ABSTRACT
Self-assembly of individual units into multicomponent complexes is a powerful approach for the generation of functional superstructures. We present the coordinative interaction of oligohistidine-tags (His-tags) with metal-organic framework nanoparticles (MOF NPs). By this novel concept, different molecular units can be anchored on the outer surface of MOF NPs in a self-assembly process generating multifunctional nanosystems. The article focuses on two main objectives: first, the detailed investigation of the assembly process and fundamental establishment of the novel functionalization concept; and second, its subsequent use for the development of biomacromolecule (e.g., peptides and proteins) delivery vehicles. Three exemplary MOF structures, MIL-88A, HKUST-1, and Zr-fum, based on different metal components, were selected for the external binding of various His-tagged synthetic peptides and recombinant or chemically H6-modified proteins. Evidence for simultaneous assembly of different functional units with Zr-fum MOF NPs as well as their successful transport into living cells illustrate the promising potential of the self-assembly approach for the generation of multifunctional NPs and future biological applications. Taking the high number of possible MOF NPs and different functional units into account, the reported functionalization approach opens great flexibility for the targeted synthesis of multifunctional NPs for specific purposes.
ABSTRACT
PURPOSE: Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. METHODS: Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. RESULTS: The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies. CONCLUSION: Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.
Subject(s)
Nanoparticles/administration & dosage , Proteins/administration & dosage , Single-Domain Antibodies/administration & dosage , Cell Line , Cell Line, Tumor , Chemistry, Pharmaceutical/methods , Cytoplasm/metabolism , Flow Cytometry/methods , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/metabolism , HeLa Cells , Humans , KB Cells , Nanoparticles/metabolism , Protein Transport , Proteins/metabolism , Single-Domain Antibodies/metabolismABSTRACT
BACKGROUND: The potential use of Fe(III) ions in biomedical applications may predict the interest of its combination with pDNA-PEI polyplexes. The present work aims at assessing the impact of this metal on pDNA complex properties. METHODS: Variations in the formation of complexes were imposed by using two types of biological buffers at different salt conditions. The incorporation of pDNA in complexes was characterised by gel electrophoresis and dynamic light scattering. Transfection efficiency and cytotoxicity were evaluated in HeLa and HUH-7 cell lines, supported by flow cytometry assays. RESULTS: Fe(III) enhances pDNA incorporation in the complex, irrespective of the buffer used. Transfection studies reveal that the addition of Fe(III) to complexes at low ionic strength reduces gene transfection, while those prepared under high salt content do not affect or, in a specific case, increase gene transfection up to 5 times. This increase may be a consequence of a favoured interaction of polyplexes with cell membrane and uptake. At low salt conditions, results attained with chloroquine indicate that the metal may inhibit polyplex endosomal escape. A reduction on the amount of PEI (N/P 5) formed at intermediary ionic strength, complemented by Fe(III), reduces the size of complexes while maintaining a transfection efficiency similar to that obtained to N/P 6. CONCLUSIONS: Fe(III) emerges as a good supporting condensing agent to modulate pDNA-PEI properties, including condensation, size and cytotoxicity, without a large penalty on gene transfection. GENERAL SIGNIFICANCE: This study highlights important aspects that govern pDNA transfection and elucidates the benefits of incorporating the versatile Fe(III) in a gene delivery system.
Subject(s)
Chlorides/metabolism , Ferric Compounds/metabolism , Plasmids/metabolism , Polyethyleneimine/metabolism , Transfection/methods , Adenosine Triphosphate/metabolism , Buffers , Chlorides/chemistry , Chlorides/toxicity , Electrophoretic Mobility Shift Assay , Energy Metabolism/drug effects , Ferric Compounds/chemistry , Ferric Compounds/toxicity , Gene Expression Regulation , Genes, Reporter , HeLa Cells , Humans , Hydrogen-Ion Concentration , Light , Luciferases/genetics , Luciferases/metabolism , Nucleic Acid Conformation , Osmolar Concentration , Plasmids/chemistry , Polyethyleneimine/chemistry , Polyethyleneimine/toxicity , Scattering, RadiationABSTRACT
Molecular medicine opens into a space of novel specific therapeutic agents: intracellularly active drugs such as peptides, proteins or nucleic acids, which are not able to cross cell membranes and enter the intracellular space on their own. Through the development of cell-targeted shuttles for specific delivery, this restriction in delivery has the potential to be converted into an advantage. On the one hand, due to the multiple extra- and intracellular barriers, such carrier systems need to be multifunctional. On the other hand, they must be precise and reproducibly manufactured due to pharmaceutical reasons. Here we review the design of precise sequence-defined delivery carriers, including solid-phase synthesized peptides and nonpeptidic oligomers, or nucleotide-based carriers such as aptamers and origami nanoboxes.
Subject(s)
DNA/metabolism , Drug Carriers , Drug Delivery Systems , Gene Transfer Techniques , Proteins/administration & dosage , RNA/metabolism , Animals , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/metabolism , Chemistry, Pharmaceutical , DNA/chemistry , DNA/genetics , Gene Expression Regulation , Humans , Proteins/chemistry , Proteins/metabolism , RNA/chemistry , RNA/geneticsABSTRACT
Molecular knowledge about schizophrenia--a psychotic, multifactorial mental disorder that affects about 1% of the population worldwide--is limited and no diagnostic biomarkers are available. The comparative proteome analysis of human brain tissue from patients with schizophrenia and healthy controls may supply useful information on both the disorder and potential biomarkers candidates. Here, we present the results of our investigation of anterior cingulate cortex samples from 11 patients and 8 controls. We used two-dimensional gel electrophoresis combined with mass spectrometry, the most traditional approach to studying the proteome, to reveal the differentially expressed proteins in schizophrenia, and western blot to validate some interesting potential biomarker candidates such as dihydropyrimidinase-like 2 and alpha-crystallin, involved in a number of processes such as cytoskeleton arrangement. Most interesting is that our additional sex-specific proteome comparison showed that male and female schizophrenia patients present different patterns of proteome regulation, for instance for the proteins aldolase C, an enzyme of glycolysis, and glutamine synthetase that synthesizes glutamine, responsible for maintain glutamate levels. Our findings not only support previous findings but also indicate areas that warrant further study in schizophrenia.
