EHOOPrAHH^ECKAa XHMH3, 2013, moM 39, № 4, c. 497-503
IN VITRO DELIVERY OF CURCUMIN WITH CHOLESTEROL-BASED
CATIONIC LIPOSOMES1
© 2013 N. Apiratikul", 2, T. Penglong* 2, K. Suksenc, S. Svasti*, A. Chairoungduac, d, B. Yingyongnarongkul", #
aDepartment ofChemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University,
Bangkapi, Bangkok 10240, Thailand bThalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhonpathom 73170, Thailand cDepartment of Physiology and Research Center of Transport Proteins for Medical Innovation, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Received September 12, 2012; in final form November 9, 2012
A new cholesterol-based cationic lipid was synthesized; liposomes prepared on its basis were evaluated as drug delivery vehicles for curcumin. Free and liposome-encapsulated curcumin cytotoxicity against HeLa, A549, HepG2, K562 and 1301 cell lines was assessed. Liposomal curcumin with ED50 values ranging from 2.5— 10 exhibited 2—8 times higher cytotoxicity than free curcumin. The synthetic cholesterol-based cationic lipid also enhanced cellular uptake of curcumin into tested cells. Cationic liposome alone showed low cytotoxicity at high doses with ED50 values of 90—210 p.M.
Keywords: cationic lipids, cholesterol-based, liposome, curcumin, drug delivery, cytotoxicity. DOI: 10.7868/S0132342313030032
INTRODUCTION
In recent years, drug delivery systems enhancing efficacy of bioactive compounds attract wide attention. These delivery systems include polymeric micelles and vesicles [1-3], dendrimers [4, 5], liposomes [6, 7], micelles [7], and nanogels [8, 9]. Among these, liposomes are considered as promising carriers for an extensive range of applications in drug as well as gene delivery [10—12]. Cationic liposomes have been studied as drug carriers also because of their selective accumulation in tumor endothelial cells [13]. Enhancement of cellular uptake of drug-loaded cationic liposomes as well as their in vitro cytotoxicity have been demonstrated and those results were attributed to the ability of cationic liposomes to interact with cells via electrostatic interaction, which could induce endocy-tosis of the liposomes and also facilitate drug release to the cytosol by endosomal escape [14]. These properties of cationic liposomes have been applied to the delivery of nucleic acid as well as cytotoxic compounds.
Curcumin (Fig. 1), a major component of turmeric, is used for centuries in Asian countries including Thailand as a spice. Curcuminoids exhibited many interesting biological activities including anti-inflam-
1 The article is published in the original.
2 These authors contributed equally to this work.
# Corresponding author: phone: +66 2 3190931; fax: +66 2 3191900; e-mail: boonek@ru.ac.th.
matory [15], antioxidant [16—18], anticancer [15, 16, 19, 20] and anti-HIV activity [21, 22]. The pharmacological safety and efficacy of curcumin makes it a potential compound for treatment and prevention of a wide variety of human diseases. However, curcumin has not yet been approved as a therapeutic agent because of problems arising from its poor bioavailability. Phospholipid-based liposomes have been demonstrated to enhance bioavailability and biological activity of curcumin [23—25]. Some of the liposome formulations incorporated cholesterol as a helper lipid for curcumin delivery [23, 25]. The synthetic cholesterol-based cationic lipids have been reported as gene delivery into mammalian cells [26—31]. Cholesterol was often used here as a lipid anchor due to its lipid bilayer stabilizing activity [32] and minimal toxicity to the treated cells. However, the use of cationic lipid with cholesterol tail to encapsulate and deliver curcumin into cells has not been reported. In this work, we report the synthesis of new cholesterol-based cationic lipid (I) (Fig. 1) and evaluation of cellular uptake and cytotoxicity of curcumin encapsulated into liposomes prepared on the basis of lipid (I).
RESULTS AND DISCUSSION
The designed cholesterol-based lipid (I) was synthesized by solution-phase chemistry (Scheme). N-(tert-Butoxycarbonyl)-1,3-diaminopropane (III) was prepared by high dilution method [33] and then trans-
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APIRATIKUL et al.
h2n ^ "nh2
2 (II) 2
BocHN v "NH2 (III) 2
BocHN ^ "N (IV) H
O
^Cl
O
A,
cr O
h2n
O
-N^O H
O
H
O
H
h2n.
O
(VII)
O
N^O H
(I)
(VI)
Scheme. Synthesis of cationic lipid (I). Reagents and conditions: (a) Boc2O, CH2Q2, rt., 89%; (b) chloroacetyl chloride, pyridine, 0°C, 81%; (c) 1,3-diaminopropane,CH2Cl2, rt., 97%; (d) Et3N, EtOH, reflux, 72%; (e) TFA, CH2Cl2, 93%.
a
b
d
e
formed into amide (IV) by condensation with chloroacetyl chloride in the presence of pyridine base at 0°C (72% yield over two steps). 3p-Cholest-5-en-3-yl-^-(3-aminopropyl)carbamate (VI) was also prepared in excellent yield by high dilution method in the same manner as the compound (III). Care must be taken for the alkylation step of amine (VI) with alkyl chloride (IV), since the dialkylation product might have taken place to give undesired product. The monoalkylation product (VII) was obtained in a good yield by refluxing compounds (VI) and (IV) in EtOH in the presence of Et3N. Finally, the Boc protecting group of derivative (VII) was removed by treating with TFA in CH2Cl2 to afford lipid (I) in 93% yield. The structures of the synthesized compounds were established on the basis of
IR, 1H, 13C NMR, and high resolution mass spectral analysis.
An important parameter to be evaluated for a liposomal delivery system is the loading efficiency. The thin-film evaporation method was used to obtain the curcumin liposomal preparation [34]. The liposome formulation composed of cationic lipid (I) and dio-leoylphosphatidylethanolamine (DOPE) at 1 : 1 (wt/wt) containing curcumin and lipid in the weight ratio of 1 : 20; this formulation was found to have moderate curcumin encapsulation efficiency (68%). We suppose that the hydrophobic nature of curcumin allows it to become incorporated into the lipid bilayer region of the liposome and improves the encapsulation efficiency. Similar observations for the hydrophobic drug have been previously reported [34, 35].
h2n
IN VITRO DELIVERY OF CURCUMIN O O
MeO HO
O
Curcumin
O
n^o
H
OMe OH
Cationic lipid (I) Fig. 1. Structures of curcumin and cholesterol-based cationic lipid (I).
90
80
70
vT 60
"u c 50
ic
tot 40
pt o 30
p
< 20
10
0
| | Liposome
Liposomal circumin
Untreated DMSO Free 30 curcumin
60 120 240
Liposome concentration, p.M
Fig. 2. Effect of empty liposomes and liposomal curcumin on apoptotic cells. The concentration of curcumin was 10.8 ^M in each liposomal curcumin formulation. The DMSO concentration was 0.5%.
The cytotoxic activity of free and liposomal encapsulated curcumin in five different human cancer cell lines, cervical epithelial adenocarcinoma (HeLa), lung epithelial adenocarcinoma (A549), liver hepatocellular carcinoma (HepG2), erythromyeloblastoid leukemia (K562), and T cell lymphoblastic leukaemia (1301), was determined. To find the optimal cur-cumin/liposome ratio, cytotoxic activities of liposomal curcumin and free liposomes were evaluated against HeLa cells. Preparations of curcumin/lipid at 1 : 5, 1 : 10, 1 : 20, 1 : 40 and 1 : 80 wt/wt ratios were prepared, the concentration of curcumin in each formulation being constant at 10.8 ^M. Empty liposomes at the same concentration were also tested for comparison. The results are shown in Fig. 2. At high weight ratio (1 : 80), cytotoxic activity of liposomal curcumin and liposomes was the same. This shows that the high concentration of liposomes was toxic to the tested cells. At curcumin/lipid ratio of 1 : 20, the concentration of liposome lipid was equivalent to 120 ^M; treatment of
tested cells with empty liposomes of the same concentration gave 25% of apoptotic cells, whereas the apoptotic cells of liposomal curcumin was at 60%. Thus, the curcumin/liposome weight ratio of 1 : 20 was used for the next experiments.
It has been reported that liposomes are toxic to various cells [36, 37]. First, we investigated whether our synthetic liposome formulation without curcumin was toxic to the tested cells. Five cell lines were treated
ED50 of curcumin, liposomal curcumin and empty liposomes
Tested preparation ED50 (^M)
HeLa A549 HepG2 K562 1310
Free curcumin 17 50 30 20 8
Liposomal curcumin 8 10 4 2.5 3.1
Empty liposomes 180 210 105 165 90
5 min 10 min
Phase Phase
contrast Fluorescence contrast Fluorescence
60 min
Phase
contrast Fluorescence
Fig. 3. Fluorescence microscopy images of HeLa cells incubated for 5, 10 and 60 min at 37°C. Row (a): PBS; row (b): 40 ^M curcumin, and row (c): 40 ^M liposome-encapsulated curcumin.
with liposomes of a range of concentrations; the results are shown in table. ED50 values of empty liposomes against HeLa, A549, HepG2, K562 and 1301 cells were 180, 210, 105, 165 and 90 |M, respectively. It was suggested that our cationic liposomes were weakly toxic to tested cells. The synthetic liposomes were also tested against normal cells, human embryonic kidney (HEK293). In contrast with the previously reported data on the cytotoxicity of cation-ic lipid to the normal cells, we have found that our liposomes were not toxic to the normal cells with the IC50 value of 600 |M (data not shown). This data suggest that our liposomes were suitable as delivery system with the minimal adverse effects on normal cells. Most of the commercial liposomes have been reported to enhance cytotoxicity of encapsulated compounds on various cells [23—25, 38]. We then investigated whether our synthetic liposomes enhance cytotoxic activity of curcumin. Cytotoxicity of liposomal and free cur-cumin was evaluated by staining with Annexin V-FITC and analyzed by flow cytometry. The results indicated that cytotoxic activity of lip
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