【作者】 周毅；

【导师】 陈建海；

【作者基本信息】 南方医科大学 ， 药理学， 2011， 博士

【摘要】 癌症严重危害人类健康,攻克癌症一直受到医疗界的关注；因此多种治疗药物及其方法孕育而生,其中基因治疗成为21世纪研究热点。基因治疗是通过插入治疗基因至病变细胞内而产生特定功能的蛋白质或抑制异常基因表达,从而治疗疾病的一种新手段。安全、高效的基因转染系统的研发成为关键。其癌细胞靶向性的研究又成为关键中的关键。目前基因载体分为两类：病毒载体和非病毒载体。非病毒载体因具有制备简单、低免疫原性且携带大分子量DNA及生物安全性高等优点而倍受青睐。在非病毒载体的制备过程中,阳离子聚合物聚乙烯亚胺(PEI,Polyethyenimine)因其有较高的转染率而被看作非病毒载体的黄金标准。PEI有线型或分枝状结构,有较高的阳性电荷,与带有阴性电荷的DNA或RNA结合能力较强,所形成的复合物可通过静置吸附等作用进入细胞。PEI通过著名的质子海绵效应,使溶酶体破裂从而将内吞的DNA释放到细胞质或者细胞核中并表达。PEI的分子量的范围很广,5000-25000 Da是目前作为基因转染载体常用的一个范围。一般说PEI的基因转染效率随着其分子量的增高而增大,但其毒性也随之增大。如分枝状PEI-25 KDa的转染效率和毒性远比10 KDa的大,反之亦然。同时有报道PEI作为基因转染载体直接体内应用会导致红细胞的聚集,并与血液中的白蛋白等非特异性结合,影响转染效率。为了降低PEI毒性,提高其转染率,通常的方法是改性PEI。改性后的PEI能提高PEI和DNA复合物的稳定性。通常聚乙二醇、环糊精和壳聚糖等高分子生物降解材料运用到PEI的结构修饰中,形成一种新的结构。比如共聚物和聚轮烷等,并通过链接配体,利用某配体与癌细胞表面高表达某受体的专属性特异结合的特点来提高靶向性,提高治疗的作用。研究目的：本研究选用三种不同分子量的PEI(0.6 KDa,10 KDa和25 KDa,均为支链)接枝到以叶酸为封端剂而制备成的多聚轮烷(PR)上,形成具有叶酸受体靶向的聚乙烯亚胺—聚轮烷超分子衍生物(FPP),探讨不同分子量PEI接枝后FPP性能和毒性的变化规律,并且研究FPP的DNA压缩性、缓冲性、抗核酸酶降解性以及转染性。从体内外评价FPP的传递性能,为临床提供安全高效的给药系统。研究方法和内容：分三大部分第一部分以叶酸为封端剂的聚轮烷-聚乙酰亚胺超分子衍生物的合成、表征和性能测试1、聚合物的合成：a、准聚轮烷(pseudo-PR)的制备；b、叶酸活性酯(FA-NHS)的制备；c、多聚轮烷(PR)的制备；d、FPP的制备。2、聚合物表征：先后使用NMR、IR和UV等对聚合物进行表征；通过1H-NMR中FPP不同组分的积分高比,求出a-环糊精(a-CD)上PEI的接枝率,FPP的平均分子量(Mw)。3、采用酸碱滴定法结合公式β=dC(HCl)/dpH计算聚合物的缓冲容量。4、琼脂糖凝胶电泳法考察不同分子量FPP, BPP和PEI-25 KDa与DNA的结合能力,及其对DNA在核酸酶I作用下的保护作用,扫描电镜观察复合物的形态。5、复合物的毒性研究：a、不同浓度的FPPs (0.6 KDa、10 KDa、25 KDa)、BPP、PEI-25 KDa载体与DNA形成的复合物对Hela细胞和16 HBE细胞的影响；b、FPPs (0.6 KDa, 10 KDa、25 KDa)、BPP (BOC-Tyr-Osu为封端剂的聚轮烷-聚乙烯亚胺)、PEI-25 KDa载体与pLuc在不同N/P比的情况下形成的复合物对Hela细胞和16 HBE细胞的影响。第二部分FPP物理、生物性能的研究1、采用碱裂解法,通过制备大肠杆菌感受态细胞、pAAV-EGFP和pLuc分别转入和重组增殖、碱裂解法纯化质粒等工序,提取质粒DNA；经琼脂糖凝胶鉴定大小及纯度。2、采用激光粒度仪测定不同分子量FPP, BPP和PEI-25 KDa分别与DNA所形成的复合物的粒径、Zeta电位。第三部分体内外复合物生物性能的研究1、体外转染率的研究：a:Luciferace评估FPPs (0.6 KDa、10 KDa、25 KDa)、BPP、PEI-25KDa载体与pLuc在不同N/P比的情况下形成的复合物对KB细胞的转染影响；b:Luciferece评估FPP(0.6 KDa)、BPP、PEI-25 KDa与pLuc(N/P=20)形成的复合物在无血清、有1%胎牛血清(FBS)和0.1%蛋白质的条件下对KB细胞的转染影响；c：流式细胞仪评估FPP(0.6 KDa)、BPP、PEI-25 KDa与pAAV-EGFP (N/P=20)形成的复合物对KB细胞和A549细胞的转染影响；d：流式细胞仪评估FPPs (0.6 KDa、10 KDa、25 KDa)、BPP、PEI-25 KDa载体与pAAV-EGFP(N/P=20)形成的复合物分别对HEK 293T、KB、LoVo、A549细胞转染影响。2、体内转染率的研究：a、动物模型的制备；b、通过瘤内注射复合物的方式,分别在24小时和48小时,luciferace检测PBS控制组,裸DNA (pLuc)组,PEI-25 KDa/pLuc,BPP/pLuc, FPP(0.6KDa)/pLuc, Lipotanmine/pLuc组的转染率。c、尾静脉注射,通过Caspase-9和Bax蛋白表达及瘤重大小的比较来检测聚合物／p53复合物的抗瘤作用。d、通过裸DNA (p53), PEI/DNA (P53), BPP/DNA(p53)和FPP(0.6KDa)/DNA(p53)组小鼠肝脏、脾脏的HE染色病理片的比较,观察载体在体内毒性的大小。结果：第一部分以叶酸为封端剂的聚轮烷-聚乙酰亚胺超分子衍生物的合成、表征1、NMR测定：a、pseudo-PR固体核磁显示：pseudo-PR中a-CD上的C1、C4的单峰消失,取而代之的是宽峰的出现。b、PR’H-NMR的显示：在6.6ppm、7.5ppm和8.5ppm处出现FA质子峰；同时,在3-4ppm之间出现a-CD上C2、C4、C3、C5、C6相连的宽峰；3.6ppm的PEG上的CH2峰,结果表明FA封端成功。c、FPP’H-NMR的显示：在6.7ppm、7.7ppm和8.7ppm处出现FA质子峰；同时,在3-4ppm之间出现a-CD上C2、C4、C3、C5、C6相连的宽峰；3.6ppm的PEG上的CH2峰,另外,在2-3ppm之间出现PEI中-(CH2-CH2-NH)-峰,表明FPP制备成功。2、IR的测定：PR在1650 cm-1显示有酰胺键形成,1605cm-1处显示有苯环结构,701.4 cm-1(苯环上的C-H),苯环特征的出现,意味着叶酸的链接成功。FPP：由于PEI氨基与a-CD中06H反应形成酯键,因此在1705 cm-1,1031.42 cm-1,有一个较强的吸收。3、UV的测定：与FA相比,FPP上FA的紫外吸收由原来的250nm、280nm红移到255nm和289nm。接枝率的测定：FPP上0.6KDa,10KDa和25 KDa的接枝率分别为86.67%,36.66%和15.33%。FPP(0.6K), FPP(10K), FPP(25K)平均分子量(Mn):85920 Da,453520Da,473520DaFPPs上各组分比值PEG:a-CD:PEI(0.6K,10K,25K)=1:20:(102,43.9,18.40)。2、不同分子量的FPP,缓冲容量随着分子量的增加而增大。3、凝胶阻滞实验显示,FPP(0.6 KDa,10 KDa和25 KDa)在N/P比2-4之间,有较好的阻滞作用,其结果稍差与PEI-25 KDa.4、聚合物对Hela、16 HBE细胞的相对生长率影响的研究结果发现：与PEI-25K相比,FPP (0.6 KDa,10 KDa和25 KDa)毒性显著性降低,FPP (0.6 KDa.10 KDa和25 KDa)、BPP的IC50 (half maximal (50%) inhibitory concentration (IC) of a substance)值分别为：(Hela:189ug/ml,178ug/ml,160ug/ml,196ug/ml; 16 HBE:193ug/ml,182ug/ml, 171ug/ml,192ug/ml。第二部分FPP物理、生物性能的研究1、采用碱裂解法制备及纯化后的pAAV-EGFP和pLuc纯度和大小合适,A260/A280 (pAAV-EGFP)=1.90±0.055; A260/A280 (PLuc=1.84±0.021;质粒分子量为5.3kb pAAV-EGFP和4.7kb的pLuc; pAAV-EGFP和pLuc质粒的浓度计算分别为278.83±1.11ug/ml和269.86±1.03ug/ml。2、当N/P比到达10时,不同分子量的FPP与DNA结合成复合物的粒径小于140nm；当N/P比升高到20时,zeta电位在20-30 Mv之间。第三部分体内外复合物生物性能的研究1、体外转染效率：首先确定N/P。当N/P=20时,FPP (0.6 KDa,10 KDa和25 KDa)的转染效率大于BPP和PEI-25K的幅度比它们在其他N/P比时的增长幅度更大。而后考察了其他转染条件。最后得出复合物最佳的转染条件：转染效率每孔加入DNA2ug;与DNA形成复合物的时间为30min；复合物在细胞上作用的时间为4h；在有血清的条件下,转染效率有显著的升高(*p<0.05(?)(?)*p<0.05),FPP(0.6K)/DNA转染效率分别是无血清的8.5倍和10倍；和Opti-MEM (blank)相比,10%-15%FBS和ALB(0.1-0.2%)转染效率分别扩大了2.5-3.5倍；在不同浓度FBS和ALD中的转染率无明显差异。2、体内转染效率：a、成功构建小鼠肿瘤模型。b、Liuciferase评估结果显示在24h FPP(0.6K) (N/P=20)的转染率与PEI-25 KDa (N/P=10) and BPP组相比分别高出6.1和5.6倍(*p<0.05 and*p<0.05),并且几乎是136倍的裸DNA组的荧光量。c、体内抗瘤活性的评估：(a)、肿瘤重量：与BPP/p53、PEI-25 KDa/p53. naked p53、PBS groups相比,FPP(0.6K)/p53组瘤重明显变小(*p<0.05,*p<0.05, **p<0.05,**p<0.05, respectively); (b)、caspase-9和bax表达：和正常对照组、PEI-25 KDa/p53 (N/P=10), BPP/p53组和FPP(0.6K) (N/P=20)/p53组相比,KB模型组中的caspase-9 and bax表达显著降低(*p<0.05,*p<0.05, *p<0.05, and*p<0.05),其中FPP/p53组显著性低于BPP/p53组(*p<0.05,*p<0.05)。d、HE染色：FPP(0.6K), FPP(10K)、FPP(25K)和裸DNA四组之间的小鼠肝脏和脾脏未见有什么变化,表明FPP(0.6K), FPP(10K)、FPP(25K)载体毒性较小。结论：1、成功构建新的非病毒载体(聚乙酰亚胺-聚轮烷,FPP)超分子聚合物；通过一系列的工艺研究得出了最佳的摩尔投料比,并且此工艺可控。2、合成的FPPs聚合物具有以下物理学特点：较好的缓冲容量；较理想的粒径、电位；较好的压缩DAN性能。3、合成的FPPs聚合物具有以下生理学特点：在体内外实验中,和PEI-25K相比,FPPs具有较低的毒性和较高的转染率。4、FPPs/DNA复合物在含有丰富叶酸受体的癌细胞上有较高的转染率。总之,FPPs系列载体具有高效的转染性、较低的毒性和叶酸受体癌细胞靶向性。更多还原

【Abstract】 Cancer is one of the disease that greatly threatened human’s health, people who pursuit in medicine is endeavoring to find a specific medicine to cure cancer. Gene therapy in the many ways becomes a 21st century hotspot. Gene therapy is an approach for human disease treatment by inserting therapeutic gene into cells in order to produce desired protein with specific functions and inhibits expression of abnormal genes. The clinical success of gene therapy depends on the development of suitable efficient and targeted gene transfer system. Gene delivery systems are classified into viral and non-viral system. Non-viral vector win us high favor for its several advantages including low immunogenicity and power of carrying high molecular weight of DNA.Poly(ethyleneimine) (PEI) who had emerged as a good candidate in the cationic Polymers is cationic polymer as non-viral gene delivery vectors used widely in research. It is repored that PEI is linear or branch polymer. There is high positive charge on its surface, so PEI can condense DNA with negative charge. PEI makes the lysosome rupture and releases the DNA swallowed into cytoplasm and nucleus for gene expression through famous protons sponge effect. The molecular weight of PEI varies from 5000-25000Da for gene delivery vectors. Higher is transgene efficieney of high molecular weight of PEI, but higher is also toxicity, or vice versa:the toxicity of low molecular weight of PEI is very low, for the transgene efficiency is very low. PEI can make erythrocyte aggregate and bind non-specificly with albumin in blood. Transgene efficieney was low when it was applied in vivo.Therefore, PEI need to be modified in order to reduce its cyctotoxicity. Usually, polyethylene glycol (PEG), cyclodextrin (CD), and chitosan (CHI) et al. in the Polymer biodegradable material were applied into modified PEI, respectively and together, such as copolymers and polyrotaxanes, what’ s more, targeted groups is grafted to the modified PEI. Then, the carrying efficiency from non-virus vectors will be enhanced.Objective:To investigate the transgene efficiency, toxicity and properties of different molecular weight of PEI-polyroaxane (FPP), a series of FPP polymers which included PEI(0.6 KDa,10 KDa,and 25 KDa, respectively) and folate terminatd polyroaxane (RP) were synthesized according to different molar ratio between different molecular weight of PEI and PR for clinic applicational delivery system.Methods and contents:three parts.One part:Synthesis and characterization of PEI-PR polymers1, Preparation of Polymers:a, Preparation of Psuedo-PR; b, Preparation of folate-NHS; c, Preparation of PR; d, Preparation of FPP.2, Characterization of polymers:The FPP composition was estimated by measuring nuclear magnetic resonance (NMR), infrared spectrum (IR), ultraviolet spectrum (UV).3, Buffer capacity was calculated by acid-base titration andβ=dC(HCl)/dpH.4, The binding ability of condense DNA and against RNase I degrate in FPPs were investigated by agarose gel electrophoresis. Morphology of FPPs/DNA complexed was observed under scanning electron microscope.5, Study of Complex toxicity:a, Influence of different concertration of FPP (0.6 KDa,10 KDa,25 KDa)/DNA、BPP/DNA and PEI-25 KDa/DNA complexes on morphology of cultured Hela and 16 HBE cells; b, Influence of different N/P ratios of FPP (0.6 KDa,10 KDa,25 KDa) /DNA, BPP/DNA, and PEI-25 KDa/DNA complexes on morphology of cultured Hela and 16 HBE cells.Two part:Preparation and biophysical properties of FPP/DNA complexes1, Using alkaline lysis method, through different kind of processes include preparation of Escherichia coli cells, re-proliferation of pAAV-EGFP or pLuc which first insert to coli cells, and extraction of plasmid pEGFP which purity and size was identified by agarose gel.2, The Zeta potential and particle size of FPPs/DNA and PEI/DNA complexes were measured by Laser Particle Size Determination.Three part:In vivo and vitro study of biological properties in complexes1, Study of transgene efficience in vitro:a, Influence of N/P ratios forming complexes on transfection efficiency of FPP(0.6K)/pLuc, FPP(10K)/pLuc, FPP(25K)/pLuc, BPP/pLuc, and PEI-25K/pLuc for KB cells by Luciferase; b, Influence of serum or without serm on transfection efficiency of FPP(0.6K)/pAAV-EGFP for KB cells; c, Influence of KB cells and A549 cells on transfection efficiency of FPP(0.6K/pAAV-EGFP by FCM; d, Influence of 293T、KB、Lovo and A549 on transfection efficiency of all complexes by FCM.2, Study of transgene efficience in vivo:a, Preparation of animal model; b, Luciferase assay:PBS, Naked DNA, PEI-25K/pLuc, BPP/pLuc, FPP(0.6KDa)/pLuc, and Lipotanmine/pLuc groups were randomly divided on bearing-tumour mice. They are killed at 24h and 48h after intratumoral injection of the complexes for luciferase assay of tumor tissue homogenates. c, The effect of anti-tumour in polymers/p53 complexes treatment were determined by tumour weight assay and gene expression of caspase-9 and bax. d, HE of heart and kidney of mice in FPP(0.6KDa), FPP(10KDa),FPP(25KDa) and Naked DNA groups after treatment 25d.ResultsOne part:Synthesis and characterization of PEI-PR (FPP) polymers1, characterization of polymers:(1), NMR:a,13C-CP/MAS NMR spectra of pseudo-PR:the spectrum shows resolved C-1 and C-4 resonances particular C-1 and C-4 adjacent to a conformationally strained glycosidic linkage are observed at 80 ppm and 98 ppm, respectively, b,1H-NMR spectra of PR:Signals corresponding to PEG were observed at 3.7 ppm ([-CH2CH2O-]n), and the C1 proton and C2-C6 protons of a-CD were identified at 4.9 ppm and 3.0-4.2 ppm, respectively. FA signals in polymers ranged from 6.5 to 8.7 ppm. c,1H-NMR spectra of FPP: Except above signals, signals from PEI ethylene protons (-CH2CH2NH-) in FPP were observed at 2.4-3.0 ppm. (2), IR:Amide bonds form in 1650 cm’and Structure of benzene in 1605cm-1 and 701.4 cm-1 indicated PR were successfully synthesised. Strong absorption in 1705 cm-1,1031.42 cm-1 for ester formation between amino of PEI and O6H of a-CD reaction indicated FPP successfully were synthesised. (3), UV: Compared FA, UV absorption in FPP shifted from 250nm and 280nm to 255nm and 289nm, respectively.Percent grating PEI on FPP(0.6 K), FPP(10 K) and FPP(25 K) were respectively 86.67%,36.66%, and 15.33%; Mw on FPP(0.6 K), FPP(10 K) and FPP(25 K) were respectively 85920 Da,453520 Da, and 473520 Da.PEG:a-CD:PEI(0.6K,10K,25K)=1:20:(102,43.9,18.40).2, Buffer capacity in different molecular weight of PEI enhanced with the increases of molecular weight, but buffer capacity in FPP decrease with the increase of molecular weight.3, Gel retardation results showed that FPP (0.6 KDa,10 and 25 KDa KDa) between N/P ratio 2 and 4 has well combined with DNA, and its result was a bit of poorer than that in PEI-25 KDa.4, Relative cell viability of Polymers on Hela and 16 HBE cells:Compared with PEI-25KDa, toxicity of FPP(0.6 KDa,10 KDa and 25 KDa) significantly was reduced. LD50 was (Hela,1285ug/ml,1256 ug/ml,1150 ug/ml,1315 ug/ml; 16 HBE,1295ug/ml, 1266 ug/ml,1210 ug/ml,1365 ug/ml).Two part:Preparation and biophysical properties of FPPs/DNA complexes1, Purity and size of pAAV-EGFP or pLuc prepared by alkaline lysis and extracted by reagent was fit the experiment request. The results were A260/A280 (pAAV-EGFP) =1.90±0.055; A260/A280 (pLuc)=1.84±0.021, plasmid molecular weight of pAAV-EGFP= 5.3kb and pLuc=4.7kb; concentration of pAAV-EGFP=278.83±1.11μg/ml and pLuc=269.86±1.03 ug/ml, respectively.2, When N/P ratio reached 10, particle sizes of different molecular weights FPPs/DNA complex were under 140nm; When N/P ratio was up to 20, zeta potential varied from 20mv to 30 mv.Three part:In vivo and vitro study of biological properties in complexes1, Study of transgene efficience in vitro:Extent of transgene efficiency of FPP (0.6 KDa, 10 and 25 KDa KDa) was the higher than those in BPP and PEI-25K When N/P=20, and extent of FPP (0.6 KDa, 10 and 25 KDa KDa) in N/P=20 was the higher than those in other N/P ratio. The best carrying conditions for complex was respectively 2ug/well DNA; the forming complexes for 30min; complex on the cells for 4h; a significant increase transgene efficiency in serum conditions (*p<0.05 and *p<0.05); transgene efficiency of FPP (0.6K)/DNA in serum was respectively the 8.5 times and 10 times that in serum-free; compared with Opti-MEM (blank), transgene efficiency of FBS (10-15%) and ALB (0.1-0.2%) were expanded by 2.5-3.5 times; no obvious difference transgene efficiency amonge different concentrations of ALD and FBS.2, Study of transgene efficience in vivo:a, Successful bearing-tumors mice model, b, Liuciferase assay showed that transgene efficiency of FPP (0.6 k) (N/P=20) was respectively 6.1 and 5.3 times those of PEI-25K (N/P=10) and BPP in 24h (*p <0.05 and*p<0.05), and was almost 136 times the amount of naked DNA. c, An antineoplastic active evaluation in vivo:(a), compared with BPP/p53, PEI-25 KDa/p53, naked p53, PBS groups, tumor heavy of FPP (0.6 K)/p53 group significantly decreased (*p<0.05,*p<0.05,**p<0.05, and**p<0.05, respectively); (b), Compared with normal control group, PEI-25 KDa (N/P=10)/p53 group, BPP/p53 group, and FPP(o.6K)(N/P=20)/p53 group, expression of caspase-9 and bax in KB model group significantly reduced (*p<0.05,*p<0.05,*p<0.05, and*p<0.05), and a significant difference between FPP/p53 and BPP/p53, it is no difference for HE of heart and kidney of mice among naked DNA, FPP (0.6K), FPP (10k), and FPP (25K) groups, indicating of low cyctotoxicity of FPP(o.6K), FPP(10K), and FPP(25K).Conclusion:1, Successful building a new non-virus carrier (PEI-polyroaxane, FPP) supramolecular polymer2, Good cushioning capacity; Ideal size and potential; Better performance of DAN compression3, In vitro and in vivo, compared with PEI -25K, FPPs have low toxicity and high transgene efficience.4, FPPs/DNA complex have high transgene efficience on the cancer cells with rich folate receptors.更多还原