| Objective: To prepare chitosan(CS) or hydroxypropyl trimethyl ammonium chloride chitosan(HACC)-modified solid lipid nanoparticles(SLNs) loading docetaxel(DTX) and to study the absorption mechanism of SLNs for their enhanced oral bioavailability.Method: DTX- loaded SLNs were prepared by an emulsion-solvent evaporation method and futher modified with CS or HACC by an e lectrostatic adsorption. The physical properties of three different types of SLNs(DTX-SLNs, CS-DTX-SLNs, HACC-DTX-SLNs) were characterized. In vitro stability of SLNs was studied in simulated gastric and intestinal fluids(SGF and SIF). The in vitro release study was carried out in phosphate-buffered saline(p H 1.2 and p H 6.8) using a dialysis bag method. X-Ray Diffraction(XRD), differential scanning calorimetry(DSC) and fourier transform infrared resonance spectroscopy(FT-IR) were used to observe the status of the drug in nanoparticles. Caco-2 and FAE cell monolayer models were established and confirmed by observing the cell morphology under inverted microscope, measuring the trans-epithelial electrical resistance(TEER), monitoring the differentiation of the alkaline phosphatase on both sides of monolayer using immunofluorescence method. The cell viability of three different types of nanoparticles was determined in Caco-2 cells using the MTT assay. The cellular uptake of SLNs in Caco-2 and FAE cell monolayer models was analyzed using HPLC, flow cytometry, fluorescence microscope and confocal microscope. In addition, the transport of SLNs was also analyzed using HPLC method. The absorption mechanism of HACC-modified SLNs was studied. In vivo pharmacokinetic study of SLNs was conducted in rats. The distribution of HACC-modified nanoparticles was observed by small animal imaging system and the gastrointestinal mucosa irritations were studied by HE staining.Result : According to the surface modification and characterization, CS/HACC-modified SLNs loading DTX were successfully prepared. The SLNs modified with HACC were stable in both SGF and SIF. The release profile of DTX from three different types of SLNs showed the controlled release manner. Caco-2 and FAE cell monolayers were successfully established through examing the related indexes. The results of MTT assay showed that three different SLNs have little toxicity to Caco-2 cells. The cellular uptake of HACC-DTX-SLNs in Caco-2 cells was higher than those of DTX-SLNs and CS-DTX-SLNs, indicating that quaternized chitosan modified SLNs can improve the cellular uptake. The transport ability of SLNs through FAE cell monolayer was stronger than through Caco-2 cell monolayer. The study of endocytosis pathway indicated that HACC-DTX-SLNs were mainly internalized in Caco-2 cells through caveolae- mediated endocytosis, and partly through clathrin- mediated endocytosis and pinocytosis. The western blot results showed that all SLNs could regulate the expression of tight junction related proteins. Especially, the negative regulation of HACC-SLNs on tight junction related proteins was most remarkable and the regulation process was reversible. The TEER values of Caco-2 cells decreased after cultured with three SLNs and the effect was much stronger for CS-DTX-SLNs and HACC-DTX SLNs. The TEER values of Caco-2 cell monolayers were gradually recovered after removing the SLNs. A 2.45- fold increase in AUC of DTX was found in HACC-DTX-SLNs group when compared with that of DTX group. HACC-C6-SLNs were mainly absorbed through the ileum after oral administration to rats, and they were uptaked by the Peyer’s patch more than the common tissue of small intestine. There was no gastrointestinal mucosa irritation of HACC-SLNs during whole experiments.Conclusion:Chitosan- modified SLNs loading DTX were successfully prepared and HACC-DTX-SLNs showed excellent stability and significantly higher oral bioavailability of DTX in various SLNs. The oral absorption mechanism of HACC-DTX-SLNs was related to the phagocytosis of M cells, compactness of tight junction and mainly caveolae- mediated endocytosis. This study provides a theoretical basis for designing novel DTX- loaded SLNs and clarifing their oral absorption mechanism. |
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