The Improved Blood-Brain Barrier Permeability of Endomorphin-1 Using the Cell-penetrating Peptide SynB3

Author:Liu Zuo

Supervisor:wang rui


Degree Year:2014





The major obstacle to targeting of drugs to the central nervous system(CNS)for therapy is the existence of the blood-brain barrier(BBB).Endomorphins(EMs)are endogenous μ-opioid receptor(MOR)agonist and involved in a wide variety of pharmacological and physiological processes including pain perception and modulation.EMs can’t be delivered into the CNS in sufficient quantity to elicit antinociception when given systemically because of the BBB,which greatly limits its application in clinic.To overcome the BBB and improve uptake of a drug in the brain,various strategies have been developed.One promising solution is to use cell-penetrating peptides(CPPs).CPPs have been widely reported as a novel technology for enhancing the delivery of therapeutic drugs,e.g.,small molecules,peptides,proteins,liposomes and nanoparticles,across cell membranes including BBB.SynB3(RRLSYSRRRF)peptide,derived from the antimicrobial peptide protegrin 1,is the most efficient CPPs in terms of delivering different moleculars across the BBB.In the present study,to improve the brain delivery of EM-1 more effectively,we explored SynB3 as a CPP carrier and developed two potential strategies for the delivery of EM-1 arcross the BBB.Our work is divided into three parts,and as follows:1.The improved BBB permeability of EM-1 using CPPs SynB3 with three different linkages.Most articles focus on the selection of CPPs that achieve efficient transport across the BBB and the expansion range of the transport molecule.However,very few examples of the various types of conjugation between biologically active cargo and CPPs have been published.In this study,EM-1 was conjugated with SynB3 via amide,maleimide and disulfide linkages.The delivery efficiency of three linkers was compared in terms of pharmacodynamics and in vitro metabolic stability.Near-infrared fluorescent and fluorescent microscopy experiments were applied to detect the brain uptake and distribution of CPP delivery qualitatively and quantitatively.After the most successful linkage was screened out,the further mechanisms were discussed.Our study demonstrated that the three conjugates produced increased analgesia compared with the free peptide and the results were further verified by NIR fluorescence imaging experiments and a microscopy fluorescence study.In particular,conjugation of EM-1 to SynB3 with the disulfide bond led to the most significant enhancement of analgesic activity compared with the other two conjugations.We concluded that compared with the other two linkages,the disulfide bond was the most efficient linkage to deliver EM-1 across the BBB and confirmed that it could be reduced at physiological conditions in the brain and release its active form.These findings indicate that for those who need to release a free drug in the brain and maintain activity,a disulfide bond might be the most efficient linkage across the BBB.2.CPPs SynB3 conjugated nanodiamond for the enhanced BBB delivery of EM-1.Nanodiamonds(NDs)are promising versatile platforms for drug delivery because they bear unique properties such as stable structure,surface modifiability,low cell toxicity and remarkable biocompatibility.The NDs can be readily functionalized for coupling with various biomolecules by a non-covalent or covalent linker and provide more efficient tools for distribution diagnosis,targeted and therapy.In this study,to further promote EM-1 cross BBB using SynB3,we constructed two strategies of conjugation by covalent bonding and non-covalent adsorption.Our results revealed that EM-1 and SynB3 were conjugated to the surface of NDs in sequence through carbodiimide coupling in order to avoid premature release in peripheral circulation and exhibit a higher antinociception effect than free EM-1.When EM-1 was non-covalently bound to NDs via physical adsorption in an aqueous solution,the experiments indicated that diluted NaOH solution could promote the loading of EM-1 on NDs.The results of desorption demonstrated a burst effect followed by slow and continuous release.EM-1 release from the NDs was rapid,probably because the drug was adsorbed on the NDs surfaces rather than entrapped into the polymeric core.In the antinociception assay,the two strategies produced increased analgesia compared with the free EM-1.It should be noted that the MPE%of the covalent bonding was significantly higher than the non-covalent adsorption,whereas the longer durations of action,the former were found to be shorter than the latter.3.Models for predicting blood-brain barrier permeation of drug.The BBB permeability is an important evaluation in drugs design and an essential part during the early drug discovery phases.Models for predicting BBB permeability of drug are mainly divided into four categories including in vivo animals model,in vitro brain tissue model,cell-or non-cell-based in vitro models and in silico modelling.We establishedin in situ brain perfusion model and parallel artificial membrane permeation model.The BBB integrity of the model was evaluated.We discussed a set of the amino oxindoles compounds for the prediction of blood-brain barrier penetration using two model and evaluate the relevance of two methods.In conclusion,we not only compared three common linkages for delivering EM-1 across BBB by a novel CPPs SynB3,but also constructed two functional penetrating peptide-conjugated ND drug delivery system to deliver EM-1 to its target site in the brain.Our study will contribute to the usefulness of peptide-mediated strategies for improving the availability and efficacy of opioid peptide drugs.The two established blood-brain barrier modelsl can help in prediction of permeation of drugs.