Advances in antimicrobial photodynamic inactivation at the nanoscale

Abstract

The alarming worldwide increase in antibiotic resistance amongst microbial pathogens necessitates a search for new antimicrobial techniques, which will not be affected by, or indeed cause resistance themselves. Light-mediated photoinactivation is one such technique that takes advantage of the whole spectrum of light to destroy a broad spectrum of pathogens. Many of these photoinactivation techniques rely on the participation of a diverse range of nanoparticles and nanostructures that have dimensions very similar to the wavelength of light. Photodynamic inactivation relies on the photochemical production of singlet oxygen from photosensitizing dyes (type II pathway) that can benefit remarkably from formulation in nanoparticle-based drug delivery vehicles. Fullerenes are a closed-cage carbon allotrope nanoparticle with a high absorption coefficient and triplet yield. Their photochemistry is highly dependent on microenvironment, and can be type II in organic solvents and type I (hydroxyl radicals) in a biological milieu. Titanium dioxide nanoparticles act as a large band-gap semiconductor that can carry out photo-induced electron transfer under ultraviolet A light and can also produce reactive oxygen species that kill microbial cells. We discuss some recent studies in which quite remarkable potentiation of microbial killing (up to six logs) can be obtained by the addition of simple inorganic salts such as the non-toxic sodium/potassium iodide, bromide, nitrite, and even the toxic sodium azide. Interesting mechanistic insights were obtained to explain this increased killing.

Keywords: Antimicrobial photodynamic inactivation; Drug-resistant microbial cells; drug delivery nanovehicles; efflux-pump inhibition; fullerenes; nanotechnology-based drug delivery; photochemical mechanisms; potentiation; titania photocatalysis; titanium dioxide photocatalysis.

Figures

Figure 1. Jablonski diagram

Ground state photosensitizer (0PS) absorbs light to form first excited singlet state (1PS) that (in addition to losing energy by fluorescence or conversion to heat) undergoes intersystem crossing to form the long lived first excited triplet state (3PS). The triplet state can undergo type I (electron transfer) photochemical reaction to form superoxide and hydroxyl radical, and/or type II (energy transfer) photochemical reaction to form singlet oxygen. These ROS can oxidatively damage and kill all known forms of microorganism.

Figure 2. Cell wall structures of Gram-positive and Gram-negative bacteria and fungi

(A) Gram-positive bacteria have a single lipid bilayer surrounded by a thick but porous layer of peptidoglycan, with teichuronic and lipoteichoic acids providing a negative charge. (B) Gram-negative bacteria have a double lipid bilayer (inner and outer membrane) separated by periplasm and peptidoglycan. The outer membrane contains porins and lipoproteins and is decorated with lipopolysaccharide chains with a negative charge. (C) Fungi have an outer cell wall composed of polysaccharides such as mannan, β-glucan, and chitin. The cells do not have a pronounced negative charge and more closely resemble eukaryotic mammalian cells.

Figure 3. Progression stages of growth of a bacterial biofilm

Initially free-floating planktonic cells settle down onto a surface that can either be inanimate or living tissue. Next secretion of extracellular matrix allows the cells to grow in place. Finally, a mature biofilm is formed complete with water channels to allow oxygen and nutrients to penetrate.

Figure 4. Tetrapyrrole absorption spectra showing porphyrins, chlorins, bacteriochlorins, and phthalocyanines

As pyrrole-ring double bonds are successively reduced starting in porphyrins and going to chlorins and bacteriochlorins, the Q-band moves to longer wavelengths and increases in size.

Figure 5. Nanovehicles that have been used to encapsulate PS

Polymeric nanoparticles are sub-μm colloidal particles designed to solubilize hydrophobic PS. (A) Nanomicelles in which amphiphilic co-polymers with hydrophobic and hydrophilic blocks self-assemble to entrap the cargo; (B) nanocapsules, in which the cargo is in solution and surrounded by a shell-like wall; (C) nanospheres, in which the cargo is dissolved, adsorbed, or dispersed throughout the matrix, attached to the surface or attached to the polymer matrix; and (D) liposomes in which an amphiphlic polymer self-assembles into a lipid bilayer that forms a unilamellar vesicle that encapsulates the cargo.

Figure 6. Gold nanoparticle-conjugated PS

(A) Gold nanoparticles with attached PS. (B) Plasmonic gold nanoparticles and nanorings. The local electric field caused by conductance electrons potentiates the optical field close to the surface especially in nanorings. (C) Potentiation of PDT by surface plasmonic enhancement of the photoactivity of an attached PS.

Figure 7. Chemical structures of some PS that have been tested for antimicrobial applications

(A) Methylene blue – a blue phenothiazinium dye. (B) Toluidine blue O – a blue phenothiazinium dye. (C) Rose Bengal – a pink halogenated xanthene dye. (D) Hypericin – a yellow naturally occurring perylenequinone from St John’s Wort. (E) BF21 – a brown/black fullerene with six cationic charges.

Figure 8. Titania photocatalysis

Schematic illustration of main processes in the photocatalytic reaction of TiO2. Nanoparticles have a sufficiently large surface area to allow this process to be efficient. Electrons are excited by UVA light from the semiconductor valence band to the conductance band. The electrons in the conductance band undergo electron transfer to oxygen to form superoxide, and the holes in the valence band react with water to form hydroxyl radicals. The ROS produced (O2·− and HO·) can kill microorganisms.

Figure 9. Upconversion nanoparticle-mediated PDT

Nanoparticles made of rare earth salts such as NaYF4 absorb CW 980-nm light and emit short wavelength 400- to 500-nm light that can excite a conjugated PS. Nine hundred and eighty-nanometer light has good tissue penetration but too low energy to excite PS.

Figure 10. Multi-drug efflux pumps found in various microbial cells

(A) Multidrug and toxic-compound extrusion (MATE) found in both Gram-positive and Gram-negative bacteria. (B) Major facilitator superfam-ily (MFS) found in both Gram-positive and Gram-negative bacteria. (C) Small multidrug resistance (SMR) found in both Gram-positive and Gram-negative bacteria. (D) Resistance nodulation division (RND) found in Gram-negative bacteria. (E) ATP-binding cassette (ABC) transporter found in fungal cells.