Gene Therapy (2002) 9, 1542–1550 2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt RESEARCH ARTICLE Folate-targeted, cationic liposome-mediated gene transfer into disseminated peritoneal tumors JA Reddy1, C Abburi1, H Hofland2, SJ Howard1, I Vlahov1, P Wils2 and CP Leamon1 1Endocyte, Inc., West Lafayette, IN, USA; and 2Gencell, Aventis Pharma, Hayward, CA, USA A folate-targeted, cationic lipid based transfection complex cated that as little as 0.01 to 0.3% of FA-Cys-PEG-PE was was developed and found to specifically transfect folate needed to produce optimal targeted expression of plasmid receptor-expressing cells and tumors. These liposomal vec- DNA. Similarly, using a disseminated intraperitoneal L1210A tors were comprised of protamine-condensed plasmid DNA, tumor model, maximum in vivo transfection activity occurred a mixture of cationic and neutral lipids, and a folic acid-cyst- with intraperitoneally administered formulations that con- eine-polyethyleneglycol-phosphatidylethanolamine (FA-Cys- tained low amounts (0.01 mol%) of the FA-Cys-PEG-PE tar- PEG-PE) conjugate. Pre-optimization studies revealed that geting lipid. Overall, folate-labeled formulations produced an inclusion of low amounts (0.01 to 0.03%) of FA-Cys-PEG- eight- to 10-fold increase in tumor-associated luciferase PE yielded the highest binding activity of expression, as compared with the corresponding non-tar- dioleoylphosphatidylcholine/cholesterol liposomes to folate geted cationic lipid/DNA formulations. These results collec- receptor-bearing cells. In contrast, higher amounts (>0.5%) tively indicate that transfection of widespread intraperitoneal of FA-Cys-PEG-PE progressively decreased cellular binding cancers can be significantly enhanced using folate-tar- of the liposomes. In vitro studies with cationic geted techniques. lipid/dioleoylphosphatidylethanolamine formulations indi- Gene Therapy (2002) 9, 1542–1550. doi:10.1038/sj.gt.3301833 Keywords: cationic liposome; gene therapy; tumor targeting; folate receptor; lipoplex Introduction (FA),12–15 transferrin16 or carbohydrates17 to liposomes for the purpose of combining the intrinsic activities of lipids Gene therapy has become an increasingly important with the receptor-mediated uptake properties of the strategy for treating a variety of human diseases, includ- applied ligand. ing inherited disorders, viral infections and cancer. A We have been interested in the use of folic acid (FA) number of studies have shown that introduction of vari- as a targeting ligand to deliver attached therapeutic and ous genes into tumor cells can lead to suppression of imaging agents to cancer cells that over-express the 1 tumor growth. A common method for delivering genetic receptor for FA.18–20 Because FA-linked cargos are material into cells is that which uses cationic liposomes. efficiently bound and internalized by folate receptor (FR)- 2 Since Brigham and co-workers first reported this tech- expressing cells, we have explored the possibility of nique, the use of cationic lipids complexed to plasmid using FA to enhance cationic liposomal vector delivery expression vectors has become one of the most promising to FR-enriched tumors. Thus, in the present study we 1,3,4 non-viral methods for in vivo gene delivery. investigated the opportunity for targeting a syngeneic Although cationic liposomal vectors mediate effective murine tumor in vivo by the regional administration of gene transfer, tissue specific in vivo DNA delivery is still a FA-labeled cationic liposomes. For this purpose, we first major challenge for gene therapy. To date, tissue-specific optimized and compared the amount of FA ligand targeting of cationic liposomal DNA has been required for optimal targeting of both neutral and cat- accomplished by two distinct techniques. The first ionic liposomes to FR-bearing cells. We then used L1210A method involves transfection of selected tissues, such as intraperitoneal tumor-bearing DBA mice to investigate 5 6 7,8 nasal epithelium, arterial endothelium, lung or the in vivo targeting potential of our FA-labeled vectors. tumors9 by locally administering the complexes within defined regions. This method has proven to be a viable option for the clinical treatment of several diseases, Results including cystic fibrosis and cancer.5,10,11 A second method used to enhance the specificity of gene therapy Optimization of folate-mediated liposome targeting is by coupling cell-binding ligands, such as folic acid Previous studies21 with FA-targeted liposomes had indi- cated that low mole fractions of a folate-polyethylenegly- col-phosphatidylethanolamine (FA-PEG-PE) conjugate Correspondence: CP Leamon, 1205 Kent Avenue, West Lafayette, IN were needed to successfully mediate liposome binding 47906, USA by FR-bearing cells. However, no investigation was ever Received 22 March 2002; accepted 16 June 2002 conducted to optimize the mole fraction of this targeting Folate-targeted gene delivery JA Reddy et al 1543 = Figure 1 Chemical structures of RPR209120, Folate-Cys-PEG3400-DSPE and Folate-Dap-Asp-Cys-PEG3400-DSPE (n 75 for PEG3400). component. Although we would have preferred to optimize this parameter using cationic liposome formu- lations, we felt that their inherent propensity to non- specifically interact with cell membranes would have adversely affected our ability to measure folate-specific cell binding. Accordingly, we determined the optimal amount of a related FA-cysteine-PEG3400-distearylphos- phatidylethanolamine (FA-Cys-PEG-PE; see Figure 1) conjugate for use in 65:35 dioleoylphosphatidylcholine/ cholesterol (DOPC/Chol) liposomes. Increasing concentrations of FA-Cys-PEG-PE were for- mulated within calcein-loaded liposomes. Formulations were then incubated with FR-positive M109 cells to allow liposome binding, and the amount of cell-associated flu- orescence was quantitated. As shown in Figure 2a, uptake of the fluorescent liposomes was found to criti- cally depend on the amount of FA-Cys-PEG-PE formu- lated in these vesicles. Cell-associated calcein fluor- escence was found to increase with increasing concentrations of FA-Cys-PEG-PE until a maximum was reached at ~0.03 mole %. However, higher mole fractions of FA-Cys-PEG-PE surprisingly caused a reduction of liposome uptake. The aforementioned observation was unexpected, but it prompted us to search for a plausible explanation. It is known that the folate molecule can form dimers, trimers and even self-assembling tubular quartets at higher con- centrations.22 Since the FR can only bind one molecule of FA,23 such self-assembled multimers of FA would be incapable of binding to the FR. Thus, we postulated that the FA moieties in liposomes containing high mole frac- tions of FA-Cys-PEG-PE might actually self-assemble, thereby preventing efficient interaction with the FR. To address this issue, we conducted additional experiments that were aimed to reduce or prevent the FA moieties from interacting with each other. First, we prepared DOPC/Chol calcein liposomes with increasing amounts Figure 2 Influence of FA-Cys-PEG3400-PE and PEG2000-PE components of the non-targetable PEG3000-DOPE component plus the on PC/Chol liposome targeting. M109 cells were incubated with fluor- apparent optimum mole percent of FA-Cys-PEG-PE escent FA-derivatized liposomes for 4 h at 37°C in the presence (ᮀ)or ᭿ (0.03%), and then incubated them with cultured M109 absence ( ) of 1 mM folic acid. Cells were then washed three times in cells. As shown in Figure 2b, M109 cells had efficiently PBS, pH 7.4, lysed in 1% Triton X-100 and cell-associated fluorescence was quantitated using a fluorimeter. (a) PC/Chol liposomes formulated bound the fluorescent FA-linked liposomes, and the pres- with increasing concentrations of FA-Cys-PEG3400-PE; (b) PC/Chol lipo- ence of up to 2 mole % of the non-targetable PEG3000- somes formulated with 0.03 mol % of FA-Cys-PEG3400-PE and increasing DOPE did not reduce their affinity for the FR-positive concentrations of PEG2000-PE. Gene Therapy Folate-targeted gene delivery JA Reddy et al 1544 cultured cells. We interpret these results to suggest that PEG-PE molecules should be available to bind to the FR the PEG component of FA-Cys-PEG-PE was not respon- when present in the liposome at Յ0.033 mole %. Interest- sible for the observed reduction in cell association of lipo- ingly, these predictions have seemingly been confirmed somes formulated with high mole percentages of FA-Cys- in our studies (see Figure 2a), where the greatest binding PEG-PE. of liposomes to FR-positive cells was observed at 0.03% Next, in an effort to reduce the potential interactions FA-Cys-PEG-PE. At concentrations greater than 0.033 of neighboring folates, perhaps via a charge repulsion mole %, the probability of folates interacting or multimer- mechanism, we synthesized a new FA-PEG-lipid conju- izing with each other likely increased with increasing gate that contained a few additional charged residues in- amounts of FA-Cys-PEG-PE, thus causing the observed between the PEG and folate moieties. Thus, the novel reduction of liposome binding to the FR-positive cells. folate-diaminopropionyl-aspartyl-cysteinyl-PEG-DOPE (FA-Dap-Asp-Cys-PEG-DOPE) component was prepared Optimization of cationic liposome/DNA complex for (see Figure 1). This conjugate was formulated at 0.01 to transgene expression in vivo 2 mole % into DOPC/Chol calcein-loaded liposomes, Before the onset of our folate-targeted in vitro and in vivo which were subsequently incubated with M109 cells for gene therapy investigations, we decided to determine the the assessment of receptor-mediated uptake. Interest- optimal formulation ratio of cationic lipid and DNA that ingly, the pattern of cell association of the FA-Dap-Asp- could manifest the greatest transfection activity in tissues. Cys-PEG-liposomes was very similar to that of FA-Cys- To accomplish this task we chose to use the novel PEG-liposomes (data not shown). Thus, decreased lipo- RPR20912026,27/DOPE formulation for DNA com- some binding to cells resulted for those formulations plexation.