Characterization and evaluation of lipid cubic particles as an oral drug delivery carrier in caco-2 cells and everted sac models in vitro

Abstract

난용성 약물을 가용화시켜 경구투여시의 생체이용률을 증가 시키기 위해 monoolein, Pluronic F-127과 유기용매로 구성된 liquid precursor 제제를 개발하였다. 이 liquid precursor 제제를 물에 분산하면 수 백 nm 크기의 cubic particle이 형성되며, 이 나노 미립자는 내부에 cubic phase라는 특이한 구조를 지닌다. Cubic phase는 그 내부에 수상과 유상이 공존하므로, 지용성 물질 및 단백질 분자가 고효율로 봉입되며, 효소와 pH에 대해 내부 약물의 안정성을 유지시킬 수 있는 장점을 지닌다. 본 실험에서는 형광물질을 함유한 cubic particle을 제조하여, 인간의 대장암 세포인 Caco-2 cell에 의한 본 미립자의 흡수를 위상차 현미경, 형광현미경, 공초점 레이저 현미경, 투과 전자 현미경으로 관찰하였다. Caco-2 cell과 cubic particle을 배양한 결과 cytosol에서 크기 2 μm 이하의 다수의 지방적이 관찰되었다. Cubic particle에 봉입된 친유성 형광물질의 흡수는 시간에 따라 증가하였으며, 흡수 정도는 미립자의 크기와 온도에 의존하였다. Cubic particle은 담즙산에 의하여 가용화되었으며, 담즙산-monoolein 혼합 미셀을 형성하였다. 담즙산에 의해 가용화될수록 랫트의 everted sac에 의한 봉입된 형광물질의 흡수가 증가하였다. 방사성동위원소를 봉입한 cubic particle을 경구투여 하였을 때, 초기 30분 후 간으로의 도달이 현저하게 증가하였다. Cubic particle은 지용성 물질을 그 내부에 고효율로 봉입시키며, 크기가 작아 표면적이 상대적으로 증가되므로, 소장에서 담즙산에 의한 가용화가 빠르고, 주성분인 모노올레인이 리파제의 소화작용 없이도 장세포로 빠르게 흡수될 수 있는 장점을 지니므로 난용성 약물을 위한 효과적인 경구 투여 시스템으로 사용될 수 있을 것이라 사료된다.;A precursor type oily liquid formulation comprising monoolein, Pluronic F-127 and organic solvent has been prepared as a carrier for lipophilic drugs. When dispersed in water, the liquid precursor formulation produces sub-micron (200-500 nm) sized lipid cubic particles. The morphology and structure of the cubic particles were characterized using small angle X-ray diffraction (SAXS) and low temperature scanning electron microscopy (LTSEM). This particle is a particulate system and has an interior structure of cubic liquid crystalline phase similar to Cubosome^?. Cubic particles were very stable in wide temperature and pH range. Various fluorescence probes were encapsulated, and encapsulation efficiency was dependent on water solubility. The encapsulation efficiency of hydrophobic probes was 100 %. Encapsulation efficiencies of hydrophilic drugs with small molecular weights were very low (40-50 %), but that of a macromolecule like protein drug was very high (ca. 75 %). Morphology change of Caco-2 cells was investigated by using the phase contrast microscopy and transmission electron microscopy. The interaction between cubic particles and human colon adenocarcinoma cells, Caco-2 cells, was studied by encapsulating pyrene in the particles. From fluorescence and confocal microscopy, blue fluorescence resulted from pyrene was localized in the lipid droplets in cell cytosol after incubation with cubic particles. The degree of pyrene absorption encapsulated in nanocubicles was dependent on particle size, incubation time and temperature. The amount of pyrene absorbed by Caco-2 cells was ca. 20 % of the total at 37℃ after an 8-h incubation. When nanocubicles with a bigger average particle size (ca. 600 nm) were applied, the uptake rate was reduced to 10 % under identical experimental conditions. The uptake was only 2 % when incubated at 4℃ with normal sized particles. The cubic particles were easily solubilized by bile salts to produce bile salt-monoolein mixed micelles. As the bile concentration increased, pyrene absorption into the enterocyte of rat everted sac increased. After incubation with cells and cubic particles, lipid droplets were observed in cytosol of enterocyte as the results of lipid absorption.