Transfection efficiency increases by incorporating hydrophobic monomer units into polymeric gene carriers

Journal of Controlled Release 68 (2000) 1–8 www.elsevier.com / locate / jconrel

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Transfection efficiency increases by incorporating hydrophobic monomer units into polymeric gene carriers Motoichi Kurisawa, Masayuki Yokoyama, Teruo Okano* Institute of Biomedical Engineering, Tokyo Women’ s Medical University, 8 -1 Kawada Shinjuku, Tokyo 162 -8666, Japan Received 18 December 1999; accepted 10 March 2000

Abstract The water soluble terpolymer, poly(N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-butylmethacrylate (BMA)) was synthesized, and its efficiency in in vitro gene transfection was evaluated. Copolymers with different compositions were synthesized by radical polymerization. For a series of copolymers containing 60 mol% of DMAEMA, the plasmid bands were retained within the gel loading slot, independent of polymer / plasmid weight ratios or BMA monomer content. In contrast, for a series of copolymers containing 20 mol% DMAEMA, plasmid bands of complexes were retarded with increasing weight ratios. For the copolymer with 10 mol% BMA content, the plasmid was completely retained within the gel loading slot. The transfection efficiency of polymer / plasmid complexes was evaluated in COS-1 cells using a pCMV-lacZ plasmid, encoding for b-galactosidase as a reporter gene. Transfection efficiency of a series of copolymers containing 20 mol% of DMAEMA varied with BMA content. The transfection efficiency of the copolymers with 0, 2, and 5 mol% of BMA was low. The transfection efficiency of the copolymers with 10 mol% of BMA was about 2-fold higher than that of the PDMAEMA control homopolymer. The transfected cells were observed at a very wide range of polymer / plasmid weight ratios. The transfection efficiency of all copolymers containing 60 mol% of DMAEMA was lower than that of the PDMAEMA homopolymer.  2000 Elsevier Science B.V. All rights reserved. Keywords: Transfection; Cationic polymers; Plasmid; Gene delivery; Hydrophobic interaction

1. Introduction Gene therapy is defined as delivery of genes for expression into a patient’s host cells to enable production of proteins to correct or moderate a disease [1]. To achieve gene delivery, safe vectors with selective and high transfection efficiency are required. A number of vectors for gene delivery have been studied. However, presently available vectors *Corresponding author. Tel.: 181-3-3353-8111; fax: 181-33359-6046. E-mail address: [email protected] (T. Okano)

are not satisfactory in terms of efficiency, selectivity and safety. Polymeric drug carrier systems [2–4] have been extensively studied and applied to various drug therapies including cancer chemotherapy, exemplified by an approved system (SMANCS) [5] and another system in clinical trials [6]. In the design of polymeric carriers, hydrophobic–hydrophilic balance of the carrier system is a very important factor, since it regulates interactions with proteins, cells and other biocomponents [7,8]. Therefore, this balance determines the biodistribution and pharmacokinetic behavior of the carrier systems. Furthermore, this hydrophobic–hydrophilic balance regulates the as-

0168-3659 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 00 )00246-7

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sociation behavior of the carrier polymers [8,9], and this association behavior considerably influences targeting characteristics. By locating a hydrophobic part and a hydrophilic part separately using a block copolymer architecture, we have obtained and extensively studied a polymeric micelle carrier system [8]. In the polymeric micelle system, efficient drug targeting was achieved [10–12] by optimizing biodistribution, pharmacokinetic behavior, and association behavior due to the well designed core (hydrophobic)–shell (hydrophilic) structure. Viral vectors are most commonly used due to their high transfection efficiency. The viral vectors, however, have disadvantages associated with their pathogenic or immunogenic properties [13]. An alternative approach to the development of gene therapy using non-viral vectors, such as cationic liposomes and cationic polymers, has been studied [14–20]. Although non-viral vectors possess advantages over viral vectors, non-viral vectors remain several orders of magnitude below viral vectors in transfection efficiency [21,22]. Generally, liposome / DNA and polymer / DNA complexes are formed by ionic interaction between polymer cationic units and DNA phosphate anions. These complexes are considered to be taken up by endocytosis [23–25]. Polymeric gene carriers may have some advantages over liposomes: (i) relatively small size and narrow distribution of complex, (ii) high stability against nuclease, and (iii) versatile control of hydrophilicity of complex by copolymerization [26]. Complexes comprising polymers and DNA are formed by ionic interactions. Such complex formation using ionic interaction leads to increased cell uptake and prevents DNA degradation by lysosomal enzymes. However, most of the polymeric gene vectors show lower transfection efficiency than cationic liposomes. One hypothesis to explain this lower efficiency is that polymer–DNA complexes have difficulty to dissociate to free DNA within cells. Formation of polymer / DNA complexes by stable ionic interactions between amino groups and phosphate groups may be strong enough to resist dissociation. From these perspectives, we focused on controlling formation / dissociation of complexes to increase transfection efficiency of non-viral vectors. We have designed gene carrier polymers both with aminocontaining and hydrophobic monomer units. Hydro-

phobic monomer units are expected to increase transfection efficiency by modulating complex interactions with cells, such as adsorption on cell surfaces and cell uptake. On the other hand, hydrophobic interactions between the hydrophobic monomer units are expected to inhibit dissociation of polymer / DNA complexes to a much lesser degree than ionic interactions between cationic units and phosphates of DNA, since DNA does not participate in hydrophobic interactions. These favorable characteristics of the hydrophobic units may lead to higher transfection efficiency than polymer systems using only ionic interactions. In this study, we synthesized new copolymers with different compositions of N-isopropylacrylamide (IPAAm), 2-(dimethylamino)ethyl methacrylate (DMAEMA) and butylmethacrylate (BMA) for application as gene carriers. In these terpolymers, BMA is the hydrophobic component. Hence, the stability of the polymer / DNA complex may be regulated by not only ionic but also hydrophobic interactions. Complexes of copolymers and plasmid DNA were transfected into model cells in vitro, and transfection efficiency was examined in relation to carrier monomer content.

2. Experimental

2.1. Materials N-Isopropylacrylamide (IPAAm, kindly provided by Kohjin, Tokyo Japan) was purified by recrystallization in hexane and dried in vacuo at room temperature. 2-(Dimethylamino)ethyl methacrylate (DMAEMA) and butylmethacrylate (BMA) were purchased from Wako (Osaka, Japan), and distilled under reduced pressure. 2,29-Azobisisobutyronitrile (AIBN, Wako, Osaka, Japan) was purified by recrystallization from methanol. Tetrahydrofuran (THF) was purified by distillation. Other chemicals were reagent-grade commercial materials and used without further purification.

2.2. Synthesis of Poly( IPAAm-co-DMAEMA-coBMA) Copolymers of IPAAm, DMAEMA, and BMA

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with different compositions were synthesized by radical copolymerization using AIBN as a radical initiator. Monomers and AIBN (0.2 mM) were dissolved in 35 ml of THF. This solution was degassed repeatedly under reduced pressure in freeze–thaw cycles and sealed with a two-way stopcock. Polymerization was carried out at 608C for 15 h. Then, the solution was poured into an excess amount of petroleum ether, and precipitated polymer was dried in vacuo. The dried polymer was dissolved in cold water and dialyzed against water using a dialysis membrane (Spectra / Por3 membrane, molecular weight cut-off (MWCO)53500) at 48C for 24 h to remove unreacted monomers. By lyophilization, the polymer was successfully obtained as white powder. Molecular weights of the copolymers were determined by gel-permeation chromatography (GPC, TOSOH, SC-8020, polystyrene standards) in DMF containing LiCl (10 mM) (elution rate: 1 ml / min) at 408C.

2.3. Transmittance measurements of copolymer solution Optical transmittance of aqueous polymer solution (2 mg / ml, 0.2 wt%) at various temperatures was measured at 500 nm with a UV-VIS spectrometer (V-530, Japan Spectroscopic, Tokyo, Japan). Sample cells were thermostated with a Peltier-effect cell holder (EHC-477, Japan Spectroscopic). Heating rate was 0.18C / min. The LCST of polymer solutions was determined as the temperature showing a 1% decrease in optical transmittance.

2.4. pKa measurements The pKa values of copolymers were determined by titration. The copolymers were dissolved in 0.9 wt% NaCl solution, acidified with 0.1 M HCl and titrated with 0.05 M NaOH.

2.5. Gel retardation assay Complexes of plasmid DNA (2 mg) with various copolymers were formed by mixing a plasmid DNA stock solution with polymer stock solutions (500 mg / ml in PBS), and the total volume was adjusted to 200 ml with PBS. The complex formation was

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carried out at 378C for 1 h. Then, 30 ml of complex solution was run on a 0.6 wt% agarose gel (100 V) and the bands were visualized by ethidium bromide staining.

2.6. Preparation of pCMV-LacZ pCMV-LacZ was kindly donated by Dr J. Lindsay Whitton of the Scripps Research Institute, La Jolla, CA, USA [27]. The plasmid was amplified in E. coli (strain DH5a) and purified by column chromatography (Qiagen  Plasmid Mega Kit, Germany). Plasmid purity was measured by OD 260 / OD 280 (OD, optical density). The ratio was between 1.85 and 1.90. Plasmid concentration was determined using the equation; 1OD 260 550 mg / ml of plasmid DNA.

2.7. Cell lines for transfection COS-1 cells (SV-40 transformed African green monkey) were purchased from Health Science Research Resources Bank (Osaka, Japan) and cultured in DMEM supplemented with 10% FBS, 100 Units / ml of penicillin, and 100 mg / ml of streptomycin in a humidified atmosphere 5% CO 2 incubator.

2.8. DNA transfection of cells COS-1 cells were seeded at a concentration of 4310 5 cells / ml, 160 ml / well, in 96-well flat-bottomed microassay plates (Falcon Co., Becton Dickinson, Franklin Lakes, NJ), 15 h before transfection experiments. Then, 4 mg of pCMV-LacZ was diluted into 100 ml of RPMI-1640. A desired amount of copolymer stock solution (500 mg / ml) was diluted into 300 ml of RPMI-1640. The complex solution was prepared by mixing 100 ml of the plasmid solution and 300 ml of the polymer solution and incubated at 378C for 1 h. Copolymer was used at copolymer / plasmid weight ratios between 1 and 30. Then, 100 ml of the complex solution was added to 96-well plates, and incubated at 378C for 1 h. After removal of the complex solution, fresh DMEM medium containing 10% FBS was added to the plate, and the cells were cultured for 47 h at 378C in a humidified 5% CO 2 atmosphere. To evaluate transfection efficiency, X-gal histochemical staining of

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transfected cells was performed for detection of expressed b-galactosidase [28]. The cells in the 96well plates were fixed with 0.25% glutaraldehyde in PBS at room temperature for 5 min, washed twice with PBS and incubated with a histochemical reaction solution (1 mg / ml X-Gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mM MgCl 2 in PBS). After a 5-h incubation in the histochemical reaction solution, all blue cells in a well were counted using microscopy.

were reduced with increasing BMA content from hydrophobic contributions of BMA groups. PIPAAm is well known to exhibit a lower critical solution temperature (LCST) at 328C [29] and has been applied extensively in thermo-responsive hydrogels [30–32], bioconjugates [33], and chromatography systems [34–36]. The LCST values were observed to be increased and decreased by incorporation of amine monomer units (DMAEMA) and hydrophobic units (BMA), respectively. This thermoresponsive character was lost by complexation with plasmid DNA, pCMV-LacZ. All copolymer solutions formed water-insoluble precipitates immediately after mixing with plasmid DNA at a concentration of 0.2 wt% even at 48C, an LCST below that of any of the copolymers. The turbid solutions containing these complex precipitates did not show any changes in solution status in a temperature range from 4 to 508C. Fig. 1 shows agarose gel electrophoresis results for copolymer / plasmid complexes. Copolymers containing 20 mol% of DMAEMA (Fig. 1a) showed DNA band retardation with increasing copolymer weight ratio. This retardation is considered due to an increase in molecular weight by complexation with copolymers as well as a decrease of phosphate anion content in the plasmid DNA by complex formation. Such a retardation was enhanced for P(IP-20DA10BM); the plasmid was completely retained within the gel loading slot at any weight ratio from 1 to 4. From these results, it is suggested the complex

3. Results and discussion

3.1. Synthesis and characterization of poly( IPAAmco-DMAEMA-co-BMA) Poly(IPAAm-co-DMAEMA-co-BMA) with various monomer ratios as gene carriers were synthesized by radical polymerization, as shown in Table 1. Copolymers were obtained with 64–79% yields and their molecular weights were in a range of 40 000– 200 000 g / mol. Here, the copolymers prepared with various monomer ratios ((DMAEMA: 20 and 60 mol% in feed) and (BMA: 0, 2, 5 and 10% in feed)) are designated as P(IP-20DA), P(IP-20DA-2BM), P(IP-20DA-5BM), P(IP-20DA-10BM), P(IP-60DA), P(IP-60DA-2BM), P(IP-60DA-5BM), and P(IP60DA-10BM), respectively. Lower critical solution temperatures (LCST) of these polymer solutions Table 1 Synthesis of poly(IPAAm-co-DMAEMA-co-BMA)a Code

PDMAEM P(IP-20DA) P(IP-20DA-2BM) P(IP-20DA-5BM) P(IP-20DA-10BM) P(IP-60DA) P(IP-60DA-2BM) P(IP-60DA-5BM) P(IP-60DA-10BM) a

Monomer content in feed (mol%)

Monomer content in feed (mol%)d

IPAAm

DMAEMA

BMA

IPAAm

DMAEMA

BMA

– 80 78 75 70 40 38 35 30

100 20 20 20 20 60 60 60 60

– – 2 5 10 – 2 5 10

– 73 71 68 63 44 38 31 21

100 27 24 22 22 56 59 62 65

– – 5 10 15 – 3 7 14

Reaction was carried out using AIBN as a radical initiator in THF for 15 h. Measured by GPC. c LCST was measured in PBS (pH 7.4) with a UV-VIS spectrometer. d Determined by 1 H-NMP spectroscopy. b

Mw b

Mw /M nb

Yield (%)

LCST c (8C)

pKa

127 000 40 000 80 000 78 000 102 000 78 000 201 000 206 000 221 000

1.75 1.87 2.01 2.07 1.84 2.31 2.08 2.09 2.22

78 78 79 72 64 69 65 67 68

– 42.4 35.0 30.9 28.4 52.1 41.5 37.3 35.4

7.6 8.0 8.0 8.0 7.6 7.6 7.6 7.7 7.6

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Fig. 1. Electrophoretic mobility of polymer / plasmid complexes on a 0.6% agarose gel electrophoresis. (a) Lane 1, plasmid DNA (2 mg); lanes 2–5, plasmid DNA (2 mg) mixed with 2, 4, 6, and 8 mg of P(IP-20DA); lanes 6–9, plasmid DNA (2 mg) mixed with 2, 4, 6, and 8 mg of P(IP-20DA-2BM); lanes 10–13, plasmid DNA (2 mg) mixed with 2, 4, 6, and 8 mg of P(IP-20DA-5BM); lanes 14–17, plasmid DNA (2 mg) mixed with 2, 4, 6, and 8 mg of P(IP-20DA-10BM). (b) Lane 1, plasmid DNA (2 mg); lanes 2–5, plasmid DNA (2 mg) mixed with 2, 4, 6, and 8 mg of P(IP-60DA); lanes 6–9, plasmid DNA (2 mg) mixed with 2, 4, 6, and 8 mg of P(IP-60DA-2BM); lanes 10–13, plasmid DNA (2 mg) mixed with 2, 4, 6, and 8 mg of P(IP-60DA-5BM); lanes 14–17, plasmid DNA (2 mg) mixed with 2, 4, 6, and 8 mg of P(IP-60DA-10BM). Plasmid DNA was visualized by ethidium bromide staining.

formation between copolymer and plasmid is controlled not only by amino group content but also by BMA hydrophobic group content. DNA condensation is considered to be due to cooperative effect of electrostatic interaction and hydrophobic interaction with the complexed sites. These results indicate that hydrophobic monomer units as well as cationic amino group-containing monomer units are important factors for complex formation with plasmid DNA. For copolymers containing 60 mol% of DMAEMA, the plasmid bands were retained within

the gel loading slot, independently of weight ratio and BMA content (Fig. 1b). This shows that amino group contents higher than 20 mol% DMAEMA initiate stronger complex formation with plasmids.

3.2. In vitro transfection of poly( IPAAm-coDMAEMA-co-BMA) /plasmid complexes Various complexes of copolymers / plasmid were formulated with a fixed amount of plasmid and various amounts of copolymers. PDMAEMA homo-

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polymer has already been developed as an efficient gene carrier [20]. A low pKa value (7.4) of PDMAEMA amino groups is believed to contribute to its high transfection efficiency [37]. Recently, Hennink et al. [38] have reported copolymers of IPAAm and DMAEMA as gene carriers. The cytotoxicity and transfection efficiency of copolymers are dependent on their monomer compositions and molecular weights. The transfection efficiency of polymer / plasmid complexes was evaluated in COS-1 cells lines using a pCMV-lacZ plasmid, encoding for b-galactosidase as a reporter gene. Fig. 2a shows the transfection

Fig. 2. Transfection efficiency of poly(IPAAm-co-DMAEMA-coBMA) / plasmid complexes. COS 1 cells were incubated with various complexes at 378C for 1 h. Histochemical reaction using b-gal was carried out at 378C for 5 h, and then all blue cells in a well were counted in a microscopic view (n53, 6S.D.) (a) s: PDMAEMA, h: P(IP-60DA), ^: P(IP-60DA-2BM), \: P(IP60DA-5BM), d: P(IP-60DA-10BM). (b) s: PDMAEMA, h: P(IP-20DA), ^: P(IP-20DA-2BM), \: P(IP-20DA-5BM), d: P(IP-20DA-10BM).

efficiency of a series of copolymers containing 60 mol% of DMAEMA with various BMA contents. Copolymers both without BMA units and with 2 mol% BMA units showed only a few transfected cells in weight ratios from 1 to 30. Many more transfected cells were observed using P(IP-60DA5BM) and P(IP-60DA-10BM), and maximum efficiency of both polymers was found at a weight ratio of 2. The transfection efficiency of all the copolymers was, however, less than that of PDMAEMA homopolymer. Fig. 2b shows transfection efficiency from copolymers containing 20 mol% of DMAEMA with various contents of BMA. The transfection efficiency of PDMAEMA homopolymer as a control was increased with increasing weight ratio until the maximum was reached at a weight ratio of 2. The transfection efficiency of the copolymers was varied with BMA content. For P(IP-20DA), P(IP-20DA2BM), and P(IP-20DA-5BM), few transfected cells were observed. In contrast, the transfection efficiency of P(IP-20DA-10BM) was considerably higher than that of the PDMAEMA homopolymer. This higher transfection efficiency was shown by the larger maximum number of transfected cells and a wider weight ratio range for transfection. A polymer / DNA weight ratio of 3, showing a maximum transfected cell number, was greater than that (weight ratio of 2) of the PDMAEMA homopolymer. Another notable finding is the significantly expanded polymer / DNA weight ratio range of the copolymer producing transfection. While PDMAEMA homopolymer did not show transfection at a weight ratio of 5, P(IP20DA-10BM) exhibited transfection up to a ratio of 30. This dramatic improvement may be related to the reduced cell toxicity of the P(IP-20DA-10BM) / DNA complex. Sato et al. [39] has reported complexes of DNA and lipoglutamate with different alkyl chains. The cell uptake and transfection efficiency of this lipid– DNA complex system are both increased with increasing hydrophobicity (increasing alkyl chain length) of the lipoglutamate. Kabanov et al. [40] compared the transfection efficiency of cationic monomer units using hydrophobic cationic monomer units in their polymer carrier system. A notable finding of our present paper is a considerable increase in transfection efficiency by incorporating

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hydrophobic monomer units (BMA) into P(IP-DA) cationic copolymers. This hydrophobic component incorporation may influence the hydrophobicity, size, and stability of DNA–polymer complexes. These physical factors are considered to contribute to increased transfection efficiency by modulating complex action with / in cells, such as surface adsorption on cells and subsequent cell uptake. Adjustment of such DNA–carrier complex hydrophobicity can be accomplished more easily for synthetic polymeric gene carriers than lipid–DNA systems by using copolymerization of a desirable amount of hydrophobic monomers. This is a significant advantage of polymeric gene carriers. In conclusion, copolymers of IPAAm, DMAEMA and BMA were synthesized and their potential as gene carriers was evaluated. Transfection efficiency was increased by hydrophobic unit incorporation. Moreover, the effective range of polymer / DNA weight ratios was significantly expanded by polymer carrier hydrophobic unit incorporation.

Acknowledgements The present study was supported by the Japan Society for the Promotion of Science, ‘Research for the Future’ Program (JSPS-RFTF96I00201) and a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan. We gratefully acknowledge Dr J. Lindsay Whitton at the Scripps Research Institute for the gift of pCMV-LacZ and Professor David W. Grainger of Colorado State University for his valuable comments.

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