Enhancement of the Uptake and Cytotoxic Activity of Doxorubicin in

Article pubs.acs.org/molecularpharmaceutics

Enhancement of the Uptake and Cytotoxic Activity of Doxorubicin in Cancer Cells by Novel cRGD-Semipeptide-Anchoring Liposomes Lucia Battistini,† Paola Burreddu,‡ Andrea Sartori,† Daniela Arosio,§ Leonardo Manzoni,§,∇ Luigi Paduano, ∥,⊥,∇ Gerardino D’Errico, ∥,⊥ Roberto Sala,#,∇ Laura Reia,# Sabrina Bonomini,○ Gloria Rassu,‡ and Franca Zanardi*,†,∇ †

Dipartimento di Farmacia, Università degli Studi di Parma, Parma 43124, Italy Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Li Punti Sassari 07100, Italy § Istituto di Scienze e Tecnologie Molecolari, Consiglio Nazionale delle Ricerche, Milano 20133, Italy ∥ Dipartimento di Scienze Chimiche, Università degli Studi di Napoli “Federico II”, Napoli 80126, Italy ⊥ CSGI−Consorzio interuniversitario per lo sviluppo dei Sistemi a Grande Interfase, Sesto Fiorentino 50019, Italy # Dipartimento di Scienze Biomediche, Biotecnologiche e Traslazionali, Università degli Studi di Parma, Parma 43126, Italy ○ Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Parma, Parma 43126, Italy ‡

S Supporting Information *

ABSTRACT: Novel liposemipeptides hanging cyclic azabicycloalkane-RGD or aminoproline-RGD terminals were synthesized and incorporated into liposomal nanoparticles cAba/ cAmpRGD-LNP5 3C/3D. Liposomes with similar composition and lacking semipeptide conjugates were constructed for comparison (LNP, 3A), and physical encapsulation of the anticancer doxorubicin drug in both targeted and untargeted liposomes was accomplished. Microstructural analysis performed by dynamic light scattering (DLS), small-angle neutron scattering (SANS), and electron paramagnetic resonance (EPR) revealed that the conjugated nanoparticles presented an average size of 80 nm and were constituted by 5 nm thick unilamellar liposome bilayer. Flow cytometry and fluorescent microscopy studies showed that 3C-DOXO and 3D-DOXO efficiently delivered the drug into the nuclei of both quiescent and proliferating cells even in a high serum concentration environment. The uptake of doxorubicin when carried by liposomes was faster than that of the free drug, and 30 min incubation was sufficient to load cell nuclei with doxorubicin. Targeted liposomes significantly induced cell death of human breast adenocarcinoma MCF7 cells (IC50 = 144 nM, 3C-DOXO; IC50 = 274 nM, 3D-DOXO), about 2- to 6-fold more potent than free doxorubicin or 3A-DOXO controls (IC50 = 527 and 854 nM, respectively). These results suggest that cAba/cAmpRGD liposomal nanoparticles hold promise for the rapid and efficient delivery of chemotherapeutic agents to αVβ3-expressing tumor cells. KEYWORDS: drug delivery, integrin ligands, liposomes, nanoparticles, RGD semipeptides

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INTRODUCTION Over the past decade, the revolutionary potential of nanometersized functional materials has become increasingly evident, particularly in those branches of medicine involving nanoparticle (NP)-mediated biomedical imaging and therapy.1−6 In molecular oncology, nanoscopic drug delivery systems may count on high-profile advantages over conventional pharmaceutical counterparts, which include protection of the cytotoxic drug against enzymatic degradation and/or rapid systemic clearance, bypassing across otherwise impermeable biological barriers, delivery of the drug to subcellular sites, possible codelivery of multiple diverse drugs, and/or imaging tools.1−10 More importantly, the large surface area of NPs enables them to host multiple copies of highly specific targeting ligands, be they either large antibodies or peptide/peptidomimetic small molecules, allowing for “active targeting”, that is enhanced accumulation at © 2014 American Chemical Society

specific tumor districts with diminished toxicity toward off-target tissues and cells.1−10 The nanotherapeutic potential has been validated in the clinic with the FDA approval of several NPs, whose preferential spatial localization within the tumor district is not actually driven by active targeting moieties and is instead ensured by passive targeting through the known “enhanced permeation and retention” effect.11−13 A major challenge in the field was recently recognized, pointing to the manufacture of clinically translable NP therapeutics, which could merge modulable and predictable physicochemical properties as dictated by the overall structure of Received: Revised: Accepted: Published: 2280

November 28, 2013 April 16, 2014 May 13, 2014 May 13, 2014 dx.doi.org/10.1021/mp400718j | Mol. Pharmaceutics 2014, 11, 2280−2293

Molecular Pharmaceutics

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exhibiting, for PTX conjugates, very attractive in vitro and in vivo antiproliferative and antitumor profiles.21−25 As an evolution of our previous work on semipeptidecontaining covalent bioconjugates21−25 and capitalizing on wide experimentation on formulation and structural characterization of supramolecular aggregates functionalized for drug delivery,26,27 we describe here the synthesis of novel cAbaRGD- and cAmpRGD-anchoring liposemipeptides and report on their incorporation into self-assembling liposomal nanoparticles (LNPs) to be exploited as αVβ3-integrin targeted drug delivery nanovectors. Preeminent purposes of the study were (1) to use simple, robust and reproducible bioconjugation chemistry to access novel and structurally defined RGD-terminating liposemipeptides; (2) to use these liposemipeptides in the formulation of novel targeted LNPs whose functional behavior as drug carrier could be reliably interpreted on the basis of a rigorous physicochemical and microstructural characterization; (3) to analyze whether and to what extent the targeting cAba/cAmpRGD ligands and the overall liposome architecture contribute in cell uptake of the entrapped doxorubicin drug with subsequent exercise of its cytotoxic action toward αVβ3-positive endothelial and tumor cells; and (4) to check whether the chemical nature of the RGD-semipeptide (cAbaRGD and cAmpRGD) could impact the characteristics and behavior of the liposomes decorated with them. Fulfilling these objectives becomes essential for further rational development of chemically defined drug delivery vectors to be assayed in clinically relevant environments.

the nanoconstruct with the effectiveness and specificity of action as imposed by the decorating targeting ligands. Meeting this challenge is a primary goal; to date, however, only a handful of targeted NPs have progressed to clinical development, based on either polymeric or liposomal cores, while no ligand-labeled NP representatives have reached the market so far.1,9 One possible explanation for slow clinical translation lies in the inherent formulation procedure leading to targeted NPs, whose ideal requisites of viability, structure reproducibility, and scalability are as much indispensable as difficult to be pursued. Uniform, reproducible synthetic procedures as well as rigorous physicochemical characterization at the molecular level are far from being established practices, especially when self-assembled supramolecular constructs based on variably sized polymeric components are involved.14 Among the most promising and popular NP constructs, liposomes stand ahead.15 Liposomes are vesicular nanoconstructs, typically sized less than 200 nm, that are considered biocompatible, biodegradable and imperceptibly immunogenic tools, and can be usefully exploited as drug delivery systems in both passive and active modalities, provided that, in the latter case, competent pharmacophores are attached to them. It is well-known that tumor endothelial cells show increased levels of expression of several cell-surface molecules that potentiate cell proliferation, invasion, and survival during tumor vascular remodeling and angiogenesis. One such molecule is the αVβ3 integrin, whose overexpression in both tumorassociated vascular endothelial cells and in various tumor types (including glioblastoma and melanoma, breast, prostate, cervical, pancreatic, and ovarian carcinomas) renders it an eligible biomarker of the cancerous disease.16 Enlightened by the early discovery that the RGD (Arg-GlyAsp) tripeptide sequence within endogenous ligands is a crucial epitope in recognition and binding toward many integrins (including αVβ3), intense and fruitful research activity followed, which resulted in the discovery of a number of specific integrin small molecule ligands comprising either truly peptide or semipeptide/peptidomimetic representatives.17,18 We gave our contribution in the field by developing two RGD semipeptide ligand series, namely, the azabicycloalkane-based ligand generation I19 and the aminoproline-based ligand generation II,20 baptized cAbaRGD and cAmpRGD, respectively (Figure 1). A

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EXPERIMENTAL SECTION Materials. All chemicals were of the highest commercially available quality and were used as received. 2-Iminothiolane hydrochloride (Traut’s reagent, Sigma-Aldrich), 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[maleimide(poly(ethylene glycol))-2000 ammonium salt [DSPE-PEG(2000)Mal, (MW 2000), Avanti Polar Lipids, USA], 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC, Avanti Polar Lipids, USA), 1-palmitoyl-2[n-(4,4-dimethyloxazolidine-N-oxyl)]stearoyl-sn-glycero-3-phosphocholine (n-PCSL, n = 5,14; Avanti Polar Lipids, USA), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2−1,3-benzoxadiazol-4-yl) ammonium salt (NBD-PE, Avanti Polar Lipids, USA), all were >99% purity and used as received. Doxorubicin hydrochloride solution (DOXO) was purchased by Sigma-Aldrich (>99% purity). Water used for liposome preparation was obtained by Millipore Elix 3 apparatus. Cell culture medium and fetal bovine serum (FBS) were from Euroclone (Milan, Italy). Culture flasks and dishes were from Corning. Sigma-Aldrich was the source of endothelial cell growth supplement (ECGS), 4,6-diamino-2-phenylindole (DAPI), Dulbecco’s modified eagle medium (DMEM), and other chemicals. The cyclic semipeptides cAbaRGD-NH 2 (1a) 19 and cAmpRGD-NH2 (1b)20 were synthesized using standard insolution peptide synthesis according to reported procedures. Compounds 1 and 2 were purified by reverse-phase highperformance liquid chromatography (HPLC) (>95% purity); their structure was certified by 1H and 13C nuclear magnetic resonance (NMR) analyses and their exact mass confirmed by mass spectrometry. Semipreparative HPLC was carried out on an Ascentis C18 column 150 × 21.2 mm, 5 μm, and analyses were performed under the conditions specified below. NMR spectra were recorded at 300 K on a Bruker AVANCE-400 or a Bruker

Figure 1. Azabicycloalkane- and aminoproline-based cyclic RGD semipeptide ligand series.

number of representatives in both series turned out to be very potent αVβ3 and/or αVβ5 integrin binders showing ligand affinities toward isolated receptors in the low nanomolar range. Importantly, covalent conjugation of these ligands with ancillary imaging-active units (e.g., DOTA, fluorescein, Cy5.5) and chemotherapeutics (e.g., paclitaxel) resulted in the construction of bioconjugates with preserved αVβ3-binding capability while 2281

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AVANCE-600 MHz spectrometer. Chemical shifts δ are expressed in ppm relative to internal Me4Si as the standard reference. Electrospray ionization (ESI) mass spectra were recorded on Bruker Esquire 3000 Plus or API 150EX apparatus; mass spectrometric analyses were performed on a Bruker Omniflex mass spectrometer or on ABSciex MALDI TOF/ TOF 4800 Plus Analyzer in the linear mode using 3,4dihydroxybenzoic acid as the matrix. Synthesis of Liposemipeptides. Synthesis of cAbaRGDPEG-DSPE (2a). To a degassed solution of cAbaRGD-NH2 (1a) (2.6 mg, 0.0034 mmol, 1 equiv) in borate buffer (pH 8 + 5 mM EDTA, 200 μL) was added a previously degassed solution of Traut’s reagent (0.93 mg, 0.0068 mmol, 2 equiv) in borate buffer (pH 8 + 5 mM EDTA, 450 μL) under argon at room temperature. The pH was checked and adjusted to pH 8 by adding 0.2 N aq NaOH, and the reaction mixture was allowed to stir and monitored by HPLC-MS. After 40 min, DSPEPEG(2000)-Mal (5.0 mg, 0.0017 mmol, 0.5 equiv) in borate buffer (pH 8 + 5 mM EDTA/CH3CN 2:1, 600 μL) was added, and the reaction mixture was stirred under argon at room temperature monitoring by TLC (CHCl3/CH3OH/H2O 12:6:1). After 2.5 h, the solvent was removed by freeze-drying, and the crude residue was purified by HPLC. For HPLC purification, the crude residue was dissolved in MeOH/i-PrOH/ H2O + 0.2% TFA 75:5:20 (1 mL). The purification was performed on an Ascentis C18 column 150 × 21.2 mm, 5 μm; solvents: (A) H2O + 0.2% TFA, (B) i-PrOH, (C) MeOH; gradient from 20% A−5% B−75% C to 20% A−65% B−15% C over 15 min, then 20% A−65% B−15% C over 15 min; flow rate 15 mL/min, λ = 216 nm. The peak of cAbaRGD-PEG-DSPE conjugate 2a was collected between 18 and 22 min, affording liposemipeptide 2a (5.3 mg, 87%) as a white foam. The formation of 2a was confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)-MS analysis (see Supporting Information, Figure S1). Synthesis of cAmpRGD-PEG-DSPE (2b). The title liposemipeptide was synthesized according to the procedure described for 2a and using cAmpRGD-NH2 (1b) (3.6 mg, 0.0060 mmol, 1 equiv) in place of 1a. The crude material was washed with Et2O (3 × 0.5 mL) to remove the unreacted DSPEPEG phospholipid and then treated with acetone (2 × 0.5 mL) to dissolve the liposemipeptide. The supernatant was removed and evaporated in vacuo affording liposemipeptide 2b (8.0 mg, 76%) as a colorless glassy solid. The formation of cAmpRGD-PEGDSPE (2b) was confirmed by MALDI-TOF-MS analysis (see Supporting Information, Figure S1). Conjugate 2b was lyophilized for subsequent liposome incorporation. Preparation of Liposomes. Liposomes of the following compositions were used in this study: (a) liposomal nanoparticles LNP (3A) composed of POPC/NBD-PE (97:3 mol/ mol); (b) LNP-doxorubicin (3A-DOXO) composed of POPC/ NBD-PE (97:3) and incorporating doxorubicin hydrochloride; (c) cAbaRGD-LNP10 (3B) composed of POPC/NBD-PE/ cAbaRGD-PEG-DSPE (87:3:10); (d) cAbaRGD-LNP5 (3C) composed of POPC/NBD-PE/cAbaRGD-PEG-DSPE (92:3:5); (e) cAmpRGD-LNP5 (3D) composed of POPC/NBD-PE/ cAmpRGD-PEG-DSPE (92:3:5); (f) cAbaRGD-LNP5-doxorubicin (3C-DOXO) composed of POPC/NBD-PE/cAbaRGDPEG-DSPE (92:3:5) and incorporating doxorubicin hydrochloride; and (g) cAmpRGD-LNP5-doxorubicin (3D-DOXO) composed of POPC/NBD-PE/cAmpRGD-PEG-DSPE (92:3:5) and incorporating doxorubicin hydrochloride. Also, liposomes POPC, POPC/cAbaRGD-PEG-DSPE (90:10 mol/mol),

POPC/cAbaRGD-PEG-DSPE (95:5), and POPC/cAmpRGDPEG-DSPE (95:5) were prepared for dynamic light scattering (DLS) and small-angle neutron scattering (SANS) studies, whereas liposomes POPC/n-PCSL/cAbaRGD-PEG-DSPE (89:1:10), POPC/n-PCSL/cAbaRGD-PEG-DSPE (94:1:5), and POPC/n-PCSL/cAmpRGD-PEG-DSPE (94:1:5) were prepared for electron paramagnetic resonance (EPR) studies; these liposomes were not numbered for simplification purposes. Liposomes were prepared by the thin film layer method. Appropriate amounts of POPC and cAbaRGD-PEG-DSPE (2a) or cAmpRGD-PEG-DSPE (2b), at the prefixed molar ratio, were dissolved in pure chloroform in order to have a total concentration of ∼1 mg mL−1. The dissolution was favored by a slight warming (∼40 °C) and a very short sonication treatment (∼5 min). Subsequently, appropriate amounts of these solutions were transferred in round-bottom glass tubes and thoroughly mixed by vortexing. A thin film was obtained through evaporation of the solvent and vacuum desiccation. The samples were then hydrated with different media, namely, pure H2O (or D2O), a 0.9 wt % NaCl solution and, finally, a pH 7.4 buffer for miming physiological conditions. This buffer was prepared by dissolving sodium dihydrogen phosphate (NaH2PO4) and disodium hydrogen phosphate (Na2HPO4) in D2O or H2O at concentrations equal to 0.0773 mol dm−3 and 0.123 mol dm−3, respectively. The pH of this pseudophysiological solution was checked to be within 0.1 pH units by means of a Radiometer pHM220 pH-meter. All the solutions were vortexed, and the suspensions were sonicated and repeatedly extruded through polycarbonate membranes of 100 nm pore size, for at least 11 times. These liposomes were stored at 4 °C. Samples prepared for EPR experiments also included 1% (mol/mol) of spin-labeled phosphatidylcholine n-PCSL (n = 5 and 14) and stored at −20 °C in ethanol solutions. Samples prepared for fluorescence microscopy were prepared as reported above by adding 3% (mol/mol) of the molecular probe NBD-PE. Doxorubicin Loading. Doxorubicin was remote-loaded in the liposome via a citric acid/sodium citrate gradient (0.3 M) following a reported procedure.28 Briefly, to a 1 mM liposomal solution, prepared as reported above, were added 20 μL of a stock doxorubicin solution (10 mg in 2 mL of NaCl 0.9 wt % water solution) in order that its final concentration was 0.1 mM. To obtain complete doxorubicin uploading (>96 wt %), the solution was kept at 65 °C for about 20 min.28 The uploading process was monitored through fluorescence measurements; see Figure S2 in the Supporting Information. For the biological assays five kinds of samples were prepared: free doxorubicin solution, POPC/NBD-PE liposome 3A, POPC/NBD-PE/cRGD liposomes 3B, 3C, and 3D, POPC/ NBD-PE-doxorubicin liposome 3A-DOXO, and POPC/NBDPE/cRGD-doxorubicin liposomes 3C-DOXO and 3D-DOXO. In all samples the NBD-PE concentration was 3 mol %. The stability of doxorubicin-containing cRGD liposomes 3C-DOXO and 3D-DOXO was followed by DLS measurements at different times (see Figure S3 in the Supporting Information). The doxorubicin-loaded liposomes were stored at 4 °C and wrapped in aluminum foil in order to preserve them from the light during all the physicochemical characterization. Physicochemical Characterization of Liposomes. Dynamic Light Scattering (DLS). Dynamic light scattering investigations were performed with a homemade setup composed by a Photocor compact goniometer, a SMD 6000 Laser Quantum 50 mW light source operating at 5325 Å and a PMT and correlator obtained from Correlator.com. All the 2282



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measurements were performed at (25.00 ± 0.05) °C by using a thermostatic bath. Zeta potential measurements were performed with a Malvern Zetasizer Nano-ZS using the technique of laser Doppler velocimetry (LDV). The diluted samples were filled into disposable cells, and the zeta potential (ζ) was determined at least three times for each type of particle systems; all the measurements were performed at (25.0 ± 0.1) °C. Cryogenictransmission electron microscopy (cryo-TEM) images were carried out at the Heinz Maier-Leibnitz Zentrum Garching, Germany on a JEOL 200 kV JEM-FS2200 with a field emission gun (FEG). A small drop of the sample solution was applied on a copper EM grid with a holey carbon film, and excess solution was blotted with a filter paper, leaving thus a thin sample film spanning the holes in the carbon film (see Supporting Information, Figure S4). Small-Angle Neutron Scattering (SANS). Small-angle neutron scattering measurements were performed at 25 °C with the SANS 2D spectrometer sited at the Rutherford Appleton Laboratory (Chilton, U.K.). SANS 2D uses neutrons of wavelength λ ranged between 2.2 and 10 Å, which are detected by a time-of-flight analysis and recorded with a 64 cm2 2D detector placed 4.1 m from the sample.29 Such setup allows collecting data in a scattering vector modulus q = 4π/λ sin(θ/2) interval ranged between 0.006 and 0.284 Å−1, where θ is the scattering angle. Samples used for the measurements were contained in 1 mm path length Hellma quartz cells, and measurements were performed at 25 °C. The time-of-flight data were corrected for the wavelength-dependent monitor spectrum, sample transmission, and detector efficiency. Incoherent scattering was removed by subtraction of data collected for a sample of D2O in a cuvette. Any residual incoherent and background scattering from the samples was taken into account by inclusion of an additional term in all the fits performed, as discussed in detail in the following. Finally, raw data collected were radially averaged and transformed in absolute scattering cross sections dΣ/dΩ using a partially deuterated polystyrene sample as the secondary standard.30 Electron Paramagnetic Resonance (EPR). EPR spectra of liposomes containing the 5-PCSL or 14-PCSL labels as aqueous suspensions were recorded on a Elexys E-500 EPR spectrometer from Bruker (Rheinstetten, Germany) operating in the X band. Capillaries containing the samples were placed in a standard 4 mm quartz sample tube. The temperature of the sample was regulated and maintained constant at 25 °C during the measurement. The instrumental settings were as follows: sweep width, 120 G; resolution, 1024 points; modulation frequency, 100 kHz; modulation amplitude, 1.0 G; time constant, 20.5 ms; sweep time, 42 s; incident power, 5.0 mW. Several scans, typically 16, were accumulated to improve the signal-to-noise ratio. Biological Evaluation. Cell Lines and Culture Conditions. Human breast adenocarcinoma MCF7 cells, human umbilical vein endothelial cells HUVECs, and human liver hepatocellular carcinoma cells HepG2 used in this study express the αV integrins.31,32 MCF7 cells were purchased from IZSLER (Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Brescia, Italy), cultured in DMEM with 2 mM glutamine and phenol red, and supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 U/mL). HUVECs were obtained by enzymatic dissociation of human umbilical vein endothelium with 0.5% Dispase solution (0.5 U/ mg) (Dispase II, Boehringer-Mannheim Italia, Milan, Italy). Cells were routinely grown in 0.1% collagen-coated dishes in

medium 199 (Me199), supplemented with 2 mM glutamine, 20% FBS, 37.5 μg/mL endothelial cell growth supplement, and 15 U/mL heparin. Cultures consisted of homogeneous endothelial populations, as determined by typical cobblestone morphology and positivity to von Willebrand’s factor and CD31/ platelet endothelial cell adhesion molecule-1 antigens. The culture medium was always renewed 24 h before the experiments. Human liver hepatocellular carcinoma cell line HepG2 were cultured in DMEM supplemented with 10% FBS. The medium was renewed every 2−3 days. Fluorescence Microscopy. Fluorescence microscopy experiments with the liposomal suspensions were conducted on cells seeded in 4-chamber culture slides (BD Biosciences EuropeBelgium). In preparation for the experiment, cells were plated at a density of 8000 cells/cm2 in 500 μL of medium per well. When the cultures had reached 50% density, cells were incubated in fresh medium consisting of DMEM supplemented with 1% or 10% FBS with the free-doxorubicin or the different liposomal formulations to achieve final doxorubicin concentration of 1 μM. At the end of the incubation time, the cells were washed 3 times with PBS (Ca−Mg) and then fixed in 3.7% paraformaldehyde for 5 min at room temperature. Nuclei were stained with a solution of DAPI in PBS (final concentration 100 ng/mL). Fluorescence was documented with Epi-fluorescence microscope Nikon Eclipse TE300I equipped with (a) UV-2A block composed of a passband excitation filter (330−380 nm) coupled to a filter barrier Longpass (420 nm wavelength cut-on); (b) G-2A block composed of an excitation filter (510−560 nm) coupled to a filter barrier Longpass (590 nm wavelength cut-on); and (c) B-2A block composed of an excitation filter (450−490 nm) coupled to a filter barrier Longpass (520 nm wavelength cut-on). Flow Cytometry. HUVECs and MCF7 were maintained in their respective medium and incubated at 37 °C until reaching a state approaching the confluence. The cells were then enzymatically harvested and suspended in a buffer containing PBS, 2 mM EDTA, 2 g/L glucose, and FBS (1%, 10% and 20%). Aliquots of the cell suspensions were incubated with the formulation to be tested at a final concentration of 1 μM of doxorubicin. The incubation was carried out at 37 °C for 30 min, after which the tubes were centrifuged (1100 rpm for 5 min). The cells were then washed, resuspended in drug-free medium, and analyzed by flow cytometry. Signals were acquired on a FACSCanto II (Becton Dickinson) after being excited by a 488 nm laser beam, with a 530 nm emission band-pass filter, to detect the green emission of fluorescent liposome and a 585 nm band-pass filter for the red emission of doxorubicin. The analysis was performed with FACS Diva software. The dye 7-AAD was added to assess viability of the cells. At least 20,000 viable events (7-AAD negative) were acquired. For competition experiments, about 105 MCF7 cells were incubated with doxorubicin-loaded targeted and untargeted liposomes (10 μM) in the presence of 20-fold molar excess (with respect to cRGD moiety) of free α V β 3 -ligand peptide c(RGDfK)17 for 1 h at 37 °C. Cells were rinsed twice with PBS and then enzymatically detached and analyzed via cytofluorimetry. Cytotoxicity Assay. The experiments with the liposomal suspensions were conducted on cells seeded into 96-well plates. In preparation for the experiment, the cells were plated at a density of 8000 cells/cm2 in 100 μL of medium per well and grown for 24 h. The cells, after washing, were exposed to different concentrations of the test compounds (1−3000 nmol/L) for 72 h in complete medium containing 1% FBS, and the vitality was 2283



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assessed with a solution of resazurin (44 μM) in complete medium. After 2 h, fluorescence was measured at 572 nm with a fluorimeter (Wallac 1420 Victor2Multilabel Counter, PerkinElmer), and the fluorescence was recorded. The mean percentage of cell survival relative to that of untreated cells and 95% confidence interval (CI) were estimated from data from three individual experiments. Cytotoxicity was expressed as percent of living cells with respect to cell grown in control medium, and the concentration of drug formulation at which cell killing was 50% was obtained by nonlinear regression analysis using the BioDataFit Online program (http://www. changbioscience.com/stat/ec50.html). Graphs were drawn with GraphPad Prism 5.0 (Graph Pad Software Inc., San Diego, CA, USA).

reminiscent of the popular cyclopentapeptide structures such as c(RGDf[NMe]V) and c(RGDfK) integrin binders, carry with them useful primary amine termini for covalent conjugation. Thus, cAbaRGD-NH2 (1a) or cAmpRGD-NH2 (1b), in turn obtained in >95% purity from the respective amino acid components according to known procedures,19,20 were coupled to 2-iminothiolane hydrochloride (Traut’s reagent) using conventional chemistry (Scheme 1). The resulting activated thiol intermediates (not shown) were reacted in situ with commercial pegylated phospholipid DSPE-PEG(2000)-Mal, furnishing bioconjugates 2a (cAbaRGD-PEG-DSPE) and 2b (cAmpRGD-PEG-DSPE) in 87% and 76% yields, respectively. Reverse-phase HPLC purification or thorough washing with appropriate solvents consigned compounds 2a and 2b, whose integrity was confirmed by MALDI-TOF analyses. The cAbaRGD and cAmpRGD lipids 2a,b were independently used as constitutive elements in the self-assembly of targeted liposomes. Liposomal nanoparticles were manufactured by the thin film layer method following the steps described in Scheme 2. In particular, the fluorescent lipid 1,2-dimyristoyl-sn-glycero-3phosphoethanolamine-N-(7-nitro-2−1,3-benzoxadiazol-4-yl) (NBD-PE, ex/em 460/530) was incorporated into LNPs (3% mol/mol) destined to in vitro biological assays. Also, spin-labeled phosphatidylcholine (n-PCSL, n = 5 and 14) was included into the liposomal samples (1% mol/mol) used for EPR experiments (vide infra). All liposome samples were homogenized by the extrusion method, stored at 4 °C, and used within 4 months. According to DLS analysis, no variation of hydrodynamic radius was observed within 4 weeks, suggesting the absence of any degradation process (vide infra). The doxorubicin upload in liposomes was performed just before the biological evaluation tests, following the procedure reported in the experimental section. A list summarizing the composition of both untargeted and targeted LNPs, which were used for biological assays, is reported in Table S1 in the Supporting Information. Physicochemical Liposome Characterization. One of the key points of this study was the punctual physicochemical investigation of the integrin-directed liposomal nanoparticles, which was performed using an experimental strategy that proved to be extremely informative on liposome aqueous suspensions.33 This was based on the combination of three techniques, namely,

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RESULTS Synthesis. Construction of αVβ3 integrin-targeted liposomes called for the synthesis of RGD-terminating lipid monomers. To this end, we started from cyclosemipeptides 1a and 1b (Scheme 1), which contain either an azabicycloalkane or a 4-aminoproline Scheme 1. Synthesis of Liposemipeptides 2a and 2b Consisting of a Semipeptide cAbaRGD or cAmpRGD Terminus Connected to a Pegylated Phospholipid Chain

amino acid residue embedded within the key RGD tripeptide sequence.19,20 These chemically robust cyclic semipeptides,

Scheme 2. Chemical Structure of Liposome Constitutive Elements and Preparation of Targeted Liposomes 3

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Figure 2. Hydrodynamic radius distribution functions obtained at 90° by means of DLS measurements for the systems displayed in the legend.

Table 1. Physicochemical Characteristics of Liposomal Nanoparticles in This Studya

liposome

D × 108 (cm2 s−1)b

⟨ID⟩c

Rh (nm)d

RPEG (nm)e

Re (nm)f

Ri (nm)f

d (nm)g

zeta-potential (mV)h

RGD numberi

LNP cAbaRGD-LNP10 cAbaRGD-LNP5 cAmpRGD-LNP5

3.17 ± 0.02 3.15 ± 0.02 3.16 ± 0.03 3.17 ± 0.02

1.08 1.06 1.05 1.05

77 ± 1 76 ± 1 77 ± 2 77 ± 1

1.24 1.24 1.24

75.8 74.8 75.8 75.8

71.2 70.3 71.3 71.1

4.6 ± 0.5 4.5 ± 0.3 4.5 ± 0.4 4.7 ± 0.4

−4.9 ± 0.5 −4.7 ± 0.6 −4.2 ± 0.3 −4.5 ± 0.5

0 ∼20000 ∼10000 ∼10000

a

The schematic representation above the table refers to RGD-bearing LNP formulations. bDiffusion coefficients of the liposomes measured through DLS. cPolydispersity of the liposomes measured through DLS. dHydrodynamic radii of the liposomes measured through DLS. eEstimated by diffusion coefficients through Stokes−Einstein equation (ref 36). fEstimated as reported in the schematic representation above the table. gBilayer thickness measured by SANS (for details see the Supporting Information). hDetermined by Malvern Zetasizer Nano-ZS. iEstimated as described in the text.

polydispersity in size in agreement with the DLS and SANS observations (Figure S4, Supporting Information). To obtain further structural characterization on the aggregates, the neutron scattering cross sections for POPC, POPC/cAbaRGD, and POPC/cAmpRGD liposome-based systems were obtained via SANS measurements. The scattering cross sections decay at small angles, displayed in Figure 3, shows the slope dΣ/dΩ ∝ q−2, which is typical for unilamellar liposomes. The region where power law dΣ/dΩ ∝ q−2 applies is due to the layers scattering (liposome bilayer). In order to extract quantitative information, scattering cross sections were modeled as arising from collections of unilamellar spherical vesicles in which the fitting parameter was the liposome bilayer thickness (see the Supporting Information).34 As reported in Table 1, for all the systems analyzed, the thickness (d) was evaluated to be about 5 nm, and no detectable differences were revealed between the liposomes containing cAbaRGD or cAmpRGD. The physicochemical characteristics of both targeted and untargeted liposomes are summarized in Table 1. The zeta potential of the particles is about −4.4 mV, demonstrating that their surface is slightly negative and that the addition of either lipopeptide does not produce a substantial change on the surface

dynamic light scattering (DLS) to estimate liposome dimensions, small-angle neutron scattering (SANS) to analyze the aggregate morphology and to estimate the thickness of the lipid bilayer, and electron paramagnetic resonance (EPR) to investigate the dynamics of the lipid hydrophobic tail in the bilayer. In Figure 2a, the hydrodynamic radius (Rh) distribution functions as obtained through DLS measurements at 90° are reported for the aqueous dispersions of LNP, cAbaRGD-LNP5, and cAmpRGD-LNP5 liposome-based systems (fixed 95:5 POPC:cRGD mol/mol ratio). The mean hydrodynamic radii ⟨Rh⟩ for all the systems (Figure 2 and Table 1) are located in the range 70−90 nm, which is the typical range of large vesicles, with no significant difference in size between the liposomes with and without doxorubicin. Furthermore, a moderate polydispersity was observed for all the aggregates, with ⟨ID⟩ values ranging between 1.05 and 1.08. Details in calculations are reported in the Supporting Information. Interestingly, all liposome sizes fitted with those required for preferred tumor accumulation by the enhanced permeation and retention effect. The shape of the particles as observed by cryo-transmission electronic microscopy (cryo-TEM) is spherical with some 2285



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Stokes−Einstein equation applied to diffusion coefficients measured on the oxyethylene glycol series.36 On this basis, the surface occupied by the ethylene glycol (48.3 nm2) is about seven times greater than that of POPC (7.2 nm2). This suggests that both sides of the leaflet are covered by the ethylene glycol chains for more than 40%. Because of the above considerations, we judged that a 5 mol % cAbaRGD- or cAmpRGD-lipid in our formulations could represent a good compromise between the need of stealth liposomes (for possible prolonging blood circulation in future in vivo experiments) and the necessity to reduce the steric interactions on the liposome surface that in turn produce destabilization of the bilayer. As a further insight on the physicochemical features of liposomal nanoparticles, the EPR spectroscopy was utilized to obtain substantial information on the acyl chains structuring the lipid bilayers.37,38 In the present study, the samples investigated were phosphatidylcholine spin-labeled on the C5 or C14 atom of the sn-2 chain (5-PCSL and 14-PCSL, respectively) incorporated in POPC, POPC/cAbaRGD, and POPC/cAmpRGD liposomes. Both mixtures were tested at 95:5 and 90:10 mol/mol ratio. 5PCSL bears the radical label close to the molecule headgroup and consequently allows to monitor the behavior of the region of the membrane inner core closer to the polar external layers. In contrast, 14-PCSL bears the radical label close to the terminal methyl group of the acyl chain, thus allowing to monitor the behavior of the more internal region of the membrane hydrophobic core. In all the systems analyzed in the present work, the 5-PCSL spectrum presented a clearly defined axial anisotropy (Figure S5 in the Supporting Information) indicating that, in all cases, the mobility of the label in the region of the bilayer just below the hydrophilic external surface was strongly reduced. In contrast, all the 14-PCSL spectra showed an almost isotropic three-line signal, indicative of a rather free motion of the radical label.

Figure 3. Scattering cross sections obtained at 25 °C by means of SANS measurements for the systems displayed in the legend. Solid lines represent the curves obtained by the fitting procedure described in the text. For better visualization, data have been multiplied by a scale factor, indicated in the plot.

charge density. RPEG was estimated by diffusion coefficients through the Stokes−Einstein equation.35,36 According to our structural parameters determination, the liposome bilayer is constituted by about 200,000 molecules (Vbilayer ≈ 2.21 × 108 Å3; Vlipid ≈ 1148 Å3), which in turn suggests that the liposomes containing 5% cAbaRGD- or cAmpRGD-units hold about 10,000 targeting RGD molecules in the external layers. Furthermore, cAbaRGD- and cAmpRGD-terminating chains are exposed beyond the aggregate surface for about 1.2 nm due to their long oxyethylene glycol chains (about 45 units). Such value was determined from their hydrodynamic radii through the

Figure 4. Cellular uptake of doxorubicin (1 μM) delivered to HepG2 cells (hepatocellular liver carcinoma; in 1% FBS DMEM at 37 °C) as monitored by fluorescence microscopy after 3 h exposure to free DOXO, untargeted 3A-DOXO (LNP-doxorubicin), and targeted 3C-DOXO (cAbaRGD-LNP5doxorubicin) and 3D-DOXO (cAmpRGD-LNP5-doxorubicin). Original magnification 10×. The acquisitions with the same filter setting were shot with an identical shutter speed. A, phase contrast; B, cell nuclei stained with DAPI; C, B2A filter; D, G2A filter. 2286



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Figure 5. Cellular uptake of doxorubicin by cytofluorimetric analysis of MCF7 cells after 30 min incubation with 1 μM free doxorubicin or 10 μM of the indicated liposomes delivering 1 μM of doxorubicin in 1% FBS DMEM at 37 °C. Channel acquisition of PE (phycoerythrin, laser excitation 488 nm; emission, band-pass filter 585/42 and 556LP dichroic filter) to detect the presence of doxorubicin (3A = LNP, 3C = cAbaRGD-LNP5, and 3D = cAmpRGD-LNP5).

Figure 6. Cellular uptake of doxorubicin by cytofluorimetric analysis of MCF7 cells after 30 min incubation with 1 μM free doxorubicin or 10 μM of the indicated liposomes delivering 1 μM of doxorubicin in 10% FBS DMEM at 37 °C. Channel acquisition of PE (phycoerythrin, laser excitation 488 nm; emission, band-pass filter 585/42 and 556LP dichroic filter) to detect the presence of doxorubicin (3A = LNP, 3C = cAbaRGD-LNP5, and 3D = cAmpRGD-LNP5).

In an attempt to quantitatively analyze the spectra, we determined the spin-label isotropic hyperfine coupling constant, aN′ , and the order parameter, S.39 The former parameter is an index of the micropolarity experienced by the nitroxide, and, in

particular, it increases with the environmental polarity, while the latter is related to the angular amplitudes of motion of the label, which in turn reflects the motion of the acyl chain segment to which the label is bound. Their values may be calculated 2287



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Figure 7. Cellular uptake of doxorubicin (1 μM) delivered to HepG2 cells (hepatocellular liver carcinoma; in 10% FBS DMEM at 37 °C) as monitored by fluorescence microscopy after 30 min and 1, 3, and 6 h exposure to 3D-DOXO (cAmpRGD-LNP5-doxorubicin). Original magnification 20×. The acquisitions with the same filter setting were shot with an identical shutter speed. A, phase contrast; B, cells nuclei stained with DAPI; C, B2A filter; D, G2A filter; E, merge DAPI + B2A + G2A; F, merge B2A (liposomes as green dots) + G2A (doxorubicin).

incubation, no detectable qualitative differences could be noted between the two RGD liposomes 3C-DOXO and 3D-DOXO versus the unfunctionalized liposome 3A-DOXO. The ability of liposomal nanoparticles to deliver doxorubicin to αVβ3-expressing cells was supported by flow cytometric experiments. Figures 5 and 6 show the cytofluorimetric analysis of MCF7 cells in 1% and 10% serum, respectively, after 30 min incubation at 37 °C with 1 μM free doxorubicin or 10 μM of the indicated liposomes able to deliver 1 μM of doxorubicin. In both instances, irrespective of serum concentration (1% vs 10%) and the liposomal formulation (targeted vs untargeted), 99% of MCF7 were loaded with the drug when delivered by liposomes; on the contrary no uptake of doxorubicin was detected when MCF7 were incubated with the free drug. Almost the same results were observed in HUVECs (Figure S6, Supporting Information), with 94−99% delivery of the drug by liposomes within 30 min incubation (untargeted 3A-DOXO 98%, cAbaRGD-targeted 3C-DOXO 99%, and cAmpRGD-targeted 3D-DOXO 94%) as compared to less than 5% positive cells in the presence of free doxorubicin. In this instance, the increasing concentration of fetal bovine serum seems to somehow penalize delivery by cAmpRGD-liposomes, and this was more evident using 20% FBS (Figure S7, Supporting Information), with 90% and 82% positive cells for RGD-directed liposomes as compared to 0% delivery for free doxorubicin and 99% positive cells for untargeted liposomes. These data suggest that a possible competition between RGD motif and serum RGD binding proteins, i.e., fibronectin, vitronectin, and thrombospondin, may exist and that a difference in the binding affinity to HUVECs between 3C-DOXO and 3D-DOXO may occur. Cellular uptake of both 3C-DOXO and 3A-DOXO in the presence of an extra amount of c(RGDfK) peptide, a known αVβ3 integrin ligand,17 was carried out using MCF7 cells. After 1 h incubation at 37 °C, delivery of doxorubicin by 3C-DOXO was reduced by about 40% pointing to a specific αVβ3 integrin involvement in its uptake. Time-Dependent Uptake of Doxorubicin. As the steady-state concentration plays a pivotal role in the systemic toxicity of doxorubicin, we wondered whether doxorubicin could enter the cells more quickly if conveyed by liposomes. The kinetic nuclear uptake of doxorubicin delivered by 3D-DOXO was visualized by

according to the relations reported in the Supporting Information. The a′N and S values obtained from the spectra of the various liposomal formulations registered at 25 °C are collected in Table S2, Supporting Information. By comparing the results obtained for 5-PCSL and 14-PCSL it was possible to observe that both a′N and S parameters decreased with increasing the depth at which the label is inserted in the bilayer, indicating that both the environmental polarity and the acyl chains ordering decreased. Concerning variations due to the presence of cAbaRGD or cAmpRGD headgroup, it was observed that the values obtained for 5-PCSL showed that a′N increased while S slightly decreased. No significant difference between the effects of the two bioconjugated molecules was observed. The evidence indicate that both cAbaRGD and cAmpRGD facilitated water molecules penetration in the more external part of the hydrophobic inner core of lipid bilayers, i.e., just below the layer constituted by the hydrophilic headgroups. At the same time, POPC/cAbaRGD and POPC/cAmpRGD bilayers resulted to be slightly less ordered than pure POPC ones. These effects have to be connected to the cAbaRGD and cAmpRGD molecular structure, which presents (i) the negative charge due to the phosphate group close to the glycerol, in contrast to POPC, which is zwitterionic, and (ii) the ethoxylic PEG chain. Indeed, even though this last chain is expected to protrude from the bilayer structure into the external aqueous medium, the bulkiness of the coil it can form could certainly induce steric repulsion that reflects in the bilayer structuring. The a′N and S values obtained for 14-PCSL appeared to be almost insensitive to the presence of cAbaRGD and cAmpRGD, indicating the central part of the bilayer hydrophobic core remains unperturbed. Biological Evaluation. Doxorubicin Uptake. The intracellular uptake and distribution of doxorubicin for the various doxorubicin-loaded liposomal formulations were first qualitatively examined by fluorescence microscopy in human liver hepatocellular carcinoma cells (HepG2), human breast adenocarcinoma cells (MCF7), and human umbilical vein endothelium cells (HUVECs). As an illustrative example, the delivery of doxorubicin to HepG2 (1% FB serum) by cAbaRGDLNP5-doxorubicin (3C-DOXO) or cAmpRGD-LNP5-doxorubicin (3D-DOXO) versus the free drug and undirected liposomes 3A-DOXO are portrayed in Figure 4. After 3 h 2288



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Figure 8. (a) Cell cycling profile of MCF7 cells cultured in 1% FBS DMEM. The P1 gated cells are mainly a quiescent population as suggested by the analysis of the cell cycle with the propidium iodide staining; on the contrary, the P2 gated cells are mainly a proliferating population. (b) MCF7−1% FBS medium analyzed after 1 h incubation with 3C-DOXO (cAbaRGD-LNP5-doxorubicin) and 3D-DOXO (cAmpRGD-LNP5-doxorubicin) delivering 1 μM doxorubicin. Gated P1 cells are smaller cells than P2 gated cells as indicated by the scatter analysis. Both cell populations are loaded with doxorubicin.

fluorescence microscope images. Figure 7 highlights the results for doxorubicin delivery to HepG2 cells by 3D-DOXO in the presence of 10% FBS from which it was evident that (i) cellular doxorubicin uptake was time dependent and (ii) the internalized drug concentrated into the nuclei where it could be barely visible already after 30 min of incubation, while HepG2 nuclei stained clearly after 3 h (for an additional set of fluorescence microscope images, see Figures S8 and S9 in the Supporting Information). For each treatment, four photos were taken of the same region with different filters to highlight the cell structures (phase contrast image, A), the DAPI-labeled nuclei (UV-2A, B), the NBD-PE-labeled liposomes (filter B2A, C), and the red fluorescent doxorubicin (filter G2A, D). Pictures C highlight both the presence of the fluorescent probe within the liposomes (bright-green coloring) and the first emission peak of doxorubicin (550 nm). At higher magnification (e.g., Figure S8, Supporting Information) the liposomes were also barely

visible as green−yellow dots colocalizing with cells; the individual green dots in particular refer to liposomes still anchored to the cell membrane but not yet merged with it, and having not yet released the drug into the cytosol. The purple color of panel E (Figures 7 and S9, Supporting Information) results from the blue of the nuclear DAPI stain and the colocalization of the red doxorubicin fluorescence; while in panel F the green fluorescence of the liposome phospholipid merged with the red doxorubicin giving orange nuances. Inspection of Figure 7 reveals that the liposomal formulation allows for a fast delivery of the drug into the nuclei where the antiblastic function mainly takes place. These observations were also confirmed in HUVE and MCF7 cells, where the RGD-liposome clearly enhanced the delivery of doxorubicin to the cells as compared to the free drug (Figure S8a,b, Supporting Information). Delivery of Doxorubicin by RGD Liposomes is Independent from the Proliferative Status of the Cell Population. Following 2289



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cytofluorimetric analysis, it became evident that MCF7 cell line encompasses at least two cell populations differing in size, and it was possible to characterize the cycling profile of both with the propidium iodide staining (Figure 8). The smaller population (gate P1 population representing the 49.4% of the total population) was mainly a resting one since the G0/G1 fraction was 82.7% of the cell population, the S phase involved 7.4% of the cells, and the G2/M was the 9.1% of the P1 gated cells. The larger population in size (gate P2 accounting for the 35.8% of the total population) was a proliferating population since the 47% of the cells were in G0/G1 phase, the 3.8% were in S phase, the 27% were in G2/M; also a polyploid fraction was detected (Figure 8a). Interestingly, both cAbaRGD- and cAmpRGD-directed liposomes 3C-DOXO and 3D-DOXO delivered the drug to 98% of gated P1 cells and to 95% and 97% of gated P2 population (Figure 8b) suggesting that the proliferative status of the cell population does not affect the uptake of doxorubicin. Cytotoxicity Assay. The cell viability was tested with the resazurin assay, after 72 h exposure of doxorubicin-responsive MCF7 cells to increasing concentrations of doxorubicin ranging from 1 to 3000 nM either in the free form or in the several liposomal formulations (Figure 9). The doxorubicin inhibitory

Figure 10. Toxicity of the different concentrations of phospholipids (POPC), expressed as % of viability with respect to untreated cells required to deliver 1, 3, 10, 30, 100, 300, 1000, and 3000 nM of doxorubicin, respectively. Bars are the means of three independent determinations with the %SD shown. A complete lack of toxicity was evident for all the liposomal formulations tested (3A = LNP, 3C = cAbaRGD-LNP5, and 3D = cAmpRGD-LNP5).

evidence have to be collected which will be reported in due course.

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DISCUSSION The past few years have witnessed a variety of strategies involving conjugation of targeting ligands to the surface of NPs in order to provide useful molecular units for recognition and interaction toward specific tumor-related tissues and cells.1−10 In particular, a handful of αVβ3 integrin-targeting small peptides including the cyclic pentapetide c(RGDfK), disulfide-cyclized dodecapeptide RGD10, and short RGD-containing peptides and peptidomimetics were selected and anchored on the surface of liposomal NPs by either functionalization of preformed lipid vesicles or covalent incorporation into lipopeptides and subsequent selfassembly.7,17,40−48 As an example,44 doxorubicin-loaded liposomes constituted by c(RGDfK)-terminating lipopeptides have been reported as being capable of enhancing the drug uptake into melanoma cells as compared to nontargeted liposomes, while remaining less “performing” than free doxorubicin. In vitro cytotoxicity assays on the same cell types revealed strict correlation between cytotoxicity of doxorubicin formulations and intracellular drug levels, with RGD-targeted liposomes being more cytotoxic than the nontargeted counterparts and less cytotoxic than free doxorubicin. In vivo studies in mice bearing B16 melanoma showed lower blood levels and concomitant higher spleen uptake for RGD-LNPs than for LNPs, while doxorubicin accumulation in the tumor area was lower for RGD-LNPs than for LNPs. Interestingly, mice receiving RGD-LNP-doxorubicin showed marked retardation in tumor growth as compared to those treated with LNPs-doxorubicin and free drug, and this therapeutic advantage was interpreted as the result of increased doxorubicin concentration in tumor cells by integrin-mediated endocytosis process. Close inspection upon the available data, however, shows some discrepancies regarding, for example, the kinetics of drug internalization within the cells and the overall cytotoxicity and/ or antitumor efficacy of passively versus actively targeted liposomes, to such a degree to dispute whether the targeting units are really necessary for efficacious biological response.15

Figure 9. Toxicity of doxorubicin concentration delivered in MCF7 by different drug formulations expressed as % of viability with respect to untreated cells. Bars are the means of three independent determinations with the %SD shown. The IC50 for each doxorubicin formulation is indicated (3A-DOXO = LNP-doxorubicin, 3C-DOXO = cAbaRGDLNP5-doxorubicin, and 3D-DOXO = cAmpRGD-LNP5-doxorubicin).

concentration values (IC50) of the different formulations were determined. The IC50 of directed liposomes 3C-DOXO and 3DDOXO were 144 nM (95% CI, 132−157 nM) and 274 nM (95% CI, 208−360 nM), respectively, which were almost 4- and 2-fold lower than that of free doxorubicin (527 nM; 95% CI, 394−704 nM), and 6- and 3-fold lower than that of undirected liposomes (854 nM; 95% CI, 576−1267 nM). Since all liposomal formulations lacking the drug did not show any cytotoxic effect (Figure 10), the cell toxicity detected was ascribed to the sole doxorubicin drug. Thus, delivery of doxorubicin through actively targeted liposomes seems to be more effective than that of free doxorubicin and untargeted liposomes in the in vitro 72 h cytotoxicity assay, and this may be attributable to a subcellular distribution of the cAbaRGD and cAmpRGD units, which fasten the delivery of the drug to the nucleus. An in-depth investigation on this subject is needed, however, and further in vitro and in vivo 2290



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this work, the better cytotoxic performance of RGD-doxorubicin LNPs as confronted to doxorubicin-LNPs could be explained by invoking a direct participation of the cAba/cAmpRGD units in integrin-mediated targeting and delivery of doxorubicin at the subcellular level.49,50 That is, the presence of multimeric RGD ligands could affect the mechanisms of LNPs internalization, with direct implications on the intracellular fate of doxorubicin. While the actual nature of this subcellular recognition event remains unclear, further investigations are needed to clarify this point. A fourth point of discussion deals with the different chemical nature of the cAbaRGD vs cAmpRGD possibly impacting the liposomal behavior; imperceptible structural variations emerged between the two series, whereas some variations of 3C-DOXO vs 3D-DOXO occurred in the uptake by HUVECs at different serum concentrations and in the cytotoxicity assay (114 vs 274 nM). The small variations of the chemical structure of the cyclic RGD semipeptides are hardly believed to substantially impact the overall LNP presentation; however, the different binding capability of the ligands toward the αVβ3 and other integrin receptors may well be expected to impact the macroscopic behavior of the targeted liposomes toward integrin-expressing cells considering the inherent multimeric display of the nanoparticles.1−6 Consequently, the evaluation and comparison between novel and diverse targeting moieties may be a task worth being pursued when designing and optimizing novel nanosized drug delivery tools. As a last point, it was established by cytofluorimetric analysis that semipeptide-labeled liposomes in this study were able to deliver doxorubicin to MCF7 cells in both quiescent and proliferating populations with equal efficiency. It is well-known that treatment with doxorubicin is beneficial to fight proliferating solid tumors, but it is not equally effective against quiescent and still clonogenic tumor cells.53,54 It was recently demonstrated that doxorubicin delivered by pegylated liposomes in combination with hexamethylenetetramine could enhance the antitumor effect due to direct action on quiescent cells.55 In our study these experiments, though preliminary, highlight the potential of the targeted LNPs as efficient nanovectors of doxorubicin to both active and dormant tumor cells. Nontargeted doxorubicin-loaded liposomes have still entered the market with success, while only a few antibody-labeled doxorubicin-loaded LNPs have advanced to phase I clinical settings.9 Clearly, for a targeted LNP to be competitive toward nontargeted counterparts, definitive advantage in terms of antitumor efficacy has to be demanded. Here, we presented novel and reproducible liposomal nanoparticle formulations bearing two different semipeptideRGD integrin binders. Their in-depth physicochemical characterization was carried out, followed by in vitro biological evaluation toward αVβ3-expressing endothelial and tumor cells. The increased cytotoxicity of doxorubicin when delivered by RGD-labeled liposomes and the rapid, almost complete uptake of doxorubicin in both proliferating and quiescent tumor cells point to the conclusion that this class of targeted liposomes deserves further investigation toward in vivo experimentation and consequent structural and formulative refinement.

Constitutive differences, i.e., the nature/number/density of the exposed targeting units, the presence/length of pegylated stabilizing components, and the inherent phospholipid composition, and differences concerning the cytotoxic cargoes and biological models all may be responsible for nonhomogeneous biological data. The basic concept here was to exploit the αVβ3-recognizing capability of cyclic AbaRGD and AmpRGD semipeptides and transfer such ability to multimeric and nanosized presentations. The purpose was to rationalize the targeting and internalizing efficacy possibly deriving by the exposed RGD units on the basis of the physicochemical characteristics of the overall nanoparticles; with the ultimate goal to evaluate the impact of such parameters on the ability of liposomal nanoparticles to selectively deliver doxorubicin in the intracellular compartment and verify the subsequent cytotoxic function. In our work, a series of resulting points may be listed and discussed. First, the physicochemical investigation performed on the different formulations decreed the presence of unilamellar liposomes possessing mean hydrodynamic radii of about 80 and 5 nm bilayer thickness, with no dramatic microstructural variations existing between targeted and untargeted NPs. Indeed, the outer leaflet sides of LNPs carrying 5 mol % cAbaRGD- or cAmpRGD-liposemipeptides resulted in a ∼ 40% coverage by ethylene glycol chains, and this was judged a good structural compromise between the need of stealth liposomes (for possible prolonging blood circulation in future in vivo applications) and to reduce the steric interactions on the liposome surface that in turn produce destabilization of the bilayer. Moreover, evidence coming from EPR studies indicate that both cAbaRGD and cAmpRGD facilitated water molecule penetration in the more external part of the hydrophobic inner core of lipid bilayers, and at the same time, POPC/cAbaRGD and POPC/cAmpRGD bilayers resulted to be slightly less ordered than pure POPC ones. Second, fluorescence microscopy and flow cytometry showed that the RGD-targeted liposomes with their doxorubicin cargo were widely and rapidly internalized into αVβ3-expressing endothelial and cancer cell lines, with kinetics much higher than free doxorubicin. No appreciable differences in uptake efficiency, instead, were registered between targeted and untargeted liposomes, nor between cAbaRGD versus cAmpRGD liposomes. This behavior may be ascribed to different internalization modalities: while free doxorubicin can slowly enter the cells by passive diffusion mechanism, liposomes are reported to be rapidly internalized into cells by endocytosis, either by αVβ3 integrin mediation (for RGD-decorated LNPs)10,44,49,50 or by nonspecific internalization process (untargeted LNPs).51,52 The αVβ3 integrin-specific internalization for cAmpRGD-LNPs was demonstrated by competition experiments using excess of the known αVβ3 integrin ligand c(RGDfK), although involvement of other RGD-recognizing integrins cannot be ruled out. Third, an in vitro 72 h cytotoxicity assay toward human breast carcinoma cells revealed that doxorubicin displayed interesting nanomolar toxicity when delivered by RGD liposomes (144 and 274 nM), which was higher than that of doxorubicin alone (527 nM) or doxorubicin delivered by untargeted LNPs (854 nM). This result appears quite surprising since one would expect the cytotoxicity data to be consistent with the internalization evidence, with at least no significant differences to be observed between targeted and untargeted liposomes. Literature data of similar experiments report controversial results,40−48 and in any case, the cytotoxic activity is strictly related to the increased drug concentration within the cell and to internalization efficiency. In

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ASSOCIATED CONTENT

S Supporting Information *

Additional information on MALDI-TOF MS analyses, liposomal formulations, doxorubicin-loading, DLS/SANS/EPR measurements, TEM images, and doxorubicin uptake of HUVE and 2291



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(8) Gunasekera, U. A.; Pankhurst, Q. A.; Douek, M. Imaging applications of nanotechnology in cancer. Target Oncol. 2009, 4, 169− 181. (9) Zhao, P.; Astruc, D. Docetaxel nanotechnology in anticancer therapy. ChemMedChem 2012, 7, 952−972. (10) Paliwal, S. R.; Paliwal, R.; Agrawal, G. P.; Vyas, S. P. Liposomal nanomedicine for breast cancer therapy. Nanomedicine 2011, 6, 1085− 1100. (11) According to this concept, accumulation of >40 KDa/10−100 nm sized particles in the tumor is attributable to the structural features of the aberrant and leaky tumor microvasculature and impaired lymphatic drainage, which facilitate extravasation of NPs from the systemic circulation to the disordered and permeable regions of tumor vasculature while delaying their clearance. (12) Line, B. R.; Mitra, A.; Nan, A.; Ghandehari, H. Targeting tumor angiogenesis: comparison of peptides and polymer-peptide conjugates. J. Nucl. Med. 2005, 46, 1552−1560. (13) Iyer, A. K.; Khaled, G.; Fang, J.; Maeda, H. Exploiting the enhanced permeability and retention effect for tumor targeting. Drug Discovery Today 2006, 11, 812−818. (14) Elsabahy, M.; Wooley, K. L. Design of polymeric nanoparticles for biomedical delivery applications. Chem. Soc. Rev. 2012, 41, 2545−2561. (15) Allen, T. M.; Cullis, P. R. Liposomal drug delivery systems: from concepts to clinical applications. Adv. Drug Delivery Rev. 2013, 65, 36− 48. (16) Desgrosselier, J. S.; Cheresh, D. A. Integrins in cancer: biological implications and therapeutic opportunities. Nat. Rev. Cancer 2010, 10, 9−22. (17) Haubner, R.; Finsinger, D.; Kessler, H. Stereoisomeric peptide libraries and peptidomimetics for designing selective inhibitors of the αVβ3 integrin for a new cancer therapy. Angew. Chem., Int. Ed. 1997, 36, 1374−1389. (18) Auzzas, L.; Zanardi, F.; Battistini, L.; Burreddu, P.; Carta, P.; Rassu, G.; Curti, C.; Casiraghi, G. Targeting αVβ3 integrin: design and applications of mono- and multifunctional RGD-based peptides and semipeptides. Curr. Med. Chem. 2010, 17, 1255−1299. (19) Manzoni, L.; Belvisi, L.; Arosio, D.; Civera, M.; Pilkington-Miksa, M.; Potenza, D.; Caprini, A.; Araldi, E. M. V.; Monferini, E.; Mancino, M.; Podestà, F.; Scolastico, C. Cyclic RGD-containing functionalized azabicycloalkane peptides as potent integrin antagonists for tumor targeting. ChemMedChem 2009, 4, 615−632. (20) Battistini, L.; Burreddu, P.; Carta, P.; Rassu, G.; Auzzas, L.; Curti, C.; Zanardi, F.; Manzoni, L.; Araldi, E. M. V.; Scolastico, C.; Casiraghi, G. 4-Aminoproline-based arginine-glycine-aspartate integrin binders with exposed ligation points: practical in-solution synthesis, conjugation and binding affinity evaluation. Org. Biomol. Chem. 2009, 7, 4924−4935. (21) Pilkington-Miksa, M.; Arosio, D.; Battistini, L.; Belvisi, L.; De Matteo, M.; Vasile, F.; Burreddu, P.; Carta, P.; Rassu, G.; Perego, P.; Carenini, N.; Zunino, F.; De Cesare, M.; Castiglioni, V.; Scanziani, E.; Scolastico, C.; Casiraghi, G.; Zanardi, F.; Manzoni, L. Design, synthesis and biological evaluation of novel cRGD-paclitaxel conjugates for integrin-assisted drug delivery. Bioconjugate Chem. 2012, 23, 1610− 1622. (22) Lanzardo, S.; Conti, L.; Brioschi, C.; Bartolomeo, M. P.; Arosio, D.; Belvisi, L.; Manzoni, L.; Maiocchi, A.; Maisano, F.; Forni, G. A new optical imaging probe targeting αVβ3 integrin in glioblastoma xenografts. Contrast Media Mol. Imaging 2011, 6, 449−458. (23) Manzoni, L.; Belvisi, L.; Arosio, D.; Bartolomeo, M. P.; Bianchi, A.; Brioschi, C.; Buonsanti, F.; Cabella, C.; Casagrande, C.; Civera, M.; De Matteo, M.; Fugazza, L.; Lattuada, L.; Maisano, F.; Miragoli, L.; Neira, C.; Pilkington-Miksa, M.; Scolastico, C. Synthesis of Gd and (68) Ga complexes in conjugation with a conformationally optimized RGD sequence as potential MRI and PET tumor-imaging probes. ChemMedChem 2012, 7, 1084−1093. (24) Drago, C.; Arosio, D.; Casagrande, C.; Manzoni, L. Bisphosphonate-functionalized cyclic Arg-Gly-Asp peptidomimetics. Arkivoc 2013, 12, 185−200. (25) Menichetti, L.; Kusmic, C.; Panetta, D.; Arosio, D.; Petroni, D.; Matteucci, M.; Salvadori, P. A.; Casagrande, C.; L’Abbate, A.; Manzoni,

MCF7 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(F.Z.) Parco Area delle Scienze 27A, 43124 Parma, Italy. Tel: 39-0521-905067. Fax: 39-0521-905006. E-mail: franca.zanardi@ unipr.it. Author Contributions ∇

These authors (L.M., L.P., R.S. and F.Z.) contributed equally to the work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR, PRIN 2010-2011, Scientific Coordinator: Prof. C. M. A. Gennari, Protocol Number 2010NRREPL_006) and Fondazione Banco di Sardegna (596/ 2011.1481). The authors thank the Jülich Centre for Neutron Science (JCNS) for provision of beam time and SANS experiments, and Dr. Marie-Sousai Appavou for cryo-TEM images. The experiments at JCNS were supported by the European Commission, NMI3.

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ABBREVIATIONS USED DAPI, 4,6-diamino-2-phenylindole; DLS, dynamic light scattering; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; DOXO, doxorubicin; DSPE, 1,2-distearoyl-sn-glycero-3phosphoethanolamine; EDTA, ethylendiaminotetraacetic acid; EPR, electron paramagnetic resonance; FBS, fetal bovine serum; FDA, food and drug administration; HPLC, high performance liquid chromatography; Mal, maleimide; MALDI-TOF, matrixassisted laser desorption/ionization-time-of-flight; NBD-PE, 1,2dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2− 1,3-benzoxadiazol-4-yl) ammonium salt; PCSL, 1-palmitoyl-2[n(4,4-dimethyloxazoline-N-oxyl)]-stearoyl-sn-glycero-3-phosphocholine; PEG, poly(ethylene glycol); POPC, 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine; PTX, paclitaxel; RGD, arginine-glycine-aspartate; SANS, small-angle neutron scattering

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