(M1330-03-17) Development, Characterization, and In Vitro Digestion of C-phycocyanin-Whey Protein Isolate-Zein Nanoparticles for Pharmaceutical Applications

Purpose: This research investigates the potential of C-phycocyanin (C-PC), a phycobiliprotein derived from cyanobacteria, for pharmaceutical and nutraceutical applications. C-PC has demonstrated promising pharmacological properties, including antioxidant, anti-inflammatory, and antidiabetic effects and potential anticancer properties (Chen et al., 2022). However, its susceptibility to rapid degradation under environmental conditions limits its technological applicability (Adjali et al., 2022). To overcome this challenge, C-PC-loaded nanoparticles were developed using the pH shifting method, with whey protein isolate (WPI) and zein as self-assembling proteins. The nanoparticles were physicochemically characterized, and a digestibility simulation assay was carried out.

Methods: Preparation of C-phycocyanin-loaded nanoparticles - The nanoparticles were obtained using the pH shifting method (Wei et al., 2021). Briefly, 0.0536 g of WPI and 0.010 g of zein were dispersed in 10 mL of water under magnetic stirring for 2 h. Then, the pH was adjusted to 12.0 with NaOH (4 M). The protein solution was kept under stirring to ensure the complete dispersion of the proteins. Finally, 0.5 mg/mL of C-PC (from Desertifilum tharense UAMC/S02) was added to the protein dispersion. The pH value was adjusted to 7.0 with HCl (1 M) to induce/promote nanoparticle self-assembly. Characterization of the protein nanoparticles - The particle size distribution and the surface electrical characteristics of both C-PC-loaded and non-loaded nanoparticles were assessed using dynamic light scattering and a particle electrophoresis instrument, respectively (Zetasizer Nano-ZS, Malvern Instruments Ltd.). The morphology of the particles was analyzed using scanning electron microscopy (TM3030Plus, Hitachi, Japan) at a voltage of 15 kV and under vacuum conditions. The encapsulation efficiency (EE %) was determined using a UV-visible spectrophotometric method (Evolution 300, Thermo Fischer, USA). An aliquot of the nanoparticle dispersion was centrifuged, and the amount of free (non-encapsulated) C-PC in the supernatant was determined by measuring the absorbances at 620 nm and 653 nm. In vitro digestion analysis of C-PC-loaded nanoparticles - The preparation of solutions to simulate the oral, gastric, and intestinal phases followed the INFOGEST protocol (Brodkorb et al., 2019). The in vitro simulated mouth digestion was performed by preparing the simulated salivary fluid (SSF) with the addition of 0.03 g/mL of mucin. Subsequently, 10 mL of SSF was combined with 10 mL of C-PC-loaded nanoparticles and incubated at 37°C for 2 minutes. Following this, 10 mL of simulated gastric fluid (SGF) containing pepsin (3.2 mg/mL) was mixed with the mouth digestion mixture and further incubated at 37°C for 2 h. Subsequently, 10 mL of the resulting gastric digestion was added to 10 mL of simulated intestinal fluid (SIF) containing trypsin (50 mg/mL), followed by incubation at 37°C for 2 h. After each digestive step, aliquots were characterized for particle size (PS), polydispersity index (PDI), surface charge (ζ), and antioxidant activity using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. Measurements were performed in triplicate.

Results: Characterization of nanoparticles - The C-PC-loaded nanoparticles showed a PS of 203.34 ± 8.37 nm, a PDI of 0.25 ± 0.01, a ζ of -16.21 ± 1.63 mV, and an EE % of 68.0 ± 7.54. Figure 1 shows the morphology of the nanoparticles obtained by SEM. The C-PC-loaded nanoparticles display different shapes and aggregate formations while maintaining sizes within the nanoscale range (between 100 and 1000 nm). In vitro digestion of C-PC-loaded nanoparticles. Figure 2 shows the changes in PS, PDI, and ζ of the nanoparticles following in vitro digestion across the oral, gastric, and intestinal phases. Notably, PS remained unaltered during the oral and gastric stages (Fig. 2a); however, an increase in PS was evident during the intestinal phase, attributed to protein hydrolysis within the nanoparticles. A similar pattern was observed for PDI (Fig. 2b), indicating an increased PS distribution during intestinal digestion, with values surpassing 0.8, indicative of nanoparticle disintegration under the activity of pancreatic proteolytic enzymes. Conversely, no significant shifts were observed in ζ (Fig. 2c), which consistently fell within the range of -15 to -20 mV. Figure 3 depicts the percentage inhibition of the DPPH radical by the C-PC-loaded nanoparticles throughout the oral, gastric, and intestinal digestion phases. The antioxidant activity of the C-PC-loaded nanoparticles increased as they traversed the different digestive processes. These findings suggest that the proteins comprising the nanoparticles undergo partial hydrolysis during the in vitro digestive process, thereby exposing peptides and proteins with inherent antioxidant properties, such as C-PC (Teixé-Roig et al., 2022).

Conclusion: C-PC-loaded biopolymeric nanoparticles were developed, demonstrating nanoscale dimensions, a uniform size distribution, negative surface charge, efficient encapsulation, and polyhedral morphology. Notably, the physicochemical properties of the nanoparticles remained stable throughout the oral and gastric phases of in vitro digestion. However, significant changes were observed following the intestinal phase, coinciding with the highest observed antioxidant activity. This indicates the potential therapeutic efficacy of C-PC-loaded nanoparticles in addressing gastrointestinal ailments such as gastric adenocarcinoma or colon cancer.

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Acknowledgements: Galindo-Pérez M.J. and Martínez-Acevedo L. acknowledge the Consejo Nacional de Humanidades Ciencias y Tecnologías (CONAHCyT) for the postdoctoral scholarship awarded.


Figure 1: Morphology of C-PC-loaded nanoparticles.


Figure 2: Changes in nanoparticle characteristics post-oral, gastric, and intestinal digestion. a) particle size; b) polydispersity index; c) Zeta potential.


Figure 3: Evaluation of the antioxidant activity (measured by DPPH radical inhibition) of the C-PC-loaded nanoparticles across the oral, gastric, and intestinal (in vitro) digestion phases.