【作者】 姚远；

【导师】 颜德岳；

【作者基本信息】 上海交通大学 ， 高分子化学与物理， 2010， 博士

【摘要】 利用NCA开环聚合法得到聚氨基酸型多肽的研究已经有数十年之久的历史，研究的领域也从早期的嵌段共聚物和相分离结构转向各类应用，比如作为微纳米功能性载体、药物载体、DNA载体、响应性凝胶等等。但是，这些体系大部分仅限于线型的多肽嵌段共聚物。本文在前人工作的基础上，合成了不同内核和支臂的超支化多肽嵌段共聚物，并研究了基于多肽构建微/纳米结构的方法，研究的领域涉及超支化多肽嵌段共聚物的合成与表征、以多肽为模板的生物矿化、在无机材料表面接枝多肽以及多肽稳定的金纳米粒子的制备和组装行为。具体研究内容和主要结论概括如下：（1）具有超支化内核的含多肽嵌段共聚物的合成和生物相容性研究。利用超支化PEI作为引发剂，使ZLys‐NCA，BLG‐NCA、Phe‐NCA和BAsp‐NCA开环聚合，得到四类具有亲水性的PEI内核，不同臂长的疏水臂的超支化多肽嵌段共聚物。通过脱除多肽侧链的保护基团，又进一步得到三类双亲水型的超支化多肽嵌段共聚物。通过1H NMR谱和FT‐IR光谱，对这些超支化多肽嵌段共聚物的结构进行了表征，并利用1H NMR谱计算了分子量、EI/多肽段百分含量等参数。利用商品化的超支化聚酯Boltorn H₂0/H40和Boc保护的苯丙氨酸，通过DCC缩合和保护基团脱除反应，得到了含部分端氨基的超支化聚酯H₂0‐NH₂和H40‐NH₂。使用它们作为大分子引发剂，令ZLys‐NCA和BAsp‐NCA开环聚合，并进一步脱除保护基团，分别得到三类疏水内核，亲水臂的超支化多肽嵌段共聚物。通过1H NMR谱和FT‐IR光谱，对这些超支化多肽嵌段共聚物的结构进行了表征，并分别利用GPC法和NMR法表征了它们的分子量、分子量分布和多肽臂长等参数。对超支化多肽嵌段共聚物的生物相容性进行了表征，结果显示聚合物的生物相容性与聚合物中可携带正电荷的基团相关，这类基团越多，生物相容性越差。通过引入含有羧基的适当长度的PGlu段和PAsp段，可以得到具备生物相容性的超支化嵌段聚合物。（2）聚赖氨酸为模板的碳酸钙生物矿化研究。以PLys为模板，通过气相扩散法制备了不同形貌的碳酸钙晶体，其中长有“腰带”的双球形碳酸钙晶体是首次报道。用SEM对长有“腰带”的双球形碳酸钙晶体形貌进行分析，结果表明，“腰带”的宽度大约为2³纳米，球体表面覆盖着具有分形结构的晶片，组成“腰带”的晶片也与之类似。LM‐Raman光谱分析结果表明，长有“腰带”的双球形碳酸钙晶体属于球文石。通过改变模板分子PLys浓度、初始pH值、钙离子浓度和反应时间等条件，还得到了双球形、扁球形、六边形和中空圆环状等一系列特殊形貌的碳酸钙晶体，它们也属于球文石。PLys控制碳酸钙矿化的过程中，分子链中存在的α螺旋起到重要的作用：在一定的pH值下，PLys吸附钙离子，并利用分子链中存在的α螺旋为模板，形成碳酸钙纳米晶。这些带有纳米晶的PLys分子链再组装成赤道平面对称的大晶核，并在不同的条件下生长成形貌各异的碳酸钙大晶体。当[Ca²⁺]和[PLys]较低时，晶核生长为无腰带的双球形晶体。[PLys]增加到一定值后，生成的是赤道区较宽的双球形晶体，随着体系pH值增加，原来的生长方向被抑制，但沿着赤道区垂直于球体方向和球体的切线（指向赤道面）方向继续诱导生长出柏树叶状，具有分形结构的晶片，最终形成长有“腰带”的双球形碳酸钙晶体。起始pH值较高，或者PLys浓度较大时，生成扁球状碳酸钙晶体，这是因为α螺旋的浓度也较高，对晶体沿极轴方向的生长有抑制作用。当钙离子浓度较高，矿化时间延长时，生成六边形碳酸钙晶体，钙离子浓度进一步提高则生成中空环状结构的碳酸钙。该研究结果表明，在自然界中，碳酸钙的矿化可能不仅仅依靠那些含有可变为负离子的残基（如天冬氨酸或谷氨酸）的多肽，多肽的二级结构在控制碳酸钙矿化中也起到非常重要的作用。（3）超支化多肽嵌段共聚物为模板的碳酸钙矿化研究。以不同聚谷氨酸段链长的PEI—PGlu分子为模板，通过气相扩散法获得了不同形貌的碳酸钙晶体。按照模板分子的差别进行了相应的研究。当以PGlu段较短的PEI‐PGlu1分子为模板时，改变模板分子和钙离子浓度，得到的是以梭状晶为主的碳酸钙晶体。随着钙离子浓度不同，晶体的形貌变化不大，随聚合物浓度不同，在聚合物含量较少时，可以得到晶面较为明显的晶体，形貌更接近拉伸菱方晶。用XRD和LM‐Raman光谱研究了晶体结构，这些晶体均为方解石。当以PGlu段长度适中的PEI‐PGlu2分子为模板时，钙离子的浓度对碳酸钙晶体的形貌可以产生很大影响。随着钙离子浓度的增加，晶体的形貌从拉伸菱方晶转化为兼具拉伸菱方晶和双球晶特征的过渡型晶体，再变为双球形晶体，最后变成球状或半球状晶体。聚合物的浓度对碳酸钙形貌的影响较小，在实验的范围内，聚合物浓度越高，越难以生成明显的大晶面。用XRD和LM‐Raman光谱研究了晶体结构，这些晶体均为方解石，伴生有一些球状的文石晶体。镁离子存在时，可以干扰方解石晶型的生成。使用不同混合溶剂也会对碳酸钙的形貌产生很大影响。这可能是溶剂对不同嵌段的溶解能力、溶剂化效应、混合溶剂对二氧化碳的溶解性以及混合溶剂对模板的形貌的影响造成的。PEI‐PGlu作为模板进行碳酸钙矿化时，PEI‐PGlu分子类似一个三维的“纳米反应器”。PEI段和PGlu段都可以对碳酸钙的形貌产生影响，以PGlu段的影响更大，它的侧链羧基可以和钙离子通过电荷复合和络合形成碳酸钙矿化的模板，而PEI的作用是吸附二氧化碳/碳酸根。PEI‐PGlu作为模板可能的机理如下：最初的诱发碳酸钙晶体生长的模板由数个被钙离子联系在一起的PEI‐PGlu分子组成，当分子为谷氨酸残基含量较少的PEI‐PGlu1时，由于Glu含量太低，无论钙离子浓度如何变化，也只能产生倾向于生成梭状的晶体的模板。当分子为谷氨酸残基含量适中的PEI‐PGlu2时，通过改变钙离子的浓度，可以改变PEI‐PGlu分子之间“交联”的程度，并依据“交联”程度的不同得到形貌不同的模板，进而生成形貌截然不同的晶体。当模板分子的PGlu比例进一步增大时，由于PGlu链段足够长，这时更倾向于把钙离子吸附在分子内，而不是在分子间共享钙离子，这种模板倾向于形成球状的碳酸钙。（4）用Graft‐From法在无机纳米材料表面修饰多肽。利用酸化和氨基化反应，得到氨基化的多壁碳纳米管MWNT‐NH₂，再用MWNT‐NH₂引发BLG‐NCA开环聚合，得到表面接枝多肽的多壁碳纳米管MWNT‐PBLG。运用透射电镜对MWNT‐PBLG进行表征，证明多肽已经接枝到多壁碳纳米管上，多肽层的厚度为4.50²2 nm。采用热重分析计算了多肽的接枝量并估算了接枝多肽的平均链长，随着投料比的不同，多肽的接枝量为3.74gPBLG/ g碳管到6.04g PBLG / g碳管不等，平均链长则在8.13¹3.2个BLG残基之间。红外光谱和XPS谱显示了与普通的PBLG相比，接枝在碳纳米管表面的PBLG的氢键作用力更弱，这可能是因为后者的一端固定在碳纳米管表面，难以形成分子间氢键造成的。溶解度测试则表明MWNT‐PBLG在强极性溶剂中分散得更好。利用硅烷偶联剂进行表面改性，得到氨基化的二氧化硅纳米微球SiO₂‐NH₂，再用它引发ZLys‐NCA开环聚合，得到表面接枝多肽的二氧化硅纳米微球SiO₂‐PZlys。运用透射电镜对SiO₂‐PZlys进行表征，证明多肽已经接枝到二氧化硅纳米微球上，多肽层的厚度为14.7²6.8 nm不等。采用热重分析计算了多肽的接枝量，随着投料比不同，多肽的接枝量为0.36g PZLys/g SiO₂⁰.23gPZLys/g SiO₂不等。两种无机纳米粒子表面接枝多肽的热重分析和透射电镜表征结果显示，接枝多肽的量并不能随着NCA:无机纳米粒子的投料比线性变化，这意味着接枝时发生的NCA开环聚合反应并非活性聚合。由于碳纳米管的比表面积比二氧化硅纳米微球大得多，而且碳纳米管还是中空的，因此单位质量的碳纳米管能够比二氧化硅纳米微球接枝更多的多肽。（5）多肽稳定的金纳米粒子的研究。使用巯基乙胺三氟乙酸盐引发ZLys‐NCA开环聚合，得到端巯基的多肽衍生物PZLys‐SH，利用不同长度的PZLys‐SH作为稳定剂，在DMF溶液中使用硼氢化钠还原法得到油溶性的金纳米粒子Au@PZLys‐SH，UV‐Vis光谱分析表明，在使用同样的PZLys‐SH时，金氯酸的量越多，所得的金纳米粒子尺寸越大，溶液的颜色越偏向蓝紫色。TEM分析表明所得的金纳米粒子尺寸为5¹0nm，拥有较为完美的晶格。以分子量较小的PZLys‐SH1为稳定剂得到的金纳米粒子还可能在水中组装为直径为数十纳米的囊泡。此外，Au@PZLys‐SH表面带有正电荷，可以与经强酸处理过，表面带有羧基的单壁碳纳米管复合。TEM照片证实这种复合物最常见的形态是多颗金纳米粒子富集于多根碳纳米管上，或是出现在碳纳米管的交汇处。使用聚赖氨酸直接在水相中还原金氯酸，得到聚赖氨酸稳定的金纳米粒子Au@PLys。这种金纳米粒子的直径为10nm左右。Au@PLys可以与侧链带有羧基的超支化多肽嵌段共聚物H40‐Phe‐PAsp复合生成胶束，胶束的直径随着Au@PLys的增加变大，增长到一定程度后，变为随着Au@PLys的增加而减小，这是胶束内增多的正电荷对胶束外壳的羧基吸引力增加的结果。同样，Au@PLys也可以与HsDNA形成复合胶束，随着Au@PLys的增多，胶束的粒径可由一百多纳米增加到七百多纳米。用TEM观察这两种胶束发现，胶束内均包含了一定量的金纳米粒子，胶束的尺度与动态光散射法测得的胶束粒径基本吻合。这两种复合胶束有望应用于生物医疗等方面的用途。更多还原

【Abstract】 Polypeptides prepared via N‐carboxyanhydride （NCA） ring‐opening polymerization have receivedintensive concern for decades. Up to date, these polypeptides and their block copolymers have beenreported to be applied as micro/nano functional carriers, bio‐compatible materials and responsivegels due to their unique properties. However, most of the reported works focused on linearpolypeptides and block polypeptide copolymers. In this dissertation, a series of hyperbranchedpolypeptide with different cores and arms were prepared, and some micro/nano structures based onpolypeptides were further constructed. The studies included the synthesis and characterization of thehyperbranched polypeptide copolymers （HPCs）, the mineralization of calcium carbonate directed bypolypeptides, polypeptides modification of multiwalled carbon nanotubes and silica nanoparticles bygraft‐from approach, and the gold nanoparticles stabilized by polypeptides and their chargecomplexes. The details and key conclusions are summarized as follows:（1） The preparation of hyperbranch cored, polypeptide armed block copolymers and the study oftheir biocompatibilities.In this chapter, hydrophilic hyperbranched polyethyleneimide （PEI） was used as initiator toprepare four kinds of HPCs with different hydrophobic polypeptide arms by ring‐openingpolymerization of N‐ε‐carbobenzoxy‐L‐Lysine NCA （ZLys‐NCA）,γ‐benzyl‐L‐glutamate NCA （BLG‐NCA）,β‐benzyl‐L‐aspartate NCA （BAsp‐NCA） and phenylalanine NCA （Phe‐NCA）. Three kinds of HPCs withdifferent hydrophilic polypeptide arms can also be obtained after deprotection. Their structures werecharacterized by FT‐IR and 1H NMR. The molecular weights and EI/peptide contents were calculatedwith the assistance of 1H NMR.Hydrophobic hyperbranched polyester Boltorn H₂0/40 were reacted with N‐butoxycarbonyl‐Lphenylalaninein the presence of dicyclohexylcarbodiimide, and then the butoxycarbonyl wasdeprotected to obtain the amino‐terminated hyperbranched polyester Boltorn H₂0‐NH₂ /H40‐NH₂.The two compounds initiated ZLys‐NCA and BAsp‐NCA ring‐opening polymerization, and then theprotective groups were removed to prepare three kinds of hydrophobic cored, hydrophilic armed HPCs.They were also characterized by FT‐IR and 1H NMR, and the molecular weights, peptide contents wereobtained by GPC and 1H NMR.The biocompatibility tests of some HPCs showed that the cytotoxicities were increased withpositive charge content. HPCs with good biocompatibility should contain proper amounts of Aspblock or Glu block, which carries carboxyl side groups.（2） The study of calcium carbonate mineralization directed by poly‐L‐Lysine.In this chapter, the novel calcium carbonate （CaCO₃） morphology, twin‐sphere with an equatorialgirdle, had been obtained under the control of poly（L‐lysine） （PLys） through gas‐diffusion method. TEM images showed the“girdle”width is 2³ nm, and the surface of the twin‐sphere are covered withfractal crystal pieces. The effect of the concentration of calcium ion and PLys, the reaction time, andthe initial pH value were investigated, and various interesting morphologies, including twin‐sphere,discus‐like, hexagonal plate and hallow structure were observed by using scanning electronicmicroscopy （SEM）. Laser microscopic Raman （LM‐Raman） spectroscopy studies indicated that all theseCaCO₃ are vaterite.This work found that PLys with side amine groups can control the mineralization of CaCO₃ just likethe acidic polypeptides. A reasonable process for forming the morphologies was discussedpreliminarily. At first, the anisotropic vaterite nanocrystals were formed under the direction of thePLys chains which contained partlyα‐helix, then the nanocrystals assembled to form a nuclear plate,and the growth from both sides of the nuclear plate resulted in the twin‐sphere. With theenvironmental pH value increasing to stronger alkaline, the conformation change of PLys chains leadto the CaCO₃ tangential oriented growth on the sphere surface and vertical oriented growth on theequatorial zone. Finally, the twin‐sphere with equatorial girdle morphology was generated. Itsuggested that alkaline polypeptide or protein may also play a suitable role in the biomineralizationprocess in nature.（3） The study of calcium carbonate mineralization directed by hyperbranched polypeptidecopolymers.In this chapter, various calcium carbonate morphologies had been obtained via gas‐diffusionmethod by the direction of PEI‐PGlu HPCs with different PGlu section length. When the template wasPEI‐PGlu1, which contained short PGlu section, the products were mainly fusiform calcite crystals.When the template was PEI‐PGlu2, which contained medium PGlu section, the morphologies ofcalcium carbonate were significantly affected by the concentration of Ca2+. At lower Ca2+concentration, the morphology of the calcium carbonate was elongated rhombohedral crystal. Withthe Ca2+concentration increasing, the morphology of the calcium carbonate turned to transitionalcrystal posses both the characters of elongated rhombohedral and twin‐sphere. If theCa2+concentration were further increased, the twin‐sphere calcium carbonate macrocrystal wasobtained. Finally, the calcium carbonate macrocrystal showed sphere morphology at highest Ca2+concentration. All these macrocrystals were determined calcite by XRD and LM‐Raman spectra. Themorphology of calcium carbonate could also be affected by Mg2+ or other mixed solvents.PEI‐PGlu served as“3D nanoreactor”in the mineralization of calcium carbonate. PEI section couldabsorb CO₂; PBLG section could conjugate with Ca2+, and“crosslink”with them to construct thetemplate. Both the two sections could affect the morphology of calcium carbonate. A possiblemechanism was described as following: the initial templates are some PEI‐PGlu molecules crosslinkedby Ca2+. The templates absorb CO₂ themselves via PEI section to generate the CaCO₃ nanocrystal. Themorphologies of the templates are determined by the length of PGlu section. PEI‐PGlu1, whichcontains short PGlu section, can only form the template to generate fusiform calcite crystals;PEI‐PGlu2, which contains medium PGlu section, can form various templates to construct CaCO₃morphologies from elongated rhombohedra crystal to sphere macrocrystal relying on the concentration of Ca²⁺. PEI‐PGlu3, which contains long PGlu section and tends to absorb Ca²⁺in onemolecular rather than share Ca²⁺ inter molecules, is a template for produce spherical macrocrystals.（4） Polypeptide modification of inorganic nanomaterials by“Graft From”approach.Covalent surface functionalization of carbon nanotubes with polypeptides is promising in possiblemedical applications. This work presented a graft‐from approach to perform the polypeptidemodification of multiwalled carbon nanotubes （MWNTs）. The raw MWNTs were amine‐functionalizedat first, then MWNT‐NH₂ was used as the initiator to initiate the ring‐opening polymerization ofBLG‐NCA, resulting in the polypeptide‐grafted MWNTs. FTIR, XPS and TGA data demonstrated that thefunctionalization was successful. The TEM images of the products showed that the thickness of thepolypeptide shell of the PBLG‐MWNT was about 4.5²2 nm. TGA analysis showed that thepolypeptide contents were 3.74⁶.04 g PBLG/g MWNTs. The average graft lengths are 8.13¹3.2 BLGsections. The polypeptide modified carbon naaotubes showed better solubility in polar solutions.According to the facile route developed here, the functionalized carbon nanotubes with other types ofpolypeptides can be fabricated easily using the corresponding NCAs.Silica nanospheres were modified by coupling agent to obtained SiO₂‐NH₂ at first, then theSiO₂‐NH₂ initiated ZLys‐NCA ring‐opening reaction, resulting in the polypeptide grafted silicananosphere SiO₂‐PZLys. TEM images showed that the thickness of the polypeptide shell is about14.7²6.8nm. Polypeptide contents were calculated 0.23⁰.36 g PZlys/g SiO₂ by TGA.The results of TGA and TEM showed that the grafted polypeptides can not increase linearly withthe NCA feed ratio. It investigated that the grafting reaction was not living polymerization. Besides,equal weight of MWNTs can graft more polypeptide than SiO₂ nanoparticles, because that the specificarea of the MWNTs is much larger than SiO₂ nanoparticles, and the MWNTs are hollow.（5） The study of gold nanoparticles stabilized by polypeptides.The mercaptoethylamine trifluoroacetate initiated ZLys‐NCA ring‐opening polymerization toobtain thiol terminated polypeptide PZLys‐SH. PZLys‐SHs with different molecular weights were usedto prepare gold nanoparticles （GNPs） Au@PZLys‐SH in DMF with HAuCl4 by the reduction of NaBH4.UV‐Vis spectra indicated that the size of GNPs grew up with the amount of HAuCl4. TEM imagesshowed the diameter of GNPs was 5¹0nm and the lattice of GNPs were perfect. GNPs stabilized byPZly‐SH1, Au@PZly‐SH1 may self‐assemble to small vesicles with tens of nanometers diameter inwater. Besides, Au@PZly‐SH can also combine with single walled carbon nanotubes （SWNTs） treatedby nitric acid because the Au@PZly‐SH possess positive charges, and the SWNTs possess carboxyl. TEMimages confirmed that the GNPs in the complexes are most absorbed on the SWNTs bundles, or thecross block of the SWNTs.HAuCl4 was reduced in situ by PLys in aqueous solution for preparing the GNPs stabilized by PLys,Au@PLys. The diameter of the Au@PLys is about 10 nm. Au@PLys can combine with hyperbranchedpolypeptide copolymer H40‐Phe‐PAsp, which contains carboxyl in arms, and form complex micelles.The diameters of the micelles are increasing with the adding of Au@PLys firstly, and then decreasewith the adding of Au@PLys. It contributes to the added positive charges carried by Au@PLys attractthe carboxyl in the micelle shell. Au@PLys can also bind with HsDNA to build complex micelles, the diameters of the micelles are increased from about 150nm to about 700nm with the adding ofAu@PLys. TEM images showed the two kinds of micelles contained several GNPs, and the diameters ofthe micelles are accorded with the results of dynamic light scatting. The two kinds of complex micellesshould be applied in biomedical in the future.更多还原