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Michal Babic1,2, Daniel Horak1,2, Pavla Jendelova2,3, Vit Herynek2,4, Vladimir Proks1,2, Vaclav Vanecek2,3, Petr Lesny2,3, Eva Sykova2,31Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq 2, 2Center for Cell Therapy and Tissue Repair, Charles University, V Uvalu 84, 3Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videnska 1083, 4Institute for Clinical and Experimental Medicine, Videnska 1958, Prague, Czech RepublicAbstract: 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.Keywords: nanoparticles, dopamine, hyaluronate, cell labeling, magnetic

Ultrathin electrospun nanofibrous membranes (NfMs) based on poly(γ-benzyl-L-glutamate) (PBLG) were prepared. Scanning electron microscopy analysis revealed the production of a high-quality, bead-free nanofibrous membrane. The membrane thicknesses, ranging from 1.7 to 4.5 μm, were found to correlate directly with membrane porosity. Raman scattering analysis was utilized to investigate the conformation of the PBLG nanofibrous membrane and to assess the effects of addition of 1 wt. % trifluoroacetic acid (TFA) into the PBLG solutions, as well as the impact of annealing at 70 °C. In addition, x-ray photoelectron spectroscopy (XPS) characterization was performed to elucidate the chemical composition of the PBLG nanofibrous membrane. The Raman and Fourier-transform infrared spectroscopy spectra indicated the characteristic α-helical conformation in both the PBLG solution and the PBLG nanofibrous membrane. Furthermore, a comparative analysis of Raman band profiles proved the absence of TFA after annealing, supporting the hypothesis of TFA evaporation post-annealing, which was subsequently confirmed by the XPS results. In addition, the results from the small punch test revealed a significant correlation between membrane thickness and stiffness, indicating that increased thickness enhances stiffness. This comprehensive study provides valuable insights into the structural and compositional properties of PBLG NfMs, laying the groundwork for future investigations into their potential applications in the field of tissue engineering. [ABSTRACT FROM AUTHOR]

In this study, we investigate the preparation of surface pattern of functional groups on poly(lactide) (PLA) surfaces through the controlled deposition of core-shell self-assemblies based on functionalized PLA-b -PEO amphiphilic block copolymers from selective solvents. Through grafting RGDS peptide onto the functionalized copolymer surface, the presented approach enables to prepare PLA surfaces with random and clustered spatial distribution of adhesive motifs. The proposed topography of the adhesion motif was proved by atomic force microscopy techniques using biotin-tagged RGDS peptide grafted on the surface and streptavidin-modified gold nanospheres which bind the tagged RGDS peptides as a contrast agent. The cell culture study under static and dynamic conditions with MG63 osteosarcoma cell line showed that the clustered distribution of RGDS peptides provided more efficient initial cell attachment and spreading, and resistance to cell detachment under dynamic culture compared to randomly distributed RGDS motif when with the same average RGDS peptide concentration. [ABSTRACT FROM AUTHOR]

Bioinks play a crucial role in tissue engineering, influencing mechanical and chemical properties of the printed scaffold as well as the behavior of encapsulated cells. Recently, there has been a shift from animal origin materials to their synthetic alternatives. In this context, we present here bioinks based on fully synthetic and biodegradable poly( α ,L-amino acids) (PolyAA) as an alternative to animal-based gelatin methacrylate (Gel-Ma) bioinks. Additionally, we first reported the possibility of the visible light photoinitiated incorporation of the bifunctional cell adhesive RGD peptide into the PolyAA hydrogel matrix. The obtained hydrogels are shown to be cytocompatible, and their mechanical properties closely resemble those of gelatin methacrylate-based scaffolds. Moreover, combining the unique properties of PolyAA-based bioinks, the photocrosslinking strategy, and the use of droplet-based printing allows the printing of constructs with high shape fidelity and structural integrity from low-viscosity bioinks without using any sacrificial components. Overall, presented PolyAA-based materials are a promising and versatile toolbox that extends the range of bioinks for droplet bioprinting., (© 2024 IOP Publishing Ltd.)

This study develops and characterizes novel biodegradable soft hydrogels with dual porosity based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers cross-linked by hydrolytically degradable linkers. The structure and properties of the hydrogels are designed as scaffolds for tissue engineering and they are tested in vitro with model mesenchymal stem cells (rMSCs). Detailed morphological characterization confirms dual porosity suitable for cell growth and nutrient transport. The dual porosity of hydrogels slightly improves rMSCs proliferation compared to the hydrogel with uniform pores. In addition, the laminin coating supports the adhesion of rMSCs to the hydrogel surface. However, hydrogels modified by heptapeptide RGDSGGY significantly stimulate cell adhesion and growth. Moreover, the RGDS-modified hydrogels also affect the topology of proliferating rMSCs, ranging from single-cell to multicellular clusters. The 3D reconstruction of the hydrogels with cells obtained by laser scanning confocal microscopy (LSCM) confirms cell penetration into the inner structure of the hydrogel and its corresponding microstructure. The prepared biodegradable oligopeptide-modified hydrogels with dual porosity are suitable candidates for further in vivo evaluation in soft tissue regeneration., (© 2023 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

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.

One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.

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., Competing Interests: Declaration of interests M.M. is a co-founder of Neurgain Technologies., (Copyright © 2022 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

A new type of hydrophilic, biocompatible, and biodegradable polypeptide nanogel depots loaded with the natural serine protease inhibitor α1-antitrypsin (AAT) was applied for the inhibition of the inflammatory mediator trypsin. Two types of nanogels were prepared from linear synthetic polypeptides based on biocompatible and biodegradable poly[ N 5 -(2-hydroxyethyl)-ʟ-glutamine- ran - N 5 -propargyl-ʟ-glutamine- ran - N 5 -(6-aminohexyl)-ʟ-glutamine]- ran - N 5 -[2-(4-hydroxyphenyl)ethyl)-ʟ-glutamine] (PHEG-Tyr) or biocompatible N α -ʟ-lysine-grafted α,β-poly[(2-propyne)-ᴅ,ʟ-aspartamide- ran -(2-hydroxyethyl)-ᴅʟ-aspartamide- ran -(2-(4-hydroxyphenyl)ethyl)-ᴅʟ-aspartamide] ( N α -Lys-NG). Both nanogels were prepared by HRP/H 2 O 2 -mediated crosslinking in inverse miniemulsions with pH and temperature-stimuli responsive behavior confirmed by dynamic light scattering and zeta potential measurements. The loading capacity of PHEG-Tyr and N α -Lys-NG nanogels and their release profiles were first optimized with bovine serum albumin. The nanogels were then used for loading and release of AAT. PHEG-Tyr and N α -Lys-NG nanogels showed different loading capacities for AAT with the maximum (20%) achieved with N α -Lys-NG nanogel. In both cases, the nanogel depots demonstrated a burst release of AAT during the first 6 h, which could be favorable for quick inhibition of trypsin. A consequent pilot in vitro inhibition study revealed that both PHEG-Tyr and N α -Lys-NG nanogels loaded with AAT successfully inhibited the enzymatic activity of trypsin. Furthermore, the inhibitory efficiency of the AAT-loaded nanogels was higher than that of only AAT. Interestingly, also non-loaded PHEG-Tyr and N α -Lys-NG nanogels were shown to effectively inhibit trypsin because they contain suitable amino acids in their structures that effectively block the active site of trypsin., (Copyright © 2022, Šálek et al.)

Research of degradable hydrogel polymeric materials exhibiting high water content and mechanical properties resembling tissues is crucial not only in drug delivery systems but also in tissue engineering, medical devices, and biomedical-healthcare sensors. Therefore, we newly offer development of hydrogels based on poly(2-hydroxyethyl methacrylate-co-2-(acetylthio) ethyl methacrylate-co-2-methacryloyloxyethyl phosphorylcholine) [P(HEMA-ATEMA-MPC)] and optimization of their mechanical and in vitro and in vivo degradability. P(HEMA-ATEMA-MPC) hydrogels differed in chemical composition, degree of crosslinking, and starting molar mass of polymers (15, 19, and 30 kDa). Polymer precursors were synthesized by a reversible addition fragmentation chain transfer (RAFT) polymerization using 2-(acetylthio)ethyl methacrylate containing protected thiol groups, which enabled crosslinking and gel formation. Elastic modulus of hydrogels increased with the degree of crosslinking (Slaughter et al., 2009) [1]. In vitro and in vivo controlled degradation was confirmed using glutathione and subcutaneous implantation of hydrogels in rats, respectively. We proved that the hydrogels with higher degree of crosslinking retarded the degradation. Also, albumin, γ-globulin, and fibrinogen adsorption on P(HEMA-ATEMA-MPC) hydrogel surface was tested, to simulate adsorption in living organism. Rat mesenchymal stromal cell adhesion on hydrogels was improved by the presence of RGDS peptide and laminin on the hydrogels. We found that rat mesenchymal stromal cells proliferated better on laminin-coated hydrogels than on RGDS-modified ones., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Horseradish peroxidase (HRP)/H 2 O 2 -mediated crosslinking of polypeptides in inverse miniemulsion is a promising approach for the development of next-generation biocompatible and biodegradable nanogels. Herein, we present a fundamental investigation of the effects of three surfactants and their different concentrations on the (HRP)/H 2 O 2 -mediated nanogelation of poly[N 5 -(2-hydroxyethyl)-l-glutamine-ran-N 5 -propargyl-l-glutamine-ran-N 5 -(6-aminohexyl)-l-glutamine]-ran-N 5 -[2-(4-hydroxyphenyl)ethyl)-l-glutamine] (PHEG-Tyr) in inverse miniemulsion. The surfactants sorbitan monooleate (SPAN 80), polyoxyethylenesorbitan trioleate (TWEEN 85), and dioctyl sulfosuccinate sodium salt (AOT) were selected and their influence on the nanogel size, size distribution, and morphology was evaluated. The most effective nanogelation stabilization was achieved with 20 wt% nonionic surfactant SPAN 80. The diameter of the hydrogel nanoparticles was 230 nm (dynamic light scattering, DLS) and was confirmed also by nanoparticle tracking analysis (NTA) which showed the diameters ranging from 200 to 300 nm. Microscopy and image analyses showed that the nanogel in the dry state was spherical in shape and had number-average diameter D n  = 26 nm and dispersity Р= 1.91. In the frozen-hydrated state, the nanogel appeared porous and was larger in size with D n  = 182 nm and Р= 1.52. Our results indicated that the nanogelation of the polymer precursor required a higher concentration of surfactant than classical inverse miniemulsion polymerization to ensure effective stabilization. The developed polypeptide nanogel was radiolabeled with 125 I, and in vivo biodistribution and blood clearance evaluations were performed. We found that the 125 I-labeled nanogel was well-biodistributed in the bloodstream, cleared from mouse blood during 48 h by renal and hepatic pathways and did not provoke any sign of toxic effects., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In the fast-developing field of tissue engineering there is a constant demand for new materials as scaffolds for cell seeding, which can better mimic a natural extracellular matrix as well as control cell behavior. Among other materials, polysaccharides are widely used for this purpose. One of the main candidates for scaffold fabrication is alginate. However, it lacks sites for cell adhesion. That is why one of the steps toward the development of suitable scaffolds for cells is the introduction of the biofunctionality to the alginate structure. In this work we focused on bone-sialoprotein derived peptide (TYRAY) conjugation to the molecule of alginate. Here the comparison study on four different approaches of peptide conjugation was performed including traditional and novel modification methods, based on 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/ N -hydroxy succinimide (EDC/NHS), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM), thiol-Michael addition and Cu-catalyzed azide-alkyne cycloaddition reactions. It was shown that the combination of the alginate amidation with the use of and subsequent Cu-catalyzed azide-alkyne cycloaddition led to efficient peptide conjugation, which was proven with both NMR and XPS methods. Moreover, the cell culture experiment proved the positive effect of peptide presence on the adhesion of human embryonic stem cells.

Injectable hydrogel scaffolds combined with stem cell therapy represent a promising approach for minimally invasive surgical tissue repair. In this study, we developed and characterized a fully synthetic, biodegradable poly( N 5 -(2-hydroxyethyl)-l-glutamine)-based injectable hydrogel modified with integrin-binding arginine-glycine-aspartic acid (RGD) peptide (PHEG-Tyr-RGD). The biodegradable hydroxyphenyl polymer precursor derivative of PHEG-Tyr was enzymatically cross-linked to obtain injectable hydrogels with different physicochemical properties. The gelation time, gel yield, swelling behavior, and storage modulus of the PHEG-Tyr hydrogels were tuned by varying the concentrations of the PHEG-Tyr precursors and horseradish peroxidase as well as the n H 2 O 2 / n Tyr ratio. The mechanical properties and gelation time of the PHEG-Tyr hydrogel were optimized for the encapsulation of rat mesenchymal stem cells (rMSCs). We focused on the 2D and 3D spreading and viability of rMSCs within the PHEG-Tyr-RGD hydrogels with different physicochemical microenvironments in vitro. Encapsulation of rMSCs shows long-term survival and exhibits cell-matrix and cell-cell interactions reflective of both the RGD concentration and hydrogel stiffness. The presented biomaterial represents a suitable biological microenvironment to guide 3D spreading and may act as a promising 3D artificial extracellular matrix for stem cell therapy.

Superporous poly(2-hydroxyethyl methacrylate- co -2-aminoethyl methacrylate) (P(HEMA-AEMA)) hydrogel scaffolds are designed for in vitro 3D culturing of leukemic B cells. Hydrogel porosity, which influences cell functions and growth, is introduced by adding ammonium oxalate needle-like crystals in the polymerization mixture. To improve cell vitality, cell-adhesive Arg-Gly-Asp-Ser (RGDS) peptide is immobilized on the N -(γ-maleimidobutyryloxy)succinimide-activated P(HEMA-AEMA) hydrogels via reaction of SH with maleimide groups. This modification is especially suitable for the survival of primary chronic lymphocytic leukemia cells (B-CLLs) in 3D cell culture. No other tested stimuli (interleukin-4, CD40 ligand, or shaking) can further improve B-CLL survival or metabolic activity. Both unmodified and RGDS-modified P(HEMA-AEMA) scaffolds serve as a long-term (70 days) 3D culture platforms for HS-5 and M2-10B4 bone marrow stromal cell lines and MEC-1 and HG-3 B-CLL cell lines, although the adherent cells retain their physiological morphologies, preferably on RGDS-modified hydrogels. Moreover, the porosity of hydrogels allows direct cell lysis, followed by efficient DNA isolation from the 3D-cultured cells. P(HEMA-AEMA)-RGDS thus serves as a suitable 3D in vitro leukemia model that enables molecular and metabolic assays and allows imaging of cell morphology, interactions, and migration by confocal microscopy. Such applications can prospectively assist in testing of drugs to treat this frequently recurring or refractory cancer.

Antifouling polymer layers containing extracellular matrix-derived peptide motifs offer promising new options for biomimetic surface engineering. In this contribution, we report the design of antifouling vascular grafts bearing biofunctional peptide motifs for tissue regeneration applications based on hierarchical polymer brushes. Hierarchical diblock poly(methyl ether oligo(ethylene glycol) methacrylate- block -glycidyl methacrylate) brushes bearing azide groups (poly(MeOEGMA- block -GMA-N 3 )) were grown by surface-initiated atom transfer radical polymerization (SI-ATRP) and functionalized with biomimetic RGD peptide sequences. Varying the conditions of copper-catalyzed alkyne-azide "click" reaction allowed for the immobilization of RGD peptides in a wide surface concentration range. The synthesized hierarchical polymer brushes bearing peptide motifs were characterized in detail using various surface sensitive physicochemical methods. The hierarchical brushes presenting the RGD sequences provided excellent cell adhesion properties and at the same time remained resistant to fouling from blood plasma. The synthesis of anti-fouling hierarchical brushes bearing 1.2 × 10 3 nmol/cm 2 RGD biomimetic sequences has been adapted for the surface modification of commercially available grafts of woven polyethylene terephthalate (PET) fibers. The fiber mesh was endowed with polymerization initiator groups via aminolysis and acylation reactions optimized for the material. The obtained bioactive antifouling vascular grafts promoted the specific adhesion and growth of endothelial cells, thus providing a potential avenue for endothelialization of artificial conduits.

Lanthanide-doped upconversion nanoparticles (UCNPs) have a unique capability of upconverting near-infrared (NIR) excitation into ultraviolet, visible, and NIR emission. Conventional UCNPs composed of NaYF 4 :Yb 3+ /Er 3+ (Tm 3+ ) are excited by NIR light at 980 nm, where undesirable absorption by water can cause overheating or damage of living tissues and reduce nanoparticle luminescence. Incorporation of Nd 3+ ions into the UCNP lattice shifts the excitation wavelength to 808 nm, where absorption of water is minimal. Herein, core-shell NaYF 4 :Yb 3+ /Er 3+ @NaYF 4 :Nd 3+ nanoparticles, which are doubly doped by sensitizers (Yb 3+ and Nd 3+ ) and an activator (Er 3+ ) in the host NaYF 4 matrix, were synthesized by high-temperature coprecipitation of lanthanide chlorides in the presence of oleic acid as a stabilizer. Uniform core (24 nm) and core-shell particles with tunable shell thickness (~0.5-4 nm) were thoroughly characterized by transmission electron microscopy (TEM), energy-dispersive analysis, selected area electron diffraction, and photoluminescence emission spectra at 808 and 980 nm excitation. To ensure dispersibility of the particles in biologically relevant media, they were coated by in-house synthesized poly(ethylene glycol) (PEG)-neridronate terminated with an alkyne (Alk). The stability of the NaYF 4 :Yb 3+ /Er 3+ @NaYF 4 :Nd 3+ -PEG-Alk nanoparticles in water or 0.01 M PBS and the presence of PEG on the surface were determined by dynamic light scattering, ζ-potential measurements, thermogravimetric analysis, and FTIR spectroscopy. Finally, the adhesive azidopentanoyl-modified GGGRGDSGGGY-NH 2 (RGDS) peptide was immobilized on the NaYF 4 :Yb 3+ /Er 3+ @NaYF 4 :Nd 3+ -PEG-Alk particles via Cu(I)-catalyzed azide-alkyne cycloaddition. The toxicity of the unmodified core-shell NaYF 4 :Yb 3+ /Er 3+ @NaYF 4 :Nd 3+ , NaYF 4 :Yb 3+ /Er 3+ @NaYF 4 :Nd 3+ -PEG-Alk, and NaYF 4 :Yb 3+ /Er 3+ @NaYF 4 :Nd 3+ -PEG-RGDS nanoparticles on both Hep-G2 and HeLa cells was determined, confirming no adverse effect on their survival and proliferation. The interaction of the nanoparticles with Hep-G2 cells was monitored by confocal microscopy at both 808 and 980 nm excitation. The NaYF 4 :Yb 3+ /Er 3+ @NaYF 4 :Nd 3+ -PEG-RGDS nanoparticles were localized on the cell membranes due to specific binding of the RGDS peptide to integrins, in contrast to the NaYF 4 :Yb 3+ /Er 3+ @NaYF 4 :Nd 3+ -PEG-Alk particles, which were not engulfed by the cells. The NaYF 4 :Yb 3+ /Er 3+ @NaYF 4 :Nd 3+ -PEG-RGDS nanoparticles thus appear to be promising as a new non-invasive probe for specific bioimaging of cells and tissues. This development makes the nanoparticles useful for diagnostic and/or, after immobilization of a bioactive compound, even theranostic applications in the treatment of various fatal diseases., (Copyright © 2020 Kostiv, Engstová, Krajnik, Šlouf, Proks, Podhorodecki, Ježek and Horák.)

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., (© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

The efficiency of solid phase extraction (SPE) of DNA on polymer particles is limited by the features of the applied solid support, such as size, hydrophilicity, and functionality and their application in SPE also requires additional steps and compounds to finally obtain sufficient amount of high-quality DNA. The present study describes a preparation of sub-micrometer monodisperse poly(methacrylic acid-co-ethylene dimethacrylate) (PME) particles by precipitation polymerization. The effect of the ethylene dimethacrylate (EDMA) crosslinker concentration on morphology and particle size, which varied from 730 to 900 nm, was investigated. The particles with 5 and 15 wt% EDMA were selected for a study of SPE of plasmid DNA under various adsorption and elution conditions, followed by the enzymatic restriction of isolated DNA to verify a quality the nucleic acid. The particles with 15 wt% EDMA were suitable for the SPE because they retained better colloidal stability during the adsorption without additional induction of DNA conformational change. The quality of isolated DNA was finally verified by enzymatic restriction by restriction endonuclease EcoRI. Moreover, the developed method using PME particles was successfully utilized for DNA isolation from Escherichia coli lysate.

Poly(glycidyl methacrylate) (PGMA) is prone to modifications with different functional groups, magnetic fluids or direct coupling with biological molecules. The purpose of this research was to synthesize new magnetically responsive particles with peroxidase-like activity. Poly(glycidyl methacrylate-co-ethylene dimethacrylate) [P(GMA-EDMA)] particles containing carboxyl groups were obtained by emulsifier-free emulsion polymerization and hydrolysis and oxidation of PGMA with KMnO 4 , resulting in poly(carboxymethyl methacrylate-co-ethylene dimethacrylate) [P(CMMA-EDMA)] particles. Thionine (Th) was also attached to the particles [(P(CMMA-EDMA)-Th] via EDC/NHS chemistry to observe its effect on electron transfer during the oxidation reaction. Finally, the particles were coated with a nitric acid-stabilized ferrofluid in methanol. The resulting magnetic particles were characterized by several methods, including scanning and transmission electron microscopy, X-ray photoelectron spectroscopy, and vibrating sample magnetometry. The effect of EDMA on the P(CMMA-EDMA) particle size and size distribution was investigated; the particle size changed from 300 to 340 nm, and the particles were monodispersed with a saturation magnetization of 11 Am 2 /kg. Finally, the effects of temperature and pH on the peroxidase-like activity of the magnetic P(CMMA-EDMA) and P(CMMA-EDMA)-Th particles were investigated. The particles, which exhibited a high activity at pH 4-6 and at ∼37 °C, represent a highly sensitive sensor component potentially useful in enzyme-based immunoassays.

Methacrylate hydrogels have been extensively used as bridging scaffolds in experimental spinal cord injury (SCI) research. As synthetic materials, they can be modified, which leads to improved bridging of the lesion. Fibronectin, a glycoprotein of the extracellular matrix produced by reactive astrocytes after SCI, is known to promote cell adhesion. We implanted 3 methacrylate hydrogels: a scaffold based on hydroxypropylmethacrylamid (HPMA), 2-hydroxyethylmethacrylate (HEMA) and a HEMA hydrogel with an attached fibronectin (HEMA-Fn) in an experimental model of acute SCI in rats. The animals underwent functional evaluation once a week and the spinal cords were histologically assessed 3 months after hydrogel implantation. We found that both the HPMA and the HEMA-Fn hydrogel scaffolds lead to partial sensory improvement compared to control animals and animals treated with plain HEMA scaffold. The HPMA scaffold showed an increased connective tissue infiltration compared to plain HEMA hydrogels. There was a tendency towards connective tissue infiltration and higher blood vessel ingrowth in the HEMA-Fn scaffold. HPMA hydrogels showed a significantly increased axonal ingrowth compared to HEMA-Fn and plain HEMA; while there were some neurofilaments in the peripheral as well as the central region of the HEMA-Fn scaffold, no neurofilaments were found in plain HEMA hydrogels. In conclusion, HPMA hydrogel as well as the HEMA-Fn scaffold showed better bridging qualities compared to the plain HEMA hydrogel, which resulted in very limited partial sensory improvement.

While many types of biomaterials have been evaluated in experimental spinal cord injury (SCI) research, little is known about the time-related dynamics of the tissue infiltration of these scaffolds. We analyzed the ingrowth of connective tissue, axons and blood vessels inside the superporous poly (2-hydroxyethyl methacrylate) hydrogel with oriented pores. The hydrogels, either plain or seeded with mesenchymal stem cells (MSCs), were implanted in spinal cord transection at the level of Th8. The animals were sacrificed at days 2, 7, 14, 28, 49 and 6 months after SCI and histologically evaluated. We found that within the first week, the hydrogels were already infiltrated with connective tissue and blood vessels, which remained stable for the next 6 weeks. Axons slowly and gradually infiltrated the hydrogel within the first month, after which the numbers became stable. Six months after SCI we observed rare axons crossing the hydrogel bridge and infiltrating the caudal stump. There was no difference in the tissue infiltration between the plain hydrogels and those seeded with MSCs. We conclude that while connective tissue and blood vessels quickly infiltrate the scaffold within the first week, axons show a rather gradual infiltration over the first month, and this is not facilitated by the presence of MSCs inside the hydrogel pores. Further research which is focused on the permissive micro-environment of the hydrogel scaffold is needed, to promote continuous and long-lasting tissue regeneration across the spinal cord lesion.

Polymer layers capable of suppressing protein adsorption from biological media while presenting extracellular matrix-derived peptide motifs offer valuable new options for biomimetic surface engineering. Herein, we provide detailed insights into physicochemical changes induced in a nonfouling poly(ethylene oxide) (PEO) brush/polydopamine (PDA) system by incorporation of adhesion ligand (RGD) peptides. Brushes with high surface chain densities (σ ≥ 0.5 chains·nm -2 ) and pronounced hydrophilicity (water contact angles ≤ 10°) were prepared by end-tethering of heterobifunctional PEOs ( M n ≈ 20 000 g·mol -1 ) to PDA-modified surfaces from a reactive melt. Using alkyne distal end group on the PEO chains, azidopentanoic-bearing peptides were coupled through a copper-catalyzed Huisgen azide-alkyne "click" cycloaddition reaction. The surface concentration of RGD was tuned from complete saturation of the PEO surface with peptides (1.7 × 10 5 fmol·cm -2 ) to values which may induce distinct differences in cell adhesion (<6.0 × 10 2 fmol·cm -2 ). Infrared reflection-absorption and X-ray photoelectron spectroscopies proved the PDA-PEO layers covalent structure and the immobilization of RGD peptides. The complete reconstruction of experimental electrohydrodynamics data utilizing mean-field theory predictions further verified the attained brush structure of the end-tethered PEO chains which provided hydrodynamic screening of the PDA anchor. Increasing the surface concentration of immobilized RGD peptides led to increased interfacial charging. Supported by simulations, this observation was attributed to the ionization of functional groups in the amino acid sequence and to the pH-dependent adsorption of water ions (OH - > H 3 O + ) from the electrolyte. Despite the distinct differences observed in the electrokinetic analysis of the surfaces bearing different amounts of RGD, it was found that the peptide presence on PEO(20 000)-PDA layers does not have a significant effect on the nonfouling properties of the system. Notably, the presented PEO(20 000)-PDA layers bearing RGD peptides in the surface concentration range 5.9 to 1.7 × 10 5 fmol·cm -2 reduced the protein adsorption from fetal bovine serum to less than 30 ng·cm -2 , that is, values comparable to the ones obtained for pristine PEO(20 000)-PDA layers.

In this study, fibrous scaffolds based on poly(γ-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 µm 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-NH 2 , using the 'click' reaction on the previously modified scaffold with 2-propynyl side-chains, was performed. Radio-assay of the 125 I-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 © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

We describe a conceptually new, microfibrous, biodegradable functional material prepared from a modified storage polysaccharide also present in humans (glycogen) showing strong potential as direct-contact dressing/interface material for wound healing. Double bonds were introduced into glycogen via allylation and were further exploited for crosslinking of the microfibers. Triple bonds were introduced by propargylation and served for further click functionalization of the microfibers with bioactive peptide. A simple solvent-free method allowing the preparation of thick layers was used to produce microfibers (diameter ca 2μm) from allylated and/or propargylated glycogen. Crosslinking of the samples was performed by microtron beta-irradiation, and the irradiation dose was optimized to 2kGy. The results from biological testing showed that these highly porous, hydrophilic, readily functionalizable materials were completely nontoxic to cells growing in their presence. The fibers were gradually degraded in the presence of cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Three-dimensional hydrogel supports for mesenchymal and neural stem cells (NSCs) are promising materials for tissue engineering applications such as spinal cord repair. This study involves the preparation and characterization of superporous scaffolds based on a copolymer of 2-hydroxyethyl and 2-aminoethyl methacrylate (HEMA and AEMA) crosslinked with ethylene dimethacrylate. Ammonium oxalate is chosen as a suitable porogen because it consists of needle-like crystals, allowing their parallel arrangement in the polymerization mold. The amino group of AEMA is used to immobilize RGDS and SIKVAVS peptide sequences with an N-γ-maleimidobutyryloxy succinimide ester linker. The amount of the peptide on the scaffold is determined using 125 I radiolabeled SIKVAVS. Both RGDS- and SIKVAVS-modified poly(2-hydroxyethyl methacrylate) scaffolds serve as supports for culturing human mesenchymal stem cells (MSCs) and human fetal NSCs. The RGDS sequence is found to be better for MSC and NSC proliferation and growth than SIKVAVS., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Starting NaYF4:Yb(3+)/Er(3+) nanoparticles with size tuned from 24 to 33 nm were prepared by high-temperature coprecipitation of lanthanide chlorides in high-boiling organic solvents. To enhance colloidal stability in aqueous medium, an aminosilica shell was introduced on the surface by hydrolysis and condensation of tetramethyl orthosilicate and (3-aminopropyl)trimethoxysilane using a reverse microemulsion technique; to form alkyne groups, reaction with 4-pentynoic acid followed. Finally, the cell adhesive and cell penetrating azidopentanoyl-GGGRGDSGGGY-NH2 (RGDS) and azidopentanoyl-GGGRKKRRQRRR-NH2 (TAT) peptides were conjugated to the upconversion particles via Cu(I)-catalyzed alkyne-azide cycloaddition. The concentrations of the peptides bound to the nanoparticle surfaces and amount of adsorbed residual Cu(I) catalyst were determined using an (125)I-radiolabeled RGDS peptide and a (64)Cu(I)-doped catalyst, respectively. Targeting and uptake of the RGDS- and TAT-conjugated NaYF4:Yb(3+)/Er(3+)&SiO2 nanoparticles by human cervix carcinoma HeLa cells were monitored by confocal microscopy. RGDS-conjugated nanoparticle probes were mainly localized on the cell plasma membrane due to specific binding of the peptide to the corresponding integrins. In contrast, the TAT-conjugated nanoparticles were able to cross the cell membrane and accumulate in the cell cytoplasm. Thus, this new peptide bioconjugation approach supported both extra- and intracellular nanoparticle uptake, enabling targeting and imaging of the specific tumor phenotypes.

We present an investigation of the preparation of highly porous hydrogels based on biodegradable synthetic poly(α-amino acid) as potential tissue engineering scaffolds. Covalently cross-linked gels with permanent pores were formed under cryogenic conditions by free-radical copolymerization of poly[N(5)-(2-hydroxyethyl)-L-glutamine-stat-N(5)-(2-methacryloyl-oxy-ethyl)-L-glutamine] (PHEG-MA) with 2-hydrohyethyl methacrylate (HEMA) and, optionally, N-propargyl acrylamide (PrAAm) as minor comonomers. The morphology of the cryogels showed interconnected polyhedral or laminar pores. The volume content of communicating water-filled pores was >90%. The storage moduli of the swollen cryogels were in the range of 1-6 kPa, even when the water content was >95%. The enzymatic degradation of a cryogel corresponded to the decrease in its storage modulus during incubation with papain, a model enzyme with specificity analogous to wound-healing enzymes. It was shown that cryogels with incorporated alkyne groups can easily be modified with short synthetic peptides using azide-alkyne cycloaddition "click" chemistry, thus providing porous hydrogel scaffolds with biomimetic features.

Protein-repulsive surfaces modified with ligands for cell adhesion receptors have been widely developed for controlling the cell adhesion and growth in tissue engineering. However, the question of matrix production and deposition by cells on these surfaces has rarely been addressed. In this study, protein-repulsive polydopamine-poly(ethylene oxide) (PDA-PEO) surfaces were functionalized with an RGD-containing peptide (RGD), with a collagen-derived peptide binding fibronectin (Col), or by a combination of these peptides (RGD + Col, ratio 1:1) in concentrations of 90 fmol/cm(2) and 700 fmol/cm(2) for each peptide type. When seeded with vascular endothelial CPAE cells, the PDA-PEO surfaces proved to be completely non-adhesive for cells. On surfaces with lower peptide concentrations and from days 1 to 3 after seeding, cell adhesion and growth was restored practically only on the RGD-modified surface. However, from days 3 to 7, cell adhesion and growth was improved on surfaces modified with Col and with RGD + Col. At higher peptide concentrations, the cell adhesion and growth was markedly improved on all peptide-modified surfaces in both culture intervals. However, the collagen-derived peptide did not increase the expression of fibronectin in the cells. The deposition of fibronectin on the material surface was generally very low and similar on all peptide-modified surfaces. Nevertheless, the RGD + Col surfaces exhibited the highest cell adhesion stability under a dynamic load, which correlated with the highest expression of talin and vinculin in the cells on these surfaces. A combination of RGD + Col therefore seems to be the most promising for surface modification of biomaterials, e.g. vascular prostheses.

The architecture and mechanical properties of a scaffold for spinal cord injury treatment must provide tissue integration as well as effective axonal regeneration. Previous work has demonstrated the cell-adhesive and growth-promoting properties of the SIKVAV (Ser-Ile-Lys-Val-Ala-Val)-modified highly superporous poly(2-hydroxethyl methacrylate) (PHEMA) hydrogels. The aim of the current study was to optimize the porosity and mechanical properties of this type of hydrogel in order to develop a suitable scaffold for the repair of spinal cord tissue. Three types of highly superporous PHEMA hydrogels with oriented pores of ~60 µm diameter, porosities of 57-68% and equivalent stiffness characterized by elasticity moduli in the range 3-45 kPa were implanted into a spinal cord hemisection, and their integration into the host tissue, as well as the extent of axonal ingrowth into the scaffold pores, were histologically evaluated. The best tissue response was found with a SIKVAV-modified PHEMA hydrogel with 68% porosity and a moderate modulus of elasticity (27 kPa in the direction along the pores and 3.6 kPa in the perpendicular direction). When implanted into a spinal cord transection, the hydrogel promoted tissue bridging as well as aligned axonal ingrowth. In conclusion, a prospective oriented scaffold architecture of SIKVAV-modified PHEMA hydrogels has been developed for spinal cord injury repair; however, to develop an effective treatment for spinal cord injury, multiple therapeutic approaches are needed., (Copyright © 2013 John Wiley & Sons, Ltd.)

The ability to tailor mechanical properties and architecture is crucial in creating macroporous hydrogel scaffolds for tissue engineering. In the present work, a technique for the modification of the pore size and stiffness of acrylamide-based cryogels is demonstrated via the regulation of an electron beam irradiation dose. The samples were characterized by equilibrium swelling measurements, light and scanning electron microscopy, mercury porosimetry, Brunauer-Emmett-Teller surface area analysis, and stiffness measurements. Their properties were compared to cryogels prepared by a standard redox-initiated radical polymerization. A (125)I radiolabeled azidopentanoyl-GGGRGDSGGGY-NH2 peptide was bound to the surface to determine the concentration of the adhesive sites available for biomimetic modification. The functionality of the prepared substrates was evaluated by in vitro cultivation of adipose-derived stem cells. Moreover, the feasibility of preparing layered cryogels was demonstrated. This may be the key to the future preparation of complex hydrogel-based scaffolds to mimic the extracellular microenvironment in a wide range of applications.

Identification and evaluation of small changes in β-amyloid peptide (Aβ) levels in cerebrospinal fluid is of crucial importance for early detection of Alzheimer's disease. Microfluidic detection methods enable effective preconcentration of Aβ using magnetic microparticles coated with Aβ antibodies. Poly(glycidyl methacrylate) microspheres are coated with α-amino-ω-methoxy-PEG5000 /α-amino-ω-Boc-NH-PEG5000 Boc groups deprotected and NH2 succinylated to introduce carboxyl groups. Capillary electrophoresis with laser-induced fluorescence detection confirms the efficient capture of Aβ 1-40 peptides on the microspheres with immobilized monoclonal anti-Aβ 6E10. The capture specificity is confirmed by comparing Aβ 1-40 levels on the anti-IgG-immobilized particles used as a control., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The efficient isolation and concentration of protein antigens from complex biological samples is a critical step in several analytical methods, such as mass spectrometry, flow cytometry and immunochemistry. These techniques take advantage of magnetic microspheres as immunosorbents. The focus of this study was on the development of new superparamagnetic polymer microspheres for the specific isolation of the tumor suppressor protein p53. Monodisperse macroporous poly(glycidyl methacrylate) (PGMA) microspheres measuring approximately 5 μm and containing carboxyl groups were prepared by multistep swelling polymerization of glycidyl methacrylate (GMA), 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA) and ethylene dimethylacrylate (EDMA) as a crosslinker in the presence of cyclohexyl acetate as a porogen. To render the microspheres magnetic, iron oxide was precipitated within their pores; the Fe content in the particles received ∼18 wt%. Nonspecific interactions between the magnetic particles and biological media were minimized by coating the microspheres with poly(ethylene glycol) (PEG) terminated by carboxyl groups. The carboxyl groups of the magnetic PGMA microspheres were conjugated with primary amino groups of mouse monoclonal DO-1 antibody using conventional carbodiimide chemistry. The efficiency of protein p53 capture and the degree of nonspecific adsorption on neat and PEG-coated magnetic microspheres were determined by western blot analysis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Superporous poly(2-hydroxyethyl methacrylate) is successfully used as a scaffold material for tissue engineering; however, it lacks functional groups that support cell adhesion. The objective of this study was to investigate the cell-adhesive properties of biomimetic ligands, such as laminin-derived Ac-CGGASIKVAVS-OH (SIKVAV) peptide and fibronectin subunits (Fn), as well as small molecules exemplified by 2-mercaptoethanol (ME) and cysteine (Cys), immobilized on a copolymer of 2-hydroxyethyl methacrylate (HEMA) with 2-aminoethyl methacrylate (AEMA) by a maleimide-thiol coupling reaction. The maleimide group was introduced to the P(HEMA-AEMA) hydrogels by the reaction of their amino groups with N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). Mesenchymal stem cells (MSCs) were used to investigate the cell adhesive properties of the modified hydrogels. A significantly larger area of cell growth as well as a higher cell density were found on Fn- and SIKVAV-modified hydrogels when compared to the ME- and Cys-modified supports or neat P(HEMA-AEMA). Moreover, Fn-modification strongly stimulated cell proliferation. The ability of MSCs to differentiate into adipocytes and osteoblasts was maintained on both Fn- and SIKVAV-modifications, but it was reduced on ME-modified hydrogels and neat P(HEMA-AEMA). The results show that the immobilization of SIKVAV and Fn-subunits onto superporous P(HEMA-AEMA) hydrogels via a GMBS coupling reaction improves cell adhesive properties. The high proliferative activity observed on Fn-modified hydrogels suggests that the immobilized Fn-subunits maintain their bioactivity and thus represent a promising tool for application in tissue engineering., (© 2013 Wiley Periodicals, Inc.)

High pressure high temperature (HPHT) nanodiamonds (NDs) represent extremely promising materials for construction of fluorescent nanoprobes and nanosensors. However, some properties of bare NDs limit their direct use in these applications: they precipitate in biological solutions, only a limited set of bio-orthogonal conjugation techniques is available and the accessible material is greatly polydisperse in shape. In this work, we encapsulate bright 30-nm fluorescent nanodiamonds (FNDs) in 10-20-nm thick translucent (i.e., not altering FND fluorescence) silica shells, yielding monodisperse near-spherical particles of mean diameter 66 nm. High yield modification of the shells with PEG chains stabilizes the particles in ionic solutions, making them applicable in biological environments. We further modify the opposite ends of PEG chains with fluorescent dyes or vectoring peptide using click chemistry. High conversion of this bio-orthogonal coupling yielded circa 2000 dye or peptide molecules on a single FND. We demonstrate the superior properties of these particles by in vitro interaction with human prostate cancer cells: while bare nanodiamonds strongly aggregate in the buffer and adsorb onto the cell membrane, the shell encapsulated NDs do not adsorb nonspecifically and they penetrate inside the cells., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The novel synthesis of a polymeric interface grown from the surface of bright fluorescent nanodiamonds is reported. The polymer enables bioorthogonal attachment of various molecules by click chemistry; the particles are resistant to nonspecific protein adsorption and show outstanding colloidal stability in buffers and biological media. The coating fully preserves the unique optical properties of the nitrogen-vacancy centers that are crucial for bioimaging and sensoric applications., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we propose a substrate-independent biomimetic modification route for the creation of antifouling polymer brushes. This modification route consists of the formation/deposition of a biomimetic polydopamine anchor layer followed by a well-controlled surface-initiated atom transfer radical polymerization of antifouling polymer brushes initiated by 2-bromo-2-methylpropanoyl groups covalently attached to the hydroxyl and amine groups present in the anchor layer. In this way, we synthesized polymer brushes of methoxy- and hydroxy-capped oligoethylene glycol methacrylate, 2-hydroxyethyl methacrylate and carboxybetaine acrylamide. Spectroscopic ellipsometry (SE) indicated well-controlled polymerization kinetics of the brushes, thus the thickness of the ultra-thin films could be precisely tuned at a nanometer scale. The covalent structure and organization of the brushes grown from the polydopamine anchor layer were accessed by infrared reflection-adsorption spectroscopy (IRRAS) while the change in hydrophilicity caused by the presence of the brush was determined by dynamic water contact angle measurements. Surface plasmon resonance as well as ex situ IRRAS and SE measurements were applied to investigate the adsorption of model protein solutions and undiluted human blood plasma to the brushes. The biomimetic brushes completely suppressed the fouling from single protein solutions and reduced the fouling from plasma to less than 3% from the fouling measured on bare gold surfaces. The proposed modification procedure is non-destructive and does not require any chemical pre-activation or the presence of reactive groups on the substrate surface. Contrary to other antifouling modifications the coating can be performed on various classes of substrates and preserves its properties even in undiluted blood plasma. This work offers a promising technology for the facile fabrication of different surface-based biotechnological and biomedical devices able to perform tailor-made functions while resisting the fouling from the complex biological media where they operate.

To minimize non-specific protein adsorption on macroporous poly(glycidyl methacrylate) and poly(2-hydroxyethyl methacrylate) microspheres containing amino and/or carboxyl groups, the microspheres are coated with α,ω-bis-carboxy poly(ethylene glycol) and amino-terminated poly(ethylene glycol-co-propylene glycol) or α-methoxy-ω-amino poly(ethylene glycol). Adsorption of bovine serum albumin (BSA), γ-globulin, (125) I-BSA, pepsin, and chymotrypsin on neat and PEGylated microspheres is determined by UV-VIS spectroscopy of supernatants and eluates or by measurement of radioactivity in an ionization chamber. Neat and PEGylated microspheres adsorb 0.8-70% and 0.02-44% of protein, respectively., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nonfouling surfaces capable of reducing protein adsorption are highly desirable in a wide range of applications. Coating of surfaces with poly(ethylene oxide) (PEO), a water-soluble, nontoxic, and nonimmunogenic polymer, is most frequently used to reduce nonspecific protein adsorption. Here we show how to prepare dense PEO brushes on virtually any substrate by tethering PEO to polydopamine (PDA)-modified surfaces. The chain lengths of hetero-bifunctional PEOs were varied in the range of 45-500 oxyethylene units (M(n) = 2000-20,000). End-tethering of PEO chains was performed through amine and thiol headgroups from reactive polymer melts to minimize excluded volume effects. Surface plasmon resonance (SPR) was applied to investigate the adsorption of model protein solutions and complex biologic medium (human blood plasma) to the densely packed PEO brushes. The level of protein adsorption of human serum albumin and fibrinogen solutions was below the detection limit of the SPR measurements for all PEO chains end-tethered to PDA, thus exceeding the protein resistance of PEO layers tethered directly on gold. It was found that the surface resistance to adsorption of lysozyme and human blood plasma increased with increasing length and brush character of the PEO chains end-tethered to PDA with a similar or better resistance in comparison to PEO layers on gold. Furthermore, the chain density, thickness, swelling, and conformation of PEO layers were determined using spectroscopic ellipsometry (SE), dynamic water contact angle (DCA) measurements, infrared reflection-absorption spectroscopy (IRRAS), and vibrational sum-frequency-generation (VSFG) spectroscopy, the latter in air and water.

A simple, versatile, protein-repulsive, substrate-independent biomimetic surface modification is presented that is based on the creation of a PEO brush on a polydopamine anchoring layer and its capacity for selective follow-up modifications with various ligands using a copper-catalyzed alkyne-azide cycloaddition reaction. The desired surface concentration of peptide biomimetic ligands can be controlled by adjusting the peptide concentration in the reaction mixture, then measuring the activity of (125)I-radiolabeled peptides that are immobilized on the substrates. The performance of the prepared substrates is tested in cell cultures with MEF cells and a human ECC line., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we propose substrate-independent modification for creating a protein-repellent surface based on dopamine-melanin anchoring layer used for subsequent binding of poly(ethylene oxide) (PEO) from melt. We verified that the dopamine-melanin layer can be formed on literally any substrate and could serve as the anchoring layer for subsequent grafting of PEO chains. Grafting of PEO from melt in a temperature range 70-110 °C produces densely packed PEO layers showing exceptionally low protein adsorption when exposed to the whole blood serum or plasma. The PEO layers prepared from melt at 110 °C retained the protein repellent properties for as long as 10 days after their exposure to physiological-like conditions. The PEO-dopamine-melanin modification represents a simple and universal surface modification method for the preparation of protein repellent surfaces that could serve as a nonfouling background in various applications, such as optical biosensors and tissue engineering.