Biogenic Polyphosphate Nanoparticles from a Marine Cyanobacterium Synechococcus sp. PCC 7002: Production, Characterization, and Anti-Inflammatory Properties In Vitro

Abstract

Probiotic-derived polyphosphates have attracted interest as potential therapeutic agents to improve intestinal health. The current study discovered the intracellular accumulation of polyphosphates in a marine cyanobacterium Synechococcus sp. PCC 7002 as nano-sized granules. The maximum accumulation of polyphosphates in Synechococcus sp. PCC 7002 was found at the late logarithmic growth phase when the medium contained 0.74 mM of KH₂PO₄, 11.76 mM of NaNO₃, and 30.42 mM of Na₂SO₄. Biogenic polyphosphate nanoparticles (BPNPs) were obtained intact from the algae cells by hot water extraction, and were purified to remove the organic impurities by Sephadex G-100 gel filtration. By using 100 kDa ultrafiltration, BPNPs were fractionated into the larger and smaller populations with diameters ranging between 30⁻70 nm and 10⁻30 nm, respectively. 4',6-diamidino-2-phenylindole fluorescence and orthophosphate production revealed that a minor portion of BPNPs (about 14⁻18%) were degraded during simulated gastrointestinal digestion. In vitro studies using lipopolysaccharide-activated RAW264.7 cells showed that BPNPs inhibited cyclooxygenase-2, inducible nitric oxide (NO) synthase expression, and the production of proinflammatory mediators, including NO, tumor necrosis factor-α, interleukin-6, and interleukin-1β through suppressing the Toll-like receptor 4/NF-κB signaling pathway. Overall, there is promise in the use of the marine cyanobacterium Synechococcus sp. PCC 7002 to produce BPNPs, an anti-inflammatory postbiotic.

Keywords: Synechococcus sp. PCC 7002; Toll-like receptors; anti-inflammation; macrophages; polyphosphate nanoparticles.

Figure 1

Characterization of biogenic polyphosphate nanoparticles (BPNPs) within the algae cells: (a) appearance of the Synechococcus 7002 culture, (b) typical transmission electron microscopic (TEM) image of thin sections of Synechococcus 7002 cells, (c) energy dispersive spectroscopy (EDS) analysis of the yellow circle region in panel b, and (d) fluorescence microscope image of Synechococcus 7002 cells stained with 4′,6-diamidino-2-phenylindole (DAPI, ×400).

Figure 2

The kinetics of (a) biomass growth, (b) inorganic polyphosphates (polyP) accumulation, and (c) residual orthophosphate concentrations at various KH₂PO₄ concentrations in the medium.

Figure 3

The accumulation of biomass and polyPs in Synechococcus 7002 after nine days of growth at various concentrations of (a) NaNO₃ and (b) Na₂SO₄ in the medium.

Figure 4

Purification and characterization of BPNPs: (a) purification of BPNPs from the cell extract of Synechococcus 7002 by Sephadex G-100 gel filtration, (b) the electrophoretic patterns for different fractions (A: the cell extract; B: peak I; C: >100 kDa fraction of peak I; D: <100 kDa fraction of peak I; E, peak II), (c,e) dynamic light scattering (DLS) size distributions and (d,f) TEM images of (c,d) larger BPNPs (L-BPNPs) and (e,f) smaller BPNPs (S-BPNPs).

Figure 5

Stability tests of BPNPs in simulated gastrointestinal digestion (GID): (a) DAPI fluorescence, (b) orthophosphate production, and (c) gel electrophoretic analysis. Data were expressed as means ± standard deviations (n = 3). The complete hydrolysis of BPNPs into orthophosphate was achieved by acid digestion (AD) in 2 N HCl at 95 °C for 40 min. Statistical differences were determined by Student’s t-test (** p < 0.01, *** p < 0.001).

Figure 6

Effects of BPNPs on (a) survivability, (b) morphological appearance (20×), and production of (c) nitric oxide (NO) (n = 6), (d) tumor necrosis factor-α (TNF-α) (n = 3), (e) interleukin-6 (IL-6) (n = 3), and (f) interleukin-1β (IL-1β) (n = 3) in RAW264.7 cells following lipopolysaccharide (LPS) stimulation. Data were expressed as means ± standard deviations, with different lowercase letters marking significant differences (p < 0.05, one-way analysis of variance (ANOVA) followed by Turkey’s Honest Significant Difference (HSD) test).

Figure 7

Effects of BPNPs on the levels of nuclear factor kappa beta (NF-κB) p65 (n = 3), cyclooxygenase-2 (COX-2) (n = 3), inducible NO synthase (iNOS) (n = 3), and Toll-like receptor 4 (TLR-4) (n = 3) in RAW264.7 cells with or without lipopolysaccharide (LPS) stimulation: (a) Western blotting analysis and (b) densitometry. Data were expressed as means ± standard deviations, with different lowercase letters marking significant differences (p < 0.05, ANOVA followed by Tukey’s HSD test).