D2001 Nature Publishing Group 0929-1903/01/$17.00/+0 www.nature.com/cgt 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 1Institute 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 AVEtechnology 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. mong the new experimental approaches to cancer Examples are vascular endothelial growth factor receptor II Atreatment, gene therapy has gained particular attention. (FLK-1, KDR),5 transforming growth factor binding 6,7 8 Clinical studies showed that cancer gene therapy can protein CD105/endoglin, vû3 integrin, and CD13/ potentially be made to work, but the lack of adequate aminopeptidase N.9 That these surface molecules can be technologies is evident. This applies, in particular, to the used for the successful targeting of drugs to the tumor blood efficiency and specificity of tumor cell transduction, vessels has been demonstrated in several studies. Thus, indicating that the design of more efficacious and site- antiangiogenic strategies have made use of antibodies selective vectors is urgently required. A second serious neutralizing KDR/FLK-1;10 ± 12 antibodies directed to problem is the poor accessibility of many tumor cells to any endoglin have been employed to direct a ricin A conjugate kind of drug, in particular vectors carrying nucleic acids, as a to the tumor vasculature;13 and a cyclic RGD peptide has been consequence of the defective tumor vasculature and the high used to target doxorubicin to the tumor site.14 Cyclic interstitial pressure.1 The concept of endothelial tumor cell RGD peptides have also been used successfully for the targeting2 is attractive because the tumor blood vessels are delivery of both viral and nonviral vectors to ECs,15 ± 19 more readily accessible than the actual tumor cell compart- and therefore, seem to be suitable for tumor EC targeting. ment. In addition, endothelial cells (ECs) are unknown to The vasculature is a highly attractive target also for gene acquire resistance to treatment,3 and the endothelium therapy because it is comprised of a large number of represents a target that is largely independent of tumor potential target cells and is easily accessible via the blood type.4 Finally, as tumor ECs are essential for the nutrition stream. Ideally, an EC-directed vector should be safe, and growth of the tumor cells, any therapy targeting tumor nontoxic, nonimmunogenic, and cell type±specific; it should ECs should have a dramatic ``bystander effect''. transduce EC cells with high efficiency; and Ð with respect Several marker proteins up-regulated in tumor endothe- to clinical applicability Ð it should offer the possibility of lium have been identified. Among these membrane- large-scale production, stability of the product, and ease of associated proteins, some are of particular interest because handling. To date, a vector combining all these features has they can be exploited in targeting the tumor vasculature. not been described. Viral vectors are often highly efficient, but safety and Received June 23, 2000; accepted November 7, 2000. immunogenicity are issues of potential concern, and the Address correspondence and reprint requests to Dr. Rolf MuÈller, Institute limited transgene size often poses a serious obstacle. of Molecular Biology and Tumor Research (IMT), Philipps University, Nonviral vectors, on the other hand, frequently face the Marburg 35033, Germany. E-mail address: [email protected] problem of low transduction efficiency. Different concepts Cancer Gene Therapy, Vol 8, No 2, 2001: pp 107±117 107 108 MUÈ LLER, NAHDE, FAHR, ET AL: TRANSDUCTION OF ENDOTHELIAL CELLS BY TARGETED ANIONIC LIPOSOMES have been developed for the generation of nonviral vectors, lamellar liposome suspension was placed in a 3-mL glass e.g., cationic lipid±DNA complexes,20 polycation±DNA vessel cooled by an ice/water mixture and sonicated for 15 complexes,21,22 and liposome-entrapped polycation-con- seconds in an MSE-Soniprep 150 (Zivy, Oberwil, Switzer- densed DNA (LPD).23 However, these complexes are often land) equipped with a titanium tip. Sonication was repeated physico-chemically or biologically unstable, serum-sensi- 10 times; each sonication period was followed by a 30- tive, or toxic as a consequence of unspecific interactions with second pause, allowing the suspension to cool. The the biological environment.23,24 Promising results have been resulting liposome suspension was extruded through obtained with Sendai virus±fused liposomes (HVJ lipo- polycarbonate membrane filters with a pore size of 50 nm somes),25 but a potential problem may be the presence of using a standard device (LiposoFast2 ) 46 purchased from viral proteins in these vectors and their large size (400 nm). Avestin (Ottawa, Canada). The liposomes were used up 2 An attractive alternative is the use of artificial virus-like months after preparation, during which time no significant envelopes (AVEs) for the encapsulation of condensed increase in liposome size could be detected. plasmid DNA.26 AVEs mimic the lipid composition of retroviruses. These natural lipids are anionic and, in contrast Targeting motif to their artificial cationic counterparts, interact only weakly Using solid-phase synthesis (Applied Biosystems Peptide with their biological environment and therefore are nontoxic. Synthesizer, Foster City, CA), a cyclic peptide with the We have taken this approach further by combining AVEs amino acid sequence CDCRGDCFC and having an with the advantages of the cationic polymer, polyethyle- 22 additional arginine at the N-terminus was synthesized. neimine (PEI), which not only condenses the DNA but Cyclic condensation of the peptide was accomplished by is also believed to act as an endosomolytic agent and to stirring an aqueous solution of the synthesized peptide under protect the DNA from cytoplasmic nucleases. In addition, access of ambient air. Completion of cyclic condensation we have equipped these artificial viral particles (AVPs) was verified by high-performance liquid chromatography. with a targeting device for activated ECs, i.e., a cyclic 27 After high-performance liquid chromatography purification, RGD peptide thought to interact with v 3 integrin. We the peptide was lyophilized and stored at 48C. refer to these targeted nonviral vectors as RGD±AVPs. Covalent attachment of RGD peptide to liposomal surface (RGD±AVE) MATERIALS AND METHODS Activation of the N-glutaryl-DPPE carboxyl group at the AVEs liposomal surface 47 was achieved by adding 3.5 mg of 1- Throughout the preparation of AVEs,synthetic phospholipids ethyl-3- (3-dimethylaminopropyl) carbodiimide to 400 L were used without further purification. 1,2-Dipalmitoleoyl- AVE and shaking the suspension for 5 hours in the dark. The sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-dio- resulting active O-acyl-intermediate reacted with added leoyl-sn-glycero-3- [phospho- L -serine] (DOPS) were RGD peptide (250 g in 150 L buffer) overnight, yielding purchased from Avanti Polar Lipids (Alabaster, AL); 1,2- a covalent coupling of the peptide to the liposomal surface. dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) was The RGD±AVEs were separated from unbound peptide by obtained from Genzyme (Liestal, Switzerland); and choles- Sephadex G25 gel permeation chromatography in Tris buffer terol was from Calbiochem (San Diego, CA). All other 10 mM, pH 7.4. Coupling efficiency was monitored using a substances were of analytical grade. fluorescently labeled derivative of the RGD peptide. For this purpose, RGD was conjugated with 5- (4,6-dichlorotriazi- Synthesis of lipid anchor nyl)aminofluorescein (5-DTAF) 48 purchased from Mole- N-glutaryl-DPPE was prepared by dissolving DPPE in cular Probes (Eugene, OR). 5-DTAF was dissolved in anhydrous chloroform. Glutaric anhydride and water-free borate buffer (pH 9) and RGD peptide