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Elastin-like polypeptide modified liposomes for enhancing cellular uptake into tumor cells
Colloids and Surfaces B: Biointerfaces 91 (2012) 130–136
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Elastin-like polypeptide modified liposomes for enhancing cellular uptake into tumor cells Kyunga Na a , Seul A Lee a , Suk Hyun Jung a , Jinho Hyun b , Byung Cheol Shin a,∗ a b
Biomaterials Research Center, Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong, Daejeon 305-600, South Korea Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, Seoul 151-742, South Korea
a r t i c l e
i n f o
Article history: Received 20 July 2011 Received in revised form 12 October 2011 Accepted 27 October 2011 Available online 2 November 2011 Keywords: Elastin-like polypeptides Liposomes Temperature-triggered drug delivery Cellular uptake
a b s t r a c t Polyethylene glycol-modified (PEGylated) liposomes have been widely used because of their long circulation time, but they have a major drawback of limited cellular uptake. In this study, liposomes modified with a thermosensitive biopolymer, elastin-like polypeptide (ELP), were prepared to enhance cellular uptake in tumor cells. Synthesized ELP exhibited an inverse transition temperature (Tt ) of 40 ◦ C in serum with hyperthermia treatment and contained a lysine residue for conjugation with 1,2dioleoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene-glycol)]-hydroxy succinamide, PEG MW 2000 (DSPE-PEG2000-NHS). ELP was covalently conjugated with liposomes encapsulating a high concentration of doxorubicin (Dox). Size and drug release properties of liposomes were investigated over a range of temperatures. ELP-modified liposomes tended to aggregate but did not show temperature-triggered release by phase transition of ELP molecules. Cellular uptake efficiency of liposomes was evaluated under normothermic and hyperthermic condition. Dox accumulation from liposomes was determined by flow cytometry and confocal microscopy. Higher internalization occurred in the ELP-modified liposomes than in ELP-unmodified liposomes. The results suggest that dehydration of ELP molecules on the liposomal surface can induce efficient cellular uptake, which can improve existing chemotherapeutic efficacy. © 2011 Elsevier B.V. All rights reserved.
1. Introduction There is a considerable amount of interest in the use of nanoparticles in biological applications, including drug delivery, medical imaging, cell sorting, biomaterial purification, and diagnostic systems [1–6]. In particular, the use of liposomes as drug delivery carriers has attracted much attention in light of the development of biomimetic membranes, which have been shown to exhibit biodegradability, immunogenicity, and low toxicity [7]. Recent studies have investigated the long circulation times and specific site-targeted delivery with liposomes in a hope to enhance therapeutic efficacy and to reduce side effects in drug delivery. For instance, a number of liposomes have been developed, including “stealth liposomes” containing (∼5%, w/w) PEGylated phospholipid to increase circulation time, liposomes functionalized with targeting ligands for specific site-targeted delivery, and stimuli-responsive liposomes exhibiting triggered drug release when subjected to external stimuli such as temperature, pH, ultrasound, and light [8–12].
∗ Corresponding author. Tel.: +82 42 860 7223; fax: +82 42 860 7229. E-mail address: [email protected] (B.C. Shin). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.10.051
PEGylated liposomes exhibit long circulation times because PEG reduces protein adsorption onto liposomes by steric hindrance. However, PEGylated liposomes have shown limited cellular uptake, and surface modification with molecules such as antibodies, ligands, proteins, and aptamers has been needed to improve cellular uptake efficacy [13,14]. Several targeting molecules are known to target specific tissues and organs, but significant efforts are still needed to discover cell-specific affinity reagents. In addition to active targeting by affinity, active targeting by external stimuli is a promising way to enhance therapeutic efficacy and to reduce side effects [15]. One of the stimuli-responsive liposomes, temperaturesensitive liposomes (TS liposomes), remains intact at 37 ◦ C, and site-specific drug release from TS liposomes by heating to 41 ◦ C was shown to occur with no damage to healthy tissue [16,17]. In addition, TS liposomes modified with thermosensitive polymers, such as poly(N-isopropylacrylamide) (pNIPAAM), released a higher concentration of drugs than unmodified TS liposomes [18,19]. However, severe side effects can result from accumulation of non-biodegradable synthetic polymers. Thus, thermosensitive polymers that are biodegradable and biocompatible must be developed as drug delivery carriers [20]. Recombinant elastin-like polypeptides (ELPs) are protein-based polymers, which undergo thermal inverse phase transition, similar to pNIPAAM. ELPs have been widely used in biological applications
K. Na et al. / Colloids and Surfaces B: Biointerfaces 91 (2012) 130–136
(e.g., scaffolds, drug carriers, or cell culture sheets) because of their high biocompatibility, biodegradability, and low immunogenicity [21–24]. Drug carriers using ELPs could enhance the drug’s accumulation at the site of the tumor and vascular permeability by hyperthermia because of the distinct features of ELPs. We developed novel nanocarriers modified with the biopolymer ELP to enhance therapeutic efficacy and to reduce side effects. ELP was designed for hyperthermia treatment and conjugation to the liposomal surface. The biosynthesized ELP was covalently conjugated with liposomes, which encapsulated a high concentration of doxorubicin (Dox). In this study, the cellular uptake efficiency of liposomes was evaluated over a range of temperatures. We found that the nanocarrier exhibited a drastic enhancement of cellular uptake by thermal inverse phase transition. Our nanocarrier system could provide a new strategy for therapeutic drug delivery into tumors. 2. Materials and methods
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and 8.6 mM Dox (1:1, v/v) were mixed and incubated for 2 h at 60 ◦ C. Finally, the mixture was dialyzed for 48 h at 4 ◦ C to remove unloaded Dox, and the liposomes were stored at 4 ◦ C until further use. 2.4. Preparation of ELP-modified liposomes Liposomes containing DSPE-PEG2000-NHS were used to prepare ELP-modified liposomes. One milliliter of liposomal suspension (14.3 mg/mL) was reacted with 1 mL of ELP solution (6.96 mg/mL) dissolved in phosphate-buffered saline (PBS; pH 7.4) at room temperature for 24 h. After the reaction, the ELP-modified liposomes were isolated by centrifugation 3 times at 9000 rpm by using Centricon (MWCO 100,000; Millipore Co., Bedford, USA). 2.5. Characterization of ELP-modified liposomes
Hydrogenated-l-␣-phosphatidylcholine (HSPC), 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), and cholesterol (CHOL) were purchased from Avanti Polar lipids (Alabaster, AL, USA), and 1,2dioleoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol)]-hydroxy succinamide, PEG MW 2000 (DSPE-PEG2000NHS) was purchased from NOF Co. (Tokyo, Japan). Doxorubicin hydrochloride (Dox) was obtained from Boryung Inc. (Seoul, South Korea). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), and 4 -6-diamidino-2-phenylindole (DAPI) were purchased from Gibco BRL/Life Technologies (New York, NY, USA).
The DSPE-PEG2000-ELP liposomes were characterized by 1 H NMR (Bruker Avance-500 FT-NMR Spectrometer; Bruker AXS, Germany). After lyophilization, the sample was dissolved in deuterated chloroform (CDCl3 ). The percentage of ELP conjugated with the liposomal surface was determined using Dox in fluorescence labeling of unreacted DSPE-PEG2000-NHS. Briefly, 1 mL of ELP-modified liposomal suspension (14.3 mg/mL) and 0.38 mg of Dox were mixed and reacted at room temperature for 24 h. After the reaction, the suspension was dialyzed using a cellulose dialysis tube (MWCO 12,000–14,000; Viskase Co.) for 48 h at 4 ◦ C and released Dox was measured by fluorescence spectrophotometry (Hitachi F-2000; Hitachi Instruments, Tokyo, Japan). The excitation and emission wavelengths for the measurement were 490 and 590 nm, respectively. The percentage of ELP conjugated on the liposomal surface was defined as follows:
2.2. Synthesis and characterization of ELP
% of ELP conjugated on liposomal surface =
2.1. Materials
The synthesis and purification of ELP has been described previously [25]. Briefly, the ELP gene coding for (Val–Pro–Gly–Val–Gly)20 was oligomerized by recursive directional ligation. ELP was expressed in the Escherichia coli strain BLR(DE)3 (Novagen, Milwaukee, WI, USA) and purified by the inverse transition cycling method, followed by freeze-drying. The purity and molecular weight were determined by silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (BioRad, Hercules, CA, USA). The inverse transition temperature of ELP was characterized by the change in optical density (turbidity) at 350 nm using UV–visible spectroscopy and was found to range between 25 and 46 ◦ C. The inverse phase transition temperature (Tt ) was the temperature at which the turbidity of the ELP solution was at 50% of its maximum. 2.3. Preparation of liposomes Dox-loaded liposomes were prepared by the thin-film hydration method and remote-loading method with an ammonium sulfate gradient, and their compositions are shown in Table 1 [26]. Briefly, lipids (104.3 mg) were dissolved in 10 mL of chloroform at 60 ◦ C and dried under vacuum in a rotary evaporator for 6 h until a thin film was formed. The thin film was hydrated with 5 mL of 250 mM ammonium sulfate solution, and the liposomal suspension was sequentially extruded 5 times through polycarbonate filters with pore sizes 200, 100, and 80 nm (Whatman, USA) by using a high pressure extruder (Northern Lipids Inc., USA). Ammonium sulfate that was not entrapped into the liposome was removed by dialysis for 48 h at 4 ◦ C using a cellulose dialysis tube (MWCO 12,000–14,000; Viskase Co., IL, USA). The liposomal suspension
1−
Fi − FR Fi − Fn
× 100 (1)
where Fi is the fluorescent intensity of Dox used in the reaction, and Fn is the fluorescent intensity of released Dox after the liposomes containing DSPE-PEG2000-NHS react with Dox. FR is the fluorescent intensity of released Dox after ELP-modified liposomes react with Dox. 2.6. Physiochemical characterization of liposomes The size and zeta potential of liposomes were determined using an electrophoretic light scattering spectrophotometer (ELSZ; Otuska, Japan) at 37 and 42 ◦ C. The amount of Dox loaded into the liposomes was determined using a UV–visible spectrophotometer (UV-mini; Shimadzu, Japan) at 497 nm, after dissolution of the liposomes in a mixture of chloroform–methanol (1:1, v/v). The loading efficiency was calculated according to the following equation: Loading efficiency (%) =
F1 × 100 Fi
(2)
where Fi is the initial concentration of Dox to be loaded, and Fl is the concentration of Dox loaded into the liposomes. 2.7. In vitro release of Dox from liposomes In vitro release of Dox from liposomes was performed in serum (50%, v/v) [18]. One milliliter (1 mg/mL) of the liposomal suspension was placed in a quartz cell and incubated for 2 h each at 37 and 42 ◦ C. The amount of Dox released from the liposomes was measured by fluorescence, with the excitation and emission wavelengths set to
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Table 1 Composition and physical properties of liposomes. Data reported are mean ± S.D. (n = 5). Liposomes
Composition
Molar ratio (mM)
Zeta potential (mV)
Modification efficiency (%)
Loading efficiency (%)
PEG-liposomes ELP-modified liposome
HSPC:CHOL:DSPE-PEG2000 HSPC:CHOL:DSPE-PEG2000-ELP
12.6:8.3:0.4 12.6:8.3:0.4
−28.75 ± 3.7 −31.80 ± 0.4
– 94 ± 0.52
99.2 ± 0.08 98.9 ± 0.13
490 and 590 nm, respectively. The release of Dox was calculated as follows: Release of Dox (%) =
(Fr − Fi ) × 100 (Ft − Fi )
(3)
where Fi and Fr are the initial and the intermediary fluorescence intensities of the liposomal suspension, respectively. Ft is the total fluorescence intensity of the liposomal suspension after dissolution of the liposomes in Triton X-100 (10%, v/v). 2.8. In vitro cellular uptake test HeLa (human cervix adenocarcinoma) cells (KCLB, South Korea) were cultured in DMEM supplemented with 10% FBS, 100 units/mL penicillin, 100 g/mL streptomycin, and 7.5 mM 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Gibco) at 37 ◦ C in a humidified atmosphere with 5.0% CO2 . For cellular uptake studies of liposomes, fluorescence intensity of Dox accumulated in HeLa cells was determined using a flow cytometry assay [27]. HeLa cells were seeded onto 24-well plates at a density of 1 × 104 cells/well and incubated for 12 h. The cells were treated with media containing liposomal solutions, with a Dox concentration of 15 g/mL. Each treatment group received a different heating regime: 30 min at 37 ◦ C (normothermia) or 10 min at 37 ◦ C, followed by 20 min at 42 ◦ C (hyperthermia). After incubation, treated media were removed, and cells were rinsed with PBS (pH 7.4). Subsequently, the cells were fixed in paraformaldehyde solution (4%, v/v) and each sample was measured using a fluorescence-activated cell sorter (FACS; Becton Dickinson, San Jose, CA, USA). A total of 10,000 events per sample were collected and analyzed with the software Cell Quest. Cellular localization of Dox originating from liposomes was investigated by treating HeLa cells with liposomal solutions containing 15 g/mL Dox. The cells were cultured on coverslips under the same conditions used for flow cytometry. After incubation, the cells were washed with PBS (pH 7.4) and fixed in paraformaldehyde solution, followed by staining with DAPI (excitation/emission: 345/458 nm) for 15 min. Finally, HeLa cells were observed using confocal laser scanning microscopy. 3. Results and discussion 3.1. Preparation and characterization of ELP and ELP-modified liposomes ELPs have been developed as drug carriers for hyperthermia treatment because of their thermosensitive properties. Thermosensitive ELPs conjugated to drugs showed 2-fold increased cellular uptake under hyperthermic conditions [28]. In this study, ELP was adopted for conjugation to the liposomal surface in order to overcome the limited cellular uptake of PEGylated liposomes. The peptide sequence of ELP was designed for covalent conjugation with liposomes containing DSPE-PEG2000-NHS (Fig. 1A). The ELP gene sequence was confirmed by DNA sequencing, and ELP was purified by inverse transition cycling method. The molecular weight of ELP was approximately 10 kDa, as measured by silver staining of bands on a SDS-PAGE gel. The Tt was determined through turbidity profiles of the ELP solution in PBS, as well as in serum, because the Tt was influenced by interaction with surrounding
Fig. 1. (A) Peptide sequences and silver-stained SDS-PAGE gels of designed ELP, and (B) turbidity profile of ELP as a function of temperature. The turbidity profiles were measured in PBS and in serum at an ELP concentration of 100 M. The data represent the mean (n = 3).
water molecules. Because serum, which provides an environment similar to that of the bloodstream for drug delivery, contains various proteins and antibiotics, the Tt of serum must be different from that of PBS. As shown in Fig. 1B, the Tt of ELP in standard buffer solution (e.g., PBS or deionized water) was 43 ◦ C, but the Tt in serum was shifted to 40 ◦ C. The transition behavior occurs by the folding and unfolding of ELP. ELP molecules exist in an unfolded state at temperatures below Tt at which the interactions between ELP molecules and water molecules are much stronger than the intramolecular interactions of ELP molecules because of hydrophobic hydration. On the other hand, ELP molecules are folded at temperatures above Tt , and strong intramolecular interactions between ELP molecules occur. Compounds in serum could inhibit interactions between ELP molecules and water molecules, causing a shift of Tt in serum. The phase transition at ∼40 ◦ C is beneficial for drug delivery through hyperthermic treatment. The preparation procedure for ELP-modified liposomes is shown in Scheme 1. Briefly, liposomes, including DSPE-PEG2000-NHS, were prepared by the thin-film hydration and extrusion methods for conjugation with amine-functionalized ELP (Scheme 1a). ELP was covalently conjugated to the liposomal surface through
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Scheme 1. Illustration of preparation of ELP-modified liposomes and proposed hypothesis of thermally induced ELP-modified liposomes. (a) Liposomes containing DSPEPEG2000-NHS, (b) ELP-modified liposomes at temperatures below Tt , and (c) ELP-modified liposomes at temperatures above Tt .
Fig. 2.
1
H NMR spectra of DSPE-PEG2000-ELP.
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Table 2 Mean diameter of liposomes and in vitro release of Dox from liposomes under normothermic and hyperthermic conditions. Data reported are mean ± S.D. (n = 5). Liposomes
PEG-liposomes ELP-modified liposome.
Mean diameter (nm)
Release of Dox (%)
37 ◦ C
42 ◦ C
37 ◦ C
42 ◦ C
109.8 ± 0.4 111.6 ± 5.2
92.8 ± 13.0 561.8 ± 201.5
0.82 ± 0.01 1.06 ± 0.52
1.31 ± 0.36 0.53 ± 0.22
the reaction of primary amine groups in ELP molecules with Nhydroxysuccinimide (NHS) esters of liposomes (Scheme 1b). The resulting DSPE-PEG2000-ELP molecule was confirmed by the analysis of 1 H NMR spectra as shown in Fig. 2. The proton peaks of PEG and DSPE were observed at 3.6 ppm and 1.2–1.3 ppm, respectively. The peaks at 1.8–2.2 ppm represented the Val and Pro residues of ELP. The ELP-modified efficiency on liposomal surface was approximately 94%, and the Dox loading efficiency of the prepared liposomes was more than 98% (Table 1). 3.2. Thermosensitive property of ELP-modified liposomes The particle diameter of all liposomes was approximately 110 nm, which was a suitable size for enhanced permeability and retention effects under normothermic condition (Table 2). In addition, liposomes exhibited a negative surface charge because of the PEG molecules in the zeta potential measurement (Table 1). However, aggregates appeared in the ELP-modified liposomes, and their average diameter increased to 600 nm under hyperthermic conditions (Fig. 3). Aggregation of liposomes was induced by interactions between ELP molecules conjugated on the liposomal surfaces at temperatures above Tt . Release experiments of Dox from prepared liposomes showed the effect of conjugation of ELP to the liposomal surface.
Fig. 3. Size distribution of ELP-modified liposomes in serum, under normothermic and hyperthermic conditions.
PEG-liposomes and ELP-PEG-liposomes did not release Dox when incubated for 2 h at 37 and 42 ◦ C (Table 2). Although contraction of ELP molecules on the liposomal surface occurred because of dehydration at temperatures above Tt ,
Fig. 4. Cellular uptake of liposomes detected by a flow cytometry assay under normothermic (blue peak) and hyperthermic (red peak) conditions. (A) PEG-liposomes, (B) ELP-modified liposomes, and (C) mean fluorescence intensity of liposomes detected by the flow cytometry assay. (a) Control, (b) PEG-liposomes, and (c) ELP-modified liposomes. The control is represented as a black peak and corresponds to cells incubated in culture media without Dox. Mean and S.D. are shown (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 5. Confocal microscopy images of HeLa cells incubated in the culture media with 15 g/mL Dox and subjected to 2 different heating regimes: (a and b) normothermia and (c and d) hyperthermia. (A) Control, (B) PEG-liposomes, and (C) ELP-modified liposomes. The control corresponds to cells incubated in the culture media without Dox. (a and c) DAPI (blue) fluorescence images, and (b and d) Dox (red) fluorescence images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
destabilization of the rigid structure of the lipid bilayer was not induced. 3.3. Cellular uptake of ELP-modified liposomes Cellular uptake is a critical factor for the development of liposomal drug delivery systems, because it is directly related to therapeutic efficacy. Although the application of PEG molecules has provided enhanced stability and relatively long circulation time for improved therapeutic effects, the steric hindrance of PEG molecules interfered with the cellular uptake of the drug systems. Herein, we hypothesized that the cell-adhesion properties of ELP-modified liposomes could be changed by hydrophilic and hydrophobic interactions of ELP at the Tt (Scheme 1b and c) because ELP-immobilized surfaces could control the ability for cell adhesion [29]. To investigate the cellular uptake of prepared liposomes, we measured Dox levels accumulated in HeLa cells by using flow cytometry. Each group was treated with 2 different heating regimes (normothermia and hyperthermia) to evaluate the thermal responsiveness of ELP. As shown in Fig. 4, Dox from PEG-liposomes and ELP-modified liposomes showed a similar mean fluorescence intensity (MFI) under normothermic condition, whereas the MFI of Dox from ELP-modified liposomes was 2.8-fold higher than that of PEG-liposomes after the temperature change from 37 to 42 ◦ C. Since PEG-liposomes and ELP-PEG-liposomes did not exhibit any temperature-triggered Dox release, the significant increase of MFI was caused by the thermosensitive property of ELP under hyperthermic conditions. As shown in Scheme 1, the dehydrated ELP contracted on the liposomal surface, thereby inducing aggregation of liposomes and reducing the hydrophilic steric barrier of PEG molecules conjugated to ELP as cell sheets [30]. The reduction of the cell-repellent property may have induced the affinity to cells, thereby resulting in higher cellular uptake [31].
The cellular localization of Dox in HeLa cells was confirmed by confocal microscopy. Fig. 5 shows confocal images of HeLa cells incubated at different temperatures. In samples treated with the liposomal suspension, a small amount of Dox was internalized into cells at 37 ◦ C, whereas Dox was rapidly internalized when the temperature increased. The change in uptake may be due to the inhibition of endocytic processes by the decreased permeability of the plasma membrane and depolarization of cells at a low temperature or by physicochemical changes of liposomes caused by thermal stress [32,33]. Although Dox from all liposomes was rapidly internalized at 42 ◦ C, Dox from ELP-modified liposomes showed higher fluorescence intensity than that of ELP-unmodified liposomes. The results indicate that dehydration of ELP contributes to enhanced cellular uptake. ELP-modified liposomes have great potential as useful drug carriers for cancer therapy because of their temperature-triggered cellular uptake properties.
4. Conclusion We applied thermosensitive ELPs to a liposomal system in order to enhance the cellular uptake of drug carriers in tumor cells. The liposomes were prepared by conjugating ELP to the surface of a liposome encapsulating a high concentration of Dox. Compared to Dox from ELP-unmodified liposomes, Dox from ELPmodified liposomes was more easily internalized into HeLa cells under hyperthermic conditions. The dehydration of ELP decreased the repulsive effect of PEG against cells by reducing the steric hindrance of PEG-liposomes, and this subsequently enhanced the cellular uptake of liposomes. The temperature-triggered cellular uptake property would be very useful for active tumor targeting. In addition, ELPs have considerable potential for use in drug delivery systems, because ELPs can be easily manipulated for responsiveness to a variety of factors, such as pH and ion concentration, and for tumor targeting by genetic engineering.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Elastin-like polypeptide modified liposomes for enhancing cellular uptake into tumor cells Kyunga Na a , Seul A Lee a , Suk Hyun Jung a , Jinho Hyun b , Byung Cheol Shin a,∗ a b
Biomaterials Research Center, Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong, Daejeon 305-600, South Korea Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, Seoul 151-742, South Korea
a r t i c l e
i n f o
Article history: Received 20 July 2011 Received in revised form 12 October 2011 Accepted 27 October 2011 Available online 2 November 2011 Keywords: Elastin-like polypeptides Liposomes Temperature-triggered drug delivery Cellular uptake
a b s t r a c t Polyethylene glycol-modified (PEGylated) liposomes have been widely used because of their long circulation time, but they have a major drawback of limited cellular uptake. In this study, liposomes modified with a thermosensitive biopolymer, elastin-like polypeptide (ELP), were prepared to enhance cellular uptake in tumor cells. Synthesized ELP exhibited an inverse transition temperature (Tt ) of 40 ◦ C in serum with hyperthermia treatment and contained a lysine residue for conjugation with 1,2dioleoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene-glycol)]-hydroxy succinamide, PEG MW 2000 (DSPE-PEG2000-NHS). ELP was covalently conjugated with liposomes encapsulating a high concentration of doxorubicin (Dox). Size and drug release properties of liposomes were investigated over a range of temperatures. ELP-modified liposomes tended to aggregate but did not show temperature-triggered release by phase transition of ELP molecules. Cellular uptake efficiency of liposomes was evaluated under normothermic and hyperthermic condition. Dox accumulation from liposomes was determined by flow cytometry and confocal microscopy. Higher internalization occurred in the ELP-modified liposomes than in ELP-unmodified liposomes. The results suggest that dehydration of ELP molecules on the liposomal surface can induce efficient cellular uptake, which can improve existing chemotherapeutic efficacy. © 2011 Elsevier B.V. All rights reserved.
1. Introduction There is a considerable amount of interest in the use of nanoparticles in biological applications, including drug delivery, medical imaging, cell sorting, biomaterial purification, and diagnostic systems [1–6]. In particular, the use of liposomes as drug delivery carriers has attracted much attention in light of the development of biomimetic membranes, which have been shown to exhibit biodegradability, immunogenicity, and low toxicity [7]. Recent studies have investigated the long circulation times and specific site-targeted delivery with liposomes in a hope to enhance therapeutic efficacy and to reduce side effects in drug delivery. For instance, a number of liposomes have been developed, including “stealth liposomes” containing (∼5%, w/w) PEGylated phospholipid to increase circulation time, liposomes functionalized with targeting ligands for specific site-targeted delivery, and stimuli-responsive liposomes exhibiting triggered drug release when subjected to external stimuli such as temperature, pH, ultrasound, and light [8–12].
∗ Corresponding author. Tel.: +82 42 860 7223; fax: +82 42 860 7229. E-mail address: [email protected] (B.C. Shin). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.10.051
PEGylated liposomes exhibit long circulation times because PEG reduces protein adsorption onto liposomes by steric hindrance. However, PEGylated liposomes have shown limited cellular uptake, and surface modification with molecules such as antibodies, ligands, proteins, and aptamers has been needed to improve cellular uptake efficacy [13,14]. Several targeting molecules are known to target specific tissues and organs, but significant efforts are still needed to discover cell-specific affinity reagents. In addition to active targeting by affinity, active targeting by external stimuli is a promising way to enhance therapeutic efficacy and to reduce side effects [15]. One of the stimuli-responsive liposomes, temperaturesensitive liposomes (TS liposomes), remains intact at 37 ◦ C, and site-specific drug release from TS liposomes by heating to 41 ◦ C was shown to occur with no damage to healthy tissue [16,17]. In addition, TS liposomes modified with thermosensitive polymers, such as poly(N-isopropylacrylamide) (pNIPAAM), released a higher concentration of drugs than unmodified TS liposomes [18,19]. However, severe side effects can result from accumulation of non-biodegradable synthetic polymers. Thus, thermosensitive polymers that are biodegradable and biocompatible must be developed as drug delivery carriers [20]. Recombinant elastin-like polypeptides (ELPs) are protein-based polymers, which undergo thermal inverse phase transition, similar to pNIPAAM. ELPs have been widely used in biological applications
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(e.g., scaffolds, drug carriers, or cell culture sheets) because of their high biocompatibility, biodegradability, and low immunogenicity [21–24]. Drug carriers using ELPs could enhance the drug’s accumulation at the site of the tumor and vascular permeability by hyperthermia because of the distinct features of ELPs. We developed novel nanocarriers modified with the biopolymer ELP to enhance therapeutic efficacy and to reduce side effects. ELP was designed for hyperthermia treatment and conjugation to the liposomal surface. The biosynthesized ELP was covalently conjugated with liposomes, which encapsulated a high concentration of doxorubicin (Dox). In this study, the cellular uptake efficiency of liposomes was evaluated over a range of temperatures. We found that the nanocarrier exhibited a drastic enhancement of cellular uptake by thermal inverse phase transition. Our nanocarrier system could provide a new strategy for therapeutic drug delivery into tumors. 2. Materials and methods
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and 8.6 mM Dox (1:1, v/v) were mixed and incubated for 2 h at 60 ◦ C. Finally, the mixture was dialyzed for 48 h at 4 ◦ C to remove unloaded Dox, and the liposomes were stored at 4 ◦ C until further use. 2.4. Preparation of ELP-modified liposomes Liposomes containing DSPE-PEG2000-NHS were used to prepare ELP-modified liposomes. One milliliter of liposomal suspension (14.3 mg/mL) was reacted with 1 mL of ELP solution (6.96 mg/mL) dissolved in phosphate-buffered saline (PBS; pH 7.4) at room temperature for 24 h. After the reaction, the ELP-modified liposomes were isolated by centrifugation 3 times at 9000 rpm by using Centricon (MWCO 100,000; Millipore Co., Bedford, USA). 2.5. Characterization of ELP-modified liposomes
Hydrogenated-l-␣-phosphatidylcholine (HSPC), 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), and cholesterol (CHOL) were purchased from Avanti Polar lipids (Alabaster, AL, USA), and 1,2dioleoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol)]-hydroxy succinamide, PEG MW 2000 (DSPE-PEG2000NHS) was purchased from NOF Co. (Tokyo, Japan). Doxorubicin hydrochloride (Dox) was obtained from Boryung Inc. (Seoul, South Korea). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), and 4 -6-diamidino-2-phenylindole (DAPI) were purchased from Gibco BRL/Life Technologies (New York, NY, USA).
The DSPE-PEG2000-ELP liposomes were characterized by 1 H NMR (Bruker Avance-500 FT-NMR Spectrometer; Bruker AXS, Germany). After lyophilization, the sample was dissolved in deuterated chloroform (CDCl3 ). The percentage of ELP conjugated with the liposomal surface was determined using Dox in fluorescence labeling of unreacted DSPE-PEG2000-NHS. Briefly, 1 mL of ELP-modified liposomal suspension (14.3 mg/mL) and 0.38 mg of Dox were mixed and reacted at room temperature for 24 h. After the reaction, the suspension was dialyzed using a cellulose dialysis tube (MWCO 12,000–14,000; Viskase Co.) for 48 h at 4 ◦ C and released Dox was measured by fluorescence spectrophotometry (Hitachi F-2000; Hitachi Instruments, Tokyo, Japan). The excitation and emission wavelengths for the measurement were 490 and 590 nm, respectively. The percentage of ELP conjugated on the liposomal surface was defined as follows:
2.2. Synthesis and characterization of ELP
% of ELP conjugated on liposomal surface =
2.1. Materials
The synthesis and purification of ELP has been described previously [25]. Briefly, the ELP gene coding for (Val–Pro–Gly–Val–Gly)20 was oligomerized by recursive directional ligation. ELP was expressed in the Escherichia coli strain BLR(DE)3 (Novagen, Milwaukee, WI, USA) and purified by the inverse transition cycling method, followed by freeze-drying. The purity and molecular weight were determined by silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (BioRad, Hercules, CA, USA). The inverse transition temperature of ELP was characterized by the change in optical density (turbidity) at 350 nm using UV–visible spectroscopy and was found to range between 25 and 46 ◦ C. The inverse phase transition temperature (Tt ) was the temperature at which the turbidity of the ELP solution was at 50% of its maximum. 2.3. Preparation of liposomes Dox-loaded liposomes were prepared by the thin-film hydration method and remote-loading method with an ammonium sulfate gradient, and their compositions are shown in Table 1 [26]. Briefly, lipids (104.3 mg) were dissolved in 10 mL of chloroform at 60 ◦ C and dried under vacuum in a rotary evaporator for 6 h until a thin film was formed. The thin film was hydrated with 5 mL of 250 mM ammonium sulfate solution, and the liposomal suspension was sequentially extruded 5 times through polycarbonate filters with pore sizes 200, 100, and 80 nm (Whatman, USA) by using a high pressure extruder (Northern Lipids Inc., USA). Ammonium sulfate that was not entrapped into the liposome was removed by dialysis for 48 h at 4 ◦ C using a cellulose dialysis tube (MWCO 12,000–14,000; Viskase Co., IL, USA). The liposomal suspension
1−
Fi − FR Fi − Fn
× 100 (1)
where Fi is the fluorescent intensity of Dox used in the reaction, and Fn is the fluorescent intensity of released Dox after the liposomes containing DSPE-PEG2000-NHS react with Dox. FR is the fluorescent intensity of released Dox after ELP-modified liposomes react with Dox. 2.6. Physiochemical characterization of liposomes The size and zeta potential of liposomes were determined using an electrophoretic light scattering spectrophotometer (ELSZ; Otuska, Japan) at 37 and 42 ◦ C. The amount of Dox loaded into the liposomes was determined using a UV–visible spectrophotometer (UV-mini; Shimadzu, Japan) at 497 nm, after dissolution of the liposomes in a mixture of chloroform–methanol (1:1, v/v). The loading efficiency was calculated according to the following equation: Loading efficiency (%) =
F1 × 100 Fi
(2)
where Fi is the initial concentration of Dox to be loaded, and Fl is the concentration of Dox loaded into the liposomes. 2.7. In vitro release of Dox from liposomes In vitro release of Dox from liposomes was performed in serum (50%, v/v) [18]. One milliliter (1 mg/mL) of the liposomal suspension was placed in a quartz cell and incubated for 2 h each at 37 and 42 ◦ C. The amount of Dox released from the liposomes was measured by fluorescence, with the excitation and emission wavelengths set to
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Table 1 Composition and physical properties of liposomes. Data reported are mean ± S.D. (n = 5). Liposomes
Composition
Molar ratio (mM)
Zeta potential (mV)
Modification efficiency (%)
Loading efficiency (%)
PEG-liposomes ELP-modified liposome
HSPC:CHOL:DSPE-PEG2000 HSPC:CHOL:DSPE-PEG2000-ELP
12.6:8.3:0.4 12.6:8.3:0.4
−28.75 ± 3.7 −31.80 ± 0.4
– 94 ± 0.52
99.2 ± 0.08 98.9 ± 0.13
490 and 590 nm, respectively. The release of Dox was calculated as follows: Release of Dox (%) =
(Fr − Fi ) × 100 (Ft − Fi )
(3)
where Fi and Fr are the initial and the intermediary fluorescence intensities of the liposomal suspension, respectively. Ft is the total fluorescence intensity of the liposomal suspension after dissolution of the liposomes in Triton X-100 (10%, v/v). 2.8. In vitro cellular uptake test HeLa (human cervix adenocarcinoma) cells (KCLB, South Korea) were cultured in DMEM supplemented with 10% FBS, 100 units/mL penicillin, 100 g/mL streptomycin, and 7.5 mM 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Gibco) at 37 ◦ C in a humidified atmosphere with 5.0% CO2 . For cellular uptake studies of liposomes, fluorescence intensity of Dox accumulated in HeLa cells was determined using a flow cytometry assay [27]. HeLa cells were seeded onto 24-well plates at a density of 1 × 104 cells/well and incubated for 12 h. The cells were treated with media containing liposomal solutions, with a Dox concentration of 15 g/mL. Each treatment group received a different heating regime: 30 min at 37 ◦ C (normothermia) or 10 min at 37 ◦ C, followed by 20 min at 42 ◦ C (hyperthermia). After incubation, treated media were removed, and cells were rinsed with PBS (pH 7.4). Subsequently, the cells were fixed in paraformaldehyde solution (4%, v/v) and each sample was measured using a fluorescence-activated cell sorter (FACS; Becton Dickinson, San Jose, CA, USA). A total of 10,000 events per sample were collected and analyzed with the software Cell Quest. Cellular localization of Dox originating from liposomes was investigated by treating HeLa cells with liposomal solutions containing 15 g/mL Dox. The cells were cultured on coverslips under the same conditions used for flow cytometry. After incubation, the cells were washed with PBS (pH 7.4) and fixed in paraformaldehyde solution, followed by staining with DAPI (excitation/emission: 345/458 nm) for 15 min. Finally, HeLa cells were observed using confocal laser scanning microscopy. 3. Results and discussion 3.1. Preparation and characterization of ELP and ELP-modified liposomes ELPs have been developed as drug carriers for hyperthermia treatment because of their thermosensitive properties. Thermosensitive ELPs conjugated to drugs showed 2-fold increased cellular uptake under hyperthermic conditions [28]. In this study, ELP was adopted for conjugation to the liposomal surface in order to overcome the limited cellular uptake of PEGylated liposomes. The peptide sequence of ELP was designed for covalent conjugation with liposomes containing DSPE-PEG2000-NHS (Fig. 1A). The ELP gene sequence was confirmed by DNA sequencing, and ELP was purified by inverse transition cycling method. The molecular weight of ELP was approximately 10 kDa, as measured by silver staining of bands on a SDS-PAGE gel. The Tt was determined through turbidity profiles of the ELP solution in PBS, as well as in serum, because the Tt was influenced by interaction with surrounding
Fig. 1. (A) Peptide sequences and silver-stained SDS-PAGE gels of designed ELP, and (B) turbidity profile of ELP as a function of temperature. The turbidity profiles were measured in PBS and in serum at an ELP concentration of 100 M. The data represent the mean (n = 3).
water molecules. Because serum, which provides an environment similar to that of the bloodstream for drug delivery, contains various proteins and antibiotics, the Tt of serum must be different from that of PBS. As shown in Fig. 1B, the Tt of ELP in standard buffer solution (e.g., PBS or deionized water) was 43 ◦ C, but the Tt in serum was shifted to 40 ◦ C. The transition behavior occurs by the folding and unfolding of ELP. ELP molecules exist in an unfolded state at temperatures below Tt at which the interactions between ELP molecules and water molecules are much stronger than the intramolecular interactions of ELP molecules because of hydrophobic hydration. On the other hand, ELP molecules are folded at temperatures above Tt , and strong intramolecular interactions between ELP molecules occur. Compounds in serum could inhibit interactions between ELP molecules and water molecules, causing a shift of Tt in serum. The phase transition at ∼40 ◦ C is beneficial for drug delivery through hyperthermic treatment. The preparation procedure for ELP-modified liposomes is shown in Scheme 1. Briefly, liposomes, including DSPE-PEG2000-NHS, were prepared by the thin-film hydration and extrusion methods for conjugation with amine-functionalized ELP (Scheme 1a). ELP was covalently conjugated to the liposomal surface through
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Scheme 1. Illustration of preparation of ELP-modified liposomes and proposed hypothesis of thermally induced ELP-modified liposomes. (a) Liposomes containing DSPEPEG2000-NHS, (b) ELP-modified liposomes at temperatures below Tt , and (c) ELP-modified liposomes at temperatures above Tt .
Fig. 2.
1
H NMR spectra of DSPE-PEG2000-ELP.
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Table 2 Mean diameter of liposomes and in vitro release of Dox from liposomes under normothermic and hyperthermic conditions. Data reported are mean ± S.D. (n = 5). Liposomes
PEG-liposomes ELP-modified liposome.
Mean diameter (nm)
Release of Dox (%)
37 ◦ C
42 ◦ C
37 ◦ C
42 ◦ C
109.8 ± 0.4 111.6 ± 5.2
92.8 ± 13.0 561.8 ± 201.5
0.82 ± 0.01 1.06 ± 0.52
1.31 ± 0.36 0.53 ± 0.22
the reaction of primary amine groups in ELP molecules with Nhydroxysuccinimide (NHS) esters of liposomes (Scheme 1b). The resulting DSPE-PEG2000-ELP molecule was confirmed by the analysis of 1 H NMR spectra as shown in Fig. 2. The proton peaks of PEG and DSPE were observed at 3.6 ppm and 1.2–1.3 ppm, respectively. The peaks at 1.8–2.2 ppm represented the Val and Pro residues of ELP. The ELP-modified efficiency on liposomal surface was approximately 94%, and the Dox loading efficiency of the prepared liposomes was more than 98% (Table 1). 3.2. Thermosensitive property of ELP-modified liposomes The particle diameter of all liposomes was approximately 110 nm, which was a suitable size for enhanced permeability and retention effects under normothermic condition (Table 2). In addition, liposomes exhibited a negative surface charge because of the PEG molecules in the zeta potential measurement (Table 1). However, aggregates appeared in the ELP-modified liposomes, and their average diameter increased to 600 nm under hyperthermic conditions (Fig. 3). Aggregation of liposomes was induced by interactions between ELP molecules conjugated on the liposomal surfaces at temperatures above Tt . Release experiments of Dox from prepared liposomes showed the effect of conjugation of ELP to the liposomal surface.
Fig. 3. Size distribution of ELP-modified liposomes in serum, under normothermic and hyperthermic conditions.
PEG-liposomes and ELP-PEG-liposomes did not release Dox when incubated for 2 h at 37 and 42 ◦ C (Table 2). Although contraction of ELP molecules on the liposomal surface occurred because of dehydration at temperatures above Tt ,
Fig. 4. Cellular uptake of liposomes detected by a flow cytometry assay under normothermic (blue peak) and hyperthermic (red peak) conditions. (A) PEG-liposomes, (B) ELP-modified liposomes, and (C) mean fluorescence intensity of liposomes detected by the flow cytometry assay. (a) Control, (b) PEG-liposomes, and (c) ELP-modified liposomes. The control is represented as a black peak and corresponds to cells incubated in culture media without Dox. Mean and S.D. are shown (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 5. Confocal microscopy images of HeLa cells incubated in the culture media with 15 g/mL Dox and subjected to 2 different heating regimes: (a and b) normothermia and (c and d) hyperthermia. (A) Control, (B) PEG-liposomes, and (C) ELP-modified liposomes. The control corresponds to cells incubated in the culture media without Dox. (a and c) DAPI (blue) fluorescence images, and (b and d) Dox (red) fluorescence images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
destabilization of the rigid structure of the lipid bilayer was not induced. 3.3. Cellular uptake of ELP-modified liposomes Cellular uptake is a critical factor for the development of liposomal drug delivery systems, because it is directly related to therapeutic efficacy. Although the application of PEG molecules has provided enhanced stability and relatively long circulation time for improved therapeutic effects, the steric hindrance of PEG molecules interfered with the cellular uptake of the drug systems. Herein, we hypothesized that the cell-adhesion properties of ELP-modified liposomes could be changed by hydrophilic and hydrophobic interactions of ELP at the Tt (Scheme 1b and c) because ELP-immobilized surfaces could control the ability for cell adhesion [29]. To investigate the cellular uptake of prepared liposomes, we measured Dox levels accumulated in HeLa cells by using flow cytometry. Each group was treated with 2 different heating regimes (normothermia and hyperthermia) to evaluate the thermal responsiveness of ELP. As shown in Fig. 4, Dox from PEG-liposomes and ELP-modified liposomes showed a similar mean fluorescence intensity (MFI) under normothermic condition, whereas the MFI of Dox from ELP-modified liposomes was 2.8-fold higher than that of PEG-liposomes after the temperature change from 37 to 42 ◦ C. Since PEG-liposomes and ELP-PEG-liposomes did not exhibit any temperature-triggered Dox release, the significant increase of MFI was caused by the thermosensitive property of ELP under hyperthermic conditions. As shown in Scheme 1, the dehydrated ELP contracted on the liposomal surface, thereby inducing aggregation of liposomes and reducing the hydrophilic steric barrier of PEG molecules conjugated to ELP as cell sheets [30]. The reduction of the cell-repellent property may have induced the affinity to cells, thereby resulting in higher cellular uptake [31].
The cellular localization of Dox in HeLa cells was confirmed by confocal microscopy. Fig. 5 shows confocal images of HeLa cells incubated at different temperatures. In samples treated with the liposomal suspension, a small amount of Dox was internalized into cells at 37 ◦ C, whereas Dox was rapidly internalized when the temperature increased. The change in uptake may be due to the inhibition of endocytic processes by the decreased permeability of the plasma membrane and depolarization of cells at a low temperature or by physicochemical changes of liposomes caused by thermal stress [32,33]. Although Dox from all liposomes was rapidly internalized at 42 ◦ C, Dox from ELP-modified liposomes showed higher fluorescence intensity than that of ELP-unmodified liposomes. The results indicate that dehydration of ELP contributes to enhanced cellular uptake. ELP-modified liposomes have great potential as useful drug carriers for cancer therapy because of their temperature-triggered cellular uptake properties.
4. Conclusion We applied thermosensitive ELPs to a liposomal system in order to enhance the cellular uptake of drug carriers in tumor cells. The liposomes were prepared by conjugating ELP to the surface of a liposome encapsulating a high concentration of Dox. Compared to Dox from ELP-unmodified liposomes, Dox from ELPmodified liposomes was more easily internalized into HeLa cells under hyperthermic conditions. The dehydration of ELP decreased the repulsive effect of PEG against cells by reducing the steric hindrance of PEG-liposomes, and this subsequently enhanced the cellular uptake of liposomes. The temperature-triggered cellular uptake property would be very useful for active tumor targeting. In addition, ELPs have considerable potential for use in drug delivery systems, because ELPs can be easily manipulated for responsiveness to a variety of factors, such as pH and ion concentration, and for tumor targeting by genetic engineering.
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