不同相对分子质量聚乙烯亚胺体外介导基因传递的研究

目的研究4种不同相对分子质量聚乙烯亚胺（PEI）作为非病毒基因载体体外介导基因传递的能力。方法采用四甲基噻唑蓝法（MTT法）测定了PEI对Hela细胞的毒性,利用琼脂糖凝胶电泳阻滞试验考察PEI与DNA的结合能力,测定PEI—DNA复合物的粒径和Zeta电位,以及考察转染率。结果PEI的细胞毒性与相对分子质量呈正相关,高相对分子质量PEI的细胞毒性远大于低相对分子质量PEI;高相对分子质量PEI在较低的N／P比时就能对DNA起到完全阻滞作用;低相对分子质量PEI与DNA形成的复合物粒径明显大于高相对分子质量的PEI;Zeta电位随着PEI相对分子质量的增大而增大,复合物的粒径和Zeta电位都与组成中的N／P比有关;相对分子质量为2000的PEI（PEI2K）在Hela细胞中的转染率最低,而相对分子质量为25000的PEI（PEI25K）的转染率最高。结论PEI的相对分子质量对其各项性能指标以及介导基因传递的能力都有较大影响。     

聚乙烯亚胺-胆固醇结合脂质微泡介导的基因转染系统

本文研究了聚乙烯亚胺-胆固醇阳离子聚合物(PEI-Chol)基因载体的性能及其结合中性脂质、聚乙二醇修饰脂质微泡介导的基因传递系统(微泡/PEI-Chol/DNA)的体外细胞基因转染效率。采用胆固醇甲酰氯与聚乙烯亚胺(Mw1800)通过酰胺化制得PEI-Chol,并运用IR、1H NMR和MADI-TOF-MS(基质辅助激光解吸附电离串联飞行时间质谱)对其结构和分子量进行测定,平均相对分子质量约为2000。采用反相挥发法制备DPPC、DSPE-PEG2000和全氟丙烷构建的粒径为2～8μm的脂质微泡,并运用自组装技术将PEI-Chol/DNA复合物与微泡融合,构建微泡/PEI-Chol/DNA(bubble/PEI-Chol/DNA)基因转染系统;以pEGFP-Cl(enhanced green fluorescent protein,增强型绿色荧光蛋白)为报告基因,用凝胶滞留试验考察PEI-Chol的DNA压缩能力,同时在A549和MCF-7两种细胞中比较以PEI-Chol为核心构建的各种载体系统的细胞毒性、抗血清沉淀作用以及细胞转染效率。结果发现,当N/P比值为4以上时,PEI-Chol能有效压缩DNA;...     

聚乙烯亚胺介导基因转染的影响因素研究

目的研究非病毒基因载体聚乙烯亚胺（PEI）介导基因转染的影响因素。方法采用透射电镜观察PEI与DNA形成复合物的形态,分别考察质粒量、复合物形成时间、复合物在细胞上作用时间和氮磷比（N/P）对转染效率的影响,以筛选较优的转染条件。结果PEI可有效地与DNA形成复合物,当质粒量为每孔2μg、复合物形成时间为30min、复合物在细胞上的作用时间为4h且N/P为20时转染效率最高。结论PEI可作为合成新型非病毒载体的骨架,但必须控制有关因素才能得到最佳的和可重复的转染结果。     

叶酸修饰的聚乙烯亚胺-聚乙二醇共聚物载体在体内外转染效率的研究

目的探讨叶酸修饰的不同相对分子质量聚乙酰亚胺构建的聚乙烯亚胺-聚乙二醇5000(PEI-PEG)作为基因载体在体内外的转染效率。方法在体外实验中,分别测试3个相对分子质量FPPs(branched PEI:0.6kDa,10kDa和Linear PEI 25kDa)在最佳N/P、有无血清、血清的浓度和叶酸受体靶向性等条件下对口腔癌上皮细胞(KB)转染率的影响;在体内实验中,FPP(0.6kDa)分别压缩pLuc-p53基因形成的复合物通过瘤内注射和尾静脉注射2种方式考察聚合物的转染效率。结果在体外实验中,转染率最佳条件分别是N/P=20、有血清且转染率随着白蛋白(ALB)或胎牛血清(FBS)浓度的增加有上升趋势;其中3个相对分子质量PEI组成的FPP转染率分别为FPP(10kDa>25kDa>0.6kDa);FPP有较强的叶酸靶向性;在体内实验中,与BPP/pLuc和PEI(25kDa)/pLuc相比,FPP(10kDa)/pLuc呈现出较高的转染率;在48h内,随着时间的推移,转染效率增加;FPP(10kDa)/p53能改善荷瘤小鼠;与裸pLuc相比,3种相对分子质量的FPPs没有毒性。结论具有靶向性的FPP非病毒载体能成为临床传递基因治疗载体的候选。     

非病毒型载体聚乙烯亚胺在基因递送应用中的研究进展

非病毒型基因载体因具有低毒性、低免疫原性及易制备等诸多优点,近年来成为制备高效低毒型基因递送载体的研究热点。聚乙烯亚胺(PEI)作为典型的非病毒型基因载体以其较好的基因压缩能力及溶酶体逃逸能力引起了广泛关注。本文对基于PEI类基因载体的体内递送过程及所面临的问题(如生物相容性低、细胞毒性、缺乏特异靶向性和目的基因释放不足等)进行了归纳与分析,并总结了相应的改进方案。此外,对基于PEI的智能响应型基因载体(内源性刺激响应型、外源性刺激响应型)的研究进展进行了综述,为开发出更加安全高效的非病毒型基因载体提供参考。     

聚乙烯亚胺的修饰及其作为基因载体的研究

目的:考察聚氧乙烯硬脂酸酯(POES)修饰聚乙烯亚胺(PEI)后聚合物的细胞毒性及其与DNA形成复合物后的载体性质。方法:使用琥珀酰亚胺碳酸脂法将POES连接在PEI上,通过1H-NMR确定接枝聚合物的结构,将接枝聚合物与DNA形成复合物后考察其琼脂糖凝胶电泳行为及测定其粒径、Zeta电位;MTT法考察接枝聚合物的细胞毒性,使用质粒pGL3-lus作为报告基因,测定虫荧光素酶活性以评价复合物对Hela细胞的转染效率。结果:1H-NMR结果表明接枝聚合物具有较高的纯度。凝胶电泳表明接枝聚合物对DNA的包裹能力随着N/P值的增加而升高,随着POES接枝数目的增大而降低。复合物的粒径小于300nm,Zeta电位随N/P值的增加而升高。接枝聚合物细胞毒性显著低于PEI,POES接枝数目低的聚合物转染效率较高。结论:POES修饰的PEI可以作为一种有效的非病毒基因载体。     

聚乙烯亚胺与壳聚糖在基因载体中的应用

基因治疗是攻克肿瘤最有潜力的方法之一,其关键是寻找可靠而有效的转基因载体。经过各种生物或化学修饰的聚乙烯亚胺和壳聚糖基因载体因转染效率高、细胞毒性低、靶向性强而成为研究热点。针对聚乙烯亚胺、壳聚糖及其衍生物应用于基因载体中存在的问题,将高转染效率的聚乙烯亚胺与高生物相容性的壳聚糖结合,可能得到兼具两者优点的新型基因载体。     

聚乙烯亚胺纳米基因递送系统的研究进展

基因治疗在恶性肿瘤、感染性疾病、自身免疫性疾病、罕见病等重大难治性疾病的治疗中表现出巨大潜力。基因递送载体是基因治疗能否成功实施的关键所在,聚乙烯亚胺（PEI）是一种被广泛研究的阳离子基因递送载体,在不同细胞系和转染条件下均展现出稳定高效的基因转染效果,其中PEI25k更被视作基因转染的“黄金标准”。为解决PEI在基因递送中存在的体内转染效率低、细胞毒性大、靶向性低和负载基因溶酶体降解等问题,该文对基于PEI设计构建新型纳米递送系统用于基因治疗的研究进展进行综述,主要包括高相对分子质量线性PEI、多糖、亲水性的聚合物和右旋糖酐修饰的PEI,交联的低相对分子质量PEI,基于PEI的无机纳米递送载体以及基于PEI的药物与基因共递送载体系统,以期为进一步构建高效低毒的基因递送系统提供理论指导。     

泊洛沙姆407修饰对聚乙烯亚胺的毒性和转染性质的影响

目的:考察泊洛沙姆407修饰对聚乙烯亚胺(PEI)的毒性和转染性质的影响.方法:使用琥珀酰亚胺碳酸脂法将P407连接在PEI的氨基上,得到新聚合物,通过1H-NMR确定新聚合物的结构,将该聚合物与DNA形成复合物,测定复合物的zeta电位,MTT法考察复合物的细胞毒性,使用质粒pGL3-lus作为报告基因,测定虫荧光素酶活性评价复合物对Hela细胞的转染效率.结果:1H-NMR结果表明合成的聚合物具有较高的纯度.复合物的Zeta电位随氮/磷比(N/P)值的增加而增高.复合物的细胞毒性随着N/P值的增加而增大,新聚合物其细胞毒性显著低于未修饰的PEI.新聚合物在高N/P值时仍能保持较高的转染效率.结论:泊洛沙姆407修饰的PEI可以作为一种有效的非病毒基因栽体.     

超声靶向微泡破碎联合半乳糖聚乙烯亚胺介导EGFP质粒转染肝癌细胞的研究

目的 探讨超声靶向微泡破碎(UTMD)联合半乳糖聚乙烯亚胺(PEI-Gal)介导pEGFP-N3质粒转染肝癌细胞HepG2的效率及对细胞增殖的影响.方法 体外培养HepG2细胞,随机分为6组:(1)空白对照组(C);(2)超声(U)+微泡(M)组;(3) PEI-Gal(P)组;(4)P+M组;(5)P+U组;(6)P+U+M组.转染24 h后荧光显微镜观察HepG2细胞中绿色荧光蛋白质的表达,流式细胞术检测转染率,MTT法检测细胞活性.结果 P+U+M组转染率为(29.16±1.78)％,明显高于其他各组(P均＜0.05),U+M组转染率最低(15.63±1.81)％.P+U+M组存活率为(83.48±0.93)％,低于U+M组(86.37±1.56)％和P+U组的(89.54±0.91)％(P＜0.01).结论 UTMD联合PEI-Gal能显著增强pEGFP-N3质粒转染肝癌细胞HepG2的效率.     

Comparative gene transfer efficiency of low molecular weight polylysine DNA-condensing peptides

Abstract: In a previous report (M.S. Wadhwa et al. (1997) Bioconjugate Chem. 8, 81–88), we synthesized a panel of polylysine-containing peptides and determined that a minimal repeating lysine chain of 18 residues followed by a tryptophan and an alkylated cysteine residue (AlkCWK₁₈) resulted in the formation of optimal size (78 nm diameter) plasmid DNA condensates that mediated efficient in vitro gene transfer. Shorter polylysine chains produced larger DNA condensates and mediated much lower gene expression while longer lysine chains were equivalent to AlkCWK₁₈. Surprisingly, AlkCWK₁₈ (molecular weight 2672) was a much better gene transfer agent than commercially available low molecular weight polylysine (molecular weight 1000–4000), despite its similar molecular weight. Possible explanations were that the cysteine or tryptophan residue in AlkCWK₁₈ contributed to the DNA binding and the formation of small condensates or that the homogeneity of AlkCWK₁₈ relative to low molecular weight polylysine facilitated optimal condensation. To test these hypotheses, the present study prepared AlkCYK₁₈ and K₂₀ and used these to form DNA condensates and conduct in vitro gene transfer. The results established that DNA condensates prepared with either AlkCYK₁₈ or K₂₀ possessed identical particle size and mediated in vitro gene transfer efficiencies that were indistinguishable from AlkCWK₁₈ DNA condensates, eliminating the possibility of contributions from cysteine or tryptophan. However, a detailed chromatographic and electrospray mass spectrometry analysis of low molecular weight polylysine revealed it to possess a much lower than anticipated average chain length of dp 6. Thus, the short chain length of low molecular weight polylysine explains its inability to form small DNA condensates and mediate efficient gene transfer relative to AlkCWK₁₈ DNA condensates. These experiments further emphasize the need to develop homogenous low molecular weight carrier molecules for nonviral gene delivery.     

Protamine sulfate enhances the transduction efficiency of recombinant adeno-associated virus-mediated gene delivery

Purpose. The purpose of this study was to evaluate glucose responsiveness in HepG2 human hepatoma cells transduced by a recombinant adeno-associated virus (rAAV) vector containing the insulin gene promoter, and to investigate the effect of protamine sulfate on rAAV-mediated gene delivery. Methods. Recombinant AAV vector, AAV.Ins.Luc. EGFP, was employed to transduce HepG2 hepatoma cells. Virus infection was carried out either in the absence or presence of protamine sulfate, followed by fluorescence microscopic examination, luciferase activity assay, and flow cytometric analysis. Electrokinetic measurements were carried out to determine the effect of protamine sulfate on zeta potential of the cells and the virus. Results. Glucose-responsive luciferase gene expression was obtained in rAAV-transduced HepG2 cells. Addition of 5 g/ml protamine reversed the zeta potential of the cells and the virus particles, leading to enhanced transgene expression in rAAV-transduced HepG2 cells. Enhancement of protamine sulfate on rAAV-mediated gene transfer was dose-dependent. Addition of more than 5 g/ml protamine resulted in a reduction of infectability of the virus. Conclusions. Glucose responsiveness in the millimolar concentration range can be obtained in rAAV-transduced HepG2 cells. Protamine sulfate, up to 5 g/ml, enhanced the rAAV transduction efficiency in HepG2 cells. The enhancement was correlated with zeta potential of the cells and the virus.     

异甘草素固体脂质纳米粒中低分子肝素钠包封率的测定

目的测定异甘草素固体脂质纳米粒中低分子肝素钠(LMWH)的包封率。方法在Britton-Robinson(B-R)缓冲溶液中,天青A与LMWH相互作用,在662 nm处测定吸光度差值(△A),用△A对LMWH浓度进行线性回归。样品同法操作,测定LMWH的包封率。结果 LMWH的回收率在90.0%～102.5%之间,线性范围为100～1 000μg/mL(r=0.999 1),异甘草素固体脂质纳米粒中LMWH的平均包封率为35%(n=5)。结论此法操作简单、准确,适用于该制剂的质量评价。     

Nanoparticle-based delivery enhances anti-inflammatory effect of low molecular weight heparin in experimental ulcerative colitis

Epithelial administration of low molecular weight heparin (LMWH) has proven its therapeutic efficiency in ulcerative colitis (UC) but still lacks of a sufficiently selective drug delivery system. Polymeric nanoparticles were used here not only to protect LMWH from intestinal degradation but also to provide targeted delivery to inflamed tissue in experimental colitis mice. LMWH was associated with polymethacrylate nanoparticles (NP) type A (PEMT-A) or type B (PEMT-B) of a size: 150?nm resulting in a maximum drug loading: 0.1?mg/mg. In a lipopolysaccharide-stimulated macrophages both, free LMWH and LMWH-NP have significantly reduced the cytokines secretion independently from cellular uptake. The in-vivo therapeutic efficiency was dose dependent as at low doses (100?IU/kg) only minor differences between free LMWH and LMWH-NP were found and the superiority of LMWH-NP became prominent with dose increase (500?IU/kg). Administration of LMWH-NP at 500?IU/kg has markedly improved the clinical activity as compared to LMWH while similarly pathophysiological indicators revealed increased therapeutic outcome in presence of NP compared to LMWH alone: Myeloperoxidase (Colitis control: 10 480?±?5335, LMWH-PEMT-A NP: 1507?±?2165, LMWH-PEMT-B NP: 382?±?143, LMWH: 8549?±?5021 units/g) and tumor necrosis factor: (Colitis control: 1636?±?544, LMWH-PEMT-A NP: 511?±?506, LMWH-PEMT-B NP: 435?±?473, LMWH: 1110?±?309?pg/g). Associating LMWH with NP is improving the anti-inflammatory efficiency of LMWH in-vivo by its protection against degradation in luminal environment and selective drug delivery. Such a combination holds promise for a highly specific therapy by its double selectivity towards the inflamed intestinal tissue. LMWH-PEMT NP have significantly improved the clinical activity in-vivo in comparison to free LMWH.     

Lyoprotection of aviscumine with low molecular weight dextrans

The aim of this research was to ascertain whether dextrans with low molecular weight will stabilize aviscumine. During freeze-drying increasing concentrations of dextran T1 (MW 1000) stabilized aviscumine. Eight percent of dextran resulted in a nearly 100% recovery of the activity and in addition a complete amorphous structure of the solid phase was obtained. By decreasing the molecular weight of the dextran from 75 to 1 kDa, the protein activity was increased by 20% in the lyophilisate. Combinations of dextran with either trehalose or mannitol showed no additional effects on stability. The improved stabilization of aviscumine using low molecular weight dextrans is explained by an increased interaction between the protein and the dextran molecules (like hydrogen bonds), whereas they are sterically hindered if larger dextran molecules are used. When the protein concentration was increased from 10 to 100 microg/ml (in formulas with 8% dextran T1), no influence on the protein activity could be found. With regard to the carbohydrate-binding activity of the protein, it was shown that the optimal content of residual water in the lyophilisate should be about 2%. Above and below this percentage a destabilization of the protein was observed. The often discussed failure of dextran as a stabilizing excipient in the freeze-drying of proteins seems to be a question of the selection of the correct molecular weight.     

Neurotrophin-like low molecular weight compounds

A large number of compounds have been reported to prevent ischemia-induced neuronal death, whereas there are few described to enhance recovery of brain functions. Since neurotrophins do not only prevent neuronal death but also protect neuronal circuits, they may be potential candidates. However, their poor penetration of the blood-brain-barrier hampers their development as therapeutic agents. In this context, low-molecular-weight compounds that possess neurite outgrowth- and neuronal survival-promoting activities may be alternative candidates. Neurite outgrowth-promoting prostaglandins, which were recently-synthesized based on the chemical structure of anti-tumor cyclopentenone prostaglandin derivatives, have been characterized by their neurotrophic effects on neurons in the central nervous system. In this paper, we present a review of these compounds as therapeutic agents against several neurodegenerative diseases.     

Conjugation of low molecular weight poly(ethylene glycol) to biphalin enhances antinociceptive profile

The objectives of this study were to examine the effect of poly(ethylene glycol) (PEG) conjugation on the tyrosine residues of biphalin to determine the proper size PEG for optimal efficacy and investigate the antinociceptive profile of PEG-biphalin against biphalin via three routes of administration. All antinociception evaluations were made using a radiant-heat tail flick analgesia meter. (2 kDa)(2) PEG-biphalin was identified as the optimal size of PEG to enhance the antinociceptive profile following intravenous administration of 685 nmol kg(-1) of biphalin or PEG-biphalin [(1 kDa)(2), (2 kDa)(2), (5 kDa)(2), (12 kDa)(2), (20 kDa)(2)]. (2 kDa)(2) PEG-biphalin displayed an area under the curve (AUC) approximately 2.5 times that of biphalin with enhanced analgesia up to 300 min postinjection. (2 kDa)(2) PEG-biphalin was equipotent to biphalin following intracerebroventricular administration (0.4 nmol kg(-1)). Both biphalin and (2 kDa)(2) PEG-biphalin were effectively antagonized with naloxone (10 mg kg(-1)) and a partial antagonistic effect was seen following pretreatment with naltrindole (20 mg kg(-1)). (2 kDa)(2) PEG-biphalin showed significantly increased potency (A(50)) when administered intravenously and subcutaneously. Additionally, (2 kDa)(2) PEG-biphalin demonstrated a significantly enhanced antinociceptive profile (AUC) via all routes of administration tested. These findings indicate that PEG conjugation to biphalin retains opioid-mediated effects observed with biphalin and is a valuable tool for eliciting potent, sustained analgesia via parenteral routes of administration.     

Combination of low molecular weight heparins with antiplatelet agents in non-ST elevation acute coronary syndromes: an update

This article reviews the use of low molecular weight heparin (LMWH) and antiplatelet agents in the treatment of unstable angina and non-ST segment elevation myocardial infarction (NSTEMI), which together account for 1 million hospitalisations annually in the US alone. Mortality and recurrent myocardial infarction (MI) in these conditions is currently approximately 8 to 16% at 1 month, and there is a need to optimise treatment further. Since their introduction, LMWHs have been shown to be successful and well tolerated in the treatment of unstable angina and NSTEMI, but differences have been seen in their efficacy compared with the parent compound, unfractionated heparin (UFH). A meta-analysis of all LMWHs, grouped, versus UFH showed equivalent efficacy and safety. The LMWHs dalteparin sodium and nadroparin calcium have independently been shown to be as effective as UFH. However, enoxaparin sodium has been shown to have greater clinical efficacy than UFH in patients with unstable angina (UA)/NSTEMI. One area of new research is patients with UA/NSTEMI who later undergo percutaneous coronary interventions (PCI), and early data suggest enoxaparin can be safely used as an anticoagulant instead of UFH in these patients. There is a wealth of data for glycoprotein (GP) IIb/IIIa receptor antagonists (abciximab, eptifibatide, lamifiban, and tirofiban), although some are conflicting. Recent meta-analyses suggest that some benefit is conferred by using these compounds, particularly in patients who undergo PCI. Recent trials have focussed on combining GP IIb/IIIa antagonists with LMWH, and although data is still scant, the ACUTE (Anti-thrombotic Combination Using Tirofiban and Enoxaparin) and ACUTE II studies indicate the safety and potential clinical benefit of combining enoxaparin with tirofiban in patients with UA/NSTEMI not undergoing PCI, compared with UFH and tirofiban. The NICE (National Investigators Collaborating on Enoxaparin) 4 study collected data on the combination of enoxaparin and abciximab in patients undergoing PCI, and both safety and efficacy data compared well with historical data collected on the use of UFH with abciximab. The more recent NICE 3 study extended this finding to the combination of enoxaparin with abciximab, tirofiban or eptifibatide. The safety of two doses of dalteparin and abciximab had also been investigated, with the higher dose the efficacious, and also with safety, in patients undergoing PCI. In addition, a GUSTO (Global Use of Strategies to Open Occluded Coronary Arteries in Acute Coronary Syndromes) IV substudy found that dalteparin had equivalent safety to UFH when co-administered with abciximab in patients not undergoing PCI. The NICE 3 and 4 trials were not randomised comparisons, and as such there results must be interpreted with caution. Recently, the CRUISE (Coronary Revascularisation Utilizing Integrelin [eptifibatide] and Single-bolus Enoxaparin) and INTERACT (Integrelin and Enoxaparin randomised assessment of Acute Coronary Syndromes Treatment) studies have provided evidence for both the safety and efficacy of enoxaparin combined with eptifibatide in non-ST elevation patients with acute coronary syndromes. A further study (SYNERGY [Superior Yield of the New strategy of Enoxaparin, Revascularization and GlYcoprotein IIb/IIIa inhibitors]) will investigate the efficacy of the combination of enoxaparin with abciximab versus that of UFH and abciximab in a large cohort of 8000 patients. The use of GP IIb/IIIa agents and LMWH in patients with UA/STEMI has led to their use in those with ST-elevation MI, and studies indicate LMWH is efficacious and can be used safely as an adjunct to thrombolysis. New studies will investigate the use of these agents in patients with STEMI not undergoing thrombolysis and we await the results of these studies.     

低分子量地黄多糖对p53基因表达的影响

研究低分子量地黄多糖（ＬＲＰＳ）对ｐ５３基因表达的影响．方法：利用定量ＰＣＲ的方法检测ｐ５３基因表达的水平．结果：ＬＲＰＳ在最适抗肿瘤有效剂量２０和４０ｍｇ·ｋｇ－１条件下，Ｌｅｗｉｓ肺癌组织内ｐ５３基因的表达水平分别是１５２和１４８，对照组为０４６，环磷酰胺组为１６０．ＬＲＰＳ能使Ｌｅｗｉｓ肺癌组织内ｐ５３基因的表达明显增加．结论：ＬＲＰＳ对抗癌基因ｐ５３表达的影响是其抗肿瘤作用的机制之一．