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

描述（由申请人提供）：随着纳米颗粒（NP）在医疗保健领域的发展，需要在体外和体内系统中测试其毒性和生物活性。本研究的目的是开发一种鸟类测试系统来监测NP的体内毒性和生物活性。这种鸟类测试系统最独特的特点是反复接触复杂的真皮组织，即生长中的羽毛（GF）。GF的注射和采集都被认为是微创手术。通过一次注射多个GF，然后在注射后的不同时间收集GF进行离体分析，可以监测注射羽毛中发生的事件。此外，血液采样和注射GF的收集可以同时对GF和血液循环中发生的事件进行定性和定量评估。因此，这种鸟类试验系统构成了一个独特的机会，可以使用微创、非终末程序来评估和监测同一个体体内NP毒性和生物活性。在鸡中，GF的被包裹的活的部分大约是一个确定的组织单位。高度8 ~ 10mm，外径2 ~ 3mm。真皮样髓被表皮层包围，含有GF的血管供应，是一个免疫活性部位，能够从循环中招募白细胞。此外，GF可以很容易地从毛囊中取出，这一过程比采集血液样本的侵入性要小，羽毛随后会再生。因此，我们假设GF可以作为适合于微创检测NP在体内生物活性的“体内试管”。该研究的主要目的是证明鸟类系统在体内监测和研究NP生物活性和相互作用的独特机会，并建立鸡作为NP生物医学应用的合适和方便的中间动物模型。具体来说，在Aim 1中，将在体外确定NP和抗原偶联NP （Ag-NP）与禽类细胞和可溶性免疫系统成分的毒性、活性和相互作用。在Aim 2中，将评估和监测GF和血液中注射NP和Ag-NP引发的生理、细胞和分子事件（如炎症过程、补体激活、细胞因子产生、血管变化）。在目标3中，将在GF和血液中评估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）对肌内注射Ag-NP的免疫反应的定性和定量方面。在Aim 4中，将通过静脉注射与Ag特异性抗体（Ab-NP）结合的np，并将目标Ag注射到GF中，探索利用GF监测np靶向递送的可能性。该项目将建立鸡作为一种合适且方便的中间动物模型，利用微创方法监测NP在体内的活性和作用。此外，所提出的研究将为NP毒性、NP分子细胞界面相互作用、NP对先天和适应性免疫反应的免疫调节作用以及NP靶向和递送提供新的知识。

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.