Jingquan Zhao

Two-photon excitation studies of hypocrellins for photodynamic therapy

The photophysical and photochemical properties of hypocrellins (HA and HB) are examined with two-photon excitations at 800 nm using femtosecond pulses from a Ti:sapphire laser. The two-photon excited fluorescence spectra of HA and HB are very similar to those obtained by one-photon excitation, which may indicate that the two-photon induced photodynamic processes of hypocrellins are similar to one-photon induced photodynamic processes. The two-photon excitation cross sections of HA and HB are measured at 800 nm as about 34.8 x 10(-50) cm(4) s/photon and 21.3 x 10(-50) cm(4) s/photon, respectively. The large two-photon cross sections of both HA and HB, suggest that the hypocrellins can be potential two-photon phototherapeutic agents. As an example for two-photon photodynamic therapy of hypocrellins, we also further examine the cell-damaging effects of HA upon two-photon illumination. Our preliminary results of cell viability test indicate hypocrellins can effectively damage the Hela cells under two-photon illumination.

Novel Surfactant-like Hypocrellin Derivatives to Achieve Simultaneous Drug Delivery in Blood Plasma and Cell Uptake

Water‐soluble derivatives of hypocrellins can be safely delivered in blood plasma but lose their photodynamic activity in vivo due to poor cell uptake, while hydrophobic derivatives retaining their activity may aggregate in the blood plasma and block vascular networks. Considering both drug delivery and biological activity, surfactant‐like hypocrellin B (HB) derivatives, sodium 12‐2‐HB‐aminododecanoate (SAHB) and sodium 11,11′‐5,8‐HB‐dimercaptoundecanoate (DMHB), were first designed and then synthesized in the current work. Both SAHB and DMHB were photoactive, generating free radicals and reactive oxygen species, as confirmed by EPR and chemical measurements. Most importantly, DMHB was not only readily soluble, allowing preparation of an intravenous injection solution at a clinically acceptable concentration, but it was also more photodynamic therapy (PDT) active to human breast carcinoma MCF‐7 cells than its parent HB under irradiation. The photodynamic activity was exactly identical to the 1O2 quantum yield and was not reduced by the improved water solubility, suggesting an independent hydrophilicity or lipophilicity. To our knowledge, this is a new strategy that possesses general significance for converting hydrophobic photosensitizers into clinically usable PDT drugs.

A novel hypocrellin B derivative designed and synthesized by taking consideration to both drug delivery and biological photodynamic activity

For making hypocrellins clinically applicable for phototherapy to vascular diseases, it is mainly focused onto finding a derivative which can be transported fluently in blood system but without serious loss of the inherent activity of its parents. Based on this consideration, a novel 17-3-amino-1-propane-sulfonic acid-HB Schiff-base (NSHB) was designed and synthesized in this work. As expected, NSHB is readily dissolved in phosphate buffered saline (PBS) or any other aqueous solvent in a concentration which is suitable for intravenous injection, while the quite higher partition coefficient (5:1) is beneficial to the affinity to biological targets. Based on EPR measurements, it is proved that the photosensitization activity of NSHB to photo-generate semiquinone anion radicals and superoxide anion radical (O*(2)(-)) is even higher than its parent HB, while the ability to generate singlet oxygen ((1)O(2)) is not seriously reduced. In addition, nearly comparable PDT activity to A549 cells for NSHB and HB confirms that the molecular design is successful and NSHB is readily delivered into target tissues via blood circulation after intravenous injection. Furthermore, the quantum yield of (1)O(2) for NSHB is as 12.5 times as that for HB under red light (600-700 nm), which is beneficial to phototherapy to solid tumors.

Effect of Structural Modification on Photodynamic Activity of Hypocrellins

Aluminum ion complexed 5,8‐di‐Br‐hypocrellin B is a new water‐soluble perylenequinonoid derivative with enhanced absorption over hypocrellin B (HB) in the phototherapeutic window (600–900 nm). Electron paramagnetic resonance and 9,10‐diphenyl‐anthracene bleaching methods were used to investigate the photosensitizing activity of [Al2(5,8‐di‐Br‐HB)Cl4]n in the presence of oxygen. Singlet oxygen, superoxide anion radical and hydroxyl radical can be generated by [Al2(5,8‐di‐Br‐HB)Cl4]n photosensitization. Singlet oxygen (1O2) is formed via energy transfer from triplet‐state [Al2(5,8‐di‐Br‐HB)Cl4]n to ground‐state molecular oxygen. 1O2 participates in the generation of a portion of superoxide anion radical (O2·−). Besides superoxide anion radical (O2·−) may originate from the electron transfer between the triplet‐state [Al2(5,8‐di‐Br‐HB)Cl4]n and the ground‐state molecular oxygen. ·OH is formed through the Fenton–Haber–Weiss reaction and the decomposition of DMPO–1O2 adduct. Compared with HB [Al2(5,8‐di‐Br‐HB)Cl4]n primarily remains and enhances the generation efficiency of superoxide anion radical and hydroxyl radical but that of singlet oxygen decreases.

PHYCOBILISOME FROM ANABAENA VARIABILIS KUTZ. AND ITS MODEL CONJUGATES

The model conjugates phycocyanin-allophycocyanin (C-PC-APC) and phycoerythrocyanin-phycocyanin-allophycocyanin (PEC-C-PC-APC) were synthesized by using a heterobifunctional coupling reagent N-succinimidyl-3-(2-pyridyldithio)propionate. The rod-core complex (αβ)6PCLRC27(αβ)3APCLC8.9 and phycobilisomes were separated from Anabaena variabilis. Energy transfer features for the conjugates and the complexes were compared. The absorption and fluorescence emission spectra indicated that the linker-peptides mediate interaction of phycobiliproteins and prompt energy transfer. The energy transfer in the conjugates was detected by fluorescence emission spectra and confirmed by the addition of dithiothreitol. The conjugates may be used as models for studying the energy transfer mechanism in phycobilisomes.

Photodynamic efficacy of liposome-delivered hypocrellin B in microvascular endothelial cells in vitro and chicken combs in vivo: a potential photosensitizer for port wine stain

Photodynamic therapy (PDT) has been proved a successful method for port wine stain (PWS), but the prolonged skin photosensitivity induced by the photosensitizers used currently seriously limits the clinical application of PDT. In this study, we investigate the feasibility of hypocrellin B (HB), a promising second-generation photosensitizer for the treatment of PWS. The photodynamic effect of liposome-delivered HB was evaluated in vitro with microvascular endothelial cells (MEC) and in vivo with chicken combs. The dark cytotoxicity and photocytotoxicity of liposomal HB in MEC were evaluated using the MTT assay. Gross and histological examinations were performed to investigate the selective occlusion of the superficial dermal microvasculature in the chicken comb. The result showed that photocytotoxicity of liposomal HB was dependent on both light dose and drug concentration. PDT with HB (0.5?1?mg?kg?1) and a light dose of 120?J?cm?2 showed selective destruction of the superficial dermal microvasculature of the chicken comb, leaving the overlying epidermis intact. This is the first study to investigate the potential efficacy of HB-PDT as a novel modality for the treatment of PWS. These findings suggest that liposomal HB is a safe and effective photosensitizer for PWS.

Synthesis of a novel hypocrellin B derivative and its photogeneration of semiquinone anion radical.

In order to improve the photosensitizing activity of HB further, the complex of 5,8-di-Br-HB with Al(3+) was first designed and synthesized in high yield. 5,8-di-Br-HB forms a 2:1 type (metal-ligand ratio) complex with Al(3+) measured by molar ratio and continuous variation methods. The new photosensitizer was characterized by UV-Vis, IR, 1H NMR and elemental analysis measurements. Based on the above experimental results, we first proposed a polymer-like structural model of the complex. The water-solubility and red absorption of the [Al(2)(5,8-di-Br-HB)Cl(4)](n) complex are both enhanced over hypocrellin B. In addition, the EPR and spectrophotometric measurements demonstrate that semiquinone anion radical of [Al(2)(5,8-di-Br-HB)Cl(4)](n) can be produced by [Al(2)(5,8-di-Br-HB)Cl(4)](n) photosensitization. The generation efficiency of ([Al(2)(5,8-di-Br-HB)Cl(4)](n))(.-) is almost equal to that of HB(.-). These results obtained indicated that [Al(2)(5,8-di-Br-HB)Cl(4)](n)was at least a favorable Type I phototherapeutic agent.

ENERGY TRANSFER IN ALLOPHYCOCYANIN HEXAMER FROM ANABAENA VARIABILIS BY TIME-RESOLVED SPECTROSCOPY

Abstract The process and mechanism for energy transfer in an allophycocyanin hexamer from Anabaena variabilis have been studied by time-resolved fluorescence spectroscopy. Deconvolution of isotropic time-resolved fluorescence spectra shows that the energy transfer time between two allophycocyanin trimers is about 14 ps, while the energy transfer time from the linker polypeptide (L cm 42 ) to the final emission is about 65 ps. The anisotropic results of time-resolved fluorescence spectra show that the energy transfer process among three ring-like ( α - 84 / α - 84 ) chromophore pairs still plays an important role with a time constant of about 45 ps. The facts support the idea that the mechanism for energy transfer in the allophycyanin hexamer should be described by Foster dipole—dipole interaction rather than by an exciton interaction.

EXCITATION ENERGY TRANSFER MECHANISMS IN ALLOPHYCOCYANIN (I) -ENERGY TRANSFER MECHANISMS IN MONOMER

The mechanisms of excitation energy transfer within allophycocyanin monomer with the theory of generalized master equation (GME) and the technique of time-resobed fluorescence anisotropic spectroscopy are studied. In the case of known information of its structure and spectra, the theory applied is based on the assumption that the coupling interaction between two chromophores is fairly weak. The theory correctly predicts the experimentlly observed rate for excitation energy transfer in allophycocyanin monomer. Based on the results, the energy transfer mechanism can be described as Förster and these processes cannot take place from the high vibrational levels of donor to acceptor.

Synthesis and energy transfer of model conjugate of phycobilisome rods

The conjugate of R-phycoerythrin (R-PE) and C-phycocyanin (C-PC) was synthesized through a heterobifunctional coupling reagent, N-succinimidyl 3-(2-pyridyldithio) propionate. The molar ratio of R-PE to C-PC was determined by absorption spectra, and the result was 2:1. The energy transfer phenomena were observed from steady-state fluorescence spectra. The calculated result showed that the energy transfer efficiency from R-PE to C-PC was 88%. The energy transfer kinetics was determined by picosecond time-resolved fluorescence spectra. The time constant of energy transfer from R-PE to C-PC was 80 ps, which was much longer than that in the rod of native phycobilisomes.