PEG修饰的聚阳离子基因载体PEG-Polycarbam-SP在COS-7细胞中转染和毒性的研究

目的：寻找一种转染效率高,细胞毒性低的非病毒基因载体,研究以人体内源性精胺为单体,以乙二醇二氯甲酸酯作为连接剂,以聚乙二醇（PEG）作为亲水基团连接剂合成亲水修饰聚阳离子载体PEG-Polycarbam-SP的基因担载效率,以及对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法：琼脂糖凝胶电泳方法考察复合物的基因担载效率,检测基因复合物的粒径和电位,以荧光素酶质粒为报告基因,研究PEG-Polycarbam-SP/DNA的复合物在COS-7细胞的转染活性,用MTT方法研究PEG-Polycarbam-SP对COS-7细胞的毒性。结果：聚合物与质粒在质量比5以后形成的复合物粒径稳定在50nm左右,Zate电位在20mV左右。COS-7细胞实验显示PEG-Polycarbam-SP具有低于PEI 25kDa的细胞毒性,同时也具有高效输送DNA的能力。结论：PEG-Polycarbam-SP是一种新型的高效、低毒,在基因治疗领域有潜在应用价值的非病毒基因输送载体。     

新型可降解PEI衍生物的体外表征及在HUVEC细胞中的毒性研究

目的:以分支状PEI 1800 Da为基本单元,以化学方法连接咪唑二醛,构建含有可降解亚胺键的新型聚乙烯亚胺衍生物基因载体。对合成的新型基因载体进行体外表征,测定其与p DNA形成的复合物的粒径和zeta电位,研究该复合物在HUVEC细胞中的细胞毒性。方法:通过有机合成方法合成新型聚乙烯亚胺衍生物,考察其与p DNA形成的复合物的粒径和zeta电位,并通过透射电镜考察复合物的形态特征,通过CCK-8方法测定复合物在HUVEC细胞中的毒性。结果:合成的新型基因载体能与p DNA复合形成200 nm左右带20 m V正电荷的纳米颗粒,有利于细胞内吞;形态特征研究表明新型基因载体能将p DNA压缩成类球形的纳米粒,大小与粒径检测结果基本一致。细胞毒性实验表明,合成的新型基因载体材料在相同质量比范围内显示出明显低于PEI25 KDa的细胞毒性。结论:合成的新型基因载体具有较低的细胞毒性,是一种具有良好应用潜力的基因输送载体。     

载基因壳聚糖纳米粒的制备及免疫增强作用的初步研究

摘 要 目的: 制备壳聚糖载基因纳米粒,并对其体外转染效率及其在小鼠体内的免疫增强效果进行初步研究。方法: 以本课题组构建的口蹄疫DNA疫苗为模型药物,采用复凝聚法制备纳米粒;用透射电镜观察形态;用纳米粒度分析仪测定粒径、多分散度和zeta电位;凝胶阻滞分析测定基因在纳米粒中的位置;用体外基因转染实验评价纳米粒的转染活性。用载基因壳聚糖纳米粒免疫雌性Balb/c小鼠,检测免疫小鼠的细胞免疫和体液免疫水平。结果: 所制备的载基因纳米粒形态规则、大多成球形,平均粒径约为150nm,多分散度＜0.26,zeta电位约为21mV;凝胶分析结果表明质粒DNA与壳聚糖分子间可以通过电性结合作用而完全结合,基因几乎全部被包裹在纳米粒内部;体外基因转染实验表明壳聚糖作为一种新型的非病毒基因递送载体能够高效传递DNA进入BHK-21细胞,基因能够在该细胞中高效表达;小鼠免疫实验表明纳米粒不仅能诱导机体产生较高的细胞免疫水平,而且体液免疫水平也显著提高。结论: 壳聚糖纳米粒能将基因递送到细胞内并且能够表达,小鼠免疫实验显示其具有良好的免疫增强效果。     

一种可降解的阳离子聚合物基因转染试剂

目的：合成一种高效、低毒的新型阳离子聚合物载体。方法：以低分子量的聚乙烯亚胺（PEI）为阳离子聚合物的基本单位,以可水解的2,4-戊二醇二丙烯酸盐（PODOA）为交联剂,合成高分子聚合物。用DNA凝胶迟滞实验验证聚合物与DNA的亲和力,以绿色荧光蛋白基因和萤光素酶检测其基因转染效率,用聚合物凝胶电泳实验鉴定其降解性,以MTT法检测其细胞毒性。结果：聚合物具有高的DNA结合亲和力、高基因转染效率,基因转染后的萤光素酶活性高达3×10^9RLU／mg,其基因转染效率相当于甚至优于目前市售的转染试剂,而细胞毒性明显低于其他试剂。该聚合物在中性环境下可降解,而在酸性条件下非常稳定。结论：合成的聚合物具有高转染效率和低细胞毒性,可能在转染和基因治疗研究中起重要作用。     

蔗糖脂肪酸酯型阳离子脂质体的制备及基因转运性能研究

该文采用蔗糖脂肪酸酯(sucrose fatty acid esters,SEs)作为助脂质与季铵盐型阳离子脂质1,2-双-[N-十四烷氧酰胺乙基-N,N-二甲基碘化铵](CTA14)制备阳离子脂质体,测定了脂质体的粒径及Zeta电位,脂质体的平均粒径为210~230 nm,Zeta电位为50~65 mV。DNA延滞实验表明,蔗糖脂肪酸酯型脂质体能够有效压缩DNA。阳离子脂质体与绿色荧光蛋白基因(plasmid green fluorescent protein-N2,p GFP-N2)结合,形成脂质体/DNA复合物,通过载入人喉癌细胞(Hep-2)和人宫颈癌细胞(Hela),观察其转染效率和细胞毒性。结果表明,阳离子脂质与SEs以质量比1:1、2:1混合制备的脂质体均能高效转染Hep-2和Hela细胞。毒性实验显示,SEs对两种细胞的毒性很小,阳离子脂质单独存在时对癌细胞具有一定的细胞毒性,随着SEs加入量的增加,脂质体对的细胞毒性也明显减小。该文进一步证实了SEs能够作为助脂质用于基因载体系统进行基因转运。     

纳米羟基磷灰石表面修饰及其转染细胞的研究

目的:探讨羟基磷灰石-聚乙烯亚胺(nHA-PEI 10KD)纳米颗粒的癌细胞基因转染效率.方法:通过透射电子显微镜(TEM)观察HA-PEI(10KD)纳米颗粒的形态及粒径,Zeta电位仪测定nHA-PEI和HA在酸、碱、中性环境中的电位,用琼脂糖凝胶电泳检测nHAP-PEI(10KD)与DNA结合的能力,MTT比色法检测nHAP-PEI(10KD)对nepG2细胞的毒性,选用增强型绿色荧光蛋白质粒pEGFP1与nHA-PEI结合后,分别转染真核细胞HepG2、Hela、SW620,计算其转染率.结果:nHA-PEI(10KD)分散程度好,粒径60-80nm,在PH7.2时,Zeta电位42.87mV,能转染实验中的细胞,转然效果最好的是HepG2细胞,其次Hela、SW620,转染率高于PEI(10KD)、nHA,但低于脂质体.结论:通过阳离子PEI修饰HA,可有效将增强型绿色荧光蛋白质粒转入HepG2细胞,HA-PEI(10KD)纳米颗粒复合物有望成为基因传递的有效栽体.     

载HCPT纳米粒的制备及其体外抗肿瘤活性研究

目的:制备载羟基喜树碱(HCPT)的PLGA-hyd-PEG-FA纳米粒(HCPT@PLGA-hyd-PEG-FA),并对其体外抗肿瘤活性进行研究。方法:采用乳化溶剂挥发法制备HCPT@PLGA-hyd-PEG-FA,通过单因素试验考察超声功率、聚合物浓度、PVA浓度、水相和油相体积比及投药量对纳米粒粒径的影响;采用zeta电位及激光粒度分析仪测定纳米粒的粒径及zeta电位,用透射电镜(TEM)观察其形态;采用透析法评价HCPT@PLGA-hyd-PEG-FA的体外释药特性;采用MTT法测定HCPT@PLGA-hyd-PEG-FA对HepG2细胞的细胞毒性。结果:HCPT@PLGA-hyd-PEG-FA平均粒径约为109±3 nm,zeta电位为-11.57 mV,载药量为5.6%,TEM显示其为球形;体外释药结果表明HCPT@PLGA-hyd-PEG-FA对HCPT的释放具有p H值依赖性;HCPT和HCPT@PLGA-hyd-PEG-FA的IC50值分别为474.6 ng/mL和286.0 ng/mL。结论:HCPT@PLGA-hyd-PEG-FA体外释药性能良好,HCPT@PLGA-hyd-PEG-FA的细胞毒性明显大于游离的HCPT,值得进一步研究。     

交联聚乙烯亚胺衍生物的构建及其转染COS-7 细胞的研究

目的:优化构建交联聚乙烯亚胺(Polyethylenemine,PEI)衍生物PEI-Bu,研究其对非洲绿猴肾成纤维细胞系(COS-7)的转染活性和细胞毒性。方法:以PEI 800Da为骨架,1,4-丁二醇二氯甲酸酯为连接剂制备聚合物PEI-Bu,琼脂糖凝胶电泳考察其复合质粒DNA的能力,MTT法检测PEI-Bu对COS-7的毒性,以荧光素酶质粒作为报告基因,测定PEI-Bu/DNA复合物在COS-7细胞的转染活性。结果:凝胶电泳表明PEI-Bu/DNA在质量比大于1时即具有复合DNA的能力,PEI-Bu的细胞毒性随浓度增大而增大,在同一浓度下PEI-Bu的细胞毒性小于PEI 25kDa,(P<0.05),PEI-Bu/DNA在质量比为5时达到最高转染活性,高于PEI 25kDa(P<0.01),并与Lipofectamine2000相当(P>0.05)。结论:PEI-Bu在COS-7细胞中是一种低细胞毒性、高转染活性的非病毒基因载体(与商业化的PEI 25kDa比较),其在基因治疗领域中具有潜在的应用前景。     

新型可降解PEI衍生物的表征及其在Brl-3A细胞中的毒性和转染研究

目的:对新型可降解高分子进行表征,研究其在Brl-3A细胞中的毒性和转染效率,以及连接剂比例对以上方面的影响。方法:通过化学方法合成不同比例PEI-Tr高分子,考察其包裹质粒DNA形成纳米颗粒的粒径和电位,以CCK-8方法考察Brl-3A细胞中的细胞毒性,以荧光素酶质粒为报告基因考察Brl-3A细胞中的转染效率。结果:PEI-Tr材料能形成200 nm以下带20 mV左右正电荷的纳米颗粒,具有较好的细胞内吞能力和溶液稳定性,细胞毒性实验证明,随着浓度增加PEI-Tr材料显示了远低于PEI-25kDa的细胞毒性,细胞转染实验表明其拥有高效输送质粒的能力。结论:PEI-Tr是一种高效低毒的可降解聚阳离子载体,在基因输送领域有很大的潜力;连接剂的比例在聚阳离子功能中起到重要作用。     

胆固醇嵌入式阳离子脂质体运载基因研究

阳离子脂质体是一种有临床应用潜力的抗肿瘤药物递药系统,助类脂能起到稳定双层膜和降低阳性成分毒性的作用,同时提供阳性类脂的细胞渗透功能。为了进一步发掘助类脂的应用潜力,该文采用胆固醇(cholesterol)作为助类脂制备阳离子脂质体,测定了脂质体的粒径及Zeta电位,脂质体的平均粒径为100~140 nm,Zeta电位为45~60 mV。脂质体分别与绿色荧光蛋白基因(pGFP-N2)、荧光素酶基因(pGL3)结合,形成脂质体/DNA复合物,通过载入人喉癌细胞(Hep-2),考察了其转染效率和细胞毒性。结果表明,阳离子类脂与胆固醇以1:1、1:2和1:4摩尔比例混合制备脂质体均能高效转染Hep-2细胞。毒性实验显示,阳离子类脂单独存在时对癌细胞具有一定的细胞毒性,随着胆固醇的加入,脂质体对细胞的毒性明显减小,与商品试剂DOTAP和Lipofectamine 2000相当。     

The transfection of Jurkat T-leukemic cells by use of pH-sensitive immunoliposomes

An effective pH-sensitive gene transfer vector has been developed utilising anionic liposomes with various formulations as a carrier of plasmid DNA (pEGlacZ, 7.6 kb) to transfect CD3 T+ lymphocytes (Jurkat cells). The plasmid DNA was condensed using poly-l-lysines with a range of molecular masses to form polyplexes that were interacted with several anionic liposome formulations to form lipopolyplexes. For targeting to the Jurkat cells, distearoylphosphatidylethanolamine (DSPE) linked to poly (ethylene glycol) molecular mass 2000 and coupled to anti-CD3 antibody was incorporated in the liposomes. The polyplexes and lipopolyplexes were characterised in terms of size, zeta potential, gel electrophoresis and electron microscopy. The gene transfer activity of the lipopolyplexes, assessed from beta-galactosidase expression, depended on the charge ratio (NH(3)+/PO(4)-) of the component polyplex and the lipid/DNA weight ratio of the lipopolyplex.     

Targeted double domain nanoplex based on galactosylated polyethylenimine enhanced the delivery of IL-12 plasmid

The objective of the present investigation was to design a targeted polyethylenimine (PEI)-based polyplex by conjugating lactose bearing galactose groups on low molecular weight PEI (LMW PEI) grafted to a high molecular weight PEI (HMW PEI) via a succinic acid linker in order to restore the amine content of the whole conjugate used for ligand conjugation. The PEI conjugate was synthesized and characterized in terms of buffering capacity, particle size, zeta potential, plasmid condensation ability, and protection of DNA against degrading enzymes. Also, the transfection efficiency and cytotoxicity were evaluated in the cell line over-expressing asialoglycoprotein receptors (ASGPRs) and compared with the cells lacking the receptors. The results demonstrated the ability of PEI conjugate in condensation of plasmid DNA and protection against enzyme degradation. The PEI conjugate formed nanoparticles of around 75 nm with higher buffering capacity compared with unmodified PEI. The polyplexes prepared by the modified PEI could increase the level of transgene up to four folds in the cells over-expressing the receptor. The results demonstrated the separation of targeting and delivery domains could be considered as a strategy to restore the amine content of the PEI molecule utilized for targeting ligand conjugation.     

A mechanistic dissection of polyethylenimine mediated transfection of CHO cells: To enhance the efficiency of recombinant DNA utilization

In this study, we examine the molecular and cellular interactions that underpin efficient internalization and utilization of polyethylenimine (PEI):DNA complexes (polyplexes) by Chinese Hamster Ovary (CHO) cells. Cell surface polyplex binding and internalization was a biphasic process, consisting of an initial rapid Phase (I), lasting approximately 15 min, followed by a slower second Phase (II), saturating at approximately 240 min post transfection. The second Phase accounted for the majority (60–70%) of polyplex internalization. While cell surface heparan sulphate proteoglycans (HSPGs) were rapidly cointernalized with polyplexes during Phase I, cell surface polyplex binding was not dependent on HSPGs. However, Phase II polyplex internalization and HSPG regeneration onto the surface of trypsinized cells occurred at similar rates, suggesting that the rate of recycling of HSPG‐containing membrane to the plasma membrane limits Phase II internalization rate. Under optimal transfection conditions, polyplexes had a near neutral surface charge (zeta potential) and cell surface binding was dependent on hydrophobic interactions, being significantly inhibited by both chemical sequestration of cholesterol from the plasma membrane and addition of nonionic surfactant. Induced alterations in polyplex zeta potential, using ferric (III) citrate to decrease surface charge and varying PEI:DNA ratio to increase surface charge, served to inhibit polyplex binding or reduce secreted alkaline phosphatase reporter expression and cell viability, respectively. To increase polyplex hydrophobicity and internalization an alkylated derivative of PEI, propyl‐PEI, was chemically synthesized. Using Design of Experiments–Response Surface Modeling to optimize the transfection process, the function of propyl‐PEI was compared to that of unmodified PEI in both parental CHO‐S cells and a subclone (Clone 4), which exhibited superior transgene expression via an increased resistance to polyplex cytotoxicity. The combination of propyl‐PEI and Clone 4 doubled the efficiency of recombinant DNA utilization and reporter protein production. These data show that for maximal efficacy, strategies to increase polyplex internalization into cells must be used in concert with strategies to offset the inherent cytotoxicity of this process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1161–1170, 2014     

A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro

Diverse polycationic polymers have been used as nonviral transfection agents. Here we report the ability of colloidal silica particles with covalently attached cationic surface modifications to transfect plasmid DNA in vitro and make an attempt to describe the structure of the resulting transfection complexes. In analogy to the terms lipoplex and polyplex, we propose to describe the nanoparticle-DNA complexes by the term "nanoplex". Three batches, Si10E, Si100E, and Si26H, sized between 10 and 100 nm and with zeta potentials ranging from +7 to +31 mV at pH 7.4 were evaluated. The galactosidase expression plasmid DNA pCMVbeta was immobilized on the particle surface and efficiently transfected Cos-1 cells. The transfection activity was accompanied by very low cytotoxicity, with LD(50) values in the milligrams per milliliter range. The most active batch, Si26H, was produced by modification of commercially available silica particles with N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, yielding spherical nanoparticles with a mean diameter of 26 nm and a zeta potential of +31 mV at pH 7.4. Complexes of Si26H and pCMVbeta plasmid DNA formed at w/w ratios of 10 were most effective in promoting transfection of Cos-1 cells in the absence of serum. At this ratio, >90% of the DNA was associated with the particles, yielding nanoplexes with a net negative surface charge. When the transfection medium was supplemented with 10% serum, maximum gene expression was observed at a w/w ratio of 30, at which the resulting particle-DNA complexes possessed a positive surface charge. Transfection was strongly increased in the presence of 100 microM chloroquine in the incubation medium and reached approximately 30% of the efficiency of a 60 kDa polyethylenimine. In contrast to polyethylenimine, no toxicity was observed at the concentrations required. Atomic force microscopy of Si26H-DNA complexes revealed a spaghetti-meatball-like structure. The surface of complexes prepared at a w/w ratio of 30 was dominated by particles half-spheres. Complex sizes correlated well with those determined previously by dynamic light scattering.     

An acid-labile block copolymer of PDMAEMA and PEG as potential carrier for intelligent gene delivery systems

Intelligent gene delivery systems based on physiologically triggered reversible shielding technology have evinced enormous interest due to their potential in vivo applications. In the present work, an acid-labile block copolymer consisting of poly(ethylene glycol) and poly(2-(dimethylamino)ethyl methacrylate) segments connected through a cyclic ortho ester linkage (PEG- a-PDMAEMA) was synthesized by atom transfer radical polymerization of DMAEMA using a PEG macroinitiator with an acid-cleavable end group. PEG- a-PDMAEMA condensed with plasmid DNA formed polyplex nanoparticles with an acid-triggered reversible PEG shield. The pH-dependent shielding/deshielding effect of PEG chains on the polyplex particles were evaluated by zeta potential and size measurements. At pH 7.4, polyplexes generated from PEG- a-PDMAEMA exhibited smaller particle size, lower surface charge, reduced interaction with erythrocytes, and less cytotoxicity compared to PDMAEMA-derived polyplexes. At pH 5.0, zeta potential of polyplexes formed from PEG- a-PDMAEMA increased, leveled up after 2 h of incubation and gradual aggregation occurred in the presence of bovine serum albumin (BSA). In contrast, the stably shielded polyplexes formed by DNA and an acid-stable block copolymer, PEG- b-PDMAEMA, did not change in size and zeta potential in 6 h. In vitro transfection efficiency of the acid-labile copolymer greatly increased after 6 h incubation at pH 5.0, approaching the same level of PDMAEMA, whereas there was only slight increase in efficiency for the stable copolymer, PEG- b-PDMAEMA.     

Cationic lipids with increased DNA binding affinity for nonviral gene transfer in dividing and nondividing cells

Effect of headgroup structure on catonic lipid-mediated transfection was investigated with either a (i) tertiary amine, (ii) quaternary amine with a hydroxyl, or (iii) quaternary amine with mesylate as headgroups. Liposomes were formulated using cholesterol or dioleoyl phosphatidyl ethanolamine (DOPE) as colipids, and transfection efficiencies were determined in rapidly dividing colon carcinoma (CT 26) and rat aortic smooth muscle (RASM) cells as well as in nondividing human pancreatic islets using luciferase and green fluorescent protein expression plasmids, pcDNA3-Luc and pCMS-EGFP, respectively. Liposome/pDNA complexes were evaluated for DNA conformational state by circular dichroism (CD), DNA condensation by electrophoretic mobility shift assay (EMSA), particle size and zeta potential by laser diffraction technique, and surface morphology by transmission electron microscopy (TEM). Encouraging transfection results were obtained with the mesylate headgroup based lipid in liposome formulations with DOPE as a colipid, which were higher than the commercially available Lipofectamine formulation. We hypothesize that the additional hydrogen bonding or covalent interactions of the headgroup with the plasmid DNA, leading to higher binding affinity of the cationic lipids to pDNA, results in higher transfection. This hypothesis is supported by TEM observations where elongated complexes were observed and more lipid was seen associated with the DNA.     

Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

Analysis and Optimization of the Cationic Lipid Component of a Lipid/Peptide Vector Formulation for Enhanced Transfection In Vitro and In Vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K₁₆GACRRETAWACG) and Lipofectin^®, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

Low-pH-sensitive poly(ethylene glycol) (PEG)-stabilized plasmid nanolipoparticles: effects of PEG chain length, lipid composition and assembly conditions on gene delivery

BACKGROUND: We have studied the effects of the poly(ethylene glycol) (PEG) chain length and acyl chain composition on the pH-sensitivity of acid-labile PEG-diorthoester (POD) lipids. The optimal conditions are described for preparation of DNA plasmid encapsulated POD nanolipoparticles (NLPs) which mediate high gene delivery activity in vitro with moderate cytotoxicity. METHODS AND RESULTS: A series of POD lipids with various PEG chain lengths (750, 2000, and 5000 Da) or acyl chains (distearoyl 18:0 or dioleoyl 18:1) were incorporated into DNA containing NLPs or model liposomes as a stealth and bioresponsive component. We investigated the collapse kinetics of the POD-stabilized liposomes when the PEG chain length was changed. We optimized a detergent dialysis method to encapsulate plasmid DNA into NLPs prepared from a mixture of the various POD lipids, cationic lipid and phosphatidylethanolamine lipid. A critical concentration (28 mM) of n-octyl-beta-D-glucopyranoside (OG) enabled high encapsulation of DNA plasmid into 100 nm particles with a neutral surface charge. The POD NLPs are stable at pH 8.5 but rapidly collapse (approximately 10 min) into aggregates at pH 5.0. In the detergent solution there is a metastable DNA-lipid intermediate that evolves into a stable NLP if the detergent is removed shortly after adding DNA to the lipid-detergent mixture. The rank order of transfection activity from NLPs containing PEG-lipid was POD 750 > POD 5000 = POD 2000 > non-pH-sensitive PEG-lipid. The particle size stability was in the reverse order. Binding of the NLPs to cells reached a maximum level by 12 hours. The POD NLPs had slightly less transfection activity but considerably lower cytotoxicity than the PEI-DNA polyplex. CONCLUSIONS: Of the PEG-orthoester lipids tested, POD 2000 is the better choice for the preparation of sterically stabilized NLPs with a small particle diameter, good stability, low cytotoxicity, and satisfactory transfection activity.