187,619 research outputs found
Plasmonic Intracellular Delivery
This chapter describes the significance of plasmonics to the field of intracellular delivery. We begin by discussing the significance of intracellular delivery, its applications in biology and medicine, and the currently available intracellular delivery techniques. Next, we discuss the field of plasmonic intracellular delivery, beginning with the discovery of optoporation. In optoporation, a laser beam is tightly focused onto a cell membrane to generate a transient pore, through which membrane-impermeable cargo can enter the cell. To improve the throughput of this technique, plasmonic materials were used for their ability to efficiently absorb laser light and generate spatially confined electric fields. Here, we describe the process by which plasmonic materials absorb laser light energy and generate plasmons. These plasmons transfer their energy to their surroundings, resulting in a rise in temperature and the subsequent creation of a bubble or shockwave. Finally, we describe how the properties of plasmons and plasmon-mediated effects facilitate cell poration for intracellular delivery
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In situ loading and delivery of short single- And double-stranded dna by supramolecular organic frameworks
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 endlabeled 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 watersoluble, 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
Acid-Labile Traceless Click Linker for Protein Transduction
Intracellular delivery of active proteins presents an interesting approach in research and therapy. We created a protein transduction shuttle based on a new traceless click linker that combines the advantages of click reactions with implementation of reversible pH-sensitive bonds. The azidomethyl-methylmaleic anhydride (AzMMMan) linker was found compatible with different click chemistries, demonstrated in bioreversible protein modification with dyes, polyethylene glycol, or a transduction carrier. Linkages were stable at physiological pH but reversible at the mild acidic pH of endosomes or lysosomes. We show that pH-reversible attachment of a defined endosome-destabilizing three-arm oligo(ethane amino)amide carrier generates an effective shuttle for protein delivery. The cargo protein nlsEGFP, when coupled via the traceless AzMMMan linker, experiences efficient cellular uptake and endosomal escape into the cytosol, followed by import into the nucleus. In contrast, irreversible linkage to the same shuttle hampers nuclear delivery of nlsEGFP which after uptake remains trapped in the cytosol. Successful intracellular delivery of bioactive ß-galactosidase as a model enzyme was also demonstrated using the pH-controlled shuttle system
Nanocarrier lipid composition modulates the impact of pulmonary surfactant protein B (SP-B) on cellular delivery of siRNA
Two decades since the discovery of the RNA interference (RNAi) pathway, we are now witnessing the approval of the first RNAi-based treatments with small interfering RNA (siRNA) drugs. Nevertheless, the widespread use of siRNA is limited by various extra- and intracellular barriers, requiring its encapsulation in a suitable (nanosized) delivery system. On the intracellular level, the endosomal membrane is a major barrier following endocytosis of siRNA-loaded nanoparticles in target cells and innovative materials to promote cytosolic siRNA delivery are highly sought after. We previously identified the endogenous lung surfactant protein B (SP-B) as siRNA delivery enhancer when reconstituted in (proteo) lipid-coated nanogels. It is known that the surface-active function of SP-B in the lung is influenced by the lipid composition of the lung surfactant. Here, we investigated the role of the lipid component on the siRNA delivery-promoting activity of SP-B proteolipid-coated nanogels in more detail. Our results clearly indicate that SP-B prefers fluid membranes with cholesterol not exceeding physiological levels. In addition, SP-B retains its activity in the presence of different classes of anionic lipids. In contrast, comparable fractions of SP-B did not promote the siRNA delivery potential of DOTAP:DOPE cationic liposomes. Finally, we demonstrate that the beneficial effect of lung surfactant on siRNA delivery is not limited to lung-related cell types, providing broader therapeutic opportunities in other tissues as well
Mechanisms underlying intracellular delivery
AuNPs are versatile systems used for different biomedical application including imaging, drug and gene delivery. These systems support the intracellular transport of active molecules, a step that is considered one of the crucial problems in drug delivery. Nevertheless, in order to design optimal multifunctional AuNPs for specific and efficient nanomedicine applications, the mechanism by which AuNPs interact with living cells must be fully understand.
The main goal of this work consisted in the assessment of the cellular uptake mechanism of 14 nm spherical AuNPs by A549 cells, through fluorescent spectroscopy and microscopy, in combination with quantitative analysis by ICP-MS. TAMRA labeled AuNPs were characterized by UV-visible and fluorescent spectroscopy and the final hydrodynamic diameter of 22.5 ± 0.33 nm was obtained by DLS. Regarding the cellular uptake studies, the AuNPs presented a fast cellular uptake kinetics reaching a saturation point after 6 hours of incubation in A549 cells. Further investigation concerning the internalization mechanism of this AuNPs was evaluated using specific inhibitors for different endocytic pathways. Optimal inhibition was achieved using chlorpromazine, inhibitor of clathrin-mediated endocytosis, resulting in a 23.5 % inhibition of AuNPs after 1 hour of incubation. This preliminary result obtained by fluorescent spectroscopy suggests that these AuNPs were predominantly uptake by clathrin-mediated endocytosis, meaning that other endocytic pathways must be involved in the cellular uptake of this AuNPs. In what cell viability is concern, the prepared AuNPs and the endocytic inhibitors revealed no significant effect on the cell viability in A549 cell line
Bioengineering bacterial outer membrane vesicles as delivery system for RNA therapeutics targeted to lung epithelial cytosols
Intact epithelia lining the airways and alveoli in the lung are essential to maintain lung function. Structural or functional damage of epithelial cells leads in severe diseases, including COPD/emphysema, ibrosis or ALI/ARDS. This central role of epithelia in pulmonary diseases identifies these cells as primary candidates for targeted therapy. With the exception of surface-expressed molecules, however, targeting intracellular components is severely restricted due to poor delivery. We aim to overcome this obstacle using topically administered, bioengineered, biocompatible bacterial outer membrane vesicles (OMVs) as recombinant drug delivery systems for novel biopharmaceuticals. Engineering recombinant surface expression of eukaryotic receptor ligands in ClearColi®, a commercial E.coli BL21 (DE3) strain deficient in lipopolysaccharide production, we have used red fluorescent protein reporters to track OMV loading, transgene expression, and eukaryotic cell trafficking. We demonstrate statistically significant differences in the levels of over 700 proteins between differentially engineered and purified OMV preps with additional differences in transcriptome and lipidome consistency. We also characterised visual and particle size differences observed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Here we report early bioadhesion and culture of re-differentiated lung epithelia. This project aims to bridge the biotechnological gap in the intracellular biopharmaceutics drug delivery challenge for respiratory epithelia through highly controlled, and scalable bio-nanotechnology process. If successful, our work will unlock intracellular imaging and therapeutics research for respiratory diseases with a significant epithelial component, paving the way for other targeting ligands and potentially non-respiratory indications. cellular uptake results in A549 culture as well as air-liquid interface
Quantitative analysis of electroporation-mediated intracellular delivery via bioorthogonal luminescent reaction
Efficient intracellular delivery is crucial for biotherapeutics, such as proteins, oligonucleotides, and CRISPR/Cas9 gene-editing systems, to achieve their efficacy. Despite the great efforts of developing new intracellular delivery carriers, the lack of straightforward methods for intracellular delivery quantification limits further development in this area. Herein, we designed a simple and versatile bioorthogonal luminescent reaction (BioLure assay) to analyze intracellular delivery. Our results suggest that BioLure can be used to estimate the amount of intracellularly delivered molecules after electroporation, and the estimation by BioLure is in good correlation with the results from complementary methods. Furthermore, we used BioLure assay to correlate the intracellularly-delivered RNase A amount with its tumoricidal activity. Overall, BioLure is a versatile tool for understanding the intracellular delivery process on live cells, and establishing the link between the cytosolic concentration of intracellularly-delivered biotherapeutics and their therapeutic efficacy
Lysosomal Delivery of Bioactive Proteins to Living Human Cells via Engineered Exosomes
Exosomes are naturally secreted nanovesicles derived from mammalian cells that are used for intercellular communication in vivo. As a result, they can potentially be used for intracellular delivery of therapeutics for disease treatment. We have developed an exosome pseudotyping approach using vesicular stomatitis virus glycoprotein (VSVG) to produce protein chimeras that optimize production of modified exosomes containing protein therapeutics and facilitate effective delivery to their target cells. To the VSVG transmembrane scaffold, we have fused both fluorescent and luminescent reporters for exosome tracking/visualization and quantification of activity respectively. Through our design, we have shown the biogenesis of VSVG modified exosomes from transfected producer cells through fluorescence imaging and the production of a VSVG-based stable cell line. In addition, we have characterized isolated engineered exosomes and shown that they exhibited the correct size, distribution, and molecular markers, while retaining the bioactivity of their protein cargo. Furthermore, we show that our engineered exosomes and their protein cargo are internalized by multiple cell lines into the endosomal and lysosomal compartments of those cells. Lastly, these modified exosomes can confer their bioactive cargo, either a luminescent reporter or puromycin resistance into these target cells. In summary, this study presents a novel approach to exosome engineering to enhance therapeutic protein loading and delivery, and more importantly, shows the delivery of modified exosomes to intracellular lysosomal compartments. This aspect leads to the assumption that in future studies, these engineered exosomes can be used as a vehicle for delivery of therapeutic proteins for treatment of lysosomal storage diseases
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Chemically Tuned Intracellular Gene Delivery by Core‐Shell Nanoparticles: Effects of Proton Buffering, Acid Degradability, and Membrane Disruption
Nanoparticles consisting of a condensed nucleic acid core surrounded by protective layers which aid to overcome extracellular and intracellular hurdles to gene delivery (i. e., core-shell nanoparticles, CSNPs) synthetically mimic viruses. The outer shells shield the core and are particularly designed to enable facilitated release of the gene payload into the cytoplasm, the major limiting step in intracellular gene delivery. The hypothetical proton sponge effect and degradability in response to a stimulus (i. e., mildly acidic pH in the endosome) are two prevailing, although contested, principles in designing effective carriers for intracellular gene delivery via endosomal escape. Utilizing the highly flexible chemical-tuning of the polymeric shell via surface-initiated photo-polymerization of the various monomers at different molecular ratios, the effects of proton buffering capacity, acid-degradability, and endosomal membrane-lysis property on intracellular delivery of plasmid DNA by CSNPs were investigated. This study demonstrated the equivalently critical roles of proton buffering and acid-degradability in achieving efficient intracellular gene delivery, independent of cellular uptake. Extended proton buffering resulted in further improved transfection as long as the core structure was not compromised. The results of the study present a promising synthetic strategy to the development of an efficient, chemically-tunable gene delivery carrier
A Comparison of Cellular Uptake Mechanisms, Delivery Efficacy, and Intracellular Fate between Liposomes and Extracellular Vesicles
A key aspect for successful drug delivery via lipid-based nanoparticles is their internalization in target cells. Two prominent examples of such drug delivery systems are artificial phospholipid-based carriers, such as liposomes, and their biological counterparts, the extracellular vesicles (EVs). Despite a wealth of literature, it remains unclear which mechanisms precisely orchestrate nanoparticle-mediated cargo delivery to recipient cells and the subsequent intracellular fate of therapeutic cargo. In this review, internalization mechanisms involved in the uptake of liposomes and EVs by recipient cells are evaluated, also exploring their intracellular fate after intracellular trafficking. Opportunities are highlighted to tweak these internalization mechanisms and intracellular fates to enhance the therapeutic efficacy of these drug delivery systems. Overall, literature to date shows that both liposomes and EVs are predominantly internalized through classical endocytosis mechanisms, sharing a common fate: accumulation inside lysosomes. Studies tackling the differences between liposomes and EVs, with respect to cellular uptake, intracellular delivery and therapy efficacy, remain scarce, despite its importance for the selection of an appropriate drug delivery system. In addition, further exploration of functionalization strategies of both liposomes and EVs represents an important avenue to pursue in order to control internalization and fate, thereby improving therapeutic efficacy
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