Studies On Oral Gastrointestinal Transport Of Solid Lipid Nanoparticles

In this study,the preparation process and physico-chemical properties of drug delivery systems using lipid nanoparticles were studied systematically.To provide basic theory and technique for the application of lipid nanoparticles as an oral drug carrier,the transport process via gastrointestinal tract and cellular membrane of lipid nanoparicles were investigated using fluorescent labeling technique.The chemical conjugate of fluorescent material and lipid material was synthesized,and was used as a fluorescent maker to label lipid nanoparticles.Using Caco-2 cell monolayer as small intestine epithelium cell model,the absorption and transport abilities of lipid nanoparticles via small intestine epithelium cell were tested by observation and determination of fluorescence intensity,and the transport mechanism and the influence factors were discussed.The absorption efficiency and route of lipid nanoparticles via gastrointestinal tract were evaluated by intragastric administration and lympha-draining technique.Using paclitaxel as a model drug,the transport and absorption process of lipid nanoparticles via gastrointestinal tract and its bioavailability were inspected.Moreover,the effects of lipid nanoparticles on the cellular membrane permeability and pharmacological action of drug were investigated.The fluorescein isothiocyanate(FITC) labeled otcadecylamine(ODA), otcadecylamine-fluorescein isothiocyanate(ODA-FITC) was synthesized and used as a fluorescence marker to be incorporated into solid lipid nanoparticles(SLN) by solvent diffusion method.The characteristic of the labeled SLN was investigated,as well as its stability in plasma and simulative intestinal fluid.The prepared ODA-FITC loaded SLN had a volume average diameter of 150～300 nm with a low polydispersity index.The loading of ODA-FITC for ODA-FITC loaded SLN was exceed 90%. Until 24 h,maximal leaked amount of ODA-FITC from the SLN under sink condition was less than 8%in plasma,and less than 3%in simulative gastrointestinal fluid of total amount of ODA-FITC loaded in the SLN,respectively.The high content and low leakage of ODA-FITC can be attributed to the same nature of stearic acid and hydrophobic chain in ODA-FITC molecule,which led to firm consolidation between them in preparation of the SLN by solvent diffusion method.Moreover,the ODA-FITC loaded stearic acid SLN concentration in blood or lymph could be simply and quickly determined by fluorescence spectrophotometer.The permeability of lipid nanoparticles through intestinal was carried out in vitro in Caco-2 cell monolayers,and the effects of lipid material,inhibitor,oleic acid,lecithin on permeability were investigated.Comparing to free FITC-ODA,FITC-ODA loaded nanoparticles can penetrate intestine cell very soon,the apparent permeability coefficient of stearic acid monostearin and monostearin nanoparticles were 7×10^-5cm/s and 4.7×10^-5cm/s,respectively,which can fulfil the need of absorption through gastrointestinal.Under the conditions of adding colchicines,chloroquine, formaldehyde,blank nanoparticles and incubate in 4℃,the penetrating rate from apical to basolateral were decreased,but there were no effect on the penetrating from basolateral to apical.The results showed that lipid nanoparticles penetrated across intestinal cell layer by combination of cytophagy and passive diffusion.The apparent permeability coefficient could be increased to 7.9×10^-5cm/s when 20%oleic acid was added in lipid nanoparticles,the penetrating from basolateral to apical was also increased.Which maybe lie on the decreased of melt point and the enhancement of oleic acid.When 20%lecithin was added in lipid nanoparticles,the apparent permeability coefficient was 8.0×10^-5cm/s,there was no effect on the penetrating from basolateral to apical at the same time.It indicated that lecithin improved the affinity to cell membrane,and made the cytophagy easier.In vivo study results showed the transport efficiency(TE) of SLN by oral administration was about 30%.The SLN could be absorbed integrately,and implies a linear absorption mechanism in gastrointestinal tract during certain range of concentration.By the external diversion experiments of the lymph,it can be seen about 77.9%of absorbed SLN was transported into systematic circulation via lymph, and the lymph was the major passage of SLN transport in gastrointestinal tract.The other part of absorbed SLN was transported directly into blood rapidly,perhaps through capillary vessel or intestinal epithelial cell paracellular.The value of TE for all dosages of oral administration is close to each other,which implies a linear absorption mechanism for SLN exists in gastrointestinal tract during certain range of concentration.It could be clearly observed the existence of SLN in blood and lymph after oral administration It can be concluded that the SLN is absorbed integrately into systematic circulation from gastrointestinal tract.When the nanoparticles was modified with PEG,the C_max of nanoparticles in blood was decreased but the t_1/2 was prolonged,the bioavailability was increased to about 50%due to the t_1/2 prolonged.The monostearin lipid nanoparticles loading paclitaxel and PEG midified monostearin lipid nanoparticles loading paclitaxel were prepared by solvent diffusion method.The PEG modification did not affect the particle size and drug encapsulation efficiency significantly.The diameters of lipid nanoparticles were between 180 and 240 nm,and the drug encapsulation efficiencies were between 83%and 95%.The results of Caco-2 cell monolayer permeation test for drug loaded lipid nanoparticles indicated.The PEG modified lipid nanoparticles could significantly increase the transport of paclitaxel via enterocyte.The permeation rate enhanced with increasing the modification ratio of PEG.The permeation rate of lipid nanoparticles loading paclitaxel with 10%PEG modification was five times than that of free drug.After the intragastric administration of lipid nanoparticles against rat,the drug content in blood increased significantly from 2h,the absorption maximum was reached between 6 and 8 h,and drug content in blood could be kept at higher level for longer time. Comparing with the intravenous injection of taxol,the absolute aboavailability of lopid nanoparticles could reach up to 24.35%.The value was 3.72 times higher than that of oral administration of free drug.The results indicated the lipid nanoparticles loading was a potential carrier for oral administration of paclitaxel.The therapeutic effect of drug is mainly implemented via the binding of drug and target.The targets of a number of drugs are inside target cells.The internalization of drug into target cells is another key factor to affect the drug activity,following absorption barrier of gastrointestinal tract.Due to the affinity between the structure of lipid nanoparticles and cellular membrane,the lipid nanoparticles could be uptaken by cells and internalize into cells,and could alter the permeability of biomembrane. Using A549 as a model cell,the cellular uptake ability of ODA-FITC labeled monostearin lipid nanoparticles,monostearin lipid nanoparticles containing oleic acid and FA-SA modified lipid nanoparticles were observed by fluorescence inverted microscopy.The results indicated the addition of oleic acid and the modification of folic acid could improve the cellular uptake of lipid nanoparticles.Comparing with taxol,the cytotoxicity of lipid nanopartcicles loading paclitaxel against SKOV3 cells were increased significantly,and further enhanced by addition of oleic acid and the modification of folic acid.The cytotoxicities were enhanced 3.0 and 4.6 times, respectively.The Multidrug Resistance(MDR) is the critical point responding for most failure of human cancer chemotherapy.The cytotoxicity of paclitaxel(PTX) against human ovarian cancer SKOV3 cells and derived multidrug resistant cells(SKOV3-TR30) was investigated.Comparing to free drug solution,encapsulated in lipid nanoparticles efficiently enhanced the cytotoxicity of PTX against SKOV3 and SKOV3-TR30 cells, the drug resistance of drug resistance cells was 46.61 times higher than drug sensitive cells.The PTX loaded lipid nanoparticles could reverse the drug resistance,the reversal power of PTX lipid nanoparticles and folic acid modified PTX lipid nanoparticles were 31.2 and 28.2 folds,respectively.