骨桥蛋白RNA干扰对人U251胶质瘤细胞在裸鼠体内生长的影响

研究下调骨桥蛋白(osteopontin,OPN)对人U251胶质瘤细胞在裸鼠体内生长的影响并探讨其对胶质瘤生长、侵袭的可能机制.应用RNA干扰技术,将OPN基因的慢病毒干扰载体LV-OPNshRNA感染U251细胞.将对照和试验组U251细胞分别接种裸鼠,建立裸鼠荷瘤模型.3周后测量肿瘤的体积、瘤重并做肿瘤组织病理切片分析;利用RT-PCR和免疫印迹法检测OPN、尿激酶型纤维蛋白酶原激活物(uPA)、基质金属蛋白酶(MMP-2、MMP-9)的mRNA和蛋白表达;免疫组化法检测肿瘤组织微血管密度和血管内皮生长因子（VEGF）表达情况. 经OPN的RNA干扰后,能显著降低肿瘤组织OPN mRNA水平及蛋白表达,有效抑制肿瘤细胞生长及侵袭能力, 肿瘤体积及重量的减小有统计学意义(P<0.05).感染组uPA、MMP-2和MMP-9的mRNA和蛋白表达明显减少, 肿瘤组织的MVD值和VEGF的表达均显著降低.上述结果表明,抑制OPN的表达能明显抑制人U251胶质瘤细胞在裸鼠体内的生长和侵袭,OPN可能通过激活uPA、MMP-2和MMP-9等蛋白酶降解细胞外基质和促进肿瘤血管生成,参与胶质瘤的生长.     

BST-2参与HCMV感染诱导的恶性胶质瘤细胞增殖和迁移

为探讨BST-2蛋白是否参与人巨细胞病毒(HCMV)感染导致的恶性胶质瘤细胞增殖和迁移,以HCMV AD169株感染U251细胞,通过细胞划痕愈合实验检测HCMV感染对U251迁移的影响;通过Western-blot方法检测HCMV感染对BST-2蛋白表达的影响;通过CCK-8、细胞划痕愈合和transwell方法检测HCMV感染后下调BST-2对U251细胞增殖和迁移能力的影响。结果显示,HCMV感染可促进U251细胞迁移并高表达BST-2,沉默BST-2后可抑制由HCMV感染诱导的细胞增殖和迁移。结果证实HCMV感染可促进胶质瘤细胞U251增殖迁移,BST-2参与了HCMV感染导致的恶性胶质瘤细胞增殖和迁移。     

Inhibition of human glioma U251 cells growth in vitro and in vivo by hydroxyapatite nanoparticle-assisted delivery of short hairpin RNAs against SATB1

Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be over-expressed in many human tumors and knockdown of SATB1 can inhibit tumor growth. The present study was designed to determine the role of SATB1 in the growth of human glioma U251 cells using the plasmid-based SATB1 short hairpin RNA (shRNA) delivered by hydroxyapatite nanoparticles in vitro and in vivo. The in vitro growth, invasion and angiogenesis assays of human glioma U251 cells were done. U251 cells tumor blocks were transplanted into the nude mice. CaCl₂-modified hydroxyapatite nanoparticles carrying shRNA-SATB1 plasmids were injected into the tumors. The apoptosis of the tumor U251 cells was examined with TUNEL assay and flow cytometer (FCM). The tumor growth and immunohistochemistry were measured. The expression level of SATB1 mRNA was investigated by RT-PCR. The expression levels of SATB1, Cyclin D1, MMP-2, VEGF, Bax and Caspase-9 protein were determined by western blot analysis. The results showed that hydroxyapatite nanoparticles-delivered shRNA-SATB1 could significantly inhibit the growth, invasion and angiogenesis of U251 cells in vitro and the growth of U251 cells in vivo. FCM results showed that Nano HAP-shRNA-SATB1-induced apoptosis (up to 67.8 %). SATB1 expression was strongly down-regulated in the tumor U251 cells. Cyclin D1, MMP-2 and VEGF were also down-regulated in the tumor tissues that also displayed significant increased in Bax expression and Caspase-9 activity. These results show that Nano HAP-shRNA-SATB1 can inhibit the growth of human glioma U251 cells in vitro and in vivo, and hydroxyapatite nanoparticles can be used for the in vitro and in vivo delivery of plasmid-based shRNAs into U251 cells.     

miR-216b-5p靶向调控BTN3A2促进胶质瘤细胞LN-229的迁移以及侵袭能力

大量证据表明microRNA（miRNA）通过靶向调控靶基因的表达从而在肿瘤侵袭与转移中发挥重要作用。然而关于microRNA-216b-5p (miR-216b-5p )通过靶向嗜乳脂蛋白第3亚家族膜蛋白A2（butyrophilin subfamily 3 member A2,BTN3A2）促进胶质瘤侵袭与转移的机制尚不明确。本研究通过GSE15824与GSE4290差异表达分析筛选出同时在2个芯片中表达上调的BTN3A2（P＜0.05）。生存曲线结果显示,高表达BTN3A2病人总生存期明显下降（P＜0.001）。表达量分析结果显示,BTN3A2表达随WHO分级升高而升高（P＜0.05）,同时1p/19q未联合缺失与IDH突变型病人BTN3A2表达升高（P＜0.001）。基因集富集分析（gene set enrichment analysis,GSEA）结果显示,BTN3A2与众多癌症相关通路有关（P＜0.05）;Western印迹结果显示,BTN3A2在7例胶质瘤组织和胶质瘤细胞系U87、U251和LN-229中表达上调,过表达miR-216b-5p （miR-216b-5p mimics）后BTN3A2蛋白表达水平降低;Transwell结果显示,转染BTN3A2干扰质粒（si-BTN3A2）和miR-216b-5p mimics后可以抑制LN 229细胞体外迁移与侵袭能力（P＜0.05）;在线预测网站证实,miR-216b-5p 为BTN3A2潜在靶基因;生存曲线结果显示,与低表达miR-216b-5p 病人相比,高表达病人生存率明显上调（P=0.025）;荧光定量RT PCR结果显示,miR-216b-5p 在胶质瘤U87、U251和LN-229细胞中表达下降（P＜0.05）;双荧光素酶结果显示,BTN3A2存在与miR-216b-5p 的结合靶点(P<005);综上所述,BTN3A2可能通过结合miR-216b-5p 促进胶质瘤细胞LN 229的迁移以及侵袭。     

P2X7 receptor antagonism inhibits tumour growth in human high-grade gliomas

Gliomas, the most common primary brain cancer, are highly infiltrative and extremely difficult to treat. Despite advancements, current treatment is limited, with patients surviving for a median of 14–15 months post-diagnosis. Previous research has demonstrated the upregulation of a purinergic receptor, P2X7R, in human gliomas. P2X7R is expressed on both glioma cells and microglia within the glioma microenvironment. It is hypothesized that P2X7R contributes to tumour growth and proliferation via immune-mediated mechanisms involving tumour cells and surrounding microglia. We sought to elucidate the role of P2X7R in a human glioblastoma cell line (U251) and on surgically resected human glioma samples. We treated U251 and human glioma cultures for 72 h with P2X7R antagonists, Brilliant Blue G (BBG), oxidized ATP (oATP) and AZ10606120. Cell counting via fluorescence confocal microscopy was conducted to assess tumour proliferation. We observed no significant reductions in tumour cell numbers following P2X7R antagonism with BBG (20 μM) and oATP (250 μM) in both U251 cells and human glioma samples. Interestingly, there was a significant reduction in tumour cell number in both U251 cells (p =?0.0156) and human glioma samples (p =?0.0476) treated with varying concentrations of AZ10606120. When compared with the conventional chemotherapeutic agent, temozolomide, AZ10606120 was also found to more effectively inhibit tumour proliferation in U251 cells (p <?0.0001). Our pilot results demonstrate a potential trophic role of P2X7R where its inhibition by AZ10606120, a potent antagonist, hinders glioma growth directly or through the inactivation of microglia. This sheds new light on P2X7R as a therapeutic target for human gliomas.

     

Oxidative stress can affect the gene silencing effect of DOTAP liposome in an in vitro translation system

Oxidative stress can affect in vitro GFP expression through its control of the gene silencing effect of the liposome prepared by 1,2-dioleoyl-3-trimethyl-ammonium propane (DOTAP). The gene silencing effect of cationic DOTAP liposome in in vitro GFP expression, especially focusing on its translation process, and the effects of oxidative stress on its silencing effect were investigated. GFP expression, initiated by mRNA, was found to be thoroughly inhibited in the presence of DOTAP liposome at concentration of more than 2.5 mM, though its inhibitory effect was reduced in the presence of hydrogen peroxide. The analyses of (i) the interaction of mRNA with DOTAP, (ii) the chemical structure of DOTAP, and (iii) the membrane fluidity of DOTAP liposome imply the possible role of gene expression by the liposome membrane and stress conditions.     

沉默ABCE1对神经胶质瘤细胞U251增殖、迁移和凋亡的影响

ABCE1是ATP结合盒蛋白亚家族成员之一,在病毒感染,细胞增殖,抗凋亡,翻译起始和核糖体生物发生等过程中有重要的作用。为了探讨ABCE1对神经胶质瘤细胞U251增殖、迁移和凋亡的作用,本研究通过实时荧光定量PCR和免疫印迹实验检测ABCE1在神经胶质瘤细胞和正常胶质细胞中的mRNA和蛋白质表达水平,结果发现ABCE1在神经胶质瘤细胞U251中的表达高于在正常胶质细胞中的表达。利用siRNA靶向沉默ABCE1后,神经胶质瘤细胞U251中ABCE1 mRNA和蛋白的表达水平均显著减少,细胞的凋亡率显著提高,细胞增殖和迁移明显受到抑制,而且细胞对化疗药物替莫唑胺的敏感性增强。此外,沉默ABCE1后,Bcl-2的mRNA和蛋白质表达水平显著下调,而Bax的mRNA和蛋白质表达水平显著上调。以上研究结果表明,ABCE1与神经胶质瘤细胞的增殖和迁移密切相关,通过siRNA靶向沉默ABCE1基因可显著降低U251细胞的增殖和迁移能力。     

Inhibition of human lung adenocarcinoma growth using survivint34a by low-dose systematic administration

Anti-apoptosis plays an important role in tumour formation and development. Survivin is a member of the inhibitor of apoptosis (IAP) family, which is a target for anti-cancer drug exploitation was replaced as development. We investigated the role of the homo dominant-negative mutant Survivin-T34A in suppressing human lung adenocarcinomas (A549). The anti-tumour activity of HSurvivinT34A plasmid was evaluated in the A549 cell line and nude mice bearing A549 subcutaneous tumours. Low-dose systemic administration was continuously used. The HSurvivinT34A plasmid (5 µg/one) complexed with a cationic liposome (DOTAP/Chol) significantly inhibited tumour growth in our model. We observed microvessel density degradation by CD31 immunohistochemistry and apoptotic cell increase by TUNEL assay, PI staining and flow cytometric analysis in the treated group. The present findings suggest that the HSurvivinT34A plasmid complexed with a cationic liposome may provide an effective approach to inhibit the growth of human lung adenocarcinomas in vitro and in vivo.     

miR-613通过靶向VEGFA抑制神经胶质瘤细胞的增殖、侵袭和血管生成

摘要 目的：旨在探究miR-613在胶质瘤中的表达及对细胞增殖、侵袭和血管生成的影响。方法：根据细胞转染将实验分组为对照miRNA组（Control组）、miR-613模拟物组（mimics组）和miR-613 mimics+VEGFA组（VEGFA组）。采用逆转录定量聚合酶链反应（RT-qPCR）检测胶质瘤细胞和组织中miR-613和VEGFA mRNA的表达水平;采用荧光素酶报告基因分析miR-613与血管内皮生长因子（VEGF）的关系;采用Western blotting检测VEGFA蛋白的表达水平;通过体外实验检测转染细胞的增殖能力、侵袭能力和管状形成能力。结果：与正常组织样本相比,胶质瘤I-II期组样本的肿瘤细胞呈现异形,具有深核染色,并且肿瘤细胞密度适度较低,而胶质瘤III-IV期组样本的肿瘤细胞的核分裂活跃,具有明显的微血管增殖和明显的细胞异型性;miR-613在胶质瘤I-IV期组织样本中显著降低（P<0.05）。在U87和U251细胞系的VEGFA-WT组中,与Control组相比,mimics组的荧光素酶活性显著降低（P<0.05）。与Control组相比,U87和U251细胞系中mimics组VEGFA的mRNA和蛋白表达水平均显著降低（P<0.05）。克隆形成实验、血管生成实验和细胞侵袭实验结果表明,与Control组相比,mimics组的克隆形成数量、细胞侵袭数、内皮细胞HUVEC的管状形成数和Ang-2蛋白表达水平均显著降低（P<0.05）;与mimics组相比,VEGFA组克隆形成数量、细胞侵袭数、内皮细胞HUVEC的管状形成数和Ang-2蛋白表达水平均显著升高（P<0.05）。结论：miR-613通过靶向VEGFA抑制了神经胶质瘤细胞的侵袭、增殖和血管生成,提示miR-613可能成为未来治疗胶质瘤的潜在靶点。     

Overexpression of Hsc70 promotes proliferation,migration, and invasion of human glioma cells

Migration and invasion are often recognized as the main reasons for the high recurrence and death rates of glioma and limit the efficacy of surgery and other antitumor therapies. In this study, we found over activation of heat shock cognate protein 70 (Hsc70) in human glioma specimens, which was closely related to glioma grade. We investigated whether Hsc70 induced the migration and invasion of glioma cells. Wound healing and transwell migration assay were used to determine the migration and invasion ability of human glioma U251 and U87 cells, in which the expression of Hsc70 was knocked down by small interfering RNA. Western blot analysis was performed to determine the expression of FAK-Src signaling in malignant glioma cells. The results showed that Hsc70 deficiency significantly retarded migration and invasion and reduced the phosphorylation of FAK, Src, and Pyk2 in U251 and U87 cells. Overall, our results indicate that the migration and invasion capacity of human brain glioma cells is at least partly induced by Hsc70-dependent activation of FAK-Src signaling.     

转录因子SOX9基因对脑胶质瘤干细胞干性维持的相关性研究

目的:应用慢病毒干扰SOX9的表达,观察其对脑胶质瘤干细胞干性维持的影响。方法:设计2条针对SOX9转录短发夹RNA(sh RNA)的DNA序列,构建慢病毒并感染胶质瘤细胞系U87、U251细胞,利用嘌呤霉素筛选稳转细胞。在转录水平及蛋白水平检测SOX9的沉默效果,利用荧光实时定量PCR(q RT-PCR)及免疫荧光染色检测相应干细胞相关标志分子SOX2、Nestin的表达差异。比较诱导形成的胶质瘤干细胞成球能力(肿瘤干细胞成球的直径),同时利用荧光实时定量PCR(q RT-PCR)及免疫荧光染色对干细胞相关标志物SOX2、Nestin的表达水平进行比较。结果:SOX9成功包装慢病毒并有效感染U87、U251细胞,实时荧光定量PCR检测其抑制率分别可降低83.74%和80.12%。并且在稳转细胞系水平相关干细胞标志分子表达含量有明显下降。在干细胞层面沉默SOX9可以明显抑制胶质瘤细胞诱导成球的直径(P0.01),同时免疫荧光显示肿瘤干细胞相关干性分子SOX2、Nestin的表达水平较对照组明显降低。结论:慢病毒感染沉默SOX9基因可以抑制胶质瘤干细胞干性的维持,为胶质瘤的生物治疗提供了重要的靶点。     

东亚钳蝎氯毒素通过下调PKM2介导的有氧糖酵解抑制神经胶质瘤细胞的增殖活性

东亚钳蝎氯离子通道毒素(Buthus martensii Karshch chlorion channel toxin,BmK CT)是一类短链神经毒素,在体内和体外均能有效抑制神经胶质瘤细胞侵袭和增殖。然而,BmK CT抑制胶质瘤细胞增殖活性的机制尚不清楚。本研究采用代谢组学的方法探索BmK CT抑制人脑神经胶质瘤细胞增殖的分子机制。首先,通过蛋白质表达纯化获得带有谷胱甘肽S转移酶(glutathione S-transferase, GST)标签的BmK CT蛋白和GST蛋白。利用圆二色谱检测两种蛋白质均具有α-螺旋二级结构,并且融合蛋白质GST-BmK CT热稳定性较好。通过噻唑蓝(methylthiazolyldiphenyl-tetrazolium bromide, MTT)法检测GST-BmK CT对神经胶质瘤细胞(U251和A172)增殖的影响。结果显示,与GST处理组相比,1μmol/L GST-BmK CT处理组对U251细胞的抑制率更明显(U251细胞19±1.1 vs. 2.25±0.95,P<0.001, 48 h; A172细胞12±1.4 vs. 2.25±1.25,P<0.001, 48 h)。利用气相色谱-质谱联用仪(gas chromatography-mass spectrometry, GC-MS)分析鉴定出细胞内的63种代谢物,通过通路分析和聚类分析证实,GST-BmK CT处理影响神经胶质瘤细胞的有氧糖酵解。根据峰面积统计数据显示,GST-BmK CT处理降低胞内丙酮酸的含量。GST-BmK CT处理组相比GST组,明显下调胞外培养基的乳酸分泌(16.33±3.78 vs. 35.6±2.31,P<0.001, 48 h),葡萄糖消耗(2.04±0.09 vs. 2.64±0.04,P<0.05, 48 h)和胞内腺苷三磷酸(ATP)含量(633.79±0.001 vs. 5392.71±266.67,P<0.05, 48 h)。结果表明,GST-BmK CT通过下调胶质瘤细胞有氧糖酵解水平降低ATP的生成。实时荧光定量PCR和蛋白质免疫印迹结果显示,GST-BmK CT降低U251细胞丙酮酸激酶(pyruvate kinase M2, PKM2)在转录水平和翻译水平的表达,进一步发现GST-BmK CT通过下调低氧诱导因子(hypoxia inducible factor-1α, HIF-1α)降低PKM2在转录水平的表达。以上结果表明,BmK CT通过下调HIF-1α的表达,降低PKM2介导的有氧糖酵解从而抑制神经胶质瘤细胞的增殖活性。     

Caudatin induces caspase-dependent apoptosis in human glioma cells with involvement of mitochondrial dysfunction and reactive oxygen species generation

Caudatin as one species of C-21 steroidal from Cynanchum bungei decne displays potential anticancer activity. However, the underlying mechanisms remain elusive. In the present study, the growth suppressive effect and mechanism of caudatin on human glioma U251 and U87 cells were evaluated in vitro. The results indicated that caudatin significantly inhibited U251 and U87 cell growth in both a time- and dose-dependent manner. Flow cytometry analysis revealed that caudatin-induced cell growth inhibition was achieved by induction of cell apoptosis, as convinced by the increase of Sub-G1 peak, PARP cleavage and activation of caspase-3, caspase-7 and caspase-9. Caudatin treatment also resulted in mitochondrial dysfunction which correlated with an imbalance of Bcl-2 family members. Further investigation revealed that caudatin triggered U251 cell apoptosis by inducing reactive oxygen species (ROS) generation through disturbing the redox homeostasis. Moreover, pretreatment of caspase inhibitors apparently weakens caudatin-induced cell killing, PARP cleavage and caspase activation and eventually reverses caudatin-mediated apoptosis. Importantly, caudatin significantly inhibited U251 tumour xenografts in vivo through induction of cell apoptosis involving the inhibition of cell proliferation and angiogenesis, which further validate its value in combating human glioma in vivo. Taken together, the results described above all suggest that caudatin inhibited human glioma cell growth by induction of caspase-dependent apoptosis with involvement of mitochondrial dysfunction and ROS generation.     

A comparison of the in vitro antimicrobial activity of liposomes containing meropenem and gentamicin

The antimicrobial activity of eight cationic, two neutral and three anionic liposome compositions containing meropenem and gentamicin was tested in vitro in broth and serum medium. The cationic formulations showed better antibacterial efficacy against both Gram-positive and Gram-negative bacteria than the anionic and neutral ones, regardless of the encapsulated drug. The most effective formulations were the cationic PC/DOPE/DOTAP 3:4:3 and PC/Chol/DOTAP 3:4:3, as the MICs with meropenem were 2 to 4 times lower than those of the free drug.     

The influence of simulated microgravity on proliferation and apoptosis in U251 glioma cells

Several studies have indicated that microgravity can influence cellular progression, proliferation, and apoptosis in tumor cell lines. In this study, we observed that simulated microgravity inhibited proliferation and induced apoptosis in U251 malignant glioma (U251MG) cells. Furthermore, expression of the apoptosis-associated proteins, p21 and insulin-like growth factor binding protein-2 (IGFBP-2), was upregulated and downregulated, respectively, following exposure to simulated microgravity. These findings indicate that simulated microgravity inhibits proliferation while inducing apoptosis of U251MG cells. The associated effects appear to be mediated by inhibition of IGFBP-2 expression and stimulation of p21 expression. This suggests that simulated microgravity might represent a promising method to discover new targets for glioma therapeutic strategy.     

Txnip基因siRNA慢病毒表达质粒构建及促进猪前体脂肪细胞分化

硫氧还蛋白互作蛋白(thioredoxin interacting protein, Txnip)是一种氧化还原调节蛋白质,与硫氧还蛋白结合并抑制其活性,调节细胞氧化还原状态,影响细胞多种生理过程,然而其在猪脂肪细胞分化中的作用尚不明确。本文设计合成3对靶向猪Txnip基因的shRNA寡核苷酸,分别连接于重组慢病毒载体pGLV_3/H_1/GFP+Puro构建siRNA表达质粒。测序验证后,与包装质粒共转染293T细胞,获得滴度1×10~8 pfu/mL的慢病毒干扰质粒。以MOI值100转染原代培养猪前体脂肪细胞,转染率均达80%以上,其中Txnip-shRNA-2转染细胞Txnip基因沉默率达75%。转染Txnip-shRNA-2的猪前体脂肪细胞用成脂分化培养液诱导后,每隔1 d检测细胞成脂分化及相关基因表达。结果发现,其分化比阴性对照质粒转染或未转染细胞显著增强(P<0.05),PPARγ和FAS mRNA表达水平显著提高(P<0.05)。本文构建siRNA慢病毒表达质粒能有效干扰猪Txnip基因表达,Txnip表达沉默可通过上调PPARγ表达促进猪前体脂肪细胞分化。本研究提示,Txnip可能是猪脂肪细胞分化的抑制因子。     

DNA pre-condensation with an amino acid-based cationic amphiphile. A viable approach for liposome-based gene delivery

A study related to the development and characterization of a new gene delivery system was performed. The approach consists in both the pre-condensation of plasmid DNA with an arginine-based cationic surfactant, arginine–N-lauroyl amide dihydrochloride (ALA), which was found not to be toxic, and the incorporation of the blood protein transferrin (Tf) into the formulations.Two cationic liposome formulations were used, one composed of a mixture of dioleoyl trimethylammoniopropane and cholesterol (DOTAP:Chol) and the other a pH sensitive formulation constituted of DOTAP, Chol, Dioleoyl phosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS).Particles with different ALA/DNA and cationic lipid/DNA charge ratios were produced and a physicochemical characterization of the systems developed was performed. DNA conformational changes in the presence of ALA were studied by Circular Dichroism (CD) and the ALA binding to DNA was followed by surface tension measurements. Insight into the structure and morphology of the various ALA-complexes (complexes composed of ALA, DNA, Tf and liposomes) was obtained by cryogenic-Transmission Electron Microscopy (cryo-TEM) and the sizes of the ALA-complexes were determined through Photon Correlation Spectroscopy (PCS). We found that the transfection capacity of these systems is directly related with the presence of ALA and the lipidic composition. Complexes based on the pH sensitive liposome formulation present better transfection profiles. The correlation between the inner structure, density and size of the ALA-complexes and their biological activity is discussed. Overall, we demonstrate that the presence of ALA improves the transfection efficiency when conjugated with cationic liposome systems.     

DNA pre-condensation with an amino acid-based cationic amphiphile. A viable approach for liposome-based gene delivery

A study related to the development and characterization of a new gene delivery system was performed. The approach consists in both the pre-condensation of plasmid DNA with an arginine-based cationic surfactant, arginine-N-lauroyl amide dihydrochloride (ALA), which was found not to be toxic, and the incorporation of the blood protein transferrin (Tf) into the formulations.Two cationic liposome formulations were used, one composed of a mixture of dioleoyl trimethylammoniopropane and cholesterol (DOTAP:Chol) and the other a pH sensitive formulation constituted of DOTAP, Chol, Dioleoyl phosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS).Particles with different ALA/DNA and cationic lipid/DNA charge ratios were produced and a physicochemical characterization of the systems developed was performed. DNA conformational changes in the presence of ALA were studied by Circular Dichroism (CD) and the ALA binding to DNA was followed by surface tension measurements. Insight into the structure and morphology of the various ALA-complexes (complexes composed of ALA, DNA, Tf and liposomes) was obtained by cryogenic-Transmission Electron Microscopy (cryo-TEM) and the sizes of the ALA-complexes were determined through Photon Correlation Spectroscopy (PCS). We found that the transfection capacity of these systems is directly related with the presence of ALA and the lipidic composition. Complexes based on the pH sensitive liposome formulation present better transfection profiles. The correlation between the inner structure, density and size of the ALA-complexes and their biological activity is discussed. Overall, we demonstrate that the presence of ALA improves the transfection efficiency when conjugated with cationic liposome systems.