The use of dopamine-hyaluronate associate-coated maghemite nanoparticles to label cells

Sodium hyaluronate (HA) was associated with dopamine (DPA) and introduced as a coating for maghemite (γ-Fe2O3) nanoparticles obtained by the coprecipitation of iron(II) and iron(III) chlorides and oxidation with sodium hypochlorite. The effects of the DPA anchorage of HA on the γ-Fe2O3 surface on the physicochemical properties of the resulting colloids were investigated. Nanoparticles coated at three different DPA-HA/γ-Fe2O3 and DPA/HA ratios were chosen for experiments with rat bone marrow mesenchymal stem cells and human chondrocytes. The nanoparticles were internalized into rat bone marrow mesenchymal stem cells via endocytosis as confirmed by Prussian Blue staining. The efficiency of mesenchymal stem cell labeling was analyzed. From among the investigated samples, efficient cell labeling was achieved by using DPA-HA-γ-Fe2O3 nanoparticles with DPA-HA/γ-Fe2O3 = 0.45 (weight/ weight) and DPA/HA = 0.038 (weight/weight) ratios. The particles were used as a contrast agent in magnetic resonance imaging for the labeling and visualization of cells

Koloidálně stabilní polypeptidové nanogely: Studie enzymem-zprostředkované nanogelace v inverzní miniemulzi

The current work presents a pivotal study of the nanogelation of the linear poly(N-5-2-hydroxypropyl-L-glutamine) polymer precursor containing tyramine (TYR) units in an inverse miniemulsion by horseradish peroxidase/H2O2-mediated crosslinking. The effects of various n(H2O2)/n(TYR) ratios on the kinetics of nanogelation in the inverse miniemulsion and on the reaction time are investigated by linear sweep voltammetry, while the formation of dityramine crosslinking is explored by fluorescence spectroscopy. The study is completed using dynamic light scattering measurements, nanoparticle tracking analysis, and cryogenic transmission electron microscopy to acquire comprehensive information about the formed nanoparticulate systems. With the optimal ratio n(H2O2)/n(TYR) = 2, the strategy yields in the high-quality 130 nm poly(amino acid)-based nanogel, which is prepared in 2 h. The nanogel is colloidally stable under different temperature and pH conditions for over 168 h. Moreover, the demonstrated nanogel is noncytotoxic for HeLa cells and human primary fibroblasts and is quickly enzymatically hydrolyzed into small fragments during a biodegradation study in human blood plasma. (c) 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 137, 48725.Tato práce představuje klíčovou studii nanogelace lineárního poly (N-5-2-hydroxypropyl-L-glutamin) polymerního prekurzoru obsahujícího jednotky tyraminu (TYR) v inverzní miniemulzi zesíťováním zprostředkovaným křenovou peroxidázou / H2O2. Účinky různých poměrů n (H2O2) / n (TYR) na kinetiku nanogelace v inverzní miniemulzi a na reakční dobu se zkoumají pomocí lineární rozmítané voltametrie, zatímco tvorba zesítění dityraminu se zkoumá fluorescenční spektroskopií. Studie je dokončena pomocí dynamických měření rozptylu světla, analýzy sledování nanočástic a kryogenní transmisní elektronové mikroskopie za účelem získání komplexních informací o vytvořených nanočásticových systémech. Při optimálním poměru n (H2O2) / n (TYR) = 2 dává strategie vysoce kvalitní nanogel na bázi 130 nm poly (aminokyseliny), který je připraven za 2 hodiny. Nanogel je koloidně stabilní za různých podmínek teploty a pH po více než 168 hodin. Kromě toho je prokázaný nanogel necytotoxický pro HeLa buňky a lidské primární fibroblasty a je rychle enzymaticky hydrolyzován na malé fragmenty během biodegradační studie v lidské krevní plazmě

Poly( amino acid)-based fibrous scaffolds modified with surface-pendant peptides for cartilage tissue engineering

In this study, fibrous scaffolds based on poly(gamma-benzyl-L-glutamate) (PBLG) were investigated in terms of the chondrogenic differentiation potential of human tooth germ stem cells (HTGSCs). Through the solution-assisted bonding of the fibres, fully connected scaffolds with pore sizes in the range 20-400 mu m were prepared. Biomimetic modification of the PBLG scaffolds was achieved by a two-step reaction procedure: first, aminolysis of the PBLG fibres' surface layers was performed, which resulted in an increase in the hydrophilicity of the fibrous scaffolds after the introduction of N-5-hydroxyethyl-L-glutamine units; and second, modification with the short peptide sequence azidopentanoyl-GGGRGDSGGGY-NH2, using the 'click' reaction on the previously modified scaffold with 2-propynylside-chains, was performed. Radio-assay of the I-125-labelled peptide was used to evaluate the RGD density in the fibrous scaffolds ( which varied in the range 10(-3) -10 pM/cm(2)). All the PBLG scaffolds, especially with density 90 +/- 20 fM/cm(2) and 200 +/- 100 fM/cm(2) RGD, were found to be potentially suitable for growth and chondrogenic differentiation of HTGSCs. Copyright (C) 2015 John Wiley & Sons, Ltd

Derivation of Sendai-Virus-Reprogrammed Human iPSCs-Neuronal Precursors: In Vitro and In Vivo Post-grafting Safety Characterization.

The critical requirements in developing clinical-grade human-induced pluripotent stem cells-derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable in vivo post-grafting differentiation, and acceptable safety profile. Here, we report on the use of manual-selection protocol for generating expandable and stable human NPCs from induced pluripotent stem cells. The hiPSCs were generated by the reprogramming of peripheral blood mononuclear cells with Sendai-virus (SeV) vector encoding Yamanaka factors. After induction of neural rosettes, morphologically defined NPC colonies were manually harvested, re-plated, and expanded for up to 20 passages. Established NPCs showed normal karyotype, expression of typical NPCs markers at the proliferative stage, and ability to generate functional, calcium oscillating GABAergic or glutamatergic neurons after in vitro differentiation. Grafted NPCs into the striatum or spinal cord of immunodeficient rats showed progressive maturation and expression of early and late human-specific neuronal and glial markers at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted brain or spinal cord samples. These data demonstrate the effective use of in vitro manual-selection protocol to generate safe and expandable NPCs from hiPSCs cells. This protocol has the potential to be used to generate GMP (Good Manufacturing Practice)-grade NPCs from hiPSCs for future clinical use

Spinal parenchymal occupation by neural stem cells after subpial delivery in adult immunodeficient rats.

Neural precursor cells (NSCs) hold great potential to treat a variety of neurodegenerative diseases and injuries to the spinal cord. However, current delivery techniques require an invasive approach in which an injection needle is advanced into the spinal parenchyma to deliver cells of interest. As such, this approach is associated with an inherent risk of spinal injury, as well as a limited delivery of cells into multiple spinal segments. Here, we characterize the use of a novel cell delivery technique that employs single bolus cell injections into the spinal subpial space. In immunodeficient rats, two subpial injections of human NSCs were performed in the cervical and lumbar spinal cord, respectively. The survival, distribution, and phenotype of transplanted cells were assessed 6-8 months after injection. Immunofluorescence staining and mRNA sequencing analysis demonstrated a near-complete occupation of the spinal cord by injected cells, in which transplanted human NSCs (hNSCs) preferentially acquired glial phenotypes, expressing oligodendrocyte (Olig2, APC) or astrocyte (GFAP) markers. In the outermost layer of the spinal cord, injected hNSCs differentiated into glia limitans-forming astrocytes and expressed human-specific superoxide dismutase and laminin. All animals showed normal neurological function for the duration of the analysis. These data show that the subpial cell delivery technique is highly effective in populating the entire spinal cord with injected NSCs, and has a potential for clinical use in cell replacement therapies for the treatment of ALS, multiple sclerosis, or spinal cord injury

Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain

Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain