NIR Light-Activated Nanoparticles for Drug and Gene Delivery

描述(申请人提供)：我们的目标是开发强大的siRNA和Micro-RNA方法来调控基础研究的基因，通过利用中空金纳米壳(HGN)的独特化学和物理特性，允许在细胞培养和线虫中高效地进行时间和空间控制。HGNs直径30-40 nm，厚度3-5 nm，金色外壳设计用于强烈吸收生理上友好的近红外光，并将这种光能转化为局部加热。HGN可以通过简单的硫醇化学很容易地结合到小分子上，靶向配体、聚合物、siRNA和DNA，或者结合到脂质体中或拴在脂质体上。水、蛋白质、脂质等不吸收近红外光，因此培养中的细胞基本上是透明的，这消除了暴露期间的损害。飞秒近红外光脉冲通过在不破坏siRNA的情况下打破硫醇键，触发硫醇结合的siRNA或其他分子从HGN释放。对于高效寡核苷酸和小分子递送的开发来说，更重要的是，通过将HGN吸收的生理友好的近红外光能转换为热，创建不稳定的微泡，机械地破裂内小体，并在几秒钟内将siRNA释放到细胞质中，可以绕过众所周知的内体逃逸瓶颈。这使得可以使用浓度低得多的HGN-siRNA结合物，极大地提高了转染率，并提供了空间和时间可控的转染法，可用于培养细胞的模式和定位线虫内的特定结构。HGN转染法与脂质体一样有效，但无毒，可用于活体。在这项计划中，我们将利用hGN-siRNA平台开发用于遥控激活或失活生物过程的掩蔽和去掩蔽技术，以设计出简单且可扩展的方法来实时地对培养细胞进行光刻图案化。我们将利用近红外光触发控制释放来研究含有HGN的脂质体中小分子的控制释放，从而使基础细胞生物学研究成为可能，包括人类干细胞分化、癌症生物学、信号转导途径和在化疗和药物输送中的应用。我们将开发多种方法，利用硫醇和二硫醇锚的组合，在不同的能量下解吸，从单一的HGN释放多种化学物质。将合成新的银纳米壳和金和银纳米棒，以探测近红外光谱的其他区域，并提供同时传递和成像的机会。这些新结构可以通过使用不同波长的近红外辐射来独立寻址。该项目充分利用HGN与生理友好的近红外光的独特相互作用，在活细胞和生物体中启动和控制生物过程，以毫秒级的速度进行精确的空间控制。

DESCRIPTION (provided by applicant): Our goal is to develop robust siRNA and micro-RNA methods to regulate genes for basic research, permitting both temporal and spatial control of transfection with high efficiency in both cell culture and C. elegans by using the unique chemical and physical properties of hollow gold nanoshells (HGN). HGNs are 30 - 40 nm diameter, 3-5 nm thick, gold shells designed to strongly absorb physiologically friendly NIR light and convert this light energy into local heating. HGN can be easily conjugated to small molecules, targeting ligands, polymers, siRNA and DNA by simple thiol chemistry, or incorporated into or tethered to liposomes. Water, proteins, lipids, etc. do not absorb NIR light, so cells in culture are essentially transparent, which eliminates damage during exposure. Femtosecond NIR light pulses trigger release of thiol-conjugated siRNA or other molecules from the HGN by breaking thiol bonds without damaging the siRNA. Even more important to the development of efficient oligonucleotide and small molecule delivery, the well-known bottleneck of endosomal escape can be bypassed by converting the physiologically friendly NIR light energy absorbed by the HGN to heat, creating unstable microbubbles that mechanically rupture endosomes and release siRNA to the cytosol within seconds. This allows much lower concentrations of HGN-siRNA conjugates to be used, greatly increases transfection efficiency, and provides spatially and temporally controlled transfection that can be used to pattern cultured cells and address specific structures within C. elegans. HGN transfection is as efficient as Lipofectamine, but is non-toxic, and can be used in living organisms. In this proposal, we will use the HGN-siRNA platform to develop masking and unmasking techniques for activating or inactivating biological processes with remote control to devise simple and scalable methods of lithographic patterning of cultured cells in real time. We will investigate controlled release of small molecules from liposomes incorporating HGN using NIR light triggering to control release, thereby enabling basic cell biological studies, including human stem cell differentiation, cancer biology, and signal transduction pathways and routes to applications in chemotherapy and drug delivery. We will develop methods to multiplex the release from a single HGN using a combination of thiol and dithiol anchors that desorbs at different energies to release of multiple chemical species. New silver nanoshells and gold and silver nanorods will be synthesized to probe other regions of the NIR spectrum and provide simultaneous delivery and imaging opportunities. These new constructs could be addressed independently by using different wavelength NIR irradiation. This project takes full advantage of the unique HGN interactions with physiologically friendly NIR light to initiate and control biological processes with precise spatial control at millisecond rates in living cells and organisms.