An Investigation Of Enzyme Specifically Responsive System For Therapeutic Proteins Intracellular Targeting Delivery

Objective:Therapeutic proteins play an important role in the cancer therapy.However,the development of protein-based cancer therapy was hindered by the poor cytoplasmic delivery of therapeutic proteins.Cell penetrating peptides（CPP）with good membrane penetration and carrying capacity provide an effective way to improve the intracellular delivery efficiency of of these proteins.To overcome the limitation of poor selectivity of CPP cells,MMP responsive,activatable cell penetrating peptides（ACPPs）ACPPs mediated intracellular therapeutic proteins delivery systems were developed hererin by taking advantage of the close correlation between matrix metalloproteinases（MMPs）and tumors.In these systems,the function of CPP activated only when the systems reached the tumor site.After being activated by the tumor associated protease MMP,the exposed CPP then mediated a potent intracellular delivery of the therapeutic protein.However,overlapping substrate specificities within the family of MMP enzymes become problematic due to their highly conserved structural topology.It results in a potential issue for a substrate to be cleaved by multiple enzymes within this family leading to the decreased targeting efficiency of these MMP responsive delivery systems.Therefore,different strategies were developed in this study to improve the specificity of MMP responsive ACPP-mediated protein drug delivery system according to the characteristics of different MMPs,aiming to improve the targeting efficiency of these therapeutic prteins delivery systemss.Content:In this study,two MMPs responsive,ACPP-mediated intracellular targeting delivery systems for therapeutic proteins were constructed.Both in vivo and in vitro investigations were carried out to confirm the feasibility and success of this innovative tumor targeting delivery system.Method:System I:This system was an MMP-2 responsive ACPP-mediated intracellular targeting delivery system for therapeutic proteins.The system is composed of two parts:the protein part is m Cherry,CPP,MMP-2 cleavage site and His-tag fusion product,the other part is the Ni Fe₂O₄nanoparticles.These two parts were self-assembled via a chelation mechanism between the His-tag of the protein part and the Ni²⁺of the Ni Fe₂O₄nanoparticles.The MMP-2 cleavage site was deeply hided inside the system due to the structure characteristics of fusion protein and the Ni Fe₂O₄nanoparticles,which resaulted in a strong steric hindrance between the system and MMPs,especially for membrane-tethered MMPs such as MT1-MMP. The fusion protein was produced by a recombinant technology,and its molecular weight and purity were characterized by SDS-PAGE.The morphology of magnetic nanoparticles was characterized by TEM.The fluorescence quenching efficiency of fusion proteins decreased by magnetic nanoparticles was investigated via fluorescence titration,which contributed to the determination of the assembly mass ratio of fusion proteins to magnetic nanoparticles.The morphology of the assembled probe system was investigated by TEM,too.The activation efficiency of the system by MMP-2 was investigated by enzyme kinetics via recombinant human MMP-2.The enhanced selectivity to tumor cell secreted MMP-2 and tumor targeting of the system were evaluated by cell imaging.To confirm the strong steric hindrance toward membrane-type MMPs,two strategies were applied.First,a truncated human MT1-MMP without transmembrane structure was expressed to evaluate the recoverable activation efficiency of the system.Second,A FRET probe with the same MMP-2 cleavage site and reduced steric hindrances effect was designed.The recoverable activation efficiency of probe to membrane-tethered MT1-MMP on HT-1080 was detected via cell-based enzyme assay. System II:This system was a MT1-MMP specifically responsive ACPP-mediated intracellular targeting delivery system for therapeutic proteins,which taking advantage of the high affinity between MT1-MMP and its specific binding peptide.Firstly,in order to enhance the specificity of the enzyme responsive module in this system,PEG Linkers with different length were introduced between the MT1-MMP substrate and binding peptide.The stimulus-response specificity was of the enzyme responsive module was examined both in vitro and in vivo via enzyme kinetics study,cell imaging and animal models.Then a deliery system,which was consist of therapeutic proteins,CPP and the optimized enzyme response module,was constructed via recombinant expression.The fluorescent protein m Cherry was used as the drug model to investigate the feasibility of the system via cell imaging at first.After that,the fluorescent protein was replaced with a therapeutic protein trichosanthin（TCS）,its antitumor activity on ovarian cells was investigated via MTT experiments.Result:System I:The expression of the fluorescent fusion protein was validated by SDS-PAGE indicating a molecular weight of about 32 k Da,which is consistent with its theoretical molecular weight.TEM results showed that the vertical diameter of the Ni Fe₂O₄magnetic nanoparticles was about 96.8±7.3 nm.The results of fluorescence quenching experiments indicated that the optimal assembly ratio of fusion protein to Ni Fe₂O₄magnetic nanoparticles was 1.0:8.3（fusion protein/Ni Fe₂O₄magnetic nanoparticles,W/W）,and the diameter of the assembled probe system was about105.6±17.5 nm,slightly larger than nanoparticles.Enzyme assay and cell-based assay claimed that the probe system could only be activated by MMP-2,but not the membrane-tethered MMP-14. System II:Enzyme kinetics analysis showed that,the combination of MT1-MMP substrate and specific binding peptide enhanced both the specificity and selectivity of MT1-MMP responsive module,and introducing a linker composed of 12 PEG subunits into these two fragments led to optimized both specificity and selectivity of MT1-MMP responsive module.Both in vitro and in vivo results revealed that with the linker composed of 12 PEG subunits,the enzyme responsive module could be effectively applied fort MT1-MMP positive tumor targeting.Base on the optimized MT1-MMP responsive module,MT1-MMP specific response therapeutic proteins intracellular targeted delivery system was designed,which consisted of three parts:the therapeutic proteins,CPP and the enzyme response module.To verify the feasibility of our designed system,the fluorescent protein m Cherry was used in as the drug model at first.Cell imaging results demonstrated that our designed system could successfully delivery therapeutic proteins into the cytosol of target cells.Then,the fluorescent protein m Cherry was replaced by the protein drug--trichosanthin（TCS）to investigate its anti-tumor activity.The cytotoxicity experiment proved that the protein drug system presented a significant inhibition on the proliferation of ovarian cancer cells.Conclusion:In this study,two MMPs responsive,ACPP-mediated intracellular targeting delivery systems for therapeutic proteins were constructed based on the characteristics of different MMP.Both in vitro and in vivo results revealed that the two systems presented enhanced MMP secificity of tumor targeting.