Induction of apoptosis in macrophages by air oxidation of dioleoylphosphatidylglycerol

Journal of Controlled Release 108 (2005) 442 – 452 www.elsevier.com/locate/jconrel

Induction of apoptosis in macrophages by air oxidation of dioleoylphosphatidylglycerol JungQhua Steven Kuo a,*, Ming-shiou Jan b, Jingyueh Jeng c, Hsuan Wen Chiu a a

Department of Biotechnology, Chia Nan University of Pharmacy and Science, 60 Erh-Jen Rd., Sec. 1, Jen-Te, Tainan 717, Taiwan b Department of Microbiology and Immunology, Medical College of Chung Shan Medical University, 110, Sec. 1, Jianguo Road, Taichung, Taiwan c Department of Industrial Safety and Hygiene, Chia Nan University of Pharmacy and Science, 60 Erh-Jen Rd., Sec. 1, Jen-Te, Tainan 717, Taiwan Received 16 February 2005; accepted 29 August 2005 Available online 23 September 2005

Abstract Dioleoylphosphatidylglycerol (DOPG) containing unsaturated sites is the target of oxidation during preparation, storage, or in vivo use of anionic liposomes. We investigated the biological effect of air oxidation of DOPG on RAW 264.7 murine macrophage-like cells. Oxidation was induced by exposing DOPG to air for 24–72 h. The extent of air oxidation was confirmed using Matrix-Assisted Laser Desorption and Ionization with Time-of-Flight (MALDI-TOF) mass spectrometry. The product of the air oxidation of DOPG was identified as the addition of one oxygen atom to one of the symmetrical fatty moieties of DOPG at m/z 814.77. The treatment of DOPG with air oxidation produced dose-dependent cytotoxicity in macrophages. RAW 264.7 cells exposed to oxidized DOPG exhibited morphological features of apoptosis, such as chromatin condensation and cell shrinkage. Typical apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated with oxidized DOPG. Flow cytometric analysis demonstrated an increase in the hypodiploid DNA population (sub-G1), indicating that DNA cleavage occurred after treatment with oxidized DOPG. In addition, we showed that pretreating RAW 264.7 cells with zVAD-fmk, a general caspase inhibitor, did not prevent apoptosis induced by oxidized DOPG, suggesting that apoptosis in macrophage cells follows a caspase-independent pathway. These results point to a need for precaution in formulating DOPG liposomes for drug delivery and therapeutic purposes. D 2005 Elsevier B.V. All rights reserved. Keywords: Dioleoylphosphatidylglycerol; Oxidation; Apoptosis; Macrophages; Cytotoxicity

Abbreviations: DOPG, dioleoylphosphatidylglycerol; MALDI-TOF, Matrix-Assisted Laser Desorption and Ionization with Time-of-Flight; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; LOOH, lipid hydroperoxide; PKC, protein kinase C; MAPKs, mitogenactivated protein kinases; MPS, mononuclear phagocyte system; LDH, lactate dehydrogenase; DHB, 2,5-dihydroxybenzoic acid; PI, propidium iodide. * Corresponding author. Tel.: +886 6 266 4911x523; fax: +886 6 266 6411. E-mail address: [email protected] (J.S. Kuo). 0168-3659/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2005.08.026

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1. Introduction Dioleoylphosphatidylglycerol (DOPG), structurally classified as a component of membrane phospholipids (phosphatidylglycerol), has been extensively studied in different fields of biomedical science and technology such as drug carriers, membrane models, and pulmonary surfactant for respiratory distress syndrome [1–3]. Phosphatidylglycerol that contains unsaturated groups as part of its molecular structure is vulnerable to oxidation during the preparation, storage, or in vivo use of liposomes [4–7]. The accompanying change in membrane structure may then influence the stability and biological properties of commonly used liposome formulations that contain unsaturated chains [8–10]. The influence of the process of lipid peroxidation on cells and liposome stability has been widely investigated, but most studies have focused on phosphatidylcholines and phosphatidylglycerol with highly unsaturated sites [5,11]. There is, however, no information about how cells respond to the oxidation of DOPG containing only one double bond in each of its fatty chains. Peroxidation of unsaturated phospholipids has been triggered by free-radical/non-radical species and resulted in the formation of lipid hydroperoxide (LOOH) [11]. Because of their increased polarity compared to parent lipids, LOOHs can disrupt cellmembrane structure and function, and become involved in the process of cell-death and survival depending on a variety of circumstances [12,13]. LOOH can also activate important protein kinases and phospholipases such as protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), phospholipase C, and calcium-activated phospholipase A2 [14,15]. LOOHs may trigger signal transduction pathways calling for either upregulation of detoxifying enzymes or apoptosis [11]. However, the exact mechanisms of lipid oxidation in signal transduction pathways are still not fully understood. Anionic liposomes deliver drugs to cells more efficiently than neutral liposomes [16,17]. Also, liposomes are primarily captured by the mononuclear phagocyte system (MPS) following intravenous injection [18]. Macrophages capture anionic liposomes through scavenger receptors [19,20]. These liposomes then affect macrophage functions by, for example, inhibiting the lipopolysaccharide-induced

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production of nitric oxide and expression of type II phospholipase A2 [21,22]. Because DOPG has been widely used in biomedical applications, we wanted to investigate whether the oxidation products of DOPG are harmful to macrophages that DOPG-containing liposomes will encounter in the body during drug delivery.

2. Materials and methods 2.1. Materials Propidium iodide (PI) and 1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (dioleoylphosphatidylglycerol (DOPG); C18:1; purity ~99%; M.W. = 797.0401 g/mol) were purchased from Sigma (St. Louis, MO, USA). The general caspase inhibitor zVAD-fmk (benzyloxycarbonyl-ValAla-Asp-fluoromethylketone) was obtained from Promega Corporation (Madison, WI, USA). 2.2. Liposome preparations DOPG was oxidized (hereafter called boxidized DOPGQ) by transferring 10 mg in 100 AL of chloroform to a glass tube and evaporating chloroform by exposing it to air for 24–72 h. The lipid residue was then dissolved in chloroform and stored at 80 8C until use. Immediately before the experiment, oxidized DOPG stored in chloroform was dried in a glass tube under a stream of N2 and dispersed in phosphate-buffered saline (PBS) solution using ultrasound for 1 min. The resulting solution was further filtered through a 0.45-Am filter and diluted to desired concentrations with culture medium before using it to incubate cells. DOPG that was not air-oxidized (hereafter called bunoxidized DOPGQ) was used as a control; it was prepared in the same manner as oxidized DOPG. 2.3. Matrix-assisted laser desorption and ionization with time-of-flight (MALDI-TOF) mass spectrometry Structural identification of DOPG oxidation was analyzed using MALDI-TOF mass spectrometry. All the mass spectra were obtained in a linear positive ion mode using an Auto FLEX RE-MALDI-TOF system

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(Bruker Daltonics, Billerica, MA, USA). The mass spectrometer was equipped with a 337-nm nitrogen laser, a 1.75-m flight, and a sample target able to accept 384 samples simultaneously. The accelerating voltage was set to 20 kV. The lipid solution (0.1 mg/ AL in chloroform) was mixed with 10 AL of 2,5dihydroxybenzoic acid (DHB, 15 mg/mL) in 70% acetonitrile / 30% water. One microliter of the mixture was applied to the sample target and air-dried for MALDI MS analysis. 2.4. Cells The murine macrophage-like cell line RAW 264.7 was maintained in medium (RPMI-1640; Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 Ag/mL) (Sigma).

Shiga, Japan). In brief, 5000 RAW 264.7 cells / 200AL/well were added with various concentrations of oxidized and unoxidized DOPG and then incubated for 1 day at 37 8C in a 5% CO2 atmosphere. After incubation, 100 AL/well of supernatant was carefully removed and transferred into an optically clear 96well plate. One hundred microliters of the reaction solution provided by the manufacturer was added to each well and incubated for 30 min in total darkness. The enzyme reaction was then stopped by adding 1 N HCl (50 AL/well). The absorbance at 490 nm was measured using an ELISA reader with a reference wavelength of 620 nm. The relative LDH release was calculated as the ratio of LDH release over control samples. Controls were treated with 1% Triton X100 and set as 100% LDH release; cells treated with culture medium only were set as 0%. 2.7. Propidium iodide (PI) nuclear staining

2.5. Cytotoxicity assays RAW 264.7 cells were seeded in 96-well plates at 20,000-cells / 100-AL/well, and then incubated overnight at 37 8C and in an atmosphere containing 5% CO2. Positive control cells were grown without adding DOPG. Various concentrations of oxidized and unoxidized DOPG were added and incubated for 24 h. To measure cell viability, 10 AL of a cell counting kit solution, a tetrazolium salt that produces a highly water-soluble formazan dye upon biochemical reduction in the presence of an electron carrier (1-Methoxy PMS) (Cell Counting Kit-8; Dojindo Laboratories, Tokyo, Japan), was added into each well and incubated for 1–4 h. The amount of the yellow formazan dye generated by dehydrogenases in cells is directly proportional to the number of viable cells in a culture medium. The absorbance at 450 nm was obtained using an ELISA reader with a reference wavelength of 595 nm. Results were reported as cell viability % (average OD / average positive control OD) F SD. 2.6. Lactate dehydrogenase (LDH) release assays LDH is a stable cytoplasmic enzyme and released into culture supernatant when cell membranes are damaged. LDH activity was assessed using an LDH cytotoxicity detection kit (Takara Bio Inc., Otsu,

Untreated and DOPG-treated RAW 264.7 cells (2  105) were incubated for 24 h and harvested. The cells were fixed with 1% paraformaldehyde for 60 min at room temperature and washed three times with 0.1% Tween 20 in PBS. After washing, the cells were incubated with PI staining solution (40 Ag/mL PI, 100 Ag/mL RNase A) for 30 min in the dark. The cells were washed five times with PBS, and then viewed under a fluorescent microscope (CKX 41; Olympus, Tokyo, Japan). 2.8. DNA fragmentation assays A procedure modified from previous studies was followed [23,24]. RAW 264.7 cells (1  106) treated with oxidized DOPG for 24 h were washed twice with PBS, suspended in 500 AL of lysis buffer (20 mM Tris, 10 mM EDTA, 0.2% TritonX-100; pH 8.0), and incubated on ice for 10 min. After centrifugation at 1200 rpm for 10 min, the cell lysate was incubated with proteinase K (Sigma; final concentration = 200 Ag/mL) to digest protein at 50 8C for 8 h, and then further incubated with RNase A (Sigma; final concentration = 100 Ag/mL) to digest RNA at 37 8C for 6 h. DNA was extracted twice, once using saturated phenol solution, and then using chloroform plus isoamyl alcohol. After centrifugation at 12,000 rpm for 10 min, glycogen (Sigma; final concentration = 20 Ag/

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mL) and an equal volume of isopropanol were added to the upper aqueous layer. The mixture was stored at 20 8C for 24 h. The extracted DNA was then dissolved in TE buffer solution (10 mM Tris, 1 mM EDTA; pH 8.0) and subjected to 2% agarose gel electrophoresis at 100 V for 30 min. The experiments were repeated three times to ensure the reproducibility of the assays. 2.9. DNA content Untreated and DOPG-treated cells (1  106) were fixed with a solution containing 70% ethanol and 30% PBS at 4 8C for 12 h. The cells were then centrifuged at 1200 rpm for 10 min to remove the fixation solution. The cell pellets were incubated with DNA staining solution (40 Ag/mL PI, 100 Ag/mL RNase A) for 30 min in the dark. Ten thousand cells per sample were analyzed using flow cytometry (FACSCalibur; Becton Dickinson, Mountain View, CA, USA). 2.10. zVAD-fmk inhibition To study the effect of zVAD-fmk on DOPG oxidation-induced apoptosis, cells (1  106) were either pretreated or untreated with 20 AM zVAD-fmk at 37 8C for 4 h, followed by treatment with oxidized DOPG at 37 8C for 12 h. Cells were then stained with PI fixation solution as described above, and analyzed using flow cytometry. The positive control of zVAD-fmk potency was confirmed by treating the cells with C2-ceramide (20 AM, N-acetylsphingosine; BIOMOL International L.P., Plymouth Meeting, PA, USA) under the same conditions as in the DOPG experiments.

rage, or in vivo use during drug delivery. In this study, oxidation was induced by exposing DOPG to air for 24–72 h. The extent of air oxidation was monitored using MALDI-TOF mass spectrometry. We investigated the interaction of DOPG after air oxidation in a macrophage cellular system. The effects of air oxidation of DOPG on cytotoxicity, cell membrane integrity, cell morphology, DNA fragmentation, DNA content, and zVAD-fmk inhibition were evaluated. We found that oxidized DOPG induced apoptosis in the RAW264.7 murine macrophage-like cell line. 3.1. MALDI-TOF mass spectrometry of the air oxidation of DOPG The chemical structure of DOPG is shown in Fig. 1. Analysis of unoxidized DOPG using MALDITOF mass spectrometry revealed predominant peaks corresponding to the protonated molecules ((DOPG + H)+ and (DOPG + Na)+) at m/z 798.83 and 820.82 with relatively few impurities (Fig. 2A). For samples with oxidized DOPG for 24 and 72 h, (DOPG + H)+ and (DOPG + Na)+ peaks, as well as a peak corresponding to the oxidation product ((DOPG + O)+ at m/z 814.77), was observed (Fig. 2B and C). Therefore, the product of the air oxidation of DOPG was identified as the addition of one oxygen atom to one of the symmetrical fatty moieties of DOPG. Furthermore, as the time of air oxidation increased from 24 to 72 h, the relative intensities of the (DOPG + O)+ peaks also increased, and the relative intensities of the (DOPG + Na)+

O

2.11. Statistical analysis Results are given as means F SD. Statistical analyses were performed using a one-way ANOVA followed by Dunnett’s test. Data were considered to differ significantly when P b 0.05.

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CH2O

C

CH2(CH2)6CH

CH(CH2)7CH3

O CHO

C

CH2(CH2)6CH

CH(CH2)7CH3

O

3. Results

CH2O

P –

DOPG containing unsaturated phospholipid sites are the targets of oxidation during preparation, sto-

OCH2CHCH2OH +

ONa

OH

Fig. 1. Chemical structure of DOPG.

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100

(M+H)+ 798.83

A. (M+Na)+ 820.82

Relative Intensity (%)

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C. (M+O)+ 814.77

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(M+Na)+ 820.82

0 790

800

810

820

m/z Fig. 2. Positive-ion MALDI-TOF mass spectrometry of oxidized or unoxidized DOPG with 0.1 mg/AL lipid concentration. (A) Control spectrum (unoxidized DOPG). (B) Spectrum of oxidized DOPG for 24 h. (C) Spectrum of oxidized DOPG for 72 h.

peaks decreased. Unless otherwise indicated, the following experiments were conducted with oxidized DOPG for 72 h. 3.2. Cell viability assays To test the effect of oxidized DOPG on cell viability, RAW 264.7 cells were treated with increasing concentrations of oxidized DOPG for 24 h, after which they were analyzed using cytotoxic assays. Cell viability decreased with increasing concentrations of oxidized DOPG (Fig. 3A). LDHrelease assays of cells treated with oxidized DOPG showed concentration-dependent decreases in membrane integrity (Fig. 3B). The characteristics of typical dose–response curves were fully expressed in the concentration range we used. There was an inverse relationship between the concentration of oxidized DOPG and cytotoxicity. The cell viability and membrane integrity of unoxidized DOPG were well maintained at the concentrations used (Fig. 3A and B).

3.3. Morphology changes of RAW 264.7 cells induced by the air oxidation of DOPG The hallmarks of apoptosis, such as chromatin condensation and cell shrinkage, developed after adding oxidized DOPG (Fig. 4C and D), whereas the nuclei of untreated cells and cells treated with unoxidized DOPG were more homogeneously stained and less intense than those of cells treated with oxidized DOPG (Fig. 4A and B). 3.4. DNA fragmentation assays In cells undergoing apoptosis, a typical ladder pattern occurs because of the activation of endogenous endonuclease, and this produces DNA fragments in multiples of about 180–200 base-pair units [25]. In the present study, DNA was extracted from RAW 264.7 cells treated with oxidized DOPG and examined using 2% agarose gel electrophoresis. Typical DNA ladders were clearly visible in cells treated with oxidized DOPG (Fig. 5, lanes 4–6),

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A. 120

Cell viability (%)

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DOPG with air oxidation

Fig. 3. Cytotoxicity assays by (A) measuring generated dehydrogenases. Positive control cells were grown without adding DOPG. Results are reported as cell viability % (average OD / average positive control OD) F SD (n = 6). (B) LDH release. The relative LDH release is calculated using the ratio of LDH release over control samples. Controls were treated with 1% Triton X-100 and set as 100% LDH release and 0% for cells with culture medium only. All results are given as means F SD (n = 6). *P b 0.05.

whereas no DNA ladders were observed in other cells (Fig. 5, lanes 2 and 3). As the concentration of DOPG increased, the relative brightness of the DNA

ladders also increased, which demonstrated that oxidized DOPG induced apoptosis in RAW 264.7 cells.

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A. Control

B. Control+DOPG

C. DOPG

D. DOPG with air oxidation (1.0038 mM)

with air oxidation (0.7528 mM)

without oxidation (1.0038 mM)

Fig. 4. Morphology changes of RAW 264.7 cells induced by treatment with oxidized or unoxidized DOPG for 24 h. Untreated cells (A) were compared with cells treated with 1.0038 mM unoxidized DOPG (B) and oxidized DOPG; (0.7528 mM (C) and 1.0038 mM (D)). The hallmarks of apoptosis, such as chromatin condensation and cell shrinkage, developed after treatment with oxidized DOPG are indicated by arrows. Images were taken at 200 magnification.

3.5. DNA content and zVAD-fmk inhibition Fluorescence staining and flow analysis of DNA have been widely used to determine cellular DNA content. A cell varies between hypodiploid and diploid DNA during the cell cycle, and flow cytometry can be used to determine its position in the cell cycle based on its DNA content [26]. In the present study, a single laser (linear PI fluorescence, 488 nm) flow cytometer was used to determine DNA strand breaks in cells treated with oxidized DOPG. After treatment with oxidized DOPG, PIstained cells showed higher hypodiploid DNA peaks than did untreated cells (Table 1), indicating that a DNA cleavage occurred after the cells were exposed to oxidized DOPG. The appearance of a sub-G1 population is characteristic of apoptotic

cells [26]. These results indicated that DNA degradation occurred after treating the cells with oxidized DOPG. To further examine the mechanisms of cell death induced by oxidized DOPG, 20 AM of zVAD-fmk was added to the culture medium for 4 h before exposing them to DOPG and determining its effect on apoptosis (Table 1). zVAD-fmk proved nontoxic to RAW 264.7 cells, excluding the apoptotic effect of zVAD-fmk itself (from the results of cytotoxicity assays; data not shown). Also, the positive control of zVAD-fmk potency was confirmed by treating cells with C2-ceramide to rule out the possibility of peptide degradation. Flow cytometric analysis showed that the percentage of the sub-G1 population of PIstained cells, pretreated with zVAD-fmk, to which oxidized DOPG was added, remained unchanged

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2

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bp 2000 bp 2000

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Fig. 5. Agarose gel electrophoresis of DNA extracted from RAW 264.7 cells treated with oxidized or unoxidized DOPG for 24 h. Lane 1: DNA marker; lane 2: control (untreated cells); lane 3: treated with 1.0038 mM unoxidized DOPG; lanes 4, 5, 6: treated with 1.0038, 0.8782, and 0.7528 mM oxidized DOPG, respectively.

compared with cells not pretreated with zVAD-fmk (Table 1). Also, zVAD-fmk did not prevent DNA fragmentation induced by oxidized DOPG (Fig. 6). This indicates that apoptosis induced by oxidized DOPG cannot be inhibited by pretreatment with

100

Fig. 6. Agarose gel electrophoresis of DNA extracted from RAW 264.7 cells pretreated with zVAD-fmk for 4 h and then treated with oxidized DOPG for 12 h. Lane 1: DNA marker; lane 2: control (untreated cells); lane 3: treatment with 20 AM zVAD-fmk; lanes 4 and 5: pretreated with 20 AM zVAD-fmk and then treated with 0.8782 and 1.0038 mM oxidized DOPG, respectively.

zVAD-fmk, suggesting a caspase-independent pathway for apoptosis.

4. Discussion Table 1 Analysis of DNA content of RAW 264.7 cells after treatment with oxidized DOPG and effect of zVAD-fmk on inhibiting apoptosis [Sub-G1 / M1] (%) Untreated 0.7528 mM oxidized DOPG 0.8728 mM oxidized DOPG 1.0038 mM oxidized DOPG 20 AM C2-ceramide 20 AM C2-ceramide + pre-incubation with 20 AM zVAD-fmk 0.8728 mM oxidized DOPG + pre-incubation with 20 AM zVAD-fmk 1.0038 mM oxidized DOPG + pre-incubation with 20 AM zVAD-fmk Results are given as means F SD (n = 6). M1 = sub-G1 + G0 / G1 + S + G2 / M.

4.53 F 2.01 17.45 F 2.23 21.58 F 2.05 25.36 F 1.92 35.61 F 2.52 3.57 F 2.74 22.57 F 1.58 25.42 F 2.28

That cationic but not anionic liposomes induce apoptosis in macrophage cells has been documented [27]. The biological effects of air oxidation of DOPG, which may be due to its relatively higher oxidative stability than other phosphatidylglycerols with highly unsaturated sites, have not been previously reported. In the present study, we demonstrated that the oxidation products of DOPG are harmful to cells that DOPG-containing liposomes will encounter in the body during drug delivery. Cis-unsaturated fatty acids are more oxidizable than trans-unsaturated fatty acids and increase their possibilities of peroxidation [28]. Also, membrane properties such as melting temperature were changed more profoundly by the

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introduction of a cis-double bond of fatty acids [29]. Therefore, our studies may describe lipid oxidation when using DOPG that contains cis-unsaturated fatty acids for drug delivery. Our method may not be 100% representative of the oxidation of lipids in aqueous systems, but it can be used as a perfect and simple model system in the study of lipid oxidation with unsaturated sites. Lipid oxidation largely occurs via free-radical mechanisms, but in aqueous systems it is typically induced by chemical reactions based on the use of organic hydroperoxides in combination with Fe+ 2 salts. The resulting undesired impurities, however, may complicate lipid identifications in mass spectra. Although unsaturated phospholipids in an aqueous dispersion can hydrolyze to form lysophospholipids and fatty acids, our method, in which DOPG was oxidized by air, was used to facilitate structural characterization by mass spectrometry for the process of lipid oxidation in liposomes containing unsaturated acyl chains, and our study focused on the oxidation of unsaturated acyl chains. In the present study, we used MALDI-TOF mass spectrometry to monitor and analyze the air oxidation of DOPG. Although this is an extremely rapid and sensitive approach, the precision of the measurements and accuracy of the data are primarily affected by the choice of an appropriate matrix (DHB) [30]. Dimers formed when we used CHC (a-cyano-4-hydroxycinnamic acid), another frequently used matrix (data not shown). Based on the molecular mass of the ion ((DOPG + O)+ at m/z 814.77) in Fig. 2, the product of air oxidation of DOPG was identified as the addition of one oxygen atom to one of the symmetrical fatty moieties of DOPG. Therefore, the oxidation products contain a mixture of predominantly unsaturated DOPG and a small portion of oxidized DOPG. We hypothesize that the change from the symmetrical/unsaturated to the asymmetric/saturated structure of one of two fatty chains of DOPG during air oxidation is responsible for apoptosis in macrophages, even though unoxidized DOPG is still the major component of the oxidation products. Generally, the uptake of liposomes by macrophages is believed to occur though adsorption of liposomes onto the cell surface and, subsequently, through endocytosis. Negatively charged liposomes such as DOPG are recognized by scavenger receptors and internalized by macrophages [19,20]. From our observations

of membrane integrity (Fig. 3B), there is no doubt that the oxidation of DOPG induces alterations in cell membranes and initiates apoptosis. Our findings may be explained by a previous study indicating that saturated but not mono-unsaturated fatty acids induce apoptosis in cardiomyocytes [31]. The relative increase of saturated fatty acids lowers the membrane fluidity, elevates the transition temperature of the membrane, and forms a major part of the process of apoptosis [32,33]. The two major cause of cell death in biological systems are necrosis and apoptosis [25]. Necrosis is related to an inflammatory and a degenerative process [34]. Cells undergoing necrosis characteristically illustrate mitochondrial swelling, lose membrane integrity, turn off metabolism, and release a cytoplasmic component that stimulates an inflammatory response. Apoptosis, also called shrinkage necrosis, is a form of programmed cell death that is characterized by cytoplasmic blebbing, condensation of nuclear chromatin, cell shrinkage, DNA fragmentation, exposure of phosphatidylserine residues on the outer leaflet, and cellular fragmentation into membrane apoptotic bodies [35]. Monocyte-derived macrophages play an important role in immune responses, atherosclerotic lesion formation, and tissue remodeling [36]. Macrophage apoptosis significantly influences these processes [37]. Exogenous and endogenous peroxides can trigger survival pathways, death pathways, and other responses in various cells [11,38]. When the extent of lipid peroxidation exceeds some repair threshold, stressed cells will undergo an apoptotic process, which is consistent with our observations of the effects of oxidized DOPG on macrophages [11]. The induction of apoptosis in macrophages by air oxidation of DOPG was confirmed by a variety of evidence. First, we showed the presence of nuclear condensation and cell shrinkage in those cells treated with oxidized DOPG (Fig. 4). Then, a DNA fragmentation assay (Fig. 5) revealed a typical apoptotic ladder, and an increase in the sub-G1 population in a flow cytometry analysis further confirmed the characteristics of apoptosis (Table 1). zVAD-fmk can irreversibly bind to the catalytic site of caspase proteases and prevent caspase-activated DNase from nicking the DNA in cultured cells [39]. Our study revealed that apoptosis cannot be blocked by the general caspase inhibitor zVAD-fmk, suggesting a caspase-indepen-

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dent pathway of apoptosis may be involved (Table 1 and Fig. 6). The cytotoxic effect of oxidized DOPG in other cell types remains to be established. In conclusion, the present study demonstrated that air oxidation of DOPG induced apoptosis in RAW 264.7 murine macrophage-like cells. These findings have important implications for precautions in formulating DOPG liposomes for drug delivery and therapeutic purposes.

[10]

[11]

[12] [13] [14]

Acknowledgment This work was supported by grant NSC 93-2216E-041-004 from the National Science Council of Taiwan.

[15] [16]

[17]

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