EHOOPrÄHH^ECRAa XHMH3, 2010, moM 36, № 3, c. 396-402
PHOTODYNAMIC ANTIMICROBIAL CHEMOTHERAPY BY LIPOSOME-ENCAPSULATED WATER-SOLUBLE PHOTOSENSITIZERS
© 2010 M. Nisnevitch*#, F. Nakonechny***, Y. Nitzan**
*Department of Chemical Engineering and Biotechnology, Ariel University Center of Samaria, Ariel, 40700, Israel; **The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University,
Ramat-Gan 52900, Israel Received June 17, 2009; in final form, October 21, 2009
Photodynamic antimicrobial chemotherapy is an alternative method for killing bacterial cells in view of the increasing problem of multi-antibiotic resistance. We examined the effect of three water-soluble photosensi-tizers (PhS): methylene blue (MB), neutral red (NR) and rose bengal (RB) on Gram-positive and Gramnegative bacteria. We compared the efficacy of PhS in their free form and encapsulated in liposomal formulations against various bacterial strains, and determined conditions for the effective use of encapsulated PhS. We found that all three PhS were able to eradicate the Gram-positive microbes Staphylococcus aureus and Sarcina lutea; and MB and RB were effective against St. epidermidis. In the case of the Gram-negative species, MB and RB were cytotoxic against the Shigella flexneri, NR-inactivated Escherichia coli and Salmonella para B, and BR was effective in killing Pseudomonas aeruginosa. None of the examined PhS showed activity against Klebsiella pneumoniae. MB and NR enclosed in liposomes gave a stronger antimicrobial effect than free PhS for all tested prokaryotes, whereas encapsulation of RB led to no increase in its activity. We suggest that encapsulation of PhS can increase the photoinactivation of bacteria.
Key words:photosensitizer; photodynamic antimicrobial chemotherapy; liposomes.
INTRODUCTION
Bacterial resistance to antibiotic treatment is a serious problem in medicine. One of the ways to overcome the difficulties of bacteria eradication is photodynamic inac-tivation (PDI) of microorganisms. The general effectiveness of PDI has been shown for a variety of Gram-positive and Gram-negative bacteria in vitro as well as for anti-tumor treatment [1—17]. In cancer treatment, the efficiency of PDI was increased by encapsulating photosensitizing agents (PhS) in biocompatible artificial phospholipid nanoparticles, also known as liposomes [18-21].
Liposomes are possible carriers for controlled drug delivery and targeting to cells. They can accommodate hydrophilic molecules in the aqueous spaces and lipophilic molecules in the lipid bilayers. A mechanism of drug delivery by liposomes was well examined for gramnegative bacteria, which is mediated by liposome-cell fusion, enhancing drug entry into bacterial cells [22, 23]. For Gram-positive bacteria a mechanism of liposome-
Abbreviations: DMPG — dimyristoyl phosphatidylglycerol; DPPC — dipalmitoyl phosphatidylcholine; EPC — egg yolk phosphatidylcholine; MB — methylene blue; MIC — minimal inhibitory concentrations; NR — neutral red; OA — octadecy-lamin; PACT — photodynamic antimicrobial chemotherapy; PDI — photodynamic inactivation; PBS — phosphate-buffered saline; PhS — photosensitizing agents; RB — rose bengal.
# Corresponding author; tel.: +972 3 906-66-06; e-mail: marinan@ ariel.ac.il.
cell interaction is assumed to be either the same as described above [24] or is mediated by "contact release", and the diffusion of liposome contents into a bacterial cell due to the hydrophobic interaction between the liposome lipid bilayer and the bacterial cell wall [6].
The action of liposome encapsulated PhS was only recently investigated by Ferro [25, 26], however, the positive effect ofliposome delivery ofantibiotics on the elimination of bacteria has been well established [23, 27—31]. The combination of photodynamic antimicrobial chemotherapy (PACT) with liposome delivery seems to have wide-ranging possibilities in providing good transport and targeting conditions for PhS acting against bacteria cells. The aim of the present work is to study the antimicrobial activity of liposomal forms ofwater-soluble PhS.
RESULTS AND DISCUSSION
A number ofexperiments were made to calibrate liposome size, composition, PhS content and illumination conditions. Liposomes of different sizes were prepared by ultrasonic treatment of polylamellar liposomes produced by vortexing of thin lipid layers in solution of PhS in the PBS. The size of the obtained vesicles was evaluated for dipalmitoyl phosphatidylcholine (DPPC) and egg phos-phatidylcholine (EPC) liposomes (Table 1) by measuring their turbidity spectra (s. Experimental).
As can be seen from Table 1, ultrasound treatment of polylamellar liposome vesicles leads to a decrease in their
average radius from >0.4 to 0.03 ^m for DPPC-based liposomes and from >0.4 to 0.25 ^m for EPC-based liposomes. The DPPC-liposomes are much smaller than the EPC ones based on the same lipid concentration and the same treatment conditions. This phenomenon can be explained by two factors — by lipid structure and by lipid phase state of liposomes. We suppose that the homogeneous composition of DPPC which contains only the saturated palmitic acid residues, enables dense lipid packing in liposome membranes, in contrast to the heterogeneous composition of EPC, which includes a number of saturated and non-saturated fatty acid residues. Such denser package leads to a formation of thinner lipid layer and consequently to smaller diameter of the DPPC unilamellar liposomes. At the temperature ofour experiments DPPC exists in a gel phase state, whereas EPC is in a liquid crystal state. Acyl chains of phospholipids are more disordered and bulky in a fluid state thus causing an increase in surface area per phospolipid molecule [32] resulting in a bigger liposomes size in the case of EPC liposomes.
The degree of PhS inclusion into vesicles was tested on a model of RB and DPPC liposomes (Table 2). RB was chosen for this estimation as it has the worst of the three PhS possibilities for liposome inclusion: RB carries a negative charge and has the largest molecular weight within the group. PhS concentration was varied at each stage of liposome preparation and non-included RB was separated from PhS-containing liposomes by size-exclusion chromatography. In addition RB/DPPC ratio in liposomes was estimated (Table 2). An increase in RB concentration led to a decrease in the degree of inclusion, whereas absolute RB amount in the aqueous liposome space showed a strong positive dependence on RB concentration. Thus liposomes having high inner concentrations of RB can be prepared using concentrated RB solution; nevertheless such liposomes have a low relative inclusion degree. The satisfactory results on the inclusion of the negatively charged RB (ca. 10—50% inclusion) promised a no less degree of inclusion ofpositive MB and electrically neutral at physiological conditions NB into the DPPC and EPC liposomes bearing a negative charge. This assumption was proved for DPPC liposomes prepared in the presence of 16 mM MB, and it was found that a degree of inclusion reached 60%.
Cell growth inhibition experiments were carried out using the three liposome-encapsulated PhS: MB, NR and RB on the Gram-positive microorganism S. lutea. Free and liposome encapsulated PhS were added to the cell culture in liquid medium and illuminated by white (luminescent) light, which overlapped the excitation spectra of each of the examined PhS (data not shown). PACT studies were assessed by cells growth inhibition estimation using the minimal inhibitory concentrations (MIC) method. All three PhS in free, as well as liposome encapsulated form eradicated the cells of S. lutea (Table 3). In the case of MB and NR, liposomal formulations inhibited cell growth more effectively than their free forms, whereas RB is equally effective encapsulated
Table 1. Characteristics of ultrasound treated DPPC and EPC liposomes
Liposome membrane composition Sonication time, s n* Vesicle radius, R (p.m)
DPPC 0 0.447 > 0.4
10 2.153 0.30
20 2.604 0.08
40 2.609 0.07
60 3.083 0.03
EPC 0 0.899 > 0.4
15 1.383
30 1.809
60 2.235 0.25
* n designates power coefficient in the equation lg — = Kk n.
Table 2. Inclusion of Rose Bengal into DPPC-based liposomes
RB concentration g/L Inclusion of RB into liposomes, % RB/DPPC ratio in liposomes, mg/g
1.5 48 24
2.5 42 35
5.0 25 42
10.0 16 54
50.0 8 125
or free. In order to keep liposomes sterile after a preparation, the PACT experiments were carried out without separation of encapsulated PhS from their free forms. As can be seen from the Fig. 1b and Table 3, even partial encapsulation of PhS led to a drastic decrease of MIC values, i.e. concentrations of free PhS needed for bacteria inactivation were several times higher than those for encapsulated PhS. Really low concentrations of free PhS present in the liposome preparations had no inhibitory effect on bacteria (under MIC concentrations), and for this reason the presence of free PhS was considered to be insignificant. "Empty" non-PhS-carrying liposomes were shown to have no inhibitory effect on bacteria growth — the MIC values of the free PhS alone and in the mixture with "empty" liposomes were the same. This fact confirmed an increase of inhibitory functioning of PhS only after their encapsulation into liposomes.
We have shown that maximal PACT effect can be achieved by PhS encapsulation in liposomes obtained under very short sonication time (Fig. a), which corresponds to the production of relatively large vesicles. Most likely as a result of sonication, polylamellar liposomes are broken into unilamellar but large vesicles, carrying a rather large volume of aqueous solution and, correspondingly, a large quantity of PhS. Longer ultrasonic treatment leads to reduction ofvesicle size, inner volume
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