Metal Chelation Modulates Phototherapeutic Properties of Mitoxantrone-Loaded Porphyrin-Phospholipid Liposomes

Abstract

Liposomes incorporating porphyrin-phospholipid (PoP) can be formulated to release entrapped contents in response to near-infrared (NIR) laser irradiation. Here, we examine effects of chelating copper or zinc into the PoP. Cu(II) and Zn(II) PoP liposomes, containing 10 molar % HPPH-lipid, exhibited unique photophysical properties and released entrapped cargo in response to NIR light. Cu-PoP liposomes exhibited minimal fluorescence and reduced production of reactive oxygen species upon irradiation. Zn-PoP liposomes retained fluorescence and singlet oxygen generation properties; however, they rapidly self-bleached under laser irradiation. Compared to the free base form, both Cu- and Zn-PoP liposomes exhibited reduced phototoxicity in mice. When loaded with mitoxantrone and administered intravenously at 5 mg/kg to mice bearing human pancreatic cancer xenografts, synergistic effects between the drug and the light treatment (for this particular dose and formulation) were realized with metallo-PoP liposomes. The drug-light-interval affected chemophototherapy efficacy and safety.

Keywords: Chemophototherapy; light-triggered; liposomes; metalloporphyrins; mitoxantrone; photobleaching; porphyrin-phospholipid; tumor ablation.

Figure 1:. Effect of metal chelation on HPPH-lipid photophysical properties in intact and lysed PoP liposomes.

A) Chemical structure of the HPPH-lipids examined. B) Fluorescence emission spectra of an equal concentration of indicated PoP liposomes in phosphate buffered saline. Liposomes were made with [DSPC:Cholesterol:DSPE-PEG2000:HPPH-lipid] at a molar ratio of [50:35:5:10] C) Fluorescence bleaching of liposomes following irradiation with 200 mW/cm² of 665 nm laser light for 60 seconds. Triton X-100 detergent (“Det.”) was added to lyse the liposomes. D) Singlet oxygen generation was assessed indirectly by examining the increase in fluorescence of singlet oxygen sensor green before and after laser irradiation.

Figure 2:. Absorbance spectra of laser-treated PoP liposomes.

Spectra of A) Free base, B) Cu, and C) Zn HPPH-lipid containing liposomes. Spectra were recorded of liposomes which were either untreated, phototreated in liposomal form, or phototreated after being lysed with a 665 nm laser at a fluence rate of 200 mW/cm² for 60 seconds. The respective Soret and Q-band peaks for the intact liposomes were: Free base: 413 and 663 nm; Cu: 425 and 650 nm; Zn: 430 and 657 nm.

Figure 3:. In vivo imaging of HPPH-lipid fluorescence pre- and post- laser exposure.

Mice were injected with equivalent doses (11 mg/kg) of free base, Cu and Zn forms of HPPH-lipid. 24 hours post injection mice were imaged and treated with a 665 nm laser at a fluence rate of 200 mW/cm² for 12.5 minutes (150 J/cm²).

Figure 4:. Drug loading and release.

Liposomes containing 10% free base, Cu, and Zn HPPH-lipid were loaded with Dox or MIT. A) Loading efficiency as assessed with gel filtration to separate free drug from liposomal drug. B) Stability in 50% serum after 24 hours and C) Light-triggered release of drugs was tested using a G-75 column for MIT and fluorescence quenching for Dox following a 10 minute laser treatment at 200 mW/cm². D) Dox release from Cu-PoP liposomes following light treatment.

Figure 5:. Kaplan-Meier survival curves for nude mice bearing MIA PaCa-2 tumors phototreated immediately post injection.

Mice were IV injected with A) 5 mg/kg mitoxantrone liposomes comprising of 10% free base, Cu, and Zn HPPH-lipid, or B) equivalent doses of empty liposomes. Mice were treated 10 minutes post injection with a 665 nm laser at 200 mW/cm² for 12.5 minutes (150 J/cm²). Mice were given a single injection and phototreatment and sacrificed when the tumor size was greater than 5 times the initial volume. Based on the log-rank test there was a statistically significant difference between all treatment groups and the saline control (P<0.05). There were no statistically significant differences between the empty and mitoxantrone loaded free base liposomes. There were statistically significant differences between the empty and loaded Cu and Zn HPPH-lipid liposomes (P<0.05)

Figure 6:. Kaplan-Meier survival curves for nude mice bearing MIA PaCa-2 tumors phototreated 24 hours post injection.

Mice were IV injected with A) 5 mg/kg mitoxantrone in 10% free base, Cu, or Zn HPPH-lipid liposomes, or B) equivalent doses of empty liposomes. Mice were treated at 24 hours post injection with a 665 nm laser at 200 mW/cm² for 12.5 minutes (150 J/cm²). Mice were given a single injection and laser treatment and were sacrificed when the tumor size was greater than 5 times the initial volume. Mice treated with the free base were all sacrificed within 24 hours of the treatment due to a reduction in body temperature or severe lethargy. Based on the log-rank test there was a statistically significant difference between both Cu and Zn groups and the saline control (P<0.05). However there was no significant differences between the mitoxantrone loaded and empty liposomes.