Stable nanovesicles formed by intrinsically planar bilayers.

HYPOTHESIS: Quatsome nanovesicles, formed through the self-assembly of cholesterol (CHOL) and cetyltrimethylammonium bromide (CTAB) in water, have shown long-term stability in terms of size and morphology, while at the same time exhibiting high CHOL-CTAB intermolecular binding energies. We hypothesize that CHOL/CTAB quatsomes are indeed thermodynamically stable nanovesicles, and investigate the mechanism underlying their formation. EXPERIMENTS: A systematic study was performed to determine whether CHOL/CTAB quatsomes satisfy the experimental requisites of thermodynamically stable vesicles. Coarse-grain molecular dynamics simulations were used to investigate the molecular organization in the vesicle membrane, and the characteristics of the simulated vesicle were corroborated with experimental data obtained by cryo-electron microscopy, small- and wide-angle X-ray scattering, and multi-angle static light scattering. FINDINGS: CHOL/CTAB quatsomes fulfill the requisites of thermodynamically stable nanovesicles, but they do not exhibit the classical membrane curvature induced by a composition asymmetry between the bilayer leaflets, like catanionic nanovesicles. Instead, CHOL/CTAB quatsomes are formed through the association of intrinsically planar bilayers in a faceted vesicle with defects, indicating that distortions in the organization and orientation of molecules can play a major role in the formation of thermodynamically stable nanovesicles.

Methods for the Characterization of Protein Aggregates.

The physicochemical characterization of protein aggregates yields an important contribution to further our understanding on many diseases for which the formation of protein aggregates is one of the pathological hallmarks. On the other hand, bacterial inclusion bodies (IBs) have recently been shown to be highly pure proteinaceous aggregates of a few hundred nanometers, produced by recombinant bacteria supporting the biological activities of the embedded polypeptides. Despite the wide spectrum of uses of IBs as functional and biocompatible materials upon convenient engineering, very few is known about their physicochemical properties.In this chapter we present methods for the characterization of protein aggregates as particulate materials relevant to their physicochemical and nanoscale properties.Specifically, we describe the use of dynamic light scattering (DLS) for sizing, nanoparticle tracking analysis for sizing and counting, and zeta potential measurements for the determination of colloidal stability. To study the morphology of protein aggregates we present the use of atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cryo-transmission electron microscopy (cryo-TEM) will be used for the determination of the internal structuration. Moreover, wettability and nanomechanical characterization can be performed using contact angle (CA) and force spectroscopic AFM (FS-AFM) measurements of the proteinaceous nanoparticles, respectively. Finally, the 4'4-dithiodipyridine (DTDP) method is presented as a way of relatively quantifying accessible sulfhydryl groups in the structure of the nanoparticle .The physical principles of the methods are briefly described and examples are given to help clarify capabilities of each technique.

Methods for Processing Protein Aggregates into Surfaces.

The processing of inclusion bodies (IBs) into surfaces is of great interest for cell culture applications due to the combined physical and biological cues these particles provide. The arrangement of these IBs into defined and tunable micropatterns can be useful for basic research purposes regarding the mechanical properties needed for cell adhesion and migration, among other responses. There are several approaches that can be used when functionalizing a substrate with IBs, regarding both the strategy used and also the kind of surface-particle interaction. The interaction between surface and IB can be mainly of three types: physisorption, electrostatic or covalent. This interaction can be controlled by depositing an appropriate self-assembled monolayer (SAM) on top of a substrate as an interface. Furthermore, several strategies can be used to immobilize IBs on surfaces in various configurations, like random deposition, micrometric printed geometries or gradient patterns.

Characterising a human endogenous retrovirus(HERV)-derived tumour-associated antigen: enriched RNA-Seq analysis of HERV-K(HML-2) in mantle cell lymphoma cell lines.

BACKGROUND: The cell-surface attachment protein (Env) of the HERV-K(HML-2) lineage of endogenous retroviruses is a potentially attractive tumour-associated antigen for anti-cancer immunotherapy. The human genome contains around 100 integrated copies (called proviruses or loci) of the HERV-K(HML-2) virus and we argue that it is important for therapy development to know which and how many of these contribute to protein expression, and how this varies across tissues. We measured relative provirus expression in HERV-K(HML-2), using enriched RNA-Seq analysis with both short- and long-read sequencing, in three Mantle Cell Lymphoma cell lines (JVM2, Granta519 and REC1). We also confirmed expression of the Env protein in two of our cell lines using Western blotting, and analysed provirus expression data from all other relevant published studies. RESULTS: Firstly, in both our and other reanalysed studies, approximately 10% of the transcripts mapping to HERV-K(HML-2) came from Env-encoding proviruses. Secondly, in one cell line the majority of the protein expression appears to come from one provirus (12q14.1). Thirdly, we find a strong tissue-specific pattern of provirus expression. CONCLUSIONS: A possible dependency of Env expression on a single provirus, combined with the earlier observation that this provirus is not present in all individuals and a general pattern of tissue-specific expression among proviruses, has serious implications for future HERV-K(HML-2)-targeted immunotherapy. Further research into HERV-K(HML-2) as a possible tumour-associated antigen in blood cancers requires a more targeted, proteome-based, screening protocol that will consider these polymorphisms within HERV-K(HML-2). We include a plan (and necessary alignments) for such work.

Correction to: Characterising a human endogenous retrovirus(HERV)-derived tumour-associated antigen: enriched RNA-Seq analysis of HERV-K(HML-2) in mantle cell lymphoma cell lines.

An amendment to this paper has been published and can be accessed via the original article.

High-Throughput Cell Motility Studies on Surface-Bound Protein Nanoparticles with Diverse Structural and Compositional Characteristics.

Eighty areas with different structural and compositional characteristics made of bacterial inclusion bodies formed by the fibroblast growth factor (FGF-IBs) were simultaneously patterned on a glass surface with an evaporation-assisted method that relies on the coffee-drop effect. The resulting surface patterned with these protein nanoparticles enabled to perform a high-throughput study of the motility of NIH-3T3 fibroblasts under different conditions including the gradient steepness, particle concentrations, and area widths of patterned FGF-IBs, using for the data analysis a methodology that includes "heat maps". From this analysis, we observed that gradients of concentrations of surface-bound FGF-IBs stimulate the total cell movement but do not affect the total net distances traveled by cells. Moreover, cells tend to move toward an optimal intermediate FGF-IB concentration (i.e., cells seeded on areas with high IB concentrations moved toward areas with lower concentrations and vice versa, reaching the optimal concentration). Additionally, a higher motility was obtained when cells were deposited on narrow and highly concentrated areas with IBs. FGF-IBs can be therefore used to enhance and guide cell migration, confirming that the decoration of surfaces with such IB-like protein nanoparticles is a promising platform for regenerative medicine and tissue engineering.

Surface-Bound Gradient Deposition of Protein Nanoparticles for Cell Motility Studies.

A versatile evaporation-assisted methodology based on the coffee-drop effect is described to deposit nanoparticles on surfaces, obtaining for the first time patterned gradients of protein nanoparticles (pNPs) by using a simple custom-made device. Fully controllable patterns with specific periodicities consisting of stripes with different widths and distinct nanoparticle concentration as well as gradients can be produced over large areas (∼10 cm2) in a fast (up to 10 mm2/min), reproducible, and cost-effective manner using an operational protocol optimized by an evolutionary algorithm. The developed method opens the possibility to decorate surfaces "a-la-carte" with pNPs enabling different categories of high-throughput studies on cell motility.

Functional protein-based nanomaterial produced in microorganisms recognized as safe: A new platform for biotechnology.

UNLABELLED: Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest. STATEMENT OF SIGNIFICANCE: The development of both natural and synthetic biomaterials for biomedical applications is a field in constant development. In this context, E. coli is a bacteria that has been widely studied for its ability to naturally produce functional biomaterials with broad biomedical uses. Despite being effective, products derived from this species contain membrane residues able to trigger a non-desired immunogenic responses. Accordingly, exploring alternative bacteria able to synthesize such biomaterials in a safe molecular environment is becoming a challenge. Thus, the present study describes a new type of functional protein-based nanomaterial free of toxic contaminants with a wide range of applications in both human and veterinary medicine.

Methods for characterization of protein aggregates.

Physicochemical characterization of protein aggregates is important on one hand, due to its large impact in understanding many diseases for which formation of protein aggregates is one of the pathological hallmarks. On the other hand, recently it has been observed that bacterial inclusion bodies (IBs) are also highly pure proteinaceous aggregates of a few hundred nanometers produced by recombinant bacteria supporting the biological activities of the embedded polypeptides. From this fact arises a wide spectrum of uses of IBs as functional and biocompatible materials upon convenient engineering but very few is known about their physicochemical properties. In this chapter we present methods for the characterization of protein aggregates as particulate materials relevant to their physicochemical and nanoscale properties. Specifically, we describe the use of infrared spectroscopy (IR) for the determination of the secondary structure, dynamic light scattering (DLS) for sizing, nanosight for sizing and counting, and Z-potential measurements for the determination of colloidal stability. To study their morphology we present the use of atomic force microscopy (AFM). Cryo-transmission electron microscopy will be used for the determination of the internal structuration. Moreover, wettability and nanomechanical characterization can be performed using contact angle (CA) and force spectroscopic AFM measurements of the proteinaceous nanoparticles, respectively. The physical principles of the methods are briefly described and examples of data for real samples and how that data is interpreted are given to help clarify capabilities of each technique.

Integrating mechanical and biological control of cell proliferation through bioinspired multieffector materials.

In nature, cells respond to complex mechanical and biological stimuli whose understanding is required for tissue construction in regenerative medicine. However, the full replication of such bimodal effector networks is far to be reached. Engineering substrate roughness and architecture allows regulating cell adhesion, positioning, proliferation, differentiation and survival, and the external supply of soluble protein factors (mainly growth factors and hormones) has been long applied to promote growth and differentiation. Further, bioinspired scaffolds are progressively engineered as reservoirs for the in situ sustained release of soluble protein factors from functional topographies. We review here how research progresses toward the design of integrative, holistic scaffold platforms based on the exploration of individual mechanical and biological effectors and their further combination.

Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles.

CD44 is a multifunctional cell surface protein involved in proliferation and differentiation, angiogenesis and signaling. The expression of CD44 is up-regulated in several types of human tumors and particularly in cancer stem cells, representing an appealing target for drug delivery in the treatment of cancer. We have explored here several protein ligands of CD44 for the construction of self-assembling modular proteins designed to bind and internalize target cells. Among five tested ligands, two of them (A5G27 and FNI/II/V) drive the formation of protein-only, ring-shaped nanoparticles of about 14 nm that efficiently bind and penetrate CD44(+) cells by an endosomal route. The potential of these newly designed nanoparticles is evaluated regarding the need of biocompatible nanostructured materials for drug delivery in CD44-linked conditions.

In vivo architectonic stability of fully de novo designed protein-only nanoparticles.

The fully de novo design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein-protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the in vivo stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids.

Two-dimensional microscale engineering of protein-based nanoparticles for cell guidance.

Cell responses, such as positioning, morphological changes, proliferation, and apoptosis, are the result of complex chemical, topographical, and biological stimuli. Here we show the macroscopic responses of cells when nanoscale profiles made with inclusion bodies (IBs) are used for the 2D engineering of biological interfaces at the microscale. A deep statistical data treatment of fibroblasts cultivated on supports patterned with green fluorescent protein and human basic fibroblast growth factor-derived IBs demonstrates that these cells preferentially adhere to the IB areas and align and elongate according to specific patterns. These findings prove the potential of surface patterning with functional IBs as protein-based nanomaterials for tissue engineering.