Masking and Triggered Unmasking of Targeting ... - ACS Publications

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Masking and Triggered Unmasking of Targeting Ligands on Liposomal Chemotherapy Selectively Suppress Tumor Growth in Vivo Amey Bandekar,† Charles Zhu,† Ana Gomez,† Monica Zofia Menzenski,‡ Michelle Sempkowski,§ and Stavroula Sofou*,† †

Department of Biomedical Engineering and Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, United States ‡ Department of Chemistry, New York University, New York, New York 10003, United States § Department of Biomedical Engineering, The College of New Jersey, Ewing, New Jersey 08628, United States S Supporting Information *

ABSTRACT: We investigated the feasibility and efficacy of a drug delivery strategy to vascularized cancer that combines targeting selectivity with high uptake by targeted cells and high bioexposure of cells to delivered chemotherapeutics. Targeted lipid vesicles composed of pH responsive membranes were designed to reversibly form phase-separated lipid domains, which are utilized to tune the vesicle’s apparent functionality and permeability. During circulation, vesicles mask functional ligands and stably retain their contents. Upon extravasation in the tumor interstitium, ligand-labeled lipids become unmasked and segregated within lipid domains triggering targeting to cancer cells followed by internalization. In the acidic endosome, vesicles burst release the encapsulated therapeutics through leaky boundaries around the phase-separated lipid domains. The pH tunable vesicles contain doxorubicin and are labeled with an anti-HER2 peptide. In vitro, anti-HER2 pH tunable vesicles release doxorubicin in a pH dependent manner, and exhibit 233% increase in binding to HER2-overexpressing BT474 breast cancer cells with lowering pH from 7.4 to 6.5 followed by significant (50%) internalization. In subcutaneous BT474 xenografts in nude mice, targeted pH tunable vesicles decrease tumor volumes by 159% relative to nontargeted vesicles, and they also exhibit better tumor control by 11% relative to targeted vesicles without an unmasking property. These results suggest the potential of pH tunable vesicles to ultimately control tumor growth at relatively lower administered doses. KEYWORDS: liposomal doxorubicin, pH tunable liposomes, masking/unmasking targeting functionality, anti-HER2 liposomal doxorubicin arising from exposed functional moieties during circulation,10 which, ultimately, substantially reduce the circulation times of liposomal carriers. Second, the significance of high specific uptake of targeted therapeutics by cancer cells is demonstrated in animal studies which show that targeted liposomal doxorubicin results in better tumor control than the nontargeted construct; this result is not due to different tumor uptake (which is identical for both constructs) but due to extensive internalization of the targeted liposomes by cancer cells in vivo.11,12 Lastly, greater killing efficacy is strongly correlated with liposomal doxorubicin exhibiting fast release kinetics of contents upon cellular

1. INTRODUCTION Cancer death rates are decreasing mainly due to early detection, but cases of advanced disease still have no cure.1 Reported strategies which aim to address the diverse challenges in drug delivery to advanced cancer focus on enhancement of targeting selectivity to cancer cells, on increasing specific cell uptake by targeted therapeutics, and/or on improving bioexposure and, therefore, killing efficacy at the target.2−4 In particular, for targeted liposomal drug carriers, several approaches are reported which all aim to improve targeting selectivity to cancer cells. These strategies utilize the masking of targeting ligands by hydrophilic polymer chains (usually PEG) during circulation followed by triggered unmasking upon localization of the lipid carrier at the tumor interstitium.5−8 The masking/unmasking strategy addresses limitations in targeting selectivity due to lack of unique molecular targets on cancer cells,9 and alleviates potential immune responses © 2012 American Chemical Society

Received: Revised: Accepted: Published: 152

May 15, 2012 September 3, 2012 November 2, 2012 November 7, 2012 dx.doi.org/10.1021/mp3002717 | Mol. Pharmaceutics 2013, 10, 152−160

Molecular Pharmaceutics

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internalization of liposomes.13 To trigger release of encapsulated therapeutics, the endosomal acidificationwhich is encountered by internalized liposomesis a mechanism commonly utilized.14−16 Development of a delivery carrier which successfully combines all of the above three properties may potentially lead to decreased administered doses and, therefore, to lower accompanying toxicities for the same or better tumor control. To simultaneously address challenges in targeting selectivity, cell uptake, and killing efficacy, we designed lipid vesicles with pH-responsive lipid membranes that form reversible phaseseparated “(nano)patterns” (resembling “lipid-rafts”) on the vesicle surface.17 We use these membranes in the form of vesicles with cancer-targeting ligands on their surface and membrane tunable properties to enable triggered binding and triggered content release. During circulation in the bloodstream (pH ∼ 7.4) lipid membranes are well mixed, and the homogeneous distribution of PEG chains sterically obstructs the targeting ligands from binding to healthy cells expressing the targeted moiety (Figure 1, left). Also, the mixed membrane

gastric, prostate)21 and of its connection to poor prognosis.22 Second, the targeted pH tunable lipid vesicles are loaded with doxorubicin. This choice is based on several preclinical and clinical studies suggesting that HER2-overexpressing (mostly breast) cancers may be especially sensitive to anthracyclinebased chemotherapy,23 while being relatively resistant to other types of chemotherapy.24 Third, specific cancer cell targeting is mediated by an anti-HER2 short peptide (KCCYSL, whose targeting efficacy has been reported before25), which is shown herein to induce significant internalization of cell associated lipid vesicles. Finally, the BT474 human breast cancer cell line and its subcutaneous xenograft tumors in nude mice were studied for two reasons: (i) BT474 cells express large numbers of HER2 antigens per cell (106), thus allowing for the evaluation of the tunable targeting selectivity; and (ii) liposome-mediated delivery of doxorubicin to these cells was shown to have therapeutic effects, and extensive studies on such xenograft breast tumors have been performed with Doxil and are used for comparison.12

2. EXPERIMENTAL SECTION 2.1. Materials. The lipids 1,2-diheneicosanoyl-sn-glycero-3phosphocholine (21PC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-[phospho-L-serine] (sodium salt) (DSPS), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Lissamine Rhodamine B sulfonyl) (ammonium salt) (DPPE-Rhodamine), and 1,2-dipalmitoyl-snglycero-3-phosphoethanolamine-N-(succinyl) sodium salt (DPPE-succinyl) were purchased from Avanti Polar Lipids (Alabaster, AL) (all lipids at purity >99%). Hybri-Care medium was purchased from ATCC (Manassas, VA). Fetal bovine serum (FBS) was purchased from Omega Scientific (Tarzana, CA). CellTiter 96 nonradioactive cell proliferation assay (MTT) was purchased from Promega Corporation (Madison, WI). Matrigel was purchased from (BD Biosciences, San Jose, CA). The amino acids Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Tyr(tBu)-OH and Knorr Amide MBHA resin (capacity 0.4 mmol/g) were all purchased from Novabiochem. Phosphate buffered saline (PBS), doxorubicin hydrochloride, Sephadex G50, Sepharose 4B, Triton-X 100, ethylenediaminetetraacetic acid (EDTA), cholesterol, and all reagents and solvents for peptide synthesis were purchased from Sigma-Aldrich (Atlanta, GA). 2.2. Lipopeptide Synthesis and Characterization. The peptide Gly-Ser-Gly-(Lys-Cys-Cys-Tyr-Ser-Leu) was prepared on a 0.10 mmol scale using standard Fmoc solid-phase peptide synthesis protocols (detailed protocol is described in the Supporting Information). For the coupling reaction, the resinattached peptide and the lipid DPPE-succinyl were reacted in DMF overnight at room temperature with continuous shaking. Cleavage from the resin and removal of all amino acid protecting groups was carried out using a cleavage cocktail followed by filtering and concentration in a rotary evaporator. Precipitated lipopeptide, using cold diethyl ether, was measured to be of purity of approximately >90% using an Agilent 1100 series capillary LCMSD Trap XCT spectrometer (detailed protocol is described in the Supporting Information). 2.3. Vesicle Formation. pH tunable vesicles composed of 21PC, DSPS, and cholesterol (8.6:0.9:0.5 mol ratio) with 3.8 mol % DSPE-PEG (unless otherwise specified) and DSPC/

Figure 1. pH tunable lipid vesicles form reversible phase-separated lipid domains, which are utilized to alter the vesicles’ apparent functionality and its membrane permeability: (left) at pH ∼ 7.4 during circulation in blood, (center) at pH ∼ 6.7−6.0 in the tumor interstitium, (right) at pH ∼ 5.5−5.0 in endosome. The targeting ligand is shown in green, and its exposure is dependent on the extent of phase separation of the vesicle’s membrane; phase separation is activated at the tumor interstitial pH (shown in the middle). Pronounced lipid packing defects on the boundaries around lipid domains result in burst release of contents at the endosomal pH (shown on the right).

at pH 7.4 results in stable retention of the encapsulated chemotherapeutic agents. At the acidic tumor interstitial pH (6.7−6.018,19), phase separation and domain formation unmasks and recruits ligand-labeled lipids, increasing local multivalency and enhancing targeting to cancer cells (Figure 1, center). Upon vesicle internalization by targeted cancer cells, in the acidic endosomal pH (∼5.020) formation of pronounced grain boundaries around the phase separated domains causes burst release of the encapsulated therapeutic agents (Figure 1, right). We have already demonstrated in vitro that targeted pH tunable lipid vesicles, with the above properties, selectively control cancer cell growth in a pH dependent manner that is attributed to the extracellular pH controlled extent of targeting followed by internalization.5 In the present study we evaluate the efficacy of the above approach in vivo in terms of targeting selectivity and killing efficacy by monitoring the extent of controlling tumor growth in a subcutaneous HER2-overexpressing BT474 breast cancer xenograft in a murine model. We made the following choices regarding the components of this approach; first, the HER2 receptor as a targeting moiety is chosen because of its variable overexpression in several types of cancer (breast, ovarian, 153

dx.doi.org/10.1021/mp3002717 | Mol. Pharmaceutics 2013, 10, 152−160


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mM NaCl, pH 2.8) for 5 min at room temperature to remove the surface bound vesicles. At the end of incubation, samples were again washed twice with ice cold PBS and resuspended in a volume of 2 mL of PBS and measured for Rhodamine intensity. Sample fluorescence intensities were corrected for light scattered from cells. 2.9. Cytotoxicity Studies on Cell Monolayers. BT474 cells were plated on 96 well plates at a density of 40,000 cells per well. Vesicles were prepared at pH 7.4, and then five different aliquots at different pH values were prepared from the parent suspension and were incubated at 37 °C and 5% CO2 for 24 h. After the incubation period, vesicles were passed through a Sepharose 4B column, eluted with isosmolar PBS at the respective pH values so as to separate the leaked contents. Cells were then incubated for a period of 24 h at different pH values with doxorubicin-containing constructs (at 0.31 or 0.16 μmol of doxorubicin/mL across all samples) at a total final volume of 0.3 mL per well. In all cases the drug-to-lipid ratio was kept constant at 0.79 μmol of doxorubicin/μmol of lipid or 0.39 μmol of doxorubicin/μmol of lipid. After 24 h of incubation, cells were washed thrice with sterile PBS and further incubated with fresh medium for a period of 48 h, i.e., one doubling time for the cell line used, at 37 °C and 5% CO2. Cell viability was evaluated by the MTT assay. 2.10. Evaluation of Cell Associated Doxorubicin. Cells were plated on 96 well plates at a density of 40,000 cells per well and incubated with different modalities at different pH values as described above. The drug-to-lipid ratio was 0.39 μmol of doxorubicin/μmol of lipid across all conditions. After 24 h of incubation, cells were gently washed thrice with sterile PBS and then trypsinized using 0.1 mL of trypsin solution (0.05% w/w). Cells were then washed by centrifugation and lysed by resuspension in 0.5 mL of distilled water followed by sonication using a Branson 1510 water sonicator (Danbury, CT). Doxorubicin associated with cells was quantified by addition of 2.5 mL of acidified isopropyl alcohol (90% isopropyl alcohol and 10% of 12 N hydrochloric acid (v/v)) and by measuring doxorubicin’s fluorescence intensity (excitation, 470 nm; emission, 592 nm). 2.11. Animal Studies. 2.11.1. Animals. Mice were housed in filter top cages and provided with sterile food and water. Animals were maintained according to the regulations of the Research Animal Resource Center (RARC) at Memorial SloanKettering Cancer Center (MSKCC), and animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC). 2.11.2. Tumor Inoculation and Assessment of Tumor Growth. Four- to six-week old female Balb/c nude mice (Taconic, Germantown, NY) were inoculated subcutaneously in the dorsum of the right thigh with 106 BT474 cells suspended in 100 μL of Matrigel (BD Biosciences, San Jose, CA). One day before inoculation, mice were subcutaneously administered estradiol (a 0.72 mg pellet per animal, Innovation Research of America, Sarasota, FL). When the tumor size reached 100 mm3, mice were randomly assigned to a treatment group (3−8 mice per group) and were then treated via tail vein injections of volumes equal to 0.1 mL. Doxorubicin containing lipid vesicles of both compositions were administered iv at 4.1 mg of doxorubicin/kg (which is significantly lower than the reported MTD of liposomal doxorubicin (18.8−22.5 mg/ kg)12) and 0.2 μmol of total lipid per dose once every week for three weeks. Empty targeted vesicles and empty nontargeted vesicles were administered at the same volume and lipid

cholesterol-based vesicles (7:3 mol ratio) with 4.8 mol % DSPE-PEG (resembling the composition of Doxil) were prepared using the extrusion method.5 For targeted vesicles of both compositions, 0.2 mol % lipopeptide was included, and, upon completion of extrusion, vesicles were eluted in PBS (pH 7.4, 300 mOsm). 2.4. Vesicle Size and Zeta Potential Measurements. Dynamic light scattering (DLS) measurements were performed at four different angles: 23.0, 30.2, 62.6, and 90° using a N4 Plus autocorrelator (Beckman-Coulter, Fullerton, CA) with a 632.8 nm He−Ne laser light source. Autocorrelation data analysis was used to evaluate the particle size distribution. Vesicles’ zeta potential was measured with a Malvern Zetasizer NanoSeries (Malvern Instruments Ltd., Worcestershire, U.K.). 2.5. Loading Vesicles with Doxorubicin. For loading doxorubicin into lipid vesicles, the method of ammonium sulfate gradient was used,5,26 and upon completion of loading, unentrapped doxorubicin was removed by a Sepharose 4B column eluted with isosmolar PBS (pH 7.4, 300 mOsm). 2.6. Cell Lines. The breast carcinoma cell line BT474 and the murine macrophage-like cell line J774 were acquired from ATCC (Manassas, VA) and cultured in Hybri-Care medium and DMEM, respectively, supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin in an incubator at 37 °C and 5% CO2. 2.7. Doxorubicin Retention by Lipid Vesicles in Serum Supplemented Media. Lipid vesicles loaded with doxorubicin as described earlier were incubated in Hybri-Care medium supplemented with 10% FBS at different pH values at 37 °C and 5% CO2. At different time points, samples from the incubating parent suspensions were removed and evaluated for the fraction of leaked doxorubicin which was removed from intact vesicles by a 10 cm Sephadex G50 column eluted with PBS (pH 7.4, 300 mOsm). Doxorubicin was quantified by addition of Triton-X 100 (5% w/v) followed by heating at 85 °C for 10 min. Samples were first cooled to room temperature before measurement of fluorescence. 2.8. Cell Binding and Internalization of Vesicles. Lipid vesicles encapsulating calcein and labeled with 1 mol % DPPERhodamine were prepared at pH 7.4. The parent vesicle suspension was divided into five aliquots, and hydrochloric acid was used to adjust pH to 7.0, 6.7, 6.5, and 6.0. The aliquots were incubated for 24 h at 37 °C and 5% CO2 to achieve the maximum possible extent of phase separation on lipid membranes. Upon completion of incubation, released calcein was removed by a 10 cm Sephadex G50 column eluted with phosphate buffer (at the respective pH, 300 mOsm). BT474 cells harvested in Hybri-Care medium supplemented with 10% FBS were washed and then resuspended at a density of 10 × 106 cells per mL in media of the above different pH values which were previously allowed to equilibrate at the respective pH values by overnight incubation at 37 °C and 5% CO2. Cell suspensions were incubated with different vesicle constructs (in 3 mL total volume, containing 30 × 106 cells and 0.31 mM lipid). At different time points, 0.3 mL of the incubated suspension was removed and suspended in 2 mL of ice cold PBS followed by washing using ice cold PBS and by resuspension in 2 mL of PBS. The fraction of lipid vesicles bound to cells was quantified by measuring the fluorescence intensity of Rhodamine−lipids (excitation, 550 nm; emission, 590 nm) using an SLM AMINCO 8000 spectrofluorometer. To evaluate the internalized vesicles, the cell suspension was incubated with 1 mL of a stripping buffer (50 mM glycine, 100 154



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Figure 2. Percentage of doxorubicin retention by lipid vesicles in serum supplemented medium over time at different pH values. (A) pH tunable lipid vesicles; (B) DSPC/cholesterol-based lipid vesicles. pH (▲) 7.4; (●) 6.5; (◇) 5.5; (□) 4.0. The error bars correspond to standard deviations of repeated measurements (two vesicle preparations, two samples per preparation per time point). The lines are guides to the eye.

Figure 3. Specific targeting of vesicles to BT474 cell suspensions in vitro over time as a function of extracellular pH. Specific cell targeting is defined as the measured cell association of targeted lipid vesicles corrected for the nonspecific cell association of nontargeted lipid vesicles of the same composition. (A) pH tunable lipid vesicles; (B) DSPC/cholesterol-based vesicles. pH (▲) 7.4; (▼) 7.0; (■) 6.7; (●) 6.5; (◆) 6.0. The error bars correspond to standard deviations of repeated measurements (two vesicle preparations, two samples per preparation per time point). The lines are guides to the eye.

resuspended in PBS, and counted using a hemocytometer. Rhodamine fluorescence intensities were measured using a spectrofluorometer and corrected for background scattering from suspended cells (detailed protocol is described in the Supporting Information).

concentration and identical administration schedule. Free doxorubicin was administered at 4.1 mg/kg at the same volume and schedule, as was saline. Tumor volume was calculated using the modified ellipsoid formula (V = (1/2)ab2), where a is the large (major) tumor diameter and b the small (minor) tumor diameter, and was monitored three times per week during the treatment period using a caliper.27,28 2.11.3. Statistical Analysis. Results are reported as the arithmetic mean of n independent measurements ± the standard deviation. Student’s t test was used to calculate significant differences in killing efficacy and tumor growth between the pH tunable and the DSPC/cholesterol-based vesicles. p-values less than 0.01 are considered to be significant. 2.12. Vesicle Association with Macrophages. Uptake by J774 macrophages of targeted and nontargeted lipid vesicles labeled with Rhodamine−lipid conjugates was evaluated using vesicle suspensions diluted in DMEM medium with 10% FBS at a final concentration of 0.3 μmol of lipid/mL. For uptake evaluation under flow conditions, a rectangular parallel plate flow chamber (Glycotech, Gaithersburg, MD) was used, and a constant flow rate of 0.1 mL/min was maintained using a PHD Ultra syringe pump (Harvard Apparatus, South Natick, MA) for four hours at 37 °C in 5% CO2. For the static incubation conditions, identical vesicle suspensions were introduced over the cell monolayers. After incubation, cells were washed,

3. RESULTS 3.1. Vesicle Characterization. The average vesicle size (diameter) used in these studies was 108 ± 3 nm (PDI = 0.05) and 104 ± 2 nm (PDI = 0.1) for pH tunable and DSPC/ cholesterol-based lipid vesicles, respectively (n = 3 independent measurements). Zeta potential values ranged from −4.1 to −5.8 mV for pH tunable lipid vesicles, and from 0.4 to 0.8 mV for DSPC/cholesterol-based lipid vesicles, respectively. Inclusion of peptide−lipid conjugates did not substantially affect vesicles’ size and zeta potential values, which were essentially constant for a least five days upon preparation (see Table S1 in the Supporting Information). The loading efficiency was not affected by the presence of peptide−lipid conjugates in the liposome preparation. 3.2. Retention of Doxorubicin by Vesicles in Vitro. Figure 2A shows increasing release of encapsulated doxorubicin with lowering pH by pH tunable vesicles. These vesicles, however, stably retain doxorubicin (90%) at pH 7.4 (filled circles) for at least two days. In particular, after six hours of 155



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Figure 4. (A, C) Viability of BT474 breast cancer cell monolayers overexpressing HER2 after treatment with pH tunable vesicles (A) or DSPC/ cholesterol-based vesicles (C) encapsulating doxorubicin in different extracellular pH values. (B, D) Internalized doxorubicin by BT474 cells delivered via pH tunable vesicles (B) or DSPC/cholesterol-based vesicles (D) incubated at different extracellular pH values under conditions identical to those of viability studies. Large symbols represent doxorubicin-loaded vesicles; small symbols denote doxorubicin-free vesicles. Filled symbols denote targeted vesicles. The drug-to-lipid ratio was kept constant at 0.39 μmol of doxorubicin/μmol of lipid (at 0.16 μmol of doxorubicin/ mL across all samples, at a total final volume of 0.3 mL per well). (large ■) anti-HER2 pH tunable vesicles with doxorubicin; (large □) nontargeted pH tunable vesicles with doxorubicin; (small ■) anti-HER2 pH tunable vesicles without doxorubicin; (small □) nontargeted pH tunable vesicles without doxorubicin; (large ●) anti-HER2 DSPC/cholesterol-based vesicles with doxorubicin; (large ○) nontargeted DSPC/cholesterol-based vesicles with doxorubicin; (small ●) anti-HER2 DSPC/cholesterol-based vesicles without doxorubicin; (small ○) nontargeted DSPC/cholesterolbased vesicles without doxorubicin; (▲) free doxorubicin.

the nonspecific cell association of nontargeted lipid vesicles of the same composition (Figure S2 in the Supporting Information). Figure 3A shows increase in specific cancer cell targeting by anti-HER2 pH tunable vesicles from only 0.4% at pH 7.4 (upward triangles) to 2.6% at pH 6.5 (circles) after four hours of incubation in serum supplemented medium. At all pH values, the fraction of targeted pH tunable vesicles that becomes internalized by BT474 cells is at least 50% of specifically bound vesicles (Figure S2 in the Supporting Information). For comparison, trastuzumab labeled vesicles (with the targeting functionality attached on the free end of PEG chains) exhibit similar (up to 65%) internalization efficacy.29,30 Specific binding of anti-HER2 DSPC/cholesterol-based vesicles does not exceed 1% of total vesicles and is independent of pH (Figure 3B); the fraction of internalized DSPC/ cholesterol-based vesicles upon specific binding is only 0.1%. 3.4. Selectivity of Cancer Cell Killing on Cell Monolayers. Figure 4A shows that anti-HER2 pH tunable vesicles that gradually unmask the binding ligand with lowering pH (large filled squares) exhibit increasing cancer cell killing with acidification of the extracellular milieu. The underlying mechanism is driven by the increasing delivery of doxorubicin into cancer cells as pH is lowered from 7.4 to 6.0 (large filled squares in Figure 4B). In particular, cell viability is 87% at pH

incubation in serum supplemented medium at pH 6.5 (open circles) and 5.5 (filled squares) only 77% and 66% of encapsulated doxorubicin is retained, respectively. At pH 4.0, indicative of the lysosomal compartment, doxorubicin retention decreases further to 51% (open squares). Contrary to pH tunable vesicles, DSPC/cholesterol-based vesicles exhibit pH independent retention ranging from 78 to 85% between pH 4.0 and 6.5, after six hours of incubation in the same conditions (Figure 2B); similarly to pH tunable vesicles, doxorubicin is stably retained to the extent of 90% at pH 7.4 (filled circles). The presence of peptide−lipid conjugate(s) does not measurably affect the retention profiles for both lipid compositions (Figures S1A and S1B in the Supporting Information). Incubation of lipid vesicles in cell conditioned medium for twenty-four hours results in identical doxorubicin retention (67%) at pH 7.4 by both lipid compositions. At the lower pH value of 6.0, the pH tunable lipid vesicles exhibit lower retention of doxorubicin (56%) relative to DSPC/cholesterolbased vesicles 63%) (Figures S1C and S1D in the Supporting Information). 3.3. Specific Targeting of Vesicles to Cells in Vitro. Figure 3A shows the pH dependence of the specific cancer cell targeting by vesicles. Specific targeting is defined as the measured cell association of targeted lipid vesicles corrected for 156



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7.4 and decreases to 66% at pH 6.0 (large filled squares, Figure 4A). For comparison, BT474 cancer cells treated with antiHER2 DSPC/cholesterol-based vesicles (without the unmasking property) encapsulating doxorubicin exhibit 90−87% viability between pH 7.4 and 6.0 (large filled circles, Figure 4C). The intracellular uptake of doxorubicin mediated by these vesicles is low and pH independent (large filled circles, Figure 4D). Greater concentrations of encapsulated doxorubicin in lipid vesicles (higher drug-to-lipid ratio) do not alter the pH dependence in killing efficacy (Figure S3 in the Supporting Information). The killing efficacy of free doxorubicin reaches a plateau with gradual acidification of the extracellular incubation media; this is attributed to its lower intracellular uptake (filled triangles, Figures 4B and 4D). Doxorubicin, being a weak base, becomes protonated at acidic pH, which results in decrease of its effective diffusivity across cell plasma membranes and, therefore, its cell permeation.31 The effect of acidic pH of the incubation media on BT474 cancer cell viability has been taken into account in Figure 4; this effect contributes up to 12% decrease in the viability of cells when exposed to acidic media during the 24 h incubation period relative to cells incubated at physiological pH (Figure S4 in the Supporting Information). 3.5. Control of Tumor Growth. In mice bearing subcutaneous HER2-overexpressing BT474 breast cancer xenografts, tumor targeting selectivity is retained by antiHER2 pH tunable vesicles resulting in more effective control of tumor growth (filled squares, Figure 5A) relative to the targeted non pH tunable DSPC/cholesterol-based vesicles (filled circles,

Figure 5A). In particular, delivery of doxorubicin by the antiHER2 pH tunable vesicles (filled squares) results in smaller tumor sizes exhibiting a constant at least 12% decrease (p = 0.0001, n = 8) after day 21 of the relative volume change (defined as (Vt − V0)/V0 × 100) compared to tumors in animals which were administered identically targeted DSPC/ cholesterol-based vesicles (filled circles in Figure 5A). The latter vesicle composition maintains the tumor size almost unchanged but smaller than the size controlled by the nontargeted composition indicating some activity of the targeting peptide which was not observed in vitro (Figure 4). The nontargeted liposomal doxorubicin of both compositions (open squares and open circles in Figure 5A) results in identically limited tumor control (n = 5). The surfaceconjugated anti-HER2 peptide does not affect tumor control as indicated by both anti-HER2 lipid vesicle compositions not containing doxorubicin (open triangles, Figure 5B).

4. DISCUSSION Liposomal forms of doxorubicin (Doxil/Caelyx) are among the most promising and exhaustingly studied approaches for cancer chemotherapy.32 While these liposomes address doxorubicin’s cardiotoxicity and improve tumor uptake, they do not address targeting selectivity12 and are certainly not designed to exhibit burst-like release profiles.3,13 Tumor targeting selectivity by functionalization of liposomes, as for example the anti-HER2 Doxil33which is perceived to be the lead of the next liposomal generation34is compromised by the fact that targeted moieties are not unique to cancer cells. However, the HER2 receptor is a frequently utilized target because it is commonly overexpressed in human cancers and associated with aggressive disease and poor clinical outcomes.35 To simultaneously improve selectivity in targeting cancer cells by functionalized liposomes encapsulating doxorubicin, enhance uptake of liposomes by cancer cells, and improve killing efficacy of delivered doxorubicin, we engineered pH tunable lipid vesicles with the following properties: (1) these vesicles mask the anti-HER2 functionality during circulation in the bloodstream and only unmask the targeting moiety upon their extravasation into the tumor interstitium; (2) upon unmasking of their targeting functionality, these vesicles bind specifically and to a high extent to cancer cells and become internalized; (3) following their cellular internalization, pH tunable vesicles release fast and extensively the encapsulated doxorubicin resulting in high killing efficacies. The underlying molecular mechanism involves lipid phase separation triggered by pH acidification. Triggering of lipid phase separation at the tumor interstitial pH is mediated by incorporating the titratable lipid phosphatidylserine (PS), which is chosen due to its apparent pKa value, which is reported to be close to or above 6.0.36,37 The pH tunable vesicles contain functional groups which are grafted directly on the lipid headgroups and are screened by the neighboring PEGylated lipids (Figure 1). This technology is general and is only limited by the size of the functional ligand which should be shorter than PEG’s length.38 We have previously shown, in a proof-of-principle study in vitro, that pH tunable lipid vesicles labeled with biotinas the targeting functionalityeffectively and reversibly mask and unmask the functional groups depending on their neighboring environment’s pH value.17 These vesicles result in pH dependent binding to and internalization by streptavidinpresenting cancer cells using a two-step (pre)targeting approach.5

Figure 5. Effect of administered vesicles containing doxorubicin on altering the tumor volume over time in a BT474 subcutaneous xenograft in a murine model. Percentage change in volume is defined as (Vt − V0) /V0 × 100 with Vt the tumor volume at time t and V0 the volume at t = 0. Squares: pH tunable vesicles. Circles: DSPC/ cholesterol-based vesicles. (■) anti-HER2 pH tunable lipid vesicles with doxorubicin (DXR) (n = 8); (□) nontargeted pH tunable lipid vesicles with DXR (n = 8); (●) anti-HER2 DSPC/cholesterol-based lipid vesicles with DXR (n = 8); (○) nontargeted DSPC/cholesterolbased lipid vesicles with DXR (n = 8); (△) targeted pH tunable lipid vesicles without DXR (n = 5); (▽) targeted DSPC/cholesterol-based lipid vesicles without DXR (n = 4); (⬡) free doxorubicin (n = 3); (◇) saline (n = 8). The error bars correspond to standard deviations of measurements in different animals (one tumor per animal). The lines are guides to the eye. Black arrows indicate drug administration; this protocol was chosen to directly compare with published studies. *p = 0.0001. 157



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vesicles. This relatively lower uptake of pH tunable vesicles, compared to the fast clearing PS-containing vesicles, is attributed to the combination of dense PEGylation (3.8 mol %) and gel-phase membranes. In contrast, the reportedly fast clearing PS-containing vesicles are composed of fluid-phase lipid membranes.40,41 However, although the in vitro macrophage uptake of PS-containing pH tunable vesicles is greater than the uptake of conventional liposomes, the tumor uptake of indium-111 labeled liposomes is measured to equal 1.52 ± 0.59% ID/g (n = 6 animals) for pH tunable vesicles and 1.80 ± 0.75% ID/g (n = 5 animals) for DSPC/cholesterol-based lipid vesicles with similar uptake by normal organs (see the Supporting Information for experimental details). A second potential mechanism regarding the observed in vivo activity of targeted vesicles not bearing the unmasking property could be the vesicles’ possible decomposition by the presence of interstitial tumor-related enzymes. The secretory phospholipase A2 (PLA2)43 is detected in several breast cancers in humans and may result in formation of lysolipids that ultimately destabilize the liposomal membrane. The enzymatic activity is increased in the presence of anionic PEGylated lipids44 such as the DSPE-PEG lipids used in the composition of DSPC/cholesterol-based vesicles. In vitro preincubation of both liposome compositions with PLA2 (see Figure S5 in the Supporting Information) shows enhancement of targeted liposome association with targeting cells. We speculate that potential disintegration of the PEGylated corona of these vesicles may occur due to enzymatic decomposition and formation of single acyl-tail PEGylated lipids. These lipids should exhibit greater CMC and may ultimately dissociate from the vesicle membrane allowing for fast exposure of the antiHER2 peptide and increase in the apparent binding of vesicles to cancer cells. In the present studies, the maximum density of 3.8 mol % of PEGylated lipid is used although greater densities could potentially further improve the circulation times of these vesicles in the bloodstream.45 The particular content of PEGylated lipid is the maximum value measured that allows for the maximum possible change in binding reactivity (233% increase in selective binding to cancer cells as shown in Figure S6 in the Supporting Information). Interestingly, although high grafting densities of PEGylated lipids tend to act against phase separation of the underlying membrane,46 the DSC thermographs of pH tunable vesicles containing 3.8 mol % PEGylated lipids (Figure S7 in the Supporting Information) clearly show formation of new phases with lowering pH. Strategies that involve nanometer sized particles for drug delivery need to address the challenge of short penetration depths in tumors (only 20−30 μm is reported for liposomes).47 Functionalization of nanocarriers decreases penetration even further (similar to the binding-site barrier effect48), and only smaller nanocarriers labeled with lower affinity ligands are reported to penetrate deeper into solid tumors.49 The pH tunable lipid vesicles used in the present studies exhibit cell binding that is increasing with deeper penetration into the more acidic tumor interstitium, therefore, significant decrease in tumor penetration due to functionalization is not expected. For such cases of vascularized tumors in which relatively low penetration of drug delivery carriers is observed, direct release of therapeutics into the tumor interstitial space has been suggested as a potential approach to improve the penetration depths of therapeutics.50,51 However, for chemotherapeutics that exhibit pH dependent diffusivities across cell mem-

In the present study, pH tunable lipid vesicles functionalized with an anti-HER2 peptide25 and encapsulating doxorubicin, using a single step administration, are demonstrated to exhibit improved control of tumor growth relative to nontargeted vesicles and relative to targeted DSPC/cholesterol-based vesicles which do not have an unmasking property. The observed improvement in tumor control is attributed to selective targeting of cancer cells within vascularized tumors by pH tunable vesicles which progressively unmask the targeting peptides upon extravasation from the bloodstream into the acidic tumor interstitium. Although not specifically measured in this model, human xenografts are reported to exhibit acidic interstitial pH values,18 and multicellular spheroids of the same cell line exhibit measurable pH gradients (between 7.5 and 6.2) in vitro.29 This stimulus is relevant to human tumors which may exhibit acidic pH values as low as 6.0 to 6.2.19 Surprisingly, the antitumor activity of targeted DSPC/ cholesterol-based vesicles, which do not possess an unmasking property with lowering pH, is significantly greater than the low killing efficacy and low cell uptake they exhibit in vitro. Two suggested mechanisms may explain this finding. First, each targeted vesicle composition may exhibit potentially different tumor uptake attributed to different circulation times. To stimulate the vesicle’s membrane response at the tumor interstitium, pH tunable lipid vesicles contain phosphatidylserine (PS). However, this lipid, when on the surface of apoptotic cells, acts as an important ligand for clearance39 that could accelerate the clearance of PS-containing liposomes.40,41 To obtain a measure of the in vivo potential clearance rates of lipid vesicles, we evaluated in vitro the uptake of lipid vesicles by J774 murine macrophage-like cells42 in monolayers using a parallel plate flow chamber or in suspension in nonstatic conditions. The uptake values by J774 of pH tunable vesicles are compared to the uptake of the longer circulating DSPC/cholesterol-based vesicleswhich exhibit blood AUCs comparable to Doxil30and of a reportedly fast clearing PS-containing lipid vesicle composition.40,41 Table 1 shows that pH tunable lipid vesicles containing PS− lipids exhibit higher uptake by J774 than the uptake of DSPC/ cholesterol-based vesicles; this value is 1.7× greater in J774 monolayers and 13× greater in suspension, but consistently lower by 3× than the uptake of the fast clearing PS-containing Table 1. Uptake of Lipid Vesicles by J774 Macrophage-like Cells in Vitro, Evaluated by the Fluorescence Intensity Associated with Cells upon Incubation with FluorescentlyLabeled Lipid Vesicles fluorescence intensity J774 in monolayer lipid vesicle composition pH tunable (nontargeted) pH tunable (targeted) DSPC/cholesterol-based (nontargeted) DSPC/cholesterol-based (targeted) PS-containing fluid membrane vesicles

J774 in suspension

static

under flow

nonstatic

569 ± 56a 659 ± 27 534 ± 24

543 ± 56 501 ± 60 381 ± 24

49 ± 14 99 ± 24 13 ± 3

498 ± 24

339 ± 27

13 ± 7

2185 ± 320

1593 ± 12

189 ± 8

a

The errors correspond to the standard deviation of independent measurements using two different vesicle preparations. 158



Article

(4) Sofou, S. Surface-active liposomes for targeted cancer therapy. Nanomedicine 2007, 2, 711−724. (5) Karve, S.; Bandekar, A.; Ali, M. R.; Sofou, S. The pH-dependent association with cancer cells of tunable functionalized lipid vesicles with encapsulated doxorubicin for high cell-kill selectivity. Biomaterials 2010, 31, 4409−4416. (6) McNeeley, K. M.; Karathanasis, E.; Annapragada, A. V.; Bellamkonda, R. V. Masking and triggered unmasking of targeting ligands on nanocarriers to improve drug delivery to brain tumors. Biomaterials 2009, 30, 3986−3995. (7) Rui, K.; Wenmin, Y.; Wanyu, L.; Qin, Y.; Tang, J.; Yuan, M.; Fu, L.; Ran, R.; Zhang, Z.; He, Q. Targeted Delivery of Cargoes into a Murine Solid Tumor by a Cell-Penetrating Peptide and Cleavable Poly(ethylene glycol) Comodified Liposomal Delivery System via Systemic Administration. Mol. Pharmaceutics 2011, 8, 2151−2161. (8) Sawant, R. M.; Hurley, J. P.; Salmaso, S.; Kale, A.; Tolcheva, E.; Levchenko, T. S.; Torchilin, V. P. “SMART” Drug Delivery Systems: Double-Targeted pH-Responsive Pharmaceutical Nanocarriers. Bioconjugate Chem. 2006, 17, 943−949. (9) Press, M. F.; Cordon-Cardo, C.; Slamon, D. J. Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene 1990, 5, 953−962. (10) Allen, T. M. Ligand-targeted therapeutics in anticancer therapy. Nat. Cancer Rev. 2002, 2, 750−763. (11) Kirpotin, D. B.; Drummond, D. C.; Shao, Y.; Shalaby, M. R.; Hong, K.; Nielsen, U. B.; Marks, J. D.; Benz, C. C.; Park, J. W. Antibody Targeting of Long-Circulating Lipidic Nanoparticles Does Not Increase Tumor Localization but Does Increase Internalization in Animal Models. Cancer Res. 2006, 66, 6732−6740. (12) 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. Anti-HER2 Immunoliposomes: Enhanced Efficacy Attributable to Targeted Delivery. Clin. Cancer Res. 2002, 8, 1172−1181. (13) Kirchmeier, M. J.; Ishida, T.; Chevrette, J.; Allen, T. M. Correlations between the rate of intracellular release of endocytosed liposomal doxorubicin and cytotoxicity as determined by a new assay. J. Lipid Res. 2001, 11, 15−29. (14) Yatvin, M. B.; Kreutz, W.; Horwitz, B. A.; Shinitzky, M. pHsensitive liposomes: possible clinical implications. Science 1980, 210, 1253−1255. (15) Simoes, S.; Moreira, J. N.; Fonseca, C.; Düzgünes, N.; Pedroso de Lima, M. On the formulation of pH-sensitive liposomes with long circulation times. Adv. Drug Delivery Rev. 2004, 56, 947−965. (16) Slepushkin, V. A.; Simoes, S.; Dazin, P.; Newman, M. S.; Guo, L. S.; Pedroso de Lima, M.; Düzgünes, N. Sterically stabilized pHsensitive liposomes. J. Biol. Chem. 1997, 272, 2382−2388. (17) Bajagur Kempegowda, G.; Karve, S.; Bandekar, A.; Adhikari, A.; Khaimchayev, T.; Sofou, S. pH-dependent formation of lipid heterogeneities controls surface topography and binding reactivity in functionalized bilayers. Langmuir 2009, 25, 8144−8151. (18) Helmlinger, G.; Yuan, F.; Dellian, M.; Jain, R. K. Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat. Med. 1997, 3, 177− 182. (19) Vaupel, P.; Kallinowski, F.; Okunieff, P. Blood Flow, Oxygen and Nutrient Supply, and Metabolic Microenvironment of Human Tumors: A Review. Cancer Res. 1989, 49, 6449−6465. (20) Mellman, I. The importance of being acid: the role of acidification in intracellular membrane traffic. J. Exp. Biol. 1992, 172, 39−45. (21) Tai, W.; Mahato, R.; Cheng, K. The role of HER2 in cancer therapy and targeted drug delivery. J. Controlled Release 2010, 146, 264−275. (22) Carey, L. A.; Perou, C. M.; Livasy, C. A.; Dressler, L. G.; Cowan, D.; Conway, K.; Karaca, G.; Troester, M. A.; Tse, C. K.; Edmiston, S.; Deming, S. L.; Geradts, J.; Cheang, M. C. U.; Nielsen, T. O.; Moorman, P. G.; Earp, H. S.; Millikan, R. C. Race, Breast Cancer

branessuch as doxorubicinacidification of the tumor interstitial pH results in lower cell permeation31 and activity52 in the more acidic core of tumors. Our in vitro studies show that, for pH values lower than 6.7, the limited cell membrane permeation of free doxorubicin may be potentially partly addressed by the pH tunable targeting mechanism of lipid vesicles; these vesicles are shown to deliver increasing amounts of doxorubicin intracellularly with lowering pH, therefore, improving the killing efficacy of delivered doxorubicin at the more acidic extracellular environment. These studies show that pH tunable lipid vesicles for targeted delivery of doxorubicin have the potential to ultimately result in effective tumor controland possibly in improved tumor managementat lower administered doses relative to nontargeted liposomal doxorubicin or to targeted liposomal doxorubicin without the unmasking property. The impact of this approach is further enhanced by its applicability to different types of advanced cancer: the proposed technologies for selective targeting and triggered release are general, and can be used for other ligand−receptor pairs and therapeutic agents.

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ASSOCIATED CONTENT

S Supporting Information *

Information on peptide synthesis, doxorubicin retention profiles by vesicles, the effect of pH and the extent of PEGylation on altering the binding profiles of vesicles to cells, and characterization of lipid phase separation by differential scanning calorimetry, and vesicle uptake by targeting cells following their preincubation with PLA2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Biomedical Engineering, and Chemical and Biochemical Engineering, 599 Taylor Road, Rutgers University, Piscataway, NJ 08854. E-mail: [email protected]. Phone: 848 445 6568. Fax: 732 445 3753. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. David Scheinberg at Memorial Sloan-Kettering Cancer Center for use of his laboratory facilities for the animal studies, and Dr. Paramjit Arora at New York University for use of his laboratory facilities and assistance with the lipopeptide synthesis. This study was supported primarily by the Susan G. Komen Foundation (Career Catalyst Award), the Wallace H. Coulter Foundation (Early Career Award in Translational Research), partially by the MRSEC Program of the National Science Foundation under Award Number DMR-0820341, and partially by the NSF REU program under award number EEC0851831.

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REFERENCES

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