Structural Stabilization Of Liposomes And Feasibility Study On Oral Delivery Of Emodin Thereof

Objective:As an excellent drug carrier,liposomes can not only increase the apparent solubility of insoluble drugs,but also promote the absorption of insoluble drugs.They also have the advantages of targeted drug delivery,enhancing efficacy and reducing toxicity/side effects.So,they are widely used in a variety of drug delivery routes.Although liposomes seem to have many advantages,they still have many problems in practical application,and the stability of liposomes is one of the most important concerns.Liposomes are mainly composed of phospholipids and cholesterol,so they are prone to oxidative degradation or lipid peroxidation due to containing unsaturated fatty acids.These reactions can occur in any process of preparation,storage and practical application,and will lead to the leakage of encapsulated drug molecules from the liposomes.Therefore,it is necessary to explore effective liposomes modification technology.Compared with surface coating and modification,the stability and sustained-release ability of liposomes can be further improved by curing liposomes.Selenium is a kind of non-metallic trace element,which is essential for human beings.It has significant safety and can contribute to a synergistic therapeutic effect with the payload.Therefore,it is a good choice for curing liposomes,and it is also desirable to further improve the properties of liposomes.Furthermore,in all kinds of natural active ingredients,emodin has liver protection,antibacterial activity,antioxidant effect,anti-inflammatory effect and anti-diabetes activity.At the same time,selenium also exhibits antioxidant effect,anti-inflammatory effect and anti-diabetic activity,so the combination of selenium and emodin may jointly cure some stubborn diseases.To this end,this study intends to address the solubility challenge of emodin by preparing liposomes,and then stabilize liposomes by selenylation to improve its gastrointestinal stability and release,whereby to enhance the oral bioavailability of emodin by structural modification of liposomes.MethodsPreformulation study of Emodin（EM）:the in vitro analysis method of emodin was established and verified,and the equilibrium solubility,oil-water distribution coefficient and stability of emodin were determined.Preparation,formulation optimization and characterization:EM-LPs were prepared by thin film dispersion method.On the base of EM-LPs,EM-Se@LPs were prepared by in situ reduction using glutathione（GSH）and sodium selenite（Na₂Se O₃）.Then,the formulation of EM-Se@LPs was optimized and validated using a combination of single-factor tests and response surface analysis.Afterwards,the particle size,ζpotential,entrapment efficiency and in vitro release characteristics of EM-Se@LPs were characterized.Oral bioavailability study:using SD rats as experimental animals,EM suspension,EM-LPs,EM-Se@LPs were administered to rats by gavage.At the pre-determined time points,approximately 250μL of blood were collected from the caudal vein of rats.Then,the plasma samples were processed to obtain plasma by anticoagulant treatment followed by centrifugation,and the plasma drug concentration was determined by liquid chromatography-tandem mass spectrometry.After that,the pharmacokinetic curves and parameters of three EM preparations were obtained and compared.Oral absorption mechanism study:using Caco-2 cells as experimental subjects,the cell uptake rate,cell internalization and cell uptake mechanism of EM-Se@LPs were explored.In addition,the physiological stability of EM-Se@LPs in rat real intestinal fluid and simulated gastric/intestinal fluids was further examined by comparing with EM-LPs and EM.ResultsAfter optimizing the formulation by response surface analysis,the prepared EM-Se@LPs particle size was approximately 126 nm with a PDI of 0.231 and aζpotential of-55.8 mv.The entrapment efficacy was determined to be 99.61%around.EM-Se@LPs were provided with higher stability than EM-LPs in a short-term survey upon storage.Under transmission electron microscope,the micromorphology of EM-Se@LPs was spherical and intact,and the distribution was also uniform.The release rate of EM from EM-Se@LPs was significantly slower than that of EM-LPs.It means selenylation substantially ameliorated the sustained release of liposomes.As per the experimental results of oral bioavailability study,the maximum plasma concentration(C_max)of EM suspensions,EM-LPs and EM-Se@LPs were 393.83 ng/m L,270.22ng/m L and 130.48 ng/m L,respectively.The results seem to indicate that the absorption of EM suspensions group is better than that of EM-LPs group and EM-Se@LPs group.After calculation through the non-compartmental model,the relative bioavailability of EM-LPs group and EM-Se@LPs group to EM suspensions group was only 66.36%and 39.99%respectively,which further confirmed that the oral absorption of EM suspensions was acceptable.In order to explain the phenomenon and explore the underlying mechanisms of EM absorption,we conducted a series of studies on the cellular uptake and physiological stability of EM solution,EM-LPs and EM-Se@LPs subsequently.The results of cellular uptake showed that macropinocytosis and clathrin-mediated endocytosis were involved in the uptake process of EM-LPs and EM-Se@LPs.The stability of liposomes in gastrointestinal fluids showed that EM-Se@LPs was more stable than EM-LPs in digestive fluids,suggesting that selenium attachment on the internal and external layers of liposomes can protect liposomes from digestion by enzymes to a certain extent.However,EM itself did not show significant intestinal first-past effect.Whatever EM in EM solution,EM-LPs or EM-Se@LPs,it all showed a good stability in digestive media,so that selenized liposomes did not exhibit prominent absorption-promoting effect in terms of emodin.ConclusionsIn this study,we constructed a kind of modified liposomes based on selenium with an ameliorative structure for oral delivery of emodin,aiming to improve its oral bioavailability.Selenylation did increase the stability of the liposomes and achieve sustained drug release in some ways.Unfortunately,there was no in vitro-in vivo correlation between optimized liposomal structure and oral absorption.The underlying reasons lie in that EM itself possesses good stability and absorbability that embarrasses the function of liposomes for absorption promotion.Although the oral bioavailability of EM is far from the expectation,this study verifies the proof-of-concept that selenium layering onto the interior and exterior of liposomes can improve the stability and sustained release thereof,which sheds a light on the structural modification and performance improvement of traditional liposomes.