钙离子诱导硫酸软骨素自组装的AFM研究

利用Ca^2 试剂掺入硫酸软骨素（Chondroitin Sulfate，CS）溶液中诱导分子链在云母表面聚集形成自组装膜，用原子力显微镜（Atomic Force Microscope，AFM）在不同的时间段研究Ca^2 试剂对硫酸软骨素自组装膜结构的影响。结果表明当CS浓度为1.2mg/ml时，Ca^2 能很好的诱导CS自组装膜的形成，这种自组装结构会随时间发生形态学改变，具有不稳定性。说明了无机小分子通过与同种大分子的表面活性基团相互作用可以诱导高分子物质的自组装，这种自组装结构是一种非平衡状态下的耗散结构。     

Self-assembled multifunctional nanotheranostics against circulating tumor clusters in metastatic breast cancer

Circulating tumor clusters(CTC) disseminating from the primary tumor are responsible for secondary tumor formation where the conventional treatments such as chemotherapy and radiotherapy does not prevent the metastasis at locally advanced stage of breast cancer. In this study, a smart nanotheranostic system has been developed to track and eliminate the CTCs before it can colonize at a new site,which would reduce metastatic progression and increase the five-year survival rate of the breast cancer...     

PEG修饰对PEI聚电解质复合物的药学特性及人乳腺癌细胞的摄取效率影响的研究

荧光素阴离子葡聚糖（DFA）和寡核苷酸（ODN）可以和带正电的聚乙亚胺（PEI）通过自组装的方式形成聚电解质复合物。为了调查PEG修饰对PEI聚电解质复合物的药学特性及细胞摄取效率的影响，我们进行了此项研究。DFA和ODN与PEI及PEI—PEG通过吹吸混匀形成聚电解质复合物。我们分别采用动态光散射法及琼脂糖凝胶电泳法对聚电解质复合物的表面性质（粒径，ξ电位），PEI及PEI—PEG延滞ODN的能力等进行了评价。MCF-7对DFA／PEI及DFA／PEI-PEG的摄取效率通过流式细胞术进行测量，并通过激光扫描聚焦显微镜（CLSM）法来观察DFA／PEI及DFA／PEI—PEG聚电解质复合物的细胞内摄作用。N／P比对ODN／PEI聚电解质复合物的粒径和ξ电位影响很大，而ODN／PEI—PEG复合物的粒径受N／P比的影响要小一些，在N／P=2～16范围内，ODN／PEI—PEG复合物的粒径在30～100nm左右。PEI和PEI—PEG在N／P比等于4时就开始出现延滞ODN的现象，彻底延滞在N／P比等于8时出现。MCF-7的细胞摄取效率与DFA浓度及转染时间呈正相关，在适宜条件下，细胞摄取率可超过99％。本实验结果显示通过隐形修饰的PEI自组装形成的聚电解质复合物可以提高其作为基因载体递送基因进入细胞的能力。     

Engineering functional anisotropy in fibrocartilage neotissues

The knee meniscus, intervertebral disc, and temporomandibular joint (TMJ) disc all possess complex geometric shapes and anisotropic matrix organization. While these characteristics are imperative for proper tissue function, they are seldom recapitulated following injury or disease. Thus, this study's objective was to engineer fibrocartilages that capture both gross and molecular structural features of native tissues. Self-assembled TMJ discs were selected as the model system, as the disc exhibits a unique biconcave shape and functional anisotropy. To drive anisotropy, 50:50 co-cultures of meniscus cells and articular chondrocytes were grown in biconcave, TMJ-shaped molds and treated with two exogenous stimuli: biomechanical (BM) stimulation via passive axial compression and bioactive agent (BA) stimulation via chondroitinase-ABC and transforming growth factor-β1. BM + BA synergistically increased Col/WW, Young's modulus, and ultimate tensile strength 5.8-fold, 14.7-fold, and 13.8-fold that of controls, respectively; it also promoted collagen fibril alignment akin to native tissue. Finite element analysis found BM stimulation to create direction-dependent strains within the neotissue, suggesting shape plays an essential role toward driving in vitro anisotropic neotissue development. Methods used in this study offer insight on the ability to achieve physiologic anisotropy in biomaterials through the strategic application of spatial, biomechanical, and biochemical cues.     

Self healing hydrogels composed of amyloid nano fibrils for cell culture and stem cell differentiation

Amyloids are highly ordered protein/peptide aggregates associated with human diseases as well as various native biological functions. Given the diverse range of physiochemical properties of amyloids, we hypothesized that higher order amyloid self-assembly could be used for fabricating novel hydrogels for biomaterial applications. For proof of concept, we designed a series of peptides based on the high aggregation prone C-terminus of Aβ42, which is associated with Alzheimer's disease. These Fmoc protected peptides self assemble to β sheet rich nanofibrils, forming hydrogels that are thermoreversible, non-toxic and thixotropic. Mechanistic studies indicate that while hydrophobic, π–π interactions and hydrogen bonding drive amyloid network formation to form supramolecular gel structure, the exposed hydrophobic surface of amyloid fibrils may render thixotropicity to these gels. We have demonstrated the utility of these hydrogels in supporting cell attachment and spreading across a diverse range of cell types. Finally, by tuning the stiffness of these gels through modulation of peptide concentration and salt concentration these hydrogels could be used as scaffolds that can drive differentiation of mesenchymal stem cells. Taken together, our results indicate that small size, ease of custom synthesis, thixotropic nature makes these amyloid-based hydrogels ideally suited for biomaterial/nanotechnology applications.     

An16-resilin: An advanced multi-stimuli-responsive resilin-mimetic protein polymer

Engineered protein polymers that display responsiveness to multiple stimuli are emerging as a promising class of soft material with unprecedented functionality. The remarkable advancement in genetic engineering and biosynthesis has created the opportunity for precise control over the amino acid sequence, size, structure and resulting functions of such biomimetic proteins. Herein, we describe the multi-stimuli-responsive characteristics of a resilin-mimetic protein, An16-resilin (An16), derived from the consensus sequence of resilin gene in the mosquito Anopheles gambiae. We demonstrate that An16 is an intrinsically disordered protein that displays unusual dual-phase thermal transition behavior along with responsiveness to pH, ion, light and humidity. Identifying the molecular mechanisms that allow An16 to sense and switch in response to varying environments furthers the ability to design intelligent biomacromolecules.     

Effect of the distribution of adsorbed proteins on cellular adhesion behaviors using surfaces of nanoscale phase-reversed amphiphilic block copolymers

In order to create suitable biocompatible materials for various tissue engineering applications, it is important to be able to understand protein adsorption and cell adhesion behaviors on the material’s surfaces. It is known that the nanoscale distribution of adsorbed proteins affects cell adhesion behaviors. However, how nanoscale structures affect cell adhesion behaviors is still unclear. Therefore, in this study, we investigate the effect of the distribution of adsorbed proteins by the phase reversal of amphiphilic block copolymers composed of protein-non-adsorptive poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and protein-adsorptive poly(3-methacryloyloxy propyltris(trimethylsilyloxy) silane) (PMPTSSi) on cell adhesion behaviors. The nanodomain structures of phase-separated block copolymers were successfully confirmed using transmission electron microscopy and atomic force microscopy. Surfaces that had PMPC dot-like domains (23 ± 4 nm) and ones that had PMPTSSi dot-like domains (25 ± 6 nm) were made. From protein adsorption and L929 cell adhesion measurements, it was found that even on surfaces with equal quantities of protein adsorption, the number of cells on surfaces with PMPC dot-like domains was larger than those with PMPTSSi dot-like domains. This suggests that the simple phase-reversal of the distribution of adsorbed proteins can be used to affect cell adhesion behaviors for designing biomaterial surfaces for tissue engineering applications.     

含FGL活性片段的自组装多肽支架材料修复大鼠脊髓损伤的研究

目的 建立大鼠脊髓损伤模型,将含FGL功能化多肽自组装神经支架材料(FGL-NS)植入到脊髓损伤局部,探讨FGL-NS神经支架材料修复脊髓损伤的效果和机制.方法 麻醉大鼠后,暴露胸10水平脊髓,用特制血管夹(夹力24g)钳夹1 min,建立大鼠胸髓钳夹损伤模型.设立空白组、对照组和实验组,分别于损伤后24h,暴露脊髓损伤局部,将2.5μl等渗葡萄糖溶液、1％ RADA-16和1％ FGL-NS多肽溶液注射入损伤局部.术后3d、1、3、5、7和9周分别采用Basso-Beattie-Bresnahan(BBB)评分评估大鼠脊髓损伤经治疗后运动功能恢复情况,术后9周取脊髓损伤部位组织,行半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3、神经丝蛋白-200 (NF-200)和胶质纤维酸性蛋白(GFAP)免疫组织化学染色评估损伤部位细胞凋亡、轴突再生和瘢痕形成情况.结果 成功建立大鼠胸髓钳夹损伤模型.BBB运动学评分大鼠脊髓损伤后经FGL-NS材料治疗后,BBB评分随时间逐渐升高,自伤后第5周起,显著高于RADA-16治疗组和空白组,大鼠运动功能显著改善.免疫组织化学染色结果显示,损伤后第9周,FGL-NS治疗组脊髓损伤局部可见大量NF-200阳性的神经元细胞[(35.32±3.12)个/视野],显著高于RADA-16组[(18.56±2.64)个/视野]和空白组[(14.83±1.43)个/视野],Caspase-3阳性的凋亡细胞[(22.45 ±2.74)个/视野]显著少于RADA-16组[(30.86 ±3.75)个/视野],而且损伤区GFAP染色的积分吸光度(IA)值为0.50±0.02,明显小于对照组(1.30 ±0.09)和空白组(1.60±0.11).结论 功能化多肽自组装神经支架材料FGL-NS能促进脊髓损伤大鼠的运动功能恢复,能减少脊髓损伤部位细胞凋亡,促进神经元再生,并减少胶质瘢痕形成.     

Cooperative coordination-mediated multi-component self-assembly of “all-in-one” nanospike theranostic nano-platform for MRI-guided synergistic therapy against breast cancer

Carrier-free multi-component self-assembled nano-systems have attracted widespread attention owing to their easy preparation, high drug-loading efficiency, and excellent therapeutic efficacy. Herein, MnAs-ICG nanospike was generated by self-assembly of indocyanine green (ICG), manganese ions (Mn²⁺), and arsenate (AsO₄^3?) based on electrostatic and coordination interactions, effectively integrating the bimodal imaging ability of magnetic resonance imaging (MRI) and fluorescence (FL) imaging-guided synergistic therapy of photothermal/chemo/chemodynamic therapy within an “all-in-one” theranostic nano-platform. The as-prepared MnAs-ICG nanospike had a uniform size, well-defined nanospike morphology, and impressive loading capacities. The MnAs-ICG nanospike exhibited sensitive responsiveness to the acidic tumor microenvironment with morphological transformation and dimensional variability, enabling deep penetration into tumor tissue and on-demand release of functional therapeutic components. In vitro and in vivo results revealed that MnAs-ICG nanospike showed synergistic tumor-killing effect, prolonged blood circulation and increased tumor accumulation compared to their individual components, effectively resulting in synergistic therapy of photothermal/chemo/chemodynamic therapy with excellent anti-tumor effect. Taken together, this new strategy might hold great promise for rationally engineering multifunctional theranostic nano-platforms for breast cancer treatment.     

Self-assembling,pH-responsive nanoflowers for inhibiting PAD4 and neutrophil extracellular trap formation and improving the tumor immune microenvironment

Self-assembling carrier-free nanodrugs are attractive agents because they accumulate at tumor by an enhanced permeability and retention (EPR) effect without introduction of inactive substances, and some nanodrugs can alter the immune environment. We synthesized a peptidyl arginine deiminase 4 (PAD4) molecular inhibitor, ZD-E-1M. It could self-assembled into nanodrug ZD-E-1. Using confocal laser scanning microscopy, we observed its cellular colocalization, PAD4 activity and neutrophil extracellular traps (NETs) formation. The populations of immune cells and expression of immune-related proteins were determined by single-cell mass cytometry. ZD-E-1 formed nanoflowers in an acidic environment, whereas it formed nanospheres at pH 7.4. Accumulation of ZD-E-1 at tumor was pH-responsive because of its pH-dependent differences in the size and shape. It could enter the nucleus and bind to PAD4 to prolong the intracellular retention time. In mice, ZD-E-1 inhibited tumor growth and metastasis by inhibiting PAD4 activity and NETs formation. Besides, ZD-E-1 could regulate the ratio of immune cells in LLC tumor-bearing mice. Immunosuppressive proteins like LAG3 were suppressed, while IFN-γ and TNF-α as stimulators of tumor immune response were upregulated. Overall, ZD-E-1 is a self-assembling carrier-free nanodrug that responds to pH, inhibits PAD4 activity, blocks neutrophil extracellular traps formation, and improves the tumor immune microenvironment.