Liposome-Cross-Linked Hybrid Hydrogels for Glutathione-Triggered Delivery of Multiple Cargo Molecules

Novel, liposome-cross-linked hybrid hydrogels cross-linked by the Michael-type addition of thiols with maleimides were prepared via the use of maleimide-functionalized liposome cross-linkers and thiolated polyethylene glycol (PEG) polymers. Gelation of the materials was confirmed by oscillatory rheology experiments. These hybrid hydrogels are rendered degradable upon exposure to thiol-containing molecules such as glutathione (GSH), via the incorporation of selected thioether succinimide cross-links between the PEG polymers and liposome nanoparticles. Dynamic light scattering (DLS) characterization confirmed that intact liposomes were released upon network degradation. Owing to the hierarchical structure of the network, multiple cargo molecules relevant for chemotherapies, namely doxorubicin (DOX) and cytochrome c, were encapsulated and simultaneously released from the hybrid hydrogels, with differential release profiles that were driven by degradation-mediated release and Fickian diffusion, respectively. This work introduces a facile approach for the development of advanced, hybrid drug delivery vehicles that exhibit novel chemical degradation

Noncovalent Modulation of the Inverse Temperature Transition and Self-Assembly of Elastin‑<i>b</i>‑Collagen-like Peptide Bioconjugates

Stimuli-responsive nanostructures produced with peptide domains from the extracellular matrix offer great opportunities for imaging and drug delivery. Although the individual utility of elastin-like (poly)­peptides and collagen-like peptides in such applications has been demonstrated, the synergistic advantages of combining these motifs in short peptide conjugates have surprisingly not been reported. Here, we introduce the conjugation of a thermoresponsive elastin-like peptide (ELP) with a triple-helix-forming collagen-like peptide (CLP) to yield ELP–CLP conjugates that show a remarkable reduction in the inverse transition temperature of the ELP domain upon formation of the CLP triple helix. The lower transition temperature of the conjugate enables the facile formation of well-defined vesicles at physiological temperature and the unexpected resolubilization of the vesicles at <i>elevated</i> temperatures upon unfolding of the CLP domain. Given the demonstrated ability of CLPs to modify collagens, our results not only provide a simple and versatile avenue for controlling the inverse transition behavior of ELPs, but also suggest future opportunities for these thermoresponsive nanostructures in biologically relevant environments

Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure

Hydrogels derived from resilin-like polypeptides (RLPs) have shown outstanding mechanical resilience and cytocompatibility; expanding the versatility of RLP-based materials via conjugation with other polypeptides and polymers would offer great promise in the design of a range of materials. Here, we present an investigation of the biochemical and mechanical properties of hybrid hydrogels composed of a recombinant RLP and a multiarm PEG macromer. These hybrid hydrogels can be rapidly cross-linked through a Michael-type addition reaction between the thiols of cysteine residues on the RLP and vinyl sulfone groups on the multiarm PEG. Oscillatory rheology and tensile testing confirmed the formation of elastomeric hydrogels with mechanical resilience comparable to aortic elastin; hydrogel stiffness was easily modulated through the cross-linking ratio. Macromolecular phase separation of the RLP-PEG hydrogels offers the unique advantage of imparting a heterogeneous microstructure, which can be used to localize cells, through simple mixing and cross-linking. Assessment of degradation of the RLP by matrix metalloproteinases (MMPs) illustrated the specific proteolysis of the polypeptide in both its soluble form and when cross-linked into hydrogels. Finally, the successful encapsulation and viable three-dimensional culture of human mesenchymal stem cells (hMSCs) demonstrated the cytocompatibility of the RLP-PEG gels. Overall, the cytocompatibility, elastomeric mechanical properties, microheterogeneity, and degradability of the RLP-PEG hybrid hydrogels offer a suite of promising properties for the development of cell-instructive, structured tissue engineering scaffolds

Measuring the Modulus and Reverse Percolation Transition of a Degrading Hydrogel

In light of the growing importance to understand and control the physical cues presented to cells by artificial scaffolds, direct, temporally resolved measurements of the gel modulus are needed. We demonstrate that an interpolation of macro- and microrheology measurements provides a complete history of a hydrogel modulus during degradation through the reverse percolation transition. The latter is identified by microrheology, which captures the critical scaling behavior of reverse percolation, a transition of key importance in controlling cell migration, implant degradation, and tissue regeneration

Aqueous Liquid–Liquid Phase Separation of Resilin-Like Polypeptide/Polyethylene Glycol Solutions for the Formation of Microstructured Hydrogels

Multiple approaches to generate microstructured hydrogels have emerged in order to control microscale properties for applications ranging from mechanical reinforcement to regenerative medicine. Here, we report new heterogeneous hybrid hydrogels comprising emerging resilin-like polypeptides (RLPs); the hydrogels can be engineered with controlled microstructure and distinct micromechanical properties via the liquid–liquid phase separation (LLPS) of aqueous solutions of the RLPs and poly­(ethylene glycol) (PEG). The microstructure in the hydrogels was captured by cross-linking a phase-separated RLP and PEG solution via a Mannich-type reaction with the cross-linker tris­(hydroxymethyl phosphine) (THP). Phase diagrams of the RLP/PEG system were generated in order to define solution parameters that would yield micron-scale domains in the hydrogels with diameters on the order of 20–90 μm; the production of RLP- and PEG-rich domains with these dimensions was confirmed via confocal microscopy. The hydrogel mechanical properties were assessed via oscillatory rheology and atomic force microscopy (AFM), with the hydrogels exhibiting a moderate bulk shear storage modulus (ca. 600 Pa) and micromechanical properties of the domains (Young’s modulus ca. 13 kPa) that were distinct from those of the matrix (ca. 6 kPa). These results demonstrate that tuning the parameters of the aqueous–aqueous phase-separated RLP/PEG solutions provides a simple, straightforward methodology for fabricating microstructured protein-containing hydrogels, without extensive material processing or purification. Given the unusual mechanical properties of the resilins, these methods potentially could be useful for engineering the micromechanical properties and cellular behavior in phase-separated protein–polymer hydrogels

Tuning the Properties of Elastin Mimetic Hybrid Copolymers via a Modular Polymerization Method

We have synthesized elastin mimetic hybrid polymers (EMHPs) via the step-growth polymerization of azide-functionalized poly­(ethylene glycol) (PEG) and alkyne-terminated peptide (AKAAAKA)₂ (AK2) that is abundant in the cross-linking domains of the natural elastin. The modular nature of our synthesis allows facile adjustment of the peptide sequence to modulate the structural and biological properties of EMHPs. Therefore, EMHPs containing cell-binding domains (CBDs) were constructed from α,ω-azido-PEG and two types of alkyne-terminated AK2 peptides with sequences of DGRGX­(AKAAAKA)₂X (AK2-CBD1) and X­(AKAAAKA)₂XGGRGDSPG (AK2-CBD2, X = propargylglycine) via a step-growth, click coupling reaction. The resultant hybrid copolymers contain an estimated five to seven repeats of PEG and AK2 peptides. The secondary structure of EMHPs is sensitive to the specific sequence of the peptidic building blocks, with CBD-containing EMHPs exhibiting a significant enhancement in the α-helical content as compared with the peptide alone. Elastomeric hydrogels formed by covalent cross-linking of the EMHPs had a compressive modulus of 1.06 ± 0.1 MPa. Neonatal human dermal fibroblasts (NHDFs) were able to adhere to the hydrogels within 1 h and to spread and develop F-actin filaments 24 h postseeding. NHDF proliferation was only observed on hydrogels containing RGDSP domains, demonstrating the importance of integrin engagement for cell growth and the potential use of these EMHPs as tissue engineering scaffolds. These cell-instructive, hybrid polymers are promising candidates as elastomeric scaffolds for tissue engineering

Sequence and Conformational Analysis of Peptide–Polymer Bioconjugates by Multidimensional Mass Spectrometry

The sequence and helical content of two alanine-rich peptides (AQK18 and GpAQK18, Gp: l-propargylglycine) and their conjugates with poly­(ethylene glycol) (PEG) have been investigated by multidimensional mass spectrometry (MS), encompassing electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) interfaced with tandem mass spectrometry (MS²) fragmentation and shape-sensitive separation via ion mobility mass spectrometry (IM-MS). The composition, sequence, and molecular weight distribution of the peptides and bioconjugates were identified by MS and MS² experiments, which also confirmed the attachment of PEG at the C-terminus of the peptides. ESI coupled with IM-MS revealed the existence of random coil and α-helical conformers for the peptides in the gas phase. More importantly, the proportion of the helical conformation increased substantially after PEG attachment, suggesting that conjugation adds stability to this conformer. The conformational assemblies detected in the gas phase were largely formed in solution, as corroborated by independent circular dichroism (CD) experiments. The collision cross sections (rotationally averaged forward moving areas) of the random coil and helical conformers of the peptides and their PEG conjugates were simulated for comparison with the experimental values deduced by IM-MS in order to confirm the identity of the observed architectures and understand the stabilizing effect of the polymer chain. C-terminal PEGylation is shown to increase the positive charge density and to solvate intramolecular positive charges at the conjugation site, thereby enhancing the stability of α-helices, preserving their conformation and increasing helical propensity

Genetically Fused Resilin-like Polypeptide–Coiled Coil Bundlemer Conjugates Exhibit Tunable Multistimuli-Responsiveness and Undergo Nanofibrillar Assembly

Peptide-based materials are diverse candidates for self-assembly into modularly designed and stimuli-responsive nanostructures with precisely tunable compositions. Here, we genetically fused computationally designed coiled coil-forming peptides to the N- and C-termini of compositionally distinct multistimuli-responsive resilin-like polypeptides (RLPs) of various lengths. The successful expression of these hybrid polypeptides in bacterial hosts was confirmed through techniques such as gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism spectroscopy and ultraviolet–visible turbidimetry demonstrated that despite the fusion of disparate structural and responsive units, the coiled coils remained stable in the hybrid polypeptides, and the sequence-encoded differences in thermoresponsive phase separation of the RLPs were preserved. Cryogenic transmission electron microscopy and coarse-grained modeling showed that after thermal annealing in solution, the hybrid polypeptides adopted a closed loop conformation and assembled into nanofibrils capable of further hierarchically organizing into cluster structures and ribbon-like structures mediated by the self-association tendency of the RLPs

Thermoresponsive Elastin‑<i>b</i>‑Collagen-Like Peptide Bioconjugate Nanovesicles for Targeted Drug Delivery to Collagen-Containing Matrices

Over the past few decades, (poly)­peptide block copolymers have been widely employed in generating well-defined nanostructures as vehicles for targeted drug delivery applications. We previously reported the assembly of thermoresponsive nanoscale vesicles from an elastin-<i>b</i>-collagen-like peptide (ELP-CLP). The vesicles were observed to dissociate at elevated temperatures, despite the LCST-like behavior of the tethered ELP domain, which is suggested to be triggered by the unfolding of the CLP domain. Here, the potential of using the vesicles as drug delivery vehicles for targeting collagen-containing matrices is evaluated. The sustained release of an encapsulated model drug was achieved over a period of 3 weeks, following which complete release could be triggered via heating. The ELP-CLP vesicles show strong retention on a collagen substrate, presumably through collagen triple helix interactions. Cell viability and proliferation studies using fibroblasts and chondrocytes suggest that the vesicles are highly cytocompatible. Additionally, essentially no activation of a macrophage-like cell line is observed, suggesting that the vesicles do not initiate an inflammatory response. Endowed with thermally controlled delivery, the ability to bind collagen, and excellent cytocompatibility, these ELP-CLP nanovesicles are suggested to have significant potential in the controlled delivery of drugs to collagen-containing matrices and tissues