Passivating Proteins in Implantable Glucose Sensors

描述(申请人提供)：血糖控制不当会导致糖尿病并发症(肾脏、视网膜、神经)。植入皮下的葡萄糖传感器可以提供连续的血糖数据，帮助预防这些并发症，并对低血糖症进行早期预警，但目前设备的使用寿命受到异物包裹的限制。这项工作的目标是开发涂层，以减少植入的葡萄糖传感器的异物反应，从而延长其使用寿命。具体地说，提出了减少单核/巨噬细胞黏附的蛋白质涂层的研究。纤维蛋白原及其巨噬细胞结合区的作用也将被探索。这些涂层的影响将在对植入大鼠皮下的葡萄糖传感器的长期研究中得到解决。 1.将创建钝化蛋白质涂层，其特征如下： A.将使用碘-125放射性标记蛋白质来测量缓冲溶液中的四种蛋白质在聚氨基甲酸酯涂层表面的吸附程度。这些蛋白质将包括von Willebrand因子、高分子激肽原、白蛋白和血红蛋白。蛋白质浓度将改变，以确定每种蛋白质的等温线，以便建立达到饱和或单层吸附所需的浓度。 B.对于每种蛋白质，将测量其对血浆置换的抵抗力。将评估减少排量的方法，包括吸附时间和吸附后停留时间的变化。 C.发现的吸附条件将根据它们在体外抑制巨噬细胞黏附和异物巨细胞形成的能力进行评估，以获得实质上抗置换的涂层。 D.葡萄糖传感器与蛋白质预吸附，在此条件下，血浆置换最少，对单核细胞黏附和异物巨细胞[FBGC]形成的抑制最大，将被送往Legacy，在那里将对它们进行评估，以评估其在体内的有用寿命。 2.将创建钝化抗体涂层，其特征如下： A.表面的聚氨酯将与纤维蛋白原预吸附，并用与纤维蛋白原的巨噬细胞结合区结合的单抗处理。 将评估戊二醛进一步稳定纤维蛋白原/抗体处理表面的能力。 C.将评估抗体对暴露在血浆中置换的抵抗力。 将评估抗体阻断在减少单核细胞黏附和FBGC形成方面的效果。 预先吸附了纤维蛋白原和抗体的葡萄糖传感器，无论有没有戊二醛，都将被准备用于传感器功能的慢性体内评估。 3.纤维蛋白原吸附在异物反应中的作用研究如下： A.在导致纤维蛋白原吸附变化的条件下，将在传感器上形成一系列气体等离子体沉积的聚氧化乙烯类涂层。 B.体外吞噬细胞在这些表面的摄取变化，并将被测量。 C.高纤维蛋白原和低纤维蛋白原的传感器表面，包括一些用抗体和戊二醛钝化的表面，将在Legacy动物实验室进行评估。 4.体外蛋白质吸附后，将评估葡萄糖传感器的功能，以评估涂层引起的任何变化。 5.传感器将在正常血糖和高血糖期间进行体内系列评估，以评估钝化蛋白质和抗体阻断的纤维蛋白原涂层对滞后时间、灵敏度、稳定性和可用的传感器寿命的影响。 6.由于未来的糖尿病用户希望最大限度地减少毛细血管血液校准，因此将对涂层传感器进行评估，以确定每周仅执行一次校准是否会导致足够的传感器准确度。 7.外植体移植后，为了更好地了解钝化蛋白置换或抗体阻断的纤维蛋白原的影响，将测量残留的放射性标记蛋白的百分比并与体内功能相关联。 8.组织学评价钝化蛋白和抗体封闭的纤维蛋白原对异物包膜性质的影响。

DESCRIPTION (provided by applicant): Inadequate control of blood glucose contributes to the complications of diabetes (renal, retinal, neural). A glucose sensor implanted subcutaneously could provide continuous glucose data, help prevent these complications, and give early warning for hypoglycemia, but the useful life of current devices is limited by foreign body encapsulation. The goal of this work is to develop coatings that will reduce the foreign body reaction to implanted glucose sensors, thereby extending their useful life. Specifically, studies of protein coatings that will reduce monocyte/macrophage adhesion are proposed. The role of fibrinogen and its macrophage-binding region will also be explored. The effect of these coatings will be addressed during chronic studies of glucose sensors implanted subcutaneously in rats. 1. Passivating protein coatings will be created and characterized as follows: a. The degree to which four proteins in buffered solutions will adsorb to polyurethane-coated surfaces will be measured using Iodine-125 radiolabeled proteins. The proteins will include von Willebrand factor, high molecular weight kininogen, albumin, and hemoglobin. Protein concentration will be varied to determine the isotherms for each protein, in order to establish concentrations needed to attain saturation or monolayer adsorption. b. For each protein, the resistance to displacement by blood plasma will be measured. Methods to reduce displacement will be evaluated, including variations in adsorption time and postadsorptive residence time. c. Adsorption conditions found to give coatings that are substantially resistant to displacement will be evaluated in regard to their ability to inhibit macrophage adhesion and foreign body giant cell formation in vitro. d. Glucose sensors preadsorbed with protein under conditions resulting in the least displacement by plasma and the greatest inhibition of monocyte adhesion and foreign body giant cell [FBGC] formation will be sent to Legacy where they will be evaluated to assess useful in vivo lifetime. 2. Passivating antibody coatings will be created and characterized as follows: a. The surface polyurethane will be preadsorbed with flbrinogen and treated with a monoclonal antibody that binds to fibrinogen's macrophage binding region. b. The ability of glutaraldehyde to further stabilize the fibrinogen/antibody-treated surfaces will be assessed. c. The resistance to displacement of the antibody by exposure to plasma will be evaluated. d. The efficacy of antibody blockade to reduce monocyte adhesion and FBGC formation will be evaluated. e. Glucose sensors preadsorbed with fibrinogen and antibody, with or without glutaraldehyde, will be prepared for chronic in vivo evaluation of sensor function. 3. The role of fibrinogen adsorption in the foreign body reaction will be studied as follows: a. A series of gas plasma-deposited polyethylene oxide-like coatings on the sensor will be created under conditions that cause variations in their fibrinogen adsorption. b. The variation of in vitro phagocyte uptake on these surfaces and will be measured. c. Sensor surfaces with high and with low amounts of fibrinogen, including some passivated with antibodies and glutaraldehyde, will be prepared for evaluation in the Legacy animal laboratory. 4. Glucose sensor function will be evaluated after protein adsorption in vitro to assess any coating-induced change. 5. Sensors will be serially evaluated in vivo during normoglycemia and hyperglycemia in order to assess the effect of passivating proteins and antibody-blocked fibrinogen coatings on lag time, sensitivity, stability, and useful sensor life. 6. Because future users with diabetes will wish to minimize capillary blood calibrations, there will be an assessment in coated sensors as to whether calibrations performed only once per week will lead to sufficient sensor accuracy. 7. After explantation, percent of remaining radiolabeled protein will be measured and correlated with in vivo function in order to better understand the effects of displacement of passivating proteins or antibody-blocked fibrinogen. 8. A histologic evaluation will be performed to assess the effect of passivating proteins and antibody blocked fibrinogen on the nature of the foreign body capsule.