壳聚糖—二氢卟吩e6-单壁碳纳米管药物输送系统的构建及体外光动力学研究
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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.
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
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[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.
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[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.