Neutral Postgrafted Colloidal Particles for Gene ... - ACS Publications

Bioconjugate Chem. 2005, 16, 608−614

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Neutral Postgrafted Colloidal Particles for Gene Delivery B. Thompson,† N. Mignet,†,* H. Hofland,‡ D. Lamons,‡ J. Seguin,† C. Nicolazzi,† N. de la Figuera,† R. L. Kuen,§ X. Y. Meng,‡ D. Scherman,† and M. Bessodes† CNRS-UMR7001/ENSCP/Gencell S.A., Aventis Pharma, 13 Quai Jules Guesdes, 94403 Vitry-Seine, France, Gencell, Aventis Pharma, 3825 Bay Center Place, Hayward, California, 94545, and Cellular and Molecular Imagery Service, Faculte´ de Pharmacie, 4 avenue de l’Observatoire, 75270 Paris Cedex 06, France.. Received March 28, 2004; Revised Manuscript Received December 3, 2004

Surface modification of cationic lipoplexes has been carried out by means of a postgrafting reaction. The original lipoplexes described comprise a cationic lipid, a neutral lipid, poly(ethylene glycol)cholesterol (with or without a targeting ligand) and DNA. Modifying their surface via a chemical, postgrafting reaction did not alter their size (∼100 nm) nor their ability to compact DNA, but did give a reduced zeta potential (∼0 mV) to afford surface neutral particles. With the modified lipoplexes nonspecific NIH3T3 cell surface binding in vitro was inhibited. Intravenous injection of the neutralized lipoplexes in mice showed decreased accumulation of the particles in the lung as compared to PEGylated cationic lipoplexes. Tumor targeting was also achieved in vivo by the addition of an RGDPEG-Cholesterol as a lipid-ligand in the postgrafted lipoplex formulation.

INTRODUCTION

Liposomes have been widely investigated for their potential clinical use, as they can be used as vectors for a various range of compounds. To allow them to efficiently associate with DNA, cationic lipids have been introduced to form the lipid bilayer. Cationic lipoplexes have been part of an expanding research area over the past decade due to their favorable DNA association as well as their cellular interactions (1). However, while they have proven very efficient in vitro they have suffered from problems in vivo, namely rapid elimination from the circulation due to interactions with proteins, opsonins, and the reticuloendothelial system (RES)1 (2). Methods to help alleviate these problems have included the incorporation of poly(ethylene glycol) (PEG) in liposome preparation. PEG, when grafted onto a lipid or cholesterol moiety and included in liposome preparation, offers colloidal stability due to its bulky, hydrophilic chain (3). Lipid-PEG has also shown to be useful at masking some of the positive charges on the surface of cationic lipoplexes. However, while PEG does indeed mask some of the positive charges, it has been found that the particles are still subject to unfavorable interactions with blood components and continue to suffer from rapid * Corresponding author. Unite´ de Pharmacologie Chimique et Ge´ne´tique, Inserm U640 - CNRS UMR 8151, Faculte´ de Pharmacie, 4 avenue de l’Observatoire, 75270 Paris cedex 06, France. [email protected]; Tel +33 (0)1 53 73 95 81; Fax +33 (0)1 43 26 69 18. † CNRS-UMR7001/ENSCP/Gencell S.A., Aventis Pharma. ‡ Gencell, Aventis Pharma. § Cellular and Molecular Imagery Service. 1 Abbreviations: CAT, chloramphenicol acetyltransferase; Chol, cholesterol; DOPE, dioleoylphosphatidylethanolamine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; ELISA, enzymelinked immunosorbent assay; EtBr, ethidium bromide; NHS, N-hydroxysuccinimide; PEG, poly(ethylene glycol); RES, reticuloendothelial system; RPR209120B, 2-(3-[bis(3-aminopropyl)amino]propylamino)-N-ditetradecylethanolamine; TEM, transmission electron microscopy.

elimination from the circulation (4). The reduction of positive charges has been achieved by the addition of negatively charged PEG-lipids which helps to afford a more neutral surface charge and allows for enhanced stability in serum (5). Other methods employed to increase the efficacity of lipoplexes in vivo have been to include a targeting moiety in conjunction with PEG-lipids in liposome preparation. One recent example includes the addition of a PEGylated, folate-bearing lipid into liposome formulation, in which folate-targeted complexes restored gene transfer activity in tumors but where the activity in the lung was reduced as compared to nontargeted lipoplexes (6). In the case of immunoliposomes, it was found that coupling an antibody to the distal ends of PEG allowed for improved target binding ability (7). Some of the more recent and efficient models have involved the preparation of PEG derivatives that are available for surface functionalization by a coupling reaction with target-specific antibodies after liposome formation. Several different chemistries have been used with success for such “postgrafting” strategies (8-11). In our case, taking advantage of the lipid cationic charges to associate DNA, grafting was carried out postlipoplex formation. Our goal was to prepare surface neutral particles by means of a “postgrafting” reaction on the surface of preformed lipoplexes. The neutrality of the lipoplexes was expected to render the particles more stable with regard to their blood circulation lifetime, which would then lead to their passive accumulation in tumor tissue. MATERIALS AND METHODS

Materials. L-R-Dioleoylphosphatidylethanolamine (DOPE), L-R-dioleoylphosphatidylethanolamine-lissamine rhodamine B (DOPE-lissamine rhodamine B), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Avanti Polar Lipids. Cationic lipid 2-(3[bis(3-aminopropyl)amino]propylamino)-N-ditetradecylcarbamoylmethylacetamide (RPR209120B) (12), cholesteryl carbonate-poly(ethylene glycol)42-OH (Chol-PEG42),

10.1021/bc040244z CCC: $30.25 © 2005 American Chemical Society Published on Web 04/30/2005

Neutral Postgrafted Colloidal Particles

and cholesteryl carbamate-poly(ethylene glycol)75(ACDCRGDCFCG)-COOH (Chol-PEG75-RGD-4C) (13) were synthesized as described in the Supporting Information. Organic solvents chloroform and ethanol were purchased from Merck. H2O for injection was purchased from Cooperative Pharmaceutique Franc¸ aise. HEPES was purchased from Sigma. Sulfo-NHS acetate and SlideA-Lyzer dialysis cassettes were purchased from Pierce. Preparation of Cationic Liposomes (thin-film method). The cationic lipid RPR209120B (16.82 mg, 20 µmol), DOPE (14.88 mg, 20 µmol), and DOPE-lissamine rhodamine B (0.52 mg, 0.4 µmol, 1% total lipid) were dissolved in chloroform, and the solvent was removed under reduced pressure on a rotary evaporator. The resulting thin film was further dried on the rotary evaporator at 5 mbar for 2 h in order to remove any remaining solvent. The film was rehydrated in 2 mL of H2O for injection by gentle rotation overnight at room temperature to afford a final concentration of 10 mM. The particles were sonicated to afford a homogeneous size distribution of approximately 100-150 nm as measured by dynamic light scattering on a Coulter N4 Plus (Coulter, Beckman). Preparation of Cationic Liposomes (ethanol injection method). The cationic lipid RPR209120B (16.82 mg, 20 µmol), DPPC (14.68 mg, 20 µmol), and DOPElissamine rhodamine B (0.52 mg, 0.4 µmol, 1% total lipid) were dissolved in absol ethanol. Their total volume was adjusted to 3.5 mL by the further addition of ethanol. This mixture was then added dropwise to 35 mL of H2O with vigorous stirring and left to stir overnight. The excess solvent was removed under reduced pressure on a rotary evaporator until only a 2 mL quantity remained (corresponding to a concentration of 10 mM). The size of the resulting lipoplexes is of the order of 90-100 nm. Postinsertion of Chol-PEG42 in Cationic Liposomes. To functionalize the surface of the liposomes, all of the PEG-lipids were introduced via a postinsertion method. The Chol-PEG42 (105 µL, 0.125 µmol) was dissolved in 40 mM HEPES pH 7.5 and added to the cationic liposomes (295 µL, 2.5 µmol) mixture dropwise with vortexing and left to incubate at 37 °C for 2 h. Preparation of DNA/Vector Lipoplexes. Lipoplexes were prepared in 5% glucose with a charge ratio (+/-) ) 5. Plasmid DNA (pCMVCAT) (500 µL, 0.5 g/L, 10% glucose) was added to RPR209120B/DOPE/Chol-PEG42 or RPR209120/DPPC/Chol-PEG42 cationic liposomes (400 µL, 5 mM total lipids) dropwise with constant vortexing. HEPES (100 µL, 1 M, pH 7.5) was then added to the complex with rapid vortexing, and the samples were left to incubate for 24 h at room temperature. The insertion of lipid-PEG into the cationic liposome prior to DNA addition was assessed by an indirect method: indeed, if the lipid-PEG was not inserted into the outer bilayer of the liposome, the complex would aggregate upon the addition of 10 mol equiv of sulfo-NHS acetate per mole of RPR209120B (i.e. 5 equiv of sulfoNHS acetate per primary amine of RPR209120B). Reaction of the Lipoplexes with Sulfo-NHS Acetate. Sulfo-NHS acetate (3.24 mg, 10 equiv, 12.5 µmol) was added to lipoplex RPR209120B/DOPE/Chol-PEG42/ DNA or RPR209120B/DPPC/Chol-PEG42/DNA (1 mL, 1 equiv, 1.25 µmol RPR209120B) buffered with 100 mM HEPES at pH 7.5. The reagent was added as a powder, and the resulting mixture was gently mixed by inverting the reaction tube several times to aid dissolution. The reaction mixture was left to incubate for 1 h at room temperature with occasional gentle mixing. The reaction complex was then transferred to Slide-A-Lyzer dialysis

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cassettes and dialyzed overnight against 5% glucose and 100 mM HEPES. Preparation of Targeting Postgrafted Lipoplexes. First, Chol-PEG42 lipids (105 µL, 0.125 µmol) were dissolved in 40 mM HEPES pH 7.5 and added to the cationic liposomes (190 µL, 2.5 µmol) mixture dropwise with vortexing and left to incubate at 37 °C for 2 h. Then, plasmid DNA (pCMVCAT) (500 µL, 0.5 g/L, 10% glucose) was added to RPR209120B/DOPE/Chol-PEG42 or RPR209120/DPPC/Chol-PEG42 cationic liposomes (295 µL, 5 mM total lipids) dropwise with constant vortexing. HEPES (100 µL, 1 M, pH 7.5) was then added to the complex with rapid vortexing, and the samples were left to incubate for 24 h at room temperature. Sulfo-NHS acetate (3.24 mg, 10 equiv, 12.5 µmol) was added to the lipoplex (895 µL, 1 equiv, 1.25 µmol of RPR209120B) buffered with 100 mM HEPES at pH 7.5; the lipoplexes were incubated and purified as described above. Finally, to prepare the RGD lipoplexes, the Chol-PEG75-RGD4C was dissolved in 40 mM HEPES pH 7.5 (105 µL, 0.125 µmol) and was added to postgrafted lipoplexes (895 µL) with vortexing, and the samples were left to incubate for 24 h at room temperature. The Chol-PEG75-RGD-4C was inserted postacetate reaction and postdialysis in order to avoid any unwanted side-reactions taking place. Size of Complexes/Zeta Potential of Complexes. The size of the complexes was determined by dynamic light scattering on a Coulter N4 Plus. Typically 10 µL of complex was added to 800 µL of H2O for injection for each sample measured, mean value are given for 90°, 1 min equilibration, 3 min run. The zeta potential measurements were performed on a Malvern Zetasizer 3000. Samples of 1 mL containing 100 µL of complex (5 mM) in 20 mM NaCl were injected in the cell and subjected to 5 × 8 s measurements. Transmission Electronic Microscopy. The lipoplexes prepared as described (Concentration divided by 10) were loaded on a Formvar/carbon copper grid 200 mesh from Agar Scientific. Uranyl acetate was used to stain the samples. Analyses were performed on a microscope JEOL, JEM 100S. In Vivo Distribution in Normal or Tumor-Bearing Mice. Six-week-old female C57B1/6 mice (Charles River) were anesthetized by intraperitoneal injection of a mix of ketamine (85.8 mg/kg; Merial) and xylazine (3.1 mg/kg; Bayer) diluted in 150 mM NaCl. A 200 µL volume of rhodamine-labeled lipoplexes (50 µg of DNA, 250 nmol cationic lipids in 5% glucose) was injected into the mouse tail vein. Blood was collected by cardiac puncture at appropriate intervals (30 min, 1, 2, and 6 h after injection), and mice were euthanized at the end of the sample taking. In the distribution study, mice were sacrificed at 30 min and at 1, 2, and 6 h after injection and the liver, spleen, and lungs were removed, weighed, and homogenized in pH 7.4 phosphate-buffered saline (PBS, 5 mL/g tissue). Experiments were conducted following NIH recommendations for animal experimentation and Aventis local ethic committee on animal care and experimentation. Rhodamine-labeled lipids were extracted according to Takeuchi et al. (14) from 100 µL of blood or tissue homogenates with 3 mL of CHCl3-MeOH (1:1, v/v) by vigorous mixing during 30 min for blood and 40 min for tissue homogenates followed by centrifugation (3000 rpm, 10 min). The fluorescence intensity corresponding to DOPE-rhodamine was assayed on the supernatant with a fluorospectrophometer (Fluoromax 2, Jobin Yvon, λexc 550 nm/λem 590 nm). The amount of lipoplexes in the blood or tissue homogenates was evaluated with a

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Scheme 1. Illustration of the Postgrafting Strategy with Sulfo-NHS Acetate

calibration curve of the initial lipoplexes containing 1% DOPE-rhodamine and expressed as the remaining percentage of injected dose. Lewis lung and colon carcinoma tumors kindly provided by Aventis Pharma (Vitry/Seine, France) were implanted subcutaneously in the right flank of six-weekold female C57B1/6 mice (Charles River), and 10-14 days later, biodistribution studies were undertaken as previously described. Reporter Gene Expression in Lungs and PO2 Colon Carcinoma. Six-week-old female C57B/16 mice (Charles River) were anesthetized by intraperitoneal injection of a mix of ketamine (85.8 mg/kg) and xylazine (3.1 mg/kg) diluted in 150 mM NaCl. A 200 µL volume of rhodamine-labeled lipoplexes (50 µg of DNA, 250 nmol of cationic lipids in 5% glucose) was injected into the mouse tail vein. Mice were sacrificed at 24 h postinjection, and lungs and PO2 colon carcinoma were harvested, weighed, and homogenized in 1 mL of PBS using an Ultra Thurax (Diax 600, Heidolph, Fisher). The samples were centrifuged (1000 rpm, 30 min, 4 °C), and the amount of Chloramphenicol acetyl transferase (CAT) transgene expressed was determined using a standard enzymelinked immunosorbent assay (ELISA) kit (Roche Diagnostics). RESULTS

Postgrafting Method. Reaction of Cationic PEGylated Lipoplexes with Sulfo-NHS Acetate. SulfoNHS acetate is traditionally used on proteins to irreversibly block primary amines and form stable amides; it is very reactive toward primary amines between pH 7-9 and less reactive toward secondary amines. The postgrafting method consists of DNA/vector lipoplex preformation followed by reaction with an excess of sulfoNHS acetate to mask the cationic charges (Scheme 1). One can predict that masking all cationic charges on the lipoplex will lead to particle aggregation due to a lack of electrostatic repulsion. The insertion of a PEG-lipid in lipoplex formulation is therefore necessary to maintain particles with a small diameter, and we have used cholesterol-PEG42 (Chol-PEG42) in the present study. The pH at which the postgrafting experiments are carried out is low enough to prevent the acetylation of DNA (a pH between 8.5 and 9.0 is required for DNA acylation). Reactions can be carried out in a buffered medium, and they proceed readily at room temperature. The successive steps involved in lipoplex preparation used in the present postgrafting study are shown (Scheme 2), and the non-

commercial lipids used in their formulation are illustrated (Scheme 3). To optimize the reaction conditions for postgrafting sulfo-NHS acetate on lipoplexes, initial experiments were carried out with various molar ratios. The following molar equivalents (1:1, 5:1, 10:1, and 50:1) of sulfo-NHS acetate versus cationic lipid were investigated (Table 1). The size of the unreacted PEGylated particles, prepared by the thin-film method, was approximately 131 nm, and their zeta potential was +16.42 mV. These results are in good agreement with previous reports. It can be shown that while the addition of PEG-lipid in lipoplex preparation does reduce the zeta potential of the particles, it does not completely mask the surface charges, and residual positive charges can still be measured (5). The size of the postgrafted particles gradually increased to approximately 189 nm (with 50 mol of sulfo-NHS acetate per mole of RPR209120B), and their zeta potential was reduced to approximately zero. In the case where lipoplexes were reacted with 50 equiv of sulfo-NHS acetate, the zeta potential of the particles was reversed to -15 mV, and we could see a partial DNA release on agarose gel electrophoresis as indicated by the ethidium bromide (EtBr) fluorescence increase (Table 1). The particles, both modified and unmodified, were stable for more than 60 days with respect to their size (approximately 150 nm for both types of particles) and their zeta potential (approximately +5 mV for particles modified with 10 mol of sulfo-NHS acetate per mole of RPR209120B and approximately +12 mV for unmodified particles) (data not shown). Acetylation Yield of Postgrafted Complexes. The lipids of postgrafted complexes were extracted and subjected to analysis (experimental details can be found in the Supporting Information). The degree of acetylation of lipid RPR209120B in the complexes was evaluated by LC-MS and it was found that the postgrafting procedure produced a mixture of compounds. Two corresponding peaks were determined, of which the major peak (65%) contained a mixture of both the starting lipid and the diacetate. The minor peak (35%) was found to be the triacetate. These results are in good agreement with the results above. A certain amount of unreacted starting lipid in the postgrafted complex is required to continue to compact DNA, and the ethidium bromide assay shows that this is indeed the case (Table 1). The proportions of diacetate and triacetate found in the extracted mixture are also what would be expected due to their reactivities and relative position within the complexes.



Scheme 2. Illustration of the Successive Lipoplex Preparation Steps Used in the Present Postgrafting Study

Scheme 3. The Cationic Lipid RPR209120B, Cholesterol-PEG42 (Chol-PEG42) and CholesterolPEG75-RGD-4C (Chol-PEG75-RGD-4C)

Table 1. Physicochemical Data Corresponding to the Reaction of Different Amounts of Sulfo-NHS Acetate on PEGylated Cationic Lipoplexesa molar ratio sulfo-NHS acetate/ cationic lipid

zeta potential (mV)

EtBr (relative fluorescence)

particle size (nm)

0 1 5 10 50

16.42 -2.19 -0.36 -2.09 -15.0

4.6 5.7 10.2 11.0 13.1

131 151 168 169 189

a The molar equivalents (1:1, 5:1, 10:1, and 50:1) of sulfo-NHS acetate versus cationic lipid RPR209120B were investigated. Lipoplexes comprised RPR209120B/DSPE-PEG42(10%)/DNA with a charge ratio (+/-) ) 5 and were prepared by the thin-film method.

Characterization of the Postgrafted Lipoplexes for Both in Vitro and in Vivo Experiments. From these results above, it was established that postgrafting up to 10 mol of sulfo-NHS acetate per mole of cationic lipid RPR209120B on the surface of preformed lipoplexes did not alter their size nor their ability to compact DNA, yet it did afford surface-neutral particles as demonstrated by their zeta potential. For biological studies, we chose to use these conditions, and we prepared postgrafted lipoplexes containing or not containing a CholesterolPEG75-RGD-4C (Chol-PEG75-RGD-4C) targeting lipid.

It was necessary to use a HEPES buffer concentration of 100 mM in order to maintain the pH at ∼7.4 during and after the reaction with sulfo-NHS acetate since it is subject to hydrolysis, which releases acetic acid and sulfoN-hydroxysuccinimide. Liposomes were prepared either by film formation followed by rehydration or by the ethanol injection method (see Materials and Methods and Scheme 2). While Chol-PEG42 was inserted in the cationic liposomes before sulfo-NHS acetate postgrafting in order to maintain the colloidal stability of the neutralized lipoplexes, the targeting lipid, Chol-PEG75-RGD-4C, was inserted after the postgrafting reaction. Size and zeta potential of the final complexes for in vivo studies were measured (Table 2). Cationic lipoplexes, prepared by the ethanol injection method, gave a measured zeta potential of +39.7 ( 1.1 mV, which was reduced to +12.5 ( 0.9 mV by addition of 5% Chol-PEG42 in the formulation. Insertion of lipidPEG42 in the film during the preparation of the liposomes led to similar results (+16 mV, Table 1). By reacting sulfo-NHS acetate with the surface amine moieties of preformed PEGylated lipoplexes, neutral


Thompson et al.

Table 2. Size and Zeta Potential Data of Non-targeted and Targeted Lipoplexes Used for Biological Experimentsa

type of complex

size (nm)

zeta potential (mV)

cationic lipoplex 109.8 ( 24.1 +39.7 ( 1.1 lipoplex + 5%Chol-PEG42 125.2 ( 50.9 +12.5 ( 0.9 lipoplex + 5%Chol-PEG42 + postgrafting 96.9 ( 29.6 +1.2 ( 0.9 211 ( 89.9 -3.4 ( 1.0 lipoplex + 5%Chol-PEG42 + postgrafting + 5%Chol-PEG75-RGD-4C a Liposomes were prepared by the ethanol injection method. Cationic lipoplex comprised RPR209120B/DPPC/DNA; lipoplex + 5%Chol-PEG42 comprised RPR209120B/DPPC/Chol-PEG42(5%)/ DNA; lipoplex + 5%Chol-PEG42 + postgrafting comprised RPR209120B/DPPC/Chol-PEG42(5%)/DNA and was reacted with sulfo-NHS acetate; lipoplex + 5%Chol-PEG42 + postgrafting + 5%Chol-PEG75-RGD-4C comprised RPR209120B/DPPC/CholPEG42(5%)/DNA, was reacted with sulfo-NHS acetate followed by insertion of Chol-PEG75-RGD-4C (5%). All above lipoplexes were prepared with a charge ratio (+/-) ) 5.

Figure 1. TEM pictures of lipoplexes obtained by negative staining. A. Liposomes (film) RPR209120B/DOPE/Chol-PEG42(5%)/DNA B. Same liposomes as in A + sulfo-NHS acetate postgrafting; C. Liposomes (ethanolic injection) RPR209120B/ DPPC/Chol-PEG42(5%)/DNA. D. Same liposomes as in C + sulfo-NHS acetate postgrafting.

particles were obtained (zeta potential ) +1.2 ( 0.9 mV). When lipoplexes were reacted with sulfo-NHS acetate, and Chol-PEG75-RGD-4C was further added to the preformed complexes, the zeta potential was reduced to -3.4 ( 1.0 mV (Table 2); this value is in agreement with the presence of two aspartic acids and a free C-terminal carboxylate function in the RGD moiety. Electronic transmission microscopy was carried out for acetylated and nonacetylated particles prepared by the thin-film and the ethanolic injection methods (Figure 1). No structural differences between postgrafted and non postgrafted particles were observed, indicating that the postgrafting process does not alter the particle structures. However, the surface of postgrafted particles appears slightly changed with enhanced lamellas. By laser diffraction size measurement, postgrafted particles appeared larger than the non postgrafted ones (within 1020 nm increase), and we obtained smaller particles by the ethanol injection method as compared to the filmhydration method. As an example, the isolated particles

in C and D represent 20 and 40 nm in diameter, respectively. On some of the particles, an outer layer probably corresponding to the PEG shielding was clearly visualized (large gray arrow on C). Lipoplex Pharmacokinetics after iv Injection. Intravenous injections of PEGylated cationic lipoplexes versus PEGylated postgrafted lipoplexes were performed in mice bearing subcutaneous 3LL Lewis Lung tumors. The postgrafted complexes, where the liposome component was prepared via the thin-film method, showed an increased amount of postgrafted complex in the circulation by a factor of 7 at the end of 30 min and by a factor of 5 after 1 h. There was also a marked decrease in the uptake of acetylated, postgrafted particles in the lung. However, there was no significant passive accumulation in the tumor (Figure 2). As already observed, most of the rhodamine signal was recovered in the RES (5). The postgrafted complexes, where the liposome component was prepared via the ethanol injection method, did not show as dramatic a difference in circulation time as compared to nonacetylated particles. The smaller size achieved with these particles might be the main reason for this. A different liposome flexibility due to the preparation might also influence this difference (15). Nevertheless, there was again a decrease in lung uptake of the acetylated particles (data not shown). Targeting with RGD-PEG-Lipid. The postgrafting strategy afforded lipoplexes with a longer circulation lifetime, but at the same time, as could be expected, diminished their cell membrane interactions required for transfection. We thus postulated that the addition of an integrin-targeting ligand in lipoplex formulation may help to restore their accumulation in the tumor. Experiments were performed on mice bearing PO2 tumors validated for their Rv β3 integrin expression in the tumor vasculature. An increased trend of lipoplex accumulation in the tumor when an RGD-4C ligand was added to the formulation was noted as compared to the same formulation of postgrafted complexes without RGD (Figure 3). Taking the Chol-PEG75-RGD-4C liposome as a control, an increase of 5- and 2-fold in the tumor were measured, 30 min and 1 h postinjection, respectively, for the liposome + Chol-PEG-RGD-4C + postgrafting particles. This increased liposome accumulation was reflected in an increased gene expression for both cationic PEGylated lipoplexes and PEGylated postgrafted lipoplexes bearing a Chol-PEG-RGD-4C targeting moiety (Figure 4). However, no statistically significant difference was observed for transgene expression between the nonneutralized or acetate-neutralized targeted lipoplexes. DISCUSSION AND CONCLUSIONS

Improving the circulation lifetime of iv injected cationic lipid/DNA complexes is of great interest. Cationic lipids are excellent vectors for DNA due to both their ability to efficiently compact negatively charged DNA at a certain charge ratio, as well as their favorable cellular interactions which lead to transfection in vitro. However, in vivo cationic lipid efficacy is hampered by their nonspecific interactions with blood components and their rapid elimination from the circulation (4). The addition of PEG in lipoplex formulation has achieved less positively charged particles, with respect to their surface charge (i.e. their zeta potential), but this has been found to be insufficient to completely mask any residual cationic charges on the lipoplex surface, which are still subject to unfavorable blood component interactions.



Figure 2. Blood, lung, RES, and tumor concentration of modified (postgrafting) versus unmodified lipoplexes. Rhodamine-lipid levels in the plasma, lung, RES, and 3LL tumor of tumor-bearing mice after systemic injection. Results are expressed as the mean ( SD. Significant difference: **: P < 0.01 and ***: P < 0.001

Figure 4. CAT expression in lung and PO2 colon carcinoma. Results are expressed as the mean of five values; error bars indicate standard error of the mean.

Figure 3. Lung and PO2 tumor concentration of targetingneutral versus targeting-PEGylated lipoplexes and neutral nontargeting lipoplexes. Rhodamine-lipid levels in the lungs and PO2 tumor of tumor-bearing mice after systemic injection. Results are expressed as the mean of five values; error bars indicate standard error of the mean.

The original postgrafting method presented here was validated as a way to compact DNA and obtain neutral particles while maintaining their size. Binding experiments performed at 4 °C on NIH3T3 cells with fluorescent particles showed no fluorescence for postgrafted complexes while nonspecific fluorescence was observed

for nonacetylated complexes (data not shown) (16). This confirmed the potential of sulfo-NHS acetate lipoplex neutralization to decrease nonspecific cell interaction. Our postgrafted formulations included PEG-lipids for steric stabilization of the particles. This strategy allowed the incorporation of a lipid-PEG-ligand without protocol modification. Incorporation of Chol-PEG75-RGD-4C into the formulation increased lipoplex tumor delivery. Although an increase in tumor transfection was observed in RGD-targeted formulations, neutralized particles were not superior to nonneutralized ones. This might result from incomplete DNA release or less efficient transfection by neutralized particles. The use of a cytotoxic drug or an antiangiogenic plasmid (17) instead of a DNA model would be an alternative and a more straightforward manner to assay the potential advantage of the present formulation for therapeutic use, as only a release at the level of the vessels would be required to starve the tumor. Integrins have been shown to be overexpressed by the inflammed endothelium (18), which would be easier to


reach for particles. It has been reported that RGDtargeted liposomes can act as a site-specific drug delivery system to tumor endothelial cells in vitro, which was demonstrated by increased binding, as well as redirecting targeted liposomes containing doxorubicin to angiogenic endothelial cells in vivo which led to inhibited tumor growth (19). Thus, in the present case, the targeted particles possibly accessed the tumoral endothelium without reaching the tumor cells. Experiments are currently being undertaken to determine the localization of the particles in and around the tumor. The postgrafting strategy reported here has succeeded in further reducing the zeta potential of cationic lipoplexes (to approximately 0 mV) to afford surface neutral particles and has allowed for a longer lifetime of the complexes in vivo. Despite increased circulation time in the blood, no passive accumulation in 3LL tumors was measured. Insertion of a targeting Chol-PEG75-RGD4C succeeded in PO2 tumor targeting and increased DNA expression. Ways of triggering DNA release in the target are now in process. ACKNOWLEDGMENT

B. Thompson thanks the European Community for the Pierre and Marie Curie grant. We thank P. Wils for very helpful discussions. We also thank S. Sable and B. Monegier (Aventis Pharma) for structural analyses. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Lipofection: a highly efficient, lipidmediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417. (2) Maurer, N., Mori, A., Palmer, L., Monck, M., Mok, K., Mui, B., Akhong, Q., and Cullis, P. (1999) Lipid-based systems for the intracellular delivery of genetic drugs. Mol. Membr. Biol. 16, 129-140. (3) Papahadjopoulos, D., Allen, T. A., Gabizon, A., Mayhew, E., Matthay, K., Huang, S., Lee, K., Woodle, M., Lasic, D., Redemann, C., and Martin, F. (1991) Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. U.S.A. 88, 1146011464. (4) Song, L., Ahkong, Q., Rong, Q., Wang, Z., Ansell, S., Hope, M., and Mui, B. (2002) Characterization of the inhibitory effect of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes. Biochim. Biophys. Acta 1558, 1-13. (5) Nicolazzi, C., Mignet, N., de la Figuera, N., Cadet, M., Torero Ibad, R., Seguin, J., Scherman, D., and Bessodes, M. (2003) Anionic poly(ethylene glycol) lipids added to cationic li-

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