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Developing genetically engineered encapsulin protein cage nanoparticles as a targeted delivery nanoplatform.

Protein cage nanoparticles are excellent candidates for use as multifunctional delivery nanoplatforms because they are built from biomaterials and have a well-defined structure. A novel protein cage nanoparticle, encapsulin, isolated from thermophilic bacteria Thermotoga maritima, is prepared and developed as a versatile template for targeted delivery nanoplatforms through both chemical and genetic engineering. It is pivotal for multifunctional delivery nanoplatforms to have functional plasticity and versatility to acquire targeting ligands, diagnostic probes, and drugs simultaneously. Encapsulin is genetically engineered to have unusual heat stability and to acquire multiple functionalities in a precisely controlled manner. Hepatocellular carcinoma (HCC) cell binding peptide (SP94-peptide, SFSIIHTPILPL) is chosen as a targeting ligand and displayed on the surface of engineered encapsulin (Encap_loophis42C123) through either chemical conjugation or genetic insertion. The effective and selective targeted delivery of SP94-peptide displaying encapsulin (SP94-Encap_loophis42C123) to HepG2 cells is confirmed by fluorescent microscopy imaging. Aldoxorubicin (AlDox), an anticancer prodrug, is chemically loaded to SP94-Encap_loophis42C123 via thiol-maleimide Michael-type addition, and the efficacy of the delivered drugs is evaluated with a cell viability assay. SP94-Encap_loophis42C123-AlDox shows comparable killing efficacy with that of free drugs without the platform's own cytotoxicity. Functional plasticity and versatility of the engineered encapsulin allow us to introduce targeting ligands, diagnostic probes, and therapeutic reagents simultaneously, providing opportunities to develop multifunctional delivery nanoplatforms.