CN103656679B - A method of preparing nanoparticles - Google Patents

A method of preparing nanoparticles Download PDF

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CN103656679B
CN103656679B CN 201310613693 CN201310613693A CN103656679B CN 103656679 B CN103656679 B CN 103656679B CN 201310613693 CN201310613693 CN 201310613693 CN 201310613693 A CN201310613693 A CN 201310613693A CN 103656679 B CN103656679 B CN 103656679B
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block
solution
dissolved
polymer
nucleic acid
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CN103656679A (en )
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金拓
葛雪梅
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上海交通大学
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Abstract

本发明提供一种纳米颗粒的制备方法,包括以下步骤:1)将聚阳离子高分子溶于超纯水或无RNase酶水配制成聚阳离子溶液,将核酸药物溶解于超纯水或者无RNase酶水配制成核酸溶液;2)将所述聚阳离子溶液加入到所述核酸溶液中,反复吹打均匀,室温下孵育,得到polyplexes;3)将嵌段高分子溶解于超纯水或无RNase酶水中配制成嵌段高分子溶液,将所述嵌段高分子溶液缓慢加入至所述步骤2)制备的polyplexes颗粒中,吹打均匀,静置,使其充分包裹,即可制得由嵌段高分子包裹的纳米颗粒。 The present invention provides a method of preparing nanoparticles, comprising the steps of: 1) a polycationic polymer is dissolved in the ultra pure water or water-free RNase enzyme formulated polycation solution, the nucleic acid drug is dissolved in ultrapure water or RNase-free enzyme formulated as aqueous nucleic acid solution; 2) the polycationic nucleic acid solution was added to the solution, uniformly repeated pipetting and incubated at room temperature to obtain polyplexes; 3) the block polymer is dissolved in ultrapure water or enzyme RNase-free water formulated as a solution polymer block, the block polymer solution was slowly added to the step 2) polyplexes prepared granules, uniformly pipetting, allowed to stand to fully wrapped, the block polymer can be prepared by the encapsulated nanoparticles. 与现有技术相比,本发明制备的纳米颗粒可以排除阳离子颗粒在体内循环的障碍,提高体内循环效率,同时,还可接枝靶向基团,实现体内病变细胞靶向,并有效提高靶向效果。 Compared with the prior art, nanoparticles prepared according to the invention can remove obstacles cationic particles circulating in the body, the body to improve cycle efficiency, while targeting group can also be a graft, to achieve targeting diseased cells in vivo, and to improve the target to the effect.

Description

一种纳米颗粒的制备方法 A method of preparing nanoparticles

[0001] 本申请是申请号为201210163285.1、发明创造名称为“一种嵌段高分子及其合成方法和纳米颗粒的制备方法”、申请日为2012年5月23日的中国发明专利申请的分案申请。 [0001] The present application is a continuation application No. 201210163285.1, entitled created as "a block polymer and its method of synthesis and preparation of nanoparticles", filed Chinese patent application points of 23 May 2012 case application.

技术领域 FIELD

[0002] 本发明涉及生物技术领域,特别涉及一种嵌段高分子及其合成方法和纳米颗粒的制备方法。 [0002] The present invention relates to the field of biotechnology, in particular, relates to a method for preparing a block polymer and its synthesis method of nanoparticles.

背景技术 Background technique

[0003] siRNA是一段21 — 25个碱基对的RNA分子,发现于单细胞生物抵御病毒侵袭的机制。 [0003] siRNA was a 21--25 base pairs of RNA molecules, found in single-celled organisms mechanism against virus attack. 单细胞生物针对入侵病毒的mRNA序列合成出一段与之互补的siRNA,主动结合mRNA,从而阻断病毒的复制。 Unicellular organism against the invasion of viral mRNA synthesis sequence complementary thereto a section of siRNA, mRNA active binding, thus blocking replication of the virus. 这种与病原体基因--对应的干扰策略如果用来开发治疗人类疾病的药物,将从根本上改变目前传统的新药发现模式,带来药物治疗技术的革命。 This pathogen gene - if the corresponding interference strategies used to develop drugs to treat human diseases, change the current traditional patterns of drug discovery will fundamentally revolutionize drug therapy technology. siRNA因其独特的靶点特异性、结构可设计性和代谢安全性,成为科学界普遍看好的下一代革命性新药的第一候选。 because of its unique target-specific siRNA, the structure can be designed and metabolic safety, became the first new drug candidate for the next generation of revolutionary scientific community generally optimistic. 然而,至目前为止,一个高效的体内输送载体的缺乏,却导致siRNA 的成药性受到了限制(Castanotto, D.&Rossi, JJThe promises and pitfallsof RNA-1nterference-based therapeutics.Nature 457,426-433(2009).)D 而目前用于核酸物质输送的载体多集中在以下几类:(1)物理导入:物理导入法是最先应用的基因导入方法,即采用电穿孔或粒子轰击技术等,将目的基因直接输送至体内或靶位的方法。 However, to date, efficient in vivo delivery vehicles a lack of siRNA has led medicine is limited (Castanotto, D. & Rossi, JJThe promises and pitfallsof RNA-1nterference-based therapeutics.Nature 457,426-433 (2009). ) D vectors currently used for the delivery of nucleic acid material more concentrated in the following categories: (1) physical introduced: the gene introduction method is a physical method of introducing the first application, i.e., by electroporation or particle bombardment techniques, etc., will direct the gene or delivery to a target site in vivo. 这些方法无需使用基因载体,但是转染效率普遍很低、操作复杂,对组织的损伤也比较大。 These methods do not need to use gene vectors, but generally low transfection efficiency, complex operation, tissue damage is relatively large. (2)病毒载体:目前对于病毒载体研究较多的是慢病毒载体、腺病毒载体,病毒载体虽然有较高的体外转染活性,然而,其免疫原性与易导致突变的缺点为体内输送带来了巨大的安全隐患。 (2) viral vectors: viral vectors for the current research is more lentiviral vector, adenoviral vector, although viral vectors have higher transfection activity in vitro, however, it is easy to cause disadvantages immunogenicity in vivo delivery mutation It brought great security risk. (3)非病毒载体:非病毒载体的优势主要在于,在保证预期的转染活性的条件下,可以大大降低病毒载体所带来的免疫原性与诸多炎症反应,其一般为以下几种载体设计:(a)阳离子脂质体;(b)聚阳离子基因载体。 (3) non-viral vectors: The main advantage is that non-viral vectors, to ensure the desired conditions of transfection activity, viral vectors can greatly reduce the immunogenicity caused by many inflammatory response, which is typically several carrier design: (a) a cationic liposome; (b) a polycationic gene carrier. 而目前研究更多的主要集中于聚阳离子基因载体与阳离子脂质体的修饰,使之适用于基因物质的靶向输送。 At present research is focused on more polycationic gene carrier modified with cationic liposomes makes it suitable for targeted delivery of genetic material. 阳离子脂质体具有较高的体内外转染活性,然而,由于表面的正电荷影响其体内的正常分布,同时,由于选用阳离子脂质,免疫原性与炎症反应在动物试验中也成为不可避免的缺点之一(Gao, K.&Huang, L.Nonviral methods for siRNA delivery.Molecular pharmaceutics6,651-658(2008).)。 Cationic liposomes have higher transfection activity in vivo, however, since the positive charges affect the normal distribution of the surface of its body, at the same time, since the choice of a cationic lipid, immunogenic and inflammatory responses in animal experiments has become unavoidable one drawback (Gao, K. & Huang, L.Nonviral methods for siRNA delivery.Molecular pharmaceutics6,651-658 (2008).). 聚阳离子基因载体目前发展已经较为成熟,在诸多文献中已有详尽的报道。 Polycationic gene carrier current development has been more mature, in many documents have been detailed report. 此外,在基因输送载体中,较为成功的实例CALAND0 Pharmaceuticals公司采用的RONDEL™技术,以与阳离子基因载体连接的环糊精、十二金刚烷为载体材料,以转铁蛋白为靶向基团对基因物质进行包裹递送,以系统给药治疗实体瘤,目前正在临床I期试验中。 Further, the gene delivery vehicle, the more successful examples CALAND0 Pharmaceuticals Company RONDEL ™ technology used to connect to the cationic cyclodextrin gene vector, twelve adamantane as a carrier material to transferrin to target a group of genetic material carried parcel delivery to system administration for the treatment of solid tumors, currently in phase I clinical trials. 然而,在结构设计中难以保证靶向基团在结构的表面,而环糊精可以减低毒性,但是此结构增多会降低转染活性,存在一个毒性与转染活性的自身设计矛盾,同时,其连接难以在体内实现无毒化降解(Davis, Μ.E.The first targeted delivery of siRNA in humansvia a self-assembling, cyclodextrin polymer-based nanoparticle:from concept toclinic.Molecular pharmaceutics6, 659-668(2009).)。 However, in the structural design is difficult to ensure that the surface structure of the targeting group, and the cyclodextrin can reduce toxicity, but this configuration can reduce an increase in transfection activity, there is a toxicity of transfection activity contradictory design itself, while its connection is difficult to achieve non-toxic in vivo degradation (Davis, Μ.E.The first targeted delivery of siRNA in humansvia a self-assembling, cyclodextrin polymer-based nanoparticle: from concept toclinic.Molecular pharmaceutics6, 659-668 (2009).).

[0004]用于治疗的核酸药物载体须以尽可能简单的结构完成以下五个步骤:A)核酸的凝聚、B)核酸对病变细胞的靶向、C)核酸的内吞逃逸、D)核酸在病变细胞浆的释放以及E)载体自身的无毒化代谢。 [0004] The pharmaceutical carrier for the nucleic acids shall be treated as a simple structure to complete the following five steps: cohesion A) a nucleic acid, escape the endocytic B) a nucleic acid targeting of diseased cells, C) a nucleic acid, D) nucleic acid in the cytoplasm of diseased release, and E) of the carrier itself non-toxic metabolites. 现有技术中,运用人体内源性单体和安全性已知的药物代谢物构建的pH响应性可降解聚阳离子以及其简单的结构高效实现了上述步骤中的A、C、D、E。 In the prior art, the use of pH-responsive monomers and endogenous human safety of the known drug metabolites constructed biodegradable polycation and its simple and efficient structure achieves the above steps A, C, D, E. 但是面对病变细胞的多样性(步骤B),其通用性却大为折扣。 But the face of diversity (Step B) diseased cells, but its versatility greatly discount. Polyplex颗粒表面膜的自组装是一项尚未妥善解决的难题。 Polyplex particle surface film is a self-assembly has not been properly solved. 中性磷脂没有吸附于Polyplex表面的化学驱动力。 No neutral phospholipid adsorbed on the surface of the chemical driving force Polyplex. Huang等人1990年代中叶报道的单价负电荷磷脂构建的Lipopolyplex (LPD 一II)表面膜的物理稳定性欠佳。 Price Huang et al reported in the mid-1990s negatively charged phospholipids built Lipopolyplex (LPD a II) poor physical stability of the surface film. 其最近报道的两价负电荷磷脂构建的Lipopolyplex虽然大幅改善了表面膜物理稳定性,外表面过多的负电荷可能影响纳米颗粒对于病变细胞的附着。 Divalent negatively charged phospholipid constructed Lipopolyplex recently reported that although a surface film greatly improved physical stability, excessive negative charge may influence the outer surface of the nanoparticles are attached to the diseased cells. 同样,很多研究提出聚阳离子载体与PEG共价连接可使得聚阳离子基因纳米颗粒表面正电荷有效屏蔽,然而,共价连接PEG后,对基因的复合能力却明显受到影响。 Similarly, many studies suggest polycation carrier and PEG may be covalently attached to the particle surface such that Jiyinnami effective shielding polycation positive charge, however, covalent connection PEG, the composite has the ability to significantly affected gene.

发明内容 SUMMARY

[0005] 本发明的第一目的在于提供一种嵌段高分子,以解决现有技术中的Polyplex颗粒表面膜的外表面存在过多的正电荷从而影响Polyplex颗粒对于病变细胞的附着,且靶向效果差的技术性问题。 [0005] The first object of the present invention is to provide a block polymer, in order to solve the problems of excess positive charge Polyplex outer surface of the particle surface film which affects the prior art Polyplex particles adhering to the diseased cells, and the target the technical problem of poor results.

[0006] 本发明的第二目的在于提供一种嵌段高分子的合成方法。 [0006] The second object of the present invention is to provide a method for synthesizing a block polymer.

[0007] 本发明的第三目的在于提供一种纳米颗粒的制备方法。 [0007] The third object of the present invention to provide a method for preparing nanoparticles.

[0008] 本发明目的通过以下技术方案实现: [0008] The object of the present invention is achieved by the following technical scheme:

[0009] 一种嵌段高分子,包括依次连接的第一嵌段、第二嵌段和第三嵌段,所述第一嵌段和所述第三嵌段为亲水嵌段,所述第二嵌段为疏水嵌段。 [0009] A polymer block comprising a first block are sequentially connected, a second block and the third block, the first block and the third block is a hydrophilic block, the The second block is a hydrophobic block.

[0010] 优选地,所述第一嵌段可选自PEG或ΡΕ0。 [0010] Preferably, the first block selected from PEG or ΡΕ0.

[0011] 优选地,所述第二嵌段可选自聚乳酸(PLA)、聚乙交酯(PGA)、聚乙交酯-丙交酯共聚物(PLGA)或聚己内酯(PCL)的一种。 [0011] Preferably, the second block may be selected from polylactic acid (PLA), polyglycolide (PGA), polyglycolide - lactide copolymer (PLGA) or polycaprolactone (PCL) a.

[0012] 优选地,所述第三嵌段选自能提供负电荷的分子或通过化学反应可与能提供负电荷的分子共价连接的化合物。 [0012] Preferably, the third block is selected to provide negatively charged molecules may be attached to the compound or to provide negatively charged molecules through covalent chemical reaction.

[0013] 优选地,所述通过化学反应可与能提供负电荷的分子共价连接的化合物包括多羟基分子,所述多羟基分子可选自甘油、乙二醇、果糖、葡萄糖、乳糖、麦芽糖、蔗糖或木糖醇的一种。 [0013] Preferably, the compound by a chemical reaction can be connected to provide negatively charged molecules covalently include polyhydroxy molecule, a polyhydroxy molecule selected from glycerol, ethylene glycol, fructose, glucose, lactose, maltose a sucrose or xylitol.

[0014] 优选地,所述能提供负电荷的分子包括多羧基化合物,所述多羧基化合物可选自苹果酸或柠檬酸。 [0014] Preferably, the negatively charged molecules can provide include polycarboxy compound, the polycarboxy compound selected from malic acid or citric acid.

[0015] 优选地,还包括靶向基团或荧光分子,所述靶向基团或所述荧光分子与所述第一嵌段连接。 [0015] Preferably, further comprising a targeting group or a fluorescent molecule, a targeting group or the fluorescent molecule is connected to the first block.

[0016] 优选地,所述靶向基团可选自蛋白、多肽、抗体或小分子靶向基团的一种或几种。 [0016] Preferably, the targeting group may be selected from one or more proteins, polypeptides, antibodies or small molecule targeting group.

[0017] 优选地,所述蛋白可选自转铁蛋白或去唾液酸糖蛋白;所述多肽可选自RGD或胰岛素;所述小分子靶向基团可选自叶酸、生物素或半乳糖的一种。 [0017] Preferably, the protein is ferritin or optional rotation asialoglycoprotein; or the RGD polypeptide selected from insulin; the small molecule targeting group selected from folic acid, biotin or galactose a.

[0018] 优选地,所述荧光分子可选自罗丹明、FITC、NBD、cy5.5或FAM的一种。 [0018] Preferably, the fluorescent molecules may be selected from one rhodamine, FITC, NBD, cy5.5 or of FAM.

[0019] 一种嵌段高分子的合成方法,包括以下步骤: [0019] A method of synthesizing a block polymer comprising the steps of:

[0020] 1)以PEG为引发剂,在Sn(oct)^催化下,在80〜140°C条件下于无水甲苯中引发开环聚合,加入己内酯,反应进行6〜24h,合成PEG-PCL嵌段; [0020] 1) PEG as an initiator in the ring-opening polymerization of Sn (oct) catalyzed initiator in dry toluene ^ conditions at 80~140 ° C, caprolactone was added, the reaction proceeds 6~24h, synthesis PEG-PCL block;

[0021] 2)以草酰氯为连接剂,先将所述步骤1)中合成的PEG-PCL嵌段溶于无水二氯甲烷中,再将PEG-PCL嵌段溶液缓慢逐滴加至草酰氯中,滴加温度为冰浴,滴加完成后恢复至室温,2〜12h后抽除溶剂及过量的草酰氯,获得中间产物:羟基端经过酰氯活化的PEG-PCL,而后将中间产物溶解于无水二氯甲烷,再将中间产物溶液逐滴加入由DMF溶解的大量麦芽三糖中,滴加温度为冰浴,滴加完成后恢复至室温,2〜12h后减压抽除溶剂,用截留分子量为1000〜10000的透析袋透析除去麦芽三糖,透析时间为12〜48h,预冻,冻干得到PEG-PCL-Maltotr1se 嵌段高分子。 [0021] 2) In oxalyl chloride as a linking agent, the first step 1) synthesized PEG-PCL block was dissolved in anhydrous dichloromethane and then PEG-PCL block was slowly added dropwise to a solution of oxalyl chloride was added dropwise to ice bath temperature, was returned to room temperature after completion of the dropwise addition, the pumping 2~12h solvent and excess oxalyl chloride was removed, to obtain an intermediate product: hydroxyl end chloride through activated PEG-PCL, and the intermediate product was dissolved after in dry methylene chloride, and then the intermediate product solution was added dropwise to a large amount of DMF maltotriose dissolved in ice bath temperature was added dropwise, warmed to room temperature after the addition was complete, the solvent under reduced pressure and after pumping 2~12h, with molecular weight cut off dialysis tubing to remove 1000~10000 maltotriose dialysis, the dialysis time 12~48h, pre-freezing, freeze-dried to give PEG-PCL-Maltotr1se block polymer.

[0022] 一种纳米颗粒的制备方法,包括以下步骤: Preparation Method [0022] A nanoparticle, comprising the steps of:

[0023] 1)将聚阳离子高分子溶于超纯水或无RNase酶水配制成聚阳离子溶液,将核酸药物溶解于超纯水或者无RNase酶水配制成核酸溶液; [0023] 1) a polycationic polymer is dissolved in the ultra pure water or water-free RNase enzyme formulated polycation solution, the nucleic acid drug is dissolved in ultrapure water or RNase-free enzyme formulated nucleic acid solution;

[0024] 2)将所述聚阳离子溶液加入到所述核酸溶液中,反复吹打均匀,室温下孵育,得到polyplexes ; [0024] 2) the polycationic nucleic acid solution was added to the solution, uniformly repeated pipetting and incubated at room temperature to obtain polyplexes;

[0025] 3)将上述的嵌段高分子溶解于超纯水或无RNase酶水中配制成嵌段高分子溶液,将所述嵌段高分子溶液缓慢加入至所述步骤2)制备的polyplexes颗粒中,吹打均匀,静置,使其充分包裹,即可制得由嵌段高分子包裹的纳米颗粒。 [0025] 3) The above-described block polymer is dissolved in a solution formulated as a block polymer or ultrapure water-free RNase enzyme, the block polymer solution was slowly added to the step 2) Preparation of granules of polyplexes the pipetting evenly, standing to fully wrapped package can be prepared by a block polymer nanoparticles.

[0026] 优选地,所述核酸药物为DNA或RNA。 [0026] Preferably, the nucleic acid agent is DNA or RNA.

[0027] 与现有技术相比,本发明的嵌段高分子可以有效的屏蔽聚阳离子基因复合物颗粒等阳离子颗粒表面的电荷,排除阳离子颗粒在体内循环的障碍,提高体内循环效率,同时,还可接枝靶向基团,实现体内病变细胞靶向,并有效提高靶向效果。 [0027] Compared with the prior art, the block polymer of the present invention can be effectively shielding the surface charge of the particles composite particles of cationic gene polycations, cationic exclude particles circulating in the body barriers, improving cycle efficiency in vivo, at the same time, graft targeting group can also achieve targeting diseased cells in vivo, and to improve the targeting effect.

附图说明 BRIEF DESCRIPTION

[0028] 图1为本发明的嵌段高分子结构及合成方法示意图; Block polymer structure [0028] Figure 1 is a schematic view of the invention and the method of synthesis;

[0029] 图2为本发明的嵌段高分子的核磁谱图; [0029] NMR spectrum of the block polymer of the present invention, FIG 2;

[0030] 图3为本发明的嵌段高分子的核磁谱图; [0030] FIG. 3 is a block polymer of the present invention, NMR spectrum;

[0031] 图4为本发明的纳米颗粒的制备示意图; [0031] Preparation of FIG. 4 schematic nanoparticles of the present invention;

[0032] 图5为本发明的纳米颗粒的制备示意图; [0032] FIG. 5 schematic nanoparticle preparation of the present invention;

[0033] 图6为本发明的纳米颗粒的荧光共定位法结构验证的示意图; Schematic structure verification method of the fluorescent nanoparticles [0033] FIG. 6 of the present invention, co-localization;

[0034]图7为本发明的纳米颗粒粒径与Zeta电位变化图(其中ABC指未经羧化的嵌段高分子,ABCH指末端经过羧化的嵌段高分子); Nanoparticles Particle Size [0034] FIG. 7 of the present invention and FIG Zeta potential change (which means non-carboxylated ABC block polymer, ABCH means through carboxylated polymer end block);

[0035] 图8为本发明的嵌段高分子的细胞毒性检测示意图; [0035] Cell toxicity assay schematic block polymer of the present invention, FIG 8;

[0036] 图9为本发明的纳米颗粒的体内毒性与循环结果示意图; Results Toxicity in vivo circulating nanoparticles [0036] FIG. 9 is a schematic view of the present disclosure;

[0037] 图10为本发明的嵌段高分子的肿瘤靶向性效果示意图。 Tumor targeting effect of block polymer [0037] FIG. 10 is a schematic view of the present disclosure.

具体实施方式 detailed description

[0038] 以下结合实施例详细说明本发明。 [0038] Example embodiments of the present invention in conjunction with the following detailed description. 实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程。 In embodiments of the present invention is a technical premise, given the specific operation and detailed embodiments. 但所举实施例并非用于限定本发明的保护范围。 It cited but examples are not intended to limit the present invention.

[0039] 本发明对嵌段高分子与polyplexes所形成的纳米颗粒的理化性质表征方法包括:透射电子显微镜、动态光散射和zeta点位测试。 [0039] The present invention physicochemical properties of the nanoparticles and a polymer block formed polyplexes characterized by comprising: transmission electron microscopy, and dynamic light scattering, zeta test point. 按本方案制备的纳米颗粒的细胞摄取、基因转染和小动物活体成像观察选用的DNA为绿色荧光蛋白质粒;相关的毒性试验选用的细胞是HepG2细胞、Hela细胞、BRL-3A细胞与SMMC-7721细胞;体内分布选用的动物是BALB/c 裸鼠。 Cellular uptake of the nanoparticles prepared according to the present embodiment, gene transfection and small animal in vivo imaging observed selected DNA as a green fluorescent protein; related toxicity were selected cell is a HepG2 cell, of Hela cells, BRL-3A cells SMMC- 7721; in vivo distribution of selected animals BALB / c nude mice.

[0040] 实施例1嵌段高分子PEG-PCL-maltotr1se-COOH的合成方法 [0040] Example 1 Synthesis of Block Copolymer PEG-PCL-maltotr1se-COOH is

[0041] 嵌段高分子PEG-PCL-maltotr1se-COOH的合成路线如图1所示。 [0041] Block Polymer Scheme PEG-PCL-maltotr1se-COOH is shown in FIG. 整个反应在无水无氧的环境中进行,取一定量的PEG、聚己内酯、辛酸亚锡至三颈瓶中,加入现制的无水甲苯,于120°C下搅拌反应24小时,反应完成后加入乙醚沉淀,而后加入二氯甲烷溶解,再用乙醚沉淀,反复三次,得到PEG-PCL嵌段高分子。 The entire reaction in anhydrous oxygen-free environment, a certain amount of PEG, polycaprolactone, stannous octoate three-necked flask, freshly made anhydrous toluene was stirred at 120 ° C for 24 hours, after completion of the reaction the precipitate was added diethyl ether, and then dissolved in dichloromethane, precipitated with ether, repeated three times, to obtain PEG-PCL block polymer.

[0042] 而后将PEG-PCL溶解于二氯甲烷中,加入过量草酰氯于反应瓶中,在无水无氧冰浴的条件下将PEG-PCL溶液逐滴加入到反应瓶中,滴加完成后恢复至室温,搅拌,12h后减压抽除剩余的草酰氯,而后加入二氯甲烷溶解末端经过酰氯活化的PEG-PCL,冰浴下将末端经过酰氯活化的PEG-PCL溶液逐滴缓慢的加入过量的麦芽三糖中(溶于少量DMF),反应在无水无氧的条件下进行,滴加完成后恢复到室温,12h后减压抽除溶剂,于截留分子量7000的透析袋中,透析24小时除去未反应的麦芽三糖。 [0042] After the PEG-PCL was dissolved in dichloromethane, excess oxalyl chloride was added to the reaction flask in an ice bath under anhydrous oxygen-free conditions PEG-PCL was added dropwise to the reaction flask dropwise addition was completed after returned to room temperature, stirred for 12h under reduced pressure pumping remaining oxalyl chloride, followed by addition of the acid chloride dissolved in dichloromethane after the end of the activated PEG-PCL, the ice bath after the end of the activated PEG-PCL acid chloride was added dropwise slowly An excess of the maltotriose (dissolved in a small amount of DMF), the reaction carried out under anhydrous oxygen-free conditions, returned to room temperature after the addition was complete, the solvent under reduced pressure pumping after 12h, in 7000 molecular weight cutoff dialysis bag, malt dialyzed for 24 hours to remove unreacted trisaccharide. 预冻,而后于冻干机中冻干得到白色粉末。 Pre-frozen, then lyophilized in a freeze dryer to give a white powder.

[0043] 将制得的白色粉末溶解于无水二氯甲烷中,加入到过量的草酰氯中,在冰浴中缓慢滴加,滴加完成后恢复至室温,搅拌反应,结束后减压除去过量草酰氯,而后加水水解,于截留分子量为3500的截留尚心管中尚心除去少量小分子片段,冻干得到终产品。 [0043] The resulting white powder was dissolved in dry dichloromethane and added to excess oxalyl chloride was slowly added dropwise in an ice bath, cooled to room temperature after completion of dropwise addition, stirring of the reaction, was removed under reduced pressure after excess oxalyl chloride, followed by hydrolysis with water, in 3500 molecular weight cutoff cutoff heart is still a small amount is still small molecule fragments core tube was removed and lyophilized to give the final product.

[0044] 其1-NMR图谱如图2、3所示:在1-NMR图谱(DMS0_d6,400MHz):其峰归属见图2、图3,其中,化学位移在4-6区间为麦芽三糖羟基氢的峰,而糖环上的骨架氢的峰在化学位移3-4之间,被高分子的峰所掩盖,所以以麦芽三糖的羟基峰作为合成结果的判定。 [0044] 1-NMR spectrum which shown in Figure 2,3: In 1-NMR spectrum (DMS0_d6,400MHz): peak attributed Figure 2, Figure 3, wherein, in the chemical shift range of 4-6 maltotriose the hydroxyl hydrogen peaks, and the peak of hydrogen on the backbone between the chemical shifts of the sugar rings 3-4, the polymer was masked peaks, the peak hydroxy maltotriose as the synthesis result of the determination. 经过羧化的嵌段高分子麦芽三糖的羟基峰部分消失或减弱,指示部分羟基被羧基取代。 After carboxylated block polymer having hydroxyl groups of the peak portion maltotriose or disappearance, indicating section hydroxy substituted carboxyl group.

[0045] 实施例2纳米颗粒的制备 Example 2 Preparation of Nanoparticles [0045] Embodiment

[0046] 取一定量的聚阳离子高分子(以PEI为例)与质粒DNA,由于考察纳米颗粒结构与电荷屏蔽情况,故选用质量比1:5 (pDNA:PEI)制备成polyplexes样品,而后加入嵌段高分子,充分复合。 [0046] A certain amount of a polycationic polymer (in Example PEI) plasmid with the DNA, nanoparticles investigated since the charge shield case structure, so the selection of mass ratio of 1: Preparation of: (PEI of pDNA) into polyplexes sample, and then 5 block polymer, a fully complex. 具体步骤见图4、5。 Figure 4, specific steps.

[0047] 实施例3纳米颗粒的表征 Example 3 Characterization of Nanoparticles [0047] Embodiment

[0048] 按照上述的制备方法制备的纳米颗粒,通荧光共定位对其进行表征,具体做法如下,将PEI与FITC通过共价键连接,并同时用nile red标记嵌段高分子的疏水PCL嵌段,将制备的荧光颗粒固定在PVA水凝胶中,并通过反复“冷冻-室温”循环进行交联固化,限制颗粒在水平面上的二维运动。 [0048] The nanoparticles prepared according to the above production method, the through colocalization were characterized, Specifically, the following, to connect the PEI by a covalent bond with FITC, while fitted with nile red marker block polymer hydrophobic PCL segment, the phosphor particles are fixed to the prepared PVA hydrogel, and by repeating - for crosslinking and curing "refrigeration temperature" cycle, two-dimensional particles restrict the movement in the horizontal plane. 观察结果见图6,红色(nile red)和绿色(FITC)在相同位置出现并重合,验证了纳米颗粒的形成。 Figure 6 observations, the red (nile red), and green (FITC) and appear in the same position coincides verified formation of nanoparticles.

[0049] 实施例4纳米颗粒的粒径与表面电位的表征 Characterization of the surface potential of the particle size [0049] Example 4 nanoparticles

[0050] 通过粒径与电位的测定对纳米颗粒结果进行表征,电位的变化,经过包裹后电位在OmV左右,同时,未经过羧化的高分子电位没有明显降低,由此可以直观证明本发明的嵌段高分子材料可以屏蔽电荷,并且粒径分布比较均匀,结果见图7。 [0050] The particle size measured by the potential of the nanoparticle characterization result, change in the potential, after wrapping around OMV, while not been carboxylated polymer without significantly reducing the potential of the potential, thereby visually demonstrate the present invention block polymer material can shield the charge, and the particle size distribution uniform, the results shown in Figure 7.

[0051] 实施例5嵌段高分子的细胞毒性的考察 [0051] Cytotoxicity inspection block polymer of Example 5

[0052] 采用MTT法测定细胞毒性,选用Η印G2、HeLa、BRL-3A、SMMC-7721细胞考察细胞毒性,以8000个/孔的细胞密度转96孔细胞板,置于37 °C 5%细胞培养箱里培养过夜。 [0052] The cytotoxicity using MTT assay, selection Η printed G2, HeLa, BRL-3A, SMMC-7721 investigated cytotoxic cells, at a cell density of 8,000 / well in 96-well cell plate is transferred, is placed 37 ° C 5% cell culture incubated overnight box. 配制l、2、3、4、6、8mg/mL的系列不同浓度的嵌段高分子溶液,每孔加入100 μ L,稀释介质是DMEM高糖培养基(无血清无酚红),从培养箱中取出96孔细胞板,吸去培养液,每孔用100 yL磷酸盐缓冲溶液冲洗一次,再弃去磷酸盐缓冲溶液,将不同浓度的嵌段高分子溶液依次加入到细胞板中,平行测定6个孔。 Formulated l, 2,3,4,6,8mg / mL series of block polymer solution of different concentrations, each well was added 100 μ L, was diluted with medium high glucose DMEM medium (serum without phenol red), from the culture box 96-well cell plate removed, the culture medium removed by aspiration, each well was washed once with 100 yL phosphate buffer solution, phosphate buffer solution and then discarded, different concentrations of block polymer solution were successively added to the cell plate, the parallel Determination of six holes. 置于细胞培养箱里培养4小时。 Cells placed in culture box cultured for 4 hours. 然后,吸去培养液,每孔用100 μ L磷酸盐缓冲溶液冲洗一次,再弃去磷酸盐缓冲溶液,每孔加入100 μ L DMEM高糖培养基(无血清无酚红)和25 μ L MTT溶液(5mg/mL),继续于培养箱里培养。 Then, the culture fluid was aspirated, 100 μ L per well was washed once phosphate buffer solution, phosphate buffer solution again was discarded, each well was added 100 μ L DMEM high glucose medium (serum without phenol red) and 25 μ L MTT solution (5mg / mL), continuing the culture in the culture box. 6小时之后,吸去培养液,每孔加入100 μ L 二甲基亚砜,放置充分溶甲赞,采用多功能酶标仪测定样品在570nm和630nm处的吸光度值(以630nm处为对照)。 After 6 hours, the culture medium was aspirated, each well was added 100 μ L of dimethylsulfoxide, placed fully dissolved formazan, using multifunctional microplate reader at 570nm and a measurement sample absorbance at 630nm (at 630nm as control in) . 经过测试可知,嵌段高分子材料毒性较低,在微克级基本无毒,结果见图8。 After testing shows that the block polymer materials less toxic, substantially non-toxic in the microgram, results shown in Figure 8.

[0053] 实施例6体内循环考察 [0053] Example 6 In vivo Investigation cycle

[0054] 按照前述方法制备的纳米颗粒,分别将polypi exes与经过包裹后的纳米颗粒经小鼠尾静脉注射,单剂量给予lmg/kg体重的pDNA质粒的复合物,其复合比例与方法同前所述,将嵌段高分子用焚光染料rhodamine共价连接标记,经过尾静脉注射后,polyplexes组的小鼠均发生急性死亡,而对于注射经过所设计的嵌段高分子包裹后的纳米颗粒的小鼠生命体征平稳,经过24小时候,颈椎脱白处死小鼠,分别取心、肝、脾、肺、肾进行冰冻切片观察,以注射等体积生理盐水组为空白对照,经过荧光显微镜观察,可以看到在肝、脾、肺处均出现了部分颗粒的聚集,结果见图9,证明经过包裹后,polyplexes表面电荷得到了有效的中和,实现了体内循环。 Before [0054] The nanoparticles prepared according to the aforementioned method, respectively polypi exes with encapsulated nanoparticles after tail vein injection via the mice were administered a single dose of lmg / kg body weight plasmid pDNA complexes, which complexes with the ratio method the, the connector block polymer labeled with a rhodamine dye covalently light burning, after tail vein injection, mice polyplexes group of acute death occurred, and for wrapping the polymer through the injection block designed nanoparticles mice stable vital signs, 24 through a child, cervical off white mice were sacrificed, are heart, liver, spleen, lung, kidney, frozen sections were observed to volume of saline for the blank control group, via fluorescence microscopy, can be seen in the liver, spleen, lung, appeared at both the part of the particle aggregation, results shown in Figure 9, prove that after wrapping, polyplexes surface charge and has been effectively achieved in vivo circulation.

[0055] 实施例7嵌段高分子的肿瘤靶向考察 Tumor [0055] embodiment of the block polymer in Example 7 Investigation targeting

[0056] 选择生物素为靶向基团与嵌段高分子进行共价连接,考察其肿瘤靶向性,将5周龄的BALB/c裸鼠在SPF级动物房饲养一周,而后以SMMC-7721细胞进行皮下肿瘤接种,待肿瘤长至200mm3后,单剂量注射pDNA的基因复合物,剂量lmg/kg体重,制备方法与比例同前,分别制备无靶向基团连接的嵌段高分子包裹的纳米颗粒与以生物素为靶向基团的高分子包裹的基因颗粒,并以rhodamine共价连接的荧光嵌段高分子进行荧光体内示踪,以同体积的生理盐水组为空白对照,进行肿瘤靶向性考察。 [0056] Select biotin covalently linked to a targeting group and a block polymer, investigate its tumor targeting, the 5-week-old BALB / c nude mice for one week in SPF grade animal room, then to SMMC- 7721 subcutaneously tumor inoculation, the tumor grew to 200mm3, a single dose injection of pDNA gene complex, the dose lmg / kg body weight, with the ratio of the former production method, a block polymer were prepared without targeting group linked parcel fluorescent nanoparticles with biotin tracing vivo gene targeting polymer particles wrapped group, and covalently attached to a fluorescent rhodamine block polymer, with the same volume of saline as the control group, for Investigation tumor targeting. 经小鼠尾静脉注射给药,分别于给药后4h、12h、24h观察小鼠肿瘤部分荧光量的蓄积,考察所制备的颗粒在肿瘤组织的靶向效果,经过靶向基团连接后的基因颗粒在肿瘤部位的蓄积量明显高于未经连接组,具体结果见图10。 Injection via the tail vein of mice, respectively, after administration of 4h, 12h, 24h mice were observed for tumor accumulation portion of the amount of fluorescence, the particle inspection targeting effect produced tumor tissue, after connecting the targeting group gene particle accumulation amount is significantly higher than the tumor site without connecting group, particularly the results shown in Figure 10.

[0057] 以上公开的仅为本申请的几个具体实施例,但本申请并非局限于此,任何本领域的技术人员能思之的变化,都应落在本申请的保护范围内。 Several [0057] The above descriptions are merely specific embodiments of the present application, but the present application is not limited thereto, anyone skilled in the art can think of variations shall fall within the scope of protection of the present application.

Claims (2)

  1. 1.一种纳米颗粒的制备方法,其特征在于,包括以下步骤: 1)将聚阳离子高分子溶于超纯水或无RNase酶水配制成聚阳离子溶液,将核酸药物溶解于超纯水或者无RNase酶水配制成核酸溶液; 2)将所述聚阳离子溶液加入到所述核酸溶液中,反复吹打均匀,室温下孵育,得到polyplexes ; 3)将嵌段高分子溶解于超纯水或无RNase酶水中配制成嵌段高分子溶液,将所述嵌段高分子溶液缓慢加入至所述步骤2)制备的polyplexes颗粒中,吹打均匀,静置,使其充分包裹,即可制得由嵌段高分子包裹的纳米颗粒; 其中,所述嵌段高分子包括依次连接的第一嵌段、第二嵌段和第三嵌段,所述第一嵌段和所述第三嵌段为亲水嵌段,所述第二嵌段为疏水嵌段; 所述嵌段高分子的合成方法包括以下步骤: 1)以PEG为引发剂,在Sn(oct)^催化下,在80〜140°C条件下于无水甲苯中引发开环聚合, 1. A method for preparing nanoparticles, characterized by comprising the steps of: 1) a polycationic polymer is dissolved in the ultrapure water or RNase-free enzyme solution was formulated as polycationic, nucleic acid or drug is dissolved in ultrapure water RNase-free water formulated as a nucleic acid enzyme solution; 2) the polycationic nucleic acid solution was added to the solution, uniformly repeated pipetting and incubated at room temperature to obtain polyplexes; 3) the block polymer is dissolved in ultrapure water or no formulating an enzyme RNase block polymer solution into water, the block polymer solution was slowly added to the step 2) polyplexes prepared granules, uniformly pipetting, allowed to stand sufficiently wrapped, can be prepared from the insert segment polymer encapsulated nanoparticles; wherein the block polymer comprises a first block are sequentially connected, a second block and the third block, the first block and the third block is a pro water block, the second block is a hydrophobic block; synthesis of the block polymer comprising the steps of: 1) PEG as initiator, at Sn (oct) ^ catalyst, at 80~140 ° under conditions of C initiator in the ring-opening polymerization of anhydrous toluene, 入己内酯,反应进行6〜24h,合成PEG-PCL嵌段; 2)以草酰氯为连接剂,先将所述步骤1)中合成的PEG-PCL嵌段溶于无水二氯甲烷中,再将PEG-PCL嵌段溶液缓慢逐滴加至草酰氯中,滴加的温度条件为冰浴,滴加完成后恢复至室温,2〜12h后抽除溶剂及过量的草酰氯,获得中间产物:羟基端经过酰氯活化的PEG-PCL,而后将中间产物溶解于无水二氯甲烷,再将中间产物溶液逐滴加入由DMF溶解的大量麦芽三糖中,滴加的温度条件为冰浴,滴加完成后恢复至室温,2〜12h后减压抽除溶剂,用截留分子量为1000〜10000的透析袋透析除去麦芽三糖,透析时间为12〜48h,预冻,冻干得到PEG-PCL-Maltotr1se嵌段高分子。 Caprolactone into the reaction proceeds 6~24h, synthesis of PEG-PCL block; 2) oxalyl chloride as a linking agent, the first step 1) synthesized PEG-PCL block was dissolved in anhydrous dichloromethane , then PEG-PCL block was slowly added dropwise to a solution of oxalyl chloride was added dropwise in an ice bath and the temperature condition was returned to room temperature after completion of the dropwise addition, the pumping 2~12h excess of oxalyl chloride and the solvent removed, to obtain an intermediate Yield: hydroxyl end chloride through activated PEG-PCL, after the intermediate product was dissolved in anhydrous dichloromethane and then the intermediate product solution was added dropwise a DMF maltotriose dissolved in a large amount, added dropwise to ice bath temperature condition recovery after completion of the dropwise addition to room temperature, the solvent under reduced pressure and after pumping 2~12h, the dialysis bag is removed 1000~10000 maltotriose molecular weight cutoff dialysis, the dialysis time 12~48h, pre-frozen and lyophilized to give PEG- PCL-Maltotr1se block polymer.
  2. 2.如权利要求1所述的纳米颗粒的制备方法,其特征在于,所述核酸药物为DNA或RNAo 2. The method for preparing nanoparticles according to claim 1, wherein said nucleic acid is DNA or medicament RNAo
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