Soonhag Kim

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.

Development of a Dual-Luciferase Reporter System for In Vivo Visualization of MicroRNA Biogenesis and Posttranscriptional Regulation

MicroRNAs (miRNAs) function in mammalian cells via translational repression or messenger RNA (mRNA) cleavage of target genes by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs. Although miRNAs are involved in cell differentiation or organ development, posttranscritptional regulation of miRNA is not well understood. Here, we developed a dual-luciferase reporter system for monitoring in vivo endogenous transcription of primary miRNA (pri-miRNA) and also the mature miRNA activity simultaneously. Methods: miR23P639/Fluc plasmid carrying firefly luciferase (Fluc) under the control of miR-23a promoter was used to monitor the transcriptional level of miR-23a, and a cytomegalovirus (CMV)/Gluc/3xPT_mir23 recombinant containing 3 copies of the target sequence of miR-23a in the 3′ UTR of Gaussia luciferase (Gluc) before the poly(A) tail was used to monitor the targeting activity of mature miR-23a. This dual-luciferase reporter system transfected to the same population of cells was used to monitor the increased transcriptional level of the pri-miR-23a reflected in the Fluc activity and the decreased Gluc activity affected by mature miR-23a action. Fluc and Gluc activities were also imaged in vivo using the respective substrates in grafted cells in the same nude mice using an in vivo bioluminescence imager. Results: In HeLa cells and undifferentiated P19 cells, the increased Fluc activity representingthe primary miR-23a transcript level reflected the resultant increase in repression of Gluc activity representing mature miR-23a activity. However, 293 cells showed Gluc activity was not repressed as much as Fluc activity was increased, suggesting a block in the posttranscriptional processing of miR-23a transcript in 293 cells. The miR-23a expression in P19 cells before and after neuronal differentiation with retinoic acid treatment showed an increase in Fluc activity and a concomitant decrease in Gluc activity in vitro. HeLa, 293 cells and undifferentiated P19 cells grafted to the nude mice showed exactly the same pattern of luciferase activities in vivo and in vitro. Conclusion: We developed a dual-luciferase reporter system to monitor expression and posttranscriptional regulation of a miR-23a in cells in vitro and in vivo. This dual-luciferase reporter system is intended to be used to monitor the expression and regulation of miRNAs noninvasively, especially to understand the differentiation of grafted cells in vivo.

Molecular beacon-based bioimaging of multiple microRNAs during myogenesis.

MicroRNAs (miRNAs, miR) are associated with multiple cellular processes and diseases. Here, we designed fluorescent DNA probes composed of stem loop-structured DNA complementary to miRNAs and fluorophore-quencher pairs [molecular beacon (MB)] to simultaneously monitor the biogenesis of miR-206 and miR-26a, which are highly expressed during myogenic differentiation. C2C12 cells were transfected with an MB targeting miR-26a and containing a 6-FAM-BHQ1 pair (miRNA-26a MB) or an MB targeting miR-206 with a Texas Red-BHQ2 pair (miRNA-206 MB). In vitro and in vivo fluorescence analysis revealed that, only in differentiated single C2C12 cell, significantly increased fluorescence signals of miRNA-26a MB, miRNA-206 MB or simultaneous incubation of both beacons were detected due to the hybridization of miR-206 or miR-26a with their respective beacons, resulting in activation of fluorescence. Our MB-based miRNA imaging system may serve as a new imaging probe for monitoring multiple miRNAs during various cellular or disease processes associated with miRNAs.

Smart magnetic fluorescent nanoparticle imaging probes to monitor microRNAs.

An imaging system that can be used to evaluate the expression levels of microRNAs during neuronal development can provide noninvasive information for investigating a variety of biological phenomena related to microRNAs (miRNAs, miRs). Herein, the development of a novel imaging platform to monitor intracellular miR124a during neuronal differentiation is reported using rhodamine-coated cobalt ferrite magnetic fluorescent (MF) nanoparticles linked to a quenching molecular system containing an miR124a binding sequence (MF-miR124a beacon). During neuronal differentiation, in vitro fluorescence signals of the MF-miR124a beacon are significantly increased under conditions where miR124a is highly expressed, and dramatically return to the original quenched fluorescence after anti-miR124a treatment. In vivo fluorescence images show enhanced fluorescence signals in mice with P19 cells within a poly-L-lactic acid scaffold after induction of neuronal differentiation. In addition, magnetic resonance (MR) images provide in vivo tracking of cells containing the MF-miR124a beacon. These studies represent the first step toward the use of nanotechnological imaging of mature miRNA, and this technique could be used for cellular tracking with a MR imaging system as well as for simultaneous monitoring of the miRNA expression pattern in vivo.

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.

Consecutive Targetable Smart Nanoprobe for Molecular Recognition of Cytoplasmic microRNA in Metastatic Breast Cancer

We report smart nanoprobe, hyaluronic acid (HA)-based nanocontainers containing miR-34a beacons (bHNCs), for the intracellular recognition of miR-34a levels in metastatic breast cancer cells, which is distinct from the imaging of biomarkers such of cell membrane receptors such as HER2. In this study, we demonstrate that a nanoscale vesicle that couples a targeting endocytic route, CD44, and a molecular imaging probe enables the efficient detection of specific miRNAs. Furthermore, bHNCs showed no cytotoxicity and high stability due to the anchored HA molecules on the surface of nanocontainers, and enables the targeted delivery of beacons via CD44 receptor-mediated endocytosis. In vitro and in vivo optical imaging using bHNCs also allow the measurement of miR-34a expression levels due to the selective recognition of the beacons released from the internalized bHNCs. We believe that the technique described herein can be further developed as a cancer diagnostic as well as a miRNA-based therapy of metastatic cancer.

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.

The role of microRNA-23b in the differentiation of MSC into chondrocyte by targeting protein kinase A signaling

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is critical for successful cartilage regeneration. Several methods have been developed to attempt to chondrogenic differentiation, because chondrogenic differentiated cells can form stable cartilage and induce expression of a cartilage-specific phenotype. In this study, we found that both H-89 and microRNA-23b induced differentiation into chondrocyte of hMSCs through down-regulation of protein kinase A (PKA) signaling. The small molecule, H-89, was identified by PCA analysis as a potential mediator of chondrogenic differentiation. H-89 induced the expression of the chondrocyte marker, aggrecan, as well as miR-23b. We searched that miR-23b regulates protein level of PKA. When miR-23b was transfected into hMSCs, chondrogenic differentiation was induced. We confirmed the target of miR-23b using a reporter gene assay. Furthermore, not only H-89 or miR-23b-treated cells, but also cell co-treated with H-89 and miR-23b differentiated into chondrocytes. Our results indicate that H-89 induces the expression of endogenous miR-23b, thereby inducing chondrogenic differentiation by negatively inhibition of PKA signaling.

In Vivo Imaging of Functional Targeting of miR-221 in Papillary Thyroid Carcinoma

MicroRNAs (miRNAs) are small, noncoding RNA molecules that control expression of target genes. The abnormally expressed miRNAs function as oncogenes or tumor suppressors in human cancer. To evaluate the abundant gene regulation of miR-221 in papillary thyroid carcinoma (PTC), we performed microarray analysis and developed a Gaussia luciferase (Gluc) reporter system regulated by miR-221. Methods: Total RNAs were isolated from pre-miR-221–treated normal human thyroid cells (HT-ori3) and anti-miR-221–treated papillary thyroid cells (NPA). Microarray analysis was performed with 44,000 probes. The messenger RNA levels of target genes regulated by miR-221 were evaluated using reverse-transcription polymerase chain reaction. Three types of cytomegalovirus (CMV)/Gluc_3′ untranslated region (UTR) of homeobox B5 (HOXB5), which included a seed sequence of mature miR-221 in the 3′ UTR of HOXB5 after the Gluc stop codon, were transfected into NPA cells, and pre-miR-221 was cotransfected with CMV/Gluc_3′ UTR of HOXB5. The Gluc activities in cells were measured by luciferase assay. Mice implanted with PTC-expressing Gluc regulated by miR-221 were monitored with bioluminescence imaging for 6 d. Results: Microarray analysis showed thousands of genes were directly and indirectly regulated by miR-221 and shifted the gene expression pattern of normal thyroid cells toward PTC. Of several genes downregulated more than 2-fold by miR-221, messenger RNA levels of HOXB5 were significantly downregulated by miR-221. Also, in vitro or in vivo Gluc activities using CMV/Gluc_3′ UTR of HOXB5 systems were downregulated dose dependently by endogenous or exogenous miR-221. Conclusion: MiR-221 overexpressed in PTC drives carcinoma gene expression patterns by directly and indirectly regulating numerous genes, including HOXB5. The bioluminescence imaging system using CMV/Gluc_3′ UTR of HOXB5 is a useful tool for noninvasive in vivo long-term monitoring of functional targeting of miR-221.