Myxococcus xanthus encapsulin cargo protein EncD is a flavin-binding protein with ferric reductase activity.

Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. Myxococcus xanthus encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe+2 to Fe+3 for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no ß-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the M. xanthus encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress.

Structure, Dynamics and Functional Implications of the Eukaryotic Vault Complex.

Vault ribonucleoprotein particles are naturally designed nanocages, widely found in the eukaryotic kingdom. Vaults consist of 78 copies of the major vault protein (MVP) that are organized in 2 symmetrical cup-shaped halves, of an approximate size of 70x40x40 nm, leaving a huge internal cavity which accommodates the vault poly(ADP-ribose) polymerase (vPARP), the telomerase-associated protein-1 (TEP1) and some small untranslated RNAs. Diverse hypotheses have been developed on possible functions of vaults, based on their unique capsular structure, their rapid movements and the distinct subcellular localization of the particles, implicating transport of cargo, but they are all pending confirmation. Vault particles also possess many attributes that can be exploited in nanobiotechnology, particularly in the creation of vehicles for the delivery of multiple molecular cargoes. Here we review what is known about the structure and dynamics of the vault complex and discuss a possible mechanism for the vault opening process. The recent findings in the characterization of the vaults in cells and in its natural microenvironment will be also discussed.

Polyvinylpyrrolidone-Coated Cubic Hollow Nanocages of PdPt3 and PdIr3 as Highly Efficient Self-Cascade Uricase/Peroxidase Mimics.

Uricase-catalyzed uric acid (UA) degradation has been applied for hyperuricemia therapy, but this medication is limited by H2O2 accumulation, which can cause oxidative stress of cells, resulting in many other health issues. Herein, we report a robust cubic hollow nanocage (HNC) system based on polyvinylpyrrolidone-coated PdPt3 and PdIr3 to serve as highly efficient self-cascade uricase/peroxidase mimics to achieve the desired dual catalysis for both UA degradation and H2O2 elimination. These HNCs have hollow cubic shape with average wall thickness of 1.5 nm, providing desired synergy to enhance catalyst's activity and stability. Density functional theory calculations suggest the PdIr3 HNC surface tend to promote OH*/O* desorption for better peroxidase-like catalysis, while the PdPt3 HNC surface accelerates the UA oxidation by facilitating O2-to-H2O2 conversion. The dual catalysis power demonstrated by these HNCs in cell studies suggests their great potential as a new type of nanozyme for treating hyperuricemia.

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.

Novel engineered HER2 specific recombinant protein nanocages for targeted drug delivery.

Protein nanocages resemble natural biomimetic carriers and can be engineered to act as targeted delivery systems, making them an attractive option for various drug delivery and biomedical applications. Our research investigated the genetic link of a specific anti-HER2 peptide (LTVSPWY) to the exposed N-terminal region of the maize (Zea mays) ferritin 1 (ZmFer1) protein nanocage, employing either a 7-amino acid (for LTVS-ZmFer1) or 16-amino acid (for LTVS-L-ZmFer1) linker. We utilized a heat treatment method to load the chemotherapeutic drug doxorubicin into the protein nanocage. The construct with the longer linker (LTVS-L) produced a greater amount of soluble protein nanocage and was selected for further experiments. The average size, polydispersity index, and zeta potential of the engineered protein nanocage were 19.01 nm, 0.168, and - 2.13 mV, respectively. The LTVS-L-ZmFer1 protein nanocage exhibited excellent thermal stability, withstanding temperatures up to 100 °C with only partial denaturation. Furthermore, we observed that cellular uptake of the LTVS-L-ZmFer1 protein nanocages in HER2-positive breast cancer cells was significantly higher compared to ZmFer1 after labeling with FITC (fluorescein isothiocyanate) (P-value = 0.0001). In addition, we observed a significant decrease in the viability of SKBR3 cells when treated with DOX-loaded LTVS-L-ZmFer1 protein nanocages compared to cells treated with DOX-loaded ZmFer1 protein nanocages. Therefore, this new treatment strategy may prove to be an effective way to reduce both the side effects and toxicity associated with conventional cancer treatments in patients with HER2-positive breast cancer.

Laser-activable murine ferritin nanocage for chemo-photothermal therapy of colorectal cancer.

Chemotherapy, as a conventional strategy for tumor therapy, often leads to unsatisfied therapeutic effect due to the multi-drug resistance and the serious side effects. Herein, we genetically engineered a thermal-responsive murine Ferritin (mHFn) to specifically deliver mitoxantrone (MTO, a chemotherapeutic and photothermal agent) to tumor tissue for the chemotherapy and photothermal combined therapy of colorectal cancer, thanks to the high affinity of mHFn to transferrin receptor that highly expressed on tumor cells. The thermal-sensitive channels on mHFn allowed the effective encapsulation of MTO in vitro and the laser-controlled release of MTO in vivo. Upon irradiation with a 660 nm laser, the raised temperature triggered the opening of the thermal-sensitive channel in mHFn nanocage, resulting in the controlled and rapid release of MTO. Consequently, a significant amount of reactive oxygen species was generated, causing mitochondrial collapse and tumor cell death. The photothermal-sensitive controlled release, low systemic cytotoxicity, and excellent synergistic tumor eradication ability in vivo made mHFn@MTO a promising candidate for chemo-photothermal combination therapy against colorectal cancer.

High-nuclearity Luminescent Lanthanide Nanocages for Tumor Drug Delivery.

There is an unmet need for easy-to-visualize drug carriers that can deliver therapeutic cargoes deep into solid tumors. Herein, we report the preparation of ultrasmall luminescent imine-based lanthanide nanocages, Eu60 and Tb60 (collectively Ln60 ), designed to encapsulate anticancer chemotherapeutics for tumor therapy. The as-prepared nanocages possess large cavities suitable for the encapsulation of doxorubicin (DOX), yielding DOX@Ln60 nanocages with diameters around 5 nm. DOX@Ln60 are efficiently internalized by breast cancer cells, allowing the cells to be visualized via the intrinsic luminescent property of Ln(III). Once internalized, the acidic intracellular microenvironment promotes imine bond cleavage and the release of the loaded DOX. DOX@Ln60 inhibits DNA replication and triggers tumor cell apoptosis. In a murine triple negative breast cancer (TNBC) model, DOX@Ln60 was found to inhibit tumor growth with negligible side effects on normal tissues. It proved more effective than various controls, including DOX and Ln60 . The present nanocages thus point the way to the development of precise nanomedicines for tumor imaging and therapy.

Integration of Protein Nanocage with CpG Motifs: A Virus-Mimicked Core-Shell Nanostructure to Ignite Antitumor Immunity.

The tumor microenvironment typically possesses immunosuppressive properties that hinder the effectiveness of antitumor immune responses, even in the context of immunotherapies. However, it is observed that pathogenic microorganisms can trigger strong immune responses during infection, offering a potential means to counteract the immunosuppressive environment of tumors. In this study, a protein nanocage called CpG@HBc nanocages (NCs) is developed, which mimics the structure of the hepatitis B virus and combines with an immunostimulatory component known as cytosine phosphoguanosine oligonucleotide (CpG). By delivering these immunostimulatory agents, CpG@HBc NCs are able to effectively reverse the suppressive tumor microenvironment, resulting in the inhibition of poorly immunogenic tumors in mice. Through high-dimensional mass cytometry (CyTOF) analysis, remarkable alterations in immune responses is observed induced by CpG@HBc. Treatment with immunogenic CpG@HBc NCs, along with co-injection of an OX40 agonist, sensitized colorectal cancer tumors to T cell immune responses, resulting in significant impairment of tumor growth and robust immune activation. Furthermore, CpG@HBc NCs induced long-term antitumor immunological memory, protecting tumor-cured mice from tumor rechallenge. Overall, these findings highlight the potential of a virus-inspired protein nanocage to mimic anti-viral immunity and offer a unique therapeutic approach for cancer immunotherapy.

MOF-Derived Mn2 O3 Nanocage with Oxygen Vacancies as Efficient Cathode Catalysts for Li-O2 Batteries.

Designing efficient and cost-effective electrocatalysts is the primary imperative for addressing the pivotal concerns confronting lithium-oxygen batteries (LOBs). The microstructure of the catalyst is one of the key factors that influence the catalytic performance. This study proceeds to the advantage of metal-organic frameworks (MOFs) derivatives by annealing manganese 1,2,3-triazolate (MET-2) at different temperatures to optimize Mn2 O3 crystals for special microstructures. It is found that at 350 °C annealing temperature, the derived Mn2 O3 nanocage maintains the structure of MOF, the inherited high porosity and large specific surface area provide more channels for Li+ and O2 diffusion, beside the oxygen vacancies on the surface of Mn2 O3 nanocages enhance the electrocatalytic activity. With the synergy of unique structure and rich oxygen vacancies, the Mn2 O3 nanocage exhibits ultrahigh discharge capacity (21 070.6 mAh g-1 at 500 mA g-1 ) and excellent cycling stability (180 cycles at the limited capacity of 600 mAh g-1 with a current of 500 mA g-1 ). This study demonstrates that the Mn2 O3 nanocage structure containing oxygen vacancies can significantly enhance catalytic performance for LOBs, which provide a simple method for structurally designed transition metal oxide electrocatalysts.

Under-Coordinated CoFe Layered Double Hydroxide Nanocages Derived from Nanoconfined Hydrolysis of Bimetal Organic Compounds for Efficient Electrocatalytic Water Oxidation.

Hierarchically structured bimetal hydroxides are promising for electrocatalytic oxygen evolution reaction (OER), yet synthetically challenging. Here, the nanoconfined hydrolysis of a hitherto unknown CoFe-bimetal-organic compound (b-MOC) is reported for the controllable synthesis of highly OER active nanostructures of CoFe layered double hydroxide (LDH). The nanoporous structures trigger the nanoconfined hydrolysis in the sacrificial b-MOC template, producing CoFe LDH core-shell octahedrons, nanoporous octahedrons, and hollow nanocages with abundant under-coordinated metal sites. The hollow nanocages of CoFe LDH demonstrate a remarkable turnover frequency (TOF) of 0.0505 s-1 for OER catalysis at an overpotential of 300 mV. It is durable in up to 50 h of electrolysis at step current densities of 10-100 mA cm-2 . Ex situ and in situ X-ray absorption spectroscopic analysis combined with theoretical calculations suggests that under-coordinated Co cations can bind with deprotonated Fe-OH motifs to form OER active Fe-O-Co dimmers in the electrochemical oxidation process, thereby contributing to the good catalytic activity. This work presents an efficient strategy for the synthesis of highly under-coordinated bimetal hydroxide nanostructures. The mechanistic understanding underscores the power of maximizing the amount of bimetal-dimer sites for efficient OER catalysis.

Column-free purification of an artificial protein nanocage, TIP60.

Protein nanocages, which have inner cavities and surface pores, are attractive materials for various applications, such as in catalysts and medicine. Recently, we produced an artificial protein nanocage, TIP60, and demonstrated its potential as a stimuli-responsive nanocarrier. In the present study, we report a simple purification method for TIP60 that can replace time-consuming and costly affinity chromatography purification. TIP60, which has an anionic surface charge, aggregated at mildly acidic pH and redissolved at neutral pH, maintaining its cage structure. This pH-responsive reversible precipitation allowed us to purify TIP60 from soluble fractions of the E. coli cell lysate by controlling the pH. Compared with conventional Ni-NTA column purification, the pH-responsive precipitation method provided purified TIP60 with similar purity (∼80%) and higher yield. This precipitation purification method should facilitate the large-scale investigation and practical use of TIP60 nanocages.

Nanocages and cell-membrane display technology as smart biomaterials.

Gold nanocages (AuNCs) have been invented and developed over two decades as biomaterial in clinical medicine with great application potential. AuNCs have a characteristic structure of porous walls with hollow interior and a compact size. This makes it possible for them to transport biomolecules or drugs with the advantages of their photothermal effects that could help further destroy germs or tumors while also regulating the release of drugs inside. Furthermore, their bioactivity and application can be broadened by using cell-membrane display technology. AuNCs have shown tremendous potential in antibacterial activity, inflammation modulation, and tissue regeneration, which is required in periodontitis and peri-implantitis treatment. Thus, this article provides an overview of AuNCs synthesis, characteristics, surface modifications, and clinical applications, aiming to serve as a reference for the design and fabrication of AuNCs-based smart materials for periodontal or peri-implant application.

B7H3 targeting gold nanocage pH-sensitive conjugates for precise and synergistic chemo-photothermal therapy against NSCLC.

BACKGROUND: The combination of drug delivery with immune checkpoint targeting has been extensively studied in cancer therapy. However, the clinical benefit for patients from this strategy is still limited. B7 homolog 3 protein (B7-H3), also known as CD276 (B7-H3/CD276), is a promising therapeutic target for anti-cancer treatment. It is widely overexpressed on the surface of malignant cells and tumor vasculature, and its overexpression is associated with poor prognosis. Herein, we report B7H3 targeting doxorubicin (Dox)-conjugated gold nanocages (B7H3/Dox@GNCs) with pH-responsive drug release as a selective, precise, and synergistic chemotherapy-photothermal therapy agent against non-small-cell lung cancer (NSCLC). RESULTS: In vitro, B7H3/Dox@GNCs exhibited a responsive release of Dox in the tumor acidic microenvironment. We also demonstrated enhanced intracellular uptake, induced cell cycle arrest, and increased apoptosis in B7H3 overexpressing NSCLC cells. In xenograft tumor models, B7H3/Dox@GNCs exhibited tumor tissue targeting and sustained drug release in response to the acidic environment. Wherein they synchronously destroyed B7H3 positive tumor cells, tumor-associated vasculature, and stromal fibroblasts. CONCLUSION: This study presents a dual-compartment targeted B7H3 multifunctional gold conjugate system that can precisely control Dox exposure in a spatio-temporal manner without evident toxicity and suggests a general strategy for synergistic therapy against NSCLC.

Insights on the molecular mechanisms of cytotoxicity induced by AS1411 linked to folate-functionalized DNA nanocages in cancer cells.

Self-assembled multivalent DNA nanocages are an emerging class of molecules useful for biomedicine applications. Here, we investigated the molecular mechanisms of cytotoxicity induced by AS1411 free aptamer, AS1411-linked nanocages (Apt-NCs) and nanocages harboring both folate and AS1411 functionalization (Fol-Apt-NCs) in HeLa and MDA-MB-231 cancer cell lines. The three treatments showed different cytotoxic efficacy and Fol-Apt-NCs resulted the most effective in inhibiting cell proliferation and inducing apoptotic pathways and ROS activation in both HeLa and MDA-MB-231 cells. RNA-seq analysis allowed to identify biological functions and genes altered by the various treatments, depending on the AS1411 route of intracellular entry, highlighting the different behavior of the two cancer cell lines. Notably, Fol-Apt-NCs altered the expression of a subset of genes associated to cancer chemoresistance in MDA-MB-231, but not in HeLa cells, and this may explain the increased chemosensitivity to drugs delivered through DNA nanocages of the triple-negative breast cancer cells.

Boron nitride nanocage as drug delivery systems for chloroquine, as an effective drug for treatment of coronavirus disease: A DFT study.

Research has shown that chloroquine (CQ) can effectively help control COVID-19 infection. B24N24 nanocage is a drug delivery system. Thus, through density functional theory, the present study analyzed pristine nanocage-CQ interaction and CQ interaction with Si- and Al -doped nanocage. The findings revealed that nanocage doping, particularly with Si and Al, yields more satisfactory drug delivery for CQ due to their greater electronic and energetic characteristics with CQ.

Artificial Protein Cage with Unusual Geometry and Regularly Embedded Gold Nanoparticles.

Artificial protein cages have great potential in a number of areas including cargo capture and delivery and as artificial vaccines. Here, we investigate an artificial protein cage whose assembly is triggered by gold nanoparticles. Using biochemical and biophysical methods we were able to determine both the mechanical properties and the gross compositional features of the cage which, combined with mathematical models and biophysical data, allowed the structure of the cage to be predicted. The accuracy of the overall geometrical prediction was confirmed by the cryo-EM structure determined to sub-5 Å resolution. This showed the cage to be nonregular but similar to a dodecahedron, being constructed from 12 11-membered rings. Surprisingly, the structure revealed that the cage also contained a single, small gold nanoparticle at each 3-fold axis meaning that each cage acts as a synthetic framework for regular arrangement of 20 gold nanoparticles in a three-dimensional lattice.

ZIF-67-Derived NiCo-Layered Double Hydroxide@Carbon Nanotube Architectures with Hollow Nanocage Structures as Enhanced Electrocatalysts for Ethanol Oxidation Reaction.

The rational design of efficient Earth-abundant electrocatalysts for the ethanol oxidation reaction (EOR) is the key to developing direct ethanol fuel cells (DEFCs). Among these, the smart structure is highly demanded for highly efficient and stable non-precious electrocatalysts based on transition metals (such as Ni, Co, and Fe). In this work, high-performance NiCo-layered double hydroxide@carbon nanotube (NiCo-LDH@CNT) architectures with hollow nanocage structures as electrocatalysts for EOR were prepared via sacrificial ZIF-67 templates on CNTs. Comprehensive structural characterizations revealed that the as-synthesized NiCo-LDH@CNTs architecture displayed 3D hollow nanocages of NiCo-LDH and abundant interfacial structure between NiCo-LDH and CNTs, which could not only completely expose active sites by increasing the surface area but also facilitate the electron transfer during the electrocatalytic process, thus, improving EOR activity. Benefiting from the 3D hollow nanocages and interfacial structure fabricated by the sacrificial ZIF-67-templated method, the NiCo-LDH@CNTs-2.5% architecture exhibited enhanced electrocatalytic activity for ethanol oxidation compared to single-component NiCo-LDH, where the peak current density was 11.5 mA·cm-2, and the jf/jb value representing the resistance to catalyst poisoning was 1.72 in an alkaline environment. These results provide a new perspective on the fabrication of non-precious metal electrocatalysts for EOR in DEFCs.

Nanocages engineered from Bacillus Calmette-Guerin facilitate protective Vγ2Vδ2 T cell immunity against Mycobacterium tuberculosis infection.

Tuberculosis (TB), induced by Mycobacterium tuberculosis (Mtb) infection, remains a top killer among infectious diseases. While Bacillus Calmette-Guerin (BCG) is the sole TB vaccine, the clumped-clustered features of BCG in intradermal immunization appear to limit both the BCG protection efficacy and the BCG vaccination safety. We hypothesize that engineering of clumped-clustered BCG into nanoscale particles would improve safety and also facilitate the antigen-presenting-cell (APC)'s uptake and the following processing/presentation for better anti-TB protective immunity. Here, we engineered BCG protoplasts into nanoscale membraned BCG particles, termed as "BCG-Nanocage" to enhance the anti-TB vaccination efficiency and safety. BCG-Nanocage could readily be ingested/taken by APC macrophages selectively; BCG-Nanocage-ingested macrophages exhibited better viability and developed similar antimicrobial responses with BCG-infected macrophages. BCG-Nanocage, like live BCG bacilli, exhibited the robust capability to activate and expand innate-like T effector cell populations of Vγ2+ T, CD4+ T and CD8+ T cells of rhesus macaques in the ex vivo PBMC culture. BCG-Nanocage immunization of rhesus macaques elicited similar or stronger memory-like immune responses of Vγ2Vδ2 T cells, as well as Vγ2Vδ2 T and CD4+/CD8+ T effectors compared to live BCG vaccination. BCG-Nanocage- immunized macaques developed rapidly-sustained pulmonary responses of Vγ2Vδ2 T cells upon Mtb challenge. Furthermore, BCG- and BCG-Nanocage- immunized macaques, but not saline controls, exhibited undetectable Mtb infection loads or TB lesions in the Mtb-challenged lung lobe and hilar lymph node at endpoint after challenge. Thus, the current study well justifies a large pre-clinical investigation to assess BCG-Nanocage for safe and efficacious anti-TB vaccination, which is expected to further develop novel vaccines or adjuvants.

A Multivalent Vaccine Based on Ferritin Nanocage Elicits Potent Protective Immune Responses against SARS-CoV-2 Mutations.

The SARS-CoV-2 pandemic has created a global public crisis and heavily affected personal lives, healthcare systems, and global economies. Virus variants are continuously emerging, and, thus, the pandemic has been ongoing for over two years. Vaccines were rapidly developed based on the original SARS-CoV-2 (Wuhan-Hu-1) to build immunity against the coronavirus disease. However, they had a very low effect on the virus' variants due to their low cross-reactivity. In this study, a multivalent SARS-CoV-2 vaccine was developed using ferritin nanocages, which display the spike protein from the Wuhan-Hu-1, B.1.351, or B.1.429 SARS-CoV-2 on their surfaces. We show that the mixture of three SARS-CoV-2 spike-protein-displaying nanocages elicits CD4+ and CD8+ T cells and B-cell immunity successfully in vivo. Furthermore, they generate a more consistent antibody response against the B.1.351 and B.1.429 variants than a monovalent vaccine. This leads us to believe that the proposed ferritin-nanocage-based multivalent vaccine platform will provide strong protection against emerging SARS-CoV-2 variants of concern (VOCs).

Controlled Uptake of an Iridium Complex inside Engineered apo-Ferritin Nanocages: Study of Structure and Catalysis.

The effect of the mutation at the core of the ferritin nanocage (apo-rHLFr) on the uptake of IrCp* has been investigated by structural and spectroscopic methods. Site-specific mutations of two polar residues viz., Asp38 and Arg52 were investigated. The uptake of IrCp* was increased by about 1.5-fold on mutation of Arg52 by His compared to the wild-type variant, while mutation of Asp38 by His had no effect on the uptake. All the variants of the Ir-embedded ferritin cages remained as stable as the wild-type analogue. These hybrid bio-nanocages of ferritin were found to efficiently catalyze transfer hydrogenation of various substituted acetophenones forming the corresponding chiral alcohols with up to 88 % conversion and 70 % enantioselectivity. An electron-withdrawing substituent on the reactant enhanced the Turnover frequency of the reaction. Molecular docking analyses suggested that the substrate binds in different orientations at the active site in different mutants of the nanocage.