Antimicrobial and antiviral activity of porphyrin photosensitization

Invited Paper

Zvi Malik, Hava Ladan, Yeshayau Nitzan and Zehava Smetana

Life Sciences Department, Bar-Ilan University, Ramat-Gan 52900, ISRAEL

ABSTRACT

The development of photodynamic therapy (PDT) has provided an effective modality against anfibiotic-resistant
bacteria and cell free viruses. The antibacterial activity of porphyrin induced photodynamic therapy shows unique
properties: I. it is independent of the antibiotic sensitivity spectrum of the treated pathogen and II it has an efficient
and non-recovering anti-microbial killing effect upon illumination of Gram positive bacteria. Bacterial PDT is
affected by the use of various sensitizers, as a general rule non-charged or positively charged molecules are
effective in photoinactivation of Staphylococcus agrees . In order to photosensitive Gram (-) bacteria such as
Pseudomonas aeuruginosa and Escherichia colt we introduced the small peptide polymyxin-B nona-peptide (PBNP)
which stimulated the translocation of porphyrin through the outer membrane of these bacteria and makes PDT
possible. Gram negative cell killing by the use of PBNP and DP broadens the antibacterial spectrum of
photodynamic inactivation and opens new horizons for this modality as a wide spectrum drug when antibiotic
resistance is the main concern. Plasmidial and chromosomal DNA damage in S. Duress and E. coil cells was
mediated by DP photosensitization. The major observation was the disappearance of the plasmid supercoiled
fraction. The chromosomal DNA was also affected and its degradation products were detected after treatment.

Porphyrin-mediated photosensitization is effective against free viruses, virus-transformed cells and the viral
contaminants in blood. Specific photodynamic inactivation of free Friend leukemia viruses and cell-associated
virions of the virus complex was attained by HPD. Our most recent results depict the PDT sensitive phases of
Herpes simplex (HSV-1, HSV-2) Varicella zoster and the nonenveloped Adeno2 viruses These viruses were treated
with different derivatives of phthalocyanines modified both by their side chains and their metal substitutes during
short time intervals of their adsorption to the target cells. Specific phthalocyanines were highly effective in
photoinactivation of the viruses during the initial stage prior to viral-endocytosis and during this process. These
new photosensitizers may act as potent viral disinfectant.

1. INTRODUCTION

Porphyrins possess a high binding-affinity to cellular components, membranes, proteins and DNAI. Living cells as
well as dead cells are stained rapidly by different porphyrins. Appropriate illumination generates an emission of red
fluorescence and generates toxic oxygen species. Cancer cells stemming from solid tumors cells and bacterial
infected tissues show preferential retention of porphyrins2 In vivo

administration of various sensitizers to tumor bearing animals and humans resulted in retention of the porphyrins in
the tumors, while the normal surrounding tissues had a low comparable porphyrin contents 2. Photodynamic
therapy of solid tumors was found highly efficient in eradication of the lnflicted tissues and the damage,-initiating
necrosis or apoptosis of photodynamic therapy, occurred within a very small time frame3. Photodynamic
interactions was described to take place wherever sensitizer, light and oxygen are simultaneously present2.
Inflammatory tissue was described to manifest similarities in porphyrin retention and therefor bacterial and viral
infected tissues may become targets for photodynamic treatment4.

2. CHARACTERISTICS OF PORPHYRIN BlNDING TO BACTERIA

Microbial cells, prokaryotic as well as eukaryotic, are believed to represent simplified models for studying
mechanisms of photosensitization by porphyrins. Nevertheless, bacterial cells present other methodological
problems. The ultrastructural differences between Gram (+), Gram (-) and yeast cells has rendered the subject of
microbial systems to be a specific research field4. The binding capacity of porphyrins to eukaryotic cells is
dependent on their hydrophobicity/hydrophilicity accountable to their side chains on the tetra-pyrrole ring, as well
as to the structure of the cellular targets such as membranes and proteins5. The cell wall of bacterial cells creates a
physical and chemical barrier that may prevent sensitizer binding to bacterial membrane or proteins. The structure /
function relationship indicates that non-charged porphyrin molecules are efficiently bound to and photodynamically
inactivate Gram (+) bacteria whereas they are bound to the outer membrane only of Gram (-) bacterial cells but do
not inactivate them. Spheroplasts of Gram (-) cells prepared by enzymatic degradation of the outer membrane and
the cell wall bind porphyrin molecules to the inner membrane and thus render them to be targets for
photodestruction6. The combined treatment of Gram (-) bacteria with the small peptide polymyxin-B nona-peptide
(PBNP) stimulates the translocation of porphyrin through the outer membrane and stimulates photosensitization7 8.
These concepts are presented in Table 1:

3. PHOTOSENSITIZATION OF BACTERIAL CELLS

The antibacterial activity of porphyrin induced photodynamic therapy shows unique properties: I It has an efficient
and non-recovering anti-microbial killing effect during illumination of Gram (+) bacteria and II is independent of
the antibiotic sensitivity spectrum of the treated pathogen9~12. The prerequisite for photosensitization of a
microbial cell is the binding of porphyrin to the cytoplasmic membrane, which is strongly pH dependent6. The
mixture of DP with hemin has a dark cytotoxic activity on S. agrees, and other Gram (+) bacteria; the effect of the
combined mixture was stronger than that of the separate constituents, and was as strong in the dark as under
illuminationl3~l7. The total inability of the Gram (+) cultures to recover from the combined treatment by
hemin-DP in the dark, suggests the possibility of the formation of an oxidizing porphyrin complex. In order to
photosensitize Gram negative bacteria such as Pseudomonas aeruginosa and E. Cole we used polymyxin-B
nona-peptide (PBNP), which stimulated the translocation of porphyrin through the outer membrane of Gram (-)
bacteria and made PDT possiblel8 19. Gram (-) cell killing by the use of PBNP and DP broadens the antibacterial
spectrum of photodynamic inactivation and opens new horizons for this modality as a wide spectrum drug when
antibiotic resistance is the main concern7s8. Table 2 reveals a characteristic experiment of photodynamic
inactivation of the bacterial cells by DP, hemin TPPS4, TMPyP and PBNP. The results clearly reveal the possibility
of bacterial PDT of Gram (+) as well as Gram (-) bacteria by appropriate conditions. For Gram (-) cells the
combined treatment with PBNP and photosensitizer is highly efficient for photodynamic inactivation of these life
threatening pathogens7 8.

4. DNA DAMAGE DURING BACTERIAL PHOTOSENSITIZATION

It was found that TMPyP and its metallo-complexes, with Mn, Fe, Co or Zn, bind to AdenineThymidine rich
regions of the pBR 322 plasmid DNA20. The outside binding of the porphyrins, by intercalating or through
hydrogen bonding to DNA, appears to respond to steric and electrostatic potential effects located in the minor
groove of the DNA21. Various effects of porphyrins on purified DNA have been described. In the presence of
oxygen and visible light the synthetic water-soluble porphyrins cleave the pBR322 purified plasmid supercoiled
DNA producing relaxed and linear DNA . In purified DNA, the photoactivated HPD caused modification of the
guanine residues. It has also been demonstrated that hemin can cause strand scission in isolated DNA only in the
presence of oxygen and mercaptoethanol22 .

In order to gain a better understanding of the potential bacterial sub-cellular targets affected by porphyrins, we
intended to examine the direct effect of DP and hemin on bacterial DNA species in vivo. Treatment of S. aureus
with photoactivated DP changed the plasmid DNA supercoiled profile and the ultrastructural appearance of the
DNA in the intact Staphylococcus aureus cells23. The chromosomal DNA was also affected by hemin and by
photosensitized DP, since chromosomal DNA degradation products were detected after treatment. In addition,
transmission electron microscopy revealed a marked change in the ultrastructural appearance of the chromosomes
of the treated cells, expressed by the formation of visible DNA fibers within the cel]s4-23. Plasmidial and
chromosomal DNA damage in E. coli cells was similarly mediated by DP photosensitization. The changes in the
plasmidial DNA profiles was time dependent. The major observation was the disappearance of the plasmid
supercoiled fraction. The results indicate that the bacterial DNA is a possible target for porphyrin's action in the
Gram (+) and Gram (-) cells. Damage caused to the bacterial DNA may contribute to the antibacterial action of
these agents. The observed perturbation of the plasmid structure and composition by porphyrins may explain
previous results24, where the survivors of porphyrin treatment have impaired antibiotic resistance to penicillin
caused by the damaged plasmids responsible for induction of 13-lactamases synthesis. Electron microscopy
depicted ultrastructural alterations in the chromosomal structure of Gram (-) bacteria induced by the photodynamic
effect. Table 3 summarizes the chromosomal and plasmid damage induced by DP and light.

5. THE CELLULAR DAMAGE IN THE PHOTOSENSITIZED BACTERIA

Ultrastructural studies disclosed that most of the cells were unable to accomplish their cell-wall synthesis and cell
division shortly after the initiation of the light phase. The disturbances in the synthesis of the membrane and
cell-wall were accompanied by the appearance of a multilamellar structure near the septum of dividing cells a
mesosome-like structure25. It has been suggested that oxygen-dependent reactions potentiated by porphyrin
photosensitization may induce cross linking of cell wall precursors D revealed as membranous-like structures9
24-75. Cell Iysis or dramatic cell decomposition are not the . initial mechanisms of bacteria] killing Lytic process
took place but never exceeded 30 /0 of the population determined by electron microscopy. Rather? inhibition of
cell propagation accompanied by quick reduction in 14C-glucose consumption was noted in porphyrin-treated and
illuminated cultures, r followed by a marked reduction in colony forming units4. Fast ionic fluxes in Staph agrees
during short --intervals of porphyrin mediated photosensitization were determined by '-ran microanalysis combined
^ with scanning electron microscopy26 27 The 5. agrees bacteria photosensitized with DP showed total K Floss as
well as marked Na effux which increased with irradiation time. this was accompanied bv the >decline of other cell
elements The prevailing K loss in bacteria during photosensitization is deduced to Lbe an immediate primary
photodynamic effect while other ionic changes are connected with the Redevelopment of cellular damage. The
overall effects are summarized in Fig 1.

6. NOVEL PiITHALOCYANINE DERIVATIVES FOR VIRAL PllOTOINACTIVATION

Photodynamic inactivation of viruses in blood and blood components using various photosensitizers and light of
appropriate wavelength is currently under intensive interest28~33. This concem is due to the continued risk of the
transmission of viruses by blood and blood products. Among these sensitizers, phthalocyanine (Pc) derivatives have
shown great promise. Pc are porphyrin-like second-generation sensitizers for photodynamic therapy (PDT) of
cancer34-35. Their intense absorption in the far red and long-lived excited triplet state are among the important
attributes that make them ideally suited for PDT In addition, by selecting appropriate metal ligands and peripheral
substituents, Pc derivatives that are powerful sensitizers for PDT cause only minimal damage to red blood cells34,
thus, making them potentially useful for viral decontamination of blood. The mechanism by which Pc inactivates
viruses is not known. Enveloped viruses mainly, have been photoinactivated by Pc. Membrane photosensitizer dyes
such as porphyrin like compounds are less mutagenic than DNA photosensitizing dyes. The viral nucleic acids are
therefore not an important target during Pc-induced photoinactivation. Rather, some protein(s) comprising the viral
envelope is probably sensitive to photoinactivation. With respect to photochemistry, it appears that viral
inactivation proceeds primarily by a type II, singlet oxygen-mediated, mechanism.

Porphyrin-mediated photosensitization is effective against free viruses, cell associated viruses, virustransformed
cells and viril contaminants in blood. Specific photodynamic inactivation of free Friend leukemia viruses and
cell-associated virions of the virus complex was attained by HPD. Our most recent results35 depict the sensitive
phases of Herpes simplex (HSV-1, HSV-2) Varicella zoster and the nonenveloped Adeno-2 viruses to
photosensitization by phthalocyanines compared to merocyanine 540 These viruses were treated with novel
derivatives of phthalocyanines modified st their side chains and metal substitutes during short time intervals of
their adsorption to the target cells. Specific phthalocyanines were highly effective in photoinactivation of the
viruses during the initial stage prior to viral-endocytosis. Other processes associated with the virus life cycle which
follow virus penetration might be also sensitive. Thus the new photosensitizers acting as potent viral disinfectants
are challenging.

The kinetics of viral photoinactivation was resolved during the stages of viral adsorption and penetration into the
host cells. Sensitivity to photoinactivation decreased progressively with time after addition of viruses to their host
cells. The viruses were most sensitive to photodynamic inactivation up to 30 min from the initiation of adsorption.
Cell-associated viruses, 45-60 min after the onset of adsorption, were highly resistant to photodynamic treatment
(PDT), with the exception of amphiphilic Pc derivatives. Thus, the mixed sulfonated Pc-naphthalocyanines
derivatives, AINSB3P and AIN2SB2P demonstrated a remarkable decontamination activity even 60 min after the
onset of adsorption. Ultrastructural examination of these photosensitized viruses demonstrated damage to the viral
envelope which prevented viral adsorption and penetration. The possible implications for viral decontamination of
blood are discussed. The non-enveloped adenovirus was found to be resistant to all the tested dyes.

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