Surfactant Impact on Interfacial Protein Aggregation and Utilization of Surface Tension to Predict Surfactant Requirements for Biological Formulations.

Biological drug products are formulated with excipients to maintain stability over the shelf life of the product. Surfactants are added to the drug product to stabilize air-water interfaces known to induce protein aggregation. Early formulation development is focused on maintaining protein conformation and colloidal stability over the course of the drug product shelf life but rarely considers stability through dose preparation and administration. Specifically, intravenous (IV) bag preparation exposes the therapeutic protein to a different solution environment concurrently diluting the stabilizing excipients that had been added to the drug product formulation. Mixing in IV bags can generate dynamic changes in the air-water interfacial area known to cause protein aggregation if not sufficiently protected. Therefore, understanding the surfactant requirements for drug product end-to-end stability in early formulation development provides critical information for a right-first-time approach to drug product formulation and robust clinical preparation. The goal of these studies was to understand if interfacial properties of proteins could predict surfactant formulation requirements for end-to-end stability. Specifically, the interfacial properties of five proteins were measured in 0.9% saline and 5% dextrose. Furthermore, shaking studies were conducted to identify the minimum surfactant concentration required to prevent subvisible and visible particle formulation in each diluent. The impact of surfactant type and concentration on particle generation and size was explored. A mathematical model was generated to predict the minimum surfactant concentration required to prevent interface-driven aggregation in each diluent based on the change in surface pressure upon exposure of the protein to the interface. The model was tested under typical IV-preparation conditions with experimental output closely matching the model prediction. By employing this model and better understanding the role of surfactants in interfacial stability, drug product development can generate robust end-to-end large molecule formulations across shelf life, dose preparation, and administration.

Eye glances towards conflict-relevant cues: the roles of anticipatory competence and driver experience.

OBJECTIVE: This paper analyzes the effects of anticipatory competence and driver experience on glance patterns towards visual cues that indicate conflict situations. BACKGROUND: Prior research has shown that experienced drivers' visual scanning patterns differ from those of novices. Experienced drivers are less erratic and more systematic in their monitoring of the environment. We have also shown in an earlier study that driving experience improves anticipatory competence in that it leads to a higher number of timely proactive actions in conflict-scenarios (avoidance actions prior to, as opposed to in reaction to a conflict). This paper investigates glance patterns specifically to relevant visual cues in conflict scenarios to determine whether glance patterns of anticipatory drivers who exhibit proactive actions differ from those who do not. It also investigates whether experienced drivers pay more attention to these cues compared to novices. METHOD: We conducted a simulator experiment with 24 experienced and 24 novice drivers. As part of the experiment, all drivers completed three distinct traffic scenarios, each with a conflict situation. RESULTS: The results show that drivers who exhibited proactive actions had more frequent and longer glances towards conflict-relevant cues than those who did not exhibit any. Similarly, experienced drivers focused on these visual cues more often, and for longer durations compared to novices. Further, experienced drivers who exhibited proactive actions looked at the cues more often compared to experienced drivers who did not exhibit any; there was no significant difference for novice drivers. CONCLUSION: These findings speak to the role of situation-specific visual cues for anticipatory competence, and to the importance of driver experience to aid in the interpretation of these cues. Future research should seek to confirm our findings in a wider variety of driving scenarios.

Supporting anticipation in driving through attentional and interpretational in-vehicle displays.

OBJECTIVE: This paper evaluates two different types of in-vehicle interfaces to support anticipation in driving: one aids attention allocation and the other aids interpretation of traffic in addition to attention allocation. BACKGROUND: Anticipation is a competency that has been shown to facilitate safety and eco-driving through the efficient positioning of a vehicle for probable, upcoming changes in traffic. This competency has been shown to improve with driving experience. In an earlier simulator study, we showed that compared to novice drivers, experienced drivers exhibited a greater number of timely actions to avoid upcoming traffic conflicts. In this study, we seek to facilitate anticipation in general and for novice drivers in particular, who appear to lack the competency. We hypothesize that anticipation depends on two major steps and that it can be supported by aiding each: (1) conscious perception of relevant cues, and (2) effective processing of these cues to create a situational assessment as a basis for anticipation of future developments. METHOD: We conducted a simulator experiment with 24 experienced and 24 novice drivers to evaluate two interfaces that were designed to aid the two hypothesized steps of anticipation. The attentional interface was designed to direct attention toward the most relevant cue. The interpretational interface represented several cues, and in addition to directing attention also aimed to aid sense-making of these cues. RESULTS: The results confirmed our hypothesis that novice drivers' anticipation performance, as measured through timely actions to avoid upcoming traffic conflicts, would be improved with either interface type. However, results contradicted our expectation that novice drivers would obtain larger improvements with the interpretational interface. Experienced drivers performed better than novice drivers to begin with and did not show any statistically significant improvements with either interface. CONCLUSION: Both interfaces improved anticipation performance for novice drivers. Future research should evaluate the effectiveness of these interfaces in a wider variety of driving conditions, such as when the driver is multitasking.

Collagen-gelatin mixtures as wound model, and substrates for VEGF-mimetic peptide binding and endothelial cell activation.

In humans, high level of collagen remodeling is seen during normal physiological events such as bone renewal, as well as in pathological conditions, such as arthritis, tumor growth and other chronic wounds. Our lab recently discovered that collagen mimetic peptide (CMP) is able to hybridize with denatured collagens at these collagen remodeling sites with high affinity. Here, we show that the CMP's high binding affinity to denatured collagens can be utilized to deliver angiogenic signals to scaffolds composed of heat-denatured collagens (gelatins). We first demonstrate hybridization between denatured collagens and QKCMP, a CMP with pro-angiogenic QK domain. We show that high levels of QKCMP can be immobilized to a new artificial matrix containing both fibrous type I collagen and heat denatured collagen through triple helix hybridization, and that the QKCMP is able to stimulate early angiogenic response of endothelial cells (ECs). We also show that the QKCMP can bind to excised tissues from burn injuries in cutaneous mouse model, suggesting its potential for promoting neovascularization of burn wounds.

On-the-resin N-terminal modification of long synthetic peptides.

Here we present a highly efficient protocol for on-the-resin coupling of fluorescent dyes or other functional groups to the N-termini of synthetic peptides prior to cleavage and deprotection. The protocol avoids expensive preactivated dyes and instead employs carboxylated dyes activated by large amounts of coupling reagents. The protocol was used to label peptides with low reactivity such as long hydrophobic peptides and peptides with strong tendencies to form sterically shielding structures or aggregates in solution. In all cases, the yields far exceeded those from commercially available preactivated compounds.

Encoding Cell-Instructive Cues to PEG-Based Hydrogels via Triple Helical Peptide Assembly.

Effective synthetic tissue engineering scaffolds mimic the structure and composition of natural extracellular matrix (ECM) to promote optimal cellular adhesion, proliferation, and differentiation. Among many proteins of the ECM, collagen and fibronectin are known to play a key role in the scaffold's structural integrity as well as its ability to support cell adhesion. Here, we present photocrosslinked poly(ethylene glycol) diacrylate (PEGDA) hydrogels displaying collagen mimetic peptides (CMPs) that can be further conjugated to bioactive molecules via CMP-CMP triple helix association. Pre-formed PEGDA-CMP hydrogels can be encoded with varying concentration of cell-signaling CMP-RGD peptides similar to cell adhesive fibronectin decorating the collagen fibrous network by non-covalent binding. Furthermore, the triple helix mediated encoding allows facile generation of spatial gradients and patterns of cell-instructive cues across the cell scaffold that simulate distribution of insoluble factors in the natural ECM.

A cellular model for the investigation of Fuchs' endothelial corneal dystrophy.

Fuchs' endothelial corneal dystrophy is the most common corneal endotheliopathy, and a leading indication for corneal transplantation in the US. Relatively little is known about its underlying pathology. We created a cellular model of the disease focusing on collagen VIII alpha 2 (COL8A2), a collagen which is normally present in the cornea, but which is found in abnormal amounts and distribution in both early and late-onset forms of the disease. We performed cellular transfections using COL8A2 cDNAs including both wild-type and mutant alleles which are known to result in early-onset FECD. We used this cell model to explore the cellular production of wild-type and mutant monomeric and trimeric collagen VIII and measured production levels and patterns using Western blotting and immunofluorescence. We studied the thermal stability of the mutated collagen VIII helices using computer modeling, and further investigated these differences using collagen mimetic peptides. The Western blots demonstrated that similar amounts of wild-type and mutant collagen VIII monomers were produced in the cells. However, the levels of trimeric collagen peptide in the mutant-transfected cells were elevated. Intracellular accumulation of trimeric collagen VIII was confirmed on immunofluorescence studies. Both the computer model and the collagen mimetic peptides demonstrated that the L450W mutant was less thermally stable than either the Q455K or wild-type collagen VIII. Thus, although both mutant collagen VIII peptides were retained intracellularly, the biochemical reasons for the retention varied between genotypes. Collagen VIII mutations, which clinically result in Fuchs' dystrophy, are associated with abnormal cellular accumulation of collagen VIII. Different collagen VIII mutations may act via distinct biochemical mechanisms to produce the FECD phenotype.

Matrix-Bound VEGF Mimetic Peptides: Design and Endothelial Cell Activation in Collagen Scaffolds.

Long term survival and success of artificial tissue constructs depend greatly on vascularization. Endothelial cell (EC) differentiation and vasculature formation are dependent on spatio-temporal cues in the extracellular matrix that dynamically interact with cells, a process difficult to reproduce in artificial systems. Here we present a novel bifunctional peptide that mimics matrix-bound vascular endothelial growth factor (VEGF) and can be used to encode spatially controlled angiogenic signals in collagen scaffolds. The peptide is comprised of a collagen mimetic domain that was previously reported to bind to type I collagen by a unique hybridization mechanism, and a VEGF mimetic domain with pro-angiogenic activity. Circular dichroism and collagen binding studies confirm the triple helical structure and the collagen binding affinity of the collagen mimetic domain, and EC culture studies demonstrate the peptide's ability to induce endothelial cell morphogenesis and network formation as a matrix-bound factor in 2D and 3D collagen scaffolds. We also show spatial modification of collagen substrates with this peptide that allows localized EC activation and network formation. These results demonstrate that the peptide can be used to present spatially directed angiogenic cues in collagen scaffolds, which may be useful for engineering organized microvasculature.

PEG-based hydrogels with collagen mimetic peptide-mediated and tunable physical cross-links.

Mechanical properties of tissue scaffolds have major effects on the morphology and differentiation of cells. In contrast to two-dimensional substrates, local biochemical and mechanical properties of three-dimensional hydrogels are difficult to control due to the geometrical confinement. We designed synthetic 3D hydrogels featuring complexes of four-arm poly(ethylene glycol) (PEG) and collagen mimetic peptides (CMPs) that form hydrogels via physical cross-links mediated by thermally reversible triple helical assembly of CMPs. Here we present the fabrication of various PEG-CMP 3D hydrogels and their local mechanical properties determined by particle tracking microrheology. Results show that CMP mediated physical cross-links can be disrupted by altering the temperature of the gel or by adding free CMPs that compete for triple helix formation. This allowed modulation of both bulk and local stiffness as well as the creation of stiffness gradients within the PEG-CMP hydrogel, which demonstrates its potential as a novel scaffold for encoding physicochemical signals for tissue formation.