Conjugation Kits for Facile Preparation of Multivalent Glyco-nanoparticles

描述(申请人提供)：碳水化合物功能化纳米颗粒，即糖纳米颗粒，具有广泛的应用范围，从碳水化合物-蛋白质相互作用的研究，体内细胞成像，生物标记，癌症诊断，以及疫苗的发现。然而，与DNA和蛋白质功能化纳米颗粒相比，糖基纳米颗粒的发展已经远远落后。目前报道的糖纳米粒很少，是通过将硫醇修饰的碳水化合物偶联到纳米粒子表面来制备的。然而，硫醇修饰碳水化合物的制备需要多步骤的合成操作，难度大，耗时长。目前，还没有可供科学和生物医学研究团体简单地将碳水化合物探针偶联到纳米颗粒表面的偶联试剂盒。在NIH SBIR项目中，ADA Technologies Inc.将使用我们专有的糖链表面结合化学技术，开发一种简单而通用的方法，将未经修饰的碳水化合物和糖蛋白一步特定地结合到金和半导体纳米颗粒上。第一阶段项目的具体目标是1)设计和生成双功能树枝状配体作为偶联剂，树枝状分子在树枝状分子上具有多个相同的酰肼偶联点和用于附着在金属纳米颗粒表面的巯基；2)开发和评估小型化微波辐射能量在树枝状修饰纳米颗粒上快速方便偶联碳水化合物的使用，并论证制备具有代表性的糖纳米粒的可行性；3)表征糖纳米粒并验证其免疫活性。第一阶段项目的成功将为科学和生物医学研究界提供一套试剂盒，用于结合具有生物意义的碳水化合物，以产生广泛的糖纳米粒。偶联试剂盒包括酰肼-树状配体功能化纳米颗粒和用于偶联碳水化合物探针的方案。用这种技术制备的糖纳米粒将具有三维多价碳水化合物展示，具有适合于探测碳水化合物与蛋白质相互作用的球形形状。在第二阶段，我们将通过制备一系列连接在不同发射波长的纳米晶上的碳水化合物探针来开发糖纳米粒文库，开发用于核磁共振试剂的磁性糖纳米粒，展示糖纳米粒作为有效的亲和标记和活体细胞标记和成像的用途，并为最终用户制定全面的操作方案。提出了一种简单而通用的方法来定制具有3D多价碳水化合物显示和球状形状的碳水化合物功能化纳米颗粒(即糖纳米粒)。这种方法允许制备具有生物意义的碳水化合物的糖纳米粒。该糖纳米粒可用作有效的亲和标记，也可用于体内细胞标记和成像。

DESCRIPTION (provided by applicant): Carbohydrate functionalized nanoparticles, i.e., the glyco-nanoparticles, have wide application ranging from studies of carbohydrate-protein interactions, in vivo cell imaging, biolabeling, cancer diagnostics, and to vaccine discovery. However, in comparison with DNA and protein functionalized nanoparticles, the development of glyco-nanoparticle has been left far behind. Currently the very few reported glyconanoparticles were prepared by conjugating thiol modified carbohydrates onto nanoparticle surfaces. However, the preparation of thiol modified carbohydrates required multi-step synthetic manipulations which are difficult and time consuming. Currently, there are no conjugation kits that are commercially available for scientific and biomedical research communities to simply conjugate carbohydrate probes onto nanoparticle surface. In this NIH SBIR project ADA Technologies Inc. will use our proprietary glycan surface conjugation chemistry to develop a simple and versatile method for one-step, site-specific conjugation of unmodified carbohydrates and glycoprotein onto Au and semiconductor nanoparticles. The specific aims of the Phase I project are 1) to design and generate a bi-functional dendron ligand as a coupling reagent where the dendron molecule bears multiple identical acyl-hydrazide coupling points for carbohydrates and a sulfhydryl for attachment to the metallic nanoparticle surface; 2) to develop and evaluate the use of miniaturized microwave radiation energy for quick and convenient coupling carbohydrates on the dendron-modified nanoparticles, and demonstrate the feasibility of preparing representative glyconanoparticles; 3) to characterize the glyco-nanoparticles and verify the immunogenic activities of the glyconanoparticles. The success of the phase I project will provide the scientific and biomedical research communities with a set of kits for conjugating biologically significant carbohydrates to generate a wide range of glyco-nanoparticles. The conjugating kits include the hydrazide-dendron ligand functionalized nanoparticles and protocols for conjugation carbohydrate probes. The glyconanoparticles prepared with this technology will have 3D polyvalent carbohydrate display, with globular shapes that are suitable for probing carbohydrate-protein interaction. In Phase II, we will develop a glyconanoparticle library by preparing a wide range of carbohydrate probes conjugated on nanocrystals with different emission wavelengths, develop magnetic glyconanoparticles for use as MRI reagents, and demonstrate the use of glyconanoparticles as an efficient affinity label and in vivo cellular labeling and imaging, and develop a full operational protocol for end-users. A simple and versatile methodology is proposed for tailoring carbohydrate-functionalized nanoparticles (i.e. glyconanoparticles) with 3D polyvalent carbohydrate display and globular shapes. This methodology allows the preparation of glyconanoparticles with biologically significant carbohydrates. The glyconanoparticles can be used as efficient affinity labels as well as for in vivo cellular labeling and imaging.