The Design Of Endomorphin-1 Prodrug Nano-delivery System Based On Peptide Self-assembling For Brain Delivery

Endomorphin-1?EM-1?is an endogenous ligand of ? opioid receptor in central nervous system?CNS?,with an ideal analgesic.It has equal analgesic effect with morphine for acute pain and is not easy to produce body tolerance,addiction and other serious side effects.However,due to the good water-solubility of EM-1 molecule,EM-1 has short half-life and poor enzymatic stability which leads difficulty to cross the blood-brain barrier?BBB?,limiting the clinical application of EM-1.In this study,we constructed a prodrug molecule nano-delivery system based on the peptide self-assembly technique in which EM-1 itself was used as part of the delivery system,namely,EM-1 was modified with lipophilic molecules with embedded single disulfide bonds as linker following by self-assembling into nanostructures.The aim is to promote brain-targeted delivery of neuropeptide drugs.Experiments were carried out by esterification of EM-1 by different lengths lipophilic alkylcarbon chains(C8,C12,C₁₈)using covalent attachment.Amphiphilic end of molecular were ligated by disulfide bond and amide bond.Eventually,EM-1 and its four amphiphilic prodrugs(abbreviated as EM-1,C8-SS-EM1,C12-SS-EM1,C₁₈-SS-EM1 and C₁₈-CONH-EM1,respectively)were synthesized and purified.The product has high purity and simple synthesis method.Four EM-1 amphiphilic prodrug molecules were self-assembled to form nanostructured aqueous solution through the traditional nano-precipitation method.The nano-morphology,particle size,secondary space structure and molecular self-assembly behavior were characterized by methods like scanning electron microscopy and particle size analyzer.The results showed that different prodrugs moleculars has different microstructures.In contrast,nano-particles with longer lipophilic carbon chain has smaller particle size,more uniform shape and more stable system.Similarly,the overall characteristics of nanostructures with isulfide bond in the molecule are relatively better than amide bond.Analgesic experiments showed that prodrug NP analgesic effects were improved relative to EM-1,indicating that the prodrug not only cross the BBB,but also release EM-1 to exert analgesic activity.Near-infrared fluorescence imaging experiments were used to visually observe whether EM-1 prodrug NP reached the brain.The results showed that the fluorescence signals of four prodrug NPs containing fluorescent probe DIR were all observed in the brain of nude mice.Signal of C₁₈-SS-EM1 NP is the strongest,indicating prodrug NP could cross BBB and the penetration ability is affected by esterification and linker.Enzymatic stability assay was used to investigate the degradation by protease in plasma and brain tissue homogenate of EM-1 and its prodrug NP.The results showed that the stability was significantly increased compared with EM-1.Relative to amide bond,the inset of isulfide bond is more likely to make EM-1 released in the brain.In vivo pharmacokinetic study was used to investigate the distribution of C₁₈-SS-EM1 NP in the body.The results showed that C₁₈-SS-EM1 could not only distributed into plasma and liver but also was absorbed into the brain.The whole experiment aims to promote EM-1 cross the BBB to exert analgesic effect.The amphiphilic prodrugs were formed by esterification of EM-1 and a nano-delivery system was formed by self-assembly techniques.Nanostructures helps to reduce the enzymatic action of peptide drugs.Esterification enhances the lipophilicity of EM-1.Application of-SS-helps NP to be stable in blood and easy break in brain.Either of these strategies can increase the brain uptake of the drug.Successful design and construction of nano-delivery system has played a triple effect.In conclusion,this experiment creatively uses the peptide drug itself as part of the delivery system,and constructs the peptide self-assembled nano-prodrug molecule by single disulfide bond.Brain targeted delivery studies for neuropeptide drugs are useful for exploring the BBB permeability of these drugs.