Preparation, characterization and in vitro anticancer testing of quercetin-loaded nanocochleates

S. A. Sonwane*, M. J. Chavan, D. P. Hase, D. S. Chumbhale, A. S. Ambare, Y. T. Bodakhe.
Department of Pharmacognosy, Amrutvahini College of Pharmacy, Sangamner 422608, Maharashtra, India.
* Corresponding author. Phone: 9766758577; E-mail address:


Background: The natural flavonoid quercetin has shown anticancer properties but it is in vivo administration remains challenging due to its poor aqueous solubility and extensive in vivo metabolism. This juncture demands an effective, controlled release formulation of quercetin would be a significant advance for the treatment of cancer.

Purpose: The objective of the current work was to prepare novel quercetin-loaded nanocochleates in order to improve its therapeutic efficacy and cytotoxicity in human breast cancer cell line MCF-7.

Methods: Quercetin-loaded nanocochleates was prepared by a trapping method. The optimized quercetin-loaded nanocochleates were evaluated for size, entrapment efficiency, in vitro quercetin release, cytotoxicity study.

Results: Stable rolled-up layers as well as a tubular structure of nanocochleates possessing particle size and encapsulation efficiency of 180 ± 3 nm and 76.69 ± 3.21%, respectively were obtained. Nanocochleates demonstrated sustained release of quercetin at physiological pH. A significant improvement in vitro anticancer towards human breast cancer MCF-7 cells was observed.

Conclusion: Developed nanocochleates markedly improved the anticancer efficacy of quercetin. The nanocochleates technology would facilitate the administration of this flavonoid in the clinical setting.

Keywords: Quercetin, soya phospatidylcholine, nanocochleates, anticancer.

1. Introduction

Natural products are preferred over synthetic drugs because of their relative safety and biocompatibility. However, the low aqueous solubility, bioavailability, poor targeting and stability hamper their clinical use. The hurdles associated with the delivery of phytopharmaceuticals raise the necessity of developing novel formulations that could overcome the delivery barriers, increase the bioaccessibility and eventually increase the therapeutic benefits [1].

Several dietary plant-derived bioactives have been linked to cancer preventive and/or therapeutic agents, including the flavonoids which occupy a central place due to their widespread abundance in dietary products such as a variety of fruits, plants and vegetables, which are usually marketed as diet supplements and relative safety [2]. They have several pharmacological actions, including anti-inflammatory, anti-oxidative, antiviral and anti-allergic activity. However, many bioactive flavonoids such as baicalein, fisetin, apigenin, luteolin and genistein possess low bioaccessibility due to their poor molecular pharmaceutical properties and their successful incorporation into pharmaceuticals and functional foods still remains a challenge. Various novel drug delivery system have been developed for flavonoids including self-microemulsifying delivery system for baicalein, fisetin and apigenin [3-5]; polymeric nanoparticles for genistein and luteolin to improve their therapeutic efficacy [6,7].

Quercetin is a naturally occurring flavone found in most edible fruits and vegetables, with the highest concentrations being found in onions, apples, and red wine [8, 9]. Quercetin has many biological activities, such as antitumor and antiproliferative effects on a wide range of human cancer cell lines and inhibition of glycolysis, macromolecule synthesis, and enzymes [10-13]. However, the poor water solubility, short biological half-life, and low oral bioavailability of quercetin hampered its application as a therapeutic agent [14-16]. 

Nanocochleates delivery vehicle are stable phospholipid precipitates derived from the interaction of anionic lipid vesicles with divalent cation (Ca2+ and Mg2+). Nanocochleates can be prepared by adding calcium chloride dropwise to preformed liposome. These are different from liposome in that they have a water-free interior, a rod-shape and a rigid structure[17,18]. This lipid based nanocochleates system bears potential for the development of innovative pharmaceuticals due to its many advantages such biocompatibility, ease and safety of production, reduced side effects and improved efficacy [19,20]. 

Non-aqueous nanocochleates structure is resistant to permeation of oxygen and consequently makes encochleated molecule less susceptible to oxidation. It also protects encochleated drug from degradation in biological fluids. Because the entire cochleate structure is a series of solid layers, components within the interior of the cochleate structure remain intact, even though its outer layers may be exposed to harsh environmental conditions or enzymes, such as in the stomach. They possess ability to target macrophase, which contains membrane phosphotidylserine (PS) receptor, which causes phagocytosis of nanocochleates to deliver the encochleated materials into the cytosol of target cell [20,21]. These novel carriers have been successfully investigated for delivery of several classes of drugs including antibiotics, antifungal, antileprosy, anticancer, protein and DNA subunit to improve their therapeutic efficacy [22-25].

To further advancement of the therapeutic utility of quercetin, the present study investigates the potential of nanocochleates as vehicle for systemic delivery of quercetin. Such formulation has not been reported previously.

2. Materials and methods

2.1 Material

Quercetin, cholesterol and dialysis bag (molecular weight cut off 12,000 D) were purchased from Sigma Aldrich Chemical Private Ltd, Bangalore, India. Soya phospatidylcholine (SPC) was gifted from Lipoid GmbH Ludwigshafen, Germany. Ethanol, ethylenediaminetetraacetic acid (EDTA) and calcium chloride were of analytical grade. Water were of HPLC grade and procured from Merck, Mumbai, India. RPMI 1940 and fetal bovine serum (FBS) were procured from GICO-RL, Invitrogen Life Technology, USA. ELISA plates of Sulforhodamine B assay (SRB) were read on the Sunrise model of Tecan, Austria. All other solvents used for study were of analytical grade.

2.2 Preparation of quercetin-loaded small unilamellar vesicle
Quercetin-loaded small unilamellar vesicles were prepared by ethanol injection method [26]. Briefly, specific amount of quercetin, cholesterol and SPC were dissolved in 2 ml of ethanol (Table 1) and heated upto the phase transition temperature (40°C) of SPC. This ethanolic solution was rapidly injected into 10 ml of the phosphate buffer (pH 7.4) under stirring at 500 rpm using Teflon-coated beads and stirring was continued until complete evaporation of ethanol under vacuum. Further, phosphate buffer was added to adjust the volume of final lipid vesicles suspension to 10 ml. Finally, the vesicles were purified by passing through a 0.22 µm membrane filter to obtain a quercetin-loaded small unilamellar vesicle suspension.

2.3 Preparation of quercetin-loaded nanocochleates 

Quercetin-loaded nanocochleates were prepared by trapping method [27]. 20µl of calcium chloride solution (0.1 M) were added drop-wise into the prepared quercetin-loaded small unilamellar vesicles under vortex. The vesicle phase immediately turned turbid because of nanocochleate formation. Precipitated nanocochleates were refrigerated at 2-8°C.

2.4 Evaluation quercetin-loaded vesicles and nanocochleates

2.4.1 Particle size and zeta potential determination

The particle size and zeta potential of quercetin-loaded vesicles and nanocochleates was determined after suitable dilutions by dynamic light scattering technique by using zetasizer Nano-ZS90 (Malvern Instruments, UK). The measurement was carried out at 633 nm using a 173° scattering angle at a temperature of 25°C. Poly dispersity index (PDI) values ranging from 0 to 1 reflects the polydispersity of the suspension with the lower value indicating a more monodispersed suspension. 

2.4.2 Encapsulation efficiency determination
The calibration curve (R2 = 0.9998) of quercetin solutions (concentration of 5 to 25 µg/ml in phosphate buffer pH 7.4) was obtained by measuring the absorbance at 378 nm by using UV Spectrophotometer (Shimadzu UV-1800, Japan). Encapsulation efficiency was determined by centrifugation (Eltek RC 4100 D, Elektrocrafts, Mumbai, India) by separating non-encapsulated quercetin from vesicular suspension at 15,000 rpm for 60 min at 4°C. The sediment vesicles were disrupted with absolute ethanol to release the entrapped drug; suitably diluted with phosphate buffer (pH 7.4) and the absorbance measured at 378 nm to calculate the encapsulation efficiency using the calibration curve equation y = 0.051x – 0.031. Here ‘y’ is the measured absorbance and ‘x’ is the concentration in µg/ml. The percent encapsulation efficiency was calculated using following Eq. (1). 

To determine of encapsulation efficiency of nanocochleates, 1ml of nanocochleates were introduced into polypropylene centrifuge tube. The tube was centrifuged at 15000 rpm for 30 min at 4°C and the supernatant and pellets were separated; 50 µl of pH 9.5 EDTA was added to the cochleate pellet to allow the opening of the cochleates into vesicles and the release of quercetin [20]. Next 1 ml of absolute ethanol was added followed by vortexing. The resulting solution was clear which was suitably diluted with phosphate buffer (pH 7.4) and absorbance determined at 378 nm. The free quercetin concentration in supernatant was also measured and percent encapsulation efficiency was calculated using following Eq.(1).

Encapsulation efficiency (%) = 
Amount of drug entrapped in the vesicle or cochleates / Total amount of the drug present × 100 (1)

2.4.3 Surface morphology 

Surface morphology of the quercetin vesicles and quercetin-loaded nanocochleates were performed by using transmission electron microscopy (TEM). To prepare the sample for TEM, a drop of diluted sample was placed onto a carbon-coated copper grid to from a thin liquid film. After excess solution was removed, the sample was examined and photographed with a PHILIPS CM 200 transmission electron microscope at an operating voltage of 20-200 kV.

2.4.4 In vitro release of quercetin from nanocochleates 

The in vitro release of quercetin from the nanocochleates were performed in phosphate buffer saline (pH 7.4) using dialysis bag diffusion method [28] and compared with free quercetin. Formulation equivalent to 2 mg of quercetin or 2 mg quercetin dispersion in dissolution medium as control were introduced into a dialysis bag, hermetically sealed and immersed into 50 ml of release medium. The entire system were kept at 37 ± 0.5°C with continuous magnetic stirring at 100 rpm/min. At predetermined intervals, sample were withdrawn and replaced with equal volume of fresh medium in order to maintain sink conditions. The absorbance of quercetin in the solution were determined using the UV spectrophotometer at 378 nm.

2.5 In vitro anticancer study

In vitro anticancer activity of free quercetin, blank nanocochleates and quercetin-loaded nanocochleates were evaluated against human breast cancer MCF-7 cells using in vitro Sulforhodamine B assay (SRB assay) [29]. The cells were cultured in RPMI 1640 medium, supplemented with 10% v/v FBS and 2 mM L-glutamine. Cells were seeded at the density of 5×103 cells per well in 96-well pates using in situ fixing agent trichloroacetic acid (TCA). After 24 h of incubation at 37°C with 100% relative humidity (RH), the growth medium was replaced with 100 µl of fresh medium containing various concentration (10-80 µg/ml) of free quercetin in DMSO (the final concentration of DMSO kept below 0.2%), drug-loaded and blank nanocochleate suspension. The culture medium without any drug formulation was used as the control. After 48 h incubation, assay was terminated by adding 50 µl of cold TCA and incubated for 60 min at 4°C. The media was removed and cells were washed with sterile PBS and air dried. 50 µl of SRB solution (0.4% w/v in 1% acetic acid) was added to each wells and further incubated for 20 min at room temperature. After staining, unbound dye was removed by washing with 1% acetic acid and plates were air dried. Bound stain was eluted with 10 mM trizma base and the absorbance was read on an Elisa plate reader at a wavelength of 540 nm with 690 nm reference wavelength. Percent growth was calculated on a plate-by plate basis for test wells relative to control wells using following Eq. (2).

Cell growth (%) = 
Average absorbance of the test well / Average absorbance of the control wells × 100 (2) 

Using the six absorbance measurements (time zero (Tz), control growth (C) and test growth in the presence of drug at various concentration levels (Ti), the percentage growth was calculated at each of the drug concentration levels. 

Percentage growth inhibition was calculated as: [(Ti - Tz) / (C - Tz)] × 100 for concentrations where Ti Tz or (Ti - Tz) is positive or zero; as [(Ti - Tz) / Tz] × 100 for concentrations where Ti Tz or (Ti - Tz) is negative. 

Growth inhibition of 50 % (GI50) was calculated from equation [(Ti - Tz) / (C - Tz)] × 100 = 50 as the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation.

3. Results and discussion

3.1 Quercetin loading into vesicles determination

With the objective of loading the maximum amount of quercetin into the lipid bilayer. For this, vesicles were prepared bearing increasing amount of quercetin (5, 10 and 15 mg) and evaluated for particle size, polydispersity index and encapsulation efficiency. As shown in Table 1, the vesicle size and the PDI increased when quercetin concentration increased from 5 mg (F1) to 10 mg (F2). However further increase (F3) resulted in low encapsulation efficiency. The formulation F2 presented good encapsulation efficiency; hence the maximum amount that can be loaded into the phospholipid bilayer was fixed at 10 mg. Also, F2 was chosen as the nanoliposomal template for nanocochleate formation. 

3.2 Evaluation of nanocochleates 

3.2.1. Particle size distribution and zeta potential

The optimized F2 showed particle size of 168 ± 3 nm with EE of 62.31 ± 4.26%. Whereas, the corresponding F2NC showed particle size of 180 ± 3 nm (Fig. 1A and B) with EE of 76.69 ± 3.41%. Both vesicles and nanocochleates showed negative zeta potential of -20.6 mV and -15.8 mV, respectively (Fig. 2A and B).

Quercetin, a lipophilic bioactive, solubilises into the lipid bilayer. The liposomal size and PDI increased with increasing quercetin concentration as the cohesive forces between the vesicles reduced due to insertion of quercetin in the bilayer. At still higher concentration of quercetin, however, the vesicle size increased but with low EE due to exclusion of the lipid components from the lipid vesicles. Unilamellar vesicles with mean diameter of 168 ± 3 nm were used to prepare the nanocochleates. Divalent cations Ca2+, Mg2+, Ba2+ and Zn2+ can be used for preparing cochleates [30]. It has been reported that Ca2+ forms a more tightly packed, highly ordered and less hydrated structure than does Mg2+ with phospholipids. Also it is required in much lower concentration than Mg2+ [31]. It is well documented that Ca2+ plays a vital role in natural membrane fusion phenomena while other cations listed above are ineffective in most such systems. Hence it is most compatible with the body. Thus, calcium is the most suitable divalent cation reported for preparing cochleates and hence used in the present work. The addition of calcium ions to the SPC and cholesterol vesicles induces fusion of lipid membranes and the formation of planar sheet which eventually coil around an initial point of folding to form cochleates cylinder. The obtained nanocochleates were larger in size and demonstrated better EE (76.69 ± 3.41%) as compared with vesicle. Our report is the first to demonstrate the quercetin encapsulation advantages of nanocochleates. Their negative zeta potential is probably due to the anionic nature of the employed SPC.

3.2.2. Surface morphology

Surface morphology revealed discrete and round structure of small unilamellar vesicles (Fig.3A). Whereas, nanocochleates showed rolled-up layers and rod structure. (Fig.3B).

3.2.3 In vitro release study

In vitro release of the quercetin from dispersion and nanocochleates were investigated by dialysis bag diffusion method and compared with free quercetin. As quercetin-loaded nanocochleates were developed aiming at its parenteral administration, the release studies were conducted in phosphate buffer saline (pH 7.4) at 37°C. Fig. 4, revealed that quercetin could freely diffuse in the dispersion form causing 100% drug release within 5 h. However, quercetin release from nanocochleates showed a biphasic pattern with initial burst release (10%) within the first 1 h followed by controlled release up to 24 h[32].

In this in vitro release study, the initial burst release may be due to the dissolution of excess drug absorbed on the surface of the nanocochleates, while the further controlled release could be caused by diffusion of the drug. The drug encapsulated into the inner core compartment stayed firmly inside the nanocochleates showing a very slow release even at sink conditions with 10% of the initially incorporated drug still being associated with the nanocochleates even after 24 h.

3.3 In vitro anticancer activity 

The in vitro anticancer activity of quercetin-loaded nanocochleates was investigated and compared with free drug in dispersion and blank-nanocochleates against human breast cancer MCF-7 cells using in vitro SRB assay. The results illustrated in Fig. 5 indicated that quercetin-loaded nanocochleates demonstrated superior anticancer activity than the free drug dispersion and blank nanocochleates. The GI50 of quercetin-loaded nanocochleates was lower (9.52 ± 0.5 µg/ml) than that of free quercetin dispersion (16.12 ± 1.2 µg/ml) and blank nanocochleates (> 70 µg/ml).

The invitro anticancer study demonstrated that the GI50 concentration of QCTNC is less than free QCT and blank NC. This can be attributed to higher accumulation of the drug via direct interaction or phagocytosis i.e fusion between nanocochleates and the cancer cell membrane followed by controlled release of the drug. Many natural membranes fusion processes involve calcium-induced perturbations of membranes containing negatively charged lipids. The Hypothesis is that contact of the calcium-rich, highly ordered membrane of a cochleate with a natural membrane causes a perturbation and reordering of the cell membrane [22]. Subsequently, a fusion occurs between the outer layer of the cochleate and the cell membrane. As a result, a small amount of the encochleated material is transferred into the cytoplasm of the target cell. The nanocochleate could then break free of the cell and be available for another fusion event, either with this or another cell. Alternatively, particularly with active phagocytic cells, the nanocochleate may be taken up by endocytosis and fuse from within the endocytic vesicle. This tumour retention effect is apparently due to the cochleates’ extravasation through the porous capillary endothelium of the tumour caused by enhanced permeability and retention effect [38]. Therefore, quercetin-loaded nanocochleates might serve as a potential carrier for quercetin. The anticancer activity of phospholipid is reported to be related to its unique apoptosis-inducing properties via the generation of reactive oxygen species or a direct perturbing effect phospholipid on the cell membrane causing fluidity and leakage [33]. This might be the reason for toxicity of blank nanocochleates to cancer at a higher concentration. 


Thus to conclude, quercetin-loaded nanocochleates formulation designed in the present study exhibiting higher drug loading, optimal encapsulation efficiency and in vitro drug release. Further, these nanocochleates exhibited better in vitro anticancer potency of quercetin. The nanocochleates technology could therefore advantageously be employed to improve the antitumor efficacy of quercetin as well as other poorly water-soluble flavonoids. Therefore, it may be useful as a promising parenteral formulation for the effective delivery of quercetin.


The authors are thankful to Dr. C. Bothiraja, Associate Professor, Poona College of Pharmacy, Bharati Vidyapeeth University, Pune, Maharashtra, India for providing technical support and encouragement during this study.

Conflict of interest

The authors declare that they have no conflict of interest.

Tables and figures:
Table 1. Characteristics of quercetin-loaded vesicles and nanocochleates formulatio

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