transfection [35]. Cationic liposomes have limited success in vivo, they show dose-dependent toxicity and pulmonary inflammation can arise as a result of reactive oxygen intermediates [36–38]. In a recent study, chemotherapeutics and MCL1-specific siRNA co-delivered using trilysine-derived cationic lipid-based liposomes was found to decrease the expression of MCL1 in the tumour tissues of keratin-forming human epidermal carcinoma (KB) cell-xenografted mice [39]. In one study of anticancer siRNA was co-formulated with a diagnostic agent in cationic liposomes for theranostic purposes [40].