scholarly journals Targeted cationic poly(D,L-lactic-co-glycolic acid) nanoparticles for gene delivery to cultured cells

2009 ◽  
Vol 14 (2) ◽  
Author(s):  
Sonsoles Díez ◽  
Itziar Miguéliz ◽  
Conchita Tros de Ilarduya
Keyword(s):  
Gene Delivery ◽  
Glycolic Acid ◽  
Cultured Cells ◽  
Interleukin 12 ◽  
High Concentration ◽  
Dna Nanoparticles ◽  
Potential System ◽  
Genes Encoding ◽  
Solvent Evaporation Process

AbstractWe developed a new targeted cationic nanoparticulate system composed of poly(D,L-lactic-co-glycolic acid) (PLGA), 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and asialofetuin (AF), and found it to be a highly effective formulation for gene delivery to liver tumor cells. The nanoparticles (NP) were prepared by a modified solvent evaporation process that used two protocols in order to encapsulate (NP1 particles) or adsorb (NP2 particles) plasmid DNA. The final particles are in the nanoscale range. pDNA loaded in PLGA/DOTAP/AF particles with high loading efficiency showed a positive surface charge. Targeted asialofetuin-nanoparticles (AF-NP) carrying genes encoding for luciferase and interleukin-12 (IL-12) resulted in increased transfection efficiencies compared to free DNA and to plain (non-targeted) systems, even in the presence of 60% fetal bovine serum (FBS). The results of transfections performed on HeLa cells, defective in asialoglycoprotein receptors (ASGPr-), confirmed the receptor-mediated endocytosis mechanism. In summary, this is the first time that asialoglycoprotein receptor targeting by PLGA/DOTAP/DNA nanoparticles carrying the therapeutic gene IL-12 has been shown to be efficient in gene delivery to liver cancer cells in the presence of a very high concentration of serum, and this could be a potential system for in vivo application.

scholarly journals PLGA Nanoparticles for Ultrasound-Mediated Gene Delivery to Solid Tumors

2012 ◽  
Vol 2012 ◽  
pp. 1-20 ◽  
Author(s):  
Marxa Figueiredo ◽  
Rinat Esenaliev
Keyword(s):  
Cardiovascular Disease ◽  
Gene Delivery ◽  
Glycolic Acid ◽  
Target Gene ◽  
Plga Nanoparticles ◽  
Efficient Tool ◽  
Targeted Gene Delivery ◽  
The Us ◽  
Novel Approaches

This paper focuses on novel approaches in the field of nanotechnology-based carriers utilizing ultrasound stimuli as a means to spatially target gene delivery in vivo, using nanoparticles made with either poly(lactic-co-glycolic acid) (PLGA) or other polymers. We specifically discuss the potential for gene delivery by particles that are echogenic (amenable to destruction by ultrasound) composed either of polymers (PLGA, polystyrene) or other contrast agent materials (Optison, SonoVue microbubbles). The use of ultrasound is an efficient tool to further enhance gene delivery by PLGA or other echogenic particles in vivo. Echogenic PLGA nanoparticles are an attractive strategy for ultrasound-mediated gene delivery since this polymer is currently approved by the US Food and Drug Administration for drug delivery and diagnostics in cancer, cardiovascular disease, and also other applications such as vaccines and tissue engineering. This paper will review recent successes and the potential of applying PLGA nanoparticles for gene delivery, which include (a) echogenic PLGA used with ultrasound to enhance local gene delivery in tumors or muscle and (b) PLGA nanoparticles currently under development, which could benefit in the future from ultrasound-enhanced tumor targeted gene delivery.

scholarly journals Inclusion of Antibodies to Cell Culture Media Preserves the Integrity of Genes Encoding RL13 and the Pentameric Complex Components during Fibroblast Passage of Human Cytomegalovirus

2018 ◽  
Author(s):  
Amine Ourahmane ◽  
Xiaohong Cui ◽  
Li He ◽  
Dirk Dittmer ◽  
Mark Schleiss ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Cytomegalovirus ◽  
Cultured Cells ◽  
Culture Media ◽  
Adaptive Mutations ◽  
Culture Passage ◽  
Genes Encoding ◽  
Cell Culture Passage

Propagation of human cytomegalovirus (CMV) in cultured cells results in genetic adaptations that confer improved growth in vitro and significant attenuation in vivo. Mutations in RL13 arise quickly during cell culture passage, while mutations in the UL128-131A locus emerge later during fibroblast passage and disrupt expression of a glycoprotein complex that is important for entry into epithelial and endothelial cells. As in vivo CMV replicates in the context of host antibodies, we reasoned that antibodies might mitigate the accumulation of adaptive mutations during cell culture passage. To test this, CMV in infant urine was used to infect replicate fibroblast cultures. One lineage was passaged in the absence of CMV-hyperimmuneglobulin (HIG) while the other was passaged with HIG in the culture medium. The former lost epithelial tropism and aquired mutations disrupting RL13 and UL131A expression, whereas the latter retained epithelial tropism and both gene loci remained intact after 22 passages. An epitheliotropic RL13+/ UL131A+ virus was isolated by limiting-dilution in the presence of HIG and expanded to produce a working stock sufficient to conduct cell tropism experiments. Thus, culture in the presence of antibodies may facilitate in vitro experiments using viruses that are genetically more authentic than has been previously possible.

scholarly journals Heat-shrinking DNA nanoparticles for in vivo gene delivery

Gene Therapy ◽  
2020 ◽  
Vol 27 (5) ◽  
pp. 196-208 ◽  
Author(s):  
Basil Mathew ◽  
Raghu Ramanathan ◽  
Nathan A. Delvaux ◽  
Jacob Poliskey ◽  
Kevin G. Rice
Keyword(s):  
Gene Delivery ◽  
Dna Nanoparticles ◽  
In Vivo Gene Delivery

scholarly journals Inclusion of Antibodies to Cell Culture Media Preserves the Integrity of Genes Encoding RL13 and the Pentameric Complex Components During Fibroblast Passage of Human Cytomegalovirus

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 221 ◽  
Author(s):  
Amine Ourahmane ◽  
Xiaohong Cui ◽  
Li He ◽  
Meaghan Catron ◽  
Dirk Dittmer ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Cytomegalovirus ◽  
Cultured Cells ◽  
Culture Media ◽  
Single Amino Acid ◽  
Adaptive Mutations ◽  
Glycoprotein Complex ◽  
Genes Encoding

Propagation of human cytomegalovirus (CMV) in cultured cells results in genetic adaptations that confer improved growth in vitro and significant attenuation in vivo. Mutations in RL13 arise quickly, while mutations in the UL128-131A locus emerge later during fibroblast passage and disrupt formation of a glycoprotein complex that is important for entry into epithelial and endothelial cells. As CMV replicates in the context of host antibodies in vivo, we reasoned that antibodies might mitigate the accumulation of adaptive mutations during cell culture passage. To test this, CMV in infant urine was used to infect replicate fibroblast cultures. One lineage was passaged in the absence of CMV-hyperimmuneglobulin (HIG) while the other was passaged with HIG in the culture medium. The former lost epithelial tropism and acquired mutations disrupting RL13 and UL131A expression, whereas the latter retained epithelial tropism and both gene loci remained intact after 22 passages. Additional mutations resulting in single amino acid changes also occurred in UL100 encoding glycoprotein M, UL102 encoding a subunit of the helicase/primase complex, and UL122 encoding the Immediate Early 2 protein. An epitheliotropic RL13+/UL131A+ virus was isolated by limiting dilution in the presence of HIG and expanded to produce a working stock sufficient to conduct cell tropism experiments. Thus, production of virus stocks by culture in the presence of antibodies may facilitate in vitro experiments using viruses that are genetically more authentic than previously available.

scholarly journals Highly compacted biodegradable DNA nanoparticles capable of overcoming the mucus barrier for inhaled lung gene therapy

2015 ◽  
Vol 112 (28) ◽  
pp. 8720-8725 ◽  
Author(s):  
Panagiotis Mastorakos ◽  
Adriana L. da Silva ◽  
Jane Chisholm ◽  
Eric Song ◽  
Won Kyu Choi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Transgene Expression ◽  
Colloidal Stability ◽  
Transfection Efficiency ◽  
Delivery Systems ◽  
Intratracheal Administration ◽  
Dna Nanoparticles ◽  
High Level

Gene therapy has emerged as an alternative for the treatment of diseases refractory to conventional therapeutics. Synthetic nanoparticle-based gene delivery systems offer highly tunable platforms for the delivery of therapeutic genes. However, the inability to achieve sustained, high-level transgene expression in vivo presents a significant hurdle. The respiratory system, although readily accessible, remains a challenging target, as effective gene therapy mandates colloidal stability in physiological fluids and the ability to overcome biological barriers found in the lung. We formulated highly stable DNA nanoparticles based on state-of-the-art biodegradable polymers, poly(β-amino esters) (PBAEs), possessing a dense corona of polyethylene glycol. We found that these nanoparticles efficiently penetrated the nanoporous and highly adhesive human mucus gel layer that constitutes a primary barrier to reaching the underlying epithelium. We also discovered that these PBAE-based mucus-penetrating DNA nanoparticles (PBAE-MPPs) provided uniform and high-level transgene expression throughout the mouse lungs, superior to several gold standard gene delivery systems. PBAE-MPPs achieved robust transgene expression over at least 4 mo following a single administration, and their transfection efficiency was not attenuated by repeated administrations, underscoring their clinical relevance. Importantly, PBAE-MPPs demonstrated a favorable safety profile with no signs of toxicity following intratracheal administration.

Composition of PLGA and PEI/DNA nanoparticles improves ultrasound-mediated gene delivery in solid tumors in vivo

2008 ◽  
Vol 261 (2) ◽  
pp. 215-225 ◽  
Author(s):  
Olga V. Chumakova ◽  
Anton V. Liopo ◽  
Valery G. Andreev ◽  
Inga Cicenaite ◽  
B. Mark Evers ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Solid Tumors ◽  
Dna Nanoparticles

Genome-wide discovery of Pax7 target genes during development

2008 ◽  
Vol 33 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Robert B. White ◽  
Melanie R. Ziman
Keyword(s):  
Dna Binding ◽  
Embryonic Development ◽  
Functional Role ◽  
Target Gene ◽  
Target Genes ◽  
Gene Selection ◽  
Cultured Cells ◽  
Expression Vectors ◽  
Genes Encoding

Pax7 plays critical roles in development of brain, spinal cord, neural crest, and skeletal muscle. As a sequence-specific DNA-binding transcription factor, any direct functional role played by Pax7 during development is mediated through target gene selection. Thus, we have sought to identify genes targeted by Pax7 during embryonic development using an unbiased chromatin immunoprecipitation (ChIP) cloning assay to isolate cis-regulatory regions bound by Pax7 in vivo. Sequencing and genomic localization of a library of chromatin-DNA fragments bound by Pax7 has identified 34 candidate Pax7 target genes, with occupancy of a selection confirmed with independent chromatin enrichment tests (ChIP-PCR). To assess the capacity of Pax7 to regulate transcription from these loci, we have cloned alternate transcripts of Pax7 (differing significantly in their DNA binding domain) into expression vectors and transfected cultured cells with these constructs, then analyzed target gene expression levels using RT-PCR. We show that Pax7 directly occupies sites within genes encoding transcription factors Gbx1 and Eya4, the neurogenic cytokine receptor ciliary neurotrophic factor receptor, the neuronal potassium channel Kcnk2, and the signal transduction kinase Camk1d in vivo and regulates the transcriptional state of these genes in cultured cells. This analysis gives us greater insight into the direct functional role played by Pax7 during embryonic development.

scholarly journals 17.4 Efficient in vivo gene delivery using chitosan/DNA nanoparticles for applications in cartilage repair

2007 ◽  
Vol 15 ◽  
pp. B74
Author(s):  
S. Methot ◽  
M. Lavertu ◽  
J. Sun ◽  
F. Smaoui ◽  
M. Jean ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Cartilage Repair ◽  
Dna Nanoparticles ◽  
In Vivo Gene Delivery

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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