A Proof-of-concept Investigation Of Enzyme Responsive System For Therapeutic Proteins Intracellular Targeting Delivery

Objective:Protein therapeutics have emerged as a significant role in treatment of a broad spectrum of diseases,however,the efficient delivery of biopharmaceutical proteins into the cytosol of target cells poses substantial challenges owing to their large size and susceptibility to degradation.In order to overcome the barrier,a matrix metalloproteinase?MMP?-responsive,CPP-mediated intracellular delivery system was developed.With the help of CPPs which have higher intracellular delivery and loading capacity and MMP-responsive cleavable sequence,the system constructed here could enhance tumor targeting ability and internalization efficiency.Content:Fusion protein and NiFe₂O₄ magnetic nanoparticles were self-assembled,via the automatic chelating effect between the nickel ions and hexahistidine tags,into the final delivery system.Herein,both in vitro and in vivo“proof-of-concept”investigations were carried out to confirm the feasibility and success of this innovative tumor targeting system.Methods:1.Fabrication and characterization of the delivery system.Fusion proteins were produced via a recombinant technology and NiFe₂O₄ magnetic nanoparticles were synthesized by hydrothermal process.NiFe₂O₄ magnetic nanoparticles were characterized by X-ray diffraction?XRD?,energy dispersive X-ray spectrometry?EDX?,transmission electron microscope?TEM?,dynamic light scattering?DLS?.The binding capacity and specific binding affinity between His-tagged protein and NiFe₂O₄-based magnetic nanoparticles were investigated,then fluorescence quenching of mCherry induced by NiFe₂O₄ magnetic nanoparticles was evaluated.2.Both in vitro and in vivo“proof-of-concept”investigations of the feasibility of designed system.1)Enzyme assay.The delivery system was incubated with recombinant human MMP-2,and the fluorescence intensity of the supernatant was recorded.2)Cell assay.In vitro activation of the delivery system by MMP-2 was evaluated by cell imaging.3)Optical in vivo imaging.The delivery/control system were administered to tumor-xenograft mice via intratumoral and tail vein injection,and the fluorescence of tumors were analyzed.3.Biosafety study of the delivery system.The cytotoxicity was studied in nontumoral cell lines of normal human umbilical vein endothelial cells?HUVEC?.The biocompatibility in vivo was further performed on KM mice which were injected with delivery system once a week,and the body weight changes and the organ coefficients were recorded,and the major organs were processed by histological examination.Results:1.Fabrication and characterization of the delivery system.SDS-PAGE assay show that fusion proteins owned a calculated molecular weight of 32 kDa.Western Blot assay confirmed the His-tag at the C-terminus of fusion proteins.XRD and EDX spectrum results confirmed that the synthesis of NiFe₂O₄ magnetic nanoparticles were successful,the hydrodynamic diameter was about 131.2±0.9 nm and average size was about 80.3±3.0 nm according to TEM.The magnetic hysteresis loop yielded a relatively high magnetic saturation value?47.51 emu/g?and superparamagnetic behavior.Binding specificity of the His-tagged protein on NiFe₂O₄ magnetic nanoparticles was proved.Finally,the optimal concentration ratio of mCherry/NiFe₂O₄ for binding was 1.0:8.3.2.Both in vitro and in vivo“proof-of-concept”investigations of the feasibility of designed system.Enzyme assay and cell assay indicated that the fabricated system could be activated by human recombinant MMP-2 and cell secreted MMP-2,respectively.In vivo study confirmed the feasibility of our designed delivery system for both magnetic targeting and tumor-activated intracellular delivery.3.Biosafety study of the delivery system.The biocompatibility of the nanoprobe was examined with the nontumoral HUVEC cell line in vitro.The results indicated that the fabricated delivery system exhibited no significant inhibitory effect on the cell growth,and the cell viability at 200?g/mL was all above 70%.Compared with control,the delivery system had no significant effect on body weight and organs of KM mice.All these results confirmed the good biocompatibility of the fabricated delivery system for application in vivo.Conclusion:An innovative,magnetic targeted cell-permeable,tumor-activatable and revivable delivery system was developed.Both in vitro and in vivo results indicated that our tumor-activatable and CPP-mediated cell-entry system deems to be an appealing platform to achieve selective intracellular protein delivery.