The Study On A Novel Oral Drug Delivery Device Liposome-in-chitosan-microspheres Loaded Ferulic Acid

Background:Liposome and chitosan microspheres were two common drug vehicles.Both had its own characterizations.Liposome had better biocompatibility.which could promote absorption of drug.but was instabile.chitosan microspheres were normally more stable.and had unique cationic property, gel forming ability and bioadhesive property. We realized that an appropriate combination of liposome and chitosan microspheres could integrate their advantages, avoid their disadvantages.and thus lead to new applications.especially in oral administration. The new drug delivery delivery system could prompt the absorption and delay the release of insoluble drug with short half-life.Objectives:In this research. Ferulic acid was selected to be model drug to prepare liposomes-in-chitosan microsphere(LICM).Liposomes loaded Ferulic acid were prepared firstly and then were incorporated into chitosan microsoheres.The preparation technology and the structure of LICM.the releasing mechanism in vitro and absorption in vivo of drug were investigared.which could provid reference for the preparation and evaluation LICM, a new drug delivery system for oral administration which integrated advantages of liposomes and chitosan microspheres.Methods and Results:1. The preparation and characterization of FA-CMFirstly.chitosan microsphere loaded Ferulic acid(FA-CM)were prepared by onic gelation method and emulsion cross-link method. The mono-varied experimental design was used to optimize technology with as indexes. As far as onic gelation method, sodium tripolyphosphate was a proper gel agent.The entrapment efficiency and drug loading could be improved significantly by decreasing the pH and volume of sodium tripolyphosphate solution.or adding the amount of Ferulic acid.The FA-CM, whose mean entrapment efficiency was 73.77±1.89%%,mean drug loading was were 8.08±0.27 % and mean size was 1.13±0.11 mm was prepared. In another preparation technology.emulsion cross-link method, the entrapment efficiency and drug loading were affected by oil composition, emulsifier composition, phase volume ratio, amount of glutaraldehyde,concentration of chitosan and amount of Ferulic acid.To obtain higher entrapment efficiency and drug loading, dimethicone was used as oil phase,2.5% chitosan solution was used as water phase.and tween80 and span 80 mixed at a ratio of 1:1 was ued as emulsifier.Reducing phase volume ratio.raising amount of glutaraldehyde and Ferulic acid could increase entrapment efficiency and drug loading.The average entrapment efficiency of optimized FA-CM was 76.33±2.07%.average drug loading was were 5.75±0.14% and average size was 88.978±12.41μm.2. The preparation and absorption of FA-LPLiposomes loaed Ferulic acid(FA-LP) were prepared by calcium acetate gradient method. The mono-varied experimental design was used to study the incubation condition of drug loading and Doehlert design was used to optimize prescription, which included the amount of Ferulic acid.the mole ratio of phospholipid to drug and the mole ratio of phospholipid to cholesterol. The average entrapment efficiency of optimized liposome was 79.97=0.54%.the average size was 187.6±11.9nm and the Zeta potential was-12.67±1.78. The absorption of FA-LP and Ferulic acid solution were compared by rat intestines recirculation experiments.The results demonstrated that liposome could promote absorption of Ferulic acid. The absorption rate of liposome in duodenum, jejunum, ileum. colon and complete intestine were as 1.78,1.73,1.97,1.57 and 1.87 times as Ferulic acid solution respectively. The absorption amount at 3h of liposome in duodenum, jejunum, ileum. colon and complete intestine were higher 63.58%,54.52%,65.62%,64.76%and 31.22% than Ferulic acid solution respectively.3. The preparation, characterization and release of FA-LICMThen FA-LP was incorporated into CM by onic gelation method and emulsion cross-link method。The effect of the concentration of chitosan. the volume ratio of chitosan solution and liposome, the concentration and pH of sodium tripolyphosphate solution or the concentration of chitosan. the volume ratio of chitosan solution and liposome. the concentration of emulsifier and the amount of glutaraldehyde on drug loading were investigated respectively.The drug loading of liposomes-in-chitosan microsphere prepared by onic gelation method(LICMi) were between 0.317% and 0.694% and increasing the pH of sodium tripolyphosphate solution or adding the amount of liposome could raise drug. loading. The drug loading of liposomes-in-chitosan microsphere prepared by emulsion cross-link method(LICMe) were between 0.426% and 0.974% and could be improved if the phase volume ratiooremulsifier concentration were reduced properly or the amount of liposome or glutaraldehyde were raised.A law was found through the release experiments in vitro.that was the release rate of liposome from LICM was near to that of drug from CM.Ferulic acid was released 39.71% at 2h.79.18% at 8h and 89.31% at 12h from CMi.Liposome was released 15-30% at 2h.55-80% at 8h and 65-90% at 12h from various LICMi.There was a remarkable burst effect in CMe and LICMe.74.7% Ferulic acid and about 70% liposome were released from CMe and LICM at 2h respectively.All these release were in accordance with weibull distribution.These verified that the release properity of CM was not changed when liposome was iocoporated into CM insead of Ferulic acid.The sizes of liposome released were raised from 115.3 mn to 330±41 nm (LICMi) or 209±18nm (LICMe).The release rate of drug from LICM was much slower than from CM. Ferulic acid was released 5-6% at 2h.30-40% at 12h and 55-75% at 24h from various LICMi.which fitted weibull distribution and zero-order kinetics.The release rates of Ferulic acid from various LICMe were 20-30%at 2h.50-60% at 12h and 65-70% at 24h, which also fitted weibull distribution. The drug releaseof LICMi was slower than LICMe.and this was in line with the fact that the size of liposome released from LICMi was larger than from LICMe. Therefore, it could be concluded that the drug release from LICM should be devided into two steps:liposome was released from LICM firstly, and then drug was released from liposome.The structure of LICM was demonstrated by scanning electron microspheres. differential scanning calorimetry and X-ray diffractometry. All microspheres were sphericity or spheroidicity with a dimater of 1.17±0.15mm for LICMi or 123.754±15.74μm for LICMe. There was much microcrystal of Ferulic acid in the transverse section of CMi,while no microcrystal was founded in the transverse section of LICMi.Liposomes were distributed in microspheres intactly.The surface of CMe was smooth and compact while the surface of LICMe was incomplete and had many pores. There was a remarkable heat absorption peak at170℃in the DSC curves of CMi or crystallization diffraction peaks in the X-ray diffractometry pattern, which demonstrated that Ferulic acid was distributed in CMi in microcrystal form. The heat absorption peak or crystallization diffraction peaks of Ferulic acid disappeared in corresponding patterns of CMe.which verified that Ferulic acid was distributed in CMi in amorphous form. The heat absorption peak or crystallization diffraction peaks of Ferulic acid also disappeared in LICMi and LICMe.Considering the release results of liposome and drug, it could be concluded that the structure of LICM was that Ferulic acid was incorporated into liposomes.and liposomes were distributed in CM intactly.4. The pharmacokinetics of FA-LICMThe pharmacokinetics was investigated by Sprague-Dawley rats. Three group of rats were admintted orally Ferulic acid. FA-CM and FA-LICM respectively. At determined time. the blood was obtained and plasma protein was precipited by methanol. The concentration of Ferulic acid in blood was analyzed by HPLC method using coumarin as internal standard, methol-0.3% acetic acid (42:58) as mobile phase at 320 nm. The data were analysed by DAS program. The tmax.MRT and t1/2βwere as follows: FA-LICM.2.5±0.354h.7.487±0.248h and 7.818±1.161h respectively;Ferulic acid.0.15±0.038h.1.365±0.091h and 1.992±0.491h:FA-CM.1±0.354h.4.171=0.149h and 4.857±0.997h. The AUC of FA-LICM was 18.331±2.846μg·L-1·h-1,which was asr 76.08 times as Ferulic acid and 2.21 times as FA-CM.Conclusion:All these studies verified that LICM integrated advantages of liposomes and chitosan microspheres, so could enhance absorption and delay drug release.which was a new drug delivery system for oral administration with good application prospersity.