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. Author manuscript; available in PMC: 2019 Aug 2.
Published in final edited form as: Curr Drug Deliv. 2016;13(5):711–719. doi: 10.2174/1567201812666151027142412

Hesperetin liposomes for cancer therapy

Joy Wolfram 1,2,#,*, Bronwyn Scott 2,#, Kathryn Boom 2, Jianliang Shen 2, Carlotta Borsoi 2, Krishna Suri 2, Rossella Grande 3, Massimo Fresta 4, Christian Celia 2,3, Yuliang Zhao 1,5, Haifa Shen 2,6, Mauro Ferrari 2,7,*
PMCID: PMC6677249  NIHMSID: NIHMS1043782  PMID: 26502889

Abstract

Hesperetin is a compound from citrus fruit that has previously been found to exert anticancer activity through a variety of mechanisms. However, the application of hesperetin to cancer therapy has been hampered by its hydrophobicity, necessitating the use of toxic solubilizing agents. Here, we have developed the first liposome-based delivery system for hesperetin. Liposomes were fabricated using the thin-layer evaporation technique and physical and pharmacological parameters were measured. The liposomes remained stable for prolonged periods of time in serum and under storage conditions, and displayed anticancer efficacy in both H441 lung cancer cells and MDA-MB-231 breast cancer cells. Furthermore, the anticancer activity was not impaired in cells expressing the multidrug resistance protein 1 (MDR-1). In conclusion, the encapsulation of hesperetin in liposomes does not interfere with therapeutic efficacy and provides a biocompatible alternative to toxic solubilizing agents, thereby enabling future clinical use of this compound for cancer therapy.

Keywords: Breast cancer, Drug delivery, Flavanone, Lung cancer, MDR-1, Natural products

1. INTRODUCTION

Hesperetin (4’-methoxy-3’,5,7-trihydroxyflavanone) is a flavanone that can be found in citrus fruit. This natural compound has multiple properties that effectively reduce cancer growth. For instance, hesperetin has been found to induce apoptosis in cancer cells [1], prevent angiogenesis [2], suppress cell migration through inhibition of transforming growth factor beta (TGF-β) signaling [3], and reduce cancer cell proliferation [4]. In addition, hesperetin has been shown to reduce glucose uptake in cancer cells by downregulating the expression of glucose transporters [5]. However, hesperetin cannot be administered as a free drug due to poor water solubility. Consequently, in vitro application of this compound requires the use of solubilizing agents [3, 5], such as dimethyl sulfoxide (DMSO), which has previously been found to display toxicity [6].

Likewise, the intravenous administration of poorly water-soluble agents generally requires the use of toxic diluents. Indeed, several hydrophobic drugs have been clinically approved as formulations containing harmful solubilizers. For example, paclitaxel is formulated with polyethoxylated castor oil (Cremophor EL) and dehydrated ethanol [7], while docetaxel is dissolved in polysorbate 80 (Tween 80) [8]. Consequently, it is common for patients to require treatment with corticosteroids and antihistamines to counteract the adverse side effects arising from the use of these solubilizing agents [9]. Although conventional cytotoxic agents can be intravenously administered with toxic diluents, this is not an option for other hydrophobic drugs that are less potent. Namely, while chemotherapeutic agents usually show efficacy at nanomolar concentrations, natural compounds derived from edible products generally display anticancer properties at micromolar concentrations. As a consequence, a substantially higher amount of natural compounds and solubilizing agents are required to achieve therapeutic efficacy. Previously, heperetin treatment has successfully been performed in vivo through oral administration with carboxymethyl cellulose [4, 10]. However, intravenous injection of hesperetin necessitates the development of other non-toxic strategies for drug delivery. In general, higher drug bioavailability can be achieved using the intravenous delivery route as compared to the oral route. Since high doses of hesperetin (micromolar) are necessary to achieve anticancer efficacy, intravenous administration could provide a means of reaching a sufficient drug concentration to halt tumor progression.

Several nanoparticle platforms, including silicon [11], metal [12], and polymeric particles [13], have been developed to improve the systemic delivery of therapeutic agents. Nevertheless, liposomes generally present a more biocompatible approach for drug administration, since lipids are naturally present in the body in high amounts. In addition to reducing toxicity, liposomes can protect the cargo, prolong blood circulation times, and reduce uptake by the immune system [14]. In particular, stealth polymers, such as polyethylene glycol (PEG), are frequently incorporated into liposomes to form a steric barrier that prevents immunological recognition and delays uptake by the reticuloendothelial system [15]. Indeed, the approval of 14 liposomal drugs [16], seven of which are intended for cancer therapy, is a clear indication of the benefits of liposomal delivery. Previously, liposomes have been used for the successful in vitro delivery of hydrophobic natural compounds [17]. Poorly water-soluble agents can usually be encapsulated in the lipid bilayer with high encapsulation efficiency [14a]. It has been shown that hesperetin interacts with phospholipid membranes, presumably through incorporation between acyl chains [18]. Here, we have characterized, developed and evaluated the first liposome-based hesperetin formulation. The liposomes consist of phosphatidylcholine (PC) and cholesterol, both of which are naturally found in the cellular membrane. In addition, PEG has been added to the formulation to decrease protein binding, thereby increasing liposomal stability.

2. MATERIALS AND METHODS

2.1. Materials

Materials were acquired from the following sources: hesperetin from MP Biomedicals; cholesterol, bovine serum albumin, DMSO, Tween 80 and Triton X-100 from Sigma-Aldrich; hydrogenated PC (HPC) from Lipoid; paraformaldehyde from Electron Microscopy Sciences; distearoyl phosphoethanolamine methoxy polyethyleneglycol 2000 (DSPE-mPEG2000) and dioleoyl phosphoethanolamine lissamine rhodamine B sulfonyl (DOPE-Liss Rhod) from Avanti Polar Lipids; Nucleopore track-etch membranes from Whatman; multidrug resistance protein-1 (MDR-1) plasmid (#10957) from Addgene; Alexa Fluor 488 phalloidin; Gibco Dulbecco’s Modified Eagle Medium (DMEM), and ProLong Gold antifade reagent with 4’,6-diamidino-2-phenylindole (DAPI) from Life Technologies; Spectra/Por Float-A-Lyzer G2 from Spectrum Laboratories; Hyclone phosphate buffered saline (PBS), Hyclone RPMI 1640 media, Hyclone fetal bovine serum (FBS), and Corning cover glasses from Thermo Fisher Scientific; CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay from Promega; Zetasizer-Nano folded capillary cells from Malvern; MDA-MB-231 breast cancer cells and H441 lung cancer cells from ATTC; paclitaxel injection (6 mg paclitaxel/527 mg Chremophor EL, 49.7% v/v dehydrated alcohol, 2 mg citric acid) from the Houston Methodist Hospital pharmacy.

2.2. Liposome preparation

Control liposomes and hesperetin liposomes were prepared by dissolving HPC, cholesterol, and DSPE-mPEG2000 (160 mg, molar ratio 6:3:1) in a round bottom flask with a methanol and chloroform solution (3 ml, 1:2 v/v). For the hesperetin liposomes, hesperetin (16 mg) was further added to the organic solvents. A Rotavapor R-215 Apparatus System from Buchi was used to create a thin lipid film (50 °C, 180 rpm, 150 mBar). The film was left to dry overnight at room temperature to ensure complete evaporation of the organic solvents. Liposomes were then formed by adding PBS (8 ml) to the round bottom flask and subjecting the formulation to three consecutive rounds of heating (3 min, 60 °C water bath) and vortex mixing (3 min). To reduce the size of the liposomes and obtain a narrow size distribution, the formulation was extruded at 60 °C through polycarbonate membranes with a LIPEX extruder from Northern Lipids. Three membranes with different pore sizes (200 nm, 100 nm, and 50 nm) were used, and the formulation was passed through each membrane eight times. Finally, liposomes were sterilized by membrane filtration (polyethersulfone, 0.22 μm) in a laminar flow cabinet. Fluorescent liposomes were prepared as described above, with the addition of DOPE-Liss Rhod (molar ratio: 0.1) to the organic solvents. The fluorescent liposomes were protected from light during fabrication and storage.

2.3. Liposome characterization

A Zetasizer Nano ZS (ZEN 3600) from Malvern Instruments was used to perform dynamic light scattering (173° backscattering) to measure the average size and polydispersity index (PDI) of hesperetin liposomes and control liposomes. Liposomes (20 μl) were mixed with Milli-Q water (1 ml) and placed in a plastic cuvette. The Zetasizer Nano ZS was also used to measure the zeta potential by laser doppler micro-electrophoresis (Smoluchowski’s theory). Liposomes (20 μl) were diluted in phosphate buffer (10 mM, pH 7.4, 1 ml) and placed in a folded capillary cell. For both size and zeta potential analysis, five measurements with ten runs each were recorded.

2.4. Hesperetin encapsulation efficiency

Control liposomes and hesperetin liposomes were collected by centrifugation (90,000 x g, 4 °C, 1 h) using an ultracentrifuge. The supernatant was retrieved for analysis and the pellet was dissolved in methanol. Serial dilutions of hesperetin in methanol or PBS (containing 0.1% Tween 80) were prepared and the absorbance was measured with a UV/Vis spectrophotometer (DU 730) at a wavelength of 287 nm, as previously reported [19]. A standard curve was created to determine the drug content in the hesperetin liposomes and supernatant. The pellet and supernatant from the control liposomes were used as blanks. The encapsulation efficiency percentage was calculated by dividing the hesperetin amount in the pellet with the hesperetin amount added during liposome fabrication. The amount of hesperetin in the supernatant was also expressed as a percentage of the total amount added during fabrication.

2.5. Storage stability

The storage stability (4 °C) of hesperetin liposomes and control liposomes was monitored over a period of 35 days, during which the size, PDI, and zeta potential were measured on a weekly basis, as described in the liposome characterization section.

2.6. Liposomal stability in serum

A simulation of the conditions present during blood circulation was formulated in order to determine the stability of control liposomes and hesperetin liposomes. Sterile conditions were maintained throughout the experiment to avoid bacterial contamination, which could interfere with the measurements. Liposomes (250 μl) were added to a serum solution (1 ml, 70% FBS in PBS). The solution was then stirred with an electronic stirrer (37 °C, 700 rpm) for 384 h. At various time points, 20 μl of the liposome mixture was removed and the size and PDI were measured, as described in the liposome characterization section.

2.7. Drug release

The release of hesperetin from the liposomes was evaluated under magnetic stirring in a simulated in vivo environment (37 °C, 700 rpm, 624 h). Hesperetin liposomes (1 ml) were placed in a pre-wetted dialysis tube with a cellulose ester membrane (Spectra/Por Float-A-Lyzer G2, 100 kD molecular weight cutoff). The dialysis tube was submerged in a solution of PBS (200 ml) with 0.1% Tween 80, which has previously been used to maintain sink conditions in liposomal drug release studies [20]. At various time periods, 1 ml of PBS was removed for analysis and replaced with an equivalent quantity of fresh solution. A standard curve was created using serial dilutions of hesperetin in PBS solution with 0.1% Tween 80. The samples were then analyzed spectrophotometrically as described in the hesperetin encapsulation efficiency section. PBS containing 0.1% Tween 80 was used as a blank. The amount of released drug was expressed as a percentage of the initial amount of drug present in the liposomes.

2.8. Cell culture

MDA-MB-231 cells and H441 cells were cultured in DMEM with 10% FBS and in RPMI-1640 media with 10% FBS, respectively. Cells were maintained at 37 °C with 5% CO2. MDA-MB-231 cells were transfected with a commercial MDR-1 plasmid, according to a previously reported procedure [21].

2.9. Fluorescence microscopy

H441 cells were grown in an 8-well chamber slide at a density of 1.05 × 104 cells/well for 24 h and then treated with fluorescent hesperetin liposomes (100 μM hesperetin) for 24 h. Cells were fixed and stained as previously reported [22]. Briefly, cells were washed, fixed with 4% paraformaldehyde, permeabilized with Triton-X-100 and blocked with 1% bovine serum albumin in PBS. The cells were then exposed to Alexa fluor 488 phalloidin and DAPI to visualize filamentous actin and nuclei, respectively. An inverted fluorescent microscope was used to detect fluorescently labeled hesperetin liposomes in cells. Images were captured using 20x and 40x magnification.

2.10. Cell viability

The cell viability of MDA-MB-231 cells and H441 cells exposed to control liposomes, hesperetin liposomes (100 μM hesperetin) or hesperetin in DMSO (100 μM hesperetin, 0.2% DMSO) was measured with a cell proliferation assay (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay) according to the manufacturer’s instructions. Cells were seeded in a 96-well plate at a density of 2 × 103 cells/well (MDA-MB-231) and 2.5 × 103 cells/well (H441) for 24 h and then exposed to the treatment groups for 72 h. For the liposome groups, the viability of cells incubated with an identical volume of PBS served as a reference value. Similarly, cells exposed to 0.2% DMSO was used as a control for the group receiving hesperetin dissolved in DMSO. The absorbance was measured with a Synergy H4 Hybrid microplate reader from BioTek. Furthermore, the effect of hesperetin on MDA-MB-231 cells expressing MDR-1 was evaluated. Cells were seeded in a 96-well plate at a density of 2 × 103 cells/well (MDA-MB-231) and 3.5 × 103 cells/well (MDA-MB-231/MDR-1) for 24 h. To confirm that the MDR-1 expressing cells had acquired drug resistance, both cell lines were exposed to varying concentrations of a paclitaxel formulation (0–210 nM) for 72 h. The MDA-MB-231/MDR-1 cells were also treated with hesperetin (100 μM in liposomes or 0.2% DMSO) for 72 h. The cell viability was measured as described above. All experiments were performed in triplicate.

2.11. Statistical analysis

T-test comparisons (two-tailed, unpaired) were performed to evaluate statistical significance.

3. RESULTS

3.1. Liposome characterization

The results from the liposome characterization experiments demonstrate that the addition of hesperetin to the liposomal bilayer did not cause any major changes in the size, heterogeneity, and charge of the particles. In particular, dynamic light scattering analysis revealed that the control liposomes and hesperetin liposomes had an average size of 65 nm and 63 nm, respectively (Figure 1a). Furthermore, the PDI index for both liposomes was under 0.06 (Figure 1b). In addition, laser doppler micro-electrophoresis measurements showed that the zeta potential of the control liposomes and hesperetin liposomes was approximately −10 mV (Figure 3c). Spectrophotometrical analysis demonstrated that ~83% and ~1% of the drug amount added during fabrication could be found in the liposome pellet and supernatant, respectively (Figure 1d). These results suggest that some of the drug was lost during the liposome extrusion process, presumably trapped in the polycarbonate membranes.

Figure 1.

Figure 1.

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Characterization of hesperetin liposomes and control liposomes. Dynamic light scattering (DLS) was used to measure the size (a) and polydispersity index (PDI) (b) of liposomes. Laser doppler micro-electrophoresis was performed to determine the zeta potential of liposomes (c). Results are expressed as the average ± standard deviation of five measurements with ten runs each. Ultracentrifugation and spectrophotometric analysis was used to determine the encapsulation efficiency (d). Results are expressed as the mean ± s.d. of three independent experiments.

Figure 3.

Figure 3.

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Stability of hesperetin liposomes and control liposomes in a simulated in vivo environment. Liposomes were incubated with 70% fetal bovine serum (FBS) at 37 °C with continuous shaking and the size (a) and PDI (b) were measured periodically. Results are expressed as the mean ± s.d. of five measurements with ten runs each.

3.2. Liposome storage and serum stability

Periodic measurements of the size, PDI, and zeta potential of liposomes stored in PBS at 4 °C showed that both control liposomes and hesperetin liposomes remained stable for at least 35 days (Figure 2). Namely, the size (Figure 2a), PDI (Figure 2b), and zeta potential (Figure 2c) did not fluctuate considerably during this time period. Moreover, the stability of liposomes exposed to serum proteins at 37 °C was monitored for 384 h. Liposomes diluted in serum displayed a smaller size (Figure 3a) and larger PDI (Figure 3b) in comparison to liposomes in PBS (Figure 1a and 1b). The liposomes displayed a single intensity peak, indicating that the serum proteins did not interfere with the size measurements. Throughout the serum incubation period, the size and PDI of control liposomes and hesperetin liposomes remained relatively consistent (Figure 3). However, there was a slight drop in size during the first hour of incubation (Figure. 3a). Overall, the liposomes remained stable both during storage conditions and upon incubation with serum.

Figure 2.

Figure 2.

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Stability of hesperetin liposomes and control liposomes under storage conditions. Liposomes were stored at 4 °C for 35 days and the size (a), PDI (b), and zeta potential (c) were measured once a week. Results are expressed as the mean ± s.d. of five measurements with ten runs each.

3.3. Drug release

The release of hesperetin from the liposomal bilayer was determined under continuous stirring at 37 °C using a dialysis tube. The amount of released drug over a period of 72 h was measured spectrophotometrically. During the first hour, hesperetin could not be detected in the dialysate reservoir (Figure 4). However, in the time frame of 2–12 h, ~38% of drug had been released in a linear fashion, where after the rate of drug release decreased (Figure 4). After 72 h, ~86% of the drug had been released from the liposomes. The release profile for hesperetin is similar to that previously reported for the hydrophobic drug tamoxifen encapsulated in liposomes [22b].

Figure 4.

Figure 4.

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Release of hesperetin from liposomes. Dialysis and spectrophotometric analysis was used to measure drug release. Results are expressed as the mean ± s.d. of three measurements.

3.4. Cellular internalization

Fluorescent microscopy was performed to visualize fluorescently labeled hesperetin liposomes in fixed H441 cells. To observe the nucleus and filamentous actin, cell were stained with DAPI and Alexa fluor 488 phalloidin, respectively. The results indicate that hesperetin liposomes were successfully internalized into cells (Figure 5). Furthermore, the liposomes primarily localized in the perinuclear region (Figure 5).

Figure 5.

Figure 5.

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Fluorescent microscopy of hesperetin liposomes in fixed cells. Fluorescently labeled liposomes (rhodamine, red) were incubated with H441 lung cancer cells for 24 h. Filamentous actin and nuclei were visualized with alexa fluor 488 phalloidin (green) and 4’,6-diamidino-2-phenylindole (DAPI, blue), respectively. Scale bar: 40 μm.

3.5. Anticancer activity

The viability of H441 and MDA-MB-231 cells in response to control liposomes and hesperetin liposomes was evaluated using a cell proliferation assay. A concentration of 100 μM was used, as this dose has previously displayed biological activity in MDA-MB-231 cells [5]. The control liposomes displayed negligible toxicity (~90–95% viability) in comparison to untreated cells (Figure 6), indicating that the delivery vehicle was biocompatible. Thereafter, the performance of hesperetin-loaded liposomes was compared to that of an equal amount of hesperetin dissolved in DMSO. In both cell lines, the liposomal and solubilized versions of hesperetin reduced the cell viability to ~60–80% (Figure 6). Notably, the hesperetin liposomes caused a greater reduction in cell viability in comparison to delivery with DMSO (P < 0.05). Next, the anticancer activity of hesperetin was evaluated in the presence of the MDR-1 protein. Firstly, MDA-MB-231 cells with and without MDR-1 were exposed to various concentrations of paclitaxel, to ensure that the transfected cells had acquired drug resistance (Figure 7a and 7b). Indeed, the MDA-MB-231/MDR-1 cells were resistant to paclitaxel at all tested concentrations (Figure 7b). The transfected cells were then incubated with liposomal hesperetin and hesperetin in DMSO. Both formulations displayed similar anticancer efficacy as in the untransfected cells (Figure 7c), indicating that the presence of MDR-1 did not interfere with hesperetin efficacy.

Figure 6.

Figure 6.

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Anticancer activity of control liposomes, hesperetin liposomes and hesperetin (100 uM) dissolved in dimethyl sulfoxide (DMSO). The viability of MDA-MB-231 breast cancer cells (a) and H441 lung cancer cells (b) exposed to the treatment groups for 72 h was measured using a proliferation assay. Cells incubated with equal volumes of phosphate buffered saline (PBS) or DMSO, were used as controls. Experiments were performed in triplicate and results are expressed as the mean ± s.d. * p < 0.05.

Figure 7.

Figure 7.

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Anticancer activity of paclitaxel and hesperetin in the presence of multidrug resistance protein 1 (MDR-1). Cell viability of MDA-MB-231 cells (a) and MDA-MB-231/MDR-1 cells (b) exposed to paclitaxel (dissolved in Chremophor EL and dehydrated ethanol) for 72 h. (c) Cell viability of MDA-MB-231/MDR-1 cells treated with hesperetin liposomes or hesperetin liposomes dissolved in DMSO (100 uM). Cells incubated with equal volumes of PBS or DMSO, were used as controls. Experiments were performed in triplicate and results are expressed as the mean ± s.d.

4. DISCUSSION

The hesperetin liposomes displayed a suitable size (<70 nm) for cancer therapeutic applications. In particular, nanosized objects generally have increased tumor accumulation due to the enhanced permeability and retention (EPR) effect [23]. This effect is mainly due to tumors having more permeable vasculature than normal tissue. In addition, certain hypovascularized and hypopermeable tumors, such as pancreatic cancer, have vascular fenestrations that are less than 100 nm in diameter [24], necessitating the use of smaller nanotherapeutics. Furthermore, the hesperetin liposomes had a PDI below 0.2, which is usually an indication of a monodisperse sample [25]. The liposomes also exhibited a slightly negative zeta potential, which is consistent with previous reports of pegyated liposomes [17].

Nevertheless, the properties of nanoparticles differ considerably when exposed to bodily fluids [26]. Therefore, it is also important to evaluate liposome characteristics in simulated in vivo conditions [27]. In this study, the stability and size of liposomes were measured in FBS at 37 °C under continuous stirring. The results revealed that the liposome size was smaller in serum than in PBS, suggesting the involvement of osmotic forces [28]. Since the liposome membrane is impermeable to proteins, water is forced out of the carrier to balance osmotic pressure, subsequently causing liposomal shrinkage [28]. Time-dependent analysis of liposome integrity in serum indicated that the liposomes did not aggregate or degrade over time. It is likely that the liposomes remained stable in FBS, due to the presence of PEG and cholesterol, which are known to provide lipid particles with structural integrity [15b, 29].

The hesperetin liposomes had an antiproliferative effect in cancer cells after 72 h, indicating that they were able to enter cells and release the payload without reducing therapeutic efficacy. Indeed, the liposomes were internalized into cells within 24 h and the majority of hesperetin was released in the same time period. Accordingly, the rate of drug release plays an important role for drug performance [14a]. For instance, if a compound is released too rapidly it could be cleared by the kidneys before reaching the target tissue. In contrast, if drug release is too slow, the concentration in the target tissue may be insufficient to have a therapeutic effect. In the case of hesperetin liposomes, the drug release occurred gradually over a period of 24 h, after which ~75% of the drug had been released. Comparatively, the clinically approved liposomal drug, Doxil, releases <10% of the cargo in the same time period [30]. However, it has been suggested that the slow drug release of Doxil impairs therapeutic efficacy [30–31], suggesting that the release rate observed with hesperetin liposomes is more favorable.

Besides drug release, the amount of bioavailable drug inside cells is an important factor for achieving therapeutic efficacy. In this context, several cancer patients develop drug resistance due to expression of MDR-1, which is responsible for pumping out xeniobiotics from the cell interior [32]. In fact, several hydrophobic natural compound drugs are susceptible to P-glycoprotein (P-gp)-driven efflux [33]. Therefore, it was important to determine whether this membrane pump was capable of removing intracellular hesperetin. Notably, while MDR-1 completely eliminated the anticancer activity of paclitaxel in breast cancer cells, the presence of this pump did not reduce the therapeutic efficacy of hesperetin, regardless of the used delivery method. Since both solubilized and liposomal formulations of hesperetin appeared unaffected by the expression of MDR-1, it is unlikely that hesperetin is a substrate for P-gp. This observation indicates that hesperetin liposomes could potentially provide an alternative treatment option for patients that have acquired resistance to conventional cancer therapeutics. In the future, it will be important to address pharmacokinetic parameters, appropriate dosing, and anticancer efficacy of these liposomes in vivo.

5. CONCLUSION

Hesperetin is a natural compound that displays multiple anticancer properties. However, the therapeutic implementation of hesperetin has been impeded, due to the hydrophobic nature of the drug. Here, we have fabricated, characterized, and evaluated the anticancer efficacy of hesperetin liposomes. Liposomes provide a means for avoiding the use of toxic solubilizing agents, which are generally required for the delivery of non-water-soluble compounds. In addition, liposomes can protect the cargo from degradation, clearance and immunological uptake. The results reveal that hesperetin liposomes are stable in serum and exhibit a suitable drug release profile. Furthermore, the liposomes were internalized into cells and displayed anticancer efficacy in breast and lung cancer cells. Notably, hesperetin liposomes cause a slightly greater reduction in cell viability than hesperetin solubilized in DMSO. Additionally, the performance of hesperetin was unaffected by the presence of the MDR-1. These results merit further investigation of hesperetin liposomes as anticancer therapeutics.

ACKNOWLEDGEMENTS

The Houston Methodist Research Institute supplied funding for this research. Partial funds were acquired from: the Ernest Cockrell Jr. Distinguished Endowed Chair (M.F.), the US Department of Defense (W81XWH-09-1-0212, W81XWH-12-1-0414) (M.F.), the National Institute of Health (U54CA143837, U54CA151668) (M.F.), Nylands nation Finland (J.W.), Victoriastiftelsen Finland (J.W.), and the Cancer Prevention Research Institute of Texas (RP121071) (M.F. and H.S.).

Footnotes

CONFLICT OF INTEREST

The authors confirm that this article has no conflict of interest.

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