Immune response induced by hematoporphyrin derivatives mediated photodynamic therapy:Immunogenic cell death and elevated costimulatory molecules

Photodynamic therapy(PDT)not only destroys tumor cells directly but also induced anti-tumor immune response through damage-associated molecular patterns(DAMPs).It is reported that anti-tumor response was associated with light dose and photosensitizer used in PDT.In this study,4T1 tumor cells were implanted on both the right and left flanks of mice.Only the right tumor was treated by HpD-PDT,while the left tumor was not irradiated.The anti-tumor immune response induced by HpD-PDT was investigated.The expression of DAMPs and costimulatory molecules induced by HpD-PDT were tested by immuno fluorescence and flow cytometry in vivo.Different light doses of PDT were designed to treat 4T1 cells.The killing effect was assessed by CCK-8 kit and apoptosis kit.The expression of DAMPs on 4T1 cells after HpDPDT were evaluated by flow cytometry,western blot and ATP kit.This study showed that CD4^(+)T,CD8^(+)T and the production of IFN-γwere increased significantly on day 10 in righttumor after PDT treatment compared with control group.HpD-PDT enhanced the expression of calreticulin(CRT)on tumor tissue.Importantly,co-stimulatory molecular OX-40 and 4-1BB were elevated on CD8^(+)T cells.In vitro,immunogenic death of 4T1 cells was induced after PDT.Besides,the expression of DAMPs increased with the increasing of energy density.This study indicates that anti-tumor immune effect was induced by HpD-PDT.The knowledge of the involvement of CRT,ATP and co-stimulatory molecules uncovers important mechanistic insight into the anti-tumor immunogenicity.It was the first time that co-stimulatory molecules were investigated and found to elevate after PDT.

THE SPECIFIC PHOTODYNAMIC EFFECTS OF MONOCLONAL ANTIBODIES CONJUGATED WITH HEMATOPORPHYRIN DERIVATIVE ON GASTRIC CANCER IN VITRO AND IN VIVO

Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD.In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxi-city this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC825. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but not by another MoAb 3G9, which combines with BGC823 at different binding sites from MoAb BB4.3.Nude mice inoculated with 2 × 10- BGC823 cells were given HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS, respectively then exposed to light. Four out of six animals treated with the HPD-BB4.3 conjugate remained tumor-free for a long period. Although two developed tumors, there was a significant difference between the HPD-BB4.3-treated group and all the control groups in tumor induction time, tumor growth rate, and survival time (p<0.001). The HPD-BB4.3 conjugate inhibited the growth of established tumors by more than 40% in comparison with control groups (p<0.05).

The Observation of the Photodynamic Effect of Hematoporphyrin Derivative (HpD) Combined with Aidiron on Ascitic Hepatoma Cells in Vitro

In this study, we found, through biochemical synthesis of DNA with the incorporation of3HTdR and morphological observation of the tumor cells, that aidiron （912）, an extract fromearthworm, had a stronger kilting effect （27%） on mouse ascitic hepatoma tumor cells (H22) invitro, and that laser-hematoporphyrin derivative （HpD） combined with 912 markedly enhancedthe kilting effect （74%） on tumor cells. By means of the fluoresoence spectrophotography we also dis-covered that the killing effect of laser-HpD-912 on tumor calls was closely related with the produc-tion of free radicals and lipid peroxidation.

Impact of the photosensitizers hematoporphyrin coated gold nanoparticles on biomphalaria alexandrina snails

The present study was done using two concentrations of the photosensitizer Hematoporphyrin coated Gold Nanoparticles (HpdGNP3) (5x10–6 , 5x10–5 mole/dicemeter–3), to evaluate their efficacy on survival rate, egg-laying capacity of Biomphalaria alexandrina snails and on histological deteriorations in their hermaphrodite gland. B. alexandrina snails were incubated for 12 hours at each tested concentration in the dark, thereafter they were exposed to direct sunlight (336.2 W/m2) for either 2 or 4 hours followed by 24 hours of recovery. Control snails were treated with these concentrations without exposure to light irradiance. Another experiment was carried out simultaneously and the snails were left for 4 weeks of recovery to evaluate their egg-laying capacity (Mx). The results indicate that 5x10–5 Mdm–3 (HpdGNPs) with 4 hours of exposure sunlight suppressed the survival rate of B. alexandrina snails by 50%. Meanwhile, control snails incubated with 5x10–5 Mdm–3 HpdGNPs were not affected and still alive (100%). For snail’s fecundity (Mx), treated snails laid low number of eggs throughout the recov-ery period (4 weeks), in comparison with that of control ones. The highest value of Mx for snails treated with 5x10–5 Mdm–3 Hpd coated GNPs was recorded at the 3rd week of recovery period, being 6.7 eggs/snail, compared to 37.6 eggs/control snail. This has a negative reflect on the reproductive rate (Ro) of treated snails as it was reduced under these conditions by 76.6% and 86.1%, respectively.Histological tests re-vealed injuries in spermatocytes, oocytes, several degenerations of B.alexandrina hermaphrodite gland then evacuations in many gonad’s cells which severely suppressed their capacity for egg-laying. It is concluded from the present work that exposing B. alexandrina snails to sublethal concentrations of the photosensiter Hpd coated GNPs (12 hours incubation, 4 hours exposure to 336.2 W/m2) significantly reduced their reproductive capacity that may have a negative reflect on schistosomiasis transmis-sion.

EFFECT OF HEMATOPORPHYRIN DERIVATIVE (HPD) PLUS LIGHT ON TRANSCRIPTION ACTIVITY IN THE NUCLEUS

To elucidate the mechanism of photodynamic damage of cells, the effect of HPD plus light on transcriptlonal ectlvlty In the mucleus Isolate from the normal rat liver was studied in vitor by 3H- UTP incorporation into RNA. Measurements of fluorescence spectrum showed that HPD was bound to the nucleus and its fluorescence Intensity Increased with the Increase of HPD concentration. The experimental results Indicated that no changes could be observed when either HPD or light was used alone. Whereas the nuclear transcription activity was found to be inhibited significantly by both HPD and light treatment, and the degree of Inhibition was dependent on the HPD concentration and the time of exposure to light. After treatment by 3 μg/ ml HPD, the inhibition rate of the nuclear transcription activity was 23% ,45% ,69% ,80% and 90%, respectively for light exposure of 2, 5, 10, 20 and 30 minutes. Our results suggested that dose-dependent decreases in the nuclear transcription activity, and marked inhibition of the activity was found in the range from 3 to 7 μg/ml HPD following exposure to light.

PURGE OF MALIGNANT CELLS FROM BONE MARROW BY HEMATOPORPHYRIN DERIVATIVES AND LIGHT EXPOSURE IN VITRO

During autologous bone marrow graft in treatment of malignant diseases, it is critical to purge malignant cells from the marrow. In the present study, the sensitivity to photodynamic inactivation of 3 leukemic cell lines was compared with their counterpart normal hematopoietic cells. After mouse leukemic L1210 cells were treated with a preparation of hematoporphyrin derivatives, YHpD, 10 μg/ml for 1 hr. and irradiated with blacklight (peak wavelength 395 nm, light intensity 0.6 mW/cm2) for 5 minutes, the survival rate of clonogenic cells decreased to <10%, while that of bone marrow granulocyte macrophage progenitor cells (CFU-GM) in DBA/2 mice remained at nearly normal level (>80%). Similar results were obtained when human leukemic HL-60 cells were compared with human CFU-GM and mouse leukemic L615 cells with CFU-GM in 615 strain mice. It is suggested that hematoporphyrin photoradiation may be useful for Iselectively killing leukemic cells in bone marrow.

Synergistic effects of focus ultrasound with different frequency and hematoporphyrin on human tumor cells

Human hematopoietic cell K 562 , human melenoma cell LiBr and human stomach cancer cells were exposed to ultrasound (US, 1.75 W/cm\+2, 1.4, 2.16 and 2.4 MHz) in vitro in the presence or absence of hematoporphyrin (Hp, 100 μg/mL). The cell damaging effects of treatments were determined by means of the Trypan Blue dye exclusion test, MTT test and FDA test. The experimental results showed that the same cell line had different sensibilities to the US of different frequencies, and different cell line had different damage at the same acoustical radiation. The combined treatment with US and Hp enhanced greatly the cell damage, and no sensibility of insonation cells to US with Hp was observed. The cell damage tests showed that the results of MTT test corresponded well with that of Trypan Blue dye test.

INQUIRY OF PHOTOSENSITIZATION MECHANISM OF YANGZHOU HEMATOPORPHYRIN DERIVATIVE(YHPD)

By the use of the advanced ESR technique and through comparing with BHPD, the characteristic of YHPD photosensitization is discussed in this paper in the respect of the primary process of photosensitization. The experiment results showed: （ⅰ） not only 1O2, but also free radicals（O2· OH and YHPD）can be formed by the aid of YHPD; and （ⅱ） as to the ability of producing 1O2, YHPD【BHPD, while for generating O2 and-OH, YHPD】BHPD. Two points are indicated: first, the photosensitized damage of YHPD is interrelated to not only 1O2, but also free radicals （O2. OH and YHPD）; second, although the photosensitized damage of YHPD is stronger than that of BHPD, yet the photosensitized damage is negatively correlated to the yield of 1O2 but positively correlated to those of O2 and OH. Based on these two points, it is suggested that activated oxygen free radicals（O2 and. OH） instead of 1O2 play the main role as instantaneously activated material in the photosensitized damage of YHPD.

NMR STUDY ON THE MAJOR COMPONENTS IN HEMATOPORPHYRIN DERIVATIVE YHPD

The hematoporphyrin derivative YHPD, a China-made product, has been clinically used in photodynamic therapy of tumors as a good photosensitizing drug. The NMR study on the structure of its major components is reported here. In terms of high performance liquid chromatography （HPLC） four major components A, B, C and D were isolated. The NMR results showed that the component A is O-acetylhematoporphyrin, B and C are two isomers of vinyldeuteroporphyrin. The spectra of 2-dimensional homonuclear correlation NMR, 2-dimensional NOE （nuclear overhauser enhancement）, 13C-NMR and off-resonance as well as FAB （fast atom bombarding） mass spectrum of component D indicate that it is a protoporphyrin dimer linked by carbon-carbon bond. This finding may providea chemical basis for understanding the difference in biological activity between YHPD and other foreign commercial HPD, as well as the composition of clincally used alkali-treated HPD and its effective component.

STUDIES ON THE EXPOSITION OF HYDROXYL RADICAL FROM HEMATOPORPHYRIN DERIVATIVE SOLUTION TO LIGHT BY SPIN-TRAPPING METHOD

That the photosensitive action of hematoporphyrin can damage tumor cells, has attracted much attention recently. However, its mechanism of action is not very clear. Some reported that the damage is due to the production of singlet oxygen （1O2）, a cy-

TIME-RESOLVED FLUORESCENCE SPECTROSCOPY OF HEMATOPORPHYRIN DERIVATIVE LASER No. 3

Ⅰ. INTRODUCTIONPhotodynamic therapy (PDT) which utilizes hematoporphyrin derivative (HpD) as a photosensitizing drug has increasingly being used for the local treatment of malignant tumors. The success of this method is attributed to the ability of HpD to accumulate to

THE ELECTRON SPIN RESONANCE STUDY ON PRIMARY PHOTOCHEMICAL REACTION OF HEMATOPORPHYRIN DERIVATIVE

The rate of oxygen consumption and the yield of free radical anion of hematoporphyrin derivative (HPD) in aqueous solutions of HPD and pyrocatechol were measured by the probe 2,2,6,6-tetramethyl4-piperidone-1-oxyl.It has been found that both singlet oxygen and free radical mechanisms exist simultaneously in primary photochemical reactions, and there is a competition between both mechanisms. When the oxygen concentration in solutions comes down to 12-14% of the stanting level, the predominant mechanism can be changed from the singlet oxygen to the free radical.Whether HPD exists in aggregation state is very important to photosensitization mechanisms.In the presence of the aggregation state of HPD, the predominant mechanism is the free radical,and photosensitization effects of HPD are all the better.

Gold nanorods with a hematoporphyrin-loaded silica shell for dual-modality photodynamic and photothermal treatment of tumors in vivo

由一个金 nanorod 核心和与 hematoporphyrin (HP ) 做的 mesoporous 硅石壳组成的 Nanocomposites (NC ) 被制作了以便由在 vivo 联合 photothermal 和光力学的治疗(太平洋夏季时间 + PTT ) 改进癌症治疗的效率。除了 810830 nm 附近的长波长的电浆子回声，制作 NC 展出了相应的一座 400-nm 吸收度山峰围住 HP，在 632.5-nm Q 乐队附近在 633-nm 刺激下面产生了汗衫氧，并且生产了在 808-nm 下面的热在红外线附近(NIR ) 激光照耀。这些形式被用于联合太平洋夏季时间 + PTT 治疗大(大约 3 cm3 ) 在有一只 xenorafted 肿瘤老鼠的 vivo 的稳固的肿瘤当模特儿。NC 直接被注入肿瘤并且与 633-nm 和 808-nm 激光同时照耀刺激联合光力学并且 NC 的 photothermal 活动。联合治疗的效率被光连贯断层摄影术评估，组织学的分析，并且由肿瘤体积生长的大小在一个 21 天的时期期间。调停 NC 的太平洋夏季时间在组织组织学并且到导致了弱变化一中等在肿瘤体积的 20% 减少。相反，联合太平洋夏季时间 + PTT 处理导致了大区域的肿瘤坏死并且在肿瘤体积导致了戏剧的减少。

血卟啉衍生物在3种肺癌细胞系中的细胞摄取和定位

目的以支气管上皮BEAS-2B细胞系为对照,比较不同肺癌细胞系(肺腺癌A549、肺鳞癌H520、肺小细胞癌H446细胞)之间血卟啉衍生物(HPD)的细胞摄取和定位差异。方法用不同浓度HPD测得的荧光值绘制HPD荧光值的标准曲线。将4种细胞分别与HPD孵育不同时间后,用多功能酶标仪检测荧光强度。进一步用流式细胞仪量化细胞摄取,比较4种细胞HPD摄取差异。用激光扫描共聚焦显微镜观察比较细胞内HPD摄取和分布情况。结果4种细胞系摄取HPD随着时间增加而增加,但HPD的累积速率有所差异。5 mg/L的HPD孵育细胞24、48 h,4种细胞内平均荧光强度差异有统计学意义(F=199.00、71.15,P<0.001),其中A549细胞内平均荧光强度显著高于H446、BEAS-2B、H520细胞,H446和BEAS-2B细胞内平均荧光强度显著高于H520细胞(P<0.05)。15 mg/L的HPD孵育细胞48 h,4种细胞内呈现出强度不等的红色荧光信号,均呈点状分布于细胞质内。结论不同类型肺癌细胞系摄取HPD存在差异,其中肺腺癌A549细胞摄取HPD的能力最强,支气管上皮BEAS-2B细胞与肺小细胞癌H446细胞次之,肺鳞癌H520细胞摄取能力最弱;HPD细胞内定位均位于细胞质内,呈点状分布。

光动力疗法治疗鲜红斑痣的临床应用

鲜红斑痣(PWS)是一种以皮肤表面毛细血管和毛细血管后微静脉的结构异常为特征的先天性进行性毛细血管畸形。这种明显的外观差异通常被认为是一种身体缺陷,随之而来的社会歧视往往会严重影响患者的情绪和身体健康。海姆泊芬(HMME)是国内新批准的用于治疗PWS的光敏剂,自2017年以来,海姆泊芬光动力疗法(HMME-PDT)被广泛用于PWS患者的治疗。HMME-PDT作为治疗PWS的一种新兴疗法,具有良好的安全性和有效性,是最有前景的治疗策略之一。本文将对HMME-PDT治疗PWS的临床问题进行概述,期待未来有更多的研究以提高治疗的有效性、安全性和舒适性。

咪喹莫特联合血卟啉光动力治疗光化性角化病的临床效果

目的:探讨光化性角化病患者采用咪喹莫特联合血卟啉光动力治疗的临床效果。方法:按照随机数字表法将2020年10月—2022年10月宜春市人民医院收治的60例光化性角化病患者分为对照组与观察组,各30例。对照组采用咪喹莫特乳膏外涂,观察组采用咪喹莫特联合血卟啉光动力方法治疗。比较两组的临床效果,治疗前后的皮损面积、VISIA皮肤检测,治疗后的复发情况,以及治疗期间不良反应发生情况。结果:观察组患者临床总有效率为93.33%,显著高于对照组的73.33%(P<0.05)。与治疗前比较,治疗后两组患者皮损面积均缩小,且观察组显著小于对照组(P<0.05)。观察组患者复发率为0,显著低于对照组的30.00%(P<0.05)。与治疗前比较,治疗后两组患者VISIA皮肤检测各项指标均升高,且观察组均高于对照组(P<0.05)。两组患者不良反应总发生率比较差异无统计学意义(P>0.05)。结论:咪喹莫特联合血卟啉光动力治疗光化性角化病效果确切,能改善皮损情况,降低患者复发风险,同时不增加不良反应发生。

光动力疗法治疗鲜红斑痣1216例临床分析

目的 分析比较血啉甲醚 (HMME)与血卟啉衍生物 (HpD)为光敏剂行光动力治疗 (PDT)鲜红斑痣的临床疗效及副反应。材料与方法 鲜红斑痣患者 12 16例 ,粉红型 13 2 % ,紫红型 6 8 3% ,增厚型 18 4%。静脉注射HpD或HMME 3～ 7mg/kg后给予铜蒸气激光、KTD/ 5 32激光或氩离子激光照射 ,功率密度 5 0～ 10 0mW/cm2 ,能量密度 90～ 5 40J/cm2 ,随访观察。结果 HMME PDT和HpD PDT对各型鲜红斑痣均能有效地消除病变颜色 ,所随访的 16 32个病灶在 2个月至 9年的随访期内未见复发。治疗后反应有 :局部水肿 5～ 7天 ,部分患者有结痂或一过性色素沉着 ,皮肤暂时性光过敏反应。HpD PDT组避光期 30～ 90天 ,HMME PDT组避光期 7～ 14天。与HpD PDT相比 ,HMME PDT具有反应轻、愈合快、不良反应少、安全度大、避光期短、色素沉着轻、护理容易、重复治疗间隔期短的特点。结论 HMME PDT与HpD PDT都能有效治疗各型鲜红斑痣 ,但HMME PDT具有不良反应少、安全度大、避光期短、护理容易。

声化学激活血卟啉诱导艾氏腹水肿瘤细胞凋亡

本实验采用频率为 2 0MHz,声强分别为 1 0W/cm2 、 1 5W /cm2 、 2 0W /cm2 等不同参数 ,研究超声激活血卟啉对艾氏腹水肿瘤细胞的杀伤作用和诱导肿瘤细胞凋亡现象。通过扫描电镜、透射电镜以及荧光显微镜观察受损后细胞形态结构的变化 ,主要表现为细胞微绒毛的减少 ,胞膜结构和通透性的改变 ,细胞器的受损以及核物质的分解、丢失 ;同时发现处理后的肿瘤细胞有核物质凝集、趋边排列以及凋亡小体的形成等细胞凋亡特征。研究中首次发现声化学激活血卟啉在对艾氏腹水肿瘤细胞杀伤的同时 ,也能诱导艾氏腹水肿瘤细胞发生凋亡 。

光动力疗法治疗鲜红斑痣的基础研究——血啉甲醚与血卟啉衍生物吸收特性的比较

目的 观察新型光敏剂———血啉甲醚 (HMME)在鸡冠皮肤组织和血管内皮细胞 (EC)的吸收特点。方法 莱亨鸡 12只 ,分为HMME组和血卟啉衍生物 (HpD)组 ,每组 6只。分别静脉注射HMME或HpD10mg kg ,每隔 10min取血 2 5ml和鸡冠皮肤组织 2 5g。培养新生儿脐带EC ,培养液中加入HMME或HpD ,孵育浓度分别为 2 0、40、6 0、80和 10 0μg ml,孵育时间为即刻、15、30、6 0、12 0、180和 2 40min。采用荧光分析法测定光敏剂含量 ,并绘制EC吸收光敏剂的浓度—含量和时间—含量关系曲线。结果 静脉注射HMME或HpD后 ,其血清浓度的峰值出现在注射后 10min ,以后随时间迅速下降 ,二者的曲线形态基本一致。HMME在鸡冠组织中的含量于注射后 10min显著高于HpD(P <0 0 1) ,以后虽然下降速率较快 ,但在注射后 80min仍显著高于HpD(P <0 0 5 )。培养EC可迅速吸收HMME和HpD ,30min达峰值的 89%以上 ;其吸收量与孵育浓度呈线性关系 ,对HMME的吸收量显著高于HpD(P <0 0 1)。结论 HMME较HpD更容易被皮肤微血管EC摄取 。