Injection of Propidium Iodide into HeLa Cells Using a Silicon Nanoinjection Lance Array

2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Zachary K. Lindstrom ◽  
Steven J. Brewer ◽  
Melanie A. Ferguson ◽  
Sandra H. Burnett ◽  
Brian D. Jensen
Keyword(s):  
Gene Therapy ◽  
Nucleic Acids ◽  
Propidium Iodide ◽  
Hela Cells ◽  
Human Cells ◽  
Advantages And Disadvantages ◽  
Culture Cells ◽  
High Viability ◽  
Promising Area ◽  
Hela Cell Culture

Delivering foreign molecules into human cells is a wide and ongoing area of research. Gene therapy, or delivering nucleic acids into cells via nonviral or viral pathways, is an especially promising area for pharmaceutics. All gene therapy methods have their respective advantages and disadvantages, including limited delivery efficiency and low viability. We present an electromechanical method for delivering foreign molecules into human cells. Nanoinjection, or delivering molecules into cells using a solid lance, has proven to be highly efficient while maintaining high viability levels. This paper describes an array of solid silicon microlances that was tested to determine efficiency and viability when nanoinjecting tens of thousands of HeLa cells simultaneously. Propidium iodide (PI), a dye that fluoresces when bound to nucleic acids and does not fluoresce when unbound, was delivered into cells using the lance array. Results show that the lance array delivers PI into up to 78% of a nanoinjected HeLa cell culture, while maintaining 78–91% viability. With these results, we submit the nanoinjection method using a silicon lance array as another promising particle delivery method for mammalian culture cells.

scholarly journals Higly fusogenic cationic liposomes transiently permeabilize the plasma membrane of HeLa cells

2007 ◽  
Vol 12 (1) ◽  
Author(s):  
Katarzyna Stebelska ◽  
Paulina Wyrozumska ◽  
Jerzy Gubernator ◽  
Aleksander Sikorski
Keyword(s):  
Plasma Membrane ◽  
Nucleic Acids ◽  
Propidium Iodide ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Membrane Integrity ◽  
Cationic Liposomes ◽  
Endosomal Membrane

AbstractCationic liposomes can efficiently carry nucleic acids into mammalian cells. This property is tightly connected with their ability to fuse with negatively charged natural membranes (i.e. the plasma membrane and endosomal membrane). We used FRET to monitor and compare the efficiency of lipid mixing of two liposomal preparations — one of short-chained diC14-amidine and one of long-chained unsaturated DOTAP — with the plasma membrane of HeLa cells. The diC14-amidine liposomes displayed a much higher susceptibility to lipid mixing with the target membranes. They disrupted the membrane integrity of the HeLa cells, as detected using the propidium iodide permeabilization test. Morphological changes were transient and essentially did not affect the viability of the HeLa cells. The diC14-amidine liposomes were much more effective at either inducing lipid mixing or facilitating transfection.

Injection Force Effects on Propidium Iodide Uptake in Nanoinjected HeLa Cells

2015 ◽  
Author(s):  
Tyler E. Lewis ◽  
Brian D. Jensen ◽  
Sandra H. Burnett
Keyword(s):  
Propidium Iodide ◽  
Dna Sequences ◽  
Hela Cells ◽  
Medical Community ◽  
Maximum Efficiency ◽  
Iodide Uptake ◽  
Advantages And Disadvantages ◽  
Area Of Interest ◽  
Slow Rise ◽  
Injection Force

The ability to deliver DNA sequences and molecular loads into the nucleus of a cell is a large area of interest in the medical community, and has numerous applications including gene therapy. Different methods of gene delivery each have their own advantages and disadvantages. Experimentation was performed using a new technique called nanoinjection to determine how force affects the efficiency and viability of the injection process. Nanoinjection, which utilizes an array of millions of solid microlances made on a 2 cm square silicon chip to inject cells, allows us to inject hundreds of thousands of cells at a time with high efficiencies. The solid nature of the lances allows them to be smaller, increasing cell viability. HeLa cells were used for testing in conjunction with an injection device that allowed for precise control of the injection force. Propidium iodide (PI), a dye that fluoresces when bound to DNA and doesn’t penetrate the membrane of living cells, was injected to monitor the efficiencies of transporting molecular loads across the cell membrane. The data was normalized to the controls for both efficiency (PI uptake) and viability to make it easier to see and determine trends. Results indicate a slow rise in injection efficiency from 0 to 1.8 Newtons of force where it reaches a maximum efficiency of 4.11 when normalized to the PI uptake of the positive controls. The data then levels, averaging an efficiency of approximately 3.1. The slow rise is likely due to more of the cells being punctured as the force increases until most have been punctured and the efficiency levels off. Viability had no clear trend, only varying between 0.97 and 1.04 when normalized to the viability of the negative controls.

Exosome as a Natural Gene Delivery Vector for Cancer Treatment

2020 ◽  
Vol 20 (11) ◽  
pp. 821-830
Author(s):  
Prasad Pofali ◽  
Adrita Mondal ◽  
Vaishali Londhe
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Nucleic Acids ◽  
Cancer Treatment ◽  
Intestinal Barrier ◽  
Gene Carrier ◽  
Gene Delivery Vector ◽  
Delivery Vector

Background: Current gene therapy vectors such as viral, non-viral, and bacterial vectors, which are used for cancer treatment, but there are certain safety concerns and stability issues of these conventional vectors. Exosomes are the vesicles of size 40-100 nm secreted from multivesicular bodies into the extracellular environment by most of the cell types in-vivo and in-vitro. As a natural nanocarrier, exosomes are immunologically inert, biocompatible, and can cross biological barriers like the blood-brain barrier, intestinal barrier, and placental barrier. Objective: This review focusses on the role of exosome as a carrier to efficiently deliver a gene for cancer treatment and diagnosis. The methods for loading of nucleic acids onto the exosomes, advantages of exosomes as a smart intercellular shuttle for gene delivery and therapeutic applications as a gene delivery vector for siRNA, miRNA and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and also the limitations of exosomes as a gene carrier are all reviewed in this article. Methods: Mostly, electroporation and chemical transfection are used to prepare gene loaded exosomes. Results: Exosome-mediated delivery is highly promising and advantageous in comparison to the current delivery methods for systemic gene therapy. Targeted exosomes, loaded with therapeutic nucleic acids, can efficiently promote the reduction of tumor proliferation without any adverse effects. Conclusion: In the near future, exosomes can become an efficient gene carrier for delivery and a biomarker for the diagnosis and treatment of cancer.

scholarly journals The Ocular Gene Delivery Landscape

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1135
Author(s):  
Bhubanananda Sahu ◽  
Isha Chug ◽  
Hemant Khanna
Keyword(s):  
Gene Therapy ◽  
Genetic Diseases ◽  
Gene Therapies ◽  
Advantages And Disadvantages ◽  
Fda Approval ◽  
New Information ◽  
Efficient Delivery ◽  
Delivery Routes ◽  
History Of ◽  
Different Parts

The eye is at the forefront of developing therapies for genetic diseases. With the FDA approval of the first gene-therapy drug for a form of congenital blindness, numerous studies have been initiated to develop gene therapies for other forms of eye diseases. These examinations have revealed new information about the benefits as well as restrictions to using drug-delivery routes to the different parts of the eye. In this article, we will discuss a brief history of gene therapy and its importance to the eye and ocular delivery landscape that is currently being investigated, and provide insights into their advantages and disadvantages. Efficient delivery routes and vehicle are crucial for an effective, safe, and longer-lasting therapy.

Expression of VL30 Vectors in Human Cells That Are Targets for Gene Therapy

1995 ◽  
Vol 209 (2) ◽  
pp. 677-683 ◽  
Author(s):  
A.K. Chakraborty ◽  
M.A. Zink ◽  
C.P. Hodgson
Keyword(s):  
Gene Therapy ◽  
Human Cells

Replication and transcription sites are colocalized in human cells

1994 ◽  
Vol 107 (2) ◽  
pp. 425-434 ◽  
Author(s):  
A.B. Hassan ◽  
R.J. Errington ◽  
N.S. White ◽  
D.A. Jackson ◽  
P.R. Cook
Keyword(s):  
Cell Cycle ◽  
Confocal Microscopy ◽  
Dna Synthesis ◽  
Fluorescent Probes ◽  
Hela Cells ◽  
Nuclear Architecture ◽  
Human Cells ◽  
Dynamic Nature ◽  
Nucleotide Triphosphates ◽  
Transcription Sites

HeLa cells synchronized at different stages of the cell cycle were permeabilized and incubated with analogues of nucleotide triphosphates; then sites of incorporation were immunolabeled with the appropriate fluorescent probes. Confocal microscopy showed that sites of replication and transcription were not diffusely spread throughout nuclei, reflecting the distribution of euchromatin; rather, they were concentrated in ‘foci’ where many polymerases act together. Transcription foci aggregated as cells progressed towards the G1/S boundary; later they dispersed and became more diffuse. Replication was initiated only at transcription sites; later, when heterochromatin was replicated in enlarged foci, these remained sites of transcription. This illustrates the dynamic nature of nuclear architecture and suggests that transcription may be required for the initiation of DNA synthesis.

scholarly journals Pseudotyping Lentiviral Vectors: When the Clothes Make the Virus

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1311 ◽  
Author(s):  
Alexis Duvergé ◽  
Matteo Negroni
Keyword(s):  
Gene Therapy ◽  
Genetic Modification ◽  
Viral Vectors ◽  
Molecular Level ◽  
Lentiviral Vectors ◽  
Human Cells ◽  
Surface Proteins ◽  
Holy Grail ◽  
Viral Therapy

Delivering transgenes to human cells through transduction with viral vectors constitutes one of the most encouraging approaches in gene therapy. Lentivirus-derived vectors are among the most promising vectors for these approaches. When the genetic modification of the cell must be performed in vivo, efficient specific transduction of the cell targets of the therapy in the absence of off-targeting constitutes the Holy Grail of gene therapy. For viral therapy, this is largely determined by the characteristics of the surface proteins carried by the vector. In this regard, an important property of lentiviral vectors is the possibility of being pseudotyped by envelopes of other viruses, widening the panel of proteins with which they can be armed. Here, we discuss how this is achieved at the molecular level and what the properties and the potentialities of the different envelope proteins that can be used for pseudotyping these vectors are.

scholarly journals Сationic liposomes as delivery systems for nucleic acids

2020 ◽  
Vol 15 (1) ◽  
pp. 7-27
Author(s):  
A. A. Mikheev ◽  
E. V. Shmendel ◽  
E. S. Zhestovskaya ◽  
G. V. Nazarov ◽  
M. A. Maslov
Keyword(s):  
Gene Therapy ◽  
Nucleic Acids ◽  
Serum Proteins ◽  
Transfection Efficiency ◽  
Biological Fluids ◽  
Genetic Material ◽  
Cationic Liposomes ◽  
Delivery Systems ◽  
Cationic Lipids

Objectives. Gene therapy is based on the introduction of genetic material into cells, tissues, or organs for the treatment of hereditary or acquired diseases. A key factor in the success of gene therapy is the development of delivery systems that can efficiently transfer genetic material to the place of their therapeutic action without causing any associated side effects. Over the past 10 years, significant effort has been directed toward creating more efficient and biocompatible vectors capable of transferring nucleic acids (NAs) into cells without inducing an immune response. Cationic liposomes are among the most versatile tools for delivering NAs into cells; however, the use of liposomes for gene therapy is limited by their low specificity. This is due to the presence of various biological barriers to the complex of liposomes with NA, including instability in biological fluids, interaction with serum proteins, plasma and nuclear membranes, and endosomal degradation. This review summarizes the results of research in recent years on the development of cationic liposomes that are effective in vitro and in vivo. Particular attention is paid to the individual structural elements of cationic liposomes that determine the transfection efficiency and cytotoxicity. The purpose of this review was to provide a theoretical justification of the most promising choice of cationic liposomes for the delivery of NAs into eukaryotic cells and study the effect of the composition of cationic lipids (CLs) on the transfection efficiency in vitro.Results. As a result of the analysis of the related literature, it can be argued that one of the most promising delivery systems of NAs is CL based on cholesterol and spermine with the addition of a helper lipid DOPE. In addition, it was found that varying the composition of cationic liposomes, the ratio of CL to NA, or the size and zeta potential of liposomes has a significant effect on the transfection efficiency.Conclusions. Further studies in this direction should include optimization of the conditions for obtaining cationic liposomes, taking into account the physicochemical properties and established laws. It is necessary to identify mechanisms that increase the efficiency of NA delivery in vitro by searching for optimal structures of cationic liposomes, determining the ratio of lipoplex components, and studying the delivery efficiency and properties of multicomponent liposomes.

scholarly journals Observation of nucleic acids inside living human cells by in-cell NMR spectroscopy

2020 ◽  
Vol 17 (0) ◽  
pp. 36-41
Author(s):  
Yudai Yamaoki ◽  
Takashi Nagata ◽  
Tomoki Sakamoto ◽  
Masato Katahira
Keyword(s):  
Nmr Spectroscopy ◽  
Nucleic Acids ◽  
Human Cells
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