Andresen, Thomas Lars

Hansen, Anders E.

Nielsen, Ole L.

Østrem, Ragnhild Garborg

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 18, 2017sPLA2 sensitive fluid phase liposomes induce severe toxicity in murine cancer modelØstrem, Ragnhild Garborg; Nielsen, Ole L.; Hansen, Anders E.; Andresen, Thomas LarsPublication date:2016Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Østrem, R. G., Nielsen, O. L., Hansen, A. E., & Andresen, T. L. (2016). sPLA2 sensitive fluid phase liposomesinduce severe toxicity in murine cancer model. Poster session presented at Clinical Nanomedicine and TargetedMedicine 2016, Basel, Switzerland.sPLA2 sensitive fluid phase liposomes induce severe toxicity in murine cancer model Ragnhild G. Østrema, Ole L. Nielsenb, Anders E. Hansenc, Thomas L. Andresena  aColloids and Biological Interfaces Group, Department of Micro- and Nanotechnology, Center for Nanomedicine and Theranostics, Technical University of Denmark, Produktionstorvet, 2800 Kgs. Lyngby, Denmark; b Dept. Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Ridebanevej 3, 1870 Frederiksberg C, Denmark; c Cluster for Molecular Imaging, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark  Summary: Tuning liposomes for secretory phospholipase A2 induced release of oxaliplatin results in enhanced anti-neoplastic effects in vitro and extensive toxicity in vivo.        Introduction  The clinical use of liposomal drug delivery vehicles is often hindered by insufficient drug release1. One compelling solution is utilizing an endogenous trigger mechanism that relies on a difference in the microenvironment of healthy and diseased tissue, such as the elevated expression of endogenous enzymes in cancerous tissues.   Secretory phospholipase A2 (sPLA2) is an interfacially active lipase which functions mainly on organized substrates, such as lipid bilayers2, and it seems to have a preference for structural defects, such as domain boundaries3,4. We hypothesized that by adjusting the level of cholesterol in anionic, unsaturated liposomes we could tune the surface defects5,6, and as such tune the enzyme sensitivity, thus obtaining liposomes with an improved therapeutic outcome and reduced side effects.  Results  Conclusion  Tuning the amount of cholesterol did not seem to considerably alter the enzyme sensitivity towards the novel sPLA2 sensitive liposomes (SSLs). Oxaliplatin loaded SSLs revealed efficient in vitro growth inhibition compared to clinically used stealth liposomes. However, treatment of nude NMRI mice induced severe toxicity, demonstrating that great caution should be implemented when using sPLA2 sensitive liposomes, and that the real utility can only be revealed in vivo.  References 1 S. Bandak, D. Goren, A. Horowitz, D. Tzemach, A. Gabizon, Pharmacological studies of cisplatin encapsulated in long-circulating liposomes in mouse tumor models, Anticancer. Drugs 10 (1999) 911–920  2 O.G. Mouritsen, T.L. Andresen, A. Halperin, P.L. Hansen, A.F. Jakobsen, U.B. Jensen, M.O. Jensen, K. Jørgensen, T. Kaasgaard, C. Leidy, A.C. Simonsen, G.H. Peters, M. Weiss, Activation of interfacial enzymes at membrane surfaces, J. Phys. Condens. Matter 18 (2006) 1293–1304  3 C. Leidy, L. Linderoth, T.L. Andresen, O.G. Mouritsen, K. Jørgensen, G.H. Peters, Domain-induced activation of human phospholipase A2 type IIA: local versus global lipid composition, Biophys. J. 90 (2006) 3165–3175  4 T.L. Andresen, S.S. Jensen, K. Jørgensen, Advanced strategies in liposomal cancer therapy: Problems and prospects of active and tumor specific drug release, Prog. Lipid Res. 44 (2005) 68–97  5 D. Marsh, Liquid-ordered phases induced by cholesterol: A compendium of binary phase diagrams, Biochim. Biophys. Acta 1798 (2010) 688–699  6 C. Mateo, A. Acuna, J. Brochon, Liquid-crystalline phases of cholesterol lipid bilayers as revealed by the fluorescence of trans-parinaric acid, Biophys. J. 68 (1995) 978–987  Figure 1: Conceptual illustration. Liposome encapsulated oxaliplatin will circulate until it encounters the fenestrated capillaries in the tumor tissue, where it extravasates. Here it encounters an elevated level of secretory phospholipase A2 (sPLA2), which hydrolyses the phosphoglycerolipids, causing release of the drug. In addition the hydrolysis products, lyso-lipids and free fatty acids, may act as permeability enhancers, thus further contributing to drug transport across the cellular membrane.  Figure 2: Enzymatic hydrolysis and release of oxaliplatin. A) POPC/POPG/Chol/DSPE-PEG2k liposomes with 0, 20 and 40 % cholesterol and HSPC/Chol/DSPE-PEG2k (Stealth) liposomes, loaded with oxaliplatin, were incubated in the presence or absence of secretory phospholipase A2 (± sPLA2) at 37 °C with gentle magnetic stirring for 0, 6 and 24 h. Release of oxaliplatin was determined by ICP-MS. Values are mean of three individual experiments ± SD. POPC/POPG/Chol/DSPE-PEG2k (35:40:20:5) liposomes were incubated with colo205 cell conditioned media (CM), human tear fluid or no enzyme at 37 °C with gentle magnetic stirring for 6 h. Hydrolysis of B) POPC and C) POPG was determined by MALDI-TOF MS. DPPC was included as internal reference. Values are mean of a minimum of three lipid extractions and three spots of each extraction.   Liposomal release of oxaliplatin is sPLA2 dependent, but only slightly cholesterol dependent   Figure 3 In vitro cytotoxicity of oxaliplatin (OxPt) loaded sPLA2-sensitive liposomes. HT-29 cells were treated with an increasing amount of OxPt loaded POPC/POPG/Chol/DSPE-PEG2k liposomes with 20 % cholesterol, either in the presence (open circles) or absence (open diamonds) of colo205 cell conditioned media containing sPLA2. OxPt in free form (closed circles) or encapsulated in HSPC/Chol/DSPE-PEG2k (Stealth) liposomes (closed diamonds) were used as controls. Cell survival was evaluated by MTS staining. Values are mean of triplicates with blank (media with MTS reagent) subtracted ± SD. All values are normalized to non-treated cells. The data is representative of minimum three separate experiments.  Figure 4:In vivo effect of sPLA2 sensitive liposomes. A) Kaplan-Meier analysis of survival. Nude NMRI mice bearing MT-3 xenograft tumors were treated with 10 mg/kg free oxaliplatin (OxPt) (dotted line), OxPt loaded POPC/POPG/Chol/DSPE-PEG2k (35:40:20:5) liposomes (dashed line) or OxPt loaded Stealth liposomes (dotted dashed line) once per week for a total of four treatments. Isotonic glucose solution (solid line) was included as control. B) Mouse treated with OxPt loaded POPC/POPG/Chol/DSPE-PEG2k (35:40:20:5) liposomes, euthanized three days post first treatment due to excessive weight loss, dehydration and subcutaneous bleedings. C) Liver section from a mouse treated with OxPt loaded POPC/POPG/Chol/DSPE-PEG2k (35:40:20:5) liposomes. The liver section displays extensive multifocal necrosis of hepatocytes (white arrows) with collapse of hepatic sinusoids and hydropical injury to the cell nucleus of the hepatocytes. Several of the hepatocytes display nuclear inclusion bodies and vacuolar degeneration (white arrowheads). Extensive inflammatory reaction is seen in the hepatic sinusoids and around necrotic regions with accumulation of granulocytes (black arrows) (HE stained FFPE 5 um). .  Liposomes show enhanced cytotoxic effect in vitro sPLA2 responsive liposomes induce severe toxicity in vivo Contact information: Ragnhild Garborg Østrem Technical University of Denmark ragga@nanotech.dtu.dk  

sPLA<sub>2</sub> sensitive fluid phase liposomes induce severe toxicity in murine cancer model

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sPLA<sub>2</sub> sensitive fluid phase liposomes induce severe toxicity in murine cancer model

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