Xiaolan Chen

Using Aptamer-Conjugated Fluorescence Resonance Energy Transfer Nanoparticles for Multiplexed Cancer Cell Monitoring

To facilitate the selection of effective therapeutic pathways and improve clinical outcomes, sensitive and simultaneous diagnosis of multiple trace biomarkers or cancer cells from complex living samples is particularly critical in the early stages of tumor development. To achieve this, we have combined the selectivity and affinity of aptamers with the spectroscopic advantages of fluorescence resonance energy transfer (FRET) nanoparticles (NPs). This has produced an aptamer-conjugated FRET NP assay that performs simultaneous multiplexed monitoring of cancer cells with the desired degree of sensitivity and selectivity. First, by changing the doping ratio of three different dyes, the FRET-mediated emission signatures could be tuned such that the nanoparticles would exhibit multiple colors upon excitation with a single wavelength. These FRET nanoparticles were then modified by a few aptamers specific for different cancer cell lines, in this case, T-cell leukemia and B-cell lymphoma. As a result, simultaneous and sensitive detection of multiple cancer cell targets was achieved. Therefore, our aptamer-conjugated FRET NPs are highly promising for potential applications in the sensitive monitoring of multiple cancer cells for biomedical research and medical diagnostics.

Multifunctional Core–Shell Upconverting Nanoparticles for Imaging and Photodynamic Therapy of Liver Cancer Cells

Lanthanide-doped upconversion nanoparticles (UCNPs) have attracted considerable attention for their application in biomedicine. Here, silica-coated NaGdF(4):Yb,Er/NaGdF(4) nanoparticles with a tetrasubstituted carboxy aluminum phthalocyanine (AlC(4)Pc) photosensitizer covalently incorporated inside the silica shells were prepared and applied in the photodynamic therapy (PDT) and magnetic resonance imaging (MRI) of cancer cells. These UCNP@SiO(2)(AlC(4)Pc) nanoparticles were uniform in size, stable against photosensitizer leaching, and highly efficient in photogenerating cytotoxic singlet oxygen under near-infrared (NIR) light. In vitro studies indicated that these nanoparticles could effectively kill cancer cells upon NIR irradiation. Moreover, the nanoparticles also demonstrated good MR contrast, both in aqueous solution and inside cells. This is the first time that NaGdF(4):Yb,Er/NaGdF(4) upconversion-nanocrystal-based multifunctional nanomaterials have been synthesized and applied in PDT. Our results show that these multifunctional nanoparticles are very promising for applications in versatile imaging diagnosis and as a therapy tool in biomedical engineering.

A Surface Energy Transfer Nanoruler for Measuring Binding Site Distances on Live Cell Surfaces

Measuring distances at molecular length scales in living systems is a significant challenge. Methods like Förster resonance energy transfer (FRET) have limitations due to short detection distances and strict orientations. Recently, surface energy transfer (SET) has been used in bulk solutions; however, it cannot be applied to living systems. Here, we have developed an SET nanoruler, using aptamer-gold nanoparticle conjugates with different diameters, to monitor the distance between binding sites of a receptor on living cells. The nanoruler can measure separation distances well beyond the detection limit of FRET. Thus, for the first time, we have developed an effective SET nanoruler for live cells with long distance, easy construction, fast detection, and low background. This is also the first time that the distance between the aptamer and antibody binding sites in the membrane protein PTK7 was measured accurately. The SET nanoruler represents the next leap forward to monitor structural components within living cell membranes.

Synthesis of magnetic, fluorescent and mesoporous core-shell-structured nanoparticles for imaging, targeting and photodynamic therapy

A synthetic method to prepare novel multifunctional core-shell-structured mesoporous silica nanoparticles for simultaneous magnetic resonance (MR) and fluorescence imaging, cell targeting and photosensitization treatment has been developed. Superparamagnetic magnetite nanoparticles and fluorescent dyes are co-encapsulated inside nonporous silica nanoparticles as the core to provide dual-imaging capabilities (MR and optical). The photosensitizer molecules, tetra-substituted carboxyl aluminum phthalocyanine (AlC4Pc), are covalently linked to the mesoporous silica shell and exhibit excellent photo-oxidation efficiency. The surface modification of the core-shell silica nanoparticles with folic acid enhances the delivery of photosensitizers to the targeting cancer cells that overexpress the folate receptor, and thereby decreases their toxicity to the surrounding normal tissues. These unique advantages make the prepared multifunctional core-shell silica nanoparticles promising for cancer diagnosis and therapy.

Superparamagnetic core-shell polymer particles for efficient purification of his-tagged proteins

Magnetic core-shell Fe3O4@SiO2@poly(styrene-alt-maleic anhydride) spheres enriched with Ni-NTA on their surface have been prepared by precipitation polymerization. The spheres have a core composed of superparamagnetic polycrystalline magnetite having a uniform size of ∼220 nm, endowing the spheres with excellent magnetic responsivity and dispersity. The shell composition of poly(styrene-alt-maleic anhydride) allows the incorporation of more Ni-NTA affinity sites onto the surface of the magnetic spheres. Owing to the multivalency effect, the separation capacity of His-tagged proteins by the as-prepared Fe3O4@SiO2@polymer/Ni-NTA composites was four times as that by Fe3O4@SiO2/Ni-NTA, making them particularly promising for the magnetic separation of low-concentration His-tagged proteins. The magnentic polymer hybrid particles also exhibited excellent performance in the direct separation of His-tagged proteins from cells lysates.

Simultaneous Photodynamic and Photothermal Therapy Using Photosensitizer-Functionalized Pd Nanosheets by Single Continuous Wave Laser

In this work, we prepared chlorin e6 (Ce6)-functionalized Pd nanosheets (Pd-PEI-Ce6) for the photodynamic and photothermal combined therapy that use a single laser. To fabricate the Pd-PEI-Ce6 nanocomposite, photosensitizer Ce6 were chemically conjugated to polyethylenimine (PEI) and the formed Ce6-PEI conjugates were then anchored onto Pd nanosheets by electrostatic and coordination interaction. The prepared Pd-PEI-Ce6 nanocomposite were about 4.5 nm in size, exhibited broad, and strong absorption from 450 to 800 nm, good singlet oxygen generation capacity and photothermal conversion efficiency, and excellent biocompability. Significantly greater cell killing was observed when HeLa cells incubated with Pd-PEI-Ce6 were irradiated with the 660 nm laser, attributable to both Pd nanosheets-mediated photothermal ablation and the photodynamic destruction effect of photosensitizer Ce6. The double phototherapy effect was also confirmed in vivo. It was found that the Pd-PEI-Ce6 treated tumor-bearing mice displayed the enhanced therapeutic efficiency compared to that of Pd-PEI, or Ce6-treated mice. Our work highlights the promise of using Pd nanosheets for potential multimode cancer therapies.

Copper sulfide nanoparticles with phospholipid-PEG coating for in vivo near-infrared photothermal cancer therapy.

In this work, small sizes of hydrophobic copper sulfide nanoparticles (CuS NPs, ∼3.8 nm in diameter) have been successfully prepared from the reaction of copper chloride with sodium diethyldithiocarbamate (SDEDTC) inside a heated oleylamine solution. These CuS NPs displayed strong absorption in the 700-1100 nm near-infrared (NIR) region. By coating CuS NPs with DSPE-PEG2000 on the surface, the as-synthesized CuS@DSPE-PEG NPs exhibited good water solubility, significant stability and biocompatibility, as well as excellent photothermal conversion effects upon exposure to an 808 nm laser. After intravenous administration to mice, the CuS@DSPE-PEG NPs were found to passively target to the tumor site, and tumor tissues could be ablated efficiency under laser irradiation. In addition, CuS@DSPE-PEG NPs do not show significant toxicity by histological and blood chemistry analysis, and can be effectively excreted via metabolism. Our results indicated that CuS@DSPE-PEG NPs can act as an ideal photothermal agent for cancer photothermal therapy.

Preparation and photodynamic therapy application of NaYF4:Yb, Tm–NaYF4:Yb, Er multifunctional upconverting nanoparticles

The preparation, characterization and application of NaYF4:Yb3+, Tm3+–NaYF4:Yb3+, Er3+ core–shell upconversion nanocrystals (UCNPs) with multiple emission peaks (e.g. 539, 654 and 802 nm) have been demonstrated in this work. The monodisperse nanocrystals were prepared via a modified thermal decomposition synthesis. The resulting UCNPs were ∼31 nm in diameter with the lanthanide ions Tm3+ and Er3+ doped in the core and the shell, respectively. Under the laser diode excitation at 980 nm, these core–shell nanocrystals give strong upconversion emissions from the visible to near-infrared (NIR) region. By coating a PEG–phospholipid (PP) layer on the surface of the nanocrystals, the as-prepared UCNPs were favorably endowed with good water solubility for the potential biological applications. Here, a photosensitizer drug of Chlorin e6 (Ce6), which has maximum absorption that overlaps with the red emission of UCNPs, was loaded on these PP-coated UCNPs (UCNP@PP) by physical adsorption. The activity of the Ce6-loaded UCNP@PP (UCNP@PP–Ce6) in photodynamic therapy of cancer cells in vitro has been fully investigated in this work. Our results indicated that these multifunctional UCNP@PP–Ce6 nanoparticles have efficient NIR-to-NIR upconversion luminescence and photodynamic therapy capabilities, which could be potentially employed as a theranostic platform for cancer treatment.

Cancer therapy improvement with mesoporous silica nanoparticles combining photodynamic and photothermal therapy.

In this work, we develop novel mesoporous silica composite nanoparticles (hm-SiO2(AlC4Pc)@Pd) for the co-delivery of photosensitizer (PS) tetra-substituted carboxyl aluminum phthalocyanine (AlC4Pc) and small Pd nanosheets as a potential dual carrier system to combine photodynamic therapy (PDT) with photothermal therapy (PTT). In the nanocomposite, PS AlC4Pc was covalently conjugated to a mesoporous silica network, and small Pd nanosheets were coated onto the surface of mesoporous silica by both coordination and electrostatic interaction. Since small Pd nanosheets and AlC4Pc display matched maximum absorptions in the 600-800 nm near-infrared (NIR) region, the fabricated hm-SiO2(AlC4Pc)@Pd nanocomposites can generate both singlet oxygen and heat upon 660 nm single continuous wavelength (CW) laser irradiation. In vitro results indicated that the cell-killing efficacy by simultaneous PDT/PTT treatment using hm-SiO2(AlC4Pc)@Pd was higher than PDT or PTT treatment alone after exposure to a 660 nm CW-NIR laser.

Nanoparticle-aptamer conjugates for cancer cell targeting and detection.

Aptamers are DNA or RNA oligonucleotide sequences that selectively bind to their target with high affinity and specificity. They are obtained using an iterative selection protocol called SELEX. Several small molecules and proteins have been used as targets. Recently, a variant of this methodology, known as cell-SELEX, has been developed for a new generation of aptamers, which are capable of recognizing whole living cells. We have used this methodology for the selection of aptamers, which show high affinity and specificity for several cancer cells. In this chapter, we describe (1) the process followed for the generation of aptamers capable of recognizing acute leukemia cells (CCRF-CEM cells) and (2) the method of enhancing the selectivity and sensitivity of these aptamers by conjugation with a dual-nanoparticle system, which combines magnetic nanoparticles (MNP) and fluorescent silica nanoparticles (FNP). Specifically, the selected aptamers, which showed dissociation constants in the nanomolar range, have been coupled to MNPs in order to selectively collect and enrich cells from complex matrices, including blood samples. The additional coupling of the aptamer to FNPs offers an excellent and highly sensitive method for detecting cancer cells. In order to prove the potential of this rapid and low-cost method for diagnostic purposes, confocal microscopy was used to confirm the specific collection and detection of target cells in concentrations as low as 250 cells. The final fluorescence of the cells labeled with the nanoparticles was quantified using a fluorescence microplate reader.