Hae Young Ko

A Nucleolin-Targeted Multimodal Nanoparticle Imaging Probe for Tracking Cancer Cells Using an Aptamer

The recent advances in molecular imaging techniques, using cancer-targeting nanoparticle probes, provide noninvasive tracking information on cancer cells in living subjects. Here, we report a multimodal cancer-targeted imaging system capable of concurrent fluorescence imaging, radionuclide imaging, and MRI in vivo. Methods: A cobalt–ferrite nanoparticle surrounded by fluorescent rhodamine (designated MF) within a silica shell matrix was synthesized with the AS1411 aptamer (MF-AS1411) that targets nucleolin (a cellular membrane protein highly expressed in cancer) using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC). This purified MF-AS1411 particle was bound with 2-(p-isothio-cyanatobenzyl)-1,4,7-triazacyclonane-1,4,7-triacetic acid (p-SCN-bn-NOTA) chelating agent and further labeled with 67Ga-citrate (MFR-AS1411). The shape and size distribution of MFR-AS1411 were characterized by transmission electron microscope (TEM). The cellular distribution of the nucleolin protein using the MFR-AS1411 nanoparticle was detected by fluorescence confocal microscopy. Phantom MR images were obtained as the concentration of MFR-AS1411 increased, using a 1.5-T MRI scanner. In vivo 67Ga radionuclide imaging and MRI were performed using a γ-camera and a 1.5-T MR imager, respectively. Results: TEM imaging revealed MF and MFR-AS1411 to be spheric and well dispersed. The purified MFR-AS1411 nanoparticle showed specific fluorescence signals in nucleolin-expressing C6 cells, compared with MFR-AS1411 mutant (MFR-AS1411mt)–treated C6 cells. The rhodamine fluorescence intensity and 67Ga activity of MFR-AS1411 were enhanced in a dose-dependent manner as the concentration of MFR-AS1411 was increased. The 67Ga radionuclide was detected in both thighs of the mice injected with MFR-AS1411, whereas the MFR-AS1411 mutant (MFR-AS1411mt) administration revealed rapid clearance via the bloodstream, demonstrating that MFR-AS1411 specifically targeted cancer cells. Bioluminescence images in the C6 cells, stably expressing the luciferase gene, illustrated the in vivo distribution. T2-weighted MR images of the same mice injected with MFR-AS1411 showed dark T2 signals inside the tumor region, compared with the MRI signal of the tumor region injected with MFR-AS1411mt particles. Conclusion: We developed a nanoparticle-based cancer-specific imaging probe using the AS1411 aptamer in vivo and in vitro. This multimodal targeting imaging strategy, using a cancer-specific AS1411 aptamer, can be used as a versatile imaging tool for specific cancer diagnosis.

In vitro derby imaging of cancer biomarkers using quantum dots.

Semiconductor quantum dots (QDs), which have broad absorption with narrow emission spectra, are useful for multiplex imaging. Here, fluorescence derby imaging using dual color QDs conjugated by the AS1411 aptamer (targeting nucleolin) and the arginine-glycine-aspartic acid (targeting the integrin alpha(v)beta(3)) in cancer cells is reported. Simultaneous fluorescence imaging of cellular distribution of nucleolin and integrin alpha(v)beta(3) using QDs enables easy monitoring of separate targets in the cancer cells and the normal healthy cells. These results suggest the feasibility of a concurrent visualization of QD-based multiple cancer biomarkers using small molecules such as aptamer or peptide ligands.

A multimodal nanoparticle-based cancer imaging probe simultaneously targeting nucleolin, integrin αvβ3 and tenascin-C proteins.

Molecular imaging of cancers has been characterized based on the sensitivity and selectivity of a single cancer probe targeting a cancer biomarker of a specific cancer cell line. Here, we designed a multimodal nanoparticle-based Simultaneously Multiple Aptamers and RGD Targeting (SMART) cancer probe targeting multiple cancer biomarkers to enhance the specificity and signal sensitivity for various cancers. Transmission electron microscopy revealed that the multimodal SMART cancer probe was spheric and well dispersed. Fluorescence, radioisotope, and magnetic resonance analysis demonstrated that the SMART cancer probe simultaneously targeting the nucleolin, integrin α(v)β(3) and Tnc proteins had dramatically enhanced specificity and signal intensity when used to target cancers from C6, NPA, DU145, HeLa and A549 cells when compared with single cancer probes conjugated with AS1411, RGD or TTA1 targeting a single cancer biomarker. The results demonstrated that the SMART cancer probe will be useful for the diagnosis of different cancers as a cancer master probe.

Development of a Quadruple Imaging Modality by Using Nanoparticles

The combination of nanotechnology with molecular imaging has great potential for the development of diagnostics and therapeutics, and multimodal imaging enables versatile applications from cell tracking in animals to clinical applications. Herein, we report a multimodal nanoparticle imaging system that is capable of concurrent fluorescence, bioluminescence, bioluminescence resonance energy transfer (BRET), positron emission tomography (PET) and magnetic resonance (MR) imaging in vivo. A cobalt-ferrite nanoparticle surrounded by rhodamine (MF) was conjugated with luciferase (MFB) and p-SCN-bn-NOTA (2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclonane-1,4,7-triacetic acid) followed by (68)GaCl(3) (magnetic-fluorescent-bioluminescent-radioisotopic particle, MFBR). Confocal microscopy revealed good transfection efficiency of MFB into cells and BRET was also observed in MFB. A good correlation among rhodamine, luciferase, and (68)GaCl(3) was found in MFBR, and the activities of each imaging modality increased dose-dependently with the amount of MFBR in the C6 cells. In vivo optical images were acquired from the thighs of mice after intramuscular and subcutaneous injections of MFBR-laden cells. MicroPET and MR images showed intense radioactivity and ferromagnetic intensities with MFBR-laden cells. The multimodal imaging strategy could be used as potential imaging tools to track cells.

Bioimaging of the unbalanced expression of microRNA9 and microRNA9* during the neuronal differentiation of P19 cells.

Generally, the 3′‐end of the duplex microRNA (miR) precursor (pre‐miR) is known to be stable in vivo and serve as a mature form of miR. However, both the 3′‐end (miR9) and 5′‐end (miR9*) of a brain‐specific miR9 have been shown to function biologically in brain development. In this study, real‐time PCR analysis and in vitro/in vivo bioluminescent imaging demonstrated that the upstream region of a primary miR9‐1 (pri‐miR9‐1) can be used to monitor the highly expressed pattern of endogenous pri‐miR9‐1 during neurogenesis, and that the Luciferase reporter gene can image the unequal expression patterns of miR9 and miR9* seen during the neuronal differentiation of P19 cells. This demonstrates that our bioimaging system can be used to study the participation of miRs in the regulation of neuronal differentiation.

A reporter gene imaging system for monitoring microRNA biogenesis

MicroRNAs (miRNAs), non-coding RNA molecules, have emerged as a part of key gene regulation, participating in a variety of biological processes such as cell development. Current research methods, including northern blot and real-time PCR analysis, have been used to quantify miRNA expression. Major disadvantages of these methods include invasive techniques, such as a tissue biopsy, and the absence of repetitive studies. In this protocol we describe a simple non-invasive imaging method for monitoring miRNAs during neurogenesis. This novel method includes the design of an miRNA reporter gene vector, cell transfection, in vitro luciferase assay and in vivo bioluminescence imaging of miRNAs. Our reporter imaging system allows for repetitive, non-invasive detection of miRNAs, illustrating the miRNA124a (miR124a)-dependent decrease of Gaussia reporter activity during neuronal differentiation. Using this method, construction of a reporter-imaging vector, in vitro and in vivo signal detection steps can be carried out in ∼10 d.

Noninvasive imaging of microRNA124a‐mediated repression of the chromosome 14 ORF 24 gene during neurogenesis

The function of microRNAs (miRNAs) is translational repression or mRNA cleavage of target genes by binding to 3′‐UTRs of target mRNA. In this study, we investigated the functions and the target genes of microRNA124a (miR124a), and imaged the miR124a‐mediated repression of chromosome 14 open reading frame24 (c14orf24, unknown function) during neurogenesis, using noninvasive luciferase systems. The expression and functions of miR124a were investigated in neuronal differentiation of P19 cells (P19 is a mouse embryonic carcinoma cell line) by qRT‐PCR and RT‐PCR. The predicted target genes of miR124a were found by searching a bioinformatics database and confirmed by RT‐PCR analysis. Remarkable repression of c14orf24 by miR124a was detected during neurogenesis, and was imaged using in vitro and in vivo luciferase systems. The expression of miR124a was highly upregulated during neuronal differentiation. Overexpression of miR124a in P19 cells resulted in a preneuronal gene expression pattern. MicroRNA124a‐mediated repression of c14orf24 was successfully monitored during neuronal differentiation. Also, c14orf24 showed molecular biological characteristics as follows: dominant expression in the cytoplasm; a high level of expression in proliferating cells; and gradually decreased expression during neurogenesis. Our noninvasive luciferease system was used for monitoring the functions of miRNAs, to provide imaging information on miRNA‐related neurogenesis and the miRNA‐regulated molecular network in cellular metabolism and diseases.

Carbon nanodot-based self-delivering microRNA sensor to visualize microRNA124a expression during neurogenesis

MicroRNAs (miRNAs, miRs) are recognized as regulators of gene expression related to cellular development and diseases. In this study, we developed a carbon nanodot (C-dot)-based miR124a molecular beacon (miR124a CMB). The C-dots were purified from candle soot (cC-dots) by thermal oxidation. The double-stranded DNA oligonucleotide containing a miR124a binding site and black hole quencher 1 (miR124a sensing oligo) was further conjugated with the cC-dots to form the miR124a CMB. P19 cells were incubated with the miR124a CMB to sense miR124a expression during neurogenesis. The physical properties of the cC-dots showed multi-color light emission with various excitation wavelengths, a broad size distribution ranging from 2 to 4 nm, a graphitic carbon core (sp2), an abundance of carboxyl groups on the surface, no evidence of cellular toxicity and a high level of self-promoted uptake into cells. The miR124a CMB showed great fluorescence quenching in the absence of miR124a. The miR124a CMB internalized into P19 cells successfully visualized a gradual increase in miR124a expression during neuronal differentiation by providing signal-on imaging activity acquired by the following mechanism: the miR124a, which was highly expressed during neurogenesis, was bound to the miR124a binding site, resulting in the detachment of the quencher from the miR124a CMB and producing fluorescence recovery. The miR124a CMB demonstrated great specificity for sensing miR124a biogenesis with the advantages of self-passivated carboxyl groups, no toxicity, and self-illumination and highly self-promoted cellular uptake which will make the sensing of other various miRNAs related to diseases easy, convenient and accurate.

A color-tunable molecular beacon to sense miRNA-9 expression during neurogenesis

A typical molecular beacon (MB) composing of a fluorophore and a quencher has been used to sense various intracellular biomolecules including microRNAs (miRNA, miR). However, the on/off-tunable miRNA MB is difficult to distinguish whether the observed low fluorescence brightness results from low miRNA expression or low transfection of the miRNA MB. We developed a color-tunable miRNA-9 MB (ColoR9 MB) to sense miR-9 expression-dependent color change. The ColoR9 MB was synthesized by a partially double-stranded DNA oligonucleotide containing a miR-9 binding site and a reporter probe with Cy3/black hole quencher 1 (BHQ1) at one end and a reference probe with Cy5.5 at the other end. The ColoR9 MB visualized CHO and P19 cells with red color in the absence of miR-9 and yellow color in the presence of miR-9. In vivo imaging demonstrated that the green fluorescence recovery of the reporter probe from the ColoR9 MB increased gradually during neuronal differentiation of P19 cells, whereas red fluorescence activity of the reference probe remained constant. These results showed the great specificity of sensing miR-9 expression- and neurogenesis-dependent color change.

Microinjection free delivery of miRNA inhibitor into zygotes

The development of gene delivery systems into embryos is challenging due to technical difficulties, delivery efficiency and toxicity. Here, we developed an organic compound (VisuFect)-mediated gene delivery system for zygotes. The VisuFect, which is hydrophilic and Cy5.5-labeled, was conjugated with poly(A) oligo (VFA). The VFA into CHO cells showed clathrin-mediated internalization and no toxicity. The VFA successfully penetrated through the zona pellucida of fertilized eggs of various species including pigs, zebrafish, drosophilas and mice. The experiment with VisuFect-mediated delivery of the miR34c inhibitor showed similar results with direct microinjection of the miR34c inhibitor by suppressing the development of zygotes up to the blastocyst stage. Noticeable features of the VisuFect will provide great benefits for further studies on gene function in sperms and embryos.