Although therapeutically active proteins are highly efficacious, their content in protective nanoparticles is often too low to elicit adequate plasma levels. A strategy to increase protein loading is the in-situ generation of calcium phosphate as a protein adsorbent. To verify this approach, a highly sensitive and reliable fluorimetric method for quantification of incorporated fluorescein-labelled bovine serum albumin (FITC-BSA) as a model protein drug was developed. Dequenching the fluorescein label by pronase E, which digests the protein backbone, and dissolving the nanoparticle matrix in acetonitrile enabled FITC-BSA quantification in the nanogram per milliliter range. This test was confirmed by a second assay involving alkaline hydrolysis of FITC-BSA and the matrix. Nanoparticles prepared with calcium phosphate contained 40 µg FITC-BSA/mg and nanoparticles without calcium phosphate only 15 µg FITC-BSA/mg, representing a 2.7-fold increase in model protein loading. In this work the nanoparticle preparation procedure was optimized in terms of size for administration in the inner ear, but the range of applications is not limited.

尽管治疗活性蛋白质非常有效，但它们在保护性纳米颗粒中的含量通常太低而无法引起足够的血浆水平。增加蛋白质负载的一种策略是原位生成磷酸钙作为蛋白质吸附剂。为了验证这种方法，开发了一种高灵敏度和可靠的荧光法，用于定量掺入的荧光素标记的牛血清白蛋白 (FITC-BSA) 作为模型蛋白药物。通过链霉蛋白酶 E 对荧光素标记进行去淬灭，蛋白酶 E 可消化蛋白质骨架，并将纳米颗粒基质溶解在乙腈中，使得 FITC-BSA 的定量达到纳克/毫升范围。该测试通过涉及 FITC-BSA 和基质的碱性水解的第二次测定得到证实。用磷酸钙制备的纳米颗粒含有 40 µg FITC-BSA/mg，而不含磷酸钙的纳米颗粒仅为 15 µg FITC-BSA/mg，代表模型蛋白载量增加了 2.7 倍。在这项工作中，纳米颗粒制备程序在内耳给药的尺寸方面进行了优化，但应用范围不受限制。