What is a stable delivery system for curcumin?

Introduction

Curcumin, with its antioxidant and anti-inflammatory properties, is widely used as a natural pigment in nutritional supplements, pharmaceuticals, and the food industry. However, limitations such as crystalline properties, low solubility, alkaline degradation, and poor oral bioavailability significantly restrict its clinical application. In this study, acylated kidney bean protein isolate (AKBPI) nanogels were synthesized via a self-assembly method and characterized by measuring protein molecular weight, particle size distribution, turbidity, zeta potential, hydrophobicity, thiol groups, and stability. The nanogel particles were characterized by Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD), Raman spectroscopy (Raman), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). The encapsulation efficiency of the curcumin-loaded nanogels was determined. These results provide an effective approach for expanding the application of chemical modification in nanogel preparation and enhancing the potential of AKBPI.

Results and Discussion

Effect of Acylation on KBPI Properties

SDS-PAGE

SDS-PAGE was used to analyze the changes in protein composition and subunits after acylation. The corresponding protein bands for each group are shown in Figure 1A. The 48 kDa subunit band corresponds to vicilin. The 30 kDa subunit band is generally considered to be a lectin. Acylation resulted in a decrease in the molecular weight of KBPI at 75, 63, 48, and 30 kDa, indicating that acylation promotes KBPI hydrolysis.

Figure 1. Effect of Acylation on the Properties of Kidney Bean Protein Isolate (KPI)

Zeta Potential

Figure 1B shows the effect of acylation on the zeta potential of KBPI and AKBPI at different pH values. The absolute zeta potential of AKBPI at pH 5 and pH 6 was significantly higher than that of KBPI (P < 0.05), which may indicate that the succinyl group partially replaces the ε-NH2 group in AKBPI, thereby increasing its electronegativity.

Fourier Transform Infrared Spectroscopy

The Fourier Transform Infrared spectra of KBPI and AKBPI exhibit five characteristic bands: amide I (1631 cm⁻¹), amide II (1533 cm⁻¹), amide III (1396 cm⁻¹), amide A (3275 cm⁻¹), and amide B (2924 cm⁻¹) (Figure 1C). The strong peak at 1631 cm⁻¹ corresponds to the antiparallel β-sheet conformation within the molecule, indicating that the β-sheet is the primary secondary structural component of KBPI. The introduction of acyl groups alters the secondary structure of KBPI, increasing its structural flexibility.

Turbidity

Figure 1D illustrates the effect of acylation on turbidity at different pH values. Within the pH range above the isoelectric point (pH 5–8), the turbidity of KBPI decreases with increasing pH, while the opposite trend is observed for AKBPI. Furthermore, the transmittance of AKBPI (79%-94%) was significantly higher than that of KBPI (43%-66.8%) (P < 0.05), possibly indicating that acylation promoted the formation of dispersed and uniform nanogels. Overall, these findings suggest that acylation can modify protein properties by increasing molecular weight, negative charge, and transmittance.

Optimization of AKBPI Nanogel Conditions

Four factors influencing nanogel formation (pH, concentration, heating time, and heating temperature) were compared to select the optimal conditions. With varying pH values, particle size and PDI initially increased and then decreased. At pH 7, the average particle size was 202.6 nm and the PDI was 0.49 (Figure 2A). At a concentration of 2 mg/mL, the smallest particle size (128.6 nm) and the PDI were 0.47 (Figure 2B). Results showed that as heating time increased from 0 to 60 min, the average particle size gradually decreased, while the average particle size of polydiphenylene ether remained stable at around 0.5. After 60 minutes of heating, the particle size was minimized (190 nm) (Figure 2C). Furthermore, when the protein was heated at 90°C, the smallest particle size (210 nm) and pDI (0.39) were achieved (Figure 2D).

Figure 2 Optimization of AKBPI Nanogel Conditions

Both non-acylated and acylated nanogels were positively charged at pH values ​​below 4.0 and negatively charged at pH 4.0–8.0. Furthermore, at pH values ​​above 4.0, the absolute zeta potential of AKBPI-nanogels was significantly higher than that of KBPI-nanogels (P < 0.05) (Figure 2E).

Characterization of KBPI-Nanogel Properties

Morphology and Zeta Potential

The optical transparency of KBPI-nanogels was improved by the acylation reaction (Figure 3A). The absolute zeta potential of AKBPI-nanogels was significantly higher than that of KBPI-nanogels (P < 0.05) (Figure 3B).

Figure 3 Effect of Acylation on the Properties of KBPI Nanogels

Free Thiol Groups and Surface Hydrophobicity

The free thiol content of AKBPI nanogels (27.94 nmol/mg) was significantly higher than that of KBPI nanogels (8.94 nmol/mg) (P < 0.05) (Figure 3C). This result indicates that the acylation reaction partially oxidizes and exposes buried cysteine ​​residues, resulting in a higher free thiol content. As shown in Figure 3D, the order of hydrophobicity from lowest to highest is: KBPI < KBPI nanogels < AKBPI < AKBPI nanogels. The hydrophobicity of AKBPI nanogels (180.35) is higher than that of KBPI nanogels. The increased surface hydrophobicity of AKBPI nanogels is the result of acylation-induced stretching of the protein molecules, which exposes internal hydrophobic groups.

Particle Size Distribution

The particle size distributions of KBPI, AKBPI, KBPI-nanogels, and AKBPI-nanogels are shown in Figure 3E. The distribution curves exhibit two peaks, with AKBPI shifting to the right relative to KBPI. As shown in Figure 3F, the average particle size of KBPI was 226.24 nm, and that of AKBPI was 258.02 nm. This is likely attributed to the increased structural flexibility of acylation, which leads to intermolecular crosslinking and the formation of aggregates. Consequently, the AKBPI-nanogels exhibited smaller particle size and PDI, and by suppressing aggregate formation, they formed a more stable system.

Structural Characterization of AKBPI-Nanogels

UV-Vis Spectroscopy

Compared with KBPI and KBPI-nanogels, the absorbance of both AKBPI and AKBPI-nanogels was significantly reduced (Figure 4A). This is likely due to the loss of native conformation due to the presence of partially unfolded structures. Furthermore, the peak-to-valley distance ratio (r = a/b) was calculated to assess changes in the microenvironment surrounding the Tyr residues. The lower r value of the AKBPI-nanogels indicates a decreased polarity of the microenvironment surrounding the Tyr residues (Figure 4B). Acetylation significantly reduced the intrinsic fluorescence intensity of both KBPI and KBPI-nanogels, with the maximum fluorescence intensity of the AKBPI-nanogels exhibiting a slight blue shift compared to the KBPI-nanogels (Figure 4C).

Figure 4 Effect of Acylation on the Structure of KBPI Nanogels

FT-IR and CD

In the FT-IR spectrum, compared with KBPI nanogels, the peak intensity at 1000 cm-1 increased in AKBPI nanogels, while the peak intensity at 1533 cm-1 decreased (Figure 4D). Acylation reduced the peak intensity at 1533 cm-1, indicating that acylation promoted peptide bond cleavage. Protein secondary structure analysis revealed that AKBPI had lower β-sheet and β-turn contents and higher α-helix and random coil contents than KBPI (Figure 4E). It is speculated that acylation weakened the intermolecular interactions of the protein, forming a highly flexible, unfolded structure and enhancing gelation ability.

XRD and Raman Spectroscopy

The XRD spectrum exhibited two characteristic diffraction peaks at 2θ = 9° and 20°. Compared with KBPI nanogels, AKBPI nanogels exhibited a broad peak at 9° and a sharp peak at 20°, as shown in Figure 4F. Furthermore, the appearance of two sharp peaks indicates that acylation alters hydrogen bonding and hydrophobic interactions, thereby increasing structural flexibility and strengthening intermolecular interactions, which contributes to the formation of a strong gel. As shown in Figure 4G, the peak intensity of AKBPI at 1600-1700 cm⁻¹ (amide I region) is significantly higher than that of KBPI.

Characterization of the Stability of AKBPI Nanogels

At pH 3-7, pH treatment had relatively little effect on AKBPI-nanogels (Figure 5A). KBPI, however, precipitated at pH 4, near its isoelectric point. AKBPI-nanogels exhibited greater transparency than KBPI-nanogels over the NaCl concentration range of 0-200 mmol/L (Figure 5B). The particle size changes of the nanogels under different NaCl concentrations were compared. The zeta potentials of all groups were negative, and the absolute zeta values ​​of AKBPI-nanogels were higher than those of KBPI gels under 50-150 mmol/L NaCl conditions (Figure 5C). As shown in Figure 5E, the particle size distribution exhibited two peaks at different dilution ratios. Compared to AKBPI-nanogels, the peak position of AKBPI-nanogels diluted 25-fold shifted to the left, while the peak position of AKBPI-nanogels diluted 10-fold remained relatively stable. These results indicate that the prepared AKBPI gel system exhibits uniform particle size and stable structure. The smaller particles of AKBPI-nanogels diluted 25-fold may be due to the lower protein content in the solution.

Figure 5 Effect of Acylation on the Stability of KBPI-Nanogels

Characterization of Curcumin-Loaded Nanogels

Encapsulation Efficiency

The effect of acylation on the morphology of KBPI nanogels and their loaded curcumin is shown in Figure 6B. Curcumin-loaded AKBPI nanogels exhibit increased transparency. The effect of acylation on curcumin-KBPI is shown in Figure 6C. The curcumin encapsulation efficiency of AKBPI nanogels (92.30%) was significantly higher than that of KBPI (82.47%) (P = 0.011). These results indicate that acylation significantly improves the curcumin encapsulation efficiency of protein nanogels (P < 0.05).

Thermal Properties

The thermal properties of curcumin-loaded KBPI/AKBPI nanogels were characterized by DSC (Figure 6D). Pure curcumin exhibited a distinct endothermic peak at a melting point of 171.0°C, indicating a highly crystalline structure. In contrast, the curcumin-loaded KBPI/AKBPI nanogel sample exhibited a gradual temperature profile with no distinct peaks, indicating amorphous curcumin alignment and successful incorporation into the protein aggregate matrix.

Figure 6 Effect of Acylation on the Surface Morphology and Curcumin Loading Profile of KBPI Nanogels

Photostability and Storage Stability

With increasing irradiation time, free curcumin exhibited high photosensitivity, with a rapid decrease in retention (Figure 7A). After 120 minutes of irradiation, curcumin retention dropped to 24.2%, indicating significant curcumin degradation. In contrast, curcumin-loaded AKBPI nanogels exhibited delayed photodegradation of curcumin, with a retention of 44.72% after 120 minutes of phototreatment. However, compared with free curcumin, the curcumin-loaded KBPI nanogels exhibited significantly lower curcumin retention. Furthermore, a 48-h storage experiment demonstrated that curcumin retention was significantly improved in both curcumin-loaded KBPI/AKBPI nanogels compared with free curcumin, with AKBPI nanogels exhibiting the strongest protective effect on curcumin retention (Figure 7B).

Figure 7 Effect of Acylation on the Stability, Antioxidant Activity, and Release Profile of Curcumin-Loaded KBPI Nanogels

Antioxidant Activity

At a curcumin concentration of 40 μg/mL, the DPPH radical scavenging rate of curcumin-loaded AKBPI nanogels was 2.05 times (16.46%) higher than that of free curcumin (Figure 7C). Similarly, at a curcumin concentration of 8 μg/mL, the ABTS radical scavenging rate of curcumin-loaded AKBPI nanogels was 1.36 times higher than that of free curcumin (Figure 7D).

In vitro gastrointestinal digestion

The in vitro release of curcumin from KBPI nanogels/AKBPI nanogels was evaluated under simulated gastric and intestinal fluid conditions. Free curcumin exhibited rapid release, while curcumin from the nanogels exhibited a slower release profile (Figure 7E). During a 6-h digestion, the release rate of free curcumin reached 95.70%, while the cumulative release rates of KBPI nanogels and AKBPI nanogels were 72.60% and 50.90%, respectively. This indicates that the nanogels significantly protect the loaded curcumin and significantly reduce its release rate.

Conclusion

In this study, AKBPI nanogels were prepared via acylation and thermally induced self-assembly techniques, and their dynamic release behavior and antioxidant capacity were evaluated. The results showed that the AKBPI nanogels exhibited excellent stability at different pH and NaCl concentrations, as well as strong storage and photostability. Furthermore, the curcumin-containing AKBPI nanogels demonstrated high encapsulation efficiency, strong photostability, high antioxidant activity, and good bioavailability. Therefore, AKBPI nanogels can serve as an innovative and ideal controlled-release delivery system for curcumin. However, further research is needed to determine the cytotoxicity, cellular immune response, and in vivo digestion of acylated protein nanogels. Future studies should prioritize the establishment of safe applications. Such efforts will broaden the application scope of proteins and provide higher-quality encapsulation and delivery materials for bioactive substances.

anna@hihealthbio.com