Preparation And Evaluation Of Isopropylidene Shikimic Acid Nanoparticles Targeting The Inflammatory Site Of Ulcerative Colitis

Ulcerative colitis(UC)is a chronic,intermittent,recurrent disease and is difficult to cure.Currently,it has become a common disease of digestive system,and is the main cause of chronic diarrhea in patients.This disease usually requires the perpetual treatment,which seriously influence the quality of life of the individuals.The marketed drugs are conventional oral colon targeting agents and enema,the former is likely to result in inaccurate targeting and impact the drug efficacy due to large differences between individuals,the latter one has poor compliance.Nanoparticles or microparticles drug carriers can achieve the topical accumulation by elevated levels of mucus production and an intense particle uptake inside the colitis tissue as a result of an enhanced permeability and the presence of a highly increased number of immune-related cells and persistent release in inflamed colonic mucosal sites.Isopropylidene shikimic acid was selected as a model drug with explicit treatment for ulcerative colitis,intends to build a nanoor microparticle drug delivery system specific targeting inflamed intestinal mucosa.There are main three parts in this paper:Firstly,coumarin 6 loaded nanoparticles was fabricated with different particle sizes and surface modifications which was used to screen the range of particle size and surface modification possessing UC inflammed intestinal mucosa specific targeting ability.Then,ISA loaded nanoparticles was fabricated with specific particle size and surface modification according to the screening results,and its in vitro drug release behavior was examined by dialysis method.Finally,the pharmacokinetics and in vivo drug release profile of ISA loaded nanoparticles were studied to evaluate the colon targeting characteristics.The main researching methods and results of this paper are summarized as follows:1.Preparation of coumarin 6 load PLGA nanoparticles.The influence factors on particle size and entrapment efficiency was studied,including preparation methods,surfactant agents,oil-water phase ratio,PLGA concentrations,ultrasonication time and based on which coumarin 6 loaded PLGA nanoparticles with different ranges of particle size was fabricated by nanoprecipitation and emulsion solvent evaporation methods using 1%PVA as surfactant.The required average size of nanoparticles and microparticleswere 114(NP100)?302(NP300)?557(NP550)?823(NP800)and1093(NP1000)nm by using centrifugation to optimize the particles size distribution,and the PD I values are 0.091?0.007?0.027?0.108 and 0.388,respectively.The range of nanoparticle size distribution was narrow,which satisfied with the size requirement of targeting screening.In order to get the coumarine 6 loaded nanoparticles with the same particle sizes and different surface modification,the 557 nm nanoparticles was rinsed repeatedly to remove the surfactant,and then incubated in the 1%HS15,1%F68,1%TPGS,1%PVA,1%Tween 80,1%Tween 20,1%SDS and 5%mannose aqueous solution for 24 h respectively,centrifuged and reconstructed with deionized water.2.Screening nanoparticle size and surface modification with which nanoparticles could specific targeting inflamed intestinal mucosa.Based on the immune phagocytosis targeting strategy,RAW 264.7 cell was selected as the in vitro models to evaluate effects of particle size and surface modifications of nanoparticles on phagocytosis of RAW 264.7.Then TNBS induced ulcerative colitis rats were administrated the nanoparticles by enema to test in vivo the targeting potential of nanoparticles with different particle size and surface modifications to the inflamed tissue.The in vitro results showed that the phagocytosis RAW 264.7 to nanoparticles presented a size-dependent relationship.As particle size gets bigger,cellular uptake increased.For the nanoparticles with different surface modification,the phagocytosis of RAW 264.7 to mannose was the strongest.The in vivo evaluation results showed that the nanoparticles NP550 accumulated mostly in the inflamed colon.The targeting indexes are ranked NP550(7.2),NP800(3.2),NP1000(2.7),NP300(1.9)and NP100(1.7).For different surface modifications,the accumulation amount of nanoparticles modified by mannose in UC colon was the most and its targeting index achieved 10.5,and the order of targeting index of other modifications of nanoparticles were Tween 20(9.6),SDS(9.2),HS 15(8.5),TPGS(8.3),PVA(7.5),Tween 80(7.4)and F68(7.2).3.Preparation,characterization and in vitro release evaluation of ISA loaded nanoparticles.A modified emulsion solvent evaporation method was finally adopted to prepare ISA loaded nanoparticles.This method using sodium alginate as the inner water phase stabilizer,dropping by CaC12 aqueous solution to the outer water phase after the preparation of W/O/W emulsion during the solvent evaporation to inhibit the diffusion of inner water solvent to the outer by Ca2+cross-linking.Taking the ISA entrapment efficiency and particle size as indicators,the prescription and preparation process of ISA nanoparticles were optimized.And the prepared nanoparticles was characterized and its in vitro release profile was evaluated as well.The prepared ISA nanoparticles presented sphere-like shape by the scanning electron microscope,the average particle size was 574.9nm,and drug loading rate was about 4.5%.The in vitro release of ISA solution and ISA nanoparticles were evaluated at 37? by dialysis method in pH 7.0 phosphate buffer.The results showed that more than 90%of ISA diffused out within 4h of ISA solution,and the slow-releasee property of ISA nanoparticles was significant,of which the release was 35.5%within 24 h and 72.3%of ISA was released within 10 days.3.Pharmacokinetics and in vivo drug release study of IS A nanoparticles in UC model rats.HPLC methods were established to determinate ISA in rat plasma,stomach,intestine,cecum,colon(including rectum)and their relative contents.The methodologies have proved that the established methods all meet with the requirement of the guideline of bioanalytical method validation(Pharmacopoeia of the people's republic of china,2015).So,these methods could be applied to the pharmacokinetics and in vivo drug release studies.The result of pharmacokinetics showed that the plasma concentration reached the peak in a short time(Tmax=0.75 h)and then decreased fast after the oral administration of ISA solution.Compared with ISA solution,the Tmax of ISA nanoparticles prolonged to 6h,and the Cmax of solution group decreased from 16.65?g·mL-1 to 7.74?g·mL-1.Due to the nanoparticles properties of slow-releasee and long retention,the MRT lengthened from 3.22 h to 9.42 h,and the AUC increased from 37.64 to 53.44 ?g·h·mL-1 which means the improvement of bioavailability.The free ISA in the gastrointestinal content was detected.For ISA solution,the quantity of ISA were low in the rectum and colon after oral administration,which proved most drugs were absorbed in the stomach and intestine or were damaged.After the oral administration of ISA nanoparticles,the amount of free ISA in the stomach and intestine was low,but it was 2.14 and 1.75 times as much as that of ISA solution in the rectum and colon due to the slow-release property of nanoparticles.Compared to the solution group,the nanoparticles group has more ISA reach the rectum and colon sites.The free ISA in the gastrointestinal tissues was also determined.After administration ISA nanoparticles,the amount of ISA in stomach and intestine was lower and higher in rectum and colon compared to that of ISA solution.Their highest distributing quantity of ISA of nanoparticles group were 4.07 and 3.58 times as much as the amount of ISA solution group.The nanoparticles group maintained at a higher concentration after 24h compared to solution group,which reflected the retention and slow-release properties of nanoparticles and further revealed the ISA nanoparticles had good colon targeting.According to the study results the pharmacokinetics and distribution of ISA in digestive tissues and contents of ISA solution,the ISA distributed to every sites of digestive tissues quickly while the ISA was absorbed in the blood.While the distributed peak in the digestive tissues showed the positive correlation with the ISA amount in the digestive content.The free ISA amount released from ISA nanoparticles in the rectum and colon was more than that from the upper digestive tract,which showed good colon targeting.The reasons might be:firstly,nanoparticles retained in the intestinal mucosa and released slowly;secondly,sodium alginate which was one of the polysaccharide materials of ISA nanoparticles which could be metabolized by colon flora and resulted in the acceleration of drug dissolution and release.In summary,nanoparticles with particle size about 550 nm and mannose modification has good UC inflamed intestinal mucosa targeting potential,and based on which,the ISA loaded nanoparticles has inflamed intestinal mucosa targeting potential was prepared.The pharmacokinetics and in vivo release studies showed it has good colon targeting behavior.