1396.full

The FASEB Journal • Research Communication

Novel peptide ligand directs liposomes toward EGF-Rhigh-expressing cancer cells in vitro and in vivo

Shuxian Song,*,1 Dan Liu,*,1 Jinliang Peng,† Hongwei Deng,‡ Yan Guo,* Lisa X. Xu,*Andrew D. Miller,§,�,2 and Yuhong Xu‡,2

*School of Life Science and Biotechnology, †Shanghai Centre for Systems Biomedicine, and ‡School ofPharmacy, Shanghai Jiao Tong University, Shanghai, China; §Imperial College Genetic Therapies Centre,Department of Chemistry, Imperial College, London, UK; and �ImuThes Limited, London, UK

ABSTRACT Epidermal growth factor receptor (EGF-R)is an important target in anticancer therapy. Here wereport how a novel EGF-R peptide ligand (D4: Leu-Ala-Arg-Leu-Leu-Thr) is identified using a computer-aideddesign approach from a virtual peptide library ofputative EGF-R binding peptides by screening againstthe EGF-R X-ray crystal structure in silico and in vitro.The selected peptide is conjugated with a polyethyleneglycol (PEG) lipid, and the lipid moiety of the peptide-PEG-lipid conjugate is inserted into liposome mem-branes by a postmodification process. D4 peptide-conjugated liposomes are found to bind to and entercells by endocytosis specifically and efficiently in vitroin a process apparently mediated by EGF-R high-ex-pressing cancer cells (H1299). In vivo, the D4 peptide-conjugated liposomes are found to accumulate in EGF-R-expressing xenograft tumor tissues up to 80 h afterintravenous delivery, in marked contrast to controls.These results demonstrate how structure-based peptidedesign can be an efficient approach to identify highlynovel binding ligands against important receptors.These data could have important consequences for thedevelopment of peptide-directed drug delivery systemswith engineered specificities and prolonged times ofaction.—Song, S., Liu, D., Peng, J., Deng, H., Guo, Y.,Xu, L. X., Miller, A. D., Xu, Y. Novel peptide liganddirects liposomes toward EGF-R high-expressing cancercells in vitro and in vivo. FASEB J. 23, 1396–1404 (2009)

Key Words: AutoDock � epidermal growth factor � computeraided design � proteomic code � PEG � lipid

Chemotherapeutics are widely used in cancer ther-apy, but drug efficacies are often undermined by seriousside effects resulting from drug toxicities to normal tis-sues. To improve the therapeutic vs. toxic effects ofchemotherapeutic agents, delivery vehicles have beendevised to enhance drug delivery to tumor tissue. This isin the hope that biodistribution to tumor tissue will alsobe improved relative to the random walk situation thatarises when small-molecule drugs (�500 Da) are admin-istered to tissue without the aid of a delivery vehicle.Several liposome-based delivery vehicles have been devel-oped for a range of anticancer drugs; some are in patient

populations, including Doxil (also known as Caelyx) (1),comprising polyethyleneglycol (PEG)-coated liposomes(stealth liposomes) with extended serum half-life and theability to gradually extravasate through the leaky vascula-tures to accumulate in tumors.

Such delivery vehicles are now being superseded by theemergence of other “targeted” delivery vehicles that in-corporate active targeting moieties on their surfaces, suchas antibodies or ligands. Antibody-targeted immunolipo-somes are being tested in preclinical as wellas clinical studies (2–6). Meanwhile, small-moleculeligands (7, 8) and peptides (9, 10) are also under consid-eration as biological targeting ligands for various deliverysystems. Special attention has been given in the literatureto ligands that interact with epidermal growth factorreceptor (EGF-R). The EGF-R is overexpressed in a widevariety of human cancers, including lung, breast, bladder,and ovarian cancers. It has also been found to associatewith various features of advanced disease with poor prog-nosis (11). Various EGF-R-targeting vectors and conju-gates have been reported for the delivery of cytotoxicdrugs, toxins, or radionuclides (12–16). Mamot andcolleagues developed an EGF-R-specific immunolipo-some and showed in a series of studies that the antibodyconferred better delivery properties and antitumorefficacies in vitro than in vivo (17–19). FortunatelyEGF-R is quite well characterized in structural terms(protein data bank entry 1nql) and provides a cleartemplate for computer-assisted design (CAD). Here wedescribe a CAD approach to the screening and discov-ery of a novel ligand appropriate for anticancer thera-peutics. The target of our approach was EGF-R. Ourgeneral approach was to prepare a CAD-designed pep-tide (with control) and then chemically couple this to adrug delivery vehicle for the characterization of deliv-ery in vitro and in vivo.

1 These authors contributed equally to this work.2 Correspondence: Y.X., School of Pharmacy, Shanghai Jiao

Tong University, 800 Dongchuan Rd, Shanghai 200240, P.R.China. E-mail: [email protected]; A.D.M., Imperial CollegeGenetic Therapies Centre, Department of Chemistry, Impe-rial College London, London SW7 2AZ, UK. E-mail:[email protected]

doi: 10.1096/fj.08-117002

1396 0892-6638/09/0023-1396 © FASEB

MATERIALS AND METHODS

Materials

Egg phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phos-phoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethyl-eneglycol 2000)(DSPE-mPEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolN-maleimido-amino(polyethyleneglycol 2000) (DSPE-PEG2000-Mal), and cholesterol were all purchased from Avanti PolarLipids (Alabaster, AL, USA). Lissamine™ rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhoda-mine DHPE) and N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl -sn-glycero-3-phosphoethanolamine(fluorescein DHPE) were purchased from Invitrogen (Carlsbad,CA, USA). I-succinimidyl 3-(2-pyridyldithio) propionate (SPDP)and tris(2-carboxyethyl) phosphine (TCEP) were purchasedfrom Pierce Biotechnology (Rockford, IL, USA). Cy5.5 mono-NHS ester was supplied by GE Healthcare (Chalfont St. Giles,UK). Doxorubicin hydrochloride was purchased from ShenzhenMain Luck Pharmaceuticals (Shenzhen, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was pur-chased from Shanghai Pufei Biotechnology (Shanghai, China).Recombinant human epithelial growth factor expressed in Esch-erichia coli was a gift from Dr. Li Zaiping (State Key Laboratory ofMolecular Biology, Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences, Shanghai, China). All otheranalytical-grade chemicals were obtained from SinopharmChemical Reagents (Shanghai, China).

Peptide design and virtual screening

The crystal structure of EGF-R in an inactive, monomericstate was downloaded from the RCSB protein data bank(code: 1nql). A structural model was built and scanned forcandidate binding pocket using the PSCAN 2.2.2 programprovided by Dr. Sheng-Hung Wang (China Medical University,Taichung, Taiwan; [email protected]). A pocket on the sur-face near the top was selected (Fig. 1). The amino acid residuestructure inside the pocket was analyzed, and a focused librarycontaining 132 hexapeptides was designed based on Mekler-Idlisamino acid pairing theory (20). This in silico library wasscreened and ranked for putative binding strength to theEGF-R surface pocket using AutoDock3 (provided byScripps Research Institute, La Jolla, CA, USA). This is auseful automated molecular docking tool; it employs anempirical scoring system and consists of two kinds of freeenergy output. One is the binding free energy that includesthe intermolecular and torsional free energies, and theother is the docking energy that includes the intermolec-ular and intramolecular energies. To speed up the calcu-lation and run the program on multiple processors, thesoftware was modified to enable parallel computing. Allcomputing was performed on an SGI Onyx3800 machine inthe Shanghai Jiaotong University supercomputing center.

Peptide synthesis

All peptides were synthesized and purified by GL Biochem(Shanghai, China). HPLC and MS were used to confirmpeptide amino acid sequences and their respective purities.

Figure 1. Dockedstructures of D4 andD4* on EGF-R.A) EGF-R structuremodel from PDB.Asterisk-filled area isthe EGF bindingsite. Area circled inred is the bindingpocket we selectedfor docking. B) Low-est energy dockedconformation of D4 inside the EGF-R pocket. Peptide is shown in the ball-stick mode. Potential hydrogen bonds are indicated as greencylinders. C) Lowest energy docked conformation of D4* inside the EGF-R pocket.

1397PEPTIDE DIRECTED LIPOSOME DELIVERY TO EGF-R

Peptide bioconjugation to DSPE-PEG-maleimide

EGF protein or D4/D4* peptides were dissolved in PBS-EDTAand mixed with SPDP (12 mol equiv) dissolved in dimethylsulfoxide for 1 h at ambient temperature and then lyophi-lized. Meanwhile, DSPE-PEG2000-Mal lipids in chloroformwere dried into a thin film and hydrated in HEPES buffer(pH 7.4; 0.4 mM). For bioconjugation, the thiolated protein orpeptides were added to TCEP buffer, incubated for 1 h at roomtemperature under nitrogen, then quickly mixed with theDSPE-PEG2000-Mal (0.2 mol equiv) solution and stirred for 16 hunder nitrogen at 10°C. Bioconjugation was judged to beessentially complete by HPLC analysis. Excess unreacted proteinor peptide was removed during liposome formulation.

Liposome preparation and ligand-conjugated lipidincorporation

Liposomes were all prepared using the dehydration-rehydra-tion method, followed by extrusion several times through100-nm membranes. Most liposomes used in this study startedwith the lipid composition of EPC/Chol/DSPE-mPEG2000

(10:5:0.5, m/m/m). For fluorescent labeling, rhodamine-DHPE or fluorescein-DHPE was included in the liposomecomposition at �0.6 mol% of total lipid composition. ForCy5.5 labeling, Cy5.5-DSPE was synthesized first by mixingCy5.5-NHS, DSPE, and triethylamine in a 3:1:3.5 (m/m/m)in chloroform and incubated in the dark for 16 h at ambienttemperature. The resulting Cy5.5-DSPE bioconjugate wasincluded in the liposome composition at �0.5 mol% of totallipid composition. Excess unreacted Cy5.5 was removed afterliposome formation by dialysis.

DSPE-PEG2000 ligands (prepared as described above) wereinserted into the EPC/Chol/ DSPE-mPEG2000 liposomes ac-cording to the procedure of Ishida et al. (21), with minormodifications (22, 23). Briefly, appropriate DSPE-PEG2000

ligand micelle (9 mol%) solutions were added to the lipo-some suspension and incubated for 1 h at 60°C. The solutionswere then dialyzed against PBS using SnakeSkinTM PleatedDialysis Tubing 10,000 MWCO (Pierce) for 4 h. To createnonligand controls, DSPE-mPEG2000 micelles were alsomixed and incubated at the same ratio to make nontargetedliposomes. All liposome preparations were prepared at astandard total lipid concentration of 5 mg/ml before use.

Cell lines and animal models

The human non-small-cell lung carcinoma cell line H1299,with high EGF-R-expressing level, was used in this study. Thecells were cultured in RPMI1640 culture medium containing10% fetal bovine serum at 37°C in a humidified atmospherecontaining 5% CO2.

H1299 xenograft mouse models were prepared by theanimal experimental center of the Shanghai Cancer Institute.They were used in the in vivo imaging experiments 3–4 wkafter tumor cell inoculation (tumor size 5–7 mm diameter)and humanely sacrificed afterward. The animal study proto-cols were approved by the Animal Study Committee ofShanghai Jiaotong University, School of Pharmacy.

In vitro binding and internalization experiments

EGF-R high-expressing H1299 cells were plated in a 35 mm-diameter culture dish (1�106 cells/dish) and cultured in RPMI1640 medium for 24–48 h. After the cell culture reached �80%confluence, samples of rhodamine-labeled ligand-conjugatedliposomes were diluted in RPMI 1640 (final vol 1 ml/sample)and added in toto into individual wells of culture dishes (final

total lipid dose: 0.2 mg/well) at either 4 or 37°C. In thecompetitive binding experiments, a 50-fold molar excess of freeD4 or EGF was added into the medium 2 h before the additionof D4-conjugated liposomes (final total lipid dose: 0.2 mg/well).The cells were incubated at the specific temperatures for 4 h,and then washed 6 times with PBS (pH 7.4). The remainingbound and internalized fluorescence lipids were visualized usinga confocal laser scanning microscope (CLSM; Zeiss LSM; CarlZeiss, Oberkochen, Germany). For the flow cytometric studies,H1299 cells were plated in 35 mm-diameter wells until cells grewto �80% confluence. After incubation with ligand-modified fluo-rescein-labeled ligand-conjugated liposomes (final total lipiddose: 0.2 mg/well) for 3 h at 37°C, cells were washed withPBS, treated with trypsin for suspension, and analyzed by aflow cytometer (BD FACS-Calibur; Becton Dickinson, Frank-lin Lakes, NJ, USA).

In vivo fluorescence imaging of peptides and peptide-directed liposomes in tumor-bearing mice

Cy5.5-DSPE-labeled D4- or D4*-conjugated liposomes wereadministered i.v. (100-�l tail-vein injection) into H1299 xeno-graft tumor-bearing mice at a standard dose of 20 mg/kg ofanimal body weight. At various time points, the mice wereanesthetized and imaged with an Optix in vivo fluorescenceimaging system (GE Healthcare). The concentration of lipo-some was considered proportional to fluorescence intensitysignals in optimum scan parameters. The representativeimages from the same most representative mouse in eachgroup were shown (n�3/group). These images were pro-cessed using the fluorescence lifetime gating for Cy5.5 (1.8–2.5 ns) to remove interference from autofluorescence.

RESULTS

Peptide library design and docking analysis

The EGF-R binding pocket targeted for CAD liganddesign was selected with the aid of the PSCAN program.

TABLE 1. Energy and sequence of top 20 peptides screened byAutoDock3, ranked according to docking energy

Dockedenergy Database SN Peptide sequence

�17.05 Part84_pep2.dlg LEU ALA ARG PHE PHE SER�16.96 Part90_pep2.dlg LEU ALA ARG LEU PHE PRO�16.45 part82_pep2.dlg LEU ALA ARG PHE PHE PRO�16.43 Part95_pep2.dlg LEU ALA ARG LEU LEU THR�16.38 part86_pep2.dlg LEU ALA ARG PHE LEU PRO�16.2 Part81_pep2.dlg LEU ALA ARG PHE PHE ALA�15.49 part88_pep2.dlg LEU ALA GLY PHE LEU PRO�15.36 Part93_pep2.dlg LEU ALA GLY PHE PHE ALA�15.31 Part85_pep2.dlg LEU ALA THR PHE LEU SER�15.31 Part74_pep2.dlg LEU ALA GLY LEU PHE PRO�15.31 Part68_pep2.dlg LEU ALA GLY PHE PHE SER�15.21 Part4_pep2.dlg LEU THR GLY PHE PHE SER�15.14 Part66_pep2.dlg LEU ALA GLY PHE PHE PRO�15.02 Part117_pep2.dlg LEU PHE THR PHE LEU ALA�14.83 Part83_pep2.dlg LEU ALA GLY PHE LEU THR�14.78 Part106_pep2.dlg LEU ALA ALA LEU PHE PRO�14.69 Part78_pep2.dlg LEU ALA GLY LEU LEU PRO�14.62 Part14_pep2.dlg LEU THR GLY LEU LEU PRO�14.61 Part72_pep2.dlg LEU ALA GLY PHE LEU SER�14.54 Part77_pep2.dlg LEU ALA GLY LEU LEU ALA

1398 Vol. 23 May 2009 SONG ET AL.The FASEB Journal

As shown in Fig. 1 (red circle), this is an easily accessiblesurface pocket on EGFR domain I, far from the EGFbinding site (labeled with asterisk symbols). Nine EGFRresidues were identified surrounding the pocket (�5 Å):ASN134, GLY177, ALA178, GLN164, CYS163, SER162,GLU110. GLU73, and ARG74, from which we selected asubgroup of 6 amino acid residues (GLN164, CYS163,SER162, GLU110, GLU73, and ARG74) as the targetresidues against which a focused peptide library wasdesigned, based on the concept of sense (S) and anti-sense (AS) peptide interactions, driven by theMekler-Idlis amino acid pairing theory (20).

The library consisted initially of 132 peptides, con-taining all combinations of AS peptides targeted at the6 EGF-R sense amino acid residues described above.For each peptide, 100 separate docking experimentswere performed, and each docking experiment wasperformed with more than 250,000 energy evaluations

using the Lamarckian genetic algorithm local searchalgorithm (24, 25). Fifty selected peptides were furtherevaluated in a refined search phase with 10 millionenergy calculations per docking experiment. Finally, 20peptides were selected based on their lowest dockingenergies (Table 1). We synthesized the first 10 peptidesfor solubility, EGFR association, and dissociation char-acteristics (results not shown) and came to the conclu-sion that peptide D4 (N-LARLLT) (Table 1) should bea good first choice. The EGFR high-expression H1299cells were used to confirm the binding of fluorescence-labeled D4 peptide, and, indeed, significant bindingwas observed. Clearly other peptides in the set could beemployed as alternative EGFR ligands, although D4worked surprisingly well. For control purposes a scram-bled peptide D4* (N-RTALLL) was prepared with anequivalent residue composition to D4 but sequence-reordered.

Figure 2. A) Synthesis scheme of peptide-PEG-DSPE.B) HPLC confirmation of the reactants.


Preparation of peptide-conjugated liposomes

The D4 and D4* peptides were first modified (N terminus)with SPDP and then conjugated to DSPE-PEG2000-Mal bystandard sulfydryl-maleimide coupling (Fig. 2A). Theresulted DSPE-PEG2000 peptide conjugate was con-firmed by reversed phase HPLC analysis (Fig. 2B). TheDSPE-PEG2000 peptide or DSPE-mPEG2000 moleculeswere incorporated into preformed liposomes (EPC/Chol/DSPE-mPEG2000) by means of the widely used PEG-lipid micelle insertion method (postmodification) (21).We determined 9 mol% of DSPE-PEG2000 peptide to beoptimal for ligand presentation and commonly used. Thefinal size distribution of the liposomes was determined byphoton correlation spectroscopy using a Zetasizer 3000H(Malvern Instruments, Worcestershire, UK). Comparedto the liposomes made right after the extrusion (�110nm), the particle sizes after the peptide lipid incorpora-tion were slightly bigger (130–150 nm) but otherwisestable for at least 3 mo storage at 4°C. HPLC analysis (asin Fig. 2B) confirmed the integrity of DSPE-PEG2000

peptide conjugates of the D4 and D4* peptides through-out the period of storage while in liposome formulations.

All liposome formulations were also found to be stablewith respect to aggregation, and degradation in thepresence of serum for at least a 12 h period; once againDSPE-PEG2000 peptide conjugate integrity also was notfound to be compromised (data not shown).

Binding and uptake of ligand-modified liposomesin H1299

The EGF-, D4-, or D4*-modified, rhodamine-labeledliposomes were tested for their binding ability to EGF-Rhigh-expressing cells in vitro. Both EGF- and D4-modi-fied liposomes bound extensively to the H1299 cells.With D4* there was almost no fluorescence exhibited(Fig. 3A). The detailed binding characteristics werefurther evaluated at different temperatures. At 4°C thefluorescence was mostly seen on cell surfaces and couldbe largely competed off by excess unlabeled free D4(Fig. 3Biii, iv). At 37°C, however, the competition effectof free D4 was less obvious (Fig. 3Bi, ii), suggestingthere might be active endocytosis and receptor turn-around after the binding of D4 to EGF-R. The presence

Figure 3. Fluorescence microscopy studies of ligand-directed liposome binding to EGF-R high-expressing cells. A) Binding ofD4-, D4*-, and EGF-targeted liposomes to H1299 cells: D4 liposomes (i), EGF liposomes (iii), D4* liposomes (v), and phasecontrast micrographs of the same fields (ii, iv, vi). B) Binding of D4-targeted liposomes to H1299 at 4 or 37°C in the presenceof 50� mole excess free ligands. i) Binding at 37°C with excess free D4. ii) Binding at 37°C without free D4. iii) Binding at 4°Cwith excess free D4. iv) Binding at 4°C without free D4. v) Binding at 37°C with excess free EGF. vi) Binding at 37°C withoutfree EGF. Scale bars � 20 �m.


of free EGF at 37°C actually affected the D4 liposomefluorescence to a certain degree, although not com-pletely (Fig. 3Bv, vi). Perhaps EGF interfered with theendocytic pathway of D4 after binding.

The D4-conjugated liposome binding and uptake byH1299 cells is also shown in cropped magnification(Fig. 4A) and in Z stack serial scan images (Fig. 4B).Lipid fluorescence may be seen in numerous endocyticvesicles inside cells. That the binding of D4 peptide toa trivial surface pocket on EGF-R far away from the EGFbinding site can initiate such cell surface binding andendocytosis is gratifying in terms of the design objec-tives of these experiments.

Comparison of binding efficiencies of D4- andD4*-conjugated liposomes

Flow cytometry was used to compare the differentbinding efficiencies of D4- and D4*-conjugated lipo-somes in larger cell populations. D4- and D4*-conju-gated liposomes were labeled with fluorescein-DSPEand incubated with H1299 cells for 3 h. As seen in Fig. 5,when the cells were gated at 101–103 FL1 value, 75% ofH1299 cells were fluorescent after D4 liposome bind-ing, but only 27% appeared to be fluorescent followingD4*-conjugated liposome binding.

Ligand-directed delivery of liposomes in vivo

D4- and D4*-conjugated liposomes with Cy5.5 dye in-cluded were injected via the tail vein into H1299 tumor-bearing mice. The entire bodies of the mice were scannedat various times after the injection, and the fluorescenceintensity from the tumor-bearing region at different timepoints was quantified. As shown (Fig. 6), D4-conjugatedliposomes accumulated gradually inside the tumor regionfrom 6 h onward. Even after 80 h, significant signal was

evident. In the case of D4*-conjugated liposomes, thefluorescence intensities in the tumor region were clearlynot as substantive. The tumor-to-background ratios in-creased from 1 h after injection all the way to 76–80 h,when a plateau was reached suggesting a gradual accumu-lation of the D4-conjugated liposomes inside tumorsfollowed by retention [enhanced permeation and reten-tion (EPR) effects] (26). Liposome circulation half-liveswere determined for Cy5.5-DSPE-labeled D4- or D4*-conjugated liposomes (and Cy5.5-DSPE-labeled peptide-free liposomes). Within experimental error, circulatoryhalf-lives were found to be approx 6 h, as judged byextrinsic fluorophore fluorescence in murine plasma as afunction of time post-administration (results not shown).Data trends suggested that Cy5.5-DSPE-labeled D4-conju-gated liposomes may have a shorter circulation half-lifecompared to Cy5.5-DSPE-labeled D4*-conjugated or pep-tide-free liposomes.

DISCUSSION

EGF-R is one of the most important anticancer targetstoday. Several successful drugs targeting EGF-Rs havebeen developed, including the tyrosine kinase inhibitorTarceva® and Iressa®, and its blocking antibodyErbitux® (27). In addition to designing drugs directlythe receptor, delivery vehicles may carry ligands specificfor EGF-R binding, either within the natural ligand,EGF, binding site, or allosteric. Many studies haveexploited the use of EGF for targeted delivery ofcytotoxic drugs, toxins, liposomes and other drug/genedelivery vehicles, and radionuclide-containing systems(12–15). However, the endogenous proproliferationeffect of EGF on cancer cells is always a concern. Twoother alternative strategies have been proposed. One isto use antibodies or antibody fragments to direct bind-

Figure 4. Internalization of D4-conjugated liposome by H1299 cells. A) Overlay of phase contrast and fluorescence images ofendocytosed D4 liposomes. B) Eleven slices from the top to the bottom of the cells using the Z-stack scan mode of confocalfluorescence microscope. Scale bars � 20 �m.


ing to EGF-R (17, 18, 28); the other is to use smallerpeptides or EGF fragments (12, 29, 30). Small peptidesthat can strongly and specifically bind to EGF-R butwith minimal immunogenicity and proproliferative effectsshould be desirable.

The search for an optimal peptide targeting ligand is achallenge, and various strategies have been designed.One strategy is to estimate and test the possible bindingfragments of the natural ligand, namely, EGF (12, 29).But in reality the peptide 3-dimensional structure maybear little relationship to what is seen in situ in a protein.Therefore retained binding affinities cannot be assumed.Another widely used approach is to screen phage displaylibraries. We recently reported the finding of a very strongpeptide binder of EGF-R based on phage screening (28).Here we took a different approach and exploited thecapability of virtual screening. There have been greatimprovements in recent years in CAD approaches to drugand ligand design, plus virtual screening (31).

In this study we developed a parallel computing algo-rithm for AutoDock3 software. AutoDock is a widely usedprogram for docking flexible ligands to macromolecules(24) that has shown some utility in the prediction ofbinding moieties mediating ligand-receptor interactions(29), and in screening compounds for lead discovery(32). Our intention was to design a focused peptidelibrary based on the structural analysis of a selectedsurface-binding cleft located in EGF-R. The library wasdesigned according to the principles of sense and anti-

sense peptide interactions to include all combinations ofAS peptide amino acid residues that are more likely tointeract with a defined set of complementary S amino acidresidues within the surface-binding cleft of EGF-R. Thebasis of S/AS peptide interactions is still controversial, butthe preferred theory of interaction is interpeptide pair-wise interactions of amino acid residues [Mekler-Idlis(M-I) pair theory] (20, 33). This interaction theory hasproven to have surprising utility and has given rise to theconcept of the proteomic code, according to which thegenetic code not only codes for the primary structure ofproteins but also has the embedded capacity to code forthrough-space interactions between amino acid residues,thereby helping to define potentially the 3-dimensionalstructures of polypeptides and protein-protein or protein-peptide interactions (20).

When the D4 peptide was modified with SPDP and thenconjugated to the distal end of DSPE-PEG2000-Mal, thenincorporated into liposomes, the resulting D4 peptide-con-jugated liposomes were able to undergo specific attachmentto and entry into EGF-R-expressing cell lines in vitro (Figs. 3and 4). Equally importantly, D4 peptide-conjugated lipo-somes apparently showed ligand-specific accumulation intoxenograft tumor post-i.v. injection (Fig. 6). Such smallanimal in vivo fluorescence imaging is a newly developedtool for monitoring the biodistribution of labeled drugs anddelivery vehicles responsible for delivering drugs. Cy5.5 isone of the most commonly used dyes in in vivo imagingstudies because of the superior tissue penetration of incident

Figure 5. FACs analysis of D4 and D4* liposome binding to H1299 cells. a) D4 fluorescein-liposome. b) D4* fluorescein-liposome. c) Overlay of a and b.

Figure 6. Fluorescence images of peptide-di-rected liposome distribution and accumulationin tumor tissues. Images shown (from left toright) are the light picture of the mouse, andfluorescence images taken at 1, 6, 12, 24, 48,and 80 h after the injection of D4- and D4*-conjugated liposomes.


radiation and low background of autofluorescence (34–36).The main advantage of this in vivo imaging technique is theopportunity for noninvasive, continuous monitoring andreduced need for animal sacrifice. In our experience, differ-ent animals in a study group may have variations in, forexample, their general health, physiology, metabolism, tu-mor size, and location, factors that will affect biodistributionand therefore experimental error. To overcome this process,multiple images can be taken at various times for the sameanimal and compared to rule out such variation artifacts.Typically, with 3 animals per experiment group, the fluores-cence intensities and detailed time points may vary amongdifferent animals, but the general trends remain consistentand reproducible. Therefore, even though the n number(n�3) is a too low for a full statistical analysis of the real-timeimaging data, we judge that the observed data trends can stillbe considered meaningful.

In accepting that this biodistribution data (Fig. 6) ismeaningful, how does it compare with the observations ofothers? Several factors have been identified that influencethe pharmacokinetic properties and extravasation EPR be-havior of liposomes equipped with antibodies or small mol-ecules (37, 38). Immune clearance against the surface con-jugated ligands has been a major concern (39, 40). Forinstance, IgG-conjugated liposomes were found to interactreadily with Fc receptors and be cleared quickly withoutreaching the tumor (41–43). Similarly, small-molecule li-gand folate conjugation has been reported to promoteenhanced recticuloendothelial system clearance of lipo-somes in vivo (8). In our case, we observed clearly thatD4-conjugated liposomes were able to accumulate morerapidly in a tumor compared with the corresponding D4*-conjugated control liposomes (pixel resolution 0.5 mm;average tumor size 5 mm). These data suggest that the D4peptide is able to confer an increased rate of EPR. However,does this suggestion make sense given what is currentlyknown about the EPR effect? We have observed such anincreased rate effect previously when Gd-chelated liposomesystems (�100 nm diameter, PEGylated with neutral charge)were prepared for magnetic resonance imaging (MRI) withand without folate targeting ligands, and then MRI moni-tored as a function of time after i.v. administration to mice(26, 44). Without targeting the folate ligand, the optimalaccumulation of Gd-chelate into xenograft tumors wasfound to take at least 24 h, whereas with the targeting ligandthe process was accelerated, and optimal accumulation ap-peared to take place between 2 and 4 h. In this instance, weconcluded that two possibilities might explain the data: 1)that the actual process of the EPR effect was accelerated bytargeting ligand, and 2) that ligand targeting does not alterthe EPR effect but enhances optimal functional delivery ofthe imaging agent.

Considering that the circulation half-lives of our D4- andD4*-conjugated liposomes (and peptide-free liposomes)were similar within experimental error (see above), then thesecond possibility seems more appropriate to account for theapparent biodistribution data observed here (Fig. 6). In-deed, Kirpotin et al. (45) have suggested that somethingsimilar occurs with antibody-targeted liposomes, and obser-vations made during studies with other ligand-targeted lipo-

some systems also concur (46, 47). Having suggested this, wedo not yet feel able to completely rule out the possibility thatthis D4 peptide ligand might not also confer some kineticenhancement on the EPR effect. This might still be possiblegiven recent reports that liposome systems without targetingligands can accumulate only weakly and more tran-siently in xenograft tumors in comparison with ligand-targeted liposome systems (47). However, further ex-periments will be required to determine to properlysubstantiate this possibility.

In summary, we have demonstrated the effective design ofan EGF-R binding peptide that was conjugated to liposomesand shown to enhance binding to and entry into EGF-R-expressing cells in vitro and vivo. Detailed investigations onthe therapeutic efficacies of such a peptide conjugated todelivery systems in various preclinical models are now under-way. We hope that our D4 peptide can be further developedinto an effective ligand that may be conjugated to a variety ofuseful delivery systems for the enhanced delivery of thera-peutic agents to tumors in clinical cancer treatment.

Funding was provided by National Science Foundation ofChina grant 30472097 and Fok Ying Tong Education Foun-dation grant 91035.

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Received for publication August 13, 2008.Accepted for publication December 4, 2008.


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