To prepare chitosan-based polymeric amphiphiles that can form nanosized core-shell structures (nanoparticles) in aqueous milieu, chitosan oligosaccharides (COSs) were modified chemically with hydrophobic cholesterol groups. The physicochemical properties of the hydrophobized COSs (COSCs) were inves...
To prepare chitosan-based polymeric amphiphiles that can form nanosized core-shell structures (nanoparticles) in aqueous milieu, chitosan oligosaccharides (COSs) were modified chemically with hydrophobic cholesterol groups. The physicochemical properties of the hydrophobized COSs (COSCs) were investigated by using dynamic light scattering and fluorescence spectroscopy. The feasibility of applying the COSCs to biomedical applications was investigated by introducing them into a gene delivery system. The COSCs formed nanosized self-aggregates in aqueous environments. Furthermore, the physicochemical properties of the COSC nanoparticles were closely related to the molecular weights of the COSs and the number of hydrophobic groups per COS chain. The critical aggregation concentration values decreased upon increasing the hydrophobicity of the COSCs. The COSCs efficiently condensed plasmid DNA into nanosized ion-complexes, in contrast to the effect of the unmodified COSs. An investigation of gene condensation, performed using a gel retardation assay, revealed that $COS6(M_n=6,040 Da)$ containing $5\%$ of cholesteryl chloroformate (COS6C5) formed a stable DNA complex at a COS6C5/DNA weight ratio of 2. In contrast, COS6, the unmodified COS, failed to form a stable COS/DNA complex even at an elevated weight ratio of 8. Furthermore, the COS6C5/DNA complex enhanced the in vitro transfection efficiency on Human embryonic kidney 293 cells by over 100 and 3 times those of COS6 and poly(L-lysine), respectively. Therefore, hydrophobized chitosan oligosaccharide can be considered as an efficient gene carrier for gene delivery systems.
To prepare chitosan-based polymeric amphiphiles that can form nanosized core-shell structures (nanoparticles) in aqueous milieu, chitosan oligosaccharides (COSs) were modified chemically with hydrophobic cholesterol groups. The physicochemical properties of the hydrophobized COSs (COSCs) were investigated by using dynamic light scattering and fluorescence spectroscopy. The feasibility of applying the COSCs to biomedical applications was investigated by introducing them into a gene delivery system. The COSCs formed nanosized self-aggregates in aqueous environments. Furthermore, the physicochemical properties of the COSC nanoparticles were closely related to the molecular weights of the COSs and the number of hydrophobic groups per COS chain. The critical aggregation concentration values decreased upon increasing the hydrophobicity of the COSCs. The COSCs efficiently condensed plasmid DNA into nanosized ion-complexes, in contrast to the effect of the unmodified COSs. An investigation of gene condensation, performed using a gel retardation assay, revealed that $COS6(M_n=6,040 Da)$ containing $5\%$ of cholesteryl chloroformate (COS6C5) formed a stable DNA complex at a COS6C5/DNA weight ratio of 2. In contrast, COS6, the unmodified COS, failed to form a stable COS/DNA complex even at an elevated weight ratio of 8. Furthermore, the COS6C5/DNA complex enhanced the in vitro transfection efficiency on Human embryonic kidney 293 cells by over 100 and 3 times those of COS6 and poly(L-lysine), respectively. Therefore, hydrophobized chitosan oligosaccharide can be considered as an efficient gene carrier for gene delivery systems.
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제안 방법
Slit width was set at 3 nm for the excitation. Based on the pyrene excitation spectra and red shift of the spectra with increasing COSC concentrations, the critical aggregation concentrations of the COSCs were measured.
The particle sizes of COS/DNA and COSC/DNA complexes and their zeta-potentials were also measured by DLS measurements. For the measurement, the carrier/plasmid DNA complexes were prepared with various carrier/DNA weight ratios of 0.5-8 (20 /ig/mL DNA in H2O). Before measurements, the prepared complexes were incubated for 30 min to ensure the complex formation.
Due to its amphiphilic characteristics, the hydrophobized COSs (COSCs) may show core-shell type micro-phase separation in aqueous milieus and the resulting nanoparticles can restore the poly-valent cationic property on the COS. Here in, we investigate the physicochemical properties of the hydrophobized COSs (COSCs) by using dynamic light scattering and fluorescence spectroscopy. Then, the potential of COSCs for gene carrier were investigated by introducing them into in vitro gene delivery system.
In this research, we prepared COS with narrow molecular weight distribution by ultrafiltration methods and modified the COSs with hydrophobic moiety of cholesterol. Owing to their amphiphilic character, the hydrophobized COSs formed core-shell type nanoparticles with fairly low critical aggregation concentration in aqueous milieu.
In this study, the self-assembled complex formations (gene condensation) of COS6 and COS6C5 with pSV-j3-gal plasmid DNA were investigated by el retardation assay. As shown in Figure 6, COS6 and COS6C5 showed different gene condensation patterns.
The particle size and size distribution of COSC nanoparticles in aqueous environment were investigated by dynamic light scattering (DLS) measurement. The DLS measurements were carried out using an ELS-800 electro phoretic LS spectrophotometer (NICOMP 380 ZLS zetapotential/particle sizer), equipped with a He-Ne laser operating at 632.8 nm at 25 ℃ and at a fixed scattering angle of 90°;. Before measurement, the COSC nanoparticles were re-dispersed in deionized water (1 mg/mL), sonicated for 30 sec, and filtered through a 0.
0 ~ 1 mg/mL, more than 5 different concentrations) and reflect index increments (dn/dc) were measured by Opt-LAB reflectometer (Wyott, USA). Then, absolute molecular weights and molecular weight distribution (PDI) of the COSs were obtained from GPC chromatogram with light scattering data (Debye plot regressions).
To overcome these problems, we prepared COSs with narrow molecular weight distribution by fractionation method using ultrafiltration technique, and modified the fractionated COSs with hydrophobic moiety of cholesterol. Due to its amphiphilic characteristics, the hydrophobized COSs (COSCs) may show core-shell type micro-phase separation in aqueous milieus and the resulting nanoparticles can restore the poly-valent cationic property on the COS.
대상 데이터
Korea. Cholesteryl chloroformate, DL-lactic acid, and pyrene were purchased from Aldrich. Ethidium bromide, and agarose were purchased from Promega (Madison, WI).
Cholesteryl chloroformate, DL-lactic acid, and pyrene were purchased from Aldrich. Ethidium bromide, and agarose were purchased from Promega (Madison, WI). The pSV-/3-gal and pEGFP-N 1 plasmid DNA were purchased from Fisher Scientific Co.
Cell Culture and in vitro Tansfection. Human embryonic kidney 293 cells (HEK 293) were purchased from American Type Culture Collection (Manassas, VA). The cells were cultivated in DMEM supplemented with 10% FBS at 37 ℃ in a humidified atmosphere containing 5% CO2.
이론/모형
Preparation of Hydrophobized COS Nanoparticles. The COSC nanoparticles were prepared by diafiltration method. Briefly, after dissolution and/or suspension of COSCs in organic solvent of DMSO (ca 10 mg/mL), the solutions were dialyzed against deionized water for 24 h.
The measurements were carried out at a higher concentration of the cac, measured by fluorescence spectroscopy. The hydrodynamic diameters of the COSC nanoparticles were calculated by the Stokes-Einstein equation.
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