Characterization of a liposomal copper(II)-quercetin formulation suitable for parenteral use
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Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a naturally derived flavonoid that is commonly found in fruits and vegetables. There is mounting evidence to suggest that quercetin has potential anticancer effects and appears to interact synergistically when used in combination with approved chemotherapeutic agents such as irinotecan and cisplatin. Unfortunately, quercetin has shown limited clinical utility, partly due to low bioavailability related to its poor aqueous solutions (< 10 μg/mL). In this study, liposomal formulations of quercetin were developed by exploiting quercetin’s ability to bind copper. Quercetin powder was added directly to pre-formed copper-containing liposomes (2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 M ratio)). As a function of time and temperature, the formation of copper-quercetin was measured. Using this methodology, a final quercetin-to-lipid (mol:mol) ratio of 0.2 was achievable and solutions containing quercetin at concentrations of > 5 mg/mL were attained, representing at least a > 100-fold increase in apparent solubility. The resulting formulation was suitable for intravenous dosing with no overt toxicities when administered at doses of 50 mg/kg in mice. Pharmacokinetic studies demonstrated that the copper-quercetin formulations had an AUC0-24H of 8382.1 μg h/mL when administered to mice at 50 mg/kg. These studies suggested that quercetin (not copper-quercetin) dissociates from the liposomes after administration. The resulting formulation is suitable for further development and also serves as a proof-of-concept for formulating other flavonoids and flavonoid-like compounds. Given that quercetin exhibits an IC50 of >10 μM when tested against cancer cell lines, we believe that the utility of this novel quercetin formulation for cancer indications will ultimately be as a component of a combination product.
KeywordsQuercetin Cancer Flavonoids Copper complexes Liposomes
One of the most common phenolics found in fruits and vegetables are the flavonoids. These phenolic compounds are believed to be one reason why fruit- and vegetable-rich diets are commonly associated with lowered risk for cancer . The most common, naturally occurring flavonoid is quercetin (3,3′,4′,5,7-pentahydroxyflavone) and it has garnered much attention as a potential anticancer agent [2, 3]. Quercetin is well-established as an effective antioxidant, protecting against damage associated with oxidative stress from free radicals or reactive oxidative species (ROS) [4, 5]. Specifically, quercetin’s antioxidant activity may be due to catalase’s ability to protect cells from oxidative stress and damage by scavenging H2O2 [4, 5, 6]. It has also been suggested that quercetin exhibits anticancer activity through mechanisms that involve induction of apoptosis. In particular, studies with A549 lung cancer cells, human glioma cells, and human hepatoma cells (HepG2) have shown that treatment with quercetin can concurrently downregulate anti-apoptotic proteins such as Bcl-2, AKT, and metallopeptidase 9 and upregulate pro-apoptotic proteins such as Bax [3, 7, 8].
Quercetin’s potential as an anticancer agent is further demonstrated in combination therapy. When used in a combination setting, quercetin appears to enhance cisplatin cytotoxicity against ovarian cancer in various murine models of cancer . The use of quercetin may also reduce cisplatin-induced ototoxicity as it was shown to protect zebrafish embryos against sensory hair cell damage engendered by cisplatin .
Due to its poor aqueous solubility, most applications of quercetin, whether in the context of clinical trials or as a supplement to therapy, rely on oral administration. However, poor dissolution in gastrointestinal fluids leads to poor gastrointestinal absorption of quercetin, resulting in low bioavailability . Therefore, despite its potential chemotherapeutic benefit, the clinical use of quercetin as an anticancer agent has remained essentially irrelevant [11, 12]. To fully explore the therapeutic potential of quercetin, there is a need to improve its solubility and various strategies have been used to achieve this including the development of more water-soluble pro-quercetin compounds that biologically convert to quercetin  as well as various formulation approaches involving the use of liposomes , polymers , or milling to produce nanocrystals . In this study, we took advantage of quercetin’s propensity to bind copper [17, 18, 19] to develop for the first time a liposomal formulation of copper-quercetin suitable for intravenous use. The resulting formulation exhibits at least a 100-fold improvement in the apparent solubility of quercetin and is suitable for intravenous administration.
Materials and methods
Quercetin, Sephadex G-50, Copper (II) sulfate, HEPES, ethylenediaminetetraacetic acid (EDTA), and sodium chloride (NaCl) were purchased from Sigma-Aldrich (St. Louis, MO). 1,2-Disteoroyl-sn-glycero-3-phosphatidylcholine (DSPC) and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). 3[H]-cholesteryl hexadecyl ether (CHE) and picofluor-15 scintillation fluid were obtained from PerkinElmer, Inc. (Boston, MA, USA). Copper (II) gluconate powder was purchased from AlfaAesar (Ward Hill, MA, USA). Sucrose was obtained from EMD Chemicals Inc. (Gibbstown, NJ, USA). Citric acid monohydrate crystals were purchased from EmScience (USA).
Determination of quercetin solubility
Two milliliters of Milli-Q water, 300 mM sucrose/20 mM HEPES (pH 7.4) (SH), or 150 mM NaCl/20 mM HEPES (pH 7.4) (HBS) was added to 10 mg of quercetin and the solution was subsequently mixed for 60 min at either 60 °C or room temperature (RT). Samples at 60 °C were cooled to room temperature prior to filtration through 0.45-μm filters to remove undissolved quercetin. The resulting clear solutions were then assayed for quercetin via UV-Vis spectroscopy (absorbance at 372 nm) on the Hewlett Packard/Agilent 8453 G1103A UV-Visible Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA).
DSPC and cholesterol were dissolved in chloroform at a 55:45 mol:mol ratio. 3[H]-CHE was added as a non-exchangeable and non-metabolizable lipid marker  prior to drying. 3H-CHE was incorporated into the chloroform mixture at a concentration of 5.0–12.5 nanocurie per μmol lipid. Chloroform was removed by drying the samples under a steady stream of nitrogen gas until the samples became thick and syrup-like. The samples were then placed under high vacuum to create a “puff” of dried lipid which was kept under vacuum for at least 3 h to remove residual chloroform. Unless stated differently, the dried lipid was hydrated in 300 mM copper sulfate (CuSO4) or 100 mM copper gluconate (CuG) (both at pH 3.5) (1 mL per 50 μmol of total lipid). To study the role of pH, lipids were hydrated in 100 mM copper gluconate (pH adjusted to 7.4 with triethanolamine (TEA)). Lipid hydration results in the formation of multilamellar liposomes and the resulting multilamellar liposomes were then extruded 10 times through two stacked 0.1 μm polycarbonate filters in a 10-mL thermobarrel extruder (Extruder™, Evonik Transferra Nanosciences, Burnaby, BC, Canada). The extruded unilamellar liposomes had a mean diameter of 110 ± 20 nm as determined by Phase Analysis Light Scattering methods (ZetaPALS, Brookhaven Instruments Corp., Holtsville, NY). To remove external copper, the sample was passed through a Sephadex G50 column pre-equilibrated with SHE buffer (300 mM sucrose, 20 mM HEPES, 15 mM EDTA) adjusted to pH 7.4. The liposome-containing fractions were subsequently passed through a second Sephadex G50 column pre-equilibrated with HBS. The final lipid concentration was determined by measuring 3[H]-CHE using liquid scintillation counting (Packard 1900 TR Liquid Scintillation analyzer). To gain an understanding of quercetin-copper stoichiometry, liposomes were also prepared with different concentrations of either CuSO4 or CuG which were adjusted to control for osmolarity using sodium sulfate or sodium chloride, respectively. Copper-free liposomes were prepared by hydrating lipids in 300 mM citric acid (pH 3.5) or SH buffer (pH 7.4). In all cases, the external buffer was exchanged to HBS (pH 7.4) prior to initiating studies with added quercetin.
Copper-quercetin formation inside liposomes
Unless otherwise specified, quercetin was added to liposomes at an initial quercetin-to-lipid ratio of 0.4 (mol/mol). The mixture was then continuously mixed with a mini stir-bar at specified temperatures ranging from RT to 60 °C and the amount of liposome-associated quercetin was measured. For these studies, 80 μL aliquots from the quercetin-liposome mixture were taken at the indicated time points and passed through 1-mL Sephadex G-50 mini-spin columns (680×g; 3 min) to separate free quercetin from liposome-associated quercetin. The excluded fraction, which appears yellow due to the formation of the copper-quercetin complex, was then analyzed for lipid, quercetin, and copper concentrations. Liposomal lipid (3[H]-CHE - tagged) was analyzed by liquid scintillation counting (10 μL of sample to 5 mL of scintillation fluid). Quercetin was measured using absorbance at 372 nm on the Hewlett Packard/Agilent 8453 G1103A UV-Visible Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) after the samples (100 μL) were diluted in acidified methanol (methanol with 3% glacial acetic acid). Copper was measured using atomic absorption spectroscopy where samples were diluted with 0.1% HNO3 (v/v with Milli-Q water) and analyzed using the AAnalyst 600 Atomic Absorbance Spectrometer (PerkinElmer, Inc. Woodbridge, ON, Canada): the samples were dried at 110 °C for 30s, heated (45 s) to 130 °C, ashed at 1200 °C for 30s, and atomized and cleaned at 2000 °C (5 s) and 2450 °C (4 s), respectively.
Characterization of copper-quercetin binding
Five milligrams of quercetin powder was dissolved in 10 mL of methanol and the resulting solution was diluted with methanol to a final quercetin concentration of 5 μg/mL. Varying concentrations of CuSO4 and CuG were then added (10 μL) to achieve copper-to-quercetin ratios of 1:8, 1:4, 1:2, 1:1, 2:1, 4:1, and 8:1. These samples were then analyzed spectrophotometrically (Hewlett Packard/Agilent 8453 G1103A UV-Visible Spectrophotometer).
In vitro serum stability assay
The indicated liposomal copper-quercetin formulations were prepared at 60 °C as described above and concentrated by tangential flow filtration (TFF) using an ultrafiltration hollow fiber column (molecular weight cutoff 500 kDa) (GE Healthcare) to achieve a final quercetin concentration of 16.5 mM (lipid concentration was 100 mM). Four hundred microliters of this sample was added to 1.6 mL of fetal bovine serum (FBS; Gibco, Burlington, ON, Canada) and mixed at 37 °C. At various time points (0, 1, 4, 8, and 24 h), 80 μL aliquots from the mixture were passed through mini-spin columns as described above. The excluded fraction was then diluted in acidified methanol and centrifuged at 10,000 rpm at 4 °C for 10 min to remove precipitated proteins. The resulting supernatant was collected and used to determine quercetin, copper, and liposomal lipid concentrations as described above.
Cryogenic electron microscopy of copper-quercetin formulations
Cryogenic electron microscopy (CEM) was performed as previously described . Particles were concentrated to a final concentration of ~ 20 mg/mL lipid and added to TEM grids, and vitreous ice was generated using an FEI Mark IV Vitrobot (FEI, Hillsboro, OR). Prepared grids were stored in liquid nitrogen until imaged. A Gatan 70° cryo-tilt transfer system pre-equilibrated to at least − 180 °C prior to insertion into the microscope was used as the sample holder. An FEI LaB6 G2 TEM (FEI, Hillsboro, OR) operating at 200 kV under low-dose conditions was used to image all samples. A bottom-mount FEI Eagle 4 K CCD camera was used to capture all images. All samples (unless otherwise stated) were imaged at a × 55,000 magnification with a nominal under-focus of 1–2 μm to enhance contrast. Sample preparation and imaging was performed at the UBC Bioimaging Facility (Vancouver, BC).
Pharmacokinetics of the copper-quercetin formulations
Preliminary pharmacokinetic studies were done in Rag2-M mice obtained from the Animal Resource Centre (ARC) at BC Cancer’s Vancouver Research Centre (Vancouver, BC, Canada). All studies were conducted in accordance with the guidelines set by the Canadian Council on Animal Care, as overseen by the University of British Columbia’s Animal Care Committee (protocols A10-0171, A10-0206, and A14-0290). All mice were placed in Optimice cages (up to 5 mice per cage) and cages were equipped with nesting materials and plastic hiding structures (“huts”) for environmental enrichment. Animals were provided with food and water ad libitum. The health status of all animals used in these studies was monitored at least once daily following an established standard operating procedure designed to assess animal health status. In particular, signs of ill health were based on body weight loss, change in appetite, and behavioral changes such as altered gait, lethargy and gross manifestations of stress. When signs of severe toxicity were present, the animals were terminated (isoflurane overdose followed by CO2 asphyxiation) for humane reasons. Necropsy was performed to assess signs of toxicity.
For injected samples, the liposomes were prepared as described above and filter-sterilized through the sterile 50-mL vacuum-driven Steriflip filtration system (Millipore, Billerica, MA, USA). Quercetin was then added to the liposomes incubated for 30 min at 60 °C then cooled to room temperature, filtered, and exchanged into HBS buffer as described above. The resulting liposomal quercetin formulation was concentrated to 5 mg/mL via TFF as described above. The filter-sterilized formulation was then injected intravenously at a quercetin dose of 50 mg/kg (injection volume was 10 μL/g body weight).
Following iv administration, animals (4 per time point) were sacrificed (isoflurane overdose followed by CO2 asphyxiation) at the indicated time points and blood was collected via cardiac puncture. The blood was then centrifuged (2500 rpm; 15 min) and plasma was collected and placed in a tube. The plasma was kept at 4 °C until all analyses were completed. Lipid and copper concentrations in plasma were determined as described above. Quercetin concentrations were measured via high-performance liquid chromatography (HPLC; Waters, Milford, MA) at an absorbance wavelength of 368 nm. Specifically, plasma samples were mixed with chilled 3% acidified methanol, then centrifuged at 14,000 rpm for 10 min to pellet precipitated plasma proteins. Twenty-five microliters of the resulting supernatant was injected onto a Symmetry C18 column (particle size 3.5 μm; 4.6 × 75 mm, Waters). The mobile phase consisted of 70% mobile phase “A” (1% triethyleneamine in water, pH 6.4 adjusted with glacial acetic acid) and 30% mobile phase “B” (100% acetonitrile). The sample temperature was maintained at 4 °C while the column temperature was set at 30 °C. Run time was 5 min per sample with a flow rate of 1 mL/min using a gradient method with the amount of organic phase increasing from 12 to 40% over 8 min.
In vitro cytotoxicity assays of quercetin alone and in combination with irinotecan
A549 lung adenocarcinoma cells were kindly provided by Dr. John Minna’s Laboratory (Dallas, TX) while BxPC3 pancreatic adenocarcinoma cells were purchased from ATCC (Manassas, VA). Both cell lines were cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Life Technologies) and 2 mM l-glutamine (Life Technologies). All in vitro experiments were performed between the third and eighteenth passages. All cell lines were tested negative for mycoplasma.
Dose-dependent viability assays were completed using quercetin and irinotecan (CPT-11) added alone and in combinations to A549 and BxPC3 cells. Briefly, cells were seeded in 384-well plates. Twenty-four hours post seeding, cells were treated with quercetin (Q) or CPT-11(C) alone and in combination at a ratio of 1:2.5 (C:Q) on A549 cells and 1:18 (C:Q) on BxPC3 cells. These ratios were selected based on the ratio of the IC50 of each drug when used alone. Cell viability was determined 72 h post-treatment using the IN Cell Analyzer 2200 (GE Healthcare, Washington, USA) as described previously . In brief, cells were stained with Hoechst for total nuclei count and ethidium homodimer I (Biotium) for dead cells at 72 h following treatment and imaged with the INCell Analyzer after a 20-min incubation at 37 °C. Cells were classified as “dead” if they showed > 30% overlap of the two stains.
Dose-response curves were constructed using Prism 6.0 (GraphPad Software, La Jolla, CA, USA) by plotting mean ± SEM from at least three independent experiments. IC50 values were interpolated from fitted curves. Combination indices (CI) were derived from the dose-response curves using CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA). CI values > 2 were considered as antagonistic; CI values < 0.8 were considered synergistic. All other CI values were considered additive.
All data are plotted using the Prism 6.0 software (Graphpad) as mean ± standard error of the mean (SEM). Pharmacokinetics data were analyzed for the elimination half-life and total drug exposure (area under the curve; AUC) using the PK Solutions software (Summit Research Services, USA).
Formation of copper-quercetin within copper-containing DSPC/Chol (55:45 mol%) liposomes
Particle size (nm) of CuSO4 liposomes and CuG liposomes incubated at 60 °C for 60 min in the absence or presence of quercetin
100 ± 10
140 ± 20
100 ± 10
160 ± 20
100 ± 10
100 ± 10
100 ± 10
100 ± 10
Additional studies were completed to assess the use of liposomes prepared in copper-free solutions and the role of using the low pH (pH 3.5) of the internal copper sulfate solution. The results, summarized in Fig. 2d, indicate low levels of quercetin association with copper-free liposome prepared using 300 mM citric acid (pH 3.5) or HBS (pH 7.4); the quercetin-to-lipid ratio was less than 0.025 which was lower than predicted based on the extrapolation made using the data in Fig. 2a. However, these data indicate that the amount of quercetin partitioning into the liposomal membrane accounts for less than 15% of the level obtained when using liposomes prepared in copper sulfate. The results also demonstrate that the level of partitioning into the lipid bilayer was not influenced by the low pH within the liposomes.
When using liposomes prepared in a pH 7.4 copper-containing solution (100 mM copper gluconate adjusted to pH 7.4 with TEA, see methods), the rate of quercetin association was lower and the maximum quercetin-to-lipid ratio achieved was lower when compared to the quercetin-to-lipid ratio achieved when using a pH 3.5 copper-containing solutions, even when adjusting for the lower copper concentrations (100 mM). However, the results, summarized in Fig. 2d, suggest that the rate of quercetin association with the CuG liposomes was influenced by the internal pH; the rate of association was faster at pH 3.5. Furthermore, Fig. 2d reiterates that quercetin association with the liposomes is dependent on the presence of encapsulated copper, but shows that the copper salt used inside the liposome affects quercetin association levels. The measured quercetin-to-lipid ratios (mol/mol) for the liposomes prepared using 100 mM CuG were as high as 0.2 and this was 2 times higher than that predicted based on liposomes prepared in CuSO4 solutions.
Suitability of the copper-quercetin liposomes as a formulation for intravenous use
Pharmacokinetics parameters of flavopiridol following intravenous administration (5 mg/kg)
AUC (μg h/mL)
Therapeutic potential of quercetin when used in combination with irinotecan
Others have highlighted that quercetin has anticancer activity and it has been argued that it can act as an apoptosis promoter through modulations of proteins such as Bcl-2 and Akt-1 [3, 7, 8]. Unfortunately, since the water solubility of quercetin is poor, its oral bioavailability is limited [10, 31] and its therapeutic utility is limited because of this. For example, a single 4-g oral dose of quercetin resulted in no detectable plasma or urine concentrations of quercetin and less than 1% of the quercetin was absorbed unaltered in the gastrointestinal tract . Importantly, this is not the only concern when considering the development of quercetin as a therapeutic to treat cancer. Most studies have shown that quercetin can only induce anticancer effects when present in high micromolar (10–20 μM) concentrations [33, 34]. If a suitable intravenous formulation of quercetin was developed, issues related to bioavailability would then be addressed and perhaps suitable concentrations of quercetin in the blood could be obtained. We recently described a formulation method for poorly soluble drugs, including flavonoids like quercetin, that considers the use of compounds that can coordinate metals and nanotechnology . In this report, liposomal formulations prepared based on quercetin-copper binding have been characterized. The results suggest that the encapsulated copper salt can influence the copper-quercetin complex that is formed. Importantly, the formulations made exhibit more than 100-fold improvements in apparent solubility and are suitable for intravenous administration.
Quercetin is difficult to prepare as an iv-injectable formulation. When described, these formulations rely on organic solvents such as dimethyl sulfoxide (DMSO) which, when used, have been shown to be toxic at many levels . Therefore, it is not surprising that efforts have been invested to develop alternative quercetin formulations. For example, when compared to free quercetin, liposomal quercetin formulations often displayed improved blood circulation and decreased rates of elimination from the plasma compartment [24, 25, 36]. In these previous studies, quercetin was formulated into liposomes passively when the liposomes are first made. Passive encapsulation into liposomes is very inefficient and requires preparation of liposomes at high lipid concentrations . In contrast, the novel copper-based methodology described here was extremely efficient, where encapsulation amount was dependent on the copper-to-quercetin ratio formed. The copper-based technology resulted in more stable and consistent formulations of quercetin. The CuSO4-based formulation showed improved pharmacokinetics compared to liposomal formulations previously described in literature with a 3-fold increase in circulation half-life (2 h versus 0.5 h) and an 18 fold increase in total drug exposure (AUC) (1.94 versus 0.106 μmol/mL) . This copper-based formulation was well-tolerated at the current maximum feasible injected dose in RAG2m mice with no acute or chronic (14 days) toxicities observed (data not shown). Additionally, necropsy reports of the animals used to assess toxicity studies were unremarkable.
In addition to liposomes, biofunctional metal-phenolic films have also been previously investigated to overcome the solubility challenges of quercetin . Bertleff-Zieschang et al. described one-step, pH-dependent coordination of quercetin with FeIII ions to form thin films and hollow capsules on solid surfaces . The formation of these capsules and films helped address the poor aqueous solubility of free flavonoids like quercetin . In this regard, the hollow iron-quercetin capsules are similar to our liposomes as they also provide a solution to solubilize quercetin. However, our liposomes present a few advantages, most notable of which is the physical shielding effects that the phospholipids of the liposomes are able to provide for the encapsulated content. As nanocarriers, liposomes are well known to act as steric barriers that may discourage interactions between entrapped compounds and plasma proteins . This property is particularly attractive for quercetin as this flavonoid is known to extensively bind human plasma protein and it has been suggested that this interaction results in poor cellular availability of quercetin . By encapsulating quercetin in liposomes, we would able to curb this activity-hindering interaction with plasma proteins. Additionally, although Bertleff-Zieschang et al. did not evaluate the pharmacokinetic profile of the hollow capsules, we believe that by shielding quercetin within liposomes, our formulation would also have improved pharmacokinetics, namely circulation time and total drug exposure, when compared to the hollow capsules of quercetin. This improved pharmacokinetic profile could enhance quercetin’s clinical value as quercetin is known to be a substrate of P-glycoprotein and its absorption rate is limited by high efflux rate .
Metal-based therapeutics made a major leap in the 1970s with the emergence of platinum-based complexes such as cisplatin, carboplatin, and oxaliplatin as widely successful, and potent therapeutics . However, efforts have been made to identify alternatives to replace platinum-based therapeutics primarily due to the many crippling toxicities that these drugs engender and copper-based therapeutics emerged as a potential replacement candidate . One of the primary reasons that copper was selected as the metal of choice was its role as an essential metal. Unlike platinum, which is not native to the human body, copper is an important trace element in the growth and development of all aerobic organisms and plays a crucial role in a number of biological pathways [42, 44]. As such, copper is tightly regulated and its homeostasis is rigorously maintained to avoid copper-related toxicities [44, 45]. In the blood, plasma protein carriers such as albumin and ceruloplasmin transport the copper absorbed into the bloodstream to the liver and the kidney for excretion and distribution . On the cellular level, copper transporters such as CTR1 and copper efflux pumps like ATP7A/B aid in the regulation of cytosolic copper levels . The presence of these intricate regulatory mechanisms helps attenuate toxicities that copper-based therapeutics may have otherwise engendered, allowing copper-based drugs to be better tolerated. In addition to having natural regulatory biological pathways, copper was also chosen as the metal of preference due to its potential to enhance anticancer activities. Disulfiram, an FDA-approved drug for treating alcoholism, paints a perfect picture of a copper-activated agent. As an anticancer agent, disulfiram has shown to have minimal anticancer activity in the absence of copper but is active in the nanomolar range against various cancer cell lines in the presence of copper [47, 48]. In the case of quercetin, forming a complex with copper did not enhance the activity of quercetin (data not shown). However, the use of copper still solved the low solubility problem that has long prevented the use of flavonoids such as quercetin in a clinical setting. Here, we have provided proof-of-concept data using copper, but we do not preclude the possibility of using other metals, such as zinc. In agreement with numerous published studies [17, 49, 50], our data suggested that quercetin forms a 2:1 ligand-to-metal complex with copper (II) ions in the presence of CuSO4 and a 1:2 ligand-to-metal complex with copper (II) ions in the presence of CuG (Fig. 5b). The different complexes formed in the presence of CuSO4 versus CuG help explain the different encapsulation efficiencies observed. Gluconic acid forms strong complexes with many transition metals including copper . The presence of a competitive copper chelator would result in the formation of a different copper complex than what is seen with CuSO4. Thus, in the presence of CuG, two quercetin molecules were needed to bind with copper ions to engender dissociation of copper ions away from gluconic acid. In contrast, sulfate does not form a complex with copper ions and the quercetin molecule can bind to two copper (II) ions. Therefore, when considering equivalent Cu (II) concentrations, CuG-containing liposomes appear to facilitate improved quercetin association compared to CuSO4-containing liposomes. However, CuG-quercetin liposomes were cleared from the plasma compartment much more rapidly compared to CuSO4-quercetin liposomes. This may have been the result of the greater variation in particle size as shown in the CEM images of CuG liposomes (Fig. 6) and the increase in particle size seen when using CuG—containing liposomes with encapsulated quercetin. The rate of liposome elimination is increased as the size of the liposome increases .
Regardless of the binding interactions, quercetin and copper-quercetin complexes are poorly soluble in aqueous solutions and this is one factor that limits quercetin’s therapeutic potential. Many studies have demonstrated that as a single agent, quercetin is not a potent chemotherapeutic agent, with effective doses at micromolar concentrations [5, 53, 54, 55]. Therefore, it can be argued that quercetin’s therapeutic value may lie in combination therapy. In fact, it has previously been shown that when used in combination, quercetin potentiates existing chemotherapeutic agents such as cytosine arabinoside, doxorubicin, and irinotecan for treatment against leukemia, breast cancer, and gastrointestinal cancer, respectively [26, 56, 57]. In particular, quercetin has been shown to work well in combination with irinotecan, demonstrating promise in the treatment of advanced non-small-cell lung cancer, with an overall response rate of up to 32% . In an initial in vitro screen, when quercetin was added concurrently with irinotecan, the interaction between the two drugs suggested additively (Fig. 9). The difference in the tissue origin and the genetic profiles between the cell lines may have accounted for this. Regardless, further studies to elucidate the therapeutic utility of quercetin combinations and to identify suitable candidates to use in combination with quercetin are needed. More studies are needed to define how a liposomal copper-quercetin formulation would be used in combination in preclinical animal models. Additionally, the use of copper complexation methods to encapsulate quercetin acts as a proof-of-concept and will now allow us to further explore utility of this copper-based technology with other promising flavonoids or flavonoid-like candidates that share similar copper-chelating properties as quercetin.
In summary, we have successfully addressed the solubility issues associated with quercetin by generation of a novel liposomal formulation of copper-quercetin. Compared to other quercetin-based formulations previously described, our formulation takes advantage of the well-characterized propensity of liposomes to enhance the circulation longevity of an associated drug. Continued efforts will be made to explore and evaluate quercetin’s value as a component in combinations for the treatment of cancer.
We thank the animal technicians from the Investigational Drug Program at the BC Cancer Agency for their help with our in vivo studies.
Dr. Gardenia Militao received financial support from CNPq (Brazilian Council for Scientific and Technological Development). Dr. Ada Leung received financial support from Canadian Institutes of Health Research (Vanier Canada Graduate Scholarship) and from the University of British Columbia (Four-Year Doctoral Fellowship). This research was funded by CCSRI (705290) and CIHR (153132).
Compliance with ethical standards
All studies were conducted in accordance with the guidelines set by the Canadian Council on Animal Care, as overseen by the University of British Columbia’s Animal Care Committee (protocols A10-0171, A10-0206, and A14-0290).
Conflict of interest
The authors declare that they have no conflicts of interest.
- 8.Pan H-C, Jiang Q, Yu Y, Mei J-P, Cui Y-K, Zhao W-J. Quercetin promotes cell apoptosis and inhibits the expression of MMP-9 and fibronectin via the AKT and ERK signalling pathways in human glioma cells. Neurochem Int. 2015;80:60–71. https://doi.org/10.1016/j.neuint.2014.12.001.CrossRefPubMedGoogle Scholar
- 28.Bansal T, Awasthi A, Jaggi M, Khar RK, Talegaonkar S. Pre-clinical evidence for altered absorption and biliary excretion of irinotecan (CPT-11) in combination with quercetin: possible contribution of P-glycoprotein. Life Sci. 2008;83(7):250–9. https://doi.org/10.1016/j.lfs.2008.06.007.CrossRefPubMedGoogle Scholar
- 29.Ramsay EC, Anantha M, Zastre J, Meijs M, Zonderhuis J, Strutt D, et al. Irinophore C: a liposome formulation of irinotecan with substantially improved therapeutic efficacy against a panel of human xenograft tumors. Clin Cancer Res. 2008;14(4):1208–17. https://doi.org/10.1158/1078-0432.ccr-07-0780.CrossRefPubMedGoogle Scholar
- 30.Waterhouse DN, Sutherland BW, Santos ND, Masin D, Osooly M, Strutt D, et al. Irinophore C™, a lipid nanoparticle formulation of irinotecan, abrogates the gastrointestinal effects of irinotecan in a rat model of clinical toxicities. Investig New Drugs. 2014;32(6):1071–82. https://doi.org/10.1007/s10637-014-0138-x.CrossRefGoogle Scholar
- 34.Srivastava S, Somasagara RR, Hegde M, Nishana M, Tadi SK, Srivastava M, et al. Quercetin, a natural flavonoid interacts with DNA, arrests cell cycle and causes tumor regression by activating mitochondrial pathway of apoptosis. Sci Rep. 2016;6:24049. https://doi.org/10.1038/srep24049.CrossRefPubMedPubMedCentralGoogle Scholar
- 40.Boulton DW, Walle UK, Walle T. Extensive binding of the bioflavonoid quercetin to human plasma proteins. J Pharm Pharmacol. 1998;50(2):243–9. https://doi.org/10.1111/j.2042-7158.1998.tb06183.x.CrossRefPubMedGoogle Scholar
- 47.Liu P, Brown S, Goktug T, Channathodiyil P, Kannappan V, Hugnot JP, et al. Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells. Br J Cancer. 2012;107(9):1488–97. https://doi.org/10.1038/bjc.2012.442.CrossRefPubMedPubMedCentralGoogle Scholar
- 58.Kelly K. Current role of irinotecan in the treatment of non-small-cell lung cancer. Oncology (Williston Park). 2002;16(9):1153–62 65; discussion 65–6 passim.Google Scholar
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