摘要
光动力疗法(Photodynamic therapy, PDT)的基本原理是光敏剂经特定波长的激光(或其它光源)激发后发生光化学反应,产生多种活性氧物质(Reactive oxygen species, ROS),包括单线态氧、超氧阴离子、羟自由基及过氧化氢等具有细胞杀伤作用。它的主要优点是选择性、不可逆的毁坏疾病组织的同时,并不损害周围的正常细胞。现有的PDT使用的光敏剂存在许多缺点,如在红光区的吸收波长短、活性氧的产量低、易产生副反应等。其中,瓶颈在于光敏剂的光谱吸收范围普遍在650 nm以下的可见光区,光源只能穿透毫米级的软组织深度,因此无法用于普遍意义的肿瘤治疗。
二氢卟吩e6(Chlorin e6, Ce6)是一种很有潜力的光敏剂。它具备663nm的吸收波长,能够产生更多的ROS,而且对皮肤的副反应也较小,因此适合用于肿瘤光动力治疗。但是Ce6水溶性差,因此限制了其在临床的开发应用。为了克服这一问题,文献报道亲水性大分子聚合物如聚乙烯吡咯烷酮(Poly(vinyl pyrrolidone), PVP)、聚乙二醇(Poly(ethylene glycol), PEG)和多肽等经常被用来提高Ce6的溶解度。但是不幸的是,这些方法的药物运载量(Drug loading content, DLC)不到15%。
单壁碳纳米管(Single wall carbon nanotubes, SWCNTs)具有大共轭结构的类石墨表面,能够运载大量的药物分子。因此,本课题采用SWCNTs作为药物载体,来构建载药量大的药物输送系统。我们构建的壳聚糖-二氢卟吩e6-单壁碳纳米管(Chitosan-Chlorin e6- SWCNTs, Chitosan-Ce6-SWCNTs)药物输送系统的载药量为128%。由于具有大π键结构,SWCNTs可以通过π-π堆积等非共价相互作用吸附具有大π键结构的Ce6,从而制备出Ce6-SWCNTs复合物。壳聚糖分子链含有大量的氨基(-NH2)和羟基(-OH),能够通过非共价相互作用缠绕在Ce6-SWCNTs表面,增加其亲水性,最终获得水溶性好、载药量大的药物输送系统。通过细胞生物学实验表明:Chitosan-Ce6-SWCNTs比单纯的Ce6更易被细胞摄取,对HeLa细胞的杀伤效果也较显著。
Photodynamic therapy (PDT) in tumors is a relatively new treatment, which involves the activation of the molecular oxygen under irradiation by light in the presence of certain photodrugs (photosensitizers) that have been selectively accumulated in the target tissue. In comparison with surgery, chemotherapy, radiation and other conventional treatment methods, PDT is considered to be a clinical treatment with high safety, few side effects, reliable repeatability, and relatively low cost. PDT relies on the application of a photosensitizer which is activated by light. The activated sensitizer reacts with the oxygen present in the tissue, which forms highly toxic radicals and then induces tissue necrosis/apoptosis. Thus photosensitizer is one of the main factors in determining the efficiency of PDT. Despite progressive development in photosensitizer, a few problems still exist, such as relatively weak absorption in the red region of visible light, poor water-solubility, and high dark toxicity. The absorption range of most photosensitizers is below 650 nm, which seriously hinders their clinical application.
Benefiting from the strong absorption band in the red light region, Chlorin e6 (Ce6) emerges as a prospective photosensitizer for PDT. Ce6 exhibits a strong absorption peak at a wavelength of 663 nm, low cytotoxicity, and almost no side effect to skin. Furthermore, it can reach deeper penetration of tissue and produce higher reactive oxygen species (ROS). For an efficient PDT, photosensitizer must travel through the blood stream with high water-stability. However, Ce6 is a hydrophobic drug and insoluble in water, which greatly reduces its PDT effects. To overcome this problem, hydrophilic macromolecules such as poly(vinyl pyrrolidone) (PVP), poly(ethylene glycol) (PEG) and various polypeptides were frequently used to improve the solubility of Ce6. Unfortunately, the drug loading content (DLC) of these approaches was lower than 15%. Furthermore, the cancer-killing effect of these complexes was usually lower than that of free Ce6. Therefore, an ideal Ce6 delivery system with good water-solubility and high DLC is still required.
In this study, an efficient Ce6 delivery system with 128% DLC was developed based on the noncovalent interactions between Ce6 and single wall carbon nanotubes (SWCNTs). Owing to nanoscale diameters, SWCNTs show a very large specific surface area, which provides the opportunity for the construction of excellent drug delivery systems with satisfied drug loading capacities. Considering the existence of large aromatic conjugation structures of Ce6 and SWCNTs, Ce6 was adsorbed onto SWCNTs by strong noncovalentπ-πinteractions with a high efficiency. Chitosan molecules chains contain a lot of amino (-NH2) and hydroxyl (-OH) groups, which can increase the water-solubility of SWCNTs. Therefor, the low molecular weight chitosan was chosen to wrap the Ce6-SWCNT complexes by noncovalentπ-πinteractions to gain water-soluble drug delivery system. The biological evaluation showed that chitosan-wrapped Ce6-SWCNT complexes (Chitosan-Ce6-SWCNTs) exhibited better cell uptake and a higher anticancer effect against HeLa cells than free Ce6.
引文
[1]丁新民,徐勤枝,顾瑛等.光动力治疗肿瘤的简史和现状.中国肿瘤2003; 12: 151-4.
[2] Triesscheijn M, Baas P, Schellens JH, et al. Photodynamic therapy in oncology. Oncologist. 2006; 11: 1034-44.
[3] Mori M, Kurado T, Obana A, et al. In vitro plasma protein binding and cellular uptake of ATX-S10(Na), a hydrophilic chlorin photosensitizer. Cancer Res 2000; 91:835-52.
[4] Engbrecht BW, Menon C, Kachur AV, et al. Photofrin-mediated photodynamic therapy induces vascular occlusion and apoptosis in a human sarcoma xenograft model. Cancer Res. 1999; 59: 4334-42.
[5] Webber J, Herman M, Kesse D, et al. Photodynamic treatment of neoplastic lesions of the gastrointestinal tract: Recent advances in techniques and results. Lang Arch Surg 2000; 385: 299-303.
[6] Oleinick NL, Evans HH. The photodynamic therapy: celluar tergets and mechanisms. Radiat Res 1998; 150: 146-56.
[7]沈彬,刘樑,赵承梅等.肿瘤光动力疗法研究进展. 2005; 19: 32-7.
[8] Star WM. Light dosimetry in vivo. Phys Med Biol 1997; 32: 763-87.
[9] Svaasand LO, Ellingsen R. Optical penetration in human intracranical tumors. Photochem Photobiol 1985; 31: 73-6.
[10] Whitehurst C, Pantelides ML, Moore JV et al. In vivo laster light disribution in human prostatic carcinoma. J Urol 1993; 151: 1411-5.
[11] Rungta A, Zheng G, Missert JR, et al. Purpurinimides as photosensitizers:effect of the presence and position of the substituents in the in vivo photodynamic efficacy. Bioorg Med Chem Lett 2000; 10: 1463-6.
[12] Walker I, Vernon DT, Brown SB. The solid-phase conjugation of purpurin-18 with a synthetic targeting peptide. Bioorg Med Chem Lett 2004; 14: 441-3.
[13]黄卓正.肿瘤光动力疗法的光敏剂研究进展.医学文选2000; 19: 955-8.
[13]许德余,张洁,陈文晖等.光动力治癌新药血卟啉单甲醚的研究.中国激光医学杂志1993; 2: 2-7.
[15] Mang TS, Allison R, Hewson G, et al. A phase II/III clinical study of tinethy letiopurpurin (Purlytin) induced photodynamic therapy for the treatment of recurrent cutaneous metastatic breast cancer. Cancer Sci Am 1998; 3: 378-83.
[16] Kaplan MJ, Somers RG, Greenberg RH, et al. Photodynamic therapy in the management of metastatic cutaneous adenocarcinomas: case reports from phase 1/2 studies using tin ethyletiopurpurin (SnET2). Surg Oncol 1998; 67: 121-5.
[17] Gossner L. Photodynamic therapy of early epithelial carcinomas and severe squamous epithelial dysplasias of the esophagus with 5-aminolevulinic acid. Z Gastroenterol 1998; 36:19-26.
[18]余建鑫.研究开发中的新型光动力治癌药物苯并卟啉衍生物单环酸A .中国激光医学杂志1998; 7 : 38-41.
[19] Richter AM, Waterfield E, Jain AK, et al. In viteo evaluation of phototoxic properties of four structurally related bebxopophyrin datives. Photochem Photobiol 1990; 52: 495-501.
[20] Morgen AR, Garbo GM, Keck RW, et al. Diels-Alder adducts of vinyl porphyrins: synthesis and in vivo photodynamic effect against a rat bladder tumor. J Med Chem 1990; 33: 1258-62.
[21] Lui H, Hruza L, Kallias N, et al. Prelininary studies on cutaneous photosensitivity in patients undergoing photodynamic therapy with benzoporphyrin derivative. Photochem photobiol 1993; 57: 190-7.
[22]俞开潮,程红,金玲.光动力治疗用酞菁类光敏剂的合成研究进展.感光科学与光化学2003, 21: 138-46.
[23]邬旭,徐基铨,陈瑱.应用三磺酸铝酞菁进行实验性光动力治疗和荧光定位诊断的初步观察.中国激光医学杂志1993; 3: 159-63.
[24]傅乃武,刘朝阳,燕利学.磺化铝酞菁光动力治疗合并高温对小鼠肝癌治疗的协同作用.中国激光医学杂志1996; 5: 27-30.
[25] Reddi E, Zhou C, Biolo R, et al. Liposome- or LDL-administered Zn(II)-phthalocyanine as a photodynamic agent for tumours. I. Pharmacokinetic properties and phototherapeutic efficiency. Br J Cancer. 1990; 61: 407-11.
[26] Milanesi C, Zhou CN, Biolo R, et al. Zn (II) -phthalocyanine as a photodynamic agent for tumors II . Studies on the mechanism of photosensitized tumor necrosis. Br J Cancer 1990; 61: 846-50.
[27]黄金陵,黄剑东,刘尔生等.酞菁配合物的结构与其光动力抗癌活性.物理化学学报2001; 17: 662-71.
[28]周世文,陈军.竹红菌素类光敏剂的生物学作用.激光杂志2001; 23: 58-60.
[29] Zhang WG. Antisense bcl-retrovirus increase the sensitivity of a human gastric adenocarcinoma cell line to photodynamic therapy. Photochem Photobiol 1999; 69: 582-6.
[30]陈祖林,唐建民,单治堂等.维拉帕米在大肠癌光动力学中增效机理的实验研究.中华消化杂志1998; 18: 96-8.
[31]黄蕾,赵福群,黄新等.光免疫偶联物的研究进展.化学进展2007; 19: 527-34.
[32]李平平,周国瑜,沈玲悦等.二氢卟酚纳米光敏剂对CAL-27细胞的光动力杀伤效应.中国口腔颌面外科杂志2011; 9: 296-300.
[33] Chin WW, Lau WK, Bhuvaneswari R, et al. Chlorin e6-polyvinylpyrrolidone as a ?uorescent marker for ?uorescence diagnosis of human bladder cancer implanted on the chick chorioallantoic membrane model. Cancer Lett 2007; 235: 127-33.
[33] Duncan R. The dawning era of polymer therapeutics. Nat Rev Drug Discov 2003; 5: 347-60.
[35] Chin WW, Lau WK, Heng PW, et al. Fluorescence imaging and phototoxicity effects of new formulation of chlorin e6-polyvinylpyrrolidone. J Photochem Photobiol B: Biol 2006; 83: 103-10.
[36] Bisland SK, Singh D, Gariépy J. Potentiation of chlorin e6 photodynamic activity in vitro with peptide-based intracellular vehicles. Bioconjugate Chem 1999; 10: 982-92.
[37] Hamblin MR, O’Donnell DA, Murthy N, et al. Polycationic photosensitizerconjugates: effects of chain length and gram classification on the photodynamic inactivation of bacteria. J Anti Chem 2002; 39: 941-51.
[38] Garcia G, Sol V, Lamarche F, et al. Synthesis and photocytotoxic activity of new chlorin-polyamine conjugates. Bioorg. Med. Chem. Lett. 2006; 16: 3188-92.
[39] Liu Z, Tabakman S, Welsher K, et al. Carbon nanotubes in biology and medicine: in vitro and in vivo detection, imaging and drug delivery. Nano Res 2009; 2 : 85-120.
[30] Zhang X, Meng L, Lu Q, Fei Z, Dyson PJ. Targeted delivery and controlled release of doxorubicin to cancer cells using modi?ed single wall carbon nanotubes. Biomaterials 2009; 30: 6041-7.
[31] Duong HM, Papavassiliou DV, Mullen KJ, et al. A numerical study on the effective thermal conductivity of biological fluids containing single walled carbon nanotubes. Int J Heat Mass Transfer 2009; 52: 5591-7.
[32] Inhet Panhuis M. Vaccine delivery by carbon nanotubes. Chem Biol 2003; 10: 897-8.
[33] Chen J, Chen S, Zhao X, Kuznetsova LV, Wong SS, Ojima I. Functionalized single-walled carbon nanotubes as rationally designed vehicles for tumor-targeted drug delivery. J Am Chem Soc 2008; 130: 16778-85.
[33] Liu Z, Davis C, Cai W, et al. Circulation and long term fate of functionalized, biocompatible single-walled carbon nanotubes in mice probed by raman spectroscopy. Proc Natl Acad Sci 2008; 105: 1410-5.
[35] Baskaran D, Mays JW, Bratcher MS. Noncovalent and nonspecific molecular interactions of polymers with multiwalled carbon nanotubes. Chem Mater 2005; 17: 3389-97.
[36] Plata DL, Gschwend PM, Reddy CM. Industrially synthesized single-walled carbon nanotubes: compositional data for users, environmental risk assessments, and source apportionment. Nanotech 2008; 19: 185706.
[37] Zhao HY, Chen C, Zhu YZ, et al. Synthesis and spectroscopic properties of functionalized single-walled carbon nanotubes with porphyrins and phthalocyanines. Chem J Chi Univ 2010; 31: 1805-10.
[1] Triesscheijn M, Baas P, Schellens JH, et al. Photodynamic therapy in oncology. Oncologist. 2006; 11: 1034-44.
[2] Mori M, Kurado T, Obana A, et al. In vitro plasma protein binding and cellular uptake of ATX-S10(Na), a hydrophilic chlorin photosensitizer. Cancer Res 2000; 91: 835-52.
[3] Engbrecht BW, Menon C, Kachur AV, et al. Photofrin-mediated photodynamic therapy induces vascular occlusion and apoptosis in a human sarcoma xenograft model. Cancer Res 1999; 59: 4334-42.
[4] Webber J, Herman M, Kesse D, et al. Photodynamic treatment of neoplastic lesions of the gastrointestinal tract: Recent advances in techniques and results. Lang Arch Surg 2000; 385: 299-303.
[5] Oleinick NL, Evans HH. The photodynamic therapy: celluar tergets and mechanisms. Radiat Res 1998; 150: 146-56.
[6] Dougherty TJ, Gomer CJ, Henderson BW, et al. Photodynamic therapy. Natl Cancer Inst 1998; 90: 889-905.
[7]黄光武,李革.光动力疗法对肿瘤诊疗的研究进展.中国医学文摘2003; 17: 166-70.
[8] Roberts WG, Shian FY, Nelson JS, et a1.In vitro character of monoaspartyl chlorin e6 and diaspartyl chlorin e6 for photodynamic therapy.J Nat Cancer Ins 1988; 80: 330-6.
[9]金晓敏,吴健.卟啉类光敏药物的研究进展.中国药物化学杂志2002; 12: 52-6.
[10] Liu Z, Tabakman S, Welsher K, et al. Carbon nanotubes in biology and medicine: in vitro and in vivo detection, imaging and drug delivery. Nano Res 2009; 2 : 85-120.
[11] Liu Z, Sun XM, Nakayama-Ratchford N, et al. Supramolecular chemistry on water-soluble carbonnanotubes for drug loading and delivery. ACS Nano 2007; 1: 50.
[12] Prato M, Kostarelos K, Bianco A. Functionalized carbon nanotubes in drug designand discovery. Acc Chem Res 2008; 31: 60-8.
[13] Debbage P. Targeted drugs and nanomedicine: present and future. Curr Pharml Design 2009; 15: 153-72.
[13] Bianco A, Kostarelos K, Prato M. Applications of carbon nanotubes in drug delivery. Curr Opin Chem Biol 2005; 9: 674-9.
[15] Klumpp C, Kostarelos K, Prato M, Bianco A.Functionalized carbon nanotubes as emerging nanovectors for the delivery of therapeutics. Biochi Biophy Acta Biomem 2006; 1758: 404-12.
[16] Lacerda L, Raffa S, Prato M, et al. Cell-penetrating CNTs for delivery of therapeutics. Nano Today 2007; 2: 38-43.
[17] Shiba K. Functionalization of carbon nanomaterials by evolutionary molecular engineering: Potential application in drug delivery systems. J Drug Targ 2006; 13: 512-8.
[18] Biju V, Muraleedharan D, Nakayama K, et al. Quantum dot-insect neuropeptide conjugates for fluorescence imaging, transfection, and nucleus targeting of living cells. Langmuir 2007; 23: 10254-61.
[19] Pantarotto D, Partidos CD, Graff R, et al. Synthesis, structural characterization, and immunological properties of carbon nanotubes functionalized with peptides. J Amer Chem Soc 2003; 125: 5650-1.
[20] Pantarotto D, Briand JP, Prato M, et al. Translocation of bioactive peptides across cell membranes by carbon nanotubes. Chem Commun 2003; 1:16-7.
[21] Hilder TA, Hill JM. Carbon nanotubes as drug delivery nanocapsules. Curr Appl Phys 2008; 8: 258-61.
[22] Bhirde AA, Patel V, Gavard J, et al. Targeted killing of cancer cells in vivo and in vitro with EGF-directed carbon nanotube-based drug delivery. Acs Nano 2009; 3: 307-16.
[23] Britz DA, Khlobystov AN. Noncovalent interactions of molecules with single walled carbon nanotubes. Chem Soc Rev 2006; 35: 637-59.
[24] Plata DL, Gschwend PM, Reddy CM. Industrially synthesized single-walledcarbon nanotubes: compositional data for users, environmental risk assessments, and source apportionment. Nanotech 2008; 19: 185706.
[25] Zhao HY, Chen C, Zhu YZ, et al. Synthesis and Spectroscopic properties of functionalized single-walled carbon nanotubes with porphyrins and phthalocyanines. Chem J Chi Univ 2010; 31: 1805-10.
[26] Li QX, Song BZ, Yang ZQ, et al. Preparation and characterization of chitosan microcapsules crosslinked by vanillin. Chinese J Pro Eng 2006; 6: 608-13.
[27] Zhang XK, Meng LJ, Wang XF, et al.Preparation and cellular uptake of pH-dependent fluorescent single-wall carbon nanotubes. Chem Eur 2010; 16: 556 -61.
[28] Zhang LW, Monteiro-Riviere NA. Lectins modulate multi-walled carbon nanotubes cellular uptake in human epidermal keratinocytes. Toxicol 2010; 23: 546 -51.
[29] Alizadeh D, Zhang, LY, Hwang, J, et al. Tumor-associated macrophages are predominant carriers of cyclodextrin-based nanoparticles into gliomas. Nano medicine-Nanotech Biology Medicine 2010; 6: 382-90.
[30] Zhang H, Guo H, Deng X, et al.Functionalization of multi-walled carbon nanotubes via surface unpaired electrons. Nanotech 2010; 21: 85706.
[31] Brown DM, Kinloch IA, Bangert U, et al. An in vitro study of the potential of carbon nanotubes and nanofibers to induce inflammatory mediators and frustrated phagocytosis. Carbon 2007; 6: 1743-56.
[32] Star A, Steuerman DW, Heath JR, et al. Starched carbon nanotubes. Adv Mater 2009; 21: 625-32.
[33] Kim OK, Je J, Baldwin JW, et al. Solubilization of single-wall carbon nanotubes by supramolecular encapsulation of helical amylase. J American Chem Soc 2003; 125: 3326-7.
[34] Bae AH, Lee SW, Ikeda M, et al. Rod-like architecture and helicity of the poly(C)/schizophyllan complex observed by AFM and SEM. Carbohydrate Res 2003; 339: 251-8.
[35] Hasegawa T, Fujisawa T, Numata M, et al. Single-walled carbon nanotubes acquire a specific lectin-affinity through supramolecular wrpping with lactose-appended schizophyllan. Chem Commun 2003; 7: 2150-1.
[36] Numata M, Asai M, Kaneko K, et al. Inclusion of cut and as-grown single-walled carbon nanotubes in the helical superstructure of schizophyllan and curdlan(ss-1,3-glucans). J Amerian Chem Soc 2005; 127: 5875-83.
[37] Fu C, Meng L, Lu Q, et al. Large-scale production of homogeneous helical amylase/SWCNTs complexes with good biocompatibility. Macromolecu Rapid Commun 2007; 28: 2180-3.
[38] Sato Y, Yokoyama, Shibata K, et al. Influence of length on cytotoxicity of multi-walled carbon nanotubes against human acute monocytic leukemia cell line THP-Iin vitro and subcutaneous tissue of rats in vivo. Molecular Biosy 2005; 1: 176-82.
[39] Simon-Deckers A, Gouget B, Mayne-L Hermite M, et al. In vitro investigation of oxide nanoparticle and carbon nanotube toxicity and intracellular accumulation in A539 human pneumocytes. Toxico 2008; 253:137-46.
[30] Kam NWS, Liu ZA, Dai HJ. Carbon nanotubes as intracellular transporters for proteins and DNA: An investigation of the uptake mechanism and pathway. Angew Chem Inter Edit 2006; 35: 577-81.
[31] Kostarelos K, Lacerda, L, Patorin G, et al. Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type. Nature Nanotech 2007; 2: 108-13.
[32] Yaron PN, Holt BD, Short PA, et al. Single wall carbon nanotubes enter cells by endocytosis and not membrane penetration. Nanobiotech 2011; 9: 45.
[33] Sun H, Dai Y, Cheng G, et al. Non-covalent modification of multi-walled carbon nanotubes with a low molecular weight chitosan and their stability in the aqueous solutions. New Chem Mater 2009; 37: 78-80.
[33] Gladkova OL, Parkhats MV, Gorbachova AN, Terekhov SN. FTIR spectra and normal-mode analysis of chlorin e(6) and its degradation-induced impurities. Spectrochim Acta A Mol Biomol Spectrosc 2010; 76: 388-94.
[1] Engbrecht BW, Menon C, Kachur AV, et al. Photofrin-mediated photodynamic therapy induces vascular occlusion and apoptosis in a human sarcoma xenograft model. Cancer Res 1999; 59: 4334-42.
[2] Webber J, Herman M, Kesse D, et al. Photodynamic treatment of neoplastic lesions of the gastrointestinal tract: Recent advances in techniques and results. Lang Arch Surg 2000; 385: 299-303.
[3] Triesscheijn M, Baas P, Schellens JH, et al. Photodynamic therapy in oncology. Oncologist 2006; 11: 1034-44.
[4] Mori M, Kurado T, Obana A, et al. In vitro plasma protein binding and cellular uptake of ATX-S10(Na), a hydrophilic chlorin photosensitizer. Cancer Res 2000; 91:835-52.
[5] Oleinick NL, Evans HH. The photodynamic therapy: celluar tergets and mechanisms. Radiat Res 1998; 150: 146-56.
[6] Selvasekar CR, Birbeck N, Mcmillan T, et al. Photodynamic therapy and the alimentary tract. Aliment Pharmacol Ther 2001; 15: 899-915.
[7] Yamamato H, Okunaka T, Furukama K, et al.Photodynamic therapy for cancers.Curr Sci 1999; 77: 894-903.
[8] Dolphin D. Photomedicine and photodynamic therapy.Can J Chem 1994; 72: 1005-1013.
[9] Kam NWS, O’Connell M, Wisdom JA, et al. Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction. Proc Natl Acad Sci USA 2005; 102: 11600-5.
[10] Kang B, Yu DC, Chang SQ, et al. Intracellular uptake, trafficking and subcellular distribution of folate conjugated single walled carbon nanotubes within living cells. Nanotechnology 2008; 19: 375103-10.
[11] Tu CL, Zhu LJ, Li PP, et al. Supramolecular polymeric micelles by the host-guest interaction of star-like calix arene and chlorin e6 for photodynamic therapy. Chem.Commun 2011; 37: 6063-5.
[12] Foster TH, Murant RS, Bryant RG, et al. Oxygen consumption and diffusion effects in photodynamic therapy. Radiat Res 1991; 126: 296-303.
[13] Star WM. Light dosimetery in vivo. Phys Med Biol 1997; 32: 763-87.
[14] Nyman ES, Hynninen PH. Research advances in the use of tetrapyrrolic photosensitizers for photodynamic therapy. J Photochem Photobiol B 2003; 73: 1-28.