Study On The Construction Of Oral Nanocrystal-loaded Lipid Carriers And The Oral Absorption Promoting Mechanism Of BCS-? Drug Etoposide

Nanocrystals(NCs)with small particle size and high drug loading capacity are considered as a feasible tool in improving oral bioavailability of insoluble drugs.The number of nanocrystal-based oral formulations in current marketed and during pharmaceutical researches is increasing annually due to the absorption promotion capability of NCs in recent years.However,most of these studies are used to improve the oral absorption of BCS-II drugs,only few studies have been conducted on oral nanocrystals of BCS-? drugs.This is because the BCS-? drugs have poor biomembrane permeation capability and susceptibility to P-glycoprotein(P-gp),simple preparation of NCs failed in boosting their oral bioavailability,and arbitrarily increasing the dissolution of BCS-? drugs may even be counterproductive.Recently,the discovery of direct and intact NC internalization into intestinal epithelial cells pointed a different way for the oral delivery of BCS-? drugs which tackled the problems faced by dissolved molecules of BCS-? drugs.To this end,the further pharmaceutical strategy of NC-based oral BCS-? drug delivery systems may focus on the“as a whole”absorption in the form of NCs rather than improving dissolution of BCS-? drugs.Based on this transformation,surface modifications such as polymer coating and stabilizer crosslinking of nanocrystals have attracted extensive attention among researchers.These modifications help maintain the intact form of NCs during its absorption through intestinal tract into the body circulation,which is largely beneficial to the oral absorption of BCS-? drugs.In recent years,it has been found that the lipid bilayer can be used as a shell to further improve the biocompatibility,cell uptake and bioavailability of nanoparticles.In addition,the presence of phospholipid bilayer tends to protect the drugs carried by extraneous particles from being rapidly released.In view of above informations,in this study,etoposide(ETO,BCS-?)was selected as the model drug,and oral nanocrystal-loaded lipid carriers(Lipo@NCs)was constructed by surface modified phospholipid bilayer and TPGS on the ETO-NCs.Besides,the feasibility and effectiveness of BCS-? in improving oral absorption were also studied.Firstly,Positive charged etoposide nanocrystals(ETO-NCs)were prepared by sonoprecipitation-high pressure homogenization method.Spherical ETO-NCs with particle size of 173.9 nm and Zeta potential of 34.6 m V were obtained through optimization of the preparation process and formula.Then,the ETO-Lipo@NCs with particle size of 220.3 nm and Zeta potential of-9.95 m V were prepared by film hydration-high pressure homogenization method.This combination of good biocompatibility of phospholipids bilayer and high drug loading capacity of nanocrystals was expected to achieve better oral absorption and improve the intestinal absorption of BCS-? drugs.The structure of ETO-Lipo@NCs was characterized by a series of methods(such as AFM,CLSM and XPS),which proved the successful coating of phospholipid bilayer on the surface of nanocrystals in ETO-Lipo@NCs.Subsequently,the in vitro release of drug from ETO-NCs and ETO-Lipo@NCs was compared.The results showed that the release rate of the drug in ETO-Lipo@NCs was significantly reduced,and was only 45.17%of ETO-NCs.This result confirmed the conjecture that Lipo@NCs are consisted of inner NCs suspended in the inner water phase and lipid bilayers encapsulated in these NCs.The inner water phase could be recognized as a saturated drug solution since drug release started from drug molecules'dissolution in the inner water phase,then a dynamic equilibrium of dissolution and precipitation was reached at the liquid-solid interface which could be served as the first release control barrier.For the dissolved drug molecules,their release is controlled by the lipid bilayer.The poor compatibility between the lipid bilayer and BCS IV drugs limited effective partition of drug to the bilayer,so the lipid bilayer would serve as the second barrier against drug release.In this way,preparation of Lipo@NCs could retard drug release and maintain NC integrity.Whereafter,the intestinal absorption capacity of ETO-Lipo@NCs was evaluated.In single-pass intestinal perfusion experiment showed that the Lipo@NCs could significantly improve the intestinal permeability and absorption of ETO.In addition,it is worth noting that the main absorption sites of ETO-Lipo@NCs are different from those of the ETO solution(ETO-Sol),which suggested the change of intestinal uptake mechanism.Then the intestinal distribution and transfer of oral Lipo@NCs were analyzed.The results showed that compared with ETO-Sol and ETO-NCs,ETO-Lipo@NCs could significantly improve the absorption rate of ETO in the intestine,thus increasing the possibility of drug absorption by oral administration.Caco-2 cells were used as a model to explore the endocytosis and intracellular trafficking behavior of ETO-Lipo@NCs.Lipo@NCs not only significantly improved the cellular uptake of nanocrystals,but also changed the process of endocytosis and transport behavior of nanocrystals.After 2 hours of administration,the cellular uptake of drugs in ETO-Lipo@NCs was 1.72-fold higher than that of ETO-NCs.By investigating the cellular uptake mechanism of ETO-NCs and ETO-Lipo@NCs,we found that ETO-Lipo@NCs were mainly internalized via the caveolae-mediated endocytosis and macropinocytosis.However,the endocytosis of ETO-NCs involves various ways,such as the the clathrin-mediated endocytosis,non-clathrin-and non-caveolae-mediated endocytosis and macropinocytosis.The intracellular trafficking behavior was studied by applying the pathway inhibitors and colocalization between organelles and NCs.It was found that the intracellular trafficking pathway of ETO-NCs and ETO-Lipo@NCs both involved the lysosomal pathway and endoplasmic reticulum/Golgi complex(ER/GC)pathway.In addition,the phospholipid bilayers could help the nanocrystals escape from lysosomes more quickly,and transport in cells faster.Finally,Caco-2 cell monolayer model was constructed to investigated the the difference in transmembrane transport ability between these ETO-formulations.The results showed that compared with ETO-Sol and ETO-NCs(2.51×10⁶ cm/s and 2.30×10⁶ cm/s),ETO-Lipo@NCs significantly increased the total transmembrane transport of drugs(4.59×10⁶ cm/s).In addition,the monitoring of transepithelial electric resistance(TEER)value of Caco-2 cell monolayer model suggested that these ETO-preparations could not transport across the Caco-2 monolayer througth the paracellular pathway.The in vivo behavior of ETO-Lipo@NCs was systematically studied and evaluated.The pharmacokinetic result showed that the AUC_0-t of ETO was significantly increased by ETO nanocrystaline loaded lipid carriers with Eudragit coating(ETO-EU@Lipo@NCs)(1688.56±231.10?g*h/L),which was 10.92-and 1.96-fold higher than that of ETO-CCs and ETO-NCs,respectively.And the oral bioavailability elevated from 1.38%ETO coarse crystals(ETO-CCs)to 15.11%.Subsequently,the subcutaneous LLC xenograft model of murine lung cancer was constructed and the in vivo anti-tumor effect of ETO-EU@Lipo@NCs was investigated.The results indicated that ETO-EU@Lipo@NCs has a good antitumor effect who showed higher biological safety compared with other ETO formulations and has almost no intestinal irritation.The tissue distribution experiment in mice also showed that there was a high drug distribution in tumor tissue after oral administration of ETO-EU@Lipo@NCs,which was more than twice as high as that in oral ETO-NCs and ETO-Sol.In conclusion,the construction of ETO nanocrystaline loaded lipid carriers in our study is quite successful.The shell-core structure of the Lipo@NCs could effectively deliver BCS-? drugs to the systemic circulation through oral absorption,which made sure that the expected oral therapeutic effect could be achieved.