Spatially resolved proteomics via tissue expansion.

Spatially resolved proteomics is an emerging approach for mapping proteome heterogeneity of biological samples, however, it remains technically challenging due to the complexity of the tissue microsampling techniques and mass spectrometry analysis of nanoscale specimen volumes. Here, we describe a spatially resolved proteomics method based on the combination of tissue expansion with mass spectrometry-based proteomics, which we call Expansion Proteomics (ProteomEx). ProteomEx enables quantitative profiling of the spatial variability of the proteome in mammalian tissues at ~160 µm lateral resolution, equivalent to the tissue volume of 0.61 nL, using manual microsampling without the need for custom or special equipment. We validated and demonstrated the utility of ProteomEx for streamlined large-scale proteomics profiling of biological tissues including brain, liver, and breast cancer. We further applied ProteomEx for identifying proteins associated with Alzheimer's disease in a mouse model by comparative proteomic analysis of brain subregions.

Chaperonin-GroEL as a Smart Hydrophobic Drug Delivery and Tumor Targeting Molecular Machine for Tumor Therapy.

The targeted delivery of hydrophobic therapeutic drugs to tumors is one of the major challenges in drug development. The use of natural proteins as drug delivery vehicles holds great promise due to various functionalities of proteins. In the current study, we exploited a natural protein, GroEL, which possesses a double layer cage structure, as a hydrophobic drug container, which is switchable by ATP binding to a hydrophilic status, to design a novel and intelligent hydrophobic drug delivery molecular machine with a controlled drug release profile. When loaded with the hydrophobic antitumor drug, Doxorubicin (Dox), GroEL was able to shield the drug from the aqueous phase of blood, releasing the drug once in the presence of a critical concentration of ATP at the tumor site. Unexpectedly, we found that GroEL has a specific affinity for the cell structural protein, plectin, which is expressed at abnormally elevated levels on the membranes of tumor cells but not in normal cells. This finding, in combination with the ATP sensitivity, makes GroEL a superior natural tumor targeting nanocarrier. Our data show that GroEL-Dox is able to effectively, and highly selectively, deliver the hydrophobic drug to fast growing tumors without overt adverse effects on the major organs. GroEL is therefore a promising drug delivery platform that can overcome the obstacles to hydrophobic drug targeting and delivery.

Fine-tuned h-ferritin nanocage with multiple gold clusters as near-infrared kidney specific targeting nanoprobe.

When stabilized and functionalized by biomolecules, noble metal (such as gold and silver) cluster-based hybrid nanocomposites have shown great promise for biomedical applications, due to their unique physiochemical properties originating from the inorganic elements and specific functionality and biocompatibility from their biological components. Although certain promise for bioimaging, biosensing, and biomimetic catalysis has been demonstrated, it is still a great challenge to integrate the defined functionality of the biomolecules with enhanced or novel physiochemical properties of the metal clusters, under control at the molecular level. Herein, based on molecular dynamics simulation of a gold (Au) cluster assembly, we designed near-infrared (NIR) fluorescent hybrid nanocomposites with multiple Au clusters within an apo H-ferritin (HFt) nanocage. The fluorescence quantum yield of near-infrared (NIR) Au-HFt is about 63.4% and the emission peak is 810 nm. The NIR Au-HFt is one of the first native protein-guided Au cluster-based nanomaterials for in vivo biowindow imaging. In vivo fluorescent imaging and quantification of Au element confirmed that Au-HFt not only retained the kidney targeting properties of HFt well (about 10 times higher Au concentration in kidney than in liver and spleen, the most common organs for nanoparticle accumulation), but also gained strong NIR imaging capability for live animals. The NIR Au-HFt showed powerful tissue penetrating ability, strong fluorescent efficiency, and excellent kidney targeting specificity. These results thus open new opportunities for kidney disease imaging and theranostic applications.

Peroxidase-like activity of apoferritin paired gold clusters for glucose detection.

The discovery and application of noble metal nanoclusters have received considerable attention. In this paper, we reported that apoferritin paired gold clusters (Au-Ft) could efficiently catalyze oxidation of 3.3',5.5'-tetramethylbenzidine (TMB) by H2O2 to produce a blue color reaction. Compared with natural enzyme, Au-Ft exhibited higher activity near acidic pH and could be used over a wide range of temperatures. Apoferritin nanocage enhanced the reaction activity of substrate TMB by H2O2. The reaction catalyzed by Au-Ft was found to follow a typical Michaelis-Menten kinetics. The kinetic parameters exhibited a lower K(m) value (0.097 mM) and a higher K(cat) value (5.8 × 10(4) s(-1)) for TMB than that of horse radish peroxidase (HRP). Base on these findings, Au-Ft, acting as a peroxidase mimetic, performed enzymatic spectrophotometric analysis of glucose. This system exhibited acceptable reproducibility and high selectivity in biosening, suggesting that it could have promising applications in the future.

Effects of nanoparticle size and gestational age on maternal biodistribution and toxicity of gold nanoparticles in pregnant mice.

Gold nanoparticles (GNPs) have considerable applications in biomedicine, such as in bio-sensing, bio-imaging, drug delivery and photothermal therapeutics. However, currently there are limited information regarding the impact of pregnancy on their biodistribution, elimination and toxicity. In this study, we investigated the biodistribution and potential toxic effects of different-sized GNPs (1.5, 4.5, 13, 30 and 70 nm in diameter) in non-pregnant and pregnant mice at different gestational ages (E5.5, 7.5, 9.5, 11.5 and 13.5). 5h after intravenous injection, GNPs exhibited size-dependent biodistribution profiles; however, regardless of size, no significant biodistribution changes were observed between non-pregnant and pregnant mice. Kinetic studies showed that 4.5 nm GNPs were primarily excreted through urine within 5h, whereas 30 nm GNPs had a more prolonged blood circulation time. No apparent toxic effects (e.g., increased mortality, altered behavior, reduced animal weight, abnormal organ morphology or reduced pregnancy duration) were observed with different-sized GNPs in pregnant mice. However, treatment with 30 nm GNPs induced mild emphysema-like changes in lungs of pregnant mice. These results indicated that the maternal biodistribution patterns of GNPs in pregnant mice depended on particle size, but not gestational age; organ-specific adverse effects may arise with treatment with some GNPs according to their size.

Effects of gestational age and surface modification on materno-fetal transfer of nanoparticles in murine pregnancy.

Nanoparticle exposure in pregnancy may result in placental damage and fetotoxicity; however, the factors that determine fetal nanoparticle exposure are unclear. Here we have assessed the effect of gestational age and nanoparticle composition on fetal accumulation of maternally-administered nanomaterials in mice. We determined the placental and fetal uptake of 13 nm gold nanoparticles with different surface modifications (ferritin, PEG and citrate) following intravenous administration at E5.5-15.5. We showed that prior to E11.5, all tested nanoparticles could be visualized and detected in fetal tissues in significant amounts; however, fetal gold levels declined dramatically post-E11.5. In contrast, Au-nanoparticle accumulation in the extraembryonic tissues (EET) increased 6-15 fold with gestational age. Fetal and EET accumulation of ferritin- and PEG-modified nanoparticles was considerably greater than citrate-capped nanoparticles. No signs of toxicity were observed. Fetal exposure to nanoparticles in murine pregnancy is, therefore, influenced by both stage of embryonic/placental maturation and nanoparticle surface composition.

Controlling assembly of paired gold clusters within apoferritin nanoreactor for in vivo kidney targeting and biomedical imaging.

Functional nanostructures with high biocompatibility and stability, low toxicity, and specificity of targeting to desired organs or cells are of great interest in nanobiology and medicine. However, the challenge is to integrate all of these desired features into a single nanobiostructure, which can be applied to biomedical applications and eventually in clinical settings. In this context, we designed a strategy to assemble two gold nanoclusters at the ferroxidase active sites of ferritin heavy chain. Our studies showed that the resulting nanostructures (Au-Ft) retain not only the intrinsic fluorescence properties of noble metal, but gain enhanced intensity, show a red-shift, and exhibit tunable emissions due to the coupling interaction between the paired Au clusters. Furthermore, Au-Ft possessed the well-defined nanostructure of native ferritin, showed organ-specific targeting ability, high biocompatibility, and low cytotoxicity. The current study demonstrates that an integrated multimodal assembly strategy is able to generate stable and effective biomolecule-noble metal complexes of controllable size and with desirable fluorescence emission characteristics. Such agents are ideal for targeted in vitro and in vivo imaging. These results thus open new opportunities for biomolecule-guided nanostructure assembly with great potential for biomedical applications.