Lycopene br Nanomicelles uptake and photocontrolled release
2.12. Nanomicelles uptake and photocontrolled release in HeLa cells
In order to study cellular uptake and location of DOX, DOX-loaded HA-NB-SC nanomicelles were co-incubated with HeLa cells for 2 h. After incubation, cells were washed with HBSS buﬀer for five times carefully and stained with DAPI at a concentration of 5 μg/mL to dis-play the cell nucleus. The cells were then imaged on a fluorescence microscopy (Olympus BX53).
In order to study photo-triggered release of nanomicelles in cells, FDA was loaded in HA-NB-SC nanomicelles. FDA-loaded nanomicelles were co-incubated with HeLa cells for 2 h, furtherly. After co-incuba-tion, cells were washed with HBSS buﬀer for five times and were ir-radiated with UV light for 1 h. The cells were then imaged on a confocal laser scanning microscope (CLSM) (Leica TCS SP8) of Life Science Research Core Services (LSRCS).
2.13. Competitive inhibition assay
For competitive inhibition experiment of DOX-loaded HA-NB-SC nanomicelles, in the presence/absence of free HA (1 mg/mL) HeLa cells were cultured with DOX loaded-HA-NB-SC nanomicelles (200 μg/mL) for 2 h at 37 °C under a 5% CO2 atmosphere. The medium was then removed and cell were washed three times with HBSS buﬀer. The cells were fixed with 4% formaldehyde. Cells were washed by HBSS five times before measurements. Then, cells were imaged on a florescence microscopy.
2.14. Statistical analysis
Results were expressed as mean ± SD. Data were analyzed by t-test with the scientific statistic software GraphPad Prism 5.01.
3. Results and discussion
3.1. Synthesis and characterization of HA-NB-SC
The synthesis of the light-responsive amphiphilic HA-NB-SC was accomplished starting from 3-nitro-4-(bromomethyl) benzoic Lycopene es-terified with stearyl alcohol in the presence of EDC and catalytic DMAP (Scheme S1). The giving bromo-ester was then conjugated to HA chain through unimolecular nucleophilic substitution (SN1) reaction in dry DMSO. The successful attachment of NB-SC to the carboxyl groups of HA was evidenced by 1H NMR, FTIR and XRD analysis, shown in Fig. 1. The characteristic peaks of HA are shown in Fig. 1A, where the N-acetyl (-NHCOCH3) groups can be identified at δ (ppm) 2.0 and glucosidic protons can be identified at δ (ppm) 3.0–4.0 (Shi et al., 2016), whereas Carbohydrate Polymers 206 (2019) 309–318
the 1H NMR spectrum of HA-NB-SC (Fig. 1B) showed newly emerged peaks at δ (ppm) 8.49, 8.10 and 7.88, which were assigned to the aromatic protons from NB linkage. These results verified the successful connection between NB-SC group and the HA backbone. Besides, FTIR analysis was also used to verify the connection. Shown in Fig. 1C and D, as compared to those of HA (Fig. 1C), the new peaks of NB-SC (Fig. 1D) appeared at ∼ 2800 cm−1, which assigned to stretching vibrations of -C-H from stearyl (Fujisawa, Saito, & Isogai, 2012; Moniri, Hantehzadeh, Ghoranneviss, & Asadi Asadabad, 2017). In order to verify the synthesis of HA-NB-SC furtherly, XRD experiments were also carried out to determine the microstructure of HA, NB-SC and HA-NB-SC (Fig. 1E). HA and HA-NB-SC exhibited no sharp diﬀraction peaks, and the intensities of their peaks were much weaker as compared to those of small molecular NB-SC. The results proved the crystalline structure of NB-SC was significantly disrupted in the amphiphilic HA-NB-SC, indicating HA was modified by NB-SC. The coupling reaction provided access to the target light-responsive amphiphilic molecule which was thereafter self-assembled into nanomicelles in aqueous medium.
The degree of substitution (DS, defined as the number of NB-SC per 100 disaccharides units of HA) of NB-SC in HA-NB-SC conjugate was calculated by measuring the content of carbon in HA, NB-SC and HA-NB-SC using elemental analysis. The results of carbon content were shown in Table S1. According to the following equation, the DS of NB-SC in HA-NB-SC conjugate was 4.98.