Monitoring nanoparticle biology in vivo: unique opportunities in the avian system

描述（由申请人提供）：随着用于医疗保健的纳米颗粒（NP）开发的进展，需要体外和体内系统来测试其毒性和生物活性。本研究的目的是建立一种禽用NP毒性和生物活性检测系统。这种鸟类测试系统最独特的特点是重复进入一个复杂的皮肤组织，即生长羽毛（GF）的纸浆。GF的注射和收集都被认为是微创手术。在注射羽毛中发生的事件可以通过一次注射多个GF，然后在注射后的不同时间收集GF用于离体分析来监测。此外，血液取样沿着注射的GF的收集允许同时定性和定量评估在GF和血液循环中发生的事件。因此，该禽类测试系统构成了使用微创、非终末程序在同一个体中评估和监测体内NP毒性和生物活性的独特机会。在鸡中，GF的被鞘的活部分是大约一个确定的组织单位。8-10直径为2-3 mm。真皮样牙髓与表皮层相邻，含有GF的血管供应，并且是能够从循环中募集白细胞的免疫活性部位。此外，GF可以很容易地从毛囊中取出，这是一个比血液样本侵入性更小的过程，羽毛随后再生。因此，我们假设，GF可以作为一个“在体内试管”，适用于微创检查的生物活性的NP在体内。拟议的研究的广泛目标是证明鸟类系统的独特机会，监测和研究NP的生物活性和相互作用在体内，并建立鸡作为一个合适的和方便的中间动物模型NP的生物医学应用。具体地，在目的1中，将在体外确定NP和抗原缀合的NP（Ag-NP）与禽类细胞和可溶性免疫系统组分的毒性、活性和相互作用。在目的2中，将在GF和血液中评估和监测通过将NP和Ag-NP注射到GF中引发的生理、细胞和分子事件（例如炎症过程、补体激活、细胞因子产生、血管变化）。在目的3中，将在GF和血液中评估对肌内注射Ag-NP的原代和记忆细胞介导的（效应细胞和活性的概况）和体液（抗体的类型和水平）免疫应答的定性和定量方面。在目的4中，将通过静脉注射与Ag特异性抗体缀合的NP（Ab-NP）和将靶Ag注射到GF中来探索使用GF监测NP靶向递送的可能性。该项目将建立鸡作为一个合适的和方便的中间动物模型，以监测NP活动和影响，在体内使用微创程序。此外，拟议的研究将提供新的知识NP毒性，NP分子细胞界面相互作用，NP对先天性和适应性免疫反应的免疫调节作用，以及NP的靶向和交付。

DESCRIPTION (provided by applicant): With the advances in nanoparticle (NP) development for use in healthcare, in vitro and in vivo systems to test their toxicity and bioactivities are needed. The goal of the proposed research is to develop an avian test-system to monitor NP toxicity and bioactivities in vivo. The most unique feature of this avian test-system is repeat access to a complex dermal tissue, namely the pulp of growing feathers (GF). Both injection and collection of GF are considered minimally invasive procedures. Events taking place in injected feathers can be monitored by injecting multiple GF at one time and then collecting GF at various times post-injection for ex vivo analysis. Moreover, blood sampling along with collection of injected GF allows for simultaneous qualitative and quantitative assessment of events taking place in GF and in the blood circulation. Hence, this avian test- system constitutes a unique opportunity to assess and monitor in vivo NP toxicity and bioactivities in the same individual using minimally invasive, non-terminal procedures. In chickens, the ensheathed living portion of a GF is a define unit of tissue approx. 8-10 mm in height with a 2-3 mm diameter. The dermis-like pulp is bordered by an epidermal layer, contains the vascular supply of the GF and is an immunologically active site capable of recruitment of leukocytes from the circulation. Moreover, GF can easily be removed by pulling it from its follicle, a procedure that is less invasive than taking a blood sample, and the feather subsequently regenerates. Hence, we hypothesized that GF can serve as an "in vivo test-tube" suitable for minimally invasive examination of biological activities of NP in vivo. The broad objective of the proposed research is to demonstrate the unique opportunities of the avian system to monitor and study NP bioactivities and interactions in vivo and to establish the chicken as a suitable and convenient intermediate animal model for biomedical application of NP. Specifically, in Aim 1, toxicity, activities and interactions of NP ad antigen-conjugated NP (Ag-NP) with avian cellular and soluble immune system components will be determined in vitro. In Aim 2, the physiological, cellular and molecular events (e.g. inflammatory processes, complement activation, cytokines production, vascular changes) initiated by injection of NP and Ag-NP into GF will be assessed and monitored in GF and blood. In Aim 3, qualitative and quantitative aspects of primary and memory cell-mediated (profiles of effector cells and activities) and humoral (type and levels of antibodies) immune responses to intramuscularly injected Ag-NP will be assessed in GF and blood. In Aim 4, the possibility of using GF to monitor NP-targeted delivery will be explored by intravenous injection of NP-conjugated with Ag-specific antibody (Ab-NP) and injection of target Ag into GF. The proposed project will establish the chicken as a suitable and convenient intermediate animal model to monitor NP activities and effects in vivo using minimally invasive procedures. In addition, the proposed research will provide new knowledge on NP toxicity, NP molecular cellular interface interactions, NP immunomodulatory effects on innate and adaptive immune responses, and on NP targeting and delivery.