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.

Gelation under stress: impact of shear flow on the formation and mechanical properties of methylcellulose hydrogels.

We demonstrate that small unidirectional applied-stresses during temperature-induced gelation dramatically change the gel temperature and the resulting mechanical properties and structure of aqueous methylcellulose (MC), a material that forms a brittle gel with a fibrillar microstructure at elevated temperatures. Applied stress makes gelation more difficult, evidenced by an increased gelation temperature, and weakens mechanical properties of the hot gel, evidenced by a decreased elastic modulus and decreased apparent failure stress. In extreme cases, formation of a fully percolated polymer network is inhibited and a soft granular yield-stress fluid is formed. We quantify the effects of the applied stress using a filament-based mechanical model to relate the measured properties to the structural features of the fibril network. The dramatic changes in the gel temperature and hot gel properties give more design freedom to processing-dependent rheology, but could be detrimental to coating applications where gravitational stress during gelation is unavoidable.

Effects of Hydrophobic Tail Length Variation on Surfactant-Mediated Protein Stabilization.

Recently, protein therapeutics have gained significant attention as a result of their enhanced selectivity and diminished side effects compared to traditional small-molecule drugs. Despite their advantages, protein formulations typically suffer from stability issues because of aggregation and denaturation during production and storage, often resulting in detrimental immune responses. Surfactants can be used to stabilize and protect proteins in solution by preventing protein adsorption onto interfaces or by forming protective structures in solution. Herein, a detailed structure-activity relationship study is described, demonstrating the role that hydrophobic tail length plays in surfactant-mediated stabilization of the model therapeutic protein IgG. The FM1000 series, originating from a surfactant scaffold that allows for easy structure modulation, was synthesized by a simple 2-step procedure. First, phenylalanine was acylated with a variety of acyl chlorides of differing lengths to yield n-acyl phenylalanine, which was then coupled to Jeffamine M1000, a polyethylene glycol-based amine, to yield the final surfactant. With this FM1000 series, it was observed that the 14 carbon-long tail surfactant (14FM1000) was optimal at preventing IgG aggregation compared to surfactants with tails that were longer or shorter. Using a combination of dynamic surface tensiometry and quartz crystal microbalance with dissipation, it was hypothesized that 14FM1000 was able to prevent IgG adsorption, and therefore aggregation, by adsorbing appreciably onto surfaces quickly. 14FM1000 had the fastest rate of initial adsorption compared to the other surfactants studied. Short-tail surfactants were slow to and did not adsorb appreciably onto surfaces, allowing IgG adsorption. Although long-tail surfactants were also slow to adsorb, allowing IgG to adsorb and aggregate, their equilibrium adsorption was strong. Additionally, 14FM1000 was the most reversibly adsorbed surfactant, likely improving its ability to desorb and adsorb quickly to transient surfaces, therefore protecting the IgG at each new hydrophobic surface and preventing aggregation. By understanding the structure-activity relationship between surfactants and protein stabilization, we move toward more efficient design of future surfactants increasing the stability and utility of important protein therapeutics.

Nonionic Surfactant Properties of Amphiphilic Hyperbranched Polyglycerols.

The surfactant properties of amphiphilic hyperbranched polyglycerols (HPGs) were investigated. The HPGs were prepared by ring-opening multibranching polymerization of glycidol using hydrophobic initiators of varying size and structure. The cloud points for all HPG surfactants were found to be >80 °C in deionized water with >1 wt % NaCl. The HPG surfactants with hydrophilic-lipophilic balance values between 16 and 18 were found to form stable octanol/water (o/w) emulsions within a 24 h period. Several surface properties, including critical micelle concentration (CMC), efficiency of surface tension reduction (pC20), effectiveness of surface tension reduction (γCMC), surface excess concentration at the CMC (Γmax), minimum area/molecule at the interface (Amin), and the CMC/C20 ratio of the HPG surfactants were measured in deionized water at 22.6 °C. In general, increasing HPG size was marked by an increase in minimum surface area per molecule (Amin) at the aqueous liquid/air interface. This increase in size also led to lower CMC and greater pC20 values of HPG surfactants prepared with Tergitol 15-S-7 initiator (HPG 5a-5d), a commercially available ethylene glycol oligomer with a branched hydrophobic tail.

Dynamic Properties of Novel Excipient Suggest Mechanism for Improved Performance in Liquid Stabilization of Protein Biologics.

To improve liquid formulation stability, formulators employ various excipients designed to stabilize protein drugs, including buffers, salts, sugars, and surfactants. One of the roles of surfactants is to protect the protein drug from surface interactions that can destabilize the protein. Protein drug products formulated with surfactants usually contain either a polysorbate or poloxamer. Even in the presence of these surfactants, protein drug stability is often insufficient, particularly because of agitation-induced aggregation. FM1000 is one of a series of surfactants containing an alkyl chain, an amino acid, and a polyetheramine. The characterization of the dynamics of FM1000 at various water/hydrophobic interfaces was compared to Polysorbate 20, Polysorbate 80, and Poloxamer 188. FM1000 stabilizes an interface 1-2 orders of magnitude faster than all three of these surfactants, even in the presence of protein. The faster dynamics leads to improved stabilization of model protein biologic drugs IgG and abatacept against agitation-induced aggregation. These results provide mechanistic understanding of the key causes and drivers of protein aggregation.

A Robust Oil-in-Oil Emulsion for the Nonaqueous Encapsulation of Hydrophilic Payloads.

Compartmentalized structures widely exist in cellular systems (organelles) and perform essential functions in smart composite materials (microcapsules, vasculatures, and micelles) to provide localized functionality and enhance materials' compatibility. An entirely water-free compartmentalization system is of significant value to the materials community as nonaqueous conditions are critical to packaging microcapsules with water-free hydrophilic payloads while avoiding energy-intensive drying steps. Few nonaqueous encapsulation techniques are known, especially when considering just the scalable processes that operate in batch mode. Herein, we report a robust oil-in-oil Pickering emulsion system that is compatible with nonaqueous interfacial reactions as required for encapsulation of hydrophilic payloads. A major conceptual advance of this work is the notion of the partitioning inhibitor-a chemical agent that greatly reduces the payload's distribution between the emulsion's two phases, thus providing appropriate conditions for emulsion-templated interfacial polymerization. As a specific example, an immiscible hydrocarbon-amine pair of liquids is emulsified by the incorporation of guanidinium chloride (GuHCl) as a partitioning inhibitor into the dispersed phase. Polyisobutylene (PIB) is added into the continuous phase as a viscosity modifier for suitable modification of interfacial polymerization kinetics. The combination of GuHCl and PIB is necessary to yield a robust emulsion with stable morphology for 3 weeks. Shell wall formation was accomplished by interfacial polymerization of isocyanates delivered through the continuous phase and polyamines from the droplet core. Diethylenetriamine (DETA)-loaded microcapsules were isolated in good yield, exhibiting high thermal and chemical stabilities with extended shelf-lives even when dispersed into a reactive epoxy resin. The polyamine phase is compatible with a variety of basic and hydrophilic actives, suggesting that this encapsulation technology is applicable to other hydrophilic payloads such as polyols, aromatic amines, and aromatic heterocyclic bases. Such payloads are important for the development of extended pot or shelf life systems and responsive coatings that report, protect, modify, and heal themselves without intervention.

Novel Surfactant Compatibility with Downstream Protein Bioprocesses.

Downstream processing of antibodies consists of a series of steps aimed at purifying the product and ensuring it is delivered to formulators structurally and functionally intact. The process can be complex and time-consuming, involving multiple filtrations, chromatography, and buffer exchange steps that can interfere with product integrity. This study explores the possibility and benefits of adding N-myristoyl phenylalanine polyether amine diamide (FM1000) as a process aid. FM1000 is a nonionic surfactant that is highly effective at stabilizing proteins against aggregation and particle formation and has been extensively explored as a novel excipient for antibody formulations. In this work, FM1000 is shown to stabilize proteins against pumping-induced aggregation which can occur while transporting them between process units and within certain processes. It is also shown to prevent antibody fouling of multiple polymeric surfaces. Furthermore, FM1000 can be removed after some steps and during buffer exchange in ultrafiltration/diafiltration, if needed. Additionally, FM1000 was compared to polysorbates in studies focusing on surfactant retention on filters and columns. While the different molecular entities of polysorbates elute at different rates, FM1000 flows through purification units as a single molecule and at a faster rate. Overall, this work defines new areas of application for FM1000 within downstream processing and presents it as a versatile process aid, where its addition and removal are tunable depending on the needs of each product.

Emerging Challenges and Innovations in Surfactant-mediated Stabilization of Biologic Formulations.

Biologics may be subjected to various destabilizing conditions during manufacturing, transportation, storage, and use. Therefore, biologics must be appropriately formulated to meet their desired quality target product profiles. In the formulations of protein-based biologics, one critical component is surfactant. Polysorbate 80 and Polysorbate 20 remain the most commonly used surfactants. Surfactants can stabilize proteins through different mechanisms and help the proteins withstand destabilization stresses. However, the challenges associated with surfactants, for instance, impurities, degradation, and potential triggering of adverse immune responses, have been encountered. Therefore, there are continued efforts to develop novel surfactants to overcome these challenges associated with traditional surfactants. Meanwhile, surfactants have also found their use in formulations of newer and novel modalities, namely, antibody-drug conjugates, bispecific antibodies, and adeno-associated viruses (AAV). This review provides an updated in-depth discussion of surfactants in the above-mentioned areas, namely mechanism of action of surfactants, a critical review of challenges with surfactants and current mitigation approaches, and emerging technologies to develop novel surfactants. In addition, gaps, current mitigations, and future directions have been presented to trigger further discussion and research to facilitate the use and development of novel surfactants.

Effects of Membrane Rheology on Leuko-polymersome Adhesion to Inflammatory Ligands.

A strategy for treating inflammatory disease is to create micro-particles with the adhesive properties of leukocytes. The underlying rheology of deformable adhesive microspheres would be an important factor in the adhesive performance of such particles. In this work the effect of particle deformability on the selectin-mediated rolling of polymer vesicles (polymersomes) is evaluated. The rheology of the polymersome membrane was modulated by cross-linking unsaturated side-chains within the hydrophobic core of the membrane. Increased membrane rigidity resulted in decreased rates of particle recruitment rather than decreased average rolling velocities. Reflective interference contrast microscopy of rolling vesicles confirmed that neither flaccid nor rigid vesicles sustained close contacts with the substrate during rolling adhesion. A variable-shear rate parallel-plate flow chamber was employed to evaluate individual vesicles rolling on substrates under different flow conditions. Analysis of the trajectories of single flaccid vesicles revealed several distinct populations of rolling vesicles; however, some of these populations disappear when the vesicle membranes are made rigid. This work shows that membrane mechanics affects the capture, but not the rolling dynamics, of adherent leuko-polymersomes.

Nanofiber-nanorod composites exhibiting light-induced reversible lower critical solution temperature transitions.

Stimuli-responsive materials are promising as smart materials for a range of applications. In this work, a photo-crosslinkable, thermoresponsive macromer was electrospun into fibrous scaffolds containing gold nanorods (AuNRs). The resulting fibrous nanocomposites composed of poly(N-isopropylacrylamide-co-polyethylene glycol acrylate) (PNPA) and PEGylated AuNRs were crosslinked and swollen in water. AuNRs strongly absorb in the near-infrared (NIR) region to generate heat, which triggered the fiber thermal transition upon NIR light exposure. During the thermal transition, scaffolds collapsed both macroscopically and microscopically, with individual fibers deswelling and pulling together. Exposure to a 1.1 W NIR laser decreased the diameter of swollen fibers by 34.7% from 1332 ± 193.3 to 868.9 ± 168.3 nm, and increased fiber density 116% from 209.5 ± 26.34 to 451.9 ± 23.68 fibers mm( - 1). This transition was dependent on the incorporation of the AuNRs, and was utilized to trigger the release of encapsulated proteins from the nanocomposite fiber mats. The expulsion of water from fibers upon NIR exposure caused the release rate of incorporated protein to increase greater than tenfold, from 0.038 ± 0.052 without external stimulus to 0.462 ± 0.227 µg protein/mg polymer/min with NIR exposure. These results suggest that light-responsive fibrous nanocomposites can be utilized in applications such as drug delivery.

Measuring Molecular Diffusion Through Thin Polymer Films with Dual-Band Plasmonic Antennas.

A general and quantitative method to characterize molecular transport in polymers with good temporal and high spatial resolution, in complex environments, is an important need of the pharmaceutical, textile, and food and beverage packaging industries, and of general interest to the polymer science community. Here we show how the amplified infrared (IR) absorbance sensitivity provided by plasmonic nanoantenna-based surface enhanced infrared absorption (SEIRA) provides such a method. SEIRA enhances infrared (IR) absorbances primarily within 50 nm of the nanoantennas, enabling localized quantitative detection of even trace quantities of analytes and diffusion measurements in even thin polymer films. Relative to a commercial attenuated total internal reflection (ATR) system, the limit of detection is enhanced at least 13-fold, and as is important for measuring diffusion, the detection volume is about 15 times thinner. Via this approach, the diffusion coefficient and solubility of specific molecules, including l-ascorbic acid (vitamin C), ethanol, various sugars, and water, in both simple and complex mixtures (e.g., beer and a cola soda), were determined in poly(methyl methacrylate), high density polyethylene (HDPE)-based, and polypropylene-based polyolefin films as thin as 250 nm.

Modular synthesis of biodegradable diblock copolymers for designing functional polymersomes.

Polymer vesicles, or polymersomes, are promising candidates for applications in drug delivery and tissue imaging. While a vast variety of polymers have been explored for their ability to assemble into polymersomes, relatively little research on the functionalization of these polymers has been reported. We present here a novel route for the synthesis of poly(caprolactone)-b-poly(ethylene glycol) (PCL-b-PEG) diblock copolymers that allows for the insertion of functional groups at the block junctions and the assembly of functional membranes. This modular synthesis has been developed on the basis of solid-phase peptide synthesis techniques and is accomplished through the formation of two peptide bonds, one between an amine-terminated PEG and the carboxyl moiety of the functional group and the other between the functional group amine and a carboxy-terminated PCL. As a demonstration of the potential utility of the resulting vesicles, we incorporated two different amino acid functional groups at the junction. 2-Nitrophenylalanine was utilized to create UV-responsive membranes in which the vesicles were destabilized and released encapsulated contents upon irradiation. A fluorescein-conjugated lysine was also utilized to create stable fluorescent membranes in which the fluorescence was built into the polymer. This method should contribute to our ability to further develop smart, functional membranes.

Addressable micropatterning of multiple proteins and cells by microscope projection photolithography based on a protein friendly photoresist.

We report a new method for the micropatterning of multiple proteins and cells with micrometer-scale precision. Microscope projection photolithography based on a new protein-friendly photoresist, poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP), was used for the fabrication of multicomponent protein/cell arrays. Microscope projection lithography allows precise registration between multiple patterns as well as facile fabrication of microscale features. Thin films of PDMP became soluble in near-neutral physiological buffer solutions upon UV exposure and exhibited excellent resistance to protein adsorption and cell adhesion. By harnessing advantages in microscope projection photolithography and properties of PDMP thin films, we could successfully fabricate protein arrays composed of multiple proteins. Furthermore, we could extend this method for the patterning of two different types of immune cells for the potential study of immune cell interactions. This technique will in general be useful for protein chip fabrication and high-throughput cell-cell communication study.

Encapsulation of catalyst in block copolymer micelles for the polymerization of ethylene in aqueous medium.

The catalytic emulsion polymerization of ethylene has been a long-lasting technical challenge as current techniques still suffer some limitations. Here we report an alternative strategy for the production of semi-crystalline polyethylene latex. Our methodology consists of encapsulating a catalyst precursor within micelles composed of an amphiphilic block copolymer. These micelles act as nanoreactors for the polymerization of ethylene in water. Phosphinosulfonate palladium complexes were used to demonstrate the success of our approach as they were found to be active for hours when encapsulated in micelles. Despite this long stability, the activity of the catalysts in micelles remains significantly lower than in organic solvent, suggesting some catalyst inhibition. The inhibition strength of the different chemicals present in the micelle were determined and compared. The combination of the small volume of the micelles, and the coordination of PEG appear to be the culprits for the low activity observed in micelles.

Preparation of Surfactant-Resistant Polymersomes with Ultrathick Membranes through RAFT Dispersion Polymerization.

Surfactant-resistant polymersomes have substantial potential to be used as delivery vehicles in industrial applications. Herein, we report the preparation of poly(ethylene oxide)-block-polystyrene copolymers with ultrahigh hydrophobic-block molecular weights through RAFT dispersion polymerization, which allows the polymerization-induced self-assembly into well-defined polymersomes with ultrathick membranes up to ∼47 nm. These ultrathick membranes significantly enhance the resistance against surfactant solubilization of the vesicles, improving the vesicles' potential for use in industrial encapsulations. Vesicle-encapsulated actives are well retained in the presence of up to 40 wt % of various anionic and nonionic surfactants, with less than 7% active leakage being observed after 30 days.

Amino-Acid-Incorporating Nonionic Surfactants for Stabilization of Protein Pharmaceuticals.

With the rapid development of protein-based pharmaceutical products over the past decade, one of the biggest challenges in product development is maintaining the structural stability of proteins during purification, processing, and storage. In this work, the design of a new class of surfactants, polyether-modified N-acyl amino acids, is presented. One surfactant from this series, containing a phenylalanine moiety, demonstrated remarkable stabilization against aggregation of several model protein drugs. Dynamic light scattering, size exclusion chromatography, and circular dichroism all show the rate of thermally accelerated protein aggregation slowed. IgG aggregation was reduced by 3-fold compared to polysorbate controls. Testing of Orencia, a prescription biologic drug for rheumatoid arthritis, demonstrated a 36% improvement in monomer retention upon heat-aging.

Trigger chemistries for better industrial formulations.

In recent years, innovations and consumer demands have led to increasingly complex liquid formulations. These growing complexities have provided industrial players and their customers access to new markets through product differentiation, improved performance, and compatibility/stability with other products. One strategy for enabling more complex formulations is the use of active encapsulation. When encapsulation is employed, strategies are required to effect the release of the active at the desired location and time of action. One particular route that has received significant academic research effort is the employment of triggers to induce active release upon a specific stimulus, though little has translated for industrial use to date. To address emerging industrial formulation needs, in this review, we discuss areas of trigger release chemistries and their applications specifically as relevant to industrial use. We focus the discussion on the use of heat, light, shear, and pH triggers as applied in several model polymeric systems for inducing active release. The goal is that through this review trends will emerge for how technologies can be better developed to maximize their value through industrial adaptation.

New frontiers for encapsulation in the chemical industry.

Encapsulation of actives comprises an area of exploration undergoing rapid growth in both academic and industrial research settings. Encapsulation processes are employed as a part of product synthesis processes for improved efficiency, enhanced stability, active ingredient compatibility, increased safety, targeted delivery, and novel performance of the end product. Such technical benefits enable producers to offer products with increased formulation complexity, access new markets, differentiate products, and improve compatibility and stability, while meeting consumer demands with improved performance, reduced costs, and new actives. In this review, we highlight several emerging academic areas of encapsulation that we believe have specific relevance to industrial formulation, with a focus on three primary areas: supramolecular encapsulation, aqueous self-assembled systems, and emulsion-based capsules. The goal of this review is to help identify the major challenges facing encapsulation technology adoption in the chemical industry, bringing focus and maximizing the potential value of ongoing research efforts.

Biodegradable Polymersomes for the Delivery of Gemcitabine to Panc-1 Cells.

Traditional anticancer chemotherapy often displays toxic side effects, poor bioavailability, and a low therapeutic index. Targeting and controlled release of a chemotherapeutic agent can increase drug bioavailability, mitigate undesirable side effects, and increase the therapeutic index. Here we report a polymersome-based system to deliver gemcitabine to Panc-1 cells in vitro. The polymersomes were self-assembled from a biocompatible and completely biodegradable polymer, poly(ethylene oxide)-poly(caprolactone), PEO-PCL. We showed that we can encapsulate gemcitabine within stable 200 nm vesicles with a 10% loading efficiency. These vesicles displayed a controlled release of gemcitabine with 60% release after 2 days at physiological pH. Upon treatment of Panc-1 cells in vitro, vesicles were internalized as verified with fluorescently labeled polymersomes. Clonogenic assays to determine cell survival were performed by treating Panc-1 cells with varying concentrations of unencapsulated gemcitabine (FreeGem) and polymersome-encapsulated gemcitabine (PolyGem) for 48 hours. 1 µM PolyGem was equivalent in tumor cell toxicity to 1 µM FreeGem, with a one log cell kill observed. These studies suggest that further investigation on polymersome-based drug formulations is warranted for chemotherapy of pancreatic cancer.

Engineering Polymersome Protocells.

The field of biomimicry is embracing the construction of complex assemblies that imitate both biological structure and function. Advancements in the design of these mimetics have generated a growing vision for creating an artificial or proto- cell. Polymersomes are vesicles that can be made from synthetic, biological or hybrid polymers and can be used as a model template to build cell-like structures. In this perspective, we discuss various areas where polymersomes have been used to mimic cell functions as well as areas in which the synthetic flexibility of polymersomes would make them ideal candidates for a biomembrane mimetic. Designing a polymersome that comprehensively displays the behaviors discussed herein has the potential to lead to the development of an autonomous, responsive particle that resembles the intelligence of a biological cell.