Development and optimization of stealth liposomal system for enhanced in vitro cytotoxic effect of quercetin

Journal of Drug Delivery Science and Technology 55 (2020) 101477

Contents lists available at ScienceDirect

Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst

Development and optimization of stealth liposomal system for enhanced in vitro cytotoxic effect of quercetin

T

Aishwarya L. Saraswat1, Timothy J. Maher∗ Department of Pharmaceutical Sciences, School of Pharmacy, MCPHS University, 179 Longwood Avenue, Boston, MA, 02115, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: Quercetin Solubility Stealth liposomes Drug delivery Cytotoxicity

The present study was designed to develop a stealth liposomal drug delivery system for a naturally occurring flavonoid, quercetin. Quercetin is a unique polyphenol with multifarious curative applications, anticancer activity being one of them. However, the poor aqueous solubility of this hydrophobic compound hinders its therapeutic effects. For this purpose, our study aimed to enhance the aqueous solubility and in vitro cytotoxic efficacy of quercetin by its incorporation in stealth liposomal formulation. Quercetin-loaded stealth liposomes were prepared with different molar ratios of egg-PC, cholesterol and DSPE-PEG2000 lipids by the traditional thin film hydration method followed by their physicochemical characterization to determine average particle size, polydispersity index (PDI) and zeta potential. The encapsulation efficiency (EE%), saturation solubility of quercetin and apparent solubility of quercetin-loaded stealth liposomes were determined using HPLC. The stealth liposomes were investigated for their stability and an optimized and stable liposomal formulation containing 5% DSPE-PEG2000 lipid was then further evaluated for its in vitro cytotoxicity in HeLa cells and compared to that of free quercetin. The average particle size, PDI, zeta potential and EE% of the optimized liposomes were found to be 110 nm, 0.169, −3.69 mV and 89%, respectively. Encapsulation of quercetin in the stealth liposomal system significantly increased its solubility by approximately 44-fold. This liposomal formulation was stable up to 21 days in terms of its physicochemical characteristics and EE%. Liposomal encapsulation of quercetin was significantly more capable of reducing cell viability of HeLa cells when compared to free quercetin. Our study suggests that this appropriately designed stealth liposomal system could be a promising carrier for encapsulation of the hydrophobic bioactive, quercetin, and significantly enhanced the aqueous solubility and escalated the cytotoxic efficacy of quercetin in cervical cancer cells.

1. Introduction Quercetin (QC) is a typical plant flavonoid derived from quercetum (oak forest), from genus Quercus and family, Fagaceae [1]. It exists in most plants, fruits and vegetables such as caper, black chokeberry, onion, tomato and lettuce and can reach levels as high as 16–25 mg per day in the human diet [2]. As shown in Fig. 1, chemically QC is 2-(3,4dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one and its molecular formula is C15H10O7 [3]. Due to its antioxidant, anti-obesity, anti-carcinogenic, anti-viral, anti-inflammatory, neuroprotective and anti-hypertensive activities, QC has attracted increasing attention [4–9]. The chemopreventive activity of QC maybe largely due to its strong antioxidant capacity [10]. QC is a potent scavenger of reactive oxygen species (ROS) such as O2−, NO• and

ONOO− [11]. These ROS induce oxidative damage and create lethal effects in cells and tissues associated with various disorders like diabetes, cardiovascular diseases and cancers [12]. The antioxidant activity of QC is attributed to its chemical structure: (a) –OH groups at positions 5 and 7 in the A ring; (b) a catechol group in the B ring; and (c) a 2,3-double bond along with a 4-oxo function in the C ring [13]. The anti-cancer effects of QC are believed to be through the inhibition of the PI3K-AKT/PKB pathway, downregulation of the expression of oncogenes and anti-oncogenes, upregulation of cell cycle control proteins, inhibition of heat shock proteins and inhibition of tyrosine protein kinases such as epidermal growth factor receptor (EGFR) [14–18]. Braganhol et al. demonstrated that QC triggers cell cycle arrest at the G2/M phase, which is mediated through the activation of the p53 tumor suppressor protein in the case of HeLa cells

∗

Corresponding author. E-mail addresses: [email protected] (A.L. Saraswat), [email protected] (T.J. Maher). 1 Present address: Aishwarya L. Saraswat, [email protected], College of Pharmacy and Health Sciences, St. John’s University, Queens, NY, United States of America https://doi.org/10.1016/j.jddst.2019.101477 Received 26 June 2019; Received in revised form 5 December 2019; Accepted 23 December 2019 Available online 24 December 2019 1773-2247/ © 2019 Published by Elsevier B.V.

Journal of Drug Delivery Science and Technology 55 (2020) 101477

A.L. Saraswat and T.J. Maher

List of abbreviations CHOL DSPE EDTA EE EGFR EPR ERK HPLC LOD LOQ MPS

MTS ODS OH PC PDI PEG PI3–K PKB PBS QC RES ROS S/N

cholesterol 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine Ethylenediaminetetraacetic acid Encapsulation efficiency Epidermal growth factor receptor Enhanced permeability and retention Extracellular signal-regulated kinase High performance liquid chromatography Limit of detection Limit of quantification Mononuclear phagocyte system

Methyltetrazolium salt Octadecylsilyl Hydroxyl Phosphatidylcholine Polydispersity index Polyethylene glycol Phosphatidylinositol 3-kinase Protein kinase B Phosphate buffer saline Quercetin Reticuloendothelial system Reactive oxygen species Signal-to-noise ratio

optimal molecular weight of PEG that can prolong the in vivo circulation time of liposomes is 2,000 Da [36]. The entrapment of a hydrophobic compound such as QC between the lipid bilayers of these stealth liposomes should overcome its solubility limitations in aqueous medium [37]. In this study, the authors developed a stealth QC-loaded liposomal formulation and explored the effect of the PEGylated lipid concentration on the physicochemical characteristics and stability of liposomes for successful in vitro delivery of QC. An optimized, stable stealth liposomal system with high encapsulation efficiency was established to incorporate poorly soluble QC, which further amplified its anti-cancer efficacy when tested in cervical cancer cells.

[19]. Quercetin has also been reported to inhibit the growth of different types of cancer cells, including colorectal, prostate, liver, pancreatic and lung, by modulation of various cellular processes [20–22]. In addition, QC has been found to exhibit selective cytotoxic activity towards cancer cells without adversely affecting normal cells [23]. In spite of these various pharmacological properties, QC has limited applications in the pharmaceutical field due to its poor aqueous solubility and physiological instability [24]. Chebil et al. reported the intrinsic solubility of QC to be < 0.01 g/L at 20 °C [25]. Additionally, the octanol-water partition coefficient (log P) of QC is 1.82 ± 0.32 [26]. These properties lead to poor bioavailability, poor permeability and instability of QC [27]. Therefore, there is a need to develop a novel delivery system for QC which could increase its solubility to obtain improved bioavailability and enhanced activity to biological systems. Liposomes are self-assembled lipid bilayer structures with hydrophilic as well as hydrophobic components, where the hydrophobic tail is shielded from water by the hydrophilic head in an aqueous environment [28]. Generally, liposomes are defined as spherical vesicles with a wide particle size range, from about 30 nm to several micrometers [29]. Liposomes are typically composed of phospholipids, and in some cases, they are comprised of cholesterol. Both phospholipids and cholesterol provide structural framework and biological stability, thereby imparting biocompatibility to these carrier systems [30]. When PEG-conjugated lipids are present in the self-assembly of liposomes, they impart steric stabilization and prolong in vivo circulation time by reducing the recognition of opsonins and liposomal clearance by the mononuclear phagocyte system (MPS) [31]. Stealth liposomes are formed when a hydrophilic polymer (PEG) linked to the external hydrophilic phospholipid head (e.g. DSPE) is present in the formation of liposomes [32]. The ability of PEG chains to prolong circulation time of drug delivery carriers like liposomes is based on its “steric stabilization” property which can further lead to low liposomal aggregation and adsorption by plasma proteins [33–35]. The

2. Materials and methods 2.1. Materials Quercetin (≥95% HPLC), Triton 100-X, Dulbecco's Modified Eagle's Medium (DMEM) and Dulbecco's phosphate buffered saline (D-PBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cholesterol (ovine wool, > 98%), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (ammonium salt) (DSPEPEG2000) and L-α-phosphatidylcholine (egg, chicken) (egg PC) lipids were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Chloroform (≥99%, molecular biology grade), orthophosphoric acid (≥85% HPLC), fetal bovine serum (FBS), penicillin/streptomycin and trypsin-EDTA were purchased from VWR (Radnor, PA, USA). Methanol (HPLC grade) was obtained from EMD Millipore (Burlington, MA, USA). Acetonitrile (HPLC grade) was obtained from J.T. Baker (Radnor, PA, USA). Phosphate buffered saline (PBS) was purchased from Boston Bio Products (Ashland, MA, USA). The cervical cancer cells (HeLa) were obtained from ATCC (Manassas, VA, USA). CellTiter 96® AQueous One Solution Cell Proliferation Assay kits for the MTS assay was purchased from Promega (Madison, WI, USA). Deionized water was obtained with a Millipore Water Purification System (Bedford, MA, USA). 2.2. Preparation and characterization of stealth liposomes QC-loaded stealth liposomes were prepared by the traditional thinfilm hydration method. Liposomes having final lipid concentrations of 5 mg/mL were prepared using the three lipids, egg-PC, cholesterol and DSPE-PEG2000 (each dissolved in chloroform having a concentration of 25 mg/mL) in three different molar ratios as indicated in Table 1, to evaluate the effect of the different molar percentages of DSPE-PEG2000 lipid (3, 5 and 7%) on the physicochemical characterization and encapsulation efficiency (EE%) of liposomes. The solution of QC was prepared in methanol (5 mg/mL) and was added when necessary to achieve the final QC concentration of 400 μM in the liposomes. Desired volumes of lipids and QC solutions were added to a culture tube followed by the removal of the organic solvent using a rotary evaporator

Fig. 1. Chemical structure of quercetin [5]. 2

Journal of Drug Delivery Science and Technology 55 (2020) 101477

A.L. Saraswat and T.J. Maher

at room temperature until reaching equilibrium. The content of QC in PBS was then analyzed using HPLC and the intrinsic solubility was determined.

Table 1 Composition of the stealth liposomal formulations at different molar ratios of lipids. Formulations

Egg-PC (%mol)

CHOL (%mol)

DSPE-PEG2000 (%mol)

Lipo-3%PEG Lipo-5%PEG Lipo-7%PEG

67 65 63

30 30 30

3 5 7

2.5. Stability studies Stealth liposomes were prepared at three different molar percentages of DSPE-PEG2000 lipid (3, 5 and 7%) and analyzed for their particle size, PDI and zeta potential at intervals of 7 days for 3 weeks. Additionally, the optimized stealth liposomes prepared with 5% of DSPE-PEG2000 lipid were analyzed for their EE% and apparent solubility at intervals of 7 days for 3 weeks in order to evaluate the stability of this formulation for performing in vitro studies. All the formulations were stored at 4 °C during this period.

(Buchi Rotavapor R-110, New Castle, DE, USA). Dried lipid film obtained was then kept under vacuum for 10 min to eliminate any residual solvent. The dried thin lipid film was then hydrated with phosphate buffered saline (10 mM, pH 7.4) followed by sonication in an ice-bath at an amplitude of 30% for a period of 10 min with pulse of 10 s using a probe sonicator (Model 100 Sonic Dismembrator, VWR). The obtained dispersion was then centrifuged at 3,500 g at 4 °C to separate the unentrapped QC from the QC-loaded liposomes. Blank stealth liposomes were also prepared at three different molar ratios of the lipids as listed in Table 1 using a similar procedure but without incorporating QC. The prepared stealth liposomes were then characterized for their average particle size, polydispersity index (PDI) and zeta potential by dynamic light scattering using a 90PLUS particle size and zeta potential analyzer (Brookhaven, NY, USA).

2.6. Cell culture Cervical cancer cells (HeLa) were cultured at 37 °C with 5% CO2 in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin solution. The cell line was sub cultured with 0.25% trypsin/ 1 mM EDTA solution when the cells reached 80–85% confluency. 2.7. In vitro cytotoxicity studies

2.3. HPLC analysis HeLa cells were seeded in 96-well plates at a density of 1x103 cells/ well and incubated for 24 h at 37 °C. Then the cells were treated with different concentrations of QC (5–200 μM) for 24, 48 and 72 h. After treatment, the cells were incubated with the MTS reagent for 1 h at 37 °C. The absorbance was then measured at 490 nm using a microplate reader (Synergia, FL, USA). Cell viability was calculated using the following equation:

An Agilent 1100 HPLC system (Agilent, CA, USA) equipped with an autosampler and a UV detector set at 370 nm was used to develop a method for quantification of QC [38]. A Hypersil ODS-C18 column (150 × 4.6 mm, 5 μm) (Alltech, CA, USA) was used for the elution of QC. The temperature of the column was maintained at 25 °C, the mobile phase composition used was acetonitrile and 0.4% v/v orthophosphoric acid at pH 2.8 in a ratio of 45:55 v/v, with a flow rate of 1 mL/min and the injection volume was 10 μL. Standard solutions of QC were prepared in methanol and used for generating the calibration curve for the developed HPLC method. The developed HPLC method was validated by determining the limit of detection (LOD) and limit of quantification (LOQ), based on the signal-to-noise ratio (S/N) method. A S/N of 3:1 is generally acceptable for estimating LOD and a S/N of 10:1 is used for estimating LOQ. The signal-to-noise ratio (S/N) was determined using the following equation:

S 2H = N h

Cell Viability % =

100%

(1) 2.8. Statistical analyses All experiments were carried out in triplicate and values expressed as mean ± SD. Statistically significant differences were determined using One-way ANOVA followed by Tukey's multiple comparison test or Dunnett's test. Grouped analyses were performed by Two-way ANOVA followed by Sidak's multiple comparison test. Statistically significant differences in all cases were defined as a p-value of < 0.05.

2.4. Encapsulation efficiency and solubility studies Samples of QC-loaded stealth liposomes were diluted in methanol and Triton X-100 followed by their injection into the HPLC system [39–41]. The amount of QC encapsulated in the liposomes was assessed using the HPLC method that was employed for establishing the standard curve of QC. The encapsulation efficiency (EE%) of QC in the stealth liposomes was calculated using the following equation:

Total weight of QC in liposomes X 100% Actual weight of QC added

(3)

To evaluate the cytotoxicity of the optimized stealth liposomal formulation, a similar method was performed and HeLa cells were treated with different concentrations of QC encapsulated in the liposomes (5–200 μM) for 24, 48 and 72 h. The cytotoxic effect of blank liposomes was also measured under the same conditions. The halfmaximal inhibitory concentration (IC50) values were then calculated.

where H is the height of the peak corresponding to QC in the chromatogram and h is the height of the background noise obtained in the chromatogram.

EE % =

(Abs intensity of treated cells − Abs of medium) x (Abs intensity of control − Abs of medium)

3. Results 3.1. Preparation and characterization of stealth liposomes The mean particle size, PDI and zeta potential of QC-loaded and blank stealth liposomes prepared with three different molar ratios of lipids are summarized in Table 2. The liposomal formulations appeared to be a turbid dispersion with no presence of visible particles or aggregates (see Supplementary material – Fig. S1). QC-loaded liposomes had an average particle size of 109.79 ± 2.92 nm, an average PDI of 0.169 ± 0.015 and an average zeta potential of −3.69 ± 1.95 mV with no statistically significant differences observed (p > 0.05) .

(2)

The concentration of QC obtained in the stealth liposomes by HPLC analysis was considered as the apparent solubility of QC in the liposomal formulation. In order to determine the saturation solubility of QC, an excess amount of QC was added to PBS at pH 7.4 followed by constant stirring 3

Journal of Drug Delivery Science and Technology 55 (2020) 101477

A.L. Saraswat and T.J. Maher

However, QC-loaded stealth liposomes demonstrated a significantly higher cytotoxic effect on HeLa cells when compared to free QC, at each of the three treatment periods. Fig. 5 represents the statistically significant decrease observed in the average IC50 values obtained for QCloaded liposomes as compared to free QC. The IC50 value of QC (> 200 μM) achieved at 24 h versus QC-loaded liposomes (184 μM); the IC50 value of QC (134 μM) achieved at 48 h versus QC-loaded liposomes (40 μM); the IC50 value of QC (65 μM) achieved at 72 h versus QCloaded liposomes (14 μM).

Table 2 Physicochemical characterization of quercetin-loaded and blank stealth liposomes prepared with different molar percentages of DSPE-PEG2000 lipid (n = 3, mean ± SD). Formulation

Particle size (nm)

Polydispersity index

Zeta potential (mV)

QC Lipo-3%PEG QC Lipo-5%PEG QC Lipo-7%PEG BLK Lipo-3% PEG BLK Lipo-5% PEG BLK Lipo-7% PEG

112.64 109.94 106.80 109.74

0.186 0.166 0.157 0.182

−4.59 −5.03 −1.45 −7.52

± ± ± ±

3.91 2.33 4.73 3.39

± ± ± ±

0.007 0.005 0.026 0.002

± ± ± ±

3.54 1.74 3.17 2.24

102.02 ± 1.53

0.166 ± 0.008

−5.23 ± 3.56

95.93 ± 0.60

0.182 ± 0.014

−4.22 ± 4.12

4. Discussion Quercetin inhibits the proliferation of cancer cell lines of different origin, induces senescence, inhibits telomerase, induces autophagy in cancer cells and can also activate immune destruction [42]. Nevertheless, the potential use of QC as a safe and effective therapeutic agent in cancer treatment is limited due to its poor aqueous solubility, low bioavailability and instability in physiological media. Previous literature has reported the formulation optimization of QCloaded liposomes prepared with dipalmitoylphosphatidylcholine (DPPC) and cholesterol to study the effect of the lipid ratio and QC concentration on the physicochemical properties with no account of any in vitro studies [43]. Cognitive effects of QC have been demonstrated via intranasal delivery of liposomal QC formulated with phosphatidylcholine (PC) and cholesterol in male wistar rats [44]. Gang et al. revealed that coating QC nanoliposomes with DSPE-PEG2000 inhibits the growth of glioma cancer cells [45]. However, there were no reports demonstrating the effect of different PEGylated lipid concentration on the physicochemical properties of the liposomal formulation. Because no investigations about the stability of various nanocarrier systems have been reported in literature, it was crucial to determine the stability of the developed QC-loaded liposomal formulation. Therefore, the primary objective of our study was to develop a unique stealth liposomal system by optimizing PEGylated lipid concentration to increase the aqueous solubility and stability of QC, as well as to determine if its cytotoxic effect in vitro was enhanced. Saturation solubility measurements performed for QC in an aqueous environment resulted in a value of 0.0025 ± 0.0002 mg/mL. This experimental value is in accordance with the value determined by King et al. where the aqueous solubility of QC was determined at different temperatures up to 100 °C and the aqueous solubility of QC was found to be 0.00215 mg/mL at 25 °C [46].

QC denotes quercetin-loaded liposomes and BLK denotes blank liposomes.

3.2. HPLC analysis A standard curve for QC was established and a linear relationship was observed between the concentration and area with a correlation coefficient (r2) of 0.9996 (see Supplementary material – Fig. S2). The LOD and LOQ of QC determined by the S/N method were found to be 0.01 and 0.039 μg/mL, respectively. 3.3. Encapsulation efficiency and solubility studies The EE% of QC-loaded stealth liposomes prepared with 3, 5 and 7% of DSPE-PEG2000 lipid were found to be 90, 88 and 81%, respectively. Fig. 2 depicts the statistically significant decrease in the EE% of the stealth liposomes with increases in the DSPE-PEG molar percentage from 3 to 5 and 7% (p < 0.01, p < 0.0001). The saturation solubility of QC was obtained to be 0.0025 ± 0.0002 mg/mL. Encapsulation of QC in the stealth liposomal system resulted in a significant increase in its solubility when compared to the free drug. As shown in Fig. 3, the solubility of QC was improved by 45, 44 and 41-fold after its encapsulation in stealth liposomes prepared with 3, 5 and 7% of DSPE-PEG2000 lipid, respectively. Also, there was a significant decrease found in the apparent solubility of QC with increases in molar percentage of DSPE-PEG2000 from 3 to 5 and 7% (p < 0.0001) in these liposomal formulations. These results are in accordance with the results obtained for the EE% of QC-loaded liposomes. 3.4. Stability studies The stability of all the QC-loaded liposomal formulations was observed during storage within 21 days at 4 °C. All liposomal formulations exhibited ideal stability in terms of particle size, PDI and zeta potential (see Supplementary material - Table S1, S2 and S3). The optimized stealth liposomes demonstrated an encapsulation of approximately 89% and apparent solubility of 0.11 mg/mL during 21 days at 4 °C, indicating the stability of this formulation for performing in vitro studies (see Supplementary material – Fig. S3). 3.5. In vitro cytotoxicity studies The cytotoxic effect of QC and QC-loaded liposomes was measured by performing the MTS cell proliferation assay in HeLa cells at 24, 48 and 72 h of treatment period. As shown in Fig. 4(a–c), QC exhibited a cytotoxic effect at higher concentrations (25–200 μM) in a concentration-dependent manner after 24, 48 and 72 h of treatment. Similarly, QC-loaded liposomes also exhibited a cytotoxic effect at all concentrations in a concentration-dependent manner (5–200 μM) after 24, 48 and 72 h of treatment (Fig. 4(d–f)). Both QC and QC-loaded liposomes exhibited a time-dependent growth suppressive activity with maximum cytotoxic effect observed at 72 h of treatment period.

Fig. 2. Effect of molar percentage of DSPE-PEG2000 lipid on encapsulation efficiency of quercetin-loaded stealth liposomes. Data represents mean ± SD (n = 3). **p < 0.01; ****p < 0.0001. 4

Journal of Drug Delivery Science and Technology 55 (2020) 101477

A.L. Saraswat and T.J. Maher

Fig. 5. Comparison of IC50 values obtained for free quercetin versus quercetinloaded stealth liposomes at 24, 48 and 72 h treatment period. IC50 value obtained for free quercetin at 24 h represents > 200 μM. Data represents mean ± SD (n = 3). **p < 0.01; ****p < 0.0001.

liposomes to avoid uptake by the RES system, enhancing the circulation time of the encapsulated drug and imparting steric stabilization to the liposomal formulation and hence the term, ‘stealth’ liposomes [31]. Depending on the grafting density of PEG lipids on the phospholipid bilayer, the PEG component can form either “mushroom” (less than 5 mol% of DSPE-PEG2000) or “brush” conformations (between 5 and 10 mol% of DSPE-PEG2000) [50]. Since PEG-conjugated lipids are incorporated into liposomes to impart steric stabilization, the concentration of PEG lipids was kept between 5 and 10 mol%, when the PEG-lipid is in the brush conformation. As the average particle size obtained for all formulations was between 105 and 115 nm with average PDI values between 0.15 and 0.20 and average zeta potential values ranging between −5.00 and −1.00 mV, the stealth liposomal formulations prepared had narrow size distribution and a neutral surface charge. Additionally, the decrease in EE% of QC-loaded liposomes from 90% to 80% with increase in molar percentage of DSPE-PEG2000 from 3% to 7% may be attributed to the ability of PEGylated lipids acting on the liposomal surface to restrict the free volume available within the lipid bilayers for carrying QC [51]. These results are in accordance with the decrease in apparent solubility of QC in stealth liposomes with increase in molar percentage of DSPE-PEG2000 lipid from 3% to 7%. Stability studies revealed that all the formulations were ideally stable for 21 days in terms of their physicochemical characterization and the optimized QC-loaded liposomes had an encapsulation efficiency of 89% within this period. Also, the apparent solubility of QC in this

Fig. 3. Effect of molar percentage of DSPE-PEG2000 lipid on apparent solubility of quercetin in stealth liposomes. Data represents mean ± SD (n = 3). ***p < 0.001; ****p < 0.0001.

Different delivery systems have previously been developed to improve the physiological properties of various pharmacologically active compounds in order to realize their health benefits [47]. Watanatorn et al. studied the encapsulation of QC in liposomes prepared with eggPC and cholesterol (1:1) [48]. These liposomes were approximately 200 nm in mean particle size, with a negative surface charge and an encapsulation efficiency of 60%–80%, demonstrating improved solubility of QC for its enhanced delivery to the central nervous system. Liposomes are biocompatible and non-toxic drug delivery carriers which can encapsulate hydrophobic compounds between its lipid bilayers to solve the challenge of its poor aqueous solubility. They release the encapsulated drug in a controlled manner and also accumulate preferentially in the tumor tissue through the EPR effect so as to improve their therapeutic efficacy and reduce their toxicity profile [49]. Stealth liposomes formulated in this study were composed of three different lipids, egg-PC, cholesterol and DSPE-PEG2000, each having its own significance in the structure and activity of liposomes. DSPEPEG2000 is a PEG-conjugated lipid that modifies the surface of the

Fig. 4. In vitro concentration and time-dependent cytotoxic effect induced by free quercetin: (a) 24 h; (b) 48 h; (c) 72 h and quercetin-loaded stealth liposomes: (d) 24 h; (e) 48 h; (f) 72 h with the representative average IC50 values. Data represents mean ± SD (n = 3). 5

Journal of Drug Delivery Science and Technology 55 (2020) 101477

A.L. Saraswat and T.J. Maher

5. Conclusion

formulation was found to be 0.11 mg/mL which results in about a 44fold increase in the solubility of QC. Moreover, when the molar percentage of DSPE-PEG2000 lipid is 5%, the PEGylated lipid chains extend from the liposomal surface to form a brush regime imparting steric stability to the liposomes [50]. Hence, liposomes prepared with 5% DSPE-PEG2000 were selected to perform the in vitro cytotoxicity studies due to their small size (~100 nm), narrow size distribution (~0.15), neutral surface charge (~-5.00 mV) and approximately 89% encapsulation efficiency as well as their stability up to 21 days. Liposomes have previously been demonstrated to be one of the approaches for enhancing the solubility of poorly water-soluble drugs, improving their bioavailability as well as for their successful delivery to the cells [52]. Hence, we postulated that encapsulation of a poorly water-soluble anticancer agent, QC, in an optimized stealth liposomal system, would enhance its aqueous solubility and physiological stability, improve its bioavailability and therefore escalate its cytotoxicity towards cancer cells. At every treatment period, free QC showed a hormetic effect in HeLa cells. Hormesis refers to a biphasic concentration-response observed in a cell or an organism on exposure to a toxin, characterized by a low concentration stimulation and a high concentration inhibitory effect [53]. The chemopreventive effects of QC observed at the lower concentration of 5 μM are likely to be mediated by its antioxidant properties [54,55]. At higher concentrations, inhibition of cell cycle regulatory pathways such as PI3K-AKT/PKB, ROS generation, activation of the p53 tumor suppressor protein and caspases activation likely contribute to its apoptotic effect observed in HeLa cells [56]. This phenomenon is also stated as the ‘quercetin paradox’ due to the dual, concentration-dependent effect of QC observed in cancer cells as previously described by Boots et al. [57]. For the 24 h treatment period, it was observed that > 200 μM of QC would be required to kill 50% of the cell population as low cytotoxicity was observed (~90% cell viability). Whereas, the IC50 value of QC-loaded liposomes was obtained as 184 μM which was significantly lower than free QC for 24 h treatment period. For the 48 h treatment period, an IC50 value of 134 μM was obtained whereas a significantly lower concentration of 40 μM was required for the QC-loaded liposomes to decrease the cell viability by 50%. For the 72 h treatment period, an IC50 value of 65 μM was obtained whereas a significantly lower concentration of 14 μM was required for the QC-loaded liposomes to decrease at least 50% of the cell population. Hence, a statistically significant decrease of the IC50 value was observed when HeLa cells were treated with QC-loaded liposomes compared to free QC. Seo et al. evaluated the cytotoxic effect of QC in breast cancer cells (BT-474) at 24, 48 and 72 h of treatment period and observed a concentration-dependent (20–100 μM) inhibition of cell viability with a time-dependent growth suppressive activity with the highest cytotoxicity observed at 72 h [58]. Similarly, with increase in the treatment incubation period, we observed an increase in the cytotoxic effect for both free QC and QC-loaded liposomes in HeLa cells. However, the cytotoxicity produced by QC-loaded liposomes was more pronounced when compared to the QC alone, based on the statistically significant difference obtained in their IC50 values at all the three treatment periods. This demonstrates the enhanced cytotoxicity achieved by encapsulating a poorly water-soluble compound, QC, in stealth liposomal system due to its increased solubility, improved bioavailability and hence, augmented therapeutic efficacy. Optimized QC-loaded stealth liposomes developed in our study have shown effective cell killing in vitro, and to the best of authors’ knowledge there have been no previous reports revealing the effect of molar percentage of DSPE-PEG2000 lipid on physicochemical properties including particle size, zeta potential and encapsulation efficiency as well as stability of liposomal QC. This is the first study reporting the successful delivery of QC in cervical cancer cells by its incorporation in the optimized unique stealth liposomal system which overcomes the barrier of its poor aqueous solubility and augments its anticancer activity for the treatment of cervical cancer.

In this study, we report the initial development and characterization of stealth liposomal formulations to incorporate QC for treatment of cervical cancer. Three liposomal formulations were developed that enabled relatively higher solubility of QC. The optimized and stable liposomal formulation significantly increased the solubility of QC by 44-fold. Cytotoxicity studies demonstrated that liposomal encapsulation of QC significantly enhanced its cytotoxic effect on cervical cancer cells when compared to free QC. In conclusion, our developed and optimized nanoliposomal formulation is a promising drug delivery carrier system that can be employed to enhance the aqueous solubility and antitumor efficacy of QC, laying the foundation for further in vitro and in vivo studies for cervical cancer therapy. Author statement The authors have revised and resubmitted the manuscript “Development and optimization of stealth liposomal system for enhanced in vitro cytotoxic effect of quercetin” in both marked-up and non-marked-up versions, abstract, and figures after addressing all of the reviewers’ comments. Declaration of competing interest The authors have confirmed that there are no conflicts of interest. Acknowledgement The authors are grateful to the Department of Pharmaceutical Sciences, School of Pharmacy, MCPHS University at Boston, for providing the financial support and necessary facilities for this research work. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jddst.2019.101477. References [1] X. Cai, Z. Fang, J. Dou, A. Yu, G. Zhai, Bioavailability of quercetin: problems and promises, Curr. Med. Chem. 20 (2013) 2572–2582. [2] S.C. Bischoff, Quercetin: potentials in the prevention and therapy of disease, Curr. Opin. Clin. Nutr. Metab. Care 11 (2008) 733–740. [3] W. Wang, C. Sun, L. Mao, P. Ma, F. Liu, L. Yang, Y. Gao, The biological activities, chemical stability, metabolism and delivery systems of quercetin: a review, Trends Food Sci. Technol. 56 (2016) 21–38. [4] M. Dueñas, S. González-Manzano, A. González-Paramás, C. Santos-Buelga, Antioxidant evaluation of o-methylated metabolites of catechin, epicatechin and quercetin, J. Pharm. Biomed. Anal. 51 (2010) 443–449. [5] S.F. Nabavi, G.L. Russo, M. Daglia, S.M. Nabavi, Role of quercetin as an alternative for obesity treatment: you are what you eat!, Food Chem. 179 (2015) 305–310. [6] S. Anandam, S. Selvamuthukumar, Fabrication of cyclodextrin nanosponges for quercetin delivery: physicochemical characterization, photostability, and antioxidant effects, J. Mater. Sci. 49 (2014) 8140–8153. [7] R. Kleemann, L. Verschuren, M. Morrison, S. Zadelaar, M.J. van Erk, P.Y. Wielinga, T. Kooistra, Anti-inflammatory, anti-proliferative and anti-atherosclerotic effects of quercetin in human in vitro and in vivo models, Atherosclerosis 218 (2011) 44–52. [8] F. Dajas, Life or death: neuroprotective and anticancer effects of quercetin, J. Ethnopharmacol. 143 (2012) 383–396. [9] J. Duarte, R. Pérez-Palencia, F. Vargas, M.A. Ocete, F. Pérez-Vizcaino, A. Zarzuelo, J. Tamargo, Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats, Br. J. Pharmacol. 133 (2001) 117–124. [10] K.A. Conklin, Dietary antioxidants during cancer chemotherapy: impact on chemotherapeutic effectiveness and development of side effects, Nutr. Cancer 37 (2000) 1–18. [11] B.H. Kim, J.S. Choi, E.H. Yi, J.K. Lee, C. Won, S.K. Ye, M.H. Kim, Relative antioxidant activities of quercetin and its structurally related substances and their effects on NF-κB/CRE/AP-1 signaling in murine macrophages, Mol. Cells 35 (2013) 410–420. [12] M. Valko, C.J. Rhodes, J. Moncol, M. Izakovic, M. Mazur, Free radicals, metals and antioxidants in oxidative stress-induced cancer, Chem. Biol. Interact. 160 (2006)

6

Journal of Drug Delivery Science and Technology 55 (2020) 101477

A.L. Saraswat and T.J. Maher

[36] N. Dos Santos, C. Allen, A.M. Doppen, M. Anantha, K.A. Cox, R.C. Gallagher, G. Karlsson, K. Edwards, G. Kenner, L. Samuels, M.S. Webb, M.B. Bally, Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: relating plasma circulation lifetimes to protein binding, Biochim. Biophys. Acta 1768 (2007) 1367–1377. [37] M. Nabi-Meibodi, A. Vatanara, A.R. Najafabadi, M.R. Rouini, V. Ramezani, K. Gilani, S.M.H. Etemadzadeh, K. Azadmanesh, The effective encapsulation of a hydrophobic lipid-insoluble drug in solid lipid nanoparticles using a modified double emulsion solvent evaporation method, Colloids Surfaces B Biointerfaces 112 (2013) 408–414. [38] N. Sanghavi, S.D. Bhosale, Y. Malode, RP-HPLC method development and validation of quercetin isolated from the plant tridax procumbens L, J Sci Innov Res JSIR 3 (2014) 594–597. [39] J.R. Jimah, P.H. Schlesinger, N.H. Tolia, Liposome disruption assay to examine lytic properties of biomolecules, Bio-protocol 7 (2017) 1–16. [40] S. Kalipatnapu, A. Chattopadhyay, Critical review membrane protein Solubilization : recent advances and challenges in solubilization of serotonin 1A receptors, IUBMB Life 57 (2005) 505–512. [41] A.M. Seddon, P. Curnow, P.J. Booth, Membrane proteins, lipids and detergents: not just a soap opera, Biochim. Biophys. Acta 1666 (2004) 105–117. [42] M. Russo, C. Spagnuolo, I. Tedesco, S. Bilotto, G.L. Russo, The flavonoid quercetin in disease prevention and therapy: facts and fancies, Biochem. Pharmacol. 83 (2012) 6–15. [43] L.R. Tefas, D.M. Muntean, L. Vlase, A.S. Porfire, M. Achim, I. Tomuță, Quercetinloaded liposomes: formulation optimization through a D-optimal experimental design, Farmacia 63 (2015) 26–33. [44] T. Tong-un, S. Muchimapura, W. Phachonpai, J. Wattanathorn, Effects of quercetin encapsulated liposomes via nasal Administration : a novel cognitive enhancer, Am. J. Appl. Sci. 7 (2010) 906–913. [45] W. Gang, W.J. Jie, Z.L. Ping, D.S. Ming, L.J. Ying, W. Lei, Y. Fang, Liposomal quercetin: evaluating drug delivery in vitro and biodistribution in vivo, Expert Opin. Drug Deliv. 9 (2012) 599–613. [46] K. Srinivas, J.W. King, L.R. Howard, J.K. Monrad, Solubility and solution thermodynamic properties of quercetin and quercetin dihydrate in subcritical water, J. Food Eng. 100 (2010) 208–218. [47] D.J. McClements, E.A. Decker, Y. Park, J. Weiss, Structural design principles for delivery of bioactive components in nutraceuticals and functional foods, Crit. Rev. Food Sci. Nutr. 49 (2009) 577–606. [48] A. Priprem, J. Watanatorn, S. Sutthiparinyanont, W. Phachonpai, S. Muchimapura, Anxiety and cognitive effects of quercetin liposomes in rats, Nanomedicine 4 (2008) 70–78. [49] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer Res. 46 (1986) 6387–6392. [50] M.L. Immordino, F. Dosio, L. Cattel, Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential, Int. J. Nanomed. 1 (2006) 297–315. [51] A.R. Nicholas, M.J. Scott, N.I. Kennedy, M.N. Jones, Effect of grafted polythylene glycol (PEG) on the size, encapsulation efficiency and permeability of vesicles, Biochim. Biophys. Acta Biomembr. 1463 (2000) 167–178. [52] A.R. Mohammed, N. Weston, A.G.A. Coombes, M. Fitzgerald, Y. Perrie, Liposome formulation of poorly water soluble drugs: optimisation of drug loading and ESEM analysis of stability, Int. J. Pharm. 285 (2004) 23–34. [53] M.P. Mattson, Hormesis defined, Ageing Res. Rev. 7 (2008) 1–7. [54] W. Wätjen, G. Michels, B. Steffan, P. Niering, Y. Chovolou, A. Kampkötter, Q.H. Tran-Thi, P. Proksch, R. Kahl, Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis, J. Nutr. 135 (2018) 525–531. [55] A. Bagde, K. Patel, A. Mondal, S. Kutlehria, N. Chowdhury, A. Gebeyehu, N. Patel, N. Kumar, M. Singh, Combination of UVB absorbing titanium dioxide and quercetin nanogel for skin cancer chemoprevention, AAPS PharmSciTech 20 (2019) 1–12. [56] S. Chirumbolo, Quercetin in cancer prevention and therapy, Integr. Cancer Ther. 12 (2013) 97–102. [57] A.W. Boots, H. Li, R.P. Schins, R. Duffin, J.W. Heemskerk, A. Bast, G.R. Haenen, The quercetin paradox, Toxicol. Appl. Pharmacol. 222 (2007) 89–96. [58] H.S. Seo, J.M. Ku, H.S. Choi, Y.K. Choi, J.K. Woo, M. Kim, I. Kim, C.H. Na, H. Hur, B.H. Jang, Y.C. Shin, S.G. Ko, Quercetin induces caspase-dependent extrinsic apoptosis through inhibition of signal transducer and activator of transcription 3 signaling in HER2-overexpressing BT-474 breast cancer cells, Oncol. Rep. 36 (2016) 31–42.

1–40. [13] C.G.M. Heijnen, G.R.M.M. Haenen, R. Minou Oostveen, E.M. Stalpers, A. Bast, Protection of flavonoids against lipid peroxidation: the structure activity relationship revisited, Free Radic. Res. 36 (2002) 575–581. [14] N. Gulati, B. Laudet, V.M. Zohrabian, R. Murali, M. Jhanwar-Uniyal, The antiproliferative effect of quercetin in cancer cells is mediated via inhibition of the PI3K-Akt/PKB pathway, Anticancer Res. 26 (2006) 1177–1181. [15] F.O. Ranelletti, N. Maggiano, F.G. Serra, R. Ricci, L.M. Larocca, P. Lanza, G. Scambia, A. Fattorossi, A. Capelli, M. Piantelli, Quercetin inhibits p21-RAS expression in human colon cancer cell lines and in primary colorectal tumors, Int. J. Cancer 85 (2000) 438–445. [16] F. Casagrande, J.M. Darbon, Effects of structurally related flavonoids on cell cycle progression of human melanoma cells: regulation of cyclin-dependent kinases CDK2 and CDK1, Biochem. Pharmacol. 61 (2001) 1205–1215. [17] G. Sliutz, J. Karlseder, C. Tempfer, L. Orel, G. Holzer, M.M. Simon, Drug resistance against gemcitabine and topotecan mediated by constitutive hsp70 overexpression in vitro: implication of quercetin as sensitiser in chemotherapy, Br. J. Canc. 74 (1996) 172–177. [18] L.T. Lee, Y.T. Huang, J.J. Hwang, P.P. Lee, F.C. Ke, M.P. Nair, C. Kanadaswam, M.T. Lee, Blockade of the epidermal growth factor receptor tyrosine kinase activity by quercetin and luteolin leads to growth inhibition and apoptosis of pancreatic tumor cells, Anticancer Res. 22 (2002) 1615–1627. [19] E. Braganhol, L.L. Zamin, A.D. Canedo, F. Horn, A.S. Tamajusuku, M.R. Wink, C. Salbego, A.M. Battastini, Antiproliferative effect of quercetin in the human U138MG glioma cell line, Anti Canccer Drugs 17 (2006) 663–671. [20] H. Kim, E.M. Seo, A.R. Sharma, B. Ganbold, J. Park, G. Sharma, Y.H. Kang, D.K. Song, S.S. Lee, J.S. Nam, Regulation of Wnt signaling activity for growth suppression induced by quercetin in 4T1 murine mammary cancer cells, Int. J. Oncol. 43 (2013) 1319–1325. [21] B.E. Shan, M.X. Wang, R. Li, Quercetin inhibit human SW480 colon cancer growth in association with inhibition of cyclin D1 and survivin expression through wnt/ beta-catenin signaling pathway, Canc. Invest. 27 (2009) 604–612. [22] K.W. Lee, A.M. Bode, Z. Dong, Molecular targets of phytochemicals for cancer prevention, Nat. Rev. Cancer 11 (2011) 211–218. [23] S.A. Chowdhury, K. Kishino, R. Satoh, K. Hashimoto, H. Kikuchi, H. Nishikawa, Y. Shirataki, H. Sakagami, Tumor-specificity and apoptosis-inducing activity of stilbenes and flavonoids, Anticancer Res. 25 (2005) 2055–2063. [24] A. Kumari, S.K. Yadav, Y.B. Pakade, B. Singh, S.C. Yadav, Development of biodegradable nanoparticles for delivery of quercetin, Colloids Surfaces B Biointerfaces 80 (2010) 184–192. [25] L. Chebil, C. Humeau, J. Anthoni, F. Dehez, J.M. Engasser, M. Ghoul, Solubility of flavonoids in organic solvents, J. Chem. Eng. Data 52 (2007) 1552–1556. [26] J.A. Rothwell, A.J. Day, M.R.A. Morgan, Experimental determination of octanolwater partition coefficients of quercetin and related flavonoids, J. Agric. Food Chem. 53 (2005) 4355–4360. [27] V. Atul Bhattaram, U. Graefe, C. Kohlert, M. Veit, H. Derendorf, Pharmacokinetics and bioavailability of herbal medicinal products, Phytomedicine 9 (2002) 1–33. [28] C.W. Su, C.S. Chiang, W.M. Li, S.H. Hu, S.Y. Chen, Multifunctional nanocarriers for simultaneous encapsulation of hydrophobic and hydrophilic drugs in cancer treatment, Nanomedicine 9 (2014) 1499–1515. [29] A. Akbarzadeh, R. Rezaei-Sadabady, S. Davaran, S.W. Joo, N. Zarghami, Y. Hanifehpour, M. Samiei, M. Kouhi, K. Nejati-Koshki, Liposome: classification, preparation, and applications, Nanoscale Res Lett 8 (2013) 102–110. [30] M. Anderson, A. Omri, The effect of different lipid components on the in vitro stability and release kinetics of liposome formulations, Drug Deliv. 11 (2004) 33–39. [31] M.C. Woodle, D.D. Lasic, Sterically stabilized liposomes, Biochim. Biophys. Acta 1113 (1992) 171–199. [32] J. Hu, J. Wang, G. Wang, Z. Yao, X. Dang, Pharmacokinetics and antitumor efficacy of DSPE-PEG2000 polymeric liposomes loaded with quercetin and temozolomide: analysis of their effectiveness in enhancing the chemosensitization of drug-resistant glioma cells, Int. J. Mol. Med. 37 (2016) 690–702. [33] K. Hristova, A. Kenworthy, T.J. McIntosh, Effect of bilayer composition on the phase behavior of liposomal suspensions containing poly(ethylene glycol)-lipids, Macromolecules 28 (1995) 7693–7699. [34] A.L. Klibanov, K. Maruyama, V.P. Torchilin, L. Huang, Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes, FEBS Lett. 268 (1990) 235–237. [35] R. Bartucci, M. Pantusa, D. Marsh, L. Sportelli, Interaction of human serum albumin with membranes containing polymer-grafted lipids: spin-label ESR studies in the mushroom and brush regimes, Biochim. Biophys. Acta 1564 (2002) 237–242.

7