Cancer is the second most frequent cause of death [1
]. Treatment of advanced stage diseases is still a major clinical problem despite the availability of a broad range of therapeutic agents with different mechanisms of anticancer activity. The discovery of cisplatin ([cis
-diammine-dichloroplatinum(II) complex]) [3
] laid the foundation for the use of metal-based compounds in the treatment of several cancers, including testicular cancer, ovarian cancer, or breast cancer. Second and third generation platinum (Pt) drugs were developed in order to reduce the dose-limiting toxicity of cisplatin and overcome the intrinsic or acquired cisplatin resistance of cancer cells [4
]. The development of further Pt-based compounds has been the subject of intensive research [5
]. In parallel, alternative metal-based complexes are also considered. Copper (Cu) chelating compounds are primary candidates for development [8
], given the strong in vitro toxicity of copper complexes. Common examples of copper chelating ligands include derivates of dithiocarbamate [11
], 8-hydroxyquinoline [12
], thiosemicarbazone [13
], and phenantroline structures [15
]. These ligands form stable and highly toxic copper complexes with persistent toxicity, even in multidrug resistant cell lines.
The essentiality of copper (Cu) is partially linked to its involvement in basic electron transfer processes through the reduction of Cu(II) to Cu(I)) [16
]. However, the same one electron exchange reaction also generates free radicals that can be harmful to cells [17
]. Cells efficiently protect their intracellular milieu by hiding copper from cellular constituents, thereby preventing the accumulation of free ionic copper. Copper chelator structures can circumvent these defensive processes. The exact mechanism by which copper increases the toxicity of certain chelator compounds is not known [13
]. The toxicity of copper chelating ionophores is usually accompanied by the increased cellular accumulation of copper [18
], which is primarily responsible for cell death [15
]. This is in contrast to the mechanism of toxicity of another group of chelators including tetrathiomolybdate, which kill cells by copper depletion [10
]. Compounds belonging to this second group do not cross cell membranes and are ionic in their chemical nature.
1,10-phenantrolines are typical copper ionofores, and copper complexes of 1,10-phenantrolines show antineoplastic properties [20
]. Both the Cu(I) and Cu(II) complexes of 2,9-dimethyl-1,10-phenantroline (neocuproine) have been shown to be potent cytotoxic agents against different cell lines [21
], and their activity was potentiated by adding Cu(II) ions to the cell culture medium [23
]. The lipophilic Cu(I)-specific chelator neocuproine is frequently used as an inhibitor of Cu-mediated damage in biological systems [26
Drug delivery systems (DDSs), such as liposomes, can be utilized to formulate Cu complexes. Liposomes can be manufactured in various sizes, depending on the desired in vivo behavior, and they can encapsulate a wide range of molecules with hydrophilic, amphiphilic, or lipophilic characteristics. Liposomes with an average diameter of 100 nm have been found to remain in circulation for a significantly longer period than liposomes of a larger size. Most DDSs target malignant tissues rather than healthy organs, even in the absence of a targeting ligand, due to the Enhanced Permeability and Retention (EPR) effect [27
]. The EPR effect is due to the poor lymphatic drainage and the defective blood vasculature of tumor tissues, which results in the extravasation of nanoparticles into the tumor area.
Although many Cu complexes have been synthesized as anticancer agents [9
], their in vivo efficacy remains questionable, and there are currently no promising copper complexes under clinical investigation. The main challenge in the clinical development is the high toxicity and low selectivity of copper complexes. Both selective targeting and toxicity reduction can be achieved by formulation strategies coupled with a homing (targeting) system. Liposomal encapsulation provides a solution to increase the concentration of cytotoxic copper complexes in the tumor, while decreasing the blood concentration and, thus, reducing general toxicity. Active targeting can be achieved by using thermosensitive liposomes and mild hyperthermia to further reduce unwanted toxicity and to enhance drug delivery [29
To date, only a few liposomal formulations containing Cu alone or Cu complex precipitates have been reported [30
]. Different chelators can be loaded into copper-containing systems at different temperatures [33
]. Liposomal formulation was most successful in the case of copper complexes forming precipitates, including clioquinol, quercetin and CX-5461 [33
]. The liposomal formulation of the copper complex of diethyldithiocarbamate showed promising in vivo results in a platinum-resistant A2780-CP ovarian xenograft model [34
Here, we describe the development and biological characterization of a drug delivery system consisting of liposomes loaded with excess copper and the water-soluble active ingredient neocuproine.
2. Materials and Methods
Throughout the experiments, deionized Milli-Q (Millipore, Molsheim, France) water with a resistivity of 18.2 MΩ·cm was used. All of the chemicals were of analytical grade, if not stated otherwise. Copper(II) sulfate, neocuproine (2,9-Dimethyl-1,10-phenanthroline) hydrochloric acid salt, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES sodium salt), ascorbic acid, bovine serum albumin (BSA), and hydrochloric acid were purchased from Sigma Aldrich Ltd. (Budapest, Hungary); NaCl 0.9% was obtained from B. Braun (Budapest, Hungary); chloroform and methanol were purchased from Reanal (Budapest, Hungary). Synthetic high-purity 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), hydrogenated soybean phosphatidylcholine (HSPC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG 2000) were obtained from Avanti Polar Lipids (Alabaster, AL, USA) or Sigma Aldrich. The chemicals were used without further purification. Disposable PD-10 and PD MidiTrap G-25 desalting columns were obtained from GE Healthcare Life Sciences (Budapest, Hungary). Concentrated nitric acid (65% w/w) and H2O2 (30% w/w) of Suprapur quality needed for the sample preparation of cell lines for Total-Reflection X-ray Fluorescence (TXRF) measurement were supplied by Merck (Darmstadt, Germany).
2.2. Preparation of Drug-Loaded Liposomes
Thermo-sensitive and thermo-resistant liposomes were prepared by the lipid film hydration and extrusion method. The lipid mixtures containing DPPC, HSPC and DSPE-PEG2000 in the weight ratios summarized in Table 1
were dissolved in chloroform and dried to a thin lipid film under a stream of N2
gas, followed by incubation overnight under vacuum to remove residual solvent.
Next, 100 mM (or 10 mM, 300 mM for sample LIPO1) of CuSO4
solution was used to hydrate the lipid films to gain a total lipid concentration of 35 mg/3 mL, while mixing with a magnetic stirrer in a 60 °C water bath for 30 min. The resulting multilamellar vesicle (MLV) suspension was subjected to five cycles of freeze-and-thaw (5 min. each, freezing in liquid nitrogen, and thawing at 60 °C) before being extruded 13 times at 60 °C through a 100 nm polycarbonate membrane filter (Whatman, Springfield Mill, UK) via a LIPEX®
Biomembranes, Burnaby, BC, Canada) to generate large unilamellar vesicles (LUVs) encapsulating the Cu salt. Residual un-encapsulated Cu was removed by size exclusion chromatography by passing 2.5 mL liposome suspension through a desalting PD-10 column and eluting with 3.5 mL 100 mM HEPES buffer (pH = 7.8), prepared from Hepes-Na salt, and setting the pH by hydrochloric acid. The pH of the buffer was manually adjusted using S20 SevenEasy™ pH-meter (Mettler Toledo, Budapest, Hungary). The resulting liposomal formulations contained the indicated copper ion solution (typically an unbuffered solution that was at pH 4.5) inside, and the HEPES buffer pH 7.8 outside [35
]. After the separation process, 0.1 M neocuproine hydrochloric acid (dissolved in MQ water) was added in 0.2:1 mol drug-to-mol phospholipid ratio and incubated overnight at room temperature for loading of the neocuproine into the liposome. The unencapsulated neocuproine, or copper-neocuproine complex was removed by loading 1 mL sample on a G-25 Midi-Trap column and eluting the purified liposome fraction with 1.5 mL sterile 0.9% NaCl solution. Dynamic Light Scattering (DLS), Microfluidic Resistive Pulse Sensing (MRPS), Differential Scanning Calorimetry (DSC), IR spectroscopy, and UV-VIS spectrophotometry were used to characterize the prepared liposomes. For the Cu determination, the liposomes were dissolved in methanol and diluted 10-fold by Milli-Q water. The copper content of the samples was measured by the TXRF method (see Section 2.12
) using 1500 ng Ga (Gallium standard solution) as an internal standard. The neocuproine content and encapsulation efficacy of the neocuproine was measured by spectrophotometry. An aliquot (20 μL) was dissolved in 50 μL methanol and then diluted with deionized water to 100 µL to release encapsulated Cu and neocuproine from liposomes. The complex was reduced with ascorbic acid and the absorption of the samples was measured at 450 nm. Calibrating solutions were prepared with the Cu-neocuproine ratio 1:1. The encapsulation efficacy was calculated as a proportion of the neocuproine administered and recovered after column filtration. Neocuproine free liposomes were also prepared for IR study, apart from these, all of the liposomes in the following are drug loaded and PEGylated.
2.3. Dynamic Light Scattering (DLS)
The mean particle diameter and size distribution function of the samples during the preparation procedure were determined using dynamic light scattering (DLS) at 20 °C. DLS measurements performed on a W130i apparatus (Avid Nano Ltd., High Wycombe, UK) and using a low-volume disposable cuvette (UVette, Eppendorf Austria GmbH, Wien, Austria), which was equipped with a diode laser (λ = 660 nm) and a side scatter detector at fixed angle of 90°. For these experiments, the samples were diluted 10-fold with 0.9% NaCl solution, data evaluation was performed with iSIZE 3.0 software (Avid-Nano), utilizing the CONTIN algorithm.
2.4. Microfluidic Resistive Pulse Sensing (MRPS)
Microfluidic resistive pulse sensing (MRPS) is the nanoscale implementation of the coulter principle in a microfluidic cartridge [36
]. The MRPS measurements were performed with a nCS1 instrument (Spectradyne LLC, Torrance, CA, USA). The samples were diluted 500-fold with BSA solution at 1 mg/mL in 0.9% NaCl, filtered through a VivaSpin 500, 300 kDa MWCO membrane filter (Sartorius, Budapest, Hungary), according to the manufacturer’s instructions. All of the measurements were performed using factory calibrated TS-300 cartridges with a measurement range from 50 nm to 300 nm.
2.5. IR Spectroscopy
The IR measurements were carried out using a Varian FTS-2000 (Scimitar Series) FTIR spectrometer (Varian Inc., Agilent, Santa Clara, CA, USA) that was equipped with a ‘Golden Gate’ single reflection diamond attenuated total reflection (ATR) accessory (Specac Ltd., Orpington, UK). 3 µL of preformed copper-neocuproine complex (1:1) or liposome samples were spread on the surface of the diamond ATR element and a thin dry film was obtained by slowly evaporation of the buffer solvent under ambient conditions. A spectral resolution of 2 cm−1 and co-addition of 64 individual spectrum were adjusted. ATR correction and buffer background subtraction were applied for all spectra. Spectral manipulations, including also curve-fitting procedure, were performed by the GRAMS/32 software package. For curve-fitting, band positions were estimated while using the second derivative, and band shapes were approximated by Gauss functions until the minimum of χ2 was reached.
2.6. Differential Scanning Calorimetry (DSC)
DSC was used to characterize thermotropic phase transitions of liposome samples. The measurements were performed on a μDSC 3 EVO (Setaram, Caluire-et-Cuire, France) instrument in the 20 °C to 60 °C temperature range with 0.2 °C/min. heating rate using 300–400 mg of the liposome samples.
2.7. Stability of the Liposomes
In order to test the stability of the liposomes 200 µL samples were centrifuged through Zeba™ Spin Desalting Columns, 40 K MWCO, 0.5 mL (Thermo Fisher, Waltham, MA, USA) after previous washing procedures according to the instructions of the manufacturer. The release of the copper-neocuproine complex from the liposomes was tested at different temperatures (temperature dependence release, each liposome was heated for 10 min by a Biosan (Biosan, Latvia) CH-100 heating block and at different timepoints after the preparation (time dependence of the stability). Absorbance was spectrophotometrically measured at 450 nm (maximal absorbance of Cu(I)-NEO complex) while using an EnSpire microplate reader (Perkin Elmer, Waltham, MA, USA). The data were normalized to unheated or freshly prepared liposomes.
2.8. Size Exclusion Chromatography (SEC)
100 µL liposome sample was incubated with 900 µL completed DMEM medium (containing 10% FBS) for four hours at 37 °C, 5% CO2 modeling cell culture conditions. 10 µL of incubated sample was injected into a Jasco HPLC system (Jasco, Tokyo, Japan) consisting of a PU-2089 pump with an UV-2075 UV/Vis detector and using a Tricorn 5/100 glass columns (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), filled with Sepharose CL-2B (GE Healthcare Bio-Sciences AB) were used, and the eluent was PBS with a flow rate of 0.5 mL/min. The UV-Vis chromatograms were collected at 450 nm wavelength corresponding to the absorption maximum of Cu(I)-neocuproine complex.
2.9. Cell Lines
The following cancer cell lines were used: HT-29 human colon adenocarcinoma and C26 mouse colon carcinoma, both obtained from ATCC (LGC Standards GmbH, Wesel, Germany) and cultured in RPMI and DMEM, respectively. RPMI and DMEM (Sigma Aldrich, Budapest, Hungary) were supplemented with 10% FBS (fetal bovine serum, Gibco, Thermo Fisher), 5 mmol/L glutamine, and 50 unit/mL penicillin and streptomycin (Life Technologies, Carlsbad, CA, USA). The cell cultures were kept in a humidified incubator at 37 °C and in 5% CO2 atmosphere. Washing steps were executed by Dulbecco’s Phosphate Buffered Saline solution (DPBS, GibcoTM, Thermo Fischer Scientific, Waltham, MA, USA), cells were trypsinized by trypsin–EDTA solution (5.0 g/L porcine trypsin and 2.0 g/L EDTA·4 Na in 0.9% v/v sodium chloride solution purchased from Sigma Aldrich).
2.10. Cell Viability Assay
Briefly, the cells were seeded into 96-well tissue culture plates (Sarstedt, Newton, USA/Orange, Braine-l’Alleud, Belgium) at a density of 5000–20,000 cells per well/ 100 µL (depending on the treatment time). The cells were allowed to attach for 12 h. Test compounds were added to achieve the required final concentration in a final volume of 200 μL per well. The liposomes (all investigated liposomes was PEGylated and drug loaded see Section 2.2
) were diluted 10× in the culture media and 100 µL was added to the cells in half dilutions. In the case where the liposomes have been heated, the diluted liposomes were heated in Eppendorf tubes while using a Biosan CH-100 heating block. After incubation (4 h, 24 h, 72 h), the supernatant was removed, cells were washed once with DPBS, and the viability was assessed by the PrestoBlue®
assay (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. The viability of the cells was spectrophotometrically measured (measuring fluorescence, excitation at 544 nm and emission at 590 nm) using an EnSpire microplate reader (Perkin Elmer). The data were normalized to untreated cells. The curves were fitted by Graph Pad Prism 8 software while using the sigmoidal dose–response (variable slope) model. Curve fit statistics were used to determine IC50
2.11. Sample Preparation for Determination of Intracellular Cu Levels
The cells were seeded into six-well culture plates (106 cells/well) in 2 mL media. Cells were incubated overnight, and the medium was changed to 2 mL FBS-free medium before the treatment. The cells were treated with different volumes (2–12 µL) of the liposomes. After a four-hour-long incubation, the cells were harvested by trypsinization. Cells were washed twice with 1 mL DPBS. The cell number was determined with a TC20™ Automated Cell Counter (Bio-Rad Laboratories, Budapest, Hungary) while using trypan blue. After the last centrifugation step, DPBS was completely removed and the cells were digested for 24 h at room temperature in 20 μL of 30% H2O2, 80 μL of 65% HNO3, and 15 μL of 10 μg/mL Ga. From the resulting solutions, 2 μL was pipetted on the quartz reflectors that were used for TXRF analysis.
2.12. Total-Reflection X-Ray Fluorescence (TXRF) Analysis
Cu content was determined by the TXRF method, as reported elsewhere [18
], using an Atomika 8030C TXRF spectrometer (Atomika Instruments GmbH, Oberschleissheim, Munich, Germany). Gallium was used as an internal standard. The stock solution of 1000 mg/L Ga was purchased from Merck (Darmstadt, Germany). The Kα line used for the determination of Cu was at 8.047 keV.
2.13. In Vivo Anti-Tumor Efficacy of Drug-Containing Liposomes
2 × 106 mouse colon carcinoma (C26) cells were injected into the left flank of 6–9 week-old male BALB/c mice from our specific pathogen-free colony subcutaneously (s.c.) in a volume of 0.2 mL serum-free media. Two weeks after injection (when the tumors were measurable), the mice were randomly and evenly divided into groups (10 mice/group). Treatment groups received 10 µL liposome/1g body weight intravenously (i.v.) on the first and eighth day of treatment. The concentration of the complex (Cu:neocuproine) was taken as 1 mM, resulting in an amount of 2.8 mg/kg of “active ingredient” calculated for the thermosensitive formulation. The dose of the encapsulated drug for the “thermoresitant” formulation was 2.6 mg/kg. The controls received equivalent volumes of sterile NaCl 0.9%. All of the animals were included in the analysis. Changes of the body weight were also determined throughout the study. No adverse events were observed during the experiment. The antitumor effects were registered by measuring tumor size with caliper twice a week. Tumor volume was calculated with the formula for a prolate ellipsoid (length × width2 × (π/6)). Eighteen days after the first treatment, the mice were euthanized, and the tumors were extracted after the experiments and then dried in an oven to a constant weight. The samples were weighed on an analytical balance. Statistical analysis was performed using Graph Pad Prism 8 software using One-Way ANOVA analyses. The animal experiments were carried out at the Department of Experimental Pharmacology, National Institute of Oncology, Budapest, Hungary, and the animal-model experiments were conducted following the standards and procedures approved by the Animal Care and Use Committee of the National Institute of Oncology, Budapest (license number: PEI/001/2574–6/2015). The Hungarian Animal Health and Animal Welfare Directorate approved all animal protocols according to the EU’s directives.
The tumors were heated to test the temperature dependent release of the liposomes in two treated animal groups. Animals were treated under anesthesia with desflurane (9% desflurane in 30% oxygen/air). Local mild hyperthermia (40–42 °C) was performed under anesthesia with a custom-made contact heating device based on direct heat conduction while using a metal rod that was connected to a temperature-controlled water bath. Intratumoral temperature was measured with optical sensors (Luxtron FOT Lab Kit, LumaSense Technologies, Inc. CA Santa Clara, CA, USA) and kept within 41–42 °C (±0.5 °C) for 20 min. The applied temperature (41–42 °C) is above the phase transition temperature of the thermosensitive liposome (HEAT SENS LIPO).
2.14. Radiolabeling of Liposomes with Cu-64 for In Vivo PET Imaging
1 mL extruded liposome sample containing HSPC (9 mg/3 mL), DPPC (21 mg/3 mL), and DSPE-PEG2000 (5 mg/3 mL) hydrated with 100 mM non-radioactive CuSO4
solution was first purified on a G-25 Midi-Trap column to remove non-entrapped Cu2+
ions. The purified liposome fraction was eluted with 1.5 mL sterile 0.9% NaCl solution and it was subsequently mixed with 200 μL (400 to 450 MBq) no carrier added [64
(produced on the cyclotron TR-FLEX, Advanced Cyclotron Systems, Inc., Richmond, BC, Canada, at the Helmholtz-Zentrum Dresden-Rossendorf). After incubation at room temperature for 60 min, 0.425 mL 100 mM HEPES buffer and 24 μL 0.1 M neocuproine hydrochloric acid (dissolved in MQ water) stock solution was added to the liposome sample (thermosensitive formulation) and incubated overnight at room temperature for loading of the neocuproine into the liposomes. Finally, the sample was purified on a PD-10 column with sterile 0.9% NaCl solution resulting in 120 to 150 MBq radiolabeled liposomes in 2 mL volume (elution profile shown in Figure S1
) with approx. 95 to 97% radiochemical purity (based on the desalting efficiency of the PD-10 column, according to the manufacturer).
2.15. Small Animal Imaging
Copper-64 (T1/2 = 12.7 h; β+, 0.653 MeV [17.8%]; β−, 0.579 MeV [38.4%]) has decay characteristics that allow for positron emission tomography (PET) imaging. NMRI-Foxn1nu/nu mice (n = 3) with U251-HRE xenografts were used for the in vivo imaging experiments. [64Cu]Cu-neocuproine liposome suspensions (8.2 ± 0.8 MBq) were injected via the tail vein. One animal received local mild heat treatment following the liposome (thermosensitive formulation) injection, while the remaining two mice served as controls. PET/CT measurements were performed with a nanoScan® PET/CT, (Mediso Ltd., Budapest, Hungary) with a system PET resolution of 0.9 mm and the detection limit of a focal signal of 0.073 mm3. All animals were anaesthetized with 1.5% isoflurane during imaging. PET data acquisition started one and four hours post-injection for the two control animals, and three hours post-injection for the animal that received mild hyperthermic treatment. The acquisition continued for 60 min. in list mode. A 5 ns coincidence window and 400–600 keV energy window was applied in 94.7 mm scan range. A three-dimensional (3D) expectation maximization (3D EM) PET reconstruction algorithm (Mediso Tera-TomoTM) was applied to produce PET images, including corrections for decay, attenuation and scatter, dead time, and randoms. The helical CT scans were acquired with 35 kV tube voltage, 300 ms exposure time, 1 mA tube current, 1:4 binning and 360 projections. The images were reconstructed with 0.12 mm isovoxel sizes. PET attenuation correction was based on the CT images. After 4 iterations the reconstruction resulted in images with 0.4 mm isovoxel size. The results of PET measurements were quantified in units of radioactivity measured per unit volume (MBq/mL). Image analysis was performed with VivoQuant software (inviCRO, Boston, MA, USA). Following registration of the PET and CT data, tumor and muscle volumes of interest (VOI) were selected by hand. The radioactivity concentrations of tumor VOI’s are presented as a ratio to the image-based whole-body radioactivity concentration. Animal whole-body volumes were determined in mm3 while using a semi-automatic image thresholding algorithm in VivoQuant based on CT densities and corrected with the PET volumes.