可精确控制药物的细胞内释放和抗癌活性的还原敏感壳可摒弃的生物可降解聚合物纳米载体
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摘要
聚酯型聚合物纳米药物载体在体内的药物释放速度通常很慢，而刺激响应性纳米药物载体则能将小分子药物或蛋白质很快释放出来。然而，在生物可降解的胶束和囊泡的研究中缺乏能够精确控制胶束和囊泡药物的释放速度的方法。
在本论文中，我们将非还原敏感型聚合物PEG-PCL和还原敏感型聚合物PEG-SS-PCL按不同比例混合制备出具有不同还原敏感度的聚合物胶束和囊泡。这类还原敏感型胶束和囊泡能够在模拟细胞内还原环境下由于PEG壳的部分摒弃，能分别精确地控制疏水小分子药物（如阿霉素DOX）或亲水大分子蛋白质药物的释放。
论文的第二章主要介绍了此类还原敏感型聚合物胶束的工作。我们利用还原敏感的PEG-SS-PCL和非还原敏感的PEG-PCL两种嵌段共聚物形成的还原敏感的胶束来装载抗癌药物阿霉素(DOX)，系统地研究了体系中双硫键含量对其还原敏感性、粒径变化、药物的释放速度以及抗癌活性等方面的影响。双硫键的含量是在制备胶束过程中通过改变PEG-PCL和PEG-SS-PCL两种嵌段共聚物的质量分数来控制的。还原触发的体外DOX释放速度、细胞内DOX的含量以及装载DOX的聚合物胶束体系对癌细胞的细胞毒性均密切依赖于胶束体系中双硫键的含量，这一结果可能是由于还原条件下PEG壳层的脱落导致在胶束内形成孔道。
论文的第三章我们设计利用还原敏感的PEG-SS-PCL和非还原敏感的PEG-PCL两种嵌段共聚物来制备还原敏感、壳可摒弃的聚合物囊泡，其在类似细胞内还原条件下可部分摒弃PEG壳而形成壁膜多孔的聚合物囊泡，通过控制壁膜上的孔道的大小、数量可用于精确控制蛋白质药物的释放。在本章中，所选的PEG-SS-PCL和PEG-PCL中，PCL的分子量要比第二章中的大，PEG和PCL的分子量分别为5和18kDa左右，有利于形成聚合物囊泡结构。我们具体研究了用该还原敏感囊泡装载蛋白质的情况，并系统地研究了双硫键含量对囊泡体系的还原敏感性、粒径变化、各种蛋白质的释放速度等方面的影响。实验结果表明，还原触发的体外蛋白质的释放速度密切依赖于囊泡体系中双硫键的含量。此外，我们也用不同分子量的蛋白质及葡聚糖初步研究了还原敏感囊泡在还原条件下壁膜的渗透性。通过体外释放曲线的拟合得到渗透率范围为0.7-12×10~(-13)m/s，还原敏感度越大即含PEG-SS-PCL成分越多的囊泡其壁膜形成的孔道对包载物质的渗透率越大，即渗透率顺序为SS100>SS70>SS50>SS30>SS0；尺寸越小的包载物质在纳米载体的渗透率也越大，即渗透率顺序为FITC-dex4k>FITC-BSA> FITC-IgG> FITC-dex40k。另外，在模拟细胞内还原条件下该还原敏感聚合物囊泡壁膜上形成的可供药物通过的孔道尺寸至少为13nm左右。
与PEG-SS-PCL胶束和囊泡在还原环境下很快发生聚集的情况不同，含有10至90wt.%PEG-PCL的PEG-SS-PCL胶束和囊泡体系在脱去亲水PEG壳后其粒径并没有明显变化。因此，还原触发的还原敏感纳米载体的亲水壳的摒弃是一种能精确控制小分子疏水抗癌药物和亲水大分子药物的细胞内释放及抗癌活性的有效措施。
Traditional polyester based micelle and polymersome nanocarriers often exhibit slowdrug release profiles in vivo, while stimuli sensitive micelles and polymersomes couldrelease drugs or proteins very quickly. However, there is lack of fine control in drug releasefrom the biodegradable micelles and polymersomes so far. In this thesis, we designedbiodegradable micelles and polymersomes based on a mixture reducible polyethyleneglycol-SS-polycaprolactone (PEG-SS-PCL) and nonreducible PEG-PCL, which exhibitedtunable DOX or protein release profiles, elegantly manipulated by varying the compositionof the two diblock copolymers.
In chapter2of the thesis, effects of disulfide contents on reduction-sensitivity, triggereddrug release as well as anti-tumor activity of shell-sheddable micelles self-assembled fromwere systematically investigated. Interestingly, in contrast to rapid aggregation ofPEG-SS-PCL micelles, mixed micelles containing10~90wt.%PEG-PCL displayed littlesize change in response to10mM dithiothreitol (DTT). The in vitro release studies showedthat under intracellular-mimicking reductive environments, DOX release rate increased withincreasing PEG-SS-PCL contents in the micelles, in which about29.4,42.7,77.9and86.9%DOX was released within12h from micelles containing30,50,70and90wt.%PEG-SS-PCL, respectively. In contrast, DOX release was minimal (<20%) undernon-reductive physiological conditions. Notably, flow cytometry displayed clear correlationbetween cellular DOX levels and PEG-SS-PCL contents in DOX-loaded micelles. Moreover,confocal laser scanning microscopy (CLSM) observations indicated progressively strongerDOX fluorescence in RAW264.7cells following12h treatment with DOX-loaded micellescontaining increasing PEG-SS-PCL contents. In addition, MTT assays in RAW264.7cellsshowed that the cytotoxicity of DOX-loaded micelles was augmented proportionally toPEG-SS-PCL contents, signifying the role of reduction-triggered “active” drug release incells.
In chapter3of the thesis, we continued the study with the polymersomes whichcomposed of PEG-SS-PCL/PEG-PCL block copolymers with high PCL molecular weight of 18kDa. We investigated the encapsulation and release of hydrophilic macromacromoleculelike proteins and dextran of the reducible polymersomes. The reducible polymersomes couldshed off PEG shell partially to form polymersomes with porous membranes underbioreducible mimicking environments, and by controlling PEG-SS-PCL content the size andthe number of the pores formed and thus the protein release could be manipulated. Theprotein loaded polymersomes with size of143-153nm (PDI0.1-0.2) could be prepared viadirect hydration method. It is found that reduction triggered protein and dextran release wasintimately dependent on the PEG-SS-PCL content in the polymersomes, possibly due to thepore formation resulting from PEGshedding under reducible conditions. By studying the invitro release of proteins and dextran of different molecular weight and size, we analyzed thepermeability of the polymersomes in the presence of10mM DTT. After fit the release datawe got the permeability of the different polymersomes for different macromolecules inbetween0.7-12×10~(-13)m/s. Moreover, high PEG-SS-PCL in the polymersomes or smallmacromolecules led to high permeability. Furthermore, the pore size formed in10mM DTTin the polymersome membranes was at lease13nm. These results have shown that the invitro drug or protein release, intracellular drug release and therefore anti-tumor activity ofnano drugs can be precisely controlled by extentof reduction-triggered shedding offhydrophilic shells.
在本论文中，我们将非还原敏感型聚合物PEG-PCL和还原敏感型聚合物PEG-SS-PCL按不同比例混合制备出具有不同还原敏感度的聚合物胶束和囊泡。这类还原敏感型胶束和囊泡能够在模拟细胞内还原环境下由于PEG壳的部分摒弃，能分别精确地控制疏水小分子药物（如阿霉素DOX）或亲水大分子蛋白质药物的释放。
论文的第二章主要介绍了此类还原敏感型聚合物胶束的工作。我们利用还原敏感的PEG-SS-PCL和非还原敏感的PEG-PCL两种嵌段共聚物形成的还原敏感的胶束来装载抗癌药物阿霉素(DOX)，系统地研究了体系中双硫键含量对其还原敏感性、粒径变化、药物的释放速度以及抗癌活性等方面的影响。双硫键的含量是在制备胶束过程中通过改变PEG-PCL和PEG-SS-PCL两种嵌段共聚物的质量分数来控制的。还原触发的体外DOX释放速度、细胞内DOX的含量以及装载DOX的聚合物胶束体系对癌细胞的细胞毒性均密切依赖于胶束体系中双硫键的含量，这一结果可能是由于还原条件下PEG壳层的脱落导致在胶束内形成孔道。
论文的第三章我们设计利用还原敏感的PEG-SS-PCL和非还原敏感的PEG-PCL两种嵌段共聚物来制备还原敏感、壳可摒弃的聚合物囊泡，其在类似细胞内还原条件下可部分摒弃PEG壳而形成壁膜多孔的聚合物囊泡，通过控制壁膜上的孔道的大小、数量可用于精确控制蛋白质药物的释放。在本章中，所选的PEG-SS-PCL和PEG-PCL中，PCL的分子量要比第二章中的大，PEG和PCL的分子量分别为5和18kDa左右，有利于形成聚合物囊泡结构。我们具体研究了用该还原敏感囊泡装载蛋白质的情况，并系统地研究了双硫键含量对囊泡体系的还原敏感性、粒径变化、各种蛋白质的释放速度等方面的影响。实验结果表明，还原触发的体外蛋白质的释放速度密切依赖于囊泡体系中双硫键的含量。此外，我们也用不同分子量的蛋白质及葡聚糖初步研究了还原敏感囊泡在还原条件下壁膜的渗透性。通过体外释放曲线的拟合得到渗透率范围为0.7-12×10~(-13)m/s，还原敏感度越大即含PEG-SS-PCL成分越多的囊泡其壁膜形成的孔道对包载物质的渗透率越大，即渗透率顺序为SS100>SS70>SS50>SS30>SS0；尺寸越小的包载物质在纳米载体的渗透率也越大，即渗透率顺序为FITC-dex4k>FITC-BSA> FITC-IgG> FITC-dex40k。另外，在模拟细胞内还原条件下该还原敏感聚合物囊泡壁膜上形成的可供药物通过的孔道尺寸至少为13nm左右。
与PEG-SS-PCL胶束和囊泡在还原环境下很快发生聚集的情况不同，含有10至90wt.%PEG-PCL的PEG-SS-PCL胶束和囊泡体系在脱去亲水PEG壳后其粒径并没有明显变化。因此，还原触发的还原敏感纳米载体的亲水壳的摒弃是一种能精确控制小分子疏水抗癌药物和亲水大分子药物的细胞内释放及抗癌活性的有效措施。
Traditional polyester based micelle and polymersome nanocarriers often exhibit slowdrug release profiles in vivo, while stimuli sensitive micelles and polymersomes couldrelease drugs or proteins very quickly. However, there is lack of fine control in drug releasefrom the biodegradable micelles and polymersomes so far. In this thesis, we designedbiodegradable micelles and polymersomes based on a mixture reducible polyethyleneglycol-SS-polycaprolactone (PEG-SS-PCL) and nonreducible PEG-PCL, which exhibitedtunable DOX or protein release profiles, elegantly manipulated by varying the compositionof the two diblock copolymers.
In chapter2of the thesis, effects of disulfide contents on reduction-sensitivity, triggereddrug release as well as anti-tumor activity of shell-sheddable micelles self-assembled fromwere systematically investigated. Interestingly, in contrast to rapid aggregation ofPEG-SS-PCL micelles, mixed micelles containing10~90wt.%PEG-PCL displayed littlesize change in response to10mM dithiothreitol (DTT). The in vitro release studies showedthat under intracellular-mimicking reductive environments, DOX release rate increased withincreasing PEG-SS-PCL contents in the micelles, in which about29.4,42.7,77.9and86.9%DOX was released within12h from micelles containing30,50,70and90wt.%PEG-SS-PCL, respectively. In contrast, DOX release was minimal (<20%) undernon-reductive physiological conditions. Notably, flow cytometry displayed clear correlationbetween cellular DOX levels and PEG-SS-PCL contents in DOX-loaded micelles. Moreover,confocal laser scanning microscopy (CLSM) observations indicated progressively strongerDOX fluorescence in RAW264.7cells following12h treatment with DOX-loaded micellescontaining increasing PEG-SS-PCL contents. In addition, MTT assays in RAW264.7cellsshowed that the cytotoxicity of DOX-loaded micelles was augmented proportionally toPEG-SS-PCL contents, signifying the role of reduction-triggered “active” drug release incells.
In chapter3of the thesis, we continued the study with the polymersomes whichcomposed of PEG-SS-PCL/PEG-PCL block copolymers with high PCL molecular weight of 18kDa. We investigated the encapsulation and release of hydrophilic macromacromoleculelike proteins and dextran of the reducible polymersomes. The reducible polymersomes couldshed off PEG shell partially to form polymersomes with porous membranes underbioreducible mimicking environments, and by controlling PEG-SS-PCL content the size andthe number of the pores formed and thus the protein release could be manipulated. Theprotein loaded polymersomes with size of143-153nm (PDI0.1-0.2) could be prepared viadirect hydration method. It is found that reduction triggered protein and dextran release wasintimately dependent on the PEG-SS-PCL content in the polymersomes, possibly due to thepore formation resulting from PEGshedding under reducible conditions. By studying the invitro release of proteins and dextran of different molecular weight and size, we analyzed thepermeability of the polymersomes in the presence of10mM DTT. After fit the release datawe got the permeability of the different polymersomes for different macromolecules inbetween0.7-12×10~(-13)m/s. Moreover, high PEG-SS-PCL in the polymersomes or smallmacromolecules led to high permeability. Furthermore, the pore size formed in10mM DTTin the polymersome membranes was at lease13nm. These results have shown that the invitro drug or protein release, intracellular drug release and therefore anti-tumor activity ofnano drugs can be precisely controlled by extentof reduction-triggered shedding offhydrophilic shells.
引文
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