ORIGINAL ARTICLE

Susceptibility of methicillin-resistant Staphylococcusaureus to photodynamic antimicrobial chemotherapywith α-D -galactopyranosyl zinc phthalocyanines:in vitro study

Zhanjuan Zhao & Yanzhou Li & Shuai Meng &

Shaozeng Li & Qiong Wang & Tianjun Liu

Received: 25 July 2013 /Accepted: 3 November 2013# Springer-Verlag London 2013

Abstract The incidence of methicillin-resistant strains ofStaphylococcus aureus (MRSA) is increasing globally, mak-ing urgent the discovery of novel alternative therapies forinfections. Photodynamic antimicrobial chemotherapy(PACT), based on oxidative damage to subcellular structures,has the advantage of circumventing multidrug resistance, andis becoming a potential therapeutic modality for methicillin-resistant bacteria. The key to PACT is photosensitization. Thisstudy demonstrates the efficiency of PACT using α-D -galactopyranosyl zinc phthalocyanines (T1–T4) for the pho-tosensitization ofMRSA, Escherichia coli , and Pseudomonasaeruginosa . Bacterial suspensions were illuminated with 650-nm light from a semiconductor laser at 0.2 W/cm2, and theenergy density was maintained at 6 J/cm2 in the presence ofdifferent concentrations of photosensitizer. The treatment re-sponse was evaluated based on the numbers of bacterialcolony-forming units. PACT with these phthalocyaninesstrongly affected MRSA, but weakly affected E. coli and P.aeruginosa . The efficiency of PACT on MRSA with thesefour phthalocyanine compounds decreased in the order T1 >T2 > T3 > T4. T1-PACT eliminated >99 % of MRSA in aconcentration range of 25–50 μM and at an energy density of6 J/cm2. Uptake measurements revealed that the PACT effectcorrelated with the bacterial uptake of the photosensitizer and

that 4–30-fold more T1 than T2–T4 was taken up by theMRSA strain, which was confirmed with laser confocal mi-croscopy. These data suggest that T1 is an efficient PACTphotosensitizer for MRSA.

Keywords MRSA . Photodynamic antimicrobialchemotherapy . Photosensitizer . Bacteria . Bactericidal effect

Introduction

Methicillin-resistant strains of Staphylococcus aureus(MRSA), which are resistant to several kinds of antibiotics,are becoming a major cause of hospital-acquired infectionthroughout the world, and are now prevalent in the commu-nity as well as in nursing and residential homes [1–3]. Skininjuries, such as cuts, abrasions, and turf burns, are susceptibleto MRSA infection, and significant morbidity can occur, withsome life-threatening sequelae [4–6]. The pathogenesis ofthese infections is multifactorial and poorly understood [7].In the clinical context, the management of patients withMRSA infections involves the systemic administration of anappropriate antibiotic and the topical application of antimicro-bial drugs to eliminate the organism from carriage sites,wounds, and burns [8]. Vancomycin and teicoplanin, whichhave been the only options for the treatment of systemicinfections of these organisms in the past, are facing seriouschallenges [9] because vancomycin-resistant S. aureus hasrecently been reported [10]. The worldwide increase in bacte-rial resistance to antibiotics makes it necessary to developantimicrobial treatments to which bacteria cannot easily de-velop resistance.

Photodynamic antimicrobial chemotherapy (PACT) is onesuch alternative chemical therapeutic strategy and has aroused

Z. Zhao :Y. Li : S. Meng :Q. Wang : T. Liu (*)Institute of Biomedical Engineering, Tianjin Key Laboratory ofBiomedical Material, Chinese Academy of Medical Sciences andPeking Union Medical College,Tianjin 300192, People’s Republic of Chinae-mail: liutianjun@hotmail.com

S. LiFirst Affiliated (304) Hospital, Chinese PLA General Hospital,Beijing 100037, China

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much attention. PACT combines harmless dyes and oxygenwith visible light to produce reactive oxygen species that arelethal to target cells [11]. Because the bactericidal activity ofPACT results from singlet oxygen and other reactive species[12–14], the development of bacterial resistance to it is im-possible [15]. PACT experiments in vitro have demonstrat-ed the effective bactericidal activity of toluidine blue Oand methylene blue as photosensitizers of MRSA [9, 16],and have stimulated even further interest in PACT. Thephotosensitizer is the crucial element in PACT. In the pastdecade, several kinds of photosensitizers have been re-por ted [17–20] , inc lud ing ca t ion ic porphyr in ,phenothiazinium, phthalocyanine, BODIPY, and toluidineblue. Phthalocyanine, with its large molar extinction ratio,long absorbance wavelength, high singlet oxygen yields,and ready availability, has received much research atten-tion as a photosensitizer in photodynamic therapies.Therefore, here, we investigated its photodynamic anti-bacterial chemotherapeutic effects.

Because sugar molecules are very popular energysources in biosystems, the modification of phthalocyaninewith sugar enhances both its cellular uptake and its watersolubility. It is relatively easy to link galactose protectedwith a ketyl moiety to other molecular moieties and itsdeprotection will produce a galactosyl complex. Practicalsynthetic methods for sugar-conjugated phthalocyanineshave been reported by several groups [21, 22]. Usingthese methods, we synthesized a series of novel α-D -galactopyranosyl zinc phthalocyanines, tethered with dif-ferent numbers (1–4) of galactopyranosyl moieties andstudied their effects in the current work. These com-pounds have several advantages, including their goodwater solubility, stability, photodynamic activity, and sim-ple synthesis. A systematic study of this system is beingconducted by our group. Molecular imaging of the effectsof this system has shown good liver targeting [23] and agood photodynamic effect in the treatment of cancer.Here, we report the effects of PACT with α-D -galactopyranosyl zinc phthalocyanines on epidemicstrains of MRSA, Escherichia coli , and Pseudomonasaeruginosa in vitro.

Materials and methods

Materials

The phthalocyanine studied in this paper was synthesizedaccording to a previously reported synthetic procedure [22].The synthetic procedure and its characterization data havebeen reported by Lv et al. [23]. For clarity, the chemicalstructure of phthalocyanine is shown in Fig. 1.

Photosensitizer and light source

The α-D-galactopyranosyl zinc phthalocyanines were dis-solved in phosphate-buffed saline (PBS) to produce a100-μM stock solution, which was stored at −20 °C in thedark before use.

Light at a wavelength of 650 nm was delivered by asemiconductor laser (7404, Intense, USA) via an optic fiberfor use in this experiment. The light spot was monitored with a5-cm diameter, and an irradiance of 200 mW/cm2as measuredby a light power meter (LM1; Carl Zeiss),

Bacterial culture

In this experiment, one clinical MRSA strain isolated at theFirst Affiliated (304) Hospital, Chinese PLA General Hospitaland standard bacterial strains E. coli (ATCC-25922) and P.aeruginosa (ATCC-27853) were obtained as a gift from the304 Hospital. Luria–Bertani (LB) mediumwas used to culturethese three bacterial strains. A single colony was used toinoculate 10 ml of liquid medium. The cells were grown at37 °C under aerobic conditions in a shaking incubator(200 rpm) until an optical density at 600 nm (OD600) ofapproximately 0.7 was reached (a 10-fold dilution was mea-sured). The cells were then harvested by centrifugation andresuspended in an equal volume of PBS [24].

PACT studies

The three strains were treated with the same procedure. Thetreatment of MRSA is described to exemplify the treatment ofall strains. Each MRSA culture was supplemented with pho-tosensitizer to a final concentration of 3.13, 6.25, 12.5, 25, or50 μM. The cultures prepared in this way were incubated at37 °C for 30 min in the dark, and then loaded into a 96-wellplate, with a total volume of 0.2 ml of culture per well. Thewells containing MRSA cultures were illuminated with anappropriate dose of light as the photoreaction, and culturesincubated in the dark under the same conditions were usedas the dark-reaction controls. After illumination, an aliquot(100 μL) was taken from each well to determine thecolony-forming units (CFU). The aliquots were seriallydiluted 10-fold in PBS to 10−1, 10−2, and 10−5 times theoriginal concentrations. Aliquots (100 μL) of each dilutionwere plated onto LB agar plates. After incubation for 18 hat 37 °C in the dark, the CFU were counted and the resultswere analyzed statistically. Each experiment was per-formed in triplicate. The surviving fractions of bacteriawere expressed as ratios of CFU produced by bacteriatreated with light and photosensitizer to CFU producedby the untreated bacteria [25].

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Imaging with confocal laser scanning microscopy

The MRSA strains were suspended in PBS to an appropriatecell density (OD600=0.7), then treated with compounds T1–T4 at a final concentration of 12.5 μM for 30 min at roomtemperature. The cells were then harvested by centrifugation(9,000×g , 1 min) and washed twice with PBS. One drop ofthis suspension was then placed onto a confocal dish andallowed to dry. Fluorescent images were taken with a confocallaser scanning microscope (LSM510; Carl Zeiss), with anexcitation wavelength of 633 nm and monitored with aband-pass filter at 650 nm. Imaging experiments were notperformed with E. coli and P. aeruginosa because their re-sponses to PACTwith this series of galactosyl phthalocyaninecompounds were weak.

Uptake study

The uptake experiment was conducted according to Soukoset al. [26]. Briefly, an aliquot (1,000 μL) of bacterial

suspension was centrifuged (9,000×g , 1 min) to remove theLB broth, then resuspended in PBS to the appropriate celldensity (OD600=0.7). Varying concentrations of T1–T4 inPBS were added to final concentrations of 3.13–50 μM. Thesuspensions were incubated at room temperature in the darkfor 30 min, then centrifuged (9,000×g , 1 min) and washedwith PBS to remove any residual photosensitizer. The cellpellets were harvested and dispersed in an aqueous solutionof 10 % sodium dodecyl sulfate (SDS) (1,000 μL) for at least24 h to release the photosensitizer taken up. The amount ofphotosensitizers taken up by the cells was measured with afluorescence assay. The fluorescence intensity of each aliquotsolution was recorded at 690 nm after excitation at a wave-length of 630 nm. A standard curve was constructed byplotting known concentrations of phthalocyanine in 10 %SDS against their fluorescence intensity. Cellular uptake wascalculated by comparing the measured values with the stan-dard curve. Uptake amount was calculated based upon thenumber of incubated photosensitizers divided by the numberof bacterial strains in incubated cells.

Fig. 1 T1: (1-α-D-galactopyranos-6-O-yl) phthalocyaninato zinc (II);T2: [2 ,9(10) o r 16(17)-b i s (α -D -ga lac topyranos-6-O-y l )phthalocyaninato] zinc (II); T3: [2, 9(10), 16(17)-tri(α-D -

galactopyranos-6-O-yl) phthalocyaninato] z inc (II ) ; T4:[2,9(10),16(17),23(24)-tetrakis(α-D -galactopyranos-6-O-yl)]phthalocyaninato zinc (II)

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Statistical analysis

The statistical analysis was performed with SPSS 19.0 PL forWindows. Analysis of variance was used to test the signifi-cance of the effects of both factors and their interactions, at a5 % significance level. All values are presented as mean ±standard deviations.

Results

Photosensitizer

Because phthalocyanine has the advantages of a long ab-sorbance wavelength, chemical stability, high 1O2 yield,and easy synthesis, so it is a good candidate for photosen-sitizer compound. However, its low solubility in water andorganic solvents is the first problem that must be resolved.To customize the four compounds (T1–T4) for antimicro-bial photodynamic therapy, we envisaged a design inwhich the sensitizer was substituted with monosaccharidefunctional groups. Until now, phthalocyanines have rarelybeen conjugated to biological molecules and only a fewexamples have been described [23]. Asialoglycoprotein(ASGP) receptors are reported to be expressed plentifullyon the surfaces of hepatoma cells and mammalian hepato-cytes, and targeting them was achieved by the introductionof galactose residues, which can bind specifically to theASGP receptors on cells [27, 28]. In this study, galactosylwas chosen to modify zinc phthalocyanine because it is awater-soluble group and a bacterial target marker. Wedesigned and synthesized a series of novel α-D -galactopyranosyl zinc phthalocyanines (T1, T2, T3, T4)with different numbers of galactose groups on one phtha-locyanine moiety and examined their structure–activityrelationships. It was clear that the galactose moiety greatlyenhanced the hydrophilicity of the phthalocyanine core.

Uptake of T1–T4 by MRSA

The uptake of the T1, T2, T3, and T4 compounds by MRSAwas dose dependent, as shown in Fig. 2a. Among the fourphotosensitizers, the T1 compound had the most pronouncedselectivity, resulting in the greatest uptake, followed by theother three phthalocyanines. At all experimental concentra-tions, MRSA bacteria took up 1–4 times more T1 than T2(P <0.001) and 7–30 times more T1 than T4. Measurement ofthe time-dependent uptake revealed the partial biocompatibil-ity of the photosensitizer.

We determined the time taken to achieve the maximumuptake of α-D -galactopyranosyl zinc phthalocyanines byMRSA, because incubation time correlates strongly with theamount of compound taken up and therefore PACTefficiency.Each of the four phthalocyanines (6.25 μM) was incubatedwith MRSA for 24 h, and the amount of phthalocyanine takenup was measured at different time points using the absorptionmethod. More data points were examined in the initial stages,when uptake was relatively rapid. Figure 2b shows that theuptake of T1 by MRSA was fastest, and that the maximumuptake of compound T1 occurredwithin 15min. However, themaximum uptake of compounds T2, T3, and T4 occurredwithin 30 min. Then, 30 min was chosen as the incubationtime for the photoreaction and dark reaction in the followingexperiment.

PACT studies

PACT efficiency is usually the combined result of the photo-sensitizer, the amount of photosensitizer taken up by the cells,the light dose, the illumination wavelength, and illuminatedtarget, so we studied each of these experimental factors one byone.

Energy dose is an important factor in PACT. To optimizethe PACT conditions, four experiments were performed: lightonly, T1 only, T1 + light, and the control. Figure 3 shows the

Fig. 2 Amounts of compoundsT1–T4 taken up by MRSA as afactor of a concentration and btime

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susceptibility of the MRSA strains to T1-PACT as a factorof the laser energy dose. In the preliminary experiment, wefound T1 at a concentration of 6.25 uM has shown excel-lent PACT effect, so, in the energy dose optimizationexperiment, the MRSA strains were incubated with6.25-μM T1 for 30 min, then exposed to different energydensities with a 650-nm laser. There was no significantdifference among the light group (Fig. 3b), T1 only group(Fig. 4b), and the control group, CFU raised high,

indicating that the laser light dose or T1 alone had noeffect or only a weak effect on the growth of the cells.However, there was a large killing effect when T1 wascombined with irradiation (T1 + light) in T1-PACT. Whenthe T1 concentration was maintained at 6.25 μM, T1-PACT was dependent on the irradiation energy, and thekilling effect was maximal for MRSA at an energy densityof 6 J/cm2. Therefore, an energy dose of 6 J/cm2 waschosen for the subsequent experiments.

Fig. 3 Effects of T1-PACT (a) orlight alone (b) on MRSA strainsas a factor of laser energy dose(c =6.25 μM; illumination at650 nm)

Fig. 4 Phototoxicity and dark toxicity of T1, T2, T3, and T4 for a–bMRSA, c–d E. coli , and e–f P. aeruginosa . Each bacterial strain wasincubated with compound in the concentration range of 3.13–50 μM for

30min, followed by exposure or not to 6 J/cm2 of 650-nm laser light. Theresults are expressed as means and standard deviations

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To study the dose–response effects of the different photo-sensitizers (T1–T4) on the test MRSA, three independent ex-periments were performed at each concentration: control, pho-tosensitizer alone, and photosensitizer + light of fixed energy.Figure 4a and b shows the PACT results obtained when theMRSA strain was incubated with increasing concentrations ofT1–T4. Clear dose-dependent lethality was observed with ex-posure to T1- or T2-PACT at concentrations of 3.13–50 μM,whereas compounds T3 and T4 were less efficient. At a con-centration of 12.5 μM, T1- or T2-PACT eradicated MRSA atrates of >99 and 88 %, respectively. The dark cytotoxicity of aphotosensitizer compound is an index of its basic chemicaltoxicity. Figure 4b shows that under dark conditions, T1 is lesstoxic in the concentration range of 3.13–12.5 μM, indicatingthat it is a good photosensitizer.Whereas T2was highly toxic toMRSA, even in the dark, T3 and T4 were less toxic, althoughtheir weak PACT effects already limit their application.

Encouraged by the effects of T1-PACT on MRSA, weexplored the generalization of this series of galactosyl phtha-locyanines to other kinds of bacterial strains, repeating thePACT experiments with E. coli (Fig. 4c and d) and P.aeruginosa (Fig. 4e and f). In the same concentration range,PACT with T1, T2, T3, or T4 had very weak effects, even at50-μMphotosensitizers, and the most efficient procedure (T1-PACT) only eradicated about 83 % of E. coli and 65 % of P.aeruginosa , whereas >99 % ofMRSAwere eradicated by T1-PACT. This large difference indicates that T1-PACT is onlyefficient for MRSA.

Confocal laser scanning microscopy images of MRSA treatedwith photosensitizers T1–T4

The microbial uptake of the photosensitizers was examinedwith confocal laser scanning microscopy. Phthalocyanineemits red fluorescence at 690 nm when excited at 633 nm,which can be readily monitored with a fluorescence micro-scope system. The images shown in Fig. 5 confirm that T1was internalized over a broad spectrum of microbes afterincubation for only 30 min. The intensity of the red fluores-cence emitted from T1 was strongest of the four phthalocya-nines, indicating that T1 uptake by MRSAwas greatest. Therelatively weak fluorescent signals from samples treated withT2, T3, or T4 are consistent with the measurements of theiruptake by MRSA.

Discussion

This study demonstrates the utility of galactosyl phthalocya-nine as a photosensitizer in the photoinactivation of bacterialstrains. The exposure of bacterial cultures to light in thepresence of compounds T1–T4, as photosensitizers, sharplyand dose-dependently reduced microorganismal viability. Of

these four sensitizers, T1 was most efficient. Although galac-tose is a popular energy source, phthalocyanine with three orfour galactose moieties is not readily taken up by cells, but hasthe reverse effect. This indicates that, in this system, galactosefunctions mainly as a polar group in tuning the lipid/waterratio, which then affects the amount of photosensitizer takenup and its combined effect with PACT. Therefore, thetargeting effect of galactose is largely irrelevant. More galac-tose moieties will increase the water solubility of the com-pound. However, whereas positively charged photosensitizershave preferential affinity for Gram-negative strains, such as E.coli and P. aeruginosa , galactose moieties, with their neutralpolar groups, have a different affinity profile, with a prefer-ence for Gram-positive strains, such as MRSA.

Successful PACT involves the optimization of a largenumber of parameters. When considering the incubation timeswith photosensitizers, Lazzeri et al. [29] reported that contactwith meso-substituted porphyrins for more than 5 min did notincrease the amount of cell-bound photosensitizer. However,in other photoinactivation experiments, contact for 30 minwas usually reported.We investigated the effects of incubationwith the photosensitizers in the dark for 10, 30, and 40 minand found that an incubation period of up to 30 min resulted inthe greatest cell lethality in MRSA.

An ideal photosensitizer must absorb light at a compatiblewavelength, with high quantum efficiency and low toxicity[30]. The photosensitizer dark effect was evaluated bycounting the viable bacteria after the strains were incubatedwith compounds T1–T4 for 30 min, the exposure time used inPACT. T2 showed relatively high dark toxicity, whereas T1,T3, and T4 were less toxic. The uptake experiment andimaging with confocal laser scanning microscopy revealedthat T1 is readily taken up by MRSA and that maximumuptake occurs within 30 min. Figure 5 confirms that T1 wasinternalized by a broad spectrum of microbes after incubationfor 30 min, whereas no distinct subcellular localization pat-terns could be discerned for T3–T4 in the MRSA strains. ThePACT effect of 25 μM T1 or T2 eradicated >99 % MRSA,which was statistically and significantly different from thecontrol group. This result is better than the effect of toluidineblue O on S. aureus , when a significant statistical differencewas only achieved with a concentration of 50 μg/ml [31].

Various types of tetrapyrrolic rings have been studied,including porphyrin derivatives, phthalocyanines, and chlo-rines [32]. Galactosyl phthalocyanine, a novel photosensitizer,was investigated here with encouraging results, which suggesta new strategy for bacterial control. Our in vitro results dem-onstrate that the exposure of MRSA to laser irradiation in thepresence of a photosensitizer reduced cell viability. The mosteffective combinations involved compounds T1 and T2 atconcentrations of 25 or 50 μM and 650-nm laser illuminationat 6 J/cm2, which reduced MRSA viability by >99 % in thestrains investigated.

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Conclusion

Our study has shown that PACT with galactosyl phthalocya-nines exerts a bactericidal effect onMRSA in vitro. An uptakeexperiment and laser confocal microscopy study revealed thatMRSA tended to decrease with the application of these pho-tosensitizers, in the order T1 > T2 > T3 > T4, so the selective

uptake of T1 makes it the most effective bactericidal photo-sensitizer. Incubation with T1 for up to 30 min produced theoptimal cell lethality in MRSA. Complete elimination wasachieved with illumination with laser light at a wavelength of650 nm and an energy dose of 6 J/cm2, with 25-μM T1 or T2as the photosensitizer. In conclusion, this new agent is animportant candidate photosensitizer in the field of

Fig. 5 Confocal laser scanningmicroscopic images of MRSAafter incubation for 30 min withT1–T4 (12.5 μM) in PBS.Excitation wavelength was633 nm. Scale bars are 1 μm. aT1, b T2, c T3, d T4, e–f control;e optical image, f confocalfluorescence image

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photodynamic antimicrobial therapy. The results describedhere should encourage further in vivo testing of these com-pounds against localized infections.

Acknowledgments This work was supported by a Peking Union Med-ical College Youth Grant to Z. Zhao (no. 2012X15), the key technologiesR & D program of Tianjin (12ZCDZSY11900) and Startup Project ofDoctor scientific research of Logistics University of CAPF (NO. WHTD201304–2).

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