Sticky Patches on Lipid Nanoparticles Enable the Selective Targeting

Jul 28, 2016 - Vesicles with clustered HER2-targeting peptides within such sticky patches (sticky vesicles) were compared to uniformly functionalized ...
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Sticky Patches on Lipid Nanoparticles Enable the Selective Targeting and Killing of Untargetable Cancer Cells Michelle Sempkowski,† Charles Zhu,† Monica Zofia Menzenski,∇ Ioannis G. Kevrekidis,∥ Frank Bruchertseifer,⊥ Alfred Morgenstern,⊥ and Stavroula Sofou*,†,‡,§ †

Department of Biomedical Engineering, ‡Department of Chemical and Biochemical Engineering, and §The Rutgers Center for Lipid Research, New Jersey Institute for Food, Nutrition, & Health, Rutgers University, 599 Taylor Road, Piscataway, New Jersey 08854, United States ∇ Department of Chemistry, New York University, New York, New York 10003, United States ∥ Department of Chemical and Biological Engineering, Program in Applied and Computational Mathematics, Princeton University, A319 Engineering Quad, Princeton, New Jersey 08544, United States ⊥ European Commission, Joint Research Centre, Institute for Transuranium Elements, P.O. Box 2340, D-76125 Karlsruhe, Germany S Supporting Information *

ABSTRACT: Effective targeting by uniformly functionalized nanoparticles is limited to cancer cells expressing at least two copies of targeted receptors per nanoparticle footprint (approximately ≥2 × 105 receptor copies per cell); such a receptor density supports the required multivalent interaction between the neighboring receptors and the ligands from a single nanoparticle. To enable selective targeting below this receptor density, ligands on the surface of lipid vesicles were displayed in clusters that were designed to form at the acidic pH of the tumor interstitium. Vesicles with clustered HER2targeting peptides within such sticky patches (sticky vesicles) were compared to uniformly functionalized vesicles. On HER2-negative breast cancer cells MDA-MB-231 and MCF7 {expressing (8.3 ± 0.8) × 104 and (5.4 ± 0.9) × 104 HER2 copies per cell, respectively}, only the sticky vesicles exhibited detectable specific targeting (KD ≈ 49−69 nM); dissociation (0.005−0.009 min−1) and endocytosis rates (0.024−0.026 min−1) were independent of HER2 expression for these cells. MDA-MB-231 and MCF7 were killed only by sticky vesicles encapsulating doxorubicin (32− 40% viability) or α-particle emitter 225Ac (39−58% viability) and were not affected by uniformly functionalized vesicles (>80% viability). Toxicities on cardiomyocytes and normal breast cells (expressing HER2 at considerably lower but not insignificant levels) were not observed, suggesting the potential of tunable clustered ligand display for the selective killing of cancer cells with low receptor densities.

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INTRODUCTION Nanoparticle-based targeted therapeutics for treatment of cancerous tumors is a major and highly promising multidisciplinary area of research.1 However, the efficacy of reportedto-date nanoparticles to selectively and effectively target cancer cells is limited by the density of targeted marker molecules (receptors) on the surface of cancer cells.2 In this context, the term efficacy is used to indicate that the binding time of the nanoparticle on the cell surface should be long enough to allow for the particle’s internalization, thereby delivering the therapeutic cargo intracellularly close to its molecular targets. In particular, the ability of uniformly (conventionally) targeted nanoparticles to provide effective targeting is limited to cancer cells expressing at least ∼220 receptors per μm2 of cell surface area (or ∼200 000 copies of targeted receptors per cell with 8.5 μm radius).2,3 This corresponds to two receptors per nanoparticle footprint (the projected area for 100 nm-diameter nanoparticles with a 3 nm PEGylated corona).4 Such a receptor © XXXX American Chemical Society

density enables the simultaneous multivalent interaction (avidity) between the neighboring cell receptors with ligands from a single nanoparticle; such a multivalent interaction is deemed necessary for successful targeting and killing. An extensively studied cell surface marker in targeted cancer therapies is the HER2 receptor because of its expression in several types of cancers (breast, ovarian, gastric, and prostate)5 and its connection to a poor prognosis.6 For breast cancer in particular, HER2-targeted therapies are applicable to only 30% of patients who have HER2-positive tumors.7 This translates to tumors with 3+ and, in some cases, 2+ HER2 levels of expression as evaluated by immunohistochemistry (IHC) or, alternatively, tumors with more than 1 000 000 and 500 000 HER2 copies per cell, respectively.8 For the 70% of breast Received: April 18, 2016 Revised: July 16, 2016

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Figure 1. (A) pH-tunable sticky vesicles (left) and conventionally functionalized nanoparticles with pH-independent uniform distributions of targeting ligands (right). Sticky vesicles are designed so that during circulation (pH 7.4) the ligands (shown in green) are uniformly distributed over the vesicle’s surface, resulting in low reactivity. In the acidic tumor interstitium (7.0 > pH > 6.0), the functionalized lipids with the targeting ligands are designed to preferentially partition within a lipid phase-separated domain (sticky patch), resulting in high reactivity even with cells with few copies of targeted receptors. Conventionally functionalized particles with ligands uniformly distributed, either directly on the surface of the particle or associated via polymer tethers (PEG), exhibit binding reactivities that are measurable only on cells with high densities of targeted receptors and undetectable on cells with low densities. (B) Intracellular distributions and characteristic confocal fluorescence images of TNBC MDA-MB-231 uptake of HER2-targeting sticky vesicles over time at pH 6.5. Per time point, n = 10 cells were analyzed and averaged. The scale bar is 50 μm.

cancer patients who have tumors designated as HER2-negative, there are no targeted therapeutic options utilizing the HER2 receptor. The term HER2-negative refers to tumors with less than 1+ HER2 expression or pH ≥ 6.0).21,22 As demonstrated before, lipid-phase separation and lipid-domain formation of the vesicle’s lipid bilayer with lowered pH is a result of the interplay of decreasing (pH-tunable) electrostatic repulsion and attractive (pHindependent) hydrogen bonding among the domain-forming lipids.19,20 We evaluate, while varying the extracellular pH, the binding efficacy and internalization, the intracellular distributions, and the cytotoxicities of sticky vesicles containing different types of clinically relevant therapeutic agents: a chemotherapeutic (doxorubicin) and a radionuclide (actinium-225). We compare these measurements to those of vesicles uniformly functionalized with the same HER2-targeting peptide and to those of vesicles uniformly functionalized via a PEG tether with an HER2-targeting monoclonal antibody (Figure 1A). The latter vesicles are similar to most of currently pursued targeted nanoparticles. The measurements are performed on breast cancer cells expressing a variety of HER2 copies (triple negative breast cancer (TNBC) cell line MDA-MB-231, HER2-negative MCF7, and HER2-positive BT-474 cell lines) and also on normal breast cells (MCF 10A) and cardiomyocytes that B

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The vesicle size distribution was measured using the Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, U.K.). Vesicles were loaded with doxorubicin using the ammonium sulfate gradient method26 or were loaded with 225Ac using calcium ionophore A23187 (as described in detail in the Supporting Information section).27 Antibody Conjugation to Vesicles. HER2-targeting monoclonal antibody Trastuzumab was conjugated to vesicles using standard 3-(2pyridyldithio)propionate (PDP)-based and succinimidyl 4-(pmaleimidophenyl)butyrate (SMPB)-based chemistry. (Detailed methods are described in the Supporting Information section.) Nonconjugated antibodies were separated from antibody-labeled vesicles by SEC. The BCA assay was used to quantify the concentration of antibodies in the purified antibody-labeled vesicle suspension. The average number of antibodies per vesicle was then calculated on the basis of the total lipid, the headgroup surface area per lipid (48 Å2 for lipids in the gel phase), and the measured mean size of vesicles. For all targeted constructs, the immunoreactivity (extent of binding in the presence of a 50-fold excess of HER2 receptors at 4 °C) was evaluated following standard protocols.27 Cell Lines. BT-474, MDA-MB-231, and MCF7 breast carcinoma cell lines and MCF 10A normal mammary epithelial cells were purchased from ATCC (Manassas, VA). Human cardiomyocytes, derived from human cardiac tissue, were purchased from Celprogen (Torrance, CA). All cells were propagated in a humidified incubator at 37 °C and 5% CO2 and were maintained in media according to vendor instructions. The 111In-radiolabeled Trastuzumab was used to evaluate the expression of HER2 receptors per cell line using, on ice, saturation experiments of fixed concentrations of cells and increasing concentrations of Trastuzumab in the absence and presence of a 50fold excess of nonradiolabeled antibody, as described before.28 Cell Association of Vesicles as a Function of Extracellular pH. Cells were incubated with DPPE-rhodamine-labeled vesicles at different extracellular pH values for 6 h in suspension in a humidified incubator at 37 °C and 5% CO2 in a ratio of 1:10 vesicles/HER2 receptors (10 × 106 cells per mL). Upon completion of incubation, aliquots were removed from the parent suspension and cells were washed three times by centrifugation and were resuspended in PBS (pH 7.4) for the measurement of the fluorescence intensity of rhodamine (excitation/emission = 550 nm/590 nm) using a Horiba Fluorolog-3 spectrofluorometer (Horiba Scientific, Edison, NJ). Depending on the rhodamine-lipid concentration, measurements were corrected for scattering by cells or cells were lysed before measurement (described in detail in the Supporting Information section). Drug Uptake by Cells, Drug Retention by Vesicles, and Vesicle Cytotoxicity. Drug uptake by cells was measured by plating cells on six-well plates at 300 000 cells per well, followed by incubation with drug-loaded vesicles (or free doxorubicin) at pH 7.4 and 6.5 for 6 h (corresponding to the minimum circulation time of liposomes). A concentration of 17.5 μg/mL of doxorubicin and on average 0.12 mM total lipid (or radioactivity levels of 0.4 μCi/mL of 225Ac and on average 0.19 mM total lipid) at a total final volume of 1 mL per well was held constant across all wells with doxorubicin- (or 225Ac-) containing constructs. After the incubation period, cells were washed three times with sterile PBS, were trypsinized, were counted, and were either lysed to detect doxorubicin uptake (by measuring its fluorescence intensity, excitation/emission = 470 nm/592 nm) or were directly measured for radioactivity by counting the γ emissions of bismuth-213 (213Bi) decay after reaching secular equilibrium (360− 480 keV) using a Cobra γ-counter (Packard Instrument Co., Inc.).27 To quantify leaked contents from vesicles during incubation with cells, vesicles loaded with doxorubicin (or 225Ac) were collected upon completion of incubation and passed through an SEC column to separate and quantify leaked doxorubicin- (or 225Ac-DOTA) (as described in detail in the Supporting Information section). For the evaluation of cytotoxicity, in parallel experiments cells were plated on 96-well plates at 10 000 cells per well and were incubated with the same constructs at pH 7.4 and 6.5 as described above. After 6 h of incubation, cells were washed three times with sterile PBS and

express HER2 at considerably lower (but not insignificant) levels.

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EXPERIMENTAL SECTION

Materials. Lipids dihenarachidoyl-sn-glycero-3-phosphocholine (21PC), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (ammonium salt) (DSPE-PEG), 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[PDP(polyethylene glycol)-2000] (ammonium salt) (PDP-PEG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (DPPErhodamine), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamineN-(succinyl) sodium salt (DPPE-succinyl) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification (all lipids at a purity >99%). Cell culture water, HybriCare medium, and EMEM were obtained from ATCC (Masassas, VA). MEBM and MEGM SingleQuots were purchased from Lonza (Basel, Switzerland). Human cardiomyocyte complete media with serum and all reagents and materials to sustain cardiomyocyte cell cultures were obtained from Celprogen (Torrance, CA). Cholesterol (Chol), doxorubicin hydrochloride, ammonium sulfate, phosphatebuffered saline (PBS), Sepharose 4B, Sephadex G-50, Triton X-100, insulin from bovine pancreas, cholera toxin from Vibrio cholerae, sodium bicarbonate, salts, tetramethylammonium acetate (TMAA), glycine, sucrose, diethylenetriaminepentaacetic acid (DTPA), calcium ionophore A23187, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glacial acetic acid, and all reagents and solvents for the synthesis of the lipopeptide were purchased from Sigma-Aldrich Chemicals (Atlanta, GA). Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA), dithiothreitol (DTT), succinimidyl 4-(pmaleimidophenyl)butyrate (SMPB), 2-propanol (IPA), trypsin, penicillin−streptomycin, and BCA protein assay were obtained from Fisher Scientific (Pittsburgh, PA). Fetal bovine serum (FBS) was purchased from Omega Scientific (Tarzana, CA). CellTiter 96 nonradioactive cell proliferation assay (MTT) was obtained from Promega Corporation (Madison, WI). The 10DG desalting columns were purchased from BioRad (Hercules, CA). Trastuzumab (Herceptin) was a generous gift from Genentech (San Francisco, CA). Lipopeptide DPPE-(linker)-peptide (DPPE-(Gly-Ser-Gly)-Lys-Cys-Cys-Tyr-SerLeu) was prepared and analyzed for purity (>90%) as described before.23 A scrambled sequence of the targeting lipopeptide (DPPE(Gly-Ser-Gly)-Cys-Leu-Lys-Tyr-Cys-Ser, purity 80%) was prepared and analyzed by Anaspec (Fremont, CA). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and p-SCN-Bn-DOTA (DOTA-SCN) were purchased from Macrocyclics (Dallas, TX). Actinium-225 (225Ac, actinium chloride) was produced from 229Th at the Institute for Transuranium Elements, Germany, as described before.24,25 Trastuzumab was purified from Herceptin, which was a generous gift from Genentech (South San Francisco, CA). Vesicle Formation and Loading with Doxorubicin or Actinium225. Vesicles were formed using the thin film hydration method. Targeted vesicles contained the following components: sticky vesicles 21PC/DPPS/Chol/DPPE-lipopeptide/DSPE-PEG lipid in a 6.54:2.80:0.47:0.19:0.01 mol ratio, vesicles with uniform peptide functionalization DPPC/Chol/DPPE-lipopeptide/DSPE-PEG lipid in a 9.34:0.47:0.19:0.01 mol ratio, and vesicles with uniform antibodytethered functionalization DSPC/Chol/DSPE-PEG/PDP-PEG lipid in a 6.67:2.86:0.29:0.19 mol ratio. The corresponding nontargeted vesicle compositions did not contain functionalized lipids. All vesicles were labeled with 1 or 5 mol % DPPE-rhodamine lipid. For doxorubicin (or actinium-225, 225Ac)-encapsulating vesicles, the dried lipid film was hydrated in 250 mM ammonium sulfate, pH 7.4 (or in 140 mM citrate buffer with 5 mg/mL DOTA and 2.1 mg/mL ascorbic acid, pH 5.0). After extrusion in a water bath at a temperature that was 10 °C above the highest transition temperature of lipids, vesicles were passed through size-exclusion chromatography (SEC) columns equilibrated with PBS at pH 7.4 or 20 mM HEPES, 250 mM sucrose at pH 7.4. C

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Langmuir further incubated with fresh medium at 37 °C and 5% CO2, and the cell viability was quantified by the MTT assay after two population doubling times (38, 38, 48, 24, and 32 h for MDA-MB-231, MCF7, BT-474, cardiomyocytes, and MCF 10A, respectively). Imaging of Intracellular Distributions of Vesicles. Breast cancer cell suspensions (106 cells/mL) were incubated with rhodaminelabeled vesicles at 37 °C and 5% CO2 in a 10:1 ratio of vesicles/HER2 receptors. To enable clear visualization of individual cell boundaries, at different time points in the incubation, cell aliquots were removed, washed three times by centrifugation with sterile PBS, and simultaneously fixed and had their nuclei stained (as described in detail in the Supporting Information section). Washed cells were then transferred to an eight-well chambered coverglass (Lab-Tek). Fixedcell precipitates forming monolayers were imaged using a Leica TCS SP2 confocal laser scanning microscope under an oil-immersion 100× objective to determine the intracellular localization of liposomes. The fraction of vesicles as a function of cytoplasmic position was determined using an image-processing erosion algorithm that draws concentric circles (of 3 pixel thickness or of 0.9 μm thickness) from the nucleus surface outward until all intracellular fluorescence has been accounted for. Characterization of Vesicles’ Effective Equilibrium Dissociation Constants (KD), Dissociation Rate Constants (koff), and Cell Internalization Rate Constants (kint). The equilibrium dissociation of all three types of targeted vesicles was measured on suspended cells, on ice, using saturation experiments of fixed concentrations of cells and increasing concentrations of targeted fluorescent vesicles (as described in detail in the Supporting Information section) in the absence and presence of a 50-fold excess of targeted nonfluorescent vesicles at two different cell concentrations. The value of an effective KD was evaluated using the law of mass action (vesiclefree + antigenfree

We report the n and p values for each table and figure panel in detail in the corresponding item caption.

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RESULTS Lipid vesicles exhibited an average size of 106 ± 4 nm in diameter (PDI = 0.075 ± 0.009, n = 10), independent of the presence or absence of functionalization. Lipo-peptide-containing vesicles were premixed to contain, on average, 1882 lipopeptides per vesicle leaflet (assuming lipid leaflet symmetry). On average (n = 3), 31 ± 3 antibodies were conjugated per vesicle. The specific immunoreactivities (binding at conditions of excess of receptors in the absence of internalization) of sticky vesicles (all performed on the HER2overexpressing BT-474 cell line) were 1.8 ± 0.2 and 2.6 ± 0.3% at pH 7.4 and 6.5, respectively (p < 0.01). Vesicles with uniform peptide labeling exhibited specific immunoreactivity values of 2.0 ± 0.2 and 1.6 ± 0.2% at pH 7.4 and 6.5, respectively, which were independent of pH (p = 0.23). Vesicles with uniform antibody labeling also exhibited pH-independent specific immunoreactivity values of 18.2 ± 3.2 and 16.5 ± 2.7% at pH 7.4 and 6.5, respectively (p = 0.62). The (fluorescently labeled) HER2-targeting antibody exhibited pH-independent immunoreactivities of 86.3 ± 5.7 and 82.8 ± 6.7% at pH 7.4 and 6.5, respectively (p = 0.53). Blocking of the HER2 receptor with the free peptide on BT-474 cells resulted in a significant decrease in the specific binding of sticky vesicles (0.1 ± 0.4 and 0.2 ± 0.3% at pH 7.4 and 6.5, respectively). Sticky vesicles labeled with a scrambled version of the targeting peptide (CLKYCS) exhibited −0.1 ± 0.3 and 0.1 ± 0.3% specific immunoreactivity on BT-474 cells at pH 7.4 and 6.5, respectively. The extent of loading of doxorubicin in vesicles was 63.4 ± 9.1% (n = 18), and that of 225Ac in vesicles was 62.7 ± 14.6% (n = 18), resulting in 0.24 ± 0.02 mole of doxorubicin per mole of lipid and 13.4 ± 3.1 Ci per mole of lipid, respectively. Both doxorubicin (Table S1) and 225Ac (Table S2) were retained to significant extents by vesicles during the 6 h incubation period with cells. The HER2 expression per cell (HER2 copies/cell), measured by 111In-labeled Trastuzumab, was (1.3 ± 0.1) × 106 for BT474 (n = 5), (8.3 ± 0.8) × 104 for MDA-MB-231 (n = 3), (5.4 ± 0.9) × 104 for MCF7 (n = 3), (1.1 ± 0.4) × 104 for cardiomyocytes (n = 2), and (4.8 ± 3.9) × 103 for MCF 10A (n = 2). Independent of HER2 expression at the extracellular pH of 6.5, sticky vesicles (Table 1A) exhibited equilibrium dissociation constants (KD) and dissociation rate constants (koff) that did not vary by more than a factor of 2 (p > 0.01) across all breast cancer cell lines. At the extracellular pH of 7.4, the composition of sticky vesicles (which were, however, not in the phase-separated form) was not active toward breast cancer cell lines with low HER2 expression (Table 1B). Uniformly functionalized vesicles were detectable only on HER2-overexpressing BT-474 breast cancer cells and exhibited pHindependent behavior (Table 1B). The effective endocytosis rates kint of sticky vesicles at the extracellular pH of 6.5 were independent of HER2 expression for the cells studied and were similar (p > 0.01) to the kint values of uniformly functionalized vesicles. The latter, however, were detectable only on the HER2-overexpressing BT-474 cells. At the extracellular pH of 7.4, sticky vesicles (not in the phaseseparated form) and uniformly functionalized vesicles exhibited

KD = koff / kon

⇐====⇒ complex) by assuming first that the measured cell-bound vesicles formed vesicle−antigen complexes (C) (and that in complexes at least one antigen (Ag) was associated with each bound vesicle) and second that at equilibrium the number of available (free) antigens was equal to the total number of antigens (Agtotal) corrected by a factor φ per complex, where φ was evaluated by the fit (Agfree = Agtotal − φC). The value of φ was assumed to represent antigens that became effectively geometrically inaccessible upon complex formation, e.g., as a result of steric obstruction by complexed vesicles (vide infra). To measure the effective dissociation rate constants koff of targeted vesicles, cells previously incubated, on ice, with fluorescently labeled vesicles in a 1:10 ratio of vesicles/HER2 receptors to result in significant levels of cell-bound vesicles were then introduced into vesicle-free solution at t = 0, and the decreasing extent of cellassociated vesicles over time was measured on aliquots of suspended cells. Dissociation from cells of nonspecifically bound (physisorbed) vesicles was evaluated by measuring, in parallel experiments, the koff of nontargeted vesicles under identical conditions. The observed fluorescence levels of specifically cell-associated vesicles vs time were first corrected by subtraction of the corresponding fluorescence intensities as a result of nonspecific cell−vesicle interactions (measured using nontargeted vesicles) and were then fitted using a single-exponential decay in time. For kint measurements, cells were preincubated with lipid vesicles, on ice, in a 1:10 ratio of vesicles/HER2 receptors for 6 h. Upon completion of incubation, cells (10 × 106 cells per mL) were introduced at 37 °C. At regular time points up to 30 min, the cellassociated vesicles (bound and internalized) were measured, followed by measurement of the internalized vesicles that were quantified by stripping off the surface-bound vesicles. The number of surface-bound vesicles significantly decreased with time; therefore, kint was extracted from the linear slope of the data representing internalized vesicles vs the sum of surface-bound vesicles in time (ln/Sur plot) (Figure S1).29 Statistical Analysis. The results are reported as the arithmetic mean of n independent measurements ± the standard deviation. For each measurement, the Student’s t test was used to calculate significant differences in behavior (e.g., in killing efficacy) between the various constructs. p values of less than 0.05 are considered to be significant. D

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exhibited perinuclear localization that became more pronounced at longer incubation times. Cell uptake of uniformly functionalized vesicles on breast cancer cells with low HER2 expression was undetectable (Figure S2_B,C). In HER2overexpressing BT-474 cells (Figure S2_D,E), the intracellular distribution of endocytosed vesicles was similar for all targeted constructs and exhibited fast perinuclear localization of internalized vesicles. Measurable specific binding and internalization on breast cancer cells with low-HER2-expression MDA-MB-231 and MCF7 (Figure S3_A,D) was observed only for sticky vesicles (black circles) exhibiting a significant, sharp increase (approximately 50%) (p < 0.01) with pH lowering from 7.4 to 6.0. Lipid vesicles with uniform functionalization, which were designed to not form phase-separated lipid domains at any pH, did not specifically associate with these cells at any pH. On cardiomyocytes and normal MCF 10A breast cells (Figure S3_J−O), specific binding and internalization of all targeted vesicle types were insignificant. Figure 2 shows that HER2-targeting sticky vesicles (black bars) loaded with doxorubicin delivered at the extracellular pH of 6.5 (used as average pH in the tumor interstitium) more chemotherapeutic agent per cell, on low HER2-expressing cells (Figure 2A,B), than any of the vesicles with uniform functionalization. This resulted in clearly greater killing effects (32.3 ± 10.4 and 39.5 ± 5.6% viability of MDA-MB-231 and MCF7 as shown in Figure 3A,B, respectively). The doxorubicin delivered to breast cancer cells with low HER2 expression by all HER2-targeting vesicles at the extracellular pH of 7.4 (Figure 2A,B) was less than 22.3 fg per cell and did not result in a decrease in cell viability (Figure 3A,B). Doxorubicin-loaded nontargeted vesicles (indicated by the dashed lines on all figures, see Figure S4) delivered on average 17.7 ± 2.4 and 18.0 ± 1.8 fg/cell at pH 7.4 and 6.5, respectively, with minimal cytotoxicities (dashed lines in Figure 3A,B; see Figure S5). On normal cells, the toxicities induced by sticky vesicles were not significant (Figure 3D,E). In agreement with cell uptake and viability studies using doxorubicin-loaded vesicles, vesicles loaded with 225Ac demonstrated similar trends in uptake and cell viability. Figure 4 shows that HER2-targeting sticky vesicles loaded with 225Ac (black bars) delivered, in acidic pH, more radioactivity per MDA-MB-231 TNBC cell (Figure 4A) and per MCF7 breast cancer cell (Figure 4B) than any of the vesicles with uniform functionalization. This resulted in greater killing effects at the extracellular pH of 6.5 (39.6 ± 15.5% in Figure 5A and 58.0 ± 11.3% in Figure 5B, respectively; * p < 0.01). On cardiomyocytes (Figure 4D) and normal breast cells (Figure 4E), the toxicities by targeted sticky vesicles were not significant.

Table 1. Equilibrium Dissociation Constants (KD), Dissociation Rate Constants (koff), and Endocytosis (Internalization) Rate Constants (kint) at (A) pH 6.5 and (B) pH 7.4 for Sticky Vesicles and Vesicles with Uniform Functionalization for Breast Cancer Cells with Low HER2 Expression (MDA-MB-231, MCF7) and with HER2 Overexpression (BT-474)a (A)

pH 6.5 KD, nM MCF7 MDAMB231 BT-474 koff, min−1 MCF7 MDAMB231 BT-474 kint, min−1 MCF7 MDAMB231 BT-474

pH 7.4 KD, nM MCF7 MDAMB231 BT-474 koff, min−1 MCF7 MDAMB231 BT-474

sticky vesicles (heterogeneously functionalized with HER2-targeting peptides) 69 ± 24 49 ± 19 115 ± 35 (t1/2, min) 0.005 ± 0.002 (140 ± 54) 0.009 ± 0.003 (82 ± 34) 0.006 ± 0.002 (123 ± 40) (t1/2, min) 0.024 ± 0.002 (29 ± 2) 0.025 ± 0.002 (27 ± 2) 0.026 ± 0.002 (27 ± 2)

vesicles uniformly functionalized with HER2-targeting peptides

vesicles uniformly functionalized with HER2-targeting Ab via PEG tethers

undetectable undetectable

undetectable undetectable

160 ± 52

5±2

undetectableb undetectableb

undetectableb undetectableb

0.004 ± 0.001 (183 ± 35)

0.005 ± 0.001 (130 ± 25)

undetectable undetectable

undetectable undetectable

0.031 ± 0.002 (23 ± 1) (B)

0.023 ± 0.004 (30 ± 4)

sticky vesicles (uniformly functionalized with HER2-targeting peptides)

vesicles uniformly functionalized with HER2-targeting peptides

vesicles uniformly functionalized with HER2-targeting Ab via PEG tethers

undetectable undetectable

undetectable undetectable

undetectable undetectable

197 ± 11 (t1/2, min) undetectableb undetectableb

215 ± 7

3±0

undetectableb undetectableb

undetectableb undetectableb

0.004 ± 0.001 (163 ± 42)

0.005 ± 0.001 (142 ± 12)

undetectable undetectable

undetectable undetectable

0.020 ± 0.001 (34 ± 2)

0.025 ± 0.004 (29 ± 4)

0.004 ± 0.001 (172 ± 21) (t1/2, min) undetectable undetectable

kint, min−1 MCF7 MDAMB231 BT-474 0.027 ± 0.001 (25 ± 1)

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DISCUSSION Targeted cancer therapies aim to improve the therapeutic index by the selective targeting of cancer cells via specific cell membrane molecules (molecular markers) while keeping toxicity at a minimum.30 However, the therapeutic benefits of targeted cancer therapies, praised as the future of cancer therapy, are currently limited to patients with tumors expressing high levels of such molecular markers. Regarding the HER2 receptor expression, approximately 20% of all breast cancer patients have triple-negative breast cancer (TNBC) with, currently, zero options for any targeted therapy.31 Efforts to broaden the inclusion of patients eligible for targeted

a

Errors correspond to standard deviations of repeated measurements (for two independent vesicle preparations). bThe measured values were comparable to those of nontargeted (physisorbed) vesicles (0.020 ± 0.004 min−1).

similar internalization rates, which were detectable only on HER2-overexpressing BT-474 cells (Table 1B). Endocytosis by breast cancer cells with low HER2 expression was detected only for the (fluorescently labeled) sticky vesicles at extracellular pH 6.5. In both MDA-MD-231 (Figure 1B) and MCF7 (Figure S2_A) cancer cells, internalized sticky vesicles E

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Figure 2. Doxorubicin uptake of the breast cancer cell lines with low-HER2-expression MDA-MB-231 (A) and MCF7 (B), of HER2-overexpressing breast cancer cell line BT-474 (C), of cardiomyocytes (D), and of MCF 10A normal breast cells (E) following incubation with HER2-targeting sticky vesicles (black bars), vesicles uniformly functionalized with HER2-targeting peptides (gray bars), and vesicles uniformly functionalized with tethered HER2-targeting antibodies (white bars). Errors correspond to standard deviations of repeated measurements (one sample per vesicle preparation, three independent vesicle preparations). * indicates p < 0.01. The dashed line represents the average doxorubicin uptake following incubation with nontargeted vesicles across all cell lines and vesicle constructs (17.7 ± 2.4 and 18.0 ± 1.8 fg/cell at pH 7.4 and 6.5, respectively). All vesicles were loaded on average with 0.18 mg of doxorubicin per mg of lipid, and cells were incubated on average with 0.10 mg of lipid per mL.

Figure 3. Cell viabilities upon chemotherapy treatment of the breast cancer cell lines with low-HER2-expression MDA-MB-231 (A) and MCF7 (B), of HER2-overexpressing breast cancer cell line BT-474 (C), of cardiomyocytes (D), and of MCF 10A normal breast cells (E) following incubation with HER2-targeting sticky vesicles (black bars), vesicles uniformly functionalized with HER2-targeting peptides (gray bars), and vesicles uniformly functionalized with tethered HER2-targeting antibodies (white bars). Errors correspond to standard deviations of repeated measurements (three samples per vesicle preparation, three independent vesicle preparations). * indicates p < 0.01. ** indicates p < 0.05. The dashed line represents the average cell viability following incubation with nontargeted vesicles across all cell lines and vesicle constructs (90.4 ± 3.5 and 91.8 ± 2.5% at pH 7.4 and 6.5, respectively). The dotted line represents the average cell viability following incubation with an identical concentration of free doxorubicin across all cell lines and vesicle constructs (7.13 ± 2.6 and 13.4 ± 3.5% at pH 7.4 and 6.5, respectively). All vesicles were loaded on average with 0.18 mg of doxorubicin per mg of lipid, and cells were incubated on average with 0.10 mg of lipid per mL.

F

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Figure 4. Radioactivity uptake of the breast cancer cell lines with low-HER2-expression MDA-MB-231 (A) and MCF7 (B), of HER2-overexpressing breast cancer cell line BT-474 (C), of cardiomyocytes (D), and of MCF 10A normal breast cells (E) following incubation with HER2-targeting sticky vesicles (white bars), vesicles uniformly functionalized with HER2-targeting peptides (gray bars), and vesicles uniformly functionalized with tethered HER2-targeting antibodies (black bars). Errors correspond to standard deviations of repeated measurements (one sample per vesicle preparation, two independent vesicle preparations). * indicates p < 0.01. ** indicates p < 0.05. The dashed line represents the average radioactivity uptake following incubation with nontargeted vesicles across all cell lines and vesicle constructs (0.23 ± 0.05 and 0.30 ± 0.03 fCi/cell at pH 7.4 and 6.5, respectively, see Figure S6). All vesicles were loaded on average with 2.55 μCi of actinium-225 per mg of lipid, and cells were incubated with 0.16 mg of lipid per mL.

Figure 5. Cell viability upon radiation treatment of the breast cancer cell lines with low-HER2-expression MDA-MB-231 (A) and MCF7 (B), of HER2-overexpressing breast cancer cell line BT-474 (C), of cardiomyocytes (D), and of MCF 10A normal breast cells (E) following incubation with HER2-targeting sticky vesicles (white bars), vesicles uniformly functionalized with HER2-targeting peptides (gray bars), and vesicles uniformly functionalized with tethered HER2-targeting antibodies (black bars). Errors correspond to standard deviations of repeated measurements (three samples per vesicle preparation, two independent vesicle preparations). * indicates p < 0.01. ** indicates p < 0.05. The dashed line represents the average cell viability following incubation with nontargeted vesicles across all cell lines and vesicle constructs (86.6 ± 8.9 and 87.2 ± 10.6% at pH 7.4 and 6.5, respectively, see Figure S7). All vesicles were loaded on average with 2.55 μCi of actinium-225 per mg of lipid, and cells were incubated with 0.16 mg of lipid per mL.

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DOI: 10.1021/acs.langmuir.6b01464 Langmuir XXXX, XXX, XXX−XXX

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geometrically smaller. Possibly, a third receptor now has time to come by. Internalization suddenly becomes a much more probable final fate for the sticky vesicle. On cells with low HER2 expression, because the HER2 receptors on the cell surface are few and far between, we postulate that we should not expect that two receptors could be lying within the projection footprint of a near-surface vesicle at any given time. This could explain why single ligand−receptor binding events on vesicles with uniformly distributed ligands were too short-lived to keep the vesicle (subject to Brownian interactions with the solvent) bound to the cell long enough to be internalized. (The koff values for uniformly functionalized vesicles were comparable to the values of nontargeted vesicles.) As evidenced by our findings, this did not seem to be the case for the sticky vesicles. The simplest mass-action model was chosen to describe the association of vesicles with cells at equilibrium (KD). The model introduces a term φ (Experimental Section and Figure S9) that represents the extent to which surface antigens become inaccessible for further binding upon formation of a complex (a vesicle bound to the cell’s surface), especially for HER2overexpressing cancer cells. The rationale for this term is based on the expected different effective distances of the actual surface (lipid surface) of different vesicle types when complexed with antigens on cells and simply originates in the presence or absence of a PEG chain used to tether targeting ligands on vesicles (Figure S9). In particular, the distance from the cell surface of vesicles functionalized with HER2-targeting antibodies via PEG tethers is expected, upon complex formation, to be a function of (a) the PEG chain length (of 2000 MW that, when in Gaussian coil conformation, extends 3.4 nm)4 and (b) the effective length of the antibody (on the order of up to 10 nm).34 This relatively long distance may enable underlying nonbound antigens to freely diffuse away from the complexed vesicle’s footprint and become available for binding with other vesicles. In contrast, the corresponding distances for vesicles functionalized with HER2-targeting peptides directly conjugated on the lipid headgroups are expected to be significantly shorter than those listed above and, in particular, shorter than the targeting peptide’s length of 3.4 nm (9 amino acids × 0.38 nm per amino acid).35 This close approach of complexed vesicles to the cell surface may obstruct the (initially) nonspecifically bound antigens lying under the bound vesicle’s footprint from further binding with different vesicles. In our simple model of HER2-overexpressing BT-474 breast cancer cells, this suggestion is supported by the fitted values of φ that were 4 and 25 for vesicles functionalized with and without a PEG tether, respectively. The value of φ = 25 coincides with the calculated average number of antigens per vesicle footprint. On breast cancer cells with low HER2 expression (MDA-MB-231 and MCF7), for sticky vesicles, functionalized with clustered ligands without a tether, the fitted value of φ was decreased to 15, which is qualitatively in the right direction for cells with lower HER2 densities. To understand the detailed, at the molecular level, binding geometry that characterizes the interactions of sticky vesicles with the HER2 receptor(s), real-time single-particle tracking studies are arguably necessary and will be pursued. We studied how the display of HER2-targeting ligands on the surface of lipid vesicles (clustered vs uniform) may alter the binding efficacy to cells with low HER2-expression levels, and we evaluated its potential to enable the HER2-targeted therapy of such cancer cells with low HER2 densities, designated as

therapies focus on the discovery of new molecular markers (e.g., EGFR and ICAM-1)32 overexpressed by cancer cells and usually do not explore alternative targeting mechanisms. In this study, we explored the potential to introduce alternative binding geometries between ligand-functionalized vesicles and receptors. These geometries exhibited successful binding at unusually low surface densities (expression levels) at which established targeted nanoparticle-based therapies fail. We designed lipid vesicles (sticky vesicles) that introduce a different geometry for binding with HER2 receptors; the vesicles exhibit on their surface a clustered display of targeting ligands triggered to form by environmental acidification. Sticky vesicles demonstrated, at pH values corresponding to the pH of tumor interstitium, selective HER2 targeting and effective killing of (a) TNBC cells MDA-MB-231 and (b) low-HER2expressing breast cancer cells MCF7. At physiological pH, they did not significantly affect cell viability. Inhibition studies on internalization by BT-474 cells and MCF7 cells demonstrated that the endocytosis of sticky vesicles followed both the clathrin and the caveolae internalization mechanisms (Figure S8). The sticky vesicles demonstrated fast perinuclear localization upon cellular internalization, enhancing the proximity of delivered doxorubicin and of 225Ac to the nucleus, which is the target site for both agents; we suspect that this improved the efficacy of at least the delivered 225Ac.33 In addition, sticky vesicles essentially did not affect the viability of cardiomyocytes and normal breast cells, which may potentially become targets of toxicity in vivo. Although the detailed molecular mechanism of interaction between sticky vesicles and HER2 receptors is not clear at this stage, two observations support our hypotheses (vide infra). First, independent of HER2 expression, the mean duration of bound sticky vesicles on the cell surface seemed to be long enough (t1/2 of dissociation ranged from 82 to 140 min) to allow for internalization of the vesicles (t1/2 of internalization ranged from 27 to 29 min), thereby increasing the delivered dose intracellularly. Second, the levels of drug specifically delivered by sticky vesicles strongly scaled with the number density of HER2 receptors on cells at low HER2 expression levels (Table S4). (Here, “specifically” is defined as the measured drug uptake corrected for nonspecific uptake, as exhibited by nontargeted vesicles in parallel experiments.) We have two hypotheses for why the observed enhanced interactions between sticky vesicles and cells with low HER2 densities caused the sticky vesicles near the cell surface to linger longer. Our first hypothesis has to do with the cooperativity between binding and transport. As the initial bond between the ligand on a sticky vesicle and the cell receptor breaks but before the Brownian water molecule collisions drive the bulky vesicle away, the proximity of many other ligands causes a new bond to form and so on, over and over, until the vesicle either moves away from the cell or becomes internalized. This gives rise to a much lower effective unbinding constant for the overall sticky vesicle−cell interaction which, as was shown by our measurements, was not dependent on HER2 expression levels. The second hypothesis complements the first. If the residence time of the singly bound (and rebound and rebound...) vesicle becomes long enough, then a far-away second receptor may “wander nearby” and bind another one of the multitude of the sticky patch ligands, resulting in an explosive prolonging of the binding time. Although each of the two receptors may rebind when unbound, the probability of both becoming unbound long enough for the vesicle to wander away becomes H

DOI: 10.1021/acs.langmuir.6b01464 Langmuir XXXX, XXX, XXX−XXX

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(2) Hendriks, B. S.; Klinz, S. G.; Reynolds, J. G.; Espelin, C. W.; Gaddy, D. F.; Wickham, T. J. Impact of Tumor HER2/ERBB2 Expression Level on HER2-Targeted Liposomal Doxorubicin-Mediated Drug Delivery: Multiple Low-Affinity Interactions Lead to a Threshold Effect. Mol. Cancer Ther. 2013, 12, 1816−1828. (3) Ghaghada, K. B.; Saul, J.; Natarajan, J. V.; Bellamkonda, R. V.; Annapragada, A. V. Folate targeting of drug carriers: A mathematical model. J. Controlled Release 2005, 104, 113−128. (4) de Gennes, P. G. Conformations of polymers attached to an interface. Macromolecules 1980, 13, 1069−1075. (5) Tai, W.; Mahato, R.; Cheng, K. The role of HER2 in cancer therapy and targeted drug delivery. J. Controlled Release 2010, 146, 264−275. (6) 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 Subtypes, and Survival in the Carolina Breast Cancer Study. J. Am. Med. Assoc. 2006, 295, 2492−2502. (7) Gradishar, W. J. Emerging approaches for treating HER2-positive metastatic breast cancer beyond trastuzumab. Ann. Oncol. 2013, 24, 2492. (8) Ross, J. S.; Fletcher, J. A.; Linette, G. P.; Stec, J.; Clark, E.; Ayers, M.; Symmans, W. F.; Pusztai, L.; Bloom, K. J. The HER-2/neu Gene and Protein in Breast Cancer 2003: Biomarker and Target of Therapy. Oncologist 2003, 8, 307−325. (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) Ballangrud, Å.; Yang, W.-H.; Palm, S.; Enmon, R.; Borchardt, P.; Pellegrini, V.; McDevitt, M.; Scheinberg, D.; Sgouros, G. Alphaparticle Emitting Atomic Generator (Actinium-225)-Labeled Trastuzumab (Herceptin) Targeting of Breast Cancer Spheroids: Efficacy versus HER2/neu Expression. Clin. Cancer Res. 2004, 10, 4489−4497. (11) Seol, H.; Lee, H. J.; Choi, Y.; Lee, H. E.; Kim, Y. J.; Kim, J. H.; Kang, E.; Kim, S.-W.; Park, S. Y. Intratumoral heterogeneity of HER2 gene amplification in breast cancer: its clinicopathological significance. Mod. Pathol. 2012, 25, 938−948. (12) Martelotto, L. G.; Ng, C. K. Y.; Piscuoglio, S.; Weigelt, B.; ReisFilho, J. S. Breast cancer intra-tumor heterogeneity. Breast Can. Res.: BCR 2014, 16, R48. (13) Avvakumova, S.; Fezzardi, P.; Pandolfi, L.; Colombo, M.; Sansone, F.; Casnati, A.; Prosperi, D. Gold nanoparticles decorated by clustered multivalent cone-glycocalixarenes actively improve the targeting efficiency toward cancer cells. Chem. Commun. 2014, 50, 11029−11032. (14) Gray, B. P.; Li, S.; Brown, K. C. From Phage Display to Nanoparticle Delivery: Functionalizing Liposomes with Multivalent Peptides Improves Targeting to a Cancer Biomarker. Bioconjugate Chem. 2013, 24, 85−96. (15) Martin, A. L.; Li, B.; Gillies, E. R. Surface Functionalization of Nanomaterials with Dendritic Groups: Toward Enhanced Binding to Biological Targets. J. Am. Chem. Soc. 2009, 131, 734−741. (16) van Dongen, M. A.; Dougherty, C. A.; Banaszak Holl, M. M. Multivalent Polymers for Drug Delivery and Imaging: The Challenges of Conjugation. Biomacromolecules 2014, 15, 3215−3234. (17) Poon, Z.; Chen, S.; Engler, A. C.; Lee, H.-i.; Atas, E.; von Maltzahn, G.; Bhatia, S. N.; Hammond, P. T. Ligand-Clustered “Patchy” Nanoparticles for Modulated Cellular Uptake and In Vivo Tumor Targeting. Angew. Chem., Int. Ed. 2010, 49, 7266−7270. (18) Kumar, S. R.; Quinn, T. P.; Deutscher, S. L. Evaluation of an 111 In-Radiolabeled Peptide as a Targeting and Imaging Agent for ErbB2 Receptor - Expressing Breast Carcinomas. Clin. Cancer Res. 2007, 13, 6070−6079. (19) Bandekar, A.; Sofou, S. Floret-shaped solid domains on giant fluid lipid vesicles induced by pH. Langmuir 2012, 28, 4113−4122. (20) Bajagur Kempegowda, G.; Karve, S.; Bandekar, A.; Adhikari, A.; Khaimchayev, T.; Sofou, S. pH-dependent formation of lipid

HER2-negative in the clinic. At low HER2-expression levels, only vesicles with clustered-ligand display (the sticky vesicles) exhibited measurable interactions with the cells. Our measurements suggest a binding geometry that allows sticky vesicles to linger long enough on the cell surface to allow for their internalization. This is also confirmed by the observed killing selectivity of sticky vesicles against cancer cells with low HER2 expression. To potentially enable additional targeting selectivity with low toxicities in vivo, the clustered display of HER2targeting ligands on the surface of sticky vesicles was designed to be environmentally responsive (pH-responsive).

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SUMMARY AND CONCLUSIONS At the limit of low HER2-expression levels on cancer cells, where uniformly functionalized nanoparticles fail to deliver adequate therapeutic doses, lipid nanoparticles with clustered display of HER2-targeting ligands (sticky vesicles) demonstrate selectivity in targeting and efficacy in killing. This is attributed to a different binding geometry between the clustered ligands and a (possibly single) cell receptor.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b01464. Detailed methods, retention of doxorubicin and 225Ac by vesicles, ln/Sur plot, intracellular distributions of vesicles, extents of cell binding and internalization of vesicles as a function of extracellular pH, cell uptake of doxorubicin and 225Ac delivered by nontargeted vesicles, endocytosis inhibition studies, calculated ratios of drug uptake per HER2 receptor, and schematic with the geometry that is represented by the term φ (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 848-445-6568. Fax: 732-445-3753. E-mail: ss1763@rci. rutgers.edu. Present Address

(M.Z.M.) Department of Immune Modulation and Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06877, United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported in part by American Cancer Society Research Scholar Grant RSG-12-044-01, National Science Foundation Grants DMR1207022, CBET1510015, and CBET1510149, and the New Jersey Commission on Cancer Research Predoctoral Fellowship (M.S.). We especially acknowledge the High Resolution Microscopy Facility, Department of Biomedical Engineering, Rutgers University.

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DOI: 10.1021/acs.langmuir.6b01464 Langmuir XXXX, XXX, XXX−XXX