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

2019 ◽  
Vol 11 (9) ◽  
pp. 362-371 ◽  
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

Abstract Non-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 nanoparticle (i-LNP)-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.

Single Cell Kinetics of Intracellular, Nonviral, Nucleic Acid Delivery Vehicle Acidification and Trafficking

2005 ◽  
Vol 16 (4) ◽  
pp. 986-994 ◽  
Author(s):  
Rajan P. Kulkarni ◽  
Swaroop Mishra ◽  
Scott E. Fraser ◽  
Mark E. Davis
Keyword(s):  
Nucleic Acid ◽  
Single Cell ◽  
Cell Kinetics ◽  
Nucleic Acid Delivery ◽  
Delivery Vehicle ◽  
Kinetics Of

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.

scholarly journals Methodologies to investigate intracellular barriers for nucleic acid delivery in non-viral gene therapy

Nano Today ◽  
2018 ◽  
Vol 21 ◽  
pp. 74-90 ◽  
Author(s):  
Lotte M.P. Vermeulen ◽  
Toon Brans ◽  
Stefaan C. De Smedt ◽  
Katrien Remaut ◽  
Kevin Braeckmans
Keyword(s):  
Gene Therapy ◽  
Nucleic Acid ◽  
Viral Gene ◽  
Nucleic Acid Delivery ◽  
Viral Gene Therapy

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 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.

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.

Peptide-Enhanced Nucleic Acid Delivery

MRS Bulletin ◽  
2005 ◽  
Vol 30 (9) ◽  
pp. 663-667 ◽  
Author(s):  
Jamie M. Bergen ◽  
Suzie H. Pun
Keyword(s):  
Nucleic Acid ◽  
Molecular Targets ◽  
Nuclease Activity ◽  
Cell Penetrating Peptides ◽  
Delivery Systems ◽  
Nucleic Acid Delivery ◽  
Surface Receptors ◽  
Endosomal Release ◽  
Nuclear Localization Sequences ◽  
Intracellular Targets

AbstractNumerous barriers, both extracellular and intracellular, hinder successful and efficient nonviral nucleic acid delivery. Due to their small size and ability to specifically recognize and interact with molecular targets, peptides can be incorporated as modular elements into synthetic nucleic acid delivery systems to overcome many of these barriers. Three classes of peptides that have frequently been integrated as components in nucleic acid delivery systems include cell-penetrating peptides (CPPs), endosomal release peptides, and nuclear localization sequences (NLSs).Various additional classes of peptides show promise for enhancing nucleic acid delivery by targeting cell surface receptors, inhibiting nuclease activity, and directing nucleic acids toward intracellular targets. In addition to a review of the various existing approaches to peptide-enhanced nucleic acid delivery, this article will discuss strategies for the development of new peptides and approaches for the incorporation of these peptides into nucleic acid delivery systems.

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.

scholarly journals Albumin Nanostructures for Nucleic Acid Delivery in Cancer: Current Trend, Emerging Issues, and Possible Solutions

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3454
Author(s):  
Rama Prajapati ◽  
Álvaro Somoza
Keyword(s):  
Nucleic Acid ◽  
Cancer Therapy ◽  
Nucleic Acids ◽  
Targeted Delivery ◽  
Transfection Efficiency ◽  
Current Trend ◽  
Nucleic Acid Delivery ◽  
Small Interfering Rnas ◽  
Emerging Issues

Cancer is one of the major health problems worldwide, and hence, suitable therapies with enhanced efficacy and reduced side effects are desired. Gene therapy, involving plasmids, small interfering RNAs, and antisense oligonucleotides have been showing promising potential in cancer therapy. In recent years, the preparation of various carriers for nucleic acid delivery to the tumor sites is gaining attention since intracellular and extracellular barriers impart major challenges in the delivery of naked nucleic acids. Albumin is a versatile protein being used widely for developing carriers for nucleic acids. It provides biocompatibility, tumor specificity, the possibility for surface modification, and reduces toxicity. In this review, the advantages of using nucleic acids in cancer therapy and the challenges associated with their delivery are presented. The focus of this article is on the different types of albumin nanocarriers, such as nanoparticles, polyplexes, and nanoconjugates, employed to overcome the limitations of the direct use of nucleic acids in vivo. This review also highlights various approaches for the modification of the surface of albumin to enhance its transfection efficiency and targeted delivery in the tumor sites.

Sign in / Sign up

Export Citation Format

Share Document