Photo dynamic therapy (PDT), a new therapeutic technology for cancers and non-tumor diseases [1, 2], consists of three essential elements: a photosensitizer, light energy (laser), and oxygen. The basic process of PDT can be described as follows. A photosensitizer molecule at the ground state (S0) is excited (K0) by a specific light wavelength to form the excited state (S1). The photosensitizer molecule at S1 can produce fluorescence (K1) and then return to S0 by internal conversion (K2) or translate to the triplet state (S3) by intersystem crossing (K3). The photosensitizer molecule at S3 can return to S0 by releasing phosphorescence (K4) or reacting with a biomolecule. There are two types of PDT reactions: a type 1 reaction, in which a direct redox (K5) and reactive oxygen species (ROS) destroys tumor tissues by damaging cellular structures and inhibiting nucleic acid synthesis, and the indirect type 2 reaction, in which the photosensitizer molecule at S3 produces singlet oxygen (1O2), which reacts with biomolecules (K6). The relationship between type 1 and 2 reactions is competitive, although the type 2 reaction is widely believed to be the main mechanism of PDT [3, 4, 5]. 1O2 can decay to triplet oxygen (3O2) by producing phosphorescence at 1260 nm (K7) or react with the photosensitizer molecule (photobleaching, K8). The phosphorescence at 1260 nm is considered as the gold standard for measurement of 1O2 (Figure 1).
The term photobleaching is generally used in photophysical and photochemical reactions to describe the reduction of a chromophore. In PDT, photobleaching refers in particular to the phenomenon of photosensitizer consumption. Photobleaching is accompanied with 1O2 production, which plays an important role in PDT. Ascencio et al.  found that photobleaching of protoporphyrin IX (PpIX) showed a direct linear correlation to necrosis of tumor tissues in rats, as high PpIX photobleaching corresponded with complete responders, whereas low photobleaching corresponded with non-responders. Johansson et al.  found that PpIX concentrations exhibited pronounced inter- and intra-tumoral variations in glioblastoma, which are directly linked to variable degrees of fluorescence intensity. Real-time monitoring of PpIX fluorescence intensity and photobleaching is considered to be a feasible and safe index for early treatment prognosis of interstitial PDT (iPDT). Hennig et al.  confirmed through computer simulations that photobleaching kinetics provided a tool for real-time monitoring of iPDT.
Many studies have reported that PDT with the same irradiation conditions and photosensitizer doses achieved discrepant therapeutic effects [6, 7, 8], which can be explained by the distribution and photobleaching of photosensitizers, oxygen partial pressure of lesions, and 1O2 productivity. Therefore, the PDT dose was recently defined as the total 1O2 output. The PDT dose can be detected directly by measuring phosphorescence at 1260 nm. However, the half-life of 1O2 is very short and, therefore, very difficult to measure accurately. Fortunately, a computational formula of 1O2 and the PDT dose was derived [9, 10] based on photobleaching and verified by in vitro experiments, which showed that photobleaching kinetics can be used to predict 1O2 production and provide references for the PDT dose.
Photobleaching plays an important role in PDT. However, the photobleaching process remains unclear, as it involves complicated photophysical and photochemical reactions that produce various photoproducts. α-(8-quinolinoxy) zinc phthalocyanine (ZnPc-F7) is a new type of amphipathic mono-substituted zinc phthalocyanine (ZnPc) with good pharmacodynamics and low toxicity (Figure 2). Preliminary studies have shown that ZnPc-F7-mediated PDT (ZnPc-F7-PDT) had excellent anti-tumor effects. The anti-psoriasis activities of amphiphilic ZnPc were also reported recently . However, the photobleaching characteristics of ZnPc-F7 remain unknown. Therefore, the aim of the present study was to investigate the photobleaching properties of ZnPc-F7 in different solutions in vitro and provide substrata for the determination of PDT dose for use in clinical research. Furthermore, the distribution and photobleaching of ZnPc-F7 in vivo was observed using an in vivo imaging system in order to provide references for anti-psoriasis studies.
2 Materials and Methods
The research related to animal use has been complied with the study protocol approved by the Institutional Animal Care and Use Committees of Materia Medica, Chinese Academy of Medical Sciences, and Peking Union Medical College (approval no. 11400700078212). All surgeries were performed under the guidance of veterinarians according to international guidelines concerning the care and treatment of experimental animals.
2.2 Animals and cells
Equal numbers of male and female Nu/Nu mice, weighing 18–22 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in individual cages in an environment with a constant temperature and humidity under a 12-h light/dark cycle with free access to food and water. HaCaT cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (1640) (HyClone Laboratories, Inc., South Logan, UT, USA) containing 10% fetal bovine serum at 37°C with 5% CO2. Cells at the logarithmic phase were used to prepare suspensions at concentration of 1 × 106/mL.
2.3 Determination of absorption and fluorescence spectra
ZnPc-F7 (Beijing Guiqianjin Medical Technology Co., Ltd., Beijing, China) was dissolved in solvent containing 96% N, N-dimethylacetamide (DMF) and 4% polyoxyethylene castor oil (CEL) to prepare a stock solution at a dose of 5.00 mg/mL. The stock solution was then diluted with normal saline (NS), pure 1640, RPMI-1640 medium containing 10% fetal calf serum (FBS), or cell suspension (CS). The absorption spectra of solutions with ZnPc-F7 concentrations of 10.00, 20.00, and 40.00 μg/mL were measured using a micro-spectrophotometer (Q5000; Quawell Technology, Inc., Palo Alto, CA, USA). Similarly, a multiple tags detector (EnSpire; Perkin Elmer, America) was used to determine the fluorescence spectra of solutions with ZnPc-F7 concentrations of 1.00, 10.00, and 100.00 μg/mL. The excitation wavelength was 670 nm.
2.4 Detection of photobleaching in different solutions
ZnPc-F7 solutions at final concentrations of 0, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, 10.00, 20.00, 40.00, and 80.00 μg/mL were added to the wells of 96-well plates. Absorbance was measured and absorbance-concentration curves were created. Then, solutions at concentrations of 0, 1.00, 2.50, 5.00, 10.00, 20.00, and 40.00 μg/mL were irradiated with a semiconductor laser unit (PDT-670; Guilin Xingda Optoelectronic Medical Instrument Co., Ltd, Guilin, China). Blank wells, solvent wells, and a non-irradiated control plate were used to eliminate deviations. The output power and spot diameter were fixed to 1.50 W and 8.00 cm, respectively. Irradiation was performed and absorbance at 3, 10, 20, 30, 40, 50, 60, 120, and 180 min was measured. After irradiation, ZnPc-F7 concentrations were calculated using absorbance-concentration curves. The extent of photobleaching was calculated according to the equation: percent (%) = (A0 – At)/A0 × 100, where A0 and At represent the absorbance before and after irradiation, respectively, and t is the irradiation time (3–180 min).
2.5 Distribution and Photobleaching of ZnPc-F7 in Nu/Nu mice
An in vivo imaging system (Caliper Life Sciences, Inc., Hopkinton, MA, USA) was used to measure the distribution and photobleaching of ZnPc-F7 in vivo. Exposure time was 10 s for X-ray and 2 min for fluorescence. The excitation wavelength was 670 nm and the emission wavelength was 790 nm. Red, green, and blue spectra were added to the fluorescence images. To measure absorption and distribution in normal animals, Nu/Nu mice were intravenously injected with 2.00 mg/kg of ZnPc-F7 and images were collected at 1, 2, 4, 6, 8, 12, and 24 h after injection. Mice were sacrificed with an overdose of pentobarbital sodium at 24 h after injection and images of organs and the dorsal skin were collected. Four mice were equally allotted to a normal control group or a model group. Imiquimod cream (IMQ; batch number, 131002; Hubei Keyi Pharmaceutic Co., Ltd, Wuhan, China) was locally applied to the dorsal skin of each mouse in the model group for 6 continuous days at a dose of 100.00 mg. Then, each mouse was intravenously injected with 2.00 mg/kg of ZnPc-F7. Images of mice at 8, 12, and 24 h after injection and images of organs after sacrifice were collected. Six mice were equally allotted to an irradiation group or a nonirradiation control group. After administration of IMQ for 6 continuous days, each mouse was intravenously injected with 2.00 mg/kg of ZnPc-F7. PDT was performed at 6 h after injection. The output power, spot diameter, and irradiation time were 0.20 W, 4.00 cm, and 10 min, respectively. The energy density was 9.60 J/cm2. Images of mice at 8 and 12 h after injection (2 and 6 h after PDT) were collected.
Local administration was used for photosensitizer-mediated PDT for treatment of psoriasis. Six mice were allocated to either an intravenous injection group or a local administration group. After administration of IMQ for 6 continuous days, 100.00 μg/mL solutions or 1% liniment of ZnPc-F7 was administrated. The final dose of ZnPc-F7 was 2.00 mg/kg. Images at 4, 6, 8, 12, and 24 h after administration were collected. Images of organs after sacrifice were also collected. ZnPc-F7 liniment was prepared as follows: 100.00 mg ZnPc-F7 was dissolved in 5.00 mL of solvent containing 96% DMF and 4% CEL; 500.00 mg of polyoxyethylene pyrrolidone-30, 0.25 mL of azone, and 0.25 mL of propylene glycol were added in order; and then, 50% ethyl alcohol was used to adjust the total volume to 10.00 mL.
2.6 Statistical analysis
Data are expressed as the mean ± standard error of the mean (SD) and analyzed using SPSS software (SPSS, Inc., Chicago, IL, USA). One-way ANOVA was used to compare inter-group differences. A probability (p) value of < 0.05 was considered statistically significant.
3.1 Comparison of absorbance and fluorescence of ZnPc-F7 in different solutions
Preliminary studies showed that ZnPc-F7 in DMF had three absorption peaks at 332, 608, and 672 nm, respectively. The use of CEL led to a shift in absorption peaks; however, it is necessary to dissolve ZnPc-F7 in water. The results showed that the absorption maximums of ZnPc-F7 in the four solutions were almost the same and emission maximums were approximately 10 nm longer than absorption maximums (Table 1). The mean values of absorption peaks were used as measurement wavelengths for absorbance measurement. Absorption and emission spectra were mirror images and characteristic absorption peaks were obvious in all four solutions. Both absorbance and fluorescence were enhanced with increasing concentrations (Figure 3). Absorbance of the four solutions at concentrations of 0–40.00 μg/mL showed good linearity; however, when the highest concentration was 80.00 μg/mL, the R2 value of FBS had obviously decreased (Figure 4) because of aggregate formation at high concentrations in FBS. Therefore, photobleaching of ZnPc-F7 was measured at a concentration range of 1.00–40.00 μg/mL.
Absorption and emission maximum of ZnPc-F7 in different solutions.
|Absorption maximum (nm)||Emission maximum (nm)|
|Solvent||10.00 μg/mL||20.00 μg/mL||40.00 μg/mL||1.00 μg/mL||10.00 μg/mL||100.00 μg/mL|
3.2 Photobleaching of ZnPc-F7 in different solutions
The energy density of light used in anti-tumor studies was 5.40 J/cm2 at an output power, spot diameter, and irradiation time of 1.50 W, 8.00 cm, and 3 min, respectively. Concentrations of ZnPc-F7 were 0.001–10.00 μg/mL in vitro and 10.00–100.00 μg/mL in vivo. After irradiation at an energy density of 5.40 J/cm2, ZnPc-F7 was bleached significantly in all four solutions with concentrations of 1.00–40.00 μg/mL. The photobleaching rates in order from fastest to slowest were FBS, CS, 1640, and NS (Figure 5). The photobleaching quantities of the four solutions increased with the initial concentration, but the percent of photobleaching was more complex. When the output power and spot diameter were fixed at 1.50 W and 8.00 cm, respectively, accumulated photobleaching quantities and photobleaching percent were increased with the irradiation time and initial concentration (Figure 5, 6 and 7). However, photobleaching percent per minute decreased with irradiation time (Figure 8). Rapid initial phases followed by a slower rate of photobleaching were observed in all four solutions.
Linear-regression analyses were performed using the irradiation time (X1) and initial concentration (X2) as independent variables, and photobleaching percent (Y) as a dependent variable. Regression formulas for NS, 1640, FBS, and CS were Y = 8.006 + 0.335 X1 + 0.338 X2 (R = 0.909, F = 107.166, Sig = 0.000); Y = 26.082 + 0.330X1 + 0.300 X2 (R = 0.811, F = 43.085, Sig = 0.000); Y = 45.545 + 0.290 X1 – 0.377 X2 (R = 0.784, F = 35.797, Sig = 0.000); and Y= 29.996 + 0.319X1 + 0.088X2 (R = 0.822, F = 46.721, Sig = 0.000), respectively. Further, with irradiation time, initial concentration, and solvents as independent variables, and photobleaching percent as a dependent variable, the fitting formula was Y = 12.625 + 5.913X1 + 0.319X2 + 0.100X3 (R = 0.798, F = 109.752, Sig = 0.000), where Y is the photobleaching percent (%), X1 is the solvent (1, 2, 3, and 4 represented NS, 1640, FBS, and CS, respectively), X2 is the irradiation time, and X3 is the initial concentration. The results suggested that the solvent was the most important factor influencing photobleaching, while the initial concentration had the lowest influence.
The half-life of photobleaching of the four solutions was calculated using time-photobleaching percent curves and described in (Table 2). Under the same irradiation conditions, ZnPc-F7 was bleached fastest in FBS and slowest in NS. The photobleaching rates of CS and 1640 were similar. ZnPc-F7 was not reduced significantly until 180 min without irradiation, as compared with the irradiation group (Figure 9). These findings suggested that ZnPc-F7 had good stability in dark environments, which might indicate low toxicities and side effects of ZnPc-F7.
Half-life periods of photobleaching in different solutions
|Solvent||Concentration μg/mL||t1/2 (min)||up limit (min)||low limit (min)|
3.3 Distribution and photobleaching characteristics of ZnPc-F7 in normal and psoriasislike mice
In normal mice, the fluorescence intensity of abdominal skin was continuously enhanced for 24 h after injection of ZnPc-F7 at a dose of 2.00 mg/kg. Fluorescence intensities of organs showed that ZnPc-F7 was mainly distributed in the liver after 24 h. High fluorescence intensities were observed in isolated skins. The distribution kinetics in different animals were similar (Figure 10). Local administration of IMQ for 6 continuous days induced obvious psoriasislike lesions in Nu/Nu mice (Figure 11). The distribution of ZnPc-F7 was homogeneous in the skin of psoriasislike mice from 8 to 24 h after injection. There were no significant differences in the change of fluorescence intensities in the skins of normal and psoriasis-like mice, with similar distributions in organs (Figure 12). Irradiation at an energy density of 9.60 J/cm2 led to significant photobleaching of ZnPc-F7 in IMQ-induced psoriasis-like mice. The fluorescence intensity of the irradiation group was obviously lower than that of the no-irradiation group at 2 and 6 h after PDT (8 and 12 h after injection) (Figure 13), suggesting that ZnPc-F7-PDT may be a viable option for the treatment of psoriasis. From 4 to 24 h, the fluorescence intensity had persistently increased in mice injected with ZnPc-F7 at a dose of 2.00 mg/kg. On the contrary, the fluorescence intensity in mice locally administrated ZnPc-F7 had continuously decreased from 4 to 24 h. After 24 h, the distributions in all organs were observably lower in the local group than in the injection group (Figure 14). These findings suggest that local administration might be an appropriate approach for ZnPc-F7 to treat skin diseases, such as psoriasis. The lower distribution in organs and faster metabolism in skin signified lower side effects and higher therapeutic effects.
Initial studies of photobleaching used dyes and chlorophyll. Of the more than 6000 papers with the term photobleaching in the PubMed database until July 2015, fewer than 200 were related to PDT. In the late 1980s, photobleaching of photosensitizers used in PDT began to receive more attention. In 1988, Moan et al.  first reported photo-induced degradation and modification of photofrin II in cells, which was followed by many other studies of photobleaching of photosensitizers [13, 14, 15]. Yang et al.  designed a multispectral fluorescence imaging system for intra-operative photobleaching detection that was tested by photofrin-PDT treatment of brain tumors. This system performed to specification under realistic operating conditions and could be used for PDT dosimetry. However, at present, there are no standards or governing principles for photobleaching research.
Fluorescence and absorbance are main detection indexes of photobleaching characteristics. Fluorescent spectrometry is often used to detect photobleaching of photosensitizers in vivo, while spectrophotometry is often used in vitro. For example, Zeng et al.  studied photoproduct formation and photobleaching of QLT0074 (a verteporfin-like photosensitizer) using fluorescence spectroscopy in BALB/c mice and proposed three parameters for prediction of PDT effects. Saw et al.  studied the influence of annexing agents on photobleaching of hypericin using both fluorescent spectrometry and spectrophotometry. In the present study, a microplate reader was used to measure absorbance to investigate the photobleaching characteristics of ZnPc-F7 in four solutions and an in vivo imaging system was used to study the distribution and photobleaching of ZnPc-F7 in mice. The results showed that ZnPc-F7 was bleached both in vitro and in vivo after irradiation with a 670 nm laser unit with irradiation doses used in pharmacodynamics studies, suggesting a basis for ZnPc-F7-PDT to react and treat diseases. ZnPc-F7 did not bleach without irradiation until 180 min, suggesting that ZnPc-F7 was stable in dark environments.
The photobleaching kinetics of ZnPc-F7 in the four solutions showed that the type of solvent, irradiation time, and initial concentration influenced the photobleaching rate. The solvent was the most important influencing factor. Photobleaching kinetics can be modeled simply using the mono-exponential function of D = D0e–βj, where D is the photosensitizer concentration, D0 is the initial concentration, J is the energy density of light (J/cm2), and β is the rate constant. This model is simple and was fitted to some experimental data. The results of the present study showed that photobleaching quantity and photobleaching percent were positively correlated with the initial concentration and irradiation, and could be modeled using a mono-exponential function. However, photobleaching kinetics could not be modeled using a mono-exponential function in an oxygen-rich environment. Solvents also influence photobleaching. So, a secondary kinetic model was proposed  based on mono-exponential decay. The substrate concentration was considered to be an important influencing factor in this model, which is consistent with the results of the present study, suggesting that the photobleaching kinetics of ZnPc-F7 in vitro fitted a secondary kinetic model.
Many studies have reported that photobleaching quantity and rate were related to therapeutic effects, but faster photobleaching is not necessarily associated with a therapeutic effect. Baran and Foster  examined the effects of the fluence rate on photobleaching of Pc 4 during PDT in BALB/c mice and found no significant correlation between fluorescence photobleaching and tumor regrowth. It appeared that photobleaching of Pc 4 was not a strong predictor of individual tumor response. To partly explain the different effects under the same irradiation conditions, Boere et al.  reported a correlation between PpIX fluorescence photobleaching rates and epithelial damage. A two-phased decay in PpIX fluorescence was identified in the response group, with a rapid initial phase followed by a slower rate of photobleaching, while rapid initial decay was not observed in non-responders, suggesting that rapid initial decay plays an important role in PDT. Obvious photobleaching of ZnPc-F7 after irradiation for 3 min at an energy density of 5.40 J/cm2 was observed in all four solutions in vitro. Irradiation for 10 min at an energy density of 9.60 J/cm2 also induced apparent photobleaching of ZnPc-F7 in psoriasis-like mice. The results of an in vitro kinetics study showed that the photobleaching rate slowed down with an increase in irradiation time, suggesting that ZnPc-F7 decayed through a two-phase process that might be advantageous for ZnPc-F7-PDT. However, fast photobleaching may lead to rapid local depletion of oxygen, which could inhibit subsequent photochemical reactions necessary for treatment . These findings suggest that the rate of photosensitizer photobleaching may not always be appropriate for monitoring of singlet oxygen availability and PDT dosimetry.
Psoriasis is a life-long chronic inflammatory disease that affects approximately 2% of the global population [23, 24]. PDT is considered an effective method for the treatment of psoriasis [25, 26]. The results of our preliminary studies showed that ZnPc-F7-PDT might be useful for psoriasis treatment. IMQ is an agonist of toll-like receptors. In the present study, IMQ was used to induce psoriasis-like lesions [27, 28, 29] in Nu/Nu mice. The distribution and photobleaching of IMQ-induced psoriasis-like mice were observed using an in vivo imaging system. The results showed that fluorescence of ZnPc-F7 in the skin of normal and psoriasis-like mice had steady increased for 24 h after injection. There was no significant difference in fluorescence in the skin and organs between normal and psoriasis-like mice. Fluorescence intensities in organs of mice the received local administration of ZnPc-F7 were lower than that in mice administrated by intravenous injection after 24 h, suggesting that ZnPc-F7 was appropriate for psoriasis treatment and local application might be an optional administration approach. An in vivo imaging system was fitted to monitor the dynamic variation of photobleaching and the distribution in animals because of the luminous characteristics of the photosensitizer. This process might could lead to the use of fewer animals and elimination of individual errors, as compared with traditional pharmacokinetic methods.
In conclusion, obvious photobleaching of ZnPc-F7 was observed both in vitro and in vivo after irradiation. Solvents, irradiation time, and initial concentration were important influencing factors of the photobleaching rate. ZnPc-F7-PDT should be considered for treatment of psoriasis. Future studies are needed to analyze photobleaching products both in vitro and in vivo, and the anti-psoriasis effects should be confirmed in animal tests.
Funding for this study was received from the National Major Drug Discovery Project of China (grant nos. 2013ZX09302302, 2012ZX09J12203, and 2012ZX09301002-001-009); the Shenzhen Key Laboratory of Drug Dependence and Safety (grant no. ZDSYS201504301045406); the Health and Family Planning Commission of Shenzhen Municipality (grant no. 201605018); and the Shenzhen Foundation of Science and Technology Innovation (grant no. JCYJ20160427185055877) for financial support.
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