Efficacy of multi-functional liposomes containing daunorubicin and emetine for treatment of acute myeloid leukaemia.

European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

Research paper

Efficacy of multi-functional liposomes containing daunorubicin and emetine for treatment of acute myeloid leukaemia Lene Myhren a, Ida Mostrøm Nilssen a,1, Valérie Nicolas b, Stein Ove Døskeland a, Gillian Barratt c, Lars Herfindal a,d,⇑ a

Department of Biomedicine, University of Bergen, Bergen, Norway Université Paris-Sud11, Plateforme d’Imagerie Cellulaire, IFR141-IPSIT, FR 92296, Chatenay-Malabry, France Insitut Galien Paris-Sud (UMR CNRS 8612), Université Paris-Sud 11, LabEx LERMIT, FR 92296, Chatenay-Malabry, France d Translational Signalling Group, Haukeland University Hospital, Bergen, Norway b c

a r t i c l e

i n f o

Article history: Received 2 January 2014 Accepted in revised form 8 April 2014 Available online xxxx Compounds studied in this article: Daunorubicin (CID: 30323) Doxorubicin (CID: 31703) Emetine (CID: 10219) Methotrexate (CID: 126941) Keywords: Leukaemia Daunorubicin Doxorubicin Emetine Methotrexate Folic acid

a b s t r a c t Despite recent advances in chemotherapy against acute myeloid leukaemia (AML), the disease still has high mortality, particularly for patients who tolerate extensive chemotherapy poorly. Nano-formulations have potential to minimise the adverse effects of chemotherapy. We present here a liposomal formulation encapsulating both the anthracycline daunorubicin (DNR) and emetine (Eme) for enhanced cytotoxic effect against AML cells. Eme could be loaded into the PEGylated liposomes together with DNR by the acid precipitation principle, with a loading efficiency of Eme at about 50% of that of DNR. The liposome surface was modified with folate to enhance drug loading into cells, giving higher cytotoxic activity. Both intracellular drug loading and cytotoxic activity could be further increased by anti-folate treatment of AML cells with methotrexate (MTX). The combination of DNR and Eme also increased drug loading in MTXtreated cells compared to DNR alone. Liposomes with both DNR and Eme were particularly efficient against AMLs with deficient p53. In conclusion, we have produced a multi-functional liposomal anti-leukaemic drug formulation designed to overcome some of the problems in anthracycline chemotherapy: (1) Combination of DNR and Eme to diminish drug resistance. (2) Using PEGylated stealth liposomes to minimise adverse side-effects. (3) Molecules on the liposomal surface target proteins on AML-cells ensure selectivity, which was enhanced by priming the leukaemia cells with MTX. Ó 2014 Published by Elsevier B.V.

1. Introduction Acute myeloid leukaemia (AML) is a hematopoietic stem cell disorder that causes excessive proliferation and rapid accumulation of myeloid precursor cells in the bone marrow. If left untreated, death occurs within weeks or months after diagnosis. AML is a heterogeneous disease with regard to disease progression and therapy response. For the sub-type acute promyelocytic leukaemia (APL, WHO classification, 2008) differentiation therapy Abbreviations: AML, Acute myeloid leukaemia; DDS, Drug delivery system; DNR, Daunorubicin; DOX, Doxorubicin; Eme, Emetine; FA, Folate; FR2, Folic acid receptor 2 (b); HEPC, Hydrogenated egg phosphatidylcholine; MTX, Methotrexate; PE, Phosphatidylethanolamine; PEG, Polyethylene-glycol; PSMA, Prostate specific membrane antigen. ⇑ Corresponding author. Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway. Tel.: +47 55 58 63 81; fax: +47 55 58 63 60. E-mail address: lars.herfi[email protected] (L. Herfindal). 1 Present address: Bergen Hospital Pharmacy, P.O. Box 1, 5021 Bergen, Norway.

with all-trans retinoic acid, often in combination with cytostatics such as arsenic trioxide and daunorubicin (DNR) has proven successful [10]. However, for most AML patients the primary treatment regime still involves high doses of chemotherapeutics such as the cell cycle-specific inhibitor cytarabine (Ara-C) in combination with the cell cycle-unspecific inhibitor anthracycline DNR [28,39]. Complete remission is reached in about 60% of AML patients between 60 and 70 years old, with 2-year disease-free survival of about 30%, while for patients above 70 years, the outcome is even worse. [25,31]. For patients younger than 60 years, complete remission is reached in 79% of patients [31]. However, the relapse risk is in the range of 45–50% in older patients, and overall, AML is the leading cause of death due to leukaemia with a 5-year relative survival below 20% [4,19,25]. There is thus a need to develop treatments with improved therapeutic efficiency that is tolerable also for weak and elderly patients. The search for novel AML therapy regimes made us undertake a study to investigate whether protein synthesis inhibitors such as

http://dx.doi.org/10.1016/j.ejpb.2014.04.002 0939-6411/Ó 2014 Published by Elsevier B.V.

Please cite this article in press as: L. Myhren et al., Efficacy of multi-functional liposomes containing daunorubicin and emetine for treatment of acute myeloid leukaemia, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.04.002

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L. Myhren et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

emetine could improve the efficacy of standard anthracycline therapy [16]. This was based on findings that AML cells show enhanced translation of mRNA coding for survival proteins upon DNR exposure [16]. As with all chemotherapy, the combination therapy DNR/protein synthesis inhibitor could include the risk of toxic side effects in normal tissue and organs. Thus, we needed to find a drug delivery system (DDS) that could spare normal tissue from excessive chemotherapy exposure, but still deliver the drugs to the AML cells. Liposomal DDS are attractive tools to overcome problems associated with chemotherapy-induced toxicity by targeting the drug to the tumour cells, and diverting it away from normal tissue, thereby increasing the therapeutic window. Doxorubicin-loaded liposomes (DoxilÒ/CaelyxÒ) are used against solid tumours to reduce cardiotoxicity [35], and many other anti-cancer liposomal formulations are in clinical trials (see [1] for a recent review). Liposomes have the advantage that drugs can be loaded during or after production, allowing loading of several drugs with different chemical properties into the same nanocarrier. In AML, the short pulses of high doses daunorubicin pose a threat to the patients due to bone marrow depletion [3] and intestinal mucositis [7]. Interestingly, the presence of p53 appears to rescue normal tissue, rather than increasing the toxic effects [18]. Thus, by designing a therapy that selectively attacks p53-deficient cells, we can minimise toxic side-effects. As a result of the protection of exposure afforded by the liposomes, it is possible to increase the therapeutic window. We wanted to create a formulation that allowed for the intracellular delivery of DNR and a protein synthesis inhibitor in one DDS. Emetine was chosen as the protein synthesis inhibitor since it has relatively equal toxic profile across the species [34] compared to cycloheximide, where the LD50 varies from 1 to 2 mg/ kg in dog and rat to 130 mg/kg in mouse [22,37]. Also, Eme is already used against protozoal infections in man [26] and approved for use in humans. We also wanted to target leukaemia cells through liposome internalisation via the folate receptor 2 (FR2) [41], and aimed to increase drug delivery by further stimulating FR2-expression in AML cells. In this study, we present a multifunctional liposomal DDS loaded with DNR and Eme for enhanced AML-cell toxicity, its surface being modified with PEG for ‘‘stealth’’ effect, and FA for targeting. Furthermore, we prove increased drug delivery to AML cells exposed to anti-folate therapy.

was next stirred at 60 °C for 20 min before extrusion at 70 °C (LIPEX™ extruder; Northern Lipids, Burnaby, Canada) through membranes (Whatman, Kent, UK) with pore size of 0.8 lm (five times), 0.4 lm (five times) 0.2 lm (double membranes, ten times) and 0.1 lm (double membranes, ten times). The liposomes were then passed through a column with G25medium Sephadex (Amersham, Uppsala, Sweden) and eluted in PBS, pH 8.0. Empty liposomes were first run through the column to avoid unspecific binding of liposomes to the gel. The liposomes were loaded with drugs based on the acid-precipitation concept [11,13]. In brief, DNR (Cerubidine, 20 mg DNR and 100 mg mannitol, Sanofi Aventis Lysaker, Norway) and emetine (Eme, Sigma–Aldrich, St. Louis, MO), were added to the liposome suspension at a ratio of 1:10 drugs:lipids (w/w). In some formulations, doxorubicin (DOX, Accord Healthcare, Gothenburg, Sweden) was used instead of DNR. DOX is the preferred anthracycline for solid tumours, whereas DNR and sometimes idarubicin (IDA) is used against AML. The liposomes and drugs were incubated for 1 h at 60 °C before gel filtration to remove non-encapsulated drug. Size and zeta potential was measured by dynamic light scattering (DLS) using a Zetasizer (Malvern Instruments Ltd. Malvern, UK). For measurement of size, the liposomes were diluted in the suspension buffer (PBS or 250 mM (NH4)2SO4) and measured three times. To measure zeta potential of loaded and unloaded liposomes, the suspension was diluted 1:10 in isotonic (5%) sucrose, since dilution with PBS gave very inconsistent results even between measurements of the same liposomal preparations. Under these conditions, we obtained similar size measurements to those recorded using PBS as suspension buffer. 2.2. Quantification of liposomal drug content by HPLC

2. Materials and methods

Liposome suspensions or solutions of DNR or Eme for standard curves were evaporated under vacuum and the residue dissolved in 0.15 ml 3:1 0.1% aqueous TFA:MeOH. The solution was next injected into a reversed phase HPLC column (Kromasil 100-5 C18 250-4.6 mm, Akzo Nobel, Sweden) connected to a Merck-Hitachi LaChrome HPLC-system (VWR, WestChester, USA) with a L-7455 diode array detector. Mobile phase A was 3:1 0.1% aqueous TFA:MeOH, and mobile phase B was acetonitrile. The flow rate was 1.0 ml/min and Eme was eluted during a 13 min isocratic mode with 100% A followed by a gradient to 100% B during 6 min, where DNR eluted. Standard curves from 0.005 to 0.12 mg/ml DNR or Eme were prepared, and drug content in liposomes quantified by integration of the peaks at 280 nm (Eme) and 480 nm (DNR).

2.1. Production of liposomes

2.3. Cell maintenance and experimental conditions

Liposomes were prepared with hydrogenated egg phosphatidylcholine (HEPC, Lipoid EPC-3, T12508 from Lipoid KG, Ludwigshafen, Germany), cholesterol (Sigma, La Jolla, CA), and PEG-ylated distearoyl phosphatidylethanolamine (PEG-PE, PEG molecular weight 2000 Da, Avanti Polar Lipids, Alabaster, Alabama, US). All lipids were dissolved in chloroform in a molar ratio of 1.81:1:0.15, HEPC:Chol:PEG-PE, and a film was made by evaporation of the lipids. Liposomes carrying folate (FA) lipids were prepared by adding distearoyl-glycero-phosphoethanolamine-NPEG5000-folate, PE-PEG-FA, Avanti Polar Lipids) at one tenth of the molar concentration of PEG-PE. Fluorescent liposomes were made by adding Rhodamine-labelled PE (Rh-PE, Molecular Probes) at concentration of one per cent of total PC. The film was rehydrated in warm (60 °C) 250 mM (NH4)2SO4 pH 6.5 at 0.5 mg lipids/ml. Between 10 and 15 glass beads (1 mm diameter) were added to facilitate the hydration by vortexing at maximum intensity until the film was totally hydrated. The sample

The NB4 [20], HL-60 (ATCC No.: CCL-240) and Molm-13 [27,33] leukaemia cell lines and the LNCaP (ATCC No.: CRL-1740) prostate cancer cells were cultured in RPMI 1640 medium enriched with 10% foetal bovine serum (FBS, Invitrogen, Carlsbad, CA). The MV4-11 cells were cultured in Iscove’s medium added 8 mM L-glutamine and 10% FBS. All cell lines were cultured in media supplemented with 100 IU/ml penicillin and 100 mg/ml streptomycin (both from Cambrex, Belgium) in a humidified atmosphere (37 °C, 5% CO2). All culture media were from Sigma (Sigma, La Jolla, CA). p53 knocked down Molm-13 cells (Molm-13 shp53) were generated by retroviral transfection for stable expression of shRNA against p53 [29]. The leukaemia cell lines represent different subclasses of AML (acute promyelocytic leukaemia (NB4 [20]), acute monocytic leukaemia with the poor prognostic factor FLT3 internal tandem duplication (Molm-13 [27]) and the commonly used HL-60 AML cell line, as well as cells with and without inducible p53 (Molm-13 and Molm-13 shp53).



For cytotoxicity studies, cells were seeded at 0.35 106/ml. The cells were exposed to the different liposomal formulations for 30– 60 min to have a stringent test for improved efficacy due to binding to FR2 or synergism from drug combinations. Moreover, in an in vivo situation, a bolus injection of liposomes would have a limited life-time in the circulation even though the liposomes are PEGylated to avoid macrophage-mediated elimination. After 30 or 60 min incubation with liposomes, the cells were washed in PBS and further incubated for 24 h before fixation in 2% formaldehyde supplemented with 2 lM DNR to visualise DNA. DNR was used to visualise DNA instead of the more common DNA probe Hoechst 33342 due to competition of binding sites in DNA between the two molecules. Cells treated with DNR or other anthracyclines will have weak or absent Hoechst 33342 staining, and we therefore enriched the fixative with additional DNR to obtain sufficient DNR fluorescence (red) to visualise DNA. Cell death was determined by microscopic evaluation of nuclear morphology. At least 200 cells were counted for each experimental parallel. Toxicity data from counting of cells with apoptotic morphology were supplemented with the WST-1 assay for metabolic activity (Roche Diagnostics, Mannheim, Germany) or CellTiter-GloÒ Luminescent Cell Viability Assay (Promega Biotech AB, Nacka, Sweden). Both assays measure metabolically active cells. The metabolic assays and counting of dead cells gave consistent data. In some experiments, cells were treated with 10 nM MTX for 2 or 3 days prior to the experiments. For study of cellular uptake of liposomes and drug by confocal microscopy, LNCaP cells (80,000/ml) were seeded on sterilised coverslips in 24-well tissue culture plates 24 h before the experiment. The formulations were added, and the cells were washed with PBS at the given time, and fixed in 2% buffered formaldehyde and left in the dark at room temperature for at least 2 h before mounting in vectashield. Confocal microscopy of LNCaP cells was done on an inverted LSM 510-Meta (Carl Zeiss, Oberkochen, Germany) using a PlanApochromat 63X/1.4 objective lens equipped with helium neon laser (543 excitation wavelength) and a 560 nm long pass emission filter. Images were acquired with the LSM 510 software (ver. 3.2). Quantification of fluorescence intensity in LNCaP cells was with the ImageJ software (http://rsbweb.nih.gov/ij/). Between 4 and 7 0.41 lm optical slices were stacked using median intensity projection, and the mean intensity of the cytoplasm or nucleus was measured. Molm-13 cells treated with 10 nM MTX for 3 days were seeded at a density of 0.35 106/ml on poly-L lysine coated coverslips before treatment with liposomes for 60 min and then washed in PBS before being mounted on glass slides and images acquired as for the LNCaP. 2.4. Immunoblotting Protein lysates were prepared from cells by lysis in RIPA buffer supplemented with Complete mini protease inhibitor (Roche Diagnostics, Mannheim, Germany). The relative protein concentration was determined by Bradford protein assay and immunoblotting was preformed as described [15]. Primary antibodies against FR2 and actin were from Abcam (Cambridge, UK) and secondary alkaline-phosphatase-conjugated antibodies (a-3687 and a-3562) were from Sigma. CDP-Star substrate was from Tropix (Bedford, MA, USA). Chemiluminescence was detected using a Luminescent Image Analyser Apparatus (LAS 3000, FujiFilm, Tokyo, Japan) and Image Gauge Software (FujiFilm, Tokyo, Japan). 2.5. Flow cytometry The uptake of liposomes by MOLM-13 cells (at 0.35 106/ml) was examined by flow cytometry on a FACS Accuri C6 (BD

3

Biosciences, San Jose, CA, USA, Accuri Cytometry). Cells were exposed to various liposomal formulations and incubated for given periods of time, before being washed in culture medium and analysed by flow cytometry. At least 10,000 non-gated events were collected and the DNR content was determined after excitation at 488 nm and sampling in the FL-1 channel. Data were analysed using AccuriC6 Software (BD Biosciences) or FlowJo Software (ver. 7.6.3, Tree Star Inc, Ashland, OR). Drug loading was quantified as either the population of cells with fluorescence intensity above that of non-treated cells, or as median fluorescence intensity of the cells gated for size (FSC).

3. Results 3.1. Emetine can be loaded into liposomes by acid precipitation The post-loading acid precipitation technique is well established for anthracyclines [11,13]. To know whether this was feasible for the protein synthesis inhibitor emetine, we performed an in silico prediction of its net charges as a function of pH (Fig. 1A). Both Eme and DNR had close to zero net charge at pH above 8, and were protonated at pH below 6. We expected therefore that the acid-precipitation technique would be suitable for loading Eme into liposomes. We reached about 55% loading efficiency of Eme, and more than 90% drug loading efficacy for DNR when adding 0.1 mg of drug/mg lipid. If we added DNR and Eme simultaneously, the loading efficiency of each drug was slightly lower (Fig. 1B). We could also manipulate the ratio of Eme:DNR in the liposomes by adding less Eme (Fig. 1B), which could be an important feature to control Eme toxicity in in vivo studies. The size (z-average) and zeta potential of the liposomes are shown in Table 1. The size did not change dramatically during loading, but we noted that the zeta potential increased after loading. This suggests that some drug was present on the outside of the liposomes. We did not observe flocculation of the liposomes during 1 month storage at +4 °C in the dark.

3.2. Folate on the liposomal surface increase uptake, drug loading and cytotoxicity in prostate cancer and AML cells To enhance AML cell drug uptake, we included FA-conjugated PE-PEG in the lipid composition. The presence of FA on the surface did not affect liposomal size, zeta potential or drug loading (Table 1, Fig. 1 and data not shown). We next wanted to find whether FA on the liposomal surface could increase liposomal uptake and drug loading in cells expressing FA-binding surface receptors such as PSMA and FR2. We first tested the PSMA-expressing LNCaP cells, and found that when FA was present on the liposome surface, there was a significant increase in liposomal uptake (Fig. 2A and B), drug delivery into the cells (Fig. 2C and D), and also ability to induce cell death (Fig. 2E). The intracellular drug distribution seen in Fig. 2C appears inconsistent with what was seen in AML cells in Fig. 5B. However, the LNCaP cells were fixed before microscopy, which could have caused leakage of DNR from endocytotic vesicles and binding to the DNA. We next tested the ability to induce cell death in FR2expressing Molm-13 AML cells. These also showed increased death when treated with FA-coated liposomes (Fig. 2F). We noted, however, that the targeting efficiency decreased after the liposomes had been stored at 4 °C in the dark for more than 3 weeks, possibly due to loss of FA, since the liposomes had the same size distribution and drug content at this time (data not shown).


4


B Emetine Daunorubicin

Net charge

2 1 0 -1 -2 4

5

6

7

8 9 pH

Drug content (µg/ml liposome solution)

A

10 11 12

80

Emetine

Daunorubicin

160

60

120

40

80

20

40 0

0 EME

1:1

2:1 5:1 DNR:Eme

Drug formulation (ratio)

DNR

1:1

2:1 DNR:Eme

5:1

Drug formulation (ratio)

Fig. 1. Loading of liposomes with emetine and daunorubicin. A: In silico predictions of net charge of emetine and daunorubicin as a function of pH. B: Content of Eme or DNR after acid precipitation of the drugs alone or in combination into folate-bearing liposomes. The drug content is given as lg drug/ml liposome solution. See Materials and methods for details on computational settings, liposomal loading and estimation of drug content. The data in B are average of 4 independent liposomal formulations and s.e.m.

Table 1 Size (z-average) and zeta potential of liposomes measured by dynamic light scattering. Size (nm)

Zeta potential (mV) Loaded (DNR + Eme)1

Unloaded

Without FA With FA

Unloaded

Z-ave

PDI

Z-ave

PDI

122 125

0.04 0.04

121 127

0.03 0.06

26 26

Loaded1 DNR

Eme

13 13

15 13

DNR + Eme 10 12

1 The drug content of loaded was: DNR + Eme: 95 lg DNR and 50 lg Eme/ml liposome suspension. Eme: 62 lg/ml liposome suspension. DNR: 110 lg/ml liposome suspension.

3.3. The combination of emetine and daunorubicin is particularly efficient towards cells with depleted p53

3.4. Increase in liposomal therapy in AML cells after anti-folate therapy and by emetine itself

Our previous study showed that combination of protein synthesis inhibitors and anthracyclines had synergistic activity towards AML cell lines in vitro and in vivo [16]. We tested therefore a panel of AML cell lines for sensitivity to the liposomes loaded with DNR, Eme or the combination of these. We found that liposomes with DNR and Eme had higher efficacy than those with only DNR in NB4 cells (Fig. 3A), but not HL-60 cells (not shown). However, Molm-13 cells responded better to the combination of emetine with either anthracycline DOX (Fig. 3D) or DNR (not shown). An interesting finding was that p53-silenced Molm-13 cells [17,29] responded better to the liposomes containing both DNR and Eme than Molm-13 with wt-p53 (Fig. 3C). It thus appears that the combination of DNR and Eme targets p53-deficient cell lines, which could be an advantage in cancer therapy. We found that giving liposomes loaded with both drugs was more efficient than giving a mixture of single drug loaded liposomes (Fig. 4A), suggesting that there was a limit for the amount of material that can be internalised by endocytosis during a given period of time. However, we observed a higher efficacy if we added the different liposomes sequentially, first DNR-loaded liposomes followed by Eme-loaded liposomes 30 min later (Fig. 4B). The original research showed that the combination of protein synthesis inhibitor and DNR had the best anti-leukaemic effect if the protein synthesis inhibitor was added about 30 min after DNR [16]. Interestingly, the increase in efficacy from sequential incubation was higher in cells with functional p53 that those with silenced p53 (Fig. 4B). We conclude that DNR and Eme loaded liposomes with folate on the surface have an enhanced capacity for death-induction in the Molm-13 AML cell line compared with liposomes without folate, or liposomes loaded with DNR alone. Also, delayed addition of Eme appears to increase cell death induction, particularly in cells with functional p53 (Fig. 4B).

We wanted to know whether it was possible to enhance uptake of liposomes by inducing FR2 expression on AML cells. This can be achieved in vitro by culturing the cells in medium free of folate, but this approach is difficult to translate into the clinic. Another approach was to use anti-folate therapy like methotrexate (MTX). MTX is already used against acute leukaemia subclasses such as acute lymphoblastic leukaemia [30]. We found that treatment with doses as low as 3 nM for 3 days increased FR2 expression in Molm13 cells (Fig. 5A). These cells showed enhanced drug loading (Fig. 5B and C), which was ascribed to liposomal uptake due to the cytoplasmic distribution of the daunorubicin fluorescence seen in live cells (Fig. 5D). As well as an MTX-dependent drug uptake in Molm-13 cells, we found that more DNR was internalised from liposomes loaded with both DNR and Eme, compared to liposomes loaded with DNR only (Fig. 5C and E). This was observed whether assessed by median fluorescence intensity (Fig. 5E) or percentage cells gated above maximum intensity in non-treated cells (Supplementary Fig. S1). In line with enhanced uptake, we also found that Molm-13 (Fig. 5F) and MV4-11 AML cells (not shown) primed with MTX were more sensitive to the liposomes, with a higher percentage apoptotic cells.

4. Discussion In this study, we demonstrate the production and in vitro efficacy of a multi functional liposomal formulation for anti-leukaemic therapy. The liposomes have PEG on their surface to avoid rapid uptake by phagocytic cells, but also folate (FA) to be recognised and internalised by leukaemia cells. Furthermore, they are loaded with two anti-leukaemic drugs, DNR and Eme, which together have shown enhanced therapeutic effect in AML models [16]. Finally, we


5


Untreated

PEG

FA-PEG 3000

Rhodamine

Mean fluorescence intensity in the cytoplasm

Surface

A

B

2500 2000 1500 1000 500 0

Untreated PEG 3000

Red fluorescence (DNR)

Surface

Mean fluorescence intensity in the nucleus

C

80 60

PEG

LNCaP prostate cancer cells Liposomes without FA

1500 1000 500

DNR Eme

PEG

FA-PEG

F Molm-13 AML cells

Liposomes with FA

40

50

Liposomes without FA

Liposomes with FA

DNR Eme

40 30 20 10

20 0

2000

Untreated

FA-PEG

Apoptotic cells (%)

Apoptotic cells (%)

100

D

2500

0

Untreated

E

FA-PEG

0 2.0 2.0 Drug content (µg/ml)

0.5

0.2

0.5

0.2

Drug content (µg/ml)

Fig. 2. Folate on the surface enhances liposomal uptake, drug loading and cell death induction in prostate and AML cells. A: LNCaP cells were incubated with PEGylated rhodamine-labelled liposomes with or without folate (FA) on their surface for 15 min, fixed and processed for confocal microscopy as described in the methods section. The median intensity projection of ten optical slices (0.41 lm thickness) was done, and the mean intensity visualised by hot red colouring in the ImageJ software. B: Quantification of mean fluorescence intensity in cells treated with the different liposomes. C: LNCaP cells were incubated with DNR-loaded PEGylated liposomes with or without FA for 15 min, fixed and processed for confocal microscopy as described in the Methods section. The fluorescence images are twenty 0.41 lm slices superimposed with median intensity in the ImageJ software. D: Mean fluorescence intensity in the nuclei of cells treated with the different liposomes measured with the ImageJ software. The data in B and D are the mean of 10–13 cells and s.e.m. The bars in A and C represent 10 lm. E and F: LNCaP (E) or Molm-13 AML cells (F) were incubated with drug-loaded liposomes for 30 min before washing and further incubation for 72 h (LNCaP) or 24 h (Molm-13). The cells were then fixed and apoptosis was assessed as described in the Methods section. The data are average of 3–4 experiments from different liposomal preparations, and s.e.m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

can prime the leukaemia cells to enhance drug loading and efficacy. Although DNR has been used as an anti-leukaemic drug for decades [30], its success is often dependent on combination with drugs such as cytarabine to overcome AML [28]. Eme was originally an anti-protozoal drug, but with limited clinical success [26]. Recently, Eme has been evaluated against several cancers [12,21], and has anti-tumour activity at nanomolar concentrations. We previously showed that DNR and protein synthesis inhibitors could have synergistic effect against several AML cell lines,

particularly when the inhibitor was presented 30–60 min after the anthracycline [16]. We could reproduce this effect if liposomes containing Eme were added 30 min after DNR-loaded liposomes. Nevertheless, liposomes loaded with both drugs were more efficient (Figs. 3 and 4), although the two drugs are presumably delivered simultaneously in this case. We also tested different timing for addition of liposomes, and found that liposomes containing both DNR and Eme were more efficient than giving two liposomal formulations simultaneously, one containing DNR and one Eme (Fig. 4A). This finding could be explained by a saturation point for the endocytotic pathway.



A

Surface

Liposomes with DNR

20

10

D

Nucleus

Empty liposomes

30 Apoptotic cells (%)

B

NB4 DNR (DNR+Eme) 10:1 (DNR+Eme) 2:1

Apoptotic cells (%)

6

Molm-13 p53 wt cells sh-p53 silenced

70 60

Eme DOX

50

(DOX+Eme)

40 30 20 10

0 DNR Eme

0.5 –

0.5 0.5 0.05 0.25

1 –

1

wt

C

1

shp53

0.1 0.5

Actin

Drug content in medium (μg/ml)

DNR

0 DOX Eme

p53

0.5 0.5

–

1

1

– 0.25

0.5

–

0.5

0.5 0.5 – 0.25

–

1

1

0.5

–

0.5

Drug content in medium (μg/ml)

−

+

− +

Fig. 3. Emetine potentiates anthracycline-induced cell death in AML cell lines. A: NB4 cells were treated with different liposomal formulations for 60 min before washing, incubation for another 24 h, and then assessed for apoptosis. B: Morphology of Molm-13 cells treated with liposomes containing DNR and Eme. The cells were fixed in 2% buffered formaldehyde (pH 7.4) enriched with additional DNR to visualise DNA as described in detail in the methods section. The bars represent 10 lm. C: Immunoblotting of p53 of Molm-13 wt and shp53 cells before and after treatment with DNR, which induces p53 transcription. D: Molm-13 cells with (wt) or without (shp53) inducible p53 were treated with different liposomal formulations for 60 min before washing, incubation for another 24 h, and then assessed for apoptotic cells. The data are the mean and s.e.m. of 3–5 separate experiments using different liposomal preparations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

A

B

70

100 Apoptotic cells (%)

Apoptotic cells (%)

60 50 40 30 20 10 0 DNR Eme

0.5 0.5 0.5

1

– 0.25 0.25

1

1

– 0.5 0.5

wt

shp53

*

*

80 60 40 20

0 DNR Eme

1.0 0.5

1.0 0.5

Drug content in medium (μg/ml) (DNR) (DNR+Eme) (DNR) + (Eme)

(DNR+Eme) (DNR) + (Eme) after 30 min

Fig. 4. Sequential delivery of liposomal anthracycline and emetine enhance efficacy. A: Molm-13 cells with silenced p53 (shp53) were given liposomes loaded with daunorubicin (DNR) and emetine (Eme), or liposomes loaded with the two drugs separately. The cells were incubated with the liposomal formulations for 30 min before washing and another 24 h incubation before fixation in formaldehyde. Cell viability was assessed by microscopy of nuclear morphology. B: Molm-13 cells with or without silenced p53 were incubated with liposomes containing DNR and emetine for 30 min before washing, or with liposomes containing DNR for 30 min, washed and thereafter with liposomes containing Eme for 30 min followed by another wash and incubation for another 24 h. The cells were then fixed and assessed for apoptosis as described under A. The data in A are the average of two separate experiments; B is average of three separate experiments and s.e.m. The asterisks indicate p < 0.05 (t-test).

The fluorescent drug (DNR) of the liposomes shows a punctate cytoplasmic pattern (Fig. 5B) resembling endocytotic vesicles, as expected, since binding to FR2 induces internalisation through endocytosis [23,24]. It is therefore probable that the main effect of MTX-priming in our study is to enhance FR2 expression (Fig. 5A), and thereby the endocytotic potential through the FR2 receptor. It appears that Eme-inclusion promotes internalisation of the liposomes, even after only five min incubation (Fig. 5E), which presumably is too rapid to be explained by inhibition of protein synthesis. We noted that liposomes loaded with drug had higher zeta-potential compared to empty liposomes (Table 1), and it could

be that Eme alters the liposomal surface in a favourable way with respect to endocytosis. Still, the enhanced uptake cannot alone explain the increased efficacy of combining DNR and Eme in one DDS, since sequential addition of liposomes with single drugs enhanced efficacy (Fig. 4B). In our system, cells with deficient p53 respond better to the combination of drugs than those with functional p53 (Fig. 3). An interesting finding is that p53 appears to repress gene expression of folate carriers [8]. This could explain why the Molm-13 cells with silenced p53 are more sensitive to the FA-liposomes. Also, p53-competent cells responded better to the sequential incubation of liposomes, underlining the importance of timing of the drugs. High doses of intravenous liposomes could have detrimental effects. It has been shown that repeated doses can lead to altered clearance of microorganisms [38] and of subsequently injected doses of liposomes [32]. This is less prevalent with PEG-decorated liposomes but even with these it has been noted that the first dose is cleared more effectively than later doses [2,14]. This can lead to new toxicities: the liposomal speciality Caelyx/Doxil can induce the so-called ‘‘hand-foot’’ syndrome as a result of accumulation in the skin [6]. Still, liposomal formulations combining DNR and Cytarabine (Ara-C) against acute leukaemia are now in clinical trials [9] underlining the potential of liposomes as drug carriers. To minimise side-effects from the liposomes themselves, it would be of interest to create a formulation containing both Eme and DNR, from which Eme is released intracellularly at a later timepoint than DNR. This could reduce the burden compared to sequential administration of two different liposomal formulations. The anthracycline-Eme liposomal formulation could also have potential towards other cancer types. We demonstrated that doxorubicin (DOX) could be loaded together with Eme using the same conditions as DNR. In contrast to DNR, which is mostly used against leukaemias, DOX is used against several solid cancers (for a review on DOX and side effects, see [36]). We achieved successful drug targeting by FA-modifications of the liposome surface, also against PSMA-expressing LNCaP cells (Fig. 2A–E). The LNCaP cells have functional p53 [5] and showed a poor response to the combination of DOX and Eme. The p53-deficient PC3-prostate cancer cell line which does not express PSMA [40] responded well to the combination when given in the medium (Supplementary Fig. S2). We thus conclude that also non-leukaemic cancer cells with both FAbinding receptors and p53 deficiency may be promising targets for the present formulation.



7

A kDa 37

DNR (2μg/ml)

B

C

DNR (2μg/ml) +Eme (0.5μg/ml)

FR2

25

Actin MTX (nM)

D

0

3

10

30

Red fluorescence (DNR)

Surface

Frequency

600

MTXtreated

Untreated

MTXtreated

Untreated

400

200

0 103

104

105

103

104

105

Fluoresence intensity (DNR uptake)

Fluorescence intensity (a.u.)

E 12000

5 min

30 min

60 min *

*

10000

**

8000

* #

6000 4000 2000 0 DNR

2

DNR:Eme DNR:Eme DNR:Eme

2 : 0.3

2 : 0.6

2:1

DNR

2


2 : 0.3

2 : 0.6

2:1

DNR

2


2 : 0.3

2 : 0.6

2:1

Liposomal formulation (μg drug / ml cell suspension) Control

MTX-treated cells

Apoptotic cells (%)

F 60

** 40

*

20 0

DNR:Eme 0.5 : 0.08

DNR:Eme 0.5 : 0.25

DNR:Eme 1: 0.15

DNR:Eme 1 : 0.5

Liposomal formulation (μg drug / ml cell suspension) Fig. 5. Methotrexate (MTX) increased folate receptor 2 expression, drug loading and efficacy in Molm-13 AML cells. A: Molm-13 cells were incubated with the given doses of MTX or vehicle for 3 days and immunoblotted for presence of FR2. B–D: Cells were incubated with liposomes loaded with DNR or DNR and Eme for 60 min, and studied by confocal microscopy (B) or flow cytometry (C and D) as described in the Methods section. B is a projection of a stack of five 1 lm optical slices. The bar represents 5 lm. E: Quantification of DNR content in MTX-primed or non-primed Molm-13 cells treated with various liposomal formulations for the given periods of time. The cells were analysed by flow cytometry and the data represent median fluorescence intensity. F: MTX-primed or non-primed Molm-13 cells were treated with the given liposomal formulations for 60 min before washing and further incubation for 24 h. The cells were assessed for apoptosis by microscopy as described in the Methods section. The data are average and s.e.m. from 3 to 4 experiments using different liposomal preparations except # which is one measurement only. Asterisks in E indicate significance at p < 0.05 (*) or 0.01 (**). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

5. Conclusions In conclusion, we have produced a multi-functional liposomal anti-leukaemic drug formulation designed to overcome some of the problems in chemotherapy including drug resistance, by adding Eme to aid cell death induction in p53-deficient cells. Adverse side-effects can be reduced by loading the drugs into ‘‘stealth’’ carriers surface-modified to target surface proteins on cancer cells. Drug delivery into the cancer cells can be enhanced by priming the leukaemia cells with MTX prior to administration of the drug formulation. We thus believe that such a formulation is promising as therapy for a sub-set of chemo-resistant AML,

but further pre-clinical investigations need to be done to fully demonstrate their clinical potential. For example, it will be imperative to demonstrate the efficacy in small animal AML models. By the use of labelled liposomes, it will be possible to follow the fate of the drug carriers, and find whether they home to AML-infiltrated tissues. This can be done with dual label in vivo imaging, using different fluorescent probes for cells and liposomes. However, based on the presented data, a therapeutic regime consisting of priming of the leukaemia cells with antifolate therapy such as MTX, followed by intravenous injection of the liposomal formulation could be used to improve antiAML therapy.


8


Author’s contributions Conception of idea, planning of experiments: LH, GB, SOD, LM. Execution of experiments: LH, LM, IMN, VN. Interpretation of data: LH, IMN, VN, SOD, GB. Preparation of manuscript: LH, LM, SOD, GB. All authors have read and approved the final version of the manuscript.

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