Membrane fluidity altering and enzyme inactivating in sarcoma 180 cells post the exposure to sonoactivated hematoporphyrin in vitro

Sonodynamic therapy (SDT) is a novel tumor therapy method. We investigated membrane fluidity, activity of the enzymes and membrane morphology in vitro post hematoporphyrin-SDT treatment. Furthermore, the potential mechanisms behind the changes in membrane fluidity and enzymic activity were discussed. Tumor cells were exposed to ultrasound at 1.75 MHz for up to 3 min in the presence and absence of hematoporphyrin. Fluorescence polarization, contents of Malonaldehyde, and levels of free fatty acid were assessed. Activity of enzymes was checked by the plumbic nitrate detection method. For the morphologic study, a scanning electron microscope was used to observe the cellular surface. Ultrasonically induced cell damage increased in the presence of HPD (from 15% to 24%). Compared with ultrasound treatment alone, the fluidity decreased from 5.037 to 3.908, malonaldehyde content and free fatty acid level increased from 0.743 nmol/mL to 0.979 nmol/mL and from 237.180 μmol/L to 730.769 μmol/L, respectively, post ultrasound combined with HPD treatment. Inactivity of adenylate cyclase and guanylate cyclase and significant deformation of the cellular surface were also observed post SDT treatment. Our results suggested that alterations in membrane modality and lipid composition played important roles in SDT-mediated inhibition of tumor growth, even inducing tumor cell death, which might be attributed to a sono-chemical activation mechanism.     

Ultrasound exposure in the presence of hematoporphyrin induced loss of membrane integral proteins and inactivity of cell proliferation associated enzymes in sarcoma 180 cells in vitro

Ultrasonically induced effects of hematoporphyrin (HPD) on cell damage and membrane protein alteration of S180 isolated tumor cells in vitro were investigated, and the potential mechanisms of sonodynamic therapy (SDT) inhibiting tumor growth were discussed. Tumor cells suspended in air-saturated PBS (pH 7.2) were exposed to ultrasound at 1.8 MHz for up to 180 s in the presence and absence of HPD. The viability of cells was determined by a trypan blue exclusion test. To estimate the damage effects of SDT on plasma membrane of tumor cells primarily, membrane integral proteins (EGFR, Ras, Fas, FasL) and cell proliferation associated enzymes (adenylate cyclase and guanylate cyclase) were checked with immunochemical methods. The results indicated that the intensity threshold for ultrasonically induced cell damage at 1.8 MHz was 3 W/cm². At this condition, the expression of the integral proteins was obviously inhibited and the activity of the enzymes was decreased post ultrasound treatment in the presence of 20 μg/ml HPD. Loss of the membrane proteins and inactivity of AC and GC post SDT was time-dependent. This paper reveals SDT can cause the loss of tumor cell membrane integral proteins and inactivity of the enzymes associated with cell proliferation which might be attributed to a sonochemical activation mechanism. The mechanisms by that tumor growth is inhibited by SDT can be understood as that the growth signaling pathway is partially interdicted and the resistance of tumor cells to the specifically activated immune cells is weakened.     

各种离子对血卟啉与牛血清白蛋白相互结合反应的影响研究

应用荧光法研究了水溶液体系中血卟啉与牛血清白蛋白分了间相互结合反应，其结合常数ＫＡ＝２．２×１０＾４，结合位置数为１．依据Ｆｏｒｓｔｅｒ非辐射能量转移机理，探讨了血卟啉与牛血清白蛋白相互结合时，其给体－受体间距离和能量转移效率，进一步研究了各种离子与牛血清白蛋白的结合反应，其结合淬灭能力大小顺序为Ｃｒ（Ⅵ）〉Ｆｅ＾３＋〉Ｃｕ＾２＋〉ＮＯ２〉Ｂｒ＾－，并探讨不同阴，阳离子及血卟啉与牛血清白蛋白间相互     

Reversed-phase high-performance thin-layer chromatography of hematoporphyrin and its nickel,copper and zinc complexes

Summary The thin-layer chromatographic mobility of hematoporphyrin IX (Hp) and its metal complexes on octyl (C₈)- or octadecyl (C₁₈)-bonded silica gel plate is described. The mobility order for those compounds is regular on both kinds of plate with various compositions of methanol-phosphate buffer mixtures as the developing solvents; thus, Cu-complex < Ni-complex < Hp (free acid) < Zn-complex. The combination of a C₁₈-plate with an 85∶15 (vol/vol) mixture of methanol and phosphate buffer (pH 3) is recommended for the successful separation of these four compounds.     

血卟啉及其金属衍生物的化学电离质谱

本研究采用直接曝热技术，分别以氢气和甲烷为反应气，测定了１０个血卟啉衍生物和１３个金属血卟啉衍生物的正、负离子解吸化学电离质谱，探讨了各种化学电离质谱法在结构测定中的应用。     

血卟啉及金属血卟啉对嘌呤衍生物识别作用的研究

采用紫外光谱法、荧光光谱法和核磁共振谱法研究了嘌呤与血卟啉及金属血卟啉的作用情况 ,并对影响因素 (酸度和离子强度等 )进行了试验。结果表明在 pH 11 2的Kolthoff缓冲溶液中 ,嘌呤有很强的荧光峰 ,且血卟啉及金属血卟啉对嘌呤有明显的识别作用 ,结合常数在 10 4～ 10 5,结合比均为 1∶1。金属血卟啉的结合常数 :KNi(Ⅱ ) HP>KCo(Ⅱ ) HP>KZn(Ⅱ ) HP>KCu(Ⅱ ) HP,同时对识别机理进行了讨论 ,提出了两点和三点作用模式的识别机理     

基于皮秒时间分辨的癌细胞荧光寿命研究

研究了用于癌症诊断与治疗的光敏剂血卟啉(hematoporphyrin derivative,HPD)的超快光动力学过程,采用超短脉冲激光光谱技术和皮秒时间相关单光子计数系统,测量了经血卟啉培养的活体癌细胞与正常细胞的皮秒时间分辨荧光光谱及荧光峰值强度随时间衰变曲线,观测到:癌细胞与正常细胞样品荧光寿命的快成分分别为150和300 ps;癌细胞与正常细胞的荧光峰值强度经12 h分别衰减10%和55%.经对测量所得的荧光衰减曲线进行分析,计算出癌细胞与正常细胞的荧光寿命分别为824和1798 ps;血卟啉在癌细胞与正常细胞样品中滞留时间分别为17天和6天.结果表明癌细胞与正常细胞对血卟啉亲和性及对血卟啉滞留的稳定性有显著差异,测量结果确认了荧光光谱技术诊断与治疗癌症的可行性,并对实时监测生物样品微弱超快荧光具有重要的指导意义和临床应用价值.     

Porphyrin derivatives having physical and chemical characteristics similar to those of the active components of hematoporphyrin derivative and with very strong photosensitizing effects

The tumour-localizing fraction of hematoporphyrin derivative (Hpd) is thought to possess an essentially diporphyrin ether structure or, alternatively, a diporphyrin ester structure, the properties of which facilitate its retention in malignant cells and its biological activity on irradiation. To elucidate this problem further, we have synthesized the dimethyl, diethyl, dipropyl, di-n-butyl and di-iso-butyl ethers of hematoporphyrin. These ethers show chromatographic properties very similar to those of the active components of Hpd. Furthermore, they are much better photosensitizers in a cellular system than are crude Hpd or Photofrin II, and, like the components of Hpd, they are taken up and retained by cells according to their degree of non-polarity.     

生理液中血卟啉溶液状态的光谱研究

本文通过对血卟啉在生理条件下（ｐＨ＝７．２，Ｉ＝０．５ｍｏｌ／ＬＮａＣｌ）水溶液中的光谱变化研究，探讨了其在溶液中的聚集状态。应用Ｋａｓｈａ理论推算出血卟啉二聚体的面－面间距离为０．５２８ｎｍ，面间夹角为２０°，平衡常数为２．８５×１０~（－６）。研究了在光作用下，血卟啉－人血清白蛋白溶液体系的光谱特性，讨论了光谱特性与血卟啉在癌瘤光化学治疗机制间的关系。