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Engineering substrate-mediated gene delivery with self-assembled monolayers and soft lithography.

机译：利用自组装单分子层和软光刻技术进行底物介导的基因传递工程。

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Substrate-mediated delivery involves the immobilization of DNA, complexed with nonviral vectors, to a biomaterial or surface that supports cell adhesion. Cells cultured on the substrate are exposed to elevated DNA concentrations within the local microenvironment, which enhances transfection. As surface properties are critical to this delivery approach, self-assembled monolayers (SAMs) of alkanethiols on gold were used to investigate the effect of surface chemistries on substrate-mediated delivery. Surface hydrophilicity and ionization affected nonspecific complex immobilization and transfection, with SAMs presenting carboxylic acid groups resulting in the greatest immobilization and transfection. Subsequent studies used SAMs to investigate the effect of surfaces presenting oligo(ethylene glycol) (EG) groups on substrate-mediated delivery. Nonspecific complex immobilization to SAMs containing combinations of EG- and carboxylic acid-terminated alkanethiols resulted in substantially greater transfection than surfaces containing no EG groups or EG groups combined with other functional groups. Transfection enhancement could not be attributed to binding or release profiles. Atomic force microscopy imaging of immobilized complexes revealed that EG groups within SAMs affected complex size and appearance, indicating the ability of these surfaces to preserve complex morphology upon binding. To control binding and release profiles, complexes were covalently linked to SAMs presenting appropriate functional groups. Covalent tethering by multiple crosslinkers resulted in lower complex binding than corresponding conditions without the crosslinker, and no transfection. The principles guiding complex immobilization and tethering could be extended to biomaterial surfaces for tissue engineering applications.; Finally, soft lithography techniques were used to pattern complex deposition and transfection, on SAMs and cell culture surfaces, for the formation of a transfected cell array, a high-throughput technique to correlate gene expression with functional cell responses. We developed an array that combines a two-plasmid system and dual bioluminescence imaging to quantitatively normalize for variability in transfection and increase sensitivity. The array was applied to quantify estrogen receptor alpha (ERalpha) activity in breast cancer cells. ER induction mimicked results obtained through traditional assay methods. Furthermore, the array captured a dose response to estrogen, demonstrating the sensitivity of bioluminescence quantification. Our system should serve as a standard for fabrication of transfected cell arrays to report on signaling pathways.
机译：底物介导的传递涉及将与非病毒载体复合的DNA固定在支持细胞粘附的生物材料或表面上。在底物上培养的细胞在局部微环境中暴露于升高的DNA浓度，从而增强了转染。由于表面性质对该传递方法至关重要，因此使用链烷硫醇在金上的自组装单分子层（SAM）来研究表面化学对基质介导的传递的影响。表面亲水性和离子化会影响非特异性复合物的固定和转染，其中SAMs具有羧酸基团，导致最大的固定和转染。随后的研究使用SAMs来研究呈现低聚（乙二醇）（EG）基团的表面对底物介导的递送的影响。与不含EG基团或与其他官能团结合的EG基团的表面相比，非特异性复合物固定至包含EG和羧酸封端的链烷硫醇的SAM的转染效果显着提高。转染增强不能归因于结合或释放特征。固定复合物的原子力显微镜成像显示，SAM中的EG基团会影响复合物的大小和外观，表明这些表面在结合后保留复合物形态的能力。为了控制结合和释放曲线，将复合物共价连接至具有适当官能团的SAM。与没有交联剂且没有转染的相应条件相比，通过多种交联剂进行的共价束缚导致复合物结合率更低。指导复杂的固定和束缚的原理可以扩展到生物材料表面以用于组织工程应用。最后，软光刻技术用于在SAM和细胞培养表面上形成复杂的沉积和转染图案，以形成转染的细胞阵列，这是一种高通量技术，可将基因表达与功能性细胞反应相关联。我们开发了一种结合了两个质粒系统和双重生物发光成像的阵列，以定量标准化转染的变异性并提高灵敏度。该阵列用于量化乳腺癌细胞中的雌激素受体α（ERalpha）活性。 ER诱导模拟了通过传统测定方法获得的结果。此外，该阵列捕获了对雌激素的剂量反应，证明了生物发光定量的敏感性。我们的系统应作为转染细胞阵列制造的标准，以报告信号传导途径。

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