Studies On Oral Absorption Mechanism Of Poorly Water-soluble Drug Nanocrystals And Polymeric Lipid Nanocarriers

Solubility and epithelial permeability are two key factors that affect oral absorption of insoluble drug.Statistics shows that approximately 60~70% of the active ingredient in discovery phase are faced with challenges including poor water solubility and poor permeability.Therefore,methods for improving drug bioavailability are still highly desirable.In this work,two factors that affect oral absorption of insoluble drug were studied:(1)a poor water soluble drug,regorafenib(BCS II)was used as a model drug.According to the poor solubility,we have designed different formulations: different particle sizes of crystalline pure drug,amorphous pure nanodrug and solid dispersion.The effect of particle size of nanocrystals on oral bioavailability was studied.(2)saquinavir(BCS IV)with poor solubility and membrane permeability was used as another model drug.We designed four formulations: Emulsion,PLGA NP,Lipid-PLGA Hybrid NP and PL-Hybrid NP.Caco-2 cell was used as cell model to evaluate their uptake and transport mechanism of different formulations.Firstly,media mill method was used to prepare regorafenib nanocrystal suspensions with different particle size(309.68±18.32 nm,568.00±26.11 nm,1324.70±848.35nm)by controlling milling time(2min,1min,10s)at milling speed of 2000 rpm.The PVA was used as stabilizer.Pure nanodrug suspensions with particle size of 214.58±18.22 nm was prepared by anti-solvent nanoprecipitation method.REG raw crystals showed a regular elongated rod-like structure observed by SEM.The wide diameter of REG raw crystals is about 20~30?m,while solid dispersion was irregular and massive particles.REG nanocrystal suspensions with different particle size showed a regular elongated rod-like structure observed by TEM.The XRPD experiment demonstrated that,pure nanodrug prepared by nanoprecipitation was in amorphous,and no diffraction peaks in X-rays were observed.REG nanocrystal suspensions prepared by media mill method had slightly decreased diffraction peaks in 7.2° and 14.5°(2?),while new diffraction peaks appeared in the range of 20~30°.When the particle size of REG nanocrystal suspensions decreased to 309.68±18.32 nm,the diffraction peaks in the range of 5~15° disappeared,but the diffraction peaks in the range of 20~30° enhanced.The results demonstrated that with prolonged milling time,crystal form of REG may have been changed.The DSC experiment demonstrated that,melting point peak of REG raw crystals was 213.94?,and when prepared as nanocrystal suspensions,the melting peak was decreased to 200?.Interestingly,a melting peak appear at 137.67? was observed,indicating that crystal form of REG APN may have been changed.The in vitro dissolution experiment showed that the drug dissolve slowly in pH4.5 acetate buffer,the accumulated release amount was less than 2% in 4h.The dissolution rate of REG nanocrystal suspensions with particle size of 568.00±26.11 nm and 1324.70±848.35 nm were increased slightly(about 6% in 4h),while REG nanocrystal suspensions with particle size of 309.68±18.32 nm(more than 50% in 4h)and pure nanodrug suspensions with particle size of 214.58±18.22nm(more than 90% in 4h)were faster notably than REG raw crystals.The oral absorption of formulations was evaluated using male SD rats as animal model.The in vivo pharmacokinetics study showed that,the AUC of pure nanodrug suspensions with particle size of 214.58±18.22 nm,REG solid dispersion and REG nanocrystal suspensions with particle size of 309.68±18.32 nm were 31.68-fold,29.06-fold and 12.98-fold respectively higher than that of REG raw crystals.Amorphous pure nanodrug suspension with particle size of 214.58±18.22 nm was better than solid dispersion(reference formulation).The results showed that the oral absorption of drugs with decreasing particle size increased dramatically,but without features of "critical diameter".Secondly,in order to study cell uptake and transport mechanism of nanoparticles,we designed four formulations by nano-precipitation method: Emulsion,PLGA NP,Lipid-PLGA Hybrid NP and PL-Hybrid NP.The average size of resulted nanoparticles were about 200 nm and negatively charged.Four formulations are rounded spherical with smooth surface observed by TEM.The in vitro dissolution experiment was conducted in pH6.8 PBS solution,the results showed that the formulations were slowly released without burst effect.Caco-2 cell was used as cell model to evaluate the uptake and transport of different formulations.The CLSM and FCAS were used to study the uptake of coumarin-6 labeled nanocarriers.The result demonstrated that PLGA NP can improve the uptake of SQV by Caco-2 cells effectively,followed by PL-Hybrid NP,Lipid-PLGA Hybrid NP and Emulsion.To assess transportation ability of formulations,Caco-2 cells cultivated over 21 days were served as monolayer model.The result of transport study showed that the Papp value of Lipid-PLGA Hybrid NP was higher than other nanocarriers,followed by PLGA NP,Emulsion and PL-Hybrid NP(P<0.05).To study the mechanism of Emulsion,PLGA NP,Lipid-PLGA Hybrid NP and PL-Hybrid NP uptake by Caco-2 cells,we inhibited different transport pathways individually using specific inhibitors.Intracellular uptake of Lipid-PLGA Hybrid NP was significantly decreased when lipid raft-dependent endocytosis was inhibited by depletion of cholesterol in the presence of M?CD.After inhibited by chlorpromazine(an inhibitor of clathrin-mediated endocytosis),nystatin(an inhibitor of caveolae/lipid raft-dependent endocytosis)and amiloride(an inhibitor of Macropinocytosis),intracellular uptake of Lipid-PLGA Hybrid NP also showed a downward trend.This result demonstrated that the internalization process occurs via distinct mechanisms acting in parallel,and different endocytosis pathways may compensate each other.However,other three formulations were only sensitive to M?CD.It shows that other three formulations were mainly through lipid-mediated endocytosis.The organelles(lysosomes and Golgi apparatus)were labeled by specific fluorescent substance,and the co-localization experiments were used to study the intracellular distribution of nanoparticles.The result showed that the colocalization coefficient of four formulations with the lysosome were less than 0.2,while the colocalization coefficient of four formulations with the Golgi were about 0.4.The permeability study in Transwell chamber and fluorescent quantitative experiment displayed that,the co-localization of Lipid-PLGA Hybrid NP and Transwell membrane pore was stronger than other three formulations.The results demonstrated that the permeability and transportation ability of Lipid-PLGA Hybrid NP was easier to exocytotic from the basolateral side than other three formulations,and thus the Papp value of Lipid-PLGA Hybrid NP was highest.