Introduction

A variety of polycations and cationic lipids within cationic DNA complexes enable the transfection of cells in culture.^1,2 Transfection in vivo, a challenging problem for cationic DNA complexes, has been notably achieved in lungs following the systemic delivery of plasmid DNA (pDNA) complexed with either cationic lipids (CL) or polyethyleneimine (PEI).³ Linear PEI (lPEI) 22 kDa in size enables higher levels of pulmonary gene expression than branched PEI (brPEI) or CL.^4,5 Interestingly, the intravenously injected PEI complexes enable expression not only in pulmonary endothelium but also in alveolar epithelium.^6,7

However, the wider utility of cationic DNA complexes for in vivo applications is limited by low gene expression and toxicity.^8,9 The intravascular route of administration, an attractive approach for widespread delivery, is particularly plagued by these confounding problems. Decreased transfection efficiency in vivo is due in part to the interaction of the polyplexes or lipoplexes with blood components such as serum proteins, which inhibit transfection.^{10,11,12,13,14} This effect is usually attributed to the opsonization of the DNA complexes with serum components.^15,16,17,18 Furthermore, intravenously injected cationic DNA complexes also encounter unintended cell types such as macrophages, monocytes, neutrophils, platelets, and erythrocytes, which are important potential mediators of toxicity.^9,19,20 Toxic manifestations of systemically administered cationic DNA complexes can range from red blood cell agglutination²¹ to potent inflammatory reaction and elevated serum levels of liver enzymes.^9,22 Several studies have attempted to avoid such adverse interactions by including polyethyleneglycol (PEG) or proteins such as albumin or transferrin in the DNA complexes.^21,23,24,25

We have previously described the formation of ternary complexes containing DNA, polycations (pC) and synthetic polyanions (pA).²⁶ We have now extended these studies and found that the addition of certain synthetic pAs such as polyacrylic acid (pAA) increased the in vitro transfection efficiency of brPEI in the presence of serum. Our initial observations with brPEI were then extended to other cationic DNA complexes containing either CL or lPEI. The pA also enhanced gene expression in lungs following tail vein injections of DNA/PEI or DNA/cationic liposome complexes in mice while reducing toxicity.

Results

Formulation of DNA/PEI complexes

As previously described, the number of positive charges per molecular weight of a pC can be experimentally measured by determining the minimal amount of pC that can maximally condense DNA.²⁷ The use of DNA covalently modified by a fluorescent group that quenches when the DNA condenses enables the condensation state to be conveniently and accurately determined. Plasmid DNA covalently labeled with Oregon Green labeling reagent (OG-DNA)²⁷ was titrated with either brPEI or lPEI by their sequential addition to the OG-DNA solution (Figure 1). Based on the inflection point for maximal quenching of DNA fluorescence, we found that 2.5 g of brPEI and 3.0 g of lPEI fully condensed 10 g of OG-DNA at the above conditions. This indicates that brPEI and lPEI polycations behave as polymers with an average molecular weight of 83 and 99 per positive charge, respectively, under the conditions of the experiment.

Figure 1.

Determination of 1:1 charge ratio for DNA/PEI complexes using fluorescence quenching of Oregon Green (OG)- labeled DNA. Aliquots of pC were sequentially added to the 10 g of OG-DNA in 0.5 ml of 10 mM HEPES, pH 7.5 directly into the cuvette. The decrease of OG fluorescence upon each pC addition was measured using a Shimadzu RF 1501 spectrofluorimeter at _ex=480  nm; _em=520 nm. The results are shown as percent decrease relative to initial fluorescence of non-condensed OG-DNA.

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We observed that the addition of a certain amount of a second pA to the binary OG-DNA/pC complex in a low salt solution caused flocculation of the whole system, indicating the formation of a ternary complex. For example, addition of 23 g of pAA (sodium salt) to the preformed binary complex of 10 g of OG-DNA and 20 g of brPEI (N/P ratio = 15) in 0.5 ml of buffered isotonic glucose solution (BIGS) resulted in clearly visible precipitation. After dissolution of the centrifuged precipitate in 1.5 M NaCl, 86% of initial OG fluorescence was recovered, thus proving the presence of the DNA in the ternary complex (data not shown).

In vitro brPEI studies

DNA complexes were formed with brPEI at a nitrogen/phosphate ratio of 15 (1:2 w/w ratio of DNA:brPEI), which is approximately a charge ratio of 8 based on the titration data above (Figure 1). This ratio was chosen because previous studies and our unpublished results have shown optimal transfection occurs in vitro with such complexes.²⁸ The share of negative charge due to the second pA addition was calculated assuming one negative charge per carboxyl group. The pA titration data are presented as a function of the pA/pC charge ratio, not counting DNA charge and using the pC charge-to-weight ratios derived above (Figure 1). The effect of pAs on the transfection efficiency of the DNA/brPEI complexes were evaluated by adding various amounts of pAA to the complexes and incubating the ternary complexes for 10 min or 3 h before applying them to HuH7 human hepatocellular carcinoma cells in serum-free Opti-MEM cell culture medium or 100% bovine serum (BS) (Figure 2).

Figure 2.

In vitro luciferase activity in HuH7 cells two days after application of pMIR48/brPEI/pAA complexes. The complexes were formulated in BIGS (10 mM HEPES, 0.29 M glucose, pH 7.5) as described in the Materials and methods and added to the cells after replacing the culture media with either serum-free Opti-MEM or 100% bovine serum. The complexes were applied to the cells 10 min or 3 h after preparation. The DNA/brPEI w/w ratio was 1:2 (N/P=15). The pA/pC ratio in the X-axis is the charge ratio between PEI and pAA only (DNA is excluded), so the first experimental point represents non-recharged, binary DNA/brPEI complex. Luciferase values represent an average two measurements.

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The presence of 100% serum during transfection with binary DNA/brPEI complex caused an approximately 1000-fold drop in luciferase activity (compare 'serum/10 min' and 'serum-free/10 min' at a pA/pC ratio of 0 in Figure 2). The addition of certain amounts of pAA to the DNA/brPEI complex (N/P ratio 15) enhanced gene transfer activity in the presence of 100% BS up to the levels that were obtained in serum-free medium. Maximum activity was obtained when the pC/pA ratios approached electroneutrality, which was inferred from the amounts of pAs added and an increase in flocculation (using visual assessment). If complexes were allowed to stand at room temperature for 3 h after mixing and before applying to the cells, they exhibited a 10-fold drop in maximum transfection activity (Figure 2). Similar data were obtained in HuH7 cells with DNA/lPEI/pAA complexes (data not shown).

The pAA also increased the percentage of cells transfected in 100% BS. Ten percent of HuH7 cells were transfected with the plasmid encoding nuclear yellow fluorescent protein (pEYFP-nuc) complexed with brPEI/pAA (pA/pC charge ratio = 0.85). No YFP expressing cells were found following incubation of binary DNA/PEI complexes with cells under the same conditions (data not shown).

Qualitatively similar results were obtained in human lung carcinoma cells A549 in terms of the effects of serum and pAA (Figure 3). In addition, the size of the triple DNA/brPEI/pAA complexes immediately before transfection was measured using dynamic light scattering. Maximum transfection coincided with near electroneutrality and flocculation of the DNA complexes (500 nm in diameter at the peak of flocculation).

Figure 3.

The effect of pAA/brPEI charge ratio on the size and in vitro gene transfer activity (luciferase expression) of pMIR48/brPEI/pAA complexes in human lung carcinoma A549 cells. The sizes of the complexes were determined for various pAA concentrations where DNA:pC ratio (1:6 w/w, N/P=45) and concentrations were constant. Transfections were performed in either 100% bovine serum or serum-free Opti-MEM. The size measurements were performed on the serum-free complexes immediately before the complexes were added to cells using dynamic light scattering (Zeta Plus Quasi Elastic Light Scattering sizer, Brookhaven Instruments). Luciferase values represent an average of two measurements.

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After observing the effect of pAA on the activity of DNA/brPEI complex in 100% BS, we studied the influence of other polycarboxylic acids on transfection activity of DNA/brPEI/pA complexes in the presence of 100% BS. We evaluated the effects on the transfection of four synthetic polycarboxylates with decreasing charge density: pAA > polyaspartic acid (pAsp) > polyglutamic acid (pGlu) > succinylated poly-L-lysine (SPLL). It was observed that the levels of enhanced transfection activity are dependent on the charge density (carboxyl/backbone distance) of the pA, with pAA demonstrating the highest effect and SPLL the lowest one (Figure 4). For all the pAs tested, the peak activity was associated with the flocculation (data not shown).

Figure 4.

Maximum gene transfer activities of DNA/brPEI complexes mixed with various pAs in HuH7 cells. Transfections were performed in 100% bovine serum. Peak activities for each pA were obtained in titration experiments similar to the one depicted in Figure 2. Abbreviations for the pAs are as follows: pAA, polyacrylic acid; pAsp, polyaspartic acid; pGlu, polyglutamic acid; SPLL, succinylated polylysine. Luciferase levels represent an average of two measurements.

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Complex-binding proteins in serum

Since the enhancement of the gene transfer activity was apparent in the presence of serum, disruption of opsonization of DNA/pC complex with serum components using pA might be responsible for the observed phenomenon. To address this hypothesis, we isolated DNA/brPEI and DNA/brPEI/pAA complexes after incubation in BS using discontinuous sucrose/metrizamide gradient ultracentrifugation and analysed serum proteins associated with the complexes using SDS PAGE (Figure 5). Fluorescent dye (Cy5)-labeled DNA was used for the complex preparation in order to visualize the DNA band in the gradient test tube. DNA/brPEI and DNA/brPEI/pAA complexes were found as flaky precipitates on the sucrose/metrizamide boundary and were easily retrievable using a Pasteur pipette. In contrast to work reported by others^16,17, our direct attempts to dissociate opsonizing proteins from DNA/pC complexes using SDS PAGE sample buffer failed. Long smears rather than distinctive protein bands appeared on the gels, rendering molecular weight determination impossible. Hence, we included an additional step of dissociating and removing DNA from these complexes by incubating them in 1.25 M NaCl and separating them by size exclusion in a Sepharose 4B-CL column in the same high salt solution. Void volume fractions containing DNA were discarded and the rest of the fractions were pooled, exhaustively dialysed against water, freeze-dried and re-solubilized in SDS PAGE sample buffer. DNA/brPEI cationic complexes, after incubation with BS, opsonized with a number of serum proteins (primary fractions with approximate molecular weights of 60, 100 and 130 kDa) (Figure 5, lane 3, see arrows). The major band with a molecular weight of 60 kDa is presumably bovine serum albumin. DNA/brPEI/pAA complexes had no detectable serum protein binding (Figure 5, lane 4).

Figure 5.

Coomassie-stained SDS PAGE gel of serum proteins isolated from DNA/brPEI and DNA/brPEI/pAA complexes. Electrophoresis was performed under non-reducing conditions. Lane 1, molecular weight ladder; lane 2, bovine serum; lane 3, proteins recovered from DNA/brPEI (1:4 w/w ratio) complexes; lane 4, proteins recovered from DNA/brPEI/pAA (pAA/brPEI charge ratio=0.64) complexes. Arrows indicate the three major proteins binding to DNA/brPEI complexes in lane 3.

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In vivo studies with brPEI and cationic lipids

Ternary complexes of pMIR48/brPEI/pAA were also evaluated for their in vivo transfection activity by injecting them into mouse tail veins and assaying luciferase activity in lungs one day later (Figure 6). Of note, is the fact that these injections are low-pressure injections done under conditions in which naked DNA delivery results in minimal activity, especially in the lungs. Triple complexes of DNA/brPEI with increasing amounts of pAA revealed a bell-shaped dependence for luciferase activity in this organ (Figure 6). For this experiment, a higher brPEI amount (1:4 w/w or N/P ratio of 30) was used in the ternary complexes (than in the in vitro experiment, Figure 2), because preliminary optimization experiments indicated that this yielded higher luciferase levels as compared to 1:2 (w/w) complexes initially used for in vitro gene transfer (Figure 2). Luciferase activity peaked at 2 ng of luciferase per mg of extracted protein when the pA/pC charge ratio was 0.67 (Figure 6). Interestingly, mortality also lessened with increasing pAA content. It was not possible to assess gene transfer activity for binary DNA/brPEI complex (at this relatively high brPEI/DNA) ratio since there were no survivors. Luciferase expression levels in other organs were at least one order of magnitude less than that in the lungs with decreasing efficiency in the following order: heart > kidney > liver = spleen. Lungs are usually observed to be the organ with the maximum transgene activity upon systemic intravenous injections of transfection complexes.^6,18

Figure 6.

Branched PEI complexes in vivo. The relationship between the amount of pAA within DNA/brPEI/pAA ternary complexes (50 g of pMIR48 and 200 g of brPEI per animal, N/P=30) and luciferase activity in mouse lung (means.e.m; n=3–7 per group). Luciferase activities were assayed in lungs one day after the ternary complexes were injected into the tail vein. Survival rates of injected animals are indicated by the filled circles.

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The addition of pAA to DNA complexed with DOTAP/cholesterol (2:1 mol/mol) cationic liposomes (CL) also enabled high pulmonary luciferase levels (Figure 7). The marked differences from DNA/brPEI experiment were the absence of apparent toxic effects (all animals survived in all groups) and overall lower gene transfer activity in the lungs. In the case of DNA/CL, the recharged complexes exhibited more extensive flocculation at certain CL/pAA ratios as compared to the preparations where brPEI was used as pC. Thus, injections of these preparations were not possible due to complete DNA precipitation.

Figure 7.

Ternary cationic lipid complexes in vivo. The relationship between the pAA-to-DOTAP charge ratio within pMIR48/DOTAP: cholesterol/pAA ternary complexes and luciferase activity in mouse lungs (means.e.m, n=3 per group). The base complex was formulated using 50/530:150 g of DNA/DOTAP: cholesterol per animal (1:5 DNA: CL charge ratio) as described in the Materials and methods. Luciferase activities were assayed in lungs one day after the ternary complexes were injected into the tail vein.

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In vivo studies with lPEI

Ternary DNA complexes containing lPEI instead of brPEI were evaluated for lung gene transfer and toxicity. As with brPEI, the inclusion of certain amounts of pAA within the pMIR48/lPEI /pAA ternary complexes (at a 0.13 pA/pC charge ratio) yielded lung luciferase levels approximately one order of magnitude greater than the levels obtained from pMIR48/lPEI binary complexes alone (Figure 8). In contrast to brPEI, the lPEI complexes did not kill the animals within 24 h although they did cause significant toxicity. The reduced toxicity of lPEI also enabled the inclusion of higher amounts of lPEI in the injectates to increase lung expression.

Figure 8.

Linear PEI complexes. The relationship between the amount of pAA within DNA/PEI/pAA ternary complexes (50 g of pMIR48 and 400 g of lPEI, N/P ratio 60) and luciferase activity in mouse lung (bars indicate mean, n=2 per group). Luciferase activities were assayed in lungs one day after the ternary complexes were injected into the tail vein. Ternary complexes with a pA/pC charge ratio of 0.13 contain 50 g of pAA.

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To investigate the toxicity of these lPEI-containing ternary complexes, lungs and livers were examined histologically 24 h after tail vein injections of ternary complexes (50 g pMIR48/400 g lPEI/50 g pAA per animal) (Figure 9a and c). In this group the lung sections stained with hematoxylin/eosin (H&E) showed perivascular and interstitial edema, blood congestion and disintegration of alveolar structures and destruction of its cellular components. Neutrophil intravascular adhesion and accumulation as well as neutrophil infiltration of edematous alveolar structures were found in this group (Figure 9a). Livers of the same animals showed extensive parenchymal damage such as bridging and scattered necrosis (green arrows in Figure 9c) and apoptosis. Consistent with the liver damage, plasma levels of the liver enzyme alanine aminotransferase (ALT), an indicator of hepatocyte toxicity, rose significantly to more than 4,000 U/l 24 h after tail vein injection of this ternary complex (Figure 10a, 'no chaser' bar).

Figure 9.

Sections of livers and lungs of mice 24 h after injection of DNA/lPEI/pAA (50/400/50 g, N/P=60) complexes without 'chaser' (a and c) and 40/240/40 g (N/P=45) with 1.5 mg pAA 'chaser'; termed 'optimal' protocol (b and d). (a) and (b) Lung paraffin sections stained with H&E. (c) and (d) Liver paraffin sections stained with H&E. Magnification for all panels is 100. Green arrows in panel (c) indicate areas of damage. These are representative sections from four animals analysed for each condition.

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Figure 10.

Serum ALT levels (a) and luciferase levels (b) in mice injected with DNA/lPEI/pAA complexes (50/400/50 g, N/P=60) via tail vein (means.d; n=3). For the 'chaser' group, a second tail vein injection of pAA (1.5 mg of pAA in 100 l BIGS) was administered 0.5 h after DNA injection. Panel (a): serum ALT levels with or without pAA chaser 24 h after injections of complexes. ALT levels are expressed as mean s.d; n=3. Panel (b): luciferase expression in lungs with or without chaser 24 h after injections of DNA/lPEI/pAA complexes. Luciferase values (ng/mg protein) are mean levels s.d; n=3.

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A 'chaser' injection of pAA reduces toxicity

In order to reduce the toxicity further, we explored the use of a 'chaser' injection of pAA 30 min after injection of the ternary complexes containing pMIR48/lPEI/pAA complexes (50/400/50 g; pA/pC charge ratio = 0.13). (Figure 11). While the 'chaser' injection of pAA did not affect the lung luciferase levels 24 h after injection (Figure 10b), they caused a substantial reduction in plasma ALT levels (Figure 10a).

Figure 11.

Serum SEAP levels (means.d, n=3–4) 24 h after tail vein injection of lPEI ternary complexes containing 50 g of pMIR85, 400 g of lPEI, (N/P=60) and varying amounts of pAA in 250 l of BIGS. A second injection of 1.5 mg of pAA in 100 l of BIGS (chaser) was administered 0.5 h post-DNA injection. The sizes of the corresponding complexes were measured immediately before injection using dynamic light scattering.

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Without 'chaser' maximal luciferase expression was observed when the pA/pC charge ratio in the ternary complexes was 0.13. Using the secreted alkaline phosphatase (SEAP) reporter gene construct pMIR85 within lPEI ternary complexes, maximum serum SEAP levels were also observed at a 0.13 pA/pC charge ratio (Figure 11), indicating that the 'chaser' did not affect the amount of pAA in the complexes required for maximum expression. Particle size of these complexes grew with increasing pAA content (Figure 11).

We further optimized several parameters of this delivery system so as to effect maximum foreign gene expression with minimal toxicity. The use of recharging and a 'chaser' injection enabled higher amounts of DNA and lPEI to be used.¹⁸ The optimized protocol involved the injection of ternary complexes containing 40 g DNA/240 g lPEI/40 g pAA (pA/pC charge ratio = 0.17) followed 30 min later by an injection of 1.5 mg of pAA 'chaser'. Compared to the ternary complexes that enabled the maximum expression in the experiment described above (albeit with some toxicity), the DNA content was reduced from 50 to 40 g, the lPEI was reduced from 400 to 240 g, pAA reduced from 50 to 40 g, and the pA/pC charge ratio increased from 0.13 to 0.17. Using this 'optimized' protocol for reduced toxicity, lung luciferase levels were around 0.5–1 ng/mg protein (10-times reduced from the previous 'maximal' results). Concomitantly, plasma ALT levels one day after injection were also diminished to approximately 200 U/l (baseline levels are 50–70 U/l, data not shown).

Using the -galactosidase reporter system, the 'optimized' protocol enabled 10% of the lung cells to be -galactosidase-positive (Figure 12b) as compared to the 35% positive cells in mice injected with the ternary complexes designed mainly for maximal expression (Figure 12a). In addition, the lungs and livers in animals injected using the 'optimized' protocol appeared significantly more normal histologically (Figure 11b and d). Some of the transfected cells, judging from their morphology and localization, are pneumocytes (Figure 12c and d, blue arrows), while others are likely endothelial cells (Figure 12d, red arrows). -Galactosidase-expressing cells were not found in non-capillary vessels or bronchial structures.

Figure 12.

-galactosidase expression in frozen lung sections of mice 24 h post-injection. (a) Mice injected with ternary complexes containing DNA/lPEI/pAA (50/400/50 g, N/P=60) and no 'chaser' injecton. (b–d) mice injected under the 'optimal' protocol using ternary complexes containing DNA/lPEI/pAA (40/240/40 g, N/P=45) and then with a 1.5 mg pAA 'chaser' 30 min later. The sections represented in (a) and (b) were only stained with X-gal to aid in the assessment of the percent of cells transfected, while the sections represented in (c) and (d) were also stained with hematoxylin to aid in the identification of the cell types. Blue arrows indicate -galactosidase-positive epithelial cells, while red arrows indicated -galactosidase-positive endothelial cells. Magnification: 200 (a and b) and 630 (c and d). These are representative sections from four mice analysed for each condition.

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Discussion

This study found that the inclusion of pAA with DNA/pC and DNA/CL complexes prevented the serum inhibition of the transfection complexes in cultured cells. The use of pAA also increased the levels of lung expression in mice in vivo substantially above the levels achieved with just binary complexes of DNA and PEI or cationic lipids. Besides transgene activity in the lung, this study also achieved therapeutically relevant levels of a foreign protein circulating in blood (ie, 750 ng/ml of SEAP, Figure 11). On the basis of our previous study, the addition of pAA to the DNA/PEI complexes 'recharges' them in terms of adding additional negative charge and altering their surface ^- potential.²⁶

It is increasingly being recognized that the toxicity of intravenously delivered cationic DNA complexes limits their animal and potential human applications.^6,18" Even when the transfection complexes do not decrease survival outright, microscopic and serologic studies can still indicate substantial toxicity, especially in the lungs and liver. Cognizant of this, we optimized the amount of pAA, lPEI, and DNA within the ternary complexes so as to enable the highest levels of expression with minimal microscopic toxicity. The use of a 'chaser' injection of pAA 30 min after injection of the ternary DNA/lPEI/pAA complexes further aided this effort to reduce toxicity while not affecting foreign gene expression. Under optimal conditions, substantial levels of lung expression were obtained (0.5–1 ng luciferase/mg protein, 10% -galactosidase-positive cells, Figure 12b), while adverse effects in lung or liver were not apparent (Figure 9b and d).

The ability for a highly charged (strong) pA such as pAA to prevent serum inhibition and enhance in vivo gene transfer while maintaining transfection competency is counter-intuitive. Several reports indicated that strong pAs (including negatively charged liposomes) inhibit gene transfer activity of DNA/CL complexes in vitro and in vivo.^29,30,31 This phenomenon is generally explained by displacement of DNA from DNA/CL complexes by such pAs.³² It is worth noting that these studies typically employed a single large dose of an excess of strong pAs to demonstrate complex disruption and consequent decrease in gene transfer activity. pA titration experiments usually were not performed.

On the other hand, the addition of pAs to DNA/pC and DNA/CL complexes may form novel structures without completely disrupting DNA interactions with the cationic molecules. For instance, alternating complexes of pC/pA form layered structures when absorbed on macrosurfaces from aqueous solutions.³³ Our previous study has demonstrated that a similar phenomenon takes place on the surface of pC-condensed DNA particles when they are further complexed with a third-layer pA.²⁶ For highly charged pAs such as pAA, ternary DNA/pC/pA complexes containing condensed DNA are present in low salt solutions and when the pA/pC charge ratio is less than one. In physiologic saline solutions, ternary complexes are still present when the pA/pC ratio is less than one, but the DNA is less condensed. Under the conditions of the present study, the ternary complexes were formed initially in BIGS, a low salt solution. Upon exposure to the physiologic salt conditions in the tissue culture plate or in vivo, DNA displacement should not occur completely below a pA/pC charge ratio of one and thereby the DNA complexes remain transfection competent. At a pA/pC charge ratio above one, separation of DNA from the pC occurs and transfection is attenuated (Figures 2 and 3).

We hypothesize that the ternary complexes are resistant to serum inhibition by their decreased proclivity to opsonization with serum proteins. Several blood proteins such as albumin or fibrinogen become associated with DNA/pC and DNA/CL and may be responsible for the serum's inhibitory effect on transfection.^{15,16,17,21,34} The effect of a strong pA on the activity of DNA/pC complexes in the serum may be explained by competition between these anionic proteins and added pAs on the surface of the cationic DNA complexes. The absence of plasma proteins associated with the recharged complex as observed by SDS PAGE supports this notion (Figure 5).

The effect of pAA on the transfection competency of DNA complexes may also involve its influence on the size of the complexes. A common point of view has been that smaller DNA particles should possess higher transfection activity since they would be more easily taken up by cells.³⁵ However, in a number of recent papers it has been demonstrated that the opposite is true: large DNA/pC complexes transfect cells in culture better than small complexes.^{5,10,12,15,36,37,38} For example, the formulation of DNA/lPEI in saline solutions increases both their size and in vitro transfection efficiency.⁵ With increasing amounts of pAA, both the size and transfection competency of DNA/PEI complexes rise as well (Figure 3). Interestingly, the binary PEI complexes became bigger by salt expressed less in vivo,⁵ while the larger-sized ternary complexes containing pAA yielded greater lung expression. Furthermore, DNA/brPEI/pAA complexes matured for 3 h transfected less efficiently than complexes formed for only 10 min (Figure 2). A similar maturation effect was observed for DNA/lPEI/pAA ternary complexes (data not shown). With time, the complexes become perhaps too large. Apparently, there is an optimal size for transfection with very large complexes being less transfection competent.

The effect of pAs on the size of the DNA complexes may be related to their effect on serum inhibition. It has been demonstrated using dynamic light scattering that the flocculation of DNA/pC complexes can be prevented by placing them into serum, most probably by adsorption of proteins.³⁸ Fluorescent DNA/lipopolyamine complexes taken up by the cells in the presence of serum are localized in smaller intracellular vesicles as compared with serum-free conditions.¹⁰ Highly charged pAs may increase transfection by avoiding the effect of serum proteins on the size of cationic DNA complexes. Naturally, in vitro and in vivo applications of DNA/PEI/pAA complexes required different ratios of the constituents. We believe that this possibly reflects the involvement of tissues and organs other than lungs in mediation of lung transfection.

The polymer pAA may have aided transfection by other mechanisms such as enhancing release of the DNA from the cationic agent.³² Polyacrylic acid does not possess any membrane activity that could directly account for its transfection-enhancing effect. Membrane-active polyanions such as influenza-derived peptides and polypropylacrylic acid have been proposed as adjuvants for transfection of cationic DNA complexes.^25,39,40 Our results emphasize that the effects of these anions on the complex structure apart from their membrane active properties need to be taken into account when analysing their effects on transfection efficiency.

The intravenous injection of cationic DNA complexes can cause not only immediate shock and death, but also delayed toxicity. The acute toxic reaction probably results from interactions of the complexes with blood components and accompanying pulmonary aggregates and clots.^8,19 The recharged complexes may have reduced acute toxicity by avoiding these immediate blood interactions and pulmonary complications (Figure 9). Animals that survive this initial shock eventually succumb from continued lung reaction and mounting liver toxicity.^9,41 These later toxic effects may be mediated through the same blood effects but also via activation of macrophages and other non-parenchymal cells in these organs (in particular Kupffer cells) after uptake of large amounts of cationic DNA complexes.⁹ The importance of CpG DNA sequences lends credence to the role of DNA uptake in the systemic toxicity of cationic DNA complexes.⁴² The greater avidity of macrophages for particulates may explain why cationic DNA complexes are significantly more toxic than DNA or cation component administered separately.⁹ More detailed studies on a role of highly charged pAs are warranted since systemic toxicity is not limited to liver enzyme levels in the blood only, but also include such issues as leukopenia and thrombocytopenia.⁹

Injection of anionic liposomes can disrupt DNA/CL complexes in the blood.³¹ Likewise, the 'chaser' pA injection may decrease the Kupffer cell uptake by disrupting the DNA/cation complexes after a saturating amount for gene expression has been taken up by lung cells. While larger amounts of pC/pA complexes may be generated in the blood as a result of the 'chaser' injection, such complexes would have lower toxicity compared to DNA/pC complexes because they do not contain DNA.

In conclusion, the judicious use of pAA within cationic DNA complexes such as ternary DNA/lPEI/pAA complexes and as a 'chaser' injection enables substantial levels of pulmonary foreign gene expression with much reduced toxicity. Future efforts could further enhance gene transfer by expediently appending targeting hydrophilic (eg, PEG) or endosomolytic groups to a synthetic pA.

Materials and methods

Materials

Luciferase-expressing plasmid pMIR48, which expresses the firefly gene (Luc+, Promega, Madison, WI, USA) under control of the human cytomegalovirus promoter and the SV40 virus polyadenylation signal, was used throughout the study unless indicated otherwise. Plasmids pCILacZ, pMIR85, and pEYFP-nuc also utilize the CMV promoter to express -galactosidase, secreted alkaline phosphatase (SEAP), and nuclear targeting, yellow-green fluorescence protein (Clontech, Palo Alto, CA, USA), respectively. Plasmid DNA (pDNA) was produced by a commercial supplier (Aldevron, Fargo, ND, USA). The following polymers were used in the study: branched poly(ethyleneimine) (brPEI, Aldrich, Mw 25 kDa), linear poly(ethylenelimine) (lPEI, Polysciences, Warrington, PA, Mw 25 kDa), succinylated poly-L-lysine (SPLL, Mw 31 kDa), poly(acrylic acid) (pAA, Aldrich, Mw 30 kDa), poly-L-(glutamic acid), (pGlu, Sigma, Mw 49 kDa), and poly-L-(aspartic acid) (pAsp, Sigma, Mw 36 kDa). SPLL was prepared by extensive succinylation of PLL with succinic anhydride.²⁶ Dioleyl-3-trimethylammoniumpropane (DOTAP) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Cholesterol was purchased from Sigma.

Polyplex preparation

The DNA/pC complexes were formed at final DNA concentrations of 0.02 and 0.2 mg/ml for in vitro and in vivo studies, respectively. All complexes were formulated in buffered isotonic glucose solution (BIGS; 5% glucose, 10 mM HEPES, pH 7.5). The complexes were formed in 1.5-ml Eppendorf tubes by 'flash' mixing of 5–50 l aliquots of DNA, PEI and pA in their corresponding stock solutions. The aliquot of pA was added carefully to the top of the DNA/PEI solution avoiding any mixing and was vortexed for approximately 10 s at maximum speed. All polyion stock solutions were prepared in BIGS except for pDNA which was dissolved in 5 mM HEPES, 0.1 mM EDTA, pH 7.5. The tubes were vortexed for 30 s upon addition of each component.

Lipoplex preparation

Cationic liposomes (CL) were prepared as follows. A DOTAP/cholesterol mixture in chloroform (2:1 mol/mol) was dried in vacuum in glass tubes. Lipids were hydrated in 20 mM HEPES, 10% glucose, pH 7.5 (2 BIGS) solution and briefly sonicated with a probe-type sonicator to yield liposomes with an average diameter of 100–200 nm (Zeta Plus Quasi Elastic Light Scattering sizer, Brookhaven Instruments). The CL suspension was mixed with an equal volume of DNA solution in water to yield a final DNA concentration of 0.2 mg/ml. pAA acid was added to the DNA/CL complex in 50 l of BIGS.

Isolation of serum proteins associated with DNA complexes

pDNA was fluorescently labeled with Cy5 LabelIT labeling reagent (Mirus Corporation, Madison, WI, USA) according to the manufacturer's specifications. DNA complexes composed of DNA/brPEI (100/200 g) and DNA/brPEI/pAA (100/200/100 g) were formulated in 0.5 ml of 5 mM HEPES, pH 7.5, as described above. These solutions were mixed with 0.5 ml of 100% bovine serum and incubated for 0.5 h at room temperature. Resulting mixtures were applied on top of an 18% sucrose (10 ml)/40% metrizamide (1 ml) discontinuous density gradient and ultracentrifuged for 30 min at 30 000 rpm using Beckman SW-41 swinging bucket rotor in order to separate bound and unbound serum components from DNA complexes. Precipitated DNA complexes were isolated from metrizamide/sucrose interface in 0.5 ml volume. These complexes were pelleted in a microcentrifuge (14 000 g, 5 min), re-dissolved in 1.5 M NaCl solution (0.5 ml) and passed through a Sepharose 4B-CL column (125 cm²) in 1.5 M NaCl to separate DNA, brPEI and serum proteins. Protein-containing fractions were pooled, exhaustively dialysed against deionized water, freeze-dried and re-dissolved in a sample buffer for SDS-PAGE. Protein SDS 7% gels were run under non-reducing conditions and were stained with Coomassie Blue.

In vitro gene transfer

Human hepatocellular carcinoma HuH7 cells were kindly provided by Dr Richard Stockert, Albert Einstein College of Medicine, NY. Human lung carcinoma A549 cells and mouse hepatoma Hepa-1clc7 cells were obtained from ATCC. The cells were maintained in DMEM medium (Mediatech, Herndon, VA, USA) supplemented with 10% fetal calf serum (FBS, Hyclone). The cultures were grown in a humidified atmosphere of 5% CO₂ in air at 37°C. The cells were seeded in 6-well plates at 40–60% confluence 24 h before transfection. Before complex application, the cells were washed once with 2 ml of Opti-MEM medium (Life Technologies, Inc.). The DNA complexes (2g/well) formulated in BIGS were added to the cells either in 2ml of Opti-MEM medium or 100% bovine serum (Hyclone, Logan, UT, USA) and incubated for 4 h at 37°C. DNA-containing media were then replaced with fresh DMEM supplemented with 10% FBS. Cells were grown for an additional 48 h before they were processed for analysis of reporter gene expression.

In vivo gene transfer

In vivo gene transfer efficiencies of the complexes were evaluated using tail vein injections in mice. Male ICR mice (18–22 g, 4–6 week old) were obtained from Harlan Sprague–Dawley (Madison, WI, USA). The DNA complexes were injected into their tail veins within approximately 3s. In some cases, 1.5 mg of pAA ('chaser') was injected into 100 l of BIGS 0.5 h post DNA administration. Animals were killed 24 h after injection. All animal studies were performed in compliance with a protocol approved by the Institutional Animal Care and Use Committee.

Reporter gene assays and histopathology

Luciferase assays in cultured cells and tissues were performed as previously described with luciferase picogram equaling 5.110^-5 RLU + 3.683 (r² = 0.992).⁴³ Protein assays for cell and tissue extracts were performed using the D_c Bio-Rad Protein Assay. The percent of cells transfected was determined by observing YFP fluorescence using an epi-fluorescence Axiovert S-100 microscope (Carl Zeiss, Goettingen, Germany). SEAP enzyme was assayed using Phospha-Light™ chemiluminiscent detection system (Applied Biosystems, Bedford, MA, USA). Serum was diluted 1:400 with substrate buffer and 100 l was added to the reaction mixture.

For the -galactosidase expression and pathological studies, all mice were killed by overdose of isoflurane. Immediately afterwards, the trachea was exposed and a 25-gauge blunt needle was inserted. One milliliter of Tissue Tek OCT compound (Miles, Elkhart, IN, USA) was slowly instilled, and the lungs were removed from the thoracic cavity.⁴⁴ Lung specimens were either embedded in OCT compound and frozen for -galactosidase analysis or fixed for 2–3 days in 10% neutral buffered formalin (VWR, Cleveland, OH, USA), routinely processed and embedded into paraffin (Sherwood Medical, St. Louis, MO, USA). Histochemical staining for -galactosidase activity was performed with an X-gal-based, -Galactosidase Staining Kit (Mirus Corporation, Madison, WI, USA). In brief, 5–7m frozen sections were prepared using Microm HM 505 N cryostat (Carl Zeiss), mounted on positively charged precleaned slides (Fisher Scientific) and air-dried overnight at RT. Before staining, the slides were fixed in 2% formaldehyde for 10 min. Then sections were washed three times with PBS, and incubated in a solution containing 1 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside), 25 mM K₃Fe(CN)₆, 25 mM K₄Fe(CN)₆(3H₂O), and 1.5 mM MgCl₂ in PBS at 37°C for 4 h. Lung sections selected for high magnification examination were counterstained with hematoxylin.

Morphometric analysis of -galactosidase-expressing cells was performed with the aid of AxioCam imaging software (Carl Zeiss). Briefly, lung images were taken under 20magnification, opened in Adobe Photoshop 5.5 (Adobe Systems Inc., San Jose, CA, USA) and enlarged to 200% and a grid with rulers was overlaid. The total number of alveolar cells and -galactosidase-expressing cells was counted in 20 random fields. Big vessels, bronchi, and intraluminal nucleated blood cells were excluded from counting.

For histopathological analysis, one slice of hepatic tissue of 5 mm thickness was excised from the middle of the left lateral lobe of each liver, fixed with neutral buffered formalin, routinely processed and embedded into paraffin. Paraffin sections (4 m thick) were mounted onto precleaned slides, and stained with hematoxylin and eosin and Mason's trichrome stain (Surgipath, Richmond, IL, USA). All sections were examined under Axioplan-2 microscope and pictures were taken with an AxioCam digital camera (both from Carl Zeiss).

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Acknowledgements

We would like to thank Mark Noble, Julia Hegge, and Stephanie Bertin for technical support and Hans Herweijer for helpful discussions. This study was funded in part by NIST ATP grant # 70NANB8H4064.