Effect Of MPEG-PLA Micelles On Cytokines By Nasal Administration Into The Brain

With the development of society,environmental changes and the degree of aging,the incidence rate of brain diseases such as epilepsy,Alzheimer's disease,depression,Parkinson's disease,cerebral ischemia,etc.,is increasing year by year.The issues of low drug concentration in brain tissue and the inaccessibility of the lesions seriously hinder the treatment of brain diseases and threaten human health.Especially on account of the existence of the blood-brain barrier,brain-targeted drug therapy of brain diseases has always been a problem.In order to improve the permeability of the blood-brain barrier and increase the intracerebral distribution of drugs,humans have come up with various methods such as preparing lipophilic prodrugs by chemical modification,applying vasoactive substances,preparing nano drug delivery systems,and bypassing-BBB administration and so on.Although the amount of drugs entering the brain has increased,the drug is faced with the problem of being quickly cleared out of the brain thus reducing the residence time of the drug in the brain.Traditionally,one of the pathway of drugs excretion in brain is that the cerebrospinal fluid in the subarachnoid space carries the drug through the sinus arachnoid granules and then be absorbed into the blood,which sequentially eliminating from the brain.The recent discovery of dural lymphatic vessels communicated with the deep cervical lymph nodes provides new ideas for the investigation of drugs efflux pathway of the brain.In this paper,the coumarin-6 mPEG-PLA micelle is prepared and its physicochemical properties,in vitro release behavior,cytotoxicity,intracerebral distribution after nasal administrationand the effects of mPEG-PLA micelles on the cytokines in vivo are surveyed comprehensively.MPEG-PLA polymer is used as the carrier material.The hydrophilic segment of the polymer consists of a certain length of polyethylene glycol(PEG),which can increase the residence time of the micelle.The coumarin-6 fluorescently labeled micelles were prepared by membrane hydration method,and the morphology and particle size of the micelles were characterized by transmission electron microscopy and laser particle size analyzer.The micelles were regular spherical and the appearance was round.The average particle size of the micelles was 31.22 nm and the PDI was 0.178.The size dispersion of particle was well and there was no statistical difference with the blank micelles(about 30.69 nm).The average encapsulation efficiency and drug loading of the coumarin-6micelles determined by high performance liquid chromatography were80.18% and 1.30%,respectively.The results of in vitro leakage test showed that the leakage of coumarin-6 in PBS solution at 37 °C and pH7.4 was lower(<4%)within 24 h,indicating that coumarin-6 was not easily leaked from the micelle thereby can accurately trace the behavior of micelles in the body,which lays the foundation for the experiment of micelles entering the body.In order to investigate the safety of nasal administration,the in vitro toxicity of Calu-3 cells loaded with coumarin-6 micelles and blank mPEG-PLA micelles was determined by MTT assay.When the micelle concentration was less than 4 mg/mL and both of them were incubated for 3 h,the cell viability was 80%-100%,indicating no cytotoxicity.When the micelle concentration reached 8 mg/mL,the cell viability of the drug-loaded micelles was 50%-80%,while the blank micelle survival rate was still higher than 80%,which indicating that the increased concentration of drug-loaded micelles produces low toxicity to cells.Meanwhile,in order to observe the intracerebral distribution of micelles,the coumarin-6 micelles were instilled through the nasal cavity,and after30 minutes of administration,the brain tissues were taken for cryosection,and the fluorescence distribution was observed under an inverted fluorescence microscope.It was found that the cerebral cortex,striatumand hippocampus had a green fluorescence distribution,indicating that the micelles entered the brain parenchyma after nasal administration.LTG drug-loaded micelles were prepared to investigate the effect of micelles on the cytokines(IL-6)after nasal administration into the brain.The average particle size of LTG mPEG-PLA micelles was 31.1 nm,which was not significantly different from that of coumarin-6 micelles and blank micelles.The rats were intranasally administered with lamotrigine micelles and normal saline.The deep cervical lymph nodes,brain tissue,jugular vein blood,and cerebrospinal fluid were taken at 30 min and 90 min,and each was determined by ELISA to survey their IL-6level respectively.After the rats were given LTG micelles for 30 minutes,there was no significant change in IL-6 levels in cerebrospinal fluid and jugular vein blood,and they were all at normal levels.In contrast.the levels of IL-6 in brain tissue and deep cervical lymph nodes were significantly increased(P<0.05),which were 2.06 and 1.62 times of the saline control group,respectively.However,90 minutes after administration,the amount of IL-6 was reduced to a level that was not significantly different from that of the saline control group.It indicates that LTG micelles may activate the immune response after nasal administration and promote the synthesis and release of IL-6 by the relevant immune cells.Later after 90 minutes,the level of IL-6 in the brain is reduced accordingly when the micelles are cleared out of the brain.A more comprehensive exploration of the effects of nasal administration on the levels of related cytokines in various tissues is investigated by nasal administration with mPEG-PLA micelles for 21 days.Plasma,cerebrospinal fluid,deep cervical lymph nodes and brain homogenate were taken after 30 min in the 21 st day of administration,and the relevant cytokine levels(IFN-?,IL-10,IL-13,IL-1?,IL-4,IL-6,TNF-?)were simultaneously determined by Luminex Bead Array Multiplex Immunoassay.The results showed that there was no significant change in the levels of various factors in plasma compared with the saline group;the levels of proinflammatory cytokines IL-1? and TNF-? in deep cervical lymph node and IL-6 in cerebrospinal fluid increased;FactorIL-10 has also been raised.This indicates that the micelles may cause the body's immune response and release pro-inflammatory factors after micelles passing into the brain,sequentially promoting the migration of the micelles through the dura mater into the deep cervical lymph nodes by mediating the proliferation of the relevant immune cells to phagocytose the micelles.At the same time,with the production of pro-inflammatory factors,the body also releases anti-inflammatory factors through a negative feedback pathway to maintain the stability of the body.In short,this study provides the new angles for the researchs on novel pharmaceutical dosage form and administration to improve the bioavailability by reducing the lymphatic efflux of the microparticle in brain and extending thier action time.