More recently, it was used for the delivery of cisplatin, but without the use of an encapsulation protocol (20). Encapsulation of molecules such as cisplatin (12,13), carboplatin (14), oxaliplatin (15), ruthenium (16,17), and gold (18,19) has been achieved using various ferritin cage disassembly or reassembly procedures. Below pH 3, the ferritin assembly may then be dissociated and reconstituted (10), enabling drugs to be trapped inside the protein “cage” (11). While, in nature, the ferritin cavity is used to store and enucleate iron, it can also be repurposed for the encapsulation of drugs or dyes (9). Ferritin is made up of 24 subunits held together by non-covalent interactions, arranged in an icosahedral cage with 2, 3, and 4-fold axes of symmetry around a central cavity (7,8). Ferritin family proteins, found ubiquitously in nature, possess many attractive features for the delivery of biotherapeutics (3–6). There is a growing trend within the field of biotherapeutics to develop molecules with a high degree of valency (1,2). The combined results of this study confirmed that Ferritin–Fab conjugates were successfully generated. Following optimization of the conjugation strategy using LC–MS, in-depth characterization was performed using SEC-MALS-QELS. Here, we describe the design, production, and characterization of a multimeric ferritin–antibody fragment conjugate. The utility of molecular cages, such as ferritin and its derivatives for applications in drug delivery, is well-known. In mammals, ferritins are composed of 24 subunits that form an icosahedron with an external hydrodynamic radius of 6 nm, and an overall molar mass of approximately 474 kDa, depending on the biological tissue from which it is derived. Multivalent, self-assembling scaffolds such as ferritin, a ubiquitous protein found in most human cell types in addition to invertebrates, higher plants, fungi and bacteria, offer an attractive “natural” alternative to polymer-based scaffolds. Although this is traditionally accomplished using polymer-based molecular scaffolds, biocompatibility and the fate of polymer by-products are often of concern. The design of molecules with a high degree of valency can be useful for receptor clustering, T-cell recruiting, agonist activation, and half-life extension.
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