Metrics & MoreArticle Recommendations CONSPECTUS: Ferritins are spherical iron storage proteins within cells that are composed of a combination of 24 subunits of two types, heavy-chain ferritin (HFn) and light-chain ferritin (LFn). They autoassemble naturally into a spherical hollow nanocage with an outer diameter of 12 nm and an interior cavity that is 8 nm in diameter. In recent years, with the constantly emerging safety issues and the concerns about unfavorable uniformity and indefinite in vivo behavior of traditional nanomedicines, the characteristics of native ferritin nanocages, such as the unique nanocage structure, excellent safety profile, and definite in vivo behavior, make ferritin-based formulations uniquely attractive for nanomedicine development. To date, a variety of cargo molecules, including therapeutic drugs (e.g., cisplatin, carboplatin, paclitaxel, curcumin, atropine, quercetin, gefitinib, daunomycin, epirubicin, doxorubicin, etc.), imaging agents (e.g., fluorescence dyes, radioisotopes, and MRI contrast agents), nucleic acids (e.g., siRNA and miRNA), and metal nanoparticles (e.g., Fe 3 O 4 , CeO 2 , AuPd, CuS, CoPt, FeCo, Ag, etc.) have been loaded into the interior cavity of ferritin nanocages for a broad range of biomedical applications from in vitro biosensing to targeted delivery of cargo molecules in living systems with the aid of modified targeting ligands either genetically or chemically. We reported that human HFn could selectively deliver a large amount of cargo into tumors in vivo via transferrin receptor 1 (TfR1)-mediated tumor-cell-specific targeting followed by rapid internalization. By the use of the intrinsic tumor-targeting property and unique nanocage structure of human HFn, a broad variety of cargo-loaded HFn formulations have been developed for biological analysis, imaging diagnosis, and medicine development. In view of the intrinsic tumor-targeting property, unique nanocage structure, lack of immunogenicity, and definite in vivo behavior, human HFn holds promise to promote therapeutic drugs, diagnostic imaging agents, and targeting moieties into multifunctional nanomedicines.Since the report of the intrinsic tumor-targeting property of human HFn, we have extensively explored human HFn as an ideal nanocarrier for tumor-targeted delivery of anticancer drugs, MRI contrast agents, inorganic nanoparticles, and radioisotopes. In particular, by the use of genetic tools, we also have genetically engineered human HFn nanocages to recognize a broader range of disease biomarkers. In this Account, we systematically review human ferritins from characterizing their tumor-binding property and understanding their mechanism and kinetics for cargo loading to exploring their biomedical applications. We finally discuss the prospect of ferritin-based formulations to become next-generation nanomedicines. We expect that ferritin formulations with unique physicochemical characteristics and intrinsic tumor-targeting property will attract broad interest in fundamental drug research and offer new op...

Precisely quantifying the membrane protein expression level on cell surfaces is of vital importance for early cancer diagnosis and efficient treatment. We demonstrate that gold nanoparticle bioconjugated by a rationally designed peptide as nanoprobe possesses selective labeling and accurate quantification capacity of integrin GPIIb/IIIa on the human erythroleukemia cell line. Through selective recognition and marking of integrin, two-photon photoluminescence of the nanoprobe is exploited for direct observation of protein spatial distribution on cell membrane. More importantly, utilizing intrinsic enzyme-like catalysis property of the nanoprobe, the expression level of integrin on human erythroleukemia cells can be quantitatively counted in an amplified and reliable colorimetric assay without cell lysis and protein extraction process. In addition, the analysis of the correlation between the gold nanoparticle and the membrane protein via relevant inductively coupled plasma mass spectrometry measurement verifies the reliability of the new analytical method. It is anticipated that this facile and efficient strategy holds a great promise for a rapid, precise, and reliable quantification of interested functional membrane proteins on the cell surface.

We reported two Au clusters with precisely controlled molecular size (AuPeptide and AuPeptide) showing different antitumor effects. In vitro, both AuPeptide and AuPeptide were well taken up by human nasopharyngeal cancer cells (CNE1 cells). However, only AuPeptide significantly induced CNE1 cell apoptosis. Further studies showed that CNE1 cells took up AuPeptide (1.98 × 10 mol/cell), and 9% of them entered mitochondria (0.186 × 10 mol/cell). As a comparison, the uptake of AuPeptide was only half the amount of AuPeptide (1.11 × 10 mol/cell), and only 1% of them entered mitochondria (0.016 × 10 mol/cell). That gave 11.6-fold more AuPeptide in mitochondria of CNE1 cells than AuPeptide. Further cell studies revealed that the antitumor effect may be due to the enrichment of AuPeptide in mitochondria. AuPeptide slightly decreased the Mcl-1 (antiapoptotic protein of mitochondria) and significantly increased the Puma (pro-apoptotic protein of mitochondria) expression level in CNE1 cells, which resulted in mitochondrial transmembrane potential change and triggered the caspase 9-caspase 3-PARP pathway to induce CNE1 cell apoptosis. In vivo, CNE1 tumor growth was significantly suppressed by AuPeptide in the xenograft model after 3 weeks of intraperitoneal injection. The TUNEL and immuno-histochemical studies of tumor tissue verified that CNE1 cell apoptosis was mainly via the Puma and Mcl-1 apoptosis pathway in the xenograft model, which matched the aforementioned CNE1 cell studies in vitro. The discovery of Au but not Au suppressing tumor growth via the mitochondria target was a breakthrough in the nanomedical field, as this provided a robust approach to turn on/off the nanoparticles' medical properties via atomically controlling their sizes.

We report for the first time seeing and counting integrin α(IIb)β3 on a single-cell level. The proposed method is based on the using of the Au cluster probe. With the fluorescent property of Au24 cluster and the specific targeting ability of peptide, our probe can directly visualize integrin α(IIb)β3 on the membrane of human erythroleukemia cells (HEL) via confocal microscopy. On the basis of the accurate formula of our probe (Au24Peptide8), the number of integrin α(IIb)β3 can be precisely counted by quantifying the gold content on a single HEL cell via laser ablation inductively coupled plasma mass spectrometry. Our results reveal that the number of integrin α(IIb)β3 on a single cell varies from 5.75 × 10(-17) to 9.11 × 10(-17) mol, because of the heteroexpression levels of α(IIb)β3 on individual cells.

Despite the precise controllability of droplet samples in digital microfluidic (DMF) systems, their capability in isolating single cells for long-time culture is still limited: typically, only a few cells can be captured on an electrode. Although fabricating small-sized hydrophilic micropatches on an electrode aids single-cell capture, the actuation voltage for droplet transportation has to be significantly raised, resulting in a shorter lifetime for the DMF chip and a larger risk of damaging the cells. In this work, a DMF system with 3D microstructures engineered on-chip is proposed to form semiclosed micro-wells for efficient single-cell isolation and long-time culture. Our optimum results showed that approximately 20% of the micro-wells over a 30 × 30 array were occupied by isolated single cells. In addition, lowevaporation-temperature oil and surfactant aided the system in achieving a low droplet actuation voltage of 36V, which was 4 times lower than the typical 150 V, minimizing the potential damage to the cells in the droplets and to the DMF chip. To exemplify the technological advances, drug sensitivity tests were run in our DMF system to investigate the cell response of breast cancer cells (MDA-MB-231) and breast normal cells (MCF-10A) to a widely used chemotherapeutic drug, Cisplatin (Cis). The results on-chip were consistent with those screened in conventional 96well plates. This novel, simple and robust single-cell trapping method has great potential in biological research at the single cell level.

Kidney disease is a worldwide health hazard. Noninvasive imaging modalities such as computed tomography are often used for diagnosis, to guide treatment, and to assess a disease state over the long-term. The physiology of the kidneys can be assessed with contrast agents. We present an albumin-stabilized Au cluster with red fluorescence and robust X-ray attenuation. In vivo studies revealed distribution of the Au clusters in the liver, spleen, and kidneys, with excretion mostly via the kidneys. Under optimal conditions, this agent can outline the anatomy of mouse kidneys on 2D and 3D computed tomography imaging, with clear visualization of the renal collecting system and ureters. This is a promising agent for kidney visualization and disease diagnosis.

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A digital microfluidic system with an innovative control structure and chip design to generate concentrations that span three to four orders of magnitude for single or multi-drug dispensing was developed.