Fusogenic pH sensitive liposomal formulation for rapamycin: Improvement of antiproliferative effect

Abstract

Context: Liposomes are increasingly employed to deliver chemotherapeutic agents, antisense oligonucleotides, and genes to various therapeutic targets.

Objective: The present investigation evaluates the ability of fusogenic pH-sensitive liposomes of rapamycin in increasing its antiproliferative effect on human breast adenocarcinoma (MCF-7) cell line.

Materials and methods: Cholesterol (Chol) and dipalmitoylphosphatidylcholine (DPPC) (DPPC:Chol, 7:3) were used to prepare conventional rapamycin liposomes by a modified ethanol injection method. Dioleoylphosphatidylethanolamine (DOPE) was used to produce fusogenic and pH-sensitive properties in liposomes simultaneously (DPPC:Chol:DOPE, 7:3:4.2). The prepared liposomes were characterized by their size, zeta potential, encapsulation efficiency percent (EE%), and chemical stability during 6 months. The antiproliferative effects of both types of rapamycin liposomes (10, 25, and 50 nmol/L) with optimized formulations were assessed on MCF-7 cells, as cancerous cells, and human umbilical vein endothelial cells (HUVEC), as healthy cells, employing the diphenyltetrazolium bromide (MTT) assay for 72 h.

Results and discussion: The particle size, zeta potential, and EE% of the liposomes were 165 ± 12.3 and 178 ± 15.4 nm, −39.6 ± 1.3, and −41.2 ± 2.1 mV as well as 76.9 ± 2.6 and 76.9 ± 2.6% in conventional and fusogenic pH-sensitive liposomes, respectively. Physicochemical stability results indicated that both liposome types were relatively stable at 4 °C than 25 °C. In vitro antiproliferative evaluation showed that fusogenic pH-sensitive liposomes had better antiproliferative effects on MCF-7 cells compared to the conventional liposomes. Conversely, fusogenic pH-sensitive liposomes had less cytotoxicity on HUVEC cell line.

• Antiproliferative effect
• breast cancer
• fusogenic pH-sensitive liposome
• rapamycin

Introduction

The mechanism of many anticancer drugs used for tumor treatment is based on cytotoxic effects. For effective anticancer chemotherapy, two points should be considered: (1) high concentrations of drug, (2) selective drug delivery to the tumor site. Cytotoxicity of non-target chemotherapy agents is a major adverse side effect that limits drug dosage and therapeutic window. Side effects can be severe and unpleasant and range from nausea and hair loss to neuropathies, neutropenia, and kidney failure (Cukierman & Khan, 2010; Iyer et al., 2013; Maeda et al., 2013).

Liposomes (lipid-based vesicles) have been widely studied as drug delivery systems due to their relative safety, structural versatility concerning size, composition, and bilayer fluidity, as well as their ability to combine with almost any molecule regardless of its structure. Due to their structure, chemical composition and colloidal size, which all can be well controlled by preparation methods, liposomes show numerous properties that may be useful in various applications (Cho et al., 2009; Meisner & Mezei, 1995; Yang et al., 2011). These properties point to several possible applications of liposomes such as their usage as solubilizers, dispersants, sustained release systems, stabilizers, protective agents, and microencapsulation systems. Since the early 1970s, hundreds of drugs, including antitumor and antimicrobial agents, chelating agents, peptide hormones, enzymes, vaccines, and genetic materials, have been incorporated into the aqueous or lipid phase of liposomes with various sizes, compositions, and other characteristics. Liposomes can be made entirely from natural substances that are non-toxic, biodegradable and non-immunogenic (Bajoria & Sooranna, 1998; Cabanes et al., 1998; Gondal et al., 1993; Hatziantoniou et al., 2006; Nagata et al., 2007; Schreier et al., 1993; Sharma & Sharma, 1997; Yagi et al., 2007).

As a targeted drug delivery system, liposomes can target specific tissues through both active, by adding additional molecules to the outer surface of the lipid bilayer, and passive targeting strategies via external stimuli such as temperature, light, magnetic fields, pulse, and pH (Akamatsu, 2009; Chandaroy et al., 2002; Cho et al., 2009; Kim & Kim, 2002; Park et al., 2013).

Considerable efforts have been made to develop liposomes that are stable under normal physiological pH but which release their contents in response to environmental pH changes. Liposomes of this type are effective, non-toxic vehicles for delivery of cytotoxic drugs, protein toxins and plasmid DNA to cultured cells. One important application of pH-sensitive liposomes is the selective release of drugs release to pathological tissues such as tumors, metastases, sites of inflammation, and ischemic areas which have considerably lower pH than that the normal tissues (Sudimack et al., 2002; Yuba et al., 2013; Zhou et al., 2012; Zignani et al., 2000). This difference could be utilized if liposomes could be constructed so that they are relatively stable in the circulating fluids at physiological pH and release encapsulated drugs when passing through areas with lower pH. The first generation of pH-sensitive liposomes is based on the cone-shaped lipid dioleoylphosphatidylethanolamine (DOPE) which introduces fusogenic property, simultaneously. The fusogenic liposome fuses with the cell membrane similar to the virus particle. This system is unique because the fusogenic liposomes can deliver their contents directly into the cytoplasm (Cho et al., 2009; Jo et al., 2007; Kono et al., 1997; Liang & Hughes, 1998; Ogawa et al., 2002; Yuba et al., 2013; Zignani et al., 2000).

Rapamycin (RAPA, C51H79NO13, MW 914.2), or Sirolimus, is a natural lipophilic macrolide antibiotic isolated from the soil bacterium, Streptomyces hygroscopicus, of samples collected from Rapa Nui (Easter Island, Chile) by Dr. Suren Seghal. It was initially studied for its antifungal properties and later for its substantial immunosuppressive effects. RAPA showed great promise as an immunosuppressive agent in transplant recipients as well as antiproliferative and anti-angiogenic agent in a variety of solid tumor treatments. Antiproliferative effects of RAPA have been demonstrated not only on lymphocytes but also on vascular and tumor cells (Barenholz, 2001; Kakegawa et al., 2003; MacDonald et al., 2000; Paghdal & Schwartz, 2007).

The mammalian target of rapamycin (mTOR) pathway controls several fundamental cell functions. mTOR has become an attractive target for anticancer drug development, since mTOR-mediated signaling is frequently up-regulated in tumor cells and inhibition of this pathway induces apoptotic and autophagic cell death in tumor cells (Costa, 2007; Mi et al., 2009; van Rossum et al., 2009). Rapamycin inhibits the in vitro proliferation of transformed cell lines of lymphoid, central nervous system, hepatic, melanocytic, osteoblastic, myogenic, renal and connective tissue origin, as well as human T-lymphotropic virus type-1 (HTLV-1)-induced proliferation of T cells, Epstein–Barr virus (EBV)-induced proliferation of B cells and cyclosporine A (CsA)-induced tumor growth. Its antitumor activity has been recently studied both preclinically and clinically in various tumors, including breast, colon, prostate, renal, lung, and pancreatic carcinomas (Bae-Jump et al., 2006, 2010; Jasinghe et al., 2008; Law, 2005; Plas & Thomas, 2009; Shen et al., 2009; Sun et al., 2008).

In this study, we investigated the selectivity and in vitro antiproliferative effect of conventional and fusogenic pH-sensitive RAPA liposomes on human breast adenocarcinoma cell line (MCF-7) and human umbilical vein endothelial cells (HUVEC), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Materials and methods

Materials

Rapamycin was purchased from Poli Pharmaceuticals Company (Lazio, Italy). DOPE and dipalmitoylphosphatidylcholine (DPPC) were purchased from Lipoid GMBH Company (Ludwigshafen, Germany). Thiazolyl blue tetrazolium bromide (MTT), l-glutamine, penicillin, streptomycin, dimethyl sulfoxide, and Sorenson buffer were purchased from Sigma-Aldrich Company (Bedford, MA). RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco (New York, NY). Cholesterol (Chol) as well as HPLC-grade methanol and acetonitrile were supplied by Merck Company (Darmstadt, Germany). All reagents were analytical grade and were purchased from Merck Company (Darmstadt, Germany).

Liposome preparation

Rapamycin liposomes were prepared by using a modified ethanol injection method as an applicable method for liposomal entrapment of lipophilic drugs such as RAPA (Ghanbarzadeh & Arami, 2013a; Ghanbarzadeh et al., in press-b). Briefly, in this method, ethanol solution of drug and lipids was injected dropwise into phosphate buffer saline (PBS) pH 7.4 under homogenizer mixing (final concentration of RAPA = 500 µg/mL). Optimized formulation of both types of liposome, after our preliminary studies including factorial design and response surface methodology approach (Ghanbarzadeh et al., in press-a), is presented in Table 1.

Table 1. Composition of two types of prepared liposomes [cholesterol (Chol), dipalmitoylphosphatidylcholine (DPPC), and dioleoylphosphatidylethanolamine (DOPE) (µmol/mL)].

Liposome type	DPPC	DOPE	Chol
Conventional	7	–	3
Fusogenic pH-sensitive	7	4.2	3

Liposome characterization

The prepared liposomes were characterized in terms of particle size (PS), encapsulation efficiency percent (EE%), polydispersity index (PI), zeta potential and in vitro release over 24 h in PBS. Particle size and zeta potential of prepared liposomes after necessary dilutions were measured using Zetasizer (Nano ZS 90; Malvern Instruments, Worcestershire, UK) in triplicate at room temperature. Prepared liposomes were separated from the free (unentrapped) drug using dynamic dialysis tube and polycarbonate filter (cutoff of 100 nm) for 24 h at 10 °C (below glass transition temperature of phospholipids). To measure the entrapped drug amount, small aliquots of liposomes (1 mL), after the separation of unentrapped drug, were diluted in 9 mL methanol, subjected to sonication until liposomes disruption and analyzed for RAPA content by HPLC. The EE% was calculated from the amount of incorporated RAPA divided by the total amount of drug used at the beginning of preparation multiplied by 100. The in vitro release rate of RAPA from the liposomal formulations was determined by using dynamic dialysis method. Liposomal dispersions (1 mL) were placed in donor chamber and dialyzed against releasing medium for achieving sink condition at 37 °C, at two pHs (7.4 and 6.5) under 100 rpm stirring for 24 h (Table 2) (Ghanbarzadeh & Arami, 2013b; Ghanbarzadeh et al., 2013a).

Table 2. Characteristics of different types of liposomes [D_24 h: drug release over 24 h].

					D24 h (%)
Liposome type	Particle size ± SD (nm)	Polydispersity index	Zeta potential ± SD (mV)	Encapsulation efficiency ± SD (%)	pH 7.4	pH 6.5
Conventional	165 ± 12.3	0.21	−39.6 ± 1.3	76.9 ± 2.6	13.2 ± 1.2	13.5 ± 0.7
Fusogenic pH-sensitive	178 ± 15.4	0.19	−41.2 ± 2.1	72.6 ± 3.2	7.2 ± 0.5	19.3 ± 0.9

Drug analysis

To analyze the RAPA concentration, a HPLC system (Beckman, FL) with a variable wavelength ultraviolet spectrophotometric detector (166 gold) set at 278 nm was used. System Gold software was used for data acquisition and system Gold nouveau software (San Francisco, CA) was used for data reporting and analysis. The mobile phase consisted of acetonitrile and ammonium acetate buffer (70:30, v/v%), eluted the Knauer column (C18, 5 µm, 4.6 × 150 mm) at a flow rate of 1.5 mL/min. The column temperature was kept at 54 °C. Calibration curves (n = 3) were linear in the range of 125–2000 ng/mL (r²> 0.991) (Islambulchilar et al., 2012).

Physicochemical stability of liposomes

To study the physicochemical stability of both types of liposomes, particle size, encapsulation EE, zeta potential and drug content were determined monthly at 4 °C and room temperature over 6 months (Table 3).

Table 3. Stability study of optimized formulations of two types of prepared liposomes during storage at 4 °C and 25 °C for 6 months.

	Particle size ± SD (nm)		Zeta potential ± SD (mV)		Encapsulation efficiency ± SD (%)
Liposome type	Initial	After 6 months	Initial	After 6 months	Initial	After 6 months	Chemical stability after 6 months ± SD (%)
Conventional
4 °C	165 ± 12.3	191 ± 20	−39.6 ± 1.3	−39.1 ± 1.6	76.9 ± 2.6	67.2 ± 2.6	83.0 ± 1.6
25 °C		243 ± 18.5		−42.3 ± 1.2		58.9 ± 3.8	72.3 ± 1.6
Fusogenic pH-sensitive
4 °C	178 ± 15.4	205 ± 16	−41.2 ± 2.1	−40.2 ± 2.1	72.6 ± 3.2	65.3 ± 2.0	85.2 ± 2.6
25 °C		269 ± 14.3		−38.6 ± 1.6		53.8 ± 2.9	76.2 ± 2.6

Cell line

The MCF-7 and HUVEC cell lines were used as an in vitro model for the study of RAPA liposomes cytotoxicity. The cells were routinely cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin G and 100 U/mL streptomycin, and incubated in 5% CO₂ at 37 °C. Subconfluent monolayer cells were detached from the culture T-75 flask by trypsin treatment, and resuspended in the fresh media. Cells were seeded at a density of 1 × 10⁴ cells per well in a 96-well flat bottom plates with a final volume of 200 µL for cell proliferation assays.

Antiproliferative study

The antiproliferative activity of the formulations was assessed by the MTT assay. The cultured cells, after an overnight period, were treated with conventional and fusogenic pH-sensitive liposomes containing rapamycin at different concentrations (10, 25, and 50 nmol/L) for 72 h. Then the incubation medium was removed and 200 µL of fresh medium containing MTT (2 mg/mL) added to each well. After incubation for 4 h at 37 °C, the culture containing MTT was removed, 200 µL of DMSO: Sorencene buffer (8:1) was added to each well. The number of viable cells in each well was analyzed using a microplate reader at 570 nm after 10 min of shaking (Biotek, ELx 800, Winooski, VT). The cell viability was expressed as the percentage of absorbance read for treated cells versus the control group.

Statistical analysis

All studies were performed in triplicate, and results were expressed as mean value ± standard deviation. A one-way ANOVA was used to compare the differences between the control group and each treated group. All statistical analyses which were performed via SPSS 17 software (SPSS Inc., Chicago, IL) with p < 0.05 considered to be significant.

Results and discussion

Preparation, characterization, and stability of liposomes

The characteristics and stability study test results of optimized formulations of conventional and fusogenic pH-sensitive liposomes are presented in Tables 2 and 3.

Factorial design was employed to formulate and screen parameters for RAPA incorporation into nano-liposomes by the ethanol injection method, which ultimately yielded a formulation with maximum EE% and release rate as well as minimum PS. Factorial design and response surface methodology approach indicated that the optimum DPPC/Chol molar ratio was 2.31 and in the case of fusogenic pH-sensitive liposomes, DPPC/DOPE molar ratio in proposed formulation was 1.67.

There are several methods to prepare liposomes and among them a suitable method should be selected based on the physicochemical properties of the entrapped drug and phospholipids which are used. Preliminary stability studies showed that RAPA was stable at high temperature (65 °C) for about 30–40 min (data not shown). Therefore, the application of ethanol injection method using homogenizer resulted in forming of liposomes with low particle size without the need for extrudation.

Results indicated that particle size and percentage of EE of prepared liposomes were in the ranges of 138.5 ± 3.5–253 ± 13.3 nm and 66.15 ± 3.8–85.05 ± 4.8%, respectively (Figure 1).

Figure 1. Transmission electron microscopy image of fusogenic pH-sensitive (A) and conventional (B) liposomes.

Zeta potential value of all liposomes immediately after preparation was in the range of −33.2 to −45.6 mV and after 6 months was in the range of −28.30 ± 4.10 to −42.15 ± 0.64 mV. The negative charge of liposomes was due to the negative charge of the liposome bilayer composition and the high values of zeta potential indicated relative stability and the absence of agglomerated particles forming.

The cumulative drug release percent over 24 h was in the range of 13.5–19.3% at pH 6.5. In vitro release study showed the incomplete and very slow release of RAPA from liposomes during 24 h which is due to its high lipophilicity of RAPA and high-phase transition temperature of DPPC (41 °C), as main phospholipid in both types of prepared formulations. In contrast, at pH 7.4, the cumulative drug release of fusogenic pH-sensitive RAPA liposomes was about 7%, indicating the pH-sensitive property of prepared fusogenic liposomes (Figure 2).

Figure 2. In vitro drug release profile of fusogenic pH-sensitive and conventional liposomes at pH 6.5 and 7.4.

At 25 °C, the particle sizes of liposomes after 6 months were increased significantly and they were in the range of 149.8 ± 5.2–225.1 ± 10.1 nm. Storing liposomes at refrigerated conditions resulted in lower increase in particle size of liposomes.

Mean EE% of liposomes after 6 months was decreased where was 75.8% of initial values.

Results also indicated that mean RAPA content of liposomes after 6 months storing at 4 °C and 25 °C was 84.6 ± 2.3 and 74.15 ± 3.6% of primary RAPA content, respectively. However, according to our pervious study, in liposomes stored for 6 months in lyophilized form at 4 °C and 25 °C, RAPA content was decreased to 92.3 ± 1.6 and 81.6 ± 2.7% of initial RAPA content, respectively (Ghanbarzadeh et al., 2013b).

Antiproliferative effect of rapamycin liposomes

Yellow MTT is reduced to purple formazan in the mitochondria of living cells. The absorbance of this colored solution can be quantified by measuring at a certain wavelength with a spectrophotometer. This reduction takes place only when mitochondrial reductase enzymes are active, and therefore, conversion can be directly related to the number of viable (living) cells. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing cell death can be deduced.

Rapamycin has shown the potential to be applied for cancer treatment. It inhibits the proliferation of various tumor cell lines in vitro. The sensitivity of MCF-7 cells to RAPA have been reported when they are used intravenously, and the mTOR rapamycin derivatives have been shown previously in human breast cancer model (Law, 2005; Martin et al., 2013; Zagouri et al., 2012). Rouf et al. (2009) also reported that the antiproliferative effects of conventional and PEGylated rapamycin liposomes were significantly more than hydroethanol solution of rapamycin.

Antiproliferative effects of both types of liposomal formulations were compared with the corresponding blank liposomes and also RAPA’s hydroethanol solution on MCF-7 and HUVEC cell lines (Figures 3 and 4). Cell viability was expressed as the ratio of the amount of formazan produced by cells treated with RAPA containing formulations to those produced by control non-treated cells. Both types of liposomes contained 10, 25, and 50 nM of RAPA. The proliferation inhibition activity of hydroethanol solution of RAPA was used as a positive control. The control blank liposomes did not show any significant antiproliferative effect on MCF-7 and HUVEC cell lines (p < 0.05) compared to control untreated groups. In the case of MCF-7 cells treated with both types of RAPA liposomes, 23-fold increases in non-viable cells were seen, compared to hydroethanol solution of RAPA, where non-viable cells increased up to 60.83 and 84.26% in the case of conventional and fusogenic pH-sensitive liposomes containing 50 nM RAPA, respectively. Between the two types of liposomal formulations, the fusogenic pH-sensitive liposomes containing RAPA had the strongest proliferation inhibition activity on MCF-7 cells which was significantly (p < 0.05) more than RAPA conventional liposomes in all concentrations which are used.

Figure 3. Antiproliferative effects on human breast adenocarcinoma (MCF-7) cells of conventional and fusogenic pH-sensitive rapamycin (RAPA) liposomes, hydroethanol (HE) blank, blank conventional liposomes (CL.Blank), blank fusogenic pH-sensitive liposome (FL.Blank), RAPA in hydroethanol (HE + drug), and RAPA-loaded conventional liposome (CL + drug) and RAPA-loaded fusogenic pH-sensitive liposomes (FL + drug) (data are the mean values of three replications, *p < 0.05 compared with CL + drug and ##p < 0.01 and ###p < 0.001 compared with HE + drug).

Figure 4. Antiproliferative effects on human umbilical vein endothelial cells (HUVEC) of conventional and fusogenic pH-sensitive rapamycin (RAPA) liposomes, hydroethanol (HE) blank, blank conventional liposomes (CL.Blank), blank fusogenic pH-sensitive liposome (FL.Blank), RAPA in hydroethanol (HE + drug) and RAPA-loaded conventional liposome (CL + drug) and RAPA-loaded fusogenic pH-sensitive liposomes (FL + drug) (data are the mean values of three replications, ***p < 0.05 compared with CL + drug and ###p < 0.001 compared with HE + drug).

However, in the case of the HUVEC cell line, due to the pH-sensitivity property, RAPA-loaded fusogenic pH-sensitive liposomes had lower cytotoxicity on normal cell line in comparison to conventional liposomes and even hydroethanol solution of RAPA.

Our study also demonstrated the dose-dependent antiproliferative effect of RAPA on breast cancer cells where increasing the concentration of RAPA from 10 nM up to 50 nM was increased the antiproliferative effects of both conventional and fusogenic pH-sensitive liposomes.

Conclusions

In the present study we prepared conventional and fusogenic pH-sensitive liposomes of rapamycin using a modified ethanol injection method as a simple, rapid, and reproducible method employing statistical experimental design. Particle size, EE, and drug release of liposomes were characterized. In vitro antiproliferative effects of optimized formulations of conventional and fusogenic pH-sensitive were studied on the MCF-7 cell line and HUVEC cells. It was shown that fusogenic pH-sensitive liposomes had higher antiproliferative effect on the MCF-7 cell line compared to conventional rapamycin liposomes. Conversely, in the case of HUVEC cells, as healthy and normal cells, fusogenic pH-sensitive liposomes had a lower cytotoxic effect in comparison to conventional liposomes. Our data showed that fusogenic pH-sensitive liposomes had higher selectivity and cytotoxicity on cancerous cell lines. This work suggests fusogenic pH-sensitive liposomes can be advantageous as carriers for anticancer drugs with localized cytotoxic effect and can reduce side effects.

Declarations of interest

The authors report no declarations of interest.

Acknowledgements

The authors would like to thank the Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.