Optimized peptide functionalization of thiol‐acrylate emulsion‐templated porous polymers leads to expansion of human pluripotent stem cells in 3D culture

Highly porous polymers produced by polymerization of the continuous phase of a high internal phase emulsion have been developed as scaffolds for 3D culture of human pluripotent stem cells. These emulsion‐templated polymerized high internal phase emulsion (polyHIPE) materials have an interconnected network of pores that provide support for the cells, while also allowing both cell ingress and nutrient diffusion. Thiol‐acrylate polyHIPE materials were prepared by photopolymerization, which, due to a competing acrylate homopolymerization process, leads to a material with residual surface thiols. These thiols were then used as a handle to allow postpolymerization functionalization with both maleimide and a maleimide‐derivatized cyclo‐RGDfK peptide, via Michael addition under benign conditions. Functionalization was evaluated using an Ellman's colorimetric assay, to monitor the residual thiol concentration, and X‐ray photoelectron spectroscopy. Maleimide was used as a model molecule to optimize conditions prior to peptide‐functionalization. The use of triethylamine as a catalyst and a mixed ethanol‐aqueous solvent system led to optimized reaction between surface‐bound thiols and maleimide. Peptide‐functionalized materials showed improved attachment and infiltration of human pluripotent stem cells over 7 days, demonstrating their promise as a scaffold for 3D stem cell culture and expansion. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019, 57, 1974–1981     

Perfluorophenyl azide functionalization of electrospun poly(para‐dioxanone)

Strategies to surface‐functionalize scaffolds by covalent binding of biologically active compounds are of fundamental interest to control the interactions between scaffolds and biomolecules or cells. Poly(para‐dioxanone) (PPDO) is a clinically established polymer that has shown potential as temporary implant, eg, for the reconstruction of the inferior vena cava, as a nonwoven fiber mesh. However, PPDO lacks suitable chemical groups for covalent functionalization. Furthermore, PPDO is highly sensitive to hydrolysis, reflected by short in vivo half‐life times and degradation during storage. Establishing a method for covalent functionalization without degradation of this hydrolyzable polymer is therefore important to enable the surface tailoring for tissue engineering applications. It was hypothesized that treatment of PPDO with an N‐hydroxysuccinimide ester group bearing perfluorophenyl azide (PFPA) under UV irradiation would allow efficient surface functionalization of the scaffold. X‐ray photoelectron spectroscopy and attenuated total reflectance Fourier‐transformed infrared spectroscopy investigation revealed the successful binding, while a gel permeation chromatography study showed that degradation did not occur under these conditions. Coupling of a rhodamine dye to the N‐hydroxysuccinimide esters on the surface of a PFPA‐functionalized scaffold via its amine linker showed a homogenous staining of the PPDO in laser confocal microscopy. The PFPA method is therefore applicable even to the surface functionalization of hydrolytically labile polymers, and it was demonstrated that PFPA chemistry may serve as a versatile tool for the (bio‐)functionalization of PPDO scaffolds.     

A degradable,porous, emulsion‐templated polyacrylate

A polyHIPE is a highly porous, emulsion‐templated polymer synthesized by polymerizing a monomer and a crosslinking comonomer in the continuous phase of a high‐internal phase emulsion (HIPE). The synthesis of degradable polyHIPE could be of interest for biomedical applications such as tissue engineering scaffolds. In this research, a poly(ε‐caprolactone) (PCL) oligomer with terminal vinyl groups was used as the crosslinking comonomer for a polyHIPE based on t‐butyl acrylate (tBA). The porous structure, properties, water absorption, and hydrolytic degradation of the polyHIPE were investigated. The polyHIPE containing 50 wt % PCL exhibited very large voids, 1 to 3 mm in diameter that resulted from the destabilization of the HIPE on addition of PCL, making the polyHIPE more suitable for tissue engineering applications. The relatively flexible PCL enhanced segmental mobility, yielding two glass transition temperatures and a significant reduction in modulus. When exposed to a 3 M aqueous solution of NaOH, the t‐butyl groups underwent hydrolysis and the PCL underwent degradation, rapidly leading to the complete disintegration of the macromolecular structure. The tBA‐based polyHIPE containing 50 wt % PCL exhibited enhanced cell adhesion, penetration, and growth indicating that it is a suitable candidate for further research and development. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2009     

Synthetic,Chemically Defined Polymer‐Coated Microcarriers for the Expansion of Human Mesenchymal Stem Cells

Mesenchymal stem cells (MSC), also called marrow stromal cells, are adult cells that have attracted interest for their potential uses in therapeutic applications. There is a pressing need for scalable culture systems due to the large number of cells needed for clinical treatments. Here, a tailorable thin polymer coating—poly(poly(ethylene glycol) methyl ether methacrylate‐ran‐vinyl dimethyl azlactone‐ran‐glycidyl methacrylate) [P(PEGMEMA‐r‐VDM‐r‐GMA); PVG]—to the surface of commercially available polystyrene and glass microcarriers to create chemically defined surfaces for large‐scale cell expansion is applied. These chemically defined microcarriers create a reproducible surface that does not rely on the adsorption of xenogenic serum proteins to mediate cell adhesion. Specifically, this coating method anchors PVG copolymer through ring opening nucleophilic attack by amine residues on poly‐l ‐lysine that is pre‐adsorbed to the surface of microcarriers. Importantly, this anchoring reaction preserves the monomer VDM reactivity for subsequent functionalization with an integrin‐specific Arg‐Gly‐Asp peptide to enable cell adhesion and expansion via a one‐step reaction in aqueous media. MSCs cultured on PVG‐coated microcarriers achieve sixfold expansion—similar to the expansion achieved on PS microcarriers—and retain their ability to differentiate after harvesting.     

Preparation of polyHIPE via CuAAC “click” chemistry and its application as a highly efficient adsorbent of Cu(II) ions

A hydrophilic emulsion‐templated porous polymer (polyHIPE) is synthesized by CuAAC “click” chemistry. Herein, a 4,4′‐diazidostilbene‐2,2′‐disulfonic acid disodium salt‐4H₂O (DAS) and tripropargylamine in the mixture of water and N,N‐dimethylformamide solution is used as external phase of the high internal phase emulsion template, and paraffin liquid is involved as the internal phase. The resulting polyHIPE has a well‐defined interconnected pore structure, which could be tailored by changing preparation parameters, such as reagent content, internal phase volume fraction, and surfactant concentration. Thermal analysis shows that the polyHIPE is stable under 180 °C. Owing to the presence of a large number of sodium sulfonate groups from the reagent DAS and the triazoles groups produced in the reaction, the polyHIPE is proved to be a highly efficient adsorbent of heavy metal ion (i.e., up to 52 mg/g for Cu(II) ions) in water. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 2129–2135     

Adsorption and adhesion studies of PdSn‐nanoparticles on protonated amine and carboxylic acid‐terminated surfaces

Electroless plating of acrylonitrile‐butadiene‐styrene‐terpolymers (ABS‐plastics) is used for decorative applications and relies on the immobilization of catalytic palladium‐tin nanoparticles. We used chemical force microscopy to measure the adhesion force of palladium‐tin nanoparticles on a patterned amine and carboxyl‐terminated surface prepared by micro‐contact printing. The kinetics of the adsorption process and the population density of the nanoparticles on amine and carboxyl‐terminated surfaces were monitored by quartz crystal microbalance with dissipation analysis. The surface chemistry was investigated by means of polarization‐modulated infrared reflection absorption spectroscopy and X‐ray photoelectron spectroscopy. Enhanced adhesion and population density of PdSn nanoparticles on protonated amine‐terminated surfaces compared with carboxyl‐terminated surfaces is observed. Copyright © 2016 John Wiley & Sons, Ltd.     

Preparation of remarkably tough polyHIPE materials via polymerization of oil‐in‐water HIPEs involving 1‐vinyl‐5‐aminotetrazole

Interconnected microcellular polymeric monoliths having unexpected high mechanical strength have been prepared using the high internal phase emulsion (HIPE) methodology. Oil‐in water concentrated emulsions of aqueous 1‐vinyl‐5‐amino [1,2,3,4]tetrazole (1‐VAT) mixed with a low molar ratio (7%) of N,N′‐methylenebisacrylamide as crosslinking agent were prepared using dodecane as dispersed phase and a mixture of hydrophilic surfactants. “Reverse” polyHIPE materials were obtained after radical copolymerization, solvent extraction, and drying. Their morphology, chemical composition, and physicochemical behavior are discussed. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 2942–2947, 2010     

Biofouling of dextran-derivative layers investigated by quartz crystal microbalance

This study reports the fouling of carboxymethyl dextran (CMD) layers in cell culture medium, fibronectin, and serum solutions. CMD layers were covalently immobilized onto amine groups available either on an n-heptylamine plasma polymer (HApp) layer or onto a polyethylenimine (PEI) coating grafted to an acetaldehyde plasma polymer (AApp) layer. The successful immobilization of the graft layers was demonstrated by X-ray photoelectron spectroscopy (XPS). Primary amines on HApp and AApp + PEI surfaces were quantified using a colorimetric assay. Quartz crystal microbalance (QCM) was used to investigate in real-time the fouling of the graft layers upon incubation in cell culture medium (RPMI), fibronectin, and foetal bovine serum (FBS) solutions. HApp, AApp and AApp + PEI layers exhibited large fouling in fibronectin and FBS solutions, while fouling in RPMI cell culture medium was not significant. Protein repellent properties of CMD layers in FBS and fibronectin have been demonstrated compared to the other tested surfaces. QCM has shown that both CMD layers were fouled to a small extent in RPMI medium.     

Combinatorial functionalization with bisurea‐peptides and antifouling bisurea additives of a supramolecular elastomeric biomaterial

The bioactive additive toolbox to functionalize supramolecular elastomeric materials expands rapidly. Here we have set an explorative step toward screening of complex combinatorial functionalization with antifouling and three peptide‐containing additives in a bisurea‐based supramolecular system. Thorough investigation of surface properties of thin films with contact angle measurements, X‐ray photoelectron spectroscopy and atomic force microscopy, was correlated to cell‐adhesion of endothelial and smooth muscle cells to apprehend their respective predictive values for functional biomaterial development. Peptides were presented at the surface alone, and in combinatorial functionalization with the oligo(ethylene glycol)‐based non‐cell adhesive additive. The bisurea‐RGD additive was cell‐adhesive in all conditions, whereas the endothelial cell‐specific bisurea‐REDV showed limited bioactive properties in all chemical nano‐environments. Also, aspecific functionality was observed for a bisurea‐SDF1α peptide. These results emphasize that special care should be taken in changing the chemical nano‐environment with peptide functionalization. © 2019 The Authors. Journal of Polymer Science Part B: Polymer Physics published by Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2019 , 57, 1725–1735     

3D and Porous RGDC‐Functionalized Polyester‐Based Scaffolds as a Niche to Induce Osteogenic Differentiation of Human Bone Marrow Stem Cells

Polyester‐based scaffolds covalently functionalized with arginine‐glycine‐aspartic acid‐cysteine (RGDC) peptide sequences support the proliferation and osteogenic differentiation of stem cells. The aim is to create an optimized 3D niche to sustain human bone marrow stem cell (hBMSC) viability and osteogenic commitment, without reliance on differentiation media. Scaffolds consisting of poly(lactide‐co‐trimethylene carbonate), poly(LA‐co‐TMC), and functionalized poly(lactide) copolymers with pendant thiol groups are prepared by salt‐leaching technique. The availability of functional groups on scaffold surfaces allows for an easy and straightforward method to covalently attach RGDC peptide motifs without affecting the polymerization degree. The strategy enables the chemical binding of bioactive motifs on the surfaces of 3D scaffolds and avoids conventional methods that require harsh conditions. Gene and protein levels and mineral deposition indicate the osteogenic commitment of hBMSC cultured on the RGDC functionalized surfaces. The osteogenic commitment of hBMSC is enhanced on functionalized surfaces compared with nonfunctionalized surfaces and without supplementing media with osteogenic factors. Poly(LA‐co‐TMC) scaffolds have potential as scaffolds for osteoblast culture and bone grafts. Furthermore, these results contribute to the development of biomimetic materials and allow a deeper comprehension of the importance of RGD peptides on stem cell transition toward osteoblastic lineage.     

Acrylic‐Acid‐Functionalized PolyHIPE Scaffolds for Use in 3D Cell Culture

This study describes the development of a functional porous polymer for use as a scaffold to support 3D hepatocyte culture. A high internal phase emulsion (HIPE) is prepared containing the monomers styrene (STY), divinylbenzene (DVB), and 2‐ethylhexyl acrylate (EHA) in the external oil phase and the monomer acrylic acid (Aa) in the internal aqueous phase. Upon thermal polymerization with azobisisobutyronitrile (AIBN), the resulting porous polymer (polyHIPE) is found to have an open‐cell morphology and a porosity of 89%, both suitable characteristics for 3D cell scaffold applications. X‐ray photo­electron spectroscopy reveals that the polyHIPE surface contained 7.5% carboxylic acid functionality, providing a useful substrate for subsequent surface modifications and bio‐conjugations. Initial bio‐compatibility assessments with human hepatocytes show that the acid functionality does not have any detrimental effect on cell adhesion. It is therefore believed that this material can be a useful precursor scaffold towards 3D substrates that offer tailored surface functionality for enhanced cell adhesion.

     

Adsorption and conformation behavior of biotinylated fibronectin on streptavidin-modified TiO(X) surfaces studied by SPR and AFM

It is well-known that protein-modified implant surfaces such as TiO(2) show a higher bioconductivity. Fibronectin is a glycoprotein from the extracellular matrix (ECM) with a major role in cell adhesion. It can be applied on titanium oxide surfaces to accelerate implant integration. Not only the surface concentration but also the presentation of the protein plays an important role for the cellular response. We were able to show that TiO(X) surfaces modified with biotinylated fibronectin adsorbed on a streptavidin-silane self-assembly multilayer system are more effective regarding osteoblast adhesion than surfaces modified with nonspecifically bound fibronectin. The adsorption and conformation behavior of biotinylated and nonbiotinylated (native) fibronectin was studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Imaging of the protein modification revealed that fibronectin adopts different conformations on nonmodified compared to streptavidin-modified TiO(X) surfaces. This conformational change of biotinylated fibronectin on the streptavidin monolayer delivers a fibronectin structure similar to the conformation inside the ECM and therefore explains the higher cell affinity for these surfaces.     

Micropatterned designs of thermoresponsive surfaces for modulating cell behaviors

The chemistry and topography of the material surfaces have an important effect on cell behaviors. In this study, we reported the preparation of thermoresponsive micropatterned surfaces (TS) and galactosylated TS for modulating the adhesion/detachment of cells. A thickness of 1 µm of poly(N‐isopropylacrylamide) grafted layer was fabricated on the polystyrene surface with microgrooves using ultraviolet‐induced copolymerization. The thick grafted layer was in favor of the interactions between cells and materials. The following immobilization of galactose ligand with specific affinity to hepatocyte onto TS promoted the adhesion of human hepatocyte line (HL‐7702 cells). The microgrooves structure could facilitate cell adhesion and regulate the oriented growth of cells. Moreover, narrow grooves accelerated the spontaneous detachment of cells only by reducing temperature. Thus, micropatterned biofunctional designs with controlled geometrical features presented in this study have sufficient biofunctional activities in facilitating cell sheet engineering and regenerative medicine. Copyright © 2013 John Wiley & Sons, Ltd.     

The surface energy of various biomaterials coated with adhesion molecules used in cell culture

This study calculates the surface energy of polystyrene tissue culture plastic, silicon, silicon dioxide and indium tin oxide, all of which have applications in tissue culture. The adhesion molecules: collagen, fibronectin, poly-L-ornithine and poly-D-lysine, were coated onto these various surfaces, and the surface energy of the coated substrates calculated. Coating with fibronectin was found to produce a monopolar acidic surface while poly-D-lysine, poly-L-ornithine and collagen coatings were found to produce monopolar basic surfaces. The calculated surface energy components of the coated materials were then used to give a quantitative determination of the magnitude of their hydrophobicity. It was concluded that collagen, polylysine and polyornithine could provide a hydrophobic or hydrophilic surface depending on the underlying substrates they were coated on. The measurement obtained for fibronectin, unlike the other adhesion molecules, was independent of the underlying surface and remained hydrophobic on all substrates tested. Wetting experiments were carried out on the coated substrates, using the tissue culture medium Dulbeccos modified eagles medium, both containing and not containing serum proteins, and saline solution. These liquids that are commonly used in tissue culture, were then used to provide information how these liquids behave on various substrates coated with the adhesion molecules. Results show that fibronectin coated surfaces represent the most phobic surface for all three liquids. The findings of this study can be used in cell manipulation studies and provide a valuable data set for the biomedical and research industries.     

Synthetic hydrogels as scaffolds for manipulating endothelium cell behaviors

Synthetic hydrogels can be used as scaffolds that not only favor endothelial cells（ECs） proliferation but also manipulate the behaviors and functions of the ECs.In this review paper,the effect of chemical structure,Young’s modulus （E） and zeta potential（ξ） of synthetic hydrogel scaffolds on static cell behaviors,including cell morphology,proliferation, cytoskeleton structure and focal adhesion,and on dynamic cell behaviors,including migration velocity and morphology oscillation,as well as on EC function such as anti-platelet adhesion,are reported.It was found that negatively charged hydrogels,poly（2-acrylamido-2-methylpropanesulfonic sodium）（PNaAMPS） and poly（sodium p-styrene sulphonate） （PNaSS）,can directly promote cell proliferation,with no need of surface modification by any cell-adhesive proteins or peptides at the environment of serum-containing medium.In addition,the Young’s modulus（E） and zeta potential（ξ） of hydrogel scaffolds are quantitatively tuned by copolymer hydrogels,poly（NaAMPS-co-DMAAm） and poly（NaSS-co-DMAAm）, in which the two kinds of negatively charged monomers NaAMPS and NaSS are copolymerized with neutral monomer,N,N-dimethylacrylamide（DMAAm）.It was found that the critical zeta potential of hydrogels manipulating EC morphology,proliferation,and motility isξ_critical= -20.83 mV andξ_critical= -14.0 mV for poly（NaAMPS-co-DMAAm） and poly（NaSS-co-DMAAm）,respectively.The above mentioned EC behaviors well correlate with the adsorption of fibronectin, a kind of cell-adhesive protein,on the hydrogel surfaces.Furthermore,adhered platelets on the EC monolayers cultured on the hydrogel scaffolds obviously decreases with an increase of the Young’s modulus（E） of the hydrogels,especially when E>60 kPa.Glycocalyx assay and gene expression of ECs demonstrate that the anti-platelet adhesion well correlates with the EC-specific glycocalyx.The above investigation suggests that understanding the relationship between physic-chemical properties of synthetic hydrogels and cell responses is essential to design optimal soft and wet scaffolds for tissue engineering.     

Preparation of Gradient Surfaces by Using a Simple Chemical Reaction and Investigation of Cell Adhesion on a Two‐Component Gradient

This article describes a simple method for the generation of multicomponent gradient surfaces on self‐assembled monolayers (SAMs) on gold in a precise and predictable manner, by harnessing a chemical reaction on the monolayer, and their applications. A quinone derivative on a monolayer was converted to an amine through spontaneous intramolecular cyclization following first‐order reaction kinetics. An amine gradient on the surface on a scale of centimeters was realized by modulating the exposure time of the quinone‐presenting monolayer to the chemical reagent. The resulting amine was used as a chemical handle to attach various molecules to the monolayer with formation of multicomponent gradient surfaces. The effectiveness of this strategy was verified by cyclic voltammetry (CV), matrix assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry (MS), MS imaging, and contact‐angle measurements. As a practical application, cell adhesion was investigated on RGD/PHSRN peptide/peptide gradient surfaces. Peptide PHSRN was found to synergistically enhance cell adhesion at the position where these two ligands are presented in equal amounts, while these peptide ligands were competitively involved in cell adhesion at other positions. This strategy of generating a gradient may be further expandable to the development of functional gradient surfaces of various molecules and materials, such as DNA, proteins, growth factors, and nanoparticles, and could therefore be useful in many fields of research and practical applications.     

Aminosilane Functionalized Aligned Fiber PCL Scaffolds for Peripheral Nerve Repair

Silane modification is a simple and cost-effective tool to modify existing biomaterials for tissue engineering applications. Aminosilane layer deposition has previously been shown to control NG108-15 neuronal cell and primary Schwann cell adhesion and differentiation by controlling deposition of ─NH₂ groups at the submicron scale across the entirety of a surface by varying silane chain length. This is the first study toreport depositing 11-aminoundecyltriethoxysilane (CL11) onto aligned Polycaprolactone (PCL) scaffolds for peripheral nerve regeneration. Fibers are manufactured via electrospinning and characterized using water contact angle measurements, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Confirmed modified fibers are investigated using in vitro cell culture of NG108-15 neuronal cells and primary Schwann cells to determine cell viability, cell differentiation, and phenotype. CL11-modified fibers significantly support NG108-15 neuronal cell and Schwann cell viability. NG108-15 neuronal cell differentiation maintains Schwann cell phenotype compared to unmodified PCL fiber scaffolds. 3D ex vivo culture of Dorsal root ganglion explants (DRGs) confirms further Schwann cell migration and longer neurite outgrowth from DRG explants cultured on CL11 fiber scaffolds compared to unmodified scaffolds. Thus, a reproducible and cost-effective tool is reported to modify biomaterials with functional amine groups that can significantly improve nerve guidance devices and enhance nerve regeneration.     

Tailorable Surface Morphology of 3D Scaffolds by Combining Additive Manufacturing with Thermally Induced Phase Separation

The functionalization of biomaterials substrates used for cell culture is gearing towards an increasing control over cell activity. Although a number of biomaterials have been successfully modified by different strategies to display tailored physical and chemical surface properties, it is still challenging to step from 2D substrates to 3D scaffolds with instructive surface properties for cell culture and tissue regeneration. In this study, additive manufacturing and thermally induced phase separation are combined to create 3D scaffolds with tunable surface morphology from polymer gels. Surface features vary depending on the gel concentration, the exchanging temperature, and the nonsolvent used. When preosteoblasts (MC‐3T3 cells) are cultured on these scaffolds, a significant increase in alkaline phosphatase activity is measured for submicron surface topography, suggesting a potential role on early cell differentiation.     

Linear Poly(methyl glycerol) and Linear Polyglycerol as Potent Protein and Cell Resistant Alternatives to Poly(ethylene glycol)

The nonspecific interaction of proteins with surfaces in contact with biofluids leads to adverse problems and is prevented by a biocompatible surface coating. The current benchmark material among such coatings is poly(ethylene glycol) (PEG). Herein, we report on the synthesis of linear polyglycerol derivatives as promising alternatives to PEG. Therefore, gold surfaces as a model system are functionalized with a self‐assembled monolayer (SAM) by a two‐step anhydride coupling and a direct thiol immobilization of linear poly(methyl glycerol) and polyglycerol. Surface plasmon resonance (SPR) spectroscopy reveals both types of functionalized surfaces to be as resistant as PEG towards the adsorption of the test proteins fibrinogen, pepsin, albumin, and lysozyme. Moreover, linear polyglycerols adsorb even less proteins from human plasma than a PEG‐modified surface. Additional cell adhesion experiments on linear poly(methyl glycerol) and polyglycerol‐modified surfaces show comparable cell resistance as for a PEG‐modified surface. Also, in the case of long‐term stability, high cell resistance is observed for all samples in medium. Additional in vitro cell‐toxicity tests add to the argument that linear poly(methyl glycerol) and polyglycerol are strong candidates for promising alternatives to PEG, which can easily be modified for biocompatible functionalization of other surfaces.     

Bioorthogonal Labeling,Bioimaging, and Photocytotoxicity Studies of Phosphorescent Ruthenium(II) Polypyridine Dibenzocyclooctyne Complexes

The synthesis, characterization, photophysics, lipophilicity, and cellular properties of new phosphorescent ruthenium(II) polypyridine complexes functionalized with a dibenzocyclooctyne (DIBO) or amine moiety [Ru(N^N)₂(L)](PF₆)₂ are reported (L=4‐(13‐N‐(3,4:7,8‐dibenzocyclooctyne‐5‐oxycarbonyl) amino‐4,7,10‐trioxa‐tridecanyl‐aminocarbonyl‐oxy‐methyl)‐4′‐methyl‐2,2′‐bipyridine bpy‐DIBO, N^N=2,2′‐bipyridine bpy ( 1 a ), 1,10‐phenanthroline phen ( 2 a ); L=4‐(13‐amino‐4,7,10‐trioxa‐tridecanylaminocarbonyl‐oxy‐methyl)‐4′‐methyl‐2,2′‐bipyridine bpy‐NH₂, N^N=bpy ( 1 b ), phen ( 2 b )). The strain‐promoted alkyne–azide cycloaddition (SPAAC) reaction of the DIBO complexes 1 a and 2 a with benzyl azide were studied. Also, the DIBO complexes 1 a and 2 a can selectively label N‐azidoglycans located on the surface of CHO‐K1 and A549 cells that were pretreated with 1,3,4,6‐tetra‐O‐acetyl‐N‐azidoacetyl‐D ‐mannosamine (Ac₄ManNAz). Additionally, the intracellular trafficking and localization of these biomolecules were monitored using laser‐scanning confocal microscopy. Interestingly, the biolabeling and cellular uptake efficiency of the DIBO complexes 1 a and 2 a were cell‐line dependent, as revealed by flow cytometry and ICP‐MS. Furthermore, the complexes showed good biocompatibility toward the Ac₄ManNAz‐pretreated cells in the dark, but exhibited photoinduced cytotoxicity due to the generation of singlet oxygen.