Ultrasmall metal alloy nanozymes mimicking neutrophil enzymatic cascades for tumor catalytic therapy.

Developing strategies that emulate the killing mechanism of neutrophils, which involves the enzymatic cascade of superoxide dismutase (SOD) and myeloperoxidase (MPO), shows potential as a viable approach for cancer therapy. Nonetheless, utilizing natural enzymes as therapeutics is hindered by various challenges. While nanozymes have emerged for cancer treatment, developing SOD-MPO cascade in one nanozyme remains a challenge. Here, we develop nanozymes possessing both SOD- and MPO-like activities through alloying Au and Pd, which exhibits the highest cascade activity when the ratio of Au and Pd is 1:3, attributing to the high d-band center and adsorption energy for superoxide anions, as determined through theoretical calculations. The Au1Pd3 alloy nanozymes exhibit excellent tumor therapeutic performance and safety in female tumor-bearing mice, with safety attributed to their tumor-specific killing ability and renal clearance ability caused by ultrasmall size. Together, this work develops ultrasmall AuPd alloy nanozymes that mimic neutrophil enzymatic cascades for catalytic treatment of tumors.

Deciphering the catalytic mechanism of superoxide dismutase activity of carbon dot nanozyme.

Nanozymes with superoxide dismutase (SOD)-like activity have attracted increasing interest due to their ability to scavenge superoxide anion, the origin of most reactive oxygen species in vivo. However, SOD nanozymes reported thus far have yet to approach the activity of natural enzymes. Here, we report a carbon dot (C-dot) SOD nanozyme with a catalytic activity of over 10,000 U/mg, comparable to that of natural enzymes. Through selected chemical modifications and theoretical calculations, we show that the SOD-like activity of C-dots relies on the hydroxyl and carboxyl groups for binding superoxide anions and the carbonyl groups conjugated with the π-system for electron transfer. Moreover, C-dot SOD nanozymes exhibit intrinsic targeting ability to oxidation-damaged cells and effectively protect neuron cells in the ischemic stroke male mice model. Together, our study sheds light on the structure-activity relationship of C-dot SOD nanozymes, and demonstrates their potential for treating of oxidation stress related diseases.

Nanozyme-strip for rapid and ultrasensitive nucleic acid detection of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic has created a huge demand for sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard for SARS-CoV-2 detection is reverse transcription-polymerase chain reaction (RT-PCR)-based nucleic acid amplification. However, RT-PCR is time consuming and requires specialists and large instruments that are unattainable for point-of-care testing (POCT). To develop POCT for SARS-CoV-2, we combined recombinase polymerase amplification (RPA) and FeS2 nanozyme strips to achieve facile nucleic acid amplification and subsequent colorimetric signal enhancement based on the high peroxidase-like activity of the FeS2 nanozymes. This method showed a nucleic acid limit of detection (LOD) for SARS-CoV-2 of 200 copies/mL, close to that of RT-PCR. The unique catalytic properties of the FeS2 nanozymes enabled the nanozyme-strip to amplify colorimetric signals via the nontoxic 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Importantly, the detection of clinical samples of human papilloma virus type 16 (HPV-16) showed 100% agreement with previous RT-PCR results, highlighting the versatility and reliability of this method. Our findings suggest that nanozyme-based nucleic acid detection has great potential in the development of POCT diagnosis for COVID-19 and other viral infections.

Ferritin nanocages for early theranostics of tumors via inflammation-enhanced active targeting.

Engineered nanocarriers have been widely developed for tumor theranostics. However, the delivery of imaging probes or therapeutic drugs to the tumor pre-formation site for early and accurate detection and therapy remains a major challenge. Here, by using tailor-functionalized human H-ferritin (HFn), we developed a triple-modality nanoprobe IRdye800-M-HFn and achieved the early imaging of tumor cells before the formation of solid tumor tissues. Then, we developed an HFn-doxorubicin (Dox) drug delivery system by loading Dox into the HFn protein cage and achieved early-stage tumor therapy. The intravenous injection of HFn nanoprobes enabled the imaging of tumor cells as early as two days after tumor implantation, and the triple-modality imaging techniques, namely, near-infrared fluorescence molecular imaging (NIR-FMI), magnetic resonance imaging (MRI), and photoacoustic imaging (PAI), ensured the accuracy of detection. Further exploration indicated that HFn could specifically penetrate into pre-solid tumor sites by tumor-associated inflammation-mediated blood vessel leakage, followed by effective accumulation in tumor cells by the specific targeting property of HFn to transferrin receptor 1. Thus, the HFn-Dox drug delivery system delivered Dox into the tumor pre-formation site and effectively killed tumor cells at early stage. IRDye800-M-HFn nanoprobes and HFn-Dox provide promising strategies for early-stage tumor diagnosis and constructive implications for early-stage tumor treatment.

Cargo loading within ferritin nanocages in preparation for tumor-targeted delivery.

Ferritins are spherical iron storage proteins within cells, composed of 24 subunits of two types, heavy-chain ferritin (HFn) and light-chain ferritin. Ferritins auto-assemble naturally into hollow nanocages with an outer diameter of 12 nm and an interior cavity 8 nm in diameter. Since the intrinsic tumor-targeting property of human HFn was first reported in 2012, HFn has been extensively explored for tumor-targeted delivery of anticancer drugs and diagnostic molecules, including radioisotopes and fluorophores, as well as inorganic nanoparticles (NPs) and chemotherapeutic drugs. This protocol provides four detailed procedures describing how to load four types of cargoes within HFn nanocages that are capable of accurately controlling cargo loading: synthesis of inorganic metal nanoparticles within the cavity of a wild-type human HFn nanocage (Procedure 1, requires ~5 h); loading of doxorubicin into the cavity of a wild-type human HFn nanocage (Procedure 2, requires ~3 d); loading Gd3+ into the cavity of a genetically engineered human HFn nanocage (Procedure 3, requires ~20 h); and loading 64Cu2+ radioisotope into the cavity of a genetically engineered human HFn nanocage (Procedure 4, requires ~3 h). Subsequent use of these HFn-based formulations is advantageous as they have intrinsic tumor-targeting capability and lack immunogenicity. Human HFn generated as described in this protocol can therefore be used to deliver therapeutic drugs and diagnostic signals as multifunctional nanomedicines.

Blastocyst-Inspired Hydrogels to Maintain Undifferentiation of Mouse Embryonic Stem Cells.

Stem cell fate is determined by specific niches that provide multiple physical, chemical, and biological cues. However, the hierarchy or cascade of impact of these cues remains elusive due to their spatiotemporal complexity. Here, anisotropic silk protein nanofiber-based hydrogels with suitable cell adhesion capacity are developed to mimic the physical microenvironment inside the blastocele. The hydrogels enable mouse embryonic stem cells (mESCs) to maintain stemness in vitro in the absence of both leukemia inhibitory factor (LIF) and mouse embryonic fibroblasts (MEFs), two critical factors in the standard protocol for mESC maintenance. The mESCs on hydrogels can achieve superior pluripotency, genetic stability, developmental capacity, and germline transmission to those cultured with the standard protocol. Such biomaterials establish an improved dynamic niche through stimulating the secretion of autocrine factors and are sufficient to maintain the pluripotency and propagation of ESCs. The mESCs on hydrogels are distinct in their expression profiles and more resemble ESCs in vivo. The physical cues can thus initiate a self-sustaining stemness-maintaining program. In addition to providing a relatively simple and low-cost option for expansion and utility of ESCs in biological research and therapeutic applications, this biomimetic material helps gain more insights into the underpinnings of early mammalian embryogenesis.

Ferritin nanocage: A promising and designable multi-module platform for constructing dynamic nanoassembly-based drug nanocarrier.

Ferritin has been widely recognized as an ideal drug delivery vehicle owing to its unique cage-like structure. Coupled with intrinsic targeting ability and excellent biosafety, ferritin-based drug delivery system, recently coined as ferritin drug carrier (FDC), has sparked great interest among researchers and shown promising application potential in the biomedical field. However, the flexibility and accuracy of traditional FDCs are limited when facing with complex disease microenvironments. To meet the fast-growing requirements for precision medicine, ferritin can serve as a designable multi-module platform to fabricate smarter FDC, which we introduce here as dynamic nanoassembly-based ferritin drug carrier (DNFDC). Compared to conventional FDC, DNFDCs directly integrate required functions into their nanostructure, which can achieve dynamic transformation upon stimuli to specifically activate and exert therapeutic functions at targeted sites. In this review, we summarize the superior characteristics of ferritin that contribute to the on-demand design of DNFDC and outline the current advances in DNFDC. Moreover, the potential research directions and challenges are also discussed here. Hopefully, this review may inspire the future development of DNFDC.

High-Performance Self-Cascade Pyrite Nanozymes for Apoptosis-Ferroptosis Synergistic Tumor Therapy.

As next-generation artificial enzymes, nanozymes have shown great promise for tumor catalytic therapy. In particular, their peroxidase-like activity has been employed to catalyze hydrogen peroxide (H2O2) to produce highly toxic hydroxyl radicals (•OH) to kill tumor cells. However, limited by the low affinity between nanozymes with H2O2 and the low level of H2O2 in the tumor microenvironment, peroxidase nanozymes usually produced insufficient •OH to kill tumor cells for therapeutic purposes. Herein, we present a pyrite peroxidase nanozyme with ultrahigh H2O2 affinity, resulting in a 4144- and 3086-fold increase of catalytic activity compared with that of classical Fe3O4 nanozyme and natural horseradish peroxidase, respectively. We found that the pyrite nanozyme also possesses intrinsic glutathione oxidase-like activity, which catalyzes the oxidation of reduced glutathione accompanied by H2O2 generation. Thus, the dual-activity pyrite nanozyme constitutes a self-cascade platform to generate abundant •OH and deplete reduced glutathione, which induces apoptosis as well as ferroptosis of tumor cells. Consequently, it killed apoptosis-resistant tumor cells harboring KRAS mutation by inducing ferroptosis. The pyrite nanozyme also exhibited favorable tumor-specific cytotoxicity and biodegradability to ensure its biosafety. These results indicate that the high-performance pyrite nanozyme is an effective therapeutic reagent and may aid the development of nanozyme-based tumor catalytic therapy.

Nanozyme for tumor therapy: Surface modification matters.

As the next generation of artificial enzymes, nanozymes have shown unique properties compared to its natural counterparts, such as stability in harsh environment, low cost, and ease of production and modification, paving the way for its biomedical applications. Among them, tumor catalytic therapy mediated by the generation of reactive oxygen species (ROS) has made great progress mainly from the peroxidase-like activity of nanozymes. Fe3O4 nanozymes, the earliest type of nanomaterial discovered to possess peroxidase-like activity, has consequently received wide attention for tumor therapy due to its ROS generation ability and tumor cell killing ability. However, inconsistent results of cytotoxicity were observed between different reports, and some even showed the scavenging of ROS in some cases. By collectively studying these inconsistent outcomes, we raise the question whether surface modification of Fe3O4 nanozymes, either through affecting peroxidase activity or by affecting the biodistribution and intracellular fate, play an important role in its therapeutic effects. This review will go over the fundamental catalytic mechanisms of Fe3O4 nanozymes and recent advances in tumor catalytic therapy, and discuss the importance of surface modification. Employing Fe3O4 nanozymes as an example, we hope to provide an outlook on the improvement of nanozyme-based antitumor activity.

Photoluminescent Spectral Broadening of Lead Halide Perovskite Nanocrystals Investigated by Emission Wavelength Dependent Lifetime.

Despite intensive efforts, the fluorescence of perovskite nanocrystals (NCs) still suffers from a poor color purity, which limits the applications in light emitting and multicolor display. A deep understanding on the fundamental of the photoluminescent (PL) spectral broadening is thus of great significance. Herein, the PL decay curves of the CsPbClxBr3-x NCs are monitored at different wavelengths covering the entire PL band. Moreover, energy relaxation time τ and radiative recombination time ß are obtained by numerical fittings. The dependences of τ and 1/ß on the detection wavelength agree well with the steady-state PL spectrum, indicating the observed PL broadening is an intrinsic effect due to the resonance and off-resonance exciton radiative recombination processes. This work not only provides a new analysis method for time-resolved PL spectra of perovskites, but also gains a deep insight into the spectral broadening of the lead halide perovskite NCs.

Natural Nanofiber Shuttles for Transporting Hydrophobic Cargo into Aqueous Solutions.

Hydrophobic biomolecules realize their functions in vivo in aqueous environments, often through a delicate balance of amphiphilicity and chaperones. Introducing exogenous hydrophobic biomolecules into in vivo aqueous systems is a challenge in drug delivery and regenerative medicine, where labile linkers, carriers, and fusions or chimeric molecules are often designed to facilitate such aqueous interfaces. Here, we utilize naturally derived silk nanofiber shuttles with the capacity to transport hydrophobic cargos directly into aqueous solutions. These nanofibers disperse in organic solvents and in aqueous solutions because of their inherent amphiphilicity, with enriched hydrophobicity and strategically interspersed negatively charged groups. Hydrophobic molecules loaded on these shuttles in organic solvent-water systems separated from the solvent after centrifugation. These concentrated hydrophobic molecule-loaded nanofibers could then be dispersed into aqueous solution directly without modification. These shuttle systems were effective for different hydrophobic molecules such as drugs, vitamins, and dyes. Improved biological stability and functions of hydrophobic cargos after loading on these nanofibers suggest potential applications in drug delivery, cosmetology, medical diagnosis, and related health fields, with a relatively facile process.

Rare earth ion Tb3+ doped natural sodium feldspar (NaAlSi3O8) Luminescent properties and energy transfer.

In this study, Tb3+-doped natural sodium feldspar (NaAlSi3O8) phosphors have been successfully prepared using high-temperature solid-state method with natural sodium feldspar as a substrate. Energy-dispersive X-ray spectrometry analysis (EDX) of NaAlSi3O8 showed that 0.03 wt% of Eu element was present, and elemental distribution mapping analysis showed that the distribution of trace Eu in minerals was aggregated. The crystal structure and luminescence properties of the natural sodium Eu-containing feldspar and synthetic sodium feldspar NaAlSi3O8:Eu3+, Tb3+ phosphors are discussed in detail. The crystal structure analysis of the samples showed that the Na+ in the natural matrix was partly replaced by the doped Tb3+. Studies on the photoluminescence properties of the samples indicate that Eu does not form a luminescent center in the natural mineral, however, the strong characteristic peak of Eu3+ at 615 nm appears after doping with Tb3+ and the peak at 615 nm increases with the increase of Tb3+ concentration. According to the above spectral results, the energy transfer from Tb3+ to Eu3+ is obtained. Through the measurement and analysis of color coordinates, it is found that with the increase of Tb3+ concentration, the luminescence color of the samples can be regulated in the green to red region. NaAlSi3O8:Eu3+ Tb3+ phosphors has potential application value.

Ferritin drug carrier (FDC) for tumor targeting therapy.

Ferritin is an iron storage protein that plays a key role in iron homeostasis and anti-oxidation of cells. Due to its unique architecture of 24 self-assembling subunits and hollow cavity capable of encapsulating drugs, and an outer surface that can be modified genetically and chemically for additional functionality, ferritin has recently emerged as a promising drug delivery vehicle. Recent research demonstrated that unmodified human heavy chain ferritin binds to its receptor, transferrin receptor 1 (TfR1), in different types of tumor tissues, including lung and breast cancer, thus highlighting the potential use of ferritin for tumor-targeting applications. In this review, we consider the many favorable characteristics of ferritin drug carriers (FDCs) for tumor drug delivery. In particular, compared with antibody-drug conjugates (ADCs), ferritin exhibits superiority in a range of attributes, including drug loading ability, thermostability, and ease of production. Thus, the emergence of FDCs may be the next step in targeted cancer therapy.

Tumor Targeting Strategies of Smart Fluorescent Nanoparticles and Their Applications in Cancer Diagnosis and Treatment.

Advantages such as strong signal strength, resistance to photobleaching, tunable fluorescence emissions, high sensitivity, and biocompatibility are the driving forces for the application of fluorescent nanoparticles (FNPs) in cancer diagnosis and therapy. In addition, the large surface area and easy modification of FNPs provide a platform for the design of multifunctional nanoparticles (MFNPs) for tumor targeting, diagnosis, and treatment. In order to obtain better targeting and therapeutic effects, it is necessary to understand the properties and targeting mechanisms of FNPs, which are the foundation and play a key role in the targeting design of nanoparticles (NPs). Widely accepted and applied targeting mechanisms such as enhanced permeability and retention (EPR) effect, active targeting, and tumor microenvironment (TME) targeting are summarized here. Additionally, a freshly discovered targeting mechanism is introduced, termed cell membrane permeability targeting (CMPT), which improves the tumor-targeting rate from less than 5% of the EPR effect to more than 50%. A new design strategy is also summarized, which is promising for future clinical targeting NPs/nanomedicines design. The targeting mechanism and design strategy will inspire new insights and thoughts on targeting design and will speed up precision medicine and contribute to cancer therapy and early diagnosis.

Ferritin variants: inspirations for rationally designing protein nanocarriers.

Ferritin, a natural iron storage protein, is endowed with a unique structure, the ability to self-assemble and excellent physicochemical properties. Beyond these, genetic manipulation can easily tune the structure and functions of ferritin nanocages, which further expands the biomedical applications of ferritin. Here, we focus on human H-ferritin, a recently discovered ligand of transferrin receptor 1, to review its derived variants and related structures and properties. We hope this review will provide new insights into how to rationally design versatile protein cage nanocarriers for effective disease treatment.

Bioengineered H-Ferritin Nanocages for Quantitative Imaging of Vulnerable Plaques in Atherosclerosis.

Inflammation and calcification concomitantly drive atherosclerotic plaque progression and rupture and are the compelling targets for identifying plaque vulnerability. However, current imaging modalities for vulnerable atherosclerotic plaques are often limited by inadequate specificity and sensitivity. Here, we show that natural H-ferritin nanocages radiolabeled with technetium-99m (99mTc-HFn) can identify and accurately localize macrophage-rich, atherosclerotic plaques in living mice using combined SPECT and CT. Focal 99mTc-HFn uptake was observed in the atherosclerotic plaques with multiple high-risk features of macrophage infiltration, active calcification, positive remodeling, and necrosis on histology and in early active ongoing lesions with intense macrophage infiltration. The uptake of 99mTc-HFn in plaques enabled quantitative measuring of the dynamic changes of inflammation during plaque progression and anti-inflammation treatment. This strategy lays the foundation of using bioengineered endogenous human ferritin nanocages for the identification of vulnerable and early active plaques as well as potential assessment of anti-inflammation therapy.

Endoscopic molecular imaging of early gastric cancer using fluorescently labeled human H-ferritin nanoparticle.

Optical imaging technologies improve clinical diagnostic accuracy of early gastric cancer (EGC). However, there was a lack of imaging agents exhibiting molecular specificity for EGCs. Here, we employed the dye labeled human heavy-chain ferritin (HFn) as imaging nanoprobe, which recognizes tumor biomarker transferrin receptor 1 (TfR1), to enable specific EGC imaging using confocal laser endomicroscopy (CLE). TfR1 expression was initially examined in vitro in gastric tumor cells and in vivo through whole-body fluorescence and CLE imaging in tumor-bearing mice. Subsequently, dye labeled HFn was topically applied to resected human tissues for EGC detection. CLE analysis of TfR1-targeted fluorescence imaging allowed distinction of neoplastic from non-neoplastic tissues (P < 0.0001), and TfR1 expression level was found to correlate with EGC differentiation degrees (P < 0.0001). Notably, the CLE evaluation correlated well with the immunohistochemical findings (κ-coefficient: 0.8023). Our HFn-nanoprobe-based CLE increases the accuracy of EGC detection and enables visualization of tumor margins and endoscopic resection.

Ferritin Nanocarrier Traverses the Blood Brain Barrier and Kills Glioma.

Over the last decades, considerable efforts have been put into developing active nanocarrier systems that cross the blood brain barrier (BBB) to treat brain-related diseases such as glioma tumors. However, to date none have been approved for clinical usage. Here, we show that a human H-ferritin (HFn) nanocarrier both successfully crosses the BBB and kills glioma tumor cells. Its principle point of entry is the HFn receptor (transferrin receptor 1), which is overexpressed in both BBB endothelial cells (ECs) and glioma cells. Importantly, we found that HFn enters and exits the BBB via the endosome compartment. In contrast, upon specifically targeting and entering glioma cells, nearly all of the HFn accumulated in the lysosomal compartment, resulting in the killing of glioma tumor cells, with no HFn accumulation in the surrounding healthy brain tissue. Thus, HFn is an ideal nanocarrier for glioma therapy and possesses the potential to serve as a therapeutic approach against a broad range of central nervous system diseases.

Fenobody: A Ferritin-Displayed Nanobody with High Apparent Affinity and Half-Life Extension.

Nanobodies consist of a single domain variable fragment of a camelid heavy-chain antibody. Nanobodies have potential applications in biomedical fields because of their simple production procedures and low cost. Occasionally, nanobody clones of interest exhibit low affinities for their target antigens, which, together with their short half-life limit bioanalytical or therapeutic applications. Here, we developed a novel platform we named fenobody, in which a nanobody developed against H5N1 virus is displayed on the surface of ferritin in the form of a 24mer. We constructed a fenobody by substituting the fifth helix of ferritin with the nanobody. TEM analysis showed that nanobodies were displayed on the surface of ferritin in the form of 6 × 4 bundles, and that these clustered nanobodies are flexible for antigen binding in spatial structure. Comparing fenobodies with conventional nanobodies currently used revealed that the antigen binding apparent affinity of anti-H5N1 fenobody was dramatically increased (∼360-fold). Crucially, their half-life extension in a murine model was 10-fold longer than anti-H5N1 nanobody. In addition, we found that our fenobodies are highly expressed in Escherichia coli, and are both soluble and thermo-stable nanocages that self-assemble as 24-polymers. In conclusion, our results demonstrate that fenobodies have unique advantages over currently available systems for apparent affinity enhancement and half-life extension of nanobodies. Our fenobody system presents a suitable platform for various large-scale biotechnological processes and should greatly facilitate the application of nanobody technology in these areas.

Stabilization of RNA Encapsulated in Silk.

The use of mRNA and miRNA as diagnostic parameters and therapeutic agents has drawn wide interest both clinically and in research. However, RNA is a labile molecule, which requires strict storage conditions, often including cold temperatures or dry environments, in order to preserve RNA integrity. Achieving this requires huge costs for storage and added difficulty in transport. To address these issues, we introduce a system to encapsulate and store it long-term in dried silk fibroin matrices. At temperatures up to 45 °C, mRNA samples stored in lyophilized silk matrices showed good stability over 1 week, as measured by real-time PCR to assess transcript recovery. While the presence of the silk interfered with the generation of cDNA required for quantitation at roughly 1% w/v (400:1 silk:RNA mass), this phenomenon was resolved by the use of commercial RNA purification kits for silk concentrations up to 4% w/v. A higher concentration of silk correlated with increased thermal protection. As an alternative to lyophilization, RNA was simply air-dried in the presence of aqueous fibroin to create storage matrices. While air-dried matrices composed of low silk content were not protective, higher concentrations were protective and progressively lost additional water over time of storage because of the overall hydrophobic nature of the system. Taken together, these findings provide a new and potentially simpler method for preserving RNA samples for long-term storage and transportation, acting primarily on a water exclusion mechanism.