Design,Preparation And Performance Evaluation Of Genetically Engineered Biomimetic Virus-based Drug Formulations

In recent years,various pharmaceutical dosage forms have developed for improving the therapeutics(e.g.,sugar,nucleic acid,protein and other functional moieties)efficacy,including enhancing their solubility,improving the stability and prolonging their half-life in the blood circulation.Inspired from the nature belongings,especially the functional characteristics of the virus itself,the intelligent pharmaceutical formulations that imitating the virus have shown outstanding advantages.Till now,they have successfully applied in various areas like biosensing,medical scaffold,vaccine preparation,and personalized treatment for a variety of diseases.From the perspective of medicine,biomimetic virus-based pharmaceutics can efficaciously bridge their own functional characteristics of virus and functional drug molecules.In this thesis,based on the comprehensive conclusion of the drug formulations that can mimic the effect of the virus,including their preparation procedure,the modification tactics,application scope,advantages and existing problems,we will evaluate the complexities,potential pitfalls,and opportunities by these strategies in future disease theranostics.To broden their applications,both the virus like particles(VLPs)of ferritin and cellular membrane vesicles were leveraged as engineering objects,and we prepared the multifunctional formulations via genetic engineering tactics.Then,not only the mimovirus vesicles that can mimic the viral transmembrane behavior but the bifunctional ferritin(RC-Fn)that has similar virus morphology have prepared,and their applications in tumor diagnosis and enhancing cartilage regeneration have studied.The main content of this paper is summarized as follows:(1)One kind of tumor microenvironment(TME)-responsive mimovirus vesicle(MVVs)is engineered to deliver azide motifs(-N3)via the membrane fusion mechanism for cancer diagnosis.As a pH-responsive functional protein,the spike vesicular stomatitis virus G-protein(VSVG)is genetically immobilized on surface of cell membrane vesicles.By virtue of the low-pH activated fusogenic peculiarity of VSVG,the azide motifs(-N3)which also presented on MVVs via metabolic engineering,can be directly anchored onto the target cells,thus reducing tumor heterogeneity and serving as the bait to amplify the tumor targeting capacity of dibenzocyclooctyne(DBCO)-modified diagnostic moieties.The potential diagnostic capability of such design is successfully confirmed in three different murine tumor models.Featuring excellent pH-sensitive fusogenic traits,the developed MVVs show great responsiveness to the slightly acidic TME of solid tumors,providing a universal platform in overcoming tumor heterogeneity for precise cancer diagnosis.(2)Aside from the genetically engineered cellular membrane vesicles for preparing virus-mimetic pharmaceutics,the ferrtin which has similar virus morphology was leveraged either.Compared with the mimovirus vesicle,the ferrtin has simple composition and controble size(about 12nm)and these characteristics are helpful for the treatment of some special diseases.For example,the dense extracellular matrix component in cartilage places higher demands on drug carriers when damage occurs because the cartilage proteoglycan network has a mesh size of≈60 nm and a side chain spacing of ≈20 nm.Based on this,a genetically engineered virus like ferritin nanocage(RC-Fn)with RGD4C peptide and WYRGRL peptide on the surface was synthesized.The RGD4C can target the integrin αvβ3 of bone marrow mesenchymal stem cells(BMSCs)and promote the proliferation,and the WYRGRL peptide has an inherent affinity for the cartilage matrix component of collagen II protein.Benefiting from and ideal size of RC-Fn nanocages for penetrating the proteoglycan network of cartilage,RC-Fn can directly enhance the enrichment of BMSCs in articular cartilage.Thus,intra-articularly injected RC-Fn with Kartogenin(KGN)loading could convert the articular cavity from a barrier into a reservoir to prevent rapid release and clearance of KGN and exogenous BMSCs,which results in efficient and persistent chondrogenesis in cartilage regeneration.In conclusion,we successfully prepared two kinds of genetically engineered biomimetic vdirus-based drug formulations:mimovirus membrane vesicles with virus behavior and viral-like ferritin with deep penetration ability in articular cartilage.Apart from the same morphology and structure characteristics like virus,these two kinds of formulations possese different particle size(about 150nm of mimovirus membrane vesicles and 12nm of ferrtin)and response characteristics in acidic environment.Benefiting from these characteristics,the mimovirus membrane vesicles can be applied in various tumors diagnosis to overcome tumor heterogeneity and the ferritin can increase the articular cartilage permeability to promote the repair of articular cartilage defects.