Guanghui Tan

Facile synthesis of chitosan assisted multifunctional magnetic Fe3O4@SiO2@CS@pyropheophorbide-a fluorescent nanoparticles for photodynamic therapy

Novel chlorin photosensitizer pyropheophorbide-a (PPA) coated multifunctional magneto-fluorescent nanoparticles Fe3O4@SiO2@CS@PPA (MFCSPPA) about 50 nm in diameter were strategically designed and prepared for photodynamic therapy (PDT) based on the good solubility and magnetic targeting of Fe3O4@SiO2 nanoparticles, excellent biocompatibility and biodegradability of chitosan (CS) polymer, and the unique fluorescence and photodynamic activity of pyropheophorbide-a. In this work, we found that magneto-fluorescent MFCSPPA has high saturation magnetization of 23.7 emu g−1, and showed super-paramagnetic properties, good dispersion in alcohol and water, excellent water-solubility, improved biocompatibility and good photoluminescence properties. In addition, we demonstrated MFCSPPA mediated singlet oxygen production in solution conditions by using 1,3-diphenylisobenzofuran (DPBF) as a fluorescence detector. Moreover, the in vitro PDT activities against human HeLa cervical cancer cells were investigated by MTT assay. The phototoxicity experiments showed that MFCSPPA has strong photodynamic therapy activity and low dark toxicity, and the cancer cell viability was reduced to 18% after treatment with PDT. Phagocytosis of MFCSPPA experiments indicated that it could be successfully taken up to some extent by HeLa cells with a suitable lipo-hydro partition coefficient and biocompatibility. Acridine orange/ethidium bromide (AO/EB) double fluorescence staining suggested that the cells are all in a state of apoptosis or necrosis after PDT treatment for 6 h. In addition, we studied the formation of reactive oxygen species in HeLa cells after MFCSPPA-PDT treatment; the results suggested that type I and type II photodynamic reactions can occur simultaneously, yet type I photodynamic reactions have a slight edge over type II. The as-prepared magneto-fluorescent MFCSPPA nanoparticles are suitable for simultaneous PDT and medical fluorescence imaging.

Preparation, characterization and in vitro photodynamic therapy of a pyropheophorbide-a-conjugated Fe3O4 multifunctional magnetofluorescence photosensitizer

Novel pyropheophorbide-a-conjugated multifunctional magnetofluorescence nanoparticles Fe3O4@SiO2@APTES@Glutaryl-PPa (MFNPs) with a mean diameter of 50 nm were strategically designed and prepared for photodynamic therapy (PDT) and medical fluorescence imaging. Chlorin photosensitizer pyropheophorbide-a (PPa) was covalently anchored on the surface of core–shell Fe3O4@SiO2@APTES nanoparticles that were prepared via a sol–gel process with a bridging glutaryl group. The phase constitution, morphology, size, chemical properties, magnetic property of the intermediates and final nanoparticles were characterized by X-ray powder diffraction, transmission electron microscopy, Fourier transform infrared spectrometer, zeta potential, vibration sample magnetometer, thermogravimetric analysis, ultraviolet-visible absorption spectra and fluorescent emission spectroscopy. These results showed that the MFNPs have good dispersibility in alcohol and water, excellent magnetization with 17.31 emu g−1 at 300 K, strong superparamagnetic and good photoluminescence property. The in vitro PDT against the human HeLa cervical cancer cell suggested that MFNPs could permeate the tumor cells quickly and possess suitable lipo-hydro partition coefficient, inducing damage and apoptotic cell death. The cancer cell viability was lowered to 10.18% after treatment with PDT. In addition, the formation of reactive oxygen species in HeLa cells after MFNPs-PDT treatment was studied, which suggested that Type I and Type II photodynamic reactions can occur simultaneously.

A Novel Photosensitizer 31,131-phenylhydrazine -Mppa (BPHM) and Its in Vitro Photodynamic Therapy against HeLa Cells

Photodynamic therapy (PDT) has attracted widespread attention due to its potential in the treatment of various cancers. Porphyrinic pyropheophorbide-a (PPa) has been shown to be a potent photosensitizer in PDT experiments. In this paper, a C-31,131 bisphenylhydrazone modified methyl pyropheophorbide-a (BPHM) was designed and synthesized with the consideration that phenylhydrazone structure may extend absorption wavelength of methyl pyro-pheophorbide-a (Mppa), and make the photosensitizer potential in deep tumor treatment. The synthesis, spectral properties and in vitro photodynamic therapy (PDT) against human HeLa cervical cancer cell line was studied. Methyl thiazolyl tetrazolium (MTT) assay showed the title compound could achieve strong inhibition of cervical cancer cell viability under visible light (675 nm, 25 J/cm2). Cell uptake experiments were performed on HeLa cells. Morphological changes were examined and analyzed by fluorescent inverted microscope. In addition, the mechanism of the photochemical processes of PDT was investigated, which showed that the formation of singlet oxygen after treatment with PDT played a moderate important role.

Synthesis and in vitro photodynamic therapy of chlorin derivative 13(1)-ortho-trifluoromethyl-phenylhydrazone modified pyropheophorbide-a.

Photodynamic therapy (PDT) is entering the mainstream of the cancer treatments recently. Pyropheophorbide-a (Pa), as a degradation product of chlorophyll-a, has been shown to be a potent photosensitizer in photodynamic therapy. In this paper, we investigated the in vitro photodynamic therapy of 131-ortho-trifluoromethyl-phenylhydrazone modified pyropheophorbide-a (PHPa) against human HeLa cervical cancer cell line, together with ultraviolet-visible spectra, fluorescence emission spectra, stability in various solvents, and single oxygen quantum yield. The results indicated that PHPa not only showed a greater molar extinction coefficient reached 4.55×104 Lmol-1cm-1, the long absorption wavelength (681nm) as we expected that makes it potential in deep tumor treatment, but also showed better stability in near neutral phosphate buffers (pH 7.4) and culture medium, as well as higher single oxygen quantum yield (ФD=40.5%) in DMF solutions. Moreover, cell experiments suggested that PHPa could be uptaken by HeLa cells successfully, and has low dark toxicity without irradiation, but remarkable photo-cytotoxicity (IC50, 1.92±0.59μM) that the inhibition rate of HeLa cells could increase up 91.4% at 30μM of PHPa after irradiation. In addition, morphological changes of HeLa cells further demonstrated that PHPa can induce damage and apoptotic cell death. Furthermore, the mechanism of photochemical processes was investigated by using specific quenching agent sodium azide (SA) and D-mannitol (DM), respectively, which showed the formation of singlet oxygen (Type II reaction mechanism) may play a predominant role, Type I and Type II photodynamic reactions could occur simultaneously in this PHPa mediated PDT process.

Magnetic iron oxide modified pyropheophorbide-a fluorescence nanoparticles as photosensitizers for photodynamic therapy against ovarian cancer (SKOV-3) cells.

Magnetic iron oxide modified pyropheophorbide-a fluorescence nanoparticles, Fe3O4@SiO2@APTES@PPa (FSAP), were designed as magnetically targeted photodynamic antineoplastic agents and prepared through continuous covalent chemical modification on the surface of Fe3O4 nanoparticles. The properties of the intermediates and the final product were comprehensively characterized by transmission electron microscopy, powder X-ray diffraction analysis, Fourier transform infrared spectroscopy, vibrating sample magnetometry, zeta potential measurement, ultraviolet-visible absorption spectroscopy, fluorescence emission spectroscopy, and thermogravimetric analysis. In this work, we demonstrated the in vitro photodynamic therapy (PDT) of FSAP against ovarian cancer (SKOV-3) cells, which indicated that FSAP could be taken up successfully and showed low dark toxicity without irradiation, but remarkable phototoxicity after irradiation. Meanwhile, FSAP had showed good biocompatibility and low dark toxicity against normal cells in the biological experiments on mouse normal fibroblast cell lines (L929 cells). In addition, in the photochemical process of FSAP mediated photodynamic therapy, the Type-II photo-oxygenation process (generated singlet oxygen) played an important role in the induction of cell damage.

The anti-cancer potency of photodynamic therapy of a novel chlorin derivative Amidochlorin p6 (ACP)

Photodynamic therapy (PDT) is a minimally invasive method in cancer treatment and has attracted considerable attention recently. In this paper, we have performed a detailed study of photodynamic activity of a chlorophyllous derivative, Amidochlorin p6 (ACP), and evaluated its potential as a photosensitizer in PDT. The singlet oxygen quantum yield (ΦΔ), the photoreaction mechanisms in PDT, the anti-photobleaching ability in phosphate buffer saline (PBS), the photocytotoxicity and dark toxicity against HeLa cells, cellular uptake and the influence on the expression of survivin and cyclin-dependent kinase (CDK2), were all investigated. The title compound showed significant photocytotoxicity and negligible toxicity in dark, and remarkable photostability. Moreover, ACP could be uptaken by HeLa cells successfully at 20 min leading to damage of cancer cells under light, during which Type I and Type II photodynamic reactions occurred simultaneously on HeLa cells in PDT treatment, and the influence of Type I (the generation of hydroxyl radicals) is slightly larger than Type II (the generation of singlet oxygen). In addition, real-time fluorescent quantitative PCR (RT-qPCR) suggested that ACP could significantly regulate the expression of survivin, which partly explained why ACP could induce the HeLa cell apoptosis and accelerate cell death.

Facile synthesis of a highly water-soluble graphene conjugated chlorophyll-a photosensitizer composite for improved photodynamic therapy in vitro

In photodynamic therapy (PDT), selection of an ideal photosensitizer and improvement of its photodynamic activities are currently of great interest. In this work, a chlorophyll-a photosensitizer derivative, p-bromo-phenylhydrazone-methyl pyropheophorbide-a (BPMppa) with a long absorption wavelength (683 nm) and a large molar extinction coefficient (7.03 × 104 M−1 cm−1), which is considered to be more suitable for the treatment of deep cancer, is loaded onto pristine graphene using a direct graphite exfoliation process via π–π stacking in water. The obtained graphene loaded photosensitizer G–BPMppa composite shows significantly improved water-solubility and dispersity in water, PBS and culture medium, and an increased singlet oxygen (1O2) quantum yield (ΦΔ = 60.55%) in DMF solution compared to free BPMppa (ΦΔ = 29.2%). In addition, cell experiments indicated that the G–BPMppa composite could be taken up by HeLa cells successfully, showing enhanced intracellular uptake behavior. Owing to its enhanced intracellular uptake and higher 1O2 quantum yield, G–BPMppa showed remarkably improved PDT efficiency (IC50: 1.36 ± 0.35 μg mL−1 of equivalent BPMppa) over free BPMppa after irradiation, but low dark toxicity without irradiation. Moreover, cell morphological changes after G–BPMppa PDT further qualitatively demonstrated that G–BPMppa could induce damage and apoptotic cell death efficiently. Furthermore, the photochemical mechanism of the G–BPMppa mediated PDT process was investigated by using specific quenching agents, sodium azide (SA, a singlet oxygen quencher) and D-mannitol (DM, a hydroxyl radical quencher); the results indicated that type I and type II photodynamic reactions could occur simultaneously, yet the type II reaction (the generation of 1O2) might play a predominant role in the G–BPMppa induced PDT process.

Folate chitosan conjugated doxorubicin and pyropheophorbide acid nanoparticles (FCDP–NPs) for enhance photodynamic therapy

We prepared new folate chitosan conjugated doxorubicin (DOX) and pyropheophorbide acid (PPa) nanoparticles (FCDP–NPs) using an ionic gelation method with tripolyphosphate (TPP) to enhance photodynamic therapy activity, based on the considerations of the long absorption wavelength (683 nm) of pyropheophorbide acid (PPa) in water and the excellent chemotherapeutic characteristics of doxorubicin (DOX) in cancer therapy. The obtained FCDP–NPs demonstrated a typical spherosome structure, a strong near infrared (NIR) absorption (705 nm) and significantly improved stability and dispersity in PBS (pH = 5, 7, 9); as well as a high singlet oxygen quantum yield (ΦΔ = 64%) compared to free PPa (ΦΔ = 59.1%). In addition, the in vitro cell experiments suggested that FCDP–NPs could be uptaken by HepG2 cells quickly and were mainly located in the cell nucleus. FCDP–NPs showed improved PDT efficiency over pure PPa and DOX at the same concentration after irradiation. Specifically, FCDP–NPs could lead to a 92% inhibition rate on HepG2 cells at 40 μg mL−1 (equal to 6 μg mL−1 DOX). However, the pure DOX showed little cytotoxicity at 6 μg mL−1, which suggests that a small amount of DOX could effectively enhance the PDT activities of PPa and lead to little “dark” cytotoxicity. Moreover, cell morphological changes after PDT treatment further indicated that FCDP–NPs could induce damage and apoptotic cell death efficiently. Finally, the photochemical mechanism of FCDP–NPs during PDT process was investigated by using specific quenching agents sodium azide (SA, a single oxygen quencher) and D-mannitol (DM, a hydroxyl radicals quencher), respectively. The results suggested that Type I and Type II photodynamic reactions can occur simultaneously, yet Type I reaction (the generation of hydroxyl radicals) might play a more important role. All these studies indicated that the FCDP–NPs could be potential nanoparticles in photodynamic cancer treatment.

The in vitro photodynamic activity, photophysical and photochemical research of a novel chlorophyll-derived photosensitizer

Chlorophyll has always been used as the leading compound for photodynamic therapy drug development. In this paper, a novel methyl pyropheophorbide-a- derived photosensitizer, 3-acetyl-3-devinyl-131-dicyanomethylene-pyropheophorbide-a was synthesized through modifications at C-131, C-3, and C-17 positions of methyl pyropheophorbide-a. The compound exhibited a longer wavelength absorption at 713 nm (in methanol) than that of methyl pyropheophorbide-a (667 nm) due to the enlarged the aromatic conjugation system by dicyanomethylene, allowing it to be potential in deep tumor treatment. Moreover, benefiting from the carboxylic group at C-17 and the acetyl group at C-3, the title compound was endowed with better water solubility than that of methyl pyropheophorbide-a. Detailed in vitro photodynamic therapy research showed ADCPPa could be uptaken by cancer cells successfully and killed the cancer cells more efficiently than the leading compound methyl pyropheophorbide-a under light (light dose 10 J/cm2) due to the high singlet oxygen quantum yield (65.98%). The excellent anti-photobleaching ability (degradation rate 1.6% in 10 min) also boosted its potential in practical application. In addition, the research has disclosed that during photochemical processes of photodynamic therapy, the formation of singlet oxygen after photodynamic therapy treatment played a major role, comparing with the formation of superoxide anion and radicals. Finally, the real time quantitative polymerase chain reaction (RT-qPCR) experiments have showed that the target compound has important regulating effect on expression of CDK2 and Survivin, consequently leading to apoptosis and cell death.

The photodynamic therapy activity of 3-(1-hydroxylethyl)-3-devinyl-131-(dicyanomethylene) pyropheophorbide-a methyl ester (HDCPPa) against HeLa cell in vitro

Photodynamic therapy (PDT) has been a potential therapeutic method for the treatment of various cancers, with photosensitizer being the key component in photodynamic therapy. In this paper, we prepared a photosensitizer 3-(1-hydroxylethyl)-3-devinyl-131-(dicyanomethylene) pyropheophorbide-a methyl ester (HDCPPa), based on chlorophyll pyropheophorbide-a according to the previous report, and systematically investigated the fluorescence emission spectrum and ultraviolet absorption spectrum HDCPPa has long absorption in the near-infrared spectral region (around 695 nm). The excitation wavelength and the emission wavelength were 415 nm and 699 nm respectively in dichloromethane, 1O2 quantum yield was 63.5%. HDCPPa also had high stability in PBS solution, DMEM cell culture medium and normal saline (NS) in vitro. After irradiation by the light of 675 nm (10 J.cm−2) for 70 min the degradation rate of HDCPPa was 12.5%, which indicated that the target compound showed high stability under light. The in vitro photodynamic therapy activities against HeLa cells were also studied, which showed that HDCPPa had extremely low dark toxicity but great phototoxicity, and the cell viability is lower than 10% under the light irradiation of 675 nm (10 J.cm−2). Moreover, HDCPPa can quickly enter the cell after being incubated with HeLa cells in less than 30 min. We also evaluated the mechanism of the photochemical reaction, which had proved that Type II is primarily responsible for the cell death. Therefore HDCPPa could serve as a very promising photosensitizer for photodynamic therapy.