Novel Thermoresponsive Nonviral Gene Vector: P(NIPAAm-co

Since viral vectors are limited due to the issues of safety, immunogenicity, and mutagenesis, there is a limited amount of genomic information, althou...
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Bioconjugate Chem. 2008, 19, 1368–1374

Novel Thermoresponsive Nonviral Gene Vector: P(NIPAAm-co-NDAPM)-b-PEI with Adjustable Gene Transfection Efficiency Han Cheng, Jing-Ling Zhu, Yun-Xia Sun, Si-Xue Cheng, Xian-Zheng Zhang,* and Ren-Xi Zhuo Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China. Received December 21, 2007; Revised Manuscript Received May 7, 2008

A thermoresponsive cationic copolymer, poly(N-isopropylacrylamide-co-N-(3-(dimethylamino)propyl)methacrylamide)-b-polyethyleneimine (P(NIPAAm-co-NDAPM)-b-PEI), was designed and synthesized as a potential nonviral gene vector. The lower critical solution temperature (LCST) of P(NIPAAm-co-NDAPM)-b-PEI in water measured by UV-vis spectroscopy was 38 °C. P(NIPAAm-co-NDAPM)-b-PEI as the gene vector was evaluated in terms of cytotoxicity, buffer capability determined by acid-base titration, DNA binding capability characterized by agarose gel electrophoresis and particle size analysis, and in vitro gene transfection. P(NIPAAm-co-NDAPM)b-PEI copolymer exhibited lower cytotoxicity in comparison with 25 kDa PEI. Gel retardation assay study indicated that the copolymer was able to bind DNA completely at N/P ratios higher than 30. At 27 °C, the mean particle sizes of P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes decreased from 1200 to 570 nm corresponding to the increase in N/P ratios from 10 to 60. When the temperature changed to 37 °C, the mean particle sizes of complexes decreased from 850 to 450 nm correspondingly within the same N/P ratio range due to the collapse of thermoresponsive PNIPAAm segments. It was found that the transfection efficiency of P(NIPAAm-co-NDAPM)b-PEI/DNA complexes was higher than or comparable to that of 25 kDa PEI/DNA complexes at their optimal N/P ratios. Importantly, the transfection efficiency of P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes could be adjusted by altering the transfection and cell culture temperature.

INTRODUCTION For human gene therapy, genes are introduced to cells to be transfected and produce therapeutic proteins (1–6). However, due to the lack of vectors that can safely and efficiently deliver nucleic acids to the target cells, gene therapy has not fulfilled its potential. Both viral and nonviral delivery systems for delivering genes into cells have been extensively investigated. Since viral vectors are limited due to the issues of safety, immunogenicity, and mutagenesis, there is a limited amount of genomic information, although they have high efficiency (7–10). Nonviral vectors such as liposomes and cationic polymers have been developed to overcome these limitations (1, 11). Watersoluble cationic polymers such as poly(ethyleneimine)s (PEIs) and their block copolymers have been studied as nonviral vectors for effective and safe gene delivery systems (11, 12). Although cationic polymers can carry much larger pieces of DNA as compared with the viral vectors, they also exhibit some problems such as the aggregation of complexes at physiological conditions and possible cytotoxicity on the target cells due to the high surface charge. The transfection efficiency and cytotoxicity of PEIs depend on the molecular weight greatly. As we know, the high molecular weight PEIs exhibit high transfection activity as well as high cytotoxicity compared with the low molecular weight PEIs. For example, the well-known commercial linear and branched 25 kDa PEIs, as an effective transfection reagent in vitro and in vivo, have high transfection efficiency but have high cytotoxicity at the same time. In order to enhance the transfection efficiency and decrease the cytotoxicity, synthetic cationic copolymers based on PEIs and stimuli-responsive * Corresponding author. Tel. & Fax: 86-27-6875 4509. E-mail address: [email protected] (X.-Z. Zhang).

polymers were investigated as nonviral gene vectors. The introduction of the stimuli-responsive polymers not only reduces the excess positive charge but also shows variable physiochemical characteristics under different cellular environments. Among various stimuli-responsive polymers, the most popular thermoresponsive polymer is poly(N-isopropylacrylamide) (PNIPAAm), which exhibits a phase transition at its lower critical solution temperature (LCST) around 32-33 °C. Several research groups reported temperature-sensitive vectors based on the PNIPAAmderived polymers (13–17). In this study, a novel thermoresponsive P(NIPAAm-coNDAPM)-b-PEI copolymer as gene vector was synthesized by a coupling reaction between P(NIPAAm-co-NDAPM)-COOH and 800 Da PEI. The copolymer as the gene vector was evaluated based on the cytotoxicity, buffer capability determined by acid-base titration, DNA binding capability characterized by agarose gel electrophoresis and particle size analysis, and in vitro gene transfection. It was found that the copolymer exhibited different binding capabilities with DNA upon temperature changes. In addition, the temperature-dependent transfection efficiency demonstrated that P(NIPAAm-co-NDAPM)b-PEI copolymer had great potential as a nonviral gene vector with low cytotoxicity and adjustable gene transfection efficiency.

EXPERIMENTAL PROCEDURES Materials. N-Isopropylacrylamide (NIPAAm) and 3-mercaptopropionic acid (MPA) were purchased from ACROS and used as received. N,N′-Carbonyldiimidazole (CDI) and branched PEIs (25 kDa and 800 Da) were purchased from Sigma and used as received. N-(3-(Dimethylamino)propyl)methacrylamide (NDAPM) was purchased from Japan Mark lot. N,N′-Dimethylformamide (DMF) was obtained from Shanghai Chemical Reagent Co. and used after distillation under reduced pressure.

10.1021/bc700478s CCC: $40.75  2008 American Chemical Society Published on Web 07/16/2008

Novel Thermoresponsive Nonviral Gene Vector

N,N′-Azobisisobutyronitrile (AIBN) was purchased from Shanghai Chemical Reagent Co., China, and used after recrystallization with 95% ethanol. Plasmid pGL-3 control with SV40 promoter and enhancer sequences encoding luciferase was obtained from Promega, Madison, WI, USA. Plasmid pEGFP-C1 encoding a red-shifted variant of wild-type green fluorescent protein (GFP) was purchased from Clontech, Mountain View, CA, USA. All other chemicals were analytical grade and used as received. Plasmid DNA Preparation and Purification. PGL-3 and EGFP-C1 plasmids were used for in vitro gene transfections. The former was transformed in E. coli JM109 and the latter was transformed in E. coli DH5R. Both plasmids were amplified in Luria-Bertani (LB) medium at 37 °C overnight at 250 rpm. Then, the plasmids were purified as described by means of EndoFree plasmid purification. The purified plasmids were diluted by TE buffer solution and stored at -20 °C. The integrity of the plasmid was confirmed by agarose gel electrophoresis. The purity and concentration of plasmids were determined by ultraviolet (UV) absorbance at 260 and 280 nm. Synthesis of P(NIPAAm-co-NDAPM)-b-PEI. P(NIPAAmco-NDAPM)-COOH was synthesized by free-radical copolymerization of NIPAAm (2.74 g) and NDAPM (150 µL) in DMF (10 mL) using AIBN (0.021 g) and MPA (0.418 g) as an initiator and a chain transfer reagent, respectively. After all the reactants were dissolved in DMF, the solution was degassed by bubbling with nitrogen for 30 min. Then the polymerization reaction was carried out at 70 °C under N2 atmosphere for 24 h. The P(NIPAAm-co-NDAPM)-COOH was obtained by precipitating the reaction mixture into diethyl ether. The precipitated polymer was isolated by filtration, dried under vacuum, and then reprecipitated from diethyl ether. Subsequently, resultant P(NIPAAm-co-NDAPM)-COOH (0.8348 g) and CDI (28 mg) were dissolved in DMSO (10 mL) and stirred for 3 h at room temperature. The solution was added quickly to a large excess of 800 Da PEI in DMSO (5 mL) with vigorous stirring. After 24 h, the product was precipitated in diethyl ether. The precipitated polymer was isolated by filtration and dried in a vacuum. Then, the dried polymer was further purified by dialysis against distilled water (MWCO 8000-12000) to remove the unreacted 800 Da PEI and other residues. Characterizations. Fourier transformed infrared (FTIR) spectra were recorded on a Lambda Bio40 UV-vis spectrometer (Perkin-Elmer). 1H nuclear magnetic resonance (NMR) spectra were recorded on a Varian Unity 300 MHz spectrometer using CDCl3 as a solvent. LCST Measurement. Optical absorbance of P(NIPAAmco-NDAPM)-COOH, P(NIPAAm-co-NDAPM)-b-PEI, and P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes at various temperatures was measured at 542 nm using a Lambda Bio40 UV-vis spectrometer (Perkin-Elmer). Sample cells were thermostatted in a refrigerated circulator bath at different temperatures from 31 to 60 °C prior to measurement. The heating rate was set at 0.1 °C/min. The LCSTs of the polymers and complexes were defined as the temperature showing a 50% change in the total optical absorbance. Cell Culture. Human embryonic kidney 293 cells (HEK293) were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin, 10000 U/mL) at 37 °C in a humidified atmosphere containing 5% CO2. Cell Viability Assay. For cell viability assay, the HEK293 cells (2500 cells/well) were seeded into 96-well plates. The cells were then incubated in culture medium containing polymer with a particular concentration for 48 h. After that, the medium was replaced with 200 µL of fresh medium, and 20 µL of sterile filtered MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg/mL) stock solution in PBS was added

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to each well, reaching a final concentration of 0.45 mg/mL. After 4 h, unreacted dye was removed by aspiration. The formazan crystals were dissolved in 200 mL DMSO per well and measured spectrophotometrically in an ELISA plate reader (model 550, Bio-Rad) at a wavelength of 570 nm. The cell survival was expressed as follows. cell viability ) (ODtreated ⁄ ODcontrol) × 100% where ODcontrol was obtained in the absence of polymers and ODtreated was obtained in the presence of polymers. Acid-Base Titration. The buffer capability of PEIs and P(NIPAAm-co-NDAPM)-b-PEI were determined by acid-base titration assay over the pH from 10 to 2 as described by Benns et al. (18, 19). Briefly, 0.2 mg/mL of each sample solution was prepared in 30 mL 150 mM NaCl solution. The pH value of the sample solution was adjusted to 10 by adding 0.1 M NaOH, and then the solution was titrated by 0.1 M HCl. During titration, the pH value of the mixture was measured using a microprocessor pH meter (pH 211, Germany). Polymer/DNAComplexFormation.P(NIPAAm-co-NDAPM)b-PEI was dissolved in NaCl solution (150 mM, pH 7.4) to form a solution with a concentration of 1 mg/mL. A plasmid DNA stock solution (120 ng/µL) was prepared in Tris-HCl buffer solution (pH 7.4). The total volumes of polycation solution and DNA stock solution were adjusted to 50 µL with 150 mM NaCl solution, respectively, and then two solutions were mixed together to form complexes with different N/P (the primary amino groups of PEI in the P(NIPAAm-co-NDAPM)b-PEI to phosphate groups) ratios. The formation of complexes was carried out for 30 min at 37 °C after vortexing polymer/ DNA mixture for 5 s. Agarose Gel Retardation Assay. The complexes at different N/P ratios were prepared by adding appropriate volumes of P(NIPAAm-co-NDAPM)-b-PEI (in 150 mM NaCl solution) to 96 ng of plasmid EGFP-C1 DNA (120 ng/µL in 40 mM TrisHCl buffer solution), and the complexes were diluted by 150 mM NaCl solution to 6 µL, then the complexes were incubated at 37 °C for 30 min. After that, the complexes were loaded on the 0.7% (w/v) agarose gel containing GelRed with Tris-acetate (TAE) running buffer at 80 V for 80 min. DNA was visualized with a UV lamp using a Vilber Lourmat imaging system (France). Particle Size Measurements. The P(NIPAAm-co-NDAPM)b-PEI/DNA complexes at various N/P ratios were prepared by adding appropriate volumes of P(NIPAAm-co-NDAPM)-b-PEI (in 150 mM NaCl solution) to 10 µg of plasmid DNA (in 40 mM Tris-HCl buffer solution), then the complexes were diluted by 150 mM NaCl solution to 1 mL and incubated at 27 and 37 °C for 30 min, respectively. The particle size was measured by Nano-ZS ZEN3600 (MALVERN Instruments) at 27 and 37 °C, respectively. Gene Transfection. For in vitro transfection study, the HEK293 cells were split one day prior to transfection and seeded in 24-well plates at a density of 5 × 104 cells/well. Before transfection, the cell culture medium was replaced with serumfree DMEM. The cells were transfected with P(NIPAAm-coNDAPM)-b-PEI/DNA complexes containing 1 µg of plasmid DNA at 25 and 37 °C for 4 h, respectively. Then, the complexes were removed and incubated in fresh DMEM with 10% FBS at 37 °C for another 48 h. Altogether, for the thermo-controlled assay, the complexes were incubated following the two different temperature programs: (1) 37 °C (4 h) f 37 °C (48 h) and (2) 27 °C (4 h) f 37 °C (48 h). Cell Tracing Assay. GelRed was used as a molecular probe to show the transport of P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes into cells. Complexes were incubated with serumfree DMEM in 24-well plates following the above two tem-

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Scheme 1. Schematic Illustration of the Synthesis of P(NIPAAm-co-NDAPM)-b-PEI

Figure 1. FTIR spectra of P(NIPAAm-co-NDAPM)-COOH (a) and P(NIPAAm-co-NDAPM)-b-PEI (b).

perature-controlled programs for 4 h. After that, the GelRed solution (at 1/5000 volume of the transfection medium) was added to 24-well plates and incubated 10 min before the serumfree DMEM was replaced by fresh DMEM with 10% FBS. Then, the cells were directly observed by an inverted microscope (TE-2000U, NIKON, JAPAN) after incubation for 4 and 12 h in DMEM with 10% FBS. Luciferase Assay. The cells were washed by PBS and lysed with the reporter lysis buffer (Promega, USA). The luciferase activity in cell extracts was measured using a luciferase assay kit (Promega, USA). Each measurement was carried out for 10 s in a single-well luminometer (Berthold Lumat LB 9507, Germany). The relative light units (RLU) were normalized by the total protein concentration of the cell extracts, and the total protein was measured with a BCA protein assay kit (PERBIO, USA). Luciferase activity was expressed as RLU/mg protein. GFP Detection. The cells which expressed green fluorescent proteins were directly observed by inverted microscope (IX 70, OLYMPUS, JAPAN).

RESULTS AND DISCUSSION Synthesis of P(NIPAAm-co-NDAPM)-b-PEI. PNIPAAm has been frequently used as a thermoresponsive segment to form block polymers with the LCST of around 32-33 °C (20). In the present study, a block copolymer of P(NIPAAm-coNDAPM)-b-PEI was prepared. The hydrophilic NDAPM units were used to adjust the LCST of P(NIPAAm-co-NDAPM)-bPEI copolymer. The synthesis of the P(NIPAAm-co-NDAPM)b-PEI copolymer involved two steps as illustrated in Scheme 1. P(NIPAAm-co-NDAPM)-COOH was synthesized by freeradical polymerization using AIBN as an initiator, and MPA as a chain transfer agent. Then, P(NIPAAm-co-NDAPM)-bPEI was prepared by coupling reaction between P(NIPAAmco-NDAPM)-COOH and PEI, using CDI as a coupling reagent. During the coupling reaction, the large excess 800 Da PEI was added to make sure that one PEI molecule would react with one P(NIPAAm-co-NDAPM)-COOH molecule, and the P(NIPAAm-co-NDAPM)-COOH would be consumed completely. The structure of polymers was confirmed by FTIR and 1H NMR spectroscopies. As shown in Figure 1, the absorbance of amide carbonyl groups in P(NIPAAm-co-NDAPM)-COOH occurred at 1630 cm-1 and the bending frequency of the amide N-H appeared at 1580 cm-1. The FTIR spectrum of P(NIPAAmco-NDAPM)-b-PEI was similar to that of P(NIPAAm-co-

Figure 2. The 1H NMR spectra of (1) P(NIPAAm-co-NDAPM)-COOH and (2) P(NIPAAm-co-NDAPM)-b-PEI in CDCl3.

NDAPM)-COOH due to the fact that the amide N-H peak of PEI overlapped the one of PNIPAAm. However, it was found that the absorbance of amide N-H in P(NIPAAm-co-NDAPM)b-PEI was greatly enhanced (Figure 1b) as compared with that of P(NIPAAm-co-NDAPM)-COOH, which indicated that the PEI was successfully grafted on P(NIPAAm-co-NDAPM)COOH. The 1H NMR spectra of P(NIPAAm-co-NDAPM)-COOH and P(NIPAAm-co-NDAPM)-b-PEI in CDCl3 are shown in Figure 2. Figure 2(1) and (2) both showed a single peak at δ 3.9-4.1 ppm, which could be attributed to the hydrogen of NCH(CH3)2 in the PNIPAAm segment. Besides, there existed a signal at around δ 1.0 ppm which could be assigned to the methyl in the PNIPAAm. In Figure 2(2), the broad peak δ 2.5-3.3 ppm was assigned to the methylene of branched 800 Da PEI. The integrals of signal δ 3.9-4.1 ppm and signal δ 2.5-3.3 ppm were 23 and 17, respectively. In addition, the molecular weights (Mn) of the polymers were calculated from the ratio of these two integrals. The Mn of P(NIPAAm-co-NDAPM)-COOH and P(NIPAAm-co-NDAPM)-b-PEI were 1.14 × 104 and 1.22 × 104, respectively. LCST Determination. The phase transition of thermoresponsive polymers in aqueous solution is attributed to a change in the hydrophilic/hydrophobic equilibrium of the polymers with respect to their hydrogen bonding between the polymer chains and water molecules. The light transmittance of a polymer solution could be examined as a function of temperature, and the LCST of the polymer was defined as the temperature producing a half-decrease of the total decrease in light transmittance. Generally, PNIPAAm itself exhibits a temperatureinduced phase transition at around 32-33 °C (20), and the complete transition occurs within a narrow temperature range. As shown in Figure 3, the LCST values of P(NIPAAm-coNDAPM)-COOH, P(NIPAAm-co-NDAPM)-b-PEI, and P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes are around 42, 38, and 37 °C, respectively. A well-recognized mechanism for the phase transition of the PNIPAAm polymer is the result of

Novel Thermoresponsive Nonviral Gene Vector

Figure 3. Thermoresponsive behavior of P(NIPAAm-co-NDAPM)COOH, P(NIPAAm-co-NDAPM)-b-PEI, and P(NIPAAm-co-NDAPM)b-PEI/DNA complexes.

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Figure 5. Agarose gel electrophoresis retardation assay of P(NIPAAmco-NDAPM)-b-PEI/DNA complexes at different N/P ratios of 0, 5, 10, 20, 30, 40, 50, and 60.

Figure 6. Particle size of P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes at N/P ratios ranging from 10 to 60 at different temperatures (27 and 37 °C). Data were shown as mean ( SD (n ) 3).

Figure 4. Acid-base titration curves of 25 kDa PEI, 800 Da PEI, and P(NIPAAm-co-NDAPM)-b-PEI in 150 mM NaCl solution.

a balance between hydrophilicity and hydrophobicity in the polymer chain. When a hydrophilic moiety is copolymerized into a PNIPAAm segment, the hydrophilic/hydrophobic balance shifts to a more hydrophilic nature, and the LCST would shift to a higher temperature accordingly (21, 22). The P(NIPAAm-co-NDAPM)-COOH and P(NIPAAm-coNDAPM)-b-PEI exhibited higher LCSTs compared with pure PNIPAAm, ascribed to the incorporation of hydrophilic NDAPM units. The fact that the LCST of P(NIPAAm-co-NDAPM)COOH was higher than that of P(NIPAAm-co-NDAPM)-b-PEI is due to the end group effect of terminal carboxylic acid groups (23, 24). Acid-Base Titration. The buffer capabilities of 25 kDa PEI, 800 Da PEI, and P(NIPAAm-co-NDAPM)-b-PEI are shown in Figure 4. It was found that the buffer capabilities of 25 kDa and 800 Da PEIs are similar, that is to say that the buffer capability is not directly correlated with the molecular weight. It was reported that the buffer capability depends on the primary, secondary, and ternary amines. Hence, the buffer capability of P(NIPAAm-co-NDAPM)-b-PEI, which is slightly lower than that of PEIs, could be ascribed to the reduction of positive charge density owing to the introduction of neutral PNIPAAm chains. Characterization of Polymer/DNA Complexes. The binding capability between the polycation and DNA is a prerequisite for the gene vector. When the DNA is condensed by the polycation, the condensed form can protect the DNA against digestion by enzymes. In addition, the compact unit facilitates the escape of the complex from endosomes into the cytoplasm, and then the DNA accumulates in the nucleus of the targeted cell where the gene is expressed. In order to study the ability of the polymer to condense DNA, the agarose gel assay was conducted. As shown in Figure 5, the plasmid DNA was retarded partly at the N/P ratio of 10. With an increase in N/P ratio, the

Figure 7. Cytotoxicity of P(NIPAAm-co-NDAPM)-b-PEI for HEK293 cells in comparison to 25 kDa PEI.

DNA binding capability of P(NIPAAm-co-NDAPM)-b-PEI was improved and the copolymer was able to bind DNA completely at N/P ratio higher than 30. Particle Size Measurement. The particle sizes of the polymer/DNA complexes at different N/P ratios at 27 and 37 °C are shown in Figure 6. The effect of the N/P ratio on the complex size shows a similar trend at 27 and 37 °C. At 27 °C, with an increase in the N/P ratio from 10 to 60, the particle size decreases from 1200 to 600 nm. At 37 °C, with an increase in N/P ratio from 10 to 60, the particle size decreases from 860 to 450 nm. It is obvious that the particle size of complexes depends on the temperature, and the particle size at 27 °C is larger than that at 37 °C at the same N/P ratio. As we know, at a higher temperature, the PNIPAAm chains collapse to form highly compact structure. At a lower temperature, the PNIPAAm chains get expanded to promote the dissociation of complexes. In the current study, at a lower N/P ratio, the complexes at both 27 and 37 °C form larger particles attributed to the weaker electrostatic interaction between P(NIPAAm-co-NDAPM)-bPEI and DNA. With increasing N/P ratio, the electrostatic interaction becomes stronger and more compact complexes are

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Figure 8. Transfection efficiency of P(NIPAAm-co-NDAPM)-b-PEI/ DNA and 25 kDa PEI/DNA complexes evaluated at N/P ratios range from 10 to 60 at two different cultured temperature programs: (1) 37 °C (4 h) f 37 °C (48 h); (2) 27 °C (4 h) f 37 °C (48 h). Data were shown as mean ( SD (n ) 3).

formed (25), and so, the particle size at 37 °C is smaller than that at 27 °C (25, 26), which implies that the particle size of P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes might be adjusted by changing the temperature. Cytotoxicity of Polymers. Low cytotoxicity is a very important requirement for gene vectors. In this study, the cytotoxicity of P(NIPAAm-co-NDAPM)-b-PEI was evaluated in HEK293 cells by MTT assay using 25 kDa PEI as the control. As shown in Figure 7, the viability of HEK293 cells treated with P(NIPAAm-co-NDAPM)-b-PEI at the concentration of 1.1 mg/mL is above 80%, indicating that the cytotoxicity of P(NIPAAm-co-NDAPM)-b-PEI is lower than that of 25 kDa PEI. Moreover, the cytotoxicity of P(NIPAAm-co-NDAPM)b-PEI increases slightly with increasing polymer concentration. The cytotoxicity of a polymer is correlated with the molecular weight as well as the cell binding between the polymer and cellular surfaces (27). The lower cytotoxicity of P(NIPAAm-

Cheng et al.

co-NDAPM)-b-PEI is attributed to the lower molecular weight of PEI and the shielding function of PNIPAAm chains, which can partially prevent the combination between P(NIPAAm-coNDAPM)-b-PEI and cellular surfaces. In Vitro Transfection. The commercially available branched 25 kDa PEI was used as the positive control due to its high transfection efficiency in vitro and in vivo. The plasmid PGL-3 was used as a reporter gene. In order to examine the influence of temperature on the transfection efficiency, the complexes were incubated following two different temperature programs: (1) 37 °C (4 h) f 37 °C (48 h) and (2) 27 °C (4 h) f 37 °C (48 h). The efficiency of P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes was evaluated at N/P ratios from 10 to 60, and the efficiency of 25 kDa PEI at the optimal ratio (N/P ) 10) was used as the control. As shown in Figure 8, the luciferase activities of P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes at various N/P ratios are 1.38 × 105 RLU/mg, 1.44 × 106 RLU/ mg, 3.11 × 107 RLU/mg, 1.72 × 107 RLU/mg, 3.21 × 107 RLU/mg, and 1.16 × 108 RLU/mg under the constant temperature program (1). However, under the condition of nonconstant temperature program (2), the luciferase activities of P(NIPAAmco-NDAPM)-b-PEI decrease 0.5-1000-fold as compared with that of the constant temperature program (1). As also shown in Figure 8, the efficiency of P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes at certain N/P ratios is higher than that of 25 kDa PEI/DNA complexes at its optimal N/P ratio under constant temperature program (1). To further illuminate the effect of temperature on the transfection efficiency, the tracing analysis was carried out using fluorescent microscopy, showing transport of the complexes into the cells. As shown in Figure 9, the GelRed labeled P(NIPAAmco-NDAPM)-b-PEI/DNA complexes initially dispersed on the cell surface after incubation in DMEM with 10% FBS for 4 h following two temperature programs (Figure 9a,c, arrows). After the complexes were incubated in DMEM with 10% FBS for 12 h, the labeled DNA was clearly restricted to the periphery

Figure 9. Cell tracing of GelRed labeled P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes in HEK293 cells at two different cultured temperature programs: (1) 37 °C (4 h) f 37 °C (48 h) for 4 h (a) and 12 h (b); (2) 27 °C (4 h) f 37 °C (48 h) for 4 h (c) and 12 h (d). The images were obtained at magnification of 100×.

Novel Thermoresponsive Nonviral Gene Vector

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Figure 10. The transfected HEK293 cells by P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes and 25 kDa PEI/DNA complexes observed by inverted microscope. The images in a, b, and c are the fluorescent images, and the images in d, e, and f are the bright-field images. The images (a, d) of P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes were obtained under the temperature program (1) with N/P ratio of 60, images (b, e) of P(NIPAAmco-NDAPM)-b-PEI/DNA complexes under the temperature program (2) with N/P ratio of 60, and images (c, f) of 25 kDa PEI/DNA complexes with N/P ratio of 10. The images were obtained at magnification of 100×.

of the nucleus following temperature program (1) (Figure 9b, asterisk). However, it was difficult to identify any labeled DNA entering the cells, and it was still dispersed on the cell surface after incubation in DMEM with 10% FBS for 12 h following temperature program (2) (Figure 9d, arrow). The results indicate that the transfection efficiency of P(NIPAAm-co-NDAPM)-bPEI/DNA complexes is adjustable through temperature control. However, the transfection result of P(NIPAAm-co-NDAPM)b-PEI/DNA complexes is partially different from that of the previously studied thermoresponsive complexes at two different temperature programs (13, 28). The difference was attributed to the different transfection methods. In this study, when the P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes were incubated in DMEM for 4 h at 37 °C, the complexes got associated compactly for cell uptake and prevention of DNA degradation; hence, the gene expression was conducted successfully. When the P(NIPAAm-co-NDAPM)-b-PEI/DNA complexes were incubated in DMEM for 4 h at 27 °C, the P(NIPAAm-coNDAPM)-b-PEI could not condense DNA compactly, as demonstrated by the particle size analysis and tracing assay. The transgene expression was limited and thus resulted in low transfection efficiency at 27 °C. In the literature reports (13, 28), the thermoresponsive complexes were first incubated for a few hours at 37 °C, and the complexes got associated tightly for cell uptake. After that, the complexes were further incubated for a couple of hours at temperatures below the LCST, resulting in the dissociation of the complexes, and then the free DNA would enter the nucleus easily. As a result, the transfection efficiency of the complexes incubated for a couple of hours at temperatures below the LCST is higher than that at temperature above the LCST. In a word, the transfection efficiency of thermoresponsive polymer/DNA complexes could be adjusted by altering the cultured temperature. In order to directly visualize the infected cells expressing GFP, EGFP-C1 encoded a red-shifted variant of wild-type GFP with optimized brighter fluorescence and higher expression in mammalian cell was used as a reporter gene. The cells expressing green fluorescence were observed by inverted microscope at the optimal N/P ratio for the polymer/DNA complexes. The transfection results are presented in Figure 10.

Figure 10a,b,c represents the fluorescent images, and Figure 10d,e,f represents the bright-field images. GFP is successfully expressed in HEK293 cells, the number of green fluorescent cells increases with increasing N/P ratio, and the nonconstant temperature program results in less GFP expression compared with the constant temperature program, which is consistent with luciferase reporter system.

CONCLUSIONS A novel thermoresponsive P(NIPAAm-co-NDAPM)-b-PEI block copolymer was prepared by the coupling reaction between P(NIPAAm-co-NDAPM)-COOH and 800 Da PEI. Compared with 25 kDa PEI, P(NIPAAm-co-NDAPM)-b-PEI copolymer exhibits much lower cytotoxicity. The particle size of P(NIPAAmco-NDAPM)-b-PEI/DNA complexes could be adjusted by varying the culture temperature. In vitro gene transfection reveals that the transfection efficiency of P(NIPAAm-coNDAPM)-b-PEI/DNA complexes is comparable to or even higher than that of 25 kDa PEI/DNA complex (N/P ) 10). Importantly, the gene transfection efficiency of P(NIPAAmco-NDAPM)-b-PEI/DNA complexes could be adjusted by altering the transfection and incubation temperature, suggesting that the P(NIPAAm-co-NDAPM)-b-PEI has great potential as an intelligent and effective nonviral gene vector.

ACKNOWLEDGMENT This work was supported by National Key Basic Research Program of China (2005CB623903), National Natural Science Foundation of China (20504024, 50633020), and Ministry of Education of China (Cultivation Fund of Key Scientific and Technical Innovation Project 707043).

LITERATURE CITED (1) Laporte, L. D., Rea, J. C., and Shea, L. D. (2006) Design of modular non-viral gene therapy vectors. Biomaterials 27, 947– 954. (2) Zhang, X. J., and Godbey, W. T. (2006) Viral vectors for gene delivery in tissue engineering. AdV. Drug DeliVery ReV. 58, 515– 534.

1374 Bioconjugate Chem., Vol. 19, No. 7, 2008 (3) Olefsky, J. M. (2000) DiabetesGene therapy for rats and mice. Nature 408, 420–421. (4) Song, S., Goudy, K., Campbell-Thompson, M., Wasserfall, C., Scott-Jorgensen, M., Wang, J., Tang, Q., Crawford, J. M., Ellis, T. M., Atkinson, M. A., and Flotte, T. R. (2004) Recombinant adeno-associated virusmediated alpha-1 antitrypsin gene therapy prevents type I diabetes in NOD mice. Gene Ther. 11, 181–186. (5) El-Aneed, A. (2004) An overview of current delivery systems in cancer gene therapy. J. Controlled Release 94, 1–14. (6) Merdan, T., Kopecek, J., and Kissel, T. (2002) Prospects for cationic polymers in gene and oligonucleotide therapy against cancer. AdV. Drug DeliVery ReV. 54, 715–758. (7) Lehrman, S. (1999) Virus treatment questioned after gene therapy death. Nature 401, 517–518. (8) Liu, Q., and Muruve, D. A. (2003) Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther. 10, 935–940. (9) Sun, J. Y., Anand-Jawa, V., Chatterjee, S., and Wong, K. K. (2003) Immune responses to adeno-associated virus and its recombinant vectors. Gene Ther. 10, 964–976. (10) Gore, M. E. (2003) Adverse effects of gene therapy, gene therapy can cause leukaemia, no shock, mild horror but a probe. Gene Ther. 10, 4–4. (11) Kunath, K., Harpe, A. V., Fischer, D., Petersen, H., Bickel, U., Voigt, K., and Kissela, T. (2003) Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery, comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine. J. Controlled Release 89, 113–125. (12) Itaka, K., Yamauchi, K., Harada, A., Nakamura, K., Kawaguchi, H., and Kataoka, K. (2003) Polyion complex micelles from plasmid DNA and poly(ethyleneglycol)-poly(L-lysine) block copolymer as serum-tolerable polyplex system, physicochemical properties of micelles relevant to gene transfection efficiency. Biomaterials 24, 4495–4506. (13) Kurisawa, M., Yokoyama, M., and Okano, T. (2000) Gene expression control by temperature with thermo-responsive polymeric gene carriers. J. Controlled Release 69, 127–137. (14) Tu¨rk, M., Dinc¸er, S., Yulugj, I. G., and Pis¸kin, E. (2004) In vitro transfection of HeLa cells with temperature sensitive polycationic copolymers. J. Controlled Release 96, 325–340. (15) Twaites, B. R., Alarco´n, C. H., Lavigne, M., Saulnier, A., Pennadam, S. S., Cunliffe, D., et al. (2005) Thermoresponsive polymers as gene delivery vectors, cell viability, DNA transport and transfection studies. J. Controlled Release 108, 472–483. (16) Twaites, B. R., Heras Alarco´n, C., Cunliffe, D., Lavigne, M., Pennadam, S., and Smith, J. R. (2004) Thermo and pH responsive polymers as gene delivery vectors, effect of polymer architecture on DNA complexation in vitro. J. Controlled Release 97, 551– 566.

Cheng et al. (17) Bisht, H. S., Manickam, D. S., You, Y. Z., and Oupicky, D. (2006) Temperature-controlled properties of DNA complexes with poly(ethylenimine)-graft-poly(N-isopropylacrylamide). Biomacromolecules 7, 1169–1178. (18) Benns, J. M., Choi, J. S., Mahato, R. I., Park, J. S., and Kim, S. W. (2000) pH-Sensitive cationic polymer gene delivery vehicle N-ac-poly(L-histidine)-graft-poly(L-lysine) comb shaped polymer. Bioconjugate Chem. 11, 637–645. (19) Benns, J. M., Mahato, R. I., and Kim, S. W. (2002) Optimization of factors influencing the transfection efficiency of folate-PEG-folate-graft-polyethylenimine. J. Controlled Release 79, 255–269. (20) Li, Y. Y., Zhang, X. Z., Cheng, H., Zhu, J. L., Cheng, S. X., and Zhuo, R. X. (2006) Self-assembled, thermoresponsive PCLg-P(NIPAAm-co-HEMA) micelles for drug delivery. Macromol. Rapid Commun. 27, 1913–1919. (21) Vernon, B., Kim, S. W., and Ba, Y. H. (2000) Thermoreversible copolymer gels for extracellular matrix. J. Biomed. Mater. Res. 51, 69–79. (22) Shibayama, M., Fujikawa, Y., and Nomura, S. (1996) Dynamic light scattering study of poly(N-isopropylacrylamide-co-acrylic acid) gels. Macromolecules 29, 6535–6540. (23) Kujawa, P., Segui, F., Shaban, S., Diab, C., Okada, Y., Tanaka, F., and Winnik, F. M. (2006) Impact of end-group association and main-chain hydration on the thermosensitive properties of hydrophobically modified telechelic poly(N-isopropylacrylamides) in water. Macromolecules 39, 341–348. (24) Xia, Y., Burke, N. A. D., and Sto¨ver, H. D. H. (2006) End group effect on the thermal response of narrow-disperse poly(Nisopropylacrylamide) prepared by atom transfer radical polymerization. Macromolecules 39, 2275–2283. (25) Cheng, N., Liu, W. G., Cao, Z. Q., Ji, W. H., Liang, D. C., Guo, G., and Zhang, J. Y. (2006) A study of thermoresponsive poly(N-isopropylacrylamide)/polyarginine bioconjugate non-viral transgene vector. Biomaterials 27, 4984–4992. (26) Sun, S. J., Liu, W. G., Cheng, N., Zhang, B. Q., Cao, Z. Q., Yao, K. D., Liang, D. H., Zuo, A. J., Guo, G., and Zhang, J. Y. (2005) A thermoresponsive chitosan-NIPAAm/vinyl laurate copolymer vector for gene transfection. Bioconjugate Chem. 16, 972–980. (27) Clements, B. A., Bai, J., Kucharski, C., Farrell, L. L., Lavasanifar, A., Ritchie, B., Ghahary, A., and Uludag, H. (2006) RGD conjugation to polyethyleneimine does not improve DNA delivery to bone marrow stromal cells. Biomacromolecules 7, 1481–1488. (28) Lavigne, M. D., Pennadam, S. S., Ellis, J., Yates, L. L., Alexander, C., and Go´recki, D. C. (2007) Enhanced gene expression through temperature profile-induced variations in molecular architecture of thermoresponsive polymer vectors. J. Gene Med. 9, 44–54. BC700478S