NMR Group Project: Preparation and Properties of Novel Multivalent Nanoparticles

肿瘤发生的一个既定标志是由于肿瘤组织中糖加工酶表达的变化而引起的异常聚糖链的生物合成。当肿瘤获得更具侵袭性的表型时，这些畸变变得更加明显。肿瘤细胞表面碳水化合物在多种肿瘤细胞的运动和转移中起着重要作用。此外，许多这些异常聚糖是肿瘤相关碳水化合物抗原（TACA），并已用于肿瘤疫苗的开发。由于大多数细胞与TACAs的相互作用尚不清楚，因此迫切需要更好地表征在这些事件中发生的特定分子相互作用。碳水化合物与大分子结合的一个很好理解的特征是多价性的概念：单体碳水化合物与蛋白质的结合非常弱，而单体的聚类将这种亲和力提高了一百万倍。我们已经在非常特定的模板上制备了重要的Thomsen-Friedenreich （Tf）抗原(Gal(β)1-3GalNAc(α)-O-Ser/Thr)，以利用这种所谓的簇糖苷效应。如上一篇报道所述，我们制备了含糖衍生物的金自组装纳米球和量子点，并报道了其功能的初步细节。我们的金纳米球在小鼠体内的实验重复了两次，结果不同。然而，这是由于使用的肿瘤细胞具有与原始细胞不同的基因组成以及使用的肿瘤数量错误造成的。我们对金颗粒进行了进一步的体外表征，发现它们的作用与单价糖分子不同。一项大型体内研究通过对不同浓度的颗粒进行对照（仅连接剂）纳米颗粒的测试，得出了相互矛盾但极具挑衅性的结果。我们似乎能够在体内抑制原发肿瘤的生长，但我们可能会促进更大流体动力学体积的颗粒的转移。在过去的一年里，一项集中的研究工作正在进行，以了解体内数据的冲突。我们认为，通过仔细研究糖的大小分布、表面密度和纳米颗粒的连接技术，我们已经解决了这个问题。我们已经证明了糖的特定立体化学对粒子自组装是有害的，并通过降低碳水化合物的表面密度来解决这个问题。我们还表明，单糖的特定表面密度可以促进或抑制艾滋病毒对某些哺乳动物细胞的附着。我们正在优化这项技术，以找到最强大的颗粒在体内应用。在上一篇报道中，我们报道了粘蛋白糖肽的合成，并开发了新的连接技术将其连接到金颗粒上。今年我们发现，由于意想不到的聚集效应，这种连接器技术在合成上是不可行的。我们制备了一个更强的连接体，并制备了4个糖肽和一个对照肽，它们与金纳米颗粒连接。进行了表征，颗粒均匀。我们已经与我们的合作者Kate Rittenhouse Olsen开始了一项疫苗接种方案，在8只老鼠身上使用两个含有MUC4重复单位糖肽的单独颗粒。此外，我们正在利用氨基酸连接体技术制备TF抗原颗粒，以探索其性质，并与单糖和单独连接体的颗粒进行比较。我们对疫苗接种研究的命运感到非常兴奋，因为这将是一个向宿主呈递抗原的新平台。

An established hallmark of tumorigenesis is the biosynthesis of aberrant glycan chains due to changes in the expression of glycoprocessing enzymes in tumor tissue. These aberrations become more marked as the tumor acquires a more aggressive phenotype. Tumor cell-surface carbohydrates play important roles in the motility and metastasis of many different cancer cells. In addition, many of these aberrant glycans are tumor-associated carbohydrate antigens (TACA) and have been used in the development of tumor vaccines. Since most of the cellular interactions with TACAs are not well understood, there is an urgent need to better characterize the specific molecular interactions that occur during these events. One feature of carbohydrate binding to macromolecules that is well understood is the concept of multivalency: Monomer carbohydrates bind to proteins very weakly while clustering of a monomer raises this affinity as much as a million-fold. We have prepared the important Thomsen-Friedenreich (Tf) antigen (Gal(beta)1-3GalNAc(alpha)-O-Ser/Thr) on very specific templates to take advantage of this so-called cluster glycoside effect. As mentioned in the last report, we have prepared gold self-assembled nanospheres and quantum dots containing sugar derivative and reported preliminary details on their function. The in vivo experiments with our gold nanospheres in mice were repeated twice with varying results. However, these were caused by the use of a tumor cells that had had a different genetic makeup than the original and an error in the amount of tumor used. We have done further in vitro characterization of the gold particles and found them to act differently than monovalent sugar molecules. A large in vivo study has yielded conflicting but highly provocative results with varying concentrations of particles being tested against control (linker-only) nanoparticles. We seem to be able to inhibit primary tumor growth in vivo, but we possibly promote metastasis with particles of larger hydrodynamic volume. In the past year, a concentrated research effort was mounted to understand the conflicting in vivo data. We feel we have solved this problem by closely examining the size distribution, surface density of sugars and the linker technology of the nanoparticles. We have shown that a specific stereochemistry of the saccharide is detrimental to particle self assembly and solved this problem with reduced surface density of the carbohydrate. We have also shown that specific surface densities of simple sugars can either promote or inhibit the attachment of HIV to certain mammalian cells. We are optimizing this technology to find the most robust particles for in vivo applications. In the last report we reported synthesis of mucin glycopeptides and developed new linker technology to attach these to gold particles. We discovered this year that that linker technology was not synthetically feasible due to unanticipated aggregation effects. We have prepared a much more robust linker and prepared 4 glycopeptides plus a control peptide that were linked to gold nanoparticles. Characterizations were performed and the particles were uniform. We have started a vaccination protocol with our collaborator Kate Rittenhouse Olsen on eight mice with two separate particles containing MUC4 repeating unit glycopeptides. In addition, we are exploring the nature of the TF antigen by preparing particles with amino acid-linker technology to compare with those of simple saccharides and linker alone. We are very excited about the fate of the vaccination studies since this would be a novel platform to present antigen to a host.