©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Folate-targeted, Anionic Liposome-entrapped Polylysine-condensed DNA for Tumor Cell-specific Gene Transfer (*)

(Received for publication, August 29, 1995; and in revised form, December 7, 1995)

Robert J. Lee Leaf Huang (§)

From the Laboratory of Drug Targeting, Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have developed a lipidic gene transfer vector, LPDII, where DNA was first complexed to polylysine at a ratio of 1:0.75 (w/w) and then entrapped into folate-targeted pH-sensitive anionic liposomes composed of dioleoyl phosphatidylethanolamine (DOPE)/cholesteryl hemisuccinate/folate-polyethlene glycol-DOPE (6:4:0.01 mol/mol) via charge interaction. LPDII transfection of KB cells, a cell line overexpressing the tumor marker folate receptor, was affected by both the lipid to DNA ratio and the lipid composition. At low lipid to DNA ratios (e.g. 4 and 6), LPDII particles were positively charged; transfection and cellular uptake levels were independent of the folate receptor and did not require a pH-sensitive lipid composition. Meanwhile, transfection and uptake of negatively charged LPDII particles, i.e. those with high lipid to DNA ratios (e.g. 10 and 12), were folate receptor-dependent and required a pH-sensitive lipid composition. The transfection activity of LPDII was lost when the inverted cone-shaped DOPE was replaced by dioleoyl phosphatidylcholine. LPDII particles with lipid to DNA ratios of 4, 6, 10, and 12 were 20-30 times more active than DNAbullet3-beta-[N-(N`,N`-dimethylethane)carbamoyl]cholesterol cationic liposome complexes in KB cells and were much less cytotoxic. On the sucrose gradient, LPDII particles had a migration rate in between those of the free DNA and the DNAbulletpolylysine complex. An electron micrograph of LPDII showed a structure of spherical particles with a positively stained core enclosed in a lipidic envelope with a mean diameter of 74 ± 14 nm. This novel gene transfer vector may potentially be useful in gene therapy for tumor-specific delivery.


INTRODUCTION

The prospect of correcting human disorders through gene therapy has recently created much excitement in the scientific community(1) . The development of suitable delivery vectors for in vivo gene transfer is a prerequisite for the clinical application of therapeutic genes. Although several highly efficient biological (viral) vectors including recombinant retroviruses(2) , adenoviruses(3) , and adeno-associated viruses (4) are available, they suffer from immunogenicity, toxicity, lack of tissue specificity, difficulty in large scale production, and potential risk of inducing tumorigenic mutations and/or generating active viral particles through recombination(5) . Synthetic vectors such as liposomes, therefore, have become an attractive alternative due to their lack of immunogenicity and restraints on the DNA they can carry, the potential for tissue-specific targeting, relative safety, and relative ease of large scale production(6, 7) .

Both cationic and anionic liposomal vectors have been evaluated for DNA delivery. Cationic liposomes, first developed by Felgner et al. in 1987, form slightly cationic complexes with the negatively charged DNA molecules which can then be taken up by cells via charge interaction(8, 9) . The disadvantages of cationic liposomal vectors are the requirement for unnatural cationic lipids, suboptimal DNA condensation, cytotoxicity, limited efficiency, lack of tissue specificity, relatively large particle size, and potential incompatibility with the biological environment, which is rich in negatively charged macromolecules, extracellular matrix components, lipoproteins, and cells. To improve DNA condensation, Gao and Huang (^1)recently designed a cationic liposomebulletpolylysinebulletDNA ternary complex (LPDI) consisting of a condensed polylysine/DNA core and a cationic lipidic envelope. These particles had a higher transfection activity than DNAbulletcationic liposome complexes but required the removal of excess cationic lipids for optimal activity.^1

pH-sensitive anionic liposomes have also been shown to mediate gene transfer but suffer from poor encapsulation efficiency due to the large size and the negative charge of the uncondensed DNA(11) . Passive encapsulation of DNA into anionic liposomes usually requires the use of a high concentration of lipids to increase the ``entrapped volume'' which leads to the generation of excessive amounts of empty liposomes. The entrapment efficiency, however, still rarely exceeds 20%. Moreover, treatments commonly used for improving liposome encapsulation or reducing particle size such as repeated freeze-thaw cycles, polycarbonate membrane extrusion, and sonication may lead to severe DNA damage(12) .

Another major focus in gene therapy vector development has been tissue-specific targeting. A variety of targeting ligands have been examined for liposome targeting including folate(13, 14, 15) . The receptor for folate has recently been identified as a tumor marker, especially among ovarian carcinomas(16, 17, 18) . Folate retains its receptor affinity when derivatized via its -carboxyl group. Folate-conjugated macromolecules and liposomes have been shown to be specifically taken up by cultured receptor-bearing tumor cells as well as by implanted xenographs derived from human tumor cells in vivo(19, 20, 21, 22) . As a low molecular weight ligand, folic acid (M(r) = 441) has the advantages of being stable and nonimmunogenic compared to their proteinacious counterparts, such as monoclonal antibodies (23) , and still having a relatively high receptor affinity (K 1 nM). Recently, folate-targeted liposomes have been successfully used in the tumor cell specific delivery of anticancer drugs and antisense oligodeoxynucleotides in vitro(24) . Also, folate-derivatized polylysinebulletDNA complexes combined with inactivate adenoviruses have been shown to mediate tumor cell-specific transfection(25) .

In this article, we describe a vector where polylysine-condensed DNA is entrapped into folate-targeted anionic liposomes via charge interaction. Due to its structural similarity to LPDI, we name this new vector LPDII (liposome-entrapped polycation-condensed DNA). (^2)LPDII differs from LPDI in that anionic lipids instead of cationic lipids are used. The description ``liposome-entrapped'' is based on structural information derived from electron micrographs of LPDII presented in this article. We find this novel DNA vector to be more efficient and less cytotoxic compared to conventional cationic liposomal vectors. We evaluated the effects of lipid/DNA ratio, lipid composition, excess free ligand, and folate receptor expression on the transfection efficiency of LPDII. We also examined the structure of LPDII by electron microscopy. The potential advantages of LPDII as a gene therapy vector are discussed.


MATERIALS AND METHODS

Folic acid, poly-L-lysine (hydrobromide, molecular mass = 25.6 kDa), and cholesteryl hemisuccinate (CHEMS) were obtained from Sigma. Dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylcholine (DOPC), and dioleoyl phosphatidylserine (DOPS) were purchased from Avanti Polar Lipids, Inc. [^3H]-Labeled cholesteryl hexadecyl ester was purchased from DuPont; 3-beta-[N-(N`,N`-dimethylethane)carbamoyl]cholesterol (DC-Chol) and folate-PEG-DOPE were synthesized as described previously (9, 21) . Luciferase assay kits were purchased from Promega. Folate-deficient modified Eagle's medium and F-12 nutrient mixture were purchased from Life Technologies, Inc.

Cell Culture

KB, a nasopharyngeal epidermal carcinoma cell line known to overexpress the folate receptor, was grown in a folate-deficient modified Eagle's medium (normal medium except without folic acid) supplemented with 10% fetal bovine serum and antibiotics (the final folate concentration in the serum-supplemented medium was approximately physiological). A Chinese hamster ovary (CHO) cell line was maintained in F-12 nutrient mixture supplemented with 10% fetal bovine serum and antibiotics. The same media (serum-free or with serum) were used during transfection experiments. Both cell lines were available through the American Type Culture Collection and were cultured in a 37 °C cell culture incubator in a humidified atmosphere containing 5% CO(2).

Preparation of Luciferase Plasmid DNA

Supercoiled plasmid pRSVL containing the firefly luciferase reporter gene under the promoter of the Rouse sarcoma virus long terminal repeat(26) , was isolated from Escherichia coli by the alkaline lysis method and twice purified by cesium chloride gradient banding(27) .

Effect of Polylysine/DNA Ratio on the Properties of the DNAbulletPolylysine Complex

DNAbulletpolylysine complex was prepared by the rapid mixing of 4 µg of pRSVL plasmid DNA with varying amounts of polylysine, each in 100 µl of deionized water. The size of the resulting complex was then determined by light scattering on a Coulter N4SD submicron particle analyzer. Elemental analysis of polylysine was performed by Galbraith Laboratories, Inc.

Preparation of Folate-targeted Anionic Liposomes

Folate-conjugated pH-sensitive liposomes with the composition DOPE/CHEMS/folate-PEG-DOPE (6:4:0.01 mol/mol) and pH-insensitive anionic liposomes with the composition DOPE/DOPS/folate-PEG-DOPE (8:2:0.01 mol/mol) or DOPC/CHEMS/folate-PEG-DOPE (6:4:0.01 mol/mol) were prepared as follows. A chloroform solution of the lipid mixture containing 20 mg of total lipid and a trace amount of ^3H-labeled cholesteryl hexadecyl ester was dried into a thin film on the wall of a 13-mm glass tube under a stream of nitrogen gas. Residual organic solvent was removed by vacuum desiccation for an additional 30 min. The lipids were then hydrated in 1 ml of sterile deionized water under vortex and sonicated in a bath-type sonicator (model G112SP1T, Laboratory Supplies Co., Inc.) with intermittent pH adjustment to 8.0 using 1 N NaOH until the mean particle diameter, as determined by light scattering, was reduced to 120 nm. The liposomes were then passed through a short Sepharose 4B gel filtration column (Pharmacia Biotech Inc.) preequilibrated in 10 mM Hepes buffer (pH 8.0). The liposome fractions were then filtered through a 0.2-µm filter and stored at 4 °C. The mean diameter of these liposomes was 120 nm. The liposomes were used within 2 months after preparation, during which period no significant changes in vesicle size were observed.

Preparation of LPDII Particles

DNAbulletpolylysine complex was prepared by rapidly mixing (under vortex) equal volumes of 0.12 µg/µl pRSVL plasmid DNA and 0.09 µg/µl polylysine in deionized water. The resulting complex, which had a polylysine/DNA ratio of 0.75:1 (w/w), was then rapidly mixed with anionic liposomes in an equal volume of deionized water to generate LPDII particles.

Preparation of DNAbulletCationic Liposome Complexes

Cationic liposomes with the composition DOPE/DC-Chol (4:6 mol/mol) were prepared as described previously and had a mean diameter of 160 nm. DNAbulletDC-Chol liposome complexes were prepared by rapidly mixing DNA (0.12 µg/µl) and cationic liposomes at a ratio of 1:6 (w/w) in deionized water, i.e. under previously optimized conditions (9) .

Transfections

Cells were plated in 24-well plates at 2.5 times 10^4 cells/well and incubated for 24 h prior to transfection, by which time the cells were 30% confluent. LPDII particles or DNAbulletcationic liposome complexes were diluted to a DNA concentration of 5 µg/ml in serum-free culture medium and were then added at 1 µg (in 200 µl) per well in triplicates. In free ligand competition studies, 1 mM folic acid was included in the transfection medium. The cells were incubated at 37 °C for 4 h, after which the transfection medium was replaced by fresh serum-containing (10%) medium. Following 48 h of additional incubation, the cells were washed with 0.5 ml of phosphate-buffered saline (130 mM NaCl, 20 mM phosphates, pH 7.4) and then lysed in 0.05% Triton X-100 (pH 7.8, containing 0.1 M Tris-HCl and 2 mM EDTA). The cell lysate was then centrifuged and the supernatant assayed for luciferase activity using a commercial kit from Promega on a AutoLumat LB953 luminometer (EG& Berthold) and protein content using a Coomassie® Plus protein assay kit (Pierce).

Cellular Uptake of DNA

pRSVL plasmid DNA was labeled with I using the Iodogen® reagent as described previously (10) , and purified by gel filtration on a Sephadex G-25 column and twice by ethanol precipitation. Labeling was confirmed by agarose gel electrophoresis and autoradiography (data not shown). DNAbulletpolylysine complexes, DNAbulletcationic liposome complexes, and LPDII particles containing iodinated DNA (with a specific activity of 0.02 µCi/µg) were prepared as described above. Cells were plated in a 24-well plate, grown overnight to 30% confluency, and transfected with the various formulations as described under ``Transfections.'' Following 4 h of incubation at 37 °C, the cells were washed twice with 0.5 ml of phosphate-buffered saline, solubilized in the lysis buffer, and assayed for radioactive DNA uptake by counting on a gamma counter.

Fractionation of LPDII Particles on a Sucrose Gradient

A discontinuous sucrose gradient was generated by layering samples containing trace amounts of I-labeled DNA (usually 50 µg total) and/or ^3H-labeled liposomes in a sucrose gradient consisting of 0.5/1/1 ml deionized water/20%/40% sucrose in a thin-wall ultracentrifuge tube. The gradient was then centrifuged at 35,000 rpm (100,000 times g) in a Beckman SW40.1 ultracentrifuge rotor for 35 min. Fractions (250 µl each) were then carefully collected from the top of the gradient and analyzed for DNA and/or lipid content by gamma and/or scintillation counting. To evaluate the transfection activity of the major DNA-containing fractions, KB cells in a 24-well plate were transfected with 1 µg/well DNA from fractions 1, 2, 6, 7, and 10 diluted in 200 µl in serum-free medium and assayed as described under ``Transfections.''

Negative Stain Electron Microscopy

For negative stain electron microscopy, 20-µl samples were applied to copper grids covered with Formvar support, which had been briefly treated in a sputter coater. The grids were then stained for 10 min with 1% uranyl acetate. Electron micrographs were then obtained on a Joel 100B electron microscope operating at 80 kV at times 72,000 magnification.


RESULTS

Preparation of LPDII

PolylysinebulletDNA complexes were prepared by rapidly mixing DNA and polylysine in deionized water. Elemental analysis of the polylysine used in this study showed a composition of C (33.88%), H (6.77%), N (12.59%), O (11.57%), and Br (28.22%), which was empirically consistent with a formula of 0.775 times HBr and 1.4 times carbon dioxide/bicarbonate per lysine unit. As shown in Fig. 1, the apparent size of the DNAbulletpolylysine complexes increased with time due to gradual aggregation at the DNA/polylysine ratio of 1:0.47 (w/w), but remained stable at either higher or lower DNA/polylysine ratios. A simple explanation is that a charge neutral complex was formed at that DNA/polylysine ratio that is prone to aggregation. However, the calculated lysine amino/DNA phosphate ratio in the aggregating complex was 0.7:1 and at neutral pH only a portion of the polylysine amino groups should be charged based on the pK(a). Formation of an ``isoelectric'' complex at this ratio was probably due to the structural organization of the condensed DNAbulletpolylysine complex in which some phosphate groups may not be accessible to polylysine. In this study, a DNAbulletpolylysine complex formed at a weight ratio of 1:0.75, i.e. with positive charge in excess, was used for DNA condensation and liposome incorporation.


Figure 1: Effect of polylysine/DNA ratio on the size of polylysinebulletDNA complexes. (Inset: time-dependent size increase of ``isoelectric'' complexes formed at the polylysine/DNA ratio of 0.47:1 w/w.) The complexes were prepared by mixing dilute solutions of polylysine and DNA in deionized water, as described under ``Materials and Methods.'' Apparent sizes were determined by light scattering. Because of the nonspherical shape of the polylysinebulletDNA complexes, the size values should only be used as a reference.



Folate-targeted LPDII particles were generated by mixing anionic liposomes composed of DOPE/CHEMS/folate-PEG-DOPE (6:4:0.01 mol/mol) and the cationic DNAbulletpolylysine (1:0.75 w/w) complexes. As shown in Fig. 2,, the apparent size of the resulting LPDII particles varied with the lipid/DNA ratio. A large particle size was observed at the lipid/DNA ratio of 8 presumably due to the formation of near ``isoelectric'' particles. At either higher or lower lipid/DNA ratios, however, smaller particles were formed possibly due to the formation of particles carrying either net positive or negative charge, the size of which remained stable at 4 °C for at least one week.


Figure 2: Effect of lipid/DNA ratio on the size of LPDII particles. LPDII particles were prepared by mixing polylysinebulletDNA (0.75:1 w/w) complexes with anionic liposomes composed of DOPE/CHEMS/folate-PEG-DOPE (6:4:0.01 mol/mol). Particle size was determined by light scattering. Due to some degree of sample heterogeneity, the size values should only be used as a reference.



Effects of Lipid/DNA Ratio and Lipid Composition on the Transfection Efficiency of LPDII Particles

KB cells were transfected with LPDII particles containing the pRSVL plasmid carrying the luciferase reporter gene. As shown in Fig. 3, DNAbulletpolylysine complexes alone had little transfection activity. However, very high transfection levels were obtained with cationic LPDII particles with lipid/DNA ratios of 4 and 6, which were not blocked by 1 mM free folic acid. Transfection was less efficient with LPDII particles at the lipid/DNA ratio of 8, probably due to particle aggregation. Anionic LPDII particles with lipid/DNA ratios of 10 and 12 also mediated efficient transfection but can be partially inhibited by 1 mM free folic acid (Fig. 3). The relatively high residual transfection activity of these particles in the presence of free folate may be explained by a much higher receptor affinity of the LPDII particles which can interact with the cellular folate receptors via multivalent binding. When the lipid/DNA ratios were further increased, there was a decrease in transfection activity. This may be due to the presence of excess lipids and, therefore, empty folate-targeted liposomes which may then compete against LPDII particles for available cellular folate receptors and lead to an increase in cytotoxicity. Control LPDII particles generated with nontargeted liposomes were only active at low lipid/DNA ratios, suggesting that transfection by LPDII particles was only receptor dependent when the over all charge was negative.


Figure 3: Effect of lipid/DNA ratio on the transfection activity of LPDII particles. LPDII particles containing the pRSVL plasmid carrying the luciferase reporter gene were prepared by mixing polylysinebulletDNA (0.75:1 w/w) complexes with anionic liposomes. KB cells were transfected and assayed for luciferase activity, as described under ``Materials and Methods.'' Formulations: folate-targeted LPDII with the lipid composition DOPE/CHEMS/folate-PEG-DOPE (6:4:0.01 mol/mol) (bullet); folate-targeted LPDII plus 1 mM free folic acid (circle); nontargeted LPDII with the lipid composition DOPE/CHEMS (6:4 mol/mol) (). The transfection activity of DNAbulletDC-Chol cationic liposome complexes was 2382 ± 729 relative light units (RLU)/µg protein. Error bar = 1 S.D. (n = 3). Luciferase activity unit: 2343 RLU is equal to the light emission of 0.02 µCi of [^14C]chloramphenicol measured by the luminometer in the Cytoscint mixture (purchased from ICN), which in turn is equivalent to 1 luciferase light unit measured under standard conditions.



The transfection activity of LPDII particles was compared to that of the DNAbulletDOPEbulletDC-Chol cationic liposome complexes. At optimal lipid/DNA ratios (e.g. 4, 6, 10, and 12), the transfection activity of LPDII particles was 20-30 times higher. This may be the result of a much higher cytotoxicity of the DNAbulletcationic liposome complexes. When more confluent (70%) cells were used in the transfection, less differences in transfection activity were observed between LPDII and DNAbulletcationic liposome complexes (data not shown).

We also analyzed the relative toxicity of LPDII particles based on the amount of extractable cellular protein in the lysate (Fig. 4). DNAbulletpolylysine complexes without lipid or LPDII particles with lipid/DNA ratios of 4, 6, 10, and 12 showed only low levels of cytotoxicity. Meanwhile, LPDII at the lipid/DNA ratios of 8 and 16, as well as the DNAbulletcationic liposome complexes, showed relatively high levels of cytotoxicity (Fig. 4). Control LPDII prepared from nontargeted liposomes showed unexpectedly high cytotoxicity, especially at high lipid/DNA ratios.


Figure 4: Effect of lipid/DNA ratio on the KB cell cytotoxicity of LPDII particles. Cytotoxicity was evaluated by protein content in the cellular extract following transfection and cell lysis (see Fig. 3legend) using untreated cells as a reference. Formulations: folate-targeted LPDII (bullet); folate-targeted LPDII plus 1 mM free folic acid (circle); nontargeted LPDII (). For cells transfected with DNAbulletDC-Chol cationic liposome complexes, the protein content was 32 ± 11% of the untreated control.



To further examine the role of folate receptor in transfection, CHO cells, a cell line that does not overexpress the folate receptor, were transfected with folate-targeted LPDII particles. Efficient transfection was observed only with cationic particles with lipid/DNA ratios of 4 and 6 but not with anionic particles with higher lipid/DNA ratios (Fig. 5). This reaffirmed the role of folate receptor in the transfection activity of anionic LPDII particles in KB cells.


Figure 5: LPDII transfection of CHO cells. CHO cells in 24-well plates were transfected with LPDII containing pRSVL and assayed for luciferase activity, as described under ``Materials and Methods.'' Formulations: folate-targeted LPDII (bullet); folate-targeted LPDII plus 1 mM free folic acid (circle).



In summary, transfection with LPDII particles was independent of the folate receptor at cationic charge ratios and receptor-dependent at anionic charge ratios. This result was in agreement with previous studies by Plank et al.(14) on a liver cell targeted lipidic DNA vector. In addition, LPDII particles also had a greater transfection activity and a lower cytotoxicity compared to DNAbulletcationic liposome complexes.

Cellular Uptake of the DNA

We quantitated the KB cell uptake of folate-targeted LPDII particles carrying I-labeled DNA. As shown in Fig. 6, at low lipid/DNA ratios (e.g. 4 and 6), DNA uptake initially decreased with an increase in the lipid/DNA ratio, presumably due to a reduction in the amount of positive charge carried by the LPDII particles resulting in a reduction in charge-mediated cellular uptake. KB cell uptake of these particles could not be blocked by 1 mM free folic acid, therefore, was probably receptor independent. At higher lipid/DNA ratios (e.g. 10 and 12), there were relatively high levels of DNA uptake, which could be partially inhibited by 1 mM free folic acid. For control LPDII particles prepared from nontargeted liposomes, the DNA uptake decreased steadily with increasing lipid/DNA ratios. These data suggested that DNA uptake and transfection were mainly mediated by charge for cationic LPDII particles with low lipid/DNA ratios and by the folate receptors for anionic LPDII particles with high lipid/DNA ratios. However, the DNA uptake level was clearly not the only determining factor, since the lipid-free DNAbulletpolylysine complexes had the highest uptake of all the LPDII formulations examined but had very little transfection activity.


Figure 6: Effect of lipid/DNA ratio on the KB cell DNA uptake. KB cells were incubated with LPDII particles containing I-labeled pRSVL plasmid and assayed for DNA uptake by cell-associated radioactivity after washing twice with phosphate-buffered saline, as described under ``Materials and Methods.'' Formulations: folate-targeted LPDII(bullet); folate-targeted LPDII plus 1 mM free folic acid (circle); nontargeted LPDII (). Uptake of DNAbulletDC-Chol cationic complexes was 0.0045 ± 0.0014 µg/µg protein which corresponds to 8.7% of the total DNA added.



The Effect of Lipid Composition on LPDII Transfection

pH-sensitive liposomes, e.g. with the composition DOPE/CHEMS (6:4, mol/mol) are stable at neutral or basic pH but become fusogenic at an acidic pH. The fusogenicity is largely a result of the inverted-cone shape of DOPE, which favors the transition of a bilayer lipid phase to a hexagonal II phase. A ``pH-sensing'' component, such as CHEMS, stabilizes a bilayer structure at neutral pH when negatively charged but not at acidic pH upon protonation. To examine the roles of DOPE and CHEMS in transfection, we prepared LPDII particles with the lipid compositions DOPC/CHEMS/folate-PEG-DOPE (6:4:0.01 mol/mol), in which DOPE was replaced by DOPC, a cylindrically shaped lipid, and DOPE/DOPS/folate-PEG-DOPE (8:2:0.01 mol/mol), in which CHEMS was replaced by DOPS, an anionic lipid with a much lower pK(a) (<4) and not protonatable at endosomal pH. At around pH 7 approximately half of the CHEMS and almost all of DOPS molecules in the liposomes should be negatively charged. Liposomes containing 20% PS should, therefore, have a similar charge density to those containing 40% CHEMS. As shown in Fig. 7, LPDII particles with the lipid composition of DOPC/CHEMS did not show transfection activity at any lipid/DNA ratios. This indicated that the fusogenic activity of DOPE was essential for the transfection activity of LPDII particles. Meanwhile, LPDII particles with the lipid composition DOPE/DOPS showed efficient transfection only at low lipid/DNA ratios where cationic particles were formed (Fig. 7). In these particles, most DOPS molecules were probably involved in complexation with the cationic polylysine/DNA core leaving membrane areas enriched with DOPE resulting in high fusogenicity. However, at high lipid/DNA ratios the particles were inactive, because the anionic groups on DOPS could not be neutralized upon endocytosis into endosomes due to its low pK(a), inhibiting fusogenicity.


Figure 7: Effect of lipid composition on the KB cell transfection activity of LPDII. Formulations: folate-targeted LPDII composed of DOPE/DOPS/folate-PEG-DOPE (8:2:0.01 mol/mol) (bullet); folate-targeted LPDII composed of DOPE/DOPS/folate-PEG-DOPE (8:2:0.01 mol/mol) plus 1 mM free folic acid (circle); folate-targeted LPDII composed of DOPC/CHEMS/folate-PEG-DOPE (6:4:0.01 mol/mol) ().



Structural Analysis of LPDII by Electron Microscopy

We examined the structures of the DNAbulletpolylysine complex and LPDII particles by negative-stain electron microscopy. As shown in Fig. 8A, DNAbulletpolylysine complexes formed at the 1:0.75 weight ratio appeared as ``rod''-shaped electron dense particles with an average thickness of 15 nm and length of 109 ± 36 nm. An electron micrograph of LPDII particles with the composition of DNA/polylysine/lipid (1:0.75:12 w/w) was shown in Fig. 8B. The majority of the particles in the picture appeared spherical and consisted of a high electron density core and low density coating. The mean diameter of these particles was 74 ± 14 nm, i.e. smaller than the empty liposomes.


Figure 8: Electron micrographs of DNAbulletpolylysine complexes LPDII particles. Samples were stained with 1% uranyl acetate and examined under electron microscope as described under ``Materials and Methods.'' Panels: A, DNAbullet polylysine (1:0.75 w/w) complexes; B, LPDII, unpurified; C, LPDII, fraction 6 on the sucrose gradient (see Fig. 10); D, LPDII, fraction 7 on the sucrose gradient. Scale bar = 200 nm (Panel A).




Figure 10: Fractionation of LPDII on a sucrose gradient. A, LPDII particles containing I-labeled DNA and ^3H-labeled lipids were fractionated on a 0/20/40% (0.5/1/1 ml) discontinuous sucrose gradient at 100,000 times g for 35 min. Ten 250-µl fractions were collected starting from the top of the gradient and counted for DNA and lipid radioactivity. I-labeled DNA (bullet); ^3H-labeled lipids (circle). B, KB cell transfection activity of the major DNA-containing fractions. C, sucrose gradient profile of free DNA (bullet), free liposomes (circle), and DNAbulletpolylysine complexes ().



One possible mechanism by which LPDII particles may have formed is illustrated in Fig. 9. In this model, DNA is first condensed into a cationically charged complex with polylysine. The cationic complex is then entrapped into anionic liposomes by spontaneous charge interaction.


Figure 9: Possible mechanism for the formation of LPDII. The targeting ligand in the current study is folate.



In Fig. 8B, we observed some very small (<20 nm), possibly lipidic, particles (arrows). In an attempt to remove these, LPDII was fractionated on a 0/20/40% discontinuous sucrose gradient. As shown in Fig. 10A, the majority of the LPDII particles migrated as an intermediate density peak at fractions 6 and 7. Free DNA and free liposomes remained mostly in fractions 1 and 2, whereas DNAbulletpolylysine complexes migrated to the last fraction in the controls (Fig. 10C). Electron micrographs of fractions 6 and 7 of the LPDII particles (Fig. 8, C and D) looked the same as those of the unpurified particles (Fig. 8B) except for the absence of the small low electron density particles. The sucrose gradient profile of LPDII (Fig. 10A) also showed a strong correlation in the distribution of I-labeled DNA and ^3H-labeled liposomes. The fractions at the top of the gradient may consist of a small amount of empty liposomes and free DNA, since the total lipid was in excess in terms of charge in these LPDII particles. The differences in the migration rate of the remaining fractions may be due to the varying degree of DNA condensation by polylysine resulting in different particles densities. Major DNA-containing fractions including 1, 2, 6, 7, and 10 were tested for KB cell transfection. As shown in Fig. 10B, very little transfection activity was observed with fractions 1 and 2, which probably consist of mostly free DNA and free liposomes. Meanwhile, fraction 6 had approximately the same transfection activity as the unpurified LPDII particles (Fig. 3). Fractions 7 and 10 had less transfection activity, possibly due to the high sucrose concentration in the transfection medium derived from the gradient. These results suggested that the small amount of contaminating particles found in unpurified LPDII particles had little or no inhibitory effects on KB cell transfection, and that purification is not required for LPDII particles prepared at a proper lipid/DNA ratio. This is in contrast with LPDI particles described by Gao and Huang,^1 which required the use of excess amounts of cationic lipids in its preparation and, therefore, the subsequent removal of excess lipids by sucrose gradient for optimal activity.


DISCUSSION

We have developed a self-assembled transfection vector in which highly condensed DNAbulletpolylysine complexes were incorporated into folate-targeted anionic liposomes. We found that these vectors were more active in transfection and less cytotoxic. Unlike DNAbulletcationic liposome complexes which usually undergo slow aggregation upon storage, our novel vectors were small in size (70-80 nm) and were not prone to aggregation when carrying a net charge. Depending on the lipid/DNA ratio, either cationic (at low lipid/DNA ratios) or anionic (at high lipid/DNA ratios) LPDII particles could be generated. The cationic particles were highly active in transfection but were not tissue specific, therefore, may be useful as a high efficiency single-vial nonspecific transfection vector. Meanwhile, the efficient receptor-dependent transfection activity of anionic LPDII particles makes them possible candidates for tissue-specific gene delivery. The requirements for folate receptor expression and pH-sensitive lipid composition strongly suggested the roles of the receptor and endocytosis in LPDII transfection.

Similar to the LPDI particles developed by Gao and Huang,^1 LPDIIs are compact spherical particles with a highly condensed DNA core. The lipidic coating on these particles may not only further stabilize the structure of the particle, protect the DNA from the nucleases, but also confer endosomal disruption activity and a site for ligand attachment. Unlike LPDI, negatively charged LPDII is targetable and do not require complicated steps of purification or the use of cationic lipids in its composition.

The design of LPDII particles eliminated potential problems associate with traditional anionic liposomal DNA vectors such as low encapsulation efficiency and generation of excessive empty liposomes. Since no unnatural cationic lipids are required, and at high lipid/DNA ratios the overall charge of the particles were anionic, LPDII particles may be somewhat more biocompatible in vivo compared to the positively charged DNA vectors. Taken together with their compact size, stability and high transfection activity, we believe that LPDII particles may potentially be useful for tissue-specific DNA delivery in vivo.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA59327, HL50256, DK44935, and CA64654. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 412-648-9667; Fax: 412-648-1945; leaf{at}prophet.pharm.pitt.edu.

(^1)
Gao, X., and Huang, L.(1996) Biochemistry35, 1027-1036.

(^2)
The abbreviations used are: LPD, liposome-entrapped polycation-condensed DNA; CHEMS, cholesteryl hemisuccinate; DC-Chol, 3-beta-[N-(N`,N`-dimethylethane)carbamoyl]cholesterol; DOPE, dioleoyl phosphatidylethanolamine; DOPC, dioleoyl phosphatidylcholine; PEG, polyethylene glycol; DOPS, dioleoyl phosphatidylserine; CHO, Chinese hamster ovary.


ACKNOWLEDGEMENTS

We thank Dr. Philip S. Low in the Department of Chemistry at Purdue University for his gift of the KB cells.


REFERENCES

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