2.2. Preparation and Characterization of Cu/Zn SOD Nanoparticles
In order to prove our hypothesis that the MR—mentioned above in G-SOD—is useful for the preparation of Cu/Zn SOD nanoparticles, G-SOD was observed under the field emission scanning electron microscope (FESEM). As shown in Figure 2
, the morphology of the nanoparticles in G-SOD is a spherical shape with a relatively uniform particle distribution between 100 and 300 nm. The inset is a magnification image of G-SOD nanoparticles, with a size close to 200 nm.
Both nanoparticle size and Zeta potential were measured using dynamic light scattering. It was found that the Zeta potential was −17.27 ± 0.59 mV and the average size was 175.86 ± 0.71 nm, which coincides with the nanoparticle pattern under FESEM observation.
The enzymatic activity of G-SOD was measured to be 1102.98 ± 31.37 U/mL. In order to verify whether this activity of SOD is derived from the SOD molecule or the free copper ion, the concentration of free copper ion in G-SOD was measured and the corresponding enzymatic activity was determined by measuring the activity of copper sulfate with the same concentration as that of the free copper ion. The results showed that the concentration of free copper ion was 2.90 ± 0.05 ppm with an enzymatic activity of 39.8 U/mL. This value only holds 3.62% of the enzymatic activity of G-SOD. Thus, the possibility that the enzymatic activity of G-SOD is from the free copper ions can be excluded, indicating that the residual activity of the protein molecule of Cu/Zn SOD is substantial after the co-incubation processing.
Next, a response surface design was used to optimize the preparation of G-SOD nanoparticles (NPs) with improved enzymatic activity, according to the principle of Box-Behnken combination design, as shown in Table 1
and Table 2
. The optimal conditions were obtained as follows: the mole ratio of Cu/Zn SOD to glucose was 1:1, reaction temperature was 75 °C and reaction time was 45 min.
After optimization, the enzyme activity of G-SOD was 22,542.9 ± 879.1 U/mL; the average particle size and Zeta potential were 168.8 ± 0.6 nm and −13.66 ± 0.55 mV, respectively. These values were close to the predicted ones of the response surface design, in which enzymatic activity, average particle size, and Zeta potential of G-SOD were expected to be 22,969.8 ± 1305.6 U/mL, 192.5 ± 6.8 nm and −14.3 ± 1.6 mV, respectively.
The enzymatic activity of the product was increased 20.43 fold by the optimization of preparation conditions, mainly because the incubation temperature decreased from 100 to 75 °C, which reduces the thermal stress of Cu/Zn SOD molecule to be deep denaturation and aggregation. This phenomenon is consistent with our previous finding that 75 °C is the transiting temperature for the denaturation of Cu/Zn SOD [13
Furthermore, gel filtration chromatography of G-SOD was carried out to separate the aggregated polymer fraction from monomer or oligomer fraction of Cu/Zn SOD, so that confirming that NPs of G-SOD exhibited meaningful enzymatic activity. As to the reactants, four peaks appeared on the chromatogram with the retention times of 5, 6.7, 16 and 43.1 min, respectively (Peak 1–4, Figure 3
A); Peak 1 and 4 were the peaks of void volume and total volume fraction, respectively. Thereby, G-SOD contained mainly fractions in Peak 2 and Peak 3. On the other hand, natural SOD showed only one peak (Peak 1) with a retention time of 17.1 min, other than the peak for total volume, on its chromatogram (Peak 2, Figure 3
B). According to the gel filtration chromatography principle stating that fractions with higher molecular weight have a shorter retention time, it was deduced that the fraction of Peak 3 should be a monomer or oligomer Cu/Zn SOD, and Peak 2 be a polymer one. Based on the results of SDS-PAGE and nanoparticle analysis of G-SOD (Figure 1
and Figure 2
), it was suggested that Peak 2 contained Cu/Zn SOD NPs. It was calculated according to their peak area in Figure 3
A that the fraction of Peak 2 and Peak 3 comprised 66.1% and 33.9% of G-SOD, respectively. Therefore, the NPs were proposed to contribute most enzymatic activity to G-SOD, because the fraction of Peak 3 possibly only exerted 33.9% enzymatic activity of original sample, but G-SOD remained more than 95% of original activity. In another word, these results implied that NPs of G-SOD had meaningful enzymatic activity.
The storage stability of G-SOD NPs after optimization is important for future application. Therefore, the enzymatic activity, particle size and Zeta potential of the G-SOD were measured for 25 days at 4 °C, to evaluate its stability of storage. It was found that the enzymatic activity of G-SOD showed almost no decrease within 25 days, remaining at a high level with an average value of 22,652.5 ± 230.8 U/mL during the period of measurement (Figure 4
A). The particle size and Zeta potential of G-SOD showed no obvious change within the detection time (Figure 4
B, C), with a mean particle size of 192.5 ± 12.5 nm and a mean Zeta potential of −13.5 ± 0.33 mV. These data indicated that the optimized G-SOD could be stably present at 4 °C for at least 25 days. Moreover, there was also no marked change in the enzymatic activity, particle size or Zeta potential of the re-dissolved sample of lyophilized G-SOD (data not shown). All these results indicate that optimized G-SOD has good storage stability.
The above data confirmed that NPs of Cu/Zn SOD could be prepared by co-incubation with glucose and, more importantly, that these NPs retained substantial enzymatic activity and good storage stability.
2.3. The Cell Uptake Efficacy of G-SOD
Different concentrations of Cu/Zn SOD and G-SOD were co-cultured with alveolar macrophage cell line NR8383 for different times, and the SOD activity of the cell lysates was measured according to our previous paper [14
]. SOD activity analysis of the cell lysates of the sample groups and control group involved in the formula of cell uptake efficacy, in order to calculate the delivery efficiency of G-SOD.
It was found that alveolar macrophage cells themselves had a SOD activity of 44.39 U/mL (this value is defined as 100% in Figure 5
), both types of SOD sample increased the intracellular SOD activity of alveolar macrophages, and the increment was related to concentration and co-culture time of the SOD samples (Figure 5
). The enzymatic activity in the alveolar macrophages co-cultured with G-SOD in the same concentration was higher than that of the Cu/Zn SOD at the time points of 3, 6 and 12 h (p
< 0.05). This indicated that the nano-form of Cu/Zn SOD found it easier to penetrate into the cells than natural Cu/Zn SOD. Additionally, it was also found that the uptake of SOD by alveolar macrophages was concentration dependent. In both the case of natural Cu/Zn SOD and G-SOD, the intracellular SOD activity of alveolar macrophages increased with the increment of the SOD concentration from 6000 to 8000 U/mL. The activity of SOD in alveolar macrophages also increased with the prolongation of co-culture time until 12 h or 6 h in the case of 6000 U/mL of G-SOD.
These results confirmed the hypothesis that the formation of Cu/Zn SOD nanoparticles by the co-incubation with glucose improved the cell uptake efficacy of SOD.