Do Won Hwang

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.

A brain-targeted rabies virus glycoprotein-disulfide linked PEI nanocarrier for delivery of neurogenic microRNA.

Recent advances in efficient microRNA (miRNA) delivery techniques using brain-targeted nanoparticles offer critical information for understanding the functional role of miRNAs in vivo, and for supporting targeted gene therapy in terms of treating miRNA-associated neurological diseases. Here, we report the rabies virus glycoprotein (RVG)-labeled non-toxic SSPEI nanomaterials capable of neuron-specific miR-124a delivery to neuron in vivo. The RVG-labeled BPEI-SS (RVG-SSPEI) nanocarrier showed less toxicity in acetylcholine receptor-positive Neuro2a cells, and electrostatic interaction of RVG-SSPEI with miR-124a exhibited optimal transfection efficacy. The RVG-SSPEI polymer specifically targeted Neuro2a using cy5.5-miR-124a mixed with RVG-SSPEI. The functional action of miR-124a oligomers released from polyplexes in the cytoplasmic region was evaluated by a reporter vector containing a miR-124a -binding sequence, and showed a significantly reduced reporter signal in a dose-dependent manner. Cy5.5-miR-124a/RVG-SSPEI- injected into mice via tail veins displayed the enhanced accumulation of miR-124a in the isolated brain. Hindrance of the efficient penetration of neuronal cells by size limitation of the miR-124a/RVG-SSPEI improved with the help of mannitol through blood-brain barrier disruption. These findings indicated that the RVG peptide combined with mannitol infusion using SSPEI polymer for neuron-specific targeting in vivo is sufficient to deliver neurogenic microRNA into the brain.

RVG peptide tethered bioreducible polyethylenimine for gene delivery to brain

The work demonstrated the successful delivery of gene to mouse brain overcoming the blood-brain barrier (BBB) through expedient vector construct having RVG peptide as targeting ligand for neuronal cells. The newly developed delivery vector was designed to impart bioreducibility for greater intracellular pDNA release, higher serum stability and efficient complexing ability by incorporating disulfide linkage, PEG and low molecular weight polyethylenimine, respectively. The physiochemical properties of the polyplex, its cytotoxicity and the in vitro transfection efficiency on Neuro2a cell were studied prior to the successful in vivo study. In vivo fluorescence assay substantiated the permeation of the pDNA loaded polymeric vector through the BBB. The RVG-mediated target-specific cellular uptake of polymeric vector was established conclusively by competitive assay.

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.

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.

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 inu2003vivo 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 inu2003vitro/inu2003vivo 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.

In Vivo Imaging of miR-221 Biogenesis in Papillary Thyroid Carcinoma

PurposeTo investigate the overexpression of miR-221 in papillary thyroid carcinoma (PTC), we developed a Gaussia luciferase (Gluc) system regulated by miR-221.ProceduresQuantities of primary or mature miR-221 in normal thyroid cells (HT-ori3) and in PTC (NPA, TPC-1) were measured by quantitative real-time polymerase chain reaction. Cytomegalovirus (CMV)/Gluc-3xPT_miR221, which included three perfect complementary target sequences repeats of miR221 in the 3′-untranslated region of Gluc, was transfected into cells with pre-miR-221 or anti-miR-221 and Gluc activities were then compared in vitro and in vivo.ResultsPrimary or mature miR-221 were overexpressed in PTC as compared with HT-ori3. In cells transfected with the Gaussia luciferase reporter system (CMV/Gluc-3xPT_miR221), Gluc activities were regulated according to miR-221 levels in vitro and in vivo.ConclusionsThese results suggest that the devised CMV/Gluc-3xPT_miR221 system may be a useful tool for monitoring quantities of endogenous miR-221 in cells or living organisms.

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.