Cancer Therapy Vol 4, 271-276, 2006

 

How could the light fluence rate influence the cure effect of photodynamic therapy?

Research Article

 

Tao Xu*, Mingzhao Li

Academy of Metrology and Quality Inspection of Shenzhen, GuangDong Pro, China, 510085

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*Correspondence: Tao Xu, Academy of Metrology and Quality Inspection of Shenzhen, GuangDong Pro, China, 5100; Phone: +86-755-26941691; Fax: +86-755-26941524; E-mail: xutao780606@126.com

Keywords: photodynamic therapy, fluence rate, microvasculature damage, RBCs column diameter, Krogh tissue model

Abbreviations: bovine serum albumin, (BSA); haematoporphyrin derivative, (HpD); optical density, (OD); Photodynamic therapy, (PDT); RBCs column diameter, (RBCCD)

 

Received: 11 May 2006; Revised: 16 November 2006

Accepted: 01 December 2006; electronically published: December 2006

 

Summary

Some works have been done in this paper to study the role of light fluence rate in the photodynamic therapy (PDT), which focuses on the influence of light fluence rate on the microvasculature damage, the cell killing and the photodynamic reaction impetus. The microvasculature damage is studied through observing the values of RBCs column diameter during the process of the HpD mediated PDT. It is found less microvasculature damage is induced by 75mW/cm2 illumination than that by 150mW/cm2, indicating that under 75mW/cm2 illumination tumor oxygen can be better preserved than 150mW/cm2. The cell killing experiment is performed in vitro and designed in the manner that cell killing rate was only influenced by light characters. We find PDT with 75mW/cm2 illumination can cause higher cell killing rate than with 150mW/cm2 illumination. In addition a PDT model of Krogh tumor is built to analyze the promotion of photodynamic reaction. We find under a same initial oxygen environment (18.4μM) the PDT stops at oxygen concentration of 3.54μM when the illuminating light is 75mW/cm2 and 5.2μM when the illuminating light is 150mW/cm2. The calculation results indicate a more powerful PDT impetus under low fluence rate than high one. The oxygen concentration in 28.4 percent of the tumor volume is higher than 5.2μM and 59.8 percent of the tumor volume is higher than 3.54μM, indicating a larger therapy range under lower fluence rate illuminating.

 

 

I. Introduction

Photodynamic therapy (PDT) is believed to act through the cytotoxic singlet oxygen, which forms when the photosensitizer is excited by light and transfers its energy to the molecular oxygen in tissues. It may be the principle way to destroy the tumor cell in PDT that the singlet oxygen will oxidize some elements of the cell. Photosensitizer, tissue oxygen and irradiation light are considered to be the three element factors of PDT (Dougherty ea al 1998; Rosenthal and Glatstain 1994). Oxygen in tissues can be easily depleted at high fluence rates while it can be preserved at low fluence rates (Henderson et al 2000). And a rule has been concluded that the higher fluence rates, the more decreasing in the oxygen concentration (Foster et al 1991; Schunck and Poulet 2000). While how oxygen can be preserved at low fluence rates is a puzzling problem. It is believed to have something to do with the microvasculature.

It is noticed at the same time that at similar oxygen environment low fluence rates cause more tumor cure than high fluence rates for the same total fluence (Sitnik et al 1998; Foster et al 1993; Veenhuizen and Stewart 1995). Are there any other reasons that cause the higher cure rate except oxygen? Maybe the fluence rate can influence the tumor cell killing ability, but it needs to be verified. Some experiments observing the microscopic cell killing rate at different fluence rates must be performed.

PDT impetus is also a guide to evaluate the efficiency of therapy. The relationship between the PDT impetus and the fluence rate should be observed. We can build a model to simulate the photodynamic reaction process. As photodynamic reaction will stop under some oxygen level (Nichols and Foster 1994), the threshold can be calculated to represent the PDT impetus. By this attempt, we can find out how the fluence rate influences the reaction process.

Works mentioned above can help us to recognize how the fluence rate can influence the photodynamic therapy effect.

 

II. Materials and Method

A. Microvasculature damage in PDT against light fluence rate

Wistar rats weighted 200±10g (Experimental Animal Center of PLA General Hospital, Beijing, China) were obtained at least 1 week before the experimentation. They were housed in a room with subdued lighting and fed with a standard pellet diet and water. Approval for this project was obtained from the Animal Experiment Center of Tianjin Medical University. A chondrosarcoma tumor cell line was transplanted into the right flank of each rat. The tumors that grew to be approximately 6-7 mm in size within about 5 days after transplantation were used in this experiment.

The animals were given the haematoporphyrin derivative (HpD) via tail vein injection at a dose of 2.5 mg kg-1. About 24 hours after the injection the rats were anaesthetized with a 50 mg kg-1 sodium pentobarbital. The RBCs column diameter (RBCCD) observation method described in (Fingar et al 1992, 1999; Xu et al 2005) was employed to investigate the tumor microvasculature. The distribution of the vascular was observed by unaided eyes and little fatty regions with arteriole and venule in pairs were chosen. The RBCCD was measured through the playback of the recorded images every 4 min for 32 min for the 75 mW/cm2 illuminating group. For the 150 mW/cm2 illuminating group it is 2 min for 16 min.

The relative RBCs column diameter RBCCDrel at time t was defined as RBCCD(t)/RBCCD(0). Ten animals were used in each different experimental group and the mean relative RBCCD at time t was thought to reflect the status of the microvasculature after some dose of illumination.

 

B. Cell killing in PDT against light fluence rate

Mammary cancer cell MDAMB 543 was obtained from the tumor laboratory of PLA General Hospital of China and was cultured in vitro with 90% RPMI 1640 incubation media (GIBCOBRL Lifetechnologies, Rockville, MD, USA), 10% bovine serum albumin (BSA) and 1% penicillin and streptomycin at 37℃. The cancer cell was laid in incubator with 5% CO2. The cultured MDAMB 543 cells were made suspension of single cell with 0.25% trypsase (Sigma, St Louis, MO, USA). The cell concentration of the suspension was 1.0×105/ml. Seven culture plates with 96 wells (Costar, Cambridge, MA, USA) were used to inoculation the suspension in a manner of 0.2 ml per well and placed in the 5% CO2 incubator at 37℃ for 16 hours. Then the supernatant culture media was removed. HpD as the photosensitizer, which was diluted by RPMI 1640 incubation media to a concentration of 0.004 mg/ml, was added to the wells. The culture plates was then placed in the 5% CO2 incubator at 37℃ and completely protected from light. About 4 hours later six of the plates were illuminated by 150mW/cm2 laser for 10s, 20s, 30s and 75 mW/cm2 laser for 20s, 40s, and 60s respectively while the other one plate was still kept in protection of light as a control group. After illuminating the culture plates were placed back in the incubator for 24 hours and then 0.1 mg diphenylterrazolium bromide (Sigma, St Louis, MO, USA) was added to the wells before another 4 hoursÕ culture. The supernatant fluid was removed when the culture was completed and 0.15 ml dimethylsufoxide was added. Culture plates were oscillation slightly for 10 min and the optical density (OD) of all the wells was measured by an Elisa (318-MC, Xinke Inc, Shanghai, China). The fraction cell surviving was defined as the ratio of the mean OD of experiment groups to the control group.

 

C. Study on the PDT impetus

The theory of oxygen diffusion from capillaries has been well established since Krogh, 1919 presented his analytical prediction of how the delivery from vessels was governed. Since that time, researchers have tried to exploit this model to better interpret the oxygen diffusion and consumption processes in tissues. It is accepted that oxygen diffuses away from the high relative concentration in the vessel to regions of tissue with lower concentration. According to these models, The equation describing PDT process can be developed as follow:

     (1)

where C (r) is the oxygen concentration at position r, D is the diffusion coefficient for oxygen in tissue, which is generally taken to be 2«10-5cm2s-1(Pogure et al, 2001). Γmet, Γpdt, s are the metabolic oxygen consumption rate, the PDT oxygen consumption rate and the supply of oxygen at each point r respectively, and all vary with C (r). According to the Michaelis-Menten model (Tang, 1933), we have deduced the expression of Γmet (Xu, 2004):

                (2)

 

Γpdt has been given in the fore studies by:

       (3)

 

kot is the bimolecular rate of triplet quenching by triplet oxygen, koa is the rate of chemical reaction between singlet oxygen and unspecified substrate [A], kos is the rate of chemical reaction between singlet oxygen and the photosensitizer, which always seems to be 0. And kp is the decay rates of the photosensitizer triplet, respectively. Γ0 is a constant that describes the initial rate of photochemical oxygen consumption and changes linearly with the light fluence rate (Nichols and Foster 1994, Geogakoudi et al 1997). Setting values of all the parameter can be found in the paper of Mitra et al, 2000. Equation (3) indicates that PDT will stop at a quite low oxygen concentration (<<kp / kot).

There is a little trouble in defining s. First, during PDT there is a similar arterial blood oxygen level with that before PDT, while the venous blood oxygen concentration decreases much after the beginning of PDT (Guyton 1981, Slonim and Hamilton, 1981). So there is a higher oxygen supply during PDT than before. Second, the higher oxygen concentration at the outer vascular wall, the higher blood oxygen supply occurs. Considering these two factors, we define s by (Xu, 2004):

 

           (4)

 

Cout(t) is the oxygen concentration at the outer vascular wall at time t after the beginning of PDT. α is a constant which can be set as 1~3.

 

III. Results

RBCCD changes of venule and arteriole both occurred under the illumination of 75mW/cm2 and 150mW/cm2 laser. Data collected from the image playback of experiment groups were summarized in Figure 1. First, the data obtained from the observation of arteriole were shown in Figure 1A, followed by the data obtained from the observation of arteriole shown in Figure1B. In Figure 1A, there was an indication that under the low fluence rate illumination there occurred less microvasculature damage than high fluence rate in the early period of PDT, while there was no significant difference between the two kinds of illumination near the end of the treatment. Arteriole RBCCDrel was obviously lower under 150mW/cm2 illumination than 75mW/cm2 when light dose is 36 J/cm2 (p=0.0031), 54 J/cm2 (p=0.0024) and 72 J/cm2 (p=0.036). The administration of light dose up to 90 J/cm2 and above seemed to make similar damages to the arteriole at both light fluence rate. For the light dose of 18 J/cm2 increase of RBCCDrel occurred under 150 mW/cm2 illumination. That might due to the physiological reaction to the high light fluence rate. The RBCCDrel of the venule decreased more quickly against the light dose than that of the arteriole as shown in Figure 1B, while no statistically significant difference in the RBCCDrel of the venule was observed under 150mW/cm2 and 75mW/cm2 illumination though one point at the light dose of 54 J/cm2 gave some peradventure. It was observed that under the illumination of 75mW/cm2 light the RBCCD of venules dropped slowly when the illuminated fluence was less than 54 J/cm2, but rapidly when between 54 J/cm2 and 72 J/cm2. Therefore we could assume that at 75 mW/cm2 the threshold energy dose causing the vessels damaged severely was between 54 J/cm2 and 72 J/cm2. Similarly, at 150 mW/cm2 that threshold might be between 36 J/cm2 and 54 J/cm2. The difference of the two thresholds might do some contribution to the exception at 54 J/cm2.

To determine the PDT induced direct MDAMB 543 cells killing at various fluence rates the survival rates were examined. The mean OD valuewas measured as the scale of the fraction surviving. For control group it was the average OD of 82 wells in the control plate. OD value of each well in the experiment plates was measured and normalized to the mean control value. For the 150mW/cm2 illuminated group the wells number of each plate was 36 and for OD 75 mW/cm2 illuminated group it was 38. The illuminating light dose of both groups was 1.5 J/cm2, 3 J/cm2, 4.5 J/cm2. Mean normalized OD value and SD of each plate were calculated and summarized in Figure 2. The surviving fraction was 0.624 versus 0.526(1.5 J/cm2), 0.516 versus 0.384(3 J/cm2), and 0.319 versus 0.219(4.5 J/cm2) under 150mW/cm2 versus 75mW/cm2 illuminating. It was recognized in a statistic way that the MDAMB 543 cells survival fractions under fluence rate of 75mW/cm2 was less than that under 150 mW/cm2, indicating more effective cell killing under lower fluence rate illumination in PDT.

 

      

 

Figure 1. RBCCDrel changes against the light dose in different fluence rates for arteriole (A) and venule (B). Values are means±SD of 10 observation results in each experiment group. Those data points that show, based on the Wilcoxon signed-ranks test, a statistically significantly difference between the 75 mW/cm2 group and 150 mW/cm2 group are indicated with an asterisk (*).

 

Figure 2. Cell fraction surviving after illuminating with light dose of 1.5 J/cm2, 3 J/cm2, 4.5 J/cm2 in PDT. Values were experimental OD normalized to the control group that was protected from light. Cell fraction surviving of the two experiment groups were significantly different (p=0.041, 0.033, 0.021).

The initial oxygen concentration at some points in a range of 100μm surrounding the vascular, including the point just at the outer wall Cout(0), could be obtained from the experimental study of Tsai et al, 1998. We expanded the range through fitting those points and got the initial oxygen distributing in a range of 1.5mm. Crank-Nicolson method was employed to solve equation (1) starting from those initial values. The variety of oxygen concentration against time t and space r was displayed by Figure 3A (fluence rate of 75mW cm-2) and Figure 3B (fluence rate of 150mW cm-2). The oxygen threshold in the photodynamic reaction was considered to be 3.54μM in Figure 3A and 5.2μM in Figure 3B. This result indicated a more powerful PDT impetus under low fluence rate than high one. It could be informed from Figure 3 that photodynamic reaction could not start in some area where the oxygen concentration was less than those thresholds. For 75mW/cm2 illuminating the inactive area was region of r>1.16mm and for 150mW/cm2 illuminating it is r>0.8mm. It is 40.2% and 71.6% of the model tumor volume respectively.

 

IV. Discussion

From this work it can be recognized that light fluence rate can improve the therapy effect through the blood oxygen supply, the cell killing rate and the reaction impetus. Low fluence rate treatments can be more effective in decreasing vascular lesions than high fluence rate treatments, if the same light fluence is applied during the PDT process. Other studies have also concluded lower fluence rate treatments can preserve the status of oxygen for a more effective PDT (Tromberg et al 1990; Foster et al 1991; Sitnik and Henderson 1998; Sitnik et al 1998), and it has been recognized that light fluence rates play a major role in the tumor oxygenation status during PDT exposure. Our work explains the mechanism of oxygen preservation under low fluence illumination in PDT to some extent. It can be concluded from Figure 1 that lower fluence brings less microvasculature damage during the PDT process, though there seems to be no difference in venules at the two different fluence rates, which may be due to the extremely vulnerable venule circulation. In theory, less damage to microvasculature indicates timely oxygen supply to recover the photochemical consumption of oxygen in PDT. As tissue oxygen is one of the elementary factors of PDT, the timely supply of oxygen is fairly important for the destruction of tumors.

Some clinical and experimental data have been reported recently pointing to a significant tumor cure effect (Robinson et al 1998; Langmack et al 2001). From the result of our cell killing experiment, we can conclude that lower fluence rate treatments may be more effective in increasing the killing rate of tumor cells, which directly indicates better local tumor control. For an experiment performed in vitro, the oxygen consumption of PDT and the photosensitizer concentration in the medium can be regarded as constant. Thus the cell killing effect may vary against the photosensitizer bleaching and singlet oxygen generating, which changes with the light fluence rate. While recently studies have reported that higher photobleaching seem to occur under illuminating of lower fluence rate both in vivo and vitro experiments (Coutier et al 2001; Finlay et al 2004). So we can only contribute the higher cell killing rate caused by lower fluence rate to higher singlet oxygen generating. In vitro experiment with sufficient oxygen supplying, sharply consumption of triplet oxygen caused by high fluence rate illuminating will increase the chance that excited singlet oxygen return to the ground state. As a result the possibility that singlet oxygen reaction with the tumor cell is decreased, means a low cell killing rate.

In the PDT process, the decay rates of the photosensitizer triplet, kp, is decided by the concentration of triplet photosensitizer. Under a higher fluence rate illuminating, more photosensitizer molecules are excited to be singlet and triplet status. High irradiance energy causes high frequence of energy level transition. Consequently the tendency of energy transferring from triplet photosensitizer to triplet oxygen is wakened. So under higher fluence rate illuminating the bimolecular rate of triplet quenching by triplet oxygen, kot, takes a lower value.

 

Figure 3. Oxygen concentration changes against time t and space r in 75 mW/cm2 (A) and 150 mW/cm2 (B) illuminating PDT. Values are deduced step by step with Crank-Nicolson method from the Krogh model tumor. C(r,0) is obtained through fitting the dates in TsaiÕs paper, 1998.

 

For the promotion of photodynamic reaction is positive going with the product of kot and oxygen concentration, there need to be a high oxygen environment to ensure the proceeding of photodynamic reaction. Similar viewpoint are presented in appendix equation (A9) by Finlay et al, 2004. As a result PDT under a higher fluence rate will stop at a higher oxygen concentration threshold. Oxygen concentration ascending showed in Figure 3 is owned to the oxygen diffusion in the model tumor. Though there is no reaction occurring, the light illuminating aggravates the oxygen diffusion to the inactive area.

 

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