Demin Duan

H-ferritin–nanocaged doxorubicin nanoparticles specifically target and kill tumors with a single-dose injection

Significance Nanoparticles capable of specifically binding to target cells and delivering high doses of therapeutic drugs with optimized safety profiles are much sought after in the nanomedical field. Here, we developed a natural H-ferritin (HFn) nanocarrier that specifically delivered a high concentration of the therapeutic drug doxorubicin (Dox) to tumor cells and significantly inhibited tumor growth with a single-dose treatment while also showing excellent biocompatibility and safety profiles in murine cancer models. Compared with the clinically approved liposomal Dox (Doxil), HFn-Dox exhibited longer median survival times and lower toxicity when administered at the same dose in all tumor models studied. An ideal nanocarrier for efficient drug delivery must be able to target specific cells and carry high doses of therapeutic drugs and should also exhibit optimized physicochemical properties and biocompatibility. However, it is a tremendous challenge to engineer all of the above characteristics into a single carrier particle. Here, we show that natural H-ferritin (HFn) nanocages can carry high doses of doxorubicin (Dox) for tumor-specific targeting and killing without any targeting ligand functionalization or property modulation. Dox-loaded HFn (HFn-Dox) specifically bound and subsequently internalized into tumor cells via interaction with overexpressed transferrin receptor 1 and released Dox in the lysosomes. In vivo in the mouse, HFn-Dox exhibited more than 10-fold higher intratumoral drug concentration than free Dox and significantly inhibited tumor growth after a single-dose injection. Importantly, HFn-Dox displayed an excellent safety profile that significantly reduced healthy organ drug exposure and improved the maximum tolerated dose by fourfold compared with free Dox. Moreover, because the HFn nanocarrier has well-defined morphology and does not need any ligand modification or property modulation it can be easily produced with high purity and yield, which are requirements for drugs used in clinical trials. Thus, these unique properties make the HFn nanocage an ideal vehicle for efficient anticancer drug delivery.

Label-free high-throughput microRNA expression profiling from total RNA

MicroRNAs (miRNAs) are key biological regulators and promising disease markers whose detection technologies hold great potentials in advancing fundamental research and medical diagnostics. Currently, miRNAs in biological samples have to be labeled before being applied to most high-throughput assays. Although effective, these labeling-based approaches are usually labor-intensive, time-consuming and liable to bias. Besides, the cross-hybridization of co-existing miRNA precursors (pre-miRNAs) is not adequately addressed in most assays that use total RNA as input. Here, we present a hybridization-triggered fluorescence strategy for label-free, microarray-based high-throughput miRNA expression profiling. The total RNA is directly applied to the microarray with a short fluorophore-linked oligonucleotide Universal Tag which can be selectively captured by the target-bound probes via base-stacking effects. This Stacking-Hybridized Universal Tag (SHUT) assay has been successfully used to analyze as little as 100 ng total RNA from human tissues, and found to be highly specific to homogenous miRNAs. Superb discrimination toward single-base mismatch at the 5′ or 3′ end has been demonstrated. Importantly, the pre-miRNAs generated negligible signals, validating the direct use of total RNA.

Nanozyme-strip for rapid local diagnosis of Ebola

Ebola continues to rage in West Africa. In the absence of an approved vaccine or treatment, the priority in controlling this epidemic is to promptly identify and isolate infected individuals. To this end, a rapid, highly sensitive, and easy-to-use test for Ebola diagnosis is urgently needed. Here, by using Fe3O4 magnetic nanoparticle (MNP) as a nanozyme probe, we developed a MNP-based immunochromatographic strip (Nanozyme-strip), which detects the glycoprotein of Ebola virus (EBOV) as low as 1 ng/mL, which is 100-fold more sensitive than the standard strip method. The sensitivity of the Nanozyme-strip for EBOV detection and diagnostic accuracy for New Bunyavirus clinical samples is comparable with ELISA, but is much faster (within 30 min) and simpler (without need of specialist facilities). The results demonstrate that the Nanozyme-strip test can rapidly and sensitively detect EBOV, providing a valuable simple screening tool for diagnosis of infection in Ebola-stricken areas.

Direct microRNA detection with universal tagged probe and time-resolved fluorescence technology

microRNAs have emerged as the central player in gene expression regulation and have been considered as potent cancer biomarkers for early disease diagnosis. Direct microRNA detection without amplification and labeling is highly desired. Here we present a rapid, sensitive and selective microRNA detection method based on the base stacking hybridization coupling with time-resolved fluorescence technology. Other than planar microarrays, magnetic beads are used as reaction platforms. In this method, one universal tag is used to report all microRNA targets. Its specificity allows for discrimination between microRNAs differing by a single nucleotide, and between precursor and mature microRNAs. This method also provides a high sensitivity down to 20 fM. Moreover, the full protocol can be completed in about 3 h starting from total RNA.

A reverse transcription-free real-time PCR assay for rapid miRNAs quantification based on effects of base stacking.

A rapid and reverse transcription-free real-time PCR microRNA assay was developed based on effects of base stacking. This microRNA assay has been shown to be highly specific to homogenous miRNAs, and the procedure can be completed within 30 min starting from total RNA.

ENHANCING DNA DETECTION SENSITIVITY THROUGH A TWO-STEP ENRICHMENT METHOD WITH MAGNETIC BEADS AND DROPLET EVAPORATION

A simple and sensitive DNA detection method has been developed through a two-step enrichment process. Trace amount of target DNA in a large volume (1 mL) is selectively separated and condensed with DNA-modified magnetic beads into a small volume (5 μL) by denaturalization. Then, the pre-enriched target DNA solution (1 μL) is transferred onto a smooth hydrophobic surface, where the target DNA is further-enriched by natural sessile droplet evaporation. Using this method, the fluorescence detection sensitivity of the target DNA can be enhanced by 3 orders of magnitude and as low as 3.91 pM of the target DNA can be detected within 2 hours.

Standardized assays for determining the catalytic activity and kinetics of peroxidase-like nanozymes.

Nanozymes are nanomaterials exhibiting intrinsic enzyme-like characteristics that have increasingly attracted attention, owing to their high catalytic activity, low cost and high stability. This combination of properties has enabled a broad spectrum of applications, ranging from biological detection assays to disease diagnosis and biomedicine development. Since the intrinsic peroxidase activity of Fe3O4 nanoparticles (NPs) was first reported in 2007, >40 types of nanozymes have been reported that possess peroxidase-, oxidase-, haloperoxidase- or superoxide dismutase–like catalytic activities. Given the complex interdependence of the physicochemical properties and catalytic characteristics of nanozymes, it is important to establish a standard by which the catalytic activities and kinetics of various nanozymes can be quantitatively compared and that will benefit the development of nanozyme-based detection and diagnostic technologies. Here, we first present a protocol for measuring and defining the catalytic activity units and kinetics for peroxidase nanozymes, the most widely used type of nanozyme. In addition, we describe the detailed experimental procedures for a typical nanozyme strip–based biological detection test and demonstrate that nanozyme-based detection is repeatable and reliable when guided by the presented nanozyme catalytic standard. The catalytic activity and kinetics assays for a nanozyme can be performed within 4 h.Nanozymes are nanomaterials that possess intrinsic enzyme-like properties and are increasingly used in various biological applications. This protocol describes standardized assays of the catalytic activity and kinetics of peroxidase-like nanozymes.

A thermodynamic model for predicting phosphate capacity of CaO-based slags during hot metal dephosphorisation pretreatment process

A thermodynamic model for predicting phosphate capacity of the CaO-based slags over a temperature range from 1750 to 1793 K (1477–1520°C) during dephosphorisation pretreatment of hot metal has been developed based on the ion and molecule coexistence theory (IMCT), i.e. the IMCT– model. The developed IMCT– model has been verified to be valid through comparing with the measured and the predicted ones by four reported models from the literature. Besides the total phosphate capacity , the respective phosphate capacity of nine dephosphorisation products as P2O5, 3FeO·P2O5, 4FeO·P2O5, 2CaO·P2O5, 3CaO·P2O5, 4CaO·P2O5, 2MgO·P2O5, 3MgO·P2O5 and 3MnO·P2O5 can also be accurately predicted by the developed IMCT– model. The formed 3CaO·P2O5 accounts for 99.20% of dephosphorisation products comparing with the generated 4CaO·P2O5 for 0.80%. The results also revealed that the effects of slag chemical composition including slag oxidisation ability, slag basicity and the comprehensive influence of iron oxides FetO and basic oxide CaO on phosphate capacity were largely different with those on phosphorus partition between the CaO-based slags and hot metal. Although and are both distinct thermodynamic quantities and related to each others provided that the entire slag system is correctly defined, phosphorus partition has much guide meaning on dephosphorisation operation than phosphate capacity . The main interest of measuring phosphate capacity from laboratory experiments can be attributed to obtain phosphorus partition through relationship between and .

Development and Application of Electromagnetic Stirring Method in Horizontal Continuous Casting

The coupling relationships between dephosphorisation and desulphurisation abilities or potentials for CaO–FeO–Fe2O3–Al2O3–P2O5 slags during secondary refining process of molten steel have been proposed as or in reducing zone at oxygen potential less than 2.27 × 10−6 Pa as well as or in oxidising zone at oxygen potential greater than 2.27 × 10−6 Pa through deleting slag oxidisation ability expressed by the comprehensive mass action concentration of iron oxides based on the ion and molecule coexistence theory (IMCT). The proposed coupling relationships between dephosphorisation and desulphurisation abilities or potentials of the slags have been verified to be valid using reported data of oxygen, phosphorus and sulphur distribution equilibria between the slags and liquid iron over a temperature range from 1811 to 1927 K (1538–1654 °C) and predicted results of dephosphorisation and desulphurisation abilities or potentials. Not only the proposed coupling relationships between dephosphorisation and desulphurisation potentials but also the correlated coupling relationships between dephosphorisation and desulphurisation abilities for the slags in reducing and oxidising zones have been verified to be indeed independent of slag oxidisation ability.

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.