3.1. Optimization of the CG-PEG-CSO-SPIONs by PBD and BBD
Based on preliminary screening experiments using PBD, 15 experimental runs were generated from five factors, four responses, and two levels with three center points (at intermediate level), as shown in
Table 1 and
Table 2. To investigate the significant factors affecting the responses, statistical evaluation was performed through ANOVA and a Pareto chart (
Figure 2). The results reveal that CTAB (X
1), CSO (X
2), and PEG (X
3) concentrations are the main significant effect to four responses in comparison with molecular weight of PEG (X
4) and CG concentration (X
5). Thus, the factors X
1, X
2, and X
3 were selected as crucial affecting factors for further optimization by a three-factor, three-level, and three-center point BBD. Quadratic regression equations and three-dimensional response surface plots (3D-RSPs) were generated for each response. Compared with other models including the linear and interaction (2FI) models, statistically significant
p-values indicated a goodness of fit for all responses, affirmed by the statistical analysis of the quadratic model (
Table 5).
The particle size (Y
1) and zeta potential (Y
2) of CG-PEG-CSO-SPIONs, according to the BBD analysis, ranged between 92 ± 32 nm and 362 ± 19 nm and from 20.2 ± 2.3 mV to 33.1 ± 1.6 mV, respectively, (
Table 4). The quadratic regression equation, which represents the relationship between the factors to the particle size and zeta potential, is shown in Equations (4) and (5), respectively:
The particle size and zeta potential of the CG-PEG-CSO-SPIONs were mainly affected by the CTAB concentration (X1) (p < 0.05), which had a positive effect on both responses. For instance, the particle size increased with increasing CTAB concentrations, which might be due to the increased level of CTAB adsorbed onto the surface of the NPs. Likewise, the zeta potential of the CG-PEG-CSO-SPIONs increased as the CTAB concentration increased (p < 0.05), presumably due to the increased formation of positively-charged micelles from the positively-charged CTAB on the surface of the negatively charged NPs resulting in charge neutralization.
The 3D-RSPs and contour plots that were used to evaluate the effects of the factors on the particle size and zeta potential are shown in
Figure 3. Contour plots indicated that the minimum particle size was obtained at the low level of all three factors, whereas the maximum zeta potential was obtained at the high level of CTAB and PEG and medium level of CSO. The ANOVA analysis of the particle size and zeta potential are summarized in
Table 5, and indicated that the data fitted well with the models for size and zeta potential (R
2 and R
2predicted values close to 1).
The LE (Y
3) and LC (Y
4) of CG-PEG-CSO-SPIONs ranged from 43.5 ± 4.6 to 86.4 ± 3.2% and from 3.1 ± 1.1 to 12.3 ± 1.3%, respectively, (
Table 4). The effect of the PEG concentration (X
3) was more remarkable than that of the CTAB and CSO concentrations, with the quadratic regression equations for both responses shown in Equations (6) and (7), respectively:
The 3D-RSPs and contour plots, as shown in
Figure 3, revealed that at a high PEG concentration and low CTAB concentration, the LE and LC were increased by up to 86.4 ± 3.2% and 7.7 ± 2.8%, respectively. Increasing the CTAB concentration decreased the LE and LC to 43.5 ± 4.6% and 3.1 ± 1.1%, respectively. The contour plots suggested that the maximum LE was obtained at a high PEG concentration, medium CSO concentration and low CTAB concentration, respectively, whereas the maximum LC was obtained at a medium concentration of all three factors. Therefore, it was concluded that the LE and LC were enhanced with increasing PEG and CSO concentrations due to the strong interaction between the drug and polymer shell coated on the surface of the NPs, which increased the chance of the drug being adsorbed onto the surface of the NPs [
24]. Furthermore, a high CTAB concentration led to increased micelle formation at the surface of the SPIONs. and so an increased particle size with a decreased specific area, which resulted in a lower drug LE and LC. The ANOVA analysis of the LE and LC is summarized in
Table 5. The R
2 value of LE and LC was 0.9919 and 0.9992, respectively, while the R
2predicted value was 0.8856 and 0.9959, respectively. All of the ANOVA analysis results were in reasonable agreement and so the design space could be navigated.
In order to obtain an improved formulation with a promising particle size, RSM optimization of the zeta potential, LE and LC was employed. Numerical optimization applying a desirability function was used to identify the optimum conditions for obtaining a minimum particle size, high zeta potential of ≥ ±20 mV and maximum LE and LC, and is shown in
Table 6. The optimal conditions were defined as 2.8 mg/mL CTAB, 0.1 mg/mL CSO, and 0.26 mg/mL PEG, giving a desirability value of 0.96. The particle size, zeta potential, LE and LC were predicted as 133 nm, 30.6 mV, 83.3%, and 8.3%, respectively, (
Table 6), which was further validated by comparison with the observed results. According to
Table 6, the LC of the optimized CG-PEG-CSO-SPIONs was found to be 7.94%, indicating that the amount of CG at 7.94 g was contained in 100 g of dried CG-PEG-CSO-SPIONs. The relatively low percentage of error (<10%) suggested that the model was suitable for the optimization of CG-PEG-CSO-SPIONs.
3.2. Characterization of the NPs
The size, morphology and particle uniformity of the SPIONs and CG-PEG-CSO-SPIONs fabricated under the optimal condition were investigated by TEM (
Figure 4). The obtained SPIONs and CG-PEG-CSO-SPIONs had a small size of around 100 nm in diameter with smooth surface and narrow size distribution.
The overlay FT-IR spectra of the CG powder, PEG-CSO-SPIONs and CDG-PEG-CSO-SPIONs are shown in
Figure 5. The spectrum of the CG powder showed its characteristic peaks at 1752 cm
−1 (C=O stretching vibration of carboxylate ester), 1508 cm
−1 (C=C vibrations), 1297 cm
−1 (C-O stretching of carboxylic acid), 1119 cm
−1 (aromatic C-C-H bending), 1028 cm
−1 (symmetrical and asymmetrical C-O-C stretching vibration), 979 cm
−1 (benzoate
trans-CH vibration), and 600 cm
−1 (
cis-CH vibration of an aromatic ring). In the case of the PEG-CSO-SPIONs and SPIONs, they showed only two characteristic peaks at 598 cm
−1 and 463 cm
−1 (Fe-O vibration). However, the characteristic PEG peaks at 2879 cm
−1 (C-H stretching of alkanes) and 1112 cm
−1 (C-O-C of ether) and the characteristic CSO peaks at 3750 cm
−1 (OH stretching vibration), 2968 cm
−1 (C-H stretching), 1625 cm
−1 (Amide I), and 1088 cm
−1 (C=O), were also observed in the spectrum of PEG-CSO-SPIONs indicating the coating of PEG and CSO on the surface of the SPIONs. In addition, most of characteristic peaks of CG were found in the spectrum of the CG-PEG-CSO-SPIONs, confirming the physical interaction of CG with the NP matrix and also suggesting that the CG structure and its integrity were not affected by PEG-CSO-SPIONs.
The XRD pattern of SPIONs, PEG-CSO-SPIONs, CG powder and CG-PEG-CSO-SPIONs are shown in
Figure 6. The sharp XRD pattern of the SPIONs revealed its characteristic peaks at 30.2°, 35.5°, 43.1°, 53.6°, 57.1° and 62.6°, respectively, indicating the relatively high crystallinity of the SPIONs [
25]. The crystalline structure of SPIONs was not changed after coating with PEG-CSO and loading with CG. Thus, the polymer coating and CG loading likely occurred at the surface of the SPOINs and did not affect their characteristics. In addition, the characteristic XRD patterns of the CG powder were not found in the pattern of the CG-PEG-CSO-SPIONs, which may indicate the amorphous state of a solid molecular dispersion or a solid solution inside the polymers coated on the surface of the SPIONPs after adsorption [
33].
The use of field magnetization to aid the uptake of SPIONs and CG-PEG-CSO-SPIONs into HT-29 cells is summarized in
Figure 7A. The magnetization of SPIONs was about 60 emu/g and was reduced to below 1% after PEG-CSO coating and CG loading. In the other words, the polymer coating and CG loading did not affect the magnetic characteristics of the SPIONs [
34]. The saturated magnetization for SPIONs and CG-PEG-CSO-SPIONs (~60 emu/g) were lower than that of the bulk iron oxide particles (~92 emu/g) [
35], and no hysteresis loop was observed, indicating the superparamagnetic behavior. Especially, the saturation remanence (M
s) and coercivity (H
c) values of the CG-PEG-CSO-SPIONs were about 0.52 emu/g and 4.11 Oe, respectively, which indicated a low residual magnetization was present when the external magnetic field was removed, and that a low intensity magnetic field is required to reduce the magnetization to zero. This may suggest that the CG-PEG-CSO-SPIONs can be separated and controlled by an external magnetic field (
Figure 7B), which would be suitable for targeted drug delivery applications.
3.3. In Vitro CG Release Kinetics from the Optimized CG-PEG-CSO-SPIONs
Figure 8 shows the cumulative release of CG from the optimized CG-PEG-CSO-SPIONs in the RM at pH 5.5 or 7.4. The fast release was observed at the first 8 h for both pH values due to the swelling of polymer layers on SPIONs, and consequently, the weakly adsorbed CG molecules on the surface of PEG-CSO-SPIONs were easily released. The CG was then slowly released from CG-PEG-CSO-SPIONs. Finally, a sustained release manner was observed with a faster release in the acidic RM comparing to the alkaline RM. This might reflect the higher solubility of CG and CSO in an acidic environment. The maximum drug release was observed at pH 5.5 and 7.4 was 89% and 66%, respectively.
The drug release profile was fitted to the kinetic models in the DDsolver software. Based on the best fit model selection criteria (highest R2adjusted and AIC with lowest MSC), the CG release from CG-PEG-CSO-SPIONs best fitted with the Korsmeyer-Peppas model at both pH 5.5 (n = 0.30, R2adjusted = 98%, AIC = 98.03, and MSC = 1.85) and pH 7.4 (n = 0.35, R2adjusted = 97%, AIC = 88.81, and MSC = 2.80). In addition, in this study n was in the range of 0.30–0.35, which indicated that Fickian diffusion was the controlling factor for CG release from the NPs.
3.4. In Vitro Protein Binding
The plasma protein binding on the surface of nanoparticles is one of the key factors that affects biodistribution, biocompatibility, and therapeutic efficacy of drug-loaded nanoparticles. In addition, the protein binding has potentially serious consequences such as hemolysis, thrombosis, and embolization [
36]. Therefore, not only is a low level of protein binding required in preclinical and human applications. In the present study, BSA was used as a model protein to investigate the non-specific protein binding level of the developed NPs. The binding capacity of BSA onto the surface of the CSO-SPIONs, PEG-CSO-SPIONs and CG-PEG-CSO-SPIONs is shown in
Figure 9A. The BSA binding capacity of the CSO-SPIONs decreased significantly after coating with PEG (
p < 0.05). Whereas it showed only a slight difference in BSA binding after loading CG onto the PEG-CSO-SPIONs. Furthermore, the highest amount of BSA bound onto the surface of CSO-SPIONs was approximately 2.9- and 3.2-fold higher than that on the PEG-CSO-SPIONs and CG-PEG-CSO-SPIONs, respectively. This phenomenon might be attributed to steric repulsion via the flexible PEG chains in solution that can then shield the CSO particles more efficiently from protein binding due to its electrostatic interaction [
21]. In addition, no significant change in the aggregation of CG-PEG-CSO-SPIONs before and after incubation with the BSA solution was noted (
Figure 9B). Although a slightly increased particle size of the NPs was observed after incubation in the BSA solution, which might be the bound layer of BSA on the surface of the NPs [
29].
3.6. Cytotoxicity Studies
The cytotoxicity of free CG, PEG-CSO-SPIONs and CG-PEG-CSO-SPIONs against HT-29 cells was examined. Serum-free CM and 0.5% (
v/
v) DMSO was used as the control for the NPs and free CG, respectively, and neither showed any significant cytotoxicity to HT-29 cells in all the assays. As shown in
Figure 11, PEG-CSO-SPIONs showed a dose-dependent cytotoxicity on HT-29 cells, whereas the free CG solution showed no cytotoxicity on HT-29 cells. Interestingly, the cytotoxic effect of the PEG-CSO-SPIONs against HT-29 cells significantly (
p < 0.05) increased after CG loading, which might be due to a synergistic effect of the magnetic NPs and CG on cell uptake. However, the toxicity of the PEG-CSO-SPIONs on HT-29 cells increased with increasing NP concentrations above 5 µg/mL (cell viability <80%), potentially due to the high release of iron ions in the intracellular space and in situ degradation [
17].
The higher toxicity of PEG-CSO-SPIONs might result from the chemopreventive activity of CSO on HT-29 cells by increasing quinine reductase (QR), glutathione-S-transferase (GST) activities, glutathione (GSH) levels, and by inhibiting the ornithine decarboxylase (ODC) activity and COX-2 expression [
37]. However, it was observed that the cytotoxicity activity of PEG-CSO-SPIONs against HT-29 cells was significantly increased by loading of CG (
p < 0.05). For example, at 5 µg/mL of CG, the viability of HT-29 cells after treatment with the PEG-CSO-SPIONs was 84% compared to 21% for CG-PEG-CSO-SPIONs, reflecting the synergistic cytotoxicity of the PEG-CSO-SPIONs and CG. At CG concentrations up to 40 μg/mL, the cytotoxicity of CG-PEG-CSO-SPIONs was significantly higher than that of free CG (
p < 0.05). For example, at 10 µg/mL of CG, the viability of HT-29 cells treated by free CG (97%) was 11-fold higher than those treated with CG-PEG-CSO-SPIONs (8.3%).