Cheng-Chung Chang

Self-assembled star-shaped chlorin-core poly(ɛ-caprolactone)–poly(ethylene glycol) diblock copolymer micelles for dual chemo-photodynamic therapies

Amphiphilic 4-armed star-shaped chlorin-core diblock copolymers based on methoxy poly(ethylene glycol) (mPEG) and poly (epsilon-caprolactone) (PCL) were synthesized and characterized in this study. The synthesized photosensitizer-centered amphiphilic star block copolymer that forms assembled micelle-like structures can be used in a photodynamic therapy (PDT)-functionalized drug delivery system. Moreover, the hydrophobic chemotherapeutic agent, paclitaxel, can be trapped in the hydrophobic inner core of micelles. In our results, the star-polymer-formed micelle exhibited efficient singlet oxygen generation, whereas the hydrophobic photosensitizer failed due to aggregation in aqueous solution. The chlorin-core micelle without paclitaxel loading exhibited obvious phototoxicity in MCF-7 breast cancer cells with 7J/cm2 or 14J/cm2 light irradiation at a chlorin concentration of 125microg/ml. After paclitaxel loading, the size of micelle increased from 71.4nm to 103.2nm. Surprisingly, these micelles were found to improve the cytotoxicity of paclitaxel significantly in MCF-7 cells after irradiation through a synergistic effect evaluated by median effect analysis. This functionalized micellar delivery system is a potential dual carrier for the synergistic combination of photodynamic therapy and chemotherapy for the treatment of cancer.

Investigation of spectral conversion of d(TTAGGG)4 and d(TTAGGG)13 upon potassium titration by a G-quadruplex recognizer BMVC molecule.

We have introduced a G-quadruplex-binding ligand, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC), to verify the major structure of d(T2AG3)4 (H24) in potassium solution and examine the structural conversion of H24 in sodium solution upon potassium titration. The studies of circular dichroism, induced circular dichroism, spectral titration and gel competition have allowed us to determine the binding mode and binding ratio of BMVC to the H24 in solution and eliminate the parallel form as the major G-quadruplex structure. Although the mixed-type form could not be eliminated as a main component, the basket and chair forms are more likely the main components of H24 in potassium solution. In addition, the circular dichroism spectra and the job plots reveal that a longer telomeric sequence d(T2AG3)13 (H78) could form two units of G4 structure both in sodium or potassium solutions. Of particular interest is that no appreciable change on the induced circular dichroism spectra of BMVC is found during the change of the circular dichroism patterns of H24 upon potassium titration. Considering similar spectral conversion detected for H24 and a long sequence H78 together with the G4 structure stabilized by BMVC, it is therefore unlikely that the rapid spectral conversion of H24 and H78 is due to structural change between different types of the G4 structures. With reference to the circular dichroism spectra of d(GAA)7 and d(GAAA)5, we suggest that the spectral conversion of H24 upon potassium titration is attributed to fast ion exchange resulting in different loop base interaction and various hydrogen bonding effects.

Selective photodynamic therapy based on aggregation-induced emission enhancement of fluorescent organic nanoparticles

Three binary molecule conjugates were designed and synthesized by conjugating a chromophore (3, 6-bis-(1-methyl-4-vinylpyridinium)-carbazole diiodide, BMVC) to mono-, bis- and trishydroxyl photosensitizers, respectively. BMVC plays the role of cancer cells recognizer; AIEE (aggregation-induced emission enhancement) generator and FRET (Fluorescence Resonance Energy Transfer) donor. The self assembling properties of these binary conjugates result in different degrees of AIEE and then achieve the formations of FONs (fluorescent organic nanoparticles), which present efficient FRET and singlet oxygen generations. Biologically, FONs-photosensitizers from these compounds were much more phototoxicities to cancer cell than to normal cell without significant dark toxicity. In addition, their intracellular fluorescent colors switching upon photo-excitation are expected to be used for further cell death biomarker applications. This improved photodynamic activity might be due to the aggregation of compounds in the cell that form FONs which can promote PDT (photodynamic therapy) and are observed in cancer cell but not normal cell.

G-Quadruplex Stabilizer 3,6-Bis(1-Methyl-4-Vinylpyridinium)Carbazole Diiodide Induces Accelerated Senescence and Inhibits Tumorigenic Properties in Cancer Cells

Carbazole derivatives that stabilized G-quadruplex DNA structure formed by human telomeric sequence have been designed and synthesized. Among them, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) showed an increase in G-quadruplex melting temperature by 13°C and has a potent inhibitory effect on telomerase activity. Treatment of H1299 cancer cells with 0.5 μmol/L BMVC did not cause acute toxicity and affect DNA replication; however, the BMVC-treated cells ceased to divide after a lag period. Hallmarks of senescence, including morphologic changes, detection of senescence-associated β-galactosidase activity, and decreased bromodeoxyuridine incorporation, were detected in BMVC-treated cancer cells. The BMVC-induced senescence phenotype is accompanied by progressive telomere shortening and detection of the DNA damage foci, indicating that BMVC caused telomere uncapping after long-term treatments. Unlike other telomerase inhibitors, the BMVC-treated cancer cells showed a fast telomere shortening rate and a lag period of growth before entering senescence. Interestingly, BMVC also suppressed the tumor-related properties of cancer cells, including cell migration, colony-forming ability, and anchorage-independent growth, indicating that the cellular effects of BMVC were not limited to telomeres. Consistent with the observations from cellular experiments, the tumorigenic potential of cancer cells was also reduced in mouse xenografts after BMVC treatments. Thus, BMVC repressed tumor progression through both telomere-dependent and telomere-independent pathways. (Mol Cancer Res 2008;6(6):955–64)

Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents

G-quadruplex (G4) is a promising target for anti-cancer treatment. In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells. The molecular engineering of a fluorescent G4 ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC), can change its major cellular localization from the nucleus to the mitochondria in cancer cells, while remaining primarily in the cytoplasm of normal cells. A number of BMVC derivatives with sufficient mitochondrial uptake can induce cancer cell death without damaging normal cells. Fluorescence studies of these anti-cancer agents in live cells and in isolated mitochondria from HeLa cells have demonstrated that their major target is mtDNA. In this study, we use fluorescence lifetime imaging microscopy to verify the existence of mtDNA G4s in live cells. Bioactivity studies indicate that interactions between these anti-cancer agents and mtDNA G4 can suppress mitochondrial gene expression. This work underlines the importance of fluorescence in the monitoring of drug-target interactions in cells and illustrates the emerging development of drugs in which mtDNA G4 is the primary target.

Special reactive oxygen species generation by a highly photostable BODIPY-based photosensitizer for selective photodynamic therapy.

We introduce a new class of photostable, efficient photosensitizers based on boron-dipyrromethene (BODIPY) derivatives that can generate singlet oxygen and super oxide simultaneously under irradiation. For compound preparation, appropriate regulation of the reaction conditions and control of specifically substituted BODIPY derivatives have been achieved. After biologically evaluating the intracellular uptake, localization, and phototoxicity of the compounds, we conclude that 3,5-dianiline-substituted BODIPY is a potentially selective photodynamic therapy candidate because its photodamage is more efficient in cancer cells than in normal cells, without apparent dark toxicity. Furthermore, direct comparison of photodamage efficacy revealed that our compound has better efficacy than Foscan and nearly equal efficacy to that of methylene blue.

Fluorescent probe for visualizing guanine-quadruplex DNA by fluorescence lifetime imaging microscopy

Abstract. The importance of guanine-quadruplex (G4) is not only in protecting the ends of chromosomes for human telomeres but also in regulating gene expression for several gene promoters. However, the existence of G4 structures in living cells is still in debate. A fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), for differentiating G4 structures from duplexes is characterized. o-BMVC has a large contrast in fluorescence decay time, binding affinity, and fluorescent intensity between G4 structures and duplexes, which makes it a good candidate for probing G4 DNA structures. The fluorescence decay time of o-BMVC upon interaction with G4 structures of telomeric G-rich sequences is ∼2.8  ns and that of interaction with the duplex structure of a calf thymus is ∼1.2  ns. By analyzing its fluorescence decay time and histogram, we were able to detect one G4 out of 1000 duplexes in vitro. Furthermore, by using fluorescence lifetime imaging microscopy, we demonstrated an innovative methodology for visualizing the localization of G4 structures as well as mapping the localization of different G4 structures in living cells.

Aggregation induced photodynamic therapy enhancement based on linear and nonlinear excited FRET of fluorescent organic nanoparticles

A binary molecule can self-assemble to form fluorescent organic nanoparticles (FONs) based on the Aggregation-Induced Emission Enhancement (AIEE) property and subsequently, presents an efficient fluorescence resonance energy transfer (FRET) to generate singlet oxygen under linear and nonlinear light sources. Biologically, this FON-photosensitizer is much more phototoxic to cancer cells than to normal cells without significant dark toxicity. Eventually, a new approach, called FON FRET-PDT or AIEE FRET-PDT, to promote the PDT effect is expected.

Induction of senescence in cancer cells by the G‐quadruplex stabilizer, BMVC4, is independent of its telomerase inhibitory activity

BACKGROUND AND PURPOSE Telomerase is the enzyme responsible for extending G‐strand telomeric DNA and represents a promising target for treatment of neoplasia. Inhibition of telomerase can be achieved by stabilization of G‐quadruplex DNA structures. Here, we characterize the cellular effects of a novel G‐quadruplex stabilizing compound, 3,6‐bis(4‐methyl‐2‐vinylpyrazinium iodine) carbazole (BMVC4).

A Dual Selective Antitumor Agent and Fluorescence Probe: the Binary BMVC–Porphyrin Photosensitizer

Photodynamic therapy (PDT) is a promising modality for the treatment of localized tumors. To cause cell death during PDT, singlet oxygen can be generated by energy transfer from the triplet state of a photosensitizer to molecular oxygen. Among various photosensitizers, porphyrin derivatives have been widely studied. 6] However, porphyrin-based sensitizers have drawbacks to their application in PDT that include poor chemical selectivity toward intended tissue targets and a lack of specific light wavelengths that are optimal for tissue penetration and chromophore excitation. To overcome these shortcomings of the traditional porphyrin chromophore, a number of binary compounds have been recently designed and prepared with a component linked to the porphyrin photosensitizer. The resulting conjugates are highly selective for cancer cells and absorb at longer wavelengths more suitable for tissue penetration. For example, Drain and co-workers synthesized porphyrin–saccharide conjugates to increase the uptake of the photosensitizer by specific cancer cells. Dichtel et al. synthesized a binary compound to enhance singlet oxygen generation via fluorescence resonance energy transfer (FRET) by two-photon excitation of the donor chromophore to the central porphyrin acceptor. Selectivity is a key feature in the design of photosensitizers for killing cancer cells without damaging normal cells. We recently synthesized a novel 3,6-bis-(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) compound with selectivity toward cancer cells over normal cells. 13] Specifically, the fluorescence of BMVC detected in cancer cells was found to be much stronger than that in normal cells. Moreover, BMVC has a large cross-section for two-photon absorption around 820 nm, a wavelength that is close to optimal for tissue penetration of the radiation needed to excite the photosensitizer. In addition, the windows of transparency for porphyrin derivatives in the range of 450–500 nm allow us to selectively excite BMVC. For the study reported herein, we combined these features into a binary porphyrin compound for selectivity in PDT. In this compound, two BMVC molecules are covalently linked to a central 5,10-bis-(4-hydroxyphenyl)-15,20-bis-(4-methoxyphenyl)porphyrin (PAP) molecule at the ortho positions, and we termed the resulting binary compound o-2B-P. The ortho position was chosen for covalent attachment, as it is known that compounds containing ortho-5,10-disubstituted water-soluble trimethylaminophenyl groups exhibit better PDT efficacy than those with para-5,15-disubstitution. The structure of o-2B-P is shown in Figure 1 A, and details of its synthesis can be found elsewhere (Supporting Information 1). We examined the efficiency of energy transfer from the excited state of BMVC to the porphyrin in o-2B-P, as well as the efficacy of singlet oxygen generation by the porphyrin in the binary compound. The absorption and fluorescence spectra of o-2B-P, PAP, BMVC, and a mixture of BMVC and PAP in DMSO are depicted in Figures 1 B and C, respectively. The absorption of o-2B-P is almost identical to that of a mixture of BMVC and PAP. The absorption is a linear sum of the absorbances of BMVC and PAP individually, indicating that there is no appreciable interaction between the two chromophores in the ground electronic states. In contrast, the fluorescence of BMVC in o-2BP is almost totally quenched, whereas the fluorescence of PAP is enhanced by at least fivefold upon excitation of o-2B-P at 470 nm. Evidently, there is efficient energy transfer from the excited state of BMVC to PAP. To evaluate the effect of photoinduced formation of singlet oxygen by o-2B-P, we applied a photochemical method using a singlet oxygen quencher, 1,3-diphenylisobenzofuran (DPBF), to verify the generation of singlet oxygen. Because the cationic meso-tetra-(4-N-methylpyridyl)porphyrin (TMPyP) molecule is itself a singlet oxygen photosensitizer, 20] we compared the yield of singlet oxygen upon irradiation of o-2B-P and TMPyP at various wavelengths. Figure 1 D shows a comparison of the absorbance of DPBF at 417 nm in the presence of o-2B-P and TMPyP in DMSO as a function of irradiation time at 470 10 nm as well as lex>580 nm selectively from a halogen lamp. Although singlet oxygen generation by o-2B-P is slightly lower than that of TMPyP at lex>580 nm, it is the singlet oxygen generated by o-2B-P, not by TMPyP, upon excitation at 470 10 nm that provides a more useful measure of the selectivity for PDT efficacy. Despite the large number of porphyrin derivatives in blood and tissues, the porphyrin absorbance at 470 10 nm is almost negligible. Accordingly, the 450–500 nm transparent windows of porphyrin derivatives [a] C.-C. Kang, Dr. C.-C. Cho, Y.-C. Lin, Prof. C.-C. Chang, Prof. T.-C. Chang Institute of Atomic and Molecular Sciences, Academia Sinica P.O. Box 23-166, Taipei 106 (Taiwan, ROC) Fax: (+ ) 886-2-23620200 E-mail : tcchang@po.iams.sinica.edu.tw [b] C.-C. Kang Taiwan International Graduate Program and Department of Chemistry National Tsing-Hua University and Academia Sinica (Taiwan, ROC) [c] Prof. C.-T. Chen Centers for Optoelectronic Biomedicine, College of Medicine National Taiwan University, Taipei (Taiwan, ROC) and Institute of Microbiology and Biochemistry, College of Life Science National Taiwan University, Taipei (Taiwan, ROC) [d] Prof. C.-C. Chang Department of Chemistry, National Chung-Hsing University Taichung (Taiwan, ROC) E-mail : ccchang555@dragon.nchu.edu.tw Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author.