Studies On The Factors Influencing The Fabrication Of Three Insoluble Components Of Tongmai Formula Drug Nanocrystals Self-stabilized Pickering Emulsions

Nanocrystals self-stabilized Pickering emulsions(NSSPE)is a new type of emulsion developed by our group firstly,which can improve the oral bioavailability of insoluble components.Our group has successfully constructed NSSPE with silybin and puerarin,respectively.However,the factors that could affect the fabrication of NSSPE have not yet been explored systematically.In this study,puerarin,tanshinone IIA and ferulic acid,which are insoluble active ingredients in Tongmai Formula,were used as model drugs.The influence of drug-oil phase properties,aqueous pH,drug concentration,and volume ratio of water to oil on the formation and stability of NSSPE were explored to reveal the important or key factors that determine the construction of NSSPE.The micro-structures of three NSSPEs were characterized by SEM,CLSM,FIM and DSC.Specific research content and results are as follows:(1)The evaluation method of NSSPEEmulsion was evaluated from the following four aspects.(1)Appearance: appearance changes of the emulsion during storing at room temperature were observed and the stability index was calculated;(2)Centrifugal stability: appearance changes were observed after centrifugation at 4000 × g for 15 min,and the absorbance of emulsion layer was measured at 500 nm;(3)Droplet size: morphology of droplets were observed by an optical microscope and droplet size was calculated by counting 200 droplets;(4)Emulsion layer drug content: the emulsion layer was separated from emulsions freshly prepared,stored at room temperature for 14 days and centrifuged at 4000 × g for 15 min.The drug content in emulsion layer was determined by HPLC.(2)Effect of properties of drug-oil phase on the preparation of NSSPECapmul C8,Fabrafil M 1944 CS,isopropyl myristate,Pzechwan Lovage Rhizome oil and olive oil were used as oil phase,and puerarin,tanshinone ?A and ferulic acid were selected as model drugs.NSSPEs were prepared by high pressure homogenization method and evaluated.The viscosity and surface tension of the oil phase,the surface energy of drugs,the particle size and Zeta potential of drug drug particles,the solubility and logP of drugs in different oil phases and three-phase contact angles were measured.The effects of all these properties of drug-oil phase on preparation of NSSPE were analyzed.It was found that viscosity of the oil phase,particle size and charge of the solid drug particle have an important effect on the preparation of NSSPE: Low oil viscosity,such as isopropyl myristate whose viscosity was close to water,was not beneficial to formation of NSSPE;The large size and low charge of drug particles,such as ferulic acid particle whose size was over several microns and Zeta potential was close to zero,is adverse to formation of NSSPE.In addition,three-phase contact angle also has some influence on the stability of NSSPE.However,properties such as solubility,logP,oil surface tension and drug surface energy,may not be related to the construction of NSSPE.Finally,mixed oil which consisted of Pzechwan Lovage Rhizome oil and Fabrafil M 1944 CS(9:1,v/v)was selected as the oil phase for subsequent experiments.(3)Effect of pH of aqueous phase on the preparation of NSSPENSSPEs of puerarin,tanshinone IIA,and ferulic acid with different pH of aqueous phase(5.0,7.0,9.0,11.0,11.5,12.0 and 13.0)were prepared and evaluated.At the same time,the particle size,Zeta potential and pH of three drug particles suspensions were determined at different pH conditions.It was found that pH of aqueous phase was another important factor influencing the fabrication and stabilization of NSSPE: NSSPE of all three drugs were more stable under alkaline conditions.The charge of drug particles of the three drugs increased significantly under alkaline conditions.The drug particles of ferulic acid and puerarin were smaller under alkaline conditions,while size of tanshinone IIA drug particles did not change obviously.The self-emulsifying ability of Pzechwan Lovage Rhizome oil and Fabrafil M 1944 CS(9:1,v/v)was also improved at alkaline aqueous phase.It is speculated that pH of aqueous phase could affect the construction of NSSPE by changing the size and charge of drug particles,as well as the self-emulsifying ability of oil.(4)Effect of drug concentration on the preparation of NSSPENSSPEs of puerarin,tanshinone IIA,and ferulic acid with different concentrations of solid drug were prepared and evaluated.The particle size,Zeta potential and pH of nanocrystal suspensions at different concentrations were measured.It was found that at a relatively high concentration flocculation took place in Pu-NSSPE,stratification was more obvious in FA-NSSPE,and absorbance of emulsion layer weakened significantly,but TanIIA-NSSPE is less affected by concentration.The reason may be that sizes of drug particles of puerarin and ferulic acid increased,charges and pH of nanocrystal suspensions decreased with the increasing of solid drug concentration,but the influencing of concentration on nanocrystal of tanshinone IIA was very low.It was inferred that solid drug concentration could influence properties and stability of NSSPE by ways of particle size,charge and pH of the drug particles suspension,and this affect may be more obvious for drugs with a certain acidity or alkalinity.(5)Effect of volume ratio of water to oil on the preparatioin of NSSPENSSPEs of puerarin,tanshinone IIA,and ferulic acid with different volume ratio(95:5,90:10,85:15,80:20,and 70:30)of water to oil were prepared and evaluated.The results showed that with the increasing of the oil proportion,the absorbance of emulsion layers raised firstly and then reduced,and seperated oil increased gradually for all three kinds of NSSPEs.These results verified that too high or too low volume ratios of water to oil were neither advantageous to the formation and stability of NSSPE.(6)Preparation and characterization of three insoluble ingredients NSSPEThe pH of aqueous phase and volume ratio of water to oil were selected to study further based on our previous studies and the levels were set at 11.5 or 12.0,90:10 or 85:15,respectively.Factorial design was used to prepared NSSPE of puerarin,tanshinone ?A and ferulic acid.The stability and drug distribution for all NSSPEs were compared.The microstructure of optimized NSSPEs was characterized by FIM?CLSM?SEM and DSC.In these four processes,Pu-NSSPE prepared by process A that the pH of the aqueous phase is 11.5 and the volume ratio of water to oil is 90:10,Tan ?A-NSSPE prepared by process D that the pH of the aqueous phase is 12.0 and the volume ratio of water to oil is 85:15,FA-NSSPE prepared by process C that the pH of the aqueous phase is 12.0 and the volume ratio of water to oil is 90:10 are still had a very good emulsion appearance and droplet morphology,no significant change in the particle size,and the drug content only slightly decreased after 90 days at room temperature..The interface adsorption rate for puerarin,tanshinone IIA,and ferulic acid were 29.4 %?84.4 % and 22.1 %,respectively.FIM?CLSM?SEM verified that nanocrystls of puerarin,tanshinone IIA,and ferulic acid all can adsorb at the oil-water interface to form a dense shell of solid particles around the droplets.DSC showed the nanocrystals adsorbed at the oil-water interface may change their crystal form compared with crude material.In this study,the effects of various factors on the fabrication and stability of puerarin,tanshinone IIA and ferulic acid NSSPE were compared,and the important factors affecting the preparation of NSSPE and its mechanism were initially revealed.