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Tetra-sulfonate phthalocyanine zinc-bovine serum albumin conjugate-mediated photodynamic therapy of human glioma Dianshuang Xu, Xiangyu Chen, Ke'en Chen, Yiru Peng, Yingxin Li, Yiquan Ke and Danhui Gan J Biomater Appl published online 31 March 2014 DOI: 10.1177/0885328214529466 The online version of this article can be found at: http://jba.sagepub.com/content/early/2014/03/31/0885328214529466
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Tetra-sulfonate phthalocyanine zinc-bovine serum albumin conjugate-mediated photodynamic therapy of human glioma
Journal of Biomaterials Applications 0(0) 1–8 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328214529466 jba.sagepub.com
Dianshuang Xu1, Xiangyu Chen2, Ke’en Chen1, Yiru Peng3, Yingxin Li4, Yiquan Ke5 and Danhui Gan1
Abstract Background: Glioma is the most common brain malignancy with poor prognosis. The current treatments for gliomas are mainly based on surgery, chemotherapy, and radiotherapy, which exhibit limited efficacy. Photodynamic therapy (PDT) using photosensitizers has been applied to glioma therapy. However, different photosensitizers usually lead to different therapeutic effects and adverse reactions. Objective: This study investigates the anti-tumor effect of photosensitizer ZnPcS4-BSA in xenograft glioma tumors. Methods: The xenograft glioma tumor model was established by inoculating nude mice with U251 cells. Tumor growth was evaluated by tumor volume, weight, and inhibition rate. Cell apoptosis was evaluated using TUNEL staining. Vascular endothelial growth factor (VEGF) expression and microvessel density were measured by immunohistochemistry. Results: Significant decreases in tumor volume and weight as well as significant increases in tumor inhibition rate, cell apoptosis, VEGF expression, and microvessel density were observed in mice in the low- and high-dose PDT groups compared to the control, irradiation alone, and photosensitizer alone groups. No significant difference in cytotoxicity was observed between control group and photosensitizer alone group. Photosensitizer ZnPcS4-BSA significantly inhibited xenograft glioma tumor growth through induction of apoptosis. Conclusion: PDT using ZnPcS4-BSA may be effective for the therapy of gliomas. Keywords ZnPcS4-BSA, glioma, photodynamic therapy, apoptosis, VEGF
Introduction Overall, brain tumors are relatively rare among tumors, and gliomas account for over 50% of primary brain tumors. The annual number of brain tumor deaths in 2011 in the UK was 2688 men and 2287 women.1 At present, the treatment of glioma mainly involves a combination of surgery, chemotherapy, and radiotherapy.2 However, the survival rates for the majority of patients with malignant glioma remain disappointingly low. The five-year survival rate is under 1%.3 This is primarily because the infiltrative nature of glioma makes treatment difficult. In addition, multidrug resistance, radioresistance, a lack of preclinical models, and a rudimentary understanding of neurooncogenetics prevent effective treatment and improved patient survival.4
Developing new drugs for the therapy of glioma is still an important part of current brain tumor research.
1
Department of Neurosurgery, First Affiliated Hospital, Jinan University, Guangzhou, Guangdong, P.R. China 2 Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, Hunan, P.R. China 3 Fujian Provincial Key Laboratory of Polymer Materials, College of Chemistry & Materials Science, Fujian Normal University, Fuzhou, Fujian, P.R. China 4 Laser medicine laboratory, Tianjin Medical University, Tianjin, P.R. China 5 Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, P.R. China Corresponding author: Xiangyu Chen, Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, P.R. China. Email: [email protected]
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The traditional method of systemic administration is quite ineffective because most chemotherapy drugs are water-soluble and have high molecular mass, making crossing the blood-brain barrier (BBB) difficult.5 Fatsoluble preparations such as nitrosourea drugs can cross the BBB easily but have high systemic toxicity.6 Photodynamic therapy (PDT) is a promising treatment for various cancers and has been widely thought to be highly selective for tumors while having little to no influence on surrounding normal tissues.7 Although first-generation photosensitizers achieved great clinical success in the field of PDT, they are mainly suitable only for the treatment of superficial lesions because their absorption spectrum is around 630 nm, and light at this wavelength can penetrate tissue to a maximum depth of 5 mm.8,9 This approach is not suitable for deep-seated tumors such as gliomas. A promising group of second-generation photosensitizers for PDT are phthalocyanines. In general, phthalocyanines exhibit deeper effective tissue penetration because their suitable light absorption region is between 600 nm to 800 nm. However, most of these compounds visibly aggregate in solution making them insoluble in water.10,11 The water-soluble phthalocyanine derivative zinc phthalocyanine tetrasulfonate (ZnPcS4) has appropriate photobiological characteristics for PDT, but selfaggregation resulting from the large hydrophobic
skeleton of ZnPcS4 can occur in most vehicles, which reduces efficiency.12 ZnPcS4-BSA is a newly developed third generation photosensitizer.13 The role of ZnPcS4-BSA in vivo in glioma tumors has not been tested. This study investigated the effects of ZnPcS4-BSA and the associated mechanisms in xenograft U251 tumors.
Materials and methods Cell culture The U251 human glioma cell line was purchased from the Laboratory Animal Center of Sun Yat-sen University. U251 cells were cultured in RPMI-1640 medium containing 10% FBS at 4 C and 5% CO2. The photosensitizer ZnPcS4-BSA (Figure 1) was prepared as previously described.13 The ZnPcS4-BSA complex was filtered with 0.22 mm membrane before use.
Xenograft glioma animal models Thirty female nude mice (4–6 weeks old, 18 2 g weight) were purchased from the Laboratory Animal Center of Southern Medical University. Animal experiments were conducted by following an approved protocol from Central South University. Every effort was
HOOC HOOC
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Figure 1. The molecular structure of ZnPcS4-BSA.
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made to minimize the number of animals used. To establish xenograft glioma tumor models, 1 108 U251 cells in 0.2 ml of saline were injected into the left armpits of mice, and then tumor growth was observed. When the tumor size reached 0.6–0.8 cm in diameter, mice were randomly divided into five groups: control group receiving saline injection; irradiation alone group receiving laser irradiation (Semiconductor laser machine BHY-670-2, wavelength: 670 nm, dosage: 200 J/cm2); photosensitizer alone group receiving photosensitizer ZnPcS4-BSA by tail vein injection (2 mg/kg); low-dose PDT group receiving ZnPcS4-BSA at the dosage of 1 mg/kg followed by laser irradiation after 4 h of no light exposure and then no light exposure for 20 h; and high-dose PDT group receiving ZnPcS4-BSA at the dosage of 2 mg/kg followed by laser irradiation after 4 h of no light exposure, and then no light exposure for 20 h. The dosages used in this study were determined by following dosages used in a previously published report.13,14 The treatments in all five groups were repeated once a week for three weeks. The longest diameters (a) and shortest diameters (b) of the tumors were measured weekly. Tumor volume (V) was calculated as V ¼ ab2/2. Tumor mass was excised, and tumor weight was recorded. The tumor inhibition rate was calculated as: Tumor inhibition rate (%) ¼ (average tumor weight of control group average tumor weight of therapy group)/average tumor weight of control group 100%. Tumors were excised and fixed in 4% paraformaldehyde for 48 h.
Immunohistochemistry and hematoxylin-eosin staining Rabbit anti-mouse VEGF antibody was purchased from Santa Cruz biotechnology (San Diego, CA, USA), and ready to use SABC IHC staining kit was purchased from Beijing Bio-Lab Materials Institute (Beijing, China). The negative control was designed by replacing the primary antibody with 5% fetal bovine serum. Immunohistochemistry of VEGF expression was performed according to the manufacture’s protocol. Briefly, 4-mm thick sections were cut from routinely paraffin-embedded tissues. The sections were deparaffinized and then incubated with peroxidase inhibitor (3% H2O2) in the dark for 15 min. Next, the sections were incubated with rabbit anti-mouse VEGF primary antibody for 60 min followed by staining with the ready-to-use SABC IHC staining kit. The slides were dehydrated with different concentrations (70– 100%) of alcohol and soaked in xylene. Ten random fields were examined per section. The percent of positively stained cells relative to the total number of cells was determined in random fields at 200.
Hematoxylin-Eosin (HE) staining was performed as previously described.15 Microvessel density (MVD) was determined by immunohistochemical staining with anti-mouse CD34 antibody (BD Science, USA). Briefly, the sections were treated as described above. The sections were then incubated with rabbit anti-mouse CD34 primary antibody for 60 min at room temperature, followed by staining with the ready-to-use SABC IHC staining kit. Microvessels were counted in 10 random fields per section at 200 by two investigators unaware of the experiment design and mean MVD was calculated.
TUNEL staining and apoptosis index (AI) calculation Apoptosis in xenograft tumor tissues was analyzed using the TUNEL (fluorescence staining) method (Mbchem, Shanghai, China) according to the manufacturer’s instructions. Slides were photographed by epifluorescence microscopy (Zeiss, Axiovert S100). To calculate apoptosis index (AI), five random fields from each section and 500 cells in each field were observed under 400 magnification. AI was calculated as the percentage of positive cells with nuclear staining.
Statistical analysis Data were presented as mean standard deviation and analyzed using the statistical package for the Social Sciences Version 17.0 (SPSS 17.0). One-way analysis of variance was used to test differences between groups, and LSD or Dunnett T3 was used for pairwise comparison. A p < 0.05 was considered statistically significant.
Results ZnPcS4-BSA treatment significantly inhibited xenograft glioma tumor growth As shown in Table 1, tumor volume was significantly lower, tumor weight was significantly lighter, and tumor inhibition rate was significantly higher in lowand high-dose PDT group compared to the control group, irradiation alone group, and photosensitizer alone group. No statistical significance was observed in tumor volume, tumor weight, or tumor inhibition rate among control group, irradiation alone group, and photosensitizer alone group (p > 0.05). Also, no significant difference in tumor volume, tumor weight, or tumor inhibition rate was observed between highdose PDT and low-dose PDT group. HE staining showed that PDT decreased the density of tumor cells and increased necrosis (Figure 2). No significant
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Journal of Biomaterials Applications 0(0) Table 1. Tumor volume, weight, and inhibition rate of gliomas (X SEM). Group
Number of animals (n)
Volume (mm3)
Weight (g)
Inhibition rate (%)
Control Irradiation alone Photosensitizer alone Low-dose PDT High-dose PDT
6 6 6 6 6
1563.434 114.329 1559.675 212.394 1539.545 136.902 763.156 52.807* 527.276 75.899*
2.074 0.111 1.929 0.074 1.914 0.071 0.877 0.056* 0.682 0.083*
0.000 6.763 7.729 57.488* 67.150*
*p < 0.001 vs. control, irradiation alone, and photosensitizer alone group.
Figure 2. HE staining of glioma tissues. Original magnification 400. (a) U251 glioma tumor tissues without treatment. (b) U251 glioma tumor tissues treated with PDT. After PDT treatment, tumor cell density decreased, neuronal cell body size decreased, number of pyknotic nuclei increased, and necrosis increased.
abnormalities in liver and kidney functions were observed.
ZnPcS4-BSA treatment significantly increased VEGF expression and MVD in xenograft glioma tumors As shown in Table 2, VEGF expression and MVD in the low-dose and high-dose PDT groups were significantly higher than that in the control, irradiation alone, and photosensitizer alone groups. The VEGF expression (Figure 3) and MVD (Figure 4) in the high-dose PDT group were significantly higher than that in the low-dose PDT group. Correlation analysis showed that VEGF expression positively correlated with MVD level (r ¼ 0.999, p < 0.001). No significant differences in the expression of VEGF and MVD were observed among control, irradiation alone, and photosensitizer alone groups.
ZnPcS4-BSA treatment significantly increased apoptosis in xenograft glioma tumors As shown in Table 3, the apoptosis index (AI) of the low-dose and high-dose PDT groups was significantly
higher than that in the control, irradiation alone, and photosensitizer alone groups. The AI of the high-dose PDT group was significantly higher than that of the low-dose PDT group (Figure 5). No significant differences in the expression of VEGF and MVD were observed among control, irradiation alone, and photosensitizer alone groups.
Discussion Developing an effective strategy to treat glioma is still a challenge for both clinicians and researchers. ZnPcS4BSA is a newly developed photosensitizer with several advantages over previous generations of photosensitizers, including an excitation wavelength that is closer to red, action time that is shorter, and metabolism that is faster.13,16–18 However, its effect in glioma tumors has not been reported. In this study, we demonstrated that ZnPcS4-BSA injection followed by laser irradiation at 670 nm wavelength significantly inhibited tumor growth in a xenograft U251 glioma tumor model. The tumor growth inhibition exhibited a dose-dependent reaction to PDT. However, no obvious toxic effects were observed in mice injected with ZnPcS4-BSA alone.
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No. of animals
VEGF
MVD
Control Irradiation alone Photosensitizer alone Low-dose PDT High-dose PDT
6 6 6 6 6
15.073 0.321 15.288 0.285 15.450 0.243 24.847 0.368* 33.038 0.390*#
24.090 0.300 23.038 0.231 23.253 0.493 46.333 0.370* 59.377 0.404*#
*p < 0.001 vs. control, irradiation alone, and photosensitizer alone group. # p < 0.05 vs. low-dose PDT group.
Figure 3. Immunohistochemistry of VEGF expression. Original magnification 400. (a) VEGF expression in control glioma tumor tissues. (b) VEGF expression in glioma tumor tissues treated with PDT. Positive cells showed brown-sepia staining in the cytoplasm (indicated by arrow).
Figure 4. Microvessel density (MVD) in glioma tissues. Original magnification 200. (a) CD34 staining of glioma tumor tissues in control group. (b) CD34 staining of glioma tumor tissues with PDT. Endothelial cells or endothelial cell clusters showed brown staining.
The study further revealed that tumor growth inhibition was associated with the induction of cell apoptosis. This study first provided evidence that ZnPcS4-BSA injection followed by laser irradiation at 670 nm may be suitable for the therapy of gliomas.
PDT is widely believed to offer a more selective approach to the treatment of brain tumors compared to other available treatment modalities.19 In general, photosensitizers can accumulate in tumor cells to a greater extent than in normal glial or neuronal cells.20
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After activation by light, photosensitizers can induce selective damage to tumor cells through the generation of ROS.7 In this study, ZnPcS4-BSA treatment without laser radiation showed no effect on cell apoptosis, VEGF expression, MVD, and tumor growth compared to the controls without ZnPcS4-BSA treatment. In contrast, significant effects in tumor cell apoptosis and tumor angiogenesis were only observed in mice treated with ZnPcS4-BSA plus laser irradiation. This suggests that ZnPcS4-BSA itself did not produce any photodynamic reaction or cytotoxicity. Also, no systemic toxicities were observed in mice treated with ZnPcS4BSA or laser radiation. Therefore, the PDT established in this study could be used for the therapy of gliomas. PDT exerts an anti-tumor role through the generation of reactive oxygen species (ROS), such as singlet oxygen, oxygen free radical, hydroxyl free radical, and hydrogen.21 ROS are produced through photochemical reaction and energy transfer of photosensitizer excited by laser irradiation at a certain wavelength.
Table 3. Apoptosis index (AI) of gliomas (X SEM). Group
Number of animals AI (%)
Control Irradiation alone Photosensitizer alone Low-dose PDT High-dose PDT
6 6 6 6 6
1.33 0.33 1.58 0.33 1.52 0.04 12.76 0.14* 19.93 0.12*#
*p < 0.001 vs. control, irradiation alone, and photosensitizer alone group.
Elevated ROS levels can lead to apoptosis and necrosis,22–25 subsequently affecting tumor growth. In this study, we demonstrated that significant apoptosis was observed in both the low- and high-dose PDT groups compared to the control groups. VEGF is closely associated with glioma vascularization. However, our study showed significant increases in VEGF expression and the density of microvessels in tumors that received lowand high-dose PDT. The elevation of VEGF expression has been widely observed in previous PDT studies.26 For example, the study by Xie et al. showed that VEGF expression in xenograft human NPC tumors increased 24 h after administering 5-ALA-PDT, but decreased 14 days after treatment.27 The mechanism of PDT-induced VEGF upregulation was proposed to be associated with PDT-induced hypoxia.28,29 VEGF expression is also induced by PDT, suggesting both favorable and deleterious effects of PDT on tumor growth. The increase in VEGF expression and MVD may suggest a side effect of PDT on tumor recurrence post-treatment. We acknowledge that there are several weaknesses to the current study. First, this is a xenograft glioma tumor model, which cannot reflect the actual depth of glioma in human brains. However, the ideal animal model of glioma has not been widely established. One of the most well-known glioma animal models is the model established by subcutaneous and intracranial inoculation of U251 glioma cells.30 Moreover, previous studies using a haematoporphrin derivative demonstrated that optimum absorption between 628 and 632 nm allows penetration depths of up to 15 mm depending on the tissue and its vascularization.3 In this study, the optimum absorption of PDT established by ZnPcS4-BSA was 670 nm. This suggests that
Figure 5. Cell apoptosis in glioma tissues. Original magnification 200. (a) TUNEL staining of glioma tumor tissues in control group. (b) TUNEL staining of glioma tumor tissues treated with PDT. Positive cells showed brown staining in the nucleus (indicated by arrow).
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ZnPcS4-BSA could be applied to glioma treatment in humans. Second, the initial tumor size at treatment is only 0.6–0.8 cm in diameter. This may not reflect the real situation of glioma in humans. This may limit the effective application of PDT with ZnPcS4-BSA to relatively early stage glioma. Third, intracranial models may be closer to the nature of glioma in the human brain and can better reflect the important role of the BBB because ZnPcS4-BSA is a rather large molecule. However, the BBB does not exist in tumor regions and the surrounding edema. In contrast, this barrier is intact in the peritumorous area where tumor cells are embedded in normal brain.31 Therefore, subcutaneous models could effectively reflect the effects of molecular size.
Conclusion PDT using the photosensitizer ZnPcS4-BSA and laser irradiation at a wavelength of 670 nm induces significantly cytotoxic effects in xenograft glioma tumor cells through induction of apoptosis. PDT using ZnPcS4BSA also stimulates angiogenesis, which may suggest a side effect of PDT on tumor recurrence post-treatment. PDT using ZnPcS4-BSA may be an optional strategy for the therapy of gliomas. Funding This study was supported by the National Natural Science Foundation of China (30901774 to CH).
Conflict of interest None declared.
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Tetra-sulfonate phthalocyanine zinc-bovine serum albumin conjugate-mediated photodynamic therapy of human glioma Dianshuang Xu, Xiangyu Chen, Ke'en Chen, Yiru Peng, Yingxin Li, Yiquan Ke and Danhui Gan J Biomater Appl published online 31 March 2014 DOI: 10.1177/0885328214529466 The online version of this article can be found at: http://jba.sagepub.com/content/early/2014/03/31/0885328214529466
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Tetra-sulfonate phthalocyanine zinc-bovine serum albumin conjugate-mediated photodynamic therapy of human glioma
Journal of Biomaterials Applications 0(0) 1–8 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328214529466 jba.sagepub.com
Dianshuang Xu1, Xiangyu Chen2, Ke’en Chen1, Yiru Peng3, Yingxin Li4, Yiquan Ke5 and Danhui Gan1
Abstract Background: Glioma is the most common brain malignancy with poor prognosis. The current treatments for gliomas are mainly based on surgery, chemotherapy, and radiotherapy, which exhibit limited efficacy. Photodynamic therapy (PDT) using photosensitizers has been applied to glioma therapy. However, different photosensitizers usually lead to different therapeutic effects and adverse reactions. Objective: This study investigates the anti-tumor effect of photosensitizer ZnPcS4-BSA in xenograft glioma tumors. Methods: The xenograft glioma tumor model was established by inoculating nude mice with U251 cells. Tumor growth was evaluated by tumor volume, weight, and inhibition rate. Cell apoptosis was evaluated using TUNEL staining. Vascular endothelial growth factor (VEGF) expression and microvessel density were measured by immunohistochemistry. Results: Significant decreases in tumor volume and weight as well as significant increases in tumor inhibition rate, cell apoptosis, VEGF expression, and microvessel density were observed in mice in the low- and high-dose PDT groups compared to the control, irradiation alone, and photosensitizer alone groups. No significant difference in cytotoxicity was observed between control group and photosensitizer alone group. Photosensitizer ZnPcS4-BSA significantly inhibited xenograft glioma tumor growth through induction of apoptosis. Conclusion: PDT using ZnPcS4-BSA may be effective for the therapy of gliomas. Keywords ZnPcS4-BSA, glioma, photodynamic therapy, apoptosis, VEGF
Introduction Overall, brain tumors are relatively rare among tumors, and gliomas account for over 50% of primary brain tumors. The annual number of brain tumor deaths in 2011 in the UK was 2688 men and 2287 women.1 At present, the treatment of glioma mainly involves a combination of surgery, chemotherapy, and radiotherapy.2 However, the survival rates for the majority of patients with malignant glioma remain disappointingly low. The five-year survival rate is under 1%.3 This is primarily because the infiltrative nature of glioma makes treatment difficult. In addition, multidrug resistance, radioresistance, a lack of preclinical models, and a rudimentary understanding of neurooncogenetics prevent effective treatment and improved patient survival.4
Developing new drugs for the therapy of glioma is still an important part of current brain tumor research.
1
Department of Neurosurgery, First Affiliated Hospital, Jinan University, Guangzhou, Guangdong, P.R. China 2 Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, Hunan, P.R. China 3 Fujian Provincial Key Laboratory of Polymer Materials, College of Chemistry & Materials Science, Fujian Normal University, Fuzhou, Fujian, P.R. China 4 Laser medicine laboratory, Tianjin Medical University, Tianjin, P.R. China 5 Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, P.R. China Corresponding author: Xiangyu Chen, Department of Radiology, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, P.R. China. Email: [email protected]
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The traditional method of systemic administration is quite ineffective because most chemotherapy drugs are water-soluble and have high molecular mass, making crossing the blood-brain barrier (BBB) difficult.5 Fatsoluble preparations such as nitrosourea drugs can cross the BBB easily but have high systemic toxicity.6 Photodynamic therapy (PDT) is a promising treatment for various cancers and has been widely thought to be highly selective for tumors while having little to no influence on surrounding normal tissues.7 Although first-generation photosensitizers achieved great clinical success in the field of PDT, they are mainly suitable only for the treatment of superficial lesions because their absorption spectrum is around 630 nm, and light at this wavelength can penetrate tissue to a maximum depth of 5 mm.8,9 This approach is not suitable for deep-seated tumors such as gliomas. A promising group of second-generation photosensitizers for PDT are phthalocyanines. In general, phthalocyanines exhibit deeper effective tissue penetration because their suitable light absorption region is between 600 nm to 800 nm. However, most of these compounds visibly aggregate in solution making them insoluble in water.10,11 The water-soluble phthalocyanine derivative zinc phthalocyanine tetrasulfonate (ZnPcS4) has appropriate photobiological characteristics for PDT, but selfaggregation resulting from the large hydrophobic
skeleton of ZnPcS4 can occur in most vehicles, which reduces efficiency.12 ZnPcS4-BSA is a newly developed third generation photosensitizer.13 The role of ZnPcS4-BSA in vivo in glioma tumors has not been tested. This study investigated the effects of ZnPcS4-BSA and the associated mechanisms in xenograft U251 tumors.
Materials and methods Cell culture The U251 human glioma cell line was purchased from the Laboratory Animal Center of Sun Yat-sen University. U251 cells were cultured in RPMI-1640 medium containing 10% FBS at 4 C and 5% CO2. The photosensitizer ZnPcS4-BSA (Figure 1) was prepared as previously described.13 The ZnPcS4-BSA complex was filtered with 0.22 mm membrane before use.
Xenograft glioma animal models Thirty female nude mice (4–6 weeks old, 18 2 g weight) were purchased from the Laboratory Animal Center of Southern Medical University. Animal experiments were conducted by following an approved protocol from Central South University. Every effort was
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Figure 1. The molecular structure of ZnPcS4-BSA.
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made to minimize the number of animals used. To establish xenograft glioma tumor models, 1 108 U251 cells in 0.2 ml of saline were injected into the left armpits of mice, and then tumor growth was observed. When the tumor size reached 0.6–0.8 cm in diameter, mice were randomly divided into five groups: control group receiving saline injection; irradiation alone group receiving laser irradiation (Semiconductor laser machine BHY-670-2, wavelength: 670 nm, dosage: 200 J/cm2); photosensitizer alone group receiving photosensitizer ZnPcS4-BSA by tail vein injection (2 mg/kg); low-dose PDT group receiving ZnPcS4-BSA at the dosage of 1 mg/kg followed by laser irradiation after 4 h of no light exposure and then no light exposure for 20 h; and high-dose PDT group receiving ZnPcS4-BSA at the dosage of 2 mg/kg followed by laser irradiation after 4 h of no light exposure, and then no light exposure for 20 h. The dosages used in this study were determined by following dosages used in a previously published report.13,14 The treatments in all five groups were repeated once a week for three weeks. The longest diameters (a) and shortest diameters (b) of the tumors were measured weekly. Tumor volume (V) was calculated as V ¼ ab2/2. Tumor mass was excised, and tumor weight was recorded. The tumor inhibition rate was calculated as: Tumor inhibition rate (%) ¼ (average tumor weight of control group average tumor weight of therapy group)/average tumor weight of control group 100%. Tumors were excised and fixed in 4% paraformaldehyde for 48 h.
Immunohistochemistry and hematoxylin-eosin staining Rabbit anti-mouse VEGF antibody was purchased from Santa Cruz biotechnology (San Diego, CA, USA), and ready to use SABC IHC staining kit was purchased from Beijing Bio-Lab Materials Institute (Beijing, China). The negative control was designed by replacing the primary antibody with 5% fetal bovine serum. Immunohistochemistry of VEGF expression was performed according to the manufacture’s protocol. Briefly, 4-mm thick sections were cut from routinely paraffin-embedded tissues. The sections were deparaffinized and then incubated with peroxidase inhibitor (3% H2O2) in the dark for 15 min. Next, the sections were incubated with rabbit anti-mouse VEGF primary antibody for 60 min followed by staining with the ready-to-use SABC IHC staining kit. The slides were dehydrated with different concentrations (70– 100%) of alcohol and soaked in xylene. Ten random fields were examined per section. The percent of positively stained cells relative to the total number of cells was determined in random fields at 200.
Hematoxylin-Eosin (HE) staining was performed as previously described.15 Microvessel density (MVD) was determined by immunohistochemical staining with anti-mouse CD34 antibody (BD Science, USA). Briefly, the sections were treated as described above. The sections were then incubated with rabbit anti-mouse CD34 primary antibody for 60 min at room temperature, followed by staining with the ready-to-use SABC IHC staining kit. Microvessels were counted in 10 random fields per section at 200 by two investigators unaware of the experiment design and mean MVD was calculated.
TUNEL staining and apoptosis index (AI) calculation Apoptosis in xenograft tumor tissues was analyzed using the TUNEL (fluorescence staining) method (Mbchem, Shanghai, China) according to the manufacturer’s instructions. Slides were photographed by epifluorescence microscopy (Zeiss, Axiovert S100). To calculate apoptosis index (AI), five random fields from each section and 500 cells in each field were observed under 400 magnification. AI was calculated as the percentage of positive cells with nuclear staining.
Statistical analysis Data were presented as mean standard deviation and analyzed using the statistical package for the Social Sciences Version 17.0 (SPSS 17.0). One-way analysis of variance was used to test differences between groups, and LSD or Dunnett T3 was used for pairwise comparison. A p < 0.05 was considered statistically significant.
Results ZnPcS4-BSA treatment significantly inhibited xenograft glioma tumor growth As shown in Table 1, tumor volume was significantly lower, tumor weight was significantly lighter, and tumor inhibition rate was significantly higher in lowand high-dose PDT group compared to the control group, irradiation alone group, and photosensitizer alone group. No statistical significance was observed in tumor volume, tumor weight, or tumor inhibition rate among control group, irradiation alone group, and photosensitizer alone group (p > 0.05). Also, no significant difference in tumor volume, tumor weight, or tumor inhibition rate was observed between highdose PDT and low-dose PDT group. HE staining showed that PDT decreased the density of tumor cells and increased necrosis (Figure 2). No significant
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Journal of Biomaterials Applications 0(0) Table 1. Tumor volume, weight, and inhibition rate of gliomas (X SEM). Group
Number of animals (n)
Volume (mm3)
Weight (g)
Inhibition rate (%)
Control Irradiation alone Photosensitizer alone Low-dose PDT High-dose PDT
6 6 6 6 6
1563.434 114.329 1559.675 212.394 1539.545 136.902 763.156 52.807* 527.276 75.899*
2.074 0.111 1.929 0.074 1.914 0.071 0.877 0.056* 0.682 0.083*
0.000 6.763 7.729 57.488* 67.150*
*p < 0.001 vs. control, irradiation alone, and photosensitizer alone group.
Figure 2. HE staining of glioma tissues. Original magnification 400. (a) U251 glioma tumor tissues without treatment. (b) U251 glioma tumor tissues treated with PDT. After PDT treatment, tumor cell density decreased, neuronal cell body size decreased, number of pyknotic nuclei increased, and necrosis increased.
abnormalities in liver and kidney functions were observed.
ZnPcS4-BSA treatment significantly increased VEGF expression and MVD in xenograft glioma tumors As shown in Table 2, VEGF expression and MVD in the low-dose and high-dose PDT groups were significantly higher than that in the control, irradiation alone, and photosensitizer alone groups. The VEGF expression (Figure 3) and MVD (Figure 4) in the high-dose PDT group were significantly higher than that in the low-dose PDT group. Correlation analysis showed that VEGF expression positively correlated with MVD level (r ¼ 0.999, p < 0.001). No significant differences in the expression of VEGF and MVD were observed among control, irradiation alone, and photosensitizer alone groups.
ZnPcS4-BSA treatment significantly increased apoptosis in xenograft glioma tumors As shown in Table 3, the apoptosis index (AI) of the low-dose and high-dose PDT groups was significantly
higher than that in the control, irradiation alone, and photosensitizer alone groups. The AI of the high-dose PDT group was significantly higher than that of the low-dose PDT group (Figure 5). No significant differences in the expression of VEGF and MVD were observed among control, irradiation alone, and photosensitizer alone groups.
Discussion Developing an effective strategy to treat glioma is still a challenge for both clinicians and researchers. ZnPcS4BSA is a newly developed photosensitizer with several advantages over previous generations of photosensitizers, including an excitation wavelength that is closer to red, action time that is shorter, and metabolism that is faster.13,16–18 However, its effect in glioma tumors has not been reported. In this study, we demonstrated that ZnPcS4-BSA injection followed by laser irradiation at 670 nm wavelength significantly inhibited tumor growth in a xenograft U251 glioma tumor model. The tumor growth inhibition exhibited a dose-dependent reaction to PDT. However, no obvious toxic effects were observed in mice injected with ZnPcS4-BSA alone.
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No. of animals
VEGF
MVD
Control Irradiation alone Photosensitizer alone Low-dose PDT High-dose PDT
6 6 6 6 6
15.073 0.321 15.288 0.285 15.450 0.243 24.847 0.368* 33.038 0.390*#
24.090 0.300 23.038 0.231 23.253 0.493 46.333 0.370* 59.377 0.404*#
*p < 0.001 vs. control, irradiation alone, and photosensitizer alone group. # p < 0.05 vs. low-dose PDT group.
Figure 3. Immunohistochemistry of VEGF expression. Original magnification 400. (a) VEGF expression in control glioma tumor tissues. (b) VEGF expression in glioma tumor tissues treated with PDT. Positive cells showed brown-sepia staining in the cytoplasm (indicated by arrow).
Figure 4. Microvessel density (MVD) in glioma tissues. Original magnification 200. (a) CD34 staining of glioma tumor tissues in control group. (b) CD34 staining of glioma tumor tissues with PDT. Endothelial cells or endothelial cell clusters showed brown staining.
The study further revealed that tumor growth inhibition was associated with the induction of cell apoptosis. This study first provided evidence that ZnPcS4-BSA injection followed by laser irradiation at 670 nm may be suitable for the therapy of gliomas.
PDT is widely believed to offer a more selective approach to the treatment of brain tumors compared to other available treatment modalities.19 In general, photosensitizers can accumulate in tumor cells to a greater extent than in normal glial or neuronal cells.20
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After activation by light, photosensitizers can induce selective damage to tumor cells through the generation of ROS.7 In this study, ZnPcS4-BSA treatment without laser radiation showed no effect on cell apoptosis, VEGF expression, MVD, and tumor growth compared to the controls without ZnPcS4-BSA treatment. In contrast, significant effects in tumor cell apoptosis and tumor angiogenesis were only observed in mice treated with ZnPcS4-BSA plus laser irradiation. This suggests that ZnPcS4-BSA itself did not produce any photodynamic reaction or cytotoxicity. Also, no systemic toxicities were observed in mice treated with ZnPcS4BSA or laser radiation. Therefore, the PDT established in this study could be used for the therapy of gliomas. PDT exerts an anti-tumor role through the generation of reactive oxygen species (ROS), such as singlet oxygen, oxygen free radical, hydroxyl free radical, and hydrogen.21 ROS are produced through photochemical reaction and energy transfer of photosensitizer excited by laser irradiation at a certain wavelength.
Table 3. Apoptosis index (AI) of gliomas (X SEM). Group
Number of animals AI (%)
Control Irradiation alone Photosensitizer alone Low-dose PDT High-dose PDT
6 6 6 6 6
1.33 0.33 1.58 0.33 1.52 0.04 12.76 0.14* 19.93 0.12*#
*p < 0.001 vs. control, irradiation alone, and photosensitizer alone group.
Elevated ROS levels can lead to apoptosis and necrosis,22–25 subsequently affecting tumor growth. In this study, we demonstrated that significant apoptosis was observed in both the low- and high-dose PDT groups compared to the control groups. VEGF is closely associated with glioma vascularization. However, our study showed significant increases in VEGF expression and the density of microvessels in tumors that received lowand high-dose PDT. The elevation of VEGF expression has been widely observed in previous PDT studies.26 For example, the study by Xie et al. showed that VEGF expression in xenograft human NPC tumors increased 24 h after administering 5-ALA-PDT, but decreased 14 days after treatment.27 The mechanism of PDT-induced VEGF upregulation was proposed to be associated with PDT-induced hypoxia.28,29 VEGF expression is also induced by PDT, suggesting both favorable and deleterious effects of PDT on tumor growth. The increase in VEGF expression and MVD may suggest a side effect of PDT on tumor recurrence post-treatment. We acknowledge that there are several weaknesses to the current study. First, this is a xenograft glioma tumor model, which cannot reflect the actual depth of glioma in human brains. However, the ideal animal model of glioma has not been widely established. One of the most well-known glioma animal models is the model established by subcutaneous and intracranial inoculation of U251 glioma cells.30 Moreover, previous studies using a haematoporphrin derivative demonstrated that optimum absorption between 628 and 632 nm allows penetration depths of up to 15 mm depending on the tissue and its vascularization.3 In this study, the optimum absorption of PDT established by ZnPcS4-BSA was 670 nm. This suggests that
Figure 5. Cell apoptosis in glioma tissues. Original magnification 200. (a) TUNEL staining of glioma tumor tissues in control group. (b) TUNEL staining of glioma tumor tissues treated with PDT. Positive cells showed brown staining in the nucleus (indicated by arrow).
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ZnPcS4-BSA could be applied to glioma treatment in humans. Second, the initial tumor size at treatment is only 0.6–0.8 cm in diameter. This may not reflect the real situation of glioma in humans. This may limit the effective application of PDT with ZnPcS4-BSA to relatively early stage glioma. Third, intracranial models may be closer to the nature of glioma in the human brain and can better reflect the important role of the BBB because ZnPcS4-BSA is a rather large molecule. However, the BBB does not exist in tumor regions and the surrounding edema. In contrast, this barrier is intact in the peritumorous area where tumor cells are embedded in normal brain.31 Therefore, subcutaneous models could effectively reflect the effects of molecular size.
Conclusion PDT using the photosensitizer ZnPcS4-BSA and laser irradiation at a wavelength of 670 nm induces significantly cytotoxic effects in xenograft glioma tumor cells through induction of apoptosis. PDT using ZnPcS4BSA also stimulates angiogenesis, which may suggest a side effect of PDT on tumor recurrence post-treatment. PDT using ZnPcS4-BSA may be an optional strategy for the therapy of gliomas. Funding This study was supported by the National Natural Science Foundation of China (30901774 to CH).
Conflict of interest None declared.
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