Study On The Design Of Puerarin-loaded Polymeric Mixed Micelles And Its Drug-delivering Behavior Based On CGMD

Many active ingredients and parts in traditional Chinese medicine have low bioavailability due to their poor water solubility or permeability or short half-life,resulting in its restricted applications.Self-assembled polymeric mixed micellar system(Polymeric Mixed micellar system,PMMs)can not only enhance drug solubility or promote oral absorption,but also prolong drug retention time in vivo,which provides a new direction for oral preparation research.However,the drug-loading mechanism and transport mechanism of PMMs is still unclear,which seriously restricts the design and development of PMMs.The self-assembly behavior of PMMs,the three-dimensional structure at mesoscopic scale,the micro-region information and the spatial distribution within PMMs haven't been explained,due to the lack of characterization technology at the molecular level;and the transport mechanism of PMMs has not been clearly explained,because the conventional fluorescent probe cannot avoid interference of free fluorescent groups.Therefore,if we can choose effective method to analysis self-assembly behavior and appropriate probe to accurately trace cell transport process of PMMs,scientific interpretation of PMMs'drug-loading mechanism and cellular transport characteristics can be provided,and then it can be revealed that how oral bioavailability and the efficacy is enhanced,to sufficiently expand PMMs'application in the field of innovative traditional Chinese medicine.This project proposes to adopt Coarse-grained Molecular Dynamics(CGMD)using the MARTINI force field to simulate the molecular dynamic self-assembly behavior of the drug-loaded FS/PMMs with different ratio of polymers,composed of Pluronic F127(F)and Solutol HS15(S),and explore drug-loading mechanism of PMMs from the molecular level;and select aggregation caused quench fluorescent probe with sensitive water environment response to track the cellular transport process of the intact PMMs particles,to elucidate the effects of FS/PMMS particles on the improvement of pharmacokinetics and pharmacodynamics.The main contents and points are as follows:1.The self-assembly kinetics of FS/PMMS system was investigated by using CGMD technology and experiments.The three-dimensional structure of FS/PMMS particles in mesoscopic scale formed at different FS ratios and the stability of the structure were investigated.Drived by hydrophobic action,the structures of FS/PMMs formed by different FS ratios are different,the system with low FS ratio will form a“coil/cluster”micelle,within which 4-6 HS15 spherical clusters were bound and coiled by“F127 belt”,and the inner region of the HS15 seed cluster is with strong hydrophobicity.Under the the double effects of“binding plus wrapping”of F127molecules,the system with high FS ratio formed a“lotus seedpod”-like structure,in which there are more secondary hydrophobic regions composed of F127 hydrophobic chain and HS15 hydrophilic chain.In addition,the aggregates formed under high FS ratio are usually more compact,while the aggregates formed under low FS ratio are looser.In this part,the relationship between the self-assembly behavior of FS/PMMs and the characteristics of the particles'internal micro regions and the stability of the particles'own structure is well explained from mesoscopic scale.2.On the basis of the previous chapter,the spatial distribution of drugs with different concentrations and different hydrophobic degrees in FS/PMMs was investigated,and the influence of drug distribution on drug loading performance(solubilization effect,drug-loading stability and in vitro release)was investigated.The results showed that the distribution of drugs in FS/PMMs obeyed the principle that the similar hydrophobicity is more likely to be accepted by each other,namely,puerarin(PUE)with relatively weak hydrophobicity tended to be distributed in the secondary hydrophobic region with relatively weak hydrophobicity,while docetaxel(DTX)with strong hydrophobicity preferred to be distributed in the strong hydrophobic region inside HS15 seed clusters.The preferred distribution region of drugs was called the comfort zone.Drug distribution is a key factor affecting the drug-loading stability of PMMs.When drug molecules are distributed in the"comfort zone",drug loading stability is good.When the drug feeding concentration is too high,some drugs will be distributed in the"uncomfortable zone",and the drug-loading stability will be reduced.However,when two different hydrophobic drugs are reasonably co-loaded,such as PUE and Daidzein(DAZ),drug molecules are more distributed in their respective"comfort zones"due to regional competition,which can improve drug-loading stability instead.Additionally,it was found that drug distribution and polymeric ratio had no significant effect on the solubility of the system,but the drug distribution was the main factor affecting the drug release rate,except when the"comfort zone"of the drug was near the outermost layer,both the PMMs'structural stability and drug distribution would affect the drug release rate.This study not only further enriched people's understanding of the relationship between molecular spatial distribution within PMMs and drug loading performance,but also provided a theoretical basis for realizing the"self-consistent"co-loading of TCM functional components.3.Drug-loaded FS/PMMs with different FS ratios,namely System-I-PUE and System-II-PUE,were prepared by thin film dispersion method.The results of unilateral intestinal perfusion in vivo showed that System-I-PUE promoted drug intestinal absorption significantly compared with Puererin suspension,while System-II-PUE showed no significant difference,indicating that the transmembrane transport mechanism of intact micellar particles with different FS ratios was different.As a result,FS/PMMs containing ACQ fluorescent probes P₄were adopted to track the cellular transfer process of PMMs with different FS ratios in Caco-2 cells.Research shows that the systems with different FS ratio have different endocytosis pathway.System-I with low FS ratio enter into the cells via the endocytosis process mediated by clathrin structure and caveolin/lipid raft structure.And giant pinocytosis mediated endocytosis process played a dominant role when the System-II particles enter into cells.After FS/PMMS particles were ingested into Caco-2 cells,no matter System-I or II,are mainly distributed in cladesin-mediated endocytic vesicles,lysosomes,endoplasmic reticulum and Golgi bodies.However,System-II has a higher probability of being acidified and degraded by lysosomes,which will affect their transport to a certain extent.This part of the study is helpful for people to understand the transport process of the intact micellar particles.4.In vivo pharmacokinetic experiments were conducted to study the effects of FS/PMMs with different FS ratios on the pharmacokinetics of PUE,namely,the pharmacokinetics of System-I-PUE and System-II-PUE.The results showed that the high loading stability of System-II could effectively shorten the elimination time of PUE,while the high cell transport volume of System-I could significantly increase the plasma drug concentration,which indicated that FS/PMMs with different FS ratios had different improvement effects on pharmacokinetic.In addition,spontaneously hypertensive rats(SHR)was chosen as a model and the intervention effect of System-II-PUE on their blood pressure was investigated.It was found that System-II-PUE was better than PUE suspension in reducing systolic and diastolic blood pressure,indicating that in addition to increasing the blood concentration,the efficacy could also be improved by extending the half-life of PUE.In this study,PMMs with different FS ratios showed different drug loading properties and intestinal absorption promoting effects,which made them different in improving the pharmacokinetic data of drugs in vivo.In conclusion,different self-assembly behaviors of PMMs will lead to differences in three-dimensional structure,internal micro-region characteristics and structural stability of particles;The distribution of drugs in FS/PMMs obeyed the principle that the similar hydrophobicity is more likely to be accepted by each other;Drug distribution will directly affect the drug delivery performance of micelles,especially the drug delivery stability and in vitro release;Different drug loading stability and intestinal absorption effect will lead to the change of different pharmacokinetic parameters,and targeted parameter improvement can usually improve the efficacy of the drug.