Cetuximab-oxaliplatin-liposomes for Epidermal Growth Factor receptor targeted chemotherapy of colorectal cancer Sara Zalba, Ana M. Contreras, Azadeh Haeri, Timo L.M. ten Hagen, I˜nigo Navarro, Gerben Koning, Mar´ıa J. Garrido PII: DOI: Reference:
S0168-3659(15)00569-6 doi: 10.1016/j.jconrel.2015.05.271 COREL 7687
To appear in:
Journal of Controlled Release
Received date: Revised date: Accepted date:
23 February 2015 8 May 2015 11 May 2015
Please cite this article as: Sara Zalba, Ana M. Contreras, Azadeh Haeri, Timo L.M. ten Hagen, I˜ nigo Navarro, Gerben Koning, Mar´ıa J. Garrido, Cetuximab-oxaliplatinliposomes for Epidermal Growth Factor receptor targeted chemotherapy of colorectal cancer, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.05.271
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 Cetuximab-oxaliplatin-liposomes for Epidermal Growth Factor receptor targeted chemotherapy of colorectal cancer
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Sara Zalba1,2,Ana M. Contreras1, Azadeh Haeri2, Timo L.M. ten Hagen2, Iñigo
1
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Navarro3, Gerben Koning2*, María J. Garrido1*
Department of Pharmacy and Pharmaceutical Technology, University of Navarra,
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31008 Pamplona, Spain; 2Laboratory Experimental Surgical Oncology, Department of Surgery, Erasmus MC Cancer Institute, Rotterdam 3015 GE, The Netherlands; 3
Department of Chemistry and Edaphology, University of Navarra, 31008 Pamplona,
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Spain
*Correspondence to: [email protected] (+31107044055, ext. 43258) [email protected] (+34948425600, ext. 806529)
ED
Abstract
Oxaliplatin (L-OH), a platinum derivative with good tolerability is currently combined
PT
with Cetuximab (CTX), a monoclonal antibody (mAb), for the treatment of certain (wild-type KRAS) metastatic colorectal cancer (CRC) expressing epidermal growth
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factor receptor (EGFR).
Improvement of L-OH pharmacokinetics (PK) can be provided by its encapsulation into
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liposomes, allowing a more selective accumulation and delivery to the tumor. Here, we aim to associate both agents in a novel liposomal targeted therapy by linking CTX to the drug-loaded liposomes. These EGFR-targeted liposomes potentially combine the therapeutic activity and selectivity of CTX with tumor-cell delivery of L-OH in a single therapeutic approach. L-OH liposomes carrying whole CTX or CTX-Fab’ fragments on their surface were designed and characterized. Their functionality was tested in vitro using four human CRC cell lines, expressing different levels of EGFR to investigate the role of CTXEGFR interactions in the cellular binding and uptake of the nanocarriers and encapsulated drug. Next, those formulations were evaluated in vivo in a colorectal cancer xenograft model with regard to tumor drug accumulation, toxicity and therapeutic activity.
ACCEPTED MANUSCRIPT In EGFR-overexpressing cell lines, intracellular drug delivery by targeted liposomes increased with receptor density reaching up to 3-fold higher levels than with nontargeted liposomes. Receptor specific uptake was demonstrated by competition with
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free CTX, which reduced internalization to levels similar to non-targeted liposomes. In
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a CRC xenograft model, drug delivery was strongly enhanced upon treatment with targeted formulations. Liposomes conjugated with monovalent CTX-Fab’ fragments showed superior drug accumulation in tumor tissue (2916.0 ± 507.84 ng/g) compared to
SC
CTX liposomes (1546.02 ± 362.41 ng/g) or non-targeted liposomes (891.06 ± 155.1 ng/g). Concomitantly, CTX-Fab’ targeted L-OH liposomes outperformed CTX-
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liposomes, which on its turn was still more efficacious than non-targeted liposomes and free drug treatment in CRC bearing mice.
These results show that site-directed conjugation of monovalent CTX-Fab’ provides targeted L-OH liposomes that display an increased tumor drug delivery and efficacy
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over a formulation with CTX and non-targeted liposomes.
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CE
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Graphical Abstract
ACCEPTED MANUSCRIPT Keywords: EGFR; targeted liposomes, oxaliplatin, Cetuximab, Fab’, Colorectal cancer PubChem: Oxaliplatin (PubChem CID: 5310940): 3-(2-Pyridyldithio)propionic-acidN-hydroxysuccinimide-ester
(PubChem
CID:
100682):
Tris(2-carboxyethyl)-
perchlorate
(PubChem
CID:
16212735):
β-
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tetramethylindocarbocyanine
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phosphine-hydrochloride (PubChem CID: 2734570): 1,1′-Dioctadecyl-3,3,3’,3’ –
Mercaptoethanol (PubChem CID: 1567): Hydrogenatedphosphatidylcholine (PubChem CID:
94190):
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[Methoxy
SC
(polyethylenglycol)-2000] (PubChem CID: 406952), Cholesterol (PubChem CID:5997): 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[Maleimide (polyethylene glycol)
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2000] (PubChem CID:406950).
Acknowledgements: Funding for this project came from the Spanish Govermment (Instituto de Salud Carlos III, ref: PS09/02512-FISS), Govermment of Navarra (ref: IIQ14334.RI1) and from University of Navarra. Sara Zalba was supported by a grant
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from the Govermment of Navarra. Authors want to thank the Imaging Unit from CIMA
1. Introduction
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(Pamplona, Spain) for their support to this work.
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Platinum derivatives are widely used in cancer chemotherapy. They are used in the chemotherapeutic treatment of approximately 70% of solid tumors, including non-small and small cell lung, breast, colorectal, gastric, pancreatic, esophageal, testicular,
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cervical, ovarian cancers and non-Hodgkin’s lymphoma [1, 2]. L-OH is a third generation platinum derivative used in first line treatment of colorectal cancer (CRC) [3]. It is one of the platinum derivatives with lower toxicity in patients [4]. However, LOH pharmacokinetics is characterized by a high volume of distribution due to its rapid and irreversible binding to erythrocytes and to plasma and tissue proteins. This causes serious limitations to reach desirable unbound drug concentrations in the target tissue as tumor and subsequent therapeutic efficacy [4]. To overcome this, novel strategies aim at modifying L-OH biodistribution, for instance by drug encapsulation in nanocarriers, particularly in liposomes [5]. Liposomal encapsulation causes a strong reduction in the volume of distribution of the loaded drug, a decrease in drug toxicity, prolonged presence of drug in circulation and enhanced tumor accumulation [6].
ACCEPTED MANUSCRIPT Two important aspects of liposomes that contribute significantly to tumor accumulation of entrapped drugs are their small size (usually around 100 nm) and surface pegylation. Both contribute to increase the circulation half-life of this formulation in blood stream
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delaying its caption by the reticulo-endothelial system and thereby, increasing the
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probability to reach the tumor [7]. The latter is promoted by the enhanced permeability and retention effect (EPR) in tumors, caused by an increased vascular permeability in
extravasation and retention at the tumor site.
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this tissue and a lack of lymphatic flow [8], allowing preferential nanoparticle
This selective retention of nanoparticles, together with a specific intracellular drug
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delivery into tumor cells, can be promoted by cell-specific targeting [9]. For this approach, one needs the presence of a specific molecule or ligand in the surface of the liposome able to recognize a receptor or antigen on the cancer cell, promoting the internalization of the complex to reach the intracellular targets. Different types of ligands are in use for nanoparticle targeting, such as vitamins, carbohydrates, growth
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factors, peptides, and monoclonal antibodies [10, 11]. In current cancer therapy, the use of targeted agents is increasing; for example, growth factor receptor inhibitors,
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molecules that regulate cell-signalling and gene expression or inhibitors of angiogenesis [12, 13].
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In recent years, L-OH combined with Cetuximab (CTX), a chimeric human-mouse antibody specific for the epidermal growth factor receptor, is the chemotherapeutic treatment of certain (wild-type KRAS) metastatic CRC expressing EGFR [14, 15].
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Binding of CTX to EGFR leads to the blockade of the receptor followed by a rapid internalization, causing not only a down regulation of receptors at the surface of the cells, but also an inhibition of EGFR, cell proliferation signals triggering of apoptosis and the activation of the immune system to destroy tumor cells [16]. This receptor is over-expressed in many solid tumors including 65-70% of CRC [17, 18]. In this study, we aim to combine L-OH chemotherapy and CTX targeted therapy of CRC by designing a L-OH-loaded liposomal formulation targeted to EGFR on CRC cells. Such an approach combines the L-OH drug delivery advantages of PEGliposomal encapsulation, with tumor cell-specific delivery by CTX on the liposomal surface. Linking whole mAb usually results in random orientation of this molecule on the liposomal surface, which limits antigen or receptor binding site exposure and also yields exposure of Fc’ domains. The latter can promote recognition by Fc’-receptors on
ACCEPTED MANUSCRIPT macrophages and faster liposome clearance from circulation as well as, induce unwanted immune reactions [19]. To overcome this, smaller targeting molecules able to recognize the receptor are coupledbut, they do not display the unwanted immune
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activity [20]. Therefore, CTX-Fab’ fragments, as a selective ligand for EGFR, were
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coupled site-specifically to the liposome. CTX-mAb and CTX-Fab’ L-OH-liposomes were prepared and characterized. Of these formulations, we investigated binding and uptake by human CRC cell lines and intracellular drug delivery. Next, we studied in
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vivo drug delivery to the tumor and therapeutic activity in CRC xenograft bearing mice.
2.1. - Drugs and chemicals
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2. Materials and methods
3-(2-Pyridyldithio)propionic-acid-N-hydroxysuccinimide-ester
(SPDP),
Tris(2-
carboxyethyl)-phosphine-hydrochloride (TCEP),1,1′-Dioctadecyl-3,3,3’,3’–tetramethylindocarbocyanine perchlorate (Dil), Vivaspin tubes (300,000 MWCO) and β-
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Mercaptoethanol were purchased from Sigma (Barcelona, Spain). The mAb CTX (Erbitux®) and L-OH (Eloxatin®) were provided by the University Clinic of Navarre
PT
(Pamplona, Spain). Hydrogenated-phosphatidylcholine (HSPC), 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[Methoxy(polyethylenglycol)-2000] and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[Maleimide
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PEG2000)
(DSPE-
(polyethylene glycol) 2000] (DSPE-PEG2000-Mal), were from Avanti Polar Lipids Inc (Alabaster, Alabama, USA) and Lipoid (Ludwigshafen, Germany) and were kindly
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provided by the Laboratory Experimental Surgical Oncology from the Erasmus MC in Rotterdam (The Netherlands). Cholesterol (CH) and bovine serum albumin (BSA) were purchased from Sigma (Barcelona, Spain). Amicon tubes with membranes of 10,000, 30,000 and 50,000 MWCO were obtained from Millipore Corporation (Billerica, MA, USA). 2.2. - Oxaliplatin liposomes Liposomes of L-OH were developed using the film hydration technique following the methodology previously described by Zalba et al [21]. Briefly, the lipids HSPC:CH:DSPE-PEG2000:DSPE-PEG2000-Mal, in a molar ratio of 1.85:1:0.12:0.03, were dissolved in a solution of chloroform:methanol [9:1 (v/v)]. The solvent was evaporated using a rotary evaporator (Büchi-R144, Switzerland) at 65ºC to obtain a lipid film, which was further dried under vacuum. The film was hydrated with a solution of L-OH (5 mg/ml) in glucose 5%, resulting a final solution of 10 mM of
ACCEPTED MANUSCRIPT lipids. The same procedure was followed to prepare the non-loaded or blank liposomes but adding glucose 5%. The solution was extruded through a 100 nm polycarbonate membrane (Mini-Extruder Set, Avanti Polar Lipids Inc., Alabaster, Alabama, USA) to
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obtain an homogeneous liposome population (LP-N).
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Non-encapsulated L-OH was removed by ultrafiltration at 2,200 g for 60 min using the Amicon system (10,000 MWCO). Liposomes were washed three times with Hepes saline solution (pH 6.7) and the final formulation was stored at 4°C until use.
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In order to prepare labelled liposomes, the fluorescent probe Dil was added to the lipid mixture [1% (w/w)] and dissolved in the solution of chloroform:methanol [9:1 (v/v)]
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together with lipids. The Dil-LP-N preparation procedure was similar to non-fluorescent liposomes. These liposomes were also used to formulate the EGFR targeted liposomes with CTX and Fab’ fragment.
2.3. - Development of immunoliposomes
2.3.1. - Liposomes coupled to Cetuximab
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The method followed to develop the immunoliposomes has been previously described by Songs and coworkers [22]. Briefly, CTX was mixed by orbital shaking with SPDP at
PT
1:12 molar ratio for 1 h at room temperature (RT). Afterwards the excess of SPDP was removed from the mixture by ultrafiltration at 2,200 g for 30 min using the Amicon
CE
system (50,000 MWCO). The ultrafiltered solution was incubated under agitation for 1 h with TCEP (1:625 molar ratio) at RT [23]. These two reactions provided thiolation of CTX, which was next co-incubated with liposomes overnight at 10ºC. The thiol groups
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behave as crosslinker with the maleimide groups of the DSPE-PEG2000-Mal lipid, forming a thioether bond, as is shown in Figure 1. The final formulation was purified by ultrafiltration at 2,200 g for 60 min using the Vivaspin system (300,000 MWCO), followed by a washout process with Hepes saline to remove the non-bound ligand. To prevent the particle aggregation, liposomes were incubated with 1 mM of L-cysteine that quenchs the free maleimide radicals avoiding the formation of disulphide bond [24]. The immunoliposome (LP-CTX) was stored at 4°C until use.
ACCEPTED MANUSCRIPT SPDP NH2
+
TCEP
SC RI PT
Maleimide
+
Pyridine2-thione
Thioether bond
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LP-CTX
Figure 1: Schematic representation of the method used for LP-CTX production
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2.3.2.- Liposomes coupled to Fab’ fragment of Cetuximab To obtain the Fab’ fragment from CTX, several steps were followed. First, the mAb was
D
hydrolized using a pepsin solution (1:20 w/w) prepared in sodium acetate (100 mM, pH 3.7). This process was carried out at 37ºC for 2 h obtaining the (Fab’)2 and the
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crystallizable fragment, Fc’. This Fc’ was removed by ultrafiltration at 2,200 g for 30 min using the Amicon system (50,000 MWCO). Afterwards the collected (Fab’)2
EP
fragments were incubated at 37°C for 30 min with a solution of 15 mM of βMercaptoethanol to obtain single Fab’ fragments, as is shown in Figure 2.These
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molecules were purified by several cycles of ultrafiltration (30,000 MWCO) and washing. To check the isolation process of Fab’ fragments and to verify the presence of the different molecules in each of the steps mentioned (whole mAb, the non-reduced (Fab’)2 fragments and the Fab’ fragment) an 8% gel SDS page was used. The induced hinge-region free thiol group in the structure of Fab’ allowed the conjugation with DSPE-PEG2000-Mal of the liposome by incubation at 10ºC overnight. The elimination of the non-coupled fragments was carried out by three cycles of ultrafiltration in Amicon tubes (50,000 MWCO) and washing with Hepes saline solution. To prevent the instability of the formulation (LP-Fab’), it was incubated with 1 mM of L-cysteine and stored at 4°C until use [24].
ACCEPTED MANUSCRIPT CTX
Fab’ fragments
(Fab’)2 fragment
S—S
β- Mercaptoethanol
pepsin
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Maleimide
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Thioether bond
+
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LP-Fab’
Figure 2: Schematic representation of the method used for LP-Fab' production
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2.3.3. - Optimization of the ligand/lipid ratio
In order to select the most adequate ligand to lipid ratio in each type of formulation, different quantities of ligand per µmol of lipid (5, 10, 20, 30, 40 and 50 µg) were tested for both ligands, CTX and Fab’. In the final formulation, the efficiency of the coupling was measured using the MicroBCATM kit (Thermo Fisher Scientific Inc, Waltham, MA
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USA). The effiency was calculated as the relation between the initial and the final amount of the ligand incorporated in the liposomes.
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In a parallel study, fluorescent liposomes (Dil-LP), previously formulated with different ligand/lipid ratios, were assayed for cell uptake in HCT-116 cell line. This human
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colorectal cancer cell line was purchased from ATCC and maintained at 37°C, 5% CO2 in RPMI containing 10% (v/v) fetal bovine serum and 1% (v/v) of PenicillinStreptomycin.
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For this study, cells were harvested by trypsinization, counted and seeded in 96-well black plates at a density of 15x103 cells/well. One day later, the culture medium was replaced by a fresh medium containing a lipid concentration of 100 μM/well of each type of fluorescent formulations, Dil-LP-CTX and Dil-LP-Fab’. After 24 h of treatment, the plates were washed twice with PBS and the fluorescence was measured (Tecan group Ltd, Maennedorf, Switzerland). The signal corresponding to the internalization with the different formulations was compared to the control basal signal. 2.4. - Characterization of liposomes The particle size and zeta potential of the formulations were analyzed by laser diffractometry using a Zetasizer Nano Series (Malvern Instruments, UK). Formulations were diluted 1:50 (v/v) in deionized water in order to ensure a convenient scattered intensity on the detector. The efficiency of L-OH encapsulation was measured by
ACCEPTED MANUSCRIPT atomic absorption spectrometry. The concentration of the lipid in each formulation was quantified using the phosphate assay method [25]. The efficiency of conjugation for the two ligands was quantified using the MicroBCATM
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kit following the manufacturing instructions.
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2.5. - Evaluation of L-OH release from the liposomal formulations
The release rate of L-OH from liposomes was characterized for the three types of formulations. Aliquots of 1 mL corresponding to 60 μg of L-OH loaded in liposomes
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were incubated at 37ºC in a dialisis tube (12,000 MWCO). The receptor medium consisted of 500 mL of isoosmotic Hepes saline solution under orbital shaking to
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maintain sink conditions during the experiment [26]. Several samples of the receptor medium were collected at 0, 10, 30, 60 min and 4, 7 and 24 h by triplicate and the L-OH quantified by atomic absorption spectrometry.
The influence of serum proteins in the stability of liposomes was assayed by their incubation in presence of 50% FBS (v/v).
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The accumulative release curve was built as the percentage of release (% R) at each assayed time applying the following formula:
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Qa 100 where, Qa represents the amount of L-OH measured in the collected % R Qt
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sample, and Qt, the total amount of the drug at the beginning of the experiment. 2.6. –In vitro studies
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2.6.1. - Cytotoxicity study Four human colon cancer cell lines, HCT-116, HT-29 and SW-480, SW-620 were purchased from ATCC and routinely maintained at standard conditions in RPMI and DMEM respectively, containing 10% (v/v) FBS and a 1% (v/v) of PenicillinStreptomycin. For this study, cells were detached by trypsinization and seeded in 96 well microtiter plates at a density of 5x103 cells/well. Twenty-four hours later, the cells were exposed for 4 h at different concentrations of L-OH, (from 0.1 to 100 µM) free and encapsulated in LP-N, LP-CTX and LP-Fab’. After drug exposure times, cells were rinsed with PBS and new fresh medium was added. Cell viability was measured at 72 h post-treatment using the Neutral Red Assay [27]. Data were expressed as concentration of L-OH that gives a 50% inhibition of cell growth compared to untreated or control cells (IC50). Blank liposomes were also tested under the same conditions. 2.6.2. – Influence of liposomes in the EGFR phosphorylation status
ACCEPTED MANUSCRIPT In order to evaluate the influence of the ligands CTX and Fab’, free or coupled to liposomes in the EGFR phosphorylation status, all formulations were assayed in HCT116 and SW-480 cell lines.
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Cells at a density of 8x105 cells/well were seeded in 6 well plates. Twenty-four hours
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later, cells were exposed for 15 min at the following treatments: 20 ng/ml of EGF, 27 μg/ml of CTX and Fab’, and 1 mM of LP-N, LP-CTX and LP-Fab’. Afterwards cells, washed twice with ice-cold PBS, were treated with 100 μl of RIPA buffer supplemented
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with sodium orthovanadate (1 mM), sodium fluoride (10 mM), β-glycerophosphate (100 mM) and Phosphatase inhibitor cocktail tablets (Roche®, Indianapolis, USA), harvested
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with a cell scrapper and incubated at 4ºC for 30 min. Lysates were centrifuged at 12,500 g for 30 min at 4ºC to collect the supernatant. The content of protein in those samples was quantified using the Micro BCATM kit. Aliquots of 40 μg of protein were loaded on the SDS PAGE (8%) and transferred to a nitrocellulose membrane by wet blotting. This membrane, incubated overnight at 4ºC in Tris buffer solution (TBS) with 1% Tween-20
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and 5% of BSA and the anti-EGFR [phospho Y1068 from abcam®(Cambridge, UK)], was washed and incubated for 1 h at RT with the secondary antibody, an anti-rabbit
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peroxidase-labelled (Cell-signalling, Danvers, USA). Blots were developed using the ECL system Super Signal ULTRA kit (Thermo Fisher Scientific Inc, Waltham, MA
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USA), and immunoreactive proteins were visualized on the high-performance chemiluminescence film (HyperfilmTM, Amersham Bioscience, Piscataway, NJ, USA). -actin was used as control.
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2.6.3. – Cellular uptake of liposomes Cell uptake in four cell lines using fluorescent liposomal formulations LP-N, LP-CTX and LP-Fab’ was characterized at two temperatures, 37 and 4ºC respectively. For this study, cells were seeded in 96 well microtiter black plates at a density of 15x103 cells/well. After 24 h, 100 µM lipid of each formulation was added to the culture medium. Cells collected at different time points, from 0.5 to 24 h, were washed twice with PBS to measure the incorporated fluorescence. In order to investigate the role of the EGFR in the uptake, a parallel study following the above protocol was carried out, but here, cells were pretreated with 100 g/mL of CTX for 1 h at 37ºC before the exposure to liposomes treatment. Additionally, cell uptake with and without CTX pretreatment was also explored by images captured by fluorescence microscopy at 40X magnification. In this experiment, 60,000 cells were
ACCEPTED MANUSCRIPT seeded in culture slides chambers (BD Falcon, Bedford, USA) and incubated for 24 h with 100 μM lipid of each type of formulation. After treatments, cells were fixed in formaldehyde 4% by incubation for 10 min at RT and preserved at 4ºC until analysis.
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Cell membrane was visualized by adding anti–alpha 1 sodium potassium ATPase
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antibody (abcam®, Cambridge, UK), and DNA in nucleus was stained with DAPI. Images were acquired using the Axio Cam MR3 video camera connected to the Zeiss Imager M1 microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with
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epifluorescence optics and Axiovison software (4.6.3.0 version). 2.7. - In vivo study
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Fifty-four female athymic nude mice weighing 20-25 g (Harlan, Barcelona, Spain) were housed in plastic cages under standard and sterile conditions (25°C, 50% relative humidity, 12 hours dark/light), with water and food at libitum. All experiments were performed according to European animal care regulations and the protocol was approved by Ethical comitee of the University of Navarra (075/07).
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A subcutaneous tumor was induced by the inoculation of 5x106 SW-480 cells in 100 μl of PBS, in the right flank of the mice. When the tumors reached aproximately a volume
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of 200 mm3, mice were randomly divided into different groups: control, free L-OH, LPN, LP-Fab’, LP-CTX. Mice were intravenously (i.v.) injected with 2.5 mg/kg dose of L-
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OH every three days during three cycles, as is shown in Figure 3. The corresponding empty formulations were also evaluated in three extra-groups by i.v. administration of
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the equivalent amount of lipids. Tumor size, measured by an electronic caliper, and the body weight were recorded every two days. Tumor volume was calculated according to the following formula: V(mm3)= 4/3π(d2xD/2), where d and D are respectively the smallest and the largest tumor diameters. At the end of the experiment, day 24, all mice were sacrified to remove the liver, spleen and the tumor to quantify the L-OH levels reached in those organs. They were homogenized in 1 mL of a mixture NaCl (150 mM)/HCl (470 mM) using the MiniBeadbeater device (Bioespec Products, Bartlesville, UK), and mixed with 3 mL of HNO3 (35%). Samples were heated for 2 h in a water bath at 65ºC and stored until use. Platinum levels were measured by atomic absorption spectometry technique.
ACCEPTED MANUSCRIPT End of experiment
Tumor inoculation
1st dose
Day 24
Day 18
SC RI PT
Day 12 Day 15
Day 0
3rd dose
Oxaliplatin quantification
2nd dose
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Figure 3. Scheme of the in vivo experimental desing. 2.8.- Statistical methods
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All data were expressed as the mean and standard deviation (SD). The statistical analysis was performed using a non-parametric test, Kruskall-Wallis to compared all treatments followed by the U of Mann Withney test to compare two by two groups. The
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D
significance level was set at p
S0168-3659(15)00569-6 doi: 10.1016/j.jconrel.2015.05.271 COREL 7687
To appear in:
Journal of Controlled Release
Received date: Revised date: Accepted date:
23 February 2015 8 May 2015 11 May 2015
Please cite this article as: Sara Zalba, Ana M. Contreras, Azadeh Haeri, Timo L.M. ten Hagen, I˜ nigo Navarro, Gerben Koning, Mar´ıa J. Garrido, Cetuximab-oxaliplatinliposomes for Epidermal Growth Factor receptor targeted chemotherapy of colorectal cancer, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.05.271
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 Cetuximab-oxaliplatin-liposomes for Epidermal Growth Factor receptor targeted chemotherapy of colorectal cancer
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Sara Zalba1,2,Ana M. Contreras1, Azadeh Haeri2, Timo L.M. ten Hagen2, Iñigo
1
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Navarro3, Gerben Koning2*, María J. Garrido1*
Department of Pharmacy and Pharmaceutical Technology, University of Navarra,
SC
31008 Pamplona, Spain; 2Laboratory Experimental Surgical Oncology, Department of Surgery, Erasmus MC Cancer Institute, Rotterdam 3015 GE, The Netherlands; 3
Department of Chemistry and Edaphology, University of Navarra, 31008 Pamplona,
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Spain
*Correspondence to: [email protected] (+31107044055, ext. 43258) [email protected] (+34948425600, ext. 806529)
ED
Abstract
Oxaliplatin (L-OH), a platinum derivative with good tolerability is currently combined
PT
with Cetuximab (CTX), a monoclonal antibody (mAb), for the treatment of certain (wild-type KRAS) metastatic colorectal cancer (CRC) expressing epidermal growth
CE
factor receptor (EGFR).
Improvement of L-OH pharmacokinetics (PK) can be provided by its encapsulation into
AC
liposomes, allowing a more selective accumulation and delivery to the tumor. Here, we aim to associate both agents in a novel liposomal targeted therapy by linking CTX to the drug-loaded liposomes. These EGFR-targeted liposomes potentially combine the therapeutic activity and selectivity of CTX with tumor-cell delivery of L-OH in a single therapeutic approach. L-OH liposomes carrying whole CTX or CTX-Fab’ fragments on their surface were designed and characterized. Their functionality was tested in vitro using four human CRC cell lines, expressing different levels of EGFR to investigate the role of CTXEGFR interactions in the cellular binding and uptake of the nanocarriers and encapsulated drug. Next, those formulations were evaluated in vivo in a colorectal cancer xenograft model with regard to tumor drug accumulation, toxicity and therapeutic activity.
ACCEPTED MANUSCRIPT In EGFR-overexpressing cell lines, intracellular drug delivery by targeted liposomes increased with receptor density reaching up to 3-fold higher levels than with nontargeted liposomes. Receptor specific uptake was demonstrated by competition with
T
free CTX, which reduced internalization to levels similar to non-targeted liposomes. In
RI P
a CRC xenograft model, drug delivery was strongly enhanced upon treatment with targeted formulations. Liposomes conjugated with monovalent CTX-Fab’ fragments showed superior drug accumulation in tumor tissue (2916.0 ± 507.84 ng/g) compared to
SC
CTX liposomes (1546.02 ± 362.41 ng/g) or non-targeted liposomes (891.06 ± 155.1 ng/g). Concomitantly, CTX-Fab’ targeted L-OH liposomes outperformed CTX-
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liposomes, which on its turn was still more efficacious than non-targeted liposomes and free drug treatment in CRC bearing mice.
These results show that site-directed conjugation of monovalent CTX-Fab’ provides targeted L-OH liposomes that display an increased tumor drug delivery and efficacy
ED
over a formulation with CTX and non-targeted liposomes.
AC
CE
PT
Graphical Abstract
ACCEPTED MANUSCRIPT Keywords: EGFR; targeted liposomes, oxaliplatin, Cetuximab, Fab’, Colorectal cancer PubChem: Oxaliplatin (PubChem CID: 5310940): 3-(2-Pyridyldithio)propionic-acidN-hydroxysuccinimide-ester
(PubChem
CID:
100682):
Tris(2-carboxyethyl)-
perchlorate
(PubChem
CID:
16212735):
β-
RI P
tetramethylindocarbocyanine
T
phosphine-hydrochloride (PubChem CID: 2734570): 1,1′-Dioctadecyl-3,3,3’,3’ –
Mercaptoethanol (PubChem CID: 1567): Hydrogenatedphosphatidylcholine (PubChem CID:
94190):
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[Methoxy
SC
(polyethylenglycol)-2000] (PubChem CID: 406952), Cholesterol (PubChem CID:5997): 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[Maleimide (polyethylene glycol)
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2000] (PubChem CID:406950).
Acknowledgements: Funding for this project came from the Spanish Govermment (Instituto de Salud Carlos III, ref: PS09/02512-FISS), Govermment of Navarra (ref: IIQ14334.RI1) and from University of Navarra. Sara Zalba was supported by a grant
ED
from the Govermment of Navarra. Authors want to thank the Imaging Unit from CIMA
1. Introduction
PT
(Pamplona, Spain) for their support to this work.
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Platinum derivatives are widely used in cancer chemotherapy. They are used in the chemotherapeutic treatment of approximately 70% of solid tumors, including non-small and small cell lung, breast, colorectal, gastric, pancreatic, esophageal, testicular,
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cervical, ovarian cancers and non-Hodgkin’s lymphoma [1, 2]. L-OH is a third generation platinum derivative used in first line treatment of colorectal cancer (CRC) [3]. It is one of the platinum derivatives with lower toxicity in patients [4]. However, LOH pharmacokinetics is characterized by a high volume of distribution due to its rapid and irreversible binding to erythrocytes and to plasma and tissue proteins. This causes serious limitations to reach desirable unbound drug concentrations in the target tissue as tumor and subsequent therapeutic efficacy [4]. To overcome this, novel strategies aim at modifying L-OH biodistribution, for instance by drug encapsulation in nanocarriers, particularly in liposomes [5]. Liposomal encapsulation causes a strong reduction in the volume of distribution of the loaded drug, a decrease in drug toxicity, prolonged presence of drug in circulation and enhanced tumor accumulation [6].
ACCEPTED MANUSCRIPT Two important aspects of liposomes that contribute significantly to tumor accumulation of entrapped drugs are their small size (usually around 100 nm) and surface pegylation. Both contribute to increase the circulation half-life of this formulation in blood stream
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delaying its caption by the reticulo-endothelial system and thereby, increasing the
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probability to reach the tumor [7]. The latter is promoted by the enhanced permeability and retention effect (EPR) in tumors, caused by an increased vascular permeability in
extravasation and retention at the tumor site.
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this tissue and a lack of lymphatic flow [8], allowing preferential nanoparticle
This selective retention of nanoparticles, together with a specific intracellular drug
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delivery into tumor cells, can be promoted by cell-specific targeting [9]. For this approach, one needs the presence of a specific molecule or ligand in the surface of the liposome able to recognize a receptor or antigen on the cancer cell, promoting the internalization of the complex to reach the intracellular targets. Different types of ligands are in use for nanoparticle targeting, such as vitamins, carbohydrates, growth
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factors, peptides, and monoclonal antibodies [10, 11]. In current cancer therapy, the use of targeted agents is increasing; for example, growth factor receptor inhibitors,
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molecules that regulate cell-signalling and gene expression or inhibitors of angiogenesis [12, 13].
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In recent years, L-OH combined with Cetuximab (CTX), a chimeric human-mouse antibody specific for the epidermal growth factor receptor, is the chemotherapeutic treatment of certain (wild-type KRAS) metastatic CRC expressing EGFR [14, 15].
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Binding of CTX to EGFR leads to the blockade of the receptor followed by a rapid internalization, causing not only a down regulation of receptors at the surface of the cells, but also an inhibition of EGFR, cell proliferation signals triggering of apoptosis and the activation of the immune system to destroy tumor cells [16]. This receptor is over-expressed in many solid tumors including 65-70% of CRC [17, 18]. In this study, we aim to combine L-OH chemotherapy and CTX targeted therapy of CRC by designing a L-OH-loaded liposomal formulation targeted to EGFR on CRC cells. Such an approach combines the L-OH drug delivery advantages of PEGliposomal encapsulation, with tumor cell-specific delivery by CTX on the liposomal surface. Linking whole mAb usually results in random orientation of this molecule on the liposomal surface, which limits antigen or receptor binding site exposure and also yields exposure of Fc’ domains. The latter can promote recognition by Fc’-receptors on
ACCEPTED MANUSCRIPT macrophages and faster liposome clearance from circulation as well as, induce unwanted immune reactions [19]. To overcome this, smaller targeting molecules able to recognize the receptor are coupledbut, they do not display the unwanted immune
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activity [20]. Therefore, CTX-Fab’ fragments, as a selective ligand for EGFR, were
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coupled site-specifically to the liposome. CTX-mAb and CTX-Fab’ L-OH-liposomes were prepared and characterized. Of these formulations, we investigated binding and uptake by human CRC cell lines and intracellular drug delivery. Next, we studied in
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vivo drug delivery to the tumor and therapeutic activity in CRC xenograft bearing mice.
2.1. - Drugs and chemicals
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2. Materials and methods
3-(2-Pyridyldithio)propionic-acid-N-hydroxysuccinimide-ester
(SPDP),
Tris(2-
carboxyethyl)-phosphine-hydrochloride (TCEP),1,1′-Dioctadecyl-3,3,3’,3’–tetramethylindocarbocyanine perchlorate (Dil), Vivaspin tubes (300,000 MWCO) and β-
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Mercaptoethanol were purchased from Sigma (Barcelona, Spain). The mAb CTX (Erbitux®) and L-OH (Eloxatin®) were provided by the University Clinic of Navarre
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(Pamplona, Spain). Hydrogenated-phosphatidylcholine (HSPC), 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[Methoxy(polyethylenglycol)-2000] and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[Maleimide
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PEG2000)
(DSPE-
(polyethylene glycol) 2000] (DSPE-PEG2000-Mal), were from Avanti Polar Lipids Inc (Alabaster, Alabama, USA) and Lipoid (Ludwigshafen, Germany) and were kindly
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provided by the Laboratory Experimental Surgical Oncology from the Erasmus MC in Rotterdam (The Netherlands). Cholesterol (CH) and bovine serum albumin (BSA) were purchased from Sigma (Barcelona, Spain). Amicon tubes with membranes of 10,000, 30,000 and 50,000 MWCO were obtained from Millipore Corporation (Billerica, MA, USA). 2.2. - Oxaliplatin liposomes Liposomes of L-OH were developed using the film hydration technique following the methodology previously described by Zalba et al [21]. Briefly, the lipids HSPC:CH:DSPE-PEG2000:DSPE-PEG2000-Mal, in a molar ratio of 1.85:1:0.12:0.03, were dissolved in a solution of chloroform:methanol [9:1 (v/v)]. The solvent was evaporated using a rotary evaporator (Büchi-R144, Switzerland) at 65ºC to obtain a lipid film, which was further dried under vacuum. The film was hydrated with a solution of L-OH (5 mg/ml) in glucose 5%, resulting a final solution of 10 mM of
ACCEPTED MANUSCRIPT lipids. The same procedure was followed to prepare the non-loaded or blank liposomes but adding glucose 5%. The solution was extruded through a 100 nm polycarbonate membrane (Mini-Extruder Set, Avanti Polar Lipids Inc., Alabaster, Alabama, USA) to
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obtain an homogeneous liposome population (LP-N).
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Non-encapsulated L-OH was removed by ultrafiltration at 2,200 g for 60 min using the Amicon system (10,000 MWCO). Liposomes were washed three times with Hepes saline solution (pH 6.7) and the final formulation was stored at 4°C until use.
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In order to prepare labelled liposomes, the fluorescent probe Dil was added to the lipid mixture [1% (w/w)] and dissolved in the solution of chloroform:methanol [9:1 (v/v)]
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together with lipids. The Dil-LP-N preparation procedure was similar to non-fluorescent liposomes. These liposomes were also used to formulate the EGFR targeted liposomes with CTX and Fab’ fragment.
2.3. - Development of immunoliposomes
2.3.1. - Liposomes coupled to Cetuximab
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The method followed to develop the immunoliposomes has been previously described by Songs and coworkers [22]. Briefly, CTX was mixed by orbital shaking with SPDP at
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1:12 molar ratio for 1 h at room temperature (RT). Afterwards the excess of SPDP was removed from the mixture by ultrafiltration at 2,200 g for 30 min using the Amicon
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system (50,000 MWCO). The ultrafiltered solution was incubated under agitation for 1 h with TCEP (1:625 molar ratio) at RT [23]. These two reactions provided thiolation of CTX, which was next co-incubated with liposomes overnight at 10ºC. The thiol groups
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behave as crosslinker with the maleimide groups of the DSPE-PEG2000-Mal lipid, forming a thioether bond, as is shown in Figure 1. The final formulation was purified by ultrafiltration at 2,200 g for 60 min using the Vivaspin system (300,000 MWCO), followed by a washout process with Hepes saline to remove the non-bound ligand. To prevent the particle aggregation, liposomes were incubated with 1 mM of L-cysteine that quenchs the free maleimide radicals avoiding the formation of disulphide bond [24]. The immunoliposome (LP-CTX) was stored at 4°C until use.
ACCEPTED MANUSCRIPT SPDP NH2
+
TCEP
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Maleimide
+
Pyridine2-thione
Thioether bond
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LP-CTX
Figure 1: Schematic representation of the method used for LP-CTX production
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2.3.2.- Liposomes coupled to Fab’ fragment of Cetuximab To obtain the Fab’ fragment from CTX, several steps were followed. First, the mAb was
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hydrolized using a pepsin solution (1:20 w/w) prepared in sodium acetate (100 mM, pH 3.7). This process was carried out at 37ºC for 2 h obtaining the (Fab’)2 and the
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crystallizable fragment, Fc’. This Fc’ was removed by ultrafiltration at 2,200 g for 30 min using the Amicon system (50,000 MWCO). Afterwards the collected (Fab’)2
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fragments were incubated at 37°C for 30 min with a solution of 15 mM of βMercaptoethanol to obtain single Fab’ fragments, as is shown in Figure 2.These
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molecules were purified by several cycles of ultrafiltration (30,000 MWCO) and washing. To check the isolation process of Fab’ fragments and to verify the presence of the different molecules in each of the steps mentioned (whole mAb, the non-reduced (Fab’)2 fragments and the Fab’ fragment) an 8% gel SDS page was used. The induced hinge-region free thiol group in the structure of Fab’ allowed the conjugation with DSPE-PEG2000-Mal of the liposome by incubation at 10ºC overnight. The elimination of the non-coupled fragments was carried out by three cycles of ultrafiltration in Amicon tubes (50,000 MWCO) and washing with Hepes saline solution. To prevent the instability of the formulation (LP-Fab’), it was incubated with 1 mM of L-cysteine and stored at 4°C until use [24].
ACCEPTED MANUSCRIPT CTX
Fab’ fragments
(Fab’)2 fragment
S—S
β- Mercaptoethanol
pepsin
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Maleimide
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Thioether bond
+
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LP-Fab’
Figure 2: Schematic representation of the method used for LP-Fab' production
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2.3.3. - Optimization of the ligand/lipid ratio
In order to select the most adequate ligand to lipid ratio in each type of formulation, different quantities of ligand per µmol of lipid (5, 10, 20, 30, 40 and 50 µg) were tested for both ligands, CTX and Fab’. In the final formulation, the efficiency of the coupling was measured using the MicroBCATM kit (Thermo Fisher Scientific Inc, Waltham, MA
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USA). The effiency was calculated as the relation between the initial and the final amount of the ligand incorporated in the liposomes.
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In a parallel study, fluorescent liposomes (Dil-LP), previously formulated with different ligand/lipid ratios, were assayed for cell uptake in HCT-116 cell line. This human
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colorectal cancer cell line was purchased from ATCC and maintained at 37°C, 5% CO2 in RPMI containing 10% (v/v) fetal bovine serum and 1% (v/v) of PenicillinStreptomycin.
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For this study, cells were harvested by trypsinization, counted and seeded in 96-well black plates at a density of 15x103 cells/well. One day later, the culture medium was replaced by a fresh medium containing a lipid concentration of 100 μM/well of each type of fluorescent formulations, Dil-LP-CTX and Dil-LP-Fab’. After 24 h of treatment, the plates were washed twice with PBS and the fluorescence was measured (Tecan group Ltd, Maennedorf, Switzerland). The signal corresponding to the internalization with the different formulations was compared to the control basal signal. 2.4. - Characterization of liposomes The particle size and zeta potential of the formulations were analyzed by laser diffractometry using a Zetasizer Nano Series (Malvern Instruments, UK). Formulations were diluted 1:50 (v/v) in deionized water in order to ensure a convenient scattered intensity on the detector. The efficiency of L-OH encapsulation was measured by
ACCEPTED MANUSCRIPT atomic absorption spectrometry. The concentration of the lipid in each formulation was quantified using the phosphate assay method [25]. The efficiency of conjugation for the two ligands was quantified using the MicroBCATM
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kit following the manufacturing instructions.
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2.5. - Evaluation of L-OH release from the liposomal formulations
The release rate of L-OH from liposomes was characterized for the three types of formulations. Aliquots of 1 mL corresponding to 60 μg of L-OH loaded in liposomes
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were incubated at 37ºC in a dialisis tube (12,000 MWCO). The receptor medium consisted of 500 mL of isoosmotic Hepes saline solution under orbital shaking to
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maintain sink conditions during the experiment [26]. Several samples of the receptor medium were collected at 0, 10, 30, 60 min and 4, 7 and 24 h by triplicate and the L-OH quantified by atomic absorption spectrometry.
The influence of serum proteins in the stability of liposomes was assayed by their incubation in presence of 50% FBS (v/v).
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The accumulative release curve was built as the percentage of release (% R) at each assayed time applying the following formula:
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Qa 100 where, Qa represents the amount of L-OH measured in the collected % R Qt
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sample, and Qt, the total amount of the drug at the beginning of the experiment. 2.6. –In vitro studies
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2.6.1. - Cytotoxicity study Four human colon cancer cell lines, HCT-116, HT-29 and SW-480, SW-620 were purchased from ATCC and routinely maintained at standard conditions in RPMI and DMEM respectively, containing 10% (v/v) FBS and a 1% (v/v) of PenicillinStreptomycin. For this study, cells were detached by trypsinization and seeded in 96 well microtiter plates at a density of 5x103 cells/well. Twenty-four hours later, the cells were exposed for 4 h at different concentrations of L-OH, (from 0.1 to 100 µM) free and encapsulated in LP-N, LP-CTX and LP-Fab’. After drug exposure times, cells were rinsed with PBS and new fresh medium was added. Cell viability was measured at 72 h post-treatment using the Neutral Red Assay [27]. Data were expressed as concentration of L-OH that gives a 50% inhibition of cell growth compared to untreated or control cells (IC50). Blank liposomes were also tested under the same conditions. 2.6.2. – Influence of liposomes in the EGFR phosphorylation status
ACCEPTED MANUSCRIPT In order to evaluate the influence of the ligands CTX and Fab’, free or coupled to liposomes in the EGFR phosphorylation status, all formulations were assayed in HCT116 and SW-480 cell lines.
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Cells at a density of 8x105 cells/well were seeded in 6 well plates. Twenty-four hours
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later, cells were exposed for 15 min at the following treatments: 20 ng/ml of EGF, 27 μg/ml of CTX and Fab’, and 1 mM of LP-N, LP-CTX and LP-Fab’. Afterwards cells, washed twice with ice-cold PBS, were treated with 100 μl of RIPA buffer supplemented
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with sodium orthovanadate (1 mM), sodium fluoride (10 mM), β-glycerophosphate (100 mM) and Phosphatase inhibitor cocktail tablets (Roche®, Indianapolis, USA), harvested
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with a cell scrapper and incubated at 4ºC for 30 min. Lysates were centrifuged at 12,500 g for 30 min at 4ºC to collect the supernatant. The content of protein in those samples was quantified using the Micro BCATM kit. Aliquots of 40 μg of protein were loaded on the SDS PAGE (8%) and transferred to a nitrocellulose membrane by wet blotting. This membrane, incubated overnight at 4ºC in Tris buffer solution (TBS) with 1% Tween-20
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and 5% of BSA and the anti-EGFR [phospho Y1068 from abcam®(Cambridge, UK)], was washed and incubated for 1 h at RT with the secondary antibody, an anti-rabbit
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peroxidase-labelled (Cell-signalling, Danvers, USA). Blots were developed using the ECL system Super Signal ULTRA kit (Thermo Fisher Scientific Inc, Waltham, MA
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USA), and immunoreactive proteins were visualized on the high-performance chemiluminescence film (HyperfilmTM, Amersham Bioscience, Piscataway, NJ, USA). -actin was used as control.
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2.6.3. – Cellular uptake of liposomes Cell uptake in four cell lines using fluorescent liposomal formulations LP-N, LP-CTX and LP-Fab’ was characterized at two temperatures, 37 and 4ºC respectively. For this study, cells were seeded in 96 well microtiter black plates at a density of 15x103 cells/well. After 24 h, 100 µM lipid of each formulation was added to the culture medium. Cells collected at different time points, from 0.5 to 24 h, were washed twice with PBS to measure the incorporated fluorescence. In order to investigate the role of the EGFR in the uptake, a parallel study following the above protocol was carried out, but here, cells were pretreated with 100 g/mL of CTX for 1 h at 37ºC before the exposure to liposomes treatment. Additionally, cell uptake with and without CTX pretreatment was also explored by images captured by fluorescence microscopy at 40X magnification. In this experiment, 60,000 cells were
ACCEPTED MANUSCRIPT seeded in culture slides chambers (BD Falcon, Bedford, USA) and incubated for 24 h with 100 μM lipid of each type of formulation. After treatments, cells were fixed in formaldehyde 4% by incubation for 10 min at RT and preserved at 4ºC until analysis.
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Cell membrane was visualized by adding anti–alpha 1 sodium potassium ATPase
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antibody (abcam®, Cambridge, UK), and DNA in nucleus was stained with DAPI. Images were acquired using the Axio Cam MR3 video camera connected to the Zeiss Imager M1 microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with
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epifluorescence optics and Axiovison software (4.6.3.0 version). 2.7. - In vivo study
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Fifty-four female athymic nude mice weighing 20-25 g (Harlan, Barcelona, Spain) were housed in plastic cages under standard and sterile conditions (25°C, 50% relative humidity, 12 hours dark/light), with water and food at libitum. All experiments were performed according to European animal care regulations and the protocol was approved by Ethical comitee of the University of Navarra (075/07).
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A subcutaneous tumor was induced by the inoculation of 5x106 SW-480 cells in 100 μl of PBS, in the right flank of the mice. When the tumors reached aproximately a volume
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of 200 mm3, mice were randomly divided into different groups: control, free L-OH, LPN, LP-Fab’, LP-CTX. Mice were intravenously (i.v.) injected with 2.5 mg/kg dose of L-
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OH every three days during three cycles, as is shown in Figure 3. The corresponding empty formulations were also evaluated in three extra-groups by i.v. administration of
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the equivalent amount of lipids. Tumor size, measured by an electronic caliper, and the body weight were recorded every two days. Tumor volume was calculated according to the following formula: V(mm3)= 4/3π(d2xD/2), where d and D are respectively the smallest and the largest tumor diameters. At the end of the experiment, day 24, all mice were sacrified to remove the liver, spleen and the tumor to quantify the L-OH levels reached in those organs. They were homogenized in 1 mL of a mixture NaCl (150 mM)/HCl (470 mM) using the MiniBeadbeater device (Bioespec Products, Bartlesville, UK), and mixed with 3 mL of HNO3 (35%). Samples were heated for 2 h in a water bath at 65ºC and stored until use. Platinum levels were measured by atomic absorption spectometry technique.
ACCEPTED MANUSCRIPT End of experiment
Tumor inoculation
1st dose
Day 24
Day 18
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Day 12 Day 15
Day 0
3rd dose
Oxaliplatin quantification
2nd dose
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Figure 3. Scheme of the in vivo experimental desing. 2.8.- Statistical methods
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All data were expressed as the mean and standard deviation (SD). The statistical analysis was performed using a non-parametric test, Kruskall-Wallis to compared all treatments followed by the U of Mann Withney test to compare two by two groups. The
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significance level was set at p
