Biotechnol. Appl. Biochem. (2003) 37, 267–271 (Printed in Great Britain) 267

Effect of Pluronic-block copolymers on the reduction ofserum-mediated inhibition of gene transfer ofpolyethyleneimine–DNA complexes

Jung-Hua Steven Kuo1

Department of Pharmacy, Chia Nan University of Pharmacy and Science, 60 Erh-Jen Rd., Sec. 1, Jen-Te, Tainan 717, Taiwan,Republic of China

Serum stability of non-viral vectors is a crucial factorfor successful in vivo gene delivery. Pluronic-blockcopolymers consisting of hydrophilic ethylene oxideand hydrophobic propylene oxide blocks were testedto prevent the reduction of serum-mediated inhibitionof gene transfer of polyethyleneimine (PEI)–DNAcomplexes in NIH/3T3 cells. The order of hydrophilic–lipophilic balance (HLB) of six different types ofPluronics used in this study was F68 > F127 > P105 >

P94 > L122 > L61. Transfection activities of NIH/3T3cells with PEI–DNA complexes containing Pluronicswith higher HLB showed marked improvement ofgene-expression levels in serum media from 10 to50 % fetal bovine serum compared with PEI–DNAcomplexes alone. Also, higher concentrations (1 and3 %) of Pluronics with higher HLB in the PEI/DNAdispersion provided a stronger steric hindrance inresisting serum components than those obtained in alower concentration (0.1 %). These results suggestedthat non-viral vectors incorporated with higher HLB ofPluronics may be used as potential vehicles for in vivodelivery of DNA.

Introduction

Delivery of nucleic acids into cells using cationic polymershas recently gained remarkable interest in the field of non-viral gene therapy due to their structural diversity, easyproduction, non-immunogenicity and safety [1]. By addingcationic polymers to DNA, the condensed complexes arespontaneously formed by electrostatic interactions, resultingin efficient transport of intact DNA into the nucleus [2].The efficiency of gene transfer depends on the structure–property relationships of cationic polymers as well as theexact mechanism used in the formation of complexes [3].Polyethyleneimine (PEI) polymers are the most commonlyused vectors in non-viral gene-delivery systems [4]. They arebelieved to provide significant buffering capacity over othercationic polymers, leading to pH inhibition of lysosomalnucleases and higher transfection efficiency [5,6].

Despite the fact that gene transfer has beenaccomplished in vitro and some in vivo, one of the majordrawbacks of non-viral vectors is that transfection efficiencyis reduced by negatively charged serum components whichpresumably neutralize the positive charges in the complexes[7]. Therefore, transfection using non-viral vectors is mostlylimited to serum-free conditions and poses a major concernfor efficient gene delivery in vivo [8]. One approach hasshown that serum inactivation associated with cationiclipids can be overcome by using non-ionic surfactants [9].In the presence of serum, these formulations containingnon-ionic surfactants demonstrated an equivalent or highertransfection efficiency in different cell lines compared withthe cationic lipids alone.

One of the effective non-ionic surfactants used,Pluronic-block copolymers, has drawn special attention inconjunction with cationic polymer formulations. Pluronic-block copolymers, also named Poloxamer or Synperonic,consist of hydrophilic ethylene oxide (PEO) and hydrophobicpropylene oxide (PPO) blocks arranged in a triblockstructure [10]. In the pharmaceutical industry, amphiphilicPluronics have been widely used as solubilizers, emulsifiers,stabilizers and adjuvants [11,12]. Also, Pluronics have beenreported to enhance the efficiency of transfection of somecationic polymers and viral vectors in vitro and in vivo[13,14]. Furthermore, previous study has demonstrated thatpolyether–PEI graft copolymers formed stable complexesin the presence of serum and exhibited a significantimprovement in gene expression in vivo [15]. Whereas freePluronic copolymer (P123) is an essential component for thiseffect, in the presence of serum, there are no data on theimportant effects of Pluronics on the in vitro transfectionactivity of these graft copolymers. Therefore, furtherstudy of the structural characteristics of Pluronics on thereduction of serum-mediated inhibition of gene transfer for

Key words: Pluronics, polyethyleneimine, serum-mediated inhibition,transfection.

Abbreviation used: DMEM, Dulbecco’s modified Eagle’s medium; HLB,hydrophilic–lipophilic balance; PEI, polyethyleneimine; FBS, fetal bovineserum; PEO, ethylene oxide; PPO, propylene oxide.

1 E-mail kuojunghua@yahoo.com.tw.

C© 2003 Portland Press Ltd

268 J.-H. S. Kuo

cationic polymers is needed. Adding Pluronics into PEI gene-delivery systems to challenge serum inactivation by usingNIH/3T3 as a model cell system is explored in this study.The results clearly show that pluronic copolymers witha higher hydrophilic–lipophilic balance (HLB) significantlyimprove the serum stability of PEI–DNA complexes.

Materials and methods

Plasmid DNAThe plasmid (pSG5lacZ), which encodes the lacZ gene forβ-galactosidase, was driven by a simian-virus-40 promoterto assess gene expression. Plasmid DNA was amplifiedin Escherichia coli and purified by column chromatography(QIAGEN-Mega kit; Qiagen, Venlo, The Netherlands). Thepurity of plasmid DNA was established by UV spectroscopy(A260/A280 ratios ranging from 1.80 to 1.89 were used).Agarose gel (0.7 %) electrophoresis analysis using restrictionenzymes showed that plasmid DNA was mainly in thesupercoiled form and one band corresponding to a size of8 kb was visible.

ChemicalsPoly(PEO)–poly(PPO)–poly(PEO) block copolymers (Plur-onic F68 and F127) were obtained from Sigma (St.Louis, MO, U.S.A.) and used without further purification.The Synperonics series (L122, P105, P94 and L61) werepurchased from Fluka Chemie GmbH (Buchs, Switzerland).PEI (800 kDa) was obtained from Sigma as a 50 % (w/v)solution. The PEI solutions were adjusted to desired aqueousconcentrations and neutralized (final pH 7.0) with HCl.

Cytotoxicity assay of PluronicsTo test the effect of Pluronics on the cell viability,NIH/3T3 murine fibroblasts were seeded in 96-well platesat 5000 cells/well, and then incubated for 1 day at 37 ◦Cand 5 % CO2. Positive control cells were grown in thepresence of fresh medium [Dulbecco’s modified Eagle’smedium (DMEM), high glucose; Gibco, Grand Island, NY,U.S.A.] supplemented with 10 % heat-inactivated fetal bovineserum (FBS; Gibco) and 100 units/ml penicillin/100 µg/mlstreptomycin (Sigma) without adding Pluronics. Variousconcentrations of Pluronics (10 µl) were added into theculture medium of the plate and incubated for 2 days.To measure the cell viability, 10 µl of the Cell CountingKit-8 solution [a tetrazolium salt which produces a highlywater-soluble formazan dye upon biochemical reductionin the presence of an electron carrier (1-methoxy-5-methylphenazinium methyl sulphate); Dojindo Laboratories,

Tokyo, Japan] was added into each well and incubated for1–4 h. The amount of the yellow-coloured formazan dyegenerated by dehydrogenases in cells is directly proportionalto the number of viable cells in a culture medium. Theattenuance at 450 nm was obtained by using a microplatereader with a reference wavelength at 595 nm. Results werereported as percentage viability (mean attenuance/meanpositive control attenuance +−S.D.) for triplicate samples.

Preparation of complexes and in vitro transfectionThe PEI–DNA complexes were prepared according totwo previously described procedures with modifications[16,17]. Briefly, 1 µg of plasmid DNA diluted in 150 mMNaCl was mixed with an appropriate amount of Pluronicsin a total volume of 50 µl. PEI (3 µg) diluted in 50 µlof 150 mM NaCl was added to previous solutions bymixing with half volumes for 10 min periods. After complexformation, total volume of the resulting solution wasadjusted to 250 µl by the addition of DMEM supplementedwith appropriate amount of FBS and 1 % antibotics.NIH/3T3 cells were seeded into 24-well cell-culture platesat a density of 3×104 cells/well and grown overnight(60–75 % confluence) at 37 ◦C and 5 % CO2. Immediatelyprior to transfection, cells were rinsed with PBS andexposed to transfection mixtures for 3 h. The transfectionmedium was then removed and replaced with DMEMsupplemented with FBS and 1 % antibotics. Then, 2 dayslater, β-galactosidase gene expression was analysed byusing combined β-Gal Assay kit (Invitrogen, Carlsbad,CA, U.S.A.) and BCA Protein Assay Reagent Kit (Pierce,Rockford, IL, U.S.A.). Results were reported as thepercentage of transfection activity of PEI–DNA complexeswithout adding Pluronics in serum-free conditions. All datawere obtained from triplicate measurements and the meanvalues +−S.D. were represented in the results. The trans-fection activity (100 %) = 546 +− 30 nmol of o-nitrophenylβ-D-galactopyranoside/incubation time per mg of protein.

Results

Cell viability assay of PluronicsStructures of the six different types of Pluronics used inthis study are illustrated in Figure 1. The order of HLBis F68 > F127 > P105 > P94 > L122 > L61. The effect ofPluronics concentrations on the relative viability of NIH/3T3is shown in Figure 2. The cell viability of all Pluronics testedis comparable with, or higher than, the positive control(100 %). The results show that all Pluronics used in the rangeof 0.01–10 % were not cytotoxic and could be safely usedfor transfection at these concentration ranges.

C© 2003 Portland Press Ltd

Effect of Pluronics on the serum-mediated inhibition of gene transfer 269

Figure 1 Chemical structure and comparison of HLB values of Pluronicsused in this study

x and z indicate the repeat units of PEO and y indicates the repeat units ofPPO.

Figure 2 Effect of Pluronics concentrations ranging from 0.01 to 10 % onthe relative viability of NIH/3T3 cells

Each point shows percentage viability (mean attenuance/mean positive controlattenuance +− S.D.) for three replicates. Positive control cells were grown in thepresence of fresh medium that did not contain any Pluronics. P94 and P105are both represented by �; the upper trace with � is P105.

Serum stabilization of PEI–DNA complexes withPluronicsCationic polyplexes and lipoplexes are often inactivated byinteractions with serum components [18,19]. Therefore theability of Pluronics to influence the serum stability of PEI–DNA complexes was tested. NIH/3T3 cells were transfectedwith the PEI–DNA (3:1, w/w) complexes with and withoutaddition of 1 % Pluronics. The choice of PEI/DNA ratiois based on the optimal gene expression in serum-freeconditions. A higher ratio of PEI/DNA resulted in highercytotoxicity on cells and a lower ratio led to lower geneexpression (results not shown). The media tested containedincreasing levels of serum from 10 to 50 % FBS. Figure 3summarizes the results of serum stabilization of PEI–DNAcomplexes in relation to Pluronics. As shown in Figure 3, the

Figure 3 Effects of FBS concentration on β-galactosidase transfectionactivity

NIH/3T3 cells were transfected with the PEI–DNA (3:1, w/w) complexes withand without 1 % Pluronics. The media assessed contained increasing levelsof serum from 0 to 50 % FBS. Results were reported as the percentage oftransfection activity of PEI–DNA complexes without adding Pluronics in serum-free conditions. All data were obtained from triplicate measurements and themeans +− S.D. were represented in the results.

PEI–DNA complexes without adding Pluronics were veryserum-sensitive and inactivated in media containing as littleas 10 % FBS. In the absence of serum, PEI–DNA complexeswith Pluronics (except L61) exhibited relatively comparableor even higher levels of transfection activity compared withPEI–DNA complexes alone, with the highest specific activityobserved in F68. As for the presence of serum (10 % FBS),the transfection activities of PEI–DNA complexes containingPluronics (except L61) obtained were higher than thoseobserved in serum-free media. Also, the total amount ofβ-galactosidase activity for Pluronics formulations slightlydecreased as the concentration of FBS increased from 20to 50 %. Transfection activity for all Pluronics tested (withthe exception of L61) greatly exceeded that of PEI–DNAalone. In the presence of serum, no obvious difference inthe level of specific activity was observed for F68, F127and P105 formulations. For P94 and L122 formulations,the levels of gene expression were lower than those withhigher HLB Pluronics in the presence of serum, but mostof the transfection activity found in serum-free conditionswas retained. As for L61, the level of specific activity wassignificantly inhibited in the presence of serum. It appearedthat the serum-sensitivity is dependent on the HLB ofPluronics. Sufficiently high HLB of Pluronics resulted inhigher serum stabilization of PEI–DNA complexes.

Effect of Pluronics concentration on serumstabilizationOne of the possible mechanisms for resisting the inhibitioneffect of serum proteins is the steric hindrance provided

C© 2003 Portland Press Ltd

270 J.-H. S. Kuo

Figure 4 Effect of Pluronics concentration (3 %) on serum stabilization

NIH/3T3 cells were transfected with the PEI/DNA (3:1, w/w) complexes withand without 3 % Pluronics. The media assessed contained increasing levelsof serum from 0 to 50 % FBS. Results were reported as the percentage oftransfection activity of PEI–DNA complexes without adding Pluronics in serum-free conditions. All data were obtained from triplicate measurements and themeans +− S.D. are represented in the results.

Figure 5 Effect of Pluronics concentration (0.1 %) on serum stabilization

All details were as for Figure 4, except that 0.1 % Pluronics was used whereindicated.

by the structure of Pluronics [9]. To test whether suchactivities are influenced by the concentration of Pluronics,two concentrations (0.1 and 3 %) were used to examinethe serum stabilization of PEI–DNA complexes by addingPluronics, and the results are shown in Figures 4 and 5. ForPEI–DNA complexes containing 3 % Pluronics (Figure 4),the levels of gene expression were close to 1 % Pluronicsformulations and no further augmentations were observed.Still, higher HLB values of Pluronics (F68, F127 andP105) formulations demonstrated higher serum-stabilitythan those with lower HLB values (P94 and L122). Also, L61formulations caused insignificant ability to resist the serum-

mediated inhibition of gene transfer of PEI–DNA complexes.The transfection activity in the presence of serum decreaseddramatically for Pluronics formulations as the concentrationdecreased to 0.1 % (Figure 5). The highest activities ofβ-galactosidase were detected at 10 % FBS for F127 andF68, which correspond to approx. 65 % of the gene-expression level observed in serum-free conditions. Also,the transfection activity for those prepared at 0.1 %Pluronics formulations decreased as the concentration ofFBS increased.

Discussion

In the present study, the effect of Pluronics on thereduction of serum-mediated inhibition of gene transferof PEI–DNA complexes was reported. PEI is known toimprove transfection activity in various cell lines and hasdemonstrated significant inhibitory effects in the presence ofserum. The effect of Pluronics on the serum stabilizationof PEI–DNA complexes is mainly determined by the value ofHLB. Among the types of Pluronics tested, ones withhigher HLB values were more effective in inhibiting seruminactivation in NIH/3T3 cells upon exposure of serum levelsup to 50 %. Pluronics with the lowest HLB, L61, did not showmuch improvement in transfection activity containing serumand serum-free conditions. Also, Pluronics have no cytotoxiceffects when added to NIH/3T3 cells at concentrationsranging from 0.01 to 10 %. These results suggest thatPluronics with higher HLB values can be used for delivery ofDNA into NIH/3T3 cells in the presence of serum.

Previous work suggests that Pluronics, such as P85,intensify non-specific endocytosis in eukaryotic cells anddramatically increase the transfection efficacy of polyplexes(nucleic acids–polymer complexes) [20]. Also, Pluronicssuch as poloxamer 407 improved the receptor-mediatedgene delivery to HepG2 and cervical cancer cells [21,22].This evidence indicated that supramolecular architectureof Pluronics may play a role in facilitating the entrance ofpolyplexes through cell membranes. It is likely that analteration or stabilization of the polyplex structure byPluronics with higher HLB values that makes it moreefficient. However, Pluronics tested in this study did notenhance the transfection activity of PEI–DNA complexesunder serum-free conditions. Instead, in the presence of10 % FBS, incorporation of higher-HLB Pluronics into PEI–DNA complexes provided higher transfection activity thanthose observed in serum-free conditions. The detailedmechanism for such unexpected activities is not fullyunderstood. On the basis of a previous report, in thepresence of 20 % FBS, transfection activity for F127- andF68-containing emulsions of lipoplexes was slightly higherthan those obtained in the absence of serum in BL-6cells [9]. Still, these results were also in accordance with

C© 2003 Portland Press Ltd

Effect of Pluronics on the serum-mediated inhibition of gene transfer 271

the observation that higher HLB of Pluronics is essential tomaintain serum stabilization for gene delivery of PEI–DNAcomplexes into NIH/3T3 cells.

The exact mixing order of Pluronics is also a criticalfactor to affect the levels of gene expression upon exposureof serum [16]. Pluronics must be added into DNA solutionsprior to complex formation; otherwise, serum inactivationwill inhibit the transfection activities of PEI–DNA complexes(J.-H. S. Kuo, Y.-H. Chen, R.-C. Lin, P.-W. Hsiao and M.-G.Tsai, unpublished work). It was believed that the hydrophilicPEO moieties in Pluronics play a role in relation to the sizeand the stability following various mixing orders [16]. Atconcentrations well above the critical micelle concentrationof Pluronics, the dynamic equilibrium between unimers andmicelles provides a favourable environment for complexstabilization [16]. Also, it was proposed that hydrophilicPEO chains provided steric-stabilization activity in DNA–lipid complexes [9]. This observation is consistent with datathat high concentrations of Pluronics (1 and 3 %) have strongresistance to the serum in transfer PEI–DNA complexes intoNIH/3T3 cells. In this research, the steric barrier providedby Pluronics at higher concentrations is sufficient to preventserum inactivation and physical instability of the dispersion.It is possible that Pluronics with higher HLB either functionby changing the structure of PEI–DNA complexes during thecomplexation process or exert their effects by complexingwith the serum rather than the polyplex. Further study isnecessary to examine and explain this mechanism of action.

Acknowledgment

I gratefully acknowledge the National Science Councilof the Republic of China (NSC 91-2216-E-041–001) forsupporting this research and also thank Dr Hsiao-Sheng Liu(Department of Microbiology and Immunology, College ofMedicine, National Cheng Kung University, Tainan, Taiwan,Republic of China) for providing pSG5LacZ and NIH/3T3cells.

References

1 De Smedt, S. C., Demeester, J. and Hennink, W. E. (2000)Pharm. Res. 17, 113–125

2 Gebhart, C. L. and Kabanov, A. V. (2001) J. Control. Release73, 401–416

3 Tang, M. X. and Szoka, F. C. (1997) Gene Ther. 4, 823–832

4 Boussif, O., Lezoualc’h, F., Zanta, M. A, Mergny, M. D.,Scherman, D., Demeneix, B. D. and Behr, J. P. (1995) Proc.Natl. Acad. Sci. U.S.A. 92, 7297–7301

5 Godbey, W. T., Wu, K. K. and Mikos, A. G. (1999) Proc. Natl.Acad. Sci. U.S.A. 96, 5177–5181

6 Godbey, W. T., Wu, K. K., Hirasaki G. J. and Mikos, A. G. (1999)Gene Ther. 6, 1380–1388

7 Felgner, P. L. and Ringold, G. M. (1989) Nature (London) 337,387–388

8 Kabanov, A. V. (1999) Pharm. Sci. Technol. Today 2, 365–372

9 Liu, F., Yang, J., Huang, L. and Liu, D. (1996) Pharm. Res. 13,1642–1646

10 Schmolka, I. R. (1977) J. Am. Oil Chem. Soc. 54, 110–11611 Alakhov, V. Y. and Kabanov, A. V. (1998) Expert Opin. Invest.

Drugs 7, 1453–147312 Bomford, R. (1981) Immunology 44, 187–19213 Kabanov, A. V., Lemieux, P., Vinogradov, S. and Alakhov, V.

(2002) Adv. Drug Deliv. Rev. 54, 223–23314 March, K. L., Madison, J. E. and Trapnell, B. C. (1995) Hum.

Gene Ther. 6, 41–5315 Nguyen, H. K., Lemieux, P., Vinogradov, S. V., Gebhart, C. L.,

Guerin, N., Paradis, G., Bronich, T. K., Alakhov, V. Y. andKabanov, A. V. (2000) Gene Ther. 7, 126–138

16 Gebhart, C. L., Sriadibhatla, S., Vinogradov, S., Lemieux, P.,Alakhov, V. and Kabanov, A. V. (2002) Bioconj. Chem 13,937–944

17 Boussif, O., Zanta, M. A. and Behr, J. P. (1996) Gene Ther. 3,1074–1080

18 Ogris, M., Brunner, S., Schuller, S., Kircheis, P. and Wagner, E.(1999) Gene Ther. 6, 595–605

19 Crook, K., Stevenson, B. J., Dubouchet, M. and Porteous, D. J.(1998) Gene Ther. 5, 137–143

20 Astafieva, I., Maksimova, I., lukanidin, E., Alakhov, V. andKabanov, A. (1996) FEBS Lett. 389, 278–280

21 Cho, C.-W., Cho, Y.-S., Lee, H.-K., Yeom, Y., Park, S.-N.and Yoon, D.-Y. (2000) Biotechnol. Appl. Biochem. 32,21–26

22 Cho, C.-W., Cho, Y.-S., Kang, B.-T., Hwang, J.-S., Park, S.-N. andYoon, D.-Y. (2001) Cancer Lett. 162, 75–85

Received 19 December 2002/12 February 2003; accepted 21 February 2003Published as Immediate Publication 21 February 2003, DOI 10.1042/BA20020123

C© 2003 Portland Press Ltd