A Carrier for Non-Covalent Delivery of Functional Beta-Galactosidase and Antibodies against Amyloid Plaques and IgM to the Brain

BackgroundTherapeutic intervention of numerous brain-associated disorders currently remains unrealized due to serious limitations imposed by the blood-brain-barrier (BBB). The BBB generally allows transport of small molecules, typically <600 daltons with high octanol/water partition coefficients, but denies passage to most larger molecules. However, some receptors present on the BBB allow passage of cognate proteins to the brain. Utilizing such receptor-ligand systems, several investigators have developed methods for delivering proteins to the brain, a critical requirement of which involves covalent linking of the target protein to a carrier entity. Such covalent modifications involve extensive preparative and post-preparative chemistry that poses daunting limitations in the context of delivery to any organ. Here, we report creation of a 36-amino acid peptide transporter, which can transport a protein to the brain after routine intravenous injection of the transporter-protein mixture. No covalent linkage of the protein with the transporter is necessary.
ApproachA peptide transporter comprising sixteen lysine residues and 20 amino acids corresponding to the LDLR-binding domain of apolipoprotein E (ApoE) was synthesized. Transport of beta-galactosidase, IgG, IgM, and antibodies against amyloid plques to the brain upon iv injection of the protein-transporter mixture was evaluated through staining for enzyme activity or micro single photon emission tomography (micro-SPECT) or immunostaining. Effect of the transporter on the integrity of the BBB was also investigated.
Principal FindingsThe transporter enabled delivery to the mouse brain of functional beta-galactosidase, human IgG and IgM, and two antibodies that labeled brain-associated amyloid beta plaques in a mouse model of Alzheimer's disease.
SignificanceThe results suggest the transporter is able to transport most or all proteins to the brain without the need for chemically linking the transporter to a protein. Thus, the approach offers an avenue for rapid clinical evaluation of numerous candidate drugs against neurological diseases including cancer. (299 words).

背景 目前，由于血脑屏障（blood-brain-barrier, BBB）带来的严重限制，众多脑部相关疾病的治疗干预仍难以实现。血脑屏障通常仅允许分子量小于600道尔顿、且正辛醇/水分配系数较高的小分子物质通过，而阻断大多数大分子的转运。不过，血脑屏障上存在的部分受体可介导同源蛋白进入脑部。借助这类受体-配体系统，已有多名研究者开发出向脑部递送蛋白的方法，其中一项核心要求是将靶蛋白与载体实体进行共价连接。但此类共价修饰需要复杂的制备及制备后化学操作，这在向任何器官递送药物的场景中都带来了极大限制。本研究构建了一种含36个氨基酸的肽转运体，将转运体与蛋白混合后进行常规静脉注射，即可将蛋白递送进入脑部，且无需将蛋白与转运体进行共价连接。
方法 本研究合成了一种肽转运体，其包含16个赖氨酸残基，以及对应载脂蛋白E（apolipoprotein E, ApoE）的低密度脂蛋白受体（low-density lipoprotein receptor, LDLR）结合域的20个氨基酸。通过酶活性染色、微型单光子发射计算机断层扫描（micro single photon emission tomography, micro-SPECT）或免疫染色，评估了将蛋白-转运体混合物静脉注射后，β-半乳糖苷酶、免疫球蛋白G（immunoglobulin G, IgG）、免疫球蛋白M（immunoglobulin M, IgM）以及抗淀粉样β斑块抗体向脑部的转运情况。此外，本研究还探究了该转运体对血脑屏障完整性的影响。
主要发现 该转运体可将功能性β-半乳糖苷酶、人源IgG与IgM，以及两种可标记阿尔茨海默病小鼠模型中脑部相关淀粉样β斑块的抗体递送至小鼠脑部。
意义 本研究结果表明，该转运体可将大多数乃至全部蛋白递送至脑部，且无需将转运体与蛋白进行化学连接。因此，该方法为快速开展针对包括癌症在内的多种神经系统疾病的候选药物临床评估提供了新途径。（全文共299词）