摘要
研究背景和目的
腺病毒(Adenovirus)具有宿主广泛、繁殖滴度高、不整合,对静止细胞也能感染及相对完全等特点,因而被广泛用于肿瘤基因治疗。但是,人群中普遍存在腺病毒中和抗体,不利于病毒载体的重复给药,制约了它在临床上的应用。此外,腺病毒对肿瘤的靶向性不足,使得感染肿瘤细胞的病毒量大大减少,减弱了杀伤肿瘤细胞的作用,增加了可能的机体毒副作用。因此,如何降低腺病毒载体的免疫原性,提高其肿瘤靶向性,成为基于腺病毒载体的肿瘤基因治疗亟需解决的问题。
当前,降低腺病毒载体免疫原性的策略主要有以下两个方面：改造腺病毒载体和免疫调控。但目前这两种方法均系抗腺病毒免疫的优化方案,未能屏蔽腺病毒免疫原性,因而免疫系统仍然对腺病毒进行免疫识别与免疫清除。以腺病毒载体外壳蛋白修饰和基因复制与表达调控为主的增强腺病毒肿瘤靶向性的策略虽然取得了进展,但无法同时解决腺病毒的免疫原性的问题。因此如何将这些改造策略有机地结合起来,以便实现多种策略协同作用,最大限度的发挥腺病毒载体作用的同时,保证它在临床应用中的安全性,将是未来研究的重点。
pHPMA是在肿瘤、骨骼及炎症等研究领域最受关注的高分子给药系统,具有生物相容性、非免疫原性、无毒性的特点,与药物接合后可以延长药物血液循环半衰期及实体瘤趋向性给药,目前通过应用pHPMA给药系统,以阿霉素为代表的多个药物已用于临床研究。近年来,Fisher等首先发现腺病毒通过pHPMA进行给药时发生免疫逃逸,证实了pHPMA可消除机体免疫系统对腺病毒的免疫识别与免疫攻击。Vladimir等在其后的研究发现pHPMA-腺病毒接合物可选择性沉积于肿瘤组织,证明了pHPMA-腺病毒接合物肿瘤靶向性给药的特点。因而,pHPMA被认为是腺病毒治疗的最佳给药系统,对提高腺病毒肿瘤靶向性,降低其免疫原性及毒副作用具有重要意义。pHPMA给药系统细胞吸收的主要障碍是细胞膜屏障,仅能通过胞吞或胞饮分布于质膜和溶酶体,难以在胞浆和胞核发挥作用,药物分子的治疗效果因而下降。目前研究的方向是开发靶向性活性分子修饰pHPMA载体的腺病毒给药系统,使pHPMA给药系统突破细胞膜屏障进入细胞浆或细胞核,提高药物的治疗效果。
pHPMA的靶向分子修饰是利用靶向活性分子改变pHPMA作为大分子吸收慢而导致的腺病毒在体内活性降低的问题,使pHPMA的被动吸收变为主动运输,提高药物的内吞速度,实现药物的可控释放。目前常用的靶向分子如配体和单抗等存在与受体作用时间长的问题,导致腺病毒在体内的循环时间延长。现在通常将穿膜作用最强的穿膜肽作为活性分子以改变pHPMA一腺病毒给药系统的跨膜转运问题。但细胞穿膜肽缺乏靶向性,如能改造细胞穿膜肽的嗜性,使之在肿瘤组织进行可控释放,则可在加速pHPMA-腺病毒给药系统跨膜转运的同时,实现肿瘤的靶向性治疗,使腺病毒达到最大程度的抗肿瘤治疗效应。但用肿瘤靶向性细胞穿膜肽修饰pHPMA-腺病毒给药系统的研究尚未见相关报道。本研究拟设计合成肿瘤靶向性细胞穿膜肽对pHPMA-腺病毒给药系统进行修饰,以期在增强穿膜效率的同时,进一步增强其主动靶向性,提高腺病毒生物治疗效应。
通过基因克隆技术,我们构建了广谱抗肿瘤腺病毒Ad. mda-7.egfp,通过化学合成的方法分别设计合成了高分子载体pHPMA及肿瘤靶向分子：可活化细胞穿膜肽(activatable cell-penetrating peptide, ACPPs),通过化学修饰的方法合成了ACPPs-pHPMA-Ad给药系统,对该系统的给药特点进行了分析与鉴定。成功构建的ACPPs-pHPMA-Ad. mda-7.egfp给药系统,不仅实现了腺病毒的免疫逃逸,同时加速了pHPMA-Ad. mda-7.egfp在靶细胞摄取,提高了pHPMA-Ad.mda-7.egfp肿瘤靶向性。因ACPPs的肿瘤靶向性设计是基于多种肿瘤细胞过表达基质金属蛋白酶的特性,故ACPPs-pHPMA-Ad. mda-7. egfp给药系统有望应用于多种肿瘤的临床分子诊断与治疗。
方法
1.抗肿瘤腺病毒Ad. mda-7.egfp的构建与鉴定
选择靶向性诱导肿瘤细胞凋亡的广谱抗肿瘤基因黑色素瘤分化相关基因7(melanoma differentiation associated gene-7, mda-7),利用RT-PCR扩增mda-7基因cDNA全长,通过基因载体克隆技术构建腺病毒Ad. mda-7. egfp,应用酶切、PCR、测序及观察细胞病理现象等方法进行鉴定。
2.抗肿瘤腺病毒Ad. mda-7.egfp的生物活性鉴定
将Ad. mda-7.egfp感染肺腺癌细胞A549后,通过绿色荧光蛋白表达检测鉴定mda-7基因表达；通过免疫组织化学方法鉴定mda-7蛋白表达及部位,通过荧光素Hoechst33342及PI双染法,在荧光显微镜下观察凋亡细胞,鉴定腺病毒Ad. mda-7.egfp诱导肿瘤细胞凋亡的活性；通过流式细胞仪检测DMEM、Ad.egfp及Ad.mda-7.egfp诱导的支气管上皮细胞HBE及肺腺癌细胞A549细胞凋亡率,鉴定Ad. mda-7.egfp选择性杀伤作用。
3.靶向分子—可活化细胞穿膜肽的合成及鉴定
以聚精氨酸RRRRRRRRN为阳离子肽段,并以具有伯氨基的氨基酸天冬酰胺为其3端,以EEEEEEEEE为阴离子肽段,以PLGLAG为MMPs的酶切位点合成肿瘤靶向性的可活化细胞穿膜肽EEEEEEEEE-PLGLAG-RRRRRRRRN,通过高效液相色谱仪测定其分子量及纯度；通过与表达MMPs的肺腺癌A549细胞、子宫颈癌GLC-82细胞、卵巢癌OVCAR3细胞、乳腺癌MCF-7细胞、肺腺癌SPCA1细胞和肝癌HepG2细胞孵育,荧光显微镜观察FITC鉴定可活化细胞穿膜肽的穿膜活性。
4.靶向分子—可活化细胞穿膜肽的生物活性鉴定
将可活化细胞穿膜肽与HBE、肺腺癌细胞A549、SW480细胞、OVCAR3细胞孵育,以荧光显微镜观察FITC及DAPI荧光,通过两种荧光图像重叠鉴定可活化细胞穿膜肽在细胞内的定位；通过流式细胞术检测FITC荧光的变化,观察温度及时间对可活化细胞穿膜肽穿膜作用的影响；通过MTT法检测|ACPPs对细胞的毒性作用；通过全波长酶标仪检测实验组与对照组A549细胞内FITC荧光量鉴定可活化细胞穿膜肽的肿瘤靶向性穿膜活性。
5. ACPPs-pc-Ad. mda-7.egfp给药系统的合成及鉴定
取pHPMA-ONp溶于25μl双蒸水中形成终浓度为10mg/ml溶液；加入含有Ad.mda-7.egfp (1010vp)的10%甘油/PBS溶液100μl,4℃反应12h形成最终的接合物ACPPs-pc-Ad. mda-7.egfp,以pc-Ad. mda-7.egfp及Ad. mda-7.egfp为对照组,以动态光散射法测量ACPPs-pc-Ad. mda-7.egfp的粒径鉴定ACPPs对pc-Ad. mda-7.egfp的修饰；将产物与肺腺癌细胞A549孵育24h,以荧光显微镜及全波长酶标仪观察egfp表达,鉴定ACPPs-pc-Ad. mda-7.egfp细胞感染能力；PI标记ACPPs-pc-Ad. mda-7.egfp后与肺腺癌细胞A549孵育12h,流式细胞仪检测PI荧光,鉴定ACPPs-pc-Ad.mda-7.egfp细胞感染能力。
6. ACPPs-pc-Ad. mda-7.egfp给药系统的免疫逃逸能力的鉴定
含人抗腺病毒抗体(NAb)血清以1：20稀释于PBS液中后,分别与Ad.mda-7. egfp及ACPPs-pc-Ad.mda-7. egfp进行孵育,将孵育前后的Ad.mda-7.egfp及ACPPs-pc-Ad.mda-7感染A549细胞48h,以全波长酶标仪于波长λex488nm与λem538nm检测四组egfp荧光表达,通过分析孵育前后ACPPs-pc-Ad.mda-7. egfp感染细胞后egfp荧光的变化,鉴定ACPPs-pc-Ad. mda-7.egfp给药系统的免疫逃逸能力。
7. ACPPs-pc-Ad.mda-7.egfp给药系统肿瘤靶向性鉴定
以104Vp/孔接种96孔板以104vp/细胞将ACPPs-pc-Ad.mda-7. egfp感染HBE、A549、SW480细胞、OVCAR3四种细胞株48h,吸弃上清,PBS冲洗三次,以100μl Triton X-100裂解细胞,全波长酶标仪检测绿色荧光蛋白表达,通过分析MMPs(?)表达细胞HBE及MMPs过表达细胞A549、SW480细胞、OVCAR3细胞内荧光表达的差异,鉴定ACPPs-pc-Ad.mda-7. egfp对过表达MMPs肿瘤细胞的靶向感染能力。
8. ACPPs-pc-Ad.mda-7.egfp给药系统的跨膜转运方式鉴定
以染料Hoechst33342对细胞染色30min,以染料PI对腺病毒进行标记15min,将pc-Ad.mda-7.egfp组及ACPPs-pc-Ad.mda-7.egfp组(104vp/细胞)感染A549细胞4h,倒置荧光显微镜下观察Hoechst33342荧光及PI荧光,通过对比两组A549细胞内PI荧光的差异,鉴定ACPPs-pc-Ad.mda-7.egfp给药系统的亚细胞分布；以(FITC) ACPPs-pc-Ad.mda-7.egfp (PI)感染A549细胞4h,倒置荧光显微镜下观察FITC荧光及PI荧光,通过两个荧光图像重叠鉴定ACPPs-pc-Ad.mda-7.egfp给药系统的跨膜转运方式；以染料PI对腺病毒进行标记15min,将pc-Ad.mda-7.egfp组及ACPPs-pc-Ad.mda-7.egfp组(104Vp/细胞)感染A549细胞,分别于20mim、2h、4h倒置荧光显微镜下观察PI荧光,通过对比两组细胞内PI荧光的差异,鉴定Ad.mda-7.egfp细胞内吞数量。
9. ACPPs-pc-Ad.mda-7.egfp靶向诱导A549细胞凋亡检测
在6孔板中接种肺癌细胞株A549,生长至40%满后,分成3组,对照组1和对照组2分别加入无血清1640, pc-Ad.mda-7.egfp (104vp/cell),实验组加入ACPPs-pc-Ad.mda-7.egfp (104vp/cell)处理4h,弃上清,以PBS液洗三次后加入培养液继续培养48h,反复PBS洗涤后加入终浓度5ug/ml的Hoechst33342,避光染色10分钟后加入终浓度为15ug/ml PI,避光染色10分钟后,Heochst33342用氪激光激发的紫外线荧光,PI用氩离子激光激发荧光,结果判断：正常细胞为低蓝光／低红光,凋亡细胞为高蓝光／低红光,坏死细胞为低蓝光／高红光。
10.统计学分析
实验数据采用SPSS13.0软件包进行统计：细胞内FITC荧光的比较采用析因设计的方差分析及单因素方差分析；细胞内gfp荧光及PI荧光的比较采用单因素方差分析；MTT结晶的比较采用单因素方差分析。
结果
1.成功构建了Ad.mda-7.egfp腺病毒表达载体
RT-PCR扩增mda-7基因cDNA全长,通过基因克隆技术构建了Ad.mda-7.egfp腺病毒,经过酶切、PCR、测序证明mda-7基因序列正确无误,通过HindⅢ及BglⅡ两个酶切位点与腺病毒基因组进行了基因重组。Ad.mda-7.egfp感染HEK293细胞后,引起细胞出现细胞变大、变圆、漂浮,呈葡萄串聚集等现象。
2. Ad. mda-7.egfp选择性诱导肿瘤细胞的凋亡
通过Ad.mda-7.egfp感染A549细胞24h可观察到绿色荧光蛋白表达；免疫组化方法检测到Ad.mda-7.egfp感染的A549细胞胞浆染色呈阳性,而Ad. Egfp组A549细胞胞浆染色呈阴性,证明了mda-7蛋白的表达。Hoechst-PI染色检测证明Ad.mda-7.egfp感染A549细胞24h可见大量高蓝色荧光及低红色荧光,为凋亡细胞荧光特征。流式细胞仪检测空白对照组、Ad. egfp及Ad.mda-7.egfp诱导HBE及A549细胞凋亡率,经单因素方差分析结果显示细胞凋亡率在A549细胞及HBE细胞组间有显著性差异(F=3627.597,P=0.000),空白对照组、Ad.egfp组、Ad.mda-7.egfp组三组间细胞凋亡率有显著性差异(F=4171.358,P=0.000),说明Ad.mda-7.egfp能够选择性诱导A549细胞凋亡。
3.靶向分子—可活化细胞穿膜肽具有细胞膜穿膜活性
合成可活化细胞穿膜肽EEEEEEEEE-PLGLAG-RRRRRRRRN,通过高效液相色谱仪测定分子量为2923.18,分子式为C116H200N48O41,纯度为99%以上。用FITC标记后的可活化细胞穿膜肽分别与肺腺癌A549细胞、SW480细胞、卵巢癌OVCAR3细胞共孵育,细胞经过洗涤固定后用荧光显微镜观察。荧光分子FITC标记的ACPPs孵育后,三种肿瘤细胞的内部均有较强的绿色荧光反应,证明了可活化细胞穿膜肽的穿膜活性。
4.靶向分子—可活化细胞穿膜肽具有肿瘤靶向性穿膜活性
流式细胞仪的荧光实验结果显示可活化细胞穿膜肽可以穿透细胞膜进入细胞内部,经析因设计的方差分析,可活化细胞穿膜肽组细胞内FITC荧光量与对照组牛血清白蛋白组相比有显著性差异(P<0.05)；全波长酶标仪检测细胞内的荧光强度显示可活化细胞穿膜肽在A549细胞内的荧光量显著高于正常细胞HBE,经析因设计的方差分析,A549细胞内荧光强度与HBE细胞内荧光强度有显著性差异(P<0.05)；细胞增殖实验证明可活化细胞穿膜肽对肿瘤细胞无明显毒性,经单因素方差分析,不同浓度的可活化细胞穿膜肽对A549细胞的毒性与对照组相比无显著差异(P>0.05)。
5.成功合成了ACPPs-pc-Ad. mda-7.egfp给药系统
合成了ACPPs-pc-Ad.mda-7.egfp接合物及pc-Ad.mda-7.egfp接合物,经动态光散射法检测,Ad.mda-7.egfp, pc-Ad.mda-7.egfp, ACPPs-pc-Ad.mda-7.egfp的粒径分别为141.8nn、189.4nm和236.4nm,证明腺病毒分别被pHPMA、 ACPPs-pHPMA修饰,粒径逐渐增加。荧光显微镜观察可见pc-Ad.mda-7.egfp接合物组A549细胞内仅见微量绿色荧光蛋白表达,ACPPs-pc-Ad.mda-7. egfp、 Ad.mda-7.egfp组所有细胞均可见强烈绿色荧光蛋白表达,经全波长酶标仪检测,pc-Ad.mda-7.egfp接合物相对荧光单位(RFU)为6.6%,Ad.mda-7.egfp、 ACPPs-pc-Ad.mda-7.egfp组RFU分别为95.9%和85.6%。经单因素方差分析,各组间RFU有显著性差异(F=894.014, P=0.000), ACPPs-pc-Ad.mda-7.egfp组RFU与Ad.mda-7.egfp组相比无显著性差异(P=0.161),与pc-Ad.mda-7.egfp组RFU有显著性差异(P=0.003),说明ACPPs-pc-Ad.mda-7.egfp具有较强感染细胞的能力。流式细胞仪分析细胞内PI荧光显示：pc-Ad.mda-7.egfp组A549细胞内PI荧光低,Ad.mda-7. egfp及ACPPs-pc-Ad.mda-7. egfp均有较强荧光,证实ACPPs-pc-Ad.mda-7.egfp具有较强感染A549细胞能力,pc-Ad.mda-7.egfp感染A549细胞能力弱。
6. ACPPs-pc-Ad. mda-7.egfp给药系统具有显著的免疫逃逸能力
将人抗腺病毒抗体(NAb)孵育前后的Ad.mda-7.egfp及ACPPs-pc-Ad.mda-7egfp感染A549细胞48h,以全波长酶标仪检测A549细胞内egfp荧光,分别为：孵育前Ad.mda-7.egfp组95.6%、ACPPs-pc-Ad.mda-7egfp组87.6%,孵育后Ad.mda-7.egfp组17.5%、ACPPs-pc-Ad.mda-7egfp组75.6%,孵育后Ad.mda-7.egfp及ACPPs-pc-Ad.mda-7egfp两组感染率皆有下降,但Ad.mda-7.egfp组显著下降,降幅达81.7%,而ACPPs-pc-Ad.mda-7egfp组仅下降13.7%。经单因素方差分析结果显示：人NAb血清孵育前后细胞内egfp荧光量有显著性差异(F=248.832,P=0.000),其中Ad.mda-7.egfp组与人NAb血清孵育前后细胞内egfp荧光量有显著性差异(F=683.132, P=0.000), ACPPs-pc-Ad.mda-7.egfp组与人NAb血清孵育前后细胞内egfp荧光量无显著性差异(F=7.324,P=0.054),说明人NAb血清对Ad.mda-7.egfp有明显清除作用,而对ACPPs-pc-Ad.mda-7.egfp则无明显清除作用,ACPPs-pc-Ad.mda-7.egfp具有免疫逃逸作用。
7. ACPPs-pc-Ad.mda-7.egfp给药系统靶向感染过表达MMPs的肿瘤细胞
以104vp/细胞将ACPPs-pc-Ad.mda-7.egfp感染HBE、A549、SW480、OVC AR3细胞四种细胞株48h,全波长酶标仪检测各组相对荧光单位(RFU)分别为：HBE(10.1)、A549(85.2)、SW480(86.2)、OVCAR3(95.6)。经单因素方差分析,三组肿瘤细胞RFU与HBE组RFU有显著性差异(P=239.827,F=0.000),说明ACPPs-pc-Ad.mda-7.egfp感染具有肿瘤靶向性,其靶向感染能力与肿瘤细胞的MMP2/9表达呈正向关系。
8. ACPPs介导了ACPPs-pc-Ad.mda-7.egfp给药系统的非内吞跨膜转运
以104vp/细胞将ACPPs-pc-Ad.mda-7.egfp(PI)感染Hoechst33342染色的A549细胞4h,倒置荧光显微镜下观察可见：两组细胞胞浆均可见均匀蓝色荧光(Hoechst33342荧光),但pc-Ad. mda-7.egfp组细胞胞浆内较少红色荧光(PI荧光),而ACPPs-pc-Ad.mda-7.egfp组细胞胞浆内可见大量红色荧光(PI荧光),证实了ACPPs-pc-Ad.mda-7.egfp的跨膜转运为非内吞方式。以104vp/细胞将(FITC) ACPPs-pc-Ad.mda-7.egfp(PI)感染A549细胞4h,倒置荧光显微镜下观察可见：细胞胞浆内同时见大量FITC荧光分布及PI荧光分布,说明ACPPs及Ad.mda-7.egfp分布于细胞胞浆,将荧光分步图像叠加,显示两种荧光分布于相同区域。证实了ACPPs介导pc-Ad.mda-7. egfp(PI)的跨膜转运。以染料PI对腺病毒进行标记15min,将pc-Ad.mda-7.egfp组及ACPPs-pc-Ad.mda-7.egfp组(104vp/细胞)感染A549细胞,于20mim、2h、4h倒置荧光显微镜下观察PI荧光,可见ACPPs-pc-Ad.mda-7.egfp组细胞内有较强PI荧光,且荧光于20mim、2h、4h逐渐增强,pc-Ad.mda-7.egfp组细胞PI荧光较少,荧光于20mim、2h、4h均较弱。相比pc-Ad.mda-7.egfp组,ACPPs-pc-Ad.mda-7.egfp组细胞内PI荧光有时间依赖性。
9. ACPPs-pc-Ad.mda-7.egfp可诱导A549细胞凋亡
培养液1640、pc-Ad.mda-7.egfp和ACPPs-pc-Ad.mda-7.egfp分别与A549细胞孵育4h,培养48h后以Hoechst33342、PI双染法观察细胞内荧光可见：空白对照组和pc-Ad.mda-7.egfp组细胞可见弥散均匀的低蓝色荧光,少见红色荧光,ACPPs-pc-Ad.mda-7.egfp组于荧光显微镜下观察到高蓝色荧光及低红色荧光,提示空白对照组和pc-Ad.mda-7.egfp组细胞凋亡数目均较少,ACPPs-pc-Ad.mda-7.egfp组细胞凋亡数目增多,说明ACPPs-pc-Ad.mda-7.egfp具有更高的诱导A549细胞凋亡的效应。
结论
1.成功构建了重组腺病毒载体Ad.mda-7.egfp,该载体能介导mda-7/IL-24基因在人肺癌细胞A549和正常支气管上皮细胞中表达。
2. Ad.mda-7.egfp能选择性诱导A549细胞凋亡,是高效的抗肿瘤腺病毒载体。
3.成功设计合成了可活化细胞穿膜肽,经流式细胞仪检测其具有明显的穿膜活性和肿瘤靶向性,对细胞无明显毒性,对细胞生长亦无明显影响。
4.成功合成了ACPPs-pc-Ad.mda-7.egfp接合物,并能有效感染细胞。
5. ACPPs-pc-Ad.mda-7.egfp能逃避抗腺病毒抗体的中和作用,并具有肿瘤靶向性,在过表达MMP2/9的肿瘤细胞其跨膜转运能力显著高于正常细胞。
6. ACPPs-pc-Ad.mda-7.egfp跨膜转运系ACPPs介导的非胞吞途径跨膜转运,药物转运速度快,分布于细胞胞浆。
7. ACPPs-pc-Ad.mda-7.egfp能有效诱导肺癌A549细胞发生凋亡,可作为肿瘤基因治疗的载体。
ACPPs-pc-Ad.mda-7.egfp可逃避免疫清除,提高腺病毒肿瘤靶向性,适用于众多实体肿瘤而无明显副作用,有望成为抗肿瘤基因治疗的理想给药方式。
BACKGROUND&OBJECTIVE
Recombinant adenoviruses are powerful vectors for gene therapy since they efficiently infect both proliferating and non-proliferating cells。However several factors restrict their usefulness:the widespread dis-tribution of the coxsackie and adenovirus receptor1(CAR), while allowing infection of a range of tissues, precludes specific in vivo targeting; most people have been exposed to natural adenovirus infection, which confers long-term immunity and may compromise effective virus delivery; even replication-deficient or 'gutless' virus can provoke immune responses, preventing repeated dosing and limiting the duration of therapeu-tic gene expression. So, we should find a way to construct a successful viral gene therapy vector which would be protected from neutralising antibodies and exhibit a preferential tropism for target cells.
The recent strategys to improve adenovirus-mediated immune response include modification of adenovirus and immune control.But neither of them could help the adenovirus evade the immune clearance from immune system. Though great progress has been made on modification of the fiber proteins to enhance infection of tumor cells,it still face the problem of immune clearance from immune system. So, the modification of adenovirus vectors, and the implications of combined strategies for the improvement of the safety will be a key in clinical applications in future cancer treatments.
HPMA polymer is a water-soluble macromolecular drugs,based on the fact that macromolecular drugs exhibit selective accumulation in tumor tissue,the term "EPR"(enhanced permeability and retention)effect in the solid tumor has been introduced.this is because1)Tumor angiogenesis results in hypervasuculature particulatly in tumor circumference where tumor growth is maximal.High vascular density provides more access for drugs in the circulating blood in the tumor.2)Tumor vasculatures become very permeable even for macromolecules and plasma protein due to factors such as tumor vascular permeability factor,bradykinin,TNF and other cytokines derived frominfiltrated leukocytes.3)A less effective function of the lymphatic drainage system is observed in tumor than normal tissue.These polymers are non-immunogenic、non-toxic and can be tailored to the characteristics of the specific target. Nevertheless,the HPMA polymer conjugates are mainly of chemotherapeutic agents. It is a feasible option and needs to be pursued to synthesize HPMA polymer-adenovirus conjugates to evaluate them on various solid tumor.
The modification of pHPMA by target molecule means that target molecule attach to the pHPMA chemically and help the pHPMA being absorbed by the cells quickly,or by the target cells. Cell penetrating peptides(CPPs) are short peptides that are able to penetrate cell membranes and translocate different cargoes into cells. ACPPs are novel vehicles for the translocation of cargo into cells, their properties make them potential drug delivery agents of interest for future use.But reseach on using the ACPPs as the delivery system of adenovirus was not reported till now.
We planed to construct a solid tumor targetted HPMA polymer-Adenovirus deliver system to strengthen the anti-tumor effect of adenovirus.so,we constructed the Ad.mda-7.egfp and demonstrated its activities,devised and synthesized the ACPP and check its ability of penetrating the cell membrane and targeting tumor cell. Synthesized the HPMA polymer and then synthesized the conjugate ACPPs-pc-Ad.mda-7.egfp, ability of the conjugate to penetrate the cell membrane and targeting tumor cell was check in vitro and in vivo. ACPPs-pc-Ad.mda-7.egfp evaded the neutralizing antibodies and help the virus targeted the solid tumor and rapid internalization.we hope the ACPPs-pc-Ad.mda-7.egfp probably develop into a wide used solid tumor targeted deliver system for gene therapy of virus.
METHODS
1. Construction of recombinant adenovirus vector carrying human mda-7/IL-24
AdMAX system was used to construct mda-7/IL-24by recombination. The virus was packaged in293cells and subsequently identified valid. A549and HBE were infected with Ad.mda-7.egfp and control virus with Ad.egfp respectively.The RNA expressing of mda-7/IL-24was indicated by RT-PCR.
2.Assay on Ad.mda-7.egfp selectively induceing growth arrest, apoptosis inA549
the GFP was check by the invert fluorescence microscope,the mda-7protein expressing was demonstrated by IHC. Hoechst and Annexin-V and Hoechst-PI staining were studied to indicate the apoptosis effect. flow cytometry were used to study tumor cell apoptosis in vitro.
3The synthesization of ACPPs and the identification of its penetrating ability
To synthesize a new ACPPs(EEEEEEEEE-PLGLAG-RRRRRRRRN),the ACPPs was labeled by FITC and the purefied,then the uptake of the ACPP was checked by Fluorescence microscopy to analysis the ability of penetrating the cell membrane.
4. The identification of tumor cells target ability of ACPPs
fluorescent cytoehemieal staining to prove the location of ACPPs inside the cell, flow cytometry were used to study the influence of temperature on ACPPs at37℃and4℃,and tumor-targeted ability of ACPPs at different dose;MTT assay was used to study effect of ACPPs on tumor cell proliferation in vitro.
5. The synthesization and the identification of ACPPs-pc-Ad. mda-7.egfp
A random copolymer (pHPMA-ONp) was prepared and Ad5coating was performed by adding25μL pHPMA-ONp (10mg/ml in H2O) to1010particles in100ml Ad.mda-7.egfp10%glycerol/PBS pH7.8, and incubated at4℃for2h to form polymer-coated adenovirus (pc-Ad.mda-7.egfp).For linkage of ACPPs, following an initial2h incubation of virus with pHPMA-ONp, ACPPs was added to a final concentration of250μg/ml and incubated for a further10h. Particle size after virus modification was measured by dynamic light scattering (DLS).Ad.mda-7.egfp, conjugates pc-Ad.mda-7.egfp and ACPPs-pc-Ad.mda-7.egfp(1×1010Ad5,50μl double distilled H2O)were mixed with950μl double distilled H2O. Particle sizing measurements were performed at a wavelength of659.0nm with a detection angle of90°at RT. cells were aliquoted at104cells into96-well plates and infected24h later with104particles per cell of virus or modified virus in200μl DMEM with10%FCS. Green fluorescent protein (gfp) expression was assessed by microscopy. For quantitation of gfp, the cells were lysed in100ml Triton X-100(0.2%v/v in100mm potassium phosphate pH7.8) in96-well plates after infection of the Ad.mda-7.egfp and the conjugates for48h,and gfp fluorescence measured at λex488nm and λem538nm in a Fluoroskan plate reader. FACS analysis was used to measure infection of the virus, A549were at2×105cells/2ml DMEM/10%FCS and incubated in6-well plates at37℃to reach90%confluence before addition of109particles Ad.mda-7.egfp, pc-Ad.mda-7.egfp or ACPPs-pc-Ad.mda-7.egfp labelled with PI, Association of PI labelled virus with cells was measured using a FACS CaliburTM flow cytometer with an argon laser set for λeX540nm and λem625nm.
6.virus neutralisation assays of ACPPs-pc-Ad. mda-7.egfp
For virus neutralisation assays human serum containing Ad5neutralizing antibodies(NAb) was diluted1:20in PBS and heated to56℃for20min to inactivate complement. Diluted serum (100μL) was then incubated with Ad. egfp and ACPPs-pc-Ad.mda-7.egfp at37℃for20min before culture medium was diluted to108particles/100μL. Cells were washed once with PBS before virus solutions were added. After48h incubation with A549cells plated in96-well plates (104cells per well), samples were assayed by measuring gfp expression,and the results expressed as the percentage of the signal obtained in the absence of human serum.
7. Assay on retargeting ability of ACPPs-pc-Ad. mda-7.egfp to alternative cancer cells
HBE,A549, SW480, OVCAR3cells were seeded into96-well plates (104cells per well) and infected24h later with104particles per cell of retargeted virus in200μL DMEM with10%FCS(A549, SW480)or200μL1640with10%FCS(HBE, OVCAR3). For quantitation, the cells were lysed in100μL Triton X-100(0.2%v/v in100mm potassium phosphate pH7.8)and gfp fluorescence measured at λex488nm and λem538nm in a Fluoroskan plate reader and expressed as relative fluorescence units (RFU). Green fluorescent protein (gfp) measured at λex488nm and λem538nm in a Fluoroskan plate reader and expressed as relative fluorescence units (RFU).
8.Intracellular distribution of ACPPs-pc-Ad. mda-7.egfp conjugates
Cells plated on sterile coverslips in6-well plates,2×105cells per well,were grown for24h to reach90%confluence. Cells were incubated with the ACPPs-pc-Ad.mda-7.egfp conjugate or pc-Ad.mda-7.egfp lacking the ACPPs for1h at37℃.For studies involving specific organelle markers Hoechst33342and Propidium iodide (PI) was followed. For studies the internalization experiments included the incubation of cells with double label conjugate (FITC)ACPPs-pc-Ad.mda-7.egfp(PI) or with pc-Ad.mda-7.egfp(PI). For the time dependence studies, cells were incubated with ACPPs-pc-Ad.mda-7.egfp(PI) or pc-Ad.mda-7.egfp(PI) for20min,2or4h. After all incubations, cells were washed thoroughly with PBS, The cells were imaged on a Nikon TI-S microscope and images recorded with a Nikon camera.
9. Assay on apoptosis of A549induced by ACPPs-pc-Ad.mda-7.egfp
Cells plated on sterile coverslips in6-well plates,2×105cells per well,were grown for24h to reach90%confluence. Cells were incubated with1640、the ACPPs-pc-Ad.mda-7.egfp conjugate or pc-Ad.mda-7.egfp lacking the ACPPs for4h at37℃,and then culture for48h. For studies involving specific organelle markers Hoechst33342for15min and Propidium iodide (PI) for10min was followed. After all incubations, cells were washed thoroughly with PBS, The cells were imaged on a Nikon TI-S microscope and images recorded with a Nikon camera.
10. Statistical methods
Experimental data were analyzed by statistical package SPSS13.0. The FITC fluorescence of the cell was analyzed by repetitive measurement analysis of variance, The gfp fluorescence and PI fluorescence of the cell was analyzed by oneway-ANOVA, The Formazan of the cell was analyzed by oneway-ANOVA.
RESULTS
1.Recombinant adenovirus vector carrying human mda-7/IL-24was constructed successfully
Ad.mda-7.egfp was successful constructed by cloning techniques,the vectorwas identified by enzyme digestion,PCR and DNA sequencing.The mda-7gene was620bp,and green fluorescent protein EGFP formed fusion protein with mda-7by restriction sites connection,the whole gene was designed properly and connected correctly.The HEK293cells infected with Ad.mda-7.egfp showed cytopathic effect (CPE).
2Ad.mda-7.egfp selectively induced growth arrest, apoptosis in A549
When A549human lung carcinoma cells were infected with104particles Ad.mda-7.egfp/cell, A549cells showed bright fluorescence in over90%of cells. The Ad.mda-7.egfp protein product was confirmed by assayed the cytoplasm with IHC, Hoechst-PI apoptosis test indicated mda-7/1L-24can induce growth suppression, apoptosis of A549in vitro while Ad.egfp has no diffirence with A549cells.The FACS assay showed that mda-7/1L-24can induce growth suppression, apoptosis of A549but not in normal bronchi epithelial cell line HBE.
3. ACPPs had the ability to penetrate the tumor cells'membrane
Peptides ACPPs was synthesized successfully,the HPLC assay show that molecular weight of ACPPs was2923.18, the formula was C116H200N48O41with99%purity. After labeled with FITC, ACPPs was added to the tumor cell A549、 GLC-82、OVCAR3、MCF-7、SPCAland HepG2for2h incubation.The result of fluorescent cytoehemieal staining showed that ACPPs could penetrating the cell membrane.
4.Peptide ACPPs had the ability to target tumor cells
The result of fluorescent cytoehemieal staining showed that ACPPs distributed in cytoplasm for2h incubation, flow cytometry assay prove that the value of the fluorescence in A549at37℃much more than that at4℃(p<0.05),and Fluoroskan plate reader assay showed that the value of the fluorescence in A549、、 SW480、OVCAR3、MDA-MB-231、and HT29much high than that of HBE and HEK293(p<0.05).The results of MTT assay proved that cytotoxicity of ACPPs to A549cells had no difference with control(DMEM with10%FCS) by treatment of ACPPs from the dose of1uM to the dose of100uM(p>0.05).
5.ACPPs-pc-Ad. mda-7.egfp was synthesized successfully
Polymer-coated Ad.mda-7.egfp was prepared by mixing HPMA copolymer with Ad5and ACPPs in10%glycerol/PBS pH7.8, resulting in polymer molecules linked to each virus particle and ACPPs. Analysis of particle size by dynamic light scattering (DLS) showed an average diameter of the unmodified virus of141.8nm, compared with189.4nm for the pc-Ad.mda-7.egfp and236.4run for the ACPPs-pc-Ad. egfp. gfp expression was measured in a fluorescence plate reader to show the ability of the virus to infect A549cells, pc-Ad.mda-7.egfp was essentially ablated by the coating procedure, while ACPPs-pc-Ad.mda-7.egfp penetrating the A549cells successfully by ACPPs showed little ablated of gfp expression. flow cytometry assay showed that uptake of Ad.mda-7.egfp by A549cells during4h incubation at37℃was effectively abolished by the presence of the polymer coat (Figure2c), while the propidium iodide could be easily checked by the penetratation of ACPPs,confirmed that pc-Ad.mda-7.egfp did not bind to cells,though ACPPs facilated the internalization of Ad.mda-7.egfp.
6.ACPPs-pc-Ad. mda-7.egfp had the ability to evade neutralising antibody
The ability of ACPPs-pc-Ad.mda-7.egfp to evade neutralising antibody was tested by determining the inhibition of adenoviral transduction. Incuba-ting modified virus in the presence of human serum which was known to contain anti-adenovirus neutralising antibodies,. Polymer coating protect-ed the virus against NAb binding, While NAb significantly inhibited Ad5transduction. shows that ACPPs-pc-Ad.mda-7.egfp is9-fold more resistant to neutralisation than Ad.mda-7.egfp Retargeted pc virus was less susceptible to inhibition of infection than the unmodified virus.
7.ACPPs-pc-Ad. mda-7.egfp retargeted alternative cancer cells
To test the feasibility of retargeting,ACPPs was chosen because it has previously prove to retarget cargoes to MMPs-overexpressed cells. targeting ACPPs were attached chemically to the reactive ester groups remaining on the surface of the pc virus and modified virus was purified from free polymer and free targeting agents using S400spin columns27-5140-01as before. After1h incubation HBE,A549, SW480, OVCAR3cells with ACPPs-pc-Ad.mda-7.egfp(PI) at37℃, The ability of ACPPs to target the MMPs-overexpressed cells was determined by measuring gfp expression. Under conditions of Ad.mda-7.egfp(PI) infecting, cellular uptake levels of ACPPs-pc-Ad.mda-7.egfp(PI) was for A549, SW480, OVCAR3and HBE cells. ACPPs-pc-Ad.mda-7.egfp showed relatively low infectivity in HBE cells,while significantly enhanced gfp expression could be seen in A549, SW480, OVCAR3cells, suggesting that selective infection in MMPs-overexpressed tumor cells should be feasible.
8.ACPPs mediated nonendocytotic transportation of pc-Ad.mda-7. egfp to the cytoplasm
Cytoplasmic import was tested by co-incubating the cells with cytoplasmic marker Hoechst33342(blue fluore-scence) and ACPPs-pc-Ad.mda-7.egfp(PI)(red fluorescence). Hoechst33342and PI on the Ad.mda-7.egfp were separately tracked by fluorescence microscopy and superimposition of the two dyes resulted in intracellular blue-red stained areas, confirming the cytoplasmic delivery of ACPPs-pc-Ad.mda-7.egfp and endocytotic uptake of pc-Ad.mda-7.egfp. Intracellular distribution of the ACPPs-pc-Ad.mda-7.egfp was studied by fluorescence microscopy.The internalization due to the ACPPs peptide accumulated in the cytoplasm as was inferred from diffuse staining visible within the cell body. FITC on the ACPPs and PI on the Ad.mda-7.egfp were separately tracked by fluorescence microscopy and superimposition of the two dyes resulted in intracellular blue-red stained areas. These results are significant as the intracellular delivery of the conjugate ACPPs-pc-Ad.mda-7.egfp was achieved by means of ACPPs.
9. ACPPs-pc-Ad.mda-7.egfp induced the apoptosis of A549cell
When A549human lung carcinoma cells were infected with104particles Ad.mda-7.egfp/cell, A549cells with1640showed low blue fluorescence in over90%of cells,while few red fluorescence can be seen; A549cells with pc-Ad.mda-7.egfp showed bright blue fluorescence in few cells,and few red fluorescence can be seen; A549cells with ACPPs-pc-Ad.mda-7.egfp showed bright blue fluorescence in most cells,anda lot of red fluorescence can be seen.
CONCLUSIONS
1. The construction of Ad.mda-7.egfp was successful, mda-7protein was expressed highly in the A549and HBE cells.
2. Ad.mda-7.egfp selectively induced apoptosis and growth suppression of A549,it maybe an useful way for cancer gene therapy.
3. The synthetization of ACPPs was successful, and it can penetrate the cell membrane
4. ACPPs can target the cancer cell with no cytotoxicity to A549cells.
5. The conjugate ACPPs-pc-Ad.mda-7.egfp was successfully syntheti-zed and characterized, ACPPs-pc-Ad.mda-7.egfp could penetrate the tumor cells'membrane.
6. ACPPs-pc-Ad. mda-7.egfp could evade neutralising antibody and retargeted alternative cancer cells
7. ACPPs mediated nonendocytotic transportation of pc-Ad. mda-7.egfp to the cytoplasm
PROSPECT
1. ACPPs-pc-Ad. mda-7.egfp could evade neutralising antibody and retargeted alternative cancer cells,and it fitted for most solid tumor cells.It will be an ideal model for cancer gene therapy.
2. ACPPs-pc-Ad. mda-7.egfp could be used to enhanced the efficiency of chemotherapy and radiotherapy.
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