The ratio imaging was conducted on fluorescent microscope
(Olympus, IX71-32PH, Shinjuku-ku, Tokyo, Japan). The PLGA microsphere was excited at 335 and 381 nm, and the images emitted at 452 and 521 nm were taken for analysis. The fluorescent intensity was analyzed using the software, WASABI V.1.4. The standard curve of ratio of fluorescent intensity vs. pH was generated by placing the LysoSensor™ Yellow/Blue dextran-loaded dextran nanoparticles at a known pH on a microscope slide. Multiple images were taken at each pH and then averaged to obtain the standard curve. Results and discussion Morphology of dextran nanoparticle The strategy for fabricating dextran nanoparticles loaded with proteins is shown in Figure 1. Briefly, proteins and PEG were dissolved in dextran solutions and aqueous solution, respectively. After these two solutions were mixed Selleck 4SC-202 to get a clear solution, the solution was frozen dried under vacuum and washed with dichloromethane 3-Methyladenine to give fine dextran nanoparticles loaded with proteins. Figure 1 The formulation strategy of fabricating the dextran nanoparticles loaded with proteins. Figure 2 shows SEM images of dextran nanoparticles loaded with BSA (DP-BSA).
DP-BSA exhibit a spherical shape, smooth surfaces, and diameters ranging from 200 to 500 nm. These results are consistent with that of the particle size analysis which shows the effective diameter of 293 nm for DP-BSA (Figure 3). Figure 2 An SEM photo of dextran nanoparticles loaded with BSA. Figure 3 The size distribution of dextran nanoparticles
loaded with BSA. Encapsulation efficiency of dextran nanoparticles As shown in Table 1, the encapsulation efficiency of dextran nanoparticles loaded with different proteins was generally larger than 98%. The recovery of proteins extracted from dextran nanoparticles ranged from 65% to 72%. Some proteins might be washed away by dichloromethane during the preparation Amino acid process. Table 1 The encapsulation efficiency and recovery of dextran nanoparticles ( n = 3) Number Protein Encapsulation efficiency(ave% ± SD) Recovery (%) (ave% ± SD) 1 BSA 99.23 ± 1.69 71.26 ± 2.06 2 GM-CSF 98.37 ± 1.27 69.16 ± 2.78 3 MYO 98.16 ± 1.55 65.57 ± 1.56 Protein aggregation during the formulation steps In order to address this novel dextran nanoparticle that may protect proteins from aggregation during the formulation Selleck Entinostat process, the BSA, GM-CSF, and G-CSF were selected as model proteins, and SEC-HPLC was used to characterize the protein extracted from the protein standard solution, dextran nanoparticle, and controlled W/O emulsion. Figure 4 shows the SEC-HPLC charts of BSA extracted from the BSA standard solution, dextran nanoparticle, and W/O emulsion. The peak of BSA samples around 9.8 and 8.2 min were ascribed to the monomer and dimer BSAs, respectively. As shown in Figure 4, only one peak corresponding to the monomer BSA was observed in the BSA solution and dextran nanoparticle.