Preparation And Application Of Nanoparticles Based On Phenylalanine Ethyl Ester Modified Sodium Alginate

Sodium alginate (SA) - phenylalanine ethyl ester (PE) conjugates (PE-SA, SP) were prepared with phenylalanine ethyl ester (PE) as the hydrophobic groups. The self-assembled behavior of the amphiphile in water solution was investigated. Vitamin B₂ (VB₂) was chosen as a model drug to fabricate the drug-loaded SP nanoparticles (VB₂-SP) and the release of VB₂ from the nanoparticles in vitro was analyzed. The human colon carcinoma cell lines (Caco-2) was used to image and measure the cell uptake of nanoparticles. The cytotoxicity of the SP nanoparticles was measured by MTT assay.A series of SP conjugates were synthesized by covalent attachment of PE to SA with amide formation with EDC and NHS mediated reaction. SP conjugates were characterized by FT-IR, 1H NMR. The degree of PE substitution (DS), as elemental analysis determined, was 3.5-4.7 PE group per hundred sugar residues of SA. The SP nanoparticals were prepared by probe sonication in aqueous media. The physicochemical properties of the SP nanoparticals were investigated with dynamic light scattering (DLS), transmission electron microscopy (TEM) and fluorescence spectroscopy. The particle size was decreased with DS increased and the mean diameter of the SP nanoparticles was in the range of 226.7-425.3nm. The TEM observation demonstrated that the SP nanoparticles were almost spherical in shape. The critical aggregation concentration (CAC) of SP nanoparticles decreased with the increasing of DS and the CAC determined for SP1, SP2 and SP3 were 0.20mg/ml, 0.12mg/ml and 0.10mg/ml, respectively.VB₂, as a model nutrient, was entrapped into the SP nanoparticles with probe sonication. The encapsulation efficiency reduced and the drug loading content increased with increasing the feed weight ratio of VB₂ to SP conjugates. The maximum drug loading content of SP1 nanoparticles was determined to 12.76±1.22%. The encapsulation efficiency and the drug loading content increased with the DS increasing. Dialysis method was used to study the release behavior of VB₂ from SP nanoparticles in vitro. VB₂ was sustained released from SP nanoparticles of different DS in 48h. The release profile was significantly influenced by the DS and the pH of the release media. The release rate decreased with the DS increasing and was lower at pH2.0 than that at pH7.4.The cytotoxicity of blank SP nanoparticles with different DS was analyzed by the MTT assay. The cellular uptake of fluorescent SP nanoparticles was assayed and visualized by using fluorescence microplate reader and confocal laser scanning microscopy (CLSM). The blank SP nanoparticles exhibited no cytotoxicity for Caco-2 cells and the cell viability was higher than 95% in 24h except SP3 in 1mg/ml. Cellular uptake of the SP nanoparticles was influenced by the DS, nanoparticle concentration, incubation time and temperature. The cellular uptake efficiency was higher with a higher DS in a lower nanoparticle concentration with longer incubation time. Cellular uptake of SP nanoparticles was inhibited at 4℃. Almost 60% nanoaparticles were internalized into cells within 4h. The laser confocal scanning microscope was used to observe Caco-2 cells uptake of SP nanoparticles, it was indicated that SP nanoparticles were taken up by Caco-2 cells.