Synthesis, Spectroscopic, and Photophysical Characterization and

Article pubs.acs.org/jmc

Synthesis, Spectroscopic, and Photophysical Characterization and Photosensitizing Activity toward Prokaryotic and Eukaryotic Cells of Porphyrin-Magainin and -Buforin Conjugates Ryan Dosselli,†,∥ Rubén Ruiz-González,‡ Francesca Moret,† Valentina Agnolon,† Chiara Compagnin,† Maddalena Mognato,† Valentina Sella,§ Montserrat Agut,‡ Santi Nonell,‡ Marina Gobbo,§ and Elena Reddi*,† †

Department of Biology, University of Padova, via U. Bassi 58/B, I-35121 Padova, Italy Institut Químic de Sarrià, Universitat Ramon Llull, via Augusta 390, E-08017 Barcelona, Spain § Department of Chemical Sciences, University of Padova, via F. Marzolo 1, I-35131 Padova, Italy ‡

S Supporting Information *

ABSTRACT: Cationic antimicrobial peptides (CAMPs) and photodynamic therapy (PDT) are attractive tools to combat infectious diseases and to stem further development of antibiotic resistance. In an attempt to increase the efficiency of bacteria inactivation, we conjugated a PDT photosensitizer, cationic or neutral porphyrin, to a CAMP, buforin or magainin. The neutral and hydrophobic porphyrin, which is not photoactive per se against Gram-negative bacteria, efficiently photoinactivated Escherichia coli after conjugation to either buforin or magainin. Conjugation to magainin resulted in the considerable strengthening of the cationic and hydrophilic porphyrin’s interaction with the bacterial cells, as shown by the higher bacteria photoinactivation activity retained after washing the bacterial suspension. The porphyrin−peptide conjugates also exhibited strong interaction capability as well as photoactivity toward eukaryotic cells, namely, human fibroblasts. These findings suggest that these CAMPs have the potential to carry drugs and other types of cargo inside mammalian cells similar to cell-penetrating peptides.

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ing.9 PDT is based on the use of a visible light-absorbing dye, called a photosensitizer (PS), that, when activated with light of appropriate wavelengths, generates reactive oxygen species (ROS) that have cytotoxic effects. PDT kills bacteria by inducing oxidative stress at multiple cellular targets,10 which makes the development of resistance very unlikely. 11 Interestingly, several authors reported that antibiotic-resistant and antibiotic-sensitive bacterial strains are equally sensitive to PDT treatment.12,13 PS charge is a major determinant of photosensitization efficacy: anionic and neutral PSs are effective only against Gram-positive bacteria, whereas cationic PSs are effective against both bacterial groups.10 This different susceptibility to PDT is determined by the lower permeability of the cellular envelope of Gram-negative bacteria compared to Gram-positive bacteria. Cationic PSs can achieve efficient interactions with the anionic charges of the cell wall components,11 ultimately resulting in a stronger photokilling efficacy when compared to anionic/neutral ones. A possible way to increase the reactivity of anionic/neutral PSs is to attach them covalently to positively charged molecules such as, as we recently proposed, cationic antimicrobial peptides.14

INTRODUCTION Currently, infectious diseases are the second leading cause of death worldwide, and this is strictly connected with the continued increase in resistance to currently available antibiotics developed by many pathogens over recent decades.1 Several classes of antibiotic-resistant pathogens are recognized as major threats to human and animal health. These include Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA),2 vancomycin-resistant Enterococcus faecalis (VRE),3 and multidrug-resistant mycobacteria.4 Even more alarming is the appearance of multi- and pan-resistant Gram-negative strains such as those of Enterobacteriaceae (Klebsiella pneumonia and Escherichia coli) with extended spectrum β-lactamase and the New Delhi metallo-β-lactamase resistance.5,6 More recently, multidrug-resistant Acinetobacter baumannii7 and Pseudomonas aeruginosa8 have become a major cause of concern in controlling infectious diseases. Although antibiotic resistance continues to expand, the number of new agents approved for clinical use has been decreasing. Thus, there is a tremendous need to develop strategies to combat antibiotic-resistant infections endowed with a low potential to stimulate resistance. Among the novel approaches that are being investigated, antimicrobial photodynamic therapy (PDT) appears promis© 2014 American Chemical Society

Received: October 25, 2013 Published: January 23, 2014 1403

dx.doi.org/10.1021/jm401653r | J. Med. Chem. 2014, 57, 1403−1415

Journal of Medicinal Chemistry

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Figure 1. Structures of the porphyrins and their peptide conjugates.

Cationic antimicrobial peptides (CAMPs) are critical components of the innate immune systems of most living organisms.15−17 CAMPs are generally short (between 15 and 60 amino acids) and show potent activity against some pathogens. Because of their wide distribution across different taxa, CAMPs can have very different structures, biological activity spectra, and modes of action against pathogens.18 Indeed, the majority of known CAMPs act by disrupting the membrane integrity of target pathogens, causing the leakage of the inner content and ultimately the death of the target microorganism.19 Typical examples of this category of CAMPs are magainin from Xenopus laevis20 and protegrin from porcine leukocytes.19 Other recurring modes of action include binding to nucleic acids (buforin), targeting of cytoplasmic proteins (apidaecin and pyrrhocoricin), stimulation of the host immune reaction (LL-37), and combination of different targets (defensins).18,19,21 These multiple modes of action are likely to interfere with the development of bacterial resistance and also to result in a broad spectrum of activity against a variety of pathogens.18,19 In a previous paper, we reported the synthesis, characterization, and antimicrobial activity resulting from the conjugation of porphyrin-type photosensitizers with apidaecin 1b.14 The conjugation resulted in a new class of photosensitizing agents with a broader spectrum of activity and improved photokilling ability compared to the original components alone. In an attempt to obtain further improved and more potent photosensitizing agents, we herein report the synthesis and characterization of conjugates between porphyrins and two of the most studied and promising CAMPs, buforin II and magainin 2. Magainin 2, a 23 amino acid CAMP, assumes an αhelical conformation in a lipid environment22,23 and shows potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, perturbing the bacterial membrane by a pore-forming mechainsm.26 Buforin II, a 21 amino acid peptide, shares a similar structure and antimicrobial properties with magainin 2, but it is considered a nonmembranolytic peptide. Similarly to apidaecin,25 buforin II translocates across the bacterial membrane and acts on intracellular targets.24

Specifically, it kills bacteria likely by inhibiting transcription or translation through interaction with nucleic acids. Both magainin 2 and buforin II have high antimicrobial efficacy21 and a broader spectrum of activity compared to apidaecin 1b.27 Therefore, because all three CAMPs have different bacterial targets and mechanisms of action, comparison of the PS conjugates generated with each of them is expected to give a more clear indication on the peptides that allow the formation of more potent photosensitizing agents for antimicrobial PDT. It is reported that these CAMPs exerts no hemolytic activity and no appreciable toxicity toward normal mammalian cells,28,29 and on the basis of this, one can expect selectivity of antimicrobial PDT when PS conjugates of these peptides are used. To assess whether the PS−peptide conjugates exert their photosensitizing activity preferentially toward bacteria while sparing normal cells of the host, we also tested phototoxicity in human normal fibroblasts.

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RESULTS AND DISCUSSION Synthesis and Characterization of the Conjugates. The new PS−peptide conjugates, 1b−c and 2d (Figure 1), were synthesized as previously reported for 1e and 2f,14 linking the properly functionalized porphyrin, 5(4′-carboxyphenyl)10,15,20-triphenylporphyrin (1a) or 5(4′-carboxyphenyl)10,15,20-tripyridylporphyrin (2a), to the N-terminal end of the peptide that is still attached to the solid support. Two analogues of the natural antimicrobial peptides were synthesized, [Leu21]magainin and Gly-buforin, to reduce possible drawbacks in the assembling of conjugates, which include the porphyrin-catalyzed oxidation of the methionine residue in magainin and the steric hindrance in the coupling step between the porphyrin and the β-branched N-terminal threonine for the buforin conjugate. As expected, methylation of the pyridinic nitrogens of the porphyrin−magainin conjugate to afford the cationic PS 2d occurred also on the π nitrogen of His because this residue was blocked only on the τ nitrogen during peptidechain assembling. After cleavage and deprotection from the solid support, the conjugates were purified by reversed-phase 1404



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Figure 2. CD spectra of magainin (A), buforin (B), and their conjugates in trifluoroethanol.

Figure 3. CD spectra of magainin (A), buforin (B), and their conjugates in Tris-NaCl buffer.

conformation from residues 12 to 20 and 5 to 10, with both regions being separated by a hinge caused by a Pro residue at position 11.31 Consistent with this, the CD spectra (in TFE) of buforin and its conjugate 1c exhibit two negative maxima localized near 222 and between 208 and 205 nm, suggesting partially ordered peptides with a population of largely helical conformers (Figures 2B and S1D). In conclusion, linking either the PS 1a (hydrophobic) or 2a (hydrophilic) to the N-terminus of magainin or buforin does not modify the propensity of the peptide backbone to adopt an amphiphilic helical structure in membrane-mimicking environments. This is an important requirement for enabling the interaction with bacterial membranes. The situation is different in aqueous environments. In buffer (Tris 10 mM and NaCl 150 mM) and in SDS micelles, the CD spectra of buforin conjugate 1c closely resemble that of the parent peptide (Figures 3B and S1B) and exhibit a broad negative maximum around 200 nm indicative of largely unstructured peptides in an aqueous environment. Moreover, in buffer, the reduced intensity of the spectrum of the conjugate with respect to that of the free peptide suggests the presence of aggregation phenomena (i.e., peptide−porphyrin and probably also porphyrin−porphyrin interactions, Figure S1C). The tendency to form aggregates in aqueous solvent is even more evident for magainin conjugate 1b, whose CD spectrum shows an intense split Cotton effect in the Soret band region (Figure S1C) and two negative Cotton effects in the peptide region (Figure 3A). These spectra suggest that the peptide chain is

HPLC and characterized by analytical HPLC, electrospray mass spectrometry, and UV−vis absorption spectroscopy. Circular Dichroism Studies. The selectivity of CAMPs against bacteria can be used to direct the PS against specific bacterial targets, improving the antimicrobial photodynamic effect while inducing little damage toward mammalian cells. As with most membrane-active peptides, magainin 2 adopts a welldefined helical structure when it interacts with bacterial membranes,23 and in buforin II, two α-helical regions connected by a proline hinge are key elements for peptide translocation and targeting of intracellular bacterial components.24 To ascertain whether the conjugation of the PS to the peptide can substantially modify its secondary structure, the conformational properties of the conjugates were investigated by CD spectroscopy in different solvents and compared to those of the parent peptide. In membrane-mimicking environments (2,2,2-trifluoroethanol, TFE, or SDS micelles), the CD spectra of PS−magainin conjugates 1b and 2d closely resemble that of the parent peptide and are characterized by two negative Cotton effects localized near 222 and 208 nm (Figures 2A and S1A). This general pattern, typical of CD spectra of predominantly righthanded α-helical peptides,30 suggests that in membranemimicking environments the conformational preferences of magainin are minimally affected by intramolecular interactions with the PS. By NMR methodologies, the solution structure of buforin was determined in a TFE/water mixture and resulted a helical 1405



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Figure 4. Absorption spectra in MeOH (red traces), PBS (blue traces), and E. coli suspensions (green traces) that are normalized to facilitate their comparison. Left panels: comparison of conjugated peptides 1b (A), 1c (B), and 2d (C) to parent porphyrins (yellow thick solid line) 1a (A, B) and 2a (C). Right panels: spectra of conjugated peptides 1b (D), 1c (E), and 2d (F) in different media. The bulk concentration is 5 μM for all compounds.

Phosphate Buffered Saline (PBS). Model porphyrin 1a is highly hydrophobic and insoluble in water and therefore no fluorescence could be recorded in PBS. On the contrary, its conjugates, 1b and 1c, are water-soluble as a consequence of the net charge conferred by the peptide at pH 7.4. Their spectroscopic and photophysical properties change substantially relative to MeOH; the absorption spectra show broader Soret bands (Figure 4, right panels), the fluorescence is strongly quenched, the decays become biexponential, and the singlet oxygen quantum yields (ΦΔ) also decrease (Table 1). These observations match those for the apidaecin conjugate14 and are consistent with aggregation phenomena as detected by UV spectroscopy (Figure 4D, E). The triplet lifetime (τT) is unusually long for a typical aqueous PS, which suggests that 1O2 is produced by the nonaggregated fraction of conjugates in which the peptide is folded around the porphyrin. Such folding favors the production of 1O2 by preventing the aggregation of the porphyrin; however, it also shields the porphyrin from oxygen, which results in a longer triplet lifetime. It must be recalled that 1O2 production requires access of O2 to the PS excited state.32 However, the lifetime of 1O2 for all conjugates in deuterated PBS (dPBS) is shorter than the value expected in this solvent (65 μs),33 indicating that the peptides quench 1O2 to some extent. The behavior of 2d is different in that there is no broadening of the Soret band as compared to MeOH (Figure 4C,F), and neither the fluorescence nor the 1 O 2 yields decrease dramatically (Table 1). This indicates that porphyrin aggregation is of lesser importance for this conjugate. The fluorescence decays are, however, biexponential, and the fluorescence spectrum is intermediate between that of the

probably folded over the hydrophobic porphyrin and the porphyrins are close to one another to reduce the exposure to the solvent. Similar aggregates were previously observed for the conjugate of apidaecin with the same PS.14 Perturbation of the peptide CD signal is less important when the neutral PS 1a is replaced by the cationic 2a. In fact, the CD spectrum of magainin conjugate 2d in an aqueous environment closely resembles that of the parent peptide and is characterized by a broad negative band around 200 nm (Figure 3A), as expected for a predominantly disordered peptide chain. Photophysical Properties of the Conjugates. The spectroscopic and photophysical properties of neutral porphyrin 1a and its conjugates with magainin and buforin, 1b and 1c, respectively, as well as cationic porphyrin 2a and its conjugate 2d with magainin were measured in aqueous and organic media and in bacterial cell suspensions to assess structural and environmental effects as well as their correlation with antibacterial activity. The data of free 1a and 2a14 were confirmed and are reproduced here for comparison. Methanol. The left panels in Figures 4 and 5 show the absorption and fluorescence spectra, respectively, of free PSs and their peptide conjugates in MeOH. In this solvent, the spectra of the PSs are not affected by conjugation to the peptides. Consistently, the fluorescence quantum yield (ΦF) values also do not change appreciably (Table 1). Conjugates show monoexponential fluorescence decay kinetics with lifetime values equal to those of free porphyrins (Table 1). In addition, no difference in behavior can be observed between the current conjugates and the previously described apidaecin−PS conjugates.14 1406



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Figure 5. Fluorescence spectra in MeOH (red solid traces), PBS (blue dotted traces), and E. coli suspensions (green dashed traces) that are normalized to facilitate their comparison. Left panels: comparison of conjugated peptides 1b (A), 1c (B), and 2d (C) to parent porphyrins (yellow thick solid line) 1a (A, B) and 2a (C). Right panels: spectra of conjugated peptides 1b (D), 1c (E), and 2d (F) in different media. The bulk concentration is 5 μM for all compounds.

Table 1. Photophysical Properties of the Peptide Conjugates and Model Compounds in MeOH and PBS λF,max (nm)

ΦF a

τS (ns) b

compound

MeOH

PBS

MeOH

1a 1b

647 647

nsg 651

0.040 0.056

ns 0.010

10.1 9.8

1c

647

650

0.052

0.008

9.6

2a

656

675

0.022

0.008

7.9

2d

654

658

0.025

0.014

8.7

PBS

MeOH

ΦΔ PBS

ns 9.6 3.8 9.5 3.8 4.2

(0.73)h (0.27) (0.82) (0.18)

8.7 (0.72) 3.1 (0.28)

MeOH

c

τΔ (μs) PBS

d

MeOH

0.63 0.68

ns 0.12

9.8 10.0

0.65

0.09

10.0

0.69

0.73

9.6

0.76

0.65

9.9

e

τT (μs) PBS

f

ns 1.9 46.4i 3.1 60.7i 3.6 60.0i 3.6 52.0i

MeOH

PBS

0.3 0.3

ns 11.9

0.4

18.8

0.3

2.0

0.5

2.6

a

Cresyl violet as standard (ΦF (MeOH) = 0.54).40 bTMPyP as standard (ΦF (PBS) = 0.017).41 cTMPyP as standard (ΦΔ (MeOH) = 0.74).42 TPPS as standard (ΦΔ (water) = 0.69).42 eLiterature value, 10.4 μs.43 fLiterature value, 3.5 μs in PBS and 65 μs in dPBS.43 gNot soluble. h Fractional amplitudes are given in parentheses. iValue in deuterated PBS d

nor 2a is capable of entering the E. coli cells, unlike tetracationic TMPyP, at long incubation times.34 Photoinactivation of Prokaryotic Cells and Uptake. The ability of the porphyrin−peptide conjugates to photosensitize the death of prokaryotic cells was studied using methicillin-resistant S. aureus (MRSA) and E. coli as models of Gram-positive and Gram-negative bacteria, respectively. The bacteria were incubated for 60 min in the dark with increasing concentrations of conjugates and then irradiated with 13.5 J cm−2 of blue light. No major differences in the photoinactivation efficiency were observed by changing the incubation time to 15 or 120 min (Figures S3 and S4). Irradiation was carried out either without washing the cells and leaving the excess of unbound conjugate in the suspension or

conjugate in MeOH and of free porphyrin 2a in buffer (Figure 5F), confirming that, in a fraction of the conjugates, the peptide perturbs the porphyrin. In this case, however, oxygen access to the porphyrin is not precluded by the peptide, which is likely the outcome of a less tight interaction owing to the three positive charges of the porphyrin. E. coli Suspensions. All of our attempts to detect differences in the photophysical properties in E. coli cell suspensions relative to PBS solutions were unsuccessful. Because the biological assays demonstrate cell binding (vide infra), we must conclude that either the majority of the conjugates remain in the aqueous phase, masking the behavior of the bound ones, or that the bound conjugates experience a microenvironment very similar to PBS. It must be mentioned that neither free PS 1a 1407



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Novel conjugates 1b and 1c were more potent in killing S. aureus upon illumination than the previously studied conjugate of the same porphyrin with apidaecin.14 This correlates well with the higher quantum yield of singlet oxygen production of the buforin and magainin conjugates in PBS relative to that of the apidaecin conjugate (Table 1 and ref 14). The quantum yield alone does not, however, explain the stronger photokilling effect of 1c relative to 1b. This might be better explained by the highest net positive charge of buforin, which facilitates tight interactions with bacteria, and its ability to drive the porphyrin toward intracellular components more sensitive to light-induced oxidation and/or more critical for cell survival than those interacting with the magainin conjugate. For instance, buforin can interact with DNA27 and therefore photodamage to this important cell component might explain, at least in part, its higher light-induced killing of S. aureus. A striking result is the observation that for both 1b and 1c the conjugate concentrations necessary to kill S. aureus are orders of magnitude higher than those of free porphyrin 1a (50 nM; Figure 6A). We have no clear explanations for this result, and, as previously reported,14 the high phototoxicity of 1a can probably be ascribed to the use of DMSO as solvent for the delivery of this hydrophobic porphyrin. However, because DMSO was not toxic per se at the concentration used, the effect most likely reflects a more efficient penetration of the porphyrin. This hypothesis can be supported by the observation that the addition of DMSO during incubation and irradiation of S. aureus with 1b or 1c did not increase the photokilling of the bacterium significantly (Figure S7). By contrast, the photosensitizing efficiency of cationic 2a was much less reduced after conjugation to magainin, and complete killing of unwashed S. aureus was observed with concentrations in the range of 0.5−1 μM. However, cell washing before irradiation dramatically reduced the photokilling efficiency of the cationic porphyrin, and at 1.5 μM, it caused only 2 and 5 log reductions of S. aureus survival, respectively, in the free form and conjugated to magainin (Figure 6B). The flow cytometry data (Figure 7) showed that magainin slightly increases the capability of the cationic porphyrin to interact with the cells, and this correlates with the higher photokilling of washed S. aureus observed for 2d relative to 2a. A similar behavior was observed with the same cationic porphyrin conjugated to apidaecin (2f in Figure 1) and was ascribed to the very weak association of 2a and its conjugates with bacterial cells and their quick removal by washing.14 E. coli. As previously reported, 1a itself, being anionic, is not active against E. coli, whereas when conjugated to buforin (1c) or magainin (1b), it showed an ability to kill E. coli upon light activation but to a different extent. At 5 μM, 1c caused complete killing (7 log reduction of survival) of both unwashed and washed E. coli, whereas 1b reduced the survival by 3 log only (Figure 8). In addition, with this bacterium, the washing out of the unbound conjugates did not reduce the photokilling effect and only marginally decreased the porphyrin red fluorescence associated with the cells, as shown by the flow cytometry measurements (Figures 7 and 8B). On the contrary, the photokilling effect of 2a and its conjugate 2d with magainin was strongly reduced after cell washing because of their weak association with the cells. However, conjugation of the cationic porphyrin to magainin limited the reduction of the bacterial photokilling caused by washing. As an example, 15 μM 2a and 2d reduced the survival of washed E. coli by 1 and 5 log, respectively. In summary, the conjugation of porphyrin 1a to

removing it by washing the cells three times with PBS before irradiation. S. aureus. Conjugates 1b and 1c caused complete killing of unwashed and washed S. aureus at concentrations of 1 and 1.5 μM, respectively (Figure 6A). The removal of the excess

Figure 6. Photoinactivation of S. aureus with increasing concentrations of 1a, 1b, 1c, 2a, and 2d. S. aureus suspensions were incubated with the compounds for 60 min and then irradiated with 13.5 J cm−2 of blue light (390−460 nm) without washing (A) or after three washes with PBS (B). The data for 1a and 2a were published in ref 14 and are shown here for comparison.

unbound conjugates did not cause any important reduction of bacterial killing (Figure 6B), suggesting that the photodynamic effect was mainly due to conjugate molecules tightly bound to the cells. Binding was confirmed by flow cytometry experiments (Figure 7) that showed distinctly higher red fluorescence from S. aureus cells incubated with 1b and 1c compared to that of the control both before and after washing (Figure 7). However, the histograms in Figure 7 showed broad Gaussian curves, indicating heterogeneous uptake and retention. In addition, two peaks were found, especially when measuring cellassociated 1b and 1c after three washes, which can be explained by the presence of more than one population of events. In fact, on the basis of the analysis of the FSC vs SSC dot plots (Figure S5), various populations were identified representing cell clusters of increasing dimensions whose formation was favored by incubation with conjugates (in particular with magainin) but not free porphyrins 1a or 2a. 1408



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Figure 7. Binding of porphyrins 1a and 2a and their conjugates to E. coli and S. aureus. The cells were incubated for 60 min with the compounds and then analyzed by flow cytometry without washing or after three washes. The concentration of the compounds was 5 and 1.5 μM for E. coli and S. aureus, respectively. The data for 1a and 2a were published in ref 14 and are reported here for comparison.

found that irradiation with 13.5 J cm−2 of blue light induced complete killing of fibroblasts that had been incubated for 60 min with 1a or 2a at concentrations of 5 and 3 μM, respectively (Figure 9A). Unexpectedly, both porphyrins showed a much higher photoactivity after conjugation to the peptides, and complete death of fibroblasts was achieved with conjugate concentrations in the nanomolar range (Figure 9B and Table 2). On the contrary, irradiation of fibroblasts that had been incubated with porphyrin 1a or 2a together with unconjugated peptides caused a decrease of viability not significantly different from that measured with the porphyrin alone (Figure S6C). Our results are different from those reported by others that showed selective photoinactivation of bacteria using protoporphyrin IX or eosin Y conjugated to a lipopolysaccharide binding peptide and the antimicrobial peptide (KLAKLAK)2, respectively.35,36 Therefore, selective targeting of bacteria over

buforin affords a very effective PS that, upon light activation, kills Gram-negative bacteria in the same concentration range of the most photoactive cationic porphyrin 2a and, contrary to this, fully retains its photosensitizing activity after repeated washing. Photoinactivation and Uptake Studies with Eukaryotic Cells. The photosensitizing activity of the porphyrin− peptide conjugates toward eukaryotic cells was studied using human normal skin fibroblasts as a cell model that well represents the host cells that might be damaged during the PDT treatment of dermatological infections. In this part of the study, we have considered the magainin and buforin conjugates as well as the previously synthesized apidaecin conjugates (1e and 2f, Figure 1) and the free porphyrins for reference. We applied the same incubation conditions and light doses used for the bacteria to the fibroblasts. As for the free porphyrins, we 1409



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Figure 9. Viability of HFFF2 cells irradiated with 13.5 J cm−2 of blue light after a 60 min incubation with increasing concentrations of PS.

Table 2. IC50 Values of Porphyrins and Porphyrin Conjugates for Fibroblasts Incubated in the Dark for 60 min and Then Irradiated with 13.5 J cm−2 of Blue Light Figure 8. Photoinactivation of E. coli with increasing concentrations of 1b, 1c, 2a, and 2d. E. coli suspensions were incubated with the compounds for 60 min and then irradiated with 13.5 J cm−2 of blue light (390−460 nm) without washing (A) or after three washes with PBS (B). The data for 2a were published in ref 14 and are shown here for comparison.

mammalian cells is possible when using conjugates formed with particular combinations of PS and peptides. It has been speculated36 that highly lipophilic PSs can compromise the targeting specificity of CAMPs because they interact with all lipid bilayers without discriminating between mammalian or bacterial membranes. This can explain the absence of selectivity of conjugates 1b, 1c, and 1e formed with the highly lipophilic porphyrin 1a. However, porphyrin 2a is very likely too hydrophilic and exhibits a very low propensity to interact with bacteria (Figure 7) and, in conjugates 2d and 2f, the peptide may lose its ability to specifically interact with bacteria membranes because it is coiled around the porphyrin molecule. It must be pointed out that none of the conjugates was toxic to fibroblasts in the dark (Figure S6A) at concentrations at least 1 order of magnitude higher than those causing complete cell death under irradiation. Similarly, the peptides alone did not significantly affect the viability of the fibroblasts both in the dark and after illumination (Figure S6B). These observations are in agreement with other findings that reported no toxicity and no hemolytic effects of the cationic peptides considered in this study.20,27−29 The fibroblasts incubated with free or conjugated porphyrins were analyzed by flow cytometry and confocal fluorescence

compound

IC50 (nM)

1a 1e 1b 1c 2a 2f 2d

1270 21 10 26 230 9 8

microscopy to determine binding and cellular localization of the conjugates relative to porphyrins 1a and 2a. The red fluorescence was much higher in cells incubated with the conjugates as compared to free porphyrins (Figures 10 and 11), confirming that conjugation to the peptide leads to a higher amount of porphyrin associated with the cells and, as a consequence, more efficient cell photosensitization. The flow cytometry analysis showed that, in general, all of the conjugates associated/entered the fibroblasts more efficiently and faster than their parent porphyrin during the incubation times of 0.5− 3 h and were totally retained by the cells after washing and incubation in PS-free culture medium (Figure 11). However, differences in uptake were particularly remarkable for the two series of compounds. The binding of conjugates 1b, 1c, and 1e was at least 1 order of magnitude higher than that of conjugates 2d and 2f, whereas differences in fibroblast photoinactivation were not so evident (Figure 9). Therefore, it must be hypothesized that several factors, whose relative importance can vary for each compound, determine the efficiency of cell photoinactivation. For conjugates 2d and 2f, the higher yield of 1410



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(Figure 10). Therefore, it is expected that following illumination the cells very likely die via necrosis caused by photodamage to the plasma membrane. The localization of the conjugates in the plasma membrane can be justified by the short incubation time used in our experiments to match the conditions used to kill bacteria. In fact, we will report elsewhere that for longer incubation times (e.g., 24 h) the porphyrins are internalized. Therefore, these CAMPs may not only have the ability to associate with eukaryotic cells but also to act as drug delivery agents capable of crossing the cell membrane, as do cell-penetrating peptides.37,38 A recent report suggests that this might indeed be the case.39 However, it must be emphasized that although the blue light used in this work is suitable for the PDT treatment of superficial bacterial infections or for skin disinfections46,47 only red light endowed with high penetration through mammalian tissues will allow for a wider PDT application with these conjugates. We anticipate that with well-tailored incubation and irradiation protocols these porphyrin−peptide conjugates will also efficiently inactivate cancer cells when activated by red light (manuscript in preparation).

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CONCLUSIONS Among new strategies to combat antibiotic-resistant infections, antimicrobial photodynamic therapy appears promising for the treatment of local infections caused by both Gram-positive and Gram-negative bacteria. In previous studies, we have shown that conjugation of hydrophobic porphyrin 1a to the CAMP apidaecin renders this porphyrin photoactive against hard-tokill Gram-negative bacteria.14,48 In this article, we have extended our investigation to porphyrin conjugates with magainin and buforin and showed that the nature of the peptide affects the phototoxic activity of the conjugate. Conjugation of 1a or 2a to magainin slightly improves the photosensitizing effect of the porphyrin with respect to the apidaecin conjugates. However, neither magainin nor apidaecin conjugates resulted in more phototoxicity than unconjugated porphyrins 1a and 2a toward MRSA and E. coli, respectively. On the contrary, buforin conjugate 1c not only was phototoxic against E. coli as the most active cationic porphyrin 2a but also was tightly bound to bacteria and fully retained its photokilling activity even after extensive cell washing. Another notable result is that despite the selective toxicity toward bacterial cells

Figure 10. Localization of porphyrin 1a and its conjugates in HFFF2 cells after a 60 min incubation. The PS concentration was 5 μM.

singlet oxygen production (0.7−0.8, Table 1 and ref 14) in aqueous media compensates for the lower uptake with respect to conjugates 1b, 1c, and 1e, rendering the fibroblast photoinactivation comparable. Fluorescence microscopy studies showed that porphyrins 1a and 2a and conjugates 2d and 2f cannot be visualized in the cells after the short incubation of 60 min because of scarce binding. On the contrary, we could detect the fluorescence of conjugates 1b, 1c, and 1e largely localized on the cell surface

Figure 11. Binding/uptake of porphyrins 1a and 2a and their conjugates to HFFF2 cells. The cells were incubated in the dark with the PS (0.5 μM) for 30, 60, or 180 min or with the PS for 60 min followed by an additional 180 min in PS-free medium (60 + 180 min) before flow cytometry analysis. 1411



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exhibited by the CAMPs used in this study28,29 their porphyrin conjugates easily interacted with eukaryotic cells, tremendously increasing the photoactivity of the bound PS toward these cells. Even with short incubation times, all conjugates induced complete killing of fibroblasts at concentrations 1 or 2 orders of magnitude lower than the corresponding free porphyrins. Although PDT is considered a selective treatment because photodamage is confined to the sites where the light-activated PS is located, the photoxicity of conjugates against fibroblasts necessitates a careful optimization of the protocols for treating cutaneous infections and a redesign of PS−CAMP conjugates to improve the targeting of prokaryotic over eukaryotic cells. Taken together, conjugation of a PS to CAMPs is a very powerful strategy to tailor their ability to interact with both prokaryotic and eukaryotic cells, which may improve the efficiency of photodynamic treatments and widen the field of their applications.

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0.05 mmol of porphyrin (1a or the pyridinic analogue), 0.05 mmol of diisopropylcarbodiimide, and 0.05 mmol of 1-hydroxybenzotriazole in DMF/CH2Cl2 (1:1 v/v). The reaction mixture was shaken overnight and then filtered to remove the excess of reagents. The resin was repeatedly washed with DMF and CH2Cl2 until the filtrate was colorless followed by drying under vacuum. Methylation of the pyridine N was carried out on the porphyrin−peptide conjugate (0.02 mmol), still attached to the solid support, by overnight treatment with a 10% solution of methyl iodide in DMF (2 mL). The excess of reagents was filtered off, and the resin was repeatedly washed with DMF and CH2Cl2 and dried under vacuum. Cleavage from the resin and deprotection of the peptide conjugates were carried out by treatment with a mixture of TFA/triisopropylsilane/water (95:2.5:2.5 v/v/v) for 1.5 h at room temperature. The filtrate, obtained from resin removal, was reduced under vacuum to a small volume, and by addition of cold ether, the PS−peptide conjugate was obtained as a green solid. The conjugates were purified to at least 95% purity by semipreparative HPLC, using a 20 min linear gradient of eluent B in A in the following range: 40−80% B (1b), 35−75% B (1c), and 30−45% (2d). 1b: yield 45%. HPLC tR 26.2 min. UV−vis (MeOH): λmax /nm (log ε/ M−1 cm−1) 414 (5.97), 512 (4.58), 547 (4.26), 589 (4.05), 643 (3.85). HRMS: [M + H]+ calcd for C160H210N34O30, 3089.59; found, 3089.95. 1c: yield 65%. HPLC tR 21.6 min. UV−vis (MeOH): λmax /nm (log ε/ M−1 cm−1) 414 (5.47), 512 (4.24), 546 (4.01), 586 (3.92), 645 (3.74). HRMS: [M + H]+ calcd for C153H214N45O28, 3131.67; found, 3131.75. 2d: yield 55%. HPLC tR 17.2 min. UV−vis (water): λmax /nm (log ε/ M−1 cm−1) 425 (4.81), 520 (3.57), 560 (3.19), 587 (3.19), 649 (2.80). HRMS: [M4+] calcd for C161H219N37O30, 787.667; found, 788.188. Spectroscopic Measurements. Absorption spectra were recorded on a double-beam Cary 6000i spectrophotometer (Varian) equipped with a 110 mm diameter integrating sphere and highperformance photomultiplier tube for diffuse transmittance measurements. Absorption coefficients were derived from the slopes of Lambert−Beer plots. Fluorescence emission spectra were recorded in a Spex Fluoromax-4 spectrofluorometer. Fluorescence decays were recorded with a time-correlated single-photon counting system (Fluotime 200) equipped with a red-sensitive photomultiplier. Excitation was achieved by means of a 405 nm LED working at a 10 MHz repetition rate. The counting frequency was always below 1%. Fluorescence decays were analyzed using the PicoQuant FluoFit 4.0 data analysis software. 1O2 phosphorescence was detected by means of a customized PicoQuant Fluotime 200 system described in detail elsewhere.45 Briefly, a diode-pumped pulsed Nd:Yag laser (FTSS355Q, Crystal Laser) working at a 10 kHz repetition rate at 532 nm (12 mW, 1.2 μJ per pulse) was used for excitation. A 1064 nm rugate notch filter (Edmund Optics) was placed at the exit port of the laser to remove any residual component of its fundamental emission in the near-IR region. The luminescence exiting from the side of the sample was filtered by a cold mirror (CVI Melles Griot) to remove any scattered laser radiation and focused on the entrance slit of a Science Tech 9055 dual-grating monochromator. A near-IR-sensitive photomultiplier tube assembly (H9170-45, Hamamatsu Photonics) was used as the detector at the exit port of the monochromator. Photon counting was achieved with a multichannel scaler (Becker&Hickl MSA 300 or PicoQuant’s Nanoharp 250). The time-resolved emission signals were analyzed using the FluoFit software to extract lifetime values. Laser flash photolysis measurements were carried out using a Q-switched Nd:YAG laser (Surelite I-10, Continuum) with right-angle geometry and an analyzing beam produced by a Xe lamp (PTI, 75 W) in combination with a dual-grating monochromator (mod. 101, PTI) coupled to a photomultiplier (Hamamatsu R928). Kinetic analysis of the individual transients was performed with software developed in our laboratory. All spectroscopic measurements were carried out in 1 cm quartz cuvettes (Hellma) at room temperature (rt). For the measurements in bacterial suspensions, bacteria were incubated in the dark under the conditions employed for the photoinactivation

EXPERIMENTAL SECTION

General Methods. All chemicals were commercial products of the best grade available, and, unless otherwise indicated, they were used directly without further purification. The starting porphyrins, 5-(4carboxyphenyl)-10,15,20-triphenylporphyrin (1a) and 5-(4-carboxyphenyl)-10,15,20-tris(4-pyridyl)porphyrin and its tris-N-methylpyridinium iodide (2a) were prepared by established methods under the Lindsey and Adler−Longo conditions, respectively. 4 4 9Fluorenylmethoxycarbonyl(Fmoc)-amino acids and all other chemicals for the solid-phase synthesis were supplied by Sigma-Aldrich. FmocSer(t-Bu)-Wang and Fmoc-Lys(Boc)-Wang were purchased from Novabiochem (Merck Biosciences). Analytical HPLC separations were carried out on a Dionex Summit dual-gradient HPLC equipped with a four-channel UV−vis detector using a Vydac 218TP54 column (250 × 4.6 mm, 5 μm, flow rate at 1.5 mL/min, W. R. Grace and Co.). Mobile phases A (aqueous 0.1% TFA) and B (90% aqueous acetonitrile containing 0.1% TFA) were used for preparing binary gradients. All analyses were carried out under gradient conditions (10−90% B in 30 min except where otherwise indicated). All crude peptides were purified to 95% or more homogeneity for analytical and other experimental purposes. Semipreparative HPLC was carried out on a Shimadzu series LC-6A chromatographer equipped with two independent pump units, a UV−vis detector, and a Vydac 218TP1022 column (250 × 22 mm, 10 μm, flow rate at 15 mL/ min). Elutions were carried out by the same mobile phases described above. All purified peptides were analyzed again by HPLC and MS. Mass spectra analyses were carried out on a Mariner API-TOF workstation (PerSeptive Biosystems Inc.) operating in positive mode. UV−vis spectra were recorded at room temperature on a Shimadzu UV-2501PC spectrophotometer or on a Lambda 5 spectrophotometer (PerkinElmer) in 0.1 or 1 cm quartz cells. Synthesis of PS−Peptide Conjugates (General Procedure). The peptide sequences were prepared on an automated Advanced Chemtech 348Ω peptide synthesizer on a 0.25 mol scale starting from Fmoc-Ser(t-Bu)-Wang or Fmoc-Lys(Boc)-Wang (resin substitution 0.63 and 0.7 mmol g−1 respectively). The tert-butyl group and 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl were used to protect the threonine and arginine side chains, respectively, and the trityl group was used for asparagine and histidine side chains. Fmoc deprotection was achieved with 20% piperidine in DMF (5 + 15 min). Couplings were performed in the presence of O-(benzotriazol-1-yl) N,N,N′,N′-tetramethyluronium hexafluorophosphate/N-hydroxybenzotriazole /N,N-diisopropylethylamine (reaction time of 45−60 min) using an excess of 4 equiv of the carboxyl component. After coupling of the last amino acid and removal of the Fmoc group, the resin was washed with DMF and CH2Cl2 and then dried under vacuum. The dried resins containing the protected amino acid sequences were used in the coupling reaction to the porphyrin derivatives. The H-peptideresin (0.025 mmol) was swollen in DMF for 1 h and then washed with DMF. To the peptidyl resin was added 600 μL of a solution containing 1412



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experiments, namely, 5 μM PS for 1 h. When required, the cells were washed once, resuspended in PBS to a final concentration of ∼1 × 107 cfu mL−1, and 3 mL of the suspensions were irradiated with 3 million laser pulses at 532 nm under gentle stirring. CD measurements were carried out on a Jasco-715 spectropolarimeter using a quartz cell with a 0.1 cm path length. The spectra were recorded at 298 K and were the average of a series of six scans made at 0.1 nm intervals in the 190− 250 and 350−550 nm regions. Sample concentrations in 10 mM Tris and 150 mM NaCl buffer (pH 7.4), TFE, and aqueous 30 mM SDS were in the range of 10−13 μM. Ellipticity is reported as mean residue ellipticity [θ]R or differential molar circular dichroic extinction coefficient Δε = εL − εR (cm2 dmol−1). Bacteria Culture. E. coli ATCC 25922 and the methicillin-resistant strain of S. aureus, ATCC BAA-44, were purchased from LGC Promochem. Cultures were maintained by 2 weeks of subcultures in brain heart infusion agar (BHI; Difco). For spectroscopic measurements, we used E. coli CECT 101 purchased from the Spanish Type Culture Collection. Bacteria Photoinactivation. For the photoinactivation experiments, the bacteria were grown overnight in BHI at 37 °C, harvested by centrifugation, washed twice, and resuspended in PBS (10 mM phosphate, 0.14 M NaCl, and 2.7 mM KCl, pH 7.3) at a density of ∼2 × 107 cells mL−1. The cell density was measured by reading the turbidity of the suspension at 650 nm in a PerkinElmer spectrophotometer (model Lambda 5). The bacteria used in the experiments were collected from cultures in the stationary phase of growth. The bacteria were incubated with different concentrations of the PSs in the dark at rt for 60 min or with a fixed PS concentration for different times. After incubation, the suspensions were (i) directly exposed to light with the unbound PS left in the suspension (no washing) or (ii) centrifuged (10 000g for 5 min), resuspended in 1 mL of PBS, and washed two additional times with PBS before illumination (three washes). For illumination, aliquots of cell samples obtained as described above were transferred into 96-well plates (200 μL/well). Samples incubated with porphyrins and their conjugates were irradiated from the bottom of the plates with blue light (390−460 nm, with a maximum at 420 nm) for a total light dose of 13.5 J cm−2. The UV 236 lamp, supplied by Waldmann Eclairage SA, used for irradiation supplies a fluence rate of 15.2 mW cm−2, as measured with the Waldmann UV meter. After illumination, aliquots of bacteria suspensions were serially diluted 10-fold in PBS, and 50 μL of appropriate dilutions were plated in duplicate onto BHI agar to determine colony forming units (cfu). Treated and untreated cells were incubated overnight at 37 °C to allow colony formation. Suspensions of bacteria exposed to PSs but kept in the dark and subjected to the same procedure applied to the irradiated suspensions were also plated onto BHI agar after appropriate serial dilutions. Controls included bacteria not exposed to any agent and bacteria exposed to light or peptide only. In addition, control experiments were also performed by incubating S. aureus with 0.05 μM 1b or 1c in the presence and the absence of an amount of DMSO equal to that used for the delivery of 0.05 μM 1a. Each experiment was performed at least three times with independent bacterial suspensions. Flow Cytometry Analysis of Bacteria. The interaction of PSs with bacteria was evaluated by flow cytometry. For these experiments, bacteria were subjected to the same treatments used for the photoinactivation experiments, but instead of being illuminated after incubation and washing, they were analyzed with a FACSCanto II flow cytometer (Becton Dickinson, BD). Samples were excited with a 488 nm laser, and fluorescence emission signals were recorded at wavelengths higher than 670 nm. The bacteria population was isolated from instrument noise by setting electronic gates on the dualparameters dot plots of forward scatter against side scatter. For each sample, 20 000 events were acquired and analyzed with FACSDiva software (BD). Samples not incubated with the PSs were used to determine the cell background fluorescence. Fibroblasts. Human fetal foreskin fibroblasts (HFFF2) were obtained from the European Collection of Cell Cultures (ECACC) and were used as a model to study phototoxicity of eukaryotic cells.

The cells were cultured as monolayer in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 2 mM L-glutamine, 1% nonessential amino acids (Life Technologies), 38 units mL−1 streptomycin, and 100 units/mL penicillin G (Sigma-Aldrich). The cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Photosensitization of Fibroblasts. The photosensitizing activity of the PSs toward fibroblasts was assessed by using the same incubation and irradiation conditions as those used for bacteria. For these experiments, the cells were seeded in 96-well plates (7 × 103 cells/well) and allowed to attach and grow for 48 h in complete culture medium. Afterward, the cells were incubated for 60 min in the dark with increasing concentrations of the PS in culture medium with 3% FBS, washed twice with PBS with Ca2+ and Mg2+, and irradiated in PBS with 13.5 J cm−2 of blue light. Immediately after irradiation, PBS was replaced with culture medium containing 10% FBS, the cells were brought back to the incubator, and after 24 h, cell viability was measured with the MTS test (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega Co.). For the test, the cell medium was replaced with 100 μL of serum-free medium and 20 μL of CellTiter 96 reagent, and the wells were incubated for 1.5 h at 37 °C. The absorbance at 492 nm was measured with Biotrak II plate reader (Amersham, GE Healthcare), and the viability of the treated cells was expressed as the percentage of the absorbance of the control cells that was taken as 100% viability. Controls included cells processed as described but not exposed to PS or light and cells exposed only to PS or light. To evaluate the contribution of the individual components in the induced cell death, the MTS assay was also used to determine the viability of cells incubated with (i) 1 μM PSs or unconjugated peptides in the dark, (ii) 1 μM unconjugated peptides and exposed to 13.5 J cm−2 of light, and (iii) 300 nM unconjugated porphyrins (1a or 2a) in combination with 300 nM unconjugated peptides and exposed to 13.5 J cm−2 of light. Binding and Release of PSs in Fibroblasts. The binding of the PSs in fibroblasts was measured by flow cytometry after 30, 60, and 180 min of incubation. HFFF2 cells were seeded in 24-well plates (3 × 104 cells/well) in complete medium, and after 48 h of growth, the cells were incubated in the dark with 0.5 μM PS in culture medium with 3% FBS. After incubation, the cells were washed with PBS, detached from the plates with a 0.25% trypsin solution (Life Technologies), centrifuged at 125g for 8 min, and resuspended in PBS for flow cytometry analysis. Ten thousand events/sample were acquired using the same excitation wavelength and emission filters used for measuring the binding of the PSs in bacteria. The release of PS from fibroblasts was assessed in cells incubated for 1 h with the PS followed by an additional 3 h in PS-free medium (60 + 180 min time point). Fluorescence Microscopy. Fibroblasts incubated for 60 min with the PS were analyzed with a Leica inverted fluorescence microscope (DM 5000B, Leica Microsystems srl) for detection and localization of the porphyrin fluorescence. Cells (1.5 × 105) were seeded in 35 mm tissue culture dishes, and after 48 h, they were incubated with the PS at 5 μM concentration. The incubated cells were washed twice with PBS with Ca2+ and Mg2+ and analyzed under the microscope using a 20× objective and filters selecting excitation and emission at 420/40 and 655/40 nm, respectively.

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ASSOCIATED CONTENT

S Supporting Information *

Circular dichroism spectra, singlet oxygen phosphorescence kinetics, data on bacteria inactivation as a function of incubation times, flow cytometry analyses of bacteria, and data on fibroblasts viability. This material is available free of charge via the Internet at http://pubs.acs.org. 1413



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AUTHOR INFORMATION

Corresponding Author

*Phone: +39 0498276335. Fax: +39 0498276300. E-mail: elena. [email protected]. Present Address ∥

Centre for Integrative Bee Research (CIBER), ARC CoE in Plant Energy Biology, MCS Building M316, The University of Western Australia, 6009 Crawley, Australia. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Barbara Biondi for ESI-MS analysis. Financial support for this research was obtained from the University of Padova, Progetto di Ateneo 2009 CPDA090338, and from the Spanish Ministerio de Economiá y Competitividad through grant nos. CTQ2010-20870-C03-01 and IT2009-0033. We thank the Italian Ministero dell’Istruzione dell’Università e della Ricerca for Mobility grant IT10H5E13A. R.R.-G. thanks the Generalitat de Catalunya (DURSI) and Fons Social Europeu for a predoctoral fellowship.

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ABBREVIATIONS USED ΦF, fluorescence quantum yield; ΦΔ, singlet oxygen quantum yield; BHI, brain heart infusion; CAMP, cationic antimicrobial peptides; cfu, colony forming units; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; MeOH, methanol; 1O2, singlet oxygen; dPBS, deuterated phosphate buffered saline; PDT, photodynamic therapy; PS, photosensitizer; TFE, 2,2,2-trifluoroethanol; TMPyP, meso-tetrakis(N-methylpyridinyl)porphyrin

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REFERENCES

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