Zhen Lei

Facile Preparation of Doxorubicin-Loaded Upconversion@Polydopamine Nanoplatforms for Simultaneous In Vivo Multimodality Imaging and Chemophotothermal Synergistic Therapy

The development of biosafe nanoplatforms with diagnostic and therapeutic multifunction is extremely demanded for designing cancer theranostic medicines. Here, a facile methodology is developed to construct a multifunctional nanotheranostic that gathers five functions, upconversion luminescence (UCL) imaging, T1-weighted magnetic resonance imaging (MRI), X-ray computed tomography (CT) imaging, photothermal therapy (PTT), and chemotherapy, into one single nanoprobe (named as UCNP@PDA5-PEG-DOX). For generating the UCNP@PDA5-PEG-DOX, a near-infrared light (NIR)-absorbing polydopamine (PDA) shell is directly coated on oleic-acid-capped β-NaGdF4:Yb(3+),Er(3+)@β-NaGdF4 upconverting nanoparticle (UCNP) core for the first time to form monodisperse, ultrastable, and noncytotoxic core-shell-structured nanosphere via water-in-oil microemulsion approach. When combined with 808 nm NIR laser irradiation, the UCNP@PDA5-PEG-DOX shows great synergistic interaction between PTT and the enhanced chemotherapy, resulting in completely eradicated mouse-bearing SW620 tumor without regrowth. In addition, leakage study, hemolysis assay, histology analysis, and blood biochemistry assay unambiguously reveal that the UCNP@PDA5-PEG has inappreciable cytotoxicity and negligible organ toxicity. The results provide explicit strategy for fabricating multifunctional nanoplatforms from the integration of UCNP with NIR-absorbing polymers, important for developing multi-mode nanoprobes for biomedical applications.

Fabricating three-dimensional carbohydrate hydrogel microarray for lectin-mediated bacterium capturing.

Herein, a three-dimensional carbohydrate modified polyacrylamide hydrogel microarray (3D carbohydrate hydrogel microarray) has been fabricated and employed as micro-reactor for capturing Escherichia coli (E. coli) by multivalent binding of concanavalin A (Con A) with O-antigen on the cellular surface of E. coli and immobilized monosaccharides on hydrogel spot, and the interactions of type 1 fimbriae of E. coli with immobilized monosaccharides. Because of the transparent performance of polyacrylamide hydrogel, the captured E. coli can be directly observed by a conventional microscope under a bright-field mode. The experimental result demonstrates that α-D-mannopyranoside (Man-α) modified hydrogel surface shows high efficiency of E. coli capturing. The 3D Man-α hydrogel microarray-based assay shows reasonable low detection limit (1.0×10(4) cells/mL) and large dynamic range (1.0×10(5) to 1.0×10(9)cells/mL) for detecting E. coli. In addition, bacterial adhesion inhibition assay has been demonstrated by the interactions of E. coli with ten saccharides, and satisfactory results have been obtained.

A novel upconversion@polydopamine core@shell nanoparticle based aptameric biosensor for biosensing and imaging of cytochrome c inside living cells.

Herein, a novel upconversion@polydopamine core@shell nanoparticle (termed as UCNP@PDA NP) -based aptameric biosensor has been fabricated for the quantitative analysis of cytochrome c (Cyt c) inside living cells, which comprises an UCNP@PDA NP, acting as an internal reference and fluorescence quenching agent, and Cy3 modified aptamer enabling ratiometric quantitative Cyt c measurement. After the hybridization of Cy3 labeled aptamer with amino-terminated single DNA on the UCNP@PDA NP surface (termed as UCNP@PDA@AP), the fluorescence of Cy3 can be efficiently quenched by the PDA shell. With the spontaneous cellular uptake of UCNP@PDA@AP, the Cyt c aptamer dissociates from UCNP@PDA NP surface through formation of aptamer-Cyt c complex, resulting in concomitant activation of the Cy3 fluorescence. High amount of Cyt c leads to high fluorescence emission, enabling direct visualization/measurement of the Cyt c by fluorescence microscopy/spectroscopy. The steady upconversion luminescent (UCL) signals can be employed not only for intracellular imaging, but also as an internal reference for evaluating intracellular Cyt c amount using the ratio of fluorescence intensity of Cy3 with the UCL intensity of UCNP. The UCNP@PDA@AP shows a reasonable detection limit (20nM) and large dynamic range (50nM to 10μM, which covers the literature reported values (1-10μM) for cytosolic Cyt c in apoptotic cells) for detecting Cyt c in buffer with excellent selectivity. In addition, the UCNP@PDA@AP has been successfully used to monitor etoposide induced intracellular releasing of Cyt c, providing the possibility for cell-based screening of apoptosis-inducing drugs.

Poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) Brushes as Peptide/Protein Microarray Substrate for Improving Protein Binding and Functionality.

We developed a three-dimensional (3D) polymer-brush substrate for protein and peptide microarray fabrication, and this substrate was facilely prepared by copolymerization of glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) monomers via surface-initiated atom transfer radical polymerization (SI-ATRP) on a glass slide. The performance of obtained poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush substrate was assessed by binding of human IgG with rabbit antihuman IgG antibodies on a protein microarray and by the determination of matrix metalloproteinase (MMP) activities on a peptide microarray. The P(GMA-HEMA) brush substrate exhibited higher immobilization capacities for proteins and peptides than those of a two-dimensional (2D) planar epoxy slide. Furthermore, the sensitivity of the P(GMA-HEMA) brush-based microarray on rabbit antihuman IgG antibody detection was much higher than that of its 2D counterpart. The enzyme activities of MMPs were determined specifically with a low detection limit of 6.0 pg mL(-1) for MMP-2 and 5.7 pg mL(-1) for MMP-9. By taking advantage of the biocompatibility of PHEMA, the P(GMA-HEMA) brush-based peptide microarray was also employed to evaluate the secretion of MMP-2 and MMP-9 by cells cultured off the chip or directly on the chip, and satisfactory results were obtained.

Fe2O3@Au core@shell nanoparticle–graphene nanocomposites as theranostic agents for bioimaging and chemo-photothermal synergistic therapy

Herein, we present the synthesis and application of a new type of graphene-based magnetic and plasmonic nanocomposite for magnetic-field-assisted drug delivery and chemo-photothermal synergistic therapy. The nanocomposites were prepared via conjugation of the PEGylated Fe2O3@Au core/shell nanoparticles (Fe2O3@Au NPs) with reduced graphene oxide (rGO). The hybrid nanostructures (rGO–Fe2O3@Au NPs) are superparamagnetic and show great photothermal conversion efficiency under 808 nm near-infrared (NIR) laser irradiation and high drug loading ability (1.0 mg mg−1). MTT cell viability assay demonstrates that the chemotherapeutic drug, doxorubicin loaded rGO–Fe2O3@Au NPs (DOX–rGO–Fe2O3@Au NPs) have synergistic interaction between photothermal therapy (PTT) and chemotherapy. Furthermore, in vitro studies using HeLa cells show that the chemo-photothermal therapeutic efficacy of DOX–rGO–Fe2O3@Au NPs can be dramatically improved by the assistance of magnetic-field-guided drug delivery.

Facile fabrication of single-phase multifunctional BaGdF5 nanospheres as drug carriers.

Multifunctional BaGdF5 nanospheres with mesoporous, luminescent, and magnetic properties have been successfully synthesized with the assistance of trisodium citrate by a hydrothermal method. The mesoporous structure is revealed by scanning electron microscope and transmission electron microscope images as well as N2 adsorption-desorption isotherm. The as-synthesized BaGdF5 nanospheres exhibit an intense broad bluish emission (centered at 450 nm) under the excitation of 390 nm, which might originate from the CO2·(-) radical-related defect produced by Cit(3-) groups. It is also shown that these BaGdF5 nanospheres brightened the T1-weighted images, suggesting that they could act as T1 contrast agents for magnetic resonance imaging. Using metformin hydrochloride as the model drug, the luminescent porous spheres show good drug storage/release capability. Furthermore, the emission intensity varies as a function of the cumulative drug release, making the drug-carrying system easily trackable and monitorable by detecting the luminescence intensity. Additionally, the paramagnetic property, originating from the unpaired electrons of Gd(3+) ions, opens the possibility of directing the magnetic targeted carrier to the pathological site by magnetic field gradient.

Spheres-on-sphere silica microspheres as matrix for horseradish peroxidase immobilization and detection of hydrogen peroxide

In this work, spheres-on-sphere (SOS) silica microspheres are prepared via a facile one-pot synthesis. After functionalization with carboxyl groups, the carboxylated SOS silica microspheres (SOS-COOH microspheres) can serve as a support for horseradish peroxidase (HRP) covalent immobilization. The obtained enzyme hybrid (SOS-COOH-HRP) is more stable under alkaline conditions than the free counterpart, and exhibits longer-term storage stability and higher resistance toward the denaturing agents such as guanidine hydrochloride (GdmCl) and urea. The Michaelis–Menten constant (Km) of the immobilized enzyme is decreased slightly while the maximum rate of reaction (Vmax) is very close to that of free HRP, resulting in the catalytic efficiency of SOS-COOH-HRP being enhanced significantly. For evaluating its utility, a SOS-COOH-HRP-based colorimetric method has been developed for selectively and sensitively detecting H2O2 both in buffer and 10% diluted human serum. Furthermore, the SOS-COOH-HRP displays excellent reusability and reproducibility in cycle analysis. The results demonstrate that the SOS-COOH-HRP has great potential for practical applications in biosensing and industrial fields.

Peptide Microarray-Based Metal Enhanced Fluorescence Assay for Multiple Profiling of Matrix Metalloproteinases Activities

Matrix metalloproteinases (MMPs) are closely associated with cancer cell invasion and metastasis. Herein, a fluorescence resonance energy transfer (FRET)-peptide microarray-based metal enhanced fluorescence (MEF) assay is proposed for multiple and sensitive profiling of MMPs activities on a novel Au/Ag@SiO2 substrate. The Au/Ag@SiO2 substrate is prepared by electroless deposition of silver on gold nanoparticle (GNP) seeds, followed by SiO2 shell coating and surface functionalization. The specific FRET peptides are spotted on the Au/Ag@SiO2 substrate to sensitively detect MMPs (MMP-2, -3, -7, -9, -14) via fluorescence recovery by the MMP cleavage of quenched peptide motifs and further enhanced by MEF. Under the optimal conditions, the limits of detection are 12.2 fg mL-1 for MMP-2, 60 pg mL-1 for MMP-3, 0.22 pg mL-1 for MMP-7, 102 fg mL-1 for MMP-9, and 0.68 ng mL-1 for MMP-14, respectively. The practicability of the FRET-peptide microarray-based MEF assay is demonstrated by profiling of multiplexed MMPs activities in various cell lines and clinical thyroid tissue samples of papillary thyroid carcinoma (PTC) patients and thyroid nodules (TN) patients, and satisfactory results are obtained.

Peptide microarray-based fluorescence assay for simultaneously detecting matrix metalloproteinases

Matrix metalloproteinases (MMPs) are believed to play an important role in tumor invasion. Herein, a peptide microarray-based fluorescence assay has been proposed for simultaneously determining the activities of MMP-2 and MMP-9 through the strong binding affinity of fluorescein isothiocyanate modified avidin (avidin-FITC) with the immobilized biotinylated peptide substrate on the microarray. In the presence of MMPs, the biotin moiety is released from the microarray by enzymatic cleavage of the peptide substrate, resulting in a significant decrease of the fluorescence signal. Under the optimal experimental conditions, the fluorescence intensity changes (ΔF%) are proportional to the concentrations of MMP-2 and MMP-9 within the ranges of 50 pg mL−1 to 50 ng mL−1 and 50 pg mL−1 to 100 ng mL−1 in the enzyme mixture, respectively. The detection limits are 45 pg mL−1 for MMP-2, and 60 pg mL−1 for MMP-9. In particular, the activities of extracellular MMP-2 and MMP-9 are determined by the peptide microarray-based fluorescence assay, and satisfactory results are obtained.

Evaluation of Matrix Metalloproteinase Inhibition by Peptide Microarray-Based Fluorescence Assay on Polymer Brush Substrate and in Vivo Assessment

Matrix metalloproteinases (MMPs) are important biomarkers and potential therapeutic targets of tumor. In this report, a peptide microarray-based fluorescence assay is developed for MMPs inhibitors evaluation through immobilization of biotin-modified peptides on the poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush-modified glass slides. After biotin is recognized with cyanine 3 (Cy3)-modified avidin (Cy3-avidin), the microarrays can produce strong fluorescence signal. The biotin moieties detach from microarray, when the biotin-modified peptide substrates are specially cleaved by a MMP, resulting in decreased fluorescence intensity of the microarray. The decreasing level of fluorescence intensity is correlated with the MMP inhibition. Nine known MMP inhibitors against MMP-2 and MMP-9 are evaluated by the assay, and the quantitative determination of inhibitory potencies (half maximal inhibitory concentration) are obtained, which are comparable with the literatures. Two biocompatible fluorogenic peptides containing MMP-specific recognition sequences and FAM/Dabcyl fluorophore-quencher pair are designed as activatable reporter probes for sensing MMP-2 and MMP-9 activities in cell and in vivo. The peptide microarray-based results are well verified by the cell inhibition assay and in vitro fluorescence imaging, and further confirmed by the in vivo imaging of HT-1080 tumor-bearing mice.