Impact of PEGylated liposomes on cytotoxicity of tamoxifen and piperine on MCF-7 human breast carcinoma cells

Abstract

Tamoxifen (TMF) is an anticancer agent used for managing estrogen receptor-positive breast cancer. It has limited therapeutic efficacy against breast cancer, which could be enhanced by the coadministration of herbal drugs like piperine (PIP). However, the hydrophobic nature of TMF and PIP restricts their therapeutic application. Therefore, the present study focuses on the impact of the anticancer activity of TMF in combination with PIP and after entrapping them into liposomes (TMF-PIP-LPs and TMF-PIP-PEG-LPs). The liposomes were prepared using the thin film hydration method. In addition, the morphology of the prepared liposomes was found spherical after SEM and TEM analyses. Further, the in vitro cytotoxicity (IC₅₀) study of pure PIP and TMF was found to be 90.3 ± 10.2 μg/mL and 40.9 ± 5.9 μg/mL, respectively. Interestingly, an improved cytotoxicity (IC₅₀) was observed when the TMF and PIP were loaded into liposomes (TMF-PIP-LPs: 21 ± 1.6 μg/mL and TMF-PIP-PEG-LPs: 10 ± 0.5 μg/mL). Also, the PEGylated liposomes showed improvement in cellular uptake as compared to liposomes without PEGylation in MCF-7 human breast carcinoma cells. Thus, the enhanced cellular uptake and improved cytotoxicity of PEGylated liposomes can be a suitable strategy for delivering TMF with PIP for breast cancer treatment.

Graphical Abstract

1. Introduction

Cancer is a disease triggered by an uncontrolled proliferation of abnormal cells in the body [1]. It disrupts the usual regulatory processes that govern cellular growth and proliferation. As per the World Health Organization report, 2.3 million women were diagnosed with cancer globally in 2020 [2]. Another report reveals that 1 in 8 women in the USA develop breast cancer, whereas 1 in 28 Indian women are likely to develop it in their lifetime [3]. Also, an estimate suggests that around 2,97,790 new cases of invasive breast cancer were diagnosed in the USA in 2023. There are currently more than 4 million women with a history of breast cancer in the USA [4]. The occurrence of breast cancer is also rising globally, and its related death is estimated to increase to 11 million by 2030 [5]. Among different types of cancers, breast cancer is a prominent cause of cancer-related deaths in women [6]. Hence, extensive treatment strategies are required to overcome the complications of breast cancer.

Currently, there are six different types of treatment for breast cancer, viz., surgery, chemotherapy, radiation therapy, immunotherapy, hormone therapy, and targeted therapy [7, 8]. Particularly in chemotherapy, the drugs used to treat breast cancer are based on hormone receptor-positive therapy. These drugs are mainly divided into several classes, such as drugs that block estrogen receptors (e.g., tamoxifen, toremifene), drugs that lower estrogen levels (e.g., letrozole, anastrozole, exemestane), and drugs that suppress ovaries (e.g., goserelin, leuprolide). It has been reported that the occurrence of estrogen receptor-positive breast cancer is high as compared to estrogen receptor-negative ones. Therefore, an anticancer agent, tamoxifen (estrogen receptor antagonist), is widely used to treat most breast cancer cases [9].

Tamoxifen (TMF) is a widely employed non-steroidal selective estrogen receptor modulator (SERM) in managing estrogen receptor-positive breast cancer [10]. Despite being a promising drug for breast cancer treatment, TMF faces specific biopharmaceutical problems, such as poor aqueous solubility and low bioavailability (30 %) [11]. Moreover, it undergoes precipitation as a free base due to the gastric environment in the stomach, which reduces systemic exposure and the formation of free radicals and toxic metabolites. Further, due to the accumulation of doses, patients are at risk of endometrial cancer, which further leads to oxidative stress-mediated hepatotoxicity. Therefore, a combinatorial approach with the herbal compound would reduce the problems and may enhance the therapeutic activity of TMF [11].

Piperine (PIP) is a known herbal compound derived from a plant alkaloid Piper longum Linn (long pepper) and Piper nigrum Linn (black pepper). The studies reveal that PIP could improve the bioavailability of coadministered drugs by up to 300 % [12]. However, PIP alone can exhibit anti-inflammatory, antioxidant, and anticancer activities. It has been reported that the G2/ M phase arrest is the main reason for the cytotoxicity of PIP in MCF-7 cells [13]. Another study reported that apoptosis and autophagy are the primary mechanisms for its anticancer activity. [14]. Interestingly, PIP may enhance the effectiveness of synthetic anticancer drugs and may inhibit further cancer progression [15]. In addition, PIP may enhance anticancer activity by reducing oxidative stress and inflammation in the tumor microenvironment [16]. In this context, the literature reveals that improved anticancer activity of TMF was observed in combination with PIP [13]. However, the poor water solubility of TMF and PIP restricts their therapeutic application in treating breast cancer. Hence, to encounter these problems, a lipid-based drug delivery system could be a suitable strategy for delivering PIP along with TMF. Among the different lipid-based delivery systems, liposomes have shown promising outcomes in exhibiting specificity toward cancer cells [17, 18].

Liposomes are spherical structures due to the self-assembly of phospholipids in solution and are widely explored in cancer treatment [19, 20]. The membrane of liposomes comprises one or more lipid bilayers (lamellas) with a hydrophilic head group and hydrophobic tail group due to the hydrocarbon chain [21]. This unique property makes them an ideal carrier for encapsulating hydrophilic and hydrophobic drugs [22]. The encapsulation of drugs in vesicle structure prevents rapid degradation and reduces systemic toxicity [23]. In addition, liposomes can be administered by intravenous, subcutaneous, intravenous, and intratumoral routes of administration for cancer treatment [24]. However, the circulation of liposomes in the body is limited, which can be overcome by adding PEGylation on the surface of liposomes [25].

Liposomes with polyethylene glycol (PEG) prolong the circulation time by preventing the interaction between liposomes and macrophages [26, 27]. The PEGylated liposomes accumulate passively in malignant tumors through the enhanced permeation and retention (EPR) effect [28, 29]. The PEGylation can improve the stability of liposomal formulations by avoiding vesicle aggregation [30]. Also, PEGylated liposomes can overcome the problem with rapid clearance of liposomes by the reticuloendothelial system, as PEG can form a hydrophilic protective layer on the liposomal surface [31]. Thus, the present study aimed to assess the impact of cytotoxicity of TMF when combined with PIP in MCF-7 human breast carcinoma cells. Moreover, liposomal formulations such as TMF-PIP-LPs and TMF-PIP-PEG-LPs were prepared to overcome the hydrophobicity of TMF and PIP. Further, the cellular uptake of the liposomal formulations and the corresponding cytotoxicity were evaluated in MCF-7 human breast carcinoma cells.

2. Materials and methods

2.1. Materials

Tamoxifen (TMF) was purchased from TCI Chemicals (Chennai, India). Piperine (PIP) and dialysis bags (molecular weight- 12 kDa) were procured from Sigma-Aldrich (Mumbai, India). Sodium acetate and cholesterol were purchased from Hi-Media (Mumbai, India). Phosphatidylcholine from soybean (SPC) was a generous gift sample of Lipoid GmbH (Ludwigshafen, Germany). DSPE-PEG₂₀₀₀ (1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene glycerol)-2000], molecular weight: 2509) was procured from Avanti Polar Lipids (Alabama, USA).

2.2. Preparation of liposomes

Liposomes (TMF-PIP-LPs) and PEGylated liposomes (TMF-PIP-PEG-LPs) were prepared using the thin-film hydration method [20]. Briefly, SPC: cholesterol: TMF: PIP (4:1:1:0.5 molar ratio), and with or without DSPE-PEG₂₀₀₀ were dissolved in a mixture of chloroform: methanol (2:1 ratio). The organic solvent was evaporated (45 °C, 100 rpm) using a rotary evaporator (IKA, Germany) to form a thin film around the bottom flask. The formed thin film was hydrated using phosphate buffer saline (PBS) of pH 7.4 for 45 minutes, followed by sonication using a probe sonicator (Vibra cell, USA) with an amplitude of 25 % (5 sec on/off) for 6 minutes. Further, the unentrapped drugs were removed through a centrifugation process to prepare TMF-PIP-LPs and TMF-PIP-PEG-LPs. Similarly, the blank liposomes (BLK-LPs) were prepared without adding the TMF and PIP and kept in a refrigerator at 4 °C until further studies.

2.3. Determination of particle size, polydispersity index (PDI), and zeta potential

The particle size, polydispersity index (PDI), and zeta potential of the liposomes (TMF-PIP-LPs) and PEGylated liposomes (TMF-PIP-PEG-LPs) were analyzed using a zeta sizer (Nano ZS, Malvern Panalytical, UK). Briefly, the samples were diluted to 50-folds with milli-Q water to avoid the multi-scattering of the prepared liposomes. The experiment was performed three times (n=3) at 25 °C, and the average particle size, PDI, and zeta potential were obtained for TMF-PIP-LPs and TMF-PIP-PEG-LPs.

2.4. Determination of drug loading and entrapment efficiency

The drug loading (% DL) drug entrapment efficiency (% EE) of PEGylated liposomes (TMF-PIP-PEG-LPs) was assessed using a centrifugal ultrafiltration method. The prepared liposomes were kept in the Amicon ultra-centrifugal filter and further centrifuged at 10,000 RPM using a centrifuge (5810R, Eppendorf, Germany) and analyzed using RP-HPLC. The % DL and % EE of liposomes were calculated using the following formula.

$$\%\text{DL}=\frac{\text{Total amount of drug}-\text{Free drug}}{\text{Total amount of drug}+\text{lipid}}\times 100$$

$$\%\text{EE}=\frac{\text{Total amount of drug}-\text{Free drug}}{\text{Total amount of drug}}\times 100$$

2.5. Morphology

The morphological features related to the shape and surface of PEGylated liposomes (TMF-PIP-PEG-LPs) were examined under a scanning electron microscope (SEM). The sample was sputter-coated with gold and analyzed under FESEM (Gemini 500, Zeiss, Germany). In addition, the morphology was confirmed using a transmission electron microscope (TEM). The sample was applied to the TEM grid and allowed to be vacuum-dried before TEM analysis (JEM-2100 PLUS (HR), Jeol).

2.6. Fourier transform infrared (FTIR) spectroscopy

The FTIR spectra of TMF, PIP, SPC, cholesterol (CHOL), physical mixture (PM) of TMF-PIP-SPC-CHOL, lyophilized blank liposomes (BLK-LPs), liposomes (TMF-PIP-LPs), and PEGylated liposomes (TMF-PIP-PEG-LPs) were studied for possible drug interaction with excipients used to prepare liposomes using an FT-IR spectrophotometer (IRAffinity-1S, Shimadzu, Japan).

2.7. Powder X-ray diffraction (XRD) analysis

The crystalline or amorphous nature of pure drugs (TMF and PIP), liposomes (TMF-PIP-LPs), and PEGylated liposomes (TMF-PIP-PEG-LPs) were studied by an X-ray diffractometer (SmartLab, Rigaku, Japan). The sample was filled in a glass substrate of 0.5 mm depth and operated at a voltage of 40 kV with 125 mA current.

2.8. In vitro drug release study

The in vitro drug release of TMF and PIP from liposomes (TMF-PIP-LPs) and PEGylated liposomes (TMF-PIP-PEG-LPs) was determined using the dialysis bag (12–1 KDa) method. The study was performed at 37 ± 0.5 °C with 100 rpm in 50 mL of drug release media such as pH 7.4 phosphate buffer saline (PBS) and pH 5.5 acetate buffer solution (to mimic tumor mass pH) [32]. The samples were withdrawn (1 mL) at different intervals, and the same volume of the respective buffer was replaced during the study. The samples withdrawn were analyzed using RP-HPLC to quantify TMF and PIP drug release.

2.9. In vitro cellular uptake study

The qualitative and quantitative cellular uptake of liposomes (TMF-PIP-LPs) and PEGylated liposomes (TMF-PIP-PEG-LPs) were evaluated in MCF-7 breast cancer cells. Briefly, MCF-7 cells were uniformly seeded into 6-well plates at 4 × 10⁴ cells per well, followed by overnight incubation. The formulations were loaded with coumarin-6 and treated with cells to evaluate cellular uptake [33]. The cells were fixed with 4 % Paraformaldehyde and then washed with PBS. Further, it was incubated with 4,6-diamidino-2-phenylindole (DAPI) and washed with PBS. The study was carried out at 2 h, 4 h, and 6 h after incubation of cells with the liposomal formulations, and fluorescence microscopy (Nikon ECLIPSE TS 100) was employed to determine the cellular internalization.

2.10. Cytotoxicity by MTT assay

The cytotoxic behavior of pure drug (TMF and PIP), liposomes (TMF-PIP-LPs), and PEGylated liposomes (TMF-PIP-PEG-LPs) were investigated in MCF-7 cells. MTT assay was carried out using a well-defined protocol [34]. The cells were grown in the T-25 flasks, and this flask (up to 75% cell growth) was trypsinized to detach the adhered cells and centrifuged to collect the pellet of cells. The cells were seeded, and the cell concentration was 1 × 10⁴ cells per well in 96-well plates, followed by incubation overnight in a CO₂ incubator. Different concentrations (1 to 80 μg/mL) of samples were prepared in culture media. Further, the cells were treated with different samples, followed by incubation. 50 μL of MTT solution was added after 24 h into each well after washing with PBS and incubated again for 4 h. To dissolve the formed formazan crystals, 150 μL of DMSO was added to each well. The absorbance values of each sample in the 96-well plates were recorded at 570 nm by a microplate reader (Omega Fluster, BMG Labtech).

2.11. Statistical analysis

The experiments were carried out at least three times (n=3), and the results were expressed in mean ± SD. The statistical analysis was performed on GraphPad Prism 8.0 software.

3. Results and discussion

3.1. Particle size and zeta potential

The particle size of liposomes plays an essential role in cancer treatment, influencing various aspects of their behavior in the body and therapeutic efficacy [35]. In this study, the prepared liposomes (TMF-PIP-LPs) and PEGylated liposomes (TMF-PIP-PEG-LPs) were characterized for the particle size using a zeta sizer. TMF-PIP-LPs exhibited a particle size and PDI of 104 ± 1.8 nm and 0.23 ± 0.05, respectively (Supplementary file: Fig. S1). Moreover, the particle size and PDI of TMF-PIP-PEG-LPs were 130.7 ± 0.5 nm and 0.12 ± 0.04, respectively (Supplementary file: Fig. S2). The prepared PEGylated liposomes exhibited a desirable particle size of less than 150 nm, which may facilitate the entry or exit of the blood vessels in the tumor microenvironment by the EPR effect [36]. In addition, extravasation from the blood circulation through tumor vasculature can increase the drug concentration within the tumor. Also, a PDI value <0.3 indicates the homogeneity of the liposomal formulations [37].

Further, the zeta potential was measured to assess the liposome’s surface charge. A positive zeta potential of +26.8 ± 0.6 mV was observed for TMF-PIP-LPs, which may be attributed to the surface localization of TMF [9, 38]. However, a negative zeta potential of −12.3 ± 0.5 mV was observed for TMF-PIP-PEG-LPs, possibly due to the addition of DSPE-PEG₂₀₀₀. Further, the storage stability of PEGylated liposomes (TMF-PIP-PEG-LPs) was studied at refrigerated conditions of 4 °C for 1 month (Fig. 1). There was no substantial increase in the particle size, PDI, and zeta potential of TMF-PIP-PEG-LPs, which indicates that liposomal formulations were stable enough to store at 4 °C for 1 month.

Fig. 1.

(A) Particle size, and (B) zeta potential analyses of liposomes (TMF-PIP-LPs) and PEGylated liposomes (TMF-PIP-PEG-LPs) using zeta sizer.

3.2. Morphology

Liposome morphology is a critical factor that directly influences the encapsulated drug’s efficiency [39]. In this study, the morphological features of the prepared PEGylated liposomes (TMF-PIP-PEG-LPs) through SEM analysis showed a spherical shape with smooth surface morphology (Fig. 2A). Moreover, the spherical shape was also confirmed using TEM analysis (Fig. 2B). Similar morphological observations were also reported by Hardiansyah et al. [40] and Wang et al. [41] in their studies related to PEGylated liposomes. According to Wang et al., the ring-like- appearance of the PEG on the surface of the liposome could not be confirmed by TEM or any other applied spectrum analysis unless it is a thick structure [41].

Fig. 2.

Morphology of TMF-PIP-PEGylated liposomes using A) scanning electron microscopy (SEM) and B) Transmission electron microscopy (TEM).

3.3. Determination of drug loading and drug entrapment efficiency

The drug loading of TMF and PIP was 2.6 ± 0.3 % and 2.7 ± 0.2 % respectively. Moreover, the percentage drug entrapment efficiency of TMF and PIP was 74.4 ± 0.7 % and 87.8 ± 0.4 %, respectively.

3.4. FTIR study

The probable interactions between the drug and excipients employed in the liposomal formulations were studied using FT-IR spectroscopy. The FT-IR spectra of the pure drug (TMF, PIP), excipients (SPC, CHOL), physical mixture (PM), and lyophilized liposomes (TMF-PIP-LPs, and TMF-PIP-PEG-LPs) analyzed are depicted in Fig. 3. The characteristic peaks of TMF were observed at 3408.22 cm⁻¹ corresponding to substituted benzene, amine N-H bending at 1591.27 cm⁻¹, ketonic C=O stretching at 1739.79 cm⁻¹, and amine C-N stretching at 1043.49 cm⁻¹ [42]. PIP exhibited characteristic peaks at 2935.17 cm⁻¹ and 1633.63 cm⁻¹, corresponding to C-H (stretch) and amide C=O (stretch). Further, the aromatic C=C (stretch) and aliphatic C-H (bend) peaks were observed in 1582 cm⁻¹ and 1441.15 cm⁻¹, respectively [43]. Cholesterol exhibited O-H stretching, CH₂ and CH₃ asymmetric stretching, and ring deformation at a characteristic peak of 3406.29 cm⁻¹, 2939.52 cm^−1, and 1053.13 cm⁻¹, respectively [44]. The fatty acid tails of SPC lipids were observed at 2924.08 cm⁻¹, 2852.72 cm⁻¹, and 1734.01 cm⁻¹, corresponding to C-H and C=O vibrations (stretch) [45]. Similarly, these C-H and C=O stretching vibrations were also observed in BLK-LPs at 2927.94 cm⁻¹, 2854.65 cm⁻¹, 1735.93 cm⁻¹ and TMF-PIP-LPs at 2929.87 cm⁻¹, 2852.72 cm⁻¹, 1735.93 cm⁻¹ respectively. In this study, no significant change in the wave number was observed in the FT-IR spectra between BLK-LPs and TMF-PIP-PEG-LPs. It suggests the absence of major chemical interactions between drugs and excipients. Similar observations were also reported in a study by Banerjee et al. between blank and drug-loaded nanoformulations [46].

Fig. 3.

FT-IR spectra of A) TMF, B) PIP, C) SPC, D) CHOL, E) Physical mixture, F) BLK-LPs, G) TMF-PIP-LPs, and H) TMF-PIP-PEG-LPs.

3.5. Powder X-ray diffraction (XRD) analysis

The crystalline or amorphous nature of the pure drugs (TMF, PIP), blank liposomes (BLK-LPs), liposomes (TMF-PIP-LPs), and PEGylated liposomes (TMF-PIP-PEG-LPs) were determined using P-XRD studies. TMF exhibited sharp diffraction peaks at 5.57°, 12.75°, 13.8°, 14.98°, 17.15°, 20.82°, 24°, and 28.1° (Fig. 4). Similarly, PIP also showed sharp diffraction peaks at 14.74°, 19.61°, 22.55°, 25.8°, and 28.21°. These diffraction peaks obtained from TMF and PIP indicated their highly crystalline nature [47]. Also, the sharp peaks in lyophilized BLK-LPs, TMF-PIP-LPs, and TMF-PIP-PEG-LPs diffractograms might be due to mannitol, a cryoprotectant used in the formulation. However, the sharp diffraction peaks of TMF and PIP are absent in TMF-PIP-LPs and TMF-PIP-PEG-LPs. The result indicates that both drugs might be converted to a noncrystalline or amorphous form after entrapping into the liposomes. Similar observations were reported in earlier studies, such as gallic acid-loaded liposomes [48] and resveratrol-loaded liposomes [49].

Fig. 4.

P-XRD diffractograms of A) TMF, B) PIP, C) BLK-LPs, D) TMF-PIP-LPs, and E) TMF-PIP-PEG-LPs.

3.6. In vitro drug release study

The in vitro drug release from the liposomes could improve the delivery of anticancer agents. It has been reported that a pH 5.5 acetate buffer mimics the tumor mass pH [9], and a pH of 7.4 mimics the pH of blood or body fluid in drug release studies. Therefore, the in vitro drug release of pure drug (TMF, PIP), liposomes (TMF-PIP-LPs), and PEGylated liposomes (TMF-PIP-PEG-LPs) was carried out in pH 7.4 phosphate buffer saline (Fig. 5A) and pH 5.5 acetate buffer (Fig. 5B) in this study. A slow drug release of TMF and PIP from the liposomal formulations was observed due to their encapsulation into the liposomes. Moreover, TMF release from the liposomes was slower than PIP. It has been reported that the drug release from the liposomes depends on encapsulated moieties [50]. Thus, the slower drug release of TMF and PIP from the liposomes could be helpful for prolonged therapeutic activity.

Fig. 5.

A) In vitro drug release in pH 7.4 phosphate buffer saline (n=3) and B) In vitro drug release in pH 5.5 acetate buffer at 37 °C (n=3). TMF and PIP release from liposomes (TMF-PIP-LPs) are denoted as TMF-LPs and PIP-LPs, respectively. Moreover, TMF and PIP release from PEGylated liposomes (TMF-PIP-PEG-LPs) are denoted as TMF-PEG-LPs and PIP-PEG-LPs, respectively.

3.7. In vitro cellular uptake study

The in vitro cellular uptake of liposomes can significantly affect the treatment outcome of various cancers. It has been reported that liposomes improve the drug uptake by cancer cells [51]. In this study, coumarin 6 was used as a dye to assess the cellular uptake of the prepared liposomes (coumarin 6-LPs) and PEGylated liposomes (coumarin 6 PEG-LPs) in MCF-7 breast cancer cells. Coumarin 6-PEG-LPs exhibited maximum fluorescence intensity as compared to coumarin 6-LPs.

A significantly (p < 0.0001) improved fluorescence intensity was observed for the PEGylated coumarin 6 PEG-LPs after 2 h, 4 h, and 6 h, respectively (Fig. 6) as compared to coumarin 6-LPs. The enhanced cellular internalization is due to lipid DSPE-PEG₂₀₀₀ in the PEGylated liposomes. A similar report was previously made by Megahed et al., where the authors observed an elevated cellular uptake for tamoxifen PEGylated nanovesicles in the breast cancer-induced model [52]. It is a fruitful and favorable observation as the PEG surface provides better uptake and can contribute to the longevity of the developed formulations. It also caters to the possibility that PEG conjugation can be further exploited for better delivery of anticancer drugs in treating breast cancer.

Fig. 6.

Qualitative cellular uptake of Coumarin 6-LPs and Coumarin 6 PEG-LPs in MCF-7 breast cancer cells after incubation at A) 2-hour, B) 4-hour, and C) 6-hour. After the treatment, the images were captured using a fluorescence microscope (NIKON, Japan), and the cellular uptake of Coumarin 6-LPs and Coumarin 6 PEG-LPs in MCF-7 breast cancer cells was quantified using ImageJ software. **** signifies p < 0.0001. Values represent mean ± SD (n = 3).

3.8. Cytotoxicity by MTT assay

The liposomes (TMF-PIP-LPs) and PEGylated liposomes (TMF-PIP-PEG-LPs) were assessed for cytotoxicity and compared to plain TMF and PIP on MCF-7 cancer cell lines. The outcome of the MTT assay revealed that pure PIP has a slight cytotoxic effect against MCF-7 cells. It was reported that the G2/ M phase arrest is the main reason for the cytotoxicity of PIP in MCF-7 cells [13]. Another study reported that apoptosis and autophagy are the primary mechanisms for its anticancer activity. [14]. In addition, the pure TMF (40.9 ± 5.9 μg/mL) showed a significant (p < 0.01) cytotoxic effect than pure PIP (Fig. 7A). The G0/ G1 phase arrest is the prime reason for the cytotoxicity of TMF in MCF-7 cells [13].

Fig. 7.

A) Cytotoxic effects of pure PIP, pure TMF, pure TMF-PIP, TMF-PIP-LPs, and TMF-PIP-PEG-LPs against MCF-7 breast cancer cells after 24 h treatment. and B) IC₅₀ values of pure PIP, pure TMF, pure TMF-PIP, TMF-PIP-LPs, and TMF-PIP-PEG-LPs against MCF-7 breast cancer cells. ***, **, and *, signify p < 0.001, p < 0.01, and p < 0.05, respectively. Values represent mean ± SD (n = 3).

The anticancer activity of TMF against MCF-7 breast cancer cell lines is probably through induction of apoptosis, cell cycle arrest, and downregulation of ERα and EGFR [13]. It is well-known that the growth inhibitory effect of TMF on ERα-positive breast cancer cells is attributed to the competitive inhibition of estrogen binding to Erα [53]. Further, the combination of TMF and PIP (TMF-PIP) was treated in MCF-7 cells, and the IC₅₀ value was significantly reduced to 31 ± 2.8 μg/mL. Although the anticancer activity of PIP was lower than that of TMF, the cytotoxicity in MCF-7 breast cancer cells significantly increased when PIP was combined with TMF. It has been reported that PIP inhibits the efflux pump of drugs, which may improve the intracellular concentration of anticancer drugs [54]. Also, PIP can modulate membrane permeability and transporters, allowing the synthetic drugs to enter cancer cells more readily. Hence, the combination of TMF and PIP could be helpful in enhancing TMF’s anticancer activity.

Moreover, TMF and PIP-loaded liposomes (TMF-PIP-LPs) showed a 1.4-fold lower IC₅₀ value (21 ± 1.6 μg/mL) than pure TMF-PIP. However, the PEGylated liposomal formulation (TMF-PIP-PEG-LPs) showed maximum cytotoxicity with the IC₅₀ value of 10 ± 0.5 μg/mL (Fig. 7B). The improved inhibition might be due to enhanced cellular uptake of PEGylated liposomal formulation as compared to non-PEGylated liposomes. Similarly, several studies observed improvement in the cytotoxicity of PEGylated formulations on the in vitro cell lines. In this regard, Steffes et al. reported a lower IC₅₀ value of paclitaxel PEGylated liposomes, which was a 1.5-fold lower IC₅₀ value as compared to the non-PEGylated ones [55]. Also, Megahed et al. observed an increase in cytotoxicity of tamoxifen PEGylated nanocarriers when compared to non-PEGylated nanocarriers [52]. Thus, PEGylated liposomes could be suitable for delivering TMF and PIP to treat breast cancer.

4. Conclusion

The liposomal formulation containing tamoxifen and piperine was prepared using the thin film hydration method. The PEGylated liposomes were found to have a particle size of <150 nm with a polydispersity index value of <0.3. The liposomes showed a spherical shape morphology. The prepared PEGylated liposomes were stable in refrigerated conditions for at least 1 month. The PEGylated liposomal formulation containing the PIP and TMF showed maximum cellular internalization as compared to liposomes without PEGylation. Further, the cytotoxicity study showed that the anticancer activity of pure TMF was higher than PIP in MCF-7 cell lines. Interestingly, the cytotoxicity of TMF was significantly increased when combined with PIP in MCF-7 cell lines. Hence, the combination of TMF and PIP can be helpful in enhancing TMF’s anticancer activity. Additionally, improved cytotoxicity (IC₅₀) was observed when the TMF and PIP were loaded into PEGylated liposomes on MCF-7 breast cancer cell lines. Thus, the enhanced cellular uptake and improved anticancer activity of PEGylated liposomal formulation can be suitable for delivering TMF when combined with PIP for breast cancer treatment. Further, in vivo studies in animal models remain an important aspect of future studies.

Highlights.

• The lipid soybean phosphatidylcholine was used to prepare tamoxifen-piperine liposomes (TMF-PIP-LPs).

• The lipid DSPE-PEG₂₀₀₀ was utilized to prepare PEGylated liposomes (TMF-PIP-PEG-LPs)_.

• The cytotoxicity of TMF was significantly increased when combined with PIP in MCF-7 cell lines.

• TMF-PIP-PEG-LPs showed enhanced cellular uptake and improved cytotoxicity as compared to TMF-PIP-LPs.