A Versatile Virus-Mimetic Engineering Approach for Concurrent Protein Nanocage Surface-Functionalization and Cargo Encapsulation.

Naturally occurring protein nanocages like ferritin are self-assembled from multiple subunits. Because of their unique cage-like structure and biocompatibility, there is a growing interest in their biomedical use. A multipurpose and straightforward engineering approach does not exist for using nanocages to make drug-delivery systems by encapsulating hydrophilic or hydrophobic drugs and developing vaccines by surface functionalization with a protein like an antigen. Here, a versatile engineering approach is described by mimicking the HIV-1 Gap polyprotein precursor. Various PREcursors of nanoCages (PREC) are designed and created by linking two ferritin subunits via a flexible linker peptide containing a protease cleavage site. These precursors can have additional proteins at their N-terminus, and their protease cleavage generates ferritin-like nanocages named protease-induced nanocages (PINCs). It is demonstrated that PINC formation allows concurrent surface decoration with a protein and hydrophilic or hydrophobic drug encapsulation up to fourfold more than the amount achieved using other methods. The PINCs/Drug complex is stable and efficiently kills cancer cells. This work provides insight into the precursors' design rules and the mechanism of PINCs formation. The engineering approach and mechanistic insight described here will facilitate nanocages' applications in drug delivery or as a platform for making multifunctional therapeutics like mosaic vaccines.

Overcoming Non-Specific Interactions for Efficient Encapsulation of Doxorubicin in Ferritin Nanocages for Targeted Drug Delivery.

Due to its beneficial pharmacological properties, ferritin (Ftn) is considered as an interesting drug delivery vehicle to alleviate the cardiotoxicity of doxorubicin (DOX) in chemotherapy. However, the encapsulation of DOX in Ftn suffers from heavy precipitation and low protein recovery yield which limits its full potential. Here, a new DOX encapsulation strategy by cysteine-maleimide conjugation is proposed. In order to demonstrate that this strategy is more efficient compared to the other approaches, DOX is encapsulated in Ftn variants carrying different surface charges. Furthermore, in contrast to the common belief, this data show that DOX molecules are also found to bind non-specifically to the surface of Ftn. This can be circumvented by the use of Tris(2-carboxyethyl)phosphine (TCEP) during encapsulation or by washing with acidic buffer. The biocompatibility studies of the resulting DOX Ftn variants in MCF-7 and MHS cancer cells shows a complex relationship between the cytotoxicity, the DOX loading and the different surface charges of Ftn. Further investigation on the cell uptake mechanism provides reasonable explanations for the cytotoxicity results and reveals that surface charging of Ftn hinders its transferrin receptor 1 (TfR-1) mediated cellular uptake in MCF-7 cells.

Condensation and Protection of DNA by the Myxococcus xanthus Encapsulin: A Novel Function.

Encapsulins are protein nanocages capable of harboring smaller proteins (cargo proteins) within their cavity. The function of the encapsulin systems is related to the encapsulated cargo proteins. The Myxococcus xanthus encapsulin (EncA) naturally encapsulates ferritin-like proteins EncB and EncC as cargo, resulting in a large iron storage nanocompartment, able to accommodate up to 30,000 iron atoms per shell. In the present manuscript we describe the binding and protection of circular double stranded DNA (pUC19) by EncA using electrophoretic mobility shift assays (EMSA), atomic force microscopy (AFM), and DNase protection assays. EncA binds pUC19 with an apparent dissociation constant of 0.3 ± 0.1 µM and a Hill coefficient of 1.4 ± 0.1, while EncC alone showed no interaction with DNA. Accordingly, the EncAC complex displayed a similar DNA binding capacity as the EncA protein. The data suggest that initially, EncA converts the plasmid DNA from a supercoiled to a more relaxed form with a beads-on-a-string morphology. At higher concentrations, EncA self-aggregates, condensing the DNA. This process physically protects DNA from enzymatic digestion by DNase I. The secondary structure and thermal stability of EncA and the EncA-pUC19 complex were evaluated using synchrotron radiation circular dichroism (SRCD) spectroscopy. The overall secondary structure of EncA is maintained upon interaction with pUC19 while the melting temperature of the protein (Tm) slightly increased from 76 ± 1 °C to 79 ± 1 °C. Our work reports, for the first time, the in vitro capacity of an encapsulin shell to interact and protect plasmid DNA similarly to other protein nanocages that may be relevant in vivo.

Escherichia coli as a New Platform for the Fast Production of Vault-like Nanoparticles: An Optimized Protocol.

Vaults are protein nanoparticles that are found in almost all eukaryotic cells but are absent in prokaryotic ones. Due to their properties (nanometric size, biodegradability, biocompatibility, and lack of immunogenicity), vaults show enormous potential as a bio-inspired, self-assembled drug-delivery system (DDS). Vault architecture is directed by self-assembly of the "major vault protein" (MVP), the main component of this nanoparticle. Recombinant expression (in different eukaryotic systems) of the MVP resulted in the formation of nanoparticles that were indistinguishable from native vaults. Nowadays, recombinant vaults for different applications are routinely produced in insect cells and purified by successive ultracentrifugations, which are both tedious and time-consuming strategies. To offer cost-efficient and faster protocols for nanoparticle production, we propose the production of vault-like nanoparticles in Escherichia coli cells, which are still one of the most widely used prokaryotic cell factories for recombinant protein production. The strategy proposed allowed for the spontaneous encapsulation of the engineered cargo protein within the self-assembled vault-like nanoparticles by simply mixing the clarified lysates of the producing cells. Combined with well-established affinity chromatography purification methods, our approach contains faster, cost-efficient procedures for biofabrication in a well-known microbial cell factory and the purification of "ready-to-use" loaded protein nanoparticles, thereby opening the way to faster and easier engineering and production of vault-based DDSs.

Dps-DNA interaction in Marinobacter hydrocarbonoclasticus protein: effect of a single-charge alteration.

DNA-binding proteins from starved cells (Dps) are members of the ferritin family of proteins found in prokaryotes, with hollow rounded cube-like structures, composed of 12 equal subunits. These protein nanocages are bifunctional enzymes that protect the cell from the harmful reaction of iron and peroxide (Fenton reaction), thus preventing DNA damage by oxidative stress. Ferrous ions are oxidized at specific iron-binding sites in the presence of the oxidant and stored in its cavity that can accommodate up to ca. 500 iron atoms. DNA-binding properties of Dps are associated with the N-terminal, positive charge rich, extensions that can promote DNA binding and condensation, apparently by a cooperative binding mechanism. Here, we describe the binding and protection activities of Marinobacter hydrocarbonoclasticus Dps using Electrophoretic Mobility Shift Essays (EMSA), and synchrotron radiation circular dichroism (SRCD) spectroscopy. While no DNA condensation was observed in the tested conditions, it was possible to determine a Dps-DNA complex formation with an apparent dissociation constant of 6.0 ± 1.0 µM and a Hill coefficient of 1.2 ± 0.1. This interaction is suppressed by the inclusion of a single negative charge in the N-terminal region by point mutation. In Dps proteins containing a ferric mineral core (above 96 Fe/protein), DNA binding was impaired. SRCD data clearly showed that no significant modification existed either in secondary structure or protein stability of WT, Q14E variant and core containing proteins. It was, however, interesting to note that, in our experimental conditions, thermal denaturation induced protein aggregation that caused artifacts in thermal denaturation curves, which were dependent on radiation flux and vertical arrangement of the CD cell.

Role of Surface RGD Patterns on Protein Nanocages in Tumor Targeting Revealed Using Precise Discrete Models.

The effectiveness of active targeting in cancer nanomedicine is becoming increasingly more debatable. Here, the role of the ligand functionalization patterns (number and distribution) on nanoparticle surfaces in tumor targeting is investigated using a 9 nm sized miniferritin protein nanocage, Dps modified with Arg-Gly-Asp (RGD) ligands whose functionalization patterns are precisely controlled. In vitro and in vivo experiments show that RGD modification endows Dps with tumor targeting capacity no matter what the surface pattern is. The tumor targeting of 2-ligand Dps, which is better than that of 1-ligand Dps, rivals or surpasses that of the 12- or 24-ligand Dps. The 12-ligand Dps with clustered RGD distribution shows 2.3 times the in vivo targeting efficiency of that with even distribution. The surface ligand pattern effects are correlated at least to receptor clustering and opsonization. This study provides insights into the understanding of the controversial findings on active tumor targeting in the literature and highlights the necessity of precise functionalization to achieve optimal active targeting in developing cancer nanomedicine.

Ferritin-based anticancer metallodrug delivery: Crystallographic, analytical and cytotoxicity studies.

The encapsulation of anticancer metal-based drugs within a protein nanocage represents a valuable strategy to improve the efficacy and selectivity of these compounds towards cancer cells. The preparation, characterization of the in vitro cytotoxicity and X-ray structures of several ferritin-metallodrug nanocomposites (mainly containing platinum-, ruthenium- and gold-based anticancer agents) are here reviewed. The molecular mechanisms of action of these Ft-metallodrug adducts are discussed and future directions in the field are outlined.

Design and Function of Engineered Protein Nanocages as a Drug Delivery System for Targeting Pancreatic Cancer Cells via Neuropilin-1.

We describe the development of neuropilin 1-binding peptide (iRGD)-nanocages that specifically target human pancreatic cancer cells in which an iRGD is joined to the surface of naturally occurring heat shock protein (HSP) cages. Using a genetic engineering approach, the iRGD domain was joined to the C-terminal region of the HSP cage using flexible linker moieties. The characteristics of the interdomain linkages between the nanocage and the iRGD domain play an important role in the specificity and affinity of the iRGD-nanocages for their target cells. An engineered L30-iRGD-nanocage with 30 amino acid linkers, (GGS)10, showed greater binding affinity for pancreatic cancer cells relative to that of other linkers. Furthermore, a moderately hydrophobic anticancer drug, OSU03012, was successfully incorporated into the L30-iRGD-nanocage by heating the mixture. The OSU03012-loaded L30-iRGD-nanocage induced cell death of pancreatic cancer cells by activating the caspase cascade more effectively than the same concentrations of free OSU03012. The iRGD-nanocages show great potential as a novel nanocarrier for pancreatic cancer-targeted drug delivery.

Self-Assembly of Heterogeneous Ferritin Nanocages for Tumor Uptake and Penetration.

Well-defined nanostructures are crucial for precisely understanding nano-bio interactions. However, nanoparticles (NPs) fabricated through conventional synthesis approaches often lack poor controllability and reproducibility. Herein, a synthetic biology-based strategy is introduced to fabricate uniformly reproducible protein-based NPs, achieving precise control over heterogeneous components of the NPs. Specifically, a ferritin assembly toolbox system is developed that enables intracellular assembly of ferritin subunits/variants in Escherichia coli. Using this strategy, a proof-of-concept study is provided to explore the interplay between ligand density of NPs and their tumor targets/penetration. Various ferritin hybrid nanocages (FHn) containing human ferritin heavy chains (FH) and light chains are accurately assembled, leveraging their intrinsic binding with tumor cells and prolonged circulation time in blood, respectively. Further studies reveal that tumor cell uptake is FH density-dependent through active binding with transferrin receptor 1, whereas in vivo tumor accumulation and tissue penetration are found to be correlated to heterogeneous assembly of FHn and vascular permeability of tumors. Densities of 3.7 FH/100 nm2 on the nanoparticle surface exhibit the highest degree of tumor accumulation and penetration, particularly in tumors with high permeability compared to those with low permeability. This study underscores the significance of nanoparticle heterogeneity in determining particle fate in biological systems.

Effects of Individual Amino Acids on the Blood Circulation of Biosynthetic Protein Nanocages: Toward Guidance on Surface Engineering.

Protein nanocages (PNCs) hold great promise for developing multifunctional nanomedicines. Long blood circulation is a key requirement of PNCs for most in vivo application scenarios. In addition to the classical PEGylation strategy, short peptides with a specific sequence screened via phage display are also very effective in prolonging the blood half-life (t1/2 ) of PNCs. However, there is a lack of knowledge on how individual amino acids affect the circulation of PNCs. Here the effects of the 20 proteinogenic amino acids in the form of an X3 or X5 tag (X represents an amino acid) are explored on the pharmacokinetics of PNCs, which lead to the formation of a heatmap illustrating the extent of t1/2 prolongation by each proteinogenic amino acid. Significantly, oligo-lysine and oligo-arginine can effectively prolong the t1/2 of strongly negatively charged PNCs through charge neutralization, while oligo-cysteine can also do so, but via a different mechanism, mediating the covalent binding of PNCs with plasma albumin as a stealth material. These findings are extendible and offer guidance for surface-engineering biosynthetic PNCs and other nanoparticles.

Manufacturing of non-viral protein nanocages for biotechnological and biomedical applications.

Protein nanocages are highly ordered nanometer scale architectures, which are typically formed by homo- or hetero-self-assembly of multiple monomers into symmetric structures of different size and shape. The intrinsic characteristics of protein nanocages make them very attractive and promising as a biological nanomaterial. These include, among others, a high surface/volume ratio, multi-functionality, ease to modify or manipulate genetically or chemically, high stability, mono-dispersity, and biocompatibility. Since the beginning of the investigation into protein nanocages, several applications were conceived in a variety of areas such as drug delivery, vaccine development, bioimaging, biomineralization, nanomaterial synthesis and biocatalysis. The ability to generate large amounts of pure and well-folded protein assemblies is one of the keys to transform nanocages into clinically valuable products and move biomedical applications forward. This calls for the development of more efficient biomanufacturing processes and for the setting up of analytical techniques adequate for the quality control and characterization of the biological function and structure of nanocages. This review concisely covers and overviews the progress made since the emergence of protein nanocages as a new, next-generation class of biologics. A brief outline of non-viral protein nanocages is followed by a presentation of their main applications in the areas of bioengineering, biotechnology, and biomedicine. Afterwards, we focus on a description of the current processes used in the manufacturing of protein nanocages with particular emphasis on the most relevant aspects of production and purification. The state-of-the-art on current characterization techniques is then described and future alternative or complementary approaches in development are also discussed. Finally, a critical analysis of the limitations and drawbacks of the current manufacturing strategies is presented, alongside with the identification of the major challenges and bottlenecks.

Carriers and Antigens: New Developments in Glycoconjugate Vaccines.

Glycoconjugate vaccines have proven their worth in the protection and prevention of infectious diseases. The introduction of the Haemophilus influenzae type b vaccine is the prime example, followed by other glycoconjugate vaccines. Glycoconjugate vaccines consist of two components: the carrier protein and the carbohydrate antigen. Current carrier proteins are tetanus toxoid, diphtheria toxoid, CRM197, Haemophilus protein D and the outer membrane protein complex of serogroup B meningococcus. Carbohydrate antigens have been produced mainly by extraction and purification from the original host. However, current efforts show great advances in the development of synthetically produced oligosaccharides and bioconjugation. This review evaluates the advances of glycoconjugate vaccines in the last five years. We focus on developments regarding both new carriers and antigens. Innovative developments regarding carriers are outer membrane vesicles, glycoengineered proteins, new carrier proteins, virus-like particles, protein nanocages and peptides. With regard to conjugated antigens, we describe recent developments in the field of antimicrobial resistance (AMR) and ESKAPE pathogens.

Decolorization and detoxification of synthetic dye compounds by laccase immobilized in vault nanoparticles.

This study presents an eco-friendly and efficient technology, using immobilized enzymes, vault-encapsulated laccases (vlaccase), for decolorization and detoxification of dyes. Vault encapsulation remarkably improved the performance of laccase at industrially relevant conditions, including neutral to alkaline pH and relatively high temperatures. Two representative anthraquinone and azo dyes, Reactive Blue 19 and Acid Orange 7, respectively, were rapidly decolorized (72% and 80%) by vlaccase treatment while natural laccase (nlaccase) achieved 40% and 32% decolorization. The toxicity of treated and untreated dyes was tested on model bacterial, algal, and insect cells. The inhibitory effects of dyes towards selected bacteria were reduced in vlaccase-treated samples. The chlorophyll synthesis in algae was less inhibited by dyes after vlaccase treatment. Furthermore, the toxicity of dye degradation products to insect cells was significantly mitigated in the vlaccase group. Collectively, these results indicate that vlaccase is a stable and strong enzymatic system for removing dyes from waters.

All-in-one biofabrication and loading of recombinant vaults in human cells.

One of the most promising approaches in the drug delivery field is the use of naturally occurring self-assembling protein nanoparticles, such as virus-like particles, bacterial microcompartments or vault ribonucleoprotein particles as drug delivery systems (DDSs). Among them, eukaryotic vaults show a promising future due to their structural features,in vitrostability and non-immunogenicity. Recombinant vaults are routinely produced in insect cells and purified through several ultracentrifugations, both tedious and time-consuming processes. As an alternative, this work proposes a new approach and protocols for the production of recombinant vaults in human cells by transient gene expression of a His-tagged version of the major vault protein (MVP-H6), the development of new affinity-based purification processes for such recombinant vaults, and the all-in-one biofabrication and encapsulation of a cargo recombinant protein within such vaults by their co-expression in human cells. Protocols proposed here allow the easy and straightforward biofabrication and purification of engineered vaults loaded with virtually any INT-tagged cargo protein, in very short times, paving the way to faster and easier engineering and production of better and more efficient DDS.

Combined Method to Remove Endotoxins from Protein Nanocages for Drug Delivery Applications: The Case of Human Ferritin.

Protein nanocages represent an emerging candidate among nanoscaled delivery systems. Indeed, they display unique features that proved to be very interesting from the nanotechnological point of view such as uniform structure, stability in biological fluids, suitability for surface modification to insert targeting moieties and loading with different drugs and dyes. However, one of the main concerns regards the production as recombinant proteins in E. coli, which leads to a product with high endotoxin contamination, resulting in nanocage immunogenicity and pyrogenicity. Indeed, a main challenge in the development of protein-based nanoparticles is finding effective procedures to remove endotoxins without affecting protein stability, since every intravenous injectable formulation that should be assessed in preclinical and clinical phase studies should display endotoxins concentration below the admitted limit of 5 EU/kg. Different strategies could be employed to achieve such a result, either by using affinity chromatography or detergents. However, these strategies are not applicable to protein nanocages as such and require implementations. Here we propose a combined protocol to remove bacterial endotoxins from nanocages of human H-ferritin, which is one of the most studied and most promising protein-based drug delivery systems. This protocol couples the affinity purification with the Endotrap HD resin to a treatment with Triton X-114. Exploiting this protocol, we were able to obtain excellent levels of purity maintaining good protein recovery rates, without affecting nanocage interactions with target cells. Indeed, binding assay and confocal microscopy experiments confirm that purified H-ferritin retains its capability to specifically recognize cancer cells. This procedure allowed to obtain injectable formulations, which is preliminary to move to a clinical trial.

Protein nanoparticles in molecular, cellular, and tissue imaging.

The quest to develop ideal nanoparticles capable of molecular, cellular, and tissue level imaging is ongoing. Since certain imaging probes and nanoparticles face drawbacks such as low aqueous solubility, increased ROS generation leading to DNA damage, apoptosis, and high cellular/organ toxicities, the development of versatile and biocompatible nanocarriers becomes necessary. Protein nanoparticles (PNPs) are one such promising class of nanocarriers that possess most of the desirable properties of an ideal nanocarrier for bioimaging applications. PNPs demonstrate high aqueous solubility, minimal cytotoxicity, and multi-cargo loading capacity. They are also amenable to surface-functionalization, as well as modulation of their hydrophobicity and hydrophilicity. The use of PNPs for bioimaging applications has made rapid advancements in the past two decades. Being comparatively less explored, the field opens up a plethora of opportunities and focus areas to engineer ideal bioimaging protein nanocarriers. The use of PNPs as carriers of their natural ligands as well as other heavy metals and fluorescent probes, along with drug molecules for combined theranostic applications has been reported. In addition, surface functionalization to impart specificity of targeting the PNPs has been shown to reduce nonspecific cellular interactions, thus reducing systemic toxicity. PNPs have been explored for their application in imaging of numerous cancers, cardiovascular diseases as well as imaging of the brain using near infrared fluorescence (NIRF) imaging, magnetic resonance imaging (MRI), X-ray computed tomography (CT), positron emission tomography (PET), single-photon emission computed tomography (SPECT), ultrasound (US), and photoacoustic (PA) imaging. This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Diagnostic Tools > In Vivo Nanodiagnostics and Imaging.

Design and biosynthesis of functional protein nanostructures.

Proteins are one of the major classes of biomolecules that execute biological functions for maintenance of life. Various kinds of nanostructures self-assembled from proteins have been created in nature over millions of years of evolution, including protein nanowires, layers and nanocages. These protein nanostructures can be reconstructed and equipped with desired new functions. Learning from and manipulating the self-assembly of protein nanostructures not only help to deepen our understanding of the nature of life but also offer new routes to fabricate novel nanomaterials for diverse applications. This review summarizes the recent research progress in this field, focusing on the characteristics, functionalization strategies, and applications of protein nanostructures.

Metal-dependent assembly of a protein nano-cage.

Short, alpha-helical coiled coils provide a simple, modular method to direct the assembly of proteins into higher order structures. We previously demonstrated that by genetically fusing de novo-designed coiled coils of the appropriate oligomerization state to a natural trimeric protein, we could direct the assembly of this protein into various geometrical cages. Here, we have extended this approach by appending a coiled coil designed to trimerize in response to binding divalent transition metal ions and thereby achieve metal ion-dependent assembly of a tetrahedral protein cage. Ni2+ , Co2+ , Cu2+ , and Zn2+ ions were evaluated, with Ni2+ proving the most effective at mediating protein assembly. Characterization of the assembled protein indicated that the metal ion-protein complex formed discrete globular structures of the diameter expected for a complex containing 12 copies of the protein monomer. Protein assembly could be reversed by removing metal ions with ethylenediaminetetraacetic acid or under mildly acidic conditions.

Elaborating a coiled-coil-assembled octahedral protein cage with additional protein domains.

De novo design of protein nano-cages has potential applications in medicine, synthetic biology, and materials science. We recently developed a modular, symmetry-based strategy for protein assembly in which short, coiled-coil sequences mediate the assembly of a protein building block into a cage. The geometry of the cage is specified by the combination of rotational symmetries associated with the coiled-coil and protein building block. We have used this approach to design well-defined octahedral and tetrahedral cages. Here, we show that the cages can be further elaborated and functionalized by the addition of another protein domain to the free end of the coiled-coil: in this case by fusing maltose-binding protein to an octahedral protein cage to produce a structure with a designed molecular weight of ~1.8 MDa. Importantly, the addition of the maltose binding protein domain dramatically improved the efficiency of assembly, resulting in ~ 60-fold greater yield of purified protein compared to the original cage design. This study shows the potential of using small, coiled-coil motifs as off-the-shelf components to design MDa-sized protein cages to which additional structural or functional elements can be added in a modular manner.

Ultrasensitive MRI detection of spontaneous pancreatic tumors with nanocage-based targeted contrast agent.

Contrast agents with greater specificity and sensitivity are required for the diagnosis of pancreatic cancers by magnetic resonance imaging (MRI). In this study, small heat shock protein 16.5 (Hsp16.5)-based nanocages conjugated to gadolinium(III)-chelated contrast agents and iRGD peptides (which target neuropilin-1 expressed on pancreatic cancer cells) were developed. To investigate whether template size influences relaxivity, nanocages with one to four hydrophobic domains were designed. MRI data showed that larger nanocages had higher T1 relaxivity than smaller nanocages, which resulted from a reduction in molecular tumbling rates caused by an increase in nanocage size, and a robust cage structure resulting from the introduction of hydrophobic domains. For in vivo MRI studies, the engineered nanocages were evaluated using the KrasG12D; Trp53R172H; Pdx-1Cre (KPC) transgenic mouse models, which develop clinically relevant pancreatic tumor under normal processes of angiogenesis, immune function and inflammation. Molecular MRI with protein nanocages was enabled to detect neuropilin-1-positive cells and to produce strong signal enhancement of spontaneous pancreatic tumors in KPC genetically engineered mouse models. Novel iRGD-modified nanocages displayed potential as a specific and sensitive MRI contrast agent for the diagnosis of pancreatic tumors for clinical translation.