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 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 Role of Lipid-Based and Polymer-Based Non-Viral Vectors in Nucleic Acid Delivery for Next-Generation Gene Therapy

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2866 ◽  
Author(s):  
Aniket Wahane ◽  
Akaash Waghmode ◽  
Alexander Kapphahn ◽  
Karishma Dhuri ◽  
Anisha Gupta ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Nucleic Acid ◽  
Messenger Rna ◽  
Viral Vectors ◽  
Cationic Lipids ◽  
Nucleic Acid Delivery ◽  
Nucleic Acid Analogs ◽  
Organ Specific ◽  
Interfering Rna

The field of gene therapy has experienced an insurgence of attention for its widespread ability to regulate gene expression by targeting genomic DNA, messenger RNA, microRNA, and short-interfering RNA for treating malignant and non-malignant disorders. Numerous nucleic acid analogs have been developed to target coding or non-coding sequences of the human genome for gene regulation. However, broader clinical applications of nucleic acid analogs have been limited due to their poor cell or organ-specific delivery. To resolve these issues, non-viral vectors based on nanoparticles, liposomes, and polyplexes have been developed to date. This review is centered on non-viral vectors mainly comprising of cationic lipids and polymers for nucleic acid-based delivery for numerous gene therapy-based applications.

scholarly journals In Situ Loading and Delivery of Short Single- and Double-Stranded DNA by Supramolecular Organic Frameworks

CCS Chemistry ◽  
2019 ◽  
Vol 1 (2) ◽  
pp. 156-165 ◽  
Author(s):  
Bo Yang ◽  
Xiao-Dan Zhang ◽  
Jian Li ◽  
Jia Tian ◽  
Yi-Peng Wu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Dna Sequences ◽  
Laser Scanning ◽  
High Stability ◽  
Intracellular Delivery ◽  
Water Soluble ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nucleic Acid Delivery

Short DNA represents an important class of biomacromolecules that are widely applied in gene therapy, editing, and modulation. However, the development of simple and reliable methods for their intracellular delivery remains a challenge. Herein, we describe that seven water-soluble, homogeneous supramolecular organic frameworks (SOFs) with a well-defined pore size and high stability in water that can accomplish in situ inclusion of single-stranded (ss) and double-stranded (ds) DNA (21, 23, and 58 nt) and effective intracellular delivery (including two noncancerous and six cancerous cell lines). Fluorescence quenching experiments for single and double end-labeled ss- and ds-DNA support that the DNA sequences can be completely enveloped by the SOFs. Confocal laser scanning microscopy and flow cytometry reveal that five of the SOFs exhibit excellent delivery efficiencies that, in most of the studied cases, outperform the commercial standard Lipo2000, even at low SOF–nucleic acid ratios. In addition to high delivery efficiencies, the water-soluble, self-assembled SOF carriers have a variety of advantages, including convenient preparation, high stability, and in situ DNA inclusion, which are all critical for practical applications in nucleic acid delivery.

Head group configuration increases the biocompatibility of cationic lipids for nucleic acid delivery

2017 ◽  
Vol 5 (28) ◽  
pp. 5597-5607 ◽  
Author(s):  
Gerile Gerile ◽  
Tsogzolmaa Ganbold ◽  
Yizheng Li ◽  
Huricha Baigude
Keyword(s):  
Nucleic Acid ◽  
Therapeutic Approach ◽  
Genetic Diseases ◽  
Genetic Material ◽  
Intracellular Delivery ◽  
Cationic Lipids ◽  
Head Group ◽  
Nucleic Acid Delivery ◽  
Group Configuration

Intracellular delivery of genetic material is a potentially powerful therapeutic approach for the treatment of genetic diseases.

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.

Functionalized graphene oxide mediated nucleic acid delivery

Carbon ◽  
2013 ◽  
Vol 51 ◽  
pp. 224-235 ◽  
Author(s):  
Sushil Kumar Tripathi ◽  
Ritu Goyal ◽  
Kailash Chand Gupta ◽  
Pradeep Kumar
Keyword(s):  
Graphene Oxide ◽  
Nucleic Acid ◽  
Nucleic Acid Delivery ◽  
Functionalized Graphene ◽  
Functionalized Graphene Oxide

scholarly journals Cationic Polyene Phospholipids as DNA Carriers for Ocular Gene Therapy

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Susana Machado ◽  
Sofia Calado ◽  
Diogo Bitoque ◽  
Ana Vanessa Oliveira ◽  
Christer L. Øpstad ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Inflammatory Responses ◽  
Transfection Efficiency ◽  
Systemic Circulation ◽  
Nucleic Acid Delivery ◽  
Rpe Cells ◽  
Recent Success ◽  
Good Target ◽  
Dna Carriers

Recent success in the treatment of congenital blindness demonstrates the potential of ocular gene therapy as a therapeutic approach. The eye is a good target due to its small size, minimal diffusion of therapeutic agent to the systemic circulation, and low immune and inflammatory responses. Currently, most approaches are based on viral vectors, but efforts continue towards the synthesis and evaluation of new nonviral carriers to improve nucleic acid delivery. Our objective is to evaluate the efficiency of novel cationic retinoic and carotenoic glycol phospholipids, designated C20-18, C20-20, and C30-20, to deliver DNA to human retinal pigmented epithelium (RPE) cells. Liposomes were produced by solvent evaporation of ethanolic mixtures of the polyene compounds and coformulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol). Addition of DNA to the liposomes formed lipoplexes, which were characterized for binding, size, biocompatibility, and transgene efficiency. Lipoplex formulations of suitable size and biocompatibility were assayed for DNA delivery, both qualitatively and quantitatively, using RPE cells and a GFP-encoding plasmid. The retinoic lipoplex formulation with DOPE revealed a transfection efficiency comparable to the known lipid references 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) and GeneJuice. The results demonstrate that cationic polyene phospholipids have potential as DNA carriers for ocular gene therapy.

scholarly journals Stabilization of Poly (β-Amino Ester) Nanoparticles for the Efficient Intracellular Delivery of PiggyBac Transposon

2021 ◽  
Vol 8 (2) ◽  
pp. 16
Author(s):  
Tina Rodgers ◽  
Nicolas Muzzio ◽  
Caleb Watson ◽  
Gabriela Romero
Keyword(s):  
Mammalian Cells ◽  
Viral Vectors ◽  
Gene Editing ◽  
Transfection Efficiency ◽  
Release Kinetics ◽  
Green Fluorescence Protein ◽  
Intracellular Delivery ◽  
Amino Ester ◽  
Piggybac Transposon ◽  
Promising Therapeutic Approach

The administration of gene-editing tools has been proposed as a promising therapeutic approach for correcting mutations that cause diseases. Gene-editing tools, composed of relatively large plasmid DNA constructs that often need to be co-delivered with a guiding protein, are unable to spontaneously penetrate mammalian cells. Although viral vectors facilitate DNA delivery, they are restricted by the size of the plasmid to carry. In this work, we describe a strategy for the stable encapsulation of the gene-editing tool piggyBac transposon into Poly (β-amino ester) nanoparticles (NPs). We propose a non-covalent and a covalent strategy for stabilization of the nanoformulation to slow down release kinetics and enhance intracellular delivery. We found that the formulation prepared by covalently crosslinking Poly (β-amino ester) NPs are capable to translocate into the cytoplasm and nuclei of human glioblastoma (U87MG) cells within 1 h of co-culturing, without the need of a targeting moiety. Once internalized, the nanoformulation dissociates, delivering the plasmid presumably as a response to the intracellular acidic pH. Transfection efficiency is confirmed by green fluorescence protein (GFP) expression in U87MG cells. Covalently stabilized Poly (β-amino ester) NPs are able to transfect ~55% of cells causing non-cytotoxic effects. The strategy described in this work may serve for the efficient non-viral delivery of other gene-editing tools.

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.

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