Preparation Of Zein Submicron Particles And Its Distribution In Vivo

Recently submicron particle delivery system has been recognized promising in pharmaceutical area.The carrier material is one of the most important factors that decide the physico-chernical properties of submicron particles.Synthetic polymers such as poly(lactic acid)(PLA)and poly(lactic-co-glycolic acid)(PLGA)have been widely studied because they are biodegradable and possess good physio-chemical properties. However,PLGA underwent bulk erosion that produced a very low pH environment within PLGA matrices,which may adversely affect sensitive therapeutic agents such as proteins or peptides.Natural polymers such as gelatin,albumin and casein presented a main drawback of a rapid solubilization in aqueous environments,resulting in fast drug release profiles.Zein is the insoluble protein of corn.As a natural polymer,zein is biocompatible and biodegradable and could be widely used.The study of zein submicron particles as drug cartier and its distribution in vivo haven't been reported in the literature until now.5-FU is an antimetabolite of the pyrimidine analog class.It is a widely used antineoplastic agent and is mainly for the therapy of gastro-intestinal cancer and liver cancer.There was no cross-resistance when it was combined with other antineoplastic agent.However,5-FU caused side effects such as reduction of white blood cell and platelet,lack of appetite,causing nausea,vomiting,diarrhea,etc.However,5-FU submicron particles will reduce the toxicity,prolong the half life,increase the bioavailability and decrease the frequency of administration of the drug.Ibuprofen is an nonsteroidal anti-inflammatory drug with analgesic and antipyretic properties.Its chemical name is 2-(p-isobutylphenyl)propionic acid.Ibuprofen has poor solubility and short half life(1.5～2h)and must be administerd frequently for keeping effective drug concentration.Moreover,ibuprofen will cause side effects such as hypopepsia, nausea,bellyache,heartburn,etc.when it is administered for a long time.For these reasons,delayed release of ibuprofen are used clinically.Literature reported that ibuprofen submicron particles were prepared for delayed release application.In the present study,different solubility of 5-FU and ibuprofen as model drugs were encapsulated into zein submicron particles using phase separation technique.The physio-chemical properties and in vitro dissolution of zein submicron particles were investigated.The distribution of zein submicron particles in mice was studied by fluorospectrophotometry after rhodamine B was encapsulated into zein submicron particles.These studies provided scientific experimental and theoretical basis for zein submicron particles as drug delivery system.The main methods and results were as follows:1.High speed centrifugation method was applied to separate free drug and the zein submicron particles.The encapsulation efficiency of the prepared submicron particles was determined by UV method.2.The preparation and formulation of 5-FU-loaded zein submicron particles were studied.The encapsulation efficiency was used to evaluate the effects of the proportion of drug and zein,zein concentration,pH of the aqueous phase,time and speed of agitation and proportion of water and ethanol.5-FU-loaded zein submicron particles were optimized using uniform experimental design concerning the encapsulation efficiency.3.The physio-chemical properties,stability and dissolution of 5-FU-loaded zein submicron particles in vitro were investigated.The formation of 5-FU-loaded zein submicron particles was studied by differential scanning calorimetry(DSC).The optimized submicron particles were sphere and homogeneous.The average particle size was 363.2nm.The encapsulation efficiency and drug loading of the optimized submicron particles were(60.7±1.74)%and(9.17±0.11)%respectively,and the zeta potential was-45.75 mV. Compared with 5-FU solution,in vitro release of 5-FU-loaded zein submicron particles in the different mediums(phosphate buffered saline pH7.4,simulated gastric fluid,simulated intestinal fluid and water)was slower.The in vitro release of 5-FU submicron particles in the different mediums could be described by double phase kinetics model and expressed by the following equations respectively:100-Q =86.95e^-0.3505t+42.91e^-0.0121t(pH7.4PBS),100-Q=100.31e^-0.2061t+50.19e-^0.0361t (simulated gastric fluid),100-Q=111.71e^-1.0714t+21.55e^-0.0454t(simulated intestinal fluid)and 100-Q=103.27e^-0.538t+27.34e^-0.0716t(water).In case of solution,the release profile of 5-FU could be described by first order kinetics model and could be expressed by the following equations:In(100-Q)=-2.29t+ 4.6044(pH7.4PBS), ln(100-Q)=-5.2894t+4.5812(simulated gastric fluid),ln(100-Q)=-6.3204t+ 4.6224(simulated intestinal fluid)and in(100-Q)=-4.0835t+4.5833(water), respectively.The stability experiment showed that 5-FU-loaded zein submicron particles precipitated when stored at 40℃for one month.At room temperature(25℃)the shape of submicron particles was unchanged after three months,while submicron particles were precipitated after six months.When 5-FU-loaded zein submicron particles are stored in refrigerator at the temperature of 4℃for six months,the encapsulation efficiency and the drug loading of 5-FU-loaded zein submicron particles changed little. Therefore,5-FU-loaded zein submicron particles could be stored at 4℃.4.The physio-chemical properties and dissolution of ibuprofen-loaded zein submicron particles in vitro were investigated.The encapsulation efficiency of the prepared submicron particles was(96.7±0.63)%.The mean particle size of ibuprofen-loaded zein submicron particles was 218.8nm and the zeta potential was 7.20mV.In vitro release of ibuprofen from submicron particles in phosphate buffered saline(pH7.4)could be described by double phase kinetics model and expressed by the following equation:100-Q=96.43e^-0.3975t+39.09e^-0.0062t.In case of solution,the release profile of ibuprofen in phosphate buffered saline(pH7.4)could be described by first order kinetics model and could be expressed by the following equation:ln(100-Q)= -1.5307t+4.7608. 5.The distribution of zein submicron particles in mice was studied.After rhodamine B was encapsulated into zein submicron particles,the distribution of rhodamine B in tissues(heart,liver,spleen,lung,kidney,plasma)was studied by fluorospectrophotometry after intravenous administration compared to rhodamine B solution.The distribution of rhodamine B in mice after administrated rhodamine B -loaded zein submicron particles was different from rhodamine B solution.Rhodamine B mainly distributed in liver,and the targeting efficiency increased 31.33% while the targeting efficiency in kidney decreased 25.32%.The concentration of rhodamine B in liver after administration of rhodamine B-loaded zein submicron particles was 2.13 times than administration of rhodamine B solution.The relative uptake rate of liver was 2.79.The results showed that zein submicron particles could be targeted to liver.At present,neither reports about the study of 5-FU-loaded zein submicron particles and ibuprofen-loaded zein submicron particles in the literature,nor reports about distribution of zein submicron particles in vivo.Our studies provided scientific experimental and theoretical basis for further study of zein submicron particles as drug delivery system.