Secretory phospholipase A₂ responsive liposomes

Abstract

Secretory phospholipase A(2) (sPLA(2)) expression is increased in several cancers and has been shown to trigger release from some lipid carriers. This study used electrospray ionization mass spectrometry (ESI-MS) and release of 6-carboxyfluorescein (6-CF) to determine the effects of sPLA(2) on various liposome formulations. Different combinations of zwitterionic [1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine, and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE)] and anionic [1,2-distearoyl-sn-glycero-3-phosphatidic acid, 1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPG), 1,2-distearoyl-sn-glycero-3-phosphatidylserine, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol) 2000 (DSPE-PEG)] phospholipids were examined. DSPG and DSPE were most susceptible to sPLA(2)-mediated degradation compared with other phospholipids. Increased 6-CF release was observed after inclusion of 10 mol % DSPE and anionic lipids into different liposome formulations. Group IIa sPLA(2)-mediated 6-CF release was less than Group III and relatively insensitive to cholesterol (Chol), whereas Chol reduced sPLA(2)-mediated release. Inclusion of DSPE-PEG increased sPLA(2)-mediated 6-CF release, whereas serum reduced lipid degradation and 6-CF release significantly. These data demonstrate that ESI-MS and 6-CF release were useful in determining the selectivity of sPLA(2) and release from liposomes, that differences in the activity of different sPLA(2) isoforms exist, and that DSPE-PEG enhanced sPLA(2)-mediated release of liposomal constituents. These findings will aid in the selection of lipids and optimization of the kinetics of drug release for the treatment of cancers and diseases of inflammation in which sPLA(2) expression is increased.

Figure 1

sPLA₂-facilitated drug release. This illustration describes the proposed mechanism of sPLA₂-mediated degradation and drug release from liposomes. Increased expression of sPLA₂ in solid tumors or other diseased tissue would enhance the degradation of phospholipids resulting in increased membrane permeability and drug release. Increased lipid degradation would be evidenced by increases in fatty acid and lysophospholipid lipid levels. Limited expression of sPLA₂ in noncancerous or diseased tissues would result in reduced lipid degradation and drug release.

Figure 2

Concentration-dependence of sPLA₂-mediated degradation of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC). The effect of sPLA₂ on degradation of DPPC (734.5 m/z) and formation of 16:0 lyosphosphatidylcholine (LPC) at 496.3 m/z and 16:0 fatty acid (FA) at 255.5 m/z were determined by ESI-MS. Data were normalized to signal intensity of DPPC, LPC, and FA in control samples. A concentration-dependent decrease in signal intensity of DPPC and increase in LPC and FA were observed. Data are represented as the percent mean intensity ± standard deviation (n = 3). Asterisk indicates a significant (p ≤ 0.05) difference compared with control.

Figure 3

Effect of Group III sPLA₂-mediated 6-CF release from (a) zwitterionic (neutral) formulations and (b) anionic formulations. (a) Percentage of 6-CF release from DSPC and DSPC/DSPE (9:1 mole ratio) formulations was assessed by fluorescent intensity changes in the media. Liposome samples (total phospholipid = 0.05 μg/mL) were treated with 0 (control) or 2.5 μg/mL Group III sPLA₂ for 0–108 h at 25°C. Fluorescence intensity was obtained by measuring the fluorescence at excitation and emission wavelengths of 480 and 510 nm, respectively. (b) Percentage of 6-CF release from anionic formulations containing DSPC with anionic phospholipids DSPA, DSPS, or DSPG (at 9:1 mole ratio). Data are represented as the mean ± standard error of the mean of at least three separate experiments (n = 5/study). Asterisk indicates significant (p≤ 0.05) difference compared with control.

Figure 4

Effect of PEG and cholesterol on Group III sPLA₂-mediated 6-CF release from modified SSL liposome formulations. The effect of cholesterol and DSPE–PEG on sPLA₂-mediated (2.5 μg/mL Group III) release of 6-CF from DSPC liposomes (total phospholipid = 0.05 μg/mL) (a) without or (b) with incorporation of 10 mol% DSPG was determined by fluorescent intensity changes in the media for 0–72 h at 25°C. Fluorescence intensity was obtained by measuring the fluorescence at excitation and emission wavelengths of 480 and 510 nm, respectively. Data are represented as the percent mean intensity ± standard error of the mean of at least three separate experiments (n = 5/study). Asterisk indicates significant (p ≤ 0.05) difference compared with control.

Figure 5

Comparison of Group IIa and Group III sPLA₂-mediated release of 6-CF from liposomes. The effect of cholesterol and DSPE–PEG on DSPC formulations (total phospholipid = 0.05 μg/mL) on 6-CF release was determined after exposure to either (a) Group III or (b) Group IIa sPLA₂ (0 to 5 μg/mL) for 6 h at 25°C. 6-CF release was assessed by fluorescent intensity changes in the media at excitation and emission wavelengths of 480 and 510 nm, respectively. Data are represented as the mean ∓ standard error of the mean of at least three separate experiments (n = 5/study). Asterisk indicates significant (p ≤ 0.05) difference compared with control.

Figure 6

Comparison of Group IIa sPLA₂-mediated release of 6-CF from standard liposome versus SPRL preparations. Formulations (total phospholipid = 0.05 μg/mL) consisting of DSPC, DSPC/DSPE alone, and DSPC or DSPC/DSPE plus Chol and DSPE–PEG were exposed to 2.5 μg/mL of Group IIa sPLA₂ for (a) 30 min or (b) 6 h at 25°C. 6-CF was assessed by fluorescent intensity changes in the media at excitation and emission wavelengths of 480 and 510 nm, respectively. Data are represented as the mean ± standard error of the mean of at least three separate experiments (n = 5/study). Asterisk indicates significant (p ≤ 0.05) difference compared with control.

Figure 7

Effect of serum on 6-CF release. The effect of FBS and sPLA₂ on the release of 6-CF from SSL (DSPC/Chol/DSPE–PEG), zwitterionic (DSPC/DSPE/Chol/ DSPE–PEG), and anionic (DSPC/DSPG/Chol/DSPE–PEG) formulations at 37°C. Fluorescence intensity was obtained by measuring the fluorescence at excitation and emission wavelengths of 480 and 510 nm, respectively. Data are represented as the mean ± standard error of the mean of at least three separate experiments (n = 5/study). Asterisk indicates significant (p ≤ 0.05) difference compared with DSPC/Chol/DSPE–PEG (SSL) formulation.