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Increased stability in plasma and enhanced cellular uptake of thermally denatured albumin-coated liposomes
Colloids and Surfaces B: Biointerfaces 76 (2010) 434–440
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Increased stability in plasma and enhanced cellular uptake of thermally denatured albumin-coated liposomes Suk Hyun Jung a,b , Sung Kyu Kim a , Soon Hwa Jung a , Eun Hye Kim a , Sun Hang Cho a , Kyu-Sung Jeong b , Hasoo Seong a , Byung Cheol Shin a,∗ a b
Biomaterials Research Center, Korea Research Institute of Chemical Technology, 100 Jang-dong, Yuseong, Deajeon, Republic of Korea Center for Bioactive Molecular Hybrids and Department of Chemistry, Yonsei University, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 15 October 2009 Received in revised form 30 November 2009 Accepted 3 December 2009 Available online 29 December 2009 Keywords: Liposome Albumin Denaturation Doxorubicin Intracellular uptake
a b s t r a c t Liposomes are nano-scale vesicles that can be used as one of drug carriers. The liposomes are, however, plagued by rapid opsonization of them and hence making their circulation time in bloodstream to be shortened. In this study, cationically charged liposomes of which surface was modified with bovine serum albumin (BSA) were prepared by using electrostatic interaction between cationic liposomes and anionically charged BSA molecules at higher pH than isoelectric point (pI) of BSA. The BSA-coated liposomes (BLs) were denatured by thermal treatment of BL at 100 ◦ C. The thermally denatured BSA-coated liposomes (DBLs) have mean particle diameter of 109 ± 1 nm. Encapsulation of model drug, doxorubicin (DOX), in the liposomes was carried out by using, so called, remote loading method and loading efficiency of DOX in liposomes was about 90%. DBL800 showed higher stability in plasma compared to Doxil® . Results of intracellular uptake evaluated by flow cytometry and confocal microscopy studies showed higher intracellular uptake of DBL800 than that of Doxil® . Consequently, the DBL, of which surface was complexed with denatured protein may be applicable as drug delivery carriers for increasing stability in plasma and enhanced cellular uptake efficacy of anticancer drugs. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Liposomes are nano-scale vesicles that can be used as chemical or biological drug carrying vehicles. Liposomes, which are mimetics of biological membranes, have been studied in field of drug delivery system because they have excellent physical, electrical and barrier properties that are given mainly by phospholipid bilayers [1–3]. The conventional liposomes are, however, plagued by rapid opsonization of them and hence making their circulation time in bloodstream to be shortened. This rapid elimination of liposomes is due to capture of them by macrophages of the reticuloendothelial system (RES) in liver and spleen [4–6]. In addition, conventional liposomes are structurally unstable due to an adsorption of blood plasma proteins to them in bloodstream during their circulation period [5,6]. Recently, to enhance the stability of liposomes by inhibiting the adsorption of blood plasma protein in bloodstream, modification of liposomal surface with a hydrophilic polymer such as polyethylene glycol (PEG) has been developed extensively (PEGylated liposomes) [7,8]. The PEGylated liposomes showed their passive targeting properties to tumors or inflamed
∗ Corresponding author. Tel.: +82 42 860 7226; fax: +82 42 860 7229. E-mail address: [email protected] (B.C. Shin). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.12.002
sites that had areas of loose endothelium. The nano-scale particles can extravasate due to the enhanced permeability and retention (EPR) effect [9–12]. However, PEG is not degradable by mammalian enzymes, resulting that it can lead to impairment of cellular function by accumulating in normal cells [13]. Therefore, there is a need to develop new method to increase bioavailability of liposomes without using PEG as a coating material on the surface of liposomes. To overcome the drawbacks of PEG, a number of liposomes having various functionalities have been studied. One of the approaches is conjugation of biocompatible materials such as albumin, heparin, chitosan and hyaluronic acid on the liposomal surface. A study was reported that pre-coating of albumin on the surface of polymeric nanoparticles increased their bioavailability [14,15]. We selected albumin as a material for preparation of new stealth liposome. Albumin is the most abundant blood plasma protein consisting about 50–55% of plasma proteins, which regulates blood volume by maintaining the osmotic pressure of blood compartment. It is one of the endogenous, non-toxic, non-immunogenic and relatively hydrophilic proteins in the body [14,15]. Albumin has 5.82 of isoelectric point (pI) and is a zwitter ion. So, albumin is cationically charged at pH 1–4 values or anionically charged at pH 6–9 values. Albumin has been extensively studied in order to develop a drug carrier especially for drug molecules having low water solubility [16]. Recently, a result was reported that introduction of albumin on
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the surface of liposomes reduced the association of serum proteins to liposomes and hence resulted in the more prolonged circulation time of PEG liposome [17]. Thermal denaturation of proteins induces loss of their solubility or enzymatic activity. Because tertiary or secondary structure of proteins is made of non-covalent bonding (Van der Waals force, charge interaction, hydrogen bond and hydrophobic interaction), thermal denaturation of proteins can turn their higher structures into primary structure of them [18–20]. Therefore, main chains of proteins become more flexible by thermal denaturation. As their bonds are broken by denaturation, water can interact with amide nitrogen and carbonyl oxygen of the peptide bonds and those interactions form new hydrogen bonds. Also, the effect of both new hydrogen bonding groups and hydrophobic groups is an increase of the amount of water molecules bound to the protein molecules in aqueous solution [21,22]. The objective of this study was to prepare thermally denatured BSA-coated liposomes (DBLs) which could increase stability in plasma and enhance cellular uptake of model drugs, doxorubicin (DOX). The stability in plasma and in vitro cellular uptake of DBL was investigated with free DOX or Doxil® (PEGylated liposomes). 2. Materials and methods 2.1. Materials l-␣-Phosphatidylcholine (soy hydrogenated, HSPC), cholesterol (CHOL), 1,2-distearoyl-sn-glycero-3-trimethylammoniumpropane (DSTAP) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-mPEG2000) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA) and doxorubicin (DOX) hydrochloride, anticancer drug, was purchased from Boryung Inc. (Seoul, South Korea). Bovine serum albumin (BSA), (s)-2-amino-3-(4imidazolyl) propionic acid (l-histidine), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and daunorubicin hydrochloride were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Doxil® was purchased from ALZA Co. (Mountain view, CA, USA). Bio-Rad protein assay reagent was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Fetal bovine serum (FBS), 4 -6-diamidino-2-phenylindole (DAPI), Minimum Essential Medium Eagle, Alpha Modification (MEM-␣) and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Gibco BRL/Life Technologies (New York, NY, USA). All other materials were of analytical grade and used without further purification. 2.2. Preparation of BSA-coated liposomes DOX-loaded liposomes were prepared according to the remote loading method by using an ammonium sulfate gradient [23,24]. Lipid compositions of the prepared liposomes were as follows: (1) conventional liposomes, HSPC:CHOL = 12.6:8.3 (molar ratio, total lipid content 12.80 mg/mL); (2) cationically charged liposomes, HSPC:CHOL:DSTAP = 12.6:8.3:1.4 (molar ratio, total lipid content 12.80 mg/mL); (3) cationically charged-PEG liposomes, HSPC:CHOL:DSTAP:DSPE-mPEG2000 = 12.6:8.3:1.4:1.1 (molar ratio, total lipid content 16.00 mg/mL). Briefly, lipids of the each composition were dissolved in 5 mL of organic solvent mixture consisting of chloroform and methanol (1:1, v/v), dried into a thin film on a rotary evaporator (Buchi Rotavapor R-200, Switzerland) and then hydrated with 250 mM ammonium sulfate solution. The liposomal solution was extruded five times through a polycarbonate filter having 200, 100 and 80 nm of pore size sequentially (Whatman, USA) by using an extruder (Northern Lipids Inc., USA). The free ammonium sulfate was removed with cellulose dialysis
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tubes (molecular weight cut-off, MWCO 12,000–14,000, Viskase Co., IL, USA) for 24 h at 4 ◦ C. Five milliliters of liposomal solution and 5 mL of DOX solution having 5 mg/mL were mixed and then incubated for 2 h at 60 ◦ C. The mixture was dialyzed with cellulose dialysis tubes (MWCO 12,000–14,000) to remove the free DOX for 48 h at 4 ◦ C. The DOX-loaded liposomes were stored at 4 ◦ C until use. The concentration of DOX in the liposomes was measured by UV–vis spectrophotometry at 497 nm (UV-mini, Shimadzu, Japan) and the loading efficiency was calculated according to the following equation: Loading efficiency (%) =
Fi × 100 Ft
(1)
where Fi is the concentration of DOX loaded in the liposomes after dissolution of DOX-loaded liposomes in organic solvent mixture consisting of chloroform, methanol and distilled water (2:1:0.05, v/v) and Ft is the initially added concentration of DOX. For preparation of BSA-coated cationic liposomes (BLs), each of 100, 200, 400, 800 and 1600 g/mL of BSA in phosphate buffered saline (PBS, pH 7.4, 1:1, v/v) solution was dropped to the prepared cationically charged liposomes at 37 ◦ C for 1 h, respectively. BL was isolated by centrifugation by using Centricon® (MWCO 100,000, Millipore Co., Bedford, USA) two times at 9000 rpm [16]. Preparation of DBL was carried out by heating of BL at 100 ◦ C for 30 min in oil bath (BW-10G, Lab. Companion, South Korea), and then cooled to room temperature. Schematic representation of cationically charged liposomes, BL and DBL was illustrated in Fig. 1. The particle size of the liposomes was measured by light scattering with a particle size analyzer (ELS-Z, Otuska, Japan) and the zeta potential of liposomes was measured by electrophoretic light scattering spectrophotometer (ELS-Z, Otuska, Japan) [16]. 2.3. Amount of BSA on the surface of cationically charged liposomes Amount of BSA on the surface of cationically charged liposomes was evaluated by Bradford assay 96-well plate [25]. Hundred microliters of BSA-coated liposomes and 100 L of Bio-Rad Protein Assay reagent (Hercules, CA, USA) were added to each well and then incubated for 10 min at 25 ◦ C. Amount of BSA was assessed by measuring absorbance at 590 nm by using microplate reader (SpectraMax 190, Molecular Device, USA). 2.4. Stability assay of liposomes in blood plasma Adsorption of protein in blood plasma protein on the surface of liposomes was evaluated by measuring the change in mean particle diameter of liposomes. One milliliter of liposomal solution was added to 3 mL of rat blood plasma and the samples were incubated for 2 days at 37 ◦ C with mild stirring. The particle size of the liposomes was measured by light scattering with a particle size analyzer (ELS-Z, Otuska, Japan) [16]. 2.5. In vitro release of DOX from liposomes In vitro releases of DOX from liposomes were investigated in PBS (pH 7.4) and acetate buffer solution (pH 5.0). Three milliliters of liposomal solution having DOX concentration of 1.5 mg/mL were sealed into cellulose dialysis tubes (MWCO 12,000–14,000) and those were incubated in 1 L of PBS and acetate buffer solution for 40 h at 37 ◦ C in thermal bath with continuous stirring. At predetermined time points, aliquots were withdrawn and the DOX concentration was measured by UV–vis spectrophotometry at 497 nm (UV-mini, Shimadzu, Japan).
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Fig. 1. Schematic representation of cationically charged liposomes, BL and DBL.
2.6. Intracellular uptake of liposomes B16F10, a murine melanoma cell line (ATCC, American type culture collection, USA), was cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS and 10 L/mL penicillin–streptomycin at 37 ◦ C in a humidified incubator containing 5% CO2 . The cells were maintained within their exponential growth phase. Intracellular uptake of liposomes was determined by flow cytometry assay [26]. B16F10 cells were transferred to 24-well tissue culture plates at 1 × 104 cells per each well and incubated for 12 h at 37 ◦ C. The culture medium was replaced with 15 g DOX/mL of liposomal solution and then incubated for 2 h in DMEM medium. The culture medium was then removed and then each well was washed with phosphate buffered saline (PBS, pH 7.4) solution. Three hundred microliters of paraformaldehyde (5%, v/v) were added to each well to fix the cells. Cell internalization of the samples was determined by flow cytometry with a FACScan (Becton Dickinson, San Jose, CA, USA). Cell-associated DOX was excited with an argon laser (488 nm) and fluorescence was detected at 560 nm. Data were collected at 10,000 gated events and analyzed with the CELL Quest software program. 2.7. Intracellular uptake of DOX by confocal microscopy A confocal fluorescent microscopy was used to compare the intracellular uptake of DOX (excitation/emission: 480/540 nm) [27]. Murine melanoma cells, B16F10 cells, were grown on coverslips to 50% confluence and incubated with free DOX, Doxil® or DBL800 diluted in culture medium (MEM-␣) at 37 ◦ C for 2 h. The cells were washed three times with PBS. Three hundred microliters of paraformaldehyde (5%, v/v) were added to fix the cells and then treated with DAPI (excitation/emission: 345/661 nm) for 15 min for nuclei staining. Fluorescent images of cells were analyzed using a LSM5 LIVE confocal microscope (Carl Zeiss, Germany). 3. Results and discussion 3.1. Physical properties of liposomes The structural consequences of DOX-loaded liposomes were investigated by measuring their mean particle diameter, zeta potential and DOX loading efficiency. Those results are summarized in Table 1. The mean particle diameter of conventional, cationically charged liposome and Doxil® was approximately 100–110 nm and DOX loading efficiency of them was 90–95%. The mean particle diameter of DBLs was approximately 105–120 nm and the DOX loading efficiency of them was 85–90%. DBLs showed lower loading efficiency approximately by 10% than conventional, cationically charged liposome and Doxil® because DOX-loaded BLs might be adsorbed on membrane of Centricon® during the isolation process of them by centrifugation. Especially, DBL1600 showed the lowest DOX loading efficiency because high concentration of BSA could induce BSA–BSA or BSA–liposome aggregation. Zeta potential value of cationically charged liposomes was 39.8 ± 2.2 mV and this value changed to −5.0 to −1.0 mV as a result of the fixation of the anioni-
Fig. 2. The amount of BSA coated on surface of various DBLs.
cally charged BSA molecules on the surface of cationically charged liposomes. These results confirmed that BSA could be completely fixed on the surface of cationically charged liposomes. The amount of BSA coated on the surface of DBL was determined by using Bradford assay method. As shown in Fig. 2, the amount of BSA coated on the liposomal surface increased according to the increase of BSA concentration from 100 to 1600 g/mL. The amount of BSA on the liposomal surface of DBL800 or DBL1600 showed high concentration of BSA that was approximately 147 or 580 g/mL liposome, respectively. These results suggest that BSA could be coated on the surface of the cationically charged liposomes through electrostatic interaction. 3.2. Stability of DBL in blood plasma The change in mean particle diameter according to incubation time of different DBL and BL800 samples which were incubated in blood plasma at 37 ◦ C are shown in Fig. 3A. The mean particle diameter of BL800 increased greatly from 108.3 ± 1.3 to 247.2 ± 3.7 nm after 48 h. The mean particle diameter of DBLs, however, increased slightly from 110 to 125 nm after 48 h of incubation. These results indicate that stability of DBL would be higher than BL in blood Table 1 Physical properties of DOX-loaded liposomes. Mean particle diameter (nm) Conventional liposome Cationically charged liposome Doxil® BL800 DBL100 DBL200 DBL400 DBL800 DBL1600
104.0 103.2 110.0 120.8 112.6 111.8 120.5 107.8 103.8
± ± ± ± ± ± ± ± ±
3.3 1.4 2.4 9.8 1.5 4.2 3.3 3.5 2.7
Zeta potential (mV) 0.3 39.8 −23.0 −3.6 −1.0 −4.4 −4.9 −1.5 −2.0
± ± ± ± ± ± ± ± ±
0.2 2.2 2.5 4.6 0.5 3.8 1.3 0.3 0.1
DOX loading efficiency (%) 90.3 93.8 95.5 93.8 82.0 84.9 89.0 83.8 61.6
± ± ± ± ± ± ± ± ±
1.2 1.6 2.4 1.6 3.2 4.5 1.2 3.2 3.1
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much higher than those of DBL800 and Doxil® . These results can be attributed to the fact that conventional and cationically charged liposomes did not contain PEG or hydrophilic macromolecules that could inhibit adsorption of protein to the surface of liposome [11,12,14,15]. Especially, protein adsorption of DBL800 was much lower than Doxil® . In case of DBL, thermal denaturation of BSA could induce the unfolding of BSA. The primary structure of BSA induced by unfolding of BSA could form new hydrogen bonds between water molecules and amide nitrogens or carbonyl oxygens of the amide bond in aqueous solution [18–20]. The primary structure of BSA makes it more flexible and hydrophobic [28,29]. Though hydrophobic character of primary structure of protein can induce its aggregation, the protein aggregation, is inhibited by newly formed hydrogen bonds with surrounding protein chains [29,30]. These indicate that the surface of thermally denatured liposomes is changed to be hydrophilic by hydrogen bonding with water molecules. The hydrophilic surface of liposomes can inhibit opsonization of them by plasma protein as PEG molecules can do [11,12]. These properties have been explained by high mobility of protein chains that is associated with conformational flexibility and water-binding ability. Moreover, thickness of the hydrophilic layer on liposome can be increased due to the chain length of hydrophilic polymers and also thick hydrophilic layer on liposomes would helpful for inhibition of opsonization of them [10,31]. The thickness of hydrophilic layer of DBL800 may be thicker than the thickness of hydrophilic layer of Doxil® because the chain length of PEG is much shorter than that of primary BSA. 3.3. Release of DOX from liposomes
Fig. 3. (A) The change of mean particle diameter of various DBLs and BL800 after incubation in blood plasma at 37 ◦ C. Mean and S.D. are shown (n = 3). (B) Amount of protein adsorbed on various liposomal DOX after incubation in blood plasma at 37 ◦ C. Amount of BSA on surface of DBL800 was 147 g/mL. Mean and S.D. are shown (n = 3).
plasma. DBL400 and DBL800 showed the smallest increase of mean particle diameter approximately from 110 to 120 nm indicating that DBL800 and DBL400 has the highest stability in plasma. However, in the case of DBL800, density of coated BSA on liposomal surface could be higher than that of DBL400 because the amount of BSA on the liposomal surface of DBL800 was approximately three times higher compared to that of DBL400, as shown in Fig. 2. Also, DBL1600 showed the lowest DOX loading efficiency, but had the highest amount of BSA on the liposomal surface of DBL, as shown in Table 1. Though there is no significant difference in diameter of DBLs which were incubated in blood plasma, DBL800 was chosen for following study because DBL800 has about three times higher amount of BSA on the liposomal surface than that of DBL400, as shown in Fig. 2. Also, DBL800 showed much higher DOX loading efficiency than that of DBL1600, as summarized in Table 1. Based on these results, DBL800 was selected as the optimum DBL for following studies. Fig. 3B shows the amount of protein adsorbed to various liposomes at 37 ◦ C in blood plasma. The average amount of protein adsorbed to conventional and cationically charged liposomes increased proportionally up to 48 h of incubation at 37 ◦ C. Protein adsorption to conventional or cationically charged liposomes was
Release properties of DOX from DBL800 were investigated at 37 ◦ C in different pH values such as phosphate buffered saline (PBS, pH 7.4) and acetate buffered solution (ABS, pH 5.0), respectively, as shown in Fig. 4. The in vitro DOX release from DBL800 showed initial burst of release up to 55% in PBS and 70% in ABS, respectively, within 10 h. On the other hand, the in vitro DOX release from Doxil® showed a comparatively constant release profile in which DOX was released to 25% in PBS and 30% in ABS within 10 h, respectively. These results can be attributable to kinds of lipids that were used for preparation of liposomes. As DBL800 has a cationic lipid, DSTAP, in the membrane of liposomes as a component of liposomes, the DSTAP can induce gaps in membrane of lipid bilayers due to repulsive force between DSTAP. Liposomal membrane of DBL800 may be weaker than that of Doxil® and therefore DBL800 can release DOX faster than Doxil® . In case of release profile in ABS as shown in Fig. 4B, either DBL800 or Doxil® showed faster DOX release than in PBS. It is considered that the acidic condition such as pH 5.0 can generally increase the solubility of DOX and hence the increased solubility of DOX would induce faster release than neutral condition such as pH 7.4. 3.4. Intracellular uptake of DBL800 evaluated by flow cytometry To investigate the intercellular uptake of DOX transfected by liposomes, the amount of DOX uptake into B16F10 cells were evaluated by flow cytometry and the results are shown in Fig. 5. Intracellular uptake of DBL800 was higher than Doxil® but it was lower than cationically charged liposomes as shown in Fig. 5. These results indicate that cationic charge of the cationically charged liposome can induce greater electrostatic interaction with B16F10 cells and hence increase internalization of the liposome by endocytosis [32,33]. Though cationically charged liposomes showed the highest cellular uptake among liposomes, it is generally known that cationically charged liposomes cleared rapidly from circulation [32,34].
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Fig. 4. (A) Release profiles of DOX from liposomes at 37 ◦ C in phosphate buffered saline (PBS, pH 7.4). Mean and S.D. are shown (n = 3). (B) Release profiles of DOX from liposomes at 37 ◦ C in acetate buffer saline (ABS, pH 5.0). Mean and S.D. are shown (n = 3).
In case of Doxil® , cellular uptake was much lower compared to DBL800 and it could be attributed to anionic charge (approximately −23.0 mV) of the surface of Doxil® . Anionic charges of liposomal surface lead to electrostatic repulsive-force with cellular membrane which can inhibit cellular uptake of liposome by endocytosis [35]. DBL800 show medium value in cellular uptake due to electrostatically neutral surface charge, compared to cationically charged liposome or Doxil® . Therefore, it is regarded that surface charge
Fig. 5. Cellular uptake of DOX-loaded liposomes by flow cytometry assay. (a) control, (b) Doxil® , (c) DBL800 and (d) cationically charged liposome. Control is a background of B16F10 cells without DOX. Mean and S.D. are shown (n = 3).
Fig. 6. Confocal microscopy images of B16F10 cells (murine melanoma cells) treated with free DOX, Doxil® and DBL800; all cells were incubated at a concentration of 6 g/mL of DOX in serum-free medium for 2 h at 37 ◦ C. (A) free DOX, (B) Doxil® and (C) DBL800. Red is fluorescence of DOX (excitation/emission: 480/540 nm). Blue is fluorescence of DAPI (excitation/emission: 345/661 nm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
of liposomal surface is the important factor for liposome-mediated transfection of the loaded drug. Also, DBL800 bind to an albumin receptor (gp60) on endothelial cells that transports the DOX into the extravascular space. The gp60 cluster on endothelial surfaces and associate with caveolin-1, leading to the formation of a caveolae that is released into the extravascular space [36]. In addition, secreted protein acidic rich in cysteine (SPARC) expression have been reported in many solid tumors [37]. SPARC protein can bind albumin and can increase intracellular uptake of DBL800 into the tumor due to such binding.
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3.5. Intracellular uptake of DBL800 observed by confocal microscopy Cellular uptake of free DOX or DOX-loaded liposomes was observed by confocal microscopy. Fig. 6 shows B16F10 cells visualized after 2 h of incubation with PBS, free DOX and DOX-loaded liposomes. Free DOX is known to have high cellular uptake and can be internalized to nucleus of tumor cells via endocytosis [26]. Though free DOX have good cellular uptake property, it is difficult to reach to the tumor site because free DOX disappears by rapid opsonization and being taken up by the RES of liver and spleen [38,39]. In case of Doxil® , cellular uptake to B16F10 cells can be observed less intense fluorescence because anionic surface charges of the surface Doxil® could induce electrostatic repulsiveforce with cellular membrane. DBL800 demonstrated enhanced cellular uptake to B16F10 cells compared to Doxil® as shown in flow cytometry study (Fig. 5). This might be due to DBL800 having neutral charge (approximately −1.5 mV) and cationic lipids which can induce electrostatic interaction with cells and facilitate DOX release into the cytosol by endosomal escape [34,40]. Also, DBL800 can bind to gp60 on endothelial cells or SPARC on tumors due to coated albumin on liposomal surface and can increase intracellular uptake of DBL800 into the tumor [36,37]. These results could indicate that DBL800 is more efficient for internalizing DOX to the cytosol of tumor cell than other surface modified liposomes. 4. Conclusions Denatured BSA-coated liposomes (DBL) as a long-circulating carrier of anticancer drug were developed and in vitro characteristics of doxorubicin (DOX)-loaded DBL were investigated. Loading efficiency of DOX, zeta potential and mean particle diameter of DBL800 were 83.8 ± 3%, −15 ± 0.3 mV and 107.8 ± 3 nm. The protein adsorption of DBL800 was much lower than BSA-coated liposome (BL) 800 and Doxil® , indicating that DBL can have higher stability in blood stream. Flow cytometry and confocal microscopy analyses revealed that DBL800 could internalize DOX efficiently to cytoplasm of tumor cells. Thus, our study suggests a new method of surface modification for stabilization of liposome and enhancement of cellular uptake of anticancer drug-loaded liposome. Our surface modification method of liposome will be expanded to human serum albumin (HSA) to eliminate the possible immune response that could be induced by BSA or denaturation of albumin. Acknowledgement This study was supported by the Ministry of Knowledge and Economy of South Korea (10016573). References [1] A. Sharma, U.S. Sharma, Liposomes in drug delivery: progress and limitations, Int. J. Pharm. 154 (1997) 123–140. [2] V.P. Torchilin, Recent advances with liposomes as pharmaceutical carriers, Nat. Rev. Drug Discov. 4 (2005) 145–160. [3] A.D. Bangham, M.M. Standish, J.C. Watkins, The first description of liposomes, J. Mol. Biol. 13 (1969) 238–252. [4] A. Gabizon, D. Papahadjopoulos, Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors, Med. Sci. 85 (1988) 6949–6953. [5] D. Liu, Biological factors involved in blood clearance of liposomes by liver, Adv. Drug Deliv. Rev. 24 (1997) 201–213. [6] S.M. Moghimi, H.M. Patel, Serum-mediated recognition of liposomes by phagocytic cells of the reticuloendothelial system—the concept of tissue specificity, Adv. Drug Deliv. Rev. 32 (1998) 45–60. [7] V.P. Torchilin, V.S. Trubetskoy, Which polymers can make nanoparticlate drug carriers long-circulating? Adv. Drug Deliv. Rev. 16 (1995) 141–155.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Increased stability in plasma and enhanced cellular uptake of thermally denatured albumin-coated liposomes Suk Hyun Jung a,b , Sung Kyu Kim a , Soon Hwa Jung a , Eun Hye Kim a , Sun Hang Cho a , Kyu-Sung Jeong b , Hasoo Seong a , Byung Cheol Shin a,∗ a b
Biomaterials Research Center, Korea Research Institute of Chemical Technology, 100 Jang-dong, Yuseong, Deajeon, Republic of Korea Center for Bioactive Molecular Hybrids and Department of Chemistry, Yonsei University, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 15 October 2009 Received in revised form 30 November 2009 Accepted 3 December 2009 Available online 29 December 2009 Keywords: Liposome Albumin Denaturation Doxorubicin Intracellular uptake
a b s t r a c t Liposomes are nano-scale vesicles that can be used as one of drug carriers. The liposomes are, however, plagued by rapid opsonization of them and hence making their circulation time in bloodstream to be shortened. In this study, cationically charged liposomes of which surface was modified with bovine serum albumin (BSA) were prepared by using electrostatic interaction between cationic liposomes and anionically charged BSA molecules at higher pH than isoelectric point (pI) of BSA. The BSA-coated liposomes (BLs) were denatured by thermal treatment of BL at 100 ◦ C. The thermally denatured BSA-coated liposomes (DBLs) have mean particle diameter of 109 ± 1 nm. Encapsulation of model drug, doxorubicin (DOX), in the liposomes was carried out by using, so called, remote loading method and loading efficiency of DOX in liposomes was about 90%. DBL800 showed higher stability in plasma compared to Doxil® . Results of intracellular uptake evaluated by flow cytometry and confocal microscopy studies showed higher intracellular uptake of DBL800 than that of Doxil® . Consequently, the DBL, of which surface was complexed with denatured protein may be applicable as drug delivery carriers for increasing stability in plasma and enhanced cellular uptake efficacy of anticancer drugs. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Liposomes are nano-scale vesicles that can be used as chemical or biological drug carrying vehicles. Liposomes, which are mimetics of biological membranes, have been studied in field of drug delivery system because they have excellent physical, electrical and barrier properties that are given mainly by phospholipid bilayers [1–3]. The conventional liposomes are, however, plagued by rapid opsonization of them and hence making their circulation time in bloodstream to be shortened. This rapid elimination of liposomes is due to capture of them by macrophages of the reticuloendothelial system (RES) in liver and spleen [4–6]. In addition, conventional liposomes are structurally unstable due to an adsorption of blood plasma proteins to them in bloodstream during their circulation period [5,6]. Recently, to enhance the stability of liposomes by inhibiting the adsorption of blood plasma protein in bloodstream, modification of liposomal surface with a hydrophilic polymer such as polyethylene glycol (PEG) has been developed extensively (PEGylated liposomes) [7,8]. The PEGylated liposomes showed their passive targeting properties to tumors or inflamed
∗ Corresponding author. Tel.: +82 42 860 7226; fax: +82 42 860 7229. E-mail address: [email protected] (B.C. Shin). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.12.002
sites that had areas of loose endothelium. The nano-scale particles can extravasate due to the enhanced permeability and retention (EPR) effect [9–12]. However, PEG is not degradable by mammalian enzymes, resulting that it can lead to impairment of cellular function by accumulating in normal cells [13]. Therefore, there is a need to develop new method to increase bioavailability of liposomes without using PEG as a coating material on the surface of liposomes. To overcome the drawbacks of PEG, a number of liposomes having various functionalities have been studied. One of the approaches is conjugation of biocompatible materials such as albumin, heparin, chitosan and hyaluronic acid on the liposomal surface. A study was reported that pre-coating of albumin on the surface of polymeric nanoparticles increased their bioavailability [14,15]. We selected albumin as a material for preparation of new stealth liposome. Albumin is the most abundant blood plasma protein consisting about 50–55% of plasma proteins, which regulates blood volume by maintaining the osmotic pressure of blood compartment. It is one of the endogenous, non-toxic, non-immunogenic and relatively hydrophilic proteins in the body [14,15]. Albumin has 5.82 of isoelectric point (pI) and is a zwitter ion. So, albumin is cationically charged at pH 1–4 values or anionically charged at pH 6–9 values. Albumin has been extensively studied in order to develop a drug carrier especially for drug molecules having low water solubility [16]. Recently, a result was reported that introduction of albumin on
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the surface of liposomes reduced the association of serum proteins to liposomes and hence resulted in the more prolonged circulation time of PEG liposome [17]. Thermal denaturation of proteins induces loss of their solubility or enzymatic activity. Because tertiary or secondary structure of proteins is made of non-covalent bonding (Van der Waals force, charge interaction, hydrogen bond and hydrophobic interaction), thermal denaturation of proteins can turn their higher structures into primary structure of them [18–20]. Therefore, main chains of proteins become more flexible by thermal denaturation. As their bonds are broken by denaturation, water can interact with amide nitrogen and carbonyl oxygen of the peptide bonds and those interactions form new hydrogen bonds. Also, the effect of both new hydrogen bonding groups and hydrophobic groups is an increase of the amount of water molecules bound to the protein molecules in aqueous solution [21,22]. The objective of this study was to prepare thermally denatured BSA-coated liposomes (DBLs) which could increase stability in plasma and enhance cellular uptake of model drugs, doxorubicin (DOX). The stability in plasma and in vitro cellular uptake of DBL was investigated with free DOX or Doxil® (PEGylated liposomes). 2. Materials and methods 2.1. Materials l-␣-Phosphatidylcholine (soy hydrogenated, HSPC), cholesterol (CHOL), 1,2-distearoyl-sn-glycero-3-trimethylammoniumpropane (DSTAP) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-mPEG2000) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA) and doxorubicin (DOX) hydrochloride, anticancer drug, was purchased from Boryung Inc. (Seoul, South Korea). Bovine serum albumin (BSA), (s)-2-amino-3-(4imidazolyl) propionic acid (l-histidine), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and daunorubicin hydrochloride were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Doxil® was purchased from ALZA Co. (Mountain view, CA, USA). Bio-Rad protein assay reagent was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Fetal bovine serum (FBS), 4 -6-diamidino-2-phenylindole (DAPI), Minimum Essential Medium Eagle, Alpha Modification (MEM-␣) and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Gibco BRL/Life Technologies (New York, NY, USA). All other materials were of analytical grade and used without further purification. 2.2. Preparation of BSA-coated liposomes DOX-loaded liposomes were prepared according to the remote loading method by using an ammonium sulfate gradient [23,24]. Lipid compositions of the prepared liposomes were as follows: (1) conventional liposomes, HSPC:CHOL = 12.6:8.3 (molar ratio, total lipid content 12.80 mg/mL); (2) cationically charged liposomes, HSPC:CHOL:DSTAP = 12.6:8.3:1.4 (molar ratio, total lipid content 12.80 mg/mL); (3) cationically charged-PEG liposomes, HSPC:CHOL:DSTAP:DSPE-mPEG2000 = 12.6:8.3:1.4:1.1 (molar ratio, total lipid content 16.00 mg/mL). Briefly, lipids of the each composition were dissolved in 5 mL of organic solvent mixture consisting of chloroform and methanol (1:1, v/v), dried into a thin film on a rotary evaporator (Buchi Rotavapor R-200, Switzerland) and then hydrated with 250 mM ammonium sulfate solution. The liposomal solution was extruded five times through a polycarbonate filter having 200, 100 and 80 nm of pore size sequentially (Whatman, USA) by using an extruder (Northern Lipids Inc., USA). The free ammonium sulfate was removed with cellulose dialysis
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tubes (molecular weight cut-off, MWCO 12,000–14,000, Viskase Co., IL, USA) for 24 h at 4 ◦ C. Five milliliters of liposomal solution and 5 mL of DOX solution having 5 mg/mL were mixed and then incubated for 2 h at 60 ◦ C. The mixture was dialyzed with cellulose dialysis tubes (MWCO 12,000–14,000) to remove the free DOX for 48 h at 4 ◦ C. The DOX-loaded liposomes were stored at 4 ◦ C until use. The concentration of DOX in the liposomes was measured by UV–vis spectrophotometry at 497 nm (UV-mini, Shimadzu, Japan) and the loading efficiency was calculated according to the following equation: Loading efficiency (%) =
Fi × 100 Ft
(1)
where Fi is the concentration of DOX loaded in the liposomes after dissolution of DOX-loaded liposomes in organic solvent mixture consisting of chloroform, methanol and distilled water (2:1:0.05, v/v) and Ft is the initially added concentration of DOX. For preparation of BSA-coated cationic liposomes (BLs), each of 100, 200, 400, 800 and 1600 g/mL of BSA in phosphate buffered saline (PBS, pH 7.4, 1:1, v/v) solution was dropped to the prepared cationically charged liposomes at 37 ◦ C for 1 h, respectively. BL was isolated by centrifugation by using Centricon® (MWCO 100,000, Millipore Co., Bedford, USA) two times at 9000 rpm [16]. Preparation of DBL was carried out by heating of BL at 100 ◦ C for 30 min in oil bath (BW-10G, Lab. Companion, South Korea), and then cooled to room temperature. Schematic representation of cationically charged liposomes, BL and DBL was illustrated in Fig. 1. The particle size of the liposomes was measured by light scattering with a particle size analyzer (ELS-Z, Otuska, Japan) and the zeta potential of liposomes was measured by electrophoretic light scattering spectrophotometer (ELS-Z, Otuska, Japan) [16]. 2.3. Amount of BSA on the surface of cationically charged liposomes Amount of BSA on the surface of cationically charged liposomes was evaluated by Bradford assay 96-well plate [25]. Hundred microliters of BSA-coated liposomes and 100 L of Bio-Rad Protein Assay reagent (Hercules, CA, USA) were added to each well and then incubated for 10 min at 25 ◦ C. Amount of BSA was assessed by measuring absorbance at 590 nm by using microplate reader (SpectraMax 190, Molecular Device, USA). 2.4. Stability assay of liposomes in blood plasma Adsorption of protein in blood plasma protein on the surface of liposomes was evaluated by measuring the change in mean particle diameter of liposomes. One milliliter of liposomal solution was added to 3 mL of rat blood plasma and the samples were incubated for 2 days at 37 ◦ C with mild stirring. The particle size of the liposomes was measured by light scattering with a particle size analyzer (ELS-Z, Otuska, Japan) [16]. 2.5. In vitro release of DOX from liposomes In vitro releases of DOX from liposomes were investigated in PBS (pH 7.4) and acetate buffer solution (pH 5.0). Three milliliters of liposomal solution having DOX concentration of 1.5 mg/mL were sealed into cellulose dialysis tubes (MWCO 12,000–14,000) and those were incubated in 1 L of PBS and acetate buffer solution for 40 h at 37 ◦ C in thermal bath with continuous stirring. At predetermined time points, aliquots were withdrawn and the DOX concentration was measured by UV–vis spectrophotometry at 497 nm (UV-mini, Shimadzu, Japan).
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Fig. 1. Schematic representation of cationically charged liposomes, BL and DBL.
2.6. Intracellular uptake of liposomes B16F10, a murine melanoma cell line (ATCC, American type culture collection, USA), was cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS and 10 L/mL penicillin–streptomycin at 37 ◦ C in a humidified incubator containing 5% CO2 . The cells were maintained within their exponential growth phase. Intracellular uptake of liposomes was determined by flow cytometry assay [26]. B16F10 cells were transferred to 24-well tissue culture plates at 1 × 104 cells per each well and incubated for 12 h at 37 ◦ C. The culture medium was replaced with 15 g DOX/mL of liposomal solution and then incubated for 2 h in DMEM medium. The culture medium was then removed and then each well was washed with phosphate buffered saline (PBS, pH 7.4) solution. Three hundred microliters of paraformaldehyde (5%, v/v) were added to each well to fix the cells. Cell internalization of the samples was determined by flow cytometry with a FACScan (Becton Dickinson, San Jose, CA, USA). Cell-associated DOX was excited with an argon laser (488 nm) and fluorescence was detected at 560 nm. Data were collected at 10,000 gated events and analyzed with the CELL Quest software program. 2.7. Intracellular uptake of DOX by confocal microscopy A confocal fluorescent microscopy was used to compare the intracellular uptake of DOX (excitation/emission: 480/540 nm) [27]. Murine melanoma cells, B16F10 cells, were grown on coverslips to 50% confluence and incubated with free DOX, Doxil® or DBL800 diluted in culture medium (MEM-␣) at 37 ◦ C for 2 h. The cells were washed three times with PBS. Three hundred microliters of paraformaldehyde (5%, v/v) were added to fix the cells and then treated with DAPI (excitation/emission: 345/661 nm) for 15 min for nuclei staining. Fluorescent images of cells were analyzed using a LSM5 LIVE confocal microscope (Carl Zeiss, Germany). 3. Results and discussion 3.1. Physical properties of liposomes The structural consequences of DOX-loaded liposomes were investigated by measuring their mean particle diameter, zeta potential and DOX loading efficiency. Those results are summarized in Table 1. The mean particle diameter of conventional, cationically charged liposome and Doxil® was approximately 100–110 nm and DOX loading efficiency of them was 90–95%. The mean particle diameter of DBLs was approximately 105–120 nm and the DOX loading efficiency of them was 85–90%. DBLs showed lower loading efficiency approximately by 10% than conventional, cationically charged liposome and Doxil® because DOX-loaded BLs might be adsorbed on membrane of Centricon® during the isolation process of them by centrifugation. Especially, DBL1600 showed the lowest DOX loading efficiency because high concentration of BSA could induce BSA–BSA or BSA–liposome aggregation. Zeta potential value of cationically charged liposomes was 39.8 ± 2.2 mV and this value changed to −5.0 to −1.0 mV as a result of the fixation of the anioni-
Fig. 2. The amount of BSA coated on surface of various DBLs.
cally charged BSA molecules on the surface of cationically charged liposomes. These results confirmed that BSA could be completely fixed on the surface of cationically charged liposomes. The amount of BSA coated on the surface of DBL was determined by using Bradford assay method. As shown in Fig. 2, the amount of BSA coated on the liposomal surface increased according to the increase of BSA concentration from 100 to 1600 g/mL. The amount of BSA on the liposomal surface of DBL800 or DBL1600 showed high concentration of BSA that was approximately 147 or 580 g/mL liposome, respectively. These results suggest that BSA could be coated on the surface of the cationically charged liposomes through electrostatic interaction. 3.2. Stability of DBL in blood plasma The change in mean particle diameter according to incubation time of different DBL and BL800 samples which were incubated in blood plasma at 37 ◦ C are shown in Fig. 3A. The mean particle diameter of BL800 increased greatly from 108.3 ± 1.3 to 247.2 ± 3.7 nm after 48 h. The mean particle diameter of DBLs, however, increased slightly from 110 to 125 nm after 48 h of incubation. These results indicate that stability of DBL would be higher than BL in blood Table 1 Physical properties of DOX-loaded liposomes. Mean particle diameter (nm) Conventional liposome Cationically charged liposome Doxil® BL800 DBL100 DBL200 DBL400 DBL800 DBL1600
104.0 103.2 110.0 120.8 112.6 111.8 120.5 107.8 103.8
± ± ± ± ± ± ± ± ±
3.3 1.4 2.4 9.8 1.5 4.2 3.3 3.5 2.7
Zeta potential (mV) 0.3 39.8 −23.0 −3.6 −1.0 −4.4 −4.9 −1.5 −2.0
± ± ± ± ± ± ± ± ±
0.2 2.2 2.5 4.6 0.5 3.8 1.3 0.3 0.1
DOX loading efficiency (%) 90.3 93.8 95.5 93.8 82.0 84.9 89.0 83.8 61.6
± ± ± ± ± ± ± ± ±
1.2 1.6 2.4 1.6 3.2 4.5 1.2 3.2 3.1
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much higher than those of DBL800 and Doxil® . These results can be attributed to the fact that conventional and cationically charged liposomes did not contain PEG or hydrophilic macromolecules that could inhibit adsorption of protein to the surface of liposome [11,12,14,15]. Especially, protein adsorption of DBL800 was much lower than Doxil® . In case of DBL, thermal denaturation of BSA could induce the unfolding of BSA. The primary structure of BSA induced by unfolding of BSA could form new hydrogen bonds between water molecules and amide nitrogens or carbonyl oxygens of the amide bond in aqueous solution [18–20]. The primary structure of BSA makes it more flexible and hydrophobic [28,29]. Though hydrophobic character of primary structure of protein can induce its aggregation, the protein aggregation, is inhibited by newly formed hydrogen bonds with surrounding protein chains [29,30]. These indicate that the surface of thermally denatured liposomes is changed to be hydrophilic by hydrogen bonding with water molecules. The hydrophilic surface of liposomes can inhibit opsonization of them by plasma protein as PEG molecules can do [11,12]. These properties have been explained by high mobility of protein chains that is associated with conformational flexibility and water-binding ability. Moreover, thickness of the hydrophilic layer on liposome can be increased due to the chain length of hydrophilic polymers and also thick hydrophilic layer on liposomes would helpful for inhibition of opsonization of them [10,31]. The thickness of hydrophilic layer of DBL800 may be thicker than the thickness of hydrophilic layer of Doxil® because the chain length of PEG is much shorter than that of primary BSA. 3.3. Release of DOX from liposomes
Fig. 3. (A) The change of mean particle diameter of various DBLs and BL800 after incubation in blood plasma at 37 ◦ C. Mean and S.D. are shown (n = 3). (B) Amount of protein adsorbed on various liposomal DOX after incubation in blood plasma at 37 ◦ C. Amount of BSA on surface of DBL800 was 147 g/mL. Mean and S.D. are shown (n = 3).
plasma. DBL400 and DBL800 showed the smallest increase of mean particle diameter approximately from 110 to 120 nm indicating that DBL800 and DBL400 has the highest stability in plasma. However, in the case of DBL800, density of coated BSA on liposomal surface could be higher than that of DBL400 because the amount of BSA on the liposomal surface of DBL800 was approximately three times higher compared to that of DBL400, as shown in Fig. 2. Also, DBL1600 showed the lowest DOX loading efficiency, but had the highest amount of BSA on the liposomal surface of DBL, as shown in Table 1. Though there is no significant difference in diameter of DBLs which were incubated in blood plasma, DBL800 was chosen for following study because DBL800 has about three times higher amount of BSA on the liposomal surface than that of DBL400, as shown in Fig. 2. Also, DBL800 showed much higher DOX loading efficiency than that of DBL1600, as summarized in Table 1. Based on these results, DBL800 was selected as the optimum DBL for following studies. Fig. 3B shows the amount of protein adsorbed to various liposomes at 37 ◦ C in blood plasma. The average amount of protein adsorbed to conventional and cationically charged liposomes increased proportionally up to 48 h of incubation at 37 ◦ C. Protein adsorption to conventional or cationically charged liposomes was
Release properties of DOX from DBL800 were investigated at 37 ◦ C in different pH values such as phosphate buffered saline (PBS, pH 7.4) and acetate buffered solution (ABS, pH 5.0), respectively, as shown in Fig. 4. The in vitro DOX release from DBL800 showed initial burst of release up to 55% in PBS and 70% in ABS, respectively, within 10 h. On the other hand, the in vitro DOX release from Doxil® showed a comparatively constant release profile in which DOX was released to 25% in PBS and 30% in ABS within 10 h, respectively. These results can be attributable to kinds of lipids that were used for preparation of liposomes. As DBL800 has a cationic lipid, DSTAP, in the membrane of liposomes as a component of liposomes, the DSTAP can induce gaps in membrane of lipid bilayers due to repulsive force between DSTAP. Liposomal membrane of DBL800 may be weaker than that of Doxil® and therefore DBL800 can release DOX faster than Doxil® . In case of release profile in ABS as shown in Fig. 4B, either DBL800 or Doxil® showed faster DOX release than in PBS. It is considered that the acidic condition such as pH 5.0 can generally increase the solubility of DOX and hence the increased solubility of DOX would induce faster release than neutral condition such as pH 7.4. 3.4. Intracellular uptake of DBL800 evaluated by flow cytometry To investigate the intercellular uptake of DOX transfected by liposomes, the amount of DOX uptake into B16F10 cells were evaluated by flow cytometry and the results are shown in Fig. 5. Intracellular uptake of DBL800 was higher than Doxil® but it was lower than cationically charged liposomes as shown in Fig. 5. These results indicate that cationic charge of the cationically charged liposome can induce greater electrostatic interaction with B16F10 cells and hence increase internalization of the liposome by endocytosis [32,33]. Though cationically charged liposomes showed the highest cellular uptake among liposomes, it is generally known that cationically charged liposomes cleared rapidly from circulation [32,34].
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Fig. 4. (A) Release profiles of DOX from liposomes at 37 ◦ C in phosphate buffered saline (PBS, pH 7.4). Mean and S.D. are shown (n = 3). (B) Release profiles of DOX from liposomes at 37 ◦ C in acetate buffer saline (ABS, pH 5.0). Mean and S.D. are shown (n = 3).
In case of Doxil® , cellular uptake was much lower compared to DBL800 and it could be attributed to anionic charge (approximately −23.0 mV) of the surface of Doxil® . Anionic charges of liposomal surface lead to electrostatic repulsive-force with cellular membrane which can inhibit cellular uptake of liposome by endocytosis [35]. DBL800 show medium value in cellular uptake due to electrostatically neutral surface charge, compared to cationically charged liposome or Doxil® . Therefore, it is regarded that surface charge
Fig. 5. Cellular uptake of DOX-loaded liposomes by flow cytometry assay. (a) control, (b) Doxil® , (c) DBL800 and (d) cationically charged liposome. Control is a background of B16F10 cells without DOX. Mean and S.D. are shown (n = 3).
Fig. 6. Confocal microscopy images of B16F10 cells (murine melanoma cells) treated with free DOX, Doxil® and DBL800; all cells were incubated at a concentration of 6 g/mL of DOX in serum-free medium for 2 h at 37 ◦ C. (A) free DOX, (B) Doxil® and (C) DBL800. Red is fluorescence of DOX (excitation/emission: 480/540 nm). Blue is fluorescence of DAPI (excitation/emission: 345/661 nm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
of liposomal surface is the important factor for liposome-mediated transfection of the loaded drug. Also, DBL800 bind to an albumin receptor (gp60) on endothelial cells that transports the DOX into the extravascular space. The gp60 cluster on endothelial surfaces and associate with caveolin-1, leading to the formation of a caveolae that is released into the extravascular space [36]. In addition, secreted protein acidic rich in cysteine (SPARC) expression have been reported in many solid tumors [37]. SPARC protein can bind albumin and can increase intracellular uptake of DBL800 into the tumor due to such binding.
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3.5. Intracellular uptake of DBL800 observed by confocal microscopy Cellular uptake of free DOX or DOX-loaded liposomes was observed by confocal microscopy. Fig. 6 shows B16F10 cells visualized after 2 h of incubation with PBS, free DOX and DOX-loaded liposomes. Free DOX is known to have high cellular uptake and can be internalized to nucleus of tumor cells via endocytosis [26]. Though free DOX have good cellular uptake property, it is difficult to reach to the tumor site because free DOX disappears by rapid opsonization and being taken up by the RES of liver and spleen [38,39]. In case of Doxil® , cellular uptake to B16F10 cells can be observed less intense fluorescence because anionic surface charges of the surface Doxil® could induce electrostatic repulsiveforce with cellular membrane. DBL800 demonstrated enhanced cellular uptake to B16F10 cells compared to Doxil® as shown in flow cytometry study (Fig. 5). This might be due to DBL800 having neutral charge (approximately −1.5 mV) and cationic lipids which can induce electrostatic interaction with cells and facilitate DOX release into the cytosol by endosomal escape [34,40]. Also, DBL800 can bind to gp60 on endothelial cells or SPARC on tumors due to coated albumin on liposomal surface and can increase intracellular uptake of DBL800 into the tumor [36,37]. These results could indicate that DBL800 is more efficient for internalizing DOX to the cytosol of tumor cell than other surface modified liposomes. 4. Conclusions Denatured BSA-coated liposomes (DBL) as a long-circulating carrier of anticancer drug were developed and in vitro characteristics of doxorubicin (DOX)-loaded DBL were investigated. Loading efficiency of DOX, zeta potential and mean particle diameter of DBL800 were 83.8 ± 3%, −15 ± 0.3 mV and 107.8 ± 3 nm. The protein adsorption of DBL800 was much lower than BSA-coated liposome (BL) 800 and Doxil® , indicating that DBL can have higher stability in blood stream. Flow cytometry and confocal microscopy analyses revealed that DBL800 could internalize DOX efficiently to cytoplasm of tumor cells. Thus, our study suggests a new method of surface modification for stabilization of liposome and enhancement of cellular uptake of anticancer drug-loaded liposome. Our surface modification method of liposome will be expanded to human serum albumin (HSA) to eliminate the possible immune response that could be induced by BSA or denaturation of albumin. Acknowledgement This study was supported by the Ministry of Knowledge and Economy of South Korea (10016573). References [1] A. Sharma, U.S. Sharma, Liposomes in drug delivery: progress and limitations, Int. J. Pharm. 154 (1997) 123–140. [2] V.P. Torchilin, Recent advances with liposomes as pharmaceutical carriers, Nat. Rev. Drug Discov. 4 (2005) 145–160. [3] A.D. Bangham, M.M. Standish, J.C. Watkins, The first description of liposomes, J. Mol. Biol. 13 (1969) 238–252. [4] A. Gabizon, D. Papahadjopoulos, Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors, Med. Sci. 85 (1988) 6949–6953. [5] D. Liu, Biological factors involved in blood clearance of liposomes by liver, Adv. Drug Deliv. Rev. 24 (1997) 201–213. [6] S.M. Moghimi, H.M. Patel, Serum-mediated recognition of liposomes by phagocytic cells of the reticuloendothelial system—the concept of tissue specificity, Adv. Drug Deliv. Rev. 32 (1998) 45–60. [7] V.P. Torchilin, V.S. Trubetskoy, Which polymers can make nanoparticlate drug carriers long-circulating? Adv. Drug Deliv. Rev. 16 (1995) 141–155.
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