scholarly journals Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4626
Author(s):  
Santhosh Chandar Maddila ◽  
Chandrashekhar Voshavar ◽  
Porkizhi Arjunan ◽  
Rashmi Prakash Chowath ◽  
Hari Krishna Reddy Rachamalla ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Nucleic Acid ◽  
Lipid Rafts ◽  
Transfection Efficiency ◽  
Vascular Diseases ◽  
Cationic Lipid ◽  
Cationic Liposome ◽  
Nucleic Acid Delivery ◽  
Liver Sinusoidal Endothelial Cells ◽  
Transfection Efficacy

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2–3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

scholarly journals Efficient Transfection of Large Plasmids Encoding HIV-1 into Human Cells—A High Potential Transfection System Based on a Peptide Mimicking Cationic Lipid

Pharmaceutics ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 805
Author(s):  
Christopher Janich ◽  
Daniel Ivanusic ◽  
Julia Giselbrecht ◽  
Elena Janich ◽  
Shashank Reddy Pinnapireddy ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Nucleic Acid ◽  
Fluorescent Protein ◽  
Cationic Lipid ◽  
Nucleic Acid Delivery ◽  
Molecular Clone ◽  
Green Fluorescent ◽  
Hiv 1

One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic.

scholarly journals Correlation of mRNA delivery timing and protein expression in lipid-based transfection

2019 ◽  
Author(s):  
A. Reiser ◽  
D. Woschée ◽  
N. Mehrotra ◽  
R. Krzysztoń ◽  
H. H. Strey ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Protein Expression ◽  
Single Cell ◽  
Serum Protein ◽  
Transfection Efficiency ◽  
Viral Gene ◽  
Nucleic Acid Delivery ◽  
Delivery Times ◽  
Time Courses ◽  
Endosomal Release

AbstractNon-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomal-release bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticles (LNPs) mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery.

scholarly journals Viral Mimicry as a Design Template for Nucleic Acid Nanocarriers

2021 ◽  
Vol 9 ◽  
Author(s):  
Ina F. de la Fuente ◽  
Shraddha S. Sawant ◽  
Mark Q. Tolentino ◽  
Patrick M. Corrigan ◽  
Jessica L. Rouge
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Molecular Mechanisms ◽  
Transfection Efficiency ◽  
Delivery Systems ◽  
Endosomal Escape ◽  
Nucleic Acid Delivery ◽  
Metastable Structure ◽  
Oligonucleotide Delivery ◽  
Genome Protection

Therapeutic nucleic acids hold immense potential in combating undruggable, gene-based diseases owing to their high programmability and relative ease of synthesis. While the delivery of this class of therapeutics has successfully entered the clinical setting, extrahepatic targeting, endosomal escape efficiency, and subcellular localization. On the other hand, viruses serve as natural carriers of nucleic acids and have acquired a plethora of structures and mechanisms that confer remarkable transfection efficiency. Thus, understanding the structure and mechanism of viruses can guide the design of synthetic nucleic acid vectors. This review revisits relevant structural and mechanistic features of viruses as design considerations for efficient nucleic acid delivery systems. This article explores how viral ligand display and a metastable structure are central to the molecular mechanisms of attachment, entry, and viral genome release. For comparison, accounted for are details on the design and intracellular fate of existing nucleic acid carriers and nanostructures that share similar and essential features to viruses. The review, thus, highlights unifying themes of viruses and nucleic acid delivery systems such as genome protection, target specificity, and controlled release. Sophisticated viral mechanisms that are yet to be exploited in oligonucleotide delivery are also identified as they could further the development of next-generation nonviral nucleic acid vectors.

Improvement of antisense oligonucleotides delivery using high hydrostatic pressurized lipoplex

2013 ◽  
Vol 1498 ◽  
pp. 3-8
Author(s):  
Tsuyoshi Kimura ◽  
Asami Sano ◽  
Kwangwoo Nam ◽  
Kazunari Akiyoshi ◽  
Yoshihiro Sasaki ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Transfection Efficiency ◽  
Cationic Liposome ◽  
Firefly Luciferase ◽  
Hek293 Cells ◽  
Luciferase Expression ◽  
Gene Suppression ◽  
Acid Therapy ◽  
Transfection Efficacy ◽  
Oligonucleotide Delivery

ABSTRACTCationic liposome (CL) is a promising vector for nucleic acid therapy. In the present study, we investigated the effect of high hydrostatic pressure (HHP) treatment to lipoplex on the lipoplex-based antisense oligodeoxynucleotides (AS-ODNs) delivery in order to improve the transfection efficacy of lipoplex. Cationic liposome consisting of DOTMA and DOPE was used. AS-ODNs were designed to inhibit the expression of firefly luciferase. The complexes of CL and AS-ODN were prepared at various C/A ratios and then pressurized hydrostatically at various atmospheres (∼10,000 atm) for 10 min (HHP treatment). After removal of pressure, the pressurized lipoplexes were used. The lipoplex with and without the HHP treatment was transferred into HeLa cells expressing firefly luciferase transiently. The luciferase activity using the HHP-treated lipoplex was decreased compared to that of the non-pressurized lipoplex. Also, for HEK293 cells expressing luciferase stably, the lipoplex with the HHP treatment could effectively suppress the luciferase expression. In order to elucidate relationship between the structure and the transfection efficiency of the HHP-treated lipoplex, the properties of the HHPtreated lipoplex were examined by various physicochemical analyses. The different physicochemical properties between the lipoplexes with and without HHP treatment were showed, suggesting that the nature of lipoplex was changed by the HHP treatment. We believe that this change of lipoplex properties by the HHP treatment affected the efficiency of gene suppression. This HHP treatment for lipoplex appears to be a promising contribution to gene and oligonucleotide delivery.

scholarly journals Graphene Oxide as 2D Platform for Complexation and Intracellular Delivery of siRNA

2018 ◽  
Author(s):  
Irene de Lázaro ◽  
Sandra Vranic ◽  
Domenico Marson ◽  
Artur Filipe Rodrigues ◽  
Maurizio Buggio ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Nucleic Acid ◽  
Gene Silencing ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Unmet Need ◽  
Sharp Decrease ◽  
Intracellular Delivery ◽  
Nucleic Acid Delivery ◽  
Primary Mouse

AbstractThe development of efficient and safe nucleic acid delivery vectors remains an unmet need holding back translation of gene therapy approaches into bedside. Graphene oxide (GO) could help bypass such bottleneck thanks to its large surface area, versatile chemistry and biocompatibility, which could overall enhance transfection efficiency while abolishing some of the limitations linked to the use of viral vectors. Here, we aimed to assess the capacity of bare GO, without any further surface modification, to complex a short double-stranded nucleic acid of biological relevance (siRNA) and mediate its intracellular delivery. GO formed stable complexes with siRNA at 10:1, 20:1 and 50:1 GO:siRNA mass ratios. Complexation was further corroborated by atomistic molecular dynamics simulations. GO:siRNA complexes were promptly internalized in a primary mouse cell culture, as early as 4 h after exposure. At this time point, intracellular siRNA levels were comparable to those provided by a lipid-based transfection reagent that achieved significant gene silencing. Time-lapse tracking of internalized GO and siRNA evidenced a sharp decrease of intracellular siRNA from 4 to 12 h, while GO was sequestered in large vesicles, which may explain the lack of biological effect (i.e. gene silencing) achieved by GO:siRNA complexes. This study underlines the potential of non-surface modified GO flakes to act as 2D siRNA delivery platforms, without the need for cationic functionalization, but warrants further vector optimization to allow effective release of the nucleic acid and achieve efficient gene silencing.

scholarly journals Manipulating fenestrations in young and old liver sinusoidal endothelial cells

2019 ◽  
Vol 316 (1) ◽  
pp. G144-G154 ◽  
Author(s):  
Nicholas J. Hunt ◽  
Glen P. Lockwood ◽  
Alessandra Warren ◽  
Hong Mao ◽  
Peter A. G. McCourt ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Insulin Resistance ◽  
Endothelial Cells ◽  
Actin Cytoskeleton ◽  
Lipid Rafts ◽  
No Synthase ◽  
Liver Sinusoidal Endothelial Cells ◽  
Sinusoidal Endothelial Cells ◽  
Nicotinamide Mononucleotide ◽  
Age Related

Fenestrations are pores within liver sinusoidal endothelial cells (LSECs) that enable the transfer of substrates (particularly insulin and lipoproteins) between blood and hepatocytes. With increasing age, there are marked reductions in fenestrations, referred to as pseudocapillarization. Currently, fenestrations are thought to be regulated by vascular endothelial growth factor and nitric oxide (NO) pathways promoting remodeling of the actin cytoskeleton and cell membrane lipid rafts. We investigated the effects of drugs that act on these pathways on fenestrations in old (18–24 mo) and young mice (3–4 mo). Isolated LSECs were incubated with either cytochalasin 7-ketocholesterol, sildenafil, amlodipine, simvastatin, 2, 5-dimethoxy-4-iodoamphetamine (DOI), bosentan, TNF-related apoptosis-inducing ligand (TRAIL) or nicotinamide mononucleotide (NMN). LSECs were visualized under scanning electron microscopy to quantify fenestration porosity, diameter, and frequency, as well as direct stochastic optical reconstruction microscopy to examine actin and NO synthase. In young and old LSECs, fenestration porosity, diameter and frequency were increased by 7-ketocholesterol, while porosity and/or frequency were increased with NMN, sildenafil, amlodipine, TRAIL, and cytochalasin D. In old mice only, bosentan and DOI increased fenestration porosity and/or frequency. Modification of the actin cytoskeleton was observed with all agents that increased fenestrations, while NO synthase was only increased by sildenafil, amlodipine, and TRAIL. In conclusion, agents that target NO, actin, or lipid rafts promote changes in fenestrations in mice LSECs. Regulation of fenestrations occurs via both NO-dependent and independent pathways. This work indicates that age-related defenestration can be reversed pharmacologically, which has potential translational relevance for dyslipidemia and insulin resistance. NEW & NOTEWORTHY We demonstrate the effects of multiple nitric oxide-dependent and -independent pharmaceutical agents on fenestrations of the liver sinusoidal endothelium. Fenestrations are reorganized in response to nicotinamide mononucleotide, sildenafil, amlodipine, and TNF-related apoptosis-inducing ligand. This work indicates that age-related defenestration can be reversed pharmacologically, which has potential translational relevance for dyslipidemia and insulin resistance in old age.

scholarly journals Scaling the effect of hydrophobic chain length on gene transfer properties of di-alkyl, di-hydroxy ethylammonium chloride based cationic amphiphiles

RSC Advances ◽  
2017 ◽  
Vol 7 (41) ◽  
pp. 25398-25405 ◽  
Author(s):  
Ankita A. Hiwale ◽  
Chandrashekhar Voshavar ◽  
Priya Dharmalingam ◽  
Ashish Dhayani ◽  
Rajesh Mukthavaram ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Gene Transfer ◽  
Transfection Efficiency ◽  
Systematic Investigation ◽  
Cationic Lipids ◽  
Nucleic Acid Delivery ◽  
Hydrocarbon Chains ◽  
Hydrophobic Chain ◽  
Transfer Properties

Asymmetric hydrocarbon chains influence the efficiency of cationic lipids based liposomes in nucleic acid delivery. A systematic investigation of role of asymmetry in transfection efficiency.

Asymmetric cationic lipid based non-viral vectors for an efficient nucleic acid delivery

RSC Advances ◽  
2016 ◽  
Vol 6 (81) ◽  
pp. 77841-77848 ◽  
Author(s):  
Rakeshchandra R. Meka ◽  
Sudhakar Godeshala ◽  
Srujan Marepally ◽  
Ketan Thorat ◽  
Hari Krishna Reddy Rachamalla ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Viral Vectors ◽  
Cationic Lipid ◽  
Cationic Lipids ◽  
Nucleic Acid Delivery

Cationic lipids have been extensively studied for their ability to complex with nucleic acids to condense and consequently deliver them into the cells.

scholarly journals Tunable Composition of Dynamic Non-Viral Vectors over the DNA Polyplex Formation and Nucleic Acid Transfection

Polymers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1313 ◽  
Author(s):  
Lilia Clima ◽  
Bogdan Florin Craciun ◽  
Gabriela Gavril ◽  
Mariana Pinteala
Keyword(s):  
Molecular Weight ◽  
Nucleic Acid ◽  
Viral Vectors ◽  
Viral Vector ◽  
Transfection Efficiency ◽  
Binding Capacity ◽  
Nucleic Acid Delivery ◽  
Force Microscopy ◽  
Transmission Electron

Polyethylene glycol (PEG) functionalization of non-viral vectors represents a powerful tool through the formation of an overall surface charge shielding ability, which is fundamental for efficient nucleic acid delivery systems. The degree of non-viral vector PEGylation and the molecular weight of utilized PEG is crucial since the excessive use of PEG units may lead to a considerable reduction of the DNA-binding capacity and, subsequently, in a reduction of in vitro transfection efficiency. Herein, we report a detailed study on a series of dynamic combinatorial frameworks (DCFs) containing PEGylated squalene, poly-(ethyleneglycol)-bis(3-aminopropyl) of different lengths, and branched low molecular weight polyethylenimine components, reversibly connected in hyperbranched structures, as efficient dynamic non-viral vectors. The obtained frameworks were capable of forming distinct supramolecular amphiphilic architectures, shown by transmission electron microscopy (TEM) and dynamic light scattering (DLS), with sizes and stability depending on the length of PEG units. The interaction of PEGylated DCFs with nucleic acids was investigated by agarose gel retardation assay and atomic force microscopy (AFM), while their transfection efficiency (using pCS2+MT-Luc DNA as a reporter gene) and cytotoxicity were evaluated in HeLa cells. In addition, the data on the influence of the poly-(ethyleneglycol)-bis(3-aminopropyl) length in composition of designed frameworks over transfection efficiency and tolerance in human cells were analyzed and compared.

scholarly journals 1,28-Di[(cholest-5-en-3β-yl)disulfanyl]-4,25-dioxo-3,8,12,17,21,26-hexaazaoctacosane tetrahydrochloride

2018 ◽  
Author(s):  
Pavel A. Puchkov ◽  
Elena V. Shmendel ◽  
Valeria D. Andreeva ◽  
Nina G. Morozova ◽  
Marina A. Zenkova ◽  
...  
Keyword(s):  
Thin Film ◽  
Gene Therapy ◽  
Nucleic Acid ◽  
Transfection Efficiency ◽  
Cationic Liposomes ◽  
Hek293 Cells ◽  
Nucleic Acid Delivery ◽  
Promising Candidate ◽  
Effective Delivery

The absence of highly effective delivery systems is a major challenge for gene therapy. Our work was aimed at the development of novel cationic liposomes possessing high transfection efficiency. For this purpose, a novel disulfide polycationic amphiphile 2S4 was synthesized. Cationic liposomes based on 2S4 and a helper lipid DOPE were formed by the thin film hydration method and exhibited effective pDNA delivery into the HEK293 cells, with a maximal transfection activity superior to that of the commercial agent Lipofectamine® 2000. Our results suggest that the polycationic amphiphile 2S4 is a promising candidate for in vitro nucleic acid delivery.

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