Porphyrin-Loaded Lignin Nanoparticles Against Bacteria: A Photodynamic Antimicrobial Chemotherapy Application

Abstract

The need for alternative strategies to fight bacteria is evident from the emergence of antimicrobial resistance. To that respect, photodynamic antimicrobial chemotherapy steadily rises in bacterial eradication by using light, a photosensitizer and oxygen, which generates reactive oxygen species that may kill bacteria. Herein, we report the encapsulation of 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphyrin into acetylated lignin water-dispersible nanoparticles ( THPP@AcLi ), with characterization of those systems by standard spectroscopic and microscopic techniques. We observed that THPP@AcLi retained porphyrin’s photophysical/photochemical properties, including singlet oxygen generation and fluorescence. Besides, the nanoparticles demonstrated enhanced stability on storage and light bleaching. THPP@AcLi were evaluated as photosensitizers against two Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa , and against three Gram-positive bacteria, Staphylococcus aureus , Staphylococcus epidermidis , and Enterococcus faecalis . THPP@AcLi were able to diminish Gram-positive bacterial survival to 0.1% when exposed to low white LED light doses (4.16 J/cm 2 ), requiring concentrations below 5 μM. Nevertheless, the obtained nanoparticles were unable to diminish the survival of Gram-negative bacteria. Through transmission electron microscopy observations, we could demonstrate that nanoparticles did not penetrate inside the bacterial cell, exerting their destructive effect on the bacterial wall; also, a high affinity between acetylated lignin nanoparticles and bacteria was observed, leading to bacterial flocculation. Altogether, these findings allow to establish a photodynamic antimicrobial chemotherapy alternative that can be used effectively against Gram-positive topic infections using the widely available natural polymeric lignin as a drug carrier. Further research, aimed to inhibit the growth and survival of Gram-negative bacteria, is likely to enhance the wideness of acetylated lignin nanoparticle applications.

Photophysical and Antibacterial Properties of Porphyrins Encapsulated inside Acetylated Lignin Nanoparticles

“…Porphyrin-loaded acetylated lignin nanoparticles were prepared as previously reported [ 20 ], and as described in detail in Materials and Methods 4.3. Nanoparticles were obtained and kept as suspensions in distilled water, during the whole characterization and evaluation process.…”

“…Usually, the hydrodynamic size takes into account the ions that may surround a particle, so can differ from the size obtained through other methods, such as microscopic observations. Previous research has demonstrated that the hydrodynamic size correlates with the size measured through transmission electron microscopy (TEM) observations, for acetylated lignin nanoparticles [ 20 ]. The obtained size, the polydispersity index (PDI) and the zeta potential ( Table 1 ) provide information of the stability and the way that nanoparticles interact.…”

“…Previous studies [ 20 ] have demonstrated that THPP encapsulated inside acetylated lignin nanoparticles conserved their main photophysical features (i.e., absorption and emission spectra), as well as their capacity to generate singlet oxygen under light irradiation. In order to investigate the impact of the metallation, the blockage of the hydroxyl moieties, and the presence of positive charges in the porphyrins, the nanoparticles library, as well as the free porphyrins, were characterized on these different parameters.…”

“…Indeed, when THPP is encapsulated, its relative intensity of the two emission bands inverted, with a higher emission band at 733 nm, instead of 663 nm, for the encapsulated porphyrin. This has been explained by the formation of the protonated species THPPH 2 2+ [ 29 ], which suggests that the microenvironment inside acetylated lignin nanoparticles is acidic [ 20 ]. Further analysis ( Supplementary Figure S4 ) revealed that encapsulated ZnTHPP and T(PrOH)PyP have similar emission profiles, with the principal emission band was found after 700 nm; this profile does not fluctuate even upon pH change in the media.…”

“…Consequently, it is not surprising the lack of examples in the literature of lignin as part of a photodynamic treatment [ 19 ]. Our latest work reported the use of acetylated lignin nanoparticles as a vehicle for 5,10,15,20-tetrakis (4-hydroxyphenyl)-21H,23H-porphine (THPP), and its efficient antibacterial effect against Gram-positive bacteria [ 20 ]. Here, we concluded that the use of acetylated lignin as vehicle might be highly advantageous, for weak and non-hydrophillic photosensitizers.…”