Targeting the Urokinase Plasminogen Activator Receptor with SyntheticSelf-Assembly Nanoparticles

Ming Wang,† Dennis W. P. M. Lowik,‡ Andrew D. Miller,†,§ and Maya Thanou*,†

Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial CollegeLondon, London SW7 2AZ, United Kingdom, Department of Organic Chemistry, Institute for Molecules and Materials,Radboud University Nijmegen, Toernooiveld 1- Huygens Building 03.016, 6525 ED Nijmegen, The Netherlands, andImuThes Ltd, Flowers Building, Armstrong Road, London SW7 2AZ, United Kingdom. Received May 13, 2008;Revised Manuscript Received November 18, 2008

Targeting specific receptors is attracting growing interest in the fields of drug delivery and gene therapy forcancer treatment. The urokinase plasminogen activator receptor (uPAR) is overexpressed on many tumors,particularly that of prostate and breast cancers. The aim of this study is to design, prepare, and characterize asynthetic self-assembled nanoparticle that presents targeting ligands at a certain conformation and molar ratio onthe surface of the particles. Here, we describe the synthesis of a novel uPAR targeting ligand consisting of an11-amino-acid sequence named U11 peptide modified with an alkyl chain to form an U11 peptide-lipid amphiphile.This peptide-lipid is inserted into the outer layer of a parent stealth liposome by post-modification to derive aU11 peptide-targeted nanoparticle. We demonstrate that the peptide moieties become separated into more singularconformations as they are inserted into a liposome membrane, rendering them to be sufficiently biologicallyactive to observe specific receptor-mediated endocytosis (RME) and delivery of plasmid DNA to uPAR positivecells (DU145 cells). The U11 peptide targeted nanoparticle transfection of DU145 cells is essentially 10-foldhigher compared to transfection achieved by nanoparticles having a scrambled peptide sequence on their surface.U11 peptide targeted nanoparticles also proved to be uPAR-specific, as they did not improve transfection levelson the uPAR-negative cell line, HEK293.

INTRODUCTION

The urokinase plasminogen activator receptor (uPAR) isoverexpressed on a variety of cancer cells, such as those of theprostate and the breast (1-3). As it is the receptor’s naturalligand, the urokinase plasminogen activator (uPA) interacts withthe cognate receptor, uPAR, to form a uPAR-uPA conjugatethat enters cells by clathrin-coated, receptor-mediated endocy-tosis (4). The uPAR-uPA conjugate is subsequently involvedin stimulating various cellular activities such as extracellularmatrix invasion (5, 6), plasminogen activation (7), cell adhesion,and metastasis (8, 9). The uPA ligand is known to be acomposite structure of three independent regions: an amino-terminal growth factor domain (termed ATF or GFD; growthfactor domain), a kringle domain, and a carboxy-terminaldomain, the region in which uPA exhibits catalytic proper-ties (10, 11). Crystallographic studies of the uPAR-uPAconjugate have revealed that the binding region of uPA foruPAR is localized at the tip of a �-hairpin loop within the GFD(12), representing amino acid residues 12-32 (10, 13). Withinthis tip are two looped structures, one comprising seven aminoacid residues (U7) and one of eleven amino acid residues (U11)(Table 1). The second loop, U11, appears to be the primaryuPAR binding motif within the uPA binding tip (14, 15).Previous studies from our laboratory have determined that theinteraction of U11 peptide with uPAR is characterized by anequilibrium dissociation constant, Kd, of 1.3-1.4 µM (16).

Considering the binding affinity along with and the RMEproperties of the uPAR-uPA complex, our expectation has beenthat the uPAR-uPA receptor-ligand system could hold potentialfor the targeting of synthetic nanoparticles, bearing therapeuticnucleic acids to either prostate or breast cancer.

Previously, uPAR-targeting has been used in gene deliveryexperiments making use of whole GDF fragments (17). Incomparison with the use of antibodies, proteins, or proteinfragments as receptor-targeting moieties, peptide-based ligandshold several advantages for receptor-mediated targeting suchas increased organizational control plus the opportunity toexclude undesirable natural biological activities (18). A numberof peptide-based ligands for targeted delivery are alreadydescribed in the literature, the most prominent examples beingthe use of Rv�3/5 integrin receptor-targeting RGD familypeptides (19, 20). Other examples include R9�1 integrin receptor-targeting peptide sequences (21) and the ApoE peptide thattargets the LDL receptor (22). uPAR-specific peptides have alsobeen used for viral vector retargeting and site-specific molecularimaging (23, 24).

Here, we describe the synthesis of a simple U11 peptide-lipidamphiphile and the formulation of that peptide-lipid intonanoparticles using our laboratory’s nomenclature and codingof each functional component. The “ABCD nanoparticle”

* To whom correspondence should be addressed. E-mail: maya.thanou@imperial.ac.uk; ming.wang05@imperial.ac.uk. Phone: +44 207 5943156.Fax: +44 207 594 5803.

† Imperial College London.‡ Radboud University Nijmegen.§ ImuThes Ltd.

Table 1. Amino Acid Sequences of Binding Epitopes within uPAwith Specificity for uPARa

peptide sequence

residue 11-32 in EGF domain CDCLNGGTC-VSNKYFSNIHWCNCU7 DCLNGGTU11 VSNKYFSNIHWscrambled ISKSVYNFWNH

a U7 and U11 indicate specific fragments within the binding regionthat exhibit high receptor affinity as synthetic peptides (15).

Bioconjugate Chem. 2009, 20, 32–4032

10.1021/bc8001908 CCC: $40.75 2009 American Chemical SocietyPublished on Web 12/19/2008

paradigm has been used to define multicomponent nanoparticlesdesigned to substantially mediate nucleic acid delivery (Figure1) (25). ABCD nanoparticles comprise a therapeutic nucleic acidsuch as pDNA (A-component) condensed into AB core particlesby means of cationic and fusogenic lipids (B-component), whichare then coated by post-modification or postcoupling procedureswith variable amounts of a stealth polymer (such as poly-(ethylene glycol), lipid-PEG amphiphiles) (C-component) toprovide stability in biological fluids. Ligands (D-component)may be similarly introduced sequentially for receptor targeting.Here, we prepare a number of nanoparticles based on ABCDsystems, to identify and promote the targeting potential of suchnanoparticles when decorated with U11 peptide-lipid am-phiphile. The molar ratio of peptide-lipid in the nanoparticleis investigated with respect to their size and transfectionefficiency. Nanoparticles are tested on uPAR overexpressingDU145 prostate cancer cells and on negative uPAR HEK293(embryonic kidney) cell line as control.

MATERIAL AND METHODS

Chemical.1,2-Dioleoyl-3-sn-phosphatidylethanolamine(DOPE),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(LissamineRhodamine B sulfonyl) (DOPE-Rho) and N-[methoxy(poly-(ethylene glycol)-2000)]-1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (MeO-PEG2000-DSPE), and N-cholesteryloxycar-bonyl-3,7-diazanonane-1,9-diamine (CDAN) were purchasedfrom Avanti Polar Lipids (Alabaster, AL). 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol lipids wereobtained from Sigma and Aldrich (UK). 4-(4-Formyl-3-meth-oxyphenoxy)butyryl AM (FMPB AM) resin, O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium (HBTU), hydroxybenzot-riazole (HOBt), and all N-protected fluorenyl methyloxycarbonyl(Fmoc)-amino acids were purchased from Novabiochem (Not-tingham, UK). N,N-Diisopropylethylamine (DIPEA), trifluoro-acetic acid (TFA), triisopropylsilane (TIPS), and piperidine wereobtained from Sigma-Aldrich (Poole, UK). Dimethylformamide(DMF) and acetonitrile were purchased from Rathburn (Walker-Burn, Scotland). Centrifugal filter device was obtained fromMillipore UK.

Biological. DU145 and HEK293 cell lines were obtained fromthe ECACC (European Collection of Cell Culture, Wiltshire,

UK). Dulbecco’s Modified Eagle Media (DMEM), Optimem,fetal calf serum (FCS), penicillin and streptomycin, phosphate-buffered saline (PBS) and trypsin-EDTA were purchased fromGibco (Invitrogen, Netherlands). Luciferase reporter gene assaykit and reporter lysis buffer 5× were obtained from Promega(Southampton, UK), and BCA total protein content assay waspurchased from Pierce (Rockford, USA). Urokinase plasmino-gen activator (uPA) was obtained from American Diagnostica(CT, USA). All plasticware, including falcon tubes and tissueculture flasks, was obtained from Falcon (Becton Dickinson,UK). 4′,6′-Diamidino-2-phenyindole, dilactate (DAPI) nuclearstaining was purchased from Sigma-Aldrich (Poole, UK).

Synthesis of U11 Peptide-Lipids. FMPB AM resin (250 mg,0.74 mmol/g loading) was combined with hexadecylamine (447mg, 1.85 mmol, 10 equiv) and dissolved in DMF (50 mL).Acetic acid (105 µL, 1.85 mmol, 10 equiv) was then added tothe flask followed by NaCNBH3 (116 mg, 1.85 mmol, 10 equiv),and the reaction was stirred at 80 °C for 3 h under reflux. Theresin was then transferred into a filtered reaction vessel andwashed with CH2Cl2, MeOH, CH2Cl2, DMF, and CH2Cl2 threetimes in the respective order. Using a few beads from thereaction, the presence of a secondary amine was confirmed bythe chloranil test (26). The U11 peptide sequence was extendedby standard Fmoc-coupling strategy. All Fmoc-amino acids wereactivated with HOBt and HBTU (3 equiv) for 45 min percoupling and the Fmoc group removed at each step using 20%piperidine in DMF. The terminal amine was capped with Ac2Oand DIPEA (3 equiv) for 45 min before cleavage off the resinusing 10 mL of 95:2.5:2.5 TFA/TIS/H2O cocktail for 5 h. Thesolution was filtered, and the TFA mixture was precipitated incold ether. The precipitate was collected by filtration, followedby resolvation in H2O, and lyophilized overnight. Purificationof peptide-lipids was carried out on a Gilson semipreparativeHPLC system at a flow rate of 10 mL/min, using a Vydac C4

protein column, with a gradient of 10% to 90% acetonitrile (20min). ES mass spectroscopy of purified peptide-lipids was [M+ H]+, 1774.

Formulations of Nanoparticles. The following lipids CDAN,DOPE, and MeO-PEG2000-DSPE were dissolved in chloroformat 5 mg mL-1, 5 mg mL-1, and 1 mg mL-1, respectively.Appropriate aliquots were combined in a round-bottom flask(5 mL) and the solvent evaporated to dryness in vacuo to forma thin lipid film on the walls of the flask. For nonstealth, coreAB nanoparticles, CDAN and DOPE lipids were combined ina 1:1 (m/m) ratio. For PEGylated nanoparticles (ABC), CDAN,DOPE, and MeO-PEG2000-DSPE lipids were combined in a(50 - x/50 - x/2x m/m/m ratio where x was between 0.25 and2.5, as required). The flask was further purged with argon gasto remove final traces of organic solvent, then hydrated withdouble-distilled H2O (ddH2O) to give a lipid dispersion, whichwas then subjected to sonication for 30 min at 40 °C (final totallipid concentration, 1 mg mL-1). After sonication, resultingcationic liposome solutions were further diluted to 0.5 mg mL-1

and pDNA (0.1 mg mL-1 in ddH2O) was then added slowly bypipet under heavy vortex mixing conditions (final lipid/pDNAratio, 12:1 w/w). To prepare targeted nanoparticles (ABCD),U11 peptide-lipid (or scrambled peptide-lipid) was dissolved(1 mg mL-1 in 20% EtOH in H2O) and an appropriate aliquot(containing 1-5 mol % of peptide-lipid as required) was addedto formulations of PEGylated (ABC) or non-PEGylated (AB)nanoparticle suspension (with different mol % of MeO-PEG2000-DSPE), and the mixture was then incubated for 1 h at 37 °C.Excess peptide-lipids left uninserted into nanoparticles wereremoved by a centrifugal filtration (MWCO ) 30 kDa) andcentrifuged at 8000 g for 6 min before the redilution of thefiltrate (containing nanoparticles) with ddH2O, and the super-natant was analyzed. The amount of uninserted peptide-lipid

Figure 1. Structure of the synthetic self-assembly ABCD-type nano-particle paradigm for nonviral vector mediated nucleic acid delivery.(Left) ABCD nanoparticles are assembled from tool kits of purpose-designed chemical components. They comprise the following concentriclayers: A, nucleic acids (siRNA, miRNA, pDNA); B, lipid envelopelayer; C, stealth/biocompatibility polymer layer; and D, biologicalrecognition ligand layer. (Right) Representation of the actual pDNA-ABCD nanoparticle system formulated as part of the reported investiga-tions here.

Urokinase Plasminogen Activator Receptor Bioconjugate Chem., Vol. 20, No. 1, 2009 33

in the supernatant was determined by measuring the intrinsicamino acid fluorescence (excitation wavelength: Iex 298 nm;emission maximum: Imax 349nm) and extrapolated against aconcentration-dependent calibration curve. Components of allpurified nanoparticles were characterized by the followingmethods: Analytical HPLC (0% to 100% acetonitrile in 20 mins,flow rate 1 mL/min) was used to determine the amount ofCDAN lipids within the purified formulation, the Stewart assayfor the determination of the amount of DOPE lipids (27) andUV-visible spectroscopy for the analysis of encapsulated DNA,using an A0.1

260 value of 30.Growth and Maintenance of Cells. DU145 and HEK293 cells

were maintained in DMEM supplemented with 10% FCS (v/v)and 1% penicillin and streptomycin (DMEM +/+) in T-25 tissueculture flasks. On confluency, media was removed by aspirationand washed with 5 mL PBS. Cells were then detached fromthe flask by addition of 1.5 mL of trypsin-EDTA and incubatedfor 10 min at 37 °C and 10% CO2. After detachment, cells werediluted with 5 mL DMEM +/+ and centrifuged to removedtrypsin solution. Cells were resuspended in 5 mL DMEM +/+,diluted 1:10 with more DMEM +/+, of which 5 mL of dilutedcells were placed in a new T-25 culture flask. DU145 cells wereused up to a passage number of 30, after which they werediscarded and a new batch defrosted.

In Vitro Transfections. uPAR-overexpressing human prostatecancer cell line (DU145) was cultured with DMEM +/+ at 37°C and 5% CO2. Cells were seeded at 4 × 104 cells/well in 24well plates (0.5 mL volume per well) and incubated for 80 h(70% confluency) before experiments. For transfections, growthmedia was removed from wells by aspiration and replaced with0.5 mL of Optimem-containing delivery systems that allowed1 µg pDNA per well. Transfections were carried out for 3 hbefore transfection media was removed by aspiration andreplaced with DMEM +/+ for further cell growth. After furtherincubation at 37 °C for 48 h, cells were lysed with 80 µL of1× reporter lysis buffer per well. One cycle of freeze-thawwas carried out before cells were scraped from wells andcentrifuged at 8000 g for 2 min to separate cellular debris.Supernatants were analyzed for luciferase transgene expressionand normalized with the BCA total protein content kit.

Characterization of Nanoparticles. Nanoparticle sizes weremeasured by photon light scattering on a Coulter Delta N4 PCSplus 440SX photon correlation spectrometer (PCS). Afterassembly, all nanoparticles were diluted to 0.1 mg mL-1 inddH2O, a concentration allowing the count rate to stabilizebetween 4 and 6 × 104 counts per second. The calculatedunimodal distribution was used as the determinant for averagediameter size. For aggregation studies, the nanoparticles werediluted in the same volume of Optimem used in preparation oftransfection cocktail. The diluted nanoparticles were thenincubated at 37 °C and average particle diameter analyzed atthe time points of 0, 30, 60, 90, and 120 min post-formulation.For aggregation studies in serum, nanoparticle solutions werediluted in fetal calf serum instead of Optimem. The diametererrors were taken as the standard deviations calculated bydynamic light scattering.

Secondary Structure Determination. Circular dichroism ex-periments were conducted using the Jasco J-715 spectropola-rimeter equipped with Jasco PCT-348WI thermostat device.Targeted nanoparticles were prepared as before, by incubationof either 1 or 5 mol % U11 peptide-lipids with nakednanoparticles formulated with 0.5 mol % of MeO-PEG2000-DSPE, at a lipid concentration of 1 mg/mL. A 0.1 mm pathlength cell was used to record spectra, values of ∆A werecollected from 250 to 190 nm at an elevated temperature of 37°C. A scan rate of 10 nm/min and a step resolution of 0.2 nmwere used throughout.

Fluorescence Imaging. Six-well plates were loaded withpresterilized circular coverslips (thickness 1 mm) and cellsseeded on top at a density of 6 × 104 cells/well. After incubationat 37 °C and 5% CO2 for 80 h (70% confluency), growth mediawas removed from wells and replaced with an aliquot ofnanoparticles (50 µg) in Optimem. Nanoparticles were incubatedwith cells for 1 or 2 h, before removal by aspiration; thereafter,coverslips were washed twice with PBS. Coverslips were thentreated with paraformaldehyde per well (1 mL, 4%v/v) andincubated at 37 °C for 20 min, followed by another PBS wash(×2) and further incubation with 1 mL glycine solution (20 mgmL-1). Cells were stained with 1 mL DAPI solution (100 nM)for 5 min at 37 °C, and after one last PBS wash, mounted face-down onto a microscopy slide with 1 drop of PBS/glycerol (1:1, v/v). Images were taken on an Olympus 251 scope.

CompetitiVe Binding of Targeted Nanoparticles in thePresence of uPA. DU145 cells were seeded in 6-well platesand incubated for 80 h before 70% confluency was achieved.Cells were washed twice with cold PBS followed by incubationat 4 °C with glycine/NaCl (0.05 M/0.1 M, pH 3) for 5 min.After incubation, HEPES/NaCl (0.5 M/0.1 M, pH 7.5) wasadded to the wells before removal of all solution and washingwith PBS. Optimem-containing 0 or 40 nM uPA was appliedto the cells and incubated at 4 °C for 30 min. Rhodamine-labeledpDNA-encapsulated nanoparticles (ABCD; with U11- or scramblepeptide) were then spiked into the cell medium to formconcentrations of 25 µg lipids/well. The cells were thenincubated for a further 30 min at 4 °C before removal of mediaand washing with PBS twice. After final washing, 1 mL of PBS(2% FCS) was added to each well, and the cells were detachedfrom the wells. The cell-associated fluorescence per well wasthen analyzed by flow cytometry using a Becton DickinsonFACS 4 Color Caliber.

RESULTS

Synthesis of U11 and Control Peptide-Lipids. Aftersynthesis on solid phase, the crude U11 peptide-lipid was thenpurified to homogeneity (>95%) by high-performance liquidchromatography (HPLC). The identity of the U11 peptide-lipid(Scheme 2, Table 1) was confirmed by electrospray massspectrometry ([M + H]+, 1774.0). The scrambled (control, Table1) peptide-lipid was prepared in an equivalent manner, retainingthe U11 amino acid residue composition, but with a reorderedsequence.

Scheme 1. Structures of Main Lipids Used in Formulation:Cationic Lipid CDAN, Fusogenic Lipid DOPE, and TargetingLipid U11 Peptide-Lipid

34 Bioconjugate Chem., Vol. 20, No. 1, 2009 Wang et al.

Formulation of Nanoparticles. Nontargeted and targetednanoparticles were prepared using the pDNA construct pEGFP-Luc (SV40 promoter) expressing the luciferase gene (luc).Cationic lipid (CDAN), neutral fusogenic lipid (DOPE), MeO-PEG2000-DSPE, and synthetic U11 peptide-lipid (or controlpeptide-lipid) were added.

The CDAN/DOPE (1:1, m/m) cationic liposomes (Scheme1) were chosen for their capacity to mediate high levels oftransfections without cellular toxicity (28). Targeted, non-PEGylated nanoparticles were prepared by the method of post-modification, involving the incubation (at 37 °C, 1 h) of standardnanoparticles with an appropriate mol % (typically 1 or 5 mol%) of synthetic U11 peptide-lipid (or scrambled peptide-lipidas required). This procedure allowed the insertion of the lipidmoieties of peptide-lipids into the outer leaflet membrane ofthe AB core nanoparticles in order to create targeted nanopar-ticles (29). Removal of excess, noninserted peptide-lipids wascarried out by centrifugal filtration (MWCO ) 30 kDa,Microcon Millipore), and gratifyingly, a very high level ofpeptide-lipid insertion (>90%) was observed using fluorescenceanalysis of the filtrate. Accordingly, no further nanoparticlepurification steps were considered necessary. These purifiedparticles were characterized by HPLC and Stewart assay toverify that CDAN and DOPE compositions, respectively, wereunaltered (comparable to initial molar ratios) by the centrifuga-tion process. Peptide-lipid content was confirmed by fluores-cence spectroscopy (excitation wavelength, Iex 298 nm; emissionmaximum, Imax 349 nm) and pDNA content was quantified byUV-visible spectroscopy (using A0.1

260 value of 30).PEGylated nanoparticles (ABC) were formulated along

similar lines by the addition of pDNA to CDAN/DOPE/MeO-PEG2000-DSPE (50 - x/50 - x/2x, m/m/m; x is 0.25 to 2.5)cationic liposomes (prepared as before) with rapid vortex mixing(final lipid/pDNA ratio 12:1, w/w, final pDNA concentration0.1 mg mL-1). Selected stealth nanoparticle systems were thenincubated (at 37 °C, 1 h) with the appropriate mol % (typically1 or 5 mol %) of synthetic U11 peptide-lipid (or scramblecontrol peptide-lipid as required). Once again, this procedureallowed for the post-modification of these nanoparticle systemswith peptide-lipids and the formation of ABCD-type nano-particles (25, 29). Excess peptide-lipid was removed once againby centrifugal filtration and the composition of purified nano-particles confirmed as described above.

Secondary Structure of U11 in Nanoparticles. Circulardichroism (CD) spectroscopy has been used widely to character-ize protein or peptide secondary structures and their conforma-tional changes (30). Ligand conformation on the surface of thenanoparticle is important as this affects the binding interactions.

Using CD spectroscopy, we report the change in organizationof U11 peptide-lipids as they are inserted into CDAN/DOPE/MeO-PEG2000-DSPE (49.75/49.75/0.5, m/m/m) PEGylatedcationic liposomes (Figure 2a,b). CD spectra of the U11peptide-lipids (1 or 5 mol %) were recorded before andimmediately after incubation with nanoparticles (without pDNA)and compared with that recorded for U11 peptide-lipidsincubated in H2O only. Incubation of 1 and 5 mol %peptide-lipids in H2O alone gave rise to spectra with significantnegative maxima at ∆A216, indicating the formation of �-sheetaggregates between the peptide bonds of the peptide-lipidmolecules. The same spectral pattern was observed on theinsertion of 5 mol % peptide-lipids into liposomal solutions,suggesting that incorporation of high concentrations of peptide-lipids into liposomes still results in �-sheet aggregation, thistime most likely on the liposome surface (Figure 2a). Insertionof U11 peptide-lipid at a lower percentage, however, generateda spectrum with fewer characteristics of �-sheet formation,indicating the disruption of the �-sheet aggregates as thepeptide-lipids are inserted into the liposomal layer (Figure 2b).

Scheme 2. - Synthesis of U11 Peptide-Lipid on Solid Phasea

a (i) C16H33-NH2, HOAc, NaCNBH3, 80 °C, 3 h. (ii) (a) Extension of peptide chain using standard Fmoc-protocol with 3 equiv Fmoc-aminoacid, HBTU (3 equiv), HOBt (3 equiv) in DMF, 1 h RT per coupling. (iii) Ac2O, DiPEA in DMF, 1 h. (iv) 95/2.5/2.5 TFA/TIS/H2O, 5 h RT.

Figure 2. Circular dichroism spectra of U11 peptide-lipids alone andwhen incorporated on the surface of optimized nanoparticles (CDAN/DOPE/MeO-PEG2000-DSPE 49.75:49.75:0.5, m/m/m) (BC system). (a)Peptide-lipids incorporated at 5 mol % on nanoparticles (2) and inH2O at equivalent concentration (grayscale 9); the spectra of liposomeswithout peptide-lipids are shown for comparison (O). (b) Peptide-lipidsincorporated at 1 mol % on nanoparticles (9) and in H2O at equivalentconcentrations ((). Negative maximum at ∆A216 indicates the presenceof �-sheet aggregates.

Urokinase Plasminogen Activator Receptor Bioconjugate Chem., Vol. 20, No. 1, 2009 35

The reduction of �-sheet character on liposomal insertion couldindicate the reorganization of the peptide-lipid aggregates intomore separated chains when incorporated into the surface ofthe targeted nanoparticles.

Optimizing Mol % of U11 Peptide-Lipid. Core ABnanoparticles (non-PEGylated) and two targeted nanoparticlesystems (comprising 1 or 5 mol % of U11 peptide-lipid) wereincubated in transfection media or in 50% fetal calf serum (tomimic in vivo circulation conditions), and particle size wasobserved as a function of time. Nanoparticles with surface-inserted peptide-lipids were clearly less resistant to aggregation,although higher mole percentages of peptide-lipids lead togreater tendencies toward aggregation (Figure 3a,b). These dataare consistent with a mechanism of aggregation promoted byhydrophobic and �-sheet-forming interactions between surfacepeptide ligands of U11-decorated nanoparticles. It appeared thatcolloidal stability of such systems was also poor for transfectionand uPAR-mediated targeting studies. Using a plasmid-basedreporter gene assay, transfection studies were performed withtargeted nanoparticles, where U11-decorated nanoparticle trans-fections were found to be much less effective than correspondinglipid/pDNA nanoparticle transfections, thereby linking thedetrimental effects of nanoparticle overaggregation with poortransfection efficiency (data not shown). Accordingly, wedecided that non-PEGylated nanoparticles were not suitable forfurther transfection and that only stable nanoparticles shouldbe used for uPAR-mediated targeting studies.

Optimizing Mol % of MeO-PEG2000-DSPE in ABCDNanoparticles. A general method for alleviating propensitytoward aggregation of nanoparticles is by introducing poly(eth-ylene glycol) (PEG) units onto the surface of such nanoparticles.Nanoparticles were prepared with 1 mol % of U11 peptide-lipidin each case, plus 1-5 mol % of MeO-PEG2000-DSPE. Byincreasing the percentage of MeO-PEG2000-DSPE from 1 mol% to 5 mol %, nanoparticle stability was indeed observed intransfection media over time. However, transfection experimentson the DU145 cell (uPAR positive cell line) indicated nosubstantial difference in transfection levels achieved by PEGy-lated-targeted nanoparticles and corresponding nontargeted

nanoparticles (data not shown). The inclusion of even only 1mol % of MeO-PEG2000-DSPE appeared to render nonfunction-ality of targeted nanoparticles, presumably due to steric blockingof surface-attached U11 peptide-lipids by neighboring PEGchains. Nanoparticles stabilized with 0.5 mol % of MeO-PEG2000-DSPE (ABCD), however, were shown to resist ag-gregation in transfection media (Figure 4), and their transfectionswere clearly enhanced over corresponding nontargeted nano-particle-mediated transfections (Figure 5a) on DU145 cells.

U11 Peptide Enhanced Transfection. U11 decorated ABCDnanoparticles were formulated with 0.5 mol % of MeO-PEG2000-DSPE and either 1 or 5 mol % of peptide-lipid (U11 sequenceor scrambled control). uPAR is a common trait found in allcancers, where levels of its expression depend on the cell type(31). Transfections were performed in two cell lines, namely,uPAR positive cells (DU145) and uPAR negative cells (non-cancerous HEK293 human embryonic kidney cell line) (32).

Figure 3. Effect of insertion of 0%, 1%, or 5% U11 peptide-lipid onthe sizes of non-PEGylated nanoparticles (comprising CDAN/DOPE1:1, m/m, and pDNA, lipid/pDNA ratio 12:1, w/w) incubated in (a)Optimem and in (b) 50% FCS over 120 min at 37 °C, as measured byPCS. Non-PEGylated nanoparticles with indicated mol % of U11peptide-lipid have greater inclination for aggregation in transfectionmedia, especially systems with high peptide-lipid loading. Diametererrors are plotted as the standard deviation calculated by PCS.

Figure 4. Effect of inclusion of U11 peptide-lipid (mol % as indicated)on PEGylated nanoparticle size with time. PEGylated, nontargetednanoparticle (comprising CDAN/DOPE 1:1, m/m, and pDNA, lipid/pDNA ratio 12:1, w/w, MeO-PEG2000-DSPE, 0.5 mol %; ABC) ortargeted nanoparticles (1 or 5 mol % U11 peptide-lipid, ABCD).Nanoparticles were incubated in Optimem over 120 min at 37 °C, asmeasured by PCS. Both nontargeted and targeted (with 1 mol % U11peptide-lipid) nanoparticles were both stable in transfection media,where the diameter was maintained under 250 nm throughout incubationtime. However, higher loading with 5 mol % peptide-lipid was stillprone to aggregation. Diameter errors are plotted as the standarddeviation calculated by PCS.

Figure 5. Luciferase transfection on uPAR-positive DU145 (a) anduPAR-negative HEK293 cell lines (b) with nanoparticles (nontargeted,comprising CDAN/DOPE 1:1, m/m, and pDNA, lipid/pDNA ratio 12:1, w/w, MeO-PEG2000-DSPE, 0.5 mol %; ABC) or targeted nanopar-ticles (with 1 or 5 mol % of U11 peptide-lipid or nonspecific scrambledpeptide-lipid; ABCD). Transfection data normalized on the value ofnontargeted nanoparticle transfections. Transfection times for cells were3 h in Optimem after which cells were incubated for 48 h before lysisand analysis for luciferase activity.

36 Bioconjugate Chem., Vol. 20, No. 1, 2009 Wang et al.

ABCD nanoparticles presenting 1 mol % U11 peptide ligandmediated the highest levels of DU145 transfection, nearly 3.5-fold higher than that by nontargeted nanoparticles (Figure 5a).Nanoparticles presenting 5 mol % U11 peptide-ligand mediatedapproximately half the levels of DU145 transfection comparedto 1 mol % nanoparticles. In comparison, the highest level ofHEK293 cell transfection was mediated by nontargeted nano-particles. On these uPAR-negative cells, ABCD targeted nano-particles presenting 1 mol % U11 peptide ligand mediatedtransfection levels that were about 50% of the control nano-particle transfection level, while targeted nanoparticles present-ing 5 mol % of U11 peptide-ligand mediated very poortransfection (Figure 5b). On uPAR-negative cell lines, the U11peptide sequence becomes nonspecific for membrane receptors,hence functioning instead as a steric shield of the surface positivecharges. In addition, as with the use of PEGylated lipids,inclusion of nonspecific polymer chains into the surface ofnanoparticles results in their impaired cell uptake and transfec-tion efficiency (33). There is a clear impression with referenceto the nontargeted nanoparticle transfections that DU145 trans-fection mediated by nanoparticles presenting 1 mol % of U11scrambled peptide is on par with HEK293 transfection mediatedby nanoparticles presenting 1 mol % of U11 peptide ligand.Therefore, there appears to be a clear U11 peptide ligandmediated enhancement of approximately one log in ABCD-typenanoparticle transfection of uPAR positive cells (DU145)compared with key controls, namely, the transfection of uPARpositive cells by nanoparticles presenting 1 mol % of U11scramble peptide, and the transfection of uPAR negative cells(HEK293) using nanoparticles presenting 1 mol % of U11ligand.

For fluorescence microscopy, 1 mol % of the fluorescentprobe lipid DOPE-Rhodamine (DOPE-Rho) was included intothe optimized targeted and nontargeted nanoparticles. Afterintroduction to DU145 cells, cell-associated rhodamine fluo-rescence was examined at 1 and 2 h post-administration (Figure6). We observed that fluorescence intensity was much greaterin cells treated with ABCD targeted nanoparticles as comparedwith control ABC nanoparticles, again another indication ofreceptor-targeted delivery by uPAR-U11 recognition. Althoughthe images obtained do not allow us to attribute the increasedfluorescence intensity solely to cell internalization, we canconfidently conclude that the targeted ABCD nanoparticles arenevertheless binding more efficiently to the cell membrane thanthe nontargeted nanoparticles.

Competitive Binding of Targeted Nanoparticles in thePresence of uPA. Following analysis by fluorescence micros-copy, flow cytometery was used to assess the uptake ofrhodamine-labeled targeted nanoparticles by DU145 cells(Figure 6e). Cell-associated fluorescence generated by bindingof scramble peptide-nanoparticles was 15% lower than that ofU11-nanoparticles, indicating a decreased amount of bindingdue to the nonspecificity of the scrambled peptide sequence.The binding of such targeted nanoparticles was challenged bypreincubation of the DU145 cells with a high concentration ofuPA, to competitively block the uPAR receptors before admin-istration of the nanoparticles. The binding study was performedat 4 °C to minimize receptor-mediated endocytosis. Using freeuPA to block uPAR, the receptor to which U11 peptides arespecific, binding of U11-nanoparticles was inhibited by 15%,whereas the binding by scramble peptide-nanoparticles wasnot affected. These data again emphasize the selectivity of U11-nanoparticles for uPAR, but also indicate the high level of

background or nonspecific interactions between the nanoparticlesand cell membranes.

DISCUSSION

The main objective of this study was to identify the potentialof U11 grafting on the surface of nanoparticles for optimumuPAR binding and cell targeting. Here, the method of post-modification was chosen for the incorporation of peptide-lipidsonto the surfaces of nanoparticles (29, 34). In order to implementthis strategy, a U11 peptide-lipid was prepared using analdehyde-modified resin (FMPB resin) for the attachment of ahexadecylamine chain to the U11 peptide moiety by a reductiveamination procedure (Scheme 2) (35, 36). Initially, 1 or 5 mol% of the U11 peptide-lipid was inserted into nanoparticleslacking a stealth/biocompatibility polymer. The most strikingaspect of the physicochemical behavior of these nanoparticleswas their propensity to aggregate through nonspecific peptide-peptide interactions. (Figure 3a,b). Nanoparticles without PE-Gylation exhibited immediate aggregation in transfection media,where diameters of over >500 nm were recorded after 30-60min incubation at 37 °C, a more than adequate demonstrationof the need for a stealth polymer such as PEG. However, theoverloading of such a sterically hindering polymer-lipid canhave deleterious effects on the targeting ability of the nanopar-ticle, caused by shielding of the targeting peptides by the

Figure 6. Uptake and binding of rhodamine-labeled optimized nano-particles. For fluorescence microscopy, DU145 cells were incubatedwith nontargeted, ABC (a,b) and targeted ABCD nanoparticles (1 mol% U11) for 1 h (a,c) and 2 h (b,d) before fixing and visualizing. Imageswere overlaid with DAPI-nuclear staining. At 1 h, the cell-associatedfluorescence appears similar in cells treated with targeted (c) andnontargeted nanoparticles (a). The difference in fluorescence is moreapparent at 2 h, where cells treated with uPAR-targeted nanoparticles(d) show increased internal rhodamine intensity, compared to corre-sponding control nontargeted nanoparticles (b), an indication of eitherincreased membrane binding or cellular internalization. Competitivebinding of rhodamine-labeled nanoparticles with free uPA, assessedby flow cytometry (e), shows the lower cell binding by scramble peptidedecorated nanoparticles, and demonstrates U11 specificity for uPARby reduced cell binding of U11 nanoparticles after blocking of uPARreceptors with 40uM uPA.

Urokinase Plasminogen Activator Receptor Bioconjugate Chem., Vol. 20, No. 1, 2009 37

polymer chains. The difficulty therefore lies within striking abalance between the molar percentages of the stealth lipid andthe targeting peptide-lipid.

In this study, we also investigated the properties of theassembled nanoparticles and the organization of peptide target-ing ligands on their surface. This poses as the first stage ofdevelopment of nanoparticles for uPAR targeting and prostatecancer drug/gene therapies. For cancer targeting, nanoparticlesrequire a diameter less than 200 nm to bypass the fenestrationfound in leaky tumor vasculature and between the tumorendothelium (37, 38). Accordingly, in the preparation of ABCD-type nanoparticles, the amount of stealth/biocompatible polymer(C-component) attached to the surface of AB core particlesshould be sufficient to prevent particle aggregation in all relevantbiological fluids, but not so overburdening as to obscure thebenefits of including a biological targeting ligand. Long chainsof linear PEG units are intended to create a spherical, stericbarrier around pDNA/lipid core nanoparticles, reducing thetendency for particle aggregation through the generation of stericrepulsion (39). The steric effect provided by PEGylation alsomasks the lipophilic nature of the core AB nanoparticle andprovides stealth properties against immune surveillance andserum aggregation, essential for in vivo applications. However,PEG at higher surface densities (>1 mol %) may interfere withthe presentation of the ligand, reducing molecular recognitionby the cognate receptor. This is in agreement with ourtransfection results using ABCD nanoparticles formulated withincreasing percentages of MeO-PEG2000-DSPE (data not shown).Much of our nanoparticle study described here was designedto find the most appropriate balance between incorporatedcomponents for the optimization of both stability and functionaldelivery of nucleic acids. In this event, this was achieved byincluding 0.5 mol % of MeO-PEG2000-DSPE and 1 mol % ofU11 peptide-lipid (Figures 4 and 5).

Nanoparticle integrity also depends on the balance of surfaceligands. Similarly, as we observed, the inclusion of biologicaltargeting ligands at higher surface densities (>1 mol %) caninduce nanoparticle aggregation and interligand �-sheet ag-gregation (Figure 2a), without furnishing any benefits fortransfection (Figure 3). Indeed, other reported studies on ligandloading have shown that lower loading rates of RGD peptide-lipid (<1 mol %) on DSPC vesicles did not reduce the bindingeffects of the ligand, nor was there reduction in the ability ofRGD moieties to induce cell adhesions on HUVEC (humanumbilicord endothelial) cells (40). Concurring with our data, itis believed that the physicochemical properties of the peptide(size, hydrophilicity) lead to particle aggregation on excessiveloading.

The incorporation of peptide-lipids into lipid membranes forpresentation of peptide ligands has been used widely in theliterature (41-45), although few studies consider the effect ofthe anchoring procedure on the secondary structure of theligand (36, 46). In our case, we investigated the effect ofintroducing peptide-lipids at high and lower molar percentagesinto the outer leaflet membrane of PEGylated cationic liposomes(Figure 2). The CD data clearly indicate that �-sheet, secondarystructures are forming when the peptide-lipids are dissolvedin aqueous solution (47, 48). However, when they are insertedat low concentrations (1 mol %), the same peptide-lipids appearto rearrange into more separated structures, as indicated by thereduction in ∆A216 in the CD spectrum. A targeted nanoparticlewith surface ligands that are more separated into individualstrands is more favorable for receptor binding. This phenomenonis only observed with peptide-lipids at low molar percentages,as the introduction of peptide-lipids into liposomes at 5 mol% showed �-sheet aggregation to the same extent as thatobserved in aqueous solution alone. This again highlights the

advantages of incorporation of peptide-lipids at lower concen-trations, where interparticle and intermolecular aggregation isprevented. Nanoparticle transfections using this formulationwere clearly superior to all control transfections (includingtransfections with nanoparticles prepared from scramble controlpeptide-lipid) (Figure 5a), thereby suggesting that the U11peptide-lipids were adopting appropriately active conformationsto enable functional receptor mediated uptake of nanoparticleswhere uPAR were presented. Further evidence for receptor-mediated uptake in uPAR positive cells DU145 was obtainedby fluorescence microscopy (Figure 6).

Previous work on ABCD nanoparticles in our laboratory hadfocused on the development of a R9�1-integrin-targetingpeptide-lipid for incorporation into nanoparticles in order toinduce receptor-mediated uptake into appropriate cell linesfollowed by efficient transfection (21). Inclusion of thepeptide-lipid into nanoparticles did indeed result in one logincrease in nanoparticle-mediated transfection of a R9�1-overexpressing cell line (in comparison with nanoparticletransfection without peptide-lipid). However, this enhancedtransfection was found to be the result of nonspecific enhancedcellular uptake processes, as inclusion of the same ABCDnanoparticles gave the same increase in transfection of a cellline without R9�1-integrin expression (in comparison withnontargeted nanoparticle transfection). Gratifyingly, the studieswith U11-nanoparticle-mediated transfection and fluorescencemicroscopy data (Figures 5 and 6) do indicate the activeinvolvement of receptor-mediated cell uptake processes and U11ligand specificity for uPAR receptor. Uptake studies in DU145cells further demonstrate the receptor specificity of the U11peptide, where the binding of U11-targeted nanoparticles wasshown to be reduced when the uPAR receptors are preblockedwith high concentrations of free uPA ligand (Figure 6e). Flowcytometry showed that the binding of nanoparticles decoratedwith scramble peptides was 15% lower than that by U11-targetednanoparticles, plus its uptake in uPAR-overexpressed cells wasnot affected by uPAR blocking. However, the maximumreduction in U11-nanoparticle binding by uPAR blocking was15%, indicating the presence of strong nonspecific interactionsthat cause the binding of the nanoparticles to the cell membranes.

The challenge of nonspecific cell interactions is also empha-sized by the relatively low transfection enhancement (3.5-fold)between transfections mediated by our leading U11-targetedABCD nanoparticles and the corresponding control nanoparticles(Figure 5a). The reason for this may well lie in the high overallcationic charge of the nanoparticle systems. Zeta potentials werefound to be on the order of +112 mV, a consequence of thehigh lipid/pDNA ratio of 12:1 (w/w) that corresponds to an N/Pratio of 4.25, assuming that each cytofectin CDAN presents anet charge of +1.7 at neutral pH (49). Cell membranes aretypically anionic, owing in part to negatively charged cell surfaceproteoglycans, with which cationic nanoparticles may interactin a nonspecific manner to trigger cellular uptake by endocytosiswithout the requirement for receptor mediation (50). Therefore,the discrimination between ABCD and ABC-type nanoparticlemediated transfections should be improved by reducing theoverall cationic charge of both nanoparticles. This could befeasible by engineering the lipid scaffold of these nanoparticlesusing alternative neutral lipids or cytofectins in order to reducethe lipid/pDNA ratio and overall mol % of cytofectin withoutimpairing transfection efficiency and nanoparticle stability withrespect to aggregation.

The design of therapeutic nanoparticles must consider bothcolloidal stability in biological fluids plus the ability to be tissue-specific. The targeting of such nanoparticles to tumor cellsinvolves the use of surface ligands that are recognized andfurther internalized by the cancer-related receptors. The primary

38 Bioconjugate Chem., Vol. 20, No. 1, 2009 Wang et al.

sequence should already have intrinsic selectivity for its targetedreceptor, but also important is the secondary conformation ofthe ligand, for maximizing receptor affinity. Here, in this studywe show that, by tuning the mol % of each lipid componentwithin the nanoparticle system, we can optimize the deliveryefficacy of tumor-targeted nanoparticles.

CONCLUSION

In this study, we present for the first time uPAR-targetednanoparticles for prostate cancer cells and we propose theexample of tailormade solutions for different biological chal-lenges. U11 is a peptide derived from the GFD in domain 1 ofuPA and is responsible for binding to the receptor due to itsepitopal folding. On the basis of this observation, we preparedU11 peptide-lipids that were inserted on the surface of thenanoparticles. It was shown that insertion of 0.5 mol % of PEGlipids induced particle stability and promoted receptor specificityfor improved cell uptake and transfection. The U11-targetingstrategy proved efficient and specific, where the U11 ABCD-type nanoparticles increased cell uptake and transfection levelsin uPAR-expressing cell lines. It is expected that optimized U11-nanoparticles for in vivo administration will show the samespecificity to uPAR expressing prostate cancers.

ACKNOWLEDGMENT

We thank The Royal Society and the EPSRC for providingthe financial support for this project.

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