Cryptococcus neoformans (Cn) is the leading cause of fungal meningitis primarily in immunosuppressed patients. Cn invades the central nervous system by overcoming the highly restricted blood-brain barrier (BBB). We previously determined that a secreted fungal metalloprotease, Mpr1, that also confers crossing ability to yeast upon CnMPR1 expression in Saccharomyces cerevisiae is central to this process. This led us to question whether Mpr1 could be engineered to function as part of a nanocarrier delivery vehicle. Here, a eukaryotic expression system produced proteolytically active Mpr1 recombinant protein that was successfully conjugated to functionalized quantum dot (QD) nanoparticles and readily internalized by brain microvascular endothelial cells. An in vitro BBB model showed QD-Mpr1 crossed the BBB significantly better than mock QD, and QD-Mpr1 did not damage BBB integrity. Internalization of QD-Mpr1 occurred by membrane invaginations and endocytic pits typical of receptor-mediated endocytosis involving clathrin-coated entry points. This study substantiates the notion that fungal mechanisms of BBB entry may be harnessed for new drug delivery platform technologies.

中文翻译：

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利用真菌金属蛋白酶Mpr1的活性，以促进通过血脑屏障的纳米载体的交叉。

新型隐球菌（Cn）是真菌性脑膜炎的主要原因，主要在免疫抑制的患者中发生。Cn通过克服高度受限的血脑屏障（BBB）侵入中枢神经系统。我们以前确定，一种分泌的真菌金属蛋白酶Mpr1，在酿酒酵母中表达CnMPR1后，还可以赋予酵母交叉能力，这一过程至关重要。这使我们提出疑问，是否可以将Mpr1设计成可作为纳米载体运载工具的一部分发挥作用。在这里，一个真核表达系统产生了蛋白水解活性的Mpr1重组蛋白，该蛋白成功地偶联到功能化量子点（QD）纳米颗粒上，并易于被脑微血管内皮细胞内化。体外BBB模型显示QD-Mpr1穿越BBB的效果明显好于模拟QD，QD-Mpr1不会破坏BBB的完整性。QD-Mpr1的内在化是由膜介导和胞吞凹坑发生的，这种膜吞吐和胞吞凹坑是典型的受体介导的内吞作用，涉及网格蛋白涂层的进入点。这项研究证实了BBB进入的真菌机制可用于新的药物递送平台技术的观点。