Search

Rheological modifiers are an important component in the development of skin cream (SC) chassis for personal skin care products (PSCPs). The viscous behavior of a PSCP is critical to its effectiveness where its uniformity and material strength impact its processing, storage, and delivery of active ingredients. Due to the mildly acidic environment of the skin, PSCPs require a SC that will assist in maintaining their material strength at low pHs. We have investigated a coiled-coil protein hydrogel system for the ability to possess pH-responsiveness, where physical cross-linking and material strength is controlled by pH relative to the isoelectric point (pI) of the protein. We recently designed a coiled-coil protein hydrogel variant, Q5, which possesses a relatively low pI that we hypothesized to have improved supramolecular assembly into a hydrogel at acidic conditions. We demonstrate that Q5 can retain a partial solution-to-gel transition at pH 6.0 and acts as a soft hydrogel by rheology. We further tested Q5 to act as a rheological modifier in a standard SC at pH 6.0 and pH 8.0 to test conditions mediated by pH changes in the skin environment. Q5 reveals the ability to uniquely increase material strength at low pH in comparison to a standard rheological modifier like hydroxyethyl cellulose (HEC), suggesting modular protein-based coiled-coil rheological modifiers can be used in PSCPs., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Phosphotriesterases (PTEs) represent a class of enzymes capable of efficient neutralization of organophosphates (OPs), a dangerous class of neurotoxic chemicals. PTEs suffer from low catalytic activity, particularly at higher temperatures, due to low thermostability and low solubility. Supercharging, a protein engineering approach via selective mutation of surface residues to charged residues, has been successfully employed to generate proteins with increased solubility and thermostability by promoting charge-charge repulsion between proteins. We set out to overcome the challenges in improving PTE activity against OPs by employing a computational protein supercharging algorithm in Rosetta. Here, we discover two supercharged PTE variants, one negatively supercharged (with -14 net charge) and one positively supercharged (with +12 net charge) and characterize them for their thermodynamic stability and catalytic activity. We find that positively supercharged PTE possesses slight but significant losses in thermostability, which correlates to losses in catalytic efficiency at all temperatures, whereas negatively supercharged PTE possesses increased catalytic activity across 25°C-55°C while offering similar thermostability characteristic to the parent PTE. The impact of supercharging on catalytic efficiency will inform the design of shelf-stable PTE and criteria for enzyme engineering., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The safe and efficient delivery of nucleic acids is crucial for both clinical applications of gene therapy and pre-clinical laboratory research. Such delivery strategies rely on vectors to condense nucleic acid payloads and escort them into the cell without being degraded in the extracellular environment; however, the construction and utilization of these vectors can be difficult and time-consuming. Here, we detail the steps involved in the rapid, laboratory-scale production and assessment of a versatile, nucleic acid delivery vehicle, known as the lipoproteoplex. In this chapter, we outline: (1) the recombinant synthesis and subsequent purification of the supercharged coiled-coil protein component known as N8; (2) the synthesis of cationic liposomes from dioleoyl-3-trimethylammonium propane (DOTAP) and sodium cholate; (3) and finally a protocol for the delivery of a model siRNA cargo into a cultured cell line., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Theranostic materials research is experiencing rapid growth driven by the interest in integrating both therapeutic and diagnostic modalities. These materials offer the unique capability to not only provide treatment but also track the progression of a disease. However, to create an ideal theranostic biomaterial without compromising drug encapsulation, diagnostic imaging must be optimized for improved sensitivity and spatial localization. Herein, we create a protein-engineered fluorinated coiled-coil fiber, Q2 TFL , capable of improved sensitivity to 19 F magnetic resonance spectroscopy (MRS) detection. Leveraging residue-specific noncanonical amino acid incorporation of trifluoroleucine (TFL) into the coiled-coil, Q2, which self-assembles into nanofibers, we generate Q2 TFL . We demonstrate that fluorination results in a greater increase in thermostability and 19 F magnetic resonance detection compared to the nonfluorinated parent, Q2. Q2 TFL also exhibits linear ratiometric 19 F MRS thermoresponsiveness, allowing it to act as a temperature probe. Furthermore, we explore the ability of Q2 TFL to encapsulate the anti-inflammatory small molecule, curcumin (CCM), and its impact on the coiled-coil structure. Q2 TFL also provides hyposignal contrast in 1 H MRI, echogenic signal with high-frequency ultrasound and sensitive detection by 19 F MRS in vivo illustrating fluorination of coiled-coils for supramolecular assembly and their use with 1 H MRI, 19 F MRS and high frequency ultrasound as multimodal theranostic agents., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Proteins are some of the most versatile and studied macromolecules with extensive biomedical applications. The natural and biological origin of proteins offer such materials several advantages over their synthetic counterparts, such as innate bioactivity, recognition by cells and reduced immunogenic potential. Furthermore, proteins can be easily functionalized by altering their primary amino acid sequence and can often be further self-assembled into higher order structures either spontaneously or under specific environmental conditions. This review will feature the recent advances in protein-based biomaterials in the delivery of therapeutic cargo such as small molecules, genetic material, proteins, and cells. First, we will discuss the ways in which secondary structural motifs, the building blocks of more complex proteins, have unique properties that enable them to be useful for therapeutic delivery. Next, supramolecular assemblies, such as fibers, nanoparticles, and hydrogels, made from these building blocks that are engineered to behave in a cohesive manner, are discussed. Finally, we will cover additional modifications to protein materials that impart environmental responsiveness to materials. This includes the emerging field of protein molecular robots, and relatedly, protein-based theranostic materials that combine therapeutic potential with modern imaging modalities, including near-infrared fluorescence spectroscopy (NIRF), single-photo emission computed tomography/computed tomography (SPECT/CT), positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound/photoacoustic imaging (US/PAI).

Engineered proteins provide an interesting template for designing fluorine-19 ( 19 F) magnetic resonance imaging (MRI) contrast agents, yet progress has been hindered by the unpredictable relaxation properties of fluorine. Herein, we present the biosynthesis of a protein block copolymer, termed "fluorinated thermoresponsive assembled protein" (F-TRAP), which assembles into a monodisperse nanoscale micelle with interesting 19 F NMR properties and the ability to encapsulate and release small therapeutic molecules, imparting potential as a diagnostic and therapeutic (theranostic) agent. The assembly of the F-TRAP micelle, composed of a coiled-coil pentamer corona and a hydrophobic, thermoresponsive elastin-like polypeptide core, results in a drastic depression in spin-spin relaxation ( T 2 ) times and unaffected spin-lattice relaxation ( T 1 ) times. The nearly unchanging T 1 relaxation rates and linearly dependent T 2 relaxation rates have allowed for detection via zero echo time 19 F MRI, and the in vivo MR potential has been preliminarily explored using 19 F magnetic resonance spectroscopy (MRS). This fluorinated micelle has also demonstrated the ability to encapsulate the small-molecule chemotherapeutic doxorubicin and release its cargo in a thermoresponsive manner owing to its inherent stimuli-responsive properties, presenting an interesting avenue for the development of thermoresponsive 19 F MRI/MRS-traceable theranostic agents.

Tremendous effort has been dedicated to the design and assembly of bioinspired protein-based architectures with potential applications in drug delivery, tissue engineering, biosensing, and bioimaging. Here, we describe our strategy to generate fibers and bionanocomposites using the coiled-coil domain of cartilage oligomeric matrix protein (COMPcc). Our construct, Q, engineered by swapping particular regions of COMPcc to optimize surface charge, self-assembles to form nanofibers. The Q protein nanofibers can efficiently bind curcumin to form robust mesofibers that can be potentially used for drug delivery and biomedical applications. In addition, using the same Q protein, we describe the biotemplation of gold nanoparticles (AuNP) in the presence and absence of the hexahistidine tag (His-tag). The Q bearing His-tag·AuNP (Q·AuNP) readily deposits on electrode surfaces, while Q without His-tag·AuNP (Qx·AuNP) stabilizes the soluble protein·gold bionanocomposites for several days without aggregating.