Revisiting the use of sPLA2-sensitive liposomes in cancer therapy

Accepted Manuscript Revisiting the use of sPLA2-sensitive liposomes in cancer therapy

Houman Pourhassan, Gael Clergeaud, Anders E. Hansen, Ragnhild G. Østrem, Frederikke P. Fliedner, Fredrik Melander, Ole L. Nielsen, Ciara K. O'Sullivan, Andreas Kjær, Thomas L. Andresen PII: DOI: Reference:

S0168-3659(17)30688-0 doi: 10.1016/j.jconrel.2017.06.024 COREL 8847

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

9 February 2017 21 June 2017 24 June 2017

Please cite this article as: Houman Pourhassan, Gael Clergeaud, Anders E. Hansen, Ragnhild G. Østrem, Frederikke P. Fliedner, Fredrik Melander, Ole L. Nielsen, Ciara K. O'Sullivan, Andreas Kjær, Thomas L. Andresen , Revisiting the use of sPLA2-sensitive liposomes in cancer therapy. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Corel(2017), doi: 10.1016/ j.jconrel.2017.06.024

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Revisiting the use of sPLA2-sensitive Liposomes in Cancer Therapy 1,2,¤

1,2,¤

1,2,3

1,2

Houman Pourhassan , Gael Clergeaud , Anders E. Hansen , Ragnhild G. Østrem , Frederik k e P. 3 1,2 4 5,6 3 1,2,* Fliedner , Fredrik Melander , Ole L. Nielsen , Ciara K. O’Sullivan , Andreas Kjær , Thomas L. Andresen . 1

US

CR

IP

T

Department of Micro- and Nanotechnology, Technical University of Denmark , Building 423, DK-2800 Kgs. 2 Lyngby, Denmark ; Centre for Nanomedicine and Theranostics, Technical University of Denmark , DK -2800 Kgs. 3 Lyngby, Denmark ; Department of Clinical Physiology, Nuclear Medicine & PET, and Cluster for Molecular Imaging, Rigshospitalet, Copenhagen University Hospital and Faculty of Health Sciences, University of 4 Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark ; Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik sberg C, Denmark ; 5 Nanobiotechnology & Bioanalysis Group, Department of Chemical Engineering, University of Rovira I Virgili, 6 Tarragona, Spain; Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain.

AN

Corresponding Author

M

*Address correspondence to [email protected]

Author Contributions ¤

AC

CE

PT

ED

Authors contributed equally to this work.

ACCEPTED MANUSCRIPT

Abstract

PT

ED

M

AN

US

CR

IP

T

The first developed secretory phospholipase A2 (sPLA2) sensitive liposomal cisplatin formulation (LiPlaCis ®) is currently undergoing clinical evaluation. In the present study we revisit and evaluate critical preclinical parameters important for the therapeutic potential and safety of platinum drugs, here oxaliplatin (L-OHP), formulated in sPLA2 sensitive liposomes. We show the mole percentage of negatively charged phospholipid needed to obtain enzyme-sensitivity for saturated systems is ≥ 25% for 16-carbon chain lipid membranes, and > 40% for 18-chain lipid membranes, which was surprising as 25% is used clinically in LiPlaCis ®. Efficient sPLA2-dependent growth inhibition of colorectal cancer cells was demonstrated in vitro, where cell membrane degradation and cytolysis depends on the sensitivity of the formulation towards the enzyme and is governed by the amount of lysolipids generated and the presence of serum proteins. We found that serum proteins did not affect the lipase activity of the enzyme towards the membranes but instead sequester the lysolipid byproducts consequently inhibiting their detergent-like cytotoxic properties. In vivo therapeutic potential and safety of the liposomes was investigated in nude mice bearing sPLA2-deficient FaDu squamous carcinoma and sPLA2-expressing Colo205 colorectal adenocarcinoma. After intravenous injections, the tumor growth was suppressed for liposomal L-OHP relative to free drug, but only a weak response was observed for both slow- and fast-releasing sPLA2-sensitive formulations compared to non-sensitive liposomes. Also, the mice did not show longer survival. In turn, for the highly sPLA2-sensitive liposomes, multiple high doses caused petechial cutaneous hemorrhages, along with multifocal hepatonecrotic lesions, suggestive of premature activation in skin and liver irrespective of sPLA2status of the tumor engraft. These results indicate that although liposomal carriers can improve the antitumor efficacy of platinum drugs, sPLA2-triggered release suffers from a narrow therapeutic index and has safety concerns.

AC

Graphical Abstract

CE

Keywords: Cancer therapy, drug delivery, liposomes, triggered release, secretory phospholipase A2, oxaliplatin.

ACCEPTED MANUSCRIPT

1. Introduction

PT

ED

M

AN

US

CR

IP

T

In the design of nanotherapeutics, controlled and site-specific drug release in diseased tissue remains one of the main challenges for the field and ideally should increase both therapeutic efficacy and minimize therapy-associated side effects. Long-circulating pegylated liposomes are designed to retain encapsulated drugs, altering drug deposition and improve efficacy and drug toxicity profiles[1,2]. Oxaliplatin (L-OHP) is a first-line chemotherapy in the combination FolFox regimen indicated for the treatment of advanced colorectal cancer[3]. In attempting to minimize the peripheral neuropathy, myelotoxicity and gastrointestinal toxicities commonly associated with L-OHP, pegylated-liposomes have been rigorously investigated for safer delivery both preclinically[4–7] and in patients[8]. Despite being well-tolerated and lowering toxicities, in many cases insufficient release rates of hydrophilic drugs at the target site has limited their therapeutic potential and more sophisticated liposomal delivery systems are therefore required[9]. Several approaches exist to trigger drug release by employing environmentally-sensitive liposomes that respond to either external stimuli, e.g. induced hyperthermia[10–12] and light in photodynamic therapy[13], or intrinsically by taking advantage of pathological changes arising in the diseased state. The elevated expression of endogenous enzymes in cancerous tissue represents a promising strategy to control and obtain a site-specific drug release intrinsically[14–16]. Apart from LiPlacis®, thermo-sensitive liposomes are among the most progressed liposome systems with triggered-release properties that have been investigated clinically, however, without sufficient succes[17]. From a therapeutic point of view secretory phospholipase A2 type IIa (sPLA2) is an attractive target, as it is overexpressed in its active form in various types of cancer including colon, breast, pancreatic, and prostate[18–22], and thereby can function as a release trigger intrinsically build-in in the tumor. Additionally, sPLA2 exhibits preferential substrate specificity for organized lipid structures (such as bilayers) over monomeric lipids in solution, making it particularly suitable for liposomal drug delivery[23]. The validity of this principle have been established in vitro[24– 27], and to some extent in vivo using mouse models[16,28].

AC

CE

The sPLA2 enzyme has a high specificity for anionic lipid membranes, e.g. composed of phosphatidylglycerol (PG)[29]. Thus, for controlled delivery purposes the sensitivity of the drug carrier to sPLA2 degradation, and thereby the level of drug release, can be modulated by producing liposomes with varied amounts of negatively charged lipids and increasing or decreasing the chain length of the fatty acids[29]. sPLA2 hydrolyzes the ester linkage of sn-2-acyls of phospholipids, which yields free fatty acids and 1acyl-lysophospholipids[9,29]. In this sense liposome membrane destabilization by sPLA2 is thought not only to liberate the encapsulated drug, but also to yield high concentrations of lysolipids and free fatty acids locally at the site of activation. In turn, these can then serve as permeability enhancers across biological membranes, or at high amounts, directly induce cellular toxicity by forming aggregated structures with detergent-like properties[16,28,30]. Thus, the carrier by itself is considered to be a prodrug. As premature activation therefore bears a risk of releasing bioactive molecules in unwanted sites, the sensitivity of the particles needs to be finely tuned in order to avoid harmful side effects. The first liposomal formulation with a sPLA2-triggered release mechanism encapsulating cisplatin, LiPlaCis®, has completed clinical phase I testing in patients with advanced or refractive solid tumors.

ACCEPTED MANUSCRIPT

US

CR

IP

T

Despite early cessation of an initial trial in 2009 owed to formulation-related safety concerns requiring reformulation, LiPlaCis® has reentered clinical testing to establish dose recommendations and is scheduled to be completed by mid-2017. In the original phase I study the formulation (DSPC:DSPG:DSPE-PEG2000 70:25:5 molar ratio) was reported to be too unstable, with high levels of platinum excreted via urine along with a high incidence of dose-related renal toxicity characteristic of free cisplatin[31]. The reason for this stability issue was not clear, but did not appear to be related to a premature activation of the liposomes during circulation by sPLA2, as no correlation could be found between serum sPLA2 levels and the plasma half-life of the particles. On the other hand, an unusually high incidence of non-dose related grade 1-2 infusion reactions (39%) was observed despite premedication with clemastine and dexamethasone and reducing infusion rates by 50%[31]. In comparison, other clinically relevant liposome formulations also induce similar symptoms, but at a much lower rate of up to 9%[31]. It was believed that this could be associated to activation of the complement system by LiPlaCis® from the observed concurrent immediate upsurge in SC5b plasma levels. Along these lines, PG-containing liposomes have been shown to have the capacity to activate the complement system[32].

2. Materials and methods

CE

2.1. Materials

PT

ED

M

AN

The aim of the present work is to revisit the potential of sPLA2-sensitive liposome formulations of platinum drugs motivated by the current clinical development stage of these systems; to get deeper insight into the sPLA2 dependency of previous developed formulations and understand the therapeutic window that is obtainable with such formulations. We report here L-OHP loaded liposomes with low- or high sensitivity towards sPLA2-triggered degradation as drug delivery nanocarriers. Hereby we compare slow- and fast-releasing formulations to non-sensitive nanocarriers, and assess their in vitro cytotoxicity against cultured cancer cells, as well as their efficacy and tolerability in vivo in mice bearing human tumors.

AC

The lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DPPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-snglycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)-2000] (DSPE-PEG2k), 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1’rac-glycerol) (LysoPPG) were purchased from Avanti Polar Lipids, Inc. (Alabama, USA). Lyophilized mixture of hydrogenated L-α-phosphatidylcholine (HSPC), cholesterol (chol), and DSPE-PEG2k (57:38:5 mol %) was acquired from Lipoid GmbH (Ludwigshafen, Germany). Oxaliplatin was purchased from Shanghai Yingxuan Chempharm (Shanghai, China). Chloroform, methanol, hydrochloric acid, HEPES, glucose, calcium carbonate, Triton X-100, Dulbecco's Modified Eagle's medium (DMEM), fetal bovine serum (FBS) and penicillin/streptavidin (pen/strep) were purchased from Sigma-Aldrich (Schnelldorf, Germany), as well as the gallium and iridium standards for ICP-MS. 2.2. Preparation of liposome vesicles

ACCEPTED MANUSCRIPT

AN

US

CR

IP

T

Liposomes were prepared following a previously reported method[33]. Briefly, accurate amounts of lipids were dissolved in chloroform:methanol (9:1 v/v), followed by solvent evaporation at room temperature (RT) under a gentle stream of nitrogen. To ensure complete solvent removal, the lipid films formed were placed under vacuum overnight. Multilamellar vesicles (MLV) were prepared by hydrating the lipids with a buffered solution (10 mM HEPES, 5% glucose, pH 7.4), containing (if desired) the encapsulate molecule, at a temperature 15ºC above the main phase transition temperature with vortexing every 5-10 min. For the preparation of calcein loaded liposomes, calcein was firstly dissolved in water with NaOH and then the pH was further adjusted to pH 7.4 prior to addition to the hydrating solution to a final calcein concentration of 20 mM. The preparation of oxaliplatin-loaded liposomes was carried out by previously dissolving 15 mg/mL oxaliplatin in the buffered solution at 65ºC for 1 h under stirring conditions. The MLV suspensions were extruded 21 times through two-stacked 100 nm pore size polycarbonate filters at 55ºC forming homogeneous large unilamellar vesicles (LUV) (< 130 nm) with a narrow size distribution (PDI < 0.1). For in vivo applications, liposomes were instead extruded using a high-pressure extrusion device (Northern Lipids Inc., Burnaby, Canada) and were sequentially downsized through 400/200/100 nm polycarbonate filters. Calcein containing liposomes were purified by gel filtration through Sephadex G-50 size exclusion column using HEPES buffer as eluent and liposomes loaded with oxaliplatin were purified by dialysis for 3 days using cassettes of 100kDa molecular cutoff and HEPES buffer containing 1mM CaCO3 (> 99% encapsulation). All liposomes were stored at 4ºC.

M

2.3. Physicochemical characterization of liposomal drug carriers

ED

2.3.1. Size and surface charge

CE

PT

Liposome hydrodynamic diameter and size distribution was analyzed using dynamic light scattering (DLS) and the vesicles surface charge was determined by zeta-potential using a ZetaPALS system (Brookhaven Instruments Corporation, New York, USA). Liposome suspensions were diluted 100-fold in filtered (0.2 µm) buffer, placed in plastic cuvettes and degassed for 5 min to expel any air in the samples before performing the DLS and zeta-potential analysis. The standard deviations were calculated from the mean data of experiments (n ≥ 3). 2.3.2. Lipid and drug concentration

AC

The lipid concentration was determined by measuring the total phosphorous content and the oxaliplatin concentration by determining the amount of platinum present in the liposomes using inductively coupled plasma mass spectrometry (ICP-MS) using a DionexTM ICS-5000+ system (Thermo ScientificTM, Dreieich, Germany). For the phosphorous measurements, samples were analyzed by diluting 5000-fold in 2% HCl containing 10 ppb of Gallium as an internal standard. To accurately measure the platinum-based chemotherapeutic, samples containing oxaliplatin were diluted 500,000-fold in 2% HCl containing 0.5 ppb of Iridium as an internal standard. 2.3.3 Phase transition temperatures High-resolution Differential Scanning Calorimetry (DSC) measurements were performed using a Q2000 calorimeter (TA Instruments, New Castle, DE) equipped to perform ascending and descending

ACCEPTED MANUSCRIPT

temperature mode operations. The lipid concentration used was 5 mM. The scan rate was 12°C/hr for both heating and cooling scans. Data were analyzed using Nano DSCRun Software v4.2.4 software. Samples were scanned several times to ensure the reproducibility of the endotherms and exotherms. 2.4. Quantification of human sPLA2 and protein content

US

CR

IP

T

Tumor xenografts were collected when sizes were ∼1000 mm 3 and dissected into 1-2 mm pieces at 4ºC using scalpels. The minced tumors were then mixed with a cocktail containing dissociation enzymes (Miltenyi biotec) and protease inhibitors (Calbiochem), and dissociated in a gentleMACS Octo Dissociator at 37ºC. Tumor interstitial fluid (IF) was separated from the cells by centrifugation for 5 min at 300g at 4 °C, and was collected and stored at -80ºC until analysis. For the plasma measurements, blood samples were taken from healthy mice (without tumors) and mice bearing Colo205 xenografts. The plasma fractions were separated by centrifugation at 300g during 5 minutes and stored at -80ºC until analysis. In vitro cultured HT-29 and Colo205 cell lines were also tested to check the amount of enzyme sPLA2 secreted.

ED

M

AN

The concentration of human sPLA2 present in different biological sources was measured using a sPLA2 (human Type IIa) enzymatic immunometric assay kit (Cayman Chemical), capable for detecting concentrations of the enzyme down to 15.6 pg/mL. Sample absorbance was measured at 405 nm using a microplate reader (Sunrise, Tecan, USA) and concentrations were extrapolated from the linear standard curve. The results are expressed as mean ± s.d. of triplicate aliquots from three different mice. The protein concentration in the samples was determined using a Pierce BCA protein assay kit (Thermo Scientific). The colorimetric reaction was measured at 570 nm using a microplate reader (Sunrise, Tecan, USA). Concentrations were determined based on the standard curve. The results are expressed as mean ± SD of triplicate aliquots from three different mice.

PT

2.5. Calcein release assay

AC

CE

Specificity and sensitivity of liposomes toward sPLA2 was determined by the calcein release assay. Fluorescent calcein was entrapped inside liposomes at a self-quenching concentration following the procedure described above and the time-resolved sPLA2-specific release of the fluorophore was recorded as a rise in the fluorescent intensity (FI). Briefly, calcein-loaded liposomes were diluted to 75 µM lipid concentration in Calcein-Release Buffer (CRB, 10 mM Hepes, 110 mM KCl, 30 µM CaCl2, 10 µM Na EDTA, pH 7.4) and transferred to a glass quartz cuvette (1mL) where the fluorescence (ex. 495 nm, em. 515 nm) was recorded under magnetic stirring at 37°C using a SLM8000 spectrophotometer (OLIS Inc., Georgia, USA). Once the fluorescence signal had stabilized for at least 1000 seconds (time 0), the sPLA2 enzyme was added to the sample (5 µL human tears or 20 µL Colo205 cell conditioned medium (CCM)) and was continuously monitored until reaching full release or for at least 2 hours. Triton X-100 (TX-100) was added to check the maximum fluorescent signal from complete release of calcein. The percentage release was calculated by the formula 100% × (F t – F0) / (FTX-100 – F0), where Ft represents the FI at a specific time point, F0 represents FI at time zero, and FTX-100 the total FI after addition of TX-100. All studies were done in triplicate. 2.6. Cell culture

ACCEPTED MANUSCRIPT

IP

T

The HT-29 human colon carcinoma and Colo205 human colon carcinoma cell lines were purchased from American Type Culture Collection (Virginia, USA). HT-29 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin (pen/strep) in a humidified 5% CO 2 atmosphere at 37°C. Colo205 cells were maintained in RPMI-1640 supplemented with 10% heat-inactivated FBS, 1% pen/strep. Both cell lines were subcultured every 23 days. Colo205 CCM was prepared under serum-starved conditions using the following procedure: (1) 15-20×106 Colo205 cells were cultured in a T-75 culture flask in complete growth medium, (2) following incubation for 48 hours the medium was exchanged with 20 ml fresh RPMI-1640 supplemented only with 1% pen/strep (no serum), and (3) following additional 48 hours of growth, the conditioned RPMI-1640 medium (denoted CCM) was collected and stored at -20°C until needed.

CR

2.7. Cell proliferation assay

PT

ED

M

AN

US

The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-te-trazolium (MTS) assay (Promega Biotech AB, Stockholm, Sweden) was used to determine the in vitro antiproliferative effect. Cells (HT-29 and Colo205) were plated in 96-well plates at a density of 1×104 cells per well in their respective complete growth media. After overnight incubation to allow cell attachment, the medium was removed and replaced with either complete growth media or Colo205 CCM containing varying concentrations of free drug (1.6-100 µM) or liposomal samples (1.6-100 µM drug, or in the case of empty vesicles, a lipid concentration equivalent to the drug-loaded liposomal sample), and the cells were further incubated for 6 hours at 37°C. After this co-incubation period, the cell media was exchanged with fresh growth media and the cells were allowed to grow for an additional 66 hours (72 h in total). After 72 h incubation the medium was removed and the cells were incubated with MTS solution until colorimetric reaction was developed within the linear range. The absorbance was read at 490 nm using a microplate reader (Sunrise, Tecan, USA). Values for cell survival are expressed as the percentage reduction in metabolically active cells relative to the solvent controls. All studies were repeated three times.

CE

2.8. Activity of sPLA2 in the presence of serum

AC

Liposomes were diluted in Colo205 CCM containing varying concentrations of FBS (0, 10 or 50%) to a final lipid concentration of 4 mM and incubated at 37°C for 6 h with gentle magnetic stirring. The 0 hour samples were prepared by mixing liposomes with heat inactivated Colo205 CCM. Lipid hydrolysis and consequent formation of lyso-PPG was analyzed by MALDI-TOF MS. Briefly, samples were mixed 1:10 with matrix/POPC (2,5-dihydroxybenzoic acid (DHB) spiked with sodium trifluoroacetate (NaTFA) in methanol as matrix with 500 µM POPC as internal reference), spotted in triplicates and analyzed by Bruker autoflex speed (Bruker Daltonics, Bremen, Germany). Observed MW: 507.4 (M+H++Na+) and 529.4 (M+2Na+). Expected MW: 507.3 (M+H++Na+) and 529.3 (M+2Na+). All peak intensities corresponding to lyso-PPG were normalized to all peak intensities corresponding to POPC. Values are mean of triplicates plus/minus standard deviation. All samples showed formation of lyso-PPG after incubation with sPLA2 (6 h), and no lyso-PPG was present before incubation (0 h). 2.9. Time-lapse microscopy

ACCEPTED MANUSCRIPT

HT-29 (3×104) cells were seeded in 8-well chamber slides (Sigma-Aldrich) with 0.3 mL of RPMI-1640 medium containing 10% FBS and pre-incubated for 24 h. Hereafter, the medium was aspirated and the liposomes (100 µL/well) were added to the cells at a final drug concentration of 100 µM in Colo205 CCM. The cells were recorded for 4 h and images were captured on a Leica TCS SP5 AOBS confocal microscope with a 20X air-objective (Heidelberg, Germany). The microscope was equipped with an incubator box and CO 2 supply for optimal growth conditions during imaging (Life Imaging Services GmbH, Basel, Switzerland).

IP

T

2.10. In vivo efficacy

AC

CE

PT

ED

M

AN

US

CR

All animal experiments were conducted according to current legislation and department policies, and were approved by the University of Copenhagen Institutional Animal Care and Use Committee (IACUC). FaDu (5×106) and Colo205 (7.5×106) cells in 100 µL culture medium were injected subcutaneously into the right flank of 6-week old female NMRI-nu mice. The mouse strain used was BomTac:NMRI-Foxn1nu from Taconic (Lille Skensved, Denmark), which has an immune system characterized by a small population of T cells; the antibody response is confined to IgM class; response to T cell dependent antigens is low; a compensatory increase in the level of natural killer cells resulting in a slightly increased level of NK cells in comparison to normal NMRI mice; and an intact complement system. Once tumor masses reached ∼50 mm 3 (FaDu) and 100 mm 3 (Colo205), animals were randomized into 5 groups of 8 mice and started the treatments. In mice-bearing FaDu tumors, mice received i.v. bolus injection via lateral tail vein of 4 or 8 mg/kg oxaliplatin every 4 days for a total of 6 doses (6q4d), free or encapsulated in sPLA2-degradable liposomes. In Colo205 tumor xenografts, mice were i.v. injected with 5 mg/kg oxaliplatin every 4 days for a total of 4 doses (4q4d), free or encapsulated in sensitive and non-sensitive sPLA2 liposomes. Control mice were injected with isotonic glucose solution at a compatible volume. Tumor sizes were measured by electronic caliper and body weight was monitored two to three times a week. The tumor volume was calculated using the formula: Volume = (length × width2) / 2, where width was the shortest measurement in millimeter. The animals were euthanized when tumor sizes reached 1000 mm 3, loss of body weight > 15% or displayed clear signs of misthriving. Kaplan-Meier plots were constructed based on the abovespecified end-points and median survival time determined for all groups and compared with a log-rank (Mantel-Cox) test. Tumor-to-control (T/C) ratios were calculated for all time points, T/C ratios < 0.15 were considered highly effective, T/C ratios between 0.15–0.45 moderately efficient and T/C ratios > 0.45 inactive. Differences were considered significant if the p value was less than 0.05. Statistical analysis were performed using GraphPad Prism version 6.0 for Mac OS X (California, USA). 2.11. Histology

Tissues were excised using sterile surgical equipment and transferred to 15 mL tube containing 4% neutral buffered formaldehyde in a 1:20 ratio between tissue and fixative. Samples were placed in the fixative for at least 24 h to ensure complete fixation. Hereafter, the samples were transferred to histocassettes and kept in 70% ethanol before becoming dehydrated and embedded in paraffin wax and sectioned into 5 µm thick sections on a microtome. The specimens were stained with hematoxylin and eosin and finally analyzed using an optical microscope.

ACCEPTED MANUSCRIPT

3. Results and Discussion 3.1 Physicochemical characterization of liposomes

M

AN

US

CR

IP

T

The enzymatic activity of sPLA2 towards liposome degradation can be significantly modulated by the composition of the lipid bilayer and its morphological and physicochemical properties. As it is of great interest to bioengineer liposomal nanocarriers that are sufficiently responsive towards sPLA2 hydrolysis to cause complete release of drug within a reasonable period of time, target release rates can be achieved by adjusting the molar ratio of constituent lipid molecules in the liposome membrane. The sPLA2-sensitive liposomes prepared were composed of DPPC:DPPG:DSPE-PEG2k (55:40:5 mol%) named DP(40), DPPC:DPPG:DSPE-PEG2k (70:25:5 mol%) named DP(25) and DSPC:DSPG:DSPE-PEG2k (55:40:5 mol%) named DS(40). As controls, non-hydrolysable liposomes were prepared from hydrogenated soybean phosphocholine (HSPC), cholesterol and DSPE-PEG2k (57:38:5 mol%) designated Stealth. The size distribution, polydispersity index, and the charge of the liposomes were characterized, along with the lipid and drug concentrations and the percentage of encapsulated drug (Table 1). Homogenous liposomes were obtained with sizes of approx. 100 nm that carried a net negative surface charge. Following purification by dialysis, all formulations exhibited drug entrapment percentages above 99% (encapsulation efficiency was approx. 8-10%, i.e. fraction of the starting material that is encapsulated) and were stable at 4ºC for at least 6 months, signifying a highly stable intravesicular retainment of L-OHP in these liposomes.

ED

Table 1. Physicochemical properties of liposomal formulations of oxaliplatin Size (nm)

PDI

Zeta-potential (mV)

L-OHP (mg/mL)

Lipid (mg/mL)

DE (%)

EE (%)

Drug-to-lipid w eight ratio

DP(25) + L-OHP

94 ± 1

0.04

-20 ± 2

1.6

16.3

99.6

10.7

1:10

DP(40) + L-OHP

98 ± 1

0.03

-26 ± 2

1.9

22.2

99.9

12.7

1:12

Stealth + L-OHP

106 ± 1

0.03

-15 ± 1

1.1

17.1

99.7

7.3

1:16

b

CE

a

PT

Formulation

a

b

Drug entrapment (DE) is the percentage of liposomal to free fraction of L-OHP. Encapsulation efficiency (EE) is the percentage of liposomal L-OHP to the total starting amount of L-OHP (15 mg/mL).

AC

The size values are the mean ± s.d. of triplicate measurements and Z-potential values the mean ± s.e. of 10 sub-measurements. Abbreviations: PDI, polydispersity index; L-OHP, oxaliplatin; DP(25), DPPC:DPPG:DSPE- PEG2k (70:25:5 mole%); DP(40), DPPC:DPPG:DSPE- PEG2k (55:40:5 mole%); Stealth, HSPC:Chol:DSPE- PEG2k (57:38:5 mole%).

3.2. Sensitivity of the liposome towards sPLA 2 drug release The sensitivity of the liposome carriers to be degraded by the sPLA2 enzyme and consequently release of their cargo was demonstrated in in vitro release studies using calcein at self-quenching concentrations loaded within the different liposome carriers. This allowed us to monitor the enzymatically-triggered release of a hydrophilic drug surrogate over time by following the increase of fluorescence signal coming from the released calcein in its de-quenched state. First, to assess the

ACCEPTED MANUSCRIPT

IP

T

expression status of sPLA2 enzyme in the various biological models used throughout the study, we performed enzyme-linked immunosorbent assay (ELISA) quantification of human sPLA2 type IIa (Table 2). Even though this does not carry information about the activation status of the enzyme, it gives a measure of how much of the total enzyme (active and inactive) that is present in the various samples. We tested the sensitivity of the liposomes against sPLA2 enzyme from two different sources, human tears and cell conditioned medium (CCM) of Colo205 cancer cells, with detected sPLA2 concentrations of 19.3 µg/mL and 185.4 ng/mL respectively (Table 2). These levels matched with reported values found in tear fluid of healthy subjects (55 ± 34 µg/mL)[34], as well as in Colo205 CCM following 72 hours of cell cultivation (75 ± 20 ng/mL)[28], thus confirming the suitability of uses these as sPLA2 enzyme sources.

a

19262 ± 2818

CCM Colo205 cell line

185.43 ± 92.46 a

CCM HT-29 cell line b

b

0.01 ± 0.01

79.42 ± 63.16

IF Colo205 xenograft

0.00 ± 0.02

Plasma healthy mice

0.01 ± 0.02

ED

Plasma Colo205 xenografted mice a

a

M

IF FaDu xenograft

0.78 ± 0.52

a

6.95 ± 0.32

AN

Human tear fluid

Protein concentration (mg/mL)

US

Human sPLA 2 concentration (ng/mL)

Enzyme source

CR

Table 2. Quantification of human sPLA2 expression and protein content in biological sources

a

0.92 ± 0.01

a

sPLA 2-to-protein w eight ratio 2

1:4 x 10

3

1:5 x 10

1.30 ± 0.04

N/A

3.10 ± 0.05

1:4 x 10

3.95 ± 0.09

N/A

45.33 ± 0.01

N/A

52.33 ± 0.04

1:7x10

4

7

Mean ± s.d. of triplicate measurements from single samples.

b

PT

IF is the soluble fraction of tumor xenografts. Measurements are the mean ± s.d. of triplicate measurements from three different animals.

CE

Abbreviations: CCM, cell conditioned medium; IF interstitial fluid; N/A, not applicable.

AC

The sPLA2 enzyme has been largely reported to have a moderate and a rapid mode of action separated by a lag-time, in which the enzyme accumulate in the lipid membrane[35]. The results presented in Figure 1A illustrate the differences in lag-time and enzyme activity of sPLA2 present in human tears, as well as in CCM of human Colo205 cancer cells, towards the calcein-loaded liposomes composed of different lipid compositions. The specificity of the enzyme from different sources was similar, as sPLA2 from tears and CCM induced comparable leakage behavior. Albeit, the substantially lower concentration of sPLA2 present in Colo205 CCM relative to tear fluid (Table 2), explains the differences observed in release kinetics between the two sources of enzyme. The molar ratio of negatively charged lipid present in the liposome membrane played a key role in the enzyme activity evaluated from calcein release profiles. In membranes composed of DPPC:DPPG:DSPEPEG2k, when 25% of the negatively charged lipid DPPG was present in the bilayer (DP(25)), only the sPLA2 from tears was able to cause partial calcein release, whereas increasing the percentage of DPPG lipid to 40% (DP(40)), the membrane became fully enzyme-degradable and calcein was

ACCEPTED MANUSCRIPT

M

AN

US

CR

IP

T

completely released within 1500 seconds irrespective of the source of enzyme (Figure 1A). More negatively charged membranes are also feasible for the sPLA2-triggered release concept, however, it may compromise in vivo performance due to the correlation between membrane charge and degree of opsonization by the immune system[32].

AC

CE

PT

ED

Figure 1. Calcein release profiles and phase transition diagrams of liposome formulations. (A) Shows the in vitro sPLA 2-dependent release kinetics of calcein from liposomal formulations incubated at 37°C. Human tear fluid and cell conditioned media (CCM) from human Colo205 cancer cells were used as enzyme sources of sPLA 2. All results are expressed relative to 100% release observed after addition of Triton X-100. Results are representative of at least three experiments. Insert shows the time-intersect for the individual formulations reaching 50% release (T50) in seconds. (B) Differential scanning calorimetric (DSC) endotherms highlighting the main phase transition temperature (Tm) of the different formulations. Abbreviations: DP(25), DPPC:DPPG:DSPE-PEG2k (70:25:5 mol%); DP(40), DPPC:DPPG:DSPE-PEG2k (55:40:5 mol%); DS(40), DSPC:DSPG:DSPE-PEG2k (55:40:5 mol%).

In addition, the sensitivity of the liposome towards sPLA2-degradation can also be tuned by the membranes physical properties. DSC of the formulations (Figure 1B) showed that liposomes made of 16-carbon chain lipids (DP(25) and DP(40)) have an overall main phase transition temperature (Tm) of 41 and 42°C respectively, and were highly sensitive towards sPLA2-degradation, whilst in 18-carbon lipid membranes (DS(40)) with higher Tm of 55°C, the enzyme activity was almost abolished, which can be explained by the gel state behavior of the membrane. This was evident from the time it took to reach 50% release (T50) of calcein with DP(40) liposomes (70.1 s) being >10 and > 30 times faster than DP(25) liposomes (857.7 s) and DS(40) liposomes (>2000 s) (Figure 1A insert).

ACCEPTED MANUSCRIPT

Also, consistent with earlier reports incorporating small amounts of pegylated lipids (5 mol% DSPEPEG2k) into the formulations for in vivo translatability, did not impede the enzymatic hydrolysis from sterically obstructing the interaction between enzyme and substrate[16,28,36]. 3.3. In vitro antiproliferative activity of L-OHP loaded sPLA2-sensitive liposomes

AC

CE

PT

ED

M

AN

US

CR

IP

T

The in vitro antiproliferative capacity of sPLA2-sensitive liposomal L-OHP formulations was investigated by MTS assay. The cytotoxic profile of free L-OHP and L-OHP loaded in sensitive DP(25) or DP(40), and in non-sensitive (Stealth) formulations were tested in sPLA2-secreting Colo205 cell line and in non-secreting HT-29 cells with added exogenous sPLA2 using CCM from Colo205 cells as a source of enzyme (Figure 2). In concordance with the liposome sensitivity of sPLA2 shown in Figure 1A, efficient growth inhibition of sPLA2 non-secreting HT-29 cells was only observed when the liposomes became activated by exogenous sPLA2 (Figure 2A). In both colorectal cancer cell lines sPLA2-sensitive liposomal L-OHP were highly cytotoxic, with drug concentrations able to inhibit cell growth by 50% (IC50) around 10 to 12.5 µM (Figure 2B). In Colo205 cells, sPLA2-sensitive liposomal L-OHP induced a comparable antiproliferative effect as the free drug, whereas in HT-29 cells the sPLA2-degradable liposomes exceeded that of unencapsulated L-OHP at identical drug concentrations.

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

AC

CE

Figure 2. In vitro evaluation of the sPLA 2-sensitive concept. (A) Lack of cytotoxicity of sPLA 2-sensitive liposomes against non-sPLA 2-secreting HT-29 cells in the absence of exogenous sPLA 2 shows the specificity of the concept towards sPLA 2 and dependence of sPLA 2 activation. (B) In vitro cytotoxicity of L-OHP in free form or encapsulated in sPLA 2-sensitive liposomes (DP(25) and DP(40)) or in non-degradable liposomes (Stealth) tested against sPLA 2-secreting Colo205 cells and sPLA 2-deficient HT-29 cells in the presence of Colo205 cell conditioned medium (CCM) containing sPLA 2. (C) Cytolytic activity of unloaded (without L-OHP) sPLA 2-sensitive liposomes arise from cellular lysis as a consequence of sPLA 2-driven hydrolysis and formation of permeability enhancing/membrane lysing components. (D) Time-lapse micrographs of HT-29 cells following 3 hours of incubation with DP(40) in the presence of sPLA 2-proficient Colo205 CCM. (E) Inhibitory effect of sPLA 2-sensitive liposomes on HT-29 cell proliferation is modulated by presence of serum components. (F) Effect of serum (1050% FBS) on the enzymatic activity of sPLA 2. Results are triplicates from one experiment (mean ± s.d.) and representative of three independent experiments. Abbreviations: L-OHP, oxaliplatin; DP(25), DPPC:DPPG:DSPE-PEG2k (70:25:5 mol%); DP(40), DPPC:DPPG:DSPE-PEG2k (55:40:5 mol%); DS(40), DSPC:DSPG:DSPE-PEG2k (55:40:5 mol%); Stealth, HSPC:Chol:DSPE-PEG2k (57:38:5 mol%).

ACCEPTED MANUSCRIPT

US

CR

IP

T

In addition, the degree of growth inhibition of HT-29 cells induced by the liposomes followed the sensitivity of the formulations towards sPLA2 hydrolysis (Figure 2B). In control experiments where HT29 cells were treated in the absence of sPLA2 (without Colo205 CCM), the sensitive liposomes showed similar antiproliferative profiles as the non-degradable Stealth formulation (Supp. Figure S1). Regardless of enzymatic activation, some antiproliferative effect was present (albeit low) for all tested liposome formulations. This was shown to correlate with an unspecific association of the nanoparticles to the tumor cells using flow cytometric analysis and fluorescently-labeled liposomes (Supp. Figure S2), and was found to depend both on the concentration of particles added and coincubation time used, indicating that these particles are taken up by the cells in a unspecific manner. Importantly, to rule out that this cytotoxicity was induced by premature leakage of the drug from the liposomes, we assessed the amount of unspecific drug leakage by separating the free and liposomal L-OHP by spinfiltration and measured the platinum content in the fractions by ICP-MS. We found that the DP(25) and DP(40) liposomes leaked negligible amounts of drug (<10% on average) after 24 h incubation at 37ºC in the presence of high serum concentrations (50% FBS) (Supp. Figure S3).

AN

Taken together, these findings illustrate an efficient and stable formulation of L-OHP in enzymesensitive liposomes that carry a desirable controlled release mechanism based on sPLA2-mediated activation. 3.4. In vitro cytolytic behavior of unloaded sPLA2-sensitive liposomes

AC

CE

PT

ED

M

Since a triggered release strategy based on sPLA2 activity is known to generate permeability enhancing lysolipids and fatty acids, we investigated their contribution to the cytotoxic effect by measuring the growth inhibition of HT-29 cells treated with unloaded sPLA2-sensitive liposomes with and without added sPLA2 (Figure 2C). Consistent with earlier reports, the empty carriers alone acted as prodrugs that upon enzymatic activation was able to induce efficient growth inhibition even in the absence of encapsulating an antiproliferative drug[16,28]. For HT-29 cells, both the DP(25) and DP(40) formulations were able to induce complete growth inhibition of the tumor cells at both molar ratios of the PG lipid in the membrane investigated. Whereas the DS(40) formulation only induced partial growth inhibition despite the presence of 40% PG. Thus, based on the evidence from the calcein release experiments and the in vitro cytotoxicity, we show that low enzyme-sensitive liposomes leads to a reduced generation of membrane permeability enhancing products that is related to the slower rate of hydrolysis, which in turn then translates to a diminished antiproliferative effect. Time-lapse micrographs of HT-29 cells during treatment with unloaded DP(40) liposomes and sPLA2containing CCM, revealed that the observed efficient growth inhibition, was in effect due to the complete disruption of the cellular membranes following 3 hours of incubation with the cells (Figure 2D). In control experiments treating the cells with non-degradable liposomes or with DP(40) liposomes in the absence of sPLA2, no cytolytic effect could be detected and membrane integrity was maintained (Supp. Figure S4). Hereby sPLA2-sensitive liposomes were confirmed to act as efficient prodrugs, and furthermore we show that their in vitro antitumor efficiency is primarily owed to generation of cell permeable byproducts from the action of the enzymatic hydrolysis. 3.5. Evaluation of the effect of serum to the sPLA2-sensitive liposomes

ACCEPTED MANUSCRIPT

CR

IP

T

Serum albumin has previously been shown to bind up to five lysophospholipids per albumin molecule[37] and therefore the presence of high amounts of plasma proteins is expected to strongly modulate the permeability enhancing properties of these components, and conversely thereby affect the effective cell lytic activity of sPLA2-sensitive liposomal carriers as well. Even though this potentially would be advantagous during blood circulation to avoid disruption of blood-borne cells, as well as ensure vessel and tissue integrity, particularly in the scenario were extra-tumoral hydrolysis occurs from the use of excessively sensitive liposomes, it may have implications for the activation of the particles inside the tumor. With respect to the intratumoral activation, plasma proteins that in normal physiology sterically are hindered from freely crossing the endothelium and extravasate into surrounding tissues and thereby help to maintain the colloidal osmotic pressure of the circulatory system, will be present in abnormally high concentrations inside the tumor interstitium in the malignant state, considering the disrupted nature of the endothelial barrier of the tumor microvasculature[38].

AC

CE

PT

ED

M

AN

US

For this reason, to address the effect of serum on the anti-proliferative effect of sPLA2-sensitive liposomes, HT-29 cells were treated with sPLA2-sensitive liposomal L-OHP together with Colo205 CCM in the absence of serum or spiked with 10% (low) or 50% (high) fetal bovine serum (Figure 2E). We demonstrate that high levels of serum proteins eliminate the cytotoxic effect of the carrier. As a positive control, pure lysoPPG was also tested with and without adding serum, and was found to exhibit an identical behavior on cell viability as seen for the unloaded liposomes (data not shown). These results support that the in vivo cytotoxic potential of generated permeability-enhancing products will not only depend on the intratumoral lipid concentration, but also on the concentration of various plasma proteins present in the interstitial compartment of the cancer. As extravasation of macromolecules such as plasma proteins and nanoparticulates across the blood endothelium varies enormously given the multitude of factors governing this process[38,39], it is difficult to predict which degree of sensitivity of the carrier towards sPLA2 will be the most therapeutically beneficial. Additionally, to rule out that the presence of serum affects the enzymatic activity of sPLA2, we measured the amount of lysolipids generated after treatment of sPLA2-sensitive liposomes with enzyme in the presence of serum using mass spectroscopy. The level of lysoPPG generated was found to be unchanged from DP(40) liposomes in the presence of serum (Figure 2F). This demonstrates that even though components in serum can sequester the generated permeability enhancing components from sPLA2-sensitive liposomes, the encapsulated drug is released as sPLA2 hydrolyzes the liposomes, irrespective of the presence of serum proteins. Of course, this process is further complicated by the presence of lipase inhibitors in a clinical setting; a feature not readily captured and accounted for here by using murine and bovine serum models 3.6. In vivo antitumor efficacy, survival and tolerability Previous findings have shown that several inbred mouse strains have an intact murine group-II sPLA2 gene, which raises the likelihood of background sPLA2 levels being present in the NMRI nude model used herein, even though the grafted tumor cells do not secrete the sPLA2 enzyme[40]. We first evaluated the efficacy and tolerability of L-OHP formulated in sPLA2-sensitive liposomes in a dose escalation study in NMRI nude mice bearing non-sPLA2-secreting FaDu squamous carcinoma. This was done to determine the baseline of the antitumor efficacy of these particle systems in a tumor

ACCEPTED MANUSCRIPT

ED

600

20

1000 800 600 400 200 0

40

60

CE

1200

AC

Tumor size (mm3)

D

0

0

Saline L-OHP 5mg/kg Stealth 5mg/kg DP(25) 5mg/kg DP(40) 5mg/kg

20 40 60 Days after tumor implantation

E

20

0

30 28 26 24 22

0 20

40

60

100

Percent survival (%)

200

40

20

F

0

20

40

60

34 32

80

Mouse weight (g)

Saline L-OHP 4mg/kg L-OHP 8mg/kg DP(40) 4mg/kg DP(40) 8mg/kg

400

0

60

34 32

Mouse weight (g)

800

80

C

M

1000

100

Percent survival (%)

B

1200

PT

Tumor size (mm3)

A

AN

US

CR

IP

T

model that lacked the capacity of stimulating specific drug release, and furthermore to establish the maximum tolerated dose including any potential unforeseen side effects occurring from premature activation by murine phospholipases. The mice received 6 i.v. doses of 4 or 8 mg/kg every 4 days, with the first dose administered upon the tumors reaching 50 mm 3 in size. L-OHP formulated in DP(40) liposomes at 4mg/kg was able to induce a minor increase in tumor growth reduction compared to mice receiving free L-OHP at equimolar doses, reaching an equivalent response rate as twice the amount of free drug injected, however, this was only a moderate effect (Figure 3A). In turn, the low antitumor effect did not give rise to an increase in survival rates (Figure 3B). Increasing the dose of L-OHP formulated in DP(40) liposomes to 8 mg/kg (equivalent to a lipid dose of 113mg/kg) was poorly tolerated and led to petechial cutaneous hemorrhages in the skin of the mice (Supp. Figure S5), along with concurrent weight loss (Figure 3C) and dehydration requiring immediate euthanasia. At this high dose, DP(40)-treated mice showed severe signs of toxicity and poor tolerance late in the course of treatment suggestive of a cumulative effect, with 3 out of 8 mice displaying massive weight loss 4 days after second and third injections, and 5 out of 8 mice presenting severe cutaneous bleedings following 4 days after the second treatment. In contrast, at 4mg/kg the mice showed no signs of toxicity from neither DP(25) nor DP(40), with 8 out of 8 mice reaching the humane end-point of tumor burden.

60 40 20

30 28 26 24 22

0 0

20 40 60 Days after tumor implantation

20

0

20 40 60 Days after tumor implantation

Figure 3. Therapeutic efficacy of sPLA 2-sensitive formulations in female nude NMRI mice bearing FaDu tumors with no-sPLA 2-expression (upper panel) and Colo205 tumors with high-sPLA 2-expression (lower panel). In FaDu xenografts, mice received 6 i.v. injections every 4 days (6q4d) of 4 or 8 mg/kg L-OHP free or loaded in highly sPLA 2-sensitive DP(40) liposomes. Colo205-bearing mice received 4 i.v. injections every 4 days (4q4d) of 5mg/kg L-OHP free or loaded in liposomes with varying degree of sensitivity towards sPLA 2 (high (DP(40)); low

ACCEPTED MANUSCRIPT

(DP(25)); non(Stealth)). (A, B) Mean tumor volumes. (B, E) Kaplan-Maier survival plots based on specified humane end-points. (C, F) Animal body weights.

ED

M

AN

US

CR

IP

T

Continued expression of sPLA2 by human Colo205 colorectal carcinoma cells following implantation in mice have previously been established [28], and was confirmed here (Table 2). This allowed us to use Colo205 as a sPLA2-proficient human cancer model. Furthermore, Colo205 xenografts have been reported to be relatively responsive towards L-OHP following three weekly i.p. injections of 5 mg/kg[41]. Taken together, Colo205 xenografts with an elevated tumor expression profile of sPLA2 and sensitivity towards L-OHP were therefore expected to serve as a suitable experimental model to evaluate the in vivo performance of the present enzyme-responsive formulations. Here we determined the efficacy of four i.v. injections of L-OHP in mice implanted with ectopic Colo205 tumors at 5 mg/kg administered every 4 days, as either unencapsulated drug or formulated in liposomes with high degree of sPLA2 sensitivity (DP(40)), low sensitivity (DP(25)), and non-sensitive highly-stable liposomes (Stealth) (Figure 3D-F). In comparison to FaDu tumors, Colo205 xenografts did exhibit marginally higher response rates towards free L-OHP following three intravenous injections, which is consistent with earlier reports[41]. Although for the liposomal formulations of L-OHP, despite a minor increased growth inhibiting effect relative to the free drug, encapsulating L-OHP within sPLA2-sensitive liposomal formulations did not improve the antitumor effect compared to nonsensitive liposomes (Figure 3D and 3E). In fact, all of the tested formulations exhibited >45% treatment-to-control ratios (%T/C) and are therefore statistically therapeutically inactive. No significant (P values >0.05) increase in survival proportions between the different treatments was obtained in the Colo205 model, as were assessed by the median survival of the groups (Figure 3E). A summary of the parameters from the Colo205 efficacy study is presented in Table 3.

PT

Table 3. Comparison of oxaliplatin and liposomal oxaliplatin antitumor activity against early-stage human Colo205 colorectal carcinoma

11, 15, 19, 23

0/9

-0.85 (21)

CE

Drug death (mice)

Median tumor volume 3 [mm on day 19 (range)]

Schedule (days)

AC

i.v. agent

Dosage (mg/kg per injection)

Mean body w eight change [g/mouse (day of nadir)]

Time for median tumor to reach 3 500 mm

Time for median tumor to reach 3 1000 mm

398 (92-1140)

21

35

69 (13)

Inactive

Opt. % T/C (day)

Comments a

Free L-OHP

5

Stealth + L-OHP

5

11, 15, 19, 23

0/9

-0.63 (17)

495 (299-1010)

19

45

49 (27)

Inactive

DP(25) + L-OHP

5

11, 15, 19, 23

0/9

-1.30 (17)

307 (137-542)

27

38

50 (30)

Inactive

DP(40) + L-OHP

5

11, 15, 19, 23

2/9

-1.31 (17)

337 (91-522)

25

45

52 (35)

Toxic

11, 15, 19, 23

0/8

-1,11 (21)

525 (298-882)

19

30

Saline

b

ACCEPTED MANUSCRIPT a

Antitumor activity based on % T/C ratios: <15% is highly efficient; 15-45% is moderately efficient; >45% is inactive.

b

Toxicity based on the total body w eight loss above 15% over 1 w eek considered to be excessively toxic according to the national animal care guidelines. 6 Colo205 colon carcinoma implanted s.c. (7.5 x 10 cells) in female NMRI-Nu/Nu. Mouse median w eights: L-OHP (25.4g); Stealth (25.8g); DP(25) (26.4g); DP(40) (26.3g). Abbreviations: L-OHP, oxaliplatin; DP(25), DPPC:DPPG:DSPE- PEG2k (70:25:5mol%); DP(40), DPPC:DPPG:DSPE- PEG2k (55:40:5mol%); DS(40), DSPC:DSPG:DSPE- PEG2k (55:40:5mol%); Stealth, HSPC:Chol:DSPE- PEG2k (57:38:5mol%).

ED

M

AN

US

CR

IP

T

With respect to the tolerability of the formulations, 2 out of 9 mice bearing Colo205 xenografts receiving four injections of a reduced dose of 5 mg/kg of DP(40), continued to display signs of poor tolerance and experienced excessive weight loss during the course of treatment (4 days after third injection and 2 days after the fourth injection, respectively). The less sensitive DP(25) formulation was on the other hand well-tolerated with none of the mice displaying systemic toxicity and was not accompanied by any weight loss (Figure 3F, Table 3). Consistent with the observations in the doseescalation study, a schedule of multiple injections at a reduced dose of 5mg/kg (equivalent to a lipid dose of 50 mg/kg for DP(25) and 58 mg/kg for DP(40), respectively) did not cause hemorrhaging in the skin of treated mice. We therefore suspect that the hemorrhages found in the skin are caused by an excessive extravasation of the particles into the skin of the mice when administering multiple large doses, and this subsequently leads to an activation of the particles by host sPLA2. In support, pegylated particles are known to accumulate in the skin of nude mice, along with the fact that sPLA2 is also present in the skin, where it plays an intricate role in maintaining the integrity of the epidermis[42]. However, further investigations are needed to address whether or not this diffuse bleeding is also implicated in the mortality of the animals.

CE

PT

These results clearly demonstrate an inability in using the highly sensitive DP(40) formulation at effective tumor growth-inhibiting doses due to dose-limiting systemic toxicities, conversely, lowering the sensitivity of the particles towards sPLA2, as seen with DP(25), the formulation becomes welltolerated yet is therapeutically inactive and indistinguishable from liposomes not carrying enzymetriggered release properties. The latter presumably being from a lack in ability to become hydrolyzed inside the tumor to a significant extent.

AC

In order to establish the underlying cause of toxicity of the DP(40) formulation, histological examination was conducted of the liver of the mice. The principle cause of death was determined to be due to multifocal peracute hepatonecrotic lesions. Analyzing the liver of a mouse treated with a single high dose of 10 mg/kg of DP(40) showed no signs of liver damage 4 days after treatment (Figure 4A), while following 3 injections of 8 mg/kg every 4 days necrotic lesions became apparent in the liver (Figure 4B). This illustrates that the cumulative accumulation of the liposomes in the liver causes the toxicity produced in the organ, and thus to the animal. On the other hand, these signs were not observed in mice treated with low sPLA2-sensitive (DP(25)) and non-degradable liposomes (Stealth) (data not shown), despite it is well-known that pegylated liposomes do accumulate in the liver to a high extent. Therefore, we believe the liver damage arises from the combined effect of high liver accumulation of the liposomes along with a high sPLA2-degradability. Since elevated levels of this enzyme is produced by hepatocytes[43], we speculate that activation of these particles from the action of hepatic sPLA2 does occur for the DP(40) formulation, resulting in the release of the encapsulated

ACCEPTED MANUSCRIPT

drug, and at the same time high local concentrations of permeability enhancing components is formed creating the observed degenerative condition. In addition, the secretion of sPLA2 into the hepatic intercellular space is further exacerbated following drug-mediated injury of the hepatic cells, which can then further deteriorate the breakdown of the tissue in a self-perpetuating and vicious cycle[44,45].

B

AN

US

CR

IP

T

A

ED

M

Figure 4. Histological examination of liver after single or multiple dosings of highly sPLA 2-sensitive liposomal LOHP. (A) No damaged regions were observed in the liver 4 days after a single injection of DP(40) at 10mg/kg. (B) Necrotic manifestations (arrows) in the liver following three injections of 8 mg/kg of DP(40) administered every 4 days. Multiple injections of high-sPLA 2-sensitive DP(40) formulations result in multifocal peracute hepatonecrotic lesions. Staining was performed with hematoxylin and eosin.

AC

CE

PT

Even though increasing the sensitivity of the formulation would lower the threshold for when the liposomes can become activated inside the tumor, it does also increase the risk of premature activation of the particles before reaching the cancerous tissue from the effect of deregulated serumlevels of sPLA2 in the diseased state. Serum sPLA2-levels are frequently seen to be elevated in cancer patients (1-14 ng/mL) compared to cancer-free subjects (1-2 ng/mL)[46–48], consequently aberrant serum levels of sPLA2 in combination with excessive sensitivity could potentially cause intravascular drug release and undesirable drug redistribution during circulation. We therefore analyzed the levels of the human isoform of secreted PLA2 that entered into the bloodstream of the mouse host from the human tumor engraft. We show that low amounts (0.26-1.30 ng/mL) of the enzyme do backflow into systemic circulation upon very high tumor burdens (∼1000 mm 3) (Table 2), which could be expected, as sPLA2 is preferentially localized in the border of colon tumors, a region of the tumor densely vascularized, and thus can facilitate systemic access[18,28,49]. Nevertheless, the levels of the enzyme entering into the bloodstream of the mouse corresponded only to the level present in healthy individuals, and therefore we do not consider this to substantially attribute to the toxicity profile of sPLA2-sensitive liposomes observed here. In support, patients treated with the clinical sPLA2formulation, LiPlaCis ®, showed no sign of premature activation during systemic circulation, as no correlation was found between serum levels of sPLA2 and the plasma half-life of the liposomes despite plasma sPLA2 levels were highly variable between the patients[31]. But further investigations are

ACCEPTED MANUSCRIPT

needed to uncover whether or not intravascular activation of highly sensitive formulations does occur and could lead to co-morbidity.

AN

US

CR

IP

T

Taken together, this data illustrate that sPLA2-mediated controlled release from liposomal drug carriers, investigated here for delivering the platinum drug L-OHP, appears to preclinically have a selflimiting effect on the antitumor efficacy by causing inadvertent liver toxicity following systemic administration. The main limitation of this system therefore being the release of membrane-active lysophospholipids and free fatty acids driven by hepatic enzymatic hydrolysis, which in particular affects the usage of highly sensitive liposome formulations designed to facilitate fast local drug release inside tumors, but more so hampers the dosage required to attain therapeutic efficacy. An interesting alternative approach of using lipase-triggered release comes from Esser et al., reporting on the preclinical utility of integrin-targeting Sn-2 lipase labile prodrugs for targeted delivery of antiangiogenic fumagillin to tumors[50]. The combination of targeted delivery and consequent internalization and intracellular hydrolysis of the prodrug could therefore from the perspective of a rational design effectually confine the release of cell lytic component to individual target cells, and thereby act as a safeguard against uncontrollable systemic activation.

4. Conclusions

CE

PT

ED

M

We did not find evidence of increased drug efficacy could be obtained by using a sPLA2-triggered drug release mechanism with high sensitivity, compared to low- or non-sensitive liposomes. In contrast, our findings suggest that sPLA2-responsive liposomes instead pose a risk of systemic toxicity from a combined effect of both their sensitivity towards the enzyme and the additive up-concentration of dosed particles accumulating in the liver following repeated dosage. Increasing the amount of PG the liposome membranes to 40% in order to reach a high rate of drug release, also increased the incidence of systemic toxicity and restrict their use for in vivo applications, whereas with 25% PG, despite no detectable systemic toxicity in our preclinical model did not produce higher growth inhibition relative to non-degradable pegylated liposomes.

AC

Overall, the sPLA2-induced drug release strategy suffers from a narrow therapeutic window, where sPLA2-sensitive liposomes are limited by drug-related liver failure whilst low-sensitive formulations, such as the clinically used LiPlaCis®, lack an actively sPLA2-controlled released mechanism, thus behaving therapeutically as conventional non-degradable pegylated formulations. This raises concerns if LiPlaCis® and similar formulations using sPLA2 as a target can benefit cancer patients.

Supplementary material In vitro HT-29 antiproliferative effect, flow cytometry cell uptake study, leakage of L-OHP from liposomes in serum at 37ºC, time-lapse of sPLA2-sensitive liposomes in the absence of sPLA2, image of petechial cutaneous hemorrhages in the skin of treated mice.

ACCEPTED MANUSCRIPT

Acknowledgments

T

This work was supported by the NaSTaR grant from ERC and the Lundbeck Foundation. Authors would like to thank Nanna Bild for her contribution to the design of the illustration used in TOC Figure, and Linda Magnusson for technical assistance in engrafting tumor xenografts in the murine models.

IP

References

K. Knop, R. Hoogenboom, D. Fischer, U.S. Schubert, Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives., Angew. Chem. Int. Ed. Engl. 49 (2010) 6288– 308. doi:10.1002/anie.200902672.

[2]

J.C. Kraft, J.P. Freeling, Z. Wang, R.J.Y. Ho, Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems., J. Pharm. Sci. 103 (2014) 29– 52. doi:10.1002/jps.23773.

[3]

T. André, C. Boni, L. Boudiaf-Mounedji, M. Navarro, J. Tabernero, T. Hickish, C. Topham, M. Zaninelli, P. Clingan, J. Bridgewater, I. Tabah-Fisch, A. de Gramont, Oxaliplatin, Fluorouracil, and Leucovorin as Adjuvant Treatment for Colon Cancer, N. Engl. J. Med. 350 (2004) 2343– 2351.

[4]

C. Yang, Z. Fu, PEG-liposomal oxaliplatin combined with nuclear factor-κB inhibitor (PDTC) induces apoptosis in human colorectal cancer cells, Oncol. Rep. 32 (2014) 1617–1621. doi:10.3892/or.2014.3336.

[5]

G. Charest, L. Sanche, D. Fortin, D. Mathieu, B. Paquette, Optimization of the route of platinum drugs administration to optimize the concomitant treatment with radiotherapy for glioblastoma implanted in the Fischer rat brain, J. Neurooncol. 115 (2013) 365–373. doi:10.1007/s11060013-1238-8.

[6]

A. Nagao, A.S.A. Lila, T. Ishida, H. Kiwada, Abrogation of the accelerated blood clearance phenomenon by SOXL regimen: Promise for clinical application, Int. J. Pharm. 441 (2013) 395– 401. doi:10.1016/j.ijpharm.2012.11.015.

[7]

S. Zalba, I. Navarro, I.F. Trocóniz, C. Tros De Ilarduya, M.J. Garrido, Application of different methods to formulate PEG-liposomes of oxaliplatin: Evaluation in vitro and in vivo, Eur. J. Pharm. Biopharm. 81 (2012) 273–280. doi:10.1016/j.ejpb.2012.02.007.

[8]

G.P. Stathopoulos, T. Boulikas, A. Kourvetaris, J. Stathopoulos, Liposomal Oxaliplatin in the Treatment of Advanced Cancer: A Phase I Study, Anticancer Res. 26 (2006) 1489–1493.

[9]

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. doi:10.1016/j.plipres.2004.12.001.

[10]

A.M. Ponce, Z. Vujaskovic, F. Yuan, D. Needham, M.W. Dewhirst, Hyperthermia mediated liposomal drug delivery., Int. J. Hyperthermia. 22 (2006) 205–13.

AC

CE

PT

ED

M

AN

US

CR

[1]

ACCEPTED MANUSCRIPT

doi:10.1080/02656730600582956. M.B. Yatvin, J.N. Weinstein, W.H. Dennis, R. Blumenthal, Design of liposomes for enhanced local release of drugs by hyperthermia, Science (80-. ). 202 (1978) 1290–1293.

[12]

J.P. May, M.J. Ernsting, E. Undzys, S.-D. Li, Thermosensitive liposomes for the delivery of gemcitabine and oxaliplatin to tumors, Mol. Pharm. 10 (2013) 4499–4508. doi:10.1021/mp400321e.

[13]

N.M. Idris, M.K. Gnanasammandhan, J. Zhang, P.C. Ho, R. Mahendran, Y. Zhang, In vivo photodynamic therapy using upconversion nanoparticles as remote-controlled nanotransducers, Nat. Med. 18 (2012) 1580–1585. doi:10.1038/nm.2933.

[14]

R. Satchi, T.A. Connors, R. Duncan, PDEPT: polymer-directed enzyme prodrug therapy, Br. J. Cancer. 85 (2001) 1070–1076.

[15]

M.T. Basel, T.B. Strestha, D.L. Troyer, S.H. Bossmann, Liposomes : A Method for Making Highly Targeted Liposomes Using, ACS Nano. 5 (2011) 2162–2175.

[16]

T.L. Andresen, J. Davidsen, M. Begtrup, O.G. Mouritsen, K. Jørgensen, Enzymatic release of antitumor ether lipids by specific phospholipase A2 activation of liposome-forming prodrugs., J. Med. Chem. 47 (2004) 1694–703. doi:10.1021/jm031029r.

[17]

E. Oude Blenke, E. Mastrobattista, R.M. Schiffelers, Strategies for triggered drug release from tumor targeted liposomes., Expert Opin. Drug Deliv. 10 (2013) 1399–410. doi:10.1517/17425247.2013.805742.

[18]

L. Tribler, L.T. Jensen, K. Jørgensen, N. Brünner, M.H. Gelb, H.J. Nielsen, S.S. Jensen, Increased expression and activity of group IIA and X secretory phospholipase A2 in peritumoral versus central colon carcinoma tissue, Anticancer Res. 27 (2007) 3179 – 3185.

[19]

S.-I. Yamashita, J.-I. Yamashita, K. Sakamoto, K. Inada, Y. Nakashima, K. Murata, T. Saishoji, K. Nomura, M. Ogawa, Increased expression of membrane-associated phospholipase A2 shows malignant potential of human breast cancer cells, Cancer. 71 (1993) 3058–3064. doi:10.1002/1097-0142(19930515)71:10<3058::AID-CNCR2820711028>3.0.CO;2-8.

[20]

T. Abe, K. Sakamoto, H. Kamohara, Y. Hirano, N. Kuwahara, M. Ogawa, Group II phospholipase A2 is increased in peritoneal and pleural effusions in patients with various types of cancer., Int. J. Cancer. 74 (1997) 245–250. doi:10.1002/(SICI)10970215(19970620)74:3<245::AID-IJC2>3.0.CO;2-Z.

[21]

H. Kiyohara, H. Egami, H. Kako, Y. Shibata, K. Murata, S. Ohshima, K. Sei, S. Suko, R. Kurano, M. Ogawa, Immunohistochemical localization of group II phospholipase A2 in human pancreatic carcinomas., Int. J. Pancreatol. 13 (1993) 49–57. doi:10.1007/BF02795199.

[22]

J. Jiang, B.L. Neubauer, J.R. Graff, M. Chedid, J.E. Thomas, N.W. Roehm, S. Zhang, G.J. Eckert, M.O. Koch, J.N. Eble, L. Cheng, Expression of group IIA secretory phospholipase A2 is elevated in prostatic intraepithelial neoplasia and adenocarcinoma., Am. J. Pathol. 160 (2002) 667–71. doi:10.1016/S0002-9440(10)64886-9.

[23]

O.G. Berg, M.H. Gelb, M.-D. Tsai, M.K. Jain, Interfacial Enzymology: The Secreted

AC

CE

PT

ED

M

AN

US

CR

IP

T

[11]

ACCEPTED MANUSCRIPT

Phospholipase A 2 -Paradigm, Chem. Rev. 101 (2001) 2613–2654. doi:10.1021/cr990139w. L. Linderoth, P. Fristrup, M. Hansen, F. Melander, R. Madsen, T.L. Andresen, G.H. Peters, Mechanistic study of the sPLA2-mediated hydrolysis of a thio-ester pro anticancer ether lipid., J. Am. Chem. Soc. 131 (2009) 12193–200. doi:10.1021/ja901412j.

[25]

T.T.T.N. Nguyen, J. Østergaard, S. Stürup, B. Gammelgaard, Investigation of a liposomal oxaliplatin drug formulation by capillary electrophoresis hyphenated to inductively coupled plasma mass spectrometry (CE-ICP-MS), Anal. Bioanal. Chem. 402 (2012) 2131–2139. doi:10.1007/s00216-011-5651-6.

[26]

U. Franzen, C. Vermehren, H. Jensen, J. Ostergaard, Physicochemical characterization of a PEGylated liposomal drug formulation using capillary electrophoresis, Electrophoresis. 32 (2011) 738–748. doi:10.1002/elps.201000552.

[27]

U. Franzen, T.T.T.N. Nguyen, C. Vermehren, B. Gammelgaard, J. Østergaard, Characterization of a liposome-based formulation of oxaliplatin using capillary electrophoresis: Encapsulation and leakage, J. Pharm. Biomed. Anal. 55 (2011) 16–22. doi:http://dx.doi.org/10.1016/j.jpba.2010.12.037.

[28]

S.S. Jensen, T.L. Andresen, J. Davidsen, P. Høyrup, S.D. Shnyder, M.C. Bibby, J.H. Gill, K. Jørgensen, Secretory phospholipase A(2) as a tumor-specific trigger for targeted delivery of a novel class of liposomal prodrug anticancer etherlipids, Mol. Cancer Ther. 3 (2004) 1451 – 1458.

[29]

N.D. Quach, R.D. Arnold, B.S. Cummings, Secretory phospholipase A2 enzymes as pharmacological targets for treatment of disease., Biochem. Pharmacol. 90 (2014) 338–48. doi:10.1016/j.bcp.2014.05.022.

[30]

J. Davidsen, C. Vermehren, S. Frokjaer, O.G. Mouritsen, K. Jørgensen, Drug delivery by phospholipase A2 degradable liposomes, Int. J. Pharm. 214 (2001) 67–69. doi:10.1016/S03785173(00)00634-7.

[31]

M.J. a de Jonge, M. Slingerland, W.J. Loos, E. a C. Wiemer, H. Burger, R.H.J. Mathijssen, J.R. Kroep, M. a G. den Hollander, D. van der Biessen, M.-H. Lam, J. Verweij, H. Gelderblom, Early cessation of the clinical development of LiPlaCis, a liposomal cisplatin formulation., Eur. J. Cancer. 46 (2010) 3016–21. doi:10.1016/j.ejca.2010.07.015.

[32]

A. Chonn, P.R. Cullis, D. V Devine, THE ROLE OF SURFACE CHARGE IN THE ACTIVATION OF THE CLASSICAL AND ALTERNATIVE PATHWAYS OF COMPLEMENT BY LIPOSOMES, (1991) 4234–4241.

[33]

M.J. Hope, M.B. Bally, G. Webb, P.R. Cullis, Production of large unilamellar vesicles by a rapid extrusion procedure: characterization of size distribution, trapped volume and ability to maintain a membrane potential., Biochim. Biophys. Acta. 812 (1985) 55 – 65.

[34]

K.M. Saari, V. V. Aho, V. Paavilainen, T.J. Nevalainen, Group II PLA2 Content of Tears in Normal Subjects, Invest. Ophtalmol. Vis. Sci. 42 (2001) 318 – 320.

[35]

H.P. Wacklin, F. Tiberg, G. Fregneto, R.K. Thomas, Distribution of reaction products in phospholipase A2 hydrolysis., Biochim. Biophys. Acta. 1768 (2007) 1036 – 1049.

AC

CE

PT

ED

M

AN

US

CR

IP

T

[24]

ACCEPTED MANUSCRIPT

K. Jørgensen, J. Davidsen, O.G. Mouritsen, Biophysical mechanisms of phospholipase A2 activation and their use in liposome-based drug delivery, FEBS Lett. 531 (2002) 23–27. doi:10.1016/S0014-5793(02)03408-7.

[37]

Y.-L. Kim, Y.-J. Im, N.-C. Ha, D.-S. Im, Albumin inhibits cytotoxic activity of lysophosphatidylcholine by direct binding., Prostaglandins Other Lipid Mediat. 83 (2007) 130–8. doi:10.1016/j.prostaglandins.2006.10.006.

[38]

D.M. McDonald, P. Baluk, Significance of Blood Vessel Leakiness in Cancer, Cancer Res. 62 (2002) 5381–5385.

[39]

R.K. Jain, Delivery of molecular and cellular medicine to solid tumors., Adv. Drug Deliv. Rev. 64 (2012) 353–365. doi:10.1016/j.addr.2012.09.011.

[40]

B.P. Kennedy, P. Payette, J. Mudgett, P. Vadas, W. Pruzanski, M. Kwan, C. Tang, D.E. Rancourt, W.A. Cromlish, A Natural Disruption of the Secretory Group II Phospholipase A2 Gene in Inbred Mouse Strains, J. Biol. Chem. 270 (1995) 22378–22385. doi:10.1074/jbc.270.38.22378.

[41]

L. Li, B. Ahmed, K. Mehta, R. Kurzrock, Liposomal curcumin with and without oxaliplatin: effects on cell growth, apoptosis, and angiogenesis in colorectal cancer., Mol. Cancer Ther. 6 (2007) 1276–82. doi:10.1158/1535-7163.MCT-06-0556.

[42]

S. Gurrieri, G. F??rstenberger, A. Schadow, U. Haas, A.G. Singer, F. Ghomashchi, J. Pfeilschifter, G. Lambeau, M.H. Gelb, M. Kaszkin, Differentiation-dependent regulation of secreted phospholipases A2 in murine epidermis, J. Invest. Dermatol. 121 (2003) 156–164. doi:10.1046/j.1523-1747.2003.12315.x.

[43]

R.M. Crowl, T.J. Stoller, R.R. Conroy, C.R. Stoner, Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response, J. Biol. Chem. 266 (1991) 2647–2651.

[44]

P.B. Limaye, U.M. Apte, K. Shankar, T.J. Bucci, A. Warbritton, H.M. Mehendale, Calpain released from dying hepatocytes mediates progression of acute liver injury induced by model hepatotoxicants, Toxicol. Appl. Pharmacol. 191 (2003) 211–226. doi:10.1016/S0041008X(03)00250-3.

[45]

M. Ito, Y. Ishikawa, H. Kiguchi, K. Komiyama, M. Murakami, I. Kudo, Y. Akasaka, T. Ishii, Distribution of type V secretory phospholipase A2 expression in human hepatocytes damaged by liver disease, J. Gastroenterol. Hepatol. 19 (2004) 1140–1149. doi:10.1111/j.14401746.2004.03435.x.

[46]

K.F. Scott, M. Sajinovic, J. Hein, S. Nixdorf, P. Galettis, W. Liauw, P. de Souza, Q. Dong, G.G. Graham, P.J. Russell, Emerging roles for phospholipase A2 enzymes in cancer., Biochimie. 92 (2010) 601–10. doi:10.1016/j.biochi.2010.03.019.

[47]

M. Menschikowski, A. Hagelgans, S. Fuessel, O. a Mareninova, V. Neumeister, M.P. Wirth, G. Siegert, Serum levels of secreted group IIA phospholipase A(2) in benign prostatic hyperplasia and prostate cancer: a biomarker for inflammation or neoplasia?, Inflammation. 35 (2012) 1113–8. doi:10.1007/s10753-011-9418-1.

AC

CE

PT

ED

M

AN

US

CR

IP

T

[36]

ACCEPTED MANUSCRIPT

A. Buhmeida, R. Bendardaf, M. Hilska, J. Laine, Y. Collan, M. Laato, K. Syrjänen, S. Pyrhönen, PLA2 (group IIA phospholipase A2) as a prognostic determinant in stage II colorectal carcinoma, Ann. Oncol. . 20 (2009) 1230–1235. doi:10.1093/annonc/mdn783.

[49]

I. Edhemović, M. Snoj, A. Kljun, R. Golouh, Immunohistochemical localization of group II phospholipase A2 in the tumours and mucosa of the colon and rectum., Eur. J. Surg. Oncol. 27 (2001) 545–8. doi:10.1053/ejso.2001.1134.

[50]

A.K. Esser, A.H. Schmieder, M.H. Ross, J. Xiang, X. Su, G. Cui, H. Zhang, X. Yang, J.S. Allen, T. Williams, S.A. Wickline, D. Pan, G.M. Lanza, K.N. Weilbaecher, Dual-therapy with αvβ3targeted Sn2 lipase-labile fumagillin-prodrug nanoparticles and zoledronic acid in the Vx2 rabbit tumor model, Nanomedicine. 12 (2016) 201–211. doi:10.1016/j.nano.2015.10.003.

AC

CE

PT

ED

M

AN

US

CR

IP

T

[48]