“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

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

Redox-Responsive “Catch and Release” Cryogels: A Versatile Platform for Capture and Release of Proteins and Cells

Macroporous cryogels are attractive scaffolds for biomedical applications, such as biomolecular immobilization, diagnostic sensing, and tissue engineering. In this study, thiol-reactive redox-responsive cryogels with a porous structure are prepared using photopolymerization of a pyridyl disulfide poly(ethylene glycol) methacrylate (PDS-PEG-MA) monomer. Reactive cryogels are produced using PDS-PEG-MA and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers, along with a PEG-based cross-linker and photoinitiator. Functionalization of cryogels using a fluorescent dye via the disulfide-thiol exchange reactions is demonstrated, followed by release under reducing conditions. For ligand-mediated protein immobilization, first, thiol-containing biotin or mannose is conjugated onto the cryogels. Subsequently, fluorescent dye-labeled proteins streptavidin and concanavalin A (ConA) are immobilized via ligand-mediated conjugation. Furthermore, we demonstrate that the mannose-decorated cryogel could capture ConA selectively from a mixture of lectins. The efficiency of protein immobilization could be easily tuned by changing the ratio of the thiol-sensitive moiety in the scaffold. Finally, an integrin-binding cell adhesive peptide is attached to cryogels to achieve successful attachment, and the on-demand detachment of integrin-receptor-rich fibroblast cells is demonstrated. Redox-responsive cryogels can serve as potential scaffolds for a variety of biomedical applications because of their facile synthesis and modification

Combretastatin A‑4 Conjugated Antiangiogenic Micellar Drug Delivery Systems Using Dendron–Polymer Conjugates

Employment of polymeric nanomaterials in cancer therapeutics is actively pursued since they often enable drug administration with increased efficacy along with reduced toxic side effects. In this study, drug conjugated micellar constructs are fabricated using triblock dendron–linear polymer conjugates where a hydrophilic linear polyethylene glycol (PEG) chain is flanked by well-defined hydrophobic biodegradable polyester dendrons bearing an antiangiogenic drug, combretastatin-A4 (CA4). Variation in dendron generation is utilized to obtain a library of micellar constructs with varying sizes and drug loadings. In particular, a family of drug appended dendron–polymer conjugates based on polyester dendrons of generations ranging from G1 to G3 and 10 kDa linear PEG were obtained using [3 + 2] Huisgen type “click” chemistry. The final constructs benefit from PEG’s hydrophilicity and antibiofouling character, as well as biodegradable nature of the hydrophobic polyester dendrons. The hydrophobic–hydrophilic–hydrophobic character of these constructs leads to the formation of flower-like micelles in aqueous media. In addition to generation-dependent subnanomolar range critical micelle concentrations, the resulting micelles possess hydrodynamic diameters suitable for passive tumor targeting through enhanced permeability and retention (EPR) effect; thereby they are suitable candidates as controlled drug delivery agents. For all constructs, <i>in vitro</i> cytotoxicities were investigated and inhibitory effect of Comb-G3-PEG on tube formation was shown on human umbilical vein endothelial cells (HUVECs)

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