“Clickable” Polymeric Nanofibers through Hydrophilic–Hydrophobic Balance: Fabrication of Robust Biomolecular Immobilization Platforms

Fabrication of hydrophilic polymeric nanofibers that undergo facile and selective functionalization through metal catalyst-free Diels–Alder “click” reaction in aqueous environment is outlined. Electrospinning of copolymers containing an electron-rich furan moiety, hydrophobic methyl methacrylate units and hydrophilic poly­(ethylene glycol)­s as side chains provide specifically functionalizable yet antibiofouling fibers that remain stable in aqueous media due to appropriate hydrophobic hydrophilic balance. Efficient functionalization of these nanofibers is accomplished through the Diels–Alder reaction by exposing them to maleimide-containing molecules and ligands. Diels–Alder conjugation based functionalization is demonstrated through attachment of fluorescein-maleimide and a maleimide tethered biotin ligand. Biotinylated nanofibers were utilized to mediate immobilization of the protein streptavidin, as well as streptavidin coated quantum dots. Facile fabrication from readily available polymers and their effective functionalization under mild and reagent-free conditions in aqueous media make these “clickable” nanofibers attractive candidates as functionalizable scaffolds for various biomedical applications

“Clickable” Nanogels via Thermally Driven Self-Assembly of Polymers: Facile Access to Targeted Imaging Platforms using Thiol–Maleimide Conjugation

Multifunctionalizable nanogels are fabricated using thermally driven self-assembly and cross-linking of reactive thermoresponsive copolymers. Nanogels thus fabricated can be easily conjugated with various appropriately functionalized small molecules and/or ligands to tailor them for various applications in delivery and imaging. In this study, a poly­(ethylene glycol)-methacrylate-based maleimide-bearing copolymer was cross-linked with a dithiol-based cross-linker to synthesize nanogels. Because of lower critical solution temperature (LCST) around 55 °C in aqueous media, these copolymers assemble into nanosized aggregates when heated to this temperature, and they are cross-linked using the thiol–maleimide conjugation. Nanogels thus fabricated contain both thiol and maleimide groups in the same cross-linked nanogels. Postgelation functionalization of the residual maleimide and thiol groups is demonstrated through conjugation of a thiol-bearing hydrophobic dye (BODIPY-SH) and <i>N</i>-(fluoresceinyl) maleimide, respectively. In addition, to demonstrate the utility of multifunctionality of these nanogels, a thiol-bearing cyclic-peptide-based targeting group, cRGDfC, and <i>N</i>-(fluoresceinyl)-maleimide-based fluorescent tag was conjugated to nanogels in aqueous media. Upon treatment with breast cancer cell lines, MDA-MB-231, it was deduced from cellular internalization studies using fluorescence microscopy and flow cytometry that the peptide carrying constructs were preferentially internalized. Overall, a facile synthesis of multifunctionalizable nanogels that can be tailored using effective conjugation chemistry under mild conditions can serve as promising candidates for various applications

Hooked on Cryogels: A Carbamate Linker Based Depot for Slow Drug Release

Poly­(ethylene glycol) (PEG) based bulk hydrogels and cryogels containing activated carbonate groups as amine reactive handles to facilitate drug conjugations through carbamate linkages were fabricated and evaluated as slow releasing drug reservoirs. As an initial approach, photopolymerization of <i>N</i>-hydroxysuccinimide (NHS)-activated carbonate functional group containing monomer and PEG-methacrylate in the presence of a cross-linker was utilized to obtain bulk hydrogels with high gel conversions. The resultant hydrogels possessed moderate water uptake (170–340%) which was dependent on the monomer ratios. These hydrogels were functionalized with an anticancer drug, namely, doxorubicin. Surprisingly, while negligible drug release was observed from the bulk hydrogels under normal pH, only about 6% drug release was observed under acidic condition. Limited swelling of these hydrogels as well as lack of porous structure as deduced from scanning electron microscopy analysis might explain the poor drug release. To enhance the drug releasing capacity of these hydrogels that might stem from the increased porosity, reactive carbonate group bearing cryogels were synthesized. Compared to the bulk hydrogels, cryogels were highly porous in structure and also possessed much higher swelling capacity (1150–1500%). As a result of these distinctions, a 7-fold enhancement in drug release was observed for the cryogel system compared to the relating hydrogel. In vitro studies demonstrated that the anticancer drug doxorubicin conjugated through carbamate linkers to the cryogels was released and proved effective against MDA-MB-231 human breast cancer cells. Overall, a novel class of slow releasing nontoxic hydrogel and cryogel scaffolds with potential applications as anticancer drug reservoirs was realized

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

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

Embedding Well-Defined Responsive Hydrogels with Nanocontainers: Tunable Materials from Telechelic Polymers and Cyclodextrins

Design, synthesis, and application of cyclodextrin (CD) containing thermoresponsive hydrogels fabricated from thiol-reactive telechelic polymers are reported. Hydrophilic polymers containing 2-hydroxyethyl methacrylate and/or di­(ethylene glycol)­methylether methacrylate monomers as side chains and thiol-reactive groups at chain ends were synthesized. A series of hydrogels was fabricated using thiol–ene conjugation of these thiol-reactive polymers with multivalent thiol-containing CDs as crosslinkers. Clear and transparent hydrogels were obtained with good conversion (79–89%) by utilizing the “nucleophilic” and “radical” thiol–ene “click” reactions. Analysis of the amount of residual thiol groups in these hydrogels using Ellman’s reagent suggested that gels with a moderately well-defined network structure were obtained. Hydrogels fabricated using different telechelic polymers were examined for their properties such as morphology, equilibrium water uptake, and rheological characteristics. Cytocompatibility of these hydrogels was ascertained by a cell viability assay that demonstrated low toxicity toward fibroblast cells. Thereafter, the CD-containing hydrogels were evaluated for the loading and controlled release of puerarin, an antiglaucoma drug. Utilization of thermoresponsive polymers as the matrix for these hydrogels allows use of temperature as a stimulus to modulate the drug release. A slower and more sustained drug release was observed at physiological temperatures compared to ambient conditions. The effect of temperature on the elasticity of the hydrogel was investigated rheologically to demonstrate that the collapse of the network structure occurs near physiological temperatures. The increased hydrophobicity and compactness of the gel matrix at higher temperatures results in a slower drug release. The strategy employed here demonstrates that tuning the matrix composition of hydrogels with well-defined network structures through appropriate choice of responsive copolymers allows design of materials with control of their physical properties and drug-release behavior

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 <i>N</i>-ethylmaleimide, a fluorescent dye BODIPY-maleimide, and a maleimide-containing biotin based ligand to direct the immobilization of streptavidin-coated quantum dots

Designing Dendron–Polymer Conjugate Based Targeted Drug Delivery Platforms with a “Mix-and-Match” Modularity

Polymeric micellar systems are emerging as a very important class of nanopharmaceuticals due to their ability to improve pharmacokinetics and biodistribution of chemotherapy drugs, as well as to reduce related systemic toxicities. While these nanosized delivery systems inherently benefit from passive targeting through the enhanced permeation and retention effect leading to increased accumulation in the tumor, additional active targeting can be achieved through surface modification of micelles with targeting groups specific for overexpressed receptors of tumor cells. In this project, nontoxic, biodegradable, and modularly tunable micellar delivery systems were generated using two types of dendron–polymer conjugates. Either an AB type dendron–polymer construct with 2K PEG or an ABA type dendron–polymer–dendron conjugate with 6K PEG based middle block was used as primary construct; along with an AB type dendron–polymer containing a cRGDfK targeting group to actively target cancer cells overexpressing α_υβ₃/α_υβ₅ integrins. A set of micelles encapsulating docetaxel, a widely employed chemotherapy drug, were prepared with varying feed ratios of primary construct and targeting group containing secondary construct. Critical micelle concentrations of all micellar systems were in the range of 10^–6 M. DLS measurements indicated hydrodynamic size distributions varying between 170 to 200 nm. An increase in docetaxel release at acidic pH was observed for all micelles. Enhanced cellular internalization of Nile red doped micelles by MDA-MB-231 human breast cancer cells suggested that the most efficient uptake was observed with targeted micelles. <i>In vitro</i> cytotoxicity experiments on MDA-MB-231 breast cancer and A549 lung carcinoma cell lines showed improved toxicity for RGD containing micelles. For A549 cell line EC₅₀ values of drug loaded micellar sets were in the range of 10^–9 M whereas EC₅₀ value of free docetaxel was around 10^–10 M. For MDA-MB-231 cell line EC₅₀ value of free docetaxel was 6 × 10^–8 M similar to EC₅₀ of nontargeted AB type docetaxel doped micellar constructs whereas the EC₅₀ value of its targeted counterpart decreased to 5.5 × 10^–9 M. Overall, in this comparative study, the targeting group containing micellar construct fabricated with a 2 kDa PEG based diblock dendron–polymer conjugate emerges as an attractive drug delivery vehicle due to the ease of synthesis, high stability of the micelles, and efficient targeting

Modular Fabrication of Polymer Brush Coated Magnetic Nanoparticles: Engineering the Interface for Targeted Cellular Imaging

Development of efficient and rapid protocols for diversification of functional magnetic nanoparticles (MNPs) would enable identification of promising candidates using high-throughput protocols for applications such as diagnostics and cure through early detection and localized delivery. Polymer brush coated magnetic nanoparticles find use in many such applications. A protocol that allows modular diversification of a pool of parent polymer coated nanoparticles will lead to a library of functional materials with improved uniformity. In the present study, polymer brush coated parent magnetic nanoparticles obtained using reversible addition–fragmentation chain transfer (RAFT) polymerization are modified to obtain nanoparticles with different “clickable” groups. In this design, trithiocarbonate group terminated polymer brushes are “grafted from” MNPs using a catechol group bearing initiator. A postpolymerization radical exchange reaction allows installation of “clickable” functional groups like azides and maleimides on the chain ends of the polymers. Thus, modified MNPs can be functionalized using alkyne-containing and thiol-containing moieties like peptides and dyes using the alkyne–azide cycloaddition and the thiol–ene conjugation, respectively. Using the approach outlined here, a cell surface receptor targeting cyclic peptide and a fluorescent dye are attached onto nanoparticle surface. This multifunctional construct allows selective recognition of cancer cells that overexpress integrin receptors. Furthermore, the approach outlined here is not limited to the installation of azide and maleimide functional groups but can be expanded to a variety of “clickable” groups to allow nanoparticle modification using a broad range of chemical conjugations

Functionalization of Reduced Graphene Oxide via Thiol–Maleimide “Click” Chemistry: Facile Fabrication of Targeted Drug Delivery Vehicles

Materials based on reduced graphene oxide (rGO) have shown to be amenable to noncovalent functionalization through hydrophobic interactions. The scaffold, however, does not provide sufficient covalent linkage given the low number of reactive carboxyl and alcohol groups typically available on the rGO. The integration of clickable groups, particularly the ones that can undergo efficient conjugation without any metal catalyst, would allow facile functionalization of these materials. This study reports on the noncovalent association of a maleimide-containing catechol (dopa-MAL) surface anchor onto the rGO. Thiol–maleimide chemistry allows thereafter the facile attachment of thiol-containing molecules under ambient metal-free conditions. Although the attachment of glutathione and 6-(ferrocenyl)­hexanethiol was used as model thiols, the attachment of a cancer cell targeting cyclic peptide, c­(RGDfC), opened the possibility of using the dopa-MAL-modified rGO as a targeted drug delivery system for doxorubicin (DOX). Although free DOX showed to be more effective at killing the human cervical cancer cells (HeLa) over human breast adenocarcinoma cancer cells (MDA-MB-231), the DOX-loaded rGO/dopa-MAL-c (RGDfC) nanostructure showed an opposite effect being notably more effective at targeting and killing the MDA-MB-231 cells. The effect is enhanced upon laser irradiation for 10 min at 2 W cm^–2. The facile fabrication and functionalization to readily obtain a functional material in a modular fashion make this clickable-rGO construct an attractive platform for various applications