Ternary Complexes Comprising Polyphosphoramidate Gene Carriers

Dec 3, 2004 - using an Orion 420A pH meter (Orion, Beverly, MA). Changes in proton ..... This project is supported by a grant from Agency for Science,...
0 downloads 0 Views 476KB Size
Download PDF
Biomacromolecules 2005, 6, 54-60

54

Ternary Complexes Comprising Polyphosphoramidate Gene Carriers with Different Types of Charge Groups Improve Transfection Efficiency† Peng-Chi Zhang,‡ Jun Wang,‡,§ Kam W. Leong,*,‡,§ and Hai-Quan Mao*,‡,| Tissue and Therapeutic Engineering Laboratory, Johns Hopkins Singapore, Singapore 117597, Singapore, Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland 21218 Received March 1, 2004; Revised Manuscript Received October 11, 2004

To understand the influence of charge groups on transfection mediated by polymer complexes, we have synthesized a series of biodegradable and cationic polyphosphoramidates (PPAs) with an identical backbone but different side chains. Our previous study showed that PPA with a spermidine side chain (PPA-SP) showed high transfection efficiency in culture, whereas PPAs with secondary, tertiary, and quaternary amino groups were significantly less efficient. To investigate whether the coexistence of 1° amino charge groups with 3° and 2° amino charge groups in the DNA/polymer complexes would enhance their transfection efficiency, we evaluated a ternary complex system containing DNA and PPAs with 1° amino groups (PPASP) and 3° amino groups (PPA-DMA) and a quaternary complex system containing DNA and PPAs with 1° and 2° and 3° amino groups (PPA-EA/PPA-MEA/PPA-DMA), respectively. Ternary complexes mediated 20 and 160 times higher transfection efficiency in COS-7 cells than complexes of DNA with PPA-SP or PPA-DMA alone, respectively. Similarly, quaternary complexes exhibited 8-fold higher transfection efficiency than PPA-EA/DNA complexes. The mechanism of enhancement in transfection efficiency by the mixture carriers appears to be unrelated to the particle size, zeta potential, or DNA uptake. The titration characterization and the transfection experiments using a proton pump inhibitor suggest that the enhancement effect is unlikely due to the slightly improved buffering capacity of the mixture over PPA-SP. This approach represents a simple strategy of developing polymeric gene carriers and understanding the mechanisms of polymer-mediated gene transfer. Introduction Cationic polymeric gene carriers have been widely used for gene delivery. Poly-L-lysine (PLL),1 poly(4-hydroxy-lproline ester),2 polyamidoamine dendrimers,3 polyethylenimine (PEI),4 and chitosan5 represent a few among the many proposed gene carriers. Compared with viral vectors, cationic polymeric gene carriers are less toxic, less immunogenic, and easier to manufacture, but also markedly less efficient. Much effort has been made to improve the transfection ability of polymeric gene carriers by molecular design and chemical modification of existing structures. Novel polymers being developed for gene delivery include polyallylamine,6 peptoids,7 polymethacrylamide,8,9 and cyclodextrin-containing polymers.10 These polymeric gene carriers vary widely in † This paper was presented at the ICMAT 2003 conference, held in Singapore June 29 through July 4, 2003. * Corresponding authors. K. W. Leong: address, 726 Ross Building, 720 Rutland Ave., Baltimore, MD 21205; tel, (410) 614-3741; fax, (410) 955-0075; e-mail, [email protected]. H.-Q. Mao: address, 102 Maryland Hall, 3400 N. Charles St. Baltimore, MD 21218; tel, (410) 516-8792; fax, (410) 516-5293; e-mail, [email protected]. ‡ Johns Hopkins Singapore. § Johns Hopkins University School of Medicine. | Johns Hopkins University.

their structures, ranging from linear to highly branched molecules that influence their complexation with nucleic acids and their transfection efficiency. Other effort involves chemically modify the polymeric carriers with ligands and peptides that can overcome one or more of the barriers in the transfection process.11,12 Although a comprehensive structure-transfection activity relationship remains to be established, a number of studies have pointed out that the type of charge groups is one of the most important structural parameters for efficient gene transfer.8,9,13-16 Recently, we designed and synthesized a series of biodegradable and cationic polyphosphoramidates (PPAs) with an identical backbone but different side chains ranging from primary to quaternary amines (PPA-SP, PPABA, PPA-EA, PPA-MEA, PPA-DMA, PPA-DEA, and PPATMA) (Scheme 1). The overall objective was to investigate the effects of different charge groups on the transfection efficiencies of the carriers.17,18 These PPA carriers with lower cytotoxicity compared with polyethylenimine (PEI) and polyl-lysine (PLL) show charge group-dependent transfection abilities and DNA binding capacities. PPAs with primary (1°) amino group (PPA-SP and PPA-EA) are the most efficient in transfecting several cell lines,17,18 while other

10.1021/bm040010i CCC: $30.25 © 2005 American Chemical Society Published on Web 12/03/2004

Polyphosphoramidate Improves Transfection Efficiency Scheme 1. Chemical Structures of Polyphosphoramidates (PPAs) Used in This Studya

Biomacromolecules, Vol. 6, No. 1, 2005 55

3° amino groups would exhibit higher transfection efficiency than a single component carrier, we prepared ternary complexes (DNA/PPA-1° amines/PPA-3° amines) and quaternary complexes (DNA/PPA-1° amine/PPA-2° amine/PPA-3° amine) and characterized their transfection efficiency in vitro (Scheme 1). The physicochemical properties of the complexes, the DNA uptake mediated by the complexes, and the pH buffering capacity of the carriers were analyzed in an attempt to elucidate the mechanism of transfection. Materials and Methods

a PPAs are a series of polymers with an identical backbone but different side chains containing primary to quaternary amino groups. The ternary complexes contain DNA and PPA-SP (with 1° amino groups) and PPADMA (with 3° amino groups). The quaternary complexes contain DNA and PPA-EA (with 1° amino groups), PPA-MEA (with 2° amino groups), and PPA-DMA.

PPAs with secondary (2°), tertiary (3°) and quaternary (4°) amino groups only achieve moderate or low levels of gene expression.18 These findings are in good agreement with results obtained in the cationic polymethacrylamide system.6,14,16 Polymethacrylamides with 1° amino groups mediate the highest transfection efficiency among all carriers with various types of charge groups.14 In the PEI system, N-quaternization of the amino groups reduces the transfection efficiency by more than 20-fold.13 While these results highlight the potency of primary amine as the charge groups for polymeric gene carriers, it has been shown that copolymers with 1° and 3° and/or 2° amino groups exhibit higher transfection efficiency.15,16 Reschel et al. showed that, of all the polymethacrylamide carriers they synthesized, the highest transfection activity was found for a copolymer carrier containing both 1° and 3° amines.16 Polyamidoamine (PAMAM) dendrimers also comprise a mixture of 1° amino groups (on the surface) and 3° amino groups (in the interior). Tang et al. reported that the fractured dendrimers (by partial hydrolysis to expose the interior 3° amino groups) show a 50-fold higher transfection efficiency than the intact dendrimers.15 These results suggest that the presence of 3° amine, and 2° amine in some cases, together with 1° amino groups could make a difference in the transfection efficiency. The mechanism of enhancement is not clear at the current stage.16,19,20 Possible causes may include high buffering capacity of the carriers (for PEI) and increased carrier flexibility (for PAMAM dendrimer) that facilitates the compaction of DNA extracellularly or swelling of the complexes intracellularly to release the DNA.15,16 A copolymer carrier containing 1°, 3°, and/or 2° amino groups, therefore, might be a superior carrier than the one with only 1° amine charge groups. A multiple-component complexes (for example, ternary complexes containing DNA with a carrier containing 1° amino groups and a carrier with 3° amino groups) may be able to mimic the copolymer carrier design. To test whether a multiple complex system containing DNA and a mixture of polymeric carriers with 1°, 2°, and

Materials. Polyphosphoramidates (PPAs) with 1° amino group (PPA-SP and PPA-EA), 2° amino group (PPA-MEA), and 3° amino group (PPA-DMA) were synthesized according to the method reported previously.17 The PPAs were purified by dialysis against water. Poly-l-lysine (PLL, MW 27 KDa), polyethylenimine (PEI, MW 25 KDa), Bafilomycin A1, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), and chloroquine diphosphate (CQ) were purchased from Sigma (St. Louis, MO). PEI was purified by dialysis against water [MWCO (molecular weight cutoff) 3500, Pierce, Rockford, IL]. [R-32P]CTP was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Cell Lines and Cell Culture. Monkey SV40-transformed kidney fibroblast cells (COS-7) and human cervix adenocarcinoma cells (HeLa) were purchased from the American Type Culture Collection (ATCC). All cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 4 mM l-glutamine, penicillin (100 units/ml), and streptomycin (100 µg/mL) in a humidified incubator (37 °C, 5% CO2). Plasmid. pRE-Luciferase plasmid is an 11.9 kb cDNA encoding firefly luciferase driven by an RSV promoter inserted into an Invitrogen pREP4 vector (a gift from Dr. R. G. Ulrich, NCI, Frederick, MD). This plasmid DNA was transformed into Escherichia coli DH5R and amplified in Terrific Broth media at 37 °C overnight with shaking at 250 rpm. The amplified plasmid DNA was purified by an endotoxin free QIAGEN Giga plasmid purification kit (QIAGEN, Valencia, CA). Purified plasmid DNA was dissolved in Tris-EDTA (TE) buffer, and its purity and concentration were determined by ultraviolet (UV) absorbance at 260 and 280 nm. All plasmids used in transfection experiments contained greater than 90% supercoiled DNA. Preparation of PPA/DNA Complexes and DNA Gel Retardation Assay. Two types of mixture carriers were prepared (Scheme 1). The first one was a binary mixture with PPA-SP (with 1° amino groups) and PPA-DMA (with 3° amino groups). The second mixture included PPA-EA (1° amine), PPA-MEA (2° amine) and PPA-DMA (3° amine). DNA/polymer complexes were formed by adding 50 µL of polymer solution containing various amounts of polymer to 50 µL of plasmid DNA solution (3 µg) in saline and vortexed for 15-30 s. PPAs in these mixtures were added in different proportions. Complexes were allowed to form for 30 min at room temperature. The complexes were

56

Biomacromolecules, Vol. 6, No. 1, 2005

used for transfection study without further purification. Electrophoretic mobility of the complexes was analyzed on 0.8% (w/v) agarose gel for 40 min at 80 V. The gel was stained with ethidium bromide (0.5 µg/ mL) and visualized on a UV transilluminator (Eagle Eye II, Stratagene, La Jolla, CA). Particle Size and Zeta-Potential Measurement. Measurements of particle size and ζ-potential of the complexes were performed using a Zetasizer® 3000HS (Malvern Instruments, Southborough, MA) at 25 °C (10 mW HeNe laser, 633 nm). The PPAs/DNA complexes were freshly prepared before test. For size measurement, scattered light was detected at a 90° angle. The mean hydrodynamic diameter was determined by cumulative analysis. ζ-Potential measurements of the complexes were carried out in the standard capillary electrophoresis cell at 25 °C. Buffering Capacity. The buffering capacity of PPA-SP, PPA-DMA, PPA-SP/PPA-DMA mixture and polyethylenimine (PEI) was measured by titration according to the method described by Tang and Szoka.21 Each carrier was dissolved at a concentration of 5 mM in a 150 mM NaCl solution. The resulting solution was incrementally titrated with 0.1 N HCl, and the acidity of the solution was followed using an Orion 420A pH meter (Orion, Beverly, MA). Changes in proton concentration were calculated from the cumulative volumes of HCl added. In Vitro Transfection Protocol and Luciferase Assay. In vitro transfection efficiency of PPA/DNA complexes in cell lines was evaluated using luciferase as a marker gene. Cells were seeded 24 h prior to transfection into a 24-well plates (Becton-Dickinson, Lincoln Park, NJ) at an initial density of 5 × 104 cells per well in 1 mL of complete medium (DMEM containing 10% FBS, supplemented with 4 mM L-glutamate, 100 U/ml penicillin and 50 µg/ mL streptomycin). At the time of transfection, the medium in each well was replaced with 1 mL of fresh serum free medium and 100 µl complexes equivalent to 3 µg of DNA were added to each well. After incubating the cells with the complexes for 3 h at 37 °C in humidified 5% CO2 atmosphere, the complex-containing medium was removed and replaced with 1 mL of fresh complete DMEM. Forty-8 h later, cells were permeabilized with 200 µl of cell lysis buffer (Promega Co., Madison, WI). The luciferase activity in cell extracts was measured using a luciferase assay kit (Promega Co., Madison, WI) on a luminometer (LUMAT LB9507, EG&G Wallac) for 10 s. The relative light units (RLU) were normalized against protein concentration in the cell extracts, which was measured using a BCA protein assay kit (Pierce, Rockford, IL). Luciferase activity in cell lysate was expressed as relative light units (RLU/mg of protein in the cell lysate). Transfection with PEI/DNA complexes or poly-l-lysine/DNA complexes was performed as positive controls using the same method. For poly-l-lysine, chloroquine (CQ) was incorporated by mixing it with the poly-llysine solution before complexation with DNA. In some cases, chloroquine was also added to the PPA solution. All transfection experiments were performed in triplicate. Transfection Experiment Involving Bafilomycin A1. The transfection experiments involving bafilomycin A1

Zhang et al.

(Sigma, St. Louis, MO) were conducted as described above except that bafilomycin A1 was added to the transfection medium together with the complexes to yield a final concentration of 175 nM or 262 nM. Cellular Association of Plasmid DNA. Plasmid DNA was labeled with [R-32P]-CTP using a nick translation kit (Life Technologies, Rockville, MD) according to manufacture’s protocols. [32P] labeled plasmid DNA was purified by ethanol precipitation. COS-7 cells were cultured in 24 well plates. [32P] pDNA-polymer complexes were produced using the same method as mentioned above. After seeding the cells for 24 h, the cells were washed twice with PBS and 1 mL of complete DMEM was added to each well. [32P] pDNA (3 µg/mL, 5.0 × 104 µCi/µl) with or without polymers was added to the cells. After incubation at 37 °C or 4 °C for 4 h, the cells were washed with PBS and then solubilized with 0.5 mL of 1 N NaOH and incubated for 2 h. The mixture was added to a PCS solution (Amersham, Arlington Heights, IL) and then measured for the [32P] radioactivity using a multipurpose scintillation counter (Beckman, Palo Alto, CA). Results and Discussion Preparation of PPA/DNA Complexes. The formation of PPA-SP/PPA-DMA/DNA complexes or PPA-EA/PPAMEA/ PPA-DMA complexes was examined by their electrophoretic mobility on an agarose gel at various ratios of amino-groups (1° + 3° in PPA-SP/PPA-DMA mixture or 1° + 2° + 3° in PPA-EA/PPA-MEA/PPA-DMA mixture) to phosphate groups (in DNA), defined as N/P ratio. The gel retardation result of PPA-SP/PPA-DMA/DNA complexes and PPA-EA/PPA-MEA/PPA-DMA/DNA complexes showed that the polymer bound the DNA efficiently at N/P ratios higher than 2 (data not shown). The particle sizes and ζ-poptentials of the PPA-SP/DNA and PPA-SP/ PPA-DMA/DNA ternary complexes at N/P ratio of 9 are shown in Figure 1. There was no difference in ζ-poptential and particle size between PPA-SP/ PPA-DMA/ DNA ternary complexes and PPA-SP/DNA complexes. At N/P ) 9, the particle sizes of these two complexes were around 250 nm and the average ζ-poptentials were about 20 mV. The particle sizes and ζ-poptentialand of the PPA-EA/ DNA and PPA-EA/ PPA-MEA/PPA-DMA/DNA quaternary complexes showed a similar trend (Figure 1). No difference was observed between PPA-EA/PPA-MEA/PPA-DMA/DNA quaternary complexes and PPA-EA/DNA complexes. At N/P ) 10, the particle sizes of these complexes were slightly smaller at around 200 nm and the average ζ-poptential and was about 24 mV. Transfection Efficiency. Transfection of PPA-SP/PPADMA/DNA Ternary Complexes in COS-7 Cells (N/P Ratio of (1° + 3°) to DNA ) 6, 9, or 12). The in vitro transfection ability of the ternary complexes was evaluated in COS-7 cells using the pRE-luciferase plasmid. The PPA with 1° (PPASP) and 3° amino groups (PPA-DMA) used for this study was chosen based on our previous work that showed PPASP yielded the highest transfection efficiency among the five PPAs, and PPA-DMA was significantly less efficient than

Polyphosphoramidate Improves Transfection Efficiency

Biomacromolecules, Vol. 6, No. 1, 2005 57

Figure 1. Average size and ζ-poptential of the ternary complexes, quaternary complexes, PPA-SP/DNA, and PPA-EA/DNA complexes. The upper panel (a) shows the average of 10 individual runs with the standard deviations. The lower panel (b) shows the average values of the ζ-poptentials calculated with the data from six individual runs.

Figure 2. Effect of different ratios of PPA-SP to PPA-DMA on the transfection efficiency of the ternary complexes at N/P ratio of 9 in COS-7 cells. The transfection efficiency was expressed as relative light units ( standard deviation (RLU/mg protein in the cell lysate, n ) 3).

PPA-SP.17 The maximum level of PPA-SP mediated gene transfection was achieved at N/P ratios between 10 and 20 without adding chloroquine diphosphate (CQ), and N/P ratios between 5 and 10 after adding CQ. In this study, we chose a ratio of amino-groups (in PPA-SP plus PPA-DMA) to phosphate groups (in DNA) of 6, 9, and 12 to investigate the effect of N/P ratio. The gene transfection results shown in Figure 2 indicate that mixtures of PPA-SP and PPA-DMA could mediate significantly higher levels of gene expression than using either polymer alone. The highest transfection efficiency mediated by ternary complexes was achieved when the N/P ratio was 9 and the ratio of PPA-SP to PPA-DMA was 80 to 20. Under this condition, transfection efficiency achieved by ternary complexes was 20 and 160 times higher

Figure 3. Effect of different ratios of PPA-SP to PPA-DMA on the transfection efficiency of ternary complexes in COS-7 cells when charge ratio of PPA-SP to DNA was fixed at 12. The transfection efficiency was expressed as relative light units ( standard deviation (RLU/mg protein in the cell lysate, n ) 3).

than PPA-SP and PPA-DMA mediated transfection, respectively, and 790 times higher than that by poly-l-lysine. Transfection of PPA-SP/PPA-DMA/DNA Ternary Complexes in COS-7 Cells (N/P Ratio of PPA-SP (1°) to DNA ) 9 or 12). Since the transfection efficiency mediated by PPA-SP is much higher than that mediated by PPA-DMA, it is possible that PPA-SP plays a much more important role than PPA-DMA in the PPA-SP/ PPA-DMA mixture. We then fixed the N/P ratio of PPA-SP to DNA at 9 or 12, and gradually increased the amount of PPA-DMA in the PPASP/PPA-DMA mixture. The results shown in Figure 3 indicate that the mixture of PPA-SP and PPA-DMA still mediated significantly higher levels of gene expression than either polymer alone. Using this mixing approach, the highest transfection level mediated by PPA-SP/PPA-DMA mixture

58

Biomacromolecules, Vol. 6, No. 1, 2005

Figure 4. Effect of chloroquine (100 µM in the transfection medium) on transfection mediated by ternary complexes at an N/P ratio of 12. The transfection efficiency was expressed as relative light units ( standard deviation (RLU/mg protein in the cell lysate, n ) 3).

Figure 5. Transfection efficiency of ternary complexes in Hela cells (N/P ratio ) 9). HeLa cells were transfected with PPA/DNA complexes in the absence or presence of 100 µM CQ. The transfection efficiency was expressed as relative light units ( standard deviation (RLU/mg protein in the cell lysate, n ) 3).

was achieved when the N/P ratio was 12 and the charge ratio of PPA-SP to PPA-DMA was 100 to 15. Under this condition, transfection efficiency achieved by PPA-SP/PPA-DMA mixture (100/30) was 4 times higher than PPA-SP-mediated transfection and 49 times higher than that by poly-l-lysine. Transfection of Ternary Complexes in COS-7 Cells in the Presence of CQ (N/P Ratio of (1° + 3°) to DNA ) 6, 9, or 12). In the presence of 100 µM of CQ, a reagent known to enhance transfection mediated by complexes trafficking through the endo-lysosomal pathway, the transfection efficiency was increased. The transfection efficiency in the presence of 100 µM CQ (supplemented in the medium) was 3- to 6-fold over that without CQ. As shown in Figure 4, in the presence of CQ, transfection efficiency achieved by PPASP/PPA-DMA mixture was 3 times higher than PPA-SP mediated transfection and 1040 times higher than that by poly-l-lysine. Transfection of Ternary Complexes in Hela Cells (N/P Ratio of (1° + 3°) to DNA ) 6, 9, or 12). The transfection efficiency was also measured against HeLa cells using PPASP/PPA-DMA/DNA ternary complexes containing pRELuciferase plasmid and CQ (Figure 5). The transfection efficiency in HeLa mediated by this mixture was about 6 times higher than PPA-SP mediated transfection and 30 times higher than that by poly-l-lysine. Transfection of PPA-EA/PPA-MEA/PPA-DMA/DNA Quaternary Complexes in COS-7 Cells and Hela Cells (N/P Ratio

Zhang et al.

Figure 6. Transfection efficiency of quaternary complexes in COS-7 cells at an N/P ratio of 10. The transfection efficiency was expressed as relative light units ( standard deviation (RLU/mg protein in the cell lysate, n ) 3).

Figure 7. Transfection efficiency of quaternary complexes in HeLa cells at an N/P ratio of 10. The transfection efficiency was expressed as relative light units ( standard deviation (RLU/mg protein in the cell lysate, n ) 3).

of (1° + 2° + 3°) to DNA ) 10). To further confirm our results, we examined a different set of mixtures containing PPA-EA with 1° amines, PPA-MEA with 2° amine and PPADMA with 3° amine (Scheme 1). In this set of PPA carriers, PPA-EA had the highest transfection efficiency.18 The transfection results shown in Figure 6 (COS-7 cells) and Figure 7 (Hela Cells) indicate that the quaternary complexes containing DNA and this PPA mixture carrier prepared under optimal conditions would mediate a higher level of gene expression than using any PPA carrier alone (p < 0.05). The highest transfection efficiency mediated by the quaternary complexes was achieved when the N/P ratio was 10 and the ratio of PPA-EA to PPA-MEA to PPA-DMA was 4:2:1. Under this condition, transfection efficiency achieved by the PPA-EA/PPA-MEA/PPA-DMA mixture was 8 times higher than the PPA-EA mediated transfection, both in COS-7 cells and Hela cells. Although the enhancement effect of transfection efficiency by the quaternary complexes was not as high as that by the ternary complexes, these results confirmed that PPA mixture carrier with 1° amine charge groups and 3° amino groups (or together with 2° amino groups) is a more efficient gene carrier than PPA with 1° amino groups alone. The results suggest that this could be a general strategy to improve the efficiency of polymer mediated gene transfection. To understand what structural or biological parameters these PPA mixture carriers have changed over single PPA carrier, we investigated the buffering capacity of the PPA-

Polyphosphoramidate Improves Transfection Efficiency

Figure 8. Titration curve for PPA-SP, PPA-DMA, and PPA-SP/PPADMA mixture (charge ratio ) 80/20). The solutions of PPA-SP, PPADMA, and PPA-SP/PPA-DMA mixture were titrated with 0.1 N HCl. Changes in proton concentration were calculated from the cumulative volumes of HCl added.

SP/PPA-DMA mixture and the cellular uptake of the complexes. Buffering Capacity. Titration CurVe. The buffering capacity of PPA-SP/PPA-DMA mixture was tested via titration with HCl compared to PPA-SP. Figure 8 shows that the buffering capacity of PPA-DMA was higher than PPASP in the range of pH4 to 6.6, which does not correlate with their individual transfection efficiencies. After mixing PPASP with PPA-DMA at a charge ratio of 80/20, the buffering capacity of the mixture matched that of PPA-DMA. The difference observed between the transfection efficiencies of PPA-SP/PPA-DMA mixture and PPA-SP alone may be attributed to the added PPA-DMA, which results in a slight increase in the buffering capacity. However, such a small increment of buffering capacity should not be significant enough to account for the enhancement of transfection efficiency. This was further confirmed with the transfection results in the presence of bafilomycin A1. Transfection in the Presence of Bafilomycin A1. If the higher transfection efficiency of the PPA-DMA/PPA-SP mixture were dependent on the buffering-based endosomolytic activity, the inhibition of the acidification of the endosomes should induce a significant decrease in the level of transfection efficiency of the mixture. We chose bafilomycin A1, a proton pump inhibitor, to test whether the transfection efficiency would decrease after the inhibition of the acidification process. Bafilomycin A1 is an antibiotic that selectively inhibits vacuolar type H+-ATPases.22,23 PEI was chosen as a control because its transfection efficiency has been demonstrated to be partially dependent on its ability to capture protons and that its efficiency decreases in the presence of bafilomycin A1.24 Figure 9 shows that the presence of bafilomycin A1 during transfection led to a 6-fold decrease of gene expression in PEI-mediated transfection, a 10-fold decrease for PPA-SP, but only 2-fold for PPA-SP/ PPA-DMA. Therefore, even in the presence of a proton pump inhibitor, the efficiency of PPA-SP/PPA-DMA mixture was still higher than that of PPA-SP alone. It suggests that the high transfection efficiency of PPA-SP/PPA-DMA mixture may not be solely due to its marginally increased buffering capacity. DNA Uptake in COS-7 Cells. Cellular uptake is another crucial step in polymer-mediated transfection. The ternary

Biomacromolecules, Vol. 6, No. 1, 2005 59

Figure 9. Effect of proton pump inhibitor bafilomycin A1 on transfection mediated by ternary complexes at an N/P ratio of 12. COS-7 cells were transfected with PEI/ DNA, PPA-SP/DNA, or ternary complexes in the presence or absence of 175 nM or 262 nM bafilomycin A1. The transfection efficiency was expressed as relative light units ( standard deviation (RLU/mg protein in the cell lysate, n ) 3).

Figure 10. DNA uptake in COS-7 cells mediated by PPA-SP/DNA, PPA-DMA/ DNA or ternary complexes. [32P]-Labeled plasmid DNA was added to cells alone or in the form of complexes at 4 and 37 °C, respectively, prior to assay. The results shown here represent the difference (in percentage) between the cell-associated radioactivity at 37 and 4 °C.

complexes might increase cellular uptake of DNA and lead to the enhanced transfection efficiency. To test this hypothesis, we examined the effects of PPA-SP/ PPA-DMA mixture on the cellular association of plasmid DNA to COS-7 cells, using [32P]-labeled DNA. After incubation at either 4 or 37 °C with the complexes containing labeled DNA, the cells were lysed with 1 N NaOH solution. Cell-associated radioactivity at 37 °C would include bound and internalized DNA. Because classical endocytosis, an energy-dependent mechanism of cell entry, is blocked at 4 °C, the cellassociated radioactivity at this temperature would be limited to cell bound material or radioactive complexes that entered by a nonendocytic, possibly fusion mechanism. Thus, the difference between the cell-associated radioactivity at 37 and 4 °C would represent uptake by endocytosis.25 DNA uptake levels mediated by ternary complexes and PPA-SP, PPADMA and naked DNA were shown in Figure 10. The amount of cell-associated radioactivity for PPA-SP/PPA-DMA mixture was similar to that for PPA-SP or PPA-DMA alone. This suggests that there was no significant difference in terms of DNA uptake between PPA-SP/PPA-DMA mixtures and PPA alone.

60

Biomacromolecules, Vol. 6, No. 1, 2005

Taken together, these results rule out the possibility that the ternary complexes enhance the transfection predominantly by any one of the following parameters: size and ζ-poptential of the particles, pH buffering capacity, or cellular DNA uptake. It seems likely that the enhancement effect is related to intracellular trafficking of the DNA, most notably, the nuclear uptake and DNA unpacking. Conclusion Using two different systemssternary complexes containing PPA-SP/PPA-DMA mixture (with 1° amino and 3° amino groups) and quaternary complexes containing PPA-EA/PPA/ MEA/PPA-DMA mixture (with 1° amino, 2° amino and 3° amino groups)swe have demonstrated that introducing multiple polymeric carriers with different charged groups into the same complexes could enhance the transfection efficiency compared with complexes comprising single PPA carrier alone. Our results also showed that the mechanism of the higher transfection efficiency mediated by this mixture appears to be unrelated to the particle size, ζ-poptential and DNA uptake. The titration characterization and the transfection experiments using a proton pump inhibitor suggest that the mechanism of enhancement is unlikely due to the slightly improved buffering capacity of the mixture over PPA-SP. Further investigation is needed to understand the exact mechanism of the increased transfection efficiency mediated by the PPA mixture gene carriers. Nevertheless, this approach represents a simple strategy of developing polymeric gene carriers and understanding the mechanisms of polymer-mediated gene transfer. Acknowledgment. This project is supported by a grant from Agency for Science, Technology and Research (A*STAR) of Singapore and Johns Hopkins Singapore. The authors thank Dr. Chou Chai for the helpful discussion. References and Notes (1) Wagner, E.; Ogris, M.; Zauner, W. AdV. Drug DeliV. ReV. 1998, 30, 97-113. (2) Putnam, D.; Langer, R. Macromolecules 1999, 32, 3658-3662. (3) Kukowska-Latallo, J. F.; Bielinska, A. U.; Johnson, J.; Spindler, R.; Tomalia, D. A.; Baker, J. R., Jr. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4897-4902.

Zhang et al. (4) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7297-7301. (5) Mao, H. Q.; Roy, K.; Troung, L.; Janes, K. A.; Lin, K. Y.; Wang, Y.; August, J. T.; Leong, K. W. J. Controlled Release 2001, 70, 399-421. (6) Boussif, O.; Delair, T.; Brua, C.; Veron, L.; Pavirani, A.; Kolbe, H. V. Bioconjugate Chem. 1999, 10, 877-883. (7) Murphy, J. E.; Uno, T.; Hamer, J. D.; Cohen, F. E.; Dwarki, V.; Zuckermann, R. N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 15171522. (8) van de Wetering, P.; Cherng, J. Y.; Talsma, H.; Crommelin, D. J.; Hennink, W. E. J. Controlled Release 1998, 53, 145-153. (9) van de Wetering, P.; Moret, E. E.; Schuurmans-Nieuwenbroek, N. M.; van Steenbergen, M. J.; Hennink, W. E. Bioconjugate Chem. 1999, 10, 589-597. (10) Gonzalez, H.; Hwang, S. J.; Davis, M. E. Bioconjugate Chem. 1999, 10, 1068-1074. (11) Midoux, P.; Monsigny, M. Bioconjugate Chem. 1999, 10, 406411. (12) Petersen, H.; Fechner, P. M.; Martin, A. L.; Kunath, K.; Stolnik, S.; Roberts, C. J.; Fischer, D.; Davies, M. C.; Kissel, T. Bioconjugate Chem. 2002, 13, 845-854. (13) Thomas, M.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 14640-14645. (14) Wolfert, M. A.; Dash, P. R.; Nazarova, O.; Oupicky, D.; Seymour, L. W.; Smart, S.; Strohalm, J.; Ulbrich, K. Bioconjugate Chem. 1999, 10, 993-1004. (15) Tang, M. X.; Redemann, C. T.; Szoka, F. C. Bioconjugate Chem. 1996, 7, 703-714. (16) Reschel, T.; Konak, C.; Oupicky, D.; Seymour, L. W.; Ulbrich, K. J. Controlled Release 2002, 81, 201-217. (17) Wang, J.; Zhang, P. C.; Lu, H. F.; Ma, N.; Wang, S.; Mao, H. Q.; Leong, K. W. J. Controlled Release 2002, 83, 157. (18) Wang, J.; Gao, S. J.; Zhang, P. C.; Wang, S.; Mao, H. Q.; Leong, K. W. Gene Ther., in press. (19) Sonawane, N. D.; Szoka, F. C.; Verkman, A. S. J. Biol. Chem. 2003, 278, 44826-44831. (20) von Harpe, A.; Petersen, H.; Li, Y. X.; Kissel, T. J. Controlled Release 2000, 69, 309-322. (21) Tang, M. X.; Szoka, F. C. Gene Ther. 1997, 4, 823-832. (22) Bowman, E. J.; Siebers, A.; Altendorf, K. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7972-7976. (23) Yoshimori, T.; Yamamoto, A.; Moriyama, Y.; Futai, M.; Tashiro, Y. J. Biol. Chem. 1991, 266, 17707-17712. (24) Kichler, A.; Leborgne, C.; Coeytaux, E.; Danos, O. J. Gene Med. 2001, 3, 135-144. (25) Colin, M.; Harbottle, R. P.; Knight, A.; Kornprobst, M.; Cooper, R. G.; Miller, A. D.; Trugnan, G.; Capeau, J.; Coutelle, C.; BrahimiHorn, M. C. Gene Ther. 1998, 5, 1488-1498.

BM040010I