Photodynamic Anti-Tumor Efficiency of Hematoporphyrin Derivative

Photodynamic therapy (PDT) is an emerging non-invasive treatment based on a combination of photochemistry and photophysics. PDT has more advantages compared with traditional chemotherapy and surgical treatment, which is more targeted, less harmful to healthy tissue [1-4]. When the photosensitizer (PS) molecule is illuminated by a light of particular wavelength, it passes from the first excited singlet state to the first excited triplet state T1, which will initiate a photochemical reaction by generating reactive free radicals or transferring its energy to the ground state oxygen molecule (3O2) to produce singlet oxygen (1O2) that can oxidate the bio-molecules of diseased cells and apoptosis or necrosis was resulted [5,6]. Photodynamic therapy was discovered nearly 100 years ago but medically approved drugs and technologies are still limited [7,8].

harmful to healthy tissue [1][2][3][4]. When the photosensitizer (PS) molecule is illuminated by a light of particular wavelength, it passes from the first excited singlet state to the first excited triplet state T1, which will initiate a photochemical reaction by generating reactive free radicals or transferring its energy to the ground state oxygen molecule (3O2) to produce singlet oxygen (1O2) that can oxidate the bio-molecules of diseased cells and apoptosis or necrosis was resulted [5,6]. Photodynamic therapy was discovered nearly 100 years ago but medically approved drugs and technologies are still limited [7,8].
The first photosensitizer used in clinics was Hematoporphyrin Derivative (HPD) which was called Photofrin or porfimer sodium. It is a mixture of six hematoporphyrin derivatives with long retention in skin which resulted in that patient need to avoid the light for 4 weeks. Hemeporfin (HMME) is a mixture of two enantiomers which was first discovered by Xu [9]. HMME has better performance than porfimer sodium. It shows low toxicity to normal tissues with rapid metabolism property. For nearly a century, the design and synthesis of hematoporphyrin derivatives are still of concern to many scientists. Here the photophysical property, cytotoxicity of new hematoporphyrin derivative BL-1 in vitro and in vivo were investigated As Photosensitizer for Photodynamic Therapy (PDT).

Singlet Oxygen
The efficiency of singlet oxygen can be used to evaluate the   (Table 1). The IC50 value of BL-1 to Eca-109 tumor cells was smaller than HMME.
These results suggested that BL-1 was a promising photosensitizer in vitro. This process promotes the growth of blood vessels and red blood cells at the tumor site. Porphyrins are coenzymes of red blood cells.
Therefore, it will preferentially accumulate in the tumor site, and has a certain tumor targeting effect [10][11][12]. The antitumor activity  (Table 2). It is suggested that the compound BL-1 exhibited efficient photodynamic antitumor efficacy on Balb/c nude mice at lower concentration.
Therefore, BL-1 is a promising antitumor PS in PDT application. Note: *P < 0.05, ***P < 0.001 represents a significant difference relative to the control group.

Instruments and Reagents
All the chemicals and reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. and Bide Pharmatech Ltd., and used without further purification. All solutions were freshly prepared with deionized water. The UV-vis absorption spectrum was recorded on an ultraviolet-visible spectrophotometer (Model V-530, Japan). Fluorescence spectrum was recorded on a fluorescence spectrophotometer (FluoroMax-4, France).

Singlet Oxygen Determination
The singlet oxygen capacity of the photosensitizer was measured by DPBF chemical oxidation. A DMSO solution containing 0.5μM PS and 20μM PBF was irradiated with a laser intensity of 622nm at 5mW/cm2, and the natural logarithm of DPBF absorption at 410nm was plotted and fitted by a first-order linear least squares model to obtain a singlet state of the photosensitive process. The rate constant of the methylene blue sensitized DPBF photooxidation reaction was converted to the 1O2 quantum yield [13].

Cell Lines and Culture Conditions
The cells were obtained from the Cell Culture Center of the Chinese Academy of Sciences. The cell-related reagents were purchased from Shanghai Mingrong Biotechnology Co., Ltd. The cells were cultured in 10% Fetal Bovine Serum (FBS), 50mg/mL penicillin and 50mg/mL. In medium culture, 5% carbon dioxide was added to a humidified incubator at 37oC. Cells in the exponential growth phase were used in each experiment.

MTT Assay
The experiment of cell phototoxicity and dark toxicity was

Conclusion
The photophysical and photochemical properties of new hematoporphyrin derivative BL-1 were determined. In vivo and in vitro tests were performed to evaluate the ability of compound BL-1 to destroy tumor. The compound exhibited long maxima absorption wavelengths and higher phototoxicity compared to HMME. BL-1 exhibited better inhibitory effect on Eca-109 tumor cells than HMME. Therefore，compound BL-1 could be a potential anti-tumor photosensitizer in photodynamic therapy.