Post-Assembly Derivatization of Electrospun Nanofibers via Strain-Promoted Azide Alkyne Cycloaddition

A primary amine-derivatized 4-dibenzocyclooctynol (DIBO) was used to initiate the ring-opening polymerization of poly­(γ-benzyl-l-glutamate) (DIBO-PBLG). This initiator yields well-defined PBLG polymers functionalized with DIBO at the chain termini. The DIBO end group further survives an electrospinning process that yields nanofibers that were then derivatized post-assembly with azide-functionalized gold nanoparticles. The availability of DIBO on the surface of the fibers is substantiated by fluorescence, SEM, and TEM measurements. Post-assembly functionalization of nanofiber constructs with bioactive groups can be facilitated easily using this process

Cascading “Triclick” Functionalization of Poly(caprolactone) Thin Films Quantified via a Quartz Crystal Microbalance

A series of mono- and multifunctionalized degradable polyesters bearing various “clickable” groups, including ketone, alkyne, azide, and methyl acrylate (MA) are reported. Using this approach, we demonstrate a cascade approach to immobilize and quantitate three separate bioactive groups onto poly­(caprolactone) (PCL) thin films. The materials are based on tunable copolymer compositions of ε-caprolactone and 2-oxepane-1,5-dione. A quartz crystal microbalance (QCM) was used to quantify the rate and extent of surface conjugation between RGD peptide and polymer thin films using “click” chemistry methods. The results show that alkyne-functionalized polymers have the highest conversion efficiency, followed by MA and azide polymers, while polymer films possessing keto groups are less amenable to surface functionalization. The successful conjugation was further confirmed by static contact angle measurements, with a smaller contact angle correlating directly with lower levels of surface peptide conjugation. QCM results quantify the sequential immobilization of peptides on the PCL thin films and indicate that Michael addition must occur first, followed by azide–alkyne Huisgen cycloadditions

2‑Hydroxyethylcellulose and Amphiphilic Block Polymer Conjugates Form Mechanically Tunable and Nonswellable Hydrogels

Herein, we report a family of mechanically tunable, nonswellable hydrogels that are based on a 2-hydroxyethylcellulose (HEC) scaffold grafted with amphiphilic diblock copolymers. Poly­[(oligo­(ethylene glycol)­methyl ether methacrylate]-<i>b</i>-poly­(methyl methacrylate) (POEGMA-<i>b</i>-PMMA) diblock copolymers of different compositions were created via RAFT polymerization using an alkyne terminated macro chain transfer agent (CTA). 2-Hydroxyethylcellulose (HEC) was modified with azide groups and the diblock copolymers were attached to the backbone via the copper-catalyzed click reaction to yield HEC-<i>g</i>-(POEGMA-<i>b</i>-PMMA) graft terpolymers. The resulting conjugates were soluble in DMF and able to form hydrogels upon simple solvent exchange in water. By increasing the concentration of the conjugates in DMF, the storage moduli of the hydrogels increased and the pore size in the gel decreased. After hydrogel formation, the structures were also found to be nonswellable (no macroscopic volume change upon incubation in water), which is an important feature for retaining size and mechanical integrity of the gels over time. Moreover, these materials were able to be electrospun into fibers that, upon hydration, formed fibrous hydrogel structures. The nonswellable and tunable mechanical properties of these materials imply great potential for a variety of applications such as personal care, active delivery, and tissue engineering

Postelectrospinning “Click” Modification of Degradable Amino Acid-Based Poly(ester urea) Nanofibers

Amino acid-based poly­(ester urea)­s (PEU) are emerging as a new class of degradable polymers that have shown promise in regenerative medicine applications. Herein, we report the synthesis of PEUs carrying pendent “clickable” groups on modified tyrosine amino acids. The pendent species include alkyne, azide, alkene, tyrosine–phenol, and ketone groups. PEUs with <i>M</i>_w exceeding to 100K Da were obtained via interfacial polycondensation methods, and the concentration of pendent groups was varied using a copolymerization strategy. The incorporation of derivatizable functionalities is demonstrated using ¹H NMR and UV–vis spectroscopy methods. Electrospinning was used to fabricate PEU nanofibers with a diameters ranging from 350 to 500 nm. The nanofiber matricies possess mechanical strengths suitable for tissue engineering (Young’s modulus: 300 ± 45 MPa; tensile stress: 8.5 ± 1.2 MPa). A series of bioactive peptides and fluorescent molecules were conjugated to the surface of the nanofibers following electrospinning using bio-orthogonal reactions in aqueous media. The ability to derivatize PEUs with biological molecules using translationally relevant chemical methods will significantly expand their use <i>in vitro</i> and <i>in vivo</i>

Enhanced Schwann Cell Attachment and Alignment Using One-Pot “Dual Click” GRGDS and YIGSR Derivatized Nanofibers

Using metal-free click chemistry and oxime condensation methodologies, GRGDS and YIGSR peptides were coupled to random and aligned degradable nanofiber networks postelectrospinning in a one-pot reaction. The bound peptides are bioactive, as demonstrated by Schwann cell attachment and proliferation, and the inclusion of YIGSR with GRGDS alters the expression of the receptor for YIGSR. Additionally, aligned nanofibers act as a potential guidance cue by increasing the aspect ratio and aligning the actin filaments, which suggest that peptide-functionalized scaffolds would be useful to direct SCs for peripheral nerve regeneration

Concentration-Dependent <i>h</i>MSC Differentiation on Orthogonal Concentration Gradients of GRGDS and BMP‑2 Peptides

Self-assembled monolayer substrates containing tethered orthogonal concentration profiles of GRGDS (glycine/arginine/glycine/aspartic acid/serine) and BMP-2 (bone morphogenetic protein) peptides are shown to accelerate or decelerate, depending on the concentrations, the proliferation and osteoblastic differentiation of human mesenchymal stem cell (<i>h</i>MSC) populations in vitro without the use of osteogenic additives in culture medium. Concurrently, the single peptide gradient controls (GRGDS or BMP-2 only) induce significantly different proliferation and differentiation behavior from the orthogonal substrates. Bone sialoprotein (BSP) and Runt-related transcription factor 2 (Runx2) PCR data acquired from <i>h</i>MSC populations isolated by laser capture microdissection correspond spatially and temporally to protein marker data obtained from immunofluorescent imaging tracking of the differentiation process. Although genomic and protein data at high concentrations area GRGDS (71–83 pmol/cm²):BMP-2 (25 pmol/cm²) reveal an implicit acceleration on the <i>h</i>MSC differentiation timeline relative to the individual peptide concentrations, most of the GRGDS and BMP-2 combinations displayed significant antagonistic behavior during the <i>h</i>MSC differentiation. These data highlight the utility of the orthogonal gradient approach to aid in identifying optimal concentration ranges of translationally relevant peptides and growth factors for targeting cell lineage commitment

Post-Electrospinning “Triclick” Functionalization of Degradable Polymer Nanofibers

4-Dibenzocyclooctynol (DIBO) was used as an initiator for the ring-opening copolymerization of ε-caprolactone and 1,4,8-trioxaspiro[4.6]-9-undecanone (TOSUO) resulting in a series of DIBO end-functionalized copolymers. Following deprotection of the ketone group, the polymers were derivatized with aminooxyl-containing compounds by oxime ligation. Mixtures of keto- and alkyne-derivatized polymers were co-electrospun into well-defined nanofibers containing three separate chemical handles. Strain-promoted azide alkyne cycloaddition (SPAAC), oxime ligation, and copper-catalyzed azide alkyne cycloaddition (CuAAC) were used to sequentially functionalize the nanofibers first with fluorescent reporters and then separately with bioactive Gly-Arg-Gly-Asp-Ser (GRGDS), BMP-2 peptide, and dopamine. This translationally relevant approach facilitates the straightforward derivatization of diverse bioactive molecules that can be controllably tethered to the surface of nanofibers

Strain-Promoted Cross-Linking of PEG-Based Hydrogels via Copper-Free Cycloaddition

The synthesis of a 4-dibenzocyclooctynol (DIBO) functionalized poly­(ethylene glycol) (PEG) and fabrication of hydrogels via strain-promoted, metal-free, azide–alkyne cycloaddition is reported. The resulting hydrogel materials provide a versatile alternative to encapsulate cells that are sensitive to photochemical or chemical cross-linking mechanisms

Peptide-Functionalized Oxime Hydrogels with Tunable Mechanical Properties and Gelation Behavior

We demonstrate the formation of polyethylene glycol (PEG) based hydrogels via oxime ligation and the photoinitiated thiol–ene 3D patterning of peptides within the hydrogel matrix postgelation. The gelation process and final mechanical strength of the hydrogels can be tuned using pH and the catalyst concentration. The time scale to reach the gel point and complete gelation can be shortened from hours to seconds using both pH and aniline catalyst, which facilitates the tuning of the storage modulus from 0.3 to over 15 kPa. Azide- and alkene-functionalized hydrogels were also synthesized, and we have shown the post gelation “click”-type Huisgen 1,3 cycloaddition and thiolene-based radical reactions for spatially defined peptide incorporation. These materials are the initial demonstration for translationally relevant hydrogel materials that possess tunable mechanical regimes attractive to soft tissue engineering and possess atom neutral chemistries attractive for post gelation patterning in the presence or absence of cells

Sequential Triple “Click” Approach toward Polyhedral Oligomeric Silsesquioxane-Based Multiheaded and Multitailed Giant Surfactants

This letter reports a sequential triple “click” chemistry method for the precise synthesis of functional polyhedral oligomeric silsesquioxane (POSS)-based multiheaded and multitailed giant surfactants. A vinyl POSS-based heterobifunctional building block possessing two alkyne groups of distinct reactivity was used as a robust and powerful “clickable” precursor for ready access to a variety of POSS-based shape amphiphiles with complex architectures. The synthetic approach involves sequentially performed strain-promoted azide–alkyne cycloaddition (SPAAC), copper-catalyzed azide–alkyne cycloaddition (CuAAC), and thiol–ene “click” coupling (TECC). Specifically, the first SPAAC reaction was found to be highly selective with no complications from the vinyl groups and terminal alkynes in the precursor. The method expands the toolbox of sequential “click” approaches and broadens the scope of synthetically available giant surfactants for further study on structure–property relationships