Self-Assembly of Cyclodextrin-Coated Nanoparticles:Fabrication of Functional Nanostructures for Sensing and Delivery.

In recent years, the bottom-up approach has emerged as a powerful tool in the fabrication of functional nanomaterials through the self-assembly of nanoscale building blocks. The cues embedded at the molecular level provide a handle to control and direct the assembly of nano-objects to construct higher-order structures. Molecular recognition among the building blocks can assist their precise positioning in a predetermined manner to yield nano- and microstructures that may be difficult to obtain otherwise. A well-orchestrated combination of top-down fabrication and directed self-assembly-based bottom-up approach enables the realization of functional nanomaterial-based devices. Among the various available molecular recognition-based "host-guest" combinations, cyclodextrin-mediated interactions possess an attractive attribute that the interaction is driven in aqueous environments, such as in biological systems. Over the past decade, cyclodextrin-based specific host-guest interactions have been exploited to design and construct structural and functional nanomaterials based on cyclodextrin-coated metal nanoparticles. The focus of this review is to highlight recent advances in the self-assembly of cyclodextrin-coated metal nanoparticles driven by the specific host-guest interaction.

Multifunctional and Transformable 'Clickable' Hydrogel Coatings on Titanium Surfaces: From Protein Immobilization to Cellular Attachment.

Multifunctionalizable hydrogel coatings on titanium interfaces are useful in a wide range of biomedical applications utilizing titanium-based materials. In this study, furan-protected maleimide groups containing multi-clickable biocompatible hydrogel layers are fabricated on a titanium surface. Upon thermal treatment, the masked maleimide groups within the hydrogel are converted to thiol-reactive maleimide groups. The thiol-reactive maleimide group allows facile functionalization of these hydrogels through the thiol-maleimide nucleophilic addition and Diels-Alder cycloaddition reactions, under mild conditions. Additionally, the strained alkene unit in the furan-protected maleimide moiety undergoes radical thiol-ene reaction, as well as the inverse-electron-demand Diels-Alder reaction with tetrazine containing molecules. Taking advantage of photo-initiated thiol-ene 'click' reactions, we demonstrate spatially controlled immobilization of the fluorescent dye thiol-containing boron dipyrromethene (BODIPY-SH). Lastly, we establish that the extent of functionalization on hydrogels can be controlled by attachment of biotin-benzyl-tetrazine, followed by immobilization of TRITC-labelled ExtrAvidin. Being versatile and practical, we believe that the described multifunctional and transformable 'clickable' hydrogels on titanium-based substrates described here can find applications in areas involving modification of the interface with bioactive entities.

Fabrication of Patterned Hydrogel Interfaces: Exploiting the Maleimide Group as a Dual Purpose Handle for Cross-Linking and Bioconjugation.

Functional hydrogels that can be obtained through facile fabrication procedures and subsequently modified using straightforward reagent-free methods are indispensable materials for biomedical applications such as sensing and diagnostics. Herein a novel hydrogel platform is obtained using polymeric precursors containing the maleimide functional group as a side chain. The maleimide groups play a dual role in fabrication of functional hydrogels. They enable photochemical cross-linking of the polymers to yield bulk and patterned hydrogels. Moreover, the maleimide group can be used as a handle for efficient functionalization using the thiol-maleimide conjugation and Diels-Alder cycloaddition click reactions. Obtained hydrogels are characterized in terms of their morphology, water uptake capacity, and functionalization. Micropatterned hydrogels are obtained under UV-irradiation using a photomask to obtain reactive micropatterns, which undergo facile functionalization upon treatment with thiol-containing functional molecules such as fluorescent dyes and bioactive ligands. The maleimide group also undergoes conjugation through the Diels-Alder reaction, where the attached molecule can be released through thermal treatment via the retro Diels-Alder reaction. The antibiofouling nature of these hydrogel micropatterns enables efficient ligand-directed biomolecular immobilization, as demonstrated by attachment of streptavidin-coated quantum dots.

Facile Fabrication of a Modular "Catch and Release" Hydrogel Interface: Harnessing Thiol-Disulfide Exchange for Reversible Protein Capture and Cell Attachment.

Surfaces engineered to "specifically capture" and "release on demand" analytes ranging from biomolecules to cells find niche applications in areas such as diagnostics and detection. Utilization of a disulfide-based linker as a building block allows fabrication of a novel hydrogel-based platform that incorporates a "catch and release" attribute. Hydrogels incorporating pyridyl disulfide groups as thiol-reactive handles were prepared by photopolymerization in the presence of a poly(ethylene glycol) (PEG)-based cross-linker. A range of bulk and micropatterned hydrogels with varying amounts of the reactive group were prepared using PEG-based monomers with different chain lengths. Thiol-containing molecules were conjugated to these hydrogels through the thiol-disulfide exchange reaction under ambient conditions with high efficiencies, as determined by UV-vis spectroscopy. Facile conjugation of a thiol-containing fluorescent dye, namely 4,4-difluoro-1,3,5,7-tetramethyl-8-[(10-mercapto)]-4-bora-3 a,4 a-diaza- s-indacene, was demonstrated, followed by its effective cleavage in the presence of dithiothreitol (DTT), a thiol-containing disulfide-reducing agent. Conjugation of a biotin-containing ligand onto the hydrogels allowed specific binding of protein extravidin when exposed to a mixture of extravidin and bovine serum albumin. The bound protein could be released from the hydrogel by simple exposure to a DTT solution. Likewise, hydrogels modified with a cell-adhesive peptide unit containing the RGD sequence acted as favorable substrates for cellular attachment. Incubation of these cell-attached hydrogel surfaces in a DTT-containing solution leads to facile detachment of cells from the surfaces, while retaining a high level of cell viability. It can be envisioned that the benign nature of these hydrogels, their facile fabrication, and modular functionalization will make them attractive platforms for many applications.

Surface-Anchored Thiol-Reactive Soft Interfaces: Engineering Effective Platforms for Biomolecular Immobilization and Sensing.

Fabrication of antibiofouling, specifically reactive polymeric coatings that undergo facile functionalization with thiol-bearing small molecules and ligands, yields effective platforms for biomolecular immobilization and sensing. Poly(ethylene glycol) (PEG)-based copolymers containing alkoxysilyl groups to enable surface-anchoring and furan-protected maleimide groups as latent thiol-reactive moieties as side-chains were synthesized. Reactive interfaces were obtained by coating these copolymers onto Si/SiO2 or glass surfaces and activating the maleimide groups to their thiol-reactive forms via thermal treatment. A series of surfaces modified with copolymers containing varying amounts of maleimide groups were synthesized. Effectiveness of surface modification was probed using Fourier transform infrared spectroscopy, contact angle goniometry, ellipsometry and X-ray photoelectron spectroscopy. Facile surface modification through thiol-maleimide conjugation was established by attachment of a thiol-containing fluorescent dye, namely BODIPY-SH. It was demonstrated that these surfaces allow spatially localized modification through microcontact printing. Importantly, the extent of surface modification could be tuned by varying the initial composition of the copolymer used for coating. Using fluorescence microscopy, it was observed that increasing amount of fluorescent dye was attached onto surfaces fabricated with copolymers with increasing amount of masked maleimide groups. Thereafter, the thiol-maleimide conjugation was utilized to decorate these surfaces with biotin, a protein-binding ligand. It was observed that though these biotinylated surfaces were able to bind Streptavidin effectively, some nonspecific binding was observed on places that were not in conformal contact with the stamp during microcontact printing. This nonspecific binding was eliminated upon neutralizing the residual maleimide units on the printed surface using thiol-containing PEG. Notably, fluorescence analysis of Streptavidin immobilized onto biotinylated surfaces fabricated using varying amounts of maleimide demonstrated that the amount of immobilized protein could be tuned by varying surface composition. It can be envisioned that facile fabrication of these maleimide-containing polymeric surfaces, their effective functionalization in a tunable manner to engineer interfaces for effective immobilization or sensing of biomolecules in a spatially controlled manner would make them attractive candidates for various biotechnological applications.

Dendrons and Multiarm Polymers with Thiol-Exchangeable Cores: A Reversible Conjugation Platform for Delivery.

Disulfide exchange reaction has emerged as a powerful tool for reversible conjugation of proteins, peptides and thiol containing molecules to polymeric supports. In particular, the pyridyl disulfide group provides an efficient handle for the site-specific conjugation of therapeutic peptides and proteins bearing cysteine moieties. In this study, novel biodegradable dendritic platforms containing a pyridyl disulfide unit at their focal point were designed. Presence of hydroxyl groups at the periphery of these dendrons allows their elaboration to multivalent initiators that yield poly(ethylene glycol) based multiarm star polymers via controlled radical polymerization. The pyridyl disulfide unit at the core of these star polymers undergoes efficient reaction with thiol functional group containing molecules such as a hydrophobic dye, namely, Bodipy-SH, glutathione, and KLAK sequence containing peptide. While conjugation of the hydrophobic fluorescent dye to the PEG-based multiarm polymer renders it water-soluble, it can be cleaved off the construct through thiol-disulfide exchange in the presence of an external thiol such as dithiothreitol. The multiarm polymer was conjugated with a thiol group containing apoptotic peptide to increase its solubility and cellular transport. In vitro cytotoxicity and apoptosis assays demonstrated that the resultant peptide-polymer conjugate had almost five times more apoptotic potential primarily through triggering apoptosis by disrupting mitochondrial membranes of human breast cancer cell line (MDA-MB-231) compared to naked peptide. The novel dendritic platform disclosed here offers an attractive template that can be modified to multiarm polymeric constructs bearing a "tag and release" characteristic.

Diels-Alder "Clickable" Polymer Brushes: A Versatile Catalyst-Free Conjugation Platform.

Polymeric brushes provide an attractive functional interface for a variety of applications in materials and biomedical sciences. Facile access to functionalized brushes can be realized through effective postpolymerization functionalization of reactive brushes. Over the past decade, efficient chemical transformations based on various "click" reactions have been employed for functionalization of polymeric brushes. This paper reports the first example of utilization of the Diels-Alder cycloaddition reaction based functionalization strategy that allows efficient conjugation of maleimide-containing molecules onto furan-containing polymer brushes under mild and reagent-free conditions. Polymers incorporating furan groups as side chains are "grafted from" silicon oxide surfaces and investigated toward their functionalization. Brushes are fabricated using atom transfer radical polymerization with varying amounts of furfuryl methacrylate to enable control over extent of functionalization, along with a poly(ethylene glycol) chain containing methacrylate as a comonomer to impart hydrophilic and antibiofouling characteristics. Functionalization of these reactive brushes were investigated through the immobilization of a model compound N-ethylmaleimide, a fluorescent dye BODIPY-maleimide, and a maleimide-containing biotin based ligand to direct the immobilization of streptavidin-coated quantum dots.

Multireactive Poly(2-oxazoline) Nanofibers through Electrospinning with Crosslinking on the Fly.

Crosslinked hydrophilic poly(2-oxazoline)-based nanofibers amenable to facile multifunctionalization are fabricated using alkene-containing poly(2-alkyl-2-oxazoline)s (PAOx) via in situ photoinitiated radical thiol-ene crosslinking during electrospinning. The resulting crosslinked nanofibers are demonstrated to be multifunctionalizable using different chemistries as they contain two functional handles, being the alkene moieties from the parent copolymer and the residual thiol groups from the tetra-thiol-based crosslinker. While the thiol groups in these nanofibers could be passivated or conjugated to install functional molecules through thiol-maleimide conjugation, the alkene groups could sequentially be modified with thiol-containing molecules using photoinitiated radical thiol-ene reactions. Utilization of the photochemically induced conjugation of thiol-bearing molecules to the alkene groups on the nanofibers is used to obtain functionalization in a spatially controlled manner.

Design and Synthesis of Water-Soluble Multifunctionalizable Thiol-Reactive Polymeric Supports for Cellular Targeting.

Design and synthesis of novel water-soluble polymers bearing reactive side chains are actively pursued due to their increasing demand in areas such as bioconjugation and drug delivery. This study reports the fabrication of poly(ethylene glycol) methacrylate based thiol-reactive water-soluble polymeric supports that can serve as targeted drug delivery vehicles. Thiol-reactive maleimide units were incorporated into polymers as side chains by use of a furan-protected maleimide containing monomer. Atom transfer radical polymerization (ATRP) was employed to obtain a family of well-defined copolymers with narrow molecular weight distributions. After the polymerization, the maleimide groups were activated to their reactive form, ready for conjugation with thiol-containing molecules. Efficient functionalization of the maleimide moieties was demonstrated by conjugation of a tripeptide glutathione under mild and reagent-free aqueous conditions. Additionally, hydrophobic thiol-containing dye (Bodipy-SH) and a cyclic peptide-based targeting group (cRGDfC) were sequentially appended onto the maleimide bearing polymers to demonstrate their efficient multifunctionalization. The conjugates were utilized for in vitro experiments over both cancerous and healthy breast cell lines. Obtained results demonstrate that the conjugates were nontoxic, and displayed efficient cellular uptake. The presence of the peptide based targeting group had a clear effect on increasing the uptake of the dye-conjugated polymers into cells when compared to the construct devoid of the peptide. Overall, the facile synthesis and highly efficient multifunctionalization of maleimide-containing thiol-reactive copolymers offer a novel and attractive class of polyethylene glycol-based water-soluble supports for drug delivery.

Indispensable platforms for bioimmobilization: maleimide-based thiol reactive hydrogels.

Poly(ethylene glycol)-based hydrogels containing thiol-reactive maleimide functional groups is prepared via a Diels-Alder/retro Diels-Alder reaction sequence using a masked maleimide monomer. Bulk and micropatterned hydrogels containing varying amounts of the thiol-reactive maleimide functional group are fabricated at ambient temperature. During the fabrication, the reactive maleimide functional group in the monomer is masked with a furan moiety and then unmasked to its reactive form via the retro-Diels-Alder reaction. The reactive maleimide groups embedded within the hydrogel are amenable to facile and efficient functionalization with thiol-containing molecules such as fluorescent dyes. Furthermore, these hydrogels are readily biotinylated using the nucleophilic thiol-ene conjugation to enable immobilization of streptavidin onto the hydrogel patterns to achieve facile bioimmobilization. Notably, the extent of functionalization of these hydrogels can be easily tailored by varying the amount of reactive handles incorporated during their fabrication.

'Clickable' hydrogels for all: facile fabrication and functionalization.

In this study, we report a facile fabrication of poly(ethylene glycol) (PEG) based bulk and micro-patterned hydrogels that are amenable to functionalization with thiol-bearing molecules using the metal-free radical 'thiol-ene' click reaction. The hydrogels were synthesized using photopolymerization of commercially available monomers, namely allyl methacrylate and PEG-methacrylate in the presence of a PEG-dimethacrylate based crosslinker. Swelling behaviour of these hydrogels could be tailored by varying the amount of the hydrophilic monomer in the feed as well as varying the length of the PEG-chain in the methacrylate monomer. Importantly, the extent of functionalization of these hydrogels could be readily tuned by varying the amount of the reactive allyl methacrylate monomer. Analysis of nitrogen content in the hydrogels after conjugation of cysteamine demonstrated that the amount of cysteamine incorporation was in correlation with the amount of allyl groups in the hydrogels. Three-dimensional hydrogel patterns were fabricated using micromolding in capillaries. Tuneable conjugation of a thiol-containing dye molecule and a ligand-mediated immobilization of streptavidin onto these hydrogel patterns were realized. It was found that the swellability of the hydrogel patterns control the diffusion of streptavidin into the interior of the hydrogel matrix. These bio-inert hydrogels could be appended with peptides to promote cellular adhesion. Furthermore, it was demonstrated that the photochemical thiol-ene based method of conjugation enables localized attachment of thiol-containing molecules within these reactive hydrogels.