新型纳米复合载体的构建及其介导NK4基因在治疗乳腺癌中的应用
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摘要
前言
随着医学分子生物学研究的深入和应用,基因治疗逐渐成为肿瘤生物治疗学中的重要组成部分,在乳腺癌的治疗中显示出良好的应用前景。目前,乳腺癌基因治疗尚存在一些问题,首先,用于研究和应用临床的基因转染方法有多种,但转染效果仍不令人满意。例如,逆转录病毒转染率较高且表达稳定,但它仅对增殖活跃的细胞才有较高的转染率,且在转染过程中可整合到宿主细胞而发生插入型突变和癌基因激活,不适于介导人体内基因的转移。腺病毒所携带的基因虽具有较高的转染率但不能整合到宿主细胞的染色体上,不能长期稳定表达,转染靶细胞后会产生免疫反应。而且,乳腺癌基因治疗缺乏特异性和高效性载体,若仅部分肿瘤细胞被导入基因,治疗效果会受到影响,而载体特异性差则会导致癌旁组织及骨髓等其他重要组织被累及。因此,加强基因治疗的靶向性、安全性,开发出高效的新载体系统已成为该领域研究的热点。
阳离子高分子聚合物聚乙烯亚胺(polyethylenimine, PEI)因其能有效浓缩DNA分子形成稳定的纳米粒子,保护DNA分子的生物活性、避免酶解、并能被细胞有效内吞,从而在细胞内实现DNA分子的解离释放和表达,已成为非病毒阳离子聚合物载体中最成功的例子,是设计新的阳离子聚合物转染载体的标准。当前研究主要从降低细胞毒性,提高转染效率及细胞靶向性等方面对PEI进行改造。最吸引人的策略之一是利用受体介导的细胞内吞作用来替代PEI和细胞间的非特异静电作用,这种主动的靶向需要使用高亲和性和特异性的配体识别结合部位的靶细胞表面的受体。透明质酸(hyaluronic acid, HA),作为抗肿瘤药物的靶向载体,能够将较小的药物分子黏附在它的网状结构中或者将药物分子接枝到透明质酸类药物的载体上,所形成的粒子或复合物与肿瘤细胞表面的受体靶向结合,使更多的药物分子进入肿瘤组织,增加抗肿瘤药在肿瘤和淋巴结中的吸收和滞留时间,从而提高药物的疗效,降低毒副作用。本实验将HA作为靶向配体修饰PEI/DNA复合物,构建HA/PEI/DNA三元纳米复合物,并对其进行表征和性能检测。
由于乳腺癌的基因治疗缺乏更为有效的目的基因,NK4作为HGF的拮抗剂,具有血管新生抑制和癌浸润抑制两大作用,这种双重作用在裸鼠乳腺癌移植试验上已获得了证实,即提示它可成为具备这两大功能的崭新抗癌药。因此本实验采用NK4为目的基因,首次构建HA/PEI/PCAGGS-NK4纳米复合物,考察其在体外对乳腺癌细胞的作用,并建立裸鼠乳腺癌移植瘤模型,观察三元复合物HA/PEI/PCAGGS-NK4在体内的抑瘤效果。
实验方法
一、HA/PEI/DNA三元纳米复合物的构建及性能检测
1、HA/PEI/DNA三元复合物的构建与表征
制备不同HA与PEI负正电荷摩尔比的HA/PEI/DNA三元复合物,琼脂糖凝胶电泳进行DNA凝胶阻滞实验,马尔文粒度分析仪检测纳米粒的粒径分布及Zeta电位,比较其在水及BSA中的粒径变化。
2、HA/PEI/DNA三元复合物的体外细胞转染实验
取乳腺癌细胞MDA-MB-231, MCF-7, MDA-MB-435,转染一定量上述不同电荷比的纳米复合物,在流式细胞仪中检测转染率。
3、乳腺癌细胞系CD44表达水平
三种乳腺癌细胞用无血清培养液培养。收集细胞的培养上清,细胞抽提物总蛋白采用BCA蛋白分析试剂盒定量。SDS-PAGE电泳后,转膜,加CD44s单克隆抗体孵育后,加入辣根过氧化物酶标记的兔抗鼠二抗,显色,以β-actin为内标。
4、三元复合物对COS-1细胞的毒性试验
对数生长期的COS-1细胞,加入不同浓度的不同HA与PEI电荷比的三元复合物,继续培养48h后,加MTT液振摇,孵育4h后加入DMSO,裂解细胞,混匀,选择570nm波长,用酶标仪测定各孔光吸收值。
二、HA/PEI/PCAGGS-NK4纳米复合物对乳腺癌细胞作用的研究
1、HA/PEI/PCAGGS-NK4纳米粒的制备与表征
制备纳米粒HA/PEI/PCAGGS-NK4与H A/PEI/PC AGGS-LacZ,进行DNA凝胶阻滞实验,测定粒径和Zeta电位,方法均同上述三元复合物的构建与表征方法。
2、Western-blot检测乳腺癌细胞NK4蛋白的表达
MDA-MB-231, MDA-MB-435, MCF-7细胞,加入用无血清培养基稀释含100μg/ml PCAGGS-NK4质粒的纳米复合物,4h后加入含有血清的培养基培养,回收各组细胞加入细胞裂解液,刮取细胞裂解物,进行SDS-PAGE分离蛋白条带,转膜后,加入NK4一抗(人HGF单抗)孵育,再加入辣根过氧化物酶标记的二抗,β-actin做对照。
3、MTT法测定细胞增殖能力
三种乳腺癌细胞过夜贴壁后,换液加入无血清培养基稀释的不同浓度HA/PEI/PCAGGS-NK4与HA/PEI/PCAGGS-LacZ纳米复合物,绘制细胞生长曲线,比较各组细胞的增殖能力。
4、Transwell法测定癌细胞侵袭能力
细胞贴壁后换液加入用无血清培养基稀释的含不同浓度PCAGGS-LacZ, PCAGGS-NK4质粒的纳米复合物,12h后用胰酶消化,接种于Transwell小室的上室,其下室中加入含10%新生小牛血清的培养液,12h后取出小室,固定,染色,高倍光镜下计数5个不同视野的穿过膜的细胞数。
5、流式细胞仪分析细胞凋亡率
将被染色分析的细胞用冷PBS洗涤二次并在恰当的染色缓冲液中重悬。吸取100ul的细胞(1×105)至试管中。加入适量的荧光标记的Annexin V试剂和PI,混匀后避光室温下孵育15min后加入400ul染色缓冲液,立即上流式细胞仪分析。取少量滴片,置荧光镜下观察细胞凋亡形态,计算凋亡、坏死百分率。
三、HA/PEI/PCAGGS-NK4对裸鼠移植瘤生长抑制作用的考察
1、建立人乳腺癌细胞裸鼠移植瘤模型与给药
BALB/c (nu/nu)裸鼠30只,每只接种0.2ml(1×107／ml MDA-MB-231)细胞于裸鼠右侧胸壁第二乳头下脂肪垫内。8d后,随机分为3组,每组10只,分别命名为：(1)对照组,即HA/PEI/PCAGGS-LacZ注射组(肿瘤及肿瘤周围多点注射200μl质粒溶液,含100μgPCAGGS-LacZ);(2)给药组,即HA/PEI/PCAGGS-NK4注射组(肿瘤及肿瘤周围多点注射200μl质粒溶液,含100μgPCAGGS-NK4);(3)阳性对照组,即阿霉素组,0.2ml(含阿霉素100μg)腹腔注射,绘制肿瘤生长曲线,抑瘤率=(1-处理组平均瘤重／对照组平均瘤重)×100%。
2、Western blot检测移植瘤细胞NK4蛋白的表达
称取于液氮中保存的组织0.2g,以预冷的PBS洗3次并用眼科剪将组织剪碎,在10倍于其体积的裂解缓冲液中迅速搅成匀浆,4℃离心10min,吸出上清液为细胞总蛋白,用Bradford法测蛋白浓度。经SDS-PAGE电泳后转膜,再用含5%脱脂奶粉的TBST液封闭,然后加入一抗、二抗孵育,用ECL发光法检测不同样品NK4蛋白表达状况。
四、统计学分析
采用SPSS11.0统计软件处理,计量资料以均数±标准差(x±s)表示,采用one-way ANOVA分析,组间比较采用LSD检验,P<0.05为差异有统计学意义。
实验结果
一、HA/PEI/DNA三元纳米复合物的构建及性能检测
1、HA/PEI/DNA三元复合物的构建与表征
制备的HA/PEI/DNA三元复合物经琼脂糖凝胶电泳,无DNA条带出现,证明其能有效结合DNA分子。随着HA量的增加,复合物粒径在双蒸水中略有增加,在BSA中的粒径无显著增大,Zeta电位值则逐渐降低,且当HA与PEI电荷比为100%时,Zeta电位呈现负值。
2、HA/PEI/DNA三元复合物的体外细胞转染实验
随着HA的加入,三元复合物在MDA-MA-231与MDA-MB-435中的转染率明显高于二元复合物PD(P<0.05),增加了2-13倍,当HA与PEI电荷比为7.5%时,三元复合物在三种细胞的转染率最高,同其他各组比较均具有显著差异(P<0.05)。
3、乳腺癌细胞系CD44表达水平
Western blotting结果表明,MDA-MB-435和MDA-MB-231的CD44s的表达水平明显高于MCF-7。平均表达水平分别为1.15±0.036,0.92±0.072和0.16±0.028。
4、三元复合物对COS-1细胞的毒性试验
结果表明：未修饰的PEI/DNA复合物(N／P,10)会引起COS-1细胞毒性,使细胞活力下降到58%。HA的加入使制备的三元复合物HA/PEI/DNA毒性大大降低,细胞存活率均达到95%以上,几乎无毒。
二、HA/PEI/PCAGGS-NK4纳米复合物对乳腺癌细胞作用的研究
1、HA/PEI/PCAGGS-NK4纳米粒的制备与表征
制备的纳米粒HA/PEI/PCAGGS-NK4与HA/PEI/PCAGGS-LacZ能有效结合DNA分子。随着HA量的增加,纳米粒在BSA中不会引起聚集,Zeta电位则逐渐降低,且当HA与PEI电荷比为100%时,Zeta电位呈现负值。
2、Western-blot检测乳腺癌细胞NK4蛋白的表达
MDA-MB-231, MDA-MB-435,和MCF-7三种细胞系内源性的HGF平均表达水平分别为0.91±0.062,0.83±0.029和0.33±0.017。三种细胞系转染100μg／mlHA/PEI/PCAGGS-NK4三元复合物后所表达的外源NK4蛋白的平均值分别为：2.23±0.039,1.91±0.062,1.30±0.018。
3、MTT法测定细胞增殖能力
空白质粒不影响细胞的增殖,随HA/PEI/PCAGGS-NK4三元复合物处理浓度的增加,细胞增殖有逐渐受到抑制的趋势,在浓度剂量为100μg／ml,200μg／ml,500μg／ml时,细胞增殖被明显抑制,与对照组比较均有显著性差异(P<0.05)。在各种HA/PEI/PCAGGS-NK4浓度下MDA-MB-231细胞增殖受抑制的程度最大(P<0.05),MDA-MB-435细胞的抑制率也明显高于MCF-7细胞(P<0.05)。
4、Transwell法测定癌细胞侵袭能力
未经转染处理空白细胞的侵袭能力为MDA-MB-231> MDA-MB-435>MCF-7,加入对照组HA/PEI/PCAGGS-LacZ处理后,随HA/PEI/PCAGGS-LacZ浓度增加,对照组与空白细胞组的细胞侵袭能力无显著性差异(P>0.05)。而给药组在加入不同浓度的HA/PEI/PCAGGS-NK4处理后,三种细胞的侵袭迁移力均有不同程度的减弱,差异均有统计学意义(P<0.05)。
5、流式细胞仪分析细胞凋亡率
未经转染处理的空白MCF-7,MDA-MB-231,MDA-MB-435细胞系,其凋亡百分率分别为2.41±0.05%,2.13±0.06%,2.32±0.09%。加入不同浓度空质粒对照组后,空质粒对照组与空白细胞组的凋亡率无显著性差异(P>0.05)。而给药组在加入不同浓度的HA/PEI/PCAGGS-NK4的复合物后,MCF-7, MDA-MB-231,MDA-MB-435三种细胞的凋亡百分率均有不同程度的升高,差异具有统计学意义(P<0.05)。
三、HA/PEI/PCAGGS-NK4对裸鼠移植瘤生长抑制作用的考察
1、建立人乳腺癌细胞裸鼠移植瘤模型与给药
各组移植瘤裸鼠一般情况较好。接种MDA-MB-231细胞8d左右,3组裸鼠均于接种部位出现微小隆起。成瘤率100%。阳性对照组裸鼠在两次给药后,个别裸鼠出现明显不适而死亡。对照组裸鼠瘤体的生长明显迅速,40天时终体积达到445.32±26.65mm3,而阳性对照组和给药组裸鼠瘤体的增长均被显著抑制(P<0.01),40天时终体积分别达到272.96±24.57 mm3,225.64±17.36 mm3。阳性对照组、给药组抑瘤率分别为：为47.75%,55.81%。
2、Western blot检测移植瘤细胞NK4蛋白的表达
空白对照组与阳性对照组均有少量HGF的表达,表达量分别为0.89±0.03,0.97±0.02,给药组表达水平为3.33±0.05,与空对照组比较蛋白表达明显增多(P<0.05)。
结论
1、所构建的HA/PEI/DNA能有效结合DNA分子,在BSA中不会引起聚集。HA的加入使纳米复合物在三种乳腺癌细胞中的转染效率均有增加,在MDA-MB-231,MDA-MB-435(高CD44S表达的)两种细胞中转染率明显升高,产生了细胞靶向性,当HA与PEI电荷比为7.5%时,HA/PEI/DNA在三种细胞中的转染率最高。三元复合物对COS-1细胞的毒性相对于PEI/DNA二元复合物大大降低,几乎无毒。
2、HA/PEI/PCAGGS-LacZ及HA/PEI/PCAGGS-NK4三元复合物在BSA中不会引起聚集,质粒的结构并不影响纳米粒的粒径及Zeta电位等物理性质。三种乳腺癌细胞系被HA/PEI／PCAGGS-NK4转染后,表达了NK4外源蛋白,随着转染复合物浓度的增加,细胞增殖有逐渐受到抑制的趋势,细胞的侵袭迁移力均有不同程度的减弱,凋亡百分率均有不同程度的增加。差异具有统计学意义(P<0.05)。
3、成功建立人乳腺癌细胞裸鼠移植瘤模型,阳性对照组(阿霉素组)和给药组(HA/PEI/PCAGGS-NK4三元复合物组)裸鼠瘤体的增长均被显著抑制(P<0.01),给药组裸鼠体内有一定水平的NK4蛋白表达。
With the in-depth study of medical molecular biology and application, gene therapy for cancer has become an important biological therapeutic method and showed good application prospects in the treatment of breast cancer. At present, gene therapy for breast cancer contains the following issues, at first, there have been several gene transfer methods for research or clinical, but the transfection efficiency is still not satisfactory. The retrovirus carriers have high transfection efficiency and a stable expression, but it is only active in proliferating cells, and it can be integrated into the host cell and occurr the insertion mutation and oncogene activation in the process of transfection, so it is unfit for human-mediated gene transfer in vivo. The gene mediated by Adeno-virus carriers can not be integrated into the host cell's chromosomes, although it has a high transfection efficiency but can not transfect the target cells with a long-term stable expression, induce immune response also. Meanwhile, lack of carrier with specificity and efficiency, if the tumor cells are only partially transfected, the treatment will be affected, while the vector with low specificity can lead to victimize cancer tissues and bone marrow and other major organizations. Therefore, gene therapy is necessary to enhance the target, safety, developing a new and efficient vector system has become a focus research.
Cationic polymer polyethyleneimine (PEI) has become the most successful example among the non-viral cationic polymer vector and has been the standards to design a new cationic polymer transfection carrier, because it can condense DNA molecules effectively to form stable nano-particles and protect the DNA molecule's biological activity, avoid hydrolysis, effective endocytosis, and dissociation to achieve the release of DNA molecules. Current research mainly focus on the lower cytotoxicity, high transfection efficiency and cell targeting to transform PEL One of the most attractive strategy is applying receptor-mediated endocytosis to replace the non-specific electrostatic interaction between PEI and the cell-cell, it is essential to use targeted ligand binding parts of the targeted receptors on cell surface with high affinity recognition and specificity. Hyaluronic acid (HA), as a targeted vector for anti-cancer drugs, can stick small drug molecules to its network structure or graft drug molecules to the carrier of hyaluronic acid-type drugs, the formed particles or complexes combined with the receptor-targeted tumor cell surface, so that more drug molecules arrive the tumor tissue, increase tumor uptake and retention time in tumor and lymph nodes, thereby enhance the efficacy of drugs and reduce the toxic or side effects. In this study, HA as a targeting ligand was to modify the PEI/DNA complexes for the first time, construct HA/PEI/DNA ternary nanocomposites, and investigate its characterization and performance.
Currently gene therapy of breast cancer need a more effective targeted gene, NK4 as an HGF antagonist, has two roles of angiogenesis inhibition and tumor invasion suppression, which has been confirmed in transplant trials of breast cancer in nude mice, it prompted that NK4 could be a new anti-cancer drug with both of the unique features.Therefore,this experiment choose NK4 gene to build HA/PEI/PCAGGS-NK4 nanocomposites for the first time, and its effects on breast cancer cells were studied in vitro, established the breast cancer xenograft model in nude mice andobserved the antitumor effect of HA/PEI/PCAGGS-NK4 ternary complex in vivo.
1. Construction and performance test of the HA/PEI/DNA ternary nanocomposites
(1) Construction and characterization of the HA/PEI/DNA ternary complexes
To prepare HA/PEI/DNA ternary complex with different charge ratios of HA and PEI, agarose gel electrophoresis, DNA gel retardation experiments, particle size distribution of nanoparticles and Zeta Potential was analyzed by Malvern, compared the particle size in BSA and in water.
(2) Cell transfection experiments of HA/PEI/DNA ternary complex in vitro.
Breast cancer cells MDA-MB-231, MCF-7, MDA-MB-435 were added a certain amount of nano-composites with different charge ratios, transfection efficiency was detected in the flow cytometer.
(3) CD44 expression was detected in breast cancer cell lines by western blot
Three kinds of breast cancer cells were cultured in serum-free culture medium. Collection of cell supernatants, total protein of cell extracts used BCA Protein Assay Kit quantitatively. After SDS-PAGE electrophoresis, transferred to NC film, plused CD44s monoclonal antibody, then added the rabbit anti-mouse secondary antibody labeled by horseradish peroxidase, color, (3-actin was used as the internal standard.
(4) COS-1 cells toxicity of the ternary complexes was determined by MTT
COS-1 cells in Logarithmic phase were managed by ternary complex with different concentrations and different charge ratio of HA and PEI, continue to culture 48 h, plused MTT solution, shaked slowly, incubated 4h, added DMSO, Selected wavelength 570nm, measured the absorbed value by UV spectrophotometry.
2.Study on the role of the HA/PEI/PCAGGS-NK4 nanocomposites in the breast cancer cells
(1) Preparation and Characterization of the HA/PEI/PCAGGS-NK4 nanoparticles Preparation of nanoparticles HA/PEI/PCAGGS-NK4 with HA/PEI/ PCAGGS-LacZ, DNA gel retardation experiments, characterized by particle size and Zeta potential, the methods were same with the HA/PEI/DNA ternary complex in section 1.
(2) NK4 protein expression in breast cancer cells was measuered by western blotting
MDA-MB-231, MDA-MB-435, MCF-7 cells, added with 100μg/ml PCAGGS-NK4 plasmid serum-free medium Nanocomposite,4h plused serum medium to culture, recovered the cell, cell lysate were scraped to conduct SDS-PAGE separation of protein bands, transfer film, add NK4 an anti-(human HGF monoclonal antibody), then add horseradish peroxidase labeled secondary antibody,β-actin as controls.
(3) Cell proliferation was determined by MTT
Three kinds of breast cancer cells were exchanged the medium containing different concentrations of nano-composite material with serum-free medium diluted PCAGGS-LacZ, PCAGGS-NK4 plasmid. Drew cell growth curve. Compared the cell proliferation of each group.
(4) Invasive ability of cancer cells was investigated by Transwell
Adherent cells were exchanged medium containing different concentrations of PCAGGS-LacZ, PCAGGS-NK4 plasmid nanocomposites, after 12h, were digested with trypsin and seeded in the upper Transwell chamber, joined the culture medium with 10%newborn calf serum to the lower room, after 12h, removed the small rooms, fixed, stained, count the number of the cells through the membrane under the microscope in five different point of view.
(5) Apoptosis rate was analyzed by flow cytometry
The pre-staining cells were washed twice with cold PBS, re-suspended in staining buffer. Took 100ul cells (1x105) to a test tube. Adding an appropriate amount of fluorescent labeled Annexin V reagents and PI, mixed in dark, after incubating for 15 minutes at room temperature, added 400ul staining buffer and immediately analyzed on a flow cytometer. Took small drops to films and observed the morphology of apoptosis under fluorescent microscope, caculated the apoptosis, necrosis percentage.
3.Investigate the effect of HA/PEI/PCAGGS-NK4 on tumor growth in nude mice
(1) Establish the xenografts of human breast cancer cell in nude mice and administration
BALB/c (nu/nu) nude mice 30, each was vaccined 0.2ml (1×107/ml MDA-MB-231) cells under the second nipple on the right chest fat pad. Afer 8d, they were randomly divided into 3 groups, named respectively as followed:(①control group, the HA/PEI/PCAGGS-LacZ injection group (multi-point injection of 200μl plasmid solution containing 100μg PCAGGS-LacZ at tumor and tumor-surrounding);②treatment group, namely, HA/PEI/PCAGGS-NK4 injection group (multi-point injection of 200μl plasmid solution containing 100μg PCAGGS-NK4 at tumor and tumor-surrounding);③positive control group, namely, adriamycin group,0.2ml (including adriamycin 100μg) injected intraperitoneally. Drawing tumor growth curve. Inhibition rate= (1-mean tumor weight of treatment group/mean tumor weight of control group)×100%.
(2) NK4 protein expression was detected in xenografts cells by western blot
Prestored in liquid nitrogen, the organization (0.2g) were take out to wash by pre-cooling PBS for three times, Shredded with scissors, milled into homogenate rapidly in 10 times of its volume lysis buffer,4℃in centrifuge 10min, aspirated supernatant of total cellular protein, the protein concentration measured by Bradford method. DS-PAGE electrophoresis, transferred to NC film, and then closed with 5% skim milk TBST solution, and then added HGF monoclonal antibody, then added the rabbit anti-mouse secondary antibody labeled horseradish peroxidase, detected NK4 protein expression in different samples with ECL chemiluminescence.
4.Statistical analysis
Applied the statistical software SPSS 11.0, the measured data showed as mean± standard deviation (x±s), one-way ANOVA analysis and LSD test were used to compare between groups, P<0.05 for the statistically significant difference.
Results
1.Construction and Performance Test of the HA/PEI/DNA ternary nanocomposites
(1) Construction and characterization of the HA/PEI/DNA ternary complexes
The prepared HA/PEI/DNA ternary complexes were managed by agarose gel electrophoresis, the results show that no DNA bands appear, the complexes can effectively combine DNA molecules. With the increasing amount of HA, the size of the composite increased gradually in the double distilled water and decreased in the BSA, Zeta potential gradually decreased, when the HA and PEI charge ratio to 100%, Zeta potential was negative.
(2) Cell transfection experiments
With adding the HA, the transfection efficiency of the ternary complexes in MDA-MA-231 and MDA-MB-435 was significantly higher than that of binary complexes PD (P<0.05), has a 2-13 times increased, when the HA and PEI charge ratio was 7.5%or 100%, the transfection rate of the ternary complexes in MCF-7, MDA-MA-231 and MDA-MB-435 was significantly higher than that of PD (P<0.05). When the HA and PEI charge ratio of 7.5%, the ternary complex had the highest transfected efficiency in the three kinds of cells compared with the other groups. Difference was significant (P<0.05).
(3) CD44 expression
The results of western blotting showed that the expression of CD44s was significantly higher in MDA-MB-435, MDA-MB-231 than in MCF-7. The average expression levels were 1.15±0.036,0.92±0.072 and 0.16±0.028.
(4) COS-1 cells toxicity
The results showed that the non-modified PEI/DNA complexes (N/P,10) will cause COS-1-cell toxicity, so that cell viability decreased to 58%. HA was added to construct the ternary complex HA/PEI/DNA which had significantly reduced toxicity, cell survival rates were 95%, almost non-toxic.
2.Study on the role of the HA/PEI/PCAGGS-NK4 nanocomposites in the breast cancer cells
(1) Preparation and Characterization of the HA/PEI/PCAGGS-NK4 nanoparticles
The prepared HA/PEI/PCAGGS-NK4, HA/PEI/PCAGGS-LacZ nanoparticles could effectively integrated DNA molecules. With the increasing amount of HA, Zeta potential decreased gradually in the BSA, nanoparticles did not cause aggregation, when the HA and PEI charge ratio was 100%, Zeta potential was negative.
(2) NK4 protein expression in breast cancer cells
The average expressive levels of endogenous HGF in MDA-MB-231, MDA-MB-435, and MCF-7 cells respectively were 0.91±0.062,0.83±0.029 and 0.33±0.017. after 100μg/ml HA/PEI/PCAGGS-NK4 was transfected in three kinds of cell lines, the average exogenous expression of NK4 protein respectively were:2.23±0.039,1.91±0.062,1.30±0.018.
(3) Cell proliferation
The results showed that:the blank plasmid does not affect cell proliferation, with the increasingconcentration of PCAGGS-NK4, cell proliferation was inhibited gradually, when the concentration of PCAGGS-NK4 arrive the dose of 100μg/ml, 200μg/ml,500μg/ml, cell proliferation was significantly inhibited compared with that of the control group(P<0.05). The HA/PEI/PCAGGS-NK4 with various concentration had the largest inhibited effect on MDA-MB-231 cells (P<0.05), and the inhibited effect in MDA-MB-435 cells was significantly higher than MCF-7 cells (P<0.05).
(4) Invasive ability of cancer cells
The invasion capacity comparison of the cells without treatment by transfection were:MDA-MB-231>MDA-MB-435>MCF-7. Invasive ability had no significant difference between cells treated with HA/PEI/PCAGGS-LacZ and the cells without treatment (P>0.05). Treated by the increasing concentrations of HA/PEI/PCAGGS-NK4, three kinds of cells had relative lower invasion capacity to a certain degree, the differences were statistically significant (P<0.05).
(5) Apoptosis rate
Apoptosis rate of the MCF-7, MDA-MB-231, MDA-MB-435 cell line without treatment by transfection were 2.41±0.05%,2.13±0.06%,2.32±0.09%. Apoptosis rate had no significant difference between cells treated with HA/PEI/PCAGGS-LacZ and the cells without treatment (P>0.05). after Treated by the increasing concentrations of HA/PEI/PCAGGS-NK4, MCF-7, MDA-MB-231, MDA-MB-435 cells had relative higher percentage of apoptosis.The difference was statistically signifiant (P<0.05).
3.Investigate the effect of HA/PEI/PCAGGS-NK4 on tumor growth in nude mice
(1) Establish the xenografts of human breast cancer cell in nude mice and administration
The xenografts in each group nude mice were generally good. After inoculated MDA-MB-231 cells for 8d, small bulge appeared at the inoculation site on three groups of nude mice. Tumorigenicity rate was 100%. The individual positive control group of nude mice died for apparent discomfort after administration. The tumor growth were significant and rapid in control group of nude mice, reach to the final volume of 445.32±26.65 after 40 days, while the tumor growth in positive control group and treatment groupof nude mice were significantly inhibited (P<0.01), final volume were respectively 272.96±24.57 and 225.64±17.36 after 40 days.Tumor inhibition rates of positive control group and treatment group were 47.75%,55.81%(compared with control group, P<0.05).
(2) NK4 protein expression in xenografts
The blank control group and the positive control group have a small expressive amount of HGF, expressive levels were 0.89±0.03 and 0.97±0.02, protein expression in treatment group was significantly increased compared with the blank control group (P<0.05), the expressive level of treatment group was up to 3.33±0.05.
Conclusions
1.HA/PEI/DNA could combined DNA molecules effectively and could not cause aggregation in the BSA. HA was added to construct the nano-composite, could significantly increase the transfection efficiency in MDA-MB-231, MDA-MB-435 (high-CD44S expression), produced cell targeting effect, when the HA and PEI charge ratio was7.5%, HA/PEI/DNA had the highest transfection efficiency in three kinds of cells.The toxicity of ternary complex on COS-1 cells was greatly reduced compared with the PEI/DNA binary complexes, showed almost non-toxic.
2.HA/PEI/PCAGGS-LacZ and HA/PEI/PCAGGS-NK4 ternary complex could not cause aggregation in the BSA, it showed that the structure of plasmid could not affect the size and Zeta potential such as physical properties of nanoparticles. Three kinds of breast cancer cell lines transfected by HA/PEI/PCAGGS-NK4 had expressed exogenous NK4 protein; cell proliferation was inhibited; cell invasive capacity was declined; apoptosis rate was increased. The differences were all statistically signifiant (P<0.05).
3.Successful established the xenografts of human breast cancer cell in nude mice, the tumor growth were significantly inhibited in positive control group (doxorubicin group) and treatment group (HA/PEI/PCAGGS-NK4 ternary complex group) nude mice,and treatment group nude mice had showed a certain level of NK4 protein expression in xenografts cells.
随着医学分子生物学研究的深入和应用,基因治疗逐渐成为肿瘤生物治疗学中的重要组成部分,在乳腺癌的治疗中显示出良好的应用前景。目前,乳腺癌基因治疗尚存在一些问题,首先,用于研究和应用临床的基因转染方法有多种,但转染效果仍不令人满意。例如,逆转录病毒转染率较高且表达稳定,但它仅对增殖活跃的细胞才有较高的转染率,且在转染过程中可整合到宿主细胞而发生插入型突变和癌基因激活,不适于介导人体内基因的转移。腺病毒所携带的基因虽具有较高的转染率但不能整合到宿主细胞的染色体上,不能长期稳定表达,转染靶细胞后会产生免疫反应。而且,乳腺癌基因治疗缺乏特异性和高效性载体,若仅部分肿瘤细胞被导入基因,治疗效果会受到影响,而载体特异性差则会导致癌旁组织及骨髓等其他重要组织被累及。因此,加强基因治疗的靶向性、安全性,开发出高效的新载体系统已成为该领域研究的热点。
阳离子高分子聚合物聚乙烯亚胺(polyethylenimine, PEI)因其能有效浓缩DNA分子形成稳定的纳米粒子,保护DNA分子的生物活性、避免酶解、并能被细胞有效内吞,从而在细胞内实现DNA分子的解离释放和表达,已成为非病毒阳离子聚合物载体中最成功的例子,是设计新的阳离子聚合物转染载体的标准。当前研究主要从降低细胞毒性,提高转染效率及细胞靶向性等方面对PEI进行改造。最吸引人的策略之一是利用受体介导的细胞内吞作用来替代PEI和细胞间的非特异静电作用,这种主动的靶向需要使用高亲和性和特异性的配体识别结合部位的靶细胞表面的受体。透明质酸(hyaluronic acid, HA),作为抗肿瘤药物的靶向载体,能够将较小的药物分子黏附在它的网状结构中或者将药物分子接枝到透明质酸类药物的载体上,所形成的粒子或复合物与肿瘤细胞表面的受体靶向结合,使更多的药物分子进入肿瘤组织,增加抗肿瘤药在肿瘤和淋巴结中的吸收和滞留时间,从而提高药物的疗效,降低毒副作用。本实验将HA作为靶向配体修饰PEI/DNA复合物,构建HA/PEI/DNA三元纳米复合物,并对其进行表征和性能检测。
由于乳腺癌的基因治疗缺乏更为有效的目的基因,NK4作为HGF的拮抗剂,具有血管新生抑制和癌浸润抑制两大作用,这种双重作用在裸鼠乳腺癌移植试验上已获得了证实,即提示它可成为具备这两大功能的崭新抗癌药。因此本实验采用NK4为目的基因,首次构建HA/PEI/PCAGGS-NK4纳米复合物,考察其在体外对乳腺癌细胞的作用,并建立裸鼠乳腺癌移植瘤模型,观察三元复合物HA/PEI/PCAGGS-NK4在体内的抑瘤效果。
实验方法
一、HA/PEI/DNA三元纳米复合物的构建及性能检测
1、HA/PEI/DNA三元复合物的构建与表征
制备不同HA与PEI负正电荷摩尔比的HA/PEI/DNA三元复合物,琼脂糖凝胶电泳进行DNA凝胶阻滞实验,马尔文粒度分析仪检测纳米粒的粒径分布及Zeta电位,比较其在水及BSA中的粒径变化。
2、HA/PEI/DNA三元复合物的体外细胞转染实验
取乳腺癌细胞MDA-MB-231, MCF-7, MDA-MB-435,转染一定量上述不同电荷比的纳米复合物,在流式细胞仪中检测转染率。
3、乳腺癌细胞系CD44表达水平
三种乳腺癌细胞用无血清培养液培养。收集细胞的培养上清,细胞抽提物总蛋白采用BCA蛋白分析试剂盒定量。SDS-PAGE电泳后,转膜,加CD44s单克隆抗体孵育后,加入辣根过氧化物酶标记的兔抗鼠二抗,显色,以β-actin为内标。
4、三元复合物对COS-1细胞的毒性试验
对数生长期的COS-1细胞,加入不同浓度的不同HA与PEI电荷比的三元复合物,继续培养48h后,加MTT液振摇,孵育4h后加入DMSO,裂解细胞,混匀,选择570nm波长,用酶标仪测定各孔光吸收值。
二、HA/PEI/PCAGGS-NK4纳米复合物对乳腺癌细胞作用的研究
1、HA/PEI/PCAGGS-NK4纳米粒的制备与表征
制备纳米粒HA/PEI/PCAGGS-NK4与H A/PEI/PC AGGS-LacZ,进行DNA凝胶阻滞实验,测定粒径和Zeta电位,方法均同上述三元复合物的构建与表征方法。
2、Western-blot检测乳腺癌细胞NK4蛋白的表达
MDA-MB-231, MDA-MB-435, MCF-7细胞,加入用无血清培养基稀释含100μg/ml PCAGGS-NK4质粒的纳米复合物,4h后加入含有血清的培养基培养,回收各组细胞加入细胞裂解液,刮取细胞裂解物,进行SDS-PAGE分离蛋白条带,转膜后,加入NK4一抗(人HGF单抗)孵育,再加入辣根过氧化物酶标记的二抗,β-actin做对照。
3、MTT法测定细胞增殖能力
三种乳腺癌细胞过夜贴壁后,换液加入无血清培养基稀释的不同浓度HA/PEI/PCAGGS-NK4与HA/PEI/PCAGGS-LacZ纳米复合物,绘制细胞生长曲线,比较各组细胞的增殖能力。
4、Transwell法测定癌细胞侵袭能力
细胞贴壁后换液加入用无血清培养基稀释的含不同浓度PCAGGS-LacZ, PCAGGS-NK4质粒的纳米复合物,12h后用胰酶消化,接种于Transwell小室的上室,其下室中加入含10%新生小牛血清的培养液,12h后取出小室,固定,染色,高倍光镜下计数5个不同视野的穿过膜的细胞数。
5、流式细胞仪分析细胞凋亡率
将被染色分析的细胞用冷PBS洗涤二次并在恰当的染色缓冲液中重悬。吸取100ul的细胞(1×105)至试管中。加入适量的荧光标记的Annexin V试剂和PI,混匀后避光室温下孵育15min后加入400ul染色缓冲液,立即上流式细胞仪分析。取少量滴片,置荧光镜下观察细胞凋亡形态,计算凋亡、坏死百分率。
三、HA/PEI/PCAGGS-NK4对裸鼠移植瘤生长抑制作用的考察
1、建立人乳腺癌细胞裸鼠移植瘤模型与给药
BALB/c (nu/nu)裸鼠30只,每只接种0.2ml(1×107／ml MDA-MB-231)细胞于裸鼠右侧胸壁第二乳头下脂肪垫内。8d后,随机分为3组,每组10只,分别命名为：(1)对照组,即HA/PEI/PCAGGS-LacZ注射组(肿瘤及肿瘤周围多点注射200μl质粒溶液,含100μgPCAGGS-LacZ);(2)给药组,即HA/PEI/PCAGGS-NK4注射组(肿瘤及肿瘤周围多点注射200μl质粒溶液,含100μgPCAGGS-NK4);(3)阳性对照组,即阿霉素组,0.2ml(含阿霉素100μg)腹腔注射,绘制肿瘤生长曲线,抑瘤率=(1-处理组平均瘤重／对照组平均瘤重)×100%。
2、Western blot检测移植瘤细胞NK4蛋白的表达
称取于液氮中保存的组织0.2g,以预冷的PBS洗3次并用眼科剪将组织剪碎,在10倍于其体积的裂解缓冲液中迅速搅成匀浆,4℃离心10min,吸出上清液为细胞总蛋白,用Bradford法测蛋白浓度。经SDS-PAGE电泳后转膜,再用含5%脱脂奶粉的TBST液封闭,然后加入一抗、二抗孵育,用ECL发光法检测不同样品NK4蛋白表达状况。
四、统计学分析
采用SPSS11.0统计软件处理,计量资料以均数±标准差(x±s)表示,采用one-way ANOVA分析,组间比较采用LSD检验,P<0.05为差异有统计学意义。
实验结果
一、HA/PEI/DNA三元纳米复合物的构建及性能检测
1、HA/PEI/DNA三元复合物的构建与表征
制备的HA/PEI/DNA三元复合物经琼脂糖凝胶电泳,无DNA条带出现,证明其能有效结合DNA分子。随着HA量的增加,复合物粒径在双蒸水中略有增加,在BSA中的粒径无显著增大,Zeta电位值则逐渐降低,且当HA与PEI电荷比为100%时,Zeta电位呈现负值。
2、HA/PEI/DNA三元复合物的体外细胞转染实验
随着HA的加入,三元复合物在MDA-MA-231与MDA-MB-435中的转染率明显高于二元复合物PD(P<0.05),增加了2-13倍,当HA与PEI电荷比为7.5%时,三元复合物在三种细胞的转染率最高,同其他各组比较均具有显著差异(P<0.05)。
3、乳腺癌细胞系CD44表达水平
Western blotting结果表明,MDA-MB-435和MDA-MB-231的CD44s的表达水平明显高于MCF-7。平均表达水平分别为1.15±0.036,0.92±0.072和0.16±0.028。
4、三元复合物对COS-1细胞的毒性试验
结果表明：未修饰的PEI/DNA复合物(N／P,10)会引起COS-1细胞毒性,使细胞活力下降到58%。HA的加入使制备的三元复合物HA/PEI/DNA毒性大大降低,细胞存活率均达到95%以上,几乎无毒。
二、HA/PEI/PCAGGS-NK4纳米复合物对乳腺癌细胞作用的研究
1、HA/PEI/PCAGGS-NK4纳米粒的制备与表征
制备的纳米粒HA/PEI/PCAGGS-NK4与HA/PEI/PCAGGS-LacZ能有效结合DNA分子。随着HA量的增加,纳米粒在BSA中不会引起聚集,Zeta电位则逐渐降低,且当HA与PEI电荷比为100%时,Zeta电位呈现负值。
2、Western-blot检测乳腺癌细胞NK4蛋白的表达
MDA-MB-231, MDA-MB-435,和MCF-7三种细胞系内源性的HGF平均表达水平分别为0.91±0.062,0.83±0.029和0.33±0.017。三种细胞系转染100μg／mlHA/PEI/PCAGGS-NK4三元复合物后所表达的外源NK4蛋白的平均值分别为：2.23±0.039,1.91±0.062,1.30±0.018。
3、MTT法测定细胞增殖能力
空白质粒不影响细胞的增殖,随HA/PEI/PCAGGS-NK4三元复合物处理浓度的增加,细胞增殖有逐渐受到抑制的趋势,在浓度剂量为100μg／ml,200μg／ml,500μg／ml时,细胞增殖被明显抑制,与对照组比较均有显著性差异(P<0.05)。在各种HA/PEI/PCAGGS-NK4浓度下MDA-MB-231细胞增殖受抑制的程度最大(P<0.05),MDA-MB-435细胞的抑制率也明显高于MCF-7细胞(P<0.05)。
4、Transwell法测定癌细胞侵袭能力
未经转染处理空白细胞的侵袭能力为MDA-MB-231> MDA-MB-435>MCF-7,加入对照组HA/PEI/PCAGGS-LacZ处理后,随HA/PEI/PCAGGS-LacZ浓度增加,对照组与空白细胞组的细胞侵袭能力无显著性差异(P>0.05)。而给药组在加入不同浓度的HA/PEI/PCAGGS-NK4处理后,三种细胞的侵袭迁移力均有不同程度的减弱,差异均有统计学意义(P<0.05)。
5、流式细胞仪分析细胞凋亡率
未经转染处理的空白MCF-7,MDA-MB-231,MDA-MB-435细胞系,其凋亡百分率分别为2.41±0.05%,2.13±0.06%,2.32±0.09%。加入不同浓度空质粒对照组后,空质粒对照组与空白细胞组的凋亡率无显著性差异(P>0.05)。而给药组在加入不同浓度的HA/PEI/PCAGGS-NK4的复合物后,MCF-7, MDA-MB-231,MDA-MB-435三种细胞的凋亡百分率均有不同程度的升高,差异具有统计学意义(P<0.05)。
三、HA/PEI/PCAGGS-NK4对裸鼠移植瘤生长抑制作用的考察
1、建立人乳腺癌细胞裸鼠移植瘤模型与给药
各组移植瘤裸鼠一般情况较好。接种MDA-MB-231细胞8d左右,3组裸鼠均于接种部位出现微小隆起。成瘤率100%。阳性对照组裸鼠在两次给药后,个别裸鼠出现明显不适而死亡。对照组裸鼠瘤体的生长明显迅速,40天时终体积达到445.32±26.65mm3,而阳性对照组和给药组裸鼠瘤体的增长均被显著抑制(P<0.01),40天时终体积分别达到272.96±24.57 mm3,225.64±17.36 mm3。阳性对照组、给药组抑瘤率分别为：为47.75%,55.81%。
2、Western blot检测移植瘤细胞NK4蛋白的表达
空白对照组与阳性对照组均有少量HGF的表达,表达量分别为0.89±0.03,0.97±0.02,给药组表达水平为3.33±0.05,与空对照组比较蛋白表达明显增多(P<0.05)。
结论
1、所构建的HA/PEI/DNA能有效结合DNA分子,在BSA中不会引起聚集。HA的加入使纳米复合物在三种乳腺癌细胞中的转染效率均有增加,在MDA-MB-231,MDA-MB-435(高CD44S表达的)两种细胞中转染率明显升高,产生了细胞靶向性,当HA与PEI电荷比为7.5%时,HA/PEI/DNA在三种细胞中的转染率最高。三元复合物对COS-1细胞的毒性相对于PEI/DNA二元复合物大大降低,几乎无毒。
2、HA/PEI/PCAGGS-LacZ及HA/PEI/PCAGGS-NK4三元复合物在BSA中不会引起聚集,质粒的结构并不影响纳米粒的粒径及Zeta电位等物理性质。三种乳腺癌细胞系被HA/PEI／PCAGGS-NK4转染后,表达了NK4外源蛋白,随着转染复合物浓度的增加,细胞增殖有逐渐受到抑制的趋势,细胞的侵袭迁移力均有不同程度的减弱,凋亡百分率均有不同程度的增加。差异具有统计学意义(P<0.05)。
3、成功建立人乳腺癌细胞裸鼠移植瘤模型,阳性对照组(阿霉素组)和给药组(HA/PEI/PCAGGS-NK4三元复合物组)裸鼠瘤体的增长均被显著抑制(P<0.01),给药组裸鼠体内有一定水平的NK4蛋白表达。
With the in-depth study of medical molecular biology and application, gene therapy for cancer has become an important biological therapeutic method and showed good application prospects in the treatment of breast cancer. At present, gene therapy for breast cancer contains the following issues, at first, there have been several gene transfer methods for research or clinical, but the transfection efficiency is still not satisfactory. The retrovirus carriers have high transfection efficiency and a stable expression, but it is only active in proliferating cells, and it can be integrated into the host cell and occurr the insertion mutation and oncogene activation in the process of transfection, so it is unfit for human-mediated gene transfer in vivo. The gene mediated by Adeno-virus carriers can not be integrated into the host cell's chromosomes, although it has a high transfection efficiency but can not transfect the target cells with a long-term stable expression, induce immune response also. Meanwhile, lack of carrier with specificity and efficiency, if the tumor cells are only partially transfected, the treatment will be affected, while the vector with low specificity can lead to victimize cancer tissues and bone marrow and other major organizations. Therefore, gene therapy is necessary to enhance the target, safety, developing a new and efficient vector system has become a focus research.
Cationic polymer polyethyleneimine (PEI) has become the most successful example among the non-viral cationic polymer vector and has been the standards to design a new cationic polymer transfection carrier, because it can condense DNA molecules effectively to form stable nano-particles and protect the DNA molecule's biological activity, avoid hydrolysis, effective endocytosis, and dissociation to achieve the release of DNA molecules. Current research mainly focus on the lower cytotoxicity, high transfection efficiency and cell targeting to transform PEL One of the most attractive strategy is applying receptor-mediated endocytosis to replace the non-specific electrostatic interaction between PEI and the cell-cell, it is essential to use targeted ligand binding parts of the targeted receptors on cell surface with high affinity recognition and specificity. Hyaluronic acid (HA), as a targeted vector for anti-cancer drugs, can stick small drug molecules to its network structure or graft drug molecules to the carrier of hyaluronic acid-type drugs, the formed particles or complexes combined with the receptor-targeted tumor cell surface, so that more drug molecules arrive the tumor tissue, increase tumor uptake and retention time in tumor and lymph nodes, thereby enhance the efficacy of drugs and reduce the toxic or side effects. In this study, HA as a targeting ligand was to modify the PEI/DNA complexes for the first time, construct HA/PEI/DNA ternary nanocomposites, and investigate its characterization and performance.
Currently gene therapy of breast cancer need a more effective targeted gene, NK4 as an HGF antagonist, has two roles of angiogenesis inhibition and tumor invasion suppression, which has been confirmed in transplant trials of breast cancer in nude mice, it prompted that NK4 could be a new anti-cancer drug with both of the unique features.Therefore,this experiment choose NK4 gene to build HA/PEI/PCAGGS-NK4 nanocomposites for the first time, and its effects on breast cancer cells were studied in vitro, established the breast cancer xenograft model in nude mice andobserved the antitumor effect of HA/PEI/PCAGGS-NK4 ternary complex in vivo.
1. Construction and performance test of the HA/PEI/DNA ternary nanocomposites
(1) Construction and characterization of the HA/PEI/DNA ternary complexes
To prepare HA/PEI/DNA ternary complex with different charge ratios of HA and PEI, agarose gel electrophoresis, DNA gel retardation experiments, particle size distribution of nanoparticles and Zeta Potential was analyzed by Malvern, compared the particle size in BSA and in water.
(2) Cell transfection experiments of HA/PEI/DNA ternary complex in vitro.
Breast cancer cells MDA-MB-231, MCF-7, MDA-MB-435 were added a certain amount of nano-composites with different charge ratios, transfection efficiency was detected in the flow cytometer.
(3) CD44 expression was detected in breast cancer cell lines by western blot
Three kinds of breast cancer cells were cultured in serum-free culture medium. Collection of cell supernatants, total protein of cell extracts used BCA Protein Assay Kit quantitatively. After SDS-PAGE electrophoresis, transferred to NC film, plused CD44s monoclonal antibody, then added the rabbit anti-mouse secondary antibody labeled by horseradish peroxidase, color, (3-actin was used as the internal standard.
(4) COS-1 cells toxicity of the ternary complexes was determined by MTT
COS-1 cells in Logarithmic phase were managed by ternary complex with different concentrations and different charge ratio of HA and PEI, continue to culture 48 h, plused MTT solution, shaked slowly, incubated 4h, added DMSO, Selected wavelength 570nm, measured the absorbed value by UV spectrophotometry.
2.Study on the role of the HA/PEI/PCAGGS-NK4 nanocomposites in the breast cancer cells
(1) Preparation and Characterization of the HA/PEI/PCAGGS-NK4 nanoparticles Preparation of nanoparticles HA/PEI/PCAGGS-NK4 with HA/PEI/ PCAGGS-LacZ, DNA gel retardation experiments, characterized by particle size and Zeta potential, the methods were same with the HA/PEI/DNA ternary complex in section 1.
(2) NK4 protein expression in breast cancer cells was measuered by western blotting
MDA-MB-231, MDA-MB-435, MCF-7 cells, added with 100μg/ml PCAGGS-NK4 plasmid serum-free medium Nanocomposite,4h plused serum medium to culture, recovered the cell, cell lysate were scraped to conduct SDS-PAGE separation of protein bands, transfer film, add NK4 an anti-(human HGF monoclonal antibody), then add horseradish peroxidase labeled secondary antibody,β-actin as controls.
(3) Cell proliferation was determined by MTT
Three kinds of breast cancer cells were exchanged the medium containing different concentrations of nano-composite material with serum-free medium diluted PCAGGS-LacZ, PCAGGS-NK4 plasmid. Drew cell growth curve. Compared the cell proliferation of each group.
(4) Invasive ability of cancer cells was investigated by Transwell
Adherent cells were exchanged medium containing different concentrations of PCAGGS-LacZ, PCAGGS-NK4 plasmid nanocomposites, after 12h, were digested with trypsin and seeded in the upper Transwell chamber, joined the culture medium with 10%newborn calf serum to the lower room, after 12h, removed the small rooms, fixed, stained, count the number of the cells through the membrane under the microscope in five different point of view.
(5) Apoptosis rate was analyzed by flow cytometry
The pre-staining cells were washed twice with cold PBS, re-suspended in staining buffer. Took 100ul cells (1x105) to a test tube. Adding an appropriate amount of fluorescent labeled Annexin V reagents and PI, mixed in dark, after incubating for 15 minutes at room temperature, added 400ul staining buffer and immediately analyzed on a flow cytometer. Took small drops to films and observed the morphology of apoptosis under fluorescent microscope, caculated the apoptosis, necrosis percentage.
3.Investigate the effect of HA/PEI/PCAGGS-NK4 on tumor growth in nude mice
(1) Establish the xenografts of human breast cancer cell in nude mice and administration
BALB/c (nu/nu) nude mice 30, each was vaccined 0.2ml (1×107/ml MDA-MB-231) cells under the second nipple on the right chest fat pad. Afer 8d, they were randomly divided into 3 groups, named respectively as followed:(①control group, the HA/PEI/PCAGGS-LacZ injection group (multi-point injection of 200μl plasmid solution containing 100μg PCAGGS-LacZ at tumor and tumor-surrounding);②treatment group, namely, HA/PEI/PCAGGS-NK4 injection group (multi-point injection of 200μl plasmid solution containing 100μg PCAGGS-NK4 at tumor and tumor-surrounding);③positive control group, namely, adriamycin group,0.2ml (including adriamycin 100μg) injected intraperitoneally. Drawing tumor growth curve. Inhibition rate= (1-mean tumor weight of treatment group/mean tumor weight of control group)×100%.
(2) NK4 protein expression was detected in xenografts cells by western blot
Prestored in liquid nitrogen, the organization (0.2g) were take out to wash by pre-cooling PBS for three times, Shredded with scissors, milled into homogenate rapidly in 10 times of its volume lysis buffer,4℃in centrifuge 10min, aspirated supernatant of total cellular protein, the protein concentration measured by Bradford method. DS-PAGE electrophoresis, transferred to NC film, and then closed with 5% skim milk TBST solution, and then added HGF monoclonal antibody, then added the rabbit anti-mouse secondary antibody labeled horseradish peroxidase, detected NK4 protein expression in different samples with ECL chemiluminescence.
4.Statistical analysis
Applied the statistical software SPSS 11.0, the measured data showed as mean± standard deviation (x±s), one-way ANOVA analysis and LSD test were used to compare between groups, P<0.05 for the statistically significant difference.
Results
1.Construction and Performance Test of the HA/PEI/DNA ternary nanocomposites
(1) Construction and characterization of the HA/PEI/DNA ternary complexes
The prepared HA/PEI/DNA ternary complexes were managed by agarose gel electrophoresis, the results show that no DNA bands appear, the complexes can effectively combine DNA molecules. With the increasing amount of HA, the size of the composite increased gradually in the double distilled water and decreased in the BSA, Zeta potential gradually decreased, when the HA and PEI charge ratio to 100%, Zeta potential was negative.
(2) Cell transfection experiments
With adding the HA, the transfection efficiency of the ternary complexes in MDA-MA-231 and MDA-MB-435 was significantly higher than that of binary complexes PD (P<0.05), has a 2-13 times increased, when the HA and PEI charge ratio was 7.5%or 100%, the transfection rate of the ternary complexes in MCF-7, MDA-MA-231 and MDA-MB-435 was significantly higher than that of PD (P<0.05). When the HA and PEI charge ratio of 7.5%, the ternary complex had the highest transfected efficiency in the three kinds of cells compared with the other groups. Difference was significant (P<0.05).
(3) CD44 expression
The results of western blotting showed that the expression of CD44s was significantly higher in MDA-MB-435, MDA-MB-231 than in MCF-7. The average expression levels were 1.15±0.036,0.92±0.072 and 0.16±0.028.
(4) COS-1 cells toxicity
The results showed that the non-modified PEI/DNA complexes (N/P,10) will cause COS-1-cell toxicity, so that cell viability decreased to 58%. HA was added to construct the ternary complex HA/PEI/DNA which had significantly reduced toxicity, cell survival rates were 95%, almost non-toxic.
2.Study on the role of the HA/PEI/PCAGGS-NK4 nanocomposites in the breast cancer cells
(1) Preparation and Characterization of the HA/PEI/PCAGGS-NK4 nanoparticles
The prepared HA/PEI/PCAGGS-NK4, HA/PEI/PCAGGS-LacZ nanoparticles could effectively integrated DNA molecules. With the increasing amount of HA, Zeta potential decreased gradually in the BSA, nanoparticles did not cause aggregation, when the HA and PEI charge ratio was 100%, Zeta potential was negative.
(2) NK4 protein expression in breast cancer cells
The average expressive levels of endogenous HGF in MDA-MB-231, MDA-MB-435, and MCF-7 cells respectively were 0.91±0.062,0.83±0.029 and 0.33±0.017. after 100μg/ml HA/PEI/PCAGGS-NK4 was transfected in three kinds of cell lines, the average exogenous expression of NK4 protein respectively were:2.23±0.039,1.91±0.062,1.30±0.018.
(3) Cell proliferation
The results showed that:the blank plasmid does not affect cell proliferation, with the increasingconcentration of PCAGGS-NK4, cell proliferation was inhibited gradually, when the concentration of PCAGGS-NK4 arrive the dose of 100μg/ml, 200μg/ml,500μg/ml, cell proliferation was significantly inhibited compared with that of the control group(P<0.05). The HA/PEI/PCAGGS-NK4 with various concentration had the largest inhibited effect on MDA-MB-231 cells (P<0.05), and the inhibited effect in MDA-MB-435 cells was significantly higher than MCF-7 cells (P<0.05).
(4) Invasive ability of cancer cells
The invasion capacity comparison of the cells without treatment by transfection were:MDA-MB-231>MDA-MB-435>MCF-7. Invasive ability had no significant difference between cells treated with HA/PEI/PCAGGS-LacZ and the cells without treatment (P>0.05). Treated by the increasing concentrations of HA/PEI/PCAGGS-NK4, three kinds of cells had relative lower invasion capacity to a certain degree, the differences were statistically significant (P<0.05).
(5) Apoptosis rate
Apoptosis rate of the MCF-7, MDA-MB-231, MDA-MB-435 cell line without treatment by transfection were 2.41±0.05%,2.13±0.06%,2.32±0.09%. Apoptosis rate had no significant difference between cells treated with HA/PEI/PCAGGS-LacZ and the cells without treatment (P>0.05). after Treated by the increasing concentrations of HA/PEI/PCAGGS-NK4, MCF-7, MDA-MB-231, MDA-MB-435 cells had relative higher percentage of apoptosis.The difference was statistically signifiant (P<0.05).
3.Investigate the effect of HA/PEI/PCAGGS-NK4 on tumor growth in nude mice
(1) Establish the xenografts of human breast cancer cell in nude mice and administration
The xenografts in each group nude mice were generally good. After inoculated MDA-MB-231 cells for 8d, small bulge appeared at the inoculation site on three groups of nude mice. Tumorigenicity rate was 100%. The individual positive control group of nude mice died for apparent discomfort after administration. The tumor growth were significant and rapid in control group of nude mice, reach to the final volume of 445.32±26.65 after 40 days, while the tumor growth in positive control group and treatment groupof nude mice were significantly inhibited (P<0.01), final volume were respectively 272.96±24.57 and 225.64±17.36 after 40 days.Tumor inhibition rates of positive control group and treatment group were 47.75%,55.81%(compared with control group, P<0.05).
(2) NK4 protein expression in xenografts
The blank control group and the positive control group have a small expressive amount of HGF, expressive levels were 0.89±0.03 and 0.97±0.02, protein expression in treatment group was significantly increased compared with the blank control group (P<0.05), the expressive level of treatment group was up to 3.33±0.05.
Conclusions
1.HA/PEI/DNA could combined DNA molecules effectively and could not cause aggregation in the BSA. HA was added to construct the nano-composite, could significantly increase the transfection efficiency in MDA-MB-231, MDA-MB-435 (high-CD44S expression), produced cell targeting effect, when the HA and PEI charge ratio was7.5%, HA/PEI/DNA had the highest transfection efficiency in three kinds of cells.The toxicity of ternary complex on COS-1 cells was greatly reduced compared with the PEI/DNA binary complexes, showed almost non-toxic.
2.HA/PEI/PCAGGS-LacZ and HA/PEI/PCAGGS-NK4 ternary complex could not cause aggregation in the BSA, it showed that the structure of plasmid could not affect the size and Zeta potential such as physical properties of nanoparticles. Three kinds of breast cancer cell lines transfected by HA/PEI/PCAGGS-NK4 had expressed exogenous NK4 protein; cell proliferation was inhibited; cell invasive capacity was declined; apoptosis rate was increased. The differences were all statistically signifiant (P<0.05).
3.Successful established the xenografts of human breast cancer cell in nude mice, the tumor growth were significantly inhibited in positive control group (doxorubicin group) and treatment group (HA/PEI/PCAGGS-NK4 ternary complex group) nude mice,and treatment group nude mice had showed a certain level of NK4 protein expression in xenografts cells.
引文
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