An interaction network approach predicts protein cage architectures in bionanotechnology.

Protein nanoparticles play pivotal roles in many areas of bionanotechnology, including drug delivery, vaccination, and diagnostics. These technologies require control over the distinct particle morphologies that protein nanocontainers can adopt during self-assembly from their constituent protein components. The geometric construction principle of virus-derived protein cages is by now fairly well understood by analogy to viral protein shells in terms of Caspar and Klug's quasi-equivalence principle. However, many artificial, or genetically modified, protein containers exhibit varying degrees of quasi-equivalence in the interactions between identical protein subunits. They can also contain a subset of protein subunits that do not participate in interactions with other assembly units, called capsomers, leading to gaps in the particle surface. We introduce a method that exploits information on the local interactions between the capsomers to infer the geometric construction principle of these nanoparticle architectures. The predictive power of this approach is demonstrated here for a prominent system in nanotechnology, the AaLS pentamer. Our method not only rationalises hitherto discovered cage structures but also predicts geometrically viable options that have not yet been observed. The classification of nanoparticle architecture based on the geometric properties of the interaction network closes a gap in our current understanding of protein container structure and can be widely applied in protein nanotechnology, paving the way to programmable control over particle polymorphism.

Protein coupled thionine acetate probed silica nanoparticles: An integrated laser-assisted therapeutic approach for treating cancer.

Herein, we report a multifaceted nanoformulation, developed by binding thionine acetate (TA) in silica matrix to form TA loaded silica nanoparticles (STA Nps), which were characterized using various physicochemical techniques. STA NPs were spherical shaped having size 40-50 nm and exhibited good heating efficiency, improved photostability and singlet oxygen production rate than TA alone. In PDT experiment, the rate of degradation for ABDMA was enhanced from 0.1367 min-1 for TA alone to 0.1774 min-1 for STA Nps, depicting an increase in the reactive oxygen species (ROS) generation ability of STA Nps. Further, the cytotoxicity of STA Nps was investigated by carrying out the biophysical studies with Calf thymus DNA (Ct-DNA) and Human Serum Albumin (HSA). The results indicated that the binding of STA Nps to Ct-DNA causes alterations in the double helix structure of DNA and as a result, STA Nps can impart chemotherapeutic effects via targeting DNA. STA Nps showed good binding affinity with HSA without compromising the structure of HSA, which is important for STA Nps sustainable biodistribution and pharmacokinetics. Based on this study, it is suggested that because of the synergistic effect of chemo and phototherapy, STA Nps can be extensively utilized as potential candidates for treating cancer.

Ultrasmall Gold Nanoparticles as Clients of Biomolecular Condensates.

Liquid-liquid phase separation (LLPS) of biopolymers to form condensates is a widespread phenomenon in living cells. Agents that target or alter condensation can help uncover elusive physiological and pathological mechanisms. Owing to their unique material properties and modes of interaction with biomolecules, nanoparticles represent attractive condensate-targeting agents. Our work focused on elucidating the interaction between ultrasmall gold nanoparticles (usGNPs) and diverse types of condensates of tau, a representative phase-separating protein associated with neurodegenerative disorders. usGNPs attract considerable interest in the biomedical community due to unique features, including emergent optical properties and good cell penetration. We explored the interaction of usGNPs with reconstituted self-condensates of tau, two-component tau/polyanion and three-component tau/RNA/alpha-synuclein coacervates. The usGNPs were found to concentrate into condensed liquid droplets, consistent with the formation of dynamic client (nanoparticle) - scaffold (tau) interactions, and were observable thanks to their intrinsic luminescence. Furthermore, usGNPs were capable to promote LLPS of a protein domain which is unable to phase separate on its own. Our study demonstrates the ability of usGNPs to interact with and illuminate protein condensates. We anticipate that nanoparticles will have broad applicability as nanotracers to interrogate phase separation, and as nanoactuators controlling the formation and dissolution of condensates.

Simple plant-based production and purification of the assembled human ferritin heavy chain as a nanocarrier for tumor-targeted drug delivery and bioimaging in cancer therapy.

Nanoparticles are used as carriers for the delivery of drugs and imaging agents. Proteins are safer than synthetic nanocarriers due to their greater biocompatibility and the absence of toxic degradation products. In this context, ferritin has the additional benefit of inherently targeting the membrane receptor transferrin 1, which is overexpressed by most cancer cells. Furthermore, this self-assembling multimeric protein can be loaded with more than 2000 iron atoms, as well as drugs, contrast agents, and other cargos. However, recombinant ferritin currently costs ~3.5 million € g-1 , presumably because the limited number of producers cannot meet demand, making it generally unaffordable as a nanocarrier. Because plants can produce proteins at very-large-scale, we developed a simple, proof-of-concept process for the production of the human ferritin heavy chain by transient expression in Nicotiana benthamiana. We optimized the protein yields by screening different compartments and 5'-untranslated regions in PCPs, and selected the best-performing construct for production in differentiated plants. We then established a rapid and scalable purification protocol by combining pH and heat treatment before extraction, followed by an ultrafiltration/diafiltration size-based separation process. The optimized process achieved ferritin levels of ~40 mg kg-1 fresh biomass although depth filtration limited product recovery to ~7%. The purity of the recombinant product was >90% at costs ~3% of the current sales price. Our method therefore allows the production of affordable ferritin heavy chain as a carrier for therapeutic and diagnostic agents, which is suitable for further stability and functionality testing in vitro and in vivo.

Secured delivery of basic fibroblast growth factor using human serum albumin-based protein nanoparticles for enhanced wound healing and regeneration.

BACKGROUND: Basic fibroblast growth factor (bFGF) is one of the critical components accelerating angiogenesis and tissue regeneration by promoting the migration of dermal fibroblasts and endothelial cells associated with matrix formation and remodeling in wound healing process. However, clinical applications of bFGF are substantially limited by its unstable nature due to rapid decomposition under physiological microenvironment. RESULTS: In this study, we present the bFGF-loaded human serum albumin nanoparticles (HSA-bFGF NPs) as a means of enhanced stability and sustained release platform during tissue regeneration. Spherical shape of the HSA-bFGF NPs with uniform size distribution (polydispersity index < 0.2) is obtained via a simple desolvation and crosslinking process. The HSA-bFGF NPs securely load and release the intact soluble bFGF proteins, thereby significantly enhancing the proliferation and migration activity of human dermal fibroblasts. Myofibroblast-related genes and proteins were also significantly down-regulated, indicating decrease in risk of scar formation. Furthermore, wound healing is accelerated while achieving a highly organized extracellular matrix and enhanced angiogenesis in vivo. CONCLUSION: Consequently, the HSA-bFGF NPs are suggested not only as a delivery vehicle but also as a protein stabilizer for effective wound healing and tissue regeneration.

Monophosphoryl lipid A-adjuvanted nucleoprotein-neuraminidase nanoparticles improve immune protection against divergent influenza viruses.

Universal influenza vaccines are urgently needed to prevent recurrent influenza epidemics and inevitable pandemics. We generated double-layered protein nanoparticles incorporating two conserved influenza antigens-nucleoprotein and neuraminidase-through a two-step desolvation-crosslinking method. These protein nanoparticles displayed immunostimulatory properties to antigen-presenting cells by promoting inflammatory cytokine (IL-6 and TNF-α) secretion from JAWS II dendric cells. The nanoparticle immunization induced significant antigen-specific humoral and cellular responses, including antigen-binding and neutralizing antibodies, antibody- and cytokine (IFN-γ and IL-4)-secreting cells, and NP147-155 tetramer-specific cytotoxic T lymphocyte (CTL) responses. Co-administration of monophosphoryl lipid A (MPLA, a toll-like receptor 4 agonist) with the protein nanoparticles further improved immune responses and conferred heterologous and heterosubtypic influenza protection. The MPLA-adjuvanted nanoparticles reduced lung inflammation post-infection. The results demonstrated that the combination of MPLA and conserved protein nanoparticles could be developed into an improved universal influenza vaccine strategy.

Role of proteins in the biosynthesis and functioning of metallic nanoparticles.

Proteins are known to play important roles in the biosynthesis of metallic nanoparticles (NPs), which are biological substitutes for conventionally used chemical capping and stabilizing agents. When a pristine nanoparticle comes in contact with a biological media or system, a bimolecular layer is formed on the surface of the nanoparticle and is primarily composed of proteins. The role of proteins in the biosynthesis and further uptake, translocation, and bio-recognition of nanoparticles is documented in the literature. But, a complete understanding has not been achieved concerning the mechanism for protein-mediated nanoparticle biosynthesis and the role proteins play in the interaction and recognition of nanoparticles, aiding its uptake and assimilation into the biological system. This review critically evaluates the knowledge and gaps in the protein-mediated biosynthesis of nanoparticles. In particular, we review the role of proteins in multiple facets of metallic nanoparticle biosynthesis, the interaction of proteins with metallic nanoparticles for recognition and interaction with cells, and the toxic potential of protein-nanoparticle complexes when presented to the cell.

Protein nanoparticle-induced osmotic pressure gradients modify pulmonary edema through hyperpermeability in acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS), caused by noncardiogenic pulmonary edema (PE), contributes significantly to Coronavirus 2019 (COVID-19)-associated morbidity and mortality. We explored the effect of transmembrane osmotic pressure (OP) gradients in PE using a fluorescence resonance energy transfer-based Intermediate filament (IF) tension optical probe. Angiotensin-II- and bradykinin-induced increases in intracellular protein nanoparticle (PN)-OP were associated with inflammasome production and cytoskeletal depolymerization. Intracellular protein nanoparticle production also resulted in cytomembrane hyperpolarization and L-VGCC-induced calcium signals, which differed from diacylglycerol-induced calcium increment via TRPC6 activation. Both pathways involve voltage-dependent cation influx and OP upregulation via SUR1-TRPM4 channels. Meanwhile, intra/extracellular PN-induced OP gradients across membranes upregulated pulmonary endothelial and alveolar barrier permeability. Attenuation of intracellular PN, calcium signals, and cation influx by drug combinations effectively relieved intracellular OP and pulmonary endothelial nonselective permeability, and improved epithelial fluid absorption and PE. Thus, PN-OP is pivotal in pulmonary edema in ARDS and COVID-19, and transmembrane OP recovery could be used to treat pulmonary edema and develop new drug targets in pulmonary injury.

Aggregation Properties of Albumin in Interacting with Magnetic Fluids.

In this study, interactions of Fe3O4 magnetic nanoparticles with serum albumin biomolecules in aqueous solutions were considered. The studies were conducted with the laser correlation spectroscopy and optical analysis of dehydrated films. It was shown that the addition of magnetite to an albumin solution at low concentrations of up to 10-6 g/L led to the formation of aggregates with sizes of up to 300 nm in the liquid phase and an increase in the number of spiral structures in the dehydrated films, which indicated an increase in their stability. With a further increase in the magnetite concentration in the solution (from 10-4 g/L), the magnetic particles stuck together and to albumin, thus forming aggregates with sizes larger than 1000 nm. At the same time, the formation of morphological structures in molecular films was disturbed, and a characteristic decrease in their stability occurred. Most stable films were formed at low concentrations of magnetic nanoparticles (less than 10-4 g/L) when small albumin-magnetic nanoparticle aggregates were formed. These results are important for characterizing the interaction processes of biomolecules with magnetic nanoparticles and can be useful for predicting the stability of biomolecular films with the inclusion of magnetite particles.

Analyzing the adhesion mechanism of FnBPA, a surface adhesin from Staphylococcus aureus on its interaction with nanoparticle.

Staphylococcus aureus expresses many Microbial Surface Recognizing Adhesive Matrix Molecules (MSCRAMM's) to recognize host extracellular matrix (ECM) molecules to initiate colonization. The MSCRAMM, fibronectin binding protein A (FnBPA), is an important adhesin for S. aureus infection. FnBPA also binds with fibrinogen (Fg) by using a unique ligand binding mechanism called dock, lock and latch. Nanoparticles, especially nanosilver particles have been widely used in a variety of biomedical applications which includes disease diagnosis and treatment, drug delivery and implanted medical device coating. In a biological system, when protein molecules encounter nanoparticle, they can be absorbed onto its surface which results in the formation of protein corona. In the present study, we have analysed the fibrinogen binding ability of rFnBPA(189-512) in the presence of silver nanoparticles by employing techniques like gel shift assay, Western blot, size exclusion chromatography, enzyme-linked immunosorbent assay, bio-layer interferometry and circular dichroism spectroscopy. The results indicate that rFnBPA(189-512) is unable to bind to Fg in the presence of a nanoparticle. This could be due to the inaccessibility of the Fg binding site and conformational change in rFnBPA(189-512). With nanoparticles, rFnBPA(189-512) undergoes significant structural changes as the ß-sheet content has drastically reduced to 10% from the initial 60% at higher concentration of the nanoparticle. Pathogenic bacteria interact with its surrounding environment through their surface molecules which includes MSCRAMMs. Therefore MSCRAMMs play an important role when bacteria encounter nanoparticles. The results of the present study suggest that the orientation of the protein during the absorption on the surface of a nanoparticle as well as the concentration of the nanoparticle, will dictate the function of the absorbed protein and in this case the Fg binding property of rFnBPA(189-512).

Insights into a Protein-Nanoparticle System by Paramagnetic Perturbation NMR Spectroscopy.

BACKGROUND: The interaction between proteins and nanoparticles is a very relevant subject because of the potential applications in medicine and material science in general. Further interest derives from the amyloidogenic character of the considered protein, ß2-microglobulin (ß2m), which may be regarded as a paradigmatic system for possible therapeutic strategies. Previous evidence showed in fact that gold nanoparticles (AuNPs) are able to inhibit ß2m fibril formation in vitro. METHODS: NMR (Nuclear Magnetic Resonance) and ESR (Electron Spin Resonance) spectroscopy are employed to characterize the paramagnetic perturbation of the extrinsic nitroxide probe Tempol on ß2m in the absence and presence of AuNPs to determine the surface accessibility properties and the occurrence of chemical or conformational exchange, based on measurements conducted under magnetization equilibrium and non-equilibrium conditions. RESULTS: The nitroxide perturbation analysis successfully identifies the protein regions where protein-protein or protein-AuNPs interactions hinder accessibility or/and establish exchange contacts. These information give interesting clues to recognize the fibrillation interface of ß2m and hypothesize a mechanism for AuNPs fibrillogenesis inhibition. CONCLUSIONS: The presented approach can be advantageously applied to the characterization of the interface in protein-protein and protein-nanoparticles interactions.

How the surface functionalized nanoparticles affect conformation and activity of proteins: Exploring through protein-nanoparticle interactions.

To understand the effect of counter ions (Na+) on the secondary conformation and functionality of the lysozyme, we have studied the interaction of lysozyme with counterion associated iron oxide nanoparticles (IONPs). The investigation was carried out at pH 7.4 and 9.0, with three different types of NPs, namely, bare IONPs, low molecular weight chitosan modified IONPs (LMWC-IONPs) and the counterion (Na+) associated sodium tripolyphosphate IONPs (STP-LMWC-IONPs) and confirmed by using various spectroscopy techniques. The difference in UV-vis absorbance (ΔA) between native and STP-LMWC-IONPs interacted hen egg white lysozyme (HEWL) was greater than that between native and NPs interacted HEWL at pH 9.0 compared with pH 7.4. Furthermore, STP-LMWC-IONPs exhibited quenching effect on lysozyme fluorescence spectrum at pH 9.0 due to binding of Na+ counterions to the protein, confirming denaturation of the latter. After HEWL interaction with STP-LMWC-IONPs (pH 9.0), CD spectra revealed a conformational change in the secondary structure of HEWL. Also, counterion induced lysozyme inactivation, due to interaction with nanoparticles at pH 9.0, was confirmed by enzymatic activity assay involving lysis of Micrococcus lysodeikticus. In conclusion, pH 9.0 was observed to be a more favorable condition, compared to pH 7.4, for the strongest electrostatic interaction between lysozyme and NPs. We postulate that the counterions in nanoparticle surface-coating can ameliorate protein misfolding or unfolding and also prevent their aggregation and, therefore, can be considered as a powerful and potential therapeutic strategy to treat incurable neurodegenerative disorders.

Specific Interaction Sites Determine Differential Adsorption of Protein Structural Isomers on Nanoparticle Surfaces.

In biological systems, nanoparticles (NPs) elicit bioactivity upon interaction with proteins. As a result of post-translational modification, proteins occur in a variety of alternative covalent forms, including structural isomers, which present unique molecular surfaces. We aimed at a detailed description of the recognition of protein isomeric species by NP surfaces. The transient adsorption of isomeric ubiquitin (Ub) dimers by NPs was investigated by solution NMR spectroscopy. Lys63- and Lys48-linked Ub2 were adsorbed by large anionic NPs with different affinities, whereas the binding strength was similar in the cases of smaller particles. After the incorporation of paramagnetic tags into NPs, the observed site-resolved paramagnetic footprints provided a high-resolution map of the different protein surfaces binding to NPs. The approach described could be extended to further protein isoforms and more specialized NP systems to allow better control of the interactions between NPs and protein targets.

Peptide ligand-mediated endocytosis of nanoparticles to cancer cells: Cell receptor-binding- versus cell membrane-penetrating peptides.

The endocytosis-mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands-human ferritin heavy chain (hFTH) nanoparticle. Twenty-four copies of a CMPP(human immunodeficiency virus-derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(αv ß3 ) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle. The quantitative level of endocytosis and intracellular localization of fluorescence dye-labeled CRBP- and CMPP-presenting nanoparticles were estimated in the in vitro cultures of integrin- and EGFR-overexpressing cancer and human dermal fibroblast cells(control). From the cancer cell cultures treated with the CMPP- and CRBP-presenting nanoparticles, it was notable that CRBPs resulted in quantitatively higher level of endocytosis than CMPP (TAT) and successfully transported the nanoparticles to the cytosol of cancer cells depending on concentration and treatment period of time, whereas TAT-mediated endocytosis localized most of the nanoparticles within endosomal vesicles under the same conditions. These novel findings provide highly useful informations to many researchers both in academia and in industry who are interested in developing anticancer drug delivery systems/carriers.

Fabrication of Polymer-Protein Hybrids.

Rapid developments in organic chemistry and polymer chemistry promote the synthesis of polymer-protein hybrids with different structures and biofunctionalities. In this feature article, recent progress achieved in the synthesis of polymer-protein conjugates, protein-nanoparticle core-shell structures, and polymer-protein nanogels/hydrogels is briefly reviewed. The polymer-protein conjugates can be synthesized by the "grafting-to" or the "grafting-from" approach. In this article, different coupling reactions and polymerization methods used in the synthesis of bioconjugates are reviewed. Protein molecules can be immobilized on the surfaces of nanoparticles by covalent or noncovalent linkages. The specific interactions and chemical reactions employed in the synthesis of core-shell structures are discussed. Finally, a general introduction to the synthesis of environmentally responsive polymer-protein nanogels/hydrogels by chemical cross-linking reactions or molecular recognition is provided.

Mild Binding of Protein to C2 N Monolayer Reveals Its Suitable Biocompatibility.

The development of biocompatible nanomaterials for smart drug delivery and bioimaging has attracted great interest in recent years in biomedical fields. Here, the interaction between the recently reported nitrogenated graphene (C2 N) and a prototypical protein (villin headpiece HP35) utilizing atomistic molecular dynamics simulations is studied. The simulations reveal that HP35 can form a stable binding with the C2 N monolayer. Although the C2 N-HP35 attractive interactions are constantly preserved, the binding strength between C2 N and the protein is mild and does not cause significant distortion in the protein's structural integrity. This intrinsic biofriendly property of native C2 N is distinct from several widely studied nanomaterials, such as graphene, carbon nanotubes, and MoS2 , which can induce severe protein denaturation. Interestingly, once the protein is adsorbed onto C2 N surface, its transverse migration is highly restricted at the binding sites. This restriction is orchestrated by C2 N's periodic porous structure with negatively charged "holes," where the basic residues-such as lysine-can form stable interactions, thus functioning as "anchor points" in confining the protein displacement. It is suggested that the mild, immobilized protein attraction and biofriendly aspects of C2 N would make it a prospective candidate in bio- and medical-related applications.

Towards interpretation of intermolecular paramagnetic relaxation enhancement outside the fast exchange limit.

In an exchanging system between major and minor species, the transverse paramagnetic relaxation enhancement rate observed on the resonances of the major species (Γ 2 (app) ) is dependent upon the exchange regime between the species. Quantitative analysis of PRE data in such systems typically assumes that the overall exchange rate k ex between the species is fast on the PRE time scale (k ex â‰« Γ2). Recently, we have characterized the kinetics of binding of the model protein ubiquitin to large (LUV) and small (SUV) unilamellar lipid-based nanoparticles or liposomes (Ceccon A, Tugarinov V, Bax A, Clore GM (2016). J Am Chem Soc 138:5789-5792). Building upon these results and taking advantage of a strong paramagnetic agent with an isotropic g-tensor, Gd(3+), we were able to measure intermolecular methyl carbon and proton PREs between paramagnetically-tagged liposomes and ubiquitin. In the limit of fast exchange (k ex â‰« Γ2) the ratio of the apparent proton to carbon methyl PREs, ((1)Hm-Γ 2 (app) )/((13)Cm-Γ 2 (app) ), is equal to the square of the ratio of the gyromagnetic ratios of the two nuclei, (γΗ/γC)(2). However, outside the fast exchange regime, under intermediate exchange conditions (e.g. when Γ2 is comparable in magnitude to k ex) the ((1)Hm-Γ 2 (app) )/((13)Cm-Γ 2 (app) ) ratio provides a reliable measure of the 'true' methyl PREs.

α-Galactosidase delivery using 30Kc19-human serum albumin nanoparticles for effective treatment of Fabry disease.

Fabry disease is a genetic lysosomal storage disease caused by deficiency of α-galactosidase, the enzyme-degrading neutral glycosphingolipid that is transported to lysosome. Glycosphingolipid accumulation by this disease causes multi-organ dysfunction and premature death of the patient. Currently, enzyme replacement therapy (ERT) using recombinant α-galactosidase is the only treatment available for Fabry disease. To maximize the efficacy of treatment, enhancement of cellular delivery and enzyme stability is a challenge in ERT using α-galactosidase. In this study, protein nanoparticles using human serum albumin (HSA) and 30Kc19 protein, originating from silkworm, were used to enhance the delivery and intracellular α-galactosidase stability. 30Kc19-HSA nanoparticles loaded with the α-galactosidase were formed by desolvation method. 30Kc19-HSA nanoparticles had a uniform spherical shape and were well dispersed in cell culture media. 30Kc19-HSA nanoparticles had negligible toxicity to human cells. The nanoparticles exhibited enhanced cellular uptake and intracellular stability of delivered α-galactosidase in human foreskin fibroblast. Additionally, they showed enhanced globotriaosylceramide degradation in Fabry patients' fibroblasts. It is expected that 30Kc19-HSA protein nanoparticles could be used as an effective tool for efficient delivery and enhanced stability of drugs.

Packaging guest proteins into the encapsulin nanocompartment from Rhodococcus erythropolis N771.

The encapsulin nanocompartment from Rhodococcus erythropolis N771 (Reencapsulin) was expressed and purified in wild-type and C-terminally His-tagged forms. Negative-stained transmission electron microscopy, field-flow fractionation combined with multi-angle light scattering and dynamic light scattering analyses showed that 60 Reencapsulin monomers were assembled as a spherical particle with a diameter of 28 nm. Heterogeneous guest proteins such as EGFP and firefly luciferase were packaged into the internal cavity of the Reencapsulin nanocompartment by fusing the C-terminal 37-amino-acid sequence of the R. erythropolis N771 DypB peroxidase to the C-terminus. Reencapsulin has the potential to package target proteins in its internal cavity and/or display them on its external surface, making it a feasible carrier for nanotechnology applications.

Optimizing the design of protein nanoparticles as carriers for vaccine applications.

Successful vaccine development remains a huge challenge for infectious diseases such as malaria, HIV and influenza. As a novel way to present antigenic epitopes to the immune system, we have developed icosahedral self-assembling protein nanoparticles (SAPNs) to serve as a prototypical vaccine platform for infectious diseases. Here we examine some biophysical factors that affect the self-assembly of these nanoparticles, which have as basic building blocks coiled-coil oligomerization domains joined by a short linker region. Relying on in silico computer modeling predictions, we selected five different linker regions from the RCSB protein database that connect oligomerization domains, and then further studied the self-assembly and stability of in vitro produced nanoparticles through biophysical characterization of formed particles. One design in particular, T2i88, revealed excellent self-assembly and homogeneity thus paving the way toward a more optimized nanoparticle for vaccine applications. FROM THE CLINICAL EDITOR: Despite the widespread use of vaccines worldwide, successful development of vaccines against some diseases remains a challenge still. In this article, the authors investigated the physic-chemical and biological properties of icosahedral self-assembling protein nanoparticles (SAPNs), which mimic viral particles, in order to utilize this technology as potential platform for future design of vaccines.