Overcoming the Inhibitory Effect of Serum on Lipofection by Increasing the Charge Ratio of Cationic Liposome to DNA

Gene Therapy (1997) 4, 950–960  1997 Stockton Press All rights reserved 0969-7128/97 $12.00 Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA

J-P Yang and L Huang Laboratory of Drug Targeting, Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

Since cationic liposome was ﬁrst developed as a lipofection ratios, ie 0–10.6. Removal of negatively charged proteins reagent, a drawback has been noted in that the efﬁciency from serum by DEAE-Sephacel column abolished the of lipofection decreases dramatically after addition of inhibitory effect of serum on lipofection. The fraction con- serum to the lipofection medium. This drawback hampers tained only negatively charged serum proteins which the application of cationic liposome for systematic delivery strongly inhibited lipofection at low charge ratios but not at of genes. In the present studies, we found that the effect higher charge ratios. Furthermore, preincubation of serum of serum on DC-chol liposome-mediated lipofection is with positively charged polylysine, which neutralized nega- dependent on the charge ratio of liposome to DNA. Serum tively charged serum proteins, eliminated the inhibitory inhibited lipofection activity of the lipoplex at low charge effect of serum on lipofection. In summary, inactivation of ratios, whereas it enhanced the lipofection activity at high cationic liposome by serum is due to negatively charged charge ratios. This phenomenon was observed using serum proteins and it can be overcome by increasing DOTAP/DOPE but not lipofectamine. Measurement of charge ratio of cationic liposome–DNA lipoplexes or by cellular association of DNA showed that serum could neutralizing the serum with polylysine. reduce the binding of lipoplex to cells at all tested charge

Keywords: liposomes; cationic liposome; lipofection; serum inhibitory; charge ratio; murine melanoma

Introduction been generated to resist serum.10 In spite of these improvements, the overall progress is still inadequate. Gene delivery system is critical for the success of gene DC-chol liposome, constituted with 3␤-(N-((N′,N′- therapy. A diverse number of vehicles including viral dimethylamino)ethane)carbamoyl)-cholesterol (DC-chol) vectors and nonviral vectors have been produced for and DOPE at a mole ratio of 6:4, was developed in this 1–3 gene delivery. Viral vectors have high transfection laboratory.11 It has been used in seven human gene ther- efficiency and have been used in many clinical trials, but apy clinical trials.4–6 The charge ratio of DC-chol lipo- they are highly immunogenic and potentially mutagenic, some to DNA has been previously proved to be critical thus nonviral vectors have gained more and more atten- for high efficiency of lipofection and the optimal charge tion recently. Among nonviral vectors, cationic liposomes ratio has been demonstrated to be approximately 2.11 4–7 are most widely used. Cationic liposomes are safe, sim- Under such optimal charge ratio, DC-chol liposome is ple and easy to produce on a large scale. A major prob- inactivated by serum. The present studies were set to lem associated with cationic liposomes is low transfection investigate the role of charge ratio of DC-chol liposome efficiency, especially in vivo. One reason is inactivation of to DNA in inhibition of lipofection by serum. We have cationic liposomes by serum, ie lipofection efficiency is further separated serum by using ion exchange chroma- decreased drastically by the addition of serum to the lipo- tography and investigated the effect of each fraction of 8 fection medium. The inactivation by serum can be cir- serum on lipofection. The results have shed some light cumvented in vitro by replacing serum-containing on the mechanism of inhibition of lipofection by serum. medium with serum-free medium, but it cannot be avoi- ded in vivo. This shortcoming limits the application of cationic liposome for systematic gene delivery in vivo. Results Much effort has recently been devoted to resolving this problem. For example, serum-resistant cationic lipids Effect of serum on lipofection 9 such as GS2888 have been synthesized and new formu- Murine melanoma BL6 cells were lipofected with 1 ␮g lations of DNA and cationic liposome lipoplexes have of pCMV-Luc complexed to varied amounts of DC-chol liposome in the absence or presence of 20% fetal bovine serum (FBS). Luciferase activity changed significantly at Correspondence: L Huang different charge ratios of lipoplexes (Figure 1). In the Received 21 January 1997; accepted 9 May 1997 absence of serum, luciferase activity was maximal at Mechanism of inhibition of lipofection by serum J-P Yang and L Huang 951

Figure 1 Lipofection of BL6 cells with DC-chol liposome/DNA complexes at different charge ratios (+/−). One microgram of pCMV-Luc plasmid and varied amounts of DC-chol liposome were used to lipofect cells in 48-well plates as described in Materials and methods. Each point represents the mean ± s.d. of three wells. charge ratios between 2.0 and 2.4 and remained high at high charge ratios (2.0 or greater) was observed under a charge ratios up to 4.0. At a charge ratio of 2.0, luciferase light microscope. After 4 h incubation with BL6 cells, DC- activity was dramatically reduced in the presence of chol liposome/DNA complex aggregated and sedi- serum. However, the inhibitory effect of serum was over- mented on to the cell surface forming visible granules come at charge ratios between 3.6 and 4.0. Moreover, at (photograph not shown). Serum seems to prevent lipo- these charge ratios, luciferase activity in the presence of plex from aggregating and sedimenting on to cells. In the serum was even greater than the maximal luciferase absence of serum, a few small granules began to appear activity observed in the absence of serum. With a further at the charge ratio of 2.0 and many large granules increase of charge ratio to 10.6, luciferase activity appeared at the charge ratio of 4.0. However, in the pres- decreased to about 3 × 106 RLU per well in the absence ence of serum, a few small granules appeared only at a or presence of serum. charge ratio higher than 4.0, while no granules were The relative cytotoxicity of lipoplex at varied charge observed at the charge ratio of 2.0. ratios was assessed by the total amount of extractable Because different cells vary greatly in terms of their cellular proteins in the cell lysate per well (data not ability for lipofection, ﬁve cell lines were lipofected with shown). In the absence of serum, lipoplexes of charge DC-chol liposome/DNA complexes of charge ratios 0.4, ratios ranging from 0.4 to 10.4 showed relatively low lev- 2.0 and 4.0 in the absence or presence of 20% FBS (Table els of cytotoxicity (ie у70% of untreated control cells). 1). At a charge ratio of 0.4, lipofection activity of the lipo- Lipoplexes with high charge ratios displayed minimal plex in all tested cells was inhibited by the addition of cytotoxicity, probably because BL6 cells have a higher tol- serum. At a charge ratio of 4.0, lipofection activity of the erance of DC-chol liposome. No cytotoxicity was lipoplex increased by 150 to 5300% with the addition of observed when cells were lipofected in the presence of serum. At a charge ratio of 2.0, lipofection activity of the serum; all wells showed greater than 90% of extracted lipoplex in the presence of serum decreased in C3, C33A proteins as compared with the untreated control. and B16-F0 cells by 40 to 90%, but was enhanced in HeLa Generally, we observed three types of DC-chol and L6 cells by 260 and 450%, respectively. Serum liposome/DNA complex which had different lipofection reduced cytotoxicity of lipoplex at a charge ratio of 4.0 activity. Lipoplexes with low charge ratios (р1.6) had (about 50% of untreated control in the absence of serum, low activity in the absence or presence of serum. Lipo- but 80% in the presence of serum). Overall, in all tested plexes with charge ratios of 2.0 to 2.4 had the highest cell lines, serum enhanced lipofection for lipoplexes with activity in the absence of serum but low activity in the high charge ratios and inhibited lipofection for those with presence of serum, ie these lipoplexes were highly inacti- low charge ratios. The point of charge ratio at which the vated by serum. Lipoplexes with high charge ratios of 3.6 lipoplex turned from serum sensitive to resistant was dif- to 4.0 had the highest activities in the presence of serum ferent in different cell lines. which were even greater than the activity in the absence The effect of serum concentration on lipofection was of serum. Three typical lipoplexes with charge ratios of examined with BL6 cells (Figure 2). At charge ratios of 0.4, 2.0 and 4.0 were then chosen for further studies. 0.4 and 2.0, lipofection activity of the lipoplexes Aggregation of DC-chol liposome/DNA complex at decreased with the increase of serum concentration. Lipo- Mechanism of inhibition of lipofection by serum J-P Yang and L Huang 952 Table 1 Lipofection of different cell lines with DC-chol liposome/DNA complexes with different charge ratios

Cell line Luciferase activity (RLU × 105/well)a

Charge ratio (±) 0.4 Charge ratio (±) 2.0 Charge ratio (±) 4.0

− serum + serum − serum + serum − serum + serum

HeLa 45 ± 819±4 105 ± 8 277 ± 21 13 ± 255±8 C3 19 ± 1 2.3 ± 6 293 ± 23 260 ± 82 183 ± 6 793 ± 29 C33A 240 ± 36 113 ± 58 3457 ± 367 1460 ± 854 2690 ± 282 4150 ± 608 B16-F0 62 ± 17 27 ± 4 110 ± 22 85 ± 23 97 ± 16 427 ± 36 L6 1.3 ± 0.5 0.76 ± 0.3 1.9 ± 0.2 8.6 ± 0.3 0.2 ± 0.9 10.6 ± 1.5

aEach data point represents the mean ± s.d. of three wells.

Figure 2 Effect of serum concentration on lipofection. BL6 cells were lipofected with DC-chol liposome/DNA lipoplexes at charge ratios of 0.4 (a), 2.0 (b) and 4.0 (c) in the presence of different serum concentrations as described in Materials and methods. Each point represents the mean ± s.d. of three wells.

fection activity of the lipoplexes with charge ratios of 0.4 the role of charge ratio in serum sensitivity is dependent and 2.0 decreased to the lowest level in the presence of 2 on cationic lipids. and 5% of serum, respectively. In contrast, the lipofection activity of the lipoplex with a charge ratio of 4.0 increased Lipofection with purified DC-chol liposome–DNA with the increase of the serum concentration. In the pres- lipoplexes ence of 5% of serum the lipofection activity increased to To exclude the possibility that free DC-chol liposome the highest level. The data indicate that the effect of may interfere with serum resistance, the DC-chol serum on lipofection was dose-dependent. liposome/DNA lipoplexes at three different charge ratios It is of interest to determine whether the above finding of 0.4, 2.0 and 4.0 were purified by discontinuous sucrose that serum inhibits lipofection at low charge ratios and gradient (0/10/20% sucrose) ultracentrifugation. The enhances lipofection at high charge ratios is limited to location of the lipoplex in the gradient varied with charge only DC-chol liposome. DOTAP/DOPE and lipofectam- ratios as shown in Figure 3a. DC-chol liposome/DNA ine were examined for serum sensitivity at different complex with a charge ratio of 0.4 banded at the bottom charge ratios (Table 2). DOTAP/DOPE showed a similar of the tube. Lipoplex with a charge ratio of 2.0 banded result as that with DC-chol liposome, but it turned to on the top of 20% sucrose layer (fraction 8). Lipoplex with serum resistant at a charge ratio (2.0) which is lower than a charge ratio of 4.0 banded at the bottom of 10% sucrose that of DC-chol liposome. In contrast, lipofectamine was layer (fraction 7). Free liposome or DNA floated to 0% always inactivated by serum regardless of the charge sucrose layer. As the charge ratio increased, the DC-chol ratio tested. Another two newly synthesized cationic liposome/DNA complex floated to a higher level in the cholesterol derivatives showed a similar result as DC- gradient and the amount of free DNA decreased. chol liposome (data not shown). These data indicate that BL6 cells were lipofected with purified DC-chol Mechanism of inhibition of lipofection by serum J-P Yang and L Huang 953 Table 2 Lipofection of BL6 cells with different cationic liposomes at different charge ratios of cationic lipid to DNA

Charge ratio Luciferase activity (RLU × 106/well)a

DOTAP–DOPE DC-chol–DOPE Lipofectamine

− serum + serum − serum + serum − serum + serum

0.4 3.6 ± 1 1.6 ± 0.5 1.6 ± 1 0.7 ± 0.2 ND ND 2.0 277 ± 2 369 ± 8 241 ± 32 8.3 ± 1.5 14 ± 3 2.8 ± 0.2 4.0 211 ± 24 275 ± 17 212 ± 26 363 ± 42 ND ND 10 ND ND 3 ± 13±0 378 ± 10 2 ± 0.1 20 ND ND ND ND 27 ± 41±0.1

aEach data point represents the mean ± s.d. of three wells. ND, not done.

Figure 3 Lipofection of BL6 cells by purified DC-chol liposome/DNA complexes with different charge ratios. (a) Distribution of 125I-labeled DNA in DC-chol liposome/DNA complexes at charge ratios (±) of 0.4, 2.0 and 4.0 in the sucrose gradient. (b) BL6 cells were lipofected with purified DC-chol liposome/DNA complexes at charge ratios (±) of 0.4, 2.0 and 4.0 as described in Materials and methods. Each point represents the mean ± s.d. of three wells. liposome/DNA complexes in the absence or presence of Cellular association of DNA serum assessed for lipofection activity (Figure 3b). For To test whether inhibition of lipofection by serum is due lipoplex with a charge ratio of 4.0, lipofection in the pres- to serum blocking the binding of lipoplexes to cells, we ence of serum was 189% relative to the lipofection in the examined the cellular association of DNA in the absence absence of serum. The addition of serum to the lipoplexes or presence of serum at different charge ratios. As shown at charge ratios of 0.4 and 2.0 reduced 20 and 47% of in Figure 4, cellular association of DNA initially increased the lipofection activity, respectively. These results were with an increase of charge ratio in the absence or pres- similar to lipofection with unpurified lipoplexes and indi- ence of serum. Lipofection activity (Figure 1) correlated cated that serum resistance of lipofection at high charge with the increase of cellular association of DNA. In the ratios was due to the difference of structure of the lipo- absence of serum, cellular association of DNA saturated plexes formed at different charge ratios but not due to at a charge ratio of 2.4, but in the presence of serum it free DC-chol liposome. It was noticed that lipofection saturated at a higher charge ratio of 4.0. The presence of activity of the purified lipoplexes was lower than that of serum shifted the plateau of DNA cellular association to unpurified lipoplexes for reasons not known at this time. a higher charge ratio, which might explain why the peak Mechanism of inhibition of lipofection by serum J-P Yang and L Huang 954

Figure 4 Cellular association of DNA. BL6 cells were lipofected with 125I-labeled DNA complexed with varied amounts of DC-chol liposome in the presence or absence of 20% FBS. Standard division of each point is less than 1%.

of lipofection activity in the presence of serum shifted to was neutral and positively charged and 6% was nega- a higher charge ratio (Figure 1). At charge ratio 2.0 or tively charged. When lipofection was performed, the lower, the presence of serum strongly inhibited the cellu- same amount of neutral and positively charged proteins lar association of DNA, in agreement with the strong (795 ␮g) or negatively charged proteins (107 ␮g) relative inhibition activity of serum for lipofection at these charge to a given concentration of serum (total 1075 ␮g) was ratios. At all charge ratios tested, serum inhibited the added. binding of DNA to cells, ie the plateau level in the pres- The DC-chol liposome/DNA complexes of charge ence of serum (about 14%) is lower than that in the ratios of 2.0 and 4.0 were chosen to lipofect BL6 cells in absence of serum (about 50%). Additionally, it was the presence of original serum, neutral and positively noticed that lipoplexes with charge ratios of 3.2 to 4.8 had charged serum proteins (fraction I), negatively charged lower cellular association of DNA in the presence of serum proteins (fraction II) or the combination of neutral serum than lipoplexes with charge ratios of 2.0 to 4.8 in and positively charged serum proteins with negatively the absence of serum, but the former showed higher lipo- charged serum proteins (fractions I and II). Results are fection activity than the latter (Figure 1). Thus, the total shown in Figure 6. Fraction I, neutral and positively amount of DNA association with cells did not correlate charged serum proteins, did not show any inhibitory with the lipofection activity. Some other factors such as effect in lipofection. Compared with lipofection in the entry of DNA into cells and intracellular distribution of absence of serum, lipofection was enhanced in the pres- DNA subsequent to DNA associated with cells may be ence of the neutral and positively charged serum proteins important. by 1.5- to four-fold in several experiments repeated in both charge ratios of 2.0 and 4.0. On the other hand, the Fractionation of serum by DEAE-Sephacel negatively charged serum proteins (fraction II) and the chromatography combination of fractions I and II inhibited lipofection to We tried to determine which components of serum block the same extent as the original serum at a charge ratio of the binding of lipoplex to cells and enhance lipofection at 2.0 but not at a charge ratio of 4.0. These data demon- higher charge ratios. Serum was fractionated by a DEAE- strated that serum inhibits lipofection due to negatively Sephacel anion exchange column as shown in Figure 5. charged serum proteins and the inhibitory effect of serum Neutral and positively charged proteins ﬂowed through can be overcome by increasing charge ratio. No differ- the column by eluting with a buffer containing 50 mm ences in luciferase activity were noted when cells were Tris-HCl, 90 mm NaCl, pH 8.0, and were combined as lipofected in the presence of original serum, serum fraction I. Negatively charged proteins were eluted from adjusted to pH 8.0 or serum dialyzed against PBS (data the column by increasing the salt concentration to 400 not shown). These data excluded the possibility that pH mm and were combined as fraction II. With a further manipulation used in serum fractionation (see Materials increase of salt concentration in the elution buffer, up to and methods) or small molecules in the serum affected 2 m, no detectable protein was eluted. The recovery of lipofection. proteins in fractions I and II was 74 and 6%, respectively. Proteins in each peak were further analyzed by SDS- Accordingly, we assumed that 74% of total serum protein PAGE (Figure 7). Compared is the protein proﬁle of each Mechanism of inhibition of lipofection by serum J-P Yang and L Huang 955

Figure 5 Fractionation of serum proteins. Serum proteins were separated by anion exchange column chromatography as described in Materials and methods. Fraction I: combined proteins that ﬂowed through the column were neutral and positively charged serum proteins. Fraction II: combined proteins eluted with the buffer containing high concentration of salt were negatively charged serum proteins.

Figure 6 Effect of fractionated serum on lipofection. BL6 cells were lipofected with DC-chol liposome/DNA complexes at charge ratios (±) of 2.0 and 4.0 in the presence of different fractions of serum or in the absence of serum. Luciferase activity in the absence of serum was standardized as 100%. No serum, lipofection in the absence of serum; Original serum, lipofection in the presence of 10% untreated serum (1075 ␮g of total proteins); Fraction I, lipofection in the presence of 795 ␮g neutral and positively charged serum proteins of fraction I; Fraction II, lipofection in the presence of 107 ␮g negatively charged serum proteins of fraction II. Fractions I and II, lipofection in the presence of 795 ␮g proteins of fraction I and 107 ␮g proteins of fraction II. Each point represents the mean ± s.d. of three wells. Mechanism of inhibition of lipofection by serum J-P Yang and L Huang 956 is sensitive to serum proteins. Lipofection activity in the presence of polylysine-treated serum was recovered with increasing of amounts of polylysine (Figure 8). When serum was pretreated with 0.1 ␮g polylysine, lipofection activity remained as low as that in the presence of untreated serum. However, when polylysine was increased to 0.3 and 0.5 ␮g, luciferase activity completely recovered to what was observed in the absence of serum (standardized as 100% luciferase activity). The data indicate that serum treated with 0.3–0.5 ␮g polylysine has no inhibitory effect on lipofection. The decrease in lipofection with a further increase of polylysine may be due to the competition between an excess of polylysine and the lipoplex. To test whether the recovery of lipofection activity by polylysine pretreatment of serum correlates to the recovery of the cellular association of DNA, cellular association of DNA was measured (Figure 8). Cellular association of DNA increased from 6 to 17% as polylysine was increased from 0 to 0.3 ␮g. With further increases of polylysine, cellular association of DNA slightly decreased, but remained at approximately 14%. In accordance with the result in Figure 4, the similar incomplete recovery of DNA association with cells was observed by the pretreatment of serum with polylysine and by using an excess of liposomes (high charge ratio). These data imply that neutralization of negatively charged proteins in serum by polylysine could reduce the blocking effect of serum on the binding of lipoplex to cells, thereby leading to higher lipofection efﬁciency.

Discussion The observation that the inhibitory effect of serum on lipofection can be overcome by increasing the charge ratio of cationic liposome to DNA is not limited to DC- chol liposome. A similar effect was shown by DOTAP/DOPE (Table 2). DOTAP and DC-chol are both monovalent cationic lipids. On the other hand, lipofectamine, which contains a multivalent cationic lipid DOSPA, did not show any charge ratio-dependent inactivation by serum (Table 2). These differences may be related to the different structures of lipoplexes containing cationic lip- Figure 7 SDS-PAGE analysis of proteins in each fraction of serum. Pro- ids with different valences.12 Liposomes containing teins were analyzed by a 4–25% gradient gel as described in Materials monovalent cationic lipids form a tubular or ‘spaghetti’ and methods. The bands shown by arrows are missing in fraction I but structure with DNA.12 These tubular structures are super- present in fraction II. The gel shown is representative of three different coiled DNA coated with a single lipid bilayer, thus the experiments. DNA is not highly condensed. Lipoplexes containing a multivalent cationic lipid such as DOSPA are highly con- fraction with the original serum, three bands shown by densed and small in size (Sorgi et al, unpublished results). arrows missed in fraction I but present in fraction II, the DNA/lipofectamine complex prepared in the presence of molecular weight of which was around 270, 137 and 95 detergent are, however, less sensitive to serum than the kDa, respectively. These proteins, singularly or in combi- lipoplex formed in the absence of detergent.10 The fact nation, may play the inhibitory role. that DC-chol liposome requires a higher charge ratio than the DOTAP/DOPE liposome to overcome the serum Neutralization of serum with polycations inhibition effect (Table 2) is probably related to the differ- Since negatively charged serum proteins inhibited lipo- ence in the structure of the amino group. DC-chol con- fection, we tested whether neutralization of the nega- tains a single tertiary amino group with an apparent pKa tively charged serum proteins by polycations such as around 7 (unpublished data), thus the real charge ratios polylysine could abolish the inhibitory effect of serum on of the lipoplex may be substantially lower than the theo- lipofection. Intact fetal bovine serum was preincubated retical ones used throughout this article. DOTAP contains with varied amounts of polylysine at room temperature a quaternary amino group which is always ionized for 10 min. In the presence of this treated serum, BL6 cells regardless of the pH of the medium. were lipofected using a DC-chol liposome/DNA lipoplex Serum reduces the binding of lipoplexes to cells at all with a charge ratio of 2.0, ie a ratio at which the lipoplex charge ratios tested (Figure 5). This may be due to the Mechanism of inhibition of lipofection by serum J-P Yang and L Huang 957

Figure 8 Effect of polylysine on lipofection. BL6 cells were lipofected with DC-chol liposome/DNA complex at a charge ratio (+/−) of 2.0 in the presence of 20% polylysine pretreated serum. Luciferase activity in the presence of polylysine pretreated serum was compared with that in the absence of serum that was standardized as 100%. Cellular association of DNA was determined after BL6 cells were lipofected with 125I-labeled DC-chol liposome/DNA complex at a charge ratio (+/−) of 2.0 in the presence of 20% polylysine pretreated serum. Each point of luciferase activity represents the mean ± s.d. of three wells and the standard derivation of cellular association of DNA was less than 1% of the mean (n = 3). nonspecific binding of serum proteins to cationic lipo- tion.17 Stimulating any step of this pathway could somes. In fact, the aggregation and sedimentation of lipo- enhance lipofection. Serum contains at least two factors plexes on to cells was observed under the microscope to that act: (1) to prevent lipoplexes from binding to the cell be reduced by the presence of serum. The binding surface; and (2) to stimulate cellular endocytosis. The for- between anionic liposomes and serum proteins has been mer was demonstrated in Figure 4. The latter was pre- reported previously.13–15 It was thought that inactivation viously demonstrated in that transferrin, insulin, and of cationic liposomes by serum is due to the neutraliz- growth factors such as EGF and M-CSF, which are ation of lipoplexes by binding with negatively charged components of serum, can stimulate endocytosis.21–23 serum proteins.16 However, no direct evidence has been Transferrin complexed with DNA and lipofectin was provided to verify this assumption. Our data showed that reported to stimulate lipofection activity dramatically.24 serum depleted of negatively charged proteins by DEAE- For lipofection with lipoplexes of low charge ratios, Sephacel no longer inhibited lipofection. Moreover, a serum blocks the binding of lipoplexes to the cell surface fraction containing negatively charged serum proteins (Figure 4). Lipoplexes with low charge ratios might be strongly inhibited lipofection. SDS-PAGE revealed that completely neutralized by serum, resulting in less DNA these proteins were of high molecular weight. Identifi- binding to the cell surface and lower lipofection cation of these proteins will require further effort. Neu- efficiency. For lipofection with lipoplexes of higher tralization of negatively charged serum proteins by poly- charge ratios, the excess of positive charge of lipoplexes lysine also resulted in the elimination of the inhibitory can at least partially overcome the neutralization effect effect of serum. Together, these data indicate that nega- of serum and mediate DNA binding to the cell surface tively charged serum proteins play a key role in inhi- (about 14% as shown in Figure 4). At this point the major bition of lipofection by serum. function of serum might be to stimulate DNA entry into The mechanism by which serum inhibits or enhances the cell via endocytosis or some unknown mechanism to lipofection is not clear. The pathway by which cationic enhance lipofection. In addition, lipofection-associated liposome mediates lipofection was believed to occur as cytotoxicity was reduced when cells were lipofected in follows: cationic liposomes form a lipoplex with DNA the presence of serum. This may relate to the fact that and mediate the lipoplex binding to the cell surface via the presence of serum reduced aggregation and sedimen- ionic interactions. DNA concentrates on the cell surface tation of the lipoplexes on to cells. Furthermore, lipo- and gains entry into the cell mainly via endocytosis and plexes with higher charge ratios protected DNA from perhaps secondarily by membrane fusion.17–20 In the cyto- nuclease digestion more effectively than lipoplexes with plasm most DNA is degraded within the lysosomes. lower charge ratios (data not shown). Therefore, lipofec- Some DNA, however, is released from the endosomes tion activity of lipoplexes with high charge ratios was with a minor portion entering the nucleus for transcrip- elevated by the addition of serum to a greater extent than Mechanism of inhibition of lipofection by serum J-P Yang and L Huang 958 the maximal lipofection activity observed in the absence was purchased from GIBCO-BRL (Gaithersburg, MD, of serum. USA). It is necessary to compare our data about preincu- Plasmid pCMV-Luc was constructed by inserting a bation of serum with polylysine with the data from DC- fragment of luciferase cDNA from pRSVL31 into HindIII– chol liposome/polylysine/DNA lipopolyplexes (LPDI) XbaI sites of pcDNA3 vector by Richard Rouse in this previously reported by Gao and Huang.25 LPDI has laboratory. Plasmid was purified by cesium chloride den- higher lipofection activity than DC-chol liposome/DNA sity gradient ultracentrifugation. complex but remained inactivated by serum. One reason is that the charge ratio of LPDI is only 2.63 which is not Calculation of charge ratio of lipoplex high enough to resist serum. Moreover, the order of The charge ratio was theoretically calculated as mole addition of components to form the ternary lipoplex ratio of DC-chol (one charge per molecule), DOTAP (one affects lipofection activity.25 The addition of polylysine to charge per molecule) or DOSPA (cationic lipid of lipofec- DC-chol liposome followed by mixing with DNA, or the tamine, five charges per molecule) to nucleotide residue addition of polylysine to DNA followed by mixing with (average MW 330). liposome can increase lipofection activity, but the addition of polylysine to the DC-chol liposome/DNA Cell culture and in vitro lipofection complex cannot. After polylysine binds with DNA, it Murine melanoma B16-F0 (ATCC, Rockville, MD, USA), probably can no longer neutralize the negatively charged human cervical carcinoma HeLa (ATCC) and C33A, rat serum proteins which are responsible for the inhibitory skeletal muscle myoblasts L6 (ATCC) were cultured in effect of serum. DMEM medium supplemented with 10% FBS (GIBCO- The ratio of liposome to DNA determines the surface BRL). Murine melanoma BL632 and murine fibroblast charge and particle size of lipoplexes, which directly C333 that was transformed with HPV16 genome and ras affect lipofection efficiency.26 Different observations gene were cultured in RPMI medium containing 10% FBS about the effect of charge ratio on lipofection were noted and antibiotics. by different investigators. Our data demonstrated that a Lipoplexes were prepared as follows: 1 ␮g DNA dis- high charge ratio of DNA/cationic liposome complex is solved in 125 ␮l of serum-free RPMI medium was mixed critical for efficient lipofection in vitro in the presence of with varied amounts of cationic liposome in equal vol- serum. In contrast, lipofection with the DNA/lipofectin umes of HBSS (GIBCO-BRL) and placed at room tem- or transfectACE complex at a lower charge ratio was perature for 5 min. Then the lipoplexes were added to reported to result in efficient lipofection of cultured CV- cells in 48-well plates with about 80% confluence. If the 1 cells in the presence of serum.27 The difference may be lipofection was performed in the presence of serum, FBS due to different cationic liposome or cell lines used in the (Hyclone laboratories, Logan, UT, USA) was added to the experiment. In animal experiments, direct injection of lipoplex solution to a final concentration as indicated in DC-chol liposome/DNA or DOTAP complex with low the text. For lipofection in the presence of polylysine- charge ratio into the tumor resulted in higher lipofection treated serum, FBS was preincubated with polylysine than injections with lipoplexes of higher charge ratios.28,29 (MW 25.7 kDa, Sigma, St Louis, MO, USA) at room tem- Direct injection into the brain of DNA/lipospermine perature for 10 min. For the lipofection with purified lipo- complex of low charge ratio also results in efficient lipoplexes, the lipoplexes containing 1 ␮g DNA were diluted fection.30 However, the tendency that a higher charge with HBSS/serum-free RPMI medium (1:1, v/v) to 250 ratio of DC-chol liposome/DNA complex results in ␮l and then added to cells. After incubation at 37°C for higher expression following tail vein injection is apparent 5 h, cells were returned to culture in the medium contain- (data not shown). In addition, among the many factors ing 10% FBS for an additional 38–42 h. Cells were then which affect the efficiency of organ lipofection by intra- lysed with 100 ␮l of lysis buffer (200 mm Tris-HCl, pH venous injection of DNA/cationic lipid complex, high 7.8, 2 mm EDTA, 0.05% Triton X-100) at room tempera- charge ratio of the lipoplex seems to be the most ture for 10 min. Ten microliters of supernatant were important one.36 These preliminary results are in agree- assayed for luciferase activity using a luciferase assay kit ment with those presented here. It seems that the require- (Promega Corporation, Madison, WI, USA) and an Auto- ments of formulations for systematic gene delivery are lumat LB 953 luminometer (EG&G berthold, Bad Wild- different from those for local delivery. Interstitial fluids bad, Germany). Luciferase activity was expressed as rela- in the tumor and brain may not contain the negatively tive light units (RLU). Protein concentration was charged proteins present in the serum. The present find- measured with Coomassie Plus Protein Assay Reagent ings should be useful in designing new formulations for (Promega Corporation). systematic gene delivery. Purification of DNA–liposome lipoplexes Sucrose gradient was generated by layering 2 ml of 10% Materials and methods sucrose on top of 2 ml of 20% sucrose in a thin ultracen- trifuge tube (Nalge Company, Rochester, NY, USA). Preparation of DC-chol liposome and plasmid DNA Twenty micrograms of DNA containing a trace amount DC-chol liposome was prepared with DC-chol:DOPE at of 125I-DNA diluted in 250 ␮l HBSS were mixed with a mole ratio of 6:4 in endotoxin-free distilled water varied amounts of DC-chol liposome in equal volume. (purchased from Abbott Laboratories, North Chicago, IL, One-half milliliter of the mixture was applied on the top USA) by sonication as previously described.11 The lipo- of a sucrose gradient. The gradient was centrifuged at somes were used within 1 month after preparation. 150 000 g for 1 h at 20°C in a Beckman SW50.1 rotor DOTAP:DOPE at 1:1 mole ratio in endotoxin-free dis- (Fullerton, CA, USA). Fractions of 450 ␮l each were care- tilled water was prepared by sonication. Lipofectamine fully collected from the top of the gradient and then Mechanism of inhibition of lipofection by serum J-P Yang and L Huang 959 measured for radioactive DNA content using a gamma- 9 Lewis JG, Lin K-Y, Kothavale A. A serum-resistant cytofectin counter. DC-chol liposomes labeled with 111In-DTPA-SA for cellular delivery of antisense oligodeoxynucleotides and floated to the top of the gradient. plasmid DNA. Proc Natl Acad Sci USA 1996; 93: 3176–3186. 10 Hofland HE, Shephard L, Sullivan SM. Formation of stable cationic lipid/DNA lipoplexes for gene transfer. Proc Natl Acad Sci Cellular association of DNA USA 1996; 93: 7305–7309. pCMV-Luc was labeled with 125I (Dupont, Wilmington, 11 Gao X, Huang L. A novel cationic liposome reagent for efficient DE, USA)34 and separated from free iodine by a spin col- lipofection of mammalian cells. Biochem Biophys Res Commun umn (Bio-Spin 30; Bio-Rad, Hercules, CA, USA). Com- 1991; 179: 280–285. pared with unlabeled DNA, the 125I-labeled DNA con- 12 Sternberg B, Sorgi FL, Huang L. New structures in lipoplex forma- tained roughly the same amount of supercoiled, open tion between DNA and cationic liposomes visualized by freeze– circled and linear DNA as examined by electrophoresis fracture electron microscopy. FEBS Lett 1994; 356: 361–366. 13 Chonn A, Semple SC, Cullis PR. Separation of large unilamellar and autoradiography. Lipoplexes prepared with trace 125 liposomes from blood components by a spin column procedure: amount of I-labeled DNA (30 000 total c.p.m.) were towards identifying plasma proteins which mediate liposome added to cells as described above. After 5 h incubation 1070 ° clearance in vivo. Biochim Biophys Acta 1991; : 215–222. at 37 C, cells were washed three times with PBS and 14 Semple SC, Chonn A, Cullis PR. Influence of cholesterol on the lysed with 500 ␮l lysis buffer at room temperature for 10 association of plasma proteins with liposomes. Biochemistry min. Cell lysate was collected and counted for radioactive 1996; 35: 2521–2525. DNA with a gamma-counter. 15 Oku N, Tokudome Y, Namba Y. Effect of serum protein binding on real-time trafficking of liposomes with different charges analyzed by positron emission tomography. Biochim Biophys Acta Fractionation of serum 1996; 1280: 149–154. Serum was fractionated with DEAE-Sephacel (Sigma) col- 16 Gao X, Huang L. Cationic liposome-mediated gene transfer. umn chromatography. DEAE-Sephacel (8 ml) was pre- Gene Therapy 1996; 2: 710–722. washed with 50 mm Tris-HCl, 2 m NaCl, pH 8.0 and equi- 17 Zabner J et al. Cellular and molecular barriers to gene transfer librated with 50 mm Tris-HCl, 90 mm NaCl, pH 8.0. Six by a cationic lipid. J Biol Chem 1995; 270: 18997–19007. milliliters of FBS, the pH of which was adjusted to 8.0, 18 Zhou X, Huang L. DNA lipofection mediated by cationic lipo- were applied to the column and then eluted with equili- somes containing lipopolylysine: characterization and mech- bration buffer. Fractions of 2.4 ml each were collected till anism of action. Biochim Biophys Acta 1994; 1189: 195–203. the A was less than 0.05. The column was then 19 Friend DS, Papahadiopoulos D, Debs RJ. Endocytosis and intra- 280 nm cellular processing accompanying lipofection mediated by cat- eluted with 50 mm Tris-HCl buffer, pH 8.0 containing 400 m ionic liposomes. Biochim Biophys Acta 1996; 1278: 41–50. m NaCl. The eluted proteins were concentrated and 20 Legendre JY, Szoka FJ. Delivery of plasmid DNA into mam- desalted by using a Centricon 10 (Amicon, Beverly, malian cell lines using pH-sensitive liposomes: comparison with MA, USA). cationic liposomes. Pharm Res 1992; 9: 1235–1242. 21 Hewlett LJ, Prescott AR, Watts C. The coated pit and macropin- SDS-polyacrylamide gel electrophoretic analysis of ocytic pathways serve distinct endosome populations. J Cell Biol serum proteins 1994; 124: 689–703. 22 Dunglison GF, Kaye PL. Endocytosis in mouse blastocysts: Proteins in each fraction were analyzed by a 4–20% characterization and quantification of the fluid phase compo- gradient SDS-polyacrylamide gel electrophoresis as nent. Mol Reprod Dev 1995; 41: 225–231. 35 described. SDS-PAGE molecular weight standard 23 Racoosin EL, Swanson JA. M-CSF-induced macropinocytosis (Sigma) was used to estimate the molecular weight of increases solute endocytosis but not receptor-mediated endo- proteins. Proteins were visualized by Coomassie blue cytosis in mouse macrophages. J Cell Sci 1992; 102: 867–880. staining as described.35 24 Cheng P-W. Receptor ligand-facilitated gene transfer: enhance- ment of liposome-mediated gene transfer and expression by transferrin. Hum Gene Ther 1996; 7: 275–282. 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