固体脂质纳米粒口服药物载体经胃肠道转运的研究
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摘要
在对固体脂质纳米粒给药系统的制备工艺与理化性质进行了系统研究的基础上,采用荧光标记技术,对脂质纳米粒通过胃肠道转运过程及其对细胞膜通透性进行了考察,为脂质纳米粒作为口服药物载体的应用提供理论与技术依据。本研究合成了一种与脂质材料嫁接的荧光物质作为脂质纳米粒的荧光标记物,利用Caco-2单层细胞为小肠上皮细胞模型,通过荧光强度的观察与测定,考察脂质纳米粒通过肠上皮细胞吸收与转运的能力,初步研究其转运的机制和影响转运的因素;通过大鼠灌胃与淋巴引流技术,考察脂质纳米粒胃肠道吸收的效率和吸收途径;以紫杉醇为模型药物,考察载药脂质纳米粒通过肠细胞转运和胃肠道吸收的过程与生物利用度;并进一步考察了脂质纳米粒对细胞膜通透性的影响,以及对药物药理作用的影响。
采用化学嫁接的方式合成一种新型的荧光素嫁接物——硬脂胺-异硫氰基荧光素(Octadecylamine-fluorescein isothiocyanate,ODA-FITC)作为荧光标记物,通过溶剂扩散法,制备荧光标记的硬脂酸、单甘酯等固体脂质纳米粒,测定其粒径、表面电位(Zeta电位)、标记物包封率,考察了标记物在模拟人工胃肠液、血液中的稳定性。采用水性溶剂扩散法制备的荧光标记固体脂质纳米粒,其粒径分布在150～300nm的范围内,呈单峰分布,且多分散系数较小。由于硬脂胺-FITC具有的18碳疏水链与脂质材料的性质相近,亲和力强,从而使两者在形成荧光标记SLN的过程中能够非常紧密地结合,不易分离泄漏。在标记的纳米粒中荧光素的包封率大于90%。体外稳定性考察表明,荧光标记SLN中的荧光素嫁接物在模拟胃肠液中泄漏非常慢,24 h泄漏量小于3%;而在血样中,24 h的泄漏量也在8%以下。
以人结肠癌上皮细胞Caco-2单层细胞为模型,进行脂质纳米粒通过肠上皮细胞的渗透试验,考察不同脂质材料的纳米粒、不同的抑制剂和渗透条件以及添加剂油酸、卵磷脂对纳米粒经肠细胞渗透的影响。与游离的荧光标记物相比,荧光标记的脂质纳米粒可迅速通过肠细胞渗透,在100μg/ml的条件下,硬脂酸与单甘酯脂质纳米粒的渗透系数分别为7×10~(-5)cm/s和4.7×10~(-5)cm/s,能够满足通过胃肠道吸收的要求;在加入抑制剂秋水仙素或氯喹、加甲醛使细胞失活、低温培养和加入未标记纳米粒等试验条件下,纳米粒从肠细胞的浆膜侧向基膜侧的转运速率明显下降,而由基膜侧向浆膜侧的渗透未受明显的影响,说明脂质纳米粒通过肠细胞的渗透是细胞内吞的主动转运和通过肠细胞层脂质通道被动扩散相结合的模式进行转运。在单甘酯脂质纳米粒的处方中添加油酸和卵磷脂可显著提高纳米粒的渗透速率,加入20%(W/W)的油酸可使渗透系数提高到7.9×10~(-5)cm/s,而油酸的加入同样也加快了脂质纳米粒从基膜侧向浆膜侧的转运,这可能是由于加入油酸后脂质的熔点下降,和油酸本身的促进吸收作用引起的;加入20%(W/W)的卵磷脂可使渗透系数提高到8.0×10~(-5)cm/s,而从基膜侧向浆膜侧的转运则无明显变化,可能是卵磷脂的加入提高了纳米粒与细胞膜的亲和性,使纳米粒的细胞摄取更容易进行。
大鼠灌胃荧光标记SLN后,在灌胃后2 h收集的血样和淋巴液中,可以清楚观察到比较强的荧光斑点。随着给药后时间的变化,血液中的纳米粒含量出现了两个峰值。第一个峰出现在1 h左右,说明纳米粒在胃肠道可以较快地被吸收;第二个浓度峰则出现在8 h左右,这可能与纳米粒进入体内后,在组织和血液之间的再分布有关。通过静脉注射荧光标记脂质纳米粒,估算得到固体脂质纳米粒生物转运效率约为30%,在实验设计的给药剂量范围内,随着剂量的增加,SLN浓度-时间曲线下面积(AUC)与给药剂量呈线性相关关系。通过淋巴引流技术,测定胃肠道转运后淋巴液中脂质纳米粒的含量,结果表明,在经胃肠道生物转运的SLN中约有77.9%的SLN经淋巴转运途径进入体循环,另有22.1%的SLN经非淋巴途径转运吸收。经PEG修饰后的脂质纳米粒大鼠灌胃后,纳米粒在血液中的峰浓度有所下降但滞留时间延长,由于在循环系统中停留的时间延长,脂质纳米粒的生物利用度可增加至约50%;随着PEG修饰比例的增加或修饰PEG分子量的增大,血液中纳米粒的峰浓度逐渐下降、滞留时间进一步延长。
采用溶剂扩散法制备紫杉醇单甘酯纳米粒和PEG修饰的紫杉醇单甘酯纳米粒,PEG修饰对载药纳米粒的粒径和紫杉醇的包封率未有明显的影响,纳米粒的粒径在180～240nm,药物包封率为83%～95%。载药纳米粒通过Caco-2单层细胞的渗透试验结果显示,经PEG修饰的脂质纳米粒载体可显著促进紫杉醇通过肠细胞的转运,其渗透速率随着PEG修饰比例的增加而增加,10%PEG修饰的紫杉醇脂质纳米粒的渗透速率是游离药物的5倍。对大鼠灌胃给予紫杉醇脂质纳米粒,在给药2h后血药浓度开始明显增加,至6～8h可产生最大吸收,血药浓度维持时间比较长,与静脉注射相比,其绝对生物利用度可达到24.35%,为游离药物直接口服给药的3.72倍,为紫杉醇制剂的口服给药提供了可能。
药物主要通过占位效应实现药物的最终疗效,大部分药物的靶点位于靶细胞内,药物分子渗透进入靶细胞内是口服药物继胃肠道吸收屏障外另一影响药物活性作用发挥的因素。脂质纳米粒利用其结构与细胞膜的亲和性,可经细胞摄取转运至细胞内,从而改变了生物膜的通透性。以A549细胞为模型细胞,用ODA-FITC标记含单甘酯纳米粒、含有油酸的脂质纳米粒和经叶酸-硬脂酸嫁接物修饰的脂质纳米粒,荧光倒置显微镜观察并比较细胞对不同脂质纳米粒的摄取能力。研究表明,随着纳米粒中油酸的加入和叶酸修饰,A549细胞对纳米粒的摄取增强;与游离药物相比,载紫杉醇脂质纳米粒对SKOV3细胞的毒性也显著增强,且随着油酸的加入和叶酸修饰,药物的毒性分别增加至3.0和4.6倍。
多药耐药(multidrug resistance,MDR)是当前临床上恶性肿瘤化疗遭遇失败的关键因素。以耐紫杉醇的卵巢癌细胞(SKOV3-TR30)考察载紫杉醇脂质纳米粒的药效,MTT法测定紫杉醇对耐药细胞SKOV3-TR30及其敏感乳细胞SKOV3的细胞毒性,耐药细胞的耐药倍数为敏感细胞的58.4倍。以紫杉醇溶液为对照,以MTT法测定载药纳米粒对耐药细胞SKOV3-TR30及其敏感乳细胞SKOV3的细胞毒性,紫杉醇载药脂质纳米粒和叶酸修饰的紫杉醇载药脂质纳米粒对耐药细胞的逆转倍数分别为31.2和28.2倍,能够逆转耐药肿瘤细胞的耐药性。
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~(-5)cm/s and 4.7×10~(-5)cm/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~(-5)cm/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~(-5)cm/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.
采用化学嫁接的方式合成一种新型的荧光素嫁接物——硬脂胺-异硫氰基荧光素(Octadecylamine-fluorescein isothiocyanate,ODA-FITC)作为荧光标记物,通过溶剂扩散法,制备荧光标记的硬脂酸、单甘酯等固体脂质纳米粒,测定其粒径、表面电位(Zeta电位)、标记物包封率,考察了标记物在模拟人工胃肠液、血液中的稳定性。采用水性溶剂扩散法制备的荧光标记固体脂质纳米粒,其粒径分布在150～300nm的范围内,呈单峰分布,且多分散系数较小。由于硬脂胺-FITC具有的18碳疏水链与脂质材料的性质相近,亲和力强,从而使两者在形成荧光标记SLN的过程中能够非常紧密地结合,不易分离泄漏。在标记的纳米粒中荧光素的包封率大于90%。体外稳定性考察表明,荧光标记SLN中的荧光素嫁接物在模拟胃肠液中泄漏非常慢,24 h泄漏量小于3%;而在血样中,24 h的泄漏量也在8%以下。
以人结肠癌上皮细胞Caco-2单层细胞为模型,进行脂质纳米粒通过肠上皮细胞的渗透试验,考察不同脂质材料的纳米粒、不同的抑制剂和渗透条件以及添加剂油酸、卵磷脂对纳米粒经肠细胞渗透的影响。与游离的荧光标记物相比,荧光标记的脂质纳米粒可迅速通过肠细胞渗透,在100μg/ml的条件下,硬脂酸与单甘酯脂质纳米粒的渗透系数分别为7×10~(-5)cm/s和4.7×10~(-5)cm/s,能够满足通过胃肠道吸收的要求;在加入抑制剂秋水仙素或氯喹、加甲醛使细胞失活、低温培养和加入未标记纳米粒等试验条件下,纳米粒从肠细胞的浆膜侧向基膜侧的转运速率明显下降,而由基膜侧向浆膜侧的渗透未受明显的影响,说明脂质纳米粒通过肠细胞的渗透是细胞内吞的主动转运和通过肠细胞层脂质通道被动扩散相结合的模式进行转运。在单甘酯脂质纳米粒的处方中添加油酸和卵磷脂可显著提高纳米粒的渗透速率,加入20%(W/W)的油酸可使渗透系数提高到7.9×10~(-5)cm/s,而油酸的加入同样也加快了脂质纳米粒从基膜侧向浆膜侧的转运,这可能是由于加入油酸后脂质的熔点下降,和油酸本身的促进吸收作用引起的;加入20%(W/W)的卵磷脂可使渗透系数提高到8.0×10~(-5)cm/s,而从基膜侧向浆膜侧的转运则无明显变化,可能是卵磷脂的加入提高了纳米粒与细胞膜的亲和性,使纳米粒的细胞摄取更容易进行。
大鼠灌胃荧光标记SLN后,在灌胃后2 h收集的血样和淋巴液中,可以清楚观察到比较强的荧光斑点。随着给药后时间的变化,血液中的纳米粒含量出现了两个峰值。第一个峰出现在1 h左右,说明纳米粒在胃肠道可以较快地被吸收;第二个浓度峰则出现在8 h左右,这可能与纳米粒进入体内后,在组织和血液之间的再分布有关。通过静脉注射荧光标记脂质纳米粒,估算得到固体脂质纳米粒生物转运效率约为30%,在实验设计的给药剂量范围内,随着剂量的增加,SLN浓度-时间曲线下面积(AUC)与给药剂量呈线性相关关系。通过淋巴引流技术,测定胃肠道转运后淋巴液中脂质纳米粒的含量,结果表明,在经胃肠道生物转运的SLN中约有77.9%的SLN经淋巴转运途径进入体循环,另有22.1%的SLN经非淋巴途径转运吸收。经PEG修饰后的脂质纳米粒大鼠灌胃后,纳米粒在血液中的峰浓度有所下降但滞留时间延长,由于在循环系统中停留的时间延长,脂质纳米粒的生物利用度可增加至约50%;随着PEG修饰比例的增加或修饰PEG分子量的增大,血液中纳米粒的峰浓度逐渐下降、滞留时间进一步延长。
采用溶剂扩散法制备紫杉醇单甘酯纳米粒和PEG修饰的紫杉醇单甘酯纳米粒,PEG修饰对载药纳米粒的粒径和紫杉醇的包封率未有明显的影响,纳米粒的粒径在180～240nm,药物包封率为83%～95%。载药纳米粒通过Caco-2单层细胞的渗透试验结果显示,经PEG修饰的脂质纳米粒载体可显著促进紫杉醇通过肠细胞的转运,其渗透速率随着PEG修饰比例的增加而增加,10%PEG修饰的紫杉醇脂质纳米粒的渗透速率是游离药物的5倍。对大鼠灌胃给予紫杉醇脂质纳米粒,在给药2h后血药浓度开始明显增加,至6～8h可产生最大吸收,血药浓度维持时间比较长,与静脉注射相比,其绝对生物利用度可达到24.35%,为游离药物直接口服给药的3.72倍,为紫杉醇制剂的口服给药提供了可能。
药物主要通过占位效应实现药物的最终疗效,大部分药物的靶点位于靶细胞内,药物分子渗透进入靶细胞内是口服药物继胃肠道吸收屏障外另一影响药物活性作用发挥的因素。脂质纳米粒利用其结构与细胞膜的亲和性,可经细胞摄取转运至细胞内,从而改变了生物膜的通透性。以A549细胞为模型细胞,用ODA-FITC标记含单甘酯纳米粒、含有油酸的脂质纳米粒和经叶酸-硬脂酸嫁接物修饰的脂质纳米粒,荧光倒置显微镜观察并比较细胞对不同脂质纳米粒的摄取能力。研究表明,随着纳米粒中油酸的加入和叶酸修饰,A549细胞对纳米粒的摄取增强;与游离药物相比,载紫杉醇脂质纳米粒对SKOV3细胞的毒性也显著增强,且随着油酸的加入和叶酸修饰,药物的毒性分别增加至3.0和4.6倍。
多药耐药(multidrug resistance,MDR)是当前临床上恶性肿瘤化疗遭遇失败的关键因素。以耐紫杉醇的卵巢癌细胞(SKOV3-TR30)考察载紫杉醇脂质纳米粒的药效,MTT法测定紫杉醇对耐药细胞SKOV3-TR30及其敏感乳细胞SKOV3的细胞毒性,耐药细胞的耐药倍数为敏感细胞的58.4倍。以紫杉醇溶液为对照,以MTT法测定载药纳米粒对耐药细胞SKOV3-TR30及其敏感乳细胞SKOV3的细胞毒性,紫杉醇载药脂质纳米粒和叶酸修饰的紫杉醇载药脂质纳米粒对耐药细胞的逆转倍数分别为31.2和28.2倍,能够逆转耐药肿瘤细胞的耐药性。
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~(-5)cm/s and 4.7×10~(-5)cm/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~(-5)cm/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~(-5)cm/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.
引文
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[15] 《药剂学》中国医药科技出版社 陆彬主编 442-443
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[1]R.H.M(u|¨)ller,M.Radtke,S.A.Wissing.Nanostructured lipid matrices for improved microencapsulation of drugs.International Journal of Pharmaceutics.242(2002),121-128.
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[4]Jenning,V.,1999.Feste Lipid-Nanopartikel(SLN) als Tr(a|¨)gersystem f(u|¨)r die dermale Applilkation von Retinol.Ph.D.thesis,FU,Berlin.
[5]Radtke,M.,2002.Nanostructured lipid carriers(NLC):Untersuchungen zur Struktur,Wirkstoffinkorporation und Stabilit(a|¨)t.Ph.D.thesis,FU,Berlin,in preparation.
[6]E.B.Souto,S.A.Wissing,C.M.Barbosa,R.H.Mailer,Development of a controlled release formulation based on SLN and NLC for topical clotrimazole delivery.International Journal of Pharmaceutics.278(2004),71-77.
[7]Nancy S.Chung,Kishor M.Wasan,Potential role of the low-density lipoprotein receptor family as mediators of cellular drug uptake.Advanced Drug Delivery Reviews.56(2004),1315-1334
[8]丁建潮,胡富强,袁弘.A549细胞对单硬脂酸甘油酯固体脂质纳米粒的摄取作用.药学学报,2004,39(11):876-880.
[9]Jenning V,Andreas FT,Gohla SH.Characterisation of a novel solid lipid nanoparticle carrier system based on binary mixtures of liquid and solid lipids.Int J Pharm,2000,199:167-177.
[10]Hu Y,Xie JW,Tong YW,Wang CH.Effect of PEG conformation and particle size on the cellular uptake efficiency of nanoparticles with the HepG2 cells.J Control Rel,2007,118:7-17.
[11]Serpe L,Catalano MG.,Cavalli R,Ugazio E,Bosco O,Canaparo R,Muntoni E, Frairia R, Gasco MR, Eandi M, Zara GP. Cytotoxicity of anticancer drugs incorporated in solid lipid nanoparticles on HT-29 colorectal cancer cell line. Eur J Pharm Biopharm 2004, 58: 673-680.
[12] Mahesh DC, Yogesh P, Jayanth P. Susceptibility of nanoparticle-encapsulated paclitaxel to P-glycoprotein-mediated drug efflux. Int J Pharm, 2006, 320: 150-156.
[2]Morazzoni P, Magistretti MJ Giachetti C, et al Comparative bioavailability of silipide, a new flavanolignan complex, in rats[J].Eur J Drug Metab Pharmacokinet, 1992,17(1):39-44.
[3] Schandalik R, Gatti G, Perucca E, et dl Pharmacokinetics of silybin following administration of silipide and silymarin in cholecystectomy patients [J], Arzneim-Forsch, 1992, 42(7):964-968.
[4] Ahn KJ, Him KM, Choi JS, et al , Effects of cyclodextrin derivatives on bioavailability of ketioprofen[J]. Drug Dev Ind Pharm, 1997, 23(4): 397-401.
[5]Tokumura T, Nanba M,Tsushima Y, et al. Enhancement of bioavailability of cinnarizine from its β-cyclodextrin complex on oral administration with.DL-phenylalanine as a competing agent [J]. J Pharm Sci. 1986,75(2):391-394.
[6] Vila Jato JL,Blanco J,Alonso MJ. et al.The effect of the molecular weight of polyethylene glycol on the bioavailability of parcetamol -polyethylene glycol solid dispersions [J]. J Pharm Pharmacol 1986,38(2): 126-128.
[7] Habib MJ,Phan MT. Owusu-Ababio G,et al .Dissolution profiles of flurbiprofen in phospholipid solid dispersions [J].Drug Dev Ind Pharm .,1998,24(11): 1077-1082.
[8]Tsuji Y,Kakegawa H,Miyataka H,et al, Pharmaceutical properties of freeze-dried formulations of egg albumin ,severel drugs and olive [J].Biol .Pharm Bull 1996,19(4):636-640.
[9] Sugimoto M, Okagaki T ,Narisawa S ,et al .Improvement of dissolution characteristics and bioavailability of poorly water -soluble drugs by novel cogrinding method using water-soluble polymer [J].Int J Pharm,1998,160(1):11-19.
[10] R.H. Muller, M. Radtke, S.A. Wissing, Nanostructured lipid matrices for improved microencapsulation of drugs, Int. J. Pharm. 242 (2002) 121-128.
[11] M. Radtke, R.H. Muller, Comparison of structural properties of solid lipid nanoparticles (SLN) versus other lipid particles, Proc. Int. Symp. Control. Rel. Bioact. Mater. 27 (2000) 309-310.
[12] P. Shahgaldian, E. Da Silva, A.W. Coleman, B. Rather, M.J. Zaworotko, Para-acyl-calix-arene based solid lipid nanoparticles(SLN):a detailed study of preparation and stability parameters, Int. J. Pharm. 253 (2003) 23-38.
[13] C. Olbrich, A. Geβner, O. Kayser, R.H. Muller, Lipid- drug conjugate (LDC) nanoparticles as novel carrier system for the hydrophilic antitrypanosomal drug diminazenediaceturate, J. Drug Target. 10 (5) (2002) 387-396.
[14] C. Olbrich, A. Geβner, O. Kayser, R.H. Muller, Lipid-drugconjugate (LDC) nanoparticles as an alternative carrier system with high drug content, Proc. Int. Symp. Control. Rel. Bioact. Mater. 27 (2000) 295-296.
[15] 《药剂学》中国医药科技出版社 陆彬主编 442-443
[16] Muranishi, S., Fujita, T., Murakami, M., et al,. Potential for lymphatic targeting of peptides, J. Control. Release, 1997,46,157-164.
[17] Porter, C.J.H., Drug delivery to the lymphatic system, Crit. Rev. Drug Carrier Syst. 1997.14, 333-393.
[18] Eldridge, J.H., Hammond, C.J., Meulbroeck, J.A., et al. Controlled vaccine release in the gut associated lymphoid tissues. I. Orally administered biodegraadable micro-spheres to target the Peyer's patches, J. Control. Release, 1990, 11,205-214.
[19] Hawley, A.E., Davis, S.S., Ilium, L., Targeting of colloids to lymph nodes: influence of lymphatic physiology and colloidal characteristics, Adv. Drug Deliv. Rev. 1995,17, 129-148.
[20] Porter, C.J.H., Charman, W.N., Uptake of drugs into the intestinal lymphat after oral administration, Adv. Drug Deliv. Rev. 1997,25, 71-89.
[21] Desai M A, Vadgama P. Estimation of effective diffusion coefficients of model solutes through gastric mucus: assessment of a diffusion chamber technique based on spectro-photometric analysis [J]. Analyst, 1991 (116): 1113—1116.
[22] Bockman D E, Winborn W B. Light and intestinal microscopy of intestinal ferritin. Observations in sensitised and non-sensitised hamsters [J]. Anat Rec, 1966(155): 603-622.
[23] Winne D, Verheyen W. Diffusion coefficient in native mucus gel of rat small intestine [J]. J Pharm Pharmacol, 1990 (42) :517-519.
[24] Volkheimer G, Schultz F H. The phenomenon of persorption [J]. Digestion, 1968 (1): 213-218.
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