基于原位聚合技术构建细胞内微环境响应型DNA递送系统北大核心CSCD

传统的非病毒载体基于分子间静电自组装作用与核酸结合,组装的复合物在体内复杂的环境中容易发生结构解离,共价结合的交联聚合物载体有望成为解决传统非病毒载体结构稳定性差的有效方案。选择N-(3-氨丙基)甲基丙烯酰胺盐酸盐、1-乙烯基咪唑、2-甲基丙烯酰氧乙基磷酸胆碱与N,N′-双(丙稀酰)胱胺作为多功能性单体,采用原位聚合方法制备包载质粒DNA(pDNA)的交联聚合物-pDNA复合物。其中,共价键为载体提供优异的结构稳定性;1-乙烯基咪唑能够响应胞内溶酶体酸性微环境,触发质子海绵效应便于复合物的溶酶体逃逸;N,N′-双(丙稀酰)胱胺的二硫键可以响应胞内高水平的谷胱甘肽(GSH),实现复合物在细胞内部选择性解聚,释放内含pDNA。研究表明,该复合物平均水合半径约135 nm,ζ电势约−6.5 mV,形貌近似球形。该复合物可在10 mg/mL肝素环境中保持结构稳定性,具有响应细胞内GSH,触发释放包载核酸分子的功能。细胞实验证明该复合物细胞毒性低。细胞摄取、转染能力强。综上所述,基于原位聚合技术制备交联聚合物载体在基因递送领域具有重要应用前景,本研究为新型基因递送载体的开发提供了新思路。     

微流控可控分步组装制备透明质酸/聚乙烯亚胺/DNA纳米复合物

阳离子聚合物/DNA形成的复合物纳米颗粒呈正电性,因此表面必须遮盖一层电中性或负电性的聚合物才能在体内应用,但如何控制遮蔽层在复合物纳米颗粒上组装,形成结构可控、尺寸均一的基因输送系统是关键.本文以基因输送系统中常见的透明质酸(HA)/聚乙烯亚胺(PEI)/DNA系统为例,探索了利用微流控芯片进行可控分步的层层自组装,制备尺寸大小均一、表面电势为负的HA/PEI/DNA纳米复合物的方法.将PEI与DNA通过第一个微流控芯片自组装得到PEI/DNA纳米复合物,该复合物颗粒在第二个微流控芯片内与HA再次组装得到HA/PEI/DNA纳米复合物.考察了微流控芯片管道的尺寸、溶液流速及流速比R、PEI与DNA氮磷比(N:P)、HA与DNA质量比(HA:DNA)等参数与所形成的纳米复合物的尺寸、均一性及表面电势的关系,并与涡旋振荡法制备的复合物进行比较.结果表明,传统的涡旋振荡法制备的HA/PEI/DNA纳米复合物尺寸偏大(340～490 nm)、均一度低(PDI,0.506～0.863);而用微流控法制备的复合物尺寸较小(190 nm)、分布更为均一(PDI=0.316).     

羟乙基纤维素/AMPS接枝共聚物与大豆分离蛋白形成聚离子复合物研究

用硝酸铈铵/硝酸(CAN/HNO3)引发了羟乙基纤维素(HEC)与2-丙烯酰胺基-2-甲基丙磺酸(AMPS)的接枝共聚反应.研究了单体浓度、引发剂浓度和反应温度对接枝率的影响.由于强酸型基团磺酸基的引入,接枝共聚物带有了阴离子电荷,在小于大豆分离蛋白等电点(pH=4.5)的缓冲溶液(pH=1.5)中,通过接枝共聚物的阴离子电荷与大豆分离蛋白(SPI)阳离子电荷之间的静电相互作用,可以形成HEC-g-PAMPS/SPI聚离子复合物.以聚离子复合物负载模型药物布洛芬(Ibuprofen),在37℃于不同pH的缓冲溶液中进行了体外释放研究.药物释放速率的测定表明,以HEC-g-PAMPS/SPI聚离子复合物作为布洛芬的载体,可以在选择性条件下实现药物可控释放的目的.     

一氧化氮供体纳米复合物的合成与性质研究

一氧化氮供体纳米复合物由谷胱甘肽(GSH)包覆的Mn掺杂的Zn Se量子点和一氧化氮(NO)光敏供体复合而成.它通过双光子激发诱导RBS分解释放NO可以实现NO的可控释放.本方法顺利实现了静电自组装,合成的纳米复合材料作为智能药物载体具有很大的开发潜力.该量子点纳米复合材料对NO释放采用双光子激发的可控释放,释放的一氧化氮表现出显著的癌症细胞杀伤活性.     

ATRP法制备聚(甲基丙烯酸氨乙酯-co-甲基丙烯酸N,N-二乙基氨乙基酯)基因载体

为得到低毒、高效的聚阳离子基因载体,以甲基丙烯酸氨乙酯(AMA)和甲基丙烯酸N,N-二乙基氨乙基酯(DEAEMA)为单体,以2-溴代异丁酸乙酯(EBIB)为引发剂,通过原子转移自由基聚合(ATRP)制备了两种聚(甲基丙烯酸氨乙酯-co-甲基丙烯酸N,N-二乙基氨乙基酯)阳离子无规共聚物(P(AMA-co-DEAEMA),简称P).琼脂糖凝胶电泳实验结果表明聚合物P作为阳离子载体可以有效地络合DNA,通过粒径仪测定的复合物粒子的尺寸在400 ～ 600 nm之间.扫描电镜观察的P/DNA复合物形貌是分散均匀的球形颗粒.以25kDa PEI为阳性参照,利用MTT比色法考察了聚合物P对HEK293T细胞的毒性.结果表明,聚合物P的细胞毒性低于25 kDa PEI的细胞毒性.以25 kDa PEI和裸质粒DNA作为参照,我们进一步考察了聚合物P与DNA形成的复合物在HEK293T细胞中的转染效率.结果表明P/DNA复合物在HEK293T细胞中的转染效率远远高于裸质粒DNA的转染效率,并且接近于25 kDa PEI/DNA复合物的转染效率.     

阳离子基因载体的pH敏感遮蔽体系的制备及表征

合成了一种pH敏感的遮蔽体系-谷氨酸苄酯/谷氨酸共聚物(PBLG-co-PGA), 用于对DNA/阳离子基因载体复合物颗粒表面正电荷的遮蔽, 以提高其在体内的稳定性. 研究表明, PBLG-co-PGA (PGA(x), x为PGA占共聚物中摩尔百分数)具有pH敏感性. 并以pH敏感点接近生理pH值的PGA(60)为遮蔽体系进行研究. PGA(60)能够对DNA/PEI(1:1)复合物颗粒表面正电荷进行有效遮蔽. 凝胶阻滞电泳显示, 用PGA(60)对DNA/PEI复合物进行不同比例遮蔽, 没有发生与DNA的链交换作用. MTT细胞毒性测试表明, PGA(60)和三元复合物DNA/PEI/PGA(60) 在测试范围内几乎没有细胞毒性. 荧光素酶转染实验表明, 部分遮蔽后转染效率有所提高; 用PGA(60)对DNA/PEI复合物完全遮蔽为负电后, 由于同细胞表面的电荷排斥作用, 三元复合物不易被细胞内吞, 导致不发生细胞转染. 因其合适的pH响应性, PGA(60)将可能成为一种能随pH值的变化, 实现对聚阳离子基因载体进行电荷遮蔽/智能释放的遮蔽材料.     

生物可裂解树状大分子组装体与智能型基因传递系统

以精氨酸外围功能化、硫辛酸内核功能化的肽类树状大分子为组装基元,在亲疏水作用驱动下形成树状大分子组装体;并通过催化量二硫苏糖醇引发组装体内核的硫辛酸残基交联而获得纳米结构稳定、胞内氧化还原可裂解的智能型基因载体系统.双功能化肽类树状大分子具有精确可控的分子结构;在水溶液中组装、交联后,形成粒径约为72 nm、电位为+30.1 m V的树状大分子组装体.树状大分子组装体能够有效压缩和保护DNA,增强DNA对核酸酶的抵抗力,且具有氧化还原响应性、实现DNA的胞内定点可控释放.树状大分子组装体与DNA复合物(A/P=20)在有血清条件下对HEK 293、Hep G2和Hela 3种细胞系的转染效率均优于阳性对照组PEI(Mw=2.5×104,N/P=10,无血清)的转染效果.同时,该树状大分子组装体还具有良好的血清耐受性及生物相容性.     

1-甲基-3-(2-甲基丙烯酰乙基)-咪唑氯离子液体/ 苯乙烯无规共聚物及其CdTe量子点复合物的合成

以偶氮二异丁腈(AIBN)为引发剂, 通过自由基聚合制备了1-甲基-3-(2-甲基丙烯酰乙基)-咪唑氯离子液体和苯乙烯的无规共聚物P(MMEIM⁺Cl^--co-St). 用红外光谱、核磁共振氢谱和凝胶渗透色谱表征了共聚物的结构. 共聚物的氯仿溶液与巯基乙酸稳定的CdTe量子点水溶液混合, 通过静电相互作用复合组装, 得到澄清透明且稳定的CdTe/P(MMEIM⁺Cl^--co-St)纳米复合物的氯仿溶液. 复合物的紫外吸收光谱、荧光发射光谱和透射电子显微镜的表征结果表明, 量子点均匀分散于共聚物中, 无团聚, 且保持了量子点的荧光性能.     

响应性阳离子聚合物及聚合物纳米胶束基因载体

人类多种疾病都与基因的结构或功能改变密切相关,基因治疗已经成为改善人类健康的新兴医学治疗手段,基因治疗的关键在于构筑高效的基因载体.发展生物可降解、具有高转染效率和低毒性的阳离子聚合物基因载体已经成为当今该领域的主要任务.本文主要介绍了对细胞内外的环境差异和外界刺激具有响应性的阳离子聚合物基因载体的合成方法,实现DNA在细胞内的有效释放;构筑不同拓扑结构的聚合物,运用聚合物的拓扑结构来调节DNA与聚合物组装复合体形貌,实现DNA的可控压缩和提高载体的转染效率;通过两亲性阳离子聚合物的自组装形成稳定的阳离子纳米胶束,增加聚合物表面电荷密度,从而有效增强聚合物和DNA结合能力,实现低正负电荷比条件下的DNA压缩和基因的高效表达.     

适配子修饰靶向PLGA纳米基因载体的构建

化学合成了功能性三嵌段复合物乳酸乙醇酸共聚物-聚乙二醇-适配子(PLGA-PEG-Apt)。使用双乳化挥发法制备包裹DNA片段的PLGA-PEG-Apt新型纳米基因药物载体,表征检测显示:制备的纳米基因载体粒径为(225.2±8.1)nm,Zeta电位约(-35.5±-3.3)mV。扫描电子显微镜下纳米颗粒形态呈圆形,表面光滑,粒径分布较均匀。纳米粒子对TFO的包封率为(25.4±3.1)%(n=3),载药量为(1.34±0.16)μg/mg。体外释放实验研究结果显示持续释放过程达23 d,且PLGA-PEG-Apt纳米粒子呈突释之后的持续缓释过程。细胞水平实验结果显示,A10适配子修饰的纳米基因载体能更多进入靶向的前列腺癌细胞株,进而发挥其抗前列腺癌增殖的作用。该研究成功制备了靶向PLGA纳米基因载体,结果满意。     

α-Amino acid derived enaminones and their application in the synthesis of N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates and other heterocycles

A new and simple synthesis of novel N-protected methyl 5-substituted-4-hydroxypyrrole-3-carboxylates, which exist in equilibrium with their 4-oxo tautomers, has been developed in two steps starting from N-protected α-amino acids. The key intermediates are enaminones, which can also be isolated, characterized, and used for the construction of other functionalized heterocycles, before they spontaneously decompose to pyrrole products. 4-Hydroxypyrroles are prone to partial aerial oxidation but can be efficiently alkylated or reduced to stable polysubstituted pyrrolidine derivatives.     

Specific intramolecular aromatic CH insertion of diazosulfonamides

The chemoselectivity in the intramolecular CH insertion of various diazosulfonamides has been experimentally studied. The results reveal that the aliphatic 1,4-, 1,5-, or 1,6-C(sp³)?H insertions of diazosulfonamides are not accessible, while the aromatic 1,5-C(sp²)?H insertion can be realized specifically by adjusting the diazo-adjacent group. In addition, the general chemoselectivities in the intramolecular CH insertions of diazosulfonyl compounds are summarized. Generally, diazosulfones undergo both aromatic 1,5-C(sp²)?H and aliphatic 1,5- and 1,6-C(sp³)?H insertions, while diazosulfonates undergo aliphatic 1,5- and 1,6-C(sp³)?H insertions. However, diazosulfonamides only undergo aromatic 1,5-C(sp²)?H insertion.     

N-Heterocyclic carbene-palladacyclic complexes: synthesis,characterization and their applications in the C-N coupling and α-arylation of ketones using aryl chlorides

N-Heterocyclic carbene-palladacyclic complexes 3 were successfully achieved in a one-pot procedure under mild conditions. The structure of 3a was unambiguously confirmed by X-ray single crystal diffraction and it was an active catalyst in the Buchwald-Hartwig amination and α-arylation of ketones even at very low catalyst loadings (0.01?mol%).     

Iodine-mediated oxidative Pictet-Spengler reaction using terminal alkyne as the 2-oxoaldehyde surrogate for the synthesis of 1-aroyl-β-carbolines and fused-nitrogen heterocycles

An efficient iodine-mediated oxidative Pictet-Spengler reaction in dimethyl sulphoxide (DMSO) using terminal alkynes as the 2-oxoaldehyde surrogate for the synthesis of aryl (9H-pyrido[3,4-b]indol-1-yl)methanones is described. The scope of the protocol includes the total synthesis of Fascaplysin, Eudistomins Y₁ and Y₂. The methodology is extended for preparing pyrrolo[1,2-a]-quinoxaline and indolo[1,5-a]quinoxaline derivatives. The utility of 1-aroyl-β-carbolines was demonstrated by performing palladium-catalyzed β-carboline directed ortho-C(sp2)-H functionalization of the phenyl ring with thiomethyl (SMe) group using DMSO as source and for accessing 4-aryl-canthin-6-ones.     

Facile synthesis of bicyclic 1-arylpyrazol-5-ones

In this Letter, we described a facile method for constructing fused bicyclic 1-arylpyrazol-5-one ring system. We employed various methylene-containing carboxylic acids as the substrates and proved that the pyrazolone ring closure requires activated methylene group in intermediate II. Accordingly, a series of structurally diversified, fused bicyclic 1-arylpyrazol-5-ones was prepared in moderate to high yields using the requisite substrates.     

A carbanion induced synthesis of highly congested pyrazole and imidazole containing heterocycles

An efficient approach to the synthesis of highly congested di, penta and hexacyclic pyrazoles as well as imidazole fragment containing novel heterocyclic molecule has been developed through a carbanion induced transformation of suitably functionalized 2H-pyran-2-ones, benzo[h]chromene and thiochromeno[4,3-b]pyrans. Due to the presence of fluorescence, we report their prime application metal sensor as off/on switching in ferric ions.     

Synthesis of pyrrolo[1,2-a]pyrazines and pyrazino[1,2-a]indoles by Curtius reaction in Morita-Baylis-Hillman derivatives

Synthesis of substituted pyrrolo[1,2-a]pyrazines and pyrazino[1,2-a]indoles from the Morita-Baylis-Hillman derivatives of acrylates via saponification followed by Curtius reaction is described.     

正丁胺对激光染料荧光谱影响的研究

用正丁胺作为碳源,采用射频辉光放电制备碳膜,选用激光染料R6G和聚乙二醇混合液作为蒸气源,采用单源热蒸发,在蒸发室与染料同时沉积得到混合膜,用拉曼光谱和红外光谱分析了碳膜的结构和键合方式,分析表明:碳膜中存在胺基团和氢原子.混合膜的荧光谱测量结果表明,认为正丁胺对染料荧光谱的影响是因为胺基和氢原子的存在.     

Selective synthesis of 4,5-dihydroimidazo- and imidazo[1,5-a]quinoxalines via modified Pictet–Spengler reaction

An efficient tandem approach for the selective synthesis of 4,5-dihydroimidazo[1,5-a]quinoxalines 6a–g and imidazo[1,5-a]quinoxalines 7a–h by the reaction of 2-imidazolyl anilines 4a–c with aryl aldehydes 5a–k under mild reaction conditions is described. Introduction of electron releasing alkyl groups in substrates 4a–b was found to be instrumental for the success of the reaction.     

Understanding the Diels-Alder reactivity of 1,2-azaborine analogues

The Diels-Alder reactivity of 1,2-heteroborines (H₄C₄B(H)X, X?=?NH, PH, AsH; O, S, Se) has been computationally explored by means of Density Functional Theory (DFT) calculations. The influence of the HB?=?X fragment on the reactivity of the system has been quantitatively analyzed in detail by means of the so-called Activation Strain Model (ASM) of reactivity. It is found that the interaction between these species and the dienophile is significantly stronger than that computed for their all-carbon isoelectronic counterpart, benzene. In addition, the strain energy plays a key role in the observed reactivity trends. The role of the aromaticity strength of these heteroarenes on the reactivity is also assessed.