The present report has demonstrated that stable, synthetic cationic bacteriochlorins are highly promising candidate photosensitizers for antimicrobial PDT. The new method for bacteriochlorin synthesis (22
) provides compounds with gem-dimethyl groups in the reduced pyrrole rings at the 8 and 18 positions. This substitution pattern locks in the bacteriochlorin macrocycle by preventing the oxidation reactions that typically occur with derivatives of naturally occurring bacteriochlorins. These reactions lead to instabilities encountered with many other bacteriochlorins previously tested for PDT activity. The versatility of the 3,13-disubstituted bacteriochlorin building blocks enables macrocycles with a variety of substituent patterns to be prepared, including the set of quaternized compounds that were studied herein.
A large number of publications have pointed out the necessity of using cationic charged photosensitizers to efficiently mediate photodynamic inhibition (PDI) of Gram-negative bacteria; however, reports that have compared structure-function relationships of photosensitizers against three different classes of microbial cell (Gram-positive bacteria, Gram-negative bacteria, and fungal yeast) are less common (30
). One striking result from the present investigation is that the photosensitizer structure that gives the maximum PDI effect is different for each class of microbial cell.
All four compounds tested were highly active against the Gram-positive S. aureus
(Fig. ). The bis-quaternized bacteriochlorin 2 was most effective, producing a remarkable 5 logs of killing at 100 nM. The other three compounds (basic, tetrakis-quaternized and hexakis-quaternized) exhibited comparable levels of cell killing that were lower than that of bacteriochlorin 2 but still quite substantial (>5 logs at 1 μM). One explanation of this finding is that there exists an optimum level of cationic charge necessary both to bind to bacterial anionic phosphate groups and to allow penetration into the bacterial cell wall, where the reactive oxygen species produced upon illumination can do most damage. Levels of cationic charge less than this optimum value (for instance, the properties of bacteriochlorin 1) will not lead to sufficient binding, and cationic charges greater than this optimum value (for instance, bacteriochlorins 3 and 4) will lead to the binding being too strong to allow greater photosensitizer penetration into the cell wall. A similar finding has been presented in two other reports by one of our groups involving comparisons of conjugates between chlorin(e6) and different sizes of polylysine chains (16
) or different sizes of polyethylenimine chains (51
). In both cases the smallest conjugate with the least cationic charges had the greatest PDI effect against S. aureus
, while the largest conjugate with the most cationic charges was the most effective against E. coli.
Maisch et al. (28
) also found that a porphyrin with two cationic groups was a better photosensitizer against S. aureus
than a molecule with four such groups.
A more straightforward structure-function relationship is found here for the effect of the bacteriochlorins against the Gram-negative E. coli (Fig. ). In particular, the greater the number of cationic quaternized groups the greater the PDI effect. Bacteriochlorin 4, with six cationic groups, kills measurable numbers of cells at 100 nM and eliminates the population at 1 μM. Bacteriochlorin 3 (four cationic groups) kills 4 logs at 1 μM, while bacteriochlorin 2 (two cationic groups) kills only 1 log at 1 μM, and bacteriochlorin 1 (no cationic groups) has no killing effect at all.
The fungal yeast C. albicans displays yet another structure-function relationship (Fig. ). Only the noncationic bacteriochlorin 1 has a high PDI killing effect, namely, elimination of the population (>6 logs) at 100 μM. The bis-cationic bacteriochlorin 2 shows a measurable 1 to 2 logs of killing at ≥1 μM, while bacteriochlorins with four (bacteriochlorin 3) or six (bacteriochlorin 4) cationic groups give no killing effect at all. The microscopy studies suggest that the noncationic bacteriochlorin 1 is able to penetrate to the interior of the fungal cells, while the cationic bacteriochlorin 2 cannot; this difference in localization and uptake explains the much greater fungicidal effect of bacteriochlorin 1. The similarity of the structure-function relationships between Candida and HeLa cells is presumably due to the fact that fungal cells are eukaryotic and to some extent resemble mammalian cells in their overall cellular structure. Because both types of cells are classified as eukaryotes, they have many component features in common, including plasma membrane, nucleus and nuclear membrane, mitochondria, endoplasmic reticulum, Golgi apparatus, and cytoskeleton.
While many authors have reported that Candida
cells are susceptible to PDI with cationic photosensitizers (9
), there are other reports that photosensitizers commonly used to kill cancer cells, such as Photofrin (5
), are also effective against yeast cells. Further study is necessary to understand the precise structural features of photosensitizer molecules for optimal PDI of fungal cells while preserving selectivity over the host mammalian cells. The overall goal of antimicrobial PDT is to be able to kill microbes that are infecting tissue after local application of the photosensitizer solution to the infected area and subsequent illumination. Thus, it is necessary to also study the PDT killing of mammalian cells that would comprise the host tissue. To this end, a human cancer cell line (HeLa cells) was investigated using the same incubation time (30 min) employed for the microbial cells. The structure-function relationship was to some extent similar to that found for C. albicans
, with only the basic bacteriochlorin (bacteriochlorin 1) giving any significant level of killing at concentrations lower than 1 μM. Therefore, selective PDT killing of bacteria (and to a lesser extent fungal cells) compared to that of mammalian cells is accomplished with quaternized bacteriochlorins, with the bis-cationic compound giving the highest selectivity for S. aureus
and the hexakis-cationic compound giving the highest selectivity for E. coli
To our knowledge there has been only one prior investigation of bacteriochlorins as antimicrobial photosensitizers. Schastak et al. (45
) compared the photodynamic killing of S. aureus
, methicillin-resistant S. aureus
(MRSA), E. coli
, and Pseudomonas aeruginosa
using a meso-substituted tetramethylpyridinium bacteriochlorin with that with a chorin(e6) derivative called Photolon. The cationic bacteriochlorin was able to kill both Gram-positive and Gram-negative bacteria, while the anionic Photolon was only able to kill Gram-positive species. Several groups have studied bacteriochlorins to kill cancer cells and to treat tumors in vivo
. The long-wavelength light between 700 and 800 nm that is absorbed by bacteriochlorins is believed to be ideally suited to penetrate living tissue due to reduced absorption by tissue chromophores and reduced Mie scattering (53
). The large extinction coefficient (>100,000 M−1
) typical of the bacteriochlorin Qy
band is also advantageous for strong absorption of near-infrared light by the photosensitizer. The Pd-containing bacteriochlorins known as TOOKAD (13
) and Stakel (2
) have been extensively investigated in laboratory studies, and, in addition, TOOKAD has been studied in clinical trials of PDT for prostate cancer (54
The photophysical studies and DFT calculations indicate that the activity differences observed among bacteriochlorins 1 to 4 must stem from cellular binding and localization effects rather than photochemical properties. Indeed, the yield of the triplet excited state (from which the reactive oxygen species is produced) is essentially identical (0.48 to 0.53) for the four bacteriochlorins, and in each case the lifetime is reduced to <1 μs in the presence of atmospheric oxygen, indicating facile excited-state quenching. Moreover, there is no specific correlation between the anticipated differences in redox properties (based on the molecular orbital energies) for the four bacteriochlorins and their PDI activities against any of the organisms studied. The only broad trend is that bacteriochlorins 1 and 2 are typically more active than bacteriochlorins 3 and 4, which, all other things being equal, would favor a mechanism of activity that involves reduction rather than oxidation of the photoexcited bacteriochlorin to the extent that electron transfer is involved.
In conclusion, bacteriochlorins with constitutive cationic charges provided by quaternized ammonium groups are highly active antibacterial photosensitizers. The hexakis-cationic bacteriochlorin 4 is capable of eliminating (>6 logs killing) both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria at the remarkably low concentration of 1 μM. Good selectivity (4 to 5 logs) for bacteria over mammalian cells is observed. Only the nonquaternized bacteriochlorin 1 shows good PDT killing of the yeast (C. albicans), and selectivity over mammalian cells is lower in this case because both cell types are eukaryotic organisms.