Also, some natural photosensitizers, are present in plants or in fungi that have been used in this area, such as: psoralen (furanocoumarins), perylenequinonoid pigments, hypericin, and hypocrellin. Nowadays, many photosensitizer types with different physicochemical and optical properties are available for photodynamic inactivation of a wide range of microorganisms.
As a rule, the chemical purity, the selective uptake and the localization inside the microorganism, the high antimicrobial efficiency, and the lack of mutagenic activity or genotoxicity are the important characteristics of an ideal photosensitizer
. Any type of these sensitizers should meet several criteria: chemical purity, tumor selectivity, fast accumulation in target tissues and rapid clearance, proper wavelengths and deeper penetration, and no dark toxicity.
In terms of solubility, photosensitizers can be classified into three main groups:
hydrophobic photosensitizers without peripheral substituents with electric charge and being slightly soluble in water or alcohol (phthalocyanines and naphthalocyanines, hematoporphyrin, hematoporphyrin derivative (HpD), porfimer sodium, and porphyrin precursors)
hydrophilic photosensitizers that contain three or more peripheral substituents with electric charge and have a high solubility in water at physiological pH.
amphiphilic photosensitizers that contain one or two peripheral substituents with electric charges, soluble in water or alcohol, at physiological pH. In their structure, there are always two regions, one hydrophobic (represented by porphyrin with electrically charged groups) and another hydrophilic
5. Anionic Photosensitizers as Anti-Viral Agent for aPDT
In recent years, many achievements have been reached in fundamental aPDT sensitizers
. The most important classes of photosensitizers tested so far are:
The first generation of photosensitizers: hematoporphyrin, hematoporphyrin derivative (HpD), porfimer sodium, and porphyrin precursors, which are not ideal photosensitizers for photodynamic therapy. Due to their complex composition, HpD does not exhibit a good photodynamic efficiency, because some of the HpD components are inactive. In addition, HpD is localized in healthy tissues, thus inducing a residual photosensitization of the whole body for almost a month after its administration.
The second generation of photosensitizers includes macrocycles as porphyrins, phthalocyanines, naphthalocyanines, benzoporphyrin derivatives, chlorines, and bacteriochlorines, with good absorption of wavelength radiation from the spectral region (650–700 nm).
The third generation of photosensitizers includes fullerene nanostructures, carbon nanotubes, bioconjugated porphyrins/phthalocyanines with DNA, or human serum albumin (HSA) biological structures.
The large majority of photosensitizers for aPDT are based on tetrapyrrolic systems, as porphyrins. They should have an excited triplet state with a sufficiently long lifespan able to lead to the production of singlet oxygen, and to have adequate chemical and physical stability. The anionic types have a strong tendency to aggregate. Although the formation of aggregates results in a reduced single singlet oxygen generation efficiency, they can promote cell penetration due to the helical spatial structure
It was discovered about 25 years ago that Gram-negative bacteria are relatively resistant to the photodynamic action of many PS, while Gram-positive bacteria and fungi are efficiently killed
. It was found that PS with a pronounced cationic charge can be very efficient at killing Gram-negative species and that this preferential effect is partly due to the fact that cationic PS bind well to the anionic Gram-negative bacterial cells, and partly due to the so-called “self-promoted uptake pathway” described by RW Hancock
by which cationic (but not anionic) PS penetrate to the interior of the bacterial cells. However, it has recently been discovered that aPDI sensitizer can be potentiated by the addition of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate
or potassium iodide to an anionic porphyrin
TSPP is a very large disk-shaped molecule with charges at the four corners and at the geometric center. In aqueous solutions, at neutral pH, the electronic absorption spectrum of TSPP is typical for free-based porphyrins (D2h symmetry) and is characterized by an intense Soret band around 420 nm and four Q bands in the range of 500–700 nm.
The formation of highly ordered TSPP aggregates at low pH values has been observed previously
, these being formed by the intermolecular electrostatic attractions between the positively charged nucleus and the negatively charged periphery of the cycle. The protonation of the two pyrrole nitrogen atoms of the porphyrin ring introduces a change in the symmetry of the molecule from a 2-fold configuration to a 4-fold configuration. Both the B band (436 nm) and the maximum Q band are red-shifted at protonation, and the color change of the solution from purple to green for free basic and deprotonated shapes, respectively, was recorded
. The structure of TSPP absorption spectra in an aqueous solution strongly depends on pH. At neutral pH, the absorption spectrum of TSPP consists of an intense Soret Band at 413 nm and four weak Q bands at 515, 550, 578, and 631 nm (Qy (1,0), Qy (0,0), Qx (1,0), Qx (0,0), respectively). In acidic solution (pH below 5.0) of low concentration, the TSPP absorption spectrum in the visible spectral region changes to a three-band spectrum composed of an intense absorption band at 645 nm, and weaker bands at 597 and 550 nm. The Soret band is red shifted to 435 nm with respect to that at neutral pH. Monoprotonated species of TSPP and dication forms (H
) in acidic solutions are expected
, and these spectral changes might be attributed to them. Further decrease of solution pH results in an appearance of the new absorption bands at 490 and 706 nm. The Soret band undergoes a slight shift to the blue and a decrease in intensity like the rest of the absorption bands belonging to the dication of TSPP (
). At pH 1.1, the TSPP absorption spectrum consists of the Soret Band at 430 nm; two intense bands at 490 and 709 nm; and three weak bands at 560, 640, and 670 nm. The band at 490 nm has a weak shoulder at 520 nm. The ratio between the intensities of both absorption bands at 490 and 709 nm and the absorption bands of dication vary depending on the total concentration of TSPP in acid solutions. Similar changes in absorption spectra can be induced by varying the ionic strength of the acidic solution of TSPP and have been assigned to the formation of J-aggregates
Absorption spectra of TSPP in aqueous solution at different pH values. (concentration 5 × 10
Due to its versatility, TSPP are now under reinvestigation, because this PS can adopt different cationic/anionic forms at different pH, temperature, concentrations, and ionic strength
. In acidic environments, new absorption bands (from 490, 706 nm) become dominant when the TSPP concentration exceeds 10
M and these are attributed to the dicationic forms of TSPP and subsequently to the aggregate forms of this porphyrin. Aggregates J are formed with monomeric dicationic molecules arranged in a dimension so that the transition moments of the monomers are parallel and the angle between the transition moment and the line joining the molecular centers is zero
. The aggregation process causes further changes in the optical spectra of TSPP. The presence of intermolecular excitonic interactions determines the division of each of the monomeric bands into a blue and a red displacement band, associated with J and H type interactions
. The band at 490 nm comes from the J (head-tail) aggregates of the porphyrin molecules. In the dicationic form, due to Coulombic static repulsion, the two central N-H + fragments in the porphyrin macrocycle are probably distorted outside the aromatic plane, as reported elsewhere
The ionized forms of porphyrins.
In contrast, the 401 nm and 422 nm bands occur in the H aggregate of porphyrin molecules
(face-to-face interaction) and occur at c > 2.5 × 10
M. H-aggregates, so named because of their band spectral, are characterized by the blue (hypochromatic) displacement in relation to the absorption band of the monomer and are found spatially by a face-to-face stacking of monomer species. In contrast, J aggregates (named after their discoverer, Scheibe Jelly) are edge or edge spatial assemblies that produce bathochromic (red) displacements,
The ionized forms of TSPP (red: dicationic form; blue: J-aggregate; black: monomer.)
Absorption bands of different species of TSPP.
Whereas UV–vis and fluorescence techniques enabled us to determine the type of aggregates formed and the size of the assemblies in solution, AFM provided direct visualization of the aggregates. AFM experiments in air at room temperature with 100 μm acquired in tapping mode for additional image processing,
3D topographic images for TSPP monomer (left) and TSPP J-aggregate (right).
Topographic studies conducted by means of atomic force microscopy at the scale of 10 μm reveal that the distribution of porphyrins varies. TSPP shows a high density of particles on the same surface. From the analysis of the 3D images of porphyrins studied, could be observed an uniform distribution of particles on the analyzed surface; their average size was 23 nm for monomer form of TSPP, which tends to form aggregates of larger sizes (73.1 nm) than the other porphyrins studied
6. Influence of Dicationic (J-Aggregates) TSPP form on aPDT
Herpes Simplex Virus (HSV) can be irreversibly and permanently made photosensitive by heterocyclic dyes so that brief exposure to visible light renders the virus non-infectious
. Photodynamic inactivation is dependent upon the dye concentration, temperature, and pH
. Membrane-photosensitizing dyes have the advantage of inactivating the virus at a site other than the genetic material
. Many systems as porphyrin derivatives have been tested in different culture cells
. Working with two viral suspensions: A type with a cell concentration 2.6–2.8 × 10
per cell standard, and B type with a cell concentration of 2.7–2.9 × 10
per cell standard. It is concluded that the survival curves of dermal HSV from rats during photosensitization with TSPP 1.377 × 10
M are the most efficient during the inactivation process of HSV. The other concentrations are not proper for this inactivation,
. This fact could be interpreted knowing different aggregated and ionized forms of TSPP
and geometrical configurations of these forms with their reduced photochemical activity, as H-aggregates
. Studies on Herpes Simplex virus type 1 (HSV-1) are useful, because without the help of HSV-1, the COVID-19 virus may not be able to cause serious illness or death in humans. This method could be a new direction for COVID treatment and immunization, either to prevent infections or to develop photoactive fabrics (e.g., masks, suits, gloves) to disinfect surfaces, under artificial light and/or natural sunlight. The use of photodynamic therapy (PDT) can be an alternative approach against SARS-CoV-2 that deserves to be explored.
The survival curves of HSV derma from rats at different TSPP concentrations. 1 = 0.274 × 10
M; 2 = 0.6885 × 10
M; 3 = 2.754 × 10
M; 4 = 5.508 × 10
M; 5 = 1.377 × 10
M up) and the HSV-derma from rats’ inactivation kinetics at different temperatures (down).
The action of this PS is highly dependent on the temperature,
. The hyperthermia (37.5 and 42 °C) can potentiate the effect of PDT, due to the temperature effect on the basic photochemical processes
Temperature dependence of TSPP.
Previous results show an increase in the reaction rate over the temperature range 15–45 °C. It can be seen that the optimum value of temperature is 37.5 °C because at this temperature TSPP could exist in the solution as a monomer and dication (J-aggregates) mixture in good agreement with other literature reports
. This porphyrin derivative demonstrates a remarkable virucidal activity upon light activation after 48 h, especially for HSV hen derma at 37.5 °C. The concentration and temperature effects were evaluated and also the time interval between dye treatment of cells and virus inoculation.
The viral membrane or protein coat might serve as a barrier to the penetration of the photosensitizing dye, and the sensitivity of the virus perhaps is determined primarily by the permeability of its exterior layer or interface with the suspending medium. The HSV envelope was found to be the major target for the photodynamic damage following dye inactivation. DNA damage is one crucial mechanism driving aPDI. aPDT leads to breaks in single-stranded and double-stranded DNA and the disappearance of the super-coiled fraction of plasmid DNA in both G
. An exemplification of the spectral interaction between TSPP with DNA, by UV-Vis spectrum (where from a new Soret band with a bathochromic shift, and a decrease of the same band), could be shown in
The spectral interaction TSPP-DNA.