Phage Lytic Protein CHAPSH3b Encapsulated in Niosomes and Gelatine Films

Abstract

Antimicrobial resistance (AMR) has emerged as a global health challenge, sparking worldwide interest in exploring the antimicrobial potential of natural compounds as an alternative to conventional antibiotics. In recent years, one area of focus has been the utilization of bacteriophages and their derivative proteins. Specifically, phage lytic proteins, or endolysins, are specialized enzymes that induce bacterial cell lysis and can be efficiently produced and purified following overexpression in bacteria. Nonetheless, a significant limitation of these proteins is their vulnerability to certain environmental conditions, which may impair their effectiveness. Encapsulating endolysins in vesicles could mitigate this issue by providing added protection to the proteins, enabling controlled release, and enhancing their stability, particularly at temperatures around 4 °C. In this work, the chimeric lytic protein CHAPSH3b was encapsulated within non-ionic surfactant-based vesicles (niosomes) created using the thin film hydrating method (TFH). These protein-loaded niosomes were then characterized, revealing sizes in the range of 30-80 nm, zeta potentials between 30 and 50 mV, and an encapsulation efficiency (EE) of 50-60%. Additionally, with the objective of exploring their potential application in the food industry, these endolysin-loaded niosomes were incorporated into gelatine films. This was carried out to evaluate their stability and antimicrobial efficacy against Staphylococcus aureus.

Keywords: antimicrobial activity; encapsulation; endolysin; gelatine films; niosomes.

Figure 1

Thin film hydration method used for the synthesis of niosomes containing the lytic protein CHAPSH3b.

Figure 2

Particle size distribution of niosomes prepared in (A) pure Milli-Q water and (B) PBS obtained by DLS.

Figure 3

TEM images of niosomes stained with 2% (w/v) phosphotungstic acid (PTA). (A) Empty niosomes in water; (B) protein-loaded niosomes in water; (C) empty niosomes in PBS; (D) protein-loaded niosomes in PBS.

Figure 4

Protein elution profiles obtained after measurement of the absorbance at 280 nm (A₂₈₀) of fractions eluted by exclusion chromatography of CHAPSH3b: 4, 8, and 12 µM in ultrapure water (A) and in PBS (B).

Figure 5

Elution profiles of fractions obtained by molecular exclusion chromatography of the solvent (ethanol), empty niosomes, and protein/niosomes/solvent after measurement at (A) different wavelengths (blue line: 224, green line: 254, and red line: 280 nm), and (B) different fractions at 254 nm. Red line: protein CHAPSH3b, green line: mixture of empty niosomes (1/3) and protein (2/3) solution; blue line: mixture of empty niosomes (2/3) and protein (1/3) solution. The green arrow indicates the CHAPSH3b fraction.

Figure 6

HPSEC chromatographs (at 280 nm) for different samples eluted in ultrapure water (A) and in PBS (B). Protein-loaded niosomes were first separated from the supernatant (blue line), and then the niosomes were broken (green line) to release CHAPSH3b. A mixture of supernatant and broken niosomes (total protein) was also analyzed (red line).

Figure 7

Analysis by SEM of gelatine films obtained with (A) PBS alone, (B) empty niosomes, (C) free protein, and (D) niosomes loaded with protein.