Cubosomes functionalized with antibodies as a potential strategy for the treatment of HER2-positive breast cancer.

Breast cancers that overexpress human epidermal growth factor receptor 2 (HER2) have poor prognosis. Moreover, available chemotherapies cause numerous side effects due to poor selectivity. To advance more effective and safer therapies for HER2-positive breast cancer, we explored the fusion of drug delivery technology and immunotherapy. Our research led to the design of immunocubosomes loaded with panobinostat and functionalized with trastuzumab antibodies, enabling precise targeting of breast cancer cells that overexpress HER2. We characterised the nanostructure of cubosomes using small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS). Moreover, we confirmed the integrity of the trastuzumab antibodies on the immunocubosomes by Fourier-transform infrared spectroscopy (FTIR) and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, we found that panobinostat-loaded immunocubosomes were more cytotoxic, and in an uptake-dependant manner, towards a HER2-positive breast cancer cell line (SKBR3) compared to a cell line representing healthy cells (L929). These results support that the functionalization of cubosomes with antibodies enhances both the effectiveness of the loaded drug and its selectivity for targeting HER2-positive breast cancer cells.

Enhanced structure/function of mTSPO translocator in lipid:surfactant mixed micelles.

TSPO is a ubiquitous transmembrane protein used as a pharmacological marker in neuroimaging. The only known atomic structure of mammalian TSPOs comes from the solution NMR of mouse TSPO (mTSPO) bound to the PK11195 ligand and in a DPC surfactant environment. No structure is available in a biomimetic environment and without PK11195 which strongly stiffens the protein. We measured the effect of different amphiphilic environments on ligand-free mTSPO to study its structure/function and find optimal solubilization conditions. By replacing the SDS surfactant, where the recombinant protein is purified, with mixed lipid:surfactant (DMPC:DPC) micelles at different ratios (0:1, 1:2, and 2:1, w:w), the α-helix content and interactions and the intrinsic tryptophan (Trp) fluorescence of mTSPO are gradually increased. Small-angle X-ray scattering (SAXS) shows a more extended mTSPO/belt complex with the addition of lipids: Dmax ∼95 Å in DPC alone versus ∼142 Å in DMPC:DPC (1:2). SEC-MALLS shows that the molecular composition of the mTSPO belt is ∼98 molecules for DPC alone and ∼58 DMPC and ∼175 DPC for DMPC:DPC (1:2). Additionally, DMPC:DPC micelles stabilize mTSPO compared to DPC alone, where the protein has a greater propensity to aggregate. These structural changes are consistent with the increased affinity of mTSPO for the PK11195 ligand in presence of lipids (Kd ∼70 µM in DPC alone versus ∼0.91 µM in DMPC:DPC, 1:2), as measured by microscale thermophoresis (MST). In conclusion, mixed lipid:surfactant micelles open new possibilities for the stabilization of membrane proteins and for their study in solution in a more biomimetic amphiphilic environment.

Structural and mechanical anisotropy in plant-based meat analogues.

The rising demand for plant-based meat analogues as alternatives to animal products has sparked interest in understanding the complex interplay between their structural and mechanical properties. The ability to manipulate the processing parameters and protein blend composition offers fundamental insights into the texturization process and holds economic and sustainable implications for the food industry. Consequently, the correlation between mechanical and structural properties in meat analogues is crucial for achieving consumer satisfaction and successful market penetration, providing comprehensive insights into the textural properties of meat analogues and their potential to mimic traditional animal produce. Our study delves into the relationship between structural and mechanical anisotropy in meat analogues produced using high moisture extrusion cooking, which involves blending protein, water, and other ingredients, followed by a controlled heating and cooling process to achieve a fibrous texture akin to traditional meat. By employing techniques such as scanning small-angle X-ray scattering, scanning electron microscopy, and mechanical testing we investigate the fibrous structure and its impact on the final texture of meat analogues. We show that textural and structural anisotropy is reflected on the mechanical properties measured using tensile and dynamic mechanical techniques. It is demonstrated that the calculated anisotropy indexes, a measure for the degree of textural and structural anisotropy, increase with increasing protein content. Our findings have significant implications for the understanding and development of plant-based meat analogues with structures that can be tuned to closely resemble the animal meat textures of choice, thereby enabling consumers to transition to more sustainable dietary choices while preserving familiar eating habits.

Pluronic F68 Micelles as Carriers for an Anti-Inflammatory Drug: A Rheological and Scattering Investigation.

Age-long ambition of medical scientists has always been advancement in healthcare and therapeutic medicine. Biomedical research indeed claims paramount importance in nanomedicine and drug delivery, and the development of biocompatible storage structures for delivering drugs stands at the heart of emerging scientific works. The delivery of drugs into the human body is nevertheless a nontrivial and challenging task, and it is often addressed by using amphiphilic compounds as nanosized delivery vehicles. Pluronics belong to a peculiar class of biocompatible and thermosensitive nonionic amphiphilic copolymers, and their self-assemblies are employed as drug delivery excipients because of their unique properties. We herein report on the encapsulation of diclofenac sodium within Pluronic F68 self-assemblies in water, underpinning the impact of the drug on the rheological and microstructural evolution of pluronic-based systems. The self-assembly and thermoresponsive micellization were studied through isothermal steady rheological experiments at different temperatures on samples containing 45 wt % Pluronic F68 and different amounts of diclofenac sodium. The adoption of scattering techniques, small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS), allowed for the description of the system features at the nanometer length scale, providing information about the characteristic size of each part of the micellar structures as a function of temperature and drug concentration. Diclofenac sodium is not a good fellow for Pluronic F68. The triblock copolymer aids the encapsulation of the drug, highly improving its water solubility, whereas diclofenac sodium somehow hinders Pluronic self-assembly. By using a simple empirical model and no fitting parameters, the steady viscosity can be predicted, although qualitatively, through the volume fraction of the micelles extracted through scattering techniques and compared to the rheological one. A tunable control of the viscous behavior of such biomedical systems may be achieved through the suitable choice of their composition.

Glycyrrhizic acid aggregates seen from a synthetic surfactant perspective.

Bio- or plant-based surfactants are a sustainable and renewable alternative to replace synthetic chemicals for environmental, drugs and food applications. However, these "green" surfactants have unique molecular structures, and their self-assembly in water might lead to complex morphologies and unexpected properties. The micellization of saponin molecules, such as glycyrrhizic acid (GA), differs significantly from those of conventional synthetic surfactants, yet these differences are often overlooked. Saponins self-assemble in complex hierarchical helical morphologies similar to bile salts, rather than the expected globular, ellipsoidal and wormlike micelles. Here, we review two potential routes for molecular self-assembly of GA, namely kinetics of crystallization and thermodynamic equilibrium, focusing on their structure as a function of concentration. Some uncertainty remains to define which route is followed by GA self-assembly, as well as the first type of aggregate formed at low concentrations, thus we review the state-of-the-art information about GA assembly. We compare the self-assembly of GA with conventional linear surfactants, and identify their key similarities and differences, from molecular and chemical perspectives, based on the critical packing parameter (CPP) theory. We expect that this work will provide perspectives for the unclear process of GA assembly, and highlight its differences from conventional micellization.

Surface Cross-Linking by Macromolecular Tethers Enhances Virus-like Particles' Resilience to Mucosal Stress Factors.

Virus-like particles (VLPs) are emerging as nanoscaffolds in a variety of biomedical applications including delivery of vaccine antigens and cargo such as mRNA to mucosal surfaces. These soft, colloidal, and proteinaceous structures (capsids) are nevertheless susceptible to mucosal environmental stress factors. We cross-linked multiple capsid surface amino acid residues using homobifunctional polyethylene glycol tethers to improve the persistence and survival of the capsid to model mucosal stressors. Surface cross-linking enhanced the stability of VLPs assembled from Acinetobacter phage AP205 coat proteins in low pH (down to pH 4.0) and high protease concentration conditions (namely, in pig and mouse gastric fluids). Additionally, it increased the stiffness of VLPs under local mechanical indentation applied using an atomic force microscopy cantilever tip. Small angle X-ray scattering revealed an increase in capsid diameter after cross-linking and an increase in capsid shell thickness with the length of the PEG cross-linkers. Moreover, surface cross-linking had no effect on the VLPs' mucus translocation and accumulation on the epithelium of in vitro 3D human nasal epithelial tissues with mucociliary clearance. Finally, it did not compromise VLPs' function as vaccines in mouse subcutaneous vaccination models. Compared to PEGylation without cross-linking, the stiffness of surface cross-linked VLPs were higher for the same length of the PEG molecule, and also the lifetimes of surface cross-linked VLPs were longer in the gastric fluids. Surface cross-linking using macromolecular tethers, but not simple conjugation of these molecules, thus offers a viable means to enhance the resilience and survival of VLPs for mucosal applications.

Hierarchical self-assembly of a reflectin-derived peptide.

Reflectins are a family of intrinsically disordered proteins involved in cephalopod camouflage, making them an interesting source for bioinspired optical materials. Understanding reflectin assembly into higher-order structures by standard biophysical methods enables the rational design of new materials, but it is difficult due to their low solubility. To address this challenge, we aim to understand the molecular self-assembly mechanism of reflectin's basic unit-the protopeptide sequence YMDMSGYQ-as a means to understand reflectin's assembly phenomena. Protopeptide self-assembly was triggered by different environmental cues, yielding supramolecular hydrogels, and characterized by experimental and theoretical methods. Protopeptide films were also prepared to assess optical properties. Our results support the hypothesis for the protopeptide aggregation model at an atomistic level, led by hydrophilic and hydrophobic interactions mediated by tyrosine residues. Protopeptide-derived films were optically active, presenting diffuse reflectance in the visible region of the light spectrum. Hence, these results contribute to a better understanding of the protopeptide structural assembly, crucial for the design of peptide- and reflectin-based functional materials.

Rheological Properties of Ionically Crosslinked Viscoelastic 2D Films vs. Corresponding 3D Bulk Hydrogels.

Ionically crosslinked hydrogels containing metal coordination motifs have piqued the interest of researchers in recent decades due to their self-healing and adhesive properties. In particular, catechol-functionalized bulk hydrogels have received a lot of attention because of their bioinspired nature. By contrast, very little is known about thin viscoelastic membranes made using similar chelator-ion pair motifs. This shortcoming is surprising because the unique interfacial properties of these membranes, namely, their self-healing and adhesion, would be ideal for capsule shells, adhesives, or for drug delivery purposes. We recently demonstrated the feasibility to fabricate 10 nm thick viscoelastic membranes from catechol-functionalized surfactants that are ionically crosslinked at the liquid/liquid interface. However, it is unclear if the vast know-how existing on the influence of the chelator-ion pair on the mechanical properties of ionically crosslinked three-dimensional (3D) hydrogels can be translated to two-dimensional (2D) systems. To address this question, we compare the dynamic mechanical properties of ionically crosslinked pyrogallol functionalized hydrogels with those of viscoelastic membranes that are crosslinked using the same chelator-ion pairs. We demonstrate that the storage and loss moduli of viscoelastic membranes follow a trend similar to that of the hydrogels, with the membrane becoming stronger as the ion-chelator affinity increases. Yet, membranes relax significantly faster than bulk equivalents. These insights enable the targeted design of viscoelastic, adhesive, self-healing membranes possessing tunable mechanical properties. Such capsules can potentially be used, for example, in cosmetics, as granular inks, or with additional work that includes replacing the fluorinated block by a hydrocarbon-based one in drug delivery and food applications.

Polymer-metal-organic framework self-assembly (PMOFSA) as a robust one-step method to generate well-dispersed hybrid nanoparticles in water.

A new process, PMOFSA, is described here, that opens the way for the one-pot straightforward and versatile manufacture of polymer-MOF nanoparticles in water. It can be expected that this study will not only expand the scope of in situ preparation of polymer-MOF nano-objects but also inspire researchers in the field to prepare a new generation of polymer-MOF hybrid materials.

Amyloid-polysaccharide interfacial coacervates as therapeutic materials.

Coacervation via liquid-liquid phase separation provides an excellent opportunity to address the challenges of designing nanostructured biomaterials with multiple functionalities. Protein-polysaccharide coacervates, in particular, offer an appealing strategy to target biomaterial scaffolds, but these systems suffer from the low mechanical and chemical stabilities of protein-based condensates. Here we overcome these limitations by transforming native proteins into amyloid fibrils and demonstrate that the coacervation of cationic protein amyloids and anionic linear polysaccharides results in the interfacial self-assembly of biomaterials with precise control of their structure and properties. The coacervates present a highly ordered asymmetric architecture with amyloid fibrils on one side and the polysaccharide on the other. We demonstrate the excellent performance of these coacervates for gastric ulcer protection by validating via an in vivo assay their therapeutic effect as engineered microparticles. These results point at amyloid-polysaccharides coacervates as an original and effective biomaterial for multiple uses in internal medicine.

Amyloid Fibrils Enhance the Topical Bio-Adhesivity of Liquid Crystalline Mesophase-Based Drug Formulations.

Despite their distinctive secondary structure based on cross ß-strands, amyloid fibrils (AF) are stable fibrous protein aggregates with features similar to collagen, one of the main components of the extracellular matrix, and thus constitute a potential scaffold for enhancing cell adhesion for topical applications. Here, the contribution of AF to skin bio-adhesivity aiming toward topical treatments is investigated. Liquid crystalline mesophase (LCM) based on phytantriol is formulated, with the aqueous phase containing either water or a solution of 4 wt% amyloid fibrils. Then resveratrol is added as a model anti-inflammatory molecule. The developed LCM presents a double gyroid Ia3d mesophase. The incorporation of AF into the LCM increases its bio-adhesive properties. In vitro release and ex vivo permeation and retention confirm the controlled release property of the system, and that resveratrol is retained in epidermis and dermis, but is also permeated through the skin. All formulations are biocompatible with L929 cells. The in vivo assay confirms that systems with AF lead to a higher anti-inflammatory effect of resveratrol. These results confirm the hypothesis that the incorporation of AF in the LCM increases the bio-adhesiveness and efficiency of the system for topical treatment, and consequently, the therapeutical action of the encapsulated drug.

Mechanical tuning of virus-like particles.

HYPOTHESIS: Virus-like particles (VLPs) are promising scaffolds for developing mucosal vaccines. For their optimal performance, in addition to design parameters from an immunological perspective, biophysical properties may need to be considered. EXPERIMENTS: We investigated the mechanical properties of VLPs scaffolded on the coat protein of Acinetobacter phage AP205 using atomic force microscopy and small angle X-ray scattering. FINDINGS: Investigations showed that AP205 VLP is a tough nanoshell of stiffness 93 ± 23 pN/nm and elastic modulus 0.11 GPa. However, its mechanical properties are modulated by attaching muco-inert polyethylene glycol to 46 ± 10 pN/nm and 0.05 GPa. Addition of antigenic peptides derived from SARS-CoV2 spike protein by genetic fusion increased the stiffness to 146 ± 54 pN/nm although the elastic modulus remained unchanged. These results, which are interpreted in terms of shell thickness and coat protein net charge variations, demonstrate that surface conjugation can induce appreciable changes in the biophysical properties of VLP-scaffolded vaccines.

Solvent modulation in peptide sub-microfibers obtained by solution blow spinning.

Peptides possess high chemical diversity at the amino acid sequence level, which further translates into versatile functions. Peptides with self-assembling properties can be processed into diverse formats giving rise to bio-based materials. Peptide-based spun fibers are an interesting format due to high surface-area and versatility, though the field is still in its infancy due to the challenges in applying the synthetic polymer spinning processes to protein fibers to peptides. In this work we show the use of solution blow-spinning to produce peptide fibers. Peptide fiber formation was assisted by the polymer poly (vinyl pyrrolidone) (PVP) in two solvent conditions. Peptide miscibility and further self-assembling propensity in the solvents played a major role in fiber formation. When employing acetic acid as solvent, peptide fibers (0.5 µm) are formed around PVP fibers (0.75 µm), whereas in isopropanol only one type of fibers are formed, consisting of mixed peptide and PVP (1 µm). This report highlights solvent modulation as a mean to obtain different peptide sub-microfibers via a single injection nozzle in solution blow spinning. We anticipate this strategy to be applied to other small peptides with self-assembly propensity to obtain multi-functional proteinaceous fibers.

Polymer induced liquid crystal phase behavior of cellulose nanocrystal dispersions.

Cellulose nanocrystals (CNCs) are a promising bio-based material that has attracted significant attention in the fabrication of functional hybrid materials. The rod-like shape and negative surface charge of CNCs enable their rich colloidal behavior, such as a liquid crystalline phase and hydrogel formation that can be mediated by different additives. This study investigates the effect of depletion-induced attraction in the presence of non-absorbing polyethylene glycol (PEG) of different molecular weights in CNC aqueous dispersions, where the polymer molecules deplete the space around particles, apply osmotic pressure and drive the phase transition. Polarized light microscopy (PLM), rheology, small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) are used to characterize the phase behavior over a time period of one month. In our results, pure CNC dispersion shows three typical liquid crystal shear rheology regimes and cholesteric self-assembly behavior. Tactoid nucleation, growth and coalescence are observed microscopically, and eventually the dispersion presents macroscopic phase separation. PEG with lower molecular weight induces weak attractive depletion forces. Tactoid growth is limited, and the whole system turns into a fully nematic phase macroscopically. With PEG of higher molecular weight, attractive depletion force becomes predominant, thus CNC self-assembly is inhibited and nematic hydrogel formation is triggered. Overall, we demonstrate that depletion induced attraction forces by the addition of PEG enable precise tuning of CNC self-assembly and phase behavior with controllable mechanical strength and optical activity. These findings deepen our fundamental understanding of cellulose nanocrystals and advance their application in colloidal systems and nanomaterials.

Multi-length scale structural investigation of lysozyme self-assembly.

Reactive amyloid oligomers are responsible for cytotoxicity in amyloid pathologies and because of their unstable nature characterizing their behavior is a challenge. The physics governing the self-assembly of proteins in crowded conditions is extremely complex and its comprehension, despite its paramount relevance to understanding molecular mechanisms inside cells and optimizing pharmaceutical processes, remains inconclusive. Here, we focus on the amyloid oligomerization process in self-crowded lysozyme aqueous solutions in acidic conditions. We reveal that the amyloid oligomers form at high protein concentration and low pH. Through multi-length scale spectroscopic investigations, we find that amyloid oligomers can further interconnect with each other by weak and non-specific interactions forming an extended network that leads to the percolation of the whole system. Our multi-length scale structural analysis follows the thermal history of amyloid oligomers from different perspectives and highlights the impact of hierarchical self-assembly of biological macromolecules on functional properties.

Mineral Crystal Thickness in Calcified Cartilage and Subchondral Bone in Healthy and Osteoarthritic Human Knees.

Osteoarthritis (OA) is the most common joint disease, where articular cartilage degradation is often accompanied with sclerosis of the subchondral bone. However, the association between OA and tissue mineralization at the nanostructural level is currently not understood. In particular, it is technically challenging to study calcified cartilage, where relevant but poorly understood pathological processes such as tidemark multiplication and advancement occur. Here, we used state-of-the-art microfocus small-angle X-ray scattering with a 5-µm spatial resolution to determine the size and organization of the mineral crystals at the nanostructural level in human subchondral bone and calcified cartilage. Specimens with a wide spectrum of OA severities were acquired from both medial and lateral compartments of medial compartment knee OA patients (n = 15) and cadaver knees (n = 10). Opposing the common notion, we found that calcified cartilage has thicker and more mutually aligned mineral crystals than adjoining bone. In addition, we, for the first time, identified a well-defined layer of calcified cartilage associated with pathological tidemark multiplication, containing 0.32 nm thicker crystals compared to the rest of calcified cartilage. Finally, we found 0.2 nm thicker mineral crystals in both tissues of the lateral compartment in OA compared with healthy knees, indicating a loading-related disease process because the lateral compartment is typically less loaded in medial compartment knee OA. In summary, we report novel changes in mineral crystal thickness during OA. Our data suggest that unloading in the knee might be involved with the growth of mineral crystals, which is especially evident in the calcified cartilage. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Potential of curcumin-loaded cubosomes for topical treatment of cervical cancer.

Cervical cancer is one of the most common cancers affecting women worldwide. There are an estimated 570.000 new cases of cervical cancer each year and conventional treatments can cause severe side effects. In this work, we developed a platform for vaginal administration of lipophilic drugs for cervical cancer treatment. We formulated mucoadhesive cubosomes for the delivery of curcumin, a lipophilic drug for cervical cancer treatment, to increase its bioavailability and local absorption. This study tests the use of cubosomes for vaginal drug administration and assesses their potential efficiency using the CAM (chick embryo chorioallantoic membrane) model. SAXS (small-angle X-ray scattering), cryo-TEM (cryo-transmission electron microscopy), and dynamic light scattering (DLS) were employed to characterise the system. With ex vivo permeation and retention studies, we find that the curcumin released from our system is retained in the vaginal mucosa. In vitro cytotoxicity assay and cellular uptake showed an increased cytotoxic effect of curcumin against HeLa cell line when incorporated into the cubosomes. The curcumin-loaded cubosomes also demonstrated an antiangiogenic effect evaluated in vivo by the CAM model.

Hierarchical Structure of Cellulose Nanofibril-Based Foams Explored by Multimodal X-ray Scattering.

Structural characterization techniques are fundamental to correlate the material macro-, nano-, and molecular-scale structures to their macroscopic properties and to engineer hierarchical materials. Here, we combine X-ray transmission with scanning small- and wide-angle X-ray scattering (sSWAXS) to investigate ultraporous and lightweight biopolymer-based foams using cellulose nanofibrils (CNFs) as building blocks. The power of multimodal sSWAXS for multiscale structural characterization of self-assembled CNFs is demonstrated by spatially resolved maps at the macroscale (foam density and porosity), at the nanoscale (foam structural compactness, CNF orientation in the foam walls, and CNF packing state), and at the molecular scale (cellulose crystallite dimensions). Specifically, we compare the impact of freeze-thawing-drying (FTD) fabrication steps, such as static/stirred freezing and thawing in ethanol/water, on foam structural hierarchy spanning from the molecular to the millimeter scale. As such, we demonstrate the potential of X-ray scattering imaging for hierarchical characterization of biopolymers.

Transformer-Induced Metamorphosis of Polymeric Nanoparticle Shape at Room Temperature.

Controlled polymerizations have enabled the production of nanostructured materials with different shapes, each exhibiting distinct properties. Despite the importance of shape, current morphological transformation strategies are limited in polymer scope, alter the chemical structure, require high temperatures, and are fairly tedious. Herein we present a rapid and versatile morphological transformation strategy that operates at room temperature and does not impair the chemical structure of the constituent polymers. By simply adding a molecular transformer to an aqueous dispersion of polymeric nanoparticles, a rapid evolution to the next higher-order morphology was observed, yielding a range of morphologies from a single starting material. Significantly, this approach can be applied to nanoparticles produced by disparate block copolymers obtained by various synthetic techniques including emulsion polymerization, polymerization-induced self-assembly and traditional solution self-assembly.

Oat Plant Amyloids for Sustainable Functional Materials.

Amyloid functional materials from amyloid fibril building blocks, produced in vitro from amyloidogenic natural proteins or synthetic peptides, show diverse functionalities ranging from environmental science and biomedicine, to nanotechnology and biomaterials. However, sustainable and affordable sources of amyloidogenic proteins remain the bottleneck for large-scale applications, and to date, interest remains essentially limited to fundamental studies. Plant-derived proteins would be an ideal source due to their natural abundance and low environmental impact. Hereby oat globulin, the primary protein of oat plant (Avena sativa), is utilized to yield high-quality amyloid fibrils and functional materials based thereof. These fibrils show a rich multistranded ribbon-like polymorphism and a fibrillization process with both irreversible and reversible pathways. The authors furthermore fabricate oat-amyloid aerogels, films, and membranes for possible use in water purification, sensors, and patterned electrodes. The sustainability footprint of oat-amyloids against other protein sources is demonstrated, anticipating an environmentally-efficient platform for advanced materials and technologies.