Abstract Papain-based nanoparticles were recently developed using radiation technologies and proven effective to generate nanosized crosslinked papain particles with preserved enzymatic activity. The applications of such nanostructured systems are expected to be similar to native papain with considerable biopharmaceutical advantages and concern drug loading among other biotechnological applications. The nature of such crosslinks and the possibility to provide simultaneous sterilization have been hypothesized but remain not totally clarified. This manuscript advances the discussion on the radiation-induced synthesis of protein nanoparticles by approaching the nature of the crosslinking and the possible contribution of bityrosine linkages and disulfide bridges to the overall nanoparticle assembly as well as the feasibility of the simultaneous sterilization process under the pre-established conditions of processing. Papain nanoparticles were synthesized and characterized according to size, proteolytic activity, bityrosine, cysteine content and molecular weight by SDS-PAGE upon sonication at 40 kHz. Bacterial identification and the sterility tests were performed in accordance with ISO 11737 prior to and after inoculating 106 CFU of Corynebacterium xerosis. Our experiments evidenced the crosslinking of rather intra- than intermolecular nature and a contribution of cysteine bridges and bityrosine linkages to the stabilization and formation of the papain nanoparticle assembly. The technique was effective to promote simultaneous crosslinking and sterilization at the established conditions of processing and may be validated in accordance with the ISO 11137.

中文翻译：

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通过伽马辐射同时分子内交联和灭菌木瓜蛋白酶纳米粒子

摘要 最近使用辐射技术开发了基于木瓜蛋白酶的纳米颗粒，并证明可有效生成具有保留酶活性的纳米级交联木瓜蛋白酶颗粒。这种纳米结构系统的应用预计类似于天然木瓜蛋白酶，具有相当大的生物制药优势，并在其他生物技术应用中关注药物负载。这种交联的性质和提供同时灭菌的可能性已被假设，但仍未完全阐明。这份手稿通过接近交联的性质和二酪氨酸键和二硫键对整个纳米粒子组装的可能贡献以及在预制下同步灭菌过程的可行性，推进了对蛋白质纳米粒子的辐射诱导合成的讨论。既定的加工条件。木瓜蛋白酶纳米颗粒被合成并根据大小、蛋白水解活性、二酪氨酸、半胱氨酸含量和分子量通过 SDS-PAGE 在 40 kHz 超声下进行表征。在接种 106 CFU 干燥棒状杆菌之前和之后，根据 ISO 11737 进行细菌鉴定和无菌测试。我们的实验证明了交联而不是分子间性质，以及半胱氨酸桥和二酪氨酸键对木瓜蛋白酶纳米颗粒组装的稳定和形成的贡献。该技术可有效促进在既定加工条件下同时进行交联和灭菌，并可根据 ISO 11137 进行验证。