Highly efficient transduction of endothelial cells by targeted ... - Nature

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Highly efficient transduction of endothelial cells by targeted artificial virus-like particles Kristina MuÈller,1 Thomas Nahde,2 Alfred Fahr,1 Rolf MuÈller,2 and Sabine BruÈsselbach 2 1

Institute of Pharmaceutical Technology and Biopharmaceutics, Philipps University, Marburg 35032, Germany; and 2Institute of Molecular Biology and Tumor Research (IMT), Philipps University, Marburg 35033, Germany.

Targeting the tumor vasculature by gene therapy is a potentially powerful approach, but suitable vectors have not yet been described. We have designed a new type of liposomal vector, based on the composition of anionic retroviral envelopes, that is serum - resistant and nontoxic. These artificial virus - like envelopes ( AVEs ) were endowed with a cyclic RGD - containing peptide as a targeting device for the avû3 - integrin on tumor endothelial cells ( ECs ) . The packaging of plasmid DNA complexed with low - molecular weight, nonlinear polyethyleneimine into these AVEs yielded artificial virus - like particles ( AVPs ) that transduced ECs with efficiencies of up to 99%. In contrast, transduction of a variety of other cell types by these RGD ± AVPs was comparably inefficient under the same experimental conditions. This EC selectivity was mediated, in part, but not exclusively, by the RGD ligand, as suggested by the reduced, but still relatively high, transduction efficiency seen with AVPs lacking RGD. The interaction of anionic lipids of the AVPs with ECs may therefore contribute to the observed selective and highly efficient transduction of this cell type. These findings suggest that the targeted AVE technology is a useful approach to create highly efficient nonviral vectors. Cancer Gene Therapy ( 2001 ) 8, 107 ± 117 Key words: Nonviral vectors; artificial virus - like envelope ( AVE ) ; artificial virus - like particle ( AVP ) ; anionic liposome; polyethyleneimine; endothelial cells; integrin targeting; cyclic RGD.

A

mong the new experimental approaches to cancer treatment, gene therapy has gained particular attention. Clinical studies showed that cancer gene therapy can potentially be made to work, but the lack of adequate technologies is evident. This applies, in particular, to the efficiency and specificity of tumor cell transduction, indicating that the design of more efficacious and site selective vectors is urgently required. A second serious problem is the poor accessibility of many tumor cells to any kind of drug, in particular vectors carrying nucleic acids, as a consequence of the defective tumor vasculature and the high interstitial pressure.1 The concept of endothelial tumor cell targeting2 is attractive because the tumor blood vessels are more readily accessible than the actual tumor cell compartment. In addition, endothelial cells ( ECs ) are unknown to acquire resistance to treatment,3 and the endothelium represents a target that is largely independent of tumor type.4 Finally, as tumor ECs are essential for the nutrition and growth of the tumor cells, any therapy targeting tumor ECs should have a dramatic ``bystander effect''. Several marker proteins up -regulated in tumor endothelium have been identified. Among these membrane associated proteins, some are of particular interest because they can be exploited in targeting the tumor vasculature. Received June 23, 2000; accepted November 7, 2000. Address correspondence and reprint requests to Dr. Rolf MuÈller, Institute of Molecular Biology and Tumor Research ( IMT ) , Philipps University, Marburg 35033, Germany. E-mail address: [email protected] - marburg.de

Cancer Gene Therapy, Vol 8, No 2, 2001: pp 107 ± 117

Examples are vascular endothelial growth factor receptor II ( FLK -1, KDR ),5 transforming growth factor binding protein CD105 /endoglin,6,7 vû3 integrin,8 and CD13 / aminopeptidase N.9 That these surface molecules can be used for the successful targeting of drugs to the tumor blood vessels has been demonstrated in several studies. Thus, antiangiogenic strategies have made use of antibodies neutralizing KDR /FLK -1;10 ± 12 antibodies directed to endoglin have been employed to direct a ricin A conjugate to the tumor vasculature;13 and a cyclic RGD peptide has been used to target doxorubicin to the tumor site.14 Cyclic RGD peptides have also been used successfully for the delivery of both viral and nonviral vectors to ECs,15 ± 19 and therefore, seem to be suitable for tumor EC targeting. The vasculature is a highly attractive target also for gene therapy because it is comprised of a large number of potential target cells and is easily accessible via the blood stream. Ideally, an EC - directed vector should be safe, nontoxic, nonimmunogenic, and cell type ±specific; it should transduce EC cells with high efficiency; and Ð with respect to clinical applicability Ð it should offer the possibility of large-scale production, stability of the product, and ease of handling. To date, a vector combining all these features has not been described. Viral vectors are often highly efficient, but safety and immunogenicity are issues of potential concern, and the limited transgene size often poses a serious obstacle. Nonviral vectors, on the other hand, frequently face the problem of low transduction efficiency. Different concepts 107

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have been developed for the generation of nonviral vectors, e.g., cationic lipid ± DNA complexes,20 polycation ± DNA complexes,21,22 and liposome- entrapped polycation - condensed DNA ( LPD ) .23 However, these complexes are often physico- chemically or biologically unstable, serum -sensitive, or toxic as a consequence of unspecific interactions with the biological environment.23,24 Promising results have been obtained with Sendai virus ±fused liposomes ( HVJ liposomes) ,25 but a potential problem may be the presence of viral proteins in these vectors and their large size ( 400 nm ) . An attractive alternative is the use of artificial virus -like envelopes ( AVEs ) for the encapsulation of condensed plasmid DNA.26 AVEs mimic the lipid composition of retroviruses. These natural lipids are anionic and, in contrast to their artificial cationic counterparts, interact only weakly with their biological environment and therefore are nontoxic. We have taken this approach further by combining AVEs with the advantages of the cationic polymer, polyethyleneimine ( PEI ),22 which not only condenses the DNA but is also believed to act as an endosomolytic agent and to protect the DNA from cytoplasmic nucleases. In addition, we have equipped these artificial viral particles (AVPs ) with a targeting device for activated ECs, i.e., a cyclic RGD peptide 27 thought to interact with v 3 integrin. We refer to these targeted nonviral vectors as RGD ±AVPs. MATERIALS AND METHODS AVEs

Throughout the preparation of AVEs, synthetic phospholipids were used without further purification. 1,2 -Dipalmitoleoyl sn -glycero- 3- phosphoethanolamine (DPPE ) and 1,2 -dioleoyl - sn-glycero -3 - [phospho - L - serine ] (DOPS) were purchased from Avanti Polar Lipids ( Alabaster, AL ); 1,2dilauroyl -sn -glycero- 3- phosphoethanolamine (DLPE ) was obtained from Genzyme (Liestal, Switzerland ); and cholesterol was from Calbiochem (San Diego, CA ). All other substances were of analytical grade. Synthesis of lipid anchor

N - glutaryl - DPPE was prepared by dissolving DPPE in anhydrous chloroform. Glutaric anhydride and water- free pyridine were added and the solution stirred at 208C for 2 days. After this period, the products were dried by applying vacuum to the solution and N -glutaryl -DPPE was purified using preparative silica gel chromatography ( Merck 60 F 254 ) in chloroform / methanol /ammonia (65 /35 /3) . Spots were dissolved in methanol, dried, and redissolved in chloroform for further use. N - glutaryl - DPPE was identified by 1 H -NMR as described previously.45 AVEs with the composition DOPS/ DLPE /cholesterol / N -glutaryl -DPPE ( 3:3:3:1 mol /mol ) were prepared as follows: a chloroform solution of the lipid mixture (total amount 10 mol ) was dried into a thin film in the inner surface of a rotating 100mL glass vessel warmed by a water bath to 308C. Residual chloroform was removed by vacuum desiccation for 15 minutes. The lipid film was hydrated either in 1 mL sterile 10 mM Tris buffer (pH 7.4) or in 1 mL sterile phosphate buffered saline ( PBS ) . After 2 hours, the resulting multi-

lamellar liposome suspension was placed in a 3- mL glass vessel cooled by an ice /water mixture and sonicated for 15 seconds in an MSE - Soniprep 150 (Zivy, Oberwil, Switzerland ) equipped with a titanium tip. Sonication was repeated 10 times; each sonication period was followed by a 30second pause, allowing the suspension to cool. The resulting liposome suspension was extruded through polycarbonate membrane filters with a pore size of 50 nm using a standard device (LiposoFast2 ) 46 purchased from Avestin (Ottawa, Canada ). The liposomes were used up 2 months after preparation, during which time no significant increase in liposome size could be detected. Targeting motif

Using solid -phase synthesis (Applied Biosystems Peptide Synthesizer, Foster City, CA ) , a cyclic peptide with the amino acid sequence CDCRGDCFC and having an additional arginine at the N -terminus was synthesized. Cyclic condensation of the peptide was accomplished by stirring an aqueous solution of the synthesized peptide under access of ambient air. Completion of cyclic condensation was verified by high -performance liquid chromatography. After high -performance liquid chromatography purification, the peptide was lyophilized and stored at 48C. Covalent attachment of RGD peptide to liposomal surface ( RGD ± AVE )

Activation of the N -glutaryl -DPPE carboxyl group at the liposomal surface 47 was achieved by adding 3.5 mg of 1ethyl - 3- ( 3 -dimethylaminopropyl ) carbodiimide to 400 L AVE and shaking the suspension for 5 hours in the dark. The resulting active O -acyl -intermediate reacted with added RGD peptide ( 250 g in 150 L buffer ) overnight, yielding a covalent coupling of the peptide to the liposomal surface. The RGD ± AVEs were separated from unbound peptide by Sephadex G25 gel permeation chromatography in Tris buffer 10 mM, pH 7.4. Coupling efficiency was monitored using a fluorescently labeled derivative of the RGD peptide. For this purpose, RGD was conjugated with 5- ( 4,6- dichlorotriazinyl )aminofluorescein (5 -DTAF ) 48 purchased from Molecular Probes ( Eugene, OR ). 5 -DTAF was dissolved in borate buffer (pH 9 ) and RGD peptide was added in a 1:4 molar ratio ( RGD /5 -DTAF ). The solution was stirred overnight and uncoupled 5 -DTAF was separated from the labeled peptide by size exclusion chromatography using Sephadex G25. The purified fluorescently labeled RGD was covalently linked to activated N - glutaryl - DPPE in AVEs as described above. Fluorescence of the labeled AVE was read using a spectrofluorimeter (model LS50B; Perkin Elmer, È berlingen, Germany ) and compared to an appropriately U diluted RGD / 5- DTAF solution. Condensation of plasmid DNA

Low -molecularweight, branched polyethyleneimine ( PEI; Lupasol G100, BASF, Ludwigshafen, Germany ) was added to 15 g of plasmid DNA up to a ratio of polyethyleneimine nitrogen:DNA phosphate of 20.7. Condensation of plasmid DNA was monitored by dye exclusion technique using Picogreen2 (Molecular Probes) as described previously.49 Cancer Gene Therapy, Vol 8, No 2, 2001


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1.0

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Figure 1. Condensation of DNA by polyethylenimine determined by dye exclusion. Six individual measurements with different DNA plasmid concentrations are shown to illustrate the high reproducibility of the method.

In another set of experiments, protamine sulfate ( USP quality; EliLilly and Co., Indianapolis, IN ) was used as condensing agent. Preparation of RGD ± AVPs

An RGD ± AVE suspension containing 60 g of lipid was added to 15 g of plasmid DNA precomplexed with PEI, as described above, and gently vortexed. The resulting AVPs are ready for transfection experiments. The indicated amounts are sufficient for one 6- cm cell culture dish. DNA condensation measurement by dye exclusion

The experimental protocol was followed as described.49 Each well of a 96 -well plate was filled with 95 L of Tris 10 mM, pH 7.5. Plasmid DNA was added at concentrations of 300 to 1333 ng /mL in each well and the Picogreen2 concentration was adjusted to a ratio of one molecule per 3.75 bp of DNA. Fluorescence spectrophotometry was performed at an excitation wavelength of 492.5 nm and an

emission wavelength of 518 nm. Appropriate amounts of PEI were added to the wells to achieve the desired N / P ratios. Size and zeta potential analysis of vector particles

Dynamic laser light scattering using a commercially available system Zetasizer 4 (Malvern Instruments, Herrenberg, Germany ) with PCS Software Version 1.26III (Malvern ) was used. Autocorrelation data were analyzed by a specialized version of CONTIN.50 Zeta potential measurements were performed on a PALS Zeta Potential Analyzer Version 3.12 ( Brookhaven Instruments, Holtsville, NY ). Cryogenic transmission electron microscopy

A suspension of AVPs was applied to a holey carbon foil grid and vitrified by flash- freezing in liquid ethane. The grids were cryo -transferred to the liquid nitrogen ± cooled cryoelectron microscope (Philips CM200 FEG, FEI, Germany ) . Images were taken at a magnification of 60,000 under liquid nitrogen conditions at 1.5 m defocus at 160 keV. Cell culture

50

Fluorescence at 520 nm (a.u.)

Figure 3. Cryogenic transmission electron microscopy ( Cryo - TEM ) of RGD ± AVPs complexed with PEI / plasmid DNA. The pictures show encapsulation of condensed DNA in a lipid layer within discrete particles. The diameter of the larger liposomes shown is approximately 140 nm.

AVE (z-av. 94 nm) DNAPEI (z-av. 146 nm) DNAPEIAVE (z-av. 182 nm)

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Diameter (nm) Figure 2. Size determination of RGD ± AVEs, PEI ± DNA, and RGD ± AVPs by photon correlation spectroscopy. The mean values for the size distributions estimated by z - average calculation are indicated.

Cancer Gene Therapy, Vol 8, No 2, 2001

Human umbilical vein endothelial cells (HUVECs ) were prepared by the method of Jaffe 51 as modified by Thornton et al.,52 and cultivated in EGM - 2 medium ( BioWhittaker Europe, Verviers, Belgium ). The colon carcinoma cell line LoVo (provided by I. Hart, London, UK ), the lung adenocarcinoma cell line A549 (obtained from K. Havemann, Marburg) , the prostate carcinoma cell line DU -145 ( obtained from G. AumuÈller, Marburg) , the choriocarcinoma cell line JEG -3 ( obtained from A. Wellstein, Georgetown University ), and the osteosarcoma cell line Saos- 2 (obtained form N. La Thangue, Glasgow ) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum ( FCS; BioWhittaker Europe) and 2 mM L - glutamine. Cells were passaged (1:5 for HUVECs and 1:10 for tumor cell lines ) by trypsinization ( 0.05% trypsin, 0.02% EDTA ) and grown at 378C in 5% CO2.

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Figure 4A. Toxicity of AVPs for HUVECs compared to Lipofectamine ( LA ) and Superfect ( SF ) . The analyses were performed at 6 and 24 hours after transfection ( see Materials and Methods for details ) . A: Identification of apoptotic cells by staining with Hoechst 33258. Control, untransfected cells.


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Figure 4B. Determination of metabolic activity by WST assay. The following amounts of DNA and transfection reagents were used ( per 3 cm well ) : AVP, 5 g DNA + 20 g RGD ± AVE; Lipofectamine, 2 g DNA + 20 g lipid; Superfect, 2 g DNA + 30 g of the transfection reagent.

WST assay

FACS analysis of v 3 expression

One day before transfection, cells were seeded in six - well plates (800,000 cells/well). Cells were incubated for 6 hours with the transfection reagents. Lipofectamine and Lipofectin were purchased from Gibco -BRL ( Eggenstein, Germany ). Effectene and Superfect were obtained from Qiagen (Hilden, Germany ) . The concentrations of the transfection reagents were optimized for transduction efficiencies and low toxicities ( see legend to Figure 4 for details ) . The medium was replaced with normal culture medium, and WST assays (Roche, Mannheim, Germany) were performed either directly or after another 18 hours. The supernatant was withdrawn; 500 L of WST diluted 1:10 in medium was added to each well and incubated for 30 minutes at 378C. Aliquots of 100 L were transferred to 96- well plates, and the plates were read at 440 nm. All measurements were performed as triplicates, and each experiment was repeated at least three times.

Cells from a 10- cm dish were detached with 0.2% EDTA ( Roche ), washed once with PBS, and incubated with the monoclonal anti ±v 3 antibody LM609 (Chemicon Int., Hofheim, Germany ) diluted 1/ 100 in PBS with 1% FCS. After washing once with PBS, cells were stained with a Cy3 - labeled secondary antibody ( goat anti ± mouse -Cy3, F ( ab) 20 fragment; Dianova, Hamburg, Germany ) diluted 1 /200 in PBS with 1% FCS. All incubations were performed on ice for 30 minutes. PBS -washed cells were analyzed with a FACSCalibur using an excitation at 550 nm. The fluorescence was amplified linearly. A minimum of 10,000 cells was analyzed. As control, unlabeled cells and cells stained with the secondary antibody only were measured.

FACS analysis of GFP expression

Cells were grown on 10- cm dishes to 70% confluence and transfected for 1 hour with AVPs made up of 45 g of DNA and 180 g of AVE. The cells were trypsinized 23 hours later, washed once with PBS, fixed in ice - cold 75% ethanol overnight at 48C, resuspended in PBS, treated with RNase A ( Roche, Mannheim, Germany; 400 g/ mL) overnight at 48C, and stained with propidium iodide (20 g/mL ) for at least 10 minutes. The cells were analyzed by flow cytometry ( FACSCalibur; Becton Dickinson, Heidelberg, Germany ) using a laser excitation at 488 nm. Cancer Gene Therapy, Vol 8, No 2, 2001

RESULTS Construction and physico - chemical properties of AVEs

Unilamellar vesicles were prepared from dry lipid films of phosphatidylethanolamine, phosphatidylserine, and cholesterol, which represent major constituents of the HIV envelope, and extruded using a LiposoFast2 extruder. The cyclic ligand with the RGD motif 27 was coupled to the liposomes via an anchor lipid containing a glutaric acid group. Coupling efficiency was 1 mol%, corresponding to 1000 peptide ligands per liposome, as judged by coupling a fluorescent derivative of the peptide to the liposomal surface. The average size of these RGD ± AVEs was 94 nm according to photon correlation spectroscopy analysis ( Fig 1 ). The

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zeta potential was 45 mV. The AVEs could be stored for > 2 months without loss of activity and did not exhibit any size increase. Plasmid DNA was condensed by mixing with small molecular mass nonlinear PEI (or protamine sulfate for comparison ). DNA condensation was already achieved at an N /P ratio of 8, as judged by dye exclusion ( Fig 2) . At an N /P ratio of 20.7, which was chosen for all subsequent experiments, DNA condensation was complete (Fig 2) . The mean size of this complex was 146 nm (Fig 1 ) . Packaging of this PEI ± DNA plasmid complex into the RGD ±AVEs gave rise to a moderate size increase and yielded particles with a mean size of 182 nm (Fig 2) . These fully assembled particles will subsequently be referred to as artificial viral particles ( AVPs ) . The expected structure of the AVPs, i.e., the encapsulation of condensed DNA in a lipid layer within discrete particles, was confirmed by cryoelectron microscopy (Fig 3 ) . The AVPs were stable for at least 350 hours in solution as measured by photon correlation spectroscopy ( data not shown ).

Ligand - dependent interaction with ECs

Toxicity for ECs

Next, we evaluated the transduction efficiency of the RGD ± AVPs with respect to (i ) transduction efficiency, ( ii ) contribution of the ligand to EC transduction, and ( iii ) cell type specificity. For this purpose, a plasmid carrying a CMV promoter -driven nuclear green fluorescent protein ( H2BGFP ) gene 28 was packaged into RGD ± AVPs. Cells were exposed for 1 hour to the RGD ± AVPs, and the fraction of green fluorescent cells was determined 23 hours later ( Fig 6) . A quantitative evaluation of the experiment showed that the majority of cells were successfully transduced by RGD ±AVPs and expressed GFP. These experiments were performed in complete cell culture medium containing 10% FCS, indicating that the RGD ± AVPs were not inhibited by serum proteins. When

To assess potential toxicities, the RGD ±AVPs were incubated with cultures of HUVECs for 6 hours and observed for another 18 hours. No indication for RGD ± AVP ± induced cell death could be detected, as suggested by the absence of apoptotic cells identified by staining with Hoechst 33258 ( Fig 4A ). In addition, no influence on metabolic activity of the cells in the presence or absence of the RGD ± AVPs was seen, as evidenced by WST assay ( Fig 4B ) . In contrast, complexes of DNA with commercially available cationic lipids (Lipofectamine, Lipofectin ) or polymers ( Effectene, Superfect) killed the majority of the cells within the same period of time (Fig 4 and data not shown) .

HUVECs were exposed to AVPs for 1 hour with or without the RGD ligand that was labeled with a fluore scent lipid (N - ( 7- nitrobenz -2 -oxa -1,3- diazol -4 -yl ) 1,2 -dihexadecanoyl - sn -glycero- 3- phospho -ethanolamine, NBD - PE ). Cells were observed directly 1 hour after incubation with the labeled AVPs (Fig 5; 1 hour ) or after another 2 hours in normal culture medium (Fig 5; 3 hours ) under a fluorescence microscope. At both time points, a considerably stronger staining was detectable in cultures that had received the RGD ±AVPs. More than 99% of these cells were stained. Confocal microscopy also showed that the staining was not confined to the cell surface, but also occurred intracellularly, indicating endosomal uptake of the RGD ± AVPs (data not shown ). These observations demonstrate a highly efficient and selective interaction of the RGD ± AVPs with ECs. Highly efficient transduction of ECs

- RGD

+ RGD

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Figure 5. Binding of AVPs to HUVECs. HUVECs were exposed for 1 hour to NBD - PE ± labeled AVPs with and without RGD ligand and analyzed by fluorescence microscopy either directly ( 1 hour ) or after another 2 hours in normal culture medium ( 3 hours ) .

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Figure 6. Transduction of HUVECs by RGD ± AVPs carrying a CMV - H2BGFP plasmid. Cells were exposed for 1 hour to the AVPs and fluorescence microscopy was performed after another 23 hours in normal medium ( left panel ) . The right panel shows the same cells stained with Hoechst 33258. Representative pictures are shown.

protamine sulfate was used as the DNA -compacting agent instead of PEI, we observed a > 50- fold decrease in transduction efficiency. Likewise, the PEI ± DNA complex, on its own (without lipid envelope ), gave poor transduction efficiencies ( < 5% transduced cells ). These results underscore the importance of the precise composition of the RGD ± AVPs established in this study and strongly suggest that PEI fulfils other roles than merely condensing the DNA. To quantitate the results, we performed FACS analyses and, for comparison, included AVPs lacking the RGD ligand. The data in Figure 7 clearly confirm the dramatic transduction efficiency already seen in the experiment shown in Figure 6. Approximately 99% of the cells receiving RGD ± AVPs showed GFP expression. In contrast, GFP expression was seen in only 40% of cells transduced with AVPs lacking the ligand, and the level of this expression was considerably lower than that seen with the ligand -carrying AVPs. Although these results clearly indicate a role for the RGD ligand in the transduction of HUVECs, the significant extent of transduction seen with the RGD -less AVPs points to an additional mechanism of EC targeting by the AVPs.

targeting contributes to the observed EC selectivity of the RGD ± AVPs.

Cell type selectivity of transduction

Finally, we analyzed the transduction of HUVECs relative to other cell types, which lack v 3 integrin expression, as determined by immunostaining analysis using antibodies recognizing the heterodimeric v 3 integrin complex. The results of this experiment are summarized in Figures 8 and 9. The data obtained with the RGD ±AVPs clearly show that the fraction of GFP positive cells was approximately 10 -fold higher with HUVECs compared to cells expressing no or low levels of v 3 integrin, including osteosarcoma cells (Saos- 2) , prostate carcinoma cells (DU -145 ), colon carcinoma cells ( LoVo) , lung adenocarcinoma cells (A549 ) , and choriocarcinoma cells (JEG -3 ). However, we also found a preferential transduction of ECs by the RGD -less AVPs (Fig 8 ). In this case, the fraction of transduced HUVECs clearly exceeded that seen with the other cell types by a factor of 5. This observation supports the conclusion that a mechanism other than RGD -mediated Cancer Gene Therapy, Vol 8, No 2, 2001

Figure 7. FACS analysis of HUVECs 24 hours after transduction with AVPs ( RGD ligand ) carrying a CMV - H2BGFP plasmid compared to untreated cells. Cells were exposed for 1 hour to the AVPs and FACS analysis was performed after another 23 hours in normal medium.

Fraction of positive cells (% of HUVEC + RGD-AVP)

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Figure 8. Transduction of different cell types with AVPs ( A ) and RGD ± AVPs ( B ) carrying a CMV promoter H2BGFP plasmid: summary of FACS analyses determining the fraction of GFP - positive cells. Cells were exposed for 1 hour to the AVPs and FACS analysis was performed after another 23 hours in normal medium. Values were calculated relative to the transduction efficiency obtained with RGD - AVP ± transfected HUVECs ( 100% ) .

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Figure 9. FACS analysis of v 3 expression in HUVECs and different tumor cell lines after indirect immunostaining as described in Materials and Methods. Bold lines: specific staining ( first antibody: v 3 - specific; second antibody: Cy3 - labeled ) ; thin line: unspecific staining ( second antibody only ) ; dotted line: unlabeled cells. The relative fraction of cells is plotted against the intensity of fluorescence.



DISCUSSION

Nonviral, lipid - based vectors have attracted particular attention in the field of gene therapy for a number of reasons, including safety, lack of antigenicity, versatility, and ease of handling. Most studies pertaining to the development of liposomal vectors have made use of cationic lipids, mostly because of their intrinsic property to complex with, and thus condense, the negatively charged DNA.29 However, liposomes with cationic lipids undergo intense reorganization after contact with DNA, and the resulting structures barely resemble liposomes.30 However, liposomes exhibiting a neutral or negatively charged surface are unable to encapsulate sufficient amounts of plasmid DNA which has a comparable hydrodynamic diameter as the liposomes themselves.29 This problem can be circumvented by precondensing the plasmid DNA with a synthetic polycation and coating this complex with negatively charged liposomal membranes. The generation of transduction -competent particles, according to this principle, has been accomplished by using polylysine -complexed DNA in conjunction with anionic and pH -sensitive lipids, the latter being required for endosomal release of the DNA.31 There are, however, a number of drawbacks associated with this approach, such as the high toxicity of the polylysine and the serum sensitivity of the particles resulting from the presence of the pH sensitive lipid CHEMS. We have used a different strategy for the construction of anionic lipid -based vectors, i.e., condensation of DNA with PEI and packing of these complexes into anionic liposomes whose composition is based on that of human immunodeficiency virus, referred to as AVEs.32 PEI appeared to be particularly suitable for this approach because of its efficient DNA -packing properties,33 its known endosomolytic property,34 its ability to protect the complexed DNA from cytoplasmic nucleases,35,36 and its claimed potential to promote nuclear entry.37,38 In the present study, we show that condensation of plasmid DNA with low - molecular -weight, branched PEI ( Fig 1 ) and packaging of this complex into AVEs gives rise to structures (AVPs ) resembling viral particles (Fig 3 ) that are nontoxic (Fig 4) and exhibit a very high transduction efficiency for ECs in the presence of serum (Figs 6 ± 8) . One of the most crucial components of these AVPs is, indeed, the nature of the condensing agent. Thus, the exchange of the low - molecularweight, branched PEI with protamine sulfate decreased the transduction efficiency by > 50- fold. Likewise, the use of high -molecularweight PEI basically abrogated the transduction of ECs (data not shown) . This observation is in agreement with the finding that low -molecularweight, branched PEI has a low toxicity for cultured cells compared to the strongly toxic high - molecular -weight PEI.39 The AVPs designed in the present study were also endowed with a targeting device for vû3 integrins which is overexpressed on tumor ECs.8 Because cultured ECs also express this integrin (see Fig 9 ) , probably because they are in a partially activated state due to the artificial culture conditions ( our unpublished observations ), these cells represent a suitable model for targeting studies. A


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cyclic RGD -containing peptide has previously been identified by phage display techniques as ligand for tumor EC targeting, presumably through interaction with vû3 integrin.27 Our data show that the presence of this peptide on the surface of the AVPs significantly increases their ability to interact with ECs ( Fig 4 ) as well as their transduction efficiency (Figs 6 ±8 ). That the RGD peptide is instrumental in EC targeting is also suggested by the observation that cell lines lacking vû3 integrin expression ( Fig 9) showed a clearly reduced transduction ( Fig 8 ). However, our data also suggest that RGD - mediated targeting in not the only way by which EC selectivity is achieved by the RGD ±AVPs. Thus, AVPs lacking the RGD ligand were also able to transduce ECs to a significant extent ( Fig 7) , and this transduction efficiency was clearly greater than that seen with cell type lacking vû3 integrin expression ( Fig 8 ). It has previously been shown that anionic lipids, such as phosphatidylserine, can bind to lipoprotein receptors.40 Such receptors are also present in ECs, and some may be expressed in a cell type ± specific fashion. Interaction of phosphatidylserine in liposomal membranes with ECs has indeed been described,41 and the adhesion of human erythrocytes to vascular endothelium has been reported to be dramatically increased by phosphatidylserine present on the erythrocyte surface.42 It is, therefore, not unlikely that a similar mechanism contributes to the selective transduction of ECs by RGD ± AVPs which contain phosphatidylserine as a major constituent. This hypothesis will be explored in more detail in future experiments. The specific composition of the targeted AVPs, in particular the use of physiologic, anionic lipids, suggests that the vector system should also be applicable in vivo and might have some advantages over previously tested liposomal vectors. With most cationic gene delivery systems, a net positive charge promotes the unspecific interaction of the cationic complex with cell surface and plasma proteins.24,43 Opsonization, destabilization, and rapid uptake of these complexes by phagocytic cells lead to diminished transduction efficiencies in vivo. Negatively charged liposomes also interact with the biological environment in a nonspecific manner, but a liposomal gene delivery system with a net negative surface potential should exhibit less nonspecific tissue uptake and a better overall biocompatibility than cationic carrier systems.43 Taken together, our data indicate that the RGD ± AVPs are nontoxic, serum -resistant, selective for ECs, and exhibit an unprecedented transduction efficiency for ECs. In principle, the same concept should also work with other ligands and cell types, and the use of tissue - specific promoters 44 could increase the selectivity of the vector system even further. We, therefore, believe that these targeted liposomal vectors represent a true advance in the field of vector development.

ACKNOWLEDGMENTS

The authors thank Dr. H. Schreier ( Sebastopol, CA ) for useful discussions and critical reading of the manuscript;

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Drs. T. Kanda and G. Wahl ( La Jolla, CA ) for the H2BGFP plasmid; Prof. T. Kissel (Marburg) for low -molecularweight, branched PEI used in initial studies; Dr. Stark (IMT, Marburg) for Cryo -TEM analysis; Dr. Krause (IMT, Marburg) for peptide synthesis; and Dr. G. Bantus ( Halle ) for valuable suggestions and help during the optimization of the lipid anchor synthesis and peptide conjugation.

REFERENCES 1. Jain RK. Delivery of molecular and cellular medicine to solid tumors. J Controlled Release. 1998;53:49 ± 67. 2. Folkman J. Fighting cancer by attacking its blood supply. Sci Am. 1996;275:150 ± 154. 3. Boehm T, Folkman J, Browder T, O'Reilly MS. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature. 1997;390:404 ± 407. 4. Arap W, Pasqualini R, Ruoslahti E. Chemotherapy targeted to tumor vasculature. Curr Opin Oncol. 1998;10:560 ± 565. 5. Plate KH, et al. Vascular endothelial growth factor and glioma angiogenesis: coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms. Int J Cancer. 1994;59:520 ± 529. 6. Burrows FJ, et al. Up - regulation of endoglin on vascular endothelial cells in human solid tumors: implications for diagnosis and therapy. Clin Cancer Res. 1995;1:1623 ± 1634. 7. Miller DW, et al. Elevated expression of endoglin, a component of the TGF ± beta - receptor complex, correlates with proliferation of tumor endothelial cells. Int J Cancer. 1999;81:568 ± 572. 8. Ruoslahti E. Fibronectin and its integrin receptors in cancer. Adv Cancer Res. 1999;76:1 ± 20. 9. Pasqualini R, et al. Aminopeptidase N is a receptor for tumor homing peptides and a target for inhibiting angiogenesis. Cancer Res. 2000;60:722 ± 727. 10. Witte L, et al. Monoclonal antibodies targeting the VEGF receptor - 2 ( Flk1 / KDR ) as an antiangiogenic therapeutic strategy. Cancer Metastasis Rev. 1998;17:155 ± 161. 11. Ramakrishnan S, Olson TA, Bautch VL, Mohanraj D. Vascular endothelial growth factor ± toxin conjugate specifically inhibits KDR / flk - 1 ± positive endothelial cell proliferation in vitro and angiogenesis in vivo. Cancer Res. 1996;56:1324 ± 1330. 12. Klement G, et al. Continuous low - dose therapy with vinblastine and VEGF receptor - 2 antibody induces sustained tumor regression without overt toxicity. J Clin Invest. 2000;105:R15 ± R24. 13. Matsuno F, et al. Induction of lasting complete regression of preformed distinct solid tumors by targeting the tumor vasculature using two new antiendoglin monoclonal antibodies. Clin Cancer Res. 1999;5:371 ± 382. 14. Arap W, Pasqualini, R, Ruoslahti E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science. 1998;279:377 ± 380. 15. Rajotte D, et al. Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display. J Clin Invest. 1998;102:430 ± 437. 16. Colin M, et al. Liposomes enhance delivery and expression of an RGD - oligolysine gene transfer vector in human tracheal cells. Gene Ther. 1998;5:1488 ± 1498. 17. Erbacher P, Remy JS, Behr JP. Gene transfer with synthetic virus ± like particles via the integrin - mediated endocytosis pathway. Gene Ther. 1999;6:138 ± 145. 18. Vigne E, et al. RGD inclusion in the hexon monomer provides adenovirus type 5 ± based vectors with a fiber

knob - independent pathway for infection. J Virol. 1999;73: 5156 ± 5161. 19. Reynolds P, Dmitriev I, Curiel D. Insertion of an RGD motif into the HI loop of adenovirus fiber protein alters the distribution of transgene expression of the systemically administered vector. Gene Ther. 1999;6:1336 ± 1339. 20. Felgner PL, et al. Lipofection: a highly efficient, lipid mediated DNA transfection procedure. Proc Natl Acad Sci USA. 1987;84:7413 ± 7417. 21. Wagner E, et al. Influenza virus hemagglutinin HA - 2 N terminal fusogenic peptides augment gene transfer by transferrin ± polylysine ± DNA complexes: toward a synthetic virus ± like gene - transfer vehicle. Proc Natl Acad Sci USA. 1992;89: 7934 ± 7938. 22. Boussif O, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethyleneimine. Proc Natl Acad Sci USA. 1995;92:7297 ± 7301. 23. Lee RJ, Huang L. Lipidic vector systems for gene transfer. Crit Rev Ther Drug Carrier Syst. 1997;14:173 ± 206. 24. Szebeni J. The interaction of liposomes with the complement system. Crit Rev Ther Drug Carrier Syst. 1998;15:57 ± 88. 25. Kaneda Y, Saeki Y, Morishita, R. Gene therapy using HVJ liposomes: the best of both worlds? Mol Med Today. 1999;5:298 ± 303. 26. Chander, R, Schreier H. Artificial viral envelopes containing recombinant human immunodeficiency virus ( HIV ) gp160. Life Sci. 1992;50:481 ± 489. 27. Pasqualini R, Koivunen E, Ruoslahti E. Alpha v integrins as receptors for tumor targeting by circulating ligands. Nat Biotechnol. 1997;15:542 ± 546. 28. Kanda T, Sullivan KF, Wahl GM. Histone ± GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr Biol. 1998;8:377 ± 385. 29. Ledley FD. Nonviral gene therapy: the promise of genes as pharmaceutical products. Hum Gene Ther. 1995;6: 1129 ± 1144. 30. Sternberg B, Sorgi FL, Huang L. New structures in complex formation between DNA and cationic liposomes visualized by freeze - fracture electron microscopy. FEBS Lett. 1994;356: 361 ± 366. 31. Lee RJ, Huang L. Folate - targeted, anionic liposome - entrapped polylysine - condensed DNA for tumor cell ± specific gene transfer. J Biol Chem. 1996;271:8481 ± 8487. 32. Schreier H, et al. ( Patho ) physiologic pathways to drug targeting: artificial viral envelopes. J Mol Recognit. 1995;8: 59 ± 62. 33. Godbey WT, Wu KK, Hirasaki GJ, Mikos AG. Improved packing of poly ( ethyleneimine ) / DNA complexes increases transfection efficiency. Gene Ther. 1999;6:1380 ± 1388. 34. Behr J. The proton sponge: a trick to enter cells the viruses did not exploit. Chimia. 1997;51:34 ± 36. 35. Liu Y, et al. Cationic liposome - mediated intravenous gene delivery. J Biol Chem. 1995;270:24864 ± 24870. 36. Liu Y, et al. Factors influencing the efficiency of cationic liposome - mediated intravenous gene delivery. Nat Biotechnol. 1997;15:167 ± 173. 37. Pollard H, et al. Polyethyleneimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. J Biol Chem. 1998;273:7507 ± 7511. 38. Godbey WT, Wu KK, Mikos AG. Tracking the intracellular path of poly ( ethyleneimine ) / DNA complexes for gene delivery. Proc Natl Acad Sci USA. 1999;96:5177 ± 5181. 39. Fischer D, et al. A novel nonviral vector for DNA delivery based on low - molecular - weight, branched polyethyleneimine: effect of molecular weight on transfection efficiency and cytotoxicity. Pharm Res. 1999;16:1273 ± 1279.



40. Fadok VA, et al. The role of phosphatidylserine in recognition of apoptotic cells by phagocytes. Cell Death Differ. 1998;5: 551 ± 562. 41. Kamps JA, Morselt HW, Scherphof GL. Uptake of liposomes containing phosphatidylserine by liver cells in vivo and by sinusoidal liver cells in primary culture: in vivo ± in vitro differences. Biochem Biophys Res Commun. 1999;256:57 ± 62. 42. Closse C, Dachary - Prigent J, Boisseau MR. Phosphatidylserine - related adhesion of human erythrocytes to vascular endothelium. Br J Haematol. 1999;107:300 ± 302. 43. Roerdink F, Wassef NM, Richardson EC, Alving CR. Effects of negatively charged lipids on phagocytosis of liposomes opsonized by complement. Biochim Biophys Acta. 1983;734: 33 ± 39. 44. Nettelbeck DM, Jerome V, Muller R. Gene therapy: designer promoters for tumour targeting. Trends Genet.2000;16:174 ± 181. 45. Ahl PL, et al. Enhancement of the in vivo circulation lifetime of L - alpha - distearoylphosphatidylcholine liposomes: importance of liposomal aggregation versus complement opsonization. Biochim Biophys Acta. 1997;1329:370 ± 382.


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46. MacDonald RC, et al. Small - volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim Biophys Acta. 1991;1061:297 ± 303. 47. Weissig V, Lasch J, Klibanov AL, Torchilin VP. A new hydrophobic anchor for the attachment of proteins to liposomal membranes. FEBS Lett. 1986;202:86 ± 90. 48. Blakeslee D, Baines MG. Immunofluorescence using dichlorotriazinylaminofluorescein ( DTAF ) : I. Preparation and fractionation of labelled IgG. J Immunol Methods. 1976;13:305. 49. Fahr A, Butt H - J, Nelles G, Welz C. Biophysical aspects of nonviral gene delivery systems. J Liposome Res. 1998;8: 9 ± 12. 50. Ruf H, Georgalis Y, Grell E. Dynamic laser light scattering to determine size distributions of vesicles. Methods Enzymol. 1989;172:364 ± 390. 51. Jaffe E. In: Jaffe E, ed. Biology of Endothelial Cells. Boston: Martinus Nijhoff; 1986:1 ± 13. 52. Thornton SC, Mueller SN, Levine EM. Human endothelial cells: use of heparin in cloning and long - term serial cultivation. Science. 1983;222:623 ± 625.