纳米材料在基因转染中的应用--Starburst PAMAM dendrimers研究现状

提高重组DNA对培养细胞及活体水平的转染效率是利用重组DNA进行预防和治疗疾病所必须解决的难题之一。一种新型阳离子多聚物00Starburst PAMAM dendrimers的出现为解决现存在难题带来了一线曙光。Starburst PAMAM dendrimers是1985年之后出现的一类新型星射状树形高分子，它们结构规整，具有呈辐射关对称的刚性球体结构，在生理条件下，Starburst PAMAM dendrimers分子具高的表面正电荷密度，能与天然状态下存在的带负电荷的生物活性物质（如核酸）发生静电相互作用形成复合物。该类树形高分子可极大增强DNA转染真核细胞的转染效率，具有许多优于其它现有转染试剂的优良特性，并具有在活体内应用的潜力。本文介绍了九十年代以来对该类树形高分子在体外培养细胞及活体水平增强DNA转染效率的研究现状并对今后的研究和应用进行了分析。     

聚酰胺-胺树状大分子的研究进展

聚酰胺-胺(PAMAM)是1985年由美国人Tomalia合成的一种树状大分子,现已广泛应用于基因载体、光电传感、废水处理、药物载体、催化剂等领域。本文综述了PAMAM树状大分子的结构和合成方法、细胞毒性,作为基因载体、药物载体所取得的成果以及PAMAM聚合物作为载体在胶质瘤治疗方面的研究进展及展望,并对今后胶质瘤放射治疗的研究方向进行了概述。     

聚酰胺-胺型纳米载体在基因治疗中的研究进展

聚酰胺-胺型树枝状高聚物可与DNA形成纳米尺寸的聚电解质复合物,具有很高的稳定性和溶解性,因此被认为是较好的基因载体.现有研究对聚酰胺一胺型树枝状高聚物介导的基因入胞机制已经较为清楚,体外研究证实该载体基因转染效率高.转基因能长期、稳定表达,且细胞毒性小;体内研究则证实在尤文肉瘤防治、心脏移植、恶性疟疫苗和肺纤维化防治等诸多疾病的基因治疗领域有较好的前景.     

聚酰胺-胺型树枝状高聚合物介导Survivin反义寡核苷酸抑制人肝癌裸鼠 移植瘤生长的作用*☆

背景：采用反义寡核苷酸等治疗方法可以抑制凋亡抑制因子Survivin 的表达,从而诱导肿瘤细胞凋亡。目前使用Survivin治疗恶性肿瘤仍存在基因载体转染效率低下、不能长期稳定表达、易被酶降解等难题。 目的：观察聚酰胺-胺型树枝状高聚合物(polyamidoamine,PAMAM)脂质体介导Survivin-反义寡核苷酸对人肝癌裸鼠移植瘤的抑制作用。 方法：以人肝癌细胞SMMC-7721裸鼠皮下注射建立人肝癌裸鼠皮下移植瘤模型,将PAMAM脂质体和PAMAM分别与Survivin-反义寡核苷酸混合得到载反义基因转染复合物。测定复合物的zeta电位及包封率。将两种载基因复合物注射道裸鼠移植瘤体内,观察两组移植瘤大小,检测移植瘤组织中survivin基因的表达。 结果与结论：PAMAM脂质体-survivin-反义寡核苷酸复合物的zeta电位高于PAMAM-survivin-反义寡核苷酸复合物(P < 0.05),基因包封率两组比较差异无显著性意义(P > 0.05)。PAMAM脂质体-survivin-反义寡核苷酸复合物治疗组裸鼠移植瘤质量及survivin蛋白表达低于PAMAM-survivin-反义寡核苷酸复合物组(P < 0.05)。提示PAMAM脂质体能将Survivin-反义寡核苷酸高效递送到人肝癌移植瘤细胞,降低survivin蛋白的表达,诱导移植瘤细胞凋亡。 关键词：聚酰胺-胺型树枝状高聚合物;脂质体;肝癌;反义寡核苷酸;Survivin doi:10.3969/j.issn.1673-8225.2012.12.020      

PEG化PAMAM树状大分子的合成及其作为药物载体的研究

研究经聚乙二醇(PEG)进行表面修饰的聚酰胺-胺树枝状大分子作为药物载体时对药物的包裹及释放能力。用经三氟乙基磺酸单甲氧基(Tresylate)活化的相对分子质量为5000的单甲氧基聚乙二醇(Tresylated MPEG-5000)对聚酰胺-胺G4.0-PAMAM树状大分子修饰,目标产物PEG化树状大分子用FT-IR1、H NMR进行结构表征。MTT研究其细胞毒性。通过包裹与释放实验研究该药物载体对抗癌药物甲氨蝶呤(MTX)的包裹及释放能力。每个修饰后的树状大分子可包裹33个抗癌药物甲氨蝶呤分子,细胞毒指数较低,释药速度减慢。修饰后的树状大分子与未PEG化的树状大分子载体相比具有更强的包裹能力,并具有一定的药物缓释功能;细胞毒性试验表明,其胞毒性与未PEG化的树状大分子相比明显偏低。     

新型阳离子磷酸胆碱聚合物可作为反义寡核苷酸基因的运输载体

背景:病毒类基因载体虽然具有较高的转染效率,其安全性一直受到人们的质疑。目的:以阳离子磷酸胆碱聚合物作为基因药物运输的载体,研究其对人类c-myc反义寡核苷酸(AS-ODN)的负载和运输能力,并对其安全性和有效性进行评价,方法:应用原子转移自由基聚合法合成具有高度生物相容性的双嵌段磷酸胆碱聚合物MPC30-DEA70,并对其溶液进行表征。用DNA凝胶电泳法对MPC30-DEA70与针对AS-ODN生成不同N/P比值的基因复合物进行表征。将MPC30-DEA70/AS-OD基因复合物转染至体外培养的HEK293细胞,检测其细胞相容性、转染效率和细胞内转运机制。结果与结论:MPC30-DEA70在pH=4.0-11.0范围内的0.01 mol/L PBS中显示出高度水溶性,其正电性随pH值降低而增强。MPC30-DEA70/AS-ODN基因复合物在DNA凝胶电泳中显示出不同程度的电泳迟滞,随电性增强而显著。MTT法检测显示MPC30-DEA70对HEK293细胞株的毒性呈剂量依赖性,高浓度下细胞毒性显著增强。流式细胞术检测发现复合物的转染随N/P比值增加而显著增强;共聚焦显微镜观察到AS-ODN分子在细胞核内的转运,提示其有效的核定位。证实新型阳离子磷酸胆碱聚合物可以有效负载和运输反义寡核苷酸,是一种高效安全的非病毒类转基因载体。     

肝靶向DNA载体乳糖化多聚乙烯基亚胺及体外转染试验

目的 研制肝靶向的DNA载体,用于干扰素的肝细胞内表达或抗病毒反义核酸的肝靶问给药。方法 以还原胺化法制备了肝靶向基因载体乳糖化多聚乙烯基亚胺（L－PEI）,并进行了体外转染试验和稳定性试验。结果 靶向载体L－PEI与DNA形成的复合物,对受体阳性的Huh－7细胞具有较高的转染效率;靶向载体乳糖化白蛋白能够竞争拮抗这种转染。靶向载体／DNA复合物在大鼠血清中具有较好的稳定性。结论 乳糖化多聚乙烯基亚胺能将DNA选择的投放于肝细胞,具有较好的应用前景。     

逆转录病毒载体介导人类G6PD基因在人红白血病细胞中的表达

目的：探讨逆转录病毒载体介导人类G6PD基因在人白血病细胞中的表达。方法：构建G6PD cDNA的逆转录病毒表达载体pLG6PDSN,转染包装细胞PA317，病毒上清感染人红白血病细胞K562，以PCR方法检测病毒载体是否整合于细胞基因组，定量法测定G6PD表达。t检验比较转染组与对照组间的表达差异。结果：酶切鉴定表明，G6PD cDNA准确插入pLXSN相应位点，载体构建成功。转染后PCR扩增NeoR基因，证明细胞DNA整合有逆转录病毒载体。转染组与对照组酶活性测定差异有显著性（P＜0．01）。结论：本试验所构建的重组载体pLG6PDSN为严重G6PD缺陷症的基因治疗提供了表达载体。     

乙酰化可降低聚酰胺-胺的细胞毒性

背景：聚酰胺-胺型树枝状高分子纳米材料已被广泛应用于药物载体的研究,但由于整代聚酰胺-胺表面有大量带正电荷的氨基,具有一定的细胞毒性。 目的：观察乙酰化对聚酰胺-胺细胞毒性的影响。 方法：①细胞增殖检测：采用MTT法检测在含0,0.125,0.25,0.5,1,2,4 μmol/L乙酰化聚酰胺-胺的培养液中人胚肾293T细胞的增殖。②细胞形态：倒置荧光显微镜观察在含4 μmol/L乙酰化聚酰胺-胺的培养液中人胚肾293T细胞的形态。③细胞周期：流式细胞术检测在含0,5,10,15,20 mg/L乙酰化聚酰胺-胺的培养液中人胚肾293T细胞的细胞周期。 结果与结论：聚酰胺-胺对293T细胞具有一定的细胞毒性,在4 μmol/L浓度下48 h的细胞存活率仅为52%,而乙酰化可显著降低聚酰胺-胺的细胞毒性(P < 0.01); 聚酰胺-胺孵育的细胞发生团缩,伸展性变差,而乙酰化聚酰胺-胺孵育的293T细胞与正常培养细胞基本一致,具有良好的伸展性;乙酰化聚酰胺-胺对细胞周期无影响,而聚酰胺-胺在20 mg/L较高质量浓度时可使细胞S期产生阻滞。表明乙酰化可以降低聚酰胺-胺的细胞毒性。     

聚乙烯亚胺用作基因转染的研究进展

 基因载体问题以及与载体相关的免疫反应、细胞毒性和安全性等问题，是基因治疗领域亟待解决的关键问题之一。聚乙烯亚胺（PEI）是阳离子聚合物非病毒载体的典型代表^[1]，是一种很早便为人所知并予以应用的有机大分子。目前，以 PEI 阳离子聚合物与 DNA 形成的 PEI/DNA 复合物已成为非病毒基因载体的研究热点。本文就近年来这方面的研究进展作简要综述。 1 PEI的特性 PEI 每 3 个原子中有 1 个胺基原子，使其具有较高正电荷密度。根据 pH 与质子作用之间的对应关系可得出：自由 PEI 的结构在生理条件下有 1/6 至 1/5 胺基发生质子化反应，从而使溶酶体肿胀破裂，从而起到“质子海绵”作用，使 PEI/DNA 复合物得以释放入胞质，很大程度上减少了 DNA 在吞噬泡内富集并进而被降解的作用，因而可以提高转染效率^[2]。     

Serum tolerance and endosomal escape capacity of histidine-modified pDNA-loaded complexes based on polyamidoamine dendrimer derivatives

Aiming to aid polyamidoamine (PAMAM, generation 4, PG4) to overcome gene delivery barriers like extrinsic serum inhibition, intrinsic cytotoxicity and lysosome digestion, histidine motifs modified PAMAM was prepared. The histidine activated PAMAM generation 4 (HPG4) was synthesized via aminolysis reaction and characterized by (1)H NMR spectrum and MALDI-TOF-MS. Cytotoxicity profiles of HPG4 on MD-MB-231 cells were significantly improved in the form of polymer and polymer/DNA complexes comparing to PG4. The luciferase protein expression level of HPG4 was 20-, 2.7- and 1.2- fold higher than that of PG4, SuperFect and PEI 25k. Most importantly, flow cytometry and gene transfection studies showed that histidine motifs of HPG4 not only acted as enhancer for faster cellular uptake, but also played an important role on enhancing serum tolerance of the system on cellular uptake and transfection. Among the serum concentrations of 10%-50%, HPG4 showed 10-100 folds higher transfection efficiency than PG4. Intracellular fate observation conducted by confocal microscope provided visual and quantitative evidence that endsomal escape efficiency of HPG4 system was higher than that of PG4. Lastly, the endosomal escape mechanism of HPG4 system was analyzed by endosome destabilization and proton pump inhibition treatment. Collectively, compared to PG4/pDNA, HPG4/pDNA showed improvement on cellular uptake, serum tolerance, cytotoxicity profile, and endosomal escape.     

Enhanced transfection efficiency of low generation PAMAM dendrimer conjugated with the nuclear localization signal peptide derived from herpesviridae

Abstract

Polyamidoamine (PAMAM) dendrimer is an extensively studied polymer in the biomedical research because of its low polydispersity, distinct molecular structure, and surface functionalities. Generally, a high-generational PAMAM dendrimer is used for gene delivery because transfection efficiency is dependent on charge density; however, an increase in charge density induces disruption of the cellular membrane, and damage to the membrane results in cytotoxicity. In this study, we selected PAMAM generation 2 to reduce the cytotoxic effect and conjugated RRILH and RRLHL sequences, nuclear localization signals (NLS) derived from herpesviridae to PAMAM generation 2. The transfection efficiency of RRILH-PAMAM G2 and RRLHL-PAMAM G2 was similar to that of polyethylenimine (PEI) in Neuro2A, HT22, and HaCaT cells, whereas their transfection efficiency was much higher than that of PEI in NIH3T3 cells. RRILH-PAMAM G2 showed relatively lower cytotoxicity than did RRLHL-PAMAM G2 in all cell lines, but the transfection capacity of the two polymers was similar. Our study shows that low-generational PAMAM dendrimer conjugated with NLS sequences has potential as an alternative to PEI in gene delivery.     

Gene delivery using dendrimer-entrapped gold nanoparticles as nonviral vectors

Development of highly efficient nonviral gene delivery vectors still remains a great challenge. In this study, we report a new gene delivery vector based on dendrimer-entrapped gold nanoparticles (Au DENPs) with significantly higher gene transfection efficiency than that of dendrimers without AuNPs entrapped. Amine-terminated generation 5 poly(amidoamine) (PAMAM) dendrimers (G5.NH₂) were utilized as templates to synthesize AuNPs with different Au atom/dendrimer molar ratios (25:1, 50:1, 75:1, and 100:1, respectively). The formed Au DENPs were used to complex two different pDNAs encoding luciferase (Luc) and enhanced green fluorescent protein (EGFP), respectively for gene transfection studies. The Au DENPs/pDNA polyplexes with different N/P ratios and compositions of Au DENPs were characterized by gel retardation assay, light scattering, zeta potential measurements, and atomic force microscopic imaging. We show that the Au DENPs can effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines as demonstrated by both Luc assay and fluorescence microscopic imaging of the EGFP expression. The transfection efficiency of Au DENPs with Au atom/dendrimer molar ratio of 25:1 was at least 100 times higher than that of G5.NH₂ dendrimers without AuNPs entrapped at the N/P ratio of 2.5:1. The higher gene transfection efficiency of Au DENPs is primarily due to the fact that the entrapment of AuNPs helps preserve the 3-dimensional spherical morphology of dendrimers, allowing for more efficient interaction between dendrimers and DNA. With the less cytotoxicity than that of G5.NH₂ dendrimers demonstrated by thiazoyl blue tetrazolium bromide assay and higher gene transfection efficiency, it is expected that Au DENPs may be used as a new gene delivery vector for highly efficient transfection of different genes for various biomedical applications.     

Evaluation of Jeffamine®-cored PAMAM dendrimers as an efficient in vitro gene delivery system

In this study, we investigated gene delivery properties of Jeffamine-cored polyamidoamine (PAMAM) dendrimers (JCPDs). The effects of dendrimer concentration, generation, and core size on the gene delivery have been analyzed. The experimental results showed that the JCPD effectively delivered plasmid DNA inside the HeLa cells, and the transfection efficiency improved considerably as the number of generation increased. The cytotoxicity of JCPD in different concentration was tested for HeLa cell line. JCPD was complexed with a lacZ gene carrying plasmid and tested for transfection efficiency using quantitative β-galactosidase expression assay. Additionally, confocal microscopy results revealed that JCPD effectively delivered green fluorescent protein-expressing plasmid into HeLa cells and produced fluorescent signal with satisfactory efficiency. The highest transfection efficiency was obtained from JCPDs G4 and G5, which mixed with expression plasmid vectors at a 10/1 weight ratio. These results indicated that under optimized conditions, JCPD can be considered as an efficient transfection reagent and can be effectively used for gene delivery applications. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2623-2628, 2012.     

A serum-resistant polyamidoamine-based polypeptide dendrimer for gene transfection

A serum tolerant polycation gene vector, G(2) PAMAM-PGlu-G(1) PAMAMs (ALA), was designed, synthesized, characterized and evaluated. A honeycomb-like molecular structure model for mechanistic explanation of ALA was postulated and discussed. Designed as a star-shaped polyamidoamine (PAMAM)-based polypeptide dendrimer through peptide bond linkages, ALA was with non-toxic low generation G(2) PAMAM (G(2)) as its central core, polyglutamate (PGlu)s as its star-shaped backbone branches and G(1) PAMAM (G(1))s as its branch grafts and peripheral terminals. IR, (1)H NMR demonstrated its successful combination. As a gene carrier, ALA exhibited good DNA binding and condensation capacity with particle size (approximately 87 nm for N/P 40, approximately 170 nm for N/P 30) and ζ-potential (approximately 16 mV for N/P 30-40), negligible cytotoxicity, exciting serum tolerant capacity and significant serum-promoted (serum-containing 56.6%>serum-free 32.7%), cell line dependent (Hek 293 > Bel 7402 > Hela), incubation period dependent (38 h > 18 h > 12 h > 9 h > 4 h > 2 h > 1 h) and sustained (peak transfection appeared at 30 h incubation) transfection efficiency. The presence of serum had not only no inhibition on, but also prominent promotion to, the transfection activity of ALA. All above features differentiated ALA clearly from most other serum-inhibitive nonviral gene carriers, and proved ALA the promising and challenging potential efficient gene vector for practical clinical application.     

Optimization of PAMAM-gold nanoparticle conjugation for gene therapy

The development of efficient and biocompatible non-viral vectors for gene therapy remains a great challenge, and exploiting the properties of both nanoparticle carriers and cationic polymers is an attractive approach. In this work, we have developed gold nanoparticle (AuNP) polyamidoamine (PAMAM) conjugates for use as non-viral transfection agents. AuPAMAM conjugates were prepared by crosslinking PAMAM dendrimers to carboxylic-terminated AuNPs via EDC and sulfo-NHS chemistry. EDC and sulfo-NHS have been utilized widely and in numerous applications such as amino acid coupling; however, their use in the coupling of PAMAM dendrimers to AuNPs presents new challenges to form effective and stable constructs for delivery that have not yet been examined. Enhanced colloidal stability and DNA condensation ability was established by probing two critical synthetic parameters: the reaction rate of the PAMAM crosslinking step, and the amine to carboxyl ratio. Based on this work, increasing the amine to carboxyl ratio during conjugation of PAMAM onto AuNPs yielded the optimal vector with respect to colloidal stability and transfection efficiency in vitro. AuPAMAM conjugates present attractive candidates for non-viral gene delivery due to their commercial availability, ease of fabrication and scale-up, high yield, high transfection efficiency and low cytotoxicity.     

Biodegradable PAMAM ester for enhanced transfection efficiency with low cytotoxicity

We synthesized biodegradable polycationic PAMAM (polyamidoamine) esters (e-PAM-R, e-PAM-K) that contain arginines or lysines at the peripheral ends of PAMAM-OH dendrimer through ester bond linkages. The PAMAM esters were readily degradable under physiological conditions (pH 7.4, 37 degrees C), with more than 50% of the grafted amino acids hydrolyzed within 5h. However, polyplexes were very stable and were hardly degraded in the endosomal pH range. Moreover, these amino-acid-modified polymers showed excellent buffering capacities between pH 5.1 and 7.4, facilitating endosomal escape of polyplexes. While the lysine-grafted PAMAM ester did not display significant improvement in transfection efficiency, the arginine-conjugated PAMAM ester-mediated transfection of a luciferase gene showed better transfection efficiency than the branched 25 kDa PEI (polyethylenimine) and PAM-R (peptide bond), and lower cytotoxicity, especially with primary cells such as HUVECs (human umbilical vein endothelial cells) and SMCs (primary rat aorta vascular smooth muscle cells). Furthermore, after DNA release, free e-PAM-R degraded completely into nontoxic PAMAM-OH and arginines by hydrolysis, which resulted in lower cytotoxicity in contrast to the poorly degradable arginine-modified PAMAM with amide bonds. These findings demonstrated that the arginine-grafted biodegradable PAMAM dendrimer, e-PAM-R, is a potential candidate as a safe and efficient gene delivery carrier for gene therapy.     

Fugene6—一种介导体外真核细胞高效率基因转染的新方法

目的 建立一种介导体外真核细胞高效率基因转染的新方法。方法 采用一种新的非脂质体基因转染试剂Fu-gene6,以人p14ARF蛋白表达载体（pCI-neo-p14ARF)为外源DNA,转染存在p14ARF基因原发缺失的人H460,A549,U251和PC-3共4个癌细胞系,通过G418筛选21d确定转染效率。结果 经转 染的4个细胞系培养孔中均长出了抗G418细胞克隆,而且这些细胞克隆的p14ARF基因PCR产物均为阳性。细胞毒性分析发现高浓度Fugene6作用下各细胞的殖活力未受影响。结论 Fugene6为一简便快速、易操作、重复性好且无细胞毒性的体外真核表达载体基因转染新系统。     

Foreword

The effect of PAMAM G3.5, PAMAM G4 and PAMAM-OH G4 dendrimers on human and bovine serum albumins has been studied by fluorescence spectroscopy at different pH and ionic strength. It has been shown that the interactions between dendrimers and proteins depend on pH and the efficiency of interactions can be regulated by changing pH. The maximal pH dependence was observed for interactions between albumins and PAMAM G4 dendrimer. At physiological pH all dendrimers affect proteins in the maximum degree. Dendrimers had no effect on N–F and N–B transitions of albumins. The effect of dendrimers on HSA was smaller than for BSA. The increase of NaCl concentration led to a decrease of interactions between dendrimers and proteins.