Differential Surface Adsorption Phenomena for Conventional and Novel Surfactants Correlates with Changes in Interfacial mAb Stabilization.

Protein adsorption on surfaces can result in loss of drug product stability and efficacy during the production, storage, and administration of protein-based therapeutics. Surface-active agents (excipients) are typically added in protein formulations to prevent undesired interactions of proteins on surfaces and protein particle formation/aggregation in solution. The objective of this work is to understand the molecular-level competitive adsorption mechanism between the monoclonal antibody (mAb) and a commercially used excipient, polysorbate 80 (PS80), and a novel excipient, N-myristoyl phenylalanine-N-polyetheramine diamide (FM1000). The relative rate of adsorption of PS80 and FM1000 was studied by pendant bubble tensiometry. We find that FM1000 saturates the interface faster than PS80. Additionally, the surface-adsorbed amounts from X-ray reflectivity (XRR) measurements show that FM1000 blocks a larger percentage of interfacial area than PS80, indicating that a lower bulk FM1000 surface concentration is sufficient to prevent protein adsorption onto the air/water interface. XRR models reveal that with an increase in mAb concentration (0.5-2.5 mg/mL: IV based formulations), an increased amount of PS80 concentration (below critical micelle concentration, CMC) is required, whereas a fixed value of FM1000 concentration (above its relatively lower CMC) is sufficient to inhibit mAb adsorption, preventing mAb from co-existing with surfactants on the surface layer. With this observation, we show that the CMC of the surfactant is not the critical factor to indicate its ability to inhibit protein adsorption, especially for chemically different surfactants, PS80 and FM1000. Additionally, interface-induced aggregation studies indicate that at minimum surfactant concentration levels in protein formulations, fewer protein particles form in the presence of FM1000. Our results provide a mechanistic link between the adsorption of mAbs at the air/water interface and the aggregation induced by agitation in the presence of surfactants.

Design Strategies to Tune the Structural and Mechanical Properties of Synthetic Collagen Hydrogels.

As an important component of biomaterials, collagen provides three-dimensional scaffolds and biological cues for cell adhesion and proliferation in tissue engineering. Recombinant collagen-like proteins, which were initially discovered in Streptococcus pyogenes and produced in heterologous hosts, have been chemically and genetically engineered for biomaterial applications. However, existing collagen-like proteins do not form gels, limiting their utility as biomaterials. Here, we present a series of rationally designed collagen-like proteins composed of a trimerization domain, triple-helical domains with various lengths, and a pair of heterotrimeric coiled-coil sequences attached to the N- and C-termini as adhesive ends. These designed proteins fold into triple helices and form self-supporting gels. As the triple-helical domains are lengthened, the gels become less stiff, pore sizes increase, and structural anisotropy decreases. Moreover, cell-culture assay confirms that the designed proteins are noncytotoxic. This study provides a design strategy for collagen-based biomaterials. The sequence variations reveal a relationship between the protein primary structure and material properties, where variations in the cross-linking density and association energies define the gelation of the protein network.

Thermoresponsive Protein-Engineered Coiled-Coil Hydrogel for Sustained Small Molecule Release.

Thermoresponsive hydrogels are used for an array of biomedical applications. Lower critical solution temperature-type hydrogels have been observed in nature and extensively studied in comparison to upper critical solution temperature (UCST)-type hydrogels. Of the limited protein-based UCST-type hydrogels reported, none have been composed of a single coiled-coil domain. Here, we describe a biosynthesized homopentameric coiled-coil protein capable of demonstrating a UCST. Microscopy and structural analysis reveal that the hydrogel is stabilized by molecular entanglement of protein nanofibers, creating a porous matrix capable of binding the small hydrophobic molecule, curcumin. Curcumin binding increases the α-helical structure, fiber entanglement, mechanical integrity, and thermostability, resulting in sustained drug release at physiological temperature. This work provides the first example of a thermoresponsive hydrogel comprised of a single coiled-coil protein domain that can be used as a vehicle for sustained release and, by demonstrating UCST-type behavior, shows promise in forging a relationship between coiled-coil protein-phase behavior and that of synthetic polymer systems.

'Reverse' Hofmeister effects on the sol-gel transition rates for an α-helical peptide-PEG bioconjugate.

We examine the dynamics of the sol-gel transition for end-functionalized linear- and 4-arm-peptides bioconjugated to poly-ethylene glycol (PEG) in aqueous environments with increasingly chaotropic (Cl- < Br- < I-) anions. A 23-amino acid peptide sequence is rationally designed to self-assemble upon folding into the ordered α-helical conformation due to the hydrophobic effect. We use Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR) to quantify the ensemble average reversible secondary structure transitions as a function of electrolyte concentration and specific ion effects along the Hofmeister series. Subsequently, microrheology is used to quantify the kinetics of the gelation process, as it relates to folding and specific ion interactions. Our key findings were non-intuitive. We observe the faster evolution of the gel transitions in systems with more chaotropic anions. For our peptides in aqueous solution, "water-structuring" ions yield faster assembly behavior with a viscoelastic exponent, n, closer to unity representing self-assemblies that are Rouse-like. In contrast, ions that are "water-breaking" resulted in smaller viscoelastic exponents where self-assembly dynamics result in a viscoelastic exponent that suggests polymer entanglements.

RGD-Targeted Liposome Binding and Uptake on Breast Cancer Cells Is Dependent on Elastin Linker Secondary Structure.

The linker between the targeting moiety and the nanoparticle is often overlooked when engineering targeted drug delivery vehicles. We hypothesized that pH-triggered conformational changes of an elastin-like peptide (ELP) linker, with repeating VPGVG sequences, could alter the binding affinity of the well-established targeting moiety arginine-glycine-aspartic acid (RGD), which is known to enhance the delivery of nanoparticles to tumor cells via integrin overexpression. The pH change from blood (pH 7.4) to the tumor environment (pH 6) was used to elicit a conformational change in the ELP linker, as described by circular dichroism. Atomic force microscopy confirmed that RGD-ELP resulted in stronger adhesion to both MDA-MB-231 and HCC1806 breast cancer cells at pH 6 relative to pH 7.4. No change in adhesion force was measured as a function of pH for the non-neoplastic MCF-10A cell line and the nontargeting GDR-ELP peptide. This translated to significant binding and uptake of RGD-ELP modified liposomes at pH 6.0 relative to pH 7.4. These results indicate that the pH-triggered conformational structure of the ELP linker shifts RGD-mediated cancer cell targeting from non-active (pH 7.4) to active (pH 6). The reversible shift in ELP secondary structure may be used to engineer targeted drug delivery vehicles with tunable uptake.

Structure-Dependent Water Responsiveness of Protein Block Copolymers.

Biological water-responsive (WR) materials are abundant in nature, and they are used as mechanical actuators for seed dispersal by many plants such as wheat awns and pinecones. WR biomaterials are of interest for applications as high-energy actuators, which can be useful in soft robotics or for capturing energy from natural water evaporation. Recent work on WR silk proteins has shown that ß-sheet nanocrystalline domains with high stiffness correlate with the high WR actuation energy density, but the fundamental mechanisms to drive water responsiveness in proteins remain poorly understood. Here, we design, synthesize, and study protein block copolymers consisting of two α-helical domains derived from cartilage oligomeric matrix protein coiled-coil (C) flanking an elastin-like peptide domain (E), namely, CEC. We use these protein materials to create WR actuators with energy densities that outperform mammalian muscle. To elucidate the effect of structure on WR actuation, CEC was compared to a variant, CECL44A, in which a point mutation disrupts the α-helical structure of the C domain. Surprisingly, CECL44A outperformed CEC, showing higher energy density and less susceptibility to degradation after repeated cycling. We show that CECL44A exhibits a higher degree of intermolecular interactions and is stiffer than CEC at high relative humidity (RH), allowing for less energy dissipation during water responsiveness. These results suggest that strong intermolecular interactions and the resulting, relatively steady protein structure are important for water responsiveness.

Compositional control of higher order assembly using synthetic collagen peptides.

We present the case of a two-component collagen peptide hydrogel that self-assembles through noncovalent electrostatic interactions. Natural collagen materials, such as those of connective tissue or the basement membrane, assemble in a hierarchic fashion. Similarly, the synthetic peptides presented here proceed from monomer to trimer to fiber and, finally, to a hydrogel. By varying stoichiometry and concentration, we are able to dissect the stages of higher order assembly. Insight gained from this study will improve the molecular design of biomimetic materials.

Modulating supramolecular assemblies and mechanical properties of engineered protein materials by fluorinated amino acids.

Here we describe the biosynthesis and characterization of fluorinated protein block polymers comprised of the two self-assembling domains (SADs): elastin (E) and the coiled-coil region of cartilage oligomeric matrix proteins (C). Fluorination is achieved by residue-specific incorporation of p-fluorophenylalanine (pFF) to create pFF-EC, pFF-CE, and pFF-ECE. Global fluorination results in downstream effects on the temperature-dependent secondary structure, supramolecular assembly, and bulk mechanical properties. The impact of fluorination on material properties also differs depending on the orientation of the block configurations as well as the number of domains in the fusion. These studies suggest that integration of fluorinated amino acids within protein materials can be employed to tune the material properties, especially mechanical integrity.

Artificial protein block polymer libraries bearing two SADs: effects of elastin domain repeats.

We have generated protein block polymer E(n)C and CE(n) libraries composed of two different self-assembling domains (SADs) derived from elastin (E) and the cartilage oligomeric matrix protein coiled-coil (C). As the E domain is shortened, the polymers exhibit an increase in inverse transition temperature (T(t)); however, the range of temperature change differs dramatically between the E(n)C and CE(n) library. Whereas all polymers assemble into nanoparticles, the bulk mechanical properties of the E(n)C are very different from CE(n). The E(n)C members demonstrate viscolelastic behavior under ambient conditions and assemble into elastic soft gels above their T(t) values. By contrast, the CE(n) members are predominantly viscous at all temperatures. All library members demonstrate binding to curcumin. The differential thermoresponsive behaviors of the E(n)C and CE(n) libraries in addition to their small molecule recognition abilities make them suitable for potential use in tissue engineering and drug delivery.

Bottom-up design of biomimetic assemblies.

Nature has evolved the ability to assemble a variety of molecules into functional architectures that can specifically bind cellular ligands. Mimicking this strategy requires the design of a set of multifaceted molecules, where elements that direct assembly were conjugated to biologically specific components. The development of functional molecular building-blocks that assemble to form compartments for therapeutics addresses the desire to have controllable morphologies that interact with biological interfaces at nanometer length scales. The practical application of such 'bottom-up' assemblies requires the ability to predict the type of aggregated structure and to synthesize molecules in a highly controlled fashion. This bottom-up approach results in a molecular platform that mimics biological systems with potential for encapsulating and delivering drug molecules.

Supramolecular assembly and small molecule recognition by genetically engineered protein block polymers composed of two SADs.

Genetically engineered protein block polymers are an important class of biomaterials that have gained significant attention in recent years due to their potential applications in biotechnology, electronics and medicine. The majority of the protein materials have been composed of at least a single self-assembling domain (SAD), enabling the formation of supramolecular structures. Recently, we developed block polymers consisting of two distinct SADs derived from an elastin-mimetic polypeptide (E) and the alpha-helical COMPcc (C). These protein polymers, synthesized as the block sequences--EC, CE, and ECE--were assessed for overall conformation and macroscopic thermoresponsive behavior. Here, we investigate the supramolecular assembly as well as the small molecule binding and release profile of these block polymers. Our results demonstrate that the protein polymers assemble into particles as well as fully or partially networked structures in a concentration dependent manner that is distinct from the individual E and C homopolymers and the E+C non-covalent mixture. In contrast to synthetic block polymers, the structured assembly, binding and release abilities are highly dependent on the composition and orientation of the blocks. These results reveal the promise for these block polymers for therapeutic delivery and biomedical scaffolds.

Structure and function of integral membrane protein domains resolved by peptide-amphiphiles: application to phospholamban.

We have used synthetic lipidated peptides ("peptide-amphiphiles") to study the structure and function of isolated domains of integral transmembrane proteins. We used 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis to prepare full-length phospholamban (PLB(1-52)) and its cytoplasmic (PLB(1-25)K: phospholamban residues 1-25 plus a C-terminal lysine), and transmembrane (PLB(26-52)) domains, and a 38-residue model alpha-helical sequence as a control. We created peptide-amphiphiles by linking the C-terminus of either the isolated cytoplasmic domain or the model peptide to a membrane-anchoring, lipid-like hydrocarbon tail. Circular dichroism measurements showed that the model peptide-amphiphile, either in aqueous suspension or in lipid bilayers, had a higher degree of alpha-helical secondary structure than the unlipidated model peptide. We hypothesized that the peptide-amphiphile system would allow us to study the function and structure of the PLB(1-25)K cytoplasmic domain in a native-like configuration. We compared the function (inhibition of the Ca-ATPase in reconstituted membranes) and structure (via CD) of the PLB(1-25) amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB(1-25)K has no effect on Ca-ATPase (calcium pump) activity, even when tethered to the membrane in a manner mimicking its native configuration, and that the transmembrane domain of PLB is sufficient for inhibition of the Ca-ATPase.