ABSTRACT
Fluorescence imaging is an indispensable tool in modern biological research, allowing simple and inexpensive color-coded visualizations of real-time events in living cells and animals, as well as of fixed states of ex vivo specimens. The accuracy of fluorescence imaging in living systems is, however, impeded by autofluorescence, light scattering, and limited penetration depth of light. Nevertheless, the clinical use of fluorescence imaging is expected to grow along with advances in imaging equipment, and will increasingly demand high-accuracy probes to avoid false-positive results in disease detection. To this end, a water-soluble and relatively safe diarylethene (DAE)-based reversible near-infrared (NIR) fluorescence photoswitch for living systems is prepared here. Furthermore, to facilitate excellent switching performance, the photoirradiation results obtained is compared using three different visible light sources to turn on NIR fluorescence through cycloreversion of DAE. While photoswitching using 589 nm light leads to slightly higher cell viability, fluorescence quenching efficiency and fatigue resistance are higher when 532 nm light with low photobleaching is used in both aqueous solution and living systems. The authors anticipate that their reversible NIR fluorescence photoswitch mediated by DAE can be beneficial for fluorescence imaging in aqueous media requiring accurate detection, such as in the autofluorescence-rich living environment.
Subject(s)
Fluorescent Dyes , Optical Imaging , Animals , WaterABSTRACT
Recently, mRNA-based therapeutics, including vaccines, have gained significant attention in the field of gene therapy for treating various diseases. Among the various mRNA delivery vehicles, lipid nanoparticles (LNPs) have emerged as promising vehicles for packaging and delivering mRNA with low immunogenicity. However, while mRNA delivery has several advantages, the delivery efficiency and stability of LNPs remain challenging for mRNA therapy. In this study, an ionizable helper cholesterol analog, 3ß[L-histidinamide-carbamoyl] cholesterol (Hchol) lipid is developed and incorporated into LNPs instead of cholesterol to enhance the LNP potency. The pKa values of the Hchol-LNPs are ≈6.03 and 6.61 in MC3- and SM102-based lipid formulations. Notably, the Hchol-LNPs significantly improve the delivery efficiency by enhancing the endosomal escape of mRNA. Additionally, the Hchol-LNPs are more effective in a red blood cell hemolysis at pH 5.5, indicating a synergistic effect of the protonated imidazole groups of Hchol and cholesterol on endosomal membrane destabilization. Furthermore, mRNA delivery is substantially enhanced in mice treated with Hchol-LNPs. Importantly, LNP-encapsulated SARS-CoV-2 spike mRNA vaccinations induce potent antigen-specific antibodies against SARS-CoV-2. Overall, incorporating Hchol into LNP formulations enables efficient endosomal escape and stability, leading to an mRNA delivery vehicle with a higher delivery efficiency.
Subject(s)
Cholesterol , Nanoparticles , RNA, Messenger , SARS-CoV-2 , Animals , Cholesterol/chemistry , Cholesterol/analogs & derivatives , Nanoparticles/chemistry , Mice , RNA, Messenger/genetics , Humans , Histidine/chemistry , Histidine/analogs & derivatives , Lipids/chemistry , COVID-19 , COVID-19 Vaccines/chemistry , Endosomes/metabolism , Female , Hemolysis/drug effects , Mice, Inbred BALB C , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , LiposomesABSTRACT
Age-related macular degeneration (AMD) is one of the leading causes of irreversible blindness, generally affecting people over 50 years of age in industrialized countries. Despite the effectiveness of anti-vascular endothelial growth factor (VEGF) therapy in attenuating the growth of new blood vessels, substantial visual improvements are rare with this complex disease. Furthermore, the current regimen of repeated monthly intravitreal injections of drugs can result in serious side effects. Combination therapies-to complement anti-VEGF alone-with a prolonged therapeutic effect and efficient delivery to the intended site are urgently needed, which could be realized through the use of carefully designed nanocarriers. To understand the physicochemical effects (e.g., size, charge, geometry) of intravitreally administered nanocarriers on their bioavailability, distribution, and targeting efficiency across multiple layers of the retina, here we prepared seven different types of surface-functionalized water-soluble dendritic nanocarriers with hydrodynamic sizes mostly under 5 nm. A similar stoichiometric amount of fluorophore was covalently attached to each of these biocompatible nanocarriers for quantitative analyses by confocal microscopy of cryosectioned healthy mouse eyes. Interestingly, at 24 h post-injection, the nanocarrier with multiple copies of glucosamine on the surface (DNSG) accumulated predominantly in the photoreceptor layer and the retinal pigment epithelium (RPE), which are speculated to be associated with AMD pathogenesis (i.e., target sites). Furthermore, extended residence at these outer retinal layers was demonstrated by DNSG, which appeared to gradually turn into micron-scale particles potentially through aggregation. Our systematic findings may provide useful guidelines for the rational design of intravitreal nanocarriers to treat vision-threatening retinal diseases, including AMD.
Subject(s)
Choroidal Neovascularization , Macular Degeneration , Pharmaceutical Preparations , Angiogenesis Inhibitors/therapeutic use , Animals , Choroidal Neovascularization/drug therapy , Intravitreal Injections , Macular Degeneration/drug therapy , Mice , RetinaABSTRACT
Recently, Bmi1 was shown to control the proliferation and self-renewal of neural stem cells (NSCs). In this study, we demonstrated the induction of NSC-like cells (NSCLCs) from mouse astrocytes by Bmi1 under NSC culture conditions. These NSCLCs exhibited the morphology and growth properties of NSCs, and expressed NSC marker genes, including nestin, CD133, and Sox2. In vitro differentiation of NSCLCs resulted in differentiated cell populations containing astrocytes, neurons, and oligodendrocytes. Following treatment with histone deacetylase inhibitors (trichostatin A and valproic acid), the potential of NSCLCs for proliferation, dedifferentiation, and self-renewal was significantly inhibited. Our data indicate that multipotent NSCLCs can be generated directly from astrocytes by the addition of Bmi1.
Subject(s)
Cell Differentiation , Multipotent Stem Cells/cytology , Neurons/cytology , Nuclear Proteins/physiology , Proto-Oncogene Proteins/physiology , Repressor Proteins/physiology , AC133 Antigen , Animals , Antigens, CD/genetics , Astrocytes/cytology , Astrocytes/drug effects , Astrocytes/metabolism , Cell Culture Techniques , Cell Differentiation/genetics , Cell Lineage/genetics , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p16/metabolism , DNA-Binding Proteins/genetics , Genetic Markers , Glycoproteins/genetics , HMGB Proteins/genetics , Intermediate Filament Proteins/genetics , Mice , Multipotent Stem Cells/metabolism , Nerve Tissue Proteins/genetics , Nestin , Neurons/metabolism , Nuclear Proteins/genetics , Peptides/genetics , Polycomb Repressive Complex 1 , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/genetics , Repressor Proteins/genetics , SOXB1 Transcription Factors , Transcription Factors/geneticsABSTRACT
Achieving accurate and efficacious tumor targeting with minimal off-target effects is of paramount importance in designing diagnostic and therapeutic agents for cancer. In this respect, nanocarriers have gained enormous popularity because of their attainable multifunctional features, as well as tumor-targeting potential by extravasation. However, once administered into the bloodstream, nanocarriers face various in vivo obstacles that may significantly impair their performance needed for clinical translation. Herein, we demonstrate a strategy to enhance tumor-targeting efficiency by embedding functionalities in the interior region of partially PEGylated nanocarriers (ca. 10 nm in diameter), intended for active or passive targeting. The cooperative impact of these topologically inner functional groups (IFGs) was marked: enhancements of >100-fold in IC50in vitro (e.g., a high-avidity ligand with cationic IFGs) and >2-fold in tumor accumulation at 2 h post-injection in vivo (e.g., a high-avidity ligand with anionic IFGs), both against the fully PEGylated counterpart. Analogous to allosteric modulators, properly employed IFGs may substantially improve the process of effectively directing nanocarriers to tumors, which is otherwise solely dependent on avidity or extravasation.
ABSTRACT
The use of computed tomography (CT) for vascular imaging is critical in medical emergencies requiring urgent diagnostic decisions, such as cerebral ischemia and many cardiovascular diseases. Small-molecule iodinated contrast media are often injected intravenously as radiopaque agents during CT imaging to achieve high contrast enhancement of vascular systems. The rapid excretion rate of these agents is overcome by injecting a significantly high dose of iodine, which can have serious side effects. Here we report a simple method to prepare blood-pool contrast agents for CT based on dendrimers for the first time using tetraiodobenzene derivatives as potent radiopaque moieties. Excellent in vivo safety has been demonstrated for these small (13-22nm) unimolecular water-soluble dendritic contrast agents, which exhibit high contrast enhancement in the blood-pool and effectively extend their blood half-lives. Our method is applicable to virtually any scaffold with suitable surface groups and may fulfill the current need for safer, next-generation iodinated CT contrast agents.
Subject(s)
Contrast Media/chemistry , Dendrimers/chemistry , Iodobenzenes/chemistry , Nylons/chemistry , Tomography, X-Ray Computed , Animals , Contrast Media/adverse effects , Contrast Media/pharmacokinetics , Dendrimers/adverse effects , Dendrimers/pharmacokinetics , HeLa Cells , Humans , Iodobenzenes/adverse effects , Iodobenzenes/pharmacokinetics , Male , Mice, Inbred C57BL , Nylons/adverse effects , Nylons/pharmacokineticsABSTRACT
A small 29 nm monodispersed silica nanoparticle 1a was synthesized as a diarylethene-based reversible fluorescence photoswitch by copolymerizing silane precursors in one-pot including 3a and 4. Reversible photoswitching of nanoparticle 1a was successfully achieved in living cells to show its potential as a highly distinguishable and safe fluorescence probe for cell tracking.
Subject(s)
Ethylenes/chemistry , Fluorescence , Nanoparticles/chemistry , Silicon Dioxide/chemical synthesis , Stem Cells/chemistry , HeLa Cells , Humans , Particle Size , Photochemical Processes , Silicon Dioxide/chemistry , Surface PropertiesABSTRACT
Photochromic compound-conjugated fluorescent fullerene-silica nanoparticles prepared by the reverse-microemulsion method was utilized for photoswitchable cellular imaging by repeatable irradiation of ultraviolet and visible light.
Subject(s)
Fluorescent Dyes/chemistry , Fullerenes/chemistry , Nanoparticles/chemistry , Silicon Dioxide/chemistry , Ultraviolet Rays , Emulsions/chemistry , HeLa Cells , Humans , Microscopy, Confocal , Nanoparticles/ultrastructureABSTRACT
In this study, we report the isolation and characterization of a population of multipotent keloid-derived mesenchymal-like stem cells (KMLSCs) from keloid scalp tissues. These KMLSCs expressed the typical mesenchymal stem cell marker proteins CD13, CD29, CD44, CD90, fibronectin, and vimentin when they were cultured in serum-containing medium and when subsequent exposure to various differentiation media resulted in their differentiation into adipocytes, osteoblasts, chondrocytes, smooth muscle cells, and angiogenic endothelial cells. When KMLSCs were cultured in neural stem culture conditions (i.e., in the presence of epidermal growth factor and fibroblast growth factor 2 in substrate-free conditions), they produced large numbers of neurospheres containing nestin-, CD133-, and SOX2-positive cells that expressed neural-crest stem cell markers. Subsequent exposure of these cells to different differentiation conditions resulted in cells that expressed neuronal cell-, astrocyte-, oligodendrocyte-, or Schwann cell-specific markers. Our study suggests that KMLSCs may be an alternative adult stem cell resource for regenerative tissue repair and auto-transplantation.