The Design Of Endomorphin-1 Nano-Micelles Delivery System Based On Peptide Self-Assembling For Brain Delivery

Endomorphin-1?Tyr-Pro-Trp-Phe-NH₂,EM-1?was an endogenous?-opioid receptor ligand with strong antinociceptive activity.Compared with morphine,EM-1has higher selectivity and affinity to?-opioid receptors,followed with avoiding severe physical and psychological dependence.But at the same time,the application of EM-1in clinical research is limited by its poor stability of enzymatic hydrolysis in vivo and its poor ability to penetrat the blood-brain barrier?BBB?.Because of its good biocompatibility and controllable degradation,peptide self-assembly has a wider application prospect than other self-assembly systems in biomedical fields such as tissue engineering,gene therapy and so on.Now it has been used in vivo tissue and nerve regeneration,cell antiproliferative agents and drug delivery systems.A series of self-assembly behavior of peptides have been reported for drug delivery including delivering peptides to penetrate BBB,the self-assembly system of peptide has many advantages,such as reducing the stability of enzymatic hydrolysis of peptides and promoting the permeability of BBB.Based on the technology of peptide self-assembly,cholesterol,a material which is safe and BBB targeting,is selected as the lipophilic part of the self-assembly.EM-1 itself is used as the hydrophilic part of the self-assembly with embedded different bonds as linkers to construct a new type of nano-micelle.The relevant experiments are summarized as follows:In the first part of the paper,four esterified peptides?Chol-SS-EM1,Chol-CONH-EM1,Chol-G₃-EM1 and Chol-PEG-EM1?were designed,synthesized and purified,the products have high yield and purity.In the second part of the paper,the morphology and physicochemical properties of nano-micelles were studied.Four different nano-micelle solutions?Chol-SS-EM1NPs,Chol-CONH-EM1 NPs,Chol-G₃-EM1 NPs and Chol-PEG-EM1 NPs?were prepared by the traditional nano-precipitation method.Then the morphology,particle size,secondary space structure and critical micelle concentration?CMC?of nano-micelles were characterized by transmission electron microscopy?TEM?,Ma Erwen particle size analyzer,circular dichroism?CD?and fluorescence spectrophotometer.The results showed that the four nano-micelles were formed with round shape and uniform particle size.In the investigation of the secondary spatial structure,the?-folding degree of the nano-micelles was higher than that of EM-1,indicating the formation of the nano-micelles further.The results of CMC investagation showed that the morphology of the nano-micelles remained intact after entering into the blood circulation.In the third part of the paper,the bio-safety,pharmacodynamics and brain targeting of four nano-micelles were studied.The hemolysis experiment was employed to investigate the safety,the result showed that the four nano-micelles have good biosafety and hemolysis rate were less than 10%.Analgesic experiments showed that four nano-micelles analgesic effects were improved compared with EM-1,Especially the Chol-SS-EM1 NP_S and Chol-PEG-EM1 NP_S displayed a strong analgesic activity.The antinociceptive effects after i.v.administration of nano-micelles were significantly reversed by naloxone but not by naloxone methiodide.These results further revealed that the observed analgesic effects may involve central?-opioid receptors.To demonstrate the delivery of nano-micelles into the mouse brain,fluorescence imaging experiments based on monitoring the fluorescence intensity of DiR were performed,the results showed that the intensity of DiR load in nano-micelles was apparently improved compared with EM-1,indicating the nano-micelles can cross the BBB further,especially Chol-SS-EM1 NP_S led to the strongest fluorescence of four nano-micelles.In the forth part of the paper,Chol-SS-EM1 NPs was employed as the representative drug to investigate the enzymatic stability and DTT reduction response.The disulfide bond was stable in the peripheral blood but easily reduced by the reductase in the brain.The enzymatic stability study includes the study of plasma enzymatic stability and the stability of brain plasma membrane enzymatic hydrolysis.The result showed that Chol-SS-EM1 NPs has higher stabilities in plasma and brain in comparing with EM-1,the introduction of disulfide bond released EM-1 in the brain and displayed exerted analgesic effect.The result of DTT reduction response study showed the process of EM-1 released from Chol-SS-EM1 NPs in the brain.In this study,we developed a short-peptide self-delivery system for brain targets with the capability to deliver EM-1 without vehicle.Cholesterol and EM-1 were selected as the estrophilic part and hydrophilic part of the self-assembly with embedded different bonds as linkers to construct a new type of nano-micelle,aiming at improving the ability of BBB penetration and exerting analgesic activity further.The nano-micelles had high enzymatic stability and high safety in comparing with traditional drug-encapsulated nano-micelles.This technology is expected to be applied to the research of peptide brain targeting and providing useful thought for the exploration of new drugs delivery system for brain.