ARTICLE pubs.acs.org/Langmuir
Reduction-Sensitive Liposomes from a Multifunctional Lipid Conjugate and Natural Phospholipids: Reduction and Release Kinetics and Cellular Uptake Bj€orn Goldenbogen,† Nicolai Brodersen,‡ Andrea Gramatica,† Martin Loew,† J€urgen Liebscher,‡ Andreas Herrmann,† Holger Egger,*,§ Bastian Budde,§ and Anna Arbuzova*,† †
Institute of Biology/Molecular Biophysics, Humboldt-University Berlin, Invalidenstrasse 42, 10115 Berlin, Germany, Institute of Chemistry, Humboldt-University Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany, § Bayer Technology Services GmbH, Process Technology, Building E41, 51368 Leverkusen, Germany ‡
bS Supporting Information ABSTRACT: The development of targeted and triggerable delivery systems is of high relevance for anticancer therapies. We report here on reduction-sensitive liposomes composed of a novel multifunctional lipidlike conjugate, containing a disulfide bond and a biotin moiety, and natural phospholipids. The incorporation of the disulfide conjugate into vesicles and the kinetics of their reduction were studied using dansyl-labeled conjugate 1 in using the dansyl fluorescence environmental sensitivity and the F€orster resonance energy transfer from dansyl to rhodamine-labeled phospholipids. Cleavage of the disulfide bridge (e.g., by tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), L-cysteine, or glutathione (GSH)) removed the hydrophilic headgroup of the conjugate and thus changed the membrane organization leading to the release of entrapped molecules. Upon nonspecific uptake of vesicles by macrophages, calcein release from reduction-sensitive liposomes consisting of the disulfide conjugate and phospholipids was more efficient than from reduction-insensitive liposomes composed only of phospholipids. The binding of streptavidin to the conjugates did not interfere with either the subsequent reduction of the disulfide bond of the conjugate or the release of entrapped molecules. Breast cancer cell line BT-474, overexpressing the HER2 receptor, showed a high uptake of the reduction-sensitive doxorubicin-loaded liposomes functionalized with the biotin-tagged anti-HER2 antibody. The release of the entrapped cargo inside the cells was observed, implying the potential of using our system for active targeting and delivery.
’ INTRODUCTION Liposomes, for example, doxorubicin stealth liposomes, are being used as vehicles to deliver drugs in anticancer therapies.16 They can encapsulate both water-soluble and hydrophobic drugs, protect drugs from degradation, and allow the codelivery of several drugs. In addition, liposomes accumulate in malignant tissues because of the enhanced permeability and retention (EPR) effect. Because of their surface properties, liposomes can be functionalized with different tumor-specific ligands for targeted delivery: liposomes functionalized with antibodies, ligands, or affibodies were shown to accumulate in respective tumors.3,7 The active and passive accumulation of nanocarriers in tumor tissues leads to a lower systemic toxicity of the drugs.2,3,8,9 Although different carriers are already in clinical use, the demand for more efficient delivery systems and a controlled triggered release only at the site of destination is very high.10 Therefore, different stimuli-sensitive systems were proposed. Giving only a few examples, phospholipase-triggered drug release from liposomal carriers,11 reduction-triggered delivery using disulfide r 2011 American Chemical Society
conjugates of poly(ethylene glycol) and phospholipids8,1214 or nucleoside-lipid based carriers,15 reduction-sensitive disulfide copolymers,1618 and redox-triggered liposomes built from a quinone-lipid conjugate were reported.19,20 Disulfide-based conjugates are considered to be promising tools for designing antitumor drug delivery systems.13,2124 Disulfide bonds are rather stable in normal healthy tissues and blood but can be reduced in tumor tissues and inside cells after endocytosis.21,23,25,26 Indeed, many tumor tissues were found to have higher GSH, thioredoxin and thioredoxin reductase concentrations than normal tissues.23,27,28 On the basis of our expertise on multifunctional nucleolipid conjugates, pore-forming peptides, and membrane organization,2932 we developed reduction-sensitive liposomes made from a mixture of natural phospholipids and new multifunctional lipidanchored disulfide conjugate 1 or 2 (Scheme 1). The incorporation Received: March 29, 2011 Revised: July 1, 2011 Published: August 05, 2011 10820
dx.doi.org/10.1021/la201160y | Langmuir 2011, 27, 10820–10829
Langmuir
ARTICLE
Scheme 1. Synthesis of Conjugates 1 and 2 Described in the Experimental Section and Reduction Products 3, 7 and 3, 9, Respectively
of the conjugates into lipid vesicles, the kinetics of the disulfide bond reduction, and dye release from vesicles were investigated. The nonspecific cellular uptake of the reduction-sensitive liposomes and the release of the cargo in cells were tested using a mouse macrophage cell line. The assembly of antibody-presenting reduction-sensitive liposomes was achieved using an antiHER2 biotin-conjugated antibody and a streptavidinbiotin block system, which is often used as a test building system because of the high-affinity binding of the biotinstreptavidin complex and the versatility with respect to the functionalization of polymerosomes.33,34 The uptake of the antibody-presenting liposomes loaded with doxorubicin, a fluorescent anthracycline antibiotic that is an anticancer drug with a strong antimitotic effect, was tested using a breast cancer cell line overexpressing the HER2 receptor.
’ EXPERIMENTAL SECTION Materials. L-R-Phosphatidylcholine (PC, egg, chicken), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-snglycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt, Rh-PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl) (ammonium salt) (Dansyl-PE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamineN-(cap biotinyl) (sodium salt, Biotinyl-PE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), L-cysteine, reduced glutathione (GSH), streptavidin, antimouse IgG-peroxidase, calcein, doxorubicin hydrochloride (DOX), and Sephadex G50 were obtained from Sigma-Aldrich (Taufkirchen, Germany). Triton X-100 was from Roth (Carl Roth, Karlsruhe, Germany). Phosphatebuffered saline (PBS), trypsin, L-glutamine, RPMI, DMEM cell culture 10821
dx.doi.org/10.1021/la201160y |Langmuir 2011, 27, 10820–10829
Langmuir mediums, fetal bovine serum (FBS), and penicillinstreptomycin were from PAA Laboratories GmbH (C€olbe, Germany). Mouse antihuman anti-p185HER2 biotin-conjugated antibody and unlabeled and FITClabeled streptavidin were from Invitrogen, Inc. (Karlsruhe, Germany). Synthesis of Conjugates 1 and 2. Disulfide conjugates 1 and 2 were synthesized as described in detail elsewhere (in preparation). See also Scheme 1 and a brief description of the synthesis below. 2-((2,3-Bis(octadecyloxy)propyl)disulfanyl)-N,N-dimethyl-N-(2-O(3-O-biotinyl-2-N-dansyl-serin-1-yl)-tetraethylenglykol-1-yl)-ethanammonium-tosylate (1). A solution of tosylate 6 (292 mg, 0.33 mmol) and disulfide 5 (80 mg, 0.11 mmol) in CHCl3 (1 mL) was stirred under argon in a sealed flask at 70 °C (2 bar) for 5 days. The solvent was stripped off by a rotary evaporator, and the remainder was purified by column chromatography (silica, 5:1 dichloromethane/MeOH, Rf = 0.2). The product was obtained as a diastereomeric mixture in 50% yield (88 mg) as a yellow solid, mp 5557 °C. 2-((2,3-Bis(octadecyloxy)propyl)disulfanyl)-N,N-dimethyl-N-(2-Obiotinyl-tetraethylen-glykol-1-yl)-ethanammonium-tosylat (2). A suspension of disulfide 5 (161 mg, 0.23 mmol) and biotin derivative 8 (332 mg, 0.58 mmol) in chloroform (1.7 mL) was put into a sealed flask under argon and heated to 70 °C under stirring for 7 days. The pressure rose to about 2 bar. The solvent was removed with a rotary evaporator, and the remainder was purified by column chromatography (silica, 5:1 dichloromethane/MeOH, Rf = 0.3) giving 115 mg (39% yield) of product 2 as a yellowish solid, mp > 190 °C under decomposition.
2-((2,3-Bis(octadecyloxy)propyl)disulfanyl)-N,N-dimethylethanamine (5). PTAD (114 mg, 0.66 mmol) was added to a solution of 3-mercapto-1,2-bis-O-(octadecyl)-propane (3)35 (400 mg, 0.66 mmol) in a mixture of dry CH2Cl2 (14 mL) and dry MeCN (5.4 mL). The resulting red solution was stirred under argon at room temperature for 30 min and then heated to 5055 °C. When the mixture had become colorless (after about 2 h), 2-dimethylaminoethanethiol hydrochloride 4 (243 mg, 1.72 mmol) was added. After being stirred at room temperature for 20 h, the solvents were removed with a rotary evaporator and the remainder was purified by column chromatography (silica, 12:1 CH2Cl2/MeOH, Rf = 0.6), providing 322 mg (68% yield) of product 5 as a colorless solid. Tosyl-biotinyl-tetraethylene Glycol (8). A suspension of (+)-biotin (1.00 g, 4.1 mmol), O-tosyltetraethylene glycol36 (1.04 g, 4.1 mmol), DCCI (1.01 g, 4.9 mmol), and DMAP (70 mg, 0.57 mmol) in CH2Cl2 (30 mL) was stirred at room temperature for 96 h. The solvent was removed with a rotary evaporator, and the remainder was purified by column chromatography (silica, 10:1 CH2Cl2/MeOH, Rf = 0.3), giving 1.03 g (43% yield) of product 8 as a colorless solid. Conjugates 1 and 2 have molecular weights of 1611 and 1291 g/mol, respectively. Preparation of Liposomes. Conjugate 1 or 2 was dissolved in 5:1 chloroform/methanol to 510 mM. Lipid mixtures were prepared in 5:1 chloroform/methanol, and a thin lipid film was formed in roundbottomed tubes by a rotary evaporator. Reduction-sensitive liposomes were produced from conjugate 1 or 2 (520 mol%), L-R-phosphatidylcholine (PC, 2030 mol%), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, 6065 mol%). Rh-PE (1 mol%) was added to lipid mixtures as an acceptor in studying the incorporation and reduction kinetics when stated. Lipid mixtures used were: 7.4:92.6 conjugate 1/PC with and without 1 mol% Rh-PE; 10:30:60, 18:27:55, 15:22:63, and 10:25:65 conjugate 1/PC/DOPE or conjugate 2/PC/ DOPE; and 20:80 conjugate 2/PC. Control-reduction-insensitive liposomes were made from 40:60 PC/DOPE, 1:99 Rh-PE/PC, 1:99 dansylPE/PC, and 1:40:60 biotinyl-PE/PC/DOPE. Note that the amount of conjugate 1 in the vesicle samples was estimated by measuring the absorption of dansyl at 330 nm and was 7.4 mol% for the experiments shown in Figure 1. Lipid films were redissolved in cyclohexane with 5% (v/v) ethanol, frozen, and lyophilized overnight. Lipids (3 μmol) were
ARTICLE
Figure 1. Fluorescence emission spectra of vesicles composed of 7.4:92.6 conjugate 1/PC with or without 1 mol% Rh-PE (red-dotted and green-dashed curves, respectively). The black curve reveals the spectrum of the PC vesicles containing 1 mol% Rh-PE. The blue dasheddotted curve reveals the spectrum after the addition of 0.1% Triton X-100 to the vesicles composed of 1:7.4:91.6 Rh-PE/conjugate 1/PC. Excitation at 330 nm, 35 °C. used to obtain 1.5 mL of 2 mM total lipid stocks in buffer. Lipid mixtures were rehydrated with calcein (70 mM calcein, 10 mM HEPES, 1 mM EDTA, pH 7.4) or KCl buffer (100 mM KCl, 10 mM HEPES, pH 7.4) at 70 °C. The suspension was mixed vigorously and subjected to five freezethaw cycles with thawing in a water bath at 70 °C and 11 extrusions through a 100 nm pore size polycarbonate filter (Nucleopore GmbH, T€ubingen, Germany) using a Mini-Extruder with a heating block (Avanti Polar Lipids, Alabaster, AL) at approximately 70 °C.37 Thin layer chromatography was used to prove that conjugates 1 and 2 could be reduced (data not shown). Vesicles with entrapped calcein were separated from free calcein by gel filtration using spin Sephadex G50 columns (1 mL polypropylene column, Qiagen, Hilden, Germany) at (100300)g for 3 min. Sephadex G50 was presoaked in the isoosmotic KCl buffer with 10 mM HEPES and 1 mM EDTA at pH 7.4. Reductionsensitive liposomes were stable and retained entrapped calcein for at least 2 weeks. Fluorescence Spectroscopy. An Aminco Bowman series 2 fluorometer with a temperature-controlled cell and continuous stirring was used. Measurements were made in 4 mL cuvettes using 1 mL samples containing a 10 to 30 μM lipid concentration. Dansyl was excited at 330 nm, and emission was measured at 520 nm. Rhodamine was excited at 560 nm, and emission was measured at 590 nm. F€orster resonance energy transfer from dansyl to rhodamine was measured with excitation at 330 nm and emission at 590 nm. Calcein was excited at 492 nm, and emission was measured at 520 nm. Time-dependent release curves were normalized using eq 1 as described elsewhere Release ðtÞ ¼
FðtÞ Fo Fmax Fo
ð1Þ
where Release (t) is the normalized release, F(t) is a raw data fluorescence curve, Fo is the fluorescence intensity before the addition of a reducing agent, and Fmax is the fluorescence intensity after the complete release of calcein upon the disruption of liposomes by Trition X-100.29,38 Time was set to 0 s at the point of addition of a reducing agent. Light scattering at 90° was measured at 550 nm on a Jobin Yvon Fluoromax-4 fluorometer (Horiba Jobin Yvon, NJ, USA) and at 600 nm on an Aminco Bowman series 2 to obtain the data shown in Figures S4 and S5 (left), respectively.
Uptake and Release Experiments in a Macrophage Cell Line. Mouse macrophage cell line J774A.1 was used to study the 10822
dx.doi.org/10.1021/la201160y |Langmuir 2011, 27, 10820–10829
Langmuir nonspecific uptake of calcein-loaded vesicles. Cells were grown to 70% confluency in 12 mL cell flasks in the presence of RPMI, 10% FBS, 5% penicillin-streptomycin, and 2 mM L-glutamine. Then cells were gently scraped off, plated in 35 mm glass-bottomed dishes (MaTek Corp., USA; 1:4 dilution), and incubated at 37 °C in 5% CO2 for 24 h. Cells were washed six times with PBS with Ca2+ and Mg2+, incubated with 25 μL of reduction-sensitive or -insensitive liposomes, 15:22:63 conjugate 2/DOPE/PC, and 37:63 DOPE/PC, and reincubated in RPMI medium at 37 °C with 5% CO2. Fluorescence Microscopy. The fluorescence microscopy images were taken with an Olympus IX-81 inverted epifluorescence microscope in 1 mL of PBS with Ca2+ and Mg2+ to avoid cell detachment during the observation. Before observation on the microscope, the cells were washed six times with PBS with Ca2+ and Mg2+. Note that cells were always incubated in a medium without phenol red to avoid the background fluorescence due to phenol red. Fluorescence-Activated Cell Sorting (FACS). FACScan (Becton Dickson Bioscience, USA) was used. Macrophages were treated with 25 μM reduction-sensitive or -insensitive liposomes (15:22:63 conjugate 2/DOPE/PC or 37:63 DOPE/PC, respectively) for 1 h. Subsequently, cells were washed six times with PBS without Ca2+ and Mg2+ and scraped off. A 200 μL sample from the cell culture were taken, treated with 1 μL of propidium iodide (PI, 1 mg/mL) to stain dead cells, and used for FACS. The data obtained were analyzed with FlowJo (Tree Star, Inc.) flow cytometry data analysis software. Only living cells, the population of cells not stained with PI, was analyzed for their green fluorescence intensity, corresponding to the uptake of loaded liposomes and the release of calcein. Enzyme-Linked Immunosorbent Assay (ELISA). Biotin-conjugated mouse antihuman p185HER-2 (50 μL, 50 μg/mL) was coupled to streptavidin (100 μg/mL) by incubation in PBS at room temperature. The conjugate was then mixed with the reduction-sensitive liposomes (60 min of incubation at room temperature, followed by 19 h of incubation at 4 °C), and the resulting construct was size-separated on a gel filtration column (Sephacryl S200 HR). Elution was carried out with PBS, and the eluate fractions were subjected to ELISA and particle count analysis. Briefly, fractions were adsorbed to a high-binding 96-well ELISA plate (Greiner Bio-One GmbH, Frickenhausen, Germany) blocked with skim milk powder and incubated with antimouse IgGperoxidase 1:1000 in Tris-buffered saline for 1 h. After a washing step (three times with Tris-buffered saline), the remaining peroxidase was detected by chromogenic detection (24.3 mM citric acid, 51.4 mM disodium phosphate, 0.04% (w/v) o-phenylene diamine, and 0.012% (v/v) hydrogen peroxide) and measurement in a UV/vis spectrophotometer (492 nm). In parallel, the particle size and count of the fractions were determined by dynamic light scattering using a Malvern Zetasizer 3000 HSA at 633 nm, 90°, and 25 °C. Antibody-Mediated Liposomes’ Uptake and Release. BT474 breast cancer cells were grown to 70% confluency in 12 mL cell flasks in DMEM with 10% FBS, 5% penicillin-streptomycin, and 2 mM L-glutamine. Then they were detached using 0.05% trypsin/EDTA and replated a day before the uptake experiment as described above for macrophages. To assemble anti-p185HER2-presenting reduction-sensitive and insensitive vesicles loaded with doxorubicin, 150 μL of vesicles, pretested for the stability and triggered release, and 25 μL of streptavidin were mixed in 1 mL PBS without Ca2+ and Mg2+ and incubated at room temperature for 30 min. For the preparation of the liposomes, the following lipid molar ratios were used: 20:20:60 conjugate 1/PC/DOPE (RS-LUVs) and 0.1:40:60 Biotinyl-PE/PC/DOPE (RI-LUVs). The samples were frozen (80 °C) overnight and lyophilized with a freeze dryer. Doxorubicin water solution (1 mL, 0.5 mg/ml) was added to the lipid mixtures. The suspension was sonified (50% interval, 20% intensity) for 10 min and subsequently subjected to five freezethaw
ARTICLE
cycles with thawing in a water bath at 70 °C and lyophilized at 0.2 mbar overnight. Doxorubicin (0.1 mL, 0.5 mg/mL) and after 5 min another portion of doxorubicin (0.4 mL, 0.5 mg/mL) were added to the lyophilized sample. A final concentration of doxorubicin of ∼3 mM entrapped in liposomes was obtained. Note that we used doxorubicin only for the detection of release, and the entrapped concentration was significantly lower than that necessary for therapeutic treatment (e.g., ∼200 mM). The obtained suspensions were then extruded 11 times through a 100 nm pore size polycarbonate filter using a Mini-Extruder with a heating block at approximately 70 °C. Vesicles with entrapped doxorubicin were separated from the free dye by gel filtration using spin Sephadex G50 columns (e.g., a 1 mL polypropylene column) for 3 min at (100300)g. Sephadex G50 was soaked in the physiological buffer. Subsequently, 25 μL of streptavidin (1 mg/mL) and 50 μL (50 μg/mL) of anti-p185HER2 biotin-conjugated antibody were added, and the mixture was incubated at 4 °C for 1 h before being applied to cells. Cells were washed four times with PBS with Ca2+ and Mg2+. Then antibody-presenting liposomes were added to the cells in PBS with Ca2+ and Mg2+, and cells were incubated at 4 °C for 1 h to minimize unspecific uptake due to energydependent endocytosis. The cells were then washed twice with PBS with Ca2+ and Mg2+, fresh medium (DMEM) was added, and the cells were incubated at 37 °C in 5% CO2 for 1 h. Before observation, the cells were washed four times with PBS with Ca2+ and Mg2+. The fluorescence microscopy images, taken with inverted epifluorescence microscope Zeiss Axiovert 100, were recorded in 1 mL of PBS with Ca2+ and Mg2+ and 50 mM HEPES to avoid cell detachment during the observation.
’ RESULTS Synthesis of Multifunctional Conjugates. Disulfide conjugates 1 and 2 were synthesized as described in detail elsewhere. Briefly, Scheme 1 shows the synthesis steps used to obtain amphiphilic conjugates 1 and 2 starting from lipophilic aminoethyl disulfide 5 and tetraethylene glycol-biotin tosylates 6 and 8 as alkylating reagents establishing a tetraalkylammonium moiety (details in the Experimental Section). Disulfide conjugates 1 and 2 each have a hydrophobic part made of two octadecyl chains, allowing incorporation into lipid bilayers. This hydrophobic part is connected via a disulfide to a polar headgroup containing a positively charged ammonium moiety. In addition, the polar headgroup was functionalized with biotin via a tetraethylene glycol linker and fluorescent dye dansyl for conjugate 1. Reductive cleavage of the disulfide moiety of 1 or 2 will produce lipophilic thioglyceroldiether 3 and hydrophilic thiol 7 bearing the charged ammonium moiety, dansyl, and biotin or 9 bearing the charged ammonium moiety and biotin. Incorporation of Conjugate 1 into Liposomes. First, using dansyl-labeled conjugate 1 we proved that disulfide conjugate 1 is incorporated into the membrane of the vesicles. Dansyl is an environmentally sensitive fluorophore that has a higher quantum yield in hydrophobic environments (e.g., when a dansyl-labeled lipid analogue is incorporated into a lipid membrane).39 The quantum yield will decrease if dansyl-labeled headgroup 7 is transferred to an aqueous solution upon cleavage of the disulfide bond. In addition, because of the overlap of dansyl emission and rhodamine excitation spectra, F€orster resonance energy transfer (FRET) from dansyl to closely located rhodamine molecules is expected. The F€orster radius of the dansyl-rhodamine FRET pair is about 4.3 nm.39,40 Phosphatidylcholine vesicles containing the FRET pair, conjugate 1, and Rh-PE; only a donor, conjugate 1; or only an acceptor, Rh-PE, were prepared in parallel. The corresponding emission spectra by the excitation of dansyl at 330 nm of the 10823
dx.doi.org/10.1021/la201160y |Langmuir 2011, 27, 10820–10829
Langmuir
Figure 2. Time traces of the dansyl fluorescence of 7.4:92.6 conjugate 1/PC vesicles (red curve, excitation at 330 nm, emission at 520 nm) and the FRET-mediated rhodamine fluorescence of 7.4:91.6:1 conjugate 1/ PC/Rh-PE vesicles (black curve, excitation at 330 nm, emission at 590 nm) at 37 °C. TCEP (16 mM) was added at about 80 s. Note that these are raw data, which are not corrected for dilution due to TCEP addition, causing a sharp drop in fluorescence directly at the time of addition.
vesicles containing conjugate 1 with and without the rhodaminelabeled phospholipid are shown in Figure 1 (red dotted and green dashed curves, respectively). When both fluorophores were incorporated, we observed a strong decrease in the dansyl fluorescence and an increase in the rhodamine fluorescence (red dotted curve) because of the strong energy transfer from dansyl on conjugate 1 to rhodamine on the phospholipid, proving the incorporation of conjugate 1 into the phospholipid membrane of the vesicles. For comparison, the black curve in Figure 1 shows the spectrum of Rh-PE in vesicles prepared without conjugate 1. The blue dashed-dotted curve shows the spectrum of the same sample as shown by the red dotted curve (containing both dansyl-labeled conjugate 1 and Rh-PE) but after the addition of Triton X-100. The addition of the detergent led to the destruction of liposomes and an increase in the distance between donor (dansyl) and acceptor (rhodamine) and therefore a decrease in energy transfer. High dansyl fluorescence in the vesicles without the acceptor (green dashed curve) indicates that conjugate 1 is incorporated into a lipid membrane. Together these results prove that dansyl on conjugate 1 was located at the membrane interface (high dansyl fluorescence) and that conjugate 1 was incorporated into the same vesicles as rhodamine-labeled phospholipids. Cryoelectron microscopy and dynamic light scattering measurements revealed that the presence of the conjugate in the lipid membrane did not change the morphology of the vesicles: reduction-sensitive liposomes prepared by extrusion through 100 nm pore size filters had diameters of 100150 nm (Supporting Information Figure S1 and below). Disulfide Reduction Kinetics. To assess the cleavage of the disulfide bonds, both the environmental sensitivity of dansyl fluorescence and the FRET between dansyl and rhodamine were used to measure the reduction kinetics (Figure 2). The red curve shows the time trace of dansyl fluorescence changes of the probe containing 7.4:92.6 conjugate 1/PC vesicles. Upon addition of the reducing agent (16 mM tris(2-carboxyethyl)phosphine, TCEP) at about 80 s, dansyl headgroups 7 of
ARTICLE
Figure 3. Normalized release of calcein from a 30 μM lipid concentration of 18:27:55 conjugate 2/PC/DOPE liposomes triggered by the addition of 1, 0.5, and 0.25 mM DTT at time 0 s and 40 °C (green, black, and red curves, respectively). The addition of reducing agent to 40:60 PC/DOPE liposomes did not induce release (cyan curve). After the release reached a plateau phase, Triton X-100 was added, leading to the complete dequenching of calcein.
conjugates 1 were detached from the membrane, leading to the decrease in fluorescence due to the lower quantum yield of dansyl in aqueous solution. The black curve represents the time trace of the rhodamine fluorescence intensity upon excitation of dansyl at 330 nm (FRET). After about 80 s, a reducing agent was added, cleaving the disulfide bridges and detaching headgroups 7 of conjugates 1 off the membrane interface, hence leading to a decrease in FRET. Although disulfide conjugate 1 was incorporated into the membrane of liposomes (Figure 1) and could be reduced by TCEP (Figure 2), no release of entrapped molecules from PC/ conjugate 1(2) liposomes could be induced by the addition of a reducing agent. Therefore, we have added unsaturated phosphatidylethanolamine, DOPE, to the lipid mixture to provoke the liposome instability upon reduction of conjugate 1 or 2. By using less than 70 mol% but at least 55 mol% DOPE, stable liposomes were formed and reduction-induced release was studied. Triggered Calcein Release. Calcein, a membrane-impermeable fluorescent dye, was encapsulated into liposomes at a high self-quenching concentration (70 mM) as a mimic of a watersoluble drug, allowing detection of the release kinetics due to the increase in fluorescence upon the dequenching of calcein due to dilution.38 Figure 3 shows the triggered release of the dye from 18:27:55 conjugate 2/PC/DOPE liposomes by DTT at 40 °C. The curves were normalized as described in the Experimental Section with the maximum fluorescence intensity measured after the addition of surfactant Triton X-100 to destroy the liposomes and hence to obtain the complete release of calcein. The release started after a lag time, denoting the time required before the onset of a rapid increase in the fluorescence signal (Figure 3). The lag time was decreasing with increasing concentration of the reducing agent (e.g., approximately 200, 120, and 50 s for 0.25, 0.5, and 1 mM DTT at 40 °C, respectively). The lag time also decreased with increasing temperature and the relative fraction of reducible conjugate 2 in the membrane (Supporting Information Figure S2). The rate of release, defined as the steepest slope of the fluorescence signal increase following the lag time, was increasing with increasing temperature, the relative fraction of reducible conjugate 2 in the membrane (Figure S2), and the 10824
dx.doi.org/10.1021/la201160y |Langmuir 2011, 27, 10820–10829
Langmuir
Figure 4. Normalized release of calcein from 30 μM 10:25:65 conjugate 2/PC/DOPE liposomes triggered by the addition at time 0 s of 30 mM L-cysteine and from 20 μM 15:22:63 conjugate 2/PC/DOPE triggered by the addition at time 0 s of 4 mM glutathione (GSH, 1.3 mg added to a 1 mL sample) shown as solid and dotted curves, respectively, at 37 °C. The addition of 30 mM L-cysteine to 40:60 PC/DOPE liposomes did not induce release (dashed curve).
concentration of the reducing agent as can be expected from simple kinetic considerations. Figure 4 shows the reduction-triggered release of calcein upon the addition of 30 mM L-cysteine (solid curve) and 1.3 mg of glutathione (GSH, dotted curve) to liposomes, respectively, at 37 °C. The addition of physiological amounts of L-cysteine or GSH41 to the conjugate 2/PC/DOPE liposomes led to the significant release of entrapped calcein. The rate of release induced upon the addition of 4 mM glutathione was slower and the final fraction of dye released was lower than the rate and release level induced upon the addition of 30 mM L-cystein. We assume that this is due to the higher rate of GSH oxidation lowering the efficient concentration of the reducing agent. Spontaneous leakage without the addition of a reducing agent from the vesicles was negligible. (For a typical spontaneous release curve, see the black dashed line in Figure 5.) These results prove that watersoluble molecules can be entrapped in the liposomes and that the reduction of the disulfide bond of conjugate 2 using physiological reducing agents induces the release of these molecules. In agreement with previously published results,13 the release of calcein from PC/DOPE-liposomes lacking the disulfide conjugates was negligible, regardless of whether one of the reducing agents was added (Figure 3, cyan curve; Figure 4, dashed curve). Comparison of Reduction and Release Kinetics. To compare the reduction and release kinetics, liposomes from 15:22:63 conjugate 1/PC/DOPE with and without entrapped calcein (70 mM) were prepared in parallel. Red solid and dotted curves in Figure 5 represent the reduction-mediated decrease of dansyl fluorescence upon addition of 33 or 9 mM TCEP, respectively. Black solid and dotted curves in Figure 5 represent the increase in calcein fluorescence due to the reduction-triggered release of the dye from the liposomes upon the addition of 33 or 9 mM TCEP, respectively. Without the addition of a reducing agent, the release of calcein from the liposomes was negligible (black dashed line). In agreement with the results described above (Figures 24), in these experiments conducted in parallel we also observed that cleavage started immediately upon addition of the reducing agent, whereas a lag phase was observed before the onset of the fast calcein release.
ARTICLE
Figure 5. Comparison of the cleavage (dansyl fluorescence decrease, red curves, right ordinate) and normalized release kinetics (calcein fluorescence dequenching, black curves, left ordinate) upon addition of 33 and 9 mM TCEP at time 0 s to 80 μM 15:22:63 conjugate 1/PC/ DOPE vesicles (solid and dotted curves, respectively). The black dotted line shows negligible calcein release from the liposomes without the addition of a reducing agent. The upper red dashed-dotted-dotted line shows a negative control: 9 mM TCEP was added to 50 μM 20:80 dansyl-PE/PC vesicles in degassed buffer. The lower red dashed line shows a decrease in the dansyl fluorescence of conjugate 1 (80 μM liposomes) due to oxidation by air when the buffer was not degassed.
The reduction kinetics was fast, hence most of the probe accessible for reduction was reduced during the slow release phase, indicating that a certain amount of the cleaved conjugate molecules is necessary to perturb the membrane of the vesicles to allow release due to the formation of transient defects and/or structural rearrangement. The upper dashed-dotted-dotted red line shows the result of a negative control: the addition of TCEP to vesicles containing dansyl-labeled phospholipid (Dansyl-PE) in degassed buffer did not change the dansyl fluorescence. Note that dansyl is sensitive to oxidation, leading to a decrease in fluorescence; therefore, when working with dansyl-labeled conjugates, buffers should be degassed and saturated with N2. The dashed red curve below shows the approximately linear decrease in dansyl fluorescence with time when the buffer was not degassed. Reduction of the conjugate disulfide bond led to the separation of the hydrophilic headgroup from the membrane surface and to the aggregation of conjugate 1, PC, and DOPEcontaining liposomes at higher lipid concentrations. Aggregation of the liposomes was observed with dynamic light scattering (not shown). Preliminary results on the pH-sensitivity of the disulfide conjugate, PC, DOPE-containing liposomes are discussed in the Supporting Information Unspecific Uptake of Liposomes by Macrophages and Release of Calcein. Macrophages were used to study cargo release from the reduction-sensitive liposomes in cells because they have a high unspecific uptake efficiency and a high reducing capability.16 First, we verified that the vesicles were stable in the cell culture medium with 020% fetal bovine serum, and the reduction-triggered release of calcein was preserved in the medium after the addition of TCEP (data not shown). Then the cells were incubated with 30 μM reduction-sensitive or-insensitive vesicles in RPMI. After 10 min and 1 or 2 h of incubation with vesicles, the cells were washed six times with PBS with Ca2+ and Mg2+. The cells incubated for 10 min had only a 10825
dx.doi.org/10.1021/la201160y |Langmuir 2011, 27, 10820–10829
Langmuir
ARTICLE
Figure 6. (a, b) Fluorescence microscopy images of macrophage cells (J774A.1) treated with calcein-containing reduction-sensitive and reductioninsensitive liposomes at 37 °C for 2 h, respectively. (c, d) Cell count versus calcein fluorescence histograms from the FACS data analysis of macrophages incubated with reduction-sensitive and reduction-insensitive liposomes, respectively, at 37 °C for 1 h. The white bars correspond to 25 μm.
low level of fluorescence inside the cells, mainly localized to the regions close to plasma membrane and small compartments, presumably endosomes, in agreement with previous studies on the uptake of reduction-sensitive polymerosomes.16 After 10 min of incubation, almost no difference between cells treated with reduction-sensitive and -insensitive liposomes was observed (Figure S3). After 1 or 2 h, however, the fluorescence intensity inside the cells increased remarkably because of the dequenching of calcein released from liposomes. Cells treated with reductionsensitive vesicles showed higher and more homogeneously spread fluorescence compared to that of the cells treated with reduction-insensitive vesicles as shown in Figure 6a,b, respectively. To quantify the release of calcein from the liposomes taken up by macrophages, we used fluorescence-activated cell sorting (FACS). Figure 6c,d shows the corresponding cell count versus fluorescence histograms for macrophages treated with reductionsensitive and -insensitive liposomes, respectively, for 1 h. A comparison of the two histograms reveals that cells incubated with reduction-sensitive liposomes showed about a 10-fold higher fluorescence than cells incubated with reduction-insensitive liposomes demonstrating that more calcein was released inside cells treated with vesicles containing conjugate 2. Uptake of Antibody-Presenting Doxorubicin-Loaded Liposomes by BT-474 Cells. To demonstrate the potential of the active targeting, we used biotinstreptavidin-specific binding to cover liposomes with an antibody against the extracellular part of the p185HER2 receptor. This receptor is known to be overexpressed in about 2030% of breast, ovarian, prostate, and gastric cancer patients, and this high expression correlates with more aggressive tumors and a poor prognosis.7,42,43 The p185HER2 receptor also represents an attractive target for antibody-based therapy because it
is stably and homogeneously overexpressed in the tumor tissues,44 whereas in normal tissues p185HER2 is expressed only at low levels.45 HER2-targeted immunoliposomes have been shown to deliver anticancer drugs specifically to p185HER2-positive BT-474 cells.46,47 The streptavidinbiotin building system was applied to allow variable targeting. Biotin was covalently attached to the headgroup of conjugates 1 and 2 (Scheme 1), and streptavidin was used as a specific linker to attach the biotinylated anti-HER2 antibody to the liposomes containing conjugate 1 or 2. Binding of streptavidin to the liposomal carriers was detected by three independent methods: by the increase in fluorescein-labeled streptavidin fluorescence upon attachment to biotin on the surface of the carriers (data not shown), by the increase in light scattering due to the aggregation of carriers via streptavidin molecules (Supporting Information Figures S4 and S5, left), and by an enzyme-linked immunosorbent assay (ELISA, Figure 7). The average liposome size was constant (110150 nm) in the eluent volume range from 0.375 to 0.8, whereas the ELISA signal first peaked and then diminished over the same elution period (black squares, Figure 7, right) in good congruence with the particle (liposome) count trend (red circles, Figure 7, right). Thus, the ELISA signal corresponds to antibody-presenting liposomes because the free antibodies were separated by chromatography as described in the Experimental Section. It can therefore be concluded that a major portion of the detected liposomes have first bound streptavidin and then biotinylated antibody; therefore, the antibody-presenting reduction-sensitive liposomes, shown as a cartoon on the left side of Figure 7, were obtained. BT-474 breast cancer cells were incubated in DMEM with antibody-presenting reduction-sensitive or -insensitive vesicles 10826
dx.doi.org/10.1021/la201160y |Langmuir 2011, 27, 10820–10829
Langmuir
ARTICLE
Figure 7. ( Right) Elution profile of reduction-sensitive the liposome/streptavidin/anti-p185HER2 complex determined by ELISA (black squares) and dynamic light scattering (red circles). Lines were drawn to guide the eye. (Left) Liposomal carrier built of phospholipids (bilayer, gray) and the multifunctional lipid conjugate (red, blue, and black), filled with small water-soluble molecules (green dots). Streptavidin tetramer (light blue) serving as a linker between biotin (red) covalently attached to the headgroup of the conjugate and the biotinylated anti-HER2 antibody (red, black).
Figure 8. (a) Fluorescence microscopy and (b) differential interference contrast images of BT-474 cells treated with doxorubicin-loaded antip185HER2 antibody-presenting reduction-sensitive liposomes at 37 °C for 1 h. White bars correspond to 25 μm. For a comparison of images of cells treated with antibody-presenting reduction-sensitive and -insensitive liposomes and reduction-sensitive liposomes without antibody, see Supporting Information Figure S6.
and vesicles without antibody, prepared as described in the Experimental Section, at 37 °C for 1 h and then washed four times with PBS before imaging. Figure 8 shows typical fluorescence and differential contrast microscopy images of BT-474 treated with antibody-presenting reduction-sensitive liposomes (Supporting Information Figure S6a,b). The partial release of doxorubicin inside the cells was observed. Cells incubated with antibody-presenting reduction-insensitive liposomes showed uptake, but doxorubicin fluorescence was merely concentrated in small granularlike structures (Supporting Information Figure S6c,d). Cells incubated with reduction-sensitive liposomes without antibody showed nonspecific uptake, which was lower than the specific uptake, and more release than for reduction-insensitive liposomes (Supporting Information Figure S6e,f). Untreated cells showed no fluorescence signal (not shown). Preliminary MTT cell viability tests revealed no toxicity of liposomes containing disulfide conjugates (2530 μM of total lipid, not shown). Together with the results described above on streptavidin and antibody binding to the liposomes containing conjugate 2, these results illustrate the possibility of the targeted delivery of a drug encapsulated by reduction-sensitive liposomes.
Scheme 2. Schematic Representation of the Multifunctional Conjugate 1
’ DISCUSSION Multifunctional Conjugates. We have developed a multifunctional amphiphilic biotin-tagged fluorescently labeled disulfide conjugate. Scheme 2 summarizes different functionalities of the conjugate: (1) hydrophobic chains offering stable incorporation into lipid membranes; (2) a disulfide bridge (yellow) allowing the cleavage of the hydrophilic headgroup and therefore destabilization of the liposomes and triggered release; (3) a 10827
dx.doi.org/10.1021/la201160y |Langmuir 2011, 27, 10820–10829
Langmuir fluorescent moiety (dansyl, blue) offering the possibility of the detection of the incorporation and the reduction kinetics; and (4) a recognition moiety (biotin, red). To the best of our knowledge, our conjugate 1 is the first to combine four different functionalities in one molecule. However, because the reducible conjugates are considered to be promising for triggered delivery, several other reduction-sensitive lipophilic conjugates and even more reduction sensitive polymers forming nanocarriers were synthesized and studied by different groups (e. g., refs 15, 23, 24, and 48 and references therein). Disulfide conjugates of drugs and carrier molecules are also under intensive investigation.49,50 M€uller et al.50 reported the suppression of human lymphocyte proliferation upon incubation with a lipophilic disulfide prodrug presumably due to the liberation of the drug as a result of the disulfide bond cleavage. The lipophilic disulfide prodrugs incubated with serum in a cell-free medium were stable, also in agreement with our observations. Reduction-Sensitive Liposomal Carriers. We provide here the proof of principle that disulfide conjugates 1 and 2 enable the formation of liposomes that (i) can be filled with cargo, (ii) recognize a biological target, and (iii) release the cargo upon triggering with physiological reducing agents. The release of calcein from liposomes containing conjugate 1 or 2 was induced using different reducing agents: final concentrations of DTT as a more potent reducing agent of 0.52 mM, of TCEP as a less potent reducing agent of 330 mM, and of L-cysteine and GSH at physiologically relevant concentrations of 30 and 4 mM, respectively, were used. Furthermore, we show that more calcein is released from reduction-sensitive than reduction-insensitive liposomes upon uptake into macrophages and that the specific targeting of our reduction-sensitive liposomes to breast cancer cells using biotinylated anti-HER2 antibody is possible. Hubbell and co-workers16 suggested that the internalization of the polymerosomes was an early and late endosome-mediated process. In agreement with this work, after 10 min of incubation both reduction-sensitive and -insensitive liposomes were taken up to similar extent and calcein was mostly localized inside the endosomes, giving a weak fluorescence signal. After 1 and 2 h, we observed a significantly higher increase in calcein fluorescence inside cells incubated with reduction-sensitive than with reduction-insensitive liposomes. This shows the accessibility of the disulfide conjugates to reduction by intracellular reducing substances leading to the efficient release of calcein and the dequenching of the fluorescence signal. Functionalization of the liposomes using the streptavidin biotin building system and the biotinylated anti-HER2 antibody allowed the efficient delivery of doxorubicin into the breast cell line known to overexpress the HER2 receptor. We used doxorubicin here as a dye, entrapped at 3 mM, giving a final concentration lower than IC50 (IC50 ≈ 10 μM51). Furthermore, the cells were imaged after 1 h of incubation, whereas the toxic effects would first be expected after 2448 h of incubation. For therapeutic applications, doxorubicin must be entrapped in carriers at a high concentration (e.g., 200 mM), giving after release a local concentration higher than IC50 . The entrapment of other drugs, subsequent uptake, release, and viability of the cells, and the toxicity of the carriers should be tested. Further development of the controlled targeted drug delivery system for applications (e.g., in cancer therapy) is necessary and promising. Mechanism of Release and Comparison of Reduction and Release Kinetics. We surmise that although the high mol% of DOPE in the carrier does not disrupt the bilayer structure it
ARTICLE
primes the membranes for instabilities, caused by sufficient reduction of disulfide conjugate 1 or 2. The reduction of conjugate 1 or 2 disulfide bond leads to a separation of the hydrophilic headgroup (thiol 7 or 9, Scheme 1) from the membrane surface. Very likely, the lipid anchor remaining in the membrane adopts an inverted conelike structure similar to that of unsaturated phosphatidylethanolamines (e.g., DOPE).52 It is known that this lipid shape interferes with the stability of the bilayer structure: at high concentrations of cone-shaped lipids, the integrity and low permeability of membranes are lost; even nonlamellar phases might be formed as reported for membranes of phosphatidylethanolamine at physiological temperatures and pH.53,54 In agreement with previous studies (e.g., reviewed in ref 55), reduction was accompanied by the subsequent collision-limited aggregation of the liposomes and release could be triggered by lowering the pH to 4.5. Very likely, the separation of the hydrophilic headgroup facilitates the aggregation of liposomes by close contact. The potential to form nonlamellar phases might also trigger fusion with adjacent membranes, which might allow the escape of the content of liposomes onto the cytosol of cells.56 A similar mechanism of triggered release has been suggested by several groups for different liposomal systems.13,19,57 For example, Thompson and co-workers reported a cryoelectron microscopy and 31P NMR study of the structural changes of the acidtriggered liposomes with decreasing pH, first showing the aggregation of the liposomes and then the formation of small condensed, presumably hexagonal, structures.57 In agreement with previous reports, the reduction kinetics is first order in the concentration of both the reducing agent and the substrate,58 whereas the release kinetics is more complex having a delay, a slow, and a fast release phase. Although further quantitative analysis of the release kinetics is necessary, our results, in particular on the lag time, indicate that a certain amount of lipophilic reduction product thioglyceroldiether 3 in the membrane was necessary to produce defects or reorganization allowing the release of entrapped molecules. Although further optimization of the reduction-sensitive liposomes (e.g., in lipid composition for enhanced stability and delivery of hydrophobic and hydrophilic substances to specific cellular organelles) is necessary, our results give the first proof of principle allowing the development of a targeted triggered delivery system based on the multifunctional lipophilic disulfide conjugates and natural phospholipids.
’ ASSOCIATED CONTENT
bS
Supporting Information. Cryoelectron microscopy image of liposomes; dependence of the lag phase on temperature; preliminary results on the pH sensitivity of the liposomes; images of macrophages after 10 min of incubation with liposomes; aggregation of liposomes by streptavidin; and fluorescence images of BT-474 cells incubated with DOX-loaded liposomes. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected];
[email protected].
’ ACKNOWLEDGMENT The J774A.1 mouse macrophage cell line was a generous gift from Prof. Dr. T. F. Meyer, MPI for Infection Biology, Berlin, 10828
dx.doi.org/10.1021/la201160y |Langmuir 2011, 27, 10820–10829
Langmuir Germany. Human breast cancer cell line BT-474 was a generous gift of Prof. Dr. H. Lage, Charite, Berlin, Germany. This work was supported by Bayer Technology Services GmbH.
’ REFERENCES (1) Allen, T. M.; Cullis, P. R. Science 2004, 303, 1818–1822. (2) Guo, X.; Szoka, F. C., Jr. Acc. Chem. Res. 2003, 36, 335–341. (3) Torchilin, V. Eur. J. Pharm. Biopharm. 2009, 71, 431–444. (4) Riehemann, K.; Schneider, S. W.; Luger, T. A.; Godin, B.; Ferrari, M.; Fuchs, H. Angew. Chem., Int. Ed. 2009, 48, 872–897. (5) Davis, M. E.; Chen, Z. G.; Shin, D. M. Nat. Rev. Drug Discovery 2008, 7, 771–782. (6) Leonard, R. C.; Williams, S.; Tulpule, A.; Levine, A. M.; Oliveros, S. Breast 2009, 18, 218–224. (7) Tai, W.; Mahato, R.; Cheng, K. J. Controlled Release 2010, 146, 264–275. (8) Maeda, T.; Fujimoto, K. Colloids Surf., B 2006, 49, 15–21. (9) Chen, Z. G. Trends Mol. Med. 2010, 16, 594–602. (10) Farokhzad, O. C.; Langer, R. ACS Nano 2009, 3, 16–20. (11) Davidsen, J.; Jorgensen, K.; Andresen, T. L.; Mouritsen, O. G. Biochim. Biophys. Acta 2003, 1609, 95–101. (12) Kirpotin, D.; Hong, K.; Mullah, N.; Papahadjopoulos, D.; Zalipsky, S. FEBS Lett. 1996, 388, 115–118. (13) Zalipsky, S.; Qazen, M.; Walker, J. A., II; Mullah, N.; Quinn, Y. P.; Huang, S. K. Bioconjugate Chem. 1999, 10, 703–707. (14) Zhang, J.; Chua, L. S.; Lynn, D. M. Langmuir 2004, 20, 8015–8021. (15) Oumzil, K.; Khiati, S.; Grinstaff, M. W.; Barthelemy, P. J. Controlled Release 2011, 151, 123–130. (16) Cerritelli, S.; Velluto, D.; Hubbell, J. A. Biomacromolecules 2007, 8, 1966–1972. (17) Sun, Y.; Yan, X.; Yuan, T.; Liang, J.; Fan, Y.; Gu, Z.; Zhang, X. Biomaterials 2010, 31, 7124–7131. (18) Ryu, J. H.; Roy, R.; Ventura, J.; Thayumanavan, S. Langmuir 2010, 26, 7086–7092. (19) Ong, W.; Yang, Y.; Cruciano, A. C.; McCarley, R. L. J. Am. Chem. Soc. 2008, 130, 14739–14744. (20) Ganta, S.; Devalapally, H.; Shahiwala, A.; Amiji, M. J. Controlled Release 2008, 126, 187–204. (21) Saito, G.; Swanson, J. A.; Lee, K. D. Adv. Drug Delivery Rev. 2003, 55, 199–215. (22) Zelikin, A. N.; Quinn, J. F.; Caruso, F. Biomacromolecules 2006, 7, 27–30. (23) Meng, F.; Hennink, W. E.; Zhong, Z. Biomaterials 2009, 30, 2180–2198. (24) Kim, E.; Kim, D.; Jung, H.; Lee, J.; Paul, S.; Selvapalam, N.; Yang, Y.; Lim, N.; Park, C. G.; Kim, K. Angew. Chem., Int. Ed. 2010, 49, 4405–4408. (25) Yang, J.; Chen, H.; Vlahov, I. R.; Cheng, J. X.; Low, P. S. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13872–13877. (26) West, K. R.; Otto, S. Curr. Drug Discovery Technol. 2005, 2, 123–160. (27) Ballatori, N.; Krance, S. M.; Notenboom, S.; Shi, S.; Tieu, K.; Hammond, C. L. Biol. Chem. 2009, 390, 191–214. (28) Arner, E. S.; Holmgren, A. Semin. Cancer Biol. 2006, 16, 420–426. (29) Arbuzova, A.; Schwarz, G. Biochim. Biophys. Acta 1999, 1420, 139–152. (30) Kurz, A.; Bunge, A.; Windeck, A. K.; Rost, M.; Flasche, W.; Arbuzova, A.; Strohbach, D.; M€uller, S.; Liebscher, J.; Huster, D.; Herrmann, A. Angew. Chem., Int. Ed. 2006, 45, 4440–4444. (31) Loew, M.; Springer, R.; Scolari, S.; Altenbrunn, F.; Seitz, O.; Liebscher, J.; Huster, D.; Herrmann, A.; Arbuzova, A. J. Am. Chem. Soc. 2010, 132, 16066–16072. (32) Pescador, P.; Brodersen, N.; Scheidt, H. A.; Loew, M.; Holland, G.; Bannert, N.; Liebscher, J.; Herrmann, A.; Huster, D.; Arbuzova, A. Chem. Commun. 2010, 46, 5358–5360. (33) Rigler, P.; Meier, W. J. Am. Chem. Soc. 2006, 128, 367–373.
ARTICLE
(34) Lin, J. J.; Ghoroghchian, P. P.; Zhang, Y.; Hammer, D. A. Langmuir 2006, 22, 3975–3979. (35) Verderone, G.; Van Craynest, N.; Boussif, O.; Santaella, C.; Bischoff, R.; Kolbe, H. V.; Vierling, P. J. Med. Chem. 2000, 43, 1367–1379. (36) Shirude, P.; Kumar, V.; Ganesh, K. Eur. J. Org. Chem. 2005, 2005, 5207–5215. (37) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55–65. (38) Allen, T. M.; Cleland, L. G. Biochim. Biophys. Acta 1980, 597, 418–426. (39) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006. (40) Stryer, L. Annu. Rev. Biochem. 1978, 47, 819–846. (41) Smith, C. V.; Jones, D. P.; Guenthner, T. M.; Lash, L. H.; Lauterburg, B. H. Toxicol. Appl. Pharmacol. 1996, 140, 1–12. (42) Hynes, N. E.; Lane, H. A. Nat. Rev. Cancer 2005, 5, 341–354. (43) Slamon, D. J.; Godolphin, W.; Jones, L. A.; Holt, J. A.; Wong, S. G.; Keith, D. E.; Levin, W. J.; Stuart, S. G.; Udove, J.; Ullrich, A.; et al. Science 1989, 244, 707–712. (44) Jones, K. L.; Buzdar, A. U. Lancet Oncol. 2009, 10, 1179–1187. (45) Press, M. F.; Cordon-Cardo, C.; Slamon, D. J. Oncogene 1990, 5, 953–962. (46) Park, J. W.; Hong, K.; Kirpotin, D. B.; Colbern, G.; Shalaby, R.; Baselga, J.; Shao, Y.; Nielsen, U. B.; Marks, J. D.; Moore, D.; Papahadjopoulos, D.; Benz, C. C. Clin. Cancer Res. 2002, 8, 1172–1181. (47) Burks, S. R.; Macedo, L. F.; Barth, E. D.; Tkaczuk, K. H.; Martin, S. S.; Rosen, G. M.; Halpern, H. J.; Brodie, A. M.; Kao, J. P. Breast Cancer Res. Treat. 2010, 124, 121–131. (48) M€uller, C. E.; Roth, H. J. Arch. Pharm. (Weinheim) 1989, 322, 343–350. (49) Lewis Phillips, G. D.; Li, G.; Dugger, D. L.; Crocker, L. M.; Parsons, K. L.; Mai, E.; Blattler, W. A.; Lambert, J. M.; Chari, R. V.; Lutz, R. J.; Wong, W. L.; Jacobson, F. S.; Koeppen, H.; Schwall, R. H.; Kenkare-Mitra, S. R.; Spencer, S. D.; Sliwkowski, M. X. Cancer Res. 2008, 68, 9280–9290. (50) M€uller, C. E.; Daniel, P. T.; Holzschuh, J.; Roth, H. J. Int. J. Pharm. 1989, 57, 41–47. (51) Liem, A. A.; Appleyard, M. V.; O’Neill, M. A.; Hupp, T. R.; Chamberlain, M. P.; Thompson, A. M. Br. J. Cancer 2003, 88, 1281–1284. (52) Cullis, P. R.; de Kruijff, B. Biochim. Biophys. Acta 1979, 559, 399–420. (53) Cullis, P. R.; de Kruijff, B. Biochim. Biophys. Acta 1978, 513, 31–42. (54) Gawrisch, K.; Parsegian, V. A.; Hajduk, D. A.; Tate, M. W.; Graner, S. M.; Fuller, N. L.; Rand, R. P. Biochemistry 1992, 31, 2856–2864. (55) Goni, F. M.; Alonso, A. Prog. Lipid Res. 1999, 38, 1–48. (56) Connor, J.; Yatvin, M. B.; Huang, L. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 1715–1718. (57) Boomer, J. A.; Inerowicz, H. D.; Zhang, Z.-Y.; Bergstrand, N.; Edwards, K.; Kim, J.-M.; Thompson, D. H. Langmuir 2003, 19, 6408–6415. (58) Whitesides, G. M.; Lilburn, J. E.; Szajewski, R. P. J. Org. Chem. 1977, 42, 332–338.
10829
dx.doi.org/10.1021/la201160y |Langmuir 2011, 27, 10820–10829
