PEGylated Liposomal Doxorubicin Targeted to α5β1-Expressing MDA

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PEGylated Liposomal Doxorubicin Targeted to α5β1-Expressing MDAMB-231 Breast Cancer Cells Kamlesh Shroff and Efrosini Kokkoli* Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455, United States S Supporting Information *

ABSTRACT: Targeting drugs selectively to cancer cells can potentially benefit cancer patients by avoiding side effects generally associated with several cancer therapies. One of the attractive approaches to direct the drug cargo to specific sites is to incorporate ligands at the surface of the delivery systems. Integrin α5β1 is overexpressed in tumor vasculature and cancer cells, thus making it an attractive target for use in drug delivery. Our group has developed a fibronectin-mimetic peptide, PR_b, which has been shown to bind specifically to integrin α5β1, thereby providing a tool to target α5β1-expressing cancer cells in vitro as well as in vivo. Our current work focuses on designing modified stealth liposomes (liposomes functionalized with polyethylene glycol, PEG) for combining the benefits associated with PEGylation, as well as imparting specific targeting properties to the liposomes. We have designed PEGylated liposomes that incorporate in their bilayer the fibronectin-mimetic peptide-amphiphile PR_b that can target several cancer cells that overexpress α5β1, including the MDA-MB-231 breast cancer cells used in this study. We have encapsulated doxorubicin inside the liposomes to enhance its therapeutic potential via PEGylation as well as active targeting to the cancer cells. Our results show that PR_b-functionalized stealth liposomes were able to specifically bind to MDA-MB-231 cells, and the binding could be controlled by varying the peptide concentration. The intracellular trafficking of the doxorubicin liposomes was examined, and within minutes after delivery the majority of them were found to be in the early endosomes, whereas after a longer period of time they had accumulated in the late endosomes and lysosomes. The functionalized liposomes were found to be equally cytotoxic as the free doxorubicin, especially at higher doxorubicin concentrations, and provided higher cytotoxicity than the nontargeted and GRGDSP-functionalized stealth liposomes. Thus, the PR_b-functionalized PEGylated nanoparticles examined in this study offer a promising strategy to deliver their therapeutic payload directly to the breast cancer cells, in an efficient and specific manner.

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INTRODUCTION Nanocarriers are promising platform for therapeutics, especially in cancer therapy. The success of anticancer therapies largely depends on the ability of the therapeutics to reach their designated cellular and intracellular target sites, while minimizing accumulation and action at nonspecific sites. Over the past decade numerous nanoparticle based drug delivery and drug targeting systems are under rigorous development.1−3 Polyethylene glycol (PEG) modification often referred as PEGylation has emerged as a common strategy to ensure stealth-shielding and long circulation of therapeutic nanocarriers, such as liposomes, micelles, and polymeric nanoparticles. PEGylated nanocarriers have become a popular delivery system and demonstrate dose dependent, log−linear kinetics, and increased bioavailability. 4−9 Furthermore, PEGylated liposomes encapsulating doxorubicin have shown good efficacy in accumulating in the tumor tissue, and diminishing some of the toxic side effects of free doxorubicin.10−14 Breast cancer has the highest incidence rates of about 30% of all types of cancer in women, and is the second largest leading cause of deaths in females with cancer.15 Doxil and Caelyx © 2012 American Chemical Society

(PEGylated liposomal doxorubicin) have emerged successfully in the treatment of solid tumors in breast carcinoma, with significant improvement in the survival rates of patients.12,13,16,17 Currently attempts are under way to combine the benefits of PEGylated nanocarriers with tumor cell-specific targeting. This type of active targeting may potentially lead to numerous advantages such as selective delivery and internalization of the nanoparticles inside the target cells to a greater extent. Bottomup approaches have become widely popular in designing such nanocarriers, allowing greater control over the formation and functionalization of the nanoparticles as well as encapsulation of the payloads, including drugs or nucleic acids.18−21 Peptidebased targeting is advantageous over other targeting approaches, such as immunoliposomes that face challenges such as immunogenicity and high cost.3,22−24 Targeting integrin α5β1, which is overexpressed in tumor vasculature and tumor cells, has led to control of tumor growth, metastasis, and tumorReceived: November 14, 2011 Revised: January 23, 2012 Published: January 23, 2012 4729

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induced angiogenesis.25−34 Additionally, ligands that can bind to integrin α5β1 with high affinity can potentially mediate cellular internalization and trafficking.35−38 The RGD peptide ligand has been widely used in the past to functionalize planar as well as 3D matrices, to promote integrin-mediated binding of several cell types. Moreover, RGD-functionalized liposomes have also shown some improvements over nontargeted systems.39,40 In one example, RGD-functionalized PEGylated liposomes targeting the αvβ3 integrin on actively proliferating endothelium showed increased cell interaction over the nonadherent nontargeted PEGylated liposomes, and RGD PEGylated liposomes encapsulating doxorubicin inhibited tumor growth in a doxorubicin-insensitive murine C26 colon carcinoma model, whereas doxorubicin in nontargeted PEGylated liposomes failed to decelerate tumor growth in mice.41 However, a concern regarding RGD peptides is that they bind many integrins, thus making it difficult to achieve specificity for an integrin of interest. Previous research in our group has shown that a model fibronectin-mimetic peptide that can specifically bind to integrin α5β1 must also contain the synergistic sequence PHSRN, separated from RGD with a linker that mimics both the distance and hydrophobicity/hydrophilicity found in the native protein between the RGD and PHSRN sequences.42,43 The fibronectin-mimetic peptide PR_b (KSSPHSRN(SG)5RGDSP) contains a KSS spacer and the RGDSP and PHSRN sequences separated by a (SG)5 peptide linker mimicking the distance (37 Å) and the hydrophobic/hydrophilic properties of fibronectin. It was coupled to a 16 carbon dialkyl tail to form the PR_b peptide-amphiphile ((C16)2-GluC2-KSSPHSRN(SG)5RGDSP).44 Our results demonstrated that PR_b surfaces and gels outperformed other fibronectinmimetic peptide sequences, as well as fibronectin surfaces and commercially available peptide hydrogels in facilitating celladhesion and proliferation, spreading, cytoskeleton formation, extracellular matrix production, enhanced FAK phosphorylation, and cyclin-D1 production.44−48 We have also demonstrated through blocking experiments with peptides and antibodies that PR_b is a specific ligand for the α5β1 integrin,44,49 with a dissociation constant of 76.3 ± 6.3 nM.48 Moreover, PR_b-functionalized nanoparticles (liposomes and polymerosomes) have shown enhanced binding, intracellular uptake, and delivery of their encapsulated load compared to nontargeted and GRGDSP-functionalized particles targeted to colon cancer cells,49−51 prostate cancer cells,52,53 and porcine islets of Langerhans.54 All these results suggest that the PR_b peptide-amphiphile is a very effective and specific ligand for the α5β1 integrin, therefore making it a promising targeting ligand for our studies. In the current study, our goal is to target MDA-MB-231 breast cancer cells with PR_b functionalized PEGylated liposomes encapsulating doxorubicin. First the expression level of integrin α5β1 on the breast cancer cells was investigated. Liposomes functionalized with PEG2000 and varying PR_b concentrations were delivered to MDA-MB-231 cells, and doxorubicin was successfully encapsulated with high payloads and was targeted to the breast cancer cells in vitro. The trafficking of the nanoparticles was visualized with confocal microscopy, and their cytotoxicity was also evaluated.

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synthesized by the Oligonucleotide and Peptide Synthesis Facility at the University of Minnesota. The dialkyl hydrocarbon tails (C16)2-GluC2 were prepared and attached to the peptide sequences to form PR_b peptide-amphiphile, (C16)2-Glu-C2-KSSPHSRN(SG)5RGDSP, and GRGDSP peptide-amphiphile, (C16)2-Glu-C2-GRGDSP, as described previously.42,44,48,55 Lipids, cholesterol (CHOL), 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC), and 1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine-N-(methoxy(polyethylene glycol)-2000)-ammonium salt) (PEG2000) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). The extruder assembly (including parts) and the 100 nm polycarbonate membranes were obtained from Avestin, Inc. (Ottawa, Canada). MDA-MB-231 breast cancer cells (human origin) were obtained from ATCC (Manassas, VA). Anti-integrin α5β1 monoclonal antibody, mouse IgG isotype control, and fluorescein isothiocyanate (FITC)-conjugated antimouse secondary antibody were purchased from Millipore, Inc. (Billerica, MA). Dulbecco’s modified Eagle medium (DMEM), antibiotics, CellLight Early Endosomes-GFP *BacMam 2.0* reagent, LysoTracker Blue DND-22 (1 mM solution in DMSO), and Hoechst 33342 nucleic acid stain were purchased from Invitrogen, Inc. (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Atlas Biologicals, Inc. (Fort Collins, CO). The bicinchoninic acid (BCA) protein assay kit was purchased from Thermo Fischer Scientific (Rockford, IL). 50 000 Da MWCO spectra/ por-7 dialysis tubing was purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA). Doxorubicin hydrochloride (suitable for florescence), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sulforhodamine B (SRB), and all other reagents were purchased from Sigma-Aldrich (Saint Louis, MO). Liposome Preparation and Characterization. Peptide-amphiphiles were dissolved in a mixture of methanol and water, while all other lipids were dissolved in chloroform. These solutions were mixed in clean round-bottom flasks in a ratio of (60 − x):35:5:x mol % DPPC:CHOL:PEG2000:peptide-amphiphile. Solvents were removed by evaporation under a gentle stream of argon at 65 °C, and lipids were dissolved again in chloroform to form a homogeneous mixture. The lipid mixture was finally dried under a gentle stream of argon at 65 °C until a uniform lipid film was formed, followed by drying under vacuum overnight. Fluorescently labeled liposomes were prepared by hydrating the lipid film with fluorescent HBSE buffer (10 mM HEPES, 150 mM NaCl, 0.1 mM EDTA, and 2 mM calcein, pH 7.4) at 65 °C and at a concentration of 10 mM total lipids. Hydrated lipid films were freeze−thawed five times, and then extruded at 65 °C for 21 cycles through two stacks of 100 nm polycarbonate membranes using a handheld extruder assembly. Calcein liposomes were purified over a Sepharose CL-4B gel filtration column to remove any unencapsulated fluorescent dye. Liposome diameter was determined by dynamic light scattering on a DLS-Zeta PALS instrument from Brookhaven Instruments Corporation. Phospholipid concentration was determined using the phosphorus colorimetric assay. The peptide amphiphile concentrations in the liposomes were determined by the BCA assay. Briefly, known amount of liposomes were disrupted with 5% triton in HBSE buffer, and reacted with the BCA assay reagents as per manufacturer’s instructions. The absorbance of the reaction product was recorded at 562 nm, and the concentrations were determined by fitting to a free PR_b standard curve. Doxorubicin liposomes were prepared using a remote loading procedure as described previously.56 Briefly, lipid films were hydrated in 120 mM ammonium sulfate solution and extruded through 100 nm polycarbonate membranes as described in the previous paragraph. The ammonium sulfate outside the liposomes was substituted with HBSE buffer by dialyzing the extruded liposome mixture through 50 000 MWCO dialysis tubing. The phospholipid and peptide contents were estimated as described previously. Doxorubicin was remotely loaded by incubating the purified liposomes with free doxorubicin (0.6 mM) at 50 °C for 24 h, and the unencapsulated doxorubicin was removed by dialysis using 50 000 MWCO dialysis tubing in HBSE buffer. Finally, the encapsulated doxorubicin was quantified by lysing a diluted liposome sample (to avoid self-quenching of doxorubicin) in 5% triton-HBSE buffer, reading the fluorescence of the doxorubicin (Ex: 485 nm, Em: 590 nm) using a fluorescence plate reader, and

MATERIALS AND METHODS

Materials. The PR_b peptide sequence (KSSPHSRN(SG)5RGDSP) and the GRGDSP peptide sequences were custom 4730

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estimating the concentration based on a standard curve for free doxorubicin. Cell Culture. The purchased frozen stock of MDA-MB-231 cells was revived and subsequently cultured in DMEM basal media supplemented with 10% FBS, 100 units/mL penicillin, and 0.1 mg/ mL streptomycin. Medium was changed every alternate day until the cells were approximately 90% confluent, and were split in the ratio of 1:3 for subsequent passages or frozen in media with 5% dimethyl sulfoxide (DMSO), biotechnology grade. Flow Cytometry. For integrin α5β1 expression studies, the confluent MDA-MB-231 cell monolayers were trypsinized (0.25% trypsin +0.1% EDTA) and resuspended in ice-cold flow buffer (FB) (phosphate-buffered saline (PBS) supplemented with 0.02% sodium azide and 2% FBS) at a concentration of 1 million/mL in 15 mL centrifuge tubes. Cells were first incubated with the primary antibody (anti-integrin α5β1) or isotype control (mouse IgG) at 4 °C for 30 min, pelleted and washed twice in ice-cold FB, then incubated again with the FITC-conjugated antimouse IgG secondary antibody for 30 min. Finally, cells were pelleted and washed twice in ice-cold FB, and flow cytometric analysis was performed immediately. For investigation of binding of liposomes to the breast cancer cells, the confluent monolayers of the MDA-MB-231 breast cancer cells were trypsinized, and cells were resuspended at 1 million/mL in growth media containing 2% FBS and liposomes at a lipid concentration of 250 μM. The cells were allowed to incubate with the liposomes for 1 h at 4 °C, pelleted and washed twice with ice-cold FB. Flow cytometric analysis was carried out immediately. For peptide blocking experiments, the protocol specified above was used, except cells were preincubated with 200 μg/mL free peptide in FB for 30 min prior to addition of the liposomes. Flow cytometric analysis for all the above experiments was performed at the flow cytometry core facility in the Cancer Research Center of the University of Minnesota. Confocal Microscopy. MDA-MB-231 cells were plated at a density of 500 cells/mm2 on glass bottom 12 well plates and allowed to adhere overnight. CellLight Early Endosomes-GFP *BacMam 2.0* reagent was added to result in a final concentration of 30 particles per cells and allowed to incubate with the cells for 18−24 h. Cells were washed twice with PBS and incubated with the LysoTracker Blue DND-22 dye at a concentration of 75 nM for 2 h. Cells were again washed twice with PBS and stained for nuclei using Hoechst 33342 at a concentration of 0.5 mM for 10 min. Finally, cells were washed three times with PBS and allowed to stay in phenol red free medium for imaging. Doxorubicin-loaded liposomes were added to the cell medium at a lipid concentration of 250 μM and imaged live under culture conditions of 37 °C and 5% CO2 at specified time points using a Zeiss Cell Observer SD Spinning Disk Confocal at the University Imaging Center of the University of Minnesota. Cytotoxicity Studies. The cytotoxicity of the free doxorubicin was determined by a SRB assay as discussed in the literature.57 Briefly, 10 μL of doxorubicin solution in 10% DMSO was added in triplicates to 96-well plates such that the resulting concentration of drug after addition of the cell suspension (190 μL) would vary from 100 to 0.05 μg/mL (in 2-fold serial dilutions). As a control, 10 μL of 10% DMSO was also added in triplicate to the same 96-well plates. MDA-MB-231 cells were trypsinized, pelleted, resuspended in growth media, added to the doxorubicin-containing wells in the 96-well plates at a seeding density of 20 000 cells/well/190 μL, and were allowed to incubate with the free doxorubicin for 72 h at 37 °C, 5% CO2. For the nogrowth control, cells were seeded at the same density and were allowed to attach to the plates for 3−4 h at 37 °C, 5% CO2. At the conclusion of the experiment, all plates were fixed by adding 100 μL cold 10% w/v trichloro acetic acid to each well and incubating at 4 °C for 1 h. Plates were then washed with slow-running tap water four times, tapped on paper towels to remove excess water, and dried using a blow dryer. 100 μL of 0.057% w/v SRB solution prepared in 1% v/v acetic acid was finally added to each well and incubated for 30 min at room temperature. Plates were then rinsed four times with 1% acetic acid solution to remove excess unbound dye, and blow-dried. 200 μL of 10 mM Tris base solution (pH 10.5) was added to each well, and the plates were shaken on a gyratory shaker for 10 min to solubilize

the protein-bound dye. Absorbance of the resulting colored solution was measured in a microplate reader at 510 nm, and the percentage of cell-growth inhibition was calculated using the following formula. The IC50 value was obtained by fitting a sigmoidal curve and obtaining the doxorubicin concentration at 50% growth inhibition.

%of control cell growth mean ODsample − mean ODno‐growth control = × 100 mean ODDMSOcontrol − mean ODno‐growth control %growth inhibition = 100 − %of control cell growth Cytotoxicity assessment of the liposomal doxorubicin was carried out using the MTT cell-viability assay. Briefly, MDA-MB-231 cells were trypsinized from culture plates, pelleted, resuspended in phenol red free growth medium, and plated at a concentration of 20 000 cells/ well/100 μL. Liposomes with doxorubicin (diluted appropriately to deliver the said amount of doxorubicin) and free doxorubicin were added to 96 well plates at final doxorubicin concentrations of 2.7, 5.4, 10.8, and 21.6 μM, and the resulting volume in each well was build up to 200 μL with phenol red free medium. In order to compare the cytotoxicities to the free doxorubicin obtained in the IC50 assay to that of the liposomal doxorubicin, cells were allowed to incubate with the free doxorubicin and liposomal doxorubicin for 72 h at 37 °C, 5% CO2. Plates were centrifuged at 200 g for 5 min, and the medium was replaced with 150 μL of freshly prepared phenol red free medium containing 0.66 mg/mL MTT reagent, and further incubated for 2 h at 37 °C, 5% CO2. Finally 150 μL of solubilizing reagent (0.1 N HCl and 10% triton in isopropyl alcohol) was added to each well, and plates were allowed to mix on a gyratory shaker for 30 min. The absorbance of each well was recorded spectrophotometrically at wavelengths of 570 and 690 nm, and the absorbance at 690 nm was subtracted as background from the readings obtained at 570 nm.

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RESULTS AND DISCUSSION The expression level of integrin α5β1 on the MDA-MB-231 breast cancer cells was investigated first. For this, the amount of binding of an antihuman integrin α5β1 antibody to the MDAMB-231 cells was investigated using flow cytometry. As a control, the binding of a mouse IgG isotype antibody was analyzed. Binding histograms of both the antibodies and the autofluorescence background of the cells are presented in Figure 1. It can be clearly seen that the binding of the mouse IgG isotype control is negligible and overlaps with the histogram of untreated cells. The binding of the anti-integrin α5β1 antibody to the breast cancer cells is over 100-fold higher

Figure 1. Expression of the α5β1 integrin on MDA-MB-231 breast cancer cells. Background fluorescence of the cells is shown as a gray shaded histogram, the binding of anti-α5β1 integrin antibody is shown in red, and the binding of the mouse IgG isotype control antibody is shown in blue. The results are representative for n = 2 but are presented only from one single experiment. 4731

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indicating that just a small amount of PR_b can potentially increase the binding ability of the stealth liposomes. Increasing the PR_b concentration in the liposomes to 3.8 mol % and 5.6 mol % significantly increased the binding levels up to 3 orders of magnitude higher than that seen for the stealth liposomes. These results are in close agreement with our previous data where 2.5 mol % PR_b in the presence of 5−6 mol % PEG2000 showed binding to colon and prostate cancer cells that, compared to stealth liposomes, was a couple of orders of magnitude higher.50,52 The flow cytometry results therefore clearly suggest that addition of the PR_b peptide-amphiphile in the liposomal formulation results in higher binding efficiencies. In order to investigate whether the binding of the PR_b liposomes was specific to the α5β1-expressing breast cancer cells, we performed blocking experiments by incubating the cells with free PR_b peptide for 30 min before delivering the liposomes and allowing them to interact with the cells for 1 h. The results of the blocking experiments are presented in Figure 3, and show that all the PR_b-functionalized PEGylated

than the binding of the isotype control, thereby indicating that integrin α5β1 is expressed in significant amounts on the breast cancer cells. The involvement and control of the integrin α5β1 receptors in invasion and metastasis of the MDA-MB-231 carcinoma cells has been reported in the literature.58,59 For example, administration of anti-α5β1 integrin antibodies was found to strongly inhibit the invasion of lung tissue by the MDA-MB-231 cells.60 The effect of the PR_b concentration on PEGylated liposomes was investigated next. We have previously examined the effect of different PEG concentrations as well as molecular weights on the binding and internalization of liposomes to different cancer cells, and our results show that higher concentrations and molecular weights contribute to increased stealth properties (i.e., less adhesion).50,52 In addition, the literature suggests that the use of PEG2000 at concentrations of around 5 mol % significantly improves the stealth properties of the liposomes and their circulation life times in vivo.5,61−65 Therefore, we have chosen to utilize 5 mol % of PEG2000 in the lipid mixture along with DPPC lipids, 35 mol % CHOL, and varying amounts of the PR_b peptide-amphiphile. Dynamic light scattering experiments suggested that the size of liposomes was between 97 and 131 nm and the preparations were sufficiently monodispersed. Particle sizes for different formulations are shown in Table S1 of the Supporting Information. To investigate the effect of the PR_b concentration on the binding of the breast cancer cells, we incubated different PR_b-functionalized PEGylated liposomes with the cells for 1 h at 4 °C and performed flow cytometry analysis. The results of this experiment are presented in Figure 2. The

Figure 3. Binding of DPPC liposomes, functionalized with 5 mol % PEG2000 and varying concentration of PR_b peptide-amphiphile, to MDA-MB-231 blocked cells. The cells were blocked with free PR_b peptide at a concentration of 200 μg/mL for 30 min at 4 °C and then incubated with the liposomes for 1 h at 4 °C. The free peptide successfully blocked the adhesion of the PR_b-functionalized PEGylated liposomes. The results are representative for n = 2 but are presented only from one single experiment.

liposomes, irrespective of their PR_b concentration, show minimal binding to the breast cancer cells that were initially blocked with the free PR_b peptide. The data suggest that the free peptide blocks the binding of the PR_b functionalized stealth liposomes and that the binding to the breast cancer cells is mediated by the binding of the PR_b peptide on the surface of the liposomes to the integrin receptors expressed on the cell surface. The PR_b peptide-amphiphile has been shown before to bind specifically to the integrin α5β1. This specificity was established by blocking the cell surface integrin α5β1 receptors with monoclonal antibodies or free PR_b peptide and subsequently testing adhesion between the PR_b surfaces and the blocked cells.44,49,50 Therefore, our previous work and the current blocking assays along with the observed α5β1 integrin expression of the breast cancer cells suggest that the interaction of the PR_b-functionalized liposomes is potentially mediated by the binding of the PR_b peptide to the α5β1 integrin. To further determine the therapeutic potential of the PR_bfunctionalized liposomes, we encapsulated doxorubicin inside the liposomes for delivery to the breast cancer cells. Doxorubicin and other anthracyclines belong to a class of weak amphipathic bases, whose encapsulation efficiencies can

Figure 2. Effect of PR_b concentration on the binding of DPPC liposomes functionalized with 5 mol % PEG2000 and delivered to MDA-MB-231 cells for 1 h at 4 °C. The binding affinity of the nontargeted PEGylated liposomes (0 mol % PR_b) is negligible, whereas significant increase in binding is observed with increasing concentrations of PR_b. The results are representative for n = 3 but are presented only from one single experiment.

histograms show that the fluorescence intensity of the nontargeted liposomes (0 mol % PR_b with 5 mol % PEG2000) overlaps with the autofluorescence of the cells, indicating that the liposomes without PR_b showed minimal binding to the breast cancer cells. This result is in agreement with previous in vivo studies, which showed that PEGylated (stealth) liposomes accumulated in the tumor but did not appear to interact with tumor cells.62,65−68 Lack of binding of the 0 mol % PR_b liposomes in our studies therefore shows that they are sufficiently shielded and prevented any nonspecific interactions. Next, liposomes with 1.6 mol % PR_b showed increased binding as compared to the 0 mol % PR_b liposomes, 4732

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compared to cell viabilities observed at the 2.7 μM doxorubicin concentration. The 4.3 and 1.7 mol % PR_b and 6.1 mol % GRGDSP liposomes encapsulating 5.4 μM doxorubicin still did a better job than the nontargeted stealth liposomes, and the 5.9 mol % PR_b liposomal doxorubicin decreased the cell viability more compared to the 6.1 mol % GRGDSP liposomes at this drug concentration. Further increasing the concentration of the free doxorubicin to 10.8 μM and 21.6 μM does not significantly lower the cell viability; however, the 5.9 mol % PR_b liposomes showed an increase in their cytotoxic potential that closely matched that of the free drug. Overall, the cell viability assay results suggest that the 5.9 mol % PR_b functionalized liposomes perform comparably to the free doxorubicin at higher concentrations and outperformed both the nontargeted and the 6.1 mol % GRGDSP-functionalized PEGylated liposomes. Higher PR_b concentrations have not been investigated since the total concentration of PEG and PR_b should not be more than approximately 10 mol %, as work in our lab showed that when higher molecular weight molecules such as PEG2000 and PR_b peptide-amphiphiles are incorporated at higher concentrations, it may result in the destabilization of the liposome membrane.50 Binding of the nanocarriers to the integrin cell receptors results in endocytosis and trafficking into endocytotic pathways that would internalize the particles into different cellular organelles. To investigate the uptake and trafficking of the PR_b liposomes inside the cells, we examined their presence in the early endosomes, late endosomes, and lysosomes. Doxorubicin was loaded at 6 μM inside liposomes to avoid self-quenching. Liposomes were functionalized with 5 mol % PEG2000 and 4.8 mol % PR_b peptide-amphiphile. The CellLight Early Endosomes-GFP cell transduction reagent was used and resulted in expression of the green fluorescence protein (GFP) in the early endosomes. The LysoTracker Blue DND-22, a blue fluorescent dye that stains acidic compartments in live cells, was used to stain all acidic vesicles (early endosomes, late endosomes, and lysosomes). These reagents allowed us to track the colocalization of the liposomal doxorubicin with the different organelles and therefore their trafficking inside the breast cancer cells. Additionally, the nuclei were stained blue with a nuclear staining dye Hoechst 33342. Confocal imaging of live cells was performed at two time points after adding the liposomal doxorubicin. After 10 min of incubating the MDA-MB-231 breast cancer cells with the liposomes (Figure 5), the liposomes (shown in red) were predominantly localized in the early endosomes (shown in green) which is clear from the colocalization of the green and red colors on this image (Figure 5 D). The presence of liposomes inside the early endosomes within 10 min of delivery to the cells (earlier time points were not examined), and the fact that work in our lab has shown that incubation of α5β1expressing cancer cells with anti-α5β1 antibodies blocked adhesion of PR_b-functionalized nanoparticles to cancer cells suggests that their uptake via receptor mediated endocytosis presumably resulting from the interaction with the α5β1 integrin receptors on the breast cancer cells.49,69,70 Figure 6 shows the trafficking of the liposomes after 80 min of incubation. The liposomes are no longer restricted to early endosomes; rather they are now localized mostly inside late endosomes and lysosomes, as evident by the colocalization of blue (all acidic vesicles) and red (liposomes) colors in the absence of colocalization between red and green (early endosomes). This colocalization with late endosomes and

be best achieved with the remote loading procedure, as discussed in the Materials and Methods section. The ammonium sulfate gradient method yielded good encapsulation efficiencies (shown in Table S2 of the Supporting Information) of the free doxorubicin in the liposomes. First we determined the cytotoxicity of free doxorubicin and its IC50 concentration against our model cancer cells using an SRB colorimetric assay. Free doxorubicin concentrations starting from 100 μg/mL (172 μM) to 0.05 μg/mL (0.08 μM) were delivered in 2-fold serial dilutions to the breast cancer cells and incubated for 72 h at 37 °C, 5% CO2. The number of living cells at the end was determined relative to the no-drug control and no-growth control, as discussed in the Materials and Methods section. From the fitting of a sigmoidal curve, the drug required to kill 50% of the cells (IC50) was found to be 2.6 ± 0.8 μM. The cytotoxicity of the PEGylated liposomal doxorubicin was evaluated next. For that liposomes containing 2-fold serial dilutions starting from 21.6 μM to 2.7 μM were incubated with the cells for 72 h and the cell viability was measured using the MTT assay. Results are plotted in Figure 4. Three trends are

Figure 4. Cell viability of the MDA-MB-231 breast cancer cells after treatment of doxorubicin that is either free form or encapsulated in DPPC liposomes functionalized with 5 mol % PEG2000 and varying concentrations of PR_b and GRGDSP peptide-amphiphiles. The free drug and the different liposomal formulations were incubated with cells for 72 h at 37 °C, 5% CO2. Values show the mean ± SD of two independent experiments (n = 2) each performed in triplicate. Z-test analysis for comparisons between free doxorubicin and the different liposomal doxorubicin formulations at each concentration: for *, p < 0.01, and for †, p > 0.01.

clearly visible; first, increasing the drug concentration (free or encapsulated) results in decreased survival of the cells; second, an increased concentration of the PR_b peptide-amphiphile on the liposomal formulations enhances the cytotoxic effect of the targeted liposomes; and third, the PR_b-functionalized PEGylated liposomes outperform both the nontargeted and the GRGDSP-functionalized formulations. The lowest drug concentration of 2.7 μM tested in its free form clearly showed cell viability levels of around 50%, which is in agreement with the IC50 values of the free drug. At the same drug concentration, however, the liposomal doxorubicin showed less cytotoxic effect. Doubling the drug concentration of the free drug to 5.4 μM decreased the cell viability to around 40%, and there was a marked improvement in the toxicity of the 5.9 mol % PR_b liposomes relative to that of the free drug as 4733

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Figure 5. Intracellular uptake and trafficking of DPPC liposomes at 37 °C and 5% CO2 functionalized with 5 mol % PEG2000, 4.8 mol % PR_b peptide-amphiphile, and loaded with 6 μM doxorubicin, 10 min after delivery to the MDA-MB-231 breast cancer cells. Confocal micrographs of the cells showing (A) intracellular location of liposomal doxorubicin in red, (B) early endosomes expressing GFP, (C) blue colored nuclei and acidic vesicles, and (D) superimposed images of A, B, and C. Panel D shows colocalization of the red colored liposomal doxorubicin preferably in the green colored early endosomes.

endosomes within minutes after delivery (Figure 5) and into late endosomes and lysosomes after 80 min (Figure 6), and finally doxorubicin was released to inhibit the proliferation of MDA-MB-231 breast cancer cells after 72 h of incubation (Figure 4).

lysosomes suggests that the drug may be active at the lower pH and follows a typical intracellular mechanism of trafficking from the early endosomes to the late endosomes and lysosomes. In fact, there is evidence in the literature that the acidic milieu of lysosome/endosomal vesicles is responsible for triggering the release of doxorubicin from liposomes.71 However, the kinetics of cellular drug binding, internalization, intracellular release of doxorubicin from the liposomes, and nuclear uptake vary from one cell line to another.71 For example, a large amount of nontargeted PEGylated liposomes was retained within the endosomes and lysosomes of HeLa cells after 2 h incubation.72 Aspargine-glycine-arginine (NGR)-functionalized PEGylated liposomal doxorubicin targeted to HT-1080 cells showed nuclear localization of doxorubicin after 60 min of incubation, whereas only a few Kaposi sarcoma-derived endothelial (SLK) cells displayed doxorubicin nuclear uptake after 60 min, as the liposomes were localized in the endosomes and lysosomes.71 Based on the in vitro results we confirmed that liposomal doxorubicin functionalized with PEG2000 and PR_b was taken up by the cells via endocytosis and transferred into early

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CONCLUSIONS MDA-MB-231 breast cancer cells showed significant expression of the integrin α5β1 and were used as our model breast cancer cell line. PEGylated liposomes prepared with different PR_b concentrations were found to be uniformly sized, and were used to target doxorubicin to the breast cancer cells. PR_bfunctionalized PEGylated liposomes showed enhanced binding as compared to the nontargeted PEGylated liposomes, and their binding efficiency increased with increasing PR_b concentration. Blocking the binding of the PR_b-functionalized liposomes with free PR_b peptide suggested that this interaction was specific and may have been mediated by the PR_b binding with the α5β1 integrin receptor. Confocal studies revealed that the liposomal doxorubicin was present in the early 4734

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Figure 6. Intracellular uptake and trafficking of DPPC liposomes at 37 °C and 5% CO2 functionalized with 5 mol % PEG2000, 4.8 mol % PR_b peptide-amphiphile, and loaded with 6 μM doxorubicin, 80 min after delivery to the MDA-MB-231 breast cancer cells. Confocal micrographs of the cells showing (A) liposomal doxorubicin in red, (B) early endosomes expressing GFP, (C) blue colored nuclei and acidic vesicles, and (D) superimposed images of A, B, and C. Panel D shows colocalization of the red liposomal doxorubicin preferably in the blue acidic vesicles and their absence in the green early endosomes, therefore suggesting that the liposomes are located in the late endosomes and lysosomes.

concentrations. This material is available free of charge via the Internet at http://pubs.acs.org.

endosomes after 10 min of incubation with the breast cancer cells and in late endosomes and lysosomes after 80 min of incubation. 72 h incubation of different doxorubicin liposomal formulations with the cells showed that nanoparticles functionalized with 5 mol % PEG2000 and 5.9 mol % PR_b performed comparably to the free doxorubicin at higher drug concentrations and outperformed both the nontargeted and the 6.1 mol % GRGDSP-functionalized PEGylated liposomes. Thus, PR_b targeting can significantly improve the binding, uptake, and cytotoxicity of PEGylated liposomes and represents a promising approach for the rapidly growing field of targeted therapeutics.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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ADDITIONAL NOTE

This work was supported by the Camille Dreyfus TeacherScholar Awards Program.

ASSOCIATED CONTENT

S Supporting Information *

Tables comparing particle sizes of 5 mol% PEG2000 liposomes functionalized with different peptide concentrations and the encapsulation efficiencies of liposomes loaded with doxorubicin and functionalized with 5 mol% PEG2000 and different peptide

Originally submitted for the “Bioinspired Assemblies and Interfaces” Special Issue, published as the January 31, 2012 issue of Langmuir (Vol. 28, No. 4). 4735

dx.doi.org/10.1021/la204466g | Langmuir 2012, 28, 4729−4736

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dx.doi.org/10.1021/la204466g | Langmuir 2012, 28, 4729−4736