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.

Protease-Triggered, Integrin-Targeted Cellular Uptake of Recombinant Protein Micelles.

Targeting nanoparticles for drug delivery has great potential for improving efficacy and reducing side effects from systemic toxicity. New developments in the assembly of materials afford the opportunity to expose cryptic targeting domains in tissue-specific microenvironments in which certain proteases are expressed. Here, recombinant proteins are designed to combine the responsiveness to environmental proteases with specific targeting. Materials made recombinantly allow complete control over amino acid sequence, in which each molecule is identically functionalized. Previously, oleosin, a naturally occurring plant protein that acts as a surfactant, has been engineered to self-assemble into spherical micelles-a useful structure for drug delivery. To make oleosins that are locally activated to bind receptors, oleosin is genetically modified to incorporate the integrin-binding motif RGDS just behind a domain cleavable by thrombin. The resulting modified oleosin self-assembles into spherical micelles in aqueous environments, with the RGDS motif protected by the thrombin-cleavable domain. Upon the addition of thrombin, the RGDS is exposed and the binding of the spherical micelles to breast cancer cells is increased fourfold.

Interdisciplinary Rehabilitation Referrals in a Concussion Clinic Cohort: An Exploratory Analysis.

OBJECTIVE: To assess the frequency and spectrum of referrals to rehabilitation disciplines in a concussion clinic population and factors associated with need for referral. DESIGN: Retrospective study. SETTING: Concussion clinic within the Physical Medicine and Rehabilitation Department of an academic medical center. PARTICIPANTS: Patients receiving physiatric management for concussion care. MAIN OUTCOME MEASURES: Referral to physical therapy (PT), occupational therapy, speech therapy (ST), neuropsychology, or any referral (Any), and reasons for referral. INTERVENTIONS: Demographic and clinical variables were analyzed for possible association with referral to rehabilitation disciplines. These independent variables included mechanism of injury, referral source, age, gender, provider, days since injury, presenting Sports Concussion Assessment Tool 2 (SCAT2) symptom score, insurance type, clinical risk factors, whether the injury was work related and whether the patient had been hospitalized. RESULTS: Among 262 patients meeting inclusion criteria, the most commonly prescribed individual therapy was physical therapy (74 patients; 28%), followed by speech therapy (60 patients, 23%), neuropsychology (27 patients, 10.3%), and occupational therapy (19 patients, 7.2%). In all, 121 (46%) of patients were referred to one or more disciplines. The most common reasons for referral were cognitive strategies (54 patients, 21%), balance/vestibular therapy (50 patients,19%), and neck pain (32 patients, 12%). Per multivariate logistic regression analysis, covariates associated with PT: age, SCAT2 symptom score, gender, provider, and (inversely) cognitive/learning disorder; ST: time elapsed since injury, gender, and referral source of internal clinic; Any: SCAT2 symptom score. Referrals did not significantly vary by mechanism of injury (sports, fall, vehicular, etc), whether work-related, or whether the patient had been hospitalized. Insurance factors were significant for PT and Any on the univariate analysis but not logistic regression. CONCLUSIONS: Relatively little has been described about the typical rehabilitation requirements of individuals recovering from concussion. Although rest and guided return to usual activities have been emphasized as mainstays of management, a large number of patients in this concussion cohort were determined to require additional rehabilitation services to assist in recovery.

Superparamagnetic iron oxide nanoparticle micelles stabilized by recombinant oleosin for targeted magnetic resonance imaging.

Recombinant surfactants present a new platform for stabilizing and targeting nanoparticle imaging agents. Superparamagnetic iron oxide nanoparticle-loaded micelles for MRI contrast are stabilized by an engineered variant of the naturally occurring protein oleosin and targeted using a Her2/neu affibody-oleosin fusion. The recombinant oleosin platform allows simple targeting and the ability to easily swap the ligand for numerous targets.

Caging metal ions with visible light-responsive nanopolymersomes.

Polymersomes are bilayer vesicles that self-assemble from amphiphilic diblock copolymers, and provide an attractive system for the delivery of biological and nonbiological molecules due to their environmental compatibility, mechanical stability, synthetic tunability, large aqueous core, and hyperthick hydrophobic membrane. Herein, we report a nanoscale photoresponsive polymersome system featuring a meso-to-meso ethyne-bridged bis[(porphinato)zinc] (PZn2) fluorophore hydrophobic membrane solute and dextran in the aqueous core. Upon 488 nm irradiation in solution or in microinjected zebrafish embryos, the polymersomes underwent deformation, as monitored by a characteristic red-shifted PZn2 emission spectrum and confirmed by cryo-TEM. The versatility of this system was demonstrated through the encapsulation and photorelease of a fluorophore (FITC), as well as two different metal ions, Zn(2+) and Ca(2+).

Noncanonical self-assembly of highly asymmetric genetically encoded polypeptide amphiphiles into cylindrical micelles.

Elastin-like polypeptides (ELPs) are a class of biopolymers consisting of the pentameric repeat (VPGαG)n based on the sequence of mammalian tropoelastin that display a thermally induced soluble-to-insoluble phase transition in aqueous solution. We have discovered a remarkably simple approach to driving the spontaneous self-assembly of high molecular weight ELPs into nanostructures by genetically fusing a short 1.5 kDa (XGy)z assembly domain to one end of the ELP. Classical theories of self-assembly based on the geometric mass balance of hydrophilic and hydrophobic block copolymers suggest that these highly asymmetric polypeptides should form spherical micelles. Surprisingly, when sufficiently hydrophobic amino acids (X) are presented in a periodic sequence such as (FGG)8 or (YG)8, these highly asymmetric polypeptides self-assemble into cylindrical micelles whose length can be tuned by the sequence of the morphogenic tag. These nanostructures were characterized by light scattering, tunable resistive pulse sensing, fluorescence spectrophotometry, and thermal turbidimetry, as well as by cryogenic transmission electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS). These short assembly domains provide a facile strategy to control the size, shape, and stability of stimuli responsive polypeptide nanostructures.

Recombinant protein-stabilized monodisperse microbubbles with tunable size using a valve-based microfluidic device.

Microbubbles are used as contrast enhancing agents in ultrasound sonography and more recently have shown great potential as theranostic agents that enable both diagnostics and therapy. Conventional production methods lead to highly polydisperse microbubbles, which compromise the effectiveness of ultrasound imaging and therapy. Stabilizing microbubbles with surfactant molecules that can impart functionality and properties that are desirable for specific applications would enhance the utility of microbubbles. Here we generate monodisperse microbubbles with a large potential for functionalization by combining a microfluidic method and recombinant protein technology. Our microfluidic device uses an air-actuated membrane valve that enables production of monodisperse microbubbles with narrow size distribution. The size of microbubbles can be precisely tuned by dynamically changing the dimension of the channel using the valve. The microbubbles are stabilized by an amphiphilic protein, oleosin, which provides versatility in controlling the functionalization of microbubbles through recombinant biotechnology. We show that it is critical to control the composition of the stabilizing agents to enable formation of highly stable and monodisperse microbubbles that are echogenic under ultrasound insonation. Our protein-shelled microbubbles based on the combination of microfluidic generation and recombinant protein technology provide a promising platform for ultrasound-related applications.

Spherical micelles assembled from variants of recombinant oleosin.

An emerging field in biomaterials is the creation and engineering of protein surfactants made by recombinant biotechnology. Protein surfactants made by recombinant biotechnology allow for complete control of the molecular weight and chemical sequence of the surfactant. The proteins are monodisperse in molecular weight, and functionalization with bioactive amino acid sequences is straightforwardly achieved through genetic engineering. We modified the naturally occurring amphiphilic plant protein oleosin by truncating a large portion of its central hydrophobic block, creating a soluble triblock surfactant. Additional variants were constructed to eliminate secondary structure and create ionic surfactants. Variants of oleosin self-assembled into spherical micelles with a diameter of ∼21 nm at concentrations above the critical micelle concentration (cmc). We found that the cmc could be manipulated through changes in the protein backbone and was correlated with changes in the protein secondary structure. Micelle size and shape are characterized with dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). Micelles were functionalized with the integrin-binding domain, RGDS, leading to a 2.9-fold increase in uptake in Ovcar-5 cells after 12 h. Oleosin surfactants present a promising platform for micellar assembly because of the ability to precisely modify the protein backbone through molecular biology, allowing for the control over the cmc and the addition of functional domains into the material.

Modular synthesis of amphiphilic Janus glycodendrimers and their self-assembly into glycodendrimersomes and other complex architectures with bioactivity to biomedically relevant lectins.

The modular synthesis of 7 libraries containing 51 self-assembling amphiphilic Janus dendrimers with the monosaccharides D-mannose and D-galactose and the disaccharide D-lactose in their hydrophilic part is reported. These unprecedented sugar-containing dendrimers are named amphiphilic Janus glycodendrimers. Their self-assembly by simple injection of THF or ethanol solution into water or buffer and by hydration was analyzed by a combination of methods including dynamic light scattering, confocal microscopy, cryogenic transmission electron microscopy, Fourier transform analysis, and micropipet-aspiration experiments to assess mechanical properties. These libraries revealed a diversity of hard and soft assemblies, including unilamellar spherical, polygonal, and tubular vesicles denoted glycodendrimersomes, aggregates of Janus glycodendrimers and rodlike micelles named glycodendrimer aggregates and glycodendrimermicelles, cubosomes denoted glycodendrimercubosomes, and solid lamellae. These assemblies are stable over time in water and in buffer, exhibit narrow molecular-weight distribution, and display dimensions that are programmable by the concentration of the solution from which they are injected. This study elaborated the molecular principles leading to single-type soft glycodendrimersomes assembled from amphiphilic Janus glycodendrimers. The multivalency of glycodendrimersomes with different sizes and their ligand bioactivity were demonstrated by selective agglutination with a diversity of sugar-binding protein receptors such as the plant lectins concanavalin A and the highly toxic mistletoe Viscum album L. agglutinin, the bacterial lectin PA-IL from Pseudomonas aeruginosa, and, of special biomedical relevance, human adhesion/growth-regulatory galectin-3 and galectin-4. These results demonstrated the candidacy of glycodendrimersomes as new mimics of biological membranes with programmable glycan ligand presentations, as supramolecular lectin blockers, vaccines, and targeted delivery devices.

Self-assembly of tunable protein suprastructures from recombinant oleosin.

Using recombinant amphiphilic proteins to self-assemble suprastructures would allow precise control over surfactant chemistry and the facile incorporation of biological functionality. We used cryo-TEM to confirm self-assembled structures from recombinantly produced mutants of the naturally occurring sunflower protein, oleosin. We studied the phase behavior of protein self-assembly as a function of solution ionic strength and protein hydrophilic fraction, observing nanometric fibers, sheets, and vesicles. Vesicle membrane thickness correlated with increasing hydrophilic fraction for a fixed hydrophobic domain length. The existence of a bilayer membrane was corroborated in giant vesicles through the localized encapsulation of hydrophobic Nile red and hydrophilic calcein. Circular dichroism revealed that changes in nanostructural morphology in this family of mutants was unrelated to changes in secondary structure. Ultimately, we envision the use of recombinant techniques to introduce novel functionality into these materials for biological applications.